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Protocol no. 105
CREATINE KINASE ACTIVITY IN RAT MYOCYTES AS A MODEL FOR ASSESSING MUSCLE IRRITATION

Primary cultures of newborn rat skeletal muscle cells can be used to evaluate the myotoxicity of various drugs.

CONTACT

Dr. Ikuo Kato Developmental Research Laboratories Shionogi & Co., Ltd. 3-1-1 Futaba-cho Toyonaka Osaka 561, Japan Tel: Japan 331-8081 Fax: Japan 332-6385

RATIONALE

A decrease in creatine kinase activity has been found to be a useful index of cytotoxicity in cultured rat skeletal muscle fibres. The depletion of creatine kinase activity following exposure to antibiotics correlates with the in vivo muscle irritation caused by these antibiotics in rabbits.

BASIC PROCEDURE

Skeletal muscle tissue is dissected from newborn rats and dissociated into a single cell suspension using a combination of enzymic digestion and repeated aspiration. This suspension is pre-incubated to remove most of the fibroblasts which adhere to the surface of the culture wells. The remaining myocytes are transferred to culture plates coated with collagen type I and incubated to allow them to differentiate into fibres. After 7 or 11 days, the cultures are exposed to the test compound for 1 hr and then intracellular creatine kinase levels are determined and the amount of test compound that causes 50% depletion (DC50) is calculated.

TEST STATUS

In-house development

CHEMICALS TESTED

Cephalosporin antibiotics Cefaloridine Cefazolin sodium Flomoxef sodium Cefamandole sodium Latamoxef sodium Cefalotin sodium

REFERENCES

  1. Harada Y, and Okamoto T (1979) Muscle irritation study on cefamandole. Chemotherapy 27: (S-5) 639-648. Harada Y, and Okamoto T (1980) Muscle irritation study on 6059-S. Chemotherapy 28: (S-7) 1226-1235.
  2. Kato I, Harihara A, and Mizushima Y (1992) An in vitro model for assessing muscle irritation of antibiotics using rat primary cultured skeletal muscle fibers. Toxicology and Applied Pharmacology 117: 194-199.
  3. Metzler C M, Elfring G L, and McEwen A J (1974) A User's Manual for NONLIN and Associated Programs Research Biostatistics, Upjohn Co.; Kalamazoo MI. Svendsen O, H¾jelse F, and Bagdon R E (1985) Tests for local toxicity of intramuscular drug preparations: Comparisons of in vivo and in vitro methods. Acta Pharmacol. Toxicol. 56: 183-190.
  4. V¾lund, A. (1978) Application of the four-parameter logistic model to bioassay: Comparison with slope ratio and parallel line models. Biometrics 34: 357-365.
  5. Williams P D, Masters B G, Evans L D, Laska D F, and Hottendorf G H (1987) An in vitro model for assessing muscle irritation due to parenteral antibiotics. Fundam. Appl. Toxicol. 9: 10-17.

IP-105 September/94