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Article Cite This: ACS Omega 2019, 4, 7938−7943
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Solubility Studies of Cyclosporine Using Ionic Liquids Paula Berton,† Manish Kumar Mishra,‡,∥ Hemant Choudhary,‡,⊥ Allan S. Myerson,§ and Robin D. Rogers*,‡ †
Chemical and Petroleum Engineering Department, University of Calgary, 2500 University Drive NW, Calgary AB T2N 1N4, Canada ‡ College of Arts & Sciences, The University of Alabama, 712 Capstone Drive, Tuscaloosa, Alabama 35401, United States § Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
ACS Omega 2019.4:7938-7943. Downloaded from pubs.acs.org by 5.101.220.39 on 05/01/19. For personal use only.
S Supporting Information *
ABSTRACT: Six ionic liquids (ILs) were selected based on their chemical and physical properties to study the solubility of cyclosporine A. Of these, cyclosporine exhibited higher room temperature solubility in 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) than in acetone, an effective molecular solvent used to solubilize and purify cyclosporine. The solubility of cyclosporine in the ILs dramatically increased at higher temperatures, a critical factor that cannot be varied in a wide range with low boiling molecular solvents. The differences in solubility were explored for cyclosporine purification. Cyclosporine was purified up to ∼93% with n-butylammonium acetate ([C4NH3][OAc]) and could be further purified to 95% using an IL/organic solvent biphasic system. After purification, cyclosporine was recovered as an amorphous solid using the ILs.
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INTRODUCTION Cyclosporine A (hereafter cyclosporine) is a neutral, lipophilic, cyclic endecapeptide with a molecular weight of 1202.635 g/ mol and low aqueous solubility (Figure 1). It is widely used as
great interest in order to facilitate their purification and establish better drug delivery systems.5−7 In this case, the solubility of the hydrophobic, non-ionizable cyclosporine is determined by its crystallinity and its interactions with the solvent. The structural conformation of this drug depends on the solvent, which in turn affects its solubility; when in an apolar environment, intramolecular hydrogen bonds stabilize the β-sheet structure, while the addition of polar solvents exposes its hydrogen bonding groups and breaks such 3D structures.8,9 These conformational changes are reflected in the differences in solubility. Cyclosporine is soluble in methylene chloride (10 mg/mLclear, colorless to faint yellow solution), >100 mg/g in ethanol, methanol, acetone, ether, acetonitrile, or chloroform; while it is slightly soluble in water (0.04 mg/g) and saturated hydrocarbons (solubility in hexane: 5.5 mg/g).8 Interestingly, its solubility in water decreases with temperature (from 101.5 to 7.3 μg/L at 5 and 37 °C, respectively10), mainly due to dehydration of amino acid residues, affecting the conformation and solubility of the drug.9 Several attempts have been made toward the isolation and purification of cyclosporine. In an earlier report, cyclosporine was extracted from a biomass broth (after inactivating the enzymes) by incubating overnight with methanol in the first step, then further extracting with ethyl acetate (EtOAc) in the second step, and finally the residue from the second step was
Figure 1. Chemical structure of cyclosporine.
an immunosuppressive agent to prevent rejection in organ and tissue transplantation during surgery by reducing the activity of the patient’s immune system.1−3 Cyclosporine is generally isolated as a complex in a nutrient medium by the cultivation of the strain of Tolypocladium inflatum fungus species.4 In comparison with cyclosporine B and cyclosporine C, the production of higher concentrations of cyclosporine depends on the type of the selected fungus. The development of strategies to solubilize poorly watersoluble active pharmaceutical ingredients (APIs) has been of © 2019 American Chemical Society
Received: March 4, 2019 Accepted: April 18, 2019 Published: May 1, 2019 7938
DOI: 10.1021/acsomega.9b00603 ACS Omega 2019, 4, 7938−7943
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purified chromatographically in two stages: (1) silica gel column (mobile phase: hexane/chloroform/methanol; 10:9:1) and (2) resin column (mobile phase: methanol).11 Other reports purified cyclosporine through crystallization with PEG400 by slow temperature reduction combined with seeding.12,13 Cyclosporine can also be purified by two-stage continuous mixed-suspension, mixed-product removal, and cooling crystallization with acetone.14,15 Still, a possible route to improvement in cyclosporine separation is to enhance the solubility and purity of cyclosporine at room temperature. An emerging research field of interest is the utilization of ionic liquids (ILs, salts with melting points below 100 °C16) in pharmaceutical applications. ILs have been exploited in the pharmaceutical industry, used in organic transformations, either as catalysts and/or solvents,17−20 to turn solid drugs into liquids, or as drug delivery systems,21 and even to control the polymorphism of an API,22,23 to name a few of their applications. ILs as potential pharmaceutical solvents to dissolve poorly soluble APIs have been investigated for a range of ILs and APIs.24,25 The goal of this work was to evaluate the solubility of cyclosporine in ILs and to develop strategies for purification at room temperature. To accomplish this goal, we chose six different ILs based on their chemical and physical properties to force interactions with cyclosporine at the molecular level.
Table 1. ILs Chosen for Study and Cyclosporine Solubilities
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RESULTS AND DISCUSSION Cyclosporine is a hydrophobic branched aliphatic connected via amido linkages into a cyclic structure.26 We, therefore, chose ILs with varied lengths of alkyl chains bearing functional groups with potential to form hydrogen bonding to (a) regulate the hydrophobicity of the medium and (b) facilitate hydrogen bonding with the amido moieties of the cyclosporine. The ILs chosen for study include n-butylammonium acetate ([C 4 NH 3 ][OAc]), n-octylammonium acetate ([C8NH3][OAc]), choline acetate ([Cho][OAc]), trihexyltetradecylphosphonium chloride ([P66614]Cl), 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), and 1-ethyl-3-methylimidazolium bistriflimide ([C2mim][NTf2]). The crude cyclosporine was obtained as an amorphous powder (Figure S1, Supporting Information) with purity of 90.8% and ca. 38 impurities as determined by high performance liquid chromatography (HPLC) (Figure S7, Supporting Information). It decomposed at 336.3 °C as determined by TGA (Figure S2, Supporting Information). In order to investigate cyclosporine’s solubility in the ILs chosen for their different physical and chemical properties (Table 1), an attempt to prepare saturated solutions was made as described in the Methods section. Saturation was achieved for [C4NH3][OAc] and [C2mim][NTf2] at 25 and 100 °C; however, solutions of [P66614]Cl and [C2mim][OAc] became too viscous to stir after reaching a certain concentration (Figure 2, right) and saturation was not achieved. Also, due to the higher melting point of [C8NH3][OAc] (mp 48 °C27) and [Cho][OAc] (mp 80−85 °C28), these ILs were studied only at 100 °C. Each final solution was filtered (hot if at elevated temperature) and a known amount of the filtrate was dissolved in acetonitrile for HPLC analysis. The cyclosporine concentrations are reported in Table 1. The solubility results (Table 1, Figure 2, left) indicate that, at 25 °C, cyclosporine was most soluble in [C2mim][OAc] where it reached 24.8 wt % before the solution became too viscous to stir. This is remarkably high in comparison with the
Values without parenthesis represent “wt %”; values in parenthesis represent mmol/L. bSolubility at 53 °C. cThese are partial solubilities, as these solutions became too viscous to stir at higher concentrations (Figure 2, right). dNA: not applicable; these ILs have melting points above 25 °C. a
Figure 2. Left: Cyclosporine solubility at 25 (gray) and 100 °C (black). Right: Solutions of (a) 24.8 wt % cyclosporine in [C2mim][OAc] after stirring at 25 °C and (b) 60.2 wt % cyclosporine in [P66614]Cl after stirring at 100 °C.
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rest of the evaluated ILs and similar to its solubility in acetone (20.3 wt %), a well-known molecular solvent for the drug.12,13 The overall trend in solubility at 25 °C followed the order [C2mim][OAc] ≈ acetone > [C4NH3][OAc] (12.3 wt %) > [P66614]Cl (5.2 wt %) > [C2mim][NTf2] ( [P66614]Cl (25.3 mmol/mol IL) > [C4NH3][OAc] (15.5 mmol/mol IL) > acetone (12.3 mmol/mol acetone) > [C2 mim][NTf2 ] (negligible). At 100 °C, the solubilities of cyclosporine in all the ILs increased dramatically. As a result of the lower viscosity as the temperature was raised, more cyclosporine could be dissolved in [C2mim][OAc] (24.8−56.4 wt % or 46.5−183.4 mmol/mol IL) before the viscosity became too high to continue. An even more dramatic increase was observed for [P66614]Cl where the amount which could be dissolved before viscosity became an issue jumped from only 5.2 wt % at 25 °C to 60.2 wt % at 100 °C (23.5 vs 652.3 mmol/mol IL, respectively). A measurable amount of cyclosporine (1.2 wt %, 3.9 mmol/mol IL) could be dissolved in the hydrophobic [C2mim][NTf2] at this temperature, while the solubility in [Cho][OAc] remained negligible (50 mg/mL,31 and in hexane which is reported to be 0.99 (Figure S6, Supporting Information). For purification studies, impurities were treated as one “dummy” impurity, and the purity of cyclosporine was calculated as in eq 1
the solutions prepared above, if no solids were observed after mixing, another 5 mg of cyclosporine was added to the solutions and further stirred for 30 min. The addition of cyclosporine and stirring steps were repeated until solid precipitation was observed after mixing. The solutions were then filtered using a syringe filter (0.45 μm) to separate the undissolved particles. The filtered homogeneous solution was weighed and dissolved in acetonitrile in a 10 mg solution:1 mL acetonitrile ratio for cyclosporine quantification using HPLC. Purification of Cyclosporine. Antisolvent Strategy. Crude cyclosporine (50 mg) was added to a 15 mL glass vial containing 1 g of [C4NH3][OAc], [C2mim][OAc], and [C4NH3][OAc] systems with excess HOAc, or C4NH2, or EtOAc. The mixtures were stirred at room temperature using a magnetic stir bar (350 rpm) to complete dissolution (ca. 1 h). After dissolution, 1 mL of water was added to precipitate the cyclosporine. The precipitates were filtered under vacuum and the filters were washed with extra water to remove any remaining IL/solvent. The precipitates were redissolved in acetonitrile and analyzed using HPLC. Liquid−Liquid Extraction Strategy. Crude cyclosporine (50 mg) was added to 15 mL glass vials containing a magnetic stir bar and 1 g of [C4NH3][OAc] or [C2mim][OAc]. The mixtures were stirred at room temperature, 350 rpm until complete dissolution (ca. 1 h). After dissolution, hexane or EtOAc was added to cyclosporine in [C2mim][OAc] solution, while hexane or ethyl ether was added to cyclosporine in [C4NH3][OAc] solutions, resulting in 1:1 (IL/organic solvent) mass ratio biphasic systems. The mixtures were vortexed for 10 s, sonicated in an ultrasonic bath for 60 min, and left on the bench for 30 min until phase separation was completed. The upper, organic phase was separated using a glass Pasteur pipet. A second portion of organic solvent was added to the IL phase and the extraction and separation steps were repeated. Both organic phases were mixed and left on the bench for evaporation. If solids appeared, these were dissolved in acetonitrile and analyzed using HPLC. The IL phase was dissolved in water and the precipitated cyclosporine was filtered, dried, and dissolved in acetonitrile for HPLC analysis. HPLC Analysis. The IL-cyclosporine solutions produced from the solubility studies and the powders recovered from the cyclosporine purification experiments were dissolved in acetonitrile, filtered through a 0.45 μm filter membrane, and injected into an Agilent 1260 series HPLC system (Agilent Technologies, Santa Clara, CA, USA). The HPLC system was equipped with a quaternary pump, an autosampler, and a multiwavelength UV−vis detector. HPLC separations were carried out on a Zorbax Eclipse XDB-C18 LC Column (250 mm × 4.6 mm, 5 μm, Agilent Technologies) at 75 °C. The flow rate was 1.5 mL/min, and the injection volume was 20 μL. Following a previously reported method,34,35 an isocratic elution was selected, using a mobile phase of 52.1% acidified H2O (0.1% phosphoric acid), 43% acetonitrile, and 4.9% tertbutyl methyl ether, with a runtime of 80 min and a detection wavelength at 210 nm. Cyclosporine calibration curves were prepared for quantitative analysis of cyclosporine dissolved and/or purified. A stock solution of cyclosporine was obtained by dissolving ∼40 mg (purified cyclosporine, 95%) in 2 mL of acetonitrile. The standard solutions were obtained by subsequent dilutions of stock solutions with acetonitrile to obtain five different concentrations in the range of 0.6−6 mg/mL. A linear calibration curve was obtained by plotting the areas of the
100% area = % areacyclosporine + % area impurities
(1)
where % area is the area under the chromatograph peak observed by HPLC.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00603. Crude cyclosporine characterization (TGA, PXRD, and HPLC chromatograms), synthesis of ILs and characterization (NMR spectra), and HPLC calibration curves for cyclosporine quantification (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Paula Berton: 0000-0003-4488-6686 Hemant Choudhary: 0000-0003-2847-3080 Allan S. Myerson: 0000-0002-7468-8093 Robin D. Rogers: 0000-0001-9843-7494 Present Addresses ∥
Pharmaceutical Materials Science and Engineering Laboratory, Department of Pharmaceutics, College of Pharmacy, University of Minnesota, 9-127B Weaver-Densford Hall, 308 Harvard Street S.E., Minneapolis, MN 55455, United States. ⊥ Joint BioEnergy Institute, 5885 Hollis St, Emeryville, CA 94608, United States. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was initiated at The University of Alabama and continued in part at McGill University. We thank the NovartisMassachusetts Institute of Technology (MIT, 5710003948) Center for Continuous Manufacturing (CCM) and Novartis International AG for financial support.
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REFERENCES
(1) Oates, J. A.; Wood, A. J. J.; Kahan, B. D. Cyclosporine. N. Engl. J. Med. 1989, 321, 1725−38. (2) Tedesco, D.; Haragsim, L. Cyclosporine: A review. J. Transplant. 2012, 2012, Article ID 230386, https://www.hindawi.com/journals/ jtrans/2012/230386/. (Last accessed Feb 28, 2019). (3) Smith, C. S.; Ortega, G.; Parker, L.; Shearer, W. T. Cyclosporin A blocks induction of tumor necrosis factor-alpha in human B lymphocytes. Biochem. Biophys. Res. Commun. 1994, 204, 383−390. (4) Dreyfuss, M.; Härri, E.; Hofmann, H.; Kobel, H.; Pache, W.; Tscherter, H. Cyclosporin A and C, new metabolites from Trichoderma polysporum. Eur. J. Appl. Microbiol. 1976, 3, 125−133. (5) Zhang, X.; Xing, H.; Zhao, Y.; Ma, Z. Pharmaceutical dispersion techniques for dissolution and bioavailability enhancement of poorly water-soluble drugs. Pharmaceutics 2018, 10, No. E74. (6) Kalepu, S.; Nekkanti, V. Insoluble drug delivery strategies: review of recent advances and business prospects. Acta Pharm. Sin. B 2015, 5, 442−453. 7942
DOI: 10.1021/acsomega.9b00603 ACS Omega 2019, 4, 7938−7943
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(7) Ting, J. M.; Porter, W. W., III; Mecca, J. M.; Bates, F. S.; Reineke, T. M. Advances in polymer design for enhancing oral drug solubility and delivery. Bioconjugate Chem. 2018, 29, 939−952. (8) Czogalla, A. Oral cyclosporine A - the current picture of its liposomal and other delivery systems. Cell. Mol. Biol. Lett. 2009, 14, 139−152. (9) Hasumi, H.; Nishikawa, T.; Ohtani, H. Effect of temperature on molecular structure of cyclosporin A. Biochem. Mol. Biol. Int. 1994, 34, 505−511. (10) Ismailos, G.; Reppas, C.; Dressman, J. B.; Macheras, P. Unusual solubility behaviour of cyclosporin A in aqueous media. J. Pharm. Pharmacol. 1991, 43, 287−289. (11) Balaraman, K.; Mathew, N. Process for the preparation of cyclosporin a from tolypocladium species. U.S. Patent, 5,656,459, Aug 12, 1997; EP 0,725,076 B1, Aug 07, 1996. (12) Giron, D.; List, M.; Richter, F.; Uike, Y.; Weber, H. P. Ciclosporin in non-solvated orthorhombic crystal form, processes for its preparation and pharmaceutical composition containing; Deutsches Patent- und Markenamt, DE 3843054C2, 1989. (13) Johnas, S. K. J.; Meents, A.; Weckert, E.; Messerschmidt, M.; Weckert, E. F. Charge density study on cyclosporine A. Acta Crystallogr D Biol Crystallogr 2009, 65, 284−293. (14) Alvarez, A. J.; Singh, A.; Myerson, A. S. Crystallization of cyclosporine in a multistage Continuous MSMPR crystallizer. Cryst. Growth Des. 2011, 11, 4392−4400. (15) Wong, S. Y.; Tatusko, A. P.; Trout, B. L.; Myerson, A. S. Development of continuous crystallization processes using a singlestage mixed-suspension, mixed-product removal crystallizer with recycle. Cryst. Growth Des. 2012, 12, 5701−5707. (16) Holbrey, J. D.; Rogers, R. D. In Ionic Liquids in Synthesis, 1st ed.; Wasserscheid, P., Welton, T., Eds.; Wiley-VCH: Weinheim, 2002; pp. 41−55. (17) Sheldon, R. Catalytic reactions in ionic liquids. Chem. Commun. 2001, 0, 2399−2407. (18) Seddon, K. R. Ionic liquids: A taste of the future. Nat. Mater. 2003, 2, 363−365. (19) Wasserscheid, P.; Stark, A. In Handbook of Green Chemistry: Green Solvents; Ionic Liquids; Anastas, P. T., Ed.; Wiley-VCH: Weinheim, 2010; Vol. 6. (20) Choudhary, H.; Berton, P.; Gurau, G.; Myerson, A. S.; Rogers, R. D. Ionic liquids in cross-coupling reactions: “liquid” solutions to a “solid” precipitation problem. Chem. Commun. 2018, 54, 2056−2059. (21) Adawiyah, N.; Moniruzzaman, M.; Hawatulaila, S.; Goto, M. Ionic liquids as a potential tool for drug delivery systems. Med. Chem. Commun. 2016, 7, 1881−1897. (22) Mukherjee, A.; Rogers, R. D.; Myerson, A. S. Cocrystal formation by ionic liquid-assisted grinding: case study with cocrystals of caffeine. CrystEngComm 2018, 20, 3817−3821. (23) Zeng, Q.; Mukherjee, A.; Müller, P.; Rogers, R. D.; Myerson, A. S. Exploring the role of ionic liquids to tune the polymorphic outcome of organic compounds. Chem. Sci. 2018, 9, 1510. (24) Mizuuchi, H.; Jaitely, V.; Murdan, S.; Florence, A. T. Room temperature ionic liquids and their mixtures: potential pharmaceutical solvents. Eur. J. Pharm. Sci. 2008, 33, 326−331. (25) Smith, K. B.; Bridson, R. H.; Leeke, G. A. Solubilities of pharmaceutical compounds in ionic liquids. Chem. Eng. Data Ser. 2011, 56, 2039−2043. (26) Vine, W.; Bowers, L. D.; Shaw, L. M. Cyclosporine: Structure, pharmacokinetics, and therapeutic drug monitoring. Crit. Rev. Clin. Lab. Sci. 1987, 25, 275−312. (27) Cláudio, A. F. M.; Pereira, J. F. B.; McCrary, P. D.; Freire, M. G.; Coutinho, J. A. P.; Rogers, R. D. A critical assessment of the mechanisms governing the formation of aqueous biphasic systems composed of protic ionic liquids and polyethylene glycol. Phys. Chem. Chem. Phys. 2016, 18, 30009−30019. (28) Choline Acetate, MSDS, Sigma Aldrich. https://www. sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country= CA&language=en&productNumber=670189&brand= ALDRICH&PageToGoToURL=https%3A%2F%2Fwww.
sigmaaldrich. com%2Fcatalog%2Fproduct%2Faldrich%2F670189%3Flang%3Den (Last accessed Feb 28, 2019). (29) Cheng, F.; Wang, H.; Chatel, G.; Gurau, G.; Rogers, R. D. Facile pulping of lignocellulosic biomass using choline acetate. Bioresour. Technol. 2014, 164, 394−401. (30) Berton, P.; Kelley, S. P.; Wang, H.; Myerson, A. S.; Rogers, R. D. Separate mechanisms of ion oligomerization tune the physicochemical properties of n-butylammonium acetate: cation-base clusters vs. anion-acid dimers. Phys. Chem. Chem. Phys. 2017, 19, 25544−25554. (31) Cyclosporin A from Tolypocladium inflatum, data sheet, Sigma Aldrich. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/ docs/Sigma/Datasheet/c1832dat.pdf (Last accessed Feb 28, 2019). (32) Zhang, Q.; Benoit, M.; De Oliveira Vigier, K.; Barrault, J.; Jérôme, F. Green and inexpensive choline-derived solvents for cellulose decrystallization. Chem. Eur J. 2012, 18, 1043−1046. (33) McCrary, P. D.; Beasley, P. A.; Gurau, G.; Narita, A.; Barber, P. S.; Cojocaru, O. A.; Rogers, R. D. Drug specific, tuning of an ionic liquid’s hydrophilic−lipophilic balance to improve water solubility of poorly soluble active pharmaceutical ingredients. New J. Chem. 2013, 37, 2196−2202. (34) Li, J.; Lai, T.-t. C.; Trout, B. L.; Myerson, A. S. Continuous crystallization of cyclosporine: Effect of operating conditions on yield and purity. Cryst. Growth Des. 2017, 17, 1000−1007. (35) Li, J.; Trout, B. L.; Myerson, A. S. Multistage continuous mixed-suspension, mixed-product removal (MSMPR) crystallization with solids recycle. Org. Process Res. Dev. 2016, 20, 510−516.
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