GMHC Treatment Issues; Vol 9, Number 9 - September 1995
Henry E. Chang

Many of the researchers hope that combination therapy will increase the time for resistant virus to emerge. In his welcoming remarks, Stefano Vella, M.D., of the Institute Superiore di Sanitê in Rome predicted that this may be "the year of constraints," meaning that HIV mutant strains may not be able to develop resistance to certain drug combinations while remaining functional and infectious.

Dual Resistance to AZT and 3TC

Therapy with the AZT/3TC combination has been reported to cause a substantial and sustained decrease in viral load, and investigators have thought that it might be impossible for HIV to be simultaneously resistant to both drugs (see Treatment Issues, December, 1994, pages 2-4 and February, 1995, pages 3-4). But laboratory and clinical findings presented in Sardinia by researchers from various groups have shown that selection of dual-resistant virus is indeed possible.

One such study, from the Medical Research Council Collaborative Center and the Glaxo-Wellcome Medicines Research Center in Great Britain was described at the Sardinia conference by Matthew Goulden, M.D. (abstract 50). That study detected HIV resistant to both AZT and 3TC in cell culture experiments that exposed an AZT-resistant clinical HIV isolate repeatedly to a constant dose of AZT and increasing doses of 3TC. The large amount of AZT required to inhibit this strain of HIV remained unchanged, while the amount of 3TC required increased by more than 1,000-fold. This change indicated the emergence of dual-resistant HIV that could replicate in the presence of both AZT and 3TC.

In an effort to determine the genetic basis of the dual resistance, Sharon Kemp, Ph.D., and colleagues at Wellcome Research Laboratories in Britain developed a test to rapidly detect HIV-derived drug resistance patterns (abstract 51). Using the mutant virus provided by Dr. Goulden, the researchers found a total of nine amino acid changes on the viral reverse transcriptase (RT) enzyme that could be responsible for the observed AZT/3TC resistance. Surprisingly, genetic changes responsible for the alteration in reverse transcriptase include ones located further downstream from where most of the "classical" mutations are found in HIV's RT gene.

A clinical study from the Antiviral Therapy Laboratory of the University of Amsterdam found further that dual-resistant virus could be detected in people one year after the addition of AZT to the regimen of symptomatic HIV-infected individuals undergoing 3TC monotherapy (abstract 52). This acquisition of AZT/3TC dual-resistant virus may in part account for the rebound of viral load back to pretreatment levels in those treated first with 3TC monotherapy and then AZT/3TC combination therapy.

The researchers cautioned that even though AZT/3TC dual resistance can occur and cause a rebound in viral load, this is not a reason to abandon AZT/3TC combination therapy. The development of resistance to one or both of the drugs does not always mean that the combination will lose its antiviral effect. And until the development of the dual-resistant virus, the combination of AZT and 3TC generally does continue to have anti-HIV activity.

These conclusions were supported by two presentations. One described the early results of an European Phase II/III trial of AZT/3TC combination therapy in AZT-experienced individuals with CD4 cell counts between 100 and 400 (abstract 54). The other concerned a North American comparative trial of high or low dose 3TC plus AZT versus AZT plus ddC in AZT-experienced individuals with CD4 cell counts between 100 and 300 (abstract 55). Both studies found that the combination therapy still produces moderate suppression of viral load after 24 weeks of therapy despite the rapid emergence of HIV with mutations conferring resistance to 3TC while the resistance mutations to AZT persisted.

Viral Resistance to Protease Inhibitors

Recent laboratory findings on viral resistance patterns reported at the Sardinia workshop provide some impetus for the design of clinical studies to evaluate "rational" combinations of protease inhibitors.

Saquinavir (Invirase): New clinical data suggest that therapy with the experimental protease inhibitor saquinavir (brand name: Invirase), developed by Hoffmann La-Roche does not cause new forms of viruses that are cross-resistant to other protease inhibitors in clinical development. Other data suggest that the combination of saquinavir and AZT or just high dose saquinavir delay the development of viral resistance to either drug.

Examination of Phase I/II trial data by Helmut Jacobsen, Ph.D., a senior scientist and group leader at Roche in Basel, found that about 50 percent of patients developed moderate resistance following one year of saquinavir as a monotherapy or in combination with AZT or AZT plus ddC (abstract 68). Genetic analysis of over 1,500 individual viral samples from these patients could discover no HIV that after exposure to saquinavir was cross-resistant to other protease inhibitors. Laboratory studies presented at the workshop by Sarah Wilson of the University of Wales also concluded that saquinavir- resistant viruses do not shrug off the antiviral effects of other unrelated protease inhibitors (abstract 66).

Researchers at Stanford University, led by Thomas Merigan, M.D., are completing a high dose saquinavir monotherapy study (abstract 73) in which HIV-positive volunteers are receiving 3,600 and 7,200 mg of saquinavir daily, two- and four-times previous doses of the drug. Jonathan Schapiro, M.D., of Stanford presented clinical data in Sardinia showing immediate and long-term viral load suppression with sustained CD4 cell increases through one year. This dose-dependent enhancement of viral load suppression correlates with a reduction in the frequency of mutations. A mixture of drug- sensitive virus and drug-resistant virus is present in patients receiving the higher doses of saquinavir even after one year of therapy.

There is, however, a lack of correlation between the appearance of drug-resistant mutations and loss of antiviral effect, which appears sooner than the mutations. The Stanford group is still looking into the causes of this phenomenon but thinks that the rebound in viral activity is related to a lack of drug reaching HIV-infected cells somewhere in the body. The researchers are convinced, based on the comparative performances of the two doses in their trial, that genetic mutations are at the root of HIV drug resistance and that higher doses retard the emergence of those mutations. They plan to release more detail and the latest data at September's Interscience Conference on Chemotherapy and Antimicrobial Agents.

VX-478: Data from cell culture experiments on resistance patterns with different protease inhibitors may provide clues for selecting inhibitors that are suitable for combination therapy. Margaret Tisdale and colleagues at Wellcome Research Labs examined the resistance patterns with five different protease inhibitors and their potential implications for combination therapy (abstract 61).

The researchers reported that some viral mutants showed cross-resistance to all the protease inhibitors examined, a finding somewhat in contradiction to the saquinavir studies noted above. Other mutant virus had increased susceptibility to certain inhibitors. For example, some HIV strains resistant to VX-478 (the Glaxo-Wellcome-Vertex protease inhibitor) were two- to five-fold more sensitive to saquinavir and to Merck's indinavir (brand name: Crixivan).

To further understand the interactions of protease inhibitors, the researchers employed another HIV strain carrying three point mutations that confer resistance to VX- 478. They subjected the virus to increasing concentrations of saquinavir. This treatment unexpectedly led to the selection of a virus that is highly resistant (50-fold decrease in susceptibility) to saquinavir but fully sensitive to VX-478.

In contrast, the same VX-478 resistant virus exposed to increasing concentrations of indinavir became resistant (ten- to twenty-fold) to indinavir while maintaining resistance to VX-478.

Indinavir (Crixivan): John Mellors, M.D., of the University of Pittsburgh Medical Center and colleagues at Merck Research Laboratories analyzed the waning effect of indinavir in HIV- infected individuals who have received either 200 mg or 400 mg indinavir four times daily over 24 weeks (abstract 71).

Thirty-three of 40 patients experienced a rebound in virus levels to pretreatment levels 24 weeks after initiating indinavir monotherapy. Increasing the dose of indinavir to 600 mg four times daily did not prevent this rebound.

It was later found that 30 of the 40 treated patients had developed resistance to indinavir by changing two particular amino acids on the protease enzyme.

The appearance of these mutations coincided with a rise in viral load toward pretreatment levels. In addition, alterations were detected in at least seventeen other amino acids in the protease enzyme. The findings provide direct evidence that the waning effect of indinavir monotherapy in patients is associated with the specific resistance patterns (specific changes in the protease's amino acid sequence).

Ritonavir: Svan Danner, M.D., and colleagues of the European- Australian Collaborative Study Group presented data on the safety and short-term antiviral activity of ritonavir (or ABT-538), an experimental protease inhibitor developed by Abbott Laboratories, in HIV-infected people (abstract 75). Eighty-four volunteers with CD4 cell counts above 50 were enrolled and randomized to receive one of four regimens of ritonavir or placebo. Trial participants on placebo were reassigned to a ritonavir regimen after four weeks.

Investigators noted a median increase of 230 in CD4 cell count after 32 weeks of treatment. At this timepoint, HIV levels in the blood were still 0.81 log (84.5 percent) below baseline in those individuals receiving the highest dose regimen (600 mg twice daily).

The researchers reported that ritonavir was generally well tolerated. However, some side effects were associated with ritonavir treatment, specifically, nausea, burning or tingling sensations in the tongue and mouth, and elevated blood levels of liver enzymes (indicating some liver impairment) or triglycerides (a sign of altered fat metabolism).

Daniel Norbeck and colleagues at Abbott Laboratories reported on a second trial (abstract 70) of individuals receiving different doses of ritonavir (300, 400, 500, or 600 mg twice daily). All trial participants experienced mean reductions in HIV plasma RNA of at least one log (90 percent) and CD4 cell count increases of at least 75 during the first two weeks of treatment. The immunologic response and reduction in viral load was greater and more sustained in those on the higher doses.

Failure to maintain at least an 80 percent decrease from pretreatment HIV levels occurred at approximately five, seven, eleven and nineteen weeks in the trial participants receiving doses of 300, 400, 500 or 600 mg of ritonavir, respectively. Genetic analyses of the HIV sequentially isolated from patients with waning responses to treatment revealed ritonavir-resistant mutations.

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