TOXICOLOGY
AND
Chronic
APPLIED
Oral
PHARMACOLOGY
Toxicity
36,301-306 (1976)
Studies of Nefopam in Rats and Dogs
Hydrochloride
MARVIN T. CASE,J. KNOX SMITHAND ROBERTA. NELSON Riker Laboratories, Inc., 3M Center, St. Paul, Minnesota 55101 Received June 6,1975; accepted December 8,197s
Chronic Oral Toxicity Studiesof Nefopam Hydrochloride in Rats and Dogs. CASE, M. T.. SMITH, J. K. AND NELSON, R. A. (1976). Toxicol. Appl. Pharmacol.36301-306. Nefopamhydrochloride, a newnon-narcotic analgesicagent, wasevaluated for chronic oral toxicity in severalrat and dog experiments.Rats weregiven 20,40, and 160mg/kg/day for 6 monthsand 1 yr; dogs receivedthe drug at dosagesof 20, 40, and 80 mg/kg/day for 6 monthsand 1 yr. The treated rats had a dose-related reduction in body weight and the malerats had dose-relatedliver changes(increasedrelative weight, pale grossappearance,and microscopicfatty changes).The dogs did not exhibit body weight or liver changes,but in the 1-yr study central nervous systemstimulation (excited behavior and sporadic convulsions) wasseenat the higher two doselevels. Human clinical studies (Gassel, 1973; Tobin and Gold, 1972; Workman and Winter,
1974) have revealed that nefopam hydrochloride has analgesic activity at oral doses of 1 to 3 mg/kg. A decrease in pain intensity, as well as a reduction of awareness to experimental pain stimuli, were achieved without sedation. A long-term safety study has been done in man and 3 mg/kg/day of nefopam hydrochloride for 12 weeks did not produce any adverse effects (Klotz, 1974). The results of several chronic oral toxicity studies of nefopam hydrochloride in rats and dogs are the subject of the present report. METHODS Rut toxicity studies.Weanling (5-6 weeks old) Carworth’ CFN (Wistar derived) rats
of both sexes were used for all studies. The animals were housed individually in wire bottom cages, 60 cages per rack, in air-conditioned humidity-controlled quarters and provided with ground Purina laboratory chow and water ad libitum. They were observed daily for appearance, behavior, and survival; body weights and food consumption were measured weekly. Nefopam hydrochloride was mixed mechanically with the
ground food on a weekly basis in the treated groups (concentration of drug adjusted weekly based on mean food consumption of previous week); the controls received untreated ground food. Three rat studies were conducted using groups of eight males and eight females per dose level in each study. In the first study, nefopam hydrochloride was given at dosages of 0, 20,40, and 80 mg/kg/day for 6 months. At this point, the high dose was increased to 160 mg/kg/day (other doses remained the same) and the 1 Carworth Farms, New York, New York. Copyright 0 1976by AcademicPress,Inc. All rights of reproduction in anyform reserved. Printed in Great Britain
301
302
CASE,
SMITH
AND
NELSON
study was continued for an additional 6 months (total of 1 yr). The second study was for 6 months at dosages of 0, 20, 40 and 160 mg/kg/day, and the third study was for 1 yr at dosages of 0,20,40, and 160 mg/kg/day. At the end of each study, blood samples were taken from six rats per sex per dose group for hemoglobin, packed cell volume, total and differential leukocyte counts, blood urea nitrogen, glucose and serum glutamic pyruvic transaminase determinations. Necropsy was performed at termination of each study on six animals per sex per dose group with the following tissues examined for gross and histopathologic changes : adrenalq2 aorta, esophagus, heart,2 intestine, kidneyq2 liver,* lungs, ovaries, pancreas, parathyroid, prostate, seminal vesicles, spleen2 stomach, testeq2 thyroid, trachea, urinary bladder and uterus.’ All tissues were fixed in Bouin’s fluid for preparation of paraffin sections and routine staining with H & E and Pollack’s trichrome. Dog toxicity studies. A 6-month oral toxicity study was conducted in conditioned young adult (1 to 2 yr old) mongrel dogs. Nefopam hydrochloride was administered as nonformulated drug in gelatin capsules 7 days a week at dose levels of 0,20,40, and 80 mg/kg/day (0, 10,20, and 40 mg/kg twice a day). Four dogs (two of each sex) comprised the O- and 80-mg/kg/day dose groups and six dogs (three of each sex) were in the 40- and 20-mg/kg/day dose groups. In the I-yr oral toxicity study, groups of eight (four per sex) of young adult (8 to 9 months) beagle dogs3 were given formulated nefopam hydrochloride tablets at dosages of 0,20,40, and 80 mg/kg/day (0, 10,20, and 40 mg/kg twice a day) 7 days a week. In both studies the dogs were observed for signs of toxicity daily and were weighed weekly, at which time drug doses were adjusted accordingly. The dogs were housed in outdoor runs (in southern California) with free access to food4 and water. In the (i-month study, the dogs were given physical examinations including ocular examinations with an ophthalmoscope prior to and at the end of the study. In the 1-yr study, physical examinations including neurological parameters (righting reflex, placing reflex, toe pinch reflex, proprioceptive-knuckle reflex) and ocular examinations were conducted before dosing and at 3,6,9, and 12 months. Fundus photographs were also taken before dosing and prior to termination. Blood samples were taken before dosing and prior to necropsy in the 6-months study and on Days -14, -7, 14, 60, 120, 180, 245, 300, and 365 in the 1-yr study. Hematologic and biochemical determinations included: erythrocyte count, hemoglobin, packed cell volume, total leukocyte count, differential leukocyte count, clotting time (Lee-White), mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, glucose cholesterol, blood urea nitrogen, bilirubin, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, alkaline phosphatase, lactic dehydrogenase, total protein, albumin, calcium, inorganic phosphate, sodium, potassium, and chloride. Urinalyses were done on urine samples collected at the start of the studies and at 6-month intervals thereafter. At the end of the studies, complete necropsy examinations were done on all dogs, and in the case of the I-yr study, the following organs were weighed: heart, liver, spleen, kidneys, adrenals, and brain. 2 Absolute and relative organ weights determined. 3 Obtained from kennel of Laboratory Research Enterprise, Inc. Kalamazoo, 4 Purina Laboratory Chow, Ralston Purina Co., St. Louis, Missouri.
Michigan.
NEFOPAM
CHRONIC
303
TOXICITY
The following tissues were fixed in 10% buffered formalin and examined microscopically following H & E and Pollack’s trichrome staining: trachea, lung, heart, aorta, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric and cervical lymph nodes, spleen, bone marrow, adrenal, pituitary, thyroid, parathyroid, liver, gall bladder, pancreas, kidney, urinary bladder, ovary, uterus, mammary gland, testes, prostate, brain, spinal cord, and eye. RESULTS
Rat toxicity studies. Compound-related deaths did not occur at any dose level in any of the three rat studies as all the treated rats survived; two control animals died due to pneumonia (one control male on each of two studies). Reduction in body weight was dose-related in all three studies but it was slight at the lower two dose levels. The highdose females had the greatest percent reduction of body weight gain. The data in Table 1 illustrate body weight changes found during 1 yr of oral administration of nefopam hydrochloride to rats. Food consumption measurements did not reveal any differences between treated rats and their respective controls. TABLE
1
BODY WEIGHT DATA OF RATS RECEIVING NEFOPAM FOR 12 MONTHS
Mean body weight (g) Dose b-uz/Wday)
Percentage of control value
Weeks
Start
8
80 79 74 78
328 301a 300” 301”
76 73 76 75
221 214 214 178”
26
Weeks
52
8
26
52
Males 0
20 40
160
516 475” 474” 450”
591 535’
92
92
90
519”
91
92
88
498”
92
87
84
338 318 297“ 240”
97 97 81
96 94 76
94 87 71
Females 0 20 40 160
296 285 278 226“
a Significant difference from controls (p -C 0.05) (Student’s t test).
Hemograms and serum analyses did not reveal any compound-related changes in the measured parameters. At necropsy, the only gross change observed was pale livers in many of the high-dose male rats in all three studies. Relative organ weights (Table 2) revealed enlarged livers in the high-dose males. Other organ weight changes were: increased relative testes weight and decreased relative uterus weight for the high-dose males and females, respectively. Compound-related organ weight changes were not found in the lower two dose groups.
304
CASE,
SMITH
AND
TABLE MEAN
ORGAN
WEIGHT
Dose
Organ
Liver Heart Spleen Kidneys Adrenals Testes Liver Heart Spleen
Kidneys Adrenals Uterus
(mg/kg/day)
DATA
2
OF RATS RECEIVING
NEFOPAM
organ weightsa
Absolute
0
NELSON
20
FOR
12 MONTHS
Relative organ weight?
40
160
0
20
Rats 17.4 1.11 0.84 2.91 33.5 2.9
40 -~.
160
21.3 1.22 0.95 3.55 37.2 3.2
Male 17.8 1.20 0.85 3.07 37.4 3.1
23.4 1.13 0.84 3.03 32.5 3.1
35.9 2.06 1.60 5.98 0.06 5.5
32.8 2.21 1.56 5.67 0.07 5.8
33.1 2.12 1.61 5.68 0.06 5.6
47.5 2.30 1.72 6.16 0.06 6.4
11.0 0.90 0.63 2.32 69.5 0.85
Female Rats 11.8 9.7 7.3 0.92 0.82 0.63 0.53 0.58 0.39 2.41 2.11 1.64 69.8 59.5 52.7 0.79 0.76 0.50
32.8 2.72 1.87 6.92 0.21 2.51
35.0 2.75 1.58 7.16 0.21 2.50
32.4 2.74 1.92 7.10 0.20 2.56
32.1 2.79 1.70 7.23 0.23 2.20
’ Absolute values for liver, heart, spleen, kidney, testes, and uterus are expressed in grams; values for adrenals are expressed in milligrams. * Relative values are expressed as milligram of organ per gram of body weight.
Microscopic examination revealed extensive fatty changes in hepatocytes of all the high-dose male rats. Mild fatty changes were found in all the mid-dose males and in several of the low-dose males. Examination of liver sections from the female rats did not reveal any fatty changes regardless of dose level. No drug-related histomorphologic changes were found in the other organs. Dog toxicity studies. Clinical signs were not observed during the 6-month toxicity study in which the dogs received nefopam hydrochloride in gelatin capsules. During the 1-yr toxicity study in which the dogs received nefopam hydrochloride in formulated tablets there was evidence of central nervous system (CNS) stimulation in the dogs receiving the high and mid-dose (80 and 40 mg/kg/day). On Day 16 of compound administration convulsions were observed in two females receiving the high dose; i.e. clonic type convulsions which lasted only a few minutes (1 to 5) and were usually preceded by nervousness and twitching of the head. Thereafter, sporadic convulsions were observed at one time or the other in all the high-dose dogs. No high-dose dog had a higher incidence of convulsions than another high-dose dog; frequently weeks to several months elapsed between the convulsions in any one dog. During the last 3 months of the I-yr study, two of the dogs receiving the mid-dose had occasional convulsions and during this time one dog receiving the mid-dose frequently would convulse when subjected to the stress of bleeding and physical examinations. The dogs receiving the high dose often had nervous apprehensive behavior during physical examinations but changes were not found in the neurological parameters. One highdose male dog died late in the 1-yr study (Day 359) and death appeared to have been preceded by convulsions.
NEFOPAM
CHRONIC
305
TOXICITY
The CNS stimulation did not have a marked adverse effect on the dogs as evidenced by normal weight gain throughout most of the study. A few of the dogs on the 6-month study had a slight weight loss during the first several weeks of compound administration but the weight was regained during subsequent weeks. The periodic ophthalmoscopic examinations and fundus photographs did not reveal any ocular effects.
LEUKOCYTIC
Leukocytic changes Leukocytosis“
CHANGES
Dose group (mdkdday) 0 20 40 80
Neutrophilia’
0 20 40 80
Lymphocytosisd
0 20 40 80
OF
TABLE 3 DOGS ON ONE-YEAR
TOXICITY
STUDY
OF NEFOPAM
Day of study -14
-7
4/8b 318 418 518 418 318 418
418 418
l/8 318 418 318
14 318
218 l/8 W 218 l/8
l/8 218
O/8
518 418
318
018
218
218 218 W
118 018 018
418
118
BTotal leukocyte counts over 18,000/mm3, b Number of dogs with change per number c Total neutrophil counts over 12,50O/mm’, d Total lymphocyte count over 5000/mm3,
W3
60
120
018 l/8 118 018 018 018 l/8 l/8 018 l/8 118 018 ‘W W-3 118 O/f3 018 O/8 l/8 218 018 W3 018 218
180
245
300
365
018 O/8 018 018 O/8 ‘W 018 018 018 118 ‘318 W3
018 018 018 l/8 O/8
018 018 018 018 O/f3 018 018 W3 O/8
018 018 018
018
O/8 l/f4 O/8 l/8 018
l/8
018 O/f3 O/8
117
018 018 018 l/7
W3 l/8 018 l/7
the upper limit for normal range in our laboratory. of dogs in group. the upper limit for normal range in our laboratory. the upper limit for normal range in our laboratory.
Drug-related effects were not found in the erythrocytic, leukocytic, blood chemistry, blood electrolyte, or urinalysis values. During the first three bleedings on the 1-yr study, several dogs in a!! groups including controls had leukocytosis with neutrophilia and lymphocytosis (Table 3). It is believed that these changes were due to the dogs being excited and not accustomed to being handled and bled. Organ weight data or gross and microscopic tissue examinations did not reveal any drug related changes in either the 6-month or I-yr study. Various sections of the brain and spinal cord were carefully examined microscopically and no morphological changes were found. DISCUSSION
The chronic oral administration of nefopam hydrochloride to rats in the diet at dosages of 20, 40, and 80-160 mg/kg/day produced some relatively minor effects, mainly at the higher doses. There was a dose-related reduction in body weight in treated animals as compared to the controls. The slower growth in the high-dose animals was the reason for two organ weight changes observed (reduced relative uterus weights in females and increased relative testes weights in males). Apparently, in slow growing male rats the testes growth is less affected than the rest of the body and increased relative testes weights result. The reverse is true in the case of the uterus in
306
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AND
NELSON
female rats. Similar changes in organ-to-body weight ratios have been reported by other workers (Schwartz et al., 1973). Dose-related liver changeswere found only in the male rats, an apparent sex difference. The liver changesconsisted of increased relative liver weight, pale gross appearance at necropsy and fatty changes on microscopic examination. Except for CNS stimulation (sporadic convulsions) at the two higher dose levels, chronic oral administration of nefopam hydrochloride at doses of 20, 40, and 80 mg/kg/day had no adverse effects in dogs. One dog receiving the high dose died after 359 days of compound administration and death appeared to have been preceded by convulsions. It is known that high dosesof nefopam hydrochloride can produce CNS stimulation and convulsions in dogs and other laboratory animals (Case et al., 1975). The reduced body weight and liver changesseenin the rats were not observed in the dogs, nor were changesfound in any other tissues.During the two predose bleedings and the first bleeding after compound administration many of the dogs (including controls) had leukocytosis with neutrophilia and lymphocytosis. Such a “physiologic” leukocytosis has been reported by Schalm (1965). It can occur when dogs have been subjected to the emotional stressesof exposure to new surroundings, people, and procedures. As the dog becomesadjusted to the new environment, the leukocyte values gradually return to normal. Toxic effects of nefopam hydrochloride occurred in both rats and dogs at dosesof 40 mg/kg/day or more. At the low dose of 20 mg/kg/day, which is several-fold greater than human therapeutic range of 1 to 3 mg/kg/day, no effects were found in the dogs; in rats, the effects were limited to minor body weight and hepatic changes. REFERENCES T., SMITH, J. K. AND NELSON, R. A. (1975).Reproductive,acuteand subacutetoxicity studieswith Nefopam in laboratory animals.Toxicol. Appl. Phurmacol. 33,46-51. GASSEL, M. M. (1973).An objective techniquefor the analysisof the clinical effectiveness and physiology of drugsin man. In New Developments in Electromyogruphy and Clinical Neurophysiology (J. E. Desmedt,ed.), Vol. 3,342359. S. Karger, Base]. KLOTZ, A. L. (1974).Long-termsafety of Nefopam hydrochloride (Acupan): A newanalgesic formulation. Current Therapeutic Res. 16,602-608. SCHALM, 0. W. (1965). Veterinary Hematology, p. 207. Lea & Febiger, Philadelphia. SCHWARTZ, E. TORNABEN, J. A. AND BOXILL, G. C. (1973).The effectsof food restriction on hematology,clinical chemistry, and pathology in the albino rat. Toxicol. Appl. Pharmacol. 25,515-524. TOBIN, W. E. AND GOLD, R. H. (1972). Nefopam hydrochloride: A novel musclerelaxant. J. Clin. Pharmacol. 12, 230-238. WORKMAN, C. AND WINTER, L., JR. (1974).A clinical evaluation of Nefopam hydrochloride (Acupan): A new analgesic.Current Therapeutic Res. 16, 609-616. CASE, M.
AND
Chronic
APPLIED
Oral
PHARMACOLOGY
Toxicity
36,301-306 (1976)
Studies of Nefopam in Rats and Dogs
Hydrochloride
MARVIN T. CASE,J. KNOX SMITHAND ROBERTA. NELSON Riker Laboratories, Inc., 3M Center, St. Paul, Minnesota 55101 Received June 6,1975; accepted December 8,197s
Chronic Oral Toxicity Studiesof Nefopam Hydrochloride in Rats and Dogs. CASE, M. T.. SMITH, J. K. AND NELSON, R. A. (1976). Toxicol. Appl. Pharmacol.36301-306. Nefopamhydrochloride, a newnon-narcotic analgesicagent, wasevaluated for chronic oral toxicity in severalrat and dog experiments.Rats weregiven 20,40, and 160mg/kg/day for 6 monthsand 1 yr; dogs receivedthe drug at dosagesof 20, 40, and 80 mg/kg/day for 6 monthsand 1 yr. The treated rats had a dose-related reduction in body weight and the malerats had dose-relatedliver changes(increasedrelative weight, pale grossappearance,and microscopicfatty changes).The dogs did not exhibit body weight or liver changes,but in the 1-yr study central nervous systemstimulation (excited behavior and sporadic convulsions) wasseenat the higher two doselevels. Human clinical studies (Gassel, 1973; Tobin and Gold, 1972; Workman and Winter,
1974) have revealed that nefopam hydrochloride has analgesic activity at oral doses of 1 to 3 mg/kg. A decrease in pain intensity, as well as a reduction of awareness to experimental pain stimuli, were achieved without sedation. A long-term safety study has been done in man and 3 mg/kg/day of nefopam hydrochloride for 12 weeks did not produce any adverse effects (Klotz, 1974). The results of several chronic oral toxicity studies of nefopam hydrochloride in rats and dogs are the subject of the present report. METHODS Rut toxicity studies.Weanling (5-6 weeks old) Carworth’ CFN (Wistar derived) rats
of both sexes were used for all studies. The animals were housed individually in wire bottom cages, 60 cages per rack, in air-conditioned humidity-controlled quarters and provided with ground Purina laboratory chow and water ad libitum. They were observed daily for appearance, behavior, and survival; body weights and food consumption were measured weekly. Nefopam hydrochloride was mixed mechanically with the
ground food on a weekly basis in the treated groups (concentration of drug adjusted weekly based on mean food consumption of previous week); the controls received untreated ground food. Three rat studies were conducted using groups of eight males and eight females per dose level in each study. In the first study, nefopam hydrochloride was given at dosages of 0, 20,40, and 80 mg/kg/day for 6 months. At this point, the high dose was increased to 160 mg/kg/day (other doses remained the same) and the 1 Carworth Farms, New York, New York. Copyright 0 1976by AcademicPress,Inc. All rights of reproduction in anyform reserved. Printed in Great Britain
301
302
CASE,
SMITH
AND
NELSON
study was continued for an additional 6 months (total of 1 yr). The second study was for 6 months at dosages of 0, 20, 40 and 160 mg/kg/day, and the third study was for 1 yr at dosages of 0,20,40, and 160 mg/kg/day. At the end of each study, blood samples were taken from six rats per sex per dose group for hemoglobin, packed cell volume, total and differential leukocyte counts, blood urea nitrogen, glucose and serum glutamic pyruvic transaminase determinations. Necropsy was performed at termination of each study on six animals per sex per dose group with the following tissues examined for gross and histopathologic changes : adrenalq2 aorta, esophagus, heart,2 intestine, kidneyq2 liver,* lungs, ovaries, pancreas, parathyroid, prostate, seminal vesicles, spleen2 stomach, testeq2 thyroid, trachea, urinary bladder and uterus.’ All tissues were fixed in Bouin’s fluid for preparation of paraffin sections and routine staining with H & E and Pollack’s trichrome. Dog toxicity studies. A 6-month oral toxicity study was conducted in conditioned young adult (1 to 2 yr old) mongrel dogs. Nefopam hydrochloride was administered as nonformulated drug in gelatin capsules 7 days a week at dose levels of 0,20,40, and 80 mg/kg/day (0, 10,20, and 40 mg/kg twice a day). Four dogs (two of each sex) comprised the O- and 80-mg/kg/day dose groups and six dogs (three of each sex) were in the 40- and 20-mg/kg/day dose groups. In the I-yr oral toxicity study, groups of eight (four per sex) of young adult (8 to 9 months) beagle dogs3 were given formulated nefopam hydrochloride tablets at dosages of 0,20,40, and 80 mg/kg/day (0, 10,20, and 40 mg/kg twice a day) 7 days a week. In both studies the dogs were observed for signs of toxicity daily and were weighed weekly, at which time drug doses were adjusted accordingly. The dogs were housed in outdoor runs (in southern California) with free access to food4 and water. In the (i-month study, the dogs were given physical examinations including ocular examinations with an ophthalmoscope prior to and at the end of the study. In the 1-yr study, physical examinations including neurological parameters (righting reflex, placing reflex, toe pinch reflex, proprioceptive-knuckle reflex) and ocular examinations were conducted before dosing and at 3,6,9, and 12 months. Fundus photographs were also taken before dosing and prior to termination. Blood samples were taken before dosing and prior to necropsy in the 6-months study and on Days -14, -7, 14, 60, 120, 180, 245, 300, and 365 in the 1-yr study. Hematologic and biochemical determinations included: erythrocyte count, hemoglobin, packed cell volume, total leukocyte count, differential leukocyte count, clotting time (Lee-White), mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, glucose cholesterol, blood urea nitrogen, bilirubin, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, alkaline phosphatase, lactic dehydrogenase, total protein, albumin, calcium, inorganic phosphate, sodium, potassium, and chloride. Urinalyses were done on urine samples collected at the start of the studies and at 6-month intervals thereafter. At the end of the studies, complete necropsy examinations were done on all dogs, and in the case of the I-yr study, the following organs were weighed: heart, liver, spleen, kidneys, adrenals, and brain. 2 Absolute and relative organ weights determined. 3 Obtained from kennel of Laboratory Research Enterprise, Inc. Kalamazoo, 4 Purina Laboratory Chow, Ralston Purina Co., St. Louis, Missouri.
Michigan.
NEFOPAM
CHRONIC
303
TOXICITY
The following tissues were fixed in 10% buffered formalin and examined microscopically following H & E and Pollack’s trichrome staining: trachea, lung, heart, aorta, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric and cervical lymph nodes, spleen, bone marrow, adrenal, pituitary, thyroid, parathyroid, liver, gall bladder, pancreas, kidney, urinary bladder, ovary, uterus, mammary gland, testes, prostate, brain, spinal cord, and eye. RESULTS
Rat toxicity studies. Compound-related deaths did not occur at any dose level in any of the three rat studies as all the treated rats survived; two control animals died due to pneumonia (one control male on each of two studies). Reduction in body weight was dose-related in all three studies but it was slight at the lower two dose levels. The highdose females had the greatest percent reduction of body weight gain. The data in Table 1 illustrate body weight changes found during 1 yr of oral administration of nefopam hydrochloride to rats. Food consumption measurements did not reveal any differences between treated rats and their respective controls. TABLE
1
BODY WEIGHT DATA OF RATS RECEIVING NEFOPAM FOR 12 MONTHS
Mean body weight (g) Dose b-uz/Wday)
Percentage of control value
Weeks
Start
8
80 79 74 78
328 301a 300” 301”
76 73 76 75
221 214 214 178”
26
Weeks
52
8
26
52
Males 0
20 40
160
516 475” 474” 450”
591 535’
92
92
90
519”
91
92
88
498”
92
87
84
338 318 297“ 240”
97 97 81
96 94 76
94 87 71
Females 0 20 40 160
296 285 278 226“
a Significant difference from controls (p -C 0.05) (Student’s t test).
Hemograms and serum analyses did not reveal any compound-related changes in the measured parameters. At necropsy, the only gross change observed was pale livers in many of the high-dose male rats in all three studies. Relative organ weights (Table 2) revealed enlarged livers in the high-dose males. Other organ weight changes were: increased relative testes weight and decreased relative uterus weight for the high-dose males and females, respectively. Compound-related organ weight changes were not found in the lower two dose groups.
304
CASE,
SMITH
AND
TABLE MEAN
ORGAN
WEIGHT
Dose
Organ
Liver Heart Spleen Kidneys Adrenals Testes Liver Heart Spleen
Kidneys Adrenals Uterus
(mg/kg/day)
DATA
2
OF RATS RECEIVING
NEFOPAM
organ weightsa
Absolute
0
NELSON
20
FOR
12 MONTHS
Relative organ weight?
40
160
0
20
Rats 17.4 1.11 0.84 2.91 33.5 2.9
40 -~.
160
21.3 1.22 0.95 3.55 37.2 3.2
Male 17.8 1.20 0.85 3.07 37.4 3.1
23.4 1.13 0.84 3.03 32.5 3.1
35.9 2.06 1.60 5.98 0.06 5.5
32.8 2.21 1.56 5.67 0.07 5.8
33.1 2.12 1.61 5.68 0.06 5.6
47.5 2.30 1.72 6.16 0.06 6.4
11.0 0.90 0.63 2.32 69.5 0.85
Female Rats 11.8 9.7 7.3 0.92 0.82 0.63 0.53 0.58 0.39 2.41 2.11 1.64 69.8 59.5 52.7 0.79 0.76 0.50
32.8 2.72 1.87 6.92 0.21 2.51
35.0 2.75 1.58 7.16 0.21 2.50
32.4 2.74 1.92 7.10 0.20 2.56
32.1 2.79 1.70 7.23 0.23 2.20
’ Absolute values for liver, heart, spleen, kidney, testes, and uterus are expressed in grams; values for adrenals are expressed in milligrams. * Relative values are expressed as milligram of organ per gram of body weight.
Microscopic examination revealed extensive fatty changes in hepatocytes of all the high-dose male rats. Mild fatty changes were found in all the mid-dose males and in several of the low-dose males. Examination of liver sections from the female rats did not reveal any fatty changes regardless of dose level. No drug-related histomorphologic changes were found in the other organs. Dog toxicity studies. Clinical signs were not observed during the 6-month toxicity study in which the dogs received nefopam hydrochloride in gelatin capsules. During the 1-yr toxicity study in which the dogs received nefopam hydrochloride in formulated tablets there was evidence of central nervous system (CNS) stimulation in the dogs receiving the high and mid-dose (80 and 40 mg/kg/day). On Day 16 of compound administration convulsions were observed in two females receiving the high dose; i.e. clonic type convulsions which lasted only a few minutes (1 to 5) and were usually preceded by nervousness and twitching of the head. Thereafter, sporadic convulsions were observed at one time or the other in all the high-dose dogs. No high-dose dog had a higher incidence of convulsions than another high-dose dog; frequently weeks to several months elapsed between the convulsions in any one dog. During the last 3 months of the I-yr study, two of the dogs receiving the mid-dose had occasional convulsions and during this time one dog receiving the mid-dose frequently would convulse when subjected to the stress of bleeding and physical examinations. The dogs receiving the high dose often had nervous apprehensive behavior during physical examinations but changes were not found in the neurological parameters. One highdose male dog died late in the 1-yr study (Day 359) and death appeared to have been preceded by convulsions.
NEFOPAM
CHRONIC
305
TOXICITY
The CNS stimulation did not have a marked adverse effect on the dogs as evidenced by normal weight gain throughout most of the study. A few of the dogs on the 6-month study had a slight weight loss during the first several weeks of compound administration but the weight was regained during subsequent weeks. The periodic ophthalmoscopic examinations and fundus photographs did not reveal any ocular effects.
LEUKOCYTIC
Leukocytic changes Leukocytosis“
CHANGES
Dose group (mdkdday) 0 20 40 80
Neutrophilia’
0 20 40 80
Lymphocytosisd
0 20 40 80
OF
TABLE 3 DOGS ON ONE-YEAR
TOXICITY
STUDY
OF NEFOPAM
Day of study -14
-7
4/8b 318 418 518 418 318 418
418 418
l/8 318 418 318
14 318
218 l/8 W 218 l/8
l/8 218
O/8
518 418
318
018
218
218 218 W
118 018 018
418
118
BTotal leukocyte counts over 18,000/mm3, b Number of dogs with change per number c Total neutrophil counts over 12,50O/mm’, d Total lymphocyte count over 5000/mm3,
W3
60
120
018 l/8 118 018 018 018 l/8 l/8 018 l/8 118 018 ‘W W-3 118 O/f3 018 O/8 l/8 218 018 W3 018 218
180
245
300
365
018 O/8 018 018 O/8 ‘W 018 018 018 118 ‘318 W3
018 018 018 l/8 O/8
018 018 018 018 O/f3 018 018 W3 O/8
018 018 018
018
O/8 l/f4 O/8 l/8 018
l/8
018 O/f3 O/8
117
018 018 018 l/7
W3 l/8 018 l/7
the upper limit for normal range in our laboratory. of dogs in group. the upper limit for normal range in our laboratory. the upper limit for normal range in our laboratory.
Drug-related effects were not found in the erythrocytic, leukocytic, blood chemistry, blood electrolyte, or urinalysis values. During the first three bleedings on the 1-yr study, several dogs in a!! groups including controls had leukocytosis with neutrophilia and lymphocytosis (Table 3). It is believed that these changes were due to the dogs being excited and not accustomed to being handled and bled. Organ weight data or gross and microscopic tissue examinations did not reveal any drug related changes in either the 6-month or I-yr study. Various sections of the brain and spinal cord were carefully examined microscopically and no morphological changes were found. DISCUSSION
The chronic oral administration of nefopam hydrochloride to rats in the diet at dosages of 20, 40, and 80-160 mg/kg/day produced some relatively minor effects, mainly at the higher doses. There was a dose-related reduction in body weight in treated animals as compared to the controls. The slower growth in the high-dose animals was the reason for two organ weight changes observed (reduced relative uterus weights in females and increased relative testes weights in males). Apparently, in slow growing male rats the testes growth is less affected than the rest of the body and increased relative testes weights result. The reverse is true in the case of the uterus in
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female rats. Similar changes in organ-to-body weight ratios have been reported by other workers (Schwartz et al., 1973). Dose-related liver changeswere found only in the male rats, an apparent sex difference. The liver changesconsisted of increased relative liver weight, pale gross appearance at necropsy and fatty changes on microscopic examination. Except for CNS stimulation (sporadic convulsions) at the two higher dose levels, chronic oral administration of nefopam hydrochloride at doses of 20, 40, and 80 mg/kg/day had no adverse effects in dogs. One dog receiving the high dose died after 359 days of compound administration and death appeared to have been preceded by convulsions. It is known that high dosesof nefopam hydrochloride can produce CNS stimulation and convulsions in dogs and other laboratory animals (Case et al., 1975). The reduced body weight and liver changesseenin the rats were not observed in the dogs, nor were changesfound in any other tissues.During the two predose bleedings and the first bleeding after compound administration many of the dogs (including controls) had leukocytosis with neutrophilia and lymphocytosis. Such a “physiologic” leukocytosis has been reported by Schalm (1965). It can occur when dogs have been subjected to the emotional stressesof exposure to new surroundings, people, and procedures. As the dog becomesadjusted to the new environment, the leukocyte values gradually return to normal. Toxic effects of nefopam hydrochloride occurred in both rats and dogs at dosesof 40 mg/kg/day or more. At the low dose of 20 mg/kg/day, which is several-fold greater than human therapeutic range of 1 to 3 mg/kg/day, no effects were found in the dogs; in rats, the effects were limited to minor body weight and hepatic changes. REFERENCES T., SMITH, J. K. AND NELSON, R. A. (1975).Reproductive,acuteand subacutetoxicity studieswith Nefopam in laboratory animals.Toxicol. Appl. Phurmacol. 33,46-51. GASSEL, M. M. (1973).An objective techniquefor the analysisof the clinical effectiveness and physiology of drugsin man. In New Developments in Electromyogruphy and Clinical Neurophysiology (J. E. Desmedt,ed.), Vol. 3,342359. S. Karger, Base]. KLOTZ, A. L. (1974).Long-termsafety of Nefopam hydrochloride (Acupan): A newanalgesic formulation. Current Therapeutic Res. 16,602-608. SCHALM, 0. W. (1965). Veterinary Hematology, p. 207. Lea & Febiger, Philadelphia. SCHWARTZ, E. TORNABEN, J. A. AND BOXILL, G. C. (1973).The effectsof food restriction on hematology,clinical chemistry, and pathology in the albino rat. Toxicol. Appl. Pharmacol. 25,515-524. TOBIN, W. E. AND GOLD, R. H. (1972). Nefopam hydrochloride: A novel musclerelaxant. J. Clin. Pharmacol. 12, 230-238. WORKMAN, C. AND WINTER, L., JR. (1974).A clinical evaluation of Nefopam hydrochloride (Acupan): A new analgesic.Current Therapeutic Res. 16, 609-616. CASE, M.
