Impact of the Dissolution Profile of the Cilostazol Cocrystal with

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Page 1 ofCrystal 37 Growth & Design

1 2 cilostazol (CLZ) 3 4 5 6 7 8 ACS Paragon Plus Environment 9 4-hydroxy 2,4-dihydroxy 2,5-dihydroxy 10 benzoic acid benzoic acid benzoic acid (2,4DHBA) (2,5DHBA) 11 (4HBA)

00

HBA (a-1) CLZ-4HBA cocrystal

00

Crystal Growth & Design Page 2 of 37

0 0 00 A 0(a-2) 4HBA5 00 00 0 0 5 (b-1) CLZ-2,4DHBA 00 ,4DHBA

10

15

20

25

1 10 15 20 25 2 cocrystal 3 0 4 00 0 5 10 15 20 25 5 2,4DHBA 00 BA (b-2) 0 6 00 0 5 10 15 20 25 7 CLZ-2,5DHBA (c-1) cocrystal 00 5DHBA 00 8 0 00 09 5 10 15 20 25 00 BA(c-2) 2,5DHBA 10 00 0 11 00 0 5 10 15 20 25 12CLZ (d) 00 13 0 0 5 10 15 20 25 00 14 (e) … Amorphous solid dispersion phous 15 00 CLZ ACS Paragon Plus Environment 16 00 10 15 20 25 0017 55 10 15 20 25 2 theta. (deg.) 18

30

30

30

30

30

30

30

30 30

Page 3 of 37 Crystal Growth & Design CLZ-4HBA cocrystal 0

CLZ-2,4DHBA cocrystal

(W/g)(W/g) flow flow Heat Heat

1 -10 2 3 4 -20 5 6 7 -30 8 9 10 -40 11 12-35 13 -50 14 15 16


Amorphous solid dispersion

x10

CLZ

80

80

100

120

140

160

180

160

180

ACS Paragon Plus Environment 100

Temperature (ºC) 120 140

Temperature (ºC)

a

1 2 3 4 5 6 7 8 9 b 10 11 12 13 14 15 16 17 18 19 20 c 21 22 23 24 25 26


ACS Paragon Plus Environment

0.020

Concentration of CLZ (mM)

Page 5 of 37 Crystal Growth &yDesign = 0.0836x + 0.0012 R² = 0.9999

0.015

y = 0.4668x + 0.0028

1 R² = 0.9781 2 3 0.010 y = 0.0357x + 0.001 4 R² = 0.9999 5 6 0.005 CLZ-4HBA cocrystal 7 CLZ-2,4DHBA cocrystal 8 CLZ-2,5DHBA cocrystal ACS Paragon Plus Environment 9 0.000 10 0.0 0.1 0.2 0.3 0.4 1/[coformer] (mM-1) 11

1.0

0.8


Concentration of CLZ (mM)

1 2 SCC-2,5DHBA 3 4 0.6 5 6 7 0.4 8 SCC-2,4DHBA 9 100.2 SCC-4HBA 11 12 CLZ 13 ACS Paragon Plus Environment 0.0 14 0.0 0.2 0.4 0.6 0.8 1.0 15 Concentration of coformer (mM)

100 7 of 37 Page Crystal Growth & Design CLZ-4HBA cocrystal CLZ-2,4DHBA cocrystal CLZ-2,5DHBA cocrystal Amorphous solid dispersion CLZ hammer-milled

a 80

Dissolved (μg/mL)

1 2 60 3 4 5 40 6 7 20 8 9 0 10 11 12 140 13 120 14 15 100 16 1780 18 1960 2040 21 2220 23 0 24 25

0

10

20

30 Time (min)

40

50

60

Dissolved (μg/mL)

b


10

20

30 40 Time (min)

50

60

Crystal Growth & Design Page 8 of 37 CLZ-4HBA cocrystal

2500

CLZ-2,4DHBA cocrystal CLZ-2,5DHBA cocrystal

Serum concentration (ng/mL)

1 2000 2 3 4 1500 5 6 1000 7 8 9 500 10 11 0 12 13

Amorphous solid dispersion CLZ hammer-milled


2

4 Time (hr)

6

8

Page 9 of 37

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Crystal Growth & Design

Impact of the Dissolution Profile of the Cilostazol Cocrystal with Supersaturation on the Oral Bioavailability Motoyasu Yoshimura*,†,§, Masateru Miyake†, Tatsuya Kawato‡, Masahiko Bando‡, Masafumi Toda†, Yusuke Kato†, Toshiro Fukami#, and Tetsuya Ozeki§ †

Formulation Research Institute and ‡Medicinal Chemistry Research Laboratories, Otsuka

Pharmaceutical Co., Ltd., Tokushima 771-0182, §Drug Delivery and Pharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 467-8603, and #

Department of Molecular Pharmaceuticals, Meiji Pharmaceutical University, Kiyose, Tokyo

204-8588, Japan. *

[email protected]

ABSTRACT: The objectives of this study are to enhance the oral bioavailability of cilostazol (CLZ), which is a poorly-soluble compound, by cocrystallization and to evaluate the correlation between the calculated solubility of the cocrystal by the solubility product (Ksp) and the complexation constant (K11) and the performance of the cocrystal. Cocrystals of CLZ with 4hydroxybenzoic

acid

(4HBA),

2,4-dihydroxybenzoic

acid

(2,4DHBA),

and

2,5-

dihydroxybenzoic acid (2,5DHBA) were prepared. Stoichiometric 1:1 structures were formed in the crystal packing of the three cocrystals according to single crystal X-ray diffraction. The calculated solubilities of the CLZ-4HBA cocrystal, CLZ-2,4DHBA cocrystal, and CLZ-


1


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Page 10 of 37

2,5DHBA cocrystal were 9.5-fold, 14.5-fold, and 34.3-fold higher than that of CLZ, respectively. Interestingly, the supersaturated dissolution profile in the non-sink condition was inverselycorrelated with the calculated solubility of the cocrystals, and the CLZ-4HBA cocrystal, which mildly enhanced the solubility compared to the other cocrystals, effectively prolonged the supersaturation. The in vivo performance correlated with the in vitro dissolution profile, and the bioavailability of the CLZ-4HBA cocrystal in beagles was also significantly enhanced even when compared to the amorphous solid dispersion. The cocrystallization of CLZ could be an effective means to enhance the bioavailability, but excessive solubility enhancement was not preferable for the CLZ cocrystal.

INTRODUCTION Recently, the number of poorly soluble drugs has been increasing in the market because hydrophobic substituents tend to be introduced to pharmaceutical compounds in order to enhance pharmacodinamic activity. The Biopharmaceutics Classification System (BCS) is proposed in order to categorize the drug type based on solubility and permeability.1 According to a previous report, approximately 40% of the market drugs and 80~90% of the R&D pipelines are poorly soluble drugs (BCS class II or class IV), and these compounds are likely to fail due to the poor solubility.2 Therefore, solubility enhancement of poorly soluble drugs is very important for pharmaceutical development, and various formulation techniques (i.e., micronized or nanonized techniques, amorphous solid dispersions, salts, cocrystals, and lipid-based formulations) have been studied in order to improve solubility properties. It is important to select the suitable formulation type for solubility improvement based on the physicochemical properties of the compound.

In the case of a highly-lipophilic compound, the bioavailability of lipid-based


2

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formulation was remarkably enhanced compared to conventional formulation or drug suspension.3-5 It has been reported that the compounds consisting of a dissociable group were able to form salt, leading to high solubility and oral absorption.6-8 Whereas amorphous solid dispersion is a useful formulation for bioavailability enhancement,9-11 the conversion to a stable crystal and the chemical degradation of the amorphous solid dispersion with high internal energy and specific volume were found to be critical.11-13 On the other hand, salts and cocrystals are prone to be physicochemically stable.14 In particular, cocrystals are very useful for undissociated and poorly-soluble compounds because it is formed with various coformers without an ionic bond, and a high potential for enhancing oral absorption has been reported.8,9,15-17 Cilostazol (CLZ) is a synthetic antiplatelet agent with a vasodilating effect.18 It is a poorly soluble compound categorized in BCS class II without dissociable groups,19 as shown in Figure 1.

Because the area under the serum concentration - time curve (AUC) of CLZ for a

conventional 50-mg tablet is significantly lower compared to an ethanol solution in human, it has been suggested that the poor solubility of CLZ limits bioavailability.20 In this research, the cocrystallization of CLZ was investigated in order to improve the poor solubility and to understand how to enhance the oral absorption. In prior investigation, cocrystal screening of CLZ was performed by liquid assisted grinding (LAG). As a result, CLZ formed cocrystals with 4-hydroxybenzoic acid (4HBA), 2,4-dihydroxybenzoic acid (2,4DHBA), and 2,5dihydroxybenzoic acid (2,5DHBA), whose structures are shown in Figure 1. In the experiment, the cocrystals were newly prepared by the slurry method under adjusted conditions because the cocrystals prepared by the LAG method may partially contain the amorphous form of CLZ, and the control of cocrystal particle size was difficult. All coformers forming cocrystals with CLZ were derivatives of hydroxybenzoic acid, and the substitution site and ratio of the hydroxyl


3


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Page 12 of 37

group influenced the solubilities of these coformers (the solubilities of 4HBA, 2,4DHBA, and 2,5DHBA were 4.9, 6.0, and 22 mg/mL at 25 °C, respectively21,22). As the solubility of the cocrystal is proportional to that of the coformer,23 the unique performances in each cocrystal were predicted even though these were formed with similar coformers. In addition, it has been reported that the coformers influenced not only the solubility but also the permeability and the oral absorption in vivo.24,25 Generally, a cocrystal forms by weak interactions, such as hydrogen bonding, between an active pharmaceutical ingredient and a coformer rather than by ionic bonding to form a salt crystal; hence, a cocrystal is likely to dissociate more quickly than a salt.14 Some of the amorphous solid dispersions or cocrystals cause not only solution-mediated transformation but also surface-mediated transformation to the more stable crystal as soon as these come in contact with liquids.26,27 Therefore, a formulation design to prevent the conversion to a stable crystal or precipitation is important for the enhancement of bioavailability.28,29

Figure 1. Chemical structures of CLZ and coformers. The purpose of this research is to clarify the correlation between the solubility enhancement of a cocrystal and the supersaturated profile and to investigate the effect of supersaturation in vitro


4

Page 13 of 37

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on the absorption in vivo, in addition to the bioavailability enhancement of CLZ by cocrystallization.

To evaluate the supersaturation of cocrystals, kinetic dissolution and

equilibrium solubility measurements were performed. The solubility product Ksp was evaluated as the equilibrium solubility of the CLZ cocrystals. If CLZ forms a stoichiometrically 1:1 cocrystal with a coformer, the solubility product Ksp is calculated by applying a linear approximation between the inverse of the coformer concentration and CLZ solubility profile to Equation 1, where K11 is the binding constant for the complex formation of CLZ/coformer in solution.30 ௄

౩౦ ሾCLZሿ୘ = ሾୡ୭୤୭୰୫ୣ୰ሿ + ‫ܭ‬ଵଵ ‫ܭ‬ୱ୮

(1)

౐

Since the hydroxybenzoic acid derivatives that form the CLZ cocrystals are acidic compounds, ionization of the coformer occurs with an increase in pH, and as a consequence, the solubility of the cocrystal exponentially increases over the pKa of the coformer, although CLZ does not dissociate over the entire pH range.23,31

The solubility products of CLZ cocrystals were

evaluated in simulated gastric fluid (SGF) because the solubility in acidic pH condition is especially important for highly-permeable CLZ to enhance oral absorption. As the ionization of the coformer can be ignored in the acidic condition, which is lower than the pKa of the coformer, the intersection of a stoichiometric 1:1 line and the theoretical solubility curve calculated by Equation 1 introducing Ksp and K11 signifies the solubility of the cocrystal.23 If the solubility of the cocrystal is higher than that of CLZ, temporary supersaturation can be detected in the kinetic dissolution. Accordingly, it is likely that the difference of solubility between the CLZ and cocrystal influences the supersaturated profile.

In this report, the correlative relationship

between the calculated solubility by the solubility product and the supersaturation in the kinetic dissolution of CLZ cocrystal was investigated.


5


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Page 14 of 37

EXPERIMENTAL SECTION Materials. CLZ was synthesized and hammer-milled in Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan).

4HBA and 2,5DHBA were purchased from Wako Pure Chemical

Industries, Ltd. (Osaka), and 2,4DHBA was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo) as the coformers.

Hypromellose, type 2910 grade TC-5E, for the formation of

amorphous solid dispersion of CLZ and the kinetic dissolution was provided by Shin-Etsu Chemical Co., Ltd. (Tokyo). Lactose monohydrate was provided from DFE Pharma Ltd. (Goch, Germany), and colloidal silicon dioxide was provided from Nippon Aerosil Co., Ltd. (Tokyo). These excipients were used to pre-mix with CLZ substances in order to improve the wettability when kinetic dissolution was performed. SGF was prepared according to USP without any addition of enzymes. All other regents for the analytical procedures were purchased from Wako Pure Chemical Industries, Ltd. (Osaka). Preparation of CLZ Cocrystal. CLZ cocrystals were prepared by the slurry method. The slurry suspension for CLZ-4HBA cocrystal was prepared by the addition of 10 g of CLZ in 40 mL of acetone containing 7.48 g of 4HBA (stoichiometric 1:2 CLZ:4HBA complex addition). Similarly, the slurry suspension for CLZ-2,4DHBA cocrystal was prepared by the addition of 10 g of CLZ in 40 mL of acetone containing 8.34 g of 2,4DHBA (stoichiometric 1:2 CLZ:2,4DHBA complex addition). These slurry suspensions were stirred at room temperature for 7 days. The slurry suspension for CLZ-2,5DHBA cocrystal was prepared by the addition of 10 g of CLZ in 15 mL of acetone containing 6.67 g of 2,5DHBA (stoichiometric 1:1.6 CLZ:2,5DHBA complex addition). As the viscosity of the slurry suspension rapidly increased as soon as CLZ was introduced in acetone with a high concentration of 2,5DHBA, it was severely shaken at 40 °C for 1 day. After that, it was stirred at room temperature for 6 days. These slurry suspensions were


6

Page 15 of 37

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filtered under reduced pressure, and the remaining solid mixtures (cocrystals) on filter paper were dried in a vacuum overnight. Preparation of Amorphous Solid Dispersion of CLZ. Then, 5 g of CLZ and 10 g of hypromellose were dissolved into 200 mL of a dichloromethane/ethanol mixed solvent (80:20, v:v). The pre-dissolved solution was spray-dried by Lab-scale Spray-dryer GB-22 (Yamato Scientific, Tokyo), and CLZ amorphous powder was prepared. The amorphous powder was vacuum-dried at 40 °C for 12 h in order to remove the residual solvent. X-ray Powder Diffraction. CLZ cocrystals, amorphous solid dispersion and host crystal were analyzed by X-ray powder diffraction measurements using X’ Pert PRO MPD (PANalytical, Almelo, The Netherlands) equipped with Cu Kα1 radiation. The tube voltage and amperage were set to 45 kV and 40 mA, respectively. Samples were scanned between 2θ of 3° and 40° with a step size of 0.04° at 1.905 step/sec. Thermal Analysis. Thermodynamic properties of CLZ cocrystal, amorphous solid dispersion and host crystal were evaluated by DSC Q2000 (TA Instruments, Newcastle, DE) equipped with a refrigerated cooling system. Then, 3-6 mg of the samples were heated at a rate of 5 °C/min under a nitrogen purge (continuously purged at a flow rate of 300 mL/min) in a closed aluminum pan. Single-crystal X-ray Diffraction. CLZ-4HBA single cocrystal was prepared in the solution dissolved 100 mg of CLZ and 374 mg of 4HBA (stoichiometric 1:10 CLZ:4HBA complex addition) in 20 mL of acetone. CLZ-2,4DHBA single cocrystal was prepared in the solution dissolved 100 mg of CLZ and 834 mg of 2,4DHBA (stoichiometric 1:20 CLZ:2,4DHBA complex addition) in 20 mL of acetone. These solutions were filtered by 0.22-µm filters, and the single crystals were slowly grown in the closed standing glass vial at room temperature. CLZ-


7


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Page 16 of 37

2,5DHBA single cocrystal was prepared in the supersaturated solution, which was prepared by heating the suspension added 4.0 g of CLZ and 16.7 g of 2,5DHBA (stoichiometric 1:10 CLZ:2,5DHBA complex addition) in 20 mL of acetone up to 70 °C and filtering it by 0.22-µm filters. The single crystal was grown in the closed standing glass vial at 30-40 °C. Well-shaped crystals were chosen and mounted on a glass fiber on a eucentric goniometer head. Singlecrystal X-ray diffraction data were collected on a Rigaku RAXIS RAPID diffractometer (Rigaku, Tokyo) using graphite monochromated radiation (λ = 0.071075 Å) at 96 K. Data collections and processing were performed with RAPID-AUTO.

The structures were solved by the direct

method (SHELXT32) and expanded using Fourier techniques. The non-hydrogen atoms were refined anisotropically. The hydrogen atoms of amide group and hydroxyl groups were found in a difference Fourier map and refined isotropically. The other hydrogen atoms were refined using a riding model. All calculations were performed using the CrystalStructure crystallographic software package and SHELXL.33 Equilibrium Solubility Measurement.

In this study, the equilibrium solubility of CLZ

cocrystal was defined as the value calculated from the solubility product Ksp and the binding constant K11 for complex formation in SGF. In order to calculate Ksp and K11, the solubility of CLZ was investigated in the SGF with various concentrations of the coformer. In reference to the solubility of the coformer, 0.2-4.0 mg/mL of 4HBA, 0.3-8.0 mg/mL of 2,4DHBA, and 0.410.0 mg/mL of 2,5DHBA were individually dissolved in the SGF.

CLZ cocrystals were

individually applied into 20 mL of each coformer solution over the saturated solubility, and they were shaken at rates of 50 Hz at 37 °C for 4 days. These suspensions were filtered by 0.22-µm filters and assayed by HPLC.


8

Page 17 of 37

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Kinetic Dissolution Profile Measurement.

The CLZ substances were mixed with a

quadruple mixture of lactose monohydrate and colloidal silicon dioxide (49:1, w:w) in order to disperse them immediately into an aqueous solution prior to performing the kinetic dissolutions. The blended powder corresponding to 10 mg or 50 mg of CLZ was applied into 100 mL of the SGF containing 1% (w/w) Hypromellose, and aliquots were taken from them at a determined interval up to 2 h while stirring at 500 rpm with the temperature being maintained at 37±1 °C. They were filtered by 0.22-µm filters and were assayed by HPLC. High-performance Liquid Chromatography (HPLC).

The CLZ samples for the

equilibrium solubility measurement and the kinetic dissolution were determined by a HPLC system (LC-2010C, Shimadzu, Kyoto) equipped with a UV detector set at 257 nm.

The

analytical column was ODS-80Ts (4.6×150 mm, 5 µm, Tosoh, Tokyo). The mobile phase, water : acetonitrile : methanol (100:70:30, v:v:v), was delivered at 1.5 mL/min. Samples were mixed in the equal volume with a 40 µg/mL solution of benzophenone in methanol as the internal standard and were introduced into HPLC system. Bioavailability Studies in Beagle Dogs. The substances corresponding to 100 mg of CLZ were introduced into 40 mL of the 1% (w/w) Hypromellose solution, and it was well-dispersed. The pre-prepared suspensions were immediately orally-administered into the beagle dogs (body weights between 8-12 kg) at 100 mg/body. A washout period of 1 week was kept between consecutive dosings. The dogs were fasted for at least 12 h before dosing and for 8 h after dosing. The dogs were allowed free access to water throughout the experiment. Blood samples (1.5 mL) were collected from a forearm vein at 0 (pre-dose), 0.5, 1, 2, 3, 4, 6 and 8 h after dosing for the oral administration. Serum samples were obtained by centrifugation of the blood samples and stored at -20 °C until use. Our investigations were performed after approval by our local


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Page 18 of 37

ethical committee at Otsuka Pharmaceutical Co. Ltd. in accordance with "Principles of Laboratory Animal Care" (NIH Publication # 85 - 23). Assay of CLZ in Serum. CLZ in serum was determined by validated LC-MS-MS (QuattroMicro, Waters, Milford) methods using OPC-3930 as the internal standard.

Briefly, after

extraction of CLZ and OPC-3930 from serum (0.1 mL) using HPLC-grade tert-butyl methyl, the organic layer was evaporated (SC210A, Thermo Scientific, Tokyo), and it was dissolved in methanol : water (50:50, v:v) at 0.2 mL. Samples were introduced into the LC-MS-MS system. The analytical column was Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm, Waters). The mobile phase, water : acetonitrile : formic acid at 50:50:0.1 (v:v), was delivered at 0.25 mL/min. The monitored ions of CLZ and OPC-3930 were m/z 370.5 as the parent ion and m/z 288.5 as the daughter ion and m/z 354.7 as the parent ion and m/z 272.5 as the daughter ion, respectively. The range of the standard curve was 10 to 5000 ng/mL, and the coefficient of variation for the standard curve ranged from 0.1 to 20.0%. Pharmacokinetic Analysis. In vivo pharmacokinetic parameters after oral administration were calculated by WinNonlin (version 6.4, Pharsight, Mountain View, CA). The area under the serum concentration - time curve (AUC) and the mean residence time for serum concentration (MRT) were calculated from 0 to 8 h by following the trapezoidal rule.

The highest

concentration of CLZ was denoted as Cmax, and the time for Cmax was defined as tmax. Statistical analyses of the PK parameters were performed by Dunnett’s test using Microsoft Excel compared to the hammer-milled CLZ.

A p-value of less than 0.05 was considered to be

significant. RESULTS AND DISCUSSION


10

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Characterization of CLZ Cocrystals and Amorphous Solid Dispersion. XRPD patterns of cocrystals prepared by the slurry method and the amorphous solid dispersion of CLZ are shown in Figure 2. All cocrystals represented different XRPD patterns from the CLZ host crystal, and the amorphous solid dispersion represented a halo pattern without a diffraction peak. The CLZ4HBA and CLZ-2,4DHBA cocrystals were likely to consist of a similar crystal construction because some analogic peaks were detected in each XRPD pattern.

Figure 2. XRPD patterns of cocrystals, amorphous solid dispersion, and reference materials. The DSC heat flows are shown in Figure 3, and the melting point and enthalpy of melting are shown in Table 1.

From the results, the melting points of the CLZ-2,4DHBA and CLZ-

2,5DHBA cocrystals were 152.9 °C and 120.1 °C, respectively, which were lower temperatures compared to CLZ, although the melting point of the CLZ-4HBA cocrystal was 161.7 °C, i.e., nearly the same melting point as CLZ. Although the melting point of the amorphous solid dispersion was detected at 156.4 °C, an exothermic peak occurred due to recrystallization of CLZ


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Page 20 of 37

before the melting point (135.9 °C). As a glass temperature Tg of the amorphous solid dispersion was detected around 40 °C of DSC curve, it was likely to be unstable, which may convert easily to the host crystal in an aqueous solution.13 As the predicted solubilities of CLZ cocrystals from the thermal properties were higher than that of CLZ (data not shown), the enhancement of solubility by cocrystallization was expected.

Figure 3. DSC curves of cocrystals, amorphous solid dispersion, and host crystal.

The

exothermic peak of the amorphous solid dispersion is separately represented as a tenfold enlarged profile. Table 1. Melting points and enthalpy values for cocrystals, amorphous solid dispersion, and host crystal. Substance

Melting

Enthalpy

point (°C)

of melting, ∆Hm


12

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(kJ/mol)a

a

CLZ-4HBA cocrystal

161.7

41.5


152.9

35.6


120.1

28.9

CLZ

159.7

51.0

The enthalpy of melting for cocrystals is normalized by moles of component molecules (drug +

coformer) per mole of cocrystal. The crystal packings of CLZ cocrystals calculated by single-X-ray crystal diffraction are shown in Figure 4. The crystal packings of CLZ-4HBA and CLZ-2,4DHBA cocrystals were similar. The 3 hydrogen bonds between CLZ and hydroxybenzoic acid, i.e., (1) amine of tetrazole - carboxylic acid, (2) amine of carbostyril group - carboxylic acid, and (3) carbonyl of carbostyril group - para-hydroxyl, were confirmed in the crystal packing. The crystal packing of the CLZ-2,5DHBA cocrystal differed from that of the CLZ-4HBA cocrystal and CLZ-2,4DHBA cocrystal. The 2 hydrogen bonds between CLZ and 2,5DHBA, i.e., (1) amine of tetrazole meta-hydroxyl and (2) carbonyl of carbostyril group - carboxylic acid, and the hydrogen bonds between carbostyrils of CLZ in the crystal packing were confirmed.

Stoichiometric 1:1

structures were formed in the all cocrystals according to the crystallographic data (Table 2).


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Page 22 of 37

Figure 4. The fragments of the crystal structures of CLZ cocrystals. H-bond interactions are represented as dashed lines. (a) represents CLZ-4HBA cocrystal, (b) represents CLZ-2,4DHBA cocrystal, and (c) represents CLZ-2,5DHBA cocrystal. Table 2. Crystallographic data for cocrystals. Compound

Formula

CLZ-4HBA

CLZ-

CLZ-

cocrystal

2,4DHBA

2,5DHBA

cocrystal

cocrystal

C20H27N5O2·

C20H27N5O2·

C20H27N5O2·


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C7H6O3

C7H6O4

C7H6O4

Formula weight

507.59

523.59

523.59

Crystal system

monoclinic

monoclinic

triclinic

Space group

P21/n

P21/c

P-1

a (Å)

10.0087(3)

9.8623(3)

6.5572(4)

b (Å)

12.2329(4)

12.2726(5)

8.0563(6)

c (Å)

20.8433(6)

21.4036(7)

25.5616(16)

α (deg)

90

90

89.046(6)

β (deg)

101.939(7)

103.442(7)

83.991(6)

γ (deg)

90

90

73.082(5)

V (Å3)

2496.76(15)

2519.66(17)

1284.67(15)

Z

4

4

2

Dcal (g·cm-3)

1.350

1.380

1.353

µ (mm-1)

9.47

9.90

9.71

F(000)

1080

1112

556

2θ max (deg)

55

55

55

Reflections collected

23710

21465

12384


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Page 24 of 37

Independent reflections, Rint

5697, 0.0319

5735, 0.0400

5859, 0.0365

R1 (I > 2.00 σ(I))

0.0377

0.0420

0.0557

R (all reflections)

0.0541

0.0649

0.0787

wR2 (all reflections)

0.0919

0.0960

0.1422

∆ρmax, ∆ρmin (eÅ-3)

0.33, –0.21

0.31, –0.24

0.34, –0.30

Equilibrium Solubility of Cocrystal.

The correlative relationship between the inverse

coformer concentration and the CLZ concentration in SGF is shown in Figure 5. The solubility product Ksp and the complexation constant K11 were calculated from the straight-line approximations and Equation 1, and these are shown in Table 3.

K11 of the CLZ-4HBA

cocrystal, CLZ-2,4DHBA cocrystal, and CLZ-2,5DHBA cocrystal were 28.0 M-1, 14.4 M-1, and 6.0 M-1, respectively.

As the K11 of all cocrystals were comparatively high, solute-solute

interaction between CLZ and hydroxybenzoic acid was predicted.30 As the K11 of the CLZ4HBA cocrystal was especially high, the rapid conversion to host crystal from CLZ-4HBA cocrystal may be prevented due to the solute-solute complex formation in aqueous solution.


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Figure 5. CLZ concentration in equilibrium with cocrystals in SGF at 37 °C as a function of the inverse coformer concentration.

The approximately linear equations, y=ax+b, and the

correlation coefficients, R2, are expressed in the figure. Table 3. The calculated solubility product, Ksp, and solution complexation constant, K11, in SGF. Cocrystals

Ksp (M2)

K11 (M-1)

CLZ-4HBA cocrystal

3.6×10-8

28.0


8.4×10-8

14.4


4.7×10-7

6.0

The theoretical phase solubility diagrams were calculated from Ksp, K11 and Equation 1, and the diagrams of all cocrystals were higher than that of CLZ (Figure 6). The solubility of each cocrystal, Scc, was represented as the intersection with the stoichiometric 1:1 concentration line. The calculated solubilities of the CLZ-4HBA cocrystal, CLZ-2,4DHBA cocrystal, and CLZ2,5DHBA cocrystal were, respectively, 9.5-fold, 14.5-fold, and 34.3-fold higher than that of CLZ (7.4 µg/mL). The calculated solubilities of the CLZ cocrystals from Ksp and K11 were correlated with the solubility of the coformer.


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Page 26 of 37

Figure 6. The calculated cocrystal solubilities as a function of coformer concentration. The solubilities of cocrystals, Scc, are expressed as the intersection with the stoichiometric concentration line. Kinetic Dissolution in SGF. The mean particle size of the three cocrystals prepared by the slurry method in this research was approximately 20 µm, which was similar to the hammermilled CLZ. The kinetic dissolutions in SGF (non-sink condition) were performed in order to evaluate the supersaturated properties of CLZ substances (Figure 7). We hypothesized that the addition of a polymer into the dissolution media was preferable for the metastable substance in order to prolong the supersaturated concentration28 because rapid recrystallization of CLZ under the supersaturated condition was presumed. Hypromellose was selected as the polymer to inhibit recrystallization effectively,34 which was pre-dissolved at 1% (w/w) into the dissolution media before dissolution testing.


18

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Figure 7. The dissolution profiles of CLZ substances in SGF containing 1% (w/w) Hypromellose at 37 °C. The drug concentrations applied into the dissolution media are (a) 100 µg/mL and (b) 500 µg/mL. The results are expressed as the mean with the bar as the S.D. (n=3). As shown in Figure 7a, the dissolution profiles of all cocrystals were higher than that of CLZ. Interestingly, the supersaturated dissolution profile was not prolonged due to the rapid conversion to host crystal as the calculated solubility of the cocrystal was excessively enhanced (although the CLZ-2,5DHBA cocrystal showed the highest solubility, the dissolution profile was especially lower than the other cocrystals). On the other hand, the CLZ-4HBA cocrystal, which mildly enhanced solubility, prolonged the supersaturation of CLZ in the kinetic dissolution. The supersaturation of the cocrystal was inversely-correlated with the calculated solubility from Ksp


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and K11.

Page 28 of 37

With regard to CLZ-2,5DHBA, even an initially rising dissolution, in which a

solubility-enhanced substance could follow, was not detected in Figure 7a.

The local

concentration of CLZ on the CLZ-2,5DHBA cocrystal surface was assumed to become significantly high due to the excessive enhancement of solubility. Therefore, it was supposed that the conversion to a stable crystal form on the surface, such as a solid-to-solid transition (or the agglomeration of cocrystal particle), immediately occurred after coming in contact with the solution, and the supersaturation in the dissolution was not detected.27 In addition, we evaluated the kinetic dissolution of a 500 µg/mL drug concentration because the excessive drug concentration exhibited negative performance for the prolongation of supersaturation (Figure 7b). Temporary supersaturation was confirmed in the dissolution test of the CLZ-2,5DHBA cocrystal, while it was not detected in the 100 µg/mL concentration condition.

This finding indicated that the supersaturation of the CLZ-2,5DHBA cocrystal

correlated with the surface area because the increase of the contact area with the solution followed the increase of drug concentration in the dissolution system.

However, the

supersaturation of the CLZ-2,5DHBA cocrystal was not sustainable, which was similar to the case of the amorphous solid dispersion. The supersaturated drug concentration of the amorphous solid dispersion rapidly rose and declined within 10 min in the kinetic dissolution, and the tendency became more conspicuous at the 500 µg/mL condition, whereas the dissolved drug concentration of the CLZ-4HBA cocrystal was maintained at more than 40 µg/mL for at least 20 min. As the nucleation of crystal and the crystal growth are proportional to the degree of supersaturation and the difference between the drug concentration in the solution and the equilibrium solubility of the host crystal, respectively,27 the excessively dissolved drug molecules boosted the conversion to the host crystal in the cases of CLZ-2,5DHBA cocrystal and


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the amorphous solid dispersion. In contrast, the moderate dissolution profile of CLZ-4HBA cocrystal, which mildly enhanced the solubility, was effective for sustainable supersaturation regardless of the drug content applied in the dissolution. In vivo Study of CLZ Cocrystals and Amorphous Solid Dispersion.

The serum

concentration profile and the PK parameter are shown in Figure 8 and Table 4, respectively. The Cmax of the CLZ-4HBA cocrystal, CLZ-2,4DHBA cocrystal, and CLZ-2,5DHBA cocrystal were 7.7-fold, 3.2-fold, and 2.0-fold higher, respectively, and the AUC of those cocrystals were 7.1fold, 3.1-fold, and 1.8-fold higher, respectively, than that of the hammer-milled CLZ. The bioavailability in vivo correlated well with the supersaturation in the kinetic dissolution; in particular, the CLZ-4HBA cocrystal, which represented the most sustainable supersaturation, considerably enhanced the bioavailability. The Cmax and the AUC of the amorphous solid dispersion were 4.2-fold and 4.2-fold higher than that of the hammer-milled CLZ, respectively. Whereas the amorphous solid dispersion showed an enormously increased drug concentration in the initial dissolution in Figure 7, rapid conversion to the host crystal was assumed, and supersaturation was less sustainable than the CLZ-4HBA cocrystal and the CLZ-2,4DHBA cocrystal. The bioavailability of the amorphous solid dispersion was approximately half of the CLZ-4HBA cocrystal; therefore, supersaturation was an important factor for the enhancement of oral absorption. However, the bioavailability of the amorphous solid dispersion was slightly higher than that of the CLZ-2,4DHBA cocrystal, contrary to the expectation based on the kinetic dissolution. The ratio of the drug to the coformer dissolved in vivo changed with dilution and absorption in the gastrointestinal tract, whereas the ratio in vitro was maintained during the dissolution. According to the calculated phase solubility diagram in Figure 6, the solubility of the CLZ cocrystal correlated with the concentration of the coformer. This may be because the


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Page 30 of 37

solubility and the supersaturated concentration of CLZ cocrystal fluctuated during the gastrointestinal transit. Therefore, the dissolution system with an absorption sink may be more adequate to evaluate the performance of a composite crystal such as a cocrystal than a closed dissolution system. The bioavailability enhancement of the amorphous solid dispersion was assumed to accompany the solubilization of drug over the saturated concentration and the inhibition of the conversion to the stable crystal form by the polymer included in the amorphous solid dispersion, in contrast to the cocrystal. While the bioavailability enhancement of the CLZ2,5DHBA cocrystal was limited due to the rapid conversion to the stable crystal, the matrix formulation with a polymer which prevents the crystallization of CLZ may improve it like the amorphous solid dispersion.

Figure 8. Serum concentration - time profile after oral administration of CLZ at a dose of 100 mg/body into fasted beagle dogs. The results are expressed as the mean with the bar as the S.D. (n=4). Table 4. Pharmacokinetic parameters.


22

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Parameter

CLZ-4HBA

CLZ-

CLZ-

Amorphous

CLZ

cocrystal

2,4DHBA

2,5DHBA

solid

hammer-

cocrystal

cocrystal

dispersion

milled

869±306

547±218

1143±184**

271±69

Cmax (ng/mL)

2074±551**

AUC (ng/mL·h)

7624±1639** 3384±1034*

1989±708

4514±1082** 1080±414

tmax (h)

1.1±0.6

2.0±0.8

1.5±0.6

1.5±0.6

1.5±0.6

MRT (h)

2.7±0.4

2.9±0.7

2.8±0.3

3.1±0.4

3.0±0.5

Results are expressed as the mean±S.D. (n=4). **

p

Impact of the Dissolution Profile of the Cilostazol Cocrystal with

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