ANTICANCER RESEARCH 32: 2807-2812 (2012)

Efficacy of Combination Using Oral Fluoropyrimidine S-1 with (SOX) against In Vivo

MAMORU NUKATSUKA1, HITOSHI SAITO2, KAZUKI SAKAMOTO3, FUMIO NAKAGAWA2, JUNJI UCHIDA1, TAKASHI KOBUNAI5, KAZUYOSHI SHIRAISHI4 and TEIJI TAKECHI5

1Oncology Medical Affairs Department, and 2Optimal Medication Research Laboratory, Tokushima Research Center, Tokushima, Japan; 3Tsukuba Research Center, Ibaraki, Japan; 4Department of Product Lifecycle Management, Tokyo, Japan; 5Oncology Medical Affairs Department, Taiho Pharmaceutical Co., Ltd., Tokyo, Japan

Abstract. Oxaliplatin is effective when used with 5- Colorectal cancer remains the third-leading cause of cancer- (5-FU) and leucovorin, or with capecitabine (COX) related death in Japan. Recently, new agents such as for the treatment of colorectal cancer. In this experiment, we camptotecin (1), oxaliplatin (2, 3), and targeted monoclonal investigated the optimal combination schedule and antitumor antibodies (bevacizumab, cetuximab, panitumumab) (4-6) activity of oral S-1 with oxaliplatin combination therapy (SOX) have been approved for clinical use. However, 5-fluorouracil against human colorectal cancer xenografts in vivo. Using (5-FU)-based combination chemotherapy remains one of the human colon cancer COL-1-bearing nude mice, oxaliplatin was most effective therapeutic agents for colorectal cancer. administered at a total dose of 8.3 mg/kg on day 1 alone, on Oxaliplatin-containing regimens, such as FOLFOX4 or day 8 alone, or in divided doses administered on days 1 and 8 mFOLFOX6, are often used clinically; however, these with S-1 (6.9 mg/kg, days 1-14). The antitumor activity of SOX, regimens require a 48-hour period of continuous intravenous administered according to the divided schedule was significantly infusion (c.v.i.) of 5-FU or leucovorin. FOLFOX4 is a superior to both monotherapies (p<0.01), and the toxicity was combination involving the bolus tolerable. However, administration on day 8 alone failed to administration of oxaliplatin in combination with the bolus significantly increase the antitumor activity, when compared administration of 5-FU and leucovorin, followed by a with that of monotherapy, while administration on day 1 alone continuous 5-FU infusion; this regimen is reportedly active was toxic in this model. Next, the efficacy of SOX was compared in patients who have been previously treated with 5-FU alone with that of COX (360 mg/kg, days 1-14). The antitumor effect or in combination with leucovorin (3). However, the of SOX was significantly superior to that of COX (p<0.01), with FOLFOX4 regimen requires at least three days of an equivalent toxicity; moreover SOX suppressed COL-1 tumor hospitalization because of the 48-hour c.v.i. and is growth for a longer period of time (2.2 times) than did COX. unsatisfactory from the perspective of quality of life (QOL). The antitumor activity of SOX against the 5-FU-resistant Oral fluoropyrimidine-containing regimens are more colorectal cancer cell line KM12C/5-FU was equivalent to that convenient, and combination therapy using capecitabine and of COX. The evaluation of intermittent SOX administration in a oxaliplatin (COX) has been used against colorectal cancer. clinical trial might be of critical value. The antitumor effects of COX are equivalent to these of FOLFOX, while the treatment regimen is more convenient than that for the FOLFOX regimen (7). However, the incidence of hand-foot syndrome is reported to be as high as This article is freely accessible online. 13% for grade 2 and 26% for grade 1, which is significantly higher than that for the FOLFOX regimen (8). Correspondence to: Mamoru Nukatsuka, Ph.D., 224-2, Ebisuno The oral fluoropyrimidine, S-1, is composed of 1 M tegafur Hiraishi, Kawauchi-Cho Tokushima-Shi, Tokushima 771-0194, (a masked form of 5-FU), 0.4 M 5-chloro-2,4-dihydroxy- Japan. Tel: +81 886656014, Fax: +81 886656554, e-mail: m- pyrimidine [gimestat; a potent inhibitor of dihydropyrimidine [email protected] dehydrogenase (DPD)], and 1 M potassium oxonate (which Key Words: Colorectal cancer, S-1, oxaliplatin, COX, growth delay mainly inhibits the phosphorylation of 5-FU in the period, COL-1 cells, capecitabine. gastrointestinal (GI) tract) (9). S-1 has been shown to be

0250-7005/2012 $2.00+.40 2807 ANTICANCER RESEARCH 32: 2807-2812 (2012) effective against various cell lines of human cancer in vivo (10) relative tumor volume (RTV) was calculated using the following and to have a potent antitumor efficacy, with a lower GI formula: RTV=(tumor volume on measured day)/(tumor volume on toxicity, for various types of cancer, including colorectal cancer day 0). On day 15, the tumor growth inhibition ratio (TGI) was calculated using the following formula: TGI=[1 – (mean tumor (11-13). Several combination using S-1 and volume of treated group)/(mean tumor volume of control group)] irinotecan (14) or cisplatin (15) are reportedly effective against ×100. The body weight change (BWC; %) was calculated as [(body gastric cancer clinically, and S-1 combined with irinotecan weight on day 15) – (body weight on day 0)]/(body weight on day (IRIS) has been reported not to be inferior to FOLFOX against 0) ×100 (%). colorectal cancer as a second-line therapy (16). The growth delay period (GDP), which indicates the difference in As an additional candidate regimen, we evaluated the period during which the RTV grew to 4 (corresponding to 50% combination therapy using oral S-1 and oxaliplatin (SOX), of the size of the control tumors at the endpoint on day 22), was determined according to a previously reported procedure (20). using a human colorectal cancer xenograft model in vivo. Toxicity was defined as a 20% or more body weight loss or toxic death. Materials and Methods Statistical analysis. The significance of the differences in the mean Chemicals. Tegafur, gimestat and potassium oxonate were RTV between the treated and control groups on day 15 was synthesized at Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan). analyzed using the Aspin-Welch two-tailed t-test. The combinational Capecitabine (N4-pentyloxycarbonyl-5’-deoxy -5-fluorocytidine) effect of S-1 and oxaliplatin was analyzed according to the closed was synthesized by KNC Laboratory, Ltd. (Kobe, Japan). testing procedure using the Aspin-Welch two-tailed t-test (21); the Oxaliplatin (SP-4-2)-[(1R, 2R)-cyclohexane-1, 2-diamine-κN, κN’] analyses were performed using the EXSAS, Ver. 7.11 software (Arm [ethanedioato(2-)-κO1, κO2] platinum) was synthesized by Systex Co., Ltd., Osaka, Japan). SINOPHARM JIANGSU Co., Ltd. (Nanjing P.R., China). The glucose solution for injection (5%) was purchased from Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan). Hydroxypropyl Results methylcellulose (HPMC) was purchased from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). All other reagents were commercially Determination of the optimal schedule and maximal available products of the highest grade. torelated dose (MTD) of oxaliplatin. S-1 alone showed significant antitumor activity against COL-1 tumor, but Tumor xenografts. The human colorectal cancer cell line COL-1 oxaliplatin alone failed to show significant antitumor activity. (17) was obtained from the Central Institute for Experimental As five out of seven mice treated with oxaliplatin at a dose of Animals (Kawasaki, Japan). A 5-FU-resistant human colorectal 8.3 mg/kg on day 1 had died before day 15, day 1 cancer cell line, KM12C/5-FU, was established in our laboratory, as administration was thought to be toxic in this model. As the described previously (18). mice treated with oxaliplatin at a dose of 8.3 mg/kg on day Antitumor activity in vivo. Four-week-old male BALB/c nude mice 8 failed to exhibit a significant increase in the antitumor were purchased from CLEA Japan Inc. (Tokyo, Japan) and were activity of S-1, day 8 administration was thought not to be housed under specific pathogen-free conditions; food and water useful. In contrast, oxaliplatin administered in a divided dose were provided ad libitum. After the animals had been in quarantine of 4.2 mg/kg on days 1 and 8 significantly increased the for one week, they were implanted subcutaneously with a human antitumor activity, compared with each monotherapy, without colorectal tumor cell line, the volume of which was approximately the death of the mice (Table I). 8 mm3. To evaluate the antitumor activity, the mice were grouped according to the tumor volume once the mean tumor volume reached about 150 to 200 mm3 (day 0). Each group consisted of 7 to Comparison of increased antitumor activity of SOX and 9 mice. COX. The antitumor activity of oxaliplatin administered in S-1 was prepared by mixing tegafur, gimestat, and potassium divided doses of 4.2 mg/kg on days 1 and 8 in combination oxonate at a molar ratio of 1:0.4:1 in 0.5% HPMC. S-1 was with an effective dosage of S-1 (6.9 mg/kg) or capecitabine administered orally at the reported effective dose of 6.9 mg/kg (10) (360 mg/kg), was compared in COL-1-bearing nude mice in once daily, for 14 consecutive days. Capecitabine, which was vivo. The COL-1 tumor volume change, after treatment with suspended in 0.5% HPMC, was administered orally at the reported effective dose of 360 mg/kg (19) once daily, for 14 consecutive SOX, and with COX is shown in Figure 1. On day 15, the days. Oxaliplatin was dissolved in 5% glucose solution and was growth-inhibitory activity of SOX was significantly superior administered intravenously. to that of COX, with a tolerable toxicity (Table II). After Firstly, oxaliplatin was administered at 8.3 mg/kg (on day 1 alone treatment, the tumor volume was measured twice a week or day 8 alone) or at 4.2 mg/kg (on days 1 and 8) in combination continuously until day 22. The period required for the RTV with S-1 (6.9 mg/kg), to COL-1-bearing nude mice, to determine to reach 4 (GDP) was 10.3 days for the SOX group and 4.9 the optimal schedule and dose of oxaliplatin to be used in days for the COX group. From these results, SOX was combination with S-1. The total dose of oxaliplatin was estimated in a pilot experiment (data not shown). expected to suppress tumor growth more effectively for The tumor diameters were measured twice a week until day 22, longer periods than COX in COL-1-bearing nude mice and the tumor volume was estimated as 0.5 × length × width2. The (Figure 1 and Table II).

2808 Nukatsura et al: Combination Chemotherapy for Colorectal Cancer Using S-1 and Oxaliplatin

Table I. Optimal schedule of oxaliplatin in combination with S-1 against human colorectal cancer, COL-1-bearing nude mice.

Group Dose Schedule Toxic Body weight change Tumor volume (%) TGI (mg/kg) death (%, mean±SD) (mm3, mean±SD)

Control – – 0 –13.9±3.5 884.1±132.6 – – S-1 alone 6.9 Days 1-14 0 –17.1±4.6 572.3±154.0 * 36.9% Oxaliplatin alone 8.3 Day 1 alone 0 –8.2±6.8 641.8±201.5 NS 28.6% 8.3 Day 8 alone 0 –12.8±1.9 820.0±304.6 NS 9.4% 4.2 Days 1, 8 0 –13.8±3.3 669.2±103.8 NS 24.7% SOX 6.9+8.3 Days 1-14+1 5/7 –15.7±6.6 419.4±164.5 NE 52.5% 6.9+8.3 Days 1-14+8 0 –18.0±1.3 505.4±81.1 * 44.1% 6.9+4.2 Days 1-14+1, 8 0 –22.5±4.2 394.5±68.8 *, # 55.1%

SOX: S-1+oxaliplatin; TGI: tumor growth inhibition; NE, not evaluable; NS, not significant vs. Control group; *p<0.01 vs. control group, by Welch t-test, #p<0.05 vs. S-1 alone group.

Table II. Antitumor activity of S-1+oxaliplatin (SOX) and capecitabine+oxaliplatin (COX) against COL-1 in vivo.

Group Dose Schedule Toxic Body weight change ΔBWC Tumor volume TGI GDP (mg/kg) death (%, mean± SD) (%) (mm3, mean and SD) (%) (days)

Control – – 0/8 –12.6±3.6 – 739.9±196.7 – 0 SOX 6.9+4.2 Days 1-14+1, 8 0/8 –19.8±2.6 * –7.3 380.1±73.1 *, # 34.9 10.3 COX 360+4.2 Days 1-14+1, 8 0/8 –19.2±3.5 * –6.6 500.5±100.6 * 25.8 4.7

ΔBWC: Body weight change of treated group –control group; TGI: tumor growth inhibition; GDP: growth delay period; *p<0.01 vs. Control group by Welch t-test, #p<0.01 vs. COX group.

Table III. Antitumor activity against KM12C/5-FU in vivo.

Group Dose Schedule Toxic Body weight change Tumor volume TGI (mg/kg) death (%, mean±SD) (mm3, mean ±SD) (%)

Control – 0/9 –8.6±6.0 1778.0±538.3 – SOX 6.9+4.2 Days 1-14+1, 8 0/9 –15.7±6.1* 1225.5±379.8** 30.2 COX 360+4.2 Days 1-14+1, 8 0/9 –17.9±5.9** 1092.7±293.5** 36.2

SOX: S-1+oxaliplatin; COX: capecitabine+oxaliplatin; TGI: tumor growth inhibition; *p<0.05 and **p<0.01 vs. Control group, by Welch t-test.

Antitumor activity toward 5-FU-resistant tumors. The tumor With this schedule, SOX had a significantly higher volume change in the 5-FU-resistant colorectal cancer antitumor activity against COL-1-bearing nude mice than KM12C/5-FU, after treatment with SOX or COX, as did COX and an equivalent activity in a KM12C/5-FU- mentioned above, is shown in Figure 2. The antitumor bearing colorectal cancer mouse model. SOX has not been activity and toxicity (body weight change) on mice shown to be inferior to COX in terms of efficacy as a following treatment with SOX was equivalent to that of first-line treatment against metastatic colorectal cancer, COX (Table III). not accompanied by hand-foot syndrome and in terms of hospitalization (22). In all previous clinical studies, Discussion oxaliplatin has been administered on the first day of treatment (23, 24); however, we found that the antitumor We evaluated a SOX-containing administration schedule activity of the divided administration of oxaliplatin was for oxaliplatin. The divided administration of oxaliplatin equivalent to day 1 administration without any increase in was optimal for increasing the antitumor activity, while toxicity. The mechanisms of the drug interactions have not obtaining a lower toxicity compared with other schedules. yet been clarified, but the divided administration did not

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Figure 1. Tumor volume changes in a human colorectal cancer cell line, Figure 2. Tumor volume changes in 5-fluorouracil (5-FU)-resistant COL-1, in vivo. Nude mice bearing COL-1 were randomized into three human colorectal cancer cell line KM12C/5-FU in vivo. Nude mice groups on day 0. S-1 (6.9 mg/kg) or capecitabine (360 mg/kg) was bearing KM12C/5-FU were randomized into three groups on day 0. S- administered orally from days 1 to 14, and oxaliplatin (4.2 mg/kg) was 1 (6.9 mg/kg) or capecitabine (360 mg/kg) was administered orally from administered intravenously on days 1 and 8. The tumor volume was days 1 to 14, and oxaliplatin (4.2 mg/kg) was administered measured twice a week until day 21. The values indicate the mean and intravenously on days 1 and 8. The tumor volume was measured twice SD of the relative tumor volume (RTV) (n=10). *p<0.01 vs. control a week until day 16. The values indicate the mean and SD of the RTV according to the Aspin Welch t-test. #Overall maximal p<0.01 according (n=9). *p<0.01 vs. control, according to the Aspin Welch t-test. to a closed testing procedure, using the Aspin-Welch t-test.

capecitabine, tegafur is metabolized not only to 5-FU, but increase the toxicity compared to the day 1 also to γ-hydroxybutyric acid and γ-butyrolactone, which administration; as such, an evaluation of the divided reportedly inhibit angiogenesis (31). Furthermore, S-1 administration of oxaliplatin may be worth considering in reportedly induces the anti-angiogenesis factor thrombo- clinical trials. spondin-1 to a greater extent than capecitabine (32, 33). Several drugs (irinotecan, gefitinib, gemcitabine, and These activities may contribute to potent antitumor effects docetaxel) reportedly increase the antitumor activity of 5- of S-1 via pathways not related to 5-FU, unlike FU or its derivatives through the down regulation of capecitabine. thymidylate thymidylate synthase (TS) (25-28), and In conclusion, SOX may be useful against colorectal oxaliplatin reportedly down regulates TS in a similar cancer in a manner equivalent to that of FOLFOX or COX manner (29). The antitumor activity of 5-FU originates but with a greater convenience and at a lower cost, and the from nucleotide imbalances through the inhibition of TS, intermittent administration of oxaliplatin may further RNA dysfunction, and the false incorporation of accelerate the effects of SOX. fluorodeoxyuridine triphosphate (FdUTP) originating from fluorodeoxyuridine monophosphate (FdUMP) into DNA. References Generally, FdUTP is metabolized to FdUMP by dUTPase; however, in the presence of oxaliplatin, which inhibits 1 Armand JP, Ducreux M, Mahjoubi M, Abigerges D, Bugat R, dUTPase, the antitumor activity of 5-FU is potentiated (30). Chabot G, Herait P, de Forni M and Rougier P: CPT-11 Both S-1 and capecitabine are metabolized to 5-FU and (irinotecan) in the treatment of colorectal cancer. Eur J Cancer exhibit a common antitumor activity. However, unlike 31A: 1283-1287, 1995.

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26 Okabe T, Okamoto I, Tsukioka S, Uchida J, Iwasa T, Yoshida T, 31 Yonekura K, Basaki Y, Chikahisa L, Okabe S, Hashimoto A, Hatashita E, Yamada Y, Satoh T, Tamura K, Fukuoka M and Miyadera K, Wierzba K and Yamada Y: UFT and its metabolites Nakagawa K: Synergistic antitumor effect of S-1 and the epidermal inhibit the angiogenesis induced by murine renal cell carcinoma, growth factor receptor inhibitor gefitinib in non-small cell lung as determined by a dorsal air sac assay in mice. Clin Cancer Res cancer cell lines: role of gefitinib-induced down-regulation of 5: 2185-2191, 1999. thymidylate synthase. Mol Cancer Ther 7: 599-606, 2008. 32 Ooyama A, Oka T, Zhao HY, Yamamoto M, Akiyama S and 27 Réjiba S, Bigand C, Parmentier C and Hajri A: Gemcitabine- Fukushima M: Anti-angiogenic effect of 5-fluorouracil-based based chemogene therapy for pancreatic cancer using Ad-dCK: drugs against human colon cancer xenografts. Cancer Lett 18: UMK GDEPT and TS/RR siRNA strategies. Neoplasia 11: 637- 26-36, 2008. 650, 2009. 33 Harada K, Supriatno, Kawashima Y, Yoshida H and Sato M: S-1 28 Hasegawa M, Miyajima A, Kosaka T, Yasumizu Y, Tanaka N, inhibits tumorigenicity and angiogenesis of human oral Maeda T, Shirotake S, Ide H, Kikuchi E and Oya M: Low-dose squamous cell carcinoma cells by suppressing expression of docetaxel enhances the sensitivity of S-1 in a xenograft model phosphorylated Akt, vascular endothelial growth factor and of human castration-resistant prostate cancer. Int J Cancer 130: fibroblast growth factor-2. Int J Oncol 30: 365-374, 2007. 431-442, 2012. 29 Yeh KH, Cheng AL, Wan JP, Lin CS and Liu CC: Down- regulation of thymidylate synthase expression and its steady- state mRNA by oxaliplatin in colon cancer cells. Anticancer Drugs 15: 371-736, 2004. 30 Wilson PM, Fazzone W, LaBonte MJ, Lenz HJ and Ladner RD: Regulation of human dUTPase gene expression and p53- Received March 29, 2012 mediated transcriptional repression in response to oxaliplatin- Revised April 22, 2012 induced DNA damage. Nucleic Acids Res 37: 78-95, 2009. Accepted April 24, 2012

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