| Cucurbitin B?CuB?is a tetracyclic triterpenoid compound extracted from cucurbitaceae plants widely present in nature,with various biological features,such as liver protective,anti-inflammatory,and anti-tumor activities,which is used clinically to treat chronic hepatitis and primary liver cancer.CuB is a poorly soluble drug with low water solubility and poor oral bioavailability,which severely limits its widespread clinical application.In order to improve its bioavailability,the group prepared a CuB solid dispersion used poloxamer407 as a carrier in the early stage.The experimental results showed that poloxamer 407 can significantly improve the dissolution and bioavailability of CuB.Meanwhile,it was found that the solid dispersion with poloxamer 407 as a carrier had obvious instability of crystal precipitation as the storage time was extended.In this study,several suitable support materials were preliminarily screened through crystal suppression and solubility tests to prepare solid dispersions with better stability.Then the solvent method was used to prepare a solid dispersion with amorphous support and semi-crystalline support,respectively.The phase characterization,dissolution,and stability investigations were performed,and the stability of CuB solid dispersions prepared by different carriers was compared and screened to obtain solid dispersions with good stability.Finally,through the influential factor test and pharmacokinetic experiment,the stability of the solid dispersion with poloxamer 407 as the carrier,and the differences in main pharmacokinetic parameters were discussed to verify the experimental results.Objective:A method for the determination of CuB content in vitro,in vivo and the dissolution test were established.Several suitable carriers were screened out by the crystal suppression test and the solubility test,and solid dispersions of different carriers were prepared and evaluated for quality.The stability of the solid dispersion was investigated to obtain a more stable solid dispersion.The CuB solid dispersion obtained was screened for influencing factors and pharmacokinetic experiments to verify related research.Methods:1.An HPLC method was established to determine the content of CuB solid dispersion and the dissolution in vitro.2.Several suitable carriers were screened out by the solubility test and crystal suppression test.CuB solid dispersions of different carriers were prepared by the solvent method.The quality of the solid dispersion was evaluated by DSC,PXRD,SEM,and FTIR and in vitro dissolution.3.Carry out related research on the stability of solid dispersion through stability test and influence factor test.4.Using liquid-mass spectrometry to study the pharmacokinetics of CuB,poloxamer 407 solid dispersions,and solid dispersions screened in rats by intragastric administration with rats as experimental subjects.Calculated the relative bioavailability and compared the differences in pharmacokinetic parameters between the two CuB solid dispersions and the CuB substance.Result:1.The content of CuB was determined by HPLC.CuB had a good linear relationship in the concentration range of 2.33 ? 81.20 ?g/ m L.The linear equation was Y=12.856X+19.341 and R2 = 0.9984.The precision test showed that the RSD of the low,medium and high concentrations were less than 2%,which indicated that the precision of the analytical method was good.The recovery rate test showed that the recovery rate was between 100.48 ?103.23%,and the RSD values were less than 2%,indicating that the recovery rate meets the requirements.The stability results showed that the CuB solution remained stable within 24 h.The concentration of CuB in the dissolution medium was determined by HPLC.The standard curve was Y = 11.038X-0.26,and R2 = 0.998.It showed that CuB had good linearity in the range of 0.735 ?11.69 ?g/m L.The precision RSD was less than 2%,which indicated that the instrument had good precision.The eluate was stable within 24 hours,and the recovery range was 102.60%?104.24%.The RSD values were less than 2%,and the recovery rate meets the requirements.2.The solubility test of CuB in different carriers showed that CuB had higher solubility in surfactant carriers such as TPGS,RH 40,PLX-407,F68,and slightly lower solubility in carriers such as PVP K30,PVP VA 64,PVP K90,PEG 6000.Crystal suppression test results indicated that PVP VA64 had the best crystal suppression ability for CuB,followed by TPGS and RH 40.PVP K30 and PLX-407 also effectively inhibited the formation of drug cores.Based on the solubility and crystal suppression test results,PVP VA64,PVP K30,TPGS,RH40,and PLX-407 can be used as carriers for solid dispersions.However,considered the physical and chemical properties of RH40,it was not suitable as a carrier for preparing solid dispersions.Therefore,PVP VA64,PVP K30,TPGS,and PLX-407 were selected as carriers of solid dispersions to prepare a single carrier solid dispersion.At the same time,in order to improve the stability of the solid dispersion,TPGS was added as a surfactant to PVP VA64 and PVP K30 materials to prepare a composite carrier solid dispersion.Different solid dispersion prepared were characterized by phase.The DSC showed that no endothermic peak of CuB was observed in several solid dispersions,and it is speculated that the drug may exist in the carrier in an amorphous state.PXRD illustrated that PVP VA64,PVP K30,and TPGS were inherently amorphous,PLX-407 had a crystalline structure.In a solid dispersion prepared with an amorphous carrier material,the drug is dispersed in the carrier material in an amorphous state.In a solid dispersion prepared with a semi-crystalline carrier,the drug exists in a partially crystalline and partially amorphous form.SEM demonstrated that CuB was a bar-shaped crystal of irregular size.The solid dispersions prepared with PVP VA64,PVP K30,TPGS amorphous carriers were block or drum structures with smooth surfaces and different sizes,and the solid dispersions prepared with semi-crystalline carriers were rough blocks and columns with rough surfaces.The FTIR results showed that the peak positions of –C= O and-OH stretching vibration peaks of CuB in the solid dispersion-shifted,indicated that there was an interaction between the drug substance and the carrier.The dissolution results in vitro indicated that the solid dispersions prepared with different carriers improved the dissolution rate and dissolution of CuB.3.The stability test results showed that the water content,dissolution,crystallinity,and interaction of the solid dispersions prepared with semi-crystalline and amorphous carriers at 25? and RH 95% all changed.Under the experimental conditions,the solid dispersion of the composite carrier was more hygroscopic than the solid dispersion of the single carrier.After a period of time,the water content increased,the crystallization phenomenon was more obvious at birth,and the dissolution rate decreased significantly,which significantly affected its stability.Semi-crystalline solid dispersion had high hygroscopicity,large crystallinity,and reduced dissolution.Comprehensive analysis,compared with PVP K30 and other carriers,the amorphous carrier PVP VA64 had less hygroscopicity,some crystals precipitate,and the dissolution rate decreased,but the changes were smaller than those of other carriers.Preliminary judgments showed that the amorphous carrier PVP VA64 and the semi-crystalline carrier PLX-407 were relatively stable,of which PVP VA64 had better stability.4.On the premise of ensuring stability,in order to prepare solid dispersions with higher dissolution and bioavailability,PVP VA64 was used as the carrier,and the optimal drug loading ratio was screened by the drug loading ratio to prepare PVP VA64 solid dispersion?CuB-PVP VA64-SD?.Tests of influencing factors under conditions of high temperature,high humidity,and strong light were performed and compared with CuB-PLX-407-SD with the same drug loading ratio.The bright light test showed that there was no significant change in the appearance,content,and dissolution of the solid dispersion.The high humidity test showed that the appearance of CuB-PVP VA64-SD was occasionally agglomerated,and its content didn't change significantly,and the dissolution rate was occasionally decreased;the appearance of CuB-PLX-407-SD was agglomerated and blocked,and the content and dissolution of the sample were significantly changed.The dissolution rate decreased significantly.The high-temperature test showed that there was no significant change in appearance,content,and dissolution of CuB-PVP VA64-SD;the content and dissolution of CuB-PLX407-SD were significantly reduced.CuB-PVP VA64-SD was less affected by high temperature,light,and high humidity,and was more stable than CuB-PLX-407-SD,but it also suggested that CuB solid dispersion should be at room temperature,dry,sealed and protected from light as much as possible.5.The content of CuB in rat plasma was determined by UPLC-MS / MS,and oleandrin was used as the internal standard.In the range of 0.1 ? 10ng/m L,the ratio of CuB to oleandrin peak area had a good linear relationship with the concentration.The inter-day precision RSD of QC samples was less than 3.71%,and the intra-day precision RSD was less than 6.16%,accuracy was less than 7%,which meet the analyze was requirements of biological samples.The matrix effects of QC samples and oleandrin were 94.81% ?97.14% and 97.89% respectively.The extraction recoveries were 95.08% ?95.88% and 95.72%,and their RSD values were less than 7%,which met the analysis requirements.The stability test showed that the rat plasma samples were stable in the autosampler?4??for 24 h,repeated freeze-thaw cycles for4 cycles,and stored at-80? for 20 days.Compared to CuB,the Cmax of the solid dispersion was higher and the peak time was shorter.The relative bioavailability of CuB-PVP VA64-SD and CuB-PLX 407-SD were 231% and386%,respectively.Conclusion:The established analytical method was simple and reliable and can be used to determine the content and dissolution of CuB.Using PVP VA64 as a carrier,the stability of the solid dispersion was better,and the stability was significantly higher than that of CuB-PLX-407-SD.Compared with the drug substance,the dissolution rate and bioavailability of the solid dispersion prepared by PVP VA64 as the carrier was improved. |
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