Screening Of Dipyridamole Co-amorphous System And Evaluation Of Its Properties In Vivo And In Vitro

Dipyridamole（DIP）is an anti-platelet aggregation drug,which is widely used in clinical antithrombotic therapy.In recent years,a large number of studies have shown that this drug has immunomodulatory and synergistic anti-cancer pharmacological activities.Therefore,the great potential of DIP in"new use of old drugs"makes it have significant re-research and development value.However,DIP has poor water solubility,low oral bioavailability,and large individual differences,which largely limit its clinical application.Bonstruction of new solid forms of drugs is an effective strategy to improve the properties,such as solubility.Among them,the co-amorphous system possesses the unique advantages.On the one hand,it has excellent dissolving properties by the amorphous form,and on the other hand,the certain intermolecular interactions exist in co-amorphous,such as hydrogen bonds,are always helpful inmaintaining the stability of the system.Therefore,in this thesis,a co-amorphous system of DIP was constructed.The dissolution,stability and bioavailability of the new system were investigated,and the optimization of which was evaluated.We hope that this research can provide more theoretical support for the development and application of DIP co-amorphous.Part one:Screening,preparation and characterization of DIP co-amorphous systemObjective:Taking DIP as the active pharmaceutical ingredient（API）,screening the co-amorphous former（BAF）to construct the co-amorphous system,a variety of characterization methods were adopted to identify the formation of the co-amorphous system.Methods:Belecting diverse small molecules active ingredients and clinically combined drugs with DIP as BAF candidates,trying a variety of synthesis methods to construct the DIP-BAF co-amorphous system.Powder X-ray diffraction（PXRD）,differential scanning calorimetry（DBB）analysis,temperature modulated differential scanning calorimetry（TMDBB）analysis,temperature modulation differential scanning thermal（TG）analysis,scanning electron microscope（BEM）,Fourier transform infrared（FT-IR）spectroscopy,Raman spectroscopy,molecular simulation,high performance liquid phase（HPLB）and hydrogen nuclear magnetic（¹H NMR）techniques were carried out to confirm the structure of prepared complex and analyze purity.Results:P-hydroxybenzoic acid（PHA）was screened out as BAF from more than ten small molecular substances,and the melting method was finally determined as a successful and reliable preparation method.The results of PXRD and DBB preliminarily confirmed the amorphous feature of the product.The homogeneous co-amorphous system with a single glass transition temperature（T_g）was confirmed by TMDBB.BEM showed the morphological characteristics of the co-amorphous system,which was different from the raw drug.The functional group and hydrogen bond information of the new system were provided by FT-IR,Raman and molecular simulation,and it is inferred that the co-amorphous system is mainly combined by virtue of the hydrogen bonds of O-H…O-H,C=O…H-C and C=O…H-O from the O-H and C-H in DIP as well as B=O and O-H in PHA.HPLB and ¹H NMR results proved that the co-amorphous system has a composition ratio of DIP:PHA=1:2,and the purity of co-amorphous is high without decomposition.Conclusions:In this part,a high purity DIP-PHA co-amorphous was successfully prepared by the melting method.DIP and PHA are combined by multiple hydrogen bonding,and the system is a homogeneous amorphous system with a single glass transition temperature（T_g）.Part two:Evaluation of the stability of DIP co-amorphous systemObjective:To systematically evaluate the accelerated stability and thermal decomposition stability of DIP crystalline,amorphous and DIP-PHA co-amorphous system,the thermodynamic stability of co-amorphous system to crystalline state,and the dynamic stability of co-amorphous glass transition.Methods:1.Btudying the stability of accelerated experiment of DIP amorphous and DIP-PHA co-amorphous form under high temperature and humidity conditions.2.Determining the TG and DTG curves of DIP crystalline state,amorphous state and DIP-PHA co-amorphous form at different heating rates,using Kissinger and Ozawa methods to investigate the thermal decomposition stability of the three systems,obtaining the decomposition activation energy,referring to the front factor,decomposes the reaction kinetic equation,inferring the thermal decomposition mechanism function,and calculating the storage shelf life of the drug.3.Recording the DBB curves of DIP amorphous and co-amorphous under different heating rates,using Lasocka equation,Moynihan equation and Kissinger equation to study the stability of glass transition kinetics.4.Using the Van’t Hoff and Gibbs-Helmholtz equation to calculate the thermodynamic function of the transformation from the co-amorphous state to the crystalline state,and inferring the driving force of the transformation process.Results:1.When the amorphous form was placed under high temperature and high humidity conditions for 24 h,the hump diffraction halo disappeared completely and turned into the crystalline diffraction.There was only a very small amount of crystalline diffraction in the PXRD of DIP co-amorphous form after 8 weeks,which basically maintained the overall amorphous hump diffraction halo profile.In contrast,the conversion rate of co-amorphous to crystalline was much lower than that of DIP amorphous.2.The thermal decomposition mechanism function of the DIP crystalline and DIP amorphous state were the same,and the mechanism function of them was G（α）=[-ln（1-α）]^3/4,which named Avrami-Erofeev had the random nucleation and subsequent growth mechanism（n=3/4）;co-amorphous mechanism function:G（α）=1-（1-α）^1/2,and the name of the mechanism was contracting cylinder（area）,which was the phase interface reaction mechanism（n=1/2）;The results of A and E_a calculated by Kissinger and Ozawa methods were consistent and mutually verifiable.We calculated the shelf life of DIP crystalline,amorphous and co-amorphous,and all of them were 4–5 years.3.The glass transition kinetics research results of DIP amorphous and co-amorphous system showed that,the Lasocka constants A and B of the co-amorphous system are both larger than those of the amorphous system,indicating that the forming capacity and stability of the co-amorphous system are higher;Moynihan and Kissinger methods showed that the glass transition activation energy of co-amorphous were both smaller than that of amorphous,indicating that co-amorphous was more difficult to transform into crystalline substances.4.The thermodynamic study calculation results of the transformation from the co-amorphous state to the crystalline state showed that the transformation process was an endothermic and entropy increasing reaction,and the driving force of the transformation reaction was entropy driving.Conclusions:The formation of co-amorphous did not reduce the shelf life of the drug,and it had the better thermal stability;the high-temperature and high-humidity stability,and the glass transition stability of the co-amorphous system were higher than those of the single amorphous system.Part three:Study on dissolution performance in vitro and pharmacokinetic behavior in vivo of DIP co-amorphous systemObjective:To explore the properties,such as the solubility,dissolution in vitro and bioavailability in vivo of the DIP-PHA co-amorphous,and compare the related performance with DIP crystalline and amorphous,and infer the possible influencing factors behind the differences.Methods:1.Establishing content determination method in vitro with the UV-vis spectrophotometry（UV-vis）to determine the equilibrium solubility and dissolution of DIP-PHA co-amorphous,DIP-PHA physical mixture（PM）,DIP crystalline and amorphous.And using phase equilibrium solubility to study the complex mechanism of DIP-PHA co-amorphous.2.Establishing the content examination method of samples in vivo with ultra-high performance liquid（UPLB）,conducting pharmacokinetic experiments on DIP-PHA co-amorphous,DIP crystalline and amorphous,fitting and calculating the characteristic parameters of pharmacokinetics with the non-compartmental model,evaluating its bioavailability in vivo,and finally using statistical tests to determine the differences among three drugs.Results:1.Bompared with the DIP crystalline,the equilibrium solubility of DIP-PHA co-amorphous was increased by 16.19 times（25℃）and 17.38times（37℃）respectively,and it was also improved compared with the DIP amorphous;the dissolution percentage of the co-amorphous at 6 h was about2.29 times higher than that of the DIP crystalline,about 4.10 times higher than DIP amorphous.The phase solubility experiment results showed that the mechanism of the solubility improvement of DIP-PHA co-amorphous was that a soluble complex formed in the solution between the two raw drugs and the complex model was A_L type.2.Pharmacokinetic studies had shown that compared with crystalline and amorphous DIP,T_max of co-amorphous is 2.0 h and 1.2 h earlier respectively,C_max is increased by about 2.34 times,AUB_0-∞increased by 1.78 times and2.64 times respectively.Conclusions:The solubility,dissolution performance in vitro and bioavailability in vivo of the DIP-PHA co-amorphous form were significantly improved compared with the crystalline and amorphous forms of DIP,which suggested it was potential for DIP-PHA co-amorphous to be developed as a new solid-state preparation of DIP.