- 8:00 am: Welcome Remarks
- 8:15 am: A. David Rodrigues, BMS: "The largely ignored world of human drug-metabolizing cytosolic sulfotransferases"
Cytosolic sulfotransferases (SULTs) catalyze the O- and N-sulfation (sulfoconjugation, sulfonation) of a wide array of drugs (e.g., cisapride, acetaminophen, minoxidil, troglitazone, salbutamol), xenobiotics (e.g., flavanoids, p-nitrophenol, umbelliferone), and “endobiotics” (e.g., steroids, neurotransmitters and thyroid hormones). Human SULTs belong to a gene superfamily of seven gene subfamilies and representative member proteins (e.g., SULT1A1, 1A3, 1E1, 2A1) are expressed in numerous tissues, exhibit a degree of substrate selectivity, and are inducible. The presentation will focus on examples of SULTs, and will describe their structure, known substrates, inhibitors, inducers, tissue expression and polymorphisms. Limitations of presently available reaction phenotyping tools will be discussed also. Overall, human cytosolic SULTs have been largely ignored by the pharmaceutical industry. However, SULTs are expressed in the gut and liver and can play a role in first pass metabolism. This is exemplified by terbutaline, isoprenaline (isoproterenol) and the oral contraceptive 17a-ethinylestradiol (EE). Possible EE drug interactions involving SULTs are explored.
- 9:15 am: Steven Wrighton, Eli Lilly: "Recent Insights into the Catalytic Properties of Carboxylesterases"
The carboxylesterases (CE) are a family of serine esterases that hydrolyze ester, amide or thioester bonds. These enzymes are important for their role in prodrug activation and xenobiotic metabolism. Although in human several forms exist, two predominate in the biotransformation of drugs with CE1 highly expressed in the liver and CE2 the dominant form in the intestine. This presentation will discuss studies to standardize assay conditions for the hydrolysis of compounds by the human CEs; comparisons of the enzyme kinetics of the hydrolysis of several probe compounds by expressed CEs from human, dog and monkey; and the genomic structure of the CEs in numerous species. Finally, the use of isolated CEs in prodrug development will be discussed.
- 10:15 am: Break
- 10:45 am: R. Scott Obach, Pfizer, Inc.: "Metabolite Toxicity: A Discussion of Recommended Guidelines and In Vitro Covalent Binding Approaches"
Considerable attention has been recently focused on the potential role of human drug metabolites in drug safety, and how to provide a risk assessment for these molecules using preclinical safety models. However, an analysis of drugs withdrawn from the market over the past 20+ years due to unacceptable safety issues suggests that the instances of the “toxic metabolite” as culprit are exaggerated. Even in those instances when it was likely that a metabolite was involved in toxicity, it was the case that the metabolite was generated in abundance in animal species used in safety evaluations of the parent drug. Toxicity arising from metabolites can be due to two mechanisms: (1) chemically stable metabolites that interact with specific enzymes or receptors (target or off-target) causing untoward effects and (2) chemically reactive metabolites that covalently modify many different macromolecule targets, some of which result in toxicity. Recently, the US FDA issued a guidance around metabolites and toxicity, which primarily focuses on the former category. In that guidance, metabolites that achieve a certain percentage of the parent exposure in circulation are deemed to require additional consideration, and if not present in adequate amounts in toxicology species, they may need direct testing in animals. Practical and logical shortcomings of this approach will be highlighted, including the limitations of definitive ADME studies in providing the data needed to make decisions. For the toxic metabolites that are chemically reactive, there has been a focus on using in vitro approaches to predict which compounds will yield these types toxicities. Assays in which nucleophiles, such as glutathione, are added to in vitro metabolism experiments to trap reactive electrophiles have become common. Alternately, the measurement of metabolism-dependent covalent incorporation of drug, typically using drug containing a radionuclide, has been described as predictive of those compounds that could be toxic through such a mechanism. While it is well established that many toxins can undergo metabolism-dependent covalent binding in vitro, the quantitative predictivity of such an approach is questionable and it has not been well-tested with non-toxins. To address this, we examined eighteen drugs (nine hepatotoxins and nine non-hepatotoxins in humans) for in vitro covalent binding in human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo covalent binding. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact paroxetine and raloxifene, two non-hepatotoxins, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total in vitro metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and non-toxic drugs based on in vitro covalent binding data, however the approach still would falsely identify some agents as potentially hepatotoxic and miss others.
Click here to view the new FDA Guidance for Industry: Safety Testing of Drug Metabolites. - 11:45 am: Lunch
- 12:45 pm: Andrew Parkinson, XenoTech "State of the art in vitro drug-drug interaction studies"
XenoTech conducts a large number of in vitro studies that are designed to assess the victim and perpetrator potential of drug candidates. During the course of these studies, we have come across drug candidates that might be described as exceptions to the rule. This presentation will highlight some of these unusual cases including: (1) drug candidates that are metabolized by “unusual” CYP enzymes, (2) drug candidates that are inducers and yet do not induce all the CYP enzymes known to be regulated by PXR and CAR, and (3) drugs that are metabolism-dependent inhibitors of CYP enzymes by virtue of their conjugation with glucuronic acid, and (4) drugs that are extensively metabolized and yet whose clearance is largely determined by hepatic transport, not hepatic metabolism.
- 1:45 pm: Amin Rostami, University of Sheffield and SimCYP, Ltd: "Prediction of Kinetics and Biotransformation of Chemicals in Humans Based on Exclusively In Vitro Data: Added Value Compared to Extrapolation from Animal Studies"
In 1984 Les Benet and colleagues wrote that “At any point in history of health care, our knowledge was considered to be quite extensive; however, in perspective, the knowledge of yesterday seems to have been very limited, just as today’s knowledge can be expected to seem one day as such”. They extended their comments by advocating for an appreciation of the physiologic constraints when applying basic scientific and mathematical concepts to pharmacokinetics. Since then efforts to predict ADME characteristics from in vitro data with the aid of modelling and simulation (M&S) have been in rise and these have formed a significant part of move towards quantitative pharmacology. Such techniques enable the researchers to take full advantage of breaking the systems (human body) into their elements and reassemble them by incorporating some of individual attributes which were not part of the original system (e.g. pharmacogenetic information, patho-physiological changes, age dependent changes etc). Nonetheless, focusing on average values is still common practice in extrapolating from in vitro data to in vivo consequences (IVIVE). Furthermore, decisions to select compounds are sometimes are made on the basis of a “single objective at a time”; this ignores the interplay between different factors. Although population pharmacokinetics in later stages informs us of the most relevant covariates of drug PK and PD, many of these co-variates can be easily identified at early stages thus requiring only “Confirmatory” evidence during the clinical studies rather than “Learning”. Our knowledge of human body may not be considered as extensive nonetheless the available knowledge is shown to be very useful despite the fact that it is underused.
Co-morbidities such as impaired renal- or hepatic function are well-known to affect the pharmacokinetics of drug ultimately resulting in altered efficacy and/or safety. Apart from these two well-known conditions many other factors such as age, gender, genetics, smoking habit, and co-medication may significantly affect PK of potential new drugs. Decision on timing of studies which specifically address the effects of covariables (e,g. as part of Phase I, Phase II or III) and their design have serious implications with respect to speed of drug development and financial investment. Determining interindividual variability in ADME is a key factor in appropriate design of clinical studies. The presentation will provide evidence that a priori identification of influential characteristics using IVIVE (rather than allometry from animals) would lead to improvement in the design of clinical studies; thereby reducing costs and the likelihood of inconclusive outcome.
- 2:45 pm: Break
- 3:15 pm: J. Brian Houston, University of Manchester: "In vitro systems to allow the prediction and likelihood of metabolism-mediated DDIs"
For the most part the routine use of in vitro (microsomal) systems for clearance and DDI prediction is successful; however exceptions persist. With the increasing importance of non-cytochrome P450 clearance mechanisms, namely transporter and direct conjugation reactions, the need for cellular rather than sub-cellular in vitro systems is evident and a number of complexities arise. For many drugs there is extensive hepato-cellular accumulation and the mechanisms responsible are not so easy to establish. For some drugs transporter activities are important, whereas for others intracellular binding is chiefly responsible, and this may be saturable and/or energy dependent. The importance of deriving the unbound hepatocellular drug concentrations in cellular systems for clearance and the selection of appropriate surrogate [I] values for DDI is necessary for in vivo predictions.
- 4:15 pm: Punit Marathe, BMS: "Prospective prediction of human exposure and efficacious dose"
Before a new chemical entity can enter clinical trials, preclinical pharmacology, pharmacokinetic and toxicology studies in animal species are mandatory. The integrated knowledge gained from these studies enables prediction of human exposure and efficacious dose and allows further assessment of therapeutic index and ability to deliver the dose in a suitable dosage form. While no single predictive method is definitive, we have utilized various methods to assess the predictive ability of each method. Such prospective predictions of human exposure and efficacious dose have a huge value in drug discovery setting rather than retroactively fitting clinical observations. Preclinical and clinical data for 35 proprietary Bristol-Myers Squibb discovery compounds (years 1997 to 2005) were collected and analyzed. In each case, exposure and efficacy in human subjects were projected at the time of nomination (for development) prior to first-in-human dosing. Projections of area under the plasma concentration-time curve (AUC) in humans involved the use of one or more methods: (1) allometric scaling of animal pharmacokinetic data; (2) clearance projection employing in vitro data (liver microsomes and hepatocytes); (3) chimpanzee as an animal model; (4) the species-invariant time method; and (5) the Css-MRT method. Whenever possible, prior clinical experience with lead compounds enabled the selection of the most appropriate method(s). Multiple approaches were also available at the time of the human efficacious dose projections: (1) efficacious exposure from animal efficacy models; (2) in vitro potency; and (3) prior experience with clinical leads. Over the eight year period described, AUC in humans was projected within 2-fold (20 out of 35 compounds; 57%), greater than 2-fold to 4-fold (11 out of 35 compounds; 32%), and greater than 4-fold (4 out of 35 compounds; 11%) of the observed value. From the available dataset, the efficacious doses were also projected within 2-fold (7 out of 10 compounds; 70%), greater than 2-fold to 4-fold (2 out of 10 compounds; 20%), and greater than 4-fold (1 out of 10 compounds; 10%) of the actual clinical dose. Overall, it was possible to project human exposure and efficacious dose within 4-fold of observed clinical values for about 90% of the compounds. The success rate truly represents our ability to predict human exposure and efficacious dose in a prospective manner at the discovery stage.
- 6:00 pm: Social Hour, followed by Dinner
- 8:00 am: Opening Remarks
- 8:15 am: Leslie Z. Benet, UCSF: "Using a Biopharmaceutics Drug Disposition Classification System as a Roadmap for Maximizing Drug Exposure"
The Biopharmaceutics Classification System (BCS) was developed to allow prediction of in vivo pharmacokinetic performance of drug products from measurements of permeability (determined as the extent of oral absorption) and solubility (determined for the highest dose strength in 250 ml of water over the pH range 1-7.5). Although the BCS is useful for characterizing drugs in Class 1 (high permeability; high solubility) for which drug dosage form dissolution alone may be amenable for waiver of in vivo bioequivalence studies, there is little predictability concerning drugs in Classes 2 (high permeability; low solubility), 3 (low permeability; high solubility) and 4 (low permeability; low solubility). In 2005 (Pharm Res 22:13-22, 2005), we suggested that a modified version of such a classification system, designated the Biopharmaceutics Drug Disposition Classification System (BDDCS), may be useful in predicting overall drug disposition including: routes of drug elimination; the effects of efflux and absorptive transporters on oral drug absorption; when transporter-enzyme interplay will yield clinically significant effects (e.g., low bioavailability and drug-drug interactions); the direction, mechanism and importance of food effects; and transporter effects on post-absorptive systemic drug concentrations following oral and intravenous dosing. These predictions are supported by a series of studies from our laboratory over the past few years investigating the effect of transporter inhibition and induction on drug exposure. In BDDCS, Classes 1 and 2 drugs are predominantly eliminated by metabolism, while Classes 3 and 4 drugs are predominantly eliminated unchanged via urinary or biliary excretion. Transporter effects will be negligible for Class 1 compounds. Efflux transporter effects will predominate in predicting the oral exposure of Class 2 compounds, while absorptive transporters will have a major influence on the oral exposure of Class 3 compounds. Transporter-enzyme interplay in both the intestine and the liver will play a major role following oral dosing of Class 2 compounds, and hepatic uptake transporters can also be very important for such drugs. We conclude by suggesting that the BDDCS, using elimination and solubility criteria, may provide predictability of drug exposure profiles for all classes of compounds.
- 9:15 am: Toshihisa Ishikawa, Tokyo Inst. Tech: "International Standardization of in vitro Assay Methods for Drug Transporters: Establishment of Transporter Working Group"
Evidence is accumulating to strongly suggest that drug transporters are major determinants of pharmacokinetics, and, by extension, drug response. There are two major families of drug transporters: solute carriers (SLC) and ATP-binding cassette (ABC) transporters. Transporters in both families are expressed in various tissues such as the intestine, brain, liver, kidney, and importantly cancer cells where they play critical roles in the absorption, distribution, excretion and cell specific delivery of drugs. The functions and substrate specificities of drug transporters have been characterized by several in vitro techniques using cells expressing the transporter genes. In particular, construction of in vitro expression systems using human transporter cDNA clones provides useful models to evaluate the substrate specificity. However, there are many factors that can affect the function as well as the expression of drug transporters.
To discuss various issues residing in the drug transporter research in vitro and in vivo, we organized an international working group for drug transporter, named “Transporter Working Group”, which involves the members of Toshi Ishikawa, Kathy Giacomini, Shiew-Mei Huang, Lei Zhang, Donald Tweedie, Joseph A. Ware, Joseph W. Polli, Kathleen M. Hillgren, Volker Fischer, Raymond Evers, Xiaoyan Chu, Dietrich Keppler, Richard Kim, and Kim L.R. Brouwer. Our goal is to prepare a White Paper on membrane transporters in drug development, with a focus on drug transporters that play a key role in drug absorption, distribution, and disposition. This White Paper will identify key transporters for which there is compelling evidence that they play a role in vivo in pharmacokinetics and drug-drug interactions. For these key transporters, in vitro experimental systems and conditions will also be discussed. We plan to present case studies from industry and FDA and obtain comments from expert panels of scientists from industry, academia and regulatory agencies.
- 10:15 am: Break
- 10:45 am: Joseph Polli, GSK: "An Integrated Approach to Identify Drug Transporters Involved in a Compound's Disposition"
Detailed elucidation of the role of drug transporters on the absorption, distribution, metabolism and excretion (ADME) of a drug candidate are often completed after the selection of the compound for initial clinical testing. Transporters can be effective barriers to drug exposure, be the rate determining step in the uptake and/or excretion of a compound or metabolite, and be a cause of drug-drug interactions. The recognition of the influence of drug transporters on disposition is driving a surge in transport-related research activities within the drug metabolism and pharmaceutical sciences. The identification of transporters that influence the disposition and safety of drugs is a new challenge for drug discovery and development programs. This presentation will highlight an integrated preclinical development approach to identify drug transporters involved in a compound's disposition. Integration of data from pharmacokinetic, mass balance, whole-body autoradiography, in situ perfusion experiments and in vitro studies are utilized to determine when and which transporter is involved in a compound's disposition. A case study involving transporter proteins (Pgp, BCRP and OATP) will be presented to illustrate this approach.
- 11:45 am: Lunch
- 12: 45 pm: Yuichi Sugiyama, University of Tokyo: "Prediction of transporter mediated drug-drug interaction based on in vitro studies"
- 1:45 pm: Kate Hillgren, Eli Lilly: "Regulatory Implications of Transporters"
Interactions between coadministered drugs (drug-drug interactions, DDI) is a major cause of hospitalization. The role of cytochrome P450 enzymes (P450) in DDIs has been extensively studied and in vitro-to-in vivo correlations for interaction potential have been established. These studies clarifying the potential for DDIs at P450s are now required studies for registration and appear in the drug label. In the last decade transporters have begun to emerge as another cause of DDIs. It has been well established in the literature that interactions of various drugs with digoxin, a narrow therapeutic index drug, occur when the clearance of digoxin in the kidney by P-glycoprotein (P-gp, MDR1, ABCB1) is blocked. Other transporters are beginning to emerge as a concern for DDIs. Since the field is relatively new, in vitro-to-in vivo correlations to predict the potential for clinically relevant DDIs at transporters have not been established. Examples of clinical DDI at transporters will be reviewed and the regulatory implications will be discussed. The proposed FDA guidance will be reviewed.
- 2:45 pm: Break
- 3:15 pm: Panel Discussion: "Drug Transporters: How important are they from a clinical and regulatory perspective?"
- 4:30 pm: Closing Remarks
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