Research on Novel Delivery System drug DMPK in New Drug Research and development and its cases Share | Hongren Report of the 14th Academic Conference on Drug and Chemical Foreign Body Metabolism of Chinese Pharmacological Society

Research content：New delivery systems for innovative drugs or new excipients usually carry out in vivo PK, tissue distribution, excretion/substance balance, metabolite identification, in vitro stability, CYP450 enzyme phenotype, CYP450 enzyme inhibition, CYP450 enzyme induction, transporter inhibition, etc. Among them, the study of CYP450 enzyme phenotype, CYP450 enzyme inhibition, CYP450 enzyme induction, and transporter inhibition depends on the frequency and cycle of administration of new excipients. Improved delivery system and gene delivery system drugs are usually studied in vivo PK and tissue distribution according to drug design ideas.

Detection method：

• Chemically modified drugs: LC-MS/MS

• Gene drugs: qPCR, ddPCR, bDNA, in vivo imaging, LBA

• Innovative drugs or new excipients: LC-MS/MS, radiolabelling

mPEG2k-PCLx polymer micelles were prepared, and their physical and chemical properties (critical micelle concentration, particle size, Zeta potential, etc.) and morphological characterization were performed to investigate their stability in vitro.

Preparation methods: thin film dispersion method, solvent volatilization method

The average particle size of the polymer micelle measured by DLS was in the range of 20 ~ 100 nm, the distribution was uniform, and the Zeta potential was neutral with the increase of molecular weight of the hydrophobic segment. The change of particle size observed by TEM is consistent with that of DLS.

• The inhibitory effect of the five micelles was not obvious or weak below CMC, and the inhibitory effect was enhanced with the increase of micelle concentration above CMC.

• When the concentration of 2 k, 3.5 k and 5 k is 0.5 mg/mL, 1 mg/mL and 5 mg/mL, respectively, the uptake activity of the three transporter subtypes can be reduced to less than 50% of the control group.

Cell localization of mPEG2k-PCL2k polymer micelles and hOCT1-3

The localization of micelles is similar to that of hOCT1-3, suggesting that micelles exert inhibition through contact with the transporter.

To further explore the possibility of interactions between polymer micelles and transporters, we used confocal imaging to observe the localization of micelles and transporters in the cell. hOCT1-3 protein was mostly located in the cell membrane of overexpressed MDCK cells, and less in the cytoplasm. After incubation with mPEG2k-PCL2k micelles loaded with Nile red, the cells showed strong red fluorescence. Most micelles adhere to the cell surface or remain in the cytoplasm, which is similar to the localization of hOCT1-3, suggesting that micelles exert inhibition through contact with the transporter.

1. Cell uptake of mPEG2k-PCLx micelles is time-dependent; In the same incubation time, the intake of the first three micelles was significantly higher than that of the last two micelles.

2. The fluorescence intensity in the cell is related to the size of micelle and the length of hydrophobic section. With the increase of particle size and length of hydrophobic section, the intracellular fluorescence intensity decreased gradually.

3, when the PCL chain length is 2k and 3.5k Da (particle size is 20-50 nm), large intracellular uptake can be observed. However, when the molecular weight of PCL reached 5k (the particle size was 50-100 nm), the intracellular micelle uptake gradually decreased with the increase of hydrophobic segment chain length. Among them, the closer the hydrophilic/hydrophobic ratio is, the smaller the diameter of the micelle is more easily taken up by the cell.

Cellular uptake of mPEG2k-PCLx micelles is time - and energy-dependent and is mediated by clathrin and foveolin. Among them, the closer the hydrophilic/hydrophobic ratio is, the smaller the diameter of the micelle is more easily taken up by the cell.

The intracellular fluorescence intensity after incubation of mPEG2k-PCL2k micelles with different concentrations for 1h is shown in the figure. Compared with the control group, the intracellular fluorescence intensity of the three kinds of cells increased gradually with the increase of micellar concentration after micellar incubation, suggesting that the effect of polymer micelles on cell membrane potential was concentration-dependent.

There was no significant difference between the single-dose micelle group and the control group, but the serum concentration of the multi-dose micelle group at 5min, 10min, 15min, 30min and 45min was significantly higher than that of the control group, AUC0-24h and Cmax were increased by 67.2% and 15.4%, and CL was decreased by 37.4%. In addition, Vss also decreased by 35.6%, and t1/2 and MRT did not change significantly.

Polymer micelles can affect intestinal absorption, liver distribution and renal excretion of metformin by inhibiting OCTs transport function.

After micellar treatment, the levels of metformin in liver and kidney could be reduced at 1h. Although there was no significant difference between 5h and 10h, there was still a downward trend, and the multi-dose group was more obvious than the single dose group. In contrast to the liver and kidney, after micellar treatment we found a significant increase in metformin levels in the small intestine. In addition, tissue/plasma concentration ratio results suggest that polymer micelles affect OCTs transport function and thus alter drug distribution characteristics.

mPEG2k-PCLx micelles showed a time-dependent distribution within 48h, mainly in the liver and kidney, and the aggregation time exceeded 24h.

• 1. Metformin reduced blood sugar levels in healthy rats compared with the control group. The hypoglycemic effect of metformin was attenuated either after single or multiple micelles administration. Compared with metformin alone, the peak blood glucose concentration of micellar combination group was significantly increased.

• 2. AUCglucose results were consistent. In the single-dose administration group, AUCglucose in the metformin group was significantly lower than that in the control group, and in the micellar combination group, AUCglucose was also lower, but there was no significant difference between the two groups. In the multi-dose administration group, AUCglucose was significantly decreased in both the metformin administration group alone and the micellar combination group. However, the reduction of AUCglucose in the combination group was significantly lower than that in the metformin group alone.

The above case study results indicate that the novel delivery system interacts with the transporter OCTs, affecting the PK and PD processes of OCTs substrate drugs in vivo, which needs further attention in the study of novel delivery systems.