The Quality Of The New Analgesic Medicine Tetrodotoxin And Its Preparation

At present,the medicines for the treatment of war trauma pain are mainly opioids,but opioids have limitations,easy addiction,easy tolerance,and many side effects on the respiratory,gastrointestinal and central nervous systems.Therefore,the development of new analgesic drugs is essential to maintain the combat effectiveness of soldiers and reduce the suffering of patients.Tetrodotoxin(TTX),as a specific sodium channel blocker,has good analgesic activity,no tolerance,no addiction,and mild and transient adverse reactions.It is expected to become the new medicine for war trauma analgesia.The purpose of this research: The quality and process of TTX active pharmaceutical ingredient(API)and powder injection were preliminarily studied,including the quality standard of TTX API,the preparation process and quality standard of TTX powder injection,and the preliminary stability investigation was carried out to provide a reference for the clinical trial data of new medicines.The main research contents are as follows:1.The quality study of TTX API: According to the relevant provisions of Chinese Pharmacopoeia(2020 Edition),the traits,identification,inspection,content determination,related substances and stability of TTX API were studied.2.The preparation process of TTX powder injection: According to the quality analysis results of TTX API and related literature patents,combined with its application scenarios,the prescription screening and preparation process optimization of TTX powder injection were carried out.3.The quality study of TTX powder injection: According to the relevant provisions of Chinese Pharmacopoeia(2020 Edition),the traits,identification,inspection,content determination,related substances and stability of TTX powder injection were studied.The results were as follows:1.The quality study of TTX API: This product was a white powder,that was soluble in acidic aqueous solution or alcohol solution,very slightly soluble in water,insoluble in organic solvents,and had a maximum UV absorption at a wavelength of191 nm.Phosphate and ammonium salts were not detected,and the drying weight loss was less than 0.1%.A method for the determination of TTX and its related substances was established.The chromatographic conditions were as follows: Agilent 1260 infinity II high performance liquid chromatograph,evaporative light scattering detector(ELSD),chromatographic column Gel Amide-80(150 mm×2 mm,5 μm);the mobile phase was acetonitrile: 0.1% formic acid solution containing 5 mmol/L ammonium acetate(70:30)(V:V).The ELSD evaporator temperature was 105°C,the gas flow rate was 1.8 L/min,the nebulizer temperature was 80°C,and the gain was 1.The column temperature was40°C,the flow rate was 0.2 m L/min,and the injection volume was 10 μL.Under this condition,TTX and related substances were completely separated,and the solvent had no effect on the content determination.The linear relationship was good,the specificity was strong,the durability was good,and the accuracy and precision were high.According to the determination results,the content limit of this product is not less than98.0%,and the sum of the impurity peak area shall not be greater than the main peak area of the control solution(1.0%).The traits,content and related substances of the product did not change significantly after accelerated test for 6 months.2.The preparation process of TTX powder injection: Through single factor investigation,the prescription and preparation process were as follows: 21.01 mg of citric acid and 29.41 mg of sodium citrate were accurately weighed and dissolved in 1000 m L of water for injection,10 mg of TTX was added and stirred for 10 min,100 mg of L-cysteine and 40 g of mannitol were added and stirred for 15 min to dissolve completely.The p H value was determined,and the content of TTX was determined by high performance liquid chromatography-diode array detection method.The solution was poured into 3 m L carton bottles,1 m L per bottle,pre-frozen at-80°C,and sublimated and dried for 48 h.Each bottle of powder injection containing about 10 μg of TTX was prepared and used with disposable mixed medicine syringes.3.The quality study of TTX powder injection: This product was a white loose block.After re-dissolution,the solution was clear and colorless without visible foreign matter.The p H value was 4.6～4.7,the average loading amount was about 0.068 g,the loading difference was ±1%,and the water content was less than 0.1%.A method for the determination of TTX powder injection and its related substances was established.The chromatographic conditions were as follows: Agilent 1260 infinity II high performance liquid chromatograph,diode array detector,detection wavelength of 200 nm,Acclaim PA2 liquid chromatographic column(250 mm×4.6 mm,5 μm),mobile phase of 50mmol/L ammonium dihydrogen phosphate: 10 mmol/L sodium heptanesulfonate(70:30)(V:V),flow rate of 1 m L/min,column temperature of 30°C.The injection volume was10 μL.Under these conditions,TTX and related substances were completely separated.Solvents and excipients had no effect on the content determination.The method had a good linear relationship,strong specificity,good durability,high accuracy and precision,and could accurately determine the content of TTX in powder injection.According to the determination results,the content of TTX in this product should be 90.0%～110.0%,and the total impurity content should not exceed 1.0%.Under the conditions of high temperature,high humidity and strong light,there was no significant change in the traits,TTX content and related substance content of this product.Under the accelerated test and long-term test(6 months),there was no significant change in the traits,TTX content and related substance content of this product.Conclusion: In this study,the quality of TTX API,the preparation process and quality of powder injection were preliminarily studied.The quality standard of TTX API was established,the preparation process of TTX powder injection was determined,and its quality standard was established.The storage conditions and validity period were determined through preliminary stability investigation,which provided a reference for the clinical trial data of new medicines.