Analytical tools and approaches for metabolite identification in early drug discovery.
ABSTRACT Determination of the chemical structures of metabolites is a critical part of the early pharmaceutical discovery process. Understanding the structures of metabolites is useful both for optimizing the metabolic stability of a drug as well as rationalizing the drug safety profile. This review describes the current state of the art in this endeavor. The likely outcome of metabolism is first predicted by comparison to the literature. Then metabolites are synthesized in a variety of in vitro systems. The various approaches to LC/UV/MS are applied to learn information about these metabolites and structure hypotheses are made. Structures are confirmed by synthesis or NMR. The special topic of reactive metabolite structure determination is briefly addressed.
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ABSTRACT: The performance of the atmospheric pressure photoionization (APPI) technique was evaluated against five sets of standards and drug-like compounds and compared to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The APPI technique was first used to analyze a set of 86 drug standards with diverse structures and polarities with a 100% detection rate. More detailed studies were then performed for another three sets of both drug standards and proprietary drug candidates. All 60 test compounds in these three sets were detected by APPI with an overall higher ionization efficiency than either APCI or ESI. Most of the non-polar compounds in these three sets were not ionized by APCI or ESI. Analysis of a final set of 201 Wyeth proprietary drug candidates by APPI, APCI and ESI provided an additional comparison of the ionization techniques. The detection rates in positive ion mode were 94% for APPI, 84% for APCI, and 84% for ESI. Combining positive and negative ion mode detection, APPI detected 98% of the compounds, while APCI and ESI detected 91%, respectively. This analysis shows that APPI is a valuable tool for day-to-day usage in a pharmaceutical company setting because it is able to successfully ionize more compounds, with greater structural diversity, than the other two ionization techniques. Consequently, APPI could be considered a more universal ionization method, and therefore has great potential in high-throughput drug discovery especially for open access liquid chromatography/mass spectrometry (LC/MS) applications.Rapid Communications in Mass Spectrometry 02/2005; 19(12):1717-24. · 2.51 Impact Factor
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ABSTRACT: Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this systemis that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used fo the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction. A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices. For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes. Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.Drug Metabolism Reviews 07/2003; 35(2-3):145-213. · 5.54 Impact Factor
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ABSTRACT: DB75 (2,5-bis(4-amidinophenyl)furan) is a promising antimicrobial agent against African trypanosomiasis and Pneumocystis carinii pneumonia. However, it suffers from poor oral activity in rodent models for both infections. In contrast, a novel prodrug of DB75, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has excellent oral activity. DB289 is currently undergoing clinical investigation as a candidate drug to treat primary stage African trypanosomiasis and Pneumocystis carinii pneumonia. In this study, metabolites of DB289 formed after incubation with freshly isolated rat hepatocytes were characterized using liquid chromatography/ion trap mass spectrometry. Administration of DB289 and octadeuterated DB289 in a 1 : 1 mixture greatly facilitated metabolite identification by providing isotope patterns with twin ions separated by 8 m/z units in the ratio 1 : 1, in the extracted ion chromatograms of molecular ions and in the product ion mass spectra of metabolites. Ten metabolites were identified. Series of O-demethylations and N-dehydroxylations led to the metabolic activation of DB289 to DB75 with the production of four intermediate phase I metabolites. Phase II glucuronidation and sulfation led to the formation of four glucuronide and one sulfate metabolites.Journal of Mass Spectrometry 05/2004; 39(4):351-60. · 3.21 Impact Factor