Effect of Metabolic Inhibitors on the Hepatic Disposition of 5-Fluorouracil after Application to the Rat Liver Surface

Abstract

We evaluated the effects of 5-fluorouracil (5-FU) metabolic inhibitors, gimeracil or uridine, on the hepatic disposition of 5-FU after application to the liver surface in rats, aiming to enhance the availability of 5-FU in the liver. 5-FU solution with or without metabolic inhibitors was applied to the rat liver surface using a cylindrical diffusion cell. The liver, blood and the remaining solution in the diffusion cell were collected at specified times, and assayed for 5-FU content. 5-FU absorption properties were not altered by addition of gimeracil and uridine. The 5-FU concentration in the diffusion cell attachment site of the rat liver (site 1) at 0.1–0.4 M ratios of gimeracil to 5-FU was significantly higher than that of the control. On the contrary, the addition of uridine did not increase the 5-FU concentration at site 1. At a 0.1 M ratio of gimeracil to 5-FU, the maximum 5-FU plasma concentration was the lowest, and the area under the 5-FU concentration–time curve at site 1 was 3.4 times greater than that of the control. We demonstrated that applying 5-FU with gimeracil to the rat liver surface could increase the availability of 5-FU in the liver.

Hepatocellular carcinoma (HCC) is the second most frequent cause of cancer-related death worldwide.¹⁾ In many cases, chronic hepatitis and hepatic cirrhosis trigger the development of HCC.²⁾ Even radical surgery, a potentially curative treatment for HCC, is limited by cancer progression and hepatic functional reserve.³⁾ In addition, HCC frequently recurs after radical surgery in the residual liver tissue because of progressive disease.⁴⁾ Although chemotherapy has been applied to treat the advanced or recurrent HCC, most of anticancer drugs cannot exert the desired effect on HCC because of high chemoresistance rate.^5,6)

Local therapies such as percutaneous ethanol injection,^7,8) intratumoral injection^9–11) and intra-arterial infusion¹²⁾ have been performed to enhance the therapeutic effect of anticancer drugs on HCC. However, optimal delivery of anticancer drugs to HCC lesion is difficult through these routes because of distribution in the normal surrounding tissue or rapid efflux into the systemic circulation from the injection site.

Therefore, we have proposed that the liver surface is a new drug administration route, and investigated the absorption and disposition characteristics of several marker compounds after direct application to the rat liver surface.^13–18) To use this route as a local HCC therapy, we showed that 5-fluorouracil (5-FU), a pyrimidine anticancer drug, was site-selectively and continuously delivered in the liver after application to the liver surface in rats.^19,20) We have also investigated other organs as an administration route of 5-FU.^21,22) Although viscous additives to 5-FU solution improved site-selective accumulation of 5-FU,²³⁾ the increase in 5-FU concentration at the application site was not large enough.

5-FU has been widely used in chemotherapy regimens against a variety of cancers such as liver,²⁴⁾ colon,²⁵⁾ stomach²⁶⁾ and pancreatic cancer.²⁷⁾ However, approximately 85% of administered 5-FU is catabolized by dihydropyrimidine dehydrogenase (DPD).²⁸⁾ 5-FU catabolism must be suppressed to enhance its anticancer effect.

DPD inhibitors have been used as adjuvants to enhance the anticancer effect of 5-FU. TS-1^® and UFT^® are oral agents that combine tegafur with the DPD inhibitors, gimeracil and uracil, respectively. TS-1^® is combined at a 0.4 molar ratio of gimeracil to tegaful. UFT^® is combined at a 4 molar ratio of uracil to tegafur. These agents could increase the availability of 5-FU by inhibiting DPD activity, leading to an improved anticancer effect by 5-FU.^29–31) Uridine was also reported to decrease the catabolism of 5-FU by inhibiting DPD.³²⁾

For liver surface application of 5-FU, DPD inhibitors are expected to enhance its concentration in the liver and keep the application site concentration high. In addition, 5-FU in combination with the DPD inhibitor would be useful to develop a pharmaceutical formulation for application of 5-FU to the liver surface to treat HCC.

In the present study, we chose gimeracil and uridine as DPD inhibitors. We examined the absorption and distribution in the liver after 5-FU application to the rat liver surface using various combination ratios of the DPD inhibitor. After determining the optimal combination ratio of the DPD inhibitor, we investigated the effect of the DPD inhibitor on the availability of 5-FU in the liver after application to the rat liver surface.

MATERIALS AND METHODS

Chemicals

5-FU was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Gimeracil and uridine were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other chemicals were of reagent grade.

Animal Experiments

All animal experiments in this study conformed to the Guidelines for Animal Experimentation at Nagasaki University (approval number: 1006100863-2, approval date: May 28, 2010). Male Wistar rats (230–270 g) were anaesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally (i.p.)). The left femoral artery was cannulated using polyethylene tubes. A cylindrical diffusion cell (i.d. 9 mm, 0.64 cm²) was attached to the liver surface (left lateral lobe) using Aron Alpha (Daiichisankyo Co., Ltd., Tokyo, Japan) after laparotomy. 5-FU (0.2 mL of 10 mg/mL solution) in an isotonic phosphate buffer (pH 7.4) in the presence or absence of gimeracil (0.05, 0.1, 0.2 and 0.4 molar ratio to 5-FU) or uridine (1.0, 2.0, 3.0 and 4.0 molar ratio to 5-FU) was added into the diffusion cell. The applied dose of 5-FU was set according to our previous study.²³⁾ A piece of aluminum foil was placed on the top of the diffusion cell to prevent evaporation.

To determine plasma 5-FU concentrations, blood samples from the left femoral artery were collected at selected times (15, 60, 120 and 180 min) and centrifuged. The solution in the diffusion cell was withdrawn at the pre-determined times to calculate the remaining amount of 5-FU in the diffusion cell. The liver was excised following perfusion with saline from the portal vein. The excised liver was divided into three sites; the region under the diffusion cell attachment site (site 1); the applied lobe except for site 1 (site 2); and the non-applied lobes (site 3). Each liver sample was weighed and homogenized using two-fold volumes of their weight in isotonic phosphate buffer (pH 7.4).

Analytical Methods

5-FU concentrations in the liver homogenate, the plasma and the solution remaining in the diffusion cell were measured using previously reported methods^33,34) with some modifications. Briefly, 300 µL of the liver homogenate, the plasma sample, or the solution remaining in the diffusion cell were mixed with a solution containing 150 µL of 20 µg/mL 5-bromouracil dissolved in isotonic phosphate buffer (pH 7.4) as an internal standard, with 500 µL of 20% anhydrous sodium sulfate, and 100 µL of 1 M sodium acetate buffer (pH 4.8). The mixtures were shaken for 10 min after addition of 4 mL of ethyl acetate and centrifuged at 1630×g for 10 min at 4°C. The organic layers (3 mL) were collected and 4 mL of fresh ethyl acetate was added to the residue. The mixtures were shaken for 10 min and centrifuged at 1630×g for 10 min at 4°C. The organic layers (4 mL) were collected and 7 mL of the mixed organic layers were evaporated. The extracted residues were dissolved in 500 µL of distilled water and washed twice with 1 mL of hexane. Samples (100 µL) were injected onto the HPLC column (Cosmosil-packed column 5C₁₈-PAQ, 4.6 mm i.d.×150 mm, Nacalai Tesque, Kyoto, Japan). A HPLC system with a variable-wavelength UV detector (SPD-10A, Shimadzu Co., Ltd., Kyoto, Japan) was used in the reverse-phase mode. The detector wavelength, flow rate, and column temperature were set at 266 nm, 0.5 mL/min, and 25°C, respectively. The mobile phase consisted of 10 mM sodium acetate buffer (pH 4.7). The absorption ratio of 5-FU after application to the liver surface in rats was calculated from the remaining amount of 5-FU in the diffusion cell.

5-FU Area under the Curve (AUC) of the Plasma or Liver Concentration–Time Profile

The 5-FU plasma and liver concentration–time profile up to 180 and 360 min after application to the rat liver surface was analyzed according to the following Eqs. 1 and 2:   

(1)

  

(2)

where t is the time (min), C_p is the plasma concentration of 5-FU (µg/mL) and AUC_p, is the area under the plasma concentration–time curve (µg×min/mL plasma). The AUC_p was calculated using the linear trapezoidal formula. C is the liver concentration of 5-FU (µg/g), and AUC, the area under the liver concentration–time curve (µg×min/g liver), was also calculated using the linear trapezoidal formula.

Statistical Analysis

Statistical comparisons were performed using Dunnett’s test after an analysis of variance for multiple groups. p<0.05 compared with the control group (in the absence of DPD inhibitor) was considered to be significant. All values were expressed as the mean value±standard error (S.E.) of at least independent different three experiments.

RESULTS

Effect of DPD Inhibitors on the Absorption of 5-FU after Application to the Liver Surface in Rats

Figure 1 shows the absorption ratio of 5-FU at 180 min after application to the rat liver surface under the various molar ratios of DPD inhibitors to 5-FU. The absorption ratios of 5-FU from the rat liver surface were approximately 70% under each condition.

Fig. 1. Absorption Ratio of 5-FU at 180 min after Application to the Rat Liver Surface at the Various Molar Ratios of Gimeracil (A) or Uridine (B) to 5-FU

Each bar represents the mean+S.E. of at least three experiments. Significantly different from the result at control: * p<0.05.

Effect of DPD Inhibitors on the Distribution of 5-FU at the Different Hepatic Sites of the Liver after Application to the Liver Surface in Rats

Figure 2 shows the liver concentration of 5-FU at 180 min using various molar ratios of DPD inhibitors to 5-FU after application to the liver surface in rats. 5-FU concentrations at site 1 using a 0.1–0.4 molar ratio of gimeracil to 5-FU were significantly increased approximately 4-fold compared with the control (Fig. 2A). The concentration ratios at site 1 using a 0.1 and 0.2 molar ratio of gimeracil to 5-FU and the control were 4.2 in both cases: the highest for each condition. However, combining uridine did not enhance 5-FU concentration at site 1 (Fig. 2B).

Fig. 2. Liver Concentration of 5-FU at 180 min after Application to the Rat Liver Surface at the Various Molar Ratios of Gimeracil (A) or Uridine (B) to 5-FU in Rats

Site 1 (closed bar), site 2 (gray bar) and site 3 (open bar). Each bar represents the mean+S.E. of at least three experiments. Significantly different from the result at site 1 of control: * p<0.05, ** p<0.01, *** p<0.001, at site 2 of control: ^† p<0.05, ^††† p<0.001, at site 3 of control: ^### p<0.001. N.D.: not detected.

Plasma Concentration Profiles of 5-FU after Application to the Rat Liver Surface in the Presence of Gimeracil

Figure 3 illustrates the plasma concentration profiles of 5-FU after application to the rat liver surface under various molar ratios of gimeracil. The maximum 5-FU plasma concentration was increased based on the molar ratio of gimeracil to 5-FU. However, the plasma concentration profiles of 5-FU were not altered by combining uridine, even in the presence of a 4.0 molar ratio to 5-FU (data not shown).

Fig. 3. 5-FU Plasma Concentration Profiles after Application to the Rat Liver Surface at the Various Molar Ratios of Gimeracil to 5-FU

Key: Molar ratio of gimeracil to 5-FU is 0 (control, ●), 0.05 (□), 0.1 (○), 0.2 (△) and 0.4 (◇). Each point represents the mean±S.E. of at least three experiments.

The AUC of the plasma concentration profile up to 180 min after administration of 5-FU (AUC_p) at control, 0.05 0.1, 0.2 and 0.4 molar ratio of gimeracil to 5-FU was calculated to be 137.5, 216.6, 346.7, 376.5 and 567.7 (µg×min/mL plasma), respectively. The AUC_p at a 0.1 molar ratio of gimeracil to 5-FU was the lowest among the 0.1–0.4 molar ratios of gimeracil to 5-FU.

Table 1 summarizes the concentration ratios of 5-FU at each site and plasma under the various molar ratios of gimeracil to 5-FU. These concentration ratios at a 0.1 molar ratio of gimeracil to 5-FU were the highest among the 0.1–0.4 molar ratios. In contrast, these concentration ratios could not be calculated in the presence of uridine, because the concentrations of 5-FU at sites 2 and 3 were below the limit of detection (data not shown).

Table 1. Ratio of 5-FU Liver Concentration at 180 min at Each Site and in the Plasma after Application to the Rat Liver Surface in the Presence or Absence of Gimeracil

	Molar ratio of gimeracil to 5-FU
	Control	0.05	0.1	0.2	0.4
Site 1/site 2	13.2	3.8	3.8	3.4	1.9
Site 1/site 3	10.4	3.8	3.5	3.5	1.9
Site 1/plasma	3.5	3.5	3.1	3.1	1.8

The distribution in the rat liver and plasma concentration profiles of 5-FU suggested that the optimal ratio of the DPD inhibitor was a 0.1 molar ratio of gimeracil to 5-FU. We then examined the effect of a 0.1 molar ratio of gimeracil on the absorption and hepatic disposition of 5-FU after application to the rat liver surface.

5-FU Absorption Profiles from the Rat Liver Surface at a 0.1 Molar Ratio of Gimeracil

Figure 4 shows a semi-log plot of the remaining amount of 5-FU in the diffusion cells after application to the rat liver surface in the presence or absence of gimeracil. The semi-log plots show straight lines for both conditions. The first-order absorption rate constant, k_a, of 5-FU from the rat liver surface in the absence or presence of a 0.1 molar ratio of gimeracil was calculated to be 5.7×10⁻³ and 5.0×10⁻³ min⁻¹, respectively.

Fig. 4. Semi-log Plot of Remaining Amount of 5-FU in the Diffusion Cell after Application to the Rat Liver Surface in the Presence or Absence of Gimeracil

Key: Control (●), 0.1 molar ratio of gimeracil to 5-FU (○). Each point represents the mean±S.E. of at least three experiments.

5-FU Liver Concentration Profiles after Application to the Rat Liver Surface at a 0.1 Molar Ratio of Gimeracil

Figure 5 shows the 5-FU liver concentration after application to the rat liver surface in the presence or absence of a 0.1 molar ratio of gimeracil. In the presence of gimeracil, 5-FU was preferentially distributed at site 1 after 15, 60, 180 and 360 min (Fig. 5B), which was similar to the control (Fig. 5A). The 5-FU concentration at site 1 was immediately increased at 15 min in the presence of gimeracil. Even after 360 min, the 5-FU concentration at site 1 remained approximately 2.5 times higher than the control.

Fig. 5. Liver Concentration of 5-FU after Application to the Rat Liver Surface

(A) Control, (B) 0.1 molar ratio of gimeracil to 5-FU. Key: Site 1 (closed bar), site 2 (gray bar), site 3 (open bar). Each point represents the mean+S.E. of at least three experiments. Significantly different from the result at site 2: ** p<0.01, *** p<0.001 or site 3: ^†† p<0.01, ^††† p<0.001 compared with site 1, respectively.

The AUC of the 5-FU liver concentration profile was calculated as an index of regional availability. Figure 6 shows the AUC of each site until 360 min after application to the rat liver surface. The AUC of the site 1 5-FU concentration–time profile at a 0.1 molar ratio of gimeracil was 3.4 times higher than that of the control. The AUC ratios of site 1 to sites 2 or 3 in the presence of a 0.1 molar ratio of gimeracil to 5-FU were 4.8 and 4.0, respectively, although the corresponding ratios in the absence of gimeracil were 5.6 and 22.5, respectively.

Fig. 6. The 5-FU Area under the Curve up to 360 min at Each Site in the Rat Liver after Application to the Liver Surface at a 0.1 Molar Ratio of Gimeracil or Control

Key: Site 1 (closed bar), site 2 (gray bar), site 3 (open bar).

DISCUSSION

We propose the liver surface as a new drug application site, and we showed that site-selective and continuous delivery of 5-FU could be accomplished by applying it directly to the rat liver surface.^13–20,23) Because 5-FU is mostly catabolized and inactivated by DPD,²⁸⁾ we expected that combining DPD inhibitors enable us to increase 5-FU availability at the administration site. In this study, we chose gimeracil and uridine as candidates for promising DPD inhibitors that can be applied to the rat liver surface. Uracil was not appropriate as a DPD inhibitor to apply together with 5-FU. That was because of difficulty in preparing the solution containing 5-FU and uracil, due to its poor solubility in water. This study aimed to examine the effect of DPD inhibitors on the hepatic disposition of 5-FU after application to the liver surface in rats, and to investigate the optimal combination ratio of the DPD inhibitor referring to TS-1^® and UFT^®.

As a first step, we studied the effect of gimeracil and uridine on the absorption properties of 5-FU after application to the rat liver surface using a cylindrical diffusion cell. The absorption ratio of 5-FU from the rat liver surface at 180 min was approximately 70% in the presence or absence of gimeracil or uridine (Fig. 1). This result suggests that gimeracil and uridine can not influence 5-FU absorption from the rat liver surface.

The 5-FU concentration at site 1 at 180 min was significantly increased by the addition of a 0.1–0.4 molar ratio of gimeracil, but the concentration was not altered by the addition of a 1.0–4.0 molar ratio of uridine (Fig. 2). Gimeracil, a stronger inhibitor of DPD than uracil,³⁵⁾ was considered to inhibit 5-FU catabolism in the rat liver, resulting in an increase in the 5-FU concentration at site 1 compared with the control. In contrast, uridine was supposed to lower DPD activity after metabolism to uracil by uridine phosphorylase in the liver. DPD-inhibition activity of uracil metabolized from uridine did not seem to be strong enough to increase 5-FU concentration at site 1. The absorption ratio of uridine from the rat liver surface at 180 min at a 4.0 molar ratio of uridine to 5-FU was 27% (data not shown). The low absorption ratio of uridine was considered to be a factor to be unchanged 5-FU concentration at site 1 in the presence of uridine. The concentration of 5-FU at site 1 at a 0.1–0.2 molar ratio of gimeracil to 5-FU was the highest (4.2 times higher) compared with control. These results suggest that addition of gimeracil to 5-FU increase the availability of 5-FU in the liver after application to the liver surface.

It has been reported that the systemic side effects caused by 5-FU were related to the plasma concentration.³⁶⁾ To reduce systemic side effects, it is necessary to keep the plasma concentration of 5-FU as low as possible. The maximum plasma concentrations of 5-FU and AUC_p were increased in the presence of gimeracil as compared with the control (Fig. 3). The exposure of 5-FU in systemic circulation was considered to be increased by inhibiting the 5-FU catabolism in the liver. In contrast, the plasma concentration of 5-FU (Fig. 3) and AUC_p at a 0.1 molar ratio of gimeracil to 5-FU was the lowest among the conditions. The concentration ratio of site 1 to site 2, site 3 or plasma at a 0.1 molar ratio of gimeracil to 5-FU was the highest among the 0.1–0.4 molar ratios of gimeracil to 5-FU (Table 1). Although these concentration ratios were lower than control, the site-selectivity of 5-FU was sufficiently high at the 0.1 molar ratio of gimeracil to 5-FU. Taking these into considerations, the 0.1 molar ratio of gimeracil to 5-FU could be an appropriate ratio to increase the availability of 5-FU in the liver after application to the liver surface.

We then focused on the effect of a 0.1 molar ratio of gimeracil to 5-FU on the absorbability and hepatic disposition of 5-FU after application to the rat liver surface. In the presence or absence of gimeracil, 5-FU was absorbed from the rat liver surface according to first-order kinetics (Fig. 4). The 5-FU k_a value in the presence of gimeracil decreased to 86% of the control. This decrease might be explained by a result of the decrease in the 5-FU concentration gradient between the applied solution and site 1.

The concentration of 5-FU at site 1 was increased immediately, and remained higher than the control until 360 min in the presence of a 0.1 molar ratio of gimeracil to 5-FU (Fig. 5). The absorption ratio of 5-FU and gimeracil from the rat liver surface at 180 min at a 0.1 molar ratio of gimeracil to 5-FU were 66% and 44% (data not shown), respectively. These results suggest that the inhibitory effect of gimeracil on 5-FU catabolism occur immediately, and sustain for at least 360 min. The AUC of the tumor 5-FU concentration–over-time profile was reported to correlate with inhibition of cancer cell proliferation.³⁷⁾ Although the AUC ratios of site 1 to sites 2 or 3 in the presence of gimeracil were lower than control, the AUC of the site 1 5-FU concentration profile in the presence of gimeracil was considerably higher than that of the control (Fig. 6). This raises the possibility that the 5-FU anticancer effect would be increased by gimeracil after application to the rat liver surface.

In our previous report, 5-FU was distributed evenly in the liver and eliminated rapidly from the liver after intravenous (i.v.) administration in rats.^19,20) On the other hand, 5-FU was preferentially distributed at site 1, and its concentration was sustained after application to the rat liver surface. AUC_p after application to the rat liver surface was lower than that of i.v. administration. Furthermore, the concentration of 5-FU at site 1, site 2 and site 3 at a 0.1 molar ratio of gimeracil to 5-FU were 2.9, 3.3 and 3.6 µg/g liver, respectively, at 180 min after its i.v. administration. AUC_p (643.3 µg×min/mL plasma) after i.v. administration was approximately twice higher than that of application to the rat liver surface. These results suggest that the 5-FU availability in the liver is enhanced with low exposure in the systemic circulation by combining gimeracil to 5-FU after application to the liver surface, compared to i.v. adminisitration.

Although the present study suggests that administering 5-FU with gimeracil directly to the liver surface can be an effective treatment for patients with HCC, clinical use of the cylindrical diffusion cell that was used to administer the 5-FU mixture is not feasible. In this study, we used the cylindrical diffusion cell to limit the 5-FU application area in the presence or absence of gimeracil or uridine, to closely examine their effect on 5-FU absorption and hepatic disposition. Therefore, pharmaceutical formulations such as sheets or hydrogel formulations are required for clinical use. Our results suggested that gimeracil is a promising candidate to add to formulations containing 5-FU for application to the liver surface in clinical for treatment of patients with liver cancer. These results are expected to be beneficial for developing formulations for clinical application of 5-FU to the liver surface.

CONCLUSION

We found that administration of 5-FU with gimeracil onto the rat liver surface could increase the availability of 5-FU in the liver. We showed that combining gimeracil and 5-FU at a 0.1 molar ratio could be the optimal ratio to apply to the rat liver surface.

Acknowledgments

This work was supported by JSPS KAKENHI Grant number 21590042. We wish to thank Yosuke Kariya and Haruna Hirata for their skilled technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J. Clin., 2011, 69–90 (2011).
• 2) Yang D, Hanna DL, Usher J, LoCoco J, Chaudhari P, Lenz HJ, Setiawan VW, El-Khoueiry A. Impact of sex on the survival of patients with hepatocellular carcinoma: a surveillance, epidemiology, and end results analysis. Cancer, 120, 3707–3716 (2014).
• 3) Raoul JL. Natural history of hepatocellular carcinoma and current treatment options. Semin. Nucl. Med., 38, S13–S18 (2008).
• 4) Carr BI. Hepatocellular carcinoma: current management and future trends. Gastroenterology, 127 (Suppl. 1), S218–S224 (2004).
• 5) Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu. Rev. Pharmacol. Toxicol., 39, 361–398 (1999).
• 6) Hirai T, Korogi Y, Ono K, Maruoka K, Harada K, Aridomi S, Takahashi M. Intraarterial chemotherapy or chemoembolization for locally advanced and/or recurrent hepatic tumors: evaluation of the feeding artery with an interventional CT system. Cardiovasc. Intervent. Radiol., 24, 176–179 (2001).
• 7) Shiina S, Tagawa K, Unuma T, Fujino H, Uta Y, Niwa Y, Hata Y, Komatsu Y, Shiratori Y, Terano A, Sugimoto T. Percutaneous ethanol injection therapy of hepatocellular carcinoma: analysis of 77 patients. AJR Am. J. Roentgenol., 155, 1221–1226 (1990).
• 8) Livraghi T, Giorgio A, Marin G, Salmi A, de Sio I, Bolondi L, Pompili M, Brunello F, Lazzaroni S, Torzilli G. Hepatocellular carcinoma and cirrhosis in 746 patients: long-term results of percutaneous ethanol injection. Radiology, 197, 101–108 (1995).
• 9) Averbach A, Stuart OA, Chang D, Sugarbaker PH. Lattice intrahepatic doxorubicin with and without in-flow occlusion: a pharmacokinetic study of direct liver injection. Eur. J. Surg. Oncol., 26, 73–79 (2000).
• 10) Mok TS, Kanekal S, Lin XR, Leung TW, Chan AT, Yeo W, Yu S, Chak K, Leavitt R, Johnson P. Pharmacokinetic study of intralesional cisplatin for the treatment of hepatocellular carcinoma. Cancer, 2011, 2369–2377 (2001).
• 11) Vogl TJ, Engelmann K, Mack MG, Straub R, Zangos S, Eichler K, Hochmuth K, Orenberg E. CT-guided intratumoural administration of cisplatin/epinephrine gel for treatment of malignant liver tumours. Br. J. Cancer, 86, 524–529 (2002).
• 12) Ando E, Tanaka M, Yamashita F, Kuromatsu R, Yutani S, Fukumori K, Sumie S, Yano Y, Okuda K, Sata M. Hepatic arterial infusion chemotherapy for advanced hepatocellular carcinoma with portal vein tumor thrombosis: analysis of 48 cases. Cancer, 95, 588–595 (2002).
• 13) Nishida K, Sato N, Sasaki H, Nakamura J. Absorption of organic anions as model drugs following application to rat liver surface in-vivo. J. Pharm. Pharmacol., 46, 867–870 (1994).
• 14) Nishida K, Sato N, Sasaki H, Nakamura J. Mechanism for drug absorption from rat-liver surface membrane: effect of dose and transport inhibitors on the pharmacokinetics of phenol red. J. Pharm. Pharmacol., 47, 227–231 (1995).
• 15) Nishida K, Sato N, Sasaki H, Nakamura J. Absorption characteristics of dextrans with different molecular weights from the liver surface membrane in rats: implications for targeting to the liver. J. Drug Target., 4, 141–150 (1996).
• 16) Nishida K, Sato N, Nakakoga Y, Mukai T, Sasaki H, Nakamura J. Effect of application volume and area on the absorption of phenol red, as a model drug, from the liver surface in rats. J. Pharm. Pharmacol., 49, 976–980 (1997).
• 17) Nakamura J, Yoshida Y, Mera K, Mukai T, Nishida K, Sasaki H. Continuous microinstillation of phenol red on liver surface for liver site-selective delivery. Biol. Pharm. Bull., 22, 713–715 (1999).
• 18) Nishida K, Yoshida Y, Mukai T, Kawakami S, Sakaeda T, Nakashima M, Sasaki H, Nakamura J. Effect of instillation method on the absorption of phenolsulphonphthalein as a model drug from the liver and small intestinal serosal surface in rats. J. Pharm. Pharmacol., 53, 1341–1346 (2001).
• 19) Nishida K, Fujiwara R, Kodama Y, Fumoto S, Mukai T, Nakashima M, Sasaki H, Nakamura J. Regional delivery of model compounds and 5-fluorouracil to the liver by their application to the liver surface in rats: its implication for clinical use. Pharm. Res., 22, 1331–1337 (2005).
• 20) Kodama Y, Fumoto S, Nishi J, Nakashima M, Sasaki H, Nakamura J, Nishida K. Absorption and distribution characteristics of 5-fluorouracil (5-FU) after an application to the liver surface in rats in order to reduce systemic side effects. Biol. Pharm. Bull., 31, 1049–1052 (2008).
• 21) Kawakami S, Horimoto T, Nishida K, Hirayama R, Mukai T, Nakashima M, Sasaki H, Sakaeda T, Nakamura J. Kidney- and site-selective delivery of 5-fluorouracil utilizing the absorption on the kidney surface in rats. Biol. Pharm. Bull., 25, 928–930 (2002).
• 22) Nakamura J, Kobayashi K, Fumoto S, Nishi J, Mukai T, Nakashima M, Sasaki H, Nishida K. Stomach- and site-selective delivery of 5-fluorouracil following its application on the gastric serosal surface in rats. Biol. Pharm. Bull., 28, 1049–1053 (2005).
• 23) Kodama Y, Horishita M, Fumoto S, Mine T, Miyamoto H, Yoshikawa N, Hirata H, Sasaki H, Nakamura J, Nishida K. Effect of viscous additives on the absorption and hepatic disposition of 5-fluorouracil (5-FU) after application to liver surface in rats. J. Pharm. Pharmacol., 64, 1438–1444 (2012).
• 24) Nagano H. Treatment of advanced hepatocellular carcinoma: intraarterial infusion chemotherapy combined with interferon. Oncology, 2010, 142–147 (2010).
• 25) Wolpin BM, Mayer RJ. Systemic treatment of colorectal cancer. Gastroenterology, 2008, 1296–1310 (2008).
• 26) Zhang YW, Zang YL, Pan H, Wei FX, Zhang YC, Shao Y, Han W, Liu HP, Wang ZY, Yang SH. Chemotherapy for patients with gastric cancer after complete resection: a network meta-analysis. World J. Gastroenterol., 20, 584–592 (2014).
• 27) Puleo F, Marechal R, Demetter P, Bali MA, Calomme A, Closset J, Bachet JB, Deviere J, Van Laethem JL. New challenges in perioperative management of pancreatic cancer. World J. Gastroenterol., 21, 2281–2293 (2015).
• 28) Aboagye EO, Saleem A, Cunningham VJ, Osman S, Price PM. Extraction of 5-fluorouracil by tumor and liver: a noninvasive positron emission tomography study of patients with gastrointestinal cancer. Cancer Res., 61, 4937–4941 (2001).
• 29) Watanabe T. Evidence produced in Japan: tegafur-based preparations for postoperative chemotherapy in breast cancer. Breast Cancer, 20, 302–309 (2013).
• 30) Lordick F, Lorenzen S, Yamada Y, Ilson D. Optimal chemotherapy for advanced gastric cancer: is there a global consensus? Gastric Cancer, 17, 213–225 (2014).
• 31) Miyamoto Y, Sakamoto Y, Yoshida N, Baba H. Efficacy of S-1 in colorectal cancer. Expert Opin. Pharmacother., 15, 1761–1770 (2014).
• 32) Tuchman M, Ramnaraine ML, O’Dea RF. Effects of uridine and thymidine on the degradation of 5-fluorouracil, uracil, and thymine by rat liver dihydropyrimidine dehydrogenase. Cancer Res., 45, 5553–5556 (1985).
• 33) Watanabe J, Hayashi Y, Iwamoto K, Ozeki S. Salivary excretion of 5-fluorouracil. I. Fluctuation of the saliva/plasma concentration ratio and salivary clearance in beagle dogs following bolus intravenous administration. Chem. Pharm. Bull., 33, 1187–1194 (1985).
• 34) Sawai Y, Yamaoka K, Ito T, Nakagawa T. Simultaneous evaluation of intestinal absorption and hepatic extraction of 5-fluorouracil using portal-systemic concentration difference by short-period double dosing in a single conscious rat. Biol. Pharm. Bull., 20, 1313–1316 (1997).
• 35) Shirasaka T, Yamamitsu S, Tsuji A, Taguchi T. Conceptual changes in cancer chemotherapy: from an oral fluoropyrimidine prodrug, UFT, to a novel oral fluoropyrimidine prodrug, S-1, and low-dose FP therapy in Japan. Invest. New Drugs, 18, 315–329 (2000).
• 36) Sato S, Hirotani Y, Ogura N, Sasaki E, Kitagawa S. Enhancing effect of N-dodecyl-2-pyrrolidone on the percutaneous absorption of 5-fluorouracil derivatives. Chem. Pharm. Bull., 46, 831–836 (1998).
• 37) Müller M, Mader RM, Steiner B, Steger GG, Jansen B, Gnant M, Helbich T, Jakesz R, Eichler HG, Blochl-Daum B. 5-Fluorouracil kinetics in the interstitial tumor space: clinical response in breast cancer patients. Cancer Res., 57, 2598–2601 (1997).