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The specific binding of [3H]5(S)hydroxy-6(R)-S-cysteinylglycyl -7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid ([3H]LTD4) to receptors on guinea pig lung parenchymal membranes and its inhibition by ICI 198,615, a representative example of a new class of leukotriene antagonists, was characterized by a receptor-ligand binding assay. [3H]LTD4 bound specifically and rapidly (Kon = 0.29 +/- 0.6 nM-1.min-1) reaching equilibrium within 15 min. The rate of binding was greatly inhibited in the presence of ICI 198,615. Excess LTD4 or ICI 198,615 slowly (t1/2 = 20 min) dissociated about 70% of the receptor-bound [3H]LTD4, whereas in combination with GTP analogs, both induced a rapid (t1/2 less than 5 min) and full dissociation. Equilibrium saturation analysis of [3H]LTD4 binding demonstrated a saturable (Bmax = 1014 +/- 174 fmol/mg) and high affinity (Kd = 0.43 +/- 0.09 nM) binding site. A high degree of stereoselectivity was demonstrated with inhibition of binding by the stereoisomers of LTD4: S,R much greater than R,R greater than R,S much greater than S,S. The rank order for inhibition of binding by peptide leukotriene was: LTD4 greater than 5(S)-hydroxy-6(R)-S-cysteinyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid much greater than 5(S)hydroxy-6(R)-S-glutathionyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (potency ratios were: 1:4:590). In competition assays, ICI 198,615 competitively inhibited binding of [3H]LTD4 (Ki = 0.27 +/- 0.16 nM) and was 2300-fold and 3100-fold more potent than LY171883 or FPL55712. These data, together with results obtained previously in functional receptor assays, illustrate that this new class of leukotriene antagonists are the most potent and selective competitive antagonists of LTD4 receptors yet described.

It has been demonstrated that leukotriene (LT)C4 is metabolized to LTD4 via the action of the enzyme gamma-glutamyl transpeptidase. LTD4 is, in turn, converted by the enzyme aminopeptidase to LTE4. In the present study, the pharmacological effects of the aminopeptidase inhibitor, L-cysteine and the gamma-glutamyl transpeptidase inhibitor, L-serine borate, on peptide LT concentration-response curves were evaluated in isolated guinea-pig trachea. L-Cysteine (3 mM) enhanced the contractile activity of both LTC4 and LTD4. L-Serine borate (45 mM) enhanced the contractile activity of LTC4 without altering the response to LTD4. In contrast, neither L-cysteine nor L-serine borate consistently altered the concentration-response curves to LTE4, histamine or carbachol, which rules out a nonspecific effect of these inhibitors on airway smooth muscle. In the absence of enzyme inhibitors the peptide LTs were equipotent; whereas in their presence the relative order of potency was LTC4 = LTD4 greater than LTE4. Incubation of isolated guinea-pig trachea with [3H]LTC4 resulted in the formation of [3H]LTD4 and [3H]LTE4 with a proportional decrease in [3H]LTC4. The bioconversion of [3H]LTC4 was blocked by L-serine borate in a concentration-related manner (IC50 = 3.4 +/- 0.5 mM, mean +/- S.E.M., n = 3) and the formation of LTE4 was blocked by L-cysteine (10 mM). The results suggest that LTC4 is converted to LTD4 and subsequently to LTE4 by isolated guinea-pig trachea. The potency of LTC4 and LTD4 is increased when their transformation to LTE4 is prevented.

The effect of the leukotriene (LT) antagonist, FPL55712, on the contractile activity of peptide leukotrienes was evaluated in the presence and absence of enzyme inhibitors of leukotriene metabolism. L-Cysteine, an inhibitor of aminopeptidase, prevents the formation of LTE4 from LTD4 and LTC4. L-Serine borate, an inhibitor of gamma-glutamyl transpeptidase prevents the conversion of LTC4 to LTD4. L-Cysteine (3mM) enhanced the contractile activity of LTC4 and LTD4. L-Serine borate (45 mM) increased selectively the contractile activity of LTC4. FPL55712 (10 microM) antagonized the contractile activity of LTC4, LTD4 and LTE4 in the absence of enzyme inhibitors. In the presence of L-serine borate, FPL55712 (10-30 microM) failed to antagonize the contractile activity of LTC4 but did antagonize LTD4 and LTE4. However, the dissociation constant (KB) for FPL55712 against LTD4 was increased by L-serine borate whereas the KB against LTE4 was not altered. In the presence of L-cysteine, FPL55712 antagonized the contractile activity of the peptide LTs. However, FPL55712 was more effective (P less than .05) in antagonizing LTD4 and LTE4 than LTC4 in the presence of L-cysteine. The data suggest that when the conversion of LTC4 to LTD4 and subsequently to LTE4 is blocked by L-serine borate FPL55712 is ineffective in antagonizing the actions of LTC4. This would indicate that LTC4 occupies a distinct LT receptor in guinea-pig trachea which is insensitive to the actions of FPL55712.

This paper describes the development a series of peptidyl trifluoromethyl ketone inhibitors of human leukocyte elastase which are found to have excellent pharmacological profiles. Methods have been developed that allow for the synthesis of these inhibitors in stereochemically pure form. Two of these compounds, 1k and 1l, have high levels of oral bioavailability in several species. Compound 1l has entered development as ZD8321 and is presently undergoing clinical evaluation. These compounds demonstrate that peptidyl trifluoromethyl ketone inhibitors can achieve high levels of oral activity and bioavailability, and therefore they may prove useful as therapeutic agents in the treatment of diseases in which elastase is implicated.

This study has examined the abilities of (+/-)-CP96345 and (+/-)-SR48968, nonpeptide antagonists selective for the tachykinin NK1 and NK2 receptors, respectively, to block bronchoconstriction caused by intravenous administration of direct-acting receptor agonists and the indirect-acting mimetics capsaicin, serotonin and 2-methyl-serotonin in the anesthetized guinea pig. The NK1 antagonist (+/-)-CP96345 was found to cause, at a maximally tolerated dose of 9 mumol/kg, an approximate 10-fold rightward shift of the dose-response curves for selective NK1 agonists substance P (SP), [Sar9,Met(O2)11]SP and Ac-[Arg6,Sar9,Met(O2)11]SP6-11 without altering responses to selective NK2 agonists neurokinin A (NKA), [Nle10]NKA4-10 or [beta-Ala8]NKA4-10. The NK2 antagonist (+/-)-SR48968 caused dose-dependent rightward shifts of the dose-response curves for the NK2 but not the NK1 agonists. Results using combinations of the receptor antagonists indicate that the NK2 agonists could cause bronchoconstriction by acting on the NK1 receptors at large doses relative to those used without antagonists. Of the agonists used here, [beta-Ala8]NKA4-10 appeared to be the most selective for the NK2 receptors. When used alone, only (+/-)-SR48968 was found to block bronchoconstriction caused by capsaicin, serotonin (after blockade of 5-HT2 receptors by LY53857) and 2-methyl-serotonin. When (+/-)-CP96345 was also given, larger additional blockade was seen with capsaicin than with serotonin or 2-methyl-serotonin as mimetic substance. Atropine caused small and variable degrees of blockade of serotonin and 2-methyl-serotonin but not of capsaicin after combinations of the two antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific binding of [3H]bradykinin (BK) to guinea pig gall bladder (GPGB) membranes was protein dependent, rapid (Kon = 0.067 min-1) with high affinity (Kd = 0.45 +/- 0.02; n = 3), saturable (Bmax = 546 +/- 56 fmol/mg of protein) and showed no cooperativity (nH = 1.19 +/- 0.08). A BK B2 receptor type was indicated by the rank order of potency for inhibition of binding by B2 antagonists, [(D)Arg-[Hyp3,Thi5,(D)Tic7-Oic8]-bradykinin (HOE140) > (D)Arg-[Hyp3,(D)HypE(transpropyl)7-Oic8]-bradykinin (NPC17731) > (D)Arg-[Hyp3,Thi5, (D)Tic7-Tic8]-bradykinin (NPC16731) > (D)Arg-[Hyp3,(D)Phe7]-bradykinin (NPC567)] and agonists (BK = kallidin = Tyr(Me)8-BK > Tyr8-BK,> Hyp4-kallidin) as well as inactivity of the B1 agonist des(Arg9)-BK. Nonhydrolyzable GTP analogs (GTP-gamma-S and guanylyl-5'-imido-diphosphate) produced 80% inhibition of specific binding suggesting receptor coupling to guanine nucleotide-binding proteins. BK increased polyphosphoinositide hydrolysis in chopped GPGB in a concentration-dependent manner (0.01-300 microM; EC50 = 414 +/- 171 nM; n = 3-9 tissues/concentration). HOE140 and NPC16731, inhibited BK-induced polyphosphoinositide hydrolysis but only the latter appeared competitive (pKb 8.09 +/- 0.19, n = 3). U73122, an inhibitor of phospholipase C pathway, also inhibited BK-induced turnover in GPGB (IC50 = 46.9 +/- 17.3 nM). BK produced a concentration-related contraction of isolated strips of GPGB. Indomethacin significantly decreased both the potency and efficacy of BK whereas thiorphan, a neutral endopeptidase inhibitor, and/or captopril, an angiotensin-converting enzyme inhibitor, enhanced potency.(ABSTRACT TRUNCATED AT 250 WORDS)

The peptide leukotrienes (LTs), LTC4, LTD4 and LTE4, produced concentration-related contraction of isolated strips of guinea pig gall bladder (GPGB) with pD2 values of 8.48 +/- 0.11, 8.26 +/- 0.22 and 7.28 +/- 0.08, respectively. In the presence of propranolol and indomethacin in epithelium-denuded GPGB, pKB values for the LT antagonist ICI198,615 vs. LTC4, LTD4 and LTE4 were, respectively: 8.61 +/- 0.12, 8.89 +/- 0.13 and 8.70 +/- 0.17. Similarly, pKB values for the LT antagonist SKF104,353 were 7.28 +/- 0.19, 7.88 +/- 0.17 and 7.45 +/- 0.17. Previous studies have shown that inhibition of LTC4 metabolism can alter the apparent affinity of the LT, especially LTC4; consequently, metabolism of [3H]LTC4 in chopped GPGB was investigated. [3H]LTC4 was converted rapidly by gamma-glutamyl transpeptidase to [3H]LTD4 with little accumulation of [3H]LTE4. The combination of acivicin (ACI) and reduced glutathione (GSH) provided complete inhibition of gamma-glutamyl transpeptidase. pD2 values for LTC4 in the presence of ACI/GSH were 8.54 +/- 0.16. pKB values for ICI198,615 and SKF104353 in the presence of ACI/GSH were, i.e., 8.42 +/- 0.14 and 7.53 +/- 0.12, respectively, vs. LTC4 and identical to those obtained in the absence of inhibitors. The mechanism(s) underlying LT-induced contraction was evaluated using the calcium channel blockers diltiazem, nifedipine and verapamil. Noncompetitive antagonism was observed against all LTs indicating the involvement of voltage-sensitive calcium channels in the contractile response. Similarly, all LTs increased polyphosphoinositide hydrolysis. These data indicate: 1) GPGB contains a single type of LT receptor similar to that in human airways and 2) LT-induced contraction appears to involve both polyphosphoinositol formation and voltage-dependent Ca++ channels.

The receptors involved in the bronchoconstriction evoked in vivo by intravenous administration to the anesthetized guinea pig of serotonin and serotonin-related agonists have been examined in this study. Animals were pretreated with indomethacin and (+/-)-propranolol to inhibit cyclooxygenase and beta adrenergic receptors, respectively, and pulmonary parameters were obtained with a Buxco pulmonary mechanics computer. Dose-dependent increases in pulmonary resistance and decreases in dynamic lung compliance were produced by serotonin, 2-methyl-serotonin, 5-methoxy-tryptamine, alpha-methyl-serotonin, 5-carboxamidotryptamine, and m-trifluoromethylphenylpiperazine (TFMPP). Responses to all agonists except 2-methyl-serotonin, a selective 5-hydroxytryptamine3 (5-HT3) agonist, were antagonized by the 5-HT2 antagonists, LY53857 and ketanserin. Zaclopride, 1 and 10 mg/kg, a selective 5-HT3 antagonist, blocked responses to 2-methyl-serotonin. A maximally effective dose of LY53857 (1 mg/kg) produced larger shifts of the dose-response curves to serotonin, 5-methoxytryptamine and alpha-methyl-serotonin than did a maximally effective dose of ketanserin (1 mg/kg). Thiorphan, 10 mg/kg, an inhibitor of neutral endopeptidase, potentiated 2-methyl-serotonin and, when studied in the presence of LY53857, also potentiated serotonin, 5-methoxytryptamine and TFMPP. After thiorphan and LY53857, responses to serotonin, but not 5-methoxytryptamine or TFMPP, were blocked by zaclopride. Capsaicin pretreatment of the animals resulted in rightward shifts of the dose-response curves to serotonin, 2-methyl-serotonin and TFMPP, but not to 5-methoxytryptamine or alpha-methyl-serotonin. Potentiation by thiorphan and antagonism by zaclopride of responses to serotonin were still evident after capsaicin pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of the epithelium on antagonism by ICI 204,219 of contractile responses to peptide leukotriene (LT) agonists was examined in guinea pig tracheal and human bronchial rings. The -log molar KB values for ICI 204,219 were found to be independent of the epithelium in both tissues. Even though uninfluenced by the epithelium, the -log molar KB values for ICI 204,219 were about 10-fold smaller in human airways than in guinea pig trachea. Removal of the epithelium from guinea pig trachea resulted in small leftward shifts of the concentration-response curves to LTC4 and LTD4 and rightward shifts of the concentration-response curves to LTE4 when examined in the presence of indomethacin. The potentiation of LTC4 and LTD4 by epithelium removal was not seen in the presence of inhibitors of the transformation of LTC4 to LTD4 and LTD4 to LTE4. The influence of the epithelium on responses to LTE4 remained in the presence of these metabolic inhibitors. The lipoxygenase inhibitors nordihydroguaiaretic acid, B755C, Rev 5901 and AA861 antagonized responses to LTE4 in the presence, but not in the absence of epithelium. In human airways, epithelium removal resulted in a small leftward shift of the concentration-response curve to LTD4 whereas responses to LTC4 and LTE4 were unaltered. This effect was not observed in the presence of indomethacin, relating it to reduced release of cyclooxygenase products. These data suggest that contractile responses of guinea pig trachea to LTE4 are modulated by LTE4-induced release of 5-lipoxygenase product(s) only when the epithelium is present.(ABSTRACT TRUNCATED AT 250 WORDS)

SK&F 78729-A, one of a series of pyranenamine compounds, was studied in several laboratory models of immediate-type hypersensitivity reactions. In vivo, SK&F 78729-A, i.v./p.o., inhibited the antigen-induced rat 48-hr passive cutaneous anaphylaxis by a mechanism other than end-organ antagonism to either histamine or serotonin. In vitro, the compound produced a concentration-related inhibition of the immunologic release of histamine and slow-reacting substance of anaphylaxis from passively sensitized fragmented rhesus monkey and canine lung, and modest inhibition of histamine release from passively sensitized fragmented rhesus monkey skin. Similarly, SK&F 78729-A inhibited the antigen-induced release of histamine from passively sensitized fragmented rat lung. Under certain conditions, SK&F 78729-A inhibited antigen-induced pathophysiologic pulmonary responses in in vivo canine and rhesus monkey models of allergic asthma. The results of these studies suggest that clinically, SK&F 78729-A should exhibit a pharmacologic profile consistent with that of a prophylactic antiallergic agent.

Contractile responses to leukotriene (LT)C4, LTD4 and LTE4 were examined in intralobar airways from human lung obtained after surgical resection. In addition, the ability of the LT receptor antagonist FPL55712 to antagonize responses to LTC4 and LTD4 was quantified (by calculating -log molar KB values) under experimental conditions designed to minimize metabolic transformation of the LTs. In the absence of drug pretreatment, the three peptide LTs were approximately equipotent and produced similar maximum degrees of contraction. L-Serine borate complex, 45 mM, used as an inhibitor of the degradation of LTC4 to LTD4 by the enzyme gamma-glutamyl transpeptidase, in paired airway segments (adjacent segments from the same branch), produced a small degree (about 3-fold) of shift to the right of the dose-response curve and reduction of the maximum response to LTC4. L-Cysteine, 3 mM, used as an inhibitor of the degradation of LTD4 to LTE4 by the enzyme aminopeptidase, in paired segments, did not alter the dose-response effects of LTD4 or appear to further alter the dose-response effects of LTC4 when applied together with L-serine borate complex in unpaired (nonadjacent) segments. The -log molar KB value for FPL55712 (about 6) was similar for antagonism of responses to both LTC4 and LTD4 in the absence or presence of treatment with the metabolic inhibitors L-serine borate complex, L-cysteine or a combination of the two treatments. The results suggest that inhibition of the enzymes involved in the pathway from LTC4 to LTE4 has little consequence in human airways because the three peptide LTs are approximately equipotent.(ABSTRACT TRUNCATED AT 250 WORDS)

The histamine H2-receptor has been implicated in the autoregulation of endogenous histamine release from actively sensitized human basophils. Consequently, the activity of metiamide (SK & F 92058), a specific histamine H2-receptor antagonist, was investigated for its effect on immunologically-induced histamine release in several in vitro models of immediate hypersensitivity. Metiamide enhanced the release of histamine from passively sensitized fragmented Rhesus monkey lung and skin when these tissues were challenged in a reversed type anaphylactic reaction with antihuman IgE. The enhancing effect of metiamide on the release of ‘slow reacting substance of anaphylaxis, from monkey lung was considerably less pronounced. In contrast, metiamide failed to significantly enhance the antigen-induced release of histamine from fragmented rat lung which had been passively sensitized with rat anti-ovalbumin serum. The data supports a species-specific enhancement of immunologic histamine release in vitro, perhaps by H2-receptor blockade on cells responsible for such release.

ICI 198,615 is a representative compound from a new class of peptide leukotriene (LT) receptor antagonists. In isolated guinea pig trachea and parenchymal lung strips, ICI 198,615 demonstrated competitive antagonism of the contractile activity of LTD4 and LTE4 with pA2 values of 10.1 to 9.5, respectively. The compound also appeared to antagonize the contractile activity of LTC4 in guinea pig trachea; however, in the presence of an inhibitor of the metabolism of LTC4 to LTD4, ICI 198, 615 provided a pKB value of 5.3, indicating weak antagonism at the LTC4 receptor. ICI 198,615 was also a potent antagonist of LTC4 and LTD4 in isolated human bronchi and pulmonary veins with pKa values of 9.75 +/- 0.32 to 9.20 +/- 0.24, respectively. When evaluated for activity on a broad variety of non-LT receptors, the compound displayed a minimal selectivity of 6310 and a maximal selectivity of greater than 125,000 for the LTE4 receptor. These in vitro studies indicate that ICI 198,615 is the most potent and selective peptide LT receptor antagonist described to date.

The in vivo pharmacology of ICI 198,615, a highly potent and selective peptide leukotriene (LT) receptor antagonist, is described. In a conscious guinea pig model, ICI 198,615 provided dose-related antagonism of aerosol LTD4-induced dyspnea when administered p.o., i.v. or by aerosol. The pharmacologic half-lives of ICI 198,615 by these routes of administration were greater than 16 hr, 68 min and 34 min, respectively. In pulmonary mechanics studies in which the effects of i.v. or aerosol administration of LTC4 and LTD4 on pulmonary resistance (Rp) or dynamic lung compliance (Cdyn) could be quantitated, ICI 198,615 provided dose-related antagonism when administered p.o., i.d, i.v. or by aerosol. The compound also antagonized LTE4-induced changes in Rp and Cdyn when administered i.v. ICI 198,615 reversed the effect of LTC4, LTD4 and LTE4 on Rp, as well as the effect of LTC4 and LTE4 on Cdyn. The compound was less able to reverse the effect of LTD4 on Cdyn. These results indicate that ICI 198,615 possesses an in vivo profile which is consonant with utility in the treatment of allergic diseases.

Immunological challenge of sensitized fragmented cynomolgus monkey lung with anti-human IgE (immunoglobulin E) induced the appearance of slow reacting substance of anaphylaxis (SRS-A) in the tissue and evoked the release of SRS-A into the Tyrode's supernatant. The elevation of tissue or residual SRS-A levels was maximal 2 min after anti-IgE challenge and remained at that plateau for at least 60 min. The released SRS-A, first detectable 3 to 5 min after challenge, achieved a plateau by 10 min. Both the released and residual SRS-A were similarly inactivated by soybean lipoxidase and were antagonized by FPL 55712 on the guinea-pig ileum. Isoproterenol, 5, 8, 11, 14-eicosatraynoic acid and SK & F 64398 all inhibited the anti-IgE induced elevation in residual SRS-A and blocked SRS-A release. Indomethacin stimulated both the elevation of residual SRS-A and the amount released. Removal of the Tyrode's supernatant containing 209 +/- 73 U of released SRS-A/g of tissue 20 min after anti-IgE resulted in the release of an additional 239 +/- 69 U/g; residual SRS-A levels remained at the plateau level. Incubation of the Tyrode's supernatant from challenged, but not control, tissue with fresh lung tissue caused a 90 +/- 7% inhibition of SRS-A release from the fresh tissue. Leukotriene D4 at 5 to 50 ng/ml (concentrations relevant to SRS-A release) showed a concentration-dependent inhibition of SRS-A release, but no effect on histamine release. Leukotriene C4 at 5 to 50 ng/mg failed to significantly alter the amount of SRS-A release. However, at 150 ng/ml, significant inhibition was observed which, in part, may have been produced by metabolism to leukotriene D4. These results demonstrate a potential role for LTD in regulating the amount of SRS-A released from monkey lung.

Nonadrenergic, noncholinergic contractile responses of guinea pig hilar bronchi to transmural electrical stimulation (TES) have been suggested to be due to release of endogenous tachykinins from capsaicin-sensitive neurons (C-fibers). Thiorphan and phosphoramidon, inhibitors of neutral endopeptidase (NEP, the major enzyme responsible for degrading tachykinins), were found to potentiate contractile responses of this isolated airway segment to TES and exogenously applied capsaicin, substance P and neurokinin A. However, the magnitude of potentiation by either inhibitor was smaller for TES and capsaicin (less than 10-fold leftward shift) than for the substrate agonists (about 100-fold leftward shift). This quantitative difference in potentiation by NEP inhibitors does not appear to be due to an influence of vasoactive intestinal peptide or calcitonin gene-related peptide, two endogenous peptides that might be released concomitantly by TES. Neither peptide caused marked effects on contractile responses to TES or tachykinins when applied to the isolated tissues. Addition of inhibitors of serine proteases, aminopeptidases, acetylcholinesterase and angiotensin-converting enzyme failed to further potentiate responses to TES in the presence of thiorphan. Therefore, the contractile response does not appear to be further modified by the activity of these peptidases. Neuropeptide gamma, but not neuropeptide K, was potentiated by thiorphan. The data suggest that peptides that are not substrates for NEP (for example, neuropeptide K) may also be released by TES from capsaicin-sensitive neurons to cause contraction. This may, at least in part, explain the quantitative difference in potentiation by NEP inhibitors of contractile responses to TES and to exogenously applied NEP-sensitive tachykinins in the guinea pig hilar bronchus.