| Gliomas occur in neuroectoderm, accounting for about40to50%of intracranial tumors. Due to the invasive growth of glioma cells, there are no obvious boundaries between the glioma and normal brain tissue. Therefore the total removal of glioma is difficult. A comprehensive treatment is generally advocated clinically, namely surgery with radiation therapy, chemotherapy, etc., which can delay recurrence and prolong survival of patients.Temozolomide (TMZ) is an antineoplastic alkylating agent for treating the gliomas in the clinical practice. The drug itself is not active but it can non-enzymatically convert to active compounds at physiological pH. Preparations of TMZ currently on the market are capsules and injectable freeze-dried powder. Adverse reactions of TMZ capsules include nausea, vomiting, and hematologic response. Patients usually take antiemetic medication before taking TMZ capsules for avoiding incomplete absorption of TMZ. The listed TMZ capsules are not suitable for surgery patients and TMZ injectable lyophilized powder solves the question. However TMZ lyophilized powder for injection used Tween80, which can cause hemolysis, allergies and other adverse reactions. Therefore, the development of new anti-glioma nano drug delivery systems became the research focuse for improving the efficacy, reducing toxicity and improving patient compliance.Liposomes are spherical closed vesicles formed by phospholipid dispersing in water, which can load water-soluble and fat-soluble drugs. As a drug carrier, liposomes may be degraded in vivo with non-toxicity and non-immunogenicity. Furthermore liposomes can have the targeting and reduce the toxicity. Therefore, liposomal drugs have been widely used with more attention.In the study, TMZ liposomes have been prepared for i.v. injection in which TMZ was as a model drug and soy lecithin and cholesterol were as materials. The research mainly includes studying equilibrium solubility and octanol-water partition coefficient of TMZ, studying the prescription and screening process of TMZ liposomes, studying physical and chemical properties of TMZ liposomes, studying in vitro evaluation of TMZ liposome and studing the pharmacokinetics and tissue distribution of TMZ liposomes.1. The research of equilibrium solubility and octanol-water partition coefficient of TMZThe equilibrium solubility of TMZ in water and phosphate buffer with pH1.75,4.32,4.97,5.57,6.06and the oil-water partition coefficient of TMZ in0.1mol/L hydrochloric acid, and phosphate buffer (pH=1.75,4.32,4.98,5.57,6.06)-octanol were determined by using UV spectrophotometry.The results showed that the equilibrium solubility of TMZ in water and phosphate buffer with pH1.75,4.32,4.97,5.57,6.06were5.255mg/mL,6.827mg/mL,6.672mg/mL,4.586mg/mL,3.473mg/mL,3.533mg/mL. TMZ is sparingly soluble in water and phosphate buffer and the equilibrium solubility of the drug decreases with the increase of pH. The octanol-water partition coefficient (denoted by1gP) of TMZ in0.1mol/L hydrochloric acid and phosphate buffer (pH1.75,4.32,4.98,5.57,6.06)-octanol were-0.603,-0.678,-0.239,0.106,0.417,1.169, so1gP increased with increasing pH, which coincided with results of the equilibrium solubility. LgP values of TMZ in PBS-octanol of were between-0.678～1.169, indicating that TMZwas poor fat-soluble and general water-soluble. The results provide the basis for new formulation and prescription design of temozolomide, and in vitro/vivo evaluation of preparations.2. The study of prescription and screening process of TMZ liposomes The content of TMZ was measured by HPLC. The results of methodological study showed that materials did not interfere with the determination of TMZ, and the method was suitable for in vitro determination of TMZ which was specific, sensitive and simple. The centrifugal ultrafiltration method was used to determine the entrapment efficiency and drug loading of TMZ liposomes, which can completely separate liposomes and free drug with simple operation, accurate and reliable results.Repeated freezing and thawing method the carrier deposition method were used to prepare TMZ liposomes. The single factor investigatin of prescription and processes of TMZ liposomes was carrying on with size and encapsulation efficiency as evaluation index in order to obtain optimal formulation and preparation process. Eventually TMZ liposomes were prepared basing on the optimal formulation and preparation process with higher encapsulation efficiency, smaller particle size and good reproducibility. The optimal formulation and preparation process:rotary evaporation speed was80rpm, rotary evaporation temperature was40℃, carrier type was sorbitol, carrier/lipid ratio was5:1, drug/lipid ratio was1:15, and the ratio of phospholipids and cholesterol was8:1. The encapsulation efficiency and drug loading of batches of TMZ liposomes prepared based on the best prescription and process were (35.45±1.48)%and (2.81±0.20)%.3. The physicochemical properties and in vitro evaluation of TMZ liposomesThe physicochemical properties of TMZ liposomes were investigated. By transmission electron microscopy (TEM), morphological appearance of TMZ liposomes has been observed, and the results showed that TMZ liposomes appeared spherical. The particle size was156.7±11.4nm measured by particle size analyzer with a narrow particle size distribution. The pH of TMZ liposomes measured by a pH meter was6.46, which met the requirements of intravenous injection.In vitro release studies of TMZ solution and TMZ liposomes were conducted by a dynamic dialysis. The results showed that TMZ released slowly from liposomes compared with the solution group. In vitro release behavior of TMZ solution was in line with Weibull equation, and in vitro release behavior of TMZ liposomes was in line with First-order kinetics and Weibull equation.The preliminary safety evaluation of TMZ liposomes was carried on using2%rabbit blood cell suspension. Different concentrations of liposomal drug suspension were prepared to study the hemolysis test. By visually observing, the test results showed no hemolysis and agglutination in test sample and TMZ liposomes can be safe as intravenous injection.4. The pharmacokinetics in rabbits and tissue distribution studies in mice of TMZ liposomesA HPLC method has been established to determine TMZ concentration in rabbit plasma and mouse tissues in this study and methodological research has been conducted. The results of methodological study showed that the established HPLC method was simple and sensitive with accurate and reliable results and endogenous substances did not interfere with the determination of TMZ.The pharmacokinetics in rabbits and tissue distribution in mice of TMZ liposomes have been studied with TMZ solution as the control. Compared with TMZ solution, the pharmacokinetic parameters of liposome group have been changed:ti/2p and MRT were13.465h and2.864h, which were3.58times and1.43times of TMZ solution; Cmax and AUC values of TMZ liposomes were6.26mg/L and14.482mg/L*h, which were1.10times and1.51times of TMZ solution. The results of pharmacokinetics study showed that prolonged in vivo circulation time and increased AUC were in favor of TMZ treatment for cancer.The tissue targeting of TMZ liposomes were studied with relative uptake rate (Re) as an evaluation index. The results showed that compared to TMZ solution, Re value of TMZ liposomes in the brain was1.22, Re values in the liver and spleen were1.337and1.306, Re values in the kidney was1.153, and Re values in the heart, lungs were0.763and0.838, which implied that TMZ liposomes increased the concentration of the drug in the brain, improved the therapeutic effect and reduced heart toxicity and lung toxicity. TMZ liposomes can change biodistribution, increase the concentration of drug at the treatment site, and reduce side effects. Furhermore TMZ liposomes avoided the use of Tween80which was contained in the listed freeze-dried powder for injection.Conclusions:In this study, TMZ liposomes can be prepared for intravenous injection. The preparation process was simple with good reproducibility, and TMZ liposomes possess stable physical and chemical properties. In vitro release tests showed that TMZ liposomes released slowly compared with TMZ solution. The results of preliminary safety trial showed TMZ liposomes had good safety without hemolysis. The results of pharmacokinetics and tissue distribution study showed that the liposomes had a slow-release effect, had a brain targeting. The drug delivery system can improve drug effiency and reduce side effects, which was promising for developing the new formulation. |
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