The Study On Pharmacokinetics Of DFP In Rats

ObjectiveDeferiprone(DFP), with its chemical name 1,2-dimethyl-3-hydroxy-4-pyrid-one, is a chemical synthesis of metal chelating agents. Deferiprone is usually used to treat iron overload in patients withβ-thalassemia. It has a good chelation with iron and aluminum as the first oral metal chelator. There were no reports about pharmacokinetics of low, middle and high concentrations, tissue distribution and plasma protein binding rate of DFP at home and abroad. The experiments established a simple, quick, accurate HPLC-UV method of DFP for the study of pharmacokinetics, tissue distribution and plasma protein binding rate of DFP in rats, which provided a theoretical basis for the further study on clinical pharmacokinetics and clinical rational drug use.Methods1 Pharmacokinetics of DFP in rats1.1 The treatment of experimental animalsFifteen wistar rats were randomly divided into three groups:low-dose group, middle-dose group and high-dose group. After three days'equilibrium, DFP was given to the rats through intragastric administration after 12 hours'fasting. Then about 0.25ml blood, from which plasma was collected, was collected from jugular sinus at different times with Heparin as anticoagulant.20μl sample was detected after the plasma had been treated.1.2 Chromatographic conditionThe separation was performed on a Diamonsil(R) C18 column (250 mm×4.6 mm, 5 5μm) and detected at 278nm. The mobile phase was a mixture of acetonitrile-natrium phosphate buffer (0.05 mol·L-1 Disodium hydrogen phosphate, adjust pH=3.0 with phosphoric acid, containing 5mmol·L-1 heptanesulfonic acid and 2 mmol·L-1 EDTA)=8:92 (v/v) at a flow rate of 1.0mL·min-1. Injection volume was 20μl, and detection run at room temperature.2 Tissue distribution of DFP in rats2.1 The treatment of experimental animalsTwenty rats were randomly divided into four groups (control group; 10 min group; 60 min group; 360 min group),and each group had 5 rats. After starvation of 12 hours, the control group was given 1 ml physiological saline, the other groups fed with DFP by intragastric administration. Each group were decapitated at different time, and organizations were taken. Samples of the tissue were rinsed by normal saline, dried by filter paper. The samples of tissue were made into 20%tissue homogenate with normal saline, then centrifuged.20μl sample was injected after the supernatant treated.2.2 Chromatographic conditionThe separation was performed on a Diamonsil(R)C18 column (250 mm×4.6 mm, 5μm) and detected at 278 nm. The mobile phase was a mixture of acetonitrile-natrium phosphate buffer (0.05 mol·L-1 Disodium hydrogen phosphate, adjust pH=3.0 with phosphoric acid, containing 5mmol-L'1 heptanesulfonic acid and 2 mmol·L-1 EDTA)=8:92 or 10:90 (v/v) at a flow rate of 1.0mL·min-1. Injection volume was 20μl, and detection run at room temperature.3 Plasma protein binding rate of DFP in ratsTen healthy male Wistar rats were decapitated for blood which was anticoagulated by heparin and made into plasma. Plasma protein binding rate of DFP was detected by equilibrium dialysis method, and 20μl sample was injected after dialysate treated.3.2 Chromatographic conditionsAs "2.2 chromatographic conditions".3.3 Drug adsorption experiment of dialysis baglml blank dialysate was added into dialysis bag. Then the dialysis bag was suspended in a wide mouth jar containing 10ml DFP dialysate of different concentrations. The stopper was bunged tightly. Then put the bottles into biochemical incubator at 37℃. After 4 hours balancing, concentrations of the drug outside the dialysis bag were measured for calculating the rate of drug absorption of dialysis membranes.3.4 The inspection of equilibrium time and the determination of plasma protein binding ratelml blank plasma was added into the dialysis bag. Then the dialysis bag was suspended in a wide mouth jar containing 10ml DFP dialysate of different concentrations. Then put the bottles in biochemical incubator at 37℃. The concentrations of inside and outside of the dialysis bag were measured at a certain time for determinating the plasma protein binding rate.4 Data processing and analysisThe compartment model and pharmacokinetic parameters were calculated by DAS 2.0 statistical analysis software. SPSS 13.0 was used for statistical description and t-test.Results1 Pharmacokinetics results of DFP in rats1.1 The results of methodology studySolution of DFP reference substance was scanned by UV-2450.278nm was selected to be the detection wavelength. The retention time of DFP was 10.8 min in the experimental conditions. It has a good correlation in the linear range from 1 to 120 mg·L-1 (r=0.9999). The absolute recovery of DFP was more than 98.7% and the relative recovery was 99.04%-100.6% at low, middle and high concentrations. The RSDs of intra-day were less than 0.74%, and those of inter-day were less than 1.16%. The RSDs were less than 1.72% after stored for 1 week at 4℃and 1 month at-20℃. The stability was good.1.2 Pharmacokinetic resultsThe pharmacokinetic process of DFP in rats was two-compartment model after ig DFP. The t1/2Ka were 18.90,16.08 and 12.69min, and Ka was at the range of 0.05-0.17 min-1 at low, middle and high concentrations. The ti/2αwere 23.33,22.22 and 20.88 min, and the V1 were 0.70,0.62 and 0.41L·kg-1 at low, middle and high concentrations. The CL were 0.017,0.021 and .016L(min-kg)-1, and the t1/2βwere 53.32,0.87 and 46.34min at low, middle and high concentrations.. The AU(o-∞,) were 2380.1,3519.4 and 8788.7mg-LL1-min after given DFP of low, middle and high concentrations, respectively. The follow parameters of low, middle and high concentrations were obtained by rosenblueth method of non compartment mode. The MRT(o-t) were 109.81,95.28 and 88.70min, and the i2z were 140.28,93.40 and 63.27 min. The Tmax were 36.00,48.00 and 54.00min, and the Cmax were 21.60,37.13 and 83.25mg·L-1. The VZ were 2.80,2.49 and 1.41L·kgg1, and the CLZ were 0.014,0.018 and 0.016L-(min·kg)-1.The AUC(0-t) were 2276.96,3580.54 and 8933.3 1mg·L-1·min, respectively.2 Tissue distribution results of DFP in rats2.1 The results of methodology studyThe retention time of DFP was 10.8min at the chromatographic conditions of small intestine, and other tissues were 7.8min The linear range of tissues were of a good correlation (r>0.9995). The absolute recoveries of DFP were more than 86.0% and the relative recoveries were 97.1%-109.6% at low, middle and high concentrations. The RSDs of intra-day were less than 3.33%, and those of inter-day were less than 4.98%. The RSDs were less than 5.49% after stored for 1 week at 4℃and 1 month at-20℃. The stabilities were good.2.2 The results of tissue distributionThe DFP was detected in each of tissues including brain and testis 10 min after ig 70mg·kg-1DFP. The concentrations of DFP were 244.8 1mg·kg-1 and 58.86 mg·kg-1 in stomach 10min and 60min after ig DFP. They were remarkably higher than those in small intestine in the same group (＜0.001). The concentrations of DFP were 359.22mg-kg-1and 56.80mg·kg-1 in liver 60 min and 360 min after ig DFP, and they were remarkably higher than those in the other tissues (＜0.001). The concentration in kidney was 10.13mg·kg-1 360 min after ig DFP, and it was remarkably higher than those in the other tissues except liver ((P<0.005).3 The results of plasma protein binding rate of DFP 3.1 The results of methodology studyThe retention time was 10.8min at the chromatographic conditions of inside dialysate, and it was 7.8min at the chromatographic conditions outside dialysate. The linear range of dialysate were of a good correlation (r>0.9998). The absolute recoveries of DFP were more than 98.71% and the relative recoveries were 99.04%-101.27% at low, middle and high concentrations. The RSDs of intra-day were less than 0.74%, and those of inter-day were less than 1.15%. The RSDs were less than 1.94% after stored for 8 hours at 37℃, and the short-term stabilities were good. The RSDs were less than 1.72% after stored for 1 week at 4℃and 1 month at-20℃, and the long-term stabilities were good.3.2 The results of plasma protein binding rateThe adsorption rates of dialysis bag were 4.53%,3.66% and 3.41% at low, middle and high concentrations, respectively. It needed 2 hours to get dialysis balancing at 37℃in biochemical incubator. The range of plasma protein binding rates were 4.25%-5.31%. There was no statistical significance among all concentration groups of plasma protein binding rates (P>0.05).Comcolutions1 The HPLC methods which were developed in this study were for the detection of DFP in rats plasma, tissue bomogenate and dialysate. They were quick, accurate, sensitive and good repeatabilities. They could be used in the studies of pharmacokinetic, tissue distribution and plasma protein binding rate of DFP in rats.2 DFP has quick absorption, wide distribution and fast elimination in rats after ig drug. The pharmacokinetic process of DFP in rats was two-compartment model.3 DFP was widely distributed in rats after ig drug. There were DFP in main tissues. It showed that DFP could pass through blood-brain barrier and blood-testis barrier.4 The plasma protein binding rate of DFP is low. So DFP is a kind of low plasma protein binding rate drug, and most of the drug molecules act on body with free type.