Stability evaluation of selected anthelmintic macrocyclic lactone compounds and formulations

Abstract

External and internal parasitic infections are a leading cause of loss of the productivity and mortality in farm animals, and illness in companion animals and subtropical human populations. Various anthelmintic drugs and their preparations are used for control of these parasites. Macrocyclic lactone (ML) compounds such as avermectins (e.g. abamectin, ivermectin, eprinomectin) and milbemycins (e.g. milbemycin oxime and moxidectin) are among most important anthelmintics, used extensively and globally. However, excessive and uncontrolled use of MLs and other anthelmintic classes in the last two decades has resulted in a global anthelmintic resistance problem. Given the lack of new molecules, anthelmintic resistance management techniques mainly rely on the use of mixtures of ML substances with anthelmintics from different chemical classes (e.g. levamisole and closantel). ML formulations with a single active pharmaceutical ingredient (API) are slightly unstable, due to their sensitivity towards pH, light and oxidation. However, formulations containing a combination of MLs and other APIs possess significantly inferior stability profiles. Furthermore, other APIs present in combination are usually at least 10-fold higher in concentration than their ML counterparts, significantly affecting the stability of the formulation and resulting in excessive unknown impurities, higher toxicity and inferior efficacy. Accordingly, this thesis aimed to evaluate selected currently used MLs and their formulations and to investigate their physico-chemical properties and degradation pathways with the ultimate aim of improving the stability of their formulations. During experiments, topical preparations (pour-on) of abamectin and eprinomectin were evaluated for stability to determine the kinetic behavior of the API in formulations. The analysis of APIs and degradation products (DPs) was carried out using newly developed and validated high-pressure liquid chromatography (HPLC) methods. In addition, new protocols were designed to isolate, purify and characterize unknown DPs using chromatography, Fouriertransform mass spectrometry (FT-MS), H/D exchange experiments, 1H, 13C and 1D/2D correlation (HMBC, COSY, DEPT, NOESY and HSQC) nuclear magnetic resonance (NMR) spectroscopy. Two major DPs found during studies on abamectin were successfully identified as 2-epi-abamectin and Δ2,3-abamectin. In the case of eprinomectin, an issue presented by the inability of the pharmacopeial method to separate an impurity that was merged with the drug peak was resolved by developing and validating a new HPLC method, followed by identification of the impurity. The results showed that this impurity was a stereoisomer 2-epi-abamectin, which had remained undetected for a decade due to exceptional similarities in UV spectrum and molecular mass (914.13 Da) with the parent compound and the fact that it eluted with the eprinomectin B1a peak. Similarly, moxidectin was investigated for stability, degradation pathways and identity/origin of the impurities presented in pharmacopeia. The stability profile of moxidectin was explored by forced degradation studies. The unknown impurities found in stress samples were isolated, purified and characterized as 23-keto-moxidectin and 3,4-epoxy-moxidectin. This project relied on a multidisciplinary collaboration of pharmaceutical scientists and chemists from the University of Auckland, Victoria University of Wellington and National Institute for Pharmaceutical Education and Research (NIPER), India, and was supported by Ancare Scientific Ltd. The scientific data collected during research are expected to improve the understanding of the chemical behavior of MLs and the findings may help in development of stable formulations of MLs in combination with other APIs. In addition, it is hoped the outcome of the eprinomectin analysis will alert USP to amend the existing monograph. The moxidectin review outcome will serve as a foundation for developing a compendial formulation monograph and the stress study results will provide support for identification of the DPs and novel degradation pathways. Furthermore, the newly developed analytical protocol will guide effective characterization of unknown DPs and establish degradation pathways.