Guinea Pig Lung Membranes Research Articles

Isolated tissue and binding studies of YM-17690, a novel and non-analogous leukotriene agonist.

YM-17690, 3-[4-carboxymethoxy-3-[p-(4-phenylbutoxy) benzamido]phenyl]propionic acid, produced a dose-dependent contraction of guinea-pig ileum and its EC50 value was 1.6 X 10(-8) M. The response was not affected by pretreatment with atropine, mepyramine, indomethacin, dazoxiben and AA-861 (a 5-lipoxygenase inhibitor), but was inhibited by FPL-55712 (an LTD4 and LTE4 antagonist). YM-17690 induced dose-dependent contractions of guinea-pig lung parenchyma and trachea with EC50 values of 3.9 X 10(-9) and 2.2 X 10(-8) M, respectively. Pretreatment of these tissues with FPL-55712 resulted in a parallel shift of the YM-17690 dose-response curves to the right. The pA2 values for FPL-55712 in lung parenchyma and trachea were 7.41 and 8.21, respectively, and the slopes of the regression lines of Schild plots were 1.00 and 1.02, respectively. YM-17690 produced a dose-dependent inhibition of [3H]LTD4 binding to guinea-pig lung membranes and its pKi value was 9.28. However, the compound showed only 25% inhibition of [3H]TLC4 binding to guinea-pig hippocampus membranes, even at 10(-5) M. These results suggest that YM-17690 is a selective leukotriene (LTD4 and LTE4) agonist and that it will therefore be a valuable tool in the study of actions of leukotrienes and for the characterization of their receptors.

Pharmacological profile of MEN91507, a new CysLT 1 receptor antagonist

MEN91507 (8-[2-( E)-[4-[4-(4-fluorophenyl)butyloxy]phenyl]vinyl]-4-oxo-2-(5-1 H-tetrazolyl)-4 H-1-benzopyran sodium salt)) potently displaced [ 3H]leukotriene D 4 binding from guinea-pig lung and dimethylsulphoxide-differentiated U937 (dU937) cell membranes ( K i 0.50±0.16 and 0.65±0.29 nM, respectively). On the other hand, MEN91507 did not display significant binding affinity for a series of receptors or channels. In functional studies on dU937 cells, MEN91507 behaved as insurmountable antagonist of leukotriene D 4-induced calcium transients, with an apparent p K B of 10.25±0.15. In anaesthetized guinea-pigs, MEN91507 antagonized in a dose-dependent manner leukotriene D 4-induced bronchoconstriction following i.v. or oral administration: the ED 50s were 3.0±0.3 and 140±90 nmol/kg, respectively. The inhibition of leukotriene D 4-induced bronchoconstriction by MEN91507 was long-lasting, since a dose of 0.6 μmol/kg produced 74% reduction of the response after 8 h from administration. Likewise, leukotriene D 4-induced microvascular leakage was antagonized by MEN91507 either following i.v. or oral administration: a significant inhibitory effect was still evident at 16 h from oral administration of a dose of 6 μmol/kg. It is concluded that MEN91507 is a potent and selective antagonist of both guinea-pig and human CysLT 1 receptors; in addition, in vivo studies on guinea-pigs indicate that MEN91507 is an orally available and long-lasting antagonist of the bronchomotor and pro-inflammatory effects induced by leukotriene D 4 through the stimulation of CysLT 1 receptors.

Binding of YM158, a new dual antagonist for leukotriene D 4 and thromboxane A 2 receptors, to guinea pig lung membranes

Arachidonic acid metabolites mediate inflammatory responses at a cellular level. The affinity of the newly synthesized compound YM158, 3-[(4- tert-butylthiazol-2-yl)methoxy]-5′-[3-(4-chlorobenzenesulfonyl)propyl]-2′-(1 H-tetrazol-5-ylmethoxy)benzanilide monosodium salt monohydrate, for leukotriene D 4 and thromboxane A 2 receptors was examined in radioligand binding assays. YM158 inhibited [ 3 H ]leukotriene D 4 and [ 3 H ]U46619 (9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F 2α) binding to guinea pig lung membrane preparations, with K i values of 0.64±0.06 nM for leukotriene D 4 and 5.0±0.88 nM for thromboxane A 2 receptors. The Hill coefficients ( n H) did not significantly differ from unity, indicating that this antagonism is competitive. In contrast, YM158 showed no affinity for several other receptors, including neurotransmitter-related (α 1-, α 2-, β-adrenoceptors, histamine, 5-HT, muscarinic, σ), C 5a, opioid, Ca 2+ channel, K + channel, protein kinase C, bradykinin, endothelin, neurokinin and platelet activating factor receptors. These studies indicate that YM158 is a highly selective dual antagonist for leukotriene D 4 and thromboxane A 2 receptors, and this has potential clinical and research applications.

Roles of histamine receptor in a guinea pig asthma model

Histamine plays an important role in broncho constriction mediated by histamine receptors which provoke bronchial asthma attack. In this study, we measured H 1 and H 2 receptors in the guinea pig lung membrane fraction and obtained the following results. The maximum binding ( B max) of H 1 receptors in the guinea pig lung membrane fraction was significantly higher in the OA-sensitized group than that in the non-sensitized group, but affinity ( K d) did not differ between the groups. Otherwise, the maximum binding ( B max) of H 2 receptors in the guinea pig lung membrane fraction was significantly lower in the OA-sensitized group than that in the non-sensitized group. But affinity ( K d) did not differ between the groups. These findings suggest a close association of Histamine receptors both H 1 and H 2 in the pathology of asthma.

A novel photoaffinity probe for the LTD 4 receptor

A novel photoaffinity probe for the leuktriene D 4 receptor (LTD 4) is described. L-745310, which is structurally related to the potent LTD 4 antagonist MK-0476 (Singulair™), was found to selectively label a 43-kDa protein in guinea-pig lung membrane previously identified as the LTD 4 receptor.

Improved discriminatory properties between human and murine tachykinin NK 1 receptors of MEN 10930: a new potent and competitive antagonist

MEN 10930 (Nα{N-[(1H)indol-3-yl-carbonyl]1-amino-cyclohexane-1-carbonyl}L-3-(2-naphthyl)alanine N-(benzyl) N methyl amide) interacts with high affinity with NK 1 tachykinin receptor expressed in human IM9 (K i = 1.0 ± 0.17 nM) and U373MG (K i = 2.8 ± 0.5 nM) cells and guinea pig lung membranes (K i = 5.9 ± 0.8 nM). MEN 10930 shows no affinity for NK i sites present in rat urinary bladder membranes up to 10 μM, resulting in more than 10 000-fold selectivity for the human NK 1 receptor. In Scatchard plots performed in IM9 cells, MEN 10930 affects the substance P affinity, without changing the B max, suggesting a competitive interaction. It shows negligible affinity for calcium channels (K i = 1.6 ± 0.6 μM), NK 2 receptor (K i = 1.5 ± 0.5 μM) and for NK 3 receptor (K i > 10 μM). Furthermore, MEN 10930 inhibits in a competitive manner the SP methyl ester-induced contractions in guinea pig ileum (pA 2 = 8.7 ± 0.08). In conclusion, MEN 10930 is a potent, selective, competitive antagonist of human, but not murine, NK 1 receptor.

Synthesis and structure-activity relationships of carboxylated chalcones: a novel series of CysLT1 (LTD4) receptor antagonists.

The synthesis and CysLT1 antagonistic activities of a new series of 2-, 3-, and 4-(2-quinolinylmethoxy)- and 3- and 4-[2-(2-quinolinyl)ethenyl]-substituted, 2'-, 3'-, 4'-, or 5'-carboxylated chalcones are described. Structure-activity relationship studies indicate a preference for the presence of a negatively charged (acidic) moiety, although in some cases nitrile or ester analogues also exhibit moderate activity. The quinoline moiety may be substituted at either the 3- or the 4-position. Replacement of this heterocycle by other aromatic groups results in compounds with comparable affinities [2-(7-chloroquinoline), 1-(1-methyl-2-benzimidazole), or 1-(2-benzothiazole)] or substantially lower activities [1-(1-ethoxyethyl)-2-benzimidazole, 2-naphthyl, or phenyl]. The quinoline and chalcone moieties may be connected by either an ethenyl or a methoxy spacer. The acidic moiety at the chalcone B ring may be attached to the 2'-, 3'-, 4'-, or 5'-position, for both the 3- and 4-substituted chalcones. There are no general patterns to specify which substitution positions gave the most potent compounds. The series contains several potent CysLT1 receptor antagonists, with K(D) values approaching the nanomolar range, as measured by the displacement of [3H]LTD4 from guinea pig lung membranes. Antagonism of LTD4-induced contraction of guinea pig ileum, the inhibition of antigen-induced contraction of guinea pig trachea in vitro, and the inhibition of LTD4-induced increase of vascular permeability in vivo are determined for chalcones with high CysLT1 receptor affinities (K(D) values below 0.1 microM). 2'-Hydroxy-4-(2-quinolinylmethoxy)-5'-(5-tetrazolyl)chalcone (14, VUF 4819) showed good activity in both in vitro and in vivo assays and has been selected for further evaluation.

Discovery of OT4003, a novel, potent, and orally active cys-LT 1 receptor antagonist

The present paper describes the structural modifications leading to the discovery of a new series of quinoline-containing cys-LT 1 receptor (LTD 4 receptor) antagonists. A structural optimization with respect to the in vitro receptor binding, the in vivo brochoconstriction, and the toxicological effect in the form of peroxisomal proliferation was performed in order to achieve the target compound OT4003. OT4003 (( S)-(+)- E-2-[2-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenylaminomethyl)phenoxy]-hexanoic acid) was found to be a potent and selective inhibitor of [ 3H]LTD 4 specific binding to guinea pig lung membranes (IC 50 2.4±1.0 nM), and also a potent, orally active, antagonist of LTD 4 induced bronchoconstriction in guinea pigs [Ed 50 0.14 (ED 16 0.1−ED 84 0.4) mg/kg; 4 h pretreatment]. The enantiomerically pure OT4003 was prepared using a short convergent synthesis, including an enzymatic resolution step.

Quinoline-substituted dihydroindoles as cysLT 1 (LTD 4 receptor) antagonists

A series of quinoline-substituted dihydroindoles has been synthesized and evaluated as antagonists of the cysLT 1 receptor. This series, exemplified by 2 (LY302905; pKi = 8.3 for inhibition of binding of 3H-LTD 4 to guinea pig lung membranes), represents reduced analogues of the corresponding indoles that were previously shown to be potent, orally active cysLT 1 receptor antagonists. These dihydroindole compounds generally displayed increased in vitro and in vivo (oral) activity.

604 Regulation of baroreceptor reflex by vasopressin in the hypertensive rats

We synthesized a novel ligand [4,5-3H-Leu9]-Neurokinin A (3H-NKA, S.A 117–144 Ci/mmol), and evaluated its binding to hamster urinary bladder membranes (HUBM). The ligand bound to HUBM in a highly-specific (94 ± 4%) and protein-dependent manner. Binding was rapid (k1 = 0.037 nM−1∗min−1) and saturable (Bmax = 1210 ± 177 fmol/mg protein), to a single population of high-affinity sites (KD = 2.41 ± 0.15 nM, nH = 0.99 ± 0.02). Binding was inhibited by non-hydrolyzable GTP analogs. Competition experiments with HUBM demonstrated the following rank order of potency: NKA > Kassinin > [β-Ala8]-NKA(4–10) > [Nle10]-NKA(4–10) = Eledoisin = NKB > Physaelamin > Substance P. The selective NK-1 and NK-3 ligands, [Sar9-Met (O2)11]-SP, (±) CP96,345 and Senktide respectively, did not inhibit binding at 10 μM, whereas, the selective NK-2 antagonists: (±) SR-48,968 ⪢ L-659,877 > R396 ⪢ MEN-10,207 > MEN-10,376, inhibited binding in a competitive manner. In contrast, the low specific binding (<30%) detected in guinea pig lung membranes, was not inhibited by selective NK-2 ligands. Over 30 ligands (0.1–10 μM) from other receptor classes, were not inhibitory. The data suggest that this new ligand binds with high-affinity and selectivity to homogeneous population of NK-2 receptors on HUBM but not on lung membranes, and is a suitable ligand to study NK-2 receptors.

Synthesis of carboxylated flavonoids as new leads for LTD 4 antagonists

Summary A series of 3′- and 5′-carboxylated chalcones, 6- or 8-carboxylated flavones and 6-carboxylated flavanones, -flavanols and -flavans were prepared. The compounds were tested for their inhibitory activities against leukotriene D 4 (LTD 4 ) induced contraction of guinea-pig ileum. A new and convenient synthetic route to 3-acetyl-2-hydroxybenzoic acid ( 1d ), a key intermediate for the synthesis of 3′-carboxy-2′-hydroxychalcones and 8-carboxylated flavones, was developed. The activities of the tested compounds ranged from 0 to 63% inhibition at 10 −5 M drug concentration against a single challenge of 10 −8 M LTD 4 . Several compounds were tested in a radioligand binding assay against [ 3 H]LTD 4 on guinea-pig lung membrane. The quinoline-containing chalcone 12 and flavone 17 were found to exhibit significant but weak affinities for LTD 4 receptors with p K D -values of 4.95 and 4.83, respectively, and are interesting lead structures for the development of rigid LTD 4 antagonists. In contrast, the rest of the compounds tested in the binding assay did not show significant displacement of the radioligand, implying that for these compounds the functional activity is probably not caused by competitive antagonism at the LTD 4 receptor. The exact mechanism of the relaxant activity remains unclear.

Synthetic and structure/activity studies on acid-substituted 2-arylphenols: discovery of 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5- hydroxyphenoxy]-propoxy]phenoxy]benzoic acid, a high-affinity leukotriene B4 receptor antagonist.

Structural derivatives of LY255283 have been studied as receptor antagonists of leukotriene B4. Substitution of the 2-hydroxyacetophenone subunit of 1 (LY255283) with a 2-arylphenol group provided entry into several new series that feature various mono- and diacidic core functionality. These new analogues, the subject of a broad structure-activity investigation, displayed significantly increased in vitro and in vivo activity as receptor antagonists of LTB4. A series of diaryl ether carboxylic acids demonstrated especially interesting activity and led to the discovery of compound 43b, 2-[2-propyl-3-[3-[2-ethyl-4-(4- fluorophenyl)-5-hydroxyphenoxy]-propoxy]phenoxy]benzoic acid (LY293111), a 2-arylphenol-substituted diaryl ether carboxylic acid which displayed potent binding to human neutrophils (IC50 = 17 +/- 4.6 nM) and guinea pig lung membranes (IC50 = 6.6 +/- 0.71 nM), inhibition of LTB4-induced expression of the CD11b/CD18 receptor on human neutrophils (IC50 = 3.3 +/- 0.81 nM), and inhibition of LTB4-induced contraction of guinea pig lung parenchyma (pKB = 8.7 +/- 0.16). In vivo, 43b demonstrated potent activity in inhibiting LTB4-induced airway obstruction in the guinea pig when dosed by the oral (ED50 = 0.40 mg/kg) or intravenous (ED50 = 0.014 mg/kg) routes. A specific LTB4 receptor antagonist, 43b had little effect on inhibiting contractions of guinea pig lung parenchyma induced by leukotriene D4 (LTD4), histamine, carbachol, or U46619. Compound 43b has been chosen as a clinical candidate and is currently in phase I studies for a variety of inflammatory diseases.

Binding sites for islet amyloid polypeptide in mammalian lung: species variation and effects on adenylyl cyclase.

Islet amyloid polypeptide (IAPP) and calcitonin gene related peptide (CGRP) share a 47% sequence homology. IAPP can interact with adenylyl cyclase coupled CGRP receptors. We have examined [125I]IAPP binding in mouse, pig, and guinea pig lung membranes in competition with IAPP, CGRP, and CGRP(8-37). Three types of site were shown by order of potency: (i) mouse, IAPP > CGRP(8-37) >> CGRP; (ii) pig, CGRP > IAPP > CGRP(8-37); and (iii) guinea pig, CGRP = IAPP = CGRP(8-37). Chemical cross-linking of [125I]IAPP and [125I]CGRP binding sites in lung demonstrated that both sites had similar molecular weights in any one species but differed across species, i.e., mouse M(r) = 70,000 and 98,000; pig M(r) = 68,000, 56,000, and 47,000; and guinea pig M(r) = 106,000 and 56,000. Adenylyl cyclase activity was stimulated by forskolin and AlCl3-NaF in rat, mouse, pig, and guinea pig membranes. Only in mouse and pig were CGRP and IAPP able to stimulate adenylyl cyclase activity. In mouse lung CGRP and IAPP stimulated adenylyl cyclase activity with EC50 values of 642 +/- 222 nM (n = 4) and 325 +/- 115 nM (n = 4), respectively. In pig lung membranes EC50 values were 5.7 +/- nM (n = 4) for CGRP and 1230 +/- 1130 nM (n = 4) for IAPP. Thus IAPP either did not stimulate adenylyl cyclase activity in these lung membranes or did so with a low potency.

(Piperidinylalkoxy)chromones: novel antihistamines with additional antagonistic activity against leukotriene D4.

A series of novel chromone derivatives, in which the chromone moiety is connected to a (diphenylmethylene)-, (diphenylmethyl)-, or (diphenylmethoxy)piperidine via an alkyloxy spacer, were synthesized as antiallergic and antiasthmatic agents. In addition to their potent antihistaminic activity, the compounds also inhibit contraction in guinea pig ileum induced by leukotriene D4. When analyzed by radioligand binding assays in guinea pig lung membranes, one of the compounds, 7-[[3-[4-(diphenylmethylene)piperidin-1- yl]propyl]oxy]-2-(5-tetrazolyl)-4-oxo-4H-1-benzopyran, showed dissociation constants (KD) of 5.62 nM and 2.34 microM for H1- and LTD4-receptors, respectively. In vivo at the dose of 10 mg/kg, the compound inhibited the histamine- and LTD4-induced increase of vascular permeability in guinea pigs by 95 and 30%, respectively. The inhibition of LTD4-induced increase in vascular permeability by the compound was increased to 56% when a dose of 50 mg/kg was employed. Similar to terfenadine, the compound does not readily occupy the brain H1-receptors when given intraperitoneally to mice, implying no sedating side effects.

Discovery and pharmacological properties of selective neurokinin-receptor antagonists, FK224 and FK888

In order to create a new drug for the treatment of respiratory diseases, such as asthma and chronic bronchitis, having a novel therapeutic mechanism, we have been trying to develop new compounds with neurokinin (NK)-receptor antagonistic effects. We used [3H]-substance P binding to guinea pig lung membrane for the first screening system and successfully discovered FK224 from a fermentation product and FK888 from chemical design studies using an octapeptide antagonist (D-Pro4,D-Trp7,9,10) SP4-11 as the parent compound. FK224 and FK888 showed different selectivities against the NK-receptor subtypes (NK1, NK2, NK3); FK888 was a highly potent NK1-selective antagonist, and FK224 was a NK1 + NK2 dual receptor antagonist. Neither compound had any activity on the NK3 receptor. In the in vivo experiments, FK224 and FK888 significantly inhibited the constriction and plasma extravasation in the airway induced by agonist injection. These compounds also showed inhibitory effects on the airway response induced by capsaicin and antidromic stimulation of vagus nerves. Furthermore, FK224 and FK888 were effective on the mucus secretion in the airway and the cough reflex induced by citric acid challenge. There were some differences in the effects of FK224 and FK888 in the in vivo experiments, and it was suggested that the NK1 receptor and NK2 receptor were mainly involved in neurogenic inflammation and airway constriction, respectively. FK224 and FK888 are now undergoing clinical studies to test the effectiveness of a NK antagonist in human respiratory diseases.

A Rapid and Selective Endothelin-Converting Enzyme Assay: Characterization of a Phosphoramidon-Sensitive Enzyme from Guinea Pig Lung Membrane

Endothelin-1 (ET-1) is the most potent vasoconstrictor hormone known to date. Biosynthesis of ET-1 from its precursor big endothelin-1 (BET-1) is mediated by endothelin-converting enzyme (ECE), a phosphoramidon-sensitive metalloprotease. We have established a simple, rapid, and selective assay for the evaluation of ECE activity. This assay is based on the quantitative determination of [ 125I]ET-1 released from (3-[ 125I] idotyrosyl 13)BET-1 by binding to the membrane-bound endothelin (ET) receptor. Using this assay we have discovered that guinea pig lung membrane (GPLGM) contains a phosphoramidon-sensitive ECE. Treatment of GPLGM with 0.06% lubrol increased ECE activity and ET binding of the membrane preparation. Lubrol-treated GPLGM (L-GPLGM) contains a high density of ET binding sites ( B max = 2000 fmol/mg protein) and shows no proteolytic activity for degredation of ET-1. At protein concentrations suitable for measurement of ECE activity (0.2 mg/ml), L-GPLGM contains a high concentration of ET receptors and shows a rapid rate of ET-1 binding to the membrane preparation (binding equilibrium in <5 min). Thus, we have utilized L-GPLGM preparation for both ECE activity and measurement of ET-1 binding in a single-step assay for the determination of enzyme activity. ECE activity of L-GPLGM was fully inhibited by phosphoramidon, 1,10-phenanthroline, and EDTA. Class-specific inhibitors of serine, cysteine, and aspartic proteases showed no significant effect on ECE activity in L-GPLGM. These results show that GPLGM contains exclusively a phosphoramidon-sensitive ECE.

High affinity [ 3H]formoterol binding sites in lung: characterization and autoradiographic mapping

Agonist binding to the β 2-adrenoceptors and its mapping were studied using the newly developed radioligand [ 3H]formoterol. The results of [ 3H]formoterol saturation binding and formoterol inhibition of [ 3H]formoterol binding were consistent with binding to a single class of receptors ( K d = 1.34 ± 0.15 nM, B max = 154.9 ± 8.0 fmol/mg protein in guinea pig lung membranes, n = 8; K d = 1.05 ± 0.17 nM, B max = 67.8 ± 8.1 fmol/mg protein in human lung membranes, n = 5) and competition assays with other agonists and antagonists disclosed only a single class of site. The nonhydrolyzable GTP analogue GTPγS caused a reduction in both K d and B max, indicating that the receptors labelled by [ 3H]formoterol are coupled to a guanine nucleotide binding regulatory protein. Receptor mapping of [ 3H]formoterol binding sites shows that β 2-adrenoceptors were widely distributed in both guinea pig and human lung, with dense labelling over airway epithelium and uniformly over alveolar walls, and sparse labelling of airway and vascular smooth muscle. In addition, submucosal glands were also sparsely labelled in human bronchus. The distribution of β 2-adrenoceptors was similar to the pattern previously described with non-selective radiolabelled antagonists in the presence of selective β 1-adrenoceptor antagonists.

Association of guinea pig lung bombesin receptors with pertussis toxin-sensitive guanine nucleotide binding proteins

The possible interaction of bombesin receptors with guanine nucleotide binding protein in guinea pig lung was studied. The non-hydrolysable GTP analogue guanosine-5′-[γ-thio]triphosphate (GTPγS) was shown to decrease [ 125I-Tyr 4]bombesin binding in a concentration-dependent manner. The specificity of this effect was assessed by examining the effects of other guanine nucleotides on this binding at a concentration of 1mM. GMP and GDP weakly inhibited [ 125I-Tyr 4]bombesin binding (2 and 19%, respectively), whereas GTP, guanosine-5′-[β-thio]triphosphate (GDPβS), and 5-guanylylimidodiphosphate (GppNHp) exhibited similar potencies, inducing 52%, 46%, and 43% inhibition of [ 125I-Tyr 4]bombesin binding respectively. Saturation experiments performed in the absence and presence of 100 μM GTPγS indicated the presence of a single population of receptors in both cases. However, the addition of GTPγS induced a marked decrease in the number of receptors (from 1.76 fmol/mg protein to 0.78 fmol/mg protein) without significantly altering the dissociation constant ( K d). These results provide evidence that bombesin receptors are coupled to a G-protein signal transduction pathway in guinea pig lung. We have further characterised this G-protein on the basis of its toxin sensitivity. Pretreatment of the lung membranes with either pertussus (10 μm/ml) or cholera toxin (50 μg/ml) was performed. Cholera toxin treatment did not affect the ability of GTPγS to inhibit [ 125I-Tyr 4]bombesin binding to guinea pig lung membranes. However, pertussis toxin treatment induced a decrease in binding and resulted in the inability of GTPγS to inhibit [ 125I-Tyr 4]bombesin binding in a concentration-dependent manner. These results suggest that bombesin receptors of the guinea pig lung interact with pertussis toxin-sensitive G-proteins.

Chiral manipulation of drug selectivity: studies on a series of terfenadine-derived dual antagonists on H1-receptors and calcium channels.

A series of terfenadine derivatives were evaluated for enantioselectivity on histamine H1-receptors and calcium channels. Whereas H1-receptors are only sterically discriminative against the benzhydryl part of the molecules, calcium channels showed enantioselectivity to either the phenylbutyl part or the benzhydryl part provided that an appropriate lipophilicity is preserved at the chiral site. It is speculated that the hydrophilicity of the butanol moiety is responsible for the lack of stereoselectivity of terfenadine enantiomers since it drives the side chain out of the stereoselective site of calcium channels, which are lipophilic. In four different test systems, (guinea-pig ileum, guinea-pig lung membranes, rat aorta and rat cortex membranes), this series of compounds generally showed about 10 times higher activity on H1-receptors than on calcium channels. By introducing a chiral center in the different parts of the molecule we were able to increase the selectivity of an enantiomer VUF4648 to calcium channels.

Expression of platelet-activating factor receptor mRNA in human and guinea pig lung.

Platelet-activating factor (PAF) has been implicated in the pathogenesis of several inflammatory pulmonary diseases, and specific binding sites have been demonstrated in human and guinea pig lung membranes by radioligand binding experiments. Both human and guinea pig PAF receptors (PAFR) have recently been cloned. We have used molecular probes to study the gene expression of PAFR in human and animal lung and in situ hybridization to determine the distribution of PAFR mRNA in peripheral lung. Northern blot analysis of total lung RNA from human lung parenchyma, using a 1.1-kb SmaI-EcoRI fragment of human PAFR cDNA or a 0.9-kb SmaI-SmaI fragment of guinea pig PAFR cDNA, demonstrated the expression of PAFR mRNA in human lung, with a single transcript of 4 kb. There was a significant increase in PAFR mRNA in the lungs of asthmatic patients and a significant decrease in PAFR mRNA in the lungs of cigarette smokers compared with normal patients. Similarly, the expression of PAFR mRNA on guinea pig and rat lung was detected as a single transcript of 3 kb, using both guinea pig and human PAFR cDNA probes. A full-length 1.8-kb human leukocyte PAFR cDNA probe was used as the DNA template for producing 35S-labeled antisense and sense cRNA probes for use in in situ hybridization studies of human peripheral lung. These studies revealed high levels of PAFR mRNA hybridization in blood vessels, moderate levels of hybridization in alveolar walls and peripheral airway smooth muscle, but no specific signal in airway epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)