On-line version ISSN 1678-4324
Braz. arch. biol. technol. vol.54 no.1 Curitiba Jan./Feb. 2011
HUMAN AND ANIMAL HEALTH
Ilknur Demir; Fazilet Zumrut Biber Muftuler*; Perihan Unak; Cigdem Acar
Department of Nuclear Applications; Institute of Nuclear Sciences; Ege University; 35100; Bornova, Izmir-Turkey
In this study, BevMab was conjugated with the bifunctional chelating agent [diethylenetriamine pentaacetic acid (DTPA)] and the product (BevMab-DTPA) was labeled with 99mTc using stannous chloride reducing method. The quality control studies of radiolabeled compound (99mTc-BevMab-DTPA) were done with Thin Layer Radio Chromatography (TLRC) and High Performance Liquid Radio Chromatography (HPLRC) methods (% 95 <) to confirm the labeling efficiency. High radiochemical yield [98.07 % ± 2.17 (n = 13)] was obtained by TLRC method. Biodistribution studies of 99mTc labeled BevMab-DTPA was run on healthy female and male Albino Wistar rats. The distribution figures demonstrated that the radiolabeled compound was eliminated through the kidneys and accumulated in urinary bladder. The values of the BevMab-DTPA uptakes were similar in heart, blood, liver and spleen in both sexes.
Key words: Angiogenesis, Bevacizumab, 99mTc
Angiogenesis, the forming of new blood vessels, plays a crucial role in many physiological and pathologic processes, including tumor growth, rheumatoid arthritis and diabetic retinopathies. Vascular endothelial growth factor (VEGF), released by tumor cells, is an important growth factor in tumor angiogenesis (Lin et al., 1999; Nagengast et al., 2007). Considering the critical role of VEGF in tumour development and recent reports on the positive impact of VEGF targeted therapy, it has became one of the most exciting fields of cancer investigation. A number of strategies have been pursued to target the VEGF, including monoclonal antibodies and soluble forms of the VEGF receptor (Jimeno and Funes, 2005). Monoclonal antibodies were used to prevent the activity of the VEGF. These molecules have been the most effective tools used in the treatment of cancer (Ruddon, 2007). Studies have focused on the use of radionuclide antibody conjugates since these reagents offer the potential for both therapy and specific nuclear imaging (Ruegg et al., 1990; Ünak, 2002). Bevacizumab is a recombinant, humanized monoclonal antibody directed against VEGF. It blocks VEGF induced tumor angiogenesis by binding, thereby neutralizing VEGF (Lin et al. 1999; Nagengast et al., 2007). It has an antitumor potential on metastatic colon carcinoma, non small cell lung cancer, breast, pancreas, prostate, ovary, urinary and central nervous system tumors (Papatsoris et al., 2005; Heinemann et al., 2009; Patel et al., 2009). The combination of anti-VEGF antibody and chemotherapy in nude mice having human cancer xenografts has greater influence than chemotherapy alone or the antibody alone (Marty et al., 2006). To date, Bevacizumab has been labeled with different radionuclides. 89Zr and 111In labeling of Bevacizumab resulted in high labeling efficiencies and adequate preservation of VEGF binding properties by using human ovarian cancer (SKOV-3) bearing athymic nude mice (Nagengast et al., 2007).
125I-Bevacizumab was developed to inhibit the effects of VEGF in the treatment of solid tumors (Lin et al., 1999). Abbas et al. (2008) used a series of bifunctional chelating agents to prepare 213Bi and 206Bi/206Bi labeled Bevacizumab. To our knowledge, there is no report on direct labeling of Bevacizumab with 99mTc using stannous chloride reducing method and biodistribution on healthy rats. Therefore, the aim of the present work was to label Bevacizumab with 99mTc and investigate the biodistribution of 99mTc labeled Bevacizumab on healthy female and male Albino Wistar rats.
MATERIALS AND METHODS
99mTcO4 was supplied by Monrol, Turkey. Bevacizumab was purchased from Roche San. A.Ş., Istanbul, Turkey. All other chemicals were supplied from Merck Chemical Co and Aldrich Chemical Co and used as supplied.
Synthesis of BevMab-DTPA
The synthesis of BevMab-DTPA was performed according to Nagengast et al., 2007 with some modifications (Nagengast et al., 2007). BevMab was purified from other excipients by ultrafiltration (Vivaspin-2 Sartarious, Germany) and diluted in water for injection to 5 mg/mL. The crude product (purified BevMab) was stored at -20 ºC. Five mg of purified Bevacizumab was conjugated with DTPA. To obtain a hydrophilic anti angiogenic compound, 5 mg of purified Bevacizumab was adjusted to pH 9.5 (50 mmol/ L Na2CO3, pH 9.5) and 50 M excess DTPA was added. After 1 h incubation at room temperature, the mixture was purified by ultracentrifugation with ammonium acetate (50 mmol/L, pH 5.5) to eliminate the excess unconjugated DTPA. Purified conjugated Bevacizumab (BevMab-DTPA) was diluted (5 mg/mL) in ammonium acetate and stored at -20 ºC in the dark until the analysis (Fig.1).
Radiolabeling procedure with 99mTc
Labeling was performed by adding 555 MBq (15 mCi) / 300 µL of 99mTcO4 and 50 µL of SnCl2 (1 mg SnCl2.2H2O in 1 mL HCl) into 100 µg (150 µL) of BevMab-DTPA solution. The pH was adjusted to 5 with 1 M NaOH solution. The reaction mixture was shaken and allowed to stand for 30 min at room temperature.
Quality control procedures
High Performance Liquid Radio Chromatography (HPLRC)
A low pressure gradient HPLC system (LC-10ATvp quaternary pump, SPD-10A/V UV detector, a syringe injector equipped with a 1 mL loop and 7-µm VP 250/21 Nucleosil 100-7 C18 column (Macherey - Nagel) was used for the the prufication experiments. The eluante was collected with a FRC-10A fraction collector (Shimadzu), equipped with a RAD501 single channel analyzer, having a Cd (Te) solid-state detector. The flow rate was set to 5.0 mL/min. A 5-µm EC 250/4.6 Nucleodur 100-5 C18 column (Macherey - Nagel) and a syringe injector equipped with a 20 µL loop was used. The flow rate was set to 1.0 mL/min. UV detection was achieved at 240 nm and 280 nm. The mobile phase system consisted of 40% water - 60% acetonitrile.
Thin Layer Radio Chromatography (TLRC)
TLRC was performed with ITLC-SG (Merck-5554) using 1.5x10 cm size plates. The samples of 99mTc-DTPA and 99mTc-BevMab-DTPA (5 µL) were applied. TLRC solvent (0.004 M ammonium acetate / methanol 9:1 v/v) was used as mobile phase 1 and SF (% 0.9 NaCl) was used as mobile phase 2 to determine the radiochemical purity. Then, the TLRC plates were counted by TLC Scanner (BioscanAR 2000). The Rf values and labeling yield were determined.
Stability study of 99mTc-BevMab-DTPA in human serum
In vitro stability of 99mTc-BevMab-DTPA in human serum was determined by incubating (66.66 µg) 100 µL of the labeled compound with 300 µL of human serum at 37 ºC. The aliquots were then analyzed at time intervals of 30, 60, 240 and 1440 minutes by TLRC after labeling and their radioactivities were measured.
Determination of the partition coefficient (logP) for the complex
Lipophilicity (logP) of the radiotracer was measured as follows: 0.1 mL of 99mTc radiolabeled compound was added to an equal volume of 1-octanol and phosphate buffer (pH 7.0) in a centrifuge tube. The resulting solution was mixed for 1 min and centrifuged for 30 min at 2500 rpm. The radioactivity of each layer was counted by Cd(Te) detector. Each measurement was repeated three times. The partition coefficient, P, was calculated using the equation; P = (cpm in octanol-cpm in background) / (cpm in buffer-cpm in background) (Biber et al., 2010). The final partition coefficient value was usually expressed as log P. Theoretical log P calculations were done with ACD/logP software (Version 6.0 for Microsoft Windows).
Biodistribution studies on female and male Albino Wistar rats
Experiments with animals were approved by the Institutional Animal Review Committee of Ege University. 99mTc labeled product was sterilized by passing through a 0.22 µm membrane filter. Then, it was injected into the tail vein of nine female and nine male albino Wistar rats (4 µg / each rat), which were 150-200 g in weight. They were maintained under controlled room conditions (22±5 ºC, 12 h of light/dark cycle), water and normal diet (adlibitum). The activity was approximately 400 µCi (0.2 mL)/ each rat. The rats were sacrificed at 60, 240 and 420 minutes post-injection under sodium penta borate anesthesia and tissues of interest were removed. Blood samples were taken, and the organs were excised. All the tissues were weighed and counted by Cd(Te) detector. The percent of radioactivity per gram of tissue weight (% injected activity / g tissue) was determined.
Differences in the mean values of the measured activities were evaluated statistically by the SPSS 13 program (Univariate Variance Analyses and Pearson Correlation). Probability values <0.05 were considered significant. Pearson correlation was carried out among different organs for 99mTc-BevMab-DTPA.
RESULTS AND DISCUSSION
Quality studies were performed by HPLRC and TLRC. HPLC chromatograms and Retention Time (Rt) values of 99mTc and 99mTc-BevMab-DTPA from Cd (Te) detector were given in Fig. 2.
HPLRC results showed that Rt value of 99mTc-BevMab-DTPA was different from 99mTc. By TLRC, Rf values of 99mTc-BevMab-DTPA, Reduced 99mTc and 99mTcO4-1 were 0.84, 0.02, 0.90 respectively when using mobile phase-1 (as seen in Table 1).
High radiochemical yield [98.07 % ± 2.17 (n = 13)] was obtained by TLRC method. The stability of labeled BevMab-DTPA in human serum was investigated at intervals of 30, 60, 240 and 1440 minutes after radiolabeling by the TLRC method. The results demonstrated that approximately 88 % of 99mTc- BevMab-DTPA existed as an intact complex in human serum up to 1440 minutes (Fig. 3).
Theoretical log P calculations for the uncharged molecule were done with ACD/logP software (Version 6.0 for Microsoft Windows) which for DTPA was -2.08 ± 0.86. Experimental logP value of 99mTc-BevMab-DTPA (-1.14 ± 0.18) was different from logP value of 99mTc-DTPA (-1.08 ± 0.20) according to ACD/lopP Algorithm programme. It is known that monoclonal antibodies are lipophilic compounds that are distributed throughout the body. The lipophilicity of BevMab was reduced by DTPA conjugation in this study.
Acar et al. (2007) have reported that, chelate charge and lipophilicity might play an important role in biologic behavior. It is also known that the bifunctional chelating agent (BFCA) can profoundly influence the biodistribution pattern and excretory route, and this in Vivo behavior can be predicted somewhat by the in vitro measurement of parameters such as stability and lipophilicity (Schmitt et al., 2005; Decristoforo et al., 2000). These results were confirmed by some others also (Ertay et al., 2005; Acar et al., 2007).
The distribution figures demonstrated that the radiolabeled compound was eliminated through the kidneys and accumulated in urine bladder in both the female and male rats. This was with 0.5 % ID/g almost the same for both the sexes. It is known that DTPA excretes through kidneys (Trejtnar and Laznicek, 2002). Conjugation of BevMab with DTPA gives the molecule a less lipophilic and more hydrophilic structure, which promotes the elimination through the kidney.
As demonstrated in Figure 4 and 5, the values of the BevMab-DTPA uptakes were similar in heart, blood, liver and spleen in both the sexes. Large intestines uptakes in the male rats were significantly higher (p<0.05). Compared to 240 min (0.07 %ID/g and 0.83 %ID/g), 11.85 times more activities were observed at 420 min post-injection (Fig. 5). Figure 5 also demonstrated that the radiolabeled compound had accumulated in the stomach together with large intestines up to 420th minutes.
No significant radioactivity uptakes were observed in the testis. In the prostate, the 60 min uptake activity was 12.25 times higher than 240 min, and 6.12 times higher than 420 min values. Statistical analysis according to Pearson corelation supported the biodistribution results (P<0.05). The highest uptake activities were observed in the stomach of both the sexes. With the DTPA, a more hydrophilic derivative of BevMab was generated.
The uptake of labeled BevMab-DTPA in stomach for the female rats (25.57%ID/g (P<0.05)) reached maximun at 60 min. The uptakes decreased with time. On the other hand, the highest uptake in the stomach for the male rats (18.22%ID/g (P<0.05)) was observed at 60 min. The stomach uptake of the female rats were significantly higher than the male rats' stomach at 60 and 240 min (Figs 4 and 5, respectively). Female rats' stomach presented 18 times higher uptake activity compared to male rats' stomach at 240 min postinjection particularly. This high stomach uptake for both the sexes could be due to the fenestration found on the capillary endothelial of endocrine organs, gastrointestinal system, kidney glomeruli and in some areas of brain (Roberts and Palade 1997; Esser et al., 1998).
99mTc-BevMab-DTPA is a less lipophilic structure which promotes the elimination through the kidney. Pillai et al. (1999) have similarly found that the compounds of carboxylic acid were more hydrophilic and eliminated through the kidney. However, the data demonstrated that the male rats had three times faster elimination in the first hour than the female rats (1.57 and 0.48%ID/g, respectively). The elimination of BevMab-DTPA was similar in both the sexes at 240 and 420 minutes.
It has been reported that VEGF is found in the heart, testis and kidneys (Jakeman et al., 1992). Lin and coworkers (1999) have demonstrated that 125I labeled BevMab showed high radioactivity at 48 h postinjection in the heart and testis of healthy rabbits, together with their urine bladder and kidney. This could be due to the reason that the hydrophilic analogues are less uptaken by the liver and lung to the hydrophobic compounds (Delpassand et al., 1996). Another report showed that 213Bi-bevacizumab had high renal uptake but low liver and spleen uptake (Abbas et al., 2008).
The radiolabeled yield of BevMab-DTPA was over 95% and it was stable during the period at 37 ºC in human serum media. Further studies with animal models with tumors and cell culture experiments should be done in order to explore if 99mTc-BevMab-DTPA could be used as an In Vivo VEGF imaging agent, early tumor detection and inhibition of tumor growth.
We thank to Resit Demir for editing the English language. This work is supported by Ege University Research Fund (contact no 2008 NBE 008).
Abbas, S. M., Song, E. Y., Raja, C., Beretov, J., Morgenstern, A., Apostolidis, C., Russell, P. J., Kearsley, J. H., Abbas, K., Allen, B. J. (2008), Preparation And Testing Of Bevacizumab Radioimmunoconjugates With Bismuth-213 And Bismuth-205/Bismuth-206. Cancer Biol. and. Ther.., 10, 1548-1555. [ Links ]
Acar, C., Teksöz, S., Ünak, P., Biber Müftüler, F. Z., Medine, E.İ. (2007), Investigation Of New Bifunctional Agents: D-Penicillamine. J. Radioanal. Nucl. Chem., 273, 641-647. [ Links ]
Acar, C., Teksöz, S., Ünak, P., Biber Müftüler, F.Z., Medine, E.İ. (2007), Somatostatin With 99mTc And Biodistribution Studies On Rats. Cancer Biother Radiopharm, 22, 748-754. [ Links ]
Biber M. F. Z., Unak P., Yolcular S., Yurt Kilcar A., Ichedef C., Enginar H. and Sakarya S. (2010), Synthesis, Radiolabeling and In Vivo Tissue Distribution of an Anti-Oestrogen Glucuronide Compound, 99mTc-TOR-G. Anticancer Res., 30, 4, 1249-1256. [ Links ]
Decristoforo, C., Melendez-Alafort, L., Sosabowski, J. (2000), 99mTc-HYNIC-[Tyr3]-octreotide For İmaging Somatostatin-Receptor-Positive Tumors: Preclinical Evaluation And Comparison With 111In-Octreotide. J. Nucl. Med., 41, 1114-1119. [ Links ]
Delpassand, S.E., Yang, J.D., Wallace, S., Cherif, A., Quadri, M.S., Price, J., Joubert, A., Inoue, T. and Podoloff, A.D. (1996), Synthesis, Biodistribution And Estrogen Receptor Scintigraphy Of Indium-111-Diethylenetriamine Pentaacetic Acid-Tamoxifen Analogue. J. Pharm. Sci., 85, 553-559. [ Links ]
Ertay, T., Unak, P., Tasc, C., et al. (2005), 99mTc-exorphin C: A New Peptide Radiopharmaceutical For Tumor İmaging. J. Radioanal. Nucl. Chem., 265, 473-479. [ Links ]
Esser, S., Wolburg, K., Wolburg, H., Breier, G., Kurzchalia, T., and Risau, W. (1998), Vascular endothelial growth factor induces endothelial fenestrations in vitro. J. Cell Biol., 140, 947-957. [ Links ]
Heinemann, V., Stintzing, S., Kirchner, T., Boeck, S., Jung, A. (2009), Clinical relevance of egfr- and kras-status in colorectal cancer patients treated with monoclonal antibodies directed against the EGFR. Cancer Treat. Rev., 35, 262-271. [ Links ]
Jakeman, L.B., Winer, J., Bennett, G.L., Altar, C.A., and Ferrara, N. (1992), Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rattissues. J. Clin. Invest., 89, 244-253. [ Links ]
Jimeno, A., and Funes, H. (2005), Membrane receptor and antiangiogenic targeted therapies in the treatment of cancer. Curr. Cancer Ther. Rev., 1, 51-57. [ Links ]
Lin, Y. S., Nguyen, C., Mendoza, J. L., Escandon, E., Fei, D., Meng, Y. G., and, Modi, N. B. (1999), Preclinical pharmacokinetics, interspecies scaling, and tissue distribution of a humanized monoclonal antibody against vascular endothelial growth factor. J. Pharmacol. Exp. Ther., 288, 371-378. [ Links ]
Marty, C.B., Lebrun, F., Awada, A., and Piccart, M.J. (2006), Monoclonal antibody-based targeted therapy in breast cancer: Current Status and Future Directions. Drugs., 66, 1577-1591. [ Links ]
Nagengast, W. B.,Vries, B. G., Hospers, G. A., Mulder, N. H., Jong, J. R., Hollema, H., Brouwers, A. H., Van, D. G. A., Perk, L. R., and Lub, H. M. N. (2007), In Vivo VEGF imaging with radiolabeled bevacizumab in a human ovarian tumor xenograft. J. Nucl. Med., 48, 1313-1319. [ Links ]
Papatsoris, A.G., Karamouzis, M.V., and Papavassiliou, A.G. (2005), Novel biological agents for the treatment of hormone-refractory prostate cancer (HRPC). Curr. Med. Chem., 12, 277-296. [ Links ]
Patel, D., Bassi, R., Hooper, A., Prewett, M., Hicklin, D. J., Kang, X. (2009), Anti-epidermal growth factor receptor monoclonal antibody cetuximab inhibits EGFR/HER-2 heterodimerization and activation. Int. J. Oncol., 34, 25-32. [ Links ]
Pillai, M.R.A., Kothari, K., Banerjee, S., Samuel, G., Suresh, M., Sarma, H. D. and Jurisson. (1999), Radiochemical studies of 99mTc complexes of modified cysteine ligands and bifunctional chelating agents. Nucl. Med. Biol., 26, 555-561. [ Links ]
Roberts, W.G., and Palade, G.E. (1997), Neovasculature induced by vascular endothelial growth factor is fenestrated. Cancer Res., 57, 765-772. [ Links ]
Ruddon R. W. (2007), Cancer Biology 4th ed., Oxford University Press., Inc, U.S.A., 207. [ Links ]
Ruegg, C.L., Anderson-Berg., W.T., Brechbiel, M.W., Mirzadeh, S., Gansow, O.A., and Strand, M. (1990), Improved In Vivo stability and tumor targeting of bismuth-labeled antibody. Cancer Res., 50, 4221-4226. [ Links ]
Schmitt, A., Bernhardt, P., Nilsson, O., et al. (2005), Differences in biodistribution between 99mTc-depreotide, 111In- DTPA-octreotide, and 177Lu-DOTA-Tyr3-octreotate in a small-cell lung cancer animal model. Cancer Biother. Radiopharm., 20, 231-236. [ Links ]
Trejtnar, F., Laznicek, M. (2002), Analysis of renal handling of radiopharmaceuticals. Q. J. Nucl. Med., 46, 181-194. [ Links ]
Unak, P. (2002), Targeted Tumor Radiotherapy, Braz. Arch Biol. Technol., 45, 117-130. [ Links ]
Received: December 22, 2009; Revised: June 29, 2010; Accepted: October 11, 2010