Assessment of drug-drug interactions: concepts and approaches

1. A priori knowledge of the enzyme inhibitory potential of new drug entities and the drug-metabolizing enzymes involved can be used in support of important decisions on the future progress of a drug in clinical development. 2. Important advances in the knowledge of human drug-metabolizing enzymes have largely fuelled the integration of in vitro drug metabolism and clinical drug interaction studies for use in drug development programmes. 3. The likelihood of correctly predicting in vivo drug-drug interactions appears highly dependent on selecting the correct enzyme inhibition model for use in deriving the inhibitor constant (Ki) and correctly determining the available concentration of inhibitor at the active site of the enzyme(s) of interest. 4. The uncertainty and inaccuracy of predicting the extent and duration of in vivo drug interactions currently stems from a lack of definitive models by which to assess likely substrate and inhibitor concentrations at the active site of metabolism. Additional issues contributing to the uncertainty of predicting drug interactions include assumptions of the contribution of presystemic drug extraction and the effect of inhibitors on the processes involved. 5. This review considers the practical aspects of in vitro enzyme inhibition studies and the use of in vitro-in vivo correlation approaches described in the literature to predict in vivo drug-drug interactions.     

In vivo approach for the evaluation of mechanism-based inhibition of cytochrome P450 3A in rats

1.?There have been no reports showing that the area under the concentration-time curve (AUC) of a probe drug is elevated due to mechanism-based inhibition (MBI) of drug-metabolizing enzymes in animals. This study ascertained that mechanism-based inhibitors reported to induce drug–drug interactions (DDIs) in humans also caused MBI in rats.

2.?Midazolam (MDZ), mainly metabolized by cytochrome P450 3A in rats, and mibefradil, which showed the most intense time-dependent inhibition among the inhibitors tested, were selected as the probe and the inhibitor, respectively. Following pretreatment of mibefradil at 24 h before MDZ administration in rats, the C_max and AUC values of MDZ were significantly elevated in comparison with the control. The free plasma concentration of mibefradil was substantially lower than the IC₅₀ value observed in the in vitro inhibition study, suggesting that the DDI was due to MBI.

3.?It is concluded that the evaluation of MBI in rats in vivo in combination with in vitro data using human enzymes could be useful to evaluate risk in clinical studies.     

Application of Chimeric Mice with Humanized Liver for Predictive ADME

Much effort to extrapolate the in vivo pharmacokinetics of drugs in human from experimental animals or in vitro studies has been made by many researchers. A urokinase-type plasminogen activator^+/+/severe combined immunodeficient transgenic mouse line, in which the liver could be replaced by more than 80% with human hepatocytes, was established recently in Japan. This chimeric mouse line is remarkable because the replacement is higher than any other chimeric mouse reported previously. Since the liver is the critical organ involved in the pharmacokinetics of drugs, human liver is essential for the development of new drugs. To predict the human drug metabolism and pharmacokinetics, human hepatocytes and liver microsomes are recognized as better tools and are frequently used. Thus, chimeric mice with humanized liver would have great advantages in studies on drug metabolism and pharmacokinetics. We have evaluated chimeric mice for studies on absorption, distribution, metabolism, and excretion (ADME). In the liver of the chimeric mice, human phase I and phase II enzymes were clarified to be expressed and to have a similar drug metabolizing capacity as the donor. Human specific metabolites could be detected in the serum, suggesting that the chimeric mice might be used as a human ADME model for both in vitro and in vivo studies. For predicting human drug interactions, enzyme induction and inhibition are serious problems. By the treatment with typical inducers, human CYP1A2 and CYP3A4 expressed in the liver of the chimeric mice had induction potencies. After the treatment with quinidine, a specific inhibitor of human CYP2D6, the area under the curve (AUC) of a CYP2D6 metabolite, 4′-hydroxydebrisoquin, was significantly decreased in the chimeric mice but not in the control mice. Therefore, it was indicated that the chimeric mice could be used for assessing the drug interactions via enzyme induction and inhibition. As well as drug metabolism, the drug excretion was demonstrated to be humanized because cefmetazole was mainly excreted in urine both in the chimeric mice and human but in feces in control uPA^?/?/SCID mice. In this review, basic researches on ADME in the chimeric mice with humanized liver are summarized and the application of the chimeric mice for predictive ADME is proposed.     

Induction of cytochrome P450 enzymes: a view on human in vivo findings

The induction of drug-metabolizing enzymes is a special case of pharmacokinetic interactions with consequences for the concurrent drug therapy. The most important enzymes affecting the pharmacokinetics of pharmaceuticals are cytochrome P450 (CYP) enzymes and their induction is often of utmost importance for the effects of the metabolized drugs. This review presents the current knowledge on the inducers of the specific CYP enzymes in humans. The focus is solely on human in vivo findings; in vitro results are referenced only when needed to interpret the induction mechanisms. As the mechanisms of CYP induction are important in understanding the effects of inducers, a concise overview of the various receptors affecting the induction of human CYP enzymes is presented.     

Mechanism-based inactivation of human cytochrome P450 enzymes: strategies for diagnosis and drug–drug interaction risk assessment

Among drugs that cause pharmacokinetic drug–drug interactions, mechanism-based inactivators of cytochrome P450 represent several of those agents that cause interactions of the greatest magnitude. In vitro inactivation kinetic data can be used to predict the potential for new drugs to cause drug interactions in the clinic. However, several factors exist, each with its own uncertainty, that must be taken into account in order to predict the magnitude of interactions reliably. These include aspects of in vitro experimental design, an understanding of relevant in vivo concentrations of the inactivator, and the extent to which the inactivated enzyme is involved in the clearance of the affected drug. Additionally, the rate of enzyme degradation in vivo is also an important factor that needs to be considered in the prediction of the drug interaction magnitudes. To address mechanism-based inactivation for new drugs, various in vitro experimental approaches have been employed. The selection of approaches for in vitro kinetic characterization of inactivation as well as in vitro–in vivo extrapolation should be guided by the purpose of the exercise and the stage of drug discovery and development, with an increase in the level of sophistication throughout the research and development process.     

Possible Drug–Metabolism Interactions of Medicinal Herbs with Antiretroviral Agents

Herbal medicines are widely used by HIV patients. Several herbal medicines have been shown to interact with antiretroviral drugs, which might lead to drug failure. We have aimed to provide an overview of the modulating effects of Western and African herbal medicines on antiretroviral drug-metabolizing and transporting enzymes, focusing on potential herb–antiretroviral drug interactions. Echinacea, garlic, ginkgo, milk thistle, and St. John's wort have the potential to cause significant interactions. In vitro and in vivo animal studies also indicated other herbs with a potential for interactions; however, most evidence is based on in vitro studies. Further pharmacokinetic studies to unveil potential Western and especially African herb–antiretroviral drug interactions are urgently required, and the clinical significance of these interactions should be assessed.     

A regulatory viewpoint on transporter-based drug interactions

1.?Pharmacokinetic drug interactions can lead to serious adverse events and the evaluation of a new molecular entity's (NME) drug–drug interaction potential is an integral part of drug development and regulatory review before its market approval. Clinically relevant interactions mediated by transporters are of increasing interest in clinical development and research in this emerging area and it has been revealed that drug transporters can play an important role in modulating drug absorption, distribution, metabolism and elimination.

2.?Acting alone or in concert with drug-metabolizing enzymes transporters can affect the pharmacokinetics and/or pharmacodynamics of a drug. The newly released drug interaction guidance by the US Food and Drug Administration (USFDA) includes new information addressing drug transporter interactions with a primary focus on P-glycoprotein (P-gp, ABCB1).

3.?This paper provides a regulatory viewpoint on transporters and their potential role in drug–drug interactions. It first outlines information that might be needed during drug development and ultimately included in new drug application (NDA) submissions to address potential transporter-mediated drug interactions. Next, it explains criteria that may warrant conduct of in vivo P-gp-mediated drug interaction studies based on in vitro assessment. In addition, it includes a review case that describes the evaluation of data suggesting a P-gp-based induction interaction.     

Current status of prediction of drug disposition and toxicity in humans using chimeric mice with humanized liver

Abstract

1.?Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans.

2.?Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice.

3.?Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans.

4.?Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans.

5.?The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.     

Carbamazepine: a 'blind' assessment of CYP-associated metabolism and interactions in human liver-derived in vitro systems

1. The ability of various in vitro systems for CYP enzymes (computer modelling, human liver microsomes, precision-cut liver slices, hepatocytes in culture, recombinant enzymes) to predict various aspects of in vivo metabolism and kinetics of carbamazepine (CBZ) was investigated. 2. The study was part of the EUROCYP project that aimed to evaluate relevant human in vitro systems to study drug metabolism. 3. CBZ was given to the participating laboratories without disclosing its chemical nature. 4. The most important enzyme (CYP3A4) and metabolic route (10,11-epoxidation) were predicted by all the systems studied. 5. Minor enzymes and routes were predicted to a different extent by various systems. 6. Prediction of a clearance class, i.e. slow clearance, was correctly predicted by microsomes, slices, hepatocytes and recombinant enzymes (CYP3A4). 7. The 10,11-epoxidation of CBZ by the recombinant CYP3A4 was enhanced by the addition of exogenous cytochrome-b5, leading to a considerable over-prediction. 8. Induction potency of CBZ was predicted in cultured hepatocytes in which 7- ethoxycoumarin O-deethylase was used as an index activity. 9. It seems that for a principally CYP-metabolized substance such as CBZ, all liverderived systems provide useful information for prediction of metabolic routes, rates and interactions.     

Aldehyde oxidase at the crossroad of metabolism and preclinical screening

Abstract

Human AOX1 is a member of the mammalian aldehyde oxidase (AOX) family of enzymes and it is an emerging cytosolic enzyme involved in phase I drug-metabolism, bio-transforming a number of therapeutic agents and xenobiotics. The current trend in drug-development is to design molecules which are not recognized and inactivated by CYP450 monooxygenases, the main drug-metabolizing system, to generate novel therapeutic agents characterized by optimal pharmacokinetic and pharmacodynamic properties. Unfortunately, this has resulted in a substantial enrichment in molecules which are recognized and metabolized by AOXs. The observation has raised interest in the generation of tools capable of predicting AOX-dependent drug-metabolism of novel molecules during the early phases of drug development. Such tools are likely to reduce the number of failures occurring at the clinical and late phase of the drug development process. The current review describes different in silico, in vitro and in vivo methods for the prediction of AOX metabolizing ability and focuses on the existing drawbacks and challenges associated with these approaches.     

Inhibition of Cytochrome P450 Enzymes by Drugs—Molecular Basis and Practical Applications

Drug-drug interactions are a major cause of hospitalization and deaths related to drug use. A large fraction of these is due to inhibition of enzymes involved in drug metabolism and transport, particularly cytochrome P450 (P450) enzymes. Understanding basic mechanisms of enzyme inhibition is important, particularly in terms of reversibility and the use of the appropriate parameters. In addition to drug-drug interactions, issues have involved interactions of drugs with foods and natural products related to P450 enzymes. Predicting drug-drug interactions is a major effort in drug development in the pharmaceutical industry and regulatory agencies. With appropriate in vitro experiments, it is possible to stratify clinical drug-drug interaction studies. A better understanding of drug interactions and training of physicians and pharmacists has developed. Finally, some P450s have been the targets of drugs in some cancers and other disease states.     

The effect of cimetidine on in vitro and in vivo microsomal drug metabolism in the rat

The effect of cimetidine on rat liver microsomal drug metabolism in vitro and in vivo was studied. Cimetidine inhibits aminopyrine N-demethylation and benzo[a]pyrene hydroxylation in a noncompetitive manner with inhibition constants between 1 and 10 mM. Benzo[a]pyrene hydroxylation in liver microsomes from 3-methylcholanthrene-pretreated rats is not appreciably inhibited by cimetidine indicating some specificity in terms of different cytochrome P-450 forms. Cimetidine gives rise to a type II spectral change with a spectral dissociation constant of about 0.1 mM. The prolonged administration of cimetidine does not result in the induction of hepatic drug metabolism. Pretreatment of rats with cimetidine prolongs aminopyrine half-life and hexobarbital sleeping time. These results demonstrate that cimetidine is an in vitro inhibitor of microsomal drug metabolism in the rat and this inhibition leads to pharmacokinetic drug-drug interactions in vivo.     

Chimeric mice with humanized liver: Application in drug metabolism and pharmacokinetics studies for drug discovery

Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery.     

Effects of steroid hormones in vitro on adrenal xenobiotic metabolism in the guinea pig

Studies were carried out to determine the effects of steroid hormones in vitro on adrenal and hepatic microsomal benzphetamine demethylation and benzo[a] pyrene hydroxylation. Testosterone inhibited adrenal drug metabolism but had no effect on hepatic enzymes, whereas 6β-hydroxytestosterone had no effect in either tissue. All of the corticosteroids tested (cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, progesterone, and 17-hydroxyprogesterone) produced a concentration-dependent inhibition of adrenal drug metabolism, but had little or no effect on hepatic metabolism. The 17-deoxy-steroids were more potent inhibitors of adrenal metabolism than were their 17-hydroxylated counterparts. Cortisol was a potent inhibitor of adrenal benzphetamine and benzo[a]pyrene metabolism, produced a type I difference spectrum in adrenal microsomes, and diminished the magnitude of the benzphetamine-induced spectrum; 6 β-hydroxycortisol had none of these effects. Prior addition of benzphetamine to adrenal microsomes reduced the size of cortisol-induced spectral change. The results demonstrate that the effects of corticosteroids in vitro are relatively specific for adrenal enzymes and established a close association between the 6 β-hydroxylase and some drug-metabolizing enzymes. Adrenal steroids may have an important role in the regulation of adrenal xenobiotic metabolism.     

Gene expression and immunochemical localization of major cytochrome P450 drug-metabolizing enzymes in bovine nasal olfactory and respiratory mucosa

Abstract

Despite tremendous advancement in the characterization of nasal enzyme expression, knowledge of the role of the nasal mucosa in the metabolism of xenobiotics is still inadequate, primarily due to the limited availability of in vitro models for nasal metabolism screening studies. An extensive knowledge of the oxidative and conjugative metabolizing capacity of the cattle (Bos taurus) olfactory and respiratory mucosa can aid in efficient use of these tissues for pre-clinical investigations of the biotransformation and toxicity of therapeutic agents following nasal administration or inhalation. Cows are also exposed to a variety of airborne pollutants and pesticides during their lifetime, the metabolism of which can have profound toxicological and ecological consequences. The aim of the present study was to characterize cytochrome P450 (CYP) enzyme expression in the bovine nasal mucosa. Amplification of the specific genes through RT-PCR confirmed expression of several CYP enzymes in bovine hepatic and nasal tissues. The results demonstrate that bovine nasal olfactory and respiratory mucosal and liver tissues express similar populations, families, and distributions of CYP enzymes, as has been previously reported with other species, including humans. Bovine ex vivo tissues can serve as a readily available reference tissue to elucidate preclinical toxico-kinetic effects resulting from exposure to substances in the environment or following drug administration.     

Identification of the human P450 enzymes involved in the in vitro metabolism of the synthetic steroidal hormones Org 4060 and Org 30659

1. The type of human P450 enzymes involved in the in vitro metabolism of Org 4060 and Org 30659, two synthetic steroidal hormones currently under clinical development by NV Organon for use in oral contraceptive and hormone replacement therapy, was investigated. 2. Both steroids were mainly hydroxylated at the 6 β -position in incubations with human liver microsomes. 3. The results from experiments with supersomes, correlation studies as well as inhibition studies with ketoconazole, a selective inhibitor of CYP3A, strongly suggest that the CYP3A family plays a significant role in the 6 β -hydroxylation of both steroids. 4. Measurements of kinetic parameters of P450 enzymes that could metabolize both steroids, combined with the fact that CYP3A4 is known to be the most abundant P450 enzyme in the human liver, indicate that CYP3A4 will be of major importance for the in vivo human metabolism of Org 4060 and Org 30659.     

Drug metabolism and drug interactions: application and clinical value of in vitro models

In vitro models of drug metabolism are being increasingly applied in the drug discovery and development process as tools for predicting human pharmacokinetics and for the prediction of drug-drug interaction risks associated with new chemical entities. The use of in vitro predictive approaches offers several advantages including minimization of compound attrition during development, with associated cost and time savings, as well as minimization of human risk due to the rational design of clinical drug-drug interaction studies. This article reviews the principles underlying the various mathematical models used to scale in vitro drug metabolism data to predict in vivo clearance and the magnitude of drug-drug interactions resulting from reversible as well as mechanism-based metabolic inhibition. Examples illustrating the predictive utility of specific in vitro approaches are critically reviewed. Commonly encountered uncertainties and sources of bias and error in the in vitro determination of intrinsic clearance and metabolic inhibitory potency, including nonspecific microsomal binding, solvent effects on enzyme activities, and uncertainties in estimating enzyme-available inhibitor concentrations are reviewed. In addition, the impact and clinical relevance of complexities such as dosing route-dependent effects, atypical multi-site kinetics of drug-metabolizing enzymes, non-cytochrome P450 determinants of metabolic clearance, and concurrent inhibition and induction, on the applicability and predictive accuracy of current in vitro models are discussed.     

Aldehyde oxidase and its importance in novel drug discovery: present and future challenges

Introduction: Aldehyde oxidases (AOXs) are molybdo-flavoenzymes that oxidize aromatic aldehydes into the corresponding carboxylic acids and heterocycles into hydroxylated derivatives. AOXs have broad substrate specificity and are present in the liver of humans and many experimental animals. These enzymes play an important role in Phase I metabolism of drugs and xenobiotics of toxicological interest.

Areas covered: Preclinical studies on the AOX-dependent metabolism of new drug candidates are problematic. Furthermore, there is a general lack of reliable in silico methodologies to predict whether a new organic molecule is an AOX substrate. In vitro systems, for the design of high- or medium-throughput screening tests, to identify AOX substrates have many limitations. In vivo studies on AOX-dependent metabolism in animal models, on the other hand, are difficult because the complement of liver AOXs in humans and popular experimental animals is different. The authors discuss the possible ways to overcome all these problems.

Expert opinion: The significance of AOXs as drug-metabolizing enzymes is increasing, as the current strategies of organic synthesis designed to avoid cytochrome P450 (CYP450)-dependent metabolism tend to enrich for new chemical structures efficiently oxidized by these enzymes. There is need for reliable methods to screen for, predict, and validate AOX-dependent metabolism of new drug candidates.     

Evaluation of metabolism-mediated herb-drug interactions

As the use of herbal medicines increases, the public health consequences of drug-herb interactions are becoming more significant. Herbal medicines share the same drug metabolizing enzymes and drug transporters, including cytochrome P450 enzymes (CYPs), glucuronosyltransferases (UGTs), and P-glycoprotein, with several clinically important drugs. Interactions of several commonly used herbal medicines, such as Ginko biloba, milk thistle, and St. John’s wort, with therapeutic drugs including warfarin, midazolam, alprazolam, indinavir, saquinavir, digoxin, nifedipine, cyclosporine, tacrolimus, irinotecan, and imatinib in humans have been reported. Many of these drugs have very narrow therapeutic indices. As the herb-drug interactions can significantly alter pharmacokinetic and pharmacodynamic properties of administered drugs, the drugs interacting with herbal medicines should be identified by appropriate in vitro and in vivo methods. A good understanding of the mechanisms of herb-drug interactions is also essential for assessing and minimizing clinical risks. In vitro methods are useful for providing mechanistic information and evaluating multiple components in herbal medicines. This review describes major factors affecting the metabolism of herbal medicines, mechanisms of herb-drug interactions mediated by CYPs and UGTs, and several in vitro methods to assess the herb-drug interactions. Finally, drug interactions of Ginkgo biloba and St. John’s wort, as representative herbal medicines, are described.     

Model for the drug–drug interaction responsible for CYP3A enzyme inhibition. I: evaluation of cynomolgus monkeys as surrogates for humans

1.?Anti-human cytochrome P450 (CYP) 3A4 antiserum completely inhibited midazolam metabolism in monkey liver microsomes, suggesting that midazolam was mainly metabolized by CYP3A enzyme(s) in monkey liver microsomes.

2.?Midazolam metabolism was also inhibited in vitro by typical chemical inhibitors of CYP3A, such as ketoconazole, erythromycin and diltiazem, and the apparent K_i values for ketoconazole, erythromycin and diltiazem were 0.127, 94.2 and 29.6?μM, respectively.

3.?CYP3A inhibitors increased plasma midazolam concentrations when midazolam and CYP3A inhibitors were co-administered orally. However, the pharmacokinetic parameters of midazolam were not changed by treatment with CYP3A inhibitors when midazolam was given intravenously. This suggests that CYP3A inhibitors modified the first-pass metabolism in the liver and/or intestine, but not systemic metabolism.

4.?The drug–drug interaction responsible for CYP3A enzyme(s) inhibition was observed when midazolam and inhibitors were co-administrated orally. Therefore, it was concluded that monkeys given midazolam orally could be useful models for predicting drug–drug interactions in man based on CYP3A enzyme inhibition.