Chemical-pharmaceutical analysis and microbiological and stability studies of daptomycin injectable preparations

Author(s)

Eliane Gandolpho Tótoli Belavenuto

• Document type: Doctoral Thesis
• Press: Araraquara. 2016-11-07.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2016-09-29
• Advisor: Hérida Regina Nunes Salgado; Sanjay Garg

Abstract

Daptomycin is the first approved member from a new class of antimicrobial agents, the cyclic lipopeptides, and presents selective action against Gram-positive bacteria, including methicillin and vancomycin resistant strains. This antimicrobial agent is commercially distributed in lyophilized powder as CubicinTM. Since daptomycin is a new drug in the market, there are few studies in the literature regarding the development of analytical methodologies and stability studies applied to this cyclic lipopeptide. In this way, researches in this area are essential and highly relevant to optimize the analysis of this drug in the industry and ensure the quality of the marketed product. This work developed and validated analytical methods for qualitative and quantitative analysis of daptomycin in pharmaceutical formulation and its stability and microbiological studies. Regarding the qualitative analysis, the physical characteristics and melting point of daptomycin were studied. The following qualitative methods were also performed: thin layer chromatography, spectrophotometry in ultraviolet (UV) region, high performance liquid chromatography (HPLC), capillary eletrophoresis (CE) and spectrophotometry in medium infrared (IR) region, which allowed the daptomycin identification. For the quantitative analysis, the following methods were developed and validated: spectrophotometry in the UV region, microbiological method by turbidimetric assay, HPLC, CE and spectrophotometry in medium infrared (IR) region. The UV spectrophotometric method was developed in a wavelength of 221 nm, with concentration linear range from 6.0 to 21.0 μg/mL, using the water as solvent. The microbiological method employed Staphylococcus aureus ATCC 6538 IAL 2082 as the microorganism-test, and presented a concentration linear range from 8.0 to 18.0 μg/mL. The HPLC method used detection wavelength of 221 nm and mobile phase consisted of water and ethanol (45:55, v/v) with the pH adjusted to 4.5 with glacial acetic acid, obtaining a retention time of 5.7 minutes. This method also showed its stability-indicating ability. The evaluated linear range was from 20.0 to 70.0 μg/mL. The CE method was performed at 200 nm with an eletrolite consisting of potassium phosphate buffer solution (15 mM), pH 7.0, yielding an average migration time of 1.7 minutes and ability to separate degradation products. The IR spectrophotometry was carried out in the spectral range from 1700 to 1600 cm-1. The linear range was from 0.2 to 0.6 mg/150 mg. The developed methods stand out for not using organic solvents in their analysis, fitting the parameters of Green Chemistry. Stability studies of daptomycin in lyophilized powder and in the pure form were conducted. The pharmaceutical product showed to be stable under the accelerated (25 oC ± 2 oC/ UR 60% ± 5%) and long term (5 ºC ± 3 ºC) studied conditions. On the other hand, daptomycin in pure form proved to be unstable in alkaline, acidic and oxidative conditions. Microbiological studies of daptomycin were also performed and daptomycin showed to be very active against strains of methicillin-resistant S. aureus, S. pneumoniae, and vancomycin-resistant enterococci. However, 15% of tested S. aureus strains (sensitive to methicillin) showed to be resistant. Regarding the synergy studies, combinations of daptomycin + LP9666 and daptomycin + NCL 195 are promising and deserve further studies in this regard. The studies conducted in this work will contribute to assess the quality, safety and therapeutic efficacy of daptomycin in lyophilized powder, in addition to expanding the scientific knowledge related to this antimicrobial agent. (AU)

• FAPESP's process: 13/02349-0 - Chemical-pharmaceutical analysis and stability studies of daptomycin injectable preparations
• Grantee: Eliane Gandolpho Tótoli Belavenuto
• Support Opportunities: Scholarships in Brazil - Doctorate