Therapeutic drug monitoring is used today in several other diseases. For example, treatments for asthma, epilepsy and certain bacterial infections sometimes employ therapeutic drug monitoring to determine the correct dosage. Several recent studies in HIV infection, too, have shown that variable blood levels of HIV drugs can have a major impact on the success or failure of antiretroviral therapy. These variations can be caused by how much drug reaches the blood, how rapidly the drug is cleared from the blood, or by metabolic interactions with other drugs. Some studies are now taking place to assess prospectively whether or not measuring antiviral drug blood levels—then individually tailoring doses to ensure continuously therapeutic levels—can reduce the incidence of treatment failure.

Since suboptimal drug concentrations are likely to lead to viral rebound, therapeutic drug monitoring might offer a way to achieve optimal concentrations and thus prevent the emergence of resistance and cross-resistance—or it might not. Therapeutic drug monitoring has already been included in French treatment guidelines (particularly in the setting of suboptimal response to protease inhibitor-containing regimens) and is available to some HIV-infected individuals in several European countries (including France, the Netherlands, and the U.K.) at a cost of $50-75 per person per drug. The clinical benefits of therapeutic drug monitoring, however, remain to be proven.

Plasma concentrations of antiretroviral drugs, particularly the protease inhibitors, vary significantly among individuals. Both the protease inhibitors and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) are metabolized through the liver by the cytochrome p450 system, creating complex two- and three-way interactions when these drugs are used together or with other drugs metabolized by the p450 system. In addition, all three classes of drugs may be affected by cellular influx and efflux systems mediated by p-glycoprotein (P-gp), which has been implicated in multi-drug resistance to cancer chemotherapy (see the accompanying article by Yvette Delph). The effects of P-gp on the distribution, metabolism and excretion of drugs, including protease inhibitors, in the body is great. Much is already known about the link between HIV drug pharmacokinetics and treatment outcomes. Let's examine what is already known about the link between HIV drug pharmacokinetics and treatment outcomes.

I. Suboptimal antiretroviral dosing can lead to treatment failure.
We already know from several early studies of protease inhibitor monotherapy and combination studies, particularly in salvage therapy, that suboptimal drug concentrations lead to therapeutic failure, resistance and cross-resistance.

a. Hard (gel) lessons of Invirase
Remember Invirase? Perhaps you'd rather not. (If you're up to the bad flashback, please refer to "Cynical Swiss Saquinavir Scam: Roche Admits Licensed Dose Suboptimal," TAGLine, 4(7), August 1997, www.treatmentactiongroup.org/tx/sqv.html). In brief, Roche rushed hard-gel saquinavir—in vitro the most potent drug in its class—to market without defining the maximum tolerated dose. This in spite of the fact that the then current hard gel capsule formulation was only 4% bioavailable. A later study, ACTG 333, showed that the insufficient concentrations of saquinavir in Invirase resulted not only in treatment failure and resistance to saquinavir, but in resistance to indinavir as well.

Later, as the drug lost market share to its more potent classmates indinavir and ritonavir, Roche belatedly conducted studies of higher Invirase doses and of a new soft gel capsule saquinavir formulation, which was dosed at a concentration able to achieve more potent and durable viral suppression. Eventually the new formulation, dubbed Fortovase, was licensed by the FDA and substantially replaced Invirase (dwindling supplies of which are being dumped by Roche in developing countries). Nevertheless, because the pill count for Fortovase, when used as a single protease inhibitor, remains daunting, it is most often used today in combination with ritonavir.

Two recent studies, ACTG 359 and VIRADAPT, showed that pharmacology can help explain treatment failure. This is different, however, from demonstrating that pharmacology—used in the clinic—can help maximize treatment success. At least one ongoing study, ATHENA, is attempting to validate the use of therapeutic drug monitoring in a prospective manner.

b. ACTG 359: A chain reaction of unintended consequences
ACTG 359 was a study of 277 pre-treated individuals with virologic failure (median viral load at baseline 31,746 copies/mL; median CD4 count 229/mm³), who were randomly assigned to take one of two double protease regimens (saquinavir with ritonavir or saquinavir with nelfinavir) plus either delavirdine, adefovir, or both. Unexpectedly, the results showed that those assigned to any of the adefovir-containing arms—with or without delavirdine—did worse than those assigned to delavirdine without adefovir.

The ACTG 359 executive summary explained that "the explanation for the inferior virologic effect in the adefovir arms may be that subjects had extensive nucleoside experience or discontinued lamivudine [3TC] upon study entry... The lack of additivity or synergy in the delavirdine plus adefovir combination arms may well be due to an adverse pharmacokinetic interaction between delavirdine and adefovir first demonstrated in the intensive pharmacokinetic substudy (ACTG 884) of the current study... [which] showed that delavirdine levels were halved when co-administered with adefovir... In addition, saquinavir levels were reduced by about half in the delavirdine plus adefovir combination arms, possibly as a direct result of the decreased delavirdine levels."

So the unexpected adefovir-delavirdine effect caused a chain reaction which, in turn, caused a delavirdine-saquinavir effect, reducing levels of both delavirdine and saquinavir. Clearly, it would have been preferable if the pharmacokinetic substudy had been carried out before ACTG 359.

c. VIRADAPT pharmacokinetic testing: Predictive but not prospective.
VIRADAPT was a prospective study designed to address whether providing genotypic resistance testing results would improve virologic responses to second-line or salvage therapy regimens. Forty-eight week follow-up data and a pharmacologic substudy were presented at the FDA Antiviral Drugs Advisory Committee meeting on 2 November 1999 by the principal investigator, Philippe Clevenbergh (l'Hôpital de l'Archet, Nice, France).

One hundred and eight individuals with viral load over 10,000 copies/mL who had been treated with protease inhibitors for at least three months and with NRTIs for at least six months were randomized to a control group (N=43) or a group which received genotypic testing results (N=65) before switching therapies. The genotyping technology used was the Visible Genetics TrueGene assay. For six months study participants were treated according to their study group, after which all study participants underwent any necessary treatment changes according to genotypic results received every three months. (A drug resistance table was provided to guide treatment switch decisions.)

The 6-12 month analysis assessed RNA changes from baseline at months 9 and 12, the proportion of individuals with RNA <200 copies/mL. Treatment groups were well-matched by baseline viral load (4.0 log¹⁰), CD4 count (210/mm³), prior antiretroviral treatment, and baseline mutation frequency.

The study results showed that protease inhibitor drug exposure was inversely correlated with plasma HIV RNA changes for all four protease inhibitors. What's more, genotypic guided therapy, protease inhibitor concentrations and primary protease mutations independently affected responses to therapy. Thus, both resistance assays and pharmacologic drug testing may be useful to improve treatment responses in experienced patients.

II. Prospective studies to validate therapeutic drug monitoring
a. Concentration-controlled vs. standard AZT+3TC+indinavir: Preliminary results favor concentration-controlled approach
Courtney Fletcher and colleagues at the University of Minnesota conducted a randomized study of concentration-controlled versus standard therapy with AZT, 3TC and indinavir in 24 individuals. Six month results were presented at the 39th ICAAC in September 1999 (abstract 322). All individuals received four weeks of standard therapy. Pharmacokinetics were carried out at Week 2, and individuals were randomized to concentration-controlled or standard therapy at Week 4.

The time to HIV RNA below the limit of quantification were 100 days for the concentration-controlled group compared to 176 days for the standard therapy group (p=0.056). The proportion of individuals reaching a plasma viral load below the limit of quantification were 91% (10 of 11) in the concentration-controlled group and 69% (9 of 13) in the standard therapy group. The investigators concluded that, "preliminary observations support the hypothesis that interpatient differences in antiretroviral drug concentrations contribute to heterogeneity in viral suppression."

b. ATHENA: Will it make a difference in the clinic?
ATHENA is an ongoing prospective randomized, controlled trial designed to evaluate whether therapeutic drug monitoring can contribute to improved virologic response. At the Noordwijk pharmacology workshop, David Burger (University Medical Center St. Radboud, Nijmegen, the Netherlands) presented the design of ATHENA. They are attempting to achieve protease inhibitor concentrations between 75-200% of the minimum effective concentration. Drug levels are taken whenever viral load and CD4 levels are monitored, and for viral failure, adverse events, and non-adherence.

What to measure? Trough levels aren't ideal; areas under the curve (AUCs) are impractical; fixed time points are arbitrary; so they are using random sampling with population pharmacokinetic curves. Six hundred individuals will be enrolled, of whom 50% will be treatment naive. Pharmacokinetic results are available to primary doctors within four weeks of sampling. Although everyone's plasma is tested, only half the study participants will be randomized to receive therapeutic drug monitoring test results and expert advice, while control group results are not reported to the primary care physician. The study is about two-thirds enrolled.

In Noordwijk, preliminary results indicated that between 26-41% of people were receiving less than 75% of the target protease inhibitor concentration, and between 5-11.5% were receiving more than 200%, depending on the protease inhibitor. Eighty-six percent of people on nevirapine were receiving adequate concentrations. Of course, no one knows whether the target concentration ratios are correct. It's too early to know whether physicians are using the information from the therapeutic drug monitoring assay to guide therapy. There are no correlations yet with viral load or resistance. The study is ongoing.

c. Discussions underway at AACTG.
The Adult AIDS Clinical Trials Group has set up a Therapeutic Drug Monitoring Working Group, involving members of the HIV Research Agenda Committee (RAC) and the Pharmacology Committee. Its role is to determine the role of therapeutic drug monitoring in AACTG studies. They are considering looking at therapeutic drug monitoring in four settings: 1) protease inhibitor naive; 2) optimization of protease inhibitor regimens; 3) drug intensification; 4) protease inhibitor virologic failure. They decided to move forward with options 1 and 3. [Odd choices.]

III. Therapeutic drug monitoring in clinical practice.
There seems to be more impetus behind therapeutic drug monitoring research in Europe than in the U.S. Some U.S. researchers have been heard to say that better drugs with longer half-lives, lower peak, and higher trough levels will take care of the problem, without necessitating the addition of another costly blood test in clinical practice. For example, coadministering low doses of ritonavir with other protease inhibitors such as amprenavir, indinavir, or saquinavir, allows the latter drugs to be taken in lower doses less often and—in the case of indinavir—without previous restrictions on food intake. This is because ritonavir, by inhibiting the cytochrome p450 enzyme system of the liver, slows down the metabolism of the other protease inhibitors, raising their trough levels, reducing their peak levels, and increasing the area-under-the-curve (AUC). However, the ritonavir/other protease inhibitor studies to date remain relatively short-term, and these combinations have yet to be added to antiretroviral treatment guidelines (with the exception of ritonavir/saquinavir, for which 72 week data are available).

Baltimore pharmacologist Charles Flexner gave an overview of this issue in the on-line Hopkins HIV Report (January 2000). He points out that therapeutic drug monitoring can be useful in certain therapeutic settings, such as with the anticoagulant drug warfarin, the anti-convulsant phenytoin, and the immunosuppressive, cyclosporin. However, continues Flexner, "Therapeutic drug monitoring is rarely useful for antibiotics... The therapeutic index for most antibiotics is high... Few studies... demonstrate a clear-cut relationship between antibiotic concentration and outcome... The most important exception is the aminoglycoside class, where therapeutic drug monitoring is used mainly to prevent toxicity." At the 12th World AIDS Conference in Geneva, two abstracts found a relationship between protease concentrations and virologic outcomes, while three found no such relationship. Flexner goes on to state that "it is essential to show, prospectively, that adjusting dose to achieve some pre-determined target concentration actually improves outcome." The Fletcher study cited above offers some preliminary evidence that this may be the case with AZT+3TC+indinavir, but its small size (N=24), and the increasing use of indinavir at the twice daily 800 milligram dose (in combination with ritonavir 100 or 200 mg BID) may reduce its clinical relevance.

Flexner notes that "some drugs (like AZT and 3TC) exert significant anti-HIV effects yet routinely have trough concentrations of zero." This is (at least in part) because these drugs are active in their intracellular, triphosphate forms, which are not measured by plasma tests. However, this point indicates that therapeutic drug monitoring may be more useful in certain drug classes, and for certain drugs, than in other classes and for other drugs.

In another timely discussion of therapeutic drug monitoring, the Medscape website (hiv.medscape.com) featured a debate between NIH pharmacokineticist Stephen Piscitelli (author of the useful and stimulating study which demonstrated that the popular herbal antidepressant St. John's wort causes dangerous reductions in indinavir concentrations, Piscitelli 2000) and Edward Acosta on the "limited value" (Piscitelli) vs. the "promise" (Acosta) of therapeutic drug monitoring in HIV infection. Piscitelli cites "at least seven substantial obstacles that suggest that therapeutic drug monitoring will have little significance in clinical practice" (see table below).

Piscitelli concludes that therapeutic drug monitoring may have a useful role in clinical trials, but "for treating individual patients, less attention should be placed on drug levels and more effort... focused on developing new drugs and strategies (ABT-378, indinavir+ritonavir, etc.) which achieve plasma concentrations well in excess of the IC95 to HIV."

Acosta responds by citing the previously mentioned saquinavir data and other protease inhibitor monotherapy studies. He concludes that "the significance of therapeutic drug monitoring for protease inhibitors in the treatment of HIV... has yet to be completely understood." He suggests that it may be useful for "random adherence checks", although most pharmacologists at the Noordwijk meeting disagreed with the concept that adherence could be measured pharmacokinetically.

This raises another issue, which is that if an individual is experiencing difficulty adhering to an antiretroviral regimen, measuring and adjusting the regimen dosage may not make much difference. Some therapeutic drug monitoring studies have been distorted by the so-called "white coat effect", in which patients not normally especially adherent dutifully take all of the pills they're supposed to in the knowledge that they're about to be monitored for drug levels. This would tend to overstate drug exposure over the long term.

The question of whether to measure adherence or drug levels, or both, to optimize therapeutic responses, or to prevent, or identify early and mitigate, antiretroviral treatment failure, remains unanswered at this time. Nor is it clear whether the best use of limited resources is to provide routine therapeutic drug monitoring in the clinic, or simply to develop therapeutic regimens which are more forgiving and have better pharmacokinetic profiles.

One thing that is clear, however, is that pharmaceutical manufacturers need to be more pro-active in providing access to their compounds for drug-drug interaction studies both before and after FDA approval.¤

A little over a year ago researchers began describing something that sounded more like science fiction than science. A sinister sounding mechanism in human cells was found to actually suck the revolutionary class of anti-HIV drugs, the protease inhibitors, out of the very cells where they were doing so much good. Not since the days of superfit multi-nucleoside resistant HIV had the therapeutic order seemed so convoluted. A cellular "protease pump?" How could this be? And might such a phenomenon explain the frequent failure of these drug regimens? If the effect turned out to be significant, would there be any way to reverse the process?

Now we're delving into serious grad school cell biology here, so the sciency talk quickly gets thick. Yvette has done her best to bring it down to earth. Her full report, complete with a deluge of references, is available on our website, www.treatmentactiongroup.org. But for now, take a deep breath and slog on.

What is P-Glycoprotein?
P-glycoprotein (P-gp) is a plasma membrane protein which acts as a localized drug transport mechanism, actively exporting drugs out of the cell. The effects of P-gp on the distribution, metabolism and excretion of drugs—including protease inhibitors—in the body is great. P-gp activity, for example, decreases the intracellular concentration of cancer drugs, enabling resistance to develop to them. The same may be true for protease inhibitors.

What is the function of P-gp?
The normal physiological function of P-gp in the absence of therapeutics or toxins is unclear. Studies of MDR-1 knock-out mice (mice bred in the lab specifically for the absence of the MDR-1 gene and, therefore, no P-gp activity) show that they have normal viability, fertility and a range of biochemical and immunological parameters. Predictably, they do have delayed kinetics and clearance of vinblastine, and they accumulate high levels of certain drugs (vinblastine, ivermectin, cyclosporin A, dexamethasone and digoxin) in their brains. The mice also demonstrated marked increases in the levels of these drugs in the testis, ovary and adrenal gland compared with wild type mice. It has been reported that some MDR-1a knock-out mice develop a severe, spontaneous intestinal inflammation similar to human inflammatory bowel disease; however, this has not been observed by other researchers.

Mechanism of action
The majority of published data suggest that P-gp acts as a transmembrane pump which removes drugs from the cell membrane and cytoplasm. It has further been proposed that P-gp acts like a hydrophobic vacuum cleaner or "flippase," transporting drugs from the inner leaflet of the plasma membrane lipid bilayer to the outer leaflet or to the external medium.

P-gp substrates and inhibitors
There have been various attempts to classify compounds based on their effect on or interaction with P-gp. A number of chemicals, including anticancer drugs, have been categorized based on their effect on ATPase activity of human P-gp. Class I compounds in low concentrations stimulate ATPase activity and in high concentrations inhibit it. Kinetic analyses show they have high affinity for the active site and low affinity for the inhibitory site. They include vinblastine, verapamil and taxol. Class II compounds stimulate ATPase activity in a dose-dependent manner without any inhibition and interact only with the active site. They include bisantrene, valinomycin and diltiazem. Class III compounds, which bind to the inhibitory site with high affinity, inhibit both basal and verapamil-stimulated ATPase activity. They include cyclosporin A, rapamycin and gramicidin D.

Some studies support a model of P-gp in which there is a region or multiple regions of interaction rather than one or two simple binding sites. Molecules interacting with P-gp may be classified as "substrate" or "antagonist." It has also been demonstrated that one possible mechanism of action for P-gp-mediated resistance to chemotherapeutic agents is through gene rearrangement.

P-gp and HIV
All HIV protease inhibitors currently in use are transported by P-gp, with affinities in the order ritonavir>nelfinavir>indinavir>saquinavir. In MDR-1a knock-out mice, plasma levels of indinavir, saquinavir and nelfinavir were 2-5 times higher compared with control mice. This strongly suggests that P-gp transport at the intestinal and/or hepatic level limits the systemic bioavailability of these drugs. The effect of P-gp on limiting oral bioavailability and tissue distribution of protease inhibitors has obvious implications for the effectiveness of protease inhibitor-containing regimens. Poor penetration of protease inhibitors into the brain, testis and other "sanctuary sites" may result in de facto compartmental mono or dual antiretroviral therapy with ongoing HIV replication and development of resistance.

Blockage of P-gp may be useful in facilitating greater intestinal absorption, bioavailability and penetration of protease inhibitors into HIV sanctuary sites as well as reduced excretion. It may also simplify protease inhibitor containing regimens by reducing the oral doses of protease inhibitors and the frequency at which they are taken. Higher protease inhibitor levels in these sites may result in greater suppression of viral replication in these sites, but they may also result in unwanted adverse effects. These effects may not be limited to protease inhibitors but may extend to other co-administered drugs. For example, the antidiarrheal agent loperamide is an opiate which acts peripherally and penetrates the brain poorly. However, in MDR-1a knock-out mice, loperamide exhibits strong morphine-like central nervous system (CNS) effects.

Transport of HIV protease inhibitors can be inhibited by P-gp inhibitors like cyclosporin A, verapamil, and PSC833. Ritonavir, saquinavir, nelfinavir and indinavir have also been shown to inhibit transport of some of the known P-gp substrates. But, with the exception of ritonavir and possibly saquinavir, the P-gp inhibiting effects of the protease inhibitors are weaker than those of the established inhibitors like verapamil or cyclosporin A.

The P-gp transport system clearly has major implications for HIV infection and its treatment. There is still much left to be understood. The effects of P-gp expression, or alterations of P-gp expression, on the immune systems of HIV-infected individuals need to be fully studied and evaluated. The impact of P-gp expression and its inhibition on protease inhibitor therapy needs to be assessed. ¤