Comparative biochemical and pharmacological characterization of a novel, NOP receptor selective hexapeptide, Ac-RYYRIR-ol.

Nociceptin/orphanin FQ (N/OFQ) is an endogenous neuropeptide, which is widely distributed in central and peripheral nervous system. Some N/OFQ sequence unrelated hexapeptides can effectively bind to the N/OFQ peptide (NOP) receptor and they were used as template for structure-activity studies that lead to discovery of the new NOP selective ligands. In the present study, the pharmacological profile of the novel hexapeptide Ac-RYYRIR-ol was investigated using various in vitro assays including receptor binding and G-protein activation in rat brain membranes, mouse and rat vas deferens, guinea pig ileum, mouse colon and Ca(2+) mobilization assay in chinese hamster ovary (CHO) cells co-expressing the human recombinant NOP receptor and the C-terminally modified Galpha(qi5) protein.

Binding studies of novel, non-mammalian enkephalins, structures predicted from frog and lungfish brain cDNA sequences.

Leu- and Met-enkephalin were the first endogenous opioid peptides identified in different mammalian species including the human. Comparative biochemical and bioinformatic evidence indicates that enkephalins are not limited to mammals. Various prodynorphin (PDYN) sequences in lower vertebrates revealed the presence of other enkephalin fingerprints in these precursor polypeptides.

Josef Rudinger Memorial Lecture 2002. Peptide related drug research.

Drug discovery directed peptide research has been pursued at the IVAX Drug Research Institute (formerly Institute for Drug Research) (IDR) since the mid 1950s. Outlined are the main projects and the most significant results, which include the first synthesis of human ACTH, the discovery of GYKI-14 166, the prototype of peptide inhibitors of thrombin, a stable anticoagulant, efegatran GYKI-14 766, and their dual acting analogues. The identification of an agonist analogue of LHRH leading to Cetrorelix, an LHRH antagonist now in clinical use, is also presented.

[Research on synthetic peptides of biological interest].

Research on synthetic peptides at the Institute for Drug Research (IDR) is exemplified by an overview of the projects that resulted in significant results. The first synthesis of oxytocin, a pituitary hormone, in 1953 launched the research on synthetic peptides all over the world. This synthesis was reproduced by Bodanszky at the IDR in 1954, then, after some improvements, the process was presented to Richter to produce synthetic oxytocin for therapeutic purposes.

Research on synthetic peptides of pharmaceutical interest at IDR.

Research on synthetic peptides at the Institute for Drug Research (IDR) is exemplified by an overview of the projects that resulted in significant results.

Peptidyl beta-homo-aspartals (3-amino-4-carboxybutyraldehydes): new specific inhibitors of caspases.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE, caspase-1) processes the IL-1 beta precursor to mature inflammatory cytokine IL-1 beta. ICE has been identified as a unique cysteine protease, which cleaves Asp-X bonds, shows resistance to E-64 (an inhibitor of most cysteine proteases) and has a primary structure that is homologous to CED-3, a protein required for apoptosis (programmed cell death) in the nematode Caenorhabditis elegans, and to mammalian cysteine proteases that initiate and execute apoptosis, e.g.

Peptidyl beta-homo-aspartals: specific inhibitors of interleukin-1 beta converting enzyme and its homologues (caspases).

Inhibition of interleukin-1 beta converting enzyme (ICE), apopain, papain, thrombin and trypsin with substrate like peptidyl L- and D-alpha-aldehydes and their L-beta-homo-aldehyde analogues was investigated. The L-beta-homo-aspartals appear to be specific inhibitors for ICE and its homologues; the other enzymes were not inhibited with such L-beta-homo aldehydes. Papain shows tolerance for D-residues at P1 depending on their chiral stability.

Active site-directed thrombin inhibitors: alpha-hydroxyacyl-prolyl-arginals, new orally active stable analogues of D-Phe-Pro-Arg-H.

D-alpha-Hydroxyacyl-prolyl-arginals, a new type of analogues of D-Phe-Pro-Arg-H (R1), have been prepared and evaluated. Unlike R1, whose terminal group is NH2, the new analogues with a terminal OH group are stable, as are the N-substituted derivatives of R1, that is, D-MePhe-Pro-Arg-H (R2), the highly potent and selective thrombin inhibitor, and Boc-D-Phe-Pro-Arg-H (R3), the much less favorable analogue. The most notable of the new analogues corresponds to the general formula D-Xaa-Pro-Arg-H, wherein Xaa means the acyl residue of mandelic acid (Man, 1), diphenyllactic acid (Dpl, 2), hexahydrophenyllactic acid (Hpl, 3), or hexahydromandelic acid (Hma, 4).

Effect of peptide aldehydes with IL-1 beta converting enzyme inhibitory properties on IL-1 alpha and IL-1 beta production in vitro.

Tripeptide and pentapeptide aldehydes as substrate-base inhibitors of cysteine proteases were designed in our laboratory for the inhibition of interleukin-1 beta converting enzyme (ICE), a recently described cysteine protease responsible for the processing of IL-1 beta. The biological effectivity of the peptide aldehydes was studied in THP-1 cells and human whole blood. The released and cell-associated IL-1 alpha and IL-1 beta levels were determined by ELISA from the supernatants and cell lysates, respectively.

Active site-directed thrombin inhibitors: alpha-hydroxyacyl-prolyl-arginals. New orally active stable analogs of D-Phe-Pro-Arg-H.

D-alpha-Hydroxyacyl-prolyl-arginals have been designed and synthesized as orally active stable analogs of D-Phe-Pro-Arg-H, the active site-directed peptidyl thrombin inhibitor prototype. Many of the new analogs possess high in vitro anticoagulant activity while having little effect on fibrinolysis. Compounds GYKI-66104 (2), -66131 (3) and -66132 (5) effectively delay the clotting time in rabbits ex vivo and prevent thrombus formation in various thrombosis models in rabbits and rats when applied in a single oral dose of 5 mg kg-1.

N-terminally protected penta- and tetrapeptide opioid antagonists based on a pentapeptide sequence found in the venom of Philippine cobra.

Opioid receptor antagonist properties of the mu opioid receptor selective peptide, BOC-Tyr-Lys-Lys-Trp-Trp-NH2 and its systematically modified analogues were determined in guinea pig ileum, mouse vas deferens and rabbit vas deferens bioassays to locate the necessary structural features to develop kappa receptor selective antagonist(s) of substantial affinity. Replacing the tyrosine residue by phenylalanine as well as increasing the lipophilicity of the C-terminal by isoamylamide substitution yielded enhanced kappa receptor affinity. The presence of the C-terminal lipophilic Trp-Trp-NH2 region is necessary as revealed from the equilibrium dissociation constant values.

The antithrombotic and anticoagulant effects of a synthetic tripeptide and recombinant hirudin in various animal models.

The pharmacologic activities of two thrombin inhibitors (D-MePhe-Pro-Arg-H, recombinant-hirudin) were compared in two animal models. The antithrombotic effect was investigated in vivo in rabbits using a modified Wessler stasis thrombosis model. During these experiments, blood was drawn for ex vivo testing to determine the coagulation profile and to determine plasma concentrations using pre-constructed calibration curves.

Screening for fibrinolysis inhibitory effect of synthetic thrombin inhibitors.

Fibrin plate assay (FPA) and thrombelastography (TEG) were used to assess the antifibrinolytic effects of D-Phe-Pro-Arg-H (1), the prototype of peptide aldehyde inhibitors of thrombin, and two of its more stable derivatives, D-MePhe-Pro-Arg-H (2) and Boc-D-Phe-Pro-Arg-H (3). Inhibition of plasmin generation by tissue plasminogen activator, urokinase and streptokinase were studied by both FPA and TEG while that of plasmin could only be examined by FPA. TEG was more sensitive than FPA in general and for the detection of streptokinase inhibition in particular.

Inhibition by D-MePhe-Pro-Arg-H (GYKI-14766) of thrombus growth in experimental models of thrombosis.

The antithrombotic action of the highly effective synthetic thrombin inhibitor D-MePhe-Pro-Arg-H (GYKI-14766) was studied in various models of experimental thrombosis. The compound administered to rats and rabbits by i.v.

Comparative studies in vitro and ex vivo on the anticoagulant effect of a reversible and an irreversible tripeptide inhibitor of thrombin.

Comparative studies on the anticoagulant effect of D-Phe-Pro-Arg-H (ALD) and D-Phe-Pro-Arg-CH2Cl (CMK) were carried out in order to estimate whether the reversible or the irreversible tripeptide inhibitor of thrombin would be more suitable to develop as a novel anticoagulant. Conventional screening assay methods in vitro were focused on the functional stability of the compounds in whole blood and blood components while ex vivo the changes in whole blood clotting time under parenteral application of the inhibitors were investigated. The efficacy of ALD relative to that of CMK was found to depend on the complexity of the test systems.

Potent agonists of growth hormone-releasing hormone. II.

Analogs of the 29-amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) or Lys-NH2 in position 29 have been synthesized by the solid-phase method, purified, and tested in vitro. Except for one peptide, all analogs contained desaminotyrosine (Dat) in position 1. All contained Nle27 in order to avoid oxidation of Met27.

In vivo anticoagulant and antiplatelet effect of D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H.

D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H synthetized in our institute were administered to mice, rats, rabbits and beagle dogs. The kinetics of the anticoagulant and antiplatelet effect was recorded by measuring various clotting parameters, platelet count and aggregation, and evaluated as proposed by Verstraete and Verwilghen. The minimum effective doses were found to be 0.

In vitro inhibition of blood coagulation by tripeptide aldehydes--a retrospective screening study focused on the stable D-MePhe-Pro-Arg-H.H2SO4.

A series of peptide aldehydes synthetized in our institute during the last 15 years were screened to detect their inhibitory effect on blood coagulation. Simple conventional clotting assays, platelet function tests and fibrinolytic methods were used to evaluate the inhibitory potency of the compounds in complex clotting systems as well as their supposed antifibrinolytic effect in vitro. Special attention was paid to the possible interactions with blood cells and plasma proteins, and to the functional stability of the inhibitors in several tissue homogenates.

Potent agonists of growth hormone-releasing hormone. Part I.

Analogs of the 29 amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) in position 29 have been synthesized by the solid phase method, purified, and tested in vitro and in vivo. The majority of the analogs contained desaminotyrosine (Dat) in position 1, but a few of them had Tyr1, or N-MeTyr1. Some peptides contained one or more additional L- or D-amino acid substitutions in positions 2, 12, 15, 21, 27, and/or 28.

Analogues of luteinizing hormone-releasing hormone containing cytotoxic groups.

In an attempt to produce better cytotoxic analogues, chemotherapeutic antineoplastic radicals including an alkylating nitrogen mustard derivative of D-phenylalanine (D-melphalan), reactive cyclopropane, anthraquinone derivatives [2-(hydroxymethyl)anthraquinone and the anticancer antibiotic doxorubicin], and an antimetabolite (methotrexate) were coupled to suitably modified agonists and antagonists of luteinizing hormone-releasing hormone (LH-RH). Analogues with D-lysine6 and D-ornithine6 or N epsilon-(2,3-diaminopropionyl)-D-lysine and N delta-(2,3-diaminopropionyl)-D-ornithine were used as carriers for one or two cytotoxic moieties. The enhanced biological activities produced by the incorporation of D amino acids into position 6 of the agonistic analogues were further increased by the attachment of hydrophobic cytotoxic groups, resulting in compounds with 10-50 times higher activity than LH-RH.

Synthesis and in vitro and in vivo activity of analogs of growth hormone-releasing hormone (GH-RH) with C-terminal agmatine.

In the search for more active analogs of human growth hormone-releasing hormone (GH-RH), 37 new compounds were synthesized by solid phase methodology, purified, and tested biologically. Most of the analogs contained a sequence of 27 amino acids and N-terminal desaminotyrosine (Dat) and C-terminal agmatine (Agm), which are not amino acids. In addition to Dat in position 1 and Agm in position 29, the majority of the analogs had Ala15 and Nle27 substitutions and one or more additional L- or D-amino acid modifications.

Prolonged inhibition of luteinizing hormone and testosterone levels in male rats with the luteinizing hormone-releasing hormone antagonist SB-75.

Inhibitory effects of the potent antagonist of luteinizing hormone-releasing hormone N-Ac-[3-(2-naphthyl)-D-alanine1,4-chloro-D-phenylalanine2,3- (3-pyridyl)-D- alanine3,D-citrulline6,D-alanine10]luteinizing hormone-releasing hormone (SB-75) free of edematogenic effects were investigated in male rats. In a study to determine the effect on luteinizing hormone levels in castrated male rats, SB-75 was injected s.c.

Oligopeptides interfering with calcium channels inhibit prolactin and growth hormone release by cultured anterior pituitary cells of the rat.

A number of oligopeptides, protected at their N termini and possessing an aldehyde residue at their C terminal amino acids, are able to inhibit 45Ca2+ influx into anterior pituitary cells grown in monolayer culture and depolarized with high extracellular potassium concentration. In addition, the same oligopeptides interfere with hormone release, especially with that produced by lactotrophs. Our findings imply that oligopeptides may represent a new class of calcium channel ligands, and the pituitary cells are sensitive targets for them.

Highly active and selective anticoagulants: D-Phe-Pro-Arg-H, a free tripeptide aldehyde prone to spontaneous inactivation, and its stable N-methyl derivative, D-MePhe-Pro-Arg-H.

D-Phe-Pro-Arg-H sulfate (GYKI-14166) is a highly active and selective inhibitor of thrombin both in vitro and in vivo. Recent studies on the stability of D-Phe-Pro-Arg-H in neutral aqueous solution at higher temperature have revealed that it is transformed into inactive 5,6,8,9,10,10a-hexahydro-2-(3'- guanidinopropyl)-5-benzyl-6-oxo- imidazo[1,2-a]pyrrolo[2,1-c]pyrazine. No such inactivation could be observed with Boc-D-Phe-Pro-Arg-H (GYKI-14451), but this compound was far less specific than the free peptide as it inhibited thrombin and, for instance, plasmin equally well.