Development of a quantification method for arginase inhibitors by LC-MS/MS with benzoyl chloride derivatization.

Arginase is an enzyme responsible for converting arginine, a semi-essential amino acid, to ornithine and urea. Arginine depletion suppresses immunity via multiple mechanisms including inhibition of T-cell and NK cell proliferation and activity. Arginase inhibition is therefore an attractive mechanism to potentially reverse immune suppression and thus has been explored as a therapy for oncology and respiratory indications. Small molecules targeting arginase present significant bioanalytical challenges for in vitro and in vivo characterization as inhibitors of arginase are typically hydrophilic in nature. The resulting low or negative LogD characteristics are incompatible with common analytical methods such as RP-ESI-MS/MS. Accordingly, a sensitive, high-throughput bioanalytical method was developed by incorporating benzoyl chloride derivatization to increase the hydrophobic characteristics of these polar analytes. Samples were separated by reversed phase chromatography on a Waters XBridge BEH C18 3.5 µm, 30 × 3 mm column using gradient elution. The mass spec was operated in positive mode using electrospray ionization. The m/z 434.1→176.1, 439.4→181.2, 334.9→150.0 and 339.9→150.0 for AZD0011, AZD0011 IS, AZD0011-PL and AZD0011-PL IS respectively were used for quantitation. The linear calibration range of the assay was 1.00-10,000 ng/mL with QC values of 5, 50 and 500 ng/mL. The qualified method presented herein exhibits a novel, robust analytical performance and was successfully applied to evaluate the in vivo ADME properties of boronic acid-based arginase inhibitor prodrug AZD0011 and its active payload AZD0011-PL.

Optimization of an Imidazo[1,2-a]pyridine Series to Afford Highly Selective Type I1/2 Dual Mer/Axl Kinase Inhibitors with In Vivo Efficacy.

Inhibition of Mer and Axl kinases has been implicated as a potential way to improve the efficacy of current immuno-oncology therapeutics by restoring the innate immune response in the tumor microenvironment. Highly selective dual Mer/Axl kinase inhibitors are required to validate this hypothesis. Starting from hits from a DNA-encoded library screen, we optimized an imidazo[1,2-a]pyridine series using structure-based compound design to improve potency and reduce lipophilicity, resulting in a highly selective in vivo probe compound 32. We demonstrated dose-dependent in vivo efficacy and target engagement in Mer- and Axl-dependent efficacy models using two structurally differentiated and selective dual Mer/Axl inhibitors. Additionally, in vivo efficacy was observed in a preclinical MC38 immuno-oncology model in combination with anti-PD1 antibodies and ionizing radiation.

Impact of Interindividual Differences in Plasma Fraction Unbound on the Pharmacokinetics of a Novel Syk Kinase Inhibitor in Beagle Dogs.

Inconsistencies in pharmacokinetic parameters between individual animals in preclinical studies are a common occurrence. Often such differences between animals are simply accepted as experimental variability rather than as indications of specific differences in animal phenotype that could lead to a different interpretation of the data. The fraction unbound in plasma is one factor influencing pharmacokinetic parameters and is typically determined using pooled plasma from multiple animals, making the assumption that there is limited population variance. However, this assumption is not often tested and may not hold true if there are polymorphisms affecting binding or variation in the concentrations of individual plasma proteins that could give rise to different fraction unbound phenotypes in individual animals. During profiling of a novel Syk inhibitor, AZ8399, striking interindividual differences in total plasma clearance and volume of distribution were observed between dogs consistent with differences in fraction unbound between animals. Determination of the fraction unbound showed a ∼5-fold difference in fraction unbound between the animals in the study. Broader analysis of individual dogs across a colony demonstrated a correlation between individual animal fraction unbound with total plasma clearance and volume of distribution. The concentrations of the common drug-binding proteins albumin and α1-acid glycoprotein in plasma were determined, and α1-acid glycoprotein levels were found to correlate with fraction unbound. Finally, single-nucleotide polymorphisms were identified at c.502 and c.522 of exon 5 of the dog α1-acid glycoprotein gene that may be correlated to the α1-acid glycoprotein concentration phenotype observed. SIGNIFICANCE STATEMENT: The current work demonstrates the potential for significant interindividual differences in plasma fraction unbound in beagle dogs and goes on to examine the underlying cause for the compound described. The findings suggest that the application of a population mean value of fraction unbound generated from a pooled sample may not always be appropriate and could introduce significant errors in scaling of in vitro clearance values, PBPK understanding, and interpretation of PKPD or toxicokinetic data in the context of unbound concentrations.

Drug metabolite identification using ultrahigh-performance liquid chromatography-ultraviolet spectroscopy and parallelized scans on a tribrid Orbitrap mass spectrometer.

RATIONALE: To capture all metabolites in metabolite identification studies, MS/MS information is required in both positive and negative ionization mode, usually involving several sample injections to gain all information about samples. A high-resolution and high mass accuracy quadrupole/linear trap/Orbitrap tribrid instrument was used to gain this information in a novel single injection 'capture-all' approach to metabolite identification. METHODS: Diclofenac, a model compound, was incubated in human and rat hepatocytes. These incubated samples were run using an ultrahigh-performance liquid chromatography/ultraviolet (UHPLC-UV) system coupled to a Thermo Fusion tribrid mass spectrometer. Five parallel scans were used: positive and negative ion full scan, data-dependent MS/MS, both high energy dissociation and collision-induced dissociation, and data-independent all ion fragmentation (AIF) spectra were collected in positive and negative ion mode. RESULTS: Nine metabolites were identified; a metabolite observed in the UV trace, but not positive ion full scan MS, was detected in the same sample injection by negative ion full scan MS. This was identified as a sulphate metabolite, and the corresponding negative ion AIF allowed for some structural elucidation. The use of a photo-diode array (PDA) detector allowed for spectral assessment in case of changes in absorbance spectra, and the subsequent semi-quantification of metabolites. CONCLUSIONS: This method provided good-quality MS/MS data across the m/z range in both positive and negative ion mode. The addition of both negative ion full scan MS and negative ion MS/MS allowed for the detection and structural elucidation of metabolites not observed in positive ion mode. The use of the PDA detector allowed for the semi-quantification of metabolites.

CYP-Mediated Sulfoximine Deimination of AZD6738.

In hepatic S9 and human liver microsomes (HLMs) the sulfoximine moiety of the ATR inhibitor AZD6738 is metabolized to its corresponding sulfoxide (AZ8982) and sulfone (AZ0002). The initial deimination to AZ8982 is nominally a reductive reaction, but in HLMs it required both NADPH and oxygen and also was inhibited by 1-aminobenzotriazole at a concentration of 1 mM. Studies conducted in a panel of 11 members of the cytochrome P450 (P450) family (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 CYP2J2, CYP3A4, and CYP3A5) confirmed that deimination was an oxidative process that was mediated largely by CYP2C8 with some CYP2J2 involvement, whereas the subsequent oxidation to sulfone was carried out largely by CYP2J2, CYP3A4, and CYP3A5. There was no measureable metabolism in flavin-containing monooxygenase (FMO) enzymes FMO3, FMO5 or NADPH cytochrome C reductase. Studies using Silensomes, a commercially available HLM in which specific members of the P450 family have been inhibited by selective mechanism-based inhibitors, showed that when CYP2C8 was inhibited, the rate of deimination was reduced by 95%, suggesting that CYP2J2 is only playing a minor role in HLMs. When CYP3A4 was inhibited, the rate increased by 58% due to the inhibition of the subsequent sulfone formation. Correlation studies conducted in HLM samples from different individuals confirmed the role of CYP2C8 in the deimination over CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A. Hence, although nominally a reduction, the deimination of AZD6738 to its sulfoxide metabolite AZ8982 is an oxidation mediated by CYP2C8, and this metabolite is subsequently oxidized to the sulfone (AZ0002) largely by CYP3A.