Michael B. Fisher

THE ROLE OF HEPATIC AND EXTRAHEPATIC UDP-GLUCURONOSYLTRANSFERASES IN HUMAN DRUG METABOLISM *,†

At present, the methods and enzymology of the UDP-glucuronosyltransferases (UGTs) lag behind that of the cytochromes P450 (CYPs). About 15 human UGTs have been identified, and knowledge about their regulation, substrate selectivity, and tissue distribution has progressed recently. Alamethicin has been characterized as a treatment to remove the latency of microsomal glucuronidations. Most UGT isoforms appear to have a distinct hepatic and/or extrahepatic expression, resulting in significant expression in kidney, intestine, and steroid target tissues. The gastrointestinal tract possesses a complex expression pattern largely containing members of the UGT1A subfamily. Thus, these forms are poised to participate in the first pass metabolism of oral drugs. The authors and others have identified a significant expression of UGT1A1 in human small intestine, an enzyme possessing considerable allelic variability and a polymorphic expression pattern in intestine. Intestinal glucuronidation therefore plays a major role not only in first pass metabolism, but also in the degree of interindividual variation in overall oral bioavailability. Due to issues such as significant genetic variability and tissue localization in first-pass organs, clearance due to UGT1A1 should be minimized for new drugs.

Discovery of CP-690,550: A Potent and Selective Janus Kinase (JAK) Inhibitor for the Treatment of Autoimmune Diseases and Organ Transplant Rejection

There is a critical need for safer and more convenient treatments for organ transplant rejection and autoimmune disorders such as rheumatoid arthritis. Janus tyrosine kinases (JAK1, JAK3) are expressed in lymphoid cells and are involved in the signaling of multiple cytokines important for various T cell functions. Blockade of the JAK1/JAK3-STAT pathway with a small molecule was anticipated to provide therapeutic immunosuppression/immunomodulation. The Pfizer compound library was screened against the catalytic domain of JAK3 resulting in the identification of a pyrrolopyrimidine-based series of inhibitors represented by CP-352,664 (2a). Synthetic analogues of 2a were screened against the JAK enzymes and evaluated in an IL-2 induced T cell blast proliferation assay. Select compounds were evaluated in rodent efficacy models of allograft rejection and destructive inflammatory arthritis. Optimization within this chemical series led to identification of CP-690,550 1, a potential first-in-class JAK inhibitor for treatment of autoimmune diseases and organ transplant rejection.

ALTERED AZT (3′-AZIDO-3′-DEOXYTHYMIDINE) GLUCURONIDATION KINETICS IN LIVER MICROSOMES AS AN EXPLANATION FOR UNDERPREDICTION OF IN VIVO CLEARANCE: COMPARISON TO HEPATOCYTES AND EFFECT OF INCUBATION ENVIRONMENT

Human liver microsomes are a reagent commonly used to predict human hepatic clearance of new chemical entities via phase 1 metabolism. Another common metabolic pathway, glucuronidation, can also be observed in human liver microsomes, although the scalability of this process has not been validated. In fact, several groups have demonstrated that clearance estimated from liver microsomes with UDP-glucuronic acid typically underpredicts the actual in vivo clearance more than 10-fold for compounds that are predominantly glucuronidated. In contrast, clearance predicted using human hepatocytes, for these same compounds, provides a more accurate assessment of in vivo clearance. We sought to characterize the kinetics of glucuronidation of the selective UGT2B7 substrate AZT (3′-azido-3′-deoxythymidine), a selective UGT2B7 substrate, in human liver microsomes (HLMs), recombinant UGT2B7, and human hepatocytes. Apparent Km values in these three preparations were 760, 490, and 87 μM, with apparent Vmax values highest in hepatocytes. The IC50 for ibuprofen against AZT glucuronidation, when run at its Km concentration in HLMs and hepatocytes, was 975 and 170 μM, respectively. Since incubation conditions have been shown to modulate glucuronidation rates, AZT glucuronidation was performed in various physiological and nonphysiological buffer systems, namely Tris, phosphate, sulfate, carbonate, acetate, human plasma, deproteinized human liver cytosol, and Williams E medium. The data showed that carbonate and Williams E medium, more physiologically relevant buffers, yielded the highest rates of AZT glucuronidation. Km observed in HLM/carbonate was 240 μM, closer to that found in hepatocytes, suggesting that matrix differences might cause the kinetic differences observed between liver preparations. Caution should be exercised when extrapolating metabolic lability via glucuronidation or inhibition of UGT enzymes from human liver microsomes, since this system appears to underpredict the degree of lability or inhibition, respectively, due in part to an apparent decrease in substrate affinity.

Physiologically Based Pharmacokinetic Modeling Framework for Quantitative Prediction of an Herb–Drug Interaction

Herb–drug interaction predictions remain challenging. Physiologically based pharmacokinetic (PBPK) modeling was used to improve prediction accuracy of potential herb–drug interactions using the semipurified milk thistle preparation, silibinin, as an exemplar herbal product. Interactions between silibinin constituents and the probe substrates warfarin (CYP2C9) and midazolam (CYP3A) were simulated. A low silibinin dose (160 mg/day × 14 days) was predicted to increase midazolam area under the curve (AUC) by 1%, which was corroborated with external data; a higher dose (1,650 mg/day × 7 days) was predicted to increase midazolam and (S)‐warfarin AUC by 5% and 4%, respectively. A proof‐of‐concept clinical study confirmed minimal interaction between high‐dose silibinin and both midazolam and (S)‐warfarin (9 and 13% increase in AUC, respectively). Unexpectedly, (R)‐warfarin AUC decreased (by 15%), but this is unlikely to be clinically important. Application of this PBPK modeling framework to other herb–drug interactions could facilitate development of guidelines for quantitative prediction of clinically relevant interactions.

Discovery, synthesis and SAR of azinyl- and azolylbenzamides antagonists of the P2X7 receptor

The discovery, of a series of 2-Cl-5-heteroaryl-benzamide antagonists of the P2X(7) receptor via parallel medicinal chemistry is described. Initial analogs suffered from poor metabolic stability and low Vd(ss). Multi parametric optimization led to identification of pyrazole 39 as a viable lead with excellent potency and oral bioavailability. Further attempts to improve the low Vd(ss) of 39 via introduction of amines led to analogs 40 and 41 which maintained the favorable pharmacology profile of 39 and improved Vd(ss) after iv dosing. But these analogs suffered from poor oral absorption, probably driven by poor permeability.