Lysozyme Hydrochloride

Lysozyme (E.C. 3.2.1.17), an about 14 kDa protein and pI 11, widely spread in nature, is present in humans mainly in milk, saliva, and intestinal mucus as a part of innate defense mechanisms. It is endowed with antimicrobial activity due to its action as an N-acetylmuramidase, cleaving the 1-4β glycosidic linkage in the peptidoglycan layer of Gram-positive bacteria. This antimicrobial activity is exerted only against a limited number of Gram-negative bacteria. Different action mechanisms are proposed to explain its activity against Gram-negative bacteria, viruses, and fungi. The antiviral activity prompted the study of a possible application of lysozyme in the treatment of SARS-CoV-2 infections. Among the different sources of lysozyme, the chicken egg albumen was chosen, being the richest source of this protein (c-type lysozyme, 129 amino acids). Interestingly, the activity of lysozyme hydrochloride against SARS-CoV-2 was related to the heating (to about 100 °C) of this molecule. A chemical-physical characterization was required to investigate the possible modifications of native lysozyme hydrochloride by heat treatment. The FTIR analysis of the two preparations of lysozyme hydrochloride showed appreciable differences in the secondary structure of the two protein chains. HPLC and NMR analyses, as well as the enzymatic activity determination, did not show significant modifications.

The aim of this work was to develop medicated chewing gums (MCGs) containing 10 mg of lysozyme hydrochloride (LH) and 20 mg of ascorbic acid (AsA) obtained by the compression method with Health in Gum® (HiG®) PWD 01 as a compressible gum base. Because of a low content of active ingredients, it was essential to choose the way of adding them to the tableting mass and evaluate their distribution homogeneity in the dosage units. The blends for compression were prepared by two methods: the first one was simple mixing of all components; the second one included the step of wet granulation of a three-component mixture - LH, sucralose and a taste additive. Flow properties of LH, AsA, HiG®, LH granules and blends for compression were studied. MCGs were evaluated according to Ph.Eur. 9.0 Chapters 2.9.5, 2.9.6 and 2.9.40. AsA and HiG® were characterized as free flowing, while LH had insufficient flow properties. Compared with a simple mixed blend, the granulation step allowed significantly improving flow properties of the final blend for compression. Unlike MCGs compressed from the simple mixed blend, MCGs prepared through the granulation step met Ph.Eur. 9.0 Chapter 2.9.40 requirements. The propriety of MCG preparation method involving the step of wet granulation also has been confirmed by mass and drug content uniformity tests.