Preclinical pharmacokinetic exploration of a novel osteoporotic quinazolinone-benzopyran-indole hybrid (S019-0385) using LC-MS/MS.

1. The current investigation was to develop and validate the LC-MS/MS method in order to analyse the various pharmacokinetic parameters of S019-0385. A sensitive, selective, and robust LC-MS/MS approach was established and validated for measuring S019-0385 in female mice plasma and tissue, using optimal multiple reaction monitoring (MRM) transition m/z 488.25/329.12 on positive mode. On a Waters Symmetry Shield C18 column, the analyte was separated using acetonitrile and deionised water with formic acid within 6 min at 0.7 mL/min. Linearity (R2 ≥ 0.99) was observed across 0.195-100 ng/mL concentration range using linear least-squares regression.2. Blood-to-plasma ratio and plasma protein drug binding (%) in mice and human was assessed and found to be less than 1 and >83%, respectively. Absolute bioavailability (%F) of S019-0385 in female Swiss mice was exhibited to be 6.90%. Percent dose excreted S019-0385 in unchanged form through urine and faecal was found to be less than 2% and 0.5%, respectively.3. Following oral administration at 5 mg/kg, the concentration of S019-0385 in tissue distribution was found to be in the order of C small intestine > C bone > C lung > C spleen > C kidney > C liver > C heart > C brain.

Hesperidin: Enrichment, forced degradation, and structural elucidation of potential degradation products using spectral techniques.

RATIONALE: Hesperidin (HES) is a well-known citrus bioflavonoid phyto-nutraceutical agent with polypharmacological properties. After 2019, HES was widely used for prophylaxis and COVID-19 treatment. Moreover, it is commonly prescribed for treating varicose veins and other diseases in routine clinical practice. Pharmaceutical impurities and degradation products (DP) impact the drug's quality and safety and thus its effectiveness. Therefore, forced degradation studies help study drug stability, degradation mechanisms, and their DPs. This study was performed because stress stability studies using detailed structural characterization of hesperidin are currently unavailable in the literature. METHODS: In the HES enrichment method crude HES was converted to its pure form (98% purity) using column chromatography and then subjected to forced degradation under acid, base, and neutral hydrolyses followed by oxidative, reductive, photolytic, and thermal stress testing (International Conference on Harmonization guidelines). The stability-indicating analytical method (SIAM) was developed to determine DPs using reversed-phase high-performance liquid chromatography (C18 column with methanol and 0.1% v/v acetic acid in deionized water [70:30, v/v] at 284 nm). Further, structural characterization of DPs was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. In addition, in silico toxicity predictions were performed using pKCSM and DataWarior freeware. RESULTS: HES was found to be susceptible to acidic and basic hydrolytic conditions and yielded three DPs in each, which were detected using designed SIAM. Of six DPs, three were pseudo-DPs (short lived), and the remaining were characterized using LC-MS/MS and NMR spectroscopy. The tentative mechanism of the formation of proposed DPs was explained. The proposed DPs were found inactive from in silico toxicity predictions. CONCLUSIONS: Hesperidin was labile under acidic and basic stress conditions. The potential DPs were characterized using LC-ESI-MS/MS and NMR spectral techniques. The proposed mechanism of formation was hypothesized. In addition, to identify and characterize the DPs, a SIAM, which has broad biomedical applications, was successfully developed.

LC-MS/MS based quantification of steroidal biomarkers in polycystic ovary syndrome induced rats.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder that causes reproductive hormones imbalance, missed periods, infertility and distributed steroidogenesis. Reportedly, during PCOS, the endogenous levels of P4 (Progesterone), 17OHP4 (17-α hydroxy progesterone), and T4 (Testosterone) were significantly altered. Thus, quantification of steroid biomarkers involved in the steroidogenesis pathway of PCOS, such as P4, 17OHP4, and T4, holds significant importance. One important drawback of current methods is steroid metabolome traceability. Without adequate traceability, the findings of these techniques will be less reliable for identifying P4, 17OHP4, and T4. These methods also need a high sample size, especially for the most important biomarker that initiates steroidogenesis. To address these challenges, we require a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for steroid biomarker analysis. Herein the present work, using validated LC-MS/MS, PCOS biomarkers were measured and compared between normal control rats and PCOS-induced rats before and after analyte administration. The experiment utilized an isocratic separation method employing an analytical C18 column. The mobile phase consisted of acetonitrile (ACN) and aqueous 0.1% formic acid (FA) in a ratio of 90:10 (v/v). The plasma samples were processed with protein precipitation (PPT) followed by the liquid-liquid extraction (LLE) method. The lower limit of quantification (LLOQ) was 0.5 ng/mL in plasma. According to USFDA criteria, the method's systematic validation took into account linearity (r2 > 0.99), accuracy and precision of intra- and inter-batch measurements, stability, biomarker recovery (60-85%) and matrix effect (<± 15%), all of which were determined to be within range ( ± 15%). The pharmacokinetic data showed that, as compared to normal rats, PCOS-induced animals had significantly higher Cmax values for 17OHP4 and T4 (∼2 fold), while lower Cmax values for P4 (∼2 fold). The present work is novel and provides scientific information to explore systematic processes involved in steroidogenesis and boost clinical applicability for PCOS therapy.

Preclinical pharmacokinetics of 4-hydroxy isoleucine using LC-MS/MS: a potential polycystic ovary syndrome phytopharmaceutical therapeutics.

Aim: To study the preclinical pharmacokinetics of 4-hydroxy isoleucine (4-HIL) targeted for polycystic ovary syndrome. Methodology: The quantitative bioanalysis of 4-HIL in different biological matrices in female Sprage-Dawley rats using LC-MS/MS. Results: At 50 mg/kg, 4-HIL had 56.8% absolute oral bioavailability. It was quickly absorbed and distributed in various tissues in order of small intestine > kidney > ovary > spleen > lung > liver > heart > brain after oral administration. Moreover, 11.07% of 4-HIL was recovered in urine and feces within 72 h. Conclusion: 4-HIL levels in vital organs were found safe, as per tissue distribution results. Hence, 4-HIL could be used as promising therapeutics for management of polycystic ovary syndrome.

Simultaneous pharmacokinetic assessment of phytopharmaceuticals in fenugreek extract using LC-MS/MS in Sprague-Dawley rats.

Fenugreek seeds are used in numerous marketed herbal formulations with therapeutic benefits. Some of its bioactive components such as 4-hydroxyisoleucine, trigonelline, raffinose, and pinitol are reported to possess potential therapeutic activities, such as antibacterial, antidiabetic, stomach stimulant, and anti-invasive, against hyperandrogenism and other allied diseases, including polycystic ovary syndrome. A fully validated, selective, and sensitive bioanalytical method for the simultaneous rapid quantification of the aforementioned bioactive components has been developed using hyphenated liquid chromatography electrospray tandem mass spectrometry. The analytes were separated within 5 min using gradient elution in a C18 column at a flow rate of 0.5 ml/min. Plasma protein precipitation technique was employed to isolate the analytes from the samples. Oral pharmacokinetic profile of the four bioactive components in Sprague-Dawley rats was further evaluated using noncompartmental analysis using Phoenix WinNonlin software.

Preclinical pharmacokinetics, CYP phenotyping, and tissue distribution study of novel anti-breast cancer candidate S-011-1559.

S-011-1559 is a tyrosine-derived novel benzoxazine CDRI molecule targeted to the oestrogen-related receptor (ER-α/ß) modulator in breast cancer. To explore the pharmacokinetics of S-011-1559, a selective and sensitive bioanalytical method using LC-MS/MS was established and validated in different biological matrices of female rats.Blood-to-plasma ratio and plasma protein binding (PPB) of S-011-1559 were found to be <1 and >97% in both rats and humans, respectively. The human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) binding was found in the range of > 68 to 45% and >14% respectively. Half-life and intrinsic clearance by microsomal stability study were found to be 28.83 min and 0.05 mL/min/mg in rats, 78.35 min and 0.036 mL/min/mg in humans, respectively. The IC50 value of S-011-1559 against CYP isoforms was revealed to moderately inhibit CYP2D6 by a reversible non-competitive mechanism.Tissue distribution of S-011-1559 on single intravenous injection at 2 mg/kg was found in the order of C lungs > C mammary gland > C spleen > C heart > C kidney > C liver > C brain.The data from the present study provides crucial information about S-011-1559 for further development as a novel potential drug candidate in modulating ER-α/ß receptors of lung and breast neoplasia.