Ramesh Mullangi

Vodobatinib (K0706) is a novel, orally bioavailable Bcr-Abl 1 tyrosine kinase inhibitor designed to overcome resistance mutations such as T3151 in chronic myeloid leukemia. While its clinical efficacy and safety are under investigation, there is limited information on vodobatinib preclinical absorption, distribution, metabolism, and excretion properties and tissue distribution. The objective of this study is to evaluate vodobatinib solubility in phosphate buffer and biorelevant media, metabolic stability in liver microsomes and hepatocytes across species, permeability using MDCK-MDR1 cells, plasma protein binding via equilibrium dialysis, CYP phenotyping and CYP inhibition. Tissue distribution was assessed in male Sprague–Dawley rats following oral administration (2.0 mg/kg), with plasma and tissue samples collected over a 24-hour period. Vodobatinib exhibited poor solubility in phosphate buffer (2.5 µM), but showed improved solubility in biorelevant media. It demonstrated high metabolic stability in liver microsomes and hepatocytes of rats and dogs, with moderate stability in humans. Permeability studies indicated low efflux liability. Plasma protein binding was extensive (>99.8%) across species. Metabolism was primarily mediated by CYP3A4 (≈70%), with minor involvement from CYP2C19 and CYP2D6. CYP inhibition was weak to moderate, suggesting a low potential for drug–drug interactions. Vodobatinib showed high distribution to the liver, intestines, and kidneys, with low brain penetration. Vodobatinib demonstrates a favorable preclinical absorption, distribution, metabolism, and excretion profile, with high metabolic stability, minimal efflux liability, and selective tissue distribution. These findings support its continued clinical development in chronic myeloid leukemia and potentially other Bcr-Abl 1 driven malignancies.

Vodobatinib is a Bcr‐Abl 1 inhibitor, currently entering into Phase 2 clinical trials as a potential drug to treat glioblastoma patients. In the present work, a validated high‐performance liquid chromatography (HPLC) detection method for the quantification of vodobatinib in rat plasma was established. Sample preparation involved liquid–liquid extraction method. The chromatographic separation of vodobatinib and the IS was accomplished on an XBridge C₁₈ column using 10 mM ammonium acetate and acetonitrile in gradient condition. The flow‐rate and injection volume were 0.8 mL/min and 20 μL, respectively. The eluent was detected at 310 nm. Vodobatinib and the IS eluted at 7.5 and 5.9 min, respectively, with a total run time of 10 min. Validation tests were performed according to FDA requirements. The established method showed good linearity in the range of 50.5–6534 ng/mL. Interday and intraday coefficients of variations were < 5.11%. Stability studies, dilution integrity, and incurred sample reanalysis met the acceptance criteria. Subsequently, the validated HPLC method was used to study the disposition kinetics of vodobatinib in rats.

A highly sensitive and rapid LC–MS/MS method was developed and validated for the quantification of dexamethasone in rat plasma and brain tissue. Protein precipitation method was used for sample preparation. The separation of dexamethasone and the IS (labetalol) was achieved on an Atlantis dC₁₈ column using an isocratic mobile phase (10 mM ammonium formate and acetonitrile, 25/75, v/v) delivered at 0.7 mL/min flow‐rate. Dexamethasone and the IS were eluted at 1.03 and 1.06 min, respectively. The MS/MS transitions monitored were m/z 393.100 → 373.100 (dexamethasone) and 329.100 → 91.100 (IS). Method validation was performed as per FDA guidelines and all parameters met the acceptance criteria. The assay was validated with a quantification range of 0.05–1046 ng/mL in both matrices. The intraday and interday precision for were in the range of 2.62–7.28 and 2.76%–6.98% and 2.24–6.85 and 2.97%–6.37%, in plasma and brain tissue, respectively. Dexamethasone was stable in a series of stability conditions in both matrices. Post‐intravenous administration to rats, dexamethasone concentrations in plasma and brain tissue were quantifiable up to 24 and 10 h, respectively. Dexamethasone half‐life was ~2.30 h. Dexamethasone exhibited low clearance and moderate volume of distribution in plasma but in brain tissue the clearance and volume of distribution were high.

Paxalisib is a pan‐PI3K and mTOR inhibitor, currently entering into Phase II clinical trials as a potential drug to treat glioblastoma patients. We report the development and validation of a high‐performance liquid chromatography (HPLC) method for the quantitation of paxalisib in mouse plasma as per the US Food and Drug Administration regulatory guidelines. From the mouse plasma, paxalisib and the internal standard (IS; filgotinib) were extracted using ethyl acetate as an extraction solvent. The chromatographic separation of paxalisib and the IS was accomplished on a Symmetry C₁₈ (250 × 4.6 mm, 5.0 μm) column maintained at 40°C using 10 mm ammonium formate and acetonitrile in gradient conditions at a 0.8 ml/min flow‐rate. The injection volume was 20 μl. The elution was monitored using a photo‐diode array detector set at λ_max 280 nm. Paxalisib and the IS eluted at 6.5 and 5.9 min, respectively with a total run time of 10 min. The calibration curve was linear over the range of 111–4,989 ng/ml. Inter‐ and intraday precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated and the results met the acceptance criteria. The validated HPLC method was extended to assess the pharmacokinetic parameters of paxalisib in mice.

Copanlisib is a dual PI3K‐δ inhibitor, used in follicular lymphoma treatment. In this research, we report a validated LC–MS/MS method for quantifying copanlisib from a mouse dried blood spot (DBS). We validated the method in line with the guidelines of the US Food and Drug Administration. The liquid–liquid extraction technique was used to extract copanlisib from the DBS discs. We used an Atlantis dC₁₈ column and isocratic mobile phase for the chromatographic separation of copanlisib and the internal standard (idelalisib). The flow was 0.90 ml/min. Under the optimized chromatographic conditions, the retentions of copanlisib and the internal standard were 0.98 and 0.93 min, respectively. Each injection total run time was 2.50 min. The MS/MS ion transitions monitored were m/z 481.31 → 128.00 and 416.10 → 176.10 for copanlisib and the internal standard (IS) idelalisib, respectively. We have used a broad calibration range (1.01–4,797 ng/ml) with a determination coefficient (r²) of 0.997. All of the evaluated parameters met the acceptance criteria. Hematocrit did not influence the DBS copanlisib concentrations. We have used the validated method to derive the intravenous pharmacokinetic parameters by quantifying copanlisib in mouse plasma.

In this study, we report the development and validation of an LC–tandem mass spectrometry method for the simultaneous quantitation of bendamustine and copanlisib in mouse plasma as per the US FDA regulatory guidelines. The sample processing involves extraction of bendamustine and copanlisib along with internal standard (IS; warfarin) from 50 μL mouse plasma using a liquid–liquid extraction method. The chromatographic separation of bendamustine, copanlisib and the IS was achieved on an Atlantis dC₁₈ column using an isocratic mobile phase (5 mM ammonium acetate:methanol, 20:80 v/v). Bendamustine, copanlisib and the IS eluted at 0.88, 1.39 and 0.74 min, respectively, with a total run time of 2.5 min. The calibration curve ranged from 3.99–2996 and 4.33–3248 ng/mL for bendamustine and copanlisib, respectively. Inter‐ and intra‐day precision and accuracy, stability in processed samples and upon storage, dilution integrity and incurred sample reanalysis were investigated for both the analytes. The intra‐ and inter‐day precisions were in the ranges of 2.01%–5.05% and 2.74%–6.13% and 1.98%–7.64 and 8.62%–9.04% for bendamustine and copanlisib, respectively. Stability studies showed that both analytes were stable on bench top for 6 h, in auto‐sampler for 24 and at −80°C for 30 days. The validated method was successfully applied to a pharmacokinetic study in mice.

Background: Darolutamide is recently approved for the treatment of non-metastatic castrate resistance prostate cancer. Hitherto, no stereoselective pharmacokinetic data have been published pertaining to darolutamide and its diastereomers in animals or humans. The key aims of the experiment were to examine darolutamide, S,S-darolutamide and S,R-darolutamide with respect to (a) assessment of in vitro metabolic stability and protein binding and (b) characterization of in vivo oral and intravenous pharmacokinetics in mice. Method: In vitro (liver microsomes stability and protein binding) and in vivo experiments (oral/intravenous dosing to mice) were carried out using darolutamide, S,S-darolutamide and S,Rdarolutamide. Besides, tissue levels of darolutamide, S,S-darolutamide and S,R-darolutamide were measured following oral and intravenous dosing. Appropriate plasma/tissue samples served to determine the pharmacokinetics of various analytes in mice. Liquid chromatography in tandem with mass spectrometry procedures enabled the delineation of the plasma pharmacokinetics, in vitro and tissue uptake data of the various analytes. Results: Chiral inversion was absent in the metabolic stability study. However, darolutamide showed profound stereoselectivity (S,S-darolutamide greater than S,R-darolutamide) after either intravenous or oral dosing. S,R-darolutamide but not S,S-darolutamide showed conversion to its antipode post oral and intravenous dosing to mice. Regardless of oral or intravenous dosing, active keto darolutamide formation was evident after administration of darolutamide, S,S-darolutamide or S,R- darolutamide. Tissue data supported the observations in plasma; however, tissue exposure of darolutamide, S,Sdarolutamide and S,R-darolutamide was much lower as compared to plasma. Conclusion: In lieu of the human pharmacokinetic data, although the administration of diastereomeric darolutamide was justified, it is proposed to delineate the clinical pharmacokinetics of S,Rdarolutamide and S,S-darolutamide relative to darolutamide in future clinical pharmacology studies.

Phosphatidylinositol 3-kinase (PI3K) inhibitors are emerging novel class of anticancer drugs. In this work, we report the development and validation of an LC-MS/MS method for the simultaneous quantitation of three PI3K inhibitors namely copanlisib, duvelisib and idelalisib in mouse plasma as per the FDA regulatory guidelines. Liquid-liquid extraction was used to extract copanlisib, duvelisib and idelalisib along with internal standard (I.S; filgotinib) from mouse plasma. Thereafter, chromatographic separation was achieved on an Ascentis Express RP-amide column using an isocratic mobile phase (5 mM ammonium acetate and acetonitrile in the ratio of 25:75, v/v) at a flow-rate of 0.5 mL/min. The analytes and I.S were detected by positive electrospray ionization tandem mass spectrometry (ESI-MS/MS) operated in multiple-reaction-monitoring (MRM) mode. The analytical time was 2.50 min in total. The calibration curve ranged from 4.41-1029, 0.70-1395 and 0.77-1544 ng/mL for copanlisib, duvelisib and idelalisib, respectively. The intra-assay accuracy and precision were ≤108 % and ≤6.23 %; ≤95.8 % and ≤13.1 %; and ≤101 % and ≤7.92 % for copanlisib, duvelisib and idelalisib, respectively and the inter-assay accuracy and precision were ≤107 % and ≤6.13 %; ≤97.6 % and ≤9.41 %; and ≤100% and ≤7.79 % for copanlisib, duvelisib and idelalisib, respectively. The validated method was successfully applied to a pharmacokinetic study in mice. GRAPHICAL ABSTRACT

AMG 510 is the first‐in‐class KRAS^G12C inhibitor, currently in phase 2 clinical trials as an orphan drug to treat non‐small cell lung cancer patients. We developed and validated a sensitive, selective, and high‐throughput HPLC‐MS/MS method for the quantitation of AMG 510 in mouse plasma per the regulatory guideline of the US Food and Drug and Administration. AMG 510 and the IS (MRTX‐1257) were extracted from mouse plasma using tert‐butyl methyl ether and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 25:75, v/v) at a flow rate of 0.65 mL/min on an Atlantis dC₁₈ column. AMG 510 and the IS eluted at ~0.95 and 0.73 min, respectively. AMG 510 and the IS were detected by positive electrospray ionization in multiple reaction monitoring using transition pair (Q1 → Q3) m/z 561.1 → 134.1 and m/z 566.5 → 98.2, respectively. Excellent linearity was achieved in the concentration range of 1.08–5040 ng/mL (r > 0.0996). No matrix effect and carryover were observed. Intra‐ and inter‐day accuracies and precisions were within the acceptance range. AMG 510 was demonstrated to be stable under the tested storage conditions. This novel method has been applied to a pharmacokinetic study in mice.

Phosphatidylinositol 3‐kinase (PI3K) inhibitors are a novel class of anticancer drugs that are approved to treat various malignancies. We report the development and validation of a HPLC method for the simultaneous quantitation of three PI3K inhibitors, namely copanlisib, duvelisib and idelalisib, in rat plasma as per the regulatory guidelines of the United States Food and Drug Administration. The method involves extraction of copanlisib, duvelisib and idelalisib along with an internal standard (IS; filgotinib) from rat plasma (100 μL) using a liquid–liquid extraction process. The chromatographic separation of the analytes was achieved using step‐wise gradient elution on a Hypersil Gold C₁₈ column. The UV detection wavelength was set at λ_max = 280 nm. Copanlisib, duvelisib, idelalisib and the IS eluted at 7.16, 12.6, 11.9 and 9.86 min, respectively, with a total run time of 15 min. The calibration curve ranged from 50 to 5000 ng/mL for all the analytes. Inter‐ and intra‐day precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated for all three analytes, and the results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.