Screening New Drugs for Immunotoxic Potential: II. Assessment of the Effects of Selective and Nonselective COX-2 Inhibitors on Complement Activation, Superoxide Anion Production and Leukocyte Chemotaxis and Migration Through Endothelial Cells

Figures & data

TABLE 1 Comparative cyclooxygenase inhibiting properties of ibuprofen, naproxen, SC-236, SC-791, and SC-245

	IC50^1 (μM)		Selectivity ratio
Drug identity	COX-1	COX-2	COX-1/COX-2
Ibuprofen	38	117	0.325^2
Naproxen	32	235	0.136^2
SC-236	15	0.004	3750
SC-791	65	0.004	16250
SC-245	> 1000	0.269	> 3717

¹IC₅₀ is the in vitro concentration required to inhibit the activity of the indicated cyclooxygenase isoform by 50%

²Maziasz et al., 2003

FIG. 1 Inhibitory effects of leupeptin on human and canine complement activities. Human and canine complement were incubated with opsonized sheep erythrocytes in the presence of leupeptin (30, 50, 100, 500, or 1000 μM) for 60 minutes. Each data point represents the mean of 6 to 9 experiments, each consisting of 3 replicate incubations. Optical density was measured at 540 nm. Data points represent absorbance expressed as percent of control (0 μ M leupeptin). Leupeptin IC₅₀ concentrations were 38.2 and 106.8 μM for human and dog sera, respectively.

FIG. 2 Inhibitory effect of staurosporine on PMA (100 ng/mL) induced superoxide anion formation by canine neutrophils. Neutrophils were exposed to 100 ng/mL PMA in the presence of 0, 0.001, 0.01, or 0.1 μM of staurosporine and optical density at 550 nm was determined. Data points represent the mean ± SD of 4 incubations, each conducted in triplicate.

TABLE 2 Responsiveness of canine leukocytes to chemoattractants¹

	Mean ± St. dev. number of cells exhibiting chemotaxis
Attractant (concentration)	Dog L	Dog O	Dog P	Dog Q	Dog R	Overall mean
Zymosan-activated dog serum (triplicate incubations)
% in medium
0	2 ± 1	11 ± 4	22 ± 10	—	—	12 ± 19
0.05	13 ± 6	47 ± 19	100 ± 2	—	—	58 ± 44
0.1	44 ± 18	125 ± 31	276 ± 8	—	—	148 ± 118
0.25	97 ± 41	183 ± 7	543 ± 61	—	—	274 ± 237
0.5	289 ± 29	451 ± 131	617 ± 120	—	—	452 ± 162
1	347 ± 42	506 ± 96	903 ± 175	—	—	585 ± 286
5	386 ± 42	576 ± 66	1171 ± 335	—	—	711 ± 410
10	289 ± 70	—	749 ± 154	—	—	519 ± 325
LTB4 (duplicate incubations except when noted)
nM in medium
0.1	7 ± 0	60 ± 15	—	3 ± 2	72 ± 10	36 ± 36
0.5	56 ± 22	135 ± 75	—	42 ± 0	226 ± 108	115 ± 85
1	117 ± 11	335 ± 11	—	76 ± 16	307 ± 4	209 ± 131
5	248 ± 5	583 ± 182	—	176 ± 42	636 ± 232	411 ± 232
10	269 ± 8	656 ± 76	—	229 ± 2	724 ± 202	470 ± 257
50	312 ± 55	846	—	300 ± 47	1032	623 ± 373
100	312 ± 133	574	—	205 ± 18	—	364 ± 190
500	201 ± 27	482	—	103 ± 5	—	262 ± 197
1000	112 ± 23	228	—	55 ± 13	—	132 ± 88

¹10⁵ freshly isolated canine leukocytes were equilibrated to 37°C in RPMI medium and then exposed to chemoattractants for 30 minutes. Cells that migrated through the filter pores to the underside of the filter were counted to assess the magnitude of chemotaxis. Data represent means ± SDs of the means of triplicate or duplicate replicates of each incubation

*Single rather than duplicate incubations.

FIG. 3 Inhibitory effects of cytochalasin B on chemotaxis of canine leukocytes toward LTB4 and ZAS. Canine leukocytes were exposed to 0.01, 0.1, 0.5, or 1.0 μM cytochalasin B then exposed to either 5 nM LTB₄ or 0.25% ZAS. Leukocytes migrating through filters were counted. Extent of migration in the absence of cytochalasin was considered to be 100% of control and effects of cytochalasin were expressed as percent of control.

TABLE 3 Effects of leupeptin, naproxen, and selective cox-2 inhibitors on canine complement activity in vitro

Test compound and concentration (μ M)	Number of replicates	Mean complement activity relative to controls (% of control ± SEM)	Statistical significance
Leupeptin
0	5	100	—
100	5	57.1 ± 4.9	< 0.0001^a
1000	5	28.3 ± 4.9	< 0.0001^a
Naproxen
0.1	4	78.6 ± 7.5	< 0.05^a
0.5	4	91.6 ± 4.9	NS^C
1	4	89.7 ± 5.0	NS
5	4	81.5 ± 6.8	NS
10	4	82.6 ± 7.7	NS
SC- 245
0.1	3	74.7 ± 3.4	0.0022^b
0.5	3	74.0 ± 1.6
1	3	73.6 ± 5.1
5	3	77.6 ± 1.5
10	3	74.6 ± 6.6
SC-236
0.1	4	86.5 ± 5.2	NS
0.5	4	89.3 ± 4.1
1	4	89.1 ± 5.1
5	4	92.4 ± 3.0
10	4	91.9 ± 4.7
SC-791
0.1	5	96.2 ± 3.4	NS
0.5	5	87.7 ± 1.4
1	5	90.2 ± 7.8
5	5	99.0 ± 5.2
10	5	89.1 ± 4.7

^aProbability computed with Dunnett's test

^bProbability computed by 1-way ANOVA

^cNS = difference from control not statistically significant (p > 0.05)

FIG. 4 Functional hemolytic complement levels in female beagle dogs dosed for up to 28 days with SC-791.

TABLE 4 Effects of staurosporine, naproxen, and selective COX-2 inhibitors on PMA-induced superoxide anion production by canine neutrophils

Test compound and concentration (μM)	Number of replicate incubations	Mean superoxide anion formed (% of control ± SEM)	Probability ANOVA
Staurosporine
0	6	100	—
0.01	6	49 ± 11.5	< 0.0001^a
0.1	5	13 ± 8.0	< 0.0001^a
SC-245
0.1	3	145 ± 22.9	0.0859
1	3	186 ± 44.6
10	3	218 ± 25.0^b
SC-236
0.1	3	110 ± 26.2	0.1510
1	3	131 ± 21.2
10	3	194 ± 44.1
SC-791
0.1	3	115 ± 10.5	0.7522
1	3	118 ± 6.7
10	3	119 ± 24.7
Naproxen
0.1	3	109 ± 28.3	0.9849
1	3	101 ± 20.0
10	3	105 ± 18.6

^aProbability computed with 1-way ANOVA and Dunnett's test

^bSignificantly greater than control (p < 0.05)

TABLE 5 Oxidative burst assessed through hydrogen peroxide production in SC-791-exposed canine neutrophils challenged with PMA¹

	Mean percent of neutrophils exhibiting fluorescence on Drug Day number
Treatment	1	14	22	28	Overall means
Stimulated with 0.1 μg/mL PMA
Control	16 ± 10	63 ± 11	15 ± 6	69 ± 9	41 ± 29
SC-791.	No samples	59 ± 14	21 ± 13	76 ± 3	52 ± 28
^2 p =	—	0.698	0.413	0.221	—
Stimulated with 1.0 μ g/mL PMA
Control	64 ± 12	78 ± 7	64 ± 7	74 ± 7	70 ± 7
SC-791	No samples	79 ± 5	74 ± 11	83 ± 3	79 ± 5
p =	—	0.700	0.172	0.062	—

¹Groups of 4 dogs were administered either empty gelatin capsules or 14 mg/kg/day of SC-791 daily for up to 28 days. Blood samples were obtained 1 hour before drug administration on the indicated day

²Percentages of control vs SC-791-exposed neutrophils fluorescing in response to PMA challenge were compared with Student's t test

TABLE 6 In vitro effects (specific migration) of naproxen and selective COX-2 inhibitors on canine leukocyte chemotaxis

		Mean specific migration ± SEM (n = 4 donor dogs per data point)
Attractant	Concentration of Test agent (μ M)	SC-236	SC-791	SC-245	Naproxen
5 nM LTB4.	0	139 ± 42	223 ± 132	180 ± 100	266 ± 170
	0.1	199 ± 95	145 ± 50	156 ± 102	194 ± 105
	1	175 ± 102	129 ± 56	149 ± 90	161 ± 94
	10	185 ± 86	127 ± 55	137 ± 82	160 ± 95
	Maximal % change	+43	−43	−24	−40
	from control
	ANOVA p value	0.2397	0.1708	0.5668	0.1316
0.25.% ZAS	0	392 ± 141	451 ± 154	283 ± 76	—
	0.1	413 ± 153	381 ± 129	328 ± 125	—
	1	505 ± 213	354 ± 142	300 ± 100	—
	10	520 ± 203	340 ± 141	348 ± 135	—
	Maximal % change from control	+32	−25	+23
	ANOVA p value	0.4700	0.0023	0.2757	—

TABLE 7 Effects of selective COX-2 inhibitors on canine and human leukocyte transmigration through endothelial monolayers

	Leukocyte migration through endothelial monolayer
Treatment regimen	Concentration in incubation well	Cell count (mean ± SD) × 1000	Cell count (percent ofcontrol ± SD)
TNFα alone	500. ng/ml	192 ± 53.4	100
TNFα + SC-236	1.3. μ M	188 ± 3	97.9 ± 29
	13.0. μ M	169 ± 55	88.3 ± 15
TNFα + SC-791	3.9. μ M	154 ± 56	80.9 ± 21
	39.2. μ M	130 ± 54.7	67.1± 20
	393. μ M	71 ± 21	37.8 ± 8
TNFα + SC-245	0.5. μ M	154 ± 42	82 ± 17
	5.2. μ M	111 ± 33	59 ± 14
	52. μ M	77 ± 30	41 ± 13
TNFα + Ibuprofen	0.15. mM	132 ± 54	68.8 ± 8
	1.5. mM	78 ± 25	42 ± 6
	7.5. mM	38 ± 26	19.6 ± 5

*Significantly less than control, ANOVA and Dunnett's test (p ≤ 0.05)