Molec. Aspects Med. Vol. 12, pp. 313-328, 1991
0098-2997/91 $0.00 + .50 ©1991 PergamonPressplc.
Pdnted in Great Britain. All dghts reserved.
CHEMOTHERAPY AGAINST HUMAN IMMUNODEFICIENCY VIRUS INFECTION David Wilks and Angus G. Dalgleish Clinical Research Centre, Watford Road, Harrow, Middlesex HA 1 3UJ, U.K.
Many compounds have been shown to have and-viral activity against the human immunodeficiency virus (HIV) in vitro, but only one, zidovudine (3'-azido,3'-deoxythymidine, AZT), has so far been licensed for clinical use. Several others are currently undergoing clinical trials of some form or other (Richman, 1988). Promising results obtained in in vitro systems led to the speedy development and deployment of AZT, but significant side effects with both AZT and other substances, such as 2',3'dideoxycytidine (ddC), have emphasised the need for careful assessment ofanti-HIV agents in vivo. The long "incubation period" of HIV infection and the nature of the patient groups most affected make the design of placebo controlled, double blind trials difficult within acceptable ethical constraints; but whilst in vitro screening of drugs, with careful delineation of any possible toxicity, is important, clinical trials remain the cornerstone of drug assessment.
Screening in vitro for Anti-viral Activity The development of new anti-HIV drugs depends largely on research into the natural history of infection and viral molecular biology. As knowledge of virus structure and function grows, so the emphasis in drug design will move from"random" screening of compounds on the basis of fortuitously observed effects or similarity to established agents, to the rational design of drugs targeted to interfere with particular phases of the viral life cycle. Both approaches, however, continue to require in vitro screening protocols, particularly in view of the absence of a cheap, widely available and acceptable animal model. Many screening protocols have some general characteristics in common: 1) a cell based assay system incorporating all stages of the viral life cycle 2) a reproducible, quantifiable, virus mediated effect which may or may not bear a relationship to established in vivo pathogenic mechanisms 3) a method of establishing the toxicity or otherwise of compounds with anti-viral actions at the concentration at which such anti-viral effects were seen.
313
314
D. Wilks and A. G. Dalgleish
Screening protocols should if possible include a variety of cell types and virus isolates, including flesh clinical isolates. Several parameters have been employed to quantify virus concentration and infectivity in tissue culture supematants including. (1) (2) (3) (4) (5)
p24 core antigen level reverse transcriptase (RT) level the capacity of HIV to induce syncytium formation in CD4+ lymphocyte cultures immunofluorescent staining of live or fixed cells detection of viral DNA by probing Southern blots, with or without amplification by the polymerase chain reaction
Both p24 and RT levels essentially measure the number of virus particles rather than infectivity (Spickett et al., 1989). Syncytium induction by HIV constitutes the basis of the syncytium inhibition assay which has been used as a sensitive, semi-quantitative measure of the anti-viral capacity of drugs, antisera and monoclonal antibodies (Popovic et al., 1984; Dalgleish et al., 1984). AZT inhibits the formation of syncytia in this assay at a concentration of 0.1 to 1 lxg/ml, and infected individuals have type-specific neutralising antibodies detectable at titres up to 1/1000. Some limited information on cellular toxicity may also be obtained by observation of cell viability in this assay system. Other screening protocols based on the quantitative estimation of p24 or RTin culture supematants employ commercial or in-house ELISAs. Formal methods of estimating cellular toxicity, for example by incorporating parallel assays assessing cellular protein metabolism and its inhibition by the agent under investigation, may also be used. Spickett et al. (1989) have shown that p24, RT and syncytium inhibition titres do not necessarily correlate one with another. RT activity may persist in culture supernatants for several days after viral replication has ceased. Defective viruses with reduced infectivity may be produced and can give rise to p24 and RT levels comparable with fully infectious forms. Minor modifications to cell culture technique, in particular the use of different concentrations and sources of foetal calf serumin infected and indicator cell cultures, can have profound effects on the results of all three assays. It is important therefore that cell culture and assay conditions are rigorously standardised if meaningful and repeatable results are to be obtained. In the case of compounds that have been designed or deduced to have a specific mechanism of antiviral action, more specific assays may be appropriate. Many protocols have been reported in the context of specific studies of putative anti-viral agents. Thus in the case ofRT inhibitors, cell free RT inhibition assays have advantages in terms of safety and convenience (Hoffman et al., 1985) Such systems do not, however, present RT in its in vivo intracellular context and thus do not address such issues as cell penetration. Screening for possible therapeutic agents to inhibit the high affinity interaction of CD4 with gp120 may in most instances be achieved using solid phase or cell-based assays using soluble recombinant CD4 (sCD4) or gp120 (rgpl20). Lundin et al. (1987) have described a specific assay measuring binding of radioiodinated native gp 120 to CD4+ cells. This assay may also be performed using labelled recombinant gp120 (Arthos et al., 1989). Considerable care must be taken to ensure that recombinant reagents retain the binding characteristics of native proteins; a recent study by Moore et al., (1990)has shown that this is not necessarily always
Chemotherapy against HIV Infection
315
the case. Proteins may deform when bound to solid phase (Soderquist et al., 1980) and procedural modifications such as capture techniques may be required to restore affinity (Moore et al., 1990). An "immunoadhesin" construct bearing the VI and V 2domains of CD4 and the constant region of human IgG 1 heavy chain has been manufactured (Capon et al., 1989). This molecule has the ability to bind gpl20 bivalently and thus with a higher functional affinity even than CD4. The presence of the Ig heavy chain allows the easy attachment of radioactive, fluorescent and enzyme labels and separation of gp120-immunoadhesin complexes by well established immunological reagents such as Protein A. Whilst the therapeutic potential of this immunoadhesin remains the subject of early clinical trials, it is clearly very useful as a laboratory tool for investigating the CD4-gpl20 interaction.
Clinical Trials of Anti-HIV Agents The prolonged "incubation period" of HIV infection, its variable clinical course and lack of real understanding of the mechanism of pathogenesis leading from infection to immunodeficiency make the design of clinical trials particularly difficult. Two unusual patterns ofclinicalinvestigation have emerged in trials of putative anti-HIV strategies. Firstly, very large numbers of compounds have been tried in very small "Phase I" studies, at a much earlier point in their development than would have occurred in the investigation of drugs to treat any other disease. Secondly, those large scale trials that have been carried out have in many cases been discontinued prematurely, either due to early differences in outcome between the treatment and placebo groups, as in the case of AZT, or due to difficulties with recruitment, follow-up and control of trial subjects. An important consequence of early discontinuation of studies is that whilst information may be gained about the early efficacy of drugs, nothing is learnt about long-term efficacy and toxicity. Now that AZT is in widespread use in asymptomatic seropositive individuals, it is difficult to envisage further trials in which one group receives only placebo treatment, except possibly as a matter of choice by trial participants; even if such a trial were to be granted ethical approval, practical difficulties with recruitment would be anticipated. In an effort to address some of these difficulties, and in particular to facilitate the design of trials which do not depend on the development of AIDS or death as endpoints, Jacobson et al. (1991) have examined the possibility of using statistical analysis of early changes in a large number of laboratory parameters as surrogate endpoints. It was found that changes in CD4+ cell count and 132-microglobulin levels at eight to twelve weeks after the start of therapy predicted a more favourable outcome. Such an approach may allow a more rapid comparison of trealrnent strategies and predictors of clinical outcome. In response to public pressure for access to drugs undergoing investigation, the Food and Drug Administration (FDA) in the USA have adopted novel procedures to expedite licensing of anti-HIV agents. Thus AZT was available to patients under a treatment investigational new drug application (IND) programme (often referred to as "fast track" approval) before it was fully licensed for clinical use. Other new drugs may be made available under "parallel track" arrangements whereby patients who are for some reason unable to take part in clinical trials of new drugs may under certain circumstances obtain them. 2',3'-dideoxyinosine (ddI) is currently being evaluated under such a programme.
Antiretroviral Therapy Antiretroviral therapy may act at many different points in the viral replicative cycle. To date most success has been obtained with agents that inhibit viral reverse transcriptase, but some promising
316
D. Wilks and A. G. Dalgleish
CD4/ __'T_\
Virusbinding
sCD4 Sulpllmedpolysacdladdes Passive immunization
/-'' '"
DNA
'l
~
. \
~ ~
AZT ddC ddl Fo~amet
~
mi,,=ur.,,,,'=p,o,J,.
~
~
Ribavarin
Virusbudding
lion
Antisense oligonucleotides
Fig. 1. The lifecycle of HIV-1: the points of action of various antiviral agents arc indicated.
agents work at other parts in the cycle. Figure 1 illustrates the viral life cycle and indicates the points of action of the substances discussed.
(1) Reverse Transcriptase Inhibitors Zidovudine (AZT) Viral reverse transcriptase is responsible for the translation of viral RNA to DNAprior to integration of the provirus into the host genome. AZT was the first RT inhibitor to undergo clinical trials and although many other deoxynucleotides have been shown to have equal or better anti-viral activity in vitro, none has yet successfully completed clinical trials. The antiretroviral activity o f A Z T w a s first reported by Mitsuya et al. (1985) who showed that it could inhibit viral replication in vitro at concentrations below 5 IxM, at which it did not affect the physiological immunological functions o f T and B cells. Phase I trials were reported in the spring of 1986 and the Phase II study, which was prematurely discontinued due to the increased rate of opportunistic infection in the placebo arm, was reported in July 1987 (Yarchoan et al., 1986; Fischl et al., 1987).
Pharmacokinetics and Action AZT is an analogue of thymidine which is phosphorylated by cellular enzymes and, as the 5'triphosphate, inhibits RT and terminates viral DNA chain elongation. It is absorbed rapidly from the
Chemotherapy against HIV Infection
317
gastrointestinal tract with peak serum concentrations at 1 hour and a bioavailability of 60%. Hepatic metabolism to an inactive and non-toxic metabolite occurs and only 19% of the parent compound is recovered from the urine. CSF penetration is good with CSF:plasma ratios of 50-100% within 4 hours of the first dose (Klecker et ai., 1987). The Phase II study in AIDS and ARC patients demonstrated that AZT (250 mg 4 hourly) could reduce mortality and the frequency of opportunistic infections and lead to increased well being, weight gain and a partial reversal of skin anergy in some patients. CD4 counts increased in the treatment group, but declined to pre-treatment levels after 12 weeks in AIDS patients, although not in patients with ARC. There was no significant influence on the incidence of Kaposi's sarcoma (KS). AZT has been shown to reduce p24 antigenaemia by greater than 90% even in patients with very high initial levels of p24 (Jackson et al., 1988). It has also been shown to have beneficial clinical and radiological effects on the neuropsychiatric manifestations of HIV infection (Yarchoan et al., 1987).
Toxicity Significant toxicity, particularly bone marrow depression, was noted during the Phase II study. (Richman et al., 1987). Nausea, myalgia, insomnia and severe headaches were significantly more common in the treatment group. Anaemia with a haemoglobin less than 7.5 g/dl occurred in 24% of the treatment group compared to 4% of placebo recipients. Macrocytosis was also common in the treatment group, although not all of the anaemia seen was macrocyfic and macrocytosis was not always associated with anaemia. Neutropenia (
0098-2997/91 $0.00 + .50 ©1991 PergamonPressplc.
Pdnted in Great Britain. All dghts reserved.
CHEMOTHERAPY AGAINST HUMAN IMMUNODEFICIENCY VIRUS INFECTION David Wilks and Angus G. Dalgleish Clinical Research Centre, Watford Road, Harrow, Middlesex HA 1 3UJ, U.K.
Many compounds have been shown to have and-viral activity against the human immunodeficiency virus (HIV) in vitro, but only one, zidovudine (3'-azido,3'-deoxythymidine, AZT), has so far been licensed for clinical use. Several others are currently undergoing clinical trials of some form or other (Richman, 1988). Promising results obtained in in vitro systems led to the speedy development and deployment of AZT, but significant side effects with both AZT and other substances, such as 2',3'dideoxycytidine (ddC), have emphasised the need for careful assessment ofanti-HIV agents in vivo. The long "incubation period" of HIV infection and the nature of the patient groups most affected make the design of placebo controlled, double blind trials difficult within acceptable ethical constraints; but whilst in vitro screening of drugs, with careful delineation of any possible toxicity, is important, clinical trials remain the cornerstone of drug assessment.
Screening in vitro for Anti-viral Activity The development of new anti-HIV drugs depends largely on research into the natural history of infection and viral molecular biology. As knowledge of virus structure and function grows, so the emphasis in drug design will move from"random" screening of compounds on the basis of fortuitously observed effects or similarity to established agents, to the rational design of drugs targeted to interfere with particular phases of the viral life cycle. Both approaches, however, continue to require in vitro screening protocols, particularly in view of the absence of a cheap, widely available and acceptable animal model. Many screening protocols have some general characteristics in common: 1) a cell based assay system incorporating all stages of the viral life cycle 2) a reproducible, quantifiable, virus mediated effect which may or may not bear a relationship to established in vivo pathogenic mechanisms 3) a method of establishing the toxicity or otherwise of compounds with anti-viral actions at the concentration at which such anti-viral effects were seen.
313
314
D. Wilks and A. G. Dalgleish
Screening protocols should if possible include a variety of cell types and virus isolates, including flesh clinical isolates. Several parameters have been employed to quantify virus concentration and infectivity in tissue culture supematants including. (1) (2) (3) (4) (5)
p24 core antigen level reverse transcriptase (RT) level the capacity of HIV to induce syncytium formation in CD4+ lymphocyte cultures immunofluorescent staining of live or fixed cells detection of viral DNA by probing Southern blots, with or without amplification by the polymerase chain reaction
Both p24 and RT levels essentially measure the number of virus particles rather than infectivity (Spickett et al., 1989). Syncytium induction by HIV constitutes the basis of the syncytium inhibition assay which has been used as a sensitive, semi-quantitative measure of the anti-viral capacity of drugs, antisera and monoclonal antibodies (Popovic et al., 1984; Dalgleish et al., 1984). AZT inhibits the formation of syncytia in this assay at a concentration of 0.1 to 1 lxg/ml, and infected individuals have type-specific neutralising antibodies detectable at titres up to 1/1000. Some limited information on cellular toxicity may also be obtained by observation of cell viability in this assay system. Other screening protocols based on the quantitative estimation of p24 or RTin culture supematants employ commercial or in-house ELISAs. Formal methods of estimating cellular toxicity, for example by incorporating parallel assays assessing cellular protein metabolism and its inhibition by the agent under investigation, may also be used. Spickett et al. (1989) have shown that p24, RT and syncytium inhibition titres do not necessarily correlate one with another. RT activity may persist in culture supernatants for several days after viral replication has ceased. Defective viruses with reduced infectivity may be produced and can give rise to p24 and RT levels comparable with fully infectious forms. Minor modifications to cell culture technique, in particular the use of different concentrations and sources of foetal calf serumin infected and indicator cell cultures, can have profound effects on the results of all three assays. It is important therefore that cell culture and assay conditions are rigorously standardised if meaningful and repeatable results are to be obtained. In the case of compounds that have been designed or deduced to have a specific mechanism of antiviral action, more specific assays may be appropriate. Many protocols have been reported in the context of specific studies of putative anti-viral agents. Thus in the case ofRT inhibitors, cell free RT inhibition assays have advantages in terms of safety and convenience (Hoffman et al., 1985) Such systems do not, however, present RT in its in vivo intracellular context and thus do not address such issues as cell penetration. Screening for possible therapeutic agents to inhibit the high affinity interaction of CD4 with gp120 may in most instances be achieved using solid phase or cell-based assays using soluble recombinant CD4 (sCD4) or gp120 (rgpl20). Lundin et al. (1987) have described a specific assay measuring binding of radioiodinated native gp 120 to CD4+ cells. This assay may also be performed using labelled recombinant gp120 (Arthos et al., 1989). Considerable care must be taken to ensure that recombinant reagents retain the binding characteristics of native proteins; a recent study by Moore et al., (1990)has shown that this is not necessarily always
Chemotherapy against HIV Infection
315
the case. Proteins may deform when bound to solid phase (Soderquist et al., 1980) and procedural modifications such as capture techniques may be required to restore affinity (Moore et al., 1990). An "immunoadhesin" construct bearing the VI and V 2domains of CD4 and the constant region of human IgG 1 heavy chain has been manufactured (Capon et al., 1989). This molecule has the ability to bind gpl20 bivalently and thus with a higher functional affinity even than CD4. The presence of the Ig heavy chain allows the easy attachment of radioactive, fluorescent and enzyme labels and separation of gp120-immunoadhesin complexes by well established immunological reagents such as Protein A. Whilst the therapeutic potential of this immunoadhesin remains the subject of early clinical trials, it is clearly very useful as a laboratory tool for investigating the CD4-gpl20 interaction.
Clinical Trials of Anti-HIV Agents The prolonged "incubation period" of HIV infection, its variable clinical course and lack of real understanding of the mechanism of pathogenesis leading from infection to immunodeficiency make the design of clinical trials particularly difficult. Two unusual patterns ofclinicalinvestigation have emerged in trials of putative anti-HIV strategies. Firstly, very large numbers of compounds have been tried in very small "Phase I" studies, at a much earlier point in their development than would have occurred in the investigation of drugs to treat any other disease. Secondly, those large scale trials that have been carried out have in many cases been discontinued prematurely, either due to early differences in outcome between the treatment and placebo groups, as in the case of AZT, or due to difficulties with recruitment, follow-up and control of trial subjects. An important consequence of early discontinuation of studies is that whilst information may be gained about the early efficacy of drugs, nothing is learnt about long-term efficacy and toxicity. Now that AZT is in widespread use in asymptomatic seropositive individuals, it is difficult to envisage further trials in which one group receives only placebo treatment, except possibly as a matter of choice by trial participants; even if such a trial were to be granted ethical approval, practical difficulties with recruitment would be anticipated. In an effort to address some of these difficulties, and in particular to facilitate the design of trials which do not depend on the development of AIDS or death as endpoints, Jacobson et al. (1991) have examined the possibility of using statistical analysis of early changes in a large number of laboratory parameters as surrogate endpoints. It was found that changes in CD4+ cell count and 132-microglobulin levels at eight to twelve weeks after the start of therapy predicted a more favourable outcome. Such an approach may allow a more rapid comparison of trealrnent strategies and predictors of clinical outcome. In response to public pressure for access to drugs undergoing investigation, the Food and Drug Administration (FDA) in the USA have adopted novel procedures to expedite licensing of anti-HIV agents. Thus AZT was available to patients under a treatment investigational new drug application (IND) programme (often referred to as "fast track" approval) before it was fully licensed for clinical use. Other new drugs may be made available under "parallel track" arrangements whereby patients who are for some reason unable to take part in clinical trials of new drugs may under certain circumstances obtain them. 2',3'-dideoxyinosine (ddI) is currently being evaluated under such a programme.
Antiretroviral Therapy Antiretroviral therapy may act at many different points in the viral replicative cycle. To date most success has been obtained with agents that inhibit viral reverse transcriptase, but some promising
316
D. Wilks and A. G. Dalgleish
CD4/ __'T_\
Virusbinding
sCD4 Sulpllmedpolysacdladdes Passive immunization
/-'' '"
DNA
'l
~
. \
~ ~
AZT ddC ddl Fo~amet
~
mi,,=ur.,,,,'=p,o,J,.
~
~
Ribavarin
Virusbudding
lion
Antisense oligonucleotides
Fig. 1. The lifecycle of HIV-1: the points of action of various antiviral agents arc indicated.
agents work at other parts in the cycle. Figure 1 illustrates the viral life cycle and indicates the points of action of the substances discussed.
(1) Reverse Transcriptase Inhibitors Zidovudine (AZT) Viral reverse transcriptase is responsible for the translation of viral RNA to DNAprior to integration of the provirus into the host genome. AZT was the first RT inhibitor to undergo clinical trials and although many other deoxynucleotides have been shown to have equal or better anti-viral activity in vitro, none has yet successfully completed clinical trials. The antiretroviral activity o f A Z T w a s first reported by Mitsuya et al. (1985) who showed that it could inhibit viral replication in vitro at concentrations below 5 IxM, at which it did not affect the physiological immunological functions o f T and B cells. Phase I trials were reported in the spring of 1986 and the Phase II study, which was prematurely discontinued due to the increased rate of opportunistic infection in the placebo arm, was reported in July 1987 (Yarchoan et al., 1986; Fischl et al., 1987).
Pharmacokinetics and Action AZT is an analogue of thymidine which is phosphorylated by cellular enzymes and, as the 5'triphosphate, inhibits RT and terminates viral DNA chain elongation. It is absorbed rapidly from the
Chemotherapy against HIV Infection
317
gastrointestinal tract with peak serum concentrations at 1 hour and a bioavailability of 60%. Hepatic metabolism to an inactive and non-toxic metabolite occurs and only 19% of the parent compound is recovered from the urine. CSF penetration is good with CSF:plasma ratios of 50-100% within 4 hours of the first dose (Klecker et ai., 1987). The Phase II study in AIDS and ARC patients demonstrated that AZT (250 mg 4 hourly) could reduce mortality and the frequency of opportunistic infections and lead to increased well being, weight gain and a partial reversal of skin anergy in some patients. CD4 counts increased in the treatment group, but declined to pre-treatment levels after 12 weeks in AIDS patients, although not in patients with ARC. There was no significant influence on the incidence of Kaposi's sarcoma (KS). AZT has been shown to reduce p24 antigenaemia by greater than 90% even in patients with very high initial levels of p24 (Jackson et al., 1988). It has also been shown to have beneficial clinical and radiological effects on the neuropsychiatric manifestations of HIV infection (Yarchoan et al., 1987).
Toxicity Significant toxicity, particularly bone marrow depression, was noted during the Phase II study. (Richman et al., 1987). Nausea, myalgia, insomnia and severe headaches were significantly more common in the treatment group. Anaemia with a haemoglobin less than 7.5 g/dl occurred in 24% of the treatment group compared to 4% of placebo recipients. Macrocytosis was also common in the treatment group, although not all of the anaemia seen was macrocyfic and macrocytosis was not always associated with anaemia. Neutropenia (
![[Microbiological diagnosis of human immunodeficiency virus infection].](/assets/img/hold.png)
