Charles S. Elmore

Metabolism And Excretion of the Dipeptidyl Peptidase 4 Inhibitor [14C]Sitagliptin in Humans

The metabolism and excretion of [14C]sitagliptin, an orally active, potent and selective dipeptidyl peptidase 4 inhibitor, were investigated in humans after a single oral dose of 83 mg/193 μCi. Urine, feces, and plasma were collected at regular intervals for up to 7 days. The primary route of excretion of radioactivity was via the kidneys, with a mean value of 87% of the administered dose recovered in urine. Mean fecal excretion was 13% of the administered dose. Parent drug was the major radioactive component in plasma, urine, and feces, with only 16% of the dose excreted as metabolites (13% in urine and 3% in feces), indicating that sitagliptin was eliminated primarily by renal excretion. Approximately 74% of plasma AUC of total radioactivity was accounted for by parent drug. Six metabolites were detected at trace levels, each representing <1 to 7% of the radioactivity in plasma. These metabolites were the N-sulfate and N-carbamoyl glucuronic acid conjugates of parent drug, a mixture of hydroxylated derivatives, an ether glucuronide of a hydroxylated metabolite, and two metabolites formed by oxidative desaturation of the piperazine ring followed by cyclization. These metabolites were detected also in urine, at low levels. Metabolite profiles in feces were similar to those in urine and plasma, except that the glucuronides were not detected in feces. CYP3A4 was the major cytochrome P450 isozyme responsible for the limited oxidative metabolism of sitagliptin, with some minor contribution from CYP2C8.

Use of radiolabeled compounds in drug metabolism and pharmacokinetic studies.

As part of the drug discovery and development process, it is important to understand the fate of the drug candidate in humans and the relevance of the animal species used for preclinical toxicity and pharmacodynamic studies. Therefore, various in vitro and in vivo studies are conducted during the different stages of the drug development process to elucidate the absorption, distribution, metabolism, and excretion properties of the drug candidate. Although state-of-the-art LC/MS techniques are commonly employed for these studies, radiolabeled molecules are still frequently required for the quantification of metabolites and to assess the retention and excretion of all drug related material without relying on structural information and MS ionization properties. In this perspective, we describe the activities of Isotope Chemistry at AstraZeneca and give a brief overview of different commonly used approaches for the preparation of (14)C- and (3)H-labeled drug candidates. Also various drug metabolism and pharmacokinetic studies utilizing radiolabeled drug candidates are presented with in-house examples where relevant. Finally, we outline strategic changes to our use of radiolabeled compounds in drug metabolism and pharmacokinetic studies, with an emphasis on delaying of in vivo studies employing radiolabeled drug molecules.

Disposition of the dipeptidyl peptidase 4 inhibitor sitagliptin in rats and dogs.

The pharmacokinetics, metabolism, and excretion of sitagliptin [MK-0431; (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine], a potent dipeptidyl peptidase 4 inhibitor, were evaluated in male Sprague-Dawley rats and beagle dogs. The plasma clearance and volume of distribution of sitagliptin were higher in rats (40–48 ml/min/kg, 7–9 l/kg) than in dogs (∼9 ml/min/kg, ∼3 l/kg), and its half-life was shorter in rats, ∼2 h compared with ∼4 h in dogs. Sitagliptin was absorbed rapidly after oral administration of a solution of the phosphate salt. The absolute oral bioavailability was high, and the pharmacokinetics were fairly dose-proportional. After administration of [14C]sitagliptin, parent drug was the major radioactive component in rat and dog plasma, urine, bile, and feces. Sitagliptin was eliminated primarily by renal excretion of parent drug; biliary excretion was an important pathway in rats, whereas metabolism was minimal in both species in vitro and in vivo. Approximately 10 to 16% of the radiolabeled dose was recovered in the rat and dog excreta as phase I and II metabolites, which were formed by N-sulfation, N-carbamoyl glucuronidation, hydroxylation of the triazolopiperazine ring, and oxidative desaturation of the piperazine ring followed by cyclization via the primary amine. The renal clearance of unbound drug in rats, 32 to 39 ml/min/kg, far exceeded the glomerular filtration rate, indicative of active renal elimination of parent drug.

[N-methyl-3H3]AZ10419369 Binding to the 5-HT1B Receptor: In Vitro Characterization and in Vivo Receptor Occupancy

Radiotracers suitable for positron emission tomography studies often serve as preclinical tools for in vivo receptor occupancy. The serotonin 1B receptor (5-HT1B) subtype is a pharmacological target used to discover treatments for various psychiatric and neurological disorders. In psychiatry, 5-HT1B antagonists may provide novel therapeutics for depression and anxiety. We report on the in vitro and in vivo evaluation of tritiated 5-methyl-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylicacid (4-morpholin-4-yl-phenyl)-amide ([N-methyl-3H3]AZ10419369), a potent 5-HT1B radiotracer. [N-methyl-3H3]-AZ10419369 showed saturable single-site high-affinity in vitro binding (guinea pig, Kd = 0.38 and human, Kd = 0.37) to guinea pig or human 5-HT1B receptors in recombinant membranes and high-affinity (Kd = 1.9 nM) saturable (Bmax = 0.099 pmol/mg protein) binding in membranes from guinea pig striatum. When [N-methyl-3H3]AZ10419369 was administered to guinea pigs by intravenous bolus, the measured radioactivity was up to 5-fold higher in brain areas containing the 5-HT1B receptor (striatum/globus pallidus, midbrain, hypothalamus, and frontal cortex) compared with the cerebellum, the nonspecific binding region. Specific uptake peaked 30 min after injection with slow dissociation from target regions, as suggested by the in vitro binding kinetic profile. Pretreatment with 6-fluoro-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylic acid [4-(4-propionyl-piperazin-1-yl)-phenyl]-amide (AZD1134) and 2-aminotetralin (AR-A000002), 5-HT1B-selective ligands, inhibited [N-methyl-3H3]AZ10419369-specific binding in a dose-dependent manner. In the guinea pig striatum, AZD1134 (ED50 = 0.017 mg/kg) occupies a greater percentage of the 5-HT1B receptors at a lower administered dose than AR-A000002 (ED50 = 2.5 mg/kg). In vivo receptor occupancy is an essential component to build binding-efficacy-exposure relationships and compare novel compound pharmacology. [N-methyl-3H3]AZ10419369 is a useful preclinical tool for investigating 5-HT1B receptor occupancy for novel compounds targeting this receptor.

Multiple Compound-Related Adverse Properties Contribute to Liver Injury Caused by Endothelin Receptor Antagonists

Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver–derived HuH-7 cells was determined using a Seahorse XFe96 analyzer. Cytochrome P450 (P450)–independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen–immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan.

PET Tracers to Study Clinically Relevant Hepatic Transporters

Transporter proteins expressed on the cell membranes of hepatocytes are directly involved in the hepatic clearance, mediating the transport of drugs and metabolites through the hepatocyte, from the bloodstream into the bile. Reduction of hepatic transporter activity (due to chemical inhibition, genetic polymorphism, or low expression) can increase systemic or liver exposure to potentially toxic compounds, causing adverse effects. Many clinically used drugs have been associated with inhibition of hepatic transporters in vitro, suggesting the potential involvement of liver transporters in drug-drug interactions (DDIs). Recently, radiolabeled hepatic transporter substrates have been successfully employed in positron emission tomography (PET) imaging to demonstrate inhibition of clinically relevant hepatic transporters. The present article briefly describes the clinical relevance of hepatic transporters followed by a review of the application of PET imaging for the determination of pharmacokinetic parameters useful to describe the transporter activity and the design, accessibility, and preclinical and clinical applications of available radiotracers. Finally, based on the analysis of the strengths and limitations of the available tracers, some criteria for the development of novel PET probes for hepatic transporters and new potential applications are suggested.

An integrated strategy for in vivo metabolite profiling using high-resolution mass spectrometry based data processing techniques

An ongoing challenge of drug metabolite profiling is to detect and identify unknown or low-level metabolites in complex biological matrices. Here we present a generic strategy for metabolite detection using multiple accurate-mass-based data processing tools via the analysis of rat samples of two model drug candidates, AZD6280 and AZ12488024. First, the function of isotopic pattern recognition was proved to be highly effective in the detection of metabolites derived from [(14)C]-AZD6280 that possesses a distinct isotopic pattern. The metabolites revealed using this approach were in excellent qualitative correlation to those observed in radiochromatograms. Second, the effectiveness of accurate mass based untargeted data mining tools such as background subtraction, mass defect filtering, or a data mining package (MZmine) used for metabolomic analysis in detection of metabolites of [(14)C]-AZ12488024 in rat urine, feces, bile and plasma samples was examined and a total of 33 metabolites of AZ12488024 were detected. Among them, at least 16 metabolites were only detected by the aid of the data mining packages and not via radiochromatograms. New metabolic pathways such as S-oxidation and thiomethylation reactions occurring on the thiazole ring were proposed based on the processed data. The results of these experiments also demonstrated that accurate mass-based mass defect filtering (MDF) and data mining techniques used in metabolomics are complementary and can be valuable tools for delineating low-level metabolites in complex matrices. Furthermore, the application of distinct multiple data-mining algorithms in parallel, or in tandem, can be effective for rapidly profiling in vivo drug metabolites.

Development of a PET radioligand for the central 5-HT1B receptor: radiosynthesis and characterization in cynomolgus monkeys of eight radiolabeled compounds.

INTRODUCTION The serotonin 1B (5-HT(1B)) receptor has been implicated in several psychiatric disorders and is a potential pharmacological target in the treatment of depression. The aim of this study was to develop a radioligand for positron emission tomography (PET) imaging of the 5-HT(1B) receptor in the primate brain in vivo. METHODS Eight carboxamide radioligands (1-8) from three different core structures were radiolabeled with carbon-11 employing N-methylation with [(11)C]methyl triflate on the piperazine structural moiety. In vivo PET evaluation of each radioligand was performed in cynomolgus monkeys and included analysis of radioactive metabolites measured in plasma using high-performance liquid chromatography. RESULTS In a total of 12 radiosynthesis of the eight radioligands, the mean decay corrected yield was 11%, and the mean specific radioactivity was 299 GBq/μmol (8075 Ci/mmol) at time of administration. Of the eight tested candidates, [(11)C]6 demonstrated the most promising in vivo characteristics, showing high binding in 5-HT(1B) receptor-rich regions and low binding in the cerebellum. When inspecting data from all eight compounds, lipophilicity appeared as a physicochemical property that could be related to favorable in vivo imaging characteristics. CONCLUSION Candidate [(11)C]6, i.e., [(11)C]AZ10419369, exhibited high binding potentials in regions known to contain 5-HT(1B) receptors and was nominated for further preclinical characterization and PET examination in human subjects.

Isotope chemistry; a useful tool in the drug discovery arsenal.

As Medicinal Chemists are responsible for the synthesis and optimization of compounds, they often provide intermediates for use by isotope chemistry. Nevertheless, there is generally an incomplete understanding of the critical factors involved in the labeling of compounds. The remit of an Isotope Chemistry group varies from company to company, but often includes the synthesis of compounds labeled with radioisotopes, especially H-3 and C-14 and occasionally I-125, and stable isotopes, especially H-2, C-13, and N-15. Often the remit will also include the synthesis of drug metabolites. The methods used to prepare radiolabeled compounds by Isotope Chemists have been reviewed relatively recently. However, the organization and utilization of Isotope Chemistry has not been discussed recently and will be reviewed herein.

[3H]Chiba-1001(methyl-SSR180711) has low in vitro binding affinity and poor in vivo selectivity to nicotinic alpha-7 receptor in rodent brain

Neuronal nicotinic acetylcholine receptor (nAChR) agonists active at the alpha‐7 (α‐7) receptor subtype are potential therapeutics for cognitive deficits in schizophrenia, Alzheimers disease, and other mental disorders. SSR180711, an α‐7 selective partial agonist, has been shown to improve preclinical cognition. A novel positron emission tomography (PET) radioligand, 11C‐Chiba1001, is a close analog of SSR180711. We labeled Chiba‐1001 with tritium in order to evaluate its utility as a preclinical radioligand tool. In vitro, the binding affinity of [3H]Chiba‐1001 at the α‐7 receptor was low (Kd = 120–180 nM) in both HEK239 cell membranes expressing human α‐7 receptor and in native rat hippocampus membranes. The α‐7 selective ligands AZD0328, ARR17779, and MLA did not inhibit [3H]Chiba‐1001 binding (Ki > 10,000 nM). In rat hippocampal membranes, Chiba‐1001 and SSR180711 inhibited [3H]Chiba‐1001 binding (Ki = 220 and 230 nM, respectively), consistent with the literature reports. The in vivo binding profile of the radioligand was examined in normal rat, wild type mouse, and α‐7 knockout mouse brain. We found that [3H]Chiba‐1001 lacks adequate and specific brain regional uptake in rat and mouse brain. No significant inhibition of the radioligand binding was obtained following pretreatment of the animal with AZ11637326, AZD0328, or MLA. Our results indicate that [3H]Chiba‐1001 has low affinity for α‐7 nAChRs in vitro and poor α‐7 regional and pharmacological selectivity in the rodent brain. Synapse, 2012.