Chemoselective Methionine Bioconjugation: Site-Selective Fluorine-18 Labeling of Proteins and Peptides.

Chemoselective methionine bioconjugation with alkyne-bearing oxaziridine and alkyne-bearing iodonium salts was investigated as a new platform for site-selective radiolabeling of proteins and peptides with fluorine-18. Alkyne-bearing sulfimide conjugates, resulting from oxaziridine modification, underwent copper-assisted alkyne-azide cycloaddition (CuAAC) with an F-labeled PEGylated azide to afford F-labeled triazoles in excellent radiochemical yields. Diazoester sulfonium salt bioconjugates, formed from alkyne-bearing 2-diazoiodonium salts, gave low yields of F-labeled triazoles and were shown to be unstable to CuAAC conditions.

Determination of Real Time in Vivo Drug Receptor Occupancy for a Covalent Binding Drug as a Clinical Pharmacodynamic Biomarker by Immunocapture-LC-MS/MS.

We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug.

Discovery of Branebrutinib (BMS-986195): A Strategy for Identifying a Highly Potent and Selective Covalent Inhibitor Providing Rapid in Vivo Inactivation of Bruton's Tyrosine Kinase (BTK).

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec family of kinases and is essential for B cell receptor (BCR) mediated signaling. BTK also plays a critical role in the downstream signaling pathways for the Fcγ receptor in monocytes, the Fcε receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as rheumatoid arthritis and lupus.

Direct Carbon Isotope Exchange through Decarboxylative Carboxylation.

A two-step degradation-reconstruction approach to the carbon-14 radiolabeling of alkyl carboxylic acids is presented. Simple activation via redox-active ester formation was followed by nickel-mediated decarboxylative carboxylation to afford a range of complex compounds with ample isotopic incorporations for drug metabolism and pharmacokinetic studies. The practicality and operational simplicity of the protocol were demonstrated by its use in an industrial carbon-14 radiolabeling setting.

A convenient strategy to overcome interference in LC-MS/MS analysis: Application in a microdose absolute bioavailability study.

Stable isotope labeled (SIL) compounds have been commonly used as internal standards (IS) to ensure the accuracy and quality of liquid chromatography-mass spectrometry (LC-MS) bioanalytical assays. Recently, the application of SIL drugs and LC-MS assays to microdose absolute bioavailability (BA) studies has gained increasing attention. This approach can provide significant cost and time saving, and higher data quality compared to the accelerator mass spectrometry (AMS)-based method, since it avoids the use of radioactive drug, high-cost AMS instrumentation and complex measurement processes.

Adnectin-drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors.

Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody-drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens-but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin-drug conjugates, an alternative platform for tumor-specific delivery of cytotoxic payloads.

Immune-modulating enzyme indoleamine 2,3-dioxygenase is effectively inhibited by targeting its apo-form.

For cancer cells to survive and proliferate, they must escape normal immune destruction. One mechanism by which this is accomplished is through immune suppression effected by up-regulation of indoleamine 2,3-dioxygenase (IDO1), a heme enzyme that catalyzes the oxidation of tryptophan to -formylkynurenine. On deformylation, kynurenine and downstream metabolites suppress T cell function.

Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics.

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages.

Synthesis of 2-(14)C-iminothiolane and 2-(13)C,(15)N-iminothiolane (Traut's reagent).

2-Iminothiolane has found utility in the growing area of antibody-drug conjugates by serving as a lysine-thiolating agent and the junction between the antibody and the cytotoxic payload during random conjugation of a monoclonal antibody. 2-(14)C-Iminothiolane was prepared from commercially available [(14)C]KCN using a four-step sequence in an overall 10% radiochemical yield. Stable-labeled 2-(13)C,(15)N-iminothiolane was also prepared from [(13)C(15)N]KCN in a similar manner.

Utilizing Internal Standard Responses to Assess Risk on Reporting Bioanalytical Results from Hemolyzed Samples.

Bioanalytical analysis of toxicokinetic and pharmacokinetic samples is an integral part of small molecule drugs development and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the technique of choice. One important consideration is the matrix effect, in which ionization of the analytes of interest is affected by the presence of co-eluting interfering components present in the sample matrix. Hemolysis, which results in additional endogenous components being released from the lysed red blood cells, may cause additional matrix interferences.

Development and validation of an LC-MS/MS assay for the quantitation of a PEGylated anti-CD28 domain antibody in human serum: overcoming interference from antidrug antibodies and soluble target.

Aim: To support drug development of a PEGylated anti-CD28 domain antibody, a sensitive and robust LC-MS/MS assay was developed for the first in-human multiple ascending dose study.

Materials & Methods: The procedure consists of a protein precipitation with acidified acetonitrile, followed by trypsin digestion of the supernatant. A surrogate peptide from the complementarity determining region was quantified with an LC-MS/MS assay using a stable isotope-labeled internal standard with flanking amino acids.

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.

Innovative use of LC-MS/MS for simultaneous quantitation of neutralizing antibody, residual drug, and human immunoglobulin G in immunogenicity assay development.

Immunogenicity testing for antidrug antibodies (ADA) faces challenges when high levels of the drug are present in clinical patient samples. In addition, most functional cell-based assays designed to characterize the neutralizing ability of ADA are vulnerable to interference from endogenous serum components. Bead extraction and acid dissociation (BEAD) has been successfully applied to extract ADA from serum samples prior to conduction of cell-based assays.

Fully validated LC-MS/MS assay for the simultaneous quantitation of coadministered therapeutic antibodies in cynomolgus monkey serum.

An LC-MS/MS assay was developed and fully validated for the simultaneous quantitation of two coadministered human monoclonal antibodies (mAbs), mAb-A and mAb-B of IgG4 subclass, in monkey serum. The total serum proteins were digested with trypsin at 50 °C for 30 min after methanol denaturation and precipitation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated with a C18 column (2.

Cytochrome P450 11A1 bioactivation of a kinase inhibitor in rats: use of radioprofiling, modulation of metabolism, and adrenocortical cell lines to evaluate adrenal toxicity.

A drug candidate, BMS-A ((N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl) 2-oxo-1,2-dihydropyridine- 3-carboxamide)), was associated with dose- and time-dependent vacuolar degeneration and necrosis of the adrenal cortex following oral administration to rats. Pretreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, ameliorated the toxicity. In vivo and in vitro systems, including adrenal cortex-derived cell lines, were used to study the mechanism responsible for the observed toxicity.

Metabolism and disposition of [14C]brivanib alaninate after oral administration to rats, monkeys, and humans.

Brivanib [(R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[1,2,4]triazin-6-yloxy)propan-2-ol, BMS-540215] is a potent and selective dual inhibitor of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. Its alanine prodrug, brivanib alaninate [(1R,2S)-2-aminopropionic acid 2-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-1-methylethyl ester, BMS-582664], is currently under development as an oral agent for the treatment of cancer. This study describes the in vivo biotransformation of brivanib after a single oral dose of [(14)C]brivanib alaninate to intact rats, bile duct-cannulated (BDC) rats, intact monkeys, BDC monkeys, and humans.

Metabolism and disposition of [14C]BMS-690514, an ErbB/vascular endothelial growth factor receptor inhibitor, after oral administration to humans.

(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514), an oral selective inhibitor of human epidermal growth factor receptors 1 (or epidermal growth factor receptor), 2, and 4, and vascular endothelial growth factor receptors 1, 2, and 3, is being developed as a treatment for patients with non-small-cell lung cancer and metastatic breast cancer. The disposition of [(14)C]BMS-690514 was investigated in nine healthy male subjects (group 1, n = 6; group 2, n = 3) after oral administration of a 200-mg dose. Urine, feces, and plasma were collected from all subjects for up to 12 days postdose.

Metabolism and disposition of [14C]BMS-690514 after oral administration to rats, rabbits, and dogs.

(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f] [1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514) is a potent inhibitor of human epidermal growth factor receptors 1, 2, and 4 and vascular endothelial growth factor receptors 1 through 3. BMS-690514 is an oral oncologic agent currently being developed for the treatment of patients with advanced non-small cell lung cancer and breast cancer. In this investigation, a series of studies was conducted to determine the biotransformation of [(14)C]BMS-690514 after oral administration to rats, rabbits, and dogs.

Metabolism and disposition of dasatinib after oral administration to humans.

SPRYCEL (dasatinib, BMS-354825; Bristol-Myers Squibb, Princeton, NJ), a multiple kinase inhibitor, is currently approved to treat chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia tumors in patients who are resistant or intolerant to imatinib mesylate (Gleevec; Novartis, Basel, Switzerland). After a 100-mg single p.o.