RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology Methyl-Beta-Cyclodextrins: The Role of Number and Types of Substituents in Solubilizing Power ´ EVA FENYVESI, JULIANNA SZEM ´ AN, KATALIN CSABAI, MILO MALANGA, LAJOS SZENTE CycloLab Cyclodextrin Research and Development Laboratory Ltd, Budapest, Hungary Received 27 November 2013; revised 5 February 2014; accepted 6 February 2014 Published online 1 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23917 ABSTRACT: Methylated cyclodextrins (CDs) are effective solubilizers of poorly soluble organic compounds. In this work, we compared various methylated -CDs concerning their structure characterized by nuclear magnetic resonance spectroscopy, composition analyzed by HPLC and solubilizing capability by using model compounds such as cholesterol, fatty acids, furosemide, tamoxifen, and amiodarone. All the commercially available methylated -CDs are mixtures of various isomers and homologues except trimethyl -CD. The effects of the degree of methylation, the composition, as well as the influence of further derivatization with ionic groups were studied. The number of methyl groups in a CD ring should be around 14 to get the highest solubility for the included guest molecules. Although the distribution of isomers and related compounds has hardly any effect at constant degree of substitution, the introduction of amino and succinyl moieties on the CD ring adds ionic interactions to the hydrophobic interactions of the inclusion complex formation, which might result in synergic effect in solubilization. C  2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1443–1452, 2014 Keywords: complexation; inclusion compounds; capillary electrophoresis; HPLC; NMR; degree of substitution; ionic cyclodextrin deriva- tives INTRODUCTION Cyclodextrins (CDs) are cyclic oligosaccharides obtained by enzymatic transformation of starch. The most common three variants, "-, $-, and (-CDs, having 6, 7, or 8 glucopyranose units, respectively, are widely used in various industries. 1–3 The inclusion complex formation of these macrocycles result- ing in enhanced solubility, stability, reduced volatility, and so on of the included guest molecules made them versatile ex- cipients in pharmaceutical formulations, food, cosmetics, and pesticides. Especially the $-CD and its derivatives are proper host molecules for many biologically active compounds. 1 Because of the low solubility of $-CD (18 g/L), its highly solu- ble derivatives are used for many purposes: the hydroxypropyl and sulfobutyl derivatives are listed in the EU and US pharma- copoeias as excipients, whereas the methyl derivatives are ap- plied in other industrial and analytical processes and products. 4 Moreover, hydroxypropyl $-CD (hydroxypropylbetadex) is ac- cepted as orphan drug for the treatment of Niemann-Pick dis- ease. By methylation of the hydroxyl groups on $-CD, products with a wide range of degree and pattern of substitution can be obtained depending on the way of preparation. 5 The degree of substitution (DS) gives the average number of substituents in a CD molecule (usually determined by nuclear magnetic res- onance, NMR), whereas the pattern gives the position of the substituents. The DS of commercially available methylated $- CDs falls in the range of 4–21. In trimethyl $-CD (TRIMEB), all the hydroxyl groups are methylated (DS 21). Correspondence to: ´ Eva Fenyvesi (Telephone: +36-1347-6075; Fax: +36-1347- 6068; E-mail: fenyvesi.e@cyclolab.hu) Dedicated to the memory of late Prof. J´ ozsef Szejtli on his 80 birthday. This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/. Journal of Pharmaceutical Sciences, Vol. 103, 1443–1452 (2014) C  2014 Wiley Periodicals, Inc. and the American Pharmacists Association Figure 1 shows the chemical formula of dimethyl $-CD hav- ing methyl groups on the C-2 and C-6 carbon atoms in each glucopyranose unit (DS 14) abbreviated as DIMEB. In the industrially produced DIMEB products, however, in addition to the heptakis(2,6-di-O-methyl)-$-CD various DIMEB isomers containing 2,6-, 2,3-, and 3,6-dimethyl glucopyranose units in various ratios as well as to a smaller extent some over- and undermethylated isomers can be found. The percentage of the target heptakis(2,6-di-O-methyl)-$-CD isomer in the product gives the value of 2,6-DIMEB content. It is usually around 50% in the industrial products, but even 98% 2,6-DIMEB con- tent was described as single isomer for capillary electrophoresis applications. 6 In this work, we characterized the composition and com- plex formation of methylated $-CD derivatives commercially available either as industrial products or as fine chemicals. The degree and pattern of substitution as well as the effect of additional ionic substituents were studied in solubilization ex- periments using cholesterol, polyunsaturated fatty acids, and ionic drugs, such as amiodarone, furosemide, and tamoxifen as model compounds of poor aqueous solubility. MATERIALS AND METHODS Materials The commercially available methylated CDs involved in this study are listed in Table 1. The ionic CD derivatives used are all the fine chemicals of CycloLab (Table 2, Fig. 2). The cholesterol, the drugs, and fatty acids were purchased from Sigma-Aldrich (Budapest, Hungary). As neutral and acidic model compounds, cholesterol and furosemide were used, respectively, whereas as basic drugs, amiodarone and tamox- ifen were used. The two long-chain fatty acids: eicosapentaenic Fenyvesi et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1443–1452, 2014 1443