| Objective: In this study,tetramethylpyrazine hydrochloride was used as a model drug,and poloxamer 407 and poloxamer 188 were used as temperature sensitive materials,screening the best prescription for temperature sensitive in situ gel,and establishing a complete in vitro and in vivo evaluation system;At the same time,the changes of plasma concentration and tissue distribution of tetramethylpyrazine hydrochloride through different routes were investigated in order to explore the feasibility of drug delivery through the eye,which provide new ideas and methods for the ocular drug delivery system.Methods: This project established an analytical method for the determination of TMPH in vitro,and investigated its basic physicochemical properties and stability;Using the cold-soluble method to prepare tetramethylpyrazine hydrochloride ophthalmic temperature sensitive in situ gel,on the basis of single factor study,the gelation temperature as the evaluation index,the prescription was optimized by the central composite design-response surface methodology(CCD-RSM);A concent determination method of tetramethylpyrazine hydrochloride ophthalmic temperature sensitive in situ gel was established and the preparation was evaluated in vitro by means of quality study,stability test,dissolution and release in vitro,eye retention time and irritation,etc;The method for analyzing the content of tetramethylpyrazine hydrochloride in plasma and tissues were established in rats;Rats were used as experimental animals and randomly divided into three groups: intravenous injection,intragastric administration group(control group)and intraocular administration group(experimental group).Each group was given the same dose(10 mg/Kg)of tetramethylpyrazine hydrochloride.The content of tetramethylpyrazine hydrochloride in plasma and tissues were determined at different sampling time points,and evaluating the bioavailability and tissue targeting for different administration routes;Three kinds of absorption enhancers(borneol,chitosan and tween 80)were added to the tetramethylpyrazine hydrochloride solution respectively,and the pharmacokinetic data of plasma and brain tissue were compared with those of intravenous and intraocular administration groups without absorption enhancers.The absorption enhancer promoting the absorption of tetramethylpyrazine hydrochloride into the blood and brain was evaluated;The concentration of tetramethylpyrazine hydrochloride in plasma and brain tissue samples were measured by HPLC after intraocular administration of tetramethylpyrazine hydrochloride ophthalmic temperature sensitive in situ gel,and evaluating the plasma bioavailability and brain targeting of this preparation.Ruselts:1.The equilibrium solubility and apparent oil/water partition coefficient of tetramethylpyrazine hydrochloride in different media were determined by HPLC.The solubility of tetramethylpyrazine hydrochloride in water was 294.56 mg/m L at 37 ?and the apparent oil/water partition coefficient was 0.1(log P).The content of API was decreased under light condition,and should be protected from light.2.The optimal ratio between temperature sensitive materials P407 and P188 was19.98%:1.64% by CCD-RSM.At the same time,0.01% benzalkonium chloride was selected as preservative and 10% Na HCO3 as p H regulator to ensure that the ophthalmic gel was sterile and p H met the requirements.3.The gel was pale yellow transparent liquid at room temperature(25 ?)and light yellow translucent semisolid when it was at ocular ambient temperature(34 ?);The p H value was about 5.0,and the gelation temperature,viscosity and content were all in compliance with the requirements;Stability test showed that the preparation was stable for a long time under normal temperature and dark conditions;The dissolution and release test of tetramethylpyrazine hydrochloride gel in vitro showed that the release of tetramethylpyrazine hydrochloride was in accordance with the zero-order kinetic process(r=0.9979),and the linear relationship between the cumulative dissolution rate of gel and the cumulative release rate of tetramethylpyrazine hydrochloride was good(r=0.9997).Drug release is mainly controlled by gel dissolution;The ocular retention time of tetramethylpyrazine hydrochloride temperature sensitive gel and solution were 60 min and 20 min,respectively.The retention time of the gel was longer than that solution,and there was no irritation to rabbit eyes after single or multiple administration.4.Methods for determination of tetramethylpyrazine hydrochloride in rat plasma and tissues was established,which met the methodological requirements;The pharmacokinetic study of tetramethylpyrazine hydrochloride solution in vivo showed that the the absolute bioavailability of drug in plasma was significantly improved in the intraocular administration group compared with the intragastric administration group(P<0.05).The absolute bioavailability of intraocular administration group(63.22%)was 3.75 times as much as the intragastric administration group(16.88%);The vaule of AUC0-t was used as an index of the distribution of tetramethylpyrazine hydrochloride in tissues.The order of AUC0-t in intravenous,intragastric and intraocular administration groups were: kidney > heart > liver > brain > spleen > lung,kidney > liver > heart >spleen > brain > lung,kidney > brain > heart > liver > spleen > lung,respectively.The relative targeting efficiency RTE(AUCig/io/AUCiv)of TMPH after intragastric and intraocular administration was calculated by taking the AUC0-t of each tissue as a reference after intravenous administration,and the target efficiency TE(AUC0-t/?AUC0-t)of each tissue was calculated by taking the sum of the AUC0-t of the respective administration routes as a reference.The results showed that RTE of tissues were 60%~105% in the intraocular administration group,and RTE of brain tissue was the highest(104.78%),which was 3.54 times higher than that intragastric administration group(29.62%).And the TE of brain tissue was 17.63%,second only to kidney tissue,which was 1.61 times higher than that of intragastric administration(10.95%).It is indicated that tetramethylpyrazine hydrochloride has certain brain targeting properties through intraocular administration and is beneficial for the treatment of brain diseases.5.When three kinds of absorption enhancers were added to tetramethylpyrazine hydrochloride solution respectively,the drug content in the plasma and brain tissues all reached the peak at 2 min after intraocular administration,which was faster than the control group(5 min).It suggested that 1% borneol,1% chitosan and 1% tween 80 could accelerate the absorption of tetramethylpyrazine hydrochloride into blood and brain.The peak concentration of tetramethylpyrazine hydrochloride in the plasma(6.33?g/m L)(P<0.05)and brain tissue(3.44 ?g/m L)(P>0.05)of 1% chitosan group was increased compared with the peak concentration of tetramethylpyrazine hydrochloride in plasma(2.96 ?g/m L)and brain tissue(1.94 ?g/m L)of the intraocular control group.After that,the drug concentration in plasma and brain tissue decreased rapidly,which may be the result of rapid transport of tetramethylpyrazine hydrochloride under the action of permeability opening.The plasma AUC0-t of the three groups with absorption enhancer were all higher than that of the intraocular control group,which significantly increased the plasma bioavailability of tetramethylpyrazine hydrochloride.For 1%borneol and 1% chitosan groups,the AUC0-t of tetramethylpyrazine hydrochloride in brain tissue were all higher than that of the intraocular control group,and the AUC0-t of1% tween 80 group was similar to that of the control group.The results indicate that the addition of absorption enhancer can increase the amount of tetramethylpyrazine hydrochloride into blood and brain to a certain extent by intraocular administration;However,if the targeting efficiency of tetramethylpyrazine hydrochloride was evaluated by AUCbrain/AUCplasma,the 1% borneol group(0.679)was slightly higher than the intraocular control group(0.662)(P>0.05),which indicated that borneol could promote the transport of tetramethylpyrazine hydrochloride to brain tissue to a certain extent.But the effect is not significant.And the ratio of AUCbrain/AUCplasma in 1%chitosan and 1% tween 80 group were lower than that in the intraocular control group,indicating that chitosan and tween 80 could not improve the brain targeting efficiency of tetramethylpyrazine hydrochloride at at this concentration.6.After intraocular administration of tetramethylpyrazine hydrochloride in situ gel,the t1/2,and AUC0-t of tetramethylpyrazine hydrochloride in plasma were 1.67 times(P<0.05),and 1.13 times(P>0.05)higher than intraocular administration of tetramethylpyrazine hydrochloride eye drop,respectively.Similarly,the t1/2,and AUC0-t of tetramethylpyrazine hydrochloride in brain were 1.48 times(P<0.05),and 1.25 times(P<0.05)higher than intraocular administration of tetramethylpyrazine hydrochloride eye drop.And the AUCbrain/AUCplasma of tetramethylpyrazine hydrochloride through intraocular administration of tetramethylpyrazine hydrochloride in situ gel was 0.725,which was 1.11 times higher than that of tetramethylpyrazine hydrochloride solution(0.662)(P<0.05).Conclusion: The tetramethylpyrazine hydrochloride eye drop could be quickly absorbed into the systemic circulation and distributed to various tissues through intraocular administration.The plasma bioavailability(63.22%)is significantly higher than intragastric administration(16.88%)(P<0.05).Meanwhile,it has a certain targeting effect on brain tissue that the value of AUCbrain/AUCplasma was 0.662.The above results indicated that tetramethylpyrazine hydrochloride could exert systemic effect through cular administration.Furthermore,when tetramethylpyrazine hydrochloride was prepared into temperature sensitive in situ gel,the plasma bioavailability(71.40%)(P<0.05)and AUCbrain/AUCplasma(0.725)(P<0.05)were improved compared with the eye drop.This may be related to the gel can prolong the retention time of tetramethylpyrazine hydrochloride in the eye. |
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