In Vitro And In Vivo Investigation Of Cyclosporine A Solid Dispersion And The Mechanistic Studies For Enhancement Of Bioavailability Using Polyoxyethylene (40) Stearate

Cyclosporine A (CyA) is a cyclic undecapeptide obtained from Tolypocladium inflatum, Fusoriumsoloni or Cylindrocapon lucidum and widely used for the inhibition of graft rejection in various organ transplantations. However, the absorption of CyA from the gastrointestinal tract is incomplete and variable. Several factors including the relatively high molecular weight, poor solubility in water (7.3μg/mL at 37℃), extensive metabolism by cytochrome P-450 3A4 in both liver and gut and effect of P-glycoprotein (P-gp)-mediated drug efflux have been suggested as the reasons for the low and variable bioavailability of CyA. According to the biopharmaceutical classification system, CyA is an example of a class II compound meaning that its oral bioavailability is determined by dissolution rate in the gastrointestinal fluid. Therefore, solid dispersion technology was utilized in this study to improve the dissolution of CyA and the possible mechanism for polyoxyethylene (40) stearate to increase the oral bioavailability was investigted.Solid dispersions containing different carriers as polyoxyethylene (40) stearate(PS), Poloxamer 407(F127), Poloxamer 188(F188) and CyA were prepared by solvent-melt method or solvent method for PVP. The solid dispersions were characterized by powder X-ray diffraction(PXRD), differential scanning calorimetry(DSC), hot-stage microscopy(HSM), infrared Fourier transform spectroscopy(FTIR) and dissolution studies. The crystalline peaks of CyA disappeared in the spectra of all the solid dispersions but showed in that of physical mixtures by using PXRD, demonstrating the amorphous state of drug in solid dispersions. The results of HSM supported the above mentioned conclusion. The solubility of CyA in aqueous solutions of PS and F127 was increased linearly with increasing amount of PS in water. Dissolution of CyA from solid dispersions prepared from PS, F127 and F188 in 0.1% of sodium lauryl sulfate was much better than that from PVP. Only solid dispersion containing PS showed a promising dissolution when using water as dissolution medium. The solid dispersions containing PS exhibited improving dissolution characteristics of CyA as the amount of carrier is increased. Solid dispersions containing PS/drug ratio (w/w) higher than 7:1 showed little or no further increase in drug release. Therefore the 7:1 ratio was selected for this investigation, as higher drug content is more suitable for practical use.The factors including the ethonal volume, rotation speed of the stirrer, water bath temperature and the particle size of the solid dispersion powder didn't influence the dissolution significantly within the experiment scope. Two percent of Aerosil was added to improve the flowability and mixed homogeneously with the solid dispersion powder before filling into the capsules and wrapped.The stability results showed that the preparation is stable up to six months under accelerate conditions and 1.5 year at 25℃/60% RH when packed in aluminum-polyethylene laminated bags. Long term stability of CyA at 25°C/60% RH is the subject of an ongoing study.In vivo oral bioavailability of CyA from the solid dispersion in Wistar rats was comparable to that from a commercial product, Sandimmun Neoral(?) (P>0.05). The derived pharmacokinetic parameters for solid dispersion and Sandimmun Neoral(?) were as follows: C_max:2348.65±495.96, 2557.38±555.09ng/mL; T_max: 2.00±0.67, 1.71±0.67h, AUC_0-60: 40283.99±5203.16, 41021.10±6239.87 ng/mLh; MRT_0-60: 17.96±1.88 å’Œ 17.97±1.29h. Statistical analyses revealed that there were nosignificant differences between the two formulations for C_max,AUC_0-60, T_max, K_a, K₁₀ and MRT(P>0.05). The experimental results illustrated that solid dispersion is a useful approach to improve the bioavailability of this poorly water-soluble drug.The bioavailability of CyA solid dispersionafter singe dosing was conducted in healthy volunteers by using Sandimmun Neoral(?) as a reference. The pharmacokinetic parameters for solid dispersion and Sandimmun Neoral(?) were as follows: C_max: 1500.3±348.8, 1742.9±361.3 ng/mL; T_max:2.63±0.55,1.39±0.49h; AUC0-24: 7993.0±2535.1, 8295.6±2295.6 ng/mLh; AUC_0-∞:8851.7±2815.0, 9258.0±2523.7ng/mLh; MRT_0-24: 6.48±1.15, 5.76±1.04h. Statistical analyses revealed that there was no significant difference between the two formulations for AUC_0-24, AUC_1-∞ and C_max of the 90% confidence interval, indicating that the two preparations were bioequivalent. T_max of CyA solid dispersion was longer that that of Sandimmun Neoral(?) (P<0.01), which com formed to the charactersitics of the two preparations.The in situ circulation method was used to investigate the relationship between the absorption of CyA and its concentration; the effects of concentration of Na⁺ and the respective accumulated absorption of CyA solid dispersion with Sandimmum Neoral(?). The results showed that there was no significant difference in the accumulated drug absorption between duodenum, jejunum and ileum (P>0.05). The drug absorption from these three sections were much higher than that from the colon (P＜0.01), indicating that CyA was mainly absorbed from the upper part of intestine. The accumulated absorption of CyA increased linearly with its concentration, meaning that a passive diffusion mechanism was dominated. The results illustrated that the preparation of solid dispersion was helpful for the drug absorption by the improvement of CyA solubility and dissolution in the gastrointestinal track. The concentration of Na⁺ didn't influence the accumulated amount of drug significantly (P<0.05). CyA in Sandimmum Neoral(?) and solid dispersion showed equal absorption levels in the intestine (P>0.05). The experiment illustrated that the enhanced solubility and dissolution by the use of solid dispersion is one of the reasons for the improved bioavailability of the preparation.The inhibitive potential of PS on CYP1A2, 2A6, 2D6 and 3A4 in human liver microsomes was evaluated. The human liver microsomes were pre-incubated with the test article at 37℃ for 15 minutes, and then incubated with selective substrates of CYP1A2, 2A6, 2D6 and 3A4 in presence of NADPH, the cofactor of cytochrome P450 oxidases, at 37℃ for 30 minutes. The formations of metabolites of CYP450 1A2, 2A6, 2D6 and 3A4 were measured by HPLC and inhibition and its kinetics were calculated by comparison with negative control respectively. The results showed that the inhibitory potential (IC₅₀) of PS on CYP1A2, 2A6, 2D6 and CYP3A4 were 0.3705 mg/mL, 1.942 mg/mL, 1.005 mg/mL and 0.6585 mg/mL respectively. These results demonstrated that PS may enhance the absorption of CyA in vivo throuth the inhibition of activity of CYP3A4.P-glycoproteins (P-gp) are the best known efluxers present in several cancer cell lines, and it is well documented that P-gp is overexpressed in Caco-2 cells. The Caco-2 cell line is used in the investigation for the study of the the characteristical uptake, transport of Rhodamine 123 (R123), a substrate of P-gp, in the presence or without the addition of PS at different concentration. The cytotoxic activity was evaluated using a standard MTT(3-(4,5-dimethylthiazol-2-y1)1,5-diphenyltetrazolium bromide) assay. The MTT results showed that PS at concentrations below 800μg/mL didn't influence the viability of Caco-2. Uptake of R123 into the Caco-2 was significantly improved by the adding of PS solution at different concentrations, which was 1.3 to 1.9 times compared to the surfactant-free control(P<0.05). Similar to uptake, the transport of R123 from apical to basolateral side was increased in the presence of PS and the enhancement factor was increased with the PS concentration before its critical micelle concentration (CMC) and decreased after that. The maximum enhancement was observed at 150μg/mL, which was 1.65 times of that of control. At the same time, the transport from basolateral to apical decreased with the same trend.The inhibitory mechanism of PS was investigated by measuing the cell membrane fluidity, cellular ATP and activity of P-gp ATPase after exposure to PS at different concentrations. The membrane fluidity increased dramatically after the treatment of PS at the concentration from 50 to 800μg/mL for 30min, and the value of the anisotropy of cell membrane was leveled off after that. The anisotry of cell membrane exposure to PS was 36.9 to 52.98% of the control. The restoration of membrane fluidity was observed after 30min following the removal of PS, indicating that the alteration of integrity of cell membrane Iipid was transient and reversable.PS at various concentrtions didn't comsume the cellular ATP after it was incubated with Caco-2 monolayers for a certain time, indicating that it didn't inhibit P-gp function by depletion of ATP.PS at various concentrtions didn't influence the activity of P-gp ATPase significantly within the experiment duration.Therefore, the inhibitory effect of PS on the efflux of P-gp maybe mainly due to the change of membrane fluidity. The inhibitory of PS on P-gp may contribute to the enhanced absorption of CyA from solid dispersion.