Screening And Evaluation Of 5-fluorouracil Cocrystals In Vitro And In Vivo

Pharmaceutical cocrystal is a supramolecular system formed by combination of active pharmaceutical ingredients（API）and biocompatible small molecule precursors,named cocrystal former（CCF）,through non-covalent weak forces such as hydrogen bonds.The advantages of cocrystallization in improving the solubility and bioavailability of API are increasing and attracted.It is generally believed that the two components in pharmaceutical cocrystal exists in the form of free monomer molecular in the dissolved state.In recent years,more and more literatures have reported that cocrystals exhibit different biological behaviors from physical mixtures（PM）of two components after dissolving.There is a synergistic effect between API and CCF at the molecular level,but the specific mechanism is rarely reported.5-Fluorouracil（5-FU）is a commonly used first-line anti-tumor drug in clinic,which is mainly used to treat skin cancer,gastrointestinal cancer,liver cancer and breast cancer.However,5-FU has such adverse factors as low permeability,short half-life,fast metabolism,obvious first pass effect,and serious untoward reactions,which limit its clinical application.Therefore,to improve the bioavailability of 5-FU and achieve synergistic activity through co-crystallization technology between 5-FU and CCF have a great significance.Based on this,this paper selects biologically active amino acids and gallic acid（GA）as CCF to screen 5-FU cocrystal.Firstly,the pharmacokinetic characteristics of 5-FU are improved by screening cocrystal.Secondly,based on the biological activity of amino acids and GA precursors,the synergistic effect and mechanism of the two components between 5-FU and CCF are investigate.Part I:Mechanism investigation of synergism between API and CCF in5-fluorouracil binary cocrystal based on cellular metabolomicsObjective:To investigate the toxicity of 5-FU,Phenylalanine（PHE）,5-FU/PHE PM,and 5-FU-PHE cocrystal on B16 F10（B16）cells,and the mechanism of the distinct toxicity between PM and cocrystal by cell metabolomics.Methods:5-FU-PHE cocrystal was prepared using solvent assisted grinding method.The cocrystal was characterized by powder X-ray diffraction（PXRD）and differential scanning calorimetry（DSC）.MTT tests were carried out to evaluate the toxicity of 5-FU,5-FU/PHE PM,and 5-FU-PHE cocrystal on B16 cells.Ultra high performance liquid chromatography quadrupole time of flight mass spectrometry（UHPLC-Q-TOF-MS/MS）was used to detect the retention time and mass spectrometry information of endogenous metabolites.Orthogonal Partial Least Squares Discriminant Analysis（OPLS-DA）model was constructed to calculate Variable Importance for the Projection（VIP）,and differential metabolites for pathway analysis were screened by t-tests and FC tests.Results:5-FU-PHE cocrystal was successfully prepared.The IC₅₀values of 5-FU,PM,and cocrystal on B16 cells were 7.61±0.46,7.55±1.65,and4.08±0.47μM,respectively,showing a statistical difference between PM and cocrystal（p<0.01）.OPLS-DA model for metabolomics was constructed and12 differential metabolites were obtained.By analyzing the metabolic pathways,it was concluded that the difference in toxicity between PM and cocrystal on B16 cells was due to the differences in purine metabolism,ether lipid metabolism,and glycerol phospholipid metabolism pathways.Conclusions:In the 5-FU-PHE cocrystal system,purine,glycerol phospholipid,and ether lipid metabolism pathways were associated with cell apoptosis and anti-proliferation.The differences between PM and cocrystal in these three pathways were the source of their differences in cytotoxicity.The metabolomics strategy provided a possible explanation for explaining the mechanism in different cytotoxicity between cocrystal and PM.Part II:Structural analysis and in vitro/in vivo evaluation of5-fluorouracil-sarcosine cocrystalObjective:To prepare 5-FU-SAR cocrystal using Sarcosine（SAR）as CCF.To obtain the single crystals and solve the crystal structure.To investigate the pharmacokinetic characteristics of cocrystal and the effect of SAR on cytotoxicity of 5-FU.Methods:5-FU-SAR cocrystal powder was prepared using solvent assisted grinding method,and single crystal was obtained using solvent slow evaporation method.The cocrystal was characterized by single crystal X-ray diffraction（SC-XRD）,PXRD,DSC,Fourier transform infrared spectroscopy（FT-IR）,and Raman spectroscopy.The solubility,dissolution,oil-water distribution coefficient,and transmembrane permeability of the cocrystal system were evaluated using the UV-VIS method.HPLC method was established to determine the concentration of 5-FU in rat plasma.By using HPLC method,the pharmacokinetic differences of 5-FU,PM,and cocrystal in SD rats as administered orally or intraperitoneally were investigated.The activity of the 5-FU-SAR cocrystal system in B16,4T1,BEL-7402,A549,and MCF-7 cells were evaluated,and the cell cycle and apoptosis were detected via flow cytometry.Results:The single crystal structure analysis showed that the cocrystal was crystallized in triclinic P-1 space group,and the cell parameters were a=7.5581（8）?,b=9.8838（11）?,c=10.4001（9）?,α=67.839（9）°,β=78.166（8）°,γ=77.063（9）°.Each asymmetric unit contained one SAR zwitterion and two 5-FU molecules.SAR and 5-FU molecules were connected to form a three-dimensional supramolecular network through various N-H···O hydrogen bonds.The solubility,permeability,and oil-water distribution coefficient of cocrystal were improved than those of 5-FU and PM.When administered orally,compared to 5-FU,the AUC of cocrystal increased from369.73±48.76μM·h to 511.03±115.59μM·h.When administered intraperitoneally,the AUC of 5-FU and cocrystal were 277.12±66.33 and406.52±119.44μM·h,respectively.Compared to 5-FU,cocrystal exhibited two components synergistic cell inhibitory activity on 4T1 and BEL-7402cells,by virtue of the induced stronger S-phase inhibition and stronger cell apoptosis.Conclusion:Single crystal of 5-FU-SAR cocrystal was obtained and the structure was solved.The cocrystal displayed superior physicochemical properties and higher bioavailability compared to 5-FU,and exhibited selective inhibition ability on different tumor cells.Part III:5-fluorouracil-arginine cocrystal:Screening,evaluation and the reduction of cardiac toxicity induced by 5-fluorouracilObjective:To apply potential NO precursors of L-Arginine（L-ARG）and D-Arginine（D-ARG）to construct cocrystal with 5-FU.To investigate the cytotoxicity,NO release efficiency,as well as the ability to alleviate cardiac toxicity induced by 5-FU in vitro and in vivo.Methods:5-FU-ARG cocrystals were prepared using solvent assisted grinding method,which then characterized by PXRD,DSC,FT-IR,UV-VIS,and Circular Dichroism（CD）techniques.Solubility,dissolution rate,oil-water distribution coefficient,and transmembrane permeability of the cocrystal system were evaluated.The pharmacokinetic of 5-FU,D-ARG/5-FU PM,and D-ARG-5-FU cocrystal in SD rats were investigated under oral and intraperitoneal injection administrations,respectively.The MTT assay was used to investigate the cell inhibitory activity of 5-FU-ARG cocrystal system in B16,4T1,BEL-7402,A549,and MCF-7 cells,and flow cytometry was used to detect cell cycle and apoptosis.Using DAF-FM DA as a probe,the NO release in cells was observed using confocal laser scanning microscope（CLSM）.Using Wistar rats as a model,the acute cardiac toxicity induced by5-FU was evaluate by intraperitoneal injection administration.The relief of ARG on cardiac toxicity was evaluated by measuring serum levels of lactate dehydrogenase（LDH）,cardiac troponin I（c Tn I）,and creatine kinase MB（CK-MB）.Results:L-ARG-5-FU and D-ARG-5-FU cocrystal were prepared.The cocrystals displayed higher optical activity in solutions with the same concentration compared to PM,indirectly proved that the existence forms of cocrystal and PM in solutions are different.That was,the hydrogen bond between API and CCF still existed in the cocrystal solution.The solubility,dissolution,and permeability of cocrystal were improved than those of 5-FU.The solubility of 5-FU in water increased from 147.47±4.52μM to 1932.22±37.7μM（L）and 1968.84±17.53μM（D）,respectively.In p H 1.2 HCl medium,the solubility ranged from 145.95μM to 1787.56±13.23μM（L）and 1757.72±24.29μM（D）,respectively.All these results showed significant differences（p<0.001）.Cocrystals induced stronger cytotoxicity on BEL-7402 cells,and enhanced inhibition on G2/M phase cells compared with5-FU.However,the toxicity of cocrystal on HUVEC cells was weaker than that of 5-FU.D-type cocrystal alleviated the cardiotoxicity of 5-FU at both cellular and animal levels.Moreover,pharmacokinetic performances of the D-type cocrystal under both oral and intraperitoneal injection administrations were significantly improved compared to 5-FU and PM.Conclusion:5-FU cocrystals were prepared using enantiomers D-ARG and L-ARG as CCFs.There was no significant difference in physicochemical properties between the two types of cocrystal in vitro.However,D-ARG-5-FU cocrystal showed significant advantages in alleviating 5-FU induced cardiac toxicity.Part IV:Screening and pharmacodynamic evaluation of 5-fluorouracil gallic acid cocrystal system in vivo and in vitroObjective:To construct a 5-FU cocrystal used gallic acid（GA）,a natural phenolic acid with antitumor activity,as CCF.To investigate the physicochemical properties,oral bioavailability and in vitro/in vivo anti-tumor activity of 5-FU-GA cocrystal.Methods:5-FU-GA cocrystal was prepared by solvent assisted grinding method,which characterized by PXRD,DSC,FT-IR,Raman spectroscopy,and Scanning Electron Microscope（SEM）.HPLC was used to determine the concentration of 5-FU to evaluate the solubility,dissolution rate,oil-water partition coefficient,and transmembrane permeability of the cocrystal system.Sprague Dawley（SD）rat model was used as model to investigate the pharmacokinetics of 5-FU,PM,and cocrystal.MTT assay was adopted to evaluate the cell inhibitory activity of 5-FU-GA cocrystal system against 4T1cells,and flow cytometry was used to detect cell cycle and apoptosis.Using acridine orange（AO）staining method,the autophagy was evaluated through observation of acidic autophagic vesicles in cells using CLSM.The antitumor activity in vivo was investigated in 4T1 tumor-bearing BALB/c mice.Results:5-FU-GA cocrystal was successfully obtained.Due to the improved oil-water partition coefficient,the permeability of cocrystal was increased.The cocrystal enhanced the inhibition of cell G0/G1 and G2/M phases and induced excessive autophagy in cells,which resulted in enhancement of anti-tumor activity in vitro.The cocrystal displayed the highest anti-tumor activity both by oral and intraperitoneal administrations.Conclusion:Compared with 5-FU,5-FU-GA cocrystal showed excellent physicochemical properties and oral bioavailability.The antitumor activity in vivo and in vitro were improved by inhibiting G0/G1 and G2/M phase and over activating autophagy.