Study On Brain Targeting And Local PK-PD Modeling Of Xiongbing Microemusion Nasal Delivery

This research is part of the project The Construction and Evaluation of local PK-PD model's on chinese medicine by Microdialysis Technique (No.30873443)-which was funded by National Nature Science Foundation of China. The research group early had developed Xiongbing microemusion delivery system, pharmacology experiments showed that it had good effect for resistance to cerebral ischemia, and studied the pharmacokinetic properties in rats after oral Xiongbing microemusion by microdialysis technique. This research is plan to study the brain targeting of Xiongbing microemusion nasal delivery and local PK-PD modeling in acute ischemia rats, at last discussed the feasibility of nasal delivery from the aspects of pharmacokinetic and pharmacodynamic respectively.The study was consisted of three parts as following as:1 Study of pharmacokinetics in awke rats'brain after intranasal administration1.1 Establish a method for microdialysis in awke ratsThe guide tube of brain probe was planted into the site of corpus striatum of the left brain, then planted small screws, at last fixed it with denture acrylic. The rats were living in standard environment 3～5 days in order to recover from surgery, and planted brain probe into guide bube when the rats were awke, which was fixed in the site of corpus striatum.1.2 Establish a method for determination the contents of Tetramethylpyrazine (TMP) in brain microdialysis samplesThe contents of TMP was determined by HPLC, the concentration and peak area of the regression equation was Y=31393X+216.64, and R2= 0.9998, TMP had a good linear relationship between the concentration of 0.02558～2.5584μg/mL, the RSD of precision was 1.04%, and the RSD of sample's stability was 1.23%, TMP minimum quantitative limit was 24pg.1.3 Study on vivo recovery and stability of brain probe in awake ratsThe effect of flow rate and concentration on vivo recovery, and the stability of vivo recovery had studied. The result showed that with the increase of flow rate (1.0～2.5μL/min), the vivo recovery was decreasing;and the concentration of TMP between 0.16～2.42μg/mL had no influence on vivo recovery;the RSD of vivo recovery in 8h was 5.56%. The parameters of sampling were determined as followings:the flow rate was 1.5μL/min, the sampling interval of early three sample was 10min, and then 20min one sample.1.4 Study on brain pharmacokinetics of Xiongbing microemulsion in awake freely-moving ratsThe feasibility of intranasal administration was preliminary evaluated with intravenous and intragaslric administration control.The concentration of TMP in brain of awke rats was determined after three ways of administration, and pharmacokinetic parameters were calculated by pharmacokinetic software DAS2.1. The results showed that the measured pharmacokinetics of free TMP concentration in brain all fitted single compartment model, the local bioavailability in brain of TMP was 18.61% after intragastric administration.It had the highest of local bioavailability as to intravenous administration, and TMP was absorbed into the brain rapidly, however, it was also metabolized rapidly, which resulted in the short of the mean residence time in brain; the local bioavailability in brain after intranasal administration was 59.18%, and the mean residence time in brain was longer, therefore it showed excellent value of research.2 Study of brain targeting in rats of Xiongbing microemulsion nasal delivery2.1 Establish two methods of planting probe in the process of two different ways of nasal deliveryThe methods of planting blood and brain probe was established in the process of two different ways of nasal delivery. Nonoperative nasal delivery:planting blood probe into the jugular vein of rat first, then fixed the rat in stereotaxic instrument of brain, and intranasal administration after brain probe was planted into the corpus striatum.Operative nasal delivery:planting blood probe first, then planted guide tube in brain, and fixed it with with denture acrylic, at last planted brain probe. The rat was fixed in supine position, and intranasal administration after making trachea cannula and ligaturing esophagus and first half of tracheal.2.2 Establish a method for determination the contents of TMP in blood and brain microdialysis samplesThe contents of TMP in blood and brain microdialysis samples were determined by HPLC, the concentration and peak area of the regression equation was Y=39802 X-748.03, and R2= 0.9999, TMP had a good linear relationship between the concentration of 0.0319～0.319μg/mL, and the specificity of this method was good.2.3 Study on stability of vivo recovery in blood and brain probeDuring rats wrer in surgical condition, the RSD of vivo recovery of blood and brain probe in 10h were respectively 2.07% and 5.08%, it meted the requirements for microdialysis experiment.2.4 The selection of methods by nasal deliveryThe ratio of AUCbrain/AUCblood (brain targeting efficiency) between nonoperative and operative nasal delivery were respectively 0.88 and 0.90, There was no obvious differences on brain targeting efficiency between them, but the dose was more accurate as to operative nasal delivery, and the drug was fully contact with the nasal mucosa,which was more helpful for study on brain targeting as to nasal delivery, so adopted operative nasal delivery.2.5 Evaluation of brain targeting of Xiongbing microemulsion nasal deliveryThe brain targeting of Xiongbing microemulsion nasal delivery was evaluated compared with intragastric and intravenous administration. The measured pharmacokinetics of free TMP concentration in blood and brain all fited single compartment model after intragastric administration, and two-compartment model after intranansal and intravenous administration. The absolute bioavailability(F) of intragastric administration was 41.89±5.16%, T1/2 and MRT0-∞of TMP in brain was approximate compared with intranansal administration, but the Cmax and AUC0-∞was significantly lesser than its, the value of drug targeting index (DTI) was 1.00, which indicated that its brain targeting was not obvious. The F of intranansal administration was 86.60±2.02%, and AUC0-∞in brain was approximate to intravenous administration, but the T1/2 and MRT0-∞was significantly prolonged, and increased by nearly 1.16,1.24 respectively.Its DTI value was 1.13, which indicated that part of TMP could be straight transported into brain by intranansal administration, which could improve the brain targeting of Xiongbing microemulsion.3 The influence factoers on brain targeting of nasal delivery3.1 The effect on brain targeting of nasal delivery from prescriptionThe ratio of AUCbrain/AUCblood after intranansal administration of TMP microemulsion and the microemulsion that was lack of borneol were respectively 0.82 and 0.85, the results showed that the extract from Rhizoma Ligustrici Chuanxiong and borneol both could improve the brain targeting, and increase AUC of TMP in brain, it could improve the brain targeting more obviously when combined with the extract from Rhizoma Ligustrici Chuanxiong and borneol, which indicated that the composing prescriptions of Xiongbing was reasonable in view of its effect on the TMP's pharmaeokinetic and brain targeting efficiency.3.2 The effect on brain targeting of nasal delivery from formulationThe measured pharmacokinetics of free TMP concentration in blood fited two-compartment model, and single compartment model in brain after intranansal administration of Xiongbing suspension liquid, its relative bioavailability was 67.34% compared with Xiongbing microemulsion, and the ratio of AUCbrain/AUCblood was 0.75, which indicated that the drug carrier of microemulsion can paly a potential role to improve concentration of TMP in brain.3.3 The effect on brain targeting of drug from anesthesia factorAfter three different ways of administration, the parameter of clearance(CL) was significantly reduced, and t1/2,MRT0-∞were all prlonged obviously, so as to Cmax,AUC, which indicated that it had obvious effect on the pharmacokinetic parameters of TMP in brain when rats were in aesthetic state. 4 Study of local cerebral PK-PD modeling of Xiongbing microemulsion4.1 Establishment of rat model of acute cerebral ischemiaThe model of acute cerebral ischemia was made by improved method of Zea Longa's thread occlusion, and checked it success or not by the way of nerve behavioral score and 2,3,5-triphenyhetrazolkml chloride(TTC)-staining. The result showed that this method can effectively block the blood of rat's middle cerebral artery, and caused focal ischemic. This surgical procedure was simple, and had a higher success rate.4.2 Establish a method of determination of animo acids'concentration in microdialysis samplesThe concentration of EAA(Excitatory Amino Acids, Glu, Asp) and IAA (Inhibitory Amino Acids, Gly,GABA, Tau) were determined by phthalie dicarboxaldehyde (OPA) pre-column derivatization and high performance liquid chromatography-fluorescence detector (HPLC-FLD). The concentration and peak area of the regression equation had a good linear relationship, and instrument's precision was favorable.4.3 Study of local cerebral PK-PD modeling of nasal deliveryRats were divided into two groups randomly in this experiment, which were respectively Xiongbing microemulsion intranasal administration group and model group, and the dose was 4mg/kg with TMP calculation. Each sample was divided into two, the one was used to calculate the concentration of TMP, which was index component in pharmacokinetics. Another was used to calculate the concentration of animo acids, which were index component in pharmacodynamics. Sample was Only used to calculate animo acids'concentration in model group. The result showed that it could reduce EAA's concentraton and improve the increase rate of IAA's concentration significantly in cerebral ischemia after nasal delivery, which can help reduce excited toxicity and protecte neural.The data was analysised by pharmacokinetic software WinNonlin4.0. 1.The result showed that the measured pharmacokinetics of free TMP concentration in brain fitted single compartment model, and the best model for pharmacodynamics was Sigmoid-Emax model. The PK-PD modeling was fitted by choosing some proper measured parameters above, and got the concentration-response equation about every index component in pharmacodynamics, which could help forecast the change rules between TMP's concentration and effect with time.From the above, the results showed that, compared with intragastric administration,it could obviously improved TMP's bioavailability in the brain of awake rats after intranasal administration.The further research indicated that the value of drug targeting index was 1.13 combined with blood and brain microdialysis technique, which could improve the brain targeting of Xiongbing Microemulsion, and The AUC0-∞values of TMP in brain were approximate between intravenous and intranansal administration.Therefor, it could be a promising alternative to traditional administration routes. The research also indicated that the composing prescriptions of Xiongbing was reasonable and the drug carrier of microemulsion could paly a potential role to improve concentration of TMP in brain. The index component in pharmacokinetics and pharmacodynamics were dererminated simultaneously in the brain of cerebral ischemia, the pharmacodynamic results showed that it could adjust the concentraton of EAA and IAA, and protecte neural. The local cerebral PK-PD modeling was established, and got the concentration-response equation about every index component in pharmacodynamics. The established methods could provide with a new idea and scientific reference for research of pharmacokinetics in brain, evaluation of brain targeting and pharmacodynamics, and research of PK-PD modeling, especially for drug with brain targeting character.