| OBJECTIVE:Recently, as the new immunosuppressive agents inhibit of comeal neovascularizationto, the effectiveness of sirolimus was of widespread concern. The inhibitory effect of rapamycin on corneal neovascularization, which was in the pathological state of corneal alkali injury, has been confirmed by some experiments. Due to the anatomical features of the eye’s blood-ocular barrier and corneal barriers, as systemic administration achieves the effective blood level, drug concentration within the eye almost can not be traced. To achieve effective drug concentration and duration is impossible. Currently, there were only sirolimus oral agents. Serious adverse reaction was emerged by oral administration. Ophthalmic formulation research was relatively backward. With high molecular weight, poor water solubility and the efflux by the system of p-gp, its absorption in local application was very poor. This study was to prepare a new ophthalmic drug delivery system:chitosan coated Hposomes-in situ gel, with some solubilization, controlled release materials which also could inhibit the p-gp system. Then to evaluate physicochemical properties, in vitro release and pharmacokinetics of the new eye drug delivery system, with the hope of providing a new, efficient and safe sirolimus eye drops. Because of the phospholipid bilayer membrane structure, Liposomes both have hydrophilic and hydrophobic, what can make it easily integrate with the biofilm. Liposomes also own advantages of the increase of corneal permeability, Sustained-release, reduction of toxicity and fluctuations in drug concentration. It has been reported that the adsorption between liposomes of the positively charged and cornea of the negatively charged, caused a stronger affinity, which would be conducive to the spreading of drug in the anterior corneal to contribute to the ocular absorption. Chitosan (of Chitosan, CS) is the only natural alkaline polysaccharide. Its derivatives are kinds of natural polymers, which was biodegradable naturally and non-toxic.They also own good biocompatibility, high charge density and mucosal adhesion. Studies showed that CS and its derivatives both could improve the membrane permeability of hydrophobic drugs and drug stability. Simultaneously, they also has a certain inhibition on the P-gp efflux. Therefore, there was a high penetrating, adhesion, and anti-P-gp efflux effect of liposomes, after it was coated by chitosan or its derivatives. In addition, the flexible liposomes and microemulsion have been researched more and more mature. Flexible liposomes are made of adding surfactants such as sodium cholate in the phospholipid bilayer of ordinary liposome. They not only have the advantages of corneal drug delivery systems, the same as the nature of conventional liposomes, but also own highly flexible and efficient penetration.Microemulsion is a transparent or translucent, lowly viscid mixed system, which is spontaneously emulsified by water phase, oil phase, surfactant. The huge specific surface area of nanoemulsion droplets formed by the microemulsion emulsifying, promotes drug absorption in the cornea, thereby enhancing the ocular bioavailability of the drug. In order to eventually filter out a novel ophthalmic sirolimus delivery system, which is of safty, good tissue compatibility and high bioavailability, flexible liposomes and microemulsion as ocular reference preparations are also studied.Finally, to combine with the phase change characteristics of in situ gel, which can extend the residence time of drug in the cornea much further to achieve the sustained release effect. Therefore, the novel ocular delivery systems studied by this experiment, were ocular sirolimus liposomes coated with chitosan-in situ gel, ocular sirolimus flexible liposomes coated with chitosan-in situ gel,and ocular sirolimus microemulsion coated with chitosan-in situ gel.Then to study formulation, process, quality, ocular irritation and pharmacokinetic dynamics evaluation of the three novel in situ gels.METHODS:Combine film dispersion method with probe sonication method to prepare liposomes, flexible liposomes, with indicators of the size of encapsulation efficiency and the difficulty degree of hydration to filter out the optimal formulation and process. At the same time, to prepare sirolimus microemulsion, to optimize formulation and process of in situ gel, and thus to make up the three novel ocular in situ gel.Then the physical and chemical properties, such as the size, zeta potential of liposomes, flexible liposomes and microemulsion, the gelling temperature, pH and viscosity of the gel formulations, were inspected. After that, to establish the HPLC methodology to determine the content of Sirolimus in three novel in situ gels, and to study the release mechanisms with no membrane stripping models. Subsequently, the ocular irritation was inspected and evaluated with rat corneas by using slit lamp microscopy combined with histological examination. Finally, rabbits ophthalmic pharmacokinetics of three in suit gels were studied (n=4).Experimental group A afforded ocular sirolimus liposomes coated with chitosan-in situ gel, experimental group B afforded ocular sirolimus flexible liposomes coated with chitosan-in situ gel, experimental group C afforded ocular sirolimus microemulsion coated with chitosan-in situ gel, and the control group was given sirolimus-castor oil eye drops(concentration as0.2%). After given samples0.5h,1h,2h,3h,4h,6h,12h,24h, aqueous humor and corneal samples were collected respectively for determining sirolimus concentrations.To analysis pharmacokinetics results by using the DAS2.1software.RESULTS:The physical and chemical parameters of the three novel in suit gels were all in line with the requirements of ophthalmic preparations. Then the high-performance liquid chromatography was established to determine the sirolimus contents and to study in vitro release models. The cumulative dissolution-release equations were get by regression analysis and all the slopes were close to1.It was shown that the main factor which influenced the release was the dissolution of the gel. In the irritation test period, there were no cloudy corneas, no inflamed and congestive iris and conjunctivas, or any other anomalies. Droping with2%sodium fluorescein to take a slit lamp microscope examination, staining reactions were not see on the rat’s corneas. And animals of the experimental group were showed no irritability, drowsiness or any other unusual behaviors during the observation period. According to the scoring criteria, every irritation score value0on each time point during the administration. Histological examination revealed:the connective tissue structure in the surface squamous epithelium of rats’corneas in the positive group of alkali injury, was damaged to shed down, and inflammatory cells were infiltrating into rats’corneas. The rats’corneas of experimental group are intact, where is no necrosis, no ulceration, and no inflammatory cell, and the connective tissue structure of the surface squamous epithelium is clearly visible. The results showed that the three in situ gels could be used for ocular drug delivery, because of their non-irritating. Fitted the compartment model with the four preparations’ pharmacokinetics datas by using the DAS2.1.1software, the results were all in line with the two-compartment model (weight coefficient1/cc).The experimental group B was5.965times the aqueous AUC (0-t) of the control group,1.206times the aqueous AUC (0-t) of the experimental group A,and1.412times the aqueous AUC (0-t) of the experimental group C. Then the experimental group B was1.925times the cornea AUC (0-t) of the control group,1.333times the cornea AUC (0-t) of the experimental group C. And duration of action of the experimental groups was significantly more than the control group. It made the experimental groups obviously play the role of controlled release formulations. The mean sirolimus concentrations of in aqueous humor and cornea of the experimental groups were all higher than which in the control group.CONCLUSION:By studying assay and in vitro release, it was initially confirmed that the characteristics of the liposomes, flexible liposomes, microemulsions, and in situ gel, were fully combined. The collaborative composite formulations were emerged.In the process of in vitro release, for any kind of the three novel in situ gels, the dissolution of in situ gels was the main factor which controlled drug release. It was derived by analyzing in vitro release data that, ocular sirolimus flexible liposomes coated with chitosan-in situ gel and ocular sirolimus liposomes coated with chitosan-in situ gel, with a better sustained-release effect could further extend the drug ocular residence time. After that, in the irritation experiments, the slit lamp and histological examination confirmed that there was no damage of the three gel formulations on the cornea. And the three novel in suit gels was safe, with no ocular irritation, which was preliminarily described. The pharmacokinetic study results showed that, compared to the experimental group A of ocular sirolimus liposomes coated with chitosan-in situ gel, the experimental group C of ocular sirolimus microemulsion coated with chitosan-in situ gel and the control group was given sirolimus-castor oil eye drops, the experimental group B of ocular sirolimus flexible liposomes coated with chitosan-in situ gel, significantly improved the ocular sirolimus bioavailability(P <0.05) among the four kinds of novel formulations, which was the most efficient, reliable and safe ocular novel dosage form, with good prospects for clinical application. |
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