Restoring the biological activity of crizanlizumab at physiological conditions through a pH-dependent aspartic acid isomerization reaction.

In this study, we report the isomerization of an aspartic acid residue in the complementarity-determining region (CDR) of crizanlizumab as a major degradation pathway. The succinimide intermediate and iso-aspartic acid degradation products were successfully isolated by ion exchange chromatography for characterization. The isomerization site was identified at a DG motif in the CDR by peptide mapping. The biological characterization of the isolated variants showed that the succinimide variant exhibited a loss in target binding and biological activity compared to the aspartic acid and iso-aspartic acid variants of the molecule. The influence of pH on this isomerization reaction was investigated using capillary zone electrophoresis. Below pH 6.3, the succinimide formation was predominant, whereas at pH values above 6.3, iso-aspartic acid was formed and the initial amounts of succinimide dropped to levels even lower than those observed in the starting material. Importantly, while the succinimide accumulated at long-term storage conditions of 2 to 8°C at pH values below 6.3, a complete hydrolysis of succinimide was observed at physiological conditions (pH 7.4, 37°C), resulting in full recovery of the biological activity. In this study, we demonstrate that the critical quality attribute succinimide with reduced potency has little or no impact on the efficacy of crizanlizumab due to the full recovery of the biological activity within a few hours under physiological conditions.

Preclinical pharmacokinetics and metabolism of MAK683, a clinical stage selective oral embryonic ectoderm development (EED) inhibitor for cancer treatment.

MAK683 (N-((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)-8-(2-methylpyridin-3-yl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine) is a potent and orally bioavailable EED inhibitor for the potential treatment in oncology. Pharmacokinetics (PK) in preclinical species are characterised by low to moderate plasma clearances, high oral exposure, and moderate to high oral bioavailability at the dose of 1-2 mg/kg.A species comparison of the metabolic pathways of MAK683 has been made using [14C]MAK683 incubations with liver microsomes and hepatocytes from rat, dog, cynomolgus monkey, and human. Overall, the in vitro hepatic metabolism pathway of MAK683 in all five species was very complex. A total of 60 metabolites with 19 metabolites >1.5% of the total integrated area in the radiochromatogram of at least one species were identified in five species (rat, mouse, dog, monkey, and human).The primary in vitro hepatic oxidative metabolism pathway identified in humans involved 2-hydroxylation of the dihydrofuran ring to form alcohol (M28), which was in a chemical equilibrium favouring the formation of its aldehyde form. The aldehyde was then oxidised to the carboxylic acid metabolite (M26) or reduced to the O-hydroxyethylphenol (M29). N-dealkylation (M1), 3-hydroxylation of the dihydrofuran ring (M27), N-oxidation of the pyridine moiety (M53), and sulphate conjugation of M28 to form M19 were also important biotransformation pathways in human hepatocytes. The above major human hepatic metabolic pathways were also observed across the animal species (rat, mouse, dog, and monkey) mostly providing precursors for the formation of other metabolites via further oxygenation, glucuronidation, and sulphation pathways.No human-specific metabolites were observed. In addition, in vivo biotransformation was also conducted in bile-duct cannulated (BDC) rat. The metabolism in BDC rat was similar to those observed the in vitro hepatocytes.

All Ion Differential Analysis Refines the Detection of Terminal and Internal Diagnostic Fragment Ions for the Characterization of Biologics Product-Related Variants and Impurities by Middle-down Mass Spectrometry.

Characterization and monitoring of post-translational modifications (PTMs) are key analytical requirements during the development of biologics. Top and middle-down (MD) approaches aim at capturing a direct snapshot of all proteoforms with their combinatorial distribution. However, classical MD data analysis is predominantly limited to the interpretation of terminal ion series and PTMs matched by mass. In this study, time-resolved deconvolution (TRD) maps were produced to detect variants and impurities in Fd, Fc/2, and LC subunits of an IgG1 consistently across multiple samples. Classical MD analysis retrieved terminal ions, suggesting a deamidation at a NN motif for a LC+1 Da species, and inconclusive information for a LC+40 Da species. Additionally, we performed differential analysis of all MS2 ions across unmodified and variant subunit spectra to focus data analysis on spectral differences and reveal diagnostic ions (present, absent, enriched, or depleted ions) before fragment assignment. This sensitive methodology was able to detect diagnostic ions in a chimeric spectrum pointing at a proline-to-histidine sequence variant (+40 Da) missed by classical MD analysis. This methodology was pivotal to unravel relevant terminal ions and internal fragments N-terminal to proline as diagnostic ions to confirm the deamidation site. Moreover, different cleavage propensities were revealed at the deamidated DN site compared to the native NN motif for terminal and internal fragments, which may be tracked as a diagnostic behavior. Differential analysis may refine the detection of novel diagnostic ions and leverage the sequence information on internal fragments for the characterization of product-related variants and impurities by MD mass spectrometry.

Evaluation of relative MS response factors of drug metabolites for semi-quantitative assessment of chemical liabilities in drug discovery.

Drug metabolism studies are performed in drug discovery to identify metabolic soft spots, detect potentially toxic or reactive metabolites and provide an early insight into potential species differences. The relative peak area approach is often used to semi-quantitatively estimate the abundance of metabolites. Differences in the liquid chromatography-mass spectrometry responses result in an underestimation or overestimation of the metabolite and misinterpretation of results. The relative MS response factors (RF) of 132 structurally diverse drug candidates and their 233 corresponding metabolites were evaluated using a capillary-liquid chromatography/high-resolution mass spectrometry system. All of the synthesized metabolites discussed here were previously identified as key biotransformation products in discovery investigations or predicted to be formed. The most commonly occurring biotransformation mechanisms such as oxygenation, dealkylation and amide cleavage are represented within this dataset. However, relatively few phase II metabolites were evaluated because of the limited availability of authentic standards. Approximately 85% of these metabolites had a relative RF in the range between 0.2 (fivefold under-prediction) and 2.0 (twofold over-prediction), and the median MS RF was 0.6. Exceptions to this included very small metabolites that were hardly detectable. Additional experiments performed to understand the impact of the MS platform, flow rate and concentration suggested that these parameters do not have a significant impact on the RF of the compounds tested. This indicates that the use of relative peak areas to semi-quantitatively estimate the abundance of metabolites is justified in the drug discovery setting in order to guide medicinal chemistry efforts. Copyright © 2017 John Wiley & Sons, Ltd.

Micropreparative isolation and NMR structure elucidation of metabolites of the drug candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from rat bile and urine.

LC-MS based drug metabolism studies are effective in the optimization stage of drug discovery for rapid partial structure identification of metabolites. However, these studies usually do not provide unambiguous structural characterization of all metabolites, due to the limitations of MS-based structure identification. LC-MS-SPE-NMR is a technique that allows complete structure identification, but is difficult to apply to complex in vivo samples (such as bile collected during in vivo drug metabolism studies) due to the presence, at high concentrations, of interfering endogenous components, and potentially also dosage excipient components (e.g. polyethylene glycols). Here, we describe the isolation and structure characterization of seven metabolites of the drug development candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from a routine metabolism study in a bile-duct cannulated rat by LC-MS-SPE. The metabolites were isolated from bile and urine by repeated automatic trapping of the chromatographic peak of each metabolite on separate Oasis HLB SPE columns. The micropreparative HPLC/MS was performed on an XBridge BEH130 C18 HPLC column using aqueous formic acid/acetonitrile/methanol as mobile phase for the gradient elution. Mass spectrometric detection was performed on a LTQ XL linear ion trap mass spectrometer using electrospray ionization. Desorption of each metabolite was performed after the separation sequence. NMR spectra ((1)H, (13)C, 2D ROESY, HSQC and HMBC were measured on a Bruker AVANCE III spectrometer (600 MHz proton frequency) equipped with a 1.7 mm (1)H{(13)C,(15)N} Bruker Biospin's TCI MicroCryoProbe™.