2021 Volume 69 Issue 11 Pages 1054-1060
In the evaluation of the druggability of candidate compounds, it was vital to predict the oral bioavailability of compounds from apparent permeability (Papp) across Caco-2 cell-culture model of intestinal epithelium cultured on commercial transwell plate inserts. The study was to investigate the transport characteristics and permeability of FL118 (10, 11-Methylenedioxy-20(S)-camptothecin) derivatives 7-Q6 (7-(4-Ethylphenyl)-10, 11-methylenedioxy-20(S)-camptothecin) and 7-Q20 (7-(4-Trifluoromethylphenyl)-10, 11-methylenedioxy-20(S)-camptothecin). Transport characteristics and permeability of the tested compounds to the small intestine were assessed at different concentrations (0.5, 1 µM) via Caco-2 cell monolayers model in vitro. Uptake studies based on Caco-2 cells, including temperatures, concentrations, and the influence of efflux transporters, were combined to confirm the transport characteristics of the tested compounds. Furthermore, cytotoxicity results showed that the concentrations used in the experiments were non-toxic and harmless to cells. In addition, The Papp of 7-Q6 was (3.69 ± 1.07) × 10−6 cm/s with efflux ratio (ER) 0.98, while the Papp of 7-Q20 was (7.78 ± 0.89) × 10−6 cm/s with ER 1.05 for apical-to-basolateral (AP→BL) at 0.5 µM, suggesting that 7-Q20 might possess higher oral bioavailability in vivo. Furthermore, P-glycoprotein (P-gp) was proved to slightly affect the accumulations of 7-Q20, while the absorption of 7-Q6 was irrelevant with P-gp and breast cancer resistant protein (BCRP) based on the cellular uptake assays. Accordingly, 7-Q6 was completely absorbed by passive diffusion, and 7-Q20 was mainly dependent on passive diffusion with being effluxed by P-gp slightly. Meanwhile, both 7-Q6 and 7-Q20 were potential antitumor drugs that might exhibit high oral bioavailability in the body.
In 2012, a novel camptothecin derivative named FL118 was confirmed as potent survivin inhibitor by high throughput screening and proved to exhibit superior antitumor efficacy in comparison with clinically anti-tumor drugs in xenograft tumor model.1) Studies have shown that the target of camptothecin was DNA topoisomerase I.2) Although FL118 was similar in structure to camptothecin, its superior anti-tumor effect was not for the topoisomerase I target inhibition. Instead, FL118 not only effectively inhibited the expression of the anti-apoptotic protein survivin in the inhibitor of apoptosis (IAP) family, but also selectively and independently inhibited the other cancer-related survival genes (Mcl-1, XIAP and cIAP2) in a p53-dependent manner, while inducing the expression of the pro-apoptotic proteins Bax and Bim.3) Additionally, FL118, nor the substrate of P-glycoprotein (P-gp) or the breast cancer resistant protein (BCRP), could effectively inhibit the drug-resistant tumor cells compared with Irinotecan and Topotecan in human tumor xenograft models.4) However, FL118 has poor solubility and low permeability. And FL118 derivatives 7-Q6 (Fig. 1a) and 7-Q20 (Fig. 1b) from our lab were designed for better permeability and enhanced activity.
Caco-2 cell monolayers model has been extensively utilized assays for permeability assessment and transport characteristics of compounds in both pharmaceutical industries and academia.5–7) Derived from human colon adenocarcinoma cells, Caco-2 cells could spontaneously differentiate into small intestinal microvilli epithelial cells over 21 d of culturing on polycarbonate membrane.8,9) More importantly, the fully polarized cell model could stably express some major absorption transporters, efflux transporters and metabolic enzymes, which were consistent with the small intestine.6,10–12) In addition, to better simulate the physiological environment in the small intestine, fasted state human intestinal fluid (Fassif) was used on the donor side and 1% bovine serum albumin (BSA) in the receiver buffer, respectively.13–15) Apart from providing sinking conditions for drugs to membrane transport, BSA was beneficial to reduce the absorption of drugs on polycarbonate membranes and transwell plates.16–18) For Caco-2 cell monolayers model, there existed a good correlation between the apparent permeability coefficient and the absorption coefficient of the human body.11,19)
Furthermore, the efficacy of chemotherapeutic drugs in the treatment of tumors depended on the extent to which the drugs were delivered to the cells, and the inadequate of the intracellular accumulation could lead to the residual tumor cells, ultimately the development of multidrug resistance.20,21) Commonly, the insufficiency was caused by the diverse efflux transporters, such as P-gp and BCRP, both of which were energy-dependent drug efflux pumps.22–24) The previous studies have shown that the mediation of P-gp and BCRP accounted for the low oral bioavailability of Topotecan and Irinotecan in clinical.25–27) Additionally, structural modification might also bring about changes in compounds transport characteristics.28) For instance, FL118, modified on the basis of camptothecin, solely depended on simple diffusion without secretion transporters (i.e., P-gp and BCRP) involved.4,29) However, 9-Q20, an analogue of FL118, was found to be not only a substrate of P-gp but also BCRP in two dimensional (2D) and 3D cell models in vitro.30) Besides, compared with raw FL118, the absorption of 7-substituted FL118 derivatives was improved via 3D cell model in vitro.31)
The current study was to explore the transport characteristics of 7-substituted FL118 derivatives 7-Q6 and 7-Q20 through intestinal permeation model in vitro. Related drug resistance and oral bioavailability would be predicted.
Dulbecco’s modified Eagle’s medium (DMEM), trypsin–ethylenediaminetetraacetic acid (EDTA), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), phosphate-buffered saline (PBS), Hank’s balanced salt solution (HBSS) and penicillin–streptomycin solution were purchased from from Solarbio (Beijing, China). Dimethyl sulfoxide (DMSO) and Lucifer yellow (LY) were obtained from Sigma (Shanghai, China). Fetal bovine serum (FBS) was purchased from Hyclone (Fisher, Spain). Fassif was purchased from Biorelevant (Shenzhen, China). All reagents for HPLC were of analytical grade. 7-Q6 and 7-Q20 (purity >99%) were synthesized by our lab. Verapamil, Gefitinib and BSA were purchased from Aladdin (Shanghai, China). Six-well cell culture plates and porous polycarbonate membrane Transwell inserts (3401) were purchased from Corning (Corning, U.S.A.).
Cell CultureThe Caco-2 cell line (No.GDC153) was purchased from CCTCC (Wuhan, China). Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin solution. Caco-2 cells were passaged every three days and were incubated in an incubator atmosphere with a relative humidity of 95%, carbon dioxide concentration of 5% and temperature at 37 °C.
Cell Viability AssayThe cytotoxicity of 7-Q6 and 7-Q20 was evaluated by MTT experiments.32) The Caco-2 cells were seeded in a 96-well plate at a cell density of 2 × 105 cells/mL and 100 µL/well. The plate with cells was placed in a 37 °C incubator (relative humidity: 95%, CO2 concentration: 5%) for 48 h until cells reaching 90% confluence. And the cells were exposed to 7-Q6 and 7-Q20 (0.2, 0.4, 0.6, 0.8, 1.0 µM) for 3 or 24 h. After the incubation, 5 mg/mL MTT (20 µL/well) was added to incubate for another 4 h. Finally DMSO (150 µL/well) was added to dissolve the formazan and mixed on the micro-oscillator. The optical density (OD) values were measured at the measuring/reference wavelength of 570/630 nm via the ELISA Plate Reader (infinite M200 Pro, TECAN). And calculate the cell viability according to Eq. (1):
 | (1) |
Where ODexperimental group referred to the 7-Q6 or 7-Q20 administration group, ODblank group referred to the medium only without cell inoculation group and the ODcontrol group referred to the cell group normally cultured with the medium.
Sample PreparationThe tested compounds were prepared with DMSO as stock solutions stored at 4 °C. The maximum concentration of working solution was 1 µM with 0.1% DMSO. For the transport assay, the tested compounds were dissolved in HBSS buffer (137.93 mM NaCl, 5.33 mM KCl, 4.17 mM NaHCO3, 0.441 mM KH2PO4, 0.338 mM Na2HPO4, 5.56 mM D-Glucose, pH 7.4) and Fassif (3.9 g/L KH2PO4, 7.7 g/L KCl, 0.3 mM Na-taurocholate and 0.075 mM egg lecithin, pH 6.5), respectively.13,15,19)
The Integrity Evaluation of Caco-2 Cells MonolayerCaco-2 cells between 35–40 passages were seeded on polycarbonate of transwell plates cultured for 21 d until they spontaneously differentiated into small intestinal epithelial cells with microvilli. The integrity of the monolayer membrane was mainly evaluated from two aspects: trans-epithelial electrical resistance (TEER) values and the transmembrane situation of LY.33)
During the 21 d culture, the TEER values of Caco-2 cell monolayers on each insert-transwell plate were monitored via Millicell ERS-2 voltameter every 3 d for better grasping the polarization and differentiation of Caco-2 cells. The TEER values were calculated as Eq. (2) below:
 | (2) |
Where TEER referred to the resistance values of cell monolayers while TEERblank control referred to the resistance values of blank polycarbonate film (without cells), and A referred to the polycarbonate membrane area of the Transwell plate (1.12 cm2).
LY utilized to evaluate the integrity of cell monolayers was a mark substance of paracellular permeability. The LY experiment was carried out after 21 d of the monolayer membrane formed but before the bi-directional transport experiments performed. First, standard solutions of LY at different concentrations (0.06, 0.13, 0.25, 0.50, 1.00, 2.00, 4.00 and 8.00 µg/mL) were prepared and the corresponding fluorescence intensity values were measured (λex = 492 nm, λem = 530 nm). Then the standard curve (y = 0.0837x + 0.0024, R2 = 0.9998) was drawn with the fluorescence intensity values as ordinate and concentrations as abscissa and the corresponding standard curve for every measurement of LY fluorescence was done. Finally, the transmembrane transport experiments of LY were carried out. Half milliliter LY solution (20 µg/mL) was added to the AP and 1.5 mL HBSS to the BL incubating for 2 h, then 0.5 mL sample was withdrawn from the BL to determine the fluorescence intensity. The concentrations were calculated from the standard curve and the corresponding apparent permeability (Papp) values were calculated by Eq. (3) to evaluate the integrity of the membrane.
Transport ExperimentsFor the absorptive study, 7-Q6 and 7-Q20 dissolved with Fassif (pH 6.5) were added to the AP as the donor side and HBSS containing 1% BSA were added to the BL as the receiver side, and vice versa for secretion experiments. The cell monolayers were exposed to 7-Q6 and 7-Q20 at different concentrations (0.5, 1 µM) for 3 h. The samples (250 µL) were taken at specific time points (90, 120, 150, 180 min) and equal volume of buffer (250 µL) was added. The samples were dertermined by HPLC. The corresponding Papp was calculated by Eq. (3)
 | (3) |
Where V referred to the volume of the solution on the receiving side (mL); A referred to the polycarbonate membrane area of the Transwell plate (1.12 cm2); C0 referred to the concentration of the tested compound (µg/mL); ∆Q/∆t referred to the rate of permeability of tested compound (µg/mL·s−1).
The efflux ratio (ER) was calculated according to Eq. (4):
 | (4) |
To form cell monolayers, a density of 2 × 105 cells/mL were seeded onto six-well plastic plates. After reaching 80 to 90% confluence, the uptake experiments were performed. Different concentrations of compounds (0.2, 0.4, 0.6, 0.8, 1.0 µM) dissolved in HBSS (containing 10 mM N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4) were exposed to Caco-2 cells at 37 or 4 °C for 3 h. Then the cells were harvested with a spatula and lysed as described by Xiao et al.34) The protein content was quantified by Coomassie Brilliant Blue method35) and the compound content was determined by HPLC method. Regarding the transport inhibitors, the cells were pretreated with Verapamil (1 µM, P-gp inhibitor) and Gefitinib (1 µM, BCRP inhibitor) before administration,36) respectively. The ratio of compound content to total protein was used to quantify drug uptake in cells.
HPLC AnalysisAll experimental samples were quantitatively analyzed by HPLC system (LC-20AT; Shimadzu, Kyoto, Japan) equipped with a SPD-20 A UV/VIS detector and a reverse-phase C18 column (5 µm, 4.6 × 250 mm; Elite, Dalian) was used. The liquid phase conditions of 7-Q6 and 7-Q20: the mobile phase was acetonitrile: 0.1% formic acid (60 : 40) with a 1.0 mL/min flow rate, the detection wavelength was 365 nm and the column temperature was 40 °C. The samples (200 µL) were pretreated by an equal volume of acetonitrile (200 µL) and centrifuged for 10 min. Then the supernatant was filtered with 0.22 µm microporous membrane and the injection volume was 20 µL.
Data AnalysisAll data were performed using GraphPad Prism version 8 and Origin® 9 software. Data were obtained from triple independent tests and expressed as mean ± standard deviation (S.D.). Statistical differences were evaluated by the two-way ANOVA. p < 0.05 was considered statistically significant.
As shown in illustration (Fig. S1, Supplementary Materials), the retention times of 7-Q6 and 7-Q20 were 10.712 and 8.780 min in the same mobile phase and ratio. No interferential peaks were observed from the chromatogram under the liquid phase condition used for the content determination of the tested compounds during the transport and uptake experiments. The calibration curves of 7-Q6 (y = 33990x + 98.282, R2 = 0.9939) and 7-Q20 (y = 33911x − 102.53, R2 = 0.9995) used for quantitative analysis showed an objective linearity at the concentration range of 0.0156 to 0.5 µM, respectively. And the corresponding standard curves for every determination of compounds content were drawn again. The relative standard deviation (RSD) of intra-day precision at 0.06, 0.25, 0.50 µM were 1.57, 0.81, 0.91% and 1.71, 1.63, 1.27% for 7-Q6 and 7-Q20, respectively (Table S1, Supplementary Materials). The RSD of inter-day precision were 1.09 to 2.86% and 1.50 to 2.02% for 7-Q6 and 7-Q20, respectively (Table S1, Supplementary Materials). And the average recovery rates were 98.12 to 100.27% and 99.01 to 101.12% for 7-Q6 and 7-Q20, respectively (Table S2, Supplementary Materials). The RSD of accuracy at 0.06, 0.25, 0.50 µM were 1.77, 0.37, 0.14% and 0.47, 0.41, 0.50% for 7-Q6 and 7-Q20, respectively (Table S3, Supplementary Materials). The RSD of stability within 24 h were no more than 15% for 7-Q6 and 7-Q20 (Table S4, Supplementary Materials).
Cell Viability AssayTo ensure that the cell membrane was intact and not damaged when exposed to the tested compounds, MTT assay was exploited to evaluate the cell viability at specific times (3 or 24 h) and concentrations (0.2, 0.4, 0.6, 0.8, 1.0 µM) used in subsequent experiments. According to the literature,33) it could basically be considered that the tested compounds were non-toxic and harmless to cells when the cell viability was more than 90%. As shown in Figs. 2a and b, 7-Q6 and 7-Q20 were almost non-toxic to Caco-2 cells within 3 h even at the maximum concentration of 1 µM. Furthermore, the cell–drug interaction time has been extended to 24 h and almost no cytotoxicity against Caco-2 cells was found.
Control: DMEM; Vehicle control: 0.1% DMSO. Data represented the mean ± S.D. from three replicates.
Caco-2 cell monolayers used for subsequent assays were evaluated by the TEER values and Papp (AP→BL) of LY, both of which were prevalently employed by tremendous researchers. It was obvious from Fig. S2 that the TEER values increased dramatically at the outset since the cells were in the proliferating state, while TEER values almost kept stable afterward as the cells were in the differentiated state, and ultimately reached at about 500 Ω·cm2. LY was utilized to test the compactness between cells and the Papp (AP→BL) values were calculated as 0.55(±0.17) × 10−6 cm/s, lower than 1 × 10−6 cm/s, demonstrating that Caco-2 cells have completed differentiation and possessed good integrity, which could be carried out for permeation experiments.
Bidirectional Transport of 7-Q6 and 7-Q20 across the Caco-2 CellTo evaluate the ability of the compounds 7-Q6 and 7-Q20 to break through the mucosal barrier, the transport experiments were subsequently carried out via Caco-2 cell monolayers model in vitro. 7-Q6 and 7-Q20 were dissolved with Fassif at different concentrations (0.5, 1 µM) respectively, 0.5 mL working medium was added to the AP side as the intestinal lumen side and 1.5 mL of HBSS containing 1% BSA was added to the BL side as the plasma side. While for secretory efflux, and vice versa. After administration, the samples that were withdrawn at prescribed time (90, 120, 150, 180 min) were quantified by HPLC. Once the experiments have been completed, the TEER values were measured immediately. And with no reducing TEER values observed, the data obtained from the permeation tests were valid.
For AP→BL, the cumulative amount of 7-Q6 and 7-Q20 transporting from the intestinal lumen side to the plasma side increased linearly with the exposure time at the concentration of 0.5 µM shown in Figs. 3a and d. Moreover, the Papp (AP→BL) of 7-Q20 (7.78 × 10−6 ± 0.89 × 10−6 cm/s) was approximately 2.1-fold higher than the Papp (AP→BL) of 7-Q6 (3.69 × 10−6 ± 1.07 × 10−6 cm/s), which indicated 7-Q20 was superior to 7-Q6 in the ability to pass through the small intestine epithelium. Simultaneously, the experiments on BL→AP were also carried out and the corresponding apparent permeability coefficients were obtained in Table 1. The Papp (BL→AP) of 7-Q6 was (3.62 ± 1.10) × 10−6 cm/s with ER 0.98 while the Papp (BL→AP) of 7-Q20 was (8.21 ± 1.11) × 10−6 cm/s with ER 1.05. And the bidirectional transport experiments of 1 µM were performed, as shown in Table 1, the Papp (AP→BL) of 7-Q6 was (4.22 ± 0.69) × 10−6 cm/s with ER 1.26, meanwhile, the Papp (AP→BL) of 7-Q20 was (6.13 ± 0.73) × 10−6 cm/s with ER 1.09. In addition, the recovery rates of 7-Q6 and 7-Q20 were relatively considerable in the permeability assays (Table 1). And to further clarify the absorption mechanism of 7-Q6 and 7-Q20, the uptake experiments were conducted subsequently.
The time course of transported amounted of 7-Q6 b) and 7-Q20 e) in secretion directions: BL-to-AP. Papp values of 7-Q6 c) and 7-Q20 f) in AP-to-BL and BL-to-AP directions. Data represented the mean ± S.D. of three replicates.
| Tested compounds | Concentration (µM) | Recovery (%) | Papp × 10−6 (cm/s) | Efflux ratio | ||
|---|---|---|---|---|---|---|
| AP-BL | BL-AP | AP-BL | BL-AP | |||
| 7-Q6 | 0.5 | 69.8 | 67.1 | 3.69 ± 1.07 | 3.62 ± 1.10 | 0.98 |
| 1 | 44.0 | 77.2 | 4.22 ± 0.69 | 5.34 ± 0.56 | 1.26 | |
| 7-Q20 | 0.5 | 89.1 | 73.5 | 7.78 ± 0.89 | 8.21 ± 1.11 | 1.05 |
| 1 | 80.2 | 92.2 | 6.13 ± 0.73 | 6.68 ± 0.44 | 1.09 | |
Data represented the mean ± S.D. of three replicates. *Papp: apparent permeability.
The uptake of 7-Q6 and 7-Q20 was mainly carried out from the aspects of temperatures, concentrations and transporter inhibitors. Caco-2 cells were exposed to different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 µM) of 7-Q6 and 7-Q20 at 37 or 4 °C for 3 h. Obviously, it could be seen from Figs. 4a and d that the uptake of 7-Q6 and 7-Q20 increased with the increasing of the concentration in concentration-dependent. Moreover, Fig. 4a showed there was almost no difference between the uptake of 7-Q6 at 37 °C and 4 °C, hinting that the absorption of 7-Q6 into cells was related to passive diffusion. However, the uptake of 7-Q20 distinctly increased at 4 °C compared with 37 °C in Fig. 4d, which suggested more than one transport pathway was involved in the absorption of 7-Q20.37) And the transporter inhibitors (Verapamil and Gefitinib) were adopted to find out whether P-gp or BCRP was involved since the absorption of camptothecin derivatives (e.g., Topotecan and Irinotecan) was usually mediated by P-gp and BCRP. As shown in Figs. 4b and c, neither P-gp inhibitors nor BCRP inhibitors exerted influence on the uptake of 7-Q6, illustrating 7-Q6 was not the substance of P-gp and BCRP. In contrast, as shown in Figs. 4e and f, with P-gp inhibitors pretreated, the accumulation of 7-Q20 in Caco-2 cells increased significantly, revealing that 7-Q20 might be the substance of P-gp.
(a and d) the Caco-2 cells were exposed to 7-Q6 and 7-Q20 (0.2, 0.4, 0.6, 0.8, 1 µM) for 3 h at 37 or 4 °C, respectively. (b, c, e and f) the Caco-2 cells were pretreated with or without Verapamil and Gefitinib (1 µM) for 30 min, then exposed to 7-Q6 and 7-Q20 (0.2, 0.4, 0.6, 0.8, 1 µM) for 3 h at 37 °C, respectively. Data represented the mean ± S.D. from three replicates. * p < 0.05 represented significantly different from the control; **** p < 0.0001 represented more significantly different from the control.
Given that FL118 exhibited exceptional anti-tumor effects, a series of novel compounds derived from FL118 were designed and synthesized by our lab. 7-Q6 and 7-Q20 were selected from FL118 derivatives we synthesized, which remained the superior anti-tumor activity like FL118 with nM level IC50.
And studies on camptothecin derivatives have shown that the structural modifications might change the way of compounds entering into cells.38) As reported that the secretion process of clinically applied camptothecin derivatives, Irinotecan and Topotecan, was mediated by P-gp and BCRP.39,40) But researchers revealed that FL118 was not the substrate of P-gp or BCRP with high permeability in Caco-2 permeability assays.26,29) However, the current study showed that the accumulation of 7-Q6 was not affected remarkably by the decreasing temperature or the transporter inhibitors interference, displaying that 7-Q6 might mainly rely on simple diffusion. Whilst secretory transporter P-gp was suggested to be involved in the absorption process of 7-Q20 since the accumulation increased significantly with decreasing temperature and P-gp inhibitor intervention, especially at high concentrations, which were also consistent with the absorption mechanism in our previous study in the 3D cell model.31)
Only by overcoming the intestinal mucosal epithelial barrier, oral drugs could enter the blood circulation and reach the target cells to exert the corresponding anti-tumor effect. The Caco-2 cell monolayers model used in our transport experiments closely simulated the physiological conditions of the small intestine. Different from the classic model, Fassif was added to the upper chamber and an additional 1% BSA was added to the lower chamber for preventing the drugs from returning to the cells or the donor side wherein a pH gradient was set with 6.5/7.4. Simultaneously, to avoid false-negatives classification, recommended low concentrations were adopted in the experiments, for high concentration easily saturating the transporters.8) The results showed that the Papp (AP→BL) of 7-Q20 (7.78 × 10−6 ± 0.89 × 10−6 cm/s) was nearly 2-fold higher than that of 7-Q6 (3.69 × 10−6 ± 1.07 × 10−6 cm/s), which suggested that 7-Q20 might exhibit higher permeability in the human small intestine in comparison with 7-Q6. The high permeability ability of 7-Q20 (6.13 × 10−6 ± 0.73 × 10−6 cm/s) seemed to be similar to the core compound FL118 (6.97 × 10−6 cm/s) reported by the previous researchers.26) And according to Biganzoli et al.41) reported, (1) Papp values < 0.2 × 10−6 cm/s, very poor absorption, bioavailability <1%, (2) Papp values between 0.2 × 10−6 and 2 × 10−6 cm/s with bioavailability between 1 and 90%, and (3) Papp values >2 × 10−6 cm/s, very good absorption, bioavailability over 90%. Therefore, the estimated absorption rates of FL118 derivatives in humans were predicted over 90%.
Regarding the bidirectional permeability experiment, it was obvious that the ER values of 7-Q6 and 7-Q20 were less than 2, which illustrated that both 7-Q6 and 7-Q20 were dependent on passive diffusion without active transporters involved, which seemed to be inconsistent with the conclusion of uptake. However, the abnormality of 7-Q20 might be due to the stronger permeability of 7-Q20, leading to a smaller ER value as mentioned in the literature.10) Hence, sometimes it was not rigorous enough to define the transport pathway of a compound based on ER values alone.42) Correspondingly, combined with cellular uptake experiments, conclusions could be drawn that 7-Q6 mainly relied on passive diffusion while 7-Q20 was dependent on passive diffusion as well as active efflux.
The permeability coefficients of 7-Q20 obtained in the transport experiment were significantly greater than that of 7-Q6 yet there was slight difference in cellular uptake, which presumably attributed to the difference of the model. Though BSA contained in the BL side could provide the driving force for the drugs to cross the membrane, the affinity to BSA varies from different compounds. Certainly, the possibility that paracellular transport was involved in the transport of 7-Q20 should not be ruled out, which requires further studies in subsequent scientific research.
The present study showed that the transport of 7-Q6 involved passive diffusion and 7-Q20 relied on passive diffusion with the efflux transporter P-gp mediating slightly. Despite the active efflux existed, the high permeability of 7-Q20 was comparable to that of FL118 in the Caco-2 penetration experiment in vitro. Therefore, 7-Q20 would be a potential candidate anti-cancer drug with high absorption ability in the body.
This work was supported by Key Research and Development Program of Zhejiang (2021C03084), National Nature Science Foundation of China (No. 31270397) and High-Level Talent Special Support Plan of Zhejiang Province (2019R52009).
The authors declare no conflict of interest.
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