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CANCER BIOTHERAPY & RADIOPHARMACEUTICALS Volume 19, Number 4, 2004 © Mary Ann Liebert, Inc.
Comparative Biodistribution of Potential AntiGlioblastoma Conjugates [111In]DTPA-hEGF and [111In]Bz-DTPA-hEGF in Normal Mice Vladimir Tolmachev,1 Anna Orlova,1 Qichun Wei,2 Alexander Bruskin,1 Jörgen Carlsson,1 and Lars Gedda1 1Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden 2 Department of Radiation Oncology, Second Hospital, Zhejiang University School of Medicine, Hangzhou, China ABSTRACT EGF-receptors (EGFR) are overexpressed in gliomas, as well as in tumors of breast, lung, and urinary bladder. For this reason, EGFR may be an attractive target for both visualization and therapy of malignant tumors using radioactive nuclides. Natural ligand of EGFR, epidermal growth factor (EGF) is a small 53-amino-acid protein. Low molecular weight of EGF may enable better intratumoral penetration in comparison to antibodies. [111In]DTPA-EGF was proposed for the targeting of glioblastoma and breast cancer, and its tumor-seeking properties were confirmed in animal studies. The aim of this study was to evaluate how the substitution of heptadentate DTPA for octadentate benzyl-DTPA (Bz-DTPA) effects the biodistribution of indium-labeled human EGF (hEGF) in normal NMRI mice. [111In]DTPA-hEGF and [111In]Bz-DTPA-hEGF, obtained by the coupling of ITC-benzyl-DTPA to hEGF, were injected into the tail vein. At 0.5, 1, 4, and 24 hours postinjection, the animals were sacrificed, and radioactivity in different organs was measured. The blood clearance of both conjugates was fast. The uptake of both conjugates in the liver, spleen, stomach, pancreas, intestines, and submaxillary gland was most likely receptor-mediated. The uptake in a majority of organs was similar. However, indium uptake in the case of [111In]DTPA-hEGF was significantly higher in the kidneys and bones. In conclusion, [111In]Bz-DTPAhEGF seems to have more favourable in vivo distribution in comparison to [111In]DTPA-hEGF. Key words: [111In]DTPA-hEGF, [111In]Bz-DTPA-heGF, tumor targeting, biodistribution, mice INTRODUCTION High-grade glioma, glioblastoma multiforme, is a lethal brain tumor. Conventional therapy, combining surgery and external beam irradiation, can provide only a few months’ survival.1,2 The proliferation of spreading cancer cells causes regrowth of the tumor, and, ultimately, the patient’s Address reprint requests to: Vladimir Tolmachev; Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University; Dag Hammarskolds weg, S-751 85 Uppsala, Sweden; Tel.: 146-18-471 34 14; Fax.: 146-18471 34 32 E-mail:
[email protected]
death. A possible solution for the selective eradication of single tumor cells was sought as a locoregional targeting of radionuclide therapy, using monoclonal antibodies against tenascin or EGFR.3–6 Promising results, such as prolonged progression-free survival and objective radiological responses, were documented in a number of clinical studies,5,7,8 but a curative effect was still not obtained. Poor diffusion of bulky antibodies in brain tissue might limit the efficiency of radioimmunotherapy of glioblastoma. The use of small, diffusible tumor-seeking agents may improve the outcome of treatment. Thus, the successful targeting of low-grade glioma distant 491
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metastases was demonstrated when small somatostatin analog [111In/90Y]DOTATOC was used.9–11 Unfortunately, a low expression of somatostatin receptors in glioblastoma multiforme precludes the use of this promising targeting vector. This urged us to search for an alternative lowmolecular-weight anti-glioma targeting conjugates. A possible target on glioblastoma tumor cells might be EGFR. An overexpression of this transmembrane protein was detected in a wide percentage of high-grade glioma.12–15 A natural ligand to EGFR, 53-amino-acids-long EGF, seems to be small enough to provide efficient diffusion through a healthy brain. In vitro experiments demonstrated the antitumor potential of 131I-labelled EGF.16 However, a poor cellular retention of radioiodine label was a major obstacle for the use of radioiodinated EGF for therapy. A DTPA-EGF conjugate17 seems to be a better targeting agent, as the use of metal chelate labeling improves an intracellular retention of radionuclides. It was demonstrated, in vitro, that such an approach increases cell-associated radioactivity in comparison to the use of radioiodinated counterpart.18,19 The selection of a radionuclide is of crucial importance for the success of radionuclide therapy. The high-energy beta-emitter yttrium-90 (T1/2 5 2.67 days), which is used in cancer treatment, is efficient against bulky tumors, but inefficient in deactivation of single spread tumor cells or their small clusters.20,21 On the opposite, nuclides that emit low-energy beta particles are well suited for the killing of spread cells.22 Indium-111 (T1/2 5 2.83 days) emits Auger and conversion electrons, having a tissue penetration of 0.02–10 mm and 200 to 500 mm, respectively, which render it as a candidate radionuclide for targeting therapy. In a number of clinical studies, 111In-labeled somatostatin analogs demonstrated an encouraging rate of objective tumor responses.23–25 A conclusion that peptide-receptor radionuclide therapy (PRRT) is feasible, also with 111In as a radionuclide has been done.24 Preclinical studies demonstrated selective cytotoxicity of [111In] DTPA-hEGF for MDA-MB-468 breast cancer cells.26 It was also found that [111In]DTPA-hEGF exhibited potent antiproliferative effects toward breast cancer cells at concentrations much lower than chemotherapeutic agents and equivalent to those produced by several Gy of high-dose-rate gamma radiation.27 Animal studies have demonstrated that [111In]DTPA-hEGF can target breast cancer xenografts in vivo28 and exhibits a strong 492
antitumor effect.29 This information led us to suppose that 111In-labelled EGF might be an efficient anti-glioblastoma targeting conjugate. A search for an EGF-chelator conjugate that stably binds not only 111In but also other radiometals prompted us to prepare [111In]benzylDTPA-EGF (Bz-DTPA-hEGF).18 In vitro characterization, using an EGFR-expressing glioma cell line U-343MGaCl2:6, showed that [111In]BzDTPA-EGF has a high affinity to EFGR (Kd value of 2.0 nM), is rapidly internalized, and the retention of cell-associated radioactivity is good. Favorable properties of [111In] Bz-DTPA-EGF enabled us to include this conjugate into a panel of possible anti-glioblastoma conjugates for further preclinical evaluation. The aim of this study was to evaluate how the substitution of heptadentate DTPA for octadentate Bz-DTPA affects the biodistribution of an indium label after the injection of hEGF-chelator conjugate in normal mice.
MATERIALS AND METHODS Materials [111In]InCl3 was from Mallinckrodt Medical B.V. (Petten, The Netherlands). All commercially available chemicals were of pro analyse grade or better. High-quality (resistance: 18 MOhm/cm3) Elga® water (VEOLIA Water Systems, Celbridge, Ireland) was used for the preparation of all solutions. Disposable NAP-5-size exclusion columns were from Pharmacia (Uppsala, Sweden) and the solid-phase extraction column, SPEC C18, were from Diagnostics, Inc. (Lake Forest, CA). Recombinant human epidermal growth factor, hEGF (Chemicon, Temecul, CA), was used in all experiments. ITC-Bz-DTPA (DTPA-benzyl-isothiocyanate) was synthesized according to a previously reported study.30 Dianhydride of DTPA (DADTPA) was purchased from Aldrich (St. Louis, MO). Coupling of Isothiocyanate-benzyl-DTPA and DTPA to hEGF hEGF solution in borate buffer, at a pH of 9.1 (8 ml, 40 mg), was added to a solution of ITC-benzyl-DTPA in the same buffer (50 ml, 1 mg/mL), and the mixture was shaken overnight. The next morning, 1 mL of borate buffer was added, and the mixture was passed through a SPEC C18 col-
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umn. The column was then washed with 0.5 mL of borate buffer, at a pH of 9.3, and 0.5 mL 5% acetonitrile in water, and the Bz-DTPA-hEGF conjugate was eluted with 0.5 mL of 50% acetonitrile in water. To change the buffer, the eluate containing Bz-DTPA-hEGF was loaded on a NAP-5 column pre-equilibrated with 0.1 M of acetate buffer, at a pH of 6.0. The separation was performed according to the manufacturer’s instructions, using acetate buffer, and the eluted high-molecular weight fraction was used for labeling. hEGF solution in borate buffer, at a pH of 9.1 (48 ml, 40 mg), was added to dianhydride DTPA (2.4-2.7 mg). The mixture was vigorously vortexed during 20 minutes, and the separation of nonconjugated DTPA was then performed in the same way as for ITC-benzyl-DTPA. A stock solution of 111In in 0.02 N HCl (5–20 MBq) was added to 6 mg of Bz-DTPA-hEGF or DTPA-hEGF in 0.1 M of ammonium acetate (pH 6.0). The reaction mixture was incubated for 1 hour at room temperature. The labeled conjugate was then purified on a NAP-5 using phosphate buffered saline (PBS) (pH 7.4). The labeled products were additionally analyzed using SPEC C18 columns. Native PAGE (7.5% Tris-HCl, Bio-Rad Laboratories, Herucles, CA) of both conjugates demonstrated the presence of a single radioactive band, confirming the uniform coupling of chelators to the amino groups of hEGF.
was injected before [111In]DTPA-hEGF or [111In]BzDTPA-hEGF. One hundred (100) mg (in 50 ml 0.9 % NaCl) nonradiolabeled recombinant hEGF was injected into the tail vein, and 0.5 hours later 30 ml [111In]DTPA-hEGF or 25 mL [111In]BzDTPA-hEGF (approximately 0.4 mg of each compound) was injected (as above). After an additional 0.5 hours, the animals were sacrificed, and the organs were collected. The weight of all organs was determined and the radioactivity was measured using an automated gamma counter with a 3-inch NaI(Tl) detector (1480 WIZARD, Wallac Oy, Turku, Finland). 111In was measured with the use of both photopeaks and the summation peak (energy setting from 140–507 keV). Organ values were calculated as a percentage of injected amount per gram of organ (% ID/g). An unpaired t test of the data was performed employing the GraphPad Prism software (GraphPad Software Inc., San Diego, CA). The differences were considered as significant if the p values were less than 0.05. RESULTS The kinetics of 111In radioactivity in the blood after an injection of [111In] DTPA-hEGF and [111In] Bz-DTPA-hEGF is shown in Figure 1. Both conjugates show quick blood clearance, with less than 1%ID/g of whole blood at 30 min
Animal Study The animal study was approved by the local Ethics Committee for Animal Research. Female NMRI mice (Möllegård, Denmark) (26–32g) were used. They were housed in a controlled environment and fed ad libitum. Mice were injected into the tail vein with 30 ml [111In]DTPA-hEGF or 25 ml [111In]BzDTPA-hEGF (approximately 100 kBq and 0.4 mg per mouse) and after 0.5, 1, 4, and 24 hours (3 animals/time) the animals were sacrificed. For anesthesia, a mixture of 1 mL Ketalar and 50 mg/mL (Pfizer, New York), 0.25 mL Rompun 5 mg/ml (Bayer, AG, Leverkusen, Germany), and 3.75 mL of water was used, injected intraperitoneally (i.p.) with 0.2 mL/10 g body weight. The heart was punctured by a preheparinized (Heparin Leo 25000 IE/ml, Leo Oharma, Malmö, Sweden) syringe using a 23-G needle. Blood, urine, and organs were collected according to a standard protocol, including 20 organs. In some mice, an excess of EGF
Figure 1. Blood kinetics of [111In] DTPA-hEGF (closed diamonds) and [111In] Bz-DTPA-hEGF (crosses). Data presented as average of three animals. Error bars reflect maximum errors.
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p.i. However, the blood-associated radioactivity after injection of [111In] Bz-DTPA-hEGF was prominently, at least twice, higher than after an injection [111In] DTPA-hEGF at all time points. Radioactivity in the urine and feces was measured at each data point, but there was no opportunity to collect whole urine and feces during this study. For this reason, information about excretion pathways is of a semiquantitative nature. We can, however, observe that a lot of indium radioactivity (up to 190%ID/g of sample) is in the urine already at earlier time points, 0.5–1 hour p.i. Urinary excretion continues to play an im-
A
portant role in radioactivity elimination throughout the whole observation period. In feces, indium radioactivity was appreciable (up to 12%ID/g of sample) at later time points, 4 and 24 hours (data not shown). In order to elucidate in which organs the uptake of labeled conjugates is receptor-mediated, a biodistribution was measured at 30 minutes with and without the preinjection of a large amount of nonlabeled hEGF. The biodistribition data are shown in Figures 2 and 3. For clearness, uptake in liver (A) and kidneys (B) is shown separately from the uptake in other organs (C). For
B
C Figure 2. Influence of pre-injection of non-labelled hEGF on biodistribution of [111In] DTPA-hEGF. Uptake in liver (A) kidneys (B) and other organs (C) is shown. Data presented as average of 3 animals. Error bars reflect maximum errors.
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B
A
C Figure 3. Influence of pre-injection of non-labelled hEGF on biodistribution of [111In] Bz-DTPA-hEGF. Uptake in liver (A) kidneys (B), and other organs (C) is shown. Data presented as average of 3 animals. Error bars reflect maximum errors.
both conjugates, the preinjection of nonlabeled hEGF caused a significant decrease of radioactivity uptake in the liver. At the same time, the uptake in the kidneys increased. Preinjection also increased blood level of radioactivity at the measurement time. Uptake of [111In] DTPA-hEGF in the spleen, pancreas, intestines, and submaxillary salivary gland was significantly decreased. A tendency toward the decrease of radioactivity in the
same organs can be observed in the case of [111In]Bz-DTPA-hEGF as well, but as statistically significant, this difference was only in the case of the pancreas. Comparative distribution of radioactivity in mice is shown in Figures 4, 5, and 6. The maximum uptake for both [111In] DTPA-hEGF and [111In] Bz-DTPA-hEGF was observed in the liver and kidneys. Accumulation in other organs was 495
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Figure 4. Biodistribution of [111In] DTPA-hEGF (closed diamonds) and [111In] Bz-DTPA-hEGF (crosses) in organs with possible receptor-mediated uptake. Data presented as average of 3 animals. Error bars reflect maximum errors.
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Figure 5. Biodistribution of [111In] DTPA-hEGF (closed diamonds) and [111In] Bz-DTPA-hEGF (crosses) in heart, lungs, muscle, and brain. Data presented as average of 3 animals. Error bars reflect maximum errors.
appreciably lower, and organs, where uptake was presumably receptor-mediated (i.e., spleen, pancreas, intestines, and submaxillary salivary gland), showed higher uptake. Accumulation of radioactivity in the brain was very low for both conjugates, below 0.1 %ID/g during whole experiments. Both in the majority of organs, where receptor-mediated uptake can be deduced (Figure 4) and in other organs (Figure 5), there was no significant difference in the accumulation of conjugates. There were, however, two important exceptions. Accumulation of radioactivity in the kidneys and bones was significantly higher in the case of [111In] DTPA-hEGF (Figure 6). The ra-
tio of areas under the curve (AUC) for [111In] DTPA-hEGF and [111In] Bz-DTPA-hEGF, determined using the GraphPad Prism software (GraphPad Software Inc.), were 2.1 for kidney and 2.6 for bone. DISCUSSION Overexpression of EGFR in many types of tumors prompts one to consider this glycoprotein as a potential target for tumor-directed radionuclide therapy. Obviously, well-documented, highlevel expression of EGFR in hepatocytes is a serious obstacle for the systemic administration of 497
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Figure 6. Biodistribution of [111In] DTPA-hEGF (closed diamonds) and [111In] Bz-DTPA-hEGF (crosses) in kidney and bone. Data presented as average of 3 animals. Error bars reflect maximum errors.
anti-EGFR–targeting conjugates. However, the use of such conjugates is probably possible, when locoregional administration of antitumor conjugates is considered. Glioblastoma multiforme is an example of a disease to be treated. It should be noted that the locoregional administration of targeting a conjugate could not preclude its leakage into the systemic circulation. Even if the surgically created resection cavity is not connected with cerebrospinal fluid (CSF) reservoirs, a diffusible targeting vector could pass there through brain tissue. Circulation of CSF would then carry the labeled compound into the bloodstream. Other reasons for leakage, such as blood-brainbarrier disruption in tumor sites can also exist.31 For this reason, whole-body pharmacokinetics is an important factor for the selection of a targeting agent aimed for locoregional treatment. Such a consideration urged us to perform comparative whole-body biodistribution of [111In] DTPAhEGF and [111In] Bz-DTPA-hEGF. Preinjection of large excess of nonlabeled EGF was performed with the aim to identify organs with a possible receptor-mediated uptake of radiolabeled hEGF. Significant decrease in uptake of [111In] DTPA-hEGF in the stomach, liver, spleen, pancreas, intestines, and submaxillary salivary gland was observed in this experiment. Reduction of uptake in the same organs was also demonstrated for [111In] Bz-DTPA-hEGF—but only in the stomach, liver, and pancreas was the difference significant, in this case. Taking into account the difficulties associated with in vivo blocking of internalizing receptors, we may suppose that 498
the [111In] Bz-DTPA-hEGF uptake in these organs is receptor-mediated. This assumption is supported by the findings of earlier researchers, who reported the expression of EGF receptors in these organs.32–38 It is evident that even partial blockage of liver receptors significantly increases kidney uptake. This may be a good illustration of two major pathways of EGF-based conjugates’ disposition in vivo: through the liver and through the kidneys. Hindrance in liver removal leaves more EGF in the blood and for kidney filtration. In principle, such a situation may open an opportunity in blood-clearance manipulation aiming for the best dosimetry in clinics. Generally speaking, radioactivity levels after an injection of both [111In] DTPA-hEGF and [111In] Bz-DTPA-hEGF were similar in a majority of organs, with two important exceptions: Uptake in the kidney and bones was appreciably higher in the case of [111In] DTPA-hEGF. Bone accumulation could be explained by the release of 111In from the chelator, because 111In ability to accumulate in the bone marrow is well known, and was even used earlier for bone-marrow scanning.36,37 On the other hand, our previous in vitro experiments with [111In] DTPAhEGF and [111In] Bz-DTPA-hEGF,18 as well as experiments with labeled antibodies done by other investigators,38 did not reveal any difference in stability between these 111In-chelator complexes during exposure to blood plasma transferrin. For example, transchelation to blood serum proteins were 5.3% and 4.6% per day for [111In] Bz-DTPA-EGF and [111In] DTPA-EGF,
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respectively, in our test setting.18 No difference in the stability of 111In complexes with either DTPA or Bz-DTPA was found in liver homogenates,39 which gives a good reason to believe that the distinction of bone accumulation was not caused by a difference in liver processing of [111In] DTPA-hEGF and [111In] BzDTPA-hEGF after receptor-mediated uptake. Although the cause for this effect is not clear, the use of Bz-DTPA enables the reduction of dose appreciably to such a radiation-sensitive organ as the bone marrow. Elevated kidney uptake of radiolabeled antibody fragments and peptides is considered as a problem in the therapeutic application of such agents.40,41 Information about renal toxicity during peptide-receptor radionuclide therapy using [90Y] DOTATOC support this.42,43 Recent investigation shows that the radiation of the shortrange (maximal 10 mm) Auger electrons of 111In originating from the cells of the proximal tubules is not harmful for the renal function during [111InDTPA]octreotide treatment of neuroendocrinal tumors.44 However, the same group stated later that the radioactivity delivered to the kidney is a major dose-limiting factor in this kind of therapy.45 For this reason, a decrease of kidney uptake in the case of [111In] Bz-DTPA-hEGF may be a big advantage for radionuclide therapy, because it would enable an increase in the injected radioactivity and, possibly, to increase a tumor dose without an elevated renal dose. Receptor-mediated uptake in the liver is probably the most worrisome factor for the use of EGFR-targeting conjugates for systemic radionuclide therapy. The situation is quite different in the case of locoregional administration. In fact, it was earlier proposed to use hepatic clearance in order to improve tumor-to-nontumor localization ratios of radiolabeled antibodies for radioimmunodetection and radioimmunotherapy.46–49 Moreover, it was found that the direction of antibody clearance to hepatocytes has advantages, in comparison to the direction to Kupffer cells, as only hepatocytes can excrete degradation products into bile.46,48 CONCLUSION In conclusion, the comparison between the [111In] DTPA-hEGF and the [111In] Bz-DTPA-hEGF biodistributions in normal mice suggests that the [111In] Bz-DTPA-hEGF conjugate may offer ad-
vantages in terms of reducing the kidney and bone accumulation of radioactivity from radiopharmaceuticals reaching the systemic circulation. ACKNOWLEDGMENTS This work was performed with financial support from the Swedish Cancer Society (Cancerfonden) and the Swedish Royal Academy of Sciences. REFERENCES 1. Lang O, Liebermeister E, Liesegang J, et al. Radiotherapy of glioblastoma multiforme. Feasibility of increased fraction size and shortened overall treatment. Strahlenther Onkol 1998;174:629. 2. Nieder C, Nestle U, Ketter R, et al. Hyperfractionated and accelerated-hyperfractionated radiotherapy for glioblastoma multiforme. Radiat Oncol Investig 1999;7:36. 3. Arista A, Sturiale C, Riva P, et al. Intralesional administration of I-131-labeled monoclonal antibodies in the treatment of malignant gliomas. Acta Neurochir (Wien) 1995;135:159. 4. Riva P, Arista A, Sturiale C, et al. Glioblastoma therapy by direct intralesional administration of I-131 radioiodine-labeled antitenascin antibodies. Cell Biophys 1994;37:24. 5. Intrathecal 131I-labeled antitenascin monoclonal antibody 81C6 treatment of patients with leptomeningeal neoplasms or primary brain tumor resection cavities with subarachnoid communication: Phase I trial results. Clin Cancer Res 1996;2:963. 6. Riva P, Franceschi G, Riva N, et al. Role of nuclear medicine in the treatment of malignant gliomas: The locoregional radioimmunotherapy approach. Eur J Nucl Med 2000;27:601. 7. Riva P, Arista A, Sturiale C, et al. Treatment of intracranial human glioblastoma by direct intratumoral administration of 131I-labeled antitenascin monoclonal antibody BC-2. Int J Cancer 1992;51:7. 8. Cokgor I, Akabani G, Kuan CT, et al. Phase I trial results of iodine-131-labeled antitenascin monoclonal antibody 81C6 treatment of patients with newly diagnosed malignant gliomas. J Clin Oncol 2000;18:3862. 9. Merlo A, Hausmann O, Wasner M, et al. Locoregional regulatory peptide receptor targeting with the diffusible somatostatin analogue 90Y-labeled DOTA0-D-Phe1Tyr3-octreotide (DOTATOC): a pilot study in human gliomas. Clin Cancer Res 1999;5:1025. 10. Hofer S, Eichhorn K, Freitag P, et al. Successful diffusible brachytherapy (dBT) of a progressive low-grade astrocytoma using the locally injected peptidic vector and somatostatin analogue [90Y]-DOTA0-D-Phe1-Tyr3octreotide (DOTATOC). Swiss Med Wkly 2001;131: 640.
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