JP2003506409A - Compositions and methods for treating HIV infection - Google Patents

Abstract

(57) Abstract: A method for suppressing or treating human immunodeficiency virus (HIV) infection is provided. The method comprises an effective amount of a pharmaceutically acceptable composition comprising a PEG-asparaginase compound or asparaginase and at least one compound selected from the group consisting of a protease inhibitor compound, a ribonucleotide reductase inhibitor compound and an HIV reverse transcriptase inhibitor compound. To a patient in need thereof.

Description

Detailed Description of the Invention

This application is a continuation application of US patent application Ser. No. 09 / 370,390 (filing date: August 6, 1999), said US application being the international patent application No. PCT / US99 / 0.
No. 2480 (filing date: February 9, 1999), which is a partial continuation application, and the international patent application is US provisional patent application No. 60 / 074,066 (filing date: February 9, 1998).
Japan, currently abandoned). TECHNICAL FIELD OF THE INVENTION The present invention comprises a PEG-asparaginase (PEG-ASNase) compound or a pharmaceutically acceptable salt thereof, optionally a protease inhibitor compound, a ribonucleotide reductase inhibitor compound and an HIV reverse transcriptase inhibitor compound. A pharmaceutical composition comprising at least one compound selected as well as a pharmaceutically acceptable carrier. The present invention is also directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which comprises a PEG-asparaginase compound or a pharmaceutically acceptable salt thereof, and optionally a protease inhibitor compound, ribo A therapeutically effective amount of at least one compound selected from the group consisting of a nucleotide reductase inhibitor compound and an HIV reverse transcriptase inhibitor compound or a pharmaceutically acceptable salt thereof is administered to a patient in need of inhibition or treatment of HIV infection. The step of administering is included.

[0002] prior art Human immunodeficiency virus (HIV) is a retrovirus, the immune system progressive destruction (acquired immune deficiency syndrome; AIDS) of a pathogen, as well as complex diseases including degeneration of the central and peripheral nervous system. This retrovirus was previously LAV, HTLV-II.
Known as I or ARV. There are various treatments for treating HIV infection, including drug combination therapies. Protease inhibitor compounds in combination with reverse transcriptase (RT) inhibitor compounds have shown success both in vitro and in vivo by patients infected with this virus. Protease inhibitor compounds interfere with the production of new infectious virus. A common feature of HIV retrovirus growth is that the precursor polyprotein is strongly post-translationally processed by virus-encoded proteases, which complete the mature viral proteins required for viral assembly and viral function. . This inhibition of processing interferes with the production of new infectious virus.

Suppression of HIV protease by a protease inhibitor suppresses processing by a virus-derived proteolytic action at a late stage of the life cycle of HIV-1 and provirus to infected T lymphocytes in the early phase of its life cycle. May be prevented from being incorporated. HIV protease inhibitors have been well documented (A. Thomasselli et al., Chimica Oggi. 6: 272.
0 (1991); T. Meek, J. Enzyme Inhibition 6: 65-98 (1992)). A common feature of retroviruses is the post-translational processing of polyproteins. This processing is done by the HIV protease enzyme encoded by the virus. This post-translational processing step results in a mature polypeptide, which subsequently aids the formation and function of the infectious virus. If this molecular processing is suppressed, normal HIV production will stop. Therefore, it was revealed that an inhibitor of HIV protease functions as an anti-HIV viral agent.

Retroviruses are widely distributed in vertebrates and are known to cause a variety of diseases in humans and animals, including HIV, leukemia and lymphoma. All retroviruses are characterized by the presence of a unique enzyme, reverse transcriptase (RT). This enzyme transcribes viral genomic RNA into a double-stranded DNA copy. Therefore, great efforts have been made to control HIV by means of suppressing the HIV reverse transcriptase required for replication of this virus (V. Merluzzi et al., "Inhibition of the
HIV-1 Replication by a Nonnucleoside Reverse Transcriptase Inhibitor ",
Science, 25, 1411 (1990)). For example, the therapeutic compound currently used, AZT, is an inhibitor of viral reverse transcriptase (US Pat. No. 4,724,232). Unfortunately, many of these compounds have problems with toxicity, lack of bioavailability (short activity in vivo), viral resistance, or a combination thereof.

HIV Reverse Transcriptase (HIV-RT) by Combination of Nucleoside-like Drugs
It was shown that these drugs alone could not completely suppress the RT function, which in turn led to the emergence of drug resistant virus strains. These escape mutant strains reestablish and abolish nucleoside analog therapy. Addition of protease inhibitor compounds to known nucleoside analog combination therapies helped reduce long term viral load.

Ribonucleotide reductase is an enzyme that is regulated in an allosteric manner,
A complex regulatory mechanism involving one or several electron transfer pathways converts nucleoside diphosphates to the corresponding deoxynucleoside diphosphates (A.
Holmgren, (Hydrogen donor system for E. coli Ribonucleoside diphosphate
reductase dependent upon gkutathione), Proc. Natl. Acad. Sci. USA, 73: 2
275-9 (1976); L. Therlander, (Reductase of Ribonucleotides), Ann. Rev. Bi
ochem. 46: 133-58 (1979); GW Ashley & J. Stubbe, (Current ideas on the c
hemical mechanism of ribonucleotide reductase), Pharmac. Ther. 30: 301-29
(1985); J. Stubbe, (Ribonucleotide Reductase: Amazing and confusing), J.
Biol. Chem. 265: 5329-32 (1990)). For the reduction of ribonucleotides by ribonucleotide reductase, DNA polymerase is deoxyribonucleotide (dN
TP) can be used in the process of DNA replication.

Ribonucleotide reductase activity has good integration with the cell growth process,
Most of NA synthesis is markedly increased in late G1 and early S phase (JG C
orey, TW Whitford Jr. (Ribonucleotide reductase and DNA synthesis in E
hrlich ascites tumor cancer cells), Cancer Res., 32: 1301-6 (1972); E. Hamm
erstan, P. Reichard, E. Saluste, (Pyrimidine nucleosides as precursors o
f pyrimidines in polynucleotides), J. Biol. Chem., 183: 105-109 (1950). D
The importance of the role of ribonucleotide reductase in NA synthesis has made reductase a target for chemotherapeutic agents. Recently, 2-hydroxy-1H-isoindole-1,3-dione (HISID) showing ribonucleotide reductase inhibitory activity
) Was discovered (P. Nandy, EJ Lien & VI Avramis, Acta Oncologica 3
3 (8): 953-61 (1994); P. Nandy, EJ Lien & VI Avramis, Rec. Adv. Chemot
h. 1: 995-996 (1994); P. Nandy, EJ Lien & VI Avramis, Med. Chem. Res.
5: 664-679 (1995)).

PEG-asparaginase (polyethylene glycosylated form of E. coli ASP)
Has been shown to be useful as a chemotherapeutic agent. In particular, PEG-asparaginase has been shown to be another alternative with a longer circulating half-life than L-asparaginase in E. coli and was useful in multidrug chemotherapy for pediatric lymphoblastic leukemia (LJ Ettinger, AG Ettinger, VI Avramis, PS Gaynon, BioDrugs, 7
(1): 30-39 (1997)). Furthermore, PEG-asparaginase may enhance anti-leukemic effects on isolated CNS recurrence (M. Malgolowkin, S. Ortega, DA Ca.
rcich, D. Steele, D. Tischer, J. Franklin, P. Nandy, A. Periclou, LJ C
ohen, VI Avramis, Proceedings of ASCO, 17 (1998)).

PEG-asparaginase is a conjugate of asparaginase and polyethylene glycol. This conjugate results from pegylation. Pegylation is the process by which polypeptides (eg, enzymes and hormones) are conjugated with polyethylene glycol, resulting in a physiologically active, non-immunogenic, water-soluble polypeptide composition. Polyethylene glycol protects the polypeptide from loss of activity. The composition can be injected into the mammalian circulatory system and is not associated with an immune response. The process of pagination is described in US Pat. No. 4,179,337 (title: "Non
-Immunogenic Polypeptide ", filed July 28, 1977, December 18, 1979
Published), which is hereby incorporated by reference. Covalent attachment of peptides to polymers is often affected by reacting the PEG-succinimide derivative with amino groups on the outside of the protein molecule. Other methods are also disclosed in US Pat. No. 4,179,337, Pollack et al., JACS, 98: 289 (1976), US Pat. No. 4,847,325 and others in the art.

[0010] It is an object applicants have to be solved by the invention is is, in the treatment of HIV infection, PEG- asparaginase (PEG-AS
Nase) acts effectively alone, and further contains a protease inhibitor compound,
It has been found to act synergistically with one or more HIV reverse transcriptase inhibitor compounds, or ribonucleotide reductase inhibitor compounds.

Therefore, in a first aspect of the invention, the invention comprises at least one PEG-asparaginase compound, optionally a protease inhibitor compound, a ribonucleotide reductase inhibitor compound and an HIV reverse transcriptase inhibitor compound. A pharmaceutical composition comprising a compound as well as a pharmaceutically acceptable carrier is intended. The present invention also provides a PEG-asparaginase compound or a pharmaceutically acceptable salt thereof for patients in need of suppression or treatment of HIV infection,
A pharmaceutically acceptable composition optionally comprising a protease inhibitor compound and at least one compound selected from the group consisting of a ribonucleotide reductase inhibitor compound and an HIV reverse transcriptase inhibitor compound or a pharmaceutically acceptable salt thereof. A method of suppressing or treating a human immunodeficiency virus (HIV) infection, comprising administering a therapeutically effective amount.

As used above and in the description of the present invention, the following terms, unless otherwise indicated, are understood to have the following meanings: "Acyl" means H A -CO- or alkyl-CO- group is meant, which alkyl group is as described herein. Preferred acyls include lower alkyl. Typical acyl groups include formyl, acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl. "Acylamino" is an acyl-NH- group in which the acyl is as defined herein.

“Alkenyl” means an aliphatic hydrocarbon containing a carbon-carbon double bond, which may be straight or branched and contain from about 2 to about 15 carbon atoms in the chain. Preferred alkenyl groups contain 2 to about 12 carbon atoms in the chain, and more preferably about 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkenyl chain. "
"Lower alkenyl" means that about 2 to about 4 carbon atoms are present in the chain and may be straight or branched. An alkenyl group may be one or more halo or cycloalkyl groups. Typical alkenyl groups are ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n.
-Includes pentenyl, heptenyl, octenyl, cyclohexylbutenyl and decenyl.

“Alkoxy” means an alkyl-O— group in which the alkyl group is as described herein. Typical alkoxy groups include methoxy, ethoxy,
Includes n-propoxy, i-propoxy, n-butoxy, and heptoxy. "Alkoxycarbonyl" means an alkyl-O-CO- group in which the alkyl group is as defined herein. Typical alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, or t-butyloxycarbonyl.

"Alkyl" means an aliphatic hydrocarbon, which may be straight or branched and is about 1
To about 20 carbon atoms in the chain. Preferred alkyl groups have from 1 to about 12 in the chain.
Including carbon atoms of. Most preferred alkyl groups contain 1 to about 3 carbon atoms in the chain. Branched means that one or more lower alkyl groups (eg, methyl, ethyl, or propyl) are attached to a linear alkyl chain. "Lower alkyl" means containing about 1 to about 3 carbon atoms in the chain which may be straight or branched. Alkyl can be substituted with one or more "alkyl group substituents" which may be the same or different and include the following groups: halo, cycloalkyl, hydroxy, alkoxy, amino. , Acylamino, aroylamino, carboxy, alkoxycarbonyl, aralkoxycarbonyl, heteroaralkoxycarbonyl, or Y ¹ Y ² NCO--, wherein Y ¹ and Y ² are each independently hydrogen, optionally substituted alkyl, An aryl substituted by,
Optionally substituted aralkyl or optionally substituted heteroalkyl, or Y ¹ and Y ² are joined together to N, whereby Y ¹ and Y ² form a 4 to 7 membered heterocyclyl). Typical alkyl groups include methyl, trifluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl,
Includes methoxyethyl, carboxymethyl, formyl, methoxycarbonylethyl, benzyloxycarbonylmethyl, pyridylmethyloxycarbonylmethyl. Preferred alkyl substituents are halo, hydroxy, alkoxy, amino, acylamino, aroylamino, carboxy, alkoxycarbonyl, aralkoxycarbonyl, heteroaralkoxycarbonyl, sulfonyl, sulfinyl, acyl, alkanoyl or Y ¹ Y ² NCO—. .

“Alkylthio” means an alkyl-S— group in which the alkyl group is as defined herein. Typical alkylthio groups include methylthio, ethylthio, i-propylthio and heptylthio. "Alkylsulfinyl" means an alkyl-SO- group in which the alkyl group is as previously defined. Preferred groups are those where the alkyl group is lower alkyl. “Alkylsulfonyl” means an alkyl-SO ₂ — group in which the alkyl group is as defined above. Preferred groups are those where the alkyl group is lower alkyl.

“Alkynyl” means an aliphatic hydrocarbon group containing a carbon-carbon triple bond, which may be straight or branched and contain about 2 to about 15 carbon atoms in the chain. Preferred alkynyl groups contain 2 to about 12 carbon atoms in the chain, and more preferably about 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups (eg methyl, ethyl or propyl) are attached to a linear alkynyl chain. "Lower alkynyl" means there are about 2 to about 4 carbon atoms in the chain which may be straight or branched. The alkynyl group may be substituted with one or more halo. Typical alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl,
Includes heptynyl, octynyl and decynyl.

“Analog” means a compound that comprises a particular compound or a chemically modified form of that class, that retains the pharmaceutical and / or pharmacological activity characteristic of the compound. . “Aralkoxy” means an aralkyl O— group in which the aralkyl group is as defined herein. Typical aralkoxy groups include benzyloxy and 1
-Or 2-naphthalenemethoxy is included. "Aralkoxycarbonyl" means an aralkyl O-CO- group in which the aralkyl group is as defined herein. A typical aralkoxycarbonyl group is benzyloxycarbonyl. "Aralkyl" means an aryl-alkyl- group in which the aryl and alkyl are as defined herein. Preferred aralkyls contain a lower alkyl moiety. Typical aralkyl groups include benzyl, 2-phenethyl and naphthalenemethyl.

“Aralkylthio” means an aralkyl-S— group in which the aralkyl group is as defined herein. A typical aralkylthio group is benzylthio. "Aryloxycarbonyl" means an aryl-O-CO- group in which the aryl group is as defined herein. Typical aryloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl. "Aroylamino" is an aroyl-NH- group in which aroyl is as defined herein. "Aroyl" means an aryl-CO- group in which the aryl group is as defined herein. Typical groups include benzoyl and 1- and 2-naphthoyl.

“Aryl” means mono- or polycyclic aromatic, having from about 6 to about 14 carbon atoms,
Preferably it contains from about 6 to about 10 carbon atoms. The aryl may be optionally substituted with one or more "ring system substituents" which may be the same or different, where the ring system substituents are as defined herein. Typical aryl groups include phenyl or naphthyl or substituted phenyl or substituted naphthyl. "Aryldiazo" means an aryl-diazo-group in which the aryl and diazo groups are as defined herein. "Aryloxy" means an aryl-O- group in which the aryl group is as defined herein. Typical groups include phenoxy and 2-naphthyloxy. "Arylsulfonyl" means an aryl -SO ₂ group, wherein the aryl group is as defined herein. "Arylsulfinyl" means an aryl-SO- group in which the aryl group is as defined herein. "Arylthio" means an aryl-S- group in which the aryl group is as defined herein. Typical arylthio groups include phenylthio and naphthylthio.

“Carboxy” means a HO (O) C- (carboxylic acid) group. “Compound of the invention” and like expressions are intended to encompass compounds of the invention as described above and, in context, prodrugs, pharmaceutically acceptable salts and solvates thereof (eg hydrated). Thing). Similarly, reference to intermediates, whether or not they are themselves claimed, are intended to encompass their salts and solvates, depending on the context. For clarity, certain cases are sometimes displayed where the context allows, but these cases are for illustration only and are not intended to exclude other cases where the context allows.

“Cycloalkenyl” means a non-aromatic monocyclic system or a non-aromatic polycyclic system, about 3
To about 10 carbon atoms, preferably about 5 to about 10 carbon atoms and further containing at least one carbon-carbon double bond. Preferred cyclic ring sizes contain about 5 to about 6 ring atoms. Cycloalkenyl is optionally the same or different 1
Substituted with one or more "ring system substituents". The ring system substituents are as defined herein. Typical monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, cycloheptenyl and the like. A typical polycyclic cycloalkenyl is norbornylenyl.

“Cycloalkyl” means a non-aromatic monocyclic or multi-aromatic polycyclic system having from about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms. Preferred cyclic ring sizes contain about 5 to about 6 ring atoms. Cycloalkyl is optionally substituted with one or more "ring system substituents" which are the same or different. The ring system substituents are as defined herein. Typical monocyclic cycloalkyls include cyclopentyl, cyclohexyl, cycloheptyl and the like. Typical polycyclic cycloalkyls include 1-decalin, norbornyl, adamant- (1- or 2-
) Includes ill. A "derivative" is a chemically modified compound that a normal chemist would consider routine, such as an ester or amide of an acid, a protecting group (such as a benzyl protecting group for an alcohol or thiol and an amine). Butoxycarbonyl group).

“Diazo” means a divalent —N═N— group. By "effective amount" is meant an amount of a compound / composition of the invention that is capable of producing the desired therapeutic effect. As defined herein, an "electron-withdrawing group" refers to a group that, when a hydrogen atom occupies the same place in a molecule, attracts electrons to itself more strongly than hydrogen (J. Marc.
h, "Advanced Organic Chemistry", 3rd Ed., John Wiley & Sons p.17 (1985))
. They include nitro, monohaloalkyl, dihaloalkyl, trihaloalkyl (e.g., CF _3), halogen, formyl, alkylsulfonyl, include groups such as alkylsulfinyl. Preferred is halogen.

By “formulation suitable for nasal or inhalation administration” is meant a formulation in a form suitable for administration nasally or by inhalation to a patient. Such formulations can include carriers in powder form, for example, with particle sizes ranging from 1 to 500 μm, including particle sizes ranging from 20 to 500 μm in increments of 5 μm (eg, 30 μm, 35 μm, etc.). When the carrier is a liquid (eg, for administration such as a nasal spray or nose drops), suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol administration can be prepared according to conventional methods and can be administered with other therapeutic agents. Inhalation therapy can be easily administered with a metered inhaler.

By “form suitable for oral administration” is meant a form of preparation suitable for oral administration to a patient. Such formulations may be, for example, as separate units, such as capsules, cachets or tablets, each containing a predetermined amount of active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid. Or an oil-in-water solution emulsion or an aqueous-in-oil emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

By “formulation suitable for parenteral administration” is meant a formulation in a form suitable for parenteral administration to a patient. Such formulations are sterile and include emulsions, suspensions, aqueous or non-aqueous injection solutions. They contain dispersants, swelling agents, and antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic, and the pH is appropriately adjusted with respect to the blood of the intended recipient.

“Formulation suitable for enteral administration” means a formulation in a form suitable for enteral administration to a patient. Such formulations, preferably in the form of suppositories, may be prepared by mixing a compound useful in this invention with a suitable nonirritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax. The excipients or carriers mentioned above are solid at normal temperatures but liquid at body temperature and thus melt in the rectal or vaginal cavity to release the active ingredient.

By “formulation suitable for systemic administration” is meant a form of preparation suitable for systemic administration to a patient. Such formulations are preferably administered by injection, including transmuscular, intravenous, intraperitoneal, and subcutaneous injection. For injection, the useful compounds of this invention are formulated in solutions, preferably physiologically compatible buffers such as Hank's solution or Ringer's solution. Furthermore, the compounds can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Systemic administration can also be performed by transmucosal or transdermal means, and the compounds can be administered orally. For transmucosal or transdermal administration, appropriate penetrants for the barriers that must penetrate are used in the formulation. Such formulations are generally known in the art and include, for example, for buccal administration bile salts and fusidic acid derivatives. In addition, surfactants can be used to enhance penetration. Transmucosal administration is by use of nasal sprays or by, for example, suppositories. For oral administration, the compounds will be formulated in conventional oral dosage forms such as capsules, tablets and tonics.

By “formulation suitable for topical administration” is meant a formulation in a form suitable for topical administration to a patient. Such formulations may be provided as topical ointments, salves, powders, sprays and inhalants, gels (water or alcohol bases), creams, or for use as a plaster as generally known in the art. It can be incorporated into a matrix base, which will allow controlled release of the compound through the transdermal barrier. When formulated as an ointment, the active ingredients can be employed with an ointment base which can be mixed with paraffin or water. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base. Formulations suitable for topical administration to the eye include eye drops. In this case the active ingredient is dissolved or suspended in a suitable carrier, in particular an aqueous solvent for the active ingredient. Formulations suitable for topical administration in the mouth include lozenges (containing the active ingredient in a flavored base (usually sucrose and acacia or tragacanth)); troches (inert bases such as gelatin and glycerin or Sucrose and acacia)) and a mouth rinse (active ingredient in a suitable liquid carrier).

By “form suitable for vaginal administration” is meant a form of preparation suitable for vaginal administration to a patient. Such formulations include pessaries, tampons, creams, gels, pastes,
They are provided as foam or spray formulations, which in addition to the active ingredient comprise such carriers as are known in the art to be appropriate.

“Heteroaralkoxycarbonyl” means a heteroaralkyl-O—CO— group in which the heteroaralkyl group is as defined herein. A typical heteroaralkoxycarbonyl group is thienylmethylcarbonyl. "Heteroaralkyl" means a heteroaryl-alkyl- group in which the heteroaryl and alkyl are as defined herein. Preferred heteroaralkyls contain a lower alkyl moiety. Typical heteroaralkyl groups will include thienylmethyl, pyridylmethyl, imidazolylmethyl and pyrazinylmethyl. "Heteroaralkylthio" means a heteroaralkyl-S- group in which the heteroaralkyl group is as defined herein. A typical heteroaralkylthio group is pyridylmethylthio.

“Heteroaryl” means a mono- or polycyclic aromatic and contains from about 5 to about 14 carbon atoms, preferably from about 5 to about 10 carbon atoms, where one or more of the rings is The two or more carbon atoms are foreign elements other than carbon, such as nitrogen, oxygen or sulfur. Preferred cyclic ring sizes contain about 5 to about 6 ring atoms. "Heteroaryl" is also
It may be substituted with one or more "ring system substituents" which may be the same or different, and these ring system substituents are as defined herein. The name aza, oxa or thia as a prefix before heteroaryl indicates that at least nitrogen, oxygen or sulfur respectively is present as a ring atom. The nitrogen atom of the heteroaryl may be a basic nitrogen atom and may also optionally be oxidized to the corresponding N-oxide.

Typical heteroaryl and substituted heteroaryl groups include pyrazinyl, thienyl, isothiazolyl, oxazolyl, pyrazolyl, flazanyl, pyrrolyl, 1,
2,4-thiadiazolyl, pyridazinyl, quinoxalinyl, phthalazinyl, imidazo [1,2-a] pyridine, imidazo [2,1-b] thiazolyl, benzoflazanyl, azaindolyl, benzimidazolyl, benzothienyl, thienopyridyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl. , Benzazaindole, 1,2,4-triazinyl, benzthiazolyl, furanyl, imidazolyl, indolyl, indolizinyl, isoxazolyl, isoquinolinyl, isothiazolyl, oxadiazolyl, pyrazinyl, pyridazinyl, pyrazolyl, pyridyl,
Included are pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl, 1,3,4-thiadiazolyl, thiazolyl, thienyl and triazolyl. Preferred heteroaryl groups include pyrazinyl, thienyl, pyridyl, pyrimidyl, isoxazolyl and isothiazolyl.

“Heteroaryldiazo” means a heteroaryl-diazo-group in which the heteroaryl and diazo groups are as defined herein. "Heteroarylsulfonyl" aryl -SO ₂ -, group in which the heteroaryl group is as defined herein. "Heteroarylsulfinyl" means an aryl-SO- group in which the heteroaryl group is as defined herein. "Heteroarylthio" means an aryl-S- group in which the heteroaryl group is as defined herein. Typical heteroarylthio groups include pyridylthio and pyrimidinylthio.

“Heterocyclenyl” means a non-aromatic mono- or polycyclic hydrocarbon containing from about 3 to about 10 carbon atoms, preferably from 5 to about 10 carbon atoms, wherein one of the rings.
One or more carbon atoms are heterogeneous components other than carbon, such as nitrogen, oxygen or sulfur atoms, which also contain at least one carbon-carbon double bond or carbon-nitrogen double bond. Preferred cyclic ring sizes contain about 5 to about 6 ring atoms. The name aza, oxa or thia as a prefix before heterocyclenyl indicates that at least a nitrogen, oxygen or sulfur atom respectively is present as a ring atom. The heterocyclenyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents are as defined herein. The nitrogen atom of a heterocyclenyl is basic. The nitrogen atom or the sulfur atom of the heterocyclenyl may also optionally be oxidized to the corresponding N-oxide, S-oxide or S, S-oxide. Is 1,2,3,4-tetrahydrohydropyridine, 1,2-dihydropyridyl, 1,4-dihydropyridyl, 1,2,3,6-tetrahydropyridine, 1,4
, 5,6-tetrahydropyrimidine, 2-pyrrolinyl, 3-pyrrolinyl, 2-imidazolinyl, 2-pyrazolinyl and the like. Typical oxaheterocyclenyl groups include 3,4-dihydro-2H-pyran, dihydrofuranyl and fluorodihydrofuranyl. Preferred is dihydrofuranyl. A typical polycyclic oxaheterocyclenyl group is 7-oxabicyclo [2.2.1] heptenyl. Preferred monocyclic thiaheterocyclenyl rings include dihydrothiophenyl and dihydrothiopyranyl, with dihydrothiophenyl being more preferred. Amidino Preferred ring system substituents include halogen, hydroxy, alkoxycarbonylalkyl, (as defined herein) carboxyalkyl or Y ¹ Y ² -N-
Is included.

“Heterocyclyl” means a mono- or polycyclic non-aromatic saturated ring, containing from about 3 to about 10 carbon atoms, preferably from about 5 to about 10 carbon atoms, the ring carbon atoms One or more of is a heterologous element other than a carbon atom, such as nitrogen, oxygen or sulfur. Preferred cyclic rings have a ring size of about 5 to about 6 ring atoms. The name aza, oxa or thia as a prefix before heterocyclyl is at least one nitrogen,
It indicates that an oxygen or sulfur atom exists as a ring atom, respectively. Heterocyclyl is optionally one or more of the same or different "ring system substituents" (
(As defined herein)). The nitrogen atom of the heterocyclyl may be a basic nitrogen atom. The nitrogen or sulfur atom of the heterocyclyl may also be optionally oxidized to the corresponding N-oxide, S-oxide, or S, S-oxide. A typical monocyclic heterocyclyl ring has piperidyl,
Pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl,
It includes tetrahydrothiophenyl, tetrahydrothiopyranyl and the like. Preferred heterocyclyl group substituents amidino, halogen, hydroxy, alkoxycarbonylalkyl, (as defined herein) carboxyalkyl or Y ¹ Y ² N- include.

“Hydrate” refers to a solvate wherein the solvent molecule is H ₂ O. "Hydroxyalkyl" means a HO-alkyl- group in which alkyl is as defined herein. Preferred hydroxyalkyls include lower alkyls. Typical hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl. "Hygroscopic" means absorption and refers to the amount or state of acquired water sufficient to affect the physical or chemical properties of a substance (Eds. J. Swarbrick & JC Boy
lan, Encyclopedia of Pharmaceutical Technology, Vol.10, p.33).

“Liquid dosage form” means that the dosage of active compound to be administered to a patient is in liquid form. For example, it refers to pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, liquid dosage forms also include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, Ethyl acetate,
Benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-
Contains butylene glycol, dimethylformamide, oil (especially cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, fatty acid ester of polyethylene glycol and sorbitan, or a mixture of these substances. You can

“MISID (PL-7)” means a compound of formula I where R1 is a methyl group and R2 is an isopropyl group. "Regulate" means directly (by binding as a ligand to a receptor)
Or indirectly (as a precursor of the ligand, or as an inducer that stimulates the production of the ligand from the precursor), induces expression of a gene that is maintained under hormonal control, or is maintained under such control It means the ability of a compound to suppress the expression of a gene that is being treated. "Patient" includes humans and other mammals.

“PEG-asparaginase” refers to asparaginase, a protein synthesis inhibitor compound conjugated with polyethylene glycol (PEG). The polyethylene glycol preferably has an average molecular weight of about 1000 to 100,000 daltons, more preferably 4000 to 400 depending on the molecular weight of the particular protein synthesis inhibiting compound used.
It has an average molecular weight of 00 Daltons. The purpose of modification is to retain biological activity,
The molecular weight of the polymer is chosen to optimize these conditions, as it is to obtain a conjugated protein that has an in vivo half-life superior to non-conjugated protein synthesis inhibitory compounds and yet has reduced immunogenicity. Preferably, the PEG homopolymer is substituted on one end with an alkyl group, although it may be unsubstituted. Preferably the alkyl groups are C ₁ -C ₄ alkyl groups, most preferably methyl. The polymer is a monomethyl substituted PEG homopolymer and has a molecular weight of about 4000 to 40,000 Daltons. Most preferably, the PEG-asparaginase is the compound sold under the name ONCASPAR by Rhone-Poulenc Rorer.

By "pharmaceutical composition" is meant a composition containing a compound of the invention and at least one ingredient selected from the group including the following, depending on the mode of administration and the nature of the dosage form: Pharmaceutically Acceptable carriers, diluents, adjuvants, excipients, vehicles such as preservatives, fillers, disintegrating agents, wetting agents, emulsifying agents, dispersing agents, sweetening agents, flavoring agents.
g agent), fragrance, antibacterial agent, antifungal agent, lubricant and suspension agent. Examples of dispersants include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth or mixtures of these substances. Prevention of the action of microorganisms is ensured by various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol and sorbic acid. In addition, it is desirable to add isotonic agents (eg sugar, sodium chloride, etc.). Prolonged absorption of the injectable drug form is obtained by the use of absorption delaying agents such as aluminum monostearate and gelatin. Examples of suitable carriers, diluents, solvents or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (eg olive oil) and injectable organic esters (eg ethyl oleate). Examples of excipients include lactose, lactose, sodium citrate, calcium carbonate, dicalcium phosphate. Examples of disintegrants include starch, alginic acid, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc as well as high molecular weight polyethylene glycols.

“Pharmaceutically acceptable” is suitable for use in contact with human and lower animal cells in appropriate medical judgment, and is inappropriate toxicity, irritation, allergic reaction, etc. Meaning that there is a reasonable profit / risk ratio. "Pharmaceutically acceptable dosage form" means a dosage form of a compound of the invention, eg tablets, dragees, powders, elixirs, syrups, liquid preparations (including suspensions), sprays, for inhalation Included are tablets, troches, emulsions, solutions, granules, capsules, and suppositories, as well as liquid preparations for injection (including liposomal preparations). Techniques and formulations are commonly found in books ("Remington's Pharmaceutical Sciences", Mack Pu).
blishing Co., Easton, PA, latest version).

As used herein, “pharmaceutically acceptable prodrug” is suitable for use in contact with human and lower animal tissues in the appropriate medical judgment,
Prodrugs of the useful compounds of this invention that have a reasonable benefit / risk ratio without inappropriate toxicity, irritation, allergic reactions, etc., and are effective in their intended use, together with prodrugs of the compounds of this invention where possible. A zwitterionic form of a compound of the invention is meant. The term "prodrug" means a compound that is rapidly transformed in vivo to yield the parent compound of the invention (eg, by hydrolysis in blood). Functional groups that are rapidly transformed in vivo (by metabolic cleavage) constitute a class of groups that react with the carboxyl groups of the compounds of the invention. They include alkanoyl (eg acetyl,
Propionyl, butyryl, etc.), unsubstituted and substituted aroyl (eg benzoyl and substituted benzoyl), alkoxycarbonyl (eg ethoxycarbonyl), trialkylsilyl (eg trimethyl- and triethysilyl), dicarboxylic acid (
Examples include, but are not limited to, monoesters formed by succinyl), and the like.

Owing to the ease with which the cleavable groups of the compounds useful in this invention can be cleaved in vivo, compounds bearing such groups function as prodrugs. Compounds with metabolically cleavable groups have the advantage that due to the presence of the metabolically cleavable group, they may show improved bioavailability as a result of the enhanced solubility and / or absorption rate of the parent compound. Have. A complete discussion of prodrugs is provided in: "Design of Prodrugs", H. Bundgaard, ed., E.
lsevier, (1985); "Methods in Enzymology", K. Widder et al., Ed., Academic
Press, 42, p.309-396 (1985); "A Textbook of Drug Design and Development"
, Krogsgaard-Larsen & H. Bundgaard, ed., Chapter 5; "Design and Applicat
ions of Prodrugs ", p.113-191 (1991);" Advanced Drug Delivery Reviews ", H.
Bundgard, 8, p. 1-38 (1992); Journal of Pharmaceutical Sciences, 77, p. 28.
5 (1988); Chem. Pharm. Bull., N. Nakaya et al., 32, p.692 (1984); "Pro-dru.
gs as Novel Delivery Systems ", T. Higuchi & V. Stella, Vol.14 of the AC
.S. Symposium Series, and Bioreversible Carriers in Drug Design, Edward
B. Roche, ed., American Pharmaceutical Association and Pergamon Press, (1
987) (these documents are incorporated herein by reference).

“Pharmaceutically acceptable salt” refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts of compounds of the present invention. These salts can be prepared in situ (in situ) during the final isolation and purification of the compound. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Typical acid addition salts include hydrobromide, hydrochloride, sulphate, bisulphate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, Lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate,
Glucoheptonate, lactiobionate, sulfamate, malonate, salicylate, propionate, methylene-bis-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyl tartarate, methane-sulfonate, ethanesulfonate, benzene Included are sulfonates, p-toluene sulfonates, cyclohexyl sulfamate and quinate lauryl sulfonates (see for example: SM Berge et al., "Pharmaceutic.
al Salts ", J. Pharm. Sci., 66: 1-19 (1977), which is incorporated herein by reference).

Base addition salts can also be prepared by reacting the purified compound in the form of the free acid separately with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminum salts. Sodium and potassium salts are preferred. Suitable inorganic base addition salts can be prepared from metal bases. These include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide. Suitable amine base addition salts can be prepared from amines having sufficient basicity to form stable salts. These amines include amines often used in the medicinal chemistry area because of their low toxicity and acceptability in medical applications, such as ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine,
Choline, N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, efunamine. , Dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids (eg lysine and arginine)
, And dicyclohexylamine and the like.

“Ring system substituent” means a substituent attached to an aromatic or non-aromatic ring system and includes: hydrogen, alkylaryl, heteroaryl, aralkyl, heteroaralkyl, hydroxy, hydroxyalkyl. , Alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroaryl Sulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, cycloalkenyl, heterocyclenyl, aryldiazo, heteroaryldi Zone, Y ¹ Y ² N-, Y ¹ Y ² N-alkyl -, Y ¹ Y ² NCO-- or Y ¹ Y ² NSO ₂ -, wherein Y ¹ and Y ² are each substituted independently hydrogen, optionally Alkyl, optionally substituted aryl, optionally substituted aralkyl or optionally substituted heteroaralkyl, or the substituent is Y ¹ Y ² N-
One is defined acyl or aroyl herein of Y ¹ and Y ² in the case of, the other Y ¹ and Y ² in as previously defined, further substituents Y ¹ Y ² NCO
- or Y ¹ Y ² NSO ₂ - For, Y ¹ and Y ² may also together with N, whereby Y ¹ and Y ² form a heterocyclyl or heterocyclenyl from 4 coupled 7-membered. When a ring system is saturated or partially saturated, "cyclic system substituent" further methylene (H ₂ C =), including an oxo (O =), thioxo (S =).

By “solid dosage form” is meant that the dosage form made up of the compounds useful in the invention is solid, eg, capsules, tablets, pills, powders, dragees or granules. In such solid dosage forms the useful compounds of this invention are mixed with at least one inert conventional excipient (or carrier) as follows: For example sodium citrate or dicalcium phosphate or (a) fillers or swelling agents such as starch, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders such as carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, syrup. Sugar and acacia, (c) humectants such as glycerol, (d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) dissolution retarders , Paraffin, (f) absorption enhancers such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glycerol monostearate, (h) adsorbents such as kaolin and bentonite, (
i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulphate, (j) opacifiers,
k) buffering agents, and agents that release a useful compound of the invention to a portion of the intestinal tract in a delayed manner.

“Solvate” means a physical association of a compound of this invention with one or more solvent molecules. This physical bond includes a hydrogen bond. In certain cases, solvates may be isolated, for example, when one or more solvent molecules are incorporated within the crystalline lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Typical solvates include hydrates, ethanolates, methanolates and the like. In a specific embodiment, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

Preferred Embodiments A preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, wherein the method comprises an effective amount of a pharmaceutically acceptable composition comprising a PEG-asparaginase compound. Is administered to a patient in need of suppression or treatment of HIV infection. Another preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a pharmaceutically acceptable composition comprising a PEG-asparaginase compound and at least one protease inhibitor compound. Is administered to a patient in need of suppression or treatment of HIV infection.

Another preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a PEG-asparaginase compound and at least one HIV reverse transcriptase inhibitor compound. Comprising administering an effective amount of a pharmaceutically acceptable composition to a patient in need of suppression or treatment of HIV infection. Another preferred embodiment of the invention is a method of inhibiting or treating a human immunodeficiency virus (HIV) infection, which method comprises a pharmaceutically acceptable PEG-asparaginase compound and at least one ribonucleotide reductase inhibitor compound. Administering an effective amount of the composition to a patient in need of suppression or treatment of HIV infection.

Another preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, the method comprising: a PEG-asparaginase compound, at least one protease inhibitor compound and at least one ribonucleotide. It comprises administering to a patient in need of suppression or treatment of HIV infection an effective amount of a pharmaceutically acceptable composition comprising a reductase inhibitor compound. Another preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, the method comprising: a PEG-asparaginase compound, at least one protease inhibitor compound and at least one HIV reverse transcriptase inhibitor. It comprises administering an effective amount of a pharmaceutically acceptable composition comprising a compound to a patient in need of controlling or treating HIV infection.

Another preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a PEG-asparaginase compound, at least one ribonucleotide reductase inhibitor compound and at least one It comprises administering to a patient in need of suppression or treatment of HIV infection an effective amount of a pharmaceutically acceptable composition comprising an HIV reverse transcriptase inhibitor compound. Another preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, the method comprising: a PEG-asparaginase compound, at least one protease inhibitor compound, at least one ribonucleotide reductase inhibitor compound. And administering an effective amount of a pharmaceutically acceptable composition comprising at least one HIV reverse transcriptase inhibitor compound to a patient in need of suppression or treatment of HIV infection. Another preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, the method comprising: a PEG-asparaginase compound;
Administering a therapeutically effective amount of at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcriptase inhibitor compounds to patients in need of inhibition or treatment of HIV infection. Including. Another preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises administering an effective amount of a pharmaceutically acceptable composition comprising asparaginase to inhibit HIV infection. Alternatively, it includes administering to a patient in need of treatment.

Another preferred embodiment of the present invention is a method of inhibiting or treating human immunodeficiency virus (HIV) infection, the method comprising asparaginase and protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV.
Administering an effective amount of a pharmaceutically acceptable composition comprising at least one compound selected from the group consisting of reverse transcriptase inhibitor compounds to patients in need of inhibition or treatment of HIV infection. Another preferred embodiment of the invention is a method of inhibiting or treating a human immunodeficiency virus (HIV) infection, which method comprises at least one ribonucleotide reductase inhibitor compound of formula I: Salt, its N-
Administering an effective amount of a pharmaceutically acceptable composition comprising oxide, a solvate thereof, acid bioisostea or a prodrug thereof to a patient in need of suppression or treatment of HIV infection:

[0056]

[0057] Wherein R1 is an alkyl, alkenyl, alkynyl or electron withdrawing group; and R2 is alkyl, alkenyl, alkynyl.

Another preferred embodiment of the present invention suppresses the production of HIV or HIV
In a method of limiting the spread of PEG, the method comprises: Exposing the HIV infected cell population to an effective amount. Another preferred embodiment of the present invention is a method of inhibiting HIV reverse transcriptase activity, the method comprising: a PEG-asparaginase compound or asparaginase;
Optionally contacting HIV reverse transcriptase with a composition comprising at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcriptase inhibitor compounds.

Another preferred embodiment of the present invention is a method of inhibiting HIV reverse transcriptase activity, which method comprises contacting HIV reverse transcriptase with a composition comprising a compound of formula I. Yet another preferred embodiment of the present invention is a method of inhibiting HIV reverse transcriptase activity, which method comprises contacting HIV reverse transcriptase with a composition comprising MISID having the formula:

[0060]

Another preferred embodiment of the present invention is a method of inhibiting HIV reverse transcriptase activity, which method comprises contacting HIV reverse transcriptase with a composition comprising a PEG-asparaginase compound. Yet another preferred embodiment of the present invention is a method of selectively inhibiting HIV-RNA production, the method comprising infecting a pharmaceutically acceptable composition comprising a PEG-asparaginase compound and at least one protease inhibitor compound with HIV infection. Including exposing the cell population. Yet another preferred embodiment of the invention is a method of selectively inhibiting HIV-RNA production, the method exposing a HIV-infected cell population to a pharmaceutically acceptable composition comprising a PEG-asparaginase compound and saquinavir. Including that.

Another preferred embodiment of the present invention is a method of selectively inhibiting HIV-RNA production, which comprises a PEG-asparaginase compound or asparaginase, and optionally a protease inhibitor compound, a ribonucleotide reductase inhibitor compound. And contacting the HIV infected cell population with a composition comprising at least one compound selected from the group consisting of: and an HIV reverse transcriptase inhibiting compound. Yet another preferred embodiment of the present invention is a method of inhibiting HIV-RNA production,
The method comprises contacting a HIV infected cell population with a composition comprising a compound of formula I.

Another preferred embodiment of the present invention is a method of inhibiting HIV-RNA production,
The method comprises contacting a HIV infected cell population with a composition comprising MISID. Yet another preferred embodiment of the present invention is a method of inhibiting HIV-RNA production,
The method comprises contacting a HIV infected cell population with a composition comprising a PEG-asparaginase compound. According to yet another preferred embodiment of the present invention, the protease inhibitor compound is selected from saquinavir, nelfinavar, enedinovia, indinavar, ritonavar, crixiban, viracept, norver and VX-478.

According to a more preferred embodiment of the present invention, the protease inhibitor compound is saquinavir. According to another preferred embodiment of the invention, the HIV reverse transcriptase inhibitor compounds are AZT (retrobar, zidovudine), ddI (bidex, didanosine), dd
It is selected from C (hibid, zarcitabine), d4T (zerit, stavudine) and 3TC (epibar, lamivudine). According to another preferred embodiment of the invention, the HIV reverse transcriptase inhibitor is A
It is ZT.

According to another preferred embodiment of the present invention, the ribonucleotide reductase inhibitor compound is selected from: hydroxyurea (HU), BW-3.
48U87, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) amidox (VF236; NSC-343341; N, 3,4-
Trihydroxybenzencarboximidamide), BILD1257 (2-
Benzyl-3-phenylpropionyl-L- (N-methyl) valyl-L-3 (methyl) valyl-L- (N4, N4-tetramethylene) asparaginyl-L- (3
, 3-tetramethylene) aspartyl-L- (4-methyl) leucine), BIL
D1357 (2-benzyl-3-phenylpropionyl-L- (N-methyl) valyl-L-3- (methyl) valyl-L- (N4, N4-tetramethylene) asparaginyl-L- (3,3-tetramethylene) Aspartic acid 1- [1 (R) -ethyl-2,2-dimethylpropylamide)], BILD1633, BILD733
(3-Phenylpropionyl-L- (N-methyl) valyl-L- [3-methyl)
Baryl-L- [3- (pyrrolidin-1-ylcarbonyl)] alanyl-L- (1
-Carboxycyclopentyl) glycyl-L- (4-methyl) leucinol),
BILD1263 (2-benzyl-3-phenylpropionyl-L- (N-methyl) valyl-L-3- (methyl) valyl-L- (N4, N4-tetramethylene)
Asparaginyl-L- (3,3-tetramethylene) aspartyl-L- (4-methyl) leucinol), BILD1351 (1- [1 (S)-[5 (S)-[3
-[(All-cis) -2,6-dimethylcyclohexyl] ureido] -2 (S
)-(3,3-Dimethyl-2-oxobutyl) -6,6-dimethyl-4-oxoheptanoylamino] -1- [1 (R) -ethyl-2,2-dimethylpropylcarbamoyl] methyl] cyclo Pentanecarboxylic acid]), CI-F-ara A (2-chloro-9- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)
Adenine, DAH (D-aspartic-beta-hydroxamate), DD
FC (2'-deoxy-2 ', 2'-difluorocytidine), Didox (VF1
47; NSC324360; N, 3,4-trihydroxybenzamide), Eu
rd (3'-ethynyl uridine), GTI2040, GTI2501, IMHA
G (1-isoquinolylmethane-N-hydroxy-N'-aminoguanine), LY
207702 (2 ', 2'-difluoro-2'-deoxyribofuranosyl-2,6
-Diaminopurine), LY295501 (N-[[3,4-dichlorophenyl)
Amino] carbonyl] 2,3-dihydro-5-benzofuransulfonamide),
MDL101731 (FMdC; KW2331; (2E) -2'-deoxy-2 '
-Fluoromethylene) cytidine), parabactin, slofena (LY1866)
41; N-[[(4-chlorophenyl) amino] carbonyl] -2,3-dihydro-1H-indene-5-sulfonamide), TAS106 (Ecyd; 3'-
Ethynyl cytidine), triapin (OCX191; OCX0191), trimidox (VF233; N, 3,4,5-tetrahydroxybenzenecarboximidamide), and compounds of formula I:

According to another preferred embodiment of the present invention, the ribonucleotide reductase inhibitor compound is a compound of formula I. According to another preferred embodiment of the present invention, the present invention is directed to a method of suppressing or treating a human immunodeficiency virus (HIV) infection, which method comprises the compound of formula I, or a pharmaceutically acceptable compound thereof. Administration of a pharmaceutically effective amount of a possible salt, its N-oxide, its solvate, its acid bioisostea or its prodrug to a patient in need of inhibition or treatment of HIV infection. In formula I, R1 is lower alkyl, lower alkenyl, lower alkynyl, or an electron withdrawing group, and R2
Is lower alkyl, lower alkenyl, lower alkynyl.

According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a compound of formula I, or a pharmaceutical agent thereof. Pharmaceutically acceptable salts, their N-oxides, their solvates, their acid bioisostea or their prodrugs are administered to patients in need of inhibition or treatment of HIV infection. In the formula of Formula I, R1 is lower alkyl, lower alkenyl, lower alkynyl, or halogen group, and R2 is lower alkyl, lower alkenyl, lower alkynyl.

According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a compound of formula I, or a medicament thereof. Pharmaceutically acceptable salts, their N-oxides, their solvates, their acid bioisostea or their prodrugs are administered to patients in need of inhibition or treatment of HIV infection. In the formula of formula I, R1 is lower alkyl or a halogen group, and R2 is lower alkyl. According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a compound of formula I, or a pharmaceutically acceptable compound thereof. Administration of a possible salt, its N-oxide, its solvate, its acid bioisostea or its prodrug to a patient in need of inhibition or treatment of HIV infection. In the formula of formula I, R1 is a halogen group and R2 is lower alkyl.

According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a compound of formula I, or a pharmaceutical agent thereof. Pharmaceutically acceptable salts, their N-oxides, their solvates, their acid bioisostea or their prodrugs are administered to patients in need of inhibition or treatment of HIV infection. In formula I, R1 is lower alkyl and R2 is lower alkyl. According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a compound of formula I, or a pharmaceutically acceptable compound thereof. Administration of a possible salt, its N-oxide, its solvate, its acid bioisostea or its prodrug to a patient in need of inhibition or treatment of HIV infection. In the formula of formula I, R1 is a bromine or chlorine atom, and R2 is a methyl group or an isopropyl group.

According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a compound of formula I, or a pharmaceutical agent thereof. Pharmaceutically acceptable salts, their N-oxides, their solvates, their acid bioisostea or their prodrugs are administered to patients in need of inhibition or treatment of HIV infection. In the formula of formula I, R1 is a methyl group and R2 is lower alkyl. According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, which method comprises a compound of formula I, or a pharmaceutically acceptable compound thereof. Administration of a possible salt, its N-oxide, its solvate, its acid bioisostea or its prodrug to a patient in need of inhibition or treatment of HIV infection. In formula I, R1 is a methyl group and R2 is an isopropyl group.

According to another preferred embodiment of the present invention, the protein synthesis inhibitor compound is PEG
-Asparaginase. According to another preferred embodiment of the invention, the protease inhibitor compound is saquinavir or enedinovia. According to another preferred embodiment of the present invention, the protease inhibitor compound is saquinavir. According to another preferred embodiment of the invention, the HIV reverse transcriptase inhibitor compound is selected from AZT and 3-TC. According to another preferred embodiment of the present invention, the ribonucleotide reductase inhibitor compound is MISID (PL-7).

According to another preferred embodiment of the present invention, the compounds used according to the present invention are administered in a simultaneous combination. According to another preferred embodiment of the invention, the compounds used according to the invention are administered continuously. According to another preferred embodiment of the invention, the compounds used according to the invention are administered continuously, preferably with a protease inhibitor, followed by PE.
A G-asparaginase compound or asparaginase is administered. According to another preferred embodiment of the invention, the compound used according to the invention is administered continuously, preferably saquinavir, followed by the PEG-asparaginase compound or asparaginase. According to another preferred embodiment of the invention, the compounds used according to the invention are consecutively, preferably saquinavir, followed by a PEG-asparaginase compound or asparaginase, followed by an HIV reverse transcriptase inhibitor compound and ribonucleotides. One or more compounds selected from the group consisting of reductase inhibitor compounds are administered in sequence.

According to another preferred embodiment of the present invention, the present invention is directed to a method of suppressing or treating a human immunodeficiency virus (HIV) infection, the method comprising: a PEG-asparaginase compound or asparaginase; , A protease inhibitor compound, a ribonucleotide reductase inhibitor compound, and at least one compound selected from the group consisting of HIV reverse transcriptase inhibitor compounds, which are administered synergistically to a patient in need of inhibition or treatment of HIV infection. including. According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, the method comprising: a PEG-asparaginase compound and at least one protease inhibitor compound. In a synergistic combination to administer to a patient in need of suppression or treatment of HIV infection. According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, the method comprising a PEG-asparaginase compound and at least one ribonucleotide reductase. Including a synergistic combination of inhibitor compounds administered to a patient in need of suppression or treatment of HIV infection.

According to another preferred embodiment of the present invention, the present invention is directed to a method of suppressing or treating a human immunodeficiency virus (HIV) infection, the method comprising a PEG-asparaginase compound and at least one It includes administering a HIV reverse transcriptase inhibitor compound synergistically in combination to a patient in need of inhibition or treatment of HIV infection. According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, the method comprising: a PEG-asparaginase compound and at least one protease inhibitor compound. And at least one ribonucleotide reductase inhibitor compound in a synergistic combination to administer to a patient in need of suppression or treatment of HIV infection.

According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating a human immunodeficiency virus (HIV) infection, which method comprises a PEG-asparaginase compound and at least one Synergistic combination of a protease inhibitor compound and at least one HIV reverse transcriptase inhibitor compound is administered to a patient in need of inhibition or treatment of HIV infection. According to another preferred embodiment of the present invention, the present invention is directed to a method of inhibiting or treating human immunodeficiency virus (HIV) infection, the method comprising a PEG-asparaginase compound and at least one ribonucleotide reductase. Synergistically combining an inhibitor compound and at least one HIV reverse transcriptase inhibitor compound, and administering to a patient in need of inhibition or treatment of HIV infection.

Yet another object of the present invention is to provide a kit containing a plurality of active ingredients (with or without carrier). The active ingredients can be effectively used to combine them to carry out the novel combination therapies of the present invention. Yet another object of the present invention is to provide novel pharmaceutical compositions which, alone or by themselves, can be used in beneficial combination therapies because they comprise a plurality of active ingredients which can be used according to the invention.

Without wishing to be bound by theory, it is believed that the present invention works by the following mechanism. T cells are the main cells in the mammalian body that HIV virus infects. T cells are considered to be the virus factory in the case of HIV infection. To reinfect new T cells, the HIV virus must enter and replicate in T cells. This viral replication and encapsulation process requires the participation of intracellular enzymes. PEG-asparaginase (a protein synthesis inhibitor unique to thymocytes) inhibits the synthesis of enzymes required for competent replication and HIV virus assembly and provides other anti-proliferative effects against the HIV virus. It has been shown to be very useful. Furthermore, the combination of PEG-asparaginase or asparaginase and one or more compounds selected from the group consisting of a protease inhibitor compound, an HIV-RT inhibitor compound and a ribonucleotide reductase inhibitor compound shows a synergistic effect, and the virus is prolonged. Reduce the load.

The intracellular mechanism by which PEG-asparaginase is believed to function is:
By blocking the supply of the amino acid asparagine (Asn), HIV-infected T
It inhibits cells from synthesizing intracellular proteins and viral proteins. Therefore, treatment of infected cells with an asparaginase such as PEG-asparaginase suppresses the synthesis of cellular and viral proteins. These proteins are proviruses (virus-derived DNA integrated into mammalian DNA)
Is required for the transcription and translation of the virus-encoded gene from Escherichia coli. Virus-derived RNA
Once is transcribed from the provirus by RNA polymerase, there are two pathways that follow. This RNA is used by ribonucleotides to produce virus-derived proteins (eg HIV-RT). Later, the same RNA is processed by the rev-protein to become HIV-1 genomic RNA. This RNA binds to already synthesized HIV-RT, and when two such molecules are present together, they constitute the novel HIV viral genomic material. HIV protease is required for processing virus-derived proteins for final viral packaging. The new HIV-1 virus can subsequently germinate from T cells as a new whole virus. Thus, protease inhibitor compounds in combination with asparaginase, such as PEG-asparaginase, can synergistically suppress the processes required for HIV-1 virus replication.

The compositions and methods of treatment of the present invention are useful for inhibiting HIV protease, preventing and treating HIV infection, and treating the resulting pathological condition (eg, AIDS). The treatment of AIDS or the prevention or treatment of HIV infection refers to a wide range of HIV infection conditions, namely AIDS, ARC (AIDS) related complications (obvious and subclinical, and acute or potentially exposed to HIV). However, it is not limited to these. For example, the compounds of the present invention may be used in the treatment of HIV infection after potential previous HIV exposures (eg, blood transfusions, fluid exchanges, bites, accidental needle sticks, and contamination with patient blood during surgery). It is useful.

In the therapeutic or prophylactic method of the present invention, a PEG-asparaginase compound or asparaginase, and optionally a protease inhibitor compound, HIV
One compound selected from the group consisting of a reverse transcriptase inhibitor compound and a ribonucleotide reductase inhibitor compound can be administered as a combination therapy in various ways, eg optionally using a dosing regimen. For example, a PEG-asparaginase compound and optionally one or more compounds selected from the group consisting of a protease inhibitor compound, an HIV reverse transcriptase inhibitor compound and a ribonucleotide reductase inhibitor compound can be administered to a patient simultaneously or separately. Can be administered at the time of. However, when they are administered at different times, they must be administered for a suitable period of time such that a pharmaceutically effective amount of both compounds is present in the patient's body so as to bring about the therapeutic effect of the present invention.

Therefore, another object of the present invention is to provide a kit for treating or preventing a physiological condition associated with HIV, wherein the kit comprises a plurality of separate containers, wherein at least the containers are One contains a PEG-asparaginase compound or asparaginase, and at least another container is one or more selected from the group consisting of a protease inhibitor compound, an HIV reverse transcriptase inhibitor compound and a ribonucleotide reductase inhibitor compound. In addition, the container optionally comprises a pharmaceutical carrier so that the kit can be effectively utilized to carry out the combination therapies of the invention.

Accordingly, another object of the present invention is to provide a kit for treating or preventing a physiological condition associated with HIV, which kit comprises a plurality of separate containers, wherein at least one of the containers is provided. One containing the compound of formula I, and at least one further container is one or two selected from the group consisting of PEG-asparaginase compounds, protease inhibitor compounds, HIV reverse transcriptase inhibitor compounds and ribonucleotide reductase inhibitor compounds. In addition to containing the compounds described above, the container optionally comprises a pharmaceutical carrier so that the kit can be effectively utilized to perform the combination therapies of the invention.

In yet another embodiment of the kit, at least one of the containers comprises a PEG-asparaginase compound without a protease inhibitor compound, an HIV reverse transcriptase inhibitor compound or a ribonucleotide reductase inhibitor compound,
Still at least another container should contain no PEG-asparaginase compound but one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transcriptase inhibitor compounds and ribonucleotide reductase inhibitor compounds. Is.

In another embodiment of the kit, at least one of said containers is free of PEG-asparaginase compound, protease inhibitor compound, HIV reverse transcriptase inhibitor compound or another ribonucleotide reductase inhibitor compound of formula I One or two selected from the group consisting of a compound, and at least another container, but not a compound of formula I, consisting of a protease inhibitor compound, an HIV reverse transcriptase inhibitor compound and another ribonucleotide reductase inhibitor compound. It should include the above compounds.

In yet another embodiment of the kit, at least one of said containers is free of PEG-asparaginase compound, protease inhibitor compound, HIV reverse transcriptase inhibitor compound or another ribonucleotide reductase inhibitor compound.
ISID (PL-7) and at least another container is MISID
It should be free of (PL-7) but should include one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transcriptase inhibitor compounds and ribonucleotide reductase inhibitor compounds.

It will be appreciated that the invention embraces all suitable combinations of the particular and preferred classes mentioned herein. The compounds of the invention (eg raw materials, intermediates or final products) are prepared according to the description herein or apply or apply known methods previously used or described in the literature. Manufactured by The compounds of formula I can be prepared by applying or adapting the known methods previously used or described in the literature. Especially in the literature (P. Nandy, EJ Lien, VI Avramis, M
ed. Chem. Res. 5: 664-679 (1995)) by known methods for preparing the derivatives of formula I.

The useful compounds of this invention are optionally supplied as salts. Pharmaceutically acceptable salts are of particular benefit as they can be administered for medical purposes. Pharmaceutically unacceptable salts are useful in the manufacturing process for isolation and purification purposes, and in some cases are useful in separating stereoisomers of compounds of the invention. The latter is especially true in the case of amine salts prepared from optically active amines.

When the useful compounds of the present invention contain a carboxy group or a sufficiently acidic bioisostere, base addition salts can be formed which, in turn, can be made into a more convenient and convenient form, and in fact the salt is used. This essentially uses the free acid form. Furthermore, when the useful compounds of this invention contain a basic group or a sufficiently basic bioisostere, an acid addition salt can be formed, which in turn can be made into a more convenient and convenient form. The use essentially uses the free base form. The aforementioned compounds useful in the present invention can also be mixed with another therapeutic compound to form a pharmaceutical composition (with or without a diluent or carrier).
Such pharmaceutical compositions provide for the simultaneous administration of the combined active ingredients, which administration results in the combination therapy of the invention.

While it is possible for the compounds of this invention to be administered alone, it is preferable to present them as a pharmaceutical composition. Useful pharmaceutical compositions of this invention for both veterinary and human use include at least one compound of the invention as defined above in one or more acceptable carriers and optionally other Included with therapeutic ingredients. In one preferred embodiment, the active ingredients required for combination therapy are only one for co-administration.
They can be combined in one pharmaceutical composition.

The choice of carrier and the content of active substance in the carrier is generally determined according to the solubility and chemical properties of the active compound, the particular mode of administration and the conditions which result in the formulation. Excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate, and disintegrants such as starch, alginic acid and certain complex silicates are used as lubricants (eg magnesium stearate, sodium lauryl sulfate and Talc) can be used to produce tablets. To make capsules, it is advantageous to use lactose and high molecular weight polyethylene glycols. If aqueous suspensions are used, they can contain emulsifying agents which facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof can also be used.

The oily phase of the emulsions of the present invention can be composed of known ingredients in a known manner. If the oily phase simply comprises an emulsifier (alternatively known as emulgent), preferably the oily phase comprises a fat or an oil, or both a fat and an oil and a mixture of at least one emulsifier. Preferably a hydrophilic emulsifier is included with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to contain both oils and fats.
Taken together, the emulsifier forms an emulsified wax with or without stabilizers and is further mixed with oils and fats to form an emulsified ointment base, which emulsifies the oily dispersed phase of the cream formulation. Form. Emulgents and emulsion stabilizers suitable for use in the formulations of the present invention include Tween 60, Tween 60, cetostearyl alcohol, benzyl alcohol, myristyl. Includes alcohol, glycerol mono-stearate and sodium lauryl sulfate.

If desired, the aqueous phase of the cream base is, for example, at least 30% (w / w) of a polyhydric alcohol (ie an alcohol with two or more hydroxy groups).
, Propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycols (including PEG400) and mixtures thereof. If desired, topical formulations may include compounds that enhance the absorption or penetration of the active ingredient into the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

The choice of suitable oil or fat for the formulation is based on achieving the desired cosmetic properties. Therefore, the cream is preferably a non-greasy, stain-free product that can be washed away and has a suitable viscosity so that it does not leak from tubes and other containers. Linear or branched monobasic or dibasic alkyl esters such as di-isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or Crodamol.
ol) A mixture of branched chain esters known as CAP can be used, but the last three are preferred. These may be used alone or in combination depending on the properties desired. Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used. With excipients such as lactose or lactose as well as high molecular weight polyethylene glycols, solid ingredients can also be used as fills in soft and hard filled gelatin capsules.

The pharmaceutical composition may be administered to a human or animal by a local or systemic administration in a suitable formulation. This local or systemic administration includes oral, inhalation, rectal, nasal, buccal, sublingual, vaginal, parenteral (subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural administration). ), Including intracisternal and intraperitoneal administration. It will be appreciated that the preferred route will vary with for example the condition of the recipient. The formulations may be prepared in unit dosage form by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations can be prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

A tablet may be made by tableting or moulding, optionally with one or more accessory ingredients. Tablets are free-flowing forms (eg powders or granules), optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant.
Can be prepared by compression on a suitable machine. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert diluent. Tablets
They may optionally be coated or scored and may be formulated to provide a sustained or controlled release of the active ingredient therein. Solid compositions for rectal administration include suppositories formulated in a known manner and containing at least one compound of the invention.

If desired and for more effective distribution, sustained release or targeting systems (eg biocompatible, biodegradable polymer matrices (eg poly (d, l-lactide coglycolide)), liposomes. , And microspheres) by incorporating or binding the compound as microcapsules and further providing continuous sustained release of the compound for a period of 2 weeks or more by subcutaneous or intramuscular depot by a technique called depot. Or it can be injected intramuscularly. The compounds can be sterilized by, for example, filtering a bacterial retention filter or by incorporating the bactericide in a sterile solid composition which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.

The actual dosage level of active ingredient in the compositions of the present invention will vary to obtain the effective dose of active ingredient to give the desired therapeutic response for the particular composition and method of administration. . The dosage level selected will thus depend on the desired therapeutic effect, the route of administration, the desired duration of treatment and other factors. The daily dosage of the compounds of the present invention administered to the host once daily or in divided doses may be, for example, from about 0.001 to about 100 mg / kg body weight per day, preferably 0.01 to 10 mg / kg. / Day. Unit dose compositions may contain sub-amounts such that they can be used to obtain daily dosages. However, the specific dose for an individual patient depends on weight, general health, sex,
It will be appreciated that it will depend on a variety of factors including diet, time and route of administration, rate of absorption and excretion, combination with other agents and the severity of the particular disorder treated.

The amount of each component administered depends on the etiology and severity of the disease, the condition and age of the patient,
It is decided by the attending physician taking into consideration the efficacy of each component and other factors. The formulations can be presented in single-dose containers or multi-dose containers (eg, sealed ampoules and vials with elastic stoppers) and can be stored in lyophilized form. In the latter case, only a sterile liquid carrier (eg water for injection) is added immediately before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

The compounds of the present invention, their method of preparation, and their biological activity will become more apparent from a scrutiny of the following examples. These examples are provided by way of illustration and should not be construed as limiting the scope of the invention. Procedures for assessing the biological activity of a compound or composition of the invention may be carried out as described herein, or used previously, or utilize or apply known procedures described in the literature. It was carried out.

Examples General Methodology for Non-Radioactive HIV Reverse Transcriptase Assay (Boehringer Mannheim ) The following is a general procedure for the HIV reverse transcriptase assay. Day 1-Samples were virus + supernatant (7 days incubation) and pellets obtained from drug flasks. These did not heat deactivate. -The sample was centrifuged at 2000g for 30 minutes at 4 ° C. 2000g was achieved using 2500 rpm. -Transfer the supernatant to a sterile labeled tube. -0.5 ml PEG solution was added. 1.2M NaCl was used as a diluent for PEG. PEG solution: 30% w / v, 30 g in 100 ml. --Fully mixed. Incubated o / n at --0 ° C. (in ice in refrigerator). Day 2--500 μl of sample was centrifuged at 8000 g for 10 minutes at 4 ° C. 8000g was achieved using 8000rpm. --Discard the supernatant. Care was taken to remove all PEG drops from the sample. -40 μl lysis buffer was added. --Pellets were completely resuspended. -Transfer the suspension to a new reaction tube. -Incubated for 30 minutes at room temperature (25 ° C). --Made standard dilutions.

[0101]

-40 μl of standard solution was transferred to a reaction tube (n = 7 × 2). --Prepared reaction buffer. The template (vial 4) was reconstituted in 430 μl of autoclaved water. Add 1 ml incubation buffer / nucleotide vial (vial 3). Add 100 μl of the reconstituted template (vial 4) to the nucleotide solution vial (
Added to vial 3). --20 μl of reaction buffer was added to all tubes, unknown and standard.
-Incubated in an incubator rack at 37 ° C for 15 hours. Day 3--Wordperfect was used to make templates for the ELISA assay. -Opened the foil packet and built the mtp (microtiter plate) module using the frame and strip provided in the kit. Unknown n =. Therefore all samples n =. Standard n =. Each strip has 8 wells, therefore Needed a strip of. Note: You need to round up to the nearest multiple of 8. -Transfer 60 μl per template from the reaction tube to the corresponding well of the mtp module. --Mtp covered with the provided cover strip.

-Incubated for 1 hour at 37 ° C in an incubator. --If necessary, wash solution was prepared. Note : The solution provided is a 10 × solution and therefore needs to be diluted with autoclaved water. ^** 1 bottle of wash solution was made by adding 225 ml of autoclaved water to the provided bottle. Mix well. It was kept on ice during the assay. -The solution was completely removed by decantation. --250μ per rinse using a 30 second soak time before decantation
The plate was washed 5 times with 1. --Anti-dig-pod working solution
It was created. ^** Anti-dig-pod working solution was made. 500 μl of autoclaved water was added to the anti-dig-pod vial (vial # 6) and stored at 4 ° C. I did not freeze. ^** Preparation of anti-dig-pod working solution The required volume was calculated. Well × 200 μl = 50 μl of anti-dig-pod solution (vial # 6) was used for each 4.95 ml conjugated dilution buffer (solution # 8). ml anti-dig-pod solution (vial # 6), Added to ml of Conjugate Dilution Buffer (solution # 8). -200 μl of anti-dig-pod working solution was added per mtp well. --Mtp covered with the provided cover strip. -Incubated for 1 hour at 37 ° C in an incubator. -The solution was completely removed by decantation. --250μ per rinse using a 30 second soak time before decantation
The plate was washed 5 times with 1. -The abts substrate solution was made using the enhancer. ^** Preparation of abts substrate solution. Abts powder mix (vial # 9) in a bottle of substrate buffer (bottle # 9).
10) was dissolved. The required capacity was calculated. Well × 200 μl = ml ^{** The} appropriate amount of enhancer was added to the solution. 1 ml of abts substrate solution (
1 mg of substrate enhancer (vial # 11) for each of bottles # 9)
It was used. mg of substrate enhancer (vial # 11) each Add to ml abts substrate solution (bottle # 9). --200 μl of abts substrate solution was added per well of mtp with enhancer. Plates were read at 405 nm (reference wavelength 490 nm) after -10, 20 and 30 minutes.

Example 1 Treatment of Combinations of PEG-ASNase Compound and Saquinavir Materials and Methods The cell line used in these studies was the CCRF / CEM / 0, human T leukemia cell line. PEG-ASNase (ONCASPAR) is Rhone-Poulenc Ro
Supplied by rer. Saquinavir was commercially available. RPMI-1640 medium (Irvine Scientific, Irvine, CA) was added to 10% fetal bovine serum (Gemini Biosourc).
e, Calabasas, CA), 5% 1M Hepes buffer and 5% non-essential amino acids (
Irvine Scientific, Irvine, CA). The drug concentrations used were as follows: PEG-ASNase IC50 alone: 0.4 IU / ml saquinavir IC50 alone: 25 μM PEG / SAQ combo IC50: 0.233 IU / ml + 15.52 μM In summary, 2 × 10 ⁶ cells / ml at 37 ° C. under 5% CO ₂ , PHA + medium for 48 hours. Further, the same number of cells were incubated in a medium containing no PHA (phytohemagglutinin) for 48 hours to serve as a negative control. At this point, cells were inoculated with HIV-1 virus by standard protocols. PEG-ASNase and / or saquinavir were added to the cells at the appropriate concentration (see above). During the 7-day exposure, control cells were resuspended in drug-free medium. On day 5, a 1 ml aliquot of medium was removed from the flask and stored under liquid nitrogen. On day 7, one or more 1 ml aliquots of medium were taken from the flask and stored under liquid nitrogen. In addition, pellet the remaining cells,
Stored at -80 ° C. The sample obtained according to this example was itemized and assayed for HIV-RT using a non-radioactive reverse transcriptase assay (Boehringer Mannehim). A calibration curve was determined and HIV-RT levels were calculated for experimental samples.

Results The first observation from the HIV-RT assay in T cell-derived samples is that there was no HIV-RT / virus in the supernatant of CEM / 0 T cells after treatment. The experimental results are shown in FIG. The T cell pellet itself was tested for intracellular HIV-RT. 0.4 IU / m
1 (approximately IC ₅₀ concentration) of PEG-ASNase showed HIV-RT inhibition of about 30%. Saquinavir, an HIV protease inhibitor compound, at 25 μM (approximately IC ₅₀ concentration) alone, depleted HIV-RT activity by approximately 70% compared to untreated control cell culture HIV-RT. Finally, we have found that PEG-AS
The simultaneous combination of Nase and saquinavir was synergistic, indicating that the IC ₅₀ concentrations of drugs in these combinations were 0.233 IU / ml and 14.5 μM, respectively. These concentrations were much lower than their respective IC ₅₀ values in CEM / 0 T cells. The combination treatment of PEG-ASNase and saquinavir inhibited intracellular HIV-RT by about 82.3% compared to untreated control values. Study after these drug treatments, since T cells did not let reduce HIV-1 in the supernatant (shed), PEG-ASNase and Saquinavir are not only synergistic against T-cells, the HIV-1 It seems to be selective and synergistic. These agents were fully inhibitory in the release of the new HIV-1 particles into the medium (equivalent to patient serum or plasma). The fact that the combination at low concentrations is more inhibitory to HIV-RT than most of the activity of the two drugs at high concentrations, saquinavir, suggests that this combination is potent at selectively inhibiting HIV at proviral levels. Suggests. These agents and their combinations suppress / inhibit HIV-RT intracellularly, suggesting that they inhibit HIV at proviral levels. In other words, the integrated HIV provirus produces mRNA, which is not translated into viral proteins and thus inhibits the production of intact viral particles that are separated in RT or medium. Therefore, on a theoretical basis, further HIV infection cannot be achieved in uninfected T cells.

Example 2: Cytotoxicity Assay Materials and Methods The human leukemia T cell line (hereinafter CEM / 0) was used in this example. PE
G-ASNase is a Rhone-Poulenc Rorer under the trade name Oncaspar (registered trademark).
Obtained from Pharmaceuticals Inc. Saquinavir was obtained from Roche laboratories under the trade name Invirase®. The RPMI-1640 medium obtained from Irvine Scientific, Irvine was added to 10% fetal bovine serum (Gemini Biosource,
Calabasas, CA), 5% 1M Hepes buffer and 5% non-essential amino acids (obtained from Irvine Scientific, Irvine, CA). Experiments were conducted to measure the cytotoxicity of saquinavir and PEG-ASNase. To measure the cytotoxicity of either compound alone, 2 × 10 ⁵ cells / m 2
1 was incubated in 24-well plates using the drug concentrations described below. PEG-ASNase: 1.0, 0.75, 0.5, 0.4, 0.3, 0.2
, 0.1, 0.03 IU / ml saquinavir: 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , 10 ⁻⁸ M Results PEG-ASN that gives rise to cytostatic conditions for CEM / 0 cells in vitro.
The ase concentration was about 0.5 IU / ml. 1 and 0.75 IU / ml PEG
-ASNase concentration caused significant cell killing and was cytotoxic to CEM / 0 cells at 72 hours. 0.03, 0.1, 0.2, 0.3 and 0.4 IU / m
The l concentration was slightly more effective in inhibiting cell growth compared to the growth rate of the control (untreated cells). However, 0.5 IU / ml PEG-ASNa
The se concentration showed a relatively flat cell growth curve. Therefore, this concentration produced a 72 hour cytostatic effect. Therefore, P containing 0.5 IU / ml
The EG-ASNase concentration range was used for the combination treatment studies. The drug, concentration and time-dependent cytotoxic effect of PEG-ASNase in this T cell line is shown. After a 72 hour incubation period, the IC ₅₀ of saquinavir in CEM / 0 cells was determined to be 26 μM. The results are shown in Figure 2. Multiple independent experiments with saquinavir showed IC ₅₀ 's of 21-28 μM. 0.001-1 μM
Saquinavir concentrations did not cause cell killing. The 10 μM concentration showed only 8.76% killing compared to the untreated control sample. The highest concentration tested, 100 μM, killed 99.50% of cells compared to control cells. Therefore, a saquinavir concentration of 1-40 μM was used in subsequent experiments to test the combination saquinavir / PEG-ASNase treatment in CEM / 0 cells.

[0107]Example 3: Study of early sequential combinations of saquinavir and PEG-ASNase Materials and methods   In this example, concentrations of saquinavir and PEG-A were used in the following ranges:
The SNase combination regimen was tested. PEG-ASNas followed by saquinavir
For systematic combination studies of e., cells were treated with the following concentrations of PEG-ASNas.
Incubated with e for 24 hours. The appropriate concentration of saquinavir in the following cells
Was added to and maintained for a further 24 hours, resulting in a total exposure time of 48 hours. The exposure is as follows
2 x 10 5 incubated in tissue culture flasks with the investigated concentrations of drug ^Five It contained cells / ml. The results are shown in Fig. 3. The drug concentrations used are as follows
Met.   PEG-ASNase: 1.020, 0.765, 0.510, 0.255 and
And 0.0255 IU / ml   Saquinavir: 40, 30, 20, 10 and 1 μM   Negative control cells included drug in all in vitro studies
The samples were incubated in the same medium for the same period of time and under the same conditions.
Cell density is measured using a Coulter Counter with a Coulter Channelyzer.
Depending on the number, each experimental flask at 24, 48 and 72 hours after incubation was
I measured it. Furthermore, a trypan blue exclusion test was performed for each experimental condition.
Cell numbers were corrected for viability cell numbers measured by Trypan Blue test and
Expressed as a percentage of physical control.   The reverse order was also tested. The results are shown in Fig. 4. Administer saquinavir and
Incubate for 24 hours, then add PEG-ASNase, add another 24 hours
Incubated for a period of time. The total exposure time was 48 hours. Except for the order of drugs,
The methodology was the same as above. Use the same methodology for simultaneous combination measures
I also tested. The results are shown in Figures 5, 6 and 7. By simultaneous combination methodologies
In the experiment, the samples were exposed to the simultaneous combination for 48 hours.result   Each treatment tested showed a synergistic effect within the particular range tested. PEG-ASN
ase followed by saquinavir continuous combination measures 1.7 after 48 hours exposure.
It showed a 2-fold synergistic effect. This is indicated by other consecutive and simultaneous measures.
Was similar to the synergistic effects that were observed. The present invention has a level that allows the survival of certain cells.
And showed the highly desirable result of giving optimum synergistic effect.

Example 4 Measurement of Amino Acid Level Materials and Methods Experiments were carried out to measure amino acid levels in cell suspensions and medium to determine the effect of PEG-ASNase on amino acid levels, especially asparagine, glutamine and aspartic acid levels. It was measured. 50 μl medium and 10 μl 1 mM aminoadopic acid
Sample) was added to 450 μl cold ethanol in a 1.5 ml microtube. The mixture was vortexed and centrifuged at 8700g for 2 minutes. The supernatant was transferred to a borosilicate glass test tube (13 x 100 mm) and freeze-dried. Samples were stored at -20 ° C until analyzed by HPLC. Prior to HPLC, sample 9
It was dissolved in a buffer containing 5% 7 nM disodium hydrogen phosphate and 5% acetonitrile. Results After exposing CEM / 0 cells to various concentrations of PEG-ASNase for 24 hours,
A significant deficiency of asparagine (Asn) was observed. Asparagine levels were less than 3.0% of untreated controls. Furthermore, 3.0% of untreated control
Dose-dependent deficiency of glutamine (gln) for levels below
-Observed in experiments using ASNase concentrations. Aspartic acid (As
p) level increased to a level showing an increase of 200-300% compared to untreated control. The results are shown in Fig. 8. The calibration curve used to calculate the HIV-RT amount is shown in Figure 8a. Even at the lowest level of PEG-ASNase tested, As after 24 hours
A deficiency of n was observed. This is consistent with PGE-ASNase being able to kill illicit T cells by lack of active amino acids, especially asparagine. Furthermore, PEG-ASNase depleted Gln levels, which is important in the mechanism of T cell destruction.

[0109] Example 5: of HIV-RNA in T-cell pellets, measuring this example of the effects of exposure to the combination of PEG-ASNase and Sa Kinabiru are the procedures and similar materials concentration as in Example 1 I went using
Exposure of HIV-RNA in T cell pellets to the combination of PEG-ASNase and saquinavir was measured. The results of this example are shown in Tables 1 and 2 and Figures 9 and 9a. It should be noted that "PEG" in Table 2 indicates PEG-ASNase. These results show that PEG-ASNase had no apparent effect on HIV-1 RNA production, but had a moderate effect on HIV-RT inhibition at day 7 in the same cell culture. (See Example 5a). Therefore, PEG-ASNase inhibited protein biosynthesis even at HIV-RT levels (see Example 5a). HIV-RNA with saquinavir alone
It had an inhibitory effect on production, and inhibited about 36% compared with the control (Fig. 9).
And 9a). The combination of PEG-ASNase and saquinavir inhibited approximately 12% of HIV-RT at synergistic low concentrations (see Example 5a). However, in the same culture, the combination of PEG-ASNase and saquinavir, at low concentrations, resulted in detectable HIV-1 up to the lower limit of 400 copies of RNA per pellet.
No RNA was provided. This data shows that protein inhibitors (PEG-ASNa
se) + HIV-1 protease inhibitor (saquinavir) acts not only synergistically, but also selectively on HIV-RT, and more importantly on HIV-1 RNA production. Shows.

Example 5a: Determination of the effect of PEG-ASNase ± saquinavir on HIV-RT levels in HIV-1 infected cell pellets This example demonstrates that HIV virus PEG-ASNase and saquinavir alone in T cell pellets, these Was performed using the same procedure and similar material concentrations as in Example 7, except that the exposure to the combination was measured. The results of the experiment are shown in Figure 2a.
It should be noted that “PEG” in Table 2a indicates PEG-ASNase. These results indicate that PEG-ASNase had a moderate effect on HIV-RT inhibition. Therefore, PEG-ASNase is
-Inhibits protein biosynthesis even at the RT level. Saquinavir alone is HIV
-It has an inhibitory effect on RT, and it inhibited about 15% in comparison. PEG-ASNa
Se and saquinavir inhibited approximately 12% of HIV-RT at synergistically low concentrations /

Example 6: Determination of HIV-RNA inhibition in CEM / 0 T cell supernatants by means of a combination of PEG-ASNase and saquinavir Example 5 shows that HIV-RT in T cell pellets is PEG-ASNase. And shows the results of exposure to the combination of saquinavir. However, the experimental procedure did not remove HIV-1 particles from the supernatant to stimulate continuous exposure of T cells to the HIV-1 virus. Therefore, HIV-1 virus was always present in the T cell culture supernatant. The combination of PEG-ASNase and saquinavir resulted in HIV-
It was found to inhibit RNA to a significant degree. In some wells,
HIV-RNA could not be quantified after treatment with the PEG + SAQ drug combination. Therefore, we have achieved complete inhibition of HIV-RNA. Supernatants of T cells from Example 5 were analyzed for HIV-RNA and the results are shown in Tables 3 and 4. It should be noted that “PEG” in Table 3 indicates PEG-ASNase. PEG alone inhibits HIV-RNA in the supernatant by about 60% and SAQ by about 6%.
Inhibited 8%. The PEG + SAQ combination reduced HIV-RNA in the supernatant by about 75% compared to untreated controls. This RNA inhibition pattern was in exact agreement with the pattern of previous experiments reporting inhibition of HIV-RT and HIV-RNA from the same experiment. Since these agents do not “kill” HIV in the supernatant, reduction of HIV-RNA can only be achieved by loss of replication-mediated infection and non-renewal by inhibition of the viral replication cycle by these agents. it can. In particular, HIV-RNA is produced intracellularly and is not exported as such or as intact HIV-virus particles into the medium.

Example 7: Measurement of the effect of PEG-ASNase ± saquinavir ± AZT ± MISID (PL-7 ) on HIV RT levels in HIV-1 infected cell pellets Materials and Methods The cell line used in this example was CEM. / 0, human T cell leukemia cell line. PEG-ASNase (ONCASPAR) was kindly provided by Rhone-Poulenc Rorer. Saquinavir was commercially available. AZT was purchased from Sigma. The ribonucleotide reductase inhibitor MISID is manufactured by Nandy P, Lien EJ, Avramis VI,
It was synthesized according to the description of Med. Chem. Res. 1995, 5: 664-679. RPMI-16
40 medium (Irvine Scientific, Irvine, CA) in 10% fetal bovine serum (Gemini
Biosource, Calabasas, CA), 5% 1M Hepes buffer and 5% non-essential amino acids (Irvine Scientific, Irvine, CA). The drug concentrations used were as follows: PEG-ASNase IC50 alone: 0.40 IU / ml saquinavir IC50 alone: 25 μM AZT: 1 μM MISID (PL-7): 0.685 μM PEG IC50 combination: 0.23 IU / ml SAQ IC50 combination: 14.52 μM In summary, 3 × 10 ⁶ cells / ml were stimulated with PHA + medium for 48 hours at 37 ° C. in 5% CO ₂ . In addition, an equal number of cells were incubated in PHA-free medium for 48 hours to serve as a negative control. At this point, cells were inoculated with HIV-1 virus by standard protocols. In this embodiment, PH
A HIV-containing supernatant from stimulated healthy human peripheral blood mononuclear cells (PBMC) was
Note that it was not removed from HA stimulated CEM / 0 cell culture. Therefore, HIV-1 virus is always present in the supernatant and the experimental conditions that stimulate the in vivo clinical conditions of newly generated and non-infectious T cells are always under constant exposure to HIV-1 particles. It was These viral particles were released by the patient's already infected T cells and / or lymph nodes. The experimental agent was added to the cells at the appropriate concentration, either simultaneously with virus inoculation or 90 minutes after virus incubation (time sufficient for T cells to infect and generate new HIV-1 virus particles). Control cells were resuspended in drug-free medium for an exposure period lasting 7 days. An aliquot of control flask medium was obtained after Rx 5 days. On day 7, three aliquots of each 1 ml medium were taken from the flask and stored under liquid nitrogen. In addition, divide the remaining cells into three,
Pelletized and stored at -80 ° C. The sample produced by this example was segmented. Cell pellets from 90 minute viral incubation flasks were used for non-radioactive reverse transcriptase assay EL.
The ISA kit (Boehringer Mannheim) was used to assay for HIV-RT. A calibration curve was determined (see Figure 10) and the HIV-RT levels of the experimental samples were calculated. The results are shown in Table 5. It should be noted that "PEG" in Table 5 indicates PEG-ASNase.

Results and Discussion HIV-RT E in samples from cell pellets of CEM / 0 T cells.
The first observation from the LISA assay was that drug treatment reduced HIV-RT activity as compared to untreated control cells. HIV-
The most dramatic inhibition of RT was caused by the nucleoside analog reverse transcriptase inhibitor AZT. Our initial experimental design and search for wild-type HIV viral particles that do not carry the HIV-RT mutation that confers resistance to AZT is described in AZ
The results showed that T alone was very active against this virus strain. PEG-ASNase or saquinavir used as monotherapy at IC ₅₀ concentrations inhibited HIV-RT by 54% and 83%, respectively. These values were similar to those measured in earlier experiments. Novel Ribonucleotide Reductase (RR) Inhibitor MISID (PL-7)
When used alone at an IC ₅₀ concentration (0.685 μM) of HIV-RT 73
It showed a% inhibition. This is the first evidence that members of this class of RR inhibitors also exhibit anti-leukemic activity as well as anti-HIV activity. The biochemical rationale for this class of compounds in the inhibition of HIV is the intracellular dN
This is due to depletion of the TP pool. Depletion or reduction of the dNTP pool will inhibit the HIV-RT function of converting HIV-RNA to proviral DNA prior to integration into mammalian genomic DNA. The combination of PEG + SAQ caused complete inhibition of HIV-RT in this example. The three drug combination AZT + PEG + SAQ caused complete inhibition of HIV-RT in 2 out of 3 wells, the value in the third well was 95.3% inhibition of control. The biochemical rationale for this drug combination is that AZT further inhibits infection by HIV-RT and that this inhibition is P
It is enhanced by the highly effective anti-HIV-RT effect of the EG + SAQ regimen. Proving that inhibition in this T cell model system infected with wild-type HIV virus was ameliorated by 3 drugs versus 2 drug treatments, as the numbers are close to 100% inhibition of HIV-RT. Is extremely difficult. AZ
In experiments with HIV that is partially resistant to T, it will appear in patients, so the procedure will justify the biochemical triad. The four drug combination MISID + AZT + PEG + SAQ caused complete inhibition of HIV-RT in two out of three wells, with the value in the third well being 95% inhibition of control. These values could be superimposed on the three drug regimens due to maximal inhibition of HIV-RT. The biochemical rationale for this combination is that the RR inhibitor MISID depletes the dNTP pool, which enhances the activity of AZT-triphosphate (AZTTP) on HIV-RT. The enhanced inhibitory effect may be additive or synergistic to the known selective synergistic effect of this protein + protease inhibitor on this virus. Since MISID alone appears to have considerable anti-HIV-RT activity, this tripartite approach can be used in experiments with HIV particles resistant to one or more drugs of these classes or in patients infected with multiresistant HIV variants. The inventors believe that this is shown to be correct. Therefore, these results are AZT + PEG + SAQ or MISID + AZT
It is shown that 3 or 4 drug treatments of + PEG + SAQ act synergistically on HIV-RT.

Example 8: Determination of synergistic effect of PEG-ASNase, saquinavir, AZT and MISID (PL- 7) in CEM / 0 cell supernatant Materials and Methods The cell lines used in this example were CEM / 0, It was a human T cell leukemia cell line. PEG-ASNase (ONCASPAR) was kindly provided by Rhone-Poulenc Rorer. Saquinavir (SQ) was commercially available. AZT was purchased from Sigma. The ribonucleotide reductase inhibitor MISID is available from Nandy P, Lien EJ, Avra
It was synthesized according to the description of mis VI, Med. Chem. Res. 1995, 5: 664-679. RPM
I-1640 medium (Irvine Scientific, Irvine, CA) with 10% fetal bovine serum (Gemini Biosource, Calabasas, CA), 5% 1M Hepes buffer and 5%.
% Enriched with non-essential amino acids (Irvine Scientific, Irvine, CA). The drug concentrations used were as follows: PEG-ASNase IC50 alone: 0.40 IU / ml saquinavir IC50 alone: 25 μM AZT: 1 μM MISID: 0.685 μM PEG IC50 combination: 0.23 IU / ml SAQ IC50 combination: 14.52 μM In summary, 3 × 10 ⁶ cells / ml. Were stimulated with PHA + medium under 5% CO ₂ at 37 ° C. for 48 hours. In addition, an equal number of cells were incubated in PHA-free medium for 48 hours to serve as a negative control. At this point, cells were inoculated with HIV-1 virus by standard protocols. In this embodiment, PH
A HIV-containing supernatant from stimulated healthy human peripheral blood mononuclear cells (PBMC) was
Note that it was not removed from HA stimulated CEM / 0 cell culture. Therefore, HIV-1 virus is always present in the supernatant and the experimental conditions that stimulate the in vivo clinical conditions of newly generated and non-infectious T cells are always under constant exposure to HIV-1 particles. It was These viral particles were released by the patient's already infected T cells and / or lymph nodes.

The experimental agent was added to the cells at the appropriate concentration, at the same time as the virus inoculation or 90 minutes after the virus incubation (a time sufficient for the T cells to infect and produce new HIV-1 viral particles). . Control cells were resuspended in drug-free medium for an exposure period lasting 7 days. An aliquot of control flask medium was obtained after Rx 5 days. On day 7, three aliquots of each 1 ml medium were taken from the flask and stored under liquid nitrogen. In addition, divide the remaining cells into three,
Pelletized and stored at -80 ° C. The sample produced by this example was segmented. Cell pellets of cell cultures from 90 minute viral incubation flasks (see Example 7) were assayed for HIV-RNA quantification assay using a non-radioactive assay kit. A calibration curve was determined and HIV-RT levels of experimental samples were calculated as above. These supernatant samples were derived from the T cell pellet culture of Example 7. The results are shown in Example 7. HIV-RT ELISA in samples from CEM / 0 T cell supernatants
The first observation from the assay is that drug treatment reduced HIV-RT activity on day 7 compared to untreated control cells on the same day.
Day 5 untreated control (1 value) has higher HIV-RTO than day 7
． D. Having had is important. The present inventors have found that all O.V.s lower than the minimum quantified concentration based on the linearity of the calibration curve. D. The value was tested. PEG-ASNas
Only one of the sample supernatants (# 7) treated with e.e. D. Value, but lower than the average value of the three untreated control culture supernatants. Greater inhibition of HIV-RT in quantitative conditions (less than 66% of the negative control value of 0.075 OD or 0.05 OD) was observed as SAQ as a single agent (Samples # 9 and 10). , AZT (Samples # 11 and 13) and MISID (Samples # 15 and 16) (see Table 6; Table 6
It should be noted that "PEG" in Table 1 indicates PEG-ASNase). HIV
Greater inhibition of −RT is due to the three samples # 17, 18 and 19 with the SAQ + PEG-ASNase combination, 3 of PEG-ASNase + SAQ + AZT.
One sample # 22 with the four drug combination and the treatment of the four drug combination was observed in all three samples # 23, 24 and 25 with PEG-ASNase + SAQ + MISID + AZT.

Our initial experimental design and search for wild-type HIV viral particles that do not carry the HIV-RT mutation that confers resistance to AZT shows that AZT alone is highly active against this viral strain. The result was shown. The novel ribonucleotide reductase (RR) inhibitor MISID, when used alone at an IC ₅₀ concentration (0.685 μM), showed significant inhibition of HIV RT as well as single agents and combinations with three other agents. Indicated. This is a result similar to cell pellets (see Example 7), where members of this class of RR inhibitors show anti-leukemic activity as well as anti-HIV activity in intracellular and T cell culture supernatant samples. That is the first evidence. The PEG + SAQ combination caused complete inhibition of HIV-RT in this example and in the experiments described above for cell pellets. In the above experiment, HIV-
There was 96% inhibition of RT (see Example 7). Combination of three drugs AZT + P
EG + SAQ caused complete inhibition of HIV-RT in 2 out of 3 wells, the value in the third well was 95.3% inhibition of control. On the other hand, in the supernatant, the same pattern was observed in Samples # 20-22. The four drug combination MISID + AZT + PEG + SAQ caused complete inhibition of HIV-RT in all three supernatant sample wells. These values are
Corresponds to measurements in cell pellets from the same experiment. These values are H
Due to maximal inhibition of IV-RT, it was possible for us to superimpose the three drug measures previously measured. The biochemical rationale for this combination is that the RR inhibitor MISID is dNT
The P pool was depleted, which resulted in AZT-triphosphate (for HIV-RT (
It means that the activity of AZTTP) is enhanced. The enhanced inhibitory effect may be additive or synergistic to the known selective synergistic effect of this protein + protease inhibitor on this virus. Since MISID alone appears to have considerable anti-HIV-RT activity, this tripartite approach is correct in experiments with HIV particles resistant to one or more drugs of these classes or in patients infected with multiresistant HIV mutants. The present inventors believe that this is shown.

Example 9: Determination of synergistic effect of PEG-ASNase, saquinavir, AZT and MISID (PL- 7) in CEM / 0 cell pellets Materials and Methods The cell lines used in this example were CEM / 0, human. It was a T cell leukemia cell line. PEG-ASNase (ONCASPAR) was kindly provided by Rhone-Poulenc Rorer. Saquinavir was commercially available. AZT was purchased from Sigma. MIS
ID, ribonucleotide reductase inhibitors are Nandy P, Lien EJ, Avramis VI
, Med. Chem. Res. 1995, 5: 664-679. RPMI-1
640 medium (Irvine Scientific, Irvine, CA) was added to 10% fetal bovine serum (Gemin
Biosource, Calabasas, CA), 5% IM Hepes buffer and 5% non-essential amino acids (Irvine Scientific, Irvine, CA). The drug concentrations used were as follows: PEG-ASNase IC50 alone: 0.40 IU / ml saquinavir IC50 alone: 25 μM AZT: 1 μM MISID: 0.685 μM PEG IC50 combination: 0.23 IU / ml SAQ IC50 combination: 14.52 μM In summary, 3 × 10 ⁶ cells / ml. Were stimulated with PHA + medium under 5% CO ₂ at 37 ° C. for 48 hours. In addition, an equal number of cells were incubated in PHA-free medium for 48 hours to serve as a negative control. At this point, cells were inoculated with HIV by standard protocols. In this example, HIV-containing supernatant derived from PHA-stimulated healthy human peripheral blood mononuclear cells (PBMC) was treated with PHA-stimulated CE.
Note that it was not removed from the M / 0 cell culture. Therefore, HIV
-1 virus was always present in the supernatant and the experimental conditions that stimulated the in vivo clinical conditions of newly generated and non-infectious T cells were always under constant exposure to HIV-1 particles. These viral particles were released by the patient's already infected T cells and / or lymph nodes.

The experimental agent was added to the cells at the appropriate concentration at the same time as the virus inoculation or 90 minutes after the virus incubation (a time sufficient for the T cells to infect and produce new HIV-1 viral particles). . Control cells were resuspended in drug-free medium for an exposure period lasting 7 days. An aliquot of control flask medium was obtained after Rx 5 days. On day 7, three aliquots of each 1 ml medium were taken from the flask and stored under liquid nitrogen. In addition, divide the remaining cells into three,
Pelletized and stored at -80 ° C. The sample produced by this example was segmented. Cell pellets of cell cultures from 90 minute viral incubation flasks (see Example 7) were assayed for HIV-RNA quantification assay using a non-radioactive assay kit. A calibration curve was determined and HIV-RT levels of experimental samples were calculated as above. The samples were derived from a culture of T cell pellets and from the same experiment as described above for the HIV-RT results. The results were examined in Example 7 (cell pellet) and Example 8 (supernatant). The first observation from the HIV-RNA quantitative assay in a sample of CEM / 0 T cells was that drug treatment reduced HIV RNA activity on day 7 compared to untreated control cells on the same day. That is. The quantitative results are shown in Table 7 and FIG. It should be noted that "PEG" in Table 7 refers to PEG-ASNase. Inhibition of HIV RNA in the quantitative period caused by AZT alone (Samples # 11-13) showed SAQ and MISID as single agents. (HIV-1 virus susceptible to AZT). The large inhibition of HIV-RT is due to the combination of SAQ + PEG-ASNase (38% of control) and PE.
G-ASNase + SAQ + AZT three drug combinations (control 30
%). Measures of combination of four drugs PEG-ASNase + SA
All three samples # 23, 24 and 25 tested with Q + MISID + AZT had the highest HIV-RNA in this example, 20% of the control. This means that the ribonucleotide reductase inhibitor MISI
D shows a significant contribution. When the novel ribonucleotide reductase (RR) inhibitor MISID was used alone at an IC ₅₀ concentration (0.685 μM), HIV as well as single agents and most importantly in combination with three other agents were used. It showed significant inhibition of RT. This is a result similar to cell pellets (see Example 6), where members of this class of RR inhibitors also show anti-leukemic activity in addition to anti-leukemic activity in intracellular and T cell culture supernatant samples. That is a recurring evidence. The biochemical rationale for this combination is MISID, RR inhibitor is dN
It is depleted of the TP pool, which enhances the activity of AZT-triphosphate (AZTTP) on HIV integration and replication. The enhanced inhibitory effect may be additive or synergistic to the known selective synergistic effect of this protein + protease inhibitor on this virus. MISID alone is quite anti-H
This tripartite method is now shown to be correct in experiments with HIV particles that are resistant to one or more agents of these classes, as they have IV-RNA activity, or in patients infected with multiresistant HIV mutants. The inventors are thinking.

Example 10: Measurement of synergistic effect of PEG-ASNase, saquinavir, AZT and MISID (PL- 7) in inhibiting HIV-RT Materials and Methods The cell lines used in this example were CEM / 0, human. It was a T cell leukemia cell line. PEG-ASNase (ONCASPAR) was kindly provided by Rhone-Poulenc Rorer. Saquinavir was commercially available. AZT was purchased from Sigma. MIS
ID, ribonucleotide reductase inhibitors are Nandy P, Lien EJ, Avramis VI
, Med. Chem. Res. 1995, 5: 664-679. RPMI-1
640 medium (Irvine Scientific, Irvine, CA) was added to 10% fetal bovine serum (Gemin
Biosource, Calabasas, CA), 5% IM Hepes buffer and 5% non-essential amino acids (Irvine Scientific, Irvine, CA). The drug concentrations used were as follows: PEG-ASNase IC50 alone: 0.40 IU / ml saquinavir IC50 alone: 25 μM AZT: 1 μM MISID: 0.685 μM PEG IC50 combination: 0.23 IU / ml SAQ IC50 combination: 14.52 μM In summary, 3 × 10 ⁶ cells / ml. Were stimulated with PHA + medium under 5% CO ₂ at 37 ° C. for 48 hours. In addition, an equal number of cells were incubated in PHA-free medium for 48 hours to serve as a negative control. At this point, cells were inoculated with HIV by standard protocols. In this example, HIV-containing supernatant derived from PHA-stimulated healthy human peripheral blood mononuclear cells (PBMC) was treated with PHA-stimulated CE.
Note that it was not removed from the M / 0 cell culture. Therefore, HIV
-1 virus was always present in the supernatant and the experimental conditions that stimulated the in vivo clinical conditions of newly generated and non-infectious T cells were always under constant exposure to HIV-1 particles. In this example, we present a control HIV-R in T cells.
Very high HIV-1 titers were observed by NA (see Example 9). These viral particles were continuously released by the patient's already infected T cells and / or lymph nodes.

The experimental agent was added to the cells at the appropriate concentration, at the same time as the virus inoculation or 90 minutes after the virus incubation (a time sufficient for the T cells to infect and produce new HIV-1 viral particles). . Control cells were resuspended in drug-free medium for an exposure period lasting 7 days. An aliquot of control flask medium was obtained after Rx 5 days. On day 7, three aliquots of each 1 ml medium were taken from the flask and stored under liquid nitrogen. In addition, divide the remaining cells into three,
Pelletized and stored at -80 ° C. Supernatant samples obtained from T cell cultures, -8
Stored at 0 ° C. The sample produced by this example was segmented. Cell pellets of cell cultures from 90 minute viral incubation flasks (see Example 9) were assayed for HIV-RNA quantitation assay using a non-radioactive assay kit. The present inventors have confirmed that quantitative HIV-R from the supernatant of these T cells.
The results of NA are disclosed. A calibration curve was determined and HIV-RT levels of experimental samples were calculated as above. Results and Discussion Samples were used to culture T cell pellets derived from the same experiment as described above for HIV-RT results (see Example 8) and quantitative HIV-RNA in T cells (see Example 9). It came from. The first observation from the HIV-RT quantitative assay in supernatant samples of CEM / 0 T cells showed that drug treatment reduced HIV RNA activity on day 7 compared to untreated control cells on the same day. It means that The quantitative results are shown in the attached table (Table 8). It should be noted that "PEG" in Table 8 indicates PEG-ASNase. Quantitative control HIV-RNA levels (viral genome copies / ml) were higher than in previous experiments and were similar to untreated control levels (214,445 in T cell pellet vs. 195,483 in supernatant).
.

The inhibition of HIV-RNA in the quantitative period caused by SAQ, AZT or MISID alone (Samples # 8-16) was non-statistically significant among these (susceptible HIV-1 to AZT. Virus). Similar% inhibition of HIV-RNA in the supernatant compared to untreated control was SAQ + PEG-A
It was observed with the combination of SNase or the combination of the three drugs PEG-ASNase + SAQ + AZT. However, measures of the combination of four drugs MISID
All three samples # 23, 24 and 25 tested with + AZT + PEG-ASNase + SAQ had the greatest HIV-RNA inhibition in this example, 50% of the control. This clearly demonstrates the significant contribution of the ribonucleotide reductase inhibitor MISID. The latter data set confirmed the initial observations of HIV-RNA inhibition shown in 20% of control T cell pellets (Example 9). Data from previous experiments and evidence in this example show that the higher the HIV titer in the supernatant remained, the lower the inhibition of the virus in T cells and supernatant. In other words, the data indicate that a) these combined measures must be provided continuously under these conditions, ie in patients with high HIV-RNA copy number and / or viremia, b) A
It is suggested that enhancement of ZT + SAQ requires enhanced activity of AZT triphosphate (AZTTP) against HIV-RT by a third RT inhibitor, such as 3TC or RR inhibitor, such as MISID or hydroxyurea. There is. A novel ribonucleotide reductase (RR) inhibitor, MISID, was used alone at an IC ₅₀ concentration (0.685 μM) to show significant HIV-R as a single agent.
It showed NA inhibition. This value was almost equal to the inhibition of SAQ or AZT. Most importantly, MISID, in combination with 3 other agents, inhibits HIV-RT in T cell pellets and supernatants (see Examples 7 and 8) and HIV-RNA remaining in the supernatants. (Table 8) showed that it was significantly useful. This is the result in T cell pellets (see Examples 6 and 9), in which members of this class of RR inhibitors have anti-leukemic as well as anti-HIV activity in intracellular and T cell culture supernatant samples. It is a recurring proof of showing. The biochemical rationale for this combination is that the RR inhibitor MISID is dNT
It is depleted of the P pool, which enhances the activity of HIV-1 reverse transcriptase, AZTTP on integration and replication. The enhanced inhibitory effect may be additive or synergistic to the known selective synergistic effect of this protein + protease inhibitor on this virus. Since MISID alone appears to have considerable anti-HIV-RNA activity, this tripartite approach is correct in experiments with HIV particles resistant to one or more drugs of these classes or in patients infected with multiresistant HIV mutants. The present inventors believe that this is shown.

Example 10: Measurement of synergism of PEG-ASNase, saquinavir, AZT and 3TC on CEM / 0 cell pellets This example was performed using the same procedure as in Example 9, using the same material concentrations. . However, 3TC was used instead of MISID. In this embodiment, PE
Exposure of HIV-RT in T cell supernatants to the combination of G-ASNase, saquinavir, AZT and 3TC was measured. The results of this example are shown in Table 9. Table 9 "P
It should be noted that "EG" refers to PEG-ASNase. In Table 9,
It was shown that saquinavir and PEG-ASNase alone inhibited HIV-RT to some extent. However, PEG-ASNase, saquinavir, AZ
The combination of T and 3TC caused a complete inhibition of HIV-RT. Example 11: Measurement of synergism of PEG-ASNase, saquinavir, AZT and 3TC on CEM / 0 cell supernatant This example was performed using the same procedure as in Example 10, using the same material concentrations. However, the exposure of HIV-RNA in T cell supernatant to the combination of PEG-ASNase, saquinavir, AZT and 3TC was measured. The results of this example are shown in Table 10.
Shown in. It should be noted that “PEG” in Table 10 indicates PEG-ASNase. In Table 10, HIV was treated with saquinavir and PEG-ASNase alone.
-RNA is inhibited to some extent (55% and 73% of control, respectively)
Was shown. The combination of PEG-ASNase, saquinavir and AZT caused large HIV-RNA inhibition (about 21% of control). However, the combination of PEG-ASNase, saquinavir, AZT and 3TC is HIV-
It caused a complete inhibition of RT (0% of control) (see Table 10). The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics.

Table 1 ^* N is the genomic copy number / ml or copy number / cell pellet of the virus in the supernatant. The HIV-1 monitor assay dynamic range is 400-750,00.
It is 0 copy / ml. The laboratory was asked if less than 400 copies / ml was needed.

Table 2 Sample list for HIV-RNA assay All 13 pellets for HIV-RNA assay.

Table 2a Sample Log MS HIV-RT Assay # 4 Calibration Curve Y = 0.5955 (X) +0.09363 r = 0.99667

Table 3 Supernatants from HIV Monitor Assay Results Supernatants ^* HIV virus was not removed from the medium. HIV-1 could not be "killed" by these agents except T cells. Therefore, PEG-A
The low level of HIV in the SNase supernatant is due to HIV loss due to infection and non-regeneration by T cells. This observation indicates that undetectable HIV RNA
Well fitted with the cell pellet data of.

Table 4 N ^* is viral genome copy number / supernatant or copy number / cell pellet. H
The dynamic range of the IV-1 monitor assay is 400-750,000 copies / ml. The laboratory was asked if less than 400 copies / ml was needed.

Table 5 Sample Log for Anti-HIV Agent Synergy Study Design CEM / 0, PHA Stimulation, 48 Hour CEM / 0, Virus Inoculation, 90 Minutes Incubation Drug Exposure Rx 7 Day Cell Pellet ELISA Assay Calibration Curve Y = 1.13X + 0.100 r = 0.999 Table 5-1

Table 5-2 ^{* If the} other two points are 0, indicates a single point decision

Table 6 Sample Logs for Anti-HIV Agent Synergy Studies Experimental Design CEM / 0, PHA Stimulation, 48 h CEM / 0, Virus Inoculation, Supernatant ELISA from Cell Suspensions 7 Days After Rx Concurrent Drug Exposure. Assay calibration curve Y = 0.73795X + 0.3006 r = 0.999962 Table 6-1

Table 7 Sample log for anti-HIV agent synergy studies Samples tested for HIV-RNA Table 8 Sample Log for Anti-HIV Agent Synergy Study Design CEM / 0, PHA stimulation, 48 hour CEM / 0, virus inoculation, 90 minute incubation and cell pellet ELISA assay 7 days after drug exposure Rx.

Table 9 Sample Log for HIV-RT ELISA Assay Experimental Design CEM / 0, PHA stimulation, 48 hour CEM / 0, virus inoculation, +/- 90 minutes drug exposure Pellets from cell suspension after 7 days Rx. And supernatant ELISA assay standard curve Y = 0.75847X + 0.07792 r = 0.99914

Table 10 Sample Log for Anti-HIV Agent Synergy Study Design CEM / 0, PHA Stimulation, 48 h CEM / 0, Virus Inoculation, 90 min Incubation, From Cell Suspension 7 Days After Drug Exposure Rx Supernatant ELISA assay HIV-RNA assay

[Brief description of drawings]

FIG. 1 shows IC50 concentrations of PEG-asparaginase or saquinavir (SA
Q) shows inhibition of HIV-RT in CEM / 0-T cells (± PHA) treated alone or in combination.

FIG. 2 depicts the cytotoxicity of saquinavir on T cells (CEM / 0) after 72 hours for different drug concentrations.

FIG. 3 shows the cytotoxicity of PEG-asparaginase and saquinavir on T cells when used alone and sequentially in combination (PEG-asparaginase followed by saquinavir) for different drug concentrations.

FIG. 4 shows single and continuous combinations (Saquinavir followed by P for different drug concentrations).
EG-asparaginase) shows their cytotoxicity on T cells.

FIG. 5 shows the cytotoxicity of PEG-asparaginase and saquinavir on T cells when used alone and in combination for different drug concentrations.

FIG. 6 shows the synergistic effect of PEG-asparaginase and saquinavir on T cells when co-combined for different drug concentrations.

FIG. 7 shows the combination index in CEM / 0 when saquinavir was followed by PEG-asparaginase and PEG-asparaginase followed by saquinavir, and when PEG-asparaginase and saquinavir were co-administered.

FIG. 8: CE after 24 hours exposure to different concentrations of PEG-asparaginase.
5 shows a decrease in asparagine, glutamine and aspartate concentrations in M / 0-T cells.

FIG. 8a shows the optical density (OD) calibration curves for various concentrations of HIV-RT.

FIG. 9 shows HIV RNA copy number per cell pellet after exposure of cells using PEG-asparaginase and saquinavir alone and in combination.

Figure 9a shows the HIV RNA copy number per cell pellet as LoglO after exposure to cells using PEG-asparaginase and saquinavir alone and in combination.

[Figure 10]   FIG. 10 shows a calibration curve for an HIV-RT ELISA assay.

FIG. 11: Monotherapy of PEG-asparaginase, saquinavir, AZT and MISID, and saquinavir and PEG-asparaginase; AZT, saquinavir and PEG-asparaginase; and MISID, AZT, saquinavir and PEG-.
1 shows an HIV-1 RNA quantitation assay of CEM-T cells treated with asparaginase combination therapy.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Cohen Lewis             United States Pennsylvania             19465 Pottstown Beachwood             Russ 100 F-term (reference) 4C076 AA95 CC29 CC41 EE23 EE59                 4C084 AA24 BA42 DC25 MA02 NA05                       NA13 ZB332 ZC192 ZC202                       ZC552

Claims (17)

[Claims] 1. A method for suppressing or treating a human immunodeficiency virus (HIV) infection, which comprises a PEG-asparaginase compound or a pharmaceutically acceptable salt thereof, and optionally a protease inhibitor compound, a ribonucleotide reductase inhibitor compound. And a therapeutically effective amount of a composition containing at least one compound selected from the group consisting of HIV reverse transcriptase-inhibiting compounds or a pharmaceutically acceptable salt thereof to a patient in need of inhibiting or treating HIV infection. And a step of administering the method. 2. The method of claim 1, wherein the at least one compound is a protease inhibitor compound. 3. The method of claim 1, wherein the at least one compound is a ribonucleotide reductase inhibitor compound. 4. The method of claim 1, wherein the at least one compound is an HIV reverse transcriptase inhibitor compound. 5. The method of claim 1, wherein the protease inhibitor compound is selected from saquinavir, nelfinavar, indinavar, enedinovia, ritonavar, crixivan, viracept, norver, and VX-478. 6. The ribonucleotide reductase inhibitor compound comprises:
Hydroxyurea (HU), BW-348U87, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) amidox (VF236;
NSC-343341; N, 3,4-trihydroxybenzenecarboximidamide), BILD1257 (2-benzyl-3-phenylpropionyl-L-
(N-Methyl) valyl-L-3- (methyl) valyl-L- (N4, N4-tetramethylene) asparaginyl-L- (3,3-tetramethylene) aspartyl-L
-(4-methyl) leucine), BILD1357 (2-benzyl-3-phenylpropionyl-L- (N-methyl) valyl-L-3- (methyl) valyl-L- (
N4, N4-tetramethylene) asparaginyl-L- (3,3-tetramethylene) aspartic acid 1- [1 (R) -ethyl-2,2-dimethylpropylamide]
), BILD1633, BILD733 (3-phenylpropionyl-L- (N
-Methyl) valyl-L- [3-methyl] valyl-L- [3- (pyrrolidine-1-
Ylcarbonyl)] alanyl-L- (1-carboxycyclopentyl) glycyl-L- (4-methyl) leucinol), BILD1263 (2-benzyl-3-
Phenylpropionyl-L- (N-methyl) valyl-L-3- (methyl) valyl-L- (N4, N4-tetramethylene) asparaginyl-L- (3,3-tetramethylene) aspartyl-L- (4- Methyl) Leucinol), BILD135
1 (1- [1 (S)-[5 (S)-[3-[(all-cis) -2,6-dimethylcyclohexyl] ureido] -2 (S)-(3,3-dimethyl-2- Oxobutyl) -6,6-dimethyl-4-oxoheptanoylamino] -1- [1 (R)
-Ethyl-2,2-dimethylpropylcarbamoyl] methyl] cyclopentanecarboxylic acid)), CI-F-araA (2-chloro-9- (2-deoxy-2-fluoro-beta-D-arabinofurano Sil) adenine, DAH (D-aspartic-beta-hydroxamate), DDFC (2'-deoxy-2 ', 2'-difluorocytidine), Didox (VF147; NSC324360; N, 3,4)
-Trihydroxybenzamide), Eurd (3'-ethynyluridine), GT
I2040, GTI2501, IMHAG (1-isoquinolylmethane-N-hydroxy-N'-aminoguanine), LY207702 (2 ', 2'-difluoro-
2'-deoxyribofuranosyl-2,6-diaminopurine), LY295501
(N-[[3,4-dichlorophenyl] amino] carbonyl) 2,3-dihydro-5-benzofuransulfonamide), MDL101731 (FMdC; KW2)
331; (2E) -2'-deoxy-2 '-(fluoromethylene) cytidine), parabactin, slofena (LY186641; N-[[(4-chlorophenyl) amino] carbonyl] -2,3-dihydro-1H-indene -5-sulfonamide), TAS106 (Ecyd; 3'-ethynylcytidine), triapin (O
CX191; OCX0191), trimidox (VF233; N, 3,4,5-
Tetrahydroxybenzenecarboximidamide), and a compound of formula I:
Or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostea or a prodrug thereof; Wherein R1 is an alkyl, alkenyl, alkynyl, or electron withdrawing group;
2 is alkyl or alkenyl. 7. The method of claim 6, wherein the ribonucleotide reductase inhibitor compound is a compound of formula I. 8. The ribonucleotide reductase compound is a compound of formula I or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostea or a prodrug thereof, wherein R1 is a lower alkyl. The method according to claim 7, which is a halogen group, and R 2 is lower alkyl. 9. The method of claim 6, wherein the ribonucleotide reductase inhibitor compound of formula I is MISID. 10. The HIV reverse transcriptase inhibitor compound is selected from AZT (retrovar, zidovudine), ddl (bidex, didanosine), ddC (hibid, salcitabine), d4T (zelito, stavudine) and 3TC (epibar, lamivudine). The method of claim 4, wherein the method comprises: 11. The method of claim 1, wherein the administration of the compound is simultaneous. 12. The method of claim 1, wherein the administration of the compound is continuous. 13. An HIV-infected cell population is exposed to an effective amount of a PEG-asparaginase compound and optionally at least one compound selected from the group consisting of a protease inhibitor compound, a ribonucleotide reductase inhibitor compound and an HIV reverse transcriptase inhibitor compound. A method of suppressing the production of HIV or limiting the spread of HIV, which comprises the step of: 14. A combination wherein the compound to be administered exhibits a synergistic action of a PEG-asparaginase compound and at least one compound selected from the group consisting of a protease inhibitor compound, a ribonucleotide reductase inhibitor compound and an HIV reverse transcriptase inhibitor compound. The method of claim 1, comprising: 15. A PEG-asparaginase compound, optionally one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transcriptase inhibitor compounds or ribonucleotide reductase inhibitor compounds, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: 16. A kit for treating or preventing a physiological condition associated with HIV, wherein the kit comprises a plurality of separate containers, at least one of which contains a PEG-asparaginase compound and at least one of said containers. Another is a kit comprising one or more compounds selected from the group consisting of a protease inhibitor compound, an HIV reverse transcriptase inhibitor compound and a ribonucleotide reductase inhibitor compound, wherein the container optionally further comprises a pharmaceutical carrier. . 17. A method for inhibiting or treating human immunodeficiency virus (HIV) infection, comprising asparaginase or a pharmaceutically acceptable salt thereof, and optionally a protease inhibitor compound, a ribonucleotide reductase inhibitor compound and HIV reversal. Administering a therapeutically effective amount of at least one compound selected from the group consisting of transcriptase-inhibiting compounds or a pharmaceutically acceptable salt thereof to a patient in need of inhibition or treatment of HIV infection. A method characterized by.