Development, Characterization, and Radiation Dosimetry Studies of F-BMS-986229, a F-Labeled PD-L1 Macrocyclic Peptide PET Tracer.

Purpose: In cancer immunotherapy, the blockade of the interaction between programmed death-1 and its ligand (PD-1:PD-L1) has proven to be one of the most promising strategies. However, as mechanisms of resistance to PD-1/PD-L1 inhibition include variability in tumor cell PD-L1 expression in addition to standard tumor biopsy PD-L1 immunohistochemistry (IHC), a comprehensive and quantitative approach for measuring PD-L1 expression is required. Herein, we report the development and characterization of an F-PD-L1-binding macrocyclic peptide as a PET tracer for the comprehensive evaluation of tumor PD-L1 expression in cancer patients.

The discovery and evaluation of [F]BMS-986229, a novel macrocyclic peptide PET radioligand for the measurement of PD-L1 expression and in-vivo PD-L1 target engagement.

Purpose: A same-day PET imaging agent capable of measuring PD-L1 status in tumors is an important tool for optimizing PD-1 and PD-L1 treatments. Herein we describe the discovery and evaluation of a novel, fluorine-18 labeled macrocyclic peptide-based PET ligand for imaging PD-L1.

Methods: [F]BMS-986229 was synthesized via copper mediated click-chemistry to yield a PD-L1 PET ligand with picomolar affinity and was tested as an in-vivo tool for assessing PD-L1 expression.

Synthesis of a dual carbon-14-labeled calcitonin gene-related peptide receptor antagonist for use in a human absorption-distribution-metabolism-elimination study.

Oral calcitonin gene-related peptide (CGRP) receptor antagonists have been shown to be effective in the acute and preventive treatment of migraine. CGRP receptor antagonists offer safety advantages over triptans because they are not active vasoconstrictors, which reduces cardiovascular risks. Bristol Myers Squibb discovered a high affinity CGRP receptor antagonist BMS-927711 for the treatment of migraine now FDA approved as Nurtec® ODT (rimegepant).

Synthesis of carbon-14 and stable isotope labeled Censavudine.

Censavudine is a nucleoside reverse transcriptase inhibitor (NRTI) explored clinically by Bristol Myers Squibb for the treatment of human immunodeficiency virus-1 (HIV-1). As part of the development process, a carbon-14 labeled analog was synthesized for use in a human absorption, distribution, metabolism, and excretion (ADME) study. A stable isotope labeled analog was also synthesized for use as a mass spectrum internal standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples.

Synthesis of tritium labeled 30-KDa PEG and conjugation to form [ H]Pegbelfermin.

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.

Synthesis and Preclinical Evaluation of a Ga-Labeled Adnectin, Ga-BMS-986192, as a PET Agent for Imaging PD-L1 Expression.

Blocking the interaction of the immune checkpoint molecule programmed cell death protein-1 and its ligand, PD-L1, using specific antibodies has been a major breakthrough for immune oncology. Whole-body PD-L1 expression PET imaging may potentially allow for a better prediction of response to programmed cell death protein-1-targeted therapies. Imaging of PD-L1 expression is feasible by PET with the adnectin protein F-BMS-986192.

Synthesis of stable-isotope-labeled N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-(3-dimethylaminopropyl)-N'-ethylurea.

N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) is a carbodiimide coupling reagent commonly used for the preparation of amides from carboxylic acids and amines. Because of initial concerns regarding the genotoxicity of EDC and its use in GMP syntheses at Bristol Myers Squibb, the quantitation of residual EDC and its by-product N-(3-dimethylaminopropyl)-N'-ethylurea (EDU) by liquid chromatography-mass spectrometry (LCMS) impurity analysis was required. These analyses required the use of stable-isotope-labeled EDC and EDU to serve as internal standards.

Nucleophilic (Radio)Fluorination of Redox-Active Esters via Radical-Polar Crossover Enabled by Photoredox Catalysis.

We report a redox-neutral method for nucleophilic fluorination of -hydroxyphthalimide esters using an Ir photocatalyst under visible light irradiation. The method provides access to a broad range of aliphatic fluorides, including primary, secondary, and tertiary benzylic fluorides as well as unactivated tertiary fluorides, that are typically inaccessible by nucleophilic fluorination due to competing elimination. In addition, we show that the decarboxylative fluorination conditions are readily adapted to radiofluorination with [F]KF.

LC-MS/MS bioanalysis of plasma 1, 14-tetradecanedioic acid and 1, 16-hexadecanedioic acid as candidate biomarkers for organic anion-transporting polypeptide mediated drug-drug interactions.

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.

Synthesis and Biologic Evaluation of a Novel F-Labeled Adnectin as a PET Radioligand for Imaging PD-L1 Expression.

The programmed death protein (PD-1) and its ligand (PD-L1) play critical roles in a checkpoint pathway cancer cells exploit to evade the immune system. A same-day PET imaging agent for measuring PD-L1 status in primary and metastatic lesions could be important for optimizing drug therapy. Herein, we have evaluated the tumor targeting of an anti-PD-L1 adnectin after F-fluorine labeling.

Synthesis of unlabelled and stable-isotope-labelled glucuronide metabolites of dapagliflozin and synthesis of stable-isotope-labelled dapagliflozin.

Two regioisomeric glucuronide metabolites of dapagliflozin (BMS-512148) were synthesized and used to elucidate the structures of dapagliflozin metabolites observed in human urine samples. The structures of the synthetic metabolites were assigned by heteronuclear multiple-bond correlation, ROESY, and total correlation spectroscopy experiments. Analogues of these metabolites containing carbon-13 as a stable label were also prepared for use as internal standards for the analysis of urine samples obtained from patients participating in clinical studies.

Tissue distribution and tumor uptake of folate receptor-targeted epothilone folate conjugate, BMS-753493, in CD2F1 mice after systemic administration.

To assess targeting of an epothilone folate conjugate (BMS-753493) to the folate receptor (FR)-overexpressed tumor in mice bearing both FR+ and FR- tumors, a series of experiments were conducted by quantitative whole-body autoradiography (QWBA) and LC-MS/MS following i.v. administration of BMS-753493 or its active moiety, BMS-748285 in mice bearing FR+ (98M109) and FR- (M109) tumors.

The syntheses of isotopically labelled CB-1 antagonists for the treatment of obesity.

BMS-725519, BMS-811064, and BMS-812204 are potent and selective central cannabinoid receptor antagonists that have been investigated for the treatment of human obesity. To further understand their biotransformation profiles, radiolabelled and stable-labelled products were required. This paper describes the utility of [ C]1,1-carbonyldiimidazole as a radiolabelling reagent for the syntheses of carbonyl-labelled [ C]BMS-725519, [ C]BMS-811064, and [ C]BMS-812204.

The synthesis and analysis of [phenyl- C(U)]BMS-770767 and [ C ]BMS-770767 for use in discovery biotransformation, human ADME and bioanalytical studies.

Type 2 diabetes is a significant worldwide health problem. To support the development of BMS-770767 as an inhibitor of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) for type 2 diabetes was required the synthesis of carbon-14-labelled material for use in metabolic profiling and for the human adsorption, distribution, metabolism and excretion (ADME) study. Initially, [phenyl- C(U)]BMS-770767 was synthesized in two steps from a late-stage intermediate and [ C(U)]2-chlorophenol to give the desired final product in 18% yield.

The syntheses of [(14) C]BMS-823778 for use in a human ADME clinical study and of [(13) CD3 (13) CD2 ]BMT-094817, a stable-isotope labeled standard of a newly detected human metabolite.

Type 2 diabetes is a significant worldwide health problem. To support the development of BMS-823778 as an inhibitor of 11β-hydroxysteroid dehydrogenase type 1 for type 2 diabetes, the synthesis of carbon-14-labeled material was required for use in a human adsorption, distribution, metabolism, and excretion (ADME) study. The HCl salt form of [(14) C]BMS-823778 was synthesized in two steps from commercially available [2-(14) C]acetone.

Synthesis of isotopically labeled daclatasvir for use in human clinical studies.

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.

Synthesis of stable isotope-labeled epothilone D using a degradation-reconstruction approach.

The stabilization of microtubules using epothilones represents a novel mechanism of action to treat Alzheimer's disease. Epothilone D is one such microtubule-stabilizing drug that has been investigated by Bristol-Myers Squibb. An important step in the development process was the synthesis of a stable isotope-labeled analog for use in bioanalytical assays to accurately quantify the concentration of the drug in biological samples.

The synthesis of 14C-labeled N-succinimidyl-3-maleimidopropionate, a linker molecule for PEGylated biologics.

Carbon-14 labeled linker molecule, N-succinimidyl-3-maleimidopropionate was prepared for disposition studies of PEGylated biologics. Our new route started with 100 mCi of carbon-14 labeled Potassium cyanide (KCN) to prepare 55 mCi of [1-(14)C]N-succinimidyl-3-maleimidopropionate, 6 in five steps. This represents a multiple of 5.

Synthesis of a stable isotopically labeled universal surrogate peptide for use as an internal standard in LC-MS/MS bioanalysis of human IgG and Fc-fusion protein drug candidates.

The synthesis of a 16-residue, stable isotopically labeled peptide is described for use as a LC-MS/MS (Liquid chromatography-mass spectrometry/mass spectrometry) internal standard in bioanalytical studies. This peptide serves as a single universal surrogate peptide capable of quantifying a wide variety of immunoglobulin G and Fc-fusion protein drug candidates in animal species used in pre-clinical drug development studies. An efficient synthesis approach for this peptide was developed using microwave-assisted solid phase peptide synthesis (SPPS) techniques, which included the use of a pseudoproline dipeptide derivative.

The synthesis of (14)C-labeled, (13)CD2-labeled saxagliptin, and its (13)CD2-labeled 5-hydroxy metabolite.

(14)C-labeled saxagliptin, (13) CD2-labeled saxagliptin, and its (13) CD2-labeled 5-hydroxy metabolite were synthesized to further support development of the compound for biological studies. This paper describes new syntheses leading to the desired compounds. A total of 3.

Synthesis of [3α-(3) H] 17α-hydroxy pregnenolone and [3α-(3) H] pregnenolone.

For the first time, [3α-(3) H] 17α-hydroxy pregnenolone (1) was synthesized through a multiple step sequence. The presence of [3β-(3) H] isomer in RP-HPLC purified product was identified by tritium NMR. The [3β-(3) H] isomer was then separated from [3α-(3) H] 17α-hydroxy pregnenolone with chiralPAK AD-H column.

An alternative and robust synthesis of [(13) C4 ]Baraclude® (entecavir).

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.

Overcoming bioanalytical challenges in an Onglyza(®) intravenous [(14)C]microdose absolute bioavailability study with accelerator MS.

Background: An absolute bioavailability study that utilized an intravenous [(14)C]microdose was conducted for saxagliptin (Onglyza(®)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [(14)C]saxagliptin were determined by accelerator MS (AMS) after protein precipitation, chromatographic separation by UPLC and analyte fraction collection. A series of investigative experiments were conducted to maximize the release of the drug from high-affinity receptors and nonspecific adsorption, and to determine a suitable quantitation range.

Characterization of the in vitro and in vivo metabolism and disposition and cytochrome P450 inhibition/induction profile of saxagliptin in human.

Saxagliptin is a potent dipeptidyl peptidase-4 inhibitor approved for the treatment of type 2 diabetes mellitus. The pharmacokinetics and disposition of [(14)C]saxagliptin were investigated in healthy male subjects after a single 50-mg (91.5 μCi) oral dose.