University of Groningen Stereoselectivity in the hepatic metabolism and transport of quaternary drugs of the morphinan type Lanting, Aleida Bartha Louisa IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2000 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Lanting, A. B. L. (2000). Stereoselectivity in the hepatic metabolism and transport of quaternary drugs of the morphinan type. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-09-2021 In this thesisthe potential of some quaternaryamine derivativesof the morphinan analoguesdextrorphan, levorphanol, dextromethorphan and levomethorphanas model compoundswas investigatedto study stereoselectivityin hepatic metabolismand transport of cationic drugs.These model compoundspossess a rigid stereochemicalstructure, resulting in major differencesin receptor affinity. We assumedthat the major influenceof the stereochemicalstructure on the receptor level might also causedifferences in carrier-mediatedtransport and biotransformation processes. During the developmentof a reversed-phaseHPLC separationsystem for the analysisof the purity of the quaternizedmorphinans (N-methyl dextrorphan, N-methyl levorphanol,N-methyl dextromethorphanand N-methyl levomethorphan)it became clear that silanophilicinteractions rvith the stationaryphase were involved.Some complicatingfactors were observed:The separationwas sensitiveto the brand and type of column material,to slightpH changesof the mobile phaseand to column overloading.However, these silanophilic interactions also played a decisiverole in the selectivityof the stationaryphase, and therefore no attempts were made to suppress the latter. SpherisorbODS-I stationaryphases appeared to be adequate in the separationof the four morphinansand were chosenfor the estimation of the purity of synthesizedquaternary ammonium morphinans (Chapter 5.1). For the analysisof biologicalsamples, a C6-bondedstationary phase and a gradient of acetonitrile in 0.02 M sodium phosphatebuffer with a low pH as a mobile phase was used.This method was able to separatethe respectiveparent compounds, their primary metabolitesand some conjugatesin a single run. The silanophilic interactionsalso causedsome troublesomeeff'ects in the bioanalysisof the metabolites of the quaternarymorphinans. First, the separationrvas affected b)'column degradationand by batch-to-batchdiff'erences in column material.Small adaptations of the mobile phasecomposition were made to compensatefor small changesin chromatographicperformance. Another effect of the involvementof silanophilic interactionswas that proteins in the biological sampleshad a detrimental effect on the columnperformance. A precolumnswitching technique on a reversedphase C8 column with a backflushstep to introduce the analyteson the HPLC column was successfullyused (Chanter5.2). To study the hepatic transport and metabolism,isolated Wistar rat liver perfusionswere performed. Experimentswith radioactivelylabelled N-methyl dextrorphan,N-methyl levorphanol,N-methyl dextromethorphan,and N-methyl levomethorphanshowed that the initial uptake of the former pair of enantiomerswas not stereoselective,but N-methyl dextromethorphanwas taken up slightlyfaster in the liver than N-methyllevomethorphan (Chapter 5.5, and Chapter5.6 resp.).Further transport stepscould not be observedbecause the compoundswere extensively metabolizedin a stereoselectiveway. Samplestaken during the liver perfusionswere analyzedwith the HPLC method describedabove. Very distinct radiochromatograms were obtained for the enantiomericpairs. Experimentswith selectiveenzl,matic LJ I hydrolysisshowed that glucuronidesand sulfatesof the morphinans were present in the samples.Further identification of the metaboliteswas accompishedby ionspray LCIMS. Liver perfusionswith stable isotope labelled compound were performed and the sampleswere analyzedwith a gradient HPLC method with the same stationary phase but with a volatile mobile phase.Both N-methyl dextrorphan and N-methyl levorphanol were found glucuronidatedand conjugatedwith glutathione.In addition, N-methyl levorphanol was hydroxylatedand subsequentlyglucuronidated, and a sulfoconjugateof N-methyl levorphanol was also found (Chapter 5.3). When sfudying the metabolism of N-methyl dextromethorphanand N-methyl levomethorphan,the O-demethylationsof both substancestook place at about the same velocity.Further metabolism stepswere glucuronidationand glutathioneconjugation for the O-demethylatedN-methyl dextromethorphanand glucuronidation,glutathione conjugation,sulfoconjugation and hydroxylationfor the O-demethylatedN-methyt levomethorphan(Chapter 5.4). The O-demethylationof N-methyl dextromethorphanand N-methyl levomethorphanmay be under control of a cytochromeP450 isozymethat is also responsiblefor the genetic polymorphismof the debrisoquinetype. In that case,the O-demethylationof the two enantiomersshould be reduced in the livers of Dark Agouti rats, which is an animal model for this geneticpolymorphism. We performed isolatedrat liver perfusionswith livers of these Dark Agouti rats to test this assumption.The O-demethl,lationof N-methyl dextromethorphanindeed was largely reduced.The O-demethylationof N-methyllevomethorphan, however, was Iargely unaffected.The velocity of the enzymaticreaction was almost identical to that in Wistar rats. Probably, other isoformsof cytochromeP450 played a role in the O-demethylationof N-methyl levomethorphan.Unfortunately, the Dark Agouti rat did not appear to be a satisfyinganimal model to study stereoselectivityin other transport phenomenathan the initial uptake into the liver of cationic drugs (Chapter 5.7). Finally, concentratingon the initial uptake of the cationic model compounds, inhibitionexperiments with procainamideethobromide and digoxinin isolatedrat liver perfusionswere performed to characterizethe type of carrier systemthat was involved in the uptake of N-methyl dextrorphan and N-methyl levorphanol.It appeared that the uptake in the liver of the enantiomersN-methyl dextrorphan and N-methyl levorphanol occurred partly via the type I transport systemand partly via the type II or multispecifictransport system.The uptake via the type II transport systemwas found to be stereoselective:The half-life of N-methyl levorphanolappeared to be about 50 % larger than that of its enantiomerN-methyl dextrorphan (Chapter 5.8). It is concludedthat the quaternaryammonium derivativesof morphinan enantiomersstudied provide interestingmodel compoundsfor the study of carrier-mediatedtransport processesfor cationic drugs.Their disadvantagefor studies with intact organs however is that extensivemetabolism occcurs at the same time and this largely complicatesthe study of concomittanttransport steps.Therefore, future srudiesshould be performed in isolatedplasma membrane vesiclesor in reconstituted membrane systemsincluding the transport proteins. Recent studiesin our laboratories using expressionsystems of these proteins, cloned by us and others, indeed demonstratestereoselectivity in membrane transport of cationic drugs..