| Diclofenac sodium belongs to the third generation of non-steroidal antipyretic,analgesic and anti-inflammatory drugs.It was developed and listed by ciba geigy pharmaceutical company in Switzerland in 1973,and approved for veterinary clinical use in China in 2016.Acetaminophen belongs to acetanilide,a kind of antipyretic and analgesic drug.It was developed by javelin pharmaceuticals and listed as an alternative to Aspirin in England in 1956.Diclofenac sodium has antipyretic,analgesic and anti-inflammatory effects,and acetaminophen has antipyretic and analgesic effects.The compound preparation composed of diclofenac sodium and acetaminophen can synergistically achieve synergistic effect and reduce the dosage,which can be used in the clinical treatment of swine fever disease.In this study,compound diclofenac sodium injection was prepared according to the injection related requirements in the Chinese Veterinary Pharmacopoeia(edition 2015)and the technical guidelines for the research of veterinary chemicals drug.The quality inspection,stability study and clinical efficacy evaluation of the preparation were carried out in order to obtain a stable,safe and effective compound diclofenac sodium injection to be used in veterinary clinic.1.Preparation of compound Diclofenac sodium injectionThe compound diclofenac sodium injection was composed of diclofenac sodium(2.5%),acetaminophen(15%)and other pharmaceutical excipients.According to the physical and chemical properties of diclofenac sodium and acetaminophen,the solvent,antioxidant and other pharmaceutical excipients were screened by single factor test.After the prescription was determined,the pH value of the injection was selected,and the preparation process of the injection was optimized.Finally,the stable compound diclofenac sodium injection was prepared.The quality of the compound diclofenac sodium injection was investigated.The compound diclofenac sodium injection was colorless or almost colorless and slightly viscous.According to the requirements of the injection,the pH,sterility,related substances,visible foreign bodies and other items of compound diclofenac sodium injection were inspected.A HPLC method was used to determine the content of main components in compound diclofenac sodium injection.The chromatographic conditions were as follows:phenylsilane bonded silica gel as filler,acetonitrile water triethylamine(40:60:0.5)as the mobile phase,adjusting the pH value to 8.0 with glacial acetic acid,column temperature 35℃,detection wavelength 254 nm.The resolution of acetaminophen peak and diclofenac sodium peak meet the requirements.The linear range of diclofenac sodium was 15～35μg/mL with y=10.402x-0.13(R2=0.9999),and the linear range of Acetaminophen was 90～210 μg/mL with y=47.336x+252.35(R2=0.9998).The recovery of diclofenac sodium was 99.8%～100.2%with RSD 0.46%;the recovery of acetaminophen was 99.2%～100.0%with RSD 0.45%.The method can be used for the determination of main components in compound diclofenac sodium injection.The stability of compound diclofenac sodium injection was investigated by influencing factor test,accelerated test and long-term test.The results showed that compound diclofenac sodium injection was stable at high temperature(60℃),but slightly sensitive to strong light(4500Lx±500Lx).So it should be kept away from light.The results of accelerated test for 6 months and long-term test for 12 months showed that pH value,visible foreign matter,related substances and content of compound diclofenac sodium injection had no significant changes.The stability test results showed that compound diclofenac sodium injection had good stability under room temperature,dark and airtight conditions.2.Safety test of compound diclofenac sodium injectionThe safety pharmacology experiment was carried out,40 healthy mice were divided into 4 groups(control group,compound diclofenac sodium injection 1 times,5 times,10 times recommended dose group)randomly to investigate the drug effect of compound diclofenac sodium injection.The results showed that compound diclofenac sodium injection was safe for mice at the recommended dose.Acute toxicity test of compound diclofenac sodium injection was carried out,60 healthy mice were divided into 6 groups randomly.Except the control group,5 test groups were intramuscularly injected with different doses of compound diclofenac sodium injection.The LD50 of mice was calculated by Bliss method.The results showed that the LD50 of compound diclofenac sodium injection was 3.5646 mL/kg(95%confidence limit=3.1609-4.0877mL/kg),about 17-18 times of the clinical recommended dose,which was safe.Muscle irritation test of compound diclofenac sodium injection:eight healthy New Zealand rabbits were used to investigate the muscle irritation of compound diclofenac sodium injection by left-right control method in the same body.The results showed that the compound diclofenac sodium injection had no obvious stimulation to the quadriceps femoris of New Zealand rabbits,which met the criteria of intramuscular injection and could be used for clinical intramuscular injection.3.Efficiency sttudy of compound diclofenac sodium injectionIn this study,a mouse model of acute lung injury was established.After LPS treatment,the expression of TNF-α,IL-1β and IL-6 in lung tissue was significantly increased.The results showed that the inhibitory effect of compound diclofenac sodium on inflammatory factors was significantly stronger than the other two.Histopathological observation showed that compound diclofenac sodium had the most obvious effect on pulmonary edema,inflammatory cell infiltration and tissue congestion after LPS treatment.The model of artificial piglet castration and the inflammatory model of piglet caused by kaolin were established respectively.70 healthy piglets were divided into seven groups(negative control group,positive control group,compound diclofenac sodium control group,acetaminophen injection control group,low,medium and high dose of compound diclofenac sodium injection group)randomly.The plasma concentrations of PGE2 and cortisol,body temperature and clinical symptoms were measured objective to evaluate the antipyretic,analgesic and anti-inflammatory effects of compound diclofenac sodium injection.The results showed that the pathological model established could be used to evaluate the efficacy of compound diclofenac sodium injection.The antipyretic and analgesic effects of middle and high dose compound diclofenac sodium injection were significantly better than those of acetaminophen injection,compound diclofenac sodium control group,and there was no significant difference between middle and high dose(p>0.05).The recommended clinical dose of compound diclofenac sodium injection was 0.04 mL/kg.4.Clinical application of compound diclofenac sodium injection in pigs60 pigs suffering from fever and pain were divided into two groups(acetaminophen injection control group and compound diclofenac sodium injection recommended dose group)randomly.The clinical efficacy of compound diclofenac sodium injection was verified by clinical symptoms and body temperature.The results showed that the compound diclofenac sodium injection at the recommended dose(0.04 mL/kg),has a better antipyretic effect on sick pigs,and the duration of efficacy was longer than Acetaminophen injection. |
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