Novel strategy for immunomodulation: Dissolving microneedle array encapsulating thymopentin fabricated by modified two-step molding technology.

Thymopentin (TP5) is commonly used in the treatment for autoimmune diseases, with a short plasma half-life (30s) and a long treatment period (7 days to 6 months). It is usually administrated by syringe injection, resulting in compromised patient compliance. Dissolving microneedle array (DMNA) offers a superior approach for transdermal delivery of biological macromolecules, as it allows painless penetration through the stratum corneum and generates minimal biohazardous waste after dissolving in the skin. Despite recent advances in DMNA as a novel approach for transdermal drug delivery, problem of insufficient mechanical strength remains to be solved. In this study, TP5-loaded DMNA (TP5-DMNA) was uniquely developed using a modified two-step molding technology. The higher mechanical strength was furnished by employing bovine serum albumin (BSA) as a co-material to fabricate the needles. The obtained TP5-DMNA containing BSA displayed better skin penetration and higher drug loading efficiency than that without BSA. The in vivo pharmacodynamics study demonstrated that TP5-DMNA had comparative effect on immunomodulation to intravenous injection of TP5, in terms of ameliorating the CD4+/CD8+ ratio, SOD activity and MDA value to the basal level. Only mild irritation was observed at the site of administration. These results suggest that the novel TP5-DMNA utilizing BSA provides an alternative approach for convenient and safe transdermal delivery of TP5, which is a promising administration strategy for future clinical application.

Determination of thymopentin in beagle dog blood by liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study.

The pentapeptide thymopentin (Arg-Lys-Asp-Val-Tyr, RKDVY) corresponds to amino acids 32-36 of the 49 amino acid immunomodulatory polypeptide, thymopoietin, whose biological activity is partially reproduced. Thymopentin is widely used in the clinic and represents a promising target for drug design but bioanalytical and pharmacokinetic data are limited due to its enzymatic instability. This paper reports a rapid and sensitive method based on liquid chromatography with tandem mass spectrometry for the determination of thymopentin in beagle dog blood. To inactivate peptidases and stabilize thymopentin, acetonitrile was added to blood samples immediately after collection followed by addition of stable isotope-labeled thymopentin as internal standard and washing with dichloromethane. Chromatography was carried out on an Ascentis Express Peptide ES-C18 column using gradient elution with methanol and aqueous 0.1% formic acid at a flow rate of 0.6 mL/min. Positive electrospray ionization mass spectrometry with selected reaction monitoring achieved linearity in the range of 1.5-800 ng/mL with good accuracy/precision and minimal matrix effects. The method was successfully applied to a pharmacokinetic study in beagle dogs after intravenous administration of 0.2 mg/kg thymopentin.

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.

Stabilized analogs of thymopentin. 1. 4,5-Ketomethylene pseudopeptides.

The pentapeptide, thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5) is known for its activity as an immunomodulating drug, but with limited half-life in plasma. In this first paper of a series of three studies, the synthesis of analogs stabilized at the peptide bond between the C-terminal amino acids via insertion of a ketomethylene moiety is described. N-Blocked pseudopeptides containing Val(k)Phe, Ala(k)Phe, and Val(k)Val units were prepared and attached to chloromethyl Merrifield resin via the carboxy terminal. Removal of the N-BOC group by trifluoroacetic acid was followed by sequential coupling with N-BOC dipeptides of aspartic acid to yield resin-bound N-BOC pseudotetrapeptides. Removal of N-BOC and coupling with N-BOC-r-N-tosylarginine followed by total cleavage of blocking groups and resin by HF afforded the target pseudopentapeptides. The analogs were found to compete favorably with thymopentin for binding to CEM cells, but binding was reduced by about 20-30% on average. All analogs showed significant enhancement of half-life versus thymopentin in mouse serum, but most showed only modest improvement in human serum. Insertion of proline or norleucine at position 2 in the chain caused a substantial increase in half-life (3-4-fold), while N-methylnorleucine conferred complete stability in the analogs.

Stabilized analogs of thymopentin. 2. 1,2- and 3,4-ketomethylene pseudopeptides.

In this second paper in a series of three studies of stable analogs of thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5), the synthesis of analogs stabilized at peptide bonds 1,2 and 3,4 via insertion of ketomethylene units is described. A tris(carbobenzyloxy)arginyl(k)norleucine pseudopeptide was synthesized and coupled to Asp-Val-Phe-resin units followed by HF cleavage to prepare Arg(k)Nle-Asp-Val-Phe analogs. Preparation of N-BOC Asp(k)Val and N-BOC Asp(k)Ala units followed by coupling to Phe- or Tyr-resin units provided resin-bound pseudotripeptide substrates for attachment of various arginyl dipeptides. Cleavage from the resin afforded 3,4-ketomethylene-stabilized pseudopeptide analogs of thymopentin. The Arg-Lys-Asp(k)Val-Phe and Arg-Lys-Asp(k)Val-Tyr analogs were more strongly bound to CEM cells than thymopentin itself. There was significant enhancement of stability in serum for the analogs, especially those containing Arg(k)Nle or Arg-NMeLys moieties at the 1,2-peptide bond.