[Stability evaluation of thymopentin in preparation process].

OBJECTIVE: To observe whether thymopentin is stable in the preparation process. METHODS: Using HPLC method, we investigated the influence of different temperature, sonification time and pH value on thymopentin. RESULTS: The optimum storage condition for preservation of thymopentin was found to be a freezing environment. Thymopentin would not be degraded in the water bath of 60 degrees C for 24 hours or in different phosphate buffer solution (PBS) (pH 2, 5, 5, 7) under 37 degrees C for 24 hours. The sonification time within 15 minutes was determined to be safe for the preparation. CONCLUSION: The experiment result indicates that thymopentin is stable during the whole preparation process.

Synthesis of [I-NaI3]] thymopoietin II and examination of its immunological effect on the uremic T-lymphocytes.

A TP II analogue, [1-Nal3] TP II, was synthesized by a conventional solution method, followed by deprotection with 1M TFMSA-thioanisole (molar ratio 1:1) in TFA in the presence of Me2Se and m-cresol as scavengers. The synthetic [1-Nal3] TP II, TP II and [Phe (4 F)3] TP II were tested for comparative effect on the impaired T-lymphocyte transformation by PHA in uremic patients suffering from recurrent infectious diseases. The synthetic analogue was found to have stronger restorative activity than those of our synthetic TP II and [Phe (4F)3] TP II.

Synthesis of [Phe(4F)3]thymopoietin II and examination of its immunological effect on the impaired blastogenic response of T-lymphocytes of uremic patients.

[Phe(4F)3]thymopoietin II was synthesized using a conventional solution method. The deprotection of the protected [Phe(4F)3]thymopoetin II was achieved by treatment with 1 M trifluoromethanesulfonic acid:thioanisole (molar ratio 1:1) in trifluoroacetic acid in the presence of dimethylselenide and m-cresol. The synthetic f1p4(4F)3]thymopoietin II and thymopoietin II were tested for effect on impaired T-lymphocyte transformation by phytohemagglutinin in uremic patients suffering from recurrent infectious diseases. The restoring activity on the impaired phytohemagglutinin stimulation of T-lymphocytes was obtained after incubation of peripheral lymphocytes isolated from uremic patients with the synthetic [Phe(4F)3]thymopoietin II. This peptide exhibited far stronger restoring effect than that of our synthetic thymopoietin II.

Solid-phase peptide synthesis and biological activity of bovine thymopoietin II (bTP-II).

Bovine thymopoietin (bTP), a 49 amino acid polypeptide, was synthesized using Merrifield's solid-phase peptide synthesis methodology. The polypeptide was purified using anion-exchange chromatography and reversed-phase HPLC and characterized by mass spectrometry and amino acid analysis of the full-length peptide and of products derived from digestion with Staphylococcus aureus V8 protease. The biological activity of the synthesized product was tested in several assay systems. Synthetic bTP was found to induce the expression of Thy 1.2 antigen on T-lymphocytes from athymic mice, in agreement with previous studies on the biological activity of endogenous bTP. Biological activity at skeletal muscle and neuronal nicotinic acetylcholine receptor sites, as reported by others for bTP, could not be confirmed in our studies. The absence of biological activity at nicotinic receptor sites may be related to the results of a recent report demonstrating the presence of a cobratoxin-like molecule in preparations of natural bTP. These data indicate that synthetic peptides have an important role for the evaluation of the specificity of the biological activity of polypeptides.

The effects of synthetic thymopoietin on binding of alpha-bungarotoxin to rat neural membranes.

Chemically synthesized bovine thymopoietin I (TPO-I) and thymopoietin II (TPO-II) were evaluated as inhibitors of 125I alpha-Bungarotoxin binding to rat brain neural membranes and found to be over 1,000 x less potent (IC50 = 10 microM) than reported for thymopoietin isolated from bovine thymus.

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.

Synthesis of human splenin (hSP) and examination of its immunological effects on the impaired T- and B-lymphocytes in uremic patients.

Human splenin (hSP) was synthesized by assembling eight peptide fragments followed by deprotection with 1M trifluoromethanesulfonic acid-thioanisole (molar ratios, 1:1) in trifluoroacetic acid in the presence of m-cresol and dimethylselenium. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulfoxide on the methionine side chain. Incubation of peripheral lymphocytes isolated from uremic patients with the synthetic hSP showed an enhancing effect on the reduced B-lymphocytes, but had no restoring effect on the impaired blastogenic response of T-lymphocytes.

Syntheses and effects of human splenin (hSP) fragment 32-48 and an analog on the reduced B-lymphocytes of uremic patients.

A heptadecapeptide, H-Arg-Lys-Ala-Val-Tyr-Val-Glu-Leu-Tyr-Leu-Gln-Ser-Leu-Thr-Ala-Glu-His-OH , corresponding to amino acids 32 to 48 of human splenin (hSP) and an analog in which the amino acid residue at position 34 is changed from Ala to Glu, were synthesized. These peptides were synthesized using conventional solution synthesis and were tested for their effect on reduced B-lymphocytes of uremic patients. Incubation of peripheral lymphocytes isolated from uremic patients with these two synthetic heptadecapeptides, hSP fragment 32-48 and [Glu34]hSP fragment 32-48, had an enhancing effect on the reduced B-lymphocytes, but synthetic bovine thymopoietin II (bTP-II) fragment 32-49 had no effect under the same conditions.

Synthesis of [Glu34]human splenin (hSP) and examination of its immunological effect on the reduced B-lymphocytes of uremic patients.

[Glu34]human splenin (hSP) was synthesized in a conventional manner by assembling ten peptide fragments followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole (molar ratio, 1:1) in trifluoroacetic acid in the presence of m-cresol and dimethylselenide. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulfoxide on the methionine side chain. Incubation of peripheral lymphocytes isolated from uremic patients with the synthetic [Glu34]hSP showed an enhancing effect on the reduced B-lymphocytes, but synthetic human thymopoietin (hTP) had no effect under the same conditions.

Splenopentin--influence on antibody formation in immunosuppressed animals and on phagocytic capability of human granulocytes.

Sublethally x-ray irradiated C57 Bl/6 Bln. mice (whole body irradiation with 600 cGy) were treated with or without a splenopentin derivative (DA SP-5: N alpha-acetyl-L-arginyl)-(N alpha-acetyl-L-lysyl)-L-glutamyl-L-valyl-L-tyrosine and compared for their capacity to produce antibodies against target sheep red blood cells. As demonstrated DA SP-5 treated mice produced antibodies earlier and in a higher level than animals untreated. Furthermore, DA SP-5 influences the phagocytic capability of human granulocytes in a dose dependent matter.

Synthesis of two biologically active fluorescent probes of thymopentin.

This study reports on the synthesis of two fluorescent analogues of thymopentin (TP-5; Arg-Lys-Asp-Val-Tyr). A fluorescein isothiocyanate labeled analogue (FITC-TP-5) and a stilbene isothiocyanate labeled analogue (SITS-TP-5) were extensively purified by ion-exchange and gel filtration chromatography. Characterization of the coupling site through amino acid analysis, dansylation and N-terminal cleavage of the fluorescent amino acid yielded results which indicated that both were mono-labeled analogues derivatized at the N-terminal. These analogues were shown to be TP-5-like in nature by their ability to induce the expression of the Thy 1.2 surface marker on nude mouse prothymocytes in both in vivo and in vitro assays. In addition, these analogues were able to inhibit the specific binding of radiolabeled TP-5 to human lymphocytes. Initial studies describing the interaction of FITC-TP-5 with human lymphocytes are shown.

Immunoregulating peptides II. In vitro effects of TP5 analogs on E-rosette formation and cell division.

The effects of seventeen synthetic analogs of thymopentin (TP-5) have been studied in the active and azathioprine-inhibited E-rosette tests. Thymopentin was gradually shortened from the C terminus to peptides and single amino acids. Thymopoietin 32-34 (Arg-Lys-Asp-RGH-0205-TP-3) (II) and thymopoietin 32-35 (Arg-Lys-Asp-Val-RGH-0206-TP-4) (I) were the most active peptides. Dipeptide Arg-Lys produced significant stimulatory effect on azathioprine (ED75) inhibited E-receptor. Treatment of azathioprine (ED75)-inhibited E-rosette forming cells (ERFC) with arginine or especially lysine increased the number of ERFC. Some of TP-4 analogs decreased further the number of ERFC decreased by azathioprine ED30. These "suppressive" peptides as well as TP-3 caused a partial arrest of K 562 cell proliferation up to 96 hours. Results suggest that TP-5 is not the smallest active fragment of thymopoietins, since peptides (TP-3 and TP-4) exhibit similar or higher T-cell membrane activation on E-receptor. Arginine, lysine, and acidic aspartyl residue seem to be a necessary basic structure to produce a cumulative chemical signal on the activity of T-lymphocytes.

Biologically active analogs of thymopentin with enhanced enzymatic stability.

Thymopentin, a synthetic pentapeptide fragment of thymopoietin (residues 32-36, Arg-Lys-Asp-Val-Tyr) is biologically active but susceptible to proteolytic digestion. Analogs were synthesized and studied for biological activity and susceptibility to peptidases. Amino acid changes were incorporated at positions known to not affect activity adversely and N-terminal acetylation and C-terminal amidation were used to increase resistance to proteolytic degradation by exopeptidases. Ac-Pro2-TP5-NH2 and Aib2-TP5-NH2 retained activity and were shown to exhibit a high degree of stability when incubated in human serum.

Syntheses and effects of [Glu34]-thymopoietin II fragment 32-45 and its analogs on the impaired T-cell transformation in a patient with common variable immunodeficiency.

[Glu34]-thymopoietin II fragment 32-45 and its three analogs were prepared by substitution of the amino acid residue at position 37. These peptides were synthesized by a conventional solution method, followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole in trifluoroacetic acid in the presence of m-cresol. These peptides were tested for their effects on impaired T-cell transformation by phytohemagglutinin in the common variable immunodeficiency. The relative potency of the synthetic [Glu34]-thymopoietin II fragment 32-45 was one-half of that of synthetic thymopoietin II fragment 32-45. Among these tetradecapeptide analogs, one analog in which Val37 was replaced by Ile exhibited a potent activity which was more than that of the synthetic [Glu34]-thymopoietin II fragment 32-45. The relative potencies of Thr37 and Tyr37 analogs were one-third and one-half, respectively of that of the synthetic [Glu34]-thymopoietin II fragment 32-45.

Syntheses and effects of a thymopoietin II fragment and its analogs on the impaired T-cell transformation in a patient with common variable immunodeficiency.

A tetradecapeptide, H-Arg-Lys-Asp-Val-Tyr-Val-Glu-Leu-Tyr-Leu-Gln-Ser-Leu-Thr-OH, corresponding to amino acids 32 to 45 of thymopoietin II and its six analogs by replacing the amino acid residue in position 37, was prepared. These peptides were synthesized using conventional synthesis in solution and were tested for their effect on impaired T-cell transformation by phytohemagglutinin (PHA) in the common variable immunodeficiency. The tetradecapeptide had increasing activity on the T-cell transformation by PHA. Among the tetradecapeptide analogs, several analogs in which Val37 was replaced by Leu or Phe exhibited potent activity which was more than that of the parent peptide fragment. On the other hand, replacement of Val37 by Pro, beta Ala, or sarcosine had no effect at concentrations as high as 3.5 X 10(-4) M. One analog whose Val37 was replaced by Gly showed activity one-third that of the parent peptide fragment. On the basis of these results, the structure-activity relationship for the tetradecapeptide is discussed.