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Abstract Nicotinamide N-methyltransferase (NNMT) is a metabolic regulator that catalyzes the methylation of nicotinamide (Nam) using the co-factor S-adenosyl-L-methionine to form 1-methyl-nicotinamide (MNA). Overexpression of NNMT and the presence of the active metabolite MNA is associated with a number of diseases including metabolic disorders. We conducted a high-throughput screening campaign that led to the identification of a tricyclic core as a potential NNMT small molecule inhibitor series. Elaborate medicinal chemistry efforts were undertaken and hundreds of analogs were synthesized to understand the structure activity relationship and structure property relationship of this tricyclic series. A lead molecule, JBSNF-000028, was identified that inhibits human and mouse NNMT activity, reduces MNA levels in mouse plasma, liver and adipose tissue, and drives insulin sensitization, glucose modulation and body weight reduction in a diet-induced obese mouse model of diabetes. The co-crystal structure showed that JBSNF-000028 binds below a hairpin structural motif at the nicotinamide pocket and stacks between Tyr-204 (from Hairpin) and Leu-164 (from central domain). JBSNF-000028 was inactive against a broad panel of targets related to metabolism and safety. Interestingly, the improvement in glucose tolerance upon treatment with JBSNF-000028 was also observed in NNMT knockout mice with diet-induced obesity, pointing towards the glucose-normalizing effect that may go beyond NNMT inhibition. JBSNF-000028 can be a potential therapeutic option for metabolic disorders and developmental studies are warranted.

A highly sensitive and selective high performance liquid chromatographic fluorescence detection method has been developed and validated for the quantification of rivastigmine in rat plasma and brain. Protein precipitation and one-step liquid-liquid extraction techniques were utilized for the extraction of RSM from brain and plasma, respectively, along with an internal standard. The chromatographic separation was achieved with a column inertsil ODS-3V and a mobile phase consisting of ammonium acetate buffer (20 mM, pH 4.5) and acetonitrile (76:24, v/v) delivered at a flow rate of 1 ml/min. The lower limit of quantitation for the developed method was 10 ng/mL for both matrices. The method was found to be accurate and reproducible and was successfully used to quantify levels of RSM in plasma and brain following intravenous administration of RSM in rats. [ABSTRACT FROM AUTHOR]

A series of exploratory investigations with multiple agents was carried out in normal rats and in rats with uranyl nitrate-induced acute renal failure to understand the disposition characteristics of intravenous topotecan (TPT) used as a model substrate. The disposition of TPT was unaltered in normal rats when treated with methotrexate, whereas treatment with probenecid increased the systemic exposure of TPT. In case of uranyl nitrate-induced acute renal failure (UN-ARF) rats, the systemic exposure of TPT was increased when compared with normal rats, whereas in UN-ARF rats treated with probenecid a further reduction in renal clearance of TPT was noted as compared with that of UN-ARF induced rats. Thus, TPT may be involved in the tubular secretory pathway when a passive glomerular filtration pathway for elimination was not possible. The disposition of TPT did not normalize in UN-ARF rats when treated with caffeine, a non-selective adenosine A1 receptor antagonist, whereas the selective adenosine A1 receptor antagonist (1,3-dipropyl-8-phenylxanthine, DPPX) normalized TPT pharmacokinetic disposition by improving renal function. Renal excretion studies demonstrated that CLR improved by almost fivefold following DPPX treatment in ARF rats. In addition, the qualitative stability/metabolism pattern of TPT in liver microsomes prepared from various groups of rats (normal rats, UN-ARF rats, rats treated with DPPX, and UN-ARF rats treated with DPPX) was found to be similar. In summary, using a pharmacokinetic tool as a surrogate, it has been shown that the pharmacokinetic disposition of TPT improved considerably upon treatment with DPPX, a selective adenosine A1 antagonist. [ABSTRACT FROM AUTHOR]

The aim of this study was to study the effect of 1-aminobenzotriazole (ABT) on in vitro metabolism, oral, and intravenous (IV) pharmacokinetics of chlorzoxazone (CZX) in rats. Enzyme kinetics of CZX was performed with rat and human liver microsomes and pure isozyme (CYP2E1) with and without ABT. The enzyme kinetics ( V max and K m ) of the formation of 6-hydroxychlorzoxazone (OH-CZX) was found to be similar among rat liver microsomes (3486  pmol  mg  protein -1   min -1 and 345  µM), human liver microsomes (3194  pmol  mg  protein -1   min -1 and 335  µM) and pure isozyme (3423  pmol  mg  protein -1   min -1 and 403  µM), but K I and K inact values for ABT towards the ability to inhibit the formation of OH-CZX from CZX varied between liver microsomes (rat: 32.09  µM and 0.12  min -1 ; human: 27.19  µM and 0.14  min -1 ) and pure isozyme (3.18  µM and 0.29  min -1 ). The novel robust analytical method was capable of quantifying CZX, OH-CZX, and ABT simultaneously in a single run, and the method was used for both in vitro and in vivo studies. Pre-treatment of rats with ABT prior to oral and IV administration of CZX significantly decreased the clearance (threefold) and consequently increased the AUC of CZX (approx. three- to fourfold). When rats were pre-treated with ABT, the formation of OH-CZX was completely blocked after oral and IV administration; however, we were able to measure OH-CZX in rats administered with CZX by oral and IV routes without pre-treatment of ABT. The oral bioavailability of CZX was ∼⃒71% when dosed alone and reached 100% under pre-treatment with ABT. The t 1/2 values of CZX was significantly prolonged for oral dosing compared with IV dosing under pre-treated conditions with ABT, suggesting an involvement of pre-systemic component in the disposition of CZX. The pharmacokinetic parameters of ABT did not change when it was dosed along with CZX (oral and IV), indicating that either CZX or OH-CZX had no effect on disposition of ABT. The plasma concentrations of ABT were above and beyond the required levels to inhibit CYP2E1 enzyme for at least 36  h post-treatment. [ABSTRACT FROM AUTHOR]

The purpose of this bridging phase I study was to characterize the toxicity, pharmacokinetics, and antitumor effects of a capsule formulation of DRF-1042, a novel camptothecin analog, in refractory solid tumor patients. DRF-1042 was given daily for 5 consecutive days for 2 weeks, repeated every 3 weeks at 81 mg/m(2). Adverse events were monitored following NCI-CTC. Blood samples were processed for bioanalysis using a validated high-performance liquid chromatography method. The pharmacokinetics of lactone and total (lactone + carboxylate) forms was determined on days 1 and 12 using a noncompartmental pharmacokinetic method. Pharmacokinetic data with the capsule formulation were compared with previously reported pharmacokinetic parameters with a suspension formulation. Efficacy was evaluated by applying World Health Organization criteria. Six patients received 10 courses of therapy. Thrombocytopenia and diarrhea were dose-limiting toxicities. The upper limit of the area under the curve of DRF-1042 (lactone and total) with the capsule formulation was higher than a suspension formulation at a similar dose on day 1 (lactone: capsule = 8.53 microMxh, suspension = 5.33 microMxh; total: capsule = 393 microMxh, suspension = 176 microMxh) and day 12 (lactone: capsule = 22.1 microMxh, suspension = 6.1 microMxh; total: capsule = 1302 microMxh, suspension = 309 microMxh). The upper limit of the area under the curve of DRF-1042 (lactone and total) was higher under fed conditions (lactone = 15.9 microMxh, total = 605 microMxh) relative to fasted conditions (lactone = 8.53 microMxh, total = 393 microMxh) on day 1. One patient experienced stable disease. The toxicity and pharmacokinetics of the capsule correlated well with the suspension. The recommended phase II dose is 81 mg/m(2). [ABSTRACT FROM AUTHOR]

The aim of this study was to characterize the pharmacokinetics and determine the absolute bioavailability and metabolism of DRF 4367, a novel COX-2 inhibitor, in mice. In addition, the in vitro metabolism of DRF 4367 was studied in mouse, rat, dog, monkey and human liver microsomes. Following oral administration, maximum concentrations of DRF 4367 were achieved after about 1?h. Upon intravenous (IV) administration, the concentration of DRF 4367 declined in a bi-exponential fashion with a terminal elimination half-life of 4.0?h. The elimination half-life was unchanged with route of administration. The volume of distribution and systemic clearance of DRF 4367 in mice were 0.80 l?h -1 ?kg -1 and 0.14 l?kg -1 , respectively, after IV administration. The absolute oral bioavailability of DRF 4367 was 44%. In all species of liver microsomes examined, the primary route of metabolism for DRF 4367 was demethylation of benzyl methoxy to form a hydroxy metabolite (M1). The formation of this metabolite was mediated by CYP2D6 and CYP2C19 enzymes. M1 was not found to possess COX-2 inhibitory activity. Chemical-inhibition studies showed that quinidine (selective for CYP2D6) and ticlopidine (selective for CYP2C19) inhibited the formation of the hydroxy metabolite of DRF 4367, whereas potent inhibitors selective for other forms of CYP did not inhibit this oxidative reaction. Upon oral or IV administration of DRF 4367 to mice, unchanged DRF 4367, M1, the O -glucuronide conjugate of M1 (M1-G) and the O -sulfate conjugate of M1 (M1-S) were identified in bile. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the effect of sucralfate pretreatment on the pharmacokinetics of rosiglitazone following a single oral dose in healthy male volunteers. After an over night fast, and according to a randomized schedule, each volunteer (n = 9) received a single oral dose of rosiglitazone 8 mg (Avandia tablets, 4 mg x 2) with or without pretreatment of sucralfate 2 g (Recolfate tablets, 1 g x 2) in an open-label crossover study with a 2-week washout period. Plasma samples were collected over a period of 24 hours at regular intervals. Safety assessment included monitoring of the vital signs, blood parameters, and ECG. No statistically significant differences (p > 0.05) were observed for any of the calculated rosiglitazone pharmacokinetic parameters in the two treatment groups. The mean parameters, AUC0-infinity and Cmax, following rosiglitazone administration alone were 3825.02 ng x h/ml and 664.47 ng/ml, respectively, and for rosiglitazone administered after pretreatment with sucralfate were 4848.19 ng x h/ml and 624.88 ng/ml, respectively. The t(max) for rosiglitazone alone and for rosiglitazone after sucralfate treatments was 1.11 and 1.67 hours, respectively. The mean elimination half-life for rosiglitazone and rosiglitazone after sucralfate treatment was 4.35 and 4.51 hours, respectively. Fraction of rosiglitazone absorbed was calculated by the Wagner-Nelson method, and no statistically significant difference (p > 0.05) was observed for the two treatments. Since sucralfate pretreatment did not show any significant difference in the pharmacokinetics of rosiglitazone, no dose adjustment is warranted for rosiglitazone when it is administered with sucralfate. [ABSTRACT FROM AUTHOR]

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 C 18 (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., (© 2023 John Wiley & Sons, Ltd.)

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 dC 18 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 (r 2 ) 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., (© 2022 John Wiley & Sons Ltd.)

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 dC 18 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., (© 2022 John Wiley & Sons Ltd.)

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 dC 18 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., (© 2020 John Wiley & Sons, Ltd.)

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 C 18 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., (© 2020 John Wiley & Sons, Ltd.)

Lumefantrine (LFN) is a chiral antimalarial drug. Enantioselective in vitro attributes and absolute oral pharmacokinetics for (-)-LFN and (+)-LFN have been characterized in mice. No stereoselectivity was seen with either of the enantiomers when compared with rac -LFN in the executed in vitro studies (solubility, metabolic stability, protein binding, permeability and blood partitioning). Post intravenous or oral administration of rac -LFN, the AUC 0-∞ and MRT of (+)-LFN was higher over (-)-LFN, which is reflected in higher clearance value for (-)-LFN. Following (-)-LFN intravenous administration to mice, the key PK parameters were comparable to (-)-LFN from rac -LFN; however, post intravenous administration of (+)-LFN alone to mice, the AUC 0-∞ was 1.3-fold higher than (+)-LFN from rac -LFN. Similarly, post oral administration of (-)-LFN to mice, both AUC 0-∞ and C max were 1.3-fold higher than (-)-LFN from rac -LFN. On other hand, (+)-LFN showed 1.4-fold higher AUC 0-∞ and 1.7-fold higher C max post oral administration over (+)-LFN from rac -LFN.

Immuno-oncology (IO) is an emerging option to treat cancer malignancies. In the last two years, IO has accounted for more than 90% of the new active drugs in various therapeutic indications of oncology drug development. Bioanalytical methods used for the quantitation of various IO small molecule drugs have been summarized in this review. The most commonly used are HPLC and LC-MS/MS methods. Determination of IO drugs from biological matrices involves drug extraction from the biological matrix, which is mostly achieved by simple protein precipitation, liquid-liquid extraction and solid-phase extraction. Subsequently, quantitation is usually achieved by LC-MS/MS, but HPLC-UV has also been employed. The bioanalytical methods reported for each drug are briefly discussed and tabulated for easy access. Our review indicates that LC-MS/MS is a versatile and reliable tool for the sensitive, rapid and robust quantitation of IO drugs., (© 2020 John Wiley & Sons, Ltd.)

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., Methods: 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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Idelalisib is a selective and second-generation PI3K-δ inhibitor, approved for the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this paper, we present a fully validated dried blood spot (DBS) method for the quantitation of idelalisib from mice blood using an LC-MS/MS, which was operated under multiple reaction monitoring mode. To the punched DBS discs, acidified methanol enriched with internal standard (IS; larotrectinib) was added and extracted using tert -butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of idelalisib and the IS was achieved on an Atlantis dC 18 column using a mixture of 10 mM ammonium formate:acetonitrile (25:75, v/v). The flow-rate and injection volume were 0.80 mL/min and 2.0 µL, respectively. Idelalisib and the IS were eluted at ~0.98 and 0.93 min, respectively and the total run time was 2.00 min. Idelalisib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 416.1→176.1 and 429.1→342.1, respectively was used for the quantitation. The calibration range was 1.01-4 797 ng/mL. No matrix effect and carry over were observed. Haematocrit did not influence DBS idelalisib concentrations. All the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mice pharmacokinetic study., Competing Interests: All authors have no conflict of interest to declare., (Thieme. All rights reserved.)

Lipids are a complex and critical heterogeneous molecular entity, playing an intricate and key role in understanding biological activities and disease processes. Lipidomics aims to quantitatively define the lipid classes, including their molecular species. The analysis of the biological tissues and fluids are challenging due to the extreme sample complexity and occurrence of the molecular species as isomers or isobars. This review documents the overview of lipidomics workflow, beginning from the approaches of sample preparation, various analytical techniques and emphasizing the state-of-the-art mass spectrometry either by shotgun or coupled with liquid chromatography. We have considered the latest ion mobility spectroscopy technologies to deal with the vast number of structural isomers, different imaging techniques. All these techniques have their pitfalls and we have discussed how to circumvent them after reviewing the power of each technique with examples.., Competing Interests: Conflict of interest : Ramesh Mullangi is Vice President, Pre-clinical Biology & DMP at Laxai Life Sciences., (Copyright © 2021 by the authors.)

Larotrectinib is a first-generation tropomyosin kinase inhibitor, approved for the treatment of solid tumors. In this paper, we present a validated dried blood spot (DBS) method for the quantitation of larotrectinib from mouse blood using HPLC-MS/MS, which was operated under multiple reaction monitoring mode. To the DBS disc cards, acidified methanol enriched with internal standard (IS; enasidenib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of larotrectinib and the IS was achieved on an Atlantis dC 18 column using 10 mm ammonium formate-acetonitrile (30:70, v/v) delivered at a flow-rate of 0.80 ml/min. Under these optimized conditions, the retention times of larotrectinib and the IS were ~0.93 and 1.37 min, respectively. The total run time was 2.50 min. Larotrectinib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transitions of 429.1 → 342.1 and 474.1 → 267.1, respectively, were used for the quantitation. The calibration range was 1.06-5,080 ng/ml. No matrix effect or carryover was observed. Hematocrit did not influence DBS larotrectinib concentrations. All of the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mouse pharmacokinetic study., (© 2020 John Wiley & Sons, Ltd.)

Different options on performing incurred sample reanalysis (ISR) on dried blood spot (DBS) cards were investigated using drugs belonging to various therapeutic areas: (a) darolutamide (to treat prostate cancer) and (b) filgotinib (to treat rheumatoid arthritis). The proposed novel methodology included the generation of half-DBS and quarter-DBS discs after initial blood collection using the full-DBS discs. Accordingly, blood collection via DBS was performed in male BALB/c mice following intravenous and oral dosing of darolutamide; in male Sprague Dawley rats following intravenous and oral dosing of filgotinib. The ISR data generated from the full-DBS disc, half-DBS disc and quarter-DBS disc were compared for the assessment of the proposed methodology. Quantification of darolutamide and filgotinib was accomplished using liquid chromatography-electrospray ionization/tandem mass spectrometry methods. Darolutamide and filgotinib ISR samples, which were collected and prepared using full-, half- and quarter-DBS discs, met the acceptance criteria for ISR analysis. In conclusion, this is the first report showing a viable tool for the performance of ISR on DBS cards. The use of quarter- or half-DBS discs would aid in not only ISR but also in long-term storage experiments of analytes because it would avoid the need for additional blood sampling in patients., (© 2020 John Wiley & Sons, Ltd.)

A selective, sensitive and rapid LC-MS/MS method has been developed and validated as per US Food and Drug Administration regulatory guidelines for the simultaneous quantitation of colchicine and febuxostat in rat plasma. Colchicine and febuxostat were extracted from the rat plasma using 10% tert-butyl methyl ether in ethyl acetate using colchicine-d 6 as an internal standard (IS). The chromatographic separation of colchicine, febuxostat and the IS was achieved using a mobile phase comprising 5 mm ammonium formate and 0.025% formic acid in acetonitrile (20:80, v/v) in isocratic mode on an Eclipse XDB-C 18 column. The injection volume and flow rate were 5.0 μl and 0.9 ml/min, respectively. Colchicine and febuxostat were detected by positive electrospray ionization in multiple reaction monitoring mode using transition pairs (Q1 → Q3) of m/z 400.10 → 358.10 and 317.05 → 261.00, respectively. The assay was linear in the ranges of 0.25-254 and 2.60-622 ng/ml for colchicine and febuxostat, respectively. The inter- and intra-day precision values were 0.58-13.0 and 1.03-4.88% for colchicine and febuxostat, respectively. No matrix or carryover effects were observed during the validation. Both analytes were stable on the bench-top, in the autosampler and in storage (freeze-thaw cycles and long-term storage at -80°C). A pharmacokinetic study in rats was performed to show the applicability of the validated method., (© 2020 John Wiley & Sons, Ltd.)

We developed and validated a simple, sensitive, selective, and reliable LC-MS/MS-ESI method for the direct quantitation of lumefantrine (LFN) enantiomers [(-)-LFN and (+)-LFN] in mice plasma as per regulatory guideline. LFN enantiomers and carbamazepine (internal standard) were extracted from mice plasma using Strata X SPE (solid-phase extraction) cartridges. Good resolution between enantiomers was achieved on a Chiralpak IA-3 column using an isocratic mobile phase (0.1% of diethyl amine in methanol), which was delivered at a flow rate of 0.8 mL/min. Detection and quantitation were performed using multiple reaction monitoring mode following the transitions m/z 530.27 → 512.30 and 237.00 → 194.00 for LFN enantiomers and the internal standard, respectively, in the positive-ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 2.39-895 ng/mL for each enantiomer. The intra- and inter-day precisions were in the range of 1.03-6.14 and 6.36-8.70 and 2.03-4.88 and 5.82-11.5 for (-)-LFN and (+)-LFN, respectively. Both (-)-LFN and (+)-LFN were found to be stable under different stability conditions. The method was successfully used to delineate stereoselective pharmacokinetics of LFN enantiomers in mice after an oral administration of rac-LFN (20 mg/kg). The pharmacokinetic results indicated that the disposition of LFN enantiomers was stereoselective in mice., (© 2020 John Wiley & Sons, Ltd.)

SAFit-1 and SAFit-2 are selective FKBP51 (FK506-binding protein 51) ligands. In this paper, we present the development and validation data of an LC-MS/MS method for the simultaneous quantitation of SAFit-1 and SAFit-2 in mice plasma as per FDA regulatory guideline. SAFit-1 and SAFit-2 along with internal standard were extracted from mice plasma using liquid-liquid extraction method. Chromatographic resolution of SAFit-1, SAFit-2 and the internal standard (warfarin) was achieved on an X-Terra phenyl column using 0.2% formic acid:acetonitrile (20:80, v/v) as an eluent, which was delivered at a flow-rate of 0.9 mL/min. The MS/MS ion transitions monitored were m/z 748.4→420.4, 803.7→384.3 and 309.2 →163.2 for SAFit-1, SAFit-2 and the internal standard, respectively. The linearity range was 2.45-2446 ng/mL for both SAFit-1 and SAFit-2. The intra- and inter-day accuracy and intra- and inter-day precision were in the range of 0.90-1.07 and 2.38-10.8%, respectively for SAFit-1; 0.97-1.15 and 0.23-12.5%, respectively for SAFit-2. Both SAFit-1 and SAFit-2 were found to be stable in stability studies (up to three freeze-thaw cycles and for long-term at -80°C for 30 days) and processed (bench-top for 3 h and in in-injector for 16 h) samples. The application of the validated method was shown in a pharmacokinetic study in mice., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Aurora B plays critical role in the process of chromosome condensation and chromosome orientation during the regulation of mitosis. The overexpression of Aurora B has been observed in several tumor types. As a part of our ongoing effort to develop Aurora B inhibitors, herein, we described the design, synthesis and evaluation of phenyl/pyridine diazepine analogs. The diazepane aniline pyrimidine (4a) was identified as an initial hit (Aurora B IC 50 6.9 µM). Molecular modeling guided SAR optimization lead to the identification of 8-fluorobenzodiazepine (6c) with single digit nM potency (Aurora B IC 50 8 nM). In the antiproliferation assay 6c showed activity across the cell lines with IC 50 of 0.57, 0.42, and 0.69 µM for MCF-7, MDA-MB 231, and SkoV3 respectively. In the in vivo PK profile. 6c has shown higher bioavailability (73%) along with good exposure (AUC of 1360 ng.h/mL)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Filgotinib is a selective JAK1 (Janus kinase) inhibitor, showed efficacy in patients suffering from moderate-to-severe rheumatoid arthritis. In this paper, we present the data on the development and validation of a sensitive, selective and high-throughput LC-MS/MS (liquid chromatography with tandem mass spectrometry) method for the quantitation of filgotinib from rat dried blood spot (DBS) cards. To the DBS disc cards, 0.2% formic acid enriched with internal standard (IS) was added and sonicated. Thereafter the extraction of filgotinib and the IS (tofacitinib) was accomplished using ethyl acetate as an extraction solvent. The resolution of filgotinib and the IS was achieved on a Gemini C 18 column with an isocratic mobile phase, which is a mixture of 0.2% formic acid:acetonitrile (20:80, v/v) at a flow-rate of 0.9 mL/min. The total run time was 2.90 min and the retention time of filgotinib and the IS was ~1.31 and 0.89 min, respectively. Filgotinib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 426.3→291.3 and m/z 313.2→149.2, respectively, for quantitation. The calibration range was 1.37-1937 ng/mL. No matrix effect and carry over were observed. All the validation parameters met the acceptance criteria. The validated method has been applied to a pharmacokinetic study in rats. A good correlation between DBS and plasma concentrations for filgotinib was observed., Competing Interests: Conflict of interest: The authors are scientists at Jubilant Biosys Ltd., (Copyright © 2020 by the authors.)

Filgotinib is a selective JAK1 (Janus kinase) inhibitor, filed in Japan for the treatment of rheumatoid arthritis. In this paper, we present the data of development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of filgotinib in mice plasma as per the FDA regulatory guideline. The method involves the extraction of filgotinib along with internal standard (IS, tofacitinib) from mice plasma (100 µL) using ethyl acetate as an extraction solvent. The chromatographic analysis was performed using an isocratic mobile phase comprising 10 mM ammonium acetate (pH 4.5) and acetonitrile (70:30, v/v) at a flow-rate of 0.8 mL/min on a Hypersil Gold C 18 column. The UV detection wavelength was set at λ max 300 nm. Filgotinib and the IS eluted at 5.56 and 4.28 min, respectively with a total run time of 10 min. The calibration curve was linear over a concentration range of 0.05 to 5.00 μg/mL ( r 2+ =≥0.992). The intra- and inter-day precision and accuracy results were within the acceptable limits. Results of stability studies indicated that filgotinib was stable on bench-top, in auto-sampler, up to three freeze/thaw cycles and long-term storage at -80°C. The validated HPLC method was successfully applied to a pharmacokinetic study in mice., Competing Interests: All the authors have no conflict of interest to declare., (© Georg Thieme Verlag KG Stuttgart · New York.)

Ceramides are known to be involved in various biological processes with their physiological levels elevated in various disease conditions such as diabetes, Alzheimer's, atherosclerosis. To facilitate the rapid screening of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 inhibition in HepG2 cells, a RapidFire coupled to tandem mass spectrometry (RF-MS/MS) method has been developed. The RF platform provides an automated solid-phase extraction system that gave a throughput of 12.6 s per sample to an MS/MS system using electrospray ionization under the positive ion mode. Chromatographic separation of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 was achieved using a ternary gradient on C 8 type E cartridge. The MS/MS ion transitions monitored were 538.2 → 264.2, 650.7 → 264.2, 648.6 → 264.2, 566.4 → 264.2, 510.4 → 264.2, 594.4 → 264.2, 622.5 → 264.2, and 552.3 → 250.2 for Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, d18:1/22:0, and the internal standard (Cer d17:1/18:0), respectively. The RF-MS/MS methodology showed an excellent performance with an average Z' value of 0.5-0.7. This is the first report of an RF-MS/MS assay for screening of ceramides which is amenable for high-throughput screening., (© 2019 John Wiley & Sons, Ltd.)

Filgotinib is a selective JAK1 (Janus kinase) inhibitor, filed in Japan for the treatment of rheumatoid arthritis. In this paper, we report a validated liquid chromatography coupled with tandem mass spectrometry for the quantification of filgotinib in rat plasma using tofacitinib as an internal standard (IS) as per the Food and Drug Administration regulatory guidelines. Filgotinib and the IS were extracted from rat plasma using ethyl acetate as an extraction solvent and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 20:80, v/v) at a flow rate of 0.9 mL/min on a Gemini C 18 column. Filgotinib and the IS were eluted at ~1.31 and 0.89 min, respectively. The MS/MS ion transitions monitored were m/z 426.3 → 291.3 and m/z 313.2 → 149.2 for filgotinib and the IS, respectively. The calibration range was 0.78-1924 ng/mL. No matrix effect and carryover were observed. Intra- and inter-day accuracies and precisions were within the acceptance range. Filgotinib was stable for three freeze-thaw cycles: on bench-top up to 6 h, in an autosampler up to 21 h, and at -80°C for 1 month. This novel method has been applied to a pharmacokinetic study in rats., (© 2020 John Wiley & Sons, Ltd.)

Copanlisib is a pan phosphatidylinositol 3-kinase (PI3K) inhibitor approved for follicular lymphoma. In this paper, we present the data of development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of copanlisib in mice plasma as per the FDA regulatory guideline. The method involves the extraction of copanlisib along with internal standard (IS, enasidenib) from mice plasma (100 μL) using ethyl acetate as an extraction solvent. The chromatographic resolution of copanlisib and the IS was achieved on a Hypersil Gold C 18 column maintained at 40 °C using a binary gradient mobile phase [10 mM ammonium formate (pH 4.0) and acetonitrile]. The flow-rate was 0.8 mL/min. For the detection of copanlisib and the IS, the photo-diode array detector was set at λ max 310 nm. Copanlisib and the IS eluted at 6.60 and 7.80 min, respectively with a total run time of 10 min. The calibration curve was linear over a concentration range of 50 to 5000 ng/mL for copanlisib (r 2 ≥ 0.998). The results of intra- and inter-day accuracy and precision studies were within the acceptable limits. Copanlisib was stable on bench-top, in auto-sampler, up to three freeze/thaw cycle and long-term storage at -80 °C. The application of the validated method was shown in a mice pharmacokinetic study., Competing Interests: Conflict of interest: The authors are scientists at Jubilant Biosys Ltd., (Copyright © 2020 by the authors.)

Cefuroxime is one of the widely used antibiotics. The objective of this study was to determine pharmacokinetics and disposition in various ocular tissues following topical (TOP), intracameral (IC) and intravitreal (IVT) administration of cefuroxime to rabbits.Following TOP, IC and IVT dosing plasma and various ocular tissues (aqueous humor (AH), vitreous humor (VH), conjunctiva, trabecular mesh (TM), lens and retina-choroid (RC)) were collected and analyzed to understand the disposition of cefuroxime. Postintravenous administration plasma samples were collected to determine the systemic pharmacokinetics.Post-TOP dosing cefuroxime concentrations were observed only in conjunctiva up to 48 h. IC administration showed cefuroxime concentrations in AH up to 8 h; in conjunctiva, TM and plasma, the concentration lasted up to 4 h and in RC and VH till 1 h. IVT administration of cefuroxime showed concentrations in all ocular tissues (up to 8 h) and lasted up to 48 h except in conjunctiva and RC.There was evidence that the mechanism(s) of cefuroxime entry into the eye by via IVT, IC and TOP routes is clearly different. The present ocular tissue data may aid clinicians for considering appropriate choice in the treatment of post-operative ocular complications due to bacterial infections including endophthalmitis.

Objective: The objective of the current research was to prepare self-micro-emulsifying drug delivery systems (SMEDDS) for BCS class II drug, nilotinib to enhance its oral bioavailability. Methodology: Different types of excipients like oil, surfactant, and co-surfactant were evaluated for drug solubility. Among the screened excipients, Capryol 90, Transcutol HP, and Tween 80 were selected as oil, co-surfactant, and surfactant, respectively, to construct a ternary phase diagram to identify a homogenous mixture. Characterization performed for the prepared SMEDDS for its particle size/droplet size, emulsification time, phase separation, droplet morphology, in vitro drug release, and oral bioavailability. Results: Prepared SMEDDS showed the highest of 87% drug release in in vitro drug release experiment. SMEDDS drug release was superior over suspension formulation, which could be attributed to oil/surfactant ratios and particle size of the SMEDDS. The acquired pharmacokinetic parameters indicate that twofold increase in systemic exposure of SMEDDS compared with nilotinib suspension formulation. A similar twofold increase in relative oral bioavailability was also observed when compared SMEDDS formulation with suspension formulation. Delayed T max (time to reach peak plasma concentrations) was observed with SMEDDS over suspension formulation, which was evident by slow rate of absorption of nilotinib from SMEDDS. Conclusion: This research demonstrated that SMEDDS could be an effective approach to improve solubility and oral bioavailability for the BCS class II poorly soluble nilotinib.

Levodropropizine (LDP) is a non-opioid anti-tussive. The stereoselective pharmacokinetics and tissue distribution (TD) of LDP vs. dextrodropropizine (DDP) have been characterized after oral and intravenous (IV) administration of LDP and rac -dropropozine in rats.Oral/IV doses of 50/5.0 mg/kg and 25/2.5 rac -dropropizine and LDP were employed. TD study focused on tissues such as liver, lung and kidney. Blood samples were collected for pharmacokinetic and TD evaluation. Validated methods were used to quantitate LDP, DDP and rac -dropropizine.No stereoselectivity in pharmacokinetics was observed between LDP vs. DDP following rac -dropropizine. However, LDP pharmacokinetics after LDP administration (oral/IV) appeared to be different compared to LDP derived from rac -dropropizine.TD data were similar between the two enantiomers regardless of oral/IV rac -dropropizine administration. When LDP alone was administered, levels were comparable to those derived for LDP from rac -dropropizine after oral/IV. However, in the lung and kidney tissues, the exposure after oral dosing was higher for LDP alone as compared to LDP from rac -dropropizine.In summary, complete characterization of stereoselective pharmacokinetics and TD of rac -dropropizine has been reported after oral/IV routes. It was evident that the presence of DDP, increased the plasma/tissue exposure of LDP which was evident after oral rac -dropropizine dosing.

In this paper, we present the incurred sample reanalysis (ISR) data for cefuroxime in various ocular tissues of rabbits. Based on the cefuroxime concentration vs. time profile in various ocular tissues, three chosen time points enabled ISR assessment. Cefuroxime was quantitated in the ocular tissues using a published liquid chromatography-electrospray ionization/tandem mass spectrometry method operated under the multiple reaction-monitoring mode in positive ion mode. Regardless of the ocular tissue, the linearity range was 12.7-2760 ng/ml with a correlation coefficient (r 2 ) of ≥0.996. All of the ISR samples representing various ocular tissues met the acceptance criteria. To the best of our knowledge, this is the first report showing the ISR of ocular tissues in any species., (© 2019 John Wiley & Sons, Ltd.)

Larotrectinib, is an orally active novel small molecule approved for the treatment of solid tumors in pediatrics and adult patients. It acts by inhibiting tropomyosin receptor kinase. In this paper, we report the development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of larotrectinib in mice plasma as per the FDA regulatory guideline. Plasma samples processing was accomplished through simple protein precipitation using acetonitrile enriched with internal standard (IS, enasidenib). The chromatographic analysis was performed using a gradient mobile phase comprising 10 mM ammonium acetate and acetonitrile at a flow-rate of 0.8 mL/min on an X-Terra Phenyl column. The UV detection wave length was set at λ max 262 nm. Larotrectinib and the IS eluted at 3.85 and 6.60 min, respectively with a total run time of 8.0 min. The calibration curve was linear over a concentration range of 0.20-5.00 μg/mL (r 2 =≥0.992). The intra- and inter-day precision and accuracy results were within the acceptable limits. Results of stability studies indicated that larotrectinib was stable on bench-top, in auto-sampler, up to three freeze/thaw cycles and long-term storage at -80°C. The validated HPLC method was successfully applied to a pharmacokinetic study in mice., Competing Interests: There are no conflict of interests to declare in connection with the contents of this manuscript., (© Georg Thieme Verlag KG Stuttgart · New York.)

Enasidenib is a selective mutant isocitrate dehydrogenase 2 inhibitor approved for the treatment of relapsed and refractory acute myeloid leukemia patients. A sensitive and rapid method has been developed and validated as per regulatory guideline for the simultaneous quantitation of enasidenib and its active metabolite, AGI-16903 in mice plasma using an LC-MS/MS. Enasidenib and AGI-16903 along with internal standard were extracted from mice plasma using simple protein precipitation method. Chromatographic resolution of enasidenib, AGI-16903 and the internal standard (close analogue of AGI-16903) was achieved on a Chromolith RP-18e column using 0.2% formic acid:acetonitrile (15:85, v/v) as an eluent, which was delivered at a flow-rate of 1.2 mL/min. The MS/MS ion transitions monitored were m/z 474.1→267.2, 402.1→188.1 and 421.0→146.1 for enasidenib, AGI-16903 and the internal standard, respectively. The linearity range was 1.01-3023 ng/mL for both enasidenib and AGI-16903. The within-run and between-run accuracy and within-run and between-run precision were in the range of - 2.29 to 2.72 (as one value is in negative side). and 4.65-9.82%, respectively for enasidenib; 0.19-10.3 and 3.22-9.22%, respectively for AGI-16903. Both enasidenib and AGI-16903 were found to be stable in stability (up to three freeze-thaw cycles and for long-term at -80°C for 30 days) and processed (bench-top for 6 h and in in-injector for 24 h) samples. Application of the validated method was shown in a pharmacokinetic study in mice., Competing Interests: The authors declare no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Quantitation of drugs used for the treatment of chronic lymphocytic leukemia in various biological matrices during both pre-clinical and clinical developments is very important, often in routine therapeutic drug monitoring. The first developed methods for quantitation were traditionally done on LC in combination with either UV or fluorescence detection. However, the emergence of LC with mass spectrometry in tandem in early 1990s has revolutionized the quantitation as it has provided better sensitivity and selectivity within a shorter run time; therefore it has become the choice of method for the analysis of various drugs. In this article, an overview of various bioanalytical methods (HPLC or LC-MS/MS) for the quantification of drugs for the treatment of chronic lymphocytic leukemia, along with applicability of these methods, is given., (© 2019 John Wiley & Sons, Ltd.)

Isocitrate dehydrogenase (IDH) inhibitors comprise a novel class of anticancer drugs, which are approved to treat acute myeloid leukemia patients having mutations on IDH1/2. We report the development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous quantitation of IDH inhibitors, namely enasidenib (EDB), ivosidenib (IDB) and vorasidenib (VDB), in mouse plasma as per the US Food and Drug Administration regulatory guidelines. The method involves extraction of EDB, IDB and VDB along with internal standard (IS; phenacetin) from mouse plasma (100 μl) using a simple protein precipitation process. The chromatographic analysis was performed on an HPLC system using a gradient mobile phase (comprising 10 mm ammonium acetate and acetonitrile in a flow-gradient) and an X-Terra Phenyl column. The UV detection wave length was set at λ max 265 nm. EDB, IDB, VDB and the IS eluted at 7.36, 8.60, 9.50 and 5.12 min, respectively, with a total run time of 10 min. The calibration curve was linear over a concentration range of 0.20-12.5 μg/ml for EDB and 0.50-12.5 μg/ml for IDB and VDB (r 2  = ≥0.998 for all of the analytes). Validation results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in mice., (© 2019 John Wiley & Sons, Ltd.)

A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of tunicamycin in rat plasma as per regulatory guideline. Chromatography of tunicamycin and the IS in the processed plasma samples was achieved on an X-Terra phenyl column using a binary gradient (mobile phase A, acetonitrile and mobile phase B, 5 mm ammonium formate) elution at a flow rate of 0.6 ml/min. LC-MS/MS was operated under the multiple reaction monitoring mode using the electrospray ionization technique in positive ion mode and the transitions of m/z 817.18 → 596.10, 831.43 → 610.10, 845.29 → 624.10, 859.23 → 638.10 and 309.24 → 163.20 were used to quantitate homologs A-D and the IS, respectively. The total chromatographic run time was 4.5 min. The correlation coefficient (r 2 ) was >0.99 for all homologs with accuracy 90.7-107.4% and precision 0.74-15.1%. The recovery of homologs was 78.6-90.2%. No carryover was observed and the matrix effect was minimal. Tunicamycin four homologs were found to be stable on the bench-top for 6 h, for up to three freeze-thaw cycles, in the injector for 24 h and for 1 month at -80°C. The applicability of the validated method has been demonstrated in a rat pharmacokinetic study., (© 2019 John Wiley & Sons, Ltd.)