American Foundation for AIDS Research, July 2001
Dave Gilden

Dual PI Combinations

The past couple of years have seen a gradual increase in the use of HAART combinations containing two protease inhibitors. At the end of last year, about 18% of those taking protease inhibitors were prescribed dual PI combinations. Such regimens usually contain ritonavir plus another protease inhibitor. Ritonavir’s function here is not primarily to further contain HIV, but rather to increase the blood levels of the second PI – and thereby decrease pill burden, simplify dosing schedules, and improve adherence to dosing schedules.

Back in the early '90s, protease inhibitor 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 in the body was only 30 minutes. Even 24-hour infusions could not maintain adequate blood levels, and they caused severe vein irritation at the infusion site. ¹ In response, Abbott's scientists fiddled with 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. This 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 protease inhibitor, ritonavir (brand name: Norvir) on March 1, 1996, two weeks before it approved indinavir (Crixivan) and less than three months after saquinavir (first marketed as Invirase and, in a later, revised formulation, as Fortovase). Even though the recommended dose was limited to 600 mg 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 (cholesterol and triglycerides), high blood levels of liver enzymes (a sign of liver damage), diarrhea, and stomach set (including regurgitation of the extraordinarily bitter ritonavir).

Indinavir and saquinavir both had their own problems. One was very familiar to ritonavir's developers at Abbott: A major hurdle that these two PIs faced was undesirably low blood levels. Saquinavir performed very well in lab tests, but in the Invirase formulation, it was absorbed poorly by the digestive tract and almost totally metabolized by the liver immediately after digestion. Only 4% of each dose reached the bloodstream. Invirase as a result was weakly effective in humans, performing about the same as ddC (Hivid).² The newer Fortovase formulation, which came on the market two years later, could deliver substantial amounts of saquinavir to the body. Fortovase results in a tenfold increase in average saquinavir levels. Aside from changing the formulation, achieving these appreciable saquinavir levels required doubling the dose to 3600 mg and ingesting six large capsules three times a day.

The dosage of indinavir (800 mg every eight hours in two or four capsules) was limited by indinavir's penchant for precipitating in the kidneys during excretion, causing a painful sludge known as nephrolithiasis. Meanwhile, the liver as well as the kidneys efficiently removes indinavir from the body. Blood levels subside by 98% before the next scheduled dose. Indinavir's minimum blood levels are therefore so low in many people that the drug's utility can be markedly impaired.

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. ³ In a one-year Dutch study of persons taking AZT/3TC/indinavir, virologic relapse (rebound in viral load) was more frequent in those with lower indinavir concentrations in the blood. ⁴ A failure rate of 33% was recorded among those with low concentrations, compared with 15% for those with higher ones. Viral breakthrough also has been especially common in persons whose suboptimal pre-PI treatment history triggered the emergence of nucleoside analog-resistant HIV. This resistance limited the effectiveness of the concomitant nucleoside analogs in indinavir-containing regimens. ⁵

Adding Ritonavir to Boost Saquinavir

As early as the spring of 1996, it was clear that because of their drawbacks, both saquinavir and ritonavir were likely to be very unpopular compared to indinavir. Yet their complementary strengths - respectively, comparative tolerability and inhibition of liver metabolism - made combining the two drugs at lower doses a feasible, attractive idea. Ritonavir raises saquinavir blood levels 20-fold and more through its inhibiting effect on liver metabolism. Conversely, saquinavir could rescue ritonavir from public opprobrium by reducing the prescribed dose of ritonavir. This would result 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. ⁶

This trial enrolled 141 HIV-positive persons with CD4 counts between 100 and 500 cells/mm³ and no prior exposure to protease inhibitors. The trial cohort initially received one of four regimens:

• 400 mg ritonavir plus 400 mg saquinavir bid;
  
• 600 mg ritonavir plus 400 mg saquinavir bid;
  
• 400 mg ritonavir plus 400 mg saquinavir tid or
  
• 600 mg ritonavir plus 600 mg saquinavir bid.

No supporting reverse transcriptase inhibitors 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 400 mg/400 mg regimen by the end of the first year, and 26 were receiving supplemental reverse transcriptase inhibitors, mostly d4T (Zerit) plus 3TC (Epivir). These figures remained stable thereafter. At week 144, 88% of the remaining participants had viral loads under 200 copies/mL. ⁷ (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 now on the twice daily 400 mg/400 mg regimen, and another 21% had added reverse transcriptase inhibitors.

But why stop at the combination of 400 mg ritonavir/400 mg 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 in other venues, they argued that the dual PI combination needed some antiviral effect from ritonavir. ⁸ Drug levels of saquinavir were still marginal for some people even when combined with ritonavir. ⁹ Also, the combined protease inhibitors might show some anti-HIV synergy 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 400 mg/400 mg doses used in the study than with combinations utilizing lower ritonavir doses.

More recently, Hoffmann-La Roche, saquinavir's developer, has taken up the argument and advocated reduced ritonavir dosages. ¹⁰ 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 1,000 mg Fortovase plus 100 mg ritonavir and a once-daily combination of 1,600 mg Fortovase plus 100 mg ritonavir. Given that saquinavir is considered more tolerable than ritonavir, these combinations may be attractive. They produce blood levels considerably above those from saquinavir alone but not high enough to create a solid barrier against the development of resistance during low points on the daily cycle. ¹¹

One recent trial with available data tested saquinavir-ritonavir at the twice-daily dose of 1,000 mg/100 mg plus efavirenz (Sustiva) and two nucleoside analogs in 32 persons. ¹² On enrollment, the volunteers were all unable to suppress HIV while taking their first PI-containing regimen. Their viral loads when entering the study were 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.

Hoffman-La Roche is sponsoring an ongoing 300-person, one-year trial comparing 1,000 mg saquinavir/100 mg ritonavir, Roche's preferred version of the combination, to 800 mg indinavir/100 mg ritonavir (both regimens taken twice daily). Enrollees may be either treatment-naive (without prior treatment) or treatment-experienced. A similar trial testing the same saquinavir-ritonavir regimen against the standard twice-daily dose of Kaletra (400 mg lopinavir/100 mg ritonavir) is in the planning stages.

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

Combining indinavir with ritonavir promises to stabilize indinavir blood levels, enabling the use of less indinavir. Lower doses of indinavir combined with ritonavir can result in higher minimum values and lower maximum values, with the upshot of less kidney toxicity and greater efficacy. Since changes in liver activity are a major source of intra- and inter-personal variability in indinavir levels, adding ritonavir could also make those levels at least somewhat more uniform from day to day and from person to person.

Merck & Co., indinavir's developer, was slow to accept this idea, however. Merck instead advocated the use of 1,200 mg twice daily as a simplified indinavir schedule. It embarked on a highly publicized trial endeavoring to demonstrate the efficacy of this regimen compared to the standard 800 mg thrice-daily one. 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 fiasco did Merck begin to approach the idea of combining indinavir and ritonavir.

Abbott, meanwhile, was hoping to repeat its success with ritonavir-enhanced saquinavir. In the spring of 1998, the company's scientists formally reported their initial findings on ritonavir/indinavir, both dosed at 400 mg twice daily. ¹³ The combination resulted in an average exposure to indinavir that was nearly the same as that attained by the standard indinavir-alone dosing schedule. Peak indinavir levels were reduced while the minimum (trough) levels between doses were increased about ten times, minimizing the window of opportunity for HIV to replicate (at least, if the HIV did not already contain drug resistance-conferring mutations). Further, taking the drugs with meals made no difference in indinavir levels. Indinavir without ritonavir cannot be taken with full meals because these interfere with its absorption.
Note: This interaction study was conducted in HIV-negative volunteers.

Later, a German study followed 92 HIV-positive, treatment-naïve volunteers who all took 400 mg ritonavir and 400 mg indinavir twice daily plus two nucleoside analogs. ¹⁴ Their 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 held up when production difficulties in June 1998 took the capsule form of ritonavir off the market for a year. This left only the vile-tasting ritonavir liquid formulation until a new capsule version of ritonavir appeared 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 8^th 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 individuals on combinations containing thrice-daily indinavir to combinations containing twice-daily indinavir-ritonavir.

The first was conducted in persons with treatment-suppressed HIV ¹⁵ It found that viral suppression continued at least through week 24 whether or not trial participants were switched from indinavir alone to ritonavir/indinavir at 400 mg/400 mg twice daily. Of the 332 persons assigned to this dual PI combination, 35% dropped out of the study compared to 31% of 83 who remained on indinavir alone. The second trial enrolled 37 persons with detectable viral loads ranging from 50 copies/mL to 50,000. ¹⁶ Their protease inhibitor was changed from standard indinavir to 400 mg indinavir plus 400 mg ritonavir. More than half of the volunteers dropped out in the course of 48 weeks. Of the 16 remaining, 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 dropout rates, especially in those receiving ritonavir. Indinavir-associated kidney problems (nephrolithiasis) or liver dysfunction (as detected by elevated bilirubin in the blood) were rare, but note that both trials enrolled only volunteers with some previous history on indinavir. People susceptible to such side effects presumably were weeded out by their poor experience with the drug. (But note that high bilirubin caused by indinavir is almost always without symptoms.)

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 - now that the company has come around to the idea of adding at least some ritonavir to its drug.

In one such study, all 72 trial participants had previously received indinavir and had had been unable to adequately suppress HIV on at least one PI-containing regimen. ¹⁷ In the trial, they received indinavir-ritonavir twice daily at doses of 800 mg/200 mg (47 volunteers) or 400 mg/400 mg (25 volunteers). They also received supporting nucleoside analogs and, in two-thirds of the cases, NNRTIs. In preliminary 12-week results, 24 of the 44 volunteers (55%) on the 800 mg/200 mg regimen with results available had viral loads under 400 copies/mL, compared to 8 of 24 (33%) on the 400 mg/400 mg 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 800 mg indinavir plus 100 mg 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/mm ³.

No advantage in viral suppression rates over indinavir alone was apparent in the indinavir-ritonavir combination, even though it enabled trial participants to forgo the midday dosing and empty stomach required by indinavir alone. After 48 weeks, virologic results in the standard indinavir and the indinavir-ritonavir arms were equivalent: about 60% on each regimen were below 50 copies/mL. 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. 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. This last difference was nearly statistically significant. Note that an ongoing German study of nucleoside analogs plus the same indinavir-ritonavir combination is also observing a high rate of nephrolithiasis (19.3% of 54 volunteers at 24 weeks). ¹⁸

One of the potential benefits of adding ritonavir to indinavir is that it may enable people to take their protease inhibitors 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 1,200 mg indinavir plus 400 mg ritonavir once a day (an onerous seven-capsule burden). They also took one or more other drugs on a once-daily basis (some combination of 3TC, d4T, nevirapine and/or efavirenz). In preliminary results out to six months, no serious protease inhibitor-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 tenfold 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 US study in ten patients indicated that lipid levels were rising in most study participants. ¹⁹ Among the six who had gone beyond six months, one had developed evidence of nephrolithiasis.

One Merck study tested a once-daily 1,200 mg indinavir-200 mg ritonavir combination in 40 treatment-naïve volunteers. ²⁰ It obtained indinavir blood levels that were only slightly lower than the 1,200 mg/400 mg studies. Eight persons dropped out of the study, and the 28 of the 32 remaining had week 24 viral loads below 400 copies/mL (21 were also 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. ²¹ This retrospective study examined the records of 431 patients taking indinavir-ritonavir in twice-daily combinations:

• 400 mg/100 mg;
  
• 400 mg/400 mg;
  
• 600 mg/100 mg;
  
• 800 mg/100 mg.

The researchers also measured blood levels of indinavir in each of these patients. Blood levels varied widely in each regimen but were usually above the threshold of efficacy. Notably, they seemed to depend on indinavir dose, but not on the ritonavir dose. There was no significant overall difference between the 400 mg/100 mg and 400 mg/400 mg indinavir-ritonavir combinations. Compared to thrice-daily 800 mg of indinavir by itself, the 400 mg/100 mg twice-daily combination achieved two times higher minimum indinavir blood levels and 2.5-fold lower maximum levels, thus promising improved efficacy and reduced toxicity.

Seventy-one patients with drug-related toxicities and high indinavir levels had their indinavir dosages reduced. As indinavir levels went down, so did side effects. Among the 29 patients 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, and in another five, the viral load did not change appreciably.

A prospective study is now in the planning stages. (In the US, the government-sponsored ACTG 5055 trial is comparing an 800 mg/200 mg combination to the 400 mg/400 mg one in 50 persons failing saquinavir, nelfinavir or amprenavir.) To the extent that it is successful, a 400 mg/100 mg indinavir-ritonavir combination would be revolutionary in terms of convenience (two capsules twice daily) as well as tolerability. It would also be relatively inexpensive - half the price of the standard 800 mg thrice-daily indinavir-alone regimen, or about the same as a single nucleoside analog.

Kaletra: Abbott Joins the Bandwagon

Last September 15, the FDA announced approval of a new anti-HIV drug, Abbott Laboratories' new protease inhibitor formulation, Kaletra. Each Kaletra capsule contains a combination of 133 mg of the new protease inhibitor lopinavir (formerly known as ABT-378) plus 33 mg of Abbott's ritonavir. Lopinavir is a very potent protease inhibitor 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. But these levels are very sensitive to the presence of ritonavir. ²²

Normally, three Kaletra capsules (a total of 400 mg lopinavir plus 100 mg 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 protease inhibitors with 400 mg 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 that needed to suppress wild-type (nonmutated) HIV by 50% (the IC₅₀ level). ²³ At those levels, lopinavir would be able to overwhelm 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-person Phase III trial compared d4T/3TC/Kaletra to d4T/3TC/nelfinavir (Viracept). At 48 weeks, 67% of those starting in the Kaletra arm had viral loads under 50 copies/mL, whereas 52% in the nelfinavir arm did so.²⁴ (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% versus 1%). (Serious elevations were defined as being at least 750 mg/dL, based on nonfasting blood samples.) The overall dropout rates were 17% on Kaletra and 24% on nelfinavir.

The results were almost as favorable in volunteers whose treatment histories included multiple protease inhibitors but no previous NNRTIs. One Kaletra salvage therapy trial enrolled 57 such persons with current viral loads over 1,000 copies/mL. ²⁵ The trial participants had to have a history of more than three months on at least two different protease inhibitors and yet still be naïve to NNRTIs such as efavirenz. They received efavirenz and Kaletra plus two individually selected nucleoside analogs. All started on a Kaletra dosage of three capsules twice daily (400 mg lopinavir/100 mg ritonavir). After the first two weeks, half of them added an extra Kaletra capsule (for a total twice-daily dosage of 533 mg lopinavir and 133 mg ritonavir), and the dose of the rest was boosted 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 dropouts (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 protease inhibitor resistance abounds. Sixty-eight percent of the volunteers had HIV with cross-resistance to three or more protease inhibitors. In September 2000, Abbott presented further data indicating that the response rate to Kaletra depended on the blood levels of lopinavir achieved relative to the lopinavir resistance present in individual trial participants. ²⁶

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 protease inhibitors. 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 protease mutations is about six. ²⁷ Another risk factor was the inability to include effective nucleoside analogs that would give lopinavir support in suppressing HIV replication.

At this past February's 8^th Conference on Retroviruses and Opportunistic Infections, it became still clearer that the high blood levels achieved by ritonavir-enhanced lopinavir cannot overcome all the effects of multiple resistance-conferring HIV mutations acquired during past protease inhibitor therapy.

In an Abbott pediatric trial, only 54% of 22 PI-experienced children achieved week 60 viral loads below 400 copies/mL compared to 79% of 76 children without prior PIs.²⁸ (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 "real-world" potential. Nearly 70% of the enrollees achieved a viral load at one or more time points that was below 500 copies/mL or at least one log (90%) below pre-Kaletra levels.²⁹ (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 count - as well as number of prior nucleoside analogs, NNRTIs and protease inhibitors. The influence of treatment history is in line with the observations in the Abbott salvage therapy trial.

Resistance to prior protease inhibitors is demonstrably detrimental to Kaletra, but 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.³⁰ The correlation between amprenavir (Agenerase) or saquinavir resistance and reduced susceptibility to lopinavir is much weaker. One trial participant was in fact rescued after Kaletra failure with an ritonavir-enhanced amprenavir regimen.

Bringing Amprenavir Back from the Edge

Amprenavir, co-developed and -marketed by GlaxoSmithKline and Vertex Pharmaceuticals, is the newest agent to make use of ritonavir's liver-inhibiting characteristics. Without ritonavir, the amprenavir dosage (1,200 mg twice daily) is limited by patients' physical inability to ingest many of the hefty 150-mg capsules. These frequently cause gagging and nausea.

In CNA2007, a salvage therapy trial conducted at the National Institutes of Health, protease inhibitor- experienced volunteers with viral loads above 500 were given abacavir, efavirenz and amprenavir - all at standard doses. In contrast to Kaletra, it reportedly was not possible to increase the amprenavir pill burden to make up for the efavirenz-induced acceleration of amprenavir's elimination. ³¹ (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 of the reasons for the trial's poor outcome, with only a quarter of the participants achieving viral loads below 500 at week 16.

The NIH researchers have since conducted a 22-person study that combined efavirenz and amprenavir at their standard doses along with 200 or 500 mg of ritonavir twice a day. ³² Both these doses had the same effect: With or without efavirenz, they increased the minimum amprenavir blood levels by more than fourfold. Maximum levels were about the same as the standard dose of amprenavir achieves without any concomitant drugs.

Raising the minimum amprenavir levels is of vital importance: In one Glaxo study, 11 of 22 individuals on standard dose amprenavir had minimum 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 protease inhibitors. ³³ (This mutation is located on codon 54 on the protease gene)

Another new Glaxo study compared the standard 1,200 mg twice-daily amprenavir regimen to ritonavir-enhanced amprenavir at doses of 600 mg amprenavir/100 mg ritonavir twice daily and 1,200 mg amprenavir/200 mg ritonavir once daily. ³⁴ These are the two most common amprenavir-ritonavir combinations. The 54 volunteers also received abacavir and 3TC, two of Glaxo's nucleoside analogs. Due to a previous regimen, nearly all started with viral loads below 400 copies/mL, and they maintained that suppression for the 12-week observation period. Once again, half 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 sixfold, whereas the maximum level - achieved soon after a new dose was taken - increased less than 50%. Either of the amprenavir-ritonavir regimens reduces the total pill burden by nearly 40% (from 16 to ten capsules per day).

The notable feature of the once-a-day ritonavir-enhanced regimen is that the amprenavir levels fall to their minimum only once 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 two-fold higher than the 600 mg /100 mg twice-daily combination (and three-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.

One potential problem with the 600 mg/100 mg 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. ³⁵ In contrast to the German study, French investigators monitoring 20 volunteers found that the 600 mg/100 mg combination did mostly resist the influence of efavirenz or nevirapine. ³⁶

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

The body's metabolism blocks protease inhibitors from reaching cells in large amounts. Countering these drugs' elimination by raising the recommended dose is not always possible because of the limits imposed by drug toxicities and patients' ability to ingest more capsules. It is therefore no wonder that successfully suppressing HIV depends on at least 90% adherence to dosing schedules ³⁷ and that even in adherent patients, protease inhibitor levels are frequently suboptimal. ³⁸

Manufacturers have now embraced adding ritonavir as one strategy for overcoming these restrictions. DuPont, Boehringer Ingelheim and Bristol Myers ³⁹ are all testing ritonavir-enhancement as part of the development program for their experimental protease inhibitors.

Glaxo and Vertex have even included a 600-person, ritonavir-enhancement trial for the new amprenavir prodrug, GW433908. They have embarked on a 330-person trial comparing the prodrug plus ritonavir to the lopinavir-ritonavir combination in Kaletra. The prodrug is a more easily absorbed version of amprenavir that should increase drug levels on its own. As it turned out, GW433908 produces the same drug levels as standard amprenavir, but with just three compact tablets and less digestive upset. ⁴⁰ 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 to single PI combinations. With the possible exception of Kaletra, there is no demonstration that adding ritonavir to another protease inhibitor results in a substantial clinical advance in controlling HIV.

As should be clear by now, 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 protease inhibitors falls on an evaluation of the difference between the minimum blood levels and the concentration of drug necessary to suppress HIV. Both these factors are measured in a variety of ways and open to a variety of interpretations.

Abbott bases its comparative data on the "inhibitory quotient" (IQ), which is the minimum blood level obtained in people divided by the IC₅₀, a lab measure of the concentration of drug that reduces HIV replication by 50%. The IQ for the 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 the 800 mg/100 mg indinavir-ritonavir combination. ⁴¹ Merck measured the IC₉₅, a much higher level of viral inhibition. It found that the indinavir trough level is 29 times the drug's IC₉₅for the 800 mg/100 mg combination and 69 times for the 800 mg/200 mg combination. ⁴²

Meanwhile, Glaxo has created the "forgiveness quotient," or (FQ). ⁴³ The FQ is defined as the IQ after missing one protease inhibitor 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. Amprenavir's longer half-life in the body makes for less dependence on absolute adherence.

By all these methods, ritonavir-saquinavir looks inferior. So it is not surprising that Hoffmann-La Roche's scientists are questioning the validity of the other companies' techniques. ⁴⁴ 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 protease inhibitor). There is a need to drop the lab estimates as much as possible and to measure real activity and real drug concentrations in average patients going about their daily lives.

None of these approaches would account for the amount of drug reaching the cells. Estimates of intercellular concentration 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 protease inhibitors as well as other undesirable molecules. Protease inhibitors 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 protease inhibitors. ⁴⁵ The extent of that P-glycoprotein inhibition has not been well characterized. And there is as yet no clinical proof that blocking P-glycoprotein will increase the protease inhibitors' anti-HIV activity. There is in fact some concern that an opposing effect will negate any possible benefit - in the lab, cells with high levels of P-glycoprotein better resist entry and infection by HIV. ⁴⁶

Further study in humans is also needed to account for the overall risks of ritonavir enhancement. Protease inhibitors have become associated with a nebulous list of long-term metabolic derangements, especially fat redistribution, or lipodystrophy, and impaired sugar processing, including diabetes. Combining a protease inhibitor with ritonavir usually leads to reduced maximum blood levels of the original protease inhibitor. These maximum levels are traditionally held accountable for acute side effects. At the same time, adding ritonavir increases the minimum concentration of the boosted protease inhibitor and, quite possibly, the overall average level. This influence on long-term toxicities has yet to be determined.

One might well wonder what are 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 escaped liver enzymes, is also elevated in trials of ritonavir-enhancement.

More immediately, ritonavir raises levels of many drugs, not just protease inhibitors. The extent of these changes is poorly characterized: The official ritonavir package insert contains a long list of interactions between ritonavir and other medications, with many of these poorly characterized even for the standard 600 mg twice-daily ritonavir dose. Among the problematic drugs are some of the common lipid-lowering agents that might ritonavir use might necessitate. Such drug-drug interactions can create surprising life-threatening conditions , as in the case of Viagra, whose total exposure is boosted 11-fold by full-dose ritonavir.

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

The bottom line is that ritonavir greatly complicates 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 act threatens to become overwhelming as patients age.

One last issue is what happens if a ritonavir-enhanced regimen should fail. The addition of the ritonavir in the regimen may create evolutionary pressure that selects for HIV resistant to the ritonavir as well as the primary protease inhibitor. Such ritonavir-associated mutations were frequently observed in one small study of persons on a 600 mg thrice-daily saquinavir (in the old, less absorbable Invirase formulation) plus 100 mg ritonavir twice daily.

Selection of such multi-PI resistant mutants might become a greater concern with passage of time: Last year, Dutch researchers published findings that saquinavir levels declined substantially in six persons on 400 mg saquinavir/400 mg ritonavir for more than a year. The median change in minimum saquinavir level was 30%, and the maximum levels were still more strongly affected. The emergence of resistant virus is favored as drug concentrations decline.

Protease inhibitors as a class have a serious flaw: Not enough gets into the blood and reaches cells. Adding ritonavir is one obvious way to get around this problem and allow the PIs to reach their 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.

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