Complex Metabolism of the Novel Neurosteroid, Ganaxolone, in Humans: A Unique Challenge for Metabolites in Safety Testing Assessment.

The human pharmacokinetics, metabolism, and excretion of [14C]-ganaxolone (GNX) were characterized in healthy male subjects (n = 8) following a single 300-mg (150 µCi) oral dose. GNX exhibited a short half-life of 4 hours in plasma, whereas total radioactivity had a half-life of 413 hours indicating extensive metabolism to long-lived metabolites. Identification of the major GNX circulating metabolites required extensive isolation and purification for liquid chromatography-tandem mass spectrometry analysis, together with in vitro studies, NMR spectroscopy, and synthetic chemistry support. This revealed that the major routes of GNX metabolism involved hydroxylation at the 16α-hydroxy position, stereoselective reduction of the 20-ketone to afford the corresponding 20α-hydroxysterol, and sulfation of the 3α-hydroxy group. This latter reaction yielded an unstable tertiary sulfate, which eliminated the elements of H2SO4 to introduce a double bond in the A ring. A combination of these pathways, together with oxidation of the 3ß-methyl substituent to a carboxylic acid and sulfation at the 20α position, led to the major circulating metabolites in plasma, termed M2 and M17. These studies, which led to the complete or partial identification of no less than 59 metabolites of GNX, demonstrated the high complexity of the metabolic fate of this drug in humans and demonstrated that the major circulating products in plasma can result from multiple sequential processes that may not be easily replicated in animals or with animal or human in vitro systems. SIGNIFICANCE STATEMENT: Studies on the metabolism of [14C]-ganaxolone in humans revealed a complex array of products that circulated in plasma, the two major components of which were formed via an unexpected multi-step pathway. Complete structural characterization of these (disproportionate) human metabolites required extensive in vitro studies, along with contemporary mass spectrometry, NMR spectroscopy, and synthetic chemistry efforts, which served to underscore the limitations of traditional animal studies in predicting major circulating metabolites in man.

Approaches to mitigate the risk of serious adverse reactions in covalent drug design.

INTRODUCTION: Covalent inhibition of target proteins using high affinity ligands bearing weakly electrophilic warheads is being adopted increasingly as design strategy in the discovery of novel therapeutics, and several covalent drugs have now received regulatory approval for indications in oncology. Experience to date with targeted covalent inhibitors has led to a number of design principles that underlie the safety and efficacy of this increasingly important class of molecules. AREAS COVERED: A review is provided of the current status of the covalent drug approach, emphasizing the unique benefits and attendant risks associated with reversible and irreversible binders. Areas of application beyond inhibition of tyrosine kinases are presented, and design considerations to de-risk covalent inhibitors with respect to undesirable off-target effects are discussed. EXPERT OPINION: High selectivity for the intended protein target has emerged as a key consideration in mitigating safety risks associated with widespread proteome reactivity. Powerful chemical proteomics-based techniques are now available to assess selectivity in a drug discovery setting. Optimizing pharmacokinetics to capitalize on the intrinsically high potency of covalent drugs should lead to low daily doses and greater safety margins, while minimizing susceptibility to metabolic activation likewise will attenuate the risk of covalent drug toxicity.

Pharmacokinetic and Metabolic Profile of Deutetrabenazine (TEV-50717) Compared With Tetrabenazine in Healthy Volunteers.

Deutetrabenazine (Austedo, Teva Pharmaceuticals) is a deuterated form of tetrabenazine. It is the first deuterated drug to receive US regulatory approval and is approved for treatment of chorea in Huntington's disease and tardive dyskinesia. Two oral single dose studies comparing deutetrabenazine (25 mg) with tetrabenazine (25 mg) in healthy volunteers evaluated the impact of deuteration on pharmacokinetics of the active metabolites, alpha-dihydrotetrabenazine (α-HTBZ) and beta-dihydrotetrabenazine (ß-HTBZ), metabolite profile, safety, and tolerability. In the two-way, cross-over study, the mean elimination half-life of deuterated total (α + ß)-HTBZ was doubled compared with nondeuterated total (α + ß)-HTBZ, with a twofold increase in overall mean exposure (area under the concentration-time curve from zero to infinity (AUC0-inf )) and a marginal increase in mean peak plasma concentration (Cmax ). In the mass balance and metabolite profiling study, there were no novel plasma or urinary metabolites of [14 C]-deutetrabenazine relative to [14 C]-tetrabenazine. Specific deuteration in deutetrabenazine resulted in a superior pharmacokinetic profile and an increased ratio of active-to-inactive metabolites, attributes considered to provide significant benefits to patients.

Safety Assessment of Acyl Glucuronides-A Simplified Paradigm.

While simple O- (ether-linked) and N-glucuronide drug conjugates generally are unreactive and considered benign from a safety perspective, the acyl glucuronides that derive from metabolism of carboxylic acid-containing xenobiotics can exhibit a degree of chemical reactivity that is dependent upon their molecular structure. As a result, concerns have arisen over the safety of acyl glucuronides as a class, several members of which have been implicated in the toxicity of their respective parent drugs. However, direct evidence in support of these claims remains sparse, and due to frequently encountered species differences in the systemic exposure to acyl glucuronides (both of the parent drug and oxidized derivatives thereof), coupled with their instability in aqueous media and potential to undergo chemical rearrangement (acyl migration), qualification of these conjugates by traditional safety assessment methods can be very challenging. In this Commentary, we discuss alternative (non-acyl glucuronide) mechanisms by which carboxylic acids may cause serious adverse reactions, and propose a novel, practical approach to compare systemic exposure to acyl glucuronide metabolites in humans to that in animal species used in preclinical safety assessment based on relative estimates of the total body burden of these circulating conjugates.

Targeted Covalent Inhibitors for Drug Design.

In contrast to the traditional mechanism of drug action that relies on the reversible, noncovalent interaction of a ligand with its biological target, a targeted covalent inhibitor (TCI) is designed such that the initial, reversible association is followed by the formation of a covalent bond between an electrophile on the ligand and a nucleophilic center in the protein. Although this approach offers a variety of potential benefits (high potency and extended duration of action), concerns over the possible toxicological consequences of protein haptenization have hindered the development of the TCI concept. Recently, approaches to mitigate the risk of serious adverse reactions to this new class of agent have emerged, thus stimulating interest in the field and leading to authorization of the first cadre of TCIs to be marketed. The covalent inhibitor approach is rapidly gaining acceptance as a valuable tool in drug discovery, and is poised to make a major impact on the design of enzyme inhibitors and receptor modulators.

Biotransformation and bioactivation reactions - 2015 literature highlights.

Since 1972, Drug Metabolism Reviews has been recognized as one of the principal resources for researchers in pharmacological, pharmaceutical and toxicological fields to keep abreast of advances in drug metabolism science in academia and the pharmaceutical industry. With a distinguished list of authors and editors, the journal covers topics ranging from relatively mature fields, such as cytochrome P450 enzymes, to a variety of emerging fields. We hope to continue this tradition with the current compendium of mini-reviews that highlight novel biotransformation processes that were published during the past year. Each review begins with a summary of the article followed by our comments on novel aspects of the research and their biological implications. This collection of highlights is not intended to be exhaustive, but rather to be illustrative of recent research that provides new insights or approaches that advance the field of drug metabolism. Abbreviations NAPQI N-acetyl-p-benzoquinoneimine ALDH aldehyde dehydrogenase AO aldehyde oxidase AKR aldo-keto reductase CES carboxylesterase CSB cystathionine ß-synthase CSE cystathionine γ-lyase P450 cytochrome P450 DHPO 2,3-dihydropyridin-4-one ESI electrospray FMO flavin monooxygenase GSH glutathione GSSG glutathione disulfide ICPMS inductively coupled plasma mass spectrometry i.p. intraperitoneal MDR multidrug-resistant NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone oaTOF orthogonal acceleration time-of-flight PBK physiologically based kinetic PCP pentachlorophenol SDR short-chain dehydrogenase/reductase SULT sulfotransferase TB tuberculosis.

The contributions of Sidney D. Nelson to drug metabolism research.

This article provides a review of Sid Nelson's key contributions to the fields of drug metabolism and toxicology over a long and distinguished career. Selected examples are discussed to illustrate the diversity of Sid's research, with an emphasis on understanding mechanistic aspects of metabolic activation processes and structure-toxicity relationships. These examples serve to illustrate the importance of emerging mass spectrometry and isotope labeling techniques in elucidating details of foreign compound metabolism at the molecular level, an area in which Sid pioneered most effectively.

The generation, detection, and effects of reactive drug metabolites.

The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently attributed to the generation of protein-reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug-drug interactions that may ensue. One important class of protein-reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.

An inducible cytochrome P450 3A4-dependent vitamin D catabolic pathway.

Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4ß,25-dihydroxyvitamin D(3) [4ß,25(OH)(2)D(3)] dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4ß,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4ß,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4ß,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.

Structural alert/reactive metabolite concept as applied in medicinal chemistry to mitigate the risk of idiosyncratic drug toxicity: a perspective based on the critical examination of trends in the top 200 drugs marketed in the United States.

Because of a preconceived notion that eliminating reactive metabolite (RM) formation with new drug candidates could mitigate the risk of idiosyncratic drug toxicity, the potential for RM formation is routinely examined as part of lead optimization efforts in drug discovery. Likewise, avoidance of "structural alerts" is almost a norm in drug design. However, there is a growing concern that the perceived safety hazards associated with structural alerts and/or RM screening tools as standalone predictors of toxicity risks may be over exaggerated. In addition, the multifactorial nature of idiosyncratic toxicity is now well recognized based upon observations that mechanisms other than RM formation (e.g., mitochondrial toxicity and inhibition of bile salt export pump (BSEP)) also can account for certain target organ toxicities. Hence, fundamental questions arise such as: When is a molecule that contains a structural alert (RM positive or negative) a cause for concern? Could the molecule in its parent form exert toxicity? Can a low dose drug candidate truly mitigate metabolism-dependent and -independent idiosyncratic toxicity risks? In an effort to address these questions, we have retrospectively examined 68 drugs (recalled or associated with a black box warning due to idiosyncratic toxicity) and the top 200 drugs (prescription and sales) in the United States in 2009 for trends in physiochemical characteristics, daily doses, presence of structural alerts, evidence for RM formation as well as toxicity mechanism(s) potentially mediated by parent drugs. Collectively, our analysis revealed that a significant proportion (∼78-86%) of drugs associated with toxicity contained structural alerts and evidence indicating that RM formation as a causative factor for toxicity has been presented in 62-69% of these molecules. In several cases, mitochondrial toxicity and BSEP inhibition mediated by parent drugs were also noted as potential causative factors. Most drugs were administered at daily doses exceeding several hundred milligrams. There was no obvious link between idiosyncratic toxicity and physicochemical properties such as molecular weight, lipophilicity, etc. Approximately half of the top 200 drugs for 2009 (prescription and sales) also contained one or more alerts in their chemical architecture, and many were found to be RM-positive. Several instances of BSEP and mitochondrial liabilities were also noted with agents in the top 200 category. However, with relatively few exceptions, the vast majority of these drugs are rarely associated with idiosyncratic toxicity, despite years of patient use. The major differentiating factor appeared to be the daily dose; most of the drugs in the top 200 list are administered at low daily doses. In addition, competing detoxication pathways and/or alternate nonmetabolic clearance routes provided suitable justifications for the safety records of RM-positive drugs in the top 200 category. Thus, while RM elimination may be a useful and pragmatic starting point in mitigating idiosyncratic toxicity risks, our analysis suggests a need for a more integrated screening paradigm for chemical hazard identification in drug discovery. Thus, in addition to a detailed assessment of RM formation potential (in relationship to the overall elimination mechanisms of the compound(s)) for lead compounds, effects on cellular health (e.g., cytotoxicity assays), BSEP inhibition, and mitochondrial toxicity are the recommended suite of assays to characterize compound liabilities. However, the prospective use of such data in compound selection will require further validation of the cellular assays using marketed agents. Until we gain a better understanding of the pathophysiological mechanisms associated with idiosyncratic toxicities, improving pharmacokinetics and intrinsic potency as means of decreasing the dose size and the associated "body burden" of the parent drug and its metabolites will remain an overarching goal in drug discovery.

The resurgence of covalent drugs.

Covalent drugs have proved to be successful therapies for various indications, but largely owing to safety concerns, they are rarely considered when initiating a target-directed drug discovery project. There is a need to reassess this important class of drugs, and to reconcile the discordance between the historic success of covalent drugs and the reluctance of most drug discovery teams to include them in their armamentarium. This review surveys the prevalence and pharmacological advantages of covalent drugs, discusses how potential risks and challenges may be addressed through innovative design, and presents the broad opportunities provided by targeted covalent inhibitors.

Managing the challenge of chemically reactive metabolites in drug development.

The normal metabolism of drugs can generate metabolites that have intrinsic chemical reactivity towards cellular molecules, and therefore have the potential to alter biological function and initiate serious adverse drug reactions. Here, we present an assessment of the current approaches used for the evaluation of chemically reactive metabolites. We also describe how these approaches are being used within the pharmaceutical industry to assess and minimize the potential of drug candidates to cause toxicity. At early stages of drug discovery, iteration between medicinal chemistry and drug metabolism can eliminate perceived reactive metabolite-mediated chemical liabilities without compromising pharmacological activity or the need for extensive safety evaluation beyond standard practices. In the future, reactive metabolite evaluation may also be useful during clinical development for improving clinical risk assessment and risk management. Currently, there remains a huge gap in our understanding of the basic mechanisms that underlie chemical stress-mediated adverse reactions in humans. This review summarizes our views on this complex topic, and includes insights into practices considered by the pharmaceutical industry.

Role of biotransformation in drug-induced toxicity: influence of intra- and inter-species differences in drug metabolism.

It is now widely appreciated that drug metabolites, in addition to the parent drugs themselves, can mediate the serious adverse effects exhibited by some new therapeutic agents, and as a result, there has been heightened interest in the field of drug metabolism from researchers in academia, the pharmaceutical industry, and regulatory agencies. Much progress has been made in recent years in understanding mechanisms of toxicities caused by drug metabolites, and in understanding the numerous factors that influence individual exposure to products of drug biotransformation. This review addresses some of these factors, including the role of drug-drug interactions, reactive metabolite formation, individual susceptibility, and species differences in drug disposition caused by genetic polymorphisms in drug-metabolizing enzymes. Examples are provided of adverse reactions that are linked to drug metabolism, and the mechanisms underlying variability in toxic response are discussed. Finally, some future directions for research in this field are highlighted in the context of the discovery and development of new therapeutic agents.

Approaches for minimizing metabolic activation of new drug candidates in drug discovery.

A large body of circumstantial evidence suggests that metabolic activation of drug candidates to chemically reactive electrophilic metabolites that are capable of covalently modifying cellular macromolecules may result in acute and/or immune system-mediated idiosyncratic toxicities in humans. Thus, minimizing the potential for metabolic activation of new drug candidates during the drug discovery and lead optimization stage represents a prudent strategy to help discover and develop the next generation of safe and effective therapeutic agents. In the present chapter, we discuss the scientific methodologies that currently are available to industrial pharmaceutical scientists for assessing and minimizing metabolic activation during drug discovery, their attributes and limitations, and future scientific directions that have the potential to help advance progress in this field. We also propose a roadmap that should help utilize the armamentarium of available scientific tools in a logical way and contribute to addressing metabolic activation issues in the drug discovery-setting in a rapid, scientifically appropriate, and resource-conscious manner.

Approaches to the assessment of stable and chemically reactive drug metabolites in early clinical trials.

The recommendations of the FDA's final Guidance document on Safety Testing of Drug Metabolites provide a framework for devising preclinical toxicology assessment paradigms, where necessary, for human metabolites of small molecule drug candidates. Importantly, these recommendations carry implications for the qualitative and quantitative analysis of circulating drug metabolites in early human trials, which typically are performed without the benefit of a radiolabeled tracer. In this perspective, an approach to these goals is outlined based on recent work at Merck Research Laboratories involving the use of ultraperformance liquid chromatography-mass spectrometry analysis, performed on a high-resolution time-of-flight mass spectrometer, of first-in-human study plasma samples. With the aid of a fractional mass filtering algorithm, drug metabolites are distinguished from endogeneous background materials and subsequently identified on the basis of their accurate masses, product ion mass spectra, and computer-assisted structure elucidation software routines. Semiquantitative analysis then is based on calibration of the MS response to each analyte with reference to radioactivity data from in vitro metabolic profiles. In the case of chemically reactive drug metabolites, which are excluded from consideration in the Guidance, a proactive approach is advocated whereby potent (low dose) drug candidates with only a limited propensity to form electrophilic intermediates are advanced into development. Overall, a decision on the need to conduct separate evaluation of the safety profile of a human drug metabolite(s) should take into consideration all of the available information on the compound of interest and be based on a case-by-case approach employing sound scientific principles.

Fractional mass filtering as a means to assess circulating metabolites in early human clinical studies.

Recent changes in the regulatory environment have led to a need for new methods to assess circulating human drug metabolites in early clinical studies with respect to their potential toxicological impact. The specific goals of such studies are to determine if the metabolites present in human plasma following administration of a drug candidate also are observed in plasma from the animal studies employed for preclinical toxicological evaluation, and to estimate corresponding exposure margins (animal:human) for the major metabolites. Until recently, the accepted best practice for the characterization of circulating drug metabolites utilized liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methodologies, in conjunction with authentic chemical standards, for the detection and quantitative analyses of metabolites predicted from both animal studies and experiments with human liver preparations in vitro. While this approach is satisfactory for anticipated biotransformation products, metabolites that were not expected to circulate in human plasma frequently escape detection. Current accurate mass instruments enable the use of the technique of fractional mass filtering to detect both expected and unexpected metabolites in a rapid, less resource-intensive and more robust manner. Application of this technology to several clinical development programs at Merck Research Laboratories has demonstrated the value of fractional mass filtering in the assessment of circulating drug metabolites in early clinical trials.

High-throughput, accurate mass liquid chromatography/tandem mass spectrometry on a quadrupole time-of-flight system as a 'first-line' approach for metabolite identification studies.

Throughput for drug metabolite identification studies has been increased significantly by the combined use of accurate mass liquid chromatography/tandem mass spectrometry (LC/MS/MS) data on a quadrupole time-of-flight (QTOF) system and targeted data analysis procedures. Employed in concert, these tools have led to the implementation of a semi-automated high-throughput metabolite identification strategy that has been incorporated successfully into lead optimization efforts in drug discovery. The availability of elemental composition data on precursor and all fragment ions in each spectrum has greatly enhanced confidence in ion structure assignments, while computer-based algorithms for defining sites of biotransformation based upon mass shifts of diagnostic fragment ions have facilitated identification of positions of metabolic transformation in drug candidates. Adoption of this technology as the 'first-line' approach for in vitro metabolite profiling has resulted in the analysis of as many as 21 new chemical entities on one day from diverse structural classes and therapeutic programs.

Minimizing metabolic activation during pharmaceutical lead optimization: progress, knowledge gaps and future directions.

Minimizing the potential for drug candidates to form chemically reactive metabolites that can covalently modify cellular macromolecules represents a rational strategy to reduce the risk of drug-induced idiosyncratic toxicity in humans. In this review, the approaches that are currently available for addressing this issue during the lead optimization phase of drug discovery, their limitations, and future scientific directions that have the potential to address these limitations are discussed.

In vitro metabolic activation of lumiracoxib in rat and human liver preparations.

Recent clinical reports have suggested that the cyclooxygenase-2 inhibitor, lumiracoxib (Prexige), may cause a rare but serious hepatotoxicity in patients. In view of the close structural resemblance between lumiracoxib and diclofenac, a widely used nonsteroidal anti-inflammatory drug whose use also has been associated with rare cases of liver injury, it is possible that the toxicity of the two agents may share a common mechanism. Because it is believed that chemically reactive metabolites may play a role as mediators of diclofenac-mediated hepatotoxicity, the present in vitro study was carried out to test the hypothesis that lumiracoxib also undergoes metabolic activation when incubated with liver microsomal preparations and hepatocytes from rats and humans. By means of liquid chromatography tandem mass spectrometry and nuclear magnetic resonance spectrometry techniques, two previously unknown N-acetylcysteine (NAC) conjugates were identified, namely, 3'-NAC-4'-hydroxy lumiracoxib (M1) and 4'-hydroxy-6'-NAC-desfluoro lumiracoxib (M2), the structures of which reveal the intermediacy of an electrophilic quinone imine species. Based on the results of studies with immunoinhibitory antibodies, it was demonstrated that the formation of M1 and M2 in human liver microsomes was catalyzed by cytochrome P450 (P450) 2C9. These findings demonstrate that lumiracoxib is subject to P450-mediated bioactivation in both rat and human liver preparations, leading to the formation of a reactive intermediate analogous to species generated during the metabolism of diclofenac.

Metabolism and toxicity of drugs. Two decades of progress in industrial drug metabolism.

The science of drug metabolism and pharmacokinetics (DMPK) has developed significantly over the past 20 years, and its functional role in today's pharmaceutical industry has matured to the point where DMPK has become an indispensable discipline in support of drug discovery and development. While contributions to the lead optimization phase of discovery efforts have been particularly noteworthy in helping to select only the best drug candidates for entry into development, it should be recognized that the scope of DMPK spans the continuum of discovery through clinical evaluation and even into the post-marketing phase; as such, the breadth of DMPK's involvement is almost unique in contemporary pharmaceutical research. This perspective summarizes notable advances in the field, many of which have been made possible by technological developments in areas such as molecular biology, genetics, and bioanalytical chemistry, and highlights the critical nature of key partnerships between Drug Metabolism, Medicinal Chemistry, and Safety Assessment groups in attempting to advance drug candidates with a low potential for causing adverse events in humans. Finally, some speculative predictions are made of the future role of DMPK in pharmaceutical research, where current advances in our mechanistic understanding of the molecular processes that control the absorption, disposition, metabolism, elimination, and toxicity of drugs and their biotransformation products will combine to further enhance the impact of DMPK in drug discovery and development.