Design, synthesis, and primary activity assays of baicalein derivatives as cyclin-dependent kinase 1 inhibitors.

Malignant tumor is a disease with high mortality. Traditional treatment methods have many disadvantages, such as side-effects, drug resistance. Because cyclin-dependent kinase 1 (CDK1) plays an indispensable role in cell cycle regulation, it became an attractive target in rational anti-cancer drug discovery. Herein, we reported a series of baicalein derivatives, which remarkably repressed the proliferation of MCF-7 tumor cells and the activity of CDK1/cyclin B kinase. Among them, compound 4a displayed better inhibition rate than flavopiridol against MCF-7 proliferation at the concentration of 50 µg/ml, comparable to compound CGP74514A, while compound 3o possessed the best activity against CDK1/cyclin B kinase (IC50  = 1.26 µM). The inhibitory activities toward the kinase well correlated with anti-proliferative activities. Molecular docking results suggested that compound 3o can interact with the key amino acid residues, E81, L83, and D146, of CDK1 through hydrogen bond just like flavopiridol does. And it can also form an extra hydrogen bond with D146 by its introduced 7-acrylate group, which flavopiridol does not have. These findings proved that baicalein derivatives can be used as CDK1 inhibitors fighting against cancer.

Efficacy of halofuginone products to prevent or treat cryptosporidiosis in bovine calves: a systematic review and meta-analyses.

A prior systematic review on the efficacy of halofuginone (HFG) treatment to prevent or treat cryptosporidiosis in bovine calves was inconclusive. We undertook an updated synthesis and meta-analyses on key outcomes for the treatment of calves with HFG. Evaluated outcomes were oocyst shedding, diarrhoea, mortality and weight gain. Experiments had to describe results for same age animals in contemporary arms. Most doses were 100-150 mcg kg-1 day-1. Results were subgrouped by study design, experiments with the lowest risk of bias and lack of industry funding. Eighteen articles were found that described 25 experiments. Most evidence came from randomized controlled trials in Europe. Significantly lower incidence of oocyst shedding, diarrhoea burden and mortality was reported when treatment started before calves were 5 days old. Most studies reported on outcomes for animals up to at least 28 days old. Publication bias was possible in all outcomes and seemed especially likely for diarrhoea outcomes. Beneficial results when HFG treatment was initiated in calves older than 5 days were also found. Prophylactic treatment to prevent cryptosporidiosis is effective in preventing multiple negative outcomes and is beneficial to calf health and will result in a reduction of environmental contamination by Cryptosporidium oocysts.

Identification, Characterization and HPLC Quantification of Process-Related Impurities in Bepotastine Besilate Bulk Drug.

Bepotastine besilate (here after referred to as BTST), chemically known as ({d(S)4[4[(4chlorophenyl) (2pyridyl) methoxy] piperidino} butyric acid monobenzene sulphonate), is a second-generation antihistamine drug. To the best of our knowledge, no studies concerning the isolation or identification of process-related impurities have been reported so far. The current study reports the development and validation of a stability-indicating RP-HPLC method for the separation and identification of 5 potential impurities in bepotastine besilate. In this experiment, the structures of 3 process-related impurities were found to be new compounds. They were characterized and confirmed by NMR and MS spectroscopy analyses. These 3 new compounds were proposed to be (S)-4-[(phenyl)-2-pyridinylmethoxy]-1-piperidinebutanoic acid,(Imp-A); 4-[(S)-(4-chlorophenyl)-2-pyridinylmethoxy]-1- piperidinebutyric acid, N-oxide (Imp-B) and (S)-4-[(4- chlorophenyl)-2-pyridinylmethoxy]-1-piperidylethane (Imp-C). In addition, an efficient optimized chromatographic method was performed on a Shimadzu Inertsil C8-3 column (150 mm×4.6 mm, 3 µm) to separate and quantify these 5 impurities. It was using 15 mmol ammonium formate buffer in water (pH adjusted to 3.8 with formic acid) and acetonitrile as the mobile phase in gradient mode. The method was developed to separate and quantify these 5 impurities obtained in the range of 0.05-0.75 µg/mL. It was validated and proven to be selective, accurate and precise and suitable. It is the first publication of identification and characterization data of the 3 new compounds. It is also the first effective HPLC method for separation and quantification of all of process-related impurities in bepotastine besilate.

An ultra HPLC-MS/MS method for quantification of hypidone hydrochloride (YL-0919) and application to a pharmacokinetic study.

Aim: Hypidone hydrochloride (YL-0919) was a novel combined selective serotonin reuptake inhibitor and 5-hydroxytryptamine receptor agonist for treatment of major depressive disorder. Quantitation of YL-0919 in plasma samples was critical for evaluation of its pharmacokinetics in clinical studies. Methodology & results: An ultra HPLC-MS/MS method has been developed and validated. Plasma samples were extracted by SPE method and then chromatographed on an Acquity BEH C18 column. Detection was performed on an API-5500 tandem mass spectrometer using positive ESI. Conclusion: A sensitive and robust method was developed and validated for quantitative analysis of YL-0919 in human plasma samples for the first time. And this novel method was successfully applied to investigate pharmacokinetic profiles of YL-0919 in Chinese healthy subjects.

Automated production of [¹8F]VAT suitable for clinical PET study of vesicular acetylcholine transporter.

Automated production of a promising radiopharmaceutical (-)-(1-(8-(2-[(18)F]fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-piperidin-4-yl)(4-fluorophenyl)methanone ([(18)F]VAT) for the vesicular acetylcholine transporter(VAChT) was achieved using a two-step procedure in a current Good Manufacturing Practices fashion. The production of [(18)F]VAT was accomplished in approximately 140 min, with radiochemical yield of ~15.0% (decay corrected), specific activity>111 GBq/µmol, radiochemical purity>99% and mass of VAT ~3.4 µg/batch (n>10). The radiopharmaceutical product meets all quality control criteria for human use, and is suitable for clinical PET studies of VAChT.

Nicotelline: a proposed biomarker and environmental tracer for particulate matter derived from tobacco smoke.

Particulate matter (PM) derived from tobacco smoke contains numerous toxic substances. Since the PM and gas phase of tobacco smoke may distribute differently in the environment and substances in them may have different human bioavailability, multiple tracers and biomarkers for tobacco smoke constituents are desirable. Nicotelline is a relatively nonvolatile alkaloid present in tobacco smoke, and therefore, it has the potential to be a suitable tracer and biomarker for tobacco smoke-derived PM. We describe experiments demonstrating that nicotelline is present almost entirely in the PM, in both freshly generated cigarette smoke and aged cigarette smoke. An excellent correlation between the mass of nicotelline and the mass of the PM in aged cigarette smoke was found. We also describe experiments suggesting that the main source of nicotelline in tobacco smoke is dehydrogenation of another little-studied tobacco alkaloid, anatalline, during the burning process. We show that nicotelline metabolites can be measured in the urine of smokers and that nicotelline can be measured in house dust from homes of smokers and nonsmokers. We conclude that nicotelline should be useful as a tracer and biomarker for PM derived from tobacco smoke.

Coupling of a high-resolution monoamine oxidase-A inhibitor assay and HPLC-SPE-NMR for advanced bioactivity profiling of plant extracts.

INTRODUCTION: Depression is a mental disease causing large personal and socio-economic problems, and new improved drugs are therefore needed. Selective monoamine oxidase A (MAO-A) inhibitors are potential anti-depressants, but discovering new MAO-A inhibitors from natural sources by bioassay-guided approaches are a lengthy and time-consuming process. New analytical technologies that allow simultaneously chemical and biological screening of extracts are therefore urgently needed. METHOD: In the present study we describe coupling of a photometric microplate-based high-resolution MAO-A inhibitor assay with a hyphenated system consisting of high-performance liquid chromatography, solid-phase extraction and tube transfer nuclear magnetic resonance (HPLC-SPE-ttNMR). The standard compound clorgyline, and an extract of black pepper (Piper nigrum L.), representing a complex plant matrix, were used for proof-of-concept. RESULTS: The work with clorgyline showed that the microplate-based high-resolution assay produced MAO-A inhibition profiles that easily allowed detection of submicrogram amounts of this selective MAO-A inhibitor. Furthermore, the HPLC-SPE-ttNMR/high-resolution MAO-A inhibition assay platform allowed identification of piperine and two piperine analogues as the main MAO-A inhibitors in the black pepper petroleum ether extract. CONCLUSION: The HPLC-SPE-ttNMR/high-resolution MAO-A inhibition assay platform is a powerful tool for fast and efficient identification of new MAO-A inhibitors from complex extracts, and promise future advancement in the search for new anti-depressants from natural sources.

Degradation of a pharmaceutical in HPLC grade methanol containing trace level formaldehyde.

An anomalous peak was observed in the HPLC/UV analysis of a developmental drug product. High resolution LC/MS revealed that the mass of this degradant was 12 Da greater than the drug substance, corresponding to a net gain of a single carbon atom. The degradant was reproduced by incubating the drug substance with formaldehyde, followed by isolation using normal phase chromatography and structure elucidation by NMR. It was determined to be an analytical artifact caused by the nucleophilic reaction of the drug substance with trace levels of formaldehyde in the methanol diluent. Typical formaldehyde levels in various grades of methanol were determined, leading to the adoption of spectrophotometric purity solvent to mitigate the recurrence of this artifact. This work demonstrates that even ppm levels of impurities in solvents can cause significant degradation of drug product and the HPLC grade solvents are not always suitable for HPLC analysis in drug product development.

Development and validation of a stability-indicating gas chromatographic method for quality control of residual solvents in blonanserin: a novel atypical antipsychotic agent.

Blonanserin is a novel atypical antipsychotic agent for the treatment of schizophrenia. Ethyl alcohol, isopropyl alcohol and toluene are utilized in the synthesis route of this bulk drug. A new validated gas chromatographic (GC) method for the simultaneous determination of residual solvents in blonanserin is described in this paper. Blonanserin was dissolved in N, N-dimethylformamide to make a sample solution that was directly injected into a DB-624 column. A postrun oven temperature at 240°C for approximately 2 h after the analysis cycle was performed to wash out blonanserin residue in the GC column. Quantitation was performed by external standard analyses and the validation was carried out according to International Conference on Harmonization validation guidelines Q2A and Q2B. The method was shown to be specific (no interference in the blank solution), linear (correlation coefficients ≥0.99998, n = 10), accurate (average recoveries between 94.1 and 101.7%), precise (intra-day and inter-day precision ≤2.6%), sensitive (limit of detection ≤0.2 ng, and limit of quantitation ≤0.7 ng), robust (small variations of carrier gas flow, initial oven temperature, temperature ramping rate, injector and detector temperatures did not significantly affect the system suitability test parameters and peak areas) and stable (reference standard and sample solutions were stable over 48 h). This extensively validated method is ready to be used for the quality control of blonanserin.

Development of chromatographic methods for the determination of genotoxic impurities in cloperastine fendizoate.

The classification of an impurity of a drug substance as genotoxic means that the "threshold of toxicological concern" (TTC) value of 1.5 µg/day intake, considered to be associated with an acceptable risk, should be the admissible limit in the raw material and that leads to new analytical challenges. In this study, reliable chromatographic methods were developed and applied as limit tests for the control of three genotoxic impurities (GTIs) in cloperastine fendizoate, drug widely used as an antitussive active pharmaceutical ingredient (API). In particular, GC-MS was applied to the determination of one alkyl halide (2-chloroethanol, 2-CE), while HPLC-DAD was selected for the analysis of two sulfonate esters (methyl p-toluenesulfonate, MPTS, and 2-chloroethyl p-toluenesulfonate, CEPTS). Regarding GC-MS, strong anion-exchange (SAX)-SPE was applied to remove fendizoate from the sample solutions, due its low volatility and its high amount in the raw material. The GC-MS analysis was performed on a Factor Four VF-23 ms capillary column (30 m × 0.25 mm I.D., film thickness 0.25 µm, Varian). Single ion-monitoring (SIM) detection mode was set at m/z 80. In the case of HPLC-DAD, a suitable optimization of the chromatographic conditions was carried out in order to obtain a good separation of the impurity peaks from the drug substance peaks. The optimized method utilizes a SymmetryShield RP(8) column (250 mm × 4.6 mm, 5 µm, Waters) kept at 50°C, with phosphate buffer (pH 3.0; 10 mM)-methanol (containing 10% ACN) (45:55, v/v) as the mobile phase, at the flow-rate of 1.7 mL/min and UV detection at 227 nm. The required sensitivity level was achieved by injecting 80 µL of sample solution, purified from fendizoate by SAX-SPE, followed by a 1:1 (v/v) dilution of the SPE eluate with water. For both GC-MS and HPLC-DAD, the method validation was performed in relation to specificity and limit of detection (LOD), as required by ICH guidelines in relation to limit assays. The developed methods were successfully applied for the determination of GTIs in five different batches of cloperastine fendizoate. In all the analyzed batches, the three target GTIs were below the concentration limit.

A comparative study of first-derivative spectrophotometry and column high-performance liquid chromatography applied to the determination of repaglinide in tablets and for dissolution testing.

A fast and reliable method for the determination of repaglinide is highly desirable to support formulation screening and quality control. A first-derivative UV spectroscopic method was developed for the determination of repaglinide in tablet dosage form and for dissolution testing. First-derivative UV absorbance was measured at 253 nm. The developed method was validated for linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ) in comparison to the U.S. Pharmacopeia (USP) column high-performance liquid chromatographic (HPLC) method. The first-derivative UV spectrophotometric method showed excellent linearity [correlation coefficient (r) = 0.9999] in the concentration range of 1-35 microg/mL and precision (relative standard deviation < 1.5%). The LOD and LOQ were 0.23 and 0.72 microg/mL, respectively, and good recoveries were achieved (98-101.8%). Statistical comparison of results of the first-derivative UV spectrophotometric and the USP HPLC methods using the t-test showed that there was no significant difference between the 2 methods. Additionally, the method was successfully used for the dissolution test of repaglinide and was found to be reliable, simple, fast, and inexpensive.

Stability indicating methods for determination of Donepezil Hydrochloride according to ICH guidelines.

Stability indicating assays for determination of Donepezil Hydrochloride in presence of its oxidative degradate were developed and validated. The first three are spectrophotometric methods depending on using zero order (D(0)), first order (D(1)) and second order (D(2)) spectra. The absorbance was measured at 315 nm for (D(0)) while the amplitude was measured at 332.1nm for (D(1)) and 340 nm for (D(2)) using deionized water as a solvent. Donepezil Hydrochloride (I) can be determined in the presence of up to 70% of its oxidative degradate (II) using (D(0)), 80% using (D(1)) and 90% using (D(2)). The linearity range was found to be 8-56 microg ml(-1) for (D(0)), (D(1)) and (D(2)). These methods were applied for the analysis of I in both powder and tablet form. Also, a spectrofluorimetric method depending on measuring the native fluorescence of I in deionized water using lambda excitation 226 nm and lambda emission 391 nm is suggested. The linearity range was found to be 0.32-3.20 microg ml(-1) using this method, I was determined in the presence of up to 90% of II. The proposed method was applied for the analysis of I in tablet form as well as in human plasma. The last method depends on using TLC separation of I from its oxidative degradate II and I was then determined spectrodensitometrically. The mobile phase was methanol : chloroform : 25% ammonia (16 : 64 : 0.1 by volume). The linearity range was found to be 2-15 microg/spot. This method was applied to the analysis of I in both powder and tablet form using acetonitrile as a solvent.

Flexible prandial glucose regulation with repaglinide in patients with type 2 diabetes.

Repaglinide is a novel, rapid-acting prandial glucose regulator. To investigate the effect of repaglinide, 1 mg before each meal, in maintaining glycaemic control in Type 2 diabetic patients who either miss a meal or have an extra meal, 25 patients were randomized to either a fixed-meal regimen of three meals/day or one of two mixed-meal regimens consisting of repeating patterns of two, three or four meals/day over a 20-day period. On the 21st day each patient received three meals. Overall glycaemic control was assessed by weekly serum fructosamine concentrations and 13-point and 37-point serum glucose profiles. Mean fructosamine concentrations decreased significantly to normal values during the treatment period (from 3.10 to 2.68 mg/dl on the fixed-meal regimen and from 3.37 to 2.85 mg/dl on the mixed-meal regimens; P < 0.05), with no statistically significant difference in glucose control between the fixed-meal and mixed-meal regimen groups. Fasting serum glucose levels decreased slightly in both groups, but were not altered by the number of meals consumed. Similarly, serum glucose profiles were not altered significantly by the number of meals consumed. Repaglinide was well tolerated, and no hypoglycaemic events were reported. Serum cholesterol levels were significantly reduced (P < 0.05) in both the fixed-meal and mixed-meal groups, as were triglyceride levels in the mixed-meal group (P < 0.05). It was concluded that meal-associated treatment with repaglinide was well tolerated irrespective of the number of meals consumed/day. Thus, since missing or postponing a meal is a realistic scenario for many individuals, repaglinide offers an oral anti-diabetic treatment which can be adjusted to suit each individual's lifestyle.

Stability of an extemporaneously compounded cisapride suspension.

The purpose of the study was to formulate a cisapride suspension that would be stable under usual storage conditions for 3 weeks. Cisapride is a new prokinetic agent that is available as a coated tablet; occasionally, however, patients require a liquid preparation of cisapride. Cisapride was formulated with propylene glycol to enhance its solubility. The suspension was buffered with sodium bicarbonate to produce a pH between 6.5 and 7.5. The formulation provided a cisapride suspension that was stable at room temperature for 3 weeks.

Ebastine in perennial allergic rhinitis.

Oral ebastine, 10 mg once daily for seven days, and placebo were compared as treatment for active perennial allergic rhinitis in 151 patients in a multicenter, randomized, double-blind trial. Ebastine treatment produced a significant reduction in the incidence and severity of most symptoms associated with perennial rhinitis. Tolerability was similar in the two treatment groups. The incidences of drowsiness and dry mouth were not more frequent in the patients treated with the active drug.

Double-blind comparison of levocabastine nasal spray with sodium cromoglycate nasal spray in the treatment of seasonal allergic rhinitis.

Nasal levocabastine (0.5 mg/mL) was evaluated for efficacy and tolerance against sodium cromoglycate (20 mg/mL) in a 2-week double-blind trial in 27 and 29 patients with seasonal allergic rhinitis. Globally at 2 weeks, the investigators found a 74% response rate in the levocabastine patients versus a 50% response rate in the cromoglycate patients (P less than .10). Sneezing responded better to levocabastine than to cromoglycate according to three efficacy indicators derived from patient diary ratings of symptom severity: sum of severity scores over the total treatment period as a percentage of the theoretical maximum sum of severity scores (median: 19% versus 41%, P = .01); percentage of symptom-free days (median: 46% versus 22%, P less than .07); percentage of days with moderate or severe symptoms (median: 0% versus 29%, P = .004). Further, the percentage of days with moderate or severe runny nose was lower than in cromoglycate patients (median: 0% versus 25%, P = .09). Although no significant differences were found for itchy nose, blocked nose, and ocular symptoms, severities tended to be generally less under levocabastine than under sodium cromoglycate. Adverse experiences were low level and of similar incidence in the two groups. It is concluded that in a q.i.d. schedule, levocabastine nasal spray is more efficacious than sodium cromoglycate in relieving sneezing and that it is equally well tolerated.

Biological profile of 2-[4-(2-N-piperidinoethoxy) phenyl]-3-phenyl (2H) benzo (b) pyran--a potent antiimplantation agent in rat.

Compound CDRI-85/287: 2-[4-(2-N-piperidinoethoxy) phenyl]-3-phenyl (2H) benzo (b) pyran has been identified as a potent antiimplantation agent in rat. A single oral dose (2.5 mg/kg body weight) of the compound administered on days 1, 2 or 3 of pregnancy or multiple dosing (0.05 mg/kg daily) on days 5-7 postcoitum effectively prevented pregnancy. When administered on days 5-7 postcoitum, it failed to interrupt pregnancy even at 20 mg/kg dose. The compound is a potent antiestrogen, with very weak uterotrophic activity; it does not induce vaginal cornification in immature ovariectomised rat. Also, it is devoid of progestational, antiprogestational, androgenic, antiandrogenic and antigonadotrophic activities. The results suggest that the compound exerts its antiimplantation acivity in rat by virtue of its antiestrogenic activity [corrected].

[Determination of the concentration of a new antiarrhythmic, flecainide, in plasma using high pressure liquid chromatography (HPLC): sample preparation with extraction columns]. / Bestimmung der Konzentration eines neuen Antiarrhythmicums, Flecainid, im Plasma durch Hochleistungsflüssigkeitschromatographie (HPLC): Probenvorbereitung durch Extraktionssäulen.

A method is described for the rapid determination of flecainid (Tambocor) in human plasma. Flecainid is removed from alkalized plasma by binding to a C-18 extraction column. After washing the column with methanol/water (1 + 1, by vol.) and acetonitrile/water (45 + 55, by vol.), the drug is extracted with methanol and determined by HPLC. Sample preparation takes about 15 minutes. With this method, flecainid can be determined directly in the concentration range 100-2000 micrograms/l, with an accuracy of 5-10% depending on the concentration. The results agree well with those obtained by the longer established but lengthy liquid extraction method.