Canine Carrageenin-Induced Acute Paw Inflammation Model and Its Response to Nonsteroidal Antiinflammatory Drugs
R. R. BROOKS, J. F. CARPENTER,S. M. JONES, T. C. ZIEGLER,AND S. F. PONG
A quantitative method for testing antiinflammatory agents in beagles has been developed, based on measurement of paw inflammation induced by a local injection of carrageenin. Carrageenin [0.5 mL of 2% (wt/vol) in saline] was injected into the plantar region of the hindpaws of pentobarbital-anesthetized beagles. Paw pressure changes registered from a water-filled balloon held on the top of the paw by a light adhesive tape wrapping were monitored for 240 min. In control dogs given 0.5% (wt/vol) methylcellulose (10 mUkg orally) just before carrageenin, paw pressure increased significantly (p < 0.05) over eightfold, from 2.9 f 0.8 mm Hg (mean % SEM, n = 29 paws) at 75 min to 26.0 + 3.5 mmHg at 240 min. The increase in paw pressure was significantly inhibited by the cyclooxygenase inhibitors, ibuprofen, indomethacin, and orpanoxin, and partially inhibited by the lipoxygenase inhibitor, phenidone, administered orally before carrageenin injection. Thus this model, with further characterization, could provide a convenient, quantitative way of assessing the efficacy of nonsteroidal antiinflammatory agents in dogs. Key Words:
Inflammation;
Carrageenin;
Ibuprofen;
Indomethacin;
Phenidone
INTRODUCTION
The search for antiinflammatory drugs has always, at some point, involved evaluation in an acute inflammation model. There are several such models using rodents, and marketed nonsteroidal antiinflammatory drugs (NSAIDs) are all active in such models. Examples include croton-oil-induced ear inflammation in mice (Izuka et al., 1981; Brooks et al., 1985a), ultraviolet light-induced skin erythema in guinea pigs (Winder et al., 1958; Peters et al., 1977), carrageenin air pouch inflammation in rats (Hambleton and Miller, 1989), and the widely used carrageenin-induced paw edema model in rats (Winter et al., 1962). Among nonrodent species, however, only knee joint synovitis in the dog has been fairly well studied. Faires and McCarty Jr, (1962) and Phelps and McCarty Jr (1966) showed that injection of sodium urate crystals into the synovial space of the dog stifle joint could induce a synovitis involving phagocytizing polymorphonuclear leukocytes. McCarty et al. (1966) ad-
From the Product Development, Norwich Eaton Pharmaceuticals, Inc., A Procter & Gamble Company, Norwich, New York. Address reprint requests to: Dr. R. R. Brooks, Product Development, Norwich Eaton Pharmaceuticals, Inc. A Proctor & Gamble Company, P. 0. Box 191, Norwich, New York 13815-0191, U.S.A. Received June 20,199O; revised and accepted December 13,199O. 275 Journal of Pharmacological
Methods
0 1991 Elsevier Science Publishing
25, 275483
0160-5402/91/S3.50
WVl)
Co., Inc., 655 Avenue of the Americas,
New York, NY 10010
276
R. R. Brooks et al.
dressed several ways of quantifying the joint response, including measurements of pressure, white blood cell accumulation, and synovial fluid pH. Rosenthale et al. (1966) showed that NSAlDs diminished symptoms (gait deficit) in this canine model. Van Arman et al. (1970), using a specially designed dog dolorimeter, showed that a good correlation existed between the efficacy of drugs in the dog synovitis model and the rat paw edema model. This model has not been widely used, most probably because of its complexity and difficulties in quantitating results, although there has been a report of using an automated microcomputer-based quantitation system (Carlson et al., 1986). We have investigated an alternative procedure in the dog. The dog is a standard species for pathology-toxicology evaluation of NSAIDs. In order to use canine toxicology data to calculate a therapeutic index, it is necessary to have a measure of efficacy in the dog. Accordingly, we have determined whether the carrageenininduced paw edema test can be adapted to the dog. Our results show that an easily quantitated edema response can be elicited in the anesthetized dog and that this response is inhibitable by NSAIDs. We have characterized the model by examining its sensitivity to orally administered strong cyclooxygenase inhibitors (ibuprofen and indomethacin), weak cyclooxygenase inhibitors [orpanoxin (Goldenberg et al., 1980)], 5-(4-chlorophenyl)-2-furanpropanoic acid [F-1067 (Short and Rockwood, 1969)], 5-(4-chlorophenyl)-2-furan-carboxylic acid IF-1292 (Mathur and Mehra, 1961)], and a mixed lipoxygenase/cyclooxygenase inhibitor [phenidone (Blackwell and Flower, 1978)J. A preliminary report of these results has appeared in the literature (Brooks et al., 1985b). MATERIALS AND METHODS Animals Male and female beagles were purchased from Marshall Farms, North Rose, New York, or were born and raised at Norwich Eaton Pharmaceuticals, Inc., Norwich, New York. At the time of use, the dogs were fasted overnight and weighed between 5.6 and 14.2 kg. Dogs were fed Lab Canine Diet 5006 (Ralston Purina Co., Richmond, Indiana) and water ad libitum. Dogs surviving at the end of the experiment were euthanized with an overdose of barbiturate anesthetic (Socumb, The Butler Co., Columbus, Ohio). Procedure Dogs were anesthetized with pentobarbital sodium, 35 mg/kg i.v. Hair was removed from both hindpaws by clipping, toenails were trimmed, and both hindpaws were lightly wrapped with nonstretching, waterproof adhesive tape (Johnson & Johnson Products, Inc., New Brunswick, New Jersey). Included in the wrap on the top of the foot were the following. 1) A tracheal tube balloon and hose were filled with water and connected to a Statham P23lD pressure transducer (Grass Instruments, Quincy, Massachusetts). Output of the transducer was recorded on a Grass Model 7B polygraph. 2) A 23-g needle inserted into the large plantar pad. After a 15-min equilibration period, 0.5 mL of 0.2% (wt/vol) carrageenin (Algin Corp. of
Carrageenin-Induced America,
New York,
New York) was injected
and the needle
Paw Inflammation
was removed.
Baseline
pressure (mmHg) was adjusted to zero at this time, again adjusted to zero 15 min later, and then was monitored for 240 min. During this time, anesthesia was maintained with pentobarbital as needed. For orpanoxin sample was collected at 240 min for determination (Andrews
experiments, a venous blood of plasma drug concentration
et al., 1982).
Dosing For acute, single-dose experiments, ibuprofen (Upjohn Company, Kalamazoo, orpanoxin, FMichigan), indomethacin (Sigma Chemical Co., St. Louis, Missouri), 1067, F-1292 (Norwich Eaton Pharmaceuticals, Inc., Norwich, New York), or phenidone
(Aldrich
Chemical
suspension in 0.5% gan) 15 min before
Co.,
Milwaukee,
Wisconsin)
was administered
orally as a
(wt/vol) methylcellulose (Dow Chemical Co., Midland, Michicarrageenin. Suspensions were prepared fresh on the day of
use. For multiple-dosing experiments, orpanoxin and F-1067 were placed in gelatin capsules and administered orally four times per day for 2 days and once on the day of the test. Carrageenin Treatment Pressure
was injected
3-4 hr later in the multiple-dose
experiments.
of Data changes
from
baseline
were
tabulated
from the polygraph
record
at 15
min intervals. The pressure change at 240 min postcarrageenin was compared between the drug-treated and methylcellulose-vehicle-treated groups by a two-tailed unpaired
t test at the 5% level of significance.
RESULTS Control
Dogs
Carrageenin injection caused an increase in paw pressure over the 240-min observation period (Figure 1). The same volume of saline alone caused no inflammation and no pressure change (data not shown). The carrageenin-induced increase was delayed in onset, ylcellulose-dosed sure increased control
remaining below 5 mmHg for the first 75 min in control, methdogs. From 75 to 240 min after carrageenin-injection, paw pres-
from 2.9 & 0.8 (mean
dog paws were
& SEM, n = 29) to 26.0
not read at every time
27-30 paws shown in Figure 1. Several experin;ents were done
to determine
period,
+ 3.5 mmHg.
accounting
the proper
Some
for the range of
dose of carrageenin
to
use in this model. The dose chosen (10 mg/dog) gave about half the maximum paw pressure increase seen in dogs injected with 40 mg of carrageenin (data not shown). Because it produced a clear and reproducible, but not a maximum pressure increase, the IO-mg/dog dose of the phlogistic agent was considered suitable for testing the effect of antiinflammatory agents on the edema response, allowing observation of both positive and negative responses. We determined the paw edema response in an initial experiment using groups of six male or female beagles that did not differ significantly in weight. Inflammatory response to carrageenin did not differ between the sexes, averaging 24.2 * 4.4
277
278
R. R. Brooks et al.
Ibuprofen
-10
i//l
L
0 '75
105
I
I
135
165
I
I
I
195
225
Time PostCarrageenin
I
I
I
(min)
FIGURE 1. Effect of a single oral dose of 0 (open circles) mg/kg methylcellulose, 10 (circles with crosses), 39 (open triangles), 78 (open squares), and 116 (open diamonds) mg/kg ibuprofen on paw swelling induced in pentobarbital-anesthetized beagles induced by subplantar injection of 0.5 ml 2% carrageenin. Test substance in methylcellulose was administered by gavage, followed 15 min later (time zero) by subplantar injection of carrageenin. Paw pressure changes are relative to a zero baseline set at 15 min after carrageenin. The number of paws is indicated by n.
TABLE 1 Pressure Increases of the Beagle Hindpaw Injected with 10 mg of Carrageenin PAW PRESSURE 240 MIN BODY WEIGHT SEX
(KC)
9.1
M M
10.7
M
11.5
M
12.9
M
13.3 11.1
M Mean
5 SEM
11.4 2 0.6
F
11.0
F
8.0
F
11.8
F
11.7
F
8.6
F
12.1
Mean Overall
-C SEM mean
Abbreviations: a Significantly
10.5 2 0.7 k SEM M, male; different
11.0 I!T 0.5 F, female. from
0 (p < 0.05, t test).
AFTER INJECTION (MMHC)
57 5 19 18 31 6 22.7 2 7.9” 42 28 28 10 15 32 25.8 ? 4.8” 24.2 t 4.4a
Carrageenin-Induced
Paw Inflammation
mmHg at 240 min after carrageenin (Table 1). Dogs of both sexes were used for subsequent work. We also assessed whether the pressure increase was a function of the size of the dog (Table I). With data from 12 control dogs, there was no significant correlation between body weight and pressure increase for left or right paws (least-squares linear correlation coefficients of -0.467 and -0.161, respectively). Effects of NSAlDs
To characterize this canine inflammation model, we examined the effects of orally administered NSAIDs. Single doses of ibuprofen significantly (p < 0.05) inhibited paw pressure increases in a dose-dependent manner. Doses of 10, 39, 78, and 116 mg/kg significantly (p < 0.05) inhibited the 240-min pressure increase by 54,77,100, and 105%, respectively (Figure 1 and Table 2) compared to the control group (0 mg/ kg) value. The last value for ibuprofen-treated dogs indicates that their paws had volumes slightly less at 240 min than their baseline volumes at IS-min postcarrageenin. The EDs0 value estimated from these data is 9.2 mg/kg p.o. lndomethacin also significantly inhibited inflammation at 9 mg/kg p.o., but was not effective at 3
TABLE 2 Effect of Orally Administered Ibuprofen, Indomethacin, Orpanoxin, and F-1067 on Carrageenin-Induced Inflammation in the Anesthetized Dog Hindpaw Model PAW PRESSURE CHANGE TREATMENT
Methylcellulose Ibuprofen
No. OF
MC/KG
PAWS
(MEAN 2 SEM
10 mL/kg 10 39 78
29
26.0 12.0 6.0 0.0 -1.3 40.2 14.3 22.0 25.0 17.5 13.7 50.7 12.4 20.8 22.9 4.0 25.7 21.2 35.0 13.0
116
lndomethacin F-1292 Phenidone
Orpanoxin
F-l 067
AT 240 MIN
ORAL DOSE
3 9 1000 3 10 20 100 300 560 1000 15” 300 560 1000 1oa
6 5 6 4 5 6 6 6 6 6 6
a 6 8
MMHG)
2 3.5 I? 3.0b r 3.7’ ” 3.56 -t 4.8b ? 8.0 t 3.16 2 7.0 f 3.1 * 3.0 2 8.0 ” 7.3 k 4.9 5 5.1 2 4.8 * 1.5b f 3.6 2 1.5 t 11.8 % 8.0
iNHIBITION (PERCENT) 0
54 77 100 105 - 5sc 45 15 4 33 47 -9sc 52 20 12 85 18
- 3sc 50
Note: In single-dose work, the compound was administered 15 min before carrageenin injection. Paw pressure change is from the zero baseline set 15 min after carrageenin. a Administered q.i.d. for 2 days and once on the day of the test 3-4 hr before carrageenin injection. b *Significantly (p < 0.05) different from methylcellulose-treated group value (t test). C Paws at 240 min had volume slightly less than their baseline volume at 15 min postcarrageenin.
279
R. R. Brooks et al.
mg/kg, where it actually produced paw volumes 55% greater than the mean paw volume of the vehicle-treated control group (not significant). At 240”min postcarrageenin, the 9-mg/kg dose of indomethacin provided significant 45% inhibition of the paw pressure increase (Table 2). Phenidone, in contrast, did not significantly inhibit the paw pressure increase in this model at oral doses of 3, 10, and 20 mg/kg, although a trend of efficacy was indicated. At 240 min postcarrageenin, inhibition by these phenidone doses was 4%, 33%, and 47%‘ respectively (Table 2). Orpanoxin even at the high doses of 100, 300, 560, and 1000 mg/kg p.o. was ineffective on acute administration. In fact, as was seen with indomethacin at a low dose, the low dose of orpanoxin increased paw swelling (not significant). Two compounds chemically related to orpanoxin, F1067 and F-1292, also failed to prevent an increase in paw pressure on single oral administration. On multiple dosing, however, orpanoxin showed antiinflammatory activity. Orpanoxin was administered at 60 mg/kg/day (15 mg/kg q.i.d.1 for 2 days and once (15 mg/kg) on the day of testing. Orpanoxin significantly suppressed increases in paw volumes at all times from 75 to 240 min. At 240 min, the paw pressure increase in orpanoxin-treated dogs was inhibited 85% (Table 2). Although not statistically significant, F-1067 at 40 mg/kg/day did give greater (50%) inhibition than that produced by single administrations of the compound (Table 2). Activity of orpanoxin was not related to plasma levels of either orpanoxin, or its active antiinflammatory metabolite, F-1292. In the single dose experiments, plasma levels of orpanoxin 4 hr after dose increased as a function of oral dose (86.8 pg/mL after the 1000 mg/kg dose; Table 3) and were higher than the metabolite F-1292 concentrations at the upper two doses. Nevertheless, orpanoxin was not effective in preventing paw inflammation on acute administration. in the multiple oral-dose experiment, the plasma concentration of orpanoxin was less than 1 kg/mL and that of F-1292 was approximately equal to that seen after acute administration of 1000 mglkg. The antiinflammatory activity of orpanoxin after this multiple oral-dose regimen may reflect tissue, rather than plasma, concentrations.
TABLE 3 Plasma levels of Orpanoxin 1292 (4 hr after dose)
MEAN rt SEM PLASMA CONCENTRATION (~GIML)
ORPANOXINDOSE
ORPANOXIN
MC/KC P.O.
1.1
300
19.6 t
560
44.7
+ 14.1
1000
86.8
f
16.9
0.8 ‘- 0.2
75 q.i.d.” ii Administered experiment
and F-
for
2
days,
day 7-8 hr before
dose level had three
dogs.
plus
F-1292 26.9
-+ 3.9
31.4
5 2.5
36.3 ” 3.1 38.8 * once
on
blood collection.
7.5 the Each
Carrageenin-Induced
Paw Inflammation
DISCUSSION
We have demonstrated that a significant inflammatory response can be elicited in the dog paw by subcutaneous injection of the irritant, carrageenin. The inflammatory response is expressed as an increase in paw volume, which can be easily and quantitatively measured as a pressure change recorded via a water-filled balloon fixed against the paw with nonexpandable tape. In our experience, the most critical variable determining reproducibility was the manner in which the paw was wrapped. Consistency in this procedure was key to obtaining reproducible pressure increases after carrageenin. It may also be possible to follow the edema response as a change in paw volume, measured by liquid displacement. The fact that the dog is anesthetized in this procedure would certainly make paw volume measurements feasible. Use of a taped-on pressure-measuring device should also allow this model to be used with conscious dogs. Although the results with antiinflammatory drugs reported here all used oral administration of the compound, this model also lends itself to assessment of intravenous efficacy of potential antiinflammatory agents. There are similarities between rat and dog versions of the carrageenin-induced paw inflammation model. The equal efficacy of carrageenin in both species in eliciting paw swelling is one point of similarity. The amount of carrageenin injected in the dog model (-1 mg/kg) was comparable to the amounts usually used in the rat 15 mg/kg (Goldenberg et al., 1980)1, but additional work would be needed to make a valid comparison. It is likely that other irritant agents besides carrageenin, for example, kaolin, zymosan, anti-IgG, and egg albumin plus anti-egg albumin, known to induce inflammation in the rat (Gemmell et al., 19791, would also work in the dog. The time course of the increase in paw pressure is similar in the two species. In the rat, maximum edema is observed 3-5 hr after carrageenin injection (Gemmell et al., 1979). In the dog, we found that the maximum pressure increase was achieved 3-4 hr after carrageenin injection (Figure 1). In rats (Holsapple and Yim, 1984) and dogs, the edema response to carrageenin is inhibited by NSAIDs, such as ibuprofen and indomethacin. For ibuprofen, our data provide an estimated EDs0 value of 9.2 mg/kg p.o., which corresponds better with the clinical dose of 8-23 mg/kg (DiRosa et al., 1971) than does the rat-model EDso value of 78 mg/kg p.o. (Goldenberg et al., 1980). Greater sensitivity of the dog versus the rat to NSAlDs has been reported for the canine sinovitis model (GarciaRafanell and Forn, 1979). If this better correlation with clinically effective doses holds for other NSAIDs, then this would be a further advantage of the dog model over the rat model. DiRosa et al. (1971) identified three phases of the edema response in rats: an initial phase mediated by histamine and serotonin; a second phase mediated by kinins; and a third phase mediated by prostaglandins. The peak in edema was shown to correlate with the peak in accumulation of polymorphonuclear leukocytes at the site of inflammation. Yet a fourth phase has been implied by the work of Holsapple and Yim (1984) based on their finding that the Iipoxygenase inhibitor, BW755c, but not the NSAIDs, aspirin and indomethacin, was effective in reducing paw swelling in rats when administered after the onset of swelling. These results implied that a fourth phase, mediated by Iipoxygenase products, existed in the rat model. These
281
282
R. R. Brooks et al.
authors also found that BW755c was even more effective in inhibiting inflammation when administered prophylactically, perhaps reflecting the weak cyclooxygenaseinhibiting property of BW755c. Although our data suggests only weak inhibition of dog paw edema by phenidone, which is a pyrazoline derivative, such as BW755c, a more potent lipoxygenase inhibitor may be required to determine if the Iipoxygenase pathway plays a significant role in the canine response. The ability of orpanoxin to inhibit paw pressure increases in this dog model after multiple oral doses, but not after a single oral dose, may reflect both the lack of potency of orpanoxin as a cyclooxygenase inhibitor and the need to accumulate orpanoxin and/or its metabolites at the inflammation site in order to see an effect. Orpanoxin has shown antiinflammatory, analgesic, and antipyretic activities in various rodent models (Goldenberg et al., 1980; Brooks et al., 1985a) and has been shown to inhibit synthesis of prostaglandins EZ and FZa by microsomal cyclooxygenase from bovine seminal vesicles (Huang and Ellis, 1981). In the in vitro cyclooxygenase assay, however, it was about loo-fold less potent than indomethacin. In the rat carrageenin-induced paw edema model, orpanoxin was about sevenfold less potent than indomethacin (Goldenberg et al., 1980). This lack of potency may account for orpanoxin’s inactivity after a single oral dose in the dog paw edema model. The possibility that the studied compounds might have an effect on paw volume in the absence of carrageenin is unlikely. No evidence of an edemagenic action has been reported for ibuprofen, indomethacin, or phenidone in preclinical or clinical studies. Both orpanoxin and F-1067 have been subject to extensive pathology-toxicology studies, including histological examination of major tissues, in these laboratories, and have shown no evidence of edemagenic actions. Neither compound had significant hemodynamic effects at the doses studied. The increased pressure responses caused by the lowest tested doses of indomethacin and orpanoxin were not statistically significant (Table 2). The canine paw edema model described here can be performed quickly and reliably to obtain quantitative information on the activity of antiinflammatory drugs in a nonrodent species. To optimize the method, we need to perform additional work to determine if there are meaningful differences in the inflammatory mechanisms and mediators at work in the dog and rat, and to characterize the canine model’s sensitivity to existing antiinflammatory agents.
REFERENCES Andrews JP, Barry DL, Crew MC (1982) Determination of orpanoxin and selected metabolites in blood and urine. Pharmacokinetics following oral administration to dogs and rats. Fed Proc 41:1337. Blackwell GJ, Flower R (1978) I-Phenyl-3-pyrazolidone: An inhibitor of cycle-oxygenase and lipoxygenase pathways in lung and platelets. Prostaglandins 16:417-425.
Brooks RR, Bonk KR, Decker GE, Miller KE (1985a) Anti-inflammatory activity of orpanoxin administered orally and topically to rodents. AgentsActions 16 :369-376. Brooks RR, Carpenter JF, Jones SM, Ziegler TC, Pong SF (1985b) Carrageenin-induced paw edema in the dog: A new canine acute inflammation model. The Pharmacologist 27(368):177. Carlson RP, Datko LJ, Welch TM, Purvis WF, Shaw
Carrageenin-Induced GW, Thompson JL, Brunner TR (1986) An automated microcomputer-based system for determining canine paw pressure quantitatively in the dog synovitis model. I Pharmacol Methods 15:95-104.
active
anti-inflammatory
Agents
Actions
Paw Inflammation steroidal
ointments.
11:254-259.
Mathur KBL, Mehra HS (1961) Acylation of 2-furoic acid in Meerwein’s diazo reaction. / Chem Sot 1961 (Part 2):2576-2578.
DiRosa M, Giroud JP, Willoughby DA (1971) Studies of the mediators of the acute inflammatory response reduced in rats in different sites by carrageenin and turpentine. / Pathol104:15-29.
McCarty DJ, Phelps P, Pyenson J (1966) Crystal-induced inflammation in canine joins. I. An experimental model with quantification of the host response. / Exp Med 124:99-114.
Faires JS, McCarty DJ Jr (1962) Acute arthritis in man and dog after intrasynovial injection of sodium urate crystals. Lancet 2:682-683.
Peters P, Cooper C, Maiorana K, Graeme ML (1977) The effect of topically applied agents on ultraviolet erytheme in guinea pigs. Agents Actions
Garcia-Rafanell J, Forn J (1979) Correlation between antiinflammatory activity and inhibition of prostaglandin biosynthesis induced by various nonsteroidal antiinflammatory agents. Arzneimittelforschung
29:630-633.
Gemmell DK, Cottney J, Lewis AJ (1979) Comparative effects of drugs on four paw edema models in the rat. Agents Actions 9:107-116. Goldenberg MM, Boise KL, llse AC (1980) Evaluation of a new anti-inflammatory/analgesic compound F-776, 5-(4-chloropenyh-B-hydroxy-2-furanpropanoic acid. Arch Int Pharmacodyn Ther 243:331-350.
Hambelton P, Miller P (1989) Studies on carrageenin air pouch inflammation in the rat. Brl fxp Patho/70:425-433.
Holsapple MP, Yim GKW (1984) Therapeutic reduction of ongoing carrageenin-induced inflammation by lipoxygenase, but not cyclooxygenase inhibitors. Inflammation 8:23-230. Huang CT, Ellis KO (1981) Inhibition of prostaglandin synthetase by F-776, 5-(4-chlorophenyhB-hydroxy-3-furan propanoic acid, a new antiinflammatory/analgesic compound. Biochem Pharmacol30:3008-3009.
lzuka Y, Endo Y, Misawa Y, Misaka E (1981) Two simple methods for the evaluation of topically
7:545-553.
Phelps P, McCarty DJ Jr (1966) Crystal-induced inflammation in canine joints. II. Importance of polymorphonuclear leukocytes. / Exp Med 12:115-126.
Rosenthale ME, Kassarich J, Schneider F Jr (1966) Effect of anti-inflammatory agents on acute experimental synovitis in dogs. Proc Sot fxp Biol Med 122:693-696.
Short FW, Rockwood GM (1969) Synthesis and interconversion of 6-aroyl-4-oxohexanoic acids and 5-aryl-2-furanpropionic acids. Anti-inflammatory agents. / Heterocyclic Chem 6:713-722. Van Arman CC, Carlson RP, Risley EA, Thomas RH, Nuss GW (1970) Inhibitory effects of indomethacin, aspirin and certain other drugs on inflammations induced in rat and dog by carrageenan, sodium urate, and ellagic acid. / Pharmacol Exp Ther 175:459-468.
Winder CV, Wax 1, Burr V, Been M, Rosiere CE (1958) A study of pharmacological influences on ultraviolet erytheme in guinea pigs. Arch Int Pharmacodyn
Ther 116:261-292.
Winter CA, Risley EA, Nuss CV (1962) Carrageenininduced edema in the hindpaw of the rat as an assay for anti-inflammatory drugs. Proc Sot Exp Biol Med
111:544-547.
283
R. R. BROOKS, J. F. CARPENTER,S. M. JONES, T. C. ZIEGLER,AND S. F. PONG
A quantitative method for testing antiinflammatory agents in beagles has been developed, based on measurement of paw inflammation induced by a local injection of carrageenin. Carrageenin [0.5 mL of 2% (wt/vol) in saline] was injected into the plantar region of the hindpaws of pentobarbital-anesthetized beagles. Paw pressure changes registered from a water-filled balloon held on the top of the paw by a light adhesive tape wrapping were monitored for 240 min. In control dogs given 0.5% (wt/vol) methylcellulose (10 mUkg orally) just before carrageenin, paw pressure increased significantly (p < 0.05) over eightfold, from 2.9 f 0.8 mm Hg (mean % SEM, n = 29 paws) at 75 min to 26.0 + 3.5 mmHg at 240 min. The increase in paw pressure was significantly inhibited by the cyclooxygenase inhibitors, ibuprofen, indomethacin, and orpanoxin, and partially inhibited by the lipoxygenase inhibitor, phenidone, administered orally before carrageenin injection. Thus this model, with further characterization, could provide a convenient, quantitative way of assessing the efficacy of nonsteroidal antiinflammatory agents in dogs. Key Words:
Inflammation;
Carrageenin;
Ibuprofen;
Indomethacin;
Phenidone
INTRODUCTION
The search for antiinflammatory drugs has always, at some point, involved evaluation in an acute inflammation model. There are several such models using rodents, and marketed nonsteroidal antiinflammatory drugs (NSAIDs) are all active in such models. Examples include croton-oil-induced ear inflammation in mice (Izuka et al., 1981; Brooks et al., 1985a), ultraviolet light-induced skin erythema in guinea pigs (Winder et al., 1958; Peters et al., 1977), carrageenin air pouch inflammation in rats (Hambleton and Miller, 1989), and the widely used carrageenin-induced paw edema model in rats (Winter et al., 1962). Among nonrodent species, however, only knee joint synovitis in the dog has been fairly well studied. Faires and McCarty Jr, (1962) and Phelps and McCarty Jr (1966) showed that injection of sodium urate crystals into the synovial space of the dog stifle joint could induce a synovitis involving phagocytizing polymorphonuclear leukocytes. McCarty et al. (1966) ad-
From the Product Development, Norwich Eaton Pharmaceuticals, Inc., A Procter & Gamble Company, Norwich, New York. Address reprint requests to: Dr. R. R. Brooks, Product Development, Norwich Eaton Pharmaceuticals, Inc. A Proctor & Gamble Company, P. 0. Box 191, Norwich, New York 13815-0191, U.S.A. Received June 20,199O; revised and accepted December 13,199O. 275 Journal of Pharmacological
Methods
0 1991 Elsevier Science Publishing
25, 275483
0160-5402/91/S3.50
WVl)
Co., Inc., 655 Avenue of the Americas,
New York, NY 10010
276
R. R. Brooks et al.
dressed several ways of quantifying the joint response, including measurements of pressure, white blood cell accumulation, and synovial fluid pH. Rosenthale et al. (1966) showed that NSAlDs diminished symptoms (gait deficit) in this canine model. Van Arman et al. (1970), using a specially designed dog dolorimeter, showed that a good correlation existed between the efficacy of drugs in the dog synovitis model and the rat paw edema model. This model has not been widely used, most probably because of its complexity and difficulties in quantitating results, although there has been a report of using an automated microcomputer-based quantitation system (Carlson et al., 1986). We have investigated an alternative procedure in the dog. The dog is a standard species for pathology-toxicology evaluation of NSAIDs. In order to use canine toxicology data to calculate a therapeutic index, it is necessary to have a measure of efficacy in the dog. Accordingly, we have determined whether the carrageenininduced paw edema test can be adapted to the dog. Our results show that an easily quantitated edema response can be elicited in the anesthetized dog and that this response is inhibitable by NSAIDs. We have characterized the model by examining its sensitivity to orally administered strong cyclooxygenase inhibitors (ibuprofen and indomethacin), weak cyclooxygenase inhibitors [orpanoxin (Goldenberg et al., 1980)], 5-(4-chlorophenyl)-2-furanpropanoic acid [F-1067 (Short and Rockwood, 1969)], 5-(4-chlorophenyl)-2-furan-carboxylic acid IF-1292 (Mathur and Mehra, 1961)], and a mixed lipoxygenase/cyclooxygenase inhibitor [phenidone (Blackwell and Flower, 1978)J. A preliminary report of these results has appeared in the literature (Brooks et al., 1985b). MATERIALS AND METHODS Animals Male and female beagles were purchased from Marshall Farms, North Rose, New York, or were born and raised at Norwich Eaton Pharmaceuticals, Inc., Norwich, New York. At the time of use, the dogs were fasted overnight and weighed between 5.6 and 14.2 kg. Dogs were fed Lab Canine Diet 5006 (Ralston Purina Co., Richmond, Indiana) and water ad libitum. Dogs surviving at the end of the experiment were euthanized with an overdose of barbiturate anesthetic (Socumb, The Butler Co., Columbus, Ohio). Procedure Dogs were anesthetized with pentobarbital sodium, 35 mg/kg i.v. Hair was removed from both hindpaws by clipping, toenails were trimmed, and both hindpaws were lightly wrapped with nonstretching, waterproof adhesive tape (Johnson & Johnson Products, Inc., New Brunswick, New Jersey). Included in the wrap on the top of the foot were the following. 1) A tracheal tube balloon and hose were filled with water and connected to a Statham P23lD pressure transducer (Grass Instruments, Quincy, Massachusetts). Output of the transducer was recorded on a Grass Model 7B polygraph. 2) A 23-g needle inserted into the large plantar pad. After a 15-min equilibration period, 0.5 mL of 0.2% (wt/vol) carrageenin (Algin Corp. of
Carrageenin-Induced America,
New York,
New York) was injected
and the needle
Paw Inflammation
was removed.
Baseline
pressure (mmHg) was adjusted to zero at this time, again adjusted to zero 15 min later, and then was monitored for 240 min. During this time, anesthesia was maintained with pentobarbital as needed. For orpanoxin sample was collected at 240 min for determination (Andrews
experiments, a venous blood of plasma drug concentration
et al., 1982).
Dosing For acute, single-dose experiments, ibuprofen (Upjohn Company, Kalamazoo, orpanoxin, FMichigan), indomethacin (Sigma Chemical Co., St. Louis, Missouri), 1067, F-1292 (Norwich Eaton Pharmaceuticals, Inc., Norwich, New York), or phenidone
(Aldrich
Chemical
suspension in 0.5% gan) 15 min before
Co.,
Milwaukee,
Wisconsin)
was administered
orally as a
(wt/vol) methylcellulose (Dow Chemical Co., Midland, Michicarrageenin. Suspensions were prepared fresh on the day of
use. For multiple-dosing experiments, orpanoxin and F-1067 were placed in gelatin capsules and administered orally four times per day for 2 days and once on the day of the test. Carrageenin Treatment Pressure
was injected
3-4 hr later in the multiple-dose
experiments.
of Data changes
from
baseline
were
tabulated
from the polygraph
record
at 15
min intervals. The pressure change at 240 min postcarrageenin was compared between the drug-treated and methylcellulose-vehicle-treated groups by a two-tailed unpaired
t test at the 5% level of significance.
RESULTS Control
Dogs
Carrageenin injection caused an increase in paw pressure over the 240-min observation period (Figure 1). The same volume of saline alone caused no inflammation and no pressure change (data not shown). The carrageenin-induced increase was delayed in onset, ylcellulose-dosed sure increased control
remaining below 5 mmHg for the first 75 min in control, methdogs. From 75 to 240 min after carrageenin-injection, paw pres-
from 2.9 & 0.8 (mean
dog paws were
& SEM, n = 29) to 26.0
not read at every time
27-30 paws shown in Figure 1. Several experin;ents were done
to determine
period,
+ 3.5 mmHg.
accounting
the proper
Some
for the range of
dose of carrageenin
to
use in this model. The dose chosen (10 mg/dog) gave about half the maximum paw pressure increase seen in dogs injected with 40 mg of carrageenin (data not shown). Because it produced a clear and reproducible, but not a maximum pressure increase, the IO-mg/dog dose of the phlogistic agent was considered suitable for testing the effect of antiinflammatory agents on the edema response, allowing observation of both positive and negative responses. We determined the paw edema response in an initial experiment using groups of six male or female beagles that did not differ significantly in weight. Inflammatory response to carrageenin did not differ between the sexes, averaging 24.2 * 4.4
277
278
R. R. Brooks et al.
Ibuprofen
-10
i//l
L
0 '75
105
I
I
135
165
I
I
I
195
225
Time PostCarrageenin
I
I
I
(min)
FIGURE 1. Effect of a single oral dose of 0 (open circles) mg/kg methylcellulose, 10 (circles with crosses), 39 (open triangles), 78 (open squares), and 116 (open diamonds) mg/kg ibuprofen on paw swelling induced in pentobarbital-anesthetized beagles induced by subplantar injection of 0.5 ml 2% carrageenin. Test substance in methylcellulose was administered by gavage, followed 15 min later (time zero) by subplantar injection of carrageenin. Paw pressure changes are relative to a zero baseline set at 15 min after carrageenin. The number of paws is indicated by n.
TABLE 1 Pressure Increases of the Beagle Hindpaw Injected with 10 mg of Carrageenin PAW PRESSURE 240 MIN BODY WEIGHT SEX
(KC)
9.1
M M
10.7
M
11.5
M
12.9
M
13.3 11.1
M Mean
5 SEM
11.4 2 0.6
F
11.0
F
8.0
F
11.8
F
11.7
F
8.6
F
12.1
Mean Overall
-C SEM mean
Abbreviations: a Significantly
10.5 2 0.7 k SEM M, male; different
11.0 I!T 0.5 F, female. from
0 (p < 0.05, t test).
AFTER INJECTION (MMHC)
57 5 19 18 31 6 22.7 2 7.9” 42 28 28 10 15 32 25.8 ? 4.8” 24.2 t 4.4a
Carrageenin-Induced
Paw Inflammation
mmHg at 240 min after carrageenin (Table 1). Dogs of both sexes were used for subsequent work. We also assessed whether the pressure increase was a function of the size of the dog (Table I). With data from 12 control dogs, there was no significant correlation between body weight and pressure increase for left or right paws (least-squares linear correlation coefficients of -0.467 and -0.161, respectively). Effects of NSAlDs
To characterize this canine inflammation model, we examined the effects of orally administered NSAIDs. Single doses of ibuprofen significantly (p < 0.05) inhibited paw pressure increases in a dose-dependent manner. Doses of 10, 39, 78, and 116 mg/kg significantly (p < 0.05) inhibited the 240-min pressure increase by 54,77,100, and 105%, respectively (Figure 1 and Table 2) compared to the control group (0 mg/ kg) value. The last value for ibuprofen-treated dogs indicates that their paws had volumes slightly less at 240 min than their baseline volumes at IS-min postcarrageenin. The EDs0 value estimated from these data is 9.2 mg/kg p.o. lndomethacin also significantly inhibited inflammation at 9 mg/kg p.o., but was not effective at 3
TABLE 2 Effect of Orally Administered Ibuprofen, Indomethacin, Orpanoxin, and F-1067 on Carrageenin-Induced Inflammation in the Anesthetized Dog Hindpaw Model PAW PRESSURE CHANGE TREATMENT
Methylcellulose Ibuprofen
No. OF
MC/KG
PAWS
(MEAN 2 SEM
10 mL/kg 10 39 78
29
26.0 12.0 6.0 0.0 -1.3 40.2 14.3 22.0 25.0 17.5 13.7 50.7 12.4 20.8 22.9 4.0 25.7 21.2 35.0 13.0
116
lndomethacin F-1292 Phenidone
Orpanoxin
F-l 067
AT 240 MIN
ORAL DOSE
3 9 1000 3 10 20 100 300 560 1000 15” 300 560 1000 1oa
6 5 6 4 5 6 6 6 6 6 6
a 6 8
MMHG)
2 3.5 I? 3.0b r 3.7’ ” 3.56 -t 4.8b ? 8.0 t 3.16 2 7.0 f 3.1 * 3.0 2 8.0 ” 7.3 k 4.9 5 5.1 2 4.8 * 1.5b f 3.6 2 1.5 t 11.8 % 8.0
iNHIBITION (PERCENT) 0
54 77 100 105 - 5sc 45 15 4 33 47 -9sc 52 20 12 85 18
- 3sc 50
Note: In single-dose work, the compound was administered 15 min before carrageenin injection. Paw pressure change is from the zero baseline set 15 min after carrageenin. a Administered q.i.d. for 2 days and once on the day of the test 3-4 hr before carrageenin injection. b *Significantly (p < 0.05) different from methylcellulose-treated group value (t test). C Paws at 240 min had volume slightly less than their baseline volume at 15 min postcarrageenin.
279
R. R. Brooks et al.
mg/kg, where it actually produced paw volumes 55% greater than the mean paw volume of the vehicle-treated control group (not significant). At 240”min postcarrageenin, the 9-mg/kg dose of indomethacin provided significant 45% inhibition of the paw pressure increase (Table 2). Phenidone, in contrast, did not significantly inhibit the paw pressure increase in this model at oral doses of 3, 10, and 20 mg/kg, although a trend of efficacy was indicated. At 240 min postcarrageenin, inhibition by these phenidone doses was 4%, 33%, and 47%‘ respectively (Table 2). Orpanoxin even at the high doses of 100, 300, 560, and 1000 mg/kg p.o. was ineffective on acute administration. In fact, as was seen with indomethacin at a low dose, the low dose of orpanoxin increased paw swelling (not significant). Two compounds chemically related to orpanoxin, F1067 and F-1292, also failed to prevent an increase in paw pressure on single oral administration. On multiple dosing, however, orpanoxin showed antiinflammatory activity. Orpanoxin was administered at 60 mg/kg/day (15 mg/kg q.i.d.1 for 2 days and once (15 mg/kg) on the day of testing. Orpanoxin significantly suppressed increases in paw volumes at all times from 75 to 240 min. At 240 min, the paw pressure increase in orpanoxin-treated dogs was inhibited 85% (Table 2). Although not statistically significant, F-1067 at 40 mg/kg/day did give greater (50%) inhibition than that produced by single administrations of the compound (Table 2). Activity of orpanoxin was not related to plasma levels of either orpanoxin, or its active antiinflammatory metabolite, F-1292. In the single dose experiments, plasma levels of orpanoxin 4 hr after dose increased as a function of oral dose (86.8 pg/mL after the 1000 mg/kg dose; Table 3) and were higher than the metabolite F-1292 concentrations at the upper two doses. Nevertheless, orpanoxin was not effective in preventing paw inflammation on acute administration. in the multiple oral-dose experiment, the plasma concentration of orpanoxin was less than 1 kg/mL and that of F-1292 was approximately equal to that seen after acute administration of 1000 mglkg. The antiinflammatory activity of orpanoxin after this multiple oral-dose regimen may reflect tissue, rather than plasma, concentrations.
TABLE 3 Plasma levels of Orpanoxin 1292 (4 hr after dose)
MEAN rt SEM PLASMA CONCENTRATION (~GIML)
ORPANOXINDOSE
ORPANOXIN
MC/KC P.O.
1.1
300
19.6 t
560
44.7
+ 14.1
1000
86.8
f
16.9
0.8 ‘- 0.2
75 q.i.d.” ii Administered experiment
and F-
for
2
days,
day 7-8 hr before
dose level had three
dogs.
plus
F-1292 26.9
-+ 3.9
31.4
5 2.5
36.3 ” 3.1 38.8 * once
on
blood collection.
7.5 the Each
Carrageenin-Induced
Paw Inflammation
DISCUSSION
We have demonstrated that a significant inflammatory response can be elicited in the dog paw by subcutaneous injection of the irritant, carrageenin. The inflammatory response is expressed as an increase in paw volume, which can be easily and quantitatively measured as a pressure change recorded via a water-filled balloon fixed against the paw with nonexpandable tape. In our experience, the most critical variable determining reproducibility was the manner in which the paw was wrapped. Consistency in this procedure was key to obtaining reproducible pressure increases after carrageenin. It may also be possible to follow the edema response as a change in paw volume, measured by liquid displacement. The fact that the dog is anesthetized in this procedure would certainly make paw volume measurements feasible. Use of a taped-on pressure-measuring device should also allow this model to be used with conscious dogs. Although the results with antiinflammatory drugs reported here all used oral administration of the compound, this model also lends itself to assessment of intravenous efficacy of potential antiinflammatory agents. There are similarities between rat and dog versions of the carrageenin-induced paw inflammation model. The equal efficacy of carrageenin in both species in eliciting paw swelling is one point of similarity. The amount of carrageenin injected in the dog model (-1 mg/kg) was comparable to the amounts usually used in the rat 15 mg/kg (Goldenberg et al., 1980)1, but additional work would be needed to make a valid comparison. It is likely that other irritant agents besides carrageenin, for example, kaolin, zymosan, anti-IgG, and egg albumin plus anti-egg albumin, known to induce inflammation in the rat (Gemmell et al., 19791, would also work in the dog. The time course of the increase in paw pressure is similar in the two species. In the rat, maximum edema is observed 3-5 hr after carrageenin injection (Gemmell et al., 1979). In the dog, we found that the maximum pressure increase was achieved 3-4 hr after carrageenin injection (Figure 1). In rats (Holsapple and Yim, 1984) and dogs, the edema response to carrageenin is inhibited by NSAIDs, such as ibuprofen and indomethacin. For ibuprofen, our data provide an estimated EDs0 value of 9.2 mg/kg p.o., which corresponds better with the clinical dose of 8-23 mg/kg (DiRosa et al., 1971) than does the rat-model EDso value of 78 mg/kg p.o. (Goldenberg et al., 1980). Greater sensitivity of the dog versus the rat to NSAlDs has been reported for the canine sinovitis model (GarciaRafanell and Forn, 1979). If this better correlation with clinically effective doses holds for other NSAIDs, then this would be a further advantage of the dog model over the rat model. DiRosa et al. (1971) identified three phases of the edema response in rats: an initial phase mediated by histamine and serotonin; a second phase mediated by kinins; and a third phase mediated by prostaglandins. The peak in edema was shown to correlate with the peak in accumulation of polymorphonuclear leukocytes at the site of inflammation. Yet a fourth phase has been implied by the work of Holsapple and Yim (1984) based on their finding that the Iipoxygenase inhibitor, BW755c, but not the NSAIDs, aspirin and indomethacin, was effective in reducing paw swelling in rats when administered after the onset of swelling. These results implied that a fourth phase, mediated by Iipoxygenase products, existed in the rat model. These
281
282
R. R. Brooks et al.
authors also found that BW755c was even more effective in inhibiting inflammation when administered prophylactically, perhaps reflecting the weak cyclooxygenaseinhibiting property of BW755c. Although our data suggests only weak inhibition of dog paw edema by phenidone, which is a pyrazoline derivative, such as BW755c, a more potent lipoxygenase inhibitor may be required to determine if the Iipoxygenase pathway plays a significant role in the canine response. The ability of orpanoxin to inhibit paw pressure increases in this dog model after multiple oral doses, but not after a single oral dose, may reflect both the lack of potency of orpanoxin as a cyclooxygenase inhibitor and the need to accumulate orpanoxin and/or its metabolites at the inflammation site in order to see an effect. Orpanoxin has shown antiinflammatory, analgesic, and antipyretic activities in various rodent models (Goldenberg et al., 1980; Brooks et al., 1985a) and has been shown to inhibit synthesis of prostaglandins EZ and FZa by microsomal cyclooxygenase from bovine seminal vesicles (Huang and Ellis, 1981). In the in vitro cyclooxygenase assay, however, it was about loo-fold less potent than indomethacin. In the rat carrageenin-induced paw edema model, orpanoxin was about sevenfold less potent than indomethacin (Goldenberg et al., 1980). This lack of potency may account for orpanoxin’s inactivity after a single oral dose in the dog paw edema model. The possibility that the studied compounds might have an effect on paw volume in the absence of carrageenin is unlikely. No evidence of an edemagenic action has been reported for ibuprofen, indomethacin, or phenidone in preclinical or clinical studies. Both orpanoxin and F-1067 have been subject to extensive pathology-toxicology studies, including histological examination of major tissues, in these laboratories, and have shown no evidence of edemagenic actions. Neither compound had significant hemodynamic effects at the doses studied. The increased pressure responses caused by the lowest tested doses of indomethacin and orpanoxin were not statistically significant (Table 2). The canine paw edema model described here can be performed quickly and reliably to obtain quantitative information on the activity of antiinflammatory drugs in a nonrodent species. To optimize the method, we need to perform additional work to determine if there are meaningful differences in the inflammatory mechanisms and mediators at work in the dog and rat, and to characterize the canine model’s sensitivity to existing antiinflammatory agents.
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