Treatment Issues, Vol 11, No 2; February 1997
Dave Gilden, with John Falkenberg and Gabriel Torres, M.D.

Similarly, the dynamics of CD4 cell proliferation tend to limit the average initial rises in CD4 counts to 100 to 150 cells/mm3 of blood. The differences between combinations are most observable in the long-term viral load endpoints and the stability of those endpoints.

The present HIV drugs are plagued by resistance. These drugs are very specific in their action, which helps to protect the body from side effects but also makes it possible for HIV to slightly alter its enzyme structures and render the drugs ineffective. Combinations of drugs require HIV to mutate at more points in its genes before it can resist all the drugs and rebound to previous viral loads. Besides a drug regimen's immediate impact on viral load, the ease with which resistance can develop is a crucial factor in determining durability of response. Reports at the conference proposed various ways to use protease inhibitor-containing combinations to build a sufficiently high "genetic barrier" against HIV breakthrough. Creating this barrier is easier in people with no prior treatment. Those with advanced disease and/or long treatment history present a more difficult problem. Their circumstances, including high viral loads, mean that the HIV within them likely has already spawned many mutants at least partially resistant to various drugs. Plus, large residual HIV levels after initiating therapy are a breeding ground for more resistance.

HAART Therapy in Advanced Disease

Nonetheless, potent HAART (for highly active antiretroviral therapy) combination therapies can be of benefit even to people with advanced AIDS and with extensive past exposure to anti-HIV therapy. This was confirmed by two studies at the Retrovirus Conference. In one (Merck protocol 039 -- abstract LB7), 320 volunteers with CD4 cell counts of less than 50 received either AZT/3TC/indinavir, indinavir alone or just AZT/3TC. All participants had at least six months of AZT but had never before taken a protease inhibitor or 3TC. The results are summarized in Table 1 (AZT/3TC/Indinavir in Advanced AIDS).

The 65 CD4 count rise at 24 weeks from indinavir monotherapy is surprising given the regimen's poor antiviral performance. The reason may be that for the first two weeks, the viral load drop from indinavir alone equaled the reduction achieved with the triple therapy, about 1.3 logs (95.0%). The median viral load in the triple therapy arm kept gradually falling at least until week 16, when it essentially stabilized. In those on indinavir alone, the median viral load rebounded after the second week, and by week 16 was only about 0.1 log (20.7%) below baseline. (The AZT/3TC arm's median viral load was stable from week 2 to week 24.) As for the CD4 counts, the two indinavir containing arms rose in parallel through the twelfth week after which the monotherapy arm declined slightly while those on triple therapy continued their rise. Other indinavir trials, too, have seen a long lag between viral load rebound and fall of CD4 count back to baseline in those on indinavir monotherapy. That process can take over a year. (Trial 039 is now continuing beyond 24 weeks with everyone on open label triple combination therapy.) Another trial looking at people in advanced disease was a pilot study conducted in Vancouver, British Colombia (abstract 234). This trial followed 21 heavily pretreated patients who were failing or intolerant to their nucleoside analog therapy and seemed to have run out of options in this drug class. The 21, who were all naive to indinavir and nevirapine, received those two drugs in combination with 3TC. Baseline CD4 count averaged only 28 (everyone was under 50), and average baseline viral load was 135,000 HIV RNA copies/ml of plasma.

This combination of a potent protease inhibitor, a rather well tolerated non-nucleoside reverse transcriptase inhibitor and a relatively benign nucleoside analog had a surprisingly positive effect as salvage therapy. In the 12 participants who had reached 20 weeks of therapy, CD4 counts were up 100 above baseline and continuing to rise, and viral load had fallen more than 1,000-fold (99.9%). However, so far only two of the 12 had undetectable viral loads using the new ultrasensitive PCR assay. Seven (58%) were undetectable by the standard PCR test that goes down only to 500 copies/ml. Three of the participants have dropped out, two due to nausea or vomiting and one because of skin rash.

Other indinavir news included the latest results from protocol 035, a trial of AZT/3TC/indinavir versus AZT/3TC versus indinavir alone in 97 adults with at least six months of prior AZT therapy. Follow-up is now out to 68 weeks in two-thirds of the group. This trial included a population that was a little less advanced than the two above: a baseline median CD4 count of 142 (one eligibility requirement was a CD4 count between 50 and 400) and a baseline median viral load of 43,000. Participants also had to have had at least six months of prior AZT treatment. The actual average past exposure to AZT was 30 months, with 80% of the participants carrying AZT-resistant HIV (by genetic analysis). The group was therefore somewhat representative of a real population with advanced diseased, in whom HIV has developed resistance to AZT during past treatment.

Trial participants assigned to receive AZT/3TC/indinavir from the beginning have exhibited stable viral load reductions, a median of 2 logs (99%) or greater, out to a year or a year and a quarter so far. Of those on treatment for 68 weeks, 18 of 21 (86%) still have plasma viral loads below the test limit of 500 HIV RNA copies/ml and 10 of 14 (71%) checked with an ultrasensitive assay are also below 50 copies/ml.

Nelfinavir: What about Cross-Resistance?

Agouron Pharmaceuticals' protease inhibitor nelfinavir (brand name Viracept) is now up for FDA licensing review. Although rumors of its imminent approval are rampant, the company's "conservative" prediction is FDA approval by June of this year. Meanwhile, Agouron researchers presented a number of reports at the Retrovirus conference, including information on the compound's efficacy when combined with AZT/3TC or d4T. In contrast to the indinavir studies above, the AZT/3TC/nelfinavir trial (Agouron protocol 511) included only people with less than one month prior AZT and no other past treatment. Their CD4 counts could be any number, and in many cases were below ten. The d4T trial (Agouron protocol 506) included both treatment-naive and -experienced people (90 naive and 218 experienced with an average of 27 months prior therapy). The trial required that their CD4 counts exceed 50. Table 2 (Two Nelfinavir Trials) summarizes the data from these two trials.

It is risky to compare the studies' data, especially given the two populations' different treatment histories. Still, the triple therapy seems superior to the double d4T/nelfinavir combination, both in terms of viral load and CD4 cell response and in the stability of that response: viral load in the d4T/nelfinavir cohorts reached a peak decline of about 1.5 logs (96.8%) and then started gradually rebounding. The viral load curves of those on AZT/3TC/nelfinavir were flat to slightly downward from week eight onward.

Most of the trial results show no significant difference in response to the 750 mg and 500 mg doses, and Agouron's application for FDA approval stated that the standard nelfinavir dose should be 500 mg thrice daily. Notice, though, that in the AZT/3TC/nelfinavir trial, 81% of those receiving 750 mg had undetectable viral loads, whereas only 65% of those receiving 500 mg reached this level. As a result of this statistically significant difference, some HIV specialists are disputing Agouron's proposed dose, asking that the 750 mg dose be specified instead since there is a sign that it better suppresses HIV. This higher dose would provide more insurance against the emergence of drug resistance and HIV rebound. Countering this argument is nelfinavir's tendency to cause diarrhea. In the triple combination trial, those on 750 mg nelfinavir had a 54% greater incidence of developing diarrhea (12 of 96 at the lower dose versus 19 of 99 volunteers at the higher).

Aside from controversies over the dose, there has been considerable discussion about nelfinavir's resistance profile. Agouron has presented data to indicate that if nelfinavir is the first protease inhibitor taken, it can profitably be succeeded by others if nelfinavir resistance emerges. There have also been claims that the converse is true -- that people who fail on other protease inhibitors due to drug resistance can still get benefit from nelfinavir. Should nelfinavir prove to have a unique resistance profile that obviates cross-resistance, it would be a very valuable addition to the anti- HIV armamentarium indeed.

Such hopes are premature at best, as the available data is scant and ambiguous. Cross-resistance between nelfinavir and other protease inhibitors first came up a year ago, during the FDA hearings on approving Abbott Laboratories' ritonavir. During its presentation the company presented a graph indicating that HIV that had mutated to protect itself from ritonavir could also resist nelfinavir to a certain extent. Agouron countered last spring with its own lab culture studies purportedly indicating that a unique, single mutation at codon 30 on the protease gene is HIV's response to exposure to nelfinavir. (This mutation switches amino acid 30 on the HIV protease enzyme so that nelfinavir has difficulty binding to the molecule.) Codon 30 mutant HIV is still fully sensitive to other protease inhibitors. However, the same nelfinavir test tube experiments found that when HIV is cultured a little further in the presence of increasing concentrations of nelfinavir, a second mutation appears that grants HIV further immunity to nelfinavir. This is the same codon 84 mutation that reduces HIV's sensitivity to ritonavir, although it is not seen that often in HIV isolated from humans taking ritonavir. (There are other, more common sets of mutations not involving codon 84 that also make HIV unsusceptable to ritonavir).

At the Retrovirus Conference, Amy Patick, Ph.D., Agouron's drug resistance expert, presented the results of several studies that she has conducted (abstract 10). The HIV isolates involved all came from participants in either the combination studies described above or earlier monotherapy studies. In the nelfinavir monotherapy trials, 36 of 54 (56%) participants analyzed had HIV with the protease codon 30 mutation by week 16. By contrast, only three of 49 (6%) of the people on AZT/3TC/nelfinavir had this mutant HIV. Sixteen such individuals who were maintained on nelfinavir for another 28 weeks (44 weeks on nelfinavir in all) had no detectable codon 84 mutation, but other, supplementary mutations did tend to accumulate. Six viral clones isolated from five people with the 30 mutation were five to 93- fold less susceptible to nelfinavir than wild type virus (the range in resistance probably arises from the supplementary mutations). All of these isolates were fully susceptible to other protease inhibitors.

But, the question remains, if someone's HIV is resistant to nelfinavir, will their HIV more rapidly become resistant to other protease inhibitors to which that person might be switched? One specific cause of concern: the supplementary mutations at codons 36, 46 and 71 are frequently seen from exposure to other protease inhibitors as well as to nelfinavir. Their presence could facilitate the development of resistance to protease inhibitors that follow nelfinavir.

And what about nelfinavir as a second-line protease inhibitor? People on one of the currently approved compounds would like to know that they can switch to nelfinavir if they start failing their present therapy. Dr. Patick presented an analysis of 23 HIV isolates obtained from people failing on indinavir, ritonavir or saquinavir. Fourteen (61%) of these isolates were still susceptible to nelfinavir in culture. The catch is that the protease inhibitor resistant HIV that had only one identified mutation was still sensitive to nelfinavir (with one mild eight-fold exception), but the virus found with two mutations (most at codon 90 plus either 82 or 84) was seven- to 75-fold resistant. Indeed, multiple mutations are more common than single ones after prolonged protease inhibitor therapy.

These data led Dr. Patick to conclude (however tentatively), "Nelfinavir failures are treatable by other protease inhibitors. Failures on other protease inhibitors with one mutation are treatable with nelfinavir, but more than one mutation is a problem -- so there is a need to switch early." Emilio Emini, M.D., who led the effort to develop indinavir at Merck would exercise greater caution about nelfinavir as either first- or second-line therapy: "You cannot say that if you start on protease inhibitor A and become resistant, the virus will still be susceptible to B, C or D," he declared. "You will get varying degrees of cross-resistance up to 100%. It all depends on the mutations generated. You don't get just one mutation. You alter the genetic background in ways that you can't see [in lab tests] because the mutations are present at too low a level. And it is from this background of minor mutations that resistance to new drugs appears."

Nelfinavir has shown itself to be a respectable protease inhibitor, but it is odd that at this late stage, with the drug before the FDA, the optimum dose is still controversial. Further, it is premature to judge how nelfinavir will be affected by cross-resistance. Certainly it is intemperate to draw conclusions on optimum protease inhibitor sequencing on the basis of 23 isolates. Further information on nelfinavir's place as follow-on protease inhibitor in the case of treatment failure may be available in the near future: Agouron is tracking the viral load response in a group of protease inhibitor treatment failures now enrolled in the nelfinavir expanded access programs.

And lest anyone think that there is no money to made from AIDS drugs: Agouron's stock price has skyrocketed from the high twenties to the mid-nineties in the past year, almost entirely on the perceived strength of nelfinavir.

Abbott Labs Tries Again

Abbott Laboratories in the meantime surprised the conference by unveiling its new protease inhibitor ABT-378 with much fanfare (abstracts 14 and 206-213). This compound has remained hidden from public view for some time (see Treatment Issues article, Protease Inhibitor New Math; May, 1996, page 5). It is highly active against HIV, about three or four times more so than Abbott's original protease inhibitor, ritonavir. The structure of ABT-378 also circumvents some of the drug-resistance mutations that afflict other protease inhibitors. For example, its activity is hardly impaired by a protease gene mutation at codon 82 that confers considerable resistance to both ritonavir and indinavir.

In general, though, ABT-378 seems to overwhelm drug resistance by its sheer potency. In combination with small amounts of ritonavir, which blocks the relatively rapid breakdown of ABT-378 by the liver, it is easy to obtain drug levels that are 50 times the levels that are effective against wild type (non-drug resistant) HIV and ten to 20 times higher than HIV with a single mutation like that at codon 82. It is nevertheless possible to generate HIV that is not inhibited by ABT-378 and is considerably less sensitive to other protease inhibitors as well. One mutant created in the laboratory by exposing HIV to increasingly high concentrations of ABT-378 had five mutations and was 338 times less sensitive to ABT-378. It also was about 20 times less sensitive to ritonavir and indinavir. Four of the five mutations also appear during analogous culture tests of Glaxo Wellcome's experimental protease inhibitor 141W94.

ABT-378 is an interesting new compound, but it does not represent a radical break with the earlier protease inhibitors. Its structure, mechanism of action and resistance mutations are comparable to those of other members of its class. A big remaining question is how feasible it is to achieve levels within humans that overwhelm any resistance on HIV's part. There are as yet no human trials to evaluate what drug levels can be attained in reality, either with ABT-378 alone or in combination with ritonavir. No information as to the new compound's toxicity is available, either. The data Abbott presented at the Retrovirus Conference at best come from rat studies, and much of the figures are derived from test-tube cell cultures or even straightforward chemical analyses in cell-free solutions.

Combining Protease Inhibitors

Although human trials of ABT-378 plus ritonavir have not yet occurred, a trial of saquinavir plus ritonavir has been continuing since last spring (unpublished presentation in session 84 by John Mellors, M.D.). The reason for combining ritonavir with saquinavir is similar to combining ritonavir with ABT-378. In both cases, ritonavir, which inhibits one of the enzyme pathways in the liver, greatly increases and stabilizes blood levels of a compound that otherwise would be rapidly metabolized. The quantities required for saquinavir are much larger than those suggested for ABT-378 and are large enough to have an anti-HIV effect of their own. This reinforcement helps to delay breakthrough of drug-resistant HIV, the more so because the two agents trigger divergent mutational patterns. Results from the ritonavir/saquinavir trial are now in hand for 20 to 24 weeks of treatment.

This trial includes four arms: 400 mg ritonavir twice daily plus 400 mg saquinavir twice daily; 600 mg ritonavir twice daily with either 400 or 600 mg saquinavir twice daily; and 400 mg three times a day of both ritonavir and saquinavir. Initial viral load averaged about 30,000 and average initial CD4 was about 300. (The trial enrolled 141 participants, all protease inhibitor naive.) For all four arms, median viral load reductions go below the limits of the viral load assay (here considered as 200 copies/ml) at four to eight weeks and stay there. This represents at least a 2 log (99%) median drop. At week 20 or 24, 60% to 85% of people on the different trial regimens had no detectable viral load. CD4 counts were up 75 to 125. There was no statistically significant difference in the effect of the various regimens, although by week 20 the group receiving ritonavir and saquinavir at 600 mg twice daily was lower than the other three in percent with undetectable viral load and in CD4 cell count rise. (This group may be less compliant due to more side effects and a more complicated dosing schedule.)

Seven participants have been allowed to add d4T/3TC after their viral loads failed to become undetectable. Six of these seven are now undetectable, following four to 16 weeks of four-drug therapy.

Another trial reported at the Retrovirus Conference (abstract 254) tried a similar four-drug approach in 32 people with refractory HIV. The participants had persistent viral loads above 5,000 copies or evidence of disease progression despite four months on two nucleoside analogs and one protease inhibitor. They received ritonavir (600 mg twice a day), saquinavir (400 mg twice a day) and two nucleoside analogs. The week 16 results were as follows: viral load down 2.2 logs (99.4% reduction from a baseline mean of 72,500 copies/ml), with 93% of participants having undetectable viral load (below 400 copies/ml), CD4 count up an average of 72 (from a baseline average 79).

Ritonavir/saquinavir by itself frequently fails to elicit a good response in advanced patients (see Treatment Issues article, Europeans Eye New Drug Cocktails; December, 1996, page 4). Throwing two more drugs into the mix may be helpful for many of these cases, assuming that a patient can absorb and tolerate extra drugs, that his or her HIV remains sensitive to these drugs, and that his or her body is still able to support at least the minimum functioning needed for survival… There are many factors that might eventually or immediately interfere, although in these two small groups, at least, treatment was initially successful for most individuals. Should Abbott have lost interest in the ritonavir/saquinavir combination due to the arrival of ABT-378, the Hoffmann La Roche protease inhibitor may have found a new partner: nelfinavir. In a preliminary trial of 13 people on saquinavir plus nelfinavir (abstract 371), nelfinavir slowed liver metabolism of saquinavir to the extent that total exposure to the latter was raised five-fold (compared to a 20-fold average increase with ritonavir). Combination therapy included 750 mg of nelfinavir thrice daily (note the dose) plus 800 mg three times a day of the new, more absorbable version of saquinavir (the "soft gel capsule" containing saquinavir formulated with special lipids which quadruple the proportion of saquinavir absorbed by the intestines). At 12 weeks, viral loads were down 2.0 logs (99%), with eight of 13 participants (62%) at undetectable levels (below 500 copies/ml). CD4 counts were up by 105.

There are 12 possible double protease inhibitor combinations just counting the three protease inhibitors on the market and nelfinavir. Many of these may be feasible combinations in terms of tolerability, drug-drug metabolic interactions and lack of cross-resistance. Bear in mind that ritonavir works with saquinavir and possibly ABT-378 above all because of ritonavir's particular metabolic properties. By blocking the hepatic breakdown of the other protease inhibitors, ritonavir raises them to highly potent levels.

Conversely, there may be interference between protease inhibitors because they compete for the same binding sites on the protease enzyme. By getting in each other's way, two protease inhibitors would have a combined effect less than the sum of their individual effects (this is termed antagonism), never mind the hoped for synergy, which occurs when the combined effects are greater than the sum of the individual ones. At the Retrovirus Conference, Merck and Roche researchers reported preliminary results indicating that indinavir administered concurrently with saquinavir raises the latter's blood levels five to eight times (abstract 608), similar to what nelfinavir does. But a team from Harvard Medical School determined that in lab culture at least, the interactive effects of indinavir and saquinavir are antagonistic, except at low doses in wild type virus (abstracts 11 and 188).

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