Determination of the Degradation Chemistry of the Antitumor Agent Pemetrexed Disodium.
Stress-testing (forced degradation) studies have been conducted on pemetrexed disodium heptahydrate (1) (LY231514·2Na·7H2O) drug substance in order to identify its likely degradation products and establish its degradation pathways. Solid samples of the drug substance were stressed under various conditions of heat, humidity, and light, and solutions of the drug substance were stressed under various conditions of heat, light, oxidation, and over a wide pH range (1-13). The stressed samples were analyzed using a gradient elution reversed-phase HPLC method. The 7 major degradation products detected in the stress-testing studies were isolated, and the structures were elucidated via spectroscopic characterization. The structures of the degradation products and their proposed mechanisms of formation indicate that 1 degrades via 2 main pathways: oxidation and hydrolysis. Of the 7 identified degradation products, 6 are proposed to result from oxidation and 1 from hydrolysis.
Enrichment of Relevant Oxidative Degradation Products in Pharmaceuticals With Targeted Chemoselective Oxidation.
The ability to produce and isolate relatively pure amounts of relevant degradation products is key to several aspects of drug product development: (a) aid in the unambiguous structural identification of such degradation products, fulfilling regulatory requirements to develop safe formulations (International Conference on Harmonization Q3B and M7); (b) pursue as appropriate safety evaluations with such material, such as chronic toxicology or Ames testing; (c) for a specified degradation product in a late-stage regulatory filing, use pure and well-characterized material as the analytical standard. Producing such materials is often a resource- and time-intensive activity, either relying on the isolation of slowly formed degradation products from stressed drug product or by re-purposing the drug substance synthetic route. This problem is exacerbated if the material of interest is an oxidative degradation product, because typical oxidative stressing (H2O2 and radical initiators) tends to produce a myriad of irrelevant species beyond a certain stress threshold, greatly complicating attempts for isolating the relevant degradation product. In this article, we present reagents and methods that may allow the rapid and selective enrichment of active pharmaceutical ingredient with the desired oxidative degradation product, which can then be isolated and used for purposes described above.
New method for analysis of enzymatic reactions: Thermolysin-catalyzed synthesis of aspartame precursor
Aspartame is an artificial sweetener which is 200 times sweeter than sucrose. A precursor of aspartame, N-(benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester (Z-APM) was synthesized and hydrolyzed with thermolysin in an aqueous monophasic system. Graphical plotting methods and/or nonlinear regression have been used to analyze enzymatic reactions because of the nonlinearity of enzymatic rate equations. There are, however, problems: the former require accurate measurement of the initial reaction rate and the latter gives no analytical information. The authors proposed a method to solve the nonlinear enzymatic rate equations with respect to the concentration of a product with the Taylor expansion, especially for the reversible Theorell-Chance mechanism (Murakami, Y., Hirata, M., and Hirata, A., J. Ferment. Bioeng., 82, 246–252, 1996). This solution, however, is complex for practical use. In this paper, a simpler solution to explain the thermolysin-catalyzed synthesis of Z-APM is obtained. New methods to determine the kinetic parameters, especially for the reversible Theorell-Chance mechanism, based on the approximated solution are proposed. The results calculated using the obtained kinetic parameters can satisfactorily explain the experimental results, which demonstrates the effectiveness of the proposed analysis. We must know the relation between the concentrations of substrates and a product, and accurately evaluate of the result obtained using Taylor expansion when we determine the kinetic parameters using our newly developed methods. The methods to determine the kinetic parameters of the reversible Theorell-Chance mechanism described in this paper is, however, expected to be applied to general enzymatic reactions.