Investigation On Quantitative Determination Of Disulfiram And Its Copper Complex In Vivo And In Vitro

Disulfiram(DSF)has long been clinically approved to treat alcohol addiction.However,it has recently been discovered to be an anticancer agent with excellent properties combined with copper ions(Cu2+).After dose ingestion of DSF,it rapidly converted to its reduced metabolite producing two molecules of Diethyldithiocarbamate(DTC),in the acidic environment,In the tumor site,in which there is plenty of Cu2+ in the cells,DTC has a solid ability to chelate with Cu2+and form a complex(DSF/Cu complex).This complex can strongly bind to a protein called Nuclear protein localization 4(NPL4)causing severe damage to the ubiquitin fusion degradation protein 1(UFD1)pathway leading to cell death.In regards to the significant role of the DSF/Cu complex in tumor treatment,it is strongly recommended to study its analytical properties.Although many analytical methods can determine disulfiram and its metabolites,it is difficult to accurately analyze the DSF/Cu complex due to its poor stability,low water solubility,and its rapid metabolism.To this end,we aim to develop a quantitative method to determine and validate DSF/Cu complex in vivo and in vitro.In this study,the in vitro detection method of the DSF/Cu complex was established by high-performance liquid chromatography(HPLC)applied with an ultraviolet(UV)detector to get more accurate results.HPLC-UV is simple to operate and has good accuracy and specificity.DSF/Cu complex has been validated as per the International Conference on harmonization(ICH)guidelines by studying its linearity,accuracy,precision,limit of detection(LOD),limit of quantification(LOQ),specificity,and stability.More importantly,we proposed an in situ DSF antitumor efficacy triggered system(DSF/Cu complex),taking advantage of a Cu-based metalorganic framework(MOF).In detail,DSF was encapsulated into Cu-MOF nanoparticles(NPs)during its formation,and the obtained NPs were coated with hyaluronic acid to enhance tumor targetability and biocompatibility.In vitro pharmacokinetics studies were performed for the MOF preparation of DSF/Cu complex and its antitumor efficacy and biosafety were investigated.Analytical instruments used in chromatography were ACQUITY UPLCTM?HPLC,equipped with cooling and autosampler,and Diode-array UV detector(DADUV).The column used was a C18 column(4.6 mm × 150 mm;particle size 5 μm).Ultraviolet-visible(UV-Vis)spectroscopy was used to determine the wavelength absorbance for each compound,and the wavelength was set at 218nm and 423nm for DSF and DSF/Cu complex,respectively.The temperature of the column was held constant at 30℃.The mobile phases were water and acetonitrile,(water:acetonitrile=20:80 V/N).Methanol is used as a solvent for needle and column washing.The flow rate was 1 mL/min,and the injection volume was 20 μL.We collected the blood sample from BALB/c mouse after a single dose injection of DSF and DSF/Cu complex for in vitro investigation.The retention time has shown to be 4 min and 7 min,respectively.The correlation coefficient was R2=0.9957 and 0.9967,respectively.The percent recovery(R%)was within the acceptable normal range of 50%-85%.The designed method of analysis was found to be selective and specific for DSF and DSF/Cu complex without any detectable impurities in plasma at the selected wavelengths.Also it was an accurate and precise method showing a good linearity at the selected concentration ranges.The pharmacokinetic studies were performed on SD female rats.In this experiment,DSF has been encapsulated into Cu-MOF NPs coated with HA to overcome the drug instability in plasma and to be able to calculate the pharmacokinetic parameters.Rats were separated into two groups(free DSF and MOF preparation of DSF/Cu complex),and they were given a single dose of Ⅳadministration through their tail.The blood sample was calculated from their ocular at different time intervals.Results has shown that the area under drug concentration in the plasma verse time curve(AUC0-t)for MOF preparation of DSF/Cu complex was ten times more than free DSF.Moreover,both DSF and MOF preparation of DSF/Cu reach their maximum concentration(Cmax)after 25 min from the administration.Pharmacodynamic experiments have been performed using 4T1 tumor-bearing BALB/c mice.Results showed that the tumor inhibition rate of MOF preparation of DSF/Cu complex reached 86%,which was significantly higher than that of free DSF and free CuCl2.The results of TUNEL staining showed that the tumor cells in the MOF preparation group produced a large area of necrosis.In addition,there was no significant change in the body weight of the mice in each group treatment,and the liver and kidney function biomarkers were all within a reasonable range.On the other hand,the group treated with free CuCl2 has shown certain cardiotoxicity and alveolar collapse due to the toxicity effect of Cu2+on cells.In general,the above results indicated that the HPLC-UV method is simple with high sensitivity and accuracy to be used for quantitative validation,and the MOF preparation of DSF/Cu complex was successfully established and proved as an effective anti-tumor therapy with excellent biological safety.