| Ketoprofen(KP), also called profenid, is practically insoluble in water, very soluble in methanol, and freely soluble in ethanol, acetone or ethyl ether. As a kind of non-selective non-steroidal anti-inflammatory drug(NSAID), it shares the pharmacological properties of other NSAIDs with analgesic, anti-inflammatory and antipyretic effects. Ketoprofen preparations are used for postoperative pain, acute renal colic, all sorts of mild and moderate cancer pain in clinical treatment (Ketoprofen is recommended as the first auxiliary drug of opioid analgesics preparations when severe cancer occurs by WHO), pain caused by gout attacks, rheumatic arthritis and chronic infectious arthritis, ankylosing spondylitis, osteoarthritis and so on. In addition, there is a certain central analgesic effect for ketoprofen. According to World Health Organization (WHO) recommended three-step analgesic ladder, ketoprofen or other NSAIDs should be the first choice for mild, moderate pain. Currently, the mainly dosage forms of ketoprofen listed in China are ordinary tablets, dispersible tablets, capsules, oral suspension and the like, but ketoprofen injection can not be found in Chinese market. Ketoprofen has major side effects of gastrointestinal after oral administration, even can develop to the serious gastrointestinal side effects such as nausea, abdominal pain, gastrointestinal bleeding, and thus ketoprofen is subject to certain restrictions in the clinical applications. Therefore, the research team led by Professor Luo Yonghuang of Southwestern University and Yichang Three Gorges Pharmaceutical Co., Ltd. have jointly researched and developed ketoprofen injection. Intramuscular injection dosing of ketoprofen can avoid directly contacting with the gastrointestinal, thereby reducing the side effect of gastrointestinal to a certain extent. What’s more, Ketoprofen injection possesses the characteristics of fast effect, high bioavailability and having no first-pass effect, for which it can be used for the patients who have oral disorder or cannot have oral dosing. Therefor, ketoprofen injection has a very good practical value and clinical application prospects.Ketoprofen injection developed by our team belong to class 3 in our country’s new drugs registration. According to the "chemical registration requirements of the classification and reporting information" of "Drug Registration" of State Food and Drug Administration’s document of No.28,2007, a study on the part of its preclinical was made to accumulate data for clinical trials and registration of ketoprofen injection. The study includes the following sections:Establishment of analytical methods:This paper established methods of determining content and related substances of ketoprofen injection. Chromatographic conditions of determining content of ketoprofen injection The chromatographic procedure may be carried out using a stainless steel column (250mm×4.6mm) packed with end-capped octadecylsilyl silica gel for chromatography (5μm) (Shimadzu Shim-pack VP-ODS C18 is suitable), a mixture of 2 volumes of freshly prepared phosphate buffer pH 3.5 (Dissolve 68.0 g of monobasic potassium phosphate in 1000 ml of water, and adjust with phosphoric acid to a pH of 3.5 ± 0.05), 43 volumes of acetonitrile and 55 volumes of water as the mobile phase with a flow rate of 1.2 ml per minute and a detection wavelength of 255 nm. The column temperature was 25℃ and the sample volume was 10μL. The column efficiency, determined from the ketoprofen peak, is not less than 2000 theoretical plates. Chromatographic conditions of determining related substances of ketoprofen injection:a detection wavelength of 233 nm and the sample volume of 20μL, other conditions are the same as the conditions of determining content of ketoprofen injection. According to the method validation, the method was proved to be simple, accurate with good repeatability and strong specificity, and is suitable for quality control of ketoprofen injection.Formula technique optimization of ketoprofen injection:The study adopted the clarity, stability and content of the injection as the investigation indicators. The optimal formula of ketoprofen injection was ultimately determined by single factor and orthogonal experiments. The optimal formula of ketoprofen injection consisted of 5% ketoprofen,3.6% arginine,2.5% benzyl alcohol, its pH was adjusted to 6.0-8.0 by an appropriate volume of citric acid solution. Meanwhile, preparation technique was screened and optimization such as activated carbon dosage(adsorption time and temperature), the liquid temperature in preparation process and sterilization conditions. The optimum production of ketoprofen injection was screened out:Weighed the water for injection with an amount of 60% of the preparation volume, placed it into a suitable container, added the prescribed dose of arginine, and stirred the solution for dissolution; added the prescribed dose of ketoprofen, and stirred the solution for thoroughly dissolution at a temperature of 50-60℃; added the prescribed dose of benzyl alcohol, stirred them till they were thoroughly mixed. Added dose of activated carbon that was 0.01% of the prescribed volume, heated to 60℃ with constant stirring and then adsorbed for 10 minutes, and then adjusted the pH to 6.5±0.5 by using appropriate amount of citric acid solution. At last, supplemented the solution with water for injection to reach a specified volume and mixed well. Vacuum suction filter while the solution was hot to remove activated carbon, and then filtered. The pH and content of ketoprofen was determined. After qualified, sealed the filtrate into a 2ml brown ampoule and sterilized the solution by 121℃ hot pressing sterilization for 12 minutes, the ketoprofen injection was finally obtained after leakage detection, light inspection, label, and packaging. Ketoprofen injection was clarity, stability, pH appropriate and accurate content which was prepared according to above the optimal formula and production. The injection strength was 2 ml:0.1 g.Study on quality standard of ketoprofen injection:Referring to the content of the limits of "ketoprofen enteric-coated capsules" in the current edition of Chinese and USP pharmacopoeia, as well as the product of three batches of samples actually measured data, the content of the limits of this product formulation defined as "ketoprofen (C16H14O3) should be labeled amount of 90.0%～110.0%"; Determination method, mainly based on the HPLC method of "ketoprofen enteric-coated capsules" and "ketoprofen liniment" standards in the current edition of Chinese Pharmacopoeia. The method has been proved to be feasible by the method validation of determination of ketoprofen; And related substances mainly referred to the standards of "Ketoprofen capsules," which contained in the current edition of the British Pharmacopoeia, namely the HPLC method, impurity A and impurity C were calculated by external standard method, unspecified impurity were calculated by main component self-compare method. The method has been proved to be feasible by the method validation. In addition, the quality of ketoprofen injection was researched in this paper, such as identification, examination and determination. As a result, identification of the project were positive; Check items were in line with the "Chinese Pharmacopoeia" item under the relevant requirements of injection; The contents and related substances of three batches of ketoprofen injection were measured by the methods established before, the results can meet the relevant requirements, which can further proved the method having high sensitivity, good reproducibility and operability.Study on stability of ketoprofen injection:The stress test, accelerated and long term test for ketoprofen injection were conducted in this paper. Stress test was consisted of the high-temperature test [(60±2)℃], strong illumination test [(4500±500)lx] and the freeze-thaw test (-10～-20℃ freezer,40℃ electric oven, three cycles). The results showed that the injection would occur degradation in the condition of strong illumination, namely, content decreased and related substances increased. Therefore, the injection should avoid the light in the process of storage and transportation. The main index such as appearance traits, color, pH, content and related substances were no significant changes in high temperature of 60℃ and freeze-thaw conditions, indicating that ketoprofen injection were stable under these conditions. Ketoprofen injection in listing packaging form were stable in the condition of accelerated and long term test conditions. The investigation of long term test is still in progress.Acute toxicity test, analgesic effect and effects on the central nervous system: Acute toxicity was observed for 14 days after a single dose of the drug in mice or rats. The analgesic test was processed by hot-plate and acetic acid-induced writhing in mice. The results of effects on the central nervous system test showed that ketoprofen could reduce locomotor activity and have a weak suppression effect on coordination movement in mice. In combination with barbiturate drugs, occured a weak synergistic effect, and extended sleep duration in male mice. In addition, ketoprofen injection have no obvious irritation on blood vessels and muscles. Hemolysis and hemagglutination phenomenon had not been seen in vitro hemolysis test yet. The results of this chapter showed that ketoprofen injection have a weak inhibitory effect on the central nervous system with a good analgesic effect and high security.Study on non-clincal pharmacokinetic:To establish a RP-HPLC method to measure ketoprofen concentrations in plasma of rats and compare the characteristics of pharmacokinetics of ketoprofen injection by intramuscular and intravenous injection. The content of ketoprofen in rats’plasma was determinated by RP-HPLC, and the data of each group was analysised by DAS 2.0 pharmacokinetic software. Results: ketoprofen were completely separated from impurities and with no other interference. The linear range of determination was 0.02 to 40μg·ml-1 with the correlation coefficient of 0.9998 for plasma concentrations of ketoprofen; The Lower limit of quantification (LLOQ) was 0.02μg·ml-1 at S/N≥5. Average extraction recoveries were 81.47%, 82.32% and 79.88%, inter-day precisions were 2.98%,3.19% and 3.43%, intta-day precisions were 6.03%,4.82% and 5.27% for high, middle and low concentrations of ketoprofen, respectively. Pharmacokinetics parameters of ketoprofen in rats afert single intramuscular and intravenous administration:T1/2z were 5.19±0.08 h and 2.95±0.14 h, Tmax were 0.33±0.01 h and 0.02±0.01 h, Cmax were 18.32±0.02 mg/L and 19.02±0.01 mg/L, AUC0-24 were 36.46±0.17 mg/L·h and 37.30±0.23 mg/L·h, respectively. In conclusion, the method was simple, accurate, with strong specificity and good reproducibility and can be used for detecting plasma concentration of ketoprofen; Ketoprofen by intramuscular injection was absorbed quickly and completely, and with high bioavailability absolutely, which could provide basic data for clinical intramuscular injection administration.
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