| In 1909, the Japanese scientist Tahara Y extracted a kind of toxin from the roe of puffer fish at first, which is named tetrodotoxin (TTX).In 1950, Yokoo A got the crystal of TTX, which is verified that it is a kind of high toxic and micromolecule non-protein toxin, and the structure is caged ortho ester. The foreign scholars have carried out a series of studies on pharmacology and clinical use for TTX. In 1972, Kishi[2] got TTX by chemosynthesis. But the capital resource of TTX is still form Fugu. According to the selection on nerve, TTX is used constantly in physiology and pharmacology field for many yeas[3～5]. Recently, more and more scientists focus on the clinical uses and hope that TTX can be developed to a good drug with anti-arhythmia, conscious-sedation, odynolysis and odynolysis. Now, scientists are developing it to the drug for quitting drug dependence.The study aim to observe the general toxicity of TTX, which is going to develop to the drug for quitting drug dependence (im, 10μg/person/once,QD, course of treatment:7~12 day) to follow the guidance of SFDA in china, we hold a series of general toxicity on TTX. We hope the date of general toxicity can provide the evidence for the doses and parameters in clinical test.The study uses the standard animals (Wistar rat, SD rat and rhesus monkey) in the acute and chronic toxicity test. We observe the general physiological index, hematology index, clinical chemistry and urine index, and we have an all round histopathologic examination on animal organ and tissue after ending of drug administration and convalescence stage. Meanwhile, we observe the irritation and haemolysis of TTX on test animals.The single dose acute intramuscular injection toxicity study in Wistar rats shows that the rats have the dyskinesia on the injection local area, the paralysis and amyasthenia are emerged with the increasing of TTX. Otherwise, the rats have toxic symptom all over the body with the increasing of TTX, which shows depressed, amyasthenia, abdominal breathing, dyspneic respiration, hyperspasmia and death so on. The result hints that the acute toxicity is the inhibition of central nerve system (including apneustic center and motor center) and the conduction blockade on nerve to muscle. The survival rats recover to normal after 7 hours from administration. The rats have no abnormal appearance and death on the following 2 weeks. On the ending of observation stage, the weight of survival rats on each group has no statistical difference. There is not abnormal appearance on the gross anatomy when the rats are mercy killed. The LD50 of male and female Wistar rats is 10.53μg/kg and 11.75μg/kg by bliss way, and the 95% of confidence interval is 9.99～11.10μg/kg and 10.91～12.65μg/kg.The Wistar rats have hypokinesia, depressed and the degrade of muscular tension so on symptom after single intravenous injection of TTX. The symptom is aggravated; even abdominal breathing, dyspneic respiration, hyperspasmia and death are emerged with the increasing of TTX. The result hints that the acute toxicity is the inhibition of central nerve system (including apneustic center and motor center) and the conduction blockade on nerve to muscle. The survival rats recover to normal after 7 hours from administration. The rats have no abnormal appearance and death on the following 2 weeks. On the ending of observation stage, the weight of survival rats on each group has no statistical difference. There is not abnormal appearance on the gross anatomy when the rats are mercy killing. The LD50 of male and female Wistar rats is 8.54μg/kg and 10.10μg/kg by bliss way, and the 95% of confidence interval is 8.10～9.00μg/kg and 9.37～10.90μg/kg.According to the above data, we calculate that the bioavailability of TTX by IM on male and female Wistar is 81.1% and 86.0%.We set 3 groups of administering TTX, 1.5, 3.0, 6.0μg/kg, meanwhile, set excipient control and blank control in chronic toxicity test on SD rats. There are 10 males and 10 females SPF SD rats in every group. The rats are administered TTX by intramuscular injection 1 time per day for 30 days. And we set 30 days recover stage. At the ending of administration and recover stage, the 50% of rats are mercy kill, hematology indexes, clinical chemistry, pathology tissue test and ophthalmology test are hold. The general clinical manifestation is observed every day and ingestion is tested every week. We find the rats in 3, 6μg/kg group have dyskinesia, paralysis and amyasthenia symptom on the posterior limb, and have hypokinesia, depressed, drowsiness and systematic amyasthenia. These kinds of symptom are aggravated with the increasing of administration times. But the rats can be recovery on the next day. Otherwise, the pelage of rats are worse with the increasing of dose. There are five rats (4 male, 1 female) have the obvious trichomadesis on the back and tail, which is keeping on until being executed. There is not any abnormal appearance on the whole stage on the other clinical observation. The body weight of animals in treated groups has no toxicity difference compare to control on whole test stage. All rats are normal on the ophthalmology examination. The change of 14 examination and 15 clinical chemistry indexes is belong to reference value. There is not any specificity change of the organs on gross anatomy; neither is on the test under microscope. There is not delayed toxicity reaction are observed on the recovery stage. There is not irritant reaction on the injection local area or hemolytic reaction all over the body. In this study, the no observed adverse effect level (NOAEL) is 1.5μg/kg for SD rats by intramuscular, the main toxicities of TTX are local and systematical conduction blockade from nerve to muscle and the inhibition on central nervous system (CNS).The single dose acute intramuscular injection toxicity study on rhesus monkey shows that the monkey in the group of 5.000μg/kg is dead. And the monkeys in the groups of 4.375μg/kg, 3.750μg/kg and 2.500μg/kg are survival. The monkey in group 5.000μg/kg began to defecate after 6 minutes from administration; amyasthenia is emerged after 14 minutes. After 16 minutes, the symptoms of palsy, little dribbling and emesis are emerged. After 23 minutes, the monkey has a weak arterial pulse, and it is on agonal. After 28minutes, the monkey is dead. The gross anatomy has not any positive finding. The monkey in group of 4.375μg/kg began to muscle weakness after 19 minutes from administration, unsteady and disable standing are emerged after 27 minutes and 34 minutes. After 50 minutes, the monkey has general fatigue more obviously, and 55 minutes, the monkey has general weakness and nuta-head. After 1.5 hours, the power of front limbs comes back gradually, but the monkey can still not stand. After 4.5 hours, the hind limbs began to recover. And after 6.5 hours, the hind limbs recover totally. The monkey in the group of 3.750μg/kg began to muscle weakness after 48 minutes from administration, can not walk. And after 4 hours, the hind limbs recover. The monkey in the group of 2.500μg/kg has not abnormal symptom. All of survival monkey has no abnormal symptom in the following 2 weeks. At the ending of test, the gross anatomy has no any abnormal finding. In this study, the approximate lethal dose (ALD) of rhesus monkey is 4.375μg/kg~5.000μg/kg, and the maximum tolerated dose (MTD) is 4.375μg/kg, and the no observed effect level(NOEL) is 2.500μg/kg.In the chronic intramuscular injection toxicity test on rhesus monkey for one month, all the monkeys on high dose group emerged emesis in the first week after 30 minutes from administration, which is disappeared in a week. The excrement and membrana mucosa has no abnormal. The body temperature, electrocardiogram and ophthalmology examination have no toxicity. The 14 hematology indexes and 17 clinical chemistry indexes are in range of normal reference value. The all monkeys'change of body weight has no toxicology sense. The ingestion quantity has no obvious change in the whole test. The pathology examination dose not find the confirm toxic target organ or change related to administration. There is not delayed toxicity is observed in the recovery phase. So we can get the no observed adverse effect level is 1.50μg /kg ,and the main toxicity of TTX is the reaction of gastrointestinal tract(just like emesia) in rhesus monkey administered TTX by IM for on month.Summarize the above, we get the LD50 of male and female Wistar rats is 10.53μg/kg and 11.75μg/kg by bliss way, and the 95% of confidence interval is 9.99～11.10μg/kg and 10.91～12.65μg/kg. The LD50 of male and female Wistar rats is 8.54μg/kg and 10.10μg/kg by bliss way, and the 95% of confidence interval is 8.10～9.00μg/kg and 9.37～10.90μg/kg. The bioavailability of TTX by IM on male and female Wistar rats is 81.1% and 86.0%. The no observed adverse effect level (NOAEL) is 1.5μg/kg for SD rats by intramuscular injection; the main toxicities of TTX are local and systematical conduction blockade from nerve to muscle and the inhibition on central nervous system (CNS). The approximate lethal dose (ALD) of rhesus monkey by intramuscular injection is 4.375μg/kg~5.000μg/kg, and the maximum tolerated dose(MTD) is 4.375μg/kg ,and the no observed effect level(NOEL) is 2.500μg/kg. the no observed adverse effect level is 1.50μg /kg ,and the main toxicity of TTX is the reaction of gastrointestinal tract(just like emesia) in rhesus monkey administered TTX by IM for on month.
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