Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.

Potent, long-acting, orally-active bradykinin antagonists for a wide range of applications.

Actions of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; BK) are mediated by constitutively expressed B2 receptors, that require the full BK peptide chain, and by B1 receptors, induced in inflammation, that use BK(1-8) as ligand. In addition to many physiological and pathophysiological functions, the growth factor activity of BK evidently allows it to act as an autocrine stimulant for small cell lung cancer. A new group of BK antagonists containing the novel amino acid a-(2-indanyl)glycine provides extremely potent broad-spectrum as well as selective antagonists for all these functions.

In vitro studies of a bradykinin B1/B2 antagonist linked to a human neutrophil elastase inhibitor; a heterodimer for the treatment of inflammatory disorders.

Inflammatory disorders typically have a complex etiology and involve a multitude of inflammatory mediators, and hence, a polytherapeutic approach to these diseases would seem appropriate. In certain chronic inflammatory conditions, we believe that bradykinin (BK) and human neutrophil elastase (HNE) are cooperatively involved. We have previously synthesized compounds with inhibitory activity toward both the BK B2 receptor and HNE. The present study describes single compounds designed to incorporate HNE inhibitory activity and BK B1 and B2 antagonist activity. A proprietary HNE inhibitor (HNEI, CP-955) was directly linked via amide bond formation to a peptide-based combined BK B1/B2 antagonist (B-9430). Three compounds were made using different linking positions in the antagonist peptide. For all compounds, B1 and B2 receptor binding in human cloned receptors was at least 10-fold less than that of B-9430, whereas in the in vitro guinea pig ileum B2 receptor functional assay, the compounds had potencies equivalent to B-9430. Compound I was found to have a fourfold increase in HNEI activity compared with CP-955, whereas compounds II and III were inactive. These data clearly demonstrate that it is possible to retain BK B1/B2 receptor antagonist and HNE activity in a heterodimer.

Potent, long-acting bradykinin antagonists for a wide range of applications.

Actions of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; BK) are mediated by constitutively expressed B2 receptors (which require the full BK peptide chain) and by B1 receptors (which require BK (1-8) as ligand) that are induced in inflammation. BK has many functions in normal and pathological physiology, including initiation of most, if not all, inflammation. BK also evidently functions as an autocrine stimulant for growth of small cell lung cancer (SCLC). A new group of BK antagonists containing the novel amino acid alpha-(2-indanyl)glycine (Igl) provides both broad-spectrum and selective antagonists for all these functions. As examples, D-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-Arg (B9430) is an extremely potent and long-acting antagonist of both B1 and B2 receptors, is stable against endogeneous kininase enzymes, and is active in various in vivo models, including by intragastric administration. Acylation of B9430 with dehydroquinuclidine-2-carboxylic acid (Dhq) gives B9562, a highly selective B2 antagonist. In contrast, Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic (B9858) is a highly potent and selective B1 antagonist. The dimer of B9430 linked at the amino terminus with suberimide is a potent selectively cytotoxic agent for SCLC cells. Results with these peptides suggest that a new generation of antiinflammatory and anticancer drugs may be at hand.

Rationally designed non-peptides: variously substituted piperazine libraries for the discovery of bradykinin antagonists and other G-protein-coupled receptor ligands.

Molecular modeling studies of potent decapeptide bradykinin antagonists suggested the de novo design of peptide mimetics based on a 1,2,3,4-tetrasubstituted 1,4-piperazin-6-one scaffold. These de novo-designed antagonists exhibited only modest potency (IC50 approximately 55 microM) on a cloned human B2 receptor and antagonist activity in an in vitro human-cell functional assay. The success of these structures led to the creation of prototype libraries based on variously substituted 1,4-piperazine scaffolds, which allowed a rapid and general search of pharmacophores attached to a piperazine scaffold. The parent piperazinedione structures and fully reduced piperazine libraries differ from recently reported diketopiperazine libraries in the use of diverse nonnatural amino acids, on-resin-submonomer synthesis to provide more diverse N-substituted structures, and the adaptation of simultaneous ring closure and resin cleavage to drive the formation of highly hindered amide bonds. Using this chemistry, a rationally directed non-peptide library of approximately 2500 N,N'-disubstituted piperazines and piperazinediones was synthesized and screened for ligand affinity on bradykinin, neurokinin, and opioid receptors. A number of lead structures were identified. Notably, a bradykinin antagonist lead, CP-2458, with good receptor selectivity and antagonist activity in human-cell assays was identified and is undergoing optimization by traditional and combinatorial methods.

Evidence of a bradykinin B1 receptor in human ileum: pharmacological comparison to the rabbit aorta B1 receptor.

Bradykinin B1 receptors have been identified in a limited number of human tissues and may have implications in pathological states of chronic inflammation. In the present study, longitudinal strips of postmortem human ileum displayed a strong contractile response to the B2 receptor agonist, bradykinin (EC50 = 7.0 nM). Noninduced ileum strips contracted only to high concentrations (1 and 10 microM) of the B1 receptor agonists, des-Arg9-BK and Lys0des-Arg9-BK. After incubation overnight at 37 degrees C the potency of des-Arg9-BK and Lys0des-Arg9-BK dramatically increased (EC50 = 183 and 13.2 nM, respectively). The increase in B1 agonist potency was inhibited by the protein synthesis inhibitor, puromycin. Similarly, rabbit aorta strips displayed a protein synthesis-dependent induction of the B1 agonist response. Incubated human ileum and rabbit aorta exhibited a reproducible response to des-Arg9-BK over time, whereas responses to Lys0des-Arg9-BK were not reproducible, having reduced potency and magnitude over time. Lys0[Leu8]des-Arg9-BK was a more potent antagonist at the B1 receptor in both tissues compared with [Leu8]des-Arg9-BK. The B2 antagonist, HOE-140, was a very weak inhibitor of the B1 response in human ileum and inactive in rabbit aorta. In conclusion, incubation of isolated human ileum overnight induces expression of a B1 receptor through a mechanism that depends on de novo protein synthesis. The potency profile of selected B1 agonists and antagonists indicates pharmacological similarities between the inducible B1 receptor in both the human ileum and rabbit aorta.

A new generation of bradykinin antagonists.

Bradykinin B2 receptors are constitutively expressed, and require the entire peptide chain of bradykinin for recognition. Expression of B1 receptors is induced in inflammation; they recognize BK-(1-8). Heretofore blockade of all the actions of bradykinin required two different antagonists, one for each class of receptors. The new antagonists described here are full chain antagonists having high potency on B2 receptors, but they are also very potent antagonists for B1 receptors. They are highly resistant to kininases and show very long action in vivo. These antagonists contain the novel amino acid alpha-(2-indanyl)glycine (IgI) at positions 5 and 7. The peptide DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg (designated B9430) shows all these desirable characteristics. It represents a new class of bradykinin antagonist peptides.

Comparative profile of novel potent bradykinin antagonists at human B1 and B2 receptors.

We have undertaken a comparative pharmacological profile of novel bradykinin antagonists on human B1 and B2 receptor binding and functional assays. We found that there was an excellent correlation between binding and functional data for the compounds at the B1 receptor. In general, although there was a good correlation between the binding and functional data at the B2 receptor there was a greater degree of scatter in the correlation, particularly with compounds that possessed both B1 and B2 binding and antagonist activity. Of the compounds that were highly selective for the B2 receptor, CP-0597 had high binding activity but showed an unexpectedly low functional potency in the human ileum. The reason for this discrepancy is unclear. In general we found that binding activity with both the B1 and B2 antagonist compounds correlated well with their functional activity.

Bradykinin receptor antagonists containing N-substituted amino acids: in vitro and in vivo B(2) and B(1) receptor antagonist activity.

We report a systematic probing of the structural requirements of the bradykinin (BK) type 2 (B(2)) receptor for antagonist activity by incorporating N-alkyl-amino acid residues at positions 7 and 8 of a potent antagonist sequence. Compound 1 (D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-N-Chg (8)-Arg(9), CP-0597)(1,2) is a potent (pA(2) = 9.3, rat uterus; pK(i) = 9.62, binding, human receptor clone) B(2) receptor antagonist devoid of in vitro B(1) antagonist activity (rabbit aorta). Compound 1 exhibits high potency (ED(50) = 29.2 pmol/kg/min, iv, rabbit) and duration of action when tested in models for in vivo B(2) antagonist activity. Although devoid of activity in a classic B(1) isolated tissue assay, B(1) antagonist activity for 1 was demonstrated in vivo, in a LPS-treated, inducible BK(1) receptor rabbit blood pressure model (ED(50) = 1.7 nmol/kg/min). D-Arg(0) of 1 can be formally replaced by an achiral arginine surrogate, without significant loss in antagonist potency on rat uterus (compound 11, B(2) pA(2) = 9.1). Antagonist 13 (Hyp(2), Nchg(8)), pK(i) = 10.2, and agonist 4 (N-methylcyclohexyl-Gly(8)), pK(i) = 10.1, also exhibited substantial binding to guinea pig ileum membrane receptors as well as a human B(2) receptor clone. Very minor structural changes in the N-alkyl amino acid residues in positions 7 and 8 can modify the activity of this class of compounds from being extremely potent antagonists to tight binding partial or full agonists. These studies have resulted in a series of compounds containing inexpensive amino acid residues but which produce broad spectrum BK receptor blocking potency and exceptional in vivo duration of action.

Renal and antihypertensive effects of a novel eukalemic diuretic, ICI 207,828.

ICI 207,828, an aminomethylphenol pyrazine derivative, produces water diuretic effects with only minimal alterations in kaliuresis in dogs and rats after oral and parenteral administration. In the dog, ICI 207,828 reached maximum activity at a dose of 10 mg/kg, p.o. This was comparable to that of hydrochlorothiazide (HCTZ) at a dose of 5 mg/kg, p.o. or higher. In the rat, a dose of 30 mg/kg, p.o. of ICI 207,828 was comparable to the maximum of water diuretic and saluretic response obtained with HCTZ at a dose of 10 mg/kg, p.o. Based upon studies using in vitro amphibian models of the mammalian nephron, ICI 207,828 appeared to act on both the thick ascending limb of the loop of Henle and the late distal nephron. In the toad bladder preparation, ICI 207,828 inhibited Na+ transport when placed on either the mucosal (amiloride-like) or serosal (thiazide-like or loop diuretic-like) sides. This compound also inhibited Cl- transport in the toad cornea preparation (loop diuretic-like). ICI 207,828 did not change plasma K+ significantly in dogs dosed for 14 days at doses having diuretic effects (5 and 10 mg/kg, p.o., daily). In contrast, HCTZ consistently decreased plasma K+, whereas amiloride increased it significantly. ICI 207,828 demonstrated antihypertensive effects in spontaneously hypertensive rats. At 30 mg/kg, p.o., b.i.d., ICI 207,828 and HCTZ produced approximately equal antihypertensive activities during a 3 1/2-day treatment period. The pharmacological profile of ICI 207,828 indicates that this compound is a potent eukalemic diuretic and antihypertensive agent in animals.

Effects of K+ channel blockers and cromakalim (BRL 34915) on the mechanical activity of guinea pig detrusor smooth muscle.

There is strong evidence that cromakalim (BRL 34915) relaxes smooth muscle by opening cell membrane K+ channels. The aim of this study was to use relatively selective K+ channel blockers to investigate 1) the K+ channel type(s) opened by cromakalim in guinea pig detrusor and 2) the role of different K+ channel types in the control of basal tension. Cromakalim produced a concentration-related relaxation (IC50 = 0.50 +/- 0.03 microM, n = 42) of 15 mM K(+)-evoked mechanical activity. The ATP-sensitive K+ channel blocker glyburide (0.3-3 microM) antagonized the effects of cromakalim in an apparently competitive manner (pA2 = 6.76). Charybdotoxin and iberiatoxin (3-30 nM), blockers of the large conductance, Ca(++)-activated K+ channel, appeared to functionally antagonize cromakalim. Apamin (1 microM) and leiurotoxin I (0.3 microM), blockers of the small conductance, Ca(++)-activated K+ channel, and noxiustoxin (0.3 microM), a blocker of squid axon delayed rectifer K+ channels, all failed to antagonize cromakalim. Cumulative administration of charybdotoxin and iberiatoxin produced marked, concentration-related stimulation of mechanical activity per se whereas glyburide, noxiustoxin, apamin and leiurotoxin I had no effect. Apamin and leiurotoxin I did stimulate mechanical activity to a small extent when administered noncumulatively, however. The results suggest that cromakalim opens ATP-sensitive K+ channels in detrusor and suggest that cromakalim does not open CA(++)-activated K+ channels and noxiustoxin-sensitive, delayed rectifier K+ channels. The marked stimulatory effects of charybdotoxin and iberiatoxin per se suggest an important role for large conductance, Ca(++)-activated K+ channels in the control of basal tension and, presumably, membrane potential in detrusor smooth muscle cells.

Use of renal artery infusion in dogs for evaluation of diuretics.

In this paper we report on a series of experiments evaluating a model of renal artery infusion (RAI) as a screening technique for comparison of new diuretics, and test some assumptions underlying its use. The femoral artery and vein of Beagle dogs were catheterized for the infusion of solutions, measurement of arterial blood pressure and the sampling of arterial blood. The left kidney was isolated through a flank incision, and a 27 ga. needle, connected to PE10 tubing, was inserted into the renal artery for administration of test diuretics. Both ureters were catheterized for collection of urine. Effects on renal function were assessed during control, drug infusion, and recovery periods. At low doses, the loop diuretics, furosemide (FUR) and MK447, increased urinary excretion within the first 15 min of infusion. The effect of muzolimine, another loop diuretic, was delayed until about 45 min after initiation of infusion. Renal function returned to baseline after cessation of drug infusion. At high doses, excretion was increased by all the loop diuretics within the first 15 min of infusion. The response to muzolimine and MK447 was prolonged well into the recovery period, while that to furosemide returned promptly to baseline. Two distal diuretics, hydrochlorothiazide and amiloride, caused a significant increase in urinary excretion at both low and high doses. The magnitude of the response was significantly less than for the loop diuretics. Low doses of the loop diuretics had very little effect on the contralateral kidney; however, at high doses the excretion rate of the contralateral kidney was significantly increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of detergents on sodium transport in toad urinary bladder.

The effects of ionic, nonionic and zwitterionic detergents (zwittergents) on apical sodium permeability of toad urinary bladder were investigated. Applied to the mucosal side at concentrations less than 1/100 of their respective critical micellar concentration (CMC), sodium lauryl sulfate, Triton X-100 and a series of sulfobetaine zwittergents reversibly increased amiloride-sensitive sodium current. These detergents decreased the current at concentrations higher than 1/100 of the CMC and caused large, irreversible increases of tissue electrical conductance at a concentration close to the CMC. Nonionic detergent Tween 80, however, stimulated the current at concentrations from 1/100 to 10,000 of its CMC. The maximum stimulation of current by each zwittergent occurred at 1/100 of its CMC, and the larger the CMC, the greater the stimulation attainable. Analysis of the dependence of current on mucosal sodium concentration in the presence of stimulating doses of a zwittergent and Tween 80 showed that the increase in current was not the result of increased apical maximum sodium permeability, but the consequence of removal of sodium self-inhibition. The amiloride dose-response curve in the presence of stimulating dose of zwittergent was shown to shift to the left and yield a smaller apparent inhibition constant as predicted on the basis of such a removal and an unaltered intrinsic amiloride blocking kinetic.

Neutral amino acid transport in embryonal carcinoma cells.

Neutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems--A, L, and ASC--although one system may make a barely measurable contribution in some cases. The characterization of N-methyl-aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single-component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+-independent leucine transport, reveals a high-affinity, single-component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenylalanine is primarily transported by Na+-dependent Systems A and ASC (83% Na+-dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+-independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+-dependency of phenylalanine transport in mouse uterine blastocysts (82% Na+-dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+-independent, BCH-inhibitable mechanism.