Hydrogen peroxide pretreatment efficiently assisting enzymatic hydrolysis of chitosan at high concentration for chitooligosaccharides

Article Cite This: Cryst. Growth Des. 2019, 19, 1942−1953

pubs.acs.org/crystal

Improving Compliance and Decreasing Drug Accumulation of Diethylstilbestrol through Cocrystallization Zhen Li,†,‡ Meiqi Li,‡,§ Bo Peng,‡ Bingqing Zhu,‡ Jian-rong Wang,‡ and Xuefeng Mei*,‡ †

College of Pharmacy, Nanchang University, Nanchang 330006, People’s Republic of China Pharmaceutical Analytical & Solid-State Chemistry Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China § University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, People’s Republic of China Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on October 22, 2019 at 13:44:03 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Diethylstilbestrol (DES), a synthetic nonsteroidal estrogen, has been prescribed for advanced breast cancer and prostate cancer. However, its poor compliance, reactive metabolite toxicity and hydrophobicity-induced drug accumulation has limited its applications. In this study, we aimed to modulate its dissolution rate and reduce reactive metabolites and drug accumulation through cocrystallization. Cocrystals of DES with isonicotinamide (INA), picolinamide (PIN), nicotinamide (NIA), urea (UREA), sarcosine (SAR), and flavone (FLA) were obtained. Different crystallization strategies result in cocrystal polymorphs for DES with INA and FLA. Intrinsic dissolution rate (IDR) characterizations in pH 2.0 buffer solution were conducted. Two assumptions (enhancing Cmax or prolonging Tmax) with the aim of improving compliance were put forward. On the basis of the IDR results (DES-NIA with a 1.5-fold increase in IDR and DES-2FLA-B with a 5.5-fold decrease in IDR) and the pharmacological activities of coformers (NIA and FLA with CYPs inhibition and UGTs stimulation effects), the pharmacokinetic behaviors of these two cocrystals were further researched. The 2-fold prolongation of Tmax in the PK profile DES-2FLA-B facilitated an improvement in compliance. In addition, the higher clearance rates and the potential to reduce oxidative metabolites in DES-2FLA-B help to decrease the drug accumulation and reduce the adverse effects of DES.

■

INTRODUCTION Cocrystals are solids composed of two or more different molecular and/or ionic compounds generally in a stoichiometric ratio.1 Recently, cocrystals have been proven to be an intriguing alternative to manipulate physicochemical properties in the field of optoelectronic communications,2 solid explosives,3,4 and pigments5,6 as well as pharmaceuticals,7−9 especially for nonionizable materials. When one of the cocrystal coformers (CCFs) at least is an active pharmaceutical ingredient (API), and the others are pharmaceutically acceptable, it is regarded as a pharmaceutical cocrystal.10 Since the majority of medicines are delivered as solid formulations, the dissolution behaviors of pharmaceutical drug products could have direct effects on the delivery and absorption performances of the medicines. Cocrystallization for APIs with limited dissolution performances could offer the potential of improving dissolution behaviors and thus potentially modulate the bioavailability.11 In addition, cocrystal engineering has been applied to the sustained release of APIs with burst effect as well, which benefits their safety and compliance performances.12 Some CCFs have pharmacological activities and even influence the activities of drug metabolic enzymes such as cytochrome P450 systems (CYPs) and UDPglucuronosyltransferases (UGTs).13−15 Thus, the involvements of these CCFs have the potential to modify the pharmacoki© 2019 American Chemical Society

netic profiles, reduce toxicity, and even have new indications.10,16 Diethylstilbestrol (DES), one of the most important synthetic estrogens, is prescribed for advanced breast cancer in postmenopausal women (15 mg/day) and prostate cancer (1−3 mg/day).17−19 Owing to the low dose, it belongs to class I according to the Biopharmaceutics Classification System (BCS) with adequate solubility (12 mg/L) and high permeability (log P = 5.07).20 However, the poor compliance, reactive metabolite toxicity, and hydrophobic-induced drug accumulation limit its applications.21,22 The oxidative metabolites of DES could covalently bond to DNA and proteins in vivo, which leads to its adverse drug reactions. The hydrophobic character also makes DES easy to accumulate in tissues. In previous studies, 11 solvates of DES were reported.23−25 Herein, we attempt to design cocrystals of DES to prolong the duration above its effective concentration in plasma, which may be favorable for overcoming its poor compliance by controlling its dissolution rate. On the basis of the DES chemical structure and the single-crystal data in CSD database, the phenolic hydrogen group could form supraReceived: December 25, 2018 Revised: January 16, 2019 Published: January 23, 2019 1942

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

Preparation of DES-2INA-A. DES and INA in a 1:2 stoichiometric ratio were ground without solvent dropping for a few minutes at room temperature. The solid phase was verified by means of XRPD. DES-2INA-A single crystals of good quality for structure determination could be obtained occasionally after slowly evaporating DES-2INA in methanol after 72 h (yield 83.97%). Anal. Calcd for C30H32N4O4: C, 70.29; H, 6.29; N, 10.93. Found: C, 70.41; H, 6.21; N, 11.01. Preparation of DES-2INA-B. DES and INA in a 1:2 molar ratio were dissolved in tetrahydrofuran and evaporated slowly under ambient conditions. Colorless blocklike crystals were obtained (yield 98.57%) for structure determination and other characterizations. Anal. Calcd for C30H32N4O4: C, 70.29; H, 6.29; N, 10.93. Found: C, 70.35; H, 6.24; N, 11.07. Preparation of DES-PIN. Equimolar amounts of DES and PIN were dissolved in methanol and evaporated slowly at room temperature. Single crystals suitable for structure determination were obtained (yield 97.91%), and cocrystal formation was verified by means of XRPD and SCXRD. Anal. Calcd for C24H26N2O3: C, 73.82; H, 6.71; N, 7.17. Found: C, 73.90; H, 6.62; N, 7.23. Preparation of DES-NIA. Equimolar amounts of DES and NIA were dissolved in methanol at 50 °C and evaporated slowly at that temperature. Colorless prismlike DES-NIA single crystals of good quality for structure determination could be obtained (yield 98.62%) after slowly evaporating DES-NIA in methanol for 72 h. Anal. Calcd for C24H26N2O3: C, 73.82; H, 6.71; N, 7.17. Found: C, 73.93; H, 6.60; N, 7.25. Preparation of DES-2NIA-MH. A 1:2 stoichiometric ratio of DES and NIA was dissolved in ethyl acetate and evaporated slowly a room temperature. Colorless prismlike crystals were obtained (yield 97.99%) for structure determination and other characterizations. Anal. Calcd for C30H34N4O5: C, 67.91; H, 6.46; N, 10.56. Found: C, 68.01; H, 6.40; N, 10.62. Preparation of DES-UREA. Equimolar amounts of DES and UREA were dissolved in ethanol and evaporated slowly under ambient conditions. Colorless platelike crystals were obtained (yield 98.43%) for structure determination and other characterizations. Anal. Calcd for C19H24N2O3: C, 69.49; H, 7.37; N, 8.53. Found: C, 69.53; H, 7.30; N, 8.59. Preparation of DES-SAR-MH. Equimolar amounts of DES and SAR were dissolved in methanol and evaporated slowly at room temperature. Colorless blocklike crystals were obtained (yield 99.01%) for structure determination and other characterizations. Anal. Calcd for C21H29NO5: C, 67.18; H, 7.79; N, 3.73. Found: C, 67.23; H, 7.72; N, 3.78. Preparation of DES-2FLA-A. A 1:2 molar ratio of DES and FLA was dissolved in methanol and evaporated slowly at room temperature. Colorless blocklike crystals were obtained occasionally (yield 98.72%). Anal. Calcd for C48H40O6: C, 80.88; H, 5.66. Found: C, 80.91; H, 5.60. Preparation of DES-2FLA-B. A 1:2 molar ratio of DES and FLA was dissolved in tetrahydrofuran and evaporated slowly at room temperature. Colorless blocklike crystals were obtained (yield 98.69%) for structure determination and other characterizations. Anal. Calcd for C48H40O6: C, 80.88; H, 5.66. Found: C, 80.93; H, 5.57.

molecular synthons with pyridinic nitrogens (O−H···Narom, 40.6%) and carbonyl oxygen (O−H···OC, 46.9%) via intermolecular hydrogen bonds in the crystal structures (Table 1). Due to the two phenolic hydroxyl groups, we Table 1. CSD Statistics of Possible Suparmolecular Synthons in DES with Phenolic Hydroxyl Functionalities

suggest that CCFs containing a pyridinic nitrogen, carboxylic acid, or amide group have the potential to form hydrogen bonds with DES molecules. Therefore, a series of CCFs with pyridinic nitrogens, carboxylic acid, and amide moiety were selected (Scheme S1). Nine cocrystals of DES with isonicotinamide (INA), picolinamide (PIN), nicotinamide (NIA), urea (UREA), sarcosine (SAR), and flavone (FLA) were successfully synthesized (Scheme 1). Among them, polymorphs existed in DES-2INA (forms A and B) and DES-2FLA (forms A and B) cocrystals. Others only lead to physical mixtures of individual components in line with X-ray powder diffraction (XRPD) characterizations. The crystal structures of these DES cocrystals were determined by single-crystal X-ray diffraction (SCXRD). The intrinsic dissolution rates (IDR) of DES as well as its cocrystals were measured in pH 2.0 buffer solution. The dissolution behavior could mainly affect the absorption phase of APIs. In addition, NIA and FLA have been reported to inhibit the activities of CYPs and stimulate the activities of UGTs in previous studies.26−29 Thus, PK experiments were performed on DES cocrystals and pure DES to confirm our conjectures.

■

EXPERIMENTAL SECTION

Materials. Diethylstilbestrol (purity >98%) was purchased from Beijing InnoChem Science&Technology Co. Ltd. Isonicotinamide (INA) and sarcosine (SAR) were purchased from Aladdin Chemistry Co. Ltd. (purity >98%). Pyridine-2-carboxamide (PIN) was purchased from Bide Pharmatech Ltd. (purity >97%). Nicotinamide (NIA) and flavone (FLA) were purchased from J&K Chemicals (purity >99%). Glibenclamide (internal standard, 97%) was purchased from Shanghai Macklin Biochemical Co., Ltd. Urea and all analytical grade solvents were purchased from the Sinopharm Chemical Reagent Co. and were used without further purification.

Scheme 1. Molecular Structures of Components Used for Cocrystal Synthesis: Diethylstilbestrol (DES), Isonicotinamide (INA), Picolinamide (PIN), Nicotinamide (NIA), Urea (UREA), Sarcosine (SAR) and Flavone (FLA)

1943

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

immediately separated by centrifugation (4 °C, 5000 rpm, 5 min) and kept frozen at −80 °C until analysis. A 500 μL portion of ethyl acetate containing 200 ng/mL glibenclamide as internal standard was added to the plasma sample (100 μL), and the samples were placed in a vortex mixer for 5 min and centrifuged for 10 min (14000 rpm). The 450 μL supernatant was dried with a drying instrument, redissolved in 100 μL of methanol, vertex-mixed for 3 min, and centrifuged for 20 min (14000 rpm). A 90 μL portion of the supernatant was transferred for analysis. A ThermoFisher TSQ QUANTIVA/DIONEX UltiMate 3000 LC-MS/MS instrument was utilized for the separation and determination of DES in plasma. The separation was operated on a Waters SunfireTM C18 column (2.1 × 100 mm, 3.5 μm) with a gradient (acetonitrile/5 mM ammonium acetate in water) from 40/60 to 95/5 in a 6.5 min run at a flow rate of 0.25 mL/min. The injection volume was 10 μL. The mass spectrometer was operated in the negative ion detection mode. The heat block temperature was maintained at 400 °C. Nitrogen was used as the nebulizing gas and drying gas. Detection and quantification were employed in the multiple reaction monitoring mode (MRM), with m/z 267.2 → 237.0 for DES and m/z 492.2 → 170.1 for IS. PK parameters were obtained on the basis of a model-independent method using a DAS 2.1.1 program.

Single-Crystal X-ray Diffraction (SCXRD). X-ray diffraction data for various cocrystals in the present study was collected on a Bruker Smart Apex II CCD diffractometer, using Mo Kα radiation (λ = 0.71073 Å) with a graphite monochromator. Data collection for all single crystals was done at 205(2) K, except the measurement for DES-NIA was conducted at 173(2) K. Data integration and scaling were completed by using the SAINT software. In addition, the SADABS program was used for multiscan absorption corrections. The structures were solved by direct methods using SHELXTL and refined with a full-matrix least-squares technique using the SHELXL-2014 program package. All non-hydrogen atoms were refined with anisotropic displacement parameters, and hydrogen atoms were placed in calculated positions and refined with a riding model. X-ray Powder Diffraction (XRPD). XRPD patterns were obtained using a Bruker D8 Advance X-ray diffractometer with Cu Kα radiation. The tube voltage and current of the generator were set to 40 kV and 40 mA, respectively. Data over the range 3−40° in 2θ were collected with a continuous scan rate of 0.1 s/step at ambient temperature. Data were imaged and integrated with RINT Rapid and peak-analyzed with Jade 6.0 software. Thermogravimetric Analysis (TGA). Thermogravimetric analysis was conducted on Netzsch TG 209F3 equipment, using nitrogen as the purge gas with a flow of 20 mL min−1. Samples were placed in open aluminum oxide pans and heated at 10 °C min−1 to 400 °C. Differential Scanning Calorimetry (DSC). The DSC experiments were performed on a DSC TA Q2000 instrument under a nitrogen gas flow of 50 mL min−1 purge. The instrument was calibrated to the temperature axis and heat flow via indium and tin. Samples weighing 2−4 mg were heated in sealed nonhermetic aluminum pans from 20 to 300 °C with a heating rate of 10 °C min−1. Polarizing Microscopy (PM). All polarizing photos were taken on a XPV-400E polarizing microscope. Crystals of DES cocrystals were filmed by using microscope (5 × 10). Intrinsic Dissolution Rate (IDR). IDR experiments were performed via a Mini-Bath dissolution apparatus equipped with a Julabo ED-5 heater/circulator. In each experiment, approximately 20 mg of the sample (DES and its cocrystals, n = 3) was compressed into a 0.07 cm2 disk in a rotating disk intrinsic dissolution die using a MiniIDR press at a pressure of 35 bar for 1 min. Only one side of the disk was exposed to the dissolution medium. The intrinsic attachment was placed in a jar of 15 mL of pH 2.0 buffer containing 0.5% Tween 80 preheated at 37 °C and stirred at 100 rpm. At time intervals (5, 10, 15, 20, 30, 40, 60, 90, 120, 180 min), 100 μL of the sample was withdrawn manually. The collected samples were diluted with equal amounts of buffer and assayed for DES concentration using HPLC. An Agilent 1260 series Infinite HPLC instrument was equipped with a ZORBAX ECLIPSE-C18 column (4.6 × 150 mm, 5 μm). An injection volume of 10 μL was used with 1 mL/min flow rate. The detection UV−vis wavelength was set at 254 nm. The samples were gradient-eluted with a mobile phase containing a mixture of methanol and water. The gradient started at 30% methanol and 70% water. After 2 min, it was changed to 80% methanol and 20% water in the following 8 min, which was maintained for 2 min. After each gradient analysis, the column was equilibrated with the started mobile phase (30% methanol). The observed retention time point for DES is 11.6 min. No overlap between peaks for DES or any CCFs was observed. In Vivo Pharmacokinetic (PK) Experiment. DES, DES-NIA, and DES-2FLA-B were employed in a rat PK study. The PK experimental protocol was approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Materia Medica and conformed to the Guide for Care and Use of Laboratory Animals. Eighteen male Sprague−Dawley rats (200−230 g) were randomly divided into three groups. The suspension formulation (0.5% CMCNa aqueous solution) was delivered as oral administration at a dose of 5 mg/kg DES (expressed as DES equivalents). The rats were allowed to have free access to water and fasted overnight before drug administration. A freshly prepared suspension formulation was immediately delivered orally to rats. Then a 200 μL blood sample was obtained from the orbital sinus and placed into heparinized tubes at 0.5, 0.75, 1, 2, 4, 6, 8, 12, and 24 h after dosing. Plasma was

■

RESULTS AND DISCUSSION X-ray Powder Diffraction (XRPD) and Single-Crystal Structure Analysis. On the basis of crystal engineering principles, phenolic hydroxyl groups tend to assemble supramolecular synthons with CCFs containing a pyridine or carbonyl group. Twenty-one CCFs were employed and resulted in nine successful examples. The formation of cocrystals could be verified by XRPD. In Figure 1, each

Figure 1. XRPD patterns of DES cocrystals.

XRPD pattern of DES cocrystals is different from either that of DES or the corresponding CCFs (Figure S1). All of the peaks displayed in the measured XRPD patterns of the DES cocrystal bulk powder are closely in accordance with those in the simulated patterns acquired from single-crystal diffraction data, which confirm the formation of high-purity phases. DES cocrystals were obtained by means of a solution evaporation approach, and all of them were subjected to single crystal-analysis. The morphologies are mostly prismlike or blocklike structures (Figure 2). Crystallographic data and Hbond parameters are summarized in Tables S1 and S2, respectively. As shown in the single-crystal structures, polymorphs were present in both DES-2INA and DES-2FLA cocrystals. DES has two hydroxyl substitutes on the benzene ring, which can act as both H-bond donors and acceptors with 1944

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

Figure 2. Polarizing microscopy images of DES cocrystals: (a) DES-2INA-A; (b) DES-2INA-B; (c) DES-PIN; (d) DES-NIA; (e) DES-2NIAMH; (f) DES-UREA; (g) DES-SAR-MH; (h) DES-2FLA-A; (i) DES-2FLA-B.

Figure 3. (a) 2D planar structure of DES-2INA-A. (b) Packing pattern of DES-2INA-A.

link with DES molecules (O2−H2···N1) to form an infinite one-dimensional (1D) linear structure. Two-dimensional (2D) planar structures are built by N−H···O interactions among these 1D lines. Then π−π interactions as well as C−H···π (3.036 Å) interactions between the aromatic hydrogen atoms and the benzene rings of DES help to form its threedimensional (3D) architecture (Figure 3b). For DES-2INA-B, it crystallized in the monoclinic C2/c space group and its asymmetric unit consisted of half of a DES molecule and one INA molecule. Even though INA dimers exist, the fold linear structures emerged (Figure 4a). This difference is due to the different twist angles of DES molecules in these two polymorphs (61.46° for DES-2INA-A, 80.42° for DES-2INA-B; Figure S2). N2−H2B···O1 and C10-H10···O2 help to form a crossed 3D packing pattern (Figure 4b,c). Different from DES-2INA (forms A and B), no dimer structures exist in DES-PIN. Instead, strong O2−H2···N1 and

CCFs. On the basis of the single-crystal structures, two dominating modes of synthons exist in DES cocrystals. Thus, we divided these DES cocrystals into two groups (groups A and B). Group A (synthon I, O−H···Narom) consists of DES2INA (forms A and B), DES-PIN, DES-NIA, and DES-2NIAMH, and group B (synthon II, O−H···Ocarbonyl) consists of DES-UREA, DES-SAR-MH, and DES-2FLA (forms A and B). During the cultivation of single crystals, two polymorphs (DES-2INA-A and DES-2INA-B) of DES-2INA were discovered. We found that DES-2INA-B could be harvested in most cases, while DES-2INA-A was only obtained once via slow evaporation from methanol solvent. Colorless prismlike DES-2INA-A crystals crystallized in the triclinic P1̅ space group. The asymmetric unit of DES-2INA-A contained one molecule of DES and two molecules of INA. In Figure 3a, two INA molecules first form typical amide dimers through an R22(8) supramolecular synthon (N2−H2B···O4). INA dimers 1945

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

Figure 4. (a) Fold linear structures in DES-2INA-B. (b) Linkages among linear structures in DES-2INA-B. (c) Packing pattern of DES-2INA-B.

O3−H1···O1 interactions between DES and ortho-substituted PIN result in the formation of a 1D linear structure (Figure 5a). These lines connect to each other via N2−H2B···O2 Hbond interactions to form 2D planar structures. Planes stacking along the b axis help the formation of its 3D architecture (Figure 5b). DES and NIA could generate cocrystals with two different stoichiometries: DES-NIA (1:1) and DES-2NIA-MH (1:2:1). These two cocrystals present obviously different crystal

arrangements relative to their basic building blocks. With 1:1 stoichiometry, the asymmetric unit of DES-NIA contains one molecule of DES and one molecule of NIA. In Figure 6a, metasubstituted NIA molecules link with DES molecules via N2− H2B··· O2 and N2−H2C···O1 interactions to form infinite wavy lines. Robust O1−H1···N1 as well as O2−H2···O3 interactions among these lines contribute to the formation of a 3D wavy architecture (Figure 6). However, in DES-2NIA-MH, the asymmetric unit consists of one DES molecule, two NIA 1946

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

molecules, and one water molecule. 1D NIA lines are formed via N−H···O interactions among staggered molecules. Then NIA lines link with water and DES molecules through H-bond interactions (N2−H2A···O2, O2−H2C···O1, O1−H1···N3, N4− H4A···O3, and O3−H3···N1) to form a sheet motif (Figure 7a). Such sheets are further extended into 3D layer structures via an O2−H2D···O5 interaction and π−π stacking (Figure 7b). For cocrystals in group B, phenolic hydroxyl groups have strong H-bond interaction swith carboxyl oxygens. In DESUREA, the hydroxyl groups of DES link with the carboxyl groups of urea (O−H···O) to form 1D lines (Figure 8a). The N−H···O interactions among these lines facilitate the formation of crossed 3D architecture (Figure 8b). SAR is present as zwitterions, as indicated by the comparable C−O bond lengths of the carboxylate in the DES-SAR-MH cocrystal structure. Unlike DES-UREA discussed above, the DES and SAR molecules do not interact with each other directly and no 1D lines exist. Instead, DES, ionic SAR, and water molecules link in turn (O2−H2···O5, O3− H3B···O2 and O3−H3A···O4) to form cyclic structures (Figure 9a). The remaining potential acceptor sites on water molecules and the donor sites of amino ions on SAR help these cyclic units further interweave into 3D fabrication by means of N1− H1B···O3 and O1−H1···O4 H-bond interactions (Figure 9b). DES and FLA cocrystallized in two polymorphs: namely, DES-2FLA-A and DES-2FLA-B (Figure 10). Similar to the single-crystal cultivation process of DES-2NIA, DES-2FLA-A single crystals were also obtained once via slow evaporation from methanol solvent. Two available hydroxyl substituents on the benzene ring of DES acting as H-bond donors connect with the carbonyl groups on the benzopyran ring of FLA

Figure 5. (a) H-bond pattern of DES-PIN. (b) Packing pattern of DES-PIN.

Figure 6. (a) H-bond pattern of DES-NIA. (b) Packing pattern of DES-NIA. 1947

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

Figure 7. (a) H-bond pattern of DES-2NIA-MH. (b) Packing pattern of DES-2NIA-MH.

Figure 8. (a) H-bond pattern of DES-UREA. (b) Packing pattern of DES-UREA.

Thermal Properties. To evaluate their physicochemical stability, thermal analysis of these nine DES cocrystals were conducted by TGA, DSC, and VT-XRPD. The DSC patterns of the DES starting material (determined according to XRPD data and compared with the literature;30 Figure S3), CCFs, and cocrystals presented in Figure S4 revealed the different thermal properties of various DES solid forms. The melting point (Tonset, in Table S3) of five DES cocrystals are found to lie between the melting point values of the individual

through O−H···Ocarbonyl interactions to form trimers in these two cocrystals. However, these two polymorphs exhibit different types of linkages among trimers. In DES-2FLA-A, adjacent trimers are further interconnected through a nonclassical C2−H2···O2 H-bond interaction. For DES-2FLA-B, a C−H···π (2.907 Å) interaction acts as the main driving force for the 3D networks. Different connection types account for the packing polymorphism in DES-2FLA cocrystals. 1948

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

Figure 9. (a) H-bond pattern of DES-SAR-MH. (b) Packing pattern of DES-SAR-MH along the b axis.

components, in accordance with most cocrystal cases.31 However, the melting points of DES-2INA-A, DES-2INA-B, DES-UREA, and DES-SAR-MH are higher than those of both starting materials. As shown in Figure S5, the TGA plots of DES-2INA-A, DES-2INA-B, DES-PIN, DES-NIA, DES-UREA, DES-2FLAA, and DES-2FLA-B reveal no significant weight loss before the melting point, confirming their unsolvated characteristics. In the case of the DSC pattern of DES-2INA-A, an additional endothermic peak with Tonset = 133.5 °C appeared (ΔH = 19.98 J/g). Since no solvate was included, this endothermic peak can be ascribed to a phase transformation. This assumption was further confirmed by VT-XRPD (Figure S6). The DES-2INA-A powder sample was held for 10 min at 160 °C and then conducted for XRPD analysis. After this heating process, additional peaks at 2θ = 4.6 and 16.7° appeared. These new peaks are identified as the characteristic peaks of DES-2INA-B. For DES-2FLA-A, an additional shoulder peak could be observed, indicating an uncompleted phase transition during the melting process. To verify this conjecture, a DES2FLA-A powder sample was heated for 20 min at 120 °C and then submitted for XRPD analysis. The emergence of characteristic peaks at 2θ = 6.2, 12.2, 12.5, and 13.5° indicated the solid-state transition from DES-2FLA-A to DES-2FLA-B (Figure S7). For DES-2NIA-MH and DES-SAR-MH, weight loss could be observed before their melting points. The thermal behaviors of DES-2NIA-MH and DES-SAR-MH are quite similar. In the case of DES-2NIA-MH, a gradual mass loss of 3.32% was observed, corresponding to the loss of 1 equiv of water molecules (calculated 3.39%). However, for DES-SAR-MH, a weight loss of 4.82% corresponding to 1 equiv of water

(calculated 4.79%) was presented. Accordingly, endothermic peaks before the melting points of DES-2NIA-MH and DESSAR-MH in the DSC diagram were exhibited (Tonset = 99.53 °C for DES-2NIA-MH and 145.04 °C for DES-SAR-MH). To analyze their phase transition processes, both DES-2NIA-MH and DES-SAR-MH powders were heated at 150 °C for 30 min and then further characterized by XRPD. For DES-2NIA-MH, new characteristic peaks at 2θ = 5.21, 10.06, 10.50, 13.57, and 14.49° are different with both DES-NIA and the individual components, which indicates the formation of a new desolvated cocrystal of DES and NIA (Figure S8). The desolvation process of DES-SAR-MH also results in a new desolvated cocrystal of DES and SAR with new characteristic peaks at 2θ = 7.15, 12.55, and 13.75° (Figure S9). Solid-State Transformations and Controlled Crystallizations. To verify the relative thermodynamic stabilities at room temperature, interconversion slurry experiments were conducted. For DES-2INA cocrystals, approximately equal amounts of forms A and B were slurried together in a methanol solution under ambient conditions for 24 h. This suspension was centrifuged and submitted for XRPD analysis. The residual solids were found to convert to DES-2NIA-B (Figure S10). The relative stability between DES-2FLA polymorphs was also investigated. A similar slurry experiment was conducted, and the remaining solids were found to transform to DES-2FLA-B (Figure S11). As a significant part in the pharmaceutical industry, polymorph control has attracted a great deal of attention. In recent decades, strategies such as crystallization from organogel and grinding (neat or liquid assisted) have been put forward to control crystallization outcomes of cocrystals.32−35 In this study, we used these aforementioned strategies in order to 1949

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

Figure 10. (a) Trimer structure in DES-2FLA cocrystals. (b) Linkage of trimers in DES-2FLA-A. (c) Linkage of trimers in DES-2FLA-B.

obtain the metastable DES-2INA-A and DES-2FLA-A. DES and CCFs (INA or FLA) with 1:2 stoichiometry (0.05 mmol:0.1 mmol) were added together. Mechanochemical cocrystallization was first applied. When neat grinding was conducted, metastable DES-2INA-A was obtained successfully. However, when solvents (MeOH or THF) were used during grinding, only the thermodynamically stable DES-2INA-B was obtained. For DES-2FLA cocrystals, only the stable DES2FLA-B was obtained whether neat or liquid-assisted grinding was applied. Under this circumstance, the pH-switchable vitamin B9 gel was used as cocrystallization media.33 VB9 in DMSO/nitromethane (2/8) solvent resulted in the formation of a faint yellow gel. After Et3N was added onto the gel surface, a rapid gel to solution phase transition was observed. White powders were obtained by filtration, and these powders were characterized by XRPD. As shown in Table 2, metastable DES2FLA-A was obtained successfully in VB9 gels, while physical mixtures were harvested in the cultivation of DES-2INA-A cocrystals. In Vitro Intrinsic Dissolution Rate (IDR). After delivery, drugs in oral preparations first dissolve in gastric and intestinal fluid to be absorbed in systemic circulation. Due to the involvement of CCFs in pharmaceutical cocrystals, changes in the dissolution behavior will manipulate the amount of API available for absorption and then modify the pharmacokinetic profiles. Therefore, in this study, the IDRs of pure DES and its

Table 2. Crystallization Experiments in the Cultivation of Metastable DES-2INA-A and DES-2FLA-Aa method VB9 gels grinding

result DES and 2INA

DES and 2FLA

DMSO/nitromethane (2/8) neat

physical mixtures DES-2INA-A

DES-2FLA-A

methanol tetrahydrofuran

DES-2INA-B DES-2INA-B

physical mixtures DES-2FLA-B DES-2FLA-B

a

The relative XRPD characterization data for each cocrystallization experiment are presented in Figure S12.

cocrystals were determined in pH 2.0 buffer solution (to simulate gastric fluids) with the presence of 0.5% Tween 80 (Figure 11 and Table 3). DES-2FLA-A is excluded from the IDR test due to its difficulty in magnification. In comparison with DES, DES-NIA presented an improvement in IDR by approximately 1.5-fold in pH 2.0 buffer solution. However, DES-SAR-MH and DES-2FLA-B exhibited a decrease in IDR of about 2-fold and 5.5-fold in pH 2.0 buffer solution, respectively. In Vivo Bioavailability. Since dissolution behaviors can strongly affect the absorption phase of APIs, we hypothesized that the strategy of enhancing Cmax (improvement in 1950

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

Figure 11. IDRs of DES and its cocrystals in pH 2.0 buffer solution with the presence of 0.5% Tween 80.

Figure 12. Plasma DES concentration−time curves of pure DES and DES-NIA and DES-2FLA-B cocrystals (data are expressed as means ± SD, n = 6).

Table 3. Summary of IDRs of DES and Its Cocrystals material

IDR pH 2.0 (μg cm−2 h−1)

DES DES-2INA-A DES-2INA-B DES-PIN DES-NIA DES-2NIA-MH DES-UREA DES-SAR-MH DES-2FLA-B

1.85 1.79 1.80 1.71 2.73 1.41 1.91 1.00 0.33

Table 4. Pharmacokinetic Parameters from the Plasma Concentration of Pure DES and DES-NIA and DES-2FLA-B Cocrystals DES material DES DES-NIA DES-2FLA-B

dissolution rate) or prolonging Tmax (decrease in dissolution rate) has a strong potential to improve drug compliance. According to the IDR results, DES-NIA with enhanced IDR, as well as DES-SAR-MH and DES-2FLA-B with reduced IDR, can be candidates. In addition, NIA and FLA could inhibit CYPs and activate UGTs, modulate the formation of metabolites, and consequently decrease the toxicity and accumulation of DES.27,36 Therefore, in vivo pharmacokinetic experiments were conducted on DES-NIA and DES-2FLA-B to confirm our previous conjectures. Pure DES was also tested for comparison. After a single oral dose of the suspensions of DES, DES-NIA, and DES-2FLA-B (equivalent to 5 mg/kg pure DES, containing 0.5% CMCNa), the pharmacokinetic parameters of DES were provided for statistical comparison and bioavailability calculations. The mean plasma concentrations of DES versus time diagrams for pure DES and DESNIA and DES-2FLA-B cocrystals are shown in Figure 12. The pharmacokinetic parameters in Table 4 were calculated from the concentration of DES. In the absorption phase, an increase in Cmax was presented in the PK profile of DES-NIA. This phenomenon verified that the higher IDR of DES-NIA could lead to a modest increase in Cmax. However, DES-NIA and pure DES present the same parameters for Tmax and similar elimination behavior within 2 h, which is unfavorable for an improvement in compliance. For DES-2FLA-B, its Tmax was 2 times longer than that of pure DES. This prolongation is correlated with its reduced IDR profile. The sustained release of DES-2FLA-B is helpful to maintain a longer time above the minimum effective concentration (MEC), which makes it have a strong potential to improve compliance. Not only the absorption phase (by altering dissolution rate) but also the involvements of CCFs have great possibilities to

Tmax (h) Cmax (μg/mL) 0.8 0.8 1.6

42.0 51.7 24.9

CL/F (mL/(h kg))

AUC0−24h (h ng/mL)

26195.5 33485.4 50788.7

145.5 118.4 77.3

modulate the elimination processes of APIs. In this study, the relative bioavailability (AUC0−24h) of DES-NIA and DES2FLA-B are about 1.2 and 2 times lower than that of pure DES. This phenomenon was attributed to the higher clearance in their PK profiles. In Figure 12, no DES could be detected 12 h after oral administration of these two cocrystals. Considering the pharmacological activities, we supposed that FLA may increase the phase II metabolites and thus decrease the accumulation of estrogens. In addition, less toxic phase I metabolites owing to the inhibitory effects of FLA on CYPs could also help to reduce the adverse effects of DES.

■

CONCLUSIONS With the aim of improving compliance and reducing drug accumulation of DES, a synthetic estrogen, cocrystals with isonicotinamide (INA), picolinamide (PIN), nicotinamide (NIA), urea (UREA), sarcosine (SAR), and flavone (FLA) were successfully prepared. Polymorphs were obtained for DES-2INA and DES-2FLA cocrystals by means of different cocrystallization methods. Single-crystal structures of all the DES cocrystals were determined by SCXRD. On the basis of their basic supramolecular motifs, we can divide these nine cocrystals into two groups (A, O−H···Narom; B, O−H··· Ocarbonyl). Furthermore, IDRs were conducted for eight DES coccrystals and pure DES for comparison. DES-NIA shows about 1.5-fold enhancement, while DES-SAR-MH and DES2FLA-B exhibit 2-fold and 5.5-fold decreases in pH 2.0 buffer solution, respectively. In order to investigate the influences of IDR and pharmacological activities of CCFs on PK performances, experiments were performed on DES-NIA and DES2FLA-B. According to the PK profiles, the Tmax of DES-2FLA1951

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

(4) Landenberger, K. B.; Matzger, A. J. Cocrystal Engineering of a Prototype Energetic Material: Supramolecular Chemistry of 2,4,6Trinitrotoluene. Cryst. Growth Des. 2010, 10, 5341−5347. (5) Bučar, D. K.; Filip, S.; Arhangelskis, M.; Lloyd, G. O.; Jones, W. Advantages of mechanochemical cocrystallisation in the solid-state chemistry of pigments: colour-tuned fluorescein cocrystals. CrystEngComm 2013, 15, 6289−6291. (6) Li, M. Q.; Li, Z.; Zhang, Q.; Peng, B.; Zhu, B.; Wang, J. R.; Liu, L. Y.; Mei, X. F. Fine tuning the colors of natural pigment emodin with superior stability through cocrystal engineering. Cryst. Growth Des. 2018, 18, 6123−6132. (7) Zhu, B.; Wang, J. R.; Zhang, Q.; Li, M. Q.; Guo, C. Y.; Ren, G. B.; Mei, X. F. Stable Cocrystals and Salts of the Antineoplastic Drug Apatinib with Improved Solubility in Aqueous Solution. Cryst. Growth Des. 2018, 18, 4701−4714. (8) Li, D. X.; Kong, M. M.; Li, J.; Deng, Z. W.; Zhang, H. L. Amine− carboxylate supramolecular synthon in pharmaceutical cocrystals. CrystEngComm 2018, 20, 5112−5118. (9) Li, J. M.; Dai, X. L.; Li, G. J.; Lu, T. B.; Chen, J. M. Constructing Anti-Glioma Drug Combination with Optimized Properties through Cocrystallization. Cryst. Growth Des. 2018, 18, 4270−4274. (10) Duggirala, N. K.; Perry, M. L.; Almarsson, O.; Zaworotko, M. J. Pharmaceutical cocrystals: along the path to improved medicines. Chem. Commun. 2016, 52, 640−655. (11) Zhu, B. Q.; Zhang, Q.; Wang, J. R.; Mei, X. F. Cocrystals of Baicalein with Higher Solubility and Enhanced Bioavailability. Cryst. Growth Des. 2017, 17, 1893−1901. (12) Chen, J. M.; Li, S.; Lu, T. B. Pharmaceutical Cocrystals of Ribavirin with Reduced Release Rates. Cryst. Growth Des. 2014, 14, 6399−6408. (13) He, H. Y.; Zhang, Q.; Wang, J. R.; Mei, X. F. Structure, physicochemical properties and pharmacokinetics of resveratrol and piperine cocrystals. CrystEngComm 2017, 19, 6154−6163. (14) Atal, C. K.; Dubey, R. K.; Singh, J. Biochemical basis of enhanced drug bioavailability by piperine: evidence that piperine is a potent inhibitor of drug metabolism. J. Pharmaco. Exp. Ther. 1984, 232, 258−262. (15) Jiang, W.; Hu, M. Mutual interactions between flavonoids and enzymatic and transporter elements responsible for flavonoid disposition via phase II metabolic pathways. RSC Adv. 2012, 2, 7948−7963. (16) Almansa, C.; Mercè, R.; Tesson, N.; Farran, J.; Tomàs, J.; PlataSalamán, C. R. Co-crystal of Tramadol Hydrochloride−Celecoxib (ctc): A Novel API−API Co-crystal for the Treatment of Pain. Cryst. Growth Des. 2017, 17, 1884−1892. (17) Miller, J. I.; Ahmann, F. R. Treatment of castration-induced menopausal symptoms with low dose diethylstilbestrol in men with advanced prostate cancer. Urology 1992, 40, 499−502. (18) Malkowicz, S. B. The role of diethylstilbestrol in the treatment of prostate cancer. Urology 2001, 58, 108−113. (19) Group, L. S. Leuprolide versus Diethylstilbestrol for Metastatic Prostate Cancer. N. Engl. J. Med. 1984, 311, 1281−1286. (20) Hughes, L. D.; Palmer, D. S.; Nigsch, F.; Mitchell, J. B. O. Why Are Some Properties More Difficult To Predict than Others? A Study of QSPR Models of Solubility, Melting Point, and Log P. J. Chem. Inf. Model. 2008, 48, 220−232. (21) Metzler, M. Diethylstilbestrol: hormonal or chemical carcinogen? Trends Pharmacol. Sci. 1982, 3, 174−175. (22) Rüdiger, H. W.; Haenisch, F.; Metzler, M.; Oesch, F.; Glatt, H. R. Metabolites of diethylstilboestrol induce sister chromatid exchange in human cultured fibroblasts. Nature 1979, 281, 392−394. (23) Görbitz, C. H.; Hersleth, H. P. Selective solvent inclusion as a tool for mapping molecular properties in crystal structures: a diethylstilbestrol example. Acta Crystallogr., Sect. B: Struct. Sci. 2000, 56, 1094−1102. (24) Carlisle, C. H.; Crowfoot, D. A determination of molecular symmetry in the αβ-diethyl-dibenzyl series. J. Chem. Soc. 1941, 0, 6− 9.

B presents 2-fold enhancement over that of pure DES, which facilitated an improvement in compliance. The higher clearance rates in DES cocrystals contribute to a decrease in drug accumulation of estrogens. Furthermore, less toxic phase I metabolites owing to the inhibitory effects of FLA on CYPs could also help to reduce the adverse effects of DES.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.8b01911. Schemes of library of coformers, XRPD patterns, crystallographic data for DES cocrystals, H-bond lengths and angles, H-bond and packing patterns, TGA diagrams, DSC diagrams, and XRPD patterns and polarizing microscopy photos for DES cocrystals (PDF) Accession Codes

CCDC 1887212−1887220 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

■

AUTHOR INFORMATION

Corresponding Author

*X.M.: e-mail, [email protected]; fax, + 86 21 50800934; tel, +86 2150800934. ORCID

Jian-rong Wang: 0000-0002-0853-7537 Xuefeng Mei: 0000-0002-8945-5794 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research work was financially supported by the Shanghai Natural Science Foundation (Grant No. 18ZR1447900), Youth Innovation Promotion Association CAS (Grant No. 2016257), CAS Key Technology Talent Program, and SANOFI-SIBS Scholarship.

■

REFERENCES

(1) Aitipamula, S.; Banerjee, R.; Bansal, A. K.; Biradha, K.; Cheney, M. L.; Choudhury, A. R.; Desiraju, G. R.; Dikundwar, A. G.; Dubey, R.; Duggirala, N.; Ghogale, P. P.; Ghosh, S.; Goswami, P. K.; Goud, N. R.; Jetti, R. R. K. R.; Karpinski, P.; Kaushik, P.; Kumar, D.; Kumar, V.; Moulton, B.; Mukherjee, A.; Mukherjee, G.; Myerson, A. S.; Puri, V.; Ramanan, A.; Rajamannar, T.; Reddy, C. M.; Rodriguez-Hornedo, N.; Rogers, R. D.; Row, T. N. G.; Sanphui, P.; Shan, N.; Shete, G.; Singh, A.; Sun, C. C.; Swift, J. A.; Thaimattam, R.; Thakur, T. S.; Kumar Thaper, R.; Thomas, S. P.; Tothadi, S.; Vangala, V. R.; Variankaval, N.; Vishweshwar, P.; Weyna, D. R.; Zaworotko, M. J. Polymorphs, Salts, and Cocrystals: What’s in a Name? Cryst. Growth Des. 2012, 12, 2147−2152. (2) Zhu, W.; Zheng, R.; Fu, X.; Fu, H.; Shi, Q.; Zhen, Y.; Dong, H.; Hu, W. Revealing the charge-transfer interactions in self-assembled organic cocrystals: two-dimensional photonic applications. Angew. Chem., Int. Ed. 2015, 54, 6785−6789. (3) Landenberger, K. B.; Bolton, O.; Matzger, A. J. Two isostructural explosive cocrystals with significantly different thermodynamic stabilities. Angew. Chem., Int. Ed. 2013, 52, 6468−6471. 1952

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953

Crystal Growth & Design

Article

(25) Busetta, B.; Courseille, C.; Hospital, M. Crystallographic Study of various solvated forms of Diethylstilbestrol. Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. 1973, 29, 2456−2462. (26) Gaudineau, C.; Auclair, K. Inhibition of human P450 enzymes by nicotinic acid and nicotinamide. Biochem. Biophys. Res. Commun. 2004, 317, 950−956. (27) Korobkova, E. A. Effect of Natural Polyphenols on CYP Metabolism: Implications for Diseases. Chem. Res. Toxicol. 2015, 28, 1359−1390. (28) Lintner, K. Cosmetic or dermopharmaceutical composition comprising at least one UDP glucuronosyl transferase (UGT) enzymes inducer. Patent WO2005IB51344, 2005. (29) Canivenc-Lavier, M. C.; Vernevaut, M. F.; Totis, M.; Siess, M. H.; Magdalou, J.; Suschetet, M. Comparative effects of flavonoids and model inducers on drug-metabolizing enzymes in rat liver. Toxicology 1996, 114, 19−27. (30) Weeks, C. M.; Cooper, A.; Norton, D. A. The crystal and molecular structure of diethylstilbestrol. Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. 1970, 26, 429−433. (31) Perlovich, G. L. Thermodynamic characteristics of cocrystal formation and melting points for rational design of pharmaceutical two-component systems. CrystEngComm 2015, 17, 7019−7028. (32) Foster, J. A.; Piepenbrock, M. O.; Lloyd, G. O.; Clarke, N.; Howard, J. A.; Steed, J. W. Anion-switchable supramolecular gels for controlling pharmaceutical crystal growth. Nat. Chem. 2010, 2, 1037− 1043. (33) Wang, J. R.; Bao, J. J.; Fan, X. W.; Dai, W. J.; Mei, X. F. pHSwitchable vitamin B9 gels for stoichiometry-controlled spherical cocrystallization. Chem. Commun. 2016, 52, 13452−13455. (34) Frišcǐ ć, T.; Jones, W. Recent Advances in Understanding the Mechanism of Cocrystal Formation via Grinding. Cryst. Growth Des. 2009, 9, 1621−1637. (35) Zhang, K. K.; Xu, S. J.; Liu, S. Y.; Tang, W. W.; Fu, X. Y.; Gong, J. B. Novel Strategy to Control Polymorph Nucleation of Gamma Pyrazinamide by Preferred Intermolecular Interactions during Heterogeneous Nucleation. Cryst. Growth Des. 2018, 18, 4874−4879. (36) Appelt, L. C.; Reicks, M. M. Soy Induces Phase II Enzymes But Does Not Inhibit Dimethylbenz[a]anthracene-Induced Carcinogenesis in Female Rats. J. Nutr. 1999, 129, 1820−1826.

1953

DOI: 10.1021/acs.cgd.8b01911 Cryst. Growth Des. 2019, 19, 1942−1953