Confirmed L - Structure Of Carnosine And Its Pharmacokinetics In Vivo And In Vitro Studies

L-carnosine is a naturally water-soluble dipeptide. It has a wide range of biological eff ects, such as anti-oxidation, the protection of membrane integrity, anti-glycation, proton buff ering and regulating the activity of macrophage functions. It has been reported that L-carno sine has therapeutic effects on human atherosclerosis, diabetes, cataracts and arthritis. It also has potential therapeutic effects on central nervous system diseases, such as Alzheimer’s di sease, Parkinson’s disease and cerebral ischemic disease. L-carnosine has not been registered domestic and abroad, it is in the research and development progress as a classⅠnew dru g. In this paper, the following researches have been done according to the requirements for new drug registeration.The chemical structure of L-carnosine raw drug, was confirmed 2-(3-aminopropanoylami no)-3-(3H-imidazol-4-yl)propanoic acid using NMR, MS, IR and UV spectra analyses. Its ph ysical and chemical properties were studied. The polarimetric method proved that L-carnosin e was L-configuration and optical pure. Thermal differential and thermo-gravimetric analyses were performed and the results showed it contained no crystal water and was heat-stable. The specific extinction coefficient was determined to be 367.The contents of L-carnosine in raw drugs were determined using UV-Vis absorption spe ctroscopy. The linear range was 6.0～22μg·mL-1 (r= 0.9997). The average recovery w as 99.6%, and the RSD was 0.6%. The established method was accurate, fast and easy. Th e results showed the contents of L-carnosine in 3 lots of raw drugs were 98.7%,98.9%an d 98.9%.The degradation dynamics of L-carnosine in simulated gastric fluids (SGF) and simulat ed intestinal fluids (SIF) were studied by a fully validated HPLC method with good repeata bility. The separation was perfomed on a Kromasil NH2 column (200 mm×4.6 mm,5 u m) with the mobile phase composed of acetonitrile and phosphate buffer solution (32 mmol·L-1 of potassium dihydrogen phosphate and 8 mmol·L-1 of dipotassium hydrogen phosphate) (50:50, v/v). The UV detector was set at 210 nm. The flow rate was 1.0 mL·min-1 and column temperature was 35℃. The injection volume was 10 pL. The linear ranges of L-c arnosine were both 10.0～120μg·mL-1 in SGF and SIF (rSGF= 0.9996 and rsw= 0.9999, respectively). The recoveries of L-carnosine in SGF and SIF were 95.0% and 99.0%, whil e the precision (RSD) were 1.2% and 3.0%, respectively. The results showed a first-order e nzymatic degradation of L-carnosine. The degradation half lifes of L-carnosine in SIF and SGF were 4.0 and 25.7, respectively.To study the absorption of L-carnosine, an HPLC method was developed for simultane ous determination of L-carnosine and phenolsulfonphthalein in rat in situ single pass intesti nal perfusion solution. The separation was perfomed on a Kromasil NH2 column (200 mm×4.6 mm,5μm) with the mobile phase composed of acetonitrile and phosphate buffer sol ution (1 mmol·L-1 of potassium dihydrogen phosphate and 0.2 mmol·L-1 of dipotassium hydr ogen phosphate) (50:50, v/v). The UV detector was set at 210 nm. The flow rate was 1.0 mL·min-1 and column temperature was room temperature. The injection volume was 10μL The linear ranges of L-carnosine and phenolsulfonphthalein were 22.6～158μg·mL-1 (r= 0.9998) and 14.2～99.4μg·mL-1 (r=0.9991), respectively. The precision values (RSD) o f L-carnosine and phenolsulfonphthalein were 1.8% and 2.8%, while the average recoveries were 100.2% and 100.3%, respectively. The variance analyses of the apparent absorption co efficients (Papp) under various pH conditions were performed.The results showed that there was a significant difference between different pH values (P<0.01).An HPLC-UV method was established for the determination of L-carnosine in rat plas ma. The plasma samples were treated by precipitation with acetonitrile. The separation was perfomed on a Kromasil NH2 column (200 mm×4.6 mm,5μm) with the mobile phase composed of acetonitrile and phosphate buffer solution (32 mmol·L-1 of potassium dihydrog en phosphate,6 mmol·L-1 of dipotassium hydrogen phosphate and 0.05% ammonia) (55:45 , v/v). The UV detector was set at 210 nm. The flow rate was 1.0 mL·min-1 and column t emperature was 35℃. The injection volume was 10μL. The linear range was 2.0μg·mL-1～2000μg·mL-1 (r=0.9993) and the lower limit of quantification was 2μg·mL-1. The method was fully validated and applied to a pharmacokinetic study of L-carnosine in rat. T he pharmacokinetic parameters were as followed, Tmax:1.3±0.5 h,t1/2:1.3±0.4 h; Cmax :1099±369μg·mL-1; AUC0-∞:2770±838μg·h·mL-1.