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Controlled recrystallization of tubular vinpocetine crystals with increased aqueous dissolution rate and in vivo bioavailability Panpan Sun, Yaping Wang, Sohrab Rohani, Ergang Liu, Shichao Du, Shijie Xu, Mingyang Chen, Zhenping Wei, and Junbo Gong Cryst. Growth Des., Just Accepted Manuscript • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 4, 2017

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Crystal Growth & Design

Controlled recrystallization of tubular vinpocetine crystals with increased aqueous dissolution rate and in vivo bioavailability

Panpan Sun 1, 2, Yaping Wang1, 2, Sohrab Rohani3, Ergang Liu1, 2, Shichao Du1, 2, Shijie Xu1, 2, Mingyang Chen1, 2, Zhenping Wei1, Junbo Gong1, 2, 4* 1

School of Chemical Engineering and Technology, State Key Laboratory of Chemical

Engineering, Tianjin University, Tianjin300072, China; 2

The Co-Innovation Center of Chemistry and Chemical Engineering of Tianjin,

Tianjin300072, China 3

Department of Chemical and Biochemical Engineering, the University of Western

Ontario, London, Ontario N6A 5B9, Canada 4

∗

The key laboratory Modern Drug Delivery and High Efficiency in Tianjin, China

Corresponding author: Junbo Gong ([email protected]), School of Chemical

Engineering and Technology, State Key Laboratory of Chemical Engineering, Tianjin University, Tianjin300072, People’s Republic of China; The Co-Innovation Center of Chemistry and Chemical Engineering of Tianjin, Tianjin300072, People’s Republic of China; Tel: 86-22-27405754, Fax: +86-022-27374971.

ACS Paragon Plus Environment


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ABSTRACT: Vinpocetine was a BCS II drug, whose clinical applications had suffered from low oral bioavailability because of its inefficient dissolution in the GI tract. As the dissolution rate depended on the surface area of drug crystals, we herein explored shape-controlled recrystallization via anti-solvent process as an excipient-free strategy to improve the bioavailability of VIN. By adjusting the water/ethanol ratio, initial VIN concentration and temperatures, morphologies of the crystalline products could be finely tuned from three-dimensional cubes and tubes, to two-dimensional frizzled plates, and finally to zero-dimensional microparticle clusters. Morphology analysis and in-situ FBRM surveillance of the growing process suggested that a diffusion-limited crystal growth mechanism was responsible for the shape variation of VIN products. Finally, we tested the in vitro dissolution efficiency as well as the in vivo bioavailability of recrystallized VIN crystals. Results manifested that the tubular crystal showed a faster dissolution behavior as compared with the raw VIN, achieving an increased AUC0-t of 484.0 ± 24.6 ng/mL·h, which was 1.3-folds of that of the raw VIN product (386.6 ± 22.8 ng/mL·h). According to our knowledge, this was one in vitro to in vivo report for bioavailability improvement of BCS II drug by applying the shape-controlled re-crystallization strategy. Keywords: Vinpocetine, Tubular crystal, Anti-solvent crystallization, In vitro dissolution, Bioavailability


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1. Introduction Vinpocetine

(VIN)

[14-ethoxycarbonyl-(3

alpha,

16

alpha-ethyl)-14,

15-eburnamenine] is the ethyl ester of apovincamine, which has been isolated from Lesser Periwinkle. As a vasodilating agent, VIN is widely used in the treatment of chronic cerebral vascular ischemia, acute stroke and central nervous system disorders.1-3 However, despite the popular applications, VIN, as with other hydrophobic APIs, suffers greatly from low clinical bioavailability because of its poor solubility in the physiological fluids. Technically, VIN belongs to BCS Class II category of drugs based on its permeability and solubility (water solubility about 5 µg/mL),4,5 demonstrating that the drug is apt to be absorbed by the GI (gastrointestinal) tract but confined by intestinal luminal drug concentration because of poor aqueous solubility. Therefore increasing the in vivo dissolution rate has been well accepted as an effective means of enhancing oral absorption, which subsequently warrants improved bioavailability of VIN.6 Various means including the application of solubilizers, nanomaterial-based drug carriers7,8, formation of co-crystal or salt9,10 and micronization technology,11 have been explored to improve the dissolution of VIN. Generally, these technics function well by increasing surface area, improving solubility and dispersity of the formulated drugs, thus is effective in dissolution improvement. Furthermore, applications of surfactants and nanomaterials can promote permeation of the encapsulated drugs across the GI membranes,12,13 which further contributes to enhanced absorption and bioavailability, thus has gained extensive attentions in formulation development in



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recent years. However, producing micro-, or even nano-sized drugs are cost- and energy- inefficient, and always need excessive surfactants to maintain the stability of the drug microparticles,14,15 which brings additional challenges on characterization, safety evaluation, and other regulatory issues as the drug pending for market approval from the pharmacy authority agency.16 Pharmaceutical crystallization is a crucial unit operation in the pharmaceutical industry. By controlling the crystallization process, we can obtain crystals with different morphologies, polymorphism, particle size/specific surface area, which together contribute to varied product physiochemical properties, such as wettability, fluidity, dissolution rate, and so on.17,18 According to the Noyes-Whitney/ Nernst-Brunner equation, the dissolution rate is strongly dependent on the surface area of the particles,19,20 therefore can be significantly raised by substantially decreasing the crystal size, which founded the fact that nano-sized drug formulations have shown superior dissolution behavior as compared to their bulk or micro-sized counterparts.14,21 However, ultra-small crystals are accompanied with high surface energy, thus are unstable during production and storage. In comparison, hollow structured crystals don’t have the aggregation problem, and possess larger surface area than solid particles with equivalent size. Therefore, the hollow structured crystals represent a cost-effective technique in improving dissolution rate of insoluble drugs. In this study, we obtained tubular VIN crystals via a controlled anti-solvent re-crystallization

method.

By

adjusting

the

crystallization

parameters

(anti-solvent/solvent volume ratio, VIN concentration, etc.), different morphologies


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including hollow cubes, curved plates and aggregated clusters could be formed, which were further investigated to depict the growth mechanism during the crystallization process. Finally, we tested the in vitro dissolution rate as well as in vivo bioavailability of VIN with different crystal structures. The present study appears to be a novel in vitro to in vivo attempt on bioavailability improvement of insoluble drugs via an excipient-free crystallization strategy.

2. Experimental section 2.1 Materials. Raw crystalline VIN (HPLC mass fraction purity of 98%) was donated by Northeast Pharmaceutical Group Co., Ltd. (China) and its chemical/crystalline structures was identified by FT-IR, PXRD, and DSC analysis (Fig. S1). Solid sodium dodecyl sulfate (SDS) and ammonium carbonate were purchased from J&K Scientific Co, Ltd (China). The experimental solvents, including ethanol and hydrochloric acid were of analytical grade and purchased from Tianjin Kewei Chemical Co., Ltd. (China), which were employed without further purification. Chromatographic grade methanol and acetonitrile were purchased from Tianjin Jiangtian Technology Development Co, Ltd (China). Distilled deionized water (< 0.5 µS·cm−1) was laboratory prepared and used throughout the measurement. 2.2 Crystallization and Process Analysis. 2.2.1

Crystallization Procedure. Tubular and other structured VIN were

obtained by re-crystallizing the raw VIN via an anti-solvent method. In details, 0.3 –



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1.5 g raw crystalline VIN was firstly dissolved in a jacketed vessel by 30 mL ethanol (good solvent); then the solution was heated to 40 – 70 °C under mild agitation (400 rpm) to get the drug fully dissolved; after that, predetermined amounts of water (18 90 mL, preheated) were poured as anti-solvent into the alcoholic pulp, which yielded rapid precipitation of the VIN crystals; 10 minutes later, the precipitates was collected by filtration and washed with cold water, then oven-dried at 40 °C to get the re-crystalized products. 2.2.2

Online Monitoring of the Crystallization Process by FRBM. Focused

Beam Reflectance Measurement (model M400LF) with iC FBRM software (Mettler-Toledo, Switzerland) was used for real-time monitoring of the crystallization process. Following a similar procedure as described above, 1 g raw VIN was recrystallized (Temperature at 70 °C, water/ethanol ratio of 0.6:1 and 3:1) in a 50 mL-vessel of EasyMax 102 Advanced Synthesis Workstation (Mettler-Toledo, Switzerland). During the crystallization, FBRM probe was placed into the VIN solution, and dynamic signals from the four major groups of VIN particles classified according to the size (< 10 µm, 10 – 50 µm, 50 – 150 µm, and 150 – 300 µm) were recorded at 2 s-interval by the FRBM workstation. 2.3 Influence factor analysis 2.3.1

Ethanol/water ratio. In order to assess the influence of ethanol/water

volume ratio on the morphology of crystallized products, different amounts of water (18 mL, 30 mL, 45 mL, 60 mL or 90 mL, corresponding to water/ethanol ratio at 0.6:1, 1:1, 1.5:1, 2:1 and 3:1, respectively) were preheated to 70 °C, which were then added


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into alcoholic VIN solution (1 g in 30 mL ethanol, 70 °C) under agitation, yielding VIN crystals with different morphologies. 2.3.2

Initial concentration. Different initial concentrations of VIN solution

were prepared by dissolving 0.3, 0.5, 1, 1.5 g raw VIN in 30 mL ethanol, into which was then added with 30 mL water (ethanol/water volume ratio at 1:1, preheated), and the crystallization process was carried out at 70 °C, as above mentioned. 2.3.3

Temperature. Temperature effect was investigated by carrying out the

crystallization (1 g/30 mL, ethanol/water ratio at 1:1) under different temperatures (40, 50, 60, 70 °C, respectively). The obtained crystalline VINs were then analyzed to depict the relevance between the product morphology with temperature. 2.3.4

Solubility and supersaturation test. The temperature dependent

solubility of VIN in ethanol, water, or in the binary solvents of different ethanol/water ratios, were measured by the isothermal saturation method.22 Correspondingly, the supersaturation of VIN under the above mentioned conditions were then calculated according to the equation: = ⁄ [unitless].23 Where being the actual concentration in solution (feeding concentration of VIN), and being the equilibrium concentration in solution (solubility of VIN) 2.4 Product characterization. 2.4.1

Powder X-ray Diffraction (PXRD). X-ray diffraction patterns of

re-crystallized VIN particles were recorded by D/MAX 2500 X-ray diﬀractometer radiated using Cu Kα (1.54) rays. The instrument parameters were set as: 2θ scanning range of 2 – 50 °C; voltage: 40 kV; current: 100 mA. Calculated PXRD pattern of



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VIN from Cambridge Crystallographic Data Centre (CCDC 1202815) was used as reference for polymorphic identification.24 2.4.2

Differential Scanning Calorimetry (DSC). Mettler-Toledo DSC 1/500

calorimeter (Mettler-Toledo, Switzerland) was used to carry out thermal analysis. The crimped aluminum pans loaded with 5 – 10 mg samples were heated under nitrogen protection, with the heating rate of 10 K/min over the range of 298.15 – 498.15 K. 2.4.3

Fourier transform infrared spectroscopy (FT-IR). FT-IR spectra of VIN

were recorded over a range of 4000 – 400 cm-1 by a Tensor 27 IR spectrometer (Bruker, Germany). 2.4.4

Scanning Electron Microscopy (SEM). The morphologies of VIN

crystals were recorded by an X650 scanning electron microscope (HITACHI, Japan). The samples were mounted with the carbon adhesive on an aluminum holder, which was then sputtered with gold and scanned at a voltage of 15 kV under vacuum. 2.5 In Vitro Dissolution Experiment. 2.5.1

Particle Size and Surface Area determination. The hydrodynamic of

raw VIN, as well as three kinds of recrystallized samples, tubes, cubes, and clustered granules, were measured by Mastersizer 3000 (Malvern Instruments Limited, UK). Generally, VIN powders were ultrasonically dispersed in deionized water, which were then loaded in the sample cell. Under vigorous stirring (1,500 rpm), the samples were then pumped through the optical cell and be analyzed. The size distribution of the tested products were calculated and given by the Malvern software which was developed based on the Mie model. Of note, size polydispersity of the testing were


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given as the span of the size distribution,25,26 which was defined as (D90–D10)/D50. Where D10, D50, and D90 are diameters at 10%, 50%, and 90% cumulative volume, respectively. Specific surface area of the crystalline VINs was quantified by the N2 adsorption method. The experiments were carried out using Quadrasorb SI (Quantachrome instruments, USA). 2.5.2

Dissolution Test. In vitro dissolution behaviors of VIN powders from

raw materials as well as the re-crystallized tubes and cubes were determined according to the USP paddle method27,28 using an RC-6 dissolution apparatus (Guoming, China). 100 mg of each sample was weighed and added into 500 mL dissolution media. According to the guideline,29 four testing media including 0.5% and 1% SDS solution, artificial gastric juice and artificial intestinal juice30,31 were used. Aqueous solution containing SDS was prepared by dissolving 0.5% or 1% SDS (m/v) in water (m/v = mass of solute/volume of solvent). 1% SDS supplemented hydrochloride buffer (pH 1.2) and phosphate buffer (pH 6.8) were used as protease-free artificial gastric and intestinal juice, respectively. The experiments were carried out under agitation (100 rpm) and maintained at 37 ± 0.5 °C. At predetermined time points (5, 15, 30, 60, 120, 240, 300 and 360 min), aliquots (10 mL) were withdrawn and replaced with equivalent volume fresh media at the same time. The samples were then passed through a Millipore filter (0.22 µm) to remove undissolved VIN, with the filtrate be measured by UV-3010 spectrophotometer (HITACHI, Japan) for the dissolved of VIN (wavelength at 315.5 nm). The dissolution assay was carried



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out in triplicates. Termination of the test would also be triggered when the percentage of dissolved drug reached 85% at two continuous sampling points or dissolution time proceeded 360 min. 2.5.3

Dissolution efficiency (DE). Dissolution efficiency (DE) was firstly

introduced by Khan (1975)32 to compare the dissolution variations among different formulations. DE was calculated based on the dissolution curve of testing drugs, defined as the AUC (area under the dissolution curve) ratio of the testing formula to drug solution (100% dissolution) at a certain time window ( t1 to t2 ). 33 Specifically, DE could be calculated according to the following function: Dissolution Efficiency (DE) =

∙

∙

× 100%

(Eq.1)

Where y referred to the weight percentage of dissolved drug from testing formulation at time t; and was that from drug solutions. Since solubilized sample was completed dissolved, dissolution curve of the control drug would be a straight line with y = 100% across the testing time window, thus giving AUC of 100% × − . 2.6 In-Vivo Pharmacokinetic Study. All animal experiments were approved by the Animal Ethics Committee of Nankai University, and complied with the “Guidelines for the Care and Use of Laboratory Animals” set by the Ministry of Health of the People’s Republic of China. Generally, Sprague Dawley rats (male, 250 g – 300 g) from the Laboratory Animal Center (Peking, China) were randomly divided into two groups (n = 6) and fasted overnight before the experiment. Testing drugs including tubular and bulk VIN


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crystals were suspended with 0.5% CMC-Na (m/v), which were then administered at the dose of 6 mg/kg by gavage method. Blood samples were withdrawn from the tail vein at 0.083, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 8.0 and 10.0 h post administration. The blood samples were immediately centrifuged at 3,500 rpm, with the supernatant collected and mixed with 4-folds volume of acetonitrile to precipitate the serum proteins. The mixture was then centrifuged at 13,000 rpm for 10 min, after which the supernatant was transferred into a new tube and blown with nitrogen to dryness. Finally, the residues were re-dissolved by 100 µl methanol and analyzed on an Agilent 1260 liquid chromatography system equipped auto-sampler (G4226A) and ultraviolet/visible (UV/VIS) detector. HPLC conditions: Column, Luna C18 (250 mm × 4.6 mm, 5 µm, Phenomenex); temperature, 30 °C; detector wavelength, 273 nm; injection volume, 20 µl; mobile phase, methanol : ammonium carbonate (10 mM): acetonitrile = 30 : 14 : 56; flow rate, 1.0 mL/min. Blood concentration of VIN was plotted against time to profile the dynamic changes of the orally administrated drug crystals. The pharmacokinetic parameters including the values of maximum concentration (Cmax), time of maximum concentration (Tmax), area under the concentration curves (AUC), and the elimination half-life T1/2 (h) were calculated using PKSolver 2.0 software,34 which was developed based on the linear trapezoidal method. 2.7 Statistical Analysis. Apart from animal study, all the other experiments were carried out in triplicates



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and be presented as mean ± S.D. Data from dissolution tests were compared following one-way analysis of variance (ANOVA) and the animal tests results were evaluated using the Student’s t-test, both of which were performed by the Graphpad Prism® software (Version 5.00).

3

Result and discussion

3.1 Fine-tuning anti-solvent crystallization for tubular VIN crystals. It was well accepted that the crystallization process was driven by supersaturation, and different extent of supersaturation could lead to variations in crystallizing rate, which could further result in diverse crystalline structures and habits.35 As for anti-solvent crystallization process, supersaturation of VIN in the system was dependent on initial concentration, crystallization temperature, and ethanol/water volume ratios, which thereby could be explored as useful parameters for process control and were critical for maintaining the quality of crystallized products.


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Fig. 1 Scanning electron microscopic photographs of crystalline products produced from different water/ethanol volume ratios. A~F represent raw VIN, and recrystallized VIN powder with water/ethanol volume ratio of 0.6:1, 1:1, 1.5:1, 2:1, 3:1, respectively. Initial VIN concentration: 1 g/30ml; Temperature: 70 °C.

3.1.1. Water/ethanol volume ratio. SEM images of the raw VIN as well as the recrystallized products from different water/ethanol ratios were shown in Fig.1. It was apparent that the morphologies of the re-crystallized VIN strongly depended on the



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volume ratios of water to ethanol–by gradually raising the water content, we could sequentially get VIN crystals of varied shapes (Fig. 1B-F). Of note, tubular VIN could be obtained only at optimal conditions where proper water content and initial drug concentrations were adopted during crystallization. For example, with the initial concentration of 1 g/30 mL, tubular crystals with terminal openings along the long axis were formed at water/ethanol ratio of 1:1 (Fig. 1C). Whereas only irregular polyhedrons were obtained when water/ethanol ratio decreased to 0.6:1 (Fig. 1B). Furthermore, increasing water content contrarily to 1.5:1, 2:1, and 3:1 resulted in more irregular shaped products like cracked tubes, frizzled plates, or aggregates of micro-particles, respectively (Fig.1D-F). By measuring solubility of VIN in the binary solvents, we found that supersaturation was very sensitive to the water/ethanol ratio changes (Table. 1 & Figure. 2): Generally, supersaturation value was only 3.37 when water/ethanol ratio of 0.6:1 was applied, which increased to 9.38 when water/ethanol volume ratio rose to 1:1. Further increasing water content would more significantly raise supersaturation to 45.09 (1.5:1), 101.05 (2:1) and 249.96 (3:1), which was 13.4-, 30.0-, and 74.2- folds, of that obtained from water/ethanol ratio of 0.6:1, respectively. Compounding together, it was suggested that ordered crystalline products (cubes, tubes, etc.) could only be obtained at moderate supersaturation. In comparison, high supersaturation (water/ethanol ratio of 1.5:1 – 3:1) caused faster nucleation and subsequently unordered crystal growth, thus resulted in the formation of numerous crystals with smaller size. The PXRD patterns of the aforementioned samples were shown in Fig. 3, depicting all the PXRD patterns of VIN samples were well matched


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with the calculated PXRD spectra (Fig. S2, Supplementary Information), suggesting all those products shared the same crystal structure. Interestingly, it was worth noting that, as compared with raw materials, a more intense signals of certain lattice planes could be found in the recrystallized samples. i.e., (1, 1, 0) plane in the cubic VIN, and (1, 1, 0) & (2, 1, 0) planes in tubular VIN, which can be attributed to crystal morphologic differences between different VIN samples.

Fig. 2 Solubility of VIN versus different temperatures in the water + ethanol solvent mixtures with different water/ethanol volume ratio: , 3:1; , 2:1; , 1.5:1; , 1:1; , 0.6:1. Tab. 1 Solubility and calculated supersaturation of VIN in binary solvents with different water/ethanol ratios Vwater : Vethanol

0.6:1

1:1

1.5:1

2:1

3:1

Solubility(mg/mL)

9.9027

3.5524

0.7393

0.3299

0.1334

Supersaturation (S)

3.37

9.38

45.09

101.05

249.96



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Fig. 3 X-ray diffraction (XRD) patterns of crystalline products produced from different water/ethanol volume ratios. A~F represent raw VIN, and recrystallized VIN powder with water/ethanol volume ratio of 0.6:1, 1:1, 1.5:1, 2:1, 3:1, respectively. Initial VIN concentration of 1 g/30ml was applied.

FBRM was used to track the real-time population changes of VIN particles of different sizes during crystallization. Generally, the crystallized particles formed in the system were divided into four groups termed as class I, II, III, and IV, which corresponded to particles in the size range of < 10 µm, 10 – 50 µm, 50 – 150 µm, and 150 – 300 µm, respectively. Since water content was one of the key shape-controlling parameters, analyzing the crystallization process under different anti-solvent/solvent volume ratios could help to reveal the kinetic differences during the crystal growth process which might provide clues to the involved mechanisms. Herein two different water/ethanol ratios (1:1 & 3:1) were applied to provide a panoramic view of the dynamic growing process as shown in Fig. 4A & Fig. 4B, respectively. It was apparent that all VIN particles formed immediately after the addition of water and displayed a sharp increase of FBRM signal in the first 20 s. Furthermore, after the acute growing phase, the population expansion of larger VIN particles (groups of III


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& IV) were slowed down and lasted till the end of the test, whereas the number of small VIN particles (groups of I & II) began to decrease in a converse way. These features shared by Fig. 4A and Fig. 4B suggested explosive nucleation happened in both conditions when instant supersaturation of the system was produced. However, despite the common trends appeared during the anti-solvent process, the kinetic growth profiles from two different water/ethanol ratios varied in several ways. As known, increasing the anti-solvent content could result in a higher supersaturation level and faster nucleation process. Consequently, substantially more particles of varied sizes were formed in the acute growing phase when water/ethanol 3:1 was applied as compared with that of 1:1 (19,200 counts/s, 14,300 counts/s, 8,700 counts/s, and 300 counts/s vs 5,200 counts/s, 12,500 counts/s, 2,300 counts/s and 20 counts/s for particles of I, II, III, and IV, respectively). Furthermore, following the acute nucleation phase (water/ethanol 3:1), the population of smaller particles less than 50 µm (I & II) rapidly decreased in the next 20 – 30 s, which accompanied by a rapid increase of FBRM signals from large particles (III & IV), suggesting aggregation of the nucleated small particles, which was manifested by the SEM results (Fig. 1F). In comparison, when lower water/ethanol ratio was used, the growth of larger particles plateaued, indicating a controlled growth process at this condition producing regularly shaped VIN crystals (Fig.1C).



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Fig. 4 On-line monitoring of the population changes of VIN particles with different sizes by Focused Beam Reflectance Measurement (FBRM). Initial VIN concentration was 1 g/30 ml, two different water/ethanol ratios of 1:1 (A), and 3:1 (B) were applied.

3.1.2. Initial VIN concentration. Similar with water/ethanol adjustment, changing initial concentration could also adjust the system supersaturation, but to a less extent level. As shown in Table 2, the system supersaturation changed proportionally to the initial concentration adjustment, with feeding VIN of 0.3 g/30 mL, 0.5 g/30 mL and 1.5 g/30 mL produced supersaturation of 2.81, 4.69, 14.07, respectively (water/ethanol = 1:1, T = 70 °C). SEM images of as-crystallized products were shown in Fig. 5, no tubular crystals were formed when the initial VIN concentration were 0.3 g/30 mL (A) and 0.5 g/30 mL (B), corresponding to lower supersaturation of 2.81 and 4.69 respectively. In contrast, solid crystals with polyhedron shape, or mixed plate and prism were obtained by beginning with VIN concentration of 0.3 g/30 mL and 0.5 g/30 mL, respectively. Further increasing the VIN concentration to 1.5 g/30 mL yielded fragmented crystals which seemed to be cracked parts of tubular products (Fig. 5C & Fig. 5D). In consistent with adjustment of water/ethanol ratios, results obtained here also suggested tubular VIN could be


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prepared with medium systemic supersaturation.

Fig. 5 Scanning electron microscopic photographs of products prepared with different initial VIN concentrations: 0.3 g/ 30 ml (A); 0.5 g/ 30 ml (B); 1 g/ 30 ml (C), and 1.5 g/30 ml (D). Water/ethanol volume ratio: 1:1; Temperature: 70 °C. Tab. 2 Calculated supersaturation of VIN in binary solvents (1:1) under different initial concentrations Initial concentration(g/30mL)

0.3

0.5

1

1.5

Supersaturation (S)

2.81

4.69

9.38

14.07

3.1.3. Temperature. The calculated supersaturation under different temperatures (Initial concentration = 1 g/30 ml, water/ethanol = 1:1) as well as SEM images of the obtained VIN products were shown in Table 3 & Figure 6, respectively. As depicted, gradual decreasing system temperature from 70 °C to 40 °C led to reduced solubility



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of VIN, thus producing an increased supersaturation from 9.38 to 40.17. Accordingly, morphologies of VIN varied with the change of temperature. For example, intact tubular crystal was obtained only at 70 °C. When the temperature dropped to 60 °C, the depression in the crystal faces became pronounced (Fig. 6C), and further decreasing the temperature to 40 – 50 °C yielded VIN products with enlarged cavity fractures(Fig. 6A & Fig. 6B), which was similar to the products obtained at higher water/ethanol ratios (Figure. 1D & Figure. 1E).

Fig. 6 Scanning electron microscopic photographs of products prepared under different temperatures: 40 °C (A); 50 °C (B); 60 °C (C), and 70 °C (D). Water/ethanol volume ratio: 1:1; Initial VIN concentration: 1 g/30ml.

Tab. 3 Solubility and calculated supersaturation of VIN in binary solvents (water/ethanol = 1:1) under different temperatures


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313.15 K

323.15 K

333.15 K

343.15 K

(mg/mL)

0.8296

1.3654

2.0252

3.5524

S

40.17

24.41

16.46

9.38

1:1

3.2 Mechanisms involved in Tubular Crystal Formation Shape-controlled crystallization has been a hot topic of investigation in recent years.18,36 However, the mechanism involved in the crystal shape formation and evolution is always complex. Meanwhile, crystal transition and polymorphism tend to occur during this multi-parameter controlled process,37,38 which makes it more challenging to get a profound understanding of the related growth mechanism. In this paper, a series of VIN crystals with different shapes have been prepared via the anti-solvent method (Fig. 1). By simply increasing the water/ethanol ratio from 0.6:1 to

3:1,

morphologies

of

the crystalline products can transformed from

three-dimensional cubes and tubes, to two-dimensional frizzled plates and finally to zero-dimensional microparticle clusters. This suggests that a 3D to 0D growth transition occurs as the anti-solvent content and the supersaturation rise.39 Interestingly, in a similar shape-controlled process for C60 crystallization, Chung and co-workers posed that 3D to 1D transition happens as supersaturation ratio decreases.40 Variations between our results and the literature may be attributed to the initial concentration difference and its related mass transfer process. As we know, crystals grow via orderly packing of the molecular unit in three dimensions. However, the intrinsic packing rates among the three dimensions are not always the same (anisotropy). When extreme low initial concentration of the material (i.e., 2 mg/mL



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for C60) is used, the resulting mass transfer will be less efficient as compared with the molecular packing process in three dimensions, thus a competition for the metastable solute source among different axis is at work. As a result, the crystals will grow in the direction along which the most stable packing occurs at low supersaturation stage, which produces one-dimensional structured product such as whiskers and wires. Subsequently the transition from 1D to 2D and 3D structure will be achieved when the mass transfer is further raised along with increased supersaturation. In our case with VIN crystallization, high initial concentration (33.3 mg/mL) will provide sufficient mass transfer for the molecular packing process and result in a preferential growth along certain directions, depending on the applied supersaturation ratios. At relatively low supersaturation ratios (0.6:1 & 1:1), mass transfer is fast enough and provides sufficient soluble VIN for the crystal packing in three dimensions, resulting in 3D crystal like cubes and squares (Fig. 1). As the water/ethanol is raised to 2:1, mass transfer becomes so fast that exceeds the packing speed limit in one dimension, while the growth rate along other dimensions still have room to raise, which leads to 2D plate like VIN. Similarly, further increase in water/ethanol ratio to 3:1 results in mass transfer faster than the crystal packing in all three dimensions and leads to unordered crystal growth. During this process, massive nuclei (0D) are formed and aggregate rapidly, producing clustered aggregates. Hollow crystals have attracted considerable attentions due to their unique properties such as high specific surface area and decreased density. For example, micro-cavities may be explored as molecular container/reactor for certain specialized


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applications. Meanwhile, as crystallization is an ordered process which generally yields homogeneous structures, the heterogeneous cavities formed within the crystals provide an opportunity to unveil the mechanisms involved in crystal formation. Of note, several mechanisms have been reported to be responsible for the hollow structure formation, including rolling up form 2D sheet, center-preferential dissolution, and diffusion limited growth, etc.41-44. Here in our experiment, the formation of hollow cavities in VIN cubes and square is more likely to be the results of diffusion differences during the crystal growth process. As reported, at high supersaturation stage, the crystal growth becomes unbalanced where a preferentially accelerated growth along the edge in comparison with center occurs because solute will usually nucleate near the edge of the crystal and then diffuses more slowly to the center of the face.42,45 This leads to a preferential thickening at the periphery and leaves the center in a starved state, which is responsible for the formation of fovea or shallow cavity, as evidenced by the tubular structure and hollow cavities formed in VIN products when water/ethanol ratio of 1:1 was applied (initial VIN concentration at 1 g/30 mL, Fig.1 C). On this account, the loosely sinks on the center of cubic faces, as well as the stepwise lowered layer appeared on the square tube surface as to the edge, all can be ascribed to the starvation flaws because of limited access of center-diffused VIN molecules.



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Scheme. 1 Proposed scheme of the growth mechanism of VIN hollow crystal.

Based on these experimental observations, a growth mechanism of hollow crystals in anti-solvent crystallization was proposed. As shown in Scheme.1, in an alcohol solution of VIN with optimized concentration (1 g/30mL), addition of water (anti-solvent, 1:1 to the volume of ethanol) yielded high supersaturation and immediate nucleation of VIN. Then the VIN molecules began to pack on the preformed nuclei, contributing to the growth of VIN crystals. As it is easier for VIN molecules to diffuse to the edges of the rapidly growing face than to the center, the growth rate in the middle of the crystal face was much lower than the edge, which led to distinct depression in the crystal faces. Similarly, the mass transfer was much slower from the solution to the center than to the outside edge of the crystals, resulting in the formation of a central cavity within the crystal. 3.3 In Vitro Dissolution Test. As discussed above, the dissolution rate was strongly dependent on particle size


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as well as surface area of the testing drugs. We first tested these two parameters by laser diffraction and nitrogen absorption method, respectively. As were shown in Fig. 5 and Table 1, VIN particle sizes from the three samples decreased in the order: Tubes (116.6 ± 42.3 µm) < Cubes (121.8 ± 40.5 µm) < Raw VIN (194.6 ± 75.4 µm). The N2 adsorption test gave a similar trend, showing the specific surface areas of the crystal samples were 0.301 m2/g (Raw VIN), 0.438 m2/g (Cube), and 0.532 m2/g (Tube), respectively.

Fig. 7 Hydrodynamic size of different VIN products.

Tab. 4 Particle size (mean ± SD) and surface area of three VIN crystals

Specific surface area

Particle size (µm)

Span

Raw

194.6 ± 75.4

1.791

0.301

Cube

121.8 ± 40.5

1.119

0.438

Tube

116.6 ± 42.3

1.267

0.532

Product

(m2/g)

*Span indicates the wide of distribution in size and polydispersity of particles

The time dependent dissolution curves of the three kinds of VIN crystals in SDS supplemented media and artificial simulation fluid with different pH values were



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shown in Fig. 8 and Fig. 9, respectively. As displayed, although the test VIN powders all shared the same crystalline form as demonstrated by the PXRD assay (shown in Fig. 3), their dissolution profiles were distinctly different. In details, recrystallized VIN tubes and cubes manifested faster dissolution than the raw material. For example, in 0.5% SDS recrystallized VIN powders dissolved immediately after being added into the dissolution media, showing 77% and 41% of the feeding VIN (corresponding to tubular and cubic VIN, respectively) had been dissolved in the media in 60 min, whereas raw VIN only reached 27% of the dissolution (Fig. 7A). Similar trends were found when 1% SDS was used, after 30 min duration, the dissolved VIN samples reached 85% (Tubes), 30% (Cubes) and 26% (Raw VIN), respectively (Fig. 7B). As in low pH (artificial gastric juice), three samples all displayed rapid dissolution rate, reaching more than 90% only after 30 min (Fig. 8A). At high pH (artificial intestinal juice), the dissolution was relatively low and tubular crystals showed obvious advantage of dissolution, showing 92% had been dissolved in 15 minutes, superior to the

Cubes

(70%)

and

raw

material

(66%).

Fig. 8 Dissolution profiles of Raw VIN, Cube and Tube in 0.5% SDS (A), and 1% SDS (B), respectively.


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Fig. 9 Dissolution profile of Raw VIN, Cube and Tube: in artificial gastric juice (A) and artificial intestinal juice (B)

The raw VIN and recrystallized crystals were compared in terms of the dissolution efficiency (DE) and the values obtained are shown in Table 5. With these recrystallized products, a significantly increased dissolution efficiency of 58.1 ± 0.6% (hollow cubes) and 80.3 ± 1.1% (square tubes) were achieved in 0.5% SDS, which is 1.4- and 1.9- folds of that from raw materials. The DE values of all samples increased when 1% SDS was applied as dissolution media, of which the calculated DE for raw and recrystallized cubic and tubular VIN were 51.4 ± 0.8%, 71.9 ± 1.0%, 92.2 ± 1.7%, respectively. As VIN was a weak basic drug, which possessed optimal dissolution efficiency in artificial gastric juice (pH 1.2) and the respective DE could achieve 86.7 ± 0.6%, 90.6 ± 0.5%, 92.9 ± 0.5% of the three samples. Tab. 5 Dissolution efficiency of products in different dissolution media

Dissolution Media

Raw

Cube

Tube

Mean ± SD (%)

Mean ± SD (%)

Mean ± SD (%)

Water with 0.5 % SDS

41.3 ± 0.2

58.1 ± 0.6***

80.3 ± 1.1***, # # #

Water with 1% SDS

51.4 ± 0.8

71.9 ± 1.0***

92.2 ± 1.7***, # # #

Artificial intestinal juice

77.4 ± 0.8

83.7 ± 0.6**

90.6 ± 0.7***, # # #

Artificial gastric juice

86.7 ± 0.6

90.6 ± 0.5**

92.9 ± 0.5***, # # #



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*Statistical significance with Raw VIN (**P < 0.01, ***P < 0.001); #Statistical significance with

Cube (# # #P < 0.001)

Interestingly, although the re-crystallized VIN cubes and square tubes had similar size (121.8 ± 40.5 nm for cubes and 116.6 ± 42.3 nm for tubes) and surface area (0.438 m2/g for cubes and 0.532 m2/g for tubes), the dissolution rate of VIN tube was significantly higher than the cubic ones. Similar trends could be obtained between the VIN tubes and the granular products (morphology shown as Fig. 1F), showing that smaller sized VIN granules possessed larger specific surface area but failed in the dissolution competition as compared with tubular VIN (Table S3). We could ascribe this dissolution discrepancy to the differences in valid surface area between the VIN tubes with the other crystalline products: For VIN cubes, although loosely sinks appeared on the center of cubic faces, the potential cavities inside cubic crystals were sealed, blocking the access of solvent into the cubic holes (Fig 1B); As for smaller sized VIN granules, despite of its large surface area, the surface energy of granular VIN was also high, which would result in serious aggregation in aqueous solutions (Fig. 1F). Thus part of the particle surface were invalid for the access of water molecules, leading to a decreased “effective” surface area and compromised dissolution. In comparison, tubular holes were available to the dissolution media, which contributed to an increased surface area, thus displayed fastest dissolution in all the test media. 3.4 In Vivo Pharmacokinetic Study. The rat plasma concentration of VIN changed over time after administration. As shown in Fig. 10, both the testing VIN samples displayed a single-peak profile after


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gavage. In details, plasma VIN increased rapidly after administration, and reached a maximum in 1 h, then gradually decreased to the baseline in the next 5 h to 6 h. Of note, tubular VIN displayed a faster absorption rate, with its peak concentration (Cmax = 267.3 ± 53.9 ng/mL, Table 6) appearing at 30 min post administration. As compared, the Cmax and Tmax of raw VIN were calculated to be 164.4 ± 23.6 ng/mL, and 1 h, respectively. The tubular VIN crystals manifested a higher AUC0→t of 484.0 ± 24.6 ng/mL·h after oral administration, which was approximately 1.3-fold of that of raw VIN crystals (386.6 ± 22.8 ng/mL·h). The results revealed that systemic absorption of VIN was improved by applying re-crystalizing the raw VIN into tubular products.

Fig.10 Plasma drug concentration vs. time of rats received raw, or recrystallized tubular VIN crystals. Each value represents the mean ± SD (n = 6). Tab. 6 Pharmacokinetic parameters of VIN after oral administrations

Evaluation parameters

Raw VIN

Tubular VIN

Dose (mg/kg)

6

6

Cmax (ng/mL)

164.4 ± 23.6

267.3 ± 53.9**

Tmax (h)

1.0

0.5

AUC0-t (ng・h/mL)

386.6 ± 22.8

484.0 ± 24.6***

Each value was represented as Mean ± SD (n = 6). *Statistical significance with Raw VIN (**P < 0.01, ***P < 0.001)



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VIN is a BCS II drug with aqueous solubility about 5 µg/mL.4 On this account, oral absorption and bioavailability of VIN can be improved by increasing its dissolution rate in the GI tract. As a base-type drug, VIN has a much higher solubility in gastric fluids than in the neutral intestinal media. Therefore the maximal absorption generally occurs in the stomach and duodenum. As a result, the plasma concentration of VIN reaches its peak value (Cmax) at 0.5 – 1 h after oral administration, which is in line with the gastric emptying time (about 0.5 – 1 h),46 suggesting gastric dissolution plays a critical role in VIN absorption. In line with this, the orally administrated VIN tubes that have a faster dissolution rate achieve a higher Cmax as compared with the bulk crystals (267.3 ng/mL vs 164.4 ng/mL). What’s more, stability assessment at 40 °C± 2 °C and 75% RH ± 5% RH both shows that the crystal structure of the recrystallized VIN remains intact after a series of consecutive tests which lasts for four weeks, suggesting the crystalline tubular products are stable at general storage conditions (Fig.S4 & Fig.S5). To be concluded, this study presents, to the best of our knowledge, the first in vitro to in vivo evidence for bioavailability improvement of BCS II drug by applying the shape-controlled re-crystallization strategy. As compared with other strategies, the crystallization method is simpler, more cost-effective, and easy to scale-up, thus may account for a useful tool in the future drug development.

4

Conclusion In this paper, a series of VIN crystals with different shapes have been prepared

via the anti-solvent method (Fig. 1). By simply adjusting the water/ethanol ratio and


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initial VIN concentration, morphologies of the crystalline products can be finely tuned from three-dimensional cubes and tubes, then to two-dimensional frizzled plates and finally to zero-dimensional microparticle clusters. PXRD analysis demonstrated all the VIN products shared the same crystal structure, yet hollow structured VIN tubes were formed only at optimized crystallizing conditions (water/ethanol of 1:1, initial VIN concentration at 1 g/30 mL). By analyzing the morphologies of the crystallized products by SEM imaging and tracking the kinetic growth process by FBRM, it was proposed that supersaturation was the key parameter promoted nucleation as well as diffusion-limited crystal growth for the formation of varied VIN morphologies. As VIN is a BCS II drug, its low bioavailability could be improved by increasing its dissolution rate. Herein two kinds of recrystallized products (VIN cubes & tubes) were chosen and assessed in four testing media (0.5% SDS, 1% SDS, artificial gastric and intestinal fluids) for their in vitro dissolution behaviors in comparison with raw VIN. Results manifested that the recrystallized tubular VIN crystal showed the fastest dissolution rate as compared with the other two kinds of crystals. In vivo pharmacokinetic study showed that tubular VIN achieved a faster higher Cmax (267.3 ± 53.9 ng/mL) in 0.5 h, whereas raw VIN reached Cmax (164.4 ± 23.6 ng/mL) in a slower pattern (Tmax = 1 h). As a result, tubular VIN achieved a significantly improved AUC0-t (484.0 ± 24.6 ng/mL·h), which was approximately 1.3-folds of that of the raw VIN products (AUC0-t = 386.6 ± 22.8 ng/mL·h). Of note, this is the first in vitro to in vivo report for bioavailability improvement of BCS II drug by applying the shape-controlled re-crystallization strategy. As compared with other strategies, the



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crystallization method is simpler, more cost-effective, and easy to scale-up, thus may account for a useful tool in the future drug development.

ASSOCIATED CONTENT Supporting Information Characteristic analysis of Vinpocetine (Figure. S1) including (A) Chemical structure of Vinpocetine; (B) X-ray diffraction; (C) differential scanning calorimetry; and (D) diffuse reflectance infrared Fourier transform spectroscopy; Comparison of The calculated PXRD pattern of Vinpocetine and X-ray diffraction (XRD) patterns of the raw VIN (Figure. S2)；Dissolution profiles of Tube and Granule samples in 0.5% SDS (A), and 1% SDS (B), respectively. (Fig. S3). Scanning electron microscopic photographs and X-ray diffraction (XRD) patterns of tubular crystal stored at 75%RH/40 °C for different times (Fig. S4 & Fig. S5).

AUTHOR INFORMATION Corresponding Author *Tel.: 86-22-27405754. Fax: 86-22-27314971. E-mail: [email protected]. Notes The authors declare no competing ﬁnancial interest

Acknowledgments


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The authors are grateful for the financial support of the National Natural Science Foundation of China (21676179, 21376164 and 91634117), the National 863 Program (2015AA021002), major project of Tianjin (15JCZDJC33200) and the Innovative Group Project 21621004.

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Table of Contents graphic (TOC) Controlled recrystallization of tubular vinpocetine crystals with increased aqueous dissolution rate and in vivo bioavailability Panpan Sun 1, 2, Yaping Wang1, 2, Sohrab Rohani3, Ergang Liu1, 2, Shichao Du1, 2, Shijie Xu1, 2, Mingyang Chen1, 2, Zhenping Wei1, Junbo Gong1, 2, 4* 1

School of Chemical Engineering and Technology, State Key Laboratory of Chemical

Engineering, Tianjin University, Tianjin300072, China; 2

The Co-Innovation Center of Chemistry and Chemical Engineering of Tianjin,

Tianjin300072, China 3

Department of Chemical and Biochemical Engineering, the University of Western

Ontario, London, Ontario N6A 5B9, Canada 4

The key laboratory Modern Drug Delivery and High Efficiency in Tianjin, China

The graphic shows the obtained vinpocetine hollow tubular crystal (right SEM image) by anti-solvent crystallization using the bulk material (left SEM image). Left curve graph illustrates tubular crystal has increased dissolution rate than the raw VIN in water with 0.5% SDS and right plasma concentration-time plots show that tubular



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VIN achieves a faster higher Cmax and an improved AUC0-t.


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Controlled Recrystallization of Tubular Vinpocetine ... - ACS Publications

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