Development Of Thymopentin And Its Ester Derivates Depot Injection

More and more peptides and proteins become main and important drugs along with the progress of genomics and protemics. However, compared with low molecular weight drugs, the distinct properties of them, such as easy degradation (e.g. by deamidation, oxidation and hydrosis), instability, low bioavailability and short half lives, make non-parenteral dosage forms not proper to deliver them. So it's a significant task to deliver proteins and peptides to target organs and tissues. Nowadays the biopharmaceutical drugs on clinical use are supplied as sterile freeze-dried powder or injectable solutions. These formulations mentioned above are administered frequently and this results in the patients'poor compliance and high-cost therapy. To improve the patients'compliance and the therapy effect and decrease the administration frequency, developing sustained release formulations for these peptides and proteins is a choice of wisdom. The other advantages include: (1) maintaining minimum effective concentration and avoiding the peak-valley phenomenon which appeared regularly in the common formulations; (2) decreasing total dose and alleviating some toxic and adverse actions; (3) decreasing the patients'costs. Therefore, injectable sustained-release preparations have been studied extensively in the last two decades in the pharmaceutics field.Thymopentin (TP5), the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr, corresponds to residues 32-36 of the thymic hormone thymopoietin. It has pleiotropic actions including selective induction of early T-cell differentiation and regulation of neuromuscular transmission. Furthermore, TP5 has been shown to have immunoregulatory actions on peripheral T cells. The product available was mainly sterile freeze-dry powder. TP5 is freely soluble in water and quickly degraded by protease and aminopeptidase in plasma. TP5 potency in vivo was enhanced by slow infusion. This suggested that the peptide was suitable for development of a sustained release formulation.The methods frequently used in the preparation of depot injection include: (1) converting a drug into insoluble salt (or ester) or insoluble complex. (2) adding macromolecular polymer such as hyaluronic acid into the injection to increase the injection's viscosity and delay the drug's spread; (3) encapsulating peptides in liposomes and obtain a sustained release formulation of peptide; (4) incorporating drugs into solid particles to obtain a extended drug release. (5) in-situ gelling system.Among these methods mentioned above, the insoluble complex is relatively simple form. The method has been widely used in the product granted market clearance. As long as the insoluble salt (or ester) could be prepared and showed certain effect, this relatively simple method will be continued to produce sustained release products in the future.At first, we tried preparing a water insoluble complex combined with thymopentin and carboxymethylcellulose sodium. But the yield was low. We only obtained less amount of product due to the high solubility of thymopentin. Meanwhile, the vitro release of the insoluble complex was affected by the release medium obviously. The peptide released rapidly in simulated tissue fluid (Hank's balanced salt solution) and almost completed in 6h. So we fail to prepare the insoluble complex directly combined with thymopentin and carboxymethylcellulose sodium. We have to look for other path to prepare sustained release formulation for thymopentin.To reduce water solubility of TP5, a chemical modification has been done. In our research, carboxyl groups in C terminal of thymopentin and side chain of aspartate are esterified. We synthesized a series of thymopentin ester derivates (ethyl, butyl, hexyl and lauryl ester). The preliminary pharmacodynamics and safety evaluated were carried out for the ester derivates. The stability of the ester derivates was investigated too. The ethyl ester was chosen for further pharmaceutics study, and at last we prepared an insoluble thymopentin ethyl ester-tannate salt complex. It shows characteristics of sustained release in vitro. So far some investigations have been made to increase the peptide's stability and efficacy, including linear peptide analogues and derivates modified by polyethylene glycol. To our knowledge, no ester derivates of thymopentin are reported.1 Synthesis, purification and identification of thymopentin ester derivates In this dissertation, the synthesis of thymopentin ester derivates was carried out by thionyl chloride method. Thionyl chloride was first dissolved in absolute ethanol. The mixture was stirred at 0°C for some time. Thymopentin was added and continued stirring at 0°C. Allowed temperature to come back at room temperature spontaneously and stirred the reaction until the TP-5 disappeared. A thin-layer chromatography (TLC) was used to detect whether the reaction completed. Ninhydrin TLC reagent spray was employed to visualize the peptides on the TLC plate. The ethyl ester reaction mixture was concentrated in vacuo to a solid. The remaining n-butanol and n-hexanol in butyl ester and hexyl ester reaction mixture were removed under vacuum by forming azeotropic mixture with water. The crude product was purified using Flash column chromatography system. Acetonitrile and TFA were removed under vacuum, followed by lyophilization. The dry product was analyzed once again. The ester derivates were found to be >98% pure and were further characterized using LC-ESI-MS. Moreover, the purified ester derivates were used for in vitro and in vivo assay.2 The study on physical properties and stability of thymopentin ester derivates Esterification modification changes physical properties of thymopentin greatly.That is to say, the liposolubility of ester derivates increased and water solubility decreased obviously. The half life of TP5 in plasma was 1.3 min and TP-ET 2.0 min. The half life of ethyl ester was almost equivalent to that of TP5. The preliminary stability test showed that the ester derivates was sensitive to light, and temperature and moisture affected the stability of ester derivates to some extent. So the ester derivates should store in cool and dark places.3 Pharmacodynamics of thymopentin ester derivatesThe in vitro immunoregulatory activity of ester derivatives was assessed by mice lymphocyte proliferation test using MTT colorimetric assay. Lymphocytes of mice spleen suspended in RPMI 1640 medium with 10% heat inactivated fetal calf serum were set up in 96-wells cell culture cluster. Under the tested concentrations, optical densities of all test wells were higher than those of blank wells. Optical densities of all wells containing thymopentin ester derivates were higher than those containing TP5. These results indicated that ester derivates showed more potent lymphocyte proliferation activity compared with TP5. Moreover, the activities of ester derivates enhanced as the alkyl chains extended.Thymopentin ethyl ester was further used in plasma superoxide dismutase (SOD) activity assay. Hydrocortisone injection was administered subcutaneously to each rat at a dose of 50 mg/kg once daily for 7 consecutive days for construction of immunosuppression rats. All the rats were fasted for 8 h before the experiments, but had free access to water. From the eighth day, the rats were given different treatment for 14 days. 6 h after the last administration, a volume of 100μl blood sample was taken from the orbit venous plexus into heparinized tubes and immediately centrifuged at 4000 revolutions per minute for 10 min. The plasma was collected and stored at -20°C until assays. For each assay, a volume of 15μl plasma sample was used. All the processing and assay validation met the requirements of the kit instruction. The results showed that the SOD values of the immunosuppressive rats were significantly lowered as compared with those of the normal control rats, indicating the immunosuppressive model was stably established. The SOD values of the immunosuppressive rats after subcutaneous administration of TP5 ethyl ester were significantly increased as compared with those of the model control group. SOD values of TP5 control group were higher than those of model group (P<0.05). But SOD values of TP5 group were lower than those of TP5 ethyl ester group with medium doses and high doses (P<0.05), equivalent to those of TP-ET group with low doses (P>0.05).4 Preliminary safety of thymopentin ester derivatesMice in test groups were injected intravenously and subcutaneously with ethyl ester (TP-ET) and hexyl ester (TP-HEX) solution. A control group received the solvent only. The solvent comprised 30% propylene glycol and 70% physiological saline solution. Drug effects were investigated by observation of body weight changes and survival rates over 15 days. Animals were observed until day 15, after which they were euthanized and subjected to a gross necropsy. The LD50 of TP-ET was >85 mg/kg. Tremors and twisting were the signs most frequently observed in the animals treated with TP-ET at 85mg/kg and 125 mg/kg. The mice were observed no obvious toxic reaction when given TP-ET 170mg/kg subcutaneously, so TP-ET was considered safe for subcutaneous injection.When the mice received a single dose of TP-HEX, Ocular proptoses and tremors usually developed and could be accompanied by tachypnea. In the most severe signs, animals lost muscle tone and righting reflex, followed by death. At the rest days of the 15-day observation period, no mice died and the survivors in all treatment groups gained weight. There were no treatment related gross necropsy findings for either scheduled euthanasia or early death animals in the study.5 Preparation and in vitro/in vivo evaluation of thymopentin ethyl ester tannate Thymopentin ethyl ester could form a stable insoluble complex with tannic acid. The complex released for a prolonged period in Hank's balanced salt solution. The rats were administered the complex oil suspension subcutaneously. The preparation showed a prolonged action period in elevating plasma SOD activity and the action maintained for about a week in vivo.The creative points of this work consist in that thymopentin ester derivates show enhanced immunoregulatory activity and increased liposolubility. This can be regarded as a new finding in chemical modification of thymopentin. On the other hand, this work provides a new example of"chemical pharmaceutics"in pharmaceutical developments.