Hydrophilic interaction liquid chromatography method for eremomycin determination in pre-clinical study.

A complex of hydrophilic interaction liquid chromatography (HILIC) methods for simple and efficient determination of eremomycin (ERM) as an active pharmaceutical ingredient (API) of a novel drug is proposed for preclinical study, which includes the dissolution test and pharmacokinetic study on the animals. A home-made HILIC silica-based stationary phase (SP) containing diol functionalities and positively charged nitrogen atoms in its structure was synthesized for this research and applied for the first time for performing the first step of preclinical study (dissolution test) of the novel ERM-containing drug. HILIC method developed using novel home-made SP allowed us to avoid any interferences from polyethylene glycol (PEG) contained in the drug matrix thus providing a unique advantage of the proposed approach over RP HPLC. The home-made SP demonstrated better chromatographic performance as compared to the tested commercially available columns with various functionalities. Different retention behaviour and mechanisms with various electrostatic impact were demonstrated for two glycopeptide antibiotics, namely, ERM and its analogue vancomycin (VAN), on the home-made SP. For the second step of the preclinical study HILIC-MS/MS method for ERM determination in rabbit plasma was developed and validated in accordance with the EMA requirements and successfully applied to the preclinical study on rabbits after intravenous and intraperitoneal drug administration. The results of dissolution test and pharmacokinetic study revealed similar in vitro solubility of ERM and VAN and low ERM bioavailability, which proved the potential safety and efficiency of the novel drug.

Eremomycin pyrrolidide: a novel semisynthetic glycopeptide with improved chemotherapeutic properties.

PURPOSE: Development of new semisynthetic glycopeptides with improved antibacterial efficacy and reduced pseudoallergic reactions. METHODS: Semisynthetic glycopeptides 3-6 were synthesized from vancomycin (1) or eremomycin (2) by the condensation with pyrrolidine or piperidine. The minimum inhibitory concentration (MIC) for the new derivatives was measured by the broth micro-dilution method on a panel of clinical isolates of Staphylococcus and Enterococcus. Acute toxicity (50% lethal dose, maximum tolerated doses), antibacterial efficacy on model of systemic bacterial infection with S. aureus and pseudoallergic inflammatory reaction (on concanavalin A) of eremomycin pyrrolidide (5) were evaluated in mice according to standard procedures. RESULTS: The eremomycin pyrrolidide (5) was the most active compound and showed a high activity against Gram-positive bacteria: vancomycin-susceptible staphylococci and enterococci (minimum inhibitory concentrations [MICs] 0.13-0.25 mg/L), as well as vancomycin-intermediate resistant Staphylococcus aureus (MICs 1 mg/L). Antimicrobial susceptibility tested on a panel of 676 isolates showed that 5 had similar activity for the genera Staphylococcus and Enterococcus with MIC90=0.5 mg/L, while vancomycin had MIC90=1-2 mg/L. The number of resistant strains of Enterococcus faecium (vancomycin-resistant enterococci) (MIC =64 mg/L) with this value was 7 (8%) for vancomycin (1) and 0 for the compound 5. In vivo comparative studies in a mouse model of systemic bacterial infection with S. aureus demonstrated that the efficacy of 5 was notably higher than that of the original antibiotics 1 and 2. In contrast to 1, compound 5 did not induce pseudoallergic inflammatory reaction (on concanavalin A). CONCLUSION: The new semisynthetic derivative eremomycin pyrrolidide (5) has high activity against staphylococci and enterococci including vancomycin-resistant strains. Compound 5 has a higher efficacy in a model of staphylococcal sepsis than vancomycin (1) or eremomycin (2). In striking contrast to natural antibiotics, the novel derivative 5 does not induce a pseudoallergic inflammatory reaction to concanavalin A and therefore has no histamine release activity. These results indicate the advantages of a new semisynthetic glycopeptide antibiotic eremomycin pyrrolidide (5) which may be a prospective antimicrobial agent for further pre-clinical and clinical evaluations.