Improvements in the Oral Absorption and Anticancer Efficacy of an Oxaliplatin-Loaded Solid Formulation: Pharmacokinetic Properties in Rats and Nonhuman Primates and the Effects of Oral Metronomic Dosing on Colorectal Cancer

Figures & data

Table 1 Inhibitors Used in the Transport Study

Inhibitor	Concentration	Function
Act D	3.2 μM	Inhibitor of ASBT-mediated transport
CFZ	10 µM	Inhibitor of OSTα/β
Chlorpromazine	32 μM	Inhibitor of clathrin-mediated endocytosis
MBCD	10 mM	Inhibitor of caveola/lipid raft-mediated endocytosis (cholesterol depletion agent)
Genistein	0.1 mM	Inhibitor of caveola/lipid raft-mediated endocytosis (nonspecific inhibitor of protein tyrosine kinase)
Amiloride	0.1 mM	Inhibitor of macropinocytosis
Cys A	10 µM	Inhibitor of P-gp-mediated efflux
Brefeldin A	90 µM	Inhibitor of the ER/Golgi pathway

Abbreviations: Act D, actinomycin D; ASBT, apical sodium-dependent bile acid transporter; CFZ, clofazimine; OST_α/β, organic solute transporter alpha/beta; MBCD, methyl-β-cyclodextrin; Cys A, cyclosporine A; P-gp, P-glycoprotein; ER, endoplasmic reticulum.

Figure 1 (A) Schematic representation of ion-pairing complex formation between OP and DLM, and the self-assembled micellar structure of ODSF in the aqueous phase. (B) Powder X-ray diffraction patterns and (C) differential scanning calorimetric thermograms of pure OP, DLM, P188, OP/DLM, ODSF, and a physical mixture of OP, DLM, and P188.

Abbreviations: OP, oxaliplatin; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol.

Figure 2 (A) Particle size, (B) zeta potential distribution, and (C) a transmission electron micrograph of ODSF micelles. Scale bar: 200 nm.

Abbreviations: DLM, N^α-deoxycholyl-l-lysyl-methylester; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol; OP, oxaliplatin; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188.

Table 2 Effective and Apparent Permeabilities of OP, OP/DLM, OP-SF, and ODSF

Test Materials	Effective Permeability (Pe, ×10^−6, cm/s)	Apparent Permeability (Papp, ×10^−6, cm/s)
OP	0.448±0.192	1.55±0.600
OP/DLM	5.56±0.140***	7.53±1.63***
OP-SF	2.87±0.149***,^###	3.07±2.06^###
ODSF	10.8±0.56***,^###,$	17.2±0.945***,^###,$

Notes: Statistics: one-way ANOVA followed by the Tukey multiple comparisons test. Effective permeability (P_e) values of OP, OP/DLM, OP-SF, and ODSF through an artificial intestinal membrane. Apparent permeability (P_app) values of OP, OP/DLM, OP-SF, and ODSF across a Caco-2 cell monolayer. Each value is mean±standard deviation (n=6). ***P<0.001, compared to OP; ^###P<0.001, compared to OP/DLM; ^$P<0.001, compared to OP-SF.

Abbreviations: OP, oxaliplatin; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; OP-SF, solid oral formulation of OP with P188 and Labrasol; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol.

Figure 3 (A) Effects of various intestinal transport inhibitors on the relative apparent permeability of ODSF across a Caco-2 cell monolayer. ***P<0.001 compared to the P_app of ODSF in the absence of all inhibitors (untreated control). (B) Prediction of a role for P-gp in intestinal absorption of ODSF. *P<0.05, **P<0.01, ***P<0.001 compared to the P_app (A to B) of ODSF in the absence of all inhibitors (untreated control); ^#P<0.05, ^###P<0.001 compared to the P_app (B to A) of ODSF in the absence of all inhibitors; ^$$$P<0.001 compared to the P_app (A to B) of ODSF in the absence of all inhibitors except Cys A. (C) P_app values with or without EGTA, in the presence or absence of all inhibitors except Cys A. **P<0.01, compared to the P_app without EGTA, in the absence of all inhibitors except Cys A; ^###P<0.001 compared to the P_app after treatment with EGTA, in the absence of all inhibitors except Cys A; ^$P<0.05 compared to the P_app without EGTA, in the presence of all inhibitors except Cys A.

Notes: Statistics: one-way ANOVA followed by the Tukey multiple comparisons test. Each value is mean±standard deviation (n=4 for each group).

Abbreviations: OP, oxaliplatin; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol; P-gp, P-glycoprotein; EGTA, ethylene glycol-bis-(2-aminoethyl ether)-N,N,N´,N´-tetraacetic acid; P_app, apparent permeability of ODSF across a Caco-2 cell monolayer; A to B, transport from apical to basal region; B to A, transport from basal to apical region; Act D, actinomycin D; CFZ, clofazimine; OST_α/β, organic solute transporter alpha/beta; MBCD, methyl-β-cyclodextrin; Cys A, cyclosporine A.

Figure 4 In vitro cytotoxic effects of OP, DLM, OP/DLM, DLM/P188, blank-ODSF, and ODSF on (A) Caco-2 and (B) Madin–Darby Canine Kidney (MDCK) cells.

Notes: Cell viability was measured using WST-8 and the growth of Caco-2 or MDCK cells compared to the untreated control group. All data are shown as means±standard deviations (n=4/group).

Abbreviations: OP, oxaliplatin; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; DLM/P188, a physical mixture of DLM with P188; blank-ODSF, solid oral formulation of DLM with P188 and Labrasol; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol.

Figure 5 In vitro cumulative percentage release of OP and ODSF at (A) pH 1.2 and (B) pH 6.8.

Notes: Values are means±standard deviations (n=6/group).

Abbreviations: OP, oxaliplatin; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol.

Table 3 Stability of ODSF

Storage Conditions	Month	Drug Content (%)	Cumulative Drug Release at 1 h (%)	Particle Size (nm)	PDI	Zeta Potential (mV)
25±2°C with 65±5% RH	0	100±1.25	97.2±2.35	133±1.47	0.147±0.020	50.3±3.02
	1	99.8±2.35	95.6±95.6	140±1.12	0.149±0.003	51.7±1.37
	3	98.7±2.58	96.0±3.48	143±2.57	0.149±0.006	50.8±0.379
	6	98.5±3.03	91.2±1.95	147±3.44	0.157±0.004	49.7±0.737
40±2°C with 75±5% RH	0	101±2.02	93.9±3.33	135±1.66	0.133±0.009	50.2±0.666
	1	97.2±1.25	95.8±4.25	140±1.02	0.137±0.001	51.0±0.416
	3	98.3±1.87	96.5±2.48	149±3.37	0.151±0.007	50.9±0.529
	6	97.6±2.21	91.4±3.75	153±2.28	0.162±0.004	50.6±0.404

Notes: Cumulative release of OP from ODSF was measured in 900 mL of 0.1 N HCl (pH 1.2) using the United States Pharmacopeia dissolution test apparatus I with a rotating speed of 100 rpm at 37±0.2°C. Each value is shown as mean±standard deviation (n=6).

Abbreviations: OP, oxaliplatin; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; OP-SF, solid oral formulation of OP with P188 and Labrasol; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol; PDI, polydispersity index; RH, relative humidity.

Table 4 Pharmacokinetic Parameters of OP in Rats After IV Injection of OP and Oral Administration of OP-S or ODSF with Various Levels of OP

Test Material	OP	OP-S	ODSF (2.5)	ODSF (10)	ODSF (20)
Administration route	IV	Oral	Oral	Oral	Oral
Dose of OP (mg/kg)	5	10	2.5	10	20
Tmax (h)	-	0.5±0.0	0.5±0.0	0.5±0.0	0.5±0.0
Cmax (μg/mL)	2.29±0.418	0.071±0.006	0.049±0.003	0.136±0.016	0.224±0.055
AUClast (μg·h/mL)	7.44±0.832	0.965±0.038	0.894±0.024	1.62±0.125	2.52±0.460
AUCinf (μg·h/mL)	11.6±1.58	3.52±1.75	3.44±1.94	2.99±0.352	4.49±0.666
Bioavailability (%)	100	6.48±0.252	24.0±0.642	10.9±0.838	8.47±1.55

Notes: Each value is mean±standard deviation (n=4). Bioavailability (%): (AUC_{last, oral}/Dose_{OP, oral})/(AUC_{last, IV}/Dose_{OP, IV})×100.

Abbreviations: OP, oxaliplatin; IV, intravenous; OP-S, OP in aqueous solution; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol; 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 6 Venous plasma concentration–time profiles of OP after a single (A) IV dose (5 mg/kg) and (B) oral administration of OP in aqueous solution (OP-S, 10 mg/kg), ODSF equivalent to 2.5 mg/kg OP [ODSF (2.5)], ODSF equivalent to 10 mg/kg OP [ODSF (10)], and ODSF equivalent to 20 mg/kg OP [ODSF (20)] to rats.

Note: Each value is mean±standard deviation (n=4/group).

Abbreviations: OP, oxaliplatin; IV, intravenous; OP-S, OP in aqueous solution; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol.

Table 5 Pharmacokinetic Parameters of OP in Monkeys After IV Injection of OP and Oral Administration of OP-S or ODSF

Test Material	OP-IV	OP-S	ODSF
Administration route	IV	Oral	Oral
Dose of OP (mg/kg)	2	10	10
Tmax (h)	-	2.00±0.00	3.00±1.00
T1/2 (h)	45.9±5.46	49.4±5.45	37.4±5.59
Cmax (μg/mL)	0.691±0.099	0.043±0.006	0.192±0.038*
AUClast (μg·h/mL)	9.17±1.96	1.05±0.182	3.97±0.659*
AUCinf (μg·h/mL)	17.8±3.52	2.07±0.219	6.44±0.869**
MRT (h)	19.6±0.254	19.7±0.124	18.4±0.049**
Bioavailability (%)	100	2.29±0.398	8.64±1.44**

Notes: Statistics: Student’s t-test. Each value is mean±standard deviation (n=3). *P<0.05, **P<0.01, compared to OP-S. Bioavailability (%): (AUC_{last, oral}/Dose_{OP, oral})/(AUC_{last, IV}/Dose_{OP, IV})×100.

Abbreviations: OP, oxaliplatin; IV, intravenous; OP-S, OP in aqueous solution; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol; 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; MRT, mean residence time.

Figure 7 Venous plasma concentration–time profiles of OP after a single IV dose of OP (OP-IV, 2 mg/kg) and oral administration of OP in aqueous solution (OP-S, 10 mg/kg) or ODSF (equivalent to 10 mg/kg OP) to monkeys.

Note: Each value is mean±standard deviation (n=3/group).

Abbreviations: OP, oxaliplatin; IV, intravenous; OP-S, OP in aqueous solution; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol.

Figure 8 In vivo analyses of the inhibitory effects of the following on tumor growth in CT26 tumor-bearing mice: biweekly intravenous administration of 3.3 mg/kg OP [OP-IV (3.3)] and 10 mg/kg OP [OP-IV (10)]; once-daily oral administration of 10 mg/kg OP in aqueous solution (OP-S); once-daily oral administration of ODSF as 2.5 mg/kg OP [ODSF (2.5)]; once-daily oral administration of ODSF as 10 mg/kg OP [ODSF (10)]; and once-daily oral administration of ODSF as 20 mg/kg OP [ODSF (20)], for 21 days. (A) Tumor volumes in mice [^aP< 0.05, ^bP<0.01, ^cP<0.001 compared to the control. ^dP<0.01, ^eP<0.001 compared to OP-IV (3.3). ^fP<0.05, ^gP<0.01 compared to OP-IV (10). ^hP<0.001 compared to OP-S (10). ⁱP<0.001 compared to ODSF (2.5)]. (B) Photographs of tumors isolated from each group on day 21. Scale bar: 10 mm. (C) Tumor weights in CT26 tumor-bearing mice on day 21 [^aP<0.05, ^bP<0.001 compared to the control. ^cP<0.05 compared to OP-IV (10). ^dP<0.05 compared to OP-S (10). ^eP<0.05 compared to ODSF (2.5)]. (D) Changes in mouse body weights during treatment [^aP<0.05, ^bP<0.001 compared to the control. ^cP<0.001 compared to OP-IV (3.3). ^dP<0.01, ^eP<0.001 compared to OP-IV (10). ^fP<0.01, ^gP<0.001 compared to OP-S (10). ^hP<0.001 compared to ODSF (2.5). ⁱP<0.05 compared to ODSF (10)]. (E) Representative cross-sectional images of isolated tumor tissues obtained 21 days after various treatments, stained as follows: With anti-CD31 antibody, indicating microvessels (brown); anti-proliferating cell nuclear antigen (PCNA) antibody, indicating proliferating cells (brown); and via fluorescent terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL), indicating apoptosis (green fluorescence). Scale bars: 50 μm for anti-CD31 antibody and anti-PCNA antibody staining; 20 μm for TUNEL staining.

Notes: Statistics: one-way ANOVA followed by the Tukey multiple comparisons test. Each value is mean±standard error of the mean (n=14/group).

Abbreviations: OP, oxaliplatin; IV, intravenous; OP-S, OP in aqueous solution; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; P188, poloxamer 188; ODSF, solid oral formulation of OP/DLM with P188 and Labrasol.

Figure 9 Antitumor effects of ODSF at various doses in HCT116 tumor-bearing mice after biweekly intravenous administration of 3.3 mg/kg OP [OP-IV (3.3)] and 10 mg/kg OP [OP-IV (10)]; once-daily oral administration of 10 mg/kg OP in aqueous solution (OP-S); once-daily oral administration of ODSF as 2.5 mg/kg OP [ODSF (2.5)]; once-daily oral administration of ODSF as 10 mg/kg OP [ODSF (10)]; and once-daily oral administration of ODSF as 20 mg/kg OP [ODSF (20)], for 21 days. (A) Tumor volumes in different groups [^aP<0.05, ^bP<0.01, ^cP<0.001 compared to the control. ^dP<0.01 compared to OP-IV (3.3). ^eP<0.05 compared to ODSF (2.5)]. (B) Photographs of tumors isolated from each group on day 21. Scale bar: 10 mm. (C) Isolated tumor weights in HCT116 tumor-bearing mice on day 21 [^aP<0.01, ^bP<0.001 compared to the control. ^cP<0.05, ^dP<0.01 compared to OP-IV (3.3). ^eP<0.05 compared to ODSF (2.5)]. (D) Changes in mouse body weight during treatment [^aP<0.001 compared to the control. ^bP<0.001 compared to OP-IV (3.3). ^cP<0.001 compared to OP-IV (10). ^dP<0.001 compared to OP-S (10). ^eP<0.01, ^fP<0.001 compared to ODSF (2.5). ^hP<0.001 compared to ODSF (10)]. (E) Representative cross-sectional images of isolated tumor tissues obtained 21 days after various treatments, stained as follows: With anti-CD31 antibody, indicating microvessels (brown); with anti-proliferating cell nuclear antigen (PCNA) antibody, indicating proliferating cells (brown); and via fluorescent terminal deoxynucleotidyl transferase-mediated dUPT nick end labeling (TUNEL), indicating apoptosis (green fluorescence). Scale bars: 50 μm for anti-CD31 antibody and anti-PCNA antibody staining, and 20 μm for TUNEL staining.

Notes: Statistics: one-way ANOVA followed by the Tukey multiple comparisons test. Each value is mean±standard error of the mean (n=13/group).

Abbreviations: OP, oxaliplatin; IV, intravenous; OP-S, OP in aqueous solution; DLM, N^α-deoxycholyl-l-lysyl-methylester; OP/DLM, ion-pairing complex between OP and DLM; ODSF, solid oral formulation of OP/DLM.