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.

Delta-opioid receptor antagonist inhibits immunomodulation by Met-enkephalin analogs.

The methionine-enkephalin (Met-enkephalin, Tyr-Gly- Gly-Phe-Met) analogs Tyr-D-Ala-Gly-MePhe-Met NHC(3)H(7)-iso (1) and Tyr-D-Ala-Gly-MePhe-Gly-NHC(3)H(7)-iso (2) have been shown to enhance human T cell proliferation in in vitro treatment. Their immunomodulatory activities were completely blocked by naloxone, an opioid antagonist. Now we demonstrate that a selective delta-opioid receptor antagonist, ICI-174,864, completely blocks enhancement of T cell proliferation by analogs (1) and (2). The T cell-stimulatory effect was only partially inhibited by the mu-receptor-selective antagonist, beta-funaltrexamine hydrochloride. The kappa-opioid receptor antagonist, nor-binaltorphimine dihydrochloride, showed no effect on T cell-proliferation stimulated by analogs (1) and (2). These observations suggest that analogs (1) and (2) of Met-enkephalin stimulate T cell proliferation predominantly via delta-opioid receptor present on T cells.

Structure-activity relationship studies of dynorphin A and related peptides.

An up-to-date review is presented covering all the available information concerning the isolation, discovery, synthesis, conformation, receptor binding characteristics, pharmacological properties and SAR studies of dynorphin A and related peptides. The potential of dynorphin A and its analogs has yet to be fully realized.

Thymopentin and splenopentin as immunomodulators. Current status.

Splenopentin (SP-5, Arg-Lys-Glu-Val-Tyr) and thymopentin (TP-5, Arg-Lys-Asp-Val-Tyr) are synthetic immunomodulating peptides corresponding to the region 32-34 of a splenic product called splenin (SP) and the thymic hormone thymopoietin (TP), respectively. TP was originally isolated as a 5-kDa (49-amino acids) protein from bovine thymus while studying effects of the thymic extracts on neuromuscular transmission and was subsequently observed to affect T cell differentiation and function. TP I and II are two closely related polypeptides isolated from bovine thymus. A radioimmunoassay for TP revealed a crossreaction with a product found in spleen and lymph node. This product, named splenin, differs from TP only in position 34, aspartic acid for bovine TP and glutamic acid for bovine splenin and it was called TP III as well. Synthetic pentapeptides (TP-5) and (SP-5), reproduce the biological activities of TP and SP, respectively. It is now evident that various forms of TPs were created by proteolytic cleavage of larger proteins during isolation. cDNA clones have been isolated for three alternatively spliced mRNAs that encodes three distinct human T cell TPs. The immunomodulatory properties of TP, SP, TP-5, SP-5 and some of their synthetic analogs reported in the literature have been briefly reviewed.

Immunomodulating activity of analogs of noninflammatory fragment 163-171 of human interleukin-1beta.

The synthetic nonapeptide Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys corresponding to the amino acid sequence 163-171 of human interleukin-1beta (IL-1beta) has been reported to retain considerable immunostimulatory activity of the native protein without the induction of the inflammatory or pyrogenic responses. Two lipophilic derivatives of this nonapeptide, one having a lauroyl residue (1) and the other having a palmitoyl residue (2) at the N-terminus of the peptide, and a more stable analog carrying D-Val residue at position 1 of the peptide (3) were synthesized with a view to find out if these structural modifications had a favorable effect on in vitro mouse thymocyte proliferation and IL-1 dependent inhibition of A375 cells. We have found that analogs (1) and (2) are active in both the tests like the parent nonapeptide. The lipophilic analog (2) is in fact, effective at a lower dose as compared to the parent nonapeptide in mouse thymocyte proliferation assay. Although the analog (3) has the ability to inhibit A375 cells, it does not stimulate mouse thymocyte proliferation in vitro. The IL-1beta fragment (163-171) and the analog (2) were further compared for their effects on pyrogenicity, blood glucose level, acute phase response and radioprotection. Unlike IL-1beta, its fragment (163-171) and the analog (2) do not induce pyrogenicity and any of the acute phase related changes such as the increase in C-reactive protein and hypoglycemia following their administration in Balb/c mice. We have found that 40% of animals treated with analog (2) survive more than 21 days after lethal irradiation as compared to 20% survivors in groups treated with recombinant IL-1beta or its nonapeptide fragment (163-171), under conditions when all the control animals died within 10 days. This study may help in designing small peptides which may be more effective and stable.

Synthesis and opioid activity of novel tetrapeptides analogous to sequence (1-4) of dermorphin.

Seven new tetrapeptides analogous to (1-4) sequence of dermorphin were synthesized and evaluated for their opioid activity. The peptides were synthesized by the solution phase method. Their opioid activity revealed that peptides II and V were the most potent in the analgesia test as well as in the peripheral assays. Peptide II was most active in the guinea pig ileum assay, whereas peptide VI was 2763 times more selective for mu-receptors.

Stimulation of IL-2 production and CD2R expression by splenopentin analogs.

Splenopentin (SP-5), is a pentapeptide corresponding to the amino acid sequence 32-36 (Arg-Lys-Glu-Val-Tyr) of the splenic hormone splenin. Its synthetic analogs: Lys-Lys-Glu-Val-Tyr(1) and D-Lys-Lys-Glu-Val-Tyr (2) have been evaluated for active T-cell rosette (CD2R), total T-cell rosette (CD2), interleukin-2 (IL-2) stimulation and effect on antibody production. SP-5 as well as both the analogs stimulated CD2R. Analogs (1) and (2) were also found to stimulate IL-2 production. These observations suggest that in vitro human NK cell augmentation with analogs (1) and (2) reported earlier may be due to enhanced IL-2 production.

Upregulation of HLA class-I gene transcription in K562 cells by analogs of splenopentin (SP-5)

We report here the capacity of three analogs of Splenopentin, to up regulate transcription of the HLA-B7 gene in the K562 cell line. These cells normally do not transcribe the HLA class I genes. The Splenopentin analogs used in this study are effective at very low molar concentrations and are non-toxic to cells at these levels. Electrophoretic Mobility Shift assays indicate that this transcriptional up regulation of HLA class I genes may be related to the appearance of novel class I promoter binding factors induced in the nuclei of treated cells.

Immunomodulation by two potent analogs of met-enkephalin.

Met-enkephalin (Tyr-Gly-Gly-Phe-Met) and its more stable analogs, Tyr-D-Ala-Gly-MePhe-Met-NHC3H gamma-iso (1) and Tyr-D-Ala-Gly-MePhe-Gly-NHC3H gamma-iso (2) significantly enhanced human T-cell proliferation in vitro after 5 days of incubation in the absence of mitogen. The activity was completely inhibited by naloxone, an opioid antagonist. These peptides significantly enhanced human active T-cell rosette (CD2R) also on in vitro treatment. Furthermore, these analogs stimulated interleukin-2 production by human peripheral blood mononuclear cells in vitro which was completely inhibited by naloxone. These observations suggest that human T-cells bear receptors for Met-enkephalin on their surface. Such findings may provide a link between the central nervous system and the immune system.

Molecular biology of opioid receptors: recent advances.

Endogenous opioid peptides and opiates like morphine produce their pharmacological effects through the membrane bound opioid receptors. These receptors belong to a superfamily of G-protein-coupled receptors, all of which possess seven membrane-spanning regions. Structure-activity relationship studies of opioids opened up new avenues for the pharmacological characterization of the opioid receptors. As a further advancement in this direction, molecular cloning has led to the identification of three different types of opioid receptors -- OP1 (delta), OP2 (kappa) and OP3 (mu) -- thereby supporting the results of earlier pharmacological studies which postulated their existence. The three opioid receptors are highly homologous. Consequent to the development of highly specific and selective agonists and antagonists, it was proposed that the three types of opioid receptors could be further categorized into different subtypes. However, the molecular biology data generated so far do not support the presence of the various subtypes of the three well-characterized opioid receptors. Recent strides towards the advancement of our knowledge relating to the molecular biology of these receptors have been reviewed in this article.

Synthesis and immunostimulant activity of novel analogs of human casein fragment (54-59).

Structural analogs of the hexapeptide sequence 54-59 (A) human casein, reported to stimulate immune response, were synthesized and evaluated for immunostimulant activity. Hexapeptide 91/409 (C), 90/649 (D) and 91/361 (E) stimulated higher antibody titre and delayed type of hyper-sensitivity (DTH) response than the natural casein hexapeptide in BALB/c mice-sheep red blood cells (SRBC) and guinea pig-ovalbumin models. These peptides also induced higher stimulation of non-specific immune response as evidenced by increase in macrophage migration index (MMI), phagocytosis of (14C) lecuine labelled Escherchia coli, incorporation of (14C)-glucosamine in peritoneal macrophages and proliferative response of mouse thymocytes. Significant suppression on the course of Plasmodium berghei infection was also observed on day 4, in the animals treated with hexapeptidse C and D.

Leishmania donovani in hamsters: stimulation of non-specific resistance by novel lipopeptides and their effect in antileishmanial therapy.

Several novel type of lipopeptides were synthesized and evaluated for their ability to stimulate non-specific resistance against Leishmania donovani infection. Peritoneal macrophages isolated from young male hamsters treated with muramyl dipeptide (MDP) and various synthetic lipopeptides (6 mg/kg i.p.) 7 days earlier, were cultured in vitro and challenged 24 h later with L. donovani promastigotes. One lipopeptide, Central Drug Research Institute (CDRI) compound 86/450, exhibited significantly higher immunostimulatory activity than MDP. Its prophylactic activity was further confirmed in hamsters by giving 2 split doses of 3 mg/kg of the compound spaced at 2 weeks, i.e. on day -7 and +7 of challenge with L. donovani amastigotes. The prophylactic effect lasted for 7 days following the last treatment with compound 86/450. The antileishmanial action of sodium stibogluconate (SAG) was also found to be enhanced by 16% in hamsters primed with compound 86/450.

Attenuation of alcohol-induced hypothermia by cyclo (His-Pro) and its analogs.

Acute administration of cyclo (His-Pro) to rats cause a dose-dependent decrease in ethanol-induced hypothermia. Bromination of the imidazole moiety of histidine in cyclo (His-Pro) resulted in a significant increase in its potency to attenuate ethanol hypothermia. In contrast, benzylation of the imidazole moiety of histidine or the substitution of one or both of the amino acids in cyclo(His-Pro) led to a total loss of its thermomodulatory activity. In conclusion, it appears from these preliminary data that it may be possible to design analogs of CHP that may be effective antagonists for ethanol hypothermia.

Novel met-enkephalin analogue: a potent systemic mu agonist antinociceptive agent.

A new met-enkephalin analogue (compound 82/205) was evaluated for its opioidergic activity in mice. The compound showed antinociception (warm water tail-flick test), tolerance, cross tolerance to morphine and physical dependence. The time course of antinociceptive effect of the compound was comparable to morphine. The antinociceptive ED50 (mumol kg-1, i.p.) values for the compound and morphine base were 5.31 and 7.59, respectively. Its antinociceptive effect was blocked by naloxone, beta-FNA (mu antagonist) and naloxonazine (mu1 antagonist) but not by ICI 174,864 (delta antagonist). Naloxone precipitated withdrawal jumpings were 2.6 times less in compound 82/205 treated mice than the morphine treated group. The new analogue compound 82/205 is a potent mu agonist antinociceptive with a possible weak dependence liability.

Immunomodulatory activity of met-enkephalin and its two potent analogs.

The effects of Met-enkephalin (Met-Enk), a delta receptor binding opioid peptide, and its more stable synthetic analogs, Tyr-D-Ala-Gly-MePhe-Met-NHC3H7-iso (1), Tyr-D-Ala-Gly-MePhe-Gly-NHC3H7-iso (2) and Tyr-D-Ala-Gly-MePhe-Gly-NHCH2C6H5 (3), on human T-cell transformation and natural killer (NK) cell cytotoxicity have been evaluated. Analogs 1 and 2 have been found to be as potent as Met-Enk in stimulating T-cell transformation and augmenting NK cell cytotoxicity. Analog 3 had no effect on T-cell transformation and NK cell cytotoxicity. Proliferative response was measured by 3H-thymidine uptake after 5 days of incubation. The kinetics of the T-cell transformation response (peak 5th day) is similar to those for in vitro T-cell responses to specific antigens rather than via polyclonal activation.

Chemotherapy of experimental filariasis: enhancement of activity profile of ivermectin with immunomodulators.

The effect of certain immunopotentiators (Freund's complete adjuvant, picroliv, tuftsin and CDRI Compound 86/448) was evaluated on exertion of antifilarial activity of ivermectin at different dose levels in cotton rats experimentally infected with Litomosoides carinii. Ivermectin alone (up to 250 micrograms/kg p.o. x 5 days) caused sterilization of most of the surviving female parasites, but had no lethal effect on adult worms. In combination with immunomodulators, ivermectin brought about significant lethal effect on adult parasites even at a dose of 1 microgram/kg x 5 days. Nevertheless, in animals receiving FCA alone, sterility was caused in > 50% of female parasites. Other immunomodulators used alone had a suppressive effect on microfilaraemia only. Immunomodulators alone or in combination with ivermectin also caused enhanced filaria-specific antibody response.

Lymphokines production by concanavalin A-stimulated mouse splenocytes: modulation by Met-enkephalin and a related peptide.

Methionine-enkephalin (ME) and its synthetic congener Tyr-D-Ala-Gly-Me-Phe-Gly-NH.C3H7-iso (82/205), in a concentration-dependent biphasic manner modulated the concanavalin A (Con A)-stimulated phagocytosis-promoting (PP)-activity elaboration in the culture supernatants of mouse splenocytes in vitro. Both these peptides at 1 x 10(-5) and 1 x 10(-6) M inhibited the production of PP activity; conversely, at 1 x 10(-7)-1 x 10(-9) M they augmented it. Peptide 82/205 was nearly 1.2-fold more inhibitory and approximately 1.8-fold more potent in augmenting the PP activity elaboration. The PP activity appeared to be due to lymphokines (LK) gamma interferon and interleukin-4 as the neutralizing concentrations of monoclonal antibodies against these LK significantly (p < 0.05) inhibited it. Cycloheximide (50.0 micrograms/ml) completely inhibited the production of LK indicating their de novo synthesis. The peptides appeared to exert their inhibitory and augmenting effects via delta- and mu-opioid receptors, respectively, as pretreatment of splenocytes with 100-fold higher (1 x 10(-3) M) concentration of naloxone was required to block their inhibitory effect; the augmenting effect was blocked by 1 x 10(-5) M only. None of the peptides or naloxone could directly stimulate the splenocytes for PP-LK elaboration.

Immunostimulant activity of a novel lipopeptide and its protective action against Leishmania donovani.

Novel lipopeptides 84/201 and 86/450 synthesized in this laboratory stimulated antibody and delayed type hypersensitivity (DTH) response to ovalbumin in guinea pigs. Lipopeptide 86/450 also stimulated antibody and DTH responses in albino mice and enhanced nonspecifically macrophage migration index (MMI), phagocytic activity and incorporation of [14C] glucosamine in peritoneal macrophages of the treated animals. Proliferative response of splenocytes from lipopeptide 86/450 treated animals was significantly higher than that from untreated controls. Peritoneal macrophages from lipopeptide 86/450 treated mice were less susceptible to Leishmania donovani promastigote invasion when co-cultured in vitro. The treated animals on challenge with L. donovani promastigote/amastigote showed 80 to 90% lower intake of infection than the control animals.

Enkephalin antisense peptides: design, synthesis, and biological activity.

The antisense mRNA complementary to the sense strand of Metenkephalin encodes the antisense peptides, His-Glu-Ala-Pro-Ile (compound 88/62). The antisense peptide and its (Gln1)-analogue (compound 88/63) have synergestic effects on the opioid activity of Met-enkephalin in the GPI test system.

Augmentation of human natural killer cells by splenopentin analogs.

Splenopentin, Arg-Lys-Glu-Val-Tyr (SP-5) and its synthetic analogs; Arg-D-Lys-Glu-Val-Tyr (pentapeptide 1), Lys-Lys-Glu-Val-Tyr (2), D-Lys-Lys-Glu-Val-Tyr (3), Arg-Lys-Gly-Val-Tyr (4), and Arg-Lys-Gln-Val-Tyr (5) have been examined for augmentation of human natural killer (NK) cell activity and human T-cell transformation response. Pentapeptides 2 and 3 were found to significantly augment the in vitro human NK cell activity. However, none of them had any effect on lymphocyte proliferative responses.