| ObjectiveTo prepare and screen the polymorphism of T-OA and isoimperatorin;The different crystal structures were characterized by Single crystal X-ray diffraction,powder X-ray diffraction(PXRD),differential scanning calorimetry(DSC),Fourier transform infrared spectroscopy(FTIR)and Scanning electron microscopy(SEM);The release behavior of the different crystal forms was evaluated in vitro dissolution experiments;The stability of different crystal forms under different temperature,humidity and grinding conditions was evaluated;To prepare the sustained-release microspheres of the amorphous form of T-OA and evaluate its pharmaceutical properties;To investigate the pharmacokinetics of isoimperatorin in plasma and evaluate the absorption in vivo after the rats were orally administered with different crystal forms of isoimperatorin in order to provide a guidance for the selection of superiority drug crystal in future.Methods1 We have prepared and screened the polymorphism of T-OA by solvent recrystallization method.Moreover,we have prepared the amorphous form of T-OA by melting method.The different crystal structures of T-OA were characterized by Single crystal X-ray diffraction,powder X-ray diffraction(PXRD),differential scanning calorimetry(DSC),Fourier transform infrared spectroscopy(FTIR)and Scanning electron microscopy(SEM).The release behavior of the different crystal forms of T-OA was evaluated in vitro dissolution experiments.2 We have prepared and screened the polymorphism of isoimperatorin by solvent recrystallization method.The different crystal structures isoimperatorin were characterized by single crystal X-ray diffraction,powder X-ray diffraction(PXRD),differential scanning calorimetry(DSC),Fourier transform infrared spectroscopy(FTIR)and Scanning electron microscopy(SEM).The release behavior of the different crystal forms of isoimperatorin was evaluated in vitro dissolution experiments.3 We have evaluated the stability of different crystal forms(T-OA and isoimperatorin)by simulating modern mass production under different conditions,including different temperature(5,50,80 and 100℃ for 24h),humidity(RH 25%、45%、65%and 75%for a week),different grinding time and grinding by adding liquid.We have prepared the microspheres of the amorphous form of T-OA by emulsion solvent evaporation method.We have conducted the orthogonal experiment and investigated the preparation process of the microspheres of T-OA by selecting drug loading and encapsulation efficiency as the index of evaluation,nd choosing the rotational speed,PVA concentration,dosing ratio and the volume ratio of oil-water phase as factors.Morover,we have estimated the pharmaceutical properties of microspheres of T-OA.The different crystal forms were characterized by powder X-ray diffraction(PXRD)in order to determine whether or not the phase transformation of crystal will be occurred.4 We have evaluated the concentration of isoimperatorin in plasma after the rats were orally administered with different crystal forms of isoimperatorin.We have established a HPLC method for the determination of the concentration of isoimperatorin in plasma.SD rats were housed in a temperature-and humidity-controlled environment(25℃,65%RH)and maintained on a 12-hour light/dark cycle for 3 days with free access to food and water before starting the experiment.Blood samples(0.5 mL)were collected from the ocular fundus veins of rats before administration and after 0.75,1,1.5,2,3,4,6,8,and 12 h(each time point 6 animal)after oral administration of two crystal forms of isoimperatorin.We have determined the blood concentration at different time points and drawn the concentration-time profiles by Excel software and calculated the pharmacokinetic parameters by Kinetica software in order to compare the absorption in vivo of different crystal forms of isoimperatorin.Results1 Form Ⅰ previously known was prepared from T-OA in acetone at 70℃,whereas FormⅡ was prepared from T-OA in the mixed solution of methanol and acetone(2:1)at 70℃.The amorphous form was prepared by melting T-OA in a crucible on a hot plate.As a comparison,form Ⅰ shows characteristic peaks at 2θ 7.8,12.3,13.1,16.7,18.2,and 20.3°.Form Ⅱ presents some obvious characteristic peaks at 2θ 6.7,11.1,12.9,16.6 and 22.3°.Form Ⅰ and Form Ⅱbelong to orthorhombic,and space group are P212121.However,the unti cell parameters of Form Ⅰ are a=8.1341(4)(?),b=11.1465(4)(?),c=36.5250(17)(?).The number of molecules in the cell is Z=4.The unti cell parameters of Form Ⅱ are a=6.7757(4)(?),b=22.7024(18)(?),c=22.7424(15)(?).The number of molecules in the cell is Z=4.The melt decomposition temperature of Form Ⅰ、Ⅱ and amorphous form are 182.17℃,180.50℃ and 179.64℃.The results shows that form Ⅱ and amorphous form all can transform to the form Ⅰ at temperature T=130℃ and T=146℃,respectively.SEM examination revealed that the form Ⅰ was rectangular crystals,while the form Ⅱ had fine column crystals.The amorphous form shows the irregular lumps.The dissolution rate of form Ⅱ is higher than form Ⅰ and the amorphous phase curve is obviously faster than other two forms.2 Form Ⅰ previously known was prepared from isoimperatorin in acetone at 50℃,whereas Form Ⅱ was prepared from bulk drug in the solution of ethanol at 50 0C.As a comparison,the previous known form Ⅰshows characteristic peaks at 20 9.9,11.1,16.4,18.5,25.0,26.1 and 27.2°.Form Ⅱ presents some obvious characteristic peaks at 2θ 9.0,20.7,20.8,21.2,25.2 and 26.7 °.Form Ⅰcrystallizes in space group P-1 with two molecules in the asymmetric unit,while form Ⅱ crystallizes with a monoclinic P21/c structure containing four molecules in the asymmetric unit.The unti cell parameters of Form Ⅰ are a=6.7200(7)(?),b=8.4829(9)(?),c=12.6357(13)(?).The unti cell parameters of Form Ⅱ are a=8.7774(3)(?),b=9.1943(3)(?),c=16.1996(6)(?).The melt decomposition temperature of Form I and Ⅱ are 107.59℃ and 108.42℃.The results shows that form Ⅰ can transform to form Ⅱ at temperature T=98℃.Large differences in spectra in this region are observed for the two forms due to conformational flexibility at side-chain:υ(=CH2)=3100-3000 cm-1 and in the methyl stretching vibration:υ(CH3)=3000-2850 cm-1.Noticeable differences can also be observed in the range of 1380 and 990 cm-1.The intensity of C=C stretching vibration at 1680-1620 cm-1 is changed.SEM examination revealed that form Ⅰ was long rod crystals,while form Ⅱ had flake crystals.The solubility of form Ⅰ is higher than form Ⅱ.3 Two crystal forms of T-OA are not suitable for manual grinding and mixing for a long time.Form Ⅰ should be controlled at about 5min during the grinding time,and grinding by adding ethanol may affect the crystal form.Form Ⅱ will gradually to Form Ⅰ in the grinding process.However,two crystal forms of imperatorin had not crystal form transition in the manual grinding and mixing for a long time and grinding by adding ethanol or water.Form Ⅰ of T-OA could keep stable at different temperatures for 24h,while Form Ⅱ will gradually to Form Ⅰ at 100℃ for 24h.Form Ⅱ of imperatorin could keep stable at different temperatures for 24h,while Form Ⅰ showed a portion of diffraction peak of Form Ⅱ at 60℃ for 24h.It will gradually to Form Ⅱ at 80℃ for 24h.Two crystal forms of T-OA and imperatorin could keep stable at different humidity,and the characteristic diffraction peaks remained stable.T-OA and PLGA were dissolved in methylene chloride(DCM)(polymer/drug,2/1(w/w)).This solution was then dispersed into an aqueous PVA solution(1%,w/v)via homogenization at 1000rpm for 2 min at room temperature.In the XRD spectra of T-OA microspheres,there were no such sharp peaks attributable to the crystalline form of T-OA in the microspheres.The microspheres were discrete spherical with porosity.It is evident that the morphology of the microspheres is homogeneous and majority of the spheres are ranging between 50 and 150 μm.4 The HPLC method of isoimperatorin in plasma was linear over the concentration range of 0.1～100μg/mL,and the lower limit of quantification was 0.1 μg/mL.The method was validated and showed good specificity and linearity,intra-day precision of 2.48%、4.98%and 6.65%,inter-day precision of 3.07%、4.61%和 5.97%,accuracy of 98.19%～112.15%,recovery of 94.48%～98.17%.5 After oral administration of different polymorphs isoimperatorin in rats,the pharmacokinetic parameters of brain tissue were analyzed with Tmax of 4.17h and 3.83h,Cmax of 1.48μg/mL,0.51μg/mL,AUC0-12h of 7.94 μg h/mL,2.28 μg h/mL,AU0-∞ of 7.97 μg h/mL,2.33 μg h/mL,MRT of 4.91h,4.96h,T1/2 of 0.95h,1.77h.Couclusion1 Two crystal forms and an amorphous form of T-OA were successfully prepared and characterized by single crystal X-ray diffraction.The results showed that crystal Ⅱ of T-OA is a kind of methanol solvation and contained one more methanol molecular.The methanol molecule which was introduced resulted in the torsional angle differences in two forms.DSC analysis showed that three solid state forms of T-OA had different melting points.Form Ⅱ and amorphous form all can transform to the form I at a certain temperature.The results of SEM showed that three solid state forms of T-OA exhibited different morphological structure.The amorphous form of T-OA had a higher dissolution rate,and the solubility of Form Ⅰ in vitro was the lowest which further verifies that the Form Ⅰ was found to be the most thermodynamically stable form.2 Two crystal forms of isoimperatorin were successfully prepared and characterized by single crystal X-ray diffraction.The results showed that two forms belonged to different crystal systems and there are differences in unit cell which leads to the difference of the molecular stacking shape and density.DSC analysis showed that two crystal forms of isoimperatorin had different melting points.Form Ⅰcan transform to the Form Ⅱ at a certain temperature.Fourier transform infrared spectroscopy(FTIR)analysis showed that there was a change in the strength and displacement of the stretching and bending vibrations of the methyl and the vinyl group.Furthermore,there was a significant difference in the stretching vibration of C-O and C=C bond position.Form Ⅰ had a higher dissolution rate in vitro experiments,which further verifies that the Form Ⅱ was found to be the most thermodynamically stable form.3 Form Ⅰ of T-OA is not suitable for manual grinding and mixing for a long time,and the alcohol grinding may affect the crystal type.It is further need to investigate the grinding time;However,the crystal form of imperatorin has not changed in manual grinding and mixing long time and grinding by adding liquid including water and alcohol.The optimum preparation process of TOA microspheres was optimized,and it was in accordance with the requirements of pharmaceutics.Stability studies showed that T-OA existed amorphous in microspheres.4 A HPLC method for the determination of the concentration of isoimperatorin was established in plasma.The peak time was 15.35min,the detection range is 0.1～10 μg/mL.The specificity of the method is good.Moreover,the accuracy,precision and quality control samples of low,medium and high can meet the quantitative requirements.5 The absorption in vivo of two crystal forms of isoimperatorin exhibited significantly differences after the rats were orally administered with different crystal forms of isoimperatorin.The absorption of Form Ⅱ had the appearance of two peak phenomena.Form Ⅰ is about 3 times as much as Form Ⅱ.However,the peak time and the average residence time showed no obvious difference.This phenomenon is consistent with the dissolution test in vitro. |
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