JP2003517428A - Multi-target hybridization nucleic acids, their preparation, compositions, formulations, kits and applications - Google Patents

Abstract

  (57) [Summary] Disclosed are antisense oligonucleotides that bind to more than one RNA target.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to adenosine-free or low-content antisense oligonucleotides (MTA oligos) for multiple targets. The agent of the present invention is effective for the prevention and treatment of diseases and symptoms associated with inflammation and airway damage, and these diseases include lung diseases and diseases that adversely affect the lungs of patients as a secondary effect. Targets targeted by the agent of the present invention include specific genes, adjacent regions of genes existing in the genome, start codons, intron-exon boundary regions, and also non-coding regions and coding regions of RNA. This RNA is a protein that encodes a certain protein, particularly a protein that is involved in diseases having multiple mediators by affecting (decreasing or strengthening) various contributing pathways. RNA that encodes Examples of diseases include lung diseases such as allergies, asthma, respiratory disorders, chest pain, cystic fibrosis, and cancers such as leukemia and colon cancer.
The agent of the present invention can be easily administered prophylactically and therapeutically in combination with other therapies.
It can also be used as an alternative therapy to a therapy with apparently harmful side effects.

Description of the Background Art Respiratory disorders associated with various diseases and conditions are quite common in the general population, but are particularly prevalent in some racial groups, such as African Americans. To be In some cases, they are associated with inflammation that exacerbates lung symptoms. For example, asthma is one of the most common diseases in industrialized countries.
The disease accounts for approximately 1% of all medical costs in the United States. Asthma has been reported to increase significantly in both epidemics and mortality over the past decade and is expected to become the predominant lung occupational disease over the next decade. Increased asthma mortality in industrialized countries may be attributed to increased reliance on beta-agonists used to treat this disease, but the cause of asthma is still poor. I don't understand.

Antisense oligonucleotides have been subjected to various theoretical studies as pharmacological agents with potential utility against human diseases. However, their practical application in human disease models has been somewhat unrealistic. One of the key problems that has hindered the effective application of antisense oligonucleotides has been the difficulty in finding an appropriate route of administration for delivering them to the site of functional expression.
Numerous in vivo experiments have been conducted with direct administration of antisense oligonucleotides to specific areas of the brain, but such applications have had only limited clinical utility due to their invasive nature. .

[0004] Systemic administration of antisense oligonucleotides also has a number of problems, but in particular, aiming at a tissue affected by a disease is inevitable due to its nature. In contrast, the lung is an excellent target for direct administration of antisense oligonucleotides, providing a non-invasive, tissue-specific pathway. In addition,
Antisense agents intended for delivery to the lung have not yet been fully developed.

Adenosine may be one of the key mediators of various lung diseases including bronchial asthma. The finding that asthmatics respond well to aerosolized adenosine and show marked bronchoconstriction, whereas normal humans do not, suggests a potential role for adenosine. Using rabbits allergic to ticks,
The rabbit animal model of asthma, which is one of the human asthma models, also responded similarly and showed marked bronchoconstriction. On the other hand, non-asthmatic rabbits showed no reaction. More recent studies using this animal model have suggested that in adenosine-induced bronchoconstriction and asthma, bronchial hyper-reactivity may occur primarily through stimulation of adenosine receptors. Adenosine has also been found to cause various unfavorable effects, including death, when administered therapeutically to patients with other diseases and previously undiagnosed symptoms of hyperreactive airways.

[0006] There are a variety of drugs for the treatment of respiratory diseases and conditions, but they generally have their limitations. Theophylline, a key agent in the treatment of asthma, is a known adenosine receptor antagonist reported to eliminate adenosine-mediated bronchoconstriction in asthmatic rabbits. 8-Cyclopentyl-1,3-dipropylxanthine (DPCP), a selective adenosine A ₁ receptor antagonist
X) is also reported to inhibit adenosine-mediated bronchoconstriction and bronchial hyperreactivity in allergic rabbits. However, currently available adenosine A ₁ receptor-specific antagonists have limited therapeutic and prophylactic applications because of their toxicity. For example, theophylline is widely used in the treatment of asthma, but it is often highly toxic due to its narrow dose range, and DPCPX is too toxic for clinical use. Despite decades of exhaustive research, the fact that specific antagonists of the adenosine receptor still cannot be used clinically demonstrates that these drugs are generally toxic. .

Various theoretical considerations have been made because antisense oligonucleotides can be useful pharmacological agents for human diseases. However, because these drugs need to be administered in large amounts, there are few discoveries of practical and effective applications in actual models of human disease, and there is still a great distance to get there.
Another consideration in the pharmacological application of these molecules concerns their route of administration. Many in vivo applications have involved the direct administration of antisense oligonucleotides to limited areas of the brain. However, such applications have limited clinical utility due to their invasive nature.

Systemic administration of antisense oligonucleotides as pharmaceuticals also has some major problems, but presents considerable difficulties, especially in targeting diseased tissues. This is because, when administered intravenously or orally, antisense oligonucleotides are necessarily diluted in the circulatory system, making it very difficult to reach therapeutic doses in target tissues. The bioavailability of orally administered antisense oligonucleotides is very low, on the order of less than about 5%.

Although antisense oligonucleotides have been used therapeutically by many people, including the present inventor, the inventor has found in previous studies that various agents and conditions could be caused by direct administration of these agents to the lungs. Was successfully treated. In many cases, other researchers have had to face problems with the delivery of DNA molecules to their targets.
Thus, the issue of route of administration will be a very important issue in treating systemic diseases and conditions, as in the case of local diseases and conditions.

As described above, an effective method is necessary for carrying out antisense treatment. In particular, there is a need for methods that can very effectively treat diseases and conditions of complex nature. Examples of the latter include diseases and conditions in which a plurality of endogenous substances that cause disease symptoms are involved alone or in combination and have complicated pathways.

SUMMARY OF THE INVENTION The present invention relates to an agent, which comprises a target gene, a genomic gene flanking region, a start codon, an intron-exon boundary region, an mRNA coding region and, for example, a 5 ′ cap. A non-coding RNA segment of a portion, its terminal portion and 3'terminal portion, that is, a poly A segment portion, and a protein known to be involved in one or more diseases and conditions, and a combined state of diseases and conditions. Includes antisense oligonucleotides that hybridize to two or more mRNAs, ie, multiple target antisense oligos (termed MTA oligos), corresponding to the entire region of the RNA, including the encoding RNA. Such mRNAs include, for example, transcription factors, stimulators and activators, interleukins, interleukin receptors, chemokines, chemokine receptors, endogenously synthesized specific and nonspecific enzymes, immunoglobulins, antibody receptors, central nervous system. System (CNS) and peripheral and non-neuronal receptors, CNS and peripheral and non-neuronal peptide transmitters, adhesion molecules, defensins, growth factors, vasoactive peptides and receptors, and polypeptides such as binding proteins Might be coded for. Also,
It may encode a polypeptide corresponding to the oncogene. The agents of the present invention contain about 15% or less adenosine (A), but are often completely devoid of adenosine, and compositions include, for example, capsules or cartridges, various dosage forms, and delivery devices. Also presented in the form of a kit with instructions for use. The amount of adenosine in the antisense oligo of the present invention may be reduced by substitution with an adenosine-like binding substitute base such as a universal base. The kit may also include other therapeutic agents and components that make up the composition. The agent of the present invention may also be provided in a state operably linked to a vector and / or incorporated in a host cell.

The agents, compositions, and formulations of the present invention include at least one target gene,
When administered to a patient suffering from a disease or condition associated with the presence of mRNA corresponding to the adjacent region of the genome or protein, it can be used for the treatment of these diseases or conditions. Administration to a patient is carried out using an MTA oligo of the invention in an effective amount such that the patient exhibits reduced production or utilization of at least one target mRNA, or increased degradation. In a preferred embodiment, at least 2
It is preferable to administer an amount of the drug that can be effective by decreasing the synthesis or availability of the target mRNA of the kind, or by increasing the degradation. Typical diseases and symptoms in the present invention are associated with respiratory disorders and inflammations, and include lung diseases, lung discomfort, and symptoms that have a negative effect on the lungs of patients. Examples of diseases and conditions that may be treated preventatively, prophylactically, therapeutically using the present invention include bronchoconstriction, inflammation,
Allergic rhinitis, acute asthma, allergies, asthma, respiratory disorders, respiratory distress syndrome,
Chest pain, cystic fibrosis, pulmonary hypertension, pulmonary stenosis, emphysema, chronic obstructive pulmonary disease (
COPDE), further leukemia, lymphoma, cancer, such as colon cancer, breast cancer, lung cancer, pancreatic cancer,
Hepatocellular carcinoma, renal cancer, melanoma, metastatic liver cancer, etc., and also cancers that may metastasize to the lung, including breast cancer and prostate cancer, or any type of cancer that has metastasized. The agents of the invention are also suitable for administration before, during and during other treatments including radiation therapy, chemotherapy, antibody therapy, phototherapy and cancer therapy, and the formation of other surgical procedures.
Also, unlike this, using the agent of the present invention, preventatively, prophylactically, therapeutically, and together with other therapeutic methods, or for a condition for which no known therapeutic method exists, itself alone, or As an alternative to treatments with serious side effects, effective administration can also be performed.

The composition of the invention may be administered transdermally or by a systemic route. These routes are oral, intracavity, intranasal, anal, vaginal, transdermal, intradermal, buccal mucosa, intravenous, subcutaneous,
Intramuscular, intratumoral, intraglandular, and by inhalation, transarterial, transvascular, and from the ear, intracranial, intratendon, and organs, such as the liver and other organs. Some include. In addition, those which are transplanted to humans or other animals, for example, vertebrates including mammals and administered through the fundus of the eye are also included. The therapy of the present invention is prophylactic or therapeutic. In a preferred embodiment, the agent of the invention is administered directly to the bronchial system of a patient for direct uptake into the lung at a concentration that is effective to reduce or inhibit the effects of the targeted lung disease or condition. Is preferred.

The agent of the present invention is produced as follows: Two or more targets, for example, a gene, a genomic flanking region, an intron-exon boundary site, etc., and a total nucleotide sequence RNA including a non-coding region RNA, and at least RNAs encoding proteins known to be involved in one disease or condition are selected; RNAs selected from the group consisting of RNAs corresponding to genes or adjacent regions of the genome and RNAs encoding target proteins A first RNA having at least 60%, preferably 80%, more preferably 90%, even more preferably about 100% homology to at least one segment of the second RNA. One segment of RNA; and one or more antisense groups for the one or more RNA segments. Synthesize a oligonucleotide. The target RNA comprises a precursor segment, a segment of spliced mRNA and a segment of another RNA molecule, which includes a non-coding region and an overlapping segment spanning the coding region and 5'-, 3'-ends, And includes the code area. In a preferred form of the invention, more than one target may be present in the same molecule. For example, in the case of an mRNA encoding a protein with multiple subunits, each MTA oligo would be found in each RNA segment encoding a different protein subunit.

[0015]   The present invention also provides MTA oligos obtainable by the method of the invention.

Detailed Description of the Preferred Embodiments The present invention provides the perspective that antisense oligonucleotides may be used therapeutically in the treatment of diseases or conditions in which there are multiple affecting pathways. Achieved by the inventor's eagerness to stand and improve the inventor's own past discoveries and the discoveries of others. In the past, the present inventor has succeeded in weakening or strengthening the influence of a specific pathway by designing an antisense oligonucleotide that binds to a specific target involved in a disease or condition, and further improved it. I thought it was possible. In this way, the inventor has initiated research to implement a strategy for novel and non-obvious multiple targets, during which the innumerable obstacles, especially the exhaustive search and selection necessary to obtain the target , And also for genomic DNA, R
Whether NA or a protein that is specifically involved in disease, it has overcome obstacles to finding the location of the desired sequence. Thereafter, the present inventor will demonstrate the present invention by applying it to a particular disease or condition, present various preferred embodiments, and specifically design a multi-targeted antisense oligonucleotide (MTA oligo) base sequence. Came to.

The multi-targeted antisense (MTA) oligonucleotides of the invention have the ability to weaken the expression of one or more target mRNAs or to increase or decrease the activity of one or more pathways. For example, about 7 present in a target mRNA
, About 10, about 12, about 15, about 18, about 21 to about 28, about 30, about 35, about 40
The method of the present invention can be carried out by first identifying all possible des adenosine (desA) antisense sequences having a single nucleic acid length of about 45, about 50, about 60 or more. . This can be achieved by searching for a segment in the target base sequence that has few or no thymidine (T), which is a nucleotide complementary to adenosine with a length of 7 or more. This search usually finds about 10 to 30 desT segments, that is, segments originally lacking thymidine or low T content segments consisting of up to or about 15% T. This segment can be used to design antisense oligonucleotides of varying lengths against an average length mRNA, ie, a typical target mRNA consisting of about 1800 nucleotides in length, followed by specific targeting. For each, a sense base sequence corresponding to a strictly complementary desA antisense base sequence can be deduced. The sense base sequence thus obtained can then be used to search for base sequences of preferred secondary targets. Alternatively, one or more databases, eg G
ENBANK and the like can be used for searching for other secondary base sequences. Thus, one can "target" in several ways, including selecting specific targets involved in one or more related diseases. Alternatively, the primary target may be selected first, then the antisense oligonucleotides (desA oligonucleotides preferred), followed by the search for secondary, tertiary, and higher targets. A typical search identifies multiple homologous secondary targets of interest from either a list of preferred secondary targets or a list of databases. That is, the technique of the present invention finds a case where natural homology exists between primary, secondary, and higher base sequences, and the specific disease involved in the target molecule from which MTA is obtained or It seeks to find a legal treatment for the condition.

The technique of the present invention is based on the design of an antisense oligo targeting mRNA involved in a disease having a pathological event in the lung airway, and at the same time, the original activity and effect of the desired oligo are obtained. It is also based on oligo-modifications, while retaining, to reduce the appearance of unwanted side effects caused by adenosine released during oligo-degradation. In this way, we target genes so that one or more antisense oligonucleotides (abbreviated as "oligos") selectively bind to the corresponding mRNA, and then, if necessary, universal bases or Adenosine analogs that cannot activate the adenosine A ₁ , A _2b or A ₃ receptors are used to reduce adenosine content. Based on past experience in this field, the inventor
One or more adenosines present in an oligonucleotide designed by selecting RNA segments with thymidine (T) deficiency or low thymidine content other than specific gene “down regulation” or vice versa Enhances the effect of the administered drug by substituting thymidine with another nucleobase, which binds to thymidine and has a function limited to adenosine, but cannot activate the adenosine receptor in the lung, so-called universal base. I thought it should be possible. Given that adenosine (A) is the nucleobase complementary to thymidine (T), when one T appears in RNA, this antisense oligo will have an A at that same position. For consistency, all RNAs and oligos are referred to herein as single strands in the 5'to 3'direction when read from left to right. It should be noted that these complementary sequences are also dealt with throughout the present invention. Furthermore, all nucleic acids and amino acids are described according to the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission, or by the known three-letter code (for amino acids).

The method of the present invention, regardless of its cause, can be used to treat a disease associated with decreased airway function in a patient. The adenosine content of the antisense agent of the present invention is low so as to prevent adenosine release when the agent is degraded in vivo. Examples of diseases that can be treated by the method of the present invention include cystic fibrosis, asthma, pulmonary hypertension and bronchovascular contraction, chronic obstructive pulmonary disease (COPD), chronic bronchitis, respiratory distress syndrome, lung cancer,
Included are metastatic lung cancer, and other diseases of the respiratory tract including those of the inflammatory response.

[0020]   Adenosine A₁, A_2a, A_2bAnd A_ThreeReceptor, CCR3 (chemo
Cain receptor), bradykinin 2B, CAM (vascular cell adhesion molecule), and
Antisense oligos for eosinophil receptors are downregulated in these genes.
It was shown to be effective in the simulation. Relief of symptoms or breathing
Some of these reduce pain and / or inflammation during treatment, such as adenosine A ₁ , A_2a, A_2b, And A_Three"Down" for all or either of the receptors
By the action of "regulation" and by CCR3 (chemokine receptor),
Bradykinin 2B, VCAM (vascular cell adhesion molecule), and eosinophil receptor
Due to the action of "down regulation". These drugs are widely distributed throughout the body.
Directly into the respiratory system by aspiration or other means without being
Preferably. This allows for techniques, systemic or other
Administration of a very low dose of the drug of the present invention as compared to the doses via common routes
And are possible. And this inevitably results in a widespread distribution of the drug in the body.
The more undesirable side effects that result can be reduced. Combination with the agent of the present invention
It has been shown that the amount of receptor protein expressed in weave is reduced. This
Like these simply interact with their target, the receptor
Rather, rather a target protein with which other drugs may interact
Reduce the amount of. In this way, the agent of the present invention is highly toxic while having low toxicity.
It can show high medicinal effect.

The adenosine receptors mentioned above are merely examples of the inventor's powerful technology. In fact, the inventors can target multiple genes in the same way, reducing or down-regulating protein expression. For example, if the target disease or symptom is breathing disorder or hypopnea, bronchoconstriction, chronic bronchitis, pulmonary bronchoconstriction, and / or hypertension, chronic obstructive pulmonary disease (COPD), allergy, asthma, cystic fibrosis, If the method of the invention is involved in respiratory distress syndrome, a cancer of the lung whether primary or metastatic, the method of the invention is a list of various potential target mRNAs, for example, in Table 1 below. Applicable to the target shown.

[0022]

[Table 1]

The oligos of the invention first select a fragment of the target nucleic acid having at least 4 contiguous nucleic acids from the group consisting of G and C, then 15% containing about 15% from the selected fragments. A first oligonucleotide of 4-60 nucleotides in length with a GC content of up to 4 is obtained. This latter step consists of a second, 4-60 nucleotide long sequence that constitutes a sequence that is antisense to the selected fragment.
This can be achieved by obtaining the oligonucleotide of Note that in this case the second oligonucleotide has an adenosine content of up to 15%, including about 15%. If the selected fragment contains at least one thymidine base, the method of the invention may further comprise replacing the adenosine base in the corresponding nucleotide of the antisense fragment with a universal base selected from the following group: Is bound to thymidine base but has antagonistic activity, and adenosine A ₁ , A _2b ,
The group consisting of an aromatic heterocyclic base having an adenosine base agonist activity of 0.3 or less in the case of A ₃ receptor, and an aromatic heterocyclic base having no agonist activity in the case of an adenosine A _2a receptor. The analog heteroaromatic base is
O, halogens, NH ₂ , SH, SO, SO ₂ , SO ₃ , COOH and branched and condensed primary and secondary amino, and O, halogen, NH ₂ , primary, secondary and tertiary amines, SH, SO , SO ₂ , SO ₃ , cycloalkyl, heterocycloalkyl, and alkyl, which may be further substituted with heteroaryl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, Arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl,
It can be selected from all pyrimidines and purines optionally substituted with alkynylaryl, arylalkyl, arylalkenyl, arylalkynyl and arylcycloalkyl. Pyrimidines and purines may be substituted at all positions, as is well known in the art. But positions 1, 2
Those substituted with 3, 4, 7, and / or 8 are preferred. And more preferable pyrimidines and purines are theophylline, caffeine, diffiline, etophylline, acephyrin, piperazine, bamifilin, enprophylline, and xanthine, which have the following structural formulas.

[0024]

[Chemical 2] Wherein R ¹ and R ² independently represent H, alkyl, alkenyl or alkynyl, R ³ is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O- cycloalkyl, O- cycloalkenyl, O- cycloalkynyl, NH 2 _- alkylamino - Ke Toki sialic km carboxymethyl - aryl and mono and dialkylaminoalkyl -
N- alkylamino -SO ₂ - aryl.

In the absence of a segment with a preferred T content, or if the preferred segment contained T, the inventor has found that the degradation product of the oligonucleotide releases adenosine to the lung tissue environment, thereby causing patient distress. Target R as a means of preventing exacerbation and / or cancellation of the beneficial effects of administration
It was proposed to reduce the amount of adenosine in the antisense oligo corresponding to thymidine (T) present in NA to less than about 15%, or to eliminate A altogether.

By way of example, the NfκB transcription factor can be selected as the primary target to search for desthymidine (desT) segments. If many desT segments are found, the antisense segment can be inferred. And perhaps about 20 or more desA antisense sequences can be obtained. If possible, select from within the mRNA of this primary target all of these antisense sequences are desA antisense sequences and, for example, as shown in Table 1 above or Table 2 below, or such as in GENBANK. It can be used to initiate a search for homologous sequences within a preferred list of secondary targets that are present in such sequence databases. Approximately 20 original d found in Nfκ transcription factor
For each of the esA antisense sequences, about 10-30 homologous sequences (secondary, tertiary, etc. targets) can be found among the other members of the group shown in Table 1 above. In some cases, the homology found in the primary target by the search is not only in the secondary target (those with homology between the primary target and the sequence of another target mRNA) but also in the tertiary target. Targets (those with homology between the primary target and the sequences of three other target mRNAs) can be found as well. However, the latter case is rare. When this happens, the antisense oligo is said to have 100% homology. However, more typically, the sequences found contain nucleic acids that have no homology within the secondary, tertiary, or quaternary sequences. In many cases, this mismatch will be sufficient to result in reduced activity of the antisense oligonucleotide, or even inactivation of the target. In some cases, the presence of only one heterologous nucleotide may be sufficient to reduce the activity of the antisense oligo. When the resulting so-called "homologous" sequences have mismatches, up to about 40% mismatches are acceptable, preferably no more than about 30%, more preferably no more than 20%, more preferably no more than 10% Is more preferable. In some cases, higher percentage mismatches are acceptable, and such oligos have "heterologous nucleotides"
4 nucleotides A that have been “modified” or present in natural DNA
, G, C, T may be substituted with "universal" bases that may be capable of forming base pairs with similar or equivalent affinity for two or more.
This "correction" step then allows the antisense oligo to bind to primary, secondary, tertiary, quaternary, etc. sequences, albeit different from those naturally occurring (preferably Bind tightly as well), creating more novel sequences.

When the NfκB transcription factor is selected as a target, a low thymidine (T) fragment or des thymidine (desT) fragment present in the mRNA or DNA is searched, and a desT segment is selected from the mRNA or DNA, and then, From these, desA antisense is produced as a complementary strand. When numerous RNAdesT segments are found, the sequence of the antisense segment can be deduced. Typically, about 10-30 and even more desA antisense sequences may be obtained. These antisense sequences may contain some or all of the desA antisense oligos corresponding to the desT segment of the target mRNA, as shown in Table 1 above and Table 2 below. If this happens, the antisense oligos found are called 100% A-free. For each of the original desA antisense oligo sequences corresponding to the target gene, the NfκB transcription factor, typically a low thymidine amount of about 10-30 sequences (RNA) can be found in the target gene or RNA. According to the invention, the selected fragment sequences are secondary, or
Secondary or quaternary sequences also contain a small number of thymidine (RNA) nucleotides. In some cases, a high adenosine content may be sufficient to reduce or inactivate the antisense oligo's activity towards its target. In accordance with the present invention, these so-called "incomplete desA" sequences preferably have an adenosine content of less than about 15%, more preferably less than about 10%, even more preferably less than 5% and some less than about 2% adenosine. It only contains. In some cases higher adenosine contents are acceptable. Such oligonucleotides are still active, especially when "modified" or adenosine nucleotide 4
This is the case when they are replaced with "universal" bases that are capable of base pairing with similar or equal affinity to two or more of the two. A universal base is defined herein as a compound that has the ability to bind thymidine and more preferably has a substantially reduced or absent binding to the adenosine receptor, more commonly an adenosine analog. Alternatively, adenosine analogs that do not activate adenosine receptors, such as adenosine A ₁ , A _{2 b} and / or A ₃ receptors, more preferably A ₁ receptors, can be used. One example of a universal base is α-deoxyribofuranosyl- (5-nitroindole), the person skilled in the art knows how to choose another, and this “correction” step is not found in nature. Unlike those produced, the antisense oligos created create newer sequences that bind to the target RNA, and more preferably with similar affinity. An example of a universal base is 2-deoxyribosyl- (5-nitroindole), and another example of a universal base is 3-nitropyrrole-2'-deoxynucleoside, 5-
Nitroindole, 2-deoxyribosyl- (5-nitroindole), 2-deoxyribofuranosyl- (5-nitroindole), 2'-deoxyinosine,
2'-deoxynebulaline, 6H, 8H-3,4-dihydropyrimido [4,5-
c] oxazin-7-one or 2-amino-6-methoxyaminopurine. In addition to the ones mentioned above, 3-nitropyrrole 2'-deoxynucleoside 2-deoxyribofuranosyl- (5-nitroindole) is a universal base which has a slightly weaker hybridization affinity but may be capable of substituting any other base. 2'-deoxyinosine, 2'-deoxynebulaline (Glen Research, Stirling, VA). A more specific mismatch repair is a "P" nucleotide, 6H, 8H-3,4-dihydropyrinide [4,5-c] [1,2] oxazine-, which can base pair with guanine (G) or adenine (A). This can be done by the use of 7-ones and also by the use of 2-amino-6-methoxyaminopurine, a "K" nucleotide capable of base pairing with cytidine (C) or thymidine (T), among others. And also those already known in the art can be used. For example, Loakers, D .; , Brown, D.M.
M. , Nucl. Acids Res. 22: 4039-4043 (1994)
Ohtsuka, E .; Et al., J. Biol. Chem. 260 (5): 2605
-2608 (1985); Lin, P .; K. T. and Brown, D.M. M.
, Ncleic Acids Res. 20 (19): 5149-5152 (1
992); Nichols R .; Et al., Nature 369 (6480): 492.
-493 (1994); Rahman, M .; S. and Humayun, N .;
Z. , Mutation Research 377 (2): 263-8 (19).
97); Amosova, O .; Et al., Nucleic Acids Res. 25
(10): 1930-1934 (1997); Loakes D .; and Br
own, D.W. M. , Nucleic Acids Res. 22 (20): 40
39-4043 (1994). The entire description of universal bases and their preparation and use for nucleic acid binding is incorporated herein as a part of this specification.

When incomplete desT sequences are present in their natural target, it is usually sufficient to select these sequences and make substitutions of about 1-3 universal bases to achieve 100% "desA".
It is possible to obtain an antisense oligo. Thus, the method of the invention is
As for antisense oligonucleotides in which one or more adenosine nucleotides, ie about 1 to 3 or more adenosine nucleotides, can be "corrected" by substituting one "universal" base, the A content To provide antisense oligonucleotides to different targets that are low or absent. Those skilled in the art know which bases act as universal bases and can substitute A with these.

The inventive approach to the design of antisense oligonucleotides will also be applicable to other diseases or conditions, including a variety of other inflammatory diseases. For example, cystic fibrosis, chronic obstructive pulmonary disease, chronic bronchitis, pulmonary hypertension, cancer, such as breast cancer, colon cancer, respiratory distress syndrome, prostate cancer, pancreatic cancer, renal cancer, lymphoma, melanoma, hepatocellular carcinoma, etc. Cancers that metastasize to the lungs are examples.

In the present invention, “treatment” of asthma and other respiratory and inflammatory symptoms or diseases
Alternatively, “treating” refers to a treatment that reduces the symptoms of respiratory and inflammatory lung diseases or conditions exhibited by a patient undergoing such treatment, and “down-regulation” refers to the production of target intracellular protein. , Secretion, or induction of reduced utility (and thus reduced concentration).

The present invention is primarily concerned with the treatment of vertebrates, among other vertebrates:
Related to mammals such as humans, non-human apes, wild and domestic animals, marine and terrestrial animals, domestic pets, and zoo animals including, for example, cats, dogs, horses, pachyderms, whales. And it is more preferred to target human patients.
A particularly preferred application of the technology of the present invention relates to animal husbandry purposes, including all types of large and small animals to which veterinarians are concerned, including wildlife, marine animals, domestic animals, zoo animals. Etc. are included. The targeted genes and proteins are preferably mammalian, and the targeted sequences are preferably of the same species as the patient being treated. In many cases it is also useful to target different species,
In particular the target RNA or gene segment has greater than about 45% homology to the patient's sequence, preferably greater than about 85% homology, and more preferably greater than about 95%.
There is more than homology. A preferred group of agents of the invention is where they are composed of des-A antisense oligos. Another preferred group is incomplete desA
This is the case when it is composed of oligonucleotides. In this case, one or more adenosine bases are replaced with universal bases.

The term “antisense” oligonucleotide generally refers to small synthetic oligonucleotides that resemble single-stranded DNA, and in the present invention, the target messenger RN.
It has been applied to the inhibition of gene expression by A (mRNA) inhibition. Milliga
n, J. F. et al. J. Med. Chem. 36 (14), 1923-1
937 (1993). In addition, in this, the part relevant to this invention is incorporated here as what comprises a part of this specification. The agents of the invention include target genes such as adenosine A ₁ , A _2a , A _2b or A ₃ receptors, CCR3 (chemical receptor 320, also known as eotaxin receptor), VCAM (
Vascular cell adhesion molecule), eonofil receptor, bradykinin 2B receptor,
And it inhibits the gene expression of many others listed in Table 1 above. This is generally the target messenger RNA (as is known in the art).
mRNA) to the coding (sense) region of the antisense oligonucleotide. The exogenously administered agents of the present invention reduce mRNA levels and protein levels encoded by their target genes and / or cause changes in the growth characteristics or shape of cells so treated. Milligan et al. (1993); Helene, C .; and Toul
me, J. Biochim. Biophys. Acta 1049, 99-1
25 (1990); Cohen, J .; S. D. Ed., Oligonucleoti
des as Anti-sense Inhibitors of Gene
Expression, CRC Press, Boca Raton, FL (
1987). This relevant part is hereby incorporated as a part of this specification. As used herein, "antisense oligonucleotide"
Is generally a short sequence of synthetic nucleotides, which (1) will hybridize under suitable hybridization conditions to any segment of the mRNA encoding the target protein. And (2) during hybridization,
This causes reduced gene expression of the target protein.

“Des adenosine” (desA) and “des thymidine” (desT) refer to oligonucleotides that are essentially devoid of adenosine (desA) or thymidine (desT), respectively. In some cases the desT sequences are naturally occurring, while in others they are obtained by replacing the unwanted nucleotide (A) with another that does not have the unwanted activity. The present invention is generally accomplished by substituting one "universal base" for A, as is already known in the art.

The mRNA sequence of the target protein is derived from the sequence of the gene that expresses this protein. For example, the sequence of the human genome adenosine A ₁ receptor and the sequence of rat and human A ₃ receptors are known. In this regard, US Pat. No. 5,32
0,962; Zhou, F .; Et al., Proc. Nat'l Acad. Sci.
(USA) 89: 7432 (1992); Jacobson, M .; A. See UK Patent Application No. 9304582.1. The sequence of the adenosine receptor A _2b is also known (Salvatore, CA, Luneau, CJ.
Johnson, R .; G. and Jacobson, M .; , Genomic
s (1995)). These relevant parts are hereby incorporated by reference as part of the present specification. The sequences of many representative target genes are also known (G
(See enBank, NIH). If the gene sequence is not yet available, the sequence can be obtained by isolating the target segment using known techniques. Once the sequence of the gene of interest and its RNA and / or its protein is known, antisense oligonucleotides can be made according to the present invention as described above to reduce production of the target protein according to standard techniques.

In one aspect of the invention, the antisense oligonucleotide is a mRN.
It has a sequence that specifically binds to a part or segment of the A molecule. This mRNA encodes a protein associated with diseases or symptoms related to respiratory disorders, pneumonia, respiratory tract disorders, bronchitis and the like. One effect of the binding is to reduce or inhibit translation of the corresponding mRNA, and thus reduce the amount of its target protein available in the lungs of the patient.

In a preferred embodiment of the invention, the phosphodiester residue of the antisense oligonucleotide is modified or replaced. Chemical analogues of the oligonucleotides with modified or substituted phosphodiester residues are particularly preferred, these analogues include methylphosphonates, phosphotriesters, phosphorothioates, phosphorodithioates or phosphoramidates, These increase the in vivo stability of the oligonucleotide of interest. The naturally occurring phosphodiester bonds of oligonucleotides are sensitive to cellular nucleases and are partially degraded. However, many of the residues presented here are, on the contrary, resistant to nuclease degradation (Milligan et al. And Cohen JSD, supra).
． See). In another embodiment of the invention, the oligonucleotide can be protected from degradation by the addition of a "3'-end cap". This "3 'end cap"
Replaces the phosphodiester bond at the 3'end of the oligonucleotide with a nuclease resistant bond. Tidd, D.M. M. and Warenius, H .;
M. , Bc. J. Cancer 60: 343-350 (1989); Sha.
w, J. P. et al. Nucleic Acids Res. 19:74
7-750 (1991). These relevant portions are hereby incorporated as part of the present specification. Thus, the phosphoramidate, phosphorothioate, and methylphosphonate linkages can all achieve the purpose of the present invention by functioning properly. The more radical the modification of the phosphodiester backbone, the more stable the resulting agents, and in many cases, their RNA.
The affinity and membrane permeability are higher (see Milligan et al., Supra). As such, the number of residues modified or substituted will vary with the need, target, and route of administration. The number is then from 1 to the number of residues present, or any number between the two. Many methods are already known for replacing the entire backbone of phosphodiesters with new bonds (Millig, supra).
See the article by an et al.). Preferred backbone analog residues include phosphorothioate, methylphosphonate, phosphotriester, thioform acetal, phosphorodithioate, phosphoramidate, formacetal boranophosphate, 3'-thioformacetal, 5'-thioether, Carbonate, 5'-
N-carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, 2'-O-methyl, sulfoxide, sulfide, hydroxylamine, methylene (methylimino) (MMI) and methyleneoxy (methylimino) (MOMI) residues Is mentioned. Phosphorothioate and methylphosphonate modified oligonucleotides are particularly preferred as they can be obtained by automated oligonucleotide synthesis (see Milligan et al., Supra). Where appropriate, the agents of the invention can be administered in the form of pharmaceutically acceptable salts or as a mixture of antisense oligonucleotides and their salts. In yet another embodiment of the invention, a mixture of different antisense oligonucleotides or their pharmaceutically acceptable salts can be administered.

The agents of the invention have the ability to cause weakened expression of the target mRNA and / or activation or weakening of certain pathways. For example, the method of the present invention uses low thymidine (
T) all possible deoxynucleotide segments or adenine (A)
This can be done by identifying all possible low amounts of the deoxynucleotide segment. These deoxynucleotide segments consist of about 7 or more mononucleotides present in the target mRNA or gene, respectively, preferably those consisting of up to about 60 mononucleotides, and those consisting of about 10 to about 36 mononucleotides. More preferably, even more preferably those consisting of about 12 to about 21 mononucleotides. The search can be accomplished by searching for 7 or more mononucleotide segments in the target sequence having a low or lacking amount of thymidine complementary to adenosine (RNA) or a low amount of adenine (gene). Typical target mRN with average size of about 1800 nucleotides
In the case of A, in most cases, this type of search can usually yield about 10-30 sequences. And these sequences naturally lack adenosine or have antisense oligos of various lengths with adenosine levels of about 40% or less. A sequence having a high content of T or A can be modified by replacing one or more A with a universal base.

The nucleotide length of the agent of the present invention may be any length. For example, the length is from about 7 to about 60, preferably about 12 to about 45, more preferably up to about 30 nucleotides, and even more preferably up to about 21, but is not limited thereto. However, they can be of other lengths, depending on the particular target and mode of delivery. The agents of the invention can be used against any and all segments of the target RNA. One preferred group of this agent is directed to the mRNA region containing the junction of intron and exon. If the agent is for the intron / exon junction, it is close enough to the junction to either completely cover the junction or to block the exon splice that enters during the process of precursor mRNA to mature mRNA. It may be necessary to be one. That is, a proximity within about 2-10 nucleotides, preferably within about 3-5 nucleotides, of the intron / exon junction is required. Other preferred antisense oligonucleotides include those that overlap the protein synthesis initiation codon and those near the 5'and 3'ends of the coding region.

Thus, the multi-targeted antisense (MTA) oligonucleotide approach of the present invention can be used in a variety of ways, including other inflammatory diseases such as cystic fibrosis, chronic obstructive pulmonary disease, chronic bronchitis, etc. It can be applied to other types of diseases or conditions. Specific examples of other diseases or conditions to which this technology can be effectively applied include pulmonary hypertension and cancer.

Table 2 below shows a number of targets to which antisense (MTA) oligonucleotides for multiple targets can be effectively applied. However, others can also be targeted.

Table 2: Cancer Target Transformation Oncogenes Therapeutic Targets ras thymidylate synthase src thymidylate synthase myc dihydrofolate reductase bcl-2 thymidine kinase dioxycytidine kinase ribonucleotide reductase preferred targets for cancer treatment. Groups may include genes associated with different types of cancer, whether related to regulation, whether involved in the production of RNA and / or proteins, or related to malignancy. There are gene groups that are generally known to An example of this is the oncogene that causes canceration. For example, ras, src, myc and bc among others
1-2 is included as such an example, but is not limited thereto. Other targets include those targeted by current chemotherapeutic agents, including, but not limited to, various enzymes, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, ribonucleotide reductase,
Etc.

For example, in the case of cancer, traditional cancer treatment methods are unsolved in order to selectively kill cancer cells while preserving normal living cells due to the destructive effects of treatment such as chemotherapy and radiation therapy. Considering this, the technique of the present invention is very important for the treatment of diseases or conditions. The techniques of the present invention provide the ability to weaken or augment multiple pathways simultaneously. This approach offers considerable advantages for cancer treatment. Using this approach, it is possible to select combinations of multiple pathways containing primary, secondary and tertiary targets that are not normally co-expressed in normal cells. Thus, by administering the agent of the present invention to a patient, toxicity can be selectively increased in cancer cells. For example, in cancer cells, all three targets are expressed, while in normal cells, It expresses only one or two of its targets, so it is less affected by the drug, or in some cases even exempt.

Thus, the target gene, the adjacent region of the genome, the starting point of protein synthesis, the intron-exon boundary, etc., or the coding region of mRNA, non-coding RNA segment, 5 ′ end, 3 ′ end, that is, poly-A segment Known to be associated with the entire region of the precursor RNA, including, and oligos that target the juxtaposition between the non-coding and coding regions, and one or more diseases or conditions or a combination of these. R corresponding to these, such as RNA encoding proteins
Two or more mRNAs are selected from the group consisting of NA to form one antisense oligonucleotide, and the present invention provides an agent comprising this antisense oligonucleotide.

Agents administered in accordance with the present invention are preferably designed as antisense targeting genes and / or RNA that are originally associated with the species to be administered. For human therapy, these agents are preferably antisense to human genes or RNA. The agent of the present invention is a naturally occurring DN
A sequences and / or RNA sequences, oligos thereof which are one base shorter than the target sequence, preferably oligonucleotides which are antisense to these fragments of at least 7 bases in length. In addition, oligo is only about 0.02%
Greater than, more preferably greater than about 0.1%, even more preferably greater than about 1%, and even more preferably about 4% adenosine nucleotides, and the adenosine nucleotides up to about 30%, more preferably up to about 15%. More preferably, up to about 10%, and even more preferably up to about 5% of oligonucleotides, or oligonucleotides lacking adenosine at all are included as agents of the invention. Further, an oligonucleotide substituted with a so-called universal base, which is capable of binding one or more adenosine nucleotides to the thymidine nucleotide but not effectively activating adenosine receptor activity, is also included in the agent of the present invention. Examples of human sequences and fragments of the antisense oligonucleotides of the present invention include the following fragments and segments shorter than these fragments, sequences of the entire gene region or mRNA coding region, exons and intron-exon junctions. There are, but are not limited to, these. These are 7, 10, 15, 18-21, 24, respectively.
It is preferably up to 27, 30, n-1 nucleotides in length. Where n is the total number of nucleotides in the sequence. These fragments can be selected from any part of the longer oligo, for example starting in the middle, 5'end, 3'end or anywhere in the original sequence. Of particular importance are low adenosine content fragments. That is, about 30% or less fragments, preferably about 15% or less, more preferably about 10% or less, even more preferably about 5% or less, and most preferably by mere selection. A fragment lacking adenosine nucleotides obtained or obtained by substituting with universal bases according to the invention. As shown here, the agent of the present invention contains, as a most preferred group, a sequence or fragment in which one or more adenosines present in the sequence are replaced by a universal base (B). Similarly, as noted above, the sequence, fragments of that sequence, and adenosine (A) present in these segments.
All shorter fragments of B-containing fragments designed by substituting for B with are also included as agents of the invention. A partial list of sequences and fragments is shown below.

Some of the examples of antisense oligonucleotide sequence fragments target the start codon of their respective genes, and in some cases adenosine is replaced by a universal base adenosine analog designated “B”. Here, "B" lacks the ability to bind to the adenosine A ₁ and / or A ₃ receptors. In fact
Such substituted nucleotides serve as "spacers". Many of the examples shown below present many fragments that overlap with one such sequence and start codon, in which the number of nucleotides n is preferably about 7, about 10, about 12, About 15, about 18, about 21 to about 28, about 35, about 40, about 50
, About 60.

In one embodiment, at least one of the mRNAs targeted by the MTA oligos of the invention is, for example, a transcription factor, a stimulatory and activator, intracellular and extracellular receptors and general peptide transmitters, intergenes, inter alia. Leukins, interleukin receptors, chemokines, chemokine receptors, endogenously synthesized specific and non-specific enzymes, immunoglobulins, antibody receptors, central nervous system (CNS), peripheral nervous system, and non-neuronal receptors, central nervous system System (CNS),
Peripheral nervous system and non-neuronal peptide transmitters, adhesion molecules, defensins, growth factors, vasoactive peptides and receptors, and binding proteins, or their mRNAs are involved in oncogenes and other diseases or conditions It corresponds to the gene of.

Examples of target proteins include eotaxin, major basic protein, preproendothelin, eosinophil cationic protein, P-selectin, STAT4,
MIP-1α, MCP-2, MCP-3, MCP-4, STAT6, c-mas
, NF-IL-6, cyclophilin, PDG2, cyclosporin A binding protein, FK5 binding protein, fibronectin, LFA-1 (CD11a / CD
18), PECAM-1, C3bi, PSGL-1, CD-34, substance P, p150, 95, Mac-1 (CD11b / CD18), VLA-4, CD.
-18 / CD11a, CD11b / CD18, C5a, CCR1, CCR2, C
Examples include CR4, CCR5 and LTB-4. However, others can be target proteins as well.

In another embodiment, the mRNA targeted by the MTA oligos of the invention
At least one of the two encodes intracellular and extracellular receptors and peptide transmitters. For example, sympathomimetic receptor, parasympathomimetic receptor, GABA receptor, adenosine receptor, bradykinin receptor, insulin receptor, glucagon receptor, prostaglandin receptor, thyroid receptor, androgen receptor, anabolic receptor, female hormone receptor, luteinizing hormone There are receptors, co-agglutinin cascade related receptors, anterior pituitary receptors, anterior pituitary peptide transmitters and histamine receptors (HisR). But others are also conceivable.

The encoded sympathomimetic and parasympathomimetic receptors include, among others, the acetylcholinesterase receptor (AcChaseR).
), Acetylcholine receptor (AcChR), atropine receptor, muscarinic receptor, epinephrine receptor (EpiR), dopamine receptor (DOPAR), norepinephrine receptor (NEpiR). Encoded by that adenosine A1 receptor as a further example of receptor, adenosine A _{2 B} receptor, adenosine A ₃ receptors, endothelin receptors A, endothelin receptor B, IgE high affinity receptor, muscarinic acetylcholine receptors, substance P receptors, histamine receptors, CC
R-1CC chemokine receptor, CCR-2CC chemokine receptor, CC
R-3CC chemokine receptor (eotaxin receptor), interleukin-1β receptor (IL-1βR), interleukin-1 receptor (IL
-1R), interleukin-1β receptor (IL-1βR), interleukin-3 receptor (IL-3R), CCR-4CC chemokine receptor, cysteine leukotriene receptor, prostanoid receptor, GATA-3
Transcription factor receptor, interleukin-1 receptor (IL-1R), interleukin-4 receptor (IL-4R), interleukin-5 receptor (
IL-5R), interleukin-8 receptor (IL-8R), interleukin-9 receptor (IL-9R), interleukin-11 receptor (IL
-11R), bradykinin B2 receptor, sympathomimetic receptor, parasympathomimetic receptor, GABA receptor, adenosine receptor, bradykinin receptor, insulin receptor, glucagon receptor, prostaglandin receptor, thyroid receptor, androgen receptor, anabolic receptor , Female hormone receptor, luteinizing hormone receptor, coaggregin cascade-related receptor, anterior pituitary receptor, and histamine receptor (H
isR). Although not listed here, others are also conceivable.

Encoded enzymes suitable for developing the MTA oligos of the present invention include: Synthase, kinase, oxidase, phosphatase, reductase, polysaccharide, triglyceride, protein hydrolase, esterase, elastase, and above all, polysaccharide, triglyceride, lipid and protein synthase are preferable. Examples of target enzymes include tryptase, inducible dinitrogen pentoxide synthase, cyclooxygenase (Cox), MAP kinase, eosinophil peroxidase, β2-adrenergic receptor kinase, leukotriene c-.
4-synthase, 5-lipoxygenase, phosphodiesterase IV, metalloprotease, tryptase, CSBP / p38MAP kinase, neutrophil elastase, phospholipase A2, cyclooxygenase 2 (Cox2), fucosyltransferase, chymase, protein kinase C, thymidylate Synthetase,
Included are dihydrofolate reductase, thymidine kinase, deoxycytidine kinase and ribonucleotide reductase. However, enzymes associated with diseases or conditions are suitable targets for the present invention.

Coded factors suitable for application of the present invention include, among others, NfκB transcription factor, granulocyte macrophage colony stimulating factor (GM-CSF), AP-1 transcription factor, GATA-3 transcription factor, monosite. Activator, Neutrophil chemotactic factor, Granulocyte /
Macrophage colony stimulating factor (G-CSF), NFAT transcription factor, platelet activating factor, tumor necrosis factor α (TNFα) and basic fibroblast growth factor. Although not specifically mentioned, other factors are also within the scope of the invention.

Intracellular adhesion molecule 1 (ICAM-1) is an adhesion molecule suitable for use in the present invention.
2 (ICAM-2), 3 (ICAM-3), vascular cell adhesion molecule (VCAM)
, Endothelial leukocyte adhesion molecule 1 (ELAM-1), neutrophil adhesion receptor, madC
AM-1 etc. Other known and (currently) unknown factors can also be targeted here.

Among cytokines, lymphokines and chemokines, preferred are interleukin 1 (IL-1), interleukin 1β (IL-1β), interleukin 3 (IL-3), interleukin 4 (IL-4), Interleukin 5 (I
L-5), interleukin 8 (IL-8), interleukin 9 (IL-9)
, Interleukin 11 (IL-11), CCR-5CC chemokine and RA
NTES ( r egulated on a ctivation, n ormal T -cell e xpressed and s ecreted)
Is. However, if others are known to be involved in the particular disease or condition to be treated, and if their activity, such as inflammation, is known, they are also suitable targets. ing.

Examples of defensins for carrying out the present invention include defensins 1, defensins 2 and defensins 3. Α4β1 selectin for selectins,
There are α4β7 selectin, LFA-1 selectin, E-selectin, P-selectin and L-selectin. Not a comprehensive list, but ra for oncogenes
There are s, src, myc, and bcl-2. However, others are also suitable for use in the present invention.

Agents administered in accordance with the present invention are preferably designed as antisense targeting genes and / or RNAs that are originally associated with the species to be administered. For human therapy, these agents are preferably antisense to human genes or RNA. The agent of the present invention is a naturally occurring DN
A sequences and / or RNA sequences, oligos thereof which are one base shorter than the target sequence, preferably oligonucleotides which are antisense to these fragments of at least 7 bases in length. In addition, oligo is only about 0.02%
Greater than, more preferably greater than about 0.1%, even more preferably greater than about 1%, and even more preferably about 4% adenosine nucleotides, and the adenosine nucleotides up to about 30%, more preferably up to about 15%. More preferably, up to about 10%, and even more preferably up to about 5% of oligonucleotides, or oligonucleotides lacking adenosine at all are included as agents of the invention. Further, an oligonucleotide substituted with a so-called universal base, which is capable of binding one or more adenosine nucleotides to the thymidine nucleotide but not effectively activating adenosine receptor activity, is also included in the agent of the present invention. Examples of human sequences and fragments of the antisense oligonucleotides of the present invention include the following fragments and segments shorter than these fragments, sequences of the entire gene region or mRNA coding region, exons and intron-exon junctions. There are, but are not limited to, these. These are 7, 10, 15, 18-21, 24, respectively.
It is preferably up to 27, 30, n-1 nucleotides in length. Where n is the total number of nucleotides in the sequence. These fragments can be selected from any part of the longer oligo, for example starting in the middle, 5'end, 3'end or anywhere in the original sequence. Of particular importance are low adenosine content fragments. That is, about 30% or less fragments, preferably about 15% or less, more preferably about 10% or less, even more preferably about 5% or less, and most preferably by mere selection. A fragment lacking adenosine nucleotides obtained or obtained by substituting with universal bases according to the invention. As shown here, the agent of the present invention contains, as a most preferred group, a sequence or fragment in which one or more adenosines present in the sequence are replaced by a universal base (B). Similarly, as noted above, the sequence, fragments of that sequence, and adenosine (A) present in these segments.
All shorter fragments of B-containing fragments designed by substituting for B with are also included as agents of the invention. A partial list of sequences and fragments is shown below.

Some examples of antisense oligonucleotide sequence fragments target the start codon of the respective gene, in some cases adenosine is replaced by a universal base adenosine analog designated “B”. Here, "B" lacks the ability to bind to the adenosine A ₁ and / or A ₃ receptors. In fact
Such substituted nucleotides serve as "spacers". Many of the examples shown below present many fragments that overlap with one such sequence and start codon, in which the number of nucleotides n is preferably about 7, about 10, about 12, About 15, about 18, about 21 to about 28, about 35, about 40, about 50
, About 60.

[0057]

[Sequence list]

In a preferred embodiment, the bond between adjacent mononucleotides is a phosphodiester bond. In another preferred embodiment, at least one mononucleotide phosphodiester residue of the antisense oligonucleotide is replaced. Specifically, methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, It is substituted with sulfoxide, sulfide, hydroxylamine, 2'-O-methyl, methylene (methylimino), methyleneoxy (methylimino), phosphoramidate residues and combinations thereof. MTA oligos having one or more phosphodiester residues substituted with one or more other residues can generally be used for extended periods, given that these residues are more resistant to hydrolysis as compared to phosphodiester residues. Up to about 10%, about 30%, about 50%, about 75% of the phosphodiester residues may be substituted, but may be 100% substituted.

The multi-targeted antisense oligonucleotide (MTA oligo) according to the present invention is
It usually consists of about 7 or more mononucleotides, sometimes up to 60 or more. The number of mononucleotides is preferably about 10 to about 36, more preferably about 12 to about 21. However, other nucleotide lengths are also suitable depending on the length of the target polymer. Examples of MTA oligos according to the present invention are shown in Table 3 below. The table contains 94 sequences (SEQ ID NOs: 2317 to 2411).

[0060]

[Table 2]

[0061]

[Table 3] NFκB: nuclear factor κB ICAM: intracellular adhesion molecule VCAM: vascular cell adhesion molecule TNF: tumor necrosis factor PAF: platelet activating factor

In the most preferred embodiment of using the MTA oligos according to the invention in the lung, the MTA
Whether the oligo is antisense to the native desthymidine sequence or the sequence substituted with one or more universal bases in the present invention, the MTA
Oligos include desadenosine oligonucleotides. Nucleotide replacement method,
The synthesis of oligonucleotides having a specific base sequence and the bases used as universal bases are known in the art and are within the knowledge of the person skilled in the art and need not be further indicated here.

In a further aspect of the agent according to the invention, the MTA oligo is operatively linked to an agent or molecule which is encapsulated or taken up by living cells. As is known in the art, this improves the uptake of the agents according to the invention. M according to the present invention
Suitable agents and molecules for use with TA oligos include transferrin, asialoglycoprotein and streptavidin, but other agents or molecules may be used.

As described above, the agent according to the present invention is also provided as a pharmaceutical composition containing an antisense oligonucleotide. The antisense oligonucleotide prevents translation by being contained in an amount effective to reduce the expression of the target mRNA by passing through the cell membrane and specifically binding to the target mRNA in the cell. Another aspect. Such compositions are provided in a suitable pharmaceutically acceptable carrier such as sterile pyrogen-free saline. The agent according to the present invention may be prepared together with a hydrophobic carrier that can pass through cell membranes such as liposomes. These liposomes are carried in a pharmaceutically acceptable aqueous carrier. The oligonucleotide may be coupled with an agent that inactivates mRNA such as ribozyme. Such oligonucleotides can be used to target specific receptors, enzymes and / or other expression products.
Alternatively, it can be administered to a patient in need of treatment that inhibits activation of proteins and / or factors. The pharmaceutical formulation may include a chimeric molecule comprising an antisense oligonucleotide linked to a molecule known to be encapsulated in cells. These oligonucleotide conjugates are utilized in the cellular uptake pathway,
Increase intracellular concentration of oligonucleotide. The molecules used in this method include macromolecules such as transferrin, asialoglycoprotein (which binds to the oligonucleotide via polylysine) and streptavidin, among others.

In the present pharmaceutical preparation, the antisense compound may be contained in lipid particles such as liposomes and fine crystals or vesicles. The structure of the particles may be any suitable one, such as a single lamella or a multiple lamella. Inclusion of antisense oligonucleotides in the liposome is a preferred embodiment. Such particles or vesicles include N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-
Particularly preferred are positively charged lipids such as trimethylammonium ethyl sulphate, or "DOTAP", but others are also suitable. Methods for preparing this type of lipid granules are well known. For example, U.S. Pat. No. 4,880,635 to Yanov et al., 4906477 to Chrono et al., 4911928 to Warrach, 49 to Warrach.
17951, Allen et al. 492016 and Feetley et al. 4921.
No. 757, etc., but the entire relevant sections for all of them are hereby incorporated as part of this specification.

The composition according to the present invention can be administered by any means that delivers the agent of the present invention to the lungs. Although the agent may be administered to the lungs of a patient by any suitable means, it is preferably administered via the respiratory system as an inhalable formulation, which also includes agents that are inhaled or inhaled by the patient. More preferred is administration in the form of an aerosol containing particles. The inhalable particles may be in the form of gas, liquid or solid, and may also contain other therapeutic and pharmaceutical ingredients.

The particles according to the present invention are preferably particles having a particle size that can be inhaled, and particles that are small enough to reach the bronchi and alveoli via the mouth and larynx during inhalation.
Generally, particles of about 0.5-10 μm in size are inhalable, but particles of other sizes may be suitable. Inclusion of non-inhalable particles of much larger size in an inhalable formulation will tend to adhere to the throat but will be swallowed. Therefore, it is desirable to minimize the amount of non-respirable particles in the aerosol. To administer to the nose,
A particle size of 10-500 μm is preferred to ensure retention in the nasal cavity.

A liquid pharmaceutical composition of an agent according to the invention for producing an inhalable preparation such as an aerosol comprises an antisense oligo and a suitable excipient or carrier, eg sterile pyrogen-free water and / or It can be prepared in combination with other known pharmaceutically or veterinary acceptable carriers. Other therapeutic ingredients can be included along with other formulation ingredients known in the art.

The solid fine particle composition containing inhalable dry particles such as the finely granulating agent of the present invention is obtained by pulverizing a dry antisense compound with a mortar and pestle, and pulverizing the finely divided composition with a screen (for example, It can be prepared by crushing or separating large agglomerates of particles through 400 mesh). The solid particulate composition comprising the antisense compound can further include dispersants and other known agents, which facilitate the formation of mists or aerosols. The preferred dispersant is lactose, which can be blended with the antisense compound in any suitable ratio, about 1: 1 w / w, although other ratios are possible. Other medications and formulations may also be included.

The aerosol of liquid particles containing the agent according to the present invention can be produced by any appropriate means such as an insufflator or a nebulizer. See, for example, US Pat. No. 4,501,729. Nebulizers are commercially available devices that either accelerate a solution or suspension of the agent through a narrow Venturi orifice, usually by compressed gas of air or oxygen, or convert it to a pharmaceutical aerosol mist by ultrasonic agitation. Suitable formulations for use in insufflators and nebulizers contain from about 0.01 to about 40%, preferably less than 20% w / w of the agent of the invention in a liquid carrier, which is usually water or a dilute aqueous alcohol solution. This aqueous alcohol solution is preferably made isotonic with body fluids by adding sodium chloride or the like. Other carriers are also suitable. If the preparation is not prepared aseptically, add methyl benzoate etc. as a preservative. Other additives include antioxidants, flavoring agents, volatile oils, buffers and surfactants.

The pharmaceutical composition provided in the present invention includes a nucleic acid containing the above-mentioned antisense oligonucleotide and one or more surfactants. Suitable surfactants or surfactant components for improving the uptake of the antisense oligonucleotides of the invention include synthetic and natural, complete and truncated Surfactant Protein A,
Surfactant protein B, surfactant protein C, surfactant protein D and surfactant protein E; disaturated phosphatidylcholine (other than dipalmitoyl), dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol,
Phosphatidylethanolamine and phosphatidylserine; phosphatidic acid, ubiquinone, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichol, sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycerol-3 -Phosphocholine,
Included are dihydroxyacetone palmitate, cytidine diphosphate (CDP) diacyl glycerol, CDP choline, choline and choline phosphate.
Further, surfactant components, namely, omega-3-fatty acid, polyenic acid, polyenoic acid, lecithin, palmitic acid, nonionic ethylene oxide / propylene oxide block copolymer, monomeric and polymeric polyoxypropylene, monomeric and polymeric poly Monomeric and polymeric polyvinylamines with oxyethylene, dextran and / or alkanoyl side chains, Brij35, Trito
nX-100 and synthetic surfactant ALEC, Exosurf,
Mention may be made of natural and synthetic lamellae, which are the natural carrier vehicles of Survan and Atvaquone. These surfactants may be used alone in the formulation or as part of a multi-component surfactant. Furthermore, it may be covalently bound to the 5'and / or 3'ends of the antisense oligonucleotide (oligo).

The compositions of the present invention can be administered by any means that delivers antisense nucleotides and detergents to the lung. The antisense compounds disclosed herein can be administered to the lungs of a patient by any suitable means, but preferably by inhalation of an aerosol consisting of inhalable particles containing the antisense compound. The inhalable particles may be in liquid or solid form and may additionally contain other therapeutic or diagnostic ingredients and other typical ingredients for the preparation of particular formulations. Examples of other agents include analgesics, specifically, acetaminophen, anireldine, aspirin, buprenorphine, butavitar, butorphanol, choline salicylate,
Codeine, dezocaine, diclofenac, diflunizar, dihydrocodeine, elcatoninine, etodolac, fenoprofen, hydrocodone, hydromorphone, ibuprofen, ketoprofen, ketorolac, levorphanol, magnesium salicylate, meclofenamate, mefenamic acid, meperidine, methadone, methadone, methodone Prazine, morphine, nalbuphine, naproxen,
Examples include opium, oxycodone, oxymorphone, pentazocaine, phenobarbital, propoxyphene, salsalate, sodium salicylate and tramadol, in addition to narcotic analgesics. See the Mosby Surgeon GenRx for this. Anti-anxiety agents are also effective and include alprazolam, bromazepam, buspirone, chlordiazepoxide, chlormezanone, chlorazepart, diazepam, halazepam, hydroxyzine, ketazozolam, lorazepam, meprobamate, oxazepam and prazepam. Anxiolytics associated with psychotic depression include chlorodiazepoxide, amitriptyline, loxapine, maprotiline and perphenazine. Non-rheumatic anti-inflammatory agents include aspirin, choline salicylate, diclofenac, diflunizar, etodolac, fenoprofen, floctafenin, flurbiprofen, ibuprofen, indomethacin, ketoprofen, magnesium salicylate, meclofenamate, mefenamic acid. , Nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, salsalate,
Mention may be made of sodium salicylate, sulindac, tenoxicam, thiaprofenic acid and tolmethine. Anti-inflammatory agents for ocular treatment include diclofenac, flurbiprofen, indomethacin, ketorolac and rimexolone (usually postoperative treatment). Non-infectious nasal anti-inflammatory agents include beclomethaxone,
Budesonide, dexamethasone, flunisolide and triamcinolone are included. Hypnotics (anti-insomnia / sleep inducers) are used to treat insomnia,
Alprazolam, bromazepam, diazepam, diphenhydramine, doxylamine, estazolam, flurazepam, harazepam, ketazolam, lorazepam, nitrazepam, prazepam, quazepam, temazepam, triazolam, zolpidem and sopiclone. Sedatives include diphenhydramine, hydroxyzine, methotrimeprazine, promethazine, propofol, melatonin and trimeprazine. Among other conditions, sedatives and agents used to treat minor seizures and tremors include amitriptyline HCl, chlordiazepoxide, amobarbital, secobarbital, aprobarbital, butabarbital, esquiovinol, glutethimide, L-tryptophan. , Mefobarbital, Methohexital Na, Midazolam Hcl, Oxazepam, Pentobarbital Na, Phenobarbital, Secobarbital Na, Thiamylal Na and many others. Agents used to treat head injury (brain injury / ischemia) include:
Enodrine HCl, a cytoprotective agent (eg, for the treatment of severe head trauma, orphan status, Warner Lambert). Examples of therapeutic agents for menopausal and menopausal syndrome (treatment) include ergotamine, belladonna alkaloids and phenobarbital. The therapeutic agents for menopausal vasomotor syndrome include clonidine, conjugated estrogen and medroxyprogesterone, estradiol, estradiol cypionate, estradiol valerate, estrogen, conjugated estrogen, esterified estrone, estropipeto and ethinyl estradiol. Therapeutic agents of the premenstrual syndrome (PMS), progesterone, progestin, gonadotropin releasing hormone, oral contraceptives, danazol, Le Pro Lido acetate and vitamin B ₆ and the like. Emotional and psychiatric therapeutic agents such as tricyclic antidepressants include amitriptyline HCl (eravir), amitriptyline HCl, perphenazine (triaville) and dozepine HCl (cinekine). As tranquilizers, antidepressants and anxiolytics, diazepam (valium), lorazepam (
Ativan), alprazolam (Xanax), SSRI's (selective serotonin reuptake inhibitor), fluoxetine HCl (Prozac), sertaline HCl
(Zoloft), paroxetine HCl (Paxil), fluvoxamine maleate (
Lvox), venlafaxine HCl (effector), serotonin, serotonin agonists (fenfluramine) and other over-the-counter (OTC) drugs.

The composition according to the invention is for example formulated as a formulation comprising inhalable particles, such as particles of a size small enough to reach the bronchi and alveoli via the nose, mouth and larynx during inhalation, Can be administered to the respiratory system. Generally, the inhalable particle size is about 0.5 to 1
It is 0 μm. The amount of non-inhalable particles in the aerosol is minimized because the non-inhalable particles contained in the aerosol adhere to the throat and are swallowed. For nasal administration, particle sizes of 10-500 μm are preferred to ensure retention in the nasal cavity.

The aerosol or mist of solid particles containing the agent according to the present invention can be similarly produced using any device that generates an aerosol or mist of solid fine particle drugs. Aerosol or mist generators are suitable for administering solid particulate medicaments. As explained above, these devices produce inhalable particles and generate a volume of aerosol or mist containing a given metered dose of drug at a rate suitable for human and animal administration. . One example of a solid particulate aerosol generator is an insufflator. Formulations suitable for administration by insufflation include finely divided powders that are delivered by the insufflator and that can be absorbed by the nasal cavity in a nasal manner. In this insufflator, the powder is contained in a capsule or cartridge, eg, a measured dose of the agent effective to effect the treatments described herein. These capsules or cartridges are usually made of gelatin or plastic and can be punctured or opened in place. The powder is delivered by air passing through the device during inhalation or by a hand pump. The powder used in the insufflator may be the agent alone or a suitable powder diluent such as lactose and a powder blend of the agent with any surfactant. This agent is usually contained in an amount of 0.01 to 100% per formulation. A second example of an aerosol generator is a metered dose inhaler. A metered dose inhaler is a pressurized aerosol dispenser and usually contains a suspension or solution formulation of the active ingredient in a liquefied propellant. When using these devices, a fine particle spray containing the active ingredient is produced by discharging the formulation through a valve suitable for delivering a fixed volume, which is usually about 10 to 150 μl, but may not. . Suitable propellants include solvents such as certain chlorofluorocarbon compounds such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and / or mixtures thereof. The formulation may further include one or more cosolvents such as ethanol, surfactants such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents. The present aerosol can be produced by an aerosol generator regardless of whether it is produced from solid particles or liquid particles, but the production rate is about 10 to 150 l /
Min, more preferably about 30-150 l / min, most preferably about 60 l / min. Aerosols containing larger amounts of drug can be administered more quickly.

As already indicated, the agent according to the present invention is also provided as a composition containing the agent and a carrier. The carrier is preferably a biologically acceptable carrier, more preferably a pharmaceutically or veterinary acceptable carrier in the form of gas, liquid, solid and mixtures thereof. These are appropriate when they differ from the intended route of administration. The composition may further comprise other agents such as other therapeutic compounds known in the art for the treatment of symptoms or diseases. Further, the composition comprises an antioxidant, a flavoring and coloring agent, a filler, a volatile oil, a buffering agent, a dispersing agent, a surfactant, an RNA inactivating agent, an antioxidant, a flavoring agent, a propellant and a preservative. It may also contain other agents known to be used in therapeutic compositions. Enzymes such as ribozymes are an example of mRNA inactivating agents.

Generally, the composition comprises about 0.0% antisense oligonucleotide per composition.
1 to about 99.99% w / w, preferably about 1 to about 40% w / w, more preferably about 5 to about 20% w / w. However, within the scope of the present invention, it is appropriate to include other components in other ranges.

The agent of the present invention is also provided as various preparations adapted to various administration methods and delivery routes. For example, transdermal formulations also include carriers suitable for delivery via the skin, mouth, nose, vagina, anus, eyes, ears, and other cavities, as well as other agents. or,
As the intradermal preparation, suppositories, creams, sustained-release preparations such as gels are considered, and these are intracranial, subarachnoid, intravascular, intrapulmonary organs by inhalation, and subcutaneous implantation as known in the art. Is administered. In certain formulations, the drug is suspended or dissolved in a solvent. In other formulations, the carrier is a hydrophobic carrier such as liposomes or lipid particles containing fine crystals or vesicles. Methods of preparing all of these formulations and the components used are known in the art and need not be further described herein. In a particularly preferred embodiment of the vesicle formulation, the vesicles consist of liposomes containing antisense oligonucleotides. Lipid vesicles are N- (1- [2,3-dioleoyloxy] propyl) -N, N, N-
Includes trimethylammonium methylsulfate and other lipids known in the art to provide proper delivery of DNA to target cells. In one aspect, the formulation comprises an inhalable formulation such as an aerosol. The agents, compositions and formulations of the present invention are provided in bulk and in unit form and also in the form of implants, solutions or suspensions, capsules or cartridges. The capsule or cartridge can be opened or pierced as is known in the art.

Further provided is a kit. It contains the delivery device and contains, in a separate container, instructions for the agent, composition or formulation according to the invention, as well as any other agents and kit components. In a preferred embodiment, the delivery device comprises a nebulizer that delivers a single or multiple doses of the agent. Nebulizers that deliver a single, fixed dose can also be provided as a single-use, disposable kit pre-loaded with the required agents in a single dose. The nebulizer may be an insufflator and the composition may be provided in a pierceable or unsealable capsule or cartridge. In another aspect, the delivery device comprises a pressurized inhaler,
The agent is in the form of a suspension or solution. This kit, in a separate container, among the additives suitable for various formulations, other therapeutic agent compounds, antioxidants, flavoring and coloring agents, fillers,
It further comprises an agent selected from the group consisting of volatile oils, buffers, dispersants, surfactants, intracellular entrapment or uptake agents, RNA inactivating agents, antioxidants, flavoring agents, propellants and preservatives. You can When adding a solvent for the present agent or other components, not only an aqueous solvent but also an organic solvent and a mixture of an organic solvent and one or more co-solvents can be used, as is known in the art.

The agent according to the present invention can be provided together with a vector for the purpose of delivery or for producing a copy thereof. The agent may be operably linked to the vector as is known in the art. The agent may be provided in host cells for MTA oligo amplification and storage.

The agent according to the present invention comprises one or more target genes; a genomic flanking region, a start codon, an intron-exon border region, etc .; or a 5′-end and 3 ′ of a poly-A segment and the like.
Corresponding to the entire base sequence of the precursor mRNA containing the terminal non-coding RNA segment and the parallel section between the coding region and the non-coding region;
It can be used alone or in the form of a composition or various preparations for treating a disease or condition corresponding to the RNA region encoding RNA and protein. The present agent is used by administering to a patient suffering from a disease or symptom in an amount effective such that the patient shows a decrease in production and availability of one or more target mRNAs or an increase in degradation. Generally, the agent is administered in an amount effective to reduce the production and availability of two or more target mRNAs or increase the degradation. The agent may be administered directly to the lungs of a patient, preferably as an inhalable aerosol. Those of skill in the art will know how to determine the dose of an agent per body weight of a patient to be treated according to the teachings of the present invention, as the agent is a multi-targeted antisense oligonucleotide Preferably, an effective amount is added so that the concentration is preferably about 0.05 to about 10 μM, more preferably about 5 μM.

The treatments provided in the present invention are suitable for treating many diseases and conditions, the application of which is limited only by the target molecules and their nucleotide sequences. One of the diseases and conditions to which the present technology is particularly effectively applied is lung diseases and conditions. For this type of application, one or more target mRNAs encode proteins such as adenosine A ₁ receptor, adenosine A ₂ B receptor, adenosine A ₃ receptor and bradykinin B ₂ receptor, although other targets are suitable. In some cases, the diseases and conditions are related to disorders of the respiratory tract of the patient, and in other cases, asthma and the like. The above target proteins are also suitable, but a target protein containing an Nf _K B transcription factor is a preferred example. Another preferred application is where the disease and condition is associated with inflammation. In this case, preferably one or more target mRNA encodes a protein selected from the group consisting of interleukins, chemokines, Rantes and their receptors. Yet another application is where the diseases and conditions are associated with allergies. In this case, preferably mRAN encodes a target selected from the group consisting of antibodies and antibody receptors. In applying the technology when the disease and condition is associated with malignancy or cancer, preferably the mRNA encodes a target selected from the group consisting of oncogenes, immunoglobulins and antibody receptors.

Depending on the target organ and tissue, the agents of the present invention can be delivered by any of a number of routes. For example, transdermal or systemic routes, more specifically oral, intracavity, intranasal, anal, vaginal, transdermal, buccal mucosa, intravenous, subcutaneous, intramuscular, intratumoral, intraglandular, transplant, dermal It can be delivered by internal and many routes of administration. Furthermore, this formulation
It may be an implant, sustained release, transdermal release, sustained release, and may be coated with one or more polymers to avoid disintegration of the agent prior to reaching the selected target. Patients which may be treated with the agent of the present invention may be generally humans and other animals, but vertebrates are particularly preferable. Among these mammals, more specifically, humans and large and small-sized wild and domesticated marine animals and domestic animals are mentioned, and humans and domesticated animals and domestic animals are preferable. In one aspect of the invention, at least one of the target mRNAs is of the same species as the patient and is preferably of human origin. However, in one aspect, mismatched nucleotides are replaced, so mismatched species can also be used.

The multi-targeted antisense oligonucleotides of the present invention can be administered in a wide dosage range. The preferred dose of this drug is about 0.005 to about 150 mg / kg of body weight, which is a single dose for acute treatment and several times a day for continuous treatment. The level of can be maintained. A preferred dose is about 0.01 to about 75 mg / kg body weight, more preferably about 1 to 50 mg / kg body weight. In this method,
It can be administered as a prophylactic or therapeutic method.

The agent according to the present invention can be manufactured as follows. That is, selecting two or more targets selected from the group consisting of genes, genomic flanking regions, mRNA and proteins known to be associated with one or more diseases or conditions; genes, genomic flanking regions, initiation codons, Targeting the entire base sequence of RNA including 5'-terminal and 3'-terminal non-coding RNA segments such as intron-exon boundary region or poly-A segment and parallel sections between coding region and non-coding region An RNA selected from the group consisting of an RNA corresponding to an RNA segment encoding a target protein; and a segment of a first RNA having at least 60% homology with a segment having at least a segment of the second RNA And combine one or more antisense oligonucleotides with one or more RNA segments. To. In a preferred embodiment, the method further comprises replacing one or more, and optionally all, of the heterologous nucleotides in the antisense oligonucleotide. In another preferred embodiment, the method further comprises substituting methylated cytosine for cytosine of one or more CpG dinucleotides present in the antisense oligonucleotide. Techniques for methylation are known in the art and need not be described further herein.

Although the specific length of the MTA oligo is determined by the length of the target and the segment contains a small number of thymidines, the antisense oligonucleotide is preferably greater than about 7 nucleotides in length and greater than about 60 nucleotides in length. The specific background chemistry can be selected by one of ordinary skill in the art based on the teachings herein and knowledge of the art in general. One factor that influences the choice of nucleotide cross-linking residues is the level of nuclease resistance desired and the particular mode of administration of each. Another factor is the need for localized treatment, which is to minimize or completely avoid side effects that may affect the therapeutic efficacy of the agent.

The present invention will be described with reference to the following examples, but the examples do not limit the present invention. In the examples, μM is micromol, ml is milliliter, μm is micrometer, mm is millimeter, cm is centimeter, ℃ is Celsius degree, μg is microgram, mg is milligram, g is gram, kg is kilogram,
M represents mol and h represents time.

[0087]

EXAMPLES Example 1 Design and Synthesis of Antisense Oligonucleotides For the design of antisense oligonucleotides for the A ₁ and A ₃ adenosine receptors, a composite secondary structure of target A ₁ receptor mRNA and target A ₃ receptor mRNA was used. Solution is required. After forming this structure, antisense nucleotides are designed for the target region of the mRNA that is thought to confer functional activity or stability on the mRNA and for the target region of the mRNA that optimally overlaps the start codon. Other targeting moieties are readily available. To demonstrate the specificity of the antisense effect, as a control in antisense experiments, an oligonucleotide is used that is not perfectly complementary to the target mRNA, but has the same nucleotide composition on a weight ratio basis.

The adenosine A ₁ receptor mRNA secondary structure was analyzed and used as described above,
A phosphorothioate antisense oligonucleotide was designed. The synthesized antisense oligonucleotide (designated HAdA ₁ AS) had the following sequence. 5'-GAT GGA GGG CGG CAT GGC GGG-3 '(SEQ ID NO: 1) As a control, a mismatch phosphorothioate antisense nucleotide (referred to as HAdA1MM1) having the following sequence was synthesized. 5'-GTA GCA GGC CGG GAT GGG GGC-3 '(SEQ ID NO: 2) The respective oligonucleotides had the same base content and substantially the same sequence structure. GENBANK (release 85.0) and EMBL (release 40.0)
A homology search in showed that this antisense oligonucleotide was specific for the human and rabbit adenosine A ₁ receptor genes and that the mismatch control was not a candidate for hybridization with known gene sequences. . The secondary structure of the adenosine A ₃ receptor mRNA was also analyzed in the same manner, were designed two phosphorothioate antisense oligonucleotides using as described above.
The first synthetic antisense oligonucleotide (HAdA3AS1) had the following sequence: 5'-GTT GTT GGG CAT CTT GCC-3 '(SEQ ID NO: 3) As a control, a mismatch phosphorothioate antisense oligonucleotide (HAdA3MM1) having the following sequence was synthesized. 5'-GTA CTT GCG GAT CTA GGC-3 '(SEQ ID NO: 4) A second phosphorothioate antisense oligonucleotide (HAdA3AS2) having the following sequence was synthesized. 5'-GTG GGC CTA GCT CTC GCC-3 '(SEQ ID NO: 5) This second oligonucleotide control (HAdA3MM2) had the following sequence: 5'-GTC GGG GTA CCT GTC GGC-3' (sequence No .: 6) These phosphorothioate oligonucleotides were synthesized using an oligonucleotide synthesizer (Applied Biosystems Model 396) and purified using NENSORB chromatography (Dupont, MD).

Example 2 In Vivo Testing of Adenosine A ₁ Receptor Antisense Oligonucleotides In an in vitro model using lung adenocarcinoma cells HTB-54, antisense oligonucleotides (SEQ ID NO: 1) against the human A ₁ receptor described above were prepared. Tested for efficacy. HTB-54 lung adenocarcinoma cells were shown to express A ₁ adenosine receptors using standard Northern blotting and receptor probes designed and synthesized in the laboratory.

HTB-54 human lung adenocarcinoma cells (106/100 mm tissue culture dish) at 5.0 μM.
Were exposed to HAdA1AS or HAdA1MM1 for 24 hours. At that time, the medium and the oligonucleotide were newly exchanged after 12 hours of incubation. Twenty-four hours after exposure to oligonucleotides, cells were harvested and their RNA was extracted by standard methods. A 21-mer probe corresponding to the region of the mRNA targeted by antisense (ie, having the same sequence as antisense but not phosphorothioated) was synthesized, HAdA1AS-treated HTB-54 cells, HAdA1MM1-treated HT.
It was used as a probe for Northern blots of RNA prepared from B-54 cells and untreated HTB-54 cells. This blot showed that HAdA1AS effectively reduced human adenosine receptor mRNA by more than 50%, whereas HAdA1MM1 had no such effect. From this result, since HAdA1AS depletes intracellular mRNA of adenosine A ₁ receptor involved in asthma,
HAdA1AS has been shown to be a good candidate for anti-asthma drugs.

Example 3 In Vivo Efficacy of Adenosine A ₁ Receptor Antisense Oligos A coincidental homology was found between the rabbit and human DNA sequences within the adenosine A ₁ gene overlapping the initiation codon. Thus, the phosphorothioate antisense oligonucleotides originally designed for use on the human adenosine A ₁ receptor could be used in the rabbit model.

New Zealand white pasteurella-free rabbits were mixed with 10% kaolin within 24 hours after birth and housed in a house dust mite (hous) of 312 antigen units / ml.
e dustmite) (D. farinae) extract (Berkeley Biolog)
icals, Berkeley, Calif.) was injected intraperitoneally to immunize. Immunization was repeated every 1 week for the first month and then every 2 weeks for the next 2 months. At the age of 3-4 months, ketamine hydrochloride (44
(mg / kg) and acepromazine maleate (0.4 mg / kg) were administered intramuscularly, anesthetized and relaxed.

The rabbit is then placed in a supine position in a comfortable position on an animal board containing a small molding pad and a 4.0 mm endotracheal tube (Mallinkrodt,
Inc. , Glens Falls, NY). A 2.4 mm outer diameter polyethylene catheter fitted with a latex balloon was passed through the esophagus and kept a constant distance from the mouth (about 16 cm) during the experimental period. F heated endotracheal tube
Attached to a leisch lung tachograph (size 00; DOM Medical, Richmond, VA), the flow rate was determined by a Gould carrier amplifier (Model).
11-4113; Valid Electronic Differential Pressure Transducer (Model DP-, driven by Gould Electronic, Cleveland, Ohio).
45161927; Validyne Engineering Corp. ,
(Northridge, Calif.). The esophageal balloon was attached to one side of the differential pressure transducer, and the outflow of the endotracheal tube was connected to the opposite side of the differential pressure transducer to record transpulmonary pressure. The flow rate was integrated to obtain a continuous respiratory volume. Also, total lung resistance (RL) and dynamic compliance (Cdyn)
Is an automatic breath analyzer (Model 6; Buxco, Sharon, CT).
Were calculated at the equal dose point and zero flow point, respectively.

Rabbits were randomly selected and on day one, P to aerosolized adenosine was used.
The value of C50 before treatment was obtained. Antisense (HAdA1AS) or mismatch control (HAdA1MM) oligonucleotides were dissolved in sterile saline to prepare solution 1.
The concentration was adjusted to 5000 μg (5 mg) per 0 ml. Rabbits were then dosed with aerosolized antisense or mismatch oligonucleotides through the endotracheal tube (approximately 5000 μg in 1.0 ml) twice daily for 4 days. Aerosols of saline, adenosine, or antisense or mismatch oligonucleotides were generated by an ultrasonic nebulizer (DeVilbiss, Somerset, PA). 80% of the aerosol particles had a diameter of less than 5 μm.

In the first arm of the experiment, four randomly selected allergic rabbits received antisense oligonucleotides and the remaining four received mismatch control oligonucleotides. PC50 values (aerosolized adenosine concentration required to reduce dynamic compliance of the bronchial airways by 50% below baseline values, in mg / ml) on the morning of day 3 before exposure to oligonucleotides In comparison with the PC50 values obtained from these rabbits.

These rabbits were crossed over at an interval of 1 week, and the rabbits previously administered with the mismatch control oligonucleotide were treated with antisense oligonucleotides, and the rabbits previously administered with antisense oligonucleotide. Received a mismatch control oligonucleotide. The treatment and measurement method was similar to that performed in the first stage of the experiment. In 6 out of 8 rabbits treated with antisense oligonucleotides, adenosine-mediated bronchoconstriction limited solubility of adenosine (
It is worth noting that up to 20 mg / ml) was not seen. The PC50 value of these rabbits was set to 20 mg / ml for calculation. Therefore, this value represents the minimum value for the antisense effect. The actual effect was higher than this value. The experimental results are shown in Table 3 below.

[0097]

[Table 4] At both arms of this experiment, increased doses of aerosolized adenosine required to reduce lung dynamic compliance by 50% were seen in rabbits treated with antisense oligonucleotides. No effect was seen on PC50 values in the case of mismatch control oligonucleotide administration. No toxicity was seen in any of the rabbits dosed with antisense or control oligonucleotides.

From the above results, it is clear that the lung has an exceptional potential as a target for antisense oligonucleotide-based therapeutic intervention in lung disease. Furthermore, the above results indicate that adenosine A ₁ receptor down-regulation significantly reduces adenosine-mediated bronchoconstriction in the asthmatic airway in a model system resembling human asthma. Bronchial hyperresponsiveness in an allergic rabbit model of human asthma is an optimal indicator of antisense intervention. Because the tissues involved in this response are near the point of contact with the aerosolized oligonucleotide, and this rabbit model accurately simulates a significant human disease.

Example 4 Specificity of A ₁ Adenosine Receptor Antisense Oligonucleotides At the end of the crossover experiment of Example 3 above, quantitative analysis of adenosine A ₁ receptor numbers was performed using airway smooth muscle from all rabbits. I went. The unaffected adenosine A ₂ receptor was simultaneously quantified as a control in viewing the specificity of the antisense oligonucleotides.

[0100]   Airway smooth muscle tissue was cut from each rabbit and the method of Kleinstein et al.
Kleinstein and H.H. Grossman, Nauny N-Schmiedebe
rg's Arch. Pharmacol. 305: 191-200 (1978)
)) Was used to prepare the membrane fraction. The relevant parts of this document are described in this specification.
It is incorporated here with some modifications as a part of. Rough preparation of cell membrane
The product was stored at 70 ° C. until the time of assay. Bradford's method for protein content
(M. Bradford, Anal. Biochem. 72,
240-254 (1976), and relevant portions of this document constitute a part of this specification.
Incorporated here as what you do). Thaw frozen cell membrane at room temperature, 0.2 U /
Incubate for 30 minutes at 37 ° C with ml of adenosine deaminase
Sex adenosine was removed. [^ThreeH] DPCPX (A₁Receptor specificity) or [ ^Three H] CGS-21680 (A₁The binding of receptor specificity was previously reported by Ali et al.
Published method (S. Ali et al., J. Pharmacol. Exp. Ther. 26.
8, Am. J. Physiol. 266, L271-277 (1994),
The relevant parts of this document are hereby incorporated as a part of this specification)
Therefore, it was measured.

Assayed by specific binding of the A ₁ specific antagonist DPCPX, the number of A ₁ receptors in rabbits treated with adenosine A ₁ antisense oligonucleotides in a crossover experiment was reduced by about 75% compared to controls. . On the other hand, the adenosine _{A 2} receptor numbers, an _{A 2} receptor-specific antagonist 2- [p- (2-
No changes were seen as a result of the assay with specific binding of carboxyethyl) -phenethylamino] -5 '-(N-ethylcarboxamido) adenosine (CGS-21680). The results are shown in Table 4 below.

[0102]

[Table 5] The above results demonstrated the efficacy of antisense oligonucleotides in treating respiratory tract disease. Since the antisense oligos described above eliminate the receptor system for adenosine-mediated bronchoconstriction, there is less need to remove adenosine from oligonucleotides. However, it is preferred to also remove adenosine from these oligonucleotides to reduce the dose required to achieve a similar effect. Other antisense oligonucleotides targeting the mRNA of proteins involved in inflammation have also been described above. Adenosine has been removed from these nucleotide contents to prevent release during degradation.

Example 5 : Antisense Oligos with Other Target Nucleic Acids This experiment was performed to show that the present invention is broadly applicable to antisense oligonucleotides (abbreviated as oligos) specific for various nucleic acid targets. The following experimental studies were performed to show that the method of the invention was designed as demonstrated by the present application and is broadly suitable for the use of antisense oligos targeted to any adenosine receptor mRNA. It was For this purpose, various antisense oligos were prepared for adenosine receptor mRNAs, such as adenosine A ₁ , A _2b and A ₃ receptor mRNAs.

Antisense oligo I was disclosed above (SEQ ID NO: 1). The other five antisense phosphorothioate oligos were designed and synthesized as described above. 1. Oligo II (SEQ ID NO: 7): Targets the adenosine A ₁ receptor but differs in region from Oligo I. 2. Oligo V (SEQ ID NO: 10): Targets the adenosine A _2b receptor.

[0105]   3. Oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9): Adenosine A _Three Target different regions of the receptor.   4. Oligo I-PD (SEQ ID NO: 1681) (phosphodiester having the same sequence as Oligo I
Stell oligo).   These antisense oligos were designed for treatment of the selected species above.
Target mRNA segments of other species have similar sequences.
Unless otherwise noted, they are generally specific to the selected species. All antisense
Suligos were prepared as described below and prepared in a rabbit model (as described in the above application).
, Dyspnea and pulmonary airway disorder found in diseases such as asthma)
Tested in vivo for bronchoconstriction, inflammation and allergies.

Example 6 Design and Sequence of Other Antisense Oligos Six oligos and their effects in a rabbit model were tested. The results are reported and discussed below. Of these oligos, 5 were selected for this test and tested in Example 1 above.
The data for oligo I (SEQ ID NO: 1) shown in ~ 4 was supplemented. Oligo I is antisense to a region of adenosine A ₁ receptor mRNA.

The oligos tested are as follows: Antisense oligos I (SEQ ID NO: 1) and II (SEQ ID NO: 7) targeting different regions of adenosine A ₁ receptor mRNA, oligo V (SEQ ID NO: 8) targeting adenosine A _2b receptor mRNA, and Antisense oligos III and IV (SEQ ID NOs: 9 and 10) targeting two different regions of adenosine A ₃ receptor mRNA. 6
The second oligo (Oligo I-PD) is the phosphodiester form of Oligo I (SEQ ID NO: 1). The design and synthesis of these antisense oligos were performed according to Example 1 above.

(I) Antisense Oligo I The antisense oligonucleotide I described in Examples 1 to 4 above targets human A ₁ adenosine receptor mRNA (EPI2010). Antisense oligo I has a nucleotide length of 21, overlaps with the start codon and has the following sequence: 5'-GAT GGA GGG CGG CAT GGC GGG-3 '(SEQ ID NO: 1) Oligo I suppresses adenosine-induced bronchoconstriction in allergic rabbits,
In addition, suppression of allergen-induced airway obstruction and bronchial hyperresponsiveness has already been shown as described above (JW Nice and WJ Mettger, (Nature, 3;
85: 721 (1977)). In addition, the relevant part of this document is used here as a part of this specification.

(II) Antisense Oligo II A phosphorothioate antisense oligo (SEQ ID NO: 7) targeting the region +936 to +956 for the start codon (start site) of rabbit adenosine A ₁ receptor mRNA was designed according to the present invention. Antisense oligo II has a nucleotide length of 21 and has the following sequence: 5'-CTC GTC GCC GTC GCC GGC GGG-3 '(SEQ ID NO: 7)

(III) Antisense Oligo III A phosphorothioate antisense oligo (SEQ ID NO: 8) other than the one shown in Example 1 above, which targets the region +3 to +22 related to the start codon start site of antisense A ₃ receptor mRNA. ) Was designed according to the present invention. Antisense oligo III has a nucleotide length of 20 and has the following sequence: 5'-GGG TGG TGC TAT TGT CGG GC-3 '(SEQ ID NO: 8)

(IV) Antisense Oligo IV Further, another phosphorothioate antisense oligo (SEQ ID NO: 9) targeting the region +386 to +401 related to the start codon (start site) of adenosine A ₃ receptor mRNA is further prepared according to the present invention. Designed Antisense oligo IV
Has a nucleotide length of 15 and has the following sequence: 5'-GGC CCA GGG CCA GCC-3 '(SEQ ID NO: 9)

(V) Antisense Oligo V A phosphorothioate antisense oligo (SEQ ID NO: 10) was designed according to the present invention targeting regions -21 to -1 with respect to the start codon (start site) of adenosine A _2b receptor mRNA. . Antisense oligonucleotide V has a nucleotide length of 21 and has the following sequence.

[0113] 5'-GGC CGG GCC AGC CGG GCC CGG-3 '(SEQ ID NO: 10)

(VI) A ₁ Mismatch Oligo Two different mismatch oligonucleotides with the following sequences were used as controls for antisense oligo I (SEQ ID NO: 1) described in Example 5 above. A ₁ MM2 5′-GTA GGT GGC GGG CAA GGC GGG
-3 ′ (SEQ ID NO: 1682) A ₁ MM3 5′-GAT GGA GGC GGG CAT GGC GG
G-3 ′ (SEQ ID NO: 1683) The antisense oligo I and the two mismatched antisense oligos have the same base content and substantially the same sequence structure. GENBANK (Release 85.0
) And EMBL (release 40.0) by homology search, antisense oligo I is specific for human and rabbit adenosine A ₁ receptor genes, and mismatch control with known human and rabbit gene sequences. It was shown not to be a candidate for hybridization.

(VII) Antisense Oligo A ₁ -PD (Oligo VI) A phosphodiester antisense oligo having the same nucleotide sequence as oligo I (oligo VI; SEQ ID NO: 1681) was designed as disclosed herein. The antisense oligo I-PD has a nucleotide length of 21, overlaps with the start codon and has the following sequence. 5'-GAT GGA GGG CGG CAT GGC GGG-3 '(SEQ ID NO: 1681)

(VIII) Control After performing the same procedure for the antisense oligo in (II), (III) and (IV) above, 5.0 ml of aerosolized sterile physiological saline was administered to each rabbit.

Example 7 : Synthesis of antisense oligo The phosphorothioate antisense oligo having the sequence described in (a) above was synthesized using an oligonucleotide synthesizer (Applied Biosystems Mo).
del 396) and NENSORB chromatography (Dup
ont, DE). TETD (tetraethyl thiuram disulfide) was used as a sulfiding agent during synthesis. Similarly, antisense oligonucleotide II (SEQ ID NO: 7), antisense oligonucleotide III (SEQ ID NO: 8)
) And antisense oligonucleotide IV (SEQ ID NO: 9) were synthesized and purified.

Example 8 Preparation of Allergic Rabbits New Zealand White Pasteurella-free rabbits were mixed with 10% kaolin within 24 hours after birth of 312 antigen units / ml of house dust mite (hous).
e dustmite) (D. farinae) extract (Berkeley Biolog)
icals, Berkeley, CA) (0.5 ml) was injected intraperitoneally to immunize. Immunization was performed by known methods (W. J. Metzger, in Lat.
e Phase Allergic Reactions, W.E. Douche, C
RC Handbook, p. 347-362, CRC Press, Boca
Raton (1990); Ali, W. J. Metzger and S. J. Mustafa, Am. J. Resp. Crit. Care Med. 149: 908 (19
94)). Note that the relevant parts of these documents are incorporated herein as a part of the present specification. Immunization is repeated weekly for the first month and then 4
Repeated every two weeks until age. These rabbits preferentially allergen-specific Ig
Produces E-antibodies and responds to aeroallergen challenge in both early and late phase asthmatic responses, and also bronchial hyperresponsiveness (BHR).
)showed that. Monthly allergen (312 unit dust mite allergen above)
Intraperitoneal administration is continued to stimulate allergen-specific IgE antibody and BHR,
Maintained. At 4 months of age, the method described by Ali et al. For the preparation of aerosol doses for sensitized rabbits (S. Ali, W. J. Metzger and S. J. Mustafa, Am.
Resp. Crit. Care Med. 149 (1994)). In addition, the relevant part of this document is used here as a part of this specification.

Dose-Response Studies Example 9 : Experimental Setup Adenosine (0-20 mg / ml), antisense oligonucleotides and 2
One of the 5 types of mismatch oligonucleotides (5 mg / ml) aerosol was separately treated with an ultrasonic nebulizer (Model 646, DeVilbiss,
(Somerset, PA). 80% of the aerosol particles had a diameter of less than 5 μm. Equal doses of aerosol were administered directly to the lung via an endotracheal tube. Rabbits were randomly selected and dosed with aerosolized adenosine. The pre-treatment value for day 1 sensitivity to adenosine was calculated as the adenosine dose that causes a 50% loss of compliance (PC ₅₀ adenosine). Administration of aerosolized antisense oligos or mismatch antisense oligos to rabbits
Via the endotracheal tube (5mg / 1.0ml), 2 minutes per dose, twice a day,
It was carried out for 2 days (total dose: 20 mg). Post-treatment PC ₅₀ values were recorded on the morning of the third day (post-treatment challenge). The results of this test are shown later in Example 21.

Example 10 : Crossover experiments In several experiments with antisense oligo I (SEQ ID NO: 1) and the corresponding mismatch control oligonucleotide A1MM2, these rabbits were crossed over at 2 week intervals. Rabbits initially dosed with the mismatch control A ₁ MM2 were dosed with antisense oligo I and rabbits initially dosed with antisense oligo I were dosed with the mismatch control A ₁ MM2 oligo.

The number of rabbits in each group was as follows. Mismatch A ₁ MM2 administration group (
In control 1), n = 7, which is due to the loss of one bird in the second control arm of the experiment due to technical difficulties. Also, the mismatch A ₁ M
In the M3 administration group (control 2), n = 4, and in the A ₁ AS antisense oligo I administration group, n = 8. Rabbits treated with A ₁ MM3 oligos were analyzed separately and
It was not used in the crossover experiment. The treatment method and measurement used after the crossover were the same as in the first step (arm) of this experiment.

In 6 out of 8 rabbits treated with antisense oligo I (SEQ ID NO: 1), a dose of adenosine up to 20 mg / ml (solubility limit of adenosine)
No C ₅₀ value was obtained. For calculation, the PC ₅₀ value of these rabbits was 20 m
It was assumed to be g / ml. Therefore, this value represents the minimum potency of the antisense oligonucleotide of the present invention. The other groups of allergic rabbits (n = 4 in each group) were administered with antisense oligo I (SEQ ID NO: 1) or A ₁ MM2 oligo at a dose of 0.
． 5 mg or 0.05 mg was used according to the above method and schedule (
Total dose: 2.0 mg or 0.2 mg). The results of this test are shown in Example 22 below.

Example 11 : Antisense oligo formulation Each antisense oligo was separately solubilized in aqueous solution and 5 mg as described for antisense oligo I (SEQ ID NO: 1) in (e) above. Divided by 4
It was administered once (total dose: 20 mg) using a nebulizer via the endotracheal tube as described above.

The results obtained for antisense oligo I and its mismatch control, as described in Example 19 below, and also by Nice and Metzger in Nature 3
85, 721-725 (1997), it was confirmed that the mismatch control was equivalent to physiological saline. Because of this finding, saline was used as a control in lung function tests with antisense oligos II, III and IV (SEQ ID NOs: 7, 8 and 9).

Example 12 : Specificity of oligo I for adenosine A ₁ receptor (receptor binding test) As described above, 20 mg of oligo I (SEQ ID NO: 1) was administered in 4 divided doses for 48 hours in a rabbit. Airway smooth muscle tissue was dissected from the first, second, and third bronchi of. The membrane fraction was determined by the method of Ali et al. (S. Ali et al., Am. J. Resp. Cri.
t. Care Med. 149: 908 (1994)). still,
Relevant parts of the membrane fraction preparation are hereby incorporated as part of the present description.

[0126]   The amount of protein was determined by the method of Bradford.
/ Ml adenosine deaminase at 37 ° C for 30 minutes
Causative adenosine was removed (MM Bradford, Anal. Bioch.
em. 72, 240-254 (1976)). In addition, the relevant parts of this document
Incorporated herein as part of the specification. [^ThreeH] DPCPX, [ ^Three H] NPC17731 or [^Three[H] CGS-21680 is bound to Jarvis
(MF Jarvis et al., Pharmaco.
l. Exptl. Ther. 251, 888-893 (1989)). still
The relevant parts of this document are hereby incorporated as a part of this specification.
The results of this test are shown in Table 8 and are discussed in Example 20 below.

Example 13 Lung Function Measurements (Compliance c _DYN and Resistance) At 4 months of age, immunized rabbits were given a mixture (1) of ketamine hydrochloride (35 mg / kg) and acepromazine maleate (1.5 mg / kg). 0.5 ml) was intramuscularly administered, anesthetized and relaxed. After induction of anesthesia, the allergic rabbit was laid in a supine position in a comfortable position on a soft animal board. After applying the ointment to prevent dryness of the eyes, the eyes were closed. Next, with 4.0 mm middle high and low cuffs (
intermediate high-low cuffed) Murphy-1 endotracheal tube (Malli
nckrodt, Inc. , Glen Falls, NY), and intubated into rabbits by the method described by Zavala and Rose (Zavara and Rose, P.
roc. Soc. Exp. Biol. Med. 144; 509-512 (197).
See 3)). In addition, the relevant part of this document is used here as a part of this specification. An OD 2.4 mm polyethylene catheter (Becton Dickinson, Clay Adams, Parsippany, NJ) fitted with a thin-walled latex balloon was passed through the esophagus and kept a constant distance from the mouth (about 16 cm) for the duration of the experiment. The endotracheal tube was attached to a heated Fleisch lung tachograph (size 00; DEM Medical, Richmond, VA), flow rate (v) was determined by a Gould carrier amplifier (Model 11-411).
3; Validyne Differential Pressure Transducer (Model DP-45-16) driven by Gould Electronics, Cleveland, Ohio.
-1927; Validyne Engineering, Northridge, CA).

The esophageal balloon was attached to one side of the Validyne differential pressure transducer,
The other side of the differential pressure transducer was attached to the outflow of an endotracheal tube to obtain transpulmonary pressure (P _tp ). Flow volume is integrated and tidal volume
Got In addition, total lung resistance (R _t ) and dynamic compliance (C _dyn ) were measured at the _isovolume point and the zero flow point. Flow rate, respiratory volume and transpulmonary pressure are 8 channels G
Recorded on an Ould 2000W radio frequency recorder and C _dys was calculated using the difference in P _tp at respiratory volume and zero flow. In addition, _{R t} is in respiratory intermediate and _{P tp (midtid}
al) Calculated as the ratio of V to lung volume. These calculations were performed automatically using a Buxco Automated Pulmonary Respiratory Analyzer (Model 6, Buxco Electronics, Sharon, CT). This calculation was previously described by Gilles et al. (Gilles et al., Arch. Int. Pharmacodyn. T.
her. 194; 213-232 (1971)). It should be noted that the relevant portions describing these calculations are incorporated herein as a part of the present specification. The results obtained for oligo II administration to allergic rabbits are shown in Example 26 below.
And consider.

Example 14 : Measurement of bronchial hyperresponsiveness (BHR) Each allergic rabbit was administered histamine by aerosol to measure the baseline of hyperresponsiveness. Saline and histamine aerosols are available from DeVilb
It was generated using an iss nebulizer (Devilbiss, Somerset, PA) for 30 seconds and dosed for 2 minutes each. 80% of the aerosol particles produced by this ultrasonic nebulizer were less than 5 microns in diameter. Histamine aerosol was administered at increasing concentrations (0.156-80 mg / ml), and lung function was measured after each dose administration. Measurements of B4R the concentration required histamine to lower to 50% _{C dyn} from baseline _{(PC 50 histamine)} (mg / ml
) Was calculated.

Example 15 : Cardiovascular Effects of Antisense Oligo I In measuring cardiac output and other cardiovascular parameters using Cardiomax ^™ , the principle of thermodilution is used, which is cold saline. After intravenous injection of a known volume, the temperature change of blood discharged from the heart is monitored. A quick single injection of cold saline into the right atrium via cannulation of the right jugular vein,
Corresponding changes in temperature of the injection and blood mixture in the aortic arch were recorded via carotid cannulation using a temperature-sensitive miniprobe.

Twelve hours after treating allergic rabbits with the aerosol of oligo I (EPI2010; SEQ ID NO: 1) as in (d) above, a mixture of 80% ketamine and 20% xylazine (0.3 ml / kg) was used. ) Was used for anesthesia. This time point was revealed by Nice and Metzger, supra (1997) SEQ ID NO: 1.
Is consistent with previous data showing the effectiveness of. In addition, related disclosure parts are hereby incorporated as a part of this specification. A thermocouple was inserted into the left carotid artery of each rabbit, pushed in 6.5 cm, and fixed with a silk ligature. Then, the right jugular vein was cannulated, and a polyethylene tube of an arbitrary length was inserted and fixed.

Thermodilution curves were determined by injecting sterile saline at 20 ° C. and using a Cardiomax ^™ II (Columbus Instruments, OH), which was used to adjust the thermocouple probe position. After adjusting the position of the thermocouple, the femoral artery and femoral vein were separated. The drug was injected through the femoral vein, and the femoral artery was used for blood pressure and heart rate measurement. After establishing a constant baseline of cardiovascular parameters, Cardiomax ^™ measurements of blood pressure, heart rate, cardiac output, total peripheral resistance and cardiac contractility were performed.

Example 16 : Sustained Oligo I Action (SEQ ID NO: 1) Adenosine was administered to 8 allergic rabbits via the endobronchial tube as described in (f) above using a nebulizer at 0.156 mg / Starting from ml, compliance was reduced by 50% (PC _{50 adenosine} ) to establish a baseline. As mentioned above, 6 of the 8 birds were administered the aerosol of SEQ ID NO: 1 (5 mg
× 4 times). The remaining two birds received the same amount of saline vehicle as a control. PC _{50 adenosine} levels were measured again starting 18 hours after the final treatment. After this, all rabbits were measured daily and continued for 10 days. The results of this test are discussed in Example 25 below.

Example 17 : Reduction of Adenosine A _2b Receptor Number by Antisense Oligo V 2.0 mg of respiratory antisense oligo V (SEQ ID NO: 10) for SD rats
) Was administered 3 times over 2 days using the inhalation chamber described above. Twelve hours after the last dose, lung parenchyma was excised and assayed for adenosine A _2b receptor binding using [311] -NECA as described by Nice and Metzger (1997). SD rats to which the same amount of physiological saline was administered served as a control. The test results are significant at p <0.05 in Student's t test. The results will be discussed in Example 28 later.

Example 18: Oligo I and its phosphodiester oligo VI (SEQ ID NO: 1)
681) Oligo I (SEQ ID NO: 1) countered the effect of adenosine, 20
Sensitivity to adenosine was removed at adenosine doses up to mg adenosine / 5.0 ml (adenosine solubility limit). On the other hand, oligo VI (SEQ ID NO: 1681), which is a phosphodiester type of the oligonucleotide sequence, was completely ineffective in the same test. The sequences of both these compounds are identical, except that in oligo I (SEQ ID NO: 1) there is a phosphorothioate residue. Both compounds were delivered as aerosols as described above and as described by Nice and Metzger (1997). In Student's t-test, p <0.
There was a significant difference of 001. The results will be discussed in Example 29 later.

Results Obtained for Antisense Oligo I (SEQ ID NO: 1) Example 19 : Results of previous studies The nucleotide sequence of Antisense Oligo I, which is specific for the adenosine A ₁ receptor, and other data are given above. did. Experimental data demonstrating the effect of Oligo I on down-regulation of adenosine A ₁ receptor number and activity is also presented above.

[0137]   Further information on the characteristics and activity of antisense oligo I can be found in J. W. Nye
And W. J. Offered by Metzger (Nature 385: 72).
1 (1997)). The relevant parts of this document relating to the results below are part of this specification.
Is incorporated herein as a component of the. By Nice and Metzger (1997)
The publication contains the following data on Antisense Oligo I (SEQ ID NO: 1):
Mu. (1) Antisense oligo I dose-dependently, as shown in Table 5 below,
-A in bronchial smooth muscle of sexual rabbits₁Decrease the number of receptors. (2) Antisense oligo I induces adenosine-induced bronchoconstriction and allergen induction.
Weakens bronchoconstriction. (3) Antisense oligo I is a standard measurement in the evaluation of bronchial hyperresponsiveness.
Is a PC_FiftyAttenuates the bronchial hyperresponsiveness obtained by histamine measurement.
From this result, as shown in Table 5 below, the anti-inflammatory activity of antisense oligo I is clearly shown.
Sex is shown. (4) Antisense oligo I is adenosine A₁Designed to target the receptor
As expected, adenosine A₁Completely specific for the receptor
. On the other hand, adenosine A, which is closely related to this_TwoReceptor or bradykinin B _Two There was no effect on the receptor regardless of dose. This result is
See Table 5 below. (5) In contrast to antisense oligo I, mismatch control molecules MM2 and M
M3 (SEQ ID NOs: 1682 and 1683) is antisense oligo I (SEQ ID NO:
It has the same base composition and the same molecular weight as 1), but has 6 and 2 mismers, respectively.
It differs from antisense oligo I depending on the strain. Same salt as antisense oligo I
These mismatch controls with the smallest possible number of mismatches while leaving the base composition
Target receptor (A₁, A_TwoAnd B_Two) Had no effect on any of the above.

These results are unpredictable, as there is no prior art using antisense oligonucleotides such as oligo I targeted to the adenosine A ₁ receptor. The results presented in this patent show the effectiveness of the claimed therapeutic agent and treatment method for diseases or symptoms related to the pulmonary airways such as bronchoconstriction, inflammation and allergy.
It clearly shows in the intended use.

Example 20 : Oligo I significantly reduces the response to adenosine challenge Receptor binding experiments are shown in Example 12 above. In addition, the results shown in Table 5 below show that adenosine A ₁ -selective ligands in the membrane isolated from airway smooth muscle of allergic rabbits treated with A ₁ adenosine receptor antisense, B ₂ bradykinin receptor antisense and mismatch [ The binding properties of ₃ H] DPCPX and the bradykinin B ₂ selective ligand [ ³ H] NPC17731 are shown.

[0140]

[Table 6]

Example 21 : Dose Response Effect of Oligo I Antisense Oligo I (SEQ ID NO: 1) was administered adenosine to rabbits in a dose-dependent manner within the dose range tested, as shown in Table 6 below. Was found to reduce the effect of.

Table 6: Dose-response effect on antisense oligo I Total dose (mg) PC50 adenosine (mg adenosine) Antisense oligo I 0.2 8.32 7.2 2.0 14.0 7.2 20 19 .5 0.34 A ₁ MM2 oligo (control) 0.2 2.51 0.46 2.0 3.13 0.71 20 3.25 0.34 The above results were tested by Student's paired t-test And was found to be statistically different (p = 0.05).

The anti-adenosine A ₁ receptor oligo I (SEQ ID NO: 1) acts specifically on the adenosine A ₁ receptor but not on the adenosine A ₂ receptor. These results are shown in Example 9 above, as well as Nice and Metzger (
1997), treatment of rabbits with antisense oligo I (SEQ ID NO: 1) or mismatch control oligo (SEQ ID NO: 1682; A ₁ MM2) (ie nebulizer and airway at 8-12 hour intervals). 5 mg x 4 doses via inner tube), and also ablated smooth muscle of the bronchus and adenosine A ₁ and adenosine A ₂ receptors determined as reported by Nice and Metzger (1997). It is derived from the numbers.

Example 22 : Specificity of oligo I (SEQ ID NO: 1) for the target gene product Oligo I (SEQ ID NO: 1) is specific for the adenosine A ₁ receptor while its mismatch control is active. can not see. FIG. 1 shows the results obtained in Example 10 above and from the crossover experiment described by Nice and Metzger (1997). Two mismatch controls (SEQ ID NOs: 1682 and 1683) are P
It showed no effect on C _{50 adenosine} levels. In contrast, administration of antisense oligo I (SEQ ID NO: 1) increased the PC _{50 adenosine} level 7-fold. The results clearly demonstrate that antisense oligo I (SEQ ID NO: 1) attenuates (decreases sensitivity) the response to exogenously administered adenosine as compared to saline controls. Also, from the results shown in Table 2 above, it is clear that the effect of antisense oligo I is dose-dependent (see the third column of Table 1).

[0145]   In addition, oligo I is adenosine A₁Adenosine A closely related to the receptor _Two Receptor and bradykinin B_TwoThat it did not induce activity at the receptor
From (see lines 2 to 10 of Table 2 above), adenosine A₁Perfect for receptors
Was shown to be specific (see the top three rows of the table).

Furthermore, the results shown in Table 2 show that antisense oligo I (SEQ ID NO: 1) reduces the sensitivity to adenosine in a dose-dependent manner. Also, two mismatch control oligonucleotides (A ₁ MM2; SEQ ID NO: 1682 and A ₁ MM)
3; SEQ ID NO: 1683), since it did not show any effect on reducing and PC 50 _{A Denoshin} value of adenosine A ₁ receptor numbers, antisense oligos I may be lowered sensitivity to antisense oligonucleotide-dependent manner adenosine Recognize.

Example 23 : Effects on Aeroallergen-induced Bronchoconstriction and Inflammation Oligo I (SEQ ID NO: 1) was shown to significantly reduce histamine-induced effects in a rabbit model compared to mismatched oligos. Antisense oligo I (SEQ ID NO: 1) for allergen-induced airway obstruction and bronchial hyperresponsiveness
And the effects of mismatch oligos (A ₁ MM2; SEQ ID NO: 1682 and A ₁ MM3; SEQ ID NO: 1682) were evaluated in allergic rabbits. The effect of antisense oligo I (SEQ ID NO: 1) on allergen-induced airway obstruction was evaluated. Antisense Oligo I significantly inhibited allergen-induced airway obstruction compared to the mismatch control, as calculated from the area under the plot curve (
55%, p <0.05; repeated measures ANOVA and Tukey's t test). The mismatch oligo A ₁ MM2 (control) was shown to have no effect on allergen-induced airway obstruction. The effect of antisense oligo I (SEQ ID NO: 1) on allergen-induced BHR was measured as described above. Antisense oligo I significantly inhibited allergen-induced BHR in allergic rabbits (61%, p <0.05; repeated measures ANOVA), as calculated from PC _{50 histamine} values, compared to the mismatch control.
, Tukey's t-test). The A ₁ MM mismatch control was shown to have no effect on allergen-induced BHR.

The above results indicate that antisense oligo I (SEQ ID NO: 1) provides effective protection against aeroallergen-induced bronchoconstriction (house dust mite). Furthermore, since antisense oligo I (SEQ ID NO: 1) showed an effect on histamine showing anti-inflammatory activity against oligo I, it was revealed that it can also be an inhibitor of dust mite-induced hyperbronchial responsiveness. It was

Example 24 : Antisense Oligo I has no adverse side effects Oligo I (SEQ ID NO: 1) was shown to have no adverse side effects to the recipient. Arterial pressure, cardiac output, stroke volume, heart rate, total peripheral resistance and cardiac contractility (dPd) after administration of 2.0 mg or 20 mg of Oligo I (SEQ ID NO: 1).
No change was seen in T). In addition, a Cardiomax ^™ device (Co
Cardiac output (C) using lumbus Instruments, Ohio
O), stroke volume (SV), mean arterial pressure (MAP), heart rate (HR), total peripheral resistance (TPR) and contractility (dPdT) were evaluated.

These results indicated that oligo I (SEQ ID NO: 1) had no deleterious effects on important cardiovascular parameters. In particular, this oligo does not cause hypotension. This finding is particularly important because other phosphorothioate antisense oligonucleotides in the past have been shown to cause hypotension in some model systems. In addition, the adenosine A ₁ receptor plays an important role in sinus conduction in the heart. Therefore, it is considered that weakening of the adenosine A ₁ receptor by antisense oligo I (SEQ ID NO: 1) causes harmful extrapulmonary activity corresponding to down-regulation of the receptor. But that doesn't happen. The antisense oligo I (SEQ ID NO: 1) does not cause any harmful pulmonary effects, and the oligo of the present invention does not show any undesired side effects and requires a small dose.

This indicates that when oligo I (SEQ ID NO: 1) is directly administered to the lung, the amount causing a harmful effect does not enter the heart. This oligo contrasts with traditional adenosine receptor antagonists such as theophylline, which pass through the lungs and cause harmful and sometimes life-threatening effects outside the lungs.

Example 25 : Long-lasting effect of Oligo I Oligo I (SEQ ID NO: 1) P was obtained on administration prior to adenosine challenge.
It showed a long-lasting effect, as demonstrated by the C ₅₀ value and resistance value. The effect duration was measured using the nebulizer via the trachea as described above (5 m
g x 4 times) adenosine antisense oligo I PC ₅₀ . The effect of this agent was significant over 1-8 days. Antisense oligo I (
When the effect of SEQ ID NO: 1) was no longer observed, saline aerosol (control) was administered to the rabbits, and PC _{50 adenosine} levels were measured again for all the rabbits. Rabbits treated with saline showed baseline PC ₅₀ adenosine levels (n = 6).

Further, the measurement of the duration of effect (for resistance) was carried out by using 6 allergic rabbits administered with 20 mg of antisense oligo I (SEQ ID NO: 1) as described above, and measuring the airway resistance as described above. It was done by measuring. Mean calculated effect duration was 8.3 for both PC ₅₀ adenosine (p <0.05) and resistance (p <0.05).
It was a day. From these results, it was unexpected that oligo I
The action of (SEQ ID NO: 1) was shown to be very long lasting.

Example 26 : Antisense Oligo II Antisense Oligo II, which targets different regions of adenosine A ₁ receptor mRNA, was found to be highly active against adenosine A ₁ mediated action. In the experiment, as described above, the antisense oligo II (SEQ ID NO: 7) was tested for the compliance and resistance values when 20 mg of antisense oligo II and physiological saline (control) were administered to two groups of allergic rabbits. ) Was administered. Compliance and resistance values were measured after administration of adenosine and saline as described in Example 13 above. The effects of the antisense oligos of the invention differed from controls with statistical significance (p <0 for compliance).
． 05 paired t-test, p <0.01 for resistance). From this result, antisense oligo I targeting adenosine A ₁ receptor
I (SEQ ID NO: 7) has been shown to effectively maintain compliance and reduce resistance upon adenosine challenge.

Example 27 : Antisense oligos III and IV Oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) are 20 mg antisense oligo III (SEQ ID NO: 8) in allergic rabbits as described above. ) And I
The effect of reducing V and the number of inflammatory cells when V (SEQ ID NO: 9) was administered separately was shown to actually specifically target the adenosine A ₃ receptor. The number of inflammatory cells was measured by counting 100 or more viable cells per washing solution 3 hours after collecting the bronchial washing solution.

The effect of antisense oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) on granulocytes and whole cells in bronchial lavage fluid was evaluated after exposure to dust mite allergens. As a result, it was shown that antisense oligo IV (SEQ ID NO: 9) and antisense oligo III (SEQ ID NO: 8) are highly likely to be anti-inflammatory agents in the asthmatic lung after exposure to dust mite allergen. . As is known to those skilled in the art, granulocytes, especially eosinophils, are the major inflammatory cells of asthma, and antisense oligo I
Administration of II (SEQ ID NO: 8) and IV (SEQ ID NO: 9) reduces that number by 40% and 66%, respectively. Furthermore, antisense oligo IV (SEQ ID NO: 9
) And III (SEQ ID NO: 8) also reduce the total number of cells in bronchial lavage fluid by 40% and 80%, respectively. This is also an important indicator of the anti-inflammatory activity of the anti-adenosine A ₃ agent of the present invention. Inflammation is known to underlie bronchial hyperresponsiveness and allergen-induced bronchoconstriction in asthma. Antisense oligonucleotides III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) both target the adenosine A ₃ receptor and can be designed to specifically target various lung receptors. It represents a new and important group of anti-inflammatory agents.

Example 28 : Antisense Oligo V Adenosine A _2b Antisense Oligo V (SEQ ID NO: 10), which targets adenosine receptor mRNA, very effectively counters adenosine A _2b mediated activity and also adenosine A _2b. It was shown to reduce the number of _2b receptors by less than half.

Example 29 : Unexpected Superiority of Substitutions to Phosphodiester Residue Oligo I-DS (SEQ ID NO: 1681) Oligo I (SEQ ID NO: 1) and I-DS (SEQ ID NO: 1681) are described above. As described above, allergic rabbits were separately administered and then challenged with adenosine. Phosphodiester oligo I-DS (SEQ ID NO: 1681) was shown to be statistically significantly less effective in combating adenosine action, whereas oligo I (SEQ ID NO: 1) was highly effective, The PC _{50 adenosine} value was 20 mg.

Example 30 : Antisense Oligo VI In the present study, the inventor designed an additional antisense phosphorothioate oligo (oligo VI) targeting the adenosine A ₁ receptor. This antisense oligo was designed for the treatment of the selected species described in the above-mentioned application, and unless the segment of adenosine receptor mRNA of other species has a sequence similar to this selected species, In contrast, it is usually specific. This antisense oligo was prepared as described below, and bronchoconstriction was performed in vivo using a rabbit model (as described in the above application, which causes dyspnea and pulmonary airway disorder observed in diseases such as asthma). Tested for inflammation and allergies.

The effect of this additional oligo in a rabbit model was tested. The results are reported and discussed below. This oligo (antisense oligo VI) was selected for this study for the purpose of supplementing the data of SEQ ID NO: 1 (oligo I), which is antisense to the adenosine A ₁ receptor mRNA described in the above application. . This additional oligo is antisense oligo VI, which targets a region of oligodendrogen A ₁ receptor that differs from oligo I. The design and synthesis of this antisense oligo was performed according to the teachings of the above application, and in particular according to Example 1.

Antisense oligo VI is a phosphorothioate designed to target the coding region from +964 to +984 for the start codon (start site) of the rabbit adenosine A ₁ receptor mRNA. Oligo VI was prepared as described in the above application. Its nucleotide length is 20. Oligo VI has the following sequence for the adenosine A ₁ receptor gene. 5'-CGC CGG CGG GTG CGG GCC GG-3 '(SEQ ID NO: ) Phosphorothioate antisense oligo VI having the sequence described in (5) above
Was synthesized on an Applied Biosystems Model 396 oligonucleotide synthesizer and subjected to NENSORB chromatography (DuPont,
Purified using DE). TETD (tetraethyl thiuram disulfide) was used as a sulfiding agent during synthesis.

Example 31 Preparation of Allergic Rabbits New Zealand White Pasteurella-free rabbits were mixed with 10% kaolin within 24 hours after birth of 312 antigen units / ml of house dust mite (hous).
e dustmite) (D. farinae) extract (Berkeley Biolog)
icals, Berkeley, CA) (0.5 ml) was injected intraperitoneally to immunize. Immunization was performed by known methods (W. J. Metzger, in Lat.
e Phase Allergic Reactions, W.E. Douche, C
RC Handbook, p. 347-362, CRC Press, Boca
Raton (1990); Ali et al., Am. J. Resp. Crit. Ca
re Med. 149: 908 (1994)).

Immunizations were repeated weekly for the first month and then every two weeks until 4 months of age. These rabbits preferentially produced allergen-specific IgE antibodies, responded to aeroallergen challenge in both early-phase and late-phase asthma responses, and also showed bronchial hyperresponsiveness (BHR). Monthly intraperitoneal administration of the allergen (the above 312 unit dust mite allergen) was continued to stimulate and maintain the allergen-specific IgE antibody and BHR. At 4 months of age, sensitized rabbits were prepared for aerosol administration as described by Ali et al. (1994) supra.

[0164]Example 32 : Adenosine aerosol preparation   Adenosine aerosol (20mg / ml) is ultrasonic nebulizer (Model)
  646, DeVilbiss, Somerset, PA)
It was 80% of the aerosol particles had a diameter of less than 5 μm. Equal amount of air
The aerosol was administered to all three rabbits directly into the lung via an endotracheal tube.   The rabbit was then dosed with aerosolized adenosine. For adenosine on the first day
The unprocessed value for sensitivity causes a 50% loss of compliance
Adenosine dose (PC_FiftyAdenosine). Aerozo for the rabbit
Administration of antisense-drug was via endotracheal tube (5 mg / 1.0 ml), 1
The administration was performed for 2 minutes per administration and twice a day for 2 days (total dose: 20 mg). PC after processing _Fifty Values were recorded on the morning of the third day (post treatment challenge). The results of this test are shown below (9
).

Example 33 Antisense Oligoformulation Each antisense oligo was separately solubilized in aqueous solution and 5 mg as described for antisense oligo I (SEQ ID NO: 1) in (e) above. A certain amount of (al
iquots) 4 times (total dose: 20 mg) via the endotracheal tube using a nebulizer as described above.

Example 34 : Oligo VI reduces response to adenosine challenge to the same or greater extent than Oligo I. Oligo VI was tested in 3 allergic rabbits with the above properties,
Prepared as described in (7) above and in the above application. Oligo VI targets a section of the coding region separate from the target of Oligo I within the A ₁ receptor. Both of these target sequences were randomly selected from a number of possible coding region target sequences.

The same treatment was performed on 3 rabbits as described above for Oligo I. That is, the rabbit was sprayed with 5 mg of oligo VI twice a day at 8 hour intervals.
Went for a day. Then, on the morning of the third day, a PC ₅₀ adenosine test was performed, and P
Compared to C ₅₀ value. This protocol is described in more detail by Nice and Metzger (Nice and Metzger, Nature 385: 72.
1-725 (1997)).

[0168]   The results obtained from 3 rabbits are shown in Table 1 below.

[Table 7] All three rabbits treated with oligo VI were completely desensitized to adenosine to measurable levels of drug as shown in Table 1 above. Thus, administration of oligo VI abrogated adenosine-induced bronchoconstriction in all three allergic rabbits. Therefore, it is considered that the actual effect of oligo VI is higher than the measured value in the experimental system used. Compared to the results of Oligo I shown above, it was shown that Oligo VI was as effective as Oligo I or better.

Example 34 : Conclusion From the results of the above tests and the results of each Example examined, all antisense nucleotides designed in accordance with the teachings of the above application counteract the effects mediated by the receptors they target, and It has been found to be very effective in reducing that effect. That is, each of two antisense oligos targeting adenosine A ₁ receptor mRNA, one antisense oligo targeting adenosine A _2b receptor mRNA, and two antisense oligos targeting A ₃ receptor mRNA are It has been shown that it is possible to counteract the effects of exogenously administered adenosine mediated by the particular receptors they target.

Furthermore, the activity of the antisense oligo of the present invention is specific for the target of the oligo and cannot inhibit another alternative target. Further, the above results showed that the adverse side effects or toxicity caused by the administration of the agent of the present invention were extremely low or none. This indicates 100% success in providing highly effective and specific agents in the treatment of bronchoconstriction and / or inflammation. The present invention also relates to all genes and their mRNAs encoding proteins involved in or associated with respiratory tract diseases,
Widely applied as well. Phosphorothioate oligonucleotides showed an unexpected superiority over phosphodiester antisense oligos in comparison to phosphodiester oligonucleotides and oligonucleotides in which the phosphodiester bond was replaced with a phosphorothioate bond.

The above examples are illustrations of the present invention, and the present invention is not limited to these examples. The invention is defined by the claims, including equivalents of the subject matter of the claims below, included in this specification.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07H 21/00 C07H 21/04 4C086 21/04 A61K 37/02 C12N 5/10 C12N 5/00 A // C12N 15/09 ZNA 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT , SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF F term (reference) 4B024 AA01 BA01 BA07 BA21 BA63 CA04 EA04 GA11 HA01 4B065 AA90X AA93Y AB01 BA02 CA24 CA27 4C057 MM01 MM04 MM09 4C076 AA11 AA19 AA22 AA24 AA31 AA53 AA93 BB21 CC15 DD09F DD19F DD38F DD41F DD63F DD70F EE13F EE30F EA41 MA41 MA04MA4 MA4A4 MA44A4 MA44A25 MA05 MA13 MA23 MA24 MA37 MA41 MA56 NA14 ZA59

Claims (58)

[Claims] 1. A target gene, a coding and non-coding region of RNA corresponding to the target gene, a start codon of the gene, a genomic flanking region, an intron-exon boundary region, a 5′-terminal, a 3′-terminal and a coding region. Two or more mRNAs selected from the group consisting of juxtaposed sections with non-coding regions and all segments of RNA encoding proteins associated with one or more diseases or conditions or mixed states thereof.
An agent containing an oligonucleotide antisense to. 2. The agent according to claim 1, wherein one of the mRNAs is a transcription factor,
Stimulators and activators, interleukins, interleukin receptors, chemokines, chemokine receptors, endogenously produced specific and non-specific enzymes, immunoglobulins, antibody receptors, central nervous system (CNS) and peripheral nerves and non-neurons An agent encoding a protein selected from the group consisting of system receptors, CNS and peripheral nerve non-neuronal peptide transmitters, adhesion molecules, defensins, growth factors, vasoactive peptides and receptors and binding proteins, or corresponding to an oncogene. 3. The agent according to claim 2, wherein the encoded receptor and peptide transmitter are sympathomimetic receptor, accessory sympathomimetic receptor, GABA receptor, adenosine receptor, bradykinin receptor, insulin receptor, glucagon receptor, A group consisting of prostaglandin receptor, thyroid receptor, androgen receptor, anabolic receptor, female hormone receptor, luteinizing hormone receptor, coaggregin cascade-related receptor, anterior pituitary receptor, anterior pituitary peptide transmitter and histamine receptor (HisR) Agent selected from. 4. The agent according to claim 2, wherein the coded sympathomimetic receptor and accessory sympathomimetic receptor are acetylcholinesterase receptor (AcChaseR), acetylcholine receptor (AcChR), atropine receptor, muscarinic receptor, epinephrine. Receptor (EpiR
), Dopamine receptor (DOPAR), taxinen receptor and norepinephrine receptor (NEpiR). 5. The agent according to claim 2, wherein the encoded enzyme is synthase, kinase, oxidase, phosphatase, reductase, polysaccharide hydrolase, triglyceride hydrolase, protein hydrolase, esterase, elastase, polysaccharide synthase, lipid synthase and An agent selected from the group consisting of protein synthases. 6. The agent according to claim 5, wherein the encoded enzyme is tryptase, inducible dinitrogen pentoxide synthase, cyclooxygenase (Cox), MA.
P kinase, eosinophil peroxidase, β2-adrenergic receptor kinase, leukotriene c-4 synthase, 5-lipoxygenase, phosphodiesterase IV, metalloproteinase, CSBP / p38MAP kinase, neutrophil elastase, phospholipase A2, cyclooxygenase 2 (C
ox-2), fucosyltransferase, IκB kinase 1 and 2, chymase, protein kinase C, thymidylate synthase, tryptase, dihydrofolate reductase, tryptase, thymidine kinase, deoxycytidine kinase and ribonucleotide reductase. Agent. 7. The agent according to claim 2, wherein the encoded factor is Nfκ.
B transcription factor, granule cell macrophage colony stimulating factor (GM-CSF), AP
-1 transcription factor, monocyte activator, neutrophil chemotactic factor, granule cell colony stimulating factor (
G-CSF), NFAT transcription factor, platelet activating factor, tumor necrosis factor α (TNF)
α) and an agent selected from the group consisting of basic fibroblast growth factor (BFGF). 8. The agent according to claim 2, wherein the encoded adhesion molecules are intracellular adhesion molecules 1 (ICAM-1), 2 (ICAM-2) and 3 (ICAM-3).
, Vascular cell adhesion molecule (VCAM), endothelial leukocyte adhesion molecule 1 (ELAM-1)
An agent selected from the group consisting of GATA transcription factor, neutrophil adhesion receptor, and madCAM-1. 9. The agent according to claim 2, wherein the encoded cytokine is
Lymphokines and chemokines are interleukin-1 (IL-1), interleukin-1β (IL-1) interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL). -5), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-1
An agent selected from the group consisting of 1 (IL-11), CCR-5CC chemokine and Rantes. 10. The agent according to claim 2, wherein the encoded receptor is adenosine A ₁ receptor, adenosine A ₂ B receptor, adenosine A ₃ receptor, endothelin receptor A, endothelin receptor B, IgE high affinity receptor, muscarinic. Acetylcholine receptor, substance P receptor, histamine receptor, CCR-1CC chemokine receptor, substance P receptor, NK-1 receptor, NK-3 receptor, CCR-2CC
Chemokine receptor, CCR-3CC chemokine receptor (eotaxin receptor), interleukin-1β receptor (IL-1βR), interleukin-1 receptor (IL-1R), interleukin-1β receptor (I
L-1βR), interleukin-3 receptor (IL-3R), CCR-4C
C chemokine receptor, cysteinyl leukotriene receptor, prostanoid receptor, interleukin-1 receptor (IL-1R), interleukin-4 receptor (IL-4R), interleukin-5 receptor (IL
-5R), interleukin-8 receptor (IL-8R), interleukin-9 receptor (IL-9R), interleukin-11 receptor (IL-1)
1R), bradykinin B2 receptor, sympathomimetic receptor, accessory sympathomimetic receptor, GABA receptor, adenosine receptor, bradykinin receptor, insulin receptor, glucagon receptor, prostaglandin receptor, thyroid receptor, androgen receptor, anabolic receptor, An agent selected from the group consisting of female hormone receptor, luteinizing hormone receptor, coaggregin cascade-related receptor, anterior pituitary receptor and histamine receptor (HisR). 11. The agent according to claim 2, wherein the encoded protein is eotaxin, major basic protein, preproendothelin, eosinophil cationic protein, P-selectin, STAT4, STAT6, c-mas, NF.
-Interleukin-6 (NF-IL-6), MIP-1α, MCP-2, MC
P-3, MCP-4, cyclophilin, PDG2, cyclosporin A-binding protein, FK5-binding protein, fibronectin, LFA-1 (CD11A
/ CD18), PECAM-1, C3bi, PSGL-1, CD-34, substance P, p150,95, Mac-1 (CD11b / CD18), VLA-4.
, CD-18 / CD11a, CD11b / CD18, C5a, CCR1, CCR
2, an agent selected from the group consisting of CCR4, CCR5 and LTB-4. 12. The agent according to claim 2, wherein the encoded defensin is selected from the group consisting of defensin 1, defensin 2 and defensin 3. 13. The agent according to claim 2, wherein the encoded selectin is selected from the group consisting of α4β1 selectin, α4β7 selectin, LFA-1 selectin, E-selectin, P-selectin and L-selectin. 14. The agent according to claim 2, wherein the mRNA is ras or src.
, An agent corresponding to the oncogene selected from the group consisting of myc and bcl-2. 15. The agent of claim 1, wherein the one or more mononucleotides linked to the phosphodiester residue of the antisense oligonucleotide are methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate. , Formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene (methylimino), methyleneoxy (methylimino), 2'-O An agent substituted with a residue selected from the group consisting of methyl, phosphoramidate residues and combinations thereof. 16. The agent according to claim 15, wherein all phosphodiester residues are substituted. 17. The agent according to claim 1, wherein the antisense oligonucleotide comprises from about 7 to about 60 mononucleotides. 18. The agent of claim 1, wherein the oligo is less than about 15% A.
Agents containing. 19. The agent according to claim 1, wherein the oligo does not contain adenosine. 20. The agent according to claim 1, wherein the antisense oligo is SEQ ID NO: ~ , SEQ ID ~ And the sequence number ~ (Fragment number ~ ) Is selected from the group consisting of oligos. 21. The agent of claim 1, which binds to thymidine base but has antagonist activity and is less than about 0.3 adenosine A ₁ receptor, adenosine A _2b receptor, adenosine A ₃ receptor. An agent in which one or more A is substituted with a universal base selected from the group consisting of a heteroaromatic base having agonist activity and a heteroaromatic base having no activity or having an agonist activity at the adenosine A _2a receptor. 22. The agent according to claim 21, wherein the heteroaromatic base is selected from the group consisting of pyrimidine and purine, and these are O, halogen, NH ₂ , S.
_{H, SO, SO 2, SO} 3, COOH and branched chain and condensation of primary and secondary amino, and O, halogen, NH _2, primary, secondary and tertiary _{amines, SH, SO, SO 2,} SO 3, Alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, which may be further substituted with cycloalkyl, heterocycloalkyl and heteroaryl,
Acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylalkyl. Agents optionally substituted with arylalkenyl, arylalkynyl and arylcycloalkyl. 23. The agent according to claim 22, wherein the pyrimidine and purine are substituted at the 1,2,3,4,7 and 8 positions. 24. The agent according to claim 23, wherein pyrimidine and purine are represented by the following formula: (In the formula, R ¹ and R ² independently represent H, alkyl, alkenyl or alkynyl, and R ³ is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl. , O- cycloalkyl, O- cycloalkenyl, O- cycloalkynyl, NH ₂ - represented by aryl) - alkylamino - Ke Toki sialic km carboxymethyl - aryl and mono and dialkylaminoalkyl -N- alkylamino -SO ₂ An agent selected from the group consisting of, theophylline, caffeine, diphylline, etophylline, acephyrin, piperazine, bamifilin, enprophylline and xanthine. 25. The agent according to claim 24, wherein the universal base is 3-nitropyrrole-2′-deoxynucleoside, 5-nitroindole, 2-deoxyribosyl- (5-nitroindole), 2-deoxyribofuranosyl. -(5-nitroindole), 2'-deoxyinosine, 2'-deoxynebulaline, 6H,
8H-3,4-dihydropyrimido [4,5-c] oxazin-7-one or 2-
An agent selected from the group consisting of amino-6-methoxyaminopurine. 26. The agent according to claim 1, wherein the methylated cytokine ( ^m C) replaces one or more CpG dinucleotides present in the oligo. 27. The agent according to claim 1, wherein the antisense oligonucleotide is encapsulated in or incorporated into a cell, or an agent functionally linked to a eukaryotic or prokaryotic vector. 28. The agent according to claim 27, wherein the agent encapsulated in or taken up by cells is selected from the group consisting of transferrin, asialoglycoprotein and streptavidin. 29. A composition comprising the agent according to claim 1 and a pharmaceutically or veterinarily acceptable carrier. 30. The composition according to claim 29, wherein the carrier is selected from the group consisting of gaseous, liquid and solid carriers. 31. The composition of claim 29, wherein the other therapeutic compound is
Selected from the group consisting of surfactants, antioxidants, flavoring and coloring agents, fillers, volatile oils, buffers, dispersants, RNA inactivating agents, antioxidants, flavoring agents, propellants and preservatives. A composition further comprising an agent. 32. The composition of claim 29, wherein the antisense oligonucleotide is present in the composition in an amount of about 0.01 to about 99.99 w / w. 33. The composition according to claim 32, which comprises a nucleic acid, a surfactant and a carrier. 34. The composition according to claim 33, wherein the surfactant is surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant protein and an active fragment thereof; disaturated phosphatidylcholine other than dipalmitoyl,
Dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, ubiquinone, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandroferone, dehydroepiandroferonic acid, dehydroepiandrofaterine, dolycholandricholine, dehydroepiandrofersteric acid, dehydroepiandrofaterine, dolycohol, dolicholsterone
Glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine, dihydroxyacetone palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, choline phosphate, synthetic lamellar vehicle of surfactant component, Omega-3-
Fatty acids, polyenic acids, polyenoic acids, lecithin, palmitic acid, nonionic ethylene oxide and / or propylene oxide block copolymers, polyoxypropylene, polyoxyethylene, dextran and / or polyvinylamines with alkanoyl side chains, Brij 35, Triton X-100. , ALEC,
A composition selected from the group consisting of Exosurf, Survan and Atvaquone. 35. A formulation comprising the composition of claim 26, wherein the carrier comprises a hydrophobic carrier. 36. The formulation of claim 35, wherein the carrier comprises lipid particles or vesicles. 37. The formulation of claim 36, wherein the vesicles comprise liposomes and the particles comprise fine crystals. 38. The preparation according to claim 35, wherein the lipid vesicle is N- (
Formulation containing 1- [2,3-dioleoyloxy] propyl) -N, N, N-trimethylammonium methylsulfate. 39. The formulation of claim 34, which comprises an inhalable formulation. 40. The formulation of claim 34, which comprises an aerosol. 41. The formulation of claim 34, which is in the form of single or multiple units. 42. The formulation of claim 34, which is in bulk form. 43. A capsule or cartridge comprising the composition of claim 26. 44. A kit comprising a delivery device and containing the composition of claim 26 and instructions for its use in a separate container. 45. The kit of claim 44, wherein the delivery device comprises a nebulizer that delivers a single dose of the formulation. 46. The kit of claim 44, wherein the nebulizer comprises an insufflator and the composition is provided in a pierceable or openable capsule or cartridge. 47. The kit according to claim 44, wherein the delivery device comprises a pressurized inhaler and the composition comprises a suspension or solution of the agent. 48. The kit of claim 44, wherein the other therapeutic agent compound, surfactant, antioxidant, flavoring and coloring agent, filler, volatile oil, buffer, dispersion in a separate container. A kit further comprising an agent, an agent encapsulated in or incorporated into cells, an RNA inactivating agent, an antioxidant, a corrigent, a propellant, and a preservative. 49. The kit according to claim 48, wherein the surfactant is surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D, and surfactant protein and active fragments thereof; disaturated phosphatidylcholine other than dipalmitoyl, Dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, ubiquinone, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandrophatherone, dehydroepiandrofasterone, dolycholandriolsterone, dehydroepiandrosterone, dolicholcholidoleic acid, dehydroepiandrofasterone, drichoic acid -Phos Fate, dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine, dihydroxyacetone palmitate,
Cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, choline phosphate, synthetic lamellar vehicle of surfactant components, omega-3
-Fatty acids, polyenic acids, polyenoic acids, lecithin, palmitic acid, nonionic ethylene oxide and / or propylene oxide block copolymers, polyoxypropylene, polyoxyethylene, dextran and / or polyvinylamines with alkanoyl side chains, Brij35, TritonX- 100, ALEC
, Exosurf, Survan and Atvaquone
A kit selected from the group consisting of: 50. The kit of claim 44, wherein the composition is provided in a capsule or cartridge. 51. A cell containing the agent according to claim 1. 52. A method of treating a disease or condition associated with mRNA corresponding to at least one target gene, genomic flanking region or protein, wherein the agent according to claim 1 is administered to a patient suffering from the disease or condition. Wherein the agent comprises an effective amount of an antisense oligonucleotide that allows the patient to exhibit decreased production or utilization or increased degradation of one or more target mRNAs. 53. The method of claim 52, wherein more than one target mRNA
A method of administering an effective amount of an agent capable of decreasing the production and utilization of, or increasing the degradation of. 54. The method of claim 52, wherein the agent is administered directly to the lungs of the patient. 55. The method of claim 54, wherein the agent is administered as an inhalable aerosol. 56. The method of claim 52, wherein the disease or condition is a lung disease or condition and the one or more target mRNAs are adenosine A ₁ receptors, adenosine A ₂ B receptors, adenosine A ₃ receptors and bradykinin. A method of encoding a protein selected from the group consisting of B2 receptors. 57. The method of claim 56, wherein the disease or condition is associated with airway obstruction in the patient. 58. The method of claim 57, wherein the disease or condition is asthma.