Symposium Objective: 

Idiosyncratic toxicity is the major cause of drug withdrawal from the market.  This three-day symposium is organized to understand the underlying mechanisms of idiosyncratic drug toxicity and evaluate the models that enable the best prediction of idiosyncratic toxicity early in drug development.  The idiosyncratic toxicity involves the target organs such as liver, bone marrow, skin and kidney.  The widely accepted concept involves the metabolic activation of drugs to reactive intermediates that covalently bind to cellular proteins that mediate critical cellular functions.  This binding is believed to be critical in eliciting immunotoxicity by producing antibodies against these cellular proteins and trigger a cascade of events ultimately leading to fatal drug reactions.  However, the nature of these reactive intermediates, the origin of these reactive intermediates and precise target proteins involved in immunotoxicity are only recently being elucidated.  In addition, the cascade of events involved in fatal drug reactions are poorly elucidated. This symposium will highlight the rapidly emerging technologies and models to predict the idiosyncratic drug toxicity in humans prior to its registration.

 Monday, June 11, 2001

 12:00 PM – 1:00 PM

 1:00 PM – 1:45 PM Welcoming Remarks – Overview of Idiosyncratic Adverse Drug Experiences (Rakesh Dixit, Merck & Co., West Point, PA)

 1:45 PM – 2:30 PM Clinical Aspects of Hypersensitivity Reactions to Abacavir (Ziagen®), a Nucleoside Reverse Transcriptase Inhibitor (Seth V. Hetherington, MD; GlaxoSmithKline, Inc.; Research Triangle Park, NC) Hypersensitivity reactions have been described for a wide variety of chemical compounds, including antiretroviral agents.  These reactions are both idiosyncratic and usually rare, and the clinical presentation may be drug-specific.  Hypersensitivity reactions to abacavir (Ziagen) occur among approximately 4% of patients with HIV-1 infection and is generally clinically distinct from those described with non-nucleoside reverse transcriptase inhibitors. We will review the clinical presentation from over 1,400-recorded cases of this reaction, and compare it to other syndromes that labeled hypersensitivity reactions to other drugs.  Management approaches have been established to promote safe and effective use of abacavir in the treatment of HIV-1 infection, and research is progressing to elucidate the mechanism of this hypersensitivity reaction.

3:00 PM – 3:45 PM QT Prolongation and Torsade de Pointes:  From Idiosyncrasy to Postmarketing Analyses (Douglas N. Shaffer, MD, MHS; Office of Postmarketing Drug Risk Assessment, U.S. Food and Drug Administration; Rockville, MD) Increasing attention has been given to iatrogenic QT prolongation and Torsade de Pointes from both public health and regulatory perspectives.  Several drugs have been withdrawn form the market due to their propensity for these adverse effects.  The goal of this presentation is to shift consideration from the molecular level (i.e. blockade of the cardiac delayed rectifier potassium channel, IKr or HERG) to the clinical sequelae of the idiosyncratic reaction.  This presentation will be clinically focused and cover historical perspectives, ongoing postmarketing initiatives, and future directions.

3:45 PM – 4:30 PM Long QT Syndrome: Unexpected Drug Induced Arrhythmia and Sudden Death.
(Craig T. January, University of Wisconsin, Madison, WI) Long QT Syndrome (LQT) is usually caused by inherited mutations or drug interactions with cardiac ion channels which can cause arrhythmias and death.  From the use of cloned human ion channels, it is now recognized that many drugs from diverse structural classes, including numerous developmental
compounds, block cardiac ion channels.  The most common target is the delayed rectifier potassium channel (IKr or the HERG channel).  This talk will review the clinical problem and basic mechanisms of drug-induced LQT.

 4:30 PM Concluding Remarks:  Fred Tonelli, RW Johnson, Raritan, NJ)

 END OF DAY

Tuesday, June 12, 2001 

 8:00 AM – 8:45 AM Role of Cytokines and Other Factors in Idiosyncratic Drug-Induced Hepatitis
(Lance R. Pohl, Ph.D.; National Heart, Lung, and Blood Institute; Bethesda, MD) Idiosyncratic drug-induced hepatitis (IDIH) is often life threatening and is believed to be a major reason for the termination of clinical trials of drugs and the removal of newly approved drugs from the market. Although the mechanisms of IDIH remain to be determined in most cases, several drugs such as halothane, tienilic acid, dihydralazine and others appear to cause IDIH by an immunopathological mechanism that is induced by immunogenic neoantigens formed by the reaction of liver proteins with reactive metabolites of drugs. Since studies of autoimmune diseases have revealed that immunosuppressive cytokines, T cells, and other factors can inhibit the induction and progression of immune reactions against autoantigens, it is conceivable that similar regulatory processes may have a role in preventing immunopathological reactions against drug-induced neoantigens in the liver. Perhaps individuals who develop IDIH may have a defect   in one or more of these immune regulatory mechanisms. Recent findings will be discussed that support this idea and suggest that similar regulatory factors may also have a role in determining the incidence of IDIH caused by non-immune mechanisms of toxicity.

8:45 AM – 9:30 AM Coenzyme A Thioesters: Reactive Intermediates of Acidic Drugs (Mark P. Grillo, Ph.D.; Pharmacia; Kalamazoo, MI) A widespread concern exists over the association of idiosyncratic hypersensitivity reactions and the clinical use of carboxylic acid-containing drugs.  An approach to understanding these rare, but sometimes fatal, reactions has been through investigations of the chemically reactive properties of unstable metabolites.  In this presentation, the role of reactive acyl-CoA thioester intermediary metabolites in the formation of covalent protein adducts will be addressed.  Synthetic, analytical, and in vitro/in vivo methods for mechanistic studies on the metabolic activation of acidic drugs to xenobiotic acyl-CoA thioesters will be reviewed.

9:45 AM – 10:30 AM New Horizons in Idiosyncratic Drug Toxicity Research:  A Biochemists Viewpoint (Peter O’Brien, University of Toronto; Toronto, CANADA) Idiosyncratic drug toxicity has been attributed to reactive drug metabolites binding to target proteins which are trafficked to the cell surface where they act as a hapten that activates the immune system and causes an toxic response (antibody or cytotoxic T cell mediated). Alternatively the stressed or compromised target cell may activate the immune system by releasing danger signals (e.g. cytokines) or undergo apoptosis without activating the immune system (i.e. tolerance). New horizons that could explain the target cell response includes an understanding of the directional trafficking of drugbound target proteins or newly synthesized stress proteins in the cell by endocytic and exocytic vesicular transport (the trans-Golgi network). Understanding the trafficking that causes the release of immune active signals by stressed target cells would be another new horizon.  Finally identifying susceptible individuals by pharmacogenomics and metabonomics could be the most useful new horizon.

 10:30 AM – 11:15 AM Migration of Microsomal Autoantigens to the Plasma Membrane (Dominique Pessayre, INSERM U481, Clichy, FRANCE) Covalent binding of CYP-generated metabolites to CYP proteins can break self tolerance and trigger immunization against both unalkylated CYP molecules and CYP-metabolite adducts. For the anti-CYP autoantibodies and anti-CYP-metabolite antibodies to possibly participate in the immune destruction of hepatocytes would require that CYP and CYP-metabolite adducts be expressed on the plasma membrane (PM). CYPs are cotranslationally inserted in the endoplasmic reticulum (ER) membrane, where they remain anchored by an N-terminal transmembrane sequence, with the rest of the molecule lying on the cytosolic face of the ER membrane. However, a fraction of newly synthesized CYPs, and also CYP-metabolite adducts, follow microtubule-mediated vesicular flow to the Golgi apparatus and then the PM. PM CYPs are located on the external surface of the PM membrane, although the reason for this inverted transmembrane topology is still unclear. PM CYPs are inducible, are associated with CYP-reductase and sustain monooxygenase activities. Autoantibodies to microsomal epoxide hydrolase (mEH) have been detected in germander-induced hepatitis. Although most mEH molecules are synthesized with a cytosolic orientation in the ER, a few mEH molecules achieve an intraluminal orientation across the ER membrane, leading to external mEH molecules after vesicular transport to the PM.

11:15 AM – 12:00PM Endoplasmic Reticulum Chaperone Proteins: Are They Important in Idiosyncratic Drug Reactions? (Alastair Cribb; University of Prince Edward Island; PEI, CANADA) Endoplasmic reticulum chaperone proteins have been identified as targets of the antibody response in idiosyncratic reactions but their role in the pathogenesis of the clinical syndrome is not clear.  In this presentation, the characteristics of these proteins, their interaction with reactive intermediates, their role in the cytoprotective response, and their induction in response to idiosyncratic toxins will be discussed. 

 1:30 PM – 2:15 PM Pharmacogenetic Determinants of Susceptibility to Idiosyncratic Drug Toxicity (J. Steven Leeder, Children’s Mercy Hospital and Clinics; Kansas City, MO) The term “idiosyncratic” is often applied to cases of severe, unanticipated drug-induced toxicity and in its strictest sense refers to determinants of susceptibility that are unique to an individual without implying any particular mechanism.  Only to a limited extent have pharmacogenetic tools have been applied to efforts aimed at identifying and characterizing the factors contributing to individual risk.  This presentation will review the current state of knowledge in this area as well as several limitations that need to be overcome. Ultimately, efficient design of pharmacogenetic studies will benefit from a clear understanding of the mechanisms leading to the clinical manifestations of idiosyncratic drug toxicity and this remains an important challenge for the immediate future.

 2:15 PM – 3:00 PM Reactive Metabolite Formation in vivo:  Interindividual Variability as a Determinant of Risk for Idiosyncratic Drug Toxicity (Christine M. Dieckhaus; University of Virginia; Charlottesville, VA) Using aplastic anemia associated with the anticonvulsant felbamate as a model, this presentation will review the accumulating evidence for the formation of reactive felbamate metabolite(s) in vivo.  Furthermore, them potential of using urinary markers of reactive metabolite formation as a ”determinant of risk” for the development of idiosyncratic toxicity during the therapeutic use of felbamate will be discussed.

4:00 PM – 4:45 PM Possible Role of Free Radical Formation in Clozapine (Clozaril) Induced Agranulocytosis (Ronald P. Mason, Ph.D., National Institute of Environmental Health Sciences/ National Institutes of Health; Research Triangle Park, NC) The use of clozapine, a unique antipsychotic drug, has been restricted due to a 1-2% incidence of drug-induced agranulocytosis.  Metabolic activation of clozapine in neutrophils or stem cells could be the molecular mechanism underlying this side effect.  Evidence for formation of a clozapine radical during the peroxidase-mediated metabolism of clozapine stems from the observation of thiyl and ascorbyl radicals in the presence of glutathione and ascorbate, respectively.  The ascorbyl radical was detected by direct ESR spectroscopy. Its steady-state concentration was significantly increased in the presence of clozapine.  Glutathionyl radical formation was demonstrated by radical trapping.  Again, the radical adduct concentration was significantly increased in the presence of clozapine.  Similarly, when oxygen consumption was measured in peroxidase systems in the presence of glutathione or NADPH, the rate of oxygen uptake was markedly enhanced upon additional of clozapine.  Thus, the data support clozapine activation to a free radical metabolite, which causes oxidative stress and may lead to adduct formation.  Further, it can be concluded from these data that radical scavengers such as ascorbic acid, when co administered with clozapine to patients, may reduce oxidative stress and protein adduct formation.

 END OF DAY 

Wednesday, June 13, 2001 

9:00 AM – 9:45 AM Bioactivation Pathways of Potential Relevance to Abacavir (ZIAGEN®) Idiosyncratic Hypersensitivity Reaction (HSR): Does Alcohol Dehydrogenase (ADH) Play a Role? (John S. Walsh, Glaxo SmithKline; Research Triangle Park NC; MJ Reese, JR Ravitch, M Pirmohamed, BK Park,) A Possible mechanism for the Bioactivation of Abacavir is proposed, based on a reactive aldehyde intermediate generated during alcohol dehydrogenase (ADH) mediated oxidation. The mechanism, Enzymology, and genetics relevant to this process were studied, leading to identification of a potential predisposing gene for the HSR.  The relevance of the ADH pathway to the HSR was studied further in vivo with animal models for metabolism, and in vitro using a proliferation assay incorporating lymphocytes from HIV-positive patients with and without a history of Abacavir hypersensitivity. 

 9:45 AM – 10:30 AM Predicting the Unpredictable: Idiosyncratic Drug Reactions (Jack Uetrecht; University of Toronto; Toronto, CANADA) Idiosyncratic drug reactions add a significant degree of uncertainty to the process of drug development and the ability to screen drugs to eliminate those that would cause such reactions would be a major advance. In order to accomplish this goal a better understanding of the basic mechanisms of such reactions is essential.  However, even at the present time there are several strategies that would likely decrease the risk that new drug candidates will be associated with a high incidence of idiosyncratic drug reactions. 

10:45 AM – 11:30 AM Evaluation of Drugs with Idiosyncratic Hepatotoxicity in Human Hepatocytes (Albert P. Li; In Vitro Technologies, Inc.; Baltimore, MD) Human hepatocytes represent a physiologically relevant experimental model for the evaluation of human drug properties.  One of our research programs the development of experimental approaches with human hepatocytes for the evaluation of idiosyncratic hepatotoxicity.  Assays are being developed based on toxicological properties found to be common among idiosyncratic drugs.  These endpoints include the following: frank cytotoxicity (alamar blue, neutral red), mitochondrial functions (MTT reduction, ATP content), reactive metabolite formation (glutathione content), and P450 induction (e.g. CYP3A induction).  Results on the application of these endpoints in human hepatocytes with drugs with known idiosyncratic hepatotoxicity and the values of these assays in the prediction of drugs with human hepatotoxic potential will be presented.

 11:30 AM – 12:15 PM In Vitro Models to Predict Idiosyncratic Drug Toxicity  (Vangala Subrahmanyam, Purdue Pharma, LP, Ardsley, NY) Idiosyncratic drug toxicity often involves multiple target organs including liver, bone marrow, kidney etc.  It is believed that reactive metabolites formed from drugs cause covalent modifications of cellular macromolecules including protein and DNA.  However, metabolic activation systems are different in different organs.  Therefore, it is expected that the nature of reactive intermediates could be different in different target organs.  Liver contains high levels of cytochrome P450 but extrahepatic organs contain very low levels of cytochrome P450.  Bone marrow and kidney contain low levels of cytochrome P450 but contain high levels of myeloperoxidase and prostaglandin H synthase respectively.  Myeloperoxidase and prostaglandin H synthase catalyze one-electron oxidation of drugs to free radicals whereas cytochrome P450 catalyzes two-electron oxidation of drugs to reactive electrophiles.  Therefore, appropriate cellular models are required in order to predict the idiosyncratic drug toxicity in various target organs.

 12:15 PM

END OF CONFERENCE

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