Akihiro Ginnaga

Comparison of effects of botulinum toxin subtype A1 and A2 using twitch tension assay and rat grip strength test

Botulinum toxin type A is used as a therapeutic agent for some spastic neurological disorders. Type A organisms have been classified into four subtypes (A1 to A4) based on the amino acid sequence variability of the produced neurotoxin. At present, commercially available preparations of the toxin belong to subtype A1. To date, no study has compared the characteristics of the biological activity of toxins from different subtypes. We compared the efficacy of A1 toxin (LL toxin or neurotoxin: NTX) with that of A2 toxin (NTX) employing the twitch tension assay using the mouse phrenic nerve hemidiaphragm and grip strength test in rats. The inhibitory effects on neuromuscular transmission of A2NTX at pH 7.4 and pH 6.8 were 1.95 and 3.73 times more potent than those of A1LL, respectively. The 50% effective doses for the administered limb, the dose which caused a 50% reduction in grip strength, i.e. ED(50), of A1LL, A1NTX, and A2NTX were calculated as 0.087, 0.060, and 0.040 U/head, respectively. These doses for the contralateral limb, i.e. TD(50), of A1LL, A1NTX, and A2NTX were calculated as 6.35, 7.54, and 15.62 U/head, respectively. In addition, the time required for A2NTX-injected rats to recover the grip strength of the contralateral limb was 17 days, while that for rats injected with A1LL was 35 days. The results indicated that A2NTX is a more potent neuromuscular blocker than A1 toxins, and suggested that A2NTX will provide a preferentical therapeutic agent for neurological disorders.

Transsynaptic inhibition of spinal transmission by A2 botulinum toxin

Botulinum toxin A (BoNT/A) blocks synaptic transmission via the cleavage of SNAP‐25. Axonal transport of BoNT/A (A1 type botulinum toxin (A1LL) and A2 type botulinum toxin (A2NTX)) from periphery to the CNS has been described in trigeminal nerve and foreleg muscles. A1LL and A2NTX were injected into the ipsilateral soleus, and their effects on ipsilateral and contralateral contractions were compared as a measure of local and systemic/transport‐mediated effects. Spinal transmission was also measured to determine axonal and transsynaptic transport of neurotoxin. A2NTX induced faster and stronger muscle relaxation than A1LL. A1LL arrived at the contralateral muscle by almost equal transport via neural pathways and by the circulation. A2NTX was mainly transported to contralateral muscles via the blood. A1LL and A2NTX were carried from peripheral to CNS and vice versa by dual antero‐ and retrograde axonal transport through either motor or sensory neurons. Our results may point to greater potential safety in A2NTX form.

Quantitative determination of biological activity of botulinum toxins utilizing compound muscle action potentials (CMAP), and comparison of neuromuscular transmission blockage and muscle flaccidity among toxins

The biological activity of various types of botulinum toxin has been evaluated using the mouse intraperitoneal LD(50) test (ip LD(50)). This method requires a large number of mice to precisely determine toxin activity, and so has posed a problem with regard to animal welfare. We have used a direct measure of neuromuscular transmission, the compound muscle action potential (CMAP), to evaluate the effect of different types of botulinum neurotoxin (NTX), and we compared the effects of these toxins to evaluate muscle relaxation by employing the digit abduction scoring (DAS) assay. This method can be used to measure a broad range of toxin activities the day after administration. Types A, C, C/D, and E NTX reduced the CMAP amplitude one day after administration at below 1 ip LD(50), an effect that cannot be detected using the mouse ip LD(50) assay. The method is useful not only for measuring toxin activity, but also for evaluating the characteristics of different types of NTX. The rat CMAP test is straightforward, highly reproducible, and can directly determine the efficacy of toxin preparations through their inhibition of neuromuscular transmission. Thus, this method may be suitable for pharmacology studies and the quality control of toxin preparations.

Quantitative determination of the biological activity of botulinum toxin type A by measuring the compound muscle action potential (CMAP) in rats.

Quantitative determination of the biological activity of botulinum toxin type A usually depends on the LD(50) method after intraperitoneal injection into mice. This method requires a large number of mice to determine the toxic activity at a high level of precision and 3-4 days to obtain the results. Techniques to replace the LD(50) method have been attempted at various institutes. As a substitute for this method, by directly measuring the inhibition of neuromuscular transmission after the administration of a toxin, a method to quantitatively assess the toxins activity by determining the compound muscle action potential (CMAP) was examined. Toxin solutions were injected into the rat gastrocnemius muscle, and that of the CMAP amplitude was determined over time. The CMAP amplitude decreased over 4 days after the injection of the toxin, and then slowly recovered. A dose-response relationship was noted for each dose, and a linear relation was observed between 0.01 and 30 U on the 1st day. From these results, we propose the CMAP as a substitute for the LD(50) method to examine the activity of toxin products as it is simple and reliable, reduces the number of experimental animals required, and lowers pain levels.

Inhibition of Membrane Na+ Channels by A Type Botulinum Toxin at Femtomolar Concentrations in Central and Peripheral Neurons

Recent studies have demonstrated that the botulinum neurotoxins inhibit the release of acetylcholine, glutamate, GABA, and glycine in central nerve system (CNS) neurons. The Na+ current (INa) is of major interest because it acts as the trigger for many cellular functions such as transmission, secretion, contraction, and sensation. Thus, these observations raise the possibility that A type neurotoxin might also alter the INa of neuronal excitable membrane. To test our idea, we examined the effects of A type neurotoxins on INa of central and peripheral neurons. The neurotoxins in femtomolar to picomolar concentrations produced substantial decreases of the neuronal INa, but interestingly the current inhibition was saturated at about maximum 50% level of control INa. The inhibitory pattern in the concentration-response curve for the neurotoxins differed from tetrodotoxin (TTX), local anesthetic, and antiepileptic drugs that completely inhibited INa in a concentration-dependent manner. We concluded that A type neurotoxins inhibited membrane Na+-channel activity in CNS neurons and that INa of both TTX-sensitive and-insensitive peripheral dorsal ganglion cells were also inhibited similarly to a maximum 40% of the control by the neurotoxins. The results suggest evidently that A2NTX could be also used as a powerful drug in treating epilepsy and several types of pain.

Spinal Central Effects of Peripherally Applied Botulinum Neurotoxin A in Comparison between Its Subtypes A1 and A2.

Because of its unique ability to exert long-lasting synaptic transmission blockade, botulinum neurotoxin A (BoNT/A) is used to treat a wide variety of disorders involving peripheral nerve terminal hyperexcitability. However, it has been a matter of debate whether this toxin has central or peripheral sites of action. We employed a rat model in which BoNT/A1 or BoNT/A2 was unilaterally injected into the gastrocnemius muscle. On time-course measurements of compound muscle action potential (CMAP) amplitudes after injection of BoNT/A1 or BoNT/A2 at doses ranging from 1.7 to 13.6 U, CMAP amplitude for the ipsilateral hind leg was markedly decreased on the first day, and this muscle flaccidity persisted up to the 14th day. Of note, both BoNT/A1 and BoNT/A2 administrations also resulted in decreased CMAP amplitudes for the contralateral leg in a dose-dependent manner ranging from 1.7 to 13.6 U, and this muscle flaccidity increased until the fourth day and then slowly recovered. Immunohistochemical results revealed that BoNT/A-cleaved synaptosomal-associated protein of 25 kDa (SNAP-25) appeared in the bilateral ventral and dorsal horns 4 days after injection of BoNT/A1 (10 U) or BoNT/A2 (10 U), although there seemed to be a wider spread of BoNT/A-cleaved SNAP-25 associated with BoNT/A1 than BoNT/A2 in the contralateral spinal cord. This suggests that the catalytically active BoNT/A1 and BoNT/A2 were axonally transported via peripheral motor and sensory nerves to the spinal cord, where they spread through a transcytosis (cell-to-cell trafficking) mechanism. Our results provide evidence for the central effects of intramuscularly administered BoNT/A1 and BoNT/A2 in the spinal cord, and a new insight into the clinical effects of peripheral BoNT/A applications.

Comparison of Systemic Toxicity between Botulinum Toxin Subtypes A1 and A2 in Mice and Rats

The adverse events caused by botulinum toxin type A (subtype A1) product, thought to be after‐effects of toxin diffusion after high‐dose administration, have become serious issues. A preparation showing less diffusion in the body than existing drugs has been sought. We have attempted to produce neurotoxin derived from subtype A2 (A2NTX) with an amino acid sequence different from that of neurotoxin derived from subtype A1 (A1NTX). In this study, to investigate whether A2NTX has the potential to resolve these issues, we compared the safety of A2NTX, a progenitor toxin derived from subtype A1 (A1 progenitor toxin) and A1NTX employing the intramuscular lethal dose 50% (im LD50) in mice and rats and the compound muscle action potential (CMAP) in rats. Mouse im LD50 values for A1 progenitor toxin and A2NTX were 93 and 166 U/kg, respectively, and the rat im LD50 values were 117 and 153 U/kg, respectively. In the rat CMAP test, the dose on the contralateral side, which caused a 50% reduction in the CMAP amplitude, that is, CMAP‐TD50, was calculated as 19.0, 16.6 and 28.7 U/kg for A1 progenitor toxin, A1NTX and A2NTX, respectively. The results indicate that A2NTX is safer than A1 progenitor toxin and A1NTX.

Retrospective survey to evaluate the safety and efficacy of Japanese botulinum antitoxin therapy in Japan.

Japanese botulinum antitoxins have been used for more than 50 years; however, their safety and therapeutic efficacy are not clear. In order to analyze the available data on botulinum antitoxin therapy in Japan, we surveyed published reports about botulism cases in which botulinum antitoxins were used, and retrospectively analyzed the safety and efficacy of the therapy. A total of 134 patients administered botulinum antitoxins were identified from published reports. Two cases of side effects (1.5%) were detected after antitoxin administration, both not fatal. The fatality rate was 9.4%, and more than 70% of the patients showed improvement in their symptoms and better clinical conditions than those not treated with antitoxins. These data suggest that the therapy with Japanese antitoxins is safe and highly effective.

Comparison of the immunogenicity of botulinum toxin type A and the efficacy of A1 and A2 neurotoxins in animals with A1 toxin antibodies

One issue with botulinum toxin type A products is a reduced therapeutic response in patients that have been injected with frequent dosing over a prolonged period. A possible cause of this is hemagglutinin, found in progenitor toxins, displaying adjuvant activity, enhancing antibody production against the toxin. We investigated whether there is any difference in immunogenicity between the LL toxin-derived subtype A1 (A1LL) and the neurotoxin-derived subtypes A1 and A2 (A1NTX and A2NTX, respectively), and investigated whether A2NTX is effective in animals which produce antibodies against A1LL. Neutralizing antibodies were detected in the A1LL-administered group; however, they were not detected in swine and rabbits administered multiple doses of A2NTX. These results indicate that A2NTX has a lower immunogenicity than A1LL. In rats with neutralizing antibodies, produced by the administration of A1LL, that were administered either A1NTX or A2NTX, A2NTX showed more potent inhibitory neuromuscular transmission than A1NTX. In human sera immunized with the botulinum toxoid vaccine (containing LL, L, and M toxoid derived subtype A1) reacted with either A1NTX or A2NTX, A2NTX showed more potent inhibitory neuromuscular transmission than A1NTX. This suggests that A2NTX has a greater therapeutic value in humans who have neutralizing antibodies against the A1 toxin.

Botulinum neurotoxin A2 reduces incidence of seizures in mouse models of temporal lobe epilepsy

Temporal lobe epilepsy often shows pharmacoresistance, and well-known anti-convulsants sometimes are not effective for blocking chronic seizures. Botulinum neurotoxins are metalloproteases that act on presynaptic proteins and inhibit neurotransmitter release in both the peripheral and central nerve systems. That is why neurotoxins may elicit an effect for the restraint of the seizures. Meanwhile, it has been suggested that a property and the stability of neurotoxin activities differ among the types A-G, in which type A neurotoxin (ANTX) is, especially, the most stable and can continue having activity for a long term. The present study therefore investigated the effects of hippocampal injections of A2NTX on seizures derived in TLE model mice, received repeated kindling stimulations in the amygdala. The injections induced complete disappearance of grand mal seizures in half of the population of amygdala kindled mice for 4 days. The injections also induced reduction of the evoked seizure level significantly for at least 18 days after injections. Taken together, these results suggest that A2NTX prevents from epileptic seizures, proposing that A2NTX is available as a new antiepileptic reagent.