HU208254B - Hiv inhibition by applying synergetic combinations of nucleoside derivatives - Google Patents

Description

The present invention is based on the inhibition of human immunodeficiency virus (HIV) replication by using nucleoside derivatives and thereby limiting the deleterious effects of HIV infection. The present invention is based on the discovery that the nucleoside derivative compounds exert a synergistic effect, such that the effect of the effective dose obtained when the agents are mixed is less than the sum of the therapeutic doses of each individual drug; increased anti-viral activity is not associated with a proportional increase in cytotoxicity; and, compared to the corresponding compounds with which the patients are treated individually, the nucleoside derivative compounds are less toxic to the infected cells.

Acquired Human Immunodeficiency Virus (HIV) is a human retrovirus that causes acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC). HIV virions, or viral particles, are spherical and have a diameter of about 1000 angstroms. The particles are covered by a lipid bilayer membrane derived from the outer membrane of the infected host cell. Viral membrane-bound glycoproteins are synthesized as a 160 kd precursor and subsequently converted to two glycoproteins, one gp41 linked to the lipid bilayer and the other called gp120 across the lipid bilayer. The envelope covers the core, which is made up of the proteins p24 and p18. The viral RNA, together with many copies of the reverse transcriptase enzyme that catalyses the binding of the viral DNA, is carried by the nucleus.

The HIV genome contains three genes encoding the components of the retroviral particles: the env gene (which encodes the envelope proteins), the gag gene (which encodes the core proteins), and the pol gene (which encodes the reverse transcriptase enzyme). The three genes are surrounded by nucleotide regions called long terminal repeats (LTRs). LTRs contain sequences involved in controlling expression of viral genes. However, unlike other retroviruses, the HIV genome contains at least five additional genes, three of which are known to have regulatory functions, and whose expression is thought to influence the pathogenicity of the virus. The tat gene encodes a protein that acts as a potential transactivator of HIV gene expression and thus plays an important role in amplifying viral replication. The reverse or trs / art gene is capable of upregulating HIV synthesis through its mechanism of repression; the germline gene allows the regulation of viral unity by selectively generated regulatory proteins or virus constituents. In contrast, the production of the nef gene or the 3'-orf gene, presumably as a secondary messenger cytoplasmic protein, which inhibits the transcription of the HIV genome, reduces the expression of the virus. The vif gene or the row gene is not essential for virus formation, but it is also critical for the successful generation of infectious virions and also affects viral transmission in vitro. The pr or R gene encodes an immunogenic protein of unknown function.

A key point in the immune pathogenesis of HIV infection is thought to be the helper / inducing subset of T lymphocytes expressing the CD4 antigen, resulting in a strong suppression of the immune system. It is likely that the killing of these immune cells by viruses is the main factor whose HIV effect manifests itself in the destruction of the immune system. The envelope glycoprotein plays an important role in the penetration of HIV into CD4 positive host cells. As we have seen, gp120 binds directly to the cellular CD4 receptor molecule, thereby producing HIV tropism on the host cell, which is expressed on the CD4 receptor, such as T helper cells (T4 cells), macrophages, and the like.

After HIV attaches to the CD4 molecule, the virus enters the body and loses its envelope. Once introduced, genomic RNA is transcribed into DNA by reverse transcriptase. The proviral DNA is then combined with the DNA of the host cell chromosome and the infection is put into a "latent" or "latent" phase. However, once activation occurs, proviral DNA is transcribed. The translational and post-translational processes in the virus result in particle attachment and release of mature virions to the cell surface.

During active replication of the virus, the CD4 + host cell is killed. However, the exact mechanism by which HIV cytopathic effects are elicited is unknown. Several mechanisms have been implicated in the immunopathogenesis of HIV infection and in examining its cytopathic effect: accumulation of a large number of unrelated viral DNA in the host cell; strong increase in cell membrane permeability as a result of the release of a large number of viruses onto the cell surface; hypotheses that HIV may induce terminal differentiation of infected T4 cells, resulting in reduced life span. There is an increasing likelihood that both the T4 molecule and the envelope of the virus will play a role in HIV-infected cells to produce a cytopathic effect through accelerated cell fusion. A characteristic feature of the cytopathology of HIV infection is the formation of polynuclear syncytia, which is produced by fusion of about 500 cells, presumably due to the induction of gp120 / gp41 envelope proteins. In contrast, HIV-infected macrophages can produce HIV for long periods without cytopathic effects; presumably, these macrophages are the main reservoir sites for HIV and may be responsible for the transmission of the virus to the central nervous system (Gartner et al., Science 233: 215-219).

To date, no cure has been found for AIDS. There are ongoing attempts to use vaccines and to control the spread of the virus in the population. However, attempts to control viral cycles in an infected patient are pointing towards the use of antiviral agents.

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Several therapies are being investigated to reduce the morbidity and mortality of HIV infection (Yarchoan et al., 1988, Scientific American 259: 110-119; Bartlett, 1988, JAMA 260: 3051-3052; De Clercq, E 1986, J. Chem. 29: 1561-1569; Yarchoan, R. and Broder, S., 1987, N. Engl. J. Med. 316: 557-564). Knowledge of the physiology of the HIV virus has provided many reasonable explanations for preventing the replication of the virus or the spread of infection. The method of the invention provides opportunities for virus attachment to the target cell, binding to the cell membrane, loss of the envelope of the viral nucleic acid, reverse transcription of the HIV HNA RNA genome, transcription and translation of viral mRNA, formation of viral proteins, and other events related to replication or infection.

Several methods have been investigated to prevent the virus from attaching to the cell membrane; they were based on inhibiting the association between the HIV envelope protein, gp120, and the CD4 antigen of a target cell, such as T lymphocytes. In addition to gpl20 and CD4 antigen binding, not only cell membrane crossing but also syncytia formation is observed (Sodroski et al., Naturre 322: 470-474; Lifson et al., 1986, Naturre 323: 725-728; Stevenson et al., 1988). Cell 53: 483-496). Smith et al. (1987, Science 238: 1704-1707) have shown that soluble CD4 antigen can block the infectivity of HIV by binding to viral particles before they encounter CD4 molecules embedded in the cell membrane. Anti-idiotypic antibodies to HIV virus associated with HIV virus in vitro are presumably having a protein structure similar to CD4 determinants (Dagleish et al., 1989, UCLA Symposium on Molecular and Cellular Biology, J. Cell Biochem. Supp. 13B). , p. 298). Furthermore, many large sulfated, negatively charged molecules, such as dextran sulfate, inhibit HIV replication and syncytium formation (Yarchoan et al., Supra).

Anti-sense oligonucleotides inhibit virally induced syncytia formation and expression of the p24 viral protein (Agrawal et al., 1988, Proc. Natl. Acad. Sci. USA 85: 7079-7083). Such synthetic nucleic acid polymers have been designed to inhibit translation of viral mRNA into protein complementary to HIV mRNA; phosphoramidate and phosphorothioate derivatives of non-sensitive oligonucleotides are essentially resistant to endonuclease degradation.

Ribavirin, including the majority of ribose and the triazole ring, is thought to play a similar role to guanosine nucleoside. It has activity against several RNA viruses in vitro, primarily by preventing the guanylation step required for capping of viral mRNA. Ribavirin has been reported to suppress HIV replication in human T lymphocyte culture (McCormick et al., The Chain, Dec. 15, 1984: 1367-1369).

Post-translational processes of viral proteins may be interrupted; Castanospermine, a plant glucosidase activity, reduces syncytia formation and the infectivity of HIV (Wall et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5644-5648).

The final step in HIV replication is to exit the host cell and infect the new cellular target. It has been found that interferon alpha suppresses in vitro replication of HIV (Ho et al., Chain, March 16, 1985: 602-604), may reduce viral delivery, and has direct anti-tumor activity in AIDS-related malignancy, against Kaposi's sarcoma. A lipid compound, AL 721, consisting of neutral glycerides, phosphatidylcholine, and phosphatidylethanolamine in a ratio of 7: 2: 1 has been shown to be able to remove cholesterol from the cell membrane and appears to reduce the infectivity of HIV (Sárin et al. , 1985, N. Engl. J. Med. 313: 1289-1290).

Several methods for controlling the infectivity of HIV have been investigated, which have been linked to inhibition of the viral reverse transcriptase enzyme. Because mammalian cells lack endogenous reverse transcriptase activity, these methods provide effective, selective inhibition of viral replication with minimal host cell cytotoxicity. Reverse transcriptase inhibitors include phosphonoformate (Sandstrom et al., Chain June 29, 1985: L1480), suramine (Mitsuya et al., Science 226: 172-174), rifabutin (or anamycin, Amand et al., Chain Jan. 11). 97, p. 97) and nucleoside derivatives, supra.

Nucleoside derivatives are modified forms of purine nucleosides (adenosine, and guanosine), and pyrimidine nucleosides (thymidine, uridine, and cytidine), which are the building blocks of RNA and DNA. Reverse transcriptase and DNA polymerase bind and accumulate nucleoside derivatives in the resulting DNA chain so that the 5 'carbon atom of the derivative is attached to the 3'-hydroxyl group of the former nucleotide (e.g., by forming a phosphodiester linkage); the following nucleotides can only be attached if the nucleoside derivatives allow their 3'-carbon atom to be attached to the 5'-phosphate group of the other nucleotide. Many nucleoside derivatives that have been studied as effective anti-HIV agents result in the termination of viral DNA replication before transcription of the entire viral genome. These derivatives lack the 3'-substituent to provide attachment to the next nucleoside and thus result in the completion of the chain.

AZT (3'-azido-2 ', 3'-dideoxy-thymidine, azidothymidine, zidovudine) is a nucleoside derivative that is effectively used in the treatment of AIDS and ARC patients; results suggest that AZT enhances the survival of AIDS patients (Fischl et al., 1987, N. Engl. J. Med. 317: 185-191; Creagh-Kirk et al., 1988, J. A. M.A. 260: 3009-3015). Results to date suggest that AZT can effectively be used to help with AIDS 3

Blue HIV-associated psoriasis (Bartlett et al., Supra), AIDS-related dementia (Schmitt et al., 1988, N. Engl. J. Med. 319: 1537-1578), and in the treatment of HIV-associated thrombocytopenia (Substitute et al., 1988, JAMA 260: 3045-3048). AZT is phosphorylated by mammalian cells to form AZT triphosphate (an analogue of thymidine triphosphate), which is thought to inhibit viral DNA production through at least two mechanisms: competitive inhibition and chain termination (Yarchoan et al., Supra). ). The reason for the development of competitive inhibition is that the reverse transcriptase of the virus binds to AZT triphosphate much more strongly than to the native nucleotide; chain termination results from the accumulation of AZT, since AZT lacks 3 'hydroxyl groups and is therefore unable to attach to the corresponding nucleotides.

Dideoxynucleotides are nucleoside derivatives that lack the 2'- and 3'-hydroxyl groups and therefore cause termination of the DNA chain. Furthermore, like AZT, the 5'-triphosphate products of 2 ', 3'-dideoxyadenosine, dideoxyguanosine, dideoxycytidine, and dideoxythymidine selectively inhibit viral reverse transcriptase and beta or gamma DNA polymerase, but do not inhibit the action of DNA polymerase alpha, the major DNA synthesizing enzyme that plays an important role in cell division (Edenberg et al., 1978, J. Biol. Chem. 253: 3273-3280 Wagar et al., 1987, Nucl. Acids Sl. 5: 1933-1946; van Der Vilet et al., Biochemistry 20: 402-408), Replication of the virus with adenosine, guanosine, inosine, cytidine, and thymidine. 2 ', 3'-dideoxynucleosides inhibit in vitro (although thymidine derivatives show less inhibitory activity); the most complete suppression of the HIV virus was observed at doses lower than 10-20 factors necessary to inhibit cell proliferation or T cell immune reactivity (Mitsuya and Bordér, 1986, Proc. Natl. Acad. Sci. USA 83: 1911-1915). Ahluwalia et al. Described an early study of the cellular pharmacology of 2 ', 3'-dideoxyinosine (1987, Biochim. Pharm. 36: 3797-3800). PCT Publication No. WO 87/01284, International Publication date March 12, 1987, for HIV with purine 2 ', 3'dideoxynucleosides such as 2', 3'-dideoxyinosine, 2 ', 3'-dideoxy -adenosine and 2 ', 3'-dideoxyguanosine, and its cytopathic effects. European Patent Publication No. 0273 277, issued July 6, 1988, to 3'-deoxythymidin-2'-ene (3'-deoxythymidine-2 ', 3'-didehydro-thymidine, d4T), of which lacking the 2 'and 3'hydroxyl groups and having a double bond on the 2' and 3 'carbon atoms refers to its use in patients infected with retrovirus. It has been found that d4T, to which 2 ', 3'-didehydro-2', 3'-dideoxy-thymidine, or 1- (2 ', 3'-dideoxy-beta-D-glycero-pent-2-enofuranosyl) - timin, inhibits reverse transcriptase activity, and exhibits anti-HIV activity comparable to AZT (Mansuri et al., J. Med. Chem., in print). However, d4T has a lower toxic effect than AZT (Mansuri et al., Supra; Ghazzouli et al., 1988, ICAAC, Abstract 1301, p. 344).

Acid-stable 2 ', 3'-dideoxy-2'-fluoronucleosides with antiviral activity are currently being addressed. See European Patent No. 0287,313 for 2 ', 3'-dideoxy-2'-fluoro-adenosine and 2', 3'-dideoxy-2'-fluoro-inosine and European Patent No. 0292023 A2, which discloses 2 ', 3'-dideoxy-2'-fluoro-arabinofuranosylcytosine (F-ddC).

Because human cells are required for the life cycle of HIV, inhibition of viral replication is often associated with a cytotoxic effect. AZT therapy involves bone marrow toxicity (Richman et al., 1987, N. Engl. J. Med. 317: 192-197) and results in a reduction in red blood cells (resulting in anemia), platelets (resulting in thrombocytopenia), and white blood cells (resulting in thrombocytopenia). leukopenia). According to Bartlett et al. (1988, JAMA 260: 3051-3052), patients treated with AZT eventually develop anemia, requiring dose reduction or transfusion, and leukopenia, which leads to a reduction in granulocyte count and thus to infection. enhances responsiveness; only about 60% of patients with ARC and AIDS can tolerate AZT therapy for over one year (Pettinelli, CB, Feinberg, J., and the AIDS Clinical Trials Group: Safety and Tolerance of Zidovudine in Patients with AIDS and advanced ARC, abstracted; Fischl, MA, and AIDS Clinical Trials Group: Safety and efficacy of two doses of zidovudine in the treatment of patients with AIDS, both presented at the Fourth International Conference on AIDS [Fourth International Conference on AIDS), Stockholm, June 15, 1988, citing Bartlett).

AZT has also been tested with other antiviral agents, such as acyclovir sodium, foscarnet sodium, interferon alpha, beta, gamma, interleukin-2, or ampligen, to avoid toxic effects. Other studies that alternate between AZT and 2 ', 3'-dideoxycytidine therapy are ongoing (Yarchoan et al., 1988, Chain 1: 76-81). Continuous administration of high doses of ddC (a relatively gentle sparing agent) over a period of 8-12 weeks results in the development of painful peripheral neuropathy; it is hoped that alternating use of AZT and ddC will reduce all toxic effects. Health researchers are currently conducting further research into an effective therapeutic approach that is clinically tolerated by AIDS patients.

The present invention is based on the inhibition of human immunodeficiency virus (HIV) replication by using nucleoside derivatives and thereby limiting the effects of HIV infection. In accordance with the present invention, the combined use of certain nucleoside derivatives may result in a stronger antiviral effect and lower cytotoxicity at lower concentrations than when each drug is used alone, thus providing a cure for HIV infection.

EN 208 254 B can be maximized and toxic side effects minimized. FIELD OF THE INVENTION The present invention relates to a composition for inhibiting HIV replication comprising 2 ', 3'-dideoxy-2', 3'-didehydro-thymidine (d4T) and 2 ', 3'-dideoxy-inosine (ddl), 2', 3 as active ingredient. '-dideoxy-beta-fluoro-inosine (F-ddI), 2', 3'-dideoxy-beta-fluoro-adenosine (FddA) or 2 ', 3'-dideoxy-beta-fluoro-cytidine (F-ddC) contain.

According to the present invention, the purine nucleoside analogue dideoxyinosine (dd1) and the pyrimidine nucleoside analogue 2 ', 3'-dideoxy-2', 3'-didehydro-thymidine (d4T) are preferably used to inhibit HIV replication. In our examples we show that dd1 and d4T have potent synergistic anti-HIV activity, a mixture of dd1 and d4T have more potent anti-HIV activity, and exhibit less cytotoxicity than the individual components.

Definitions and abbreviations

The following terms, singular or plural, have the following meanings. Combination index (Cl): A numerical form used for the synergistic and antagonistic effects of a mixture of drugs, where Cl is less than one, synergism is greater than one, antagonism, and if equal to one, additivism. The value of Cl can be obtained using an isobolugram equation by computer simulation according to Chou and Talalay (1984, Adv. Enz. Reg. 22: 27-55, and New Avenues in Developmental Cancer Chemotherapy, Harrap and Connors, eds. Bristol Miller Symposium Series, Academic Press, pp. 37-64; Hartshom et al., 1987, Antimicrobial Agents and Chemotherapy 31: 168172).

The mixing index can be determined from the following equation:

(D) i (P) ₂ alpha x (P), x (P) ₂ (Dx), ⁺ (DX) 2 ⁺ (Dx), x (D _{x) i} where (D _x ) i is dose, which alone produces an x% effect, and (D) the dose of substance 1 which, when mixed with (D) _2, produces the same x% effect. Similarly, (D _{x) 2} is the dose of the second material, which itself to x% of the effect created, and (D) ₂ to the dose of the second substance which is a mixture with the same x% of the effect (D) _r digested create it. As described by Hartshom et al. (See above), the slope of the dose-range curve reflects the mutually exclusive (ie similar mechanism of action) or non-mutually exclusive (ie independent mechanism of action) effects of the individual substances.

If the effects of each substance are mutually exclusive, alpha = 0 (that is, Cl is the sum of the two terms); if the effects of each substance are not mutually exclusive, alpha = 1 (ie Cl is the sum of the three terms).

Synergism: the effect obtained when two or more substances are used, which is greater than the effect achieved with each substance used individually. For antiviral activity, synergism is interpreted as the highest achievable antiviral activity.

Tissue Culture Inhibitory Dose (TCID ₅₀ ): the amount of virus required to reduce viral expression to a certain degree, i.e. 50%.

Tissue Culture Toxic Dose (Tissue Culture Toxic Dose, or TCTD ₅₀ ): The amount of active ingredient that reduces the number of cells in a tissue culture by a specific amount, i.e., 50%.

Description of the figure

Figure 1: Antiviral activity curves of d4T (dashed line), ddl (dotted line), and d4T + ddl (solid line) at the same total concentration, measured by p24 gag binding, and values expressed as percent inhibition.

The present invention is based on the inhibition of human immunodeficiency virus (HIV) replication by using synergistic nucleoside derivatives, thereby limiting the effects of HIV infection. At a comparable amount of the drug according to the invention, the mixture of each nucleoside derivative compound exhibits a stronger anti-HIV activity but less cytotoxicity than the nucleoside derivative used alone.

Although it is not the duty or duty of the inventors to explain the mechanism of action of the invention, when two or more nucleoside derivatives are used together, one of which replaces a naturally occurring nucleoside in reverse transcriptase activity, consequently, reverse transcription is eliminated by preventing the elongation of the nascent DNA), it increases significantly.

For a clear understanding of the invention, without limiting it, the present invention is subdivided into three parts: (1) nucleoside derivative compounds; (2) an in vitro assay for demonstrating the HIV inhibitory activity of nucleoside derivative compounds; and (3) therapeutic use of nucleoside derivative compounds to inhibit HIV.

The nucleoside derivatives used in the compositions of the present invention include, without limitation: 2 ', 3'-dideoxyadenosine (ddA); 2 ', 3'-dideoxy guanosine (ddG); 2 ', 3'-dideoxyinosine (ddl); 2 ', 3'-dideoxycytidine (ddC); 2 ', 3'-dideoxy-thymidine (dd T); 2 ', 3'-dideoxy-2', 3'-didehydro-thymidine (d4T), and 3'-azido-2 ', 3'-dideoxy-thymidine (AZT). It is also possible to use halogenated nucleoside derivatives which are preferably 2 ', 3'-dideoxy-2'-fluoronucleosides, such as, without limitation, 2', 3'-dideoxy-2'-fluoro-adenosine; 2 ', 3'-dideoxy-2'-fluoro-inosine; 2 ', 3'-dideoxy-2'-fluorocytosine; and the 2 ', 3'-dideoxy-2', 3'-didehydro-2'-fluoronucleosides may be, for example, without limitation, 2 ', 3'

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2 ', 3'-didehydro-2'-fluorothymidine (Fd4T). Preferably, the 2 ', 3'dideoxy-2'-fluoronucleosides are those in which the fluorine is attached in the beta configuration, and are preferably, without limitation, the following: 2,3-dideoxy-2'-beta-fluoroinosine (F -ddI), and 2 ', 3'-dideoxy-2'beta-fluorocytosine (F-ddC).

The use of 2 ', 3'-dideoxyinosine (ddl) and 2', 3'-dideoxy2 ', 3'-didehydro-thymidine (d4T) is one of the most preferred embodiments of the invention. The use of 2 ', 3'-dideoxy-2'-beta-fluoroinosine (F-ddI) and 2', 3'-dideoxy-2 ', 3'-didehydro-thymidine (d4T) is also a preferred embodiment of the invention. its appearance. For a detailed description of the use of 2 ', 3'-dideoxy-2'-fluoronucleosides and 2', 3'dideoxy-2 ', 3'-didehydro-2'-fluoronucleosides, see U.S. Patent No. 1,2001,1991 to Mansuri. and Martin, November 12, 1987, which is incorporated herein by reference in its entirety.

In order to evaluate the appropriate therapeutic efficacy of the nucleoside derivative compounds, the anti-viral activity of the mixtures was assayed by methods well known in the art. For example, the inhibition of HIV cytotoxicity, syncytia formation, reverse transcriptase activity, or viral RNA or protein production may be tested in vitro.

Antiviral and / or cytotoxic activity can be assayed over a wide range of concentrations of each of the constituents of the nucleoside derivative compounds and assayed for induction as described in 6.1. , see below. A preferred method for demonstrating the anti-HIV activity of nucleoside derivative compounds is described in Section 5.2. and Chapter 6, see below. Further, in vivo antiviral activity of nucleoside derivative compounds may be assayed using an animal model of AIDS, such as monkey immunodeficiency virus (Kanki et al., 1985, Science 230: 951-954); however, because of the different animal species (or different cell types within a species) that differ in the degree to which each drug is phosphorylated with different potency, the results obtained with each species should be treated with particular caution for the other species (or different cell types within that species). extrapolate (Yarchoan et al., 1987, N. Engl. J. Med. 316: 557-564; Wagar, 1984, J. Cell Physiol. 121: 402-408; Furman et al., 1986, Proc. Natl. Acad. Sci. 83: 833-7; Ono et al., 1979, Biochem. And Biophys. Commun. 88: 1255-1262. Therapeutic and cytotoxic doses of said compounds can be determined and compounds exhibiting the strongest synergism, with the highest antiviral activity and / or the lowest cytotoxicity, can be used for human purposes.

The inhibitory activity of the nucleoside derivative compounds was assayed using an in vitro assay system, such as that described in Example 6. The efficacy of each selected nucleoside derivative compound can be evaluated by its relative inhibitory activity: (a) inhibition of relative syncytia formation, and (b) inhibition of in vitro production of HIV particles in HIV-infected cells using a HIV-infected cell line. Such cell lines are usually myelocyte / monocyte derived T cells or other cell types having genes transfected with CD4 cells. If the nucleoside derivatives are coupled to a suitable target molecule, such as a monoclonal antibody, hormone or growth factor, etc. coupled, the target cell line expresses the surface antigen or receptor of the corresponding cell bound to the nucleoside derivative.

To determine the efficacy of selected nucleoside derivative compounds, the target cell line is cultured in vitro, infected with HIV, and treated with various doses of the nucleoside derivative before, during and after infection (e.g., limited dilution techniques). The inhibitory effect on the virion can be measured as described above, for example (a) by measuring the production of HIV, the content of HIV proteins, such as culture medium gag25 viral core protein, or reverse transcriptase; (b) induction of HIV antigens in cells by immunofluorescence; or (c) visual assessment of a decrease in syncytia training. The inhibitory activity of the nucleoside derivative compounds refers to the inhibitory activity and the inhibitory dose of the tested nucleoside derivative compounds.

The synergistic nucleoside derivative compounds of the present invention may be used in vivo to inhibit syncytia formation and to prevent the production of HIV virions, and thus to prevent the development of HIV in patients exposed to HIV. An effective amount of the nucleoside derivative, in admixture with suitable pharmaceutical carriers, may be administered by appropriate routes including, but not limited to, injections (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous), or administration via epithelial or mucocutaneous layers (e.g., oral mucosa). , rectal or vaginal epithelial layer, nasopharyngeal mucosa, intestinal mucosa, etc.); etc

Nucleoside derivatives, when mixed with suitable pharmaceutical carriers, linked to a carrier or target molecule (e.g., antibodies, hormones, growth factors, etc.) and / or accumulated in liposomes, microcapsules, should be checked for release prior to in vivo treatment.

The synergistic nucleoside derivative compounds may also be used in other treatments associated with HIV infection. For example, without limitation, the synergistic nucleoside derivative compounds may be used in combination with other antiviral compounds that inhibit reverse transcriptase activity (e.g., AZT); water-soluble CD4, or monoclonal antibodies that inhibit HIV cell wall absorption; or other cytokines and growth factors (e.g., interferon alpha or beta; tumor necrosis factor; culture stimulating factor1 [colony stimulating factor-1], etc.); and substances6

These are used to treat other microorganisms or viruses that cause opportunistic infections in AIDS patients.

The synergistic nucleoside derivative compounds of the present invention can be used to test the efficacy of various drugs in vitro. In this regard, it would be beneficial to expose bone marrow samples from AIDS patients to the in vitro effects of the drug, compound, or diet to identify, for example, whether killing the virus still saves healthy cells or stimulates renal remodeling. . However, bone marrow samples from AIDS patients show extremely poor bone marrow production in vitro. This is probably due to the HIV precursor CD 34 cells. As a result, it was difficult to evaluate the efficacy of various in vitro tested drugs. The use of a synergistic nucleoside derivative compound in such a system to inhibit HIV enhances bone marrow formation in vitro and thus allows for the monitoring of several other processes and in vitro effects of the active ingredient on bone marrow.

Example

Use of dideoxy-didehydro-thymidine and dideoxydnosine

The experiment presented below illustrates the in vitro inhibitory effect of dideoxy inosine (dd1) and dideoxy didehydro-thymidine (d4T) on T-cell target cell culture in HIV infection.

Materials and Methods

The cells and the virus

CEM-F cells initially form a well-established T lymphocyte cell line derived from acute human lymphoblastic leukemia. Available from American Culture Collection as CCRF-CEM cells (ATCC. No. CCL119).

The human immunodeficiency virus strain (HIV) was obtained from Dr. Luc Montagnier, Institut Pasteur, Paris, France. The virus was adapted to CEM-F cells and stored in small aliquots under liquid nitrogen. Virus titer was determined every two to three weeks and expressed as TCID ₅₀ (Tissue Culture Inhibitory Dose) is the amount of virus required to reduce viral expression to a certain degree, i.e. 50%. In the sections presented below, viral doses of 50 TCID _{50 were} used.

Both CEM-F cells and HIV were cultured in LAV / CEM medium. RPMI 1640 supplemented with 1% glutamine, 100 U / ml penicillin, 100 pg / ml streptomycin, 2 pg / ml polybrene, and 10% fetal calf serum was used.

Nucleoside derivatives of d4T and addl

Dideoxy-didehydro-thymidine (d4T, BMY27 8573/8, lot # 26630-23A) and dideoxy-inosine (ddl,

BMY40900, Lot # 25 879-46-1) was obtained from the Pharmaceutical Research and Development Division of Bristol-Myers Company. Both materials were suspended in 2-3 tablets of dimethyl sulfoxide (DMSO, D-8779, Sigma Chem. Co.) and LAV / CEM medium. The suspension was sonicated (Microultrasonic Disrupter, Kontes) and adjusted to a final concentration of 1 mM. Each dilution was prepared from a 1 mM stock solution and two independent experiments were performed to investigate the effect of the compounds on HIV. In the first series, the active ingredients were mixed by keeping one of them at a constant concentration while the other at a different concentration. For example, the concentration of d4T was maintained at 0.01, 0.1, 1.0, or 10 μΜ, while the concentration of ddl varied between 0.1, 1.0, 10, and 100 μΜ. In this system, the proportions of the active compounds were different in each mixture. In order to determine more precisely the potential synergism between d4T and ddl, in the second series of experiments, the active compounds were mixed at a ratio of 1: 5, starting with 10 and 50 μΜ and diluted twice. Data were evaluated using Dose-Effect Analysis with Microcomputers software (Chou, J., and T-C. Chow, Elsevier-Biosoft Publishers, 1986). The same procedure was used to determine the toxicity of d4T and ddl to non-infectious cells, except that in the constant ratios experiment, the ratio of active ingredients was set to 1: 1 and 100 μΜ of each substance was used as an initial concentration. Azidothymidine (AZT, BMY27 755/7, horse # 2300-38) was used as a positive control in the assays.

Determination of the proliferative effect of d4T and ddl

For each assay, 2x10 ⁴ CEM-F cells / well were plated in a 96-well plate. Cells were mixed for each of the different combinations, and each measurement was repeated four times. The total volume / well was 250 μΐ. The plates were incubated for 6 days at 37 ° C in 5% CO 2. On day 6, cells were labeled with 1 gCi / well 2 in ³ HTdR (New England Nuclear Corp., specific activity 6.7 Ci / mmol), collected on a glass wool filter, and scintillation counter (Hartzmann, RJ, Segall, M.). , Bach, ML, 1971, Histocompatibility matching (VI. Miniaturization of mixed leukocyte culture test: A preliminary report. Transplantation, 11: 268-273). Results are expressed as% of control ³ HTdR uptake of cells incubated without drug. Results were expressed as a toxic dose of 50% tissue culture (Tissue Culture Toxic Dose, or TCTD50), the amount of active compound expressed in μg which reduced the number of cells in tissue culture by 50% relative to control.

HU 208 254 B

Inhibition of HIV replication

CEM-F cells were plated at 2x10 ⁴ CEM-F cells / well in 96-well plates and shaken well with virus TCTD _{50 for} 45 min. After 45 minutes, the nucleoside derivatives were introduced into the wells and incubated as described for proliferation. The presence of the viral antigen in the supernatant was measured after six days using the antigen capture ELISA assay (presented below). The assay uses two monoclonal antibodies against the p24gag virus protein (Hu, SH et al., 1987, Natur. 328: 721-723; and Kinney Thomas, E. et al., 1988, AIDS 2: 25-29).

Antigen capture procedure elisa

Microtiter plates (96-well plates) were coated with two monoclonal antibodies: 25-2 (ATCC # 9407) and 25-3 (ATCC # 9408), each at a dilution of 1: 2500. These antibodies (capture reagents) are specific for the p24, p40, and p55 HIV gag proteins.

The conjugate used was horseradish peroxidase (HRP) conjugated to human IgG purified from seropositive individual serum. Absorbance (450/630 nm) was measured after addition of substrate [tetramethylbenzidine (TMB)]. OD values can be divided into three categories: Experimental values = values obtained for wells containing cells, virus inoculum, and nucleoside derivative (s); Control values = values obtained for wells containing cells and virus; and Background values = values obtained for wells containing only virus inoculum. The background values were subtracted from the OD values obtained in each case. The antiviral activity of the nucleoside derivative (s) is expressed as a percentage of control p24gag binding. For example, if a value of 20% is obtained, this means that viral replication measured indirectly via the p24gag binding is inhibited by 80%.

Syncytia training

Before the supernatant was collected for antigen capture ELISA, cells were visually examined. This verified that the infection was indeed occurring and also verified the condition of the cells. Syncytia, although not counted, are easy to observe, and their amount in each well is strongly conspicuous.

Monitoring of cytotoxicity

The toxicity of D4T and DDI was checked against CEM-F host cells (ATCC CCL119) D. CEMF is a T-cell line expressing CD4 receptors, which makes it an appropriate host cell for HIV infections. The effect of d4T and ddl on the proliferation of uninfected cells was measured by the incorporation of thymidine into d4T and uninfected CEM-F cells treated with dd1.

Inhibition of HIV nucleoside derivative by compounds

In vitro assays for the p25gag gene, in the case of a mixture of d4T with dd1, showed a strong synergistic effect on the inhibition of p25gag expression and a stronger antiviral activity than the individual components tested. The results of p25gag binding assays are summarized in Table I below. The results obtained with d4T mixed with ddl and the individual components tested at the same total concentration are shown in Figure 1. Table I shows the results of a series of experiments conducted to achieve the optimal concentration and ratios of d4T and ddl required for maximum antiviral activity. The test was performed over a wide range of concentrations, and the results were evaluated by logarithmic increase in the concentration of each derivative in several compounds using a "checkerboard" sample.

/. spreadsheet

Percent inhibition of viral replication by d4T and ddl *

D4T concentration (uM) ddl concentration (μΜ) 0 0.1 1 10 100 0 - 1 3 2 86 0.01 2 25 13.38 83 0.1 1 26 11 13 76 1 24 52 52 75 78 10 87 92 88 92 94

*

P25gag bindings based on

Using d4T or ddl in combination with AZT at 1:10:50 concentrations resulted in a smaller synergistic effect than the synergism obtained when d4T and ddl were mixed (see Table III).

Nucleoside derivatives

The results of cytotoxicity measurements of ³ H-thymidine accumulation show that the d4T and ddl used in the compound do not show greater cytotoxicity to LEM-F cells, despite the increased cytotoxicity of the virus, than the compounds tested alone at the same concentration. The results of the measurements were analyzed over a wide concentration range of d4T and ddl, or in a 1: 1 mixture of d4T and ddl, and were analyzed in Table II. is shown in Table II.

HU 208 254 Β

II. spreadsheet

Effect of increasing concentrations of d4T and ddl on CEM percent cytotoxicity *

D4T concentration (μΜ) ddl concentration (μΜ) 0 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 75

* Based on 3H-thymidine uptake

The data presented show that the d4T and dd1 nucleoside derivatives have a strong synergistic antiviral activity when used in a compound. For example, with reference to the data in Table I, 1 μΜ of d4T resulted in a 24% inhibition of virus activity and 10 μΜ of ddl resulted in a 2% inhibition of virus activity; however, a mixture of 1 μΜ d4T with 10 μd ddl resulted in 75% inhibition of HIV activity. Figure 1 is a graph showing the antiviral activity of d4T, dd1, and (d4T + ddl) at the same total nucleoside concentration; the figure clearly shows the synergistic effect of d4T and ddl.

The Combination Index (Cl) is a numerical form used for synergistic and antagonistic effects of drug mixtures. The value of Cl can be obtained using an isobologram equation by computer simulation according to Chou's procedure, see 3.1. section. If the Cl value is less than one, we are talking about synergism (i.e., the whole value is greater than the sum of the constituents), if greater than one, antagonism (i.e., the whole value is less than the sum of the constituents); integer equals sum of creators). In III. Table IV shows the Cl of antiviral activity of (d4T + ddl), (d4T + AZT), and (ddl + AZT) at a 1:10:50 concentration of AZT, d4T and ddl.

III. spreadsheet

Cl values for anti-HIV activity are 50, 70, and 90% inhibition of the virus

Dry Illet 50% 70% 90% d4T + ddI 0,024 0.049 0.16 AZT + d4T 0.53 0.43 0.30 ddl + AZT 0.77 0.74 0.75

Due to the synergistic relationship between d4T and dd1, a suitable viral inhibitory effect can be achieved even at lower nucleoside concentrations. II. Table 4 shows that increased antiviral activity of d4T and dd1 does not result in increased cytotoxicity and thus selective antiviral activity can be achieved. In addition, the results of experiments to generate a TD ₅₀ (toxic dose) value using d4T and ddl alone or in combination, while the TD ₅₀ values obtained using d4T and ddl alone were above 100 μΜ, d4T and ddl gave 730 μΜ (data not shown). The Cl of cytotoxicity of 1:10:10 for AZT, d4T, and ddl indicates that the concentration required to achieve 90% HIV inhibition is much lower for d4T and ddl than for the same virocidal concentration (d4T + ddl). ), (d4T + AZT), and (ddl + AZT), respectively (see Table IV). Thus, d4T and ddl are much more virocidal than d4T and dd1, respectively, and (d4T + ddl) is also slightly toxic. Thus, the therapeutic prospects for the treatment of HIV infections by using nucleoside derivative compounds are much broader; during therapy, patients can achieve anti-HIV activity without dangerous and painful complications.

ARC. spreadsheet

Cytotoxicity Cl values at 50 and 90% viral inhibition at nucleoside concentrations

Compound 50% 90% d4T + ddl 2.17 6.40 AZT + d4T 2.45 1.66 AZT + ddl 2.21 2.37

PATENT CLAIMS

Claims (4)

PATENT CLAIMS A process for the preparation of a composition for inhibiting HIV replication, characterized in that 2 ', 3'-didehydro-thymidine (d4T) and 2', 3'-dideoxy-inosine (dd1), 2 ', At least 3'-dideoxy-beta-fluoroinosine (Fddl), 2 ', 3'-dideoxy-beta-fluoroadenosine (F-ddA) or 2', 3'-dideoxy-beta-fluorocytidine (F-ddC) in at least together with a pharmaceutically acceptable carrier, to form a standard dosage form. (Priority: 09.09.1989) 2. A process according to claim 1 wherein the active ingredient is d4T and dd1. (Priority: March 17, 1989) 3. The process of claim 2, wherein d4T and dd1 are used in a weight ratio of from 1: 1 to 1:10. (Priority: March 17, 1989) 4. A process according to claim 3, wherein d4T and dd1 are used in a weight ratio of 1: 5.