David Aharony

Determination of SRS-A release from guinea-pig lungs by a radioimmunoassay

A sensitive radioimmunoassay for leukotrienes (LTs) has been developed. Rabbits were immunized with a conjugate of LTD4 and bovine serum albumin, prepared by using 1,5-difluoro-2,4-dinitrobenzene as the coupling agent. The assay can detect 0.045 pmol LTD4 at a final plasma dilution of 1:72. 50% displacement of bound 3H-LTD4 was obtained with 0.43 +/- 0.03 pmol LTD4. LTC4, LTE4 and LTF4 cross-react 159%, 57% and 85%, respectively, whereas LTB4, 5-HETE and prostaglandins did not. The assay was validated by measuring the antigen-induced release of LTs from sensitized guinea-pig chopped lungs. High correlation (0.9434, p less than 0.05) was found when LTs were simultaneously determined by this assay and a bioassay on guinea pig ileum.

Kinetic studies on the inactivation of 5-lipoxygenase by 5(S)-hydroperoxyeicosatetraenoic acid

The oxygenation of arachidonic acid (AA) by guinea-pig neutrophil 5-lipoxygenase terminates prematurely at a substrate utilization of only 50%. In the presence of dithiothreitol (DTT), reaction progress continues longer but still terminates prematurely, at about 70% substrate turnover. The addition of more substrate during the first 60 seconds of the initial reaction resulted in continued product formation. However, at times after 120 seconds, the addition of more AA could not produce additional product formation. Together, these results indicate a time-dependent (t1/2 = 0.5-1.0 min), irreversible loss of enzyme activity. To determine if the product 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) mediates the inactivation, it was tested for its ability to irreversibly inhibit the enzyme and found to inactivate 5-lipoxygenase with Ki = 0.05 +/- 0.01 microM and ki = 1.4 +/- 0.4 min-1. DTT changed the apparent affinity of 5-HPETE (Ki = 0.33 +/- 0.09 microM) but had no effect on the rate of inactivation (ki = 1.26 +/- 0.62 min-1). In contrast, the hydroxy derivative of 5-HPETE, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), is a reversible, time-independent inhibitor with Ki = 6.3 +/- 0.9 microM regardless of DTT. The ability of thiols to protect 5-lipoxygenase from production inactivation is due, at least in part, to a non-enzymatic reaction between DTT and 5-HPETE that converts the hydroperoxy acid to a material that can no longer inactivate the enzyme.

In vitro metabolism of [3H]-peptide leukotrienes in human and ferret lung: A comparison with the guinea pig

Fragmented lung tissue prepared from human, ferret and guinea pig converted [3H]-leukotriene C4 ([3H]-LTC4) to [3H]-LTD4 and [3H]-LTE4. [3H]-LTD4 was the major product recovered from incubations with human and guinea pig lung, whereas [3H]-LTE4 was the predominant metabolite in ferret. Kinetic analysis in human lung yielded a much faster rate for the conversion of [3H]-LTC4 to [3H]-LTD4+E4 than for the conversion of [3H]-LTD4 to [3H]-LTE4. In all three species serine-borate complex blocked the metabolism of [3H]-LTC4, whereas L-cysteine blocked the metabolism of [3H]-LTD4. These studies demonstrate that guinea-pig and human lungs have a similar metabolic pattern and capacity which is dissimilar to the ferret.

Biochemical and pharmacological characterization of ICI 198,615: a peptide leukotriene receptor antagonist

ICI 198,615 is one representative of a new chemical class of peptide leukotriene receptor antagonists that are the most potent and selective described to date. ICI 198,615 antagonized LTC4-, LTD4- and LTE4-induced increases in cutaneous vascular permeability in the guinea pig, with i.v. ED50 values of 0.083, 0.11 and 0.067 mumol/kg, respectively. Against LTD4, ICI 198,615 was 615 and 415 times more potent than LY 171883 and FPL 55712, respectively. L-Serine borate, an inhibitor of the metabolism of LTC4 to LTD4, did not influence the antagonism by ICI 198,615 of LTC4-induced increases in cutaneous vascular permeability. The compound both inhibited and reversed aerosol ovalbumin antigen-induced increases in pulmonary resistance in passively sensitized guinea pigs, but demonstrated little ability to inhibit or reverse ovalbumin antigen-induced decreases in dynamic lung compliance. At concentrations ranging from 10(-8) to 10(-5) M, ICI 198,615 had no significant effect on either the spontaneous or ovalbumin antigen-induced release of histamine, peptide leukotrienes, thromboxane B2 or 6-keto-prostaglandin F1 alpha from chopped guinea pig lung. At 10 microM, the compound did not inhibit 5-, 12- or 15-lipoxygenase. Finally, ICI 198,615 antagonized LTD4-induced increases in TxB2 release from chopped guinea-pig lung.

Inhibition by REV-5901 of leukotriene release from guinea-pig and human lung tissue in vitro☆

The 5-lipoxygenase inhibitor REV-5901 [alpha-pentyl-3-(2-quinolinylmethoxy)benzene-methanol] was evaluated for effects on mediator release in vitro from fragmented guinea-pig and human lung. In guinea-pig lung, REV-5901 inhibited the antigen-induced release of immunoreactive leukotriene D4 (iLTD4) with an IC50 of 9.6 +/- 2.9 microM and immunoreactive leukotriene B4 (iLTB4) with an IC50 of 13.5 +/- 2.2 microM. REV-5901 also inhibited the calcium ionophore-induced release of immunoreactive leukotrienes from human lung in vitro with IC50 values of 11.7 +/- 2.2 MicroM versus peptide leukotrienes and 10.0 +/- 1.1 microM versus iLTB4. The inhibition of release of iLTD4 and iLTB4 with similar IC50 values suggests that REV-5901 acts by inhibiting 5-lipoxygenase in this system. At concentrations as high as 50 microM, REV-5901 did not inhibit the release of thromboxane B2 (TxB2), 6-keto-prostaglandin-F1 alpha (6-keto-PGF1 alpha), or histamine from either lung. The lack of effect on TxB2 and 6-keto-PGF1 alpha indicates that REV-5901 did not inhibit phospholipase A2, cyclooxygenase, or thromboxane synthetase. Inhibition of leukotriene release by REV-5901 could not be reversed by washing. Among various 5-lipoxygenase inhibitors, the order of potency for inhibition of iLTD4 release from guinea-pig lung was AA-861 greater than REV-5901 greater than phenidone greater than nafazatrom greater than NDGA greater than BW755C. These findings suggest that REV-5901 is a selective and relatively potent inhibitor of leukotriene release from lung tissue in vitro.

Inhibition of antigen-induced contraction of guinea pig trachea by ICI 198,615

We examined the extent of peptide leukotrienes involvement in ovalbumin-induced contraction of guinea pig trachea isolated from animals passively sensitized with antiovalbumin antibodies. Antigen challenge resulted in a concentration-dependent (EC50 = 10 +/- 3 ng/ml, X +/- S.E.M., n = 6) and prolonged (greater than 60 min) contraction of guinea pig trachea. The maximal contractile response was directly proportional to the quantity of sensitizing antibodies. The maximal response (but not EC50 for ovalbumin) was significantly (P less than 0.01) enhanced by indomethacin (20%), only slightly (10%) inhibited by the histamine H1-antagonist pyrilamine and unaffected by the platelet-activating factor (PAF) antagonist CV-3988. The potent and selective leukotriene antagonist ICI 198,615 partially (45%) inhibited the antigen-induced contraction in a concentration-related fashion (1-300 nM). Even at concentrations that abolish responses to leukotrienes, ICI 198,615 did not further inhibit the response, nor could other leukotriene antagonists (i.e. LY 171883 or FPL 55712) inhibit more than 50% of this response. In contrast, the 5-lipoxygenase inhibitors AA-861 and REV 5901, also a leukotriene antagonist, abolished the contractile response. Taken together, the results suggest that in guinea pig trachea, leukotrienes, in combination with other lipoxygenase metabolites, mediate a major part of the response to antigen. In contrast, PAF, histamine and prostaglandins appear to play only a minor role.

Azepines and piperidines with dual norepinephrine dopamine uptake inhibition and antidepressant activity.

Herein, we describe the discovery of inhibitors of norepinephrine (NET) and dopamine (DAT) transporters with reduced activity relative to serotonin transporters (SERT). Two compounds, 8b and 21a, along with nomifensine were tested in a rodent receptor occupancy study and demonstrated dose-dependent displacement of radiolabeled NET and DAT ligands. These compounds were efficacious in a rat forced swim assay (model of depression) and also had activity in rat spontaneous locomotion assay.

4-Alkylpiperidines related to SR-48968: Potent antagonists of the neurokinin-2 (NK2) receptor

A series of 4-alkylpiperidine derivatives related to the potent neurokinin-2 (NK2) receptor antagonist SR-48968 (1) is described. Simple aliphatic derivatives were found to be poorly active, but appropriate placement of an alcohol functional group afforded compounds that were of similar activity to 1. Several representatives in this series, such as the 4-(1-hydroxy-1-ethylpropyl)piperidine (14), were found to exhibit oral activity in a model of labored abdominal breathing in guinea pigs. These results expand the latitude of substituents available in this region of this series of NK2 receptor antagonists.

Substituted 2,4-diaminoquinazolines and 2,4-diamino-8-alkylpurines as antagonists of the neurokinin-2 (NK2) receptor

Abstract Modification of the heterocyclic nucleus of a lead pyrrolopyrimidine (1) found to be active as an antagonist at the neurokinin-2 (NK2) receptor is described. Compounds based on the purine nucleus (3) were found to be particularly interesting, and were modified in the C(2), C(4) and C(8) substituents to afford compounds with high potency.

Specific binding of 3H-ICI 198,615, a potent LTD4 antagonist, to guinea pig cardiac ventricular membranes.

Peptido-leukotrienes (LTs) elicit myocardial depression in several mammalian species, and radioligand binding assays with 3H-LTC4 and 3H-LTD4 have provided evidence of putative receptor sites on guinea pig cardiac ventricular membranes (GPCVM). Our objective was to characterize specific binding of 3H-ICI 198,615, a potent and selective LTD4 antagonist, to the 155,000 X g pellet of GPCVM. 3H-ICI 198,615 (0.01-3.8 nM) showed high specific binding (85-90% of total), which was protein dependent, saturable (Bmax = 4914 +/- 706 fmol/mg protein, n = 3), of high affinity (Kd = 4.3 +/- 0.8 nM, n = 3) and without cooperativity. Equilibrium binding was achieved by 20 minutes and could be rapidly reversed by addition of excess unlabeled ICI 198,615 or FPL55712. Competition studies with 3H-ICI 198,615 against several LTD4 antagonists produced an order of potency: ICI 198,615 much greater than SKF102922 greater than FPL55712 greater than or equal to LY171883. Addition of divalent cations caused a concentration dependent decrease in specific binding apparently due to a reduction in affinity. Binding was not influenced by the guanine nucleotide analogs GTP gamma S and Gpp(NH)p, EDTA, or a multitude of diverse non-LT receptor agonists and antagonists. These data provide evidence supporting the existence of specific and high affinity binding sites for 3H-ICI 198,615 in GPCVM.