Dual Protease Inhibitors Gain Ground

The past couple of years have seen a marked increase in the use of combinations of highly active antiretroviral therapy (HAART) that contain two protease inhibitors (PIs), usually ritonavir (Norvir) plus another PI. Ritonavir's function is not primarily to further contain HIV but rather to increase the blood levels of the second PI, decrease pill burden, simplify dosing schedules, and thereby improve drug adherence.

Back in the early 1990s, PI developers at Abbott Laboratories faced a major problem. Their initial molecule, A77003, potently blocked HIV in the test tube but broke down rapidly in the body. Its half-life was only 30 minutes (the half-life is the time required for half of the amount of an agent to be eliminated from the body). Even 24-hour infusions could not maintain adequate blood levels, and they caused severe vein irritation at the infusion site. In response, Abbott's scientists altered the structure of their molecule. (Among other things, they changed the hexagonal carbon-nitrogen rings at each end to pentagonal rings that also included a sulfur atom.) The change yielded a molecule that was more than a potent inhibitor of the HIV protease enzyme. It also strongly inhibited the CYP3A4 liver enzyme that breaks down A77003 as well as many other drugs.

The U.S. Food and Drug Administration (FDA) approved Abbott's revised PI, ritonavir, on March 1, 1996, two weeks before it approved indinavir (Crixivan) and less than three months after saquinavir (first marketed as Invirase and later, in a revised formulation, as Fortovase). Even though the recommended dose was 600mg twice daily, ritonavir found few customers due to its wide range of disturbing side effects at that dose. These included numbness around the lips and on patches of skin, increases in blood lipids (fats), high blood levels of liver enzymes (a sign of liver damage), diarrhea, and stomach upset (including regurgitation of the extraordinarily bitter ritonavir).

Indinavir and saquinavir both had their own problems. One was familiar to ritonavir's developers at Abbott: relatively low blood levels. Saquinavir performed very well in lab tests, but in the Invirase formulation, it was absorbed inadequately in the body (only 4% of each dose was absorbed) and almost totally metabolized by the liver immediately after digestion. Invirase was therefore weakly effective in humans, performing about the same as ddC (Hivid) monotherapy. The newer Fortovase formulation, which came on the market two years later, was absorbed to a greater extent than Invirase, but this required ingesting six large capsules three times a day.

The dosage of indinavir (800mg every eight hours in two or four capsules) was limited by indinavir's penchant to concentrate and precipitate, or collect in solid form, in the kidneys during excretion, causing a painful condition known as nephrolithiasis, or kidney stones. Meanwhile, the liver as well as the kidneys efficiently remove indinavir from the body such that blood levels subside by 98% before the next dose is scheduled to be taken. Indinavir's minimum blood levels therefore are so low in some people that the drug's effectiveness can be markedly impaired; for this reason the drug must be taken every eight hours around the clock. While monitoring drug levels (also known as therapeutic drug monitoring, or TDM) may be potentially beneficial, no therapeutic window -- the precise range of drug levels that are effective without being toxic -- has yet been determined for PIs or any of the other anti-HIV drug classes. And if intracellular drug levels of PIs (i.e., drug levels within cells) turn out to be more important than blood levels of the drugs, monitoring blood levels will not be useful. (For more information on TDM, see "Therapeutic Drug Monitoring" in the Autumn 2000 BETA.)

Indinavir levels also vary greatly from person to person and even in the same person from day to day. A recent study of the AZT (Retrovir)/3TC (Epivir)/indinavir combination in Thailand found that indinavir blood levels were of marginal efficacy in a quarter of the trial participants. In a one-year Dutch study of persons taking AZT/3TC/indinavir, virologic relapse (increases in viral load) was more frequent in those with lower indinavir levels in the blood (33%) compared with those with higher indinavir levels (15%). Viral breakthrough also has been especially common in those with pre-HAART treatment histories that triggered the formation of drug-resistant HIV and limited the effectiveness of supporting nucleoside analogs (NRTIs) in indinavir-containing regimens.

Adding Ritonavir to Boost Saquinavir

As early as the spring of 1996, it was clear that because of their drawbacks, both saquinavir (as Invirase) and ritonavir (as a single PI therapy) were likely to be very unpopular compared with indinavir. Yet their complementary strengths -- comparative tolerability and inhibition of hepatic (liver) metabolism -- made combining the two drugs at lower doses an attractive idea. Ritonavir could enhance saquinavir through its inhibiting effect on hepatic metabolism, which raises saquinavir blood levels 20-fold and more. This allowed for a significant dose reduction of saquinavir from 600mg three times a day to 400mg twice a day, resulting in fewer side effects. By April 1996, a ritonavir/saquinavir trial was enrolling volunteers under the supervision of University of Ottawa AIDS specialist William Cameron, M.D.

This trial enrolled 141 HIV-positive persons with CD4 cell counts between 100 and 500 cells/mm3 and no prior exposure to PIs. The trial cohort initially received one of four regimens: 400mg ritonavir/400mg saquinavir twice daily; 600mg ritonavir/400mg saquinavir twice daily; 600mg ritonavir/600mg saquinavir twice daily; or 400mg ritonavir/400mg saquinavir three times daily. No supporting NRTIs were used at first, but the trial participants could receive these extra drugs if their viral loads were not below 200 copies/mL at week 12 or if they later rebounded above this threshold. (After week 48, supporting drugs could be added at the physician's discretion.) Ritonavir and/or saquinavir dosages could be reduced in case of intolerance.

At week 48, about 90% of volunteers remaining in the trial had viral loads below 200 copies/mL regardless of their initial regimen. A quarter of the original cohort had dropped out of the trial. Of those remaining, almost half (52 of 106 persons) were on the twice-daily 400mg/400mg regimen by the end of the first year, and 26 were receiving supplemental NRTIs, mostly d4T(Zerit) plus 3TC. These figures remained stable thereafter. At week 144, 88% of the remaining participants had viral loads under 200 copies/mL. (By an intent-to-treat analysis that considers dropouts to be participants with viral breakthroughs, this percentage goes down to 69%.) Sixty percent of the continuing cohort was on the twice-daily 400mg/400mg regimen, and another 21 had added NRTIs. The 600mg/600mg and three-times-daily regimens had very high rates of intolerability.

But why stop at the combination of 400mg ritonavir/400mg saquinavir? Wouldn't further reducing the ritonavir while increasing the saquinavir to compensate result in a more tolerable regimen that is at least as effective? Abbott scientists have been opposing such suggestions since 1996. At a community meeting held that year during the XI International Conference on AIDS in Vancouver, Canada, and elsewhere, they argued that the dual PI combination needed some antiviral effect from ritonavir. Drug levels of saquinavir were still marginal for some people even when combined with ritonavir. Also, the combined PIs might show some anti-HIV synergy (enhanced effects) because somewhat different mutational patterns are required to reduce HIV susceptibility to each of these drugs. The genetic barrier to drug resistance and treatment failure may therefore be greater at the 400mg/400mg doses used in the study. However, Hoffmann-La Roche, saquinavir's developer, has more recently taken up the argument and advocated reduced ritonavir dosages. Since the introduction of Fortovase, Roche has tried to reduce this formulation's pill burden and thrice-daily schedule through the use of several "mini" doses of ritonavir. Roche researchers are currently testing a twice-daily combination of 1,000mg saquinavir plus 100mg ritonavir and a once-daily combination of 1,600mg saquinavir plus 100mg ritonavir.

One recent trial with available data tried saquinavir/ritonavir at the twice-daily dose of 1,000mg/100mg plus efavirenz (Sustiva) and two NRTIs in 32 persons. On enrollment, the volunteers were all unable to suppress HIV while taking their first PI-containing regimen, with baseline viral loads over 5,000 copies/mL. None of them had ever taken saquinavir or any non-nucleoside reverse transcriptase inhibitor (NNRTI). At week 48, 56% had viral loads below 50 copies/mL.

Roche is sponsoring an ongoing 300-person, one-year trial comparing 1,000mg saquinavir/100mg ritonavir, Roche's preferred dosing of the combination, with 800mg indinavir/100mg ritonavir, both twice daily, in treatment-naive (without prior treatment) and treatment-experienced volunteers. A similar trial testing the same saquinavir/ritonavir regimen against the standard twice-daily dose of Kaletra (400mg lopinavir/100mg ritonavir) is in the planning stages.

Antiretroviral Drugs: Dietary Restrictions

No dietary restrictions
Taken on an empty stomach
Taken with a light snack
Taken with a full meal


(Lopinavir + Ritonavir)











(AZT + 3TC)




Although medications in the "No dietary restrictions" column do not have specific dietary requirements, taking them with food may help reduce gastrointestinal side effect.

*avoid taking with a high-fat meal

**can also be taken with a low-fat snack

Once-Daily and Twice-Daily Regimens with Indinavir Plus Ritonavir

Combining ritonavir with indinavir has been shown to stabilize indinavir blood levels, resulting in less variability. In general, lower doses of indinavir (combined with ritonavir) could result in less kidney toxicity and greater efficacy. Since changes in liver activity (i.e., metabolic processes) are a main source of intra- and inter-personal variability in indinavir levels, adding ritonavir might also make those levels at least somewhat more uniform from day to day and from person to person (although there are many other unidentified reasons for pharmacokinetic variability within and among persons). Merck & Co., indinavir's developer, initially was reluctant to endorse the indinavir/ritonavir combination. Instead, Merck advocated the use of 1,200mg twice daily as a simplified indinavir schedule. It embarked on a highly publicized trial endeavoring to demonstrate the efficacy of this regimen compared with the standard dose of 800mg three times daily. However, early results from the trial showed a significantly higher rate of viral breakthrough in those taking twice-daily indinavir. Merck halted the trial in September 1997, and the company warned the public against twice-daily indinavir, which had become increasingly popular. Only after this unfortunate situation did Merck begin to approach the idea of combining indinavir and ritonavir.

Abbott, meanwhile, hoped to repeat its success with ritonavir-enhanced saquinavir. In the spring of 1998, the company's scientists formally reported their initial findings on indinavir/ritonavir, both dosed at 400mg twice daily. The combination resulted in an average exposure to indinavir that was nearly the same as with the standard indinavir-alone dosing schedule. Peak indinavir levels were reduced while the minimum (trough) levels between doses were increased about ten-fold, minimizing the window of opportunity for HIV to replicate (at least, if the HIV contained no drug resistance-conferring mutations, and assuming perfect adherence and drug absorption). Further, taking the drugs with meals made no difference in indinavir levels. Indinavir without ritonavir cannot be taken at mealtimes because full (high-caloric) meals interfere with its absorption.

Note that this interaction study was conducted in HIV-negative volunteers. Later, a German study followed 92 HIV-positive, treatment-naive volunteers who all took 400mg indinavir and 400mg ritonavir twice daily along with two NRTIs. The average baseline viral load was 401,000 copies/mL. Forty-eight weeks worth of data were available for 88 of the participants, including 24 who dropped out. About 90% of the continuing trial participants, or 70% of the entire 88, had viral loads below 80 copies/mL at this 48-week point. Development of ritonavir-enhanced indinavir was delayed when production difficulties in June 1998 caused Abbott to take the capsule form of ritonavir off the market for a year. This meant that consumers were forced to take the vile-tasting liquid form of ritonavir until a new capsule version became available in the summer of 1999.

This past year has seen an abundance of new data on various dosage combinations of these two PIs, particularly at the 8th Conference on Retroviruses and Opportunistic Infections (CROI), held February 4-8 in Chicago. That conference featured reports on two Abbott-sponsored studies that evaluated switching persons on combinations containing three-times-daily indinavir to combinations containing twice-daily indinavir/ritonavir.

The first was conducted in subjects with treatment-suppressed HIV. It found that viral suppression continued at least through week 24, whether or not the participants were switched from indinavir alone to indinavir/ritonavir at 400mg/400mg twice daily. Of the 332 persons assigned to this dual PI combination, 35% dropped out of the study compared with 31% of 83 who remained on indinavir alone. The second trial enrolled 37 persons with detectable viral loads, ranging from 50 to 50,000 copies/mL. Their PI was changed from standard indinavir to 400mg indinavir plus 400mg ritonavir. More than half of the volunteers dropped out. Of the 16 who remained for the full 48 weeks, 38% had viral loads below 50 copies/mL. In both these studies, high blood lipid levels were the only commonly observed serious toxicity and contributed greatly to the drop-out rates, especially in those receiving ritonavir. Indinavir-associated kidney problems (nephrolithiasis) or elevated bilirubin were rare, but note that both trials enrolled only volunteers who already had some previous history on indinavir. People susceptible to such side effects (e.g., persons who had developed nephrolithiasis while previously on the drug) presumably were excluded from the studies.

The high discontinuation rates among those taking indinavir/ritonavir in these two trials were mostly due to drug-related side effects. Analogous with saquinavir/ritonavir, patient discontent may be moderated by lowering the ritonavir dose while increasing that of indinavir. This is a strategy advanced by many recent Merck-sponsored studies.

In one such study, all 72 trial participants had previously received indinavir and had not been able to adequately suppress HIV on at least one PI-containing regimen. In the trial, they received indinavir/ritonavir twice daily at doses of 800mg/200mg (47 volunteers) or 400mg/400mg (25 volunteers). They also received supporting NRTIs and, in two-thirds of the subjects, NNRTIs. In preliminary 12-week results, 24 of the 44 volunteers (55%) on the 800mg/200mg regimen with results available had viral loads under 400 copies/mL compared with 8 of 24 (33%) on the 400mg/400mg combination.

The Thai study alluded to above recruited 106 volunteers whose only previous therapy was AZT plus either ddI (Videx) or ddC. The volunteers received AZT, 3TC, and indinavir with or without ritonavir for 48 weeks. The indinavir/ritonavir combination consisted of 800mg indinavir plus 100mg ritonavir, both twice daily. Median baseline viral load was 10,000 copies/mL while the median CD4 cell count at study entry was 168 cells/mm3.

No advantage in viral suppression rates over indinavir alone was apparent in the indinavir/ritonavir combination, even though it enabled participants to forego the midday dosing and empty stomach requirements required by indinavir alone. After 48 weeks, virologic results in the standard indinavir and the indinavir/ritonavir arms were equivalent: about 60% had fewer than 50 copies/mL of HIV RNA. Adverse event rates were also nearly the same, with the indinavir/ritonavir arm having a slightly higher incidence of such indinavir-related side effects as nephrolithiasis and high bilirubin levels. (Note that high bilirubin alone due to indinavir almost never causes symptoms.) Sixteen percent of the indinavir arm and 18% of the indinavir/ritonavir arm dropped out of the trial while adverse events required dose reductions or treatment interruptions in 36% of those in the indinavir arm and 44% of those on indinavir/ritonavir. An ongoing German study of NRTIs plus the same indinavir/ritonavir combination is also observing a high rate of nephrolithiasis (19.3% of 54 volunteers at 24 weeks).

One of the potential benefits of adding ritonavir to indinavir is that it may enable people to take their PIs only once a day. One Dutch study examined this possibility in 11 persons who had a history of not adhering to regimens with more complicated dosing schedules. The study also enrolled five treatment-naive individuals who strongly preferred a once-daily regimen. The study participants were prescribed 1,200mg indinavir plus 400mg ritonavir once a day (a total of seven capsules). They also took at least one other once-daily drug (some combination of 3TC, d4T, nevirapine [Viramune], and/or efavirenz; note that once-daily dosing of nevirapine and NRTIs except ddI is considered experimental). In preliminary results out to six months, no serious PI-related toxicities were observed. Starting with a median viral load of 280,000 copies/mL, eight of the first nine volunteers had viral loads below 500 copies/mL at six months.

But the daily ten-fold swings in indinavir levels observed in this study meant that those levels frequently exceeded the indinavir toxicity thresholds observed in the Thai study. Early results from a similar U.S. study in ten persons indicated that lipid levels were rising in most study participants. Among the six who had continued beyond six months, one had developed evidence of nephrolithiasis.

One Merck study tested a once-daily 1,200mg indinavir/200mg ritonavir combination in 40 treatment-naive volunteers. It obtained indinavir blood levels that were only slightly lower than the 1,200mg/400mg studies. Eight persons dropped out of the study, and at week 24, 28 of the remaining 32 had viral loads below 400 copies/mL (21 also were below 50 copies/mL). One volunteer had evidence of nephrolithiasis, and lipid levels (especially cholesterol) were generally rising by the end of six months.

Finally, a French study has pushed the indinavir/ritonavir combination to new extremes in an attempt to find a nontoxic, convenient formulation. This retrospective, multicenter study examined the records of 431 persons taking indinavir/ritonavir in various twice-daily combinations: 400mg/100mg (103 persons); 400mg/400mg (83 persons); 600mg/100mg (76 persons); and 800mg/100mg (169 persons). (According to the researchers, some physicians had lowered indinavir doses in some of their patients due to adverse events.) The researchers also measured blood levels of indinavir in each of these persons; blood levels varied widely in each regimen but usually were above the threshold of efficacy. Notably, they seemed to depend on the indinavir dose, but not on the ritonavir dose. There was no significant overall difference between the 400mg/100mg and 400mg/400mg indinavir/ritonavir combinations. Compared with three-times-daily 800mg of indinavir by itself, the 400mg/100mg twice-daily combination achieved two times higher minimum indinavir blood levels and 2.5-fold lower maximum levels, thus potentially leading to improved efficacy and reduced toxicity.

In the French study, 71 subjects with drug-related toxicities and high indinavir levels had their indinavir dosages reduced. As indinavir dosages decreased, so did the frequency of side effects and the possibility of viral breakthrough. Among the 29 subjects with documented virologic results, 22 had viral loads that either remained below 200 copies/mL or newly attained that level. Viral loads in two persons decreased one log (90%) but remained above 200 copies/mL, and in another five, viral load did not change appreciably.

A prospective study is now in the planning stages. (In the U.S., the government-sponsored ACTG 5055 trial is comparing an 800mg/200mg combination twice daily with 400mg/400mg in 50 persons not achieving viral suppression with saquinavir, nelfinavir [Viracept], or amprenavir [Agenerase] as mono-PI therapy.) To the extent that it is successful, and given the data from the French study mentioned earlier, a 400mg/100mg indinavir/ritonavir combination would be revolutionary in terms of convenience (two capsules twice daily) as well as tolerability, which would presumably improve adherence. It would also be relatively inexpensive -- half the price (in U.S. dollars) of the standard 800mg three-times-daily indinavir-alone regimen, or about the same as a single NRTI.

One recurrent theme about the 400mg/400mg twice-daily regimen of indinavir/ritonavir is the near-zero rate of kidney stones, unlike all the other combination doses. And this appears to be the case even without fluid supplementation that is standard for all the other twice-daily indinavir/ritonavir doses and the FDA-approved three-times-daily dosing of indinavir without ritonavir.

Kaletra: Abbott Joins the Bandwagon

This past September 15, the FDA announced approval of a new anti-HIV drug, Abbott Laboratories' new PI formulation, Kaletra. Each Kaletra capsule contains a combination of 133mg of the new PI lopinavir (formerly known as ABT-378) plus 33mg of Abbott's ritonavir. Lopinavir is a highly potent PI that is active against some PI-resistant HIV. But without the added ritonavir, lopinavir blood levels -- never very high to begin with -- decline to negligible levels in four or five hours. Yet these levels are very sensitive to the presence of ritonavir.

Normally, three Kaletra capsules (a total of 400mg lopinavir plus 100mg ritonavir) are taken twice daily. Abbott itself has thus bought into the ritonavir "mini-dose" strategy, abandoning its former insistence on the need to enhance other PIs with 400mg of ritonavir.

Abbott says that the ritonavir in the twice-daily regimen also leads to plasma levels of lopinavir that are at least 75-fold higher than those needed to suppress normal, wild-type (nonmutated) HIV by 50% (the IC50 level, after correcting for protein binding). At those levels, lopinavir may be able to suppress HIV that contains mutations conferring some resistance to the drug. These claims are based on testing in laboratory cell cultures; the ultimate proof is how well Kaletra performs in humans.

In persons without prior treatment, a 653-subject Phase III trial compared d4T/3TC/Kaletra with d4T/3TC/nelfinavir. At 48 weeks, 67% of those starting in the Kaletra arm had viral loads under 50 copies/mL compared with 52% in the nelfinavir arm. (These figures come from an intent-to-treat analysis that counted as treatment "failures" those who altered their initial therapy.)

Toxicity-related dropouts were low in both trial arms: by week 40, 2% of those taking Kaletra and 3% of those taking nelfinavir had discontinued for this reason. The most common serious side effects were diarrhea (about 15% in each arm) and nausea (7% for Kaletra and 4% for nelfinavir). Blood lipid levels increased in both trial arms. The Kaletra arm had significantly more cases of serious triglyceride elevations compared with the nelfinavir arm (9% vs 1%); serious elevations were defined as being at least 750mg/dL, based on nonfasting blood samples. The overall dropout rates for any reason were 17% on Kaletra and 24% on nelfinavir.

The results were almost as favorable in volunteers whose treatment histories included multiple PIs, provided they had not received any NNRTIs. One Kaletra salvage therapy trial enrolled 57 such persons with current viral loads over 1,000 copies/mL. The trial participants had to have a history of more than three months on at least two different PIs and yet still be naive to NNRTIs such as efavirenz. They received efavirenz and Kaletra plus two individually selected NRTIs. All started on a Kaletra dosage of three capsules twice daily (400mg lopinavir/100mg ritonavir). After the first two weeks, half of them added an extra Kaletra capsule (for a total twice-daily dosage of 533mg lopinavir and 133mg ritonavir), while the other half did so at week 24. (Adding the extra capsule overrides the effect of efavirenz, which stimulates the liver's degradation of lopinavir and other drugs.)

At the end of 48 weeks, 56% of the trial participants had viral loads below 50 copies/mL (this result was arrived at through the same sort of intent-to-treat analysis as the one above). The most common side effects were diarrhea and asthenia (lack of energy). Blood lipid levels (both triglycerides and cholesterol) increased by about one quarter during the 48 weeks. Among the ten drop-outs (a rate of 17.5%), three left the study due to drug-related toxicities.

Abbott took this trial's outcome to show that Kaletra is a highly effective drug even in situations where PI drug resistance is common. Sixty-eight percent of the volunteers had HIV with cross-resistance to three or more PIs. This past September, Abbott presented further data indicating that the response rate to the combination formulation (Kaletra) depended on the blood levels of lopinavir relative to the lopinavir resistance present in individual trial participants.

Lopinavir resistance in turn was not associated with any definite set of HIV mutations, but with a number of the standard HIV mutations that confer resistance to other PIs. The company analysis indicated that the risk of lopinavir failure increases as more and more of these mutations are present in the HIV protease gene. The chance of lopinavir "failure" reaches 50% when the number of baseline protease mutations is about six. Another risk factor was the inability to include effective NRTIs that would give lopinavir support in suppressing HIV replication.

At this past February's CROI, however, it became clear that even the high blood levels achieved by ritonavir-enhanced lopinavir cannot overcome all the effects of multiple resistance-conferring HIV mutations acquired during past PI therapy. In an Abbott pediatric trial, only 54% of 22 PI-experienced children achieved week 60 viral loads below 400 copies/mL compared with 79% of 76 children without prior PI experience. (Note that viral suppression is much harder to achieve in children.)

The record of the Kaletra expanded access program, which enrolled 8,733 persons needing Kaletra to construct a new treatment regimen, indicates lopinavir's potential. At some point, 70% of the enrollees achieved a viral load below 500 copies/mL or at least one log (ten-fold) below pre-Kaletra levels. (Using a stricter standard, only about 40% of total enrollees ever dropped below 500 copies/mL on the viral load assay.) Such success depended on baseline viral load and baseline CD4 cell count -- as well as number of prior NRTIs, NNRTIs, and PIs, which reflected baseline drug resistance. The influence of treatment history is in line with the observations in the Abbott salvage therapy trial.

Resistance to prior PIs is demonstrably detrimental to Kaletra's effectiveness, but how detrimental it is varies according to the PI: Abbott has found a high degree of cross-resistance to lopinavir in trial participants whose HIV had developed resistance to ritonavir and/or indinavir. The correlation between amprenavir or saquinavir resistance and reduced susceptibility to lopinavir is much weaker. One trial participant achieved viral suppression with a ritonavir-enhanced amprenavir regimen after disappointing results with Kaletra.

Bringing Amprenavir Back from the Edge

Amprenavir, co-developed and co-marketed by GlaxoSmithKline and Vertex Pharmaceuticals, is the latest agent to partake of ritonavir's advantages. Without ritonavir, the amprenavir dosage (1,200mg, eight capsules, twice daily) is limited by the individual's physical inability to ingest many of the hefty 150-mg capsules, which frequently cause gagging and nausea. In CNA2007, a salvage therapy trial conducted at the National Institutes of Health (NIH), PI-experienced volunteers with viral loads above 500 copies/mL were given standard abacavir (Ziagen), efavirenz, and amprenavir. In contrast with Kaletra, it reportedly was not possible to increase the amprenavir pill burden to make up for the efavirenz-induced acceleration of amprenavir's elimination (efavirenz decreased the peak level of amprenavir by 46% and the minimum level by 59% in a pilot study done in preparation for CNA2007). The loss of amprenavir potency is one reason for the trial's poor outcome, with only a quarter of the participants achieving viral loads below 500 copies/mL at week 16.

The NIH researchers have since conducted a 22-person study that combined efavirenz and amprenavir at their standard doses along with 200mg or 500mg of ritonavir twice per day. Both of these doses had the same effect: with or without efavirenz, they increased the minimum amprenavir blood levels by more than four-fold. Maximum levels were about the same as those achieved with the standard dose of amprenavir without any concomitant drugs.

Raising the minimum amprenavir levels is of vital importance: in one Glaxo study, 11 out of 22 individuals on standard-dose amprenavir had minimum drug levels below the threshold (220 nanograms/mL) for triggering development of amprenavir resistance via a mutation that also confers moderate cross-resistance to most other PIs (this mutation is located on codon 54 of the protease gene).

Another new Glaxo study compared the standard 1,200mg twice-daily amprenavir regimen with ritonavir-enhanced amprenavir at doses of 600mg amprenavir/100mg ritonavir twice daily and 1,200mg amprenavir/200mg ritonavir once daily. These are the two most common amprenavir/ritonavir combinations being studied. The 54 volunteers also received abacavir and 3TC, two of Glaxo's NRTIs. Due to a previous regimen, nearly all subjects started with viral loads below 400 copies/mL, and they maintained that suppression for the 12-week observation period. Once again, about half of the 15 persons in the amprenavir-alone arm had minimum blood levels of amprenavir that were below 220 nanograms/mL. Both the ritonavir-enhanced doses raised this minimum amprenavir level nearly six-fold, whereas the maximum level -- achieved soon after a new dose was taken -- increased less than 50%. Either of these regimens has a significantly lower pill burden compared with amprenavir alone.

The notable feature of the once-a-day ritonavir-enhanced regimen is that the amprenavir levels fall to their minimum only one time in a 24-hour cycle rather than twice. For that reason, the average amprenavir level in the blood over the course of a day is more than two-fold higher than the 600mg/100mg twice-daily combination (and nearly four-fold greater than that of amprenavir without ritonavir enhancement). Whether this higher average blood level leads to better efficacy or worse side effects remains to be determined. The once-daily regimen is less forgiving, however, with a longer period of low amprenavir levels if one dose is missed, when compared with the twice-daily regimen.

One potential problem with the 600mg/100mg combination arises when amprenavir/ritonavir is taken together with efavirenz or the NNRTI nevirapine, which also accelerates amprenavir's metabolism. In that case, amprenavir levels again become marginal according to a German study of five volunteers. In contrast with the German study, French investigators monitoring 20 volunteers found that the 600mg/100mg combination mostly curbed the influence of efavirenz or nevirapine.

The German study also tried a different twice-daily dose combination, 450mg amprenavir and 200mg ritonavir, which is much easier to take than the NIH combination described above. The 450mg/200mg combination reversed the efavirenz/nevirapine effect in 12 volunteers, resulting in amprenavir levels on the order of those achieved in five persons not on NNRTIs. They also approximated the amprenavir levels achieved in the Glaxo study of the twice-daily 600mg/100mg and once-daily 1,200mg/200mg combinations without efavirenz or nevirapine.

What Is the Real Benefit?

One of the problems encountered by people taking PIs and researchers trying to understand their action in the body is that PIs often do not reach their target cells in large amounts. Countering this difficulty by raising the recommended dose is not always possible because of the limits imposed by drug toxicities and the individual's capacity to ingest more capsules. It is therefore no wonder that successfully suppressing HIV depends on at least 95% adherence to dosing schedules and that even in adherent persons -- to the extent that PI levels have been studied -- PI levels may be suboptimal.

Manufacturers have now embraced adding ritonavir as one strategy for overcoming these restrictions. DuPont, Boehringer Ingelheim, and Bristol-Myers are all testing ritonavir enhancement as part of the development program for their experimental PIs.

Glaxo has even included a 600-person, ritonavir-enhancement trial for the new amprenavir prodrug, GW433908. The prodrug is a more easily absorbed version of amprenavir that should presumably increase drug levels on its own. As it turns out, GW433908 produces the same drug levels in the blood as standard amprenavir with just three compact capsules and less digestive upset. Increasing the dose does not raise these blood levels, perhaps because an upper limit to the digestive system's ability to absorb the drug has been reached.

In this glut of different combinations and different doses, it is difficult to say which is more effective or safer. There have been few trials that compare ritonavir-enhanced regimens with single PI combinations. With the possible exception of Kaletra, there is no demonstration that adding ritonavir to another PI results in a substantial clinical advance in controlling HIV.

Clearly, the field is fraught with commercial implications, and each company is struggling to produce data that support higher doses of its own drug and less ritonavir. With little clinical data, the basis for devising and ranking ritonavir-enhanced PIs falls on an evaluation of the difference between the minimum blood levels and the concentration of drug necessary to suppress HIV. Both these measurements are derived in a variety of ways and are open to a variety of interpretations.

Abbott bases its comparative data on the inhibitory quotient (IQ), or the minimum blood levels obtained in vivo (in the body) divided by the IC50, a lab measure of the concentration of drug that reduces HIV replication by 50%. The IQ for lopinavir in the Kaletra combination is about 75. In the case of indinavir/ritonavir, Abbott claims that minimum levels of indinavir are raised to about 25 times the IC50 for wild-type HIV for the 800mg/100mg indinavir/ritonavir combination. Merck measured the IC95, a much higher level of viral inhibition (95%). It found that the indinavir trough (minimum) level is 29 times the drug's IC95 for the 800mg/100mg combination and 69 times for the 800mg/200mg combination.

Meanwhile, Glaxo has created the so-called forgiveness quotient, or FQ. The FQ is defined as the IQ after missing one PI dose and just before taking the next scheduled one -- i.e., using blood levels 24 hours after the last dose for a twice-a-day regimen and at 48 hours for a once-a-day regimen. By this measure, amprenavir/ritonavir turns out to be the best combination, according to their calculations. Amprenavir's longer half-life in the body makes for somewhat less dependence on absolute adherence.

Depending upon which measurement (IQ or FQ) is used, saquinavir/ritonavir may be inferior in terms of drug concentrations achieved. So it is not surprising that Roche's scientists are questioning the validity of the other companies' techniques. They point out that different methodologies can lead to wide differences in the estimate of minimum blood levels, inhibitory concentrations, and binding to blood proteins (which locks up most of the ingested PI). These researchers suggest de-emphasizing laboratory estimates as much as possible and measuring real activity and real drug concentrations in average persons going about their daily lives.

Yet none of these approaches would account for the amount of drug reaching cells. In the final analysis, intracellular drug levels appear to be of paramount importance, and estimates of intracellular drug concentrations take the evaluation of ritonavir-enhancement a giant step further into the realm of speculation. Intracellular concentration depends on the activity of P-glycoprotein, which exists on cell membranes to expel PIs as well as other "undesirable" drugs. PIs are affected by this cellular pump to varying extents, both in the target white blood cells and in the digestive system, where P-glycoprotein on cell walls reduces the absorption of PIs.

There is evidence that ritonavir blocks P-glycoprotein activity parallel to its blocking of the CYP3A4 liver enzyme that breaks down PIs. The extent of that P-glycoprotein inhibition in various cell types has not been well characterized. And there is as yet no clinical proof that blocking P-glycoprotein will increase the PIs' anti-HIV activity. There is in fact some concern that an opposing effect will negate any possible benefit -- in the laboratory, cells with high levels of P-glycoprotein better resist entry and infection by HIV.

Further study in humans is also needed to account for the overall risks of ritonavir enhancement. PIs have become associated with a number of long-term metabolic side effects, especially fat accumulation, or lipodystrophy, and impaired sugar processing (though only rare cases of diabetes). Combinations of ritonavir with other PIs usually lead to reduced maximum blood levels of the boosted PIs, which are traditionally held accountable for most side effects. At the same time, ritonavir increases the overall exposure and minimum concentration of the boosted PIs, while the effect on long-term toxicities has yet to be determined. Some experts are also concerned that low-dose ritonavir exposure may lead to resistance against ritonavir, hence to other PIs.

One might well wonder what the long-term effects of ritonavir are on the liver, which may be central to lipodystrophy due to its lipid and sugar regulatory functions. Ritonavir is noted specifically for increasing blood lipids, which raises the risk of cardiovascular disease. In addition to increased lipid levels, the incidence of liver inflammation, as measured by blood levels of liver enzymes, is also elevated in trials of ritonavir-enhancement.

More immediately, ritonavir raises levels of many drugs, not just PIs. The extent of these changes is not well-defined: the official ritonavir package insert contains a long list of interactions between ritonavir and other medications, a good number of which are poorly characterized even for the standard 600mg twice-daily ritonavir dose. Among the problematic drugs are some of the common lipid-lowering agents that ritonavir use might necessitate. Such drug-drug interactions can create life-threatening situations, as in the case of sildenafil (Viagra), whose total exposure, or area-under-the-curve (AUC), is boosted 11-fold by full-dose ritonavir. (According to Cristina Gruta, Pharm.D., of San Francisco General Hospital, the sildenafil dosage is limited to 25mg every 48 hours in persons taking a ritonavir-containing regimen to minimize the risk of serious, potentially life-threatening toxicity. Persons taking sildenafil with any ritonavir-containing regimen are encouraged to talk with their providers about drug interactions.)

Still less is known about P-glycoprotein inhibition. Inhibiting P-glycoprotein might allow more drugs and undesirable substances into the body and into cells. It would also circumvent blood-brain and maternal-fetal (womb) barriers that provide special protection in these vital areas.

The bottom line is that low-dose ritonavir often improves blood levels of the boosted PI, yet may also complicate the medical management of persons with HIV, who may be taking a variety of medications for other physical and psychological conditions both related and unrelated to their HIV infection. This medical juggling often increases as people with HIV age, since more medications are often necessary due to other, non-HIV-related chronic conditions.

One last issue is what might happen if a ritonavir-enhanced regimen should fail. The addition of ritonavir in the regimen may create evolutionary pressure that selects for HIV that is resistant to ritonavir as well as to the boosted PI, particularly if viral suppression is not achieved. Such ritonavir-associated mutations were frequently observed in one small study of persons taking 600mg saquinavir three times daily (in the old, less absorbable Invirase formulation) plus 100mg ritonavir twice daily. Selection of resistant mutants might become a greater concern with the passage of time: last year, Dutch researchers published findings that saquinavir levels declined substantially in six persons on 400mg saquinavir/400mg ritonavir for more than a year. The median change in minimum saquinavir level was 30%, and the maximum levels were still more strongly affected. Only one subject had a detectable viral load over the course of the study, suggesting an increase in metabolic resistance to saquinavir among the six participants rather than problems with adherence.

PI drugs as a class have a serious flaw: not enough drug gets into the blood and reaches cells. Adding ritonavir is one obvious way to get around this problem and allow the boosted PI to reach its antiretroviral potential. But with the exception of Kaletra, little is established concerning the proper doses or dosing schedule for a ritonavir-enhanced combination. Still less is known about each combination's comparative value, and even less about its long-term effects. Ritonavir enhancement is now popular, especially for second-line therapies, but it has become so as a result of word-of-mouth rather than rigorously designed studies.

Nevertheless, it should be noted that the expert panel of physicians advising the Department of Health and Human Services (DHHS) has included ritonavir plus either saquinavir or indinavir among the "strongly recommended" first-line agents in the U.S. treatment guidelines for adults and adolescents updated this past February (see www.hivatis.org, Table 12).

Most of the ritonavir plus second PI dosing combinations discussed in this report should be considered experimental. Not all changes in side effects or drug concentrations (minimum, maximum, and AUC) were listed for each dosing combination, particularly in combination therapy with an NNRTI. Always consult with a physician about medication dosing before changing any drug regimen.

Dave Gilden is Director of Treatment Information Services at amfAR.

Selected Sources

Bangsberg, D.R. and others. Adherence to HAART predicts progression to AIDS. 8th Conference on Retroviruses and Opportunistic Infections. Chicago. February 4-8, 2001. Poster 483.

Bernstein, B. and others. Absence of resistance to Kaletra (ABT-378/r) observed through 48 weeks of therapy in antiretroviral-naive subjects. 8th CROI. Poster 453.

Brun, S. and others. Patterns of protease inhibitor cross-resistance in viral isolates with reduced susceptibility to ABT-378. 8th CROI. Poster 452.

Burger, D. and others. Both short-term virological efficacy and drug-associated nephrotoxicity are related to indinavir (IDV) pharmacokinetics (PK) in HIV-1-infected Thai patients. 8th CROI. Poster 730.

Burger, D. and others. Indinavir pharmacokinetics are related to efficacy and toxicity. 7th European Conference on Clinical Aspects and Treatment of HIV-Infection. Lisbon. October 23-27, 1999. Poster 826.

Cadman, J. The great salvage therapy drug juggle. GMHC Treatment Issues 12(4): 1-6. April 1998.

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