CN1045791A

CN1045791A - Use the cooperative compositions HIV inhibiting of nucleoside derivates

Info

Publication number: CN1045791A
Application number: CN90102298A
Authority: CN
Inventors: 维拉·布兰可芬; 理查德·J·怀特
Original assignee: Oncogen LP
Current assignee: Oncogen LP
Priority date: 1989-03-17
Filing date: 1990-03-17
Publication date: 1990-10-03
Also published as: OA09555A; HU208254B; FI914367A0; JPH04504850A; KR920700653A; CA2050473A1; WO1990011081A1; HUT57988A; DD301787A9; GR900100189A; PT93449A; NZ232912A; GR1000618B; YU53790A; EP0464137A4; NO913659L; IL93783A0; NO913659D0; HU902898D0; EP0464137A1

Abstract

The present invention relates to unite use has synergistic nucleoside derivates HIV inhibiting (HIV) to duplicate, thus the method that restriction HIV infects.In a particular, with fast cry of certain animals nucleoside analog dideoxyinosine and pyrimidine nucleoside analoys 2 ', 3 '-dideoxy-2 ', 3 '-two dehydrogenation thymidines (d4T) merge to use, and show very strong synergistic activity and have reduced cytotoxic activity to mammalian cell.

Description

Use the cooperative compositions HIV inhibiting of nucleoside derivates

The present invention relates to use the composition HIV inhibiting (HIV) of nucleoside derivates to duplicate, thus the consequence that restriction HIV infects.Key of the present invention is that the nucleotide derivative of finding the associating use has synergy, and like this, the associating effective dose of these medicaments is lower than the summation of the single medicine therapeutic dose that is used for individuality; The raising of antiviral activity and without Cytotoxic corresponding increase; In fact, compare when giving same compound respectively, the composition of nucleotide derivative is littler to the toxicity of non-infected cells.

Human immunodeficiency virus (HIV) is a kind of human inverse transcription virus, it is believed that it is the pathogenic factor of the relevant syndromes with AIDS of the acquired immunodeficiency disease day after tomorrow (AIDS) (ARC).HIV virus particle or virion are diameters about 1000 Spheroplast.Particle is surrounded by the adventitia derivative bimolecular lipid membrane of one deck by infected host cell outward.Studies show that viromembrane is a kind of capsid glycoprotein, it synthesizes, is processed to then two kinds of glycoprotein as the 160kd precursor: cross over the gp41 of double-layer of lipoid and extend to the outer gp120 of double-layer of lipoid.Capsid is wrapped in a core that is made of p24 and p18 protein.Carry viral RNA in the core, and several copies of ThermoScript II (this enzyme is used for the assembling of catalysis viral DNA).

The HIV assortment of genes has its coding capsid protein matter of three gene: env(of coding counter-transcription-ing virus particle composition), its coding core protein of gag() and its ThermoScript II of encoding of pol().The both sides of these three genes are connected to the Nucleotide stretch section that is called long terminal repeat (LTR).LTR has comprised the order of the expressional function with control virogene.But different with other retroviruss is, the genome of HIV comprises 5 episomes at least, known wherein 3 regulatory function is arranged, think that its expression can influence the mechanism of causing a disease of virus.A kind of protein that the strong deactivation agent function of HIV genetic expression is arranged of tat genes encoding, therefore the amplification to virus replication plays an important role.Rev or trs/art gene can be by bringing into play anti-checking mechanism synthesizing to adjusted HIV; Rev can make the HIV virus that is integrated optionally produce and regulate albumen or virus particle composition.On the contrary, nef or 3 '-orf gene then can be regulated the expression of virus downwards by producing a kind of plasmosin matter, and this protein may suppress that HIV is genomic to transcribe by second information.Vif or sor gene pairs virus particle form unimportant, but it is very crucial to producing the infectious virus particle effectively, and can influence virus in external propagation.The immunogenic protein of pr or a kind of unknown function of R genes encoding.

The important foundation that it is believed that the immune pathogenesis of HIV infection is the exhaustion of auxiliary/inducing cell subgroup (it can express CD4 albumen) in the T lymphocyte, and causes extremely immunosuppression.The virus of these immunocytes is killed and wounded and is considered to weaken the principal element of HIV to the influence of immunity system.Capsid glycoprotein enters in the positive host cell of CD4 at HIV and plays an important role.Known gp 120 parts can directly combine with cell CD4 acceptor molecule, thereby cause the taxis of HIV to the host cell of expressing the CD4 acceptor (as t helper cell (T4 cell) scavenger cell etc.).

After HIV was attached on the CD4 molecule, virus entered in the cell and sloughs adventitia.In case enter in the cell, geneome RNA promptly is transcribed into DNA under the catalysis of ThermoScript II.Proviral DNA is integrated in the host chromosome DNA then, infects this moment and just is in " dormancy " or latent period.In case after activating, provirus is promptly transcribed.Translate and translate post-treatment and cause the virus assembling, and sophisticated virion is stretched by cell surface.

When the virus particle Active Replication, common host CD + ₄Cell promptly is killed.But HIV brings into play its cytopathic effect cutter reason Shang Buming Liao really.Proposed that many immunity about the HIV infection are caused a disease and the mechanism of cell pathology effect: the viral DNAs of a large amount of not integration are infected intracellular gathering; The permeability of cytolemma significantly increases when a large amount of viruses break away from cell surface; Or infer that HIV may induce and do not break up the end of infected T4 cell and shortened cell survival.More and more evidences shows that CD4 molecule and viral capsid promote cytogamy in some way in the cell that HIV infects, and works in cytopathic effect.In the cytopathology that HIV infects, principal character is to form multinucleated syncytia, and it is for being the nearly syzygys of 500 cells of gp 120/gp 41 capsid protein inductive.Otherwise the scavenger cell that HIV infects then may produce HIV continuously, and does not have cytopathic effect in long-time; Therefore think that scavenger cell is the main bank of HIV, and may be responsible for virus is transported in the central nervous system (Gartner et al., 1986, Science 233: 215-219).

Can't cure AIDS (AIDS) up to now.The propagation of vaccine control virus on probation in the crowd at present.But controlling, the interior lysis of infected patient body then mainly is to use antiviral agent.

In recent years, in order to reduce HIV incidence of infection and mortality ratio, carried out exploration (Yarchoan et al., 1988, the Scientific American 259: 110-119 of multiple therapy methods; Bartlett, 1988, J.A.M.A.260: 3051-3052; De Clercq, E., 1986, J, Med.Chem.29: 1561-1569; Yarchoan, R.and Broder, S., 1987, N.Engl.J.Med.316: 557-564).According to the knowledge of the physiology aspect of relevant HIV virus, designed that many viral interferences are duplicated or the means of transmission of infection.Intervention effect may stop potentially virus and target cell in conjunction with, merge with cytolemma, the exposing of viral nucleic acid, the reverse transcription of HIV rna gene group become transcribing of DNA, virus mRNA or translate, be assembled into sophisticated virus particle, or other and duplicate or process that infectivity is relevant.

Several prevention HIV and cytolemma bonded method have been tested; These methods are intended to suppress HIV capsid glycoprotein gp 120 and the upward interaction between T4 antigen of target cell (as helper T cell).Not only to pass through cytolemma relevant with virus in the combination of gp 120 and T4 antigen, and with synplasm form relevant (Sodroski et al., Nature 322: 470-474; Lifson et al., 1986, Nature 323: 725-728; Stevenson et al., 1988, Cell 53: 483-496).(1987, Science 238: point out that 1704-1707) before virion ran into the CD4 molecule that is embedded in the cytolemma, the solubility T4 antigen was blocked the HIV infection by combining with virion for people such as Smith.In addition, shown anti-alloantibody at CD4 antibody, may combine (Dalgleish et al. in external with HIV virus by the similar protein configuration of processing and CD4 decision base, 1989, UCLA Symposia on Molecular and Cellular Biology, J.Cell Biochem.Supp.13B, p.298).Moreover, shown that several big, the Sulfated elements with negative charge that comprise dextran can duplicate and synplasm formation (Yarchoan et al., document is the same) in external preventions HIV.

Found that oligonucleotide anti-significance can suppress synplasm formation and p24 protein expression (Agrawal et al, 1988, the Proc.Natl.Acad.Sci.USA.85: 7079-7083) of virus induction.These of design and virus mRNA complementary synthesizing ribonucleotide polymer can suppress virus mRNA and be translated into protein; The phosphoramidate and the thiophosphatephosphorothioate that form antisense oligonucleotide have produced the compound that can resist the endonuclease enzyme liberating in fact.

It is believed that the Ribavirin that contains a ribose and a triazole ring can play the effect of guanosine analogue.It has the activity in several RNA viruses of external antagonism, thinks that tentatively its viral interference mRNA adds the necessary guanosine acidification step of cap process.According to reports, Ribavirin can suppress (Mccormick et al., The Lancet, Dec.15,1984: 1367-1369) of duplicating of HIV in human T lymphocyte's culture.

Another method is the proteinic post-treatment process of translating of break virus; The castanospermine (Castanospermine) of having found to have the plant glycosidase activity can reduce synplasm and form, and reduces HIV infectivity (Wall et al., 1988, Proc.Natl.Acad.Sci.USA.85: 5644-5648).

Final step during HIV duplicates comprises by coming out in the host cell and infecting new target cell.Found that interferon-alpha can duplicate (Ho et al. in vitro inhibition HIV, Lancet, March 16,1985: 602-604), suppress viral budding, and shown that it has direct anti-tumor activity to Kaposi sarcoma (a kind of malignant change relevant with AIDS).Proved that the lipoid substance AL721 that is made up of with 7: 2: 1 ratios neutral glycerine ester, phosphatldylcholine and phosphatidyl ethanolamine has the ability of extraction cholesterol from cytolemma, and can reduce infectivity (the Sarin et al. of HIV, 1985, N.Engl.J.Med.313: 1289-1290).

Tested the method that several control HIVs relevant with suppressing viral reverse transcriptase infect.Because mammalian cell lacks the endogenous retrovirus activity, so these methods can optionally suppress virus replication host cell being brought under the toxic situation of the smallest cell.The medicament that can suppress reversed transcriptive enzyme comprises phosphine carbamoyl ester (Sandstrom et al., Lancet, June 29,1985: L1480). Suramine (Mitsuya et al., 1984, Science 226: 172-174), Rifampin (or ansamycin, Amand et al, Lancet, Jan.11,1986, nucleoside derivates p.97) and hereinafter described.

Adorned nucleotide derivative comprises purine (VITAMIN B4 and guanine) and pyrimidine (thymus pyrimidine, uridylic and the cytosine(Cyt)) Nucleotide that constitutes RNA and DNA.Reversed transcriptive enzyme and archaeal dna polymerase will make nucleoside derivates in conjunction with and mix in the newborn DNA chain, condition is that 5 ' carbon of derivative can be attached on 3 ' hydroxyl of previous Nucleotide (promptly forming phosphodiester bond); If nucleoside derivates contains a gene that makes on 5 ' phosphoric acid that its 3 ' carbon is connected to another Nucleotide, then continue after Nucleotide can be coupled with.Many nucleoside derivates as potential anti-HIV modifier of studying entering before viral genome is decrypted, can make viral dna replication ripe termination the in early stage occur.These derivatives lack and can cause chain termination with next nucleosides bonded substituting group.

The nitrogen base-2 of AZT(3 '-repeatedly ', 3 '-Didansine, repeatedly nitrogen base thymidine, zidovudine) be proved to be a kind of treatment AIDS and the effective nucleoside derivates of ARC; Data shows that AZT can make patient's AIDS survival rate double (Fischl et al., 1987, N.Engl.J.Med.317: 185-191; Greagh-Kirk et al., 1988, J.A.M.A.260: 3009-3015).The Prima Facie Evidence prompting, AZT is for treatment children acquired immune deficiency syndrome (AIDS), relevant psoriasis (the Bartlett et al. of HIV, document is the same), relevant dementia (the Schmitt et al. of AIDS, 1988, N.Engl.J.Med.319: 1573-1578) (the Pottage et al. of relevant thrombocytopenia with HIV, 1988, J.A.M.A.260: may be effective 3045-3048).AZT is formed triphosphoric acid AZT(by the mammalian cell phosphorylation, and it is the analogue of triphosphoric acid thymidine), it is believed that it can suppress the generation of viral DNA at least by two kinds of mechanism: i.e. competitive inhibition and chain termination (Yarchoan et al., above-mentioned document).The inhibition of competing property is because of more tight in the binding ratio natural nucleotide of viral reverse transcriptase and triphosphoric acid AZT; And chain termination is because thereby AZT shortage 3 ' hydroxyl not can be incorporated on the Nucleotide of back.

Di-deoxynucleoside is 2 ' and 3 ' carbon residue on lack the nucleotide derivative of hydroxyl, therefore cause the DNA chain termination.In addition, the same with AZT, 2 ', 3 '-5 ' triphosphoric acid product of DIDEOXYADENOSINE, dideoxy guanosine, zalcitabine, Didansine optionally suppresses β and γ archaeal dna polymerase in viral reverse transcriptase and the cell, but function (the Edenberg et al. of main DNA synthetic enzyme-archaeal dna polymerase α of being utilized of interference cell interkinesis not, 1978, J.Biol.Chem.253: 3273-3280; Wagar et al., 1978, Nucl.Acids Res.5: 1933-1946; Van der Vilet et al., 1981, Biochemistry 20: 2628-2632; Wagar et al., 1984, J.Cell.Physiol.121: 402-408).2 of adenosine, guanosine, Trophicardyl, cytidine and thymidine etc. ', 3 '-the di-deoxynucleoside derivative can be vitro inhibition HIV virus replication (but the inhibition activity of thymidine derivative be less); When being lower than suppressor T cell propagation or immunoreactivity required dosage 10 to 20 times, promptly can be observed HIV virus and be suppressed (Mitsuya and Broder 1986, Proc.Natl.Acad.Sci.USA.83: 1911-1915) basically fully.People such as Ahluwalia to 2 ', 3 '-cellular pharmacology of dideoxyinosine carried out preliminary research (1987, Biochem.Pharm.36: 3797-3800).The open WO of PCT No. 87/01284 (international open day on March 12nd, 1987) relate to comprise 2 ', 3 '-dideoxyinosine, 2 ', 3 '-dideoxy guanosine and 2 ', 3 '-DIDEOXYADENOSINE interior 2 ', 3 '-infectivity and the cytopathic effect of dideoxy purine nucleoside vitro inhibition HIV.Open day on July 6th, 1988 of european patent application 0273277() relate to using and lack 2 ' and 3 ' hydroxyl, and 2 ' and 3 ' carbon atom between have 3 of a pair of key '-Didansine-2 '-ketone (3 '-deoxidation-2 ', 3 '-two dehydrogenation thymidine d4T) patient of treatment retroviral infection.Also find D4T promptly 2 ', 3 '-two dehydrogenations-2 ', 3 '-Didansine or 1-(2,3-dideoxy-β-D-glycerine-penta-2-alkene furyl glycosyl) thymus pyrimidine can suppress hiv reverse transcriptase, and have and the much the same HIV (human immunodeficiency virus)-resistant activity of AZT (Mansuri et al., J.Med.Chem., wait to publish).And the toxicity of D4T is than AZT littler (Mansuri et al., above-mentioned document; Ghazzouli et al., 1988, ICAAC, Abstract 1301, p.344)

Recently the someone reported acid acceptance 2 that antivirus action is arranged ', 3 '-dideoxy-2 '-the fluorine nucleosides.Described 2 for 0287313A2 number as european patent application is open ', 3 '-dideoxy-2 '-fluorine adenosine and 2 ', 3 '-dideoxy-2 '-the fluorine Trophicardyl; European patent application has been described a kind of fluorinated pyrimidine derivative open 0292023A2 number, promptly 2 ', 3 '-dideoxy-2-fluoro-arbinofuranose base cytosine(Cyt) (F-ddc).

Because HIV dependence people cell is supplied with it and finished necessary condition of life cycle, usually be associated with cytotoxic effect so suppress virus replication.The AZT treatment is often with bone marrow toxicity (Richman et al., 1987, N.Engl.J.Med.317: 192-197), the result makes red corpuscle, thrombocyte and white corpuscle generate minimizing, causes anaemia, thrombocytopenia and leukopenia.According to people such as Bartlett (1988, JAMA, 260: statistics 3051-3052) among the patient of AZT treatment, has 40% anaemia and granulocytopenia finally to occur, thereby has increased the chance that infects, and needs to reduce dosage or blood transfusion; After treatment 1 year, have only about 60% ARC and patient AIDS can continue to tolerate AZT(Pettinelli, C.B., Feinberg, J., and the AIDS Clinical Trials Group:Safety and tolerance of zidovudine in patients with AIDS and advanced ARC, abstracted; Fischl, M.A., and the AIDS Chinical Trials Group:The safety and efficacy of two doses of Zidovudine in the treatment of patients with AIDS, abstracted; Two documents are all preceding the delivering of the 4th international AIDS meeting (Stockholm, on June 15th, 1988), and are quoted from by Bartlett).

For fear of these toxic actions, tested to unite and used AZT and other antiviral agents such as acyclovir sodium, foscarnet sodium, interferon alpha, β or γ, interleukin II or ampligen.With 2 ', 3 '-zalcitabine (ddc) also carry out with the research work of AZT alternating treatment (Yarchoan et al., 1988, Lancet 1: 76-81).Its toxicity to marrow of use high dosage ddc(is less relatively continuously) after 8 to 12 weeks, the very peripheral nerve pathology of pain usually appears; So expectation is used alternatingly AZT and ddC, reduces to greatest extent with the toxicity with both.The investigator is trying to explore AIDS therapy a kind of not only effective in cure but also that patient is tolerated.

The present invention relates to utilize the synergy HIV inhibiting (HIV) of nucleoside derivates to duplicate, thus the method that control HIV infects.According to the present invention, unite some nucleoside derivates of use with low concentration, the comparable medicine that is used alone demonstrates stronger antiviral effect and littler toxicity, thereby can improve curative effect to greatest extent and reduce the effect of paying.

In a particular of the present invention, can use purine nucleoside analogs dideoxyinosine (ddI) and pyrimidine nucleoside analoys 2 ', 3 '-dideoxy-2, ', 3 '-two dehydrogenation thymidines (d4T) suppress HIV and duplicate.Embodiment shows that ddI and d4T have showed very strong collaborative anti HIV-1 virus activity; Compare with being used alone compound, unite and use ddI and d4T can produce bigger anti-HIV effect, and have less cytotoxicity.

In another embodiment of the present invention, can unite and use fluorinated nucleoside analogue and d4T inhibition HIV to duplicate.For example, can unite use purine nucleoside analogs 2 ', 3 '-dideoxy-2-β-fluoroinosine (F-ddI) and d4T.

Term used herein (no matter being odd number or plural number) has following specific implication:

Association index (CI): the numeric expression of collaborative or antagonistic action during for drug combination, wherein CI is collaborative less than this numerical value representative, greater than this numeric representation antagonism, equal then expression effect addition.The CI value is according to Chou and the described method (1984 of Talalay, Adv.Enz.Reg.22: 27-55 and 1987, in " New Avenues in Developmental Cancer Chemotherapy ", Harrap and Connors, eds., Bristol-Myers Symposium Series, Academic Press, PP.37-64; Hartshorn et al., 1987, Antimicrobial Agent and Chemotherapy 31: 168-172) use constant amplitude to penetrate that figure equation (isobologram equation) and computer simulation system obtain.

Available following equation is determined association index:

CI = ((D) ₁)/((Dx) ₁) + ((D) ₂)/((Dx) ₂) + (α(D) ₁(D) ₂)/((Dx) ₁(Dx) ₁)

Wherein (Dx) ₁Be to make with medicament 1 produce x% separately to render a service required dosage, (D) ₁Be medicament 1 with (D) ₂Unite and produce the required dosage of x% effectiveness when using.Equally, (Dx) ₂Be to make with medicament 2 produce x% separately to render a service required dosage, (D) ₂Be and (D) ₁Drug combination produces the same required dosage of rendeing a service.As described in people such as Hartshorn, dosage-curative effect slope of a curve shows whether medicament has mutual exclusiveness effect (as the similar mode of action) or mutual nonexcludability effect (as the mode of action independently).If these medicaments are mutually exclusive, then α be 0(be CI be two and); If be non-exclusive each other between medicament, then α be 1(be CI be three and).

Synergy: two or more material combined action have bigger effect when any than single.With regard to antiviral activity, synergistic mechanism should constitute bigger antiviral activity; Then cause average bigger cytotoxicity with regard to cytotoxicity.

It (is TCID that tissue culture suppresses dosage ₅₀): make expressing viral reduce to the required virus quantity of given percentage ratio (promptly 50%).

The tissue culture toxicity dose (is TCTD ₅₀): the reduced number that makes tissue culture cells is to the required medication amount of given percentage ratio (promptly 50%).

Fig. 1 shows the broken broken line of the d4T(of the equal total concn that records according to the p25gag binding capacity), the ddI(dotted line) or the d4T+ddI(solid line) antiviral activity, the result represents with percent inhibition.

The present invention relates to utilize the synergy of nucleoside derivates to suppress duplicating of HIV, thus the consequence that restriction HIV infects.According to the present invention, unite and use some nucleoside derivates stronger antiviral efficacy to be arranged than the indivedual nucleoside derivates of independent use, littler cytotoxicity is arranged simultaneously.

Though the applicant does not have responsibilities and obligations to explain the mechanism of action of the inventive method, but it may be by two or more nucleoside derivates are provided, and wherein any may replace naturally occurring nucleosides in the viral reverse transcriptase activity, like this, these derivatives will mix the possibility that (also stops transcriptive process,reversed by the prolongation that stops nascent DNA then) in the viral DNA order has just increased greatly.

For fully open, will be divided into three parts to content of the present invention and describe: (1) nucleoside derivates is united use; (2) prove that nucleoside derivates unites the in vitro tests that the HIV of use suppresses effect, and the treatment of (3) HIV inhibition nucleoside derivates associating is used.

According to the present invention, the nucleoside derivates that can unite use includes but are not limited to 2 ', 3 '-DIDEOXYADENOSINE (ddA), 2 ', 3 '-dideoxy guanosine (ddG), 2 ', 3 '-dideoxyinosine (ddI), 2 ', 3 '-zalcitabine (ddC), 2 ', 3 '-Didansine (ddT), 2 ', 3 '-dideoxy-2 ', 3 '-Didansine (d4T) and 3 '-the nitrogen base-2 that changes ', 3 '-Didansine (AZT).Can use halogenated nucleoside derivates in addition, particularly 2 ', 3 '-dideoxy-2 '-the fluoro nucleosides, it includes but are not limited to 2 ', 3 '-dideoxy-2-fluoro adenosine, 2 ', 3 '-dideoxy-2 '-fluoroinosine, 2 ', 3 '-dideoxy-2 '-the fluoro cytidine, and 2 ', 3 '-dideoxy-2 ', 3 '-dideoxy-2 '-the fluoro nucleosides, it includes but are not limited to 2 ', 3 '-dideoxy-2 ', 3 '-two dehydrogenations-2 '-fluoro thymidine (Fd4T).Be used for of the present invention 2 ', 3 '-dideoxy-2 '-the fluoro nucleosides preferably wherein fluorine bond be beta comfiguration, it includes but are not limited to 2 ', 3 '-dideoxy-2 '-β-fluoro adenosine (F-ddA), 2 ', 3 '-dideoxy-2 '-β-fluoroinosine (F (Yarchoan and Broden, 1987, N.Engl.J.Med.316: 557-564; Wagar, 1984, J.Cell.Physiol.121: 402-408; Furman et al., 1986, Proc.Natl.Acad.Sci.USA.83: 833-7; Ono et al., 1979, Biochem.and Biophys.Res.Commun.88: 1255-1262).Can determine the treatment and the cytotoxicity dosage of these compositions by test, and consider to use those that high antiviral active and/or minimum Cytotoxic drug combination mode are arranged human body.

Can use vitro detection system (any detection system as mentioned below) to test the inhibition activity of these nucleoside derivates compositions.For example, the target cell system that can use an energy to be infected by HIV forms and (b) produces the effectiveness that HIV particulate relative capacity is estimated selected any nucleoside derivates composition outside infected intracellular according to its inhibition (a) synplasm.In general, this class clone can be T cell or myelocyte/monocytic series or by the other types cell of the gene transfection of CD4.In addition, if nucleoside derivates is connected on " hitting target " molecule such as monoclonal antibody, hormone, the somatomedin etc., then the acceptor of the molecule that is connected with this nucleoside derivates or suitable cell-surface antigens should be able to be expressed by target cell system.

Be to estimate the effectiveness of selected nucleoside derivates associating, can infect the vitro culture target cell, with HIV and before infection, in the course of infection and infect the back and handle it (as use limiting dilution technique) with the nucleoside derivates of various dose.Can estimate the generation of HIV as HIV protein such as 25gag viral core protein or reversed transcriptive enzyme in (1) detection substratum by the method for having stated; (b) detect the antigenic inducing action of HIV in the pair cell with immunofluorescence technique; Or (c) reduction that forms of visual inspection synplasm, with the retarding effect of estimating virus particle is produced.Nucleoside derivates unite the ability that suppresses HIV be by tested nucleosides-ddI) and 2 ', 3 '-dideoxy-2 '-β-fluoro cytidine (F-ddC).

Unite use 2 ', 3 '-dideoxyinosine (ddI) and 2 ', 3 '-dideoxy-2 ', 3 '-two dehydrogenation thymidines have been represented a preferred embodiment of the present invention.Unite use 2 ', 3 '-dideoxy-2 '-β-fluoroinosine (F-ddI) and 2 ', 3 '-dideoxy-2 ', 3 '-two dehydrogenation thymidines (d4T) have also been represented the preferred embodiments of the invention.Relevant 2 ', 3 '-dideoxy-2 ', 3 '-two dehydrogenations-2 '-fluoro nucleosides and 2 ', 3 '-dideoxy-2 '-detailed description of fluoro nucleosides, can apply for that in awaiting the reply jointly of submission on November 12nd, 1987 document is classified this paper reference as No. 120051 referring to Sterzycki, Mansuri and Martin.

To use the potential treatment of nucleoside derivates to render a service in order estimating to unite, can to test the antiviral activity of these associating medicaments by currently known methods.For example, can form in the inhibition HIV of vitro detection nucleotide composition cytotoxicity, synplasm, the ability of reverse transcriptase activity, and viral RNA or proteinic generation etc.

Can be according to method hereinafter described, each the tested nucleic acid derivative that obtains very wide concentration is united the antiviral and/or cytotoxic activity when using and is calculated association index.The preferred method that has hereinafter related to the anti-HIV usefulness of proof nucleoside derivates associating.In addition, can use the animal model system of relevant AIDS, as simian immunodeficiency virus (SIV) system (Kanki et al., 1985, Science 230: 951-954), the antiviral activity that uses nucleoside derivates is united in vivo test; But because the effectiveness difference of allogenic animal (and even with different cells of a kind of animal) when making these medicine phosphorylations not, so in the time of will deriving from the result of a kind of animal (or homozoic a kind of cell type) and seemingly shift another kind of animal (or cell type) onto, inhibition activity that should very careful derivative composition and inhibitor scale show.

According to the present invention, can use synergistic nucleoside derivates composition to stop the generation of plasmodial formation and HIV virus particle in vivo, thereby suppress the progress that HIV infects in the patient body.Can use and the suitable nucleoside derivates of the effective dose of medical carrier preparation by any suitable route of administration, these approach comprise but are not only limited to injection (as intravenously, intraperitoneal intramuscular, subcutaneous etc.) or by epithelium or mucous membrane (or oral mucosa, rectum or vagina epithelium lining cell, mucous membrane of nasopharynx, intestinal mucosa etc.) absorption etc.

In addition, nucleoside derivates may be mixed in in any suitable pharmacology carrier, is connected to carrier or hits target molecule (as antibody hormone, somatomedin etc.) and go up and/or mix in the preparation of liposome, microcapsule and sustained release.

In another embodiment, can infect with treatment HIV having synergistic nucleoside derivates composition and other medicaments to unite use.For example, can with synergistic nucleoside derivates and other antiviral compounds (as AZT) that suppress reverse transcriptase activity are arranged, with solubility CD4 or suppress monoclonal antibody that HIV absorbs or unite use on cell with other cell activators and somatomedin (as interferon alpha, β or γ, tumour necrosis factor, interleukin II, granulocyte/monocyte G CFS, colony-stimulating factor-1 etc.) and with antagonism opportunistic infection AIDS patient's other microorganisms or the medicament of virus.

In another embodiment of the present invention, can use the effectiveness of synergistic nucleoside derivates in the in vitro tests different pharmaceutical.With regard to this point; the practice is by obtaining the marrow sample in the AIDS patient body easily; and make it to accept the processing of tested medicine, compound or regime, thereby identify that those can kill virus and protect normal cell, maybe will stimulate marrow to form medicine, compound or the therapy of colony again.Yet the medullary cell that derives from the AIDS patient is very little at the colony of external formation, and this may be because due to precursor CD34 cell infected by HIV.Therefore in the external effectiveness that just is difficult to maybe can not judge various tested medicines.In such detection system, there is synergistic nucleotide derivative to suppress HIV as use, then will increase the formation of external medullary cell colony, thereby can screen various additive methods and medicine based on external marrow activity.

Following dideoxy two dehydrogenation thymidines (d4T) and dideoxyinosine (ddI) the vitro inhibition HIV of experimental results show that infects the effectiveness of the target cell in T cell source.

Material and method

1) cell and virus

CEM-F is obtained from acute human lymphoblast leukemia patient at first, and has represented the T lymphoid stem cells clone of building strain.It is preserved in American type culture collection (ATCC No.CCL119) as the CCRF-CEM cell.

Human immunodeficiency virus's (HIV) LAVBRU virus strain is by Institute Pasteur, Paris, and Luc doctor Montagnier of France provides.This virus is tamed in the CEM-F cell, and is stored in the liquid nitrogen with little equal portions.Virus titer is every measuring once in two to three weeks and with TCID ₅₀(tissue culture suppresses dosage; Even expressing viral reduces by 50% required virus quantity) represent it.In each experiment hereinafter described, all use 50TCID ₅₀This viral dosage.

CEM-F cell and HIV virus all are grown in the LAV/CEM substratum.This substratum is made up of the RPMI1640 that has added 1% glutamine, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, 2 μ g/ml Polybrene and 10% foetal calf serum.

2) nucleoside derivates D4T and DDI

Dideoxy two dehydrogenation thymidine (d4T, BMY27857-3/8, lot numbers ^#26630-23A) and dideoxyinosine (ddI, BMY40900, lot number ^#25879-46-1) the medical research and development portion by Bristol-Myers company provides.Two kinds of compounds are suspended in 2-3 again and drip in dimethyl sulfoxide (DMSO) (DMSO, D-8779, Sigma Chem.Co.) and the LAV/CEM substratum.Supersound process (Microultrasonic disrupter, Kontes) this suspension and make its final concentration reach 1mM then.Carry out serial dilution by this 1mM storing solution, to carry out two groups of tests that are intended to anti-HIV effect of uniting of trial drug.In first group, hybrid medicine is so that wherein a kind of maintenance constant density, and another kind of medicine then has different concentration.For example d4T remains on 0.01,0.1,1 or 10 μ M, and the concentration range of ddI is 0.1,1,10 to 100 μ M.In this group experiment, the drug ratios in each mixture is all inequality.In order to measure the degree of synergy between d4T and the ddI more accurately, in second group of experiment, medicine was with 1: 5 mixed, and initial concentration is 10 and 50 μ M and does the twice dilution.In all diluents, the ratio of two kinds of medicines all remains unchanged.Use " microcomputer dosage-effect analysis " software (Chou, J., and T-C.Chow, Elsevier-Biosoft Publishers, 1986) to calculate relevant data.Use two kinds of same methods to estimate d4T and ddI to the toxicity of the cell of infection not simultaneously, different is that the ratio of medicine is 1: 1, and the initial concentration of two kinds of medicines is 100 μ M in waiting drug ratios experiment.In all tests, all with nitrogen base thymidine (AZT, the BMY27755/7 lot number of changing ^#2300-38) as positive control.

3) mensuration of D4T and DDI multiplication effect

Every aperture shop applies 2 * 10 in 96 aperture flat boards ⁴Individual CEM-F cell.Cell is mixed with variant composition, and every group is repeated four parts.The cumulative volume of every aperture is 250 μ l.Dull and stereotyped in 37 ℃ and 5%CO ₂Under kept 6 days.At the 6th day with 1 μ Ci/ aperture ³HTdR(New England Nuclear Corp., specific activity 6.7Ci/mmol) with cell marking 4 hours, collect on the glass fiber filter and counting (Hartzman in scintillometer, R.J., Segall, M., Bach, M.L., and Bach, F.H., 1971, Histocompatibility matching.VI.Miniaturization of the mixed Leukocyte culture test:A preliminary report.Transplantation, 11: 268-273).Result's percentage of the cellular control unit that is incubated when not having medicine ³The HTdR intake is represented.In addition, also available tissue culture toxicity dose 50(TCTD ₅₀) expression, it has been represented to compare with control group and has made cell number reduce by 50% necessary medication amount (micrograms).

4) the HIV inhibition of duplicating

With 2 * 10 ⁴The concentration of individual cell/aperture is applied CEM-F cell shop in 96 aperture flat boards, and and 50TCID ₅₀Virus was mixed 45 minutes.In each aperture, add nucleoside derivates after 45 minutes and detection method is described to be incubated by above breeding.When insulation finished in the 6th day, use in the following antigen capturing ELISA method test supernatant liquor to have or not virus antigen to exist.Used in this method two kinds of antiviral core protein p24gag monoclonal antibody (Hu, S.H.et al., 1987, Nature 328: 721-723; Kinney Thomas, E.et al., 1988, AIDS 2: 25-29).

5) antigen is captured detection method

In this method, use two kinds of monoclonal antibody: 25-2(ATCC with 1: 2500 times of dilution ^#9407) and 25-3(ATCC ^#9408) bag is by 96 aperture microtiter plates.These antibody (capture reagent) are special to p24, p40 and p55HIVgag protein.Use be connected with horseradish peroxidase, by the human IgG of purifying in serotype positive person's the serum as indicator.Add substrate tetramethyl benzidine (TMB) back and measure light absorption ratio (450/630nm).OD value reading is divided three classes: experimental value=and be the numerical value that contains the aperture of cell, virus inoculation thing and nucleoside derivates; Control value=be the numerical value (100%) that contains the aperture of cell and virus; Background values=for only containing the numerical value of the aperture of virus inoculation thing.All OD values all should deduct background values.The antiviral effect of representing nucleoside derivates with the percentage p24gag combination rate of control group.For example, be 20% as this numerical value, promptly mean by p25gag to be suppressed in conjunction with the virus replication effect 80% that records indirectly.

6) synplasm forms

Be used for seeing earlier and looking into each The Small Well, with generation of determining to infect and the state of checking cell before antigen captures the supernatant liquor of detection method in collection.Synplasm is easy to observe, although can not count, the difference of synplasm number is clearly between each aperture.

7) cytotoxicity analysis

Can be to host cell CEM-F(ATCC CCL119) detect the toxicity of D4T and DDI.CEM-F is a kind of from forming the T clone of expressing the CD4 acceptor, therefore being to be suitable for the host target cell that HIV infects.Estimate D4T and DDI influence by intracellular the mixing of the CEM-F that detects the not infection that thymidine handles at D4T and DDI to the propagation of non-infected cells.

The result

1) unites the restraining effect of use nucleoside derivates to HIV

Vitro detection p25gag albumen shows, d4T and ddI share suppressing p25gag has very strong synergy in expressing, and this has represented the antiviral activity when being used alone compound bigger.Shown result in the table 1 to the combination test of p25gag; Shown among Fig. 1 that under the condition of same total concn d4T and ddI share and single difference with a kind of medicine.

Table 1 has provided a series of result of experiment, wherein is related to obtain maximum antiviral activity, the optimal concentration of d4T and DDI and concentration ratio thereof.Use " checker " test model, estimate the logarithmic increment of the concentration of each derivative in the various combinations, tested very wide concentration range.

Table 1

D4T and ddI are to the percent inhibition of virus replication ^*

The concentration (μ M) of the concentration of D4T (μ M) ddI

0 1 1 10 100

0 - 1 3 2 86

.01 2 25 13 38 83

.1 1 26 11 13 76

1 24 52 52 75 78

10 87 92 88 92 94

^*Root p25gag binding capacity records

When d4T or ddI and AZT being united (concentration is 1: 10: 50) when using, the synergy that is reached with unite the synergy that uses d4T and ddI little (see Table III and part is discussed).

2) unite the use nucleoside derivates and reduced cytotoxicity

Detect ³The cytotoxicity detection method of H-thymidine incorporation shows, unites and uses d4T and ddI ratio to use arbitrary compound of same concentration little to the cytotoxicity that the CEM-F cell produces separately, but virus resistance is increased more to some extent.Table has shown the d4T, the ddT that use wide concentration range or the test-results that d4T and ddI were done that is used in combination with 1: 1 ratio in the II.

The table II

The d4T of cumulative concentration and/or ddI are to the percentage cytotoxicity of CEM ^*

The concentration (μ m) of the concentration of D4T (μ m) ddI

0 1 1 10 100

0 - - 1 1 1

1 1 1 2 3 1

10 1 3 3 9 14

100 19 26 31 28 30

^*According to ³H-thymidine incorporation records

Discuss

Above-mentioned data show that nucleotide derivative d4T and ddI unite to show when using very strong antiviral synergy.For example, by the table I as can be seen, 1 μ Md4T can suppress 24% virus activity, and 10 μ M ddI can suppress 2% virus activity; But 1 μ M d4T and 10 μ M ddI share, and then above-mentioned inhibiting rate can reach 75%.The curve display of Fig. 1 when the Nucleotide total concn is identical the antiviral activity of d4T, ddI and d4T+ddI, and the synergistic effect when clearly showing d4T and ddI and share.

Association index (CI) is the collaborative and antagonistic action that is produced with the medication combined use of numeric representation.The CI value is by the described method of Chou (seeing above), and the is used thermal radiation survey sheet equation (isobologram equation) of Denging and microcomputer simulation program obtain.The CI value less than certain numerical value interval scale collaborative (promptly sum greater than its each several part and), CI value greater than certain numerical value interval scale antagonism (promptly sum greater than its each several part and), the equal with it then role of delegate addition of CI value (promptly total equal its each several part with).The table III shows when AZT, d4T and ddI concentration ratio are 1: 10: 50, (d4T+ddI), (d4T+AZT) and (ddI+AZT) the CI value of antivirus action.

The table III

When being had 50%, 70% and 90% inhibiting rate, virus obtains

The anti-HIV effect CI value of compound

50% 70% 90%

D4T+DDI 0.024 0.049 0.16

D4T+AZT 0.53 0.43 0.30

AZT+DDI 0.77 0.74 0.75

Because the conspiracy relation between d4T and the ddI, so use low concentration to produce effective restraining effect to virus.As show shown in the II, increase when d4T and ddI share antiviral activity with and without Cytotoxic increase, therefore obtained the effect that selectivity increases antiviral activity.In addition, about single TD with d4T and ddI or when uniting two kinds of medicines of use ₅₀The result of study of value (toxicity dose) shows, when using d4T and ddI separately, and TD ₅₀Be 100 μ M, and unite TD when using d4T and ddI ₅₀Be that 730 μ M(data not shown go out).Unite use AZT, d4T and ddI with 1: 10: 10 concentration ratio, the CI value of its cytotoxic effect discloses, unite and use d4T and ddI to suppress the active required concentration of 90%HIV, than using (d4T+AZT) or (ddI+AZT) obtaining the identical required concentration much smaller (seeing Table IV) of the effect of killing the virus.Therefore, unite use d4T and ddI not only than independent use they the time the stronger effect of killing the virus is arranged, and cytotoxicity reduces.Unite and use these nucleoside derivates to provide new method for treatment HIV infects, make patient be safe from danger and painful situation under obtain the result of treatment that anti-HIV infects.

The table IV

The cytotoxicity CI value that under the nucleosides concentration that is equivalent to 50% and 90% viral inhibiting rate, obtains

Compound 50% 90%

D4T+DDI 2.17 6.40

D4T+AZT 2.45 1.66

AZT+DDI 2.21 2.37

Claims

1, preparation suppresses the method for compositions of HIV, and being characterised in that to unite to use has synergistic nucleoside derivates.

2, be 2 according to the process of claim 1 wherein that the nucleoside derivates that uses is at least a ', 3 '-di-deoxynucleoside.

3, according to the process of claim 1 wherein one of nucleoside derivates of using be 2 ', 3 '-dideoxyinosine.

4, according to the process of claim 1 wherein one of nucleoside derivates of using be 2 ', 3 '-dideoxy-2 ', 3 '-two dehydrogenation thymidines.

5, according to the process of claim 1 wherein one of nucleoside derivates of using be 2 ', 3 '-dideoxy-2 '-the fluoro nucleoside derivates.

6, according to the method for claim 5, wherein the fluoro nucleoside derivates be 2 ', 3 ' ,-dideoxy-2 '-fluoroinosine.

7, according to the method for claim 6, wherein the fluoro nucleoside derivates be 2 ', 3 '-dideoxy-2 '-β-fluoroinosine (F-ddI).

8, according to the method for claim 5, wherein the fluoro nucleoside derivates be 2 ', 3 '-dideoxy-2 '-the fluoro adenosine.

9, method according to Claim 8, wherein the fluoro nucleoside derivates be 2 ', 3 '-dideoxy-2 '-β-fluoro adenosine (F-ddA).

10, according to the method for claim 5, wherein the fluoro nucleoside derivates be 2 ', 3 '-dideoxy-2 '-the fluoro cytidine.

11, according to the method for claim 10, wherein the fluoro nucleoside derivates be 2 ', 3 '-dideoxy-2 '-β-fluoro cytidine (F-ddC).

12, according to the process of claim 1 wherein that at least a nucleoside derivates receives on second molecule with chemical bond-linking.

13, according to the process of claim 1 wherein have synergistic nucleoside derivates be 2 ', 3 '-dideoxyinosine (ddI) and 2 ' 3 '-dideoxy-2 ', 3 '-two dehydrogenation thymidines (d4T).

14, according to the method for claim 13, wherein the ratio of d4T and ddI is 1: 1 to 1: 10.

15, according to the method for claim 13, wherein the ratio of d4T and ddI is 1: 5.

16, according to the process of claim 1 wherein synergistic nucleoside derivates be 2 ', 3 '-dideoxy-2 '-β-fluoroinosine (F-ddI) and 2 ', 3 '-dideoxy-2 ', 3 '-two dehydrogenation thymidines (d4T).

17, according to the process of claim 1 wherein synergistic nucleoside derivates be 2 ', 3 '-dideoxy-2 '-β-fluoro adenosine (F-ddA) and 2 ', 3 '-dideoxy-2 ' 3 '-two dehydrogenation thymidines (d4T).

18, according to the process of claim 1 wherein synergistic nucleoside derivates be 2 ', 3 '-dideoxy-2 '-β-fluoro cytidine (F-ddC) and 2 ', 3 '-dideoxy-2 ', 3 '-two dehydrogenation thymidines (d4T).