Ž .Immunopharmacology 43 1999 187–194www.elsevier.comrlocaterimmpharm

Pharmacological profile of LF 16-0687, a new potent non-peptidebradykinin B receptor antagonist2

Didier Pruneau ), Jean Luc Paquet, Jean Michel Luccarini, Evelyne Defrene,ˆChantal Fouchet, Rose-Marie Franck, Bruno Loillier, Claude Robert, Pierre Belichard,´

Herve Duclos, Beatrice Cremers, Pierre Dodey´ ´Receptor Pharmacochemistry Group, Centre de Recherche, Laboratoires Fournier, 50 Rue de Dijon, 21121 Daix, France

Accepted 3 June 1999

Abstract

Ž ww wwŽ . x x x x w ww Ž .LF 16-0687 1- 2,4-dichloro-3- 2,4-dimethylquinolin-8-yl oxy methyl phenyl sulfonyl -N- 3- 4- aminoiminomethyl -x x x Ž . .phenyl carbonylamino propyl -2 S -pyrrolidinecarboxamide has been selected from a large-scale medicinal chemistry

w3 x Ž .program for further development. In competition binding studies using H bradykinin BK , LF 16-0687 bound to thehuman, rat and guinea-pig recombinant B receptor expressed in CHO cells giving K values of 0.67 nM, 1.74 nM and 1.372 i

Ž . Ž .nM, respectively. It also bound to the native BK B receptor from human umbilical vein HUV , rat uterus RU and2Ž .guinea-pig ileum GPI giving K values of 0.89 nM, 0.28 nM and 0.98 nM, respectively. It inhibited BK-induced IP1, IP2i

and IP3 formation in INT407 cells yielding pK values of 8.5, 8.6 and 8.7, respectively. In isolated organs experiments, LFB

16-0687 behaved as a competitive antagonist of BK-mediated contractions giving p A values of 9.1 in HUV, 7.7 in RU and2

9.1 in GPI. Binding and functional studies performed over 40 different receptors revealed that LF 16-0687 was selective forthe BK B receptor. A continuous intravenous infusion of LF 16-0687 antagonized in a dose-dependent manner and with a2

rapid onset of action BK-induced hypotensive response. Subcutaneous administration of LF 16-0687 at 1.1 mmolrkg to ratsŽ . Ž . Ž .markedly reduced BK-induced edema of the stomach y69% , duodenum y65% and pancreas y56% . q 1999 Elsevier

Science B.V. All rights reserved.

Keywords: Bradykinin B receptors; Non-peptide antagonists; Pharmacology; Rat receptors; Guinea-pig receptors; Human receptors2

1. Introduction

Ž .Bradykinin BK and kallidin are endogenouspeptides which participate to a wide range of inflam-

AbbreÕiations: BBB, Blood brain barrier; BK, Bradykinin;GPI, Guinea-pig ileum; IPs, Phosphoinositosides; HUV, Humanumbilical vein; RU, Rat uterus; TBI, Traumatic brain injury

) Corresponding author. Tel.: q33-3-80-44-75-39; fax: q33-3-80-44-76-00; e-mail: d.pruneau@fournier.fr

Ž .matory conditions Bhoola et al., 1992; Hall, 1992 .Most of the physiological effects of BK are mediated

Žby B receptors including pain Dray and Perkins,2. Ž1993 , vasodilatation Regoli and Barabe, 1980;´

.D’Orleans-Juste et al., 1989 and increase of vascu-´Žlar permeability Unterberg et al., 1984; Wahl et al.,

.1993 . Therefore, there is a wide interest in develop-ing selective and potent antagonists of the BK B2

receptor as potential therapeutic agents. Recently,FR173657 was described as a new chemical entity

0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0162-3109 99 00128-9

( )D. Pruneau et al.r Immunopharmacology 43 1999 187–194188

selectively binding to B receptors and potently ant-2Žagonizing in vivo BK-induced responses Aramori et

.al., 1997; Asano et al., 1997 . Others chemicalderivatives belonging to the same chemical serieswere subsequently disclosed including FR167344 and

ŽFR165649 Aramori et al., 1997; Inamura et al.,.1997; Asano et al., 1998 . In the present paper, we

describe the pharmacological profile of LF 16-0687Ž ww wwŽ . x1- 2,4-dichloro-3- 2,4-dimethylquinolin-8-yl oxy -

x x x w ww Ž .methyl phenyl sulfonyl -N- 3- 4- aminoiminomethylx x x Ž .phenyl carbonylamino propyl -2 S -pyrrolidinecar-

.boxamide which is so far the most potent compoundof a novel series of non-peptide B receptor antago-2

nists.

2. Methods

2.1. In Õitro studies

Cloning and stable expression of the human andrat B receptor was performed as previously de-2

Ž .scribed Pruneau et al., 1998a . The rat B receptor2

cDNA subcloned in pRCrCMV was kindly providedŽby Prof. J. Navarro University of Texas Medical.Branch, Galveston, TX .

Based on the nucleotide sequence of the cDNA asŽ .described by Farmer et al. 1998 , the coding region

of the guinea-pig B receptor was isolated by PCR2Ž .using the cDNA from guinea-pig ileum GPI as a

template and appropriate 5X-primer 49-mers and 3X-primer 36-mers oligonucleotides. The cDNA encod-ing the guinea-pig receptor was subcloned in a bi-

Ž .cistronic eukaryotic vector Rees et al., 1996 . CHOcells were transfected with the cDNA containing

Ž .vector 1 mgrplate of 35 mm in diameter usingSuperfect Reagent from Quiagen.

Cell membranes were obtained as previously de-Ž .scribed Pruneau et al., 1998a, 1999 . Protein con-

centration was determined according to the methodŽ .of Bradford 1976 using a Bio-Rad protein assay

kit. Competition-binding studies were conducted asw3 x Ž .previously described using H -BK 0.1 to 5 nM

Ž .Pruneau et al., 1998a, 1999; Paquet et al., 1999 .Additional competition binding assays of LF 16-

0687 at 1 mM were performed in human and non-human cell membrane preparations for adenosine A1

and A , a - and a -adrenoceptors, b -, b -and b -2 1 2 1 2 3

adrenoceptors, angiotensin AT and AT , cholecys-1 2

tokinin CCK and CCK , dopamine D , D and D ,1 2 1 2 3

endothelin ET and ET , GABA and GABA ,A B A B

glutamate AMPA, histamine H , H and H ,1 2 3

leukotriene BLT and CysLT , acetylcholine mus-1Žw3 xcarinic M and M and nicotinic H nicotine as a1 2

.non-selective radioligand , tachykinin NK , NK and1 2Žw3 xNK , neuropeptide Y H neuropeptide Y as a non-3

. Žselective radioligand , neurotensin NST , opioid m,1w3 xd and k; H naloxone as a non-selective radioli-

.gand , 5-hydroxytryptamine 5-HT , 5-HT , 5-1A 1D

HT , 5-HT and 5-HT , vasopressin V and V2A 3 4 1 2

receptors and for L-type Ca2q channels, N-type Ca2q

channels, ATP-sensitive K channels, voltage-depen-dent K channels, Ca2q-dependent K channels, Nachannels sites 1 and 2 and Cl channels using appro-priate radiolabeled ligands and standard methods.LF 16-0687 was also tested at 1 mM against an-giotensin-converting enzyme and cyclooxygenase 2.

To measure the effect of LF 16-0687 on BK-Ž .induced production of phosphoinositosides IPs ,

INT407 cells grown in 12-well plates were labeledw3 xfor 18 h with 1 mCirml H myo-inositol in serum

Ž .free medium 199 Gibco, Cergy-Pontoise, France .Different components of IPs were separated and

Žmeasured as previously described Paquet et al.,.1999 .

2.2. Isolated organs experiments

Ž . Ž .Human umbilical vein HUV , rat uterus RUand GPI were isolated, prepared and set up in organ

Žbaths as previously reported in detail Pruneau et al.,.1995, 1998a, 1999; Paquet et al., 1999 . LF 16-0687

or its respective vehicle was added at various con-centrations 15 min before cumulative addition ofBK.

2.3. In ÕiÕo experiments

Effects of LF 16-0687 against BK-induced hy-potensive response was evaluated in anesthetized ratsaccording to the previously described methodŽ .Pruneau et al., 1999 .

Ž .Male Sprague–Dawley rats 80–100 g were usedfor BK-induced edema experiments. Catheters were

( )D. Pruneau et al.r Immunopharmacology 43 1999 187–194 189

implanted in both right and left jugular veins forEvans blue dye injection and BK infusion, respec-

Ž .tively. Evans blue dye 20 mgrkg was injected as aŽ .bolus and 5 min later BK 0.56 nmolrkgrmin was

Ž .infused for 20 min. LF 16-0687 1.1 mmolrkg or itsŽ .vehicle saline were given subcutaneously 60 min

before starting BK infusion. At the end of BK infu-sion, 40 ml of saline was infused through the ascend-ing aorta and the animals were sacrificed. The wholestomach, duodenum and pancreas were then dis-sected out, carefully dried up, weighed and desic-cated for 16 h in a vacuum oven at 608C, weighedagain and incubated in formaldehyde for 24 h at558C. The Evans blue dye content of tissues wascalculated from a standard calibration spectrophoto-metric curve.

2.4. Analysis of data

Binding competition data and concentration–re-sponse curves for IPs hydrolysis and BK-inducedcontractions were analysed using GraphPADInPlotŽ .GraphPAD Software, San Diego, CA . Equilibrium

Ž .dissociation constant K , inhibitory binding con-DŽ .stant K , IC , pK and p A values were calcu-i 50 B 2

Ž .lated as previously described Paquet et al., 1999 .Statistical analysis were performed using Statview

Ž .Abacus Concept, Palo Alto, CA . A one-way analy-sis of variance followed by a Student’s t-test wasused to establish significant differences between Ki

values. A P-0.05 was considered as statisticallysignificant.

2.5. Drugs

w3 x Ž . w3 xH -BK 90 to 120 Cirmmol and H -des-10 w 9 x Ž .Arg - Leu -kallidin 74 to 77 Cirmmol were from

Ž .New England Nuclear Les Ullis, France . MER-ŽGETPA DL-2-mercaptomethyl-3-guanidinoethyl-

. Žthiopropanoic acid was from Calbiochem La Jolla,.CA . All molecular biology and cell culture reagents

Ž .were from Boehringer-Mannheim Meylan, France .Ž .Other chemicals were from Sigma St. Louis, MO .

Ž ww wwŽLF 16-0687 1- 2,4-dichloro-3- 2,4-dimethyl-. x x x x w wwquinolin-8-yl oxy methyl phenyl sulfonyl -N- 3- 4-

Ž . x x xaminoiminomethyl phenyl carbonylamino propyl -Ž . . Ž .2 S -pyrrolidinecarboxamide Fig. 1 was synthe-

Ž .sised at Laboratoires Fournier Daix, France . This

Fig. 1. Chemical structure of LF 16-0687.

compound was used as a dichlorhydrate or dimesy-late salt and dissolved in water.

3. Results

3.1. Binding studies

We have successfully established cell lines stablyexpressing the human, rat and guinea-pig B recep-2

w3 xtor which bound H BK giving K values of 0.64DŽ . Ž ."0.08 nM ns9 , 0.46"0.09 nM ns6 and

Ž .0.09"0.04 nM ns4 , and B of 2283"325,max

2265"526 and 1492"301 fmolrmg protein, re-spectively. In corresponding cell membrane prepara-

w3 xtions, LF 16-0687 competed with H BK givingŽ .calculated K values of 0.67"022 nM ns4 ,i

Ž . Ž .1.74"0.68 nM ns3 and 1.37"0.32 nM ns4for the recombinant human, rat and guinea-pig B2

Ž .receptor, respectively see Fig. 2 .w3 xBinding characteristics of H BK to membrane

preparations of HUV, RU and GPI have been de-Ž .scribed in details Pruneau et al., 1998a, 1999 . LF

w3 x16-0687 inhibited the binding of H BK giving KiŽ .values of 0.89"0.22 nM ns3 , 0.28"0.07 nM

Ž . Ž .ns3 and 0.98"0.18 nM ns3 in HUV, RUŽ .and GPI membranes, respectively Fig. 2 .

3.2. SelectiÕity studies

LF 16-0687, at concentrations up to 10 mM, didw3 x 10 w 9 xnot alter the binding of H -des-Arg - Leu kallidin

in membranes from 293 cells stably expressing thehuman kinin B receptor according to the previously1

Ž .described method Bastian et al., 1997 .

( )D. Pruneau et al.r Immunopharmacology 43 1999 187–194190

w3 xFig. 2. Competition binding of H BK by LF 16-0687 to membranes of CHO cells expressing cloned rat, human and guinea-pig receptorand of RU, HUV and GPI. K values are inserted. Values represent mean"S.E.M. from three to four experiments.i

Selectivity of LF 16-0687 was further assessed bytesting in 40 receptor binding, two enzyme and eightion channel assays. LF 16-0687 at 1 mM had nosignificant effect in this battery of assays, except forthe human muscarinic M and M receptor binding2 1

assay in which it had an IC of 0.30 mM and 0.4650

mM, respectively.

3.3. Functional assays

BK increased in a concentration-dependent man-Ž .ner the production of IPs in INT407 cells Fig. 3 .

LF 16-0687 at 100 nM markedly shifted to the rightresponse-curves to BK without significantly affect-ing the maximum. Calculated pK values of LFB

Ž . Ž .16-0687 were 8.5"0.1 ns3 , 8.6"0.1 ns3Ž .and 8.7"0.1 ns3 for IP1, IP2 and IP3 response-

Ž . Žcurve Fig. 3 . As described Pruneau et al., 1995,.1998a, 1999 , BK contracted HUV, RU and GPI. LF

16-0687 produced a concentration-dependent right-ward shift of the contraction curve to BK in the three

Ž .tissue preparations Fig. 4 . LF 16-0687 behaved as acompetitive antagonist giving p A values of 9.1"2

0.2, 7.7"0.3 and 9.1"0.5 in HUV, RU and GPI,

Fig. 3. Inhibitory effect of LF 16-0687 on BK-induced formation of IPs in INT407 cells. Values represent mean"S.E.M. from threeexperiments.

( )D. Pruneau et al.r Immunopharmacology 43 1999 187–194 191

Fig. 4. Concentration–contraction curves of BK in the HUV, RU and GPI in the absence and presence of LF 16-0687. Schild plot curves areinserted. Values represent mean"S.E.M. from four to six experiments.

respectively. Schild plot slopes were not differentfrom unity.

In the anesthetized rat, BK produced a short-last-ing decrease of arterial blood pressure. A continuousintravenous infusion of LF 16-0687 antagonized in adose-dependent manner the hypotensive response to

Ž .BK Fig. 5 . Interestingly, the maximum inhibitoryeffect was obtained 5 min after the beginning ofinfusion indicating a rapid onset of action of LF

Ž .16-0687 data not shown .BK increased the content of Evans blue dye in the

Ž .pancreas)stomach)duodenum Fig. 6 . A pre-

( )D. Pruneau et al.r Immunopharmacology 43 1999 187–194192

Fig. 5. Inhibitory effects of an intravenous infusion of LF 16-0687against BK-induced hypotensive response in the rat. Values repre-sent mean"S.E.M. from six to seven experiments.

treatment with LF 16-0687 at 1.1 mmolrkg reducedŽ .BK-induced edema of the pancreas y56% , stom-

Ž . Ž . Ž .ach y69% and duodenum y65% Fig. 6 .

4. Discussion

In the present study, we have shown that LF16-0687 is one of the most potent non-peptide antag-onist of the human B receptor so far described. This2

compound did not bind to the B receptor or to a1

range of other G-protein coupled receptors, with theexception of muscarinic M and M receptors for2 1

which a moderate binding affinity of LF 16-0687

was noted. We also showed that the compoundantagonized BK-mediated responses in vivo.

Kinins are active fragments produced by enzy-matic cleavage of large precursors, kininogens, whichare circulating proteins activated in a number ofpathophysiological situations. In this respect, kininsare considered as important mediators of inflamma-

Ž .tion Bhoola et al., 1992; Hall, 1992 . Most of theacute proinflammatory effects of kinins includingvasodilatation, increase of vascular permeability, ac-tivation and sensitization of primary afferent C- and

ŽAd-fibers are mediated by B receptors for review,2

see Bhoola et al., 1992; Dray and Bevan, 1993;.Walker et al., 1995 . This may explain the outstand-

ing efforts made to discover potent and selective B2Žreceptor antagonists. In this regard, Hoe 140 D-Arg-

w 3 5 7 8 x .Hyp , Thi , D-Tic , Oic -BK has long been con-sidered as the gold-standard B receptor antagonist.2

Recently, the pharmacological profile of novel potentnon-peptide B receptor antagonists, FR1736572Ž .Aramori et al., 1997; Asano et al., 1997 , FR167344Ž .Aramori et al., 1997; Inamura et al., 1997 and

Ž .FR165649 Asano et al., 1998 was disclosed.FR167344 was described as an orally active and

Žlong-acting B receptor antagonist Inamura et al.,2.1997 .

BK dilates the cerebral vasculature in an endothe-Ž .lium-dependent manner Sobey et al., 1997 and

Ž . Žopens the blood brain barrier BBB Wahl et al.,

Fig. 6. Inhibition by LF 16-0687 of Evans blue dye extravasation induced by BK in the pancreas, duodenum and stomach. 1.1 mmolrkg LF16-0687 was administered subcutaneously 40 min before a continuous 20-min infusion of BK. Values represent mean"S.E.M. from fiveexperiments.

( )D. Pruneau et al.r Immunopharmacology 43 1999 187–194 193

.1993 . In addition, all the components of thekallikrein–kinin system have been identified in the

Ž .brain Walker et al., 1995 and B receptors appear2

widely distributed in neurons from specific brainŽareas, as well as in astrocytes Hosli and Hosli,¨ ¨

.1993; Raidoo and Bhoola, 1997 . Targeting cerebralŽ .edema secondary to traumatic brain injury TBI as a

potential therapeutic application, we have beensearching for novel non-peptide BK B receptor2

antagonists. They need to display a high potency andselectivity for the receptor, exhibit a short onset ofaction, be sufficiently soluble for intravenous admin-istration, and potentially able to cross the BBB. Thefirst prototype compound was LF 16-0335 which hada K value of 0.84 nM for the cloned human Bi 2

receptor and a p A value of 8.3 in the HUV con-2

tracted by BK and therefore, partially fulfilled theŽ .above-criteria Pruneau et al., 1998a . In the present

study, we report the pharmacological profile of LF16-0687 which is approximately nine times morepotent than LF 16-0335 inhibiting functional re-sponses mediated by human and guinea-pig B re-2

Ž .ceptors Pruneau et al., 1998a, 1999 .LF 16-0687 was also independently evaluated for

its effects against brain edema in an animal model ofTBI. In a preliminary account of these resultsŽ .Pruneau et al., 1998b , we showed that a continuousinfusion of LF 16-0687, starting 1 h after closedhead trauma in rats, reduced by 64% the increase ofwater content in the injured hemisphere measured 24h later. Although it is not known yet if LF 16-0687crossed the BBB, these results, taken together withthose from the literature, favor an important detri-mental role of BK in the formation of brain edemafollowing head injury. Additional experimental stud-ies are underway to further evaluate the potentialvalue of LF 16-0687 as a novel therapeutic approachof TBI.

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