- Email: [email protected]
Single-dose and multiple-dose intravenous toxicity studies of BMY-25282 in rats
FUNDAMENTALANDAPPLIEDTOXICOLOGY9,90-109(1987)
Single-Dose and Multiple-Dose intravenous Toxicity Studies of BMY-25282 in Rats C. L. BREGMAN, C. R. COMERESKI, R. A. BUROKER, H. MADISSOO, AND G. H. HOTTENDDRF Bristol-Myers
Company,
Pathology
Pharmaceutical and Toxicology
Single-Dose and Multiple-Dose MAN,C.L.,COMERESIU,C.R.,BUROKER, DORF, G. H. (1987). Fundam.
R. S. HIRTH,
Research and Development Division, Syracuse, New York 13221-4755
Intravenous Toxicity Studies of BMY-25282
Department
of
in Rats. BREG
R. A.,HIRTH,R.S.,MADISSOO,H.,ANDHOTTEN-
Appl. Toxicol. 9, 90-109. Singledose and multiple-dose (daily X5 and weekly X5) intravenous toxicity studies in rats were conducted to determine the possible acute and delayed toxicity of BMY-25282 (7-N-(dimethylaminomethylene) mitomycin C), a potential anticancer drug. Rats in the single-dose study received either 0.05,0.25, or 0.50 mgl kg (0.3, 1.5, or 3.0 mg/m’) of BMY-25282; rats in the daily X5 multiple-dose study received doses of 0.005,0.025, or 0.050 mg/kg (0.03, 0.15, and 0.3 mg/m2) of BMY-25282 once each day for 5 days; and rats in the weekly X5 multiple-dose study received 0.05 mg/kg of BMY25282. All doses were in 0.1% Pluronic F-68 diluent. Acute toxicities included gastrointestinal epithelial necrosis, myelosuppression, and splenic lymphoid depletion in the high and intermediate dose groups in the single-dose study and myelosuppression in the high dose group of the daily X5 multiple-dose study. One death in a high dose male of the single-dose study was attributed to acute gastrointestinal and lymphoid toxicity. Between the interim necropsy on Day 5 or 9 and termination of the 9-week dose-free observation period, 9/20 rats of the high and intermediate dose groups of the single-dose study and 4/10 high dose rats in the daily X5 multiple-dose study died, primarily due to hydrothorax and congestive heart failure caused by delayed, drugrelated myocardial degeneration. The most prominent drug-related histopatbologic changes observed in rats of both the single-dose study and the daily X5 studies were myocardial degeneration (cardiomyopathy), glomerulopathy with tubular degeneration, and necrotizing arteritis. These three changes, observed at 0.5 and 0.25 mg/kg in the single-dose study and at 0.05 mg/ kg/day in the multiple-dose (daily X5) study, were delayed in onset and irreversible. Drug-related tubular degeneration and slight glomerulopathy were observed in male BMY-25282-treated rats in the weekly X5 study, but cardiotoxicity, pulmonary arteritis, hydrothorax, and lethality were not observed. The diluent, Pluronic F-68, was not associated with any morphologic or clinicopathologic changes. A single-dose of 0.05 mg/kg or 5 daily doses of 0.025 and 0.005 mg/kg of BMY-25282 were considered nontoxic doses in rats. A cumulative dose of 0.25 m&kg, which caused cardiotoxicity in the daily X5 study, was not cardiotoxic in the weekly X5 study. These results indicate that the delayed cardiotoxicity of BMY-25282 is schedule dependent. o 1987 Society of Toxicology.
BMY-25282, 7-N-(dimethylaminomethylene)-mitomycin C, is one of a novel series of amidino mitomycin derivatives. BMY25282 has been shown to have markedly superior antitumor effects compared to mitomycin C in a number of in vivo mouse tumor models and is considered to be two to four times more potent than mitomycin C (Bradner et al., 1985). 0272-0590187 $3.00 Copyright 0 1987 by the Society of Toxirology. All rights of reproduction in any form reserved.
The single-dose and multiple-dose (daily X5 and weekly X5) intravenous toxicity studies in rats reported herein were conducted to assessthe acute and delayed toxicity of BMY25282 and its diluent Pluronic F-68 and to establish nontoxic doses for different treatment schedules. The design of the single-dose and the daily X5 multiple-dose studies was based on National Cancer Institute guide90
BMY-25282
TOXICITY
91
IN BATS
TABLE 1 BMY-25282 Dosing schedule
Dose budoWe
~SESIN~TTOXICI~%KJD~ES
Dose (mg/m*/day)
Doseas%of mouse LDIO”
Total cumulative dose b-m&)
Single dose
0.5 0.25 0.05
3.00 1.50 0.30
67 33 7
0.5 0.25 0.05
Daily x5
0.05 0.025 0.005
0.30 0.15 0.03
27 13 3
0.25 0.125 0.025
Weekly ~5
0.05
0.30
-b
0.25
a Single-dose mouse LD 10 was 1.49 mg/kg. The daily X5, multiple-dose LD 10 was 0.37 mg/kg. b A weekly x5 multiple-dose mouse lethality study was not conducted.
lines for the preclinical evaluation of anticancer drugs (Crout, 1981). METHODS Experimental design. In each of the single-dose and multiple-dose (daily ~5) studies, 100 rats (50 males and 50 females) were divided into five groups of 10 males and 10 females each. In the single-dose study, rats of three groups received a single intravenous injection via the caudal vein of either 0.05,0.25, or 0.50 mg/kg of BMY25282 in 0.1% Pluronic F-68 diluent. In the multipledose study, rats of three groups received either 0.005, 0.025, or 0.05 mg/kg/day of BMY-25282 in 0.1% Pluronit F-68 diluent via the caudal vein for 5 days. In each study, a fourth group of rats served as negative controls and was administered intravenous injections of Water for Injection, USP. A fifth group in each study served as vehicle controls and was administered either one or five injections of0. 1% Pluronic F-68 diluent. An interim necropsy on 5 rats/sex/group was conducted 4 or 5 days after the last dose. The remaining animals were sacrificed after a 9-week, drug-free observation period. In the weekly x5 multiple-dose study, 5 males and 5 females were administered 0.05 mg/kg of BMY-25282 in 0.1% Pluronic F-68 diluent intravenously once per week for 5 consecutive weeks. Another 5 males and 5 females served as negative controls and were administered five injections of 0.1% Pluronic F-68 diluent according to the same schedule. The last dose of BMY25282 was followed by a IO-week observation period. Necropsies were performed on all rats at the termination of the observation period. The doses used in these rat studies are listed in Table 1 in terms of mg/kg, mg/m2, and as percentages of the
mouse LDlO. Single-dose and daily x5 multiple-dose intravenous lethality studies in Swiss-Webster mice had been previously conducted in our laboratory to determine the LD50 and LD 10 values. The combined LD 10 for both males and females in the single-dose study and the daily X5, multipledose study were 1.49 mg/kg and 0.37 mgfkgfday, respectively. Test materials. The vehicle, Pluronic F-68, a block polymeric nonionic surfactant, was prepared as a 0. I %
TABLE 2 MORTALITYANDTIMEOFDEATHOF BMY-25282-TREATEDRATS
Number of deaths Dose (mg/kg)
M
Single-dose
0” 0.05 0.25 0.50
O/20 o/10 l/IO 3/10
O/20 o/10 3/10 35,50, 5 I,60 2110 7,45,60,63 (X2)
Daily x5
0” 0.005 0.025 0.050
o/20 o/10 O/10 2/10
O/20 o/10 O/10 2/10
29,49,62,64
WeeklyX5
Ob 0.05
O/5 015
Of5 Of5
-
Schedule
F
Day of death of individual rats
’ Two control groups (water control and vehicle control) were combined for this table. ’ Control group received vehicle (Pluronic F-68).
BREGMAN
92
ET AL.
TABLE 3 SUMMARY OF MAJOR CLINICOPATHOLOGIC CHANGES IN BMY-~~~~~-TREATED Single-dose study
RATS Weekly X5 study
Daily x5 study
Dose of BMY-25282 (mg/kg): Number ofratsexamined”:
Oh 40
0.05 20
0.25 20
0.50 20
Ob 40
0.005 20
0.025 20
0.05 20
0’ 10
0.05 10
Increased BUN Increased creatinine Hypoproteinemia Proteinuria Anemia Leukocytosis (neutrophilia) Leukopenia, neutropenic and lymphopenic Reticulocytopenia, early Reticulocytosis, late Elevated aspartate aminotransferase
-d -
-
+ + + + + +
+ + + + + +
-
-
-
+ + + -
NM -
+ + NM -
-
-
+ -
+ +
-
-
-
-
-
+ -
-
-
+
+
-
-
+
+
-
-
a Each dose group was composed of equal numbers of male and female rats. b Two control groups, one given water and the other given Pluronic F-68 vehicle, were combined for this table. ‘Control group received Pluronic F-68 vehicle. d -, no drug-related changes observed; +, drug-related changes observed, NM, parameters not measured in rats of this treatment group.
solution in sodium citrate and citric acid. BMY-25282 was dissolved in the 0. I % Pluronic F-68 vehicle at concentrations ranging from 0.005 to 0.25 mg/ml. The resulting solutions were administered intravenously at 2.0 ml/kg except for the 0.005 mg/ml solution which was given at 1.Oml/kg. The solutions were prepared immediately prior to dosing each day and were used within the 3-hr time period for which solution stability had been established. Dosing solutions were assayed for BMY25282 content on Day 1 of each study. Animals and animal husbandry. Sprague-Dawley (Crl:CD(SD)BR) rats (Charles River Breeding Laboratories, Inc., Wilmington, MA), which were 6 weeks old upon arrival, were used. The rats were subjected to a general health examination and a conditioning period of 2 weeks prior to study inception. Healthy rats selected for the study were subdivided by sex, ranked by body weight, and randomly divided into groups having approximately equal average body weights. Rats were individually identified with numbered ear tags and housed in individual wire-bottomed stainless-steel cages in temperature-controlled rooms. Ground food (Purina Laboratory Chow, Ralston Purina, St. Louis, MO) and water were offered ad libitum except as necessary to obtain blood and urine samples for clinical pathology determinations. In life observations, food intake, and body weights. Rats were observed daily for signs of toxicity and changes
in general health and behavior. Body weights were determined prior to drug administration, daily during drug administration in the multiple-dose studies, and once a week thereafter in all studies. General health and ophthalmological examinations were conducted on each rat prior to study inception and prior to scheduled necropsies. Food intake was determined once after the end of the treament period and once a week thereafter. Hematology. Blood samples for hematologic determinations were collected via the caudal vein from 5 rats/ sex/group on Days 4, 11, 35, and 60 in the single-dose study and on Days 8, 16,43, and 66 in the multiple-dose (daily X5) study, and on Days 35,64, and 84 in the multiple-dose (weekly X 5) study. Hematologic determinations included hemoglobin (Hgb) concentration, hematocrit (Hct), total erythrocytes (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLAT), and total leukocyte counts (WBC), all of which were determined using automated methods.’ Additionally, differential leukocyte count, absolute neutrophil and lymphocyte counts, and reticulocyte count (Brecher, 1949) were performed. ’ Ortho ELT-8/ds, Westwood, MA.
Ortho Diagnostic Systems, Inc.,
BMY-25282
TOXICITY
93
IN RATS
TABLE 4
Single-dose study Dose of BMY-25282 (mg/kg): Number ofratsexamined”: Heart Myocardial degeneration Perivasculitis, slight Ventricular hypertrophy Lung Arteritis Arterial medial hypertrophy Interstitial pneumonia with fibrosis Kidney Glomerulopathy Tubular degeneration Arteritis Thymus Lymphoid depletion Spleen Lymphoid depletion Pancreas, testis, epididymis, cecum, and/or colon Arteritis
Weekly X5 study
Daily X5 study
Ob 20
0.05 10
0.25 10
0.50 10
Ob 20
0.005 10
0.025 10
0.05 10
0’ 10
0.05 10
0 0 0
0 0 0
10 8 2
9 8 6
0 0 0
-d -
0 0 0
8 4 2
0 0 0
0 0 0
0
0
3
5
0
-
0
2
0
0
0
0
5
4
0
-
0
4
0
0
0
0
7
I
0
-
0
2
0
0
0 0 0
0 1 0
8 6 1
9 I 0
0 0 0
0 3 0
9 I 0
0 1 0
2 5 0
0
0
2
6
0
-
0
3
-
-
0
0
3
3
0
-
0
3
-
-
0
0
1
0
0
-
0
1
-
-
0 0 0
a Each dose group was composed of equal numbers of male and female rats. b Two control groups, one given water and the other given Pluronic F-68 vehicle, were combined for this table. ’ Control group received the vehicle, Pluronic F-68. d Organ of that dose group was not scheduled for examination. Clinicalchemistries. Urea nitrogen (BUN), fastingglucase (FBS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, total protein (TP), albumin (ALB), creatine kinase (CK), sodium, and potassium were measured in 5 rats/sex/group in each study at the interim and final necropsies on serum collected via cardiac puncture in the single-dose and daily X5 studies.2,3BUN and ALT were also performed on Days 11 and 35 in the single-dose study and on Days 16 and 43 in the multiple-dose study. Serum globulin fraction (GLOB) and albumin to globulin ratios were computed using TP and ALB data. Creatinine, BUN, ALT, AST, and total protein were performed on Days 35,64, and 84 of the weekly X5, multiple-dose study.
’ Centrifichem System 600, Baker Instruments, Allentown, PA. 3 Coming Model 450 Flame Photometer, Coming Scientific Instruments, Medfield, MA.
Urinalysis. Pooled urine samples of 5 male or 5 female (or surviving) rats which had been placed in metabolism cages for 18 hr were collected prior to each of the scheduled necropsies during the single-dose and multiple-dose (daily X5) studies. Eighteen-hour volume, specific gravity, pH, protein, ketones, glucose, bilirubin, and occult blood were determined on all samples! Urine sediment was microscopically examined for erythrocytes, leukocytes, epithelial cells, bacteria, and casts. Pathological examinations. All rats from all three studies were necropsied. Moribund rats and those at scheduled necropsies were euthanatized with an intraperitoneal injection of sodium pentobarbital. Representative sections of adrenal gland, bone, bone marrow, brain, cecum, colon, duodenum, epididymis, esophagus, eye, heart, ileum, jejunum, kidney, liver, lung, mammary
4Ames Urinalysis System, Miles Laboratories, Inc., Ames Division, Elkhart, IN.
94
BREGMAN
gland, mesenteric lymph node, ovary, pancreas, parathyroid, pituitary, prostate, salivary gland, seminal vesicles, skeletal muscle, testis, thyroid gland, trachea, urinary bladder, and uterus were collected from each rat of the appropriate sex in the single-dose and multiple-dose (daily X5) studies and were fixed in 10% neutral buffered formalin. Prior to fixation, the weights of the heart, liver, adrenal glands, thyroid glands, gonads, and uterus or prostate with seminal vesicles were recorded for each rat necropsied at scheduled necropsies. Representative sections from only heart, lung, and kidney were collected from rats of the multiple-dose (weekly X5) study and no organ weights were measured. Representative sections of organs and tissues were processed using conventional methods and were stained with hematoxylin and eosin. Statisticalevaluation.In life body weight gains, food intake, terminal body weights, and organ weight data (collected at each scheduled necropsy) were evaluated using a multiple t statistic computed with a pooled error term, at an overall significance level of p < 0.05. The Bonferroni inequality was used when sample sizeswithin all groups were not equal; the Dunnett procedure was used when all group sizes were equal (Kirk, 1968).
RESULTS
In Life Observations In the single-dose and daily X5 multipledose studies, drug-related clinical signs including emaciation, hunched body position, rough hair coat, and whole body discoloration (suggestive of anemia) were seen in rats treated with a single dose of 0.25 or 0.50 mg/ kg and with five daily doses of 0.05 mg/kg of BMY-25282. Respiratory distress was observed only in rats that subsequently died. Liquid feces were noted in one male treated with 0.50 mg/kg that died on Day 7. All clinical signs, except liquid feces, were delayed in onset and appeared in the latter half of the observation periods. Lower food intakes were noted in the latter half of the studies in rats of the single-dose study that received 0.25 or 0.50 mg/kg and in rats of the daily X5 multiple-dose study that received 0.05 m&kg/day. No drug-related clinical signs or changes in food intake were observed in rats in the
ET AL.
weekly X5, multiple-dose study or in any rat that was treated with 0.1% Pluronic F-68 diluent alone.
In Life Body Weights In the single-dose study, significant body weight losses occurred in 0.25 mg/kg males and 0.50 mg/kg males and females prior to the interim necropsy on Day 5. Lower body weights persisted in these rats until the termination of the study. Lower body weights, which were delayed in onset, were also observed in 0.05 mg/kg rats of the daily X5 multiple-dose study. Body weights of rats treated with 0.05 mg/kg of BMY-25282 on a weekly X5 schedule were comparable to those of controls. There were no vehicle-related changes in the vehicle control groups.
Mortality Deaths occurred in 4 of 20 (1 male and 3 females) of the 0.25 mg/kg dose group and 5 of 20 (3 males and 2 females) of the 0.50 mg/ kg group in the single-dose study (Table 2). With the exception of the death of one 0.50 mg/kg male that occurred on Day 7, all deaths occurred between Days 35 and 63. Deaths occurred in 2 of 10 males and 2 of 10 females of the 0.05 mg/kg group of the daily ~5, multiple-dose study on Days 29,49, 62, and 64. No deaths occurred in the weekly X5, multiple-dose study.
Organ Weightsand Organ to Body Weight Ratios There were no absolute or relative organ weight changes, which were considered treatment related, observed at the interim necropsy of the single-dose study (on Day 5) or the daily X5, multiple-dose study (on Day 9).
BMY-25282
TOXICITY
IN RATS
95
FIG. 1. BMY-25282-related cardiomyopathy in a female rat 9 weeks after a single dose of 0.5 mg/kg. Note marked vacuolation within sarcoplasm and between cardiac myocytes.
At the terminal necropsy of the single-dose study, mean relative heart weights of males and females of the 0.5 and 0.25 mg/kg dose groups were significantly increased. In the daily X5, multiple-dose study, mean relative heart and liver weights of 0.05 mgf kg males were significantly increased at the termination of the study. No vehicle-related effects on absolute or relative organ weights were seen in the vehicle control group of either study. Hematology Major drug-related clinicopathologic changes for all three studies are summarized in Table 3. Slight anemia was present in a few rats of the 0.5 and 0.25 mg/kg groups of the singledose study on Days 4,35, and 60, but not on Day 11. Anemia was accompanied by reticulocytopenia on Day 4. On Day 60, 0.5 mg/
kg female rats exhibited mild reticulocytosis. Slight drug-related increases in total leukocyte count with increases in relative and absolute neutrophil counts were observed in 0.5 and 0.25 mg/kg rats. No significant BMY-25282~related changes occurred in hematologic parameters in any treatment group of the daily X 5, multiple-dose study at any of the four sampling periods. In the weekly X5, multiple-dose study, BMY-25282-treated rats, especially females, exhibited drug-related decreases in mean total WBC count, absolute neutrophil count, and absolute lymphocyte count. Absolute neutropenia was noted on Day 35, but was not observed at subsequent sampling periods on Days 64 and 84. The incidence of absolute lymphopenia peaked on Day 64 and had subsided by Day $4. No vehicle-related changes were noted in hematologic parameters in vehicle control rats of any of the three studies.
96
BREGMAN
ET AL.
FIG. 2. Right ventricular myocardium of a female rat 9 weeks after a single dose of 0.5 mg/kg of BMY25282. Cardiomyopathy is characterized by myocytolysis, cellular pleomorphism, and dilatation of small vessels.
Clinical Chemistries Drug-related alterations in clinical chemistries in the single-dose study included progressive elevations of mean BUN in the 0.5 and 0.25 mg/kg groups beginning on Day 35, slight to mild proportional elevations of mean creatinine values in 0.5 mg/kg females and 0.25 mg/kg males at the termination of the study, slight dose-related decreases in total protein on Day 68 in the 0.5 and 0.25 mg/ kg groups, and elevation of aspartate aminotransferase in a few rats of the 0.5 and 0.25
mg/kg groups on Day 68. Other possibly drug-related alterations in clinical chemistries included hyponatremia in the 0.5 mg/kg group on Day 5 and decreased glucose values in 0.5 mg/kg females on Days 5 and 68. Drug-related alterations of clinical chemistries in the daily ~5, multiple-dose study included elevation of mean BUN in the 0.05 mg/kg males and females on Day 43 and in 0.05 mg/kg males on Day 68, elevated mean creatinine in 0.05 mg/kg males on Day 67, and elevation of AST on Day 67 in 1 of 3 remaining 0.05 mg/kg males and 3 of 5 of the 0.025 mg/kg females.
BMY-25282
TOXICITY
FIG. 3. Slight perivasculitis near a focus of cardiomyopathy single dose of0.5 mg/kg of BMY-25282.
In the weekly X5, multiple-dose study, drug-related alterations in clinical chemistries included a low incidence of abnormally elevated BUN and creatinine values in BMY25282~treated rats at each of the three sampling times. Urinalysis
Urinalysis revealed no drug-related findings on Day 5 in the single-dose study. However, on Day 60, urinalysis revealed proteinuria in males and females of 0.5 and 0.25 mg/
IN RATS
97
in the heart of a male rat 9 weeks after a
kg groups. Examination of urine sediment at that time revealed an increased number of leukocytes in males and females of the 0.5 mg/kg group and in males of the 0.25 mg/kg group. Epithelial cell numbers were increased in the urine sediments of males and females of both 0.5 and 0.25 mg/kg groups. Occult blood was increased in the urine of 0.25 mg/ kg females. Proteinuria was the only drug-related change observed in the urinalysis of the daily X5, multiple-dose study. Proteinuria occurred in 0.05 mg/kg males and females on Day 67.
98
BREGMAN
ET AL.
FIG. 4. Swollen glomerulus of a male rat 9 weeks after a single dose of 0.5 mg/kg of BMY-25282. ment membranes of capillaries are markedly thickened by homogeneous eosinophilic material.
Vehicle-related changes in urinalytical data were not observed in vehicle control rats of either study.
GrossPathologicalFindings At the interim necropsy on Day 5 of the single-dose study, watery gastrointestinal tract contents and perineal soiling in a few females of the 0.5 mg/kg group were the only BMY-25282-related, grossly visible changes. At the terminal necropsy of the single-dose study and in rats that died during the observation period, hydrothorax was frequently observed in the 0.5 and 0.25 mg/kg groups. Hydrothorax was considered the cause of death (secondary to congestive heart failure) in rats that died. Cardiac changes including enlargement, thickening or dilatation of the ventricles, tan myocardial foci, and hydropericardium were observed in rats of the 0.5 and 0.25 mg/kg groups. Other drug-related gross
Base-
findings observed in these two groups included diffuse tan or light brown discoloration and roughened, capsular surfaces of renal cortices; thymic atrophy; enlargement with hyperemia of perithymic lymph nodes; tail lesions; congestion in multiple organs; subcapsular hemorrhages and interlobar adhesions in the liver; atrophy of seminal vesicles, prostate, and uterus; rough hair coat; bloody nasal discharge; and perineal soiling. In the daily X5, multiple-dose study, no BMY-25282-related gross changes were observed in any rat at the interim necropsy on Day 9. All 4 rats that died prior to the termination ofthe daily ~5, multiple-dose study exhibited marked hydrothorax. These 4 rats also exhibited other signs of congestive heart failure, including ventral subcutaneous edema, pulmonary edema, and congestion in multiple organs. Other possibly drug-related, grossly visible changes in these 4 rats were heart en-
BMY-25282
TOXICITY
IN RATS
99
FIG. 5. Glomerulopathy in a male rat 9 weeks after a single dose of 0.5 mg/kg of BMY-25282. Fibrin or fibrinoid material is deposited along Bowman’s capsule, in vessels, and thickened membranes (arrows). Martius-scarlet-blue stain.
largement, uterine atrophy, subpleural petechiae, tan hepatic foci, petechiae in gastric glandular mucosa, bloody incrustations of the muzzle and forepaw, and yellow perineal soiling. Drug-related, grossly visible changes at the termination of the daily X5, multiple-dose study were limited to the high dose BMY25282-treated group. Grossly visible cardiac lesions were cardiac enlargement, thickening of ventricular myocardium, and pale myocat-dial foci. Other grossly visible changes included tan, pulmonary mottling, and pale discoloration and roughening of renal cortices. No grossly observable, drug-related lesions were present in the lung or heart of the BMY25282-treated rats of the weekly X5, multiple-dose study. However, swollen kidneys with roughened, mottled, tan and brown cortices were observed in 1 of 5 BMY-25282 treated males and were considered suggestive of BMY-25282-related nephrotoxicity.
No vehicle-related gross pathological changes were observed in vehicle control rats in any of the three studies.
HistopathotogicFindings in the Single-Dose Study Drug-related histopathologic changes observed in rats necropsied on Day 5 included glandular degeneration and necrosis in stomach, jejunum, ileum, cecum, and colon; bone marrow hypocellularity; and splenic lymphoid depletion. These toxicities were observed in the 0.5 and 0.25 mg/kg groups. At the terminal necropsy on Day 68, cardiotoxic lesions due to BMY-25282 were observed in nearly all rats of both sexes in the 0.5 and 0.25 mg/kg groups. A variety of cardiotoxic changes including ventricular hypertrophy, mural thrombosis, endarteritis, focal necrosis, perivasculitis, hemorrhage, congestion, and endocarditis were observed in the two dose
BREGMAN
ET AL.
FIG. 6. Chronic tubular degeneration and glomerulopathy in a rat 9 weeks after the administration of a single dose of BMY-25282. Tubular basement membranes are thickened and epithelial cells are atrophic. Glomerulus at top is enlarged and sclerotic. Occasional tubules are dilated.
groups; however, myocardial degeneration (cardiomyopathy) occurred with the greatest incidence (Table 4). The cardinal feature of the BMY-25282-related myocardial degeneration was focal to dilhrse vacuolation, both sarcoplasmic and intercellmar, throughout the myocardium of both ventricles (Fig. 1). Degenerate myocytes were further characterized by pleomorphism, focal variations in staining and loss of striations. Actual loss of myocytes and slight interstitial edema were observed in a few severely affected foci (Fig. 2). A slight, d&se increase in mononuclear cells and mast cells was frequently observed in degenerate foci, the increase in mononuclear and mast cells was often prominent around small vascular structures and constituted a slight perivascuhtis (Fig. 3). The earliest time point at which cardiomyopathy was observed was on Day 35 in a 0.25 mg/ kg female that died prior to the termination of the study.
Glomerulopathy caused by BMY-25282 administration occurred in most rats of both sexes in the 0.5 and 0.25 mg/kg groups. The earliest time point at which glomerulopathy was observed was on Day 45 in a 0.5 mg/kg male. Glomerulopathy was characterized microscopically by dense, diffuse, lightly eosinophilic, proteinaceous deposits along basement membranes in the glomerular tuft (Fig. 4). This deposition caused glomeruli to appear swollen. Occasionally, the glomerular deposits were deeply eosinophilic resembling fibrinoid material (Fig. 5). Glomerulopathy was not accompanied by infiltrations of leukocytes into the affected glomeruli. Frequently, rats with glomerulopathy also exhibited slight to mild, multifocal tubular degeneration (Fig. 6). Tubular degeneration was most severe in kidneys that also had the most severe glomerular lesions.
BMY-25282
TOXICITY
IN RATS
101
FIG.7. Pulmonary panarteritis in a female rat 9 weeks after a single dose of 0.5 mg/kg of BMY-25282. The lesion is characterized by intimal deposits of fibrin and infiltration of neutrophils, endothelial cell proliferation, and marked adventitial infiltration by leukocytes.
Drug-related arteritis (panarteritis) occurred sporadically in the 0.5 and 0.25 mg/kg groups. Arteritis was most commonly observed in the lung, but was also observed in the cecum, colon, and kidney. Pulmonary arteritis was characterized by hypertrophy of the tunica media, focal endothelial destruction, focal endothelial proliferation, subintimal infiltration with neutrophils and mononuclear cells, occasional subintimal deposits of fibrin, and severe adventitial fibrosis and infiltration with mononuclear cells (Fig. 7). Occasionally, the internal elastic membrane was disrupted. Adventital inflammation frequently extended into adjacent alveoli. Marked, diffuse necrotizing panarteritis of the cecum occurred in 1 male rat of the 0.25 mg/kg group (Fig. 8). The lesion involved several submucosal arteries and morphologically resembled the arteritis observed in the lung. The lesion also possessed two additional morphological features, which were fibrinoid
necrosis of the tunica media and periadventitial infilration with numerous eosinophils. Arteritis at this site was associated with severe submucosal edema and inflammation and multiple mucosal hemorrhages. Multifocal arteritis of the kidney, which also occurred in 1 male rat of the 0.25 mg/kg group, was characterized by prominent fibrinoid necrosis in many small arteries and arterioles in the outer cortex (Fig. 9). Other apparently drug-related histopathologic changes were observed in rats of the 0.5 and 0.25 mg/kg groups at the final necropsy. These included pulmonary arterial medial hypertrophy; slight to mild, fibrosing interstitial pneumonia; congestion and edema in numerous organs; focal skeletal muscle degeneration, mineralization, and inflammation (Fig. 10); hepatic subcapsular hemorrhages; lymphoid depletion in spleen and thymus; resolving inflammation of the tail at injection sites; at-
102
BREGMAN
ET AL.
qi >m.#-
i”-
FIG. 8. Artel ritis in submucosal vessel of cecum of a male rate treated with 0.25 mg/kg of BMY-25282, after a 9-week observation period.
rophy of the uterus and pancreas; and a decrease in secretion volume in the prostate and seminal vesicles. Atrophy of the uterus and pancreas and decreased secretion volume in prostate and seminal vesicles were attributed to the declining nutritional state of rats in affected dose groups rather than to a direct drug effect. Declining nutritional status was reflected by decreased body weights and organ weights of individual rats in affected dose groups. The cause of lymphoid depletion of spleen and thymus observed in rats of the terminal necropsy was not immediately clear. Lym-
phoid depletion was not detected by differential leukocyte counts or by absolute lymphocyte counts in rats of this study. Tail lesions were probably due to inadvertent perivascular injection of BMY25282 and the drug’s apparent, locally irritating properties. The bone marrow and gastrointestinal toxicities observed at the interim necropsy were not observed in any rat which survived beyond Day 7. Vehicle-related histopathologic changes were not observed in any rat treated with 0.1% Pluronic F-68 diluent alone.
BMY-25282
TOXICITY
IN RATS
FIG. 9. Arteritis in a renal vessel of a male rat 9 weeks after a single dose of 0.25 mg/kg of BMY-25282. Deeply stained fibrin deposits are present on the intimal surface.
FIG. 10. Multifocal skeletal muscle (psoas) degeneration, mineralization, rat 9 weeks after a single dose of03 mg/kg of BMY-25282.
and intlammation
in a male
104
BREGMAN
ET AL.
FIG. 11, Arteritis in the pancreas of a male rat 9 weeks after five consecutive daily doses of 0.05 mg/kg of BMY-25282.
FIG. 12. Arteritis in the testis of a rat 9 weeks after five consecutive daily doses of 0.05 mg/kg of BMY25282.
BMY-25282
TOXICITY
IN RATS
105
FIG. 13. Arteritis in the epididymis of a rat 9 weeks after five consecutive daily doses of 0.05 mg/kg of BMY-25282.
Histopathologic Multiple-Dose
Findings Study
in the Daily
X5
Drug-related histopathologic findings at the interim (Day 9) necropsy were limited to mild bone marrow hypocellularity in 1 male rat of the 0.5 mg/kg group. At the final necropsy on Day 67, however, several drug-related histopathologic changes were observed. All of these changes at that time were limited to the 0.05 mg/kg group. Drug-related cardiotoxicity was observed in 8/ 10 rats of the 0.05 mg/kg group. Cardiotoxicity was constituted of three related cardiac lesions: myocardial degeneration, ventricular hypertrophy, and slight nonsuppurative perivasculitis. The morphologic appearance of the BMY-25282-related cardiomyopathy in this study was similar to that in the singledose study. Severely affected myocardial foci also appeared more vascular than normal, perhaps because myolysis had allowed vascu-
lar channels more room in which to expand. Multiple hemorrhages were observed in these foci. Foci of myocardial degeneration appeared in both ventricles without a particular affinity for either ventricle. Myocardial degeneration was first observed on Day 29 of this study in a high dose (0.05 mg/kg) female. Cardiomyopathy in that rat was slightly different from that of other affected rats in that the degenerate foci were infiltrated with a greater number of inflammatory cells than were degenerate foci of rats that died or were sacrificed later in the study. Drug-related glomerulopathy was observed in 9 of 10 rats of the 0.05 mg/kg group. Glomerulopathy was morphologically similar to that observed in rats of the single-dose study. Occasionally changes in the glomerular tuft were accompanied by a thickened, sclerotic Bowman’s capsule. As in the singledose study, multifocal tubular degeneration was frequently observed concomitantly with
106
BREGMAN
glomerulopathy. Glomerulopathy was first observed in a high dose male that died on Day 49. Drug-related arteritis (panarteritis) was observed in pulmonary arteries of 2/ 10 rats of the 0.05 mg/kg group and in the pancreas, testis, and epididymis of 1 high dose male rat (Figs. 1 l- 13). The histopathologic appearance of the arteritis in this study closely resembled the arteritis observed in the singledose study. In the lung, pancreas, and testis, arteritis was associated with inflammation, necrosis, or atrophy in adjacent tissue. Other apparently drug-related histopathologic changes which occurred in the 0.05 mgf kg group at the termination of the multipledose study included pulmonary arterial medial hypertrophy; pulmonary edema and focal hemorrhage; slight interstitial pneumonia with fibrosis; hepatic centrolobular fatty change, atrophy, and necrosis; hepatic subserosa1 hemorrhages; lymphoid depletion in spleen and thymus; congestion in multiple organs; uterine atrophy; and subcutaneous edema. No vehicle-related histopathologic changes were observed in rats treated with 0.1% Pluronic F-68 diluent alone.
HistopathologicFindings in the WeeklyX5, Multiple-DoseStudy Drug-related histopathologic changes were not observed in the heart or lungs of BMY25282-treated rats. Drug-related increases in the incidence and/or severity of tubular degeneration and glomerulopathy were observed in BMY252828-treated male rats. Slight to mild thickening of glomerular membranes was observed in 2 of 5 BMY-25282-treated males, while slight to severe tubular degeneration was present in 5 of 5 BMY-25282-treated males and in 1 of 5 control males. DISCUSSION The toxicity of BMY-25282 in rats, as shown in this report, varies significantly from
ET AL.
the toxicity of BMY-25282’s parent compound, mitomycin C, as it has been previously reported (Philips et al., 1960) and demonstrated in a pilot study (unpublished data, Bristol-Myers Co., 1984). Although both drugs share certain cytotoxic, acute toxicities, including gastrointestinal epithelial necrosis and degeneration, splenic lymphoid depletion, and bone marrow hypocellularity, significant differences in chronic toxicity exist between the two compounds. The delayed irreversible cardiomyopathy, glomerulopathy, and arteritis caused by BMY-25282 administration have not been shown with mitomycin C administration, either acutely or after a 9-week dose-free observation period. The heart was the major target organ of BMY-25282 in both the single-dose and daily X5, multiple-dose studies of this report. Of the various cardiotoxic lesions, myocardial degeneration (cardiomyopathy) was considered the most important in terms of its high incidence and its lethality. BMY-25282-related cardiomyopathy was considered to be the primary cause of congestive heart failure as evidenced by hydrothorax and, to a lesser extent, by pulmonary congestion and edema, subcutaneous edema, hydropericardium, and congestion of multiple organs in rats that died prior to the termination of both studies. In both studies, cardiomyopathy was delayed in onset and irreversible. Although mitomytin C has been reported to produce myocarditis in Wistar rats (Levillain and Cluzan, 1973) endocardial and pericardial hemorrhages in dogs (Philips et al., 1960) and radiomimetic myocardial changes in man (Suzuki and Niibe, 1975) mitomycin C-related cardiac changes and BMY-25282-related cardiomyopathy were morphologically and biologically dissimilar. Indeed, the morphology of BMY-25282-related myocardial degeneration strongly resembled the chronic myocardial degeneration caused by anthracyclines in the rat (Buyniski and Hirth, 1980; Olson and Capen, 1978; Mettler et al., 1977), mouse (Bertazzoli et al., 1979; Rosenoff et al., 1975) rabbit (Olson et al., 1974), and human (Le-
BMY-25282
TOXICITY
frak et al., 1975; Halazun et al., 1974). Hydrothorax, delayed onset, and chronic course are other features of myocardial degeneration shared by both BMY-25282 toxicity and anthracycline toxicity. A major difference between the two drug-induced, delayed chronic cardiotoxicities in rats is that anthracycline toxicity depends upon the administration of multiple low doses (Chalcroft et al., 1973; Philips et al., 1975), which are cumulative, whereas, BMY-25282 cardiotoxicity can be induced by a single dose. Several of the other chronic toxicities reported in the singledose and daily X5, multiple-dose studies were considered to be the result of cardiac damage and subsequent heart failure. These included pulmonary arterial medial hypertrophy, pulmonary edema and focal hemorrhage, slight interstitial pneumonia with slight fibrosis, hepatic changes (fatty change, atrophy, necrosis, and subserosal hemorrhages), subcutaneous edema, and congestion in multiple organs. It may be useful to note that aspartate aminotransferase and creatine kinase, enzymes which may indicate muscle damage, were neither predictive of nor diagnostic for myocardial damage at 4 days after dosing or at approximately 9 weeks after dosing. In the case of CK, no drug-related elevations were seen at either the early or late sampling time in either study. The absence of elevated CK in the face of grossly and microscopically evident cardiomyopathy may indicate that (1) the type of myocyte damage did not result in measurable leakage of CK or perhaps (2) the initial insult to myocardial tissue, the resultant leakage of CK, and clearance of CK all occurred at a time between the two sampling times. Although AST was elevated in some individual rats of the high and intermediate dose groups of each study, AST elevations correlated imperfectly with rats that had microscopic evidence of cardiomyopathy. Glomerulopathy was another prominent BMY-25282~related toxicity in both studies that has not been reported with mitomycin C administration. The incidence of this lesion
IN RATS
107
was high in the 0.5 and 0.25 mg/kg groups of the single-dose study and in the 0.5 mg/kg group of the daily X5, multiple-dose study. Tubular degeneration, which has been reported to be a chronic liability of mitomycin C (Philips et al., 1960) was frequently associated with BMY-25282-related glomerulopathy. Tubular degeneration usually occurred in conjunction with glomerulopathy and its severity tended to parallel the severity of glomerulopathy. The hypoproteinemia observed in the single-dose study and the increased BUN and creatinine levels and proteinuria which were observed in both studies were attributed primarily to glomerular damage and secondarily to tubular damage. The hypoproteinemia in the single-dose study was characterized by slightly decreased albumin and normal globulin concentrations, thus resulting in a slight decrease in albumin to globulin ratio. Neither the decrease in serum protein nor the decrease in albumin was considered to be severe enough to affect intravascular osmotic pressure in hypoproteinemic rats. Therefore, the hydrothorax observed at necropsy was considered to be primarily due to cardiomyopathy rather than to nephrotic syndrome alone. Glomerulopathy in these two studies was delayed in onset. The pathogenesis of the lesion is unclear. The morphology of the lesion, however, bears a certain resemblance to glomerulonephritis in chronic serum sickness, which is caused by the circulation and subsequent deposition of foreign protein in soluble immune complexes. Arteritis, while occurring less frequently in both studies than cardiotoxicity or nephrotoxicity, was considered a significant BMY25282-related toxicity for three reasons. First, arteritis showed the potential to occur in multiple organs in the same individual rat. Arteritis occurred in the pancreas, testis, and epididymis of one individual, in the kidney, colon, and cecum of another individual, and only in the lung of several other rats. Second, in each of the organs where arteritis occurred, arteritis was associated with various degrees
108
BREGMAN
of inflammation, necrosis, or atrophy. In the lung, the most frequently involved organ, several vessels throughout this organ were involved, thus presenting the potential for widespread pulmonary damage due to infarction and embolism. Third, arteritis as a clinical entity is difficult to diagnose and monitor, especially considering the confusing, complicated sets of clinical signs that can occur with arteritis present in multiple, randomly affected organs. As in the case of glomerulopathy and cardiomyopathy, arteritis was delayed in onset. Likewise, the pathogenesis of the lesion is unknown, although clearly related to treatment with BMY-25282. Also, as in the case of glomerulopathy, the histopathologic appearance of BMY-25282-related arteritis resembles that observed in serum sickness (immune complex disease). As previously mentioned, BMY-25282-related cardiotoxicity closely resembles anthracycline-caused cardiotoxicity, although the anthracyclines are not chemically similar to BMY-25282. Anthracycline and BMY25282 toxicities also share at least three other toxic lesions, namely, chronic sclerotic glomerulopathy, tubular degeneration, and arteritis. Rats administered daunomycin, like those administered BMY-25282, developed diffusely fibrotic glomeruli; some of which were obliterated, while some had foci of fibrinoid necrosis. These rats also had arteritis in the kidney, pancreas, heart, skeletal muscle, and intestinal mesentery (Sternberg et al., 1972). Glomerular sclerosis associated with severe tubular and interstitial changes has been reported in rats treated with Adriamycin (Bertani et al., 1983; Mettler et al., 1977; Philips et al., 1975) and with carminomycin (Hirth, 1980). Hypocellular swollen glomeruli expanded by eosinophilic mesangial deposits have also been observed in rats treated with daunorubicin (Deprez-DeCampeneere et al., 1982). However, vacuolation of glomerular epithelial cells, which is reported to be a prominent feature of chronic Adriamycin-induced glomerular damage in
ET
AL.
rats (Giroux et al., 1984), was not a feature of BMY-25282-related glomerulopathy. Comparison of the three studies of this report illustrates that BMY-25282 toxicity is schedule dependent. The dosing schedule in the weekly X5, multiple-dose study produced less toxicity than the single-dose schedule at 0.25 mg/kg or the daily ~5 schedule using 0.05 mg. The latter two schedules produced cardiotoxicity, pulmonary toxicity, arteritis, and a greater degree of nephrotoxicity than the weekly X5 study, which produced only nephrotoxicity. In each study, the total cumulative dose was 0.25 mg/kg for the pertinent dose group.
REFERENCES BERTANI, T., ROCCHI, G., MECCA, G., SACCHI,G., AND REMUZZI, G. (1983). Adriamycin-induced chronic proteinuria: A new model of glomerular focal sclerosis (abstract). Kidney Int. 23, 192. BERTAZZOLI, C., BELLINI, O., MAGRINI, U., AND ToSANA, M. G. (1979). Quantitative experimental evaluation of Adriamycin cardiotoxicity in the mouse. Cuncer Treat. Rep. 63, 1877-1883. BRADNER, W. T., ROSE, W. C., SCHURIG, J. E., FLORCZYK, A. P., HUFTALEN, J. B., AND CATINO, J. J. (1985). Antitumor activity and toxicity in animals of BMY-25282, a new mitomycin derivative. Cancer Res. 45,6475-648 1. BRECHER, G. ( 1949). New methylene blue as a reticulocyte stain. Amer. J. Clin. Pathol. 19,895896. Bristol-Myers Company (1984). A Multiple Dose Intravenous Toxicity Study of Mitomycin C in Rats, Study Code No. BL-6782-001-I l-83, unpublished data. Pharmaceutical Research and Development Division, Department of Pathology and Toxicology, Evansville, IN. BUYNISKI, J. P., AND HIRTH, R. S. (1980). Anthracycline cardiotoxicity in the rat. In Anthracyclines: Current Status and New Developments (S. T. Crooke and S. D. Reich, Eds.), pp. 157-170. Academic Press, New York. CHALCROFT, S. C. W., GAVIN, J. B., AND HERDSON, P. B. (1973). Fine structural changes in rat myocardium induced by daunorubicin. Pathology 5,99-105. CROUT, J. R. ( 198 1). FDA Memorandum ofJuly 9,1981. Director, Bureau of Drugs (J. R. Crout), to Associate Director for New Drug Evaluation (M. J. Finkel). DEPREZ-DE~AMPENEERE, D., JAENKE, R., AND TROUET, A. (1982). Comparative cardiac and renal
BMY-25282
TOXICITY
toxicity of daunorubicin in the rat and rabbit. Cancer Treat. Rep. 66,395-391. GIROUX, L., SMEESTERS,C., BOURY, F., FAURE, M. P., AND JEAN, G. (1984). Adriamycin and AdriamycinDNA nephrotoxicity in rats. Lab. Invest. 50, 190-196. HALAZUN, J. F., WANGER, H. R., GAETA, J. F., AND SINKS, L. F. (1974). Daunorubicin cardiotoxicity in children with acute lymphocytic leukemia. Cancer 33, 545-554.
HIRTH, R. S. (1980). Other toxicities associated with anthracyclines in animal systems. In Anthracyclines: Current Status and New Developments (S. T. Crooke and S. D. Reich, Eds.), pp. 221-240. Academic Press, New York. KIRK, R. E. (1968). Experimental Design: Proceduresfor Behavioral Sciences, pp. 69-98. Brooks/Cole, Belmont, CA. LEFRAK, E. A., PITHA, J., ROSENHEIM, S., O’BRYAN, R. M., BURGESS,M. A., AND GOTTLIEB, J. A. (1975). Adriamycin (NSC- 123 127) cardiomyopathy. Cancer Chemother. Rep. 6,203-208. LEVILLAIN, R., AND CLUZAN, R. (1973). Cardiac toxicity of antimitotic drugs. In Proceedings ofthe 3rd Meeting the European Association for Cancer Research, Heidelberg, p. 100. METTLER, F. P., YOUNG, D. M., AND WARD, J. M. (1977). Adriamycin-induced cardiotoxicity (cardio-
IN RATS
109
myopathy and congestive heart failure) in rats. Cancer Res.37,2105-2713. OLSEN, H. M., AND CAPEN, C. C. (1978). Chronic cardiotoxicity of doxorubicin (Adriamycin) in the rat: Morphologic and biochemical investigations. Toxicol. Appl. Pharmacol. 44,605-6 16. OLSEN, H. M., YOUNG, D. M., PRIEUR, D. J.. LEROY, A. F., AND REAGAN, R. L. (1974). Electrolyte and morphologic alterations of myocardium in Adriamytin-treated rabbits. Amer. J. Pathof. 77,439-450. PHILIPS, F. S., GILLADOGA, A., MARQUARDT, H., STERNBERG, S. S., AND VIDAL, P. M. (1975). Some observations on the toxicity of Adriamycin (NSC123127). Cancer Chemother. Rep. 6,111-181. PHILIPS, F. S., SCHWARTZ, H. S., AND STERNBERG,S. S. (1960). Pharmacology of mitomycin C. 1.Toxicity and pathologic effects. Cancer Res. 20, 1354- 136 1. ROSENOFF,S. H., OLSEN, H. M., YOUNG, D. M., BosTICK, F., AND YOUNG, R. C. (1975). Adriamycin-induced cardiac damage in the mouse: A small-animal model of cardiotoxicity. J. Natl. Cancer Inst. 55, 19 l194.
STERNBERG, S. S., PHILIPS, F. S., AND CRONIN, A. P. (1972). Renal tumors and other lesions in rats following a single intravenous injection of daunomycin. CancerRes. 32, 1029-1036. SUZUKI, Y., AND NIIBE, H. (1975). Radiation-induced heart injury: Radiopathological study. Kita Kanto Igaku 25,395-407.
Single-Dose and Multiple-Dose intravenous Toxicity Studies of BMY-25282 in Rats C. L. BREGMAN, C. R. COMERESKI, R. A. BUROKER, H. MADISSOO, AND G. H. HOTTENDDRF Bristol-Myers
Company,
Pathology
Pharmaceutical and Toxicology
Single-Dose and Multiple-Dose MAN,C.L.,COMERESIU,C.R.,BUROKER, DORF, G. H. (1987). Fundam.
R. S. HIRTH,
Research and Development Division, Syracuse, New York 13221-4755
Intravenous Toxicity Studies of BMY-25282
Department
of
in Rats. BREG
R. A.,HIRTH,R.S.,MADISSOO,H.,ANDHOTTEN-
Appl. Toxicol. 9, 90-109. Singledose and multiple-dose (daily X5 and weekly X5) intravenous toxicity studies in rats were conducted to determine the possible acute and delayed toxicity of BMY-25282 (7-N-(dimethylaminomethylene) mitomycin C), a potential anticancer drug. Rats in the single-dose study received either 0.05,0.25, or 0.50 mgl kg (0.3, 1.5, or 3.0 mg/m’) of BMY-25282; rats in the daily X5 multiple-dose study received doses of 0.005,0.025, or 0.050 mg/kg (0.03, 0.15, and 0.3 mg/m2) of BMY-25282 once each day for 5 days; and rats in the weekly X5 multiple-dose study received 0.05 mg/kg of BMY25282. All doses were in 0.1% Pluronic F-68 diluent. Acute toxicities included gastrointestinal epithelial necrosis, myelosuppression, and splenic lymphoid depletion in the high and intermediate dose groups in the single-dose study and myelosuppression in the high dose group of the daily X5 multiple-dose study. One death in a high dose male of the single-dose study was attributed to acute gastrointestinal and lymphoid toxicity. Between the interim necropsy on Day 5 or 9 and termination of the 9-week dose-free observation period, 9/20 rats of the high and intermediate dose groups of the single-dose study and 4/10 high dose rats in the daily X5 multiple-dose study died, primarily due to hydrothorax and congestive heart failure caused by delayed, drugrelated myocardial degeneration. The most prominent drug-related histopatbologic changes observed in rats of both the single-dose study and the daily X5 studies were myocardial degeneration (cardiomyopathy), glomerulopathy with tubular degeneration, and necrotizing arteritis. These three changes, observed at 0.5 and 0.25 mg/kg in the single-dose study and at 0.05 mg/ kg/day in the multiple-dose (daily X5) study, were delayed in onset and irreversible. Drug-related tubular degeneration and slight glomerulopathy were observed in male BMY-25282-treated rats in the weekly X5 study, but cardiotoxicity, pulmonary arteritis, hydrothorax, and lethality were not observed. The diluent, Pluronic F-68, was not associated with any morphologic or clinicopathologic changes. A single-dose of 0.05 mg/kg or 5 daily doses of 0.025 and 0.005 mg/kg of BMY-25282 were considered nontoxic doses in rats. A cumulative dose of 0.25 m&kg, which caused cardiotoxicity in the daily X5 study, was not cardiotoxic in the weekly X5 study. These results indicate that the delayed cardiotoxicity of BMY-25282 is schedule dependent. o 1987 Society of Toxicology.
BMY-25282, 7-N-(dimethylaminomethylene)-mitomycin C, is one of a novel series of amidino mitomycin derivatives. BMY25282 has been shown to have markedly superior antitumor effects compared to mitomycin C in a number of in vivo mouse tumor models and is considered to be two to four times more potent than mitomycin C (Bradner et al., 1985). 0272-0590187 $3.00 Copyright 0 1987 by the Society of Toxirology. All rights of reproduction in any form reserved.
The single-dose and multiple-dose (daily X5 and weekly X5) intravenous toxicity studies in rats reported herein were conducted to assessthe acute and delayed toxicity of BMY25282 and its diluent Pluronic F-68 and to establish nontoxic doses for different treatment schedules. The design of the single-dose and the daily X5 multiple-dose studies was based on National Cancer Institute guide90
BMY-25282
TOXICITY
91
IN BATS
TABLE 1 BMY-25282 Dosing schedule
Dose budoWe
~SESIN~TTOXICI~%KJD~ES
Dose (mg/m*/day)
Doseas%of mouse LDIO”
Total cumulative dose b-m&)
Single dose
0.5 0.25 0.05
3.00 1.50 0.30
67 33 7
0.5 0.25 0.05
Daily x5
0.05 0.025 0.005
0.30 0.15 0.03
27 13 3
0.25 0.125 0.025
Weekly ~5
0.05
0.30
-b
0.25
a Single-dose mouse LD 10 was 1.49 mg/kg. The daily X5, multiple-dose LD 10 was 0.37 mg/kg. b A weekly x5 multiple-dose mouse lethality study was not conducted.
lines for the preclinical evaluation of anticancer drugs (Crout, 1981). METHODS Experimental design. In each of the single-dose and multiple-dose (daily ~5) studies, 100 rats (50 males and 50 females) were divided into five groups of 10 males and 10 females each. In the single-dose study, rats of three groups received a single intravenous injection via the caudal vein of either 0.05,0.25, or 0.50 mg/kg of BMY25282 in 0.1% Pluronic F-68 diluent. In the multipledose study, rats of three groups received either 0.005, 0.025, or 0.05 mg/kg/day of BMY-25282 in 0.1% Pluronit F-68 diluent via the caudal vein for 5 days. In each study, a fourth group of rats served as negative controls and was administered intravenous injections of Water for Injection, USP. A fifth group in each study served as vehicle controls and was administered either one or five injections of0. 1% Pluronic F-68 diluent. An interim necropsy on 5 rats/sex/group was conducted 4 or 5 days after the last dose. The remaining animals were sacrificed after a 9-week, drug-free observation period. In the weekly x5 multiple-dose study, 5 males and 5 females were administered 0.05 mg/kg of BMY-25282 in 0.1% Pluronic F-68 diluent intravenously once per week for 5 consecutive weeks. Another 5 males and 5 females served as negative controls and were administered five injections of 0.1% Pluronic F-68 diluent according to the same schedule. The last dose of BMY25282 was followed by a IO-week observation period. Necropsies were performed on all rats at the termination of the observation period. The doses used in these rat studies are listed in Table 1 in terms of mg/kg, mg/m2, and as percentages of the
mouse LDlO. Single-dose and daily x5 multiple-dose intravenous lethality studies in Swiss-Webster mice had been previously conducted in our laboratory to determine the LD50 and LD 10 values. The combined LD 10 for both males and females in the single-dose study and the daily X5, multipledose study were 1.49 mg/kg and 0.37 mgfkgfday, respectively. Test materials. The vehicle, Pluronic F-68, a block polymeric nonionic surfactant, was prepared as a 0. I %
TABLE 2 MORTALITYANDTIMEOFDEATHOF BMY-25282-TREATEDRATS
Number of deaths Dose (mg/kg)
M
Single-dose
0” 0.05 0.25 0.50
O/20 o/10 l/IO 3/10
O/20 o/10 3/10 35,50, 5 I,60 2110 7,45,60,63 (X2)
Daily x5
0” 0.005 0.025 0.050
o/20 o/10 O/10 2/10
O/20 o/10 O/10 2/10
29,49,62,64
WeeklyX5
Ob 0.05
O/5 015
Of5 Of5
-
Schedule
F
Day of death of individual rats
’ Two control groups (water control and vehicle control) were combined for this table. ’ Control group received vehicle (Pluronic F-68).
BREGMAN
92
ET AL.
TABLE 3 SUMMARY OF MAJOR CLINICOPATHOLOGIC CHANGES IN BMY-~~~~~-TREATED Single-dose study
RATS Weekly X5 study
Daily x5 study
Dose of BMY-25282 (mg/kg): Number ofratsexamined”:
Oh 40
0.05 20
0.25 20
0.50 20
Ob 40
0.005 20
0.025 20
0.05 20
0’ 10
0.05 10
Increased BUN Increased creatinine Hypoproteinemia Proteinuria Anemia Leukocytosis (neutrophilia) Leukopenia, neutropenic and lymphopenic Reticulocytopenia, early Reticulocytosis, late Elevated aspartate aminotransferase
-d -
-
+ + + + + +
+ + + + + +
-
-
-
+ + + -
NM -
+ + NM -
-
-
+ -
+ +
-
-
-
-
-
+ -
-
-
+
+
-
-
+
+
-
-
a Each dose group was composed of equal numbers of male and female rats. b Two control groups, one given water and the other given Pluronic F-68 vehicle, were combined for this table. ‘Control group received Pluronic F-68 vehicle. d -, no drug-related changes observed; +, drug-related changes observed, NM, parameters not measured in rats of this treatment group.
solution in sodium citrate and citric acid. BMY-25282 was dissolved in the 0. I % Pluronic F-68 vehicle at concentrations ranging from 0.005 to 0.25 mg/ml. The resulting solutions were administered intravenously at 2.0 ml/kg except for the 0.005 mg/ml solution which was given at 1.Oml/kg. The solutions were prepared immediately prior to dosing each day and were used within the 3-hr time period for which solution stability had been established. Dosing solutions were assayed for BMY25282 content on Day 1 of each study. Animals and animal husbandry. Sprague-Dawley (Crl:CD(SD)BR) rats (Charles River Breeding Laboratories, Inc., Wilmington, MA), which were 6 weeks old upon arrival, were used. The rats were subjected to a general health examination and a conditioning period of 2 weeks prior to study inception. Healthy rats selected for the study were subdivided by sex, ranked by body weight, and randomly divided into groups having approximately equal average body weights. Rats were individually identified with numbered ear tags and housed in individual wire-bottomed stainless-steel cages in temperature-controlled rooms. Ground food (Purina Laboratory Chow, Ralston Purina, St. Louis, MO) and water were offered ad libitum except as necessary to obtain blood and urine samples for clinical pathology determinations. In life observations, food intake, and body weights. Rats were observed daily for signs of toxicity and changes
in general health and behavior. Body weights were determined prior to drug administration, daily during drug administration in the multiple-dose studies, and once a week thereafter in all studies. General health and ophthalmological examinations were conducted on each rat prior to study inception and prior to scheduled necropsies. Food intake was determined once after the end of the treament period and once a week thereafter. Hematology. Blood samples for hematologic determinations were collected via the caudal vein from 5 rats/ sex/group on Days 4, 11, 35, and 60 in the single-dose study and on Days 8, 16,43, and 66 in the multiple-dose (daily X5) study, and on Days 35,64, and 84 in the multiple-dose (weekly X 5) study. Hematologic determinations included hemoglobin (Hgb) concentration, hematocrit (Hct), total erythrocytes (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLAT), and total leukocyte counts (WBC), all of which were determined using automated methods.’ Additionally, differential leukocyte count, absolute neutrophil and lymphocyte counts, and reticulocyte count (Brecher, 1949) were performed. ’ Ortho ELT-8/ds, Westwood, MA.
Ortho Diagnostic Systems, Inc.,
BMY-25282
TOXICITY
93
IN RATS
TABLE 4
Single-dose study Dose of BMY-25282 (mg/kg): Number ofratsexamined”: Heart Myocardial degeneration Perivasculitis, slight Ventricular hypertrophy Lung Arteritis Arterial medial hypertrophy Interstitial pneumonia with fibrosis Kidney Glomerulopathy Tubular degeneration Arteritis Thymus Lymphoid depletion Spleen Lymphoid depletion Pancreas, testis, epididymis, cecum, and/or colon Arteritis
Weekly X5 study
Daily X5 study
Ob 20
0.05 10
0.25 10
0.50 10
Ob 20
0.005 10
0.025 10
0.05 10
0’ 10
0.05 10
0 0 0
0 0 0
10 8 2
9 8 6
0 0 0
-d -
0 0 0
8 4 2
0 0 0
0 0 0
0
0
3
5
0
-
0
2
0
0
0
0
5
4
0
-
0
4
0
0
0
0
7
I
0
-
0
2
0
0
0 0 0
0 1 0
8 6 1
9 I 0
0 0 0
0 3 0
9 I 0
0 1 0
2 5 0
0
0
2
6
0
-
0
3
-
-
0
0
3
3
0
-
0
3
-
-
0
0
1
0
0
-
0
1
-
-
0 0 0
a Each dose group was composed of equal numbers of male and female rats. b Two control groups, one given water and the other given Pluronic F-68 vehicle, were combined for this table. ’ Control group received the vehicle, Pluronic F-68. d Organ of that dose group was not scheduled for examination. Clinicalchemistries. Urea nitrogen (BUN), fastingglucase (FBS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, total protein (TP), albumin (ALB), creatine kinase (CK), sodium, and potassium were measured in 5 rats/sex/group in each study at the interim and final necropsies on serum collected via cardiac puncture in the single-dose and daily X5 studies.2,3BUN and ALT were also performed on Days 11 and 35 in the single-dose study and on Days 16 and 43 in the multiple-dose study. Serum globulin fraction (GLOB) and albumin to globulin ratios were computed using TP and ALB data. Creatinine, BUN, ALT, AST, and total protein were performed on Days 35,64, and 84 of the weekly X5, multiple-dose study.
’ Centrifichem System 600, Baker Instruments, Allentown, PA. 3 Coming Model 450 Flame Photometer, Coming Scientific Instruments, Medfield, MA.
Urinalysis. Pooled urine samples of 5 male or 5 female (or surviving) rats which had been placed in metabolism cages for 18 hr were collected prior to each of the scheduled necropsies during the single-dose and multiple-dose (daily X5) studies. Eighteen-hour volume, specific gravity, pH, protein, ketones, glucose, bilirubin, and occult blood were determined on all samples! Urine sediment was microscopically examined for erythrocytes, leukocytes, epithelial cells, bacteria, and casts. Pathological examinations. All rats from all three studies were necropsied. Moribund rats and those at scheduled necropsies were euthanatized with an intraperitoneal injection of sodium pentobarbital. Representative sections of adrenal gland, bone, bone marrow, brain, cecum, colon, duodenum, epididymis, esophagus, eye, heart, ileum, jejunum, kidney, liver, lung, mammary
4Ames Urinalysis System, Miles Laboratories, Inc., Ames Division, Elkhart, IN.
94
BREGMAN
gland, mesenteric lymph node, ovary, pancreas, parathyroid, pituitary, prostate, salivary gland, seminal vesicles, skeletal muscle, testis, thyroid gland, trachea, urinary bladder, and uterus were collected from each rat of the appropriate sex in the single-dose and multiple-dose (daily X5) studies and were fixed in 10% neutral buffered formalin. Prior to fixation, the weights of the heart, liver, adrenal glands, thyroid glands, gonads, and uterus or prostate with seminal vesicles were recorded for each rat necropsied at scheduled necropsies. Representative sections from only heart, lung, and kidney were collected from rats of the multiple-dose (weekly X5) study and no organ weights were measured. Representative sections of organs and tissues were processed using conventional methods and were stained with hematoxylin and eosin. Statisticalevaluation.In life body weight gains, food intake, terminal body weights, and organ weight data (collected at each scheduled necropsy) were evaluated using a multiple t statistic computed with a pooled error term, at an overall significance level of p < 0.05. The Bonferroni inequality was used when sample sizeswithin all groups were not equal; the Dunnett procedure was used when all group sizes were equal (Kirk, 1968).
RESULTS
In Life Observations In the single-dose and daily X5 multipledose studies, drug-related clinical signs including emaciation, hunched body position, rough hair coat, and whole body discoloration (suggestive of anemia) were seen in rats treated with a single dose of 0.25 or 0.50 mg/ kg and with five daily doses of 0.05 mg/kg of BMY-25282. Respiratory distress was observed only in rats that subsequently died. Liquid feces were noted in one male treated with 0.50 mg/kg that died on Day 7. All clinical signs, except liquid feces, were delayed in onset and appeared in the latter half of the observation periods. Lower food intakes were noted in the latter half of the studies in rats of the single-dose study that received 0.25 or 0.50 mg/kg and in rats of the daily X5 multiple-dose study that received 0.05 m&kg/day. No drug-related clinical signs or changes in food intake were observed in rats in the
ET AL.
weekly X5, multiple-dose study or in any rat that was treated with 0.1% Pluronic F-68 diluent alone.
In Life Body Weights In the single-dose study, significant body weight losses occurred in 0.25 mg/kg males and 0.50 mg/kg males and females prior to the interim necropsy on Day 5. Lower body weights persisted in these rats until the termination of the study. Lower body weights, which were delayed in onset, were also observed in 0.05 mg/kg rats of the daily X5 multiple-dose study. Body weights of rats treated with 0.05 mg/kg of BMY-25282 on a weekly X5 schedule were comparable to those of controls. There were no vehicle-related changes in the vehicle control groups.
Mortality Deaths occurred in 4 of 20 (1 male and 3 females) of the 0.25 mg/kg dose group and 5 of 20 (3 males and 2 females) of the 0.50 mg/ kg group in the single-dose study (Table 2). With the exception of the death of one 0.50 mg/kg male that occurred on Day 7, all deaths occurred between Days 35 and 63. Deaths occurred in 2 of 10 males and 2 of 10 females of the 0.05 mg/kg group of the daily ~5, multiple-dose study on Days 29,49, 62, and 64. No deaths occurred in the weekly X5, multiple-dose study.
Organ Weightsand Organ to Body Weight Ratios There were no absolute or relative organ weight changes, which were considered treatment related, observed at the interim necropsy of the single-dose study (on Day 5) or the daily X5, multiple-dose study (on Day 9).
BMY-25282
TOXICITY
IN RATS
95
FIG. 1. BMY-25282-related cardiomyopathy in a female rat 9 weeks after a single dose of 0.5 mg/kg. Note marked vacuolation within sarcoplasm and between cardiac myocytes.
At the terminal necropsy of the single-dose study, mean relative heart weights of males and females of the 0.5 and 0.25 mg/kg dose groups were significantly increased. In the daily X5, multiple-dose study, mean relative heart and liver weights of 0.05 mgf kg males were significantly increased at the termination of the study. No vehicle-related effects on absolute or relative organ weights were seen in the vehicle control group of either study. Hematology Major drug-related clinicopathologic changes for all three studies are summarized in Table 3. Slight anemia was present in a few rats of the 0.5 and 0.25 mg/kg groups of the singledose study on Days 4,35, and 60, but not on Day 11. Anemia was accompanied by reticulocytopenia on Day 4. On Day 60, 0.5 mg/
kg female rats exhibited mild reticulocytosis. Slight drug-related increases in total leukocyte count with increases in relative and absolute neutrophil counts were observed in 0.5 and 0.25 mg/kg rats. No significant BMY-25282~related changes occurred in hematologic parameters in any treatment group of the daily X 5, multiple-dose study at any of the four sampling periods. In the weekly X5, multiple-dose study, BMY-25282-treated rats, especially females, exhibited drug-related decreases in mean total WBC count, absolute neutrophil count, and absolute lymphocyte count. Absolute neutropenia was noted on Day 35, but was not observed at subsequent sampling periods on Days 64 and 84. The incidence of absolute lymphopenia peaked on Day 64 and had subsided by Day $4. No vehicle-related changes were noted in hematologic parameters in vehicle control rats of any of the three studies.
96
BREGMAN
ET AL.
FIG. 2. Right ventricular myocardium of a female rat 9 weeks after a single dose of 0.5 mg/kg of BMY25282. Cardiomyopathy is characterized by myocytolysis, cellular pleomorphism, and dilatation of small vessels.
Clinical Chemistries Drug-related alterations in clinical chemistries in the single-dose study included progressive elevations of mean BUN in the 0.5 and 0.25 mg/kg groups beginning on Day 35, slight to mild proportional elevations of mean creatinine values in 0.5 mg/kg females and 0.25 mg/kg males at the termination of the study, slight dose-related decreases in total protein on Day 68 in the 0.5 and 0.25 mg/ kg groups, and elevation of aspartate aminotransferase in a few rats of the 0.5 and 0.25
mg/kg groups on Day 68. Other possibly drug-related alterations in clinical chemistries included hyponatremia in the 0.5 mg/kg group on Day 5 and decreased glucose values in 0.5 mg/kg females on Days 5 and 68. Drug-related alterations of clinical chemistries in the daily ~5, multiple-dose study included elevation of mean BUN in the 0.05 mg/kg males and females on Day 43 and in 0.05 mg/kg males on Day 68, elevated mean creatinine in 0.05 mg/kg males on Day 67, and elevation of AST on Day 67 in 1 of 3 remaining 0.05 mg/kg males and 3 of 5 of the 0.025 mg/kg females.
BMY-25282
TOXICITY
FIG. 3. Slight perivasculitis near a focus of cardiomyopathy single dose of0.5 mg/kg of BMY-25282.
In the weekly X5, multiple-dose study, drug-related alterations in clinical chemistries included a low incidence of abnormally elevated BUN and creatinine values in BMY25282~treated rats at each of the three sampling times. Urinalysis
Urinalysis revealed no drug-related findings on Day 5 in the single-dose study. However, on Day 60, urinalysis revealed proteinuria in males and females of 0.5 and 0.25 mg/
IN RATS
97
in the heart of a male rat 9 weeks after a
kg groups. Examination of urine sediment at that time revealed an increased number of leukocytes in males and females of the 0.5 mg/kg group and in males of the 0.25 mg/kg group. Epithelial cell numbers were increased in the urine sediments of males and females of both 0.5 and 0.25 mg/kg groups. Occult blood was increased in the urine of 0.25 mg/ kg females. Proteinuria was the only drug-related change observed in the urinalysis of the daily X5, multiple-dose study. Proteinuria occurred in 0.05 mg/kg males and females on Day 67.
98
BREGMAN
ET AL.
FIG. 4. Swollen glomerulus of a male rat 9 weeks after a single dose of 0.5 mg/kg of BMY-25282. ment membranes of capillaries are markedly thickened by homogeneous eosinophilic material.
Vehicle-related changes in urinalytical data were not observed in vehicle control rats of either study.
GrossPathologicalFindings At the interim necropsy on Day 5 of the single-dose study, watery gastrointestinal tract contents and perineal soiling in a few females of the 0.5 mg/kg group were the only BMY-25282-related, grossly visible changes. At the terminal necropsy of the single-dose study and in rats that died during the observation period, hydrothorax was frequently observed in the 0.5 and 0.25 mg/kg groups. Hydrothorax was considered the cause of death (secondary to congestive heart failure) in rats that died. Cardiac changes including enlargement, thickening or dilatation of the ventricles, tan myocardial foci, and hydropericardium were observed in rats of the 0.5 and 0.25 mg/kg groups. Other drug-related gross
Base-
findings observed in these two groups included diffuse tan or light brown discoloration and roughened, capsular surfaces of renal cortices; thymic atrophy; enlargement with hyperemia of perithymic lymph nodes; tail lesions; congestion in multiple organs; subcapsular hemorrhages and interlobar adhesions in the liver; atrophy of seminal vesicles, prostate, and uterus; rough hair coat; bloody nasal discharge; and perineal soiling. In the daily X5, multiple-dose study, no BMY-25282-related gross changes were observed in any rat at the interim necropsy on Day 9. All 4 rats that died prior to the termination ofthe daily ~5, multiple-dose study exhibited marked hydrothorax. These 4 rats also exhibited other signs of congestive heart failure, including ventral subcutaneous edema, pulmonary edema, and congestion in multiple organs. Other possibly drug-related, grossly visible changes in these 4 rats were heart en-
BMY-25282
TOXICITY
IN RATS
99
FIG. 5. Glomerulopathy in a male rat 9 weeks after a single dose of 0.5 mg/kg of BMY-25282. Fibrin or fibrinoid material is deposited along Bowman’s capsule, in vessels, and thickened membranes (arrows). Martius-scarlet-blue stain.
largement, uterine atrophy, subpleural petechiae, tan hepatic foci, petechiae in gastric glandular mucosa, bloody incrustations of the muzzle and forepaw, and yellow perineal soiling. Drug-related, grossly visible changes at the termination of the daily X5, multiple-dose study were limited to the high dose BMY25282-treated group. Grossly visible cardiac lesions were cardiac enlargement, thickening of ventricular myocardium, and pale myocat-dial foci. Other grossly visible changes included tan, pulmonary mottling, and pale discoloration and roughening of renal cortices. No grossly observable, drug-related lesions were present in the lung or heart of the BMY25282-treated rats of the weekly X5, multiple-dose study. However, swollen kidneys with roughened, mottled, tan and brown cortices were observed in 1 of 5 BMY-25282 treated males and were considered suggestive of BMY-25282-related nephrotoxicity.
No vehicle-related gross pathological changes were observed in vehicle control rats in any of the three studies.
HistopathotogicFindings in the Single-Dose Study Drug-related histopathologic changes observed in rats necropsied on Day 5 included glandular degeneration and necrosis in stomach, jejunum, ileum, cecum, and colon; bone marrow hypocellularity; and splenic lymphoid depletion. These toxicities were observed in the 0.5 and 0.25 mg/kg groups. At the terminal necropsy on Day 68, cardiotoxic lesions due to BMY-25282 were observed in nearly all rats of both sexes in the 0.5 and 0.25 mg/kg groups. A variety of cardiotoxic changes including ventricular hypertrophy, mural thrombosis, endarteritis, focal necrosis, perivasculitis, hemorrhage, congestion, and endocarditis were observed in the two dose
BREGMAN
ET AL.
FIG. 6. Chronic tubular degeneration and glomerulopathy in a rat 9 weeks after the administration of a single dose of BMY-25282. Tubular basement membranes are thickened and epithelial cells are atrophic. Glomerulus at top is enlarged and sclerotic. Occasional tubules are dilated.
groups; however, myocardial degeneration (cardiomyopathy) occurred with the greatest incidence (Table 4). The cardinal feature of the BMY-25282-related myocardial degeneration was focal to dilhrse vacuolation, both sarcoplasmic and intercellmar, throughout the myocardium of both ventricles (Fig. 1). Degenerate myocytes were further characterized by pleomorphism, focal variations in staining and loss of striations. Actual loss of myocytes and slight interstitial edema were observed in a few severely affected foci (Fig. 2). A slight, d&se increase in mononuclear cells and mast cells was frequently observed in degenerate foci, the increase in mononuclear and mast cells was often prominent around small vascular structures and constituted a slight perivascuhtis (Fig. 3). The earliest time point at which cardiomyopathy was observed was on Day 35 in a 0.25 mg/ kg female that died prior to the termination of the study.
Glomerulopathy caused by BMY-25282 administration occurred in most rats of both sexes in the 0.5 and 0.25 mg/kg groups. The earliest time point at which glomerulopathy was observed was on Day 45 in a 0.5 mg/kg male. Glomerulopathy was characterized microscopically by dense, diffuse, lightly eosinophilic, proteinaceous deposits along basement membranes in the glomerular tuft (Fig. 4). This deposition caused glomeruli to appear swollen. Occasionally, the glomerular deposits were deeply eosinophilic resembling fibrinoid material (Fig. 5). Glomerulopathy was not accompanied by infiltrations of leukocytes into the affected glomeruli. Frequently, rats with glomerulopathy also exhibited slight to mild, multifocal tubular degeneration (Fig. 6). Tubular degeneration was most severe in kidneys that also had the most severe glomerular lesions.
BMY-25282
TOXICITY
IN RATS
101
FIG.7. Pulmonary panarteritis in a female rat 9 weeks after a single dose of 0.5 mg/kg of BMY-25282. The lesion is characterized by intimal deposits of fibrin and infiltration of neutrophils, endothelial cell proliferation, and marked adventitial infiltration by leukocytes.
Drug-related arteritis (panarteritis) occurred sporadically in the 0.5 and 0.25 mg/kg groups. Arteritis was most commonly observed in the lung, but was also observed in the cecum, colon, and kidney. Pulmonary arteritis was characterized by hypertrophy of the tunica media, focal endothelial destruction, focal endothelial proliferation, subintimal infiltration with neutrophils and mononuclear cells, occasional subintimal deposits of fibrin, and severe adventitial fibrosis and infiltration with mononuclear cells (Fig. 7). Occasionally, the internal elastic membrane was disrupted. Adventital inflammation frequently extended into adjacent alveoli. Marked, diffuse necrotizing panarteritis of the cecum occurred in 1 male rat of the 0.25 mg/kg group (Fig. 8). The lesion involved several submucosal arteries and morphologically resembled the arteritis observed in the lung. The lesion also possessed two additional morphological features, which were fibrinoid
necrosis of the tunica media and periadventitial infilration with numerous eosinophils. Arteritis at this site was associated with severe submucosal edema and inflammation and multiple mucosal hemorrhages. Multifocal arteritis of the kidney, which also occurred in 1 male rat of the 0.25 mg/kg group, was characterized by prominent fibrinoid necrosis in many small arteries and arterioles in the outer cortex (Fig. 9). Other apparently drug-related histopathologic changes were observed in rats of the 0.5 and 0.25 mg/kg groups at the final necropsy. These included pulmonary arterial medial hypertrophy; slight to mild, fibrosing interstitial pneumonia; congestion and edema in numerous organs; focal skeletal muscle degeneration, mineralization, and inflammation (Fig. 10); hepatic subcapsular hemorrhages; lymphoid depletion in spleen and thymus; resolving inflammation of the tail at injection sites; at-
102
BREGMAN
ET AL.
qi >m.#-
i”-
FIG. 8. Artel ritis in submucosal vessel of cecum of a male rate treated with 0.25 mg/kg of BMY-25282, after a 9-week observation period.
rophy of the uterus and pancreas; and a decrease in secretion volume in the prostate and seminal vesicles. Atrophy of the uterus and pancreas and decreased secretion volume in prostate and seminal vesicles were attributed to the declining nutritional state of rats in affected dose groups rather than to a direct drug effect. Declining nutritional status was reflected by decreased body weights and organ weights of individual rats in affected dose groups. The cause of lymphoid depletion of spleen and thymus observed in rats of the terminal necropsy was not immediately clear. Lym-
phoid depletion was not detected by differential leukocyte counts or by absolute lymphocyte counts in rats of this study. Tail lesions were probably due to inadvertent perivascular injection of BMY25282 and the drug’s apparent, locally irritating properties. The bone marrow and gastrointestinal toxicities observed at the interim necropsy were not observed in any rat which survived beyond Day 7. Vehicle-related histopathologic changes were not observed in any rat treated with 0.1% Pluronic F-68 diluent alone.
BMY-25282
TOXICITY
IN RATS
FIG. 9. Arteritis in a renal vessel of a male rat 9 weeks after a single dose of 0.25 mg/kg of BMY-25282. Deeply stained fibrin deposits are present on the intimal surface.
FIG. 10. Multifocal skeletal muscle (psoas) degeneration, mineralization, rat 9 weeks after a single dose of03 mg/kg of BMY-25282.
and intlammation
in a male
104
BREGMAN
ET AL.
FIG. 11, Arteritis in the pancreas of a male rat 9 weeks after five consecutive daily doses of 0.05 mg/kg of BMY-25282.
FIG. 12. Arteritis in the testis of a rat 9 weeks after five consecutive daily doses of 0.05 mg/kg of BMY25282.
BMY-25282
TOXICITY
IN RATS
105
FIG. 13. Arteritis in the epididymis of a rat 9 weeks after five consecutive daily doses of 0.05 mg/kg of BMY-25282.
Histopathologic Multiple-Dose
Findings Study
in the Daily
X5
Drug-related histopathologic findings at the interim (Day 9) necropsy were limited to mild bone marrow hypocellularity in 1 male rat of the 0.5 mg/kg group. At the final necropsy on Day 67, however, several drug-related histopathologic changes were observed. All of these changes at that time were limited to the 0.05 mg/kg group. Drug-related cardiotoxicity was observed in 8/ 10 rats of the 0.05 mg/kg group. Cardiotoxicity was constituted of three related cardiac lesions: myocardial degeneration, ventricular hypertrophy, and slight nonsuppurative perivasculitis. The morphologic appearance of the BMY-25282-related cardiomyopathy in this study was similar to that in the singledose study. Severely affected myocardial foci also appeared more vascular than normal, perhaps because myolysis had allowed vascu-
lar channels more room in which to expand. Multiple hemorrhages were observed in these foci. Foci of myocardial degeneration appeared in both ventricles without a particular affinity for either ventricle. Myocardial degeneration was first observed on Day 29 of this study in a high dose (0.05 mg/kg) female. Cardiomyopathy in that rat was slightly different from that of other affected rats in that the degenerate foci were infiltrated with a greater number of inflammatory cells than were degenerate foci of rats that died or were sacrificed later in the study. Drug-related glomerulopathy was observed in 9 of 10 rats of the 0.05 mg/kg group. Glomerulopathy was morphologically similar to that observed in rats of the single-dose study. Occasionally changes in the glomerular tuft were accompanied by a thickened, sclerotic Bowman’s capsule. As in the singledose study, multifocal tubular degeneration was frequently observed concomitantly with
106
BREGMAN
glomerulopathy. Glomerulopathy was first observed in a high dose male that died on Day 49. Drug-related arteritis (panarteritis) was observed in pulmonary arteries of 2/ 10 rats of the 0.05 mg/kg group and in the pancreas, testis, and epididymis of 1 high dose male rat (Figs. 1 l- 13). The histopathologic appearance of the arteritis in this study closely resembled the arteritis observed in the singledose study. In the lung, pancreas, and testis, arteritis was associated with inflammation, necrosis, or atrophy in adjacent tissue. Other apparently drug-related histopathologic changes which occurred in the 0.05 mgf kg group at the termination of the multipledose study included pulmonary arterial medial hypertrophy; pulmonary edema and focal hemorrhage; slight interstitial pneumonia with fibrosis; hepatic centrolobular fatty change, atrophy, and necrosis; hepatic subserosa1 hemorrhages; lymphoid depletion in spleen and thymus; congestion in multiple organs; uterine atrophy; and subcutaneous edema. No vehicle-related histopathologic changes were observed in rats treated with 0.1% Pluronic F-68 diluent alone.
HistopathologicFindings in the WeeklyX5, Multiple-DoseStudy Drug-related histopathologic changes were not observed in the heart or lungs of BMY25282-treated rats. Drug-related increases in the incidence and/or severity of tubular degeneration and glomerulopathy were observed in BMY252828-treated male rats. Slight to mild thickening of glomerular membranes was observed in 2 of 5 BMY-25282-treated males, while slight to severe tubular degeneration was present in 5 of 5 BMY-25282-treated males and in 1 of 5 control males. DISCUSSION The toxicity of BMY-25282 in rats, as shown in this report, varies significantly from
ET AL.
the toxicity of BMY-25282’s parent compound, mitomycin C, as it has been previously reported (Philips et al., 1960) and demonstrated in a pilot study (unpublished data, Bristol-Myers Co., 1984). Although both drugs share certain cytotoxic, acute toxicities, including gastrointestinal epithelial necrosis and degeneration, splenic lymphoid depletion, and bone marrow hypocellularity, significant differences in chronic toxicity exist between the two compounds. The delayed irreversible cardiomyopathy, glomerulopathy, and arteritis caused by BMY-25282 administration have not been shown with mitomycin C administration, either acutely or after a 9-week dose-free observation period. The heart was the major target organ of BMY-25282 in both the single-dose and daily X5, multiple-dose studies of this report. Of the various cardiotoxic lesions, myocardial degeneration (cardiomyopathy) was considered the most important in terms of its high incidence and its lethality. BMY-25282-related cardiomyopathy was considered to be the primary cause of congestive heart failure as evidenced by hydrothorax and, to a lesser extent, by pulmonary congestion and edema, subcutaneous edema, hydropericardium, and congestion of multiple organs in rats that died prior to the termination of both studies. In both studies, cardiomyopathy was delayed in onset and irreversible. Although mitomytin C has been reported to produce myocarditis in Wistar rats (Levillain and Cluzan, 1973) endocardial and pericardial hemorrhages in dogs (Philips et al., 1960) and radiomimetic myocardial changes in man (Suzuki and Niibe, 1975) mitomycin C-related cardiac changes and BMY-25282-related cardiomyopathy were morphologically and biologically dissimilar. Indeed, the morphology of BMY-25282-related myocardial degeneration strongly resembled the chronic myocardial degeneration caused by anthracyclines in the rat (Buyniski and Hirth, 1980; Olson and Capen, 1978; Mettler et al., 1977), mouse (Bertazzoli et al., 1979; Rosenoff et al., 1975) rabbit (Olson et al., 1974), and human (Le-
BMY-25282
TOXICITY
frak et al., 1975; Halazun et al., 1974). Hydrothorax, delayed onset, and chronic course are other features of myocardial degeneration shared by both BMY-25282 toxicity and anthracycline toxicity. A major difference between the two drug-induced, delayed chronic cardiotoxicities in rats is that anthracycline toxicity depends upon the administration of multiple low doses (Chalcroft et al., 1973; Philips et al., 1975), which are cumulative, whereas, BMY-25282 cardiotoxicity can be induced by a single dose. Several of the other chronic toxicities reported in the singledose and daily X5, multiple-dose studies were considered to be the result of cardiac damage and subsequent heart failure. These included pulmonary arterial medial hypertrophy, pulmonary edema and focal hemorrhage, slight interstitial pneumonia with slight fibrosis, hepatic changes (fatty change, atrophy, necrosis, and subserosal hemorrhages), subcutaneous edema, and congestion in multiple organs. It may be useful to note that aspartate aminotransferase and creatine kinase, enzymes which may indicate muscle damage, were neither predictive of nor diagnostic for myocardial damage at 4 days after dosing or at approximately 9 weeks after dosing. In the case of CK, no drug-related elevations were seen at either the early or late sampling time in either study. The absence of elevated CK in the face of grossly and microscopically evident cardiomyopathy may indicate that (1) the type of myocyte damage did not result in measurable leakage of CK or perhaps (2) the initial insult to myocardial tissue, the resultant leakage of CK, and clearance of CK all occurred at a time between the two sampling times. Although AST was elevated in some individual rats of the high and intermediate dose groups of each study, AST elevations correlated imperfectly with rats that had microscopic evidence of cardiomyopathy. Glomerulopathy was another prominent BMY-25282~related toxicity in both studies that has not been reported with mitomycin C administration. The incidence of this lesion
IN RATS
107
was high in the 0.5 and 0.25 mg/kg groups of the single-dose study and in the 0.5 mg/kg group of the daily X5, multiple-dose study. Tubular degeneration, which has been reported to be a chronic liability of mitomycin C (Philips et al., 1960) was frequently associated with BMY-25282-related glomerulopathy. Tubular degeneration usually occurred in conjunction with glomerulopathy and its severity tended to parallel the severity of glomerulopathy. The hypoproteinemia observed in the single-dose study and the increased BUN and creatinine levels and proteinuria which were observed in both studies were attributed primarily to glomerular damage and secondarily to tubular damage. The hypoproteinemia in the single-dose study was characterized by slightly decreased albumin and normal globulin concentrations, thus resulting in a slight decrease in albumin to globulin ratio. Neither the decrease in serum protein nor the decrease in albumin was considered to be severe enough to affect intravascular osmotic pressure in hypoproteinemic rats. Therefore, the hydrothorax observed at necropsy was considered to be primarily due to cardiomyopathy rather than to nephrotic syndrome alone. Glomerulopathy in these two studies was delayed in onset. The pathogenesis of the lesion is unclear. The morphology of the lesion, however, bears a certain resemblance to glomerulonephritis in chronic serum sickness, which is caused by the circulation and subsequent deposition of foreign protein in soluble immune complexes. Arteritis, while occurring less frequently in both studies than cardiotoxicity or nephrotoxicity, was considered a significant BMY25282-related toxicity for three reasons. First, arteritis showed the potential to occur in multiple organs in the same individual rat. Arteritis occurred in the pancreas, testis, and epididymis of one individual, in the kidney, colon, and cecum of another individual, and only in the lung of several other rats. Second, in each of the organs where arteritis occurred, arteritis was associated with various degrees
108
BREGMAN
of inflammation, necrosis, or atrophy. In the lung, the most frequently involved organ, several vessels throughout this organ were involved, thus presenting the potential for widespread pulmonary damage due to infarction and embolism. Third, arteritis as a clinical entity is difficult to diagnose and monitor, especially considering the confusing, complicated sets of clinical signs that can occur with arteritis present in multiple, randomly affected organs. As in the case of glomerulopathy and cardiomyopathy, arteritis was delayed in onset. Likewise, the pathogenesis of the lesion is unknown, although clearly related to treatment with BMY-25282. Also, as in the case of glomerulopathy, the histopathologic appearance of BMY-25282-related arteritis resembles that observed in serum sickness (immune complex disease). As previously mentioned, BMY-25282-related cardiotoxicity closely resembles anthracycline-caused cardiotoxicity, although the anthracyclines are not chemically similar to BMY-25282. Anthracycline and BMY25282 toxicities also share at least three other toxic lesions, namely, chronic sclerotic glomerulopathy, tubular degeneration, and arteritis. Rats administered daunomycin, like those administered BMY-25282, developed diffusely fibrotic glomeruli; some of which were obliterated, while some had foci of fibrinoid necrosis. These rats also had arteritis in the kidney, pancreas, heart, skeletal muscle, and intestinal mesentery (Sternberg et al., 1972). Glomerular sclerosis associated with severe tubular and interstitial changes has been reported in rats treated with Adriamycin (Bertani et al., 1983; Mettler et al., 1977; Philips et al., 1975) and with carminomycin (Hirth, 1980). Hypocellular swollen glomeruli expanded by eosinophilic mesangial deposits have also been observed in rats treated with daunorubicin (Deprez-DeCampeneere et al., 1982). However, vacuolation of glomerular epithelial cells, which is reported to be a prominent feature of chronic Adriamycin-induced glomerular damage in
ET
AL.
rats (Giroux et al., 1984), was not a feature of BMY-25282-related glomerulopathy. Comparison of the three studies of this report illustrates that BMY-25282 toxicity is schedule dependent. The dosing schedule in the weekly X5, multiple-dose study produced less toxicity than the single-dose schedule at 0.25 mg/kg or the daily ~5 schedule using 0.05 mg. The latter two schedules produced cardiotoxicity, pulmonary toxicity, arteritis, and a greater degree of nephrotoxicity than the weekly X5 study, which produced only nephrotoxicity. In each study, the total cumulative dose was 0.25 mg/kg for the pertinent dose group.
REFERENCES BERTANI, T., ROCCHI, G., MECCA, G., SACCHI,G., AND REMUZZI, G. (1983). Adriamycin-induced chronic proteinuria: A new model of glomerular focal sclerosis (abstract). Kidney Int. 23, 192. BERTAZZOLI, C., BELLINI, O., MAGRINI, U., AND ToSANA, M. G. (1979). Quantitative experimental evaluation of Adriamycin cardiotoxicity in the mouse. Cuncer Treat. Rep. 63, 1877-1883. BRADNER, W. T., ROSE, W. C., SCHURIG, J. E., FLORCZYK, A. P., HUFTALEN, J. B., AND CATINO, J. J. (1985). Antitumor activity and toxicity in animals of BMY-25282, a new mitomycin derivative. Cancer Res. 45,6475-648 1. BRECHER, G. ( 1949). New methylene blue as a reticulocyte stain. Amer. J. Clin. Pathol. 19,895896. Bristol-Myers Company (1984). A Multiple Dose Intravenous Toxicity Study of Mitomycin C in Rats, Study Code No. BL-6782-001-I l-83, unpublished data. Pharmaceutical Research and Development Division, Department of Pathology and Toxicology, Evansville, IN. BUYNISKI, J. P., AND HIRTH, R. S. (1980). Anthracycline cardiotoxicity in the rat. In Anthracyclines: Current Status and New Developments (S. T. Crooke and S. D. Reich, Eds.), pp. 157-170. Academic Press, New York. CHALCROFT, S. C. W., GAVIN, J. B., AND HERDSON, P. B. (1973). Fine structural changes in rat myocardium induced by daunorubicin. Pathology 5,99-105. CROUT, J. R. ( 198 1). FDA Memorandum ofJuly 9,1981. Director, Bureau of Drugs (J. R. Crout), to Associate Director for New Drug Evaluation (M. J. Finkel). DEPREZ-DE~AMPENEERE, D., JAENKE, R., AND TROUET, A. (1982). Comparative cardiac and renal
BMY-25282
TOXICITY
toxicity of daunorubicin in the rat and rabbit. Cancer Treat. Rep. 66,395-391. GIROUX, L., SMEESTERS,C., BOURY, F., FAURE, M. P., AND JEAN, G. (1984). Adriamycin and AdriamycinDNA nephrotoxicity in rats. Lab. Invest. 50, 190-196. HALAZUN, J. F., WANGER, H. R., GAETA, J. F., AND SINKS, L. F. (1974). Daunorubicin cardiotoxicity in children with acute lymphocytic leukemia. Cancer 33, 545-554.
HIRTH, R. S. (1980). Other toxicities associated with anthracyclines in animal systems. In Anthracyclines: Current Status and New Developments (S. T. Crooke and S. D. Reich, Eds.), pp. 221-240. Academic Press, New York. KIRK, R. E. (1968). Experimental Design: Proceduresfor Behavioral Sciences, pp. 69-98. Brooks/Cole, Belmont, CA. LEFRAK, E. A., PITHA, J., ROSENHEIM, S., O’BRYAN, R. M., BURGESS,M. A., AND GOTTLIEB, J. A. (1975). Adriamycin (NSC- 123 127) cardiomyopathy. Cancer Chemother. Rep. 6,203-208. LEVILLAIN, R., AND CLUZAN, R. (1973). Cardiac toxicity of antimitotic drugs. In Proceedings ofthe 3rd Meeting the European Association for Cancer Research, Heidelberg, p. 100. METTLER, F. P., YOUNG, D. M., AND WARD, J. M. (1977). Adriamycin-induced cardiotoxicity (cardio-
IN RATS
109
myopathy and congestive heart failure) in rats. Cancer Res.37,2105-2713. OLSEN, H. M., AND CAPEN, C. C. (1978). Chronic cardiotoxicity of doxorubicin (Adriamycin) in the rat: Morphologic and biochemical investigations. Toxicol. Appl. Pharmacol. 44,605-6 16. OLSEN, H. M., YOUNG, D. M., PRIEUR, D. J.. LEROY, A. F., AND REAGAN, R. L. (1974). Electrolyte and morphologic alterations of myocardium in Adriamytin-treated rabbits. Amer. J. Pathof. 77,439-450. PHILIPS, F. S., GILLADOGA, A., MARQUARDT, H., STERNBERG, S. S., AND VIDAL, P. M. (1975). Some observations on the toxicity of Adriamycin (NSC123127). Cancer Chemother. Rep. 6,111-181. PHILIPS, F. S., SCHWARTZ, H. S., AND STERNBERG,S. S. (1960). Pharmacology of mitomycin C. 1.Toxicity and pathologic effects. Cancer Res. 20, 1354- 136 1. ROSENOFF,S. H., OLSEN, H. M., YOUNG, D. M., BosTICK, F., AND YOUNG, R. C. (1975). Adriamycin-induced cardiac damage in the mouse: A small-animal model of cardiotoxicity. J. Natl. Cancer Inst. 55, 19 l194.
STERNBERG, S. S., PHILIPS, F. S., AND CRONIN, A. P. (1972). Renal tumors and other lesions in rats following a single intravenous injection of daunomycin. CancerRes. 32, 1029-1036. SUZUKI, Y., AND NIIBE, H. (1975). Radiation-induced heart injury: Radiopathological study. Kita Kanto Igaku 25,395-407.