Preclinical Pharmacokinetic Study Of NAP

Objective:Previous studies have shown that the silkworm protease?NAP?has a good antithrombotic and defibrinating function,and has obvious proliferation inhibition effects on leukemia cells and non-small cell lung cancer cells,so it is expected to be developed into a clinical drug.In this paper,preliminary pharmacokinetic studies of trichoenzymes were carried out from three aspects:drug absorption,distribution and excretion.Fluorescent labeling and characterizing of Nereis active protease?NAP?,On this basis,We studied the regulation and pathways of NCI-H1299 cells'uptake of NAP,Where NAP is distributed in the cell.The lung absorption of NAP was simulated by Calu-3 cell monolayer model;the pharmacokinetic parameters,tissue distribution and excretion of NAP were studied after intravenous injection and pulmonary administration in rats.To lay the foundation for further clinical pharmacokinetics.Methods:The labeling is completed by nucleophilic reaction of FITC and NAP in an alkaline solution,and then the free FITC is removed by dialysis method and organic solvent precipitation method.FITC-NAP were characterized by thin-layer chromatography,infrared spectrum,ultraviolet spectrum and fluorescence spectrum.The fluorescence substitute ratio of FITC-NAP was measured by ultraviolet spectrophotometry.The activity of NAP and FITC-NAP were compared by measuring the enzymatic reaction kinetic parameters.Cell viability was evaluated by MTT assay.The uptake mechanism of NCI-H1299 cells to FITC-NAP was studied by fluorescence microscopy.Fluorescence microscopy was used to qualitatively observe the uptake of FITC-NAP by NCI-H1299 cells.Calu-3 cells were used to establish a lung model to simulate the absorption of the drug in the lungs,and the microfilaments of the cell monolayer and Calu-3 cells were observed by measuring transmembrane resistance,marker acidic protein expression,and inverted microscope and transmission electron microscopy.Cell-to-cell tight junctions were used to verify the success of the cell monolayer model;MTT assay was used to determine the highest concentration of NAP in cell model transport experiments,and then NAP was quantified by Elisa to study transit time and NAP concentrations on NAP in Calu-3 Cell monolayer model transport effects,and calculate cumulative transport and apparent permeability coefficients.Rats were administered by intravenous and pulmonary administration.Blood samples were taken from the tail vein at different time points to determine the plasma concentration and the pharmacokinetic parameters were calculated.After 60 minutes of administration,the rats were sacrificed and the hearts were removed.Liver,spleen,lung and kidney.After homogenization,the supernatant was centrifuged,and the distribution of NAP in the tissues of rats was studied by quantitative detection of NAP by Elisa method.After intravenous administration of the rats,the urine of the rats was collected at different time points,and the supernatant was centrifuged,and the NAP was quantitatively detected by the Elisa method to study the excretion of NAP in the rats.Results:Each molecule of the labelled product approximately contained 1.78molecules of FITC,The optimal fluorescence excitation and emission wavelengths of FITC-NAP were 490 and 515 nm,Infrared spectroscopy showed that the structural characteristics of the polysaccharide did not significantly change after labeling.The activity of NAP after fluorescent labeling was not significantly affected.the uptake of FITC-NAP in NCI-H1299 cells was positively correlated with drug concentration and incubation time.The fluorescence intensity of the phenylene oxide group was lower than that of the FITC-NAP group and there was a significant difference?P<0.01?.This shows that the uptake mechanism of NCI-H1299 cells to FITC-NAP was endocytosis.Further studies has found that the uptake of NAP by NCI-H1299 cells is a combination of clathrin-dependent endocytosis and caveolin/lipoprotein-mediated endocytosis.Fluorescence microscopy revealed that more cellular uptake FITC-NAP over time,and FITC-NAP mainly distributed in the cell membrane and cytoplasm,no distributed in the nucleus.The Calu-3 model transport experiment found that the transport of NAP was time-and concentration-dependent.The transport speed of the apical side?A?to the bottom side?B?was much higher than that of the reverse transport,and the transport amount was also significantly higher than the bottom side?B?to the apical side.?A?,indicating that the transport of NAP is directional,and the absorption is greater than the efflux.The Papp?A-B?of NAP was?5.49±0.60?×10^-7 cm·s^-1,which indicated that NAP was a relatively less absorbed drug after pulmonary administration.The Tmax of rats after intravenous administration was?10±0?min,and the Tmax after pulmonary administration was?190±24.495?min.The AUC_0-t values of intravenous administration and pulmonary administration were?10628.947±1759.428?ng/mL*min and?6574±166.536?ng/mL*min,the results showed that the absorption of NAP lungs was slow,the absorption was relatively poor,and the bioavailability was relatively low.However,the elimination half-life t_1/2 of NAP after intravenous injection was?39.027±12.121?min,and the elimination half-life t_1/2after pulmonary administration was?63.834±44.697?min,indicating that the lung administration had better sustained release.The effect is that the retention time in the body is longer than that of intravenous injection,and the elimination is slower.Studies have shown that rats have the highest distribution in the kidney after intravenous injection of NAP,indicating that NAP is mainly distributed to the kidney and eliminated by the elimination of urinary excretion.Conclusion:The establishment of this fluorescence labeling method will facilitate further research on the absorption and metabolism,functions and underlying mechanisms of NAP and its interactions with other biomolecules.the uptake of FITC-NAP in NCI-H1299cells was positively correlated with drug concentration and incubation time.the uptake of NAP by NCI-H1299 cells is a combination of clathrin-dependent endocytosis and caveolin/lipoprotein-mediated endocytosis.The Calu-3 model transport experiment showed that NAP was a relatively less absorbed drug after pulmonary administration.By comparing the pharmacokinetic parameters of intravenous injection and pulmonary administration,it was found that the absorption of NAP lungs was slow,the absorption was relatively poor,and the bioavailability was relatively low,but the pulmonary administration had a better sustained release.The effect is that the residence time in the body is longer and the elimination is slower than intravenous injection.After intravenous injection,NAP is mainly distributed to the kidney and eliminated by the elimination of urinary excretion.