Studies On The Quality Control And Pharmacokinetics Of Rutin Deca(H-) Sulfate Sodium

Rutin deca(H-) sulfate sodium (RDS) is a novel polysubstituted flavone, possess very good activity as inhibitor of the complement system of warm-blooded animals and can be used in the therapeutic treatment of certain immunological diseases. It was also found that this compound possesses potential anti-HIV activity. In this work, we study on its quality control, drug metabolism and pharmacokinetics.1. Elucidation of RDS structure and quality controlElement analysis, IR, UV, ¹H-NMR, ¹³C-NMR and MS spectrum of RDS were determined and all the chemical shifts of H and C have been assigned based on ¹H-¹H COSY, ¹H-¹³C COSY and DEPT. The chemical structure of RDS was confirmed to be that of aimed compound, 2-[3,4-bis(sulfooxy)phenyl]-3-[[6-O-(6-deoxy-2,3,4-tri-sulfo -α-L-mannopyranosyl)-2,3,4-tri-O-sulfo-β-D-glucopyranosyl]oxy]-5,7-bis(sulfooxy)-4H-1-benzopyran-4-one,decasodium salt.Some physical-chemical properties of RDS, such as description, solubility, chloride, iron, arsenic, heavy metals, absorptivity, loss of dry, moisture and organic volatile impurities et al were investigated. RDS is light yellow powder, soluble in water, chloride < 0.02%, iron < 0.002%, arsenic < 2 ppm, heavy metals < 30 ppm, ε254 nm= 15512.4, ε319nm=13090.4, loss of dry 12.4%, moisture 11.15%. Organic volatile impurities of ethanol, ether, acetone, TEA, n-butanol and DMA were determined.An ion-pairing high-performance liquid chromatographic method was been developed to separate RDS and rutin nona(H-) sulfonate sodium (RNS). After selectedwavelength, ion-pairing type, TBAB concentration, pH value and ion strength in mobile phase, the chromatographic conditions used were optimized: Agilent Cs column was used as analytical column. The mobile phase consisted of acetonitrile and 25 mmolL"1 phosphate buffer solution containing 20 mmolL"1 TBAB (pH7.5) (48:52, v/v) with a flow-rate 1.0 mL-min"1. Detection wavelength was set at 254 run. The linear range of calibration curve was 0.5 200.0 ug-mL'1. The assay recoveries were about 94.7%. The intra-day and inter-day RSD were less than 3.5% and LOQ was 0.5 ± 0.036 ug-mL1 (n = 5). The method developed was simple, precise, and reproducible, and has been applied to determined RDS content and its raated substances.The results from stress test showed RDS is not stable when exposed to high temperature (40 V and 60 °C) and high humidity (RH 75 ±5% and RH 90+5%) but stable exposed to light (4500 ± 500 lx) for 10 days2. RDS pharmacokinetics studies in vivoAn ion-pairing coupled with solid-phase extraction technique (IP-SPE) was developed to extract RDS from rat plasma samples. 0.2 mol-L"1 of tetrabutyl ammonium bromide buffer (pH 8.0) was used as the ion-pairing extraction reagent and LC-18 as SPE sorbet. In addition, an ion-pairing HPLC method was established for the specific determination of RDS in plasma. The calibration curve was linear from 1.0 to 200.0 ug-mL"1. The regression equation was y = 0.3944* - 0.1093, R = 0.9997. The extraction recoveries from rat plasma were 60.00 62.53%, the limit of quantitation was 1.0 ± 0.1 ugmL'1 ( n = 5). The intra- and inter-day precision did not exceed 8.9% RSD. The stability under various conditions (three freeze-thaw cycles, stored at room temperature for 24 h and stored at - 24 °C for 4 weeks) showed that RDS was stable.The assay was applied to RDS pharmacokinetic studies in rats after iv via caudal vein of 5 % 20^ 100 mg-kg'1 RDS. The plasma concentration-time profiles were fitted using 3p97 software. The pharmacokinetic parameters of terminal half-life (fi/2p) and terminal coefficient (Kp) were 205.9 ± 11.1 min and 0.0034 ± 0.0002 min'1,respectively. The area under concentration-time (AUC) of 5^ 20^ 100 mg-kg"1 RDS iv rats were 3391.0, 14838.8 and 88598.2 jigminmL"1, respectively. The relationship of dose and AUC of RDS was linear within the dosage range. This suggested that the disposition of RDS in rats belong to linear kinetics and the pharmacokinetic parameters of RDS were dose independent.An ion-pairing coupled with solid-phase extraction technique (IP-SPE) was developed to determine RDS in rat urine and bile samples. The regression equations were R = 0.1362 C - 0.1187(R = 0.9981) in urine and R = 0.1232 C - 0.0753(R = 0.9995) in bile, respectively. The extraction recoveries in urine and bile were 57.44 61.07%, and the accuracies were 98.0 102.1%. After iv RDS 20 mg-kg'1 to 12 rats, the biliary excretion of parent drug amount to only 0.3181 ± 0.2087% of dosage in 36 h, and the urinary excretion 86.0 + 6.1% in 36 h.3. RDS metabolism studies in vitroThere were no significant inbibitive interactions between RDS and several major CYP450, CYP1A2, 2B, 2C9, 2C19, 2D6 and 3A4. (IC50?100 umolL"1). RDS was slightly metabolized in rat liver microsomes induced with BNF, Dex and PB.Ultrafiltration techniques were applied to determine the protein binding of RDS in plasma (rat, beagle dog and human), human serum albumin (HSA) and ai-acid glycoprotein (AGP). The mean protein binding in rat, beagle dog and human plasma of RDS were 80 90%, in which the range of concentration of RDS was 5 to 100 ug-mL'1. The protein binding to HSA was 85.7 + 1.3% and 14.0 ± 3.2% to AGP. The /-test results showed significant difference between HSA and AGP (P < 0.001).4. RDS metabolism in intestinal flora and elucidation of metabolite structureAn ion-pairing coupled with solid-phase extraction technique (IP-SPE) was developed to determine RDS in rat and human intestinal flora in vitro incubation. Metabolic rates of RDS incubate for 24 h in rat and human intestinal flora were 73.5 + 6.7% and 24.5 ± 3.5%, respectively. Three metabolites (M2, M3 and M4) were observed from intestinal flora samples.The metabolites of RDS in rat intestinal flora in vitro were studied. After IP-SPE, IP-RP-HPLC, freeze drying, glucosans, activated carbon and filtration, one compound (M3) related to metabolites were separated from intestinal flora. From HPLC, UV, DAD, 'H-NMR, 13C-NMR and MS spectrum, structure of metabolite M3 was identified. The structure of M3 was confirmed as 5,7-bis(de-sulfooxy) - rutin deca(H-) sulfate sodium. Compare the results of incubation in sulfatase and in rat intestinal flora, metabolites structure of Ml, M2 and M4 were proposed.