RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology Crystal Structure and Physicochemical Characterization of Ambazone Monohydrate, Anhydrous, and Acetate Salt Solvate MARIETA MURESAN-POP, 1 DARIO BRAGA, 2 MIHAELA M. POP, 1 GHEORGHE BORODI, 1 IRINA KACSO, 1 LUCIA MAINI 2 1 National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca R-400293, Romania 2 Dipartimento di Chimica G. Ciamician, Universit` a di Bologna 40 126, Bologna, Italy Received 10 April 2014; revised 7 August 2014; accepted 11 August 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24151 ABSTRACT: The crystal structures of the monohydrate and anhydrous forms of ambazone were determined by single-crystal X-ray diffraction (SC-XRD). Ambazone monohydrate is characterized by an infinite three-dimensional network involving the water molecules, whereas anhydrous ambazone forms a two-dimensional network via hydrogen bonds. The reversible transformation between the monohydrate and anhydrous forms of ambazone was evidenced by thermal analysis, temperature-dependent X-ray powder diffraction and accelerated stability at elevated temperature, and relative humidity (RH). Additionally, a novel ambazone acetate salt solvate form was obtained and its nature was elucidated by SC-XRD. Powder dissolution measurements revealed a substantial solubility and dissolution rate improvement of acetate salt solvated form in water and physiological media compared with ambazone forms. Also, the acetate salt solvate displayed good thermal and solution stability but it transformed to the monohydrate on storage at elevated temperature and RH. Our study shows that despite the requirement for controlled storage conditions, the acetate salt solvated form could be an alternative to ambazone when solubility and bioavailability improvement is critical for the clinical efficacy of the drug product. C  2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: ambazone; antimicrobial; X-ray diffractometry; crystal structure; polymorphism; acetate salt solvate; proton transfer; dissolution rate; stability INTRODUCTION Poor physicochemical properties of pharmaceutical compounds can be improved by obtaining different crystalline forms includ- ing polymorphs, hydrates, salts, and cocrystals of active phar- maceutical ingredients (APIs). Salts of APIs are used when the API is not sufficiently soluble or stable due to the fact that dif- ferent salt forms may have different profiles, or bioavailability properties. 1–4 Ambazone, [4-(2-(diaminomethylidene) hydrazinyl)phenyl] iminothiourea or (p-benzoquinone amidinohydrazone thiosemi- carbazone), is the active ingredient of Faringosept, 5 an anti- septic for the oral cavity because it is active in a number of pathogens that trigger infections of the mouth and upper res- piratory tract. Moreover, ambazone has been used for a long time as a medicine for bacteriostatic activity, allowing the re- placement of antibiotics in topical treatment of oral pharyngeal infections. 6–11 Further studies showed a possible anticancer ac- tivity of ambazone because of the amidinohydrazone group be- ing an anticancer agent. 12,13 The commercial form of ambazone used in the pharmaceutical industry is the monohydrate form, which is slightly soluble in water and has a variable 35%– 50% oral bioavailability affecting the therapeutic effect of the Faringosept tablets. The molecular structure of ambazone (Fig. 1) is characterized by a thiosemicarbazone group and an amidinohydrazone group, these two groups allow the existence of several tautomers, and Correspondence to: Lucia Maini (Telephone: +39-051-2099597; Fax: +39-051- 2099456; E-mail: l.maini@unibo.it) This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/. Journal of Pharmaceutical Sciences C  2014 Wiley Periodicals, Inc. and the American Pharmacists Association two of those are shown in Figure 1. Only one conformation of the thiosemicarbazone group is observed in the crystal struc- tures reported in the Cambridge Structure Database (CSD), whereas two different tautomers are observed for the amidino- hydrazone group. The hydrogen position of amidinohydrazone group in tautomer A (which is commonly reported in sketches of ambazone) is present in only one structure (RAGNAQ) 14 in the CSD, whereas the disposition of the hydrogen atoms in the tautomeric conformation B is present in nine struc- tures (AZGIY, HAJFIP, MAXYOZ, 15 MAXYUF, 15 MENMEY, 16 NTRGUA, 17 RAGNEU, 14 TANPEE, 18 WASPIP 19 ). The C–N dis- tances observed in the structures of ambazone monohydrate (AMB·H 2 O) and ambazone anhydrous forms (AMB) are compa- rable with the ones reported for the tautomeric disposition B and this conformation is adopted in this paper. A total of six salts of ambazone have been reported in literature, 20–25 but no crystal structure is reported so far. In this article, we present the first crystal structures of the monohydrate and anhydrous forms of ambazone together with their characterization and relative stability investigation. Also, we report a novel acetate salt solvate form of ambazone [AMBAc]·HAc and its potential use in an oral drug product. EXPERIMENTAL Materials The API ambazone monohydrate was purchased from Microsin S.A. (Bucharest, Romania) and was used without further purifi- cation. Acetic acid glacial and all other chemicals were supplied by Sigma-Aldrich (Bucharest, Romania) and were of reagent grade. Muresan-Pop et al., JOURNAL OF PHARMACEUTICAL SCIENCES 1