Mechanistic Study of HPMC-Prolonged Supersaturation of

Communication pubs.acs.org/crystal

Mechanistic Study of HPMC-Prolonged Supersaturation of Hydrocortisone Xiaotong Yang, Boyuan Shen, and Yanbin Huang* Key Laboratory of Advanced Materials (MOE), Department of Chemical Engineering, Tsinghua University, Beijing 100084, China S Supporting Information *

ABSTRACT: Supersaturation formulation is an effective way to improve the oral bioavailability of poorly water-soluble drugs, and water-soluble polymers are commonly used to prolong the supersaturation. However, the mechanism for such supersaturation prolonging effect is not clearly understood. Using the hydrocortisone/hydroxypropyl methylcellulose (HPMC) pair as a model system, we found that HPMC prolongs hydrocortisone supersaturation by inducing the formation of a metastable crystal polymorph of higher solubility, hence lowering the actual supersaturation level in the solution. A possible mechanism is proposed to explain the selective polymorph formation in the presence of polymers.

concentration is very low and has little effect on the bulk solution viscosity.20 Though all these proposed mechanisms are reasonable, our understanding of the polymer-prolonged supersaturation is far from being complete, as exemplified by the fact that there is no general principle available to identify the optimal polymers based simply on the drug chemical structure and properties. Therefore, more mechanistic studies are still needed. In this manuscript, we studied the hydrocortisone/hydroxypropyl methylcellulose (HPMC) system in detail and proved that HPMC prolonged hydrocortisone supersaturation by making the drug precipitate crystallize in a metastable polymorph, suggesting that polymers can interfere with the drug crystallization process beyond inhibiting nucleation and crystal growth. Hydrocortisone (Scheme 1) is a steroid hormone with low solubility, and it is known to have three polymorphs (Forms I, II, and III).22 The commercially available hydrocortisone crystal

The dissolution profile of drugs has a great influence on their oral bioavailability. However, many recently developed drugs are poorly water-soluble.1 Various strategies such as solid dispersion, nanosizing, and cocrystals are used to improve the drug dissolution.2,3 Most of these approaches can be explained with the “spring and parachute” concept;4,5 i.e., by means of these technologies, drugs first dissolve rapidly to generate a supersaturated solution (“the spring”), and more importantly, this thermodynamically unstable supersaturation state needs to be maintained for an extended period of time for effective drug absorption (“the parachute”).6,7 Without the prolonged supersaturation, drugs would quickly precipitate, and the advantages of these solubilization technologies would be significantly compromised. Various additives, especially water-soluble polymers, have been used to prolong the supersaturation.3,8−10 The proposed mechanisms in which polymers can prolong the supersaturation of drugs include (1) increasing drug solubility, i.e., polymers may interact with drug molecules to form water-soluble complex (similar to drug−cyclodextrin complexation) and hence increase the equilibrium solubility of the drugs and lower the true supersaturation level;11,12 (2) inhibiting crystal nucleation, i.e., polymers may interact with the drug clusters (or amorphous precipitate) and, via mechanisms not yet clearly understood, prevent crystal nuclei formation and hence maintain a supersaturation level at least equal to the amorphous drug solubility;13−16 (3) inhibiting crystal growth, i.e., polymers may adsorb on the drug crystal surface, occupy the crystal growth sites, and form a barrier to inhibit drug diffusion, and hence keeps more drug molecules in the supersaturated solution;13,16−21 (4) increasing solution viscosity and hence slowing down the drug molecule mobility.12,20 This last one seems not that important as the supersaturation-prolonging effect is usually already very significant even when the polymer © 2015 American Chemical Society

Scheme 1. Chemical Structure of Hydrocortisone

Received: December 8, 2014 Revised: January 9, 2015 Published: January 12, 2015 546

DOI: 10.1021/cg501784n Cryst. Growth Des. 2015, 15, 546−551

Communication

Crystal Growth & Design

unnecessary to separate supernatant and precipitate and enable continuous monitoring. As shown in Figure 1, with the initial concentration of 6 mg/ mL (supersaturation level about 20, defined as S = C/Cs, where

is in Form I, which is also the most stable crystal form at room temperature.22 In 1996, Loftsson and co-workers reported that low concentrations (0.10−0.25 wt %) of HPMC significantly increased the aqueous solubility of hydrocortisone, while polyvinylpyrrolidone (PVP) had a much less effect.11 In their study, favorable drug−polymer interactions were proposed to be the reason for HPMC-enhanced hydrocortisone solubility. With their results in mind, we studied in detail the hydrocortisone precipitation behavior in the presence of a low concentration of HPMC as well as PVP. Materials and Methods. 2,2-Dimethyl-2-silapentane-5-sulfonic acid sodium salt (DSS), poly(vinylpyrrolidone) (PVP, MW 40 000 Da), and hydroxypropyl methylcellulose (HPMC, MW ∼10 000 Da, methoxy content 29 wt %, hydroxypropyl content 7 wt %, equivalent to Methocel E, viscosity grade 6 cP) were purchased from SigmaAldrich. Hydrocortisone (HCS) was purchased from Alfa Aesar. Dimethyl sulfoxide (DMSO) and acetonitrile were purchased from Beijing Chemical Company. All reagents were of analytical grade and used as received. (a) Concentration Determination. The precipitation process of HCS from different solution was monitored by 1H NMR (JNM-ECA300, JEOL), and DSS was added as the internal standard for quantification. As a typical example, HCS was first dissolved in DMSO-d6 at the concentration of 150 mg/mL. HPMC or PVP was predissolved in D2O at the concentration of 2 mg/mL (with a DSS concentration of 0.48 mg/mL). Then, 40 μL HCS/DMSO-d6 solution was added into 1 mL D2O (or the polymer/D2O solution) to make the initial drug concentration as 6 mg/mL. After mixing with a turbine-mixer for 20 s, 500 μL of the supersaturating HCS solution was transferred to a NMR tube and the 1H NMR spectrum was obtained every 3 min. The HCS concentration was determined by comparing the integral area of HCS at 0.81 ppm to that of DSS at 0.00 ppm. All experiments were repeated 3 times at room temperature. (b) Precipitate Characterization. To analyze the precipitate solid, the precipitation experiments were repeated (using deionized water instead of D2O), and the solid from different solutions at specific time points was collected via centrifugation. After being dried in a vacuum overnight, the precipitate was characterized by powder X-ray diffraction (PXRD, D8 ADVANCE, Bruker) at room temperature with a scan speed of 2° 2θ/min under Cu Kα radiation. Polarized optical microscopy (XS-402, Nikon) equipped with a digital camera (WVGP420, Panasonic) and a hot stage (KEL-XMT-3100, WEITU, China) was used to observe the morphology and thermal behavior of the collected HCS solid. The thermal behavior of the dried precipitate was also characterized by differential scanning calorimetry (DSC-60, SHIMADZU) from 50 to 230 °C with a heating rate of 10 °C/min. (c) Solubility Determination. The solubility of different HCS crystal forms in aqueous solutions was also measured by 1H NMR. The HCS raw material was in crystal Form I, and Form II was prepared following the protocol in the literature.22 Unfortunately, we could not obtain pure Form III following the same protocol. As detailed below, Form III was instead obtained by precipitating HCS in 2 mg/mL HPMC aqueous solution, and the resulting crystals were washed thoroughly with deionized water to remove as much adsorbed HPMC as possible. Excess amounts of HCS in different crystal forms (I, II, or III) were then added into different solutions (D2O, 2 mg/mL PVP-D2O solution and 2 mg/mL HPMC-D2O solution). After being stirred for 72 h at room temperature, the supernatant was collected via centrifugation for 1H NMR measurement to determine the solubility, and the residual crystals were collected for PXRD tests. The solubility tests were also repeated three times for each condition.

Figure 1. 1H NMR-visible concentration profiles of HCS in different solutions (n = 3): (▲): in the 2 mg/mL HPMC solution; (⧫): in the 2 mg/mL PVP solution; (■): in the heavy water.

Cs is the equilibrium concentration of a specific crystal form), HCS precipitated quickly from the solution without polymers, while HPMC (at the concentration of 2 mg/mL) kept the initial supersaturation level for at least 20 min, after which the HCS concentration started to decrease at a rate much slower than that without polymer. After 95 min, the HCS concentrations were 3.62 and 1.02 mg/mL with and without the HPMC, respectively. To compare, in the PVP solution, HCS started to precipitate shortly with the concentration curve falling in between those with and without HPMC, and reached 1.90 mg/mL after 95 min. The morphology of the precipitates taken from different solutions after 4 h of precipitation was observed under polarized optical microscopy (POM). As shown in Figure 2, the precipitates from water are plate-shaped, while all the crystals obtained from the HPMC solution are rod-shaped. The crystals obtained from the PVP solution have two types of morphology: rod-shaped and granular. PXRD demonstrated that the HCS crystals obtained from deionized water and from the HPMC solution are in Form I and Form III respectively, while those from the PVP solution are a mixture of these two crystal forms (Figure 3). As demonstrated in the literature, HCS Form III is metastable and will transform to more stable crystal forms at around 190 °C on heating.22 Differential scanning calorimetry (DSC) further confirmed the existence of HCS Form III from the PVP and HPMC groups, as evidenced by the exothermic peak in the DSC thermograms at around 190 °C (Figure 4, more clearly in the inset). On further heating, the new crystals from the HPMC and PVP groups melted at 218.0 and 225.1 °C respectively, while the precipitate from the water group melts at 223.7 °C. The reason for the lower melting point observed in the HPMC group is unclear, but maybe the existence of HPMC made the crystal transformation incomplete or less perfect.

Results and Discussion. The precipitation kinetics of HCS in aqueous solutions was monitored using the 1H NMR method,23 and DSS was used as internal standard24 for quantification. 1H NMR assay is good at detecting the true concentration of the drug molecules in the supersaturated solution, as drug molecules in the precipitate are invisible in NMR when the precipitate size is larger than 20 nm,23,25 thus making it 547

DOI: 10.1021/cg501784n Cryst. Growth Des. 2015, 15, 546−551

Communication

Crystal Growth & Design

Figure 2. POM pictures of HCS precipitates after 4 h of precipitation, from left to right: (A) deionized water, scale bar 100 μm; (B) 2 mg/mL PVP solution, scale bar 150 μm; (C) 2 mg/mL HPMC solution, scale bar 150 μm.

Figure 4. From the top to the bottom, DSC thermograms of HCS precipitates obtained from deionized water, 2 mg/mL PVP solution, and 2 mg/mL HPMC solution. The heating curves of HCS obtained from PVP solution and HPMC solution showed an exothermic peak at around 190 °C, while the one of the water group did not (more clearly shown in the inset).

reported in the literature, for example, in the dipyridamole system.26,27 We then tried to determine the minimum HPMC concentration required for the polymorph change of the HCS precipitate. The precipitates from solutions (initial HCS concentration 8 mg/mL) with a series of HPMC concentrations (from 0.001 mg/mL to 5 mg/mL) were collected after 4 h of precipitation and analyzed by PXRD. As shown in Figure 5, significant characteristic diffraction peaks of HCS Form I can be observed when the HPMC concentration is lower than 1 mg/mL, suggesting that the minimal HPMC concentration to completely inhibit the formation of HCS Form I is between 1 and 2 mg/mL. Interestingly, even at the lowest HPMC concentration tested (0.001 mg/mL), significant fractions of the HCS precipitate are still in Form III. Next, we studied the stability of Form III in different aqueous solutions. HCS Form III did not transform to other polymorphs in water even after 10 days at room temperature (Figure S2, Supporting Information). In contrast, when a mixture of HCS Form I and III (i.e., HCS crystals precipitated from the 0.01 mg/mL HPMC solution, Figure 5E) was incubated in water or in 0.01 mg/mL HPMC solutions for 72 h, the HCS Form III disappeared and likely transformed to Form I. However, when the same crystal mixture was incubated in 2 mg/mL HPMC solution, no obvious crystal transformation was observed (Figure S3). Therefore, it can be concluded that (a)

Figure 3. PXRD patterns of the obtained crystals after 4 h of precipitation from (A) 2 mg/mL PVP solution; (B) 2 mg/mL HPMC solution; (C) deionized water (initial HCS concentration 6 mg/mL); and the calculated patterns of HCS crystals of (D) Form III and (E) Form I based on the crystallography data in ref 22.

Hot-stage POM observation was also consistent with the DSC results (Figure S1, Supporting Information). Therefore, the presence of HPMC in the solution completely changed the polymorph of the precipitate HCS crystal, while PVP did so incompletely. Similar phenomena (i.e., polymers changing crystal polymorph of drug precipitate) have been 548

DOI: 10.1021/cg501784n Cryst. Growth Des. 2015, 15, 546−551

Communication

Crystal Growth & Design

Figure 5. PXRD patterns of the obtained HCS crystals after 4 h of precipitation from solutions with different HPMC concentrations (initial HCS concentration 8 mg/mL). The HPMC concentrations from bottom to top are (A) 5 mg/mL, (B) 2 mg/mL, (C) 1 mg/mL, (D) 0.1 mg/mL, (E) 0.01 mg/mL, and (F) 0.001 mg/mL respectively.

respectively, representing only about 6% increase caused by the HPMC. Again, the solubility data in PVP solutions fell in between the two systems. For comparison, we also determined the solubility of Form II. It should be noted that, for HCS Form II, the measured solubility data in D2O and 2 mg/mL PVP solution are little different from that of Form I, while the solubility in the HPMC solution is much higher than that of Form I. The PXRD patterns of the residual solid showed that the HCS Form II had transformed to Form I in D2O and the PVP solution, while no obvious crystal form transformation was observed in the HPMC solution (Figure S4, Supporting Information). Therefore, the measured solubility of HCS Form II in water and PVP solution was actually the solubility of Form I, and similar to the Form III case, HPMC can inhibit the crystal form transition of Form II. For HCS Form III, no crystal form transformation occurred in all three solutions tested (Figure S5, Supporting Information), and hence the measured solubility represented the true solubility of Form III. The solubility of HCS Form III in the 2 mg/mL HPMC solution is much higher (by 60%) than that of Form I without polymer additives. With the same apparent HCS concentrations (e.g., 6 mg/mL), the true supersaturation level of the two

in the absence of crystal Form I, the HCS crystal Form III is apparently stable in water even without polymer additives; (b) in the presence of Form I, Form III will disappear, but this transformation can be inhibited with enough HPMC in the solution. The solubility of different polymorphs of HCS crystal in D2O is determined again by 1H NMR (Table 1). For Form I, its solubility in D2O is about 0.295 mg/mL, while the presence of 2 mg/mL HPMC increased it by about 40% to 0.410 mg/mL. For HCS crystal Form III, its solubility in D2O and in the 2 mg/mL HPMC solution are 0.446 and 0.475 mg/mL, Table 1. Measured Solubility of HCS Polymorphs in Different Solutions (unit: mg/mL, N = 3) D2O 2 mg/mL PVP solution 2 mg/mL HPMC solution a

Form I

Form II

Form III

0.295 ± 0.020 0.352 ± 0.014

0.282 ± 0.023a 0.354 ± 0.016a

0.446 ± 0.016 0.462 ± 0.007

0.410 ± 0.005

0.491 ± 0.034

0.475 ± 0.007

Please see text for details. 549

DOI: 10.1021/cg501784n Cryst. Growth Des. 2015, 15, 546−551

Communication

Crystal Growth & Design

Figure 6. Schematic diagram of the precipitation process (a) in solution without polymer and (b) in the 2 mg/mL HPMC solution.

study suggests that polymers can interfere with the drug crystallization process with a mechanism different from inhibiting nucleation and crystal growth. In previous studies,29−31 insoluble polymer fibers or microgels were used as heterogeneous nuclei for crystalline polymorph selection. However, the polymers used in our study are water-soluble and enable crystal polymorph selection by changing the actual supersaturation level in the solution and hence changing the nucleation rate of different polymorphs.

systems are actually very different (12.6 and 20.3, respectively). Therefore, we proposed that the mechanism of the HPMC prolonged supersaturation level of HCS is likely that HPMC enables the drug to precipitate into a crystal polymorph of higher solubility and hence lowers the actual supersaturation level of the solution. It deserves more discussion on how exactly HPMC influences the polymorph of HCS precipitate. There are two possible scenarios: (1) HCS Form III may appear no matter there is HPMC in the solution or not, but without HPMC, it transforms quickly into Form I, while HPMC inhibits the crystal form transition; (2) HPMC interferes with the nucleation process and different crystal nuclei form in deionized water and HPMC solution. To test these two scenarios, we collected the precipitate from deionized water after 15 min of precipitation and analyzed it by PXRD. Both characteristic diffraction peaks of HCS Form I and Form III were identified (Figure S6, Supporting Information), which means that Form III crystal initially formed even without HPMC but disappeared later when there was not enough HPMC to inhibit the crystal form transformation. On the basis of these results, we proposed the following physical picture of the HCS precipitation process (Figure 6). When drug molecules crystallize from the supersaturated solution, the driving force, i.e., the changes of Gibbs free energy, is proportional to ln(S), and the nucleation energy barrier is proportional to {ln(S)}−2.28 In the deionized water, crystal nuclei of both HCS Form I and Form III formed at the early stage of precipitation. Later, with the existence of Form I, HCS Form III crystals transformed to Form I, probably via the dissolution-and-recrystallization route. While in the HPMC solution, the solubility of HCS Form I increased significantly, which resulted in a lower driving force and higher nucleation energy barrier for Form I crystallization. In contrast, HPMC had much less effect on the solubility of Form III and hence also had little effect on its crystal nucleation rate. Therefore, in the presence of enough HPMC, the balance was tipped completely to Form III, which became the only crystals observed. In the meantime, the transformation from Form III to Form I was too slow in the absence of Form I seeds, and hence only Form III could be found in the 2 mg/mL HPMC solution. Conclusion. In this study, the mechanism that HPMC prolongs the supersaturation of HCS was investigated. The mechanism is that HPMC enables the drug to precipitate into a metastable crystal polymorph of higher solubility and hence lowers the actual supersaturation level in the solution. This

■

ASSOCIATED CONTENT

* Supporting Information S

Figures S1−S6. This material is available free of charge via the Internet at http://pubs.acs.org/.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +86-1062797572. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was supported by Natural Science Foundation of China (Project No. 50873056) REFERENCES

(1) Thayer, A. M. Chem. Eng. News 2010, 88, 13−18. (2) Babu, N. J.; Nangia, A. Cryst. Growth Des. 2011, 11, 2662−2679. (3) Williams, H. D.; Trevaskis, N. L.; Charman, S. A.; Shanker, R. M.; Charman, W. N.; Pouton, C. W.; Porter, C. J. Pharmacol. Rev. 2013, 65, 315−499. (4) Guzmán, H.; Tawa, M.; Zhang, Z.; Ratanabanangkoon, P.; Shaw, P.; Mustonen, P.; Gardner, C.; Chen, H.; Moreau, J.-P.; Almarsson, Ö .; Remenar, J. AAPS J. 2004, 6, T2189. (5) Guzmán, H. R.; Tawa, M.; Zhang, Z.; Ratanabanangkoon, P.; Shaw, P.; Gardner, C. R.; Chen, H.; Moreau, J. P.; Almarsson, Ö .; Remenar, J. F. J. Pharm. Sci. 2007, 96, 2686−2702. (6) Brouwers, J.; Brewster, M. E.; Augustijns, P. J. Pharm. Sci. 2009, 98, 2549−2572. (7) Gao, P.; Morozowich, W. Expert Opin. Drug Delivery 2006, 3, 97−110. (8) Ilevbare, G. A.; Liu, H.; Edgar, K. J.; Taylor, L. S. Cryst. Growth Des. 2012, 12, 6050−6060. (9) Warren, D. B.; Benameur, H.; Porter, C. J.; Pouton, C. W. J. Drug Target. 2010, 18, 704−731. (10) Xu, S.; Dai, W.-G. Int. J. Pharm. 2013, 453, 36−43. (11) Loftsson, T.; Fridriksdottir, H.; Gudmundsdottir, T. K. Int. J. Pharm. 1996, 127, 293−296.

550

DOI: 10.1021/cg501784n Cryst. Growth Des. 2015, 15, 546−551

Communication

Crystal Growth & Design (12) Terebetski, J. L.; Michniak-Kohn, B. Int. J. Pharm. 2014, 475, 536−546. (13) Raghavan, S. L.; Trividic, A.; Davis, A. F.; Hadgraft, J. Int. J. Pharm. 2001, 212, 213−221. (14) Anwar, J.; Boateng, P. K.; Tamaki, R.; Odedra, S. Angew. Chem., Int. Ed. 2009, 121, 1624−1628. (15) Ilevbare, G. A.; Liu, H.; Edgar, K. J.; Taylor, L. S. Cryst. Growth Des. 2013, 13, 740−751. (16) Alonzo, D. E.; Raina, S.; Zhou, D.; Gao, Y.; Zhang, G. G.; Taylor, L. S. Cryst. Growth Des. 2012, 12, 1538−1547. (17) Simonelli, A. P.; Mehta, S. C.; Higuchi, W. I. J. Pharm. Sci. 1970, 59, 633−638. (18) Hasegawa, A.; Taguchi, M.; Suzuki, R.; Miyata, T.; Nakagawa, H.; Sugimoto, I. Chem. Pharm. Bull. 1988, 36, 4941−4950. (19) Ziller, K. H.; Rupprecht, H. Drug Dev. Ind. Pharm. 1988, 14, 2341−2370. (20) Usui, F.; Maeda, K.; Kusai, A.; Nishimura, K.; Yamamoto, K. Int. J. Pharm. 1997, 154, 59−66. (21) Somasundaran, P.; Krishnakumar, S. Colloid Surf. A-Physicochem. Eng. Asp. 1997, 123−124, 491−513. (22) Suitchmezian, V.; Jeß, I.; Näther, C. J. Pharm. Sci. 2008, 97, 4516−4527. (23) Friesen, D. T.; Shanker, R.; Crew, M.; Smithey, D. T.; Curatolo, W. J.; Nightingale, J. A. Mol. Pharmaceutics 2008, 5, 1003−1019. (24) Tiers, G. V.; Coon, R. I. J. Org. Chem. 1961, 26, 2097−2098. (25) LaPlante, S. R.; Carson, R.; Gillard, J.; Aubry, N.; Coulombe, R.; Bordeleau, S.; Bonneau, P.; Little, M.; O’Meara, J.; Beaulieu, P. L. J. Med. Chem. 2013, 56, 5142−5150. (26) Chauhan, H.; Hui-Gu, C.; Atef, E. J. Pharm. Sci. 2013, 102, 1924−1935. (27) Hsieh, Y. L.; Box, K.; Taylor, L. S. J. Pharm. Sci. 2014, 103, 2724−2735. (28) Mullin, J. W. Crystallization, 4th ed.; Butterworth-Heinemann: Oxford, U.K., 2001. (29) Diao, Y.; Whaley, K. E.; Helgeson, M. E.; Woldeyes, M. A.; Doyle, P. S.; Myerson, A. S.; Hatton, T. A.; Trout, B. L. J. Am. Chem. Soc. 2012, 134, 673−684. (30) Price, C. P.; Grzesiak, A. L.; Matzger, A. J. J. Am. Chem. Soc. 2005, 127, 5512−5517. (31) Lang, M.; Grzesiak, A. L.; Matzger, A. J. J. Am. Chem. Soc. 2002, 124, 14834−14835.

551

DOI: 10.1021/cg501784n Cryst. Growth Des. 2015, 15, 546−551