The clinical significance of surrogate markers
CD4 counts and HIV RNA levels do predict rate of disease progression
People in the HIV/AIDS community have long debated the merits of using surrogate markers like absolute CD4 counts and HIV RNA levels to assess disease stage and/or gauge the effects of treatment. The data from two recent studies may help resolve this issue -- and provide practitioners with a clearer picture of the clinical utility of these widely used laboratory tests.
Phillips et al. examined the impact of nucleoside analog therapy on CD4 counts and HIV RNA levels in peripheral blood, in an attempt to correlate those two markers with disease progression. These investigators evaluated the clinical data from two studies that used ZDV and 3TC in combination. A total of 620 patients participated in those studies, and all of the subjects were followed for at least one year. CD4 and HIV RNA levels were assessed every four weeks throughout the study period.
The authors found that desirable changes in these two surrogate markers -- increases in CD4 count, decreases in HIV RNA level -- correlated with a diminished relative hazard for progression to a diagnosis of AIDS or a new C.D.C. stage of AIDS. These findings led Phillips et al. to conclude that therapy-induced reductions in viral burden contribute substantially to the clinical improvement seen in patients treated with nucleoside analogs. Changes in CD4 counts also correlated with changes in clinical condition, but the association was not as strong.
Phillips and coworkers also found a correlation between clinical status and surrogate markers in peripheral blood -- a finding which reinforces the theory that HIV activity in peripheral blood is a reflection of what is occurring in the body as a whole.
This additional evidence that HIV RNA levels are a good gauge of clinical status and disease progression makes it all the more important to know how accurate such assays are. Practitioners will therefore be interested in a recently published analysis of HIV quantitation assays in individuals with relatively high CD4 counts. Paxton et al. evaluated 45 subjects, all participants in a vaccine trial, in an attempt to ascertain the degree of variability among several HIV RNA assays that are currently in clinical use. Two groups were examined: 30 patients with CD4 counts above 600 cells/mm3 who were not receiving antiretroviral therapy, and 15 patients with CD4 counts between 400 and 550 cells/mm3 who were on ZDV monotherapy. The participants were followed for more than one year, during which time their viral burden was assessed at regular intervals by three assay methods: proviral DNA, viral RNA, and cell-associated infectious virus.
All of these means of measuring HIV RNA correlated well with one another in individual patients. However, assay that measure plasma viral RNA levels were judged to be the most consistent and reliable. The authors conclude that this method of quantifying viral load is the best of the currently available clinical tools for tracking an individual patient's response to antiretroviral treatment.
Phillips AN, Eron J, Bartlett J, Kuritzkes DR, Johnson VA, Gilbert C et al. Correspondence between the effect of zidovudine plus lamivudine on plasma HIV level/CD4 lymphocyte count and the incidence of clinical disease in infected individuals.AIDS 1997;11:169-75.
Paxton WB, Coombs RW, McElrath MJ, Keefer MC, Hughes J, Sinangil F et al. Longitudinal analysis of quantitative virologic measures in human immunodeficiency virus-infected subjects with >400 CD4 lymphocytes: implications for applying measurements to individual patients.J Infect Dis 1997;175:247-54.
Dr. Paul A. Volberding, editor-in-chief of HIV Newsline, comments:
It was CD4 counts which first identified and characterized what came to be called the acquired immunodeficiency syndrome, and since that time clinicians have been using CD4 lymphocyte depletion as an indirect means of diagnosing AIDS and HIV infection, tracking disease progression, and gauging response to therapy. Since early last year, when HIV RNA assays became commercially available, practitioners who treat patients with HIV disease have been able to measure viral load -- and response to therapy -- directly, using this valuable new clinical tool (see "The HIV RNA Assay," Vol. 2, No. 2, pages 27-30; and "Guidelines for the Clinical Use of the HIV RNA Assay," the PULL OUT AND SAVE feature in that same issue).
The work of Phillips et al. confirms the clinical utility of both the CD4 count and the HIV RNA assay in staging patients and assessing their response to antiretroviral therapy. The work of Paxton et al. suggests that assays that measure plasma RNA levels offer clinicians the most reliable means of determining the level of viral activity in a particular patient. As such, they provide practitioners with the best method of measuring how rapidly and how completely a given patient is responding to a given therapeutic regimen.
In treating MAC, less may be more
Adding clofazimine to clarithromycin and ethambutol does not improve symptoms or survival
Last year we reported the preliminary results from a clinical trial that randomized AIDS patients with MAC to one of two multidrug regimens: clarithromycin (500 mg b.i.d.) plus ethambutol (15 mg/kg/d) with or without clofazimine at a dose of 100 mg per day (see "Fine-tuning the medical management of MAC," Vol. 2, No. 2, pages 34-35). Those results have now been peer-reviewed and published in the journal AIDS, with clarifications and additional observations. The investigators find no advantage to the three-drug regimen for any of the following parameters: development of negative blood cultures for mycobacterial organisms, time to relapse of bacteremia, improvement of constitutional symptoms (such as fevers and night sweats), and adverse reactions to medications.
We previously noted that a significantly higher percentage of patients randomized to the three-drug MAC regimen dropped out of the study due to adverse reactions, but the published report amends this observation: 11% of those on two drugs and 14% of those receiving clofazimine left the study because of adverse reactions attributed to the medications, a difference that was not statistically significant.
More patients randomized to the clofazimine arm died during the study (Figure). Further analysis of the results reveals that patients randomized to three drugs had higher mycobacterial colony counts than patients in the two-drug arm. The median number of colony forming units (CFU) was 152/mL blood in patients in the two-drug cohort versus 1907/mL blood in those who also got clofazimine.
The investigators assert that this imbalance was due to chance. The higher mortality rate noted among clofazimine recipients (61% vs. 38%) may have been due to their higher baseline CFU levels rather than to the toxicity of clofazimine. While other investigators have found an association between mortality and mycobacterial load in AIDS patients, the authors suggest that the issue is unresolved. In any case the increased mortality in the three-drug arm is another argument against including clofazimine in an anti-MAC regimen.
Chaisson RE, Keiser P, Pierce M, Fessel WJ, Ruskin J, Lahart C et al. Clarithromycin and ethambutol with or without clofazimine for the treatment of bacteremic Mycobacterium avium complex disease in patients with HIV infection.AIDS 1997;11:311-317.
Dr. David Hardy, a member of the editorial advisory board of HIV Newsline and the author of "Recent Advances in Prophylaxis for MAC" (Vol. 1, No. 3, pages 52-57), comments:
One particularly noteworthy finding of this study is that only one patient in the trial developed MAC strains that were resistant to clarithromycin. Other investigators have reported substantially higher levels of clarithromycin resistance to MAC in patients who received this drug as prophylactic monotherapy. (In a small pilot study, the incidence of resistance, when clarithromycin was used as sole prophylaxis against recurrent MAC, was 40% among patients with breakthrough infections.) In the study by Chaisson et al., participants received a combination of drugs from the start. The authors speculate that the addition of ethambutol to the regimen may have prevented the emergence of clarithromycin-resistant strains of MAC.
Is resistance always a bad thing?
Repeated mutation may eventually render HIV ineffective
Recent in vitro studies involving two experimental protease inhibitors shed new light on the phenomenon of viral resistance to these drugs. Investigators grew laboratory and clinical strains of HIV in the presence of these potent new protease inhibitors in order to select out strains that were resistant to the two investigational drugs. These resistant strains were subsequently analyzed in several ways to assess their viability.
Croteau and coworkers used protease enzymes extracted from the mutant HIV strains they had developed to create HIV clones that were highly resistant to these new protease inhibitors. The catalytic efficiency of these mutant viral strains was found to be significantly lower than that of unaltered versions of the HIV protease enzyme. This may indicate that HIV does not always adapt to selective pressures as successfully as conventional wisdom has held that it does.
As importantly, these mutant strains of HIV appeared to be less virulent than wild-type virus. In the test tube, viral strains with a single mutation did not manifest impaired growth properties, but strains that exhibited between four and seven mutations at the active protease enzyme site propagated at a lower rate. So substantial was this growth differential that when the highly mutant clones were mixed with wild-type viral strains in cell cultures, the mutants completely disappeared.
These findings lend support to a report, published by Kaplan et al. in 1993, which suggested that HIV might actually lose its ability to infect CD4 cells if it mutated often enough in response to antiretroviral assault. Kaplan and coworkers demonstrated that subtle defects in the protease gene -- defects that resulted from incubating the virus with protease inhibitors at the concentrations achieved in treated patients -- dramatically altered the ability of these clones to reproduce in host cells. This team noted that the altered morphology they observed in these mutant viral strains was associated with reduced infectivity.
Croteau G, Doyon L, Thibeault D, McKercher G, Pilote L, Lamarre D. Impaired fitness of human immunodeficiency virus type 1 variants with high-level resistance to protease inhibitors. J Virol 1997; 71 (2): 1089-96.
Kaplan AH, Zack JA, Knigge M, Paul D, Kempf DJ, Norbeck DW, Swanstrom R. Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles. J Virol 1993; 67 (7): 4050-5.
Dr. James O. Kahn, one of the authors of "The Problem of Protease Resistance," comments:
The findings of Croteau et al. suggest that protease inhibitors may retard viral growth in an unexpected way. We know that this class of drugs interferes with viral reproduction at the last stage of the virion's life cycle -- by disabling a gene that is critical to viral reproduction. What the in vitro data collected by these investigators indicate is that protease inhibitors may also provoke the emergence of mutant versions of the virus that are "unfit" for survival. This phenomenon would explain the continuing clinical stability of patients even after they develop drug-resistant strains of HIV -- and it would be an argument for continuing patients on therapy after resistance does develop.
Enhancing saquinavir levels with ritonavir
Two-drug combination overcomes chief limitation of saquinavir, its low bioavailability
The protease inhibitor saquinavir has the lowest bioavailability of the four F.D.A.-approved protease inhibitors, and research is currently underway to correct this limitation. In the meantime, there is mounting evidence that combining saquinavir with another approved protease inhibitor, ritonavir, may partially ameliorate this problem.
Saquinavir and ritonavir are both metabolized in the liver, mostly by enzymes that make up the cytochrome p450 system. Since the level of cytochrome p450 enzymes in the liver is limited, it is possible to saturate all of the active sites on those enzymes. The theory behind saquinavir-ritonavir combination therapy is that taking these two protease inhibitors concurrently will saturate enough active sites to increase the amount of time it takes the body to eliminate the drugs -- and this, in turn, will keep more active, unmetabolized drug in the bloodstream.
This is apparently what happens, at least in the test tube. Kempf et al. exposed human live cells to combinations of ritonavir and the following drugs: saquinavir, indinavir, and nelfinavir. The investigators noted that the metabolism of each drug was decreased in the presence of ritonavir, resulting in higher concentrations of the second drug over a longer period of time. Interestingly enough, the level of ritonavir did not change when it was administered in these combination regimens.
This study also included a clinical component: eight volunteers were given a single dose of saquinavir (600 mg) with or without ritonavir (200 mg). In two subjects who were deemed "typical", the peak concentration of saquinavir rose from .1g/mL to .3g/mL when the drug was coadministered with ritonavir -- a substantial improvement in peak levels. Furthermore, circulating levels of saquinavir were detected 40 hours after the two-drug combination was administered -- a substantial improvement in the drug's half life. One implication of these observations is that the bioavailability of protease inhibitors is limited by metabolism rather than absorption from the gut.
Kempf DJ, Marsh KC, Kumar G, Rodrigues D, Denissen JF, McDonald E, et al. Pharmacokinetic enhancement of inhibitors of the human immunodeficiency virus protease by coadministration with ritonavir. Antimicrob Agents and Chemother 1997; 41 (3): 654-60.
Dr. Harold A. Kessler, the author of "The Next Generation of Antiretroviral Agents" and a member of the editorial advisory board of HIV Newsline, comments:
The work of Kempf et al. suggests several ways in which we may be able to enhance the clinical utility of the protease inhibitors that are currently available to us. First, the data collected by these researchers reinforce the conviction -- widely held among experts in the field, but supported by very little clinical evidence -- that certain protease inhibitors, taken concurrently, have synergistic activity against HIV. Second, the evidence that saturation of the active sites on cytochrome p450 enzymes leads to higher plasma concentrations of active drug has important clinical implications. We know that drug-resistant strains of HIV develop when plasma levels of those drugs fall below therapeutic levels. If combination therapy leads of higher concentrations and long half-life, this could thwart the emergence of resistant strains.
It is also possible that combining protease inhibitors might permit less frequent dosing, which would doubtless enhance compliance. The authors observe that ritonavir and saquinavir make a particularly attractive combination because of the limited cross- resistance seen between these drugs.
New macrolide inhibits late-stage HIV replication
First compound in its class shows strong in vitro antiretroviral activity
Preliminary data suggest that disulfide-containing macrolides might nicely complement currently available antiretrovirals agents. The first of these compounds, SRR-SB3, is distinct from other antiretroviral agents in that it does not specifically inhibit reverse transcriptase, protease, or any other viral enzyme. Furthermore, it has no effects on syncytia formation and does not block the binding of HIV to target cells. Instead, SRR-SB3 forms a complex that has a chemical binding motif known as a "zinc finger." Zinc fingers are a biochemical gripping mechanism that chemicals use to attach themselves to chosen molecules.
HIV uses these finger-like projections to bind zinc and to accomplish certain other biological activities. SRR-SB3 dislodges zinc from the pocket in which the virus normally holds it, thereby interfering with the assembly and disassembly of HIV's nucleocapsid proteins.
It is believed that the observed antiretroviral activity of SRR-SB3 is a function of the macrolide's ability to hamper late-stage viral replication, a theory supported by the fact that the drug does not inhibit the level of proviral DNA in cell lines exposed to it. Inhibition of proviral DNA would imply antiretroviral action at an earlier stage of HIV infection.
This purported mechanism of action is consistent with laboratory findings that this drug thwarts the virus in chronically infected cells (whereas most antiretroviral agents are effective only in acutely infected cells). Another quality that makes SRR-SB3 an attractive adjuvant therapy is that it demonstrates activity against strains of HIV that are resistant to nucleoside analogs, protease inhibitors, and/or non-nucleoside reverse transcriptase inhibitors.
The authors speculate that SRR-SB3 itself may be immune to the development of viral resistance, since the zinc finger motif it targets is highly conserved among viral strains. There was also some suggestion that this macrolide may have additive and/or synergistic effects when combined with nucleoside analogs.
Although SRR-SB3 did cause some cell toxicity at high doses, the authors point out that studies in mice did not reveal adverse effects. They also postulate that the drug does not have a global effect on zinc finger binding in normal cells, only in those that harbor HIV. Look for this drug and others in its class to undergo further development.
Witvrouw, M, Balzarini J, Pannecouque C, Jhaumeerl-Laulloo S, Este JA, Schols D et al. SRR-SB3, a disulfide-containing macrolide that inhibits a late stage of the replicative cycle of human immunodeficiency virus. Antimicrob Agents Chemother 1997; 41 (2): 262-8.
A novel NNRTI with activity against resistant strains
Another entrant in a rapidly expanding field
The class of antiretroviral agents known as non-nucleoside reverse transcriptase inhibitors is extremely active against the target enzyme but is vulnerable to the rapid development of resistance. Two NNRTIs have been approved this year: nevirapine and delavirdine. If resistance to these drugs becomes clinically significant -- a development that seems likely, given our clinical experience with these drugs and with antiretroviral agents in general -- alternatives will be needed. The potential candidates include four novel NNRTIs, now in the early stages of development, that show considerable activity against strains of HIV that are resistant to nevirapine and delavirdine.
Oxathiin carboxanilide derivatives have been identified as powerful antiretroviral agents. They target reverse transcriptase, but use a different mechanism of inhibition than the nucleoside analogs. As might be expected of RT inhibitors, they are active in cells that are acutely infected. The specificity of oxathiin carboxanilide derivatives for HIV-1 is reflected in their inability to inhibit HIV-2 or simian immunodeficiency virus. The four candidate drugs are referred to as UC10, UC40, UC781 and UC82.
The hallmark of these drugs is their potent activity against HIV strains that have developed resistance to other NNRTIs. This characteristic may portend a role for these drugs in combination with nevirapine and delavirdine, since these second-generation NNRTIs may prove capable of inhibiting the emergence of resistant strains.
These investigational drugs also demonstrate considerable versatility in terms of the cellular environments in which they can function. Their antiviral effects were duplicated in B lymphocytes, monocytes, and macrophages as well as T lymphocytes. Importantly, these UC derivatives are active against HIV found in the peripheral blood of HIV-infected persons, meaning that clinical as well as laboratory strains of HIV are susceptible to these new drugs. As further evidence of their potential clinical utility in combination antiretroviral regimens, these UC derivatives demonstrated significant synergism when mixed with ZDV.
Buckheit RW, Snow MF, Fliakas-Boltz v, Kinjerski TL, Russell JD, Pallansch LA et a. Highly potent oxathiin carboxanilide derivatives with efficacy against nonnucleoside reverse transcriptase inhibitor-resistant human immunodeficiency virus isolates. Antimicrob Agents Chemother 1997; 41 (4): 831-7.
Dr. Harold A. Kessler, the author of "The Next Generation of Antiretroviral Agents" and a member of the editorial advisory board of HIV Newsline, comments:
One of these candidate drugs has emerged as a particularly promising antiretroviral agent. UC781 had high activity against HIV in vitro and has been shown to completely inhibit viral isolates that have become resistant to other reverse transcriptase inhibitors, except when the mutant strains had developed multiple amino acid substitutions. These effects were seen at drug concentrations that were non-cytotoxic. In fact, Buckheit et al. did not observe any toxic effects to cells in their in vitro studies, a finding consistent with the drug's extremely specific attraction for the RT enzyme.
In addition, UC781 has a wide therapeutic index, which means that the effective therapeutic dose is much lower than the observed toxic dose. For obvious reasons, clinicians feel more comfortable prescribing new drugs to their patients when the therapeutic index of those drugs is wide, knowing that there is a reduced likelihood of adverse events.
Some level of resistance to UC derivatives was seen by these investigators. Interestingly enough, these resistant strains proved susceptible to other NNRTIs, suggesting a potentially reciprocal therapeutic combination.
Approval sought for new formulation of saquinavir
Soft gelatin capsule has up to nine times the bioavailability of old formulation
Hoffmann-LaRoche, maker of saquinavir (Invirase®), has applied to the F.D.A. for permission to market a new formulation of their protease inhibitor. This version, which comes in a soft gelatin capsule, has eight or nine times the bioavailability of the old formulation when given at the doses used in clinical trials. Low bioavailability has been the chief limitation of saquinavir, which has an acceptable side effects profile even at doses substantially higher than the standard therapeutic dose. Ongoing clinical trials are testing this new formulation of saquinavir in combination with nucleoside analogs, NNRTIs, and other protease inhibitors (see "Enhancing saquinavir levels with ritonavir").