Solubilization of Vorinostat by Cyclodextrins
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Journal of Clinical Pharmacy and Therapeutics (2009) 34, 1–6 doi:10.1111/j.1365-2710.2009.01095.x ORIGINAL ARTICLE Solubilization of vorinostat by cyclodextrins Y. Y. Cai* BSc,C.W.Yap*PhD,Z.Wang*BEng,P.C.Ho*PhD,S.Y.Chan*PhD, K. Y. Ng* PhD,Z.G.Ge PhD MBBS and H. S. Lin* PhD *Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore and Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China using RM-b-CD as an oral absorption enhancer. SUMMARY Molecular simulation appeared to be a useful tool Background: Vorinostat (suberoylanilide hydro- for the selection of appropriate CD as excipient for xamic acid) is the first histone deacetylase inhib- drug delivery. itor approved by US FDA for use in oncology. However, as a hydrophobic acid, its limited Keywords: cyclodextrin, inclusion complex, mole- aqueous solubility poses a problem for parenteral cular simulation, phase solubility, vorinostat delivery. Such limited solubility may also affect its oral bioavailability. INTRODUCTION Objective: The aim of this study was to evaluate whether cyclodextrins (CDs), common excipients Vorinostat, also known as suberoylanilide used in pharmaceutical industry, could increase hydroxamic acid or N-hydroxy-N¢-phenyloctane- the aqueous solubility of vorinostat. diamide (Fig. 1), is a histone deacetylase inhibitor Methods: The actual aqueous solubility of originally developed by Marks and Breslow for its vorinostat was investigated by phase-solubility anti-neoplastic effects (1). Vorinostat effectively method. Molecular simulation was employed to induces cell cycle arrest, cell differentiation and ⁄ or predict the interaction energy and preferred apoptotic cell death in various transformed cells at orientation of vorinostat in CD cavities. micromolar level (1–3). The clinical efficacy of Results: Phase-solubility studies indicated that vorinostat in oncology had been demonstrated and )1 the solubility of vorinostat (7Æ24 · 10 mM) the drug was approved by US FDA for the treat- was substantially increased when complexed ment of cutaneous T cell lymphoma in October with various CDs, in the following order: 2006 (1, 4). The applications of vorinostat in other randomly methylated-b-cyclodextrin (RM-b-CD) > solid or haematological malignancies are currently hydroxypropyl-b-cyclodextrin (HP-b-CD) > a-cyclo- under extensive clinical investigations (2, 5). dextrin > hydroxypropyl-a-cyclodextrin > Hydroxy- Besides anti-neoplastic effects, the anti-inflamma- propyl-c-cyclodextrin > c-cyclodextrin. RM-b-CD tory activity of vorinostat has been documented in 300 mM increased vorinostat solubility to 70Æ8mM, preclinical models of various auto-immune disor- almost two orders of magnitude higher than the ders, including inflammatory bowel diseases, baseline solubility. Such findings were in good rheumatoid arthritis and systemic lupus erythe- agreement with the results obtained from molec- matosus (2, 6). Moreover, vorinostat was shown to ular simulation. ameliorate motor deficits in a mouse model of Conclusion: CDs, particularly RM-b-CD and HP-b- Huntington’s disease (7). CD, increased vorinostat’s solubility. Future studies could be focused on the application of H HP-b-CD in parenteral delivery of vorinostat or O N O Received 9 December 2008, Accepted 28 April 2009 Correspondence: Hai-Shu Lin, Department of Pharmacy, Faculty HN of Science, National University of Singapore, 10 Kent Ridge OH Crescent, Singapore 119260. Tel.: +65 6516 6537; fax: +65 6779 1554; e-mail: [email protected] Fig. 1. Molecular structure of vorinostat. Ó 2009 The Authors. Journal compilation Ó 2009 Blackwell Publishing Ltd 1 2 Y. Y. Cai et al. Vorinostat is a hydrophobic acid with limited propyl-c-cyclodextrin (HP-c-CD) (degree of sub- aqueous solubility (approximately 0Æ2mg⁄ mL), and stitution: approximately 0Æ8) was obtained from therefore difficult to formulate for parenteral Research Biochemicals International (Natick, MA, delivery. In an early phase I clinical trial, vorinostat USA). HP-b-CD (degree of substitution: approxi- was administered as a 2-h infusion (8). The mately 0Æ6) was donated by Roquette (Lestrem, intravenous infusion solution was prepared by France). a-cyclodextrin (a-CD), hydroxypropyl- dissolving vorinostat with sodium hydroxide and a-cyclodextrin (HP-a-CD; degree of substitu- adjusting the pH value to 11Æ2 with hydrochloric tion: approximately 0Æ5–0Æ8), RM-b-CD (degree of acid (8). Such a high pH value makes this formula- substitution: approximately 1Æ8) and c-cyclodextrin tion unsuitable for routine clinical practice as tedious (c-CD) were generous gifts from Wacker (Burghau- preparation procedures are required. Moreover, the sen, Germany). Chromatographic grade methanol limited aqueous solubility of vorinostat may also and acetonitrile were obtained from Tedia (Fairfield, affect its oral bioavailability. Therefore, it is of OH, USA). Formic acid was purchased from interest to develop new formulations of vorinostat Fluka Chemika (Buchs, Switzerland). Pure water for both oral and parenteral use. (18Æ2 MW cm at 25 °C) was generated from a Milli- Cyclodextrins (CDs), cyclic oligosaccharides pore Direct-QÒ ultra-pure water system (Billerica, derived from starch, are well known for their MA, USA) and used throughout the study. ability to form inclusion complexes with small molecules and portions of large compounds (9). High performance liquid chromatography (HPLC) Such inclusion complexes usually offer increased assay aqueous solubility, improved oral bioavailability and enhanced chemical stability. Moreover, the High performance liquid chromatography assays parenteral safety of hydroxypropyl-b-cyclodextrin were performed for the phase-solubility study. A (HP-b-CD) had been well documented and HP-b- Shimadzu (Kyoto, Japan) 2010A liquid chromato- CD-based intravenous formulations of itraconazole graphy was used for the analysis. The separation and mitomycin are routinely used in the clinic (9, 10). was performed on a reversed-phase HPLC column Therefore, HP-b-CD may be a suitable excipient for (Zorbax Eclipse XDB-C18, 3 · 250 mm, 5 lm; parenteral delivery of vorinostat. Furthermore, in a Agilent, Palo Alto, CA, USA), protected by a pilot study, Hockly et al. solubilized vorinostat with mechanical filter (Rheodyne, Cotati, CA, USA). The HP-b-CD and supplied it as a therapeutic agent with assay was performed at 40 °C through isocratic drinking water to mice with motor deficits (7). delivery of the mobile phase, consisting of acetoni- However, the phase solubility and physicochemical trile – 0Æ1% formic acid (22 : 78, v ⁄ v). The flow rate properties of such inclusion complexes have not was set at 0Æ8mL⁄ minand 10 lL samplewas injected been studied. Moreover, randomly methylated- into the HPLC system for each run. Vorinostat was b-cyclodextrin (RM-b-CD), which usually is better at quantified by measuring UV absorbance at 241 nM, improving aqueous solubility and oral bioavailabil- and calibrated through an external standard method. ity of the complexed drug, has never been studied. The calibration curve was linear (R2 >0Æ999) within Therefore, it is hypothesized that both HP-b-CD, the range of 1Æ0–50Æ0 lg ⁄ mL. The intra-day and RM-b-CD as well as other CD could be appropriate inter-day variations were all less than 4%. excipients for the delivery of vorinostat. To confirm this hypothesis, phase-solubility and molecular Phase-solubility study simulation of the inclusion complexes prepared with various CDs were investigated in this study. The intrinsic aqueous solubility of vorinostat in purified water was first assessed. Vorinostat–CD inclusion complexes were then prepared by a me- MATERIALS AND METHODS thod previously reported (11). An excess amount of vorinostat (20 mg ⁄ mL, except 30 mg ⁄ mL in RM-b- Materials CD) was added to CD solutions at different Vorinostat was purchased from Toronto Research concentrations (a-CD: 10, 20, 40, 80, 120 mM; c-CD Chemicals (North York, ON, Canada). Hydroxy- and HP-c-CD: 10, 20, 50, 100, 150 mM; HP-a-CD, Ó 2009 The Authors. Journal compilation Ó 2009 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 34, 1–6 Solubilization of vorinostat by cyclodextrins 3 HP-b-CD and RM-b-CD: 10, 20, 30, 50, 100, 200, minimized by MMFF94s force field in the afore- 300 mM). The suspension was then shaken on a mentioned software. After minimization, docking horizontal rotary shaker at a speed of 200 rpm for experiments were carried out on all molecules using 2 days. Subsequently, it was filtered through a the ‘Dock’ module in SYBYL. This allowed the 0Æ20-lM syringe filter (Sartorius, Hanover, Germany) vorinostat to move within the energy field of the CDs to obtain a clear solution. The concentration of and vice versa, in order to find the most preferable vorinostat in inclusion complex solutions was binding geometries. The interaction energy was determined by HPLC assay. The apparent inclusion computed for each of the complexes in their most rate constant (K1:1) was calculated with the favourable conformation. equation established by Higuchi and Connors (12): Slope1 K1 : 1 ¼ ; RESULTS AND DISCUSSION S0 ð1 À Slope1Þ Phase-solubility study where Slope1 is slope of the phase-solubility curve and S0 is the solubility of vorinostat in pure water. Phase-solubility study is a traditional approach used to measure the stability constants. In addition, Dilution and re-dissolution study of it also gives insight into the stoichiometry of the vorinostat-HP-b-CD complex equilibrium (14). In this study, the intrinsic aque- In order to examine the suitability of HP-b-CD as a ous solubility of vorinostat in pure water was parenteral excipient to deliver vorinostat, vorino- assessed and found to be 191Æ4 lg ⁄ mL (7Æ24 · )1 stat-HP-b-CD solution (10 mg vorinostat was fully 10 mM), a value consistent with the ‘Full Pre- dissolved in 1 mL 300 mM HP-b-CD) was diluted scription Information’ provided by its developer with isotonic phosphate buffer (pH = 7Æ4) to pro- (4). The results were plotted and the phase- duce 5, 10, 50, 100 and 500 dilutions.