RELATIONSHIP BETWEEN MEDIAN PARALYSIS DOSE (PD₅₀) AND MEDIAN LETHAL DOSE (LD₅₀) OF BOTULINUM TOXIN TYPE A IN MICE.

Mônica Colombini (Vac. Anaeróbicas/IBu); José Marcos Oliveira (Vac. Anaeróbicas/IBu); Patrícia dos Santos Carneiro (Serv. Bact./IBu); José Amaral Prado (Lab.P.Liofiliz./IBu); Hisako Gondo Higashi (D. Bioindustrial/IBu); Sally Müller Affonso Prado (Serv. Bact./IBu); Aryene Góes Trezena (Vac. Anaeróbicas/IBu)

Introduction:  Botulinum toxins are paralytic neurotoxins known for its therapeutic and cosmetic potential. Intramuscular injection of purified botulinum toxin, mainly type A, is the treatment of choice for facial wrinkles and a number of muscle hyperactivity syndromes. The international Unit (IU) of botulinum toxin has been defined as the lethal dose 50% (LD₅₀) in mice what reflects the toxic activity of botulinum toxin but does not reflect its pharmacological properties. On the other hand, the regional flacid paralysis reflects the mechanism of action of botulinum toxin in the clinical setting. Objective: This study was performed to compare an assay to assess the paralyzing activity (paralysis dose 50% - PD₅₀) with the assay to measure the LD₅₀. Methods: Three independent experiments were conducted for lyophilized preparations of 100 IU or of 50 IU. In all experiments it was used the botulinum toxin type A produced at Instituto Butantan. For PD₅₀ different dilutions of botulinum toxin were injected into the gastrocnemius muscle of NIH mice whereas for LD₅₀ they were injected in NIH mice intraperitoneally. In both experiments the mice were observed by 96 hours and the percentage of paralyzed and death animals was determined at each dose. The PD₅₀ was defined as the inverse of the toxin dilution that caused complete local paralysis in 50% of injected animals while the LD₅₀ was determined as the inverse of the toxin dilution that caused death in 50% of injected animals. For calculating the PD₅₀ and LD₅₀ Probit analysis was performed. Results and discussion: Botulinum type A toxin preparations were analyzed in different times for 180 days after lyophilization process. During the observation time the LD₅₀ ranged 19.2% (100 IU) or 26.6% (50 IU) while the PD₅₀ ranged 13.9% (100 IU) or 15.2% (50 IU). These results indicated that the PD₅₀ activity is more stable than the LD₅₀ activity. The more concentrated preparations showed less variation in LD₅₀ and PD₅₀ during the time studied. The linear regression plots showed high correlation coefficient (r=0.98) between LD₅₀ and PD₅₀ assays.