Study On The Degradation Products Of Amphotericin B And The Unification Between HPLC Purity And Microbiological Potency

The antibiotics are the class of substances which are produced in the living process of the bacteria, mold, or other microorganisms, they can selectively restrain or kill the microorganisms or the tumor cells at a low density. From 1941 when the penicillin was applied in the clinic till now, there are thousands of kinds of antibiotics found by the effort of microbiologist and biochemist. As the data based on the Center for Disease Control and Prevention (CDC) showed, there are more than one hundred and fifty kinds of antibiotics used in the clinic currently, including antineoplastic agents, antifungal agents, antiviral agents, anti gram-negative bacillus agents and so on. Antibiotics can be classified into the following categories by the structure:penicillins, cephalosporins, fosfomycins, vancomycins, rifomycins, polymyxins, amino sugars, tetracyclines, macrolides etc. Polyene macrolides play a very important role in the clinic as an essential part of the antibiotics. Amphotericin B, which belongs to the polyene macrolides is a conventional agent used for the cure of systemic fungal infections. The solubility of polyene macrolides is very poor, and the aggregation may occur. Due to the complicated structure, unstable chemical property, the research about polyene macrolides is relatively slow. After the review of the clinical use of amphotericin B, it was found that it is still an irreplaceable agent for curing the systemic fungal infections, so the scientists are keeping on the exploration and research on the detention, side effect, the pharmacokinetics and clinical application of amphotericn B. The polyene antifungal antibiotics, an antibiotic family amphotericin B belonging to, selectively associate ergosterol that located in fungal cell membrane to form transmembrane channels. As a result, cellular components such as potassium and magnesium ions, glucose, and amino acids, pass through the channels. There are various methods for the determination of amphotericin B, except for the Microbiological Potency assay, there are HPLC method and spectrophotometry method etc. However, in respect of its impurities control research, the further effort is needed. Amphotericin B was first isolated from Streptomyces nodosus in 1956 and has been used in the clinic since 1960 to treat a great variety of systemic fungal infections caused by Candida albicans, Cryptoccus neoformans and Aspergillus. Its molecular formula is C47H73NO17. For some serious deeply infected fungus diseases such as cereospinal menigitis of Cryptococcus neoformans, invasive aspergillosis, particularly the treatment of immunodeficiency or seriousagranulocytosis, and some local fungus, amphotericin B is still needed and irreplaceable. Amphotericin B is an important antifungal antibiotic referred to as a "golden standard" due to its high and broad spectrum of activity. In view of the importance of amphotericin B in clinical application, in this paper, the degradation products of amphotericin B have been researched systematically to explore for as much as possible information about the impurities. Different degradations of amphotericin B were obtained under different conditions to speed up the degradation reactions, then they were purified through the proper means, and the UV, NMR, MS data were determined to identify the structures for them. For one of the degradation products named amphotericin B (5), the systemic research has been done, including the primary activity test by Microbiological Potency assay, and the primary toxicity test on zebrafish animal model. The other major research content in this paper is purifying some pure amphotericin B sample by preparative HPLC, establishing a fully new HPLC method and doing the systematic methodology validation. A conclusion that the proposed HPLC method can replace the microbiological assay for potency determination of amphotericin B can be drawn after the method validation and the unification between potency and purity. In this way, the routine quality control for amphotericin B can become faster, simpler and more reliable.