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Pharmacology at the 2009 Conference on Retroviruses and Opportunistic Infections
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By: Stephen Becker MD
The 16th CROI was notable for the small number of pharmacology presentations. However among the presentations several were of considerable potential importance. In his Bernard Fields Lecture during the Opening Session, Bob Silicaino reviewed his landmark work on viral persistence and latent reservoirs (summarized in the natap report by David Margolis), and connected this to his current investigations defining drug potency. Early phase studies of possible alternatives to ritonavir as a pharmacokinetic enhancer were presented by scientists from both Gilead Sciences and Sequoia Pharmaceuticals. Finally a novel means of measuring intracellular concentrations of nucleoside reverse transcription inhibitor was presented by Charles Flexner and colleagues. While none of these presentations will permit clinicians new tools or agents immediately, all have great potential impact for the development of new antiretroviral agents and an enhanced understanding of how to optimally utilize current and future therapies in the treatment of HIV infection.
Non-Ritonavir Pharmacokinetic Enhancers
Initially developed as a protease inhibitor in the early 1990s, ritonavir's use to the field of antiretroviral therapeutics is in the enhancement (or boosting) of other PIs. Ritonavir (RTV) has complex pharmacologic activity that results in the elevation of coadministered PI levels. This has resulted in the enhanced therapeutic potency of the coadministered PI, a notable reduction in the development of PI-associated resistance mutations, and greater ease of dosing. These favorable attributes are achieved by several actions of RTV including enhanced oral bioavailability, slowing of coadministered drug metabolism, and reduction of drug (efflux) transport from target cells. These actions are mediated through RTV's inhibition of the predominant drug metabolizing enzyme, CYP3A4, and xenobiotic transporter, P-glycoprotein (P-gp) located in critical tissue compartments of the gut, liver and lymphocytes. However RTV's inhibitory activity is not limited to CYP3A4 or P-gp, but rather extends to other drug metabolizing enzymes and cellular transport mechanisms, leading to complex and often unpredictable drug-drug interactions. Further the pharmacologic effects of RTV are not limited to enzyme and transporter inhibition, but also include induction of many of these same drug metabolizing enzymes and cellular transporters. The generalized effect of RTV on other enzyme and transporter systems is one aspect that has prompted investigation of alternative approaches to enhance PI drug exposures.
With few exceptions, HIV PIs are given with RTV in doses that range from 100mg to 400mg per day. As patients and clinicians are well aware the considerable benefits of PI enhancement often come at a cost. Clinically relevant gastrointestinal intolerance, adverse effects on lipid and glucose metabolism, and in its oral solution formulation used by young children with HIV, a taste so odious that descriptions are not appropriate for a professional report, occur with a frequency that may limit use. High cost, and until recently the requirement for refrigeration have further constrained use, particularly in resource poor settings. The search for a RTV substitute is nearly as old as the as the agent itself and has included other approved drugs, or natural compounds such as grapefruit juice, which inhibit CYP3A4 metabolism. None have proven as effective as RTV itself. The reports by scientists from Gilead Sciences and Sequoia Pharmaceutical are therefore viewed with considerable interest.
As defined by both groups a suitable RTV alternative would possess several qualities:
• A small molecule with good oral bioavailability and heat stability
• Specificity as a CYP3A4 inhibitor with little or no effect on other CYP450 or conjugative enzyme systems
• No inhibition of P-gp or other major cellular transport mechanisms
• No induction of CYP450 or conjugative enzyme systems
• No inherent anti-HIV activity
• No adverse effect on lipid or carbohydrate metabolism
• High solubility permitting an oral solution for use in pediatrics
Brian Kearney from Gilead Sciences presented data on their PK enhancer, GS-9350 (abstract 40). Selected aspects of the pre-clinical program were summarized. This includes a greater specificity of GS-9350 for CYP3A4 inhibition compared to RTV, lessened enzyme induction (measured by effect of the agent on the nuclear receptor PXR) and less effect on adipocytes, mediators of lipid and glucose abnormalities seen with RTV and other PIs. Indeed GS-9350 has 5-fold less in vitro activity on adipocytes than RTV, though it maintains significantly greater activity than the lipid and glucose 'neutral' PI atazanavir. What effect on lipids and glucose metabolism will be noted when the drug is dosed for a longer period of time remains to be seen. Phase I data was also summarized describing the short-term safety, pharmacokinetics and CYP3A4 inhibitory activity of varying doses of the drug. Inhibition of CYP3A4 was determined by use of the standard midazolam probe. GS-9350 has an effect on 3A4 comparable to RTV at doses that could be used clinically. Finally, preliminary data was presented of a co-formulated pill containing GS-9350, elvitegravir, tenofovir and emtricitabine. As readers are aware, to date elvitegravir has been boosted by RTV during its ongoing clinical development, and while it is premature to imagine that 9350 will substitute for RTV, it is clear from a physicochemical perspective that the agent can be co-formulated with other ART.
Robert Guttendorf from Sequoia Pharmaceuticals, a small East Coast biotech presented data on their pharmacokinetic enhancer, SPI-452 (abstract 41). Perhaps reflecting the resource differential of a small biotech compared to those of well established pharma, the Sequoia presentation was more modest in scope. However, from early pre-clinical and phase I data SPI-452 appears to be a viable RTV substitute. Like the Gilead compound, SPI-452 has 3A4 inhibitory activity akin to that of RTV, lacks anti-HIV activity, is not an inhibitor of P-gp and is not an apparent inducer of oxidative or conjugative enzyme systems. SPI-452 has been studied in vitro with all of the current HIV PIs as well as an anti-HCV PI. Studies in animals demonstrate PI boosting effects similar to those of RTV. Clinical studies in healthy volunteers have evaluated the effect of SPI-452 on coadministered saquinavir, darunavir and atazanavir. Data to date show enhanced PI exposures with no undue or unanticipated safety signal.
Sequoia regards these findings as proof of concept, and plan to progress the compound through further human clinical trials
The reported successful early stage development of RTV alternatives is encouraging. Clearly both compounds require further clinical evaluation to determine safety and efficacy, a process that will take several years. Further complicating development is the regulatory pathway for such agents. Session co-chair Kimberly Struble, from the US FDA, indicated that these pharmacokinetic enhancers - though lacking anti-HIV activity - would be evaluated in the US by the Antivirals Division in a fashion consistent with typical antiretroviral agents. Whether either GS-9350 or SPI-452 will be developed as a stand-alone agent, or co-formulated with other ARTs, or both remains to be seen. The future development of both agents will assuredly be watched with interest.
Defining Antiretroviral Potency - The Role of Dose Dependency
The Bernard Fields Lecture was given by Bob Silicaino from Johns Hopkins University School of Medicine. Summarizing more than a decade of sentinel work on viral persistence (see natap report by David Margolis), Silicaino followed this discussion with work that currently involves his laboratory and collaborators. Attempting to better define and delineate concepts that assess antiretroviral potency he described an approach that adds dose-dependent antiviral response to the more traditional measures of IC50 and the inhibitory quotient. This approach was first published in 2008 (Shen, Nature Medicine).
The definition of antiviral potency is more complex than it might seem. Traditional measures such as the IC50 (the drug concentration that yields 50% of the maximum inhibitory effect) and the inhibitory quotient or IQ (the ratio of plasma drug concentration to IC50) have been employed for numerous antimicrobial agents since the 1980s. More recently viral decay curves, using both viral RNA reduction, and time to maximal RNA reduction, have been presented as an additional measures of putative potency. In the clinical realm however potency is complicated by factors such as medication compliance, drug-related toxicity, comorbid conditions and drug-drug interactions. Using the IC50 and IQ Silicaino added the effect of the slope of dose dependency to the consideration of drug potency. As traditionally understood dose-dependency is the relationship between drug dosage and pharmacodynamic effect. Dose-dependency is plotted as the slope of drug concentration vs. antiviral activity, and is traditionally expressed in a semi-log fashion. In this form, despite similar IC50 values and slope of dose response, near 100% inhibition is achieved at higher drug concentrations for most agents. The drugs appear to have identical activity. When using a log-log plot instead, however, drugs with similar IC50 or IQ now demonstrate marked differences in the slope of dose response at clinically relevant drug concentrations. This observation is important and may prove to be clinically relevant.
Based on these observations the group developed a novel and highly sensitive in vitro assay to better define actual drug activity. Called the instantaneous inhibitory potential, or IIP, this metric yields a measure of the log reduction of HIV RNA noted in a single round of viral replication in the presence of drug. This assay appears far more sensitive than previous measure of drug activity. To give an idea of the assay's sensitivity, both entecavir and acyclovir - neither of which were felt to have anti-HIV activity at the time of their development - demonstrate anti-HIV activity, confirming recent clinical observations. To demonstrate the power of this approach Silicaino pointed out that the IIP of a drug with a slope of 3 has 10,000 times greater potency than a drug with a slope of 1. When the IIP was used to measure current antiretrovirals the log reduction of HIV RNA was approximately 1-4 logs for the NRTIs, 4-6 logs for NNRTIs and up to 10 logs for certain PIs. For the integrase inhibitors values were close to 1. While these observations perhaps help explain the superiority of PI and NNRTI-based ART when compared to triple NRTI therapy, the apparent lack of potency of the integrase agents in this assay is at odds with recent clinical experience. There may be several explanations for this. First, on a purely mechanistic basis NRTIs and integrase inhibitors (IIs) target the interaction between a single molecular complex of the viral enzyme and nucleic acid, mediating the key processes of chain termination for the NRTI, or strand transfer for the II. NNRTIs and PIs, by contrast target the enzymes directly. In essence, there is 'more' target substrate available for inhibition by NNRTI or PI agents than for either NRTIs or IIs, and this translates to higher IIP values. A second explanation, the stage of viral replication, may also account for lower IIPs among the class of IIs. Strand transfer inhibition occurs at a later stage in viral replication where the second phase compartment of replication is smaller for the II class than other classes. The clinical performance of the IIs also demonstrate that while more precise in vitro assays may prove to explicate critical aspects of drug activity, that the situation in the clinic, with multiple agents acting at differing stages in the viral life cycle, is not only more complex , but more telling in the end.
Silicaino concluded his remarks with the suggestion that this approach may have application in interpreting drug resistance and in the fashioning of subsequent therapeutic regimens. To be clinically useful the approach will require an estimate of the combined effect of multiple agents, an issue that has limited current resistance testing as well. The approach outlined by Silicaino may also prove to have application for hepatitis C and other viral diseases, vaccine effector responses, and therapies for other non-viral infectious diseases.
Measurement of Intracellular NRTI Levels
From the time of their development it has been appreciated that the active moiety of nucleoside reverse transcription inhibitors is the intracellular tri-phosphate (TP). The ability to measure NRTI-TP during the past several years has lead to a more complete understanding of intracellular pharmacokinetics and pharmacodynamics. Indeed the dosing schedules of several approved agents have been revised based on determination of the intracellular half-life of the TP. These measurements have been time consuming, expensive, required large volumes of blood and limited in the level of TP detection. Charles Flexner in collaboration with investigators from York, UK and GlaxoSmithKline (abstract 704) reported on the use of a novel technique to quantify NRTI-TP. Using Accelerator Mass Spectrometry (AMS), radio-labeled NRTI, in this instance 14carbon-labeled zidovudine, the investigators established that the method is capable of detecting levels of ZDV-TP to 10-21, considerably more sensitive than current measures. The application of AMS is potentially substantial for both currently available and investigational agents allowing determination of intracellular metabolites in numerous tissue compartments and cellular subsets. One can envision use of such techniques in the development programs of microbicides or in the development of agents used for pre-exposure prophylaxis.
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1] The IIP does not predict the magnitude or durability of viral suppression, outcomes that are based on the more complex mix of pharmacokinetics, volume of distribution, compartment penetration, barriers to resistance as well as numerous host-defined factors. One pharmacokinetic factor that illustrates this point well is the effect of drug half-life (t1/2). Changes in the plasma drug concentration have a profound effect on the IIP. The IIP drops rapidly for drugs with a short t1/2 (indinavir and saquinavir, both agents with very high IIPs), while those agents with a longer t1/2 (efavirenz, atazanavir and darunavir) maintain high IIPs even following a missed dose.
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