Protease inhibitors (PI) may be monitored for two key reasons: to ensure that drug levels are high enough to suppress HIV and to allow dose alterations when high levels of drug lead to toxicity.

Ensuring efficacy

To ensure the efficacy of protease inhibitors, tests can identify people who do not achieve an adequate PI trough level. (A trough level is the lowest concentration or C_min of a drug in the blood over one dosing period e.g. 12 hours).

Drug monitoring generally involves a one-off blood sample taken in the morning, just before the first dose of the day is due, when the concentration of the drug in the blood will be at its lowest. The earliest this test should be done is around two weeks after starting the drug when blood levels should have stabilised.

People whose trough level is too low then have the option to modify their treatment, for example by continuing with their current combination and adding a small amount of ritonavir. Ritonavir inhibits the effects of both P450 and P-gp, and this is why its use in combination with other PIs raises the blood levels of the other drug.

An alternative application for drug level monitoring is to identify users of ritonavir who are absorbing the drug quickly and getting very high peak levels just after a dose, which may cause unpleasant side-effects. In these circumstances there is the option to split the ritonavir dose (change from 600mg or 7.5ml twice daily to 300mg or 3.75ml four times daily) to iron out the peaks.

The Athena study, carried out in the Netherlands, has suggested that therapeutic drug monitoring of nelfinavir levels is valuable. After one year of follow-up, 81% of those who received therapeutic drug monitoring and dose adjustment where necessary had undetectable viral load, compared with 59% of those who did not receive dose adjustments. People in the dose adjustment arm may have received up to three dose adjustments, since TDM was also carried out after a dose adjustment to check that nelfinavir levels had improved. After the first measurement, patients were advised on the importance of taking nelfinavir with food. If blood levels were still sub-optimal after the second or third measurements, the dose of nelfinavir was increased.

One unanswered question about this study is the proportion of individuals whose plasma nelfinavir levels normalised without the need for dose adjustment, following a discussion with their doctor regarding the need to follow dosing and food instructions carefully. How much of the benefit of TDM was derived from spotting that a patient had problems with adherence and subsequently devoting time to addressing those problems? Would it be cheaper in the long run to do this with all patients rather implement therapeutic drug monitoring?

The Athena study also looked at people receiving indinavir, and the results from this arm of the study suggest that dose adjustment based on TDM also resulted in a significantly greater chance of undetectable viral load in the TDM arm after one year (75% vs 48%), and a significantly higher rate of discontinuation due to toxicity in those receiving indinavir alone or IDV/RTV 800/100mg twice daily who did not receive TDM results and subsequent dose adjustments (Burger 2001).

The PharmAdapt study, on the other hand, showed no benefit from therapeutic drug monitoring carried out four weeks after switching to a new protease inhibitor on the basis of a genotypic resistance test. However, this study has a number of flaws which may not make it applicable to clinical practice. The sample size may have been too small to detect differences between the two arms in this randomised study. Also, the cut-off points at which dose adjustment might be necessary were derived from the drug levels necessary to inhibit wild-type virus, not drug resistant virus. Finally, a four-week delay between testing and dose modification in patients with drug resistance may not have been helpful, since this delay would have increased the risk that additional drug resistance mutations could develop.

The Genophar study also showed no short-term benefit to therapeutic drug monitoring when used in conjunction with the results of genotypic resistance testing to optimise drug dosing at week 8 of treatment (Bossi 2004).

Side-effects and monitoring

With the growing concern regarding drug-related toxicity and the impact this has on peoples ability to take antiretroviral therapy in the longer term, drug monitoring is increasingly being used to look for a link between side-effects and high drug concentrations. As discussed in Variability in protease inhibitor levels in Anti-HIV therapy: Testing drug levels, high concentrations of certain drugs in the blood may worsen the severity of side-effects. Doctors may also use drug monitoring to check that lower dosages with improved tolerability are still effective against HIV.

Consequently, therapeutic drug monitoring may be used to determine an appropriate dosage which achieves adequate levels while minimising side-effects.

Drawbacks of using drug monitoring to assess protease inhibitor levels

A number of factors may distort the results of therapeutic drug monitoring (TDM), particularly in relation to PIs.

For example, levels of indinavir can vary during the menstrual cycle (Adams), and levels of alpha-1 acid glycoprotein (AAG) can also influence PI levels (Sadler). AAG levels rise during periods of infection, stress and injury, speeding the clearance of PIs from the blood.

The timing of the sample is also critical, because food can cause drug levels to vary. Stephen Piscitelli of the National Institutes of Health Clinical Pharmacokinetics Research Laboratory suggests that the best time to draw blood for TDM is just after dosing if a drug has been taken with food because a meal will delay absorption. Testing after dosing and food will reveal the true trough level reached before absorption. However, other pharmacologists question this advice, arguing that all PIs are dosed on a full stomach because this improves absorption, and that is the reason for current advice to test before the first dose of the day, and before concentrations begin to rise again.

The usefulness of one-off testing to assess PI concentrations has been questioned by a team of researchers who found one trough sample did not correspond with drug concentrations established from repeated testing (Merry). Another study found that the bodys daily cycle affects concentrations of nelfinavir with higher trough levels seen in the morning compared to the evening (Burger). If full pharmacokinetic testing is required, then TDM may be more difficult to integrate into routine clinical practice.

There is also recent evidence that intracellular levels of the protease inhibitors do not necessary accord with blood plasma levels (Hennessy).

Finally, even if drug concentrations appear to be optimal (according to pre-defined standards drawn from pharmacokinetic studies, drug concentrations may not be adequate to inhibit virus with reduced sensitivity to a drug. For example, if peak drug concentrations are just about adequate, this may result in effective inhibition in an individual with wild-type virus, but viral rebound in an individual with virus that exhibits a four-fold loss of sensitivity to the drug.

Quality control

A quality control system for therapeutic drug monitoring has recently been established in Europe following studies which showed considerable variability in the quality of drug level testing.

Nijmegen University Medical Centre in the Netherlands organised the first international comparison study to determine how much variation exists in drug level measurements from different laboratories.

The researchers sent plasma samples from HIV-negative volunteers, spiked with three different, standardised doses of indinavir, nelfinavir, ritonavir and saquinavir, to 10 laboratories worldwide. Measurements were considered to be correct if they fell within 20% of the concentration of drug that should have been present based on the original weight of drug dissolved in the plasma sample. This method had been chosen to minimise interpersonal variation in drug metabolism.

Indinavir was the drug with the most consistent detection rate; 86% of measurements were correct. In contrast, ritonavir was poorly measured; only 42% of measurements were correct. For saquinavir and nelfinavir, two-thirds (65%, 67%) of measurements were correct.

Only one laboratory got all the measurements correct. In all cases the ability to detect low concentrations correctly was worse than for other concentrations (Aarnoutse 2002).

Despite the significant levels of inaccuracy, the Dutch study showed a marked improvement in quality compared to a study of nelfinavir drug level monitoring from five US labs which found a five-fold variation between labs.