Unveiling Pharmacokinetics and Drug Interaction of Linagliptin and Pioglitazone HCl in Rat Plasma Using LC-MS/MS.

The linagliptin (LIN) and pioglitazone HCl (PIO) combination, currently undergoing phase III clinical trials for diabetes mellitus treatment, demonstrated significant improvements in glycemic control. However, the absence of an analytical method for simultaneous determination in biological fluids highlights a crucial gap. This underscores the pressing need for sensitive bioanalytical methods, emphasizing the paramount importance of developing such tools to advance diabetes management strategies and enhance patient care. Herein, a sensitive reverse-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for simultaneous determination of LIN and PIO in rat plasma using alogliptin as an internal standard. Chromatographic separation was performed on an Agilent Eclipse Plus C18 (4.6 × 100 mm, 3.5 µm) using an isocratic mobile phase system consisting of ammonium formate (pH 4.5) and methanol using an acetonitrile-induced protein precipitation technique for sample preparation. Multiple reaction monitoring in positive ion mode was used for quantitation of the precursor to production at m/z 473.2 → 419.9 for LIN, 357.1 → 134.2 for PIO, and 340.3 → 116.1 for ALO. The linearity range was 0.5 to 100 and 1 to 2000 ng/mL for LIN and PIO, respectively. The developed method was validated as per US-FDA guidelines and successfully applied to clinical pharmacokinetic and drug-drug interaction studies with a single oral administration of LIN and PIO in rat plasma. Pharmacokinetic parameters of LIN were significantly influenced by the concomitant administration of PIO and vice versa. Molecular modeling revealed the significant interaction of LIN and PIO with P-glycoprotein. Therefore, the drug-drug interaction between LIN and PIO deserves further study to improve drug therapy and prevent dangerous adverse effects.

2-Phenylquinazolin-4(3H)-one scaffold as newly designed, synthesized VEGFR-2 allosteric inhibitors with potent cytotoxicity through apoptosis.

New derivatives of 2-phenylquinazolin-4(3H)-one were designed, synthesized, and biologically evaluated as potent allosteric kinase inhibitors with in situ cytotoxicity against MCF-7 and HepG2 cells. Compounds 15 and 18 among the proposed compounds showed promising antiproliferative activity against MCF-7 (IC50 = 1.35 µM) and HepG2 cells (IC50 = 3.24 µM), comparable to sorafenib, with IC50 values of 3.04, 2.93 µM, respectively, according to in situ cytotoxicity testing. Comparing compounds 15 and 18 to sorafenib, the in vitro VEGFR-2 inhibitory activity displayed encouraging selective efficacy with IC50 values of 13, 67, and 30 nM, respectively. Results of VEGFR-2 inhibition at various ATP concentrations proved that there was no statistically significant difference between the IC50 values, which improved the non-ATP competitive binding. Compound 15 caused apoptotic breast cancer cell death with 55.11-fold cell-cycle arrest at the S-phase, where it affected the apoptosis-mediated genes through upregulating P53, Bax, caspases 3, 8, and 9 and downregulating the antiapoptotic gene Bcl-2. A molecular docking study was conducted to confirm the binding of the designed compounds to the allosteric site of VEGFR-2 in DFG-out mode, leaving the ATP-binding pocket unoccupied when superimposed to the pose of sorafenib. The designed molecules showed resealable binding affinity toward the DFG loop and the allosteric site. Hence, the 2-phenylquinazolin-4(3H)-one derivative constitutes intriguing starting points for designing apoptotic-inducing drugs.

New Antiproliferative Triflavanone from Thymelaea hirsuta-Isolation, Structure Elucidation and Molecular Docking Studies.

In this study isolates from Thymelaea hirsuta, a wild plant from the Sinai Peninsula of Egypt, were identified and their selective cytotoxicity levels were evaluated. Phytochemical examination of the ethyl acetate (EtOAc) fraction of the methanolic (MeOH) extract of the plant led to the isolation of a new triflavanone compound (1), in addition to the isolation of nine previously reported compounds. These included five dicoumarinyl ethers found in Thymelaea: daphnoretin methyl ether (2), rutamontine (3), neodaphnoretin (4), acetyldaphnoretin (5), and edgeworthin (6); two flavonoids: genkwanin (7) and trans-tiliroside (8); p-hydroxy benzoic acid (9) and ß sitosterol glucoside (10). Eight of the isolated compounds were tested for in vitro cytotoxicity against Vero and HepG2 cell lines using a sulforhodamine-B (SRB) assay. Compounds 1, 2 and 5 exhibited remarkable cytotoxic activities against HepG2 cells, with IC50 values of 8.6, 12.3 and 9.4 µM, respectively, yet these compounds exhibited non-toxic activities against the Vero cells. Additionally, compound 1 further exhibited promising cytotoxic activity against both MCF-7 and HCT-116 cells, with IC50 values of 4.26 and 9.6 µM, respectively. Compound 1 significantly stimulated apoptotic breast cancer cell death, resulting in a 14.97-fold increase and arresting 40.57% of the cell population at the Pre-G1 stage of the cell cycle. Finally, its apoptosis-inducing activity was further validated through activation of BAX and caspase-9, and inhibition of BCL2 levels. In silico molecular docking experiments revealed a good binding mode profile of the isolates towards Ras activation/pathway mitogen-activated protein kinase (Ras/MAPK); a common molecular pathway in the development and progression of liver tumors.

Jaceidin Flavonoid Isolated from Chiliadenus montanus Attenuates Tumor Progression in Mice via VEGF Inhibition: In Vivo and In Silico Studies.

Phytochemical study of Chiliadenus montanus aerial parts afforded six compounds; Intermedeol (1), 5α-hydroperoxy-ß-eudesmol (2), 5,7-dihydroxy-3,3',4'-trimethoxyflavone (3), 5,7,4'-trihydroxy-3,6,3'-trimethoxyflavone (jaceidin) (4), eudesm-11,13-ene-1ß,4ß,7α-triol (5) and 1ß,4ß,7ß,11-tetrahydroxyeudesmane (6). These compounds were identified based on their NMR spectral data. The isolated compounds were tested for their cytotoxicity against liver cancer cell line (HepG2) and breast cancer cell line (MCF-7). Jaceidin flavonoid (4) exhibited the highest cytotoxic effect in vitro. Therefore, both of jaceidin and C. montanus extract were evaluated for their in vivo anti-tumor activity against Ehrlich's ascites carcinoma (EAC). Compared to control group, jaceidin and C. montanus extract decreased the tumor weight, improved the histological picture of tumor cells, lowered the levels of VEGF and ameliorate the oxidative stress. Molecular docking and in silico studies suggested that jaceidin was a selective inhibitor of VEGF-mediated angiogenesis with excellent membrane permeability and oral bioavailability.

Pharmacophore modeling, 3D-QSAR, synthesis, and anti-lung cancer evaluation of novel thieno[2,3-d][1,2,3]triazines targeting EGFR.

Two series of thieno[2,3-d][1,2,3]triazine derivatives were designed, synthesized, and biologically evaluated as potential epidermal growth factor receptor (EGFR) inhibitors targeting the non-small-cell lung cancer cell line H1299. Most of the synthesized compounds displayed IC50 values ranging from 25 to 58 nM against H1299, which are superior to that of gefitinib (40 µM). 3-(5,6,7,8-Tetrahydro-7H-cyclohexa[4:5]thieno[2,3-d]-1,2,3-triazin-4-ylamino)benzene-1,3-diamine (6b) achieved the highest cytotoxic activity against H1299 with an IC50 value of 25 nM; it had the ability to decrease the EGFR concentration in H1299 cells from 7.22 to 2.67 pg/ml. In vitro, the IC50 value of compound 6b was 0.33 nM against EGFR, which is superior to that of gefitinib at 1.9 nM and erlotinib at 4 nM. The three-dimensional quantitative structure-activity relationships and molecular modeling studies revealed comparable binding modes of compound 6b, gefitinib, and erlotinib in the EGFR active site. The in silico ADME (absorption, distribution, metabolism, and excretion) prediction parameters of this compound revealed promising pharmacokinetic and physicochemical properties. Moreover, DFT (density functional theory) calculations showed the high reactivity of compound 6b toward the EGFR compared with other compounds. The designed compound 6b might serve as an encouraging lead compound for the discovery of promising anti-lung cancer agents targeting EGFR.

Design, synthesis, biological evaluation and molecular modeling study of new thieno[2,3-d]pyrimidines with anti-proliferative activity on pancreatic cancer cell lines.

Pancreatic cancer is one of the most challenging diseases with seven months only as median survival time due to its poor prognosis. Several enzymes are blamed for the progress of pancreatic cancer especially, platelet-derived growth factor receptors (PDGFRs), this in turn makes them promising targets for its treatment. In this study, twenty eight new compounds based on thieno[2,3-d]pyrimidine scaffold were synthesized as anti-pancreatic cancer agents mimicking the benzofuro[3,2-d]pyrimidine derivative, amuvatinib. Various linkers including amides, esters, ketones, urea and thiourea derivatives were utilized to study their effect on the anti-proliferative activity of these compounds. Most of the tested compounds revealed good cytotoxic activities against pancreatic carcinoma cell line PANC-1. Compound 9d showed the highest cytotoxicity with an IC50 value of 5.4 µM. Furthermore, 9d showed excellent platelet derived growth factor receptor (PDGFR-α) inhibitory activity, with IC50 value 0.155 µM. Docking study was carried out into PDGFR-α active site which showed comparable binding mode to that of FDA approved PDGFR-α inhibitor, imatinib. 3D-Quantitative structure activity relationship (QSAR) model was built up with five-featured pharmacophore which could be implemented for emerging effective lead structures. These compounds could serve as a new chemotype for discovering new agents for pancreatic cancer therapy.

Simple and rapid HPLC method for simultaneous determination of atenolol and chlorthalidone in spiked human plasma.

A simple, sensitive and rapid chromatographic method was developed and validated for the simultaneous quantification of atenolol and chlorthalidone in human plasma using hydrochlorothiazide as internal standard (IS). The method utilized proteins precipitation with acetonitril as the only sample preparation involved prior to reverse phase-HPLC. The analytes were chromatographed on Shim-pack cyanopropyl column with isocratic elution with 10 mM KH2PO4 (pH 6.0) - methanol (70:30, v/v) at ambient temperature with flow rate of 1 mL min(-1) and UV detection at 225 nm. The chromatographic run time was less than 10 min for the mixture. The calibration curves were linear over the range of 0.1-10 µg mL(-1). The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The within- and between-day accuracy and precision were found to be within acceptable limits <15%. The analytes were stable after three freeze-thaw cycles (deviation <15%). The proposed method was specific for the simultaneous determination of atenolol and chlorthalidone in human plasma where there was no interference from endogenous biological substances.

Development and validation of an LC method for simultaneous determination of amiloride hydrochloride and hydrochlorothiazide in human urine.

An HPLC method with photometric detection has been developed for determination of a binary mixture of amiloride hydrochloride and hydrochlorothiazide in human urine using chlorthalidone as the internal standard. Reversed-phase chromatography was performed at room temperature on a cyanopropyl column with the mobile phase consisting of a 10 mM KH2PO4 solution (pH 4.5)-methanol (70 + 30, v/v) at a flow rate of 1 ml/min. The detector was set at 214 nm. The total analysis time was 10 min. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability, and re-injection reproducibility. The procedure shows good accuracy, repeatability, and selectivity. Moreover, the method was applied directly to urine that had not undergone prior treatment. The intra- and interday coefficients of variation for all compounds were below 4%, and the method was highly accurate, with a relative error for all compounds that was below 8%. No interference from endogenous compounds in urine samples was found. The proposed method, which is rapid, simple, and does not require any separation steps, has been successfully applied to the assay of human urine containing amiloride hydrochloride and hydrochlorothiazide.

Development and validation of a reversed-phase high-performance liquid chromatographic method for the simultaneous determination of amiloride hydrochloride, atenolol, hydrochlorothiazide, and chlorthalidone in their combined mixtures.

A high-performance liquid chromatographic method was developed for the simultaneous determination of 2 ternary mixtures containing amiloride hydrochloride, atenolol, hydrochlorothiazide, and chlorthalidone used in hypertension therapy. The use of cyanopropyl column results in satisfactory separation of both mixtures. The mobile phase consisted of 10 mM KH2PO4 buffer (pH 4.5) and methanol in a ratio of (75 + 25% v/v), at a flow rate of 1 mL/min. UV detector was operated at 275 nm. Calibration graphs were linear in the concentration ranges of 2-10, 20-200, 10-100, and 5-50 microg/mL for amiloride hydrochloride, atenolol, hydrochlorothiazide, and chlorthalidone, respectively. Intraday and interday precision values (relative standard deviation) were <1.13 for mixture I (amiloride hydrochloride, atenolol, chlorthalidone), and <0.93 for mixture II (amiloride hydrochloride, atenolol, hydrochlorothiazide). The method was successfully applied for the determination of the 2 combinations in laboratory-prepared mixtures and commercial pharmaceutical formulation with high accuracy and precision. Statistical comparison of the results with those of the published methods showed excellent agreement and indicates no significant difference between them.