CHIRALITY 4351355 (1992)
Optical Isomers of a Leukotriene B4 Antagonist Have Differential Effects on Granulocyte Diapedesis in the Guinea Pig Dermis DONALD J. FRETLAND, DEBORAH L. WIDOMSKI, CHARLES P. ANGLIN, STELLA w, AND STEVAN W.D W c Departments of ImmunoInfimmatory Diseases Research and Chemktiy, Searle Research and Development, Skokie. Uknois
ABSTRACT Leukotriene B4 (LTB4) is a proinflammatory product of arachidonic acid metabolism that has been implicated in a number of inflammatory diseases. When injected intradermally into the guinea pig, LTB4 has been shown to elicit a dose-dependentinfiltration of granulocytes as assessed by the level of the neutrophil marker enzyme myeloperoxidase. SC-41930 [7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-~-l-benzopyran-2-carboxylicacid] is a potent LTB4 receptor antagonist. When compounds were coadministered along with LTB4 (35 ng) into the dermal site, racemic SC-41930,( + )-SC-41930, and ( - )-SC-41930each inhibited granulocyte accumulation with ED,, values of 340 f 30,98 f 5.7, and lo00 f 142 ng, respectively; when given intravenously inhibited with EDm values of 0.5 f 0.06,0.3 f 0.04, and 1.4 f 0.19 mg/kg, respectively; and when given intragastrically inhibited with EDM values of 1.7 f 0.20, 1.4 f 0.23, and 3.0 f 0.41 mg/kg, respectively. 0 1992 Wiley-Liss, Inc.
KEY WORDS: leukotriene B4, SC-41930, dermis, granulocyte, stereoselectivity,guinea pig INTRODUCTION [Crl:(HA)BR], 250-400 g body weight, were purchased from LTB4 is a product of arachidonic acid metabolism by the Charles River Laboratories, Wilmington, Massachusetts. SC5-lipoxygenasepathway. LTB4 has been shown to be a potent 41930 (lot CD-227-159A) and ( + )- and ( - )-isomers were synneutrophil activator and has been proposed as an important thesized at G.D. Searle & Co., Skokie, Illinois. The optical isomediator of inflammation. In vitro, LTB4 was found to be mers were found to be >98.5% pure as determined by equipotent to the bacterial peptide f-Met-Leu-Pheand the Ck analytical HPLC. Resolution of the optical isomers utilized the fragment of complement in inducing neutrophil chemokinesis, following conditions: column, Daicel Chiralcel OD (4.6 mm x chemotaxis, aggregation, and adherence. Injected LTB, in- 25 cm); mobile phase, hexane/2-propanol/formic acid duces granulocyte accumulation within the skin of the guinea (85:15:Ol); flow rate, 1.0 ml/min; temperature, 35°C; injection pig ear, in rabbit skin, and into aqueous humor.57 In addition, volume, 10 pl; detection at 254 nm. The isomers were found to intradermal injection of LTB4 in humans produced neutrophil have the following rotation: (+)-SC-41930[ct]g = + 17.0" f = -22.0' & 3.0 (c infiltration as determined by punch biopsy histological analy- 3.0" (c 1.056; CHCl3); (-)-SC-41930 1.068, CHC13). sis. Myeloperoxidase (MPO), a neutrophil marker enzyme, has LTB4-Induced PMN Chemotaxis in Guinea Pig Dermis previously been used as a quantitative index for tissue inMethods for intradermal LTB4 stimulation of neutrophil flammation in the gastrointestinal tract and in skin. lCl2 To assess the in vivo activity of LTB,, neutrophil infiltration into chemotaxis have been previously reported. l3 A solution of the guinea pig skin was quantitated by the presence and degree LTB, (350 ng/ml) was made up in 0.9% NaC1. LTB,, 35 ng, was injected intradermally with a 26-gauge allergist's needle of MPO activity. into the shaved backs of guinea pigs lightly anesthetized with SC-41930[7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-l-benzopyran-2-carboxylic acid] is pentobarbital. Appropriate intradermal vehicle controls were a potent antagonist of the LTB4 receptor on granulocytes, that used throughout. After 4 h, the animals were killed by carbon is effective orally 13J4 (Fig. 1). SC-41930 and its optical isomers dioxide anoxia, excorticated, and skin frozen at - 70°C. Skin were evaluated in vivo for inhibitory effects on LTB4-induced punches (12 mm diameter) were processed to determine MPO granulocyte chemotaxis in the guinea pig dermis by a number levels as previously reported. lo Punches were minced, homogenized with detergent-buffer,sonicated, and freeze-thawed. Exof routes. MPO was measured spectrophotometrically used tracted MATERIALS AND METHODS o-dianisidine hydrochloride as the substrate. Data were reMaterials and Animals corded as the absorbance (460 nm) at 15 min and expressed as LTB4 was purchased from BioMol, Plymouth Meeting, Pennsylvania, and was 97% pure by analytical HPLC. Received for publication January 13, 1992; accepted March 5, 1992. o -Dianisidine hydrochloride was purchased from Sigma, Address reprint requests to Donald J. Fretland, Immunolnflammatory Diseases St. Louis, Missouri. Cavines: male Hartley guinea pigs Research, Searle R&D, 4901 Searle Parkway, Skokie, IL 60077. sts
0
1992 Wiley-Liss, Inc.
354
FRETLAND ET AL. p2H
Fig. 1. Chemical structure of SC-41930. *The chiral center.
mean percent inhibition f SEM of myeloperoxidaseactivity or mean myeloperoxidase (unitslg) f SEM. h u g Treatment Several aspects of potential inhibitory activity of SC-41930 and its isomers were examined. When coinjected with LTB4, drugs were first dissolved in ethanol such that the final concentration of ethanol was never greater than 1%. When used intravenously, drugs were first dissolved in DMSO, again such that the final concentration of DMSO was never more than 1Yo and given 1 min before the intradermal injection of LTB,. When used intragastrically, drugs were given as a sonicated suspension in 1% aqueous methyl cellulose and administered 30 min before intradermal injection of LTB4.Appropriate vehicle controls were utilized throughout. Statistical Analysis Statistical evaluation of doseresponse studies was by linear regression analysis after analysis of variance. Analysis between groups was by Student’s t test with significance ascribed when P < 0.05. All statistical tests were conducted using the Minitab statistical program mounted on a Digital Equipment Corp. VAX/VMS/11/785 computer.22 RESULTS AND DISCUSSION
Intradermally injected LTB4 (35 ng) elicited its well-documented, time-dependent increase in neutrophil infiltration, as assessed by MFQ activity, that peaked at 4 h and slowly returned to background levels by 36 h. When various doses of LTB4 (0-100 ng/site) were injected intradermally and MPO assessed at 4 h (Table l), there was a dose-related neutrophil TABLE 1. Effect of intradermal leukotriene B4a concentration on dermal myeloperoxidase content Leukotriene B, (ng)
0 10 35 100
Myeloperoxidase* 0.021 0.047 0.376 0.597
f 0.007 f 0.024”s f 0.041* f 0.054**
“Various doses of LTB, were injected intradermally in the guinea pig. Four hours later animals were killed and dermal biopsies processed for myeloperoxidase content. *Valuesrepresent mean myeloperoxidase (unitslg) f SEM of N = 6. Statistical analysis by Student’s t test; values significantly different to no-dose (vehicle only) at *P
Optical Isomers of a Leukotriene B4 Antagonist Have Differential Effects on Granulocyte Diapedesis in the Guinea Pig Dermis DONALD J. FRETLAND, DEBORAH L. WIDOMSKI, CHARLES P. ANGLIN, STELLA w, AND STEVAN W.D W c Departments of ImmunoInfimmatory Diseases Research and Chemktiy, Searle Research and Development, Skokie. Uknois
ABSTRACT Leukotriene B4 (LTB4) is a proinflammatory product of arachidonic acid metabolism that has been implicated in a number of inflammatory diseases. When injected intradermally into the guinea pig, LTB4 has been shown to elicit a dose-dependentinfiltration of granulocytes as assessed by the level of the neutrophil marker enzyme myeloperoxidase. SC-41930 [7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-~-l-benzopyran-2-carboxylicacid] is a potent LTB4 receptor antagonist. When compounds were coadministered along with LTB4 (35 ng) into the dermal site, racemic SC-41930,( + )-SC-41930, and ( - )-SC-41930each inhibited granulocyte accumulation with ED,, values of 340 f 30,98 f 5.7, and lo00 f 142 ng, respectively; when given intravenously inhibited with EDm values of 0.5 f 0.06,0.3 f 0.04, and 1.4 f 0.19 mg/kg, respectively; and when given intragastrically inhibited with EDM values of 1.7 f 0.20, 1.4 f 0.23, and 3.0 f 0.41 mg/kg, respectively. 0 1992 Wiley-Liss, Inc.
KEY WORDS: leukotriene B4, SC-41930, dermis, granulocyte, stereoselectivity,guinea pig INTRODUCTION [Crl:(HA)BR], 250-400 g body weight, were purchased from LTB4 is a product of arachidonic acid metabolism by the Charles River Laboratories, Wilmington, Massachusetts. SC5-lipoxygenasepathway. LTB4 has been shown to be a potent 41930 (lot CD-227-159A) and ( + )- and ( - )-isomers were synneutrophil activator and has been proposed as an important thesized at G.D. Searle & Co., Skokie, Illinois. The optical isomediator of inflammation. In vitro, LTB4 was found to be mers were found to be >98.5% pure as determined by equipotent to the bacterial peptide f-Met-Leu-Pheand the Ck analytical HPLC. Resolution of the optical isomers utilized the fragment of complement in inducing neutrophil chemokinesis, following conditions: column, Daicel Chiralcel OD (4.6 mm x chemotaxis, aggregation, and adherence. Injected LTB, in- 25 cm); mobile phase, hexane/2-propanol/formic acid duces granulocyte accumulation within the skin of the guinea (85:15:Ol); flow rate, 1.0 ml/min; temperature, 35°C; injection pig ear, in rabbit skin, and into aqueous humor.57 In addition, volume, 10 pl; detection at 254 nm. The isomers were found to intradermal injection of LTB4 in humans produced neutrophil have the following rotation: (+)-SC-41930[ct]g = + 17.0" f = -22.0' & 3.0 (c infiltration as determined by punch biopsy histological analy- 3.0" (c 1.056; CHCl3); (-)-SC-41930 1.068, CHC13). sis. Myeloperoxidase (MPO), a neutrophil marker enzyme, has LTB4-Induced PMN Chemotaxis in Guinea Pig Dermis previously been used as a quantitative index for tissue inMethods for intradermal LTB4 stimulation of neutrophil flammation in the gastrointestinal tract and in skin. lCl2 To assess the in vivo activity of LTB,, neutrophil infiltration into chemotaxis have been previously reported. l3 A solution of the guinea pig skin was quantitated by the presence and degree LTB, (350 ng/ml) was made up in 0.9% NaC1. LTB,, 35 ng, was injected intradermally with a 26-gauge allergist's needle of MPO activity. into the shaved backs of guinea pigs lightly anesthetized with SC-41930[7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-l-benzopyran-2-carboxylic acid] is pentobarbital. Appropriate intradermal vehicle controls were a potent antagonist of the LTB4 receptor on granulocytes, that used throughout. After 4 h, the animals were killed by carbon is effective orally 13J4 (Fig. 1). SC-41930 and its optical isomers dioxide anoxia, excorticated, and skin frozen at - 70°C. Skin were evaluated in vivo for inhibitory effects on LTB4-induced punches (12 mm diameter) were processed to determine MPO granulocyte chemotaxis in the guinea pig dermis by a number levels as previously reported. lo Punches were minced, homogenized with detergent-buffer,sonicated, and freeze-thawed. Exof routes. MPO was measured spectrophotometrically used tracted MATERIALS AND METHODS o-dianisidine hydrochloride as the substrate. Data were reMaterials and Animals corded as the absorbance (460 nm) at 15 min and expressed as LTB4 was purchased from BioMol, Plymouth Meeting, Pennsylvania, and was 97% pure by analytical HPLC. Received for publication January 13, 1992; accepted March 5, 1992. o -Dianisidine hydrochloride was purchased from Sigma, Address reprint requests to Donald J. Fretland, Immunolnflammatory Diseases St. Louis, Missouri. Cavines: male Hartley guinea pigs Research, Searle R&D, 4901 Searle Parkway, Skokie, IL 60077. sts
0
1992 Wiley-Liss, Inc.
354
FRETLAND ET AL. p2H
Fig. 1. Chemical structure of SC-41930. *The chiral center.
mean percent inhibition f SEM of myeloperoxidaseactivity or mean myeloperoxidase (unitslg) f SEM. h u g Treatment Several aspects of potential inhibitory activity of SC-41930 and its isomers were examined. When coinjected with LTB4, drugs were first dissolved in ethanol such that the final concentration of ethanol was never greater than 1%. When used intravenously, drugs were first dissolved in DMSO, again such that the final concentration of DMSO was never more than 1Yo and given 1 min before the intradermal injection of LTB,. When used intragastrically, drugs were given as a sonicated suspension in 1% aqueous methyl cellulose and administered 30 min before intradermal injection of LTB4.Appropriate vehicle controls were utilized throughout. Statistical Analysis Statistical evaluation of doseresponse studies was by linear regression analysis after analysis of variance. Analysis between groups was by Student’s t test with significance ascribed when P < 0.05. All statistical tests were conducted using the Minitab statistical program mounted on a Digital Equipment Corp. VAX/VMS/11/785 computer.22 RESULTS AND DISCUSSION
Intradermally injected LTB4 (35 ng) elicited its well-documented, time-dependent increase in neutrophil infiltration, as assessed by MFQ activity, that peaked at 4 h and slowly returned to background levels by 36 h. When various doses of LTB4 (0-100 ng/site) were injected intradermally and MPO assessed at 4 h (Table l), there was a dose-related neutrophil TABLE 1. Effect of intradermal leukotriene B4a concentration on dermal myeloperoxidase content Leukotriene B, (ng)
0 10 35 100
Myeloperoxidase* 0.021 0.047 0.376 0.597
f 0.007 f 0.024”s f 0.041* f 0.054**
“Various doses of LTB, were injected intradermally in the guinea pig. Four hours later animals were killed and dermal biopsies processed for myeloperoxidase content. *Valuesrepresent mean myeloperoxidase (unitslg) f SEM of N = 6. Statistical analysis by Student’s t test; values significantly different to no-dose (vehicle only) at *P
