Vesicular phospholipid gels using low concentrations of phospholipids for the sustained release of thymopentin: pharmacokinetics and pharmacodynamics.

Vesicular phospholipid gels (VPGs) with high concentrations of phospholipids are used as implantable depots for sustained release of drugs due to high viscosity. This study aimed to investigate VPGs with low concentrations of phospholipids for subcutaneous injection and sustained release in vivo. A small peptide, thymopentin, was selected and incorporated into various VPG formulations. The VPG viscosity was greatly increased with higher concentrations of phospholipids (E80) and thus VPGs based on low lipid contents are more suitable for injection. Additionally, VPGs loaded with 5-hydroxy-fluorescein-thymopetin (5-FAM-TP5-VPGs) were developed and their pharmacokinetic profile was investigated in vivo. After subcutaneous injection, the release time of 5-FAM-TP5 was 216 h for 5-FAM-TP5-VPGs (containing 300 mg/g lipid), which was much longer than that of 5-FAM-TP5 solution. The therapeutic efficacy of TP5-VPGs (containing 300 mg/g lipid) after subcutaneous administration once a week was demonstrated to be comparable to that of TP5 solution injected subcutaneously once daily for 7 days. In conclusion, TP5-VPGs with low lipid content (300 mg/g) displayed sustained release properties in vivo that may serve as a sustained delivery system for subcutaneous injection.

Multivesicular liposomes for the sustained release of thymopentin: stability, pharmacokinetics and pharmacodynamics.

The objective of the present study was to investigate the storage stability of thymopentin multivesicular liposomes (TP5-MVLs) prepared with different emulsifiers, and to study the pharmacokinetics and pharmacodynamics of the produced TP5-MVLs in vivo. The stability studies of TP5-MVLs indicated that MVLs particles prepared with mixed emulsifiers (Myrj52:solutolHS15 = 2:3) were stable at the storage temperature of 4 +/- 2 degrees C within 3 months. In addition, FITC-TP5-loaded MVLs was prepared for pharmacokinetic studies that after subcutaneous administration, the fluorescence signal lasted for about 5 days in plasma demonstrating that the rate of drug release from MVLs was very slow. The pharmacodynamic studies indicated that the therapeutic efficacy of TP5-MVLs after subcutaneous administration once every four days was the same as free TP5 solution after intravenous or subcutaneous administration once daily. In conclusion, MVLs, which possessed great storage stability, can be utilized to reduce the administration frequency of TP5, and therefore, served as a promising sustained release delivery system for polypeptide.

An LC-MS/MS assay to determine plasma pharmacokinetics of cyclic thymic hexapeptide (cTP6) in rhesus monkeys.

A robust and simple method for absolute quantification of a novel bidirectional immunomodulatory drug candidate, cyclic thymic hexapeptide (cTP6), in rhesus monkey plasma was developed and validated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Plasma proteins were precipitated by adding four volumes of acetonitrile. Peptides in the supernatant were separated by liquid chromatography on an Agilent Zorbax Eclipse Plus-C18 chromatographic column with gradient elution using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) at 0.2 mL/min. The analytes were identified by triple quadrupole mass spectrometry in positive ion-mode. The assay was linear over a concentration range of 10-5000 ng/mL for cTP6, with a lower limit of quantification (LLOQ) of 10 ng/mL. Intra- and inter-day precision of the assay at three concentrations were 1.51-7.70% with accuracy of 95.1-104.2%. The average recovery of cTP6 for three concentration levels was 59.6-64.0%. No significant matrix effect was observed. Peptide cTP6 was detected in plasma of live rhesus monkeys up to 6-8h after intra-muscular injection. The half-life was 2.24-2.95 h. The result revealed a nonlinear pharmacokinetic response to increasing doses of cTP6 (100, 200, 500 µg/kg). For the multiple dose study of cTP6, the drug did not accumulate during daily administration at 100 µg/kg for 7 consecutive days in rhesus monkeys.

Box-Behnken optimization design and enhanced oral bioavailability of thymopentin-loaded poly (butyl cyanoacrylate) nanoparticles.

This study was done to prepare thymopentin (TP5)-loaded poly (butyl cyanoacrylate) nanoparticles (TP5-PBCA-NPs) and evaluate thier efficacy for oral delivery. TP5-PBCA-NPs were prepared by emulsion polymerization, and the formulation was optimized based on Box-Behnken experimental design. The physico-chemical characteristics of TP5-PBCA-NPs were evaluated using transmission electron microscopy (TEM), malvern zetasizer, Fourier transform infra-red spectroscopy (FT-IR) and differential scanning calorimetry (DSC). The encapsulation efficiency, enzymatic degradation and release behavior of TP5-PBCA-NPs in various media were evaluated using a high-performance liquid chromatography (HPLC) method. The pharmacodynamic studies on oral administration of TP5-PBCA-NPs were performed in FACScan flow cytometry. An optimum formulation consisted of 0.7% poloxamer 188 (Pol), 0.6% dextran-70 (Dex), 0.1% sodium metabisulfite (Sm), 0.1% TP5 and 1% (v/v) n-butyl cyanoacrylate. The particle size and zeta potential of optimized TP5-PBCA-NPs was 212 nm and -22.6 mV respectively with 82.45% encapsulation efficiency. TP5 was entrapped inside the nanoparticles in molecular dispersion form. The release of TP5 from PBCA-NPs was pH dependent; the cumulative release percentage in 0.1 M HCI for 4 hours was less than 16% while it was more than 80% in pH6.8 PBS. The PBCA-NPs could efficiently protect TP5 from enzymatic degradation; the remained percentage of TP5 encapsulated in PBCA-NPs was 58.40% after incubated with trypsin in pH6.8 PBS for 4 h while it was only 32.29% for free drug. In the oral administration study in vivo, the lowered T-lymphocyte subsets values were significantly increased and the raised CD4+/CD8+ ratio was evidently reduced compared with that of TP5 solution (p < 0.05), and the improvement of bioavailability was dose-dependent. These results indicated that the PBCA nanoparticles may be a promising carrier for oral delivery of TP5.

The influence of lipid characteristics on the formation, in vitro release, and in vivo absorption of protein-loaded SLN prepared by the double emulsion process.

PURPOSE: To study the influence of lipid characteristics on the formation, in vitro release, and in vivo absorption of solid lipid nanoparticles (SLN) prepared by the double emulsion method. METHODS: Stearic acid (SA), octadecyl alcohol (OA), cetyl palmitate (CP), glyceryl monostearate (GM), glyceryl palmitostearate (GP), glyceryl tripalmitate (GT), and glyceryl behenate (GB) were selected as the representatives of different kinds of lipids, insulin and thymopentin (TP5) were selected as the model protein drugs. Before preparation, the contact angles between water and lipids were determined to investigate their hydrophobicity. The influence of lipid hydrophobicity or lipid solution viscosity on the preparation of primary emulsion, double emulsion, and SLN were studied by evaluating the particle size, state, and stability of the systems. CP-SLN, GT-SLN, and GP-SLN were selected to be loaded with insulin and TP5 for the in vitro release and in vivo absorption examination. After oral administration to diabetic rats, the pharmacological availability (PA) of insulin-CP-SLN, insulin-GP-SLN, and insulin-GT-SLN were determined. RESULTS: The hydrophobicity order of the lipids was GM

Inhalable microparticles as carriers for pulmonary delivery of thymopentin-loaded solid lipid nanoparticles.

PURPOSE: Microparticles containing solid lipid nanoparticles (SLNs) are receiving increased attention as carriers for the lung delivery of the SLNs. Thus, we aim to prepare the hybrid microparticles and thoroughly evaluate their feasibility for the pulmonary drug delivery. METHODS: The microparticles were prepared by co-spray-drying the thymopentin (TP5)-loaded SLNs with bulking agents. Thereafter, we systematically estimated the potential of the microparticles as the carriers for the pulmonary delivery of the SLNs, including the investigations of their characteristics, aerodynamic properties, pharmacokinetics and pharmacodynamics. RESULTS: The spherical and hollow microparticles presented a size of 4.1 +/- 0.1 microm and a low tap density of 0.175 +/- 0.02 g/cm(3). In addition, the microparticles showed a high aerosolization efficiency (emitted dose of 98.0% +/- 1.23% and respirable fraction of 51.07% +/- 1.21%). Furthermore, the SLNs could be easily recovered from the microparticles without essential changes on their characteristics and the drug release behavior. The pharmacokinetic and pharmacodynamic studies suggested that, compared to i.v. TP5 solution, the bioavailability and therapeutic efficacy of TP5 were remarkably strengthened after the pulmonary administration of the microparticles. CONCLUSIONS: Taken together, we believe the microparticles were suitable for inhalation and possessed an ample potential for the pulmonary delivery of the SLNs.

Synthesis, characterization and biological activities of thymopentin ethyl ester.

The ethyl ester derivative of thymopentin was synthesized and characterized. The biological activities of thymopentin were evaluated by mouse spleen lymphocyte proliferation test and superoxide dismutase activity assay. Compared with thymopentin, the synthesized ethyl ester showed more potent immunoregulation activity in the MTT assay and anti-oxidation activity in the immune-suppressed rat model induced by hydrocortisone. The half life of the ester derivate in rat plasma determined by HPLC was slightly longer than that of thymopentin. The ester derivate gained advantages in activity and stability compared to thymopentin.

[Preparation of sustained release multivesicular liposome for thymopentin and preliminary study on its pharmacokinetics in rats].

To optimize the formulation and preparation method of multivesicular liposome of thymopentin and to investigate its pharmacokinetics in rats, the multivesicular liposome of thymopentin was prepared by double emulsification method and the formulation was optimized by orthogonal design. The release characteristics of thymopentin from multivesicular liposome in PBS (pH 7.4) and in plasma were investigated. The multivesicular liposome of thymopentin labeled with fluorescein isothiocyanate was prepared by double emulsification method. Its pharmacokinetics was evaluated following intramuscular injection in rats. The optimal formulation of multivesicular liposome of thymopentin were formulated with 7.5% glucose in aqueous phase and 2.25 mol x L(-1) triolein, 2.68 mol x L(-1) DPPG and 16.96 mol x L(-1) DOPC in organic phase. The entrapment efficiency of the multivesicular liposome of thymopentin was above 85% and the mean particle size was about 22 microm. The in vitro release of thymopentin from multivesicular liposome in PBS (pH 7.4) and in plasma was found to be in a sustained manner. The release curves were fitted to Higuchi equation. The pharmacokinetics following intramuscular injection of the multivesicular liposome of thymopentin labeled with fluorescein isothiocyanate in rats showed that the peak concentration of thymopentin was lower and elimination of it was slower significantly than that of thymopentin labeled with fluorescein isothiocyanate solution in the same dose. The plasma concentration of thymopentin maintained above quantitative limitation at 120 h after administration of multivesicular liposome of thymopentin. The optimized formulation and preparation technology of multivesicular liposome of thymopentin with higher entrapment efficiency are feasible with good reproducibility. Multivesicular liposome of thymopentin showed significant sustained-release property following intramuscular injection in rats.

Preparation and evaluation of poly-butylcyanoacrylate nanoparticles for oral delivery of thymopentin.

Thymopentin (Tp5) was loaded in poly-butylcyanoacrylate (PBCA) nanoparticles (NP) in order to enhance the oral bioavailability of Tp5. PBCA-Tp5-NP was prepared by nanoprecipitation methods. Dialyzing membrane method was employed to examine the in vitro release of PBCA-Tp5-NP in PBS, and Tp5 samples in the release medium were detected by HPLC. The cell proliferation test ((3)H-thymidine) was conducted to verify the PBCA-Tp5-NP bioactivity in vitro. The pharmacodynamical studies were performed on preimmunoinhibited rats and in flow cytometer. The size and the entrapment efficiency of PBCA-Tp5-NP were 178 +/- 39 nm and 92.21 +/- 1.08%, respectively. In vitro release data show that less than 60% Tp5 was released from lyophilized PBCA-Tp5-NP while 80% Tp5 was released from the colloidal PBCA-Tp5-NPs in 48 h. The proliferation test showed that PBCA-Tp5-NP had the similar effect as Tp5. The in vivo data showed that oral PBCA-Tp5-NPs had similar function as what intravenous Tp5 did. The oral bioavailability of Tp5 could be enhanced by PBCA nanoparticles. PBCA-Tp5-NP had the property of sustained-release and the efficacy of Tp5 was not changed after formulation.

[Research on thymopentin-loaded N-trimethyl chitosan nanoparticles administered through mouth].

OBJECTIVE: To prepare a peroral thymopentin-loaded N-trimethyl chitosan chloride-nanoparticle (Tp5-TMC-NP) ,and observe the pharmacodynamic action when the Tp5-TMC-NP is taken by way of the mouth. METHODS: N-trimethyl chitosan chloride was first synthesized, and then Tp5-TMC-NP was prepared with the formulation technology optimized by the Central Composite Design. The influence of Tp5-TMC-NP on the ratio of CD4+/CD8+ of T-lymphocytes were determined by flow cytometer. RESULTS: The regular global Tp5-TMC-NP prepared with the optimized formulation craft had the mean diameter of 110.6 nm and got the entrapment efficiency of 78.8%. The ratio of lymphocyte CD4+/CD8+ of Wistar rat administered with Tp5-TMC-NP perfusing stomach had 2.59 times higher than that with Tp5. CONCLUSION: Taken orally the Tp5-TMC-NP has much higher efficiency than Tp5.

Lectin-conjugated PLGA nanoparticles loaded with thymopentin: ex vivo bioadhesion and in vivo biodistribution.

The conjugation of lectins onto PLGA nanoparticles has been demonstrated to effectively improve the intestinal absorption of thymopentin. In this study, thymopentin-loaded nanoparticles made from fluorescein isothiocyanate labeled PLGA were modified with wheat germ agglutinin (WGA). The specific bioadhesion of nanoparticles on rat intestinal mucosa was studied ex vivo. An important increase of interaction between WGA-conjugated nanoparticles and the intestinal segments was observed compared with that of the unconjugated one (p<0.05). Fluorescence photomicrographs confirmed the bioadhesion of WGA-conjugated nanoparticles on intestinal villous epithelium as well as Peyer's patches. Biodistribution of nanoparticles was evaluated using tissues obtained from rats, to which nanoparticles were orally administered. The highest amount of WGA-conjugated nanoparticles was detected in small intestine, suggesting an increase of intestinal bioadhesion and endocytosis. The systemic uptake was as high as 6.48-13.4% of dose at 1 day and 7.32-15.26% at 7 days, which representing an increase of almost 1.4-3.1 fold across the intestine compared to <4.9% of the unconjugated one. The enhanced uptake was related to the increasing of WGA density on nanoparticles. These results further revealed the promising potential of lectin-conjugated nanoparticles on the improvement of intestinal bioadhesion and absorption for oral drug delivery.

Thymopentin-loaded pH-sensitive chitosan nanoparticles for oral administration: preparation, characterization, and pharmacodynamics.

Thymopentin, a potent immunomodulating drug, was incorporated into pH-sensitive chitosan nanoparticles prepared by ionic gelation of chitosan with tripolyphosphate anions and then coated with Eudragit S100 to improve the stability and the oral bioavailability. Nanoparticles particle size and zeta potential were measured by photo correction spectroscopy and laser Dopper anemometry. Its morphology was examined by environment scan electron microscope. The encapsulation efficiency and the release in vitro were determined by HPLC. Enzymatic stabilization was expressed by the enzymatic degradation of aminopeptidase. Biological activity of TP5 loaded in nanoparticles was assayed by lymphocyte proliferation test in vitro and the immune function (CD4+/CD8+) of irradiated rat in vivo. The results obtained demonstrated that the average sizes of pH-sensitive chitosan nanoparticles were 175.6 +/- 17 nm, the zeta potential was 28.44 +/- 0.5 mV and the encapsulation efficiency was 76.70 +/- 2.6%. The cumulative release percentages of thymopentin from the pH-sensitive nanoparticles were 24.65%, 41.01%, and 81.44% incubated in different medium, 0.1 N HCl, pH 5.0 PBS, and pH 7.4 PBS, respectively. The pH-sensitive chitosan nanoparticles could efficiently protect TP5 from enzymatic degradation and prolong the degradation half-time of TP5 from 1.5 min to 15 min. It was demonstrated from the lymphocyte proliferation test that the nanoparticle-encapsulated TP5 still kept its biological activity. In immunosuppression rats, the lowered T-lymphocyte subsets values were significantly increased and the raised CD4+/CD8+ ratio was evidently reduced. These results indicated that pH-sensitive chitosan nanoparticles may be used as the vector in oral drug delivery system for TP5.

Pharmacokinetics, toxicity of nasal cilia and immunomodulating effects in Sprague-Dawley rats following intranasal delivery of thymopentin with or without absorption enhancers.

Thymopentin (TP 5), a synthetic pentapeptide, has been used in clinic as a modulator for immnuodeficiencies through intramuscular administration. The objectives of this study was to investigate the pharmacokinetics using normal rats and toxicity of nasal cilia as well as immunomodulating effects using immunosuppression rats after intranasal delivery of thymopentin with or without an absorption enhancer. The absorption extent of fluorescein isothiocyanate (FITC) labeled TP 5 via nasal delivery at a single dose is significantly improved by incorporating sodium deoxycholate, Brij 35 and chitosan, respectively. FITC-TP 5 can also be absorbed to such an extent ranging from 15 to 28% after intranasal administration of FITC-TP 5 alone, FITC-TP 5 with sodium caprylate, or with bacitracin, respectively. After seven consecutive days multiple dosing, TP 5 formulation with sodium deoxycholate or Brij 35 caused apparently injury to nasal cilia, indicating these two enhancers would not be appropriate for nasal delivery. Results from superoxide dismutase activity, maleic dialdehyde, T-lymphocyte subsets (CD3+, CD4+, CD8+ and CD4+/CD8+ ratio) analyses suggest that all the selected enhancers improve the modulating effects of TP 5 in the immunosuppression rats. On an overall evaluation, intranasal TP 5 alone, TP 5 with chitosan, or TP 5 with bacitracin formulation may be suitable for the future clinical application.

Vaccine adjuvants: A new hope for effective immunization

Two important issue regarding the use of immunization to control infections and malignancies in the futureare: 1) the need to render poorly immunogenic, often highly purified, antigens more effective; and 2) the desire to direct the immune response in specific ways to achieve the most relevant response for each disease. The first issue can be solved by a broad range of vaccine adjuvants. The second requires careful selection among the adjuvants to allow directing of the immune response in the most appropriate manner. For exemple, in different settings expansion of a B cell response, cytotoxic T cell response, or enhancement of either a Th1 or Th2 subset response may be desired. These goals are accomplished by the use of several newly developed non-cytokine adjuvants, or by direct injection of the relevant cytokines. Some non-cytokine molecular adjuvants and cytokines used as adjuvants have already been proven effective in animal models and/or in clinical trials. Here, we review the present state of art in the use of vaccine adjuvants for control of various infections diseases.