JP2008516996A - Antiviral oligonucleotide - Google Patents

Abstract

Described are random sequence oligonucleotides with antiviral activity and their use as antiviral agents. In many cases, the oligonucleotide has a nucleotide length greater than 40 and includes chemical modifications such as, for example, a modified internucleotide linkage and a modification at the 2 ′ position of the ribose moiety. Also described are uses for the prevention or treatment of viral infections in humans and animals, and use for the prevention or treatment of cancers caused by human and animal oncoviruses. These uses typically provide a pharmacologically acceptable therapeutically effective amount of at least one oligonucleotide that acts in a sequence-independent manner on a human or animal in need of such treatment. Administration.

Description

FIELD OF THE INVENTION The present invention relates to oligonucleotides having antiviral activity and their use as therapeutics in viral infections caused by human and animal viruses, cancers caused by oncogene viruses and other diseases whose etiology is virus-based. .

BACKGROUND OF THE INVENTION The following description is provided solely to aid the reader's understanding and is not intended to allow any of the described information or cited documents to constitute prior art to the present invention. Absent.

  Many serious infections that afflict humanity are caused by viruses. Many of these diseases are often fatal, including rabies, smallpox, leukomyelitis, viral hemorrhagic fever, hepatitis, yellow fever, immunodeficiency, and various encephalitis. Other diseases are important in that they are contagious, like influenza, measles, mumps and chickenpox, as well as respiratory and gastrointestinal disorders, and cause acute discomfort. Things like rubella and cytomegalovirus can cause birth defects. Finally, there are viruses known as tumor viruses that can cause cancer in humans and animals.

  Among viruses, Herpesviridae is very interesting. The herpesviridae is a ubiquitous class of dodecahedron double-stranded DNA viruses. Only 8 of the over 100 herpesviridae (HHV) families that have been characterized infect humans. The best known of these are herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (varicella or shingles), cytomegalovirus (CMV) And Epstein-Barr virus (EBV). Herpes viruses are highly prevalent in humans, affecting at least one third of the world's population, and 70-80% of the population in the United States has some herpes infection. While the pathology of herpes infections is usually not dangerous, as is the case with HSV-1, which usually only causes lesions that immediately heal around the mouth and face, these viruses are more dangerous It is also known to be causative and varies from genital ulcers and secretions to fatal infections that can lead to encephalitis (15% mortality) and disseminated infection (40% mortality) .

  Herpesviruses are highly disseminated in nature and highly pathogenic to humans. For example, Epstein-Barr virus (EBV) is known to cause infectious monocytosis in late childhood or adolescence or early adulthood. Prominent features of acute infectious monocytosis are sore throat, fever, headache, lymphadenopathy, tonsil hypertrophy and atypical lymphocyte division in peripheral blood. Other signs often include mild hepatitis, spleen enlargement and encephalitis. EBV is also implicated in cancer: Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC). In the epidemic region of Equatorial Africa, BL is the most common childhood malignancy, accounting for approximately 80% of cancers in children. NPC is modestly observed in North American whites, but is one of the most common cancers that occurs in the age of 25-55 years in southern China. Like cytomegalovirus, EBV is associated with lymphoproliferative disease after transplantation, which can be a fatal complication of chronic immunosuppression after solid organ or bone marrow transplantation.

  For example, other diseases involving skin and eye infections such as central choroiditis or keratoconjunctivitis are also associated with HSV. In the United States, approximately 300,000 ocular HSV infections are diagnosed per year.

  AIDS (Acquired Immunodeficiency Syndrome) is caused by the human immunodeficiency virus (HIV). By killing or damaging cells of the body's immune system, HIV progressively destroys the body's ability to fight infection and certain cancers. Currently, around 42 million people around the world live with HIV / AIDS. In 2002, a total of 3.1 million people died from HIV / AIDS-related causes. The ultimate goal of anti-HIV drug therapy is to prevent the virus from regenerating and damaging the immune system. Substantial progress has been made in the fight over HIV over the past 15 years, but treatment still evades medicine. Today, physicians have a number of antiviral agents in three different drug classes to manage this disease. Usually two or three classes of drugs are formulated in various combinations known as HAART (Highly Active Antiretroviral Therapy). HAART therapy usually includes two nucleoside reverse transcriptase inhibitors with a third agent, either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor. Clinical trials have shown that HAART is the most effective means of reducing viral load and minimizing the potential for drug resistance.

  Although HAART has been shown to reduce the amount of HIV in the body, commonly known as viral load, tens of thousands of patients have encountered serious problems with this therapy. Some side effects are serious and include abnormal fat metabolism, kidney stones, and heart disease. Other side effects such as nausea, nausea and insomnia, for example, are less severe, but remain a problem for HIV patients who need chronic medication for life.

  Currently approved anti-HIV agents enter CD4 + T cells infected with HIV and act by blocking the function of viral enzymes, either reverse transcriptase or protease. HIV needs these enzymes to regenerate. However, HIV is often mutated to prevent reverse transcriptase or protease inhibitors from working against these resistant strains. Once resistance develops, the viral load increases and forces the ineffective drug to be replaced with another antiretroviral agent. Unfortunately, when a virus becomes resistant to one drug in a class, the effectiveness of another drug in that class is also reduced. This phenomenon, known as cross-resistance, occurs because many anti-HIV agents act in a similar manner. The occurrence of drug cross-resistance is highly undesirable because it reduces the treatment options available to the patient.

  Therefore, there is a great need for the development of other antiviral agents effective against HIV that act through other mechanisms of action where the virus does not develop resistance. Considering recent data that 1 in 10 newly diagnosed HIV patients in Europe are already infected with HIV strains that are already resistant to at least one of the approved drugs on the market. Is becoming particularly important.

  Respiratory polynuclear virus (RSV) causes infection of the upper and lower respiratory tract. It is a negative-strand envelope RNA virus and is highly infectious. It affects young children in general and is the most common cause of infant lower airway disease. RSV infection is usually associated with moderate to severe cold-like symptoms. However, severe lower respiratory tract disease can occur at any age, especially in elderly or immunocompromised patients. Children with severe infection may need oxygen therapy and, in certain cases, mechanical ventilation. According to the National Medical Association, an increasing number of children are hospitalized for bronchiolitis, often due to RSV infection. RSV infection is also responsible for approximately one third of community-related respiratory viral infections in patients at bone marrow transplant centers. In the elderly population, RSV infection has recently been recognized as very similar to influenza virus infection in severity.

  Influenza (INF), also known as flu, is a contagious disease caused by influenza viruses. It attacks the human respiratory tract (nose, throat and lungs). In the United States, an average of about 36,000 people die each year from influenza, and 114,000 people are hospitalized for influenza each year. In recent years, the emergence of strains with higher pathogenicity (eg, bird flu) that have previously been found only in animals has become a more serious concern.

  In any infectious disease, the effectiveness of a given therapy often depends on the host's immune response. This is especially true for herpes viruses because the ability of all herpesviruses to establish a latent infection results in a very high incidence of reactivated infections in immunocompromised patients. In kidney transplant patients, 40-70% reactivate latent infection with HSV and 80-100% reactivate CMV infection. Such virus reactivation is also observed in AIDS patients.

  Hepatitis B virus (HBV) is a DNA virus belonging to the hepatitis virus family of viruses. HBV causes hepatitis B in humans. It is estimated that 2 billion people are infected worldwide (1 in 3). About 350 million people remain chronically infected and an estimated 1 million people die each year from hepatitis B and its complications. HBV causes lifelong infection, cirrhosis, liver cancer, liver failure and death. Viruses are transmitted through blood and body fluids. This can occur through direct contact between blood, unprotected sexual intercourse, the use of filthy needles, and from a woman who is infected during childbirth to a newborn. Most healthy adults (90%) who are infected recover and make protective antibodies against future hepatitis B infections. A small number (5-10%) are unable to clean up the virus and suffer from chronic infections, but up to 90% and 50% of young children suffer from chronic infection when infected by the virus. α-interferon is the most frequent type of therapeutic agent used. Serious side effects including flu-like symptoms, depression, rash, other reactions and abnormal blood counts are associated with this treatment. Another treatment option includes 3TC, which has a number of side effects associated with its use. In recent years, an increasing number of reports have shown that patients treated with 3TC express resistant strains of HBV. This is particularly problematic in patient populations co-infected with HBV and HIV. There is clearly an urgent need to develop new antiviral agents against this virus.

  Hepatitis C virus (HCV) infection is the most common blood-borne chronic infection in the United States, with more than 4 million infected cases. This common viral infection is the leading cause of cirrhosis and liver cancer in the United States and is now the main reason for liver transplantation. Recovery from infection is uncommon, with approximately 85 percent of infected patients becoming chronic virus carriers and 10 to 20 percent developing cirrhosis. Currently, it is estimated that 170 million people worldwide are chronic carriers. According to the Centers for Disease Control, every year in the United States alone, 8,000-10,000 people die from chronic hepatitis C and about 1,000 people undergo liver transplants. There is no vaccine available for hepatitis C. Long treatments with interferon alpha or a combination of interferon and ribavirin are effective only in about 40 percent of patients and produce significant side effects.

  Today, the prospects for treating viral infections are generally not good. In general, treatment for viruses has only mediocre efficacy and is associated with strong side effects that prevent administration of effective doses or prevent long-term treatment. Three clinical situations that illustrate these problems are herpes virus, HIV and RSV infection.

  In the case of herpesviruses, there are five main therapies currently approved for use in the clinic: idoxuridine, vidarabine, acyclovir, foscarnet and ganciclovir. Although having limited effectiveness, these treatments also have side effects. Allergic reactions have been reported in 35% of patients treated with idoxuridine, vidarabine causes gastrointestinal disorders in 15% of patients, and the nucleoside analogs acyclovir, foscarnet and ganciclovir are host cells. Affects DNA replication. In the case of ganciclovir, neutropenia and thrombocytopenia have been reported in 40% of AIDS patients treated with this drug.

  There are a number of different drugs currently available to treat HIV infection, all of which are powerful enough to require a large amount of supplemental medication in order for the patient to obtain a reasonable quality of life. It is related to side effects. Additional problems with drug-resistant strains of HIV (problems also found in herpesviridae infections) usually require periodic changes in treatment cocktails, and in some cases make infections extremely difficult to treat.

  Treatment of RSV infection in infants is another example of the urgent need for new drug development. In this case, the usual treatment is delivery of ribavirin by inhalation using a particulate aerosol in an isolation tent. Not only does ribavirin have a mild effect, but its use is associated with serious side effects. In addition, since ribavirin is known for its teratogenicity, the potential for drug spills has caused great concern to hospital staff.

  The newly developed antiviral drugs under development have the following three characteristics: Improved efficacy, 2. Clearly, it is highly desirable to incorporate a reduced risk of side effects and a mechanism of action that is difficult for viruses to overcome by mutation.

  Attempts have been made to inhibit specific viruses by various antisense techniques.

  Zamecnik et al. Used ON specifically targeting reverse transcriptase primer sites and splice donor / acceptor sites (Zamecnik et al., (1986) Proc. Natl. Acad. Sci. USA 83: 4143) ( Goodchild & Zamecnik, 1989, US Pat. No. 4,806,463)

  Crooke and coworkers (Crooke et al., (1992) Antimicrob. Agents Chemother., 36: 527-532) described antisense to HSV-1.

  (1993, US Pat. No. 5,248,670) reported an antisense oligonucleotide having a Cat sequence and having anti-HSV activity that hybridizes to the HSV-1 gene, UL13, UL39, and UL40.

  Kean et al. (Biochemistry, 34: 14617-14620, 1995) reported a survey of antisense methylphosphonate ligomers as anti-HSV agonists.

  Peyman et al. (Biol. Chem. Hoppe. Seyler, Mar, 376: 195-198, 1995) have reported specific antisense oligonucleotides for HSV-1 IE110 and UL30 mRNA for their antiviral properties.

  Oligonucleotides or oligonucleotide analogs targeting the mRNA of CMV encoding IE1, IE2 or DNA polymerase were reported by Anderson et al. (1997, US Pat. No. 5,591,720).

  (1999, US Pat. No. 5,952,490) described a modified oligonucleotide that has a conserved G quartet sequence and a sufficient number of flanking nucleotides and significantly inhibits the activity of a virus such as HSV-1. .

  Jairath et al. (Antiviral Res., 33: 201-213, 1997) reported an antisense oligonucleotide against RSV.

  Torrence et al. (1999, US Pat. No. 5,998,602) describe a compound containing an antisense complementary to a single-stranded portion of an RSV antigenome strand (mRNA strand), a linker, and an oligonucleotide activator for RNase L. reported.

  Qi et al. (Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 14: 253-256, 2000) reported that they investigated an antisense PS-oligonucleotide (PS-ON) in Coxsackievirus B3.

  WO9203051 (Roizman and Maxwell) describes a methylphosphonate antisense oligomer that is complementary to an essential region of the HSV viral genome or its mRNA transcript and exhibits antiviral activity.

  Guanosine / thymidine or guanosine-rich phosphorothioate oligodeoxynucleotides (GT-PS-ON) have been reported to have antiviral activity. The article states that "Several different PS-containing GT-rich ONs (B106-140, I100-12 and G106-57) all 26 or 27 nt in length from 36 (B106-96, B106-97) or 45 nt It was effective in lowering the titer of HIV-2 as well as GT Rich ON (Table 4). (Fennewald et al., Antiviral Res., 26: 37-54, 1995)

  US Pat. No. 6,184,369 describes anti-HIV, anti-HSV and anti-CMV oligonucleotides containing a high proportion of guanosine bases. In a preferred embodiment, the oligonucleotide has a tertiary structure, which is stabilized by a guanosine tetramolecule. In a further embodiment, the oligonucleotide composition of this invention has two or more types of two adjacent deoxyguanosines. This patent claims a G-rich oligodeoxynucleotide (ODN) that includes at least two G residues at at least two positions.

  Cohen et al. (US Pat. Nos. 5,264,423 and 5,276,019) describe inhibition of HIV replication, and more particularly, used to prevent replication of foreign nucleic acids in the presence of normal living cells. PS-ODN analogs that can be used are described. Cohen et al. Describe the antiviral activity of antisense PS-ODN specific for viral sequences. They also described examining the PS-ODNs of 14, 18, 21, and 28-mer poly A, poly T and poly C, indicating the antiviral effect of these PS-ODNs.

  Matsukura et al. (Matsukura et al., Proc. Natl. Acad. Sci. USA, 84: 7706-7710, 1987) later published the results described in the Cohen et al. US patent.

  Gao et al. (Gao et al., J. Biol. Chem, 264: 11521-11526, 1989) examine PS-ODN of poly A, poly T and poly C at sizes of 7, 15, 21 and 28 nucleotides. Describes that PS-ODN inhibits HSV-2 replication.

  Archambault, Stein and Cohen (Archambault et al., Arch. Virol., 139: 97109, 1994) report that 28 nucleotide PS-ODN poly C is not effective against HSV-1.

  Stein et al. (Stein et al., AIDS Res. Hum. Retrovir., 5: 639-646, 1989) published results on additional data on anti-HIV ODN, generally 21-28 nucleotides in length.

  Marshal et al. (Marshall et al., Proc. Natl. Acad. Sci. USA, 89: 6265-6269, 1992) describe the anti-HIV-1 effect of phosphorothioate and phosphorothioate poly C oligos 4 to 28 nucleotides in length. ing.

  Stein and Cheng (Stein et al., Science, 261: 1004-1012, 1993) mentioned in a review article the antiviral activity of a non-specific ODN of 28 nucleotides, “The anti-HIV properties of PS oligos are It is significantly affected by non-sequence-specific effects, ie the inhibitory effect is independent of the base sequence ”.

  Among the review papers, Lebedeva and Stein (Lebedeva et al., Annul. Rev. Pharmacol., 41: 403-419, 2001), various nonspecifics that bind the activity of PS-ODN, including viral proteins Have reported a specific protein. They state that "these molecules are extremely biologically active and relatively easy to mistake artifacts for antisense."

  Rein et al. (US Pat. No. 6,316,190) reported a GT-rich ON decoy that binds to a nucleocapsid of HIV that can be linked to a fusion partner and used as an antiviral compound. Similarly, Campbell et al. (Campbell et al., J. Virol., 73: 2270-2279, 1999), PO- with a TTGGT motif that binds specifically to the nucleocapsid of HIV but is not related to antiviral activity. Reported ODN.

  Feng et al. (Feng et al., J. Virol., 76: 11757-11762, 2002) bind to the nucleocapsid of recombinant HIV but have no reference to data on anti-HIV activity A (n) and TG (n ) PO-ODN.

Antisense ODNs developed as anticancer agents, antiviral agents, or to treat other diseases are usually approximately 20 nucleotides in length. In the review paper (Stein, CA, J. Clin. Invest., 108: 641-644, 2001) “The length of the antisense oligonucleotide must be optimized: the antisense oligonucleotide is either too long or too short If it is too much, the element of specificity is lost, and it is now confirmed that the optimal length of an antisense oligonucleotide is roughly 16-20 nucleotides. " Similarly, in another review paper (Crooke, ST., Methods Enzymol., 313: 3-45, 2000), “phosphorothioate oligonucleotides are approximately −2.2 per unit compared to RNA and RNA duplex formation. It has a low T _m, which means that in order to be effective in vitro, phosphorothioate oligonucleotides usually must be 17-20 mer in length.

  Caruthers and co-workers (Marshall et al., Proc. Natl. Acad. Sci. USA, 89: 6265-6269, 1992) are concerned with 12-mer polycytidine-PS2-ODN and 14-mer PS2-ODN. The anti-HIV activity of phosphorodithioate ODN was reported. Another size was not examined for anti-HIV activity. They also reported inhibitors of HIV reverse transcriptase (RT) for 12, 14, 20, and 28-mer polycytidine PS2-ODN. Later (Marshal et al., Science, 259: 1564-1570, 1993) reported results showing sequence specific inhibition of HIV RT. The same group published data on PS2-ODN in several patents. US Pat. Nos. 5,218,103 and 5,684,148 describe the structure and synthesis of PS2-ODN. US Pat. Nos. 5,452,496, 5,278,302, and 5,695,979 describe inhibition of HIV RT for PS2-ODN of 15 bases or less. US Pat. Nos. 5,750,666 and 5,602,244 describe the antisense activity of PS2-ODN.

  Oligonucleotides modified at the 2 ′ position of ribose and their use in antisense strategies have been evaluated, for example, as described in the references cited below.

  lnoue and coworkers (Inoue et al. (1985) Nucleic Acid Res. 16: 165168) describe the synthesis and properties of oligos (2′-O-methyl ribonucleotides). The same group (Inoue et al. (1987) FEBS Letter 215: 327-330) reported that RNase H-mediated mRNA cleavage occurs when all oligonucleotides contain 2'-O-methyl ribonucleotides. Using mixed oligonucleotides, i.e., oligonucleotides with unmodified and 2'-O-methyl ribonucleotides, the investigators have sequenced the nucleic acid complex formed by RNA and 2'-O-methyl ribonucleotides. Specific RNAse H hydrolysis has been reported.

  Using fully 2'-O-methylated and phosphorothioated oligonucleotides that do not support RNase H-mediated cleavage of the target mRNA, active antisense oligonucleotides promote ICAM-1 expression through an RNase H-dependent mechanism. It was determined whether to inhibit (Chiang et al., (1991) J. Biol. Chem. 266: 18162-18171). They stated that these antisense oligonucleotides can be useful as therapeutic agents.

  The antisense activity of oligonucleotides with 2′-sugar modifications including 2′-O-methyl, 2′-O-propyl, 2′-O-pentyl and 2′-fluoro was analyzed. Evaluation of the antisense activity of uniformly 2′-modified oligonucleotides revealed that these compounds were not at all effective in inhibiting gene expression. Activity was restored when the compound contained an extension of at least 5 2'-deoxyribonucleotide residues. This minimal deoxyribonucleotide length correlated perfectly with the minimum length required for effective RNase H activity in vitro (Monia et al., 1993, J. Biol. Chem. 268: 14514.).

  Yu et al. ((1996) Bioorganic. Med. Chem. 4: 1685-1692) described hybrid antisense oligonucleotides with mixed backbones containing phosphorothioate, phosphodiester or 2'-O-methyl modified sugar moieties, p24 ELISA quantification. Reported to have specific anti-HIV activity measured by.

  Phosphorothioated 2'-O-methyl-oligoribonucleotide targets an aberrant splice site of IVS2-654 pre-mRNA expressed in mammalian cells stably transformed with its mutant human β-globulin gene In this case, it has been reported that accurate splicing is effectively restored (Sierakowska, et al (1996) Proc. Natl. Acad. Sci. USA 93: 12840-12844.).

  A review, Agrawal ((1999) Biochim. Biophys. Acta 1489: 53-68), shows that for optimal activity, antisense oligonucleotides not only have increased stability for nucleases or high affinity for target RNA. Suggests that various properties need to be combined. Such properties include RNase H activity. In a later review, Agrawal and Kandimalla ((2000) Mol. Med. Today-6: 72-81) found that mixed backbone oligonucleotides containing 2'-O-methyl modifications were improved including their RNAse H activity. Because of its unique properties, it has become an option for second generation antisense oligonucleotides. Antisense oligos are assumed to have certain important properties, such as the ability to bind to target RNA and activate RNAse H (Agrawal and Kandimalla, 2001, Current Cancer Drug Target 1: 197-209.). In most antisense approaches, target RNA cleavage by RNase H is desired to increase antisense efficacy (Kurreck, 2003, Eur. J. Biochem. 270: 1628-1644.).

  Numerous studies have described the use of 2′-O-methoxyethyl modifications in antisense oligonucleotides. An example is a study using gap 2 ′ modified oligonucleotide antisense described in Zellweger et al. ((2001) J. Pharmacol. Experimental Therapeutics 298: 934-940). Another example shows inhibition of translation initiation complex formation using RNase H-independent 2'-O-methoxyethyl antisense (Baker et al. 1997) J. Biol. Chem. 272: 1994-12000.).

  Kuwasaki et al. (2003) J. Antimicrob. Chemother. 51: 813-819 is a highly nuclease-resistant dimer containing a 2'-O-methyl group on the nucleoside and a sulfur group on the internucleotide linkage. Describes the structure of the body hairpin anosine-quadruplex and its anti-HIV-1 activity in cultured cells.

Mou and Gray (2002) (Nucleic Acid Res. 30: 749-758) show that the addition of 2'-O-methyl modification improves nonspecific protein binding properties compared to typical phosphorothioate-DNA oligomers. It shows that it decreases. The protein binding affinity of g5p for the 36mers oligonucleotide is: dA ₃₆ <rA ₃₆ <2'-O-MeA ₃₆ <S-rA ₃₆ <<S-2'-O-MeA ₃₆ <S-dA ₃₆ (where d = deoxy, r = ribo, 2′-O-Me = 2′-O-methyl, S = phosphorothioate). This order is reported to Kandimalla et al. ((1998) Bioorganic Med Chem Lett. 8: 2103-2108) for non-specific binding of plasma proteins such as human serum albumin, γ-globulin and fibrinogen with respect to these oligomer modifications. The order of S-RNA <<S-2'-O-MeRNA<S-DNA was consistent.

  US Pat. No. 5,591,623 and US Pat. No. 5,514,788 describe compositions and methods for the treatment and diagnosis of potentially therapeutic diseases through modulation of intracellular adhesion molecule synthesis or metabolism. According to a preferred embodiment, oligonucleotides capable of specific hybridization with nucleic acids encoding intracellular adhesion genes are described. The present invention describes the synthesis of 2′-O-methyl phosphorothioate oligonucleotides and their use as antisense.

  U.S. Pat.Nos. 5,652,355, 6,143,881 and 6,346,614 resist nucleolytic degradation, form stable duplexes with RNA or DNA and activate RNase H when hybridized with RNA Hybrid oligonucleotides (including deoxy- and ribonucleotide segments) are described. One of the properties of phosphorothioate 2′-O-methyl-oligonucleotide has been shown to be that it does not activate RNase H. In one aspect, the present invention provides hybrid oligonucleotides that are effective in inhibiting the expression of viruses, pathogenic organisms or cellular genes. A feature of the oligonucleotide according to this aspect of the invention is the presence of deoxyribonucleotides. The oligonucleotide according to the invention comprises at least one deoxyribonucleotide. The nucleotide sequence of the oligonucleotide according to this aspect of the invention is complementary to a nucleic acid sequence derived from a virus, pathogenic organism or cellular gene.

  US Pat. No. 5,591,721 and US Pat. No. 6,608,035 describe methods for down-regulating gene expression in animals by oral administration of oligonucleotides whose nucleotide sequences are complementary to the target gene. Thus, due to the properties described in the patent, such oligonucleotides are said to be therapeutically useful due to their ability to control or down-regulate the expression of certain genes in animals. Hybrid DNA / RNA oligonucleotides useful in the methods of the invention resist nucleolytic degradation, form stable duplexes with RNA or DNA, and preferably activate RNase H when hybridized with RNA. Oligonucleotides according to the present invention have been reported to be effective in inhibiting the expression of various genes in viruses, pathogenic organisms or in inhibiting the expression of cellular genes. Thus, an oligonucleotide according to the method of the invention has a nucleotide sequence that is complementary to a nucleic acid sequence derived from a virus, pathogenic organism or cellular gene.

  In US Pat. No. 6,608,035, a phosphorothioate oligonucleotide is not stable in the stomach after 6 hours, but contains a 2′-O-methyl ribonucleotide at the 3 ′ and 5 ′ ends and a deoxyribonucleotide internally. The data show that phosphorothioate oligonucleotides are more stable in the stomach but are partially degraded.

SUMMARY OF THE INVENTION The present invention finds that oligonucleotides (ON), such as oligodeoxynucleotides (ODN), including highly modified oligonucleotides, can have broadly adaptable, sequence-independent antiviral activity. About. Advantageous modifications include modified internucleotide linkages and 2′-modifications. In order to have antiviral activity, the oligonucleotide need not be complementary to any viral sequence or have a specific nucleotide distribution. Such oligonucleotides may be prepared as randomers, so there are at most several copies of any particular sequence in the preparation, for example in the preparation of a 15 micromolar randomer with a length of 32 or more nucleotides. I think that.

  In addition, the inventors have discovered that oligonucleotides of different lengths have different antiviral effects. For example, the length of an antiviral oligonucleotide that produces the maximum antiviral effect when modified with phosphorothioate internucleotide linkages is typically in the range of 40-120 nucleotides. In view of this discovery regarding the antiviral properties of oligonucleotides, the present invention provides oligonucleotide antiviral agents that can have activity against a number of different viruses and that can also be selected as a broad range of antiviral agents. To do. Such antiviral drugs are particularly advantageous in view of the limited options of currently available antiviral treatments.

  Accordingly, ONs of the invention, such as ODNs, are therapeutic or therapeutic for the treatment or prevention of viral infections, or the treatment or prevention of tumors or cancers induced by viruses such as oncoviruses (e.g. retroviruses, papillomaviruses and herpesviruses) As well as the treatment or prevention of other diseases whose etiology is based on viruses. Such treatment applies to many types of patients and treatments including, for example, prevention or treatment of viral infections in human and animal immunosuppressed patients.

  A first aspect of the present invention is that antiviral oligonucleotides in which antiviral properties occur essentially in a sequence independent manner, eg, in a mode of action that is not sequence complementary, such as purified oligonucleotides, and such oligonucleotides. Relates to a formulation containing

  Oligonucleotides useful in the invention can be of various lengths, e.g. at least 6, 10, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 38, It may have a nucleotide length of 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160 or more. Similarly, oligonucleotides may be used in a range that defines any two of the above values as including the end of the range, such as 10-20, 20-30, 20-40, 30-40, 30- It may have a nucleotide length in the range of 50, 40-50, 40-60, 40-80, 50-60, 50-70, 60-70, 70-80, 60-120 and 80-120. In certain embodiments, the minimum length or length range is combined with any other oligonucleotide details listed herein for the antiviral oligonucleotide.

  Antiviral nucleotides may include various modifications, such as stabilizing modifications, and thus may include at least one modification within the phosphodiester linkage and / or on the sugar and / or base. For example, the oligonucleotide may comprise one or more phosphorothioate linkages, phosphorodithioate linkages and / or methylphosphonate linkages. Different modified bonds that are chemically compatible can be combined, for example, modifications in which the synthesis conditions are chemically compatible. While modified linkages are useful, oligonucleotides can include phosphodiester linkages, such as at least one phosphodiester linkage, or at least 5, 10, 20, 30% or more phosphodiester linkages. Further useful modifications are modifications at the 2 ′ position of the sugar, 2′-O-alkyl modifications such as 2′-O-methyl modifications, 2′-amino modifications, 2′-halo such as 2′-fluoro. Modifications; including but not limited to acyclic nucleotide analogs. Other modifications are known in the art and can be used. In certain embodiments, the oligonucleotide has a modified bond, eg, phosphorothioate, has a 3′- and / or 5′-cap; contains a terminal 3′-5 ′ bond; the oligonucleotides are linked by a linker Or a chain consisting of two or more oligonucleotide sequences.

  The invention further modifies the nature of the oligonucleotide at one or more nucleotide residues to provide higher stability, lower interaction with serum, higher cellular uptake, higher interaction with viral proteins. 1 selected from the group consisting of improved ability to formulate for delivery, detectable signal, higher antiviral activity, better pharmacokinetic properties, specific tissue distribution, lower toxicity Alternatively, an oligonucleotide is provided that is bound or conjugated to a molecule that acquires multiple properties.

  In certain embodiments, the oligonucleotide comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 100% modified linkages such as phosphorothioate, phosphorodithioate and / or methylphosphonate. .

  In certain embodiments, at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95%, or all nucleotides, are at the 2 ′ position of ribose, for example 2′-OMe, 2′-F. , Modified with 2'-amino.

  In certain embodiments, the modified linkage is combined with a 2′-modification of the oligonucleotide, eg, at least 30% modified linkage and at least 30% 2′-modification; or at least 40% and 40%, at least 50%, respectively. Combined as 50%, at least 60% and 60%, at least 70% and 70%, at least 80% and 80%, at least 90% and 90%, 100% and 100%. In certain embodiments, the oligonucleotide has at least 30, 40, 50, 60, 70, 80, 90 or 100% modified linkage and at least 30, 40, 50, 60, 70, 80, 90 or 100% 2 Wherein the embodiment includes a combination of the percentage of each modified bond listed and the percentage of 2'-modification (e.g., at least 50% modified bonds and at least 80% 2'- Modification, and at least 80% modified bonds and 100% 2'-modification). In each particular embodiment of the described combination proportions, the modified bond is a phosphorothioate bond; the modified bond is a phosphorodithioate bond; 2′-modification is 2′-OMe; 2′-modification is 2 '-Fluoro; 2'-modification is a combination of 2'-OMe and 2'-fluoro; the modified bond is a phosphorothioate bond and the 2'-modification is 2'-OMe; the modified bond is a phosphorothioate bond And the 2'-modification is 2'-fluoro; the modified bond is a phosphorodithioate bond and the 2'-modification is 2'-OMe; the modified bond is a phosphorodithioate bond and 2 The '-modification is 2'-fluoro; the modified bond is a phosphorodithioate bond and the 2'-modification is a combination of 2'-OMe and 2'-fluoro. In certain embodiments of the oligonucleotides described herein that combine a certain percentage of modified linkages and a certain percentage of 2'-modifications, the oligonucleotide is at least 15, 20, 25, 30, 35, 40, 50, 60 , 70, 80, 90, 100, 110 or 120 nucleotides in length, or a length within a range defining any two of the specified lengths as including the end of the range.

  In certain embodiments, all internucleotide linkages except 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 internucleotide linkages, and / or the 2 ′ position of the ribose moiety are, for example, It is modified by phosphorothioate, phosphorodithioate or methylphosphonate linkages and / or by 2'-OMe, 2'-F, and / or 2'-amino modifications of the ribose moiety.

  In some embodiments, the oligonucleotide comprises at least 1, 2, 3 or 4 ribonucleotides, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90% or 100% Contains ribonucleotides.

  In certain embodiments, the oligonucleotide is present in the strand (ie, forming part of the strand backbone) and / or as a side chain portion, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10 or more, or up to 5, 10, 20% or more of the chain and / or side chain moieties containing non-nucleotide groups.

  In certain embodiments, the oligonucleotide has no self-complementary sequence longer than 5, 8, 10, 15, 20, 25, 30 nucleotides; the oligonucleotide has catalytic activity, eg, cleavage activity on RNA. No; oligonucleotides do not induce RNAi mechanisms.

  In certain embodiments, the oligonucleotide binds to one or more viral proteins; the sequence of the oligonucleotide (or a portion thereof, eg, at least 20, 30, 40, 50, 60, 70% or more sequences) Is derived from the viral genome; the activity of oligonucleotides with sequences derived from the viral genome is not superior to random-length or random oligonucleotides of the same length; the oligonucleotide comprises a portion complementary to the viral sequence and the virus The sequence of the oligonucleotide is derived from a viral packaging sequence or other viral sequence involved in aptamer interaction; unless otherwise indicated, the oligonucleotide sequence is A (x ), C (x), G (x), T (x), U (x), l (x), AC (x), AG (x), AT (x), AU (x), CG (x ), CT (x), CU (x), GT (x), GU (x), TU (x), Al (x), IC (x), IG (X), IT (x) IU (x) Where x is 2, 3, 4, 5, 6, ... 60 ... 120 (in certain embodiments, the oligonucleotide is at least 15, 20, 25, 29, 30, 32, 34, 35, 36, 38, 40, 45, 46, 50, 60, 70, 80, 90, 100, 110, 120, 140 or 160 nucleotides in length, or any two of the listed values, The length within the range defined as including the end of the range, or the length of the identified repetitive sequence is at least within the length or length range specified immediately above); For example, each combination of the above monomers and / or dimers comprises a combination of repetitive sequences (eg, the repetitive sequences specified above), including each combination repeated 2, 3, or 4 times at a time. The oligonucleotide is single stranded (RNA or DNA); The oligonucleotide is double stranded (RNA or DNA); the oligonucleotide comprises at least one G quartet or CpG moiety; the oligonucleotide comprises a part complementary to the viral mRNA and at least 29, 37 or 38 Nucleotide length (or other length as specified above); at least one non-Watson-Crick oligonucleotide and / or at least one participating in non-Watson-Crick binding with one other nucleotide One nucleotide, and / or at least one nucleotide that cannot base pair with another nucleotide; the oligonucleotide is a random oligonucleotide, the oligonucleotide is a randomer, or includes a randomer moiety, such as at least 5, 10, 15, 20, 25 30, 35, 40 or more continuous oligonucleotides, or comprising a randomer moiety having a length specified above with respect to the oligonucleotide length, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90% or all nucleotides are randomers; the oligonucleotide alters the properties of the oligonucleotide at one or more nucleotide residues, eg, higher stability (in serum Stability or in a particular solution), lower interaction with serum, higher cellular uptake, higher interaction with viral proteins, improved ability to be formulated for delivery, detection Bind to molecules that provide possible signals, improved pharmacokinetic properties, specific tissue distribution, and / or lower toxicity It is compounded.

  Phosphorothioated ON containing only (or at least primarily) pyrimidine nucleotides, including cytosine and / or thymidine and / or other pyrimidines, is resistant to low pH, and polycytosine oligonucleotides are resistant to numerous nucleases. Have been found to exhibit increased resistance and thereby provide two important properties for oral administration of antiviral ON. Thus, in certain embodiments, the oligonucleotide has at least 80, 90, or 95 or 100% modified internucleotide linkages (eg, phosphorothioate or phosphorodithioate) and has a pyrimidine content greater than 50%. More than 60%, more than 70%, more than 80%, more than 90% or 100%, i.e. it is a pyrimidine oligonucleotide or has a cytosine content of more than 50%, more than 60%, 70 Greater than%, greater than 80%, greater than 90% or 100%; ie a polycytosine oligonucleotide. In certain embodiments, the length is at least 29, 30, 32, 34, 36, 38, 40, 45, 50, 60, 70 or 80 nucleotides, or 20-28, 25-35, 29-40, 30- 40, 35-45, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75 or 70-80, or any two of the listed values within the range The length within the range defined as including the end point of. In certain embodiments, the oligonucleotide is at least 50, 60, 70, 80 or 90% cytosine; at least 50, 60, 70, 80 or 90% thymidine (and has a total pyrimidine content as listed above). Get). In certain embodiments, the oligonucleotide comprises any of the listed proportions of cytosine and thymidine and the remaining pyrimidine nucleotides are the other of cytosine and thymidine. Also, in certain embodiments, the oligonucleotide is at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40 or more consecutive pyrimidine nucleotides, such as C nucleotides, T nucleotides, or CT dinucleotides Includes pairs. In certain embodiments, the pyrimidine oligonucleotide consists solely of pyrimidine nucleotides; comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-pyrimidine moieties; 1-5, 6- 10, 11-15 or at least 16 non-pyrimidine backbone moieties; at least one 1-20, 1-5, 6-10, 11-15 or 16-20 non-nucleotide moieties; at least one Contains 1-20, 1-5, 6-10, 11-15 or 16-20 purine nucleotides. Preferably, in embodiments where non-nucleotide moieties are present, the linkage between such moieties, or between such moieties and the nucleotide, is a gel under conditions appropriate to the size and chemistry of the oligonucleotide. As assessed by electrophoresis, it is at least 25, 35, 50, 70, 90 or 100% resistant to acidic conditions, similar to PS binding of 40-mer polyC oligonucleotides.

  Oligonucleotides may be used in combination, for example as a mixture. Such combinations or mixtures can include, for example, at least 2, 3, 4, 5, 10, 20, 50, 100, 1000, 10,000, 100,000, 1,000,000 or more different oligonucleotides, such as any of the oligonucleotides Combinations of are described herein. Such combinations or mixtures can have, for example, different sequences, and / or different lengths, and / or different modifications, and / or different linkers or complex molecules. In certain embodiments of such combinations or mixtures, the plurality of oligonucleotides have a minimum length or have a length within the range as described above for oligonucleotides. In certain embodiments of such combinations or mixtures, at least one, multiple, or each oligonucleotide may have any other properties detailed herein for individual antiviral oligonucleotides (compatible) Any combination).

  In certain embodiments, the sequence of the oligonucleotide is not completely complementary to any equal length portion of the target virus genomic sequence, or to any equal length portion of the target virus genomic sequence. Have less than 95, 90, 80, 70, 60 or 50% complementarity. The oligonucleotide sequence does not consist essentially of a poly A, poly C, poly G, poly T, G quartet or TG rich sequence.

  As used in connection with the oligos of the present invention, the term “TG rich” refers to an antiviral oligonucleotide sequence comprising at least 50 percent T and G nucleotides, or at least as specified. 60, 70, 80, 90 or 95%, or 100% T and G.

  In related aspects, the present invention provides at least two, eg, at least 2, 3, 4, 5, 7, 10, 50, 100, 1000, 10,000, 100,000, 1,000,000 or more different as described herein. A mixture of antiviral oligonucleotides containing antiviral oligonucleotides is provided.

  As used herein in connection with oligonucleotides or other substances, the term “antiviral” refers to viral in a system, otherwise in a system suitable for the formation of infectious viral particles of at least one virus. Refers to the effect of the presence of oligonucleotides or other substances that inhibit the production of particles, ie reduce the number of infectious virus particles formed. In certain embodiments of the invention, the antiviral oligonucleotide is assumed to have antiviral activity against a number of different viruses.

  The term “antiviral oligonucleotide formulation” refers to a preparation containing at least one antiviral oligonucleotide adapted for use as an antiviral agent. The formulation contains one or more oligonucleotides and may contain other substances that do not interfere with the use of the formulation as an antiviral agent in vivo. Such other materials include, but are not limited to, diluents, excipients, carrier materials, and / or other antiviral materials.

  As used herein, the term “pharmaceutical composition” refers to an antiviral oligonucleotide formulation containing a physiologically or pharmaceutically acceptable carrier or excipient. Such compositions can also include other ingredients that do not render the composition unsuitable for administration to a desired subject, eg, a human.

  In the context of the present invention, unless otherwise limited, the term “oligonucleotide (ON)” means an oligodeoxynucleotide (ODN) or oligodeoxyribonucleotide or oligoribonucleotide. Thus, “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA), and / or analogs thereof. The term includes naturally occurring nucleobases, oligonucleotides consisting of covalent bonds (backbones) between sugars and nucleosides, as well as oligonucleotides having non-naturally occurring moieties that function similarly. Such modified or substituted oligonucleotides may be preferred over the natural form due to the desired properties, such as improved cellular uptake, improved affinity for nucleic acid targets and increased stability in the presence of nucleases. . Examples of modifications that can be used are described herein. Oligonucleotides containing backbone and / or other modifications can also be referred to as oligonucleosides.

  In the context of the present invention, the expression “modified internucleotide linkage” refers to a linkage between nucleotides or nucleotide analogues of an oligonucleotide that is different from the phosphodiester linkages commonly found in naturally occurring polynucleotides. Examples are phosphorothioate linkages, phosphorodithioate linkages and methylphosphonate linkages.

  A specific length detail of an oligonucleotide, eg, at least 20 nucleotides long, means that the oligonucleotide comprises at least 20 linked nucleotides. If not explicitly denied, the oligonucleotide may also contain additional non-nucleotide moieties that form part of the backbone of the oligonucleotide chain. Unless otherwise indicated, when there is a non-nucleotide moiety in the backbone, at least 10 linked nucleotides are contiguous.

  As used in connection with antiviral formulations, pharmaceutical compositions, or other materials, the expression “adapted for use as an antiviral agent” means that the substance exhibits an antiviral effect, such as in a human subject. It does not contain any component or substance that renders the substance inappropriate for use in inhibiting virus production in an in vivo system for administration to a subject.

  As used herein in connection with the antiviral activity of an antiviral oligonucleotide, a “sequence-independent mode of action” is a specific biological activity (eg, antiviral activity) where the oligonucleotide Independence from specific oligonucleotide sequences. For example, activity does not depend on sequence-dependent hybridization, such as antisense activity or specific sequences that cause sequence-dependent aptamer interactions. Similarly, the expression “mode of action that is not sequence complementary” indicates that the mechanism by which the substance exhibits an antiviral effect is not due to hybridization of complementary nucleic acid sequences, eg, an antisense effect. Conversely, “sequence-complementary mode of action” means that the antiviral effect of the substance is involved in the hybridization of complementary nucleic acid sequences or sequence-specific aptamer interactions. Thus, if the antiviral activity of a substance is “due to a sequence-independent mode of action” or the activity is indicated as “not primarily due to a sequence-complementary mode of action”, the activity of the oligonucleotide is Means meeting at least one of the four tests provided in the specification (see Example 10). In certain embodiments, the oligonucleotide meets Test 1, Test 2, Test 3, Test 4 or Test 5; the oligonucleotide is a combination of two tests, namely Test 1 and 2; Test 1 and 3; Test 1 and 4, test 1 and 5, test 2 and 3, test 2 and 4, test 2 and 5, test 3 and 4, test 3 and 5, or test 4 and 5; oligonucleotide is a combination of three tests, Test 1, 2 and 3, test 1, 2 and 4, test 1, 2 and 5, test 1, 3 and 4, test 1, 3 and 5, test 2, 3 and 4, test 2, 3 and 5, Satisfy tests 3, 4 and 5; oligonucleotide meets all of tests 1, 2, 3 and 4.

  As used herein in connection with administration of antiviral agents, the term “subject” refers to an animal such as a mammal, eg, a human, a non-human primate, a cow, a pig, a sheep, a horse, Refers to higher organisms, including dogs and cats; birds (Birds); and plants, such as fruit trees.

  A related aspect relates to an antiviral oligonucleotide randomer or a randomer formulation containing at least one randomer, wherein the antiviral activity of the randomer occurs primarily in a sequence independent manner, for example by a mode of action that is not sequence complementary. Such randomer formulations include, for example, a mixture of randomers of different lengths, such as at least 2, 3, 5, 10 or more different lengths, or include other mixtures described herein. obtain.

  As used herein in connection with oligonucleotide sequences, the term `` random '' characterizes a sequence or ON that is not complementary to viral mRNA, is selected so as not to form a hairpin, and has a Paris therein. Does not contain any nucleotide sequence. When the term “random” is used in the context of the antiviral activity of an oligonucleotide directed to a particular virus, the term implies the absence of complementarity to the viral mRNA of the particular virus. The absence of complementarity can be more widespread, for example, multiple viruses, viruses from specific virus families or infectious human viruses.

  In the present application, the term “lander” shall mean single-stranded DNA having fluctuations (N), such as NNNNNNNNNN, at each position. Each base is synthesized as a fluctuation so that this ON actually exists as a collection of different randomly generated sequences of substantially the same size. It is recognized that the preparation of such randomers usually produces a distribution of sizes around a certain length (mainly shorter); unless explicitly denied, in this context The preparation is considered as a randomer of a certain length.

  The expression “derived from a viral genome” has a nucleotide sequence whose specific sequence has at least 70% identity to the viral genomic nucleotide sequence or its complement (eg, identical or complementary to such viral genomic sequence). Or a corresponding RNA sequence. In certain embodiments of the invention, the term indicates that the sequence is at least 70% identical to the viral genome sequence of the particular virus to which the oligonucleotide is directed or its complementary sequence. In certain embodiments, identity is at least 80, 90, 95, 98, 99 or 100%.

  The invention also provides a therapeutically effective amount of a pharmacologically acceptable antiviral oligonucleotide, or an oligonucleotide as described herein, eg, of at least 6 nucleotides in length, or other lengths listed herein. , Wherein the antiviral activity of the oligonucleotide occurs primarily in a sequence independent manner, eg, by a mode of action that is not sequence complementary, and contains a pharmaceutically acceptable carrier I will provide a. In certain embodiments, the oligonucleotides or combinations or mixtures of oligonucleotides are as specified above for individual oligonucleotides or combinations or mixtures of oligonucleotides. In certain embodiments, the pharmaceutical compositions are approved for administration to humans or non-human animals, such as non-human primates.

  In certain embodiments, the pharmaceutical composition is adapted for the treatment, control or prevention of diseases associated with viral pathogenesis; adapted for the treatment, control or prevention of prion diseases; intraocular administration, oral digestion, Suitable for enteral administration, inhalation, intradermal, subcutaneous, intramuscular, intraperitoneal, intrathecal, intratracheal, or intravenous infusion, or local delivery.

  In certain embodiments, the pharmaceutical composition is administered by oral digestion, oral mucosal delivery, intranasal instillation or spray, intraocular injection, subconjunctival injection, eye drop, ear drop, inhalation, intratracheal injection or spray, intrabronchial injection or Nebulization, intrapleural injection, intraperitoneal injection, perfusion or lavage, intrathecal injection or perfusion, intracranial injection or perfusion, intramuscular injection, intravenous injection or perfusion, intraarterial injection or perfusion, intralymphatic injection or perfusion, subcutaneous Infusion or perfusion, intradermal injection, topical skin application, organ perfusion, local application during surgery, intratumoral injection, topical application, gastric infusion perfusion or irrigation, intestinal injection or perfusion, intestinal infusion perfusion or irrigation, Rectal infusion perfusion or lavage, rectal suppository or enema, urethral suppository or infusion, intravesical infusion perfusion or lavage, or intraarticular injection Is selected from the group consisting of, it may be formulated for delivery by a manner which is not limited thereto.

  In certain embodiments, the composition comprises, for example, a delivery system targeted to a specific cell or tissue; a liposomal formulation, another antiviral drug, such as a non-nucleotide antiviral polymer, an antisense molecule, siRNA or a small molecule drug. contains.

In certain embodiments, the antiviral oligonucleotide, oligonucleotide preparation, oligonucleotide formulation or antiviral pharmaceutical composition is a target virus (e.g., any particular virus, or virus as indicated herein) in vitro. Group virus) is 10, 5, 2, 1, 0.50, 0.20, 0.10, 0.09.0.08, 0.07, 0.75, 0.06, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015 or 0.01 μM. Or an IC ₅₀ of less than that.

  In certain embodiments of the formulation, pharmaceutical composition, use for prevention or treatment, and method for prevention or treatment, the composition or formulation is adapted for treatment, control, or prevention of a disease associated with viral pathogenesis. Suitable for treatment, control or prevention of prion diseases; delivery mode selected from the group consisting of intraocular, oral digestion, enteral, inhalation, or intradermal, subcutaneous, intramuscular or intravenous infusion Further comprising a molecule that increases or can be associated with a specific cell; further comprising at least one other antiviral agent in combination; and In combination with / or further antiviral polymers.

  In certain embodiments, the pharmaceutical composition contains at least one polypyrimidine oligonucleotide as described herein. Given the resistance to low pH found for polypyrimidine oligonucleotides; in certain embodiments, the composition is adapted for delivery to acidic sites in vivo, such as oral or vaginal delivery.

  In certain embodiments of compositions and formulations for oral administration containing such polypyrimidine oligonucleotides, the composition or formulation is a suspension in powder, granules, microparticles, nanoparticles, water or a non-aqueous medium. Alternatively, it is prepared in the form of a solution, emulsion (eg, microemulsion), capsule, gel capsule, sachet, tablet or small tablet. In certain embodiments, thickeners, flavoring agents, diluents, emulsifiers, dispersion aids or binders may be included. In some embodiments, the oral formulation comprises the oligonucleotide of the invention as one or more penetration enhancers, surfactants and / or chelating agents, such as, but not limited to, fatty acids and / or Its esters or salts (e.g. arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin Glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglyceride, diglyceride or a pharmaceutically acceptable salt thereof (eg, sodium), bile acid and / or salt thereof ( For example, chenodeoxycholic acid (CDCA) and urso Ursodeoxychenedeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycholic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, tauro-24, 25 -Dihydro-sodium fusidate, sodium glycodihydrofusidate) Some embodiments are permeation enhancer combinations, eg fatty acids / salts such as the sodium salt of lauric acid, capric acid and UDCA And a bile acid / salt combination, further exemplary penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.

  In certain embodiments where the oligonucleotides of the invention are prepared in granular form (including spray-dried particles) or complexed to form microparticles or nanoparticles, polyamino acids; polyimines; polyacrylates; polyalkylacrylates, poly Oxetane, polyalkyl cyanoacrylate; selected from cationized gelatin, albumin, starch, acrylate, polyethylene glycol (PEG) and starch; polyalkyl cyanoacrylate; DEAE-derivatized polyimine, porlan, cellulose, and starch, or more Specifically, chitosan, N-trimethytchitosan, poly-L-lysine, polyhistidine, polyorithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P (TDA E), polyaminostyrene (e.g., p-amino), poly (methyl cyanoacrylate), poly (ethyl cyanoacrylate), poly (butyl cyanoacrylate), poly (isobutyl cyanoacrylate), poly (isohexyl cyanoacrylate), DEAE -Methacrylate, DEAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly (D, L-lactic acid), poly (DL-lactic acid-glycolic acid (PLGA), alginate And complexing agents selected from polyethylene glycol (PEG) are used, but are not limited thereto.

  In certain embodiments, the composition is adapted for vaginal administration. In such embodiments, the composition may be prepared in the form of tablets, solutions, creams, gels, suppositories, but is not limited thereto.

  In certain embodiments, the composition is adapted for topical administration.

  As used herein, the term “polypyrimidine oligonucleotide” or “pyrimidine oligonucleotide” means an oligonucleotide comprising more than 50% pyrimidine nucleotides.

  As used with respect to in vivo administration of the oligonucleotides and compositions of the invention, the term “acidic site” means a site below pH 7. Examples include stomach (generally pH 1-2), vagina (generally pH 4-5, but may be lower), and to a lesser extent skin (generally pH 4-6).

  As used herein, “compatible with oral delivery” and similar expressions means that the composition is well tolerated to acidic pH and has a clinically excessive loss of activity, such as gastric Indicates that it can be administered orally without excessive loss of less than 50% (or indicated as less than 40%, 30%, 20%, 10% or 5%) due to acidity due to acidity.

  As used herein, “compatible with vaginal administration” and similar expressions refer to the extent to which the composition is clinically unacceptable when the composition is properly administered (eg, specific 50%, 40%, 30%, 20%, less than 10% or 5%) during the retention time of at least 1 hour (or specified) in the vagina (excluding absorbed substances) If at least 2 hours, 4 hours, 8 hours, 12 hours, 1 day or 2 days). Such composition retention may be due to a number of different factors or combinations of factors such as, for example, physical form or adhesive properties.

  As used herein in connection with antiviral oligonucleotides and formulations, etc. in connection with a particular virus or group of viruses, the term “target” is used to block a virus or group of viruses. Indicates that an oligonucleotide is selected. When used in connection with a particular tissue or cell type, the term means that the oligonucleotide, formulation, or delivery system is preferentially present in or near a particular tissue or cell type. And / or preferentially selected to exhibit an antiviral effect.

  As used herein, the term “delivery system” refers to an oligonucleotide that contacts the intended location in vivo when combined with an oligonucleotide (eg, an antisense oligo, siRNA, or oligonucleotide described herein). And / or extend the duration of its presence in the target by at least 20, 50 or 100%, or more, for example, compared to the amount and / or duration in the absence of the delivery system And / or an ingredient that prevents or reduces interactions that cause side effects.

  As used herein in connection with antiviral drugs and other drugs or test compounds, the term “small molecule” means a molecule having a molecular weight of 1500 daltons or less. In some cases, the molecular weight is 1000, 800, 600, 500 or 400 daltons or less.

  In another aspect, the present invention provides a kit comprising at least one antiviral oligonucleotide, antiviral oligonucleotide mixture, antiviral oligonucleotide formulation or antiviral pharmaceutical composition, the kit comprising such an oligonucleotide. Nucleotides, oligonucleotide mixtures or oligonucleotide preparations are contained in a labeled package, where the antiviral activity of the oligonucleotide occurs mainly in a sequence-independent manner, for example by a mode of action that is not sequence complementary, The upper label indicates that the antiviral oligonucleotide can be used against at least one virus.

  In certain embodiments, the kit comprises a pharmaceutical composition containing at least one antiviral oligonucleotide described herein. In one embodiment, the kit comprises a mixture of at least two different antiviral oligonucleotides. In one aspect, the antiviral oligonucleotide is compatible with the in vivo use of the animal, and / or the label indicates that the oligonucleotide or composition is acceptable and / or approved for use in an animal; Is a mammal such as a human or a non-human mammal such as a cow, pig, ruminant, sheep, or horse; the animal is a non-human animal; the animal is a bird, the kit is Approved for use in animals, eg, humans, by regulatory agencies such as the US Food and Drug Administration or equivalent.

  In another aspect, the invention provides a method for selecting an antiviral oligonucleotide, eg, an antiviral oligonucleotide that is not sequence complementary, for use as an antiviral agent. The method synthesizes a plurality of different random oligonucleotides, tests the activity of the oligonucleotide to inhibit the ability of the virus to produce infectious virions, and is used at an pharmaceutically acceptable level. Selecting an oligonucleotide having the following activity:

  In certain embodiments, different random oligonucleotides comprise different lengths of randomers; random oligonucleotides may have different sequences or have a common sequence, sequences of multiple shortest oligos; and / or different Random oligonucleotides comprise a plurality of oligonucleotides comprising randomer segments that are at least 5 nucleotides in length, or different random oligonucleotides comprise a plurality of randomers of different lengths. Other oligonucleotides described herein, for example with respect to antiviral oligonucleotides, can be tested in specific systems.

  In yet another aspect, the invention provides a method of preventing or treating a viral infection in a subject, said method comprising at least a therapeutically effective amount described herein in a subject in need of such treatment. Administering a kind of pharmacologically acceptable oligonucleotide, for example an oligonucleotide that is not sequence complementary at least 6 nucleotides in length, or an antiviral pharmaceutical composition or formulation or mixture containing such an oligonucleotide By administering. In a further aspect, the present invention provides a use for the prevention or treatment of a viral infection, said use comprising at least one therapeutically effective amount as described herein in a subject in need of such treatment. Administering a pharmacologically acceptable oligonucleotide, e.g. an oligonucleotide that is not sequence complementary at least 6 nucleotides in length, or an antiviral pharmaceutical composition or formulation or mixture containing such an oligonucleotide By doing. In certain embodiments, the virus can be any virus listed herein suitable for suppression using the present invention; the infection is a disease indicated herein as being associated with a viral infection. A subject is a subject of the type indicated herein, eg, a human, a non-human animal, a non-human mammal, a bird, a plant, etc .; a treatment is associated with a viral disease, or a viral etiology Related diseases, such as those indicated in the “Background of the Invention” herein.

  In yet another aspect, the present invention provides a method for preventing or treating a viral infection in an acidic environment in a subject, the method comprising a therapeutically effective amount of at least one of a subject in need of such treatment. Administration of a pharmacologically acceptable antiviral pharmaceutical composition of the invention, said composition being adapted for administration to an acidic site in vivo.

  In yet another aspect, the present invention provides use in the prevention or treatment of a viral infection in an acidic environment in a subject, the use comprising at least one therapeutically effective amount in a subject in need of such treatment. Administering a pharmacologically acceptable antiviral pharmaceutical composition of the invention, the composition being adapted for administration to an acidic site in vivo.

  In certain embodiments, an antiviral oligonucleotide (or oligonucleotide formulation or pharmaceutical composition) described herein is administered; administration is performed in a manner as described herein. A delivery system or method as described herein is used; the viral infection is an infection of a DNA virus or RNA virus; the virus is a parvoviridae, papovaviridae, adenoviridae, herpesviridae, Poxviridae, Hepadnaviridae or Papillomaviridae; viruses are Arenaviridae, Bunyaviridae, Calciviridae, Coronaviridae, Filoviridae, Flaviridae, Orthomyxoviridae, Paramyxoviridae , Picornaviridae, Reoviridae, La Grape Rusaceae, Retroviridae or Togaviridae; Herpesviridae virus is EBV, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7 or HHV-8; The virus is a respiratory multinuclear virus (RSV); the virus is a parainfluenza-3 virus; the virus is an influenza virus, such as influenza A; the virus is The virus is a smallpox virus or cowpox virus; the virus is a coronavirus; the virus is a SARS virus; the virus is a West Nile virus; the virus is a hantavirus; Is a parainfluenza virus; the virus is a Coxsackie virus; the virus is a rhinovirus; The virus is a dengue virus; the virus is a hepatitis C virus; the virus is an Ebola virus; the virus is a Marburg virus; the virus is a Lassa fever virus; The varicella-zoster virus; the virus is Epstein-Barr virus; the virus is human herpesvirus 6A or 6B; the virus is HBV; the virus is a parainfluenza virus; the virus is human meta The virus is Rift Valley fever virus; the virus is Crimea-Congo hemorrhagic fever virus; the virus is Western equine encephalitis virus.

  In certain embodiments, the oligonucleotides of the invention target influenza viruses.

  In certain embodiments, the oligonucleotide is a polypyrimidine oligonucleotide (or a formulation or pharmaceutical composition containing such a polypyrimidine oligonucleotide), for example for oral or vaginal administration as described herein. Can fit.

  Similarly, in a related aspect, the invention provides a method for the prophylactic treatment of cancer caused by oncoviruses in humans or animals, the method comprising at least 6 humans or animals in need of such therapy. A therapeutically effective amount of at least one pharmacologically acceptable random oligonucleotide of nucleotide length (or other length as described herein), or a formulation or pharmaceutical composition containing such an oligonucleotide. By administering. In one embodiment, a mixture of oligonucleotides of the invention.

  Similarly, in a related aspect, the present invention provides use for the prophylactic treatment of cancer caused by oncovirus in a human or animal, the use being directed to a human or animal in need of such treatment. A therapeutically effective amount of at least one pharmacologically acceptable random oligonucleotide at least 6 nucleotides in length (or other length described herein), or a formulation or pharmaceutical containing such an oligonucleotide By administering the composition. In one embodiment, a mixture of oligonucleotides of the invention is administered.

  In certain embodiments, the oligonucleotide is as described herein for the present invention, eg, has a length as described herein; the administration method as described herein is used. Uses as described herein are used; delivery systems as described herein are used.

  The term “therapeutically effective amount” refers to an amount sufficient to provide a therapeutically or prophylactically significant reduction in the production of infectious viral particles when administered to a typical subject of the intended type. In aspects involving antiviral oligonucleotide administration to a subject, typically the oligonucleotide, formulation or composition should be administered in a therapeutically effective amount.

  In certain embodiments, including oligonucleotide formulations, pharmaceutical compositions, methods of treatment and prevention, and / or uses for treatment and prevention described herein, an oligonucleotide having a sequence-independent mode of action comprises Not associated with transfection agents; oligonucleotides having a sequence-independent mode of action are not encapsulated within liposomes and / or non-liposomal lipid particles. In certain embodiments, an oligonucleotide having a sequence-independent mode of action is present in a pharmaceutical composition, or an antiviral oligonucleotide that acts primarily in a sequence-dependent mode of action, such as an antisense oligonucleotide or It is administered with siRNA (simultaneously or sequentially). Here, oligonucleotides having a sequence-dependent mode of action can be associated with transfection agents and / or encapsulated in liposomes and / or non-liposomal lipid particles.

  In another aspect, the discovery that sequence-independent, e.g., non-sequence complementary, interactions results in effective antiviral activity is the detection of viral components, one or more viral proteins (e.g., infected host organisms). Screening methods for identifying compounds that alter the binding of oligonucleotides to (extracted or purified from, or produced by, recombinant methods). For example, the method can involve determining whether a test compound reduces binding of the oligonucleotide to one or more viral components.

  The term “screening” as used herein refers to assaying a plurality of compounds to determine whether they possess the desired property. The plurality of compounds can be, for example, at least 10, 100, 1000, 10,000 or more test compounds.

  In certain embodiments, any of a variety of assay formats and detection methods can be used to identify such binding changes. For example, the oligonucleotide is contacted with a viral component in the presence and absence of the compound to be screened (e.g., in a separate reaction) and compared to the absence of the compound in the presence of the compound in the viral component of the oligo. Measure whether a difference in binding to occurs. The presence of such differences indicates that the compound alters the binding of the random oligonucleotide to the viral component. Alternatively, competitive displacement can be used, such as binding the oligonucleotide to the viral component and measuring displacement by the added test compound, or conversely, binding the test compound and measuring displacement by the added oligonucleotide. .

In certain embodiments, the oligonucleotide is as described herein for the antiviral oligonucleotide; the oligonucleotide is at least 6, 8, 10, 15, 20, 25, 29, 30, 32, 34, 36 , 38, 40, 46, 50, 60, 70, 80, 90, 100, 110 or 120 nucleotides in length, or at least other lengths specified herein for antiviral oligonucleotides, or Having a length within the range defining any two of the values as including the end of the range; the test compound is a small molecule. A test compound has a molecular weight of less than 400, 500, 600, 800, 1000, 1500, 2000, 2500 or 3000 Daltons, or any two of the above values are defined as including endpoints of that range A viral extract or viral component is derived from the viruses listed herein; at least 100, 1000, 10,000, 20,000, 50,000 or 100,000 compounds are screened; In vitro, it has an IC ₅₀ equal to or less than 10, 5, 2, 1, 0.500, 0.200, 0.100, 0.075, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015 or 0.01 μM.

  The present invention further provides the oligonucleotides shown in Table 21.

  The present invention further includes REP 1001, REP 2001, REP 3007, REP 2004, REP 2005, REP 2006, REP 2007, REP 2008, REP 2017, REP 2018, REP 2020, REP 2021, REP 2024, REP 2036, A20, G20 , C20, REP 2029, REP 2031, REP 2030, REP 2033, REP 2055, REP 2056, REP 2057, REP 2060, and REP 2107.

  The term “viral component” as used herein refers to a product encoded by a virus or a product produced by a host cell infected as a result of a viral infection. Such components can include proteins and other biomolecules. Such viral components can be obtained, for example, from viral cultures, infected host organisms such as animals or plants, or viral sequences in recombinant systems (prokaryotes or eukaryotes), as well It can be produced from a synthetic protein having an amino acid sequence corresponding to the protein encoded by the virus. The term “virus culture extract” refers to an extract from cells infected by a virus, including virus-specific products. Similarly, "viral protein" refers to a virus-specific protein that is normally encoded by a virus, but also refers to a virus-specific protein that can be partially encoded by a host sequence as a result of viral infection.

  In a related aspect, the present invention provides antiviral compounds identified by the above methods, for example, novel antiviral compounds.

  In a further aspect, the present invention relates to a pool of oligonucleotides from a pool of oligonucleotides, for example by contacting the pool of oligonucleotides with at least one viral component bound to a stationary phase medium and recovering the oligonucleotide bound to the viral component. Methods are provided for purifying oligonucleotides that bind to at least one viral component. In general, recovery involves the replacement of oligonucleotides from viral components. The method can also include sequencing and / or antiviral activity testing of recovered oligonucleotides (ie, oligonucleotides bound to viral proteins).

  In certain embodiments, the pool-bound oligonucleotide is displaced from the stationary phase medium by any suitable method, eg, using an ionic displacement body, and the displaced oligonucleotide is recovered. Typically, for various substitution methods, increasing the stringency so that the oligonucleotides are uniformly replaced in order of high binding affinity (eg, salt-like substitution agents so that there is a stepped or continuous gradient) Can be performed by increasing the concentration of In many cases, low stringency washing is performed to remove weakly bound oligonucleotides and one or more fractions containing displaced, more strongly bound oligonucleotides are recovered. In some cases, it may be desirable to select a fraction containing oligonucleotides that bind very strongly (eg, oligonucleotides in fractions resulting from substitution under more stringent substitution conditions) for further use.

  Similarly, the invention relates to at least one viral component by contacting a pool of oligonucleotides with one or more viral proteins and amplifying and concentrating the oligonucleotides bound to the viral proteins to enrich the oligonucleotide pool. Methods are provided for concentrating oligonucleotides from a pool of binding oligonucleotides. Contacting and amplification can be performed multiple times, for example, 1, 2, 3, 4, 5, 10 or more additional times, using the previous round of concentrated oligonucleotide pool as the next pool of oligonucleotides. . The method can also include sequencing of the oligonucleotides in the enriched oligonucleotide pool and antiviral activity testing following one or more times of contacting and amplification.

  The method includes, for example, substituting an oligonucleotide from a viral component (eg, a viral protein bound to a solid phase medium) by various techniques, as described above, using, for example, a displacing agent. As indicated above, it may be advantageous to select oligonucleotides that bind more strongly for further use, eg, further rounds of binding and amplification. The method can further include selecting and further using one or more concentrated oligonucleotides, eg, high affinity oligonucleotides. In certain embodiments, selection can include the elimination of oligonucleotides having sequences complementary to the host genomic sequence (eg, human) for the particular virus of interest. Such exclusion includes, for example, using a sequence placement program (eg, a BLAST search) to compare the oligonucleotide sequence with a sequence from a particular host in a sequence database, and at the same or a specific level as the host sequence. And excluding oligonucleotides having sequences with identity. The elimination of such host complementarity sequences and / or selection of one or more oligonucleotides that are not complementary to the host sequence can also be performed for other aspects of the invention.

  In the previous method of identifying, purifying or concentrating oligonucleotides, the oligonucleotides are of the type as described herein. The above methods are advantageous, for example, for identifying, purifying or enriching high affinity oligonucleotides from oligonucleotide randomer preparations.

  In a related aspect, the invention relates to one or more oligonucleotides identified using any of the previous methods for identifying, purifying or enriching antiviral oligonucleotides from an initial oligonucleotide pool. One or more viral proteins, wherein the oligonucleotide in the oligonucleotide preparation is higher than the average binding affinity of the oligonucleotide in the original oligonucleotide pool Exhibits an average binding affinity for.

  In certain embodiments, the average binding affinity of the oligonucleotide is at least 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold greater than the average binding affinity of the oligonucleotides in the original oligonucleotide pool. Higher or even higher; median binding affinity is at least 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold compared to the median binding affinity of the original oligopool In that case, the median is the middle value.

  In yet another aspect, the present invention provides an antiviral polymer mix comprising at least one antiviral oligonucleotide and at least one non-nucleotide antiviral polymer. In certain embodiments, the oligonucleotide is an oligonucleotide as described herein for an antiviral oligonucleotide and / or the antiviral polymer is an antiviral polymer as described herein. Yes, or else is an antiviral polymer known in the art, or an antiviral polymer specified below.

  In yet another aspect, the invention provides an oligonucleotide randomer, wherein the randomer is at least 6 nucleotides in length. In certain embodiments, the randomer has a length as specified above for the antiviral oligonucleotide; the randomer includes at least one phosphorothioate linkage, and the randomer includes at least one phosphorodithioate linkage or Including other modifications as listed herein; a randomer oligonucleotide includes at least one non-randomer fragment (eg, a fragment complementary to a selected viral nucleic acid sequence), which is an oligonucleotide Can have a length as specified above; the randomer is at least 5, 10, 15, 20, 50, 100, 200, 500 or 700 μM, 1, 5, 7, 10, 20, 50, In preparations or pools of preparations containing 100, 200, 500 or 700 mM or 1 M randomer, or as an inclusive endpoint Are in a range defined by taking two different values from or synthesized with one of the listed scales or scale ranges.

  Similarly, the present invention provides a method of preparing an antiviral randomer by synthesizing at least one randomer, eg, a randomer as described above.

  As indicated above, for any aspect involved in viral infection or risk of viral infection or targeting to a particular virus, in certain embodiments, the virus is as listed above.

  The expression “human and animal viruses” is generally intended to encompass, without limitation, DNA viruses and RNA viruses. DNA viruses include, for example, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, Hepatitis Viridae, and Papillomaviridae. RNA viruses include, for example, Arenaviridae, Bunyaviridae, Calciviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Reoviridae, Rhabdoviridae, Retroviridae, or Togaviridae.

  In connection with altering the properties of an oligonucleotide by binding or conjugating with another molecule or moiety, the property alteration is assessed relative to the same oligonucleotide without the bound or complexed molecule or portion. .

  Additional aspects will be apparent from the detailed description and from the claims.

Detailed Description of Preferred Embodiments The present invention relates to the identification and use of antiviral oligonucleotides that act by a sequence-independent mechanism and includes the discovery of use against a number of viruses, where antiviral activity is greater In oligonucleotides, typically 40 nucleotides or longer in length.

  According to the present invention there is provided an oligonucleotide comprising at least one modified internucleotide linkage, said oligonucleotide having antiviral activity against the target virus, said activity being largely sequence independent. Acts in a mode of action.

  According to the present invention, there is provided an oligonucleotide wherein at least 50% of the nucleotides in the oligonucleotide are modified at the 2 ′ position of the ribose moiety and at least 50% of the internucleotide linkage is modified, the oligonucleotide comprising: Have antiviral activity against the target virus, which acts largely in a sequence-independent manner of action. In one embodiment, they are 50% and 80%, respectively. In one embodiment, they are 80% and 80%, respectively. In one embodiment, they are 90% and 90%, respectively. In one embodiment, they are 100% and 100%, respectively.

Length and Non-Self Complementarity The present invention further provides oligonucleotides that are at least 15 nucleotides in length. In one embodiment, at least 20 nucleotides in length. In one embodiment, at least 25 nucleotides in length. In one embodiment, at least 30 nucleotides in length. In one embodiment, at least 35 nucleotides in length. In one embodiment, at least 40 nucleotides in length. In one embodiment, at least 45 nucleotides in length. In one embodiment, at least 50 nucleotides in length. In one embodiment, at least 60 nucleotides in length. In one embodiment, at least 80 nucleotides in length.

  The present invention further provides oligonucleotides 20-30 nucleotides in length. In one embodiment, 30-40 nucleotides in length. In one embodiment, 40-50 nucleotides in length. In one embodiment, 50-60 nucleotides in length. In one embodiment, an oligonucleotide 60-70 nucleotides in length. In one embodiment, 70-80 nucleotides in length.

  The invention further provides oligonucleotides that do not have a self-complementary sequence of contiguous nucleotides greater than 5. In one embodiment, more than 10 contiguous nucleotides. In one embodiment, greater than 20 contiguous nucleotides.

  The present invention further provides oligonucleotides that do not have catalytic activity.

Random The invention further provides an oligonucleotide having antiviral activity against a target virus, the sequence of the oligonucleotide not being complementary to any part of equal length of the target virus genomic sequence.

  The present invention further provides oligonucleotides that are not complementary to any part of equal length of the genomic sequence of the human pathogenic virus.

  The present invention further provides oligonucleotides that are not complementary to any part of equal length of the genomic sequence of a human pathogenic virus as sequenced on January 1, 2005.

  The present invention further provides oligonucleotides that are not complementary to any portion of equal length in the human genomic sequence.

  The present invention is further not complementary to any part of equal length of the genomic sequence of one or more animals selected from the group consisting of cattle, horses, pigs, sheep, birds, dogs, cats and fish. Oligonucleotides are provided.

Restriction of RNA and other strand moieties The present invention further provides oligonucleotides in which at least 30% of the nucleotides are ribonucleotides. In one embodiment, at least 50% of the nucleotides are ribonucleotides. In one embodiment, at least 70% of the nucleotides are ribonucleotides. In one embodiment, at least 80% of the nucleotides are ribonucleotides. In one embodiment, at least 90% of the nucleotides are ribonucleotides. In one embodiment, all nucleotides are ribonucleotides.

  The invention further provides an oligonucleotide comprising 1 to 4 non-nucleotide strand moieties.

Randomers The present invention further provides at least 10 consecutive nucleotides of a randomer sequence. In one embodiment, at least 20 nucleotides of the randomer sequence. In one embodiment, at least 30 nucleotides of the randomer sequence. In one embodiment, at least 40 nucleotides of the randomer sequence.

  The present invention further provides oligonucleotides that are randomer oligonucleotides.

Homopolymer The present invention further provides an oligonucleotide comprising a homopolymer sequence consisting of at least 10 consecutive A nucleotides. In one embodiment, at least 10 consecutive T nucleotides. In one embodiment, at least 10 consecutive U nucleotides. In one embodiment, at least 10 consecutive C nucleotides. In one embodiment, at least 10 consecutive G nucleotides. In one embodiment, at least 10 consecutive I nucleotide analogues.

Heterodimers The present invention further provides oligonucleotides comprising a polyAT sequence at least 10 nucleotides in length. In one embodiment, a poly AC sequence that is at least 10 nucleotides in length. In one embodiment, a poly AG sequence that is at least 10 nucleotides in length. In one embodiment, a poly AU sequence that is at least 10 nucleotides in length. In one embodiment, a polyAI sequence that is at least 10 nucleotides in length. In one embodiment, a poly GC sequence that is at least 10 nucleotides in length. In one embodiment, a poly GT sequence that is at least 10 nucleotides in length. In one embodiment, a polyGU sequence that is at least 10 nucleotides in length. In one embodiment, a polyGI sequence that is at least 10 nucleotides in length. In one embodiment, a poly CT sequence that is at least 10 nucleotides in length. In one embodiment, a polyCU sequence that is at least 10 nucleotides in length. In one embodiment, a polyCI sequence that is at least 10 nucleotides in length. In one embodiment, a polyTI sequence that is at least 10 nucleotides in length.

Modified linkage comprising ps and ps2 The present invention further provides an oligonucleotide wherein the modified linkage is selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage and a boranophosphate linkage.

  The invention further provides oligonucleotides in which at least 50% of the internucleotide linkages are modified linkages. In one embodiment, at least 80% of the internucleotide linkages are modified linkages. In one embodiment, at least 90% of the internucleotide linkages are modified linkages. In one embodiment, all internucleotide linkages are modified linkages.

  The invention further provides oligonucleotides wherein at least 50% of the internucleotide linkages are phosphorothioate linkages. In one embodiment, at least 80% of the internucleotide linkages are phosphorothioate linkages. In one embodiment, at least 90% of the internucleotide linkages are phosphorothioate linkages. In one embodiment, all internucleotide linkages are phosphorothioate linkages.

  The invention further provides oligonucleotides in which at least 50% of the internucleotide linkages are phosphorodithioate linkages. In one embodiment, at least 80% of the internucleotide linkages are phosphorodithioate linkages. In one embodiment, all internucleotide linkages are phosphorodithioate linkages.

2′-Modification, Combination with Modified Bond The present invention further provides an oligonucleotide comprising at least one phosphodiester bond. In one embodiment, the oligonucleotide comprises at least 10% phosphodiester linkage. In one embodiment, the oligonucleotide comprises at least 20% phosphodiester linkage.

  In one embodiment, at least 50% of the nucleotides in the oligonucleotide are modified at the 2 ′ position of the ribose moiety. In one embodiment, at least 60% of the nucleotides in the oligonucleotide are modified at the 2 ′ position of the ribose moiety. In one embodiment, at least 70% of the nucleotides in the oligonucleotide are modified at the 2 ′ position of the ribose moiety. In one embodiment, at least 80% of the nucleotides in the oligonucleotide are modified at the 2 ′ position of the ribose moiety. In one embodiment, at least 90% of the nucleotides in the oligonucleotide are modified at the 2 ′ position of the ribose moiety. In one embodiment, 100% of the nucleotides in the oligonucleotide are modified at the 2 ′ position of the ribose moiety.

  The present invention further provides oligonucleotides in which at least 50% of the internucleotide linkages in the oligonucleotide are modified and at least 50% of the nucleotides are modified at the 2 ′ position of the ribose moiety. In one embodiment, at least 60% of the internucleotide linkages within the oligonucleotide are modified and at least 60% of the nucleotides are modified at the 2 ′ position of the ribose moiety. In one embodiment, at least 70% of the internucleotide linkages within the oligonucleotide are modified and at least 70% of the nucleotides are modified at the 2 ′ position of the ribose moiety. In one embodiment, at least 80% of the internucleotide linkages within the oligonucleotide are modified and at least 80% of the nucleotides are modified at the 2 ′ position of the ribose moiety. In one embodiment, all internucleotide linkages within the oligonucleotide are modified and all nucleotides are modified at the 2 ′ position of the ribose moiety.

  The invention further provides an oligonucleotide wherein at least 15% of the nucleotides in the oligonucleotide comprise a 2′-0Me moiety at the 2 ′ position of the ribose moiety. In one embodiment, at least 20% of the nucleotides in the oligonucleotide comprise a 2′-0Me moiety at the 2 ′ position of the ribose moiety. In one embodiment, at least 30% of the nucleotides in the oligonucleotide comprise a 2′-0Me moiety at the 2 ′ position of the ribose moiety. In one embodiment, at least 50% of the nucleotides in the oligonucleotide comprise a 2′-0Me moiety at the 2 ′ position of the ribose moiety. In one embodiment, at least 60% of the nucleotides in the oligonucleotide comprise a 2′-0Me moiety at the 2 ′ position of the ribose moiety. In one embodiment, at least 70% of the nucleotides in the oligonucleotide comprise a 2′-0Me moiety at the 2 ′ position of the ribose moiety. In one embodiment, at least 80% of the nucleotides in the oligonucleotide comprise a 2′-0Me moiety at the 2 ′ position of the ribose moiety. In one embodiment, at least 90% of the nucleotides in the oligonucleotide comprise a 2′-0Me moiety at the 2 ′ position of the ribose moiety. In one embodiment, all nucleotides in the oligonucleotide comprise a 2′-0Me moiety at the 2 ′ position of the ribose moiety.

Various Features The present invention further provides an oligonucleotide that is a chain consisting of two or more oligonucleotide sequences linked by a linker.

  The invention further modifies the nature of the oligonucleotide at one or more nucleotide residues to provide higher stability, lower interaction with serum, higher cellular uptake, higher interaction with viral proteins. 1 selected from the group consisting of improved ability to prepare for delivery, detectable signal, higher antiviral activity, better pharmacokinetic properties, specific tissue distribution, lower toxicity Alternatively, an oligonucleotide is provided that is bound or conjugated to a molecule that acquires multiple properties.

  The invention further provides oligonucleotides that are double stranded.

  The invention further provides an oligonucleotide comprising at least one base that is double-stranded or single-stranded and is capable of hybridizing via non-Watson-Crick interactions.

  The invention further provides an oligonucleotide comprising a portion that is complementary to a viral mRNA.

  The invention further provides an oligonucleotide that binds to one or more viral components.

  The invention further provides an oligonucleotide that interacts with one or more host components, which interaction results in inhibition of viral activity or production.

  The invention further provides an oligonucleotide wherein at least part of the sequence is derived from the viral genome.

  The present invention further provides an oligonucleotide having antiviral activity, wherein at least part of the sequence is derived from the viral genome and the mode of action is largely non-sequence complementary.

  The invention further provides an oligonucleotide wherein at least part of the sequence is derived from a viral packaging sequence or other viral sequences involved in aptamer interactions.

  The invention further provides an oligonucleotide wherein at least part of the sequence is involved in aptamer interaction with a viral component or a host component or both.

Activity Level The present invention further provides oligonucleotides having an IC ₅₀ of 0.10 μm or less for the target virus. In one embodiment, the oligonucleotide has an IC ₅₀ of 0.05 μm or less for the target virus. In one embodiment, the oligonucleotide has an IC ₅₀ of 0.025 μm or less for the target virus. In one embodiment, the oligonucleotide has an IC ₅₀ of 0.015 μm or less for the target virus.

Target Virus The present invention further provides oligonucleotides that target DNA viruses. In one embodiment, an RNA virus. In one embodiment, a member of the Herpesviridae family. In one embodiment, HSV-1. In one embodiment, HSV-2. In one embodiment, CMV. In one embodiment, a member of the Hepadnaviridae family. In one embodiment, HBV. In one embodiment, a member of the Parvoviridae family. In one embodiment, a member of the Poxviridae family. In one embodiment, a member of the Papillomaviridae family. In one embodiment, a member of the adenoviridae family. In one embodiment, a member of the Retroviridae family. In one embodiment, HIV-1. In one embodiment, HIV-2. In one embodiment, a member of the Paramyxoviridae family. In one embodiment, RSV. In one embodiment, parainfluenza virus. In one embodiment, a member of the Bunyaviridae family. In one embodiment, a hantavirus. In one embodiment, a member of the Picornaviridae family. In one embodiment, Coxsackie virus. In one embodiment, rhinovirus. In one embodiment, a member of the Flaviviridae family. In one embodiment, yellow fever virus. In one embodiment, dengue virus. In one embodiment, West Nile virus. In one embodiment, hepatitis C virus. In one embodiment, a member of the Filoviridae family. In one embodiment, Ebola virus. In one embodiment, Marburg virus. In one embodiment, a member of the Orthomyxoviridae family. In one aspect, an influenza virus. In one embodiment, a member of the Togaviridae family. In one embodiment, a member of the Coronaviridae family. In one embodiment, a member of the Reoviridae family. In one embodiment, a member of the Rhabdoviridae family. In one embodiment, a member of the Arenaviridae family. In one embodiment, a member of the Calciviridae family. In one embodiment, varicella-zoster virus. In one embodiment, Epstein-Barr virus. In one embodiment, herpes virus 6A or 6B. In one embodiment, a member of the Hepadnaviridae family. In one embodiment, a human metapneumovirus. In one aspect, Rift Valley virus. In one embodiment, Crimea Congo hemorrhagic fever virus. In one embodiment, western equine encephalitis virus. In one embodiment, Lassa fever virus.

Oligonucleotides The present invention further comprises an oligonucleotide comprising at least 20 linked nucleotides, wherein at least 80% of the linkages are modified; and at least 80% of the nucleotides comprise a 2′-modification of the ribose sugar moiety. provide. In one embodiment, the oligonucleotide has antiviral activity.

  In one embodiment, at least 90% of the internucleotide linkages are modified. In one embodiment, all internucleotide linkages are modified. In one embodiment, at least 90% of the nucleotides contain a 2′-modification of the ribose sugar. In one embodiment, every nucleotide comprises a 2′-modification of the ribose sugar.

  The invention further provides an oligonucleotide wherein the 2′-modification is a 2′-OMe substitution. In one embodiment, at least 90% of the nucleotides contain 2′-OMe substitutions. In one embodiment, all nucleotides contain 2′-OMe substitutions.

  The invention further provides an oligonucleotide wherein the 2'-modification is a 2'-methoxyethoxy substitution. In one embodiment, at least 15% of the nucleotides contain 2'-methoxyethoxy substitution. In one embodiment, at least 50% of the nucleotides contain 2′-methoxyethoxy substitution. In one embodiment, at least 90% of the nucleotides contain 2'-methoxyethoxy substitution. In one embodiment, all nucleotides contain 2′-methoxyethoxy substitutions.

  The present invention further provides oligonucleotides that are at least 40 nucleotides in length. In one embodiment, at least 50 nucleotides in length. In one embodiment, at least 60 nucleotides in length. In one embodiment, at least 80 nucleotides in length.

  The present invention further provides oligonucleotides of 30-40 nucleotides in length. In one embodiment, 40-50 nucleotides in length. In one embodiment, 50-60 nucleotides in length. In one embodiment, 60-70 nucleotides in length. In one embodiment, 70-80 nucleotides in length.

  The invention further provides nucleotides that do not have a self-complementary sequence of more than 5 consecutive nucleotides. In one embodiment, more than 10 contiguous nucleotides. In one embodiment, more than 20 contiguous nucleotides.

  The present invention further provides oligonucleotides that do not have catalytic activity.

Chain Part Restriction The present invention further provides an oligonucleotide further comprising 1 to 4 non-nucleotide chain moieties.

Mixtures The invention further provides an oligonucleotide mixture comprising a mixture of at least two different antiviral oligonucleotides of the invention. In one embodiment, at least 10 different antiviral oligonucleotides. In one embodiment, at least 100 different antiviral oligonucleotides. In one embodiment, at least 1000 different antiviral oligonucleotides. In one embodiment, at least 106 different antiviral oligonucleotides.

  The invention further provides a mixture wherein the plurality of different oligonucleotides are at least 10 nucleotides in length. In one embodiment, it is at least 20 nucleotides in length. In one embodiment, it is at least 30 nucleotides in length. In one embodiment, it is at least 40 nucleotides in length. In one embodiment, it is at least 50 nucleotides in length. In one embodiment, it is at least 60 nucleotides in length. In one embodiment, it is at least 70 nucleotides in length. In one embodiment, it is at least 80 nucleotides in length. In one embodiment, it is at least 120 nucleotides in length.

Pharmaceutical Composition The present invention further comprises an antiviral pharmaceutical comprising a therapeutically effective amount of at least one pharmacologically acceptable antiviral oligonucleotide, polypyrimidine or oligonucleotide mixture, and a pharmaceutically acceptable carrier. The antiviral activity of the oligonucleotides or oligonucleotides in the mixture arises primarily through a sequence-independent mode of action.

  The present invention further provides antiviral pharmaceutical compositions adapted for the treatment, control or prevention of diseases associated with viral pathogenesis.

  The invention further provides an antiviral pharmaceutical composition adapted for the treatment, control or prevention of prion diseases.

  The invention is further of a mode selected from the group consisting of intraocular, oral digestion, enteral, inhalation, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, subarachnoid injection, intratracheal injection and intravenous injection. An antiviral pharmaceutical composition adapted for delivery by is provided.

  The present invention further provides an antiviral pharmaceutical composition further comprising a delivery system. In one embodiment, the delivery system targets a specific cell or a specific tissue. In one embodiment, the composition further contains at least one other antiviral agent in combination. In one embodiment, the composition further comprises a combination of non-nucleotide antiviral polymers. In one embodiment, the composition contains a combination of antiviral antisense oligonucleotides. In one embodiment, the composition further comprises an antiviral RNAi-derived oligonucleotide. In one embodiment, the antiviral RNAi-derived oligonucleotide is an siRNA.

The present invention further provides antiviral pharmaceutical compositions having an IC ₅₀ of 0.10 μM or less for the target virus. In one embodiment, an antiviral pharmaceutical composition is provided that has an IC ₅₀ of 0.05 μM or less for the target virus. In one embodiment, an antiviral pharmaceutical composition is provided that has an IC ₅₀ of 0.025 μM or less for the target virus. In one embodiment, an antiviral pharmaceutical composition is provided that has an IC ₅₀ of 0.015 μM or less for the target virus.

Kits The present invention further includes at least one antiviral oligonucleotide, mixture or antiviral pharmaceutical composition in a labeled package, wherein the antiviral activity of the oligonucleotide is primarily non-sequence complementary. A kit is provided that occurs and the label on the package indicates that the antiviral oligonucleotide can be used against at least one virus.

  The present invention further provides a kit comprising a mixture of at least two different antiviral oligonucleotides.

  The present invention further provides kits approved for human use by regulatory agencies.

  The present invention further provides kits that have been approved for use in regulatory animals by at least one non-human animal. In one embodiment, the non-human animal is a primate. In one embodiment, the non-human animal is a cat. In one embodiment, the non-human animal is a cow. In one embodiment, the non-human animal is a sheep. In one embodiment, the non-human animal is a dog. In one embodiment, the non-human animal is a pig. In one embodiment, the non-human animal is a horse.

Method of treatment The invention further provides the use of at least one oligonucleotide according to the invention, or a pharmaceutical composition according to the invention, in the manufacture of a medicament for the prevention or treatment of a viral infection in a subject.

  In one embodiment, the subject is a human. In one embodiment, the subject is a non-human animal. In one embodiment, the non-human animal is a primate. In one embodiment, the non-human animal is a cat. In one embodiment, the non-human animal is a cow. In one embodiment, the non-human animal is a sheep. In one embodiment, the non-human animal is a dog. In one embodiment, the non-human animal is a pig. In one embodiment, the non-human animal is a horse. In one embodiment, the subject is a plant.

  The invention further relates to the use of at least one oligonucleotide according to the invention, or a pharmaceutical composition according to the invention in the manufacture of a medicament for the prophylactic treatment of cancer caused by oncoviruses in humans or non-human animals. I will provide a.

  In one embodiment, the oligonucleotide is administered to a human. In one embodiment, the oligonucleotide is administered to an animal that is not human. In one embodiment, the non-human animal is a primate. In one embodiment, the non-human animal is a cat. In one embodiment, the non-human animal is a cow. In one embodiment, the non-human animal is a sheep. In one embodiment, the non-human animal is a dog. In one embodiment, the non-human animal is a pig. In one embodiment, the non-human animal is a horse.

Polypyrimidine oligo-related

  The invention further provides an oligonucleotide comprising at least 50% pyrimidine residues. In one aspect, at least 80%. In one aspect, at least 90%. In one embodiment, there are only pyrimidine residues.

  The present invention further provides an oligonucleotide wherein the pyrimidine residue is a cytosine residue. In one aspect, a thymine residue. In one embodiment, it is a cytosine residue or a thymine residue.

  The present invention further comprises a therapeutically effective amount of at least one pharmacologically acceptable polypyrimidine oligonucleotide or polypyrimidine oligonucleotide mixture, and a pharmaceutically acceptable carrier, wherein the oligonucleotide or the mixture An antiviral pharmaceutical composition is provided in which the antiviral activity of the oligonucleotides of the present invention occurs primarily through a sequence independent mode of action. In one embodiment, the oligonucleotide comprises at least one modified internucleotide linkage.

In one aspect, the composition is adapted for administration to an acidic site in vivo.
In one embodiment, the composition further contains a penetration enhancer.
In one embodiment, the composition further contains a surfactant.
In one embodiment, the composition is in powder form.
In one embodiment, the composition is in granular form.
In one embodiment, the composition is in particulate form.
In one embodiment, the composition is in nanoparticulate form.
In one embodiment, the composition is in the form of a suspension or solution.
In one embodiment, the composition is in the form of an emulsion.
In one embodiment, the composition is adapted for oral administration.
In one embodiment, the composition is adapted for vaginal administration.
In one embodiment, the composition contains at least one poly C oligonucleotide.
In one embodiment, the composition contains at least one poly T oligonucleotide.
In one embodiment, the composition contains at least one poly CT oligonucleotide.
In one embodiment, the composition is approved for human administration.
In one embodiment, the composition is approved for administration to a mammal.
In one aspect, the composition is approved for administration to a non-mammal.

  The present invention further provides a pharmaceutical composition comprising at least one pharmacologically acceptable polypyrimidine oligonucleotide adapted for administration to an acidic site in vivo for preventing or treating viral infection in a subject. For use in the manufacture of a medicament.

  In one embodiment, the subject is a human. In one embodiment, the subject is a mammal. In one embodiment, the subject is a non-mammal.

  As described in “Background of the Invention”, the antiviral activity of a number of antisense oligonucleotides (ON) has been tested. However, these antisense ONs are sequence specific and are typically about 16-20 nucleotides in length.

As shown in the results of Example 1 and Example 2, the antiviral effect of random PS-ON is not sequence specific. Given the volume and concentration of PS-ON used in these studies, it is theoretically almost impossible to have more than one copy of a particular random sequence in the mixture. This means that these PS-ON randomers cannot have an antisense effect. In the latter case, assuming that the antiviral effect was caused by the sequence specificity of PS-ON, such an effect needs to be caused by only one molecule, and it does not seem possible. For example, 40 with respect to the base length of ON randomers, any particular sequence in the population, theoretically exists only 1/4 ⁴⁰ or 4.1 × 10 ^-41 Total fragments. Based on the assumption that 1 mole = 6.022 × 10 ²³ molecules, our largest scale synthesis is currently carried out on a 15 micromole scale, and it is believed that not all possible sequences exist, and Each sequence is most likely to exist as only one copy. Of course, those skilled in the art applying the teachings of the present invention may use ONs having such sequence-specific ON sequences, but take advantage of the sequence-independent activities discovered in the present invention. Thus, the present invention is not limited to non-sequence complementary ONs, but claims of those disclosed in the prior art for sequence-specific antisense and RNAi (eg, siRNA) ON to treat viral infections. Give up the right.

  For applicable viruses (including, for example, those whose data are described herein), as the size of the randomer increases, the oligonucleotide length increases to 40 nucleotides and even more than 40 nucleotides. Increases antiviral ability. Due to the limitations of current phosphoramidite-based oligonucleotide synthesis, it must be pointed out that relatively large PS-ONs (eg 80- and 120-mers) are considerably contaminated with fragments smaller than the desired size. Don't be. The relatively weak effects (on a base-by-base basis) seen with relatively large oligos (80 and 120 bp) can reflect the relatively low concentration of full-length randomers in these populations and are available at the appropriate site It can also reflect a decline in sex. A relatively large randomer (> 40 bases) of appropriate purity (eg, at least 75% full length) is synthesized or purified, and / or delivery of any of these ONs provides, for example, targeted release or sustained release It would be possible to achieve much increased antiviral activity if facilitated by the system.

  In the present invention, a randomer (or other antiviral oligonucleotide as described herein) can block viral replication by several mechanisms including, but not limited to: 1. Prevent virion absorption or receptor interaction to prevent infection 2. Dope viral assembly or package viral genome in capsid (competitive packaging with viral DNA or RNA 3. Incomplete virions 3. While the packaging or capsid protein interacts with other structural proteins, it disrupts or prevents the formation of the capsid, thereby inhibiting viral release or incomplete Release virions, 4. bind to key viral components and inhibit or reduce their activity, 5. Binds to key host components required for virus growth.

  Without being limited to the mechanisms for achieving viral inhibition, as indicated above, there are several possible mechanisms that can explain and / or predict the inhibitory properties of ON against viral replication. The first of these is that the general aptamer effect of ON binds them to either cell surface or viral proteins to prevent viral absorption and fusion. The size threshold for the effect is considered to be the result of a predetermined cumulative charge required for interaction.

  A second possible mechanism is that ON can function intracellularly by inhibiting viral packaging and / or assembly. An ON that exceeds a predetermined size threshold can compete with or interfere with normal capsid / nucleic acid interactions to prevent packaging of the functional viral genome into a new virus. Alternatively, ON can prevent normal capsid formation to prevent normal virus germination, alter virus stability, or prevent proper virion degradation by internalization.

  Although the mechanism of action is not yet fully proven, the results of the assay indicate that the ON has a greater resistance to virus inhibition compared to clinically relevant substances, acyclovir, ganciclovir, ribavirin and protease inhibitors. Shown to get. Thus, the ON according to the present invention can be used for the treatment or prevention of viral infection. Viral infections to be treated can be caused by human, animal and plant viruses. In order to increase the stability and / or activity of the antiviral oligonucleotides, chemical modification of the oligonucleotides can be advantageously used. Methoxylation and other modifications at the 2 'position of ribose on RNA stabilizes RNA against nucleases, minimizes protein binding found in phosphorothioated nucleic acids, and increases the melting point of these oligos with target sequences It has been proven. 2'-O methylation and other 2'-modifications are currently used to improve the properties of antisense oligonucleotides, but if these modifications are present on all riboses, the oligonucleotide It does not elicit H activity and makes fully 2′-modified oligonucleotides relatively weak candidates for antisense activity. This uses 2'-O methyl and other 2 'modified "gapmers" that contain 2' modifications only at both ends of the oligonucleotide and maintain the ability of the oligo to activate RNase H It is the result. According to our knowledge, non-sequence anti-specific oligonucleotides with phosphorothioate linkages and 2'-O-methyl or other 2 'modified ribonucleotides on each ribose sugar of the oligonucleotide are reported. It has not been.

  As described herein, the inventors have found that the 40 base PS-ON randomer is a potent inhibitor of several different viruses. The inventors have provided a non-limiting indication that a thioated backbone may confer increased hydrophobic properties to the ON randomer, thereby allowing interaction with the hydrophobic domain of the ON randomer's viral fusion protein. Propose a hypothesis. These hydrophobic domains are believed to be essential for the membrane fusion activity of a number of different viruses including HSV, HIV, influenza, RSV and Ebola. In the case of HIV, such hydrophobic domains have been used as targets for the development of fusion inhibitors.

  Thus, incorporation of phosphorothioate linkages and ribonucleotide modifications, including 2'-O-methyl and other 2 'sugar modifications, into the oligonucleotides of the invention is useful for improving the properties of sequence-nonspecific antiviral oligonucleotides. is there. The results show that modification of the 2 'position of each ribose in PS-ON does not significantly alter its antiviral activity, but reduces the general interaction of PS-ON with serum proteins and significantly reduces resistance to low pH To improve. These properties are expected to improve pharmacokinetic performance and reduce the toxic side effects normally seen with PS-ON. For example, these pH tolerances make PS-ON more suitable for oral delivery. Also, the reduction in interaction with these serum proteins is expected to improve its pharmacokinetic behavior without affecting antiviral activity. Thus, an oligonucleotide having phosphorothioation of each bond and modification at the 2 ′ position of the ribose of each ribonucleotide, for example 2′-O-methyl modification, has antiviral activity and is a conventional antisense oligonucleotide Does not cause RNase H activity, which is a desired property, but is completely unnecessary for the activity described in the present invention. The results also show that the modification at position 2 of each ribose of PS-ON increases the nuclease resistance of ON compared to PS-ON that contains the same modification but is modified at both ends only (gapmer). Is shown. Gapmers are preferentially used in antisense technology. The nuclease resistance of PS-ON containing a modification at the '2-position of each ribose should exhibit beneficial properties such as improved pharmacokinetics and / or oral availability.

  In addition, PS-ONs containing modifications at the 2 ′ position of each ribose exhibit the desired properties, and PS-ONs that are modified at a number of the 2 ′ positions of ribose, such as at least 50% modification of ribose, and more PS-ON, preferably containing 80% or more modifications, would also exhibit the desired properties.

  As described above, the activity of the oligonucleotide does not target the nucleic acid by hybridization because the randomer does not have, for example, antisense activity. The inventors are therefore convinced that oligonucleotides target proteins. Adding 2'-O-methylribose modification to phosphorothioate oligonucleotides reduces protein binding activity (Kandimalla et al., 1998, Bioorganic Med Chem Lett. 8: 2103-2108; Mouet al., 2002, Nucleic Acid Res. 30: 749-758), these modifications were expected to reduce antiviral activity. Surprisingly, the inventors have found that adding 2'-O-methyl ribose modifications to phosphorothioate oligonucleotides does not affect antiviral activity.

  Described herein are the results of the assay for a number of different oligonucleotides. Unless otherwise indicated, the tested oligonucleotides have a 2'-H moiety (2'-deoxy) and are therefore ODN. However, the sequence-independent activity of the present invention is not limited to oligonucleotides containing such 2′-H moieties, but 2′-OH moieties as well as other 2′-modifications such as 2′-O-methyl. And also in oligonucleotides containing nucleotides with 2'-fluoro.

In the description herein, a number of abbreviations are used including:
Selected abbreviation
ON: Oligonucleotide
ODN: oligodeoxynucleotide
PS: phosphorothioate
PS2: phosphorodithioate
PRA: plaque reduction assay
PFU: Plaque formation unit
INF A: Influenza A virus
HIV: Human immunodeficiency virus (including both HIV-1 and HIV-2 if not specified)
HSV: Herpes simplex virus (including both HSV-2 and HSV-3 if not specified)
RSV: Respiratory polynuclear virus
COX: Coxsackie virus
DHBV: duck hepatitis B virus

Broad spectrum antiviral activity In accordance with the conclusions discussed above and the data reported herein, random ON and ON randomers can be used to replicate viral genome assembly and / or packaging and / or capsid formation. It appears to have a broad spectrum of antiviral activity with the virus. Therefore, to test this hypothesis, several PS-ONs of different sizes were selected and tested in various cell models of viral infection. A number of such tests are described herein in the Examples, including tests with CMV, HIV-1, RSV, Coxsackievirus B2, DHBV, Hantavirus, parainfluenza virus, and vaccinia virus; Also included are tests in HSV-1 and HSV-2 described in Examples 1 and 2.

Conclusions on broad spectrum antiviral activity Efficacy studies with various viruses reveal that random ONs and randomers exhibit inhibitory properties against a variety of different viruses. In addition, these studies indicate the conclusion that larger randomers show greater efficacy for viral inhibition than smaller randomers. This suggests a common size-dependent and / or charge-dependent mechanism for random ON or ON randomers in all encapsidating viruses.

  HSV and CMV are both double-stranded DNA viruses of the herpesvirus family, while HIV is a retrovirus-derived RNA virus, and RSV is a paramyxovirus-derived RNA virus. Given the fact that ON randomers can inhibit the viral function of a variety of different viruses, the following mechanisms are reasonable without being limited to the mechanisms listed above: A) ON / ON randomers are aptamers Inhibits viral function through effects and prevents viral fusion with the plasma membrane; and / or B) ON / ON randomers prevent virion assembly or packaging of viral DNA into the capsid or Doping results in defective virions; and / or C) ON / ON randomers interfere with host proteins or components required for viral assembly and / or packaging and / or gene expression is doing.

Prerequisites for antiviral activity Since randomized DNA sequences appear to be sufficient for viral inhibition, whether antiviral activity can be maintained in the absence of phosphorothioate, and efficacy depends on random sequence or viral genome It is interesting to see which of the specific sequences found in can be enhanced by selection.

  Therefore, DNA and RNA modifications were examined for their effects on the antiviral efficacy of randomers. Since randomers act through a sequence-independent, eg non-sequence complementary mechanism, these experiments were designed to examine minor changes in nucleic acid composition and charge distribution in antiviral efficacy.

To test whether ONs containing different nucleotide / nucleoside modifications can block HSV-1, REPs 2024, 2026, 2059 and 2060 were tested in HSV-1PRA as described in the Examples. REP 2024 (PS-ON with 2-O-methyl modification on ribose on 4 bases at both ends of ON), REP 2026 (PO-ON with methyl phosphonate modification at the bond between 4 bases at both ends of ON), REP 2059 (20 base RNA PS-ON randomer) and REP 2060 (30 base RNA PS-ON randomer) showed anti-HSV-1 activity. The assay was performed in VERO cells as a plaque reduction assay using HSV-1 (KOS strain). PS-ON was tested at increasing concentrations. IC ₅₀ values calculated from linear regression were 0.14, 3.41, 1.36 and 0.80, respectively.

  In the latter example, if the antiviral effect occurs only with ON consisting of a DNA phosphorothioate backbone, such an effect will occur with only one molecule. However, other backbones and modifications also provided positive antiviral activity. Of course, one of ordinary skill in the art applying the teachings of the present invention can also use different compounds ON. Modifications of ON, such as but not limited to phosphorothioate, may be beneficial for antiviral activity. This is most likely due to the required charge and / or DNA stabilization requirements of the ON both in the medium and in the cell, and may also depend on the chirality of the PS-ON. .

  Compound REP 2026 exhibited antiviral activity while having a central portion containing unmodified PO nucleotides and four methylphosphonate linkages at both ends protecting from degradation. This indicates that PO-ON can be used as an antiviral agent while protecting against degradation. This protection can be achieved by modifying the nucleotides at the termini and / or using a suitable delivery system as described below.

  In general, the sequence composition of the DNA used, whether randomer, random or specific HSV-1 sequences, has little impact on overall effectiveness. However, at medium length, the HSV-1 sequence was almost three times stronger than the random sequence. This data shows that while specific antisense exists functionally for specific HSV sequences, the sequence-independent mechanism elucidated herein (non-antisense mechanism) predominates this activity. This suggests that this part may be expressed. Indeed, as ON grows to 40 bases, essentially all antiviral activity can be attributed to sequence-independent (eg, non-antisense) effects.

Low toxicity randomer
One goal with ON randomers is to reduce toxicity. In animals, different sequences are known to elicit different responses such as general toxicity, interactions with serum proteins and interactions with the immune system (Monteith et al., Toxicol. Sci., 46: 365-375,1998). Thus, a mixture of ONs may reduce the toxic effect because the level of any particular sequence is very low, so the possibility of significant interaction by sequence or nucleotide composition is low.

Pharmaceutical Compositions ONs of the present invention may be in the form of therapeutic compositions or formulations useful for treating (or preventing) viral diseases, in human or non-human animals and / or specific Can be approved by regulatory authorities for use against other viruses or virus groups. These ONs may be used as part of a pharmaceutical composition when combined with a physiologically and / or pharmaceutically acceptable carrier. The characteristics of the carrier may depend on the route of administration. The pharmaceutical composition of the present invention may also contain other active factors and / or agents that improve the activity.

  Administration of ONs of the present invention used in pharmaceutical compositions or formulations or used to practice methods of treating animals can be performed in a variety of conventional ways, such as intraocular, oral ingestion, enteral, It can be done by inhalation or infusion into the skin, subcutaneous, intramuscular, intraperitoneal, subarachnoid, intratracheal, or intravenous.

  The pharmaceutical composition or oligonucleotide formulation of the present invention further includes, for example, without limitation, rifampin, ribavirin, pleconaril, cidofovir, acyclovir, penciclovir, ganciclovir, valacyclovir, famciclovir, foscarnet, vidarabine, amantadine, zanamivir, Oseltamivir, resquimod, anti-protease, pegylated interferon (Pegasys ™) anti-HIV protease (eg, lopinivil, saquinivir, amprenavir), HIV fusion inhibitor, nucleotide HIV RT inhibitor (eg, AZT, lamivudine, abacavir) , Non-nucleotide HIV RT inhibitors, doconosol, interferons, butylated hydroxytoluene (BHT) and chemotherapeutic agents for the treatment of viral diseases such as hyperlysine. For example, such additional factors and / or agents may be included in the pharmaceutical composition to produce a synergistic effect with the ON of the present invention.

  The pharmaceutical composition or oligonucleotide formulation of the present invention may further include, for example, without limitation, polyanionic agents, sulfated polysaccharides, heparin, dextran sulfate, polypentosane sulfate, polyvinyl alcohol sulfate, acemanane, polyhydroxycarboxylate. , Cellulose sulfate, polymers containing sulfonated benzene rings or naphthalene rings and naphthalene sulfonate polymers, acetylphthaloyl cellulose, poly-L-lysine, sodium caprate, cationic amphiphiles, cholic acid, etc. A polymer may be contained. It is known that polymers sometimes affect virion entry in cells by binding or adsorbing to virions themselves. This property of the antiviral polymer can be useful in competing with ON for binding or adsorption to virions, resulting in increased intracellular activity of ON compared to extracellular activity.

Exemplary lipid encapsulation and delivery
PS-ON (and oligonucleotides containing other modified linkages) are more resistant to endogenous nucleases than natural phosphodiesters, but are not completely stable and degrade slowly in blood and tissues. A limitation in the clinical application of PS oligonucleotide drugs is their propensity to activate complement by iv administration. In general, liposomes and other delivery systems reduce drug toxicity, prolong plasma residence time, and allow greater outflow to diseased tissue by extravasation of the carrier through the hyperpermeable vasculature. By delivering active drugs, the therapeutic index of drugs including ON is improved. In addition, in the case of PS-ON, lipid encapsulation prevents possible interactions with protein binding sites during circulation (Klimuk et al. (2000) J Pharmacol Exp Ther 292: 480-488).

According to the results obtained by the inventors described herein, approaches include, but are not limited to, lipophilic molecules, polar lipids, liposomes, monolayers, bilayers, small molecules Using delivery systems like vesicles, programmable fusion vesicles, micelles, cyclodextrins, PEG, ion injection, powder injection and nanoparticles (such as PIBCA, PIHCA, PHCA, gelatin, PEG-PLA) Delivers ON, and / or antisense and siRNA oligonucleotides as described herein. The use of such delivery systems provides one or more of the following advantages, including but not limited to: reduced toxicity of active compounds in animals and humans, reduced IC ₅₀ , drug delivery Prolonged duration of action from a point of view and protection from non-specific binding of oligonucleotides to serum proteins.

  Thus, the inventors have shown that the antiviral activity of PS-ON randomers increases with increasing size. Furthermore, this activity correlates with increased affinity for viral proteins (in viral lysates). Since it is well known in the art that phosphorothioate modifications increase affinity in protein-DNA interactions, we use the same FP-based assay as measuring interaction with viral lysates. Thus, the increasing ability of PS-ON randomer to bind fetal bovine serum (FBS) was tested. In this assay, 250 ug of non-heat inactivated FBS was conjugated with a fluorescently labeled 20 base PS-ON randomer under conditions of saturated binding (mP value). Increasing sizes (REP 2003, REP 2004, REP 2006 and REP 2007) of unlabeled PS-ON randomers were used to compete with FBS labeled bait interactions. The results of this study clearly show that the affinity for FBS increases as the size of the PS-ON randomer increases. This result suggests that the most active antiviral PS-ON will also bind to proteins with the highest affinity.

  However, the main therapeutic problems of phosphorothioate antisense oligonucleotides are as described by Kandimalla and co-workers (Kandimalla et al. (1998) Bioorg. Med. Chem. Lett. 8: 2103-2108) It is known in the art to be a side effect mainly due to increased interaction with proteins (especially serum proteins). Thus, in some cases it may be beneficial to use an appropriate delivery system that can deliver antiviral ON to the site of action while preventing its interaction with serum proteins. In addition, it may be beneficial to use a delivery system suitable for combined use of the sequence-independent ON of the present invention with other types of antiviral ONs such as antisense oligonucleotides and siRNAs.

  To show the predetermined effect of the delivery system, we tested two different delivery technologies based on liposomes: Cytofectin and DOTAP. We measured the protection of REP 2006 from interaction with serum proteins by DOTAP and cytofenctin in our in vitro FP-based interaction assay. Unencapsulated REP 2006 was able to compete with serum for bound fluorescent oligos, but once REP 2006 was encapsulated with either DOTAP or cytofenctin, it could no longer compete for serum binding. there were. These data suggest that encapsulation results in better pharmacokinetic behavior with protection of the oligo from serum interaction and reduced side effects.

  We have also demonstrated the delivery of PS-ON randomer REP 2006 (encapsulated with either cytofenctin and DOTAP) to 293A cells in the presence of high concentrations of serum (50%). It was measured by measuring the intracellular concentration of labeled REP 2006 using a quantitative method. These results indicate that such delivery agents increased the intracellular concentration of REP 2006, and in the case of DOTAP, the level of intracellular REP 2006 was significantly increased after 24 hours. Finally, we measured the protection of REP 2006 from serum protein interactions by DOTAP and cytofenctin in our in vitro FP-based interaction assay. Unencapsulated REP 2006 was able to compete with serum for bound fluorescent oligos, but once REP 2006 was encapsulated with either DOTAP or cytofenctin, it could no longer compete for serum binding. there were. These data suggest that encapsulation will result in better pharmacokinetic behavior with protection of the oligo from serum interaction and reduced side effects.

  Similarly, to demonstrate the effect of oligonucleotide lipid encapsulation, we expose cultured cells to fluorescently labeled randomers and then test the fluorescence intensity in lysed cells after two rounds of washing. Thus, the uptake of additional PS-ON randomers was monitored. Intracellular uptake of cells exposed to 250 nM REP 2004-FL was tested without a delivery system and encapsulated within one of the following lipid-based delivery systems: Lipofectamine ™ (Invitrogen), Polyfect ™ ) (Qiagen) and Oligofectamine ™ (Invitrogen). After 4 hours, the cells were washed twice with PBS and lysed using MPER lysis reagent (PROMEGA). The relative fluorescence yield from the same number of exposed cells with and without lipid system was detected. We observed that in the presence of all three drugs tested, there was a significant increase in intracellular PS-ON concentration compared to the case without the delivery system.

  According to test results, the use of a delivery system serves to protect the oligonucleotide from serum interactions, reduces side effects, increases tissue distribution, and / or significantly increases intracellular delivery of ON. Can do.

  Another potential benefit in using the delivery system is that with infectious virions, such as adsorption, to prevent amplification of viral infections through different mechanisms such as increased cell invasion of virions. Protect ON from interaction.

  Another approach is to achieve cell-specific delivery by associating the delivery system with a molecule that enhances affinity with specific cells, such molecules without limitation, antibody, receptor Body, ligand, vitamins, hormones and peptides.

  Additional options for delivery systems are provided below.

Combined ON
In certain embodiments, ONs of the invention are modified in a number of ways without compromising their ability to inhibit viral replication. For example, ON is bound to or complexed with another moiety at one or more of its nucleotide residues. Thus, the modification of the oligonucleotides of the invention increases activity, increases cell distribution, increases transport across the cell membrane specifically or non-specifically, or protects against degradation or excretion, or is advantageous. It may include chemically linking the oligonucleotide to one or more moieties or conjugates that provide the property. Such advantageous properties include, for example, lower serum interactions, higher virus-protein interactions, the ability to be formulated for delivery, detectable signal, improved pharmacokinetic properties, and lower toxicity. Can be mentioned. Such conjugated groups can be covalently linked to functional groups such as primary or secondary hydroxyl groups. For example, the conjugated moiety may include steroid molecules, non-aromatic lipophilic molecules, peptides, cholesterol, bis-cholesterol, antibodies, PEG, proteins, water-soluble vitamins, fat-soluble vitamins, another ON, or ON activity and / or Mention may be made of any other molecule that improves bioavailability.

  More specifically, exemplary conjugated groups of the present invention include interfering substances, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, SATE, t-butyl-SATE, groups that enhance the pharmacodynamic properties of oligomers, And groups that enhance the pharmacokinetic properties of the oligomer. Typical conjugated groups can include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, fluorescent nucleobases and dyes. .

  In the context of the present invention, groups that enhance pharmacodynamic properties include groups that enhance the degradation resistance of oligomers and / or groups that protect against serum interactions. In the context of the present invention, groups that enhance pharmacokinetic properties include groups that improve oligomer uptake, distribution, metabolism or excretion. Exemplary conjugated groups are described in International Patent Application No. PCT / US92 / 09196, filed Oct. 23, 1992, which is incorporated herein by reference.

  Conjugate moieties include, for example, cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86: 6553-6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let. , 4: 1053-1060, 1994), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 660: 306-309, 1992, Manoharan et al., Bioorg. Med) Chem. Let., 3, 2765-2770, 1993), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 20: 533-538, 1992), aliphatic chains such as dodecadiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 10: 1111-1118, 1991, Kabanov et al., FEBS Lett., 259: 327-330, 1990, Svinarchuk et al., Biochimie, 75, 49-54, 1993), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-o-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 36: 3651-3654,1995; Shea e t al., Nucl. Acids Res., 18: 3777-3783, 1990), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 14: 969-973, 1995), or adamantane acetic acid (Manoharan et al ., Tetrahedron Lett., 36: 3651-3654, 1995), pearl mityl moiety (Mishra et al., Biochim. Biophys. Acta, 1264, 229-237, 1995), or octadecylamine or hexylaminocarbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol Exp. Ther., 277: 923-937, 1996), but not limited thereto.

  The oligonucleotide may be, for example, without limitation, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-planoprofen, carprofen, dansylsarcosine, 2,3,5 -Active drug substances such as triodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethacin, barbiturates, cephaloproline, sulfa drugs, antidiabetics, antibacterials or antibiotics May be conjugated.

  Typical U.S. patents describing the preparation of typical oligonucleotide conjugates include, for example, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541 313; ; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582,4 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371, 241, 5,391 723; 5,416,203; 5,451, 463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; No. 5,688,941, each of which is incorporated herein by reference in its entirety.

  Another approach is to use anti-viral properties such as lipophilic pro-oligonucleotides by modification with charge-neutralizing adducts such as, for example, s-acetylthio-ethyl or s-pivacylylthio-ethyl. ON is to be prepared (Vives et al., Nucl. Acids Res., 27: 4071-4076, 1999). Such modifications have been shown to be beneficial for ONs that increase the uptake of ONs into cells and are therefore active in cells.

Designing non-specific ONs In another approach, an antiviral ON is designed that exhibits a low, preferably the lowest possible homology to the human (or other target organism) genome. The goal is to obtain an ON with minimal toxicity due to interaction with human or animal genomic sequences and mRNA. The first step produces a sequence of the desired length of ON, for example, manually or more commonly using a computer program, by aligning nucleotides, A, C, G, T in a random fashion . The second step compares the sequence of ON with a library of human sequences, such as GenBank and / or Ensemble Human Genome Database. If desired, sequence generation and comparison can be repeated to identify sequences having the desired low homology with the target genome. Desirably, the sequence of ON has the lowest possible homology with the entire genome, while preferably minimizing self-interaction.

Nonspecific ON with antisense activity
In another approach, an antiviral non-specific sequence portion is attached to the antisense sequence portion to enhance the final ON activity. Non-specific parts of ON are described in the present invention. The antisense portion is complementary to the viral mRNA.

Non-specific ON with G-rich motif activity
In another approach, an antiviral non-specific sequence moiety is attached to a motif moiety that improves the activity of the final ON. Non-specific parts of ON are described in the present invention. Non-limiting examples of the motif portion include CpG, G quartet, and / or CG described in the literature as stimulators of the immune system. Agrawal et al., Curr. Cancer Drug Targets, 3: 197-209, 2001.

Non Watson Crick ON
Another approach is to use an ON composed of one or more types of non-Watson-Crick nucleotides / nucleosides. Such ONs can mimic PS-ONs with some of the following properties similar to PS-ONs: (a) global changes; (b) inter-unit space; (c) chain Length; (d) net dipole due to accumulation of negative charge on one side; (e) ability to bind protein; (f) ability to bind viral protein; (g) ability to enter cells; (H) Acceptable therapeutic index, (i) Antiviral activity. ON has a preferred phosphorothioate backbone, but is not limited thereto. Examples of non-Watson-Crick nucleotide / nucleosides are Kool, Acc. Chem. Res., 35: 936-943, 2002 and Takeshita et al., J., describing ON containing a synthetic abasic site. Biol. Chem., 262: 10171-10179, 1987.

Antiviral polymers Another approach is to use polymers that mimic the activity of phosphorothioate ON. As described in the literature, some anionic polymers have been shown to have antiviral inhibitory activity. These polymers belong to several classes: (1) polysaccharide sulfates (dextrin and dextran sulfate; cellulose sulfate); (2) polymers containing sulfonated benzene or naphthalene rings and sulfonated naphthalene polymers. (3) polycarboxylate (acrylic acid polymer); and acetylphthaloylcellulose (Neurath et al., BMC Infect. Dis., 2:27, 2002) and (4) abasic oligonucleotide (Takeshita et al., 1987, J. Biol. Chem., 262: 10171-10179). Other examples of non-nucleotide antiviral polymers are described in the literature. The polymers described herein mimic the PS-ON described herein and have the following properties similar to PS-ON: (a) chain length; (b) one side A net dipole due to the accumulation of negative charge at (c) the ability to bind to a protein; (d) the ability to bind to a viral protein; (e) an acceptable therapeutic index; (f) antiviral activity. In order to mimic the effect of PS-ON, the antiviral polymer may preferably be a polyanion that exhibits a similar space between its units, as compared to PS-ON. It may have the ability to enter cells alone or in combination with a delivery system.

Antiviral activity of double-stranded PS-ON Random sequences and their complements (either PS modified or not) are fluorescently labeled as described elsewhere and described in the present invention. The ability to bind to HSV-1 and HIV-1 purified by fluorescence polarization is examined. Hybridization was verified by acrylamide gel electrophoresis. Unmodified REP2017 (2017U) had no binding activity in HSV-1 or HIV-1 lysates, either single stranded (ss) or double stranded (ds). However, PS-modified REP2017 was able to interact with HSV-1 and HIV-1 either single-stranded or double-stranded.

  According to our results described herein, the approach is to use double stranded ON as an effective antiviral agent. Preferably, such ONs have a phosphorothioate backbone, but other and / or enhance antiviral activity and / or stability and / or delivery properties as described herein for single chain ONs. There may also be additional modifications.

In vitro assays for drug discovery An in vitro assay is developed based on fluorescence polarization to measure the ability of PS-ON to bind to viral components, such as viral proteins. When protein (or another interactor) binds to a fluorescently labeled bait, the three-dimensional tumbling of the bait is delayed in solution. This tumbling delay is measured by the intrinsic rise in the polarization of the excited light derived from the labeled bait. Thus, increased polarization (reported as an unmeasured measure [mP]) correlates with increased binding.

  One methodology is to use PS-ON randomers labeled as FITC at the 3 'end with FITC using an inflexible linker (3'-(6-fluorescein) CPG) as the bait. The PS-ON randomer is diluted to 2 nM in assay buffer (10 mM Tris, pH 7.2, 80 mM NaCl, 10 mM EDTA, 100 mM b-mercaptoethanol and 1% Tween 20). The oligo is then mixed with the appropriate interactor. In this case, we have 0.5M KCl and 0.5% Triton X-100 (HSV-1 and HIV-1) or 10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.1% Triton X-100 (RSV Sucrose gradient purified HSV-1 (Maclntyre strain), HIV-1 (Mn strain) or RSV (A2 strain) lysate suspended in Following interaction with the bait, the complex is challenged with various unlabeled PS-ONs to assess the ability to displace the bait from the complex.

  In the preliminary test, three baits of different sizes were used; 6 (REP 2032-FL), 10 (REP 2003-FL) and 20 bases (REP 2004-FL), and bait HSV-1, HIV-1 and The ability to interact with RSV lysate was tested. Viral lysate binding to different sized baits was measured by fluorescence polarization. In the presence of any virus lysate, the extent of binding was dependent on the size of the bait used, and in the presence of virus lysate, 2004-FL showed the greatest shift in mP (binding). The inventors have found that it is similar to the antiviral efficacy of size-dependent PS-ON randomers. This bait was then used to evaluate the ability of different sized PS-ONs to compete for bait and lysate interactions.

  The interaction of REP 2004-FL with HSV-1, HIV-1 and RSV lysates was challenged with increasing sizes of PS-ON. Measurement of the affinity of PS-ON randomer for virus lysates was detected by fluorescence polarization. Challenge complex formation with HSV-1 lysate, HIV-1 lysate or RSV lysate with increasing concentrations of REP 2003, REP 2004, REP 2006 or REP 2007 using REP 2004-FL as bait did. For each virus lysate tested, we found that REP 2003 could not competitively remove the bait from the lysate. The bait interaction was very strong, as evidenced by the relatively weak competition drawn by the REP 2004 (unlabeled bait) competitor. However, it was observed that as the size of competitor PS-ON increased beyond 20 bases, its bait replacement capacity was also enhanced. This indicates that the affinity for protein components in the virus lysate increases as the size of the PS-ON randomer increases. This phenomenon reflects the increased antiviral activity of larger PS-ON randomers against HSV-1, HSV-2, CMV, HIV-1 and RSV.

  The similarity between the potency in bait competition and the antiviral activity of PS-ON randomers indicates that the typical of this assay is a good predictor of antiviral activity. Because this assay is robust, easy to perform, and very stable, it is based on the ability to interact with one or more components, for example, viral proteins, rather than based on the identification of a specific target. It is a very good candidate for high throughput screening to identify new antiviral molecules.

  The exemplary methods described herein use fluorescence polarization to measure interaction with virus lysates, but numerous techniques for monitoring protein interactions are known in the art, It can be used in this method. They include surface plasmon resonance, fluorescence resonance energy transfer (FRET), enzyme immunosorbent assay (ELISA), gel electrophoresis (for mobility shift measurements), isothermal titration and differential scanning calorimetry, and column chromatography. However, it is not limited to them. These other different techniques can be applied to measure the interaction of ON with viral lysates or components and may thus be useful in screening for compounds with antiviral activity.

  The methods described herein are used to screen new compounds from libraries of interest, eg, synthesized by combinatorial chemistry or isolated by purification of natural materials. A) to determine the appropriate size, modification and backbone of the new ON; b) to investigate new molecules containing the new polymer; c) to identify new ONs or specific compounds for new compounds Used to predict virus susceptibility; or d) to determine a set of compounds to maximally inhibit a particular virus.

The large affinity of the lysate with the large size PS-ON randomer is due to the fact that the antiviral mechanism of action of the PS-ON randomer is associated with one or more viral protein components that prevent either virion infection or replication correction. It is based on the interaction. It also suggests that this interaction is charge (size) dependent and sequence independent. Because these PS-ON randomers have size-dependent activity across multiple viruses across several different families, we believe that PS-ON is a common, charge-dependent protein-protein interaction, protein -Suggests interfering with DNA / RNA interactions and / or molecule-molecule interactions. These interactions can include (but are not limited to):
Interaction between individual capsid subunits during capsid formation.
Interaction between capsid / nucleocapsid protein and viral genome.
Interaction between budding capsid and glycoprotein.
Interaction between glycoprotein and receptor during infection.
Interaction between other key viral components involved in virus replication.

  These multiple simultaneous inhibitors of protein-protein interactions represent a novel mechanism of antiviral inhibition.

Effect of sequence composition of PS-ON on viral lysate interactions We monitored the ability of different sequences of PS-ON to interact with several viral lysates. In each case, a 20mer PS-ON is labeled with FITC at the 3 'end as previously described herein. The PS-ON examined consisted of A20, G20, T20, C20, AC10, AG10, TC10, TG10, REP 2004 and REP 2017. Each of these sequences was diluted to 4 nM with assay buffer and incubated in the presence of 1 ug of HSV-1, HIV-1 or RSV lysate. The interaction was measured by fluorescence polarization.

  The profile of interaction with all sequences tested is similar for all virus lysates, indicating that the nature of the binding interaction is very similar. The ability of 20mer PS-ONs with different sequence compositions (A20, C20, G20, T20, AC10, AG10, TC10, TG10, REP 2004, REP 2017) to bind to viral lysates was measured by fluorescence polarization. PS-ON3 ′ labeled with FITC was incubated in the presence of 1 ug of HSV-1, HIV-1 or RSV lysate. Among each lysate, PS-ON of uniform composition (A20, G20, T20, C20) was the weakest interactant, and among these, A20 was the weakest interactant with a significant difference. For the other PS-ONs tested, all except TG10 showed similar strong interactions, which were consistently the strongest interaction in each lysate. The binding profiles of these PS-ONs were similar for all three virus lysates.

Target identification of PS-ON randomers in HIV-I The ability of PS-ON randomers to bind to purified HIV-1 protein was tested by fluorescence polarization as described in Example 9. The amount of purified HIV-1p24 or purified HIV-1 gp41 was increased and reacted with REP 2004-FL. The present invention and others note that there is a protein concentration dependent shift in fluorescence polarization that shows an interaction with both of these proteins.

  A range of sizes of PS-ON randomers were also tested for their ability to bind to these proteins using fluorescent versions of REP 2032, REP 2003, REP 2004, REP 2006 and REP 2007. We have observed that for p24 there is no size-dependent interaction with p24, but we have a PS of greater than 20 bases for randomers of less than 20 bases. We observed increased binding of the -ON randomer to gp41. This suggests that when the length of PS-ON randomers exceeds 20 bases, multiple copies of gp41 can bind to individual randomers and increase their polarization.

  This is a significant finding as it reveals the greater ability of ON to sequester structural proteins during viral synthesis and limits its utility for new virion formation.

High Affinity Oligonucleotides Another approach is to concentrate or purify ONs with a higher affinity for viral components, such as viral proteins, than the average affinity in the standard pool of ONs. Thus, the method exhibits a high affinity for one or more viral components, for example providing one or more non-sequence complementary ONs having a three-dimensional shape that contributes to such a high binding affinity. The rationale is that ON can be folded into a three-dimensional shape that acts as a linear molecule in association with virus components while enhancing interaction with such virus components. Without being limited to a specific technique, high affinity ON can be purified or concentrated by the following method.

One method of purifying high affinity ONs or multiple high affinity ONs is to use a stationary phase medium with viral proteins attached as the affinity matrix to bind the ONs, then increasing stringent conditions. It can be eluted with (for example, increasing concentrations of salt or other chaotropic agent, and / or increasing temperature and / or pH changes). Such a method can be performed, for example, by:
(A) loading a pool of ONs into an exchange column having viral proteins or several viral proteins or virus lysates bound to a stationary phase;
(B) displace (elute) bound ON from the column, eg, by using a displacer solution such as increasing salt solution;
(C) collecting ON fractions eluted at different salt concentrations;
(D) Clone and sequence ON eluted from different fractions, more preferably from higher salt concentration fractions, so that the ON eluted at high salt concentrations has a high affinity with viral proteins. And (e) a binding assay and / or a virus inhibition assay, eg, a ON sequence sequenced in a fluorescence polarization binding assay and / or a cellular virus inhibition assay and / or an animal virus inhibition assay as described herein. Check for activity.

In a second example, high affinity ONs can be purified using methods derived from and modified from the SELEX methodology (Morris et al., Biochemistry, 95: 2902-2907, 1998). One implementation of such a method can be performed as follows.
(A) To provide a starting pool of ONs that are a population of synthetic random ONs containing a vast number of sequences, eg, 10 ¹⁴ to 10 ¹⁶ different sequences. Each ON molecule contains a fragment of random sequence adjacent to the primer-binding sequence at each end to facilitate the polymerase chain reaction (PCR). Since the nucleotide sequence of essentially every molecule is unique, a huge number of structures are sampled in a population. These structures determine the biochemical properties of each molecule, such as the ability to bind to a given viral target molecule;
(B) contacting ON with a viral protein or several viral proteins or lysates;
(C) Select ONs that are bound to viral proteins using a partitioning technique that can partition unbound and unbound ONs, such as the original gel shift and nitrocellulose filtration. The bound species can be physically separated from the unbound species by any of these methods, and the most bound sequences can be preferentially recovered. Also, to select ONs that bind to small proteins, it is desirable to bind the target to a solid support and use that support as an affinity purification matrix. Those molecules that do not bind are washed away, those that bind are eluted with the free target, and again again physically separate the bound and unbound species.
(D) amplifying the eluted bound ON using PCR with a primer that hybridizes to the flanking sequence of the ON;
(E) Steps (b), (c) and (d) are repeated multiple times (ie multiple cycles or multiple enrichments and Amplification) Repeat. After several cycles of enrichment and amplification, the population is dominated by sequences that exhibit the desired biochemical properties:
(F) cloning and sequencing one or more ONs selected from the enrichment cycle, eg, the last cycle; and (g) an assay, eg, a fluorescence polarization binding assay as described herein, and / or To examine the binding and / or activity of ON sequenced in cell virus inhibition assays and / or animal virus inhibition assays.

  Another approach is to apply a modification of the split synthesis methodology to 1-bead 1-PS as described in Yang et al., Nucl. Acids Res., 30 (e132): 1-8, 2002. -ON and 1-Bead 1-PS2-ON are created. Binding and selection of specific beads to viral proteins can be performed. PCR-based identification tags of selected beads can be used to sequence both nucleobases and locate thioate / dithioate bonds. This approach allows rapid and convenient identification of PS-ON or PS2-ON that exhibits potent antiviral properties that bind to viral proteins.

  Once the specific sequence of high affinity that binds to the viral protein is determined (eg, by individual amplification and sequencing), one or more such high affinity sequences are selected and synthesized (eg, , Either chemical synthesis or enzymatic synthesis), provides for the preparation of high affinity ONs that can be modified to improve activity, including improving pharmacokinetic properties. Such a high affinity ON can be used in the present invention.

Prion disease Another approach is used in another aspect of the invention for the treatment, control or prevention of prion disease. This fatal neurodegenerative disease is infectious and affects both humans and animals. Structural changes to cellular prion protein, PrPC scrapie isoform, and PrPSC are considered essential steps in the onset and progression of prion disease. Amyloid polymers are involved in the neuropathology of prion diseases.

  Incubation of prion protein fragments with double stranded nucleic acids results in amyloid fibril formation (Nandi et al., J. Mol. Biol., 322: 153-161, 2002). ONs that have an affinity for proteins, such as phosphorothioates, are used to compete with or inhibit the interaction of double-stranded nucleic acids with PrPC, thus stopping amyloid polymer formation. Such ONs of different sizes and different compositions can be used in appropriate delivery forms to treat patients with prion disease or to prevent high-risk situations. Such interfering ONs can be identified by measuring the amyloid polymer folding change, as described by Nandi et al., (Supra) in the presence of a test ON.

Possible viral pathogenesis Another approach is another aspect of the invention for the treatment or prevention of a disease or condition with a possible viral pathogenesis, as described without limitation in the examples below. Used in. Viruses are putative causative agents in diseases and conditions that are not related to the primary infection of the virus. For example, arthritis can occur in HCV (Olivieri et al., Rheum. Dis. Clin. North Am., 29: 111-122, 2003), parvovirus B19, HIV, HSV, CMV, EBV and VZV (Stahl et al., Clin Rheumatol., 19: 281-286, 2000). Other viruses have also been identified as playing a role in various diseases. For example, influenza A in Parkinson's disease (Takahashi et al., Jpn. J. Infect. Dis., 52: 89-98, 1999), coronavirus, EBV and other viruses in multiple sclerosis (Talbot et al., Curr Top Microbiol. Immunol., 253, 247-71, 2001), EBV, CMV and HSV-6 in chronic fatigue syndrome (Lerner et al., Drugs Today, 38: 549-561, 2002), asthma (Walter et al , J. Clin. Invest., 110: 165-175, 2002) and Paramyxovirus in Paget's disease, and HBV, HSV and influenza in Guillain-Barre syndrome.

  Because of these etiologies, inhibition of related viruses using the present invention can delay, slow down or prevent the corresponding disease or condition, or at least a portion of the disease from developing.

Oligonucleotide Modifications and Synthesis As indicated in the summary above, modified oligonucleotides are useful in the present invention. Such modified oligonucleotides include, for example, oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having a modified main chain include those that retain a phosphorus atom in the main chain and those that do not have a phosphorus atom in the main chain.

  Such modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, 3′-alkylene phosphonates, 5 ′ -Methyl or other alkyl phosphonates including alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidites including 3'-amino phosphoramidites and aminoalkyl phosphoramidites, thionophosphoramidites, thiono Alkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, carvalanyl phosphate borano-phosphates with conventional 3 ', 5' linkages, 2 ', 5' linked analogs, and one or more Between nucleotides If found 3'～3 ', 5'～5' include those having a polarity obtained by inverting a bond or 2'～2 '. Oligonucleotides with reversed polarity typically have a single 3 'to 3' linkage with most 3 'internucleotide linkages, ie abasic (losing nucleobase or having a hydroxyl group instead) And a single inverted nucleoside residue. Various salts, mixed salts and free acid forms are also mentioned.

  Preparation of oligonucleotides having linkages containing phosphorus as shown above is described, for example, in U.S. Pat.Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697; and 5,625,050 The entirety is incorporated herein.

Some typical oligonucleotide backbones that do not include phosphodiesters include short alkyl or cycloalkyl internucleotide bonds, mixed heteroatoms and alkyl or cycloalkyl internucleotide bonds, or one or more short heteroatoms. Or a main chain formed by a heterocycle internucleotide linkage. These include morpholino linkages (partially formed from the nucleoside sugar moiety), siloxane backbones, sulfides, sulfoxides and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioform Acetyl backbone; ribocetyl backbone; alkene-containing backbone; sulfamate backbone; methyleneimino and methylenehydrazino backbone; sulfonate and sulfonamide backbone; amide backbone; and mixed N, O, S and CH ₂ The thing which has the other thing which has these component parts is mentioned. Particularly advantageous are backbone bonds that include one or more charged moieties. Examples of U.S. patents describing the preparation of prior oligonucleotides include U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269; 5,677,439;

Modified oligonucleotides may also contain one or more substituted sugar moieties. For example, such oligonucleotides may have the following 2 ′ modifications: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N- alkynyl; or O- alkyl -O- 1 one to be able to encompass alkyl, in which alkyl, alkenyl and alkynyl, alkyl or C ₂ -C ₁₀ of C ₁ -C ₁₀ not substituted or substituted It may be alkenyl and alkynyl, or 2'-o- (o-carboran-1-yl) methyl. Specific examples are O [(CH ₂ ) _n O] _m CH ₃ , O (CH ₂ ) -OCH ₃ , O (CH ₂ ) _n NH ₂ , O (CH ₂ ) _n CH ₃ , O (CH ₂ ) _n ONH ₂ and O (CH ₂ ) _n ON [CH ₂ ) _n CH ₃ ] ₂ , where n and m are 1 to 10. Other exemplary oligonucleotides include the following 2 ′ modifications: C ₁ -C ₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN , Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, It includes one of a substituted silyl, a reporter group, an interfering substance, a group for improving the pharmacokinetic properties of the oligonucleotide, or a group for improving the pharmacodynamic properties of the oligonucleotide. Examples include 2′-methoxyethoxy (2′-O—CH ₂ CH ₂ OCH ₃ ), also known as 2′-O- (2-methoxyethyl) or 2′-MOE (Martin et al., Helv. Chim. Acta., 78: 486-504, 1995), ie, alkoxyalkoxy groups; 2'-dimethyl-aminoxyethoxy, ie, O (CH ₂ ) ₂ ON (CH ₃ ), also known as 2'-DMAOE ₂ groups; and 2′-dimethylaminoethoxyethoxy (also known as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), ie 2′-O—CH ₂ —O—CH ₂ —N (CH ₂ ) ₂ is included.

Other modifications include locked nucleic acids (LNA) in which the 2′-hydroxy group is attached to the 3 ′ or 4 ′ carbon of the sugar ring, thereby forming a bicyclic sugar moiety. The bond can be a methylene (—CH ₂ —) group bridging the 2 ′ oxygen atom and the 4 ′ carbon atom, where n is 1 or 2. LNA and its preparation are described in WO 98/39352 and 99/14226, which are hereby incorporated by reference in their entirety.

  Other modifications include sulfur-nitrogen bridge modifications such as locked nucleic acids as described in Orum et al., (2001) Curr. Opin. Mol. Ther., 3: 239-243. .

Other modifications include 2'-methoxy (2'-O-CH ₃ ), 2'-methoxyethyl (2'O-CH ₂ -CH ₃ ), 2'-ethyl, 2'-ethoxy, 2'- Aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ), 2'-allyl (2'-CH ₂ -CH = CH ₂ ), 2'-O-allyl (2'-O-CH ₂ -CH = CH ₂ ) and 2 ′ fluoro (2′-F).

  The 2 ′ modification may be in the arabino position (up) or in the ribo (down) position. Similar modifications may be made at other positions on the oligonucleotide, particularly at the 3 ′ position of the sugar on the 3 ′ terminal nucleotide or at the 2′-5 ′ linked oligonucleotide and at the 5 ′ position of the 5 ′ terminal nucleotide. May be. Oligonucleotides may have sugar mimetics as cyclobutyl moieties instead of pentofuranosyl sugars. Typical U.S. patents describing the preparation of such modified sugar structures include, for example, U.S. Pat.Nos. 4,981, 957; 5,118,800; 5,319,080; 5,359,044; 5,393, 878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567, No. 811; 5,576,427; 5,591, 722; 5,597,909; 5,610,300; 5,627, 053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792, 747; and 5,700,920.

  Still other modifications include ON concatamers consisting of multiple oligonucleotides linked by a linker. A linker may consist of, for example, modified nucleotide and non-nucleotide units. In some cases, the linker provides a degree of freedom for the ON concatemer. The use of such ON concatamers can provide an easy way to synthesize the final molecule by linking smaller oligonucleotide building blocks to obtain the desired length. For example, a 12 carbon linker (C12 phosphoramidite) is used to link two or more ON concatamers to provide length, stability and freedom.

As used herein, “unmodified” or “natural” bases (nucleobases) include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine ( C) and uracil (U) are included. Oligonucleotides may also include base modifications or base substitutions. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and 6-methyl and other alkyl derivatives of guanine, adenine and guanine 2-propyl and other alkyl derivatives of 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH ₃ ) uracil and other alkyls of cytosine and pyrimidine bases Derivatives, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines And guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines , 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine Bases and natural bases may be included. Additional modified bases include phenoxazine cytidine (1H-pyrimido [5,4-b] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido [5,4-b ] [1,4] benzothiazin-2 (3H) -one), substituted phenoxazine cytidine (eg 9- (2-aminoethoxy) -H-pyrimido [5,4-b] [1,4] benzoxazine -2 (3H) -one), carbazole cytidine (2H-pyrimido [4,5-b] indol-2-one), pyridoindole cytidine (H-pyrido [3 ′, 2 ′: 4,5] pyrrolo [ And tricyclic pyrimidines such as G-clamp such as 2,3-d] pyrimidin-2-one). Modified bases may include those in which purine or pyrimidine is replaced by other heterocycles such as, for example, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Additional nucleotides include those described in US Pat. No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, p858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990. , Englisch et al., Angewandte Chemie, International Edition, 30: 613, 1991, and Antisense Research and Applications, Chapter 15 Sanghvi YS, p289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993.

  Another modification includes a phosphorodithioate linkage. Phosphorodithioate ON (PS2-ON) and PS-ON have similar binding affinity for proteins (Tonkinson et al., Antisense Res. Dev., 4: 269-278, 1994) (Cheng et al., J. Mol. Recogn., 10: 101-107, 1997) and know that the potential mechanism of action of ON is binding to viral proteins, It is desirable to include phosphorodithioate in the antiviral ON described in the present invention.

  Another approach to modify ON is described in Yu et al., Bioorg. Med. Chem., 8: 275-284, 2000 and Inagawa et al., FEBS Lett., 25: 48-52, 2002. Is to produce a stereo-defined or stereo-enriched ON. The ON prepared by conventional methods consists of a mixture of diastereomers due to asymmetry around the phosphorus atom involved in internucleotide linkage. This can affect the stability of binding between ON and viral components such as viral proteins. Previous data has shown that protein binding is significantly sterically dependent (Yu et al.). Thus, by using stereo-defined or stereo-enriched ON, it was possible to improve the binding properties of the protein and improve the antiviral efficacy.

  Modification incorporation as described above can be utilized in a number of different incorporation patterns and levels. That is, specific modifications need not be included at each nucleotide or bond in the oligonucleotide, and various modifications can be utilized in combination, even on a single oligonucleotide or single nucleotide.

  By way of example and according to the above description, modified oligonucleotides containing phosphorothioate or dithioate linkages can include one or more substituted sugar moieties, particularly 2'-ethyl, 2'-ethoxy, 2'- in the sugar moiety. Including, but not limited to, methoxy, 2'-aminopropoxy, 2'-allyl, 2'-fluoro, 2'-pentyl, 2'-propyl, 2'-dimethylaminooxyethoxy and 2'-dimethylaminoethoxyethoxy Modifications that are not made may also be included. The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-fluoro. Similar modifications can be made at other positions on the oligonucleotide, especially on the 3 'terminal nucleotide or in the 2'-5' linked oligonucleotide at the 3 'position of the sugar and the 5' position of the 5 'terminal nucleotide. Good. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Furthermore, the ON may have a structure consisting of a glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone or a part thereof (Zhang L, et al (2005) J. Am. Chem. Soc. 127 (12): 4174-5). Such GNAs may contain phosphorothioate linkages and may contain only pyrimidine bases.

Oligonucleotide Synthesis The oligonucleotides can be synthesized using methods known in the art. For example, an unsubstituted or substituted phosphodiester (P = O) oligonucleotide can be synthesized on an automated DNA synthesizer (eg, Applied Biosystems model 380B) using standard phosphoramidite reagents with oxidation by iodine. . For phosphodiester oligonucleotides, except replacing the standard oxidation bottle with acetonitrile solution of 0.2M 311-1,2-benzodithiol-3-one 1,1-dioxide for stepwise thioation of phosphite linkages Thus, phosphorothioate (P = S) can be synthesized. The thioate waiting step can be increased to 68 seconds, followed by a capping step. After cleavage from a CPG column and deblocking in concentrated ammonium hydroxide at 55 ° C (18 hours), the oligonucleotide can be purified by precipitating twice from 0.5 M NaCl solution with 2.5 volumes of ethanol. it can.

  Phosphinate oligonucleotides can be prepared as described in US Pat. No. 5,508,270; alkyl phosphonate oligonucleotides can be prepared as described in US Pat. No. 4,469,863; US Pat. No. 5,610,289 3′-deoxy-3′-methylene phosphonate oligonucleotides can be prepared as described in US Pat. Nos. 5,625,050; phosphonates as described in US Pat. Nos. 5,256,775 and 5,366,878. Loamidite oligonucleotides can be prepared; published PCT applications, PCT / US94 / 00902 and PCT / US93 / 06976 (published as WO94 / 17093 and 94/02499, respectively) Alkylphosphonothioate oligonucleotides can be prepared as described in US Pat. No. 5,476,925, as described in US Pat. No. 5,476,925. 3′-amino phosphoramidite oligonucleotides can be prepared; phosphotriester oligonucleotides can be prepared as described in US Pat. No. 5,023,243; US Pat. Nos. 5,130,302 and 5,177,198 Boranophosphate oligonucleotides can be prepared as described in US Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240, and 5,610,289. , Methylenemethylimino-linked oligonucleotides that are also identified as MMI-linked oligonucleotides, methylenedimethylhydrazo-linked oligonucleotides that are also identified as MDII-linked oligonucleotides, and methylenecarbonylamino bonds that are also identified as amide-3 linked oligonucleotides Oligonucleotide and amide-4-linked oligo Methyleneaminocarbonyl-linked oligonucleotides that are also identified as gonnucleosides can be prepared, as well as mixed backbone compounds having, for example, another MMI and P═O or P═S linkage; US Pat. No. 5,264,562 and the like Form acetal and thioform acetal linked oligonucleotides can be prepared as described in US Pat. No. 5,264,564; and ethylene oxide linked oligonucleotides can be prepared as described in US Pat. No. 5,223,618. Each cited patent and patent application is hereby incorporated by reference in its entirety.

Oligonucleotide preparations and pharmaceutical compositions The oligonucleotides can be prepared into oligonucleotide preparations or pharmaceutical compositions. Thus, the present oligonucleotides can be used for other molecules, molecular structures or compounds such as liposomes, receptor targeting molecules, oral, enteral, topical or other formulations to aid uptake, distribution and / or absorption. It may be mixed with a mixture, encapsulated, conjugated, or otherwise associated. Typical U.S. patents describing the preparation of formulations that assist in such uptake, distribution and / or absorption include, for example, U.S. Pat.Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; , 721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; _5,469,854;, 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and No. 5,595,756 can be mentioned, respectively Which is incorporated herein by reference in its entirety.

  The oligonucleotides, formulations and compositions of the present invention are capable of providing (directly or indirectly) biologically active metabolites or residues thereof when administered to animals, including humans. It includes certain pharmaceutically acceptable salts, esters or salts of such esters. Thus, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

  The term “prodrug” refers to a therapeutic agent prepared in an inactive form that is converted into the active form (ie, drug) in the body or cells by the action of endogenous enzymes or other chemicals and / or conditions. . In certain embodiments, prodrug versions of the oligonucleotides are disclosed in Gosselin et al. WO 93/24510 and Imbach et al. WO 94/26764 and incorporated herein by reference in their entirety. Prepared as a SATE [(S-acetyl-2-thioethyl) phosphate] derivative according to the method disclosed in US Pat. No. 5,770,713.

  The term “pharmaceutically acceptable salt” refers to a physiologically and pharmaceutically acceptable salt of the compound, ie, retains the desired biological activity of the parent compound and imparts undesirable toxic effects thereto. Say salt that does not give. Many of such pharmaceutically acceptable salts are known and can be used in the present invention.

  For oligonucleotides, useful examples of pharmaceutically acceptable salts include, for example, salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine; For example, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid; for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, Organics such as citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, arginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, polygalacturonic acid Salts formed with acids; and like chlorine, bromine and iodine Salts formed from elemental anions but not limited thereto.

  The invention also encompasses pharmaceutically acceptable compositions and formulations containing the antiviral oligonucleotides of the invention. Such pharmaceutically acceptable compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the place to be treated. For example, administration may be local (mucosal including ocular and vaginal and rectal delivery); may be into the lungs by inhalation or inhalation of powder or aerosol by nebulizer; intrabronchial; intranasal Epithelial and transdermal; oral; or parenteral. Parenteral administration includes intravenous or intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or, for example, intracranial administration such as subarachnoid or intraventricular.

Pharmaceutically acceptable compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous solutions, powders or oily bases, thickeners and the like may be necessary or desirable. Coated condoms and gloves are also useful. Preferred topical formulations are those in which the oligonucleotides of the invention are admixtures with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Can be mentioned. Preferred lipids and liposomes include neutral (eg dioleoyl phosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), negative (eg dimyristoyl phosphatidylglycerol DMPG) and cationic (eg di Oleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The oligonucleotide may be encapsulated within the liposome or may form a complex with it, particularly with a cationic liposome. Alternatively, the oligonucleotide may form a complex with a lipid, particularly a cationic lipid. Preferred fatty acids and esters include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurate, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, or alkyl esters of C _{1 to 10} (e.g., isopropyl myristate stearate - DOO IPM), monoglyceride, diglyceride, or a pharmaceutically Include, but are not limited to, chemically acceptable salts.

  Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sacks, tablets or microtablets. Thickeners, flavorings, diluents, emulsifiers, dispersion aids or binders may be desirable. A preferred oral formulation is one in which the oligonucleotide of the invention is mixed with one or more penetration enhancer surfactants and chelating agents. Typical surfactants include fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. Typical bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxy chenedodecholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycolic acid, glycodeoxycholic acid, taurochol Examples include acids, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Typical fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin Glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acrylic carnitine, acrylic choline, monoglyceride, diglyceride or a pharmaceutically acceptable salt thereof (for example, sodium). Also preferred are combinations of penetration enhancers, such as fatty acids / salts used in combination with bile acids / salts. Particularly preferred combinations are lauric acid, capric acid and UDCA sodium salt. Further typical penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The oligonucleotides of the invention may be delivered orally in the form of granules containing spray-dried particles or may be conjugated to form microparticles or nanoparticles. Oligonucleotide complexing agents include polyamino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxyethanes, polyalkyl cyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) And polyalkylcyanoacrylates; DEAE-derivatized polyimines, porlans, cellulose and starch. Particularly advantageous complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (for example , P-amino), poly (methyl cyanoacrylate), poly (ethyl cyanoacrylate), poly (butyl cyanoacrylate), poly (isobutyl cyanoacrylate), poly (isohexyl cyanoacrylate), DEAE-methacrylate, DEAE-hexyl acrylate , DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly (D, L-lactic acid), poly (DL-lactic acid-co-glycolic acid (PLGA), alginate and polyethylene glycol (PEG) ) It is.

  Compositions for vaginal delivery include, for example, gels, creams, tablets, pills, capsules, suppositories, films, or any other pharmaceutically acceptable form that adheres to the mucosa and is not easily washed away. It can have various forms. In addition, a great variety of different formulations for vaginal delivery are described in the art, for example in US Pat. No. 4,615,697 and US Pat. No. 6,699,494, which are hereby incorporated by reference in their entirety. Be incorporated.

  In addition, additives (such as those described in US Pat. No. 4,615,697) may be combined in the formulation for maximum or desired efficacy of the delivery system or for patient comfort. Such additives include, for example, lubricants, plasticizers, preservatives, gel forming agents, tablet forming agents, pill forming agents, suppository forming agents, film forming agents, cream forming agents, tablet disintegrating agents, coatings. , Binders, vehicles, colorants, taste and / or fragrance modifiers, wetting agents, viscosity modifiers, pH modifiers, and similar agents.

  In certain embodiments, the composition may comprise a crosslinked polycarboxylic acid polymer formulation generally described in US Pat. No. 4,615,697. In general, in such embodiments, at least 80 percent of the monomers of the polymer in such formulations should contain at least one carboxyl functional group. The cross-linking agent must be present in an amount that provides sufficient bioadhesion to keep the system attached to the target epithelial surface for a sufficient amount of time and to achieve the desired dosage.

  For vaginal administration, such formulations remain attached to the epithelial surface for at least about 24-48 hours. These results may be measured clinically over various time periods by testing the pH drop of the sample from the vagina due to the continuous presence of the polymer. This preferred level of bioadhesion is usually obtained when the crosslinker is present at about 0.1 to 6.0 weight percent of the polymer, with about 1.0 to 2.0 weight percent being most preferred as long as an adequate level of bioadhesion is obtained. . Bioadhesion can also be measured using a commercially available surface tensiometer used for measuring adhesive strength.

  The polymer formulation can be adjusted to control the release rate by varying the amount of crosslinker in the polymer. Suitable crosslinkers include divinyl glycol, divinyl benzene, N, N-diallylacrylamide, 3,4-dihydroxy-1,5-hexadiene, 2,5-dimethyl-1,5-hexadiene and similar agents.

  A preferred polymer for use in such formulations is Polycarbophil, U.S.P., commercially available from BF Goodrich Specialty Polymers of Cleveland, Ohio under the trade name NOVEON.RTM.-AA1. In the United States Pharmacopeia, 1995, United States Pharmacopeial Convention, Inc., Rockville, Md., Pages 1240-41, it is shown that polycarbophil is polyacrylic acid crosslinked with divinyl glycol.

  Other useful bioadhesive polymers that can be used in such drug delivery system formulations are mentioned in US Pat. No. 4,615,697. For example, these include polyacrylic acid polymers crosslinked with, for example, 3,4-dihydroxy-1,5-hexadiene, and polymethacrylic polymers crosslinked with, for example, divinylbenzene. Typically, these polymers will not be used in their salt form because they reduce bioadhesive ability. Such bioadhesive polymers can be produced by conventional free radical polymerization methods using initiators such as benzoyl peroxide, azobisisobutyronitrile, and the like. An exemplary formulation of a useful bioadhesive is provided in US Pat. No. 4,615,697.

  Compositions and formulations for parenteral, intrathecal or intraventricular administration are limited to buffers, diluents and, for example, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Sterile aqueous solutions may also be included that may also contain additives that are not.

  The pharmaceutical compositions of the present invention may include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be formed from a variety of ingredients including, but not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

  Conveniently, the pharmaceutical formulations of the invention that may be presented in unit dosage form may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with the pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and, if necessary, shaking the product.

  The compositions of the invention may be formulated into a number of possible dosage forms including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

  In one embodiment of the invention, the pharmaceutical composition is formulated and used as a foam. Pharmaceutical foams include, but are not limited to, dosage forms such as emulsions, microemulsions, creams, jellies and liposomes. Although fundamentally similar in nature, these dosage forms differ in the components and concentrations of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

Emulsion The formulations and compositions of the present invention may be prepared as an emulsion and formulated. Emulsions are usually heterogeneous systems, usually in the form of droplets with a diameter greater than 0.1 μm, with one liquid dispersed in the other. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (lids.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et at., In Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems that contain immiscible liquid phases that are intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w / o) or oil-in-water (o / w) types. When the aqueous phase is finely divided and dispersed into a large amount of oil phase as fine droplets, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, if the oil phase is finely divided and dispersed into a large amount of aqueous phase as fine droplets, the resulting composition is called an oil-in-water (o / w) emulsion. Emulsions may contain additional components in addition to the dispersed phase and the active agent present as a liquid in the aqueous phase, oil phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes and antioxidants may also be present in the emulsion if desired. The pharmaceutical emulsion may be a plurality of emulsions composed of more than two phases such as, for example, oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w). Such complex formulations often offer certain advantages that simple biphasic emulsions do not provide. Multiple emulsions in which individual oil droplets of an o / w emulsion enclose small aqueous droplets constitute a w / o / w emulsion. Similarly, a system of oily droplets encapsulated in water globules stabilized with an oily continuous phase provides an o / w / o emulsion.

  Emulsions are characterized by little or no thermodynamic stability. Often the dispersed or discontinuous phase of the emulsion is well dispersed and maintained in this form via the emulsifier or viscosity means of the formulation to the external or continuous phase. As in the case of an emulsion type ointment base or cream, any phase of the emulsion may be semi-solid or solid. Another means of stabilizing the emulsion involves the use of emulsifiers that may be incorporated into any phase of the emulsion. Emulsifiers may be broadly classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorbent bases and finely dispersed solids (Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker ( Eds.), 1988, Marcel Dekker Inc., New York, NY, volume 1 p.199).

  Synthetic surfactants, also known as surface activators, have found wide applicability in emulsion formulations and have been described in the literature (Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p. 199 ). Surfactants are usually amphiphilic and include a hydrophilic portion and a hydrophobic portion. The ratio between the hydrophilic and hydrophobic properties of a surfactant is called the hydrophilic / lipophilic balance (HLB) and is a valuable tool for classifying and selecting surfactants in the preparation of a formulation. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and zwitterionic (Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker ( Eds.), 1988, Marcel Dekker Inc., New York, NY, Vol. 1 p.285).

  Naturally occurring emulsifiers used in emulsion formulations include lanolin, honey, phosphatide, lecithin and acacia. Absorbent bases retain the properties of semisolids such as anhydrous lanolin and hydrophilic petrolatum, but have hydrophilic properties to absorb water and form w / o emulsions. Finely divided solids are also used in particular in combination with surfactants and as good emulsifiers in various preparations. These include polar inorganic solids such as heavy metal hydroxides, non-expanded clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and carbon Or a non-polar solid such as glyceryl tristearate.

  A wide variety of non-emulsifying materials are included in the emulsion formulation and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, wetting agents, hydrophilic colloids, preservatives and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker ( Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York , NY, volume 1, p. 199).

  Hydrophilic colloids or hydrocolloids include naturally occurring rubbers and synthetic polymers such as polysaccharides (eg acacia, agar, alginic acid, carrageenan, guar gum, karaya gum and tragacanth), cellulose derivatives (eg carboxy Methylcellulose and carboxypropylcellulose), and synthetic polymers such as carbomers, cellulose ethers and carboxyvinyl polymers. They disperse in water or swell in water, forming a strong inter-surface coating around the dispersed phase droplets and forming a colloidal solution that stabilizes the emulsion by increasing the viscosity of the internal phase.

  Because emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that readily support microbial growth, these emulsions often incorporate preservatives. Preservatives that are included and commonly used in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent degradation of the formulation. Antioxidants used include tocopherols, alkyl gallate, butylated hydroxyanisole, free radical scavengers such as butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and citric acid, Antioxidant synergists such as tartaric acid and lecithin may also be used.

  The application of emulsion formulations via the dermal, oral and parenteral routes and methods for their preparation have been reviewed in the literature (Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker Inc. , New York, NY, Vol.1 p.199). Emulsion formulations for oral delivery are very widely used because of their ease of formulation, absorption and bioavailability (Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.) , 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins and high fat nutrients are among the substances commonly administered orally as o / w emulsions.

  In one embodiment of the invention, the oligonucleotide composition is formulated as a microemulsion. A microemulsion may be defined as a system of water, oil and amphiphile, which is an optically single isotropic, thermodynamically stable solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman Rieger and Banker (Eds.), 1988, Marcel Dekker Inc., New York, NY, volume 1, p245). Usually, a microemulsion is prepared by first dispersing the oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, typically an intermediate chain length alcohol, to form a transparent system. To do. Thus, microemulsions are also described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids that are stabilized by an intersurface coating of surface-activating molecules (Leung and Shah, in Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., VCH Publishers, New York, p.185-215, 1989). In general, microemulsions are prepared by a combination of 3 to 5 components including oil, water, surfactant, surfactant synergist and electrolyte. Whether the microemulsion is a water-in-oil (w / o) or oil-in-water (o / w) type depends on the properties of the oil and surfactant used and the polar head and carbonization of the surfactant molecule. Depends on the structure and geometric packing of the hydrogen tail (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., P.271, 1985).

  A phenomenological approach using phase diagrams has been extensively studied and comprehensive knowledge of microemulsion formulation methods has been given to those skilled in the art (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.) , 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY , volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilization of water-insoluble drugs in naturally formed thermodynamically stable droplet formers.

  Surfactants used in the preparation of microemulsions include ionic surfactants, nonionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, alone or surfactant synergists In combination with tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquiolate (SO750) decaglycerol dekaorate (DA0750), but not limited thereto. Surfactant synergists are usually short chain alcohols such as ethanol, 1-propanol and 1-butanol, which penetrate the surfactant coating due to voids created between the surfactant molecules, As a result, it acts to increase the fluidity between the surfaces by creating a disordered coating. However, microemulsions may be prepared without the use of surfactant synergists, and alcohol-free self-emulsifying emulsion systems are known in the art. The aqueous phase may usually be water, aqueous drug solutions, glycerol, PEG300, PEG400, polyglycerols, propylene glycol, and ethylene glycol derivatives. The oil phase consists of Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated poly Examples may include, but are not limited to, glycolated C8-C10 glycerides, vegetable oils and silicone oils.

  Microemulsions are of particular interest in terms of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to enhance the oral bioavailability of drugs containing peptides (Constantinides et al., Pharmaceutical Research, 11: 1385-1390, 1994; Ritschet, Met / i. Find. Exp. Clin. Pharmacol., 13: 205, 1993). Microemulsions improve drug solubilization, protect drugs from enzymatic hydrolysis, potentially improve drug absorption by surfactant-induced changes in membrane fluidity and permeability, easy preparation, solid administration Provides the benefits of easy oral administration in form, improved clinical efficacy and reduced toxicity (Constantinides et al., Pharmaceutical Research, 11: 1385, 1994; Ho et al., J. Pharm. Set., 85 : 138-143, 1996). Often the microemulsions may form spontaneously when the ingredients are brought together at room temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions are also effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention improve the local cellular uptake of oligonucleotides and nucleic acids in the gastrointestinal tract, vagina, buccal cavity and other places of administration, as well as systemic oligonucleotide and nucleic acids from the gastrointestinal tract. It is expected to promote absorption.

  The microemulsions of the present invention also contain additional ingredients and additives such as sorbitan monostearate (Grill 3), labrasol, and penetration enhancers that improve the properties of the formulation and enhance the absorption of the oligonucleotides and nucleic acids of the present invention. You may contain. The penetration enhancers of the present invention can be classified as belonging to one of five broad categories, surfactants, fatty acids, bile salts, chelating agents and non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92).

Liposome In addition to the microemulsions that have been investigated and used for drug formulation, there are a number of organized surfactant structures. These include monolayers, micelles, bilayers and vesicles. Vesicles provide the specificity and extended duration of action for drug delivery. Thus, as used herein, the term “liposomes” refers to vesicles composed of amphiphilic lipids arranged in a spherical bilayer, ie liposomes are formed from lipophilic substances. A monolayer or multilayer having a membrane and an aqueous interior. The aqueous portion usually contains the composition to be delivered. In order to cross intact mammalian skin, lipid vesicles should pass through a series of micropores, each less than 50 nm in diameter, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use liposomes that are very easily deformable and that can pass through such micropores. Additional factors for liposomes include changes in lipid surface and aqueous volume of the liposomes.

  A further advantage of liposomes is that liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; The ability to protect the drugs contained in the compartment from metabolism and degradation (Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker Inc., New York, NY, volume 1, p245) It is done.

  For topical administration, there is evidence that liposomes present several advantages over other formulations. Such benefits include reducing side effects associated with high systemic absorption of the administered drug, increasing the accumulation of the drug administered at the desired target, hydrophilic and hydrophobic to the skin. It is mentioned that it is both. Compounds containing analgesics, antibodies, hormones, and high molecular weight DNA are administered to the skin, generally targeting the upper epithelium.

  Liposomes are roughly divided into two types. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA to form stable complexes. The positively charged DNA / liposome complex binds to the negatively charged cell surface and is internalized into the endosome. Due to the acidic pH within the endosome, the liposomes rupture and release their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

  Liposomes that are pH sensitive or negatively charged capture DNA rather than complex with DNA. Since DNA and lipids are similarly charged, a reciprocal rather than complex formation occurs. Thus, DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used, for example, to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

  One of the main types of liposome compositions includes phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, whereas anionic membrane fusogenic liposomes are formed primarily from dioleyl phosphatidylethanolamine (DOPE). Another type of liposome is formed from phosphatidylcholine (PC) such as, for example, soy PC and egg PC. Another type is formed from phospholipids and / or phosphatidylcholines and / or cholesterol.

  Several studies have evaluated the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin alleviated skin herpes wounds, but delivery via other means (eg, as a solution or as an emulsion) was ineffective (Weiner et al. , Journal of Drug Targeting, 1992, 2,405-410). Additional studies investigated the effectiveness of interferon administered as part of a liposomal formulation for interferon administration using an aqueous system and concluded that the liposomal formulation is superior to aqueous administration (du Plessis et al. , Antiviral Research, 1992, 18, 259-265).

  Non-ionic liposome systems have also been investigated, particularly in systems containing non-ionic surfactants and cholesterol, which have established convenience in drug delivery to the skin. Nonionic liposome formulation comprising Novason ™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome ™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) Was used to deliver cyclosporin A into the dermis of mouse skin. The results showed that such nonionic liposome systems are effective in promoting the deposition of cyclosporin A in different layers of the skin (Hu et al., STP Pharma Sci., 1994, 4,6,466 ).

Liposomes also encompass “sterically stabilized” liposomes, the term as used herein refers to liposomes containing one or more specialized lipids, and specialized lipids. When incorporated into liposomes, it has a longer circulation life than liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the lipid portion of the vesicle formation include glycolipids, such as monosialoganglioside G _M1 1 or more, or hydrophilic, such as polyethylene glycol (PEG) moiety Derivatized with one or more of the functional polymers. Without being bound by a particular theory, for sterically stabilized liposomes containing lipids derivatized with gangliosides, sphingomyelin, or PEG, the circulation life extension of these sterically stabilized liposomes is This is thought to be due to a decrease in reticuloendothelial (RES) uptake into cells (Allen et al., FEBS Lett., 1987, 223,42; Wu et al., Cancer Research 1993, 53, 3765).

Various liposomes containing one or more glycolipids are described in Papahadjopoulos et al., Ann. NY Acad. Sci., 1987, 507: 64, (monosialoganglioside G _M1 , galactocerebroside sulfate and phosphatidylinositol); Gabizon et al. , Proc Natl Acad Sci USA, 1988 , 85,6949;..... Allen et al, U.S. Patent No. 4,837,028 and International application Publication WO 88/04924 (sphingomyelin and gangliosides G _M1 or sulphate galacto Cerebroside esters); Webb et al., US Pat. No. 5,543,152 (sphingomyelin); Lim et al., WO 97/13499 (1,2-sn-dimyristoylphosphatidylcholine).

Liposomes containing lipids derivatized with one or more hydrophilic polymers and methods of preparation are described, for example, in Sunamoto et al., Bull. Chem. Soc. Jpn., 1980, 53, 2778 (nonionic surfactants, 2C ₁₂ 15G including PEG moiety);.. Illum et al, FEBS Lett, 1984, a hydrophilic coating of polystyrene particles by 167,79 (polymer glycol); Sears, U.S. Patent No. 4,426,330 and ibid. No. 4,534,899 (polyalkylene Glycols (eg, synthetic phospholipids modified by attachment of the carboxyl group of PEG); Klibanov et al., FEBS Lett., 1990, 268, 235 (phosphatidylethanolamine derivatized with PEG or PEG stearate (PE) ); Blume et al., Biochimica et Biophysica Acta, 1990, 1029: 91 (phospholipids derivatized with PEG, eg, DSPE-PEG formed from a combination of distearoylphosphatidylethanolamine (DSPE) and PEG) ; Fisher, Europe State Patent Nos. EP 0 445 131 B1 and WO 90/04384 (PEG moieties covalently attached to the outer surface of liposomes); Woodle et al., US Pat. Nos. 5,013,556 and 5,356,633, and Martin et al. al., US Patent No. 5,213,804 and European Patent No. EP0496813B1 (liposome composition containing PE derivatized with 1-20 mole percent PEG); Martin et al., WO 91/05545 and US Patent 5,225,212 and Zalipsky et al., WO 94/20073 (liposomes containing a number of other lipid-polymer conjugates); Choi et al., WO 96/10391 (modified with PEG) Liposomes containing ceramide lipids); Miyazaki et al., US Pat. No. 5,540,935 and Tagawa et al., US Pat. No. 5,556,948 (liposomes containing PEG that can be further derivatized with functional moieties on their surface) Are listed.

  Liposomes containing nucleic acids are described, for example, in Thierry et al., WO 96/40062 (method of encapsulating high molecular weight nucleic acids in liposomes); Tagawa et al., US Pat. No. 5,264,221 (protein-bound, RNA Rahman et al., US Pat. No. 5,665,710 (method of encapsulating oligodeoxynucleotides in liposomes); Love et al., WO 97/04787 (liposomes containing antisense oligonucleotides) It is described in.

  Another type of liposome, transfersomes, are highly deformable lipid aggregates that are attractive for drug delivery vehicles (Cevc et al., 1998, Biochim. Biophys. Acta, 1368 (2): 201-15). Transfersomes may be described as highly deformable lipid droplets that can penetrate through pores smaller than the droplets. Transfersomes are applicable to the environment in which they are used, for example, they are shape-applicable, self-repairing, often reach the target without disruption and self-load. Transfersomes can be made, for example, by adding a surface edge activator, usually a surfactant, to a standard liposome composition.

Surfactants Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank many different types of natural and synthetic surfactants is by using the hydrophilic / lipophilic ratio (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means of classifying the various surfactants used in the formulation (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, NY, 1988 p.285).

  If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical products and cosmetics and can be used over a wide range of pH values, with typical HLB values ranging from 2 to about 18 depending on the structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters; and fatty alcohol ethoxylates, propoxylated Nonionic alkanolamides such as alcohols are included, and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are those most commonly used in the class of nonionic surfactants.

  Surfactant molecules that have a negative charge when dissolved or dispersed in water are classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acylamides of amino acids, esters of sulfuric acids such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isothionates, Examples include acyl laurate and sulfosuccinate and phosphate. Alkyl sulfates and soaps are the most commonly used anionic surfactants.

  Surfactant molecules that have a positive charge when dissolved or dispersed in water are classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines, with quaternary ammonium salts being most frequently used.

  Surfactant molecules that can have either a positive charge or a negative charge are classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

  The use of surfactants in pharmaceutical products, formulations and emulsions is reviewed in Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y., 1988, p.285.

Penetration enhancers In some embodiments, penetration enhancers are used in or with the composition to enhance delivery of nucleic acids, particularly oligonucleotides, to the animal skin or through mucosa. Most drugs exist in solution in both ionized and non-ionized forms. However, usually only fat-soluble or lipophilic drugs easily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to helping non-lipophilic drugs cross cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

  Penetration enhancers are classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of these classes of penetration enhancers is described in detail below.

  Surfactant: In the context of the present invention, a surfactant (or “surface activator”), when dissolved in an aqueous solution, reduces the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, The result is a chemical component that enhances the absorption of oligonucleotides through the mucosa. These penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). As well as perfluoro compound emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40,252), each of which is incorporated herein by reference in its entirety.

Fatty acids: Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dica Plate, tricaplate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, carprilic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, them C _1-10 alkyl esters of (eg, methyl, isopropyl and t-butyl) and their mono- and diglycerides (ie, oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al. , Critical Reviews in Therapeutic Drug Carrier Systems, 1991 , p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654). The entirety is incorporated herein by reference.

  Bile salts: The physiological role of bile includes promoting the dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38, Goodman & Gilman's The Pharmacological Basis of Therpeutics, 9th Ed., Hardman et al., Eds , McGraw-Hill, New York, 1996, p.934-935). Various natural bile salts and synthetic derivatives thereof act as penetration enhancers. Thus, the term “bile salt” encompasses any naturally occurring component of bile and any synthetic derivatives thereof. Examples of the bile salt of the present invention include cholic acid (or a pharmaceutically acceptable sodium salt thereof, sodium cholate), dihydrocholic acid (sodium dihydrocholate), deoxycholic acid (sodium deoxycholate), glycol. Acid (sodium glycolate), glycolic acid (sodium glycolate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid ( Examples include sodium chenodeoxycholic acid), ursodeoxycholic acid (UDCA), tauro-24,25-dihydro-fusidic acid sodium (STDHF), sodium glycodihydrofusidic acid and polyoxyethylene-9-lauryl ether (POE). That. (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther, 1992, 263, 25; Yamashita et al., J. Pharm: Sci., 1990, 79, 579-583).

  Chelating agent: In this context, a chelating agent can be considered a compound that forms a complex with it to remove metal ions from the solution, resulting in enhanced absorption of the oligonucleotide through the mucosa. With regard to its use as a penetration enhancer in the present invention, most of the DNA nucleases that have been characterized require a divalent metal ion for the catalyst and are inhibited by the chelating agent, so that the chelating agent is a DNA degrading enzyme inhibitor. Has the additional advantage of acting (Jarrett, J. Chromatogr., 1993, 618, 315-339). Without limitation, chelating agents include disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (eg, sodium salicylate, 5-methoxysalicylate and homovanillate), N-acyl derivatives of collagen, laureth of beta diketone. -9 and N-aminoacyl derivatives (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7,1-33; Buur et al , J. Control Rel., 1990, 14, 43-51).

  Non-chelating non-surfactant: As used herein, compounds that enhance the penetration of non-chelating non-surfactants do not have significant chelating and surfactant activity and are digestive system It is a compound that enhances the absorption of oligonucleotides through the mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of such penetration enhancers include unsaturated cyclic urea 1-alkyl and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92) and diclofenac sodium, Non-steroidal anti-inflammatory agents such as indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

  Agents that enhance the uptake of oligonucleotides at the cellular level may be added to the pharmaceutical compositions and other compositions and formulations of the present invention. For example, cationic lipids such as lipofectin (Junichi et al., US Pat. No. 5,705,188), cationic glycerol derivatives, and polylysine (Lollo et al., PCT application, WO 97/30731) Such polycationic molecules are also known to enhance uptake of oligonucleotides into cells.

  Of nucleic acids administered using other agents including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azones, and terpenes such as limonene and menthone. Penetration may be increased.

Carrier Certain compositions of the present invention also incorporate a carrier compound in the formulation. As used herein, “carrier compound” or “carrier” refers to a nucleic acid or analog thereof, which is inactive (ie, does not have its own biological activity), eg, biology It is recognized as a nucleic acid by an in vivo process that degrades the bioavailability of a biologically active nucleic acid by degrading the active nucleic acid or promoting its removal from the circulation. Co-administration of nucleic acid and carrier compound often results in an excess of the latter, but results in a substantial reduction in the amount of nucleic acid recovered in the liver, kidney, or other storage location outside the circulation. For example, the recovery of phosphorothioate in liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2-disulfonic acid. (Miyao et al., Antisense Res. Dev., 1995, 5,115-121; Takakura et al., Antisense & Nucl Acid Drug Dev., 1996, 6,177-183), each of which is incorporated herein by reference in its entirety.

Excipient In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other agent for delivering one or more nucleic acids to an animal. A pharmaceutically inert vehicle, usually a liquid or a solid. Pharmaceutical carriers are generally selected from the viewpoint of the intended mode of administration and provide the desired bulk, concentration, etc. when combined with nucleic acids and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include binders (eg, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (eg, lactose and other sugars, microcrystalline cellulose, pectin, gelatin, sulfuric acid) Calcium, ethyl cellulose, polyacrylate or calcium hydrogen phosphate, etc .; lubricants (eg magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, benzoic acid Sodium, sodium acetate, etc.); disintegrants (eg, starch, sodium starch glycolate, etc.); and wetting agents (eg, sodium lauryl sulfate, etc.). Not.

  Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like However, it is not limited to these.

  Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or solutions of nucleic acids in liquid or solid oil bases. . The solution may contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not adversely react with nucleic acids can be used.

Other Pharmaceutical Composition Ingredients The composition may additionally contain other ingredients conventionally found in pharmaceutical compositions at the level of application established in the art. Thus, for example, the composition may contain additional, compatible, pharmaceutically active substances such as, for example, anti-itch agents, astringents, local anesthetics or anti-inflammatory agents, or Additional useful for physically formulating various dosage forms of the compositions of the invention, such as, for example, dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners and stabilizers. A substance may be contained. However, such materials should not unduly interfere with the biological activity of the components of the composition of the invention when added. Sterilizes the formulation and, if desired, adjuvants that do not adversely interact with the nucleic acid of the formulation, eg, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring Mixing agents, flavoring agents and / or aromatic substances.

  Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran, and / or stabilizers.

  Certain embodiments of the present invention provide pharmaceutical compositions comprising (a) one or more antiviral oligonucleotides and (b) one or more other chemotherapeutic agents that function by different mechanisms. Examples of such chemotherapeutic agents include daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bischloroethylnitrosourea, busulfan, mitomycin C, actino Mycin D, mitramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmethamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosourea, nitrogen mustard, melphalan, cyclophosphamide , 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, de Xycofomycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP -16), trimethrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES), but are not limited thereto. See generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., Eds., Rahway, NJ. When used with a compound of the invention, individually (eg, 5-FU and oligonucleotide), sequentially (eg, 5-FU and oligonucleotide for a period, followed by MTX and oligonucleotide) or one or more such Such chemotherapeutic agents may be used in combination with chemotherapeutic agents (eg, 5-EU, MTX and oligonucleotides, or 5-FU, radiation therapy and oligonucleotides). Anti-inflammatory drugs include, but are not limited to, non-steroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs include, but are not limited to, ribavirin, cidofovir, vidarabine, acyclovir and ganciclovir. It is not limited, You may combine with the composition of this invention. See generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. Berkow et al., Eds., 1987, Rahway, NJ., Pages 2499-2506. Other non-oligonucleotide chemotherapeutic agents may also be used together or in sequence with two or more compounds within the scope of the present invention.

Examples Example 1: Herpes simplex virus Herpes simplex virus (HSV) affects a significant proportion of the human population. It has been found in the present invention that random ON or ON randomers inhibit the infectivity of viruses such as HSV. Using HSV cellular replication assay in VERO cells (susceptible to HSV-1 (KOS strain) and HSV-2 (MS2 strain) infection), single-stranded PS-ON complementary to HSV replication origin HSV-1 and HSV-2 replication was inhibited. Surprisingly, the control PS-ON complementary to the start of human (343ARS) and plasmid (pBR322 / pUC) also inhibited viral infectivity. Experiments with random-sequence PS-ON and PS-ON randomers demonstrated that inhibition of viral infection increased with increasing ON size. These data indicate that ON is a powerful antiviral agent useful for the therapeutic treatment of viral infections.

  The inventors theorized that a possible mechanism to prevent the spread of viruses such as HHV is to prevent its DNA replication. With this in mind, phosphorothioate oligonucleotides (ON) complementary to the HSV1 and HSV2 origins of replication were introduced into infected cells. These ONs produced DNA triplets at the viral origin of replication, preventing the association of necessary trans-acting factors and viral DNA replication. Surprisingly, results are presented herein that show that in an experimental paradigm, the ability of ON to inhibit viral infection increases as its size (length) increases.

Inhibition of HSV-1 The ability of PS-ON to inhibit HSV-1 was measured in a plaque reduction assay (PRA). Cultured immortalized African green monkey kidney cells (VERO) in MEM (minimum essential medium) supplemented with gentamicin, vancomycin and amphotericin B plus 10% fetal bovine serum at 37 ° C, 5% CO ₂ did. Cells were seeded in 12-well plates at a density that yielded confluent monolayer cells after 4 days of growth. When confluence is reached, the medium is replaced with one containing only 5% serum and the above complements, and then the cells are placed in the presence of the test compound for 90 minutes in HSV-1 (KOS strain, approximately 40-60 PFU total) Exposed to. After exposure to the virus, the medium was changed to a new “layered” medium containing 5% serum, 1% human immunoglobulin, the above complements and test compounds. Three to four days after infection, plaque counts were performed following formalin fixation and cresyl violet staining of infected cultures.

  All ONs (except as otherwise noted) were prepared at the University of Calgary, Core DNA Service Lab 1 or 15 μM synthesis scale (see Table 21) and 50 cm Sephadex G -25 column for deprotection and desalting. Analyzing the ON obtained by UV shadow gel electrophoresis, about 95% full length, n-1 and n-2 oligos and up to 5% short oligo species (these are likely to have random deletions) It was confirmed to contain. For random oligo synthesis, adenine, guanosine, cytosine and thymidine amidites were mixed together in equimolar amounts to maximize the randomness of incorporation at each ON position during synthesis.

To test the ability of HSV-1 inhibition by PS-ON, REP 1001, 2001 and 3007 are tested with HSV-1 PRA. Since PS-ON is directed to the origin of replication in HSV, it was assumed that only REP 2001 would show any activity (the other two are directed to the origin of replication in humans and plasmids ). However, all three PS-ONs showed anti-HSV-1 activity. The test was performed by plaque reduction assay performed in VERO cells using HSV-1 (KOS strain). Infected cells were treated with increasing concentrations of REP 1001, REP 2001, or REP 3007. IC ₅₀ values calculated from linear regression of the assay results were 2.76, 0.77, and 5.33 micromolar, respectively. Furthermore, the effectiveness of the anti-HSV-1 effect was found to depend on the size of the oligo.

To confirm the size-dependence and relative sequence-independence on the anti-HSV-1 activity of PS-ON, we have determined that various sizes of PS-ON (REP 2002, 2003, 2004, 2005 And 2006) were tested with the antiviral drug acyclovir. These PS-ONs are inactive with respect to sequence-specific effects by synthesizing each base as a “fluctuation” (N) and making each PS-ON actually a group with different random sequences of the same size. These PS-ONs are referred to as “Randamers”. Plaque reduction assay was performed in VERO cells using HSV-1 (KOS strain). Infected cells are treated with increasing concentrations of REP 2001, REP 2002 or REP 3003, REP 2004, REP 2005, REP 2006 and acyclovir. IC ₅₀ values were calculated from linear regression of assay data. The relationship between the size of PS-ON for HSV-1 and IC ₅₀ was determined by plotting the IC ₅₀ value against the specific size of each PS-ON tested that showed anti-HSV-1 activity. The IC ₅₀ of acyclovir was used as a reference for clinical correlations. We have oligos of 10 bases or less that have no detectable anti-HSV-1 activity, but effectiveness also increases as the size of PS-ON increases beyond 10 bases. (IC ₅₀ decreased). We also noted that PS-ONs greater than 20 bases have significantly lower IC ₅₀ values compared to the clinically acceptable anti-HSV-1 drug, acyclovir.

To further define the effective size range of PS-ON for anti-HSV-1 activity, we tested 10-120 base PS-ON randomers over a wider range of sizes. Plaque reduction assay was performed in VERO cells using HSV-1 (KOS strain). A wide range of PS-ON randomers were tested with increasing concentrations of REP 2003, REP 2009, REP 2010, REP 2011, REP 2012, REP 2004, REP 2006, REP 2007 and REP 2008. IC ₅₀ values were calculated from linear regression. We found that 12 bases and larger oligos have detectable anti-HSV-1 activity, and potency against HSV-1 increases with increasing PS-ON randomer length to at least 120 bases. Also found. However, the increase in potency with increasing bases in size is less with PS-ON randomers larger than 40 bases.

In order to compare the potency of non-PS-ON randomers, random sequence PS-ON and HSV-1 specific sequence PS-ON, we have identified these three types for ONs of size 10, 20 and 40 bases. The modification of was tested. Plaque reduction assay was performed in VERO cells using HSV-1 (KOS strain). Unmodified ON, PS-ON with random sequence, and PS-ON targeting HSV-1 start codon IE110 were tested at increasing concentrations. ON were REP 2013, REP 2014, REP 2015, REP 2016, REP 2017, REP 2018, REP 2019, REP 2020 and REP 2021. IC ₅₀ values were calculated from linear regression. In this system, the unmodified ON randomer has no detectable anti-HSV-1 activity at the size tested. Both the random sequence and the HSV-1 specific sequence PS-ON show size-dependent anti-HSV-1 activity (none of these modifications are active at 10 bases). According to the comparison of random sequence, HSV-1 specific sequence and randomer PS-ON, there was an increase in anti-HSV-1 activity due to HSV-1 specific sequence in PS-ON of 20 base length, but in 40 base length All modifications, randomers, random sequences or HSV-1 specific sequences have been shown to be equally effective against HSV-1.

To the best of our knowledge, this is the first time that an IC _{50 for} HSV-1 as low as 0.059 μM and 0.043 μM has been reported for PS-ON.

Example 2: Inhibition of HSV-2
The ability of HS--2 inhibition by PS-ON is measured by PRA. Immortalized African green monkey kidney (VERO) cells are cultured in MEM supplemented with gentamicin, vancomycin and amphotericin B and 10% fetal bovine serum at 37 ° C. and 5% CO ₂ . Cells are seeded in 12-well plates at a density that gives a confluent monolayer of cells 4 days after growth. When confluency is reached, the medium is changed to contain only 5% serum and the above supplements, and the cells are then transferred to HSV-2 (MS2 strain, total about 40-60 PFU) in the presence of the test compound. Exposure for 90 minutes. After virus exposure, the medium is replaced with fresh “overlay” medium containing 5% serum, 1% human immunoglobulin, the above supplements and test compounds. Three to four days after infection, plaque counts are performed after infected cultures are formalin fixed and cresyl violet stained.

To test the ability of HSV-2 inhibition by PS-ON, REP 1001, 2001 and 3007 are tested with HSV-2 PRA. Using HSV-2 (MS2 strain), infected cells were treated with increasing concentrations of REP 1001, REP 2001 or REP 3007 to perform plaque reduction assays in human neuroblasts. IC ₅₀ values were calculated from linear regression. If the inhibitory activity is due to antisense or other sequence-complementary mechanisms, only REP 2001 is expected to show any activity, which indicates that this PS-ON is directed to the origin of replication within HSV-1 / 2 (The other two are directed to the origin of replication in humans and plasmids, respectively). All three PS-ONs showed anti-HSV-2 activity. Furthermore, the effectiveness of the anti-HSV-2 effect depends on the size of the PS-ON and not on the sequence.

In order to confirm the size-dependence and sequence-independence on the anti-HSV-2 activity of PS-ON, we have determined that various sizes of PS-ON (REP 2001, 2002, 2003, 2004, 2005 and 2006). These PS-ONs are inactive with respect to sequence-specific effects by synthesizing each base as a “fluctuation” (N) and making each PS-ON actually a population with random sequences of the same size. These PS-ONs are called “Randamers”. When testing these PS-ONs with HSV-2PRA, we found that oligos of 10 bases or less had no detectable anti-HSV-2 activity, but the size of PS-ON was 10 We find that as we increase larger than the base, the effectiveness also increases (IC ₅₀ reduction). We also noted that PS-ONs greater than 20 bases have significantly lower IC ₅₀ values compared to the clinically acceptable anti-HSV-2 drug, Acyclovir ™.

To the best of our knowledge, this is the first time that an IC _{50 for} HSV-2 as low as 0.012 μM has been reported for PS-ON.

In order to determine whether a non-sequence specific composition has an effect on the antiviral activity of ON, the anti-HSV1 activity of several PS-ONs with equivalent size but different sequence composition Tested with HSV-1 PRA. The PS-ONs tested were REP 2006 (N20), REP 2028 (G40), REP 2029 (A40), REP 2030 (T40) and REP 2031 (C40). IC ₅₀ values generated from HSV-1 PRA indicate that REP 2006 (N40) was the most active of all sequences tested, while REP 2029 (A40) was the least active. We also note that all other PS-ONs have significantly lower activity compared to N40 and are ordered N40>C40>T40>A40> G40 in terms of potency.

  We also tested the efficacy of different PSONs with various sequence compositions using two different nucleotides. PS-ON randomer (REP 2006) has significantly higher potency against HSV-1 compared to AC20 (REP 2055), TC20 (REP 2056) or AG20 (REP 2057) , Ordered as follows: N40> AG> AC> TC. This data suggests that the antiviral effect is not sequence complementary and that the non-specific sequence composition (ie C40 and N40) has a stronger antiviral activity. We have observed that while this phenomenon retains the intrinsic protein binding ability, sequences such as C40, A40, T40 and G40 probably have some restricted tertiary formation formed within these sequences. We propose that the structure can be explained by the fact that it binds with a smaller number of viral proteins with high affinity. On the other hand, due to the random nature of N40, N40 maintains the ability to bind to a wide range of antiviral proteins with high affinity, which contributes to its strong antiviral activity.

Example 3: Inhibition of CMV
The ability of PS-ON to inhibit CMV is measured by plaque reduction assay (PRA). This assay is identical to the assay used for anti-HSV-1 and anti-HSV-2 tests, except that CMV (strain AD169) was used as the virus inoculum and human neuroblast was used as the cell host. .

To test the size-dependence and sequence-independence on the anti-CMV activity of PS-ON, we tested various sizes of PS-ON randomers. Plaque reduction assay was performed in VERO cells using CMV (AD169 strain). Infected cells were treated with increasing concentrations of REP 2004 (a) or REP 2006 (b). IC ₅₀ values were calculated from linear regression and the relationship between PS-ON size and IC ₅₀ versus CMV was determined by plotting IC ₅₀ values against the specific size of each PS-ON tested. When testing these PS-ON at CMV PRA, the present inventors have found that as the size of the PS-ON is increased, it discovers that effectiveness also increases (IC ₅₀ decreases).

To further clarify the effective size range of PS-ON for anti-CMV activity, we tested 10-80 base PS-ON randomers over a wider range of sizes. We also treated with several clinically acceptable small molecule CMVs (ganciclovir, foscarnet and cidofovir) and two versions of CMV retinitis commercially available antisense treatment (Vitravene ™; University of Also synthesized and marketed by Calgary). Plaque reduction assay was performed in VERO cells using CMV (AD169 strain). Three clinical CMV treatments were tested: ganciclovir, foscarnet and cidofovir. Increasing concentrations of REP 2003, REP 2004, REP 2006, and REP 2007 also tested a wide range of PS-ON randomer sizes. Finally, REP 2036 (Vitravene) synthesized indoors and commercially available was tested. IC ₅₀ values were calculated from linear regression. We have found that an increase in PS-ON randomer size results in increased potency, while this effect saturates at about 40 bases. Furthermore, 20, 40 and 80 base PS-ON randomers all have significantly higher potency compared to any small molecule therapy tested. In addition, 40- and 80-base PS-ON randomers have higher potency compared to Vitravene ™.

To the best of our knowledge, this is the first time that an IC _{50 for} CMV as low as 0.067 μM has been reported for PS-ON.

Example 4: Inhibition of HIV-1
Two different assays measured the ability of PS-ON to inhibit HIV-1.

Cytopathic effect (CPE)
To report the degree of cellular metabolism, the cytopathic effect is monitored using MTT dye. Human lymphocytes (MT4) immortalized at 37 ° C. and 5% CO ₂ are cultured in MEM supplemented with antibiotics with 10% fetal bovine serum. Cells are seeded in medium containing the appropriate test compound in a 96 well plate and incubated for 2 hours. After preincubation with the test compound, HIV-1 (strain NL4-3) was added to the wells (0.0002TCID ₅₀ / cell). After 6 days of additional incubation, CPE is monitored by MTT conversion. Cytotoxicity is measured by incubating the drug for 6 days in the absence of virus seeding. To convert the MTT absorption value to% viability, the absorption in uninfected untreated cells is set to 100% and the absorption of infected untreated cells is set to 0%.

Replication assay (RA)
HIV replication capacity is monitored in immortalized human fetal kidney cells (293A). Two plasmids are co-transfected into these cells. One plasmid contains the recombinant wild-type HIV-1 genome (NL4-3) with the env gene intervening luciferase expression cassette (identified as CNDO strain), the other plasmid is murine leukemia virus (MLV) Contains the env gene derived from These two plasmids provide in trans any protein factor that produces mature chimeric virus with all components derived from HIV-1, except for the protein product provided in trans from the env gene of MLV . The virions produced from these cells are infectious and replicate, but they cannot produce the next generation of infectious virions due to the lack of the env gene.

  Twenty-four hours after transfection, these cells are trypsinized and seeded in 96-well plates. After the cells attach, the medium is washed and replaced with medium containing the test compound. Virus production proceeds in another 24 hours. The supernatant is then collected and used to reinfect intact 293A cells. The original cells infected with the luciferase gene product are identified. The number of luciferase positive cells is a measure of the extent of replication and / or infection by recombinant HIV-1. Numerous clinically approved anti-HIV-1 drugs by using the HIV-1 genome with several point mutations known to induce resistance to several different classes of anti-HIV drugs This assay is also applied to examine resistance to. Percent inhibition is set as 100% when there are no detectable luciferase positive cells, 0% positive cells in infected cells, and untreated cells as controls.

To test the size-dependence and sequence-independence on the anti-HSV-1 activity of PS-ON, we tested various sizes of PS-ON randomers. CPE assay was performed in MT4 cells using HIV-1 (NL4-3 strain). Infected cells were treated with increasing concentrations of REP 2004 or REP 2006. IC ₅₀ values were calculated from linear regression. Cytotoxicity profiles in uninfected MT4 cells for REP 2004 and REP 2006 were measured. The inventors have found that as PS-ON size increases, the effectiveness also increases (IC ₅₀ decreases). The inventors also noted that PS-ON randomers did not show significant toxicity to host cells in this assay.

To the best of our knowledge, this is the first time that an IC _{50 for} HIV-1 as low as 0.011 μM has been reported for PS-ON.

  To further elucidate the effective size range of PS-ON for anti-HIV-1 activity, we made more by RA with wild-type HIV-I (recombinant NL4-3 (CNDO)). PS-ON randomers of 10-80 bases over a wide range of sizes were tested. Replication assays were performed in 293A cells using recombinant wild type HIV-1 NL4-3 (CNDO strain). In addition, we tested four protease inhibitors currently in clinical use (amprenavir, indinavir, lopinavir and saquinavir). Infected cells were treated with increasing concentrations of amprenavir, indinavir, lopinavir, saquinavir, REP 2003, REP 2004, REP 2006 and REP 2007. We found that PS-ON randomers with 10 bases and larger have anti-HIV-1 activity, and potency against HIV-1 increases with increasing length of PS-ON randomers, but about 40 Found to be saturated with base. Furthermore, the 40- and 80-base PS-ON randomers were almost equivalent to the efficacy with the four clinical controls.

To the best of our knowledge, this is the first time that an IC _{50 for} HIV-1 as low as 0.014 μM has been reported for PS-ON.

  In order to test the ability of PS-ON randomers to inhibit HIV drug-resistant strains, we reproduced the above test using a recombinant MDRC4 strain of HIV-1. This recombinant strain exhibits significant resistance to at least 16 of all classes of clinically acceptable drugs: nucleotide RT inhibitors, non-nucleotide RT inhibitors and protease inhibitors. The inventors have found that all PS-ON randomers tested function similarly to resistant strains as to wild type strains. However, three of the four protease inhibitors have reduced potency against mutant strains, so 40- and 80-base PS-ON randomers are more potent against this resistant strain compared to these drugs. It was shown that.

Example 5: Inhibition of RSV
The ability of PS-ON randomers to inhibit RSV is measured by monitoring CPE with Alamar Blue (an indirect indicator of cellular metabolism). Human laryngeal carcinoma (Hep2) cells are cultured in MEM supplemented with 5% fetal calf serum at 37 ° C. and 5% CO ₂ . Cells are seeded in 96-well plates at a density that yields a confluent monolayer of cells 5-6 days after growth. The day after plating, cells were infected with RSV (A2 strain, 10 ^8.2 TCID ₅₀ / mL) in reduced volume in the presence of test compound for 2 hours. After inoculation, the medium was changed and supplemented with the test compound. Six days after infection, CPE was monitored by measuring the fluorescence conversion of alamar blue. The toxicity of test compounds in Hep2 cells was monitored by treatment of uninfected cells for 7 days and measurement of alamar blue conversion in these cells. The measurement value of alamar blue in uninfected and untreated cells was set to 100% survival, and the measurement value in infected and untreated cells was set to 0% survival.

To confirm the size dependence and sequence independence on the anti-RSV activity of PS-ON, we tested various sizes of PS-ON randomers. In addition, we tested a clinically acceptable treatment for RSV infection, ribavirin (Virazole ™). CPE assay was performed in Hep2 cells using RSV (A2 strain). Infected cells are treated with increasing concentrations of REP 2004, REP 2006, REP 2007, or ribavirin. IC ₅₀ values are calculated from linear regression and reported on each graph. The cytotoxicity profile in non-infected Hep2 cells for REP 2004, REP 2006, REP 2007 or ribavirin was measured. The inventors have found that as PS-ON randomer size increases, effectiveness also increases, but saturates at a size of about 40 bases. We also noted that the 20, 40 and 80 base PS-ON randomers have significantly lower IC ₅₀ values compared to the clinically acceptable anti-RSV drug, ribavirin. Ribavirin showed significant toxicity (therapeutic index = 2.08), while PS-ON randomer showed no toxicity in Hep2 cells.

To the best of our knowledge, this is the first time that an IC _{50 for} RSV-1 as low as 0.015 μM has been reported for PS-ON.

Example 6: Inhibition of Coxsackievirus type B2
The ability of the PS-ON randomer to inhibit COX B2 is measured by monitoring CPE with Alamar Blue (an indirect indicator of cellular metabolism). Rhesus monkey kidney (LLC-MK2) cells are cultured in MEM supplemented with 5% fetal calf serum at 37 ° C. and 5% CO ₂ . Cells are seeded in 96-well plates at a density that yields a confluent monolayer of cells 5-6 days after growth. The day after plating, cells were infected with COX B2 (Ohio-1 strain, 10 ^7.8 TCID ₅₀ / mL) in reduced volume in the presence of test compound for 2 hours. After inoculation, the medium was changed and supplemented with the test compound. Six days after infection, CPE was monitored by measuring the fluorescence conversion of alamar blue. Toxicity of test compounds in LLC-MK2 cells was monitored by treatment of uninfected cells for 7 days and measurement of alamar blue conversion in these cells. The measurement value of alamar blue in uninfected and untreated cells was set to 100% survival, and the measurement value in infected and untreated cells was set to 0% survival.

  We tested the anti-COX B2 activity of REP 2006 in the COX B2 CPE assay. CPE assay was performed in LLC-MK2 cells using Coxsackievirus type B2 (Ohio-1 strain). Infected cells were treated with REP 2006 at increasing concentrations. The cytotoxicity profile in LLC-MK2 cells for REP 2006 was measured. The present inventors have found that this PS-ON randomer exhibits some slight toxicity in LLC-MK2 cells, while infecting LLC-MK2 cells can be rescued to some extent from COX B2 infection.

Example 7: Inhibition of vaccinia virus We used a vaccinia infection model as an indicator of the efficacy of our compounds against poxviruses, including smallpox virus. The ability of PS-ON randomer to inhibit vaccinia is measured by monitoring CPE with Alamar Blue (an indirect indicator of cellular metabolism). Vero cells are cultured in MEM supplemented with 5% fetal calf serum at 37 ° C. and 5% CO ₂ . Cells are seeded in 96-well plates at a density that yields a confluent monolayer of cells 5-6 days after growth. The day after plating, cells were infected with vaccinia (10 ^7.9 TCID ₅₀ / mL) in reduced volume in the presence of test compound for 2 hours. After inoculation, the medium was changed and supplemented with test compounds (all 10 μM except for cidofovir used at 50 μM). Supernatant was collected 5 days after infection. VERO cells were reinfected with 1: 100 diluted supernatant and 7 days after reinfection, CPE was monitored by measuring fluorescence conversion of alamar blue to determine viral load in the supernatant.

  We tested various sizes of PS-ON randomers (REP 2004, 2006 and 2007). In addition, we tested a known effective treatment for vaccinia infection, cidofovir (Vistide ™). Indirect measurement of viral load in infected supernatants from vaccinia infected VERO cells was determined by measuring CPE in naive cells induced by these supernatants. REP 2004, 2006 and 2007 were tested at 10 μM, while cidofovir was tested at 50 μM. When tested in the vaccinia CPE assay, we all treated with REP 2004, 2006 and 2007 showed antiviral activity (i.e., reinfection showed a decrease in CPE in the supernatant). However, this activity was found to be weak compared to that observed for cidofovir.

Example 8: Inhibition of DHBV, parainfluenza 3 virus and hantavirus
Since DHBV, parainfluenza type 3 virus and hantavirus do not readily produce measurable plaques or CPE, we have demonstrated the efficacy of REP 2006 within these viruses by detecting fluorescence focus forming units (FFFU). Tested. In this assay, REP 2006 (at a final concentration of 10 μM) is mixed with the virus, which is then adsorbed onto the cells. After adsorption, the infected cells are further incubated for 7-14 days, at which time they are fixed in methanol. The region of viral replication is detected by immunofluorescence microscopy against the appropriate viral antigen. Specific experimental conditions and results for each of the three viruses tested are shown in Table 1 below.

Table 1: Inhibition of DHBV, parainfluenza 3 virus and hantavirus

This initial data shows that at 10 μM, REP 2006 is effective at inhibiting DHBV, parainfluenza 2 and hantaviruses. The present inventors have found that by a given potent responses in the preliminary test, expect that an IC ₅₀ value would be lower. These data support the efficacy of PS-ON randomers for treatment of human infections with hantavirus and hepatitis B (closely associated with DHBV) and may require differential diagnosis to initiate treatment. Provide principles for the acute treatment of childhood bronchitis due to RSV and parainfluenza type 3.

Example 9: Current Non-Responsive Viruses To date, we have not used delivery systems, drug combinations or chemical modifications in the following viral systems shown in Table 2, PS-ON randomers (up to 10 μM). ) No detectable antiviral efficacy.

(Table 2) Virus system

  Under the current test procedure, we did not demonstrate activity. However, the lack of demonstrated antiviral activity may be due to the limitations of the particular assay used. Further studies are ongoing to demonstrate the efficacy of these viruses.

  Our evidence shows that the charge properties of PS-ON are important in inhibiting several different families of viruses, so we have We hope this charge-dependent mechanism has the potential to inhibit the activity of all encapsidated viruses. The reasoning for this is that the antiviral efficacy against those viruses listed in Example 9 is not detected, because the interaction between PS-ON and the structural proteins of these viruses during replication of these viruses This suggests that it may not be strong enough to prevent protein interactions. In this case, one way to achieve efficacy against these viruses is to alter the charge properties of the DNA or antiviral polymer (e.g., to replace the phosphorothioate linkage of the DNA with a phosphorodithioate) so that their affinity for viral proteins. Is to increase.

Example 10: Inhibition of influenza A
The ability of PS-ON to inhibit influenza A virus (INF) is measured by plaque reduction assay (PRA). Immortalized canine kidney (MDCK) cells are cultured in MEM with 10% fetal calf serum supplemented with gentamicin, vancomycin and amphotericin B at 37 ° C. and 5% CO ₂ . Cells are seeded in 6-well plates at a density that yields a confluent monolayer of cells after 6 days of growth. When confluency is reached, the medium is changed to contain only the above supplements, and the cells are then exposed to INF A (H3N2 strain, about 35-70 PFU total) for 60 minutes in the presence of the test compound. After virus exposure, the medium is replaced with fresh medium containing only the drug. 24 hours after infection, the medium is again replaced with 4% albumin, 0.025% DEAE dextran, 2 mg / mL TPCK-treated trypsin and 0.8% sheep plaque agarose, overlay medium containing the above supplements and no test compound. To do. Two to three days after infection, infected cultures are formalin fixed and cresyl violet stained before plaque counting.

  We tested the anti-INF A activity of various PS-ON randomers in the INF A PRA assay. We have found that only REP 2006 shows any measurable antiviral activity, but this activity is significant (see Table 3 below).

(Table 3) Activity of PS-ON randomers against INF A (H3N2)

  Since only the largest randomer appears to have some activity, we understand that the activity of the randomer in many other viruses was size dependent. They tested the antiviral activity of randomers distributed over a larger size using a wider dilution range. We found that for other viruses we tested, the anti-INF A activity of randomers became stronger as their length increased, but for randomers over 40 bases in length, the activity We found that there was no significant increase.

(Table 4) Size-dependent anti-INF A activity of PS-ON randomer

  In order to determine the mechanism of action of REP 2006, we attempted to measure the effect of adding REP 2006 (IC99 concentration) at various times before, during and after infection. In this experiment, we observed that the addition of REP 2006 completely inhibited INF A activity even after 5 hours (300 minutes) after infection (see table below). These results indicate that at least a significant portion of the effect of REP 2006 on influenza occurs after infection. Since PS-ON randomers do not enter cells easily, PS-ON randomers will also interfere with viruses that bud from host cells.

(Table 5) Effect of REP 2006 on addition time versus INF A activity

Example 11: Testing to Determine that Oligonucleotides Act Mostly in a Sequence Independent Mode of Action We hereby describe the effect that the antiviral activity of the ON of the invention is sequence independent. Proved to be caused by the style. Of course, one skilled in the art can prepare sequence-specific ONs, eg, antisense ONs that target specific viral mRNAs, and incorporate all phosphorothioate and 2′-O-methyl modifications. However, such ONs are sequence independent of the ON modifications we have described herein and the activity of such modified ONs we have demonstrated herein. It is thought that it can benefit from the fact that there is. Thus, ON has sequence-independent activity if it meets any one of the criteria of the five tests outlined below, ie if a substantial part of its function is due to sequence-independent activity. It is considered a thing. The ON used in the following tests can be prepared according to the general methodology described in Example 12 for the synthesis of PS-ON.

Test # 1-Effect of Partial Degeneration of Candidate ON on Its Antiviral Efficacy This test serves to measure the antiviral activity of a candidate ON sequence when part of its sequence is degenerate. A degenerate version of a candidate ON having the same chemical structure is considered to have sequence-independent activity if it retains its activity as described below. Candidate ONs are degenerated according to the following rules.

L = number of candidate ON bases
X = number of bases on each end of the degenerate oligo (although it has the same chemical structure as the candidate ON).
When L is an even number, X = integer (L / 4).
When L is an odd number, X = integer ((L + 1) / 4).
X must be greater than or equal to 4.

If a candidate ON is determined to have antiviral activity against a member of the herpesviridae, retrovirus or paramyxovirus family, the virus strain preferably indicated for that virus family is used herein, preferably IC ₅₀ generation is performed by the described assay. If a candidate ON is determined to have antiviral activity against a member of a particular viral family not listed above, an IC ₅₀ value is generated by an antiviral efficacy test accepted by the pharmaceutical industry. IC ₅₀ values are generated by 3 or more points within the linear range of the dose response curve using a minimum of 7 concentrations of the compound. These tests are used to compare the candidate ON IC ₅₀ to its degenerate counterpart. Partial degeneracy IC ₅₀ of ON is 5 times smaller case than the original candidate ON (based on the measurement of minimum 3 times the standard deviation does not exceed 15% of the average), ON is largely sequence-independent It is considered to work according to the mode of action.

Test # 2—Comparison of Antiviral Activity of Candidate ON and ON Randomer This test serves to compare the antiviral efficacy of candidate ON with that of a randomer ON of equivalent size and chemical structure within the same virus.

If a candidate ON is determined to have antiviral activity against a member of the herpesviridae, retrovirus or paramyxovirus family, the virus strain preferably indicated for that virus family is used herein, preferably IC ₅₀ generation is performed from the described assay. If a candidate ON is determined to have antiviral activity against a member of a particular viral family not listed above, an IC ₅₀ value is generated by an antiviral efficacy test accepted by the pharmaceutical industry. IC ₅₀ values are generated by 3 or more points within the linear range of the dose response curve using a minimum of 7 concentrations of the compound. This test is used to compare the candidate ON IC ₅₀ to ON randomers with equivalent size and chemistry. If the ON randomer IC ₅₀ is less than 5 times that of the candidate ON (based on a minimum of 3 measurements, the standard deviation does not exceed 15% of the mean), the candidate ON is largely due to a sequence-independent mode of action. It is considered to work.

Test # 3-Comparison of antiviral activity of candidate ONs in two non-homologous viruses of the same virus family It serves to compare the potency of the candidate ON against a second virus that has no homology but belongs to the same virus family. For example, if the candidate ON is reported to have activity against HSV, its activity against HSV is compared with its activity against CMV or VZV. A comparison of the relative activity of candidate ONs in the target virus and the second virus was obtained in the two viruses using the activity of the ON randomer of the same length and chemical structure in both viruses This is accomplished by normalizing the candidate ON IC ₅₀ values.

Thus, if a candidate ON is determined to have antiviral activity against a given virus, one of the assays described herein or the art for the herpesviridae, retrovirus or paramyxovirus family Other assays known in the above are used to determine IC ₅₀ production within this virus. Similarly, using one of the assays described herein or an assay accepted by the pharmaceutical industry, for viruses that do not have homology to the candidate ON sequence but whose genome belongs to the same virus family Perform IC ₅₀ generation on candidate ON for the second virus. IC ₅₀ is also generated for randomers with equivalent size and chemistry for each virus. The IC ₅₀ of the ON randomer for the two viruses is used to normalize the IC ₅₀ value of the candidate ON for the two viruses as follows:

First (homologous) by applying the equivalent algebraic converted into IC ₅₀ of candidate ON and ON randomers within virus, the IC ₅₀ of randomer 1.

The second (non-homologous) by applying the equivalent algebraic converted into IC ₅₀ of candidate ON and ON randomers within virus, the IC ₅₀ of randomer 1.

Phase candidates IC ₅₀ of ON at the same the virus pair differences multiples of IC ₅₀ of candidate ON in heterologous in viruses, candidate ON of the conversion IC ₅₀ of candidate ON in heterologous in viral phases at the same within the viral Calculate by dividing by the conversion IC ₅₀ .

If the difference in IC ₅₀ fold difference between the two viruses is less than 5, the candidate ON is considered to act largely by a sequence-independent mode of action.

Test # 4-Antiviral Activity of Candidate ONs in Different Virus Families This test serves to determine whether the candidate ON has drug-like activity in the virus, where the sequence of the candidate ON is It is not homologous to any part of the viral genome and the virus belongs to a different family. Thus, using one of the assays described herein for the herpesviridae, retrovirus or paramyxovirus family, the sequence of the candidate ON to be tested is not homologous to any part of the viral genome used. Test candidate ON like no. A minimum of 7 concentrations of candidate ON is used to generate IC ₅₀ values with 3 or more points within the linear range of the dose response curve. If the resulting dose response curve shows drug-like activity (typically appears as a decaying or sigmoid curve, showing a decrease in antiviral efficacy with decreasing concentrations of the candidate ON) and generated from the curve If the IC ₅₀ is less than 10 μM, the candidate ON is considered to have drug-like activity. When a candidate ON is considered to have drug-like activity in a different family of viruses that cannot have sequence-dependent antisense activity because the candidate ON is not complementary, it is largely sequence-independent Are considered to work in different modes of action.

Test # 5-Extracellular Antiviral Activity of Candidate ON The current results we show indicate that the sequence-independent antiviral activity of ON occurs extracellularly. State-of-the-art ON technology teaches that since ON cannot easily penetrate cells, they need to be delivered across the cell membrane with a suitable carrier to exhibit antisense activity. Thus, by definition, the antiviral activity of antisense ON depends on intracellular delivery for activity. If a particular sequence-specific candidate ON has antiviral activity when used naked in vitro (and therefore has poor intracellular penetration), it may benefit from the sequence-independent nature of the ON described in the present invention. It seems to receive.

  If the sequence-specific candidate ON is complementary to a portion of the HSV-1, HIV-1 or RSV genome, the presence of the sequence-independent antiviral activity of the candidate ON is determined by a suitable assay described below. Can be determined. If the candidate ON is complementary to a virus that is not HSV-1, HIV-1 or RSV, the antiviral activity of the candidate ON is determined using an assay accepted by the pharmaceutical industry.

Using a suitable assay, compare the antiviral activity of the naked candidate ON with the antiviral activity of the encapsulated candidate ON (for transfer) (identical for both naked and encapsulated candidate ON). With concentration). Activity is measured by dose response curves using more than 7 concentrations, at least 3 of which fall within a linear range containing 50% inhibition of viral activity. IC ₅₀ (concentration that reduces viral activity by 50%) is calculated by linear regression of the linear range of the dose response curve defined above. IC ₅₀ of bare candidate ON is less than five times the IC ₅₀ of encapsulated candidate ON, the candidate ON activity, mostly deemed to act by sequence-independent mode of action.

Thresholds used in these tests The purpose of these tests was to determine whether the ON would benefit from or utilize the sequence-independent antiviral properties of the ON described herein and be sequence-independent. It is determined by rational analysis whether it acts in an activity. Of course, any person skilled in the art will be able to measure the difference in antiviral activity between two compounds, assuming that the entire methodology of the test, in particular the inherent variability of the antiviral test method, is the threshold of the activity of the compound. You will recognize that it is not possible to conclude reliably if the difference is set to 2 or 3 times. This is due to the fact that variability from experiment to experiment with the same compound in these assays can give an IC ₅₀ that varies within this range. Therefore, in order to reasonably confirm that there is indeed a difference between the activities of two compounds (e.g., two ONs), we have at least five-fold between the IC _{50 of the} compounds. Is set as a threshold value. This threshold ensures the reliability of the test evaluation described above.

The threshold values described in tests 1 to 3 and 5 above are the default threshold values. Other thresholds may be used for the above comparative tests 1-3 and 5 if specifically indicated. Thus, for example, where indicated, thresholds that determine whether ON is acting with sequence-independent activity are 10 times, 8 times, 6 times, 5 times, 4 times, 3 times, 2 times, 1.5 times It can be either double and identical. The threshold values described in Test 4 above are also the default threshold values. In particular, the threshold for determining whether ON has sequence-independent activity in Test 4 is 10 μM, 5 μM, 1 μM, 0.8 μM, 0.6 μM, 0.5 μM, 0.4 μM, 0.3 μM, 0.2 μM Or an IC ₅₀ of less than 0.1 μM.

  Similarly, even if the initial settings are sufficient to satisfy any one of the five tests above, ON is specifically indicated if any two (e.g., tests 1 and 2, 1 and 3, 1 and 4, 1 and 5, 2 and 3, 2 and 4, 2 and 5, 3 and 4, and 3 and 5), any three (e.g., tests 1 and 2 and 3, 1 and 2 and 4, 1 and 2 and 5, 1 and 3 and 4, 1 and 3 and 5, 2 and 3 and 4, and 2 and 4 and 5), any four of the tests (eg, 1 and 2 and 3 and 4, 1 and 2) And 3 and 5, and 2 and 3, and 4 and 5) may need to be met with a default threshold or, if specifically indicated, with another threshold shown above.

Example 12 Methodology The following method is provided for application to the test described in Example 11.

Oligonucleotide Synthesis The oligonucleotides of the present invention can be synthesized by methods well known in the art. For example, unsubstituted and substituted phosphodiester (P = O) oligonucleotides can be synthesized with iodine on an automated DNA synthesizer (e.g., Applied Biosystems model 380B or Akta Oligopilot 100) using standard phosphoramidite chemical structures. It can be synthesized by oxidation. Phosphorothioate (P = S) replaces the standard oxidation bottle with a 0.2M solution of 311-1,2-benzodithiol-3-one 1,1-dioxide in acetonitrile, stepwise for phosphite binding It can be synthesized in the same manner as a phosphodiester oligonucleotide, except that it is thioated. The thioate waiting step may be extended to 68 seconds, followed by a capping step. After cleaving from the support column and deblocking in concentrated ammonium hydroxide at 55 ° C. (18 hours), the oligonucleotide can be purified by precipitating twice from 0.5 M NaCl solution with 2.5 volumes of ethanol.

Antiviral Assay for Herpesviridae Plaque reduction for Herpesviridae is performed as follows:
For HSV-1 or HSV-2, VERO cells (ATCC # CCL-81) are heated in a 12-well tissue culture plate (NUNC or equivalent) at 37 ° C. and 5% CO ₂ with 10% heat. Grow to confluence in the presence of inactivated fetal bovine serum and MEM supplemented with gentamicin, vancomycin and amphotericin B. Once confluency was reached, the medium was supplemented with either HSV-1 (KOS strain, total 40-60 PFU) or HSV-2 (MS2 strain, total 40-60 PFU) and 5% fetal bovine serum detailed above Change to contain antibiotics. After allowing the virus adsorption to proceed for 90 minutes, the cells are washed and replaced with fresh “overlay” medium containing 5% fetal bovine serum and 1% human immunoglobulin. Three to four days after adsorption, cells are fixed with formalin and plaques are counted after formalin fixation and cresyl violet staining.

For CMV, human neuroblasts are grown as detailed for VERO cells in the HSV-1 / 2 assay. Media components and adsorption / overlay procedures are the same with the following exceptions:
1. During adsorption, use CMV (AD169 strain, total 40-60 PFU) for cell infection.
2. Replace 1% human immunoglobulin with 4% sieve plaque agarose in overlay medium.

  For other herpesviridae, tests are performed in the plaque assay described above using a suitable cellular host and 40-60 PFU virus during adsorption.

  This test is only effective if there are plaques identifiable in the absence of the compound at the end of the test.

In this test, the IC ₅₀ is the concentration at which 50% plaque is present compared to the untreated control.

  The compound to be tested is present during adsorption and overlay.

Antiviral Assay for Retrovirus The assay for retrovirus HIV-1 is performed by detecting total p24 in the supernatant of HIV-1-infected cells by ELISA performed as follows.

PBMCs were isolated from fresh human blood of screened donors that were seronegative for HIV and HBV. Peripheral blood cells were pelleted / washed 2-3 times by low speed centrifugation and resuspension in PBS to remove contaminating platelets. The washed blood cells were then diluted 1: 1 with Dulbecco's phosphate buffered saline (PBS), and the lymphocyte separation solution (Mediatech, Inc. LSM; cellgro®; Density 1.078 +/- 0.002 g / mL; Cat. # 85-072-CL) Overlaid on 14 mL and centrifuged at 600 × g for 30 minutes. The band-shaped PBMC was gently sucked from the obtained interface, and then washed twice with PBS by low speed centrifugation. After the final wash, cells were counted by trypan blue exclusion and reconstituted at 1 × 10 ⁷ cells / mL in RPMI 1640 supplemented with 15% fetal bovine serum (FBS), 2 mM L-glutamine, 4 μg / mL PHA-P. Suspended. Cells were incubated for 48-72 hours at 37 ° C. After incubation, the PBMCs are centrifuged and resuspended in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL gentamicin and 20 U / mL recombinant human IL-2. It became cloudy. PBMC were maintained at a concentration of in the medium 1 to 2 × 10 ⁶ cells / mL, until use in assay protocol, the medium was replaced every two weeks. Monocytes were depleted from the culture as a result of adsorption to the tissue culture flask.

For standard PBMC assays, PHA-P stimulated cells from at least 2 normal donors are pooled and diluted to a final concentration of 1 × 10 ⁶ cells / mL in fresh medium, 96 well round bottom Plating was performed in a microplate well at 50 μL / well (5 × 10 ⁴ cells / well). Test drug dilutions were prepared at 2 × concentration in microtiter tubes and 100 μL of each concentrate was placed in appropriate wells of a standard format. After a 2 hour preincubation period (cell + drug), 50 μL of a stock dilution of virus was placed in each test well (final MOI 0.1). Wells containing only cells and virus were used for virus control. Using the MTS assay system (described below), separate plates with the virus removed were prepared identically for drug cytotoxicity testing. PBMC cultures were maintained for 7 days post infection, at which time cell-free supernatant samples were collected and assayed for reverse transcriptase activity as described below.

  The p24 ELISA kit was purchased from Coulter Electronics. The assay is performed according to the manufacturer's instructions. Control curves are generated in each assay to accurately quantify the amount of p24 antigen in each sample. Data are acquired by spectrophotometric analysis at 450 nm using a Molecular Devices Vmax plate reader. The final density is calculated from the optical density value.

  This test is valid only if p24 accumulation is present in the tissue culture supernatant of infected untreated cells.

In this test, the IC ₅₀ is the concentration at which the detectable amount of p24 is 50% of the p24 present in the untreated control.

  The compound to be tested is present in the medium during and after adsorption.

Antiviral assay for Paramyxoviridae
For RSV, CPE measurement is performed as follows.

Hep2 cells were plated in 96 well plates and grown overnight at 37 ° C. with 5% CO ₂ in MEM with 5% fetal calf serum. The next day, cells are infected with RSV (A2 strain in 100 ul / well, 10 ^8.2 TCID50 / mL) for 2 hours by adsorption. After adsorption, the medium is changed and after 7 days of growth, CPE is measured by conversion of the alamar blue dye to its fluorescent adduct by living cells.

  This test is only valid when the CPE measurement in infected cells in the absence of compound (measured by Alamar Blue conversion) is 10% of the conversion measured in uninfected cells.

To compare IC _{50 s} , 100% CPE is set to the conversion level found in infected cells and 0% CPE is set to the conversion level found in uninfected cells. IC ₅₀ is therefore the concentration of compound that produces 50% CPE.

  The compound to be tested is present in the medium during and after absorption.

Example 13: 2′-O methylated phosphorothioated randomers show potent antiviral activity with increased pH tolerance and decreased serum protein binding We have herein described that PS-ON randomers are Indicates that they do not act through sequence-specific mechanisms (ie, their activity does not require binding to nucleic acids and the activity is not due to sequence-specific aptamer effects). The inventors further in this example show their antiviral activity of oligonucleotides combining unmodified bonds, phosphorothioate bonds, 2'-O methyl modified ribose and unmodified ribonucleotides on a 40 base randomer, Shows effects on serum protein interactions and chemical stability.

  All randomers are prepared at the University of Calgary Core DNA Services lab using standard solid phase, batch synthesis, 1 or 15 □ mol synthesis scale, deprotected, and on a 50 cm Sephadex G-25 column. Desalted.

Immortalized canine kidney (MDCK) cells supplemented with gentamicin, vancomycin and amphotericin B at 37 ° C. and 5% CO ₂ for antiviral activity testing in influenza A (INF A), 10% bovine Incubate in MEM with fetal serum. Cells are seeded in 6-well plates at a density that yields a confluent monolayer 6 days after growth. Once confluency is reached, the medium is changed to contain only the above supplements, and the cells are then exposed to INF A (H3N2 strain, about 35-70 PFU total) for 60 minutes. After virus exposure, the medium is replaced with a new medium containing only the drug. Two to three days after infection, plaque counts are performed after formalin fixation and cresyl violet staining of infected cultures.

For antiviral testing in HSV, immortalized African green monkey kidney (VERO) cells were supplemented with gentamicin, vancomycin and amphotericin B at 37 ° C. and 5% CO ₂ with MEM supplemented with 10% fetal calf serum Incubate in. Cells are seeded in 12-well plates at a density that gives a confluent monolayer of cells 4 days after growth. When confluency is reached, the medium is changed to contain only 5% serum and the above supplements, and then the cells are placed in HSV-1 (KOS strain, total about 40-60 PFU) for 90 minutes in the presence of the test compound To expose. After virus exposure, the medium is replaced with fresh “overlay” medium containing 5% serum, 1% human immunoglobulin, the above supplements and test compounds. Three to four days after infection, infected cultures are formalin fixed and cresyl violet stained and plaque counts are performed.

  To measure serum protein interactions, phosphorothioate randomers labeled with FITC (bait) at the 3 ′ end were added to assay buffer (10 mM Tris, pH 7.2, 80 mM NaCl, 10 mM EDTA, 100 mM b-mercaptoethanol and 1% Dilute to 2 nM in tween 20). The oligo is then mixed with an appropriate amount of FBS that has not been heat inactivated. Following the randomer-FBS interaction, the complexes are challenged with various unlabeled randomers to assess their ability to displace the baits from the complex. The displaced bait is measured by fluorescence polarization. A displacement curve was used to determine Kd.

  The pH tolerance was measured by incubating the randomer in PBS adjusted to the appropriate pH with HCl. After 24 hours of incubation, the samples were neutralized with 1M TRIS, pH 7.4, run on a denaturing acrylamide gel and visualized following EtBr staining.

  For these experiments, we compared the behavior of different modified randomers: REP 2006, REP 2024, REP 2107, REP 2086 and REP 2060 (see Table 6 in this example). The antiviral activity of these randomers was tested for antiviral activity within HSV and influenza A by plaque reduction assay (see Table 7 in this example). Within these two viruses, REP 2006, 2024, and 2107 have similar and potent antiviral activity, REP 2060 exhibits significant anti-HSV activity, and REP 2086 Below, it had no detectable antiviral activity within both HSV-1 and influenza A.

N＝非修飾デオキシリボヌクレオチド、非修飾結合
N＝非修飾デオキシリボヌクレオチド、ホスホロチオエート結合
N＝2'-Oメチル修飾リボース、非修飾結合
N＝2'-Oメチル修飾リボース＋ホスホロチオエート結合
N＝非修飾リボヌクレオチド＋ホスホロチオエート結合 (Table 6) Description of randomer

N = unmodified deoxyribonucleotide, unmodified bond
N = unmodified deoxyribonucleotide, phosphorothioate linkage
N = 2'-O methyl modified ribose, unmodified bond
N = 2'-O methyl modified ribose + phosphorothioate linkage
N = unmodified ribonucleotide + phosphorothioate linkage

Table 7: Antiviral activity of various randomers in HSV and influenza A

  The relative affinity of these randomers for serum proteins was determined as described above. The results of these experiments show that REP 2107 has a lower affinity for serum proteins than REP 2006 or REP 2024 (see Table 8 in this example), and there is no interaction between REP 2086 and serum proteins. It was not detected. Furthermore, at competitive saturation, REP 2107 was less effective at replacing bound baits compared to REP 2006 or REP 2024 (see Table 9 in this example).

(Table 8) Serum protein affinity of various randomers

(Table 9) Replacement of baytrandamer in saturation

  Finally, we tested the pH stability of these randomers within the range of pH 1 to pH 7 during incubation for 24 hours at 37 ° C. REP 2006 and REP 2024 showed significant degradation at pH 3 and were completely degraded at pH 2.5, while REP 2107, 2086 and 2060 were completely stable at pH 1 after 24 hours of incubation.

  These results reproduce the results of our previous study that phosphorothioation of ON randomers is highly beneficial in their antiviral activity. The inventors further noted that the incorporation of 2′-O methyl modifications within the PS-ON randomer may prevent the anti-reactivity of these molecules even though each ribose within the PS-ON randomer contains a 2′-O-methyl modification. Indicates that it does not affect virus activity. Furthermore, fully 2'-O-methylated and fully phosphorothioated randomer (REP 2107) has relatively weak interaction with serum proteins and significantly increased resistance to hydrolysis induced by low pH Has been.

Example 14: PS-ON acts in large part by an extracellular mode of action Prior art teaches that the use of delivery agents that increase the intracellular concentration of PS-ON would be beneficial to their activity is doing. We show here that the antiviral activity of PS-ON acts largely extracellularly and therefore would not accept a major benefit by facilitating the transfer of intracellular delivery agents.

  In this example, we use PS-ONs made from deoxyribonucleotides (DNA) that do not contain other modifications such as ribonucleotides (RNA) or 2′-O-methyl modifications. . Since these molecules are well known in the art to not easily penetrate cells in vitro without the aid of a delivery system or transfection agent, especially in the case of antisense activity, this data represents further modification. It is safe to think that it applies to PS-ON that supports

  For measurement of cell delivery, HeLa cells are cultured under standard conditions and then naked or with a delivery agent (in this case DOTAP [1,2-dioleoyl-3-trimethylammonium-propane], cationic lipid ) And incubated with fluorescently labeled REP 2006 (FL-REP 2006, 3 'fluorescein isothiocyanate-conjugated 40 base PS-ON randomer). After various times of incubation, the cells were washed thoroughly with PSB to remove any non-internalized ON and then the cells were lysed. The level of intracellular ON in the cell lysate was measured with a fluorescent plate reader.

  Antiviral efficacy with HSV-1 and naked REP 2006 in influenza, and REP 2006 encapsulated with DOTAP and PEI (polyethyleneimine) was measured as described above.

  The duration of action of REP 2006 during the HSV-1 infection cycle was measured as described above, but REP 2006 was added at various times before, during and after infection. Within HIV-1, REP 2006 was measured by addition to HIV-LTR-β-gal HeLa cells at various times before, during and after infection. HIV-1 infection was monitored by a colorimetric assay for β-gal production using absorption photometry.

  Initially, we measured that DOTAP and PEI can deliver fluorescent REP 2006 into cells (see Table 10). This data showed that both DOTAP and PEI can deliver FL-2006 (and REP 2006 by reasoning) into the cells.

Table 10: Intracellular concentration of FL-REP 2006 with and without delivery (pmol / cell)

  Next, we measured the activity of HSV-1, encapsulation within influenza A (DOTAP or PEI) REP 2006 (see Table 11 and Table 12 in this example). These results indicated that encapsulated REP 2006 did not have detectable antiviral activity in both HSV-1 and influenza.

Table 11: Activity of encapsulated REP 2006 in ICV-1 (IC ₅₀ , μM)

Table 12: Activity of PEI-encapsulated REP 2006 in influenza A (inhibition of% plaque formation)

  Finally, a study of the addition time within HSV-1 and HIV-1 was conducted. Here, REP 2006 was added at various times before, during and after infection. These results indicate that REP 2006 is most effective in both viruses when present before or during infection, and REP 2006 is a fusion / entry inhibitor in HSV-1 and HIV-1. It showed that.

  These results indicate that the antiviral activity of PS-ON supporting additional modifications such as, but not limited to, REP 2006 and ribonucleotides (RNA) or 2'-O-methyl is primarily extracellular. It shows what happens.

Example 15: REP 2107 exhibits excellent nuclease resistance 40mer randomers of various chemical structures were evaluated for 4 hours at 37 ° C for their ability to resist degradation by various nucleases (Table 13 of this example) reference). Although most chemical structures were resistant to more than one nuclease, only REP 2107 was resistant to all four nucleases tested. REP 2024 (with a 2'-O methyl modification at the 4 ribose at each end of the molecule) showed the same resistance profile as its parent molecule REP 2006 and was sensitive to S1 nuclease degradation, but 2107 (fully It is important to note that 2′-O methyl modified) was resistant to this enzyme. These results suggest that of the oligonucleotides tested, REP 2107 would be most effective in resistance to nuclease degradation in blood.

Table 13: Resistance to various nucleases with different randomer chemical structures

Example 16: Phosphorothioated polypyrimidine ON exhibits acid resistance and nuclease resistance
To measure the degree of ON of acid resistance of ON, various 40 base ONs with different chemical structures and / or sequences are incubated for 24 hours at 37 ° C. in PBS buffered to different pH values. The degradation of these ONs was evaluated by urea polyacrylamide gel electrophoresis (see Table 14).

  The results of these studies indicate that randomer ONs (including both pyrimidine and purine nucleotides) are resistant to acidic pH only when they are fully 2'-O-methylated. Our data showed that even the partial 2′-O-methylated ON (gapmer, REP 2024) did not show any significant increase in acid resistance compared to fully phosphorothioated ON. Even fully phosphorothioated randomers do not show increased pH tolerance compared to unmodified ON. In contrast, we have phosphorothioated 40mer ON containing only the pyrimidine nucleotide cytosine (poly C, REP 2031) or thymidine (poly T, REP 2030) or poly TC heteropolymer (REP 2056). (REP 2107) or not (REP 2086) was found to have acid resistance equivalent to the fully 2′-O-methylated randomer. In contrast to polypyrimidine oligonucleotide results, phosphorothioated oligonucleotides containing only the purine nucleotide adenosine (poly A, REP 2029) or any adenosine or guanosine nucleotide (REP 2033, 2055, 2057) compared to unmodified DNA And did not show any high acid resistance.

  These results indicate that the preferred method described in the prior art to achieve high acid resistance compared to phosphorothioated ON is a 2′-O-methyl modification (or other 2′-ribose modification) or other This is significant because of the modification. The data of the present invention show that when ON is polypyrimidine (i.e. containing only pyrimidine nucleotides [e.g. cytosine or thymidine homopolymer, or cytosine and thymidine heteropolymer]) to achieve pH resistance and nuclease resistance. , 2'-O-methyl ribose modification or other 2'-ribose modifications are not required. The presence of purines (A or G) in the presence of pyrimidines can also make ON acid labile.

PII＝ホスホジエステラーゼII、S1＝S1ヌクレアーゼ、Exo1＝エキソヌクレアーゼ1、PS＝全結合がホスホロチオエート化、2'OMe＝全リボースが2'Oメチル化、+++＝分解なし、++＝5%未満の分解、-/+＝90%より多い分解、−＝完全に分解 Table 14: Acid stability of various 40mer ONs

PII = Phosphodiesterase II, S1 = S1 nuclease, Exo1 = Exonuclease 1, PS = Total linkage phosphorothioated, 2'OMe = Total ribose 2'O methylation, +++ = No degradation, ++ = less than 5% Decomposition,-/ + = more than 90% decomposition,-= complete decomposition

  To determine the range of ON nucleotide compositions and modifications for nuclease resistance, various 40 base ONs with different nucleotide compositions and modifications were incubated at 37 ° C. for 4 hours in the presence of various endonucleases and exonucleases. These ON degradations were evaluated by urea polyacrylamide gel electrophoresis.

  The results of these studies show that Randamer ON is one of the four total enzymes tested (phosphodiesterase II [Sigma], S1 nuclease [Fermentas] only when they are fully phosphorothioated and fully 2'-O-methylated. ], Bal31 [New England Biolabs] and exonuclease 1 [New England Biolabs]) (see Table 15). Deletion of any of these modifications within the randomer increased sensitivity to one or more nucleases tested. We have fully phosphorothioated, the partial 2'-O-methylated randomer (REP 2024) is equivalent to REP 2006 in nuclease resistance, and for achieving optimal nuclease resistance, phosphorothioated ON It has been found that 2'-O-methylation may be required for each nucleotide. However, the inventors have found that phosphorothioated 40mer polypyrimidine polycytosine (Poly C, REP 2031) has nuclease resistance equivalent to fully phosphorothioated, fully 2'0 methylated randomer (REP 2107). .

  These results teach that the prior art is the preferred way to increase the nuclease resistance of phosphorothioated ON is to add 2'-O-methyl modifications, other 2'-ribose modifications or other modifications Is significant. This new data shows that when ON is fully phosphorothioated and consists of a pyrimidine homopolymer, 2'-O-methyl or other 2'-ribose modifications or any other modification is required to increase nuclease resistance It means not to.

PII＝ホスホジエステラーゼII、S1=S1ヌクレアーゼ、Exo1＝エキソヌクレアーゼ1、PS＝全結合がホスホロチオエート化、2'OMe＝全リボースが2'Oメチル化、−＝完全に分解、++++＝分解なし、PS＝ホスホロチオエート、2'OMe＝リボースの2'-O-メチル修飾 Table 15: Nuclease resistance of various 40mer ONs

PII = phosphodiesterase II, S1 = S1 nuclease, Exo1 = exonuclease 1, PS = total linkage phosphorothioated, 2'OMe = total ribose 2'O methylation,-= completely degraded, ++++ = no degradation , PS = phosphorothioate, 2'OMe = 2'-O-methyl modification of ribose

  These results indicate that phosphorothioated ONs containing only pyrimidine nucleotides, including cytosine and / or thymidine and / or other pyrimidines, are resistant to low pH and phosphorothioated ON containing only cytosine nucleotides are superior nucleases It is resistant and shows two important properties for oral administration of antiviral ON. Thus, a high pyrimidine nucleotide content of antiviral ON is advantageous to provide resistance to low pH, and a high cytosine content is advantageous to provide improved nuclease resistance. For example, in certain embodiments, the pyrimidine content of such oligonucleotides is greater than 50%, greater than 60%, or greater than 70%, or greater than 80%, or greater than 90% or 100%. . Furthermore, these results indicate the possibility of a treatment method by oral administration of a therapeutically effective amount of at least one pharmaceutically acceptable ON composed of pyrimidine nucleotides. These results also indicate the possibility of ON containing high levels of pyrimidine nucleotides as a component of antiviral ON formulations.

Example 17: Sequence-independent broad activity of ON in vivo We here describe six different animal models in which a 40 base sequence-independent PS-ON randomer was virally infected. It has a strong antiviral activity in (see Table 16). The 40 base PS-ON randomer was introduced into animals by multiple routes of administration including subcutaneous, intraperitoneal and aerosol (inhalation). These data strongly support the therapeutic potential of sequence-independent ON as a broad spectrum antiviral agent.

ND＝測定せず Table 16: PS-ON randomers have potent broad spectrum antiviral activity in vivo

ND = not measured

Example 18: Oligonucleotides have antiviral activity against a broad spectrum of viruses We have here demonstrated that 40 PS-ON randomers have antiviral activity against 13 viral families in vitro. Shown (see Table 17).

Table 17: PS-ON randomers have broad antiviral activity in vitro

Example 19: In vivo and in vitro anti-influenza activity of PS-ON
To further evaluate the anti-influenza activity of ON, REP 2006 was tested against different strains of influenza by a hemagglutination assay. As shown in Table 18, REP 2006 showed a wide range of anti-influenza activity.

Table 18: Widespread antiviral activity of REP 2006 against multiple strains of influenza

  To evaluate the potential of ON as a drug for influenza treatment, REP 2006 was tested in a mouse model of influenza infection. REP 2006 was prepared in two concentrations in distilled water for injection and aerosolized by spraying, where the exhaust was connected to Anderson's cascade chamber. 20 g Balb / c mice were exposed to aerosolized randomer 1 daily for 30 minutes using 10 mL of various concentrations of REP 2006 in an aerosol chamber. Mice were infected intranasally with approximately 100 TCID of influenza A (H3N2, A / Hong Kong / 68), animals were sacrificed 4 days after infection, and pulmonary virus titers were measured by hemagglutination assay. As shown in Table 19, REP 2006 showed potent anti-influenza activity in vivo.

SDA＝小滴エアロゾル、IP＝腹腔内、SC＝皮下、^**は、12時間間隔で投与される2つの1日量 Table 19: Efficacy of REP 2006 against influenza A in vivo

SDA = droplet aerosols, IP = intraperitoneal, SC = subcutaneous, ^**, the amount of two daily administered at 12 hour intervals

Example 20: Phosphorothioated polypyrimidine ON exhibits improved antiviral activity in an acidic environment in vivo Polypyrimidine ON's tolerance to low pH and their ability to become active drugs at lower pH in vivo To do so, REP 2031 (PS Poly C) was tested in the HSV-2 intravaginal mouse model. A group of female Swiss Websters were administered by subcutaneous injection 0.1 mL of a suspension containing 3 mg of medroxyprogesterone acetate 7 and 1 day before the viral challenge to increase the susceptibility of intravaginal HSV-2 infection. It was. The vaginal cavity was wiped twice with a cotton swab, first with a wet type 1 swab with a calcium alginate tip and then with a dry swab. Animals were treated with 15 μL of either the candidate solution or a placebo control using a continuous pipette. Five minutes later, the animals were inoculated by instillation of 15 μL of suspension containing HSV-2, 186 strain 10 ⁴ pfu. Vaginal wipe samples were collected from all animals 2 days after inoculation and stored frozen (-80 ° C) in culture until assayed for the presence of virus. Mice were evaluated daily for signs of symptomatic infection until 21 days after inoculation, including hair loss and erythema around the perineum, chronic urinary incontinence, hindlimb paralysis and death. Animals that did not develop symptoms were defined as infected if the virus was isolated from a vaginal wipe sample collected 2 days after inoculation. The results showed that polypyrimidine REP 2031 has antiviral activity in an acidic environment such as the vagina or stomach in this example (Table 20).

(Table 20) Vaginal efficacy of ON against HSV-2

  All patents and other references cited in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each reference, including any tables and figures, individually and entirely. As if incorporated herein by reference in their entirety.

  Those skilled in the art will readily and fully appreciate that the present invention has been successfully applied to obtain the results and advantages mentioned, as well as those inherent therein. The methods, variations and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the scope of the invention and are defined by the claims.

  It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. For example, changes can be made to the synthesis conditions and the composition of the oligonucleotide. Accordingly, such additional embodiments are within the scope of the present invention and claims.

  The invention described in the description, suitably described herein, may be practiced in the absence of any elements, limitations not specifically disclosed herein. Thus, for example, in each example herein, any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions employed are used as descriptive terms and are not limiting terms, and exclude the equivalents, or portions thereof, of the features shown and described in the use of such terms and expressions. Although not intended, it will be recognized that various modifications are possible within the scope of the claimed invention. Thus, although the invention has been specifically disclosed with preferred embodiments and optional features, modifications and changes to the concepts disclosed herein may be relied upon by those skilled in the art, and such modifications and changes may be It should be understood that it is deemed to be within the scope of the present invention as defined by the appended claims.

  Further, where features and aspects of the invention are described in terms of a Markush group or other alternative group, one of ordinary skill in the art will recognize that the invention is thereby a member of an individual or subgroup of the Markush group or other group. You will understand that

  Also, on the contrary, if not stated, additional aspects are described by utilizing any two different values as the endpoints of the range when various numerical values are provided for the aspects. Such ranges are also within the scope of the described invention.

  Accordingly, additional embodiments are within the scope of the invention and are within the scope of the claims.

(Table 21) Oligonucleotide description

Claims (76)

  An oligonucleotide in which at least 50% of its nucleotides in the oligonucleotide are modified at the 2 'position of the ribose moiety and at least 50% of its internucleotide linkage is modified and has antiviral activity against the target virus , Oligonucleotides whose activity acts in a largely sequence-independent manner.   An oligonucleotide in which at least 50% of its nucleotides in the oligonucleotide are modified at the 2 'position of the ribose moiety and at least 80% of its internucleotide linkage is modified and has antiviral activity against the target virus The oligonucleotide of claim 1, wherein the activity acts in a largely sequence independent manner.   An oligonucleotide in which at least 80% of its nucleotides in the oligonucleotide are modified at the 2 'position of the ribose moiety and at least 80% of its internucleotide linkage is modified and has antiviral activity against the target virus The oligonucleotide of claim 1, wherein the activity acts in a largely sequence independent manner.   An oligonucleotide in which at least 90% of its nucleotides in the oligonucleotide are modified at the 2 'position of the ribose moiety and at least 90% of its internucleotide linkage is modified and has antiviral activity against the target virus The oligonucleotide of claim 1, wherein the activity acts in a largely sequence independent manner.   An oligonucleotide in which at least 100% of its nucleotides in the oligonucleotide are modified at the 2 'position of the ribose moiety and at least 100% of its internucleotide linkage is modified and has antiviral activity against the target virus The oligonucleotide of claim 1, wherein the activity acts in a largely sequence independent manner.   The oligonucleotide according to any one of claims 1 to 5, wherein the modified bond is selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond and a boranophosphate bond.   The oligonucleotide according to any one of claims 1 to 5, wherein at least 50% of the nucleotides in the oligonucleotide comprise a 2'-OMe moiety at the 2 'position of the ribose moiety.   The oligonucleotide according to any one of claims 1 to 5, wherein at least 50% of the nucleotides in the oligonucleotide comprise a 2'-methoxyethoxy substitution at the 2 'position of the ribose moiety.   The oligonucleotide according to any one of claims 1 to 8, which is at least 30 nucleotides in length.   10. The oligonucleotide according to any one of claims 1 to 9, comprising a homopolymer sequence of at least 10 consecutive nucleotides selected from the group consisting of A, T, U, C, G and I.   At least 10 nucleotides in length selected from the group consisting of poly AT, poly AC, poly AG, poly AU, poly AI, poly GC, poly GT, poly GU, poly GI, poly CT, poly CU, poly CI and poly TI The oligonucleotide according to any one of claims 1 to 9, comprising the sequence of:   The oligonucleotide according to any one of claims 1 to 11, wherein at least 15% of the nucleotides in the oligonucleotide comprise a 2'-methoxyethoxy or 2'OMe substitution at the 2 'position of the ribose moiety.   The oligonucleotide according to any one of claims 1 to 12, which is a chain consisting of two or more oligonucleotide sequences linked by a linker.   Modify oligonucleotide properties at one or more nucleotide residues to increase stability, lower interaction with serum, higher cellular uptake, higher interaction with viral proteins, formulated for delivery One or more properties selected from the group consisting of improved ability to detect, detectable signal, higher antiviral activity, better pharmacokinetic properties, specific tissue distribution, lower toxicity 14. The oligonucleotide according to any one of claims 1 to 13, which is bound or conjugated to a molecule to be acquired.   The oligonucleotide according to any one of claims 1 to 14, which is double-stranded.   The oligonucleotide according to any one of claims 1 to 15, which targets a DNA virus or an RNA virus.   Herpesviridae, HSV-1, HSV-2, CMV, varicella-zoster virus, Epstein-Barr virus, human herpesvirus 6A and 6B, hepadnaviridae, HBV, parvoviridae, poxviridae, papillomaviridae, Adenoviridae, Retroviridae, HIV-1, HIV-2, Paramyxoviridae, RSV, Parainfluenza virus, Human metapneumovirus, Bunyaviridae, Hantavirus, Rift Valley fever virus, Crimea-Congo hemorrhagic fever virus, Picornaviridae, Coxsackie virus, Rhinovirus, Flaviviridae, Yellow fever virus, Dengue virus, West Nile virus, Hepatitis C virus, Filoviridae, Ebola virus, Marburg virus, Orthomyxoviridae, Influenza virus , Targeting members of the group consisting of Togaviridae, Western equine encephalitis virus, Coronaviridae, Reoviridae, Rhabdoviridae, Arenaviridae, Lassa fever virus and Calciviridae The oligonucleotide according to claim 1.   REP 1001, REP 2001, REP 3007, REP 2004, REP 2005, REP 2006, REP 2007, REP 2008, REP 2017, REP 2018, REP 2020, REP 2021, REP 2024, REP 2036, A20, G20, C20, REP 2029 , REP 2031, REP 2030, REP 2033, REP 2055, REP 2056, REP 2057, REP 2060 and an oligonucleotide as shown in any one of REP 2107.   19. An oligonucleotide mixture comprising a mixture of at least two different antiviral oligonucleotides according to any one of claims 1-18.   19. An oligonucleotide mixture comprising a mixture of at least 10 different antiviral oligonucleotides according to any one of claims 1-18. An antiviral pharmaceutical comprising a therapeutically effective amount of at least one pharmacologically acceptable antiviral oligonucleotide as defined in any one of claims 1-18; and a pharmaceutically acceptable carrier. Composition.   A kit comprising at least one antiviral oligonucleotide as defined in any one of claims 1-18 in a labeled package, wherein the antiviral activity of the oligonucleotide is primarily sequence complementary. A kit produced by no mode of action, wherein the label on the package indicates that the antiviral oligonucleotide can be used against at least one virus.   23. The kit of claim 22, comprising a mixture of at least two different antiviral oligonucleotides.   An antiviral pharmaceutical composition comprising a therapeutically effective amount of at least one pharmacologically acceptable polypyrimidine oligonucleotide and a pharmaceutically acceptable carrier, wherein the antiviral activity of the oligonucleotide is primarily sequence independent Antiviral pharmaceutical composition resulting from a mode of action.   25. The antiviral pharmaceutical composition of claim 24, wherein the oligonucleotide comprises at least one modified internucleotide linkage.   26. A composition according to any one of claims 24 or 25, wherein the composition is formulated for administration to an acidic site in vivo.   27. A composition according to any one of claims 24-26, adapted for oral, vaginal or topical administration.   28. The composition of any one of claims 24-27, comprising at least one poly C oligonucleotide.   28. The composition of any one of claims 24-27, comprising at least one poly T oligonucleotide.   28. The composition of any one of claims 24-27, comprising at least one poly CT oligonucleotide.   Oligonucleotides that have been modified with at least 50% of their internucleotide linkages having antiviral activity against the target virus, the activity acting largely in a sequence independent manner of action, at least 80% Oligonucleotides comprising the pyrimidine residues of   32. The oligonucleotide of claim 31, wherein at least 80% of the internucleotide linkages are modified.   32. The oligonucleotide of claim 31, wherein at least 80% of the internucleotide linkages are modified and have 100% pyrimidine residues.   32. The oligonucleotide of claim 31, wherein at least 100% of the internucleotide linkages are modified and have at least 80% pyrimidine residues.   32. The oligonucleotide of claim 31, wherein at least 100% of the internucleotide linkages are modified and have 100% pyrimidine residues.   36. The oligonucleotide according to any one of claims 31 to 35, wherein the modified bond is selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond and a boranophosphate bond.   36. The oligonucleotide according to any one of claims 31 to 35, wherein the modified bond is a phosphorothioate bond.   The oligonucleotide according to any one of claims 31 to 37, wherein the pyrimidine residue is a cytosine residue.   The oligonucleotide according to any one of claims 31 to 37, wherein the pyrimidine residue is a thymine residue.   38. The oligonucleotide according to any one of claims 31 to 37, wherein the pyrimidine residue is a cytosine residue or a thymine residue.   41. The oligonucleotide according to any one of claims 31 to 40, wherein the oligonucleotide is at least 30 nucleotides in length.   41. The oligonucleotide according to any one of claims 31 to 40, wherein the oligonucleotide is at least 40 nucleotides in length.   43. The oligonucleotide according to any one of claims 31 to 42, wherein at least 15% of the contained nucleotides contain a 2'-methoxyethoxy or 2'-OMe substitution at the 2 'position of the ribose moiety.   44. The oligonucleotide according to any one of claims 31 to 43, wherein the oligonucleotide is a chain consisting of two or more oligonucleotide sequences linked by a linker.   At one or more nucleotide residues, modify the properties of the oligonucleotide to be formulated for higher stability, lower interaction with serum, higher cellular uptake, higher interaction with viral proteins, delivery One or more properties selected from the group consisting of improved ability to detect, detectable signal, higher antiviral activity, better pharmacokinetic properties, specific tissue distribution, lower toxicity 45. The oligonucleotide according to any one of claims 31 to 44, which is bound or conjugated to a molecule to be acquired.   The oligonucleotide according to any one of claims 31 to 45, wherein the oligonucleotide is double-stranded.   47. The oligonucleotide according to any one of claims 31 to 46, which targets a DNA virus or an RNA virus.   Herpesviridae, HSV-1, HSV-2, CMV, varicella-zoster virus, Epstein-Barr virus, human herpesvirus 6A and 6B, hepadnaviridae, HBV, parvoviridae, poxviridae, papillomaviridae, Adenoviridae, Retroviridae, HIV-1, HIV-2, Paramyxoviridae, RSV, Parainfluenza virus, Human metapneumovirus, Bunyaviridae, Hantavirus, Rift Valley fever virus, Crimea-Congo hemorrhagic fever virus, Picornaviridae, Coxsackie virus, Rhinovirus, Flaviviridae, Yellow fever virus, Dengue virus, West Nile virus, Hepatitis C virus, Filoviridae, Ebola virus, Marburg virus, Orthomyxoviridae, Influenza virus 47, targeting members of the group consisting of Togaviridae, Western equine encephalitis virus, Coronaviridae, Reoviridae, Rhabdoviridae, Arenaviridae, Lassa fever virus and Calciviridae The oligonucleotide according to claim 1.   49. An oligonucleotide mixture comprising a mixture of at least two different antiviral oligonucleotides according to any one of claims 31-48.   49. An oligonucleotide mixture comprising a mixture of at least 10 different antiviral oligonucleotides according to any one of claims 31-48. 49. An antiviral pharmaceutical comprising a therapeutically effective amount of at least one pharmacologically acceptable antiviral oligonucleotide as defined in any one of claims 31 to 48; and a pharmaceutically acceptable carrier. Composition.   52. The composition of claim 51, formulated for administration to an acidic site in vivo.   53. A composition according to any one of claims 51 to 52, adapted for oral, vaginal or topical administration.   49. A kit comprising in a labeled package at least one antiviral oligonucleotide as defined in any one of claims 31 to 48, wherein the antiviral activity of the oligonucleotide is primarily sequence complementary. A kit produced by no mode of action, wherein the label on the package indicates that the antiviral oligonucleotide can be used against at least one virus.   55. The kit of claim 54, comprising a mixture of at least two different antiviral oligonucleotides.   An antiviral pharmaceutical composition comprising a therapeutically effective amount of at least one pharmacologically acceptable polypyrimidine oligonucleotide and a pharmaceutically acceptable carrier, wherein the antiviral activity of the oligonucleotide is primarily sequence independent An antiviral pharmaceutical composition resulting from a mode of action; and a pharmaceutically acceptable carrier.   57. The antiviral pharmaceutical composition of claim 56, wherein the oligonucleotide comprises a modified internucleotide linkage.   58. The composition of claim 56 or 57, formulated for administration to an acidic site in vivo.   59. A composition according to any one of claims 56 to 58, which is in powder form.   59. A composition according to any one of claims 56 to 58, adapted for oral, vaginal or topical administration.   59. A composition according to any one of claims 56 to 58, comprising at least one poly C oligonucleotide.   59. A composition according to any one of claims 56 to 58, comprising at least one poly T oligonucleotide.   49. Use of at least one pharmacologically acceptable oligonucleotide as defined in any one of claims 1-18 and 31-48 in the manufacture of a medicament for the prevention or treatment of a viral infection in a subject.   49. Use of at least one pharmacologically acceptable oligonucleotide as defined in any one of claims 1-18 and 31-48 for the prevention or treatment of a viral infection in a subject.   Use of a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide mixture as defined in claim 19, 20, 49 or 50 in the manufacture of a medicament for the prevention or treatment of a viral infection in a subject.   Use of a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide mixture as defined in claim 19, 20, 49 or 50 for the prevention or treatment of a viral infection in a subject.   A virus in a subject of a therapeutically effective amount of at least one pharmacologically acceptable antiviral pharmaceutical composition as defined in any one of claims 21, 24-30, 51-53 and 56-62. Use in the manufacture of a medicament for the prevention or treatment of infection.   A virus in a subject of a therapeutically effective amount of at least one pharmacologically acceptable antiviral pharmaceutical composition as defined in any one of claims 21, 24-30, 51-53 and 56-62. Use for prevention or treatment of infection.   49. A therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as defined in any one of claims 1-18 and 31-48 resulting from an oncovirus in a human or non-human animal. Use in the manufacture of a medicament for the prophylactic treatment of cancer.   49. A therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as defined in any one of claims 1-18 and 31-48 resulting from an oncovirus in a human or non-human animal. Use for preventive treatment of cancer.   A therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide mixture as defined in claim 19, 20, 49 or 50, for the prevention of cancer caused by oncovirus in a human or non-human animal. Use in the manufacture of a medicament for treatment.   A therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide mixture as defined in claim 19, 20, 49 or 50 for the prevention of cancer caused by oncoviruses in humans or non-human animals. Use for treatment.   A therapeutically effective amount of at least one pharmacologically acceptable antiviral pharmaceutical composition as defined in any one of claims 21, 24-30, 51-53, and 56-62, of human or Use in the manufacture of a medicament for the prophylactic treatment of cancer caused by an oncovirus in a non-human animal.   A therapeutically effective amount of at least one pharmacologically acceptable antiviral pharmaceutical composition as defined in any one of claims 21, 24-30, 51-53, and 56-62, of human or Use for the prophylactic treatment of cancers caused by oncoviruses in non-human animals.   A subject of a therapeutically effective amount of at least one pharmacologically acceptable antiviral pharmaceutical composition as defined in any one of claims 51 to 53 adapted for administration to an acidic site in vivo. In the manufacture of a medicament for the prevention or treatment of viral infections in an acidic environment.   A subject of a therapeutically effective amount of at least one pharmacologically acceptable antiviral pharmaceutical composition as defined in any one of claims 51 to 53, adapted for administration to an acidic site in vivo. Use for the prevention or treatment of viral infections in acidic environments.