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SC27 prediction of in vivo drug metabolism in humans from in vitro data
Short Communications / European Journal o f Pharmaceutical Sciences 2 (1994) 8 9 - 9 7
SC25 PREDICTION OF DRUG METABOLISM:N-OXYGENATIONAND RETROREDUCTION OF THE N-OXYGENATED METABOLITES B. Clement, R. Lomb PharmaceuticalInstitute,Christian-AlbrechtsUniversityKiel, 24118Kiel, Germany
For predictions about the functionalisation (phase 1 metabolism) of drugs, the nitrogen atoms present are of major importance since an electron can very easily be abstracted from this heteroatom in the course of a catalytic reaction with the cytochrome P-450 enzyme system (P-450). A further reason for this significance is the fact that many nitrogen-containing groups possess sufficient nuelcophilicity to attack the oxygenating form of the flavine-containing monooxygenase (FMO), namely a 4ot-hydroperoxyfiavine. Even so, many nitrogen-containing functional groups are considered to be metabolically stable, as has mainly been demonstrated by in vivo investigations. For the example of the N-hydroxylation reactions of amidines, guanidines, and amidinohydrazones by P-450 and the retroreductions (N-dehydroxylation) of the resultant primary products, it has been shown that the initially formed Noxygenated metabolites are very easily transformed back into the starting materials with the result that this metabolic conversion can be missed. Investigations of structure-activity relationships have shown that, in contrast to previous concepts, N-hydruxylations by P-450 are also possible when the structural prerequisites for the N-dealkylation reaction are present. The retroreductions (N-dehydruxylation) of the formed metabolites (amidoximes, N-hydruxyguanidines, N-hydroxyamidinohydrazones) are catalysed by an enzyme system consisting of cytochrome bs, cytochrome b 5 reductase, and a third enzyme which has now been purified to homogeneity. It was found that some of the N-hydroxylaled metabolites possess a genotoxic potential. In this context, the retroreductions can be considered as detuxifieations. On the whole, particular attention must be directed to metabolic cycles in the sense of N-oxygenation and retroreduction during predictions on the metabolism of drugs possessing nitrogen-containing functional groups.
SC27 PREDICTION OF IN VNO DRUG METABOLISMIN HUMANSFROM IN VITRO DATA H.K. Kroemer Dr. MargareteFischer-Boseh-lnstituth~rKlinischePharmakologie,70~76Stultgafl,Germany
One major factor in modulating the dose vs. concentration relationship of drugs is interindividual variability in the activity of drug metabolizing enzymes. Thus, administration of the same dose may result in variable concentrations and therefore elicit variable effects. Characterization of the enzyme involved in a certain metabolic step would allow for a prediction of interindividual variability of this pathway once the variability of expression of this enzyme is known in a given population. Identification of the en~me involved can be achieved by estimating the maximum rate of formation ~x,.~, ~ in the microsomal fraction of different human livers and correlating V ~ to the amount of different enzymes as derived from Western blotting. Subsequently, specific antibodies directed against single enzymes are used to inhibit metabolite formation. The extent of inhibition allows to determine whether one or multiple enzymes catalyze this pathway. Finally, stable expression of single enzymes enables a qualitative judgement whe,,her a metabolite is formed by a specific enzyme. A more complex scenario arises if a drug is metabolized via different routes at different rates. In order to quantify the relative contribution of individual pathways the :mrinsic cIearance of each metabolic step needs to be calculated from the ratio of V~, and the apparent affinity (Km). Ranking of the instrinsic clearance data allows for a prediction of the relative contribution of each pathway to the metabolic clearance of the drug. The approach described in here has been successfully applied using verapamil and propafenone as model compounds. The relative contribution of individual metabolic pathways, stereoselectivity in metabolism and metabolic enantiomer/enantiomer interactions have been evaluated in vitro and the validity of predictions from these in vitro data has been investigated in in vivo trials. Supported by the Robert Bosch foundation, Stuttgart, Germany
97
SC26 ASSESSMENTOF HUMAN CYTOCHROME P-450"ISOENZYMESPECIFICITY R. Gasser, A. Viger-Chougnet PharrneResearch,PreclinicalDivision,F. Hoffmann-LaRoche.4002Basel,Switzerland
It is advantageous to identify specific P-450s x,,hich interact ",,,ith drug candidates prior to their entrance into clinical trials or the market for several reasons: a drug metabolized by a polymorphic enzyme may lead to adverse reactions in those patients who lack these enzymes, or it might interact with other compounds metabolized by the same enzyme. In addition, metabolism by a highly variable human enzyme might reduce therapeutic efficiency or increase the incidence of adverse side effects. Various approaches can be used to determine which P..450s are contributing to a particular metabolic reaction. The validity of the use of selective chemical inhibitors rests on the specifity of the different substrates and inhibitors. These however are often poorly characterized in published reports, and the results therefore have to be evaluated cautiously. For example, several CYP2EI inhibitors (e.g disulfiram, dihydrocapsaicin) and substrates (e.g. chlorzoxazone, p-nitropbeool) reported to be "rather" specific do also competitively inhibit CYP 3A4 mediated reactions (e.g. midazolam hydroxylation). Recombinant P-450s can be used to confirm the results of inhibition-competition studies, but the product formation rates should be high enough to detect them. In our experience human liver microsomes are between 15-80 times more active than recombinant preparations commercially available. Where no recombinant P..450 is available, as in the case of CYP 2C19, involved in the polymorphic hydroxylation of S-mephenytoin, phenotyped human liver preparations can be used to correlate in v~tro activities with the metabolism of a test compound. Using this approach the formation of a lantam metabolite of moolobemide (Aurorix) was shown to involve the polymorphic CYP 2C 19 isoenzyme. Finally, as candidate drugs enter the clinical development phase, we need to consider which products are likely to be co-prescribed. The potentidal of these compounds to interact with the metabolism of the candidate drug can be assessed m vitro, as the basis for specific and focused trials m vivo.
SC25 PREDICTION OF DRUG METABOLISM:N-OXYGENATIONAND RETROREDUCTION OF THE N-OXYGENATED METABOLITES B. Clement, R. Lomb PharmaceuticalInstitute,Christian-AlbrechtsUniversityKiel, 24118Kiel, Germany
For predictions about the functionalisation (phase 1 metabolism) of drugs, the nitrogen atoms present are of major importance since an electron can very easily be abstracted from this heteroatom in the course of a catalytic reaction with the cytochrome P-450 enzyme system (P-450). A further reason for this significance is the fact that many nitrogen-containing groups possess sufficient nuelcophilicity to attack the oxygenating form of the flavine-containing monooxygenase (FMO), namely a 4ot-hydroperoxyfiavine. Even so, many nitrogen-containing functional groups are considered to be metabolically stable, as has mainly been demonstrated by in vivo investigations. For the example of the N-hydroxylation reactions of amidines, guanidines, and amidinohydrazones by P-450 and the retroreductions (N-dehydroxylation) of the resultant primary products, it has been shown that the initially formed Noxygenated metabolites are very easily transformed back into the starting materials with the result that this metabolic conversion can be missed. Investigations of structure-activity relationships have shown that, in contrast to previous concepts, N-hydruxylations by P-450 are also possible when the structural prerequisites for the N-dealkylation reaction are present. The retroreductions (N-dehydruxylation) of the formed metabolites (amidoximes, N-hydruxyguanidines, N-hydroxyamidinohydrazones) are catalysed by an enzyme system consisting of cytochrome bs, cytochrome b 5 reductase, and a third enzyme which has now been purified to homogeneity. It was found that some of the N-hydroxylaled metabolites possess a genotoxic potential. In this context, the retroreductions can be considered as detuxifieations. On the whole, particular attention must be directed to metabolic cycles in the sense of N-oxygenation and retroreduction during predictions on the metabolism of drugs possessing nitrogen-containing functional groups.
SC27 PREDICTION OF IN VNO DRUG METABOLISMIN HUMANSFROM IN VITRO DATA H.K. Kroemer Dr. MargareteFischer-Boseh-lnstituth~rKlinischePharmakologie,70~76Stultgafl,Germany
One major factor in modulating the dose vs. concentration relationship of drugs is interindividual variability in the activity of drug metabolizing enzymes. Thus, administration of the same dose may result in variable concentrations and therefore elicit variable effects. Characterization of the enzyme involved in a certain metabolic step would allow for a prediction of interindividual variability of this pathway once the variability of expression of this enzyme is known in a given population. Identification of the en~me involved can be achieved by estimating the maximum rate of formation ~x,.~, ~ in the microsomal fraction of different human livers and correlating V ~ to the amount of different enzymes as derived from Western blotting. Subsequently, specific antibodies directed against single enzymes are used to inhibit metabolite formation. The extent of inhibition allows to determine whether one or multiple enzymes catalyze this pathway. Finally, stable expression of single enzymes enables a qualitative judgement whe,,her a metabolite is formed by a specific enzyme. A more complex scenario arises if a drug is metabolized via different routes at different rates. In order to quantify the relative contribution of individual pathways the :mrinsic cIearance of each metabolic step needs to be calculated from the ratio of V~, and the apparent affinity (Km). Ranking of the instrinsic clearance data allows for a prediction of the relative contribution of each pathway to the metabolic clearance of the drug. The approach described in here has been successfully applied using verapamil and propafenone as model compounds. The relative contribution of individual metabolic pathways, stereoselectivity in metabolism and metabolic enantiomer/enantiomer interactions have been evaluated in vitro and the validity of predictions from these in vitro data has been investigated in in vivo trials. Supported by the Robert Bosch foundation, Stuttgart, Germany
97
SC26 ASSESSMENTOF HUMAN CYTOCHROME P-450"ISOENZYMESPECIFICITY R. Gasser, A. Viger-Chougnet PharrneResearch,PreclinicalDivision,F. Hoffmann-LaRoche.4002Basel,Switzerland
It is advantageous to identify specific P-450s x,,hich interact ",,,ith drug candidates prior to their entrance into clinical trials or the market for several reasons: a drug metabolized by a polymorphic enzyme may lead to adverse reactions in those patients who lack these enzymes, or it might interact with other compounds metabolized by the same enzyme. In addition, metabolism by a highly variable human enzyme might reduce therapeutic efficiency or increase the incidence of adverse side effects. Various approaches can be used to determine which P..450s are contributing to a particular metabolic reaction. The validity of the use of selective chemical inhibitors rests on the specifity of the different substrates and inhibitors. These however are often poorly characterized in published reports, and the results therefore have to be evaluated cautiously. For example, several CYP2EI inhibitors (e.g disulfiram, dihydrocapsaicin) and substrates (e.g. chlorzoxazone, p-nitropbeool) reported to be "rather" specific do also competitively inhibit CYP 3A4 mediated reactions (e.g. midazolam hydroxylation). Recombinant P-450s can be used to confirm the results of inhibition-competition studies, but the product formation rates should be high enough to detect them. In our experience human liver microsomes are between 15-80 times more active than recombinant preparations commercially available. Where no recombinant P..450 is available, as in the case of CYP 2C19, involved in the polymorphic hydroxylation of S-mephenytoin, phenotyped human liver preparations can be used to correlate in v~tro activities with the metabolism of a test compound. Using this approach the formation of a lantam metabolite of moolobemide (Aurorix) was shown to involve the polymorphic CYP 2C 19 isoenzyme. Finally, as candidate drugs enter the clinical development phase, we need to consider which products are likely to be co-prescribed. The potentidal of these compounds to interact with the metabolism of the candidate drug can be assessed m vitro, as the basis for specific and focused trials m vivo.