| Gastrodin(Gas) is the major and bioactive component in Tianma(Gastrodia elata Bl.) and has sedative,anticonvulsive and neuroprotective effects.It has been approved as a drug for the treatment of neurasthenia,dizzy,headache and adjunctive therapy to epilepsy in China.The results of recent clinical trials showed that it was efficient in treatment of patients with vascular dementia.Currently,the marketed preparations of Gas are intramuscularly,intravenously or orally administered,with injection as the most common dosage form.The target organ of Gas is brain,but it is difficult to pass blood-brain barrier (BBB) because of its water-solubility.There has a controversy about the active form of Gas.Some experts believe only the metabolite of Gas(p-hydroxybenzyl alcohol, HBA) can permeate through the blood-brain barrier(BBB) and have pharmacologic effect.Others think both of Gas and HBA have pharmacologic effect.But the dose of Gas is high in clinic in order to achieve pharmacologic activity because the amount of Gas permeation through BBB to brain is too low.The elimination of Gas is fast and the accumulation is very low in body.In order to keep effect,it is needed to administer several times each day in clinic.Although the toxicity of Gas is low,the frequent injection can increase the patient's pain and oral administration has some gastrointestinal side effect.Since little was known about the neuropharmacokinetics and brain metabolism of Gas,it would be meaningful to investigate the brain pharmacokinetics of Gas and HBA and their brain target in Sprague-Dawley(SD) male rat.In this paper,we also studied the cell transport of Gas and HBA. 1.Metabolism in vitro(1) The metabolism of Gas in rat brain,liver and kidney homognateThe metabolism of Gas in brain,liver and kidney was studied in rat tissue homogenate.A HPLC method was established to determine the metabolite,HBA concentration after incubation.HBA was analyzed on Diamonsil C18 column(4.6 mm×250mm,5μm),with a mobile phase consisting of methanol-water(2.5:97.5,v/v), and detected with UV detector at 221nm.The flow rate was set at 1.0 mL/min.The temperature of the column oven was maintained at 33℃.The assay was linear over the concentration range of 0.30—18.96μg/mL in brain homogenate,0.30—9.48μg/ mL in liver homogenate and 0.59—75.86μg/mL in kindey homogenate(n=3, r2＞0.999).The intra-and inter-day precision over these ranges were not more than 9.6%and 12.1%,respectively.The Accuracy was in the range of 97.5%—106.9%. The lower limit of quantification(LLOQ) was 0.290±0.045μg/mL,0.307±0.030μg/mL and 0.627±00.047μg/mL in brain,liver and kidney homogenate(n=5), respectively.The results showed the velocity constants of formation metabolite,HBA,were 0.0305,0.0121 and 0.1409μg/mL/min in brain,liver and kidney,respectively.The clearance rates of Gas were 29.7±2.1,10.4±0.8 and 299.5±22.7 L/min/g(×10-6) in the brain,liver and kidney,respectively.The metabolism rate of Gas was slow in brain and liver,while it was fast in kidney.(2) The metabolism of Gas in rat different brain regionsThe metabolism of Gas was studied in six different brain regions(the cerebellum,thalamus,pons and medulla oblongata,frontal cortex,hippocampus and striatum).In brain homogenate,the formation velocity of HBA was fast in the cerebellum, thalamus,pons and medulla oblongata,and slow in the frontal cortex,hippocampus and striatum.The velocity constants of the formers were about 1.5 times higher than those of the latters.Clearance rates of Gas were 45.9±7.4,39.6±5.6 and 24.4±3.0 L/min/g(×10-6) in cerebellum,thalamus and cortex,respectively.The results suggested that Gas is stable in non-enzyme system and can be metabolized to HBA in brain,liver and kidney homogenate in vitro.And Gas is metabolized most rapidly in the kidney homogenate.The metabolism rates of Gas in the cerebellum,thalamus,pons and medulla oblongata are faster than other three brain regions.2.Brain pharmacokinetics and metabolism of Gas after intravenous administrationA HPLC method was established to determine Gas and its metabolite HBA after Gas intravenous(i.v.) administration(200 mg/kg).The samples were analyzed on a Diamonsil C18 column(5μm,250 mm×4.6 mm) with a mobile phase consisting of acetonitrile-water(5%acetonitrile for brain microdialysate,2.5%acetonitrile for plasma and cerebrospinal fluid(CSF)),and detected with a UV detector at 221 nm.No peaks interfered with the analytes in the chromatograms of plasma and brain samples.The calibration curves of Gas were linear over the concentration range of 0.28-571.70μg/mL in plasma,0.16-40.02μg/mL in CSF and 0.07-17.86μg/mL in microdialysate(n=5,r2＞0.999).The LLOQ of Gas was 0.269±0.028μg/mL in plasma,0.147±0.011μg/mL in CSF and 0.072±0.008μg/mL in microdialysate (n=6).The intra-and inter-day precision of Gas over these ranges were not more than 12.4%.The Accuracy was 101.4%,96.6%,103.8%in plasma,CSF and microdialysate,respectively.The calibration curves for HBA were also linear over the range of 0.15-2.36μg/mL in plasma,0.07-1.18μg/mL in CSF and 0.04-0.59μg/mL in brain microdialysate(n=3,r2＞0.999).The LLOQ of HBA was 0.139±0.015μg/mL,0.072±0.006μg/mL in CSF and 0.038±0.005μg/mL in brain microdialysate(n=6).The intra-and inter-day precision of HBA over these ranges were not more than 13.7%. The Accuracy was 99.7%,98.8%,101.6%in plasma,CSF and microdialysate, respectively.SD male rats were administered with Gas at a dose of 200 mg/kg via the femoral vein.The blood sample was taken from the tail vein and CSF sample was collected using the cistern puncture.The microdialysis was applied in the frontal cortex (coordinates:AP 2.1,ML 2.0,DV 1.0) and hippocampus(AP-6.0,ML-4.6,DV 3.0), or thalamus(AP-3.0,ML 1.0,DV 4.5) and cerebellum(AP-11.0,ML-1.3,DV 2.0) according to the Paxinos and Watson atlas.Each probe was subjected to in vitro recovery studies before in vivo experiments for validation.The mean value of Gas recovery for all of the microdialysis probes was 0.209±0.018 at a microdialysate flow rate of 2.5μl/min at 37℃.The results from distribution of Gas in rat showed that the levels of Gas declined rapidly after drug administration and the entry of Gas into the brain was rapid. However,the ratios of AUCbrain/AUCplasma were not high.The individual ratios of the AUC in the CSF,frontal cortex,hippocampus,thalamus and cerebellum to the AUC in the plasma were 4.8±2.4%,3.3±1.2%,3.0±0.7%,3.3±1.3%and 6.1±1.9%, respectively.The AUC in the cerebellum was significantly higher than that in other brain regions(P＜0.05).The concentrations of HBA,the main metabolite of Gas,were very low in both of the CSF and plasma.3.Brain pharmacokinetics and metabolism of Gas after duodenum admininstraionSD male rats were administered with Gas via duodenum at a dose of 200 mg/kg and the samples of blood,CSF and brain microdialysate were collected at the indicated time points.The mean value of Gas recovery for the microdialysis probes was 0.233±0.021 at a microdialysate flow rate of 2.0μl/min at 37℃. The results showed Tmax in plasma and CSF was similar.The Cmax and AUC were the biggest in cerebellum among four brain regions.The individual ratios of the AUC in the CSF,frontal cortex,hippocampus,thalamus and cerebellum to the AUC in the plasma were 4.9±1.2%,2.4±1.0%,2.5±0.8%,2.6±0.8%and 4.7±2.2%, respectively.The absolute bioavailability of Gas in plasma after duodenum admininstration was 55.0%.The metabolism of Gas in both of brain and plasma were low.The AUC of HBA in brain and plasma after oral was higher than that after i.v.. The AUC of HBA in CSF and plasma were 2.3 and 1.4 times compared with those after i.v..4.Brain pharmacokinetics of Gas after intranasal and intravenous admininstraionThe pharmacokinetic behavior of Gas in rat plasma and CSF after intranasal (i.n.) and intravenous(i.v.) administration(50 mg/kg) was investigated.A HPLC method for the determination of Gas in rat CSF and plasma was developed and validated.The assay was linear over the concentration range of 0.156—9.985μg/mL in CSF and 0.390-99.85μg/mL in plasma(n=5,r2＞0.999),and intra-and inter-day precision over these range were not more than 6.4%.The LLOQ in CSF and plasma were 0.148±0.008μg/mL and 0.397±0.020μg/mL(n=6), respectively.The mean accuracy in plasma and CSF were 100.4%and 100.6%, respectively.Intranasal administration of Gas provided a comparable AUC in CSF compared with the intravenous administration.But Gas level in plasma was very low.The ratios of AUC values of intranasal to intravenous administration were 8.8%and 105.5%in plasma and CSF,respectively.The drug targeting index(DTI) was 12.3. Intranasal administration of Gas is a promising alternative to traditional administration.Olfactory mucosa did present another pathway for transport Gas to the brain. 5.Brain pharmacokinetics of HBA after intravenous administraionA HPLC method for the determination of Gas in rat cerebrospinal fluid and plasma was developed and validated.A mixture of acetonitrile-water was employed as a mobile phase,with a flow rate of 1.0 mL/min(3%acetonitrile for the plasma and 4%acetonitrile for the CSF).The assay was linear over the concentration range of 0.07-37.73μg/mL in CSF and 0.23-30.34μg/mL in plasma(n=3,r2＞0.999).The intra-and inter-day precision over these ranges were not more than 9.4%.The mean accuracy were 100.5%and 105.5%in CSF and plasma,respectively.The LLOQ in CSF and plasma were 0.074±0.007μg/mL and 0.258±0.016μg/mL(n=5), respectively.SD male rats were administered with HBA or Gas at 50mg/kg via i.v..The result of pharmacokinetics of HBA after i.v.(50 mg/kg) showed that t1/2 in CSF and plasma was 12.4±3.1 and 9.2±1.7 min,respectively,which was only 35%of those of Gas with same dosage.The concentration of HBA was declined faster than that of Gas.The Cmax and AUC of HBA in CSF were higher than those in plasma,while the Cmax and AUC of Gas in CSF were much lower than those in plasma.6.Brain pharmacokinetics of HBA after i.n.and i.v.admininstraionSD male rats were administered with HBA at a dose of 10 mg/kg via i.v.or i.n.. The results showed that HBA rapidly enters CSF after administration.There was no significant difference in AUCCSF or AUCplasma between i.n.and i.v.administration.In conclusion,HBA can be absorbed into the systemic circulation rapidly and completely after i.n.administration.Intranasal HBA is a promising alternative to intravenous administration. 7.The cell transport of Gas or HBA on Caco-2,MDCK and MDCK-MDR1 cellsThe cell transport of Gas or HBA was studied on Caco-2,MDCK and MDCK-MDR1 cells which were seeded on polycarabonate microporous membrane filters and allowed to grow to confluence as an in vitro model to assess the membrane permeability properties of drugs.The result from the transport experiment of Caco-2 cells showed that oral absorbed availability of Gas may be poor and related to its poor liposolubility,while HBA can be absorbed well.The result from the transport experiment of MDCK and MDCK-MDR1 cells showed Gas permeated cells very difficult.The R ratio of Gas was lower than 2 and it suggested that Gas is not the substrate of P-gp.HBA permeated cells easily.HBA had no significant effect on the efflux ratio of Rho123 and its R ratios was also lower than 2,which suggested HBA is not the substrate or inhibitor of P-gp.The results from two cells transport experiments showed that Gas is not the substrate of P-gp.HBA can be absorbed well and not the substrate or inhibitor of P-gp.The mechanism of non-P-pg mediated effiux may be existed at low HBA concentration and can be reversed by high concentration of HBA.
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