- Email: [email protected]
Effects of some isoxazolpyrimidine derivatives on nitric oxide and eicosanoid biosynthesis
Life Sciences,
Vol.
66, No. 9, pp. PL 125131,200O
Copyright Prmted PII
ELSEVIER
0 2000 in the USA.
Elsevier
0024-3205100&see
SOO24-3205(99)00658-x
Science
Inc.
All rights reserved front matter
4 cc
EFFECTS OF SOME ISOXAZOLPYRIMIDINE DERIVATIVES EICOSANOID BIOSYNTHESIS
ON NITRIC OXIDE AND
J.M. Quintela3, C. Peinador3,
‘Department of Pharmacology, University of Valencia, Spain. 2Dept. of Organic Chemistry. University of Santiago, Spain. Dept.of Fundamental Chemistry. University of La Coruha, Spain. (Submitted May 2 I, 1999; accepted June 17, 1999; received in final form September 28, 1999)
Abstract.
The inhibitory effect of some isoxazolpyrimidine derivatives on iNOS and COX-2 endotoxin induction in mouse peritoneal macrophages has been studied. Three of these compounds inhibited nitrite and PGE2 accumulation in a concentration dependent-manner at pM range. None of these active compounds affected iNOS, COX-2, COX-1 or PLA2 activities, although some reduced iNOS or COX-2 expression. Besides, no effect was observed on human neutrophil inflammatory responses (LTB4 biosynthesis and superoxide or elastase release). Active compounds were assayed by oral administration in the mouse air pouch model, where they inhibited nitrite accumulation without affecting PGE2 levels or leukocyte migration. 0 2000 Elsevier Science Inc. Key Words:
isoxazolpyrimidine,
nitric oxide, PGE,, macrophages,
!z
-I F g d
PHARMACOLOGYLET-TERS Accelerated Communicalion
A. Vidal’, M.L. Ferrartdiz’, A. Ubeda’, I. Guillen’, R. Riguer$, M.J. Moreira3, M.J. Alcaraz’
P E
mouse air pouch
Macrophages play a crucial role in modulating the initiation and perpetuation of the inflammatory response. One means by which macrophages modulate inflammation is via their capacity to elaborate biological mediators, like nitric oxide (NO) and prostaglandins (PGs), which have numerous cardiovascular and inflammatory effecs (1). NO is formed from L-arginine by NO synthase (NOS) (2). Cyclooxygenase (COX) is the first enzyme in the pathway in which arachidonic acid is converted to PGs, prostacyclin and thromboxane A2 (TXA2) (3). NOS and COX exist in at least two isofotms. The constitutive isoforms, COX-1 and cNOS, are present in many types of cells, while the inducible isoforms COX-2 and iNOS are expressed after stimulation of cells with a variety of agents including endotoxin (bacterial lipopolysaccharide, LPS) and cytokines (2;4;5). Since the induction of COX-2 and iNOS results in the increased synthesis of PGs and NO, selective modulation of these mediators overproduction might represent a therapeutic goal in different inflammatory pathologies. Many compounds described as inhibitors of iNOS are nitrogen-containing heterocycles (6); this prompted us to assay some 3-methyl-4-R-6-dimethylamineisoxazolo[5,4-d]pyrimidine derivatives (IOPs)(Fig. 1) on NO production and iNOS activity. Corresponding Author: Dr. A. Ubeda. Department of Pharmacology, Faculty of Pharmacy. Avda. Vicent Andre% Estelles s/n, 46100 Burjassot, Valencia, Spain.
P
PL-126
IOPs on NO and Eicosanoid Biosynthesis
Vol. 66, No. 9,200O
IOPs were evaluated on nitrite and PGE2 production in LPS-stimulated mouse peritoneal macrophages, as an index of iNOS and COX-2 induction. We also studied their effects on the enzymatic activity of iNOS, COX-2, COX-1, cytosolic phospholipase A2 (cPLA2) and secretory phospholipase A2 (sPLA& as well as on the release of LTB4, elastase or superoxide by human neutrophils. Active compounds were assayed by oral administration in the mouse air pouch model of inflammation. R:
-OCH3
IOP-1
--N-l --cy -@J
IGP_2
-N
IOP-3
-r+--_
3
Fig. 1 Structural formula of isoxazolpyrimidine
IOP-4
derivatives (IOPs).
Materials and methods Drugs. IOPs have been prepared starting from the appropiate b-enaminonitrile,5-amino-4-cyano3-methylisoxazole and dichloromethyleneammonium chloride (7;8). Isolation and culture of mouse peritoneal macrophages. Peritoneal macrophages were obtained from female mice weighing 25-308. Cells were haversted by peritoneal lavage 4 days after i.p. inyection of Iml of 10% thioglicolate broth. Cells were resuspended in RPMI-1640 medium supplemented with 10% foetal bovine serum and incubated at 37°C for 2 h. The adherent cells were stimulated with E.coZi LPS (lOug/ml) for 18h The mitochondrial dependent reduction of 3(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) to formazan was used to asses the possible cytotoxic effects of test compounds (9). Quantification of nitrite and PGE2 levels. Nitrite as index of NO production, was assayed fluorimetrically in microtiter plates (10). The amount of nitrite was obtained by extrapolation from a standard curve with sodium nitrite as a standard. PGE2 levels were measured by radioimmunoassay (RIA) (11). CO&I. Human platelets were sonicated and microsomes were prepared by centrifugation at 2,000 x g for 5 min at 4°C followed by centrifugation of the supernatant at 100,000 x g for 100 min at 4°C. Microsomes (20 pg of protein/tube) were incubated with arachidonic acid and test compound or vehicle and TXB2 levels were determined by RIA (12). COX-2. Mouse peritoneal macrophages were incubated with E. coli LPS (10 us/ml) at 37°C for 24 h and were scraped. After centrifugation the cells were sonicated and microsomes were prepared as above. Microsomes (40 ug of protein/tube) were used as a source of COX-2 and PGE2 synthesis was determined by RIA (12).
Vol. 66, No. 9,200O
IOPs on NO and Eicosanoid
Biosynthesis
PL-127
iNOX High speed (100,000 x g) supernatants from sonicated mouse peritoneal macrophages were obtained as described above. NOS activity was determined in supernatants by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline (13). Western blot analysis. Mouse peritoneal macrophages obtained as described above were collected after 18h with lysis buffer (1% Triton X-l 00, 1% deoxicolic acid, 20 mM NaCl and 25 mM Tris; pH 7.5) and were used to assay COX-2 and iNOS expression using specific antibodies (14). sPLAz assay. sPLA1 was assayed by using [3H]oleate labeled membranes of E. coli (15). Bee venom and human recombinant synovial enzymes were used as sources of sPLA2. cPLA,J assay. cPLA2 was prepared from human monocytic U937 cells (Cell Collection, Department of Animal Cell Culture, C.S.I.C., Madrid, Spain) (12). cPLA2 activity was measured as the release of radiolabeled arachidonic acid according to the method of Clark et al (16). Synthesis and release of LTBd by human neutrophils. Leukocytes were obtained as previously described (12). Viability was greater than 95% by the trypan blue exclusion test. A suspension of human neutrophils (5x106/ml) was preincubated with test compounds or vehicle and then stimulated with 1 uM A23 187. The LTB4 levels in supernatants were measured by RIA (12). Chemiluminescence. Neutrophils (2.5x106/ml) were mixed with luminol (40 uM) and stimulated with 12-0-tetradecanoyl phorbol 13-acetate (TPA, 1 uM). The chemiluminescence was recorded in a Microbeta Trilux counter (Wallac, Turku, Finland) after 7 min. (12). Elastase release by human neutrophils. Neutrophils (2.5x106/ml) were preincubated with test compound or vehicle for 5 min and then stimulated with cytochalasin B (10 uM) and N-formylL-methionyl-L-leucyl-L-phenylalanine (FMLP, 10 nM). Elastase activity was estimated in supernatants as p-nitrophenol release (12). Mouse air pouch model. Air pouch was performed in female Swiss mice (25-308) as previously described (12). Tested compounds were administered orally, lh before and 8h after zymosan inyection at doses of 50, 35 and 25 mg/kg. 24h after zymosan administration, the animals were killed by cervical dislocation, and the exudate in the pouch was collected with lml of saline. Leukocytes present in exudates were measured using a Coulter Counter. After centrifugation of exudates (1,200g at 4°C for lOmin), the supernatants were used to measure nitrite and PGE2 levels as described above. Statistical analysis. The results are presented as mean f S.E.M.; n represents the number of experiments. Inhibitory concentration 50% (I&O) values were calculated from at least 4 significant concentrations (n=6) by linear regression analysis using Graph Pad Prism II. The level of statistical significance was determined by analysis of variance (ANOVA) followed by Dum-rett’s t-test for multiple comparisons. Results Inhibition of nitrite and PGE2 accumulation. Compounds IOP- 1, IOP-4 and IOP-5 inhibited the accumulation of both metabolites in LPS-stimulated peritoneal macrophages in a concentrationdependent manner, as shown in Table I. These compounds were more potent than the reference NOS inhibitor aminoguanidine (AG) on nitrite accumulation but showed less potency on PGE2
IOPs on NO and Eicosanoid
PL-128
Vol. 66, No. 9, 2000
Biosynthesis
levels than the reference COX-2 inhibitor NS-398. IOP-2 and IOP-3 were inactive at 10pM. Tested compounds did not affect cellular viability, as assessed by mitochondrial reduction of MTT after 18h challenge (data not shown), indicating that they were not cytotoxic. Active compounds were selected for further studies. Table I Effect of IOPs derivatives on nitrite and PGEz accumulation KS0 on nitrite accumulation IOP-1 IOP-2 IOP-3 IOP-4 IOP-5 AG NS-398
ICJOon PGEl accumulation
(FM)
(PM)
1.2(0.7-I .7) Inactive at 10 FM Inactive at IO pM 0.7 (0.1-1.4) 2.9 (3.7-2.8) N.D. 3.1 (1.3-5.4) nM
7.3 (7.0-7.6) Inactive at IO pM Inactive at IO pM 4.6 (4.2-5.0) 4.3 (3.9-4.8) 25.1 (22.9-28.8) N.D.
Data show means f S.E.M. (n=6-10). .N.D., not determined. AC: aminoguanidine
iNOS, COX-1 and COX-2 activities. As shown in Table II, none of these active compounds at the concentration of 10 PM, significantly affected the activity of iNOS measured in the cytosolic fraction of LPS-stimulated peritoneal macrophages, COX-2, or COX-I , measured in microsomes from LPS-treated mouse macrophages or human platelets, respectively. Table II Effect of IOPs on iNOS, COX-2 and COX-1 activities
in vitro
iNOS (pm01 citrullinei mg x min)
cox-2 (@ml
Control IOP-I (10 PM)
1331.3 + 156.4 1332.0 + 157.3
8.4 rt 0.4 7.6 zk0.5
36. I f 2.2 30.5 * 1.4
lop-4
27.0 zk 1.8
PGE*)
cox-I (ng/ml TXB?)
(10 PM)
1341.0 + 144.1
7.9 k 0.6
IOP-5 (IO PM)
1330.0 + 151.9
10.9 + 1.6
33.6 f 3.9
AG (100 PM)
160.7 + 18.2 **
N.D.
N.D.
NS 398 (10 PM)
N.D.
4.7 k 0.3**
N.D.
lndomethacin
N.D.
N.D.
2.7 + 0.2**
(IO PM)
Data show means f S.E.M. (n = 6-10). **P
iNOS and COX-2 expression on LPS-stimulated peritoneal macrophages. Compound IOP-4 caused a great reduction of iNOS expression without affecting COX-2 protein levels (Fig. 2). Nevertheless, compound IOP-5 mainly affected COX-2 expression.
iNOS
c
1
4
5
Dex
Fig. 2 Effects of IOPs on iNOS and COX-2 expression in mouse peritoneal macrophages. C: control; Dex: Dexamethasone. Representative of two experiments.
Vol. 66, No. 9, 2000
IOPs
on NO and Eicosanoid Biosynthesis
PL-129
cPLA2 and sPLA2 activities. None of these active compounds at 1OpM significantly affected the activity of cPLAz measured in the cytosolic fraction of LPS-stimulated U937 monocytes, or sPLA2 from bee venom or human synovial sPLA2 (data not shown). Synthesis of LTBd by human neutrophils. No cytotoxic effects of IOPs derivatives were observed at the concentrations used in our study. Addition of calcium ionophore A23 187 to human neutrophils promoted a significant generation of LTB4 which was not inhibited by these compounds (data not shown). Elastase and superoxide release from human neutrophils. These compounds did not blocked the degranulation of human neutrophils activated with FMLP in cytochalasin-pretreated cells that was determined by elastase release. Besides, none of them inhibited the chemiluminescence response induced by stimulation of neutrophils with TPA (data not shown). Mouse air pouch. Active compounds administered at 50 mg/kg (p.0.) caused a significant reduction of nitrite levels in the exudate of the mouse air pouch (Fig. 3), without affecting PGE2 levels or leukocyte migration (data not shown). Compound IOP-4 showed an inhibition higher than 50% on nitrite levels, with an EDso of 32.4 (29.0-35.8) mg/kg. Mice treated with IOPs did not show any sign of toxicity.
100 .;
**
75
2 ; H 9 0
-
IOP-I
-
IOP-5
rzmJ IOP-4 50 25 0 50
50
50
35
25
Dose (mg/kg) Fig. 3 Effect of IOPs on nitrite levels in the exudate of the mouse air pouch. Nitrite levels from saline- (1.1kO.l rig/ml) and zymosan-injected mice (80.1f3.8 @ml). Discussion The induction of iNOS and COX-2 results in a great increase in the synthesis of NO and PGE2, which contribute to the pathophysiology of different inflammatory processes. It has been described an NO-mediated increase in the production of proinflammatory PGs in macrophages and other cells, that may result in an exacerbated inflammatory response. Thus, NOS inhibitors could modulate inflammatory processes by the dual inhibition of NO biosynthesis and related PG generation (17;18). Nevertheless, the situation in vivo is quite complex and the interactions between both pathways can vary according to the response considered (18-21). In this study we show that some isoxazolpyrimidine derivatives can prevent the biosynthesis of these mediators as observed by the reduction on nitrite and PGEz accumulation in culture medium of LPS-stimulated murine peritoneal macrophages. This effect is not related to a
PL-130
IOPs on NO and Eicosanoid Biosynthesis
Vol. 66, No. 9,200O
direct inhibition of enzyme activities, which suggest that these compounds act at the level of induction/expression of iNOS and COX-2. Western blots assays showed that IOP-4 inhibited iNOS expresion and IOP5 reduced COX-2 expression. Thus, reduction of PGE2 production by IOP-1 or IOP-4, and inhibition of NO generation by IOP-1 or IOP-5 are due to mechanisms other than inhibition of protein expression. Some possibilities may be an interference with the availability of substrate, although they were inactive on several PLAz activities, or an alteration in the structure or function of these inducible enzymes. In other in vitro experiments, these isoxazolpyrimidine derivatives did not affect human neutrophil inflammatory responses, since neither release of lysosomal enzymes, nor superoxide or LTB4 synthesis was inhibited by these compounds. We have also assayed active compounds in the mouse air pouch model of inflammation, by oral administration. The inflammatory response induced by zymosan is a complex process where different types of cells (PMNs, mononuclear cells) migrate into the exudate besides cells lining the pouch. Coinduction of iNOS and COX-2 enzymes has been demonstrated in the later phase (from 12 h after challenge), resulting in high levels of NO and PGE2. Besides, at this later phase COX-1 is still present and may contribute to PG production (14). Although NO may have an anti-adhesive role (22), IOPs inhibited NO production without affecting cellular migration in the mouse air pouch after 24 h injection with zymosan. In this in vivo model, IOP-1, IOP-4 and IOP-5 reduced nitrite levels. The most active compound was IOP-4, which also inhibited iNOS expression in vitro in peritoneal macrophages. Nevertheless, IOPs did not show any effect on PGE2 levels in the mouse air pouch injected with zymosan. In addition to possible complex interactions with the different biochemical pathways involved in this model, this discrepancy with in vitro effects could be due to pharmacokinetic modification of these IOPs. On the other hand, lack of effect on COX-1 activity may be considered a positive feature since ulcerogenic effects of non steroidal antiinflammatory drugs have been related to a reduced PG synthesis by COX-1 inhibition (23). Our study indicates that some isoxazolpyrimidine derivatives are potent inhibitors of nitrite and PGE2 production in endotoxin-stimulated macrophages, which can be related to a reduced expression of iNOS and COX-2 in some cases. These products were also active on nitrite levels by oral administration, in the mouse air pouch model of inflammation. This group of compounds may offer new drugs for the modulation of NO production in different inflammatory and NO-related pathologies. Acknowledgements This work was supported by grant SAF97-0249, C.I.C.Y.T., Spanish Ministerio de Education y Ciencia and by grant XUGA 10308B95 and 20908B97. A. Vidal thanks Generalitat Valenciana for a scholarship. We wish to thank Dr. S.J. Foster (Zeneca Pharmaceuticals, Macclestield, Cheshire, UK) for the kind gift of antibody against LTB+ References 1. 2. 3. 4.
E. MOILANEN and H. VAPAATALO, Ann.Med. 27 359-367 (1995). S. MONCADA and A. HIGGS, N.Engl.J.Med. 329 2002-2012 (1993). J. VANE and R. BOTTING, FASEB J. 189-96 (1987). S.T. REDDY and H.R. HERSCHMAN, J.Biol.Chem. 269 15473-15480 (1994).
Vol. 66, No. 9,200O
IOPs on NO and Eicosanoid Biosynthesis
5. J. MACMICKING, Q.W. XIE and C. NATHAN, Annu.Rev.Immunol. 15 323350 (1997). 6. J.M. FUKUTO and G. CHAUDHURI, Annu.Rev.Pharmacol.Toxicol. 35 165194 (1995). 7. J.M. QUINTELA and C. PEINADOR, Tetrahedron 53 8269-8272 (1997). 8. C. PEINADOR, L. BOTANA, M. ESTEVEZ and R. RIGUERA, Bioorg.Med.Chem. 5 1543-1553 (1997). 9. S.S. GROSS and R. LEVI, J.Biol.Chem. 267 25722-25729 (1992). 10. T.P. MISKO, R.J. SCHILLING, D. SALVEMINI, W.M. MOORE and M.G. CURRIE, AnalBiochem. 214 11-16 (1993). Il. M.A. MORONEY, M.J. ALCARAZ, R.A. FORDER, F. CAREY and J.R.S. HOULT, J.Pharm.Pharmacol. 40 787-792 (1988). 12. V. ESCRIG, A. UBEDA, M.L. FERRANDIZ, J. DARIAS, J.M. SANCHEZ, M.J. ALCARAZ and M. PAYA, J.Pharmacol.Exp.Ther. 282 123-131 (1997). 13. J.A. MITCHELL, H. SHENG, U. FORSTERMANN and F. MURAD, Br.J.Pharmacol. 104 289-291 (1991). 14. I. POSADAS, M.C. TERENCIO, I. GUILLEN, M.L. FERRANDIZ, J. COLOMA, M. PAYA, and M.J. ALCARAZ, Naunyn-Schmiedeberg’s Arch.Pharmacol. (in press) (1999). 15. R. FRANSON, P. PATRIARCA and P. ELSBACH, J.Lipid Res. 15 380-388 (1974). 16. J.D. CLARK, N. MILONA and J.L. KNOPF, Proc.Natl.Acad.Sci.USA 87 7708-7712 (1990). 17. D. SALVEMINI, T.P. MISKO, J.L. MASFERRER, K. SEIBERT, M.G. CURRIE and P. NEEDLEMAN, Proc.Natl.Acad.Sci.USA 90 7240-7244 (1993). 18. D. SALVEMINI, P.T. MANNING, B.S. ZWEIFEL, K. SEIBERT, J. CONNOR, M.G. CURRIE, P. NEEDLEMAN and J.L. MASFERRER, J.Clin.Invest. 96 301-308 (1995). 19. J.R. VANE, J.A. MITCHELL, I. APPLETON, A. TOMLISON, D. BISHOPBAILEY, J. CROXTALL and D.A. WILLOUGHBY, Proc.Natl.Acad.Sci.USA 91 2046-2050 (1994). 20. M. PAYA, P. GARCIA PASTOR, J. COLOMA and M.J. ALCARAZ, Br.J.Pharmacol. 120 1445-1452 (1997). 21. L.C. HAMILTON and T.D. WARNER, Br.J.Pharmacol. 125 335-340 (1998). 22. J.E. CARTWRIGHT, A.P. JOHNSTONE and G.St.J. WHITLEY, Br.J.Pharmacol. 120 146-152 (1997). 23. J.R. VANE and R.M. BOTTING, Am.J.Med. 104 2S-8s (1998).
PL-131
Vol.
66, No. 9, pp. PL 125131,200O
Copyright Prmted PII
ELSEVIER
0 2000 in the USA.
Elsevier
0024-3205100&see
SOO24-3205(99)00658-x
Science
Inc.
All rights reserved front matter
4 cc
EFFECTS OF SOME ISOXAZOLPYRIMIDINE DERIVATIVES EICOSANOID BIOSYNTHESIS
ON NITRIC OXIDE AND
J.M. Quintela3, C. Peinador3,
‘Department of Pharmacology, University of Valencia, Spain. 2Dept. of Organic Chemistry. University of Santiago, Spain. Dept.of Fundamental Chemistry. University of La Coruha, Spain. (Submitted May 2 I, 1999; accepted June 17, 1999; received in final form September 28, 1999)
Abstract.
The inhibitory effect of some isoxazolpyrimidine derivatives on iNOS and COX-2 endotoxin induction in mouse peritoneal macrophages has been studied. Three of these compounds inhibited nitrite and PGE2 accumulation in a concentration dependent-manner at pM range. None of these active compounds affected iNOS, COX-2, COX-1 or PLA2 activities, although some reduced iNOS or COX-2 expression. Besides, no effect was observed on human neutrophil inflammatory responses (LTB4 biosynthesis and superoxide or elastase release). Active compounds were assayed by oral administration in the mouse air pouch model, where they inhibited nitrite accumulation without affecting PGE2 levels or leukocyte migration. 0 2000 Elsevier Science Inc. Key Words:
isoxazolpyrimidine,
nitric oxide, PGE,, macrophages,
!z
-I F g d
PHARMACOLOGYLET-TERS Accelerated Communicalion
A. Vidal’, M.L. Ferrartdiz’, A. Ubeda’, I. Guillen’, R. Riguer$, M.J. Moreira3, M.J. Alcaraz’
P E
mouse air pouch
Macrophages play a crucial role in modulating the initiation and perpetuation of the inflammatory response. One means by which macrophages modulate inflammation is via their capacity to elaborate biological mediators, like nitric oxide (NO) and prostaglandins (PGs), which have numerous cardiovascular and inflammatory effecs (1). NO is formed from L-arginine by NO synthase (NOS) (2). Cyclooxygenase (COX) is the first enzyme in the pathway in which arachidonic acid is converted to PGs, prostacyclin and thromboxane A2 (TXA2) (3). NOS and COX exist in at least two isofotms. The constitutive isoforms, COX-1 and cNOS, are present in many types of cells, while the inducible isoforms COX-2 and iNOS are expressed after stimulation of cells with a variety of agents including endotoxin (bacterial lipopolysaccharide, LPS) and cytokines (2;4;5). Since the induction of COX-2 and iNOS results in the increased synthesis of PGs and NO, selective modulation of these mediators overproduction might represent a therapeutic goal in different inflammatory pathologies. Many compounds described as inhibitors of iNOS are nitrogen-containing heterocycles (6); this prompted us to assay some 3-methyl-4-R-6-dimethylamineisoxazolo[5,4-d]pyrimidine derivatives (IOPs)(Fig. 1) on NO production and iNOS activity. Corresponding Author: Dr. A. Ubeda. Department of Pharmacology, Faculty of Pharmacy. Avda. Vicent Andre% Estelles s/n, 46100 Burjassot, Valencia, Spain.
P
PL-126
IOPs on NO and Eicosanoid Biosynthesis
Vol. 66, No. 9,200O
IOPs were evaluated on nitrite and PGE2 production in LPS-stimulated mouse peritoneal macrophages, as an index of iNOS and COX-2 induction. We also studied their effects on the enzymatic activity of iNOS, COX-2, COX-1, cytosolic phospholipase A2 (cPLA2) and secretory phospholipase A2 (sPLA& as well as on the release of LTB4, elastase or superoxide by human neutrophils. Active compounds were assayed by oral administration in the mouse air pouch model of inflammation. R:
-OCH3
IOP-1
--N-l --cy -@J
IGP_2
-N
IOP-3
-r+--_
3
Fig. 1 Structural formula of isoxazolpyrimidine
IOP-4
derivatives (IOPs).
Materials and methods Drugs. IOPs have been prepared starting from the appropiate b-enaminonitrile,5-amino-4-cyano3-methylisoxazole and dichloromethyleneammonium chloride (7;8). Isolation and culture of mouse peritoneal macrophages. Peritoneal macrophages were obtained from female mice weighing 25-308. Cells were haversted by peritoneal lavage 4 days after i.p. inyection of Iml of 10% thioglicolate broth. Cells were resuspended in RPMI-1640 medium supplemented with 10% foetal bovine serum and incubated at 37°C for 2 h. The adherent cells were stimulated with E.coZi LPS (lOug/ml) for 18h The mitochondrial dependent reduction of 3(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) to formazan was used to asses the possible cytotoxic effects of test compounds (9). Quantification of nitrite and PGE2 levels. Nitrite as index of NO production, was assayed fluorimetrically in microtiter plates (10). The amount of nitrite was obtained by extrapolation from a standard curve with sodium nitrite as a standard. PGE2 levels were measured by radioimmunoassay (RIA) (11). CO&I. Human platelets were sonicated and microsomes were prepared by centrifugation at 2,000 x g for 5 min at 4°C followed by centrifugation of the supernatant at 100,000 x g for 100 min at 4°C. Microsomes (20 pg of protein/tube) were incubated with arachidonic acid and test compound or vehicle and TXB2 levels were determined by RIA (12). COX-2. Mouse peritoneal macrophages were incubated with E. coli LPS (10 us/ml) at 37°C for 24 h and were scraped. After centrifugation the cells were sonicated and microsomes were prepared as above. Microsomes (40 ug of protein/tube) were used as a source of COX-2 and PGE2 synthesis was determined by RIA (12).
Vol. 66, No. 9,200O
IOPs on NO and Eicosanoid
Biosynthesis
PL-127
iNOX High speed (100,000 x g) supernatants from sonicated mouse peritoneal macrophages were obtained as described above. NOS activity was determined in supernatants by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline (13). Western blot analysis. Mouse peritoneal macrophages obtained as described above were collected after 18h with lysis buffer (1% Triton X-l 00, 1% deoxicolic acid, 20 mM NaCl and 25 mM Tris; pH 7.5) and were used to assay COX-2 and iNOS expression using specific antibodies (14). sPLAz assay. sPLA1 was assayed by using [3H]oleate labeled membranes of E. coli (15). Bee venom and human recombinant synovial enzymes were used as sources of sPLA2. cPLA,J assay. cPLA2 was prepared from human monocytic U937 cells (Cell Collection, Department of Animal Cell Culture, C.S.I.C., Madrid, Spain) (12). cPLA2 activity was measured as the release of radiolabeled arachidonic acid according to the method of Clark et al (16). Synthesis and release of LTBd by human neutrophils. Leukocytes were obtained as previously described (12). Viability was greater than 95% by the trypan blue exclusion test. A suspension of human neutrophils (5x106/ml) was preincubated with test compounds or vehicle and then stimulated with 1 uM A23 187. The LTB4 levels in supernatants were measured by RIA (12). Chemiluminescence. Neutrophils (2.5x106/ml) were mixed with luminol (40 uM) and stimulated with 12-0-tetradecanoyl phorbol 13-acetate (TPA, 1 uM). The chemiluminescence was recorded in a Microbeta Trilux counter (Wallac, Turku, Finland) after 7 min. (12). Elastase release by human neutrophils. Neutrophils (2.5x106/ml) were preincubated with test compound or vehicle for 5 min and then stimulated with cytochalasin B (10 uM) and N-formylL-methionyl-L-leucyl-L-phenylalanine (FMLP, 10 nM). Elastase activity was estimated in supernatants as p-nitrophenol release (12). Mouse air pouch model. Air pouch was performed in female Swiss mice (25-308) as previously described (12). Tested compounds were administered orally, lh before and 8h after zymosan inyection at doses of 50, 35 and 25 mg/kg. 24h after zymosan administration, the animals were killed by cervical dislocation, and the exudate in the pouch was collected with lml of saline. Leukocytes present in exudates were measured using a Coulter Counter. After centrifugation of exudates (1,200g at 4°C for lOmin), the supernatants were used to measure nitrite and PGE2 levels as described above. Statistical analysis. The results are presented as mean f S.E.M.; n represents the number of experiments. Inhibitory concentration 50% (I&O) values were calculated from at least 4 significant concentrations (n=6) by linear regression analysis using Graph Pad Prism II. The level of statistical significance was determined by analysis of variance (ANOVA) followed by Dum-rett’s t-test for multiple comparisons. Results Inhibition of nitrite and PGE2 accumulation. Compounds IOP- 1, IOP-4 and IOP-5 inhibited the accumulation of both metabolites in LPS-stimulated peritoneal macrophages in a concentrationdependent manner, as shown in Table I. These compounds were more potent than the reference NOS inhibitor aminoguanidine (AG) on nitrite accumulation but showed less potency on PGE2
IOPs on NO and Eicosanoid
PL-128
Vol. 66, No. 9, 2000
Biosynthesis
levels than the reference COX-2 inhibitor NS-398. IOP-2 and IOP-3 were inactive at 10pM. Tested compounds did not affect cellular viability, as assessed by mitochondrial reduction of MTT after 18h challenge (data not shown), indicating that they were not cytotoxic. Active compounds were selected for further studies. Table I Effect of IOPs derivatives on nitrite and PGEz accumulation KS0 on nitrite accumulation IOP-1 IOP-2 IOP-3 IOP-4 IOP-5 AG NS-398
ICJOon PGEl accumulation
(FM)
(PM)
1.2(0.7-I .7) Inactive at 10 FM Inactive at IO pM 0.7 (0.1-1.4) 2.9 (3.7-2.8) N.D. 3.1 (1.3-5.4) nM
7.3 (7.0-7.6) Inactive at IO pM Inactive at IO pM 4.6 (4.2-5.0) 4.3 (3.9-4.8) 25.1 (22.9-28.8) N.D.
Data show means f S.E.M. (n=6-10). .N.D., not determined. AC: aminoguanidine
iNOS, COX-1 and COX-2 activities. As shown in Table II, none of these active compounds at the concentration of 10 PM, significantly affected the activity of iNOS measured in the cytosolic fraction of LPS-stimulated peritoneal macrophages, COX-2, or COX-I , measured in microsomes from LPS-treated mouse macrophages or human platelets, respectively. Table II Effect of IOPs on iNOS, COX-2 and COX-1 activities
in vitro
iNOS (pm01 citrullinei mg x min)
cox-2 (@ml
Control IOP-I (10 PM)
1331.3 + 156.4 1332.0 + 157.3
8.4 rt 0.4 7.6 zk0.5
36. I f 2.2 30.5 * 1.4
lop-4
27.0 zk 1.8
PGE*)
cox-I (ng/ml TXB?)
(10 PM)
1341.0 + 144.1
7.9 k 0.6
IOP-5 (IO PM)
1330.0 + 151.9
10.9 + 1.6
33.6 f 3.9
AG (100 PM)
160.7 + 18.2 **
N.D.
N.D.
NS 398 (10 PM)
N.D.
4.7 k 0.3**
N.D.
lndomethacin
N.D.
N.D.
2.7 + 0.2**
(IO PM)
Data show means f S.E.M. (n = 6-10). **P
iNOS and COX-2 expression on LPS-stimulated peritoneal macrophages. Compound IOP-4 caused a great reduction of iNOS expression without affecting COX-2 protein levels (Fig. 2). Nevertheless, compound IOP-5 mainly affected COX-2 expression.
iNOS
c
1
4
5
Dex
Fig. 2 Effects of IOPs on iNOS and COX-2 expression in mouse peritoneal macrophages. C: control; Dex: Dexamethasone. Representative of two experiments.
Vol. 66, No. 9, 2000
IOPs
on NO and Eicosanoid Biosynthesis
PL-129
cPLA2 and sPLA2 activities. None of these active compounds at 1OpM significantly affected the activity of cPLAz measured in the cytosolic fraction of LPS-stimulated U937 monocytes, or sPLA2 from bee venom or human synovial sPLA2 (data not shown). Synthesis of LTBd by human neutrophils. No cytotoxic effects of IOPs derivatives were observed at the concentrations used in our study. Addition of calcium ionophore A23 187 to human neutrophils promoted a significant generation of LTB4 which was not inhibited by these compounds (data not shown). Elastase and superoxide release from human neutrophils. These compounds did not blocked the degranulation of human neutrophils activated with FMLP in cytochalasin-pretreated cells that was determined by elastase release. Besides, none of them inhibited the chemiluminescence response induced by stimulation of neutrophils with TPA (data not shown). Mouse air pouch. Active compounds administered at 50 mg/kg (p.0.) caused a significant reduction of nitrite levels in the exudate of the mouse air pouch (Fig. 3), without affecting PGE2 levels or leukocyte migration (data not shown). Compound IOP-4 showed an inhibition higher than 50% on nitrite levels, with an EDso of 32.4 (29.0-35.8) mg/kg. Mice treated with IOPs did not show any sign of toxicity.
100 .;
**
75
2 ; H 9 0
-
IOP-I
-
IOP-5
rzmJ IOP-4 50 25 0 50
50
50
35
25
Dose (mg/kg) Fig. 3 Effect of IOPs on nitrite levels in the exudate of the mouse air pouch. Nitrite levels from saline- (1.1kO.l rig/ml) and zymosan-injected mice (80.1f3.8 @ml). Discussion The induction of iNOS and COX-2 results in a great increase in the synthesis of NO and PGE2, which contribute to the pathophysiology of different inflammatory processes. It has been described an NO-mediated increase in the production of proinflammatory PGs in macrophages and other cells, that may result in an exacerbated inflammatory response. Thus, NOS inhibitors could modulate inflammatory processes by the dual inhibition of NO biosynthesis and related PG generation (17;18). Nevertheless, the situation in vivo is quite complex and the interactions between both pathways can vary according to the response considered (18-21). In this study we show that some isoxazolpyrimidine derivatives can prevent the biosynthesis of these mediators as observed by the reduction on nitrite and PGEz accumulation in culture medium of LPS-stimulated murine peritoneal macrophages. This effect is not related to a
PL-130
IOPs on NO and Eicosanoid Biosynthesis
Vol. 66, No. 9,200O
direct inhibition of enzyme activities, which suggest that these compounds act at the level of induction/expression of iNOS and COX-2. Western blots assays showed that IOP-4 inhibited iNOS expresion and IOP5 reduced COX-2 expression. Thus, reduction of PGE2 production by IOP-1 or IOP-4, and inhibition of NO generation by IOP-1 or IOP-5 are due to mechanisms other than inhibition of protein expression. Some possibilities may be an interference with the availability of substrate, although they were inactive on several PLAz activities, or an alteration in the structure or function of these inducible enzymes. In other in vitro experiments, these isoxazolpyrimidine derivatives did not affect human neutrophil inflammatory responses, since neither release of lysosomal enzymes, nor superoxide or LTB4 synthesis was inhibited by these compounds. We have also assayed active compounds in the mouse air pouch model of inflammation, by oral administration. The inflammatory response induced by zymosan is a complex process where different types of cells (PMNs, mononuclear cells) migrate into the exudate besides cells lining the pouch. Coinduction of iNOS and COX-2 enzymes has been demonstrated in the later phase (from 12 h after challenge), resulting in high levels of NO and PGE2. Besides, at this later phase COX-1 is still present and may contribute to PG production (14). Although NO may have an anti-adhesive role (22), IOPs inhibited NO production without affecting cellular migration in the mouse air pouch after 24 h injection with zymosan. In this in vivo model, IOP-1, IOP-4 and IOP-5 reduced nitrite levels. The most active compound was IOP-4, which also inhibited iNOS expression in vitro in peritoneal macrophages. Nevertheless, IOPs did not show any effect on PGE2 levels in the mouse air pouch injected with zymosan. In addition to possible complex interactions with the different biochemical pathways involved in this model, this discrepancy with in vitro effects could be due to pharmacokinetic modification of these IOPs. On the other hand, lack of effect on COX-1 activity may be considered a positive feature since ulcerogenic effects of non steroidal antiinflammatory drugs have been related to a reduced PG synthesis by COX-1 inhibition (23). Our study indicates that some isoxazolpyrimidine derivatives are potent inhibitors of nitrite and PGE2 production in endotoxin-stimulated macrophages, which can be related to a reduced expression of iNOS and COX-2 in some cases. These products were also active on nitrite levels by oral administration, in the mouse air pouch model of inflammation. This group of compounds may offer new drugs for the modulation of NO production in different inflammatory and NO-related pathologies. Acknowledgements This work was supported by grant SAF97-0249, C.I.C.Y.T., Spanish Ministerio de Education y Ciencia and by grant XUGA 10308B95 and 20908B97. A. Vidal thanks Generalitat Valenciana for a scholarship. We wish to thank Dr. S.J. Foster (Zeneca Pharmaceuticals, Macclestield, Cheshire, UK) for the kind gift of antibody against LTB+ References 1. 2. 3. 4.
E. MOILANEN and H. VAPAATALO, Ann.Med. 27 359-367 (1995). S. MONCADA and A. HIGGS, N.Engl.J.Med. 329 2002-2012 (1993). J. VANE and R. BOTTING, FASEB J. 189-96 (1987). S.T. REDDY and H.R. HERSCHMAN, J.Biol.Chem. 269 15473-15480 (1994).
Vol. 66, No. 9,200O
IOPs on NO and Eicosanoid Biosynthesis
5. J. MACMICKING, Q.W. XIE and C. NATHAN, Annu.Rev.Immunol. 15 323350 (1997). 6. J.M. FUKUTO and G. CHAUDHURI, Annu.Rev.Pharmacol.Toxicol. 35 165194 (1995). 7. J.M. QUINTELA and C. PEINADOR, Tetrahedron 53 8269-8272 (1997). 8. C. PEINADOR, L. BOTANA, M. ESTEVEZ and R. RIGUERA, Bioorg.Med.Chem. 5 1543-1553 (1997). 9. S.S. GROSS and R. LEVI, J.Biol.Chem. 267 25722-25729 (1992). 10. T.P. MISKO, R.J. SCHILLING, D. SALVEMINI, W.M. MOORE and M.G. CURRIE, AnalBiochem. 214 11-16 (1993). Il. M.A. MORONEY, M.J. ALCARAZ, R.A. FORDER, F. CAREY and J.R.S. HOULT, J.Pharm.Pharmacol. 40 787-792 (1988). 12. V. ESCRIG, A. UBEDA, M.L. FERRANDIZ, J. DARIAS, J.M. SANCHEZ, M.J. ALCARAZ and M. PAYA, J.Pharmacol.Exp.Ther. 282 123-131 (1997). 13. J.A. MITCHELL, H. SHENG, U. FORSTERMANN and F. MURAD, Br.J.Pharmacol. 104 289-291 (1991). 14. I. POSADAS, M.C. TERENCIO, I. GUILLEN, M.L. FERRANDIZ, J. COLOMA, M. PAYA, and M.J. ALCARAZ, Naunyn-Schmiedeberg’s Arch.Pharmacol. (in press) (1999). 15. R. FRANSON, P. PATRIARCA and P. ELSBACH, J.Lipid Res. 15 380-388 (1974). 16. J.D. CLARK, N. MILONA and J.L. KNOPF, Proc.Natl.Acad.Sci.USA 87 7708-7712 (1990). 17. D. SALVEMINI, T.P. MISKO, J.L. MASFERRER, K. SEIBERT, M.G. CURRIE and P. NEEDLEMAN, Proc.Natl.Acad.Sci.USA 90 7240-7244 (1993). 18. D. SALVEMINI, P.T. MANNING, B.S. ZWEIFEL, K. SEIBERT, J. CONNOR, M.G. CURRIE, P. NEEDLEMAN and J.L. MASFERRER, J.Clin.Invest. 96 301-308 (1995). 19. J.R. VANE, J.A. MITCHELL, I. APPLETON, A. TOMLISON, D. BISHOPBAILEY, J. CROXTALL and D.A. WILLOUGHBY, Proc.Natl.Acad.Sci.USA 91 2046-2050 (1994). 20. M. PAYA, P. GARCIA PASTOR, J. COLOMA and M.J. ALCARAZ, Br.J.Pharmacol. 120 1445-1452 (1997). 21. L.C. HAMILTON and T.D. WARNER, Br.J.Pharmacol. 125 335-340 (1998). 22. J.E. CARTWRIGHT, A.P. JOHNSTONE and G.St.J. WHITLEY, Br.J.Pharmacol. 120 146-152 (1997). 23. J.R. VANE and R.M. BOTTING, Am.J.Med. 104 2S-8s (1998).
PL-131