Experimental methods for immunization and challenge in antigenicity studies in guinea pigs.

Optimal experimental methods for antigenicity studies in guinea pigs were investigated on: (1) the effects of different immunizing methods using complete or incomplete Freund's adjuvants (CFA or IFA), and various injection sites, the number of immunizations, the immunizing doses, and the immunizing periods, (2) the relationship between the severity of active systemic anaphylaxis (ASA) reactions and passive cutaneous anaphylaxis (PCA) titers, (3) positive control for oral administration, and (4) the effects of incubation mixture of drug and serum protein as the challenge for the ASA assay. The following results provided useful information for designing more appropriate methods for antigenicity studies: (1) The optimal immunization method for benzylpenicillin (PcG), cephaloridine, 2,4,6-trinitrobenzene sulfonic acid and adriamycin, which were selected as positive controls for low molecular medicines in this experiment, involved subcutaneous administration of 1 ml of a test substance in CFA (1st immunization) or IFA (2nd and 3rd immunizations) at two doses, 1 and 10 mg/animal, 3 times at 2-week intervals on the back of a guinea pig. Blood collection for PCA assay was needed 2 weeks after the last immunization, and ASA assay, 1 or 2 days after the blood collection. (2) The insensitivity of ASA reactions in bovine serum albumin-immunized animals with very high PCA titers was overcome by increasing the challenge antigen dose from 1 to 10 mg/animal. (3) Most animals administered lysozyme at 0.1, 1 or 10 mg/animal by gavage for 2 weeks or more showed ASA and PCA reactions. (4) Incubation of a mixture of 20 mg/ml of PcG and 2 mg/ml of guinea pig serum albumin for 4 hr was the most effective as challenge for the induction of ASA reaction in PcG-immunized guinea pigs.

[Toxicological study of amikacin following intravenous drip infusion. 1. Acute toxicity in rats, rabbits and dogs, and subacute toxicity in rabbits].

Acute toxicity of amikacin sulfate (AMK) was studied in rats, rabbits and dogs following 1 hour drip intravenous infusion (1 hour d.i.v.), and compared with those after intravenous injection (i.v.). Subacute toxicity was studied in rabbits following 1 hour d.i.v. of AMK at doses of 400, 200, 100 and 25 mg/kg for 36 days. Acute toxicity: Acute toxicity of AMK by 1 hour d.i.v. was extremely diminished as compared with that by i.v. judging from LD50, the toxicity was approximately 1/6 in rats and approximately 1/2 in rabbits and dogs. No difference was observed between males and females. Subacute toxicity: 1. No death was found in all rabbits. 2. At the doses of 25 mg/kg and 100 mg/kg, there were no significant changes or signs recognized as toxicological effects of AMK. 3. At the dose of 200 mg/kg, very slight renal damages were observed histopathologically in all the 5 rabbits. 4. At the dose of 400 mg/kg, slight or moderate renal damages were observed in all the rabbits. Those were mainly dilatation of renal proximal tubules, hydropic swelling and degeneration of renal proximal tubular epithelium. No significant impairment effect of AMK was found in heart, lung, liver and other organs. 5. From these results, it is considered that the maximum no effect dose from a view point of safety is about 100 mg/kg in this study.

[Toxicological study of amikacin following intravenous drip infusion. 2. Subacute toxicity in dogs after intravenous injection].

Subacute toxicity of amikacin sulfate (AMK) was investigated with 50 Beagle dogs (male 25, female 25). As a substitute for drip intravenous infusion (d.i.v.), AMK was administered by intravenous injection (i.v.) twice a day, 1 hour apart, for 38 days at daily doses of 400, 200, 100 mg/kg and 25 mg/kg. 1. At the doses of 25 mg/kg and 100 mg/kg, there were no significant changes or signs recognized as toxicological effects of AMK. 2. At the dose of 200 mg/kg, slight renal damages were observed in 3 of 5 males and 2 of 5 females. These were slight elevation of urea N and creatinine, and slight degeneration or regeneration of the renal proximal tubules. 3. At the dose of 400 mg/kg, renal damages were noted in all the dogs. Two males and 1 female died after 14-17 days' treatment and the remainders were sacrificed for inspection after 18-20 days' treatment. In any dogs, the adverse findings were mainly observed in the renal proximal tubules but not in the glomeruli. In the other organs, there were no significant impairments. 4. From these results, it is considered that the maximum no effect dose of AMK is 100 mg/kg.