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Mohamed W Attwa, Ali S Abdelhameed, Adnan A Kadi Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi ArabiaCorrespondence: Ali S Abdelhameed Tel +966 1146 98314Fax +966 1146 76 220Email asaber@ksu.edu.saBackground: Rociletinib (CO-1686; RLC) is a new, small molecule that is orally administered to inhibit mutant-selective covalent inhibitor of most epidermal growth factor receptor (EGFR)-mutated forms, including T790M, L858R, and exon 19 deletions, but not exon 20 insertions. Non–small-cell lung cancer (NSCLC) with a gene mutation that encodes EGFR is sensitive to approved EGFR inhibitors, but usually resistance develops, which is frequently mediated by T790M EGFR mutation. RLC is an EGFR inhibitor found to be active in preclinical models of EGFR-mutated NSCLC with or without T790M.Methods: In silico drug metabolism prediction of RLC was executed with the aid of the WhichP450 module (StarDrop software package) to verify its metabolic liability. Second, a fast, accurate, and competent LC-MS/MS assay was developed for RLC quantification to determine its metabolic stability. RLC and bosutinib (BOS) (internal standard; IS) were separated using an isocratic elution system with a C18 column (reversed stationary phase).Results: The developed LC-MS/MS analytical method showed linearity of 5– 500 ng/mL with r2 ≥ 0.9998 in the human liver microsomes (HLMs) matrix. A limit of quantification of 4.6 ng/mL revealed the sensitivity of the analytical method, while the acquired inter- and intra-day accuracy and precision values below 4.63% inferred the method reproducibility. RLC metabolic stability estimation was calculated using intrinsic clearance (20.15 μL/min/mg) and in vitro half-life (34.39 min) values.Conclusion: RLC exhibited a moderate extraction ratio indicative of good bioavailability. The developed analytical method herein is the first LC-MS/MS assay for RLC metabolic stability.Keywords: rociletinib, metabolic stability

Ali S Abdelhameed, Mohamed W Attwa, Adnan A Kadi Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Kingdom of Saudi ArabiaCorrespondence: Ali S Abdelhameed Tel +966 1146 98314Fax +966 1146 76 220, Email asaber@ksu.edu.saBackground: Ensartinib (ESB) is a novel anaplastic lymphoma kinase inhibitor (ALK) with additional activity against Abelson murine leukemia (ABL), met proto-oncogene (MET), receptor tyrosine kinase (AXL), and v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1) and is considered a safer alternative for other ALK inhibitors. ESB chemical structure contains a dichloro-fluorophenyl ring and cyclic tertiary amine rings (piperazine) that can be bioactivated generating reactive intermediates.Methods: In vitro metabolic study of ESB with human liver microsomes (HLMs) was performed and the hypothesis of generating reactive intermediates during metabolism was tested utilizing trapping agents to capture and stabilize reactive intermediates to facilitate their LC-MS/MS detection. Reduced glutathione (GSH) and potassium cyanide (KCN) were utilized as trapping agents for quinone methide and iminium intermediates, respectively.Results: Four in vitro ESB phase I metabolites were characterized. Three reactive intermediates including one epoxide and one iminium intermediates were characterized. ESB bioactivation is proposed to occur through unexpected metabolic pathways. The piperazine ring was bioactivated through iminium ions intermediates generation, while the dichloro-phenyl group was bioactivated through a special mechanism that was revealed by LC-MS/MS.Conclusion: These findings lay the foundations for additional work on ESB toxicity. Substituents to the bioactive centers (piperazine ring), either for blocking or isosteric replacement, would likely block or interrupt hydroxylation reaction that will end the bioactivation sequence.Keywords: in vitro phase-I metabolites, iminium intermediates, epoxide intermediates, hum

Mohamed W Attwa, Ali S Abdelhameed, Nasser S Al-Shakliah, Adnan A Kadi 1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi ArabiaCorrespondence: Ali S AbdelhameedDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi ArabiaTel +966 1146 70237Fax +966 1146 76220Email asaber@ksu.edu.saPurpose: Tandutinib (MLN518 or CT 53518) (TND) is a novel, oral, small-molecule inhibitor of type III receptor tyrosine kinases utilized for the treatment of acute myeloid leukemia (AML).Materials and Methods: In silico prediction of hepatic drug metabolism for TND was determined using the StarDrop® WhichP450™ module to confirm its metabolic liability. Second, an efficient and accurate LC-MS/MS method was established for TND quantification to evaluate metabolic stability. TND and entrectinib (ENC) (internal standard; IS) were resolved using an isocratic elution system with a reversed stationary phase (C8 column).Results: The established LC-MS/MS method exhibited linearity (5– 500 ng/mL) with r2 ≥ 0.9999 in the human liver microsomes matrix. The method sensitivity was indicated by the limit of quantification (3.8 ng/mL), and reproducibility was revealed by inter- and intraday precision and accuracy (below 10.5%). TND metabolic stability estimation was calculated using intrinsic clearance (22.03 μL/min/mg) and in vitro half-life (29.0 min) values.Conclusion: TND exhibited a moderate extraction ratio indicative of good bioavailability. According to the literature, the approach developed in the present study is the first established LC-MS/MS method for assessing TND metabolic stability.Keywords: tandutinib, metabolic stability assessment, in vitro half-life, validated LC-MS/MS methodology

Mohamed W Attwa, 1, 2 Adnan A Kadi, 1 Hany W Darwish 1, 3 1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; 2Students’ University Hospital, Mansoura University, Mansoura 35516, Egypt; 3Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, EgyptCorrespondence: Hany W Darwish Tel +966 1146 77343Fax +966 1146 76 220Email hdarwish@ksu.edu.saIntroduction: Larotrectinib (VITRAKVI) is an orally potent tropomyosin receptor kinase (Trk) inhibitor that acts by competitive inhibition of all corresponding receptor kinases. It demonstrated a marked response rate (75%) and robust anticancer activity in Trk fusion-positive patients. This response is independent of cancer type, age and gender.Methods: In this study, an efficient and accurate LC-MS/MS analytical method was developed for Larotrectinib (LRB) quantification in addition to evaluation of its metabolic stability. LRB and lapatinib (LTP) (which is chosen as an internal standard; IS) were eluted utilizing an isocratic mobile phase with a reversed phase elution system (C 18 column).Results and Discussion: The linearity range of the established method was 5– 500 ng/mL (r 2 ≥ 0.9999) in the human liver microsomes (HLMs) matrix. Various parameters were calculated to validate the method sensitivity (limit of quantification was 5 ng/mL) and reproducibility (inter and intra-day accuracy and precision were below 3% in all samples) of our methodology. For evaluation of LRB metabolic stability in HLMs matrix, in vitro half-life (48.8 min) and intrinsic clearance (14.19 μL/min/mg) were computed.Conclusion: Accordingly, we can conclude that LRB is a moderate extraction ratio drug when compared with other tyrosine kinase inhibitors (TKIs). According to our knowledge, the discussed procedure in this study is the first LC-MS/MS analytical method for evaluating LRB metabolic stability.Keywords: larotrectinib, human

Haitham AlRabiah,1 Adnan A Kadi,1 Haya I Aljohar,1 Mohamed W Attwa,1 Nasser S Al-Shakliah,1 Sabry M Attia,2 Gamal AE Mostafa1,3 1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11459, Saudi Arabia; 2Department of Pharmacology Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; 3Micro-Analytical Laboratory, Applied Organic Chemistry Department, National Research Center, Dokki, Cairo, EgyptCorrespondence: Gamal AE Mostafa, Email gmostafa@ksu.edu.saBackground: Dovitinib (TKI 258) is a small-molecule multi-kinase inhibitor for the treatment of different types of cancer. There is currently no validated method for its quantitative determination; therefore, we aimed to develop a reliable method to assay dovitinib.Method and Results: An electrospray ionization tandem mass spectrometry (ESI-MS/MS) method was used to separate dovitinib using an analytical C18 column (50 × 2.1 mm, 1.8 μm) at 25°C. Bosutinib was used as the internal standard (IS). Dovitinib was extracted from mouse plasma using a precipitation procedure. The mobile phase consisted of 10 mM ammonium formate: acetonitrile (68:32, v/v, pH 4.3) run at a rate of 0.3 mL min− 1. MS detection was performed in the positive ion mode. Multiple reaction monitoring transitions were 393→ 337 and 393→ 309 for dovitinib, and 530→ 141 and 530→ 113 for bosutinib. The investigated method was validated as a bio-analytical method based on FDA guidelines. The linearity of the developed method was over the range of 5– 500 ng mL,− 1 coefficient of determination (r2= 0.9998). The average intra-day recovery and relative standard deviation (RSD) of the quality control (QC) sample were 97.24% and 1.32%, whereas the overall inter-day accuracy and precision were 97.99% and 0.54%, respectively. Dovitinib was stable during sample storage and handling conditions. Furthermore, the di