Isolation, Characterization, and Toxicity Study of Stress Degradation Products of Pranlukast Hydrate.

Pranlukast hydrate (PRN), a cysteinyl leukotriene receptor antagonist (CysLT1), is used to treat bronchial asthma. The objective of this study is to perform the isolation, characterization, and toxicity analysis of stress degradation products of PRN. In high-performance liquid chromatography (HPLC), the separation was achieved using a Phenomenex Gemini C18 (250 × 4.6 mm, 5 µ) column; the ammonium format buffer (50 mM), pH 4, with formic acid: acetonitrile (50:50, v/v) was used as a mobile phase at a flow rate of 1.25 mL/min; and the photodiode array detector was used for detection at 230 nm. The drug was subjected to stress degradation as per ICH Q1A (R2) and ICH Q1B guidelines. The drug was found to be labile in alkaline (62.48% degradation) and photolytic (liquid state) (7.67% degradation) conditions, whereas the drug was found to be stable in acidic, peroxide, photolytic (solid state), and thermal conditions. The characterization of the drug and its degradation products was achieved using liquid chromatography-electrospray ionization-quadrupole time of flight tandem mass spectrometry (LC-ESI-QTOF-MS/MS), and the degradation mechanism was proposed. There were two degradation products observed in alkaline conditions (DP6 and DP9), whereas six novel degradation products were observed in photolytic degradation products (DP1, DP3, DP4, DP5, DP7, and DP10). The developed method was successfully validated as per the ICH Q2 (R1) guideline. The isolation of the alkaline degradation product DP9 was performed using preparative HPLC, and it was found to be 96.8% pure degradation product. The characterizations of the isolated degradation product (DP9) and procured impurity were performed using MS/MS, NMR, and FTIR. The mass of the procured impurity and DP9 were observed to be 404 and 500 Da, respectively. The in vitro cytotoxicity study of the procured impurity and DP9 was conducted using a 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using an A549 cell line, and they were found to be cytotoxic at concentrations above 62.5 and 250 µg/mL, respectively. Furthermore, an in silico toxicity study was performed to predict the toxicity of all the major characterized degradation products of PRN using admetSAR software version 2.0. DP1, DP2, DP6, and DP10 were found to be hepatotoxic, mutagenic according to the micronucleus test, and aquatic toxic. We can conclude that the drug should be kept away from the direct exposure of light and the toxicity levels of DP1, DP2, DP6, and DP10 should be reduced below 0.1% to avoid their toxic effect.

Discovery and Optimization of DNA Gyrase and Topoisomerase IV Inhibitors with Potent Activity against Fluoroquinolone-Resistant Gram-Positive Bacteria.

Herein, we describe the discovery and optimization of a novel series that inhibits bacterial DNA gyrase and topoisomerase IV via binding to, and stabilization of, DNA cleavage complexes. Optimization of this series led to the identification of compound 25, which has potent activity against Gram-positive bacteria, a favorable in vitro safety profile, and excellent in vivo pharmacokinetic properties. Compound 25 was found to be efficacious against fluoroquinolone-sensitive Staphylococcus aureus infection in a mouse thigh model at lower doses than moxifloxacin. An X-ray crystal structure of the ternary complex formed by topoisomerase IV from Klebsiella pneumoniae, compound 25, and cleaved DNA indicates that this compound does not engage in a water-metal ion bridge interaction and forms no direct contacts with residues in the quinolone resistance determining region (QRDR). This suggests a structural basis for the reduced impact of QRDR mutations on antibacterial activity of 25 compared to fluoroquinolones.

Development and Validation of a Stability Indicating LC-MS/MS Method for the Determination of Clenbuterol HCl.

Clenbuterol hydrochloride (CLT), ß2 adrenergic agonist is used as a bronchodilator in the therapeutic treatment of asthma. It is important to know the stability behaviour of the drug in different degradation conditions as per ICH Q1A (R2) guidelines for safety and efficacy purpose. The main objective of the study is to develop and validate stability indicating LC-MS/MS method for the determination of Clenbuterol HCl. The separation was achieved using Phenomenex Gemini NX C18 (250*4.6 mm, 5 µ) column and the mobile phase consisting of ammonium acetate buffer (5 mM), 0.15% triethylamine (TEA), pH 7.5 with acetic acid: methanol (70:30, v/v) at flow rate 1 ml/min. The detection was done using PDA detector at 245 nm. The validation was performed as per ICH Q2 (R1) guideline. The drug was subjected to stress degradation conditions as per ICH Q1A (R2) guidelines. The significant degradation was observed in acidic (8.78%) and sunlight (liquid) (9%) condition while no degradation was observed in neutral, basic, oxidation and thermal condition. The drug and its degradation products were characterized using LC-MS/MS and the proposed degradation mechanism was communicated. The developed method was found to be stability-indicating, simple, specific, selective, sensitive, linear, accurate, robust and precise and used as a routine analysis in quality control laboratory.

Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria.

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region.

Impurity Profile of Bronchodilators used in Asthma: A Critical Review.

BACKGROUND: Asthma is defined as a heterogeneous disease usually characterized by chronic airway inflammation (GINA 2016) affecting almost 334 million people worldwide (Global asthma report 2014). Treatment of asthma with a long-acting bronchodilator is important because it reduces the symptoms that occur at night or in the early morning and it is very effective to use as a long term control medication for asthma by preventing asthmatic symptoms. The main objective of this review is to describe the impurity profile and force degradation studies for three major classes of bronchodilators namely ß2-adrenoceptor agonists, muscarinic receptor antagonists and xanthine. Unidentified and potential toxic impurities are hazardous to health, so in order to increase the safety of drug therapy; impurities should be identified and determined by selective analytical methods. METHODS: Different conditions for degradations like hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermolytic have been discussed in detail for bronchodilators. Furthermore, it is discussed with the name along with number of impurities and degradants present in different matrices including its clinical implication. The name as well as structures of all the observed impurities in different bronchodilators is included, which can aid in impurity profiling. Various analytical methods, including Chromatographic techniques like TLC; HPTLC; HPLC; GC, Spectroscopic techniques like UV; IR; NMR; MS and hyphenated techniques like GC-MS; LC-MS; CE-MS; SFC-MS; LC-NMR; CENMR; LC-FTIR has been used for the identification and quantification of impurities. A general scheme has been presented for the impurity profiling. RESULT: Nineteen articles, six patents and fifteen drugs are included in this review. In that, majority (7) of papers are based on HPLC-UV, 5 papers are based on LC-MS, 2 papers are based on LC-MS-NMR, 1 paper is based on LC-NMR, 1 paper is based on GC-MSNMR, 1 paper is based on GC-UV and 1 paper is based on TLC-UV technique for isolation and characterization of impurities. In salbutamol, 7 degradants were found by LC-MS as compare to 4 degradants by HPLC-UV. In bambuterol, 12 degradants were found by LC-MS-NMR as compare to 4 degradants by LC-MS. CONCLUSION: After a thorough literature search, LC-MS and LC-MS-NMR techniques are found most useful for impurity profiling. In future, LC-DAD-NMR-MS, CE-ESI-FTICR- MS can also be explored for the isolation and characterization of impurities.

Treatment outcome of patients having extensively drug-resistant tuberculosis in Gujarat, India.

BACKGROUND: To investigate the factors affecting treatment outcome of extensively drug resistant tuberculosis (XDR-TB) in Gujarat, India. METHODS: A prospective, observational study was conducted on patients with XDR TB from January 2012 to October 2016. Details of demography, clinical symptoms, sputum/culture and radiological examination, drug treatment, adverse drug reactions (ADRs) and treatment outcome were recorded in pretested case record form (CRF). Data was analyzed using Fisher's exact test and paired student's t test. RESULTS: Out of 112 patients, 83 (74%) were men and 29 (26%) were women and majority of belonged to age group of 16 - 45 years. Majority of patients (79%) received standardized treatment. A total of 61 (54%) patients converted to sputum culture negative by 12 months and out of these, 49 turned sputum culture negative within initial 6 months of treatment. Successful treatment outcome was seen in 29 (25.89 %). Age ≤40 years (P<0.05), body mass index > 18.5 (P<0.05) and sputum/culture conversion at three month (P<0.001) were positive predictors for successful treatment outcome, while tobacco chewing habit (P<0.05) and alcohol consumption (P<0.05) were negative predictors for the successful treatment outcome. Out of 83 (74.1 %) patients with unsuccessful treatment outcome, 58 (51.78 %) died, 11 (9.82 %) were defaulter and 10 (8.92 %) were treatment failure. Factors positively associated with death were very low BMI (< 18.5), concomitant diseases and harmful personal habits. CONCLUSIONS: Treatment outcome of XDR TB patients is extremely poor with high mortality rate.

Critical Analysis of Cardiovascular and Central Nervous System Fixed Dose Combinations Available in Indian Market.

INTRODUCTION: Fixed Dose Combinations (FDCs) are being increasingly used to improve compliance and achieve greater benefits of the two or more active ingredients given together than the corresponding individual drug components given separately. AIM: To analyse the rationality of Cardiovascular (CV) and Central Nervous System (CNS) FDCs available in Indian market. MATERIALS AND METHODS: CVS and CNS FDCs, enlisted in Indian Drug Review, 2014, were analysed by a pretested validated eight point criteria tool. Each FDC was assessed for number of active pharmacological ingredients, approval by regulatory authority, listing in WHO Essential Medicine List. While efficacy, safety, pharmacokinetic, pharmacodynamic interactions and advantages of each FDC were analysed by literature search. The total score of the tool was 12 and score ≥7 was considered rational. FDCs were divided in four groups as per rationality and DCGI approval. ANOVA was used for statistical analysis and p<0.05 was considering statistically significant. RESULTS: Out of 152 FDCs, 107 were CV and 45 belonged to CNS group and 40 had documented evidence of efficacy and safety. Majority of FDCs showed advantage of being convenient by reducing pill count and only 32 showed reducing adverse drug reactions. Out of 107 CV FDCs, 46 were rational and 61 were irrational with a mean rationality score of 6.72±2.82 (CI- 95 %, 3.90 - 9.54). While out of 45 CNS FDCs, 8 were rational and 37 were irrational with a mean rationality score of 6.22±2.08 (CI - 95 %, 4.14 - 8.30). A significant difference in mean rationality score of group A (DCGI approved + rational) was observed as compared to group B (DCGI approved + irrational) and group C (DCGI unapproved + rational) as compared to group D (DCGI unapproved + irrational) (p<0.05). CONCLUSION: The absence of watertight pre-requisite, critical analysis of the scientific validity of the formulations and 'convenience' category has resulted into proliferation of irrational FDCs. This calls for strict regulatory approval process to avoid miserable FDC scenario in the country.