A fifty-two-week chronic toxicity study of halopredone acetate (THS-201) in dogs.

In order to evaluate the long-term-safety of halopredone acetate (THS-201: 17 alpha, 21-diacetoxy-2-bromo-6 beta, 9 alpha-difluoro-11 beta-hydroxy-1, 4-pregnadiene-3, 20-dione), a 52-week chronic toxicity study was performed on the basis of its local accumulation in dogs. In doses of 0.1, 0.5 and 2.5 mg/kg, THS-201 was injected into the right knee joint in both sexes of dogs every 2 weeks for 39 weeks and withdrawn for 13 weeks. In this study, the below slight local changes were observed in both sexes of dogs treated with 2.5 mg/kg/2 weeks of THS-201: focal loss of hair of the injection site, lesser stain in cartilage matrix of articular cartilage and meniscus in light microscopic examinations, and irregular thickness and elongation of collagen fibers, roughness of fibrous density and decrement of proteoglycans in electron microscopic examinations. In conclusion, systemic adverse effects were not observed in any dogs treated with THS-201.

[Mutagenicity test of halopredone acetate].

The mutagenicity of halopredone acetate (THS-201) was investigated by means of reverse mutation test in seven bacterial strains (S. typhimurium TA100, TA1535, TA98, TA1538, TA1537 and E. coli WP2, WP2 uvrA) and chromosomal aberration test in cultured Chinese hamster cells (CHL). In the reverse mutation test, no significant increase of revertant was observed at dose levels from 50 to 5000 micrograms/plate in the absence and presence of mammalian metabolic activation system. THS-201 caused no increase of chromosomal aberrants at dose levels of 1.6, 8.0, 40.0 and 200 micrograms/ml irrespective of metabolic activation. These results indicated that THS-201 has no mutagenic activity.

[Study on toxicity of halopredone acetate. (I) Acute toxicity study in mice and rats].

Since halopredone acetate (THS-201), a synthetic corticosteroid, is expected to be used clinically for intra-articular injections because of its long-lasting activity in the synovial bursa, its acute toxicity was compared with that of triamsinolone acetonide (TA) and methylprednisolone acetate (MPA). The test animals were Jcl: ICR mice and Jcl: Wistar rats, and drugs were administered orally, intraperitoneally and subcutaneously. The LD50 values of THS-201 both in mice and rats were estimated more than 5000 mg/kg at each route, and these are for above larger than those of TA or MPA. Moreover, in oral and subcutaneous administration of THS-201, no severe toxic signs were observed either in mice or in rats. In intraperitoneal injection, a few of mice and rats died after showing several clinical signs and suppression of body weight gain, and their autopsy revealed atrophy of thymus, spleen and adrenal, induction of infection and hemorrhage in digestive tract. On the other hand, the mice and rats administered TA or MPA revealed the severe toxic signs such as loss of hair gloss, marked emaciation, decrease in spontaneous movement, anemia, bloated face, decrease or suppression of body weight gain, atrophy of thymus, spleen and adrenal, severe induction of infection and lesions in digestive tracts. Accordingly, it is concluded that the acute toxicity of THS-201 in mice and rats was lower than that of TA or MPA.

[Study on toxicity of halopredone acetate. (II). Subacute toxicity study in rats].

Halopredone acetate (THS-201), a synthetic corticosteroid, is expected to be used clinically for intra-articular injection because of its long-lasting activity in the synovial bursa. Subacute toxicity study was carried out on THS-201 by using Jcl: Wistar rats. THS-201 was subcutaneously administered to the rat in doses of 0.1, 0.5, 2.5 and 12.5 mg/kg/day, with the periods for administration and recovery being 3 and 2 months, respectively. Methylprednisolone acetate (MPA) was used for the positive control in dose of 0.5 mg/kg/day. In the group of THS-201 12.5 mg/kg, the below findings were observed: thinning of lumbar hair, swelling of injection site, suppression in body weight gain and decrease of food consumption. The lesions to lymphatic system were indicated by the examinations of peripheral blood, autopsy and histopathology. Slight changes in erythrocytic and biochemical values were seen, and foreign body granuloma was observed in injected subcutis. These findings, exception for lesion of injection site, were almost recovered after the 2 month-recovery period. In the group of THS-201 2.5 mg/kg, some of the changes were noted slightly. In the groups of THS-201 0.5 and 0.1 mg/kg, any toxic changes attributable to THS-201 were not observed. On the other hand, in the group of MPA 0.5 mg/kg, similar findings noted in THS-201 12.5 mg/kg group were observed, but these changes were recovered almost completely at the end of the 2 month-recovery period. It was concluded that the non-toxic dose and the defined toxic dose of THS-201 in this study were 0.5 and 2.5 mg/kg/day, respectively.

[Study on toxicity of halopredone acetate. (III). Chronic toxicity study in rats].

Chronic toxicity study of halopredone acetate (THS-201), a synthetic corticosteroid, was carried out using Jcl: Wistar rats. THS-201 was subcutaneously administered to the rat in doses of 0.02, 0.1, 0.5 and 2.5 mg/kg/day, with the periods for administration and recovery being 12 and 2 months, respectively. In the group of THS-201 2.5 mg/kg, the below findings were observed: thinning of lumbar hair, suppression in body weight gain, decrease of food consumption in both sexes and decrease of water consumption in male. The lesions to lymphatic system were indicated by the examinations of hemogram, organ weight and histopathology. A few changes in urinary and biochemical values were seen, and foreign body granuloma was observed in the injected subcutis. After 2 month-recovery period, above-mentioned findings almost disappeared. In the group of THS-201 0.5 mg/kg, some of the changes were noted slightly and disappeared after the recovery period. In the groups of THS-201 0.1 and 0.02 mg/kg, any toxic changes attributable to THS-201 were not observed. It was concluded that the non-toxic dose and the defined toxic dose of THS-201 in this study were 0.1 and 0.5 mg/kg/day, respectively.

[Teratogenicity study on halopredone acetate in rats].

A teratogenicity study of halopredone acetate (THS-201), a synthetic corticosteroid, was carried out using Jcl: Wistar rats. Pregnant rats were treated subcutaneously in doses of 0.1, 0.5, 2.5 and 12.5 mg/kg/day, from day 7 to day 17 of gestation. Two-thirds of pregnant rats were killed on day 20 of gestation to examine the development of fetuses, and remaining rats were allowed to litter naturally in order to investigate the postnatal development of offspring. The results obtained from the present study were as follows. During the gestation and lactation periods, there occurred a decrease in the maternal body weight gain in 2.5 and 12.5 mg/kg groups. No influences of THS-201 administration were observed on gestation, delivery and lactation of dams. No external, visceral and skeletal anomalies attributable to THS-201 were observed in the fetuses. THS-201 administration did not have any influences on viability and development, various functions such as reflex response, learning and reproductive performance of F1 generation, and further on development of F2 generation. Therefore, it was concluded that the non-effect dose of THS-201 for the reproduction of dams and development of F1 generation was 12.5 mg/kg/day.

[Chronic toxicity study on difluprednate in dogs].

The chronic toxicity of a new topical glucocorticoid, difluprednate (DFBA) was studied in Beagle dogs. DFBA ointment (0.05%) was percutaneously treated to the back of dogs at daily doses of 125, 12.5 and 1.25 micrograms/kg for 6 months. The local effects of DFBA In the treated area, thinning of the skin and inhibition of the fur-growth were observed with scale and erythema. The skin showed histological atrophy of the epidermis, a decrease of the adipose tissue and atrophy of the adnexa. These changes returned to normal after the 2-month withdrawal period. The systemic effects of DFBA In the 125 micrograms/kg group, the following changes were observed, although neither death nor severe symptoms occurred: General observations were seen an increase of water intake and urinary volume. A decrease of lymphocytes and eosinophils, and an increase of neutrophils were observed in the hematological examination. There were high sodium and low potassium levels, and an increase of alkaline phosphatase and gamma-glutamyltranspeptidase activities in the biochemical examination. The organ weights showed a decrease of the thymus, adrenals, prostate and ovaries, and an increase of the liver and kidney. An atrophy of the lymphatic tissues and adrenal cortex, retardation of the sexual maturation, glycogen deposit in the hepatic cells, slight degeneration of the renal tubuli, and slight thinning of the sternum and non-treated skin were noted in the pathological examination. These changes returned to normal after the 2-month withdrawal period. In the 12.5 micrograms/kg group, the atrophic changes in the thymus, adrenal and non-treated skin appeared slight. In the 1.25 micrograms/kg group, no changes were found. Conclusively, all the local and systemic changes observed by DFBA in this study were due to the already known pharmacological effects of glucocorticoids. It is considered that a 12.5 micrograms/kg dosage is similar to a non-effect dose.

[Studies on antigenicity of difluprednate--antigenicity of difluprednate, phototoxicity and photocontact sensitivity of difluprednate ointment and cream].

The antigenicity of difluprednate, phototoxicity and photocontact sensitivity of difluprednate ointment and cream were studied in guinea pigs and mice. The results in this study were as follows. Guinea pigs immunized with difluprednate emulsified with Freund's complete adjuvant did not exhibit the systemic anaphylaxis and the delayed type hypersensitivity by the elicitation with either difluprednate alone or difluprednate-BSA conjugate. The antisera obtained from these guinea pigs did not show the positive response in the homologous 4-hour PCA reaction and the passive hemagglutination test against the challenge of the same antigens described above. In the maximization test, guinea pigs immunized with difluprednate did not show the contact sensitivity when elicited with difluprednate suspended in saline. Guinea pigs applied difluprednate ointment did not show positive contact sensitivity by the elicitation with difluprednate ointment and ointment base. The antisera obtained from mice immunized with difluprednate and difluprednate-OVA conjugate absorbed to Al (OH)3 gel did not give the positive response in the 72-hour PCA reaction against the challenge of either difluprednte alone or difluprednate-BSA conjugate. In the phototoxicity and photocontact sensitivity test, 0.05% difluprednate ointment and 0.05% difluprednate cream did not show positive reactions in guinea pigs, although very slight erythema was caused by the primary irritancy of cream base, with or without irradiation of ultraviolet ray (320-400 nm).

Effects of intraarticular injection of halopredone diacetate on the articular cartilage of rabbit knees: a comparison with methylprednisolone acetate.

Halopredone diacetate, which is a new synthetic corticosteroid drug showing local retention, and methylprednisolone acetate in common clinical use were injected into the knee joints of adult rabbits to compare the effects of the two drugs on the articular cartilage. The dosage of one injection was 1.75 mg (H-1 group) and 8.75 mg (H-5 group) for halopredone diacetate and 1.4 mg (M-1 group) and 7.0 mg (M-5 group) for methylprednisolone acetate. The drugs were injected into the right knee joint once a week, 12 times in total. One week after the last injection, the steroid crystals remaining in the knee joint were observed in all rabbits in the H-1 and H-5 groups. White deposits were seen locally on a part of the cartilage in all the rabbits in the H-1, H-5, and M-5 groups but not in the M-1 group. These white deposits were observed as cystic lesions by light microscopy and contained abundant hydroxyapatite. Other histologic findings in the articular cartilage included fissure, hypocellularity, and a decrease of proteoglycan in each group. However, no distinct difference was noted between the H-1 group and the M-1 group as regards the histological-histochemical grades of the cartilage on the medial tibial condyle or in the electron microscopic findings of the cartilage on the medial femoral condyle. The same was true for the H-5 and M-5 groups. These results show that repeated intraarticular injections of these two drugs cause severe cartilage lesions to the same degree, except for the intracartilaginous white deposits of rabbit knees.