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.

[Concentrations of MMP-1, TIMP-1, MMP-1/TIMP-1 and I CTP complexes in follicular fluid as related to fertilization rate in women treated with in-vitro fertilization]. / Stezenie MMP-1, TIMP-1, kompleksów MMP-1/TIMP-1 i I CTP w plynie pecherzykowym w odniesieniu do wskaznika zaplodnienia komórek jajowych u kobiet leczonych metoda zaplodnienia pozaustrojowego.

OBJECTIVES: Metalloproteinases and their inhibitors appear to control connective tissue remodelling during follicular rupture. The aim of the study was to establish if oocytes fertilisation rate after ovulation induction depends on the concentrations of MMP-1, its inhibitor TIMP-1, MMP-1/TIMP-1 complex and I CTP in follicular fluid (FF). MATERIAL AND METHODS: FF were collected from 37 infertile patients undergoing ovulation induction using either short or long protocol. FF was obtained 36 hours after administration of hCG (Pregnyl). The level of MMP-1, TIMP-1, MMP-1/TIMP-1 complex were measured using ELISA kits and I CTP, E2, FSH, LH, using RIA assay kits. RESULTS: Statistically significant (p < 0.05) difference was found in TIMP-1, E2, and FSH concentration, being higher in the group with more than 75% fertilisation rate: TIMP-1 728.8 + 100.1 ng/ml vs 666.3 + 94.5 ng/ml; E2 477.3 +/- 160.0 ng/ml vs 368.0 +/- 190.0 ng/ml and FSH 7.27 +/- 1.45 mIU/ml vs 6.24 +/- 1.6 mIU/ml. CONCLUSIONS: Statistically significant increase in TIMP-1 concentration observed among patients with fertilisation rate above 75% indicates an important role of this substance in ovulation process.

Stimulation of nonspecific resistance by thymopentin and its analogs against Leishmania donovani infection in hamsters.

Thymopentin and its analogs have been synthesized by the solution phase method of peptide synthesis and evaluated for their prophylactic efficacy against L. donovani infection in hamsters. Thymopentin and some of the analogs were found to stimulate nonspecific resistance of the host against Leishmania donovani infection in hamsters.

Isothermal and nonisothermal decomposition of thymopentin and its analogs in aqueous solution.

PURPOSE: The degradation kinetics of thymopentin (RKDVY) and its analogs (RKDVW and RPDVY) in aqueous solution was studied by isothermal and nonisothermal methods. METHODS: The isothermal decomposition of thymopentin and its analogs was investigated as a function of pH (2-10), temperature (37, 57, and 80 degrees C) and ionic strength (micro = 0.02 to 1). Nonisothermal decomposition studies were performed using a linear temperature programmer. The temperature increasing rate was set to 0.25 degrees C per hour and the temperature interval varied from 40 to 88 degrees C. RESULTS: The decomposition of thymopentin and its analogs followed first order kinetics. The dependence of the rate constant on temperature followed a linear Arrhenius plot. This indicated that the degradation mechanism of thymopentin and its analogs might be the same within the temperature range studied. The energies of activation were found to be in close agreement for the isothermal and nonisothermal studies, suggesting that the nonisothermal studies may save considerable amount of time in the early stages of drug development. The logK-pH profile of thymopentin suggests that maximum stability is achieved in the pH range of 6-8. CONCLUSIONS: These results indicate that the nonisothermal methodology provides an attractive alternative to isothermal methods, as it requires a much lower amount of both material and time, to determine the peptide stability and to estimate the shelf-life for peptide pharmaceutical preparations.

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.

Stabilized analogs of thymopentin. 3. Evaluation of ketomethylene pseudopeptides for antiarthritic properties.

This study analyzed the role of ketomethylene pseudopeptides of thymopentin as potential agents for the treatment of arthritis. The analogs were tested in vivo using assessment of inflammation and antibody production in the mouse type II collagen arthritis model and the rat adjuvant arthritis model. The compounds were also tested for immune-potentiating activity in vitro using induction of the lymphocyte marker, Thy-1.2, in mouse spleen cells and stimulation of T-cell proliferation. The results show that certain of the compounds exhibit disease-remitting properties for arthritis as evidenced by reduction of paw swelling in the mouse and rat models and decreased incidence of disease in the mouse model. The active compounds were dose specific and represented a range in efficacy. In spite of effects on arthritis, type II collagen antibody levels were not altered in the mouse model. Selected compounds also exhibited immune potentiating properties as evidenced by induction of Thy-1.2 expression and stimulation of T-cell proliferation. The absence of effect of the compounds on type II collagen antibody production suggests that the antiarthritic activity of the effective compounds results from alteration of cell-mediated immunity.

Comparison of the photochemical behavior of four different photoactivatable probes.

The most commonly used photoaffinity labeling probes are compared, which are aryl azides, aryl diazirines, alpha-diazocarbonyls and benzophenone-derivatives. The compounds were used under identical conditions and crosslinking efficiency, influence of water, irradiation requirements, and by-products were investigated. Using the pentapeptide thymopentin (TP5) as a model system, we synthesized four analogues by solid-phase peptide synthesis and partially N-terminal modification to obtain [p-(3-trifluoromethyl)diazirinophenylalanine5]TP5, [p-benzoylphenylalanine5]TP5, 4-azidobenzoyl-TP5 and 2-diazo-3,3,3-trifluoropropionyl-TP5. The peptides were characterized by HPLC and ion-spray mass spectroscopy. Irradiation of the peptides with two different ultraviolet sources was carried out in water, n-propanol and water/n-propanol to imitate both hydrophobic and hydrophilic peptide/protein-interactions as well as the influence of the aqueous environment. Analysis of the products with HPLC, ion-spray MS, HPLC-MS and HPLC-CID-MS revealed that (Tmd)Phe is a highly potent carbene-precursor, which can be transformed easily into uniform crosslinking products by smooth photolysis. However, the electrophilic nature of the intermediate causes a high tendency to react with water molecules. The 4-azidobenzoyl group showed comparable crosslinking efficiency, but the probability to create non-uniform irradiation products (e.g. through rearrangement) is higher, whereas the reaction with water is less dominant. In contrast, Bpa was found to have an extremely low affinity to react with water, whereas prolonged UV irradiation is needed to get complete rearrangement into a variety of products. As the absorption band of alpha-diazocarbonyls at around 350 nm possesses a low extinction coefficient, 2-diazo-3,3,3-trifluoropropionyl-TP5 could not be activated at all with the optimized irradiation conditions that we have chosen for our comparative studies.

Further investigations on thymopentin-like fragments of HLA-DQ and their analogs.

Recently we showed that the fragments of HLA-DQ with the Thr-Pro-Gln-Arg-Gly-Asp-Val-Tyr-Thr and Gln-Arg-Gly-Asp-Val-Tyr-Thr sequences strongly suppress the immune response, while their shorter analogs, Arg-Gly-Asp-Val, Arg-Gly-Asp-Val-Tyr and Gln-Arg-Gly-Asp-Val-Tyr, show very weak stimulatory activity with respect to humoral immunological response. The fragments contain the sequence which is very similar to thymopentin (pentapeptide Arg-Lys-Asp-Val-Tyr, an active fragment (32-36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the Arg-Gly-Asp (RGD) sequence, known as an inhibitor of adhesion processes. In the present study we found that a hexapeptide: Arg-Gly-Asp-Val-Tyr-Thr is the smallest size fragment of HLA-DQ having both cellular and humoral immunosuppressive activity. We also found that linear and cyclic fragments of HLA-DQ do not affect cell line production of various cytokines, what suggests that the mechanism of interactions of these peptides with the immunological system is different as compared with most other known immunosuppressors.

IRI-514, a synthetic peptide analogue of thymopentin, reduces the behavioral response to social stress in rats.

Thymopentin, a synthetic pentapeptide (Arg-Lys-Asp-Val-Tyr) corresponding to amino acids 32-36 of the thymic polypeptide thymopoietin, has been reported to block adrenocorticotrope responses to stress. The purpose of the present study was to explore potential antistress properties of a synthetic analogue of thymopentin, IRI-514 (Ac-Arg-Pro-Asp-Phe-NH2) using a behavioral response to a stressor. The behavioral response to social conflict stress (resident-intruder paradigm) was evaluated by the elevated plus-maze test of anxiety in adult Wistar rats. A single subcutaneous (SC) administration of IRI-514, 48 h before stress, dose-dependently reversed the anxiety-like behavior induced by the social stress. The effect of IRI-514 was present over an extended period (24-72 h) following SC administration and was maximally effective at a dose of 1 mg/kg. These results indicate that IRI-514 has a long-lasting modulatory effect on behavioral responses to a stressor, and suggest that thymopoietin-derived peptides may have a role in modulating both behavioral and neuroendocrine responses to stress.

Immunological properties of the thymopentin-like fragments of HLA-DQ.

Class II human leukocyte antigens (HLA-II) are cell surface alpha beta heterodimers (M(r) approximately 60,000) that play a pivotal role in the immune response by presenting peptides derived from environmental antigens to the T-cell receptor. A 167-171 fragment of the beta 2-chain of the HLA-DQ molecule consists of the sequence RGDVY, which is very similar to thymopentin (pentapeptide RKDVY, an active fragment (32-36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the RGD sequence, known as an inhibitor of adhesion processes. We synthesized and investigated the immunomodulatory activity of series of peptide fragments of HLA-DQ containing thymopentin-like sequences. The results indicate that all synthesized peptides suppress the cellular immune response. However, RGDV, RGDVY and QRGDVY show very weak stimulatory activity in humoral immunological response tests. In contrast to the shorter peptides, the nonapeptide fragment of HLA-DQ, TPQRGDVYT, shows significant immunosuppressive activity in all tests. A possible role of these fragments of the polypeptide chain of HLA-DQ in the regulation of HLA functions is discussed.

Immunomodulatory activity of a potent thymopentin analog: disulphide bridged beta-mercaptopropionyl-arginyl-lysyl-aspartyl-valyl-tyrosyl-cysteine amide.

The immunomodulatory activity of the disulphide bridged heptapeptide Mpa-Arg-Lys-Asp-Val-Tyr-Cys-NH2 (CTP-1), containing the thymopentin (TP5) sequence (Arg-Lys-Asp-Val-Tyr), was investigated by different immunological tests. The activity of CTP-1 was compared with the activities of two other disulphide bridged TP5 analogs: Mpa-Arg-Lys-D-Pro-Val-Tyr-Cys-NH2 (CTP-2) and Mpa-Arg-Lys-Asp-Val-Pro-Cys-NH2 (CTP3). The results obtained indicate that from the whole series of TP5 analogs CTP-1 may be of interest from the point of view of its prospective medical use. The results of autologous rosette forming cells (ARFC-test), as well as the direct examination of the influence of CTP-1 on the interleukin-1 (IL-1) production by P-388 D1 cell line evidence that the mechanism of CTP-1 action involves stimulation of IL-1 production. The results of E-rosette assay, ARFC test, and imuran test show that the activity of CTP-1 is stronger than that of TP5.

Folding and secretion of Saccharomyces cerevisiae carboxypeptidase Y are influenced by fusion with short heterologous peptides.

High levels of secreted yeast carboxypeptidase Y can be obtained in yeast by regulated overexpression under the control of the GAL1 promoter. Carboxypeptidase Y was investigated as a potential carrier for expression of heterologous oligopeptides. Coding sequences for two pentapeptides, Hepp (H-Asp-Ser-Asp-Pro-Arg-OH) and a thymopentin analogue (H-Arg-Pro-Asp-Val-Tyr-OH), and an analogue of salmon calcitonin, were inserted into, or added on to, the coding sequence for carboxypeptidase Y, and the plasmids were introduced into a yeast mutant that mis-sorts vacuolar proteins. Translation efficiency of mRNA from the expression plasmids encoding hybrid carboxypeptidase Y was apparently not influenced by the insert. However, secretion of the hybrid proteins was lower than that of wild-type carboxypeptidase Y. A major fraction of the hybrid proteins accumulated intracellularly as a form characteristic for the endoplasmic reticulum. The results suggest that the inserted peptides influenced the secretion through the position and sequence effect on post-translational events. Furthermore, studies on folding properties indicated that the in vitro refolding capacity of the hybrid proteins was reduced. Thus, in the case of insertions, the transition from an unfolded chain to the correctly folded protein was likely to be disfavoured by misfolding. However, the tendency towards misfolding could be partially suppressed by changing the insertion site, and secretion was most effective in the cases of C-terminal fusions.

Biologically active conformations of thymopentin. Studies with conformationally restricted analogs.

Four cyclic analogs of thymopentin were synthesized and evaluated for biological activity on the human T cell line CEM. Three of these conformationally restricted analogs were biologically active. The one analog which most closely mimicked the conformation predicted from NMR and theoretical energy minimization calculations proved to be inactive. These studies establish that the biologically active conformations of thymopentin differ from the most probable conformation predicted from solution NMR and theoretical energy minimization studies.

Design of a novel thymopoietin analogue based on conformational analyses.

A novel cyclic peptide c(Arg-Pro-Asp-D-Val-Tyr) related to thymopentin--the immunostimulant pentapeptide contained in thymic hormones--was designed on the basis of theoretical computer modeling. We applied molecular dynamics/energy minimization techniques and restrained molecular dynamics to determine the preferred conformation of this peptide. The linear precursor of the peptide is biologically active and probably exists in a highly motile dynamical equilibrium of different conformations. Our calculations show that the cyclic peptide consists of a single conformational family containing a beta turn at position Pro 2. Experimental support for this conclusion was derived from 2-D NOE data in aqueous solution for the closely related analogue c(Arg-Lys-Glu-D-Val-Tyr). Synthesis and biological testing of the cyclic peptide is therefore indicated.