By Harvey S. Bartnof, MD, Staff Physician at AVERI, AIDS Virus Education and Research Institute in San Francisco, California

PART 2:
     Triple PI Drug Therapy;
     HIV/HCV Co-Infection & Nelfinavir TDM;
     Other Nelfinavir Reports not in Part 1

Triple PI Regimen: Amprenavir plus Kaletra (lopinavir/ritonavir) Benefits Described
There have been a few anecdotal reports from clinicians about the benefits of the triple PI (protease inhibitor) drug combination of amprenavir/lopinavir/ritonavir (Agenerase/Kaletra) as "salvage" therapy for treatment-experienced patients who have few, if any treatment options. However, there have been essentially no reports at Conferences or Workshops. There were two reports about this triple PI drug combination at the 2nd Pharmacology Workshop. Researchers have expressed interest in this particular combination since lopinavir (in Kaletra with ritonavir) has a very high "inhibitory quotient" (minimum lopinavir blood concentration ¸ "IC50" or inhibitory concentration to block 50% of HIV growth) meaning it's effective against PI-resistant virus, while amprenavir has a low rate of cross-resistance with other PI drugs.

The first report was authored by Dr. Xavier Duval and colleagues of Bichat Hospital in Paris, France. Dr. Duval reported their observations in 14 patients (100% men) who started the triple PI drug regimen in combination with 1-3 NRTI drugs (nucleoside analogs), 57% of whom also took an NNRTI (non-nucleoside) drug. The patients had significant previous experience with anti-HIV therapy, a mean of 80 months (more than 6 years) with a mean PI drug history duration of 44 months (more than 3 years). None had even taken lopinavir, but 14% had taken amprenavir. All patients had taken at least two HAART regimens previously with a median 7 anti-HIV drugs. Previously, 86% had an AIDS diagnosis (CDC stage C). The mean CD4 count was 118 cells per microliter, with a nadir (lowest ever) of 34 cells (range 4-200 cells) per microliter. The mean viral load was 4.7 log (50,118) copies per milliliter. Previous NNRTI drug experience was present in 57%.

The drug combination regimens were as follows. All patients took standard lopinavir/ritonavir (Kaletra) twice-daily dosing. All patients also took twice-daily amprenavir: dosing was 450 mg for 64%, 650 mg for 7%, and 750 mg for 29%. Additional ritonavir (Norvir, in addition to that in Kaletra) 100 mg twice daily was also part of the regimen in 64%, including 36% at baseline and 28% one month into the study. Among the 57% who also took an NNRTI drug, 36% took efavirenz (Sustiva) and 21% nevirapine (Viramune).

Baseline drug resistance information was as follows. The median number of resistance mutations to amprenavir was 3 (range 0-6), with 43% having predicted genotypic resistance. The median number of resistance mutations to lopinavir was 5 (range 0-8), with 36% having predicted genotypic resistance. Phenotype resistance to amprenavir was predicted for 36%, with a median "protein-corrected IC90" of 354 ng/mL (approximately 3-fold decreased susceptibility from "wild-type" of 120 ng/mL). Phenotype resistance to lopinavir was predicted for 57%, with a median "protein-corrected IC90" of 585 ng/mL (approximately 13-fold decreased susceptibility compared to "wild-type" of 45 ng/mL). Among the 8 patients who took an NNRTI drug as a part of the regimen, 13% had genotypic resistance.

Dr. Duval also reported the baseline "inhibitory quotient" (IQ defined above), which is the relationship between the lowest blood concentration of drug measured in the patient and the phenotypic resistance of the patient's HIV isolate (here as the "protein-corrected IC90"). The median baseline IQ for amprenavir was 2 (range 0.9-6.1), while that for lopinavir was 13. (range 0.7-339).

The results were as follows. After a median follow-up of 7 months, 71% achieved an undetectable viral load (limit 200 copies per milliliter). However, for amprenavir-naïve patients, the rate was 83% (limit 200 copies), with 100% being undetectable using a limit of less than 500 copies per milliliter. (One patient each had levels of 208 copies after 10 months and 447 copies after 5 months.) A poor virologic outcome occurred for the two patients with previous amprenavir experience, with viral loads of 6,700 and 78,000 copies per milliliter, respectively, after 6 months. The median CD4 count increase was 103 cells per microliter. Undetectability was not reported by subdividing patients into those who did and did not take NNRTI therapy. Minimum mean amprenavir and lopinavir drug levels were reported, but they were not separated by those who were and were not taking an NNRTI drug. Yet Dr. Duvall reported that when compared to historical control patients taking amprenavir (APV) plus ritonavir, adding lopinavir/ritonavir (as Kaletra) to APV reduced the minimum concentration of amprenavir 3-fold, and that additional ritonavir (100 mg twice daily) did not offset that decrease (see also next report below). However, in these triple PI drug studies, the additional ritonavir did increase minimum lopinavir concentrations 41-75%. Dr. Duvall also concluded that these triple PI drug combinations still had inhibitory quotients greater than 1 for amprenavir and lopinavir in most of these heavily treatment-experienced patients. Adverse events were not reported. These preliminary results in a small number of patients with only moderate follow-up show benefits for these triple PI drug combinations with 1-3 NRTI drugs, with or without an NNRTI drug. Additional follow-up and more reports of this combination are necessary before definitive conclusions can be drawn.

In a separate presentation, overlapping authors from the same institution reported more detailed drug level changes with the triple PI drug combination (amprenavir/lopinavir/ritonavir = APV/LPV/RTV), with or without efavirenz (Sustiva, NNRTI drug). In the retrospective study of 28 "salvage" patients whereby Dr. G. Peytavin was the lead author, it is possible that many of the patients from the Duvall study above were included. (Dr. Duvall is a co-author of this Peytavin study). Overall, the results showed that when compared to amprenavir levels with twice daily APV 600 mg plus RTV 100 mg, trough APV levels are decreased 220-420% when 450-600 mg twice daily of APV is added to standard dosing of LPV/RTV (as Kaletra). This effect was not modified much whether or not efavirenz was added. However, even with that reduction, APV levels were still at least 2-fold greater than with standard APV mono PI dosing (1,200 mg twice daily, without ritonavir and without efavirenz.) Interestingly, adding an additional dosing of RTV (twice-daily 100 mg) (i.e., in addition to the RTV already in Kaletra) not only did not increase the APV minimum concentration, but led to an approximate 20% decrease! LPV levels with APV/LPV/RTV dosing were decreased by 15-36% (statistical "trend"), but this was reported to be significant only when efavirenz was added, leading to LPV levels that decreased by 42%. In contrast to APV levels above, when an additional RTV dose (100 mg twice daily, in addition to that in Kaletra) was added, LPV levels increased by 22%. In all of these drug levels, inter-patient variability was quite high. Not reported were potential changes in peak or total ("area-under-the-curve") concentrations of either LPV or APV.

Dr. Peytavin concluded that there was a "reciprocal drug-drug interaction" in that lopinavir induced the metabolism of amprenavir and to a lesser degree, APV induced the metabolism of LPV. Also, ritonavir did not counteract the enzymatic induction of APV by LPV, whether the total RTV dose was 100 or 200 mg, twice daily. The researchers also concluded that prospective studies are necessary to evaluate this "reciprocal interaction," including "pharmacokinetic" (drug levels) and clinical (virologic, side effects) outcomes. They say that this would especially be the case for patients with multiple PI drug "failures" that would include phenotype resistance information to clarify the significance of these new findings.

Despite these interesting and encouraging reports with triple PI drug combinations of APV/LPV/RTV plus NRTI drugs with or without an NNRTI drug, these should still be considered highly experimental and risky. Much additional research is necessary in this area before recommendations can be made or generalized.

References
Duval X and others. Inhibitory quotient and efficacy of amprenavir-lopinavir containing HAART in heavily pretreated HIV infected patients. Abstract and poster 5.11 at the 2nd International Workshop of Clinical Pharmacology of HIV Therapy; April 2-4, 2001; Noordwijk, the Netherlands.

Furfine ES, Berry MM and others. Virological and pharmacokinetic rationale for a new salvage regimen: amprenavir (600 mg)/saquinavir (800 mg)/ritonavir (100 mg) twice daily. Abstract and poster presentation 16 at the 3rd International Workshop on Salvage Therapy for HIV Infection; April 12-14, 2000; Chicago; Illinois and Antiviral Therapy 2000; 5 (Suppl. 2): page 14.

Peytavin G and others. Amprenavir plasma concentrations are dramatically decreased by association with ABT378/r in HIV-infected patients. Abstract and poster 1.14 at the 2nd International Workshop of Clinical Pharmacology of HIV Therapy; April 2-4, 2001; Noordwijk, the Netherlands.

HIV/HCV Co-Infected Patients and Nelfinavir TDM (Therapeutic Drug Monitoring)

• Minimum nelfinavir drug levels were found to be high.
• 1-2 dose reductions up to 60 days did not change levels.
• Levels of active nelfinavir metabolite M8 were low, before and after dose reductions.
• Authors acknowledge, "therapeutic range has not been defined."
• Some attendees questioned whether dose reductions were appropriate.

The only presentation at the Workshop that specifically addressed HIV/HCV (hepatitis C virus) co-infected patients was authored by Dr. D. Landman of Bichat-Cl Bernard Hospital in Paris, France. The presenting author was Dr. G. Peytavin. TDM (Therapeutic Drug Monitoring) was monitored prospectively in the LIVIR IMEA 014 Study. At that institution, 20-30% of HIV patients are co-infected with HCV. The goal was to achieve a blood plasma (no cells) minimum nelfinavir concentration (12 hours after the last dose) between 0.3-1.0 mg per liter. TDM was performed every two weeks for two months. For blood concentrations greater than 1.0 mg/liter, the recommendation was to decrease each nelfinavir dose by at least 250 mg.

All 18 patients had had previous anti-HIV therapy for at least six months, were started on nelfinavir (Viracept) plus two NRTI drugs, and 94% had HCV co-infection. The 18 patients included: 28% women; 17% with cirrhosis (severe scarring) on liver biopsy and/or by laboratory or clinical evidence; 83% with chronic liver disease on liver biopsy plus liver enzymes ALT, AST greater than 1.5 times the upper normal limit; and 11% who also had HBV (hepatitis B virus) co-infection. Past or current injection drug use was present in 44%, while past or current alcoholism was present in 39%. The median baseline CD4 count was 475 cells per microliter, with a viral load of 200 copies per milliliter. Median HCV RNA was not reported.

The results after two weeks revealed that all patients but one had a minimum nelfinavir blood level greater than the upper target of 1.0 mg/liter, with a median 2.4 mg/liter. Between days 30-60, one or two dose decreases was undertaken for those patients with high levels. Specifically, on day 60, the twice daily dosing was 1,000 mg for 50%, 750 mg for 22%, "ongoing" for 17%, and unchanged for 11% (non-adherent). Yet, on day 60, the median minimum nelfinavir level showed no change, at 2.5 mg/liter. Moreover, 87% still had a level greater than 1.0 mg/liter, when nelfinavir and "M8" (active nelfinavir metabolite) were included. On day 15, the ratio of M8 to nelfinavir was 18%, compared to a normal ratio of 30%. This was due to low M8 levels and high nelfinavir levels, possibly associated with decreased liver conversion to M8. CD4 counts, HIV RNA and liver tests were "stable." The authors reported that tolerance was optimal, without specifics of adverse events.

The authors concluded that the "primary objective" of therapeutic levels of nelfinavir "was not reached because the reduction of nelfinavir dose was probably insufficient, the study duration was not long enough, and a nelfinavir dose-concentration linear [direct] relationship was not conserved [observed]." However, they acknowledge that the "specific therapeutic range had not been defined [and that] the relationship between nelfinavir plasma concentrations and adverse effects has not been defined." They acknowledge that they did not test for a genetic variation in the liver P450 CYP2C19 (enzyme responsible for converting nelfinavir to M8). An overall conclusion was, "TDM is useful for HIV-infected patients with chronic liver disease treated with nelfinavir." Potential changes in HCV RNA were not reported.

This reviewer and other attendees were not convinced of the overall concluding statement above. During the question-and-answer session, people queried whether dose reductions of nelfinavir were appropriate in these patients, since there was no evidence of toxicity, per se and since the therapeutic range has not been unequivocally established. The authors did not report that the patients, 94% of whom had "high" drug levels of nelfinavir with standard dosing had excess nelfinavir toxicity. At least in terms of liver enzyme toxicity, those levels were reported to be stable during the 60-day period, even with dose reductions, yet with unchanged blood levels of nelfinavir and M8. Others commented that the "maximum tolerated dose" of nelfinavir may be higher than standard, FDA-approved dosing and that without toxicity, higher levels might lead to greater anti-HIV potency. Notwithstanding these limitations, nonetheless, addressing TDM in HIV/HCV co-infected patients certainly is an important issue, particularly the varying levels of nelfinavir and M8. Previously, only low levels of nelfinavir have been associated with HIV virologic "failure;" high levels have not been associated with toxicity.

Other Nelfinavir TDM Reports
In a separate presentation, Dr. Peytavin reported that low nelfinavir blood levels were significantly associated with HIV viral rebound in a retrospective, substudy "multivariate" analysis of the APROCO (ANRS EP 11) Study. In APROCO (1,283 patients taking their first PI drug regimen), early responders (less than 500 copies per milliliter at month 4) had a higher rate of viral rebound up to 19 months later if they had been randomized to nelfinavir combination therapy than those randomized to other PI drug combination therapy (all mono-PI drug). Dr. Peytavin and colleagues found that the "relative risk" of rebound among nelfinavir patients was 1.8 if drug levels were low (less than 1.0 mg/liter, p=0.05) and 4.2 if drug levels were "undetectable" (less than 0.03 mg/liter, p=0.01). A total of 160 out of 231 treatment-naïve patients randomized to nelfinavir were included who had information about the length of time since the last nelfinavir dose. The median time after nelfinavir dosing was 4 hours. Blood concentration analysis included nelfinavir and its active metabolite M8, with median levels of 1.7 mg/liter for the former and 2.1 mg/liter for nelfinavir plus M8. At month 4, 83% had a nelfinavir blood level greater than 1.0 mg/liter. Viral rebound occurred in 31% (50 of 160). The multivariate analysis also found that viral rebound was associated with higher baseline HIV RNA, lower baseline CD4 counts and any previous anti-HIV therapy (versus treatment-naïve patients), but not with patient-reported adherence to dosing. Diarrhea rates were similar among those with therapeutic and low nelfinavir blood levels. The retrospective findings further substantiate the findings that low nelfinavir levels are associated with poorer, longer-term virologic outcome (see also Part I report). Anti-HIV contributions from the NRTI (nucleoside analog) drugs were not considered.

Nelfinavir Diarrhea Not Associated With Reduced Antiviral Activity

In a separate presentation, P. Hsyu, PhD and colleagues from Agouron Pharmaceuticals found no correlation between blood levels of nelfinavir and the presence of diarrhea, a common side effect of the drug. Also, he found that the presence of diarrhea was not correlated with anti-HIV efficacy after 24 or 48 weeks of nelfinavir combination therapy in 99 patients. In addition, he concluded that nelfinavir absorption from the gastrointestinal (stomach-colon) tract and anti-HIV efficacy "are not adversely affected by the presence of treatment-related diarrhea." Patients took the original dosing of nelfinavir, 750 mg 3-times daily plus the NRTI drugs AZT (Retrovir) and 3TC (Epivir). Blood levels of nelfinavir were measured immediately prior to and 2 hours after nelfinavir dosing at study weeks 2 and 8. Previously, there had been some concern that diarrhea might negatively affect nelfinavir absorption and possibly anti-HIV efficacy.

In a fourth presentation about nelfinavir, researchers from the HIV Netherlands Australia Thailand Research Collaboration reported, "Monitoring of nelfinavir [blood] concentrations is warranted in [newborns] to ensure adequate exposure to this drug." Also, blood levels (pharmacokinetics) were "highly variable," and showed an overall decrease at day 7. A total of 24 Thai newborns were treated with nelfinavir twice daily (15, 30, or 45 mg per kilogram) plus 2 NRTI drugs (ddI [Videx] and d4T [Zerit]). Blood levels were monitored during a 4-week period. Interestingly, there was a trend towards higher blood levels in girls than boys. Dr. van Heeswijk proposed a therapeutic nelfinavir threshold concentration of 400-700 ng/mL. The lead author was Dr. C. Rongkavilit of Bangkok, Thailand, while the presenting author was Rolf P.G. van Heeswijk, PharmD, PhD of the International Antiviral Therapy Evaluation Center in Amsterdam, The Netherlands.

Another presentation about nelfinavir drug levels was reported by Dominique Breilh, PharmD, PhD of Bordeaux University Hospital in France. Dr. Breilh reported that minimum nelfinavir drug levels were significantly associated with virologic outcome after a mean of 16 months of nelfinavir combination therapy. Her study included a mixed group of 154 HIV positive patients (20% women), including 31% who were treatment-naïve, 27% who were PI drug-naïve and 41% who were PI drug-experienced. A starting nelfinavir dose of 750 mg 3-times daily was taken by 45%, with the remainder taking 1,250 mg twice daily. The drug regimen also included 2 NRTI drugs, while 8% also took an NNRTI drug. The median baseline CD4 count was 276 cells (247 cells per microliter in treatment-naïve), with a viral load of 4.0 log (10,473 copies) (5 log copies per milliliter in naïve). Virologic success was defined by achieving an HIV RNA level less than 50 copies per milliliter within 3 months and remaining there during follow-up, while virologic "failure" was defined as not achieving undetectability within 3 months or rebounding thereafter.

The results after a median of 16 months showed that 43% achieved virologic success, with no significant difference when comparing nelfinavir dosing arms. In a "univariate" statistical analysis, higher median nelfinavir levels (including each of minimum, maximum and "AUC" or area-under-the-curve concentrations) were significantly associated with virologic success. The median minimum nelfinavir level was 1.8 mg/L in those with virologic success and 0.7 mg/L in virologic "failures" (p<0.001). In a statistical "multivariate" analysis, virologic failure was significantly associated with each of the following: a lower minimum nelfinavir concentration ("odds ratio" [OR] 5.8, p<0.0001); lower maximum nelfinavir concentration (OR 1.2, p=0.015); a lower baseline CD4 count (less than 200 cells versus greater than 350 cells, OR 4.0, p=0.036); and number of baseline genotypic mutations in HIV's RT (reverse transcriptase) and PR (protease) enzymes (more than 4 versus 4 or less, OR 3.9, p=0.009). "M8" (active metabolite of nelfinavir) levels were not reported. Dr. Breilh also discussed how a "population pharmacokinetic estimates" model was used to calculate nelfinavir concentrations in her patients, as she presented at last year's ICAAC meeting (40th Interscience Conference on Antimicrobial Agents and Chemotherapy, 2000, Toronto, Canada).

The feasibility of once-daily nelfinavir (NFV) plus ritonavir (RTV) was reported by Dr. R. Aarnoutse and colleagues of University Medical Center in Nijmegen, The Netherlands. Once-daily anti-HIV regimens have gained increasing interest as a possibility for easier adherence or for "directly-observed therapy" for certain injection drug-using or other marginalized populations. A total of 27 healthy volunteers (44% women) were included in the 15-day study. Taken with food, the once daily doses of NFV/RTV were: 2,000/200 mg (arm 1); 2,000/400 mg (arm 2); and 2,500/200 mg (arm 3).

Results were as follows. When compared to a control arm of 1,250 mg NFV twice daily without RTV, the mean minimum concentration of NFV (plus active metabolite "M8") was increased by 92% (arm 1), by 51% (arm 2) and by 63% (arm 3). However, only arm 1 participants had a majority (7 of 8) with a minimum concentration of NFV plus M8 that was greater than the "proposed" minimum of 1.0 mg/liter. (Note that arm 2 with a higher RTV dose [400 mg] and arm 3 with a higher NFV dose [2,500 mg] both had slightly lower minimum concentrations than arm 1. This suggests "non-linear" kinetics and that actual drug levels need to be measured with new unique drug dosings.) When compared to the control arm, arms 1, 2 and 3 had total NFV plus M8 exposures ("area-under-the-curve" or total concentration) that were increased 100%, 64% and 86%, respectively. (Note a similar trend to previous parenthesis sentences regarding arms 2 and 3 versus arm 1.) And, when compared to controls, the maximum concentration of NFV plus M8 in arms 1, 2 and 3 were increased by: 95%, 66% and 100%, respectively. Ritonavir was found to increase the M8/nelfinavir ratio, suggesting increased conversion from nelfinavir to M8 in the liver.

Ritonavir strongly increased M8/NFV ratio, so NFV and M8 concentrations were added up and NFV-M8 PK (geometric mean + 95% CI) are presented. The antiviral effect of NFV is believed to come from both M8 (metabolite) and NFV. Nelfinavir M8 metabolite is a product of Nelfinavir metabolism. It is believed that both the M8 and NFV have anti-viral effect, but it may be uncertain how much effect each has. The authors said inter-patient (between different patients) variability was wide:

There were 5 premature withdrawals due to adverse events: 3 patients due to rash (one in each dosing arm), one due to increased liver enzymes and fatigue, and one associated with "precautionary withdrawal." Other tolerability was rated as good, and diarrhea was "unexpectedly mild in nature." There were increases in liver enzymes and cholesterol. The authors concluded that the once daily regimen of nelfinavir 2,000 plus ritonavir 200 mg deserves further study in HIV positive patients.

References
Aarnoutse R and others. Multiple dose pharmacokinetics and tolerability of once daily nelfinavir and ritonavir combinations in healthy volunteers. Abstract and oral presentation 1.3 at the 2nd International Workshop of Clinical Pharmacology of HIV Therapy; April 2-4, 2001; Noordwijk, the Netherlands.

Breilh D and others. Population pharmacokinetic of nelfinavir in 156 HIV-infected patients. Abstract 1656 at the 40th ICAAC (Interscience Conference on Antimicrobial Agents and Chemotherapy); September 17-20, 2000; Toronto, Ontario (Canada).

Hsyu P and others. Correlation of efficacy, nelfinavir pharmacokinetics and diarrhea in treatment-naïve HIV positive patients receiving nelfinavir, zidovudine and lamivudine. Abstract and poster 4.4 at the 2nd International Workshop of Clinical Pharmacology of HIV Therapy; April 2-4, 2001; Noordwijk, the Netherlands.

Landman R, Peytavin G and others. Therapeutic drug monitoring (TDM) of nelfinavir in a prospective study (LIVIR, IMEA 014) in HIV-HCV co-infected patients with chronic liver disease. Abstract and oral presentation 6.4 at the 2nd International Workshop of Clinical Pharmacology of HIV Therapy; April 2-4, 2001; Noordwijk, the Netherlands.

Pelligrini JL, Breilh D and others. Virological response to nelfinavir-containing regimens: analysis of individual pharmacokinetic parameters and drug resistance mutations. Abstract and oral presentation 5.5 at the 2nd International Workshop of Clinical Pharmacology of HIV Therapy; April 2-4, 2001; Noordwijk, the Netherlands.

Peytavin G and others. Relationship between nelfinavir plasma concentrations and late viral failure in APROCO (ANRS EP 11) cohort study. Abstract and poster 5.13 at the 2nd International Workshop of Clinical Pharmacology of HIV Therapy; April 2-4, 2001; Noordwijk, the Netherlands.

Rongkavilit C, van Heeswijk RPG and others. The safety and pharmacokinetics of nelfinavir in a dose-escalating study in HIV-1-exposed newborn infants: HIV-NAT 007. Abstract and oral presentation 3.1 at the 2nd International Workshop of Clinical Pharmacology of HIV Therapy; April 2-4, 2001; Noordwijk, the Netherlands.