Current Drug Discovery Technologies - Volume 7, Issue 3, 2010

Improvement in research and development (R&D) productivity via leveraging the probability of technical success to efficiently bring the medicine to the market is a determining factor in maintaining a growing and sustainable pharmaceutical industry. The modern technology advance in drug metabolism and pharmacokinetics (DMPK) plays a key role in the drug discovery and development paradigms in selecting therapeutic drug candidates with better drug-like properties which in turn contribute to productivity growth. This special issue of Current Drug Discovery Technologies focuses on the emerging techniques in DMPK and acknowledges the efforts from contributors in making this issue possible. Benjamin B, Barman TK, Chaira T and Paliwal JK at the Department of Drug Metabolism & Pharmacokinetics, Ranbaxy Research Laboratories, proposed various physiological-pharmacokinetic-based models addressing absorption, tissue distribution and clearance via integration of the individual compound property to physiological properties to predict drug-like behavior. These prediction models should be able to provide a new direction of application for lead optimization process to avoid the need for performing a larger number of in vitro experiments with new chemical entities. Knowledge of the concentrations of the administrated drug candidates to induce an oxidative stress response that disrupts cell or tissue function will definitely help in the assessment of the safety window and potential liability upon co-administration with other components. Vickers AEM, Fisher RL and Sinclair JR from Drug Safety Evaluation, Allergan Inc., describe that human liver slices can be successfully used to investigate compound induced pathways of oxidative stress, which can be augmented in the presence of reduced glutathione (GSH) levels as an example. The authors demonstrate that generation of a stressed model via modulation of the GSH levels with both L-buthionine-S-sulfoximine (BSO) or acetaminophen (APAP) reflected the consequences of a poor liver GSH status, as well as human variability and the extent of response evident amongst the human donor livers. Valerio LG and Long A from Center for Drug Evaluation and Research, U.S. Food and Drug Administration and Lhasa Limited, respectively, present the use of a commercial software program to perform predictions in silico on seventeen hepatotoxic drugs for determining human-specific drug metabolites which might lead to chronic toxicity and cause potentially serious implications for clinical drug-drug interactions. The authors suggest that the proposed computational tools help support not only the development of therapeutics but also the safety assessment in identifying drug metabolites early to protect patients prior to initiating clinical studies. The primary site responsible for drug metabolism is normally the liver. So, the liver-derived in vitro systems such as slices, microsomes, S9 fractions, and hepatocytes are generally considered as an effective model for predictions of metabolic stability, enzyme induction, hepato-biliary transport, and hepatotoxicity for new chemical entities during lead optimization processes. Sahi J, Grepper S and Smith C from ADME/Tox Division of Life Technologies review traditional and newer in vitro approaches such as siRNA using primary hepatocytes to extrapolate clinical hepatic metabolism, transport and toxicity. Transporters are responsible to translocate substrates such as drugs against a concentration gradient across biological membranes which have been demonstrated to have an important role in controlling drug disposition and in the protection from toxic compounds. Inhibition and/or induction of transporter and drug metabolism enzymes may lead to drug-drug interaction. Lu C, Liao M, Cohen L and Xia CQ from Drug Metabolism and Pharmacokinetics department of Millennium Pharmaceuticals, Inc. assesses emerging in vitro methods with drug-drug interaction models for evaluating CYP450 and transporter-mediated issues in this review. Higher throughput bioanalytical methods for the simultaneous analysis of drug components and their metabolites with different chemical properties in complex biological samples are essential for supporting various drug discovery experiments. Hsieh Y at Drug Metabolism and Pharmacokinetics department of Merck Research Laboratories reviews hyphenation of hydrophilic interaction liquid chromatography (HILIC) with tandem mass spectrometry (MS/MS) techniques, which cover a wider range of pharmaceutical compounds in support of drug research as compared with traditional reversed-phase HPLCMS/ MS methods. It was concluded that any improvement in the design of HILIC columns and the instrumentation for MS detection should enhance the chance of success in extending the applications range of HILIC-MS/MS assays.

There have been major strides in the development of novel ways of investigating drug like properties of new chemical entities (NCE) in the last three decades. Identification of ideal properties of a clinical candidate that would give a clinical proof of concept (POC) is the most critical step in the discovery process. Besides the biological dose-response relationship, the pharmacokinetic parameters play the most vital role in influencing the therapeutic response or toxicity of NCE. While there are numerous techniques to understand various drug-like properties individually, the behavior of an NCE in a dynamic in vivo system which influences its therapeutic or toxic effects is a composite function of its various physicochemical and pharmacokinetic parameters. This implies the need to understand the collective influence of various measured parameters, and knowing how variations made in them can result in favorable pharmacodynamic or toxicokinetic properties. Understanding this behavior holds the key to a successful and time efficient lead optimization process. Physiological based pharmacokinetic models (PBPK) are of great interest in this context as they involve a natural way of integrating the individual compound property to physiological properties, providing a rational approach to predict drug like behavior (an ideal behavior identified may be addressed generally as Target Product Profile) in vivo. In the current review, various physiological pharmacokinetic models addressing absorption, tissue distribution and clearance are discussed along with their application in integrating various physicochemical and ADME parameters to predict human pharmacokinetics helping lead optimization.

Glutathione (GSH) levels are modulated in human liver slices to evaluate if drug induced liver injury is enhanced by a poor liver GSH status. Liver slice GSH levels were decreased by: 1) BSO (L-buthionine-S-sulfoximine) to inhibit GSH synthesis, and by 2) APAP (acetaminophen) which consumes GSH via conjugation to a metabolite. In this study, methimazole (MMI) liver injury was evaluated in the presence of a poor GSH status. MMI was selected because its structural thione moiety is linked with hepatotoxicity and during metabolism GSH is co-oxidized. MMI (500-1000 M) affected oxidative stress pathways and mitochondrial function, resulting in lower liver slice GSH and ATP levels. Co-incubation of MMI with BSO or APAP led to further decreases of GSH and ATP levels in some human livers, at time points and concentrations not detected with MMI alone. Variation in human response was evident and demonstrated that some subjects with a poor liver GSH status could be further compromised with high MMI concentrations. MMI induced an up-regulation of gene expression linked with the GSH pathway, mitochondrial GSH and inflammation. Co-treatment of MMI with BSO induced a mixed response of oxidative stress related genes and an up-regulation of heat shock genes. The combination of MMI with APAP increased the expression of genes involved with oxidative stress and anti-oxidant defense, likely to protect the cells from mitochondrial injury. In summary, MMI induces oxidative stress at high concentrations, which can be augmented when liver GSH levels are decreased by the co-administration of some drugs or health status.

In this study we employed the use of the Meteor computational software program to perform predictions in silico on 17 hepatotoxic drugs for determining human-specific drug metabolites. Congruence of the in silico predictions from a qualitative standpoint of drug metabolite structures was established by comparison to human in vivo drug metabolic profiles characterized in publically available clinical studies. A total of 87 human-specific metabolites were identified from the 17 drugs. We found that Meteor's positive predictions included 4 out of the 9 reported major metabolites (detected in excreta at a level of >10% of the administered p.o. dose) and 10 out of the 15 major phase II metabolites giving a total of 14 correctly predicted drug metabolite structures out of 23 major metabolites. A significant level of unconfirmed positive predictions resulted and discussion on reasons for this is presented. An example is given whereby the in silico metabolism prediction succeeded to predict the putative toxic pathway of one of the drugs whilst conventional rodent liver microsomal assays failed to predict the pathway. Overall, we describe a reasonable simulation of human metabolic profiling using this in silico method with this data set of hepatotoxic drugs now withdrawn from the market. We provide an in-depth and objective discussion of this first of its kind validation test using clinical study data with interest in the prediction human-specific metabolism. Further research is discussed on what areas need to be investigated to improve upon the predictive data. The strong potential of this method to predict human-specific drug metabolites suggests the utility of this computational tool to help support not only the discovery development of therapeutics but also the safety assessment in identifying drug metabolites early to protect patients prior to initiating clinical studies.

The liver is the primary site of metabolism for most drugs. Its major roles include detoxification of the systemic and portal blood, and production and secretion of critical blood and biliary components. A number of liver-derived in vitro systems, such as slices, primary and immortalized hepatocytes, microsomes and S9 fractions are used to assess the metabolism and potential toxicity of new chemical entities. Over the past decade, primary hepatocytes have become a standard in vitro tool to evaluate hepatic drug metabolism, cytochrome P450 (P450) induction, and drug interactions affecting hepatic metabolism. While earlier, hepatocytes were used in suspension for metabolic stability evaluations, more recent studies have demonstrated the added value of using these over longer terms in primary culture. Primary hepatocyte cultures are particularly useful in the evaluation of low turn-over compounds. Hepatic transporter studies are recommended for drug candidates that are predominantly eliminated through the bile. An appropriate strategy is to use primary hepatocytes to assess uptake, followed by singly transfected cell lines to identify the specific transporter(s) involved. Primary hepatocytes can also be used to assess biliary clearance to enable improved hepatic clearance predictions. Newer technologies such as siRNA can be used to knock out specific transporters for more predictive evaluations of potential clinically- based drug-drug interactions. In vitro safety (toxicology) studies have historically been conducted using cell lines. There is increasing evidence that co-cultures of primary hepatocytes and Kupffer cells would be more predictive of the in vivo outcome, as this system provides the complete complement of drug metabolizing enzymes, transcription factors and cytokines necessary to get a more in vivo-like toxicological response. In this review, we will discuss standard and novel in vitro approaches for using primary hepatocytes to extrapolate clinical hepatic metabolism, transport and toxicity.

Drug-drug interactions DDI comprise a significant cause of morbidity and mortality worldwide as they may lead to adverse clinical events, result in decrease/inactivation of the therapeutic effect of a drug, may enhance drug toxicity and indirectly compromise treatment outcomes and adherence. Drug transporters and drug metabolism enzymes govern drug absorption, distribution, metabolism, and elimination (ADME). Inhibition or induction of transporter and drug metabolism enzymes can alter the ADME of a co-administered drug, which may lead to drug-induced toxicity or lack of efficacy. This review assesses our current understanding of the in vitro methods of evaluating CYPs and transporter- mediated DDI. The DDI prediction models based on in vitro assays are also discussed in this review. The applications, advantages and limitations of each method are also addressed in this review.

Hydrophilic interaction chromatography (HILIC) offers a difference in selectivity as compared to traditional normal phase and reversed-phase liquid chromatography and therefore has great potential for the separation of a variety of pharmaceuticals. This review is devoted to summarizing HILIC coupled to tandem mass spectrometric (MS/MS) methods for the rapid, sensitive and accurate determinations of small molecules employed for supporting drug discovery. The perspectives and challenges on performing HILIC-MS/MS assays are also presented.