Simultaneous determination of sulfamethoxazole, trimethoprim and bromhexine in veterinary formulation using high performance liquid chromatography (HPLC)

Objetivo: desarrollar y validar un método simple, sensible y rápido para la determinación simultánea de sulfametoxazol (SMT), trimetoprima (TMP) y bromhexina (BMX) en formulación veterinaria por cromatografía líquida de alta resolución de acuerdo con las directrices de validación y control. Guía para la calidad analítica de medicamentos en productos alimenticios y medicamentos veterinarios, RDC 166/2017 y guías internacionales Conferencia Internacional sobre Armonización y Asociación Internacional de Químicos Analíticos Oficiales. Materiales y métodos: la separación se realizó en una columna analítica ThermoScientific® C18 AcclaimTM120 (4,6 x 250 mm, 5 µm), con caudal de 0,7 mL min-1 y detección a 245 nm, 265 nm y 271 nm, para BMX, SMT y TMP, respectivamente. Todas las mediciones se realizaron en metanol:agua (84:16 v/v; pH 3,0). Las curvas analíticas fueron lineales (r > 0,9997) en el rango de concentración de 15.0 a 30.0 µg-mL-1 para SMT, 3.0 a 9.0 µg-mL-1 para TMP y 0,5 a 2,0 µg-mL-1 para BMX. Resultados: el método demostró ser preciso con coeficientes de variación por debajo del límite máximo de 2,0%, robusto, sin influencia significativa de las variaciones utilizadas en el análisis, exacto (recuperación >99%) y selectivo, en la evaluación de la interferencia de adyuvantes Conclusión: por lo tanto, el método desarrollado demostró ser adecuado para los análisis de control de calidad de rutina para la determinación simultánea de SMT, TMP y BMX en formulaciones farmacéuticas.

SUMMARY Aim: To develop and to validate a simple, sensitive and fast method for the simultaneous determination of sulfamethoxazole (SMT), trimethoprim (TMP) and bromhexine (BMX) in veterinary formulation by high performance liquid chromatography according to the guidelines of the Validation and Control Guide for analytical quality of medicines in food products and veterinary medicines, RDC 166/2017 and international guides International Conference on Harmonization and International Association of Official Analytical Chemists. Materials and methods: The separation was performed on a ThermoScientific® C18 AcclaimTM120 analytical column (4.6 X 250 mm, 5 µm), with a flow rate of 0.7 mL min-1 and detection at 245 nm, 265 nm and 271 nm, for BMX, SMT and TMP, respectively. All measurements were performed in methanol: water (84:16 v/v; pH 3.0). The analytical curves were linear (r > 0.9997) in the concentration range of 15.0 to 30.0 µg-mL-1 for SMT, 3.0 to 9.0 µg-mL-1 for TMP and 0.5 to 2.0 µg-mL-1 for BMX. Results: The method proved to be accurate, with coefficients of variation below the maximum limit of 2.0%, robust, without significant influence of the variations used in the analysis, exact (recovery >99%) and selective, in the assessment of interference from adjuvants. Conclusion: Therefore, the developed method proved to be suitable for routine quality control analyzes for the simultaneous determination of SMT, TMP and BMX in pharmaceutical formulations.

Objetivo: desenvolver e validar um método simples, sensível e rápido para a determinação simultânea de sulfametoxazol (SMT), trimetoprima (TMP) e bromexina (BMX) em formulação veterinária por cromatografia líquida de alta eficiência de acordo com as diretrizes do Validation and Control Guia de qualidade analítica de medicamentos em produtos alimentícios e medicamentos veterinários, RDC 166/2017 e guias internacionais Conferência Internacional de Harmonização e Associação Internacional de Químicos Analíticos Oficiais. Materiais e métodos: a separação foi realizada em coluna analítica ThermoScientific® C18 AcclaimTM120 (4,6 X 250 mm, 5 µm), com vazão de 0,7 mL min-1 e detecção em 245 nm, 265 nm e 271 nm, para BMX , SMT e TMP, respectivamente. Todas as medições foram realizadas em metanol:água (84:16 v/v; pH 3,0). As curvas analíticas foram lineares (r > 0,9997) na faixa de concentração de 15,0 a 30,0 µg-mL-1 para SMT, 3,0 a 9,0 µg-mL-1 para TMP e 0,5 a 2,0 µg-mL-1 para BMX. Resultados: o método mostrou-se preciso, com coeficientes de variação abaixo do limite máximo de 2,0%, robusto, sem influência significativa das variações utilizadas na análise, exato (recuperação >99%) e seletivo, na avaliação da interferência de adjuvantes. Conclusão: portanto, o método desenvolvido mostrou-se adequado para análises de controle de qualidade de rotina para a determinação simultânea de SMT, TMP e BMX em formulações farmacêuticas.

Potential Mechanistic Investigation of Bromhexine for the Treatment of COVID-19 / 中国药学杂志

OBJECTIVE: To investigate antiviral mechanism of bromhexine for the treatment of COVID-19. METHODS: Based on the existing literature, the infection pathways of new coronavirus(SARS-CoV-2) were systematically summarized by us, and used the SWISSDOCK molecular simulation method to carry out virtual screen systematically for key targets and marketed drugs. RESULTS: TMPRSS2/ACE2 pathway was found to be the most efficient and probably the major pathway for SARS-CoV-2 virus to infect the lung and other tissue by us. Bromhexine, an expectorant, can strongly inhibit TMPRSS2 protease (EC50:0.75 μmol•L-1) in vitro. Bromhexine has few adverse effects and also has the beneficial effects of promoting the release and maintenance of endogenous active substances in the lung, alveolar function, and promoting sputum excretion, which is suitable for use together with other COVID19 medication and therapies. CONCLUSION: Bromhexine has a unique potential antiviral mechanism, and clinical research should be conducted to play its role in the prevention, treatment and prognosis of COVID19.

Clinical study of 3g/L sodium hyaluronate eye drops with bromhexine hydrochloride tablets for dry eye / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM:To investigate the clinical efficacy of 3g/L sodium hyaluronate eye drops combined with bromhexine hydrochloride tablets on the treatment of dry eye. ·METHODS: Totally 200 patients with dry eye were randomly divided into the control group (n= 100) and observation group (n=100). Patients in two groups were given 3g/L sodium hyaluronate eye drops and physiotherapy. On the basis of this, the observation group were treated with bromhexine hydrochloride tablets. The inflammatory factors (IL-6, IL-10, TNF-α and IL-1β) levels and ocular symptom scores (OSDI, BUT, S Ⅰ t, FL) in the two groups were compared between before and after treatment. And the clinical efficacy and adverse reactions were evaluated. ·RESULTS: After treatment, the IL-6, IL-10, TNF-α, IL-1β, OSDI and FL scores in two groups were significantly lower than those before treatment, and BUT and SⅠt were significantly higher than those before treatment. Moreover, the improvement degree of the above indexes in the observation group were better than those in the control group, showing statistically significant difference (P<0.05). The total effective rate of the observation group was higher than that of the control group (χ2=5.531,P=0.019), but there was no significant difference in the incidence of adverse reactions between the two groups (χ2=0.307,P=0.579). ·CONCLUSION:As for the patients with dry eye, the combination of 3g/L sodium hyaluronate eye drops with bromhexine hydrochloride tablets can significantly decrease the level of inflammatory factors, improve the eye symptoms and the clinical total efficiency, without increasing treatment-related adverse effects.

Formula Optimization of Bromhexine Hydrochloride Dry Powder Inhalations by Central Composite Design-response Surface Methodology / 中国药师

Objective: To optimize the formula of bromhexine hydrochloride dry powder inhalations (BH DPIs). Methods: BH DPIs were prepared by freezing-drying combined with an air-jet milling method. Three factors, including the weights of mannitol (X1), leucine (X2) and poloxamer 188 (X3) in the formula were known to be associated with the quality of BH DPIs. A central composite design was used to investigate the effects of the three factors on the response angle (Y1), fine particle fraction (FPF, Y3) and aerody-namic diameter (Y4). Response surface and overlay contour plot were delineated according to the best-fit mathematic models. Opti-mum formula was selected by overlay contour plot. Results: The quantitative relationships between the three factors and the three re-sponses were obtained. The optimal formula was mannitol﹕leucine﹕poloxamer 188 (2. 4: 2. 22: 0. 05) in the excipients. The pre-dicted and observed values of the optimum formula were similar. Conclusion: The multi-objective simultaneous optimization of the for-mula of BH DPIs is achieved by central composite design-response surface methodology.

Study on the Quality and Stability of Bromhexine Hydrochloride Dry Powder Inhalations / 中国药师

Objective: To study the quality and stability of bromhexine hydrochloride dry powder inhalations (BH DPIs). Meth-ods: BH DPIs were prepared by freeze-drying with an air jetting method. The humidity and critical relative moisture of the inhalations were investigated. The aerodynamic particle size ( Dae) and water content were also analyzed. The powder morphology was observed under a scanning electronic microscope. The fine particle fraction (FPF) and delivered dose uniformity (DDU) of BH DPIs were de-termined. The stability of the inhalations was determined by accelerated testing and long-term testing. Results: The prepared BH DPIs exhibited the following properties: the critical relative humidity was 65% ; Dae was <5 μm; the water content was less than 2% ; FPF was >30% ,and DDU was good according to the requirement of Chinese Pharmacopoeia. The accelerated testing and long-term testing both indicated promising stability of BH DPIs. Conclusion: Bromhexine hydrochloride dry powder inhalations are stable, and suitable for the use in pulmonary delivery system.

Biotransformation of bromhexine by Cunninghamella elegans, C. echinulata and C. blakesleeana

Abstract Fungi is a well-known model used to study drug metabolism and its production in in vitro condition. We aim to screen the most efficient strain of Cunninghamella sp. among C. elegans, C. echinulata and C. blakesleeana for bromhexine metabolites production. We characterized the metabolites produced using various analytical tools and compared them with mammalian metabolites in Rat liver microsomes (RLM). The metabolites were collected by two-stage fermentation of bromhexine with different strains of Cunninghamella sp. followed by extraction. Analysis was done by thin layer chromatography, high performance thin layer chromatography, Fourier transform infrared spectroscopy, high performance liquid chromatography and Liquid chromatography–mass spectrometry. The role of Cytochrome P3A4 (CYP3A4) enzymes in bromhexine metabolism was studied. Fungal incubates were spiked with reference standard – clarithromycin to confirm the role of CYP3A4 enzyme in bromhexine metabolism. Three metabolites appeared at 4.7, 5.5 and 6.4 min retention time in HPLC. Metabolites produced by C. elegans and RLM were concluded to be similar based on their retention time, peak area and peak response of 30.05%, 21.06%, 1.34%, and 47.66% of three metabolites and bromhexine in HPLC. The role of CYP3A4 enzyme in metabolism of bromhexine and the presence of these enzymes in Cunninghamella species was confirmed due to absence of peaks at 4.7, 5.4 and 6.7 min when RLM were incubated with a CYP3A4 enzyme inhibitor – clarithromycin.

Analysis of related substances in bromhexine hydrochloride tablets / 中国药学杂志

OBJECTIVE: To evaluate the status of related substances in bromhexine hydrochloride tablets and to find the existing problems. METHODS: 95 batches of samples from six domestic manufacturers were tested using statutory methods combined with exploratory research, and the results were evaluated by statistical analysis. RESULTS: The related substances in 95 batches of bromhexine hydrochloride tablets met the requirement of current specification. The total amount was not more than 0.4%. The exploratory studies on domestic tablets, raw materials and tablets produced by original manufacturer revealed that 12 impurities were detected in the samples from six domestic manufacturers, which originated mainly from the raw material. The species and contents of the impurities in domestic samples were different with those in the product of original manufacturer. The structure of the unknown impurity with the highest content was identified by LC-MS/MS, and it was judged to be produced in the manufacturing process of the raw material. CONCLUSION: The related substances in domestic bromhexine hydrochloride tablets are in good control overall and have met the requirement of current statutory specification. The impurities originate mainly from the raw material, so improving the quality of the materials at the source is the critical factor for promoting the quality level of bromhexine hydrochloride tablets.

Bromhexine for acute bronchitis: A systematic review / 中国药学杂志

OBJECTIVE: To evaluate the effectiveness and safety of bromhexine for acute bronchitis. METHODS: Cochrane Library , PubMed, Embase, ISI, CBMdisc, CNKI, VIP and WanFang database were retrieved. Systematic review, meta-analysis or randomized controlled trials (RCT) comparing bromhexine with placebo, ambroxol, tanreqing and N-acetylcysteine for acute bronchitis were included. Quality assessment and Meta-analysis were performed for RCT that met the inclusion and exclusion criteria. In addition, the conclusions of the systematic review and Meta-analysis in this aspect were referred. RESULTS: Ten RCTs met the inclusion criteria, all in Chinese. No systematic review or Meta-analysis was retrieved. Five RCTs of bromhexine versus placebo were included. There was no significant difference in clinical overall efficacy [RR=1.22, 95% CI (0.88,1.69), P=0.24]. The pulmonary rale vanishing time and cough vanishing time of bromhexine were obviously shorter than that of placebo with significant difference [MD=-2.32, 95% CI (-3.34, -1.29), P < 0.00001; MD=-2.85, 95%CI(-3.12, -2.59), P < 0.00001]. Significant difference was demonstrated in adverse reaction incidence [RR=17.00, 95% CI(1.01,286.82), P=0.05]. Three RCT of bromhexine versus ambroxol were included. Outcomes of these studies could not be combined. One RCT of bromhexine versus Tanreqing were included. Meta-analysis could not be performed. One RCT of bromhexine versus N-acetylcysteine were included. Meta-analysis could not be performed. CONCLUSION: Based on current evidence, the effectiveness of bromhexine in decurtating course of pulmonary rale and cough are superior to placebo. Clinical overall efficacy is similar between bromhexine and placebo. High risk of adverse reaction incidence still exists with bromhexine. Ambroxol, Tanreqing and N-acetylcysteine exhibit better effectiveness than bromhexine, but more studies are needed to testify the results from the small quantity of studies. Copyright 2012 by the Chinese Pharmaceutical Association.

RP HPLC Method for the determination of Tamsulosin in bulk and Pharmaceutical Formulations.

A simple, sensitive, precise and specific reverse phase high performance liquid chromatographic method was developed and validated for the determination of Tamsulosin in bulk and tablet dosage forms. It was found that the excipient in the tablet dosage forms does not interfere in the quantification of active drug by proposed method. The HPLC separation was carried out by reverse phase chromatography on Shimadzu HPLC, 10-At detector with hypersil ODS C18 Column 250 X 4.6 mm (particle size of 5μ) and constant flow pump. Rheodyne injector with 20 μl loop with a mobile phase composed in the ratio acetonitrile: (0.05M) KH2PO4 buffer (45:55) at flow rate 1.8 ml /min. The detection was monitored at 240nm. The calibration curve for Tamsulosin was linear from 10-50g/ml and internal standard (Bromhexine) 10g/ml were prepared by suitable dilutions of the stock solution with appropriate mobile phase. The interday and intraday precision was found to be within limits. The proposed method has adequate sensitivity, reproducibility and specificity for the determination of Tamsulosin in bulk and its tablet dosage forms. LOD and LOQ for Tamsulosin were found to be 0.495 and 0.461.Accuracy (recoveries: 98.5-98.55%) and reproducibility were found to satisfactory.

Pharmacokinetic interaction between cefaclor and bromhexine in healthy Chinese volunteers / 中南大学学报(医学版)

Objective To determine the pharmacokinetic interaction between cefalor and bromhexine in healthy Chinese volunteers. Methods Twelve subjects received a cefaclor (CEF) treatment, a bromhexine (BHX) treatment, and a co-treatment of CEF and BHX with a 3 × 3 Latin square design. The wash-out time between periods was 14 days. The plasma and urine drug concentrations of CEF and BHX were detected by HPLC-UV and LC/MS, respectively. Results All the 12 volunteers completed the study. There were no significant differences in AUC0-t and Cmax of CEF in logarithm between the single administration group of CEF and the co-administration group of CEF with BHX. Two one sided t-test showed that CEF was bioequivalent in the 2 groups. There were no significant differences in tmax, MRT, t1/2, and Clr between the 2 groups. Vd/F was significantly lower in the single CEF group than in the co-administration group of CEF and BHX. There were no significant differences of AUC0-t and Cmax of BHX in logarithm between the single administration group of BHX and the co-administration group of BHX with CEF. Two one sided t-test showed that BHX was bioequivalent in the 2 groups. There were no significant differences in tmax, MRT, t1/2, Vd/F, and Clr between the 2 groups. Conclusion There is no significant pharmacokinetic parameter change in the drug absorption, metabolism, and excretion, but Va/F of CEF significant increases in the co-administration of CEF with BHX. The co-administration of CEF and BHX has no adverse drug interaction. The increase of Vd/F may be a favorable drug interaction, which may be the mechanism of the synergistic effect of the 2 drugs.