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The Food and Drug Administration recently approved the new drug avacopan for a relatively rare disease, anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis. Avacopan is an antagonist of receptor-1 for anaphylatoxin C5a (C5aR 1 ) that is the first one to meet all expectations of an orally bioavailable drug. Pharmacological effects of C5a on vascular tissue are reviewed; they are essentially indirect, via resident or infiltrating leukocytes, and largely mediated by vasoconstrictor prostanoids that are potentially thrombogenic. The in vivo acute neutropenic effect of C5a and various responses of isolated neutrophils to the peptide have been exploited in the preclinical development of avacopan, but not the prominent hemodynamic responses. Possible clinical risks and extension of therapeutic C5aR 1 blockade are discussed. Therapeutic intervention on the blood-derived peptide C5a and on its G protein coupled receptor for specific forms of vascular injury contrasts with other current research approaches in vascular pathology, such as investigating the roles of cytokines and intracellular signaling., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The centrally acting antitussive opiate derivative, noscapine, has been claimed to be a non-competitive bradykinin B 2 receptor antagonist. Raloxifene, a selective estrogen receptor modulator, was predicted to bind the bradykinin B 2 receptor and to exert a partial agonist activity. These intriguing claims suggest that new molecular scaffolds ("chemotypes") may be identified for small molecule ligands of kinin receptors and that some off-target effects of noscapine or raloxifene may be mediated by bradykinin B 2 receptors. An established contractile bioassay for ligands of the bradykinin B 2 receptor, the isolated human umbilical vein, was exploited to characterize the inhibitory effect of noscapine and raloxifene on the B 2 receptor-mediated contractile response to bradykinin. Observed effects were compared with those of the peptide antagonist icatibant, a potent, selective and competitive B 2 receptor antagonist. Our results indicate that neither noscapine (2.5 µM) nor raloxifene (20 µM) behave as B 2 receptor antagonists in concentrations that vastly exceeded an effective concentration of the control antagonist, icatibant; further, none of these drugs had direct contractile effects. It is suggested that the previously reported B 2 receptor inhibitory effect of noscapine, a putative sigma-receptor agonist, might result from an indirect physiological antagonism, while raloxifene did not appear to have any significant affinity for the B 2 receptors., (Copyright © 2022 Elsevier B.V. All rights reserved.)

PHA-022121 is a novel small molecule bradykinin B 2 receptor antagonist, in clinical development for the treatment and prevention of hereditary angioedema attacks. The present study describes the in vitro pharmacological characteristics of PHA-022121 and its active metabolite, PHA-022484 (M2-D). In mammalian cell lines, PHA-022121 and PHA-022484 show high affinity for the recombinant human bradykinin B 2 receptor with K i values of 0.47 and 0.70 nM, respectively, and potent antagonism of the human bradykinin B 2 receptor with K b values of 0.15 and 0.26 nM, respectively (calcium mobilization assay). Antagonist potency at the recombinant cynomolgus monkey bradykinin B 2 receptor is similarly high (K b values of 1.42 and 1.12 nM for PHA-022121 and PHA-022484, respectively), however, potency at rat, mouse, rabbit and dog bradykinin B 2 receptors is at least 100-fold lower than the potency at the human receptor for both compounds. In the human umbilical vein contractility assay, both PHA-022121 and PHA-022484 show a potent, surmountable and reversible B 2 antagonist activity with pA 2 values of 0.35 and 0.47 nM, respectively. The in vitro off-target profile of PHA-022121 and PHA-022484 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B 1 receptor. It is concluded that PHA-022121 is a novel, low-molecular weight, competitive antagonist of the human bradykinin B 2 receptor with high affinity, high antagonist potency, and high selectivity. It is about 20-fold more potent than icatibant at the human bradykinin B 2 receptor as assessed using recombinant or endogenously expressed receptors., (Copyright © 2022 Pharvaris, GmbH. Published by Elsevier B.V. All rights reserved.)

Bradykinin-related peptides, the kinins, are blood-derived peptides that stimulate 2 G protein-coupled receptors, the B 1 and B 2 receptors (B 1 R, B 2 R). The pharmacologic and molecular identities of these 2 receptor subtypes will be succinctly reviewed herein, with emphasis on drug development, receptor expression, signaling, and adaptation to persistent stimulation. Peptide and non-peptide antagonists and fluorescent ligands have been produced for each receptor. The B 2 R is widely and constitutively expressed in mammalian tissues, whereas the B 1 R is mostly inducible under the effect of cytokines during infection and immunopathology. The B 2 R is temporarily desensitized by a cycle of phosphorylation/endocytosis followed by recycling, whereas the nonphosphorylable B 1 R is relatively resistant to desensitization and translocated to caveolae on activation. Both receptor subtypes, mainly coupled to protein G G q , phospholipase C and calcium signaling, mediate the vascular aspects of inflammation (vasodilation, edema formation). On this basis, icatibant, a peptide antagonist of the B 2 R, is approved in the management of hereditary angioedema attacks. This disease is the therapeutic showcase of the kallikrein-kinin system, with an orally bioavailable B 2 R antagonist under development, as well as other agents that inhibit the kinin forming protease, plasma kallikrein. Other clinical applications are still elusive despite the maturity of the medicinal chemistry efforts applied to kinin receptors., Competing Interests: Declaration of Competing Interest FM. served as a consultant and received research funds from for Pharvaris B.V. There are no other competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In human peripheral blood neutrophils or in myeloid PLB-985 cells differentiated towards a neutrophil-like phenotype, the peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-norleucyl-L-tyrosyl-L-leucyl-fluorescein isothiocyanate (f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC) binds to and activates formyl peptide receptor1 (FPR1) and is submitted to receptor-mediated endocytosis (microscopy, cytofluorometry). This peptide may be considered a C-terminally extended version of f-Met-Leu-Phe which carries a fluorescent cargo into cells. By analogy to other peptide hormones for which we have evaluated epitope-tagged agonists as carriers of antibody cargoes, we have designed and evaluated f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, C-terminally extended with the 10-residue myc tag. This peptide is as potent as f-Met-Leu-Phe to compete for f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC uptake by PLB-985 cells, but did not mediate (10-1000nM) the internalization of the fluorescent anti-myc monoclonal antibody 4A6 added to the extracellular fluid at ~7nM (microscopy). The nonfluorescent version of the antibody (28nM) acts as a pre-receptor antagonist of f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, but not of f-Met-Leu-Phe (superoxide release assay in differentiated PLB-985 cells). A further prolonged analog, f-Nle-Leu-Phe-Nle-Tyr-Lys-(Asn-Gly)5-myc, designed to decrease the possible steric hindrance between FPR1 and the bound anti-myc antibody, has little affinity for the receptor, precluding a direct assessment of this issue. Thus, the relatively low-affinity anti-myc antibody used at a high concentration functionally behaves as a selective pre-receptor antagonist of the agonist f-Nle-Leu-Phe-Nle-Tyr-Lys-myc., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Whether peptidases present in vascular cells can activate prodrugs active on vascular cells has been tested with 2 potential latent ligands of the histamine H1 receptor (H1R). First, a peptide consisting of the antihistamine cetirizine (CTZ) condensed at the N-terminus of ε-aminocaproyl-bradykinin (εACA-BK) was evaluated for an antihistamine activity that could be revealed by degradation of the peptide part of the molecule. CTZ-εACA-BK had a submicromolar affinity for the BK B2 receptor (B2R; IC50 of 590 nM, [(3)H]BK binding competition), but a non-negligible affinity for the human H1 receptor (H1R; IC50 of 11 μM for [(3)H]pyrilamine binding). In the human isolated umbilical vein, a system where both endogenous B2R and H1R mediate strong contractions, CTZ-εACA-BK exerted mild antagonist effects on histamine-induced contraction that were not modified by omapatrilat or by a B2R antagonist that prevents endocytosis of the BK conjugate. Cells expressing recombinant ACE or B2R incubated with CTZ-εACA-BK did not release a competitor of [(3)H]pyrilamine binding to H1Rs. Thus, there is no evidence that CTZ-εACA-BK can release free cetirizine in biological environments. The second prodrug was a blocked agonist, L-alanyl-histamine, potentially activated by aminopeptidase N (APN). This compound did not compete for [(3)H]pyrilamine binding to H1Rs. The human umbilical vein contractility assay responded to L-alanyl-histamine (EC50 54.7 μM), but the APN inhibitor amastatin massively (17-fold) reduced its apparent potency. Amastatin did not influence the potency of histamine as a contractile agent. One of the 2 tested latent H1R ligands, L-alanyl-histamine, supported the feasibility of pro-drug activation by vascular ectopeptidases., (© 2013.)

The blood vessels are one of the important target tissues for the mediators of inflammation and allergy; further cytokines affect them in a number of ways. We review the use of the isolated blood vessel mounted in organ baths as an important source of pharmacological information. While its use in the bioassay of vasoactive substances tends to be replaced with modern analytical techniques, contractility assays are effective to evaluate novel synthetic drugs, generating robust potency and selectivity data about agonists, partial agonists and competitive or insurmountable antagonists. For instance, the human umbilical vein has been used extensively to characterize ligands of the bradykinin B(2) receptors. Isolated vascular segments are live tissues that are intensely reactive, notably with the regulated expression of gene products relevant for inflammation (e.g., the kinin B(1) receptor and inducible nitric oxide synthase). Further, isolated vessels can be adapted as assays of unconventional proteins (cytokines such as interleukin-1, proteases of physiopathological importance, complement-derived anaphylatoxins and recombinant hemoglobin) and to the gene knockout technology. The well known cross-talks between different cell types, e.g., endothelium-muscle and nerve terminal-muscle, can be extended (smooth muscle cell interaction with resident or infiltrating leukocytes and tumor cells). Drug metabolism and distribution problems can be modeled in a useful manner using the organ bath technology, which, for all these reasons, opens a window on an intermediate level of complexity relative to cellular and molecular pharmacology on one hand, and in vivo studies on the other., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Bradykinin (BK)-related peptides stimulate two major classes of receptors, B1 and B2. The B1 receptor (B1R) plays an important role in various pathophysiological states including chronic inflammation, pain, hypotension, trauma and proliferation of cancer. Therefore, there is interest in the development of highly potent peptide BK B1R antagonists. We previously developed a highly potent and selective BK B1R receptor antagonist, B9958 (Lys-Lys-[Hyp3, CpG5, d-Tic7, CpG8]des-Arg9-BK) (Hyp, trans-4-hydroxyproline; CpG, alpha-cyclopentylglycine; Tic, tetrahydroisoquinoline-3-carboxylic acid). We now report on new BK B1R antagonist analogs of B9958 with N-terminal basic residues in the d-configuration, or Lys-, Orn- derivatives (NiK, epsilon-nicotinoyllysine; PzO, 3-pyrazinoylornithine) and/or having hindered unusual amino acids at position 5 (Igl, alpha-(2-indanyl)glycine). These changes were designed to prevent enzyme degradation while keeping an acceptable affinity. However, these new analogs do not show higher B1R antagonist activity than B9958, but its N-terminal acylated derivative with a bulky and hydrophobic 2,3,4,5,6-pentafluorocinnamic acid (F5c), B10324, retains a B1R antagonist activity close to that of B9958 and, in addition, has high inhibition in vivo against lung cancer (SCLC, 86 %) and moderate inhibition against prostate cancer (PC3, 43%) xenografts. This class of compounds offers hope for the development of new BK antagonist peptide drugs for lung or prostate cancer.

Rhinitis of allergic and viral origin is often self-treated by patients with locally applied vasoconstrictor decongestant drugs. In turn, prolonged use of these agents produce an inflammatory condition termed rhinitis medicamentosa. Cationic drugs are sequestered into cells via various mechanisms, including mitochondrial concentration and V-ATPase-driven trapping in vacuoles that swell by an osmotic mechanism. We hypothesized that receptor-independent endomembrane sequestration of topically applied concentrated alpha-adrenoceptor agonists (decongestants, mydriatics) could contribute to their toxicity and prolonged duration of action. The morphological and functional effects of phenylephrine and xylometazoline on rabbit aortic smooth muscle cells were examined and their possible sequestration evaluated using the contractility of rabbit aorta rings. Synthetic agonists produced V-ATPase-dependent cell vacuolization (prevented by bafilomycin A1; xylometazoline 250 microM, phenylephrine 2.5 mM). V-ATPase-mediated cytotoxicity was slow (24 h; phenylephrine only, 5-10 mM); a rapid xylometazoline-induced cytotoxicity (> or =500 microM, 4 h) correlated to mitochondrial functional alterations. Xylometazoline had slower contraction and relaxation kinetics than the other alpha-adrenoceptor agonists in the aorta; bafilomycin pre-treatment influenced its kinetics (accelerated contraction and relaxation) and concentration-effect relationship (potentiation). V-ATPase-driven sequestration contributed to a component of the tissue reservoir of both phenylephrine and xylometazoline as assessed by aortic rings contracted with the concentrated agonists and subsequently washed. Phenylephrine and xylometazoline caused the V-ATPase-dependent cytopathology at a fraction of the usual topical concentrations; this form of sequestration influenced the toxicity and pharmacology of individual agents.

Analgesic and anti-inflammatory applications for non-peptide bradykinin (BK) B2 receptor antagonists have been documented in rats. However, very large species differences in affinity were also noted within this class of drugs, making the preclinical development of relevant drugs difficult. Bradyzide is a potent antagonist at the rat B2 receptor, but a weak one at the human receptor; a series of analogues in which the diphenylmethyl moiety of this drug has been substituted with dibenzosuberane have been reported to gain potency at the human B2 receptor, with some loss of affinity at the rat receptor. The present experiments have been performed in order to verify that the novel series of dibenzosuberane B2 receptor antagonist optimized for affinity in the human species are effective in the isolated human umbilical vein contractility assay. Bradyzide, its analog compound (S)-14c and the dibenzosuberane compounds (S)-14d and 19c surmountably antagonized BK-induced contraction (pA2 values of 5.42, 6.48, 7.42 and 7.53, respectively). In the rabbit jugular vein contractility assay, the pA2 of compound 19c was smaller than 5. Potency at the recombinant rabbit B2 receptor was generally decreasing in the series of four drugs (Ki in a [3H]BK competition assay to recombinant receptors of 0.78, 0.77, 10.2 and 44.4 nM, respectively); these four compounds did not displace [3H]Lys-des-Arg(9)-BK binding from human B1 receptors expressed by smooth muscle cells. The dibenzosuberane compound 19c, verified to functionally antagonize the vascular B2 receptor, is an example of a drug unusually specific for the human form of the receptor.

Two types of receptors (B1R, B2R) for kinins are defined in mammalian species. Comparative experiments involving recombinant fusion proteins consisting of rabbit B1R or B2R fused to GFP-related proteins are exploited to study the regulation of the response to kinins at the receptor level. The following points will be briefly reviewed and supported by some novel data. (1) The constitutive B2Rs are internalized upon agonist stimulation, but completely recycled to the cell surface; however, B2R destruction can be achieved following limited proteolysis (extracellular trypsin, neutrophil proteases), a plausible down-regulation mechanism in pathology. (2) The inducible B1Rs, stimulated by des-Arg9-kinins, are not phosphorylated nor internalized upon agonist stimulation, but rather undergo a reversible redistribution to caveolae-related rafts. B2Rs are also subjected to this translocation, but only transiently (before endocytosis). (3) Based on the analysis of rabbit aortic smooth muscle cells, B1R induction by cytokines is dependent on nuclear factor KB in rabbit vascular tissue, but exogenous kinins acting on either receptor type do not induce B1R expression.