- Email: [email protected]
131I]iodo-2′-deoxyuridine to rat hepatoma by using lipiodol emulsion
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Improved targeting of 5-[125I/131I]iodo-20-deoxyuridine to rat hepatoma by using lipiodol emulsion Hung-Man Yua, Hsin-Pei Yeha, Tien-Kui Changa, Kuang-Liang Huangb, Kuo-Tang Chuangb, Ren-Shen Liuc, Shyh-Jen Wangd, Jeng-Jong Hwanga, Kwan-Hwa Chie, Fu-Du Chena,f, Wuu-Jyh Linb, Chin-Hsiung Chenb, Hsin-Ell Wanga, a
Institute of Radiological Sciences, National Yang-Ming University, Taipei, Taiwan ROC b Institute of Nuclear Energy Research, Taoyuan, Taiwan, ROC c Department of Nuclear Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC d Department of Nuclear Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, ROC e Division of Radiation Therapy and Oncology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, ROC f Central Taiwan University of Science and Technology, Taichung, Taiwan, ROC Available online 20 September 2006
Abstract This study aims to assess whether emulsion of [125/131I]IUdR and lipiodol (IUdR/LP) can improve delivery of IUdR into hepatoma. Methods: In vitro release profile of IUdR from IUdR/LP to serum was performed. IUdR/LP was injected into N1-S1 hepatomabearing SD rat via hepatic artery and IUdR/normal saline (IUdR/NS) was used for comparison. Biodistribution, autoradiography, imaging and tumor DNA incorporation assay were performed. The radioactive metabolites in plasma and urine were analyzed. Radiation doses to tumor and organs were estimated. Results: IUdR released from lipiodol into serum was fast. There were longer retention, more DNA incorporation and higher radiation dose of IUdR in the tumor by using IUdR/LP. IUdR/LP deposited deep in the hepatomas. Only free iodide was found in the plasma and urine after injection of IUdR/LP. Conclusions: Hepatic artery injection of IUdR/LP emulsion could definitely enhance the tumor cell uptake and incorporation to DNA of *IUdR, prolong the tumor retention time and increase radiation dose to tumor. IUdR/LP may be an effective therapeutic agent for the treatment of hepatic tumors. r 2006 Elsevier B.V. All rights reserved. PACS: 47.63.mh; 79.20.Fv; 87.58.b; 87.58.Ji; 87.58.Sp Keywords: Hepatoma; Iododeoxyuridine; Auger electron; Lipiodol; Radionuclide therapy
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world [1]. Since hepatic tumors received blood predominately from the hepatic artery, intra-arterial (i.a.) treatment offers opportunities of selectively targeting the tumors [2]. Lipiodol, an ethyl ester of poppy seed oil fatty acids, has been found to accumulate Corresponding author. Tel.: +886 2 28267215; fax: +886 2 28201095.
E-mail address: [email protected] (H.-E. Wang). 0168-9002/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nima.2006.08.089
selectively in the hepatomas and remain there for a long time [3]. Lipiodol has been commonly used as a vehicle for targeted treatment of HCCs when labeled with radioisotopes [4] or mixed with chemotherapeutic agents [5]. When Auger electron emitters, such as 125I and 123I, are in the form of 5-iodo-20 -deoxyuridine (IUdR) and are incorporated into DNA, it becomes highly cytotoxic [6,7]. Intravenously (i.v.) administrate radioiodinated IUdR (*IUdR), however, is unlikely to be useful as an antitumor agent because of its non-specific uptake by all proliferating cells and its rapid dehalogenation. Previous studies in
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animals and humans have shown that efficient tumor targeting can be obtained after locoregional administration of *IUdR [8,9]. Based on the extreme radiotoxicity of *IUdR and the selective accumulation of lipiodol in hepatic tumor, we prepared a lipiodol emulsion mixed with *IUdR (IUdR/ LP) and demonstrated the improved tumor targeting of IUdR/LP after injection through the hepatic artery in a rat hepatoma model. The results of our study indicated that *IUdR/LP would be a useful agent for the treatment of hepatic tumors.
injection. Tissue samples of rats were embedded in paraffin and sequentially sectioned (5 mm), coated with emulsion (NTB-2, Eastman Kodak Company, Rochester, NY), dried and stored at 4 1C in light-tight boxes. Following exposure of at least 4 weeks, the emulsion were developed (D-19, Eastman Kodak Company, Rochester, NY), fixed (Polymax T, Eastman Kodak Company, Rochester, NY) and finally were stained with hematoxylin and eosin, dehydrated and mounted in Permount.
2. Materials and methods
The incorporation of [125I]IUdR into tumor DNA was determined after i.a. injection of [125I]IUdR/NSand [125I]IUdR/LP (11.1 MBq per rat). Tumor samples of rats were digested with enzyme cocktail solution. The radioactivity in DNA was determined using the method previously described [10].
2.1. Preparation of [125/131I]IUdR/NS and [125/ I]IUdR/LP
131
[125/131I]IUdR was synthesized as described previously [10] and purified with HPLC. *IUdR was resuspended in physiological saline and lipiodol to afford IUdR/NS and IUdR/LP, respectively. 2.2. Release profile of IUdR from IUdR/LP 1 mL of [131I]IUdR/LP (3.7 MBq) was introduced in 1 mL of prewarmed (37 1C) FBS. The mixture was placed on an orbital shaker (100 rpm). A sample (250 mL) of serum was withdrawn at every 5 min interval and the radioactivity was determined with a dose calibrator (CRC15R, Capintec, NJ). 2.3. Cell culture and N1-S1 Hepatoma-bearing rat model The N1-S1 hepatoma cells cultured and hepatomabearing Male Spraque–Dawley rat model were detailed in our previously report [4]. 2.4. Biodistribution of [125I]IUdR/NS and [125I]IUdR/LP The animal experiments were approved by the Institutional Animal Care and Use Committee of the National Yang-Ming University. The biodistribution studies was detailed in our previously report [4]. 2.5. Planar gamma imaging After i.a. injection of 11.1 MBq [131I]IUdR/NS or [ I]IUdR/LP, animals were anesthetized with isoflurane and static images were obtained at 48 h post injection on a gamma camera (Apex 400, Elscint, Israel) equipped with a high-energy pinhole collimator. The images were acquired using a preset-time acquisition mode (10 min). 131
2.6. Autoradiography After i.a. injection of 11.1 MBq [125I]IUdR/NS or [ I]IUdR/LP, animals were sacrificed at 48 h post 125
2.7. Incorporation of [125I]IUdR into nucleic acid
2.8. Determination of [131]IUdR and its metabolite in plasma and urine Venous blood samples were drawn at various time period (1–30 min) after i.v. and i.a. injection of [131]IUdR/ NS or [131]IUdR/LP (11.1 MBq). The plasma was analyzed using the HPLC system described above. Urine samples were analyzed by the same procedures. 2.9. Radiation dosimetry The diameter of the tumor cell and nucleus were measured 7 and 6 mm, respectively. The S-phase cell in hepatic tumor was proposed 15%. Following the radiation ¯ h ¼ Sti S i Þ, all the specific t values on dose formula ðD each source organ were derived from the biodistribution study, DNA incorporation study and mathematical model. The specific S value on each target–source organ pair was obtained from MIRDIII program and ‘‘MIRD cellular S value’’ [11]. Both the conventional and cell nucleus-level radiation dose of hepatic tumor were estimated as the tumor dose. 3. Results The percentage radioactivity in serum increased from 40% at 5 min to 100% at 30 min indicated that in vitro extraction of [131I]IUdR from lipiodol to serum could be considerably fast (Fig. 1A). The radioactivity accumulated in the tumor was much higher in IUdR/LP group (Table 2) than that in IUdR/NS group (Tables 1 and 2). The tumorto-normal liver ratio reached maximum (3.85) at 24 h and (63.88) at 0.5 h after IUdR/NS and IUdR/LP injection, respectively. Except liver, the radioactivity in normal tissues was about the same grade compared with that of IUdR/NS group (data not shown). The scitigraphic images of [131I]IUdR/LP clearly delineated the hapatoma (Fig. 2B), but gave only blurred images for [131I]IUdR/
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NS (Fig. 2A). In autoradiographic study, the radioactivity retained in tumor in the IUdR/LP group (Fig. 3B) was higher than that in the IUdR/NS group (Fig. 3A). In IUdR/LP group, [125I]IUdR was delivered into the inner areas of the tumor, whereas only cells on the periphery of the tumor were labeled in the IUdR/NS group. The autoradiograms of the normal tissue (liver, lung, stomach, small intestine and bone marrow) sections showed only background silver grain density (data not shown). The IUdR/LP group exhibited much higher radioactivity incorporation in the DNA of tumor cell than that of the IUdR/NS group (Table 3). For IUdR/LP group, the
accumulated radioactivity (mBq) in DNA per tumor cell increased from 0.022 at 1 h to 1.497 at 72 h post injection. At 30 min after i.v. or i.a. injection of IUdR/NS, no more [131]IUdR can be detected in the plasma (Fig. 1B). For IUdR/LP, even at the first minute after i.a. injection, there was no intact [131]IUdR, only free 131I was found in the plasma. In all urine samples, the radioactive species was represented solely by free 131I. The retention time of
Fig. 1. (A) The in vitro release profile of [131I]IUdR from lipiodol to serum. (B) Percentage radioactivity of intact [131I]IUdR in plasma samples after i.v. injection of [131I]IUdR/NS (E), after i.a. injection of [131I]IUdR/ NS ( ) or [131I]IUdR/LP (m).
Fig. 2. Planar gamma images of SD rats bearing N1S1 hepatoma at 48 h after i.a. injection of 11.1 MBq [131I]IUdR/NS (A) and [131I]IUdR/LP (B). Selective uptake of [131I]IUdR in hepatoma was clearly observed in lipiodol group.
Table 1 Biodistribution of [125I]IUdR in tumor and other normal tissues of N1S1 hepatoma-bearing SD rats after intra-arterial injection of 3.7 MBq IUdR/NS (n ¼ 5) Organ
%ID/g7SD 0.5 h
1h
4h
12 h
24 h
48 h
72 h
Blood (B) Lung Liver (L) Tumor (T)
0.9970.02 0.5370.06 0.4270.01 0.5070.02
0.8870.05 0.4670.06 0.3370.04 0.4170.05
0.7970.20 0.3970.07 0.3070.06 0.4270.11
0.3270.13 0.1770.06 0.1270.04 0.2370.10
0.0870.06 0.0570.04 0.0370.02 0.1270.11
0.0170.00 0.0170.00 0.0170.00 0.0170.01
0.0070.00 0.0070.00 0.0070.00 0.0170.01
T/B T/L
0.50 1.17
0.46 1.26
0.54 1.42
0.72 2.00
1.62 3.85
1.93 2.53
6.003 2.050
Table 2 Biodistribution of [125I]IUdR in tumor and other normal tissues of N1S1 hepatoma-bearing SD rats after intra-arterial injection of 3.7 MBq IUdR/LP (n ¼ 5) Organ
%ID/g7SD (h) 5 min
15 min
0.5 h
1h
4h
12 h
24 h
48 h
72 h
Blood (B) Lung Liver (L) Tumor (T)
2.0170.46 0.9170.18 2.2970.66 63.52715.45
1.5070.11 0.8370.06 1.5670.80 55.86735.79
1.1170.21 0.6170.07 0.5770.06 36.23715.74
0.9770.20 0.4570.15 0.4870.12 24.28710.64
0.7370.09 0.3870.08 0.4070.09 6.3472.54
0.5270.26 0.2470.12 0.2470.15 0.7770.49
0.1070.06 0.0970.04 0.1170.06 0.3570.11
0.0170.01 0.0170.01 0.0270.01 0.1170.02
0.0070.00 0.0170.01 0.0470.03 0.17070.134
T/B T/L
31.63 27.77
37.15 35.75
32.71 63.88
25.16 50.78
8.68 15.79
1.48 3.22
3.53 3.29
9.60 5.35
74.82 4.37
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Fig. 3. Autoradiographs of thin section from tumor of N1-S1 hepatoma-bearing rat at 48 h after i.a. injection of 11.1 MBq [125I]IUdR/NS (A) and [125I]IUdR/LP (B).
Table 3 The average radioactivity incorporated in DNA per tumor cell after i.a. injection of 11.1 MBq [125I]IUdR/NS and [125I]IUdR/LP Time after injection (h)
1 4 24 48 72
mBq/DNA of cell [125I]IUdR/NS
[125I]IUdR/LP
0.00270.001 0.00670.002 0.00970.004 0.00570.002 Not analyzed
0.02270.012 0.02870.010 0.60370.294 1.25270.598 1.49770.758
*IUdR in tumor (Table 4) was much longer for IUdR/LP (86.52 h) than that for IUdR/NS (9.37 h). The radiation dose to tumor was almost ten folds higher for IUdR/LP (6.84 Gy/MBq) than that for IUdR/NS (0.69 Gy/MBq). The relatively low liver dose from both IUdR/NS (0.0008 Gy/MBq) and IUdR/LP (0.0011 Gy/ MBq) indicated minimal radiation injury to normal organs via i.a. injection. 4. Discussion In this study, [125/131I]IUdR was mixed with lipiodol to give a clear and sterilized IUdR/LP emulsion. The hydrophilicity of IUdR facilitated the extraction of it from lipiodol to serum. The release profile of IUdR/LP in serum (Fig. 1A) suggested the facile in vivo release of IUdR from IUdR/LP to tissues where lipiodol retained. The IUdR/LP was i.a. injected to optimize the accumulation of IUdR in
tumor and to minimize its incorporation into normal tissues. The results of biodistribution (Table 2) showed that the radioactivity accumulated in tumor declined considerably with time within 4 h after i.a. of IUdR/LP, which was in agreement with the fast in vitro release profile of IUdR from IUdR/LP to serum. However, the incorporation of IUdR into DNA of tumor cells (Table 3), increased significantly by using IUdR/LP. It is noteworthy that except for liver, the radioactivity in normal tissues was almost of the same magnitude for IUdR/LP and IUdR/NS. Lipiodol increased the tumor uptake, both in tissue and DNA levels, of IUdR while sparing the normal tissues from radiation injury. In autoradiography study, only the peripheral cells of the tumor were labeled in IUdR/NS group (Fig. 3A), whereas IUdR was delivered into the inner areas of the tumor in IUdR/LP group (Fig. 3B). Taniguchi et al. [12] have shown that the delivery of BUdR to liver tumors was enhanced after i.a. infusion of BUdR/lipiodol suspension. They showed that hepatic tumors had cells incorporated BUdR in their deepest area. Our findings suggested that, due to the tumor vessel embolization with lipiodol, IUdR was incorporated into cells closed to the tumor vessel (Fig. 3B) and the angiogenesis status of hepatic tumor is critical to the effective tumor targeting of IUdR. Mariani et al. [9] have shown a virtually 100% first-pass hepatic deiodination of unincorporated [123I]IUdR after i.a. infusion of [123I]IUdR in patients with liver metastases from colorectal cancer. Our study showed that even at 1 min after i.a. injection of IUdR/LP, the only radioactive species that could be detected in blood was [131I]I. So IUdR/LP can
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Table 4 Estimated radiation dose to tumor and liver after i.a. injection of [131I]IUdR/NS and [131I]IUdR/LP (radiation dose unit: Gy/MBq) Formulation
131
[ I]IUdR/NS [131I]IUdR/LP
Tumor
Liver
Tumor/liver dose ratio
Residence time (h)
Radiation dose
Residence time (h)
Radiation dose
9.37 86.52
0.694 6.835
4.41 5.85
0.0008 0.0011
minimize the systemic uptake of [131I]IUdR by normal tissues. Due to the prolonged retention of IUdR/LP in tumor, the estimated radiation dose (Table 4) to tumor for IUdR/LP was almost ten folds higher than that for IUdR/ NS, whereas the doses to liver were both very low. This suggested that lipiodol was washed out easily from normal liver tissue and was regarded generally safe as radionuclide vehicle.
5. Conclusion This study showed that lipiodol, as a vehicle of n.c.a. *IUdR, can improve the targeting, specific accumulation and radiation dose of *IUdR to the tumor cells. IUdR/LP has been demonstrated as a potential therapeutic agent for the treatment of hepatic tumors.
Acknowledgments This work was supported by grants from the National Science Council (NSC 90-2314-B-010-030, NSC 93-3112-B010-019, NSC 94-2623-7-010-001).
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References [1] A.P. Venook, J. Clin. Oncol. 12 (1994) 1323. [2] W.T. Leung, W.Y. Lau, S. Ho, M. Chan, N. Leung, J. Lin, K.C. Ho, C. Metreweli, P.J. Johnson, A.K. Li, J. Nucl. Med. 35 (1994) 1313. [3] B. Lambert, K. Bacher, L. Defreyne, F. Gemmel, H. Van Vlierberghe, J.M. Jeong, R.A. Dierckx, C. Van de Wiele, H. Thierens, F. De Vos, J. Nucl. Med. 46 (2005) 60. [4] S.J. Wang, W.Y. Lin, W.Y. Lui, M.N. Chen, Z.T. Tsai, G. Ting, J. Nucl. Med. 37 (1996) 332. [5] T. Konno, Cancer 66 (1990) 1897. [6] P.K. LeMotte, J.B. Little, Cancer Res 44 (1984) 1337. [7] V.R. Narra, R.W. Howell, R.S. Harapanhalli, K.S. Sastry, D.V. Rao, J. Nucl. Med. 33 (1992) 2196. [8] G. Mariani, P. Collecchi, S. Baldassarri, L. Di Luca, S. Buralli, G. Fontanini, J. Baranowska-Kortylewicz, S.J. Adelstein, A.I. Kassis, J. Nucl. Med. 37 (Suppl) (1996) 16S. [9] G. Mariani, S.D. Sacco, D. Volterrani, L. Di Luca, S. Buralli, R. Di Stefano, J. Baranowska-Kortylewicz, D. Bonora, F. Matteucci, S. Ricci, C.R. Bellina, A. Falcone, P.A. Salvadori, F. Mosca, S.J. Adelstein, A.I. Kassis, J. Nucl. Med. 37 (Suppl) (1996) 22S. [10] K.H. Chi, H.E. Wang, Y.S. Wang, S.L. Chou, H.M. Yu, Y.H. Tseng, I.M. Hwang, W.Y. Lui, J. Nucl. Med. 45 (2004) 478. [11] S. Murty Goddu, D.V. Rao, W.E. Bolch, R.W. Howell, L.G. Bouchet, MIRD Cellular S Values, Society of Nuclear Medicine, Reston, VA, 1997. [12] H. Taniguchi, T. Daidoh, Y. Shioaki, T. Takahashi, Cancer 71 (1993) 50.
Nuclear Instruments and Methods in Physics Research A 569 (2006) 538–542 www.elsevier.com/locate/nima
Improved targeting of 5-[125I/131I]iodo-20-deoxyuridine to rat hepatoma by using lipiodol emulsion Hung-Man Yua, Hsin-Pei Yeha, Tien-Kui Changa, Kuang-Liang Huangb, Kuo-Tang Chuangb, Ren-Shen Liuc, Shyh-Jen Wangd, Jeng-Jong Hwanga, Kwan-Hwa Chie, Fu-Du Chena,f, Wuu-Jyh Linb, Chin-Hsiung Chenb, Hsin-Ell Wanga, a
Institute of Radiological Sciences, National Yang-Ming University, Taipei, Taiwan ROC b Institute of Nuclear Energy Research, Taoyuan, Taiwan, ROC c Department of Nuclear Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC d Department of Nuclear Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, ROC e Division of Radiation Therapy and Oncology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, ROC f Central Taiwan University of Science and Technology, Taichung, Taiwan, ROC Available online 20 September 2006
Abstract This study aims to assess whether emulsion of [125/131I]IUdR and lipiodol (IUdR/LP) can improve delivery of IUdR into hepatoma. Methods: In vitro release profile of IUdR from IUdR/LP to serum was performed. IUdR/LP was injected into N1-S1 hepatomabearing SD rat via hepatic artery and IUdR/normal saline (IUdR/NS) was used for comparison. Biodistribution, autoradiography, imaging and tumor DNA incorporation assay were performed. The radioactive metabolites in plasma and urine were analyzed. Radiation doses to tumor and organs were estimated. Results: IUdR released from lipiodol into serum was fast. There were longer retention, more DNA incorporation and higher radiation dose of IUdR in the tumor by using IUdR/LP. IUdR/LP deposited deep in the hepatomas. Only free iodide was found in the plasma and urine after injection of IUdR/LP. Conclusions: Hepatic artery injection of IUdR/LP emulsion could definitely enhance the tumor cell uptake and incorporation to DNA of *IUdR, prolong the tumor retention time and increase radiation dose to tumor. IUdR/LP may be an effective therapeutic agent for the treatment of hepatic tumors. r 2006 Elsevier B.V. All rights reserved. PACS: 47.63.mh; 79.20.Fv; 87.58.b; 87.58.Ji; 87.58.Sp Keywords: Hepatoma; Iododeoxyuridine; Auger electron; Lipiodol; Radionuclide therapy
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world [1]. Since hepatic tumors received blood predominately from the hepatic artery, intra-arterial (i.a.) treatment offers opportunities of selectively targeting the tumors [2]. Lipiodol, an ethyl ester of poppy seed oil fatty acids, has been found to accumulate Corresponding author. Tel.: +886 2 28267215; fax: +886 2 28201095.
E-mail address: [email protected] (H.-E. Wang). 0168-9002/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nima.2006.08.089
selectively in the hepatomas and remain there for a long time [3]. Lipiodol has been commonly used as a vehicle for targeted treatment of HCCs when labeled with radioisotopes [4] or mixed with chemotherapeutic agents [5]. When Auger electron emitters, such as 125I and 123I, are in the form of 5-iodo-20 -deoxyuridine (IUdR) and are incorporated into DNA, it becomes highly cytotoxic [6,7]. Intravenously (i.v.) administrate radioiodinated IUdR (*IUdR), however, is unlikely to be useful as an antitumor agent because of its non-specific uptake by all proliferating cells and its rapid dehalogenation. Previous studies in
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animals and humans have shown that efficient tumor targeting can be obtained after locoregional administration of *IUdR [8,9]. Based on the extreme radiotoxicity of *IUdR and the selective accumulation of lipiodol in hepatic tumor, we prepared a lipiodol emulsion mixed with *IUdR (IUdR/ LP) and demonstrated the improved tumor targeting of IUdR/LP after injection through the hepatic artery in a rat hepatoma model. The results of our study indicated that *IUdR/LP would be a useful agent for the treatment of hepatic tumors.
injection. Tissue samples of rats were embedded in paraffin and sequentially sectioned (5 mm), coated with emulsion (NTB-2, Eastman Kodak Company, Rochester, NY), dried and stored at 4 1C in light-tight boxes. Following exposure of at least 4 weeks, the emulsion were developed (D-19, Eastman Kodak Company, Rochester, NY), fixed (Polymax T, Eastman Kodak Company, Rochester, NY) and finally were stained with hematoxylin and eosin, dehydrated and mounted in Permount.
2. Materials and methods
The incorporation of [125I]IUdR into tumor DNA was determined after i.a. injection of [125I]IUdR/NSand [125I]IUdR/LP (11.1 MBq per rat). Tumor samples of rats were digested with enzyme cocktail solution. The radioactivity in DNA was determined using the method previously described [10].
2.1. Preparation of [125/131I]IUdR/NS and [125/ I]IUdR/LP
131
[125/131I]IUdR was synthesized as described previously [10] and purified with HPLC. *IUdR was resuspended in physiological saline and lipiodol to afford IUdR/NS and IUdR/LP, respectively. 2.2. Release profile of IUdR from IUdR/LP 1 mL of [131I]IUdR/LP (3.7 MBq) was introduced in 1 mL of prewarmed (37 1C) FBS. The mixture was placed on an orbital shaker (100 rpm). A sample (250 mL) of serum was withdrawn at every 5 min interval and the radioactivity was determined with a dose calibrator (CRC15R, Capintec, NJ). 2.3. Cell culture and N1-S1 Hepatoma-bearing rat model The N1-S1 hepatoma cells cultured and hepatomabearing Male Spraque–Dawley rat model were detailed in our previously report [4]. 2.4. Biodistribution of [125I]IUdR/NS and [125I]IUdR/LP The animal experiments were approved by the Institutional Animal Care and Use Committee of the National Yang-Ming University. The biodistribution studies was detailed in our previously report [4]. 2.5. Planar gamma imaging After i.a. injection of 11.1 MBq [131I]IUdR/NS or [ I]IUdR/LP, animals were anesthetized with isoflurane and static images were obtained at 48 h post injection on a gamma camera (Apex 400, Elscint, Israel) equipped with a high-energy pinhole collimator. The images were acquired using a preset-time acquisition mode (10 min). 131
2.6. Autoradiography After i.a. injection of 11.1 MBq [125I]IUdR/NS or [ I]IUdR/LP, animals were sacrificed at 48 h post 125
2.7. Incorporation of [125I]IUdR into nucleic acid
2.8. Determination of [131]IUdR and its metabolite in plasma and urine Venous blood samples were drawn at various time period (1–30 min) after i.v. and i.a. injection of [131]IUdR/ NS or [131]IUdR/LP (11.1 MBq). The plasma was analyzed using the HPLC system described above. Urine samples were analyzed by the same procedures. 2.9. Radiation dosimetry The diameter of the tumor cell and nucleus were measured 7 and 6 mm, respectively. The S-phase cell in hepatic tumor was proposed 15%. Following the radiation ¯ h ¼ Sti S i Þ, all the specific t values on dose formula ðD each source organ were derived from the biodistribution study, DNA incorporation study and mathematical model. The specific S value on each target–source organ pair was obtained from MIRDIII program and ‘‘MIRD cellular S value’’ [11]. Both the conventional and cell nucleus-level radiation dose of hepatic tumor were estimated as the tumor dose. 3. Results The percentage radioactivity in serum increased from 40% at 5 min to 100% at 30 min indicated that in vitro extraction of [131I]IUdR from lipiodol to serum could be considerably fast (Fig. 1A). The radioactivity accumulated in the tumor was much higher in IUdR/LP group (Table 2) than that in IUdR/NS group (Tables 1 and 2). The tumorto-normal liver ratio reached maximum (3.85) at 24 h and (63.88) at 0.5 h after IUdR/NS and IUdR/LP injection, respectively. Except liver, the radioactivity in normal tissues was about the same grade compared with that of IUdR/NS group (data not shown). The scitigraphic images of [131I]IUdR/LP clearly delineated the hapatoma (Fig. 2B), but gave only blurred images for [131I]IUdR/
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NS (Fig. 2A). In autoradiographic study, the radioactivity retained in tumor in the IUdR/LP group (Fig. 3B) was higher than that in the IUdR/NS group (Fig. 3A). In IUdR/LP group, [125I]IUdR was delivered into the inner areas of the tumor, whereas only cells on the periphery of the tumor were labeled in the IUdR/NS group. The autoradiograms of the normal tissue (liver, lung, stomach, small intestine and bone marrow) sections showed only background silver grain density (data not shown). The IUdR/LP group exhibited much higher radioactivity incorporation in the DNA of tumor cell than that of the IUdR/NS group (Table 3). For IUdR/LP group, the
accumulated radioactivity (mBq) in DNA per tumor cell increased from 0.022 at 1 h to 1.497 at 72 h post injection. At 30 min after i.v. or i.a. injection of IUdR/NS, no more [131]IUdR can be detected in the plasma (Fig. 1B). For IUdR/LP, even at the first minute after i.a. injection, there was no intact [131]IUdR, only free 131I was found in the plasma. In all urine samples, the radioactive species was represented solely by free 131I. The retention time of
Fig. 1. (A) The in vitro release profile of [131I]IUdR from lipiodol to serum. (B) Percentage radioactivity of intact [131I]IUdR in plasma samples after i.v. injection of [131I]IUdR/NS (E), after i.a. injection of [131I]IUdR/ NS ( ) or [131I]IUdR/LP (m).
Fig. 2. Planar gamma images of SD rats bearing N1S1 hepatoma at 48 h after i.a. injection of 11.1 MBq [131I]IUdR/NS (A) and [131I]IUdR/LP (B). Selective uptake of [131I]IUdR in hepatoma was clearly observed in lipiodol group.
Table 1 Biodistribution of [125I]IUdR in tumor and other normal tissues of N1S1 hepatoma-bearing SD rats after intra-arterial injection of 3.7 MBq IUdR/NS (n ¼ 5) Organ
%ID/g7SD 0.5 h
1h
4h
12 h
24 h
48 h
72 h
Blood (B) Lung Liver (L) Tumor (T)
0.9970.02 0.5370.06 0.4270.01 0.5070.02
0.8870.05 0.4670.06 0.3370.04 0.4170.05
0.7970.20 0.3970.07 0.3070.06 0.4270.11
0.3270.13 0.1770.06 0.1270.04 0.2370.10
0.0870.06 0.0570.04 0.0370.02 0.1270.11
0.0170.00 0.0170.00 0.0170.00 0.0170.01
0.0070.00 0.0070.00 0.0070.00 0.0170.01
T/B T/L
0.50 1.17
0.46 1.26
0.54 1.42
0.72 2.00
1.62 3.85
1.93 2.53
6.003 2.050
Table 2 Biodistribution of [125I]IUdR in tumor and other normal tissues of N1S1 hepatoma-bearing SD rats after intra-arterial injection of 3.7 MBq IUdR/LP (n ¼ 5) Organ
%ID/g7SD (h) 5 min
15 min
0.5 h
1h
4h
12 h
24 h
48 h
72 h
Blood (B) Lung Liver (L) Tumor (T)
2.0170.46 0.9170.18 2.2970.66 63.52715.45
1.5070.11 0.8370.06 1.5670.80 55.86735.79
1.1170.21 0.6170.07 0.5770.06 36.23715.74
0.9770.20 0.4570.15 0.4870.12 24.28710.64
0.7370.09 0.3870.08 0.4070.09 6.3472.54
0.5270.26 0.2470.12 0.2470.15 0.7770.49
0.1070.06 0.0970.04 0.1170.06 0.3570.11
0.0170.01 0.0170.01 0.0270.01 0.1170.02
0.0070.00 0.0170.01 0.0470.03 0.17070.134
T/B T/L
31.63 27.77
37.15 35.75
32.71 63.88
25.16 50.78
8.68 15.79
1.48 3.22
3.53 3.29
9.60 5.35
74.82 4.37
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Fig. 3. Autoradiographs of thin section from tumor of N1-S1 hepatoma-bearing rat at 48 h after i.a. injection of 11.1 MBq [125I]IUdR/NS (A) and [125I]IUdR/LP (B).
Table 3 The average radioactivity incorporated in DNA per tumor cell after i.a. injection of 11.1 MBq [125I]IUdR/NS and [125I]IUdR/LP Time after injection (h)
1 4 24 48 72
mBq/DNA of cell [125I]IUdR/NS
[125I]IUdR/LP
0.00270.001 0.00670.002 0.00970.004 0.00570.002 Not analyzed
0.02270.012 0.02870.010 0.60370.294 1.25270.598 1.49770.758
*IUdR in tumor (Table 4) was much longer for IUdR/LP (86.52 h) than that for IUdR/NS (9.37 h). The radiation dose to tumor was almost ten folds higher for IUdR/LP (6.84 Gy/MBq) than that for IUdR/NS (0.69 Gy/MBq). The relatively low liver dose from both IUdR/NS (0.0008 Gy/MBq) and IUdR/LP (0.0011 Gy/ MBq) indicated minimal radiation injury to normal organs via i.a. injection. 4. Discussion In this study, [125/131I]IUdR was mixed with lipiodol to give a clear and sterilized IUdR/LP emulsion. The hydrophilicity of IUdR facilitated the extraction of it from lipiodol to serum. The release profile of IUdR/LP in serum (Fig. 1A) suggested the facile in vivo release of IUdR from IUdR/LP to tissues where lipiodol retained. The IUdR/LP was i.a. injected to optimize the accumulation of IUdR in
tumor and to minimize its incorporation into normal tissues. The results of biodistribution (Table 2) showed that the radioactivity accumulated in tumor declined considerably with time within 4 h after i.a. of IUdR/LP, which was in agreement with the fast in vitro release profile of IUdR from IUdR/LP to serum. However, the incorporation of IUdR into DNA of tumor cells (Table 3), increased significantly by using IUdR/LP. It is noteworthy that except for liver, the radioactivity in normal tissues was almost of the same magnitude for IUdR/LP and IUdR/NS. Lipiodol increased the tumor uptake, both in tissue and DNA levels, of IUdR while sparing the normal tissues from radiation injury. In autoradiography study, only the peripheral cells of the tumor were labeled in IUdR/NS group (Fig. 3A), whereas IUdR was delivered into the inner areas of the tumor in IUdR/LP group (Fig. 3B). Taniguchi et al. [12] have shown that the delivery of BUdR to liver tumors was enhanced after i.a. infusion of BUdR/lipiodol suspension. They showed that hepatic tumors had cells incorporated BUdR in their deepest area. Our findings suggested that, due to the tumor vessel embolization with lipiodol, IUdR was incorporated into cells closed to the tumor vessel (Fig. 3B) and the angiogenesis status of hepatic tumor is critical to the effective tumor targeting of IUdR. Mariani et al. [9] have shown a virtually 100% first-pass hepatic deiodination of unincorporated [123I]IUdR after i.a. infusion of [123I]IUdR in patients with liver metastases from colorectal cancer. Our study showed that even at 1 min after i.a. injection of IUdR/LP, the only radioactive species that could be detected in blood was [131I]I. So IUdR/LP can
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Table 4 Estimated radiation dose to tumor and liver after i.a. injection of [131I]IUdR/NS and [131I]IUdR/LP (radiation dose unit: Gy/MBq) Formulation
131
[ I]IUdR/NS [131I]IUdR/LP
Tumor
Liver
Tumor/liver dose ratio
Residence time (h)
Radiation dose
Residence time (h)
Radiation dose
9.37 86.52
0.694 6.835
4.41 5.85
0.0008 0.0011
minimize the systemic uptake of [131I]IUdR by normal tissues. Due to the prolonged retention of IUdR/LP in tumor, the estimated radiation dose (Table 4) to tumor for IUdR/LP was almost ten folds higher than that for IUdR/ NS, whereas the doses to liver were both very low. This suggested that lipiodol was washed out easily from normal liver tissue and was regarded generally safe as radionuclide vehicle.
5. Conclusion This study showed that lipiodol, as a vehicle of n.c.a. *IUdR, can improve the targeting, specific accumulation and radiation dose of *IUdR to the tumor cells. IUdR/LP has been demonstrated as a potential therapeutic agent for the treatment of hepatic tumors.
Acknowledgments This work was supported by grants from the National Science Council (NSC 90-2314-B-010-030, NSC 93-3112-B010-019, NSC 94-2623-7-010-001).
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