Improving permeability and oral absorption of mangiferin by phospholipid complexation

Fitoterapia 93 (2014) 54–61

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Improving permeability and oral absorption of mangiferin by phospholipid complexation Hequn Ma, Hongming Chen, Le Sun, Lijin Tong, Tianhong Zhang ⁎ Shenyang Pharmaceutical University, No.103, Wenhua Road, Shenyang, China

a r t i c l e

i n f o

Article history: Received 9 September 2013 Accepted in revised form 28 October 2013 Available online 9 November 2013 Keywords: Mangiferin Phospholipid complex Physicochemical properties Intestinal absorption Pharmacokinetics

a b s t r a c t Mangiferin is an active ingredient of medicinal plant with poor hydrophilicity and lipophilicity. Many reports focused on improving aqueous solubility, but oral bioavailability of mangiferin was still limited. In this study, we intended to increase not only solubility, but also membrane permeability of mangiferin by a phospholipid complexation technique. The new complex's physicochemical properties were characterized in terms of scanning electron microscopy (SEM), differential scanning calorimetry (DSC), infrared absorption spectroscopy (IR), aqueous solubility, oil–water partition coefficient and in vitro dissolution. The intestinal absorption of the complex was studied by the rat in situ intestinal perfusion model. After oral administration of mangiferin–phospholipid complex and crude mangiferin in rats, the concentrations of mangiferin were determined by a validated RP-HPLC method. Results showed that the solubility of the complex in water and in n-octanol was enhanced and the oil–water partition coefficient was improved by 6.2 times and the intestinal permeability in rats was enhanced significantly. Peak plasma concentration and AUC of mangiferin from the complex (Cmax: 377.66 μg/L, AUC: 1039.94 μg/L*h) were higher than crude mangiferin (Cmax: 180 μg/L, AUC: 2355.63 μg/L*h). In view of improved solubility and enhanced permeability, phospholipid complexation technique can increase bioavailability of mangiferin by 2.3 times in comparison to the crude mangiferin. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The bioavailability of oral drug depends on its dissolution and membrane permeability. The BCS in FDA guidelines classifies drug substances into four categorizes based on their solubility and permeability [1]. Bioavailability of drugs with poor dissolution is due to either low water-solubility or large particle size. Therefore their bioavailability can be improved by dissolution enhance methods like salt formation [2], solid dispersion [3], encapsulation in cyclodextrins [4], micronization [5] and so on. Strategies to mitigate poor permeability include formulating with bile salts, surfactants, phospholipids, medium-chain glycerides, fatty acids, and mixed micelles [6].Among the absorption enhancers, some were known to cause membrane damage [7,8].

⁎ Corresponding author. Tel.: +86 24 23984159; fax: +86 24 23986320. E-mail address: [email protected] (T. Zhang). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.10.016

Mangiferin (2-β-D-glucopyranosyl-1, 3, 6, 7-tetrahydroxyxanthone) is a natural polyphenol compound (Fig. 1). It is distributed in Anemarrhenaasphodeloides (Liliaceae family), Mangiferaindica L. (Anacardiaceaefamily) and Belamcandachinensis (L) DC (BelamcandaAdans) [9,10]. Mangiferin has a xanthone nucleus (dibenzo-γ-pirone) and belongs to glycosylated xanthones [11]. Mangiferin has attracted considerable interest due to its potent antioxidant [12], antitumor, antiviral [13], antidiabetic [14], anti-inflammatory and immunomodulatory activities [15]. According to recent reports, memory enhancement [16] positive impact on hyperlipidemia [17,18] and hepatic-protective effects [19] could make mangiferin a promising drug. However, mangiferin's poor bioavailability restricts its clinical application, and there are no products on the market yet. It is reported that the solubility of mangiferin was 0.111 mg/mL [20] and oral bioavailability was only 1.2% [21]. The lipophilicity of mangiferin is also poor [22] which

H. Ma et al. / Fitoterapia 93 (2014) 54–61

OH

Pharmaceutical and Biological Products. The other chemical reagents were of analytical grade and used as received. OH

O

OH

OH OH O

2.2. Preparation of mangiferin–phospholipid complex

O

HO

HO

55

OH

Fig. 1. Structure of mangiferin.

results in poor intestinal membrane permeability and low oral absorption. Zhou had prepared the inclusion compound of mangiferin-HP-β-CD [23] to improve the solubility of mangiferin, but its effect on oral bioavailability was not reported. A novel mangiferin calcium salt has been prepared and reported to have improved dissolution and bioavailability was improved by 1.9 times [24]. However all the studies focused on the improvement of dissolution, but another potential root-cause of low bioavailability is the poor permeability of mangiferin. The proper dosage form of mangiferin should improve not only solubility, but also membrane permeability of mangiferin. Phospholipid as the major composition of the bio membrane has good biocompatibility. A drug–phospholipid complex is formed by interaction of phospholipid with a functional group of the drug. Advantages of drug–phospholipid complex include not only enhancing solubility of drugs with low aqueous solubility (phospholipid as a kind of surfactant enhances the solubility) but also the potential to improve permeability of low membrane penetration (phospholipid as a carrier carrying drug penetrating bio-membrane) [25]. Previous studies have demonstrated that phospholipid complex can improve the lipophilicity and the therapeutic efficacy of certain drugs with poor oral bioavailability [26,27]. In recent years, the oral bioavailability of silybin, curcumin and oxymatrine has been enhanced by phospholipid complexation and their human studies have been reported [27–29]. Therefore in this present study, for the purpose of improving the bioavailability of mangiferin, we prepared mangiferin–phospholipid complex. The physicochemical properties and intestinal absorption potential of mangiferin–phospholipid complex were characterized. The concentration of mangiferin in rat plasma was determined respectively after oral administration of mangiferin and mangiferin–phospholipid complex equivalent to 25 mg/kg of mangiferin. And finally in vitro/in vivo correlation (IVIVC) was studied.

The complex was prepared with mangiferin and Lipoid E80 at a molar ratio of 1:1. The required amounts of mangiferin were placed in a 100 mL round-bottom flask and dissolved in 80% ethanol. Then Lipoid E80 was dissolved in ethanol by ultra-sonication. The mixture was refluxed at temperature of 60 for 2 h. After evaporating off ethanol under vacuum at 40 °C, we stored it within the sealed container for drying. The dried residues were kept in glass bottle, flushed with nitrogen and stored at − 20 °C. 2.3. Determination of the content of mangiferin in the complex The content of mangiferin in the phospholipid complex was determined by HPLC method as follows. Approximately 14.25 mg of phospholipid complex and 5 mg of mangiferin reference standard were respectively dissolved in 50 mL of solvent A (methanol: water = 60:40, v/v), and 10 μL aliquot of the two solutions was injected into a HPLC system. The stationary phase was SepaxGP-C8 (4.6 mm × 250 mm, 5 μm). The mobile phase was 0.05% (v/v) phosphoric acid and acetonitrile at a ratio of 85:15. The elution was carried out with a flow rate of 1.0 mL/min at 25 °C, and a wavelength of 258 nm was used for detection. 2.4. Differential scanning calorimetry (DSC) The samples were sealed in the aluminum crimp cell and heated at the rate of 10 °C/min from 0 to 300 °C in nitrogen atmosphere. The peak transition onset temperature of mangiferin, phospholipid, physical mixture of mangiferin and phospholipid (1:1), and mangiferin–phospholipid complex were determined and compared. 2.5. Infrared absorption spectroscopy (IR) Mangiferin, phospholipid, physical mixture of mangiferin and phospholipid (1:1), and mangiferin–phospholipid complex were identified and compared by the infrared spectrophotometric method with potassium bromide as supporter. 2.6. Scanning electron microscopy (SEM) Mangiferin and mangiferin–phospholipid complex and its physical mixture (1:1) were coated with platinum in a sputter coater and their surface morphology was viewed and photographed.

2. Materials and methods

2.7. Solubility studies and assay of oil–water partition coefficient

2.1. Materials

2.7.1. Solubility studies Solubility determinations of mangiferin of crude mangiferin, phospholipid complex and physical mixture (1:1) were carried out by adding excess of mangiferin material, phospholipid complex and physical mixture (1:1) to 6 mL of water or n-octanol in sealed glass container. Each sample was performed in triplicate. After 48 h of shaking in the shaker

Mangiferin was purchased from Shan' xi Biology Technique Ltd., purity 90%. Lipoid E80 (trade name of phospholipid) was purchased from Shanghai Dongshang Biology Technique Ltd., purity 80%. Reference standard of mangiferin was obtained from the National Institute for the Control of

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at 37 °C, the liquid was centrifuged at 15,000 rpm for 10 min. Then the supernatant was filtered through a 0.22 μm membrane. For solubility in water, 10 μL aliquot of the filtrate was injected into HPLC system directly. For solubility in n-octanol, the filtrate was diluted five times with methanol, and 10 μL aliquot of the resulting solution was injected into HPLC system. The HPLC conditions were the same as that of the mangiferin content determination (see 2.3). 2.7.2. Oil–water coefficient 2 mL of the mangiferin saturated n-octanol (water saturation) was shaken for 48 h, and 2 mL of water (saturated-octanol) was added. The miscible liquid was agitated for 0.5 h, and concentration in each phase was determined by HPLC method after standing for layering [30]. Each sample was carried out in triplicate. 2.8. Dissolution studies The dissolution studies were carried out according to a dissolution test apparatus of China Pharmacopoeia (2010 edition, paddle method) [31].Three kinds of dissolution medium (pH 1.2 HCl, pH 6.8 phosphate buffer saline and water, 900 mL) were continuously stirred at 75 rpm at 37 °C. Crude mangiferin and its phospholipid complex, which is equivalent to 50 mg of mangiferin, were packed into capsules separately, and were added into the stirred dissolution medium at the beginning of the study. To make sure the capsule was immersed totally, the capsule was putted into a mechanical spring. 10 mL of solution was withdrawn and filtered before equal amount of fresh medium was supplemented at the time point of 5, 10, 20, 30, 45, 60 min according to China Pharmacopoeia 2010. All the filtrated sample solutions were determined by HPLC with methods above (see 2.3). 2.9. Rats intestinal absorption studies The animal experiments in this paper were all approved and carried out in accordance with the Guide for Care and Use of Laboratory Animals [32]. Male SD rats weighing 240–280 g were used for all perfusion studies. Prior to each experiment, the rats were fasted overnight (12–18 h) with free access to water. Animals were randomly assigned to experimental groups. The procedure for the in situ single-pass intestinal perfusion followed previously published reports [33,34]. Rats were anesthetized with an intraperitoneal injection of 0.3 mL/100 g of 25% urethane solution. With infrared light to keep the animal warm, the abdomen was opened by a midline incision of 3–4 cm. About 10 cm intestine segment was carefully exposed and cannulated on two ends. After the area was cleaned by normal 37 °C normal saline solution, one end was connected to the peristaltic pump and the other end was connected to a collecting vial. Four intestine segments were examined totally—Duodenum: starting from Pylorus; Jejunum: 15 cm away from pylorus; Ileum: 20 cm above Caecum; Colon: following the Caecum. Each segment is done using 3 rats. Care was taken to avoid disturbance of the circulatory system, and the exposed segment was kept moist with 37 °C normal saline solution. The perfusion buffer

consisted of Krebs–Ringer buffer solution [35] and the 25 μg/mL mangiferin/mangiferin–phospholipid complex equivalent to 25 μg/mL of mangiferin. All perfusion solutions were placed in 37 °C water bath to maintain temperature. At the start of the study, the test perfusion buffer solution was first perfused for 1 h at a flow rate of 0.3 mL/min, in order to ensure steady state conditions. After reaching steady state, the entry and exit vials were replaced by pre-weighted empty vials swiftly, every 15 min interval for 105 min (15, 30, 45, 60, 75, 90 and 105 min). The replaced vials were weighted again and injected to HPLC for concentration determination. Following the termination of the experiment, the length of each perfused intestinal segment was accurately measured. The net water flux in the single-pass rat intestinal perfusion studies, resulting from water absorption in the intestinal segment, was determined by gravimetric method. The measured Cout/Cin ratio was corrected for water transport according to the following equation: 0

C out C out Q out ¼  C 0 in C in Q in

ð1Þ

Where Cout is the concentration of mangiferin in the outlet sample, Cin is the concentration of mangiferin in the inlet sample, Qout and Qin are the volume of exit sample and entry sample (hypothesis: the density of perfusion buffer is 1.0 g/mL at exit and entry). The effective permeability (Peff) through the rat gut wall in the single-pass intestinal perfusion studies and rate constraint of drug absorption (Ka) were determined in the following equation:  Ka ¼

1−

 C out Q out Q C in Q in V

ð2Þ

 0  C out C 0 in 2πrl

−Q ln P eff ¼

ð3Þ

where Q is the perfusion buffer flow rate, C′out/C′in is the ratio of the outlet concentration and the inlet or starting concentration of the tested drug that has been adjusted for water transport via Eq (1), R is the radius of the intestinal segment,

HO

OH HO

OH

O

OH O O O

O

O

OH OH HO

OH OH

Fig. 2. Structure of rutin.

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Table 1 Solubility and oil–water partition coefficient of mangiferin, mangiferin–phospholipid complex and the physical mixture at 37 °C. Samples

Solubility in water (mg/mL)

Solubility in n-octanol (mg/mL)

Oil–water partition coefficient

Mangiferin Physical mixture of mangiferin and phospholipid (1:1) Mangiferin–phospholipid complex

0.162 ± 0.006 0.179 ± 0.007

0.014 ± 0.003 0.013 ± 0.001

0.530 ± 0.031 0.863 ± 0.010

0.214 ± 0.013

0.415 ± 0.020

3.256 ± 0.098

Values are mean ± SD. (n = 3).

and L is the length of the intestinal segment. V is the volume of perfused intestinal segment. 2.10. Rat bioavailability experiments 2.10.1. Chromatography The plasma concentration of mangiferin was determined by Shimadzu HPLC. The stationary phase, SepaxGP-C8 (4.6 mm × 250 mm, 5 μm), was kept at 25 °C. The mobile phase was a mixture of acetonitrile and 0.05% phosphoric acid (18:82). The flow rate was 1.0 mL/min. Effluent was monitored at 258 nm. Two peaks (mangiferin, rutin (Fig. 2)) were detected at 5.5 and 11.8 min. 2.10.2. Plasma sample preparation and validity The rats were anaesthetized with ether, and 300 μL of blood was taken from the eye ground veins at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24 h after oral administration. The plasma was obtained after centrifugation (10 min, 5000 rpm), and was stored at − 20 °C until analyzed. 100 μL of the plasma sample was mixed with 50 μL internal standard (IS) solution (20 μg/mL). Subsequently, 250 μL methanol was added and vortexed for 1 min, followed by centrifugation at 15,000 rpm for 10 min for two times. 20 μL of the supernatant was injected into the HPLC system for analysis. 2.10.3. Pharmacokinetic study of mangiferin and mangiferin–phospholipid complex in rats Twelve male SD rats (body weight 200–220 g) divided randomly into two groups were fasted for 12 h, but free access to water. One group was given mangiferin suspension orally at a dose of 25 mg/kg, while the other group was given mangiferin–

Fig. 3. DSC thermograms exhibited the peak transition onset temperature of mangiferin (a), phospholipid (b), physical mixture of mangiferin and phospholipid (1:1) (c) and mangiferin–phospholipid complex (d).

phospholipid complex suspension orally at equivalent to 25 mg/kg of mangiferin. Both were prepared in normal saline. Pharmacokinetic parameters were calculated using DAS software. The relative bioavailability was the ratio of AUC (0 → t) of mangiferin–phospholipid complex and mangiferin. 3. Results and discussion 3.1. Preparation, content and solubility of mangiferin–phospholipid complex In order to use a small amount of phospholipids, the complex was prepared by mangiferin and Lipoid E80 at a molar ratio of 1:1. From the physical appearance, the mangiferin–phospholipid complex was in semi-solid state. Content of mangiferin in the complex was as high as 35.02% (w/w). The complex had higher solubility in water or n-octanol than crude mangiferin and physical mixtures. Especially in n-octanol, solubility of the complex was about 30 times higher than crude mangiferin and then the oil–water partition coefficient of complex is 6 times higher than mangiferin (Table 1). These results indicated the complex increased the solubility of mangiferin in the lipid phase. Furthermore, the complex may increase the intestinal membrane permeability of mangiferin, which is probably the rate-limiting step for oral absorption of mangiferin. 3.2. Differential Scanning Calorimetry (DSC) Fig. 3 showed the DSC curves of mangiferin, phospholipid, physical mixture and mangiferin–phospholipid complex.

Fig. 4. Infrared radiation (IR) of mangiferin (a), phospholipid (b), physical mixture of mangiferin and phospholipid (1:1) (c) and mangiferin– phospholipid complex (d).

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3.3. Infrared absorption spectroscopy (IR) Fig. 4 showed the IR spectra of mangiferin, phospholipid, physical mixture and mangiferin–phospholipid complex. In the case of mangiferin–phospholipid complex, the specific peak of mangiferin at 3350 cm− 1 (characteristic peak of phenolic hydroxyl group), and the specific peak of phospholipid at 2250 cm− 1 (characteristic peak of azide) disappeared. However, the specific peak of phospholipid around 3000 cm− 1 (stretching vibration of C–H) still existed. These results indicated that phenolic hydroxyl group of mangiferin interacted with quaternary amine nitrogen in the phospholipid to form mangiferin–phospholipid complex, and nonpolar part of phospholipid remained unchanged. 3.4. Scanning Electron Microscopy (SEM) The surface morphology of mangiferin–phospholipid complex as examined by SEM is shown in Fig. 5. With × 500 magnification, crude mangiferin material appeared column shape; mangiferin was uniformly dispersed and parceled by phospholipid in the mangiferin phospholipid complex; the shape of mangiferin clearly appeared in the physical mixture. 3.5. Dissolution studies

Percentage release rate (%)

Fig. 6 showed the dissolution of mangiferin from mangiferin– phospholipid complex and crude mangiferin material in HCl (pH 1.2), water and phosphate buffer saline (pH 6.8) solutions, 100

water

80 60 40 20 0

0

20

40

60

40

60

40

60

Mangiferin had only one sharp peak at 272.16 °C. Phospholipid had two different kinds of endothermal peaks. The first one (161.33 °C) was mild due to the hot movement of phospholipid polar parts, and the second peak (251.25) was relatively sharp which was considered as the transition point from gel state to liquid crystal state. DSC of mangiferin– phospholipid complex showed that the endothermal peaks of mangiferin and phospholipid disappeared, and the mild one of phospholipid decreases to 150.18 °C. But physical mixture of mangiferin and phospholipid still exhibited the endothermal peak at 269.23 °C, nearly the same as the onset temperature of mangiferin. This result showed that mangiferin interacts with the polarity parts of phospholipid molecule, and the carbon– hydrogen chain in phospholipid could rotate freely and then enwrap the polarity parts of mangiferin [36].

pH=6.8

100 80 60 40 20 0 0

20

time Percentage release rate (%)

Fig. 5. Scanning electron microscopic pictures of mangiferin–phospholipid complex (a), mangiferin (b) and physical mixture of mangiferin and phospholipid (c) at × 500 magnification.

Percentage release rate (%)

time

pH=1.2

100 80 60 40 20 0 0

20

time Fig. 6. Dissolution curves of mangiferin–phospholipid complex( ) and crude mangiferin( ) in water, phosphate buffer saline (pH = 6.8) and HCl (pH = 1.2) solutions. (mean ± SD).

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Table 2 The Ka and Peff values of mangiferin and mangiferin–phospholipid complex in four intestinal segments. Mangiferin Ka Duodenum Jejunum Ileum Colon

0.92 0.79 0.46 0.50

Mangiferin–phospholipid complex Peff

± ± ± ±

0.21 0.63 0.15 0.10

1.96 1.74 0.71 0.78

Ka ± ± ± ±

0.39 0.54 0.24 0.01

Improvement

Peff

4.49 3.8 2.7 4.67

± ± ± ±

0.20 1.22 0.49 0.05

22.27 23.06 6.5 25.18

± ± ± ±

1.50 6.19 1.70 0.98

Ka

Peff

4.9 4.8 5.9 9.34

11.4 13.25 9.2 32.28

Values are mean ± SD. (n = 3).

respectively. The dissolution performances of mangiferin from the mangiferin–phospholipid complex in the three dissolution media were slower than crude mangiferin material, because of the viscous state of the complex.

18.7 times higher than that of crude mangiferin at duodenum, jejunum, ileum and colon, respectively. The Peff values of mangiferin–phospholipid complex were 11.4, 13.25, 9.2 and 64.6 times higher than that of crude mangiferin in Duodenum, Jejunum, Ileum and Colon, respectively.

3.6. Rat intestinal absorption studies The membrane permeability of mangiferin for the complex and crude drug was evaluated at four intestinal segments of rats by in situ intestinal perfusion model. In this study, gravimetry method was adopted in a rat single-pass intestinal perfusion model which is more simple and reliable. The volume was calculated by the conversion of mass. Peff and Ka values of mangiferin were improved significantly in four intestine segments to different extent by phospholipid complex (Table 2). The Ka values of mangiferin–phospholipid complex were 4.9, 4.8, 5.9 and

Fig. 7. HPLC chromatograms of blank plasma (a); The blank plasma sample spiked with mangiferin(0.075 μg/mL) and rutin (20.00 μg/mL) (b); Plasma sample 12 h after a p.o. administration of mangiferin (c). Peak I: mangiferin, Peak II: rutin.

3.7. Pharmacokinetics studies in rats Mangiferin in plasma was completely separated from the endogenous components under developed and validated analytical conditions (Fig. 7). Typical equation of the calibration curves was: y = 6 × 10− 4 x + 6.85 × 10− 3, r = 0.99997 (25 ng/mL–5000 ng/mL). The method recoveries of high, middle and low concentration were 87.0%, 88.8%, 84.7%, respectively. The recovery of the IS (Internal Standard) was as high as 94.3%. The accuracy and precision of intra-days or intra-days were satisfactory, and the lower limit of quantification (LLOQ) was 25 ng/mL. Fig. 8 showed the mean plasma concentration–time curve of mangiferin in rats after oral administration of mangiferin–phospholipid complex and crude mangiferin at a single dose equivalent to 25 mg/kg of mangiferin. Evidently, the average Cmax increased from 180.90 to 377.66 μg/mL after oral administration of crude mangiferin and phospholipid complex, respectively. The elimination half-life of mangiferin was increased from 4.49 to 9.31 h− 1 for crude mangiferin and phospholipid complex (Table 3). The complex maintained the effective concentration of mangiferin for a long

Fig. 8. Mean plasma concentration–time curve of mangiferin in rats after oral administration of crude mangiferin and mangiferin–phospholipid complex equivalent to 25 mg/kg of mangiferin (n = 6), respectively. ) represents mangiferin; ( ) represents mangiferin–phospholipid ( complex. Each data point is given as mean ± SD.

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Table 3 The main pharmacokinetic parameters of mangiferin and mangiferin–phospholipid complex in rats (n = 6). Parameters

Unit

Mangiferin

Mangiferin–phospholipid complex

AUC0–24 h AUC0–∞ t1/2z Tmax Cmax

μg/L*h μg/L*h h h μg/L

1039.94 1522.65 4.49 3.00 180.90

2355.63 4304.69 9.31 3.00 377.66

Values are mean ± SD. (n = 3).

period of time in rat blood with a higher relative bioavailability of 230.00%. 4. Conclusions In the present study, we successfully prepared mangiferin– phospholipid complex by a simple solvent-evaporation method. DSC curve and IR spectroscopy of phospholipid complex showed that mangiferin and phospholipid cooperatively interacted to form a new complex. Solubility studies showed there was a higher solubility in water (1.4 times) and n-octanol (about 30 times) for phospholipid complex than crude mangiferin. In comparison with crude mangiferin, the oil– water partition coefficient was improved by 6.2 times and the intestinal permeability in rats was enhanced significantly in four intestine segments to different extents. The average Cmax value of the phospholipid complex was 2.3-fold greater than crude mangiferin. The in vitro dissolution profile is inconsistent with the in vivo pharmacokinetics. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors are grateful to the Analytical Department of Shenyang Pharmaceutical University. Thanks also go to the Animal Center of Shenyang Pharmaceutical University for assistance with the animal studies. References [1] FDA. Draft - guidance for industry: waiver of in vivo bioavailability and bioequivalence studies for immediate release solid oral dosage forms containing certain active moieties/active ingredient based on a biopharmaceutics classification system. US Department of Health, Food and Drug Administration, Center for Drug Evaluation and Research BP2; January 1999. [2] Adamson. Physical chemistry of surfaces, 40. New York: Wiley-Interscience; 1999. [3] Serajuddin ATM. Solid dispersions of poorly water-soluble drugs: early promises, subsequent problems, and recent breakthroughs. J Pharm Sci 1999;88:1058–66. [4] Li J, Xiao H, Li J, Zhong YP. Drug carrier systems based on water-soluble cationic β-cyclodextrin polymers. Int J Pharm 2004;278:329–42. [5] AllesiP Cortesi A, Kikic I, Valli M, Foster NR, MacNaughton SJ, Colombo I. Micronization processes: RESS of progesterone, Atti del III Congresso: I fluid supercriticie lelo roapplicazioni. Trieste 1995:213–20. [6] Le Cluyse EL, Sutton SC. In vitro models for selection of development candidates. Permeability studies to define mechanisms of absorption enhancement. Adv Drug Deliv Rev 1997;23:163–83.

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