Preclinical Studies Of Tetrodotoxin Pellets And Tetrodotoxin Lidocaine Compound Preparation

Tetrodotoxin is mainly extracted from the organs of puffer fish.It is a highly selective blocker for voltage-gated sodium channel.It has analgesic,rehabilitated and antiarrhythmic effect.At present,tetrodotoxin is a hotspot of innovative drug research.[3H]Tetrodotoxin was prepared by chemical isotopic labeling method and used to study on pharmacokinetics in rats in vivo.The extremely low content tetrodotoxin preparations,the immediate-release pellets and enteric-coated sustained-release pellets were prepared by fluidized-bed spray irrigation and used to study on the analgesic effect in animals.The compound preparation of tetrodotoxin and lidocaine hydrochloride was prepared and it has completely reversed the mortality by ventricular arrhythmia.The research is of great value for overcoming the technical difficulties to make breakthroughs of the non-clinical pharmacokinetics of tetrodotoxin,and to expand clinical application and indications for tetrodotoxin.The main research contents were as follows:1.Preparation of[3H]tetrodotoxinTetrodotoxin was oxidized by DCC to get 11-oxo-tetrodotoxin,which was then reduced by Boron tritiated sodium to get 11-[3H]labeled tetrodotoxin.The product was purified by HPLC separation,and confirmed by LC/(+)ESI-FTMS analysis,showing that the labeled products has the same retained time and mass spectral fragmentation regularity as tetrodotoxin standard.The radioactive purity of[3H]tetrodotoxin was 90.71%.2.Pharmacokinetic studies of[3H]tetrodotoxin using rat modelMale and female rats had similar pharmacokinetic characteristics according to the total plasma radioactivity.In the dry plasma the Mean Retention Time(MRT)of total plasma radioactivity was 1.62 hr,and the estimated average terminal half-life(t1/2)was 2.31 hr.The total recovery of[3H]telrodotoxin radioactivity after 0-72 hr intravenous administration in rats was 69.35%.The distribution and elimination of male and female rats were similar according to the total radioactivity.The drug was distributed in the organs except for the testis,epididymis and the brain.The stomach,kidneys and lungs had the relatively high concentration,and the peak time was half an hour for most tissues and organs.Using high-performance liquid chromatography combined with low energy radionuclide detection technology,the tetrodotoxin metabolite spectrum for plasma,urine and fecal samples were obtained.One metabolite which was an oxidant of tetrodotoxin has been identified by LC-MS,which provides possible metabolic pathway of tetrodotoxin.3.The preparation of immediate-release and enteric coated sustained-release tetrodotoxin pelletsThe determination method for the in vitro released tetrodotoxin pellets was established.The prescription and preparation process for immediate-release and enteric coated sustained-release pellets were established.The immediate-release pellet with sucrose pellets as a drug carrier was prepared by fluidized bed spray irrigation.The preparation process has high yield,drug content uniformity and advantages of rapid drug release,which met the quick release preparation standard.The enteric coated sustained-release tetrodotoxin pellets were coated sequentially by Eudragit NE30D as sustained release film,HPMC as an isolation film,and Eudragit L30D-55 as an enteric soluble film with fluidized coating preparation process.The preparation of pellets samples can stable release for 12 hr,in accordance with the standard of enteric sustained-release preparation.4.Acute toxicity and analgesic effect study for tetrodotoxin immediate-release and enteric coated sustained-release pelletsThe LD50 for the tetrodotoxin immediate-release pellets was 517.43 ?g·kg-1 via oral administration to male and female SD rats.The analgesic effect was measured by hot plate test,the results showed that the analgesic effect began 30 mins after i.g.administration at the dosage of 40,60 and 80 ?g-kg-1,.The effect maintained 1.5-3 hr,and the 80 ?g-kg-1 had the strongest effect.In acetic acid induced rat twist test,there is a good time-effect and dose-effect relationship.The results showed that the analgesic effect began at about 15 to 30 mins after i.g.administration at the dosage of 20,30 and 40 ?g-kg-1.The effect maintained 3 hr.The ED50 was about 21.04 ?g-kg-1.The treatment index was 24.59,which showed greatly enhanced safety compared to muscle injection and subcutaneous injection preparations.The LD50for the enteric coated sustained-release pellets was 840.13 ?g·kg-1.This was 1.62 times as the immediate-release preparation,which showed enhanced safety.The analgesic effect maintained 3-12 hr after i.g.administration at the dosage of 40,60 and 80 ?g·kg-1 according to the hot plate test.In acetic acid induced rat twist test,the analgesic effect began at about 1.5-9 hr after i.g.administration at the dosage of 20,40,60 and 80 ?g kg-1.The effective rate was at about 50%with the strongest effect at about 3-6 hr.5.The preparation and animal study of the antiarrhythmic injection compound with lidocaine and tetrodotoxinThe freeze-dried powder of tetrodotoxin and lidocaine hydrochloride compound for injection was prepared and the antiarrhythmic study was carried out comparing the compound preparation to single antiarrhythmic drugs with the rat model.The results showed that the preparation of compound(composed of 1 ?g tetrodotoxin and 5mg lidocaine hydrochloride)completely reversed the mortality by ventricular arrhythmia comparing to the single antiarrhythmic drug.As a results,the compound preparation is suitable for the treatment of lethal ventricular arrhythmia.