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Enhancing bioavailability of dihydromyricetin through inhibiting precipitation of soluble cocrystals by a crystallization inhibitor Chenguang Wang, Qing Tong, Xiaolong Hou, Shenye Hu, Jianguo Fang, and Changquan Calvin Sun Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00591 • Publication Date (Web): 19 Jul 2016 Downloaded from http://pubs.acs.org on July 21, 2016

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Crystal Growth & Design

Enhancing

bioavailability

of

dihydromyricetin

through

inhibiting

precipitation of soluble cocrystals by a crystallization inhibitor

Chenguang Wang, 1, 2 Qing Tong, 1 Xiaolong Hou, 1 Shenye Hu, 2 Jianguo Fang,1, * and Changquan Calvin Sun2,*

1

Department of Pharmacy, Tongji Hospital affiliated with Tongji Medical College, Huazhong University

of Science and Technology, Wuhan 430030, PR China 2

Pharmaceutical Materials Science and Engineering Laboratory, Department of Pharmaceutics, College

of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA

*Corresponding authors Jianguo Fang No 1095, Jiefang Road, Wuhan, Hubei, 430030, P.R China. E-mail: [email protected] Tel: 027-83649095

Changquan Calvin Sun, Ph.D. 9-127B Weaver-Densford Hall, 308 Harvard Street S.E. Minneapolis, MN 55455, USA Email: [email protected] Tel: 612-624-3722, Fax: 612-626-2125

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Abstract Highly soluble cocrystals can be used to improve bioavailability of a poorly soluble drug, through generating supersaturation, when absorption is limited by drug dissolution. Dihydromyricetin (DMY) is a biopharmaceutics classification system (BCS) IV drug, exhibiting dissolution limited absorption. Two novel soluble cocrystals of (±)DMY with caffeine and urea were prepared, and their physicochemical properties were evaluated for suitability in formulation development. Although having a much higher solubility than (±)DMY, both cocrystals undergo rapid precipitation during dissolution and form the poorly soluble (±)DMY dihydrate in aqueous media. This negates the potential advantage offered by the high solubility of the two cocrystals in enhancing the dissolution rate and in vivo bioavailability. To solve this problem, we have systematically evaluated suitable crystallization inhibitors to maintain the supersaturation generated by cocrystals dissolution over a prolonged period of time. At 37 °C, an approximately five fold enhancement in oral bioavailability of (±)DMY was achieved when both cocrystals were dosed with 2.0 mg/mL polyvinylpyrrolidone K30 solution than (±)DMY dihydrate suspended in 0.5 mg/mL carboxymethylcellulose sodium solution. This study demonstrates that the use of a highly soluble cocrystal along with an appropriate crystallization inhibitor is a potentially effective formulation strategy for improving oral bioavailability of poorly soluble BCS IV drugs.


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Introduction Dihydromyricetin (DMY) is a natural flavonoid isolated from a traditional Chinese medicine, Ampelopsis grossedentata. Studies suggest a strong intrinsic activity and efficacy of DMY for treating a number of diseases, including hypertension, chronic pharyngitis, and alcohol intoxication.1 Recently, a double-blind randomized clinical trial showed that DMY can also benefit patients with nonalcoholic fatty liver disease.2

Despite its therapeutic promise, DMY is faced with the problem of low oral

bioavailability, which is less than 10 % in rat.3 The low bioavailability is presumably caused by the combined effects of its low solubility (0.2 mg/mL at 25 °C)4 and poor permeability (Peff = 1.84 ± 0.37 ×10-6 cm/s),5 which place DMY into class IV of the biopharmaceutics classification system (BCS). In fact, poor absorption has been identified as the major reason of the generally poor bioavailability of flavonoids.6,7 For Class IV compounds, improving either solubility or permeability has the potential to improve their clinical performance.8 To ensure the maximum absorbable dose (334 mg) of DMY with oral administration, the solution concentration should exceed 4.8 mg/mL,9 which is approximately six times the equilibrium solubility of DMY (0.82 mg/mL, dihydrate) at 37 °C in water. Because of their good stability, solid formulations are generally preferred over liquid formulations. Among possible solid forms, soluble salts are commonly used to address poor solubility problem of drugs. However, DMY is chemically unstable in alkaline aqueous solutions, thus excluding the use of salts for improving solubility and bioavailability. Amorphous solid dispersions, although effective for improving the biopharmaceutical performance of poorly soluble compounds, are faced with the physical instability problem. Other formulation approaches typically used to improve the apparent solubility of compounds, such as surfactants and cyclodextrins, are not effective in improving absorption because they are coupled with a lower apparent permeability.10,11

Prodrugs has the potential to

simultaneously improve both solubility and permeability, but the new molecular structures require additional efforts in chemical synthesis, characterization, and formulation development.



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Cocrystallization is one means of crystal engineering by utilizing noncovalent interactions, such as hydrogen bonding, π−π interactions, and halogen bonds.12 The multiple hydrogen bond acceptors and donors on DMY (Figure 1) render it a suitable candidate for cocrystals formation.

Among other

pharmaceutical applications, highly soluble cocrystals have been used to improve bioavailability of poorly soluble drugs.13 We, therefore, sought to solve the poor solubility problem of DMY using the cocrystallization approach.

However, a major impediment to successful application of the

cocrystallization strategy is the rapid precipitation of the poorly soluble parent drug crystals during dissolution in vivo.14,15

This phenomenon is analogous to the disproportionation of soluble salts.

Maintaining a high drug concentration in media, where drug is in either molecular form or colloidal aggregates, is critical for improving the solubility limited drug absorption.16

Desupersaturation by

precipitation of the poorly soluble drug from the solution impacts the in vivo bioavailability of soluble cocrystals. The attainable degree and duration of a supersaturation state strongly depend on both the physicochemical properties of the drug molecule and the presence of polymer/surfactant additives in the medium.17 For a given drug that readily crystallizes from a supersaturated solution, it is essential to use an effective crystallization inhibitor in the formulation to prevent premature precipitation of the drug. Hence, it is a common practice to screen polymers for inhibiting the desupersaturation process in the dissolution of amorphous solid dispersions (ASD). Although cocrystals differ from ASDs in the sense that dissolved coformers may significantly alter drug precipitation kinetics and the maximum achieve supersaturation ratio,18 it is still reasonable to expect that the use of crystallization inhibitors along with a soluble cocrystals also can be effective in achieving a sustained highly supersaturated drug solution, which leads to improved bioavailability of the drug.18 Although promising, there are only limited efforts in this direction.19,20 We hypothesized that for DMY, prolonging the supersaturated concentration during dissolution of a soluble cocrystal with a crystallization inhibitor can attain higher bioavailability than using the cocrystal alone.

The objectives of this investigation were to 1) identify and characterize soluble


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cocrystals of DMY suitable for formulation development; 2) investigate the polymer type and amount required for effectively inhibiting DMY crystallization; 3) verify effectiveness of the bioavailability enhancement by the integrated cocrystals-inhibitor strategy in rats. EXPERIMENTAL SECTION Materials (±) DMY (purity > 98%) was purchased from Kangbaotai Fine Chemical Inc. (Hubei, China). The identity and purity of this compound were verified by HPLC, H-NMR, and PXRD. Racemic nature of this compound was determined by both chiral HPLC and polarimetry. Caffeine, urea, pepsin, and pancreatin were purchased from Sigma-Aldrich (Missouri, USA).

Low-substituted hydroxypropyl

cellulose (L-HPC), polyvinylpyrrolidone (PVP) K30, hydroxypropylmethyl cellulose acetate succinate (HPMCAS-HF), Soluplus, and Kollidon VA 64 were received from BASF (Geismar, Germany). Sodium dodecyl sulfate (SDS) was purchased from J.T. Baker Inc. (Phillipsburg, NJ, USA). Polyox N750 was purchased from Dow Chemical Company. Carboxymethylcellulose Sodium (CMC-Na) was purchased from FMC Biopolymer (Philadelphia, USA).

The internal standard for pharmacokinetic analysis,

quercetin, was purchased from National Institutes for Food and Drug Control (Beijing, China). K2-EDTA was purchased from Ruite Biotechnology Co., Ltd. (Guangzhou, China). Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the USP 35/NF 30. For SGF, 2.0 g of sodium chloride and 3.2 g of pepsin were dissolved in 7.0 mL of concentrated hydrochloric acid and diluted with distilled water to 1000 mL to form a solution with pH of approximately 1.2. For SIF, 6.8 g of monobasic potassium phosphate was first dissolved in 250 mL of water. Then, 77 mL of 0.2 N sodium hydroxide and 500 mL of water were added and mixed along with 10.0 g pancreatin. The solution was adjusted to pH 6.8 with either 0.2N sodium hydroxide or 0.2N hydrochloric acid and then diluted with water to 1000 mL. All solvents were HPLC grade.

Methods 5 ACS Paragon Plus Environment


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Synthesis of cocrystals A mixture of 1:1 stoichiometric amount of DMY (0.02 mol) and coformer (0.02 mol) was suspended in 100 mL of acetonitrile in a covered glass beaker and stirred with a magnetic stirring bar under ambient conditions for 24 h. The resulting solid was then recovered by vacuum filtration and dried in oven at 60°C for 6 h. Powder X-Ray Diffraction (PXRD) Powders were analyzed on a Siemens 5005 powder diffractometer with Cu Kα radiation (1.54056 Å), with two-theta pre-calibrated using a silicon standard. Samples were scanned from 5 to 30° two theta with a step size of 0.02° and a dwell time of 0.5 s/step. The tube voltage and amperage were set at 45 kV and 40 mA, respectively. Fourier transformation infrared spectroscopy (FT-IR) FT-IR spectra of the powder samples were collected using a high resolution FT-IR spectrometer (VERTEX 70, Bruker Optics Inc., Billerica, MA, USA). For each sample, 32 scans were averaged. IR data in the range of 4000-600 cm-1 at a resolution of 4 cm-1 were processed using OPUS software (v5.5, Bruker Optics Inc., Billerica, MA, USA). Thermal analyses Powder samples (3∼5 mg) were loaded into Tzero hermetic sealed aluminum pans and heated from 25 to 250 °C with a heating rate of 10 °C/min on a differential scanning calorimeter (Q2000, TA Instruments, New Castle, DE, USA) under a continuously nitrogen purge (flow rate of 50 mL/min). The instrument was equipped with a refrigerated cooling system and pre-calibrated for temperature and enthalpy using high purity indium. Samples (∼5 mg) were heated in an open aluminum pan, on a thermogravimetry analyzer (Model Q50, TA Instruments, New Castle, DE, USA) from room temperature to 300 °C at 10 °C/min under 50 mL/min dry nitrogen purge. 6 ACS Paragon Plus Environment

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Dynamic water vapor sorption isotherm (DVS) Water sorption profiles of the materials were obtained by using an automated moisture balance (Intrinsic DVS, Surface Measurement Systems Ltd., Allentown, PA, USA) at 25 °C. The nitrogen flow rate was 50 mL/min. The sample was equilibrated at each step with the equilibration criteria of either dm/dt ≤ 0.003% or maximum equilibration time of 6h. Once one of the criteria was met, the relative humidity (RH) was changed to the next target value. After a sample was dried in situ by purging with N2 until a constant weight was obtained, water sorption isotherm from 0% to 95% RH with a step size of 5% RH was obtained. Intrinsic dissolution rate The intrinsic dissolution rate (IDR) was measured using the rotating disc method.21,22 Each powder was compressed at a force of 1000 lb, using a custom-made stainless steel die, against a flat stainless steel disc for 1 min to prepare a pellet (round, 6.39 mm in diameter). The resulting pellet had a visually smooth surface that was coplanar with the surface of the die. While rotating at 300 rpm, the die was immersed in 500 mL of the dissolution medium at 37 °C in a water-jacketed beaker. An UV−Vis fiber optic probe (Ocean Optics, Dunedin, FL) was used to continuously monitor the UV absorbance of the solution at λ=310 nm, which was used to obtain concentration-time profiles based on a previously constructed concentration – absorbance standard curve. This wavelength does not correspond to the peak absorbance of (±)DMY at 290 nm but it avoids interference from UV absorbance by coformers. Particle size analysis Particle size distribution (PSD) of (±)DMY dihydrate and two new cocrystals was measured in triplicate using a laser scattering particle sizer (Mastersizer 2000 with a Scirocco dry module; Malvern Instruments Ltd., Worcestershire, UK), operated with an inlet air pressure of 1.75 bar and a feed rate of 46 %.



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Solubility measurements The solubility of different (±)DMY solid forms in 0.1 N HCl aqueous solution was determined by equilibrating excess solid (50 mg) in 20 mL of the medium at 25 °C under stirring at 600 rpm (SP131015Q Digital stirring hotplates, Thermo Fisher Scientific. Inc., USA).

An aliquot of the

suspension was taken at predetermined time points, 5, 10, 20, 30, 45, 60, 90, 120, 150 min, and one week, and were passed through 0.22 µm membrane filters.

The filtrates were diluted with water to an

appropriate concentration for assay (DU 530 UV/Vis spectrophotometer; Beckman Coulter, Chaska, Minnesota) at λ =310 nm. The filtered residues were recovered from the filter membrane and analyzed by DSC and PXRD to identify the solid form. The apparent solubility in SGF and SIF were measured using a shake-flask method, where an excess amount (~30 mg) of material was suspended in 5 mL of the SGF or SIF and shaken at 37 °C using the QYC-200 isothermal oscillator (Shanghai CIMO instrument manufacturing Co., Ltd). Since (±)DMY is chemically unstable in neutral and alkaline solution (see Figure S1), (±)DMY concentrations in SIF and SGF after three days were determined by HPLC, instead of UV/Vis, for accuracy. The suspensions were filtered through 0.45 µm membranes and the filtrates were diluted with water to an appropriate concentration for HPLC analysis (Waters Alliance e2695, Waters Corporation, USA), using a unit equipped with a VP-ODS column (4.6 mm×250 mm, 5µm, Shimadzu Corporation, Japan) and a DAD detector (2998 photodiode array detector, Waters Corporation, USA).

Mobile phase A was 0.1%

phosphoric acid aqueous solution, whereas mobile phase B was acetonitrile. The chromatographic separation was achieved with initial solvent ratio of 80:20 (A:B). The solvent ratio was gradually changed to 88:12 over 5 min and maintained between 5 and 8 min. Then, the mobile phase was gradually changed to 65:35 over 7 min and subsequently held constant between 15 and 37 min. A flow rate of 0.8 mL/min was used, with an injection volume of 20 µL and wavelength scan from 200 to 400 nm. The column temperature was maintained at 40 °C. Analysis was performed using the Empower 3 data processing software. All experiments were triplicated. 8 ACS Paragon Plus Environment

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Desaturation of supersaturated solutions Drug concentration – time profiles of supersaturated (±)DMY solutions (with a supersaturation ratio of 6.25) were determined at 25 °C. Supersaturation ratio is defined as the solution concentration divided by thermodynamic solubility at a given temperature. Supersaturated solutions were prepared by a ‘solvent shift’ method. Briefly, 0.5 mL of a 400 mg/mL N,N-dimethylformamide (DMF) solution of (±)DMY was quickly dispensed into 100 mL of a 0.1 N HCl aqueous solution, with and without a polymer, while stirred using a magnetic stirrer at 600 rpm on a stirring hotplate (Thermo Fisher Scientific. Inc., USA). A 2 mL aliquot was withdrawn using a syringe at 5, 10, 20, 30, 45, 60, 90, 120, 150 min time points and passed through a 0.22 µm membrane filter. The filtrates were then diluted with water to an appropriate concentration for UV assay (DU 530 UV/Vis spectrophotometer; Beckman Coulter, Chaska, Minnesota) at λ =290 nm. Induction time measurement The impact of polymer on the crystallization of (±)DMY from the solution was also evaluated by measuring the nucleation induction time using a previously described method.23 A supersaturated solution (supersaturation ratio of 4.88) was generated by titrating 4mL of concentrated (±)DMY solution in DMF (500 mg/mL) to 500 mL of 0.1 N HCl aqueous medium while stirred at 600 rpm, using a magnetic stirring bar, at 37°C. The onset time of nucleation was determined from the absorbance data at λ=600 nm, which rises sharply with time once nucleation occurs, by the means of a fiber optic probe (Ocean Optics, Dunedin, FL). Although only single runs were performed in solutions of other polymers, the induction time experiments in water and PVP K30 solutions were triplicated. Dynamic light scattering (DLS) The size distribution of the colloid, formed by a supersaturated (±)DMY solution, was determined with DLS (Zetasizer Nano ZS90, Malvern Instruments, Worcestershire, UK). The scattered light was detected at an angle of 90 ° from the incident light. The solution was stirred at 600 rpm, and the 9 ACS Paragon Plus Environment


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temperature was held constant at 37 °C using a circulating water bath. Supersaturated solutions of (±)DMY were obtained by either the ‘solvent shift’ method or by dissolving a suitable amount of cocrystals. All measurements were duplicated and the average sizes were reported. Effect of polymer concentration on precipitation of (±)DMY cocrystals A cocrystal powder equivalent to 100 mg of (±)DMY was suspended in 20 mL of aqueous solutions with variable concentrations of PVP, 0.1 - 5.0% (wt%), under vigorous stirring at 37 °C for 6h. Then, the entire mixture was vacuum filtered through 3 µm filter paper (Whatman, grade 6; Hangzhouwohua, ZheJiang, China).

Absence of solid residuals suggests that the added solid was

completely dissolved. Powder dissolution of cocrystals The in vitro powder dissolution was measured in triplicate using the relatively gentle paddle method (100 rpm, 900 mL of degassed water) at 37 ± 0.5 °C (ZRS-8G, TJDX Company. LTD, China). A suitable amount of powder, 4.5 g of (±)DMY (or equivalent for cocrystals), was suspended in the dissolution vessel. An aliquot (5 mL) was taken at 5, 10, 20, 30, 40, 50, 60, 90, 120, 180, 240, and 300 min, filtered through a 0.45 µm membrane, and quantified at wavelengths of 310 nm by UV spectroscopy (UV-2450, Shimadzu, Japan). When the 2 mg/mL PVP K30 solution was used as the dissolution medium, aliquots were vacuum filtered through 3 µm filter paper placed in a Buchner funnel because they was extremely hard to pass through 0.45 µm membrane filters. In Vivo Pharmacokinetic studies Male Sprague–Dawley rats (230–300 g, 7–8 weeks) were obtained from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). All the rats had free access to food and water in a controlled environment for one week. The animal experiments were carried out in accordance with the Guidelines for Animal Experimentation of Tongji Medical College, Huazhong University of Science


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and Technology (Wuhan, China) approved by the Animal Ethics Committee of the institution. The rats were fasted overnight and randomized into groups of 3 to receive different formulations of (±)DMY, 1) (±)DMY dihydrate suspended in 0.5 mg/mL carboxymethylcellulose sodium (CMC-Na) solution; 2) (±)DMY cocrystal suspended in 0.5 mg/mL CMC-Na solution; 3) (±)DMY cocrystal suspended in 2.0 mg/mL PVP-K30 solution. After stirring for 5 min at 37°C to obtain a uniform suspension with target concentration of 5.0 mg/mL, an appropriate volume (~4 mL) was delivered via oral gavage at a dosage of 75 mg/kg body weight. This dose was slightly lower than a previous PK study of (±)DMY dihydrate, where < 1% absolute bioavailability was observed when 100 mg/mL dose was used.3 Approximately 200 µL blood samples were obtained from tail veins and placed into tubes pretreated with K2- EDTA before dosing and at 0.08, 0.16, 0.32, 0.50, 0.67, 0.83, 1, 2, 3, 4, 5 and 6 h after dosing. Blood samples were immediately centrifuged at 5000 rpm for 15 min. All the plasma samples were stored at −20 °C until they were analyzed. The plasma samples were prepared for analysis by acetonitrile precipitation. The concentrations of (±)DMY in plasma samples were determined by LC-MS following experimental details previously reported.3 The pharmacokinetic data were analyzed by PKsolver 2.0 (China Pharmaceutical University, Nanjing, China),24 using the non-compartmental method to calculate the peak plasma concentration (Cmax), time to reach Cmax (Tmax), and area under the plasma concentration - time curve (AUC). Student’s t-tests were performed using OriginPro 8.0 (OriginLab Corporation, MA, USA) to evaluate the significance of differences between groups. Differences with a p value < 0.05 were considered to be statistically significant.

Results and discussion 1. Synthesis of cocrystals



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The cocrystal screening experiments revealed two new solid forms of (±)DMY with “GRAS” compounds, caffeine and urea (Figure. 1). In the PXRD patterns of both solid forms, (±)DMY-caffeine and (±)DMY-urea, characteristic peaks of the starting materials are absent while new peaks appeared (Figure. 2a). FT-IR spectra of the solid forms have characteristic peaks of both reactants with slight peak shifts, suggesting hydrogen bonding interactions between (±)DMY and the coformers (Figure. 2b). Characteristic IR bands of these solids are summarized in Table S1. Similar flavonoid compounds of myricetin,25 quercetin,26 epigallocatechin-3-gallate,27 and epicatechin28 form 1:1 cocrystals with caffeine through ring A hydroxyl O−H···O=C hydrogen bond. The O=C stretching frequency of caffeine (1659 cm-1) and urea (1624 cm-1) shift to 1636 cm-1 and 1651 cm-1, respectively.

The hydroxyl stretch

absorption in (±)DMY (3571 cm-1) also shifted in both new crystal forms, indicating its involvement in hydrogen bonding. The DSC thermograms of (±)DMY-caffeine and (±)DMY-urea cocrystals show single melting endotherms, with onset temperatures of 141.25 oC (△Hf = 57.52 J/g) and 203.65 oC (△Hf =154.2 J/g), respectively. These melting events are distinct from those of pure (±)DMY (252.61 oC, △Hf =142.2 J/g), caffeine form II (236.29 oC, △Hf =104.6 J/g), and urea (134.43 oC, △Hf =235.3 J/g). Note that the small peak in the caffeine DSC trace with an onset at 147.40 oC and △H =18.70 J/g corresponds to caffeine Form II to I transition. The distinct melting points and associated enthalpies indicate different lattice strengths of the new solid forms. When heated, (±)DMY-caffeine and (±)DMY-urea showed a weight loss starting at 159.22 oC and 190.85 oC, which correspond to decomposition of these new phases. Collectively, the PXRD, FTIR, DSC, and TGA data show that the (±)DMY forms molecular complexes with urea and caffeine. It is generally accepted that cocrystal, rather than salt, will exclusively form when ∆pKa (= pKa(base)- pKa(acid)) < 0.29 The pKa of caffeine (-0.13 to -1.22), urea (0.18), and (±)DMY (6.8),30 lead to ∆pKa of -6.93 and -6.62 for (±)DMY-caffeine and (±)DMY-urea, respectively. Thus, (±)DMY-caffeine and (±)DMY-urea are cocrystals since proton transfer is unlikely in both systems. Chiral HPLC and polarimetry data confirmed that both cocrystals contain racemic (±)DMY, i.e., cocrystallization was not enantiomer discriminative.


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2. Assessment of cocrystal physicochemical properties The main motivation for the cocrystals screen is to improve the performance of drugs through structure modifications.31

However, it is not yet possible to reliably predict the physicochemical

properties of crystals even when their structures are known. Therefore, it is important to characterize key pharmaceutical properties of each new solid form for its suitability to support successful development of tablet products. 2.1. Moisture sorption behavior Moisture sorption profiles of (±)DMY anhydrate and the two new cocrystals at 25 oC are shown in Figure 3a. The step change in the (±)DMY profile, commencing at 10% RH and progressing through 90% RH, is typical of hydrate formation. However, the 7.3% water at 10% RH is lower than the expected 11.25% weight gain for complete conversion to the dihydrate. The discrepancy indicates incomplete conversion (~65%) of (±)DMY to the dihydrate under the experimental conditions employed. The percentage weight gain gradually increased with increasing RH and eventually reached 11.25% at ~90% RH, indicating complete conversion to the dihydrate. The stability of the hydrate above 10% RH explains why (±)DMY powders are commercially available as the dihydrate. The (±)DMY-caffeine cocrystal absorbs little moisture at RH below 45% (weight gain < 0.8%), but it absorbs 3-5% water between 45 – 75% RH and deliquesces at > 75% RH, which was visually verified. The (±)DMY-urea cocrystal showed a gradual increase in weight up to 95% RH (3% weight gain) without showing a step change in weight, suggesting the absence of hydrate formation.

2.2 Intrinsic dissolution, powder dissolution, and apparent solubility



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The intrinsic dissolution profiles of different crystal forms of (±)DMY in deionized water at 37 oC are displayed in Figure 4b with initial intrinsic dissolution rates (IDR) summarized in Table S2. The IDR of (±)DMY-caffeine cocrystal is 2.04±0.10 µg·cm-2·min-1. However, the IDRs of (±)DMY anhydrate, (±)DMY dihydrate, and (±)DMY-urea cocrystal are not significantly different, all are ~0.12 µg·cm-2·min1

. This is attributed to their rapid phase conversion to (±)DMY dihydrate when exposed to water, which

was confirmed by PXRD analysis (Figure S2).

Similar behavior was observed for curcumin-

phloroglucinol cocrystal, which did not show improvement in dissolution rate because of the same phenomenon of rapid precipitation on crystal surfaces.22 This phenomenon is also responsible when soluble solid forms, such as highly soluble salts, do not exhibit enhanced dissolution rate and bioavailability.32 The initial IDR of (±)DMY-caffeine cocrystal was approximately 17 times greater than that of (±)DMY (Fitting line 1, Figure 3b). However, the IDR rapidly decreased after the first two minutes. In the time period of 30-60 min, the dissolution rate was identical to that of the (±)DMY dihydrate (Fitting line 2, Figure 3b), which suggests a phase change during the course of the dissolution experiment. This is consistent with the observed (±)DMY-caffeine cocrystal phase conversion when suspended in water (Figure S2b). Mass median diameter size of (±)DMY dihydrate, (±)DMY-caffeine and (±)DMY-urea cocrystal particle were 3.89±0.02, 4.37±0.01, 7.45±0.90 µm. The powder dissolution profiles (Figure 3c), using 50 mg of crystals in 20 mL 0.1 N HCl at 25 oC, show that the maximum solution concentrations was reached within 20 min for both cocrystals (1.061 and 0.625 mg/mL for (±)DMY-caffeine and (±)DMY-urea, respectively). After one week, (±)DMY concentration in the (±)DMY-urea sample (0.35 mg/mL) was close to (±)DMY dihydrate solubility (0.32 mg/mL) but was 28% higher in the (±)DMY-caffeine sample (0.41 mg/mL). The (±)DMY concentrations in SGF at 37 oC were 0.46±0.03, 0.79±0.02, and 1.41±0.17 mg/mL, when (±)DMY dihydrate, (±)DMY-urea, and (±)DMY-caffeine were suspended for 3 days. Similarly, (±)DMY concentrations in SIF were 0.33±0.02, 0.94±0.11, and 1.41±0.05 mg/mL for the three crystal forms (Figure 3d).

Therefore, both cocrystal powders could generate solutions of higher


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concentrations than (±)DMY in both SGF and SIF and maintained some degree of supersaturation for at least 3 days. However, the magnitude of improvement was still less than what is desired for improving bioavailability. Overall, (±)DMY-caffeine is a promising solid form because of its high aqueous solubility and slow precipitation kinetics. The improved IDR (~17 fold) and 3-4 fold greater solution concentration in SGF and SIF, when compared to (±)DMY dihydrate, suggest that it is likely useful for improving bioavailability of (±)DMY. However, since intestinal tract is the site of absorption for most drugs, maintaining a longer period of the supersaturation by using an inhibitor may further improve bioavailability of (±)DMY. The metastable solubility of the (±)DMY-urea cocrystal is unknown due to its fast precipitation during dissolution. However, (±)DMY-urea cocrystal can still be useful in achieving higher bioavailability of (±)DMY if effective inhibition of the precipitation process can be achieved. We, therefore, explored ways of inhibiting the solution-mediated conversion to (±)DMY from both cocrystals. 3. Screen for a suitable (±)DMY crystallization inhibitor The effectiveness of the potential crystallization inhibitors, including polymers and surfactants (Figure 4), was initially evaluated at 0.1 mg/mL concentration at 25 oC. At this concentration, neither the polymers nor the surfactant significantly impact the aqueous solubility of DMY at 25 or 37 oC (Table S3). The supersaturation ratio of 6.25 was attained by adding 0.5 mL of the concentrated (±)DMY solution (400 mg/mL) in DMF to 100 mL of each potential inhibitor solution. The desupersaturation profiles (Figure 5), with and without potential inhibitors, were used to assess effectiveness of inhibitors. (±)DMY concentration did not decrease to the solubility of the thermodynamically stable (±)DMY dihydrate after 300 min, indicating slow desupersaturation process.

Among the eight potential inhibitors studied,

Soluplus and SDS promoted the crystallization, a phenomenon likely due to the decreased interfacial energy that favors nucleation of (±)DMY dihydrate from solution.33 HPMCAS, HPC, and polyox slightly inhibited the crystallization process.

PVP K30, PVPVA, and HPMC effectively inhibited the



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crystallization of (±)DMY and maintained a constant solution concentration over a period of at least 5h. Interestingly, concentrations of filtered solutions containing the three effective inhibitors were ~30% lower than the initial (±)DMY concentration of 2 mg/mL. The lower experimental concentration than expected concentration indicates the formation of particles larger than 0.22µm, the size of the filter membrane pores.

We excluded the possibility of absorption of (±)DMY to filter membrane because

concentration of DMY solution in water was the same after membrane filtration (Figure 5).

The mechanism of crystallization inhibition was further evaluated by the complementary nucleation induction time experiments using 0.1 mg/mL polymer concentration and at a supersaturation ratio of 4.88 at 37 oC. The use of a lower supersaturation ratio here was intended to minimize the formation of colloid observed at (±)DMY supersaturation ratio of 6.25. More importantly, the (±)DMY concentration corresponding to the 4.88 supersaturation ratio is close to the target (±)DMY concentration for optimum absorption. Under this condition, the precipitation commenced in 4.64 ± 0.34 minute (n = 3) in absence of a polymer (Figure 6). The small error bar indicates the excellent repeatability of this method. The induction times were 54 min and 350 min in 0.1 mg/mL of HPMC and PVPVA solutions, respectively. In these experiments, only one experiment was carried to save the material since we did not further pursue them as inhibitors. At a supersaturation ratio of 6.25 at 25 oC, 0.1 mg/mL of HPMC and PVPVA prevented the (±)DMY precipitation from a (±)DMY solution but allowed the formation of a colloid of (±)DMY.

At the lower supersaturation ratio of 4.88 but higher temperature (37 oC),

precipitation was observed in presence of 0.1 mg/mL of either PVPVA or HPMC (Figure 6). However, no precipitation was observed in 0.1 mg/mL PVP K30 (n=3). Instead, the system appeared milky (Figure 6), which indicates the formation of a colloid. The colloid does not cause absorbance of the long wavelength light, 600 nm in this case, due to the Tyndall effect.34 Colloidal (±)DMY particle size distribution shifted to the smaller end with time, with volume mean diameter changing from 85.74 nm at 5 min, to 36.74 nm at 30 min, and then to 30.14 nm at 60 min (Figure 7). In summary, PVP K30 is the most effective crystallization inhibitor for (±)DMY among those examined in this work. Neither solution viscosity

35


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nor thermodynamic solubility of (±)DMY dihydrate was significantly changed at 0.1 mg/mL PVP solution. Therefore, the likely mechanism for maintaining supersaturation by PVP K30 is through unique intermolecular interactions that interfere with nucleation and growth of (±)DMY crystals. 4. Powder dissolution in presence of PVP K30 Having identified that PVP K30 can effectively inhibit (±)DMY crystallization, the potential solubility advantages of cocrystals in the presence of 0.1 mg/mL PVP K30 at 37 oC was determined. At 25 oC only 68% of the (±)DMY-caffeine and 30% of the (±)DMY-urea dissolved without PVP K30 (Figure 3c). To study dissolution behavior in 0.1 mg/mL PVP K30, we used 100 mg cocrystals and 20 mL of the medium to minimize the amount of excess solid, which may otherwise induce rapid precipitation of (±)DMY by the way of heterogeneous nucleation. (±)DMY-caffeine cocrystal reached a plateau of ~2.75 mg/mL (92 % dissolved) after 20 min, which was maintained for at least 4 h (Figure 8). Since no solid residue remained after 20 min, the 8% discrepancy between the expected and experimental (±)DMY concentration suggests that some colloidal particles larger than the 0.22 µm membrane pores were filtered out. The (±)DMY-urea cocrystal reached a maximum concentration of 3.56 mg/mL (89 % dissolved) after 5 min but gradually decreased to ~1.3 mg/mL (32 % dissolved) after 4 h (Figure 8). Solid particles remain observable throughout the experiment. The decline in (±)DMY concentration after 5 min corresponds to the formation of the less soluble (±)DMY dihydrate. Overall, the use of 0.1 mg/mL of PVP K30 (Figure 8) led to a considerable improvement in maximum (±)DMY solution concentration by dissolution of the cocrystals over that without using a polymer at 25 oC (Figure 4c). It is reasonable to expect that the precipitation of the (±)DMY-urea cocrystal maybe be further slowed, or even eliminated, if a sufficiently high concentration of PVP K30 is used in the dissolution medium. When 0.3- 2.0 mg/mL PVP K30 was used, both (±)DMY-caffeine and (±)DMY-urea cocrystals completely dissolved to form 5.0 mg/mL (±)DMY solutions under vigorous stirring (600 rpm).



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Therefore, maximum (±)DMY concentration of both cocrystals must be at least 5.0 mg/mL (±)DMY in these media at 37 oC. Fast stirring leads to faster dissolution of cocrystals, which minimizes the chance of heterogeneous nucleation of (±)DMY dihydrate. However, earlier experiments at 600 rpm are likely unrealistic in the context of dissolution in gastrointestinal track. We, therefore, further explored the feasibility of attaining 5.0 mg/mL (±)DMY concentration by dissolving the two cocrystals under the more gentle hydrodynamic condition of 100 rpm. In absence of polymer, dissolution profile of (±)DMY dihydrate in water rose to 0.60 mg/mL in 10 min and then slowly approached the aqueous solubility of (±)DMY dihydrate at 37 oC, 0.82 mg/mL (determined after one week of equilibration) (Figure 9). The dissolution profile of (±)DMY-caffeine cocrystal reached a maximum (±)DMY concentration of 4.96 ±0.10 mg/mL at 30 min and then gradually decreased. Similarly, a maximum (±)DMY concentration of 4.13 ±0.19 mg/mL was achieved at 5 min for the (±)DMY-urea cocrystal, followed by a gradual decline. Thus, the solubility of (±)DMY-caffeine and (±)DMY-urea cocrystals are at least 6.05 and 4.80 fold of the solubility of (±)DMY dihydrate in the polymer free medium at 37 oC. However, in absence of PVP K30, the crystallization of the less soluble (±)DMY dihydrate negates advantages offered by the highly soluble cocrystals in enhancing the in vitro dissolution rate. In the 2.0 mg/mL PVP K30 solution, both (±)DMY-caffeine and (±)DMY-urea reached plateau (±)DMY concentration values of 5.0 mg/mL (Figure 9). We have found that the presence of PVP K30, up to 2.0 mg/mL, did not affect solubility of (±)DMY dihydrate. However, higher polymer concentration in the range of 0.3-2.0 mg/mL slowed down the initial dissolution rate likely because of the elevated solution viscosity. Importantly, no desupersaturation was observed for at least 300 min, which is more than sufficient for ensuring absorption of the dissolved drug in vivo. Therefore, the preparation of target 4.8 mg/mL (±)DMY solutions for optimum absorption is feasible by dissolving either cocrystal in 2 mg/mL PVP K30 solution.


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In these highly supersaturated (±)DMY solutions prepared by dissolving cocrystals in 2 mg/mL PVP K30, colloids with volume mean size of 396.9 nm ((±)DMY-urea cocrystal) and 403.2 nm ((±)DMY-caffeine cocrystal) were present (Figure 10). The significantly larger colloidal droplet size from dissolving cocrystals than those formed by pure (±)DMY from solvent shift method (Figure 7) may have been a result of higher PVP K30 concentration, the presence of coformers, stirring intensity, or any of their combinations. 5. Pharmacokinetics of the (±)DMY and (±)DMY cocrystals The (±)DMY control group exhibited a plasma concentration – time profile similar but slightly better than the literature data (Figure S3). 3 The higher Cmax and shorter tmax may be attributed the higher temperature (37 oC) used in this study to prepare formulations for dosing animals than the earlier study (room temperature). The overall AUC is not significantly different (Table 1). Figure 11a shows the pharmacokinetics profiles of (±)DMY and (±)DMY-urea cocrystal dosed with a vehicle with and without PVP K30. The (±)DMY-urea cocrystal alone did not improve the bioavailability when dissolved in 0.5% CMC aqueous solution.

This agrees with the observation that IDR of (±)DMY-urea cocrystal is

comparable to that of the (±)DMY dihydrate because of rapid surface precipitation of the urea cocrystal (Figure 3b).

When (±)DMY-urea was dosed using a 2.0 mg/mL PVP aqueous solution vehicle,

significant improvement in Cmax and AUC(0-6h) was attained, with 4.59 and 4.93 fold increase over (±)DMY dihydrate control group, respectively (Table 1).

This is consistent with the significantly

improved dissolution behaviors of (±)DMY-urea than (±)DMY dihydrate in presence of 2 mg/mL of PVP K30 (Figure 9). Two peaks in the profile indicate possible occurrence of enterohepatic cycle, which has been observed in pharmacokinetic profiles of other flavonoid compounds, e.g., quercetin and epigallocatechin-3-gallate.36 Figure 11b shows the pharmacokinetics profile of (±)DMY-caffeine using different formulations. When (±)DMY-caffeine dosed with polymer free vehicle, Cmax and AUC(0-6h) were 3.58 and 3.94 fold of



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that of (±)DMY control group, respectively (Table 1). The pharmacokinetic profiles of two (±)DMYCAF groups, with and without PVP K30 in the vehicle, could not be meaningfully distinguished because of the high variability in data, in part due to the small number of animal tested (n=3). In the group of (±)DMY-caffeine dosed with 2 mg/mL PVP as the vehicle, bioavailability in one rat was much lower, ~1/4 AUC compared with other two rats. It is likely, pharmacokinetic studies using a larger number of rats may show significantly improved AUC when 2.0 mg/mL PVP K30 is used. Nevertheless, the significantly improved pharmacokinetic profile using (±)DMY-caffeine cocrystal alone suggests that the brief maintenance (about 30 min) of ≥ 4.8 mg/mL (±)DMY concentration by dissolving this cocrystal (Figure 9) already has an impact on improving bioavailability of (±)DMY. When 2.0 mg/mL PVP K30 solution was used as a dosing vehicle, which maintains the 5 mg/mL (±)DMY concentration in vitro (Figure 9), Cmax and AUC(0-6h) are 4.26 and 5.26 fold of (±)DMY dihydrate control, respectively (Table 1). The results support the effectiveness of improving biopharmaceutical performance of a poorly soluble drug by using highly soluble cocrystals combined with polymeric nucleation inhibitors. Without using an inhibitor, precipitation of the soluble cocrystal into less soluble drug crystals may fail to improve bioavailability, as shown by (±)DMY-urea.

Conclusions Although cocrystallization has been utilized to improve the bioavailability of poorly water soluble BCS II drugs, there are few examples on more challenging BCS IV drugs. Moreover, there is scarce information on reaping solubility advantage of soluble cocrystals to improve bioavailability by preventing cocrystal precipitation during dissolution using crystallization inhibitors. Using (±)DMY as an example, we have shown that cocrystallization can be used to improve physiochemical and biopharmaceutics properties of BCS IV drugs. The use of appropriate crystallization inhibitors was effective for addressing the problem of precipitation of highly soluble cocrystals in aqueous media. This integrated strategy of


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using a suitable crystallization inhibitor along with soluble cocrystal can both achieve and maintain the desired supersaturation for improving bioavailability.

Supporting Information Summary of characteristic IR peaks and bands and intrinsic dissolution rates of different DMY solid forms; solubility in the presence of polymer or surfactant; representative HPLC chromatographs of DMY saturated solutions in SGF and SIF for 1 week at 37 oC; Powder X-ray diffraction patterns of samples slurried samples; pharmacokinetic profiles of DMY control in this study and literature data. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements We are grateful for resources from the University of Minnesota through the Minnesota Supercomputing Institute. Some of the experiments were performed at the University of Minnesota I.T. Characterization Facility, which receives partial support from the NSF through the NNIN program. We thank Mr. Dong Xiang, Mr. Yu Xia and Ms. Xuejia Xie for their help with the pharmacokinetics data collection.

REFERENCES (1) Hou, X.; Tong, Q.; Wang, W.; Wei, X.; Shi, C.; Fang, J. Life. Sci. 2015, 130, 38-46. (2) Chen, S.; Zhao, X.; Jing, W.; Li, R.; Yu, Q.; Wang, X.; Gao, Y.; Shu, F.; Yong, Z.; Peng, L. Pharmacol. Res. 2015, 99, 74-81. (3) Tong, Q.; Hou, X.; Fang, J.; Wang, W.; Wei, X.; Xu, L.; Xie, X.; Shi, C. J. Pharmaceut. Biomed. 2015, 114, 455-461.



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(4) Ruan, L. P.; Yu, B. Y.; Fu, G. M.; Dan, N. Z. J. Pharmaceut. Biomed. 2005, 38, 457-64. (5) Ruan, L. P.; Chen, S.; Yu, B. Y.; Zhu, D. N.; Cordell, G. A.; Qiu, S. X. Eur. J. Med. Chem. 2006, 41, 605-610. (6) Jr, M. E.; Kandaswami, C.; Theoharides, T. C. Pharmacol. Rev. 2000, 52, 673-751. (7) Hu, M. Mol. Pharm. 2007, 4, 803-806. (8) Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R. Pharm. Res. 1995, 12, 413-420. (9) 334 mg was maximum absorption dose (MAD), the 28% absorption was the average value based on the ref 5, which predict Fa was 21-35% in human. Cs was calculated by MAD/(250mL*0.28) (10) Dahan, A.; Miller, J. M.; Hoffman, A.; Amidon, G. E.; Amidon, G. L. J. Pharm. Sci. 2009, 99, 2739-49. (11) Miller, J. M.; Avital, B.; Krieg, B. J.; Carr, R. A.; Borchardt, T. B.; Amidon, G. E.; Amidon, G. L.; Arik, D. Mol. Pharm. 2011, 8, 1848-1856. (12) Duggirala, N. K.; Perry, M. L.; Almarsson, Ö.; Zaworotko, M. J. Chem. Commun. 2016, 52, 640-655. (13) Sun, C.C. Expert Opin. Drug Deliv. 2013, 10: 201-213. (14) Bak, A.; Gore, A.; Yanez, E.; Stanton, M.; Tufekcic, S.; Syed, R.; Akrami, A.; Rose, M.; Surapaneni, S.; Bostick, T. J. Pharm. Sci. 2008, 97, 3942–3956. (15) Ullah, M.; Hussain, I.; Sun, C. C. Drug Dev. Ind. Pharm. 2015, 42, 969-976. (16) Raina, S. A.; Zhang, G. G. Z.; Alonzo, D. E.; Wu, J.; Zhu, D.; Catron, N. D; Yi, G.; Taylor, L. S. J. Pharm. Sci. 2014, 103, 2736–2748. (17) Raina, S. A.; Alonzo, D. E.; Zhang, G. G. Z.; Gao, Y.; Taylor, L. S. Mol. Pharm. 2014, 11, 3565-3576. (18) Taylor L.S.; Zhang G.G.Z. Adv. Drug Deliv. Rev. (2016), Adv Drug Deliv Rev. 2016, 101, 122-142. (19) Childs, S. L.; Kandi, P.; Lingireddy, S. R. Mol. Pharm. 2013, 10, 3112-3127. (20) Remenar, J. F.; Peterson, M. L.; Stephens, P. W.; Zhong, Z.; Zimenkov, Y.; Hickey, M. B. Mol. Pharm. 2007, 4, 386-400. (21) Wang, C.; Perumalla, S.; Lu, R.; Fang, J.; Sun, C.C. Sweet berberine. Cryst. Growth Des. 2016, 16, 933–939. (22) Chow, S. F.; Shi, L.; Ng, W. W.; Leung, K. H. Y.; Nagapudi, K.; Sun, C. C.; Chow, A. H. L. Cryst. Growth Des. 2014, 14, 5079-5089. (23) Kuldipkumar, A.; Kwon, G. S.; Zhang, G. G. Z. Cryst. Growth Des. 2006, 7, 234-242. (24) Zhang, Y.; Huo, M.; Zhou, J,; Xie, S. PKSolver: Comput. Meth. Prog. Bio. 2010, 99, 306-314. (25) Hong, C.; Xie, Y.; Yao, Y.; Li, G.; Yuan, X.; Shen, H. Pharm Res, 2015, 32, 47-60. (26) Smith, A. J.; Kavuru, P.; Wojtas, L.; Zaworotko, M. J.; Shytle, R. D. Mol. Pharm. 2011, 8, 1867-1876. (27) Sinha, A. S.; Maguire, A. R.; Lawrence, S. E. Cryst. Growth Des. 2015, 15, 984-1009. (28) Smith, A. J.; Kavuru, P.; Arora, K. K.; Kesani, S.; Tan, J.; Zaworotko, M. J.; Shytle, R. D. Mol. Pharm. 2013, 10, 2948-2961. (29) Childs S, Stahly G, Park A. Mol. Pharm. 2007, 4, 323-338. (30) pKa for caffeine given as a range of values based the book of Profiles of Drug Substances, Excipients and Related Methodology, volume 33: Critical Compilation of pKa Values for Pharmaceutical Substances. Academic Press; Pka for urea given by "pKa Data" Williams, R. and pKa for (±)DMY calculated from ACD/lab. (31) Sun, C. C. J. Pharm. Sci. 2009, 98, 1671-87. (32) Hawley, M. Mol. Pharm. 2010, 7, 1441-1449. (33) Rodríguez-Hornedo, N.; Murphy, D. J. Pharm. Sci. 2004, 93, 449–460. (34) Agarwal, P.; Berglund, K. A. Cryst. Growth Des. 2004, 4, 479-483. (35) Patel, D. D.; Anderson, B. D. Mol. Pharm. 2014, 11, 1489-1499. (36) Moon, Y. J.; Morris, M. E. Mol Pharm. 2007, 4, 865-72.


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Table

Table 1. Pharmacokinetic parameters for (±)DMY and (±)DMY cocrystals dosed at 75 mg/kg (n = 3)a Parameters

(±)DMY REFb

(±)DMY Control

(±)DMYcaffeine

(±)DMYurea

(±)DMYcaffeine with PVP 862.2±352.2

(±)DMYurea with PVP 927.3±353.4

Cmax 81.3±17.2 202.0±43.9 723.0±338.9 127.2±37.5 (ng/mL) Tmax 2.67±1.5 0.4±0.2 1.0±0.7 0.9±0.2 2.9±1.6 0.8±0.5 (h) AUC(0-6h) 20.50±5.9 23.2±11.2 91.5±18.8 16.8±2.7 122.2±64.5 114.4±28.4 (mg×min/L) a Cmax, maximum plasma concentration; Tmax, time of maximum plasma concentration; AUC(0-6h), calculated area under the curve from 0 to 6h. b (±)DMY REF was dosed at 100 mg/kg and suspended the (±)DMY dihydrate in 0.5 % CMC-Na aqueous solution at room temperature. Data are from ref. 3, n = 6.



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Figures

Figure 1. Molecular structures of dihydromyricetin (DMY), caffeine, and urea


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(a)

(c)

(b)

(d)

Figure 2. Characterization of (±)DMY cocrystals, compared with urea and caffeine anhydrate and (±)DMY anhydrate: a) PXRD patterns, b) FT-IR spectra, c) DSC, and d) TGA



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(a)

(c)

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(b)

(d)

Figure 3. The physicochemical properties of (±)DMY cocrystals compared with (±)DMY: a) moisture sorption isotherm at 25 oC, b) intrinsic dissolution profiles in water at 37 oC, c) powder dissolution profiles in 0.1 N HCl at 25 oC, d) apparent solubility in SGF and SIF at 37 oC


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Figure 4. Molecular structures of polymers and surfactant used in this study



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Figure 5. Desupersaturation curves of (±)DMY (2 mg/mL, 6.25 supersaturation ratio) from 0.1 HCl aqueous solution containing 0.1 mg/mL of various potential crystallization inhibitors at 25°C. The horizontal line marks the saturation solubility of (±)DMY dihydrate.


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Figure 6. Induction time measurement of (±)DMY, at supersaturation ratio of 4.88, from 0.1 mg/mL polymer solution at 37°C.



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Figure 7. Evolution of (±)DMY colloidal droplets with time for a 5.0 mg/mL (±)DMY aqueous solution containing 0.1 mg/mL PVP K30 at 37°C.


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Figure 8. Powder dissolution profiles of 100 mg of (±)DMY cocrystals and the dihydrate at 37 °C in 0.1 mg/mL PVP K30 solution in 0.1 N HCl. The broken line indicates thermodynamic solubility of (±)DMY dihydrate in water at 37 °C.



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Figure 9. Powder dissolution profiles of (±)DMY cocrystals with and without 2 mg/mL of PVP K30 in water at 37°C. The dissolution profile of crystalline (±)DMY dihydrate in water is included as a reference. The data represent the mean value with error bar (n=3).


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2 mg/mL PVP

Figure 10. Colloidal droplet size distribution of 5.0 mg/mL (±)DMY solution formed by dissolving the two cocrystals in a 2 mg/mL PVP K30 aqueous solution at 37°C.



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(a)

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(b)

Figure 11. Pharmacokinetic profiles of (±)DMY, (±)DMY cocrystals in the absence and presence of PVP K30 pre-dissolved at a concentration of 2 mg/mL in 0-6 h (n=3).


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For Table of Contents Use Only

Enhancing

bioavailability

of

dihydromyricetin

through

inhibiting

precipitation of soluble cocrystals by a crystallization inhibitor Chenguang Wang, Qing Tong, Xiaolong Hou, Shenye Hu, Jianguo Fang, and Changquan Calvin Sun

TOC graphic

Synopsis Dihydromyricetin ((±)DMY) is a biopharmaceutics classification system (BCS) IV drug, exhibiting dissolution limited absorption. cocrystals with caffeine and urea.

A systematic screen identified two 1:1 soluble

The use of either soluble cocrystal in presence of a

crystallization inhibitor led to an approximately five fold enhancement in oral bioavailability of (±)DMY.


Enhancing Bioavailability of Dihydromyricetin ... - ACS Publications

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