Chemo-enzymatic Synthesis Of TP-5 And Preparation Of Liposome As A Sustained-release Dosage Form

Thymopoietin is a polypeptide hormone produced by Thymus, consisting of 49 amino acids. The pentapeptide thymopentin (TP-5) Arg-Lys-Asp-Val-Tyr, corresponding to amino acids 32-36 of thymopoietin, appears to represent the active site of thymopoietin, and it has all the biological activities of the native Thymopoietin.TP-5 has many biological functions, it can induce the secretion of many hormones, mediate the cytodifferentiation of T-cell, regulate immune system and so on. Studies show that Thymopentin has clinical efficiency in the treatments of rheumatoid arthritis and atopic dermatitis, and genital warts. It also can be used as adjunctive therapy of genital system infection and chemotherapy & radiotherapy. TP-5 has the biological characteristics of being immunonormalizing which, whether the immune disbalance be in the direction of hyper- or hyporesponsiveness, points to its potential utility in human diseases characterized by immune disbalance. Researches have proved that TP-5 can cure lymphadenopathy of drug addicts, inhibit the accumulation of hyperoxide in the cells, and relieve the insulin dependence of diabetic. A large amount of researches indicate that TP-5 is safe in treatments and can be widely used in therapy and adjuvant therapy. Therefore, the characteristic TP-5 has been attracting much attention of investigators.So far, all of commercially available thymopentin (TP-5) are synthesized TP-5 by chemical methods. Generally, chemical method is suitable for synthesis the moderate length peptide on a laboratory scale with high cost. In contrast, enzymatic method is suitable for synthesis short peptides. As compared with the chemical method, the important benefits of enzymatic peptide synthesis are:a) the mild conditions of the reaction; b) the high regiospecifity of enzyme allowing the use of minimally protected substrates; c) the reaction being stereospecificity without racemization. At present, many hydrophobic small peptides were synthesized in high yield using proteases in organic media as largely reported. However, the formation of peptide bonds between hydrophilic amino acids faced many problems. Hydrophilic substrate has low solubility in hydrophobic organic solvents, hindering us to use hydrophobic organic solvents where the enzyme usually shows better activity and selectivity than in hydrophilic organic solvents. Hydrophilic organic solvents are suitable as the reaction media to enhance the solubility of hydrophilic substrates. However, hydrophilic solvents are often harmful to enzyme because it has a greater tendency to strip the tightly bound essential water from the enzyme molecules. The appropriate reaction system should be selected considering the balance of the solubility of substrates and enzymatic activity.In this study, TP-5 is a good mode of hydrophilic small peptides. Considering the solubility of hydrophilic substrates, we selected hydrophilic organic solvents such as ethanol, acetonitrile as the reaction media. A new and practical enzymatic procedure for peptide synthesis using alcalase as a biocatalyst was studied. Chen et al. successfully synthesized a number of dipeptides and tripeptides in anhydrous alcohol with higher yields. The industrial alkaline protease alcalase is a proteolytic enzyme prepared from submerged formation of a selective strain of Bacillus licheniformis. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg (alkaline protease A), which is one of the most thoroughly characterized bacterial proteases. The alkaline protease alcalase has a high protease activity and stability in alcohols, acetonitrile, DMF, etc. At the same time it is also cost-effective. Therefore, the enzyme alcalase was our first choice for synthesis of the precursor dipeptide Bz-Arg-Lys-NH2,Z-Asp-Val-NH2 and the precursor tripeptide Z-Asp-Val-Tyr-OH of TP-5 under kinetic control condition in water-organic cosolvents systems. In addition, for the synthesis of the precursor dipeptide Bz-Arg-Lys-NH2 of TP-5, trypsin would be the suitable catalyst considering its substrate specificity. The synthesis reaction conditions were optimized by examining the effects of several factors on the yields of peptide products including water content, temperature, pH, and reaction time on the yields.When the precursor dipeptide Bz-Arg-Lys-NH2 as the left side precursor dipeptide of TP-5 was synthesized, the enzyme alcalase was tested also. Using trypsin as the catalyst, the optimum conditions for the synthesis of Bz-Arg-Lys-NH2 were Bz-Arg-OEt (50 mM), Lys-NH2 (350mM),triethylamine (28μl/ml), in ethanol/0.1M This-HCl pH8.0 buffer system (80:20,V/V) , 35℃, 4h with the maximum yield of 76.1%; Using Alcalase as the catalyst for the synthesis of Bz-Arg-Lys-NH2, the optimum conditions were Bz-Arg-OEt (50 mM), Lys-NH2 (350mM),triethylamine (28μl/ml), in acetonitrile/DMF/0.1M Na2CO3-NaHCO3 pH 10.0 buffer system(80:10:10, V/V/V), 35℃, 6h with the maximum yield of 71.1%. The above results indicate that trypsin as the catalyst is better than alcalse for the synthesis of Bz-Arg-Lys-NH2 due to its better substrate specificity.When the precursor dipeptide Z-Asp-Val-NH2 of TP-5 was synthesized, it is difficult to select a suitable enzyme with good the substrate specificity. We tested the enzyme alcalse which has wider substrate specificity. The optimum conditions for Z-Asp-Val-NH2 synthesis using Alcalase as the catalyst were Z-Asp-OMe (0.05M),Val-NH2 (0.25M), triethylamine (14μl/ml), in acetonitrile /0.1M Na2CO3-NaHCO3 pH 10.0 buffer system (90:10, V/V) , 35℃, 5h with the dipeptide yield of 63%.When synthesizing the right side precursor tripeptide Z-Asp-Val-Tyr-OH of TP-5 with Z-Asp-OMe as the acyl donor and Val-Tyr-OH as the nucleophile, alcalse was used on the bases of the synthesis of Z-Asp-Val-NH2. The optimum conditions for Z-Asp-Val-Tyr-OH synthesis were Z-Asp-OMe (50 mmol), Val-Tyr-OH (150 mmol), triethylamine(50μl/ml), in acetonitrile/0.1M Na2CO3-NaHCO3 pH 10.0 buffer system (85:15, V/V) , 35℃, 2.5h with the tripeptide yield of 71.3%.In this study, the precursor tripeptide of thymopentin and the free thymopentin were synthesized also by liquid-phase method and solid-phase method, respectively. For the synthesis of Thymopentin, the resin of HMP was used as the solid support. Firstly, Fmoc-Tyr(OBut)-OH was linked to the resin of HMP. Secondly, Fmoc-Val-OH, Fmoc-Asp(OBut)-OH, Fmoc-Lys(Bos)-OH, and Fmoc-Arg(Mtr)-OH were linked one by one. Lastly, the peptide segment was cut with TFA. The yield of TP-5was 30% calculated from the first amino acid anchored to resin.TP-5 have small molecular weight and can be easily hydrolyzed by proteases, and its half life in vivo is very short. Some efforts had been made to increase circulating time and enhance their stability by modification of TP-5 structure. For instance, TP-5 repetitive sequence, TP-5-conjugated with polyethylene glycol or CM-chitin, TP-5-containing pseudopeptide and peptidomimetics were synthesized. However, TP-5 amounts in these linear peptides contained are limited. In this paper, TP-5 liposome was designed. Liposome is a new drug delivery dosage form. It has been widely used in medical area. Liposome has some characters such as prolonged action, target effect, slow release, less administration frequency. TP-5 liposome was prepared by reverse phase evaporation method. The impact of every factor on the encapsulation efficiency was investigated, and the factors were optimized by orthogonal design The optimum conditions for TP-5 liposome preparation: the mass ratio of egg phosphatidylcholine to cholesterol 5:1, TP 15mg, the volume of phosphate buffer 10ml, sonicating for 5min. Its mean size was 340nm. The highest encapsulation efficiency was 58.21%. The TP-5 liposome should be stored under lower temperature, because the stability is not good enough under higher temperature. There is no any phenomenon of layering and emulison damage after storing under 4℃for 2 months.