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Non-alcoholic steatohepatitis (NASH) is the most chronic liver condition in the western population and is fueled by the obesity and type 2 diabetes epidemic. Pegbelfermin (1), a PEGylated human fibroblast growth factor 21 (FGF21) analogue, has previously been shown to improve markers of metabolism and liver fibrosis in obese patients with type 2 diabetes. Radiolabeled Pegbelfermin was needed to access the accumulation of intact drug and metabolized PEG. In an effort to accomplish both goals with one labeled synthesis, the isotopic label was positioned in the PEG. A total of 21 mCi of tritium labeled Pegbelfermin was synthesized having a specific activity of 21.6 Ci/mmol for use in animal studies.

Non-alcoholic steatohepatitis (NASH) is the most chronic liver condition in the western population and is fueled by the obesity and type 2 diabetes epidemic. Pegbelfermin (1), a PEGylated human fibroblast growth factor 21 (FGF21) analogue, has previously been shown to improve markers of metabolism and liver fibrosis in obese patients with type 2 diabetes. Radiolabeled Pegbelfermin was needed to access the accumulation of intact drug and metabolized PEG. In an effort to accomplish both goals with one labeled synthesis, the isotopic label was positioned in the PEG. A total of 21 mCi of tritium labeled Pegbelfermin was synthesized having a specific activity of 21.6 Ci/mmol for use in animal studies.

Aim: Coproporphyrins (CP-I and CP-III) have been identified as possible biomarkers to predict human hepatic organic anion-transporting polypeptides-mediated-drug-interactions for a new drug entering clinical development. Results: The method is applicable to quantify plasma CP-I and CP-III within 0.078–15.0 nM. The results identify and address a number of challenges encountered with porphyrin assays such as photodegradation and interferences. To overcome interferences from ubiquitous porphyrins, a surrogate matrix was used to prepare calibration standards. Quality controls were prepared in plasma and surrogate matrix to ensure parallelism between surrogate matrix and plasma. Conclusion: A robust UHPLC–MS/MS assay was developed and validated for CP-I and CP-III in plasma, and is currently applied to clinical studies to confirm suitability of Coproporphyrins as a potential substitute for drug–drug interaction study.

Daclatasvir is a novel hepatitis C virus NS5A inhibitor developed by Bristol-Myers Squibb and marketed as Daklinza®. The need to support the development of daclatasvir required the synthesis of carbon-14 labeled material for use in human absorption, distribution, metabolism, and excretion studies. A total of 7.53 mCi of [(14) C]-daclatasvir was synthesized in eight steps from commercially available [(14) C]-copper cyanide. The radiochemical purity was 99.6%, and specific activity was 3.86 μCi/mg. To support a human absolute bioavailability study, 5.56 g of [(13) C2 , (15) N4 ]-daclatasvir was synthesized in four steps.

Aim: A robust LC–MS/MS assay was developed to quantify endogenous 1, 14-tetradecanedioic acid (TDA) and 1, 16-hexadecanedioic acid (HDA) in human plasma as potential biomarkers for evaluating drug–drug interactions mediated by the hepatic drug transporters, organic anion-transporting polypeptides. Results: This assay was validated using fit-for-purpose approach over standard curve range of 2.5–1000 nM for TDA and HDA using analyte-free charcoal-stripped human plasma as the surrogate matrix. Chromatographic separation condition was successfully optimized to separate TDA from an interference peak while maintaining both analytes in neutral forms to minimize carryover issue. Conclusion: The described assay is currently applied to a clinical study for evaluating TDA/HDA as potential substitute biomarkers for drug–drug interaction studies.

Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1 ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2 ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.

Bristol-Myers Squibb and others are developing drugs that target novel mechanisms to combat Alzheimer's disease. γ-Secretase inhibitors are one class of potential therapies that have received considerable attention. (R)-2-(4-Chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide (Avagacestat) is a γ-secretase-inhibiting drug that has been investigated by Bristol-Myers Squibb in preclinical and clinical studies. An important step in the development process was the synthesis of a carbon-14-labeled analog for use in a human absorption, distribution, metabolism, and excretion study and a stable isotope labeled analog for use as a standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon-14 labeled Avagacestat was synthesized in seven steps in a 33% overall yield from carbon-14 labeled potassium cyanide. A total of 5.95 mCi was prepared with a specific activity of 0.81 μCi/mg and a radiochemical purity of 99.9%. (13) C6 -Labeled Avagacestat was synthesized in three steps in a 15% overall yield from 4-chloro[(13) C6 ]aniline. A total of 585 mg was prepared with a ultraviolet purity of 99.9%.

Stable isotope-labeled [(13) C4 ]entecavir (1) was prepared in 11 steps. Commercially available [(13) C]guanidine hydrochloride and diethyl[1,2,3-(13) C3 ]malonate were condensed to yield 2-amino[2,4,5,6-(13) C4 ]pyrimidine-4,6-diol (8). This was converted to the desired purine (7) in five steps. Introduction of the chiral epoxide was followed by subsequent deprotection to give [(13) C4 ]entecavir (1), in an overall yield of 5.7% from labeled precursors. The chemical purity of the title compound was determined to be >99% by HPLC. The isotopic distribution was determined by mass spectrometry to be 282[M + 4], 98.4%; 281[M + 3], 1.6%; and 278[M + 0]

1. Asunaprevir (ASV, BMS-650032), a highly selective and potent NS3 protease inhibitor, is currently under development for the treatment of chronic hepatic C virus infection. This study describes in vivo biotransformation in humans and the identification of metabolic enzymes of ASV.2. Following a single oral dose of [14C]ASV to humans, the majority of radioactivity (>73% of the dose) was excreted in feces with In vitro studies with human cDNA expressed CYP enzymes and with human liver microsomes (HLM) in the presence of selective chemical inhibitors demonstrated that ASV was primarily catalyzed by CYP3A4 and CYP3A5.

A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

This session is a continuation of Session 1. A number of methods detailing the synthesis of tritiated compounds as well as a discussion on the handling of tritium on a multi-Curie scale are presented. In addition, the descriptions of a number of preparations of individual isotopically labelled compounds are detailed. Copyright © 2010 John Wiley & Sons, Ltd.

Tipranavir or Aptivus® is a non-peptidic protease inhibitor approved for the combination treatment with ritonavir of HIV infection. Tipranavir labeled with radioactive and stable isotopes of carbon was required for drug metabolism (excretion, distribution, and absorption) studies and to develop bioanalytical methods needed for the support of clinical studies. [7-14C]-Benzoic acid and uniformly labeled benzoic acid (ring-13C6 99 at% 13C) were used to prepare [14C]- and [13C6]-labeled tipranavir, respectively. Radioactively labeled tipranavir was prepared with a specific activity of 54 mCi/mmol (2GBq/mmol); it was necessary to dilute its specific activity with unlabeled tipranavir to 28 mCi/mmol (46.45 µCi/mg) because of its instability. The N-hydroxyl metabolite (12) and the glucuronide conjugate (13), the most abundant metabolites of tipranavir (when administered in conjunction with ritonavir) were also synthesized. Copyright © 2008 John Wiley & Sons, Ltd.

Daclatasvir is a first-in-class, potent, and selective inhibitor of the hepatitis C virus nonstructural protein 5A replication complex. In support of nonclinical studies during discovery and exploratory development, liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance were used in connection with synthetic and radiosynthetic approaches to investigate the biotransformation of daclatasvir in vitro and in cynomolgus monkeys, dogs, mice, and rats. The results of these studies indicated that disposition of daclatasvir was accomplished mainly by the release of unchanged daclatasvir into bile and feces and, secondarily, by oxidative metabolism. Cytochrome P450s were the main enzymes involved in the metabolism of daclatasvir. Oxidative pathways included δ-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. Despite robust formation of the resulting metabolite in multiple systems, rates of covalent binding to protein associated with metabolism of daclatasvir were modest (55.2-67.8 pmol/mg/h) in nicotinamide adenine dinucleotide phosphate (reduced form)-supplemented liver microsomes (human, monkey, rat), suggesting that intramolecular rearrangement was favored over intermolecular binding in the formation of this metabolite. This biotransformation profile supported the continued development of daclatasvir, which is now marketed for the treatment of chronic hepatitis C virus infection.

Daclatasvir is a novel hepatitis C virus NS5A inhibitor developed by Bristol-Myers Squibb and marketed as Daklinza®. The need to support the development of daclatasvir required the synthesis of carbon-14 labeled material for use in human absorption, distribution, metabolism, and excretion studies. A total of 7.53 mCi of [(14) C]-daclatasvir was synthesized in eight steps from commercially available [(14) C]-copper cyanide. The radiochemical purity was 99.6%, and specific activity was 3.86 μCi/mg. To support a human absolute bioavailability study, 5.56 g of [(13) C2 , (15) N4 ]-daclatasvir was synthesized in four steps.

Lysophosphatidic acids (LPAs) are biologically active signaling molecules involved in the regulation of many cellular processes and have been implicated as potential mediators of fibroblast recruitment to the pulmonary airspace, pointing to possible involvement of LPA in the pathology of pulmonary fibrosis. LPAs have been measured in various biological matrices and many challenges involved with their analyses have been documented. However, little published information is available describing LPA levels in human bronchoalveolar lavage fluid (BALF). We therefore conducted detailed investigations into the effects of extensive sample handling and sample preparation conditions on LPA levels in human BALF. Further, targeted lipid profiling of human BALF and plasma identified the most abundant lysophospholipids likely to interfere with LPA measurements. We present the findings from these investigations, highlighting the importance of well-controlled sample handling for the accurate quantitation of LPA. Further, we show that chromatographic separation of individual LPA species from their corresponding lysophospholipid species is critical to avoid reporting artificially elevated levels. The optimized sample preparation and LC/MS/MS method was qualified using a stable isotope-labeled LPA as a surrogate calibrant and used to determine LPA levels in human BALF and plasma from a Phase 0 clinical study comparing idiopathic pulmonary fibrosis patients to healthy controls.

Bristol-Myers Squibb and others are developing drugs that target novel mechanisms to combat Alzheimer's disease. γ-Secretase inhibitors are one class of potential therapies that have received considerable attention. (R)-2-(4-Chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide (Avagacestat) is a γ-secretase-inhibiting drug that has been investigated by Bristol-Myers Squibb in preclinical and clinical studies. An important step in the development process was the synthesis of a carbon-14-labeled analog for use in a human absorption, distribution, metabolism, and excretion study and a stable isotope labeled analog for use as a standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon-14 labeled Avagacestat was synthesized in seven steps in a 33% overall yield from carbon-14 labeled potassium cyanide. A total of 5.95 mCi was prepared with a specific activity of 0.81 μCi/mg and a radiochemical purity of 99.9%. (13) C6 -Labeled Avagacestat was synthesized in three steps in a 15% overall yield from 4-chloro[(13) C6 ]aniline. A total of 585 mg was prepared with a ultraviolet purity of 99.9%.

Four members of a novel class of pyrrolo[2,3-d]pyrimidines that show potential for the treatment of asthma and neurodegenerative disorders, have been prepared with radioisotope labels and in one case with multiple stable isotope labels for ADME studies as part of the drug development process. The syntheses utilize an isotopically labeled 2,4,6-trisubstituted pyrimidine as a common building block, readily prepared from isotopically labeled urea. Cyclizations of the pyrimidine with bromo-ketones generate the ring fused pyrrolo[2,3-d]-pyrimidines with elegant efficiency as demonstrated by the preparation of structures 4, 5, 8 and 12. Copyright © 2001 John Wiley & Sons, Ltd.

Cytochrome P450 (CYP) enzymes are responsible for much of the phase I oxidative metabolism observed in vivo. Many important pharmaceutical compounds are metabolized by CYP. Co-administration of a drug with another agent can alter the efficacy or the toxicity, especially in cases where drug clearance depends primarily on the CYP metabolism. Compounds that induce or inhibit the CYP activity are often used in drug–drug interaction studies. Midazolam is one such compound that is routinely used in drug–drug interaction studies because it is a known substrate for CYP3A enzymes. The synthesis of this important tool molecule has been documented, but unfortunately a detailed preparation of carbon-14-labelled midazolam has not been reported in the literature. This paper describes a two-step synthesis leading to [14C]midazolam. A total of 4.5 mCi of [14C]midazolam was obtained having a specific activity of 120.1 µCi/mg (39.12 mCi/mmol). The radiochemical purity as determined by HPLC was 99.8% and the overall radiochemical yield was 9%. Copyright © 2009 John Wiley & Sons, Ltd.

The title compound is a potent and selective 5-HT 1A receptor agonist with clinical potentials for treating anxiety and depression. It has been labeled with stable and radioactive isotopes to support ADME studies in animals and human subjects. Its carboxamide group was labeled with C-14, C-13, or C-13, N-15, and O-18. To provide a radioligand for investigating the receptor binding characteristics of the compound, we also labeled it with tritium in the N-propyl group to obtain the high specific activity of 86.7 Ci/mmol.

Stable isotope-labeled [(13) C4 ]entecavir (1) was prepared in 11 steps. Commercially available [(13) C]guanidine hydrochloride and diethyl[1,2,3-(13) C3 ]malonate were condensed to yield 2-amino[2,4,5,6-(13) C4 ]pyrimidine-4,6-diol (8). This was converted to the desired purine (7) in five steps. Introduction of the chiral epoxide was followed by subsequent deprotection to give [(13) C4 ]entecavir (1), in an overall yield of 5.7% from labeled precursors. The chemical purity of the title compound was determined to be99% by HPLC. The isotopic distribution was determined by mass spectrometry to be 282[M + 4], 98.4%; 281[M + 3], 1.6%; and 278[M + 0],0.1%.

(R)-5-(Methylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (1) is a dopamine agonist which shows selectivity for the D2 receptor subtype, and is of interest as a potential drug for the treatment of Parkinson's disease. An asymmetric epoxidation approach has been used to prepare 1 in eleven steps (15% overall yield) from 8-nitroquinoline. An advanced intermediate in this synthesis, tert-butyl (R)-methyl(8-amino-1,2,3,4-tetrahydro-3-quinolinyl)carbamate (10), has been reacted with [ 14 C]phosgene to provide a two-step synthesis of 1 labeled with carbon-14 at the C-2 position (236 μCi/mg). Bromination of 1 gave the dibromo analogue 12b which was reduced in the presence of tritium gas to give 1 labeled with tritium at the C-6 and C-7 positions (28.5 Ci/mmol). In addition to providing syntheses for labeled forms of the drug which are useful in drug disposition and receptor binding studies, this approach also provides a convenient synthesis for the unlabeled form of drug

A strategy of using selected reaction monitoring (SRM) mass spectrometry for evaluating oral absolute bioavailability with concurrent intravenous (i.v.) microdosing a stable isotopically labeled (SIL) drug was developed and validated. First, the isotopic contribution to SRM (ICSRM) of the proposed SIL drug and SIL internal standard (IS) was theoretically calculated to guide their chemical synthesis. Second, the lack of an isotope effect on drug exposure was evaluated in a monkey study by i.v. dosing a mixture of the SIL and the unlabeled drugs. Third, after the SIL drug (100 μg) was concurrently i.v. dosed to humans, at T(max) of an oral therapeutic dose of the unlabeled drug, both drugs in plasma specimens were simultaneously quantified by a sensitive and accurate SRM assay. This strategy significantly improves bioanalytical data quality and saves time, costs, and resources by avoiding a traditional absolute bioavailability study or the newer approach of microdoses of a radio-microtracer measured by accelerator mass spectrometry.

Several α,β-unsaturated ketoximes R1CH=CHC(=NOH)R2 were nitrosated using butyl nitrite in aqueous ethanol in the presence of copper(II) sulfate and pyridine. The product distribution varied depending on whether the oxime hydroxyl group was syn or anti with respect to the carboncarbon double bond. The anti-oximes gave the copper complexes of 1-hydroxypyrazole 2-oxides in high yields. The isomeric syn-oximes gave lower yields of the pyrazole complexes along with 4-oximino-4,5-dihydroisoxazole derivatives. For the syn-oximes where R1 is phenyl and R2 is either methyl or ethyl, conversion of the oximes to the parent ketones was also observed. The results may be explained by processes involving N-nitrosonitrone intermediates.

Nitrosation of the oximes of 3-bromo-3-penten-2-one, 3-bromo-4-phenyl- 3-buten-2-one, and 2-bromo-1,3-diphenyl-2-propen-1-one using sodium ni- trite in acetic acid gave low yields of 4-pyrazolone 1,2-dioxides. Nit- rosation using butyl nitrite in the presence of copper(II) sulfate and pyridine in aqueous ethanol produced insoluble copper complexes from which 3,5-dimethyl-, 3-methyl-5-phenyl-, and 3,5-diphenyl-4-bromo-1-hy- droxypyrazole 2-oxides could be liberated by treatment with dilute po- tassium hydroxide, filtration, and acidification of the filtrate. High yields were obtained with the first two oximes, but, presumably due to unfavorable stereochemistry of the oxime, the diphenyl derivative gave a lower yield of the complex, accompanied by 4-bromo- and 4-nitro-3,5- diphenylisoxazole and 4-oximino-3,5-diphenyl-4,5-dihydroisoxazole

Profiling of rat plasma using a highly sensitive LC-ARC-MS technique showed that [(3)H] mefenamic acid was metabolized to several products, including a sulfate conjugate and a hydroxylated analogue as major metabolites. This technique of detecting low levels of radioactivity in plasma was superior to previously used methods, such as beta-RAM detectors.

Our study objectives were: To quantitatively determine the effect of radiolabel instability on terminal phase radioactive tissue residues in a multiple dose tissue distribution study. To quantitatively compare tissue residue artifacts (non drug-related radioactivity) from two chemically-distinct radiolabel locations. To conduct a definitive multiple dose tissue distribution study using the better of the two radiolabeled compounds. We compared the excretion and tissue distribution in rats of [(14)C]linezolid, radiolabeled in two different locations, after 7 consecutive once daily [(14)C] oral doses. The radiolabels were in the acetamide (two carbon) and oxazolidinone (isolated carbon) functional groups. Terminal phase tissue residue and excretion data were compared to data from rats dosed orally with [(14)C]sodium acetate. Drug-related radioactivity was excreted rapidly over 24 h. After a single dose, the acetamide and oxazolidinone radiolabel sites both gave 3% of dose as exhaled (14)CO(2). After 7 daily [(14)C] oral doses, terminal phase radioactive tissue residues were higher from the acetamide radiolabel, relative to the oxazolidinone radiolabel, and were primarily not drug-related. In the definitive tissue distribution study, low concentrations of drug-related radioactivity in skin and thyroid were observed. We conclude that although small amounts of radiolabel instability do not significantly affect single dose tissue radioactivity C(max) and AUC, artifacts arising from radiolabel instability can prolong the apparent terminal phase half life and complicate study data interpretation. When possible, it is always preferable to use a completely stable radiolabel site.