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Current research opinion

Current Medical Research and Opinion Vol. 15: Supplement, 1999 A Critical Analysis of the Pharmacology of AZT and its Use in AIDS
Eleni Papadopulos-Eleopulos (1), Valendar F. Turner (2), John M. Papadimitriou (3), David Causer (4), Helman Alphonso (5) and Todd Miller (6) (1) Corresponding author, Biophysicist, Department of Medical Physics, Royal Perth Hospital, Wellington Street, Perth 6001, Western Australia (2) Consultant Emergency Physician, Department of Emergency Medicine, Royal Perth Hospital (3) Professor of Pathology, University of Western Australia (4) Senior Physicist, Department of Medical Physics, Royal Perth Hospital (5) Head, Department of Research, Universidad Metropolitana Barranquilla, Colombia (6) Assistant Scientist,Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida, USA The triphosphorylated form of the nucleoside analogue 3'-azido-3'-deoxythymidine (Zidovudine, AZT) is claimed to interrupt the HIV replication cycle by a selective inhibition of viral reverse transcriptase, thereby preventing the formation of new proviral DNA in permissive, uninfected cells. Given that initial HIV infection of an individual instigates abundant HIV replication from inception until death, and that the life of infected T-cells is only several days, the administration of AZT should lead both in vitro and in vivo (i) to decreased formation of proviral DNA; and thus (ii) to decreased frequencies of 'HIV isolation' (detection of p24 or reverse transcription or both) in stimulated cultures/cocultures of T-cells from seropositive individuals; (iii) to decreased synthesis of HIV p24 and RNA ('antigenaemia', 'plasma viraemia', 'viral load') ultimately resulting in low or absent levels of all three parameters; and (iv) to a perfect and direct correlation between all these parameters. A critical analysis of the presently available data shows that no such evidence exists, an outcome not unexpected given the pharmacological data on AZT. HIV experts all agree that only the triphosphorylated form of AZT (AZTTP) and not the unphosphorylated form administered to patients, nor its mono- or diphosphate, is the active agent. Furthermore, the mechanism of action is the ability of AZTTP to halt the formation of HIV-DNA (chain termination). However, although this claim was posited from the outset, AZT underwent clinical trials and was introduced as a specific anti-HIV drug many years before there were any data proving that the cells of patients are able to triphosphorylate the parent compound to a level considered sufficient for its putative pharmacological action. Notwithstanding, from the evidence published since 1991 it has become apparent that no such phosphorylation takes place and thus AZT cannot possess an anti-HIV effect. However, the scientific literature does elucidate: (i) a number of biochemical mechanisms which predicate the likelihood of widespread, serious toxicity from use of this drug; (ii) in vitro data proving that AZT has significant antibacterial and antiviral properties which confound interpretation of its effects when administered to patients. Based on all these data it is dificult if not impossible to explain why AZT was introduced and still remains the most widely recommended and used anti-HIV drug. Introduction
Any drug used in the treatment of patients suffering from an infectious disease relies upon evidence obtained from both in vitro and in vivo studies proving beyond reasonable doubt that: 1. The patients are infected with a specific microbial agent and the agent is the cause of the 2. The drug inhibits the agent or its biological effects. 3. The drug is non-toxic or its toxicity is less detrimental than its benefits. The claim that HIV plays a causative role in AIDS has been questioned by many individuals (1–18). In fact, there is considerable doubt that the presently available data prove that AIDS patients, those at risk or other individuals are infected with a unique retrovirus HIV (3,7,8,10–12,17–19). None the less, for the purpose of the present discussion it will be assumed that such laboratory tests as 'HIV isolation', 'plasma viraemia', 'p24', 'p24 antigenaemia', 'HIV RNA' and 'proviral' 'HIV DNA' are all HIV specific and thus are proof of infection with a unique, exogenously acquired retrovirus, HIV. The retroviral theory of AIDS asserts that the cycle of HIV replication begins with fusion of HIV to permissive cells and the introduction of HIV into the cell. Inside the cell the viral RNA is reverse transcribed into DNA, which is then inserted into the cellular DNA as the 'HIV provirus'. The process of reverse transcription is catalysed by an enzyme said to be viral specific known as reverse transcriptase. Subsequently, the DNA provirus is transcribed into viral RNA, which in turn is translated into viral proteins. Finally, RNA and proteins are assembled into viral particles which are released from the cell membrane, whereupon the newly produced viral particles infect fresh cells and the replicative cycle repeats. Although the previous conviction was that the production of HIV from proviral DNA involved prolonged virological latency, at present HIV experts assert 'high-level viral replication from the time of initial infection until death'; that is, HIV infected T-cells are killed from inception (20,21). According to the HIV experts, AZT in its triphosphorylated form is a selective inhibitor of viral reverse transcriptase, inhibiting the generation of proviral HIV DNA and thus interrupting the cycle of new cellular infection while leaving intact the production of virus from cells already infected. Since virus production from infected T-cells is soon exhausted by their short lifespan ('half-life of about 1.6 days'), it can be predicted that the administration of AZT will be followed by a rapid reduction in all HIV parameters ('HIV isolation', 'plasma viraemia', 'p24', 'p24 antigenaemia', 'HIV RNA' and 'proviral' 'HIV DNA') and indeed to the complete absence of infected T-cells. AZT is the first drug introduced to treat HIV infection and still remains the most frequently used drug for this purpose. The design, execution and interpretation of the clinical studies of AZT, administered either alone or in combination with other drugs, have been questioned by many authors. From the time of its introduction into clinical practice, John Lauritsen and Peter Duesberg have thoroughly and critically analysed the clinical trials of AZT and have consistently argued that the drug has no clinical benefits but is severely toxic - 'Poison by Prescription' (22), 'AIDS by prescription' (1). Recently, many other authors have expressed doubts in relation to the trials and the clinical usefulness of AZT (23,24). Because of this, the clinical data will not be further analysed here, and instead the present analysis will concentrate on evaluating the data which are said to affirm AZT as an anti-retroviral agent. AZT is a thymidine analogue in which the 3'-hydroxy (–OH) group is replaced by an azido (=N) group. The 3'-hydroxy group is absolutely necessary for the triphosphorylated nucleotides to be attached to the growing DNA chain. Because in AZT this group is missing, once AZT becomes attached to the DNA chain, no further growth can take place; that is, 'the DNA chain is terminated' (25). For AZT, as for the natural nucleotides, to be attached to the DNA chain – that is, to act as a DNA chain terminator – it must first be triphosphorylated. However, although AZT given to patients is not triphosphorylated, it is said that AZT, like the natural nucleotides, is triphosphorylated by cells. Since AZT has been used routinely in clinical practice for over 10 years, one would expect that at present there would be ample evidence which proves that cells are able to metabolise AZT to its active form to levels sufficient to inhibit the replication of HIV both in vitro and in vivo and that the drug indeed inhibits the replication of HIV. A. Anti-HIV Effects of AZT – in Vitro
The introduction of AZT to treat HIV infected individuals is based on two studies conducted by researchers from the National Cancer Institute, Duke University Medical Center, and the Wellcome Research Laboratories. In the first study, reported by Mitsuya et al. in October 1985 (26), the effects of AZT on two HIV parameters, p24 and reverse transcriptase (RT), were investigated in cell cultures. It was concluded that AZT 'was a selective and potent inhibitor of human T-cell lymphotropic virus type III'. In the second study, published by Furman et al. in November 1986 (27), the only HIV parameter studied was reverse transcriptase. These authors reported that 'The reverse transcriptase was much more sensitive to inhibition than was the DNA polymerase a of H9 cells. The IC50 values for the viral reverse transcriptase were 0.7 mM with poly(rA).oligo(dT)12–18 and 2.3mM with activated calf thymus DNA as primer-templates. In contrast, an IC50 value of 260 mM was determined for azidothymidine triphosphate with the H9 DNA polymerase a when activated calf thymus DNA was used as primer-template.' Based on the evidence from these two in vitro studies, the authors introduced AZT into clinical practice. In fact the first two clinical trials of AZT were commenced before the publication of the Furman et al. study. However: 1. In vitro data cannot be extrapolated in vivo. The authors themselves emphasised 'that the activity of an agent against viruses in vitro does not ensure that the agent will be clinically useful in treating viral diseases. Toxicity, metabolic features, bioavailability, and other factors could negate the clinical utility of a given agent'. Because of this, the introduction of a drug in clinical practice is usually preceded by experiments to gain such data in animals. Such data on AZT, in addition to providing information on the anti-HIV effects of the drug, may have also provided useful information on the bioavailability, cellular triphosphorylation and toxicity of AZT. However, the first data on the bioavailability of AZT were not obtained until the first clinical trial, where it was found that the maximum plasma concentration was reached about 1 h after the oral administration of AZT and was 1.5 –2 mmol/l with a 2 mg/kg dose, and 4–6 mmol/l with a dose of 10 mg/kg. The 'plasma disappearance had a half-life of approximately 1 h'. By giving 10 mg/kg of AZT every 4 h, 'plasma drug levels were maintained above 1 mmol/l' (28). At present, such a dose would be considered prohibitively toxic by most, if not all, HIV/AIDS researchers. None the less, these authors reported that 'Treatment was not limited by side-effects, the commonest of which were headaches and depression of white-cell counts.' No data on the triphosphorylation of AZT were obtained. 2. In their 1985 paper, Mitsuya et al. reported that 'a substantial level [14,000 cpm] of reverse transcriptase activity could be detected in the supernatants of normal PBM exposed to HTLVIIIB in the absence of A509U [AZT]… Inhibition was observed at doses as low as 0.005 mM and was marked [8,000 cpm] at 0.05 mM. Complete inhibition was achieved at doses of 0.5 mM and more'. However: (a) although the authors stated that 'the unphosphorylated compound [AZT] does not inhibit reverse transcriptase per se' and the AZT used was not triphosphorylated, no data were presented that the AZT used in their experiments ('Structure I', 'A509U') was triphosphorylate d by the cells; (b) in the two studies, AZT was introduced at the time of infection of the cultures, while patients are infected for many months or years before treatment. (c) one year after the publication of the two studies researchers from Yamaguchi University and Hokkaido University, Japan, reported that AZT 'did not show any effect in the HTLV-III-producing cell line Molt-4/HTLV-III', which was infected before the introduction of AZT (29). (d) in a study published in 1987 by researchers from the University of North Carolina, H9 and Jurkat cells were pretreated with concentrations of AZT ranging from 0.5 to 100 mM, infected and maintained in drug-containing medium. Discussing their findings and those of others, the authors wrote: 'Although AZT may be primarily a competitive inhibitor for RT, acts as a chain terminator, and perturbs nucleoside triphosphate pools within the cells, our results showed that complete DNA copies of the viral genome were formed in the presence of AZT. Since further steps in the virus life cycle (e.g. production of mRNA and progeny viral RNA) dependent on cellular RNA polymerase were not affected by the drug, virus production could then ensue. These proposed effects of the drug on aspects of the viral replication cycle are supported by a report that virus production is not suppressed in cells already producing HIV. Whether virus spread occurs by cell-free virus or by cell-to-cell contact, cultures treated with 25 mM AZT eventually produced as much virus as the non-drug-treated infected cultures. These results were confirmed by the detection of unintegrated viral DNA in drug-treated H9 cultures when they began producing virus at high levels. The unintegrated viral DNA in these drug-treated cultures was present in quantities similar to those in non-drug-treated infected cultures' (30). 3. As mentioned, in the 1986 Furman et al. paper, the IC50 AZT 'values for the viral reverse transcriptase were 0.7 mM with poly (rA).oligo(dT)12–18 and 2.3 mM with activated calf thymus DNA as primer-templates'. These results were obtained by using 'purified HIV reverse transcriptase'. However, the ability of AZT to inhibit 'purified' enzyme does not prove the same effect will be observed on RT present in cells or in the viral particle. 4. HIV reverse transcriptase was 'purified' as follows: '750 ml of culture fluid harvested from HIV-infected H9 cells was centrifuged at 18,000 rpm for 90 min in an R19 rotor (Beckman) to pellet virus. Enzyme was extracted by incubating the virus pellet in buffer A [50 mM Tris.HC1, pH 7.9/0.25% Nonidet P-40/20 mM dithiothreitol/50% (vol/vol) glycerol] containing 1 mM EDTA, 500 mM KC1, and 0.5% deoxycholate. The enzyme was partially purified by passing the extract through a DEAE -cellulose column (3 x 10cm) previously equilibrated with buffer A. Fractions containing enzyme activity were dialysed against buffer B [50 mM Tris.HC1, pH 7.9/50 mM NaCl/1 mM EDTA/1 mM dithiothreitol/ 20% glycerol] and were further purified by phosphocellulose chromatography. The peak fractions were pooled and dialysed against buffer B containing 50% glycerol. To the dialysed enzyme, bovine serum albumin was added to give a final concentration of 1 mg/ml. The enzyme was characterised as HIV reverse transcriptase based on its cation, salt, pH, and template requirements (5)'. However, by this method it is not possible to say that one has a purified HIV enzyme or any purified enzyme, viral or cellular. As far as the claim of characterisation as 'HIV reverse transcriptase' is concerned, Reference 5, cited by the authors, is a paper published by Jay Levy and his colleagues in 1985 where they present results which 'indicate specific characteristics of the RT of ARV', namely 'The RNA-dependent DNA polymerase of the AIDS-associated retrovirus (ARV) gives highest activity with the synthetic template, poly(rA).oligo(dT) and prefers Mg2+ over Mn2+ as a divalent cation', '100– 200 mM KCl' as the monovalent cation, 'the major peak occurring at pH 8.0. A change in 0.2 pH units from 8.0 in either direction did not dramatically affect the reaction sensitivity' (31). However, all cellular DNA polymerases can use Mg2+ as the divalent cation and KCl as a monovalent cation and can be active at the pH of 8.0. As far as the template poly(A).oligo(dT) is concerned, it is sufficient to mention that: (a) the template-primer A(n).dT15 can be transcribed not only by RT but by all the cellular DNA polymerases, a, b and g32. In fact, in 1975, an International Conference on Eukaryotic DNA polymerases, which included Baltimore and Gallo33 defined DNA polymerase g, 'a component of normal cells' (34), 'found to be widespread in occurrence' (32), whose activity can be increased by many factors including PHA stimulation (35), as the enzyme which 'copies A(n).dT15 with high efficiency but does not copy DNA well' (33); (b) in a paper published in 1984 by French researchers including Barre-Sinoussi, Montagnier and Chermann, it was shown that cellular DNA polymerases can also use Mg2+ as a divalent cation, KCl as a monovalent cation, including 200 mM KCl and a pH of 7.8. They also showed that enzymes from non-infected lymphocytes (especially DNA polymerase b) also used poly(rA).oligo(dT) as template primer (36). (c) Thus it is impossible to claim that the 'purified' enzyme which was inhibited by the drug was 'HIV reverse transcriptase', and not a cellular reverse transcriptase or any of the other cellular DNA polymerases. Indeed, given the facts that: (i) the existence of 'HIV reverse transcriptase' was proven following the demonstration of revers e transcription of a particular synthetic RNA template-primer; (ii) the same template -primer, under the same experimental conditions, can be reverse transcribed by cellular DNA polymerases; one can plausibly argue that at present no proof exists for the existence of a specific retroviral enzyme. 5. Even if the 'purified' enzyme which transcribed poly(rA).oligo(dT)12–18 and 'activated calf thymus DNA' was HIV RT, just because the drug inhibited the transcription of this primer-template it does not mean that it will have the same or similar effect when the template is the HIV genome. That the template-primer to be transcribed plays a significant role is best illustrated by the finding that 'The IC50 values for the viral reverse transcriptase were 0.7 mM [of triphosphorylated AZT] with poly (rA).oligo(dT)12–18 and 2.3mM with activated calf thymus DNA as primer template'. 6. Because 'Azidothymidine triphosphate inhibited HIV reverse transcriptase about 100 times better than it inhibited the H9 polymerase a, with activated calf thymus DNA as template', the authors of the first two in vitro studies concluded that AZT was 'a selective' inhibitor of HIV RT. However, polymerase a is not the only cellular DNA polymerase. For some unknown reason, these authors did not present data on the two other cellular DNA polymerases, polymerase b and g. However, in 1990 Mitsuya and his associates, discussing the effects of nucleoside analogues in general, wrote: 'Several 2',3'-dideoxynucleoside 5'-triphosphates have been extensively studied and have higher affinities for HIV reverse transcriptase than for cellular DNA polymerase a, although cellular DNA polymerases b and g (mitochondrial DNA polymerase) appear to be sensitive to the dideoxynucleoside 5'-triphosphates. The activity against mitochondrial DNA polymerase might explain certain side effects, such as a toxic mitochondrial myopathy in individuals receiving long-term AZT therapy' (37). B. Phosphorylation of AZT
In determining the inhibition of the HIV RT by AZT, Furman et al., in addition to not using cells or even 'pure HIV' but 'purified' enzyme, also did not use AZT in the form administered to patients. Instead, they used the triphosphorylated form of AZT (AZTTP), the only form of AZT accepted to have an anti-retroviral effect. (For their experiments 'The mono-, di-, and triphosphates of azidothymidine were prepared from azidothymidine by published methods'.) Apparently, to overcome this predicament, they conducted experiments to prove that cells are capable of phosphorylating AZT to AZTTP. For this, 'H9 cells were infected with HTLV-IIIB' and incubated with 50mM AZT for 24 h either during infection or 'through the replication cycle of the virus', that is at days 3, 6 and 9 after infection. One non-infected H9 culture was also cultured with the same concentration of AZT for 24 h. The phosphorylated derivatives of AZT were measured using High-Performance Liquid Chromatography (HPLC). They reported that 'High concentrations of azidothymidine monophosphate were detected in the uninfected and the HIV-infected H9 cells, whereas the levels of the diphosphate and triphosphate were low. By 24 h these phosphorylated derivatives had accumulated maximally. Increasing the time that the cells were exposed to the drug did not result in higher levels of phosphorylated derivatives'. The level of AZTTP reported was 1.5 pmol per 106 cells (1.8 mM) in the non-infected culture and 0.9 (1.1); 1.0 (1.3); 1.7 (2.0); and 0.9 (1.1) in the four infected cultures. In other words, the level of AZT phosphorylated to AZTTP by the H9 cells was not sufficient to induce even a 50% inhibition of the 'purified HIV RT' when the non-synthetic, 'activated calf thymus DNA' was used as template-primer. To determine the decrease in the levels of the phosphorylated derivatives of azidothymidine, after removal of the drug from the incubation medium at day 5 after infection, cells were incubated for 24 h with 50 mM AZT, after which the cells were washed and the incubation was continued in a drug-free culture medium. The level of AZTTP was determined at time 0, 0.5, 1, 2 and 4 hours after removal of the drugs and was reported as being 7.2, 5.2, 1.9, 1.7 and 1 mM respectively. As can be seen, the level of AZTTP reported in the H9 cells not only did not decrease after the cells were washed as one would expect but, at least for the first 2 h, was if anything higher than when the drug was present. Even if one cell line phosphorylates AZT to levels of AZTTP sufficient to inhibit the HIV RT, it does not mean that other cell lines will be able to produce the same effect. Indeed, by 1988 researchers from the US National Institutes for Health, including Samuel Broder, in collaboration with researchers from Belgium showed that the phosphorylation of AZT to AZTTP was dependent on the type of cell as well as the length of time during which the cells are incubated with the drug. The human lymphocyte ATH8 and human lymphoblast Molt/4F cells were incubated with 5 mM AZT for 5, 24 and 48 h. The level of AZTTP was 0.6, 0.4 and 0.2 mM in the Mo lt/4F cells at 5, 24 and 48 h. The respective levels in the ATH8 cells was 0.2; 0.1; < 0.1 mM (38). In a study published in 1991 by researchers from Sweden, resting and PHA stimulated PBMC from 31 healthy individuals and 5 HIV seropositive individuals were incubated with different concentrations of either radioactive or non-radioactive AZT. The phosphorylated AZT metabolites were quantified by HPLC. The authors reported: 'It was only possible to measure the di- and triphosphorylates when the cells had been labelled with radioactive AZT, while the monophosphate was detectable by ultraviolet (UV) absorbance even after incubation with non-labelled AZT'. The stimulated PBMC were incubated with 0.08, 0.16, 0.8 and 1.6 mM AZT. The quantity of AZTTP found in these cultures was: 0.12 ± 0.06, 0.17 ± 0.09, 0.20 ± 0.15 and 0.32 ± 0.19 nmol/10 cells, respectively (nmol/10 cells = pmol/10 cells). For the non-stimulated PBMC the results for only two concentrations of AZT, 0.8 and 1.6 mM, are reported. In these cultures the AZTTP was found to be 0.002 ± 0.001 and 0.003 ± 0.002 nmol/109 cells, respectively. In other words, cells which are stimulated form approximately 100 times the amount of the triphosphorylated compound compared with cells which are unstimulated. They also measured the half-life of AZT phosphorylated metabolites. For this the cells were cultured with AZT for 4 h after which the drug was washed and the cells cultured in drug-free medium. The half-lives of AZTMP, AZTDP and AZTTP in stimulated cells were 2.3 ± 0.7 h, 2.5 ± 0.6 h and 2.8 ± 0.6 h, respectively. 'The half-life of intracellular AZTMP in resting PBMC was also measured and was determined to be 1.5 ± 0.2 h. Because of the low incorporation of radioactivity in the azidothymidine di- and triphosphate pools of the resting PBMC it was impossible to determine the half-life of these two metabolites… An approximately 20% variation in the amount of product found in stimulated cells from different individuals was found… The corresponding variation in resti ng PBMC was 50%… The intra-individual variation measured in subjects analysed repeatedly at 2 –4 different occasions was also around 20%'. They reported the following results from five seropositive individuals: 'PBMC from 5 HIV+ patients (1 classified as asymptomatic, 3 as ARC, and 1 as AIDS, respectively) were incubated with AZT.we found stimulation by PHA of the PBMC only in the asymptomatic case. These cells thus accumulated AZTMP, AZTDP and AZTTP (18, 0.2 and 0.01 nmol/10 cells, respectively, after incubation for 4 h with 1.6 mM AZT). In the ARC and AIDS cases no stimulation was observed after 72 h. Resting PBMC from all 5 patients accumulated azidometabolites (1.6 mM AZT gave 0.05–0.42 nmol AZTMP/10 cells), which would correspond to what was found with PBMC from HIV subjects'. No mention is made of the level of AZT diphosphate or AZTTP in the cells from ARC and AIDS patients (39). Even if all human cells phosphorylated AZT to AZTTP with high efficiency under in vitro condition, it does not follow that the same effect would be observed in vivo. In other words the finding in vitro cannot be extrapolated to the situation in vivo. In fact, it is paramount that such evidence be obtained from AIDS patients and HIV seropositive individuals, not healthy volunteers. Indeed, given that: (a) The toxicity of AZT was recognised long before the AIDS era; (b) It is recognised that the antiretroviral effect of AZT is conferred only by its triphosphorylated form; it is inconceivable to contemplate the introduction of AZT in clinical practice before there is proof that AZT is triphosphorylated in HIV positive individuals to a level necessary to inhibit viral RT. Yet this seems to be the case, since the first results of in vivo phosphorylation of AZT did not appear until the 1990s. Even then, although the then available in vitro evidence showed that no relationship existed between AZT concentration and the level of phosphorylated AZT or the total AZT phosphates level, or the triphosphate levels, for some unknown reason researchers from well known institutions such as the University of Cincinnati and Division of AIDS, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, continued to report total AZT phosphorylates and not its active form, AZTTP (40–42). In 1991, Takuo Toyoshima and his colleagues from the University of Tokyo and Research Institute, Sankyo Co., pointed out that 'for the better understanding of pharmacokinetics of AZT, it is necessary to gain an insight into the metabolism of AZT, especially into the intracellular concentrations of AZT-TP', but 'concentrations of these metabolites in peripheral blood mononuclear cells have not been measured in patients with acquired immunodeficiency syndrome'. They performed both in vitro and in vivo experiments. For the in vitro experiments they used the MT-4 and Molt-4 cell lines. For the in vivo experiments, 'A patient with AIDS and an asymptomatic carrier (AC) received 200 mg of AZT orally, and blood samples (15 ml each) were drawn 1 and 4 h after ingestion of the drug'. Using the MT -4 cell line they found that 'intracellular concentrations of AZT-MP increased as concentrations of AZT in the medium were elevated; a concentration of 6770 pmol/10 cells was attained when 10 mM of AZT was present in the medium. Concentrations of AZT-DP and AZT-TP were one to two orders of magnitude lower than those of AZT-MP, and seemed to level off when the concentrations of AZT were higher than 5 and 2 mM, respectively. 'In MT-4 and Molt-4 cells incubated with 5 mM AZT, concentrations of AZT-MP increased time dependently, while the AZT-DP/ AZT-MP ratios decreased with time'. They concluded, 'These data suggest that high dose of AZT may not necessarily increase intracellular concentration of AZT-TP'. From their experiments they reported that 'Concentrations of AZT-MP in PBMCs from a patient with AIDS and an AC were 260 and 500 pmol/10 cells after 1 h, and 260 and 240 pmol/10 cells after 4 h, respectively. Those of AZT-DP were measured in an AC only; 6.5 pmol/10 cells after 1 h, and 3.9 pmol/10 cells after 4 h. Those of AZT-TP were 56 and 15 pmol/10 cells after 1 h, and 17 and 13 pmol/10 cells after 4 h, for a patient with AIDS and an AC, respectively' (41). In the same year Herbert Kuster and his colleagues from the University Hospital Zurich wrote: 'Serum pharmacokinetics of AZT have been studied extensively: however, no data about the extent and kinetics of in vivo phosphorylation are available. To date the intracellular anabolism of AZT and of other dideoxynucleosides has been examined only in vitro using radiolabeled compounds. A detailed knowledge about the phosphorylation is important for several reasons. First, there is a documented variability of AZT phosphorylation in various cell systems, and data from in vitro experiments cannot necessarily be extrapolated to the in vivo situation. Second, interindividual differences in drug metabolism are well known in clinical medicine for a variety of compounds…. Finally, a better understanding of the in vivo pools and pharmacokinetics of intracellular AZT-TP might lead to improved drug schedules for individual patients. Thus we developed a method to measure the intracellular anabolites of AZT in whole blood from patients treated with this drug'. They also performed both in vitro and in vivo experiments. For the in vitro experiments IL-2 stimulated PBMC from a healthy HIV-negative individual were incubated with 3H labelled and unlabelled AZT. The following findings were reported: 'In PBMC cultured in the presence of 2 mmol/1 [3H] AZT for 24 h, concentrations of AZT-MP, AZT-DP and AZT-TP were 193, 1.3, and 2.0 pmol/10 PBMC, respectively, after ficoll-hypaque density-gradient centrifugation. If cells were harvested by simple centrifugation, concentrations of 215, 1.7 and 1.6 pmol/10 PBMC were found. PBMC of the same donor treated under identical conditions but with unlabelled AZT yielded concentrations of 198 pmol/10 PBMC for AZT-MP, 1.8 pmol/10 PBMC for AZT-DP, and In the in vivo study: 'Blood samples were obtained from three patients on long term oral therapy with 250 mg of AZT every 12 h. AZT nucleotides were determined before and 1, 2, and 4 h after administration of the drug. No phosphorylation products were found before administration. Intracellular concentrations of AZT-MP after 1–2 h were 0.9–1.4 pmol/10 PBMC and then declined to 0.3–1.1 pmol/10 PBMC after 4 h. AZT-DP and AZT-TP reached concentrations of 0.3– 0.5 pmol/10 PBMC after 1–2 h and could not be detected after 4 h in any of the three patients'44. In 1992, researchers from Johns Hopkins University, Baltimore, stressed that the 'In vitro studies of Zidovudine (ZDV) phosphorylation may not accurately reflect the in vivo dose–response relationship, which is crucial to determining the relationship between ZDV exposure, efficacy, and toxicity. Quantification of intracellular levels of ZDV-TP, which is the active metabolite, and defining the time course of ZDV-TP formation and degradation, are of paramount importance for understa nding the relationships between intracellular levels of ZDV-TP and antiviral activity'. Commenting on their own work and that of other researchers, the authors wrote: 'Attempts at measuring ZDV and its phosphorylated anabolites have been reported by Toyoshima et al., who utilised a high-pressure liquid chromatography (HPLC) system with column switching and UV detection. Kuster et al., using a coupled HPLC-radioimmunoassay (RIA) method, also measured ZDV and ZDV phosphates in HIV-infected patients. These methods however have not been thoroughly validated and they lack the sensitivity (limit of detection, 0.1 pmol/10 peripheral blood mononuclear cells [PBMC]) needed for the study of the time course of ZDV anabolism. Our study describes the development and validation of a specific and sensitive assay for measurement of ZDV and its phosphorylated anabolites from PBMCs of ZDV-treated HIV-infected patients'. For their in vitro assay they used the Molt-4 cell line and PBMC, which they cultured with 2 mM of AZT. The levels of ZDV-TP were 1.6 ± 0.7 pmol/10 cells in the Molt-4 cells and 0.011 ± 0.002 pmol/10 cells in the PBMC. In vivo they studied six infected patients who were receiving ZDV. 'The duration of previous ZDV therapy at the time of the study ranged from 1 to 8 months'. Two hours after a 300 mg oral dose, 'The mean concentrations (± standard deviation) of parent and of mono-, di-, and triphosphates were 0.15 ± 0.08, 1.4 ± 1, 0.082 ± 0.02, and 0.081 ± 0.13 pmol/10 PBMC, respectively (1 pmol/10 PBMC represents a concentration of approximately 1 mM). Concurrent serum ZDV concentrations were between 1.3 and 7.1 mM' (45). In a study published in 1994, 'ZDV-TP in PBMCs and plasma ZDV concentrations were measured in 12 HIV-infected adult volunteers receiving ZDV at St Jude Children's Research Hospital. All 12 volunteers studied were administered a single 100- or 500-mg oral dose of ZDV. Plasma ZDV concentrations and intracellular ZDV-TP levels were determined at 1, 2, 4, and 6 h after administration of the drug'. The authors reported that: 'Median intracellular ZDV-TP levels ranged from 5 to 57 and 42 to 92 fmol/10 cells in volunteers administered 100 and 500 mg of ZDV, respectively' (46) (1 fmol = 10–3 pmol). Michael Barry and his associates from the University of Liverpool's Department of Pharmacology and Therapeutics published two papers, one in 1994 and the other in 1996. In the first study five seronegative volunteers and 12 HIV-positive patients were given 250 mg AZT and blood samples were taken at 0, 1, 2, 4 and 6 h following drug administration. 'Three patients were asymptomatic [Centers for Disease Control and Prevention (CDC) group II] and nine had AIDS'. In the seronegative volunteers the mean ZDV-TP levels were 0.04, 0.03, 0.02 and 0.06 pmol/10 cells at 1, 2, 4 and 6 h respectively. In the patients the corresponding values were: 0.05, 0.06, 0.06 and 0.04 pmol/10 cells. Commenting on their findings, the authors wrote: 'A concentration-dependent block in the formation of ZDV-DP and ZDV-TP from ZDV-MP has been observed in activated PBMC. These in vitro findings are consistent with the results we obtained in 12 HIV-seropositive patients administered ZDV 250 mg, where ZDV-MP was the main metabolite found in PBMC. Interestingly, the concentrations of ZDV-TP in both HIV-seropositive patients and seronegative volunteers were comparable. In both groups there were subjects in whom ZDV-TP levels could not be detected. Although a more sensitive assay would be useful it is difficult at present to envisage an RIA with a detection limit much below that achieved in this and previous studies' (47) [our italics]. The 1996 study was designed to determine 'The effect of ZDV dose on the formation of intracellular phosphorylated metabolites', which 'may help define the optimum daily dose of ZDV, which is still unknown' [our italics]. Ten 'patients (ZDV-experienced) received, in random order, two dose regimens: ZDV 300 mg twice daily (600 mg per day) and ZDV 100 mg three times daily (300 mg per day) for 6 days. Therefore, all patients were at steady state ZDV therapy on attending the department for pharmacokinetic study on day 7. The study days were separated by at least 14 days…. On the study day patients arrived at 0800 h after an overnight fast. They ingested 100 or 300 mg ZDV at 0900 h according to the dose regime,' and blood was taken at 0, 1, 2, 4, 6 and 12 h after drug administration. When they compared the maximum concentration of ZDV in the plasma (Cmax) and the area under the ZDV concentration time curve (AUC0–12h) for the two doses, they found that: 'The 300 mg dose produced an increase in Cmax (2.59 ± 0.52 versus 0.7 ± 0.14 mmol/l) and AUC0–12h (4.59 ± 0.79 versus 1.42 ± 0.51 mmol/l x h)'. The time at which Cmax was obtained, Tmax, was not significantly different. 'For total intracellular ZDV phosphate metabolites the AUC0–12h was doubled (7.64 ± 3.67 versus 3.71 ± 1.83 pmol/106 cells x h) in patients taking 300 mg compared with 100 mg. The AUC0–12h for ZDV-MP was significantly increased at the higher dose (6.47 ± 3.14 versus 2.77 ± 1.70 pmol/106 cells x h)… However, there was marked intersubject variability in the AUC0–12h for ZDV-DP (0.52 ± 0.32 versus 0.56 ± 0.57 pmol/10 cells x h) and ZDV-TP (0.42 ± 0.42 versus 0.61 ± 0.81 pmol/10 cells x h) with wide 95% confidence intervals on the differences in mean values, following ZDV 100 and 300 mg, respectively'. The mean Cmax and Tmax for AZT-TP were almost the same for both doses and were approximately 0.07 pmol/10 cells and 2 h respectively. Discussing their findings, the authors wrote: 'Consistent with previous reports, we found a weak correlation between plasma concentration of ZDV and intracellular metabolites. Total phosphorylation appears to be a saturable process, and therefore increases in plasma ZDV concentration do not result in parallel increases in total phosphate concentrations. As ZDV-TP inhibits viral reverse transcriptase, its measurement (or more precisely the ratio of ZDV-TP to thymidine triphosphate) is more likely to provide satisfactory dose–response relationships for ZDV. In this study, the AUC0–12h for ZDV-TP did not differ significantly following the 100 or 300 mg ZDV dose. With the evidence that saturation of ZDV phosphorylation occurs after administration of 100 mg ZDV and with the half-life of intracellular phosphates being approximately 4 h, the ability of the lower 100 mg dose to produce similar active drug, ZDV-TP and lower ZDV-MP (potentially toxic) suggests that ZDV 100 mg 8-hourly may be preferable to ZDV 300 mg 12-hourly. However, we also recognise that the antiviral effect of ZDV is ultimately dependent on the ratio of ZDV-TP to thymidine triphosphate, and we are aiming in future studies to measure the levels of both triphosphate anabolites' (48). In an article published in Nature Medicine 1997, one reads that 'Azidothymidine triphosphorylate (AZT-TP) inhibits the viral RT by competing with endogenous thymidine triphosphorylate (TTP). The extent of inhibition, therefore, depends as much on the interplay of AZT-TP and TTP concentrati ons as on the concentrations of their respective intermediates, and the degree to which they themselves serve as substrates for the two kinases. Although AZT is converted to AZT-MP with nearly the same efficiency as the thymidine is converted to TMP, the conversion of AZT-MP to AZT-TP is less than one percent the efficiency of the TMP to TTP conversion. The end result is an accumulation of high concentrations of the inactive AZT-MP but not of the active AZT-TP' (49). Lately, several research groups have put forward proposals to account for the inability to achieve 'effective concentration of AZT-TP within cell sufficient to suppress HIV replication' (50–52), while others have reported that the herpes simplex virus type 1 thymidine kinase improves AZT triphosphorylation and suggested that 'gene transfer might be envisioned for genetic pharmacomodulation of antiviral drugs' (53). Comments
Whatever the reason(s), the fact remains that, for AZT to have an anti -HIV effect, it must be triphosphorylated (28), but this is insignificant in vivo. In addition, the triphosphorylated form is deemed responsible for its toxicity (1,2). In their 1986 paper Philip Furman and his research colleagues from the National Cancer Institute, Duke University and Wellcome Laboratories (27) reported that, under ideal conditions, 'The IC50 values for the viral reverse transcriptase were 0.7mM with poly(rA).oligo(dT)12–18, and 2.3 mM with activated calf thymus DNA as primer-templates'. In their first clinical trial (28) they acknowledged that 'a minimal level for an in vitro antiviral effect' is 'above 1mmol/l' of AZTTP. However, such levels of AZT triphosphorylation are not obtained even under ideal, in vitro conditions, and the level of AZT triphosphorylation in vivo is even lower. This means that, as has been generally accepted to date, neither the well known toxic effects of AZT nor any antiretroviral effects can be due to its action as a DNA chain terminator. The question then is, how does AZT produce its toxic effects as well as its anti-HIV effects, if any? Although AZT is not efficiently triphosphorylated it is very efficiently mono-phosphorylated. The mono-phosphorylation of AZT could act as an inhibitor of phosphorylation of cellular constituents, including cellular nucleotides. Indeed, in 1986 Furman and his associates showed that, in vitro, exposure of cells to 50mM of AZT for 72 h led to a decrease of approximately 95% in dTTP and dCTP and a decrease of approximately 63% in dGTP. This decrease in the triphosphorylated nucleotides in its turn will lead to decreased cellular DNA synthesis. In the presence of such a profound, global reduction in the concentrations of the naturally occuring nucleotides, one would predict untoward effects on many tissues, especially those with the most rapid cellular turnover including the gut and the bone marrow. Indeed, 'a characteristic feature of zidovudine therapy is an elevated MCV [mean corpuscular red cell volume]' (54), and 'The antiviral agent zidovudine (AZT), used for treating the human immunodeficiency virus (HIV), often causes severe megaloblastic anemia' (55), anaemia 'caused by impaired DNA synthesis' (55). It is a well known fact that AZT inhibits mitochondrial DNA (mtDNA) replication. However, since the level of AZT triphosphorylation is negligible, this effect cannot be due to AZT acting as a DNA chain terminator. In their effort to explain the AZT mitochondrial toxicity, researchers from the University of Nagoya studied the mtDNA of mice given either 1mg/kg/day or 5mg/kg/day of AZT orally for 4 weeks. Their findings, published in 1991, 'suggest that the oxygen damage of mtDNA is the primary cause of mitochondrial myopathy with AZT therapy… oxidative damage of mtDNA can be accumulated during even short period of AZT administration'. They concluded: 'The animal model of mitochondrial myopathy with AZT administration reported here seems to be useful for elucidating the mechanism of mtDNA mutations leading to myopathy. However, for AIDS patients, it is urgently necessary to develop a remedy substituting this toxic substance, AZT' (56). The cellular toxicity of AZT was extensively studied by researchers from the State University of New York. In 1996, summarising their findings, they wrote: 'Prior to the commencement of the present study, although strong evidence existed that many ddNs, including AZT, could inhibit mtDNA replication, we had not yet substantiated our hypothesis that such inhibition would result in the impairment of oxidative phosphorylation. Nor had we yet demonstrated a caus e-and-effect relationship between the AZT inhibition of mtDNA replication (or its consequence, an impairment of oxidative phosphorylation) on the one hand and an inhibition of cell growth on the other. Thus, the possibility had not been eliminated that AZT was exerting some general cytotoxic effect on the cell, which resulted in an inhibition of cell growth, and this, in turn, was leading to an inhibition of mtDNA replication. We noticed that the beginning of the AZT-induced inhibitory effect on cell growth occurred at a relatively short time after AZT addition to the medium, a period of time too short to account for the effect to have been brought about by an inhibition of mtDNA replication. This observation led to studies of the early metabolic events that occur upon exposure of the cells to AZT'. In these studies the authors found that: 'mitochrondria isolated from cells grown in the presence of pharmacological levels of AZT (5mM) for 5 days and tested for their ability to carry out oxidative phosphorylation showed a marked decrease in ability to synthesize ATP… Further studies of this phenomenon in which the frequency of sampling the medium was in hours rather than days… showed early changes in O2 uptake, lactate synthesis, ATP level, and number of mitochondria per cell. Some of these changes, particularly that of ATP level, were observable as early as 3 h after exposure to AZT and, judging from the precipitous decline of the ATP/cell curve between 0 and 3 h, may have begun earlier than that. The 3 h time interval, equivalent to only 7% of the doubling time of the AZT-treated cells, is far too short a period of time to account for the effect brought about by an inhibition of mtDNA replication' (57). In a study published in 1997, researchers from several French institutions 'compared the effects of AZT, ddI and ddC on proliferation, differentiation, lipid accumulation, lactate production and mitochondrial enzyme activities in cultured human muscle cells'. They reported that: 'All 3 compounds induced a dose-related decrease of cell proliferation and differentiation. AZT seemed to be the most potent inhibitor of cell proliferation. AZT, ddI and ddC induced cytoplasmic lipid droplet accumulations, increased lactate production and decreased activities of COX (complex IV) and SDH (part of complex II)' (COX=cytochrome c oxidase; SDH=succinate dehydrogenase). Summarising their findings they wrote: 'In conclusion, AZT, ddI and ddC all exert cytotoxic effects on human muscle cells and induce functional alterations of mitochondria possibly due to mechanisms other than the sole mtDNA depletion' (58). At present, evidence also exists which shows that AZT is rapidly reduced by compounds containing sulphydryl (–SH); that is, AZT oxidises the –SH groups (59). Ample evidence also exists which shows that oxidation in general (and of –SH in particular) and decreased levels of ATP may lead to many laboratory and clinical abnormalities, including wasting, muscular atrophy, anaemia, damage to the liver and kidney, decreased cellular proliferation, cancer and immunodeficiency (8,19). Since patients who are at risk of AIDS are exposed to many oxidising agents (8) and are known to have low –SH levels (60,61) one would expect AZT to have particularly toxic effects in these individuals – and the sicker the patient the more toxic the drug. That this is the case was accepted by researchers from the National Cancer Institute, Wellcome Laboratories and Abbott Laboratories as far back as 1988: 'Azidothymidine, however, is associated with toxicities that can limit its use. These toxicities are particularly troublesome in patients with established AIDS; the use of azidothymidine is often limited in this population' (62). Despite these caveats it is possible that, if a thymidine analogue is to be administered to patients with AIDS or to those at risk, at least part of its toxicity may be eliminated by substituting the 3'-OH group with a –SH-group instead of an azido (=N) group. Yuzhakov et al. have performed such experiments and shown that the resulting compound inhibits 'HIV RT' (63). C. Anti-HIV Effect of AZT
1. HIV experts agree that AZT produces its anti-HIV effects only by inhibiting the reverse transcription of the 'HIV RNA' into 'HIV proviral DNA'; 2. The same experts also agree that only triphosphorylated AZT can inhibit the synthesis of 3. The AZT given to patients is not triphosphorylated; 4. The triphosphorylation of AZT in HIV seropositive and AIDS patients, if any, is significantly lower than the concentration needed to inhibit RT even in the most ideal conditions; the inescapable conclusion is that AZT, as given to patients, cannot have an anti-HIV affect. How is it then possible to reconcile this fact with the claim that HIV is an anti-HIV drug? The only way of proving the antiretroviral effect of AZT is to determine its effect on HIV isolated from tissues of infected, treated patients. The correct procedure, used for over half a century to achieve isolation of retroviruses (64,65), requires: 1. Culture of putatively infected tissues. 2. Purification of specimens by density gradient ultracentrifugation. 3. Electron micrographs of particles exhibiting the morphological characteristics and dimensions of retroviral particles at the sucrose density gradient of 1.16 gm/ml containing nothing else, not even particles of other morphologies or dimensions. 4. Proof that such particles contain reverse transcriptase. 5. Analysis of the particles' proteins and RNA, and proof that these are unique. 6. Proof that 1–5 are properties only of putatively infected tissues and cannot be induced in 7. Proof that such particles replicate into identical particles and are thus infectious. This procedure has never been used to prove the antiretroviral effects of AZT, or for any other purpose, including proving the existence of HIV. Instead, the antiretroviral effects of AZT have been studied by observing its effects on: 1. 'HIV isolation', defined as detection of RT and the 'HIV p24' protein in stimulated cultures/cocultures of tis sues obtained from treated patients. Most often, the effects on 'HIV isolation' are merely detection of just one of these phenomena. 2. 'HIV antigenaemia', by which is meant reaction of proteins present in patient sera with 3. Estimation of 'viral load', defined by HIV researchers as the quantity of 'HIV RNA' molecules in a sample of patient plasma, or detection of p24 in plasma cultures. However, RT is not specific to retroviruses and p24 and 'HIV RNA' have never been shown to belong to a particle, viral or non-viral, much less to a unique retroviral particle, HIV (7,10,12,17,18). In fact, at present there is ample evidence which shows that these parameters are not HIV specific (66–71). This means that, even if AZT has an effect on these three parameters, such evidence cannot be considered as proof that AZT has an anti-HIV effect. If, on the other hand, there is no proof that AZT significantly effects these three parameters, then it would be impossible to claim that AZT has an anti-HIV effect. 1. HIV Isolation
By design, the role of AZT is not to inhibit HIV expression (activation) but to inhibit the reverse transcription of the HIV-RNA into new proviral DNA. In other words, if AZT has anti-HIV effects, then the first thing one would observe is a decrease in the HIV-DNA which in its turn would lead to a decrease in the rate of HIV isolation. In 1986 the researchers from the National Cancer Institute, Duke University and Wellcome Research Laboratories, published their results of the Phase I clinical trial of AZT in 19 patients with AIDS or AIDS related complex. 'All patients received test doses of AZT. They were then given AZT intravenously for 14 days according to the following regimens: 1 mg/kg every 8 h for patients 1–4 (regimen A), 2.5 mg/kg every 8 h for patients 5–10 (regimen B), 2.5 mg/kg every 4 h for patients 11–15 (regimen C), and 5 mg/kg every 4 h for patients 16–19 (regimen D). Each dose was administered over a period of 1 h. Patients 1, 2, 3 and 12 received additional intravenous doses for another 7–14 days. Except for patients 2 and 12 who were withdrawn from the study, the patients next received 4 weeks of oral therapy at twice the intravenous dose'. For the four patients treated with regimen A, the authors reported: 'Virus detected while on IV therapy, but not at end of oral'; 'Virus detected sporadically'; 'Decreased virus during initial AZT administration'; 'Virus not detected on day 0 or on AZT'. With regimen B: in one patient, 'Low levels of virus detected early, then negative'; another patient, 'Low levels of virus at entry, then virus not detectable'. In the remaining 4 patients, 'Virus detected throughout'. Regimen C: in 3 patients, 'Virus detected sporadically'; for one patient results were not available; and for another they reported, 'Virus detected during first 2 weeks, but not after'. With Regimen D: in 2 patients, 'Virus not detected on day 0 or on AZT'; for one, 'Virus detected on day 0 and day 7, but not after'; and for the other, 'Virus detected on day 0, but not on AZT'. Discussing their results, they wrote: 'For most of the patients on regimens A–C, virus continued to be detected in cultures established during therapy but virus was not detected in cultures established from any of the 4 patients on regimen D after 2 weeks of therapy. [From 2 of these 4 patients, they could not isolate HIV even before AZT administration.] In 2 of these patients (nos 16 and 18) virus cultures established at entry had been positive, which suggests that the failure to isolate virus was related to the administration of AZT…One patient (no. 15) on regimen C, also became virus negative while on AZT' (28). On the basis of the findings in the Phase I clinical trial, a multicentre 'double-blind, randomised placebo-controlled trial intended to last 24 weeks. to evaluate the safety and efficiency of AZT in the treatment of a well-defined group of subjects with AIDS or AIDS-related complex' was conducted by Margaret Fischl and her associates. AZT was given to 145 patients, 250 mg every 4 h; 137 received placebo. Blood was collected, in addition to other tests, 'for detection of anti-HIV antibody by enzyme-linked immunosorbent assay, for measurement of serum p24 antigen levels (Abbot Laboratories, Chicago) and for isolation of HIV from peripheral-blood lymphocytes (8)'. Reference 8 in this extract is a paper by Levy and his colleagues, who apparently consider that just the detection of reverse transcription is synonymous with HIV isolation. In this 'double-blind' study, 'Drug therapy was temporarily discontinued or the frequency of doses decreased to one capsule every eight hours or longer if severe adverse reactions were noted. The study medication was withdrawn if unacceptable toxic effects or a neoplasm requiring therapy developed. Subjects in whom an opportunistic infection developed were withdrawn from the study only if therapy with another experimental medication was required or if antimicrobial therapy might have resulted in serious additive toxic effects. Twenty-seven subjects had completed 24 weeks of the study, 152 had completed 16 weeks, and the remainder had completed at least 8 weeks'. Fischl and her colleagues reported that 'HIV was isolated at entry in 57 percent of the AZT group and 58 percent of the placebo group. No statistically significant differences in isolation rates were noted between the two groups during the study'. Discussing this finding, the authors wrote: 'The lack of a measurable effect on virus isolation from peripheral-blood lymphocytes may have been due to the activation of latent virus in cells by the culture techniques or by the failure of AZT to inhibit virus replication. Nevertheless, the ability to culture virus from many patients after several months of therapy indicates that such patients are still infectious and should be counseled to continue to follow appropriate practices to prevent the transmission of HIV' (72). In 1988, Antonella Surbone and her associates from the National Cancer Institute, Wellcome Research Laboratories, Abbott Laboratories and the Rush–Presbyterian–St. Luke's Medical Center treated 8 patients (4 with AIDS and 4 with ARC) with AZT and acyclovir. Patients received 100 mg AZT orally every 4 h for 7 days, followed by 100 mg of AZT and 800 mg of acyclovir orally every 4 h for an additional 9 weeks. 'In four patients, virus isolation was attempted at the initiation of therapy and during treatment. Human immunodeficiency virus could be detected by culture of mitogen-stimulated lymphocytes throughout the treatment period in Patient 6; virus was detected during treatment in Patients 3 and 4, who were negative at entry. and no virus could be detected at entry or during therapy in one patient' (62). In 1990, Ann Collier and her associates from several institutions in the USA, including the University of Washington and the University of California, 'conducted a Phase II open-label, dose-escalating trial to evaluate the clinical and antiviral effects of zidovudine at low (300 mg daily, 28 subjects), medium (600 mg, 24 subjects), and high (1500 mg, 15 subjects) doses, either with or without acyclovir (4.8 g) by random assignment'. From 402 individuals screened they enrolled only 67. 'Most exclusions were due to the absence of HIV antigenemia or viremia or to ineligible CD4 counts [<200/uL]. The study was divided into three phases: an initial 12-week period, an elective extension phase of varying duration (from the end of the first 12 weeks until April 1989), and an 8-week crossover phase involving a new dose of zidovudine. During the crossover phase, the subjects who had received 300-mg or 600-mg doses of zidovudine were given 1500 mg per day, and those who had received 1500-mg doses were given 300 mg per day. The subjects randomly assigned to acyclovir received it throughout the study'. For some unknown reason, data on HIV isolation were given only for the first 12 weeks. 'Of the 38 subjects who had plasma viremia before entry [only] 25 had quantifiable titers. The mean (± SD) log10 plasma titer on day 0 was 2.5 ± 0.9. Mean plasma virus titers decreased by 1.80 during the first 12 weeks. No dose of zidovudine caused plasma viremia to disappear, but the magnitude of the decrease in plasma titers was similar for all doses of zidovudine. Thirty-nine of the 40 subjects who had peripheral-blood mononuclear cells cultured for HIV tested positive. The proportion with positive cultures was similar in all groups during the study' (73). (The authors fail to explain how it is possible to obtain a decrease in plasma viraemia with a drug like AZT which, by definition, inhibits only the quantity of proviral DNA and not the transcription of DNA into RNA; that is, any reduction in plasma viraemia is related to a decrease in HIV proviral DNA, the latter reflected by a decreased frequency of HIV isolation from cells.) In a study published in 1997 by researchers from several institutions from the USA, 'Two groups of subjects were recruited on the basis of CD4 cell count, antiretroviral therapy, and lack of cell-free virus in plasma at entry. Group A cons isted of HIV-1 infected subjects with >600 CD4 cells/mL before enrollment (n=30); group B subjects had initial CD4 cell counts of 400–550 (n=15). All group B subjects received zidovudine monotherapy (500–600mg/day) for >= 6 months before enrollment and continued to receive zidovudine monotherapy for the duration of the study… At study entry, HIV-1 was isolated by the quantitative microculture method from 12 (86%) of 14 subjects in group B versus 15 (56%) of 27 in group A', although the patients from group B had received AZT. Furthermore, 'the titer of cell-associated virus increased over time', in group B but not in group A (74). 2. HIV DNA
According to the HIV model of AIDS pathogenesis, in the years following infection the concentration of infected mononuclear cells in the blood progressively increases, eventuating in very high levels of infected cells – that is, proviral DNA concentration, 'viral burden' – followed by viral expression and cellular death; that is, acquired immune deficiency. Given the general acceptance of this theory, one would assume that at present there is ample evidence to prove (i) the model; (ii) that AZT decreases the number of infected cells. However, these do not appear to be the case. According to American researchers from the California State Department of Health, University of California and the Departments of Epidemiology and Biostatistics and Laboratory Medicine, University of San Francisco, 'Surprisingly, most of the data supporting the above model are based on cross-sectional studies or short term follow-up studies of small numbers of patients.' To overcome this deficiency, these researchers tested the peripheral blood mononuclear cells (PBMC) of 9 rapid-, 9 intermediate- and 10 non-progressors whose date of seroconversion was not known at entry to the study using 'HIV-1 DNA gag polymerase chain reaction'. The same test was then repeated after five years. To their surprise, the number of infected PBMCs at entry was low in all groups, 73 (approx. 1–85)/106 PBMC, 160 (approx. 10–500)/106 PBMC, and 330 (approx. 10–1000)/106 PBMC in non-, intermediate - and rapid-progessors respectively. Even more surprising was their finding 'that there was little or no change in the concentration of HIV DNA positive cells from study entry to the 5-year follow-up visit for most subjects' in all three groups. In fact, 'the concentration of circulating HIV DNA positive cells' in at least two subjects from each group decreased in time, although none of the patients had anti-retroviral treatment. They also studied serial samples collected immediately before and after seroconversion for 18 subjects; samples were collected at 6-month intervals. 'In all subjects the concentration of HIV-1 infected PBMCs established shortly after seroconversion remained remarkably stable for up to 5 years', including in subjects whose CD4 cell counts declined (from 1049 to 46 cells/ml and 1063 to 276 cells/ml, one of whom developed PCP). In fact, on inspection of the graph depicting the results for the first 12 months for 15 of the subjects, it is easily seen that the 'HIV-1 infected cell burdens' fluctuated over time and that one patient 'had a substantially higher viral burden on the initial polymerase chain-reaction positive sample relative to the subsequent samples', although the patient did not receive anti-retroviral treatment (75). The fact that when patients are treated with the drug AZT the frequency of HIV isolation is not diminished means that AZT does not affect the level of proviral DNA. In a paper published in 1994, researchers from the AIDS Research Center, Department of Veterans' Affairs Medical Center, Palo Alto and the Center for AIDS Research, Stanford University, discussing their proviral DNA findings and those of others, wrote: 'Donovan et al. found that proviral DNA copy number was constant in six patients who had multiple samples taken during a 5–14 month period while on zidovudine (ZDV) therapy. We have also shown that there was no significant change in provirus level in four patients who were followed for a mean of 13 months' (76). That AZT does not have any effects on the proviral DNA has been confirmed by other researchers (77). Since, contrary to its putative action, proviral DNA remains unaffected by AZT treatment, and since AZT does not affect the expression of HIV, one would expect the drug to have no effect on the p24 antigenaemia and 'HIV RNA'. 3. p24 Antigenaemia
If p24 is an HIV protein, and if the cause of ARC and AIDS is HIV, then one would expect at least these patients, if not all HIV seropositive patients, to have high levels of p24 antigenaemia. If AZT is an anti-HIV drug, then the concentration of p24 should decrease in all patients who are treated with AZT. The decrease should be observed only in treated patients. As mentioned, in their 1987 'double-blind, placebo-controlled trial', Margaret Fischl and her associates had 145 patients who received AZT and 137 who received placebo. 'Thirty-six AZT recipients and 40 placebo recipients were found to have detectable serum p24 antigen. Of these, 28 in each group had both a serum specimen obtained at entry and specimens obtained later in which changes in antigen level could be evaluated. Statistically significant decreases from the serum level of p24 antigen at entry were found among AZT recipients at weeks 4, 8 and 12 (overall, P < 0.05). Similar trends were also noted at weeks 16 and 20, but the numbers of subjects were small for statistical analysis' (72). In 1988, several studies were published in which the relationship between AZT treatment and p24 was examined. In the study by Surbone et al. mentioned above, 'Serum obtained at periodic intervals from the patients was assayed for HIV p24 antigen using an enzyme-linked immunosorbent assay (Abbott Laboratories.). Patients 4 and 6 had detectable serum p24 antigen at entry; in each of these patients, p24 could no longer be detected at week 10 of therapy. The other four patients had no detectable HIV p24 antigen either at entry of during treatment', although the patients had either AIDS or ARC (62). In a letter to Lancet, researchers from the University of Amsterdam wrote: 'An in vitro study has lately demonstrated resumption of virus production in HIV-infected T lymphocytes in the continued presence of initially highly inhibitory doses of zidovudine. As indicated by HIV antigen levels in two patients we have treated, a similar resumption of antigen production may occur after prolonged zidovudine treatment. Both were HIV-Ag seropositive (Abbott enzyme immunoassay) AIDS patients and were treated with 200 mg zidovudine 4-hourly. Serum HIV-Ag concentrations fell rapidly below the cut-off level for the assay [50 pg/ml]. However, despite continuation of the same drug regimen and patient compliance with the therapy, HIV-Ag serum levels subsequently rose in both patients. Neither had diarrhoea or clinical evidence of malabsorption' (78). In the same year, in yet another study by researchers from the University of California, Burroughs Wellcome and Abbott Laboratories, the authors noted that 'Clinical testing of drugs potentially active against the human immunodeficiency virus (HIV) has been seriously impeded by the lack of a reproducible quantitative method of estimating viral burden. We have investigated the clinical utility of an antigen capture assay for the HIV gag gene product p24 in patients undergoing treatment with zidovudine. Previous studies have shown that HIV gag or core antigen can be detected with greater frequency in patients with more advanced HIV infection, and presence of antigen is a predictor of disease progression in initially asymptomatic HIV seropositive homosexual men and hemophiliacs. In addition, HIV antigen can be reliably quantitated in picogram amounts allowing the possibility of dose-effect observations. We previously reported the use of a serum HIV core antigen (HIV-Ag) capture assay in a preliminary study of the in vivo antiviral effect of zidovudine (5). We describe results of a larger study of serum HIV-Ag levels in patients enrolled in the multicenter phase II trial of zidovudine for the treatment of acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC)'. (Reference 5 cited in this extract is the 1987 paper by Fischl et al.) 'Two hundred eighty-two subjects with either severe ARC (weight loss or oral candidias is and fever, leukoplakia, lymphadenopathy, night sweats or herpes zoster) or a recent episode of Pneumocystis carinii pneumonia were recruited for the study… Subjects were randomly assigned to receive either 250 mg of zidovudine or placebo every 4 hours in a double-blind fashion. Dose modifications were made at the investigators' discretion based on toxicity. Median duration of treatment was 16 weeks.One hundred fifty-eight subjects, 83 treated with zidovudine and 75 given placebo, had serum samples available for testing and are the subject of this article. The prevalence of HIV-Ag at any time in subjects from whom baseline samples were available was 43% for zidovudine-treated individuals and 48% for placebo recipients . The prevalence of HIV-Ag varied by diagnosis: 59% of subjects with AIDS have detectable HIV-Ag vs 37% of those with ARC'. Their findings are presented in a table and also are discussed in the text. In the text, one reads: 'Thirty-one zidovudine and 32 placebo recipients who were HIV-Ag positive had a baseline and at least one additional sample available to evaluate changes in HIV-Ag levels according to treatment. Median HIV-Ag levels in zidovudine patients declined significantly with treatment, falling from 111 pg/mL at entry to 46 pg/mL at four weeks, and stabilizing at that level through 16 weeks. In contrast, HIV-Ag levels in placebo recipients varied little over time with a nonsignificant increment at 16 weeks. Fifty-nine percent of zidovudine-treated patients who were initially HIV-Ag positive became HIV-Ag negative during therapy compared with only 7% of placebo treated subjects (P < .0001)'. (However, it is obvious from the data presented in the table that at least one patient from each group was HIV-Ag negative already at day 0). Commenting on their findings, the authors wrote: 'The use of HIV-Ag assay to monitor patients treated with zidovudine is limited by the prevalence of antigenemia in patients with AIDS and ARC. As previously reported, a greater proportion of our patients with AIDS, 59%, had HIV-Ag present compared with patients with ARC, where the prevalence was 37%. Approximately half the patients in each treatment group (zidovudine or placebo) were HIV-Ag positive during the course of the trial' (79). In a study by researchers from University of Illinois and Abbott Laboratories, 16 patients with AIDS or ARC were enrolled 'using criteria applied in the national placebo-controlled trial. One half of the patients were randomised to receive zidovudine in an initial dose of 250 mg orally every 4 h. Changes in dosage were made by protocol definition based on reduction in leukocytes, hemoglobin, hematocrit, or platelets. Positive cultures were identified by the presence of HIV antigen (predominantly p24) in the culture supernatant (EIA) (Abbott Laboratories.). Serum or plasma was tested for HIV antigen by the same EIA. Antigenemia was found in the initial serum specimen from 11 and in serial specimens during the study from 12 of 16 patients with AIDS or severe AIDS-related complex. Three of the four antigen-negative patients had detectable serum anti-p24 antibody. Among patients who had antigenemia on entry, 8 could be characterised as having a high level (greater than 100 pg/mL), 4 had a low level (15 to 65 pg/mL), and 4 had no detectable antigenemia.' Three out of the 4 patients with no detectable antigenaemia, 3/4 with low level and 2/8 with high level of antigeaemia were treated with AZT; and 1/4 with no detectable antigenemia, 1/4 with low level and 6/8 with high level of antigenaemia were given placebo (can this be said to be a randomised placebo-controlled study for p24?). 'Treatment was always begun with 250 mg or 200 mg every 4 h. The regimen consistently reduced the serum level of HIV antigen. Doses of 100 mg every 4 h or 250 mg every 8 h often permitted an increase in the serum level of HIV antigenemia. Cultures for HIV were nearly always positive in many patients with antigenemia regardless of the level' (80). Three-hundred and sixty-five consecutive patients with ARC (80) or AIDS (285) who were eligible for AZT treatment by the Claude Bernard Hospital AZT Committee were followed up for a mean of 31 weeks. 'The full dose of AZT was 200 mg orally every 4 h. Patients with haemoglobin below 9 g/dl and/or PMN count below 1000/ml were treated with 200 mg 8-hourly. For patients treated with the full dose the dosage was reduced to 200 m g 8-hourly if PMN count dropped to less than 1000/ml, haemoglobin to less than 9 g/dl, platelets to less than 50,000/ml, or if non-haematological side-effects occurred. AZT treatment was temporarily interrupted when PMN count fell below 750/ml, haemoglobin below 7 g/dl, or if non-haematological side-effects could not be tolerated'. Antigenaemia analysis 'was restricted to the 52 patients with detectable p24 antigen (cut-off level 40 U/ml) before treatment who could be maintained on the full-dose or half-dose regimen for at least 12 months. The patients were stratified by pretreatment p24 antigen level (200 or more, or less than 200 U/ml). For patients with p24 antigen at 200 U/ml or above, the relative decrease was similar in the full-dose and the half-dose groups. The 16 full-dose patients were still p24 antigen positive at month 1, and only 1 was negative at month 2; none of the 7 half-dose patients became p24 antigen negative at months 1 or 2. Conversely, for patients with pretreatment p24 antigen level less than 200 U/ml, the relative decrease was significantly greater (ANOVA and t-test, p < 0.02) in the patients treated at full-dose than in those treated at half-dose. Of the 19 full-dose patients 9 (47%) and 10 (53%) became p24 antigen negative at months 1 and 2; only 2/10 and 3/10 half-dose patients were p24 antigen negative at months 1 and 2'. Discussing their findings in general and of antigenaemia in particular, the authors wrote: 'Generally, in our series, full-dose AZT for 2 months did not eliminate antigenemia in patients with pretreatment p24 levels of 200 U/ml or higher. in AIDS and ARC patients, the rationale for adhering to high-dose regimens of AZT, which in many instances heads to toxicity and interruption of treatment, seems questionable' (81). In the 1990 study by Collier et al. (73) discussed earlier, of 67 patients enrolled, 51 (76%) had antigenaemia before treatment with AZT. 'Forty of the 47 subjects who completed 12 weeks of therapy continued treatment for a median of 29 additional weeks. Only 13 of 37 of the subjects positive for HIV antigen (28%) became negative during this period. The proportion in whom HIV antigenemia resolved after therapy was 32% in the 300 mg group, 21% in the 600 mg group, and 33% in the 1500 mg group. The median change in the level of HIV antigen was 82% in the low-dose group, 71% in the medium-dose group, and 74% in the high-dose group (P not significant). The decrease or increase in the dose of zidovudine had no effect on levels of HIV antigen during the eight-week crossover period. Among the subjects positive for HIV antigen, there was a 50% decrease in the level of antigen during the first 12 weeks in 76% of the 25 treated with zidovudine alone and in 79% of the 24 treated with the combination (p not significant)'. In a study published in 1994, Victor DeGruttola and many of his associates, including Margaret Fischl, Paul Volberding and the Aids Clinical Trials Group Virology Laboratories, from several institutions from the USA pointed out that 'The primary clinical end points for evaluation of antiretroviral therapies in phase II and III studies are the development of AIDS-related complex (ARC) or AIDS or death. As therapy is initiated earlier in the course of human immunodeficiency virus type 1 (HIV-1) infection, there is an increased need for surrogate markers for clinical end points that can be used as early indicators of therapeutic efficacy'. In their study they 'investigated whether changes in serum p24 antigen levels can be used as a surrogate marker for clinical end points in phase II and III studies by examining whether pretreatment and follow-up serum p24 antigen measurements predicted subsequent clinical end points in three completed phase III clinical trials of zidovudine in persons with HIV-1 infection or AIDS'. The first trial 'was a randomized, open-label trial evaluating a reduced dose of zidovudine in 524 subjects with AIDS and a first episode of Pneumocystis carinii pneumonia (PCP)… Randomized subjects (262 in each group) received zidovudine at 1500 or 600 mg/day. Serum p24 antigen levels were measured before treatment and at weeks 8, 16, 24, 48, 64, 80, 96, 112 and 128 of treatment'. Because only 406 patients had a pretreatment serum p24 antigen measurement, the analysis was restricted to those patients. Only 65% of these patients with AIDS and PCP 'had measureable pretreatment concentration of serum p24 antigen (>= 10 pg/mL)' ('Estimated concentrations of serum p24 antigen < 10 pg/ml were considered to be negative'). Of the 203 patients on 600 mg/day AZT, 69 had a negative pretreatment p24 antigen. In the follow-up period 5 had no further measurements, 53 remained negative and 11 became positive. Of the 134 who had a pretreatment positive p24 antigen level, 21 had no follow-up measurement, 73 had > 50% decrease, 25 had <= 50% decrease and 15 had an increased p24 antigen level. Of the 203 patients who received 1500 mg/day AZT, 73 had a negative p24 antigen level. Of those, 14 had no follow-up measurements, 51 remained negative and 8 became positive. Of the 130 who were positive, 23 had no follow-up measurement, 74 had 50% decrease, 17 had <= 50% decrease and 16 had increased p24 antigen levels. The survival of the 406 patients 'was unrelated to the pretreatment concentration of p24 antigen in serum, and among those with available pretreatment antigen data there was no difference in survival. Changes during treatment were not associated with reduced mortality'. The second study was a 'randomized, double-blind, placebo-controlled' study consisting of 713 subjects with 'mildly symptomatic HIV-1 infection and CD4+ cell counts of 200–800/mm'(3)… Serum p24 antigen levels were measured before treatment and at weeks 4, 8, 16, 24, 40, 52, 76 and 88 of treatment. In this 'mildly symptomatic' study, 150 (24%) of the 637 patients with a pretreatment serum p24 antigen measurement had >= 10 pg/ml'. Of the 238 patients who were given placebo and who had a negative pretreatment p24 antigen, 9 had no follow-up measurement, 206 remained negative and 23 became positive. Of the 71 patients who were positive, 4 had no follow-up measurements, 5 had > 50% decrease, 25 had < 50% decrease and 37 an increase. Of the 249 patients treated with AZT (dose not given) and who had a negative pretreatment p24 antigen, 11 had no follow-up measurement, 220 remained negative and 18 became positive. Of the 79 patients who had a positive pretreatment p24 antigen, 5 had no follow-up measurement, 41 had 50% decrease, 28 had < 50% decrease and 5 had an increase in their p24 antigen level. In this study, having measurable serum p24 antigen before treatment 'about doubled the risk of developing advanced ARC or AIDS or dying. regardless of treatment'. Changes in the p24 levels 'were marginally associated with increased time until more advanced disease'. The third study 'was a randomised, double-blind, placebo-controlled trial of two dosages of zidovudine in 1323 asymptomatic HIV-1 infected subjects who had CD4+ cell counts of < 500/mm3….Serum p24 antigen levels were measured before treatment and at weeks 8, 16, 32, 48 and 64 of treatment…. Of 683 asymptomatic subjects, 123 (18%) with a pretreatment serum p24 antigen measurement had >= 10 pg/mL'. Of 204 individuals who were given placebo and who had negative pretreatment p24 antigen, 34 had no follow-up measurements, 155 remained negative and 15 became positive. Of 35 who had positive p24 antigen, 6 had no follow-up measurement, 7 had 50% decrease, 8 had <= 50% decrease and 14 had an increased level of p24 antigen. Of 356 individuals who were treated with AZT there was no difference in the results of the 500 or 1500 mg dosages and because of this the results were combined, 58 had no follow-up measurement, 287 remained negative and 11 became positive. Of 88 individuals with a positive pretreatment p24 antigen, 19 had no follow-up measurement, 38 had > 50% decrease, 21 had <= 50% and 10 had an increased p24 antigen level. In this trial, changes in the p24 antigen levels 'were not associated with increased time until progression". According to the authors of this study, serum p24 antigen is 'a specific marker of HIV-1 replication' but their study shows that 'much of the clinical improvement with zidovudine must be due to some other drug effect not mediated through p24'; that is, virus replication, viral load (82). 4. Virus Quantitation
If HIV is the cause of AIDS, the appearance of immune deficiency and of the clinical syndrome should be preceeded by an increase in the 'viral load' and not vice versa. AZT treatment should lead to a significant decrease, if not to a complete elimination, of the 'viral load'. Two methods have been used to quantify HIV in plasma, viral load. 1. Plasma culture. Plasma from infected individuals is cultured with normal stimulated PBMC and the p24 in cultures measured. However, according to an article published in 1996 in Nature Medicine by some of the best known workers in the fields of HIV research and viral treatment, including Saag, Shaw, Volberging, Coombs, 'fewer than 50% of patients with CD4+ counts greater than 200 cell/ul had positive plasma cultures, and inherent biologic variability in virus quantitation required that a 25-fold (approximately 1.4 log) increase was seen before it was likely to be clinically meaningful' (83). 2.HIV RNA. To the quantitation of HIV performed by measuring p24 in cultures, a test apparently introduced by David Ho (84), he and many others have added a test in which 'HIV RNA' in plasma is quantified. The three assays frequently used to quantify the 'viral load' are reverse transcription-polymerase chain reaction (RT-PCR), nucleic acid sequence-based amplification (NASBA) and branched chain DNA (bDNA). To assess the impact of the assays used and of 'genetic variability in HIV-1 RNA quantification', researchers from France 'evaluated three commercial kits by using a panel of HIV-1 isolates representing clades A to H…. These isolates were expanded in culture. Virus was collected by ultracentrifugation and resuspended in HIV-seronegative plasma. To standardize the quantities of virus to similar levels in each preparation, the p24 antigen was determined and the volume adjusted so that each specimen contained approximately 10pg of p24 antigen per ml'. The 'HIV-1 RNA copies' per ml of plasma obtained were as indicated in Table 1. HIV-1 STRAIN
These results prove that 'quantification of HIV-1 RNA is highly influenced' by the 'HIV-1 clade' and the test kit used. Indeed, given their data it is virtually impossible to make any sense at all of 'viral load' findings (85). There are two practical reasons for measuring plasma RNA levels: 1. The RNA levels and its changes are said to predict disease progression. However, in a paper published in 1997 by researchers from the Walter Reed Army Institute of Research and the Henry M. Jackson Foundation, the authors wrote: 'Whereas levels of cell-free viral RNA were shown in cross-sectional studies to vary over 1 to 2 logs with disease progression, four recent longitudinal studies have revealed a more complex view of viral RNA dynamics. Although all these reports have shown approximately 1 log higher levels of initial cell-free RNA from rapid versus slow progessors, in three of these studies cell-free RNA levels showed a < 1 log increase in the majority of rapid progressors. In contrast, Mellors and co-workers showed a > 1 log plasma RNA increase in three patients but a < 1 log RNA change in two of five patients studied intensively in their report'. In Michael and colleagues' study, there were 17 patients who were rapid progressors and 20 slow. They reported that 'The mean ± SD for the initial serum RNA (expressed in log10 copies/ml) in the rapid progressor (4.07 ± 0.53) exceeded that for the slow progressors (3.07 ± 1.25) [note the large SD in the latter group]. Dynamics of serum viral burden in rapid progressors reveal two distinct patterns. Serum viral burden changes of < 0.5 log were previously shown to be consistent with biological variation. This level of variance was used to sort the rapid progressors into two groups [contrary to the HIV theory of AIDS]. Seven rapid progressors show a <= 0.5 log change in viral burden (static group), and 10 showed a > 0.5 log increase in viral burden (increase group) over time' (one patient 0.6 log; 3 patients 0.7 log, 2 patients 0.8 log; 1 patient 0.9 log; 1 patient 1 log and 2 patients 1.4 log) (86). 2. To determine the effects of treatment. According to the 1997 British HIV Association guidelines for antiretroviral treatment of HIV seropositive individuals, 'If the viral load has not fallen by about 1 log 8–12 weeks after treatment initiation consideration should be given to modify therapy' (87). In their 1996 paper in Nature Medicine, Saag, Shaw, Coombs and their associates stated that 'A three-fold or greater sustained reduction (> 0.5 log) of the plasma HIV RNA levels is the minimal response indicative of an antiviral effect. return of HIV RNA levels to pretreatment values (or to within 0.3–0.5 log of the pretreatment value), confirmed by at least two measurements, is indicative of drug failure', and that 'Zidovudine monotherapy results in a median 0.7 log decrease in plasma HIV RNA level within two weeks, which returns toward baseline values by 24 weeks (19,20).' At least the claim regarding the effects of AZT on the RNA level is not substantiated by the presently available data, not even in the two studies which they are citing. Reference 19 in the above extract is a 1996 paper by William O'Brien and his associates from the Veteran Affairs Cooperative study group of AIDS. In this 'blinded study' the authors made a 'comparison of immediate with deferred zidovudine therapy… All the patients in the immediate-therapy group received open-label zidovudine for the entire study period, whereas those in the deferred-therapy group received placebo until their CD4+ lymphocyte counts fell below 200 cells per cubic millilitre or an AIDS-defining illness developed, when they were switched to open-label zidovudine'. In the immediate therapy group the maximum decrease, which is reached at two months, was about 0.6 log. By 12 weeks the RNA level returns to the baseline value, and at 24 months it is about 0.25 log above the baseline. In the deferred therapy group the RNA level never dropped below the baseline value88. Reference 20 is a 1996 paper by Coombs et al. In this study, 'In total, 913 subjects who had received at least 16 weeks of previous zidovudine therapy were enrolled in ACTG protocol 116B/117 and followed a mean of 48 weeks for disease progression defined as a new AIDS event or death. A subset of subjects for whom plasma samples were obtained for HIV-1 RNA quantitation were enrolled throughout the distribution of randomization dates for all subject participants enrolled. These subjects were followed in the study for a median of 304 days (range, 12–736). Plasma samples were available from 100 subjects at baseline; 71 of them had plasma samples at week 4, 72 at week 8, 66 at week 12, and 49 at week 24.' The only data given on changes of the RNA level with therapy are the following: 'The plasma HIV-1 RNA level declined by a median of 0.2 log10 during therapy for subjects who were switched to didanosine (figure 2A) but not for those who continued zidovudine (figure 2B)'. In figure 2B, where the effect of AZT treatment on the RNA level is shown, the level is always above the baseline (89). In a paper published in 1993 by Ann Collier and her associates, including Coombs and Fischl, the authors conducted 'a clinical trial to characterize the safety and efficiency of a range of doses using combination zidovudine and didanosine therapy compared with zidovudine therapy alone'. From 25 out of the 69 patients in their study they took sequential plasma samples at 0, 12 and 24 weeks and the plasma HIV-1 RNA was determined 'by a semiquantitative assay'. They reported that: 'Seventeen patients had a one log or more decrease in virion-associated HIV-1 RNA copy number during therapy, 7 had no change, and 1 had an increase. Nine patients had a decrease in virion RNA from pretreatment leve ls at both 3 and 6 months, 5 had a decrease between 3 and 6 months, and 3 had a decrease at 3 months that was not sustained at 6 months. Of 17 patients who had a decrease in plasma RNA titers, 15 were treated with a combination regimen. Overall, 15 (83%) of 18 patients receiving combination regimens had a decrease in plasma HIV-1 RNA titers compared with 2 (29%) of 7 patients receiving zidovudine alone' (90). In their well known 1993 study 'High Levels of HIV-1 in Plasma During All Stages of Infection Determined by Competitive PCR', Piatek, Saag, Shaw and their associates reported that 'Sixty-six consecutive enrolled HIV-1-infected subjects representing all stages of infection [Centers for Disease Control (CDC) Stages I to IV] and ten HIV-1 seronegative healthy donors were evaluated for virion-associated HIV-1 RNA by QC -PCR. Infected subjects were also tested for culturable virus and for p24 antigen with both standard and immune complex dissociation (ICD) test procedures'. They reported that the 'RNA copy numbers ranged from 1.00 x 102 to 2.18 x 107 HIV-1 RNA copies per millilitre of plasma. The average decline in HIV-1 RNA among the ten patients treated with AZT was 11-fold, whereas the average decline associated with resolution of the acute retroviral syndrome in six patients was 72-fold'. They also claimed to have found a correlation between plasma HIV-1 RNA and 'virus titers measured by endpoint dilution culture'. Given their finding that: 'Whereas the QC-PCR method quantified virion-associated HIV-1 RNA in all 66 patients tested, virus culture and standard p24 antigen assays were much less sensitive, with positive results in 4/20 and 5/20 subjects with CD4+ T-cell counts >500 per cubic millimeter, 6/18 and 7/18 subjects with CD4+ T-cell counts of 200 to 500 per cubic millimeter, and in 22/28 and 24/28 subjects with CD4+ cells fewer than 200 per cubic millimeter, respectively', it is difficult to see how such correlation can be determined (91). In a paper published in 1995, Joseph Eron and his associates for the North American HIV Working Party 'studied two doses of lamivudine in combination with zidovudine in patients with little or no prior antiretroviral therapy who had 200 to 500 CD4+ cells per cubic millimeter'. In this study, 'The greatest mean reductions in the plasma concentration of HIV-1 RNA were 0.52 ± 0.04 log in the zidovudine-only group, 1.19 ± 0.07 log in the lamivudine-only group, 1.56 ± 0.10 log in the low-dose combination-therapy group, and 1.55 ± 0.09 log in the high-dose combination-therapy group' (92). In a paper published in 1996, David Katzenstein and his associates in the AIDS Clinical Trials Group Study, 175 Virology Study Team, determined the relationship of virological and immunological factors to clinical progression. The virology subgroup comprised 391 subjects. Blood was collected on 'two occasions, at least 72 h apart, during the 14 days preceding treatment, to determine plasma HIV RNA concentrations; the geometric mean of these two measurements was defined as the baseline value. Plasma HIV RNA concentrations were measured at weeks 8, 20 and 56 provided that the subjects continued to receive the assigned treatment'. Eighty-nine subjects were treated with AZT only, 107 with didanosine only, 102 with AZT plus didanosine, and 93 with AZT plus zalcitabine. In this study, the 'mean baseline plasma HIV RNA concentration was 4.20 log (15,791 copies per milliliter), and the values ranged as high as 6.61 log. For 80 percent of the subjects, the difference in the log concentration between the two baseline measurements was less than 0.26 and for 90 percent it was less than 0.41'. No data are given for the other 10%. The presence of symptoms such as oral hairy leukoplakia, candidiasis or herpes zoster was significantly associated with increased HIV RNA concentration. 'Homosexuality was associated with a significantly higher plasma concentration of HIV RNA (p = 0.002), and intravenous drug use with a significantly lower concentration (p = 0.003). Women had significantly lower plasma HIV RNA concentrations (p < 0.001), as did black subjects (p = 0.013). . Antiretroviral treatment before entry into the study was associated with lower CD4 cell counts and a higher rate of the presence of syncytium-inducing phenotype, but not with differences in plasma HIV RNA concentrations. Measurements made eight weeks after the start of treatment revealed significant differences in the response of plasma HIV RNA concentrations to antiretroviral therapy among the treatment groups. There was a mean decrease of 0.26 ± 0.06 log (45%) in the HIV-RNA concentration in 65 subjects who received zidovudine alone, a decrease of 0.65 ± 0.07 (78%) in 87 subjects who received didanosine alone, a decrease of 0.93 ± 0.10 (88%) in 81 subjects who received zidovudine plus didanos ine, and a decrease of 0.89 ± 0.06 (87%) in 76 subjects who received zidovudine plus zalcitabine. Subjects without a history of antiretroviral treatment who took zidovudine alone had a mean reduction at week 8 of 0.47 log; subjects with that history had a mean reduction of 0.02'. During the follow-up, 48 (12%) of the 391 subjects 'were given a diagnosis of AIDS or died; and 28 (7%) died. A decrease of 1.0 log in the concentration of HIV RNA from baseline to week 8 was associated with a significant lowering to 0.35 in the hazard ratio for AIDS or death (i.e., 65% reduction in the risk of AIDS or death). There was a 90% reduction in the risk of progression of disease associated with a reduction of 1.0 log in the plasma HIV RNA concentration between baseline and week 56'. (How was it possible to determine such relationships when only a small percentage of patients developed AIDS or died, and even a smaller proportion if any of these patients had a decrease of 1.0 log at week 8 and nobody at week 56?) Discussing their finding, the authors wrote: 'The presence of lower baseline plasma HIV RNA concentrations among women and among intravenous drug users is an interesting but unexplained observation. However, risk factors for HIV infection, sex, ethnic group, and a history of previous antiretroviral treatment were not independently associated with differences in clinical outcome. Neither are the clinical results of ACTG 175 fully explained by the overall comparison of the changes in HIV RNA concentrations in the different treatment regimens. Therapy with didanosine alone led to clinical results comparable to those with the combination of zidovudine and didanosine, although patients treated with the latter regimen had a clearly larger mean decrease in plasma HIV RNA concentrations. The reduction in plasma HIV RNA concentrations after treatment with zidovudine plus zalcitabine was similar to that after zidovudine plus didanosine, yet the latter regimen was more effective in the subjects with a history of antiretroviral therapy, and similar results have been observed in a recently reported study of combination therapies in subjects with more advanced disease, but without a history of antiretroviral therapy… These differences point to the importance of other factors in the treatment of HIV infection (93). In a study published in the same year (1996), researchers from Spain and Belgium conducted a 6-month follow-up study in 46 patients previously treated for at least 6 months with AZT plus zalcitabine (ddC) who were subsequently allocated to receive either ZDV/ddC/3TC (15 patients), ZDV/3TC (15 patients), or to continue with the ZDV/ddC regimen (16 patients). 'Maximum mean decrease in VL [plasma HIV-1 RNA] was achieved at week 4 in the ZDV/ddC/3TC (–0.64 log) and ZDV/3TC (–0.72 log) groups. At week 12 and 24 the decrease in the ZDV/ddC/3TC group was 0.41 log and 0.45 log, respectively. The corresponding values for the ZDV/3TC group were 0.16 log and 0.15 log. In the ZDV/ddC group there was a continuous increase in the plasma HIV-1 RNA level, and at week 24 was 0.36 log above the baseline level (94). In 1996 there was also a paper by Ann Collier and her associates for the AIDS Clinical Trials Group. Because, in patients treated with RT inhibitors, 'the disease eventually progresses to the acquired immunodeficiency syndrome (AIDS) despite the use of these agents', the authors 'studied the safety and efficacy of saquinavir, an HIV-protease inhibitor, given with one or two nucleoside antiretroviral agents, as compared with the safety and efficacy of a combination of two nucleosides alone'. The patients were given either saquinavir plus AZT and zalcitabine or AZT plus either saquinavir or zalcitabine. The study lasted 24 weeks, with an option of an additional 12 to 32 weeks. The plasma HIV-1 RNA was quantified by using two methods, branched chain DNA and the quantitative polymerase-chain-reaction amplification by the RT method. The mean plasma RNA levels were given in separate graphs for the two methods. The average decrease in patients treated with saquinavir plus AZT was 0.1 log; for the zalcitabine plus AZT group, 0.39 log; and for the group which received the three-drug combination, 0.68 log. In addition to the plasma RNA, Collier and her colleagues also determined the quantity of 'HIV in PBMCs'. 'In the quantitative analysis of HIV in PBMCs, the titer of infectious units per million cells was calculated for each sample28'. In reference 28 they cite Susan Fiscus et al.95 who used a method, 'Quantitative cell microculture assay (QMC)', where the quantity of HIV in a co-culture is determined by measuring p24. 'Six serial dilutions of each subject's PBMC, starting at a concentration of 106 PBMC, were cocultured in duplicate with 106 HIV-seronegative donor PBMC that had been prestim ulated with PHA for 1–3 days according to standard procedures. A culture was scored as positive if > 30 pg/mL HIV-1 p24 antigen (Abbott, Abbott Park, IL) was present in the supernatant.Several dilution schemes, including 2-fold, 5-fold, and 10-fold serial dilutions of the subjects' PBMC, were tested as part of the early development of the QMC method for the quantitation of virus load. However, since all dilution schemes used 106 PBMC/ well as one of the serial dilutions tested, an algorithm was calculated to express the results as infectious units per million cells (IUPM). The median change in log10 IUPM (hereafter called log IUPM) from study entry with treatment over time was determined'. They reported: 'At baseline, 107 (98%) of the 109 evaluatable subjects had cultivatable HIV-1 from at least 1 PBMC specimen. For 94 subjects, 2 independent baseline blood specimens, drawn a median of 10 days apart, were available for PBMC HIV-1 culture. Duplicate specimens were both positive in 78 (83%), discordant in 15 (16%), and negative in 1 (1%) case. For the baseline comparison (ignoring the effect of censoring i.e. failing to reach a dilution end point), 56 (60%) of 94 duplicate specimens differed in HIV-1 titer by <= 1 log IUPM; 20 (21%) of 94 differed by >1 but <2 log IUPM; and 18 (19%) of 94 differed by >=2 log IUPM. In an analysis that accounted for the effect of censoring, the within-person SD with paired baseline PBMC culture data was 0.72 log IUPM '. Using Fiscus et al.'s methods, Collier and her colleagues found in their patients that 'The mean titer of HIV in PBMCs decreased by 0.8 log in the three-drug group, as compared with no change in the saquinavir-zidovudine group and a change of less than 0.4 log in the zalcitabine-zidovudine group. Zalcitabine and zidovudine lowered titers more than did saquinavir and zidovudine (P=0.004). The patients assigned to three-drug therapy had titers that remained below baseline longer than those of the patients assigned to saquinavir and zidovudine, although over time, even in the three-drug group, there was a gradual return toward the baseline titer'. Regarding the clinical outcome, they reported that 'No statistically significant differences were found among the three regimens with respect to any clinical or laboratory measure during either the first 24 weeks or the overall study. One of the interesting observations was that the suppressive effect of the three-drug combination on viral load, as measured by quantitative microculture of PBMCs, HIV RNA titers, and effects on serum activation markers, appeared to be more durable than the elevation of CD4+ counts. That the antiviral response was sustained longer than the CD4+ cell response raises intriguing questions about the association between quantitative measures of HIV, immune activation, and CD4+ cell counts. Nonetheless, these results suggest that the combination of saquinavir, zalcitabine, and zidovudine should be further investigated in long-term studies' (96).

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