Antiviral therapy against HIV infection

Antiviral therapy against HIV infection

Journal of the American Academy of Dermatology Resnick et ai. 9. Greenspan 18, Mastrucci MT, Leggot Pl, et al. Hairy leukoplakia in a child [LetterJ...

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Journal of the American Academy of Dermatology

Resnick et ai. 9. Greenspan 18, Mastrucci MT, Leggot Pl, et al. Hairy leukoplakia in a child [LetterJ. AID8 1988;2:143. 10. Schofer H, Ochsendorf FR, Helm EB, et al. Treatment of oral "hairy" leukoplakia in AIDS patients with vitamin A acid (topically) or acyclovir (systemically) [Letter]. Dermatologica 1987;174:150-1. 11. Herbst lS, Morgan J, Raab-Traub N, et at. Comparison of the efficacy ofsurgery and acyclovir in the treatment of oral hairy leukoplakia. 1 AM ACAD DERMATOL 1989;21:753-6. 12. Schidt M, Greenspan D, Daniels TE, et al. Clinical and histologic spectrum of oral hairy leukoplakia. Oral Surg Oral Med Oral Pathol 1987;64:716-20. 13. Lupton GP, James WD, Redfield RR, et a!. Oral hairy leukoplakia. A distinctive marker ofhuman T -eel I Iymphotropic virus type III (BTLV-III) infection. Arch Dermatol 1987;123:624-8. 14. Conant MA. Hairy leukoplakia: a new disease of the oral mucosa. Arch DermatoI1987;IZ3:585-7. 15. Eversole LR, Stone CE, Beckman AM. Detection of EBV and HPV DNA sequences in oral "hairy" leUkoplakia by in situ hybridization. 1 Med Viral 1988;26:271-7. 16. Reed KD, Fowler CB, Brannon RB. Ultrastructural detection of herpes- type virions by negative staining in oral hairy leukoplakia. Am J Clin Pathol 1988;90:305-8. 17. Greenspan 18, Greenspan D, Lennette ET, et al. Replication of Epstein-Barr virus within the epithelial cells of oral "hairy" leukoplakia, an AIDS-associated lesion. N Engl J Med 1985;313:1564-71.

18. Desgranges C, de-The G, Ho JHC, et al. Neutralizing EBV-specific IgA in throat washings of nasopharyngeal carcinoma (NPC) patients. Int J Cancer 1977;19:627-33. 19. Bunker ML, Chewning L, Wang SE, eta!. Dermatophilus congolensis and "hairy" leukoplakia. Am J Clin Pathol 1988;89:683-7. 20. Friedman-Kien AE. Viral origin of hairy leukoplakia. Lancet 1986;2:694-5. 21. Newman C, Polk BF. Resolution of oral hairy leukoplakia during therapy with 9-(l,3-dihydroxy-2-propoxymethyl) guanine (DHPG). Ann Intern Med 1987;107:348-50. 22. Greenspan D, Greenspan lS, DeSouza Y, et aI. Efficacy of BWA515U in treatment of EBV infection in hairy leukoplakia. J Dent Res 1987;66: 184. 23. Kessler HA, Benson CA, Urbanski P. Regression of oral hairy leukoplakia during zidovudine therapy. Arch Intern Moo 1988;148:2496-7. 24. Phelan J, Klein R8. Resolution of oral hairy leukoplakia during treatment with azidothymidine. Oral Surg Oral Moo Oral PathoI1988;65:717-20. 25. Elion GB. Mechanism of action and selectivity of acyclovir. Am 1 Med 1982;73(suppl):7-12. 26. Pagano 18, Dalta AK. Perspectives on interactions of acyclovir with Epstein-Barr and other herpes viruses. Am J Moo 1982;73(suppl):18-25. 27. Pagano 18, Sixbey JW, Lin 1. Acyclovir and Epstein-Barr virus infection. 1 Antimicrob Chemother 1983;12(suppl): 113-21.

Antiviral therapy against HIV infection Hiroaki Mitsuya, MD, Robert Yarchoan, MD, Seiji Hayashi, MD, and Samuel Broder, MD Bethesda, Maryland The replication of human immunodeficiency virus (HIV) can be suppressed in vivo by drugs chosen on the basis of their selective in vitro antiviral activity. Such suppression can confer prolonged survival and improved quality oflife in patients with already established HIV infection. The clinical benefits indicate that targeted therapy for acquired immunodeficiency syndrome based on the emerging knowledge of replicative cycle of HIV is an attainable goal. (J AM ACAD DERMATOL 1990;22:1282-94.)

The acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV), which hasthe capacity to replicate within critical cells of the immune system, particularly in monocytes/ macrophages and helper T cells). 1-4 The From the Clinical Oncology Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health. Reprint requests: Hiroaki Mitsuya, MD, The Clinical Oncology Program, Building 10, Room 13N248, National Cancer Institute, Bethesda MD 20892. 16/0/19115

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purpose of this article is to summarize possible therapeutic interventions that are based on the emerging knowledge of the life cycle of HIV and progress in the development of drugs against AIDS. The different stages in the life cycle of HIV present a variety of targets for antiviral agents. 5 These are summarized in Table I. Reverse transcriptase is one of the most attractive targets and there have been notable clinical successes, especially with 3'-azido-2',3'-dideoxythymidine, better known as zidovudine or azidothymidine (AZT).6-9 The testing of new antiretroviral agents depends on the

Volume 22 Number 6, Part 2 June 1990

Antiviral therapy against HIV infection 1283 rev

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availability of rapid and sensitive in vitro screening systems. In 1984, we developed rapid in vitro systems and made a commitment to screen drugs in large numbers for potential anti-HIV effects. 10, II In February 1985, in collaboration with the Wellcome Research Laboratory, we established that a dideoxynucleoside analog, AZT, is potent against HIV in vitro. 6 The system we developed almost accurately predicted the target in vivo therapeutic concentrations of this drug. During the next 2 years, AZT was administered to patients with advanced AIDS and produced immunologic and clinical improvement (including neurologic) in such patients and prolonged the survival of certain patients. 7-9 For certain nucleoside analogs, a great deal of knowledge in terms of structure/activity relationships has now emerged. 5,11-14 It is worth stressing that effective therapy of HIV infections may well depend on a combination of therapeutic strategies without relying on any single agent for two reasons: (I) emergence of drug-resistant strains might be less likely and (2) efficacy could be enhanced and toxicity lessened. Once the virus has been controlled, it may be possible to introduce immunopotentiative and adoptive cellular therapy to repair the immune damage. POSSIBLE TARGETS IN THE LIFE CYCLE OF HIV FOR THERAPEUTIC INTERVENTION HIV as a retrovirus HIV belongs to a family of retroviruses whose members catalyze the conversion of genomic RNA to DNA in their life cycle (i.e., at one step of their cycle of replication, genetic information flows from RNA to DNA, reverse or "retro" direction). A crucial step in viral replication is the reverse transcrip-

Table I. Some stages of HIV replication that may theoretically be targets for therapeutic intervention Binding to target-cell-CD4 Phosphorylation of CD4 Fusion of virus with target cell Entry into target cell and exposure of functional RNA as a ribonucleoprotein Transcription of RNA to DNA by reverse transcriptase Degradation of RNA by RNase activity (encoded by viral pol gene) Migration into the nucleus Integration of DNA into host genome Transcriptional efficiency/translation of viral RNA Ribosomal frameshifting to produce the gag-pol fusion protein Viral component production, modification, maturation, and encapsidation Transport of nucleocapsid to the plasma membrane Viral budding packaging

tion of the viral RNA to produce linear doublestranded proviral DNA. In this process, the termini of the RNA genome are duplicated to give terminal structures in the DNA that are called long terminal repeats (LTRs). The viral genome is expressed from the proviral DNA. The LTR, which has enhancer/ promoter and polyadenylation signals, serves to initiate and regulate the expression of the viral genes. HIV is the most complex retrovirus studied. The virus has at least nine known genes (Fig. 1), and its replication requires a complex sequence of steps, each of which might be a target for therapeutic intervention. 5, 13 Before the recognition of human pathogenic retroviruses, retroviruses were known to contain a standard set of genes, called gag, pol, and

Journal of the American Academy of Dermatology

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Fig. 2. Antiviral activity of soluble CD4 and chimeric CD4-IgG proteins against HIV-1 in vitro. Susceptible T cells (ATH8; 2 X 105) were exposed to high multiplicity of infection of HIV-111I Band cultured in the presence and absence of CD4 proteins (solid bars). Control cells were similarly treated but not exposed to the virus (open bars). Total viable cells were counted by trypan blue exclusion method on day lOin culture. Note that chimeric proteins [CD4(2hi and CD4(4}yl represent chimeric molecules consisting of the two distal and all four Ig-like domains of CD4, respectively, fused to the constant region of IgG I heavy and light chains] exhibit virtually the same antiviral effect against HIV-ll1lB as "plain" soluble CD4. (From Capon DJ, Chamow SM, Mordenti J, et al. Designing CD4 immunoadhesins for AIDS therapy. Reprinted by permission from Nature 1989;337:525-31. Copyright 1989, Macmillan Magazines Limited.)

env, as basic components of a replicating genome. Reverse transcriptase (the viral DNA polymerase) that catalyzes this step is encoded by the pol gene of the virus, a gene that is conserved to a considerable extent in its amino acid and nucleotide sequences among all retroviruses. IS The reverse transcriptase gene is one component of the pol gene, and in general this gene is expressed as a large molecular weight fusion protein that includes a protease, reverse transcriptase, ribonuclease H (RNase H), and an endonuclease (integrase). Posttranslational cleavage of this fusion protein is thought to yield appropriate peptide subunits as functioning molecules (vide infra). HIV, in common with previously known animal retroviruses, has as its major structural components a core of genomic RNA; group-specific antigen (gag) proteins that playa role both in the structure of the core and assembly of the virion on the membrane of the host cell; a lipid bilayer; and an outside envelope glycoprotein. As the virus is expressed in a given cell, it manufactures a gag-pol fusion protein. This retroviral fusion protein (gag-pol) can then undergo posttranslational cleavage events mediated by viral protease to fonn active gag and pol products. Various retroviruses, including HIV, use a riboso-

mal frameshift to translate the starting gag-pol polyprotein. 16 The specific functions of several of nine genes are still not known or are not completely understood. One such gene, vif (virion infectivity protein), has recently been linked to the ability to replicate by a pathway of cell-free virion infect ionp,18 The vif gene encodes a 23 kd protein that appears to playa crucial role in the efficient generation of infectious virus. HIVs that contain nonfunctioning mutant vif genes are quite limited in their capacity to establish stable infection in vitro. Therefore, it is conceivable that drugs or biologics could be developed to interfere with vif and thereby attenuate the pathogenicity of HIV infection. Other as yet incompletely characterized genes are two newly identified genes, vpu and vpr, that are actively expressed in HIVinfected cells. 19, 20 Perhaps products of these two genes playa role in controlling the replication of the virus. Studies on the function of these genes are still at an early stage, however. CELL BINDING AND ENTRY

The first step in the infection of a cell by HIV is its binding to the target cell receptor, CD4 molecule. The process of specific binding between the CD4 re-

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Antiviral therapy against HIV infection 1285

Fig. 3. Inhibition of syncytia formation by dextran sulfate. Uninfected CD4+ Molt-4 cells were cocultured with IlIV-I-producing G 11 cells for 24 hours in the presence (A) or absence (B) of 6.25 }.tmol/L dextran sulfate.

ceptor and parts of the viral envelope glycoprotein' may be vulnerable to attack by antibodies either to the virus or to the receptor, and, theoretically, certain chemicals or small peptides could be designed to occupy the receptor of the target cell or the receptor of the virus and accomplish inhibition of the infectivity of the virus. A genetically engineered soluble form of the CD4 protein can exert a potent antiviral effect against HIV-l (Fig. 2).21-25 Such a construct would be expanded to provide false attachment sites for the virus and prevent normal viral attachment to the CD4+ target cell. This soluble CD4 is undergoing phase I clinical trials. The use of soluble CD4 as a therapeutic approach against HIV infection is still evolving. For example, the plasma half-life of soluble CD4 appears to be short. To overcome this problem, "chimeric" mole-

cules have been created by genetically combining the CD4 protein and the constant region of human IgG.26 These chimeric CD4 molecules, designated immunoadhesins, retain antiviral activity against HIV-I comparable to "plain" soluble CD4 (Fig. 2), and also retain the capacity to bind Fc receptors with high affinity. Furthermore, immunoadhesins acquire a substantially longer plasma half-life compared with soluble CD4. 26 Although it is not yet clear to what extent the addition of immunoglobulin components can bring about advantages, once the structure requirements are met for the optimal effectiveness for clinical application, modified CD4 species could be potent antiviral agents against HIV infection. Soluble CD4 may also be combined to a toxin or radionuclide so that one could specifically destroy

1286 Mitsuya et al.

and eliminate HIV-infected cells. Chaudhary and co-workers27 have recently generated a fusion protein, CD4-PE40, which has been shown to specifically attack and destroy HIV-infected cells. More studies ofthese CD4 proteins of"second generation" are presently under way. Dextran sulfate, with a molecular weight of approximately 8000, which has been given orally to patients as an anticoagulant or antilipemic agent, has recently been shown to block the binding of HIV virions to CD4+·target cells, inhibit syncytial formation, and exert a potent inhibitory effect against HIV (Fig. 3).28-31 Recent data, however, suggest that dextran sulfate is poorly absorbed, and sufficient inhibitory doses are not achieved by oral administration. It also remains to be determined how quickly this agent is degraded in human plasma and whether it is effective against the virus in the presence of 100% plasma in vivo. Nonetheless, perhaps dextran sulfate could be viewed as a prototype for a large class of anionic polysaccharides (of both natural and synthetic origins) that can block viral replication in vitro, but it should be stressed that cautious interpretation of such in vitro data is required. Another target is the envelope protein itself. Although there can be considerable variation in the protein from one viral isolate to another, the range of alterations in the binding site is most likely constrained by the need to bind to CD4, which is relatively constant in structure. An antibody directed against this site might bind to and neutralize most strains of HIV, and perhaps kill infected cells as they begin to express envelope antigens, so that spread of virus to uninfected cells can be reduced. In this context, monoclonal antibodies to the envelope protein could have a therapeutic role in patients with AIDS or related diseases. A murine monoclonal antibody against gp120 has been generated and shown to inhibit the infectivity of certain strains of HIV-l (type-specific) and to block syncytial formation. 32 A potential difficulty of this approach, however, is that virally infected cells could make infectious cellto-cell contacts that are beyond the reach ofhumoral antibodies. (Even viral mutants, which are defective in vifand are thereby poorly transmitted by cell-free virion infection, may still be transmitted by a process of cell-to-cell spread.) Thus, antibodies might not gain access to relevant epitopes under certain circumstances. Also, AIDS can occur in the face of what in vitro appears to be neutralizing antibodies to

Journal of the American Academy of Dermatology

HIV. Whether this occurs because the titers of such antibodies are low or because such antibodies do not block epitopes that mediate in vivo cytopathogenicity is under investigation. One must at least consider that humoral immunity per se does not protect the host, and what protection there is is mediated by cellular immunity. After binding to a cell, HIV enters the CD4+ target cell by an incompletely understood fusion process mediated by specific sites in the gp41 portion of the envelope (the fusogenic domain). Binding of HIV virions to CD4 brings about phosphorylation of the CD4 molecule, probably via protein kinase. 33 The process of viral entry cannot be completed without such CD4 phosphorylation, suggesting a potential target for future therapies. Another theoretical target is the stage of "uncoating" of the virus after it enters a target cell. At this stage, the virus may lose a significant portion of its envelope coat and functional RNA is released into the cytoplasm, most likely as a ribonucleoprotein complex (each virion is thought to convey a dimer of two identical genomic RNA subunits into the cytoplasm). Pharmacologic agents that interfere with these steps might eventually be developed. Viral RNA is then used as a template for the DNA synthesis by reverse transcriptase. HIV reverse transcriptase uses a lysine transfer RNA as a primer to make a negative strand DNA copy, thus forming an RNA-DNA hybrid. Reverse transcriptase possesses an inherent ribonuclease H (RNase H) activity that specifically degrades the RNA of the RNA-DNA hybrid. The C-terminal region of the reverse transcriptase protein is believed to be a domain with RNase H activity (Fig. 1). Theoretically, inhibition of this process would suppress viral replication because an effective and orderly degradation of the viral RNA is a requirement of effective conversion of genomic RNA to proviral DNA. The reverse transcriptase then catalyzes the production of a positive-strand DNA, and eventually a double-stranded viral DNA is formed. The double-stranded viral DNA can then migrate to the nucleus by an as yet poorly characterized mechanism. It is possible that this nuclear migration could be blocked by drugs or biologics. It has recently been shown that HIV-1 reverse transcriptase is highly error prone. The high error rate of HIV-1 reverse transcriptase in vitro translates to approximately 5 to 10 errors per HIV-I genome per round of replication in vivo. 34,35 This high

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error rate may be, at least in part, responsible for the hypermutability of HIV. Thus, the emergence of drug-resistant HIV variants should always be con~ sidered possible. The reverse transcriptase of HIV has been purified and seems to exist as a p51 and a p66 molecule. 36 Large quantities of the HIV reverse transcriptase have become readily available because the enzyme has been expressed in bacteria and yeast by recombinant DNA technology. 37, 38 This should aid the testing of potential reverse transcriptase inhibitors. Two drugs, suramin lO and HPA-23,39 which were chosen early on for clinical trials because they inhibit reverse transcriptase (and were already being given to human beings for other purposes), have not appeared to confer clinical benefits. Only a limited range of doses and schedules of administration have been tested, however. Nevertheless, agents that inhibit reverse transcriptase have the most likelihood of achieving an immediate clinical impact. Several potent agents that inhibit this enzyme are either already available, are already available for the therapy of other viral diseases (e.g., phosphonoformate),40 or are being developed specifically for AIDS on the basis of in vitro screening,

Integration, latency, and reactivation The proviral DNA then becomes integrated into the genome of the host cell. After migration into the nucleus, a linear form of proviral DNA is integrated into the cellular genome, which is medicated by the in protein (integrase) or the viral endonuclease. The capacity of retroviruses to integrate into the genome of host target cells was first thought to render retroviral diseases inherently untreatable. In the future, drugs might be developed to interfere with the viral endonuclease. Later in the viral life cycle, perhaps upon activation of the infected cell by physiologic signals, including antigens or regulatory interleukins such as GM-CSF in monocytes!macrophages,41 the proviral DNA is transcribed to mRNA (and viral genomic RNA) by host cell RNA polymerases. Indeed, immunologic activation ofT cells by antigenic or mitogenic stimuli has been correlated with induction of a DNA binding protein (NF-KB) that in turn activates the enhancer element in the viral LTR and increases HIV expression in synergy with another gene product (tat). 42 Proteins encoded by a variety of DNA viruses, such as herpes simplex virus type I or adenovirus, are also able to activate expression of

Antiviral therapy against HIV infection 1287 HIV; it is theoretically possible that infection by such viruses (including the newly identified virus called human herpesvirus type 6)43 may directly contribute to the activation of HIV by mechanisms independent of immune stimuli. 44-46 It is also possible that HTLV-I (or HTLV-II) can produce transcriptional activating factors that potentiate transcription of HIV when both viruses coinfect the same cell. Coinfection has been reported. 47 ,48 Whatever the route of transcription inhibition is, the viral RNA is subsequently translated to form viral proteins, again using the biochemical apparatus of the host cell. Retroviruses use a novel mechanism for the translation of certain genes. For example, they can synthesize a single gag-pol polyprotein from two separate reading frames on an RNA template: reliable ribosomalframeshifting occurswithin the HIVI gag-pol overlap region.J7 The coupling of these reading frames absolutely requires that the ribosome correctly shifts from one reading frame to another, something that mammalian cells are not thought to do as part of a physiologic genetic translation process. It is theoretically possible that specific chemicals or antibiotic-like agents could interrupt this process of ribosomal frameshifting, leading to impaired viral expression. HIV has a regulatory gene (tat) coded for a diffusible protein that markedly enhances the expression of other viral genes and viral replication. 49 The tat gene, like the tax genes of the first two known human pathogenic retroviruses, HTLV-I and HTLV-II (formerly called tat-I and tat-II genes, respectively), is so called because it was originally thought to mediate a transactivation of transcription, that is, it worked through a mechanism that affected the transcription of genes not in direct proximity to itself. The mechanism of transcription is not yet clear. Evidence suggests that tat may bring about increases in protein synthesis that appear to exceed the increase in RNA expression. 49-51 This suggests that tat operates through some type of posttranscriptional mechanism, including potentiation of translational efficiency. However, tat may also affect transcription but not translation.52 Experiments in in vitro transcription, on the other hand, suggest that tat works at the level of transcriptional initiation.53 Whatever the precise mechanisms are, this protein is thought to provide the virus with a positive feedback loop by which a viral product can amplify the production of new virions. The tat protein is small (86 amino acids) with a cluster of pos~

1288 Mitsuya et al. itively charged amino acids, and it is thought to affect the synthesis ofviral products by influencing the viral LTRs. The predicted amino acid sequence of tat contains two highly conserved domains rich in cysteine residues and arginine/lysine residues. Recent analyses have revealed that the tat protein forms a dimer mediated by binding to a metal (cadmium or zinc) that exerts primary effects in the cysteine-rich domain. Frankel and Pab054 and others have recently found that the tat protein is taken up by cells and subsequently transactivates the viral promoter to enhance the expression ofthe virus. This observation raises the possibility that, under certain conditions, the tat protein might function as a "viral growth factor" to stimulate the viral expression in latently infected cells. It may be possible to find drugs that inhibit the production of tat protein, the binding of tat protein to nucleic acid, the dimerization, or the uptake of tat protein by cells, and thus to inhibit the replication of HIV. With a different reading frame, the rev gene of HIV produces a different small (116 amino acids), positively charged protein, which is thought to function as a second essential transacting factor in viral replication. 55 , 56 Again, drugs that bind or inactivate this protein would be expected to inhibit viral replication. In the absence of this second regulatory factor, gag- and env-encoded protein synthesis is severely diminished. The suggested mechanism of action of the gene was first described as an abrogation of negative regulatory effects on translation of viral mRNA encoding HIV structural proteins (hence the name art for antirepression transactivator).55 It is possible that tat controls rev, which in turn regulates the activity of gag-pol and env. Experiments by Feinberg et al. 56 suggested that the rev gene product balances the amount of spliced and unspliced viral RNA to permit the preservation of gag-pol and env mRNA (hence the alternative name, trs for transacting regulator of splicing). The rev gene might prevent excessive splicing of viral RNA in infected cells by affecting the pathways for RNA transcript out of the nucleus.37Drugs or chemicals that interfere with the function of the rev gene might block the formation of full-length, genomic viral RNA in infected cells because these large molecules would be spliced out of existence. Recent studies by Matsukura et a1. 58 have shown that nuclease-resistant phosphorothioate analogs of various oligodeoxynucleotides (oligodeoxynucleotides that contain a sulfur in place of one of the two inter-

Journal of the American Academy of Dermatology

nucleotide nonbridging oxygens), in the form of an antisense construct against rev, can suppress replication of the virus in chronically HIV-infected T cells. This "antisense" oligomer drastically reduced the amount of unspliced (genomic) transcripts. The precise antiviral mechanism of phosphorothioate oligomers containing antisense construct is not yet completely understood. Whether some of these phosphorothioate analogs work through a process of hybridization arrest or affect other stages of viral replication is now under study. HIV has an additional regulatory gene, designated nef(negative regulatory factor), which slows the transcription of the viral genome. 59 This gene is thought to be responsible for the ability of HIV to be dormant in the genome of the host cell by suppressing expression of all viral genes. 60 The nefprotein is found mainly in the cytoplasm of the host cell in contrast to the tat and rev proteins in the nucleus. The nef protein probably functions through intermediary molecules derived from the host cells. This protein is a myristylated GTP-binding phosphoprotein with features similar to the cellular src and ras oncogene products. 61 The three regulatory genes described perhaps form a network that controls the replication of the virus. Although the precise regulatory mechanisms in the replication of HIV are matters offuture study, it is clear that this retrovirus has evolved an astonishingly complex system of genetic regulation. Perhaps this is because of the race between viral replication within a T cell and the destruction of the cell that the virus has commandeered, leaving no tolerance for inefficiency or improper timing in the synthesis of viral components. We can expect that the very complexity of the virus will contribute to its defeat from a clinical point of view. Drugs that interfere with the structure and function of retroviral mRNA could be of therapeutic value in AIDS. One drug, ribavirin, is believed to act as a guanosine analog that interferes with the 5' -capping of viral mRNA in other viral systems, and perhaps could be useful in retrovirally induced disorders. 62 To date, however, there are no convincing data that this drug is useful in the treatment of HIV infection. Therapeutic interventions that target the stages of transcription/translation of the viral genome would use another approach, "antisense" oligodeoxynucleotides, as described previously in this article. Basically, this approach employs short sequences of

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Number 6, Part 2 June 1990

Antiviral therapy against HIV infection 1289

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DNA (or DNA that is chemically modified to enable better cell penetration and resistance to enzymatic degradation) whose base pairs are complementary to a vital segment of the viral genome. Such antisense oligodeoxynucleotides could block expression of the viral genome through a kind of hybridization arrest of translation or possibly could interfere with the binding of a regulatory protein such as tat or rev. 57 ,63 Plans are under way to develop antisense phosphorothioate oligodeoxynucleotides in the National Cancer Institute. Protein production and assembly

The final steps in the replicative cycle of HIV involve crucial secondary processing of certain viral proteins by a protease (a function of one of the pol gene products) and myristylating (the covalent attachment of the ionic form of myristic acid, a naturally occurring carbon 14-saturated fatty acid) and glycosylating enzymes (provided by the host) as a preclude to assembly of infectious virions. These stages could also be possible targets for developing antiviral agents against HIV.64 For example, viruses produced in the presence of inhibitors of trimming glucosidase have recently been shown to have aberrantly glycosylated gp120, less syncytia formation, and reduced infectivity of target cells. 65 Finally, retroviruses are released by a process of viral budding, which may be inhibited by interferons or drugs that induce interferon production. Interferons are thought to block several stages, as well as the replication, of HIV. ACTIVITY OF DIDEOXYNUCLEOSIDE DERIVATIVES AGAINST HIV

2' ,3' -Dideoxynucleosides

In 1985, we found that a broad family of dideoxynucleoside analogs, even at large viral doses, can

completely inhibit in vitro HIV replication, and its capacity to destroy T cell cultures, at concentrations that are ten- to twentyfold lower than those that impair the proliferation and survival of target cells.5,6,11,12 Several related compounds, including didehydro-dideaxythymidine, have also been shown to have potent activity against HIV in vitro. 66 The structures of four representative 2',3'-dideoxynucleoside derivatives are depicted in Fig. 4. In several cases, studies of some of these compounds have been done during the past 20 years or so, and in a triphosphate form, some of 2',3'-dideoxynuc1eosides have routinely been used as reagents for the Sanger DNA-sequencing procedure. 67 The dideoxynuc1eoside analogs are of special interest because they prove that a simple chemical modification of the sugar moiety can predictably convert a normal substrate for nucleic acid synthesis into a compound with a potent capacity to inhibit the replication and cytopathic effect of HIV, at least in vitro. 5, 6, 12 Although these drugs may have the same ultimate mechanism of action, they behave as different agents from a clinical and phannacologic point of view. For example, AZT suppresses HIV replication in vivo,7-10 and its major toxicity is bone marrow suppression. 7, 68, 69 A closely related drug, 2' ,3'dideoxycytidine (ddC), can also suppress HIV replication in vivo, but its major toxicity is a dosedependent peripheral neuropathy,?0,71 This side effect may be significantly reduced by employing regimens that have drug-free rest periods. We have administered a regimen ofAZT and ddC; each drug is given for 7-day periods to patients with AIDS and AIDS-related complex.70 Preliminary results from this trial suggest that the toxicity of both agents can be reduced in this alternating regimen. Indeed, some patients have tolerated this regimen for more than 80 weeks without developing either bone marrow

Journal of the American Academy of Dermatology

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CONCENTRATION (11M) Fig. 5. In vitro inhibition of the infectivity and cytopathic effect of HIV-1 by combinations of 3'-azido-2' ,3' -dideoxythymidine (AZT) plus 2',3' -dideoxycytidine (ddC) or 2',3'dideoxyadenosine (ddA). CD4+ ATH8 cells (2 X 105) (ref. 6, 11) were exposed to a high multiplicity of infection of HIV-llllB and cultured in the presence or absence of drugs (solid bars). Control cells were similarly treated but not exposed to the virus (open bars). Total viable cells were counted by trypan blue exclusion method on day 9 in culture. Ten combinations of AZT and ddC (for AZT, om, 0.05, 0.1, 0.5, and 1 ,umoljL for ddC, 0.05 and 0.1 JLIDol/L) and 15 combinations of AZT and ddA (for AZT, 0.01; 0.05,0.1,0.5, and 1 /-Lm; for ddA, 0.5, 1, and 2 jim) were tested. Quantitative analysis of data obtained using combination indices (CI) (ref. 91; kindly analyzed by Dr. Ting-Chao Chou) revealed that the majority of combined drugs showed synergism (S. Hayashi and H. Mitsuya, unpublished). Figure illustrates representative data in each combination: panel A: AZT plus ddC; panel B: AZT plus ddA. (Reprinted by permission from Nature 1989;337:525-31. Copyright 1989, Macmillan Magazines Limited.)

suppression or neuropathy (Yarchoan et a1., unpublished data). Another member of the dideoxynuc1easide family, 2',3'-dideoxyinosine (ddI), has very recently been shown to have an antiviral effect

against HIV-l in patients with AIDS and ARC in escalating dose phase I trials at the National Cancer Institute and several other medical centers. Patients receiving ddI had improvement in immuno-

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logic function and evidence of a decrease of viral load. The most notable adverse effects were peripheral neuropathy and acute pancreatitis. The latter side effect can be lethal. However, doses up to 9.6 mg/kg have been tolerated in some patients for more than 18 months without toxicity (R. Yarchoan et al., unpublished data). Although there are many issues related to the antiretroviral effects of 2' ,3'-dideoxynucleosides that are as yet not resolved, it would appear that as they are successively phosphorylated in the cytoplasm of a target cell by yield 2',3'-deoxynucleosidt?-5'triphosphates, they become analogs of the 2'-deoxynuc1eoside-5'-triphosphates that are the natural substrates for cellular DNA polymerases and reverse transcriptase. 68, 73 It is generally thought that nuc1eoside-5'-triphosphates do not cross cell membranes and are not active as drugs because of their ionic character and comparatively low lipophilicity. Such analogs could compete with the binding of normal nuc1eotides to DNA polymerases (with high relative affinity for reverse transcriptase), or could be incorporated into DNA and bring about DNA chain termination because normal 5'--3 phosphodiester linkages cannot be completed. 5 Some dideoxynucleotide analogs, at concentrations that are achievable in human cells, can certainly serve as substrates for the HIV reverse transcriptase to elongate a DNA chain by one residue, after which the DNA chain is terminated. 74 At doses that are not toxic for mammalian cells, pyrimidine and purine dideoxynucleoside analogs can inhibit the in vitro replication and pathogenic effects of a range of animal and human retroviruses, even when the pathogenic effect being monitored (transformation) requires only a single round of replication. Moreover, with certainlentiviruses, these drugs can reduce the in vitro viral infectivity by more than five orders of magnitude. 75 We have found that two dideoxynucleosides, 2',3'-dideoxycytidine (ddC) and 3'-azido-2',3 / -dideoxythymidine (AZT; vide infra) can block the replication of other human pathogenic retroviruses, HTLV-I, 76 and HIV-2, 77 in vitro. Thus it appears that dideoxynudeosides can have an effect against a number of human and animal retroviruses if target cells can phosphorylate these compounds; however, the emergence of drugresistant HIV variants must always be considered possible. (By analogy to herpes simplex, pol mutations might lead to attenuation in pathogenicity of the mutant virus.) In fact, the site-specific mutagen-

Antiviral therapy against HIV infection 1291 esis in the pol gene can render the reverse transcriptase less sensitive to AZT-5'-triphosphate.7 8 Such mutations may well affect the capacity of the virus to utilize the normal corresponding nucleotides efficiently and thus might provide a net selective advantage to the virus. More recently, Larder and his colleagues79 have reported that AZT-resistant HIV variants were isolated from patients with AIDS and ARC who had received AZT for 6 months or more. These HIV variants exhibited in vitro decreased sensitivity to AZT with a lOO-fold increase of 50% inhibitory dose (ID so ), as compared to the virus isolated from the patients before AZT therapy. The emergence of these resistant viruses apparently does not appear to correlate with the clinical status of the patients, however, but more investigation is required. 79 In any event, it should be emphasized that investigators and clinicians must not rely on any single agent or approach but instead must attack the virus at multiple stages in the HIV replicative life cycle. Such combination strategy could lead not only to a far better outcome (and reduced side effects than with any single agent or approach) (Fig. 5) but also to minimization of the development of drug-resistant HIV variants.

AZT Synthesized about 26 years ago by Horwitz et aI., and shown to inhibit C-type murine retrovirus replication in vitro by Ostertag et al. more than 17 years ago,80 no medical application of 3'-azido-2' ,3' dideoxythymidine (AZT) had emerged before our studies. We found that AZT is a very potent in vitro inhibitor of HIV replication, that it protects susceptible target T cells against the HIV cytopathic effect in vitro, and that it has an antiretroviral effect against widely divergent strains of HIV. 6 AZT undergoes anabolic phosphorylation in human T cells to AZT-5'-triphosphate, which can compete with thymidine-5' -triphosphate (TTP)81 and serve as a chain-terminating inhibitor of HIV reverse transcriptase. In that sense, AZT parallels the other dideoxynucleosides. In a phase I study, AZT was found to induce clinical, immunologic, and virologic benefits in patients with severe HIV infection. 7 After the phase I study, large multicenter placebocontrolled trials were conducted in 1986 in patients with AIDS and ARC, and AZT was proved to prolong the survival and improve the clinical status of such patients. 9 In March 1987, AZT was approved as a prescription drug in the United States for adults

Journal of the American Academy of Dermatology

1292 Mitsuya et al. who have a history of cytologically confirmed Pneumocystis carinii pneumonia or an absolute decrease in the number of peripheral blood helper/inducer lymphocytes «200/mm3). The development of AZT could not have taken place without a commitment to the controlled trial methodology of drug testing. AZT can produce significant bone marrow suppression (e.g., megaloblastic anemia) that can be a serious side effect in patients with advanced AIDS,7,69 while it seems to be less of a problem in patients with earlier disease. 82 This feature of the drug might lend itself to regimens that combine AZT with agents that do not exhibit the same marrow-depressing capacity, such as dideoxycytidine.70, 71 Although AZT has prominent side effects in some patients,7,69 it is noteworthy that this drug lacks cardiac, renal, and hepatic toxicity, and AZT may also at least partially improve HIV-related dementias, 8 especially in children. 83 AZT represents only a first step in developing practical chemotherapy against pathogenic human retroviruses. A number of studies have been undertaken to determine whether AZT can be administered to other subsets of patients with HIV infection. Other regimens have also been explored, including AZT with acyclovir,5, 84 AZT with interleukin 2, or AZT with a-inteferon. 85 In the long term, the true value of AZT may be as a validation of the key assumptions underlying antiretroviral strategies f9r intervening against established AIDS, From a practical point of view, the development of AZT has already stimulated research for identifying other more effective antiviral drugs. Several unique antiviral agents, for example, acyclic nucleoside analogs such as adenallene, cytallene,86 PMEA [9-(phosphonylmethoxyethyl) adenine],87,88 cyclobut-A and _G,89 and tetrahydro-imidazo[4,5, 1,jk][ 1, 4]-benzodiazepin-2(lH)-one and -thione (TIBO) derivatives unrelated to any other known antiviral agents,90 have recently been identified. More research is needed for these classes of compounds. REFERENCES 1. Gottlieb MS, Schroff R, Schanker HM. Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men. Evidence of a new acquired cellularimmunodeficiency. N Engl J Med 1981;305:1425-31. 2. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T cell lymphotropic virus from a patient at risk for acquired immunodeficiency syndrome (AIDS). Science 1983;220:868-71. 3. Popovic M, Sarngadharan MG, Read E, et al. Detection, isolation, and continuous production of cytopathic retrovi-

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