| 2,5-dihydroxymethyl-3,6-dimethylpyrazine(liguzinediol)is a compound with less toxicity and heart safety which can be used for the treatment of acute heart failure.Previous pharmacokinetic studies have shown that liguzinediol in rats and beagle dogs has a shorter half-time and a faster clearance rate.In order to expand the application of Liguzinediol and apply it to the treatment of chronic heart failure,we expect to study its sustained-release preparations.There are many kinds of sustained-release preparations,among which the multi cell formulation is more stable than the single cell dosage forms and the dosage is flexible.Therefore,the sustained release pellets are selected.Firstly,the properties of Liguzinediol were studied to provide the basis for the formulation design of sustained-release pellets.According to the results of preparation of Liguzinediol containing pellets,by single factor test and orthogonal test were used to optimize the formulation.The target size of pellets yield as the evaluation index,to determine the optimum conditions of preparation of pellet cores,and amplification.Preparation of Liguzinediol sustained-release pellets on the basis of this,the optimization of single factor and central composite design of formulation and process,to the target size and yield of pellets cumulative release rate as the evaluation index,to determine the optimal conditions for the preparation of Liguzinediol sustained-release pellets,and amplification.In vivo evaluation of the prepared Liguzinediol sustained release pellets was carried out as follows:1.Preparation of liguzinediol sustained release pelletsA rapid,sensitive and accurate HPLC-PDA method for the determination of liguzinediol concentration was established to study the basic physicochemical properties of liguzinediol.The test solution was centrifuged at 12000r·min-1 for 5min and the supernatant was taken.The samples were analyzed by HPLC-PDA method,chromatographic conditions were Elite Hypersil ODS column(250mm × 4.6mm,5μm).The mobile phase was methanol-water(32:68),the flow rate was 1mL·min-1,the column temperature was 30℃,and the chromatographic conditions were as follows:Detection wavelength:278nm,Injection volume:10μL.The concentration of liguzinediol was in good linear relationship within 12.51～400.4μg·mL-1(r=1.000).Repeatability,precision,accuracy are in accordance with the methodological requirements.And it applied to the dissolution of liguzinediol pellets and the determination of solubility in different pH conditions.The results showed that liguzinediol pellets have good solubility in water.2.Preparation of liguzinediol pelletsThe liguzinediol pellets were prepared by fluidized bed method.The factors affecting the formulation of liguzinediol pellets prepared by fluidized bed method were investigated by single factor.The types and dosage of binder,the type and size of blank pellets were investigated.The optimum conditions of fluidized bed preparation of Liguzinediol pellets is 0.5%HPMCE5 as binder,using 0.7-0.85mm blank sucrose as raw materials and adopts pellets into the air inlet 28 m3·h-1,the temperature is 35℃,atomization pressure is 0.4bar,coating liquid flow rate is 3rpm.To determine the optimum conditions,the scale-up experiments were carried out to investigate the properties of the powders.The results showed that the pellets prepared by the best prescription and the technology had good properties and repeatability.3.Preparation and amplification of liguzinediol sustained release pelletsLiguzinediol sustained-release pellets were prepared by using liguzinediol pellets(fluidized bed method),the factors and process factors were optimized.Preparation of Liguzinediol sustained-release pellets by fluidized bed method,using Eudragit NE30D as coating material,superfine talc powder as pore forming agent,antistatic agent with twelve sodium dodecyl sulfate as solvent,preparation of liguzinediol sustained-release pellets in water,and the atomization pressure,the process of coating liquid flow velocity and aging time were investigated and the optimal preparation conditions of liguzinediol sustained-release pellets for determining.The coating concentration is 6%,anti sticking agent is 60%,antistatic agent isl%,coating liquid atomization pressure is 0.4bar and flow rate is 2rpm.The optimum conditions were used to amplify the liguzinediol sustained-release pellets,and the process of the pellets and sustained-release tablets were also amplified.The results showed that the three processes had good repeatability.4.The study on the pharmacokinetics of different dosage forms of liguzinediol in beagle dogsA sensitive and rapid LC-ESI-MS/MS method was established for simultaneous determination of concentrations of liguzinediol and its two major metabolites in beagle dogs.This method was applied to pharmacokinetics in beagle dogs of liguzinediol pellets,sustained-release pellets and sustained-release tablets.The internal standard Antipyrine were added to plasma samples of beagle dogs.Methanol was added to precipitate protein.After high-speed centrifugation,the supernatant was taken for analysis.The shim-pack XR-ODS column(2.0 mm ×50 mm,2.2 μm),Shim-pack GVP-ODS guard column(2.0 × 5 mm,4.6μm)were used.The column temperature was 40℃.The mobile phase was methanol-0.1%formic acid gradient elution.The flow rate was 0.5 mL·min-1.Mass spectrometry was carried out using ESI ion source and multiple reaction monitoring mode(MRM)for positive ion detection.The selectively detect ion pairs of the liguzinediol(M0),M1,M2 and internal standard was m/z 169.1→122.1,m/z 183.0→137.0,m/z 345.1→169.1,m/z 188.9→56.0.A methodological study was carried out on the prototype and metabolites.The plasma concentrations of liguzinediol and metabolites M1,M2 are within a certain range of linearity.Methodological studies are in line with the requirements.Intraday and Interday precisions(RSD)were less than 15%.The recovery rate,extraction recovery,dilution reliability,stability,matrix effect,and residue were in accordance with the requirements of biological sample analysis.Six male beagle dogs were divided into three groups.Each group has two beagle dogs.Respectively,liguzinediol pellets,sustained-release pellets and sustained-release tablets were given to each group by oral administration.The dose was 55 mg.Blood is collected at different points in time.After 7 days of clearance,the experiment was crossed and was repeated for 2 times.The plasma concentrations at different times after administration was determined using the established LC-MS/MS method.The half-time of oral administration of liguzinediol pellets,sustained-release pellets and sustained-release tablets were 0.784 ± 0.757,4.132 ± 3.2,1.934 ±1.232h.The peak time were 0.708 ± 0.102,3 ± 2.53,1±0.548h,respectively.By comparison,both the half-time and the peak time showed that sustained-release pellets>sustained-release tablets>pellets.Simultaneously,two major metabolites showed similar regularity,indicating that the prepared sustained-release pellets has a good sustained-release effect. |
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