Thesis
Reference
Population pharmacokinetics and pharmacogenetics of antiretroviral drugs
ARAB, Mona
Abstract
The population pharmacokinetic approach has proved its usefulness for Therapeutic Drug Monitoring (TDM) and it allows in particular, the quantification of pharmacokinetic variability, and offers the possibility to detect clinically useful predictors of treatment outcomes. In that perspective, we performed a first pharmacogenetic-based population pharmacokinetic analysis of efavirenz in HIV infected individuals. We demonstrated that functional alleles of CYP2B6 were associated with higher Efavirenz exposure. We proposed an innovative non-linear model to describe the relationship between phenotypes and functional allelic variants and accordingly predict Efavirenz exposure. We developed Pop-PK models for darunavir, etravirine and raltegravir. We identified demographic and environmental factors affecting drug disposition. We simulated compare drug exposure under several dosage regimens. Finally, we derived reference pharmacokinetic curves for 7 widely used antiretroviral drugs based on an innovative concept of meta-analysis that could represent useful tools for clinicians to interpret the value of an individual plasma [...]
ARAB, Mona. Population pharmacokinetics and pharmacogenetics of antiretroviral drugs. Thèse de doctorat : Univ. Genève, 2012, no. Sc. 4400
URN : urn:nbn:ch:unige-184576
DOI : 10.13097/archive-ouverte/unige:18457
Available at:
http://archive-ouverte.unige.ch/unige:18457
Disclaimer: layout of this document may differ from the published version.
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Section des Sciences Pharmaceutiques Dr. Chantal Csajka Pharmacie Hospitalière et Clinique
Population Pharmacokinetics
& Pharmacogenetics of Antiretroviral Drugs
Thèse
présentée à la Faculté des sciences de l’Université de Genève
pour obtenir le grade de Docteur ès sciences, mention sciences pharmaceutiques
par
Mona ARAB ALAMEDDINE
de
Tripoli - Liban
Thèse N°4400
Genève – 2012
REMERCIEMENTS
Je tiens à exprimer ma plus sincère reconnaissance au Dr. Chantal Csajka qui m’a offert l’opportunité d’effectuer ce projet au sein de son groupe. Je la remercie pour sa confiance, sa disponibilité et son soutien tout au long de ma thèse.
Je remercie profondément le Professeur Thierry Buclin qui m’a transmis ses compétences en Pharmacologie Clinique et plus spécifiquement en Pharmacocinétique, ce domaine très intéressant dans lequel j’ai choisi de continuer ma carrière. Je le remercie également pour son encadrement et ses conseils ainsi que pour ses remarques pertinentes et son enthousiasme.
Merci au groupe du laboratoire de Pharmacologie Clinique pour tous les dosages médicamenteux sans lesquels ce travail n’aurait pas pu être effectué.
Un grand merci au groupe de l’institut de Microbiologie pour leurs analyses génétiques qui ont rendu les résultats de ce travail beaucoup plus précieuses et utiles.
Je remercie tous mes collègues de la division de Pharmacologie Clinique du CHUV, Monia, Ursula, Monique, Verena, Catherine, Delphine, Nicolas, Alice, Françoise, Laura, Aline, Hélène, Eric, Haythem, Manuel et Elyes. Je leur dis merci pour votre amitié et votre bonne humeur et merci pour les bons moments de rigolade. J’adresse un merci particulier à Ali Maghraoui pour son soutien informatique et son encouragement permanent.
Enfin, je remercie mes amis, ma famille et j’exprime toute ma reconnaissance à mon
mari Said et mes deux enfants Zein et Omar pour leur amour et leur soutien durant
ces quatre ans.
ABSTRACT
Population Pharmacokinetics and Pharmacogenetics of Antiretroviral Drugs The giant advances in the field of antiretroviral therapy (ART) has drastically decreased the HIV related morbidity and mortality. On the other hand, ART is a lifelong treatment; therefore, many factors should be taken into consideration in order to ensure therapy success, in particular drug tolerability that directly influences patient’s adherence and treatment success.
Despite the large pharmacokinetic variability reported for antiretroviral drugs, ART is prescribed at standard dosage. Therefore, dosage individualization based on the identification of demographic, environmental and genetic factors affecting drug disposition is can be valuable in a significant number of HIV-infected patient to ensure treatment efficacy and tolerability.
Therapeutic Drug Monitoring (TDM) can be used to ascertain whether treatment failure is caused by intrinsic resistance or by insufficient drug levels, or whether toxicity might be related to excessive concentrations. Despite routine use of TDM is not yet recommended, it can have an important place in some situations to optimize ART response. The population pharmacokinetic (Pop-PK) approach has proved its usefulness for TDM and has gained increasing recognition over the years because it allows in particular, the quantification of inter-individual and intra-individual variability, and offers the possibility to detect clinically useful demographic and genetic predictors of treatment outcomes.
In that perspective, we performed a first pharmacogenetic-based population pharmacokinetic analysis of efavirenz (EFV) in HIV infected individuals. This study aimed at characterizing the joint impact of genetic polymorphisms in the main (CYP2B6) and accessory metabolic pathways (CYP2A6, CYP3A4/A5) involved in EFV elimination. We demonstrated that functional alleles of CYP2B6 were associated with higher EFV exposure. In addition, we ascertained the importance genetic variations in EFV accessory metabolic pathways in EFV disposition. We proposed an innovative non-linear model to describe the relationship between phenotypes and functional allelic variants and accordingly predict EFV exposure.
This study provided new insights in the understanding of the mechanisms of genetic influences and can be used to build up rational dosage guidance according to multiple genetic polymorphisms.
We then developed the first Pop-PK models of new generation antiretroviral compounds including darunavir, etravirine and raltegravir. The pharmacokinetic modeling allowed us to identify some demographic and environmental factors affecting drug disposition.
Furthermore, we integrated pharmacogenetic data in the Pop-PK models that enabled us to assess the impact of some known genes on the variability and to test the potential influence of new genes on drug disposition. We performed model-based simulations to predict and compare the exposure profile of the drug under several dosage regimens.
In the last part of this thesis, we aimed at exploiting the available Pop-PK models to develop clinically useful tools for TDM. In that respect, we performed a systematic review of published Pop-PK models and derived reference pharmacokinetic curves for 7 widely used antiretroviral drugs based on an innovative concept of meta-analysis. These reference curves were thought to be well representative of all the existing data and could represent useful tools for clinicians to interpret the value of an individual plasma concentration whenever needed.
In conclusion, the population pharmacokinetic analyses have contributed to a better understanding of the demographic and genetic sources of variability that may lead to extreme drug exposure. Using a meta-analysis approach, we have developed reference pharmacokinetic curves that can be useful for TDM-guided dose adaptation. Both approaches are likely to optimize ART efficacy and reduce its toxicity in a significant number of HIV-infected patients.
RÉSUMÉ
Pharmacocinétique de Population et Pharmacogénétique des médicaments antirétroviraux
Depuis l’introduction de la thérapie antirétrovirale (ART), la morbidité et la mortalité liées au VIH ont considérablement diminué. D'autre part, l'ART est un traitement à vie et de nombreux facteurs doivent être pris en considération afin d'assurer le succès de la thérapie, en particulier la tolérance des médicaments qui influence directement l'adhésion du patient et le succès du traitement. Malgré la grande variabilité pharmacocinétique connu pour les médicaments antirétroviraux, ceux-ci sont toujours prescrits à des posologies standard. Par conséquent, l'individualisation du traitement basée principalement sur l'identification des facteurs démographiques, environnementaux et génétiques susceptibles de moduler l'élimination des médicaments pourrait être bénéfique pour un grand nombre de patients infectés par le virus afin d'assurer l'efficacité et la bonne tolérance du traitement.
Le suivi thérapeutique des médicaments (TDM) représente une approche efficace pour piloter l’ART. Le TDM permet d’une part de vérifier si l’échec thérapeutique est du à une exposition sous-optimale aux médicaments ou à la présence d’une souche résistante, d’autre part il permet de contrôler si l’intolérance aux médicaments est provoquée par des concentrations excessives. Bien que l’utilité du TDM des ART dans la routine clinique n’ait pas été démontrée dans des études randomisées, le suivi des concentrations joue un rôle important dans certaines situations afin d'optimiser la réponse au traitement. La pharmacocinétique de population (Pop-PK) a pu démontrer son utilité pour le TDM car elle permet de quantifier des variabilités interindividuelles et intra-individuelles dans ces concentrations, et de détecter les facteurs démographiques et génétiques qui influencent la réponse thérapeutique.
Dans cette perspective, nous avons effectué la première analyse de pharmacocinétique- pharmacogénétique de population pour l’efavirenz chez les patients infectés par le VIH.
Cette étude visait à étudier l'impact simultané des polymorphismes génétiques impliqués dans les voies métaboliques principales (CYP2B6) et secondaires (CYP2A6, CYP3A4/A5) de l'élimination de l'EFV. Nous avons démontré que les individus ayant un polymorphisme génétique du CYP2B6 présentaient une exposition élevée à l’efavirenz. En outre, nous avons constaté que les variations génétiques des voies métaboliques secondaires ont une influence significative sur les taux plasmatiques d’efavirenz surtout en présence de polymorphisme génétique du CYP2B6. Nous avons proposé un modèle non-linéaire qui décrit la relation entre les phénotypes et les variantes génétiques et qui pourrait en
conséquence prédire les taux d’efavirenz. Cette étude a ouvert de nouvelles perspectives dans la compréhension des influences génétiques sur la pharmacocinétique et peut être utilisée pour orienter l’ajustement posologique d’efavirenz en fonction des polymorphismes génétiques.
Ce travail a permis également à développer les modèles de pharmacocinétique de population pour les nouvelles molécules telle que le darunavir, l'étravirine et le raltégravir.
Ces modèles ont permit d’identifier certains facteurs démographiques et environnementaux influençant le métabolisme de ces médicaments. Par ailleurs, nous avons intégré les données pharmacogénétiques dans les modèles PK-Pop qui afin de mesurer l'impact de certains gènes connus sur la variabilité pharmacocinétique et de d’évaluer l'influence potentielle de nouveaux gènes sur l'élimination de ces médicaments. Nous avons effectué des simulations basées sur des modèles pour prédire et comparer les profils d'exposition sous plusieurs schémas posologiques.
Finalement, nous avons effectué la première revue systématique des modèles de pharmacocinétique de population dans le but d’en tirer une utilité et une application clinique.
En se basant essentiellement sur le concept de la méta-analyse, nous avons dérivé des courbes pharmacocinétiques de référence pour 7 médicaments antirétroviraux souvent prescrits. Ces courbes de référence pourraient être représentatives de tous les modèles publiés dans la littérature et pourraient donc servir d'outils cliniques pour interpréter la valeur d'une concentration plasmatique chez un patient.
En conclusion, les modèles de pharmacocinétique de population ont contribué à une meilleure compréhension des facteurs démographiques et génétiques qui déterminent la variabilité pouvant ainsi conduire à des concentrations trop basse ou trop élevées des médicaments. En utilisant une approche de méta-analyse, nous avons développé des courbes de pharmacocinétiques de référence qui peuvent être utiles pour l'adaptation de dose guidée par le TDM. Ces deux approches sont susceptibles d'optimiser l'efficacité ART et de réduire sa toxicité dans un nombre significatif de patients infectés par le VIH.
TABLE OF CONTENT
CHAPTER 1: INTRODUCTION 1
Overview ... 1
HIV transmission and life cycle ... 1
Infection with HIV and progression to AIDS ... 2
Antiretroviral treatment ... 3
Factors Affecting Antiretroviral Drug Treatment ... 5
Population Pharmacokinetic Modeling and TDM of ART ... 5
Current Challenges to ART ... 6
Objective of the thesis ... 7
CHAPTER 2: LITERATURE REVIEW 11
Abstract ... 11
Introduction ... 13
Non Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) ... 14
Protease Inhibitors (PIs) ... 18
Integrase Inhibitors ... 24
Conclusion ... 26
CHAPTER 3: POPULATION PHARMACOKINETICS 41 CHAPTER 3.1: EFAVIRENZ 45
Abstract ... 47
Introduction ... 47
Material and Methods ... 48
Results ... 54
Discussion ... 62
CHAPTER 3.2: RALTEGRAVIR 69
Abstract ... 71
Introduction ... 71
Materials and methods ... 72
Results ... 76
Discussion ... 84
Supplementary Material ... 87
CHAPTER 3.3: ETRAVIRINE 97
Abstract ... 99
Introduction ... 99
Materials and Methods ... 100
Results ... 104
Discussion ... 112
Supplementary material ... 114
CHAPTER 3.4: DARUNAVIR 121
Abstract ... 123
Introduction ... 123
Materials and methods ... 125
Results ... 130
Discussion ... 137
Supplementary material ... 140
CHAPTER 4: TDM OF ANTIRETROVIRALS 145
Introduction ... 147
Material & Methods ... 148
Results ... 153
Discussion ... 185
CHAPTER 5: DISCUSSION AND CONCLUSION 197
1
CHAPTER 1: INTRODUCTION
Overview
Human Immunodeficiency Virus (HIV) is a genetically related member genus of the Lentivirus genus of the Retroviridae family that primarily infects white blood cells [1]. In 1983, HIV was confirmed to be the causative agent of the Acquired Immune Deficiency Syndrome (AIDS) [2], that was first reported in 1981 as a rare form of pneumonia and skin cancer in the United States of America [3]. The structure of HIV is depicted in Figure 1.
Figure 1: Structure of Human immune deficiency virus. From:www.yale.edu/HIV/hivstructure.html
HIV transmission and life cycle
HIV is transmitted sexually, maternofetally and by exposure to infected blood [4, 5]. HIV envelope gp120 protein binds to CD4, and enables the membrane proximal portion of the envelope gp41 subunit to bind to the CCR5 or CXCR4 coreceptor of the T cells and macrophages, thus triggerring the fusion of HIV envelope with the plasma membrane (Figure 2). HIV can also enter target cells by endocytosis. Once the viral core is released into the cytosol, HIV reverse transcriptase converts RNA into DNA within the reverse transcription complex that will later mature into the preintegration complex. The latter delivers reverse transcribed HIV DNA to the nucleus for chromosomal integration. Once the integration of
2
viral genomic DNA into the host chromosome is achieved, viral transcription is activated to produce three major transcripts like host RNA that are later translated to generate Gag-Pol gene products and incorporated as genomic RNA into nascent virions at cell membrane sites where the envelope and capsid proteins assemble before budding [6]. During budding, protease cleaves the precussor protein to liberate the individual Gag and Pol component that t will later mature to form their typical cone shaped inner part of the virion [1].
Figure 2: Model of the HIV life cycle and known interactions between HIV and innate immunity. HIV RNA and nascent capsid protein (CA) can be detected in dendritic cells (left) by TLR7 and an unknown sensor, respectively. HIV DNA can be detected in CD4 T cells and macrophages (right) by an unknown cytosolic DNA sensor that signals through STING and TBK1. The host factor TREX1 inhibits innate immune detection of HIV DNA by metabolizing nonproductive RT products. Detection of HIV by any of these three innate immune pathways activates IFN genes. From Yan et al. Curr Opin Immunol. 2011 Feb;23(1):21-8.
Infection with HIV and progression to AIDS
Clinical manifestations of HIV infection range from asymptomatic state to life-threatening cancers and opportunistic infections. After seroconversion, acute HIV infection usually occur within 2 to 6 weeks in 10% to 20% of the individuals and is manifested as a mononucleosis- like syndrome that may include fever, myalgia, arthralgia, headache, photophobia, diarrhea, sore throat, lymphadenopathy, and maculopapular rash. Some of these patients as well as some asymptomatic patients may develop lymphadenopathy. The condition may then progress to AIDS related complex characterized by long lasting fever, weight loss persistent
3
diarrhea, oral candidiasis and multidermatodomal herpes simplex. Most patients with AIDS related complex as well as those with persistent lymphadenopathy and even asymptomatic patients develop full blown AIDS characterized by opportunistic infections, Kaposi sarcoma and B-cell lymphomas [4, 5].
Antiretroviral treatment
It is undoubted that during the last 25 years, the introduction of highly potent antiretroviral drugs has transformed HIV from a deadly disease into a chronic manageable condition. The United Nations program on HIV/AIDS (UNAIDs) [7] lately reported that between 2001 and 2009, the incidence of HIV has dropped by more than 25% in 33 countries, including 22 sub- Saharan African countries. Furthermore, the number of annual AIDS-related deaths worldwide is steadily decreasing from the peak of 2.1 million in 2004 to an estimated 1.8 million in 2009.
Currently, there are 25 approved antiretroviral drugs [8], with different mechanism of actions (Figure 3):
1. Protease inhibitors (PI’s): atazanavir, lopinavir, ritonavir, amprenavir, fosamprenavir, saquinavir, indinavir, nelfinavir, tipranavir, and darunavir
2. Non nucleoside reverse transcriptase inhibitors (NNRTI’s): efavirenz, delavirdine, nevirapine, etravirine and rilpivirine
3. Nucleoside reverse transcriptase inhibitors (NRTI’s): abacavir, didanosine, emtricitabine, lamivudine stavudine, zidovudine, zalcitabine and tenofovir
4. Fusion inhibitors (FI’s): enfuvirtide
5. Co-receptor inhibitors (CCRI’s): maraviroc 6. Integrase inhibitors (II’s): raltegravir
These drugs must be taken in combination regimens to achieve optimal clinical outcomes in terms of long term virologic efficacy and good tolerability [9].
In order to choose a treatment regimen, the clinicians must consider many pharmacologic aspects of treatment such as virologic efficacy, HIV resistance-testing and drug toxicity, in addition to patient’s characteristics including quality of life, co-morbidity, drug-drug interactions and pill burden [9]. Based on the results reported in randomized controlled trials, the internationsl guidelines strongly recommend 4 treatment regimens for initiating ART [9]:
• Efavirenz + tenofovir+ emtricitabine
• Atazanavir (boosted with ritonavir) + tenofovir + emtricitabine
• Darunavir (boosted with ritonavir) + tenofovir + emtricitabine
• Raltegravir + tenofovir + emtricitabine
4
There are also other optional combination regimens, however, they have lower viral efficacy, higher toxicity or potential for drug interactions; they are usually chosen when the preferred regimens do not suit the patient’s individual situation [9].
Figure 3. Replicative cycle of human immunodeficiency virus (HIV), highlighting the principal targets for therapeutic intervention: (co-)receptor interaction; virus–cell fusion; reverse transcription (by reverse transcriptase); integration; and proteolytic processing (by viral protease). Picture from: De Clercq et al. International Journal of Antimicrobial Agents 33 (2009) 307–320
NNRT’s & NRTI’s II’s
CCRI’s
& FI’s
PI’s
5
Factors Affecting Antiretroviral Drug Treatment
Despite the success of highly potent antiretroviral therapy (ART), a significant percentage of patients do not achieve adequate suppression of plasma HIV RNA or experience drug toxicity [10]. Besides poor adherence to therapy, treatment failure has been attributed to environmental factors (drug-drug, drug-food interactions), pharmacokinetic heterogeneity in absorption, metabolism, and elimination of the drug and genetic variations in the metabolizing enzymes (e.g. polymorphisms of cytochrome P450). All these factors lead to highly variable drug disposition rates and to marked inter-individual differences in circulating drug concentrations under a given dosage regimen. Therefore, dosage individualization based on previous knowledge of the demographic and genetic predictive factors of plasma concentrations may improve both the efficacy and the tolerability of antiretroviral drugs.
HIV therapy involves many compounds and is highly complex. As variation in drug pharmacokinetics directly affects pharmacodynamics and therapy outcomes, it becomes challenging to identify sources of variability. Besides demographic factors, drug interactions and adherence, there is increasing evidence that genetic polymorphisms of metabolizing enzymes and transporter proteins of antiretroviral drugs, characterized by a variable expression or function, play a crucial role in determining drug exposure and explain a significant part of inter-individual variability [11]. Consequently pharmacogenetics of anti-HIV drugs can be predictive of clinical response [12] and is a valuable tool for optimizing ART outcomes.
Population Pharmacokinetic Modeling and TDM of ART
Drug concentrations correspond to a composite phenotypic trait, the consequence of complex pharmacogenetic and non-genetic factors influencing drug transport and metabolism. Thus, for many therapeutic agents, the precise and accurate determination of drug levels is an essential component of the Therapeutic Drug Monitoring (TDM) and represents the only mean to ascertain whether treatment failure is caused by intrinsic resistance or by insufficient drug levels, or whether toxicity occurs because of excessive concentrations or of a form of idiosyncrasy [13, 14].
In HIV therapy, attaining adequate plasma concentrations is crucial as suboptimal exposure can lead to the emergence of resistance and high concentrations may lead to toxicity. The role of TDM in improving virologic response is however a subject of a big controversy.
Currently, TDM of antiretroviral drugs is implemented in routine practice in some European countries [15, 16], yet it is not the case for the United States [9] where TDM is only recommended in some specific situations such as drug interactions, co-infections,
6
suboptimal compliance, pathophysiological alterations. TDM may also improve ART response in subgroups of patients like pediatric and adolescent patients, pregnant women and patient with renal or hepatic failure [9, 17-19]. Among the existing classes of antiretroviral drugs, TDM of first generation NNRTI’s and PI’s appears to have some clinical benefit for the management of antiretroviral treatment in an increasing number of patients [20] and recommended minimal trough concentrations associated with virologic efficacy in patients infected with the wild-type virus are readily available for them [9]. Although no clear association between drug exposure and therapy outcomes has been demonstrated for more recent antiretroviral compounds and despite the absence of target minimal concentrations, TDM interventions may still be useful to optimize ART outcomes for some patients taking raltegravir, etravirine and darunavir mainly due to the potential of drug interactions and the large reported pharmacokinetic variability where the clinical judgment alone may not be sufficient to get hold of satisfactory response.
Population pharmacokinetics (Pop-PK) is the study of variability in plasma concentrations of a given drug in a population under standard dose regimen. It allows, in particular, the characterization of the dose-concentration-effect/toxicity relationships, quantification of inter- individual and intra-individual variability and the identification of demographic and genetic factors that are in part responsible for this variation, thus offering the possibility to detect clinically useful predictors of treatment outcomes. Population pharmacokinetic modeling has gained increasing recognition over the years [21] and several well established tools, in particular the NONMEM® [22] (Non-linear Mixed Effect Modeling) software, have been developed for such purposes.
Current Challenges to ART
Given the high prevalence of toxicity in HIV therapy [23], the factors implicated in drug- induced morbidities should be identified in order to improve the safety, tolerability and adherence to the treatments.
The emergence of viral resistance to the older drugs has urged the development of new molecules active against the resistant strains. Significant advances in ART landscapes have been achieved since the introduction of the second generation PI’s darunavir, and the next generation NNRTI’s etravirine, as well as the highly potent integrase inhibitor raltegravir.
These compounds are pharmacologically powerful and can achieve viral suppression even in highly experienced patients with multiple viral resistances; in addition they are characterized by an improved toxicity profile compared to older molecules [24, 25]. However, we do not dispose of Pop-PK of these newer molecules so far.
7
Antiretroviral drugs are characterized by high and unexplained pharmacokinetic variability.
Furthermore, many phase 1 and phase 2 enzymes as well as drug transporters known with highly variable expression are involved in ART disposition. These Variations could significantly affect exposure and consequently clinical outcomes. Therefore, incorporating pharmacogenetics in the Pop-PK model has a significant importance in identifying sources of variability to attain adequate exposure.
Objective of the thesis
The overall objective of this research is to expand the current knowledge about pharmacokinetic, demographic and genetic factors influencing antiretroviral drug disposition and consequently outcomes in HIV-infected individuals.
To that Endeavour, the goals of our thesis were:
1. To perform an extensive search of the literature in order to provide a comprehensive review that outlines the pharmacokinetics and metabolic profile of 3 classes of antiretroviral drugs, with emphasis on the influence of pharmacogenetics on drug exposure and concentration-effect relationships.
2. To perform Pop-PK analysis of efavirenz, as well as newly introduced darunavir, raltegravir and etravirine in order to characterize their pharmacokinetic profiles and variability in HIV-infected individuals and to identify potential demographic and environmental influencing factors on their disposition and elimination. A main focus of these analyses consisted in incorporating the pharmacogenetic information in the Pop-PK models in order to quantify the influence of the variants of known genes on drug exposure, and to discover new potential genes involved in the drug elimination.
3. To develop a tool to aid the interpretation of antiretroviral drug levels measured within the frame of TDM. In that intent, we performed a systematic review of Pop-PK analyses of 3 classes of antiretroviral drugs with the objectives of deriving average pharmacokinetic parameters that were later used to draw out reference percentile curves.
These findings are expected to give way for improving ART clinical management and optimizing treatment response.
8 References
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15. Stöhr W, Back D, Dunn D, Sabin C, Winston A, Gilson R, et al. Factors influencing lopinavir and atazanavir plasma concentration. Journal of Antimicrobial Chemotherapy 2010 January 1, 2010;65(1):129-37.
16. DiFrancesco R, Rosenkranz S, Mukherjee AL, Demeter LM, Jiang H, DiCenzo R, et al. Quality assessment for therapeutic drug monitoring in AIDS Clinical Trials Group (ACTG 5146): a multicenter clinical trial. Therapeutic drug monitoring 2010 Aug;32(4):458-66.
17. Liu X, Ma Q, Zhang F. Therapeutic drug monitoring in highly active antiretroviral therapy.
Expert Opinion on Drug Safety 2010;9(5):743-58.
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20. Back D, Gibbons S, Khoo S. An update on therapeutic drug monitoring for antiretroviral drugs.
Therapeutic drug monitoring 2006 Jun;28(3):468-73.
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CHAPTER 2: LITERATURE REVIEW
Abstract
Besides therapeutic effectiveness, drug tolerability is a key issue for treatments that must be taken indefinitely. Given the high prevalence of toxicity in HIV therapy, the factors implicated in drug-induced morbidities should be identified in order to improve the safety, tolerability and adherence to the treatments. Current approaches have focused almost exclusively on parent drug concentrations; whereas recent evidence suggests that drug metabolites resulting from complex genetic and environmental influences can also contribute to treatment outcome.
Pharmacogenetic variations have shown to play a relevant role in the variability observed in antiretroviral drug (ART) exposure, clinical response and sometimes toxicity. The integration of pharmacokinetic, pharmacogenetic and metabolic determinants will more likely address current therapeutic needs in patients.
This chapter offers a concise description of three classes of antiretroviral drugs’ metabolic profile and a comprehensive summary of the existing literature on the influence of pharmacogenetics on their pharmacokinetics and metabolic pathways, and the associated drug or metabolite toxicity.
Antiretroviral drug toxicity in relation to pharmacokinetics, metabolic profile and pharmacogenetics
M.Arab-Alameddine1,2, A. Telenti3, T. Buclin1, L.A. Décosterd1 and C.Csajka1,2
1Division of Clinical Pharmacology and Toxicology, University Hospital Center, University of Lausanne
2Department of Pharmaceutical Sciences, University of Geneva, University of Lausanne; Switzerland
3Institute of Microbiology, University Hospital Center, University of Lausanne, Switzerland
Expert Opin. Drug Metab. Toxicol. (2011) 7(5):609-62
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Introduction
Important advances in the anti-retroviral therapy and development of new target molecules have been achieved within the last 20 years. Besides therapeutic effectiveness, drug tolerability is an important issue for HIV lifelong treatment. Given the high prevalence of toxicity in HIV therapy 1, efforts must be pursued to improve not only antiretroviral efficacy but also the safety and tolerability of the treatments. Those elements represent, in addition to treatment simplification, an important element to promote adherence. Nowadays, treatment modifications occurring during the management of HIV infection are more often triggered by adverse effects than by efficacy issues 2.
Patient management could benefit from approaches integrating pharmacogenetics and applied clinical pharmacokinetics to better understand and predict drug exposure, toxicity and clinical response. Most investigations have focused on the parent drug solely, considering it as the best pharmacokinetic marker of antiretroviral drug exposure, and in case of higher levels, of toxicity. However, drug metabolites resulting from complex genetic and environmental influences can also contribute to treatment outcomes. For most drugs, metabolism is a detoxification process, but it can constitute in certain cases the rate-limiting step in drug toxicity. For some drugs in other therapeutic areas, the accumulation of reactive metabolites due to reduced elimination have been implicated in the occurrence of tissue toxicity, carcinogenicity, teratogenicity, and/or immune-mediated injuries 3. Unfortunately, integration of this aspect with pharmacogenetics and pharmacokinetics has attracted little attention in the field of antiretroviral therapy. Distinct metabolite profiles could modulate toxicity, tolerability and outcome of antiretroviral therapy.
This review focuses on current knowledge of the metabolic pathways of Protease Inhibitors, Non Nucleoside Reverse Transcriptase Inhibitors and Integrase Inhibitors and the implication and mechanism of parent and metabolite toxicity. Current knowledge of the main characteristics of their pharmacokinetics along the pharmacogenetic-attributed variation in drug exposure and potentially toxicity will be outlined. Finally, the use of metabolites as markers of metabolic capacity will be addressed. The metabolic pathways of the reviewed antiretroviral drugs, their metabolites, as well as their genetic variants and the respective clinical consequences are summarized in table 1.
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Non Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
NEVIRAPINE
Nevirapine (NVP) was the first NNRTI approved by the U.S. Food and Drug Administration. It is used either in combination with other antiretroviral agents for the treatment of HIV-1 infection or in a single prophylactic dose to prevent mother to child transmission. Due to its low cost, NVP is one of the most widely prescribed antiretroviral drugs in resource-limited countries. NVP use is limited by its FDA Black Box warning for idiosyncratic liver toxicity and skin hypersensitivity.
After oral administration, NVP is rapidly and readily absorbed with an absolute bioavailability of around 93%. Peak plasma concentrations are attained within approximately 4 hours following a single 200 mg dose. Extent of absorption does not appear to be altered by concomitant anti-acid administration or food intake. The molecule is highly lipophilic, crosses the placenta, is excreted into breast milk and is about 60% bound to plasma proteins 4. NVP induces hepatic cytochromes (CYP) CYP3A4 and CYP2B6 mediating its own degradation, therefore auto-induction increases NVP apparent oral clearance by approximately 1.5- to 2- fold, and decreases the terminal phase half-life, from 45 hours following a single dose intake to 25-30 hours under continuous treatment. Induction is complete within 28 days 5, 6 and steady state NVP plasma concentration is then reached 7.
NVP undergoes significant oxidative metabolism followed by glucuronidation to water soluble conjugates which are primarily cleared in the urine. Renal excretion of the unchanged parent drug is insignificant 6. 2 and 12-hydroxynevirapine (OH-NVP) formation is predominantly mediated by CYP3A4, while CYP2B6 is involved in the formation of 3- and 8-OH-NVP 8. Secondary oxidation of 12-OH-NVP yields carboxy-nevirapine 6.
NVP has been associated with a significant incidence of hepatotoxicity (3%) and cutaneous adverse reactions (9%) with occasional life threatening side effects such as Stevens-Johnson syndrome or toxic epidermal necrolysis transition syndrome (0.3%) 9. The onset of these reactions occurs usually during the first 6 weeks of treatment 10, therefore, close clinical and laboratory monitoring is strictly required to quickly identify life threatening adverse events. Rash and liver toxicity have been reported to be more frequently observed in female with a low body mass index 9.The causal element of NVP idiosyncratic toxicity is not determined and multiple mechanistic pathways have been proposed to explain this adverse reaction. It has been advocated that drug toxicity could be generated through reactive intermediates which produce cellular damage by alkylating human DNA, thus resulting in hypersensitivity reactions 11, 12. Furthermore direct immune mediated mechanisms have also
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been suggested since NVP is contraindicated in patients with high CD4+ count 4. Prediction of increased risk of NVP-associated hypersensitivity 13 and hepatotoxicity 14 has been proposed with the use of the human leucocyte antigen (HLA) class II allele HLADRB*0101.
HLA-B*3505 is another candidate that could possibly be implicated in NVP induced skin rash in Asian patients 15.
Several in vitro and in vivo approaches have been used to elucidate the involvement of NVP metabolites in the idiosyncratic drug reactions. CYP3A activity was used as a marker for NVP toxicity, demonstrating that rats pretreated with CYP3A inducers such as dexamethasone or NVP exhibited increased hepatotoxicity compared to naïve animals, thus implying the role of metabolites in liver injury 16. Skin rash and hepato-toxicity were observed in brown rats secondary to the formation of the antigen quinine methide; This metabolite is thought to be generated either via the sulfoconjuguaison of 12-OH-NVP directly in the skin or by cytochromes mediated formation in the liver 17. NVP incubation with NADPH- supplemented microsomes together with glutathione, a tripeptide found in all mammalian tissues and used as trapping agent for reactive intermediates 18, revealed the P450-mediated formation of a glutathione-mediated sulfhydryl conjugate that could initiate hepatotoxicity 19. Using a synthetic model, Antunes et al. 20-22 successfully demonstrated that phase II esterification of 12-OH-NVP to electrophilic derivatives yielded covalent DNA adducts that could potentially play a role in the hepatocarcinogenicity of NVP in vivo. Recently, Srivastava et al 23 provided the first evidence of NVP bioactivation in human patients by isolating and quantifying NVP mercaptopurates in urine, an approach that can be particularly useful for the assessment of human exposure to environmental and biogenic toxic compounds.
The current available knowledge is not conclusive regarding the association between higher NVP or NVP metabolites exposure and toxicity. A study in 49 case-control pairs did not find a relationship between NVP induced toxicities and higher blood levels of NVP or 12-OH-NVP in patients at week 4 of treatment 24, even though higher blood levels of the 12-OH NVP were found in a sub-group at higher risk of skin rash, that is, women and patients co-treated with prednisone. The role of genetic polymorphisms in NVP-induced toxicity was evaluated in two studies. The first reported that a genetic polymorphism of CYP2B6 at position G516T is associated with greater NVP plasma exposure, and might therefore lead to toxicity 25-27. The second study showed that variation in the MDR1 position 3435C>T allele was associated to a decreased risk of NVP hepatotoxicity 28, 29. Recent observations suggest that CYP3A5 6986A>G and MDR1 3435C>T could be correlated to elevation in liver transaminases 30.
16 EFAVIRENZ
Efavirenz (EFV) is among the preferred regimens for first line therapy in HIV treatment guidelines because it exhibits excellent virological and immunological response 31, 32. Once daily dosage promotes adherence, which is a crucial element since first generation NNRTI have a low barrier to resistance that rapidly develops after virologic failure, and is mainly attributed to a single mutation in the reverse transcriptase 32-34.
EFV is readily absorbed with a time to peak plasma concentration reached within 3 to 5 hours. Bioavailability is increased upon administration with high caloric/fat meal, where a small increase in area under the concentration curve (AUC) (17-22 %) and in maximal concentration (39%-51%) was observed compared to fasted conditions. EFV is approximately 99.5-99.75% bound to plasma proteins, predominantly to albumin. EFV is a dose-dependent inducer of CYP2B6, CYP3A4 and P-gp 35-38 and is a CYP2B6 reversible inhibitor as well 39. Due to auto-induction, terminal elimination half-life drops from 52-76 hours after single dose to 40-55 hours after multiple dosing with 200-400 mg daily for 10 days.
EFV is extensively metabolized primarily by hepatic CYP2B6 with partial involvement of CYP3A4 and CYP2A6 40. CYP2B6 mediated conversion to 8-OH-EFV accounts for 77 to 92% of overall EFV clearance, whereas only 7 to 23% are eliminated as 7-OH-EFV essentially via CYP2A6. The metabolite 8,14-dihydroxyefavirenz is also detected in vitro and in vivo and is formed either by direct 14-hydroxylation of 8-OH-EFV or the sulfate of 8-OH- EFV 40, 41. Hydroxylated EFV metabolites undergo further conjugation with glucuronic acid forming N-glucuronide-EFV. EFV is excreted in urine (14 – 34%) mainly as metabolites and in feces (16 – 61%) mainly as parent compound. Recently, EFV metabolite profiling was used as a novel tool to enable the quantification of the metabolic capacity of the primary and secondary metabolic pathways and consequently their importance in drug detoxification 40. It was used as well to identify the behavior of accessory metabolic pathways in case of decrease or loss of function of the major metabolizing enzyme 42.
EFV is characterized by its frequent induction (25 -70 %) of central nervous system (CNS) side effects. Early CNS symptoms include dizziness, depression, anxiety, irritability, headache and sleep disturbance 31, 32, 43 which occur at therapy initiation and usually resolve spontaneously after 2 to 4 weeks of treatment. Symptoms may however persist longer and lead to at least 4 to 10% of treatment discontinuation in some patients 44-46. It has been suggested that neuropsychological symptoms are correlated to plasma EFV concentrations
47, with plasma levels higher than 4 mg/L being associated with higher frequency of CNS side effects 48, 49; a dosage reduction in such patient was shown to prevent discontinuation due to
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toxicity 50. However, CNS toxicity is not uniformly found at higher EFV levels 51 and might therefore be attributed to variations in EFV metabolite concentrations or in CNS penetration.
The relationship between metabolites and toxicity in vivo has not been studied. EFV metabolite-induced nephrotoxicity was solely detected in rats and involved glutathione adducts, proven responsible for renal tubular cell necrosis 52, 53, 54.
Since EFV major metabolizing enzyme CYP2B6 and minor pathways CYP2A6 and CYP3A4/3A5 are highly polymorphic, the influence of CYP2B6 genetic variants on EFV exposure and consequently on EFV induced CNS side effects was extensively studied.
CYP2B6 allelic variation (CYP2B6*6, *11,*15, *18, *27, *28, *29), associated with loss or decreased function or expression accounted for most of the inter-individual differences in EFV clearance and yielded higher EFV exposure55. An association has been reported between CYP2B6 polymorphism and early occurrence and higher frequency of CNS side effects with a resulting treatment discontinuation and increased emergence of virologic failure 56. 27, 57-59. Some evidence is in favor of the importance of CYP2A6 and CYP3A4/A5 accessory pathways in EFV disposition, independently from CYP2B6, or in case of loss of function of this pathway 27, 40, 42, 60. CYP3A4*1B, CYP3A4_rs4646437 and CYP3A5*3 as well as CYP2A6 allelic variants (*2, *4, *1H, *1J, *5, *7, *9, *10, *12, *13, *15, *17, *19, *34) explained a small but relevant part of the inter-patient variability in drug exposure and might also affect the metabolic profile of this drug 27, 60, 61. A genetic association study showed that individual with CYP2B6, CY2A6 and CYP3A4 loss of function genotypes experienced higher discontinuation rates due to toxic events that individuals with only CY2B6 polymorphism.
These results suggest that EFV genotyping might represent a useful tool in predicting toxicity and consequently in treatment individualization Others did not find an effect of CYP3A polymorphism on EFV exposure without assessing the correlation between plasma EFV and CNS side effects 27, 60, 62-64. The role of MDR1 in EFV kinetic and toxicity profiles appears to be controversial as well 27, 59, 60, 62, 64, 65. Whether variations in EFV metabolite levels induced by genetic polymorphisms are related to toxicity remains to be established.
ETRAVIRINE
Etravirine (ETV) is the first member of the second generation NNRTI; it has a potent activity against wild type and mutant HIV-1 strains and is characterized by an increased resistance barrier. It is prescribed as a second line therapy in treatment experienced patient with HIV-1 mutations and resistance to either EFV or NVP, since ETV retains antiviral activity in presence of several common NNRTI resistance associated mutation 66. ETV is neither recommended for treatment naïve patients nor for pediatric patients 67-70.
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ETV absolute bioavailability is not yet known. Maximal concentrations are reached within 2.5 to 4 hours. ETV absorption is not affected by agents increasing gastric pH; on the other hand, an increase in AUC by approximately 50 % was reported following the administration of a meal. ETV is highly bound to plasma proteins (99.9%), principally to albumin and to alpha-1-acid glycoprotein (AAG) 69 .
ETV undergoes hepatic metabolism by CYP3A4, CYP2C9 and CYP2C19 and is a CYP3A4 inducer and an inhibitor of CYP2C9 and CYP2C19 and P-gp 71. Methylhydroxylation of the dimethylbenzonitrile to form mono or dihydroxy–ETV accounts for the majority of ETV metabolism whereas hydroxylation of the dimethylbenzonitrile without the methyl groups plays only a minor role. Glucuronide conjugates of these metabolites are also detected.
Elimination occurs in a large portion in feces (85%) and bile (11%) while only 1% is recovered in urine. ETV has a long elimination half-life of 30 to 40 hours. Although once and twice daily dosing results in comparable exposure 72, evidence for once daily dosing is considered not yet sufficient 69, 73. ETV metabolites have minimal anti-HIV activity compared to the parent drug 72 and are mainly excreted in urine 73.
The most commonly reported side effect is a mild to moderate rash, which occurs in 10% of patients at the beginning of the treatment and resolves spontaneously within 1 to 2 weeks on continued ETV. Post-marketing experience revealed rare cases of severe Stevens-Johnson including hepatic failure, life threatening conditions and even fatalities 70, 73, 74. So far, no evidence of causal relationship between the occurrence of ETV side effects and plasma concentrations of parent drug, toxic metabolites has been identified to date.
Pharmacogenetics of ETV metabolizing enzymes has been recently analyzed 75 suggesting that carriers of CYP2C9*3 AND CYP2C19*2 had 44% and 20% lower ETV clearance respectively. The clinical influence of these genetic polymorphisms was not clear as it was confounded by the opposite effect of the co-administered medications on ETV disposition.
Protease Inhibitors (PIs)
LOPINAVIR
Lopinavir (LPV) is used in adults and children 76, 77 and remains a commonly prescribed PI in pregnancy 68. It is co-formulated with low dose RTV as a booster, thus increasing its plasma AUC 77- to 100-fold by inhibiting P-gp and first pass inactivation 78-80.
After multiple dosing, time to peak concentration occurred after approximately 4 hours.
Administration of LPV/RTV with a moderate to high fat meal improved bioavailability, with a
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more relevant effect for the liquid formulation. At steady state, LPV is approximately 98-99%
bound to both AAG and albumin with a higher affinity for AAG 81. At a dosing of 400 mg + RTV 100mg twice daily, the apparent elimination half life ranges from 4 to 6 hours 81, 82. LPV is both a substrate and inhibitor of CYP3A family.
LPV is metabolized by CYP3A4 and CYP3A5 to 3 major metabolites, 4-OXO-LPV and 2 epimeric 4-OH-LPV with other minor metabolites. Even though 4-OXO-LPV and the 2 OH-epimers are the main metabolites, their total measured radioactivity in plasma is relatively small and they exhibit only minor HIV protease inhibition capacity 83, 84. LPV derivatives are primarily excreted in the feces with only a minor role of renal excretion (10.4%). Nearly 20% of the dose is excreted unchanged in the feces, while renal elimination of unchanged compound accounts for less than 3% after multiple dosing.
Moderate to severe diarrhea, nausea and vomiting as well as severe (grade 3 to 4) laboratory abnormalities, such as elevated cholesterol, triglycerides and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are reported in >2% of patients treated with LPV/RTV 81, 82, 85, 86. LPV exposure has been suggested to be directly correlated to drug tolerability and therapeutic efficacy: a target trough concentration of 1 mg/L68 is required to achieve viral suppression and a threshold of 4 mg/L is required when five or fewer LPV baseline resistance mutations are present 87. A higher incidence of nausea and diarrhea were observed in patients taking once daily 800/200 mg LPV/RTV compared to twice daily 400/100 mg after 3 months of treatment 81. Further results suggest that high RTV boosting leads to higher LPV exposure and is associated with more frequent nausea and vomiting 88.
The impact of genetic polymorphisms on LPP/RTV elimination was recently investigated 57, 89. Genetic variations in solute carrier SLCO1B1*4 induced a marked increase in LPV clearance compared to carriers of the reference allele, thus reducing drug exposure. Another variant of this transporter SLCO1B1*5 that is characterized by impaired drug transport activity, induced a small decrease in LPV clearance. Genetic variation in the ATP-Binding Cassette subfamily C member 2 (ABCC2) genes encoding the Multidrug resistance-related protein MRP2 was associated with impaired LPV elimination as well related to a decreased biliary efflux of the drug. It is worth noting that despite the inhibitory action of the associated low dose RTV, the effect of CYP3A allelic variants on LPV clearance remained significantly relevant. Genetic variations in the efflux pump P-gp encoded by the Multidrug resistance 1 gene (MDR1) MDR1 3435C>T and 2677G>T polymorphism did not influence LPV plasma levels 90. Interestingly, ultra rapid metabolizers of CYP2D6, a cytochrome partially involved in RTV disposition, were found to have lower RTV and LPV concentrations91. Although variations in
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drug exposure consequent to genetic polymorphisms were identified for this drug, the relationship between LPV, metabolites and toxicity profile has not been investigated so far.
ATAZANAVIR
Excellent bioavailability, favorable lipid profile and gastrointestinal tolerability in addition to low pill burden, are the advantages that ensured for atazanavir (ATV) a relevant role in HIV pharmacotherapy. It is the preferred PI for the treatment of HIV+ naïve patient 92. It is administered in both adults and children, most frequently with low dose RTV as a booster.
ATV is rapidly absorbed and maximal concentrations are reached within approximately 2 -3 hours. Moderate to high fat meals improve ATV bioavailability and increase AUC (35 to 70%). ATV absorption requires an acidic medium, and the co-administration of proton pump inhibitors and antacids significantly lowers ATV plasma concentrations. ATV is extensively bound (86%) to α-1-acid glycoprotein and to albumin 93-95. ATV is an inhibitor of CYP3A4, UGT1A1 and a weak inhibitor of CYP2C8 and P-gp 94-97. After a single 400 mg ATV dose, small fractions of unchanged drug are recovered in feces (20%) and urine (7%). Steady state mean elimination half-life is approximately 7 hours following a light meal 93, 98, 99.
ATV is extensively metabolized in the liver by CYP3A4 into 5 metabolites, one N-dealkylation product, two metabolites resulting from carbamate hydrolysis, a hydroxylated product, and a keto-metabolite. Their concentrations are positively correlated with that of parent compound but none was found to have a pharmacological activity.
ATV inhibits uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) resulting in elevated indirect bilirubin, which occurs in 20% to 59% of exposed patients, while clinical jaundice is observed in 1 to 3% of patients 100 . Hyperbilirunemia is a dose-dependent side effect 93, 101. It was suggested that trough concentrations above than 850 ng/ml could be associated with greater risk of increased bilirubin levels and therefore toxicity 102. It has not been established whether ATV metabolites could play a further role in this drug toxicity.
Polymorphism of the promoter of the gene encoding UGT1A1, UGT1A1*28 predicts benign hyperbilirunemia and jaundice – the Gilbert syndrome 103-105. Since hyperbilirunemia is positively correlated to ATV exposure, and high bilirubinemia is influenced by the activity of the drug transporter MDR1-encoded P-gp, the relationship between polymorphisms in MDR1 and ATV plasma levels has been explored. Strong association was found between MDR1 34>35 allelic variants and elevated boosted and unboosted ATV plasma levels in patients 104,
106. Homozygocity of decreased function UGT1A1 alleles (*28/*28 or *28/*37) was associated with a increased risk of treatment discontinuation du to toxicity 57. On the other hand, such a
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correlation could not be formally confirmed by another study although a similar trend was observed 90.
DARUNAVIR
Darunavir (DRV) is a second generation PI that has more potent and higher binding to both wild type and multidrug resistant HIV-1 strains enzymes than other drugs in the same class
107, 108. Moreover, DRV was found to have superior antiviral efficacy at 96 weeks and significantly lower incidence of gastrointestinal intolerance and lower increase in triglyceride and total cholesterol compared to first generation protease inhibitors 107, 109, 110.
DRV is rapidly absorbed from the gastrointestinal track, with a time to peak concentration reached within approximately 2.5 to 4 hours. Food is known to increase DRV solubility thus increasing both AUC (42%) and Cmax (35%). DRV is 95% bound to plasma protein mainly to AAG and to a lesser extent to albumin 107, 111. RTV co-administration with DRV results in an 11 fold increase in DRV exposure; the exact underlying mechanism could be mediated by either direct CYP3A4 inhibition or bile acid transport inhibition 112.
DRV undergoes extensive oxidative metabolism predominantly by CYP3A. Unboosted DRV metabolites are formed mainly through carbamate hydrolysis, isobutyl aliphatic hydroxylation, and aniline aromatic hydroxylation, to a lesser extent by benzylic aromatic hydroxylation, and only to a minor extent by glucuronidation. RTV boosting produces significant inhibition of carbamate hydrolysis, isobutyl aliphatic hydroxylation, and aniline aromatic hydroxylation, but does not affect benzylic aromatic hydroxylation, and accelerates elimination of glucuronide conjugates 113. All of these metabolites have only 10% activity against wild-type virus compared to parent drug 107, 109.
DRV is generally well tolerated and the most commonly reported side effects (> 5%) are mainly diarrhea, nausea, headache and rash, resulting in 4.7% treatment discontinuation.
Some concerns about 0.5% of patient developing DRV induced hepatitis were recently raised
107. The available data so far suggest that DRV toxicity is not dose dependant.
RITONAVIR
Ritonavir (RTV) is among the first approved protease inhibitors. It is an excellent antiretroviral drug, but at high therapeutic dose is not well tolerated mainly due to gastro-intestinal complaints such as diarrhea, vomiting and abdominal pain. Moreover, RTV causes body fat redistribution as well as abnormal lipids, hepatic and pancreatic laboratory test 114. Despite its clinical effectiveness against HIV virus, adverse effects lead to drug discontinuation in 30%
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of patient. The exact mechanism of RTV toxicity has not been demonstrated, however, it has been suggested that covalent binding of unidentified reactive RTV metabolites/intermediates to proteins might play a role in RTV intolerance 115, 116. Currently, RTV is no longer used as an antiretroviral drug, but is principally administered in a small sub- therapeutic dose as a pharmacoenhancer of other protease inhibitors to improve their systemic exposure 117, 118 . RTV absolute bioavailability is not determined; after a dose of 600 mg, maximal concentration is reached within 2 hours in fasting conditions and 4 hours in fed condition.
RTV is 98-99% bound to plasma proteins mainly to albumin and AAG114.
It is mainly metabolized by CYP3A into 5 oxidative metabolites. The isopropylthiazole oxidation metabolite which is the major and active RTV metabolite is also produced by CYP2D6. Unchanged drug is recovered in the feces (34%) and urine (3.5%). Steady state is achieved within 2 weeks 118. RTV is a powerful inhibitor of CYP3A4 and CYP3A5 119 and to a less extent of CYP2D6. It is a substrate and a weak inducer of CYP3A4, CYP2C9 and CYP2C19 120. Finally RTV is a substrate, inhibitor 121 and inducer of P-glycoprotein 122.
The effect of pharmacogenetics on RTV pharmacokinetics and consequently on the pharmacokinetics of the co-administered protease inhibitor were recently addressed.
Individuals with genetic polymorphism of ABCC2 were found to have lower RTV clearance that indirectly affected the co-administered LPV levels 89. Furthermore, individuals exhibiting the CY2D6 ultra-rapid phenotype had lower RTV levels and consequently lower CYP3A4 inhibition, which resulted in lower concentrations of the co-administered LPV 123.
SAQUINAVIR
The oldest protease inhibitor Saquinavir (SQV) has good antiviral efficacy and a better triglyceride profile than other protease inhibitors 124, 125. However, due to its high pill burden that negatively affects adherence, it is considered as an alternative protease inhibitor 68. We will focus in this review on the currently marketed SQV mesylate formulated as hard gelatin capsules and film coated tablets. These 2 formulations are bioequivalent, have less gastrointestinal side effects, smaller pill size and easier storage compared to the previously formulated soft gelatin capsules. The virologic efficacy of SQV requires the co-administration of low doses RTV, which improve its pharmacokinetic profile and decrease pill burden 68, 126 SQV mesylate is poorly absorbed as it is a P-gp substrate126; absolute bioavailability is around 4%, SQV exposure is considerably increased following a moderate to high fat meal.
Maximal concentrations are reached within 1 hour under fasted conditions and 3 hours under
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fed conditions. SQV is well distributed and 98% is bound to plasma protein 127, while cerebrospinal penetration is negligible 128.
SQV undergoes extensive intestinal and hepatic first pass metabolism 129, 130. SQV is metabolized by CYP3A, primarily CYP3A4 to numerous inactive mono and dihydroxy metabolites129. Biliary excretion is the major elimination pathway responsible for almost the entire SQV elimination with less than 1% renal excretion 127 . It is an inhibitor of CYP3A4 131 and CYP3A5 119 and a weak inhibitor of CYP3A7 132, with some inhibitory effect on CYP2C9
133 and is also reported to be a P-gp inhibitor 134.
SQV has a favorable tolerability profile and 3% of patients discontinued SQV due to side effects 126. The most commonly reported side effects are mainly nausea, vomiting, diarrhea and abdominal pain in addition to elevations in cholesterol and triglycerides levels as well as body fat abnormalities 124, 126, 127.
Plasma SQV concentrations are correlated to therapeutic efficacy in terms of viral suppression and CD4+ count 135, 136. Trough concentrations ranging between 100-250 ng/ml are required to maintain therapeutic efficacy and prevent viral failure 68. Higher maximal concentrations were also reported to be associated with better immunologic response but also with adverse events 137. Many studies reported higher SQV exposure in female patients, even after correction for body weight. This gender difference could be attributed to lower expression or function of CYP3A and P-gp in females. The pharmacokinetic difference between genders is directly reflected on SQV pharmacodynamics, where a greater proportion of women achieved viral suppression compared to men 138, 139 .
The implication of pharmacogenetics of SQV metabolizing enzymes in the inter-subject variability in SQV exposure was explored. Some studies found that CYP3A5*1 carriers have higher SQV clearance and metabolites level and lower plasma concentrations than CYP3A5*3 carriers 55, 138, 140-142. On the other hand no association was found between CYP3A4/5 or P-gp genetic polymorphisms and SQV exposure 126. Unfortunately, the correlation between SQV plasma concentration and therapeutic outcomes in terms of efficacy or toxicity was not evaluated in these studies.
FOSAMPRENAVIR
Fosamprenavir (FPV) is the pharmacologically inactive, water soluble, phosphate ester prodrug of the highly lipophilic protease inhibitor amprenavir. FVP was formulated to increase bioavailability, decrease pill burden of amprenavir and consequently enhance patient’s compliance 143. Boosting with low dose RTV is recommended but not required, first to
