Article pubs.acs.org/molecularpharmaceutics
A Strategy for the Improvement of the Bioavailability and Antiosteoporosis Activity of BCS IV Flavonoid Glycosides through the Formulation of Their Lipophilic Aglycone into Nanocrystals Yan Li,†,‡ Shaoping Sun,§ Qi Chang,∥ Lei Zhang,∥ Gengnan Wang,∥ Weixing Chen,‡ Xiaoqing Miao,† and Ying Zheng*,† †
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China ‡ Pharmaceutical and Biological Examination Department, Patent Examination Cooperation Center of The Patent Office, SIPO, Beijing 100088, China § School of Chemistry & Chemical Engineering & Materials Science, Heilongjiang University, Harbin 150080, China ∥ Institute of Medicinal Plant Development, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100193, China ABSTRACT: Epimedium-derived flavonoid glycosides are widely used for the prevention of osteoporosis, but these compounds generally exhibit poor membrane permeability and oral absorption. To address these limitations, the bioactive lipophilic aglycone icaritin (ICT) was selected and successfully developed into nanocrystals (ICTN) through the antisolvent-precipitation method. After the parameters in the preparation of ICTN were optimized, the morphology, crystallinity, adsorption of the stabilizers on the ICT surface, and the dissolution of the resulting nanocrystals were characterized. The pharmacokinetics in rat and the in vitro antiosteoporosis activity of serum withdrawn after the oral administration of ICTN to rats on mouse osteoblastic cells were evaluated. Consistent with its good performance in stabilizing the ICT nanosuspension, atomic force microscopy showed that hydroxypropyl methylcellulose (HPMC) exhibits better adsorption on the ICT surface compared with other stabilizers. Needleshaped crystals (∼220 nm in diameter) with a high drug loading (∼90%) were generated when 0.16 mL of the ICT acetone solution (10 mg/mL) was injected quickly into 2 mL of the HPMC solution (0.02%, w/w) under ultrasonication for 10 s at room temperature. The thermal analysis demonstrated that the majority of the particles are in their crystalline forms, similarly to the unformulated ICT. After oral administration, ICTN exhibited a faster dissolution rate and significantly faster absorption, as supported by the increased AUC0−36h and Cmax and the reduced Tmax of these nanocrystals compared with the raw suspension (p < 0.05). Compared with blank serum, enhanced proliferation and differentiation activities were observed when serum withdrawn after the oral administration of ICTN in rat was incubated with osteoblast MC3T3-E1 cells. The present delivery system could provide a new promising strategy for BCS IV glycoside of flavonoids or other natural products by formulation of their bioactive lipophilic aglycone forms to enhance oral absorption and in vivo bioactivity. KEYWORDS: icaritin (ICT), glycoside, nanocrystal, antisolvent-precipitation, oral bioavailability, osteoporosis
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INTRODUCTION Herba Epimedii, which is called Yinyanghuo in Chinese, is a widely used herbal medicine listed in the Chinese Pharmacopoeia. Traditionally, Herba Epimedii is taken orally to treat impotence and prevent osteoporosis. The main bioactive components in Herba Epimedii are flavonoid glycosides with one or two sugar moieties, such as icariin and icariside II (Figure 1).1 However, due to its poor solubility (∼15 μg/mL in water at 25 °C2), poor membrane permeability (∼10−7 cm/s), interaction with efflux transporters, including BCRP and MRP2,3 and extensive hydrolysis through the removal of one or more sugar moieties in the small intestine, the oral bioavailability of icariin in rats is only 12.02%.4 Because the © 2013 American Chemical Society
membrane permeability is the limiting step in the oral absorption of the flavonoid glycosides in Herba Epimedii, suitable formulation strategies need to be considered to simultaneously enhance their dissolution rate and membrane permeability. Based on our previous study on the BCS IV compound oleanolic acid (OA), which has an oral bioavailability of 0.7% in rats, the amorphization of OA sodium salt with polymeric PVP-40, which was used as a stabilizer, and Received: Revised: Accepted: Published: 2534
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stable ICT nanocrystal for in vitro and in vivo tests. Because ICT has extensive metabolism in vivo,6 the serum pharmacology method was utilized to investigate the antiosteoporosis activity of the serum, which containing its metabolites, after the oral administration of icaritin nanocrystals (ICTN) to rats. To achieve this aim, the bottom-up antisolvent-crystallization method was utilized. The effects of stabilizers, the concentration of the ICT and stabilizer solution, the volume of ICT solution injected, and the duration of the ultrasonication on the particle size and the short-term physical stability were investigated. The optimized formulation was further characterized in terms of its morphology, crystallinity, thermal behavior, dissolution, oral bioavailability in rats, and serum antiosteoporosis activity on osteoblast cells.
Figure 1. Chemical structures of icariin, icariside II, and their aglycone icaritin.
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sodium caprate, which was used as a wetting agent and penetration enhancer, through a spray freeze-drying processing could yield kinetically stable solid dispersions with a superior dissolution performance. However, the oral bioavailability of the product was only enhanced approximately 2-fold compared to unformulated OA, which indicates that only a limited enhancement could be obtained by the above formulation approach for BCS IV compounds (e.g., solid dispersion). A new formulation strategy is required to solve the problem of the absorption of BCS IV compounds. As a bioactive aglycone form and a major intestinal metabolite of icariin and icariside II, icaritin (ICT, Figure 1), which belongs to the group of BCS II compounds, is considered an alternative form that will be developed in the present study. Compared with its glycoside form, icaritin exhibited more potent activity than icariin for enhancing the differentiation and proliferation of osteoblasts in vitro5 and considerably better oral bioavailability (35% in rat estimated by total plasma drug concentrations after enzymatic hydrolysis6) as an unformulated drug suspension. Although ICT is a promising aglycone form of icariin/icariside II, its high lipophilicity (logP ∼ 67) and its poor aqueous solubility ( 0.80). The effects of these two parameters on the particle size are illustrated in Figure 5. In addition, confirmation studies using the other five different conditions were used to compare the experimental and theoretical values. The results showed that the experimental data consistently agreed with the theoretical prediction (data not shown). Considering the feasibility of scale up, a relatively high concentration of drug with a 2538
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Figure 5. Effects of the injected volume of icaritin acetone solution and the concentration of HPMC E3 on the particle size of the icaritin nanosuspension (n = 3).
Figure 6. (a) Scanning electron microscopy of raw ICT crystals and (b) transmission electron microscopy of formulated ICT nanosuspension.
In Vitro Dissolution. Figure 9 shows the dissolution profiles of the nanocrystals and the raw crystals in vitro over 120 min. The raw crystal dissolved very slowly, and approximately half of the drug was released within 2 h. In contrast, the ICT nanocrystals exhibited a significantly enhanced dissolution rate, in which 90% of the drug was dissolved within 5 min, and all of the crystals were dissolved within 15 min. The significant dissolution enhancement of the ICT nanocrystals may be due to the reduced particle size and the corresponding increased specific surface area. Anti-Osteoporosis Activity. Compared with the control group, the treatment of MC3T3-E1 cells with ICT did not stimulate proliferation (Figure 10a) but enhanced the in vitro osteoblast differentiation, as evidenced by the ALP activity (Figure 10b). We next investigated whether serum metabolites after the oral administration of ICTNs play a role in the
proliferation and differentiation of osteoblasts. After 72 h of in vitro culture, the MTT assay demonstrated that serum withdrawal at 10 min and 1.5 h significantly stimulated cell proliferation by 120.1 ± 9.0% and 116.6 ± 9.9%, respectively, compared with the control (p < 0.05, Figure 10c). Moreover, the ALP activities of cells treated with serum withdrawn at 10 min and 1.5 h were significantly higher than those of the blank serum group (p < 0.05, Figure 10d). These results suggest that the metabolites of ICT in the serum after the oral administration of the formulated nanocrystals improved the proliferation and differentiation of osteoblasts. Bioavailability Studies in Rats. To investigate the in vivo performance of the nanocrystals, their oral absorptions were compared to that of the unformulated drug in SD rats. As extensive phase II metabolism occurred, the parent ICT could only be detected within 2 h after the oral administration. However, 2539
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DISCUSSION
The use of surfactants, including Tween 80 and SDS, as stabilizers in the nanosuspension increased the concentration of ICT in its solubilized form (data not shown). The increased solubility during the preparation and storing process is usually more prone to Ostwald ripening,13 which results in particle growth and may lead to poor physical stability during storage. In addition, due to the presence of nonuniformly distributed particles in the initial preparation, as indicated by the relatively high PDI, the short physical stabilities of the ICT nanosuspension were poor when PVA and PVP360 were used as the stabilizers. The large difference in their size can induce different saturation solubilities and concentration gradients, consequently leading to Ostwald ripening, which may explain the rapidly increased particle size observed during storage. In this study, we utilized atomic force microscopy to investigate the adsorption of stabilizers on the ICT surface and to understand the role of the stabilizers on the physical stability of the resulting nanosuspension formulations. As an important required precondition, a smooth surface was generated using mica to provide a planar drug substrate, which was used for the study of the adsorption behavior of surfactants and polymers on the substrate. As shown in Figure 3, HPMC resulted in a better surface coverage through the extension of the molecules in the chain-like pattern. Compared to the other polymers tested, the relatively low HLB of HPMC (10−12) represents its relatively high hydrophobicity. It has been suggested that hydrophobic stabilizers tend to have a higher probability of interacting with a hydrophobic drug surface and thus possibly achieving a smaller particle size.17 However, F68 and SDS are relatively hydrophilic compared to HPMC E3 and did not interact well with the ICT surface. The low level of interaction with these polymers was revealed through the shape of the structures formed. It is assumed that the level of interaction between the stabilizer and the ICT surface could better mimic the binding properties between the stabilizer and the nanocrystal in nanosuspensions.18 More effective coverage of the ICT may lead to the protection of the nanocrystals through the inhibition of flocculation, aggregation, and/or crystal growth. Therefore, HPMC plays a key role in controlling the particle morphology and inhibiting particle growth in the nanosuspension formulation due to its extensive and effective coverage of the ICT nanocrystals. This hypothesis may explain why the HPMC-based formulations exhibited superior particle size and physical stability during storage compared with the other polymer-based formulations. It was also observed that the ultrasonication time did not affect the particle size, and a longer duration did not reduce the particle size (data not shown). The formation of a stable suspension with the smallest particle size requires a high nucleation rate and a low growth rate. The rapid mixing of the solvent with a large amount of antisolvent induced by ultrasonication resulted in a high nucleation rate.19 Simultaneously, the rapid mixing can dilute the high concentration in the vicinity of the nucleating crystals, which can reduce the growth rate.20 Therefore, nucleation and crystal generation occurred within the least ultrasonication time, and a short duration of ultrasonication can achieve this effect thorough subsequent dilution. In general, successful nanosuspensions can be achieved when the stabilizer is capable of wetting the surface and effectively covering the surface of the newly formed drug particles. In the absence of a sufficient amount of stabilizer, the high surface energy of the nanometer-sized drug particles would tend to result in agglomeration or aggregation. However, too much
Figure 7. DSC patterns: (a) raw crystals; (b) physical mixture (the weight ratio of icaritin to HPMC E3 is 15:1); (c) nanocrystals; (d) HPMC E3.
Figure 8. X-ray powder diffraction patterns: (a) raw crystals; (b) physical mixture (the weight ratio of icaritin to HPMC E3 is 15:1); (c) nanocrystals; (d) HPMC E3.
Figure 9. Dissolution profiles for (▲) raw icaritin and (◆) nanocrystals of icaritin (n = 3).
a relatively high concentration of ICT could be detected after the enzymatic hydrolysis with β-glucuronidase/sulphatase.6 The pharmacokinetic parameters of the two groups are shown in Table 1. It can be observed that the nanocrystals exhibited a significantly increased AUC0−12 and Cmax and a much shorter Tmax compared with the unformulated ICT (p < 0.05). The AUC0−12 and Cmax of the nanocrystals were 2.0- and 4.7-fold higher than those of the raw crystals, respectively, likely due to the enhanced dissolution rate of the formulated nanocrystals. 2540
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Figure 10. (a) Effect of ICT on the proliferation of MC3T3-E1 cells, as measured by the MTT assay. The cells were cultured with different concentrations of ICT for 72 h. (b) Effect of ICT on osteoblastic differentiation of MC3T3-E1 cells. The cells were cultured with different concentrations of ICT for 4 days. (c) Effect of rat serum collected at different time points on the proliferation of MC3T3-E1 cells, as measured by the MTT assay. The cells were cultured for 72 h in the presence of the drug-containing serum collected at different times (10 min, 1.5 h, and 24 h) after the oral administration of ICTN at a dose of 30 mg/kg. (d) Effect of rat serum collected at different time points on the differentiation of MC3T3-E1 cells. The MC3T3-E1 cells were cultured for 4 days in the presence of drug-containing serum collected at different times (10 min, 1.5 h, and 24 h) after the oral administration of ICTN at a dose of 30 mg/kg. Estradiol was used as the positive control. The ALP activity was determined through the pNPP method and normalized to the protein content. Each bar represents the mean ± SD (n = 6). *p < 0.05 vs control, **p < 0.01 vs control.
the fact that, under a fast nucleation rate, the drug solute lacks sufficient time to accurately incorporate into the growing crystal lattice to form perfect crystals.20 Therefore, the resulting nanocrystals may lack the long ordered structures, which will result in the absence of the intensive peaks observed with the raw drug crystals through XRPD. The bioavailability of poorly water-soluble drugs in a nanosuspension form can be improved with an increased specific surface area available for dissolution, as described by the Noyes−Whitney equation,21 and an increased saturation solubility, as explained by the Kelvin and Ostwald−Freundlich equations.22 The pharmacokinetic study showed that the free form of ICT could only be detected for a short period in the rat plasma, and most of the ICT was rapidly transformed into its metabolites in the GI tract/liver. Therefore, a serum pharmacological testing method was used to address whether the ICTNs exhibited biological activity after oral administration. In this experiment, the effects of the metabolites contained in the serum on the growth and differentiation of MC3T3-E1 cells were investigated. The highest biological activity was observed when the ICT concentration reached Cmax in the serum at 10 min. Because the drug-containing serum obtained after the oral administration of ICTN need to be diluted in cell culture
Table 1. Pharmacokinetic Parameters of Raw Icaritin and Nanosuspension after Oral Administration at 5 mg/kg to SD rats (n = 6) pharmacokinetic parameters
raw icaritin (Chang et al., 2012)
icaritin nanosuspension
Tmax (h) Cmax (μg/mL) t1/2 (h) AUC0−36h (μg·h/mL) MRT (h) Rel.Ba (%)c
1.00 ± 0.00 0.49 ± 0.12 10.16 ± 3.33 5.43 ± 1.60 13.76 ± 1.11 100
0.17 ± 0.00b 2.29 ± 0.99b 6.00 ± 2.06a 10.96 ± 2.09b 13.96 ± 3.57 201.74
a
p < 0.05 in two-tailed Student’s t test compared with raw icaritin. p < 0.01 in two-tailed Student’s t test compared with raw icaritin. c Relative bioavailability compared with raw icaritin. b
HPMC E3 would lead to high viscosity, which would promote Ostwald ripening.9 As shown in the DSC and XRPD results (Figures 7 and 8), the values for the crystallinity calculated by these two methods are not consistent. Taking the TEM results into consideration, we hypothesize that XRPD may underestimate the crystallinity of the nanocrystals. This underestimation may be explained by 2541
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(6) Chang, Q.; Wang, G. N.; Li, Y.; Zhang, L.; You, C.; Zheng, Y. Oral absorption and excretion of icaritin, an aglycone and also active metabolite of prenylflavonoids from the Chinese medicine Herba Epimedii in rats. Phytomedicine 2012, 19 (11), 1024−1028. (7) Li, Y.; Sun, S. P.; Zheng, Y. Determination of partition coefficients of Icaritin-II and Icaritin based on the HPLC retention time. Chin. Pharm. J. 2011, 47, 122−126. (8) Lakshmi, P.; Kumar, G. A. Nano-suspension technology: a review. Int. J. Pharm. Pharm. Sci. 2010, 2 (Suppl 4), 35−40. (9) Merisko-Liversidge, E.; Liversidge, G. G.; Cooper, E. R. Nanosizing: a formulation approach for poorly-water-soluble compounds. Eur. J. Pharm. Sci. 2003, 18 (2), 113−120. (10) Verma, S.; Gokhale, R.; Burgess, D. J. A comparative study of top-down and bottom-up approaches for the preparation of micro/ nanosuspensions. Int. J. Pharmaceutics 2009, 380, 216−222. (11) Gao, L.; Zhang, D.; Chen, M. drug nanocrystals for the formulation of poorly soluble drugs and its application as a potential drug delivery system. J Nanopart. Res. 2008, 10, 845−862. (12) Junghanns, J. U.; Muller, R. H. Nanocrystal technology, drug delivery and clinical applications. Int. J. Nanomed. 2008, 3 (3), 295− 309. (13) Patravale, V. B.; Date, A. A.; Kulkarni, R. M. Nanosuspensions: a promising drug delivery strategy. J. Pharm. Pharmacol. 2004, 56 (7), 827−840. (14) Verma, S.; Huey, B. D.; Burgess, D. J. Scanning Probe Microscopy Method for Nanosuspension Stabilizer Selection. Langmuir 2009, 25 (21), 12481−12487. (15) Xia, D. N.; Quan, P.; Piao, H. Z.; Piao, H. Y.; Sun, S. P.; Yin, Y. M.; Cui, F. D. Preparation of stable nitrendipine nanosuspensions using the precipitation-ultrasonication method for enhancement of dissolution and oral bioavailability. Eur. J. Pharm. Sci. 2010, 40 (4), 325−334. (16) Matteucci, M. E.; Hotze, M. A.; Johnston, K. P.; Williams, R. O. Drug nanoparticles by antisolvent precipitation: Mixing energy versus surfactant stabilization. Langmuir 2006, 22 (21), 8951−8959. (17) Peltonen, L.; Hirvonen, J. Pharmaceutical nanocrystals by nanomilling: critical process parameters, particle fracturing and stabilization methods. J. Pharm. Pharmacol. 2010, 62 (11), 1569−79. (18) Verma, S.; Gokhale, R.; Burgess, D. J. A comparative study of top-down and bottom-up approaches for the preparation of micro/ nanosuspensions. Int. J. Pharmaceutics 2009, 380 (1−2), 216−222. (19) Dalvi, S. V.; Dave, R. N. Analysis of nucleation kinetics of poorly water-soluble drugs in presence of ultrasound and hydroxypropyl methyl cellulose during antisolvent precipitation. Int. J. Pharmaceutics 2010, 387 (1−2), 172−179. (20) Rabinow, B. E. Nanosuspensions in drug delivery. Nat. Rev. Drug Discovery 2004, 3 (9), 785−796. (21) Noyes, A. A.; Whitney, W. R. The rate of solution of solid substances in their own solutions. J. Am. Chem. Soc. 1897, 19, 930− 934. (22) Kesisoglou, F.; Panmai, S.; Wu, Y. H. Nanosizing - Oral formulation development and biopharmaceutical evaluation. Adv. Drug Delivery Rev. 2007, 59 (7), 631−644.
medium for the in vitro antiosteoporosis study, the oral dose levels used to prepare the drug-containing serum are higher than the dose level used in the PK study. After hydrolysis, the total concentrations of ICT in the rat plasma sampled at 10 min, 1.5 h, and 24 h after the oral administration of ICTN were 3726 ± 1253 ng/mL, 2856 ± 1094 ng/mL, and 501 ± 484 ng/mL, respectively. In the pharmacokinetic study, the total concentrations of ICT in the rat plasma after hydrolysis at 10 min, 1.5 h, and 24 h after the oral administration of ICTN were 2,288 ± 990 ng/mL, 752 ± 400 ng/mL, and 365 ± 137 ng/mL, respectively. After the drug-containing serum in the first group was diluted 4-fold for subsequent cell culture studies, the concentration of total ICT at each time point was slightly lower but still comparable to the corresponding concentration in the pharmacokinetic study. Moreover, an in vivo antiosteoporosis study needs to be performed in the future to show the in vivo superiority of the formulation.
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CONCLUSIONS ICT nanosuspensions of ∼200 nm with a uniform size distribution and a high drug loading of 93.8% were successfully prepared using the antisolvent-crystallization method. The ICTN demonstrated good short-term physical stability, enhanced dissolution, and improved bioavailability compared with the unformulated ICT. Moreover, the serum collected from rats that received an oral administration of ICTN, which contained the metabolites of ICT, exhibited good antiosteoporosis activity. The present delivery system could provide a new promising strategy for BCS IV glycoside of flavonoids or other natural products by formulation of their bioactive lipophilic aglycone and also their in vivo metabolites forms into nanocrystals with high drug loading to enhance oral bioavailability and in vivo bioactivity.
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AUTHOR INFORMATION
Corresponding Author
*Institute of Chinese Medical Sciences, University of Macau 3/ F, Rm 204A, Block 3, Av. Padre Tomás Pereira, S.J. Taipa Macao SAR, China. Tel.: 853-83974687; fax: 853-28841358; email:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The financial support from the Macao Science and Technology Development Fund (FDCT Fund Project No.: 008/2007/A1 and 044/2011/A2) is gratefully acknowledged. The authors would like to thank Dr. Wang Yancai for his helpful discussion.
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REFERENCES
(1) Chen, X. J.; Guo, B. L.; Li, S. P.; Zhang, Q. W.; Tu, P. F.; Wang, Y. T. Simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and high-performance liquid chromatography. J. Chromatogr., A 2007, 1163 (1−2), 96−104. (2) Wang, Y.-l.; Jia, X.-b.; Chen, Y.; Li, X. Determination of equilibrium solubility of icariin and its apparent oil/water partition coefficient. China J. Trad. Chin. Med. Pharm. 2008, 23, 777−779. (3) Chen, Y.; Zhao, Y. H.; Jia, X. B.; Hu, M. Intestinal Absorption Mechanisms of Prenylated Flavonoids Present in the Heat-Processed Epimedium koreanum Nakai (Yin Yanghuo). Pharm. Res. 2008, 25 (9), 2190−2199. (4) Ye, L. K.; Chen, J. M.; Liu, S. H.; Li, G. X. Pharmacokinetics of icariin in rats. Chin. Pharm. J. 1999, 34, 33−36. (5) Huang, J.; Yuan, L.; Wang, X.; Zhang, T. L.; Wang, K. Icaritin and its glycosides enhance osteoblastic, but suppress osteoclastic, differentiation and activity in vitro. Life Sci. 2007, 81 (10), 832−840. 2542
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