Rationalizing Salvage Therapy
Drug-resistant HIV strains are a major cause of treatment failure in the management of HIV infection. These resistant viral strains can evolve whenever the virus is not fully suppressed by a particular drug treatment regimen. Long-term therapy with suboptimal drug regimens, poor drug absorption, or noncompliance with existing regimens can lead to the accumulation of viral gene mutations associated with resistance to particular drugs and/or drug combinations. Work underway at Stanford University Medical Center's Center for AIDS Research aims to design salvage therapies for patients harboring resistant HIV. Determining the specific genetic and drug susceptibility profiles of the patients' HIV is key to this effort.
Sequence Analysis and Database Entry
The process for developing new salvage therapies unfolds in four principal steps. The first involves sequencing the viral strains from heavily treated patients who are failing their current regimen and entails mapping out the entire protease gene and the reverse transcriptase gene's first 300 codons or "positions" (each one encoding a single amino acid on the RT enzyme). Establishing the genetic mutations related to drug resistance is known as genotypic analysis and indicates a given HIV isolate's "genotypic resistance."
In the second step, the sequences of these two genes are analyzed for resistance mutations and entered into a computer database. At present, this database contains nearly 5,000 sequences from more than 1,000 patients, with some patients having submitted several sequence analyses to the database. The sequences are from the GenBank (a repository for genetic sequences from HIV isolates that is maintained at Los Alamos National Laboratory), published reports on HIV and local clinics. The Stanford database is geared primarily to specialized researchers because of its focus on raw sequence data. With the treatment history for every sequenced patient included in the database, this information helps associate mutations with known treatment regimens. Researchers can also quantify the frequency of specific mutational patterns.
Among 75 recent patient referrals, all of whom had received at least four reverse transcriptase inhibitors, 10% were found to have the Q151M reverse transcriptase mutation and 63% were found to have the M41L, M184V and T215Y mutations. (Genetic mutations are described as the change at a certain numbered amino acid in an enzyme protein arising from the genetic switch. The first letter represents the original, "wild type" amino acid and the final letter represents the amino acid substitution arising from the genetic switch. For example, "M41L" means that leucine has replaced methionine as the forty-first amino acid in reverse transcriptase.) Similarly, three protease gene mutational profiles were predominant in patients who had received at least three protease inhibitors. Each of these three genotypic profiles exhibited different combinations of protease gene mutations: (1) at positions 48, 54, 82; (2) at positions 36/46, 84, 90; or (3) at positions 46/54, 82, 90.
Knowing the amount and type of existing drug-induced mutations prior to initiation of the salvage therapy is useful for designing an appropriate salvage regimen. Although the database currently correlates sequences with treatment history, the plan is to expand the project to also correlate sequences with lab tests indicating susceptibility and clinical outcome.
Drug Susceptibility of Resistant Viral Strains
The degree of resistance arising from suspect gene mutations can be measured directly by virus-cell culture assays and is termed "phenotypic" resistance. In the third step of the process, researchers test the activity of both approved and experimental antiretrovirals and try to correlate the results with specific mutations. These in vitro drug susceptibility assays have revealed a high degree of cross-resistance within each of the three classes of currently available anti-HIV drugs: protease inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside analogs.
Cross-resistance affected new compounds in clinical development, too. For instance, a multinucleoside-resistant viral isolate containing resistance mutations V75I, F77L, F116Y and Q151M was not only highly resistant to AZT, d4T, ddI, 3TC, 1592U89 (abacavir) and foscarnet, but also showed small reductions in susceptibility to F-ddA, adefovir and PMPA. Likewise, multidrug-resistant clinical isolates with resistance mutations M41L, D67N, M184V, L210W, T215Y and K219N were resistant to AZT, d4T, ddI, 3TC and abacavir and displayed decreased susceptibility to F-ddA. These viruses were still sensitive to foscarnet, adefovir and PMPA. Due to their lack of resistance mutations to NNRTIs, both of these isolates were found to be susceptible to inhibition by available and experimental NNRTIs.
A high prevalence of cross-resistance between currently available protease inhibitors and experimental protease inhibitors also was detected. Viral strains containing one of the three protease gene mutation profiles were found to be resistant to indinavir, nelfinavir, saquinavir and the experimental protease inhibitor 141W94 (amprenavir). The susceptibility of viral strains containing two of these genotypic profiles (48, 54 and 82 or 36/46, 84 and 90) were further tested against five other experimental protease inhibitors (DG-35, DG-43, palinavir, GS3333 and SD146). These strains were found to be resistant to all of these new protease inhibitors, except one. The one inhibitor active against these isolates was the SD146, a compound DuPont Merck bought from Searle. This drug has solubility problems and has not progressed to clinical testing.
Activity of Drug Combinations Against Resistant Viral Strains
Having established the anti-HIV activity of single drugs, a final step in the process involves utilizing cell culture assays to assess the activity and interaction of promising drug combinations against resistant viral isolates. These assays check combinations of either only experimental drugs or experimental drugs plus approved drugs. Certain antiretroviral drug enhancers such as hydroxyurea are also combined with available drugs and experimental drugs. Hydroxyurea inhibits cellular DNA formation and has been shown to increase the anti-HIV activity of the nucleoside analog ddI.
These analyses help to identify promising drug combinations for salvage therapies to attack highly resistant HIV strains. The Stanford lab studies found that two-drug combinations of PMPA or adefovir with one of the NNRTIs (nevirapine, delavirdine or efavirenz [Sustiva]) synergistically inhibited, or exhibited enhanced activity against, either the "multidrug-" or "multinucleoside-" resistant viral strains. Additional studies showed that the presence of hydroxyurea at low, clinically tolerated, concentrations boosted the anti-HIV activity of ddI, adefovir and PMPA against resistant and susceptible clinical isolates (although resistance to ddI was not completely overcome in the case of the "multidrug-resistant" HIV).
New Clinical Trial
To further elaborate this model for developing salvage therapies, the Center for AIDS Research has now begun a clinical trial based on the lab findings concerning various drug combinations' activity against resistant HIV isolates. Patients whose HIV has specific reverse transcriptase and protease gene mutations are enrolled in the trial and will receive a quadruple drug combination containing adefovir, efavirenz, ddI and hydroxyurea. The 24 volunteers will be monitored closely over a period of six months for drug toxicity and changes in viral RNA levels, CD4 cell counts and gene mutations.
This article was provided by Gay Men's Health Crisis. It is a part of the publication GMHC Treatment Issues. Visit GMHC's website to find out more about their activities, publications and services.