Pharmacokinetic Assessment of Absorptive Interaction of Oral Etoposide and Morphine in Rats

Abstract

Etoposide and morphine are well known P-glycoprotein (P-gp) substrates. The pharmacokinetic effect of morphine on plasma etoposide concentration after the oral concomitant use of etoposide and morphine in rats was assessed using a population analysis approach. A P-gp substrate quinidine and the anticholinergic drug propantheline were also administered with etoposide to compare with the effects of morphine. Plasma etoposide concentration after oral administration was well described using a linear 2-compartment open model with first-order kinetic absorption from the intestine, although a flip-flop phenomenon was shown. After administration of etoposide with morphine, an increased concentration and extended time at maximum concentration were observed compared with the administration of etoposide alone. However, coadministered quinidine significantly increased the maximum concentration without changing the time of the peak concentration of etoposide. Coadministered propantheline significantly extended the time at maximum concentration, although no changes in the peak concentration of etoposide were observed. These coadministered drugs resulted in different pharmacokinetic parameters of etoposide and acted as a significant covariate. That is, morphine and quinidine significantly increased the bioavailability of etoposide believed to be due to competitive P-gp inhibition in the intestine. In contrast, morphine and propantheline decreased the absorption rate constant and were associated with the suppression of enterokinesis. These results indicate that it is necessary to understand the effects on P-gp as well as have information on other effects on the gastrointestinal tract, such as enterokinesis suppression, and to appropriately assess the pharmacokinetic interactions of the combined oral use of P-gp substrate drugs.

Etoposide (ETP) is an effective agent in cancer therapy acting as a specific inhibitor of DNA topoisomerase II. Its cytotoxic action is reversible, saturable at high concentrations, and cell cycle-specific. For small cell lung cancer and malignant lymphoma, ETP is given orally for 5 consecutive days followed by a 3-week rest period. ETP glucuronide is a major metabolite of ETP,¹⁾ and CYP 3A is also associated with ETP metabolism.^2,3) ETP has also been reported to be a substrate of P-glycoprotein (P-gp).²⁾ P-gp is widely understood to pump toxicants out of the body and is widely located in the plasma membranes of cells in the intestine, liver, renal tube, and other tissues.⁴⁾ The P-gp expression in the intestine suppresses the oral bioavailability of ETP, though the species difference of contribution of P-gp on the intestinal uptake was reported.⁵⁾ Inhibitory effects by drugs coadministered with ETP on the P-gp efflux and also on CYP3A metabolism in the intestine resulted in increased plasma ETP concentrations after oral administration.⁶⁾

Morphine (MOR) is an opioid analgesic and is used orally for the treatment of moderate to severe cancer pain. Therefore, it is possible that a patient receiving ETP treatment will be treated with MOR simultaneously. MOR is also reported to be a substrate of P-gp.⁷⁾ Although a competitive interaction on the absorptive processes could be predicted when these drugs are coadministered orally, there are few investigations regarding the variation in pharmacokinetic profiles of ETP and MOR because of their interactions at P-gp in the intestine.

This study was designed to reveal the effects on ETP pharmacokinetics after oral administration of MOR in rats. The effects of MOR on plasma ETP concentrations were compared with those of quinidine (QIN), a potent P-gp substrate,⁸⁾ and propantheline (PPT), an anticholinergic drug, using population pharmacokinetic analysis.

MATERIALS AND METHODS

Chemicals

ETP, QIN sulfate, and PPT bromide were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). MOR sulfate hydrate was kindly supplied by Shionogi & Co., Ltd. (Osaka, Japan). Saline was obtained from Otsuka Pharmaceuticals (Tokyo, Japan). All other reagents and solvents were commercial products of reagent grade.

Animal Surgery

Fifty-six male Wistar rats (Japan SLC Inc., Shizuoka, Japan, 240–370 g) were used (n=40 and 16 for the oral or intravenous administration, respectively). Rats were housed in constant environmental facilities (temperature, 24±1°C; humidity, 55±10%), exposed to 12 : 12 h light-dark cycles (06 : 00 h/18 : 00 h) for more than 1 week, and allowed free access to a standard diet and tap water. On the day before the experiment, rats were lightly anesthetized with ethyl ether and surgically implanted with a SP10 catheter (Natsume Seisakusho, Tokyo, Japan) connected to a PE50 (Clay Adams, Parsippany, NJ, U.S.A.) catheter in the femoral vein for drug intravenous administration. Rats were also implanted with a Phicon tube (Fuji Systems, Tokyo, Japan) connected to a PE50 catheter (Clay Adams) in the jugular vein for blood sampling. Both catheters were externalized through the back in the neck region and secured. For the oral administration study, a stomach catheter was inserted temporarily into the stomach. Unless otherwise specified, all animal experiments were performed under non-restraining and non-anesthetic conditions and in a fasting state. These animal experiments were approved by the Animal Experimentation Committee of the Osaka University of Pharmaceutical Sciences.

Animal Experiments

ETP was dissolved in 54% polyethylene-glycol 400 (PEG) solution. MOR and QIN were dissolved in the PEG solution with ETP. PPT was dissolved in saline. ETP (20–40 mg/kg) was orally administered with or without MOR (30 mg/kg) and QIN (4 mg/kg). PPT (6 mg/kg) was administered intraperitoneally 2 h before the oral administration of ETP. Blood samples were withdrawn from the jugular vein at designated postdose intervals. The blood samples were transferred into tubes containing heparin and then centrifuged (6000×g for 3 min). The isolated plasma was stored at −20°C until analysis. Plasma ETP concentrations were determined using an HPLC method. Briefly, 100 µL of plasma were extracted with a mixture of methanol/chloroform. The organic phase was dried under nitrogen and reconstituted with 100 µL of mobile phase. A 50 µL sample was injected into the HPLC system (Shimadzu, Kyoto, Japan) in which electrochemical detection (ECD; 850 mV; Eicom Co., Kyoto, Japan) was used in the oral administration study and UV detection (λ=278 nm; Shimadzu) was used in the intravenous (i.v.) administration study. The mobile phase consisted of 25 mmol/L citrate buffer (pH 2.4) and acetonitrile (7 : 3, v/v) for the ECD method, or water and acetonitrile (7 : 3, v/v) for the UV method. The quantitation limits were 0.01 and 0.1 µg/mL, respectively. For the intravenous administration study, ETP (20 and 40 mg/kg) was injected through the femoral vein. QIN (4 mg/kg) was orally administered with ETP. MOR (0.2 µmol/kg/min) was intravenously infused 2 h before and after the ETP injection. ETP (30 mg/kg) was orally administered to one animal (body weight 300 g) with both QIN (4 mg/kg) and PPT (6 mg/kg), and plasma concentrations were determined to comparing with the model-simulated values. In the preliminary experiment, plasma MOR concentration reached the steady state of approximately 2 µmol/L within 2 h after infusion and was almost equal to the maximum plasma MOR concentration after oral administration of 30 mg/kg MOR. PPT (6 mg/kg) was intraperitoneally administered 2 h before the ETP injection. Plasma MOR concentration was determined using an LC/MS method.

Pharmacokinetic Model Analysis

The concentration-time data of ETP were analyzed by a nonlinear mixed effect model method using NONMEM software (version VI) to evaluate the pharmacokinetic interaction with QIN, MOR, and PPT. The first-order method was employed throughout the analysis. Both one-compartment and two-compartment open models were investigated using the Akaike information criterion (AIC).⁹⁾ Interindividual variability for pharmacokinetic parameters and residual variability were estimated using an exponential error model. Effects of the coadministered drugs on pharmacokinetic parameters of ETP were evaluated as covariates using the base model. Model comparisons were based on objective function values (OFVs) in NONMEM using the likelihood ratio test. The significance level was set at p<0.05, which corresponds to a reduction of 3.84 in OFV to discriminate between the two nested structural models after the inclusion of one additional parameter. Goodness-of-fit (GOF) and visual predictive check (VPC) plots were performed to evaluate the adequacy of the final model. VPC plots were generated by simulating 1000 individual profiles for ETP 30 mg/kg oral administration using the estimated parameters. The 95% predictive interval and median were determined to assess the bias and predictive performance of modeling. The 95% predictive interval was constructed by 2.5th and 97.5th percentiles of simulated values for each sampling point. Plasma ETP concentrations after concomitant oral use of ETP, QIN, and PPT were predicted using the fixed parameters for QIN and PPT in the final model.

RESULTS

Pharmacokinetic Profiles after Coadministration of ETP with QIN, MOR, PPT

The pharmacokinetics of ETP after oral administration of ETP is shown in Fig. 1. A dose dependency was observed in the investigated dose range of 20–40 mg/kg (Fig. 1A). The time at maximum concentration was approximately 30 min after administration, and the concentration decreased to less than 0.1 µg/mL at 180–270 min after administration. A nonlinear kinetics was not shown, because the dose-normalized plasma concentration–time curves were similar in the doses range (data not shown). Thus, the ETP 30 mg/kg was used in the following coadministration studies. Figure 1B shows the effect of QIN coadministration in the time courses of plasma ETP concentration. Compared with the control, the peak concentration significantly increased. However, no obvious change in the time at maximum concentration occurred. Figure 1C shows the effect of MOR coadministration in the time courses of plasma ETP concentration. Significantly increased concentration and extended time at maximum concentration were observed compared with the control. PPT slightly extended the time at maximum concentration without affecting the peak concentration (Fig. 1D). A large individual variability of plasma ETP concentrations was observed in all oral treatments, especially MOR coadministration. The pharmacokinetics of ETP after intravenous bolus administration of ETP is shown in Fig. 2. A two-exponential decline of ETP level was elicited, and the concentrations increased in a dose-dependent manner (Fig. 2A). No obvious effects of the coadministered drugs in the concentration–time profiles of ETP were observed (Figs. 2B–D).

Fig. 1. Time Courses of Plasma ETP Concentration after Oral Administration of ETP in Rats

A: Administration of ETP alone at 20 mg/kg (∆), 30 mg/kg (○) and 40 mg/kg (□) (n=18); B: Effect of QIN (4 mg/kg, p.o., ▲) coadministration (n=9); C: Effect of MOR (30 mg/kg, p.o., ●) coadministration (n=6); D: Effect of PPT (4 mg/kg, i.p., ■) coadministration (n=7). The points represent data for an individual animal.

Fig. 2. Time Courses of Plasma ETP Concentration after Intravenous Bolus Administration of ETP in Rats

A: Administration of ETP alone at 20 mg/kg (∆) and 40 mg/kg (□) (n=6); B: Effect of QIN (4 mg/kg, p.o., ▲) coadministration (n=4); C: Effect of MOR (0.2 µmol/kg/min, i.v. infusion, ●) coadministration (n=3); D: Effect of PPT (4 mg/kg, i.p., ■) coadministration (n=3). The points represent data for an individual animal.

Population Pharmacokinetic Analysis of the Interaction

Population pharmacokinetic analysis was performed using the individual plasma ETP concentrations and the concomitant treatments as covariates. Using data of the oral and intravenous control studies, the linear one- and two-compartment models with a first-order kinetic absorption were assessed by the AIC value (−113.437 vs. −210.076). Furthermore, a two-compartment model with an absorptive lag time significantly reduced OFV by 63.221 (p<0.05, the likelihood ratio test). Therefore, a two-compartment model with a first-order kinetic absorption and a lag time was selected as the basic model in subsequent analyses. Figure 3 shows the effects of concomitant administered drugs on absorptive parameters (absorption rate constant, k_a; bioavailability, F) estimated by the empirical Bayesian method in the basic model. Different effects were observed for these drugs, although a large interindividual variability was shown for the QIN and MOR groups compared with the control and PPT groups. The covariate adjustments that were investigated are summarized in Table 1. For QIN and MOR, their concomitant use affected both k_a and F and was statistically significant (Models Q1–4 and M1–4). The effect of PPT coadministration on k_a was also significant (Model P1). Therefore, the full model that adopted the concomitant use of QIN, MOR, and PPT (covariates; WQ, WM and WP) for k_a and F was employed as the final model:

Fig. 3. Correlation between Individual Estimates for Absorptive Parameters and the Treatment

Parameters estimated by Bayes analysis in the basic model. The points represent data for an individual animal.

Table 1. Summary of Analysis for the Absorptive Interactions of ETP and Coadministered Drugs

Model	Tested equation	OFV	ΔOFV	p Value
Base model	ka=θ1	−313.050	—	—
	F=θ6
	lag=θ7
Q1	ka=θ1×θ8WQ	−455.912	142.862	<0.01
Q2	F=θ6×θ8WQ	−372.009	58.959	<0.01
Q3	lag=θ7×θ8WQ	−313.733	0.683	NS
Q4	ka=θ1×θ8WQ	−478.795	165.745	<0.01
	F=θ6×θ9WQ
M1	ka=θ1×θ8WM	−333.793	20.060	<0.01
M2	F=θ6×θ8WM	−383.255	69.522	<0.01
M3	lag=θ7×θ8WM	ND	—	—
M4	ka=θ1×θ8WM	−455.401	142.351	<0.01
	F=θ6×θ9WM
P1	ka=θ1×θ8WP	−364.042	50.992	<0.01
Final model	ka=θ1×θ8WQ×θ10WM×θ12WP	−629.715	—	—
	F=θ6×θ9WQ×θ11WM

ΔOFV: change in objective function value (OFV) compared with the base model; WQ=0 for dosing without QIN and WQ=1 for dosing with QIN; WM=0 for dosing without MOR and WM=1 for dosing with MOR; WP=0 for dosing without PPT and WP=1 for dosing with PPT; NS: not significant; ND: not determined.

  

  

The estimated population parameters are listed in Table 2. As shown by the GOF plots (Fig. 4) and VPC plots (Fig. 5), the final model adequately described the pharmacokinetic profiles of ETP. Using the final model, Fig. 6 shows the simulated plasma ETP concentration-time profile after oral administration of ETP (30 mg/kg; body weight, 300 g) with both QIN (4 mg/kg) and PPT (6 mg/kg). The simulated profile well explained the observed data as shown by the plots. This profile was similar to MOR coadministration, which demonstrated a slightly increased C_max and t_max.

Table 2. Population Parameters of the Final Model

	Estimated values	95% CI	CV (%)
ka (min−1)=θ1×θ8WQ×θ10WM×θ12WP
	θ1=0.0109	(0.00786–0.0139)
	θ8=3.70	(2.48–4.93)
	θ10=0.233	(0.134–0.332)
	θ12=0.561	(0.452–0.670)
ωka			46.0
CL (mL/min)=θ2
	θ2=6.16	(5.32–7.00)
ωCL			18.2
V1 (mL)=θ3
	θ3=78.4	(63.7–93.1)
Q (mL/min)=θ4
	θ4=5.12	(3.41–6.83)
V2 (mL)=θ5
	θ5=151	(123–179)
F=θ6×θ9WQ×θ11WM
	θ6=0.0390	(0.0299–0.0481)
	θ9=1.25	(0.923–1.58)
	θ11=5.16	(1.91–8.41)
ωF			31.9
lag (min)=θ7
	θ7=3.30	(0.556–6.04)
Residual variability			31.9

CI: confidence interval; CV: coefficient of variation for interindividual variability or residual variability: k_a; absorption first-order rate constant; CL; total elimination clearance; Q: clearance for the peripheral compartment: V₁ and V₂: distribution volumes of the central and peripheral compartments: F: bioavailability; lag: lag time for intestinal absorption: ω_ka, ω_CL and ω_F: interindividual variability for k_a, CL and F.

Fig. 4. Goodness-of-Fit Plots of the Final Model of ETP after Oral Administration

Plot of observed versus population (PRED) and individual (IPRED) predicted ETP concentrations (A and B) and weighted residues (WRES) versus population predicted ETP concentration (PRED) and time (C and D).

Fig. 5. Visual Predictive Check of the Final Model for Oral ETP Administration Alone (A) and Coadministration with QIN (B), MOR (C) and PPT (D)

Points represent the observed data. The solid line shows the population-predicted median profile, and the dashed lines show the 95% prediction intervals.

Fig. 6. Comparison of the Simulated Plasma ETP Concentration–Time Course and the Observed Data after Oral ETP Administration with Both QIN and PPT

The solid and dashed lines are the predicted population median and the 95% prediction intervals, respectively. Plots represent the observed data.

DISCUSSION

We aimed to quantitatively characterize pharmacokinetic interactions of plasma ETP concentration after oral administration of ETP with MOR. QIN and PPT were also coadministered with ETP for comparison with the effect of MOR. ETP is mainly eliminated by hepatic metabolism in rodents.¹⁰⁾ The variability in total clearance of ETP may be principally related with the hepatic intrinsic clearance and plasma protein binding rather than the hepatic blood flow because the total clearance of 5.08 mL/min was estimated in the preliminary experiment compared with a hepatic blood flow of 14.7 mL/min.¹¹⁾ ETP is an acidic drug and mainly binds to albumin in blood. In contrast, QIN and MOR are basic drugs and mainly bind to α₁-acid glycoprotein. Orally coadministered QIN and MOR with ETP caused an increase in plasma ETP concentration. However, intravenously coadministration did not affect the ETP disposition (Figs. 1, 2). These results indicate that orally administered QIN and MOR affected the absorptive pathway of ETP in the intestinal tract, but not its hepatic metabolism and elimination. Because these drugs are a P-gp substrate, the enhanced absorption is most likely due to competitive inhibition of the P-gp-mediated elimination process. PPT and MOR maintained ETP concentration in plasma after oral administration of ETP compared with QIN (Fig. 1). PPT slows both gastric emptying time and small intestinal transit. However, the relevancy for P-gp interaction has not been reported. The combined use of ETP and PPT delayed the time of maximal ETP concentration without a significant increase in the area under the concentration-time curve of ETP in patients.¹²⁾ MOR was reported to prolong the gastric emptying time significantly at a dose that can cause analgesia.¹³⁾ In our study, analgesic effects were observed during the 3-h intravenous MOR infusion (30 mg/kg) in rats (data not shown). Significant analgesic effects were also shown after oral coadministration of MOR (30 mg/kg) and ETP (10 mg/kg) in mice.¹⁴⁾ ETP was reported to be absorbed from both the upper and lower intestine.¹⁵⁾ Therefore, prolonged ETP concentration–time course by PPT and MOR might be related to the suppression of enterokinesis. These hypotheses were verified using the population pharmacokinetic approach.

The population pharmacokinetic approach revealed the low oral bioavailability (3.90%) of ETP from PEG400 solution in the control study (Table 2). The absolute bioavailability from PEG8000 solution was approximately 10% after oral administration of 20 mg/kg ETP in rats.¹⁶⁾ However, a higher bioavailability of 44% was shown for the oral administration of ETP in patients despite dosing using capsules.^12,17) These different bioavailabilities may be caused by a variation in the amount and variety of transporters for the absorption and elimination of ETP in the intestine. Although ETP fundamentally have a nonlinear kinetics in terms of the absorption process, it was not obvious in the investigated dose range. Flip-flop kinetics were observed to be common in children with lymphoblastic leukemia,¹⁸⁾ rats and monkeys.⁵⁾ In our study, the estimated k_a value (0.0109 min⁻¹) was obviously smaller than the first-order elimination rate constant k₁₀ (0.0781 min⁻¹) calculated from CL/V₁ after administration of ETP alone (Table 2). Flip-flop kinetics were also observed even in conditions of an increased absorption rate by QIN coadministration (k_a: 0.0403 min⁻¹). In the general pharmacokinetics with a rapid k_a and a slow k₁₀ (k_a >> k₁₀), a change in k_a could not lead to large significant variations in drug plasma concentration profiles. A large variation in the plasma concentration profile might also be observed in children administered ETP with MOR orally. Furthermore, the quantitative analysis characterized the interaction by coadministered drugs as shown in Table 2 seems reasonable. QIN accelerated the absorptive rate (θ8) and increased bioavailability (θ9). This is sufficient to describe the inhibitory effect of QIN on the elimination mediated by P-gp in the intestinal tract. In contrast, the suppressive effect of PPT on the absorptive rate (θ12) indicates the suppression of enterokinesis without inhibition of P-gp. However, MOR decreased the absorptive rate (θ10) and increased bioavailability (θ11). This could reasonably explain the possibility of inhibitory effects on P-gp elimination in the intestinal tract as well as the suppressive effects on enterokinesis. Therefore, we simulated the MOR-like effects on plasma ETP concentration using the population parameter estimates for QIN and PPT from the final model. The predicted plasma ETP concentration profile well explained the observed values and showed similar properties after ETP administration in combination with MOR (Fig. 6). This simulation indicates that suppression of enterokinesis masks the effects of P-gp inhibition. That is, suppressed enterokinesis can lead to the inhibition of increases in plasma ETP concentration after oral administration, even if the coadministered drug significantly inhibits the activity of P-gp in the intestine. To appropriately assess the pharmacokinetic interactions of the combined oral use of P-gp substrate drugs, it is necessary to not only measure apparent drug concentrations in plasma and evaluate in vitro data for P-gp inhibition but to also understand information for other effects on the gastrointestinal tract, such as the suppression of enterokinesis. If the substrate shows flip-flop pharmacokinetics, the contribution of enterokinesis would be higher.

In conclusion, the effect of MOR on plasma ETP concentrations after oral coadministration of ETP and MOR was evaluated and compared with QIN or PPT coadministration in rats. The effect was explained by an increase in the bioavailability and decrease in the absorptive rate of ETP from the intestinal tract. These were characterized by two different specificities of MOR; one is the substrate of P-gp, such as QIN, and the other is the suppression of enterokinesis, such as PPT. The in vivo inhibitory effect of MOR on the P-gp elimination system in the intestine might be more important contrary to expectations based on the increased plasma ETP concentration alone.

Acknowledgment

This study was supported in part by a “High-Tech Research Center” Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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