European Journal of Pharmacology, 216 (1992) 315-318 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
315
EJP 21072
Short communication
PF-5901 inhibits gastrointestinal platelet-activating factor synthesis in vivo C o r y M. H o g a b o a m , D o n n a D o n i g i - G a l e a, T. S c o t t S h o u p e a a n d J o h n L. W a l l a c e Gastrointestinal Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada and ~ International Molecular Discocery, Purdue Frederick Company, Norwalk, CT, USA Received 16 January 1992, accepted 14 April 1992
PF-5901, a novel anti-inflammatory compound, has previously been shown to inhibit the synthesis of platelet-activating factor (PAF) by peritoneal mast cells in vitro. The purpose of the present study was to assess the effects of this compound on PAF synthesis in vivo using two animal models which are characterized by elevated levels of gastrointestinal PAF synthesis. In the first study, rats were infected with Nippostrongylus brasiliensis and killed 17 days later. Groups of infected rats were given PF-5901 at doses ranging from 0.1 to 100 mg/kg, and 12 h later the effects on jejunal PAF synthesis were determined. PF-5901 reduced PAF synthesis in a concentration-dependent manner, with inhibition reaching 95% at the highest dose tested. In the second study, groups of rats were orally pretreated with PF-5901 (100 mg/kg) or vehicle 3 or 12 h prior to induction of endotoxic shock by i.v. administration of lipopolysaccharide from Salmonella typhosa. PAF synthesis in various regions of the gastrointestinal tract was assessed 15 min later. PF-5901 significantly reduced the hemoconcentration, but not the hypotension associated with endotoxic shock. Moreover, this compound significantly inhibited PAF synthesis in all tissues studied when a 12 h pretreatment time was used, and in all tissues except the duodenum when a 3 h pretreatment time was used. These studies demonstrate that PF-5901 inhibits gastrointestinal PAF synthesis in two in vivo models. This compound may therefore be a useful pharmacological tool for future studies on the role of PAF in inflammatory processes, and the effects of PF-5901 on PAF synthesis may contribute to its anti-inflammatory properties.
PAF (platelet-activating factor, PAF-acether); Gastrointestinal; Inflammation; Endotoxic shock; Parasitic infection
1. Introduction Platelet-activating factor (PAF) is considered a significant contributor to many pathophysiological conditions affecting the gastrointestinal tract. Many studies have demonstrated the ability of this inflammatory mediator to induce gastrointestinal ulceration and to mediate the gastrointestinal damage associated with endotoxic and hemorrhagic shock (reviewed by Wallace, 1990). While the exact mechanism by which P A F mediates gastrointestinal damage is unclear, it is known that P A F activates platelets and neutrophils, is a potent chemotactic agent for neutrophils and eosinophils and modifies intestinal smooth muscle contractility (Braquet et al., 1987).
Correspondence: J.L. Wallace, Department of Medical Physiology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada. Tel. 1.403.220 4539, fax 1.403.270 3353.
Evidence supporting a role for P A F in gastrointestinal ulceration and inflammation is derived from studies utilizing a variety of PAF receptor antagonists in experimental models. On the other hand, few studies have utilized inhibitors of PAF synthesis as tools to investigate the role of P A F in these disorders, mainly because such inhibitors have not been available. We have recently demonstrated that PF-5901, an inhibitor of leukotriene synthesis (Van Inwegen et al., 1987), inhibits P A F synthesis by activated peritoneal mast cells in vitro (Hogaboam et al., 1992). This action was shared by a structurally related compound (Wy-50,295), and was shown to occur independent of effects of these agents on leukotriene synthesis (Hogaboam et al., 1992). In the present study, the effects of PF-5901 on PAF synthesis have been assessed using two in vivo rat models which are characterized by elevated gastrointestinal P A F synthesis: endotoxic shock (Whittle et al., 1987) and intestinal infection with the parasite N. brasiliensis (Hogaboam et al., 1991).
316
2. Materials and methods
2.1. Animals Male, Wistar (200-250 g) and Sprague-Dawley rats (300-400 g) were obtained from Charles River Canada Inc. (St. Constant, QC, Canada). Male, New Zealand white rabbits (2-3 kg) were obtained from Riemans Fur Ranches Ltd. (St. Agatha, ON, Canada). The rats were housed at a temperature of 25°C on a 12 h light/dark cycle and had free access to food and water. Rabbits were individually housed in standard cages under similar conditions. The experimental procedures used in this study are in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the Animal Care Committee of the University of Calgary.
2.2. Nippostrongylus brasiliensis infection model S p r a g u e - D a w l e y rats were infected with N. brasiliensis as described in detail previously (Hogaboam et aI., 1991). Briefly, rats were lightly anesthetized with CO 2 and 3000 third stage larvae in 0.5 ml of phosphate-buffered saline (PBS; pH 7.4) were injected s.c. into the scruff of the neck. Sixteen days later the rats were divided into five groups of five and given PF-5901 orally at doses ranging from 0.1 to 100 mg/kg. The control group received the vehicle in the same manner. An additional group of five rats which had not been infected with N. brasiliensis was also studied. The rats were killed 12 h later and samples (approximately 150-200 rag) of the jejunum (7 cm from the ligament of Treitz) were excised for determination of PAF synthesis. We have previously shown that this time point in the N. brasiliensis infection corresponds with significantly elevated jejunal PAF synthesis (Hogaboam et al., 1991).
occurs in the gastrointestinal tract at this time point following LPS administration (Whittle et al, 1987). Groups of five rats each were orally treated with PF5901 (100 m g / k g ) or the vehicle at 3 or 12 h prior to LPS administration.
2.4. Measurement of PAF synthesis PAF synthesis was determined following incubation of the tissue samples in 1 ml of 0.25% bovine serum albumin (BSA)/0.9% saline (w/v), subsequent extraction, thin-layer chromatography and platelet aggregation bioassay. Each of these procedures have been described in detail previously (Hogaboam et al., 1991; Parente and Flower, 1985). Rabbit platelets were isolated from arterial blood, washed twice in the presence of prostacyclin (300 ng/ml), then treated with 10 /~g/ml of indomethacin. The concentrations of PAF in each sample was then determined by the ability of the sample to cause platelet aggregation. Each sample was retested in the bioassay with platelets pretreated with a specific PAF receptor antagonist, WEB 2086 (10 p.M; Casals-Stenzel et al., 1987), to confirm that the platelet aggregating activity of the sample was attributable to PAF. This antagonist did not affect the ability of platelets to respond to other agonists (A23187 or thrombin), but completely inhibited aggregation induced by the samples. It should be noted that the concentrations of PF-5901 used in this study did not directly interfere with platelet aggregation induced by authentic PAF.
2.5. Statistical analyses All data are expressed as the means +_ S.E.M. Comparisons between groups were made using an analysis of variance followed by the Newman-Keuls test. An associated probability (P value) of 5% or less was considered significant.
2.3. Endotoxic shock model 2. 6. Materials Wistar rats were anesthetized with sodium pentobarbital (60 m g / k g i.p.) and the right carotid artery was cannulated for continuous monitoring of systemic blood pressure (BP) and for sampling of blood for determination of initial and final hematocrit. Lipopolysaccharide (LPS) from Salmonella typhosa (50 m g / kg) or the vehicle (0.9% saline) was administered as a bolus via a tail vein. Fifteen minutes later the rats were exsanguinated and gastrointestinal tissue was removed from the stomach (corpus), duodenum, jejunum (15 cm distal to the pylorus) and ileum (proximal to the ileocecal valve) for determination of PAF synthesis. It has previously been shown that significant PAF synthesis
PF-5901 (a-pentyl-3-(2-quinolinylmethoxy)-benzene-methanol) was provided by The Purdue Frederick Company (Norwalk, CT, USA) and was prepared as a suspension in 1% carboxymethylcellulose. LPS (S. typhosa) was obtained from Sigma Chemical Company (St. Louis, MO, USA) and was dissolved in isotonic saline at a concentration of 25 m g / m l . Sodium prostacyclin was a gift from Burroughs-Welcome Canada (Montreal, Canada) and was stored ( - 20°C) in aliquots dissolved in 0.25 mM Tris buffer (pH 8.4). WEB 2086 was a gift from Boehringer Ingelheim (Germany) and was dissolved in methanol.
317 3. Results
1250 -
3.1. N. brasiliensis infection
=
P A F synthesis by samples of jejunum from rats killed 17 days post-infection was dose dependently inhibited by PF-5901 (fig. 1). At the highest dose tested, PF-5901 reduced P A F synthesis by approximately 95% compared to the vehicle-treated control group. Indeed, the levels of P A F synthesis by samples taken from rats which had received the 50 and 100 m g / k g doses of PF-5901 were significantly lower than those of jejunal samples from normal (uninfected) rats (292 _+ 33 p g / m g ) . 3.2. Endotoxic shock In vehicle-treated control rats, LPS administration resulted in a profound decrease in systemic arterial BP which recovered to resting levels within 5 rain. This recovery was transient, however, as BP subsequently decreased by 53 +_ 24 mm Hg below resting levels at 15 min post-LPS. LPS administration also resulted in a marked elevation in hematocrit (45.6 + 5.6%) above basal. Figure 2 illustrates the levels of PAF synthesis by samples of various gastrointestinal tissues excised 15 min after administration of LPS. In rats treated with PF-5901 (100 m g / k g p.o.) at 3 or 12 h prior to LPS, the changes in hematocrit were significantly (P < 0.05) reduced. The 3 h pretreatment group had a change in hematocrit of 18 _+ 3% while that in the 12 h group was 24 _+ 4%. PF-5901 pretreatment failed to significantly affect the changes in BP induced by LPS, but did significantly inhibit gastrointestinal P A F synthesis (fig. 2). The only tissue in which
1000 •
~- 75o
= a "-_ '500250 ~ 0 '~ ~' ~=*~
C o n tr ol
0.1
* , * 10
50
Dose of P F - 5 9 0 1
100 (mg/kg)
Fig. 1. D o s e - d e p e n d e n t effects of PF-5901 on jejunal P A F synthesis
in N. brasiliensis-infected rats. Groups of five rats each were infected with N. brasiliensis. On the 16th day after infection, they received PF-5901 orally at doses ranging from 0.1 to 100 mg/kg. Values are expressed as means_+S.E.M. Asterisks denote a significant difference from the vehicle-treated control group (P < 0.05). The mean PAF synthesis by samples of jejunum from non-infected rats was 292 ± 33 pg/g.
n¢~
1000-
• Control []3 h post PF-5901 [ ] 12 h post PF-5901
750 -
o~ r-
500
u_ < 13_
250 0
t
Stomach
Duodenum
Jejunum
Gastrointestinal
Ileum
site
Fig. 2. Effects of PF-5901 on gastrointestinal PAF synthesis in endotoxin-treated rats. Groups of five rats each were treated with PF-5901 (100 mg/kg p.o.) at 3 or 12 h prior to endotoxin administration, while the control group received the vehicle. Values are expressed as means_+S.E.M. Asterisks denote significant differences from the control group (P < 0.05).
P A F synthesis was not significantly inhibited by PF5901 was the duodenum, and this only in the case of the 3 h pretreatment group.
4. Discussion This study demonstrates that PAF synthesis by gastrointestinal tissues can be significantly inhibited by oral administration of the quinoline-based compound PF-5901. This effect was evident in both the endotoxic shock and Nippostrongylus brasiliensis infection models. In the latter, the effects of PF-5901 were shown to be dose-dependent. These results therefore confirm in in vivo systems what we have previously demonstrated in vitro using isolated mast cells (Hogaboam et al., 1992). Confirmation of in vitro activity of PF-5901 is important because numerous compounds have previously been shown to inhibit P A F synthesis in vitro, but are subsequently found to be inactive in in vivo models of inflammation. For example, corticosteroids can inhibit the synthesis of P A F in vitro (Parente and Flower, 1985), but we were unable to demonstrate any effects of dexamethasone on gastrointestinal PAF synthesis in an endotoxic shock model similar to that used in the present study (Ibbotson and Wallace, 1989). Furthermore, ketotifen has been reported to inhibit PAF synthesis in vitro (Joly et al., 1987), but we have been unable to demonstrate an effect of this compound on P A F synthesis in various in vivo models (unpublished). The mechanism through which PF-5901 inhibits P A F synthesis is not known. In our earlier in vitro studies (Hogaboam et al., 1992), effects of this compound on PAF synthesis could be clearly distinguished from el-
318 f e c t s o n l e u k o t r i e n e synthesis. I n a d d i t i o n , a n u m b e r o f o t h e r s t r u c t u r a l l y u n r e l a t e d l e u k o t r i e n e s y n t h e s i s inh i b i t o r s f a i l e d to a f f e c t P A F synthesis. It is u n l i k e l y that PF-5901 inhibits PAF synthesis through effects on p h o s p h o l i p a s e A 2 , s i n c e this c o m p o u n d did n o t a f f e c t P G D 2 s y n t h e s i s by i s o l a t e d m a s t cells ( H o g a b o a m et al., 1992). P F - 5 9 0 1 has b e e n s h o w n to h a v e a n t i - i n f l a m m a t o r y p r o p e r t i e s in e x p e r i m e n t a l colitis ( W a l l a c e et al., 1992). T h e r e s u l t s o f this s t u d y s u g g e s t t h a t e f f e c t s o n P A F s y n t h e s i s c o u l d c o n t r i b u t e to t h e s e a n t i - i n f l a m m a t o r y effects. M o r e o v e r , P F - 5 9 0 1 m a y b e a u s e f u l t o o l for f u t u r e s t u d i e s in w h i c h t h e r o l e o f P A F in g a s t r o i n t e s t i nal i n f l a m m a t i o n is e x a m i n e d .
Acknowledgements This work was supported by grants from the Medical Research Council of Canada (MRC), Purdue Frederick Inc. and the Alberta Heritage Foundation for Medical Research (AHFMR). C.M.H. is supported by an AHFMR Studentship. J.L.W. is an AHFMR Medical Scholar and MRC Scientist.
References Braquet, P., L. Touqui, T.Y. Shen and B.B. Vargaftig, 1987, Perspectives in platelet-activating factor research, Pharmacol. Rev. 39, 97.
Casals-Stenzel, J., G. Muacevic and K.H. Weber, 1987, Pharmacological actions of WEB 2086, a specific antagonist of platelet-activating factor, J. Pharmacol. Exp. Ther. 241, 974. Hogaboam, C.M., A.D. Befus and J.L. Wallace, 1991, Intestinal platelet-activating factor synthesis during Nippostrongylus brasiliensis infection in the rat, J. Lip. Med. 4, 211. Hogaboam, C.M., D. Donigi-Gale, T.S. Shoupe, E.Y. Bissonnene, A.D. Befus and J.L. Wallace, 1992, Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds, Br. J. Pharmacol. (in press). Ibbotson, G.C. and J.L. Wallace, 1989, Inhibitory effects of dexamethasone in endotoxic shock and its relation to PAF-acether synthesis in the gastrointestinal tract and lung, J. Lip. Med. 1, 273. Joly, F., G. Bessou, J. Benveniste and E. Ninio, 1987, Ketotifen inhibits PAF-acether biosynthesis and /3-hexosaminodase release in mouse mast cells stimulated with antigen, Eur. J. Pharmacol. 144, 133. Parente, L. and R.J. Flower, 1985, Hydrocortisone and macrocortin inhibit the zymosan-induced release of lyso-PAF from rat peritoneal leucocytes, Life Sci. 36, 1225. Van Inwegan, R.G., A. Kwandwala, R. Gordon, P. Sonnino, S. Courts and S. Jolly, 1987, REV-5901: an orally effective peptidoleukotriene antagonist, detailed biochemical/pharmacological profile, J. Pharmacol. Exp. Ther. 241, 118. Wallace, J.L., 1990, Lipid mediators of inflammation in gastric ulcer, Am. J. Physiol. 258, G1. Wallace, J.L., C.M. Keenan, D. Donigi-Gale and T.S. Shoupe, 1992, Exacerbation of experimental colitis by nonsteroidal anti-inflammatory drugs is not related to elevated leukotrienes B 4 synthesis, Gastroenterology 102, 18. Whittle, B.J.R., N.K. Boughton-Smith, I.R. Hutcheson, J.V. Esplugues and J.L. Wallace, 1987, Increased intestinal formation of PAF in endotoxin-induced damage in the rat, Br. J. Pharmacol. 92, 3.
315
EJP 21072
Short communication
PF-5901 inhibits gastrointestinal platelet-activating factor synthesis in vivo C o r y M. H o g a b o a m , D o n n a D o n i g i - G a l e a, T. S c o t t S h o u p e a a n d J o h n L. W a l l a c e Gastrointestinal Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada and ~ International Molecular Discocery, Purdue Frederick Company, Norwalk, CT, USA Received 16 January 1992, accepted 14 April 1992
PF-5901, a novel anti-inflammatory compound, has previously been shown to inhibit the synthesis of platelet-activating factor (PAF) by peritoneal mast cells in vitro. The purpose of the present study was to assess the effects of this compound on PAF synthesis in vivo using two animal models which are characterized by elevated levels of gastrointestinal PAF synthesis. In the first study, rats were infected with Nippostrongylus brasiliensis and killed 17 days later. Groups of infected rats were given PF-5901 at doses ranging from 0.1 to 100 mg/kg, and 12 h later the effects on jejunal PAF synthesis were determined. PF-5901 reduced PAF synthesis in a concentration-dependent manner, with inhibition reaching 95% at the highest dose tested. In the second study, groups of rats were orally pretreated with PF-5901 (100 mg/kg) or vehicle 3 or 12 h prior to induction of endotoxic shock by i.v. administration of lipopolysaccharide from Salmonella typhosa. PAF synthesis in various regions of the gastrointestinal tract was assessed 15 min later. PF-5901 significantly reduced the hemoconcentration, but not the hypotension associated with endotoxic shock. Moreover, this compound significantly inhibited PAF synthesis in all tissues studied when a 12 h pretreatment time was used, and in all tissues except the duodenum when a 3 h pretreatment time was used. These studies demonstrate that PF-5901 inhibits gastrointestinal PAF synthesis in two in vivo models. This compound may therefore be a useful pharmacological tool for future studies on the role of PAF in inflammatory processes, and the effects of PF-5901 on PAF synthesis may contribute to its anti-inflammatory properties.
PAF (platelet-activating factor, PAF-acether); Gastrointestinal; Inflammation; Endotoxic shock; Parasitic infection
1. Introduction Platelet-activating factor (PAF) is considered a significant contributor to many pathophysiological conditions affecting the gastrointestinal tract. Many studies have demonstrated the ability of this inflammatory mediator to induce gastrointestinal ulceration and to mediate the gastrointestinal damage associated with endotoxic and hemorrhagic shock (reviewed by Wallace, 1990). While the exact mechanism by which P A F mediates gastrointestinal damage is unclear, it is known that P A F activates platelets and neutrophils, is a potent chemotactic agent for neutrophils and eosinophils and modifies intestinal smooth muscle contractility (Braquet et al., 1987).
Correspondence: J.L. Wallace, Department of Medical Physiology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada. Tel. 1.403.220 4539, fax 1.403.270 3353.
Evidence supporting a role for P A F in gastrointestinal ulceration and inflammation is derived from studies utilizing a variety of PAF receptor antagonists in experimental models. On the other hand, few studies have utilized inhibitors of PAF synthesis as tools to investigate the role of P A F in these disorders, mainly because such inhibitors have not been available. We have recently demonstrated that PF-5901, an inhibitor of leukotriene synthesis (Van Inwegen et al., 1987), inhibits P A F synthesis by activated peritoneal mast cells in vitro (Hogaboam et al., 1992). This action was shared by a structurally related compound (Wy-50,295), and was shown to occur independent of effects of these agents on leukotriene synthesis (Hogaboam et al., 1992). In the present study, the effects of PF-5901 on PAF synthesis have been assessed using two in vivo rat models which are characterized by elevated gastrointestinal P A F synthesis: endotoxic shock (Whittle et al., 1987) and intestinal infection with the parasite N. brasiliensis (Hogaboam et al., 1991).
316
2. Materials and methods
2.1. Animals Male, Wistar (200-250 g) and Sprague-Dawley rats (300-400 g) were obtained from Charles River Canada Inc. (St. Constant, QC, Canada). Male, New Zealand white rabbits (2-3 kg) were obtained from Riemans Fur Ranches Ltd. (St. Agatha, ON, Canada). The rats were housed at a temperature of 25°C on a 12 h light/dark cycle and had free access to food and water. Rabbits were individually housed in standard cages under similar conditions. The experimental procedures used in this study are in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the Animal Care Committee of the University of Calgary.
2.2. Nippostrongylus brasiliensis infection model S p r a g u e - D a w l e y rats were infected with N. brasiliensis as described in detail previously (Hogaboam et aI., 1991). Briefly, rats were lightly anesthetized with CO 2 and 3000 third stage larvae in 0.5 ml of phosphate-buffered saline (PBS; pH 7.4) were injected s.c. into the scruff of the neck. Sixteen days later the rats were divided into five groups of five and given PF-5901 orally at doses ranging from 0.1 to 100 mg/kg. The control group received the vehicle in the same manner. An additional group of five rats which had not been infected with N. brasiliensis was also studied. The rats were killed 12 h later and samples (approximately 150-200 rag) of the jejunum (7 cm from the ligament of Treitz) were excised for determination of PAF synthesis. We have previously shown that this time point in the N. brasiliensis infection corresponds with significantly elevated jejunal PAF synthesis (Hogaboam et al., 1991).
occurs in the gastrointestinal tract at this time point following LPS administration (Whittle et al, 1987). Groups of five rats each were orally treated with PF5901 (100 m g / k g ) or the vehicle at 3 or 12 h prior to LPS administration.
2.4. Measurement of PAF synthesis PAF synthesis was determined following incubation of the tissue samples in 1 ml of 0.25% bovine serum albumin (BSA)/0.9% saline (w/v), subsequent extraction, thin-layer chromatography and platelet aggregation bioassay. Each of these procedures have been described in detail previously (Hogaboam et al., 1991; Parente and Flower, 1985). Rabbit platelets were isolated from arterial blood, washed twice in the presence of prostacyclin (300 ng/ml), then treated with 10 /~g/ml of indomethacin. The concentrations of PAF in each sample was then determined by the ability of the sample to cause platelet aggregation. Each sample was retested in the bioassay with platelets pretreated with a specific PAF receptor antagonist, WEB 2086 (10 p.M; Casals-Stenzel et al., 1987), to confirm that the platelet aggregating activity of the sample was attributable to PAF. This antagonist did not affect the ability of platelets to respond to other agonists (A23187 or thrombin), but completely inhibited aggregation induced by the samples. It should be noted that the concentrations of PF-5901 used in this study did not directly interfere with platelet aggregation induced by authentic PAF.
2.5. Statistical analyses All data are expressed as the means +_ S.E.M. Comparisons between groups were made using an analysis of variance followed by the Newman-Keuls test. An associated probability (P value) of 5% or less was considered significant.
2.3. Endotoxic shock model 2. 6. Materials Wistar rats were anesthetized with sodium pentobarbital (60 m g / k g i.p.) and the right carotid artery was cannulated for continuous monitoring of systemic blood pressure (BP) and for sampling of blood for determination of initial and final hematocrit. Lipopolysaccharide (LPS) from Salmonella typhosa (50 m g / kg) or the vehicle (0.9% saline) was administered as a bolus via a tail vein. Fifteen minutes later the rats were exsanguinated and gastrointestinal tissue was removed from the stomach (corpus), duodenum, jejunum (15 cm distal to the pylorus) and ileum (proximal to the ileocecal valve) for determination of PAF synthesis. It has previously been shown that significant PAF synthesis
PF-5901 (a-pentyl-3-(2-quinolinylmethoxy)-benzene-methanol) was provided by The Purdue Frederick Company (Norwalk, CT, USA) and was prepared as a suspension in 1% carboxymethylcellulose. LPS (S. typhosa) was obtained from Sigma Chemical Company (St. Louis, MO, USA) and was dissolved in isotonic saline at a concentration of 25 m g / m l . Sodium prostacyclin was a gift from Burroughs-Welcome Canada (Montreal, Canada) and was stored ( - 20°C) in aliquots dissolved in 0.25 mM Tris buffer (pH 8.4). WEB 2086 was a gift from Boehringer Ingelheim (Germany) and was dissolved in methanol.
317 3. Results
1250 -
3.1. N. brasiliensis infection
=
P A F synthesis by samples of jejunum from rats killed 17 days post-infection was dose dependently inhibited by PF-5901 (fig. 1). At the highest dose tested, PF-5901 reduced P A F synthesis by approximately 95% compared to the vehicle-treated control group. Indeed, the levels of P A F synthesis by samples taken from rats which had received the 50 and 100 m g / k g doses of PF-5901 were significantly lower than those of jejunal samples from normal (uninfected) rats (292 _+ 33 p g / m g ) . 3.2. Endotoxic shock In vehicle-treated control rats, LPS administration resulted in a profound decrease in systemic arterial BP which recovered to resting levels within 5 rain. This recovery was transient, however, as BP subsequently decreased by 53 +_ 24 mm Hg below resting levels at 15 min post-LPS. LPS administration also resulted in a marked elevation in hematocrit (45.6 + 5.6%) above basal. Figure 2 illustrates the levels of PAF synthesis by samples of various gastrointestinal tissues excised 15 min after administration of LPS. In rats treated with PF-5901 (100 m g / k g p.o.) at 3 or 12 h prior to LPS, the changes in hematocrit were significantly (P < 0.05) reduced. The 3 h pretreatment group had a change in hematocrit of 18 _+ 3% while that in the 12 h group was 24 _+ 4%. PF-5901 pretreatment failed to significantly affect the changes in BP induced by LPS, but did significantly inhibit gastrointestinal P A F synthesis (fig. 2). The only tissue in which
1000 •
~- 75o
= a "-_ '500250 ~ 0 '~ ~' ~=*~
C o n tr ol
0.1
* , * 10
50
Dose of P F - 5 9 0 1
100 (mg/kg)
Fig. 1. D o s e - d e p e n d e n t effects of PF-5901 on jejunal P A F synthesis
in N. brasiliensis-infected rats. Groups of five rats each were infected with N. brasiliensis. On the 16th day after infection, they received PF-5901 orally at doses ranging from 0.1 to 100 mg/kg. Values are expressed as means_+S.E.M. Asterisks denote a significant difference from the vehicle-treated control group (P < 0.05). The mean PAF synthesis by samples of jejunum from non-infected rats was 292 ± 33 pg/g.
n¢~
1000-
• Control []3 h post PF-5901 [ ] 12 h post PF-5901
750 -
o~ r-
500
u_ < 13_
250 0
t
Stomach
Duodenum
Jejunum
Gastrointestinal
Ileum
site
Fig. 2. Effects of PF-5901 on gastrointestinal PAF synthesis in endotoxin-treated rats. Groups of five rats each were treated with PF-5901 (100 mg/kg p.o.) at 3 or 12 h prior to endotoxin administration, while the control group received the vehicle. Values are expressed as means_+S.E.M. Asterisks denote significant differences from the control group (P < 0.05).
P A F synthesis was not significantly inhibited by PF5901 was the duodenum, and this only in the case of the 3 h pretreatment group.
4. Discussion This study demonstrates that PAF synthesis by gastrointestinal tissues can be significantly inhibited by oral administration of the quinoline-based compound PF-5901. This effect was evident in both the endotoxic shock and Nippostrongylus brasiliensis infection models. In the latter, the effects of PF-5901 were shown to be dose-dependent. These results therefore confirm in in vivo systems what we have previously demonstrated in vitro using isolated mast cells (Hogaboam et al., 1992). Confirmation of in vitro activity of PF-5901 is important because numerous compounds have previously been shown to inhibit P A F synthesis in vitro, but are subsequently found to be inactive in in vivo models of inflammation. For example, corticosteroids can inhibit the synthesis of P A F in vitro (Parente and Flower, 1985), but we were unable to demonstrate any effects of dexamethasone on gastrointestinal PAF synthesis in an endotoxic shock model similar to that used in the present study (Ibbotson and Wallace, 1989). Furthermore, ketotifen has been reported to inhibit PAF synthesis in vitro (Joly et al., 1987), but we have been unable to demonstrate an effect of this compound on P A F synthesis in various in vivo models (unpublished). The mechanism through which PF-5901 inhibits P A F synthesis is not known. In our earlier in vitro studies (Hogaboam et al., 1992), effects of this compound on PAF synthesis could be clearly distinguished from el-
318 f e c t s o n l e u k o t r i e n e synthesis. I n a d d i t i o n , a n u m b e r o f o t h e r s t r u c t u r a l l y u n r e l a t e d l e u k o t r i e n e s y n t h e s i s inh i b i t o r s f a i l e d to a f f e c t P A F synthesis. It is u n l i k e l y that PF-5901 inhibits PAF synthesis through effects on p h o s p h o l i p a s e A 2 , s i n c e this c o m p o u n d did n o t a f f e c t P G D 2 s y n t h e s i s by i s o l a t e d m a s t cells ( H o g a b o a m et al., 1992). P F - 5 9 0 1 has b e e n s h o w n to h a v e a n t i - i n f l a m m a t o r y p r o p e r t i e s in e x p e r i m e n t a l colitis ( W a l l a c e et al., 1992). T h e r e s u l t s o f this s t u d y s u g g e s t t h a t e f f e c t s o n P A F s y n t h e s i s c o u l d c o n t r i b u t e to t h e s e a n t i - i n f l a m m a t o r y effects. M o r e o v e r , P F - 5 9 0 1 m a y b e a u s e f u l t o o l for f u t u r e s t u d i e s in w h i c h t h e r o l e o f P A F in g a s t r o i n t e s t i nal i n f l a m m a t i o n is e x a m i n e d .
Acknowledgements This work was supported by grants from the Medical Research Council of Canada (MRC), Purdue Frederick Inc. and the Alberta Heritage Foundation for Medical Research (AHFMR). C.M.H. is supported by an AHFMR Studentship. J.L.W. is an AHFMR Medical Scholar and MRC Scientist.
References Braquet, P., L. Touqui, T.Y. Shen and B.B. Vargaftig, 1987, Perspectives in platelet-activating factor research, Pharmacol. Rev. 39, 97.
Casals-Stenzel, J., G. Muacevic and K.H. Weber, 1987, Pharmacological actions of WEB 2086, a specific antagonist of platelet-activating factor, J. Pharmacol. Exp. Ther. 241, 974. Hogaboam, C.M., A.D. Befus and J.L. Wallace, 1991, Intestinal platelet-activating factor synthesis during Nippostrongylus brasiliensis infection in the rat, J. Lip. Med. 4, 211. Hogaboam, C.M., D. Donigi-Gale, T.S. Shoupe, E.Y. Bissonnene, A.D. Befus and J.L. Wallace, 1992, Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds, Br. J. Pharmacol. (in press). Ibbotson, G.C. and J.L. Wallace, 1989, Inhibitory effects of dexamethasone in endotoxic shock and its relation to PAF-acether synthesis in the gastrointestinal tract and lung, J. Lip. Med. 1, 273. Joly, F., G. Bessou, J. Benveniste and E. Ninio, 1987, Ketotifen inhibits PAF-acether biosynthesis and /3-hexosaminodase release in mouse mast cells stimulated with antigen, Eur. J. Pharmacol. 144, 133. Parente, L. and R.J. Flower, 1985, Hydrocortisone and macrocortin inhibit the zymosan-induced release of lyso-PAF from rat peritoneal leucocytes, Life Sci. 36, 1225. Van Inwegan, R.G., A. Kwandwala, R. Gordon, P. Sonnino, S. Courts and S. Jolly, 1987, REV-5901: an orally effective peptidoleukotriene antagonist, detailed biochemical/pharmacological profile, J. Pharmacol. Exp. Ther. 241, 118. Wallace, J.L., 1990, Lipid mediators of inflammation in gastric ulcer, Am. J. Physiol. 258, G1. Wallace, J.L., C.M. Keenan, D. Donigi-Gale and T.S. Shoupe, 1992, Exacerbation of experimental colitis by nonsteroidal anti-inflammatory drugs is not related to elevated leukotrienes B 4 synthesis, Gastroenterology 102, 18. Whittle, B.J.R., N.K. Boughton-Smith, I.R. Hutcheson, J.V. Esplugues and J.L. Wallace, 1987, Increased intestinal formation of PAF in endotoxin-induced damage in the rat, Br. J. Pharmacol. 92, 3.
