A nanostructured liquid crystalline formulation of 20(S)-protopanaxadiol with improved oral absorption

Fitoterapia 84 (2013) 64–71

Contents lists available at SciVerse ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

A nanostructured liquid crystalline formulation of 20(S)-protopanaxadiol with improved oral absorption Xin Jin a, b, c, Zhen-hai Zhang a, Song-lin Li c, E. Sun a, Xiao-bin Tan a, Jie Song a, Xiao-bin Jia a, b,⁎ a b c

Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, PR China College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, PR China Department of Pharmaceutical Analysis and Metabolomics, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, PR China

a r t i c l e

i n f o

Article history: Received 27 August 2012 Accepted in revised form 11 September 2012 Available online 21 September 2012 Keywords: 20(S)-protopanaxadiol Cubosome Caco-2 cell monolayer Rat intestinal perfusion model Bioavailability

a b s t r a c t As with many other anti-cancer agents, 20(S)-protopanaxadiol (PPD) has a low oral absorption. In this study, in order to improve the oral bioavailability of PPD, the cubic nanoparticles that it contains were used to enhance absorption. Therefore, the cubic nanoparticle loaded PPD were prepared through the fragmentation of the glyceryl monoolein (GMO)/poloxamer 407 bulk cubic gel and were verified by transmission electron microscope, small angle X-ray scattering and differential scanning calorimetry. The in vitro release of 20(S)-protopanaxadiol from these nanoparticles was less than 5% at 12 h. And then Caco-2 cell monolayer model was used to evaluate the absorption of PPD in vitro. Meanwhile the rat intestinal perfusion model and bioavailability were also estimated in vivo. The results showed that, in the Caco-2 cell model, the PPD-cubosome could increase the permeability values from the apical (AP) to the basolateral (BL) of PPD at 53%. The result showed that the four-site rat intestinal perfusion model was consistent with the Caco-2 cell model. And the result of a pharmacokinetic study in rats showed that the relative bioavailability of the PPD-cubosome (AUC0–∞) compared with the raw PPD (AUC0–∞) was 169%. All the results showed that the PPD-cubosome enhanced bioavailability was likely due to the increased absorption by the cubic nanoparticles rather than by the improved release. Hence, the cubic nanoparticles may be a promising oral carrier for the drugs that have a poor oral absorption. © 2012 Elsevier B.V. All rights reserved.

1. Introduction 20(S)-protopanaxadiol (PPD, Fig. 1) is the aglycone of PPD-containing ginsenosides such as ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Rg3 and Rh2. PPD is the metabolite of them by losing the attached sugar moieties in the gastrointestinal tract through a series of deglycosylation steps [1–3]. Moreover, PPD exhibits powerful pleiotropic anti-cancer effects in several cancer cell lines and possessed the capability of inhibiting metastasis [4–7]. Further investigations discover that PPD is the most

⁎ Corresponding author at: Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, PR China. Tel.: +86 25 856378091; fax: +86 25 85637809. E-mail address: [email protected] (X. Jia). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2012.09.013

potent anticancer activity of ginsenosides [8–11]. As such it is currently undergoing phase I clinical trials in China. However, PPD is a biopharmaceutics classification system (BCS) IV drug of poor water-solubility and poor permeability. And the pharmacokinetics of PPD shows that its low oral bioavailability is poor [12]. Unfortunately, there are few carriers used to increase the oral bioavailability of PPD [12]. Therefore, the new carrier is required to improve dissolution and permeation simultaneously to improve the oral bioavailability of PPD. Colloidal dispersions of bicontinuous cubic phases formed in the monoolein/poloxamer/water system have been proposed as potential drug carriers [13,14], as they (i) are bioadhesive, (ii) present a permeation enhancer as the structure forming lipid (glyceryl monooleate), and (iii) afford the ability to incorporate compounds independently of their solubility, and

X. Jin et al. / Fitoterapia 84 (2013) 64–71

OH

65

HO

HO

Fig. 1. The structure of PPD.

sustain their delivery [15–17]. The emulsification of the cubic lipid phases in water results in the production of cubosome, which can be defined as the nanoparticulate dispersion systems. It has been demonstrated that the dispersed particles retain the internal structure of the bulk phase and its properties. In comparison with the bulk gel, the dispersions present some advantages, such as a larger surface area and high fluidity (low viscosity) [18,19]. In this study, PPD was loaded in a new delivery system-selfassembled nanostructure of liquid crystalline particle to increase the absorption.

2. Materials and methods

2.3. Preparation of GMO-based dispersions by fragmentation Cubic nanoparticle was prepared through the fragmentation of GMO/poloxamer 407 bulk cubic gel. GMO and poloxamer 407 (9:1) were melted at 60 °C in a hot water bath, after which PPD was added and stirred continuously to dissolve. Milli-Q deionized water was added gradually and vortex mixed to achieve a homogenous state. After the equilibration for seven days at room temperature, an optically isotropic cubic phase gel was formed. After the addition of deionized water, the cubic gel was disrupted by mechanical stirring. This dispersion was subsequently homogenized by passing five cycles through a high-pressure homogenizer (Avestin Em-C3, Ottawa, Canada) at 670 bar. The final dispersions of cubic nanoparticle were stored at room temperature until required.

2.1. Instruments and materials 2.4. Physicochemical characterization of cubosome 20(S)-PPD (99.8%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China). Glyceryl monoolein (GMO, RYLOTMMG19) was kindly gifted by Danisco Ingredients (Brabrand, Denmark). Poloxamer 407 (Lutrol® F127) was obtained from BASF (Ludwigshafen, Germany). Cloned Caco-2 TC7 cells were a kind gift from Dr Ming Hu of INSERM U178 (Houston, America). Hanks' balanced salt solution (HBSS; powder form) was purchased from Sigma-Aldrich (St. Louis, MO). Milli-Q water (Millipore, Bedford, MA) was used throughout the study. Acetonitrile and methanol were of chromatographic grade (Merck Company Inc., UN). All other reagents were of analytical grade.

2.2. Animal experiments Male Sprague–Dawley rats weighing between 200 and 250 g were obtained from the SLEK Lab Animal Center of Shanghai (Shanghai, China). Animals were housed under standard conditions of temperature, humidity, and light. Food and water were provided ad libitum. The rats were fasted overnight before the day of the experiment. All animal care measures and experimental procedures were conducted according to the Guiding Principles in the Use of Animals in Toxicology, as adopted in 1989, revised in 1999 and amended in 2008 by the Society of Toxicology (SOT, 2008).

2.4.1. Particle size distribution, zeta potential and encapsulation efficiency Particle size distribution (Z-average), polydispersity (PDI) and zeta potential of the dispersions were determined using photon correlation spectroscopy (PCS) (Malvern Zetasizer 3000, Malvern, UK). Measurements were performed at 25 °C, and the results presented are the mean of three successive measurements of at least three independent samples. Samples were diluted with water to adjust the signal level. The morphological evaluation was performed by transmission electron microscopy (TEM, JEM-1200EX, Japan). The drug encapsulation efficiency was determined by ultrafiltration. A 1 ml aliquot of cubosome was transferred to microcolumn coupled with an ultrafilter, which was then centrifuged at 4000 rpm for 30 min. The amount of drug loaded in the cubosome was calculated as the difference between the total amount used in preparation of the cubosome and the amount in the filtrate, as determined by UPLC. The drug encapsulation efficiency was calculated according to: E:E: ¼ ½ðWtotal –Wfree Þ=Wtotal   100%≈½ðCtotal Vtotal –Cfree Vfree Þ=Ctotal Vtotal   100% E.E. is the drug encapsulation efficiency; Wtotal is the total amount of the drug in the cubosome; Wfree is the amount of the drug in the filtrate; Ctotal is the concentration of the drug in the

66

X. Jin et al. / Fitoterapia 84 (2013) 64–71

cubosome; Cfree is the concentration of the drug in the filtrate; Vtotal is the volume of the drug in the cubosome; and Vfree is the volume of the drug in the filtrate. 2.4.2. SAXS sample preparation and analysis The SAXS measurements were carried out using NanoStar (Bruker AXS GmbH, Germany), which consisted of a ‘Hi-Star’ 2D-detector, a 3 collimator SAXS system, a 3 kW high voltage generator, cross-coupled Goebel mirrors and copper X-ray radiation at 5000 W. In Cu Kα radiation, the wavelength is 1.54 Å (λ = 0.154 nm), and the scattering angle (2θ) ranged from 0.2° to 2.8°. The measurement was carried out in a vacuum at 25 ± 0.1 °C. The samples were equilibrated at 25 °C for 30 min prior to the collection of the scattering patterns for 60 min. The diffraction patterns obtained were converted to plots of intensity versus q-value, which enabled the identifications of the peak positions, and their conversion to Miller Indices. The one-dimensional scattering function I(q), where q is the length of the scattering vector, defined by q = (4π / λ) sinθ, λ being the wavelength and 2θ the scattering angle. The peak positions in I(q) versus q plots were indexed to Miller indices against known space groups to identify the phase structure. The mean lattice parameter, a, was calculated from the interplanar distance, d using the appropriate scattering law for the phase structure. For cubic phases, a = d(h2 + k2 + l2) 1/2, where h, k and l are the Miller indices for the particular structure present. The lattice constant of the respective cubic phase was calculated from the spacing d of the first reflection observed by a = d √ 2 and given by d = 2π / q. 2.4.3. Differential scanning calorimetry (DSC) Samples were sealed in aluminum crimp cells and heated at a rate of 10 °C/min from 30 °C to 500 °C in a nitrogen atmosphere (DSC-60, Shimadzu, Japan). The peak transition maximum temperatures were compared using a Thermal Analyzer (TA-60WS, Shimadzu, Japan). 2.5. Release of PPD from cubic nanoparticles A preliminary study on the PPD release from the cubic nanoparticles of the same formulation for the further pharmacokinetic study (Table 1) was carried out using the dynamic dialysis method in a ZRS-8G release tester (Tianjin, China). Two milliliters PPD-loaded cubic nanoparticles (4 mg PPD equivalent) were sealed in dialysis bags (14,000 MWCO, Millipore, Boston, MA, USA) and immersed in 100 ml aqueous medium containing 0.2% (w/v) sodium lauryl sulfate (SDS) thermostatically maintained at 37±0.5 °C and stirred at a revolution speed of 100 rpm. At time intervals of 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10, and 12 h, 0.2 ml of medium was withdrawn and filtered through a 0.45 μm nylon film. The PPD content was then determined by UPLC as described above. Since the solubility of PPD in 0.2% SDS Table 1 Physico-chemical properties of PPD cubosome (n = 3). Preparations

PPD-cubosome a

Average size

Zeta potential

Polydispersity index

E.E.a

(nm)

(mV)

(PDI)

(%)

101.3 ± 9.2

−15.7 ± 1.3

0.158 ± 0.013

93.7 ± 2.6

E.E. the encapsulation efficiency of PPD.

solution was about 0.921 mg/ml, the sink conditions were maintained throughout the release test. A nearly complete release within 1 h was observed for the saturated PPD solution indicating a negligible hindering effect of the dialysis bag on the PPD release. 2.6. Transport experiments in the Caco-2 cell culture model The Caco-2 TC7 cell line was cultured in the laboratories of Dr Ming Hu of INSERM U178 (Houston, America) similar to the wild-type Caco-2 cells and more stable during the passage since it is a cloned cell line [20]. The culture conditions for growing Caco-2 cells were as a reference [21–23]. Briefly, after the aspirating culture medium, the cell monolayer was washed three times with 37 °C blank HBSS (pH 7.4). The transepithelial electrical resistance (TEER) values of the cell monolayer then measured to be more than 350 Ω × cm2. The monolayer was incubated with the pH 7.4 blank HBSS for 1 h after which the incubation medium was aspirated. A solution containing the compound was then loaded on to the apical or basolateral side. The amounts of the transported compound were measured as a function of time by UPLC. Donor samples (400 μl) and receiver samples (400 μl) were taken at different times in triplicate, followed by the addition of 400 μl drug donor solution to the donor side or 400 μl of the blank buffer to the receiver side. When comparing the permeability of PPD and its preparations, each compound was used at the same concentration (30 μM) and the samples were taken at 0, 1, 2, 3, and 4 h after the incubation. To each transport sample (400 μl), Rh2 (60 μM) was added as an internal standard and a preservative, and then the resulting mixture vortexes for 30 s. The liquid was analyzed by UPLC after filtrating. At the end of the transport experiment, the integrity of the monolayer was monitored by the TEER value to ensure there was no significant change. 2.7. The four-site rat intestinal perfusion experiment A single-pass perfusion method was used. Four segments of the intestine (duodenum, upper jejunum, terminal ileum, and colon) were perfused simultaneously with a perfusate containing 30 μM PPD or PPD-loaded cubic nanoparticles. To keep the temperature of the perfusate constant, the inlet cannulae was insulated and kept warm by a 37 °C circulating water bath. A flow rate of 0.20 mL/min was used, and the perfusate samples were collected every 30 min. The outlet concentrations of PPD in the perfusate were determined by UPLC. 2.8. Oral bioavailability studies Blood was taken from the eye ground veins of the rats through the cannulated tube at times 0, 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0 and 12.0 after PPD and its preparations by oral administration (PPD 2 mg/kg), respectively. The plasma obtained after centrifugation (5 min, 3000 rpm) was stored at − 20 °C before it was analyzed. In 2.0 ml polyethylene centrifuge tube, an aliquot of 100 μl plasma sample was added and spiked with a 10 μl IS solution. After the vortex-mixing for 30 s, 1.0 ml of ethyl acetate was added into each tube and the mixture was vigorously vortexed for 3 min. The organic and aqueous phases were separated by centrifugation at 13,000 rpm

X. Jin et al. / Fitoterapia 84 (2013) 64–71

for 15 min. One ml upper organic phase was transferred to another tube and evaporated to dryness at 25 °C using a Thermo Savant SPD 2010 Speed Vac System (Thermo Electron Corporation, USA). The residue was dissolved in 100 μl of the methanol, and vortex mixed for 1 min. After centrifugation at 15,000 rpm for 15 min, 20 μl supernatant was injected into the HPLC–MS system for analysis. 2.9. Chromatographic conditions and mass spectrometric conditions The UPLC method was used to determine the concentration of PPD in the transport samples. The conditions for UPLC analysis of PPD of the transport samples were as follows: system, Waters Acquity UPLC with a photodiode array detector and Empower software; and column, Agilent Poroshell 120 EC-C18 column (100 mm×3.0 mm, 2.7 μm) (Agilent, Milford, MA, USA). The mobile phase consisted of (A) acetonitrile and (B) aqueous solution. The UPLC elution condition was optimized as follows: 0–5 min (62%A–65%A). Column temperature was maintained at 30 °C. The injection volume of the sample was 8 μl. The flow rate was set at 0.4 ml/min. The detection wavelength was set at 203 nm. The retention times for the internal standard and PPD were 1.91, 3.80 min, respectively. Plasma separation was performed using the Agilent Zorbax SB-C18 column (150 mm× 2.1 mm i.d., 5 μm) at 25 °C with a mobile phase of methanol/water (95:5, v/v) at a flow rate of 0.4 ml/min. In APCI source, the probe was set at 4.0 kV and the temperature of the probe was kept at 350 °C. Nitrogen served as nebulizer gas (flow rate: 10 l/min). Mass spectra were obtained at a dwell time of 0.2 and 1 s in SIM and scan modes, respectively. Masses were scanned from m/z 300 to 500, where the major fragment ions were detected. In the SIM mode positive ions of PPD and Rh2 were monitored at m/z 425 and 318, respectively. 2.10. Data analysis The rate of transport is usually obtained from amounts transported versus the time curve using linear regression. The permeability of PPD was thus calculated using the following equation [24]:

P app ¼

V dC 1 dM   S  C dt S  C dt

ð1Þ

where V is the volume of the receiver (2.5 ml), S is the surface area of the cell monolayer (4.2 cm 2), C is the initial concentration, hence dC is the rate of concentration change in dt the receiver side, and dM the rate of the drug transport. The dt rate of the drug transport was obtained by linear regression analysis (a Microsoft Excel function). The Peff of PPD was calculated using the following equation in the four-site rat intestinal perfusion model [25]:

P eff ¼

′

Q lnðCin =Cout Þ 2πrL

ð2Þ

where Q is the flow rate in ml/min, r the radius of the intestine, L is the length of the perfused intestinal segment (cm), and (Cin/Cout)’ is the concentration ratio corrected for the fluid flux.

67

2.11. Statistical analysis One-Way ANOVA with Tamhane's post hoc analysis was used to analyze the data for multiple comparisons when the variance was shown to be unequal because this method is less likely to generate type II errors. One-Way ANOVA with Tukey's posthoc analysis method was not used here to avoid type II errors. Unpaired Student's t test (Microsoft Excel) was used to analyze the data when there were only two groups in the experiments. The prior level of significance was set at 5%, or p b 0.05. 3. Result and discussion 3.1. Characterization of cubosome formulations 3.1.1. Particle size, zeta potential, encapsulation efficiency and transmission electron microscopy As can be seen in Table 1, the particle diameters for GMO PPD-cubosome were 101.3 ± 9.2 nm (polydispersity index of b0.2), and zeta potential (mV) − 15.7 ± 1.3. Furthermore, because of the lipophilicity of GMO, the encapsulation efficiency (E.E. %) of PPD in the cubosome was above 90%, which meant that most of the drugs were encapsulated in the cubic nanoparticles. The ideal particle size properties for GMO cubosome were also obtained; the transmission electron microscope (TEM) morphology was that of the monodisperse cubic particles (Fig. 2). 3.1.2. Phase boundary determination using small angle x-ray scattering (SAXS) SAXR was performed to determine the structural formulation of the cubosome. Several Bragg peaks showed the plot of intensity versus q (A−1) obtained from the SAXS diffraction patterns. It showed the data obtained from the bulk cubic phase of the PPD-cubosome. The diffraction peaks were observed with relative positions at ratios of reflections which were d1:d2:d3:d4: d5:d6 (√2:√3:√4:√6:√8:√9), which can be indexed to the Miller indices, in accordance with the Pn3m space group, indicating a diamond cubic phase [26]. 3.1.3. Differential scanning calorimetry (DSC) Differential scanning calorimetry was a rapid and reliable way to screen PPD, GMO, F127, their mixture and the preparation of the PPD-cubosome compatibility. As shown in Fig. 3, the DSC thermograms of PPD (a), GMO and F 127 (b) physical mixture (c) and the PPD-cubosome (d). It could be inferred that in the cubosome, the endothermic peaks of PPD that disappeared would indicate that the cubosome was formed. 3.2. In vitro release As a result (Fig. 4), the cumulative percent release of free PPD is about 70% at 12 h. However, the overall release of PPD from cubic nanoparticles was less than 5% at 12 h. It may be because the high affinity of PPD with the hydrophobic domain in the cubic phase made it difficult to be released from the nanoparticles. Since the overall release of PPD from cubic nanoparticles was negligible, we did not take out an

68

X. Jin et al. / Fitoterapia 84 (2013) 64–71

Fig. 2. Photographs of the PPD‐cubosome by TEM.

model of human intestinal absorption. It has been previously used in several oral absorption studies [27,28]. The PPD-cubosome significantly (p b 0.05) increased the absorptive direction (apical to the basolateral side) transport of PPD; absorptive permeability of (1.15±0.31)×10−6 cm/s compared to control (1.76±0.55)×10−6 cm/s (53% increase). However, the secretory direction (basolateral to the apical side) transport of PPD had no significant effect (Table 2). Comparing with the permeability of PPD, forming the PPD-cubosome could

extensive study on the release of other formulations in this study.

3.3. Transport of PPD and its cubosome across the Caco-2 cell monolayer The Caco-2 cell culture model which is routinely used to investigate drug absorption is recognized by FDA as a viable

a b c

d 50

100

150

200

250

300

350

400

450

500

Temperatuer/oC Fig. 3. The DSC thermograms of PPD (a), GMO and F 127 (b), physical mixture (c), and the PPD-cubosome (d).

90

69

3.5

80

PPD

70

PPD-cubosome

PPD PPD-cubosome

3 2.5

60 50

2

40

Peff

cumulative percent released (%)

X. Jin et al. / Fitoterapia 84 (2013) 64–71

30

1.5

20

1

10 0.5

0 0

2

4

6

8

10

12

Time (h)

0 duodenum

jejunum

ileum

colon

Fig. 4. Release profiles of PPD and the PPD-cubosome (n = 3).

promote the permeability. This could be mainly due to the characteristics of the cubosome, which appeared to be bioadhesive and is a permeation enhancer as the structure forming lipid leading to increased membrane permeability. 3.4. The influence of the PPD-cubosome in the four-site rat intestinal perfusion model The Peff obtained for PPD in the four-site rat intestinal perfusion were presented in Fig. 5. With the PPD-cubosome, the Peff of PPD were 2.49 ± 0.23, 1.98 ± 0.21, 1.75 ± 0.17 and 1.24 ± 0.21 in the duodenum, jejunum, ileum and colon, respectively. In its cubosomes, the Peff of PPD was increased significantly in the four-site rat intestine (p b 0.05). The four-site rat intestinal perfusion model measures the disappearance of the drug from the perfused intestinal segment. PPD could be absorbed by enterocytes directly, and the amounts that were absorbed in the duodenum were much higher than in the jejunum, ileum and colon (pb 0.05). The cubosome carrier had an effect on the PPD permeability obtained in this study, and especially had a higher effect in the duodenum, jejunum and ileum in comparison to the colon. The result showed that the four-site rat intestinal perfusion model was consistent with the Caco-2 cell model.

Fig. 5. Comparison of the permeability of PPD in different intestinal segments. Data presented as mean ± S.D.; n = 4; *p b 0.05 versus absence of the PPD group.

analysis is shown in Table 3, the average value of Cmax is 73.45 ng/ml after the oral administration of PPD with a Tmax of about 85 min. However, the average value of Cmax was 100.14 ng/ml after the oral administration of the PPD-cubosome with a Tmax of about 125 min. There was a significant difference in the time taken to reach the peak concentration (Tmax) of PPD between the control group (85 min) and the PPD-cubosome (125 min). But any effect on the t1/2 was not significantly observed. It could indicate that forming the PPD-cubosome could increase the absorption and has a delayed release rate [29,30]. The average value of AUC0–∞ of PPD and the PPD-cubosome in rats were 28.01 and 47.44 mg/l∗ min, respectively. The PPD-cubosome was significantly higher than the control. And the AUC0–∞ of the PPD-cubosome was 1.69 multiples than those of PPD. SAXR and TEM are usually used to characterize the internal structure of the dispersed cubic particles [31]. The TEM images in this study showed a typical ordered cubic texture which was further confirmed by SAXR. Guest molecules generally influences the self-assembly structure properties. And DSC may also prove that most of the drug was encapsulated in the cubic nanoparticle.

3.5. Pharmacokinetics of PPD and its cubosome after oral administration

Table 2 Permeabilities (Papp) and efflux ratios. Absorptive permeability was expressed as A–B, whereas secretory permeability was expressed as B–A. Data expressed as mean±SD (n=3). Efflux ratio was Papp (B–A)/Papp (A–B). Compound

Papp × 10−6(cm/s) A–B

PPD PPD-cubosome

1.15 ± 0.31 1.76 ± 0.55⁎

Data presented as mean ± S.D.; n = 3. ⁎ p b 0.05 vs. PPD group.

Efflux ratio

PPD

120

cubosomes

100 80 60 40 20 0 0

B–A 2.45 ± 0.46 2.44 ± 0.33

Concentration (ng/ml)

140

Fig. 6 showed the sample equivalent to 2 mg/kg of PPD and the PPD-cubosome were orally administered to rats (n= 6), respectively. From the above profiles, a summary of statistical

2.13 1.38⁎

200

400

600

800

1000

1200

1400

1600

time (min) Fig. 6. The plasma concentration–time curve of PPD in rats after the oral administration of PPD and the PPD-cubosome (2 mg/kg, PPD). Data are presented as mean± S.D. (n = 6).

70

X. Jin et al. / Fitoterapia 84 (2013) 64–71

Table 3 Pharmacokinetic parameters of PPD and PPD-cubosome (2 mg/kg, p.o.) in rats (n = 6). Parameters

PPD

PPD-cubosome

AUC0–t (mg/l ∗ min) AUC0–∞ (mg/l ∗ min) MRT0–t (min) MRT0–∞ (min) CLz (l/min/kg) t1/2 (min) Tmax (min) Cmax (ng/ml)

25.76 ± 4.38 28.01 ± 4.02 361.18 ± 51.43 489.76 ± 63.44 0.071 ± 0.012 324.01 ± 33.49 85 ± 20.61 73.45 ± 13.56

43.37 ± 6.39⁎ 47.44 ± 7.21⁎ 396.18 ± 43.45⁎ 531.79 ± 60.28⁎ 0.068 ± 0.021 372.59 ± 49.41 125 ± 29.49⁎ 100.14 ± 19.40⁎

Abbreviations: AUC, area under concentration–time curve; MRT, mean residence time; CLz, mean the clearance; Tmax, time to maximum plasma concentration; Cmax, maximum plasma concentration. Data represent the mean ± SD. ⁎ p b 0.05, statistical significance: versus 2 mg/kg PPD.

In its normal state, the intestinal membrane is relatively permeable to the particles in the nanometer size range. The carrier in the nanometer size range can be absorbed by fluid-phase pinocytosis which also results in the invagination of the membrane and vesicle formation. Both processes are energy dependent and saturable. And the extent and rate of particle absorption are mainly dictated by the size and surface properties of the carrier. The increase in the relative bioavailability of the PPDcubosome compared with PPD might be determined by two major factors: On one hand, the in vitro Caco-2 cell monolayer model study showed that the apical to basal permeability of PPD formulated in the cubosomes exhibited a 1.53-fold increase relative to the raw PPD. Some researchers found that the lipid bilayer of the cubosomes was very similar to the microstructure of the cell membrane [32,33]. It is reasonable to suppose that the cubic nanoparticle-associated drugs can be transported across the epithelial membrane enhancing drug absorption. Whether or not the cubic nanoparticles can be transported intact across the cell membranes has yet to be determined however, they are known to possess lyotropic properties. This property makes it easier for the nanoparticles to penetrate the “unstirred water layer” [34–36] and make a closer contact with the cell membranes. Meanwhile the cubic nanoparticles are isotropic and fragmentation results in the formation of smaller cubic nanoparticles that retain the properties of their “parental” nanoparticles. Here, the secondary cubic nanoparticles or derivative vehicles like micelles may facilitate further contact with the cell membrane and facilitate absorption.

4. Conclusion In this article aimed to improve the oral bioavailability of PPD, the cubic nanoparticle was used to enhance absorption. In the Caco-2 cell model, the cubosome could increase the permeability values from the apical to the basolateral of PPD. The result showed that the four-site rat intestinal perfusion model was consistent with the Caco-2 cell model. And the result of a pharmacokinetic study in the rats showed that the relative bioavailability of the PPD-cubosome (AUC0–∞) compared with the raw PPD (AUC0–∞) was 169%. The novel carrier has increased

the absorption designed to be suitable to the low absorption compounds. Acknowledgments This study was financially supported by the National Natural Science Foundation of China (No. 81274068), the Administration of Traditional Chinese Medicine of Jiangsu Province (LZ11066) and the open fund from the Key Laboratory of New Drug Delivery System of Chinese Materia Medica in Jiangsu Province Academy of Traditional Chinese Medicine (No. 2011NDDCM02003), as well as the Foundation for High-level Talent on six areas of the Jiangsu province, the Suzhou City Science and Technology Development Plan in 2012 (ZXY2012009) and the Key Laboratory of Traditional Chinese Medicine Oral Preparation of Chinese Traditional Medicine Release System (2011NDDCM01001). References [1] Bae EA, Han MJ, Kim EJ, Kim DH. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch Pharm Res 2004;27:61-7. [2] Tawab MA, Bahr U, Karas M, Wurglics M, Schubert-Zsilavecz M. Degradation of ginsenosides in humans after oral administration. Drug Metab Dispos 2003;31:1065-71. [3] Xie HT, Wang GJ, Sun JG, Tucker I, Zhao XC, Xie YY, et al. High performance liquid chromatographic-mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solid-phase extraction for pharmacokinetic studies. J Chromatogr B Analyt Technol Biomed Life Sci 2005;818:167-73. [4] Dong H, Bai LP, Wai WVK, Zhou H, Wang JR, Liu Y, et al. The in vitro structure-related anti-cancer activity of ginsenosides and their derivatives. Molecules 2011;16:10619-30. [5] Li G, Wang Z, Sun Y, Liu K, Wang Z. Ginsenoside 20(S)-protopanaxadiol inhibits the proliferation and invasion of human fibrosarcoma HT1080 cells. Basic Clin Pharmacol Toxicol 2006;98:588-92. [6] Liu GY, Bu X, Yan H, Jia WW. 20S-protopanaxadiol-induced programmed cell death in glioma cells through caspase-dependent and -independent pathways. J Nat Prod 2007;70:259-64. [7] Zhu GY, Li YW, Tse AKW, Hau DKP, Leung CH, Yu ZL. 20(S)-protopanaxadiol, a metabolite of ginsenosides, induced cell apoptosis through endoplasmic reticulum stress in human hepatocarcinoma HepG2 cells. Eur J Pharmacol 2011;668:88-98. [8] Popovich DG, Kitts DD. Structure–function relationship exists for ginsenosides in reducing cell proliferation and inducing apoptosis in the human leukemia (THP-1) cell line. Arch Biochem Biophys 2002;406:1-8. [9] Usami Y, Liu YN, Lin AS, Shibano M, Akiyama T, Itokawa H. Antitumor agents. 261. 20(S)-protopanaxadiol and 20(S)-protopanaxatriol as antiangiogenic agents and total assignment of 1H NMR spectra. J Nat Prod 2008;71:478-81. [10] Wang W, Zhao YQ, Rayburn ER, Hill DL, Wang H, Zhang RW. In vitro anti-cancer activity and structure–activity relationships of natural products isolated from fruits of Panax ginseng. Cancer Chemother Pharmacol 2007;59:589-601. [11] Yu Y, Zhou O, Hang Y, Bu XX, Jia W. Antiestrogenic effect of 20Sprotopanax-adiol and its synergy with tamoxifen on breast cancer cells. Cancer 2007;109:2374-82. [12] Han MH, Chen J, Chen SL, Wang X. Development of a UPLC-ESI-MS/MS assay for 20(S)-Protopanaxadiol and pharmacokinetic application of its two formulations in rats. Anal Sci 2010;26:749-53. [13] De Waard H, De Beer T, Hinrichs WL, Vervaet C, Remon JP, Frijlink HW. Controlled crystallization of the lipophilic drug fenofibrate during freeze-drying: elucidation of the mechanism by in-line Raman spectroscopy. APPS J 2010;12:569-75. [14] Lai J, Lu Y, Yin ZN, Hu FQ, Wu W. Pharmacokinetics and enhanced oral bioavailability in beagle dogs of cyclosporine A encapsulated in glyceryl monooleate/poloxamer 407 cubic nanoparticles. Int J Nanomedicine 2010;5:13-23. [15] Gopal GR, Shailendra SR, Swarnlata SR. Cubosomes: an overview. Biol Pharm Bull 2007;30:350-3. [16] Guo CY, Wang J, Cao FL, Lee RJ, Zhai GX. Lyotropic liquid crystal systems in drug delivery. Drug Discov Today 2010;15:23-4.

X. Jin et al. / Fitoterapia 84 (2013) 64–71 [17] Zeng N, Gao X, Hu Q, Song Q, Xia H, Liu Z, et al. Lipid-based liquid crystalline nanoparticles as oral drug delivery vehicles for poorly water-soluble drugs: cellular interaction and in vivo absorption. Int J Nanomed 2012;7:3703-18. [18] Siekmann B, Bunjes H, Koch MH, Westesen K. Preparation and structural investigations of colloidal dispersions prepared from cubic monoglyceride-water phases. Int J Pharm 2002;244:33-43. [19] Esposito E, Eblovi N, Rasi S, Drechsler M, Di Gregorio GM, Menegatti E, et al. Lipid-based supramolecular systems for topical application: a preformulatory study. AAPS PharmSci 2003;18:E30. [20] Caro I, Boulenc X, Rousset M, Meunier V, Bourrié M, Julian B, et al. Characterisation of a newly isolated Caco-2 clone (TC-7), as a model of transport processes and biotransformation of drugs. Int J Pharm 1995;166:147-58. [21] Zumdick S, Deters A, Hensel A. In vitro intestinal transport of oligomeric procyanidins (DP 2 to 4) across monolayers of Caco-2 cells. Fitoterapia 2012;7:1210-7. [22] Gu LL, Li N, Li QR, Zhang Q, Wang CY, Zhu WM, et al. The effect of berberine in vitro on tight junctions in human Caco-2 intestinal epithelial cells. Fitoterapia 2009;4:241-8. [23] Hansawasdi C, Kawabata J. α-Glucosidase inhibitory effect of mulberry (Morus alba) leaves on Caco-2. Fitoterapia 2006;8:568-73. [24] Jin X, Zhang ZH, Sun E, Tan XB, Zhu FX, Li SL, et al. Preparation of icariside II-phospholipid complex and its absorption across Caco-2 cell monolayers. Pharmazie 2012;67:293-8. [25] Cook TJ, Shenoy SS. Intestinal permeability of chlorpyrifos using the single-pass intestinal perfusion method in the rat. Toxicology 2003;184: 125-33. [26] Li G, Shun H, Shen JQ, Zhu JB, Zhu CL, Zhang XX, et al. Self-assembled liquid crystalline nanoparticles as a novel ophthalmic delivery system for dexamethasone: improving preocular retention and ocular bioavailability. Int J Pharm 2010;396:179-87.

71

[27] Coyuco JC, Liu YJ, Tan BJ, Chiu NC. Functionalized carbon nanomaterials: exploring the interactions with Caco-2 cells for potential oral drug delivery. Int J Nanomedicine 2011;6:2253-63. [28] Wahlang B, Pawar YB, Bansal AK. Identification of permeability-related hurdles in oral delivery of curcumin using the Caco-2 cell model. Eur J Pharm Biopharm 2011;77:275-82. [29] Nguyen TH, Hanley T, Porter CJ, Boyd BJ. Nanostructured liquid crystalline particles provide long duration sustained-release effect for a poorly water soluble drug after oral administration. J Control Release 2011;153:180-6. [30] Boyd BJ, Khoo SM, Whittaker DV, Davey G, Porter CJ. A lipid-based liquid crystalline matrix that provides sustained release and enhanced oral bioavailability for a model poorly water soluble drug in rats. Int J Pharm 2007;340:52-60. [31] Gustafsson J, Ljusberg-Wahren H, Almgren M, Larsson K. Submicron particles of reversed lipid phases in water stabilized by a nonionic amphiphilic polymer. Langmuir 1997;13:6964-71. [32] Lenhardt T, Vergnault G, Grenier P, Scherer D, Langguth P. Evaluation of nanosuspensions for absorption enhancement of poorly soluble drugs: in vitro transport studies across intestinal epithelial monolayers. APPS J 2008;10:435-8. [33] Thomson AB, Schoeller C, Keelan M, Smith L, Clandinin MT. Lipid absorption: passing through the unstirred layers, brush-border membrane, and beyond. Can J Physiol Pharmacol 1993;71:531-55. [34] Katneni K, Charman SA, Porter CJ. An evaluation of the relative roles of the unstirred water layer and receptor sink in limiting the in-vitro intestinal permeability of drug compounds of varying lipophilicity. J Pharm Pharmacol 2008;60:1311-9. [35] Korjamo T, Heikkinen AT, Mönkkönen J. Analysis of unstirred water layer in in vitro permeability experiments. J Pharm Sci 2009;98:4469-79. [36] Kesisoglou Filippos, Mitra Amitava. Crystalline nanosuspensions as potential toxicology and clinical oral formulations for BCS II/IV compounds. APPS J 2012, http://dx.doi.org/10.1208/s12248-012-9383-0.