The objective of the current study was to evaluate the mechanism of absorption and metabolism of a PEGylated peptide, MRL-1 (46 kDa), after s.c. dosing in dogs and rats. Thoracic lymph duct-cannulated (LDC) dog and rat models were developed that allowed continuous collection of lymph for up to 8 days. When [(3)H]MRL-1 was administered s.c. to LDC dogs, ∼73% of the administered radioactivity was recovered in pooled lymph over a period of 120 hours, suggesting that lymphatic uptake is the major pathway of s.c. absorption for this peptide. In agreement with these data, the systemic exposure of radioactivity related to [(3)H]MRL-1 in LDC dogs was decreased proportionately when compared with that in noncannulated control dogs. After i.v. dosing with [(3)H]MRL-1 in LDC dogs, 20% of the administered radioactivity was recovered in pooled lymph over 168 hours, suggesting some level of recirculation of radioactivity related to [(3)H]MRL-1 from the plasma compartment into the lymphatic system. Experiments conducted in the LDC rat model also resulted in similar conclusions. Analysis of injection site s.c. tissue showed significant metabolism of [(3)H]MRL-1, which provides an explanation for the <100% bioavailability of therapeutic proteins and peptides after s.c. dosing. After s.c. dosing, the major circulating components in plasma were the parent peptide and the PEG-linker [(3)H]MRL-2. The metabolism profiles in lymph were similar to those in plasma, suggesting that the loss of peptide was minimal during lymphatic transport. After i.v. dosing in rats, [(3)H]MRL-1 was metabolized and excreted primarily in the urine as metabolites.

Here we report an efficient synthesis of (14)C-labeled 6-[2-(dimethylamino)ethyl]-14-(1-ethylpropyl)-5,6,7,8-tetrahydroindolo-[2,1-alpha][2,5]benzodiazocine-11-carboxylic acid (1) using the Curtius rearrangement as a key step. The synthesis was initiated by converting the unlabeled aryl carboxylic acid to an aryl amine via Curtius rearrangement reaction. The resulting aryl amine was then converted to an aryl iodide which was coupled with zinc [(14)C] cyanide to form an aryl [(14)C] nitrile. Subsequent hydrolysis yielded the (14)C-labeled carboxylic acid [(14)C]-1. This overall process represents a mild and potentially general approach for conversion of unlabeled carboxylic acids to isotopically labeled carboxylic acids.

A radiolabeled photo-affinity probe containing a diazirine group was designed and synthesized based on an atypical leukotriene (LT) biosynthesis inhibitor to determine whether this type of inhibitor (such as 5) interacts with a novel protein, critical for LT biosynthesis. One of the key transformations was to introduce a tritium label in the thiazole ring. This was eventually achieved by treating the iodinated compound with tritium, Pt/C (sulfided) and triethylamine in anhydrous aprotic solvent, to give the tritiated compound with a specific activity of 303 GBq/mmol. The affinity probe will be used to identify the proteins and protein complexes with which this type of molecules interacts with in inflammatory cells.

Because glucocorticoids and the neurokinin-1 receptor antagonist aprepitant influence CYP3A4 activity, this study assessed whether aprepitant added to a 5-HT3 antagonist and glucocorticoid would affect CYP3A4 induction.

The pharmacokinetics and metabolism of anacetrapib (MK-0859), a novel cholesteryl ester transfer protein inhibitor, were examined in rats and rhesus monkeys. Anacetrapib exhibited a low clearance in both species and a moderate oral bioavailability of ∼38% in rats and ∼13% in monkeys. The area under the plasma concentration-time curve in both species increased in a less than dose-proportional manner over an oral dose range of 1 to 500 mg/kg. After oral administration of [14C]anacetrapib at 10 mg/kg, ∼80 and 90% of the radioactive dose was recovered over 48 h postdose from rats and monkeys, respectively. The majority of the administered radioactive dose was excreted unchanged in feces in both species. Biliary excretion of radioactivity accounted for ∼15% and urinary excretion for less than 2% of the dose. Thirteen metabolites, resulting from oxidative and secondary glucuronic acid conjugation, were identified in rat and monkey bile. The main metabolic pathways consisted of O-demethylation (M1) and hydroxylation on the biphenyl moiety (M2) and hydroxylation on the isopropyl side chain (M3); these hydroxylations were followed by O-glucuronidation of these metabolites. A glutathione adduct (M9), an olefin metabolite (M10), and a propionic acid metabolite (M11) also were identified. In addition to parent anacetrapib, M1, M2, and M3 metabolites were detected in rat but not in monkey plasma. Overall, it appears that anacetrapib exhibits a low-to-moderate degree of absorption after oral dosing and majority of the absorbed dose is eliminated via oxidation to a series of hydroxylated metabolites that undergo conjugation with glucuronic acid before excretion into bile.

Carbon-14 labeled sodium formate was utilized for the preparation of a radiolabeled tracer to support the development of a Maxi-K channel blocker. Key to the process was a palladium-catalyzed hydroxycarbonylation of a bromoarene for installation of the radiolabel.

Anacetrapib is a novel cholesteryl ester transfer protein inhibitor being developed for the treatment of primary hypercholesterolemia and mixed dyslipidemia. The absorption, distribution, metabolism, and excretion of anacetrapib were investigated in an open-label study in which six healthy male subjects received a single oral dose of 150 mg and 165 microCi of [(14)C]anacetrapib. Plasma, urine, and fecal samples were collected at predetermined times for up to 14 days postdose and were analyzed for total radioactivity, the parent compound, and metabolites. The majority of the administered radioactivity (87%) was eliminated by fecal excretion, with negligible amounts present in urine (0.1%). The peak level of radioactivity in plasma (approximately 2 microM equivalents of [(14)C]anacetrapib) was achieved approximately 4 h postdose. The parent compound was the major radioactive component (79-94% of total radioactivity) in both plasma and feces. Three oxidative metabolites, M1, M2, and M3, were detected in plasma and feces and were identified as the O-demethylated species (M1) and two secondary hydroxylated derivatives of M1 (M2 and M3). Each metabolite was detected at low levels, representing <or=14% of the radioactivity in plasma or fecal samples. In vitro data indicated that anacetrapib is metabolized mainly by CYP3A4 to form M1, M2, and M3. Overall, these data, along with those from other preclinical and clinical studies, indicate that anacetrapib probably exhibits a low-to-moderate degree of oral absorption in humans and the absorbed fraction of the dose is eliminated largely via CYP3A4-catalyzed oxidative metabolism, followed by excretion of metabolites by the biliary-fecal route.

During our effort to design a receptor binding assay to aid in the elucidation of the molecular mechanism of ezetimibe, we prepared a sulfur-35 containing radioligand which exhibits improved potency over the glucuronide conjugate of ezetimibe in both native enterocyte brush border membranes and membranes from cells expressing recombinant NPC1L1. Herein, we describe the different synthetic strategies which were used to obtain this compound as well as its effectiveness in the aforementioned assay.

PURPOSE:Gaboxadol, a selective extrasynaptic agonist of the delta-containing gamma-aminobutyric acid type A (GABAA) receptor, is excreted in humans into the urine as parent drug and glucuronide conjugate. The goal of this study was to identify the UDP-Glucuronosyltransferase (UGT) enzymes and the transporters involved in the metabolism and active renal secretion of gaboxadol and its metabolite in humans.Methods. The structure of the glucuronide conjugate of gaboxadol in human urine was identified by LC/MS/MS. Human recombinant UGT isoforms were used to identify the enzymes responsible for the glucuronidation of gaboxadol. Transport of gaboxadol and its glucuronide was evaluated using cell lines and membrane vesicles expressing human organic anion transporters hOAT1 and hOAT3, organic cation transporter hOCT2, and the multidrug resistance proteins MRP2 and MRP4.Results. Our study indicated that the gaboxadol-O-glucuronide was the major metabolite excreted in human urine. UGT1A9, and to a lesser extent UGT1A6, UGT1A7 and UGT1A8, catalyzed the O-glucuronidation of gaboxadol in vitro. Gaboxadol was transported by hOAT1, but not by hOCT2, hOAT3, MRP2, and MRP4. Gaboxadol-O-glucuronide was transported by MRP4, but not MRP2.Conlusion. Gaboxadol could be taken up into the kidney by hOAT1 followed by glucuronidation and efflux of the conjugate into urine via MRP4.

Recent clinical reports have suggested that the cyclooxygenase-2 inhibitor, lumiracoxib (Prexige), may cause a rare but serious hepatotoxicity in patients. In view of the close structural resemblance between lumiracoxib and diclofenac, a widely used nonsteroidal anti-inflammatory drug whose use also has been associated with rare cases of liver injury, it is possible that the toxicity of the two agents may share a common mechanism. Because it is believed that chemically reactive metabolites may play a role as mediators of diclofenac-mediated hepatotoxicity, the present in vitro study was carried out to test the hypothesis that lumiracoxib also undergoes metabolic activation when incubated with liver microsomal preparations and hepatocytes from rats and humans. By means of liquid chromatography tandem mass spectrometry and nuclear magnetic resonance spectrometry techniques, two previously unknown N- acetylcysteine (NAC) conjugates were identified, namely, 3'-NAC-4'-hydroxy lumiracoxib (M1) and 4'-hydroxy-6'-NAC-desfluoro lumiracoxib (M2), the structures of which reveal the intermediacy of an electrophilic quinone imine species. Based on the results of studies with immunoinhibitory antibodies, it was demonstrated that the formation of M1 and M2 in human liver microsomes was catalyzed by cytochrome P450 (P450) 2C9. These findings demonstrate that lumiracoxib is subject to P450-mediated bioactivation in both rat and human liver preparations, leading to the formation of a reactive intermediate analogous to species generated during the metabolism of diclofenac.

C-14-Labeled farnesyl-protein transferase inhibitor 1a was required for drug metabolism studies. The first approach was to synthesize C-14-labeled 1b using Zn((CN)-C-14)(2) in a Pd-catalyzed cyanation. A second approach was to prepare labeled 1c with the C-14-label in the piperazine ring. All metabolites from 1b were non-radioactive whereas the same metabolites from 1c were all radioactive and quantifiable.

Absorption of dietary cholesterol in the proximal region of the intestine is mediated by Niemann-Pick C1-like protein (NPC1L1) and is sensitive to the cholesterol absorption inhibitor ezetimibe (EZE). Although a correlation exists between EZE binding to NPC1L1 in vitro and efficacy in vivo, the precise nature of interaction(s) between NPC1L1, EZE, and cholesterol remain unclear. Here, we analyze the direct relationship between EZE analog binding to NPC1L1 and its influence on cholesterol influx in a novel in vitro system. Using the EZE analog [(3)H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. It is noteworthy that whereas two cell lines (CaCo-2 and HepG2) commonly used for studying NPC1L1-dependent processes express almost undetectable levels of NPC1L1 at the cell surface, polarized Madin-Darby canine kidney (MDCKII) cells endogenously express 4 x 10(5) [(3)H]AS sites/cell under basal conditions. Depleting endogenous cholesterol with the HMG CoA reductase inhibitor lovastatin leads to a 2-fold increase in the surface expression of NPC1L1, supporting the contention that MDCKII cells respond to changes in cholesterol homeostasis by up-regulating a pathway for cholesterol influx. However, a significant increase in surface expression levels of NPC1L1 is necessary to characterize a pharmacologically sensitive, EZE-dependent pathway of cholesterol uptake in these cells. Remarkably, the affinity of EZE analogs for binding to NPC1L1 is almost identical to the IC(50) blocking cholesterol flux through NPC1L1 in MDCKII cells. From a mechanistic standpoint, these observations support the contention that EZE analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.

In support of a research program aimed at discovering purine-related anticancer drug candidates, a method for the (14)C-labeling of pyrazolopyrimidines utilizing the readily available labeled starting material, sodium [(14)C]formate, has been developed with a good overall yield. This new method was proven to be general in the preparation of other related [4.3.0] heterocycles containing N, O, and S atoms. A concise synthesis of a model compound, 8-aza-7-deaza-5'-[(14)C]noraristeromycin, was achieved utilizing this methodology as a key step.