Preclinical Toxicity Evaluation of Tepoxalin, a Dual Inhibitor of Cyclooxygenase and 5-Lipoxygenase, in Sprague–Dawley Rats and Beagle Dogs

FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.

33, 38–48 (1996)

0141

Preclinical Toxicity Evaluation of Tepoxalin, a Dual Inhibitor of Cyclooxygenase and 5-Lipoxygenase, in Sprague–Dawley Rats and Beagle Dogs1 E. V. KNIGHT,2 J. P. KIMBALL, C. M. KEENAN, I. L. SMITH,* F. A. WONG,* D. S. BARRETT, A. M. DEMPSTER, W. G. LIEUALLEN, D. PANIGRAHI, W. J. POWERS, AND R. J. SZOT Departments of Drug Safety Evaluation and *Drug Metabolism, The R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869–0602 Received October 10, 1995; accepted April 26, 1996

tinal side effects, within the estimated therapeutic dose range, distinguishes tepoxalin from most marketed anti-inflammatory drugs.

Preclinical Toxicity Evaluation of Tepoxalin, a Dual Inhibitor of Cyclooxygenase and 5-Lipoxygenase, in Sprague–Dawley Rats and Beagle Dogs. KNIGHT, E. V., KIMBALL, J. P., KEENAN, C. M., SMITH, I. L., WONG, F. A., BARRETT, D. S., DEMPSTER, A. M., LIEUALLEN, W. G., PANIGRAHI, D., POWERS, W. J., AND SZOT, R. J. (1996). Fundam. Appl. Toxicol. 33, 38–48.

q 1996 Society of Toxicology

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of therapeutic compounds that are widely used to prevent inflammation (Xie et al., 1992; Greaves, 1987). Gastrointestinal toxicity is a common adverse effect of NSAIDs, causing symptoms of gastrointestinal bleeding, erosions, and ulcers (Schoen and Vender, 1989; Khokhar, 1984), in addition to nephrotoxicity and cutaneous reactions. It is also known that NSAID sensitivity to gastrointestinal effects differs between species (i.e., dog ú rat ú monkey), at comparable dose level and duration of treatment (Brooks et al., 1993). The ability of NSAIDs to inhibit prostaglandin synthesis, via the cyclooxygenase (CO)-dependent pathway, was suggested as the underlying mechanism for both the anti-inflammatory effects and the gastrointestinal ulceration (Vane, 1971; Gyires, 1994). In some inflammatory diseases, such as rheumatoid arthritis, significant concentrations of the 5lipoxygenase (LO) pathway metabolites, 5-s-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B4 (LTB4), have been detected at the site of inflammation (Weinblatt et al., 1992). The failure of most NSAIDs to block the production of LO-derived inflammatory mediators may account for their limited efficacy seen in diseases such as rheumatoid arthritis as well as their side effects. By virtue of its dual CO and LO inhibitory activity, tepoxalin is distinguished from most marketed NSAIDs, which are pure CO inhibitors (Wallace et al., 1993). Similar to NSAIDs, tepoxalin inhibits prostaglandin, thromboxane, and prostacyclin production and, thus, has anti-inflammatory properties (Anderson et al., 1990). Moreover, tepoxalin’s inhibition of production of LTB4 not only contributes to its anti-inflammatory activity, but may prevent further joint destruction in rheumatoid arthritis models and lessen the gastrointestinal side effects associated with gastric ulceration within the therapeutic dose range (Wallace et al., 1993; An-

Tepoxalin [5-(4-chlorophenyl)-N-hydroxy-1-(4-methoxyphenyl)N-methyl-1H-pyrazole-3-propanamide] is an orally active anti-inflammatory agent, which inhibits both cyclooxygenase and 5-lipoxygenase activities. The oral toxicity of tepoxalin was evaluated in 1and 6-month rat (up to 50 mg/kg/day) and dog (up to 150 mg/kg bid) studies. In rats, increased liver weight, centrilobular hypertrophy, and hepatic necrosis were observed at dosages §20 mg/kg/day. Renal changes indicative of analgesic nephropathy syndrome (i.e., papillary edema or necrosis, cortical tubular dilatation) were seen at §15 mg/ kg. In rats treated for 1 month, these hepatic and renal effects were largely reversible after a 1-month recovery period. Gastrointestinal erosions and ulcers were seen in female rats given 40 mg/kg/day for 6 months. Changes in clinical pathology parameters included decreases in red blood cell count, hemoglobin, and hematocrit mean values; elevation in platelet counts; and an increase in prothrombin and activated partial thromboplastin times. Mild increases in alanine aminotransferase, aspartate aminotransferase, and cholesterol were also noted in rats. Decreased erythrocyte parameters, increased leukocyte counts, and decreased total protein, albumin, and/or calcium were noted in some dogs in the 300 mg/kg/day group following 6 months of dosing. Small pyloric ulcerations were seen at 100 and 300 mg/kg/day dosages for up to 6 months. In both rats and dogs, no accumulation of tepoxalin or its carboxylic acid metabolite was detected in plasma following multiple dosing over a range of 5 to 50 mg/kg/day for rats and 20 to 300 mg/kg/day for dogs. Plasma concentrations of the carboxylic acid metabolite were severalfold higher than those of the parent compound. The no-effect dosages in rats (5 mg/kg/day) and dogs (20 mg/kg/day) were approximately one and six times the ED50 (3.5 mg/kg), respectively, for inhibition of inflammatory effects in the adjuvant arthritic rat without gastric mucosal damage. In terms of severity, the relative lack of gastrointes1 Presented in part at the 34th annual meeting of the Society of Toxicology, Baltimore, MD, March 5–9, 1995 (Toxicologist 15, 370, 1995). 2 To whom correspondence should be addressed.

0272-0590/96 $18.00 Copyright q 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.

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FIG. 1. Structures of (a) tepoxalin and (b) carboxylic acid metabolite.

derson et al., 1990). Tepoxalin’s inhibition of CO and LO has been shown in several in vitro and ex vivo assays. Tepoxalin produced dose-related inhibition of the edematous response in the adjuvant arthritic rat assay (ED50 Å 3.5 mg/ kg, po), a model predictive of clinical efficacy (Argentieri et al., 1990). Tepoxalin also inhibited edema influx of inflammatory cells into the knee joint space of arthritic rabbits and dogs with a corresponding reduction of LTB4 production in synovial fluid (Argentieri et al., 1990). This report describes the oral toxicity of tepoxalin in 1and 6-month studies in Sprague–Dawley rats (up to 50 mg/ kg/day) and beagle dogs (up to 150 mg/kg bid). The relative effects of tepoxalin on clinical signs, pharmacokinetics, and hematological, uroanalytical, biochemical, and histopathological findings are presented for these two species. METHODS Test and control materials. Tepoxalin, 3-[5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-pyrazolyl]-N-hydroxy-N-methylpropanamide, and 0.5% hydroxypropyl methylcellulose (HPMC) Premium F4M dosage forms were prepared by The R. W. Johnson Pharmaceutical Research Institute (Raritan, NJ). The chemical structures of tepoxalin and its carboxylic acid hydrolysis product, 3-[5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-pyrazolyl]propanoic acid, which will be referred to as the acid metabolite, are shown in Fig. 1. The test and control articles were assayed for verification of concentration and identity, stability, absence of test article in control article, content, uniformity, density, pH, and particle size and were determined to be acceptable for use in nonclinical studies. The test article was micronized (particle size of õ2.9 mm) and prepared in 0.5% HPMC Premium F4M at concentrations necessary for delivery of appropriate dosages in a volume of 10 ml/kg (rat) or 1–2 ml/kg (dog) body wt. Dose volumes were adjusted weekly for changes in body weight. Rats. Male and female Crl:CD (SD)BR (VAF) rats, approximately 8 weeks of age at initiation of dosing, and weighing 151–257 g (females) and 242–373 g (males), were obtained from Charles River Laboratories, Inc. (Kingston, NY). All rats were housed individually in stainless-steel cages. The room was maintained at a temperature of 64–777F with a relative humidity of 30–73%, and had a 12-hr light/dark cycle. Rats were used in accordance with USDA guidelines for humane care. Food (Purina Certified Rodent Chow Meal No. 5002; Purina Mills Inc., St Louis, MO) and water were given ad libitum. Dogs. Immunized male and female beagle dogs, 7–9 months old at initiation of dosing and weighing 6.3–9.5 kg, were obtained from Marshall Farms (North Rose, NY). The dogs were individually housed in climatecontrolled indoor runs with a 12-hr light/dark cycle. All dogs had water ad libitum and were fed a measured amount of Purina Certified Canine Chow 5007 (Purina Mills, Inc.) 1–2 hr after the second daily dose.

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Study Design Rats. Rats were dosed orally daily with tepoxalin for 1 month at dosages of 0 (vehicle control), 15, 25, 35 and 50 mg/kg and for 6 months (with a 3-month interim euthanasia) at 0, 5, 10, 20, and 40 mg/kg. In the 1-month study, 10 rats/sex/group were dosed except for the control and 50 mg/kg groups, which had 15/sex/group. The additional 5 rats/sex from the control and 50 mg/kg groups were maintained for a 1-month recovery period. Ancillary (6 rats/sex) groups were used for evaluation of plasma concentrations of tepoxalin and its carboxylic acid metabolite on Days 1 and 28 of the study. In the 6-month study with the 3-month interim euthanasia), the initial 25 rats/sex/group was reduced to 15 rats/sex/group at 3 months from early mortality or necropsied at 3 months of dosing. Ancillary (3 rats/sex) groups were included for evaluation of parent drug and its carboxylic acid metabolite on Days 1, 92, and 184 of study. All remaining rats were necropsied after 6 months of dosing. Dogs. From an initial pilot study, it was observed that there was a plateau in plasma concentrations for both tepoxalin and the acid metabolite above a single dose of 200 mg/kg. In addition, at dosages below 200 mg/ kg, plasma concentrations did not increase proportionally with increasing dose, indicating that the absorption of tepoxalin in dogs was limited from oral administration. Consequently, dogs were dosed orally twice daily to maximize exposure during the 1-month study and during the 6-month study with a 3-month interim euthanasia. Dosages for both studies were 0 (vehicle control), 10, 50, and 150 mg/kg/dose for totals of 0, 20, 100, and 300 mg/ kg/day, respectively. In the 1-month study, the control and 300 mg/kg/day groups consisted of 5 dogs/sex/group, and the other two groups (20 and 100 mg/kg/day) consisted of 3 dogs/sex/group. The additional 2 dogs/sex/ group in the control and 300 mg/kg/day groups were maintained for a 1month recovery period. Plasma concentrations of the parent compound and its acid metabolite were determined on Days 1 and 22 of the study. In the 6-month study (4/sex/group) with a 3-month interim euthanasia (3/sex/ group), plasma concentrations of tepoxalin and its acid metabolite were determined on Days 1, 80, and 179. For all rat and dog studies, clinical signs of toxicity, body weights, and food consumption were determined at scheduled intervals throughout the study. All animals were observed daily for survival. Ophthalmoscopic examinations were performed predose and at termination of dosing in the 1-month rat and predose, prior to the interim euthanasia, and prior to termination in the 6-month rat study with 3-month interim euthanasia. Clinical pathology specimens were collected prior to termination of dosing and after the recovery period for the 1-month rat study. In the 6-month rat study, specimens (blood or urine) were collected from 20 rats/ sex/group prior to the 3-month interim euthanasia, and from all survivors at study termination for hematology, coagulation, clinical chemistry, and urinalysis measurements. In the dog studies, electrocardiographic, ophthalmoscopic, and physical examinations were performed on all dogs predose and prior to termination of dosing for the 1-month study and predose, prior to interim euthanasia, and prior to termination for the 6-month study with the 3-month interim euthanasia. Clinical pathology specimens were collected for the evaluation

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of hematology,3 coagulation,4 clinical chemistry,5 and urinalysis6 parameters predose and prior to termination of dosing and recovery periods for the 1month study. For the 6-month study, specimens were collected predose and at 1.5, 3, and 6 months. Blood was collected from the retroorbital sinus of fasted rats under CO2:O2 (70:30% v/v) anesthesia or from the jugular vein of fasted awake dogs at periodic intervals prior to, during, or postdosing for clinical pathology analyses. Urine specimens were collected overnight in the presence of thymol preservative. Clinical pathology. Hematology, coagulation, and clinical chemistry parameters were analyzed by routine laboratory procedures and included differential (percent and absolute), hematocrit (HCT), hemoglobin (HB), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), platelet count (PLT), red blood cell count (RBC), white blood cell count (WBC), activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen (only in 6-month dog study), alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (T. Bili), calcium (Ca), chloride (Cl), cholesterol (CHOL), creatinine (CREAT), g-glutamyltransferase (GGT), glucose, phosphorus (P), potassium (K), sodium (Na), thyroxine (T4), total protein (T. PROT), triglycerides (TRIG), urea nitrogen (UREA N), and uric acid ([UA] only in 6-month dog study). At study termination, rats were euthanized by carbon dioxide asphyxiation and dogs were exsanguinated while under barbiturate (thiamylal sodium) anesthesia. Complete necropsies were performed on all animals. Absolute organ weights were recorded for adrenal glands, brain, heart, kidneys, liver, ovaries, pituitary, testes, thyroid/parathyroid and prostate/ seminal vesicles (dogs). All collected tissues were fixed in 10% neutral buffered formalin. Tissues to be examined were embedded in paraffin, sectioned, and stained with hematoxylin–eosin–phloxine. The following tissues were examined microscopically in all control and high-dosage groups with the exception of the 1-month dog study with 1-month recovery in which all animals were examined: adrenal glands, aorta (thoracic), bone (femur/stifle), bone marrow (femur/stifle), brain, epididymis, esophagus, eyes, gall bladder (dog), heart, intestine (duodenum, jejunum, ileum, cecum, colon) kidneys, lacrimal gland, liver, lung, lymph node (mesenteric and mandibular), mammary gland (inguinal), sciatic nerve, ovary, pancreas, parathyroid gland, pituitary gland, prostate, salivary gland (submaxillary), skeletal muscle, skin (inguinal), spinal cord (thoracic), spleen, stomach, testis, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, and vagina. In addition, selected organs (kidney, liver, prostate, and stomach) were examined in all remaining animals. Plasma Drug/Metabolite Concentration Determinations Plasma concentrations of both tepoxalin and its acid metabolite were determined during the 1- and 6-month dog and rat studies. In the rat studies, blood was collected into heparinized syringes approximately 2 hr after dosing from 3 rats/sex/dosage. The collections were obtained from the inferior vena cava using ether anesthesia and the blood was transferred to microcentrifuge tubes. In the dog studies, 5-ml blood samples were collected in heparinized tubes from the jugular vein immediately prior to (0 hr) and approximately 1, 2, 4.5, 6, 7, and 24 hr following the first of the two daily doses which were separated by a 5-hr interval. During analytical method

3

Technicon H-1E Hematology Analyzer, Miles Inc. Diagnostics Division, Tarrytown, NY. 4 ACL-300 Plus Coagulation Analyzer, Instrumentation Laboratories, Lexington, MA. 5 Hitachi 717 Chemistry Analyzer, Boehringer Mannheim Corp., Indianapolis, IN. 6 Ames Clinitek 200 Urine Chemistry Analyzer, Miles Inc., Diagnostics Division, Elkhart, IN.

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validation, tepoxalin was found to be less stable in rat plasma than in dog plasma. Thus, samples were processed for analysis immediately on collection from rats. For dogs, the plasma was stored at 0207C prior to analysis. Plasma concentrations of both tepoxalin and its acid metabolite were determined using either of two validated high-performance liquid chromatographic (HPLC) methods. A single liquid–liquid extraction with methylene chloride was used to isolate the analytes from plasma samples. The HPLC system consisted of a Hitachi (Danbury, CT) pump and a Shimadzu (Columbia, MD) autosampler. In the rat studies, detection was accomplished with a MacPherson (Acton, MA) 7750 fluorescence detector equipped with a high-sensitivity attachment at 290 nm (excitation) and 420 nm (emission). In the dog studies, detection was accomplished with a Shimadzu SPD-10A UV–Vis detector at 254 nm. Data were integrated using a HP 3350A Laboratory Automation System (Hewlett Packard, Avondale, PA). A 10-mm Inertsil (Keystone, Bellefonte, PA) C18 analytical column (4.6 1 15 mm) was used to separate the analytes from endogenous coextractants. The compounds were eluted isocratically at ambient temperature with a mobile phase consisting of 0.01 M 1-octanesulfonic acid buffer (pH adjusted to 5.0 with phosphoric acid):acetonitrile:methanol:tetrahydrofuran (43:17:20:20, v:v:v:v). The flow rate was 0.9 ml/min. Using this procedure, the retention times of tepoxalin and its acid metabolite were 5.4 and 3.8 min, respectively. The detection limit for both compounds was 0.25 mg/ml. Data Analyses All quantitative data were checked for aberrant values. A test to determine variance homogeneity was performed. If the data passed that test, then one-way analysis of variance was used to assess overall differences among group means. If overall differences were indicated, Dunnett’s comparison to control was performed (Dunnett, 1964). The Jonckheere – Terpstra test was performed to test for an increasing/decreasing trend in response with increased dose. All tests were conducted at the 1 and 5% two-sided risk levels. Incidence tables were generated to summarize qualitative data (clinical observations and clinical pathology, gross pathology, and histopathology).

RESULTS

Mortality and Clinical Observations In the 1-month rat study, one treatment-related death occurred (Day 22) in the 50 mg/kg group intended for the measurement of plasma concentration. One death occurred in the 15 mg/kg group 1-month rat study, and 12 deaths occurred across all dosage groups in the 6-month rat study. These deaths were attributed to either dosing or blood collection accidents and were not considered to be drug related. No deaths occurred in the dog studies. In the rat and dog studies, there were no drug-related changes in body weight, food consumption, or ophthalmoscopic and electrocardiographic (dog only) examinations; however, administration of tepoxalin to beagle dogs resulted in whitish to yellowish discoloration of the feces mainly in the 100 and 300 mg/kg/day groups (at 3 months) and mainly in the 300 mg/kg/day group (at 6 months) which was attributed to unabsorbed drug. Clinical Pathology Rat studies. The effect of tepoxalin administration after 1 or 6 months of treatment on the clinical pathology parame-

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TABLE 1 Selected Hematology and Clinical Chemistry Parameters (Mean { SE) for Rats Receiving Tepoxalin for 1 Montha Treatment Monthb RBC (11012/liter) Males Females HB (g/dl) Males Females HCT (%) Males Females CHOL (mg/dl) Males Females

a b

Control

15 mg/kg

25 mg/kg

35 mg/kg

50 mg/kg

1 2 (Recovery) 1 2 (Recovery)

7.82 8.05 7.52 7.54

{ { { {

0.09 0.21 0.13 0.15

7.83 { 0.15 NA 7.33 { 0.18 NA

7.60 { 0.14 NA 7.22 { 0.08 NA

7.52 { 0.12 NA 7.03 { 0.07** NA

7.52 8.05 6.88 7.98

{ { { {

0.11 0.21 0.09** 0.10*

1 2 (Recovery) 1 2 (Recovery)

16.39 16.15 16.17 15.97

{ { { {

0.21 0.34 0.24 0.07

16.40 { 0.28 NA 15.80 { 0.16 NA

16.05 { 0.23 NA 15.38 { 0.12 NA

15.91 { 0.17 NA 15.07 { 0.15** NA

16.02 16.15 14.90 16.34

{ { { {

0.16 0.34 0.26** 0.16

1 2 (Recovery) 1 2 (Recovery)

50.90 49.93 49.88 49.50

{ { { {

0.76 0.82 0.78 0.75

50.53 { 0.71 NA 48.82 { 0.57 NA

49.23 { 0.61 NA 47.99 { 0.41 NA

49.38 { 0.51 NA 47.07 { 0.38** NA

48.74 49.78 46.47 51.10

{ { { {

0.46 0.55 0.70** 0.37

1 2 (Recovery) 1 2 (Recovery)

56.70 70.40 70.60 80.20

{ { { {

2.80 5.80 4.20 8.40

67.50 { 3.50 NA 73.80 { 3.30 NA

65.70 { 5.00 NA 88.90 { 5.70* NA

67.20 { 4.70 NA 105.40 { 6.10** NA

73.10 57.60 99.90 69.60

{ { { {

4.10 4.60 4.70** 4.90

Significant difference from control (Dunnett’s test): *p £ 0.05, **p £0.01. n Å 10/sex at 1 month; n Å 5/sex at 2 months (Recovery).

ters for rats included decreases for RBC, hemoglobin, and hematocrit mean values and increased cholesterol mean values in the 35 and 50 mg/kg dosage groups after 1 month of dosing (Table 1). These changes were not observed in rats from the 50 mg/kg recovery group at the end of the 1month recovery period. Cholesterol values for the 50 mg/kg recovery group returned to normal values comparable to those of controls. The apparent decrease (Table 1) compared with controls was attributed to mild elevation for single male and female control rats. In the 6-month (with a 3-month interim) rat study, decreases in mean RBC, HB, and HCT values for females at the interim and at termination and an increase in prothrombin and activated partial thromboplastin times for males at termination were observed (Table 2). Although group means were comparable, elevations in platelet counts for some individual female rats were noted at the interim and at termination in the 10, 20, and 40 mg/kg groups. Slight increases in alanine aminotransferase, aspartate aminotransferase, and cholesterol occurred in some individual female rats in the 20 and 40 mg/kg groups. Urinalysis revealed an increase in urinary protein content for some individual 20 mg/kg females and for rats of both sexes in the 40 mg/kg group at the 6-month scheduled termination (Table 3). Dog studies. No drug-related changes were observed in clinical pathology parameters in the 1-month dog study. In the 6-month dog study, treatment with tepoxalin resulted in

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two females in the 300 mg/kg/day group with mild decreases in RBC, HB, and HCT values and increases in WBC and neutrophil counts after 6 months of treatment (Table 4). These female dogs also had mild to moderate decreases in serum total protein, albumin, and/or Ca values at Week 6 and 3 and 6 months which were considered to be related to drug treatment (Table 5). No urinalysis parameters were affected by tepoxalin administration in either the 1- or 6month dog studies. Drug Absorption In mammals including rats and dogs, tepoxalin undergoes both rapid and extensive in vivo conversion to its carboxylic acid hydrolysis product (see Fig. 1). Consequently, for both of these species, plasma concentrations of the acid metabolite were substantially higher and remained in the systemic circulation for a longer period than the parent compound. As might be expected for a compound metabolized in this fashion, intrasubject variability was high and, due to the limited number of animals sampled in these studies, data from female and male groups were combined. In both the 1- and 6-month studies, plasma tepoxalin and acid metabolite concentrations at the completion of the studies appeared to be similar to those seen on the first day of dosing across the dosage range evaluated. Therefore, representative kinetic data from these studies are reported to show the exposure of the animals to tepoxalin and metabolite. Although the

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TABLE 2 Selected Hematology Parameters (Mean { SE) for Rats Receiving Tepoxalin for 3 and 6 Monthsa Treatment Monthb RBC (11012/liter) Males Females HB (g/dl) Males Females HCT (%) Males Females PT (sec) Males Females APTT (sec) Males Females

a b

Control

5 mg/kg

10 mg/kg

3 (Interim) 6 3 (Interim) 6

8.09 8.73 7.90 8.12

{ { { {

0.09 0.11 0.13 0.13

8.52 8.72 7.90 8.12

{ { { {

0.09** 0.14 0.13 0.12

3 (Interim) 6 3 (Interim) 6

14.97 15.78 15.49 15.69

{ { { {

0.13 0.15 0.20 0.24

15.44 15.44 15.30 15.73

{ { { {

3 (Interim) 6 3 (Interim) 6

45.21 47.89 47.59 47.43

{ { { {

0.49 0.46 0.60 0.74

46.08 46.77 46.99 47.99

3 (Interim) 6 3 (Interim) 6

15.75 13.53 12.81 12.59

{ { { {

0.66 0.19 0.38 0.20

3 (Interim) 6 3 (Interim) 6

19.44 14.20 15.81 13.22

{ { { {

1.05 0.46 0.65 0.34

20 mg/kg

40 mg/kg

8.36 8.96 7.79 8.04

{ { { {

0.12 0.14 0.06 0.10

8.26 8.62 7.76 7.89

{ { { {

0.07 0.14 0.08 0.16

8.35 8.65 7.47 7.60

{ { { {

0.09 0.12 0.09** 0.17*

0.17 0.18 0.15 0.17

15.30 16.01 15.29 15.41

{ { { {

0.15 0.20 0.10 0.20

15.40 15.84 15.30 15.18

{ { { {

0.13 0.18 0.14 0.28

15.34 15.49 14.80 14.54

{ { { {

0.13 0.17 0.20* 0.30**

{ { { {

0.52 0.51 0.51 0.55

45.50 48.07 47.05 46.67

{ { { {

0.54 0.51 0.30 0.60

46.20 47.18 47.11 45.81

{ { { {

0.48 0.56 0.45 0.79

45.98 46.42 45.69 44.52

{ { { {

0.37 0.60 0.60 0.89*

14.67 13.45 13.85 12.72

{ { { {

0.60 0.19 1.26 0.18

16.29 13.71 13.00 12.48

{ { { {

0.86 0.22 0.48 0.23

15.60 13.99 13.07 12.23

{ { { {

0.93 0.34 0.43 0.13

16.95 14.94 12.21 11.85

{ { { {

1.03 0.29** 0.18 0.19*

18.38 13.38 18.16 12.48

{ { { {

1.17 0.45 2.86 0.32

19.06 15.08 15.73 12.92

{ { { {

1.09 0.61 0.95 0.40

18.61 14.95 16.16 13.11

{ { { {

0.84 0.65 0.72 0.37

19.97 16.42 15.67 12.98

{ { { {

1.12 0.32** 0.50 0.36

Significant difference from control (Dunnett’s test): * p £ 0.05, **p £ 0.01. n Å 20/sex at 3 months (interim); n Å 15/sex at 6 months.

absolute bioavailability of tepoxalin was not determined, this suggests that the absorption of tepoxalin from oral dosing remains constant over time (for at least out to 1 year of dosing; unpublished findings). Rat studies. Plasma concentration data for the 6-month study are indicated in Table 6. Mean plasma tepoxalin concentrations 2 hr after dosing were below the limits of quantification (0.25 mg/ml) in the 5 and 10 mg/kg dosage groups and did not exceed 0.81 mg/ml at 40 mg/kg. Mean plasma concentrations of the acid metabolite were detectable at all dosage levels and ranged from 1.39 { 0.49 to 10.70 { 3.68 mg/ml. Dog studies. Oral administration of tepoxalin to dogs resulted in dose-related increases in plasma concentrations of both parent compound and its acid metabolite (Fig. 2). Mean peak concentrations normally occurred within 1–2 and 1–3 hr following the first of the two daily doses for tepoxalin and metabolite, respectively. Maximum plasma concentrations for the acid metabolite were around fourfold greater than those of tepoxalin for all three dosage levels. A rapid decline in tepoxalin concentrations was observed after the peak absorption had been achieved with no detectable levels at 24 hr, whereas the acid metabolite was elimi-

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nated at a much slower rate and could still be detected at low concentrations in the plasma 24 hr postdose. However, no unexpected accumulation of the acid metabolite occurred on multiple dosing. Organ Weights and Anatomic Pathology Rat studies. In the 1-month study, dose-related increases in liver weights up to 28% over control weights occurred in all dosage groups except 15 mg/kg males. Although hepatic necrosis was present in some rats, neither this nor any other microscopic findings appeared to be related to liver weight differences. Necropsy observations, some of which were confirmed microscopically, revealed an increased incidence of discolored foci in the mucosa of the stomach of rats in the 50 mg/kg dosage group. Although microscopically, the incidence of stomach erosion(s) appeared to be slightly higher in the 25 (females) and 50 mg/kg dosage groups, their relationship to tepoxalin administration is uncertain because there was no qualitative difference between the lesions in the vehicle control and drug-treated rats. Microscopic evaluation of rats in the 1-month study revealed drug-related changes in the liver and kidney. Females exhibited hepatic necrosis in 1 of 10 and 4 of 10 rats in

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TABLE 3 Selected Clinical Chemistry and Urinalysis Parameters (Mean { SE) for Rats Receiving Tepoxalin for 3 and 6 Monthsa Treatment Monthb ALT (U/liter) Males Females AST (U/liter) Males Females CHOL (mg/dl) Males Females Urinalysis: Protein (mg/dl) Males Females

a b

Control

5 mg/kg

10 mg/kg

20 mg/kg

40 mg/kg

3 (Interim) 6 3 (Interim) 6

42.5 43.9 43.3 59.2

{ { { {

1.3 2.6 3.9 7.2

40.1 46.0 63.6 59.9

{ { { {

2.2 2.9 14.7 8.4

37.5 40.9 53.2 66.5

{ { { {

1.5 2.3 12.2 10.3

43.4 42.0 46.9 61.1

{ { { {

1.8 1.3 7.4 6.8

43.4 42.0 35.4 75.2

{ { { {

1.8 1.3 2.7 12.8

3 (Interim) 6 3 (Interim) 6

103.1 103.7 105.3 114.5

{ { { {

3.9 4.1 4.7 9.3

106.3 92.4 115.5 122.8

{ { { {

6.0 2.8 14.4 15.9

103.1 99.4 114.5 133.2

{ { { {

4.3 5.8 12.5 13.7

98.9 95.6 103.3 116.1

{ { { {

3.4 5.7 9.0 9.2

103.0 92.6 89.9 153.5

{ { { {

5.7 5.0 6.2* 26.3

3 (Interim) 6 3 (Interim) 6

65.2 78.7 92.0 107.3

{ { { {

3.6 4.2 4.0 6.7

67.1 79.9 93.7 111.0

{ { { {

4.0 6.4 5.3 7.2

62.0 77.7 86.8 96.9

{ { { {

2.8 4.6 5.2 5.7

68.9 76.9 98.3 127.5

{ { { {

4.7 4.1 5.1 10.8

66.9 78.4 103.5 145.1

{ { { {

3 (Interim) 6 3 (Interim) 6

66.6 93.6 25.3 56.9

{ { { {

6.9 14.6 13.8 39.8

78.3 133.5 19.6 43.7

{ { { {

8.8 26.1 3.8 17.2

82.9 79.1 21.8 17.6

{ { { {

7.5 7.3 9.0 5.6

78.8 82.0 13.0 111.4

{ { { {

9.7 9.8 1.7 68.1

91.9 123.7 24.3 138.4

{ { { {

3.1 5.0 4.9 7.3** 9.0 20.1 5.9 73.3

Significant difference from control (Dunnett’s test): *p £ 0.05, **p £ 0.01. n Å 20/sex at 3 months (Interim); n Å 15/sex at 6 months.

the 25 and 50 mg/kg groups, respectively; however, livers appeared to be normal after a 1-month recovery period. Kidney changes included tubular dilatation in the cortex, chronic progressive nephropathy, and renal papillary edema or necrosis in both males and females. Renal papillary edema or necrosis was observed in 0 of 10, 1 of 10, 2 of 10, and 5 of 10 male rats and 1 of 10, 2 of 10, 1 of 10, and 3 of 10 female rats treated with 15, 25, 35, and 50 mg/kg tepoxalin, respectively. The severity of chronic progressive nephropathy in both male and female rats, a lesion usually associated with aging, was also slightly accentuated in tepoxalin-treated rats. One female rat in the 50 mg/kg dosage group developed marked chronic interstitial nephritis and papillary necrosis which may be drug related. Renal papillary edema was still seen in 1 of 5 rats/sex (50 mg/kg) after the recovery period. Liver weights were significantly increased by approximately 20% in female rats dosed at 20 and 40 mg/kg for 3 and 6 months. The increased weight was associated with centrilobular hypertrophy of hepatocytes observed in 3 of 10 and 6 of 10 female rats dosed with 20 and 40 mg/kg, respectively, for 3 months. After 6 months, 1 of 15, 2 of 15, and 7 of 15 female rats dosed with 10, 20, or 40 mg/kg respectively, had this change in the liver. Drug-related changes were observed in the kidney. Papillary edema was observed in 2 of 10 males dosed with 20 mg/kg and 7 of 10 male and 2 of 10 female rats dosed with 40 mg/kg for 3 months. After 6 months, only 1 of 15 males

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dosed with 40 mg/kg had papillary edema. Compared with controls, an increased incidence of chronic progressive nephropathy was observed in 40 mg/kg females at 3 months and in male rats at this dosage level after 6 months. Gastric erosions were observed in one female rat dosed with 40 mg/kg for 6 months. Ulcers of the cecum were observed in one 40 mg/kg female after 3 months and two 40 mg/kg females after 6 months of treatment. One additional 40 mg/kg female rat exhibited acute inflammation of the cecum. One 40 mg/kg female rat had an ulcer in the ileum as well as in the cecum. Dog studies. In the 1-month study, there were no microscopic findings in any organs that were considered to be related to drug treatment. In the 6-month study, however, one male dog dosed with 300 mg/kg/day had two mucosal ulcers and associated inflammation in the pylorus after 3 months of dosing. In addition, pyloric ulceration occurred in two females dosed with 300 mg/kg/day after 6 months of dosing. One female dosed with 100 mg/kg/day and one male dosed with 300 mg/kg/day showed gross evidence of ulceration, but this was not confirmed microscopically. A number of microscopic changes were seen sporadically and were considered to be incidental findings, unrelated to administration of tepoxalin. DISCUSSION

The results of these studies indicate that tepoxalin was well tolerated for up to 6 months in rats and dogs at dosages

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TABLE 4 Selected Hematology Parameters (Mean { SE) for Dogs Receiving Tepoxalin for 3 and 6 Monthsa Treatment Monthb RBC (11012/liter) Males

Females

HB (g/dl) Males

Females

HCT (%) Males

Females

WBC (1109/liter) Males

Females

a b

Control

20 mg/kg/day

100 mg/kg/day

300 mg/kg/day

Predose 1.5 (Interim) 3 (Interim) 6 Predose 1.5 (Interim) 3 (Interim) 6

6.41 6.51 7.03 7.11 6.25 6.95 7.03 6.47

{ { { { { { { {

0.02 0.14 0.26 0.42 0.24 0.20 0.20 0.32

6.21 6.80 7.01 6.43 6.59 6.87 6.29 6.44

{ { { { { { { {

0.15 0.11 0.16 0.28 0.14 0.26 0.63 0.31

6.34 6.47 6.64 6.15 7.00 6.84 6.69 6.40

{ { { { { { { {

0.24 0.18 0.15 0.08 0.19 0.24 0.38 0.15

6.50 6.84 7.29 6.37 6.60 6.55 6.98 6.25

{ { { { { { { {

0.13 0.16 0.10 0.17 0.19 0.46 0.26 0.54

Predose 1.5 (Interim) 3 (Interim) 6 Predose 1.5 (Interim) 3 (Interim) 6

14.17 14.73 15.49 16.58 14.33 16.17 15.99 15.88

{ { { { { { { {

0.45 0.30 0.54 0.94 0.45 0.47 0.34 0.83

13.90 15.67 15.70 15.45 14.73 15.83 14.13 15.45

{ { { { { { { {

0.31 0.34 0.31 0.30 0.33 0.57 1.34 0.76

14.29 15.01 15.19 14.55 15.24 15.24 14.71 14.73

{ { { { { { { {

0.58 0.32 0.40 0.30 0.26 0.29 0.84 0.46

14.56 15.66 16.26 15.58 14.83 15.26 15.73 14.55

{ { { { { { { {

0.40 0.36 0.26 0.27 0.35 0.29 0.66 1.53

Predose 1.5 (Interim) 3 (Interim) 6 Predose 1.5 (Interim) 3 (Interim) 6

40.99 40.80 47.27 49.00 41.19 45.11 49.31 47.85

{ { { { { { { {

1.39 0.97 1.66 2.34 1.20 1.38 0.96 2.34

40.00 43.37 47.81 45.80 42.44 43.79 43.23 46.25

{ { { { { { { {

0.97 0.91 0.93 2.15 0.91 1.61 4.11 2.06

40.99 41.50 46.34 43.45 43.26 41.89 45.47 44.73

{ { { { { { { {

1.82 0.87 1.20 0.92 0.87 0.85 2.49 1.06

42.24 43.30 50.26 46.78 42.81 42.03 48.53 44.33

{ { { { { { { {

1.17 0.91 0.78 0.63 0.93 3.06 1.91 4.39

Predose 1.5 (Interim) 3 (Interim) 6 Predose 1.5 (Interim) 3 (Interim) 6

11.89 9.40 9.81 9.30 9.67 10.09 10.53 10.55

{ { { { { { { {

0.90 0.56 0.41 0.88 0.66 1.06 0.72 1.24

11.89 10.41 9.41 10.05 9.66 10.30 10.86 8.35

{ { { { { { { {

0.84 0.93 0.72 0.57 1.17 1.14 1.15 1.07

11.34 12.66 10.43 11.55 10.54 11.00 8.69 9.85

{ { { { { { { {

0.79 1.70 1.12 0.63 0.81 0.55 0.83 0.95

11.93 11.54 12.16 10.88 9.80 12.31 11.50 15.85

{ { { { { { { {

0.75 0.93 1.24 0.75 0.62 0.99 1.45 3.89

There were no significant differences from control. n Å 7/sex at 1.5 and 3 months (Interim); n Å 4/sex at 6 months.

in excess of the ED50 (3.5 mg/kg) for inhibition of inflammatory effects in the adjuvant arthritic rat, with reduced gastric mucosal damage. In the rat studies, dosage-related histomorphological changes were observed in the liver, kidneys, stomach, ileum, and/or cecum at dosages of 10 mg/kg and higher, whereas in the dog studies gastrointestinal effects were observed at 100 mg/kg and higher dosages. In the 1month rat study, dose-related increased liver weights were observed in all tepoxalin groups except 15 mg/kg males. Hepatic necrosis was observed in female rats at dosage levels of 25 and 50 mg/kg after 1 month of dosing; however, the livers of rats dosed with 50 mg/kg appeared normal after 4 weeks of recovery. Administration of tepoxalin for 6 months resulted in minimal to mild hypertrophy of centrilobular he-

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patocytes in female rats given 10, 20, or 40 mg/kg. This morphological change was associated with a significant increase in liver weights at the 20 and 40 mg/kg dosage levels, an approximately 20% increase over controls. Two 40 mg/ kg females dosed for 6 months had minimal centrilobular necrosis, which was not associated with increased AST or ALT activities. Generally, centrilobular hypertrophy is characterized by an increase in one or more subcellular organelles and is considered to be an adaptive rather than a toxic response (Popp and Cattley, 1991). It was the only druginduced histomorphological change at the 10 mg/kg dosage level. Some anti-inflammatory drugs have been shown to alter aryl hydrocarbon hydroxylase activity and cytochrome P450 content of rodent liver microsomes (Mostafa et al.,

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TABLE 5 Selected Clinical Chemistry Parameters (Mean { SE) for Dogs Receiving Tepoxalin for 3 and 6 Monthsa Treatment Monthb CA (mg/dl) Males

Females

T. PROT (g/dl) Males

Females

ALB (g/dl) Males

Females

a b

Control

20 mg/kg/day

100 mg/kg/day

Predose 1.5 (Interim) 3 (Interim) 6 Predose 1.5 (Interim) 3 (Interim) 6

10.51 10.89 10.27 10.33 10.80 11.06 10.40 10.08

{ { { { { { { {

0.07 0.13 0.14 0.05 0.17 0.09 0.12 0.09

10.50 10.86 10.29 10.23 10.73 10.91 10.07 10.10

{ { { { { { { {

0.10 0.14 0.14 0.15 0.08 0.23 0.23 0.12

10.90 10.47 10.29 10.03 10.73 10.83 10.14 10.20

{ { { { { { { {

0.12 0.14 0.12 0.11 0.11 0.22 0.16 0.08

Predose 1.5 (Interim) 3 (Interim) 6 Predose 1.5 (Interim) 3 (Interim) 6

5.54 5.73 5.69 5.58 5.30 5.56 5.53 5.30

{ { { { { { { {

0.08 0.13 0.11 0.09 0.06 0.06 0.09 0.13

5.56 5.99 5.74 5.93 5.37 5.63 5.36 5.33

{ { { { { { { {

0.10 0.21 0.12 0.15 0.07 0.12 0.16 0.06

5.44 5.34 5.29 5.23 5.64 5.64 5.27 5.40

{ { { { { { { {

Predose 1.5 (Interim) 3 (Interim) 6 Predose 1.5 (Interim) 3 (Interim) 6

3.42 3.70 3.53 3.48 3.44 3.79 3.63 3.40

{ { { { { { { {

0.05 0.04 0.06 0.09 0.09 0.10 0.09 0.11

3.37 3.86 3.46 3.33 3.39 3.76 3.19 3.30

{ { { { { { { {

0.05 0.04 0.06 0.09 0.05 0.08 0.15 0.07

3.40 3.46 3.29 3.10 3.46 3.69 3.27 3.20

{ { { { { { { {

300 mg/kg/day

11.04 10.94 10.47 10.15 10.87 10.69 10.19 9.60

{ { { { { { { {

0.18* 0.19 0.13 0.16 0.11 0.27 0.20 0.42

0.11 0.12 0.18 0.14 0.06** 0.14 0.12 0.07

5.63 5.79 5.59 5.38 5.56 5.41 5.24 4.83

{ { { { { { { {

0.07 0.14 0.08 0.05 0.08* 0.22 0.13 0.32

0.07 0.06** 0.14 0.09 0.05 0.06 0.13 0.08

3.51 3.71 3.46 3.28 3.57 3.54 3.34 2.93

{ { { { { { { {

0.06 0.06 0.06 0.06 0.06 0.21 0.12 0.28

Significant difference from control (Dunnett’s test): *p £ 0.05, **p £ 0.01. n Å 7/sex at 1.5 and 3 months (Interim); n Å 4/sex at 6 months.

1990). Interestingly, increased liver weight of approximately 20% was observed in female rats after 1 year of treatment with tepoxalin and the increased weight was not associated with an increase in total cytochrome P450 content or peroxi-

some proliferation (unpublished data). No adverse hepatic effects were noted in any of the dog studies. Renal papillary edema or necrosis related to tepoxalin administration was seen in female rats at 15, 25, 35, and 50

TABLE 6 Tepoxalin and Carboxylic Acid Metabolite Plasma Concentrations (mg/ml) in Rat 2 hr Postdose Dosage (mg/kg/day)

Tepoxalin Day 1 Day 92 Day 184 Acid metabolite Day 1 Day 92 Day 184

5

10

20

40

BQLa BQL BQL

BQL BQL BQL

0.70 { 0.53 0.52 { 0.58 0.34 { 0.52

0.72 { 0.40 0.30 { 0.17 0.81 { 0.58

1.39 { 0.49 2.80 { 0.93 1.81 { 0.78

1.40 { 0.49 3.51 { 1.35 3.13 { 1.32

5.24 { 2.16 6.22 { 3.83 4.49 { 3.00

5.04 { 2.58 5.85 { 2.64 10.70 { 3.68

Note. Each value represents the group mean { SD, combined sex, for six rats. a Below quantitation limit (0.25 mg/ml).

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FIG. 2. Representative mean profiles in dog plasma for (a) tepoxalin and (b) carboxylic acid metabolite.

mg/kg and in male rats at 25, 35, and 50 mg/kg after 1 month of treatment. At the end of the 1-month recovery period, evidence of renal papillary edema was still present in 1 of 5 rats/sex dosed with 50 mg/kg. Renal papillary edema was also noted in male and female rats at 20 and 40 mg/kg after 6 months of treatment. This lesion is compatible with the analgesic nephropathy characteristic of NSAIDs (Alden and Frith, 1991). Rats are particularly susceptible to the development of renal papillary edema and/or necrosis. An increased incidence of nephropathy was noted for 40 mg/kg female rats after 3 months. The increased urinary protein levels noted at the 40 mg/kg dosage level after 6 months may be associated with the increased incidence of

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chronic progressive nephropathy observed in male rats. No adverse renal effects were noted in any of the dog studies. This is considered very unique in view of the sensitivity of the canine species to many of the marketed NSAIDS. Erosions and ulcers of the gastrointestinal tract are commonly observed with NSAIDs (Walker, 1985; Levi and Shaw-Smith, 1994; Bjarson and Macpherson, 1989); however, in the 1-month rat study, there was no qualitative difference between stomach erosions in the vehicle control and tepoxalin-treated groups. With continuous tepoxalin treatment over 6 months, only 1 of 15 female rats at 40 mg/kg had stomach erosions. In the intestinal tract, at 40 mg/kg, 1 of 10 female rats dosed for 3 months and 1 of 15 female

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TOXICITY OF TEPOXALIN IN RATS AND DOGS

rats dosed for 6 months had an ulcer of the ileum. Two of the 15 females dosed for 6 months with 40 mg/kg had ulcers in the cecum and one additional female rat had a focal area of acute inflammation. While the usual location for NSAIDinduced changes is the upper gastrointestinal tract, some NSAIDs have been noted to produce lesions in the lower intestinal tract (Walker, 1985; Bjarson and Macpherson, 1989); however, in an earlier dose range-finding study, at a higher dosage of 75 mg/kg tepoxalin, there was a higher incidence of ulcers of the stomach, which is a primary target organ in the rat (unpublished data). This was possibly attributed to the level of the acid metabolite, which has been characterized as a pure CO inhibitor. There were no consistent histomorphological correlates for the anemia noted clinically in the 6-month rat study. Administration of NSAIDs can result in anemia associated with gastrointestinal blood loss (Bjarson and Macpherson, 1989). In this study, however, only 4 of 25 female rats given 40 mg/kg had morphological evidence of gastrointestinal erosions or ulcerations. Nevertheless, similar changes in erythrocyte parameters were observed in the 1-month rat study at dosages of 35 and 50 mg/ kg but were not seen in the 50 mg/kg dosage group after 1 month of recovery. In the 6-month dog study, small pyloric ulcers were seen (either grossly or microscopically) in one 100 mg/kg/day (one female) and three 300 mg/kg/day (one male and two females) group dogs. In addition, a similar ulcer was seen in one 300 mg/kg/day group dog at the 3month interim euthanasia. Although not directly correlated with histopathological findings, the hematological changes observed for high-dosage female dogs were suggestive of potential gastrointestinal blood loss secondary to gastric irritation. Cyclooxygenase-inhibiting anti-inflammatory agents are known to cause extensive gastric irritation in the dog (Bertram, 1991); however, the mild and sporadic nature of the changes seen in the stomachs of dogs in this study in the presence of compound absorption is considered noteworthy. In fasted rats, tepoxalin induced lesions in 50% [ulcerogenic dose 50 (UD50)] of the animals tested at a dose of 173 mg/kg. In contrast, the UD50 for naproxen was 1.0 mg/kg at 3 hr postdose and 5.0 mg/kg at 16 hr postdose (unpublished data). BF-389, a dual CO/LO inhibitor, had a reported UD50 of 520 mg/kg/day in rats (Wong et al., 1992). CI-986, another CO/LO dual inhibitor, showed low ulcerogenic potential relative to other NSAIDs when tested in dogs, monkeys, and rats (Robertson et al., 1993). It has been suggested that the reduced ulcerogenic activity of dual CO/LO inhibitors may be due to the ability to inhibit leukotriene synthesis (Rainsford, 1987; Guslandi, 1987); however, NSAID-induced gastric mucosal damage is not consistently accompanied by stimulation of leukotrienes formation. Lee and Feldman (1992) reported no significant changes in mucosal leukotriene C4 synthesis and content and no correlation between changes in mucosal LTB4 synthesis and the extent of mucosal damage in aspirin-induced acute gastric mucosal

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injury in rats. Also, MK-571, a leukotriene D4 receptor antagonist, and MK-886, a leukotriene biosynthesis inhibitor, did not reduce mucosal lesions induced by aspirin, suggesting that leukotriene production is not a crucial factor in the development of indomethacin-induced gastric mucosal erosion (Lee and Feldman, 1992). Mediators of NSAIDinduced mucosal injury are probably different from those involved in mucosal damage induced by various necrotizing agents such as ethanol (Peskar, 1991). Peskar (1991) also reported that stimulation of leukotriene C4 formation in the rat stomach, induced by ethanol, could be blocked by MK886 without any evidence for inhibition of gastric damage. Ford-Hutchison et al. (1993) showed that MK-886 had no significant effect on indomethacin-induced gastrointestinal lesions in the rat. In conclusion, tepoxalin was remarkably well tolerated for up to 6 months in rats and dogs at dosages in excess of the ED50 for inhibition of inflammatory effects in the adjuvant arthritic rat without gastric mucosal damage. The fact that only a mild effect of tepoxalin on the gastric mucosa and no other indication of progression in toxicity was observed between 3 and 6 months, in both rats and dogs, has been particularly impressive when compared with most NSAIDs. Further work is required to clarify the implications of these findings and the possible risk of human exposure. ACKNOWLEDGMENTS The authors thank the Drug Safety Evaluation and Drug Metabolism Associate Staff for their technical support. Special thanks to Basil McKenzie and Harris Mosher for critically reviewing the manuscript.

REFERENCES Alden, C. L., and Frith, C. H. (1991). Urinary system. In Handbook of Toxicologic Pathology (W. M. Haschek and C. G. Rousseaux, Eds.), pp. 347–348. Academic Press, New York. Anderson, D. W., Argentieri, D. C., Ritchie, D. M., Katz, L. B., Shriver, D. A., Rosenthale, M. E., and Capetola, R. J. (1990). Gastrointestinal (GI) profile of tepoxalin (TX), an orally active dual cyclooxygenase (CO)/lipoxygenase (LO) inhibitor with potent antiinflammatory activity. FASEB J. 4, A1122. Argentieri, D. C., Anderson, D. W., Ritchie, D. M., Rosenthale, M. E., and Capetola, R. J. (1990). Tepoxalin (RWJ 20485) inhibits prostaglandin (PG) and leukotriene (LT) production in adjuvant arthritic rats and in dog knee joints challenged with sodium urate and immune complexes. FASEB J. 4, A1122. Bertram, T. A. (1991). Gastrointestinal tract. In Handbook of Toxicologic Pathology (W. M. Haschek and C. G. Rousseaux, Eds.), p. 217. Academic Press, New York. Bjarson, I., and Macpherson, A. (1989). The changing gastrointestinal sideeffect profile of non-steroidal anti-inflammatory drugs. Scand. J. Gastroenterol. 24(Suppl. 163), 56–64. Brooks, R. R., Moorehead, T. J., and Pong, S. F. (1993). Gastric toxicity and prostaglandin content in rats dosed with two chemically similar nonsteroidal anti-inflammatory agents. Proc. Soc. Exp. Biol. Med. 202, 233– 238.

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Rainsford, K. D. (1987). The effects of 5-lipoxygenase inhibitors and leukotriene antagonists on the development of gastric lesions induced by nonsteroidal antiinflammatory drugs in mice. Agents Actions 21, 316–319. Robertson, D. G., Loewen, G., Walsh, K. M., Dethloff, L. A., Sigler, R. S., Dominick, M. A., and Urda, E. R. (1993). Subacute and subchronic toxicology studies of CI-986, a novel antiinflammatory compound. Fundam. Appl. Toxicol. 20, 446–455. Schoen, R. T., and Vender, R. J. (1989). Mechanisms of nonsteroidal antiinflammatory drug-induced gastric damage. Am. J. Med. 86, 449–458. Vane, J. R. (1971). Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature (London) 231, 232–235. Walker, F. S. (1985). Azapropazone and related benzotriazines. In Antiinflammatory and Anti-rheumatic Drugs. Vol. II: Newer Anti-inflammatory Drugs (K. D. Rainsford, Ed.), pp. 1–32. CRC Press, Boca Raton, FL. Wallace, J. L., McCafferty, D., Carter, L., McKnight, W., and Argentieri, D. (1993). Tissue selective inhibition of prostaglandin synthesis in rat by tepoxalin: Anti-inflammatory without gastropathy? Gastroenterology 105, 1630–1636. Weinblatt, M. E., Kremer, J. M., Coblyn, J. S., Helfgott, S., Maier, A. L., Petrillo, G., Henson, B., Rubin, P., and Sperling, R. (1992). Zileuton, a 5-lipoxygenase inhibitor in rheumatoid arthritis. J. Rheum. 19, 1537– 1541. Wong, S., Lee, S. J., Frierson, M. R., III, Proch, J., Miskowski, T. A., Rigby, B. S., Schmolka, S. J., Naismith, R. W., Kreutzer, D. C., and Lindquist, R. (1992). Antiarthritic profile of BF-389: A novel antiinflammatory agent with low ulcerogenic liability. Agents Actions 37, 90– 98. Xie, W., Robertson, D. L., and Simmons, D. L. (1992). Mitogen-inducible prostaglandin G/H synthase: A new target for nonsteroidal antiinflammatory drugs. Drug. Dev. Res. 25, 249–265.

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