Intestinal transport mechanism and in vivo anticancer efficacy of a solid oral formulation incorporating an ion-pairing complex of pemetrexed with deoxycholic acid derivative

Figures & data

Table 1 Inhibitors with different functions used in the transport study and their concentrations

Inhibitors	Concentrations	Functions
Act D	3.2 μM	Inhibitor of ASBT-mediated transport
Chlorpromazine	32 μM	Inhibitor of clathrin-mediated endocytosis
MβCD	10 mM	Inhibitor of caveola/lipid raft-mediated endocytosis (cholesterol depletion agent)
Genistein	0.1 mM	Inhibitor of caveola/lipid raft-mediated endocytosis (broad inhibitor of protein tyrosine kinase)
Amiloride	0.1 mM	Inhibitor of macropinocytosis
Cyclosporine A	10 mM	Inhibitor of P-gp mediated efflux
Brefeldin A	90 μM	Inhibitor of ER/Golgi pathway

Abbreviations: Act D, actinomycin D; MβCD, methyl-β-cyclodextrin; ASBT, apical sodium-dependent bile acid transporter; P-gp, P-glycoprotein; ER, endoplasmic reticulum.

Figure 1 (A) Schematic illustration of ion-pairing complex formation between PMX and DCK, and self-assembled micelle formation of PMX/DCK-OP in the aqueous phase. (B) Transmission electron micrograph of PMX/DCK-OP micelles. Scale bar represents 200 nm.

Abbreviations: PMX, pemetrexed; DCK, N^α-deoxycholyl-L-lysyl-methylester; PMX/DCK, ion-pairing complex between PMX and DCK; PMX/DCK-OP, oral powder formulation of PMX/DCK.

Table 2 Effective and apparent permeability of PMX, PMX/DCK, and PMX/DCK-OP

Test material	Effective permeability (Pe, ×10^−6, cm/s)^a	Apparent permeability (Papp, ×10^−6, cm/s)^b
PMX	0.290±0.080	1.78±0.118
PMX/DCK	10.5±1.97***	5.97±1.38*
PMX-OP	1.44±0.180	3.45±0.531
PMX/DCK-OP	40.6±1.48***^,###,$$$	14.7±3.98***^,###,$$$

Notes: Statistics: one-way ANOVA followed by Tukey’s multiple-comparison test. ^aEffective permeability (P_e) of PMX, PMX/DCK, PMX-OP, and PMX/DCK-OP through an artificial intestinal membrane. ^bApparent permeability (P_app) of PMX, PMX/DCK, PMX-OP, and PMX/DCK-OP across a Caco-2 cell monolayer. Each value represents the mean ± standard deviation (n=6). *P<0.05, ***P<0.001 compared to PMX; ^###P<0.001 compared to PMX/DCK; ^$$$P<0.001 compared to PMX-OP.

Abbreviations: PMX, pemetrexed; DCK, N^α-deoxycholyl-L-lysyl-methylester; PMX/DCK, ion-pairing complex between PMX and DCK; PMX-OP, oral powder formulation of PMX; PMX/DCK-OP, oral powder formulation of PMX/DCK.

Figure 2 (A) Relative apparent permeability of PMX/DCK-OP across a Caco-2 cell monolayer after incubation with various transport inhibitors and (B) with or without EGTA in the presence or absence of all inhibitors except cyclosporine A.

Notes: Each value represents the mean ± standard deviation (n=6). **P<0.01, ***P<0.001 compared to the P_app of the untreated control group. ^###P<0.001 compared to the apparent permeability of PMX/DCK-OP after treatment with all inhibitors except cyclosporine A in the absence of EGTA. ^$$$P<0.001 compared to the P_app of PMX/DCK-OP after treatment with EGTA in the presence of all inhibitors except cyclosporine A.

Abbreviations: PMX, pemetrexed; DCK, N^α-deoxycholyl-L-lysyl-methylester; PMX/DCK, ion-pairing complex between PMX and DCK; PMX/DCK-OP, oral powder formulation of PMX/DCK; Act D, actinomycin D; MβCD, methyl-β-cyclodextrin; ASBT; EGTA, ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; P_app, apparent permeability of PMX/DCK-OP across a Caco-2 cell monolayer.

Figure 3 Schematic diagrams of possible specific and nonspecific transport pathways, and predicted intracellular trafficking and transcytosis of PMX/DCK-OP across an intestinal epithelium: (1) ASBT-mediated transport; (2) caveola/lipid raft-mediated endocytosis; (3) macropinocytosis; (4) paracellular transport; and (5) transcellular passive diffusion.

Abbreviations: PMX, pemetrexed; DCK, N^α-deoxycholyl-L-lysyl-methylester; PMX/DCK, ion-pairing complex between PMX and DCK; PMX/DCK-OP, oral powder formulation of PMX/DCK; ASBT, apical sodium-dependent bile acid transporter; IBABP, intestinal bile acid-binding protein; OST_α, organic solute transporter alpha; OST_β organic solute transporter beta; ER, endoplasmic reticulum.

Table 3 Pharmacokinetic parameters of PMX in rats after intravenous injection of PMX and oral administration of PMX, PMX/DCK, or PMX/DCK-OP

Test material	PMX	PMX	PMX/DCK	PMX/DCK-OP
Administration	Intravenous	Oral	Oral	Oral
Dose of PMX (mg/kg)	5	25	25	25
Tmax (h)	-	0.63±0.25	0.50±0.00	0.50±0.00
T1/2 (h)	0.35±0.08	2.50±0.45	4.05±2.02	2.47±0.37
Cmax (μg/mL)	15.3±6.10	1.28±0.17	4.58±2.63	5.95±3.23
AUClast (μg/mL)	9.38±4.17	2.35±0.27	4.13±1.29	9.27±3.25**^,#
AUCinf (μg/mL)	9.41±4.22	2.77±0.21	4.90±1.73	10.2±2.94***^,#
Bioavailability (%)		5.02±0.57	8.80±2.89	19.8±6.93**^,#

Notes: Statistics: one-way ANOVA followed by Tukey’s multiple-comparison test. Each value represents the mean ± standard deviation (n=4). Bioavailability (%), (AUC_{last, oral}/Dose_{PMX, oral})/(AUC_{last, intravenous}/Dose_{PMX, intravenous})×100. **P<0.01, ***P<0.001 compared to PMX; ^#P<0.05 compared to PMX/DCK.

Abbreviations: PMX, pemetrexed; DCK, N^α-deoxycholyl-L-lysyl-methylester; PMX/DCK, ion-pairing complex between PMX and DCK; PMX/DCK-OP, oral powder formulation of PMX/DCK; T_max, time to reach maximum plasma concentration; T_1/2, half-life of plasma concentration; C_max, maximum plasma concentration; AUC_last, area under the plasma concentration-time curve from zero to the time of the last measurable plasma concentration; AUC_inf, area under the plasma concentration-time curve from zero to infinity.

Figure 4 Venous plasma concentration-time profiles of PMX after (A) single intravenous administration of PMX (5 mg/kg) and (B) oral administration of PMX (25 mg/kg), PMX/DCK (equivalent to 25 mg/kg of PMX), or PMX/DCK-OP (equivalent to 25 mg/kg of PMX) in aqueous solution to rats.

Notes: Each value represents the mean ± standard deviation (n=4 for each group).

Abbreviations: PMX, pemetrexed; DCK, N^α-deoxycholyl-L-lysyl-methylester; PMX/DCK, ion-pairing complex between PMX and DCK; PMX/DCK-OP, oral powder formulation of PMX/DCK.

Figure 5 Efficacy of in vivo tumor growth inhibitory effects in B16F10 tumor-bearing mice after biweekly intravenous administration of 20 mg/kg PMX (PMX-IV), intraperitoneal administration of 10 mg/kg anti-PD1 (Anti-PD1) once every 3 days, once-daily oral administration of PMX/DCK-OP as 20 mg/kg PMX (PMX/DCK-OP), and combined treatment with once-daily oral administration of PMX/DCK-OP as 20 mg/kg PMX and intraperitoneal administration of 10 mg/kg anti-PD1 (PMX/DCK-OP+Anti-PD1) once every 3 days for 14 days. (A) Tumor volume in mice (**P<0.01, ***P<0.001). (B) Variation of body weight in mice during treatment. (C) Tumor weight in B16F10 tumor-bearing mice (**P<0.01, ***P<0.001 compared to the control group). (D) Photographs of tumors isolated from each group on day 15. Scale bar represents 10 mm.

Notes: Statistics: one-way ANOVA followed by Tukey’s multiple-comparison test. Each value represents the mean ± standard deviation (n=10 for each group).

Abbreviations: PMX, pemetrexed; DCK, N^α-deoxycholyl-L-lysyl-methylester; PMX/DCK, ion-pairing complex between PMX and DCK; PMX/DCK-OP, oral powder formulation of PMX/DCK.

Figure 6 Representative cross-sectional images of isolated tumor tissues stained with (A) anti-CD31 antibody for microvessels (brown), (B) proliferating cell nuclear antigen (PCNA) for proliferating cells (brown), and (C) fluorescent terminal deoxynucleotidyl transferase-mediated dUPT nick end labeling (TUNEL) for apoptosis (green fluorescence) in the tumor tissues taken 14 days after treatment with various modes.

Notes: The treatment modes were biweekly intravenous administration of 20 mg/kg PMX (PMX-IV), intraperitoneal administration of 10 mg/kg anti-PD1 (Anti-PD1) once every 3 days, once-daily oral administration of PMX/DCK-OP as 20 mg/kg of PMX (PMX/DCK-OP), and combined once-daily oral administration of PMX/DCK-OP as 20 mg/kg of PMX with once every 3 days intraperitoneal administration of 10 mg/kg anti-PD1 (PMX/DCK-OP + Anti-PD1). Scale bars represent 50 μm for anti-CD31 antibody and PCNA staining, and 20 μm for TUNEL staining, respectively.

Abbreviations: PMX, pemetrexed; DCK, N^α-deoxycholyl-L-lysyl-methylester; PMX/DCK, ion-pairing complex between PMX and DCK; PMX/DCK-OP, oral powder formulation of PMX/DCK.

Figure S1 In vitro cytotoxic effect of Act D on Caco-2 cells.

Notes: Cell viability was measured using WST-8 and the growth of Caco-2 cells compared to the untreated control group. All data are expressed as means ± standard deviation (n=6).

Abbreviation: Act D, actinomycin D.