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 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 . 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 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 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.

2009 The Authors. Journal compilation 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 , 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-Q 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,

2009 The Authors. Journal compilation 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 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. The diluted solubility diagram of vorinostat complexed in solutions were centrifuged at 10 000 g for 10 min various types of CDs is shown in Fig. 2. Several and carefully observed for precipitates; 2 mL of qualitative observations can be made from the vorinostat–HP-b-CD solution (10 mg ⁄ mL) was diagram; significant increases in apparent aqueous freeze-dried overnight. The dried product was then solubility of vorinostat were obtained when the re-dissolved in isotonic phosphate butter and made concentrations of RM-b-CD, HP-b-CD, HP-a-CD up to 2 mL. The re-dissolved solutions were checked and a-CD were increased. Most significantly, RM- for the presence of re-precipitates. Both procedures b-CD increased the aqueous solubility of vorinostat were carried out in triplicate. by approximately 100-folds when vorinostat was complexed in 300 mM RM-b-CD, resulting in a maximal solubility of 70Æ8mM (18Æ7mg⁄ mL). Molecular simulation A molecular simulation study was performed to Phase-solubility 80 study the complexation of vorinostat with the various CDs. Software SYBYL version 7Æ2 (Tripos

Co., St Louis, MO, USA) was used for the study. ) 60

M α-CD Dimethyl-b-cyclodextrin (DM-b-CD) was adopted HP-α-CD β to facilitate determination of stable structure of 40 HP- -CD RM-b-CD as RM-b-CD (degree of substitution = 1Æ8) RM-β-CD γ is a mixture of different structures. Four 2-hy- -CD Vorinostat (m HP-γ-CD droxypropyl groups were added on the primary 20 hydroxyl groups of a-CD and b-CD, as shown by Mura et al., to simulate the structure of HP-a-CD 0 (degree of substitution: 0Æ5–0Æ8) and HP-b-CD 0 100 200 300 400 Cyclodectrin (mM) (degree of subtitution: 0Æ6), respectively (13). The structures of vorinostat, a-CD, HP-a-CD, HP-b-CD, Fig. 2. Phase-solubility study of vorinostat in various DM-b-CD, c-CD and HP-c-CD were individually CDs.

2009 The Authors. Journal compilation 2009 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 34, 1–6 4 Y. Y. Cai et al.

Similarly, the solubility of vorinostat was increased 500 ⁄ M in this study. From these values, it could be to 43Æ2mM (11Æ4mg⁄ mL) in the presence of 300 mM foreseen that upon intravenous administration, HP-b-CD. In comparison, HP-c-CD increased the HP-b-CD, a-CD or HP-a-CD would not alter the aqueous solubility of vorinostat only 4-fold. At pharmacokinetic profile of vorinostat. The mecha- HP-c-CD concentration higher than 100 mM, the nisms of drug release from the CD complexes have enhancement in solubility of vorinostat was less also been studied. It has been reported that after than twice. The aqueous solubility of vorinostat intravenous administration, the major driving force beyond 150 mM a-CD and 150 mM c-CD cannot be for dissociation of weakly to moderately bound determined because of limitation in the intrinsic drugs in the inclusion complex is simple dilution aqueous solubility of these CDs. (15). In most cases, the release of drug molecules The phase-solubility curves of HP-a-CD, a-CD, from CD complexes is rapid and quantitative (15). HP-b-CD, RM-b-CD and HP-c-CD were linear Drug ⁄ CD complexes are continually forming and 2 (R >0Æ99) and displayed a typical AL type of dissociating with lifetimes of milliseconds or less phase-solubility profile, indicating that the (15). Thus, this equilibrium kinetics should not have complex formation is first order with respect to the a significant impact on the elimination kinetics. CD and may be first or higher orders with respect Moreover, is also able to to the drug (14). As the slopes of the phase-solu- induce drug dissociation from the CD-drug com- bility curves are smaller than 1 in all the CDs, the plex. Besides, vorinostat binds weakly with CDs but formation of a 1 : 1 inclusion complex is suggested moderately with plasma proteins (approximately between vorinostat and the various CDs (14). This 71%) (4). Therefore, HP-b-CD, a-CD or HP-a-CD justifies the tabulation of the apparent inclusion should work as a parenteral vehicle for vorinostat rate constant (K1:1), calculated with the equation without changing its pharmacokinetic profile. established by Higuchi and Connors (12). However, such postulation needs to be confirmed in The tabulated apparent inclusion rate constant an actual pharmacokinetic study.

(K1:1) values (shown in Table 1) indicated that the complexation affinity between vorinostat and the Dilution and re-dissolution of various CDs are in the following order: RM-b- vorinostat-HP-b-CD complex CD > HP-b-CD > a-CD > HP-a-CD > HP-c-CD > c-CD. Hence, in aqueous solutions of vorinostat No precipitation was observed in any of the and CDs, the free drug molecules are in equilib- samples with 5-, 10- 50-, 100- and 500-fold dilutions rium with the drug molecules entrapped within the with isotonic phosphate buffer (pH value of 7Æ4). cavities. On increasing the concentration of the The freeze-dried product prepared from the solu- CDs, more vorinostat molecules will transfer from tion of vorinostat in 300 mM HP-b-CD was re-dis- the aqueous solution to the hydrophobic cavities of solved in purified water and then made up to the CD, increasing the apparent solubility of vori- 2 mL. A clear colourless solution of vorinostat was nostat. formed and no re-precipitation occurred. No covalent bond is generated during the In current clinical practice, vorinostat is only formation of the inclusion complex. Therefore, the available in the form of 100 mg capsule with a binding affinity between CD and vorinostat was daily oral dose of 400 mg (4). Assuming its oral not exceptionally strong. In fact, the K1:1 values bioavailability is 43% (16), the intravenous dose of between the CDs and vorinostat were all less than vorinostat would be approximately 200 mg. The aforementioned amount of vorinostat could be Table 1. Inclusion rate constant between vorinostat and administered intravenously in 20 mL of 300 mM CDs HP-b-CD. As the clinical safety for intravenous administration of HP-b-CD has been documented, HP- HP- RM- HP- a parenteral formulation of vorinostat formulated a-CD a-CD b-CD b-CD c-CD with HP-b-CD is feasible. Such a formulation would provide an alternative dosing route for Cyclodextrin 188Æ7 123Æ4 218Æ3 414Æ844Æ0 )1 vorinostat and might improve its therapeutic K (M ) 1:1 usage in oncology. Vorinostat is well known for

2009 The Authors. Journal compilation 2009 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 34, 1–6 Solubilization of vorinostat by cyclodextrins 5

Total energy Steric energy Electrostatic Inclusion complex (kcals ⁄ mol) (kcals ⁄ mol) energy (kcals ⁄ mol)

Vorinostat-DM-b-CD )34.5 )17.559 )17.019 Vorinostat -HP-b-CD )33.2 )17.509 )15.766 Vorinostat -a-CD )32.4 )17.910 )14.564 Vorinostat -HP-a-CD )31.4 )17.588 )13.835 Vorinostat - HP-c-CD )30.2 )14.950 )15.320 Vorinostat - c-CD )29.1 )10.709 )18.444

Table 2. Interaction energies for DM-b-CD, dimethyl-b-cyclodextrin; HP-b-CD, hydroxypropyl-b-cyclodextrin; the complexes formed by vorinostat a-CD, a -cyclodextrin; HP-a-CD, hydroxypropyl- a-cyclodextrin; and various CDs HP- c –CD, hydroxypropyl-c-cyclodextrin; c-CD, c –cyclodextrin. its ability to provide synergistic anti-cancer effects mode 2, the benzene ring of the simulated vorino- with conventional chemotherapeutic agents in cell stat molecule was inserted into the CD cavity and culture models (3). Furthermore, the recent onco- allowed to dock freely. After several attempts, the logical trials of vorinostat indicated its values in preferred docking mode was found to be mode 1. combination (5). Diarrhoea, nausea Fig. 3 shows the hypothetical structures of the and vomiting are common side-effects of cancer complexes between vorinostat and the various chemotherapy. The oral bioavailability of vorino- CDs. As aforementioned, the alkyl chain of stat can be affected because of these problems and vorinostat is enclosed by the CD and its aromatic the therapeutic efficacy may be affected. A par- ring situated at the rim of the CD cavity. The enteral formulation of vorinostat could overcome simulated diagrams suggested that vorinostat and such issues. Oral administration of capsules is also the various CDs undergo a 1 : 1 stoichiometric not convenient for the small children or comatose interaction. Interaction energies of the complexes, patients. Under such circumstance, an oral liquid including its steric and electrostatic energies, are of vorinostat formulated with HP-b-CD or RM-b- tabulated and presented in Table 2. The magni- CD may be useful. tude of the interaction energy indicates the strength of interaction between vorinostat and the respective CD molecule. The strengths of interac- Molecular simulation study tion are decreasing in the following order: vori- Two different inclusion modes were attempted nostat-DM-b-CD > vorinostat-HP-b-CD > vorinostat- when the docking was performed. In mode 1, the a-CD > vorinostat-HP-a-CD > vorinostat-HP-c-CD > alkyl chain was introduced into the CD cavity. In vorinostat-c-CD.

(a)Side view (b)Side view with electron surface (c)Top view (d) Top view with electron surface

α-CD

HP-α-CD

HP-β-CD

RM-β-CD Fig. 3. Hypothetical structure of the vorinostat-CD inclusion complex.

2009 The Authors. Journal compilation 2009 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 34, 1–6 6 Y. Y. Cai et al.

The results obtained from the molecular simu- 3. Bolden JE, Peart MJ, Johnstone RW (2006) Anticancer lation study were consistent with the findings in activities of histone deacetylase inhibitors. Nature the phase-solubility study. This suggests that Reviews Drug Discovery, 5, 769–784. molecular modelling accurately predicted the dif- 4. FDA (2006) Available at: http://www.accessdata. ferences in binding affinity between vorinostat and fda.gov/scripts/cder/drugsatfda/index.cfm?fuseac tion=Search.Label_ApprovalHistoryapphist (access- the various CDs. Molecular simulation appears to ed 08 December 2008). be useful for the selection of appropriate CD as 5. ClinicalTrials.gov (2009). A Service of the US excipient for drug delivery. CDs are generally National Institute of Health. Available at: http://www. used to improve the aqueous solubility of guest clinicaltrials.gov. (accessed 04 April 2009). drug molecules. However, different CDs show 6. Lin HS, Hu CY, Chan HY et al. (2007) Anti-rheumatic different binding affinities for a specific guest activities of histone deacetylase (HDAC) inhibitors molecule. Therefore, it is time-consuming to in vivo in collagen-induced arthritis in rodents. identify the most appropriate combination of CD British Journal of Pharmacology, 150, 862–872. and guest drug experimentally. With the rapid 7. Hockly E, Richon VM, Woodman B et al. (2003) development of computational chemistry, the Suberoylanilide hydroxamic acid, a histone deacet- stoichiometric interaction and the interaction ylase inhibitor, ameliorates motor deficits in a mouse energies of the complexes could be predicted model of Huntington’s disease. Proceedings of the National Academy of Sciences of the United States of in silico. Based on such information, the combina- America, 100, 2041–2046. tion with good binding affinity could be selected 8. Kelly WK, Richon VM, O’Connor O et al. (2003) for further investigation. Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered CONCLUSION intravenously. Clinical Cancer Research, 9, 3578–3588. 9. Davis ME, Brewster ME (2004) Cyclodextrin-based Cyclodextrins, namely a-CD, HP-a-CD, HP-b-CD, pharmaceutics: past, present and future. Nature RM-b-CD and HP-c-CD increased the apparent Reviews Drug Discovery, 3, 1023–1035. aqueous solubility of vorinostat in a concentration- 10. Willems L, van der Geest R, de Beule K (2001) dependent manner. RM-b-CD and HP-b-CD Itraconazole oral solution and intravenous formula- enhanced the solubility of vorinostat most. Future tions: a review of and pharma- studies could focus on HP-b-CD for parenteral codynamics. Journal of Clinical Pharmacy and Therapeutics, 26, 159–169. delivery of vorinostat or on RM-b-CD as an oral 11. Lin HS, Chean CS, Ng YY, Chan SY, Ho PC (2000) absorption enhancer. Molecular simulation was 2-hydroxypropyl-beta-cyclodextrinincreasesaqueous useful for the selection of an appropriate CD. Fur- solubility and photostability of all-trans-retinoic acid. ther investigations of the biopharmaceutical prop- JournalofClinicalPharmacyandTherpeutics,25,265–269. erties of such formulations are warranted. 12. Higuchi T, Connors KA (1965) Phase-solubility techniques. Advances in Analytical Chemical Instru- ments, 4, 117–212. ACKNOWLEDGEMENT 13. Mura P, Bettinetti G, Melani F, Manderioli A (1995) The authors are grateful for the financial support Interaction between naproxen and chemically modi- from Agency for Science, Technology and Research, fied beta-cyclodextrins in the liquid and solid state. Republic of Singapore (BMRC 06 ⁄ 1 ⁄ 21 ⁄ 19 ⁄ 441). European Journal of Pharmaceutical Sciences, 3, 347–355. 14. Brewster ME, Loftsson T (2007) Cyclodextrins as pharmaceutical solubilizers. Advanced Drug Delivery REFERENCES Reviews, 59, 645–666. 15. Stella VJ, Rao VM, Zannou EA, Zia VV (1999) 1. Marks PA, Breslow R (2007) Dimethyl sulfoxide to Mechanisms of drug release from cyclodextrin vorinostat: development of this histone deacetylase complexes. Advanced Drug Delivery Reviews, 36, 3–16. inhibitor as an anticancer drug. Nature Biotechnology, 16. Kelly WK, O’Connor OA, Krug LM et al. (2005) Phase I 25, 84–90. study of an oral histone deacetylase inhibitor, sube- 2. Choo QY, Ho PC, Lin HS (2008) Histone deacetylase roylanilide hydroxamic acid, in patients with advan- inhibitors: new hope for rheumatoid arthritis? ced cancer. Journal of Clinical Oncology, 23, 3923–3931. Current Pharmaceutical Design, 14, 803–820.

2009 The Authors. Journal compilation 2009 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 34, 1–6