Discovery of new 2-(3-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)thiazole derivatives with potential analgesic and anti-inflammatory activities: In vitro, in vivo and in silico investigations.

Joining the global demand for the discovery of potent NSAIDs with minimized ulcerogenic effect, new pyrazole clubbed thiazole derivatives 5a-o were designed and synthesized. The new derivatives were initially evaluated for their analgesic activity. Eight compounds 5a, 5c, 5d, 5e, 5f, 5h, 5m, and 5o showed higher activity than Indomethacin (potency = 105-130 % vs. 100 %). Subsequently, they were picked for further evaluation of their anti-inflammatory activity, ulcerogenic liability as well as toxicological studies. Derivatives 5h and 5m showed a potential % edema inhibition after 3 h (79.39 % and 72.12 %, respectively), with a promising safety profile and low ulcer indices (3.80 and 3.20, respectively). The two compounds 5h and 5m were subjected to in vitro COX-1 and COX-2 inhibition assay. The candidate 5h showed nearly equipotent COX-1 inhibition (IC50 = 38.76 nM) compared to the non-selective reference drug Indomethacin (IC50 = 35.72 nM). Compound 5m expressed significant inhibitory activities and a higher COX-2 selectivity index (IC50 = 87.74 nM, SI = 2.05) in comparison with Indomethacin (SI = 0.52), with less selectivity than Celecoxib (SI = 8.31). Simulation docking studies were carried out to gain insights into the binding interaction of compounds 5h and 5m in the vicinity of COX-1 and COX-2 enzymes that illustrated the importance of pyrazole clubbed thiazole core in hydrogen bonding interactions. The thiazole motif of compounds 5h and 5m exhibited a well orientation toward COX-1 Arg120 key residue by hydrogen bonding interactions. Compound 5h revealed an additional arene-cation interaction with Arg120 that could rationalize its superior COX-1 inhibitory activity. Compounds 5h and 5m overlaid the co-crystallized ligand Celecoxib I differently in the active site of COX-2. Compound 5m showed an enhanced accommodation with binding energy of - 6.13 vs. - 1.70 kcal/mol of compounds 5h. The naphthalene ring of compound 5m adopted the Celecoxib I benzene sulfonamide region that is stabilized by hydrogen-arene interactions with the hydrophobic sidechains of the key residues Ser339 and Phe504. Further, the core structure of compound 5m, pyrazole clubbed thiazole, revealed deeper hydrophobic interactions with Ala513, Leu517 and Val509 residues. Finally, a sensitive and accurate UPLC-MS/MS method was developed for the simultaneous estimation of some selected promising pyrazole derivatives in rat plasma. Accordingly, compounds 5h and 5m were suggested to be promising potent analgesic and anti-inflammatory agents with improved safety profiles and a novel COX isozyme modulation activity.

Insight on novel oxindole conjugates adopting different anti-inflammatory investigations and quantitative evaluation.

Background: A dual COX/5-LOX strategy was adopted to develop new oxindole derivatives with superior anti-inflammatory activity. Methods: Three series of oxindoles - esters 4a-p, 6a-l and imines 7a-o - were synthesized and evaluated for their anti-inflammatory and analgesic activities. Molecular docking and predicted pharmacokinetic parameters were done for the most active compounds. A new LC-MS/MS method was developed and validated for the quantification of 4h in rat plasma. Results: Compounds 4h, 6d, 6f, 6j and 7m revealed % edema inhibition up to 100.00%; also, 4l and 7j showed 100.00% writhing protection. Compound 4h showed dual inhibitory activity with IC50 = 0.0533 and 0.4195 µM for COX-2 and 5-LOX, respectively. Molecular docking rationalized the obtained biological activity. The pharmacokinetic parameters of 4h from rat plasma were obtained.

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Exploring new imines as anti-inflammatory COX and 5-LOX inhibitors with an improved pharmacokinetic profile.

Background: Dual COX/5-LOX inhibition is a bright strategy for developing new potent and safe anti-inflammatory agents. Methods: New imines were synthesized and evaluated for their anti-inflammatory activity. The most active compounds were further investigated for their safety profile. Their molecular docking and physicochemical parameters were assessed. A new LC-MS/MS method was developed for the quantification of compound 4d in rat plasma. Results: Synthesized compounds were found to have anti-inflammatory activity (77-88% edema inhibition). In addition, 4d, 5m and 7d showed analgesic activity (92.50, 95.71 and 96.28% protection, respectively). 4d showed dual COX-2/5-LOX activity. Molecular docking expected the binding pattern of compounds in COX-1, COX-2 and 5-LOX active sites. The in vivo pharmacokinetic parameters of compound 4d were also obtained.

Plackett-Burman and face-centered composite designs for development and optimization of chromatographic method for the simultaneous determination of glycopyrronium, indacaterol and mometasone in their fixed dose combination inhaler - Green profile assessment.

A novel simple, specific, sensitive, accurate and precise reversed phase high performance liquid chromatographic method (RP-HPLC/UV) was developed and validated for the simultaneous estimation of Glycopyrronium bromide (GLY), Indacaterol acetate (IND) and Mometasone furoate (MOF) in pure form, in laboratory prepared mixtures and in pharmaceutical dosage form. Experimental design methodology was applied by using Plackett-Burman and face-centered composite designs to achieve the best resolution with minimum experimental trials. The designed model was statistically analyzed, graphically presented by surface plots and the relationships between coefficients of the derived polynomial equations were interpreted. Chromatographic separation was achieved on Inertsil ODS C18 column (250 ×4.6 mm, 5 µm) at ambient temperature using a mobile phase composed of methanol: 0.1% glacial acetic acid (pH4) in a gradient elution at a flow rate 1 mL /min. UV detection was carried out at 233 nm. Response was found to be linear in the concentration range of 20-120 µg /mL with regression coefficient (r2 = 0.999) for GLY, 50-300 µg /mL with regression coefficient (r2 = 0.9995) for IND and 50-300 µg /mL with regression coefficient (r2 = 0.9998) for MOF. The method was validated as per ICH guidelines and satisfactory results were achieved. The method was successfully applied for the analysis of the cited drugs in their fixed dose combination (FDC) pharmaceutical formulation. Statistical comparison between the results obtained by the proposed method and the reference methods for GLY, IND and MOF showed no significant difference. The developed method could be implemented in quality control aspects of the cited drugs. Four green metrics were used to evaluate the new RP-HPLC/UV method's greenness and compare it to other published techniques.

Development and Bio-Analytical Validation of Chromatographic Determination Method of Rufinamide in Presence of its Metabolite in Human Plasma.

Rufinamide (RF), antiepileptic drug, is biotransformed to inactive metabolite. Frequent plasma monitoring is required for dose adjustment. This work is concerned with the development and validation of a sensitive and selective RP-HPLC method for the quantitative determination of RF in spiked human plasma in the presence of its main metabolite. Lacosamide was selected as internal standard. Preparation of plasma samples involved precipitation of plasma proteins using methanol. Isocratic elution mode was applied and the chromatographic separation was performed on Prontosil CN column (5 µm, 250 × 4.6 mm). Good resolution was achieved using acetonitrile: water (10:90, v/v, adjusted with 0.01 N aqueous solution of o-phosphoric acid to pH = 3) as a mobile phase at a flow rate of 1 mL/min, and UV detection was carried out at 210 nm. Linearity was observed over the concentration range of 0.5-50 µg/mL of RF in plasma. Bio-analytical validation of the developed method was carried out in accordance to the European Medicines Agency guidelines. The accuracy ranged from 95.97 to 114.13%, and the coefficient of variation of the assay intra-day and inter-day precision did not exceed 10%. The samples were stable under the employed experimental conditions. In conclusion, the findings of the present study revealed its usefulness for therapeutic drug monitoring, assessment of drug pharmacokinetics and application for bioequivalence study.

Determination of Rufinamide in the Presence of 1-[(2,6-Difluorophenyl)Methyl]-1H-1,2,3-Triazole-4 Carboxylic Acid Using RP-HPLC and Derivative Ratio Methods as Stability Indicating Assays to Be Applied on Dosage Form.

BACKGROUND: Rufinamide is a triazole derivative that is structurally dissimilar to other marketed antiepileptic drugs, has been assumed a marketing authorization, by the European Union and FDA, for use as a complementary therapy for seizures associated with Lennox-Gastaut syndrome. OBJECTIVE: This work is concerned with development of two methods for determination of rufinamide (RUF) in presence of 1-[(2,6-difluorophenyl)methyl]-1H-1,2,3-triazole-4 carboxylic acid as its alkaline degradation product in dosage form. METHODS: The first method was capable of determing RUF in the presence of its alkaline degradation product and in dosage form. Kromasil C8 column and mobile phase consisting of acetonitrile-water (50:50, v/v) were used and UV detection at 210 nm. In the second method, first derivative ratio spectrophotometry, RUF was determined by measuring peak amplitude at 269.5 nm over 5-30 µg/mL. RESULTS: The linearity range of RUF was 10-90 µg/mL for HPLC method covering its therapeutic range with r2 = 0.9999. Forced degradation under alkaline conditions was carried out, the degradation product was isolated and its structure was confirmed. Both methods were validated in accordance to ICH guidelines. Statistical analysis revealed no significant difference between obtained results and reported ones. CONCLUSION: The present study is useful for therapeutic drug monitoring and routine analysis of RUF in quality control laboratories. HIGHLIGHTS: Kinetics of the alkaline degradation of RUF was studied by following the concentration of the remaining drug until complete degradation was achieved.

Novel spectrophotometric methods for the determination of Leflunomide and Diacerein in binary mixtures.

Two novel spectrophotometric methods were presented in this work using ethanol as a solvent. The first method was the ratio difference spectrophotometric method [RDSM], in which the amplitude difference between two selected wavelengths on the ratio spectra were recorded and used for estimation of each of Leflunomide LEF in mixture with its alkaline induced degradate DEG and also for Diacerein DIA determination in mixture with Aceclofenac ACEC without interference from the other component in the mixture. The second method is the ratio subtraction coupled with constant multiplication [RS-CM], where LEF was determined in its mixture with its alkaline degradate DEG at 261 nm which is considered as a stability indicating assay. In addition to simultaneous determination of Diacerein DIA and Aceclofenac ACEC in their mixtures at 257 and 277 nm, respectively, by the second method without previous separation. Linearity was shown over the concentration range of [1.5-15 µg/ml] for LEF, [1-11 µg/ml] for DIA and [2.5-25 µg/ml] for ACEC, by both proposed methods. Leflunomide was found to be completely degraded when subjected to alkaline degradation producing one alkaline product. Validation of the suggested methods was conducted according to ICH guidelines, concerning precision, accuracy, repeatability. The suggested spectrophotometric methods were statistically compared to reference methods showing no significant difference. The suggested spectrophotometric methods are considered to be simple, sensitive and could be easily applied in quality control laboratories instead of LC methods.

Simultaneous ultraperformance liquid chromatography/tandem mass spectrometry determination of four antihypertensive drugs in human plasma using hydrophilic-lipophilic balanced reversed-phase sorbents sample preparation protocol.

Therapeutic drug monitoring of angiotensin-converting enzyme inhibitors has a great impact on blood pressure control in patients with heart failure and hepatic and renal impairment. To provide an efficient tool for drug assessment in plasma, a UPLC-MS/MS method was developed for simultaneous determination of benazepril hydrochloride, fosinopril sodium, captopril and hydrochlorothiazide in human plasma samples. Solid phase extraction was applied for sample preparation using OASIS® hydrophilic-lipophilic balanced reversed-phase sorbents cartridges. Chromatographic separation was performed using an Agilent SB-C18 column and methanol-0.1% formic acid in water (95:5, v/v) as mobile phase, at flow rate 0.3 mL/min. Detection was accomplished using a tandem mass spectrometer. The method was validated according to US Food and Drug Administration guidelines. It showed good linearity over concentration ranges 5-400 ng/mL for benazepril hydrochloride, fosinopril sodium and hydrochlorothiazide and 100-3500 ng/mL for captopril. CV% values were <13.92% whereas the mean accuracy ranged from 94.50 to 113.82% for quality control samples and their extraction recoveries ranged from 90.60 to 99.38%. In conclusion, the present study revealed method selectivity and sensitivity; it can be applied for estimation of angiotensin converting enzyme inhibitors and hydrochlorothiazide in human plasma for dose adjustment and therapeutic drug monitoring.

Smart spectrophotometric methods based on normalized spectra for simultaneous determination of alogliptin and metformin in their combined tablets.

Alogliptin (ALO) and metformin (MET) are coformulated for the treatment of type II diabetes mellitus. ALO is estimated at its λmax 277 nm (0D), while MET was determined accurately by four spectrophotometric methods with minimum manipulation steps based on normalized division spectrum namely; ratio difference, advanced amplitude modulation, first derivative ratio (1DD) and mean centering of the ratio spectra spectrophotometric methods. Linearity was acceptable over the concentration ranges of 5-40 and 2-16 µg/mL for ALO and MET, respectively. Accuracy and precision of the suggested methods were found to be within the acceptable limit. The specificity was inspected by analyzing laboratory prepared mixtures of the above drugs and their pharmaceutical preparation. The results of proposed and reported methods were statistically compared showing no significant difference regarding accuracy and precision. The developed methods could be applied for routine analysis of the cited drugs in quality control laboratories.

Simultaneous HPLC Determination of Betamethasone Esters-Containing Mixtures: Analysis of Their Topical Preparations.

Topical pharmaceutical preparations containing betamethasone esters are widely prescribed for treatment of severe inflammatory skin conditions. Some betamethasone esters-containing preparations are formulated with either an antibacterial or an antifungal agent or a vitamin D3 derivative. A fast reversed-phase high-performance liquid chromatography method has been developed for the simultaneous determination of three betamethasone esters-containing binary mixtures along with the excipients of their dosage forms using clobetasone butyrate as internal standard. The first mixture was betamethasone valerate and fusidic acid (Mixture I) with chlorocresol as preservative. The second mixture was betamethasone dipropionate (BTD) and clotrimazole (Mixture II) with benzyl alcohol as preservative. The third mixture was BTD and calcipotriol monohydrate (Mixture III). Optimized chromatographic separation was achieved on a Discovery® C18 (4.6 × 250 mm, 5 µm) column, using water: acetonitrile (35:65, v/v) as mobile phase at flow rate of 1 mL/min with UV detection at 230 nm. The method was validated according to ICH guidelines. The regression coefficients were > 0.999 for all drugs. The method was successfully applied for the determination of the studied drugs in bulk, synthetic mixtures and dosage forms. The developed method is accurate, sensitive, selective and precise and can be used for routine analysis in quality control laboratories.

Determination of four antiepileptic drugs in plasma using ultra-performance liquid chromatography with mass detection technique.

Status epilepticus (SE) is considered the second most frequent neurological emergency. Its therapeutic management is performed using sequential antiepileptic drug regimens. Diazepam (DIA), midazolam (MID), phenytoin (PHT) and phenobarbital (PB) are four drugs of different classes used sequentially in the management of SE. A sensitive, selective, accurate and precise method was developed and validated for simultaneous determination of the four antiepileptic drugs in human plasma. Their separation and quantification were achieved using ultra-performance liquid chromatography (UPLC) with mass detection using carbamazepine as internal standard (IS). For the first three drugs and the IS, UPLC-MS/MS with electrospray ionization working in multiple reaction monitoring mode was used at the following transitions: m/z 285 → 193 for DIA; m/z 326 → 291 for MID; m/z 253 → 182 for PHT; and m/z 237 → 194, 237 → 192 for IS. For the fourth drug (PB), a molecular ion peak of PB [M + H] + at m/z 233 was used for its quantitation. The method was linear over concentration ranges 5-500 ng/mL for DIA and MID and 0.25-20 µg/mL for PHT and PB. Bioanalytical validation of the developed method was carried out according to European Medicines Agency guidelines. The developed method can be applied for routine drug analysis, therapeutic drug monitoring and bioequivalence studies.

In vitro metabolism study of a novel P38 kinase inhibitor: in silico predictions, structure elucidation using MS/MS-I.

AIM: Metabolism study of PH-797804, a promising newly developed drug for treatment of chronic inflammation which inhibits P38 mitogen-activated protein kinase. MATERIALS & METHODS: Susceptibility of PH-797804 to metabolism was first investigated using SMARTCyp and Xenosite web servers. Molecular docking of the drug into CYP3A4 crystal structures evaluated binding interactions with active site. The predicted results were confirmed by in vitro incubation with rat S9 fraction. Metabolites of PH-797804 were identified by MS/MS. RESULTS: A hydroxy metabolite and a cysteine/glutathione conjugate were detected. Computational prediction of reactive site of PH-797804 was conducted. CONCLUSION: The probable cysteine/glutathione adduct is indicative of potential drug chemical reactivity with potential to damage DNA and may provide guidance to the design of analogs with minimum toxicity.

Simultaneous determination of amlodipine besylate and atorvastatin calcium in binary mixture by spectrofluorimetry and HPLC coupled with fluorescence detection.

Caduet tablets are novel prescription drug that combines amlodipine besylate (AM) with atorvastatin calcium (AT). A spectrofluorimetric and an HPLC-fluorescence detection methods were developed for simultaneous determination of both drugs in tablets. In the spectrofluorimetric method, native fluorescence of AM and AT were measured in methanol at 442 and 369 nm upon excitation at 361 and 274 nm, respectively. The emission spectrum of each drug reveals zero value at the emission wavelength of the other drug, thus allowing their simultaneous determination without interference. In the HPLC method, separation of AM and AT was achieved within 8 minutes on a C18 column using acetonitrile:phosphate buffer (0.015 M, pH 3) (45:55, v/v) as the mobile phase. Fluorescence detection was carried out using excitation wavelengths 361 and 274 nm and emission wavelengths 442 and 378 nm for AM and AT, respectively. Excellent linearity was observed. Careful validation proved advantages of the new methods: high sensitivity, accuracy, selectivity and suitability for quality control laboratories.

Utility of Experimental Design in Pre-Column Derivatization for the Analysis of Tobramycin by HPLC-Fluorescence Detection: Application to Ophthalmic Solution and Human Plasma.

A novel, selective, and sensitive reversed phase high-performance liquid chromatography (HPLC) method coupled with fluorescence detection has been developed for the determination of tobramycin (TOB) in pure form, in ophthalmic solution and in spiked human plasma. Since TOB lacks UV absorbing chromophores and native fluorescence, pre-column derivatization of TOB was carried out using fluorescamine reagent (0.01%, 1.5 mL) and borate buffer (pH 8.5, 2 mL). Experimental design was applied for optimization of the derivatization step. The resulting highly fluorescent stable derivative was chromatographed on C18 column and eluted using methanol:water (60:40, v/v) at a flow rate of 1 mL min(-1). A fluorescence detector (λex 390 and λem 480 nm) was used. The method was linear over the concentration range 20-200 ng mL(-1). The structure of the fluorescent product was proposed, the method was then validated and applied for the determination of TOB in human plasma. The results were statistically compared with the reference method, revealing no significant difference.