RESUMEN
Clinically used botulinum neurotoxins (BoNTs) are natural products of Clostridium botulinum. A novel, recombinant BoNT type A1 (rBoNT/A1; IPN10260) has been synthesized using the native amino acid sequence expressed in Escherichia coli and has previously been characterized in vitro and ex vivo. Here, we aimed to characterize rBoNT/A1 in vivo and evaluate its effects on skeletal muscle. The properties of rBoNT/A1 following single, intramuscular administration were evaluated in the mouse and rat digit abduction score (DAS) assays and compared with those of natural BoNT/A1 (nBoNT/A1). rBoNT/A1-injected tibialis anterior was assessed in the in situ muscle force test in rats. rBoNT/A1-injected gastrocnemius lateralis (GL) muscle was assessed in the compound muscle action potential (CMAP) test in rats. The rBoNT/A1-injected GL muscle was evaluated for muscle weight, volume, myofiber composition and immunohistochemical detection of cleaved SNAP25 (c-SNAP25). Results showed that rBoNT/A1 and nBoNT/A1 were equipotent and had similar onset and duration of action in both mouse and rat DAS assays. rBoNT/A1 caused a dose-dependent inhibition of muscle force and a rapid long-lasting reduction in CMAP amplitude that lasted for at least 30 days. Dose-dependent reductions in GL weight and volume and increases in myofiber atrophy were accompanied by immunohistochemical detection of c-SNAP25. Overall, rBoNT/A1 and nBoNT/A1 exhibited similar properties following intramuscular administration. rBoNT/A1 inhibited motoneurons neurotransmitter release, which was robust, long-lasting, and accompanied by cleavage of SNAP25. rBoNT/A1 is a useful tool molecule for comparison with current natural and future modified recombinant neurotoxins products.
Asunto(s)
Toxinas Botulínicas Tipo A/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Proteínas Recombinantes/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Inyecciones Intramusculares , Ratones , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Tamaño de los Órganos , Ratas , Proteína 25 Asociada a Sinaptosomas/efectos de los fármacos , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
Collagen-induced arthritis in rats is associated with inflammatory polyarthritis, sharing clinical and pathological features with those of human rheumatoid arthritis (RA). Described in this unit is a protocol for consistently inducing arthritis in female Lewis rats by immunizing them with bovine type II collagen (CII) emulsified in complete Freund's adjuvant. This model is of value not only in defining the underlying pathogenesis of RA, but also as a tool for evaluating pharmacological strategies for treating this condition.