Research ArticleBasic Science Investigations

Synthesis and In Vitro and In Vivo Evaluation of Hypoxia-Enhanced 111In-Bombesin Conjugates for Prostate Cancer Imaging

Zhengyuan Zhou, Nilesh K. Wagh, Sunny M. Ogbomo, Wen Shi, Yinnong Jia, Susan K. Brusnahan and Jered C. Garrison
Journal of Nuclear Medicine September 2013, 54 (9) 1605-1612; DOI: https://doi.org/10.2967/jnumed.112.117986

Abstract

Receptor-targeted agents, such as gastrin-releasing peptide receptor (BB2r)–targeted peptides, have been investigated extensively in preclinical and clinical studies. In an attempt to increase the effectiveness of diagnostic or radiotherapeutic agents, we have begun to explore the incorporation of the hypoxia-selective prodrug 2-nitroimidazole into receptor-targeted peptides. Hypoxia is a well-known characteristic of many solid tumors, including breast, prostate, and pancreatic cancers. The aim of this approach is to use the hypoxia-trapping capability of 2-nitroimidazoles to increase the retention of the agent in hypoxic, BB2r-positive tumors. We have demonstrated that incorporation of one or more 2-nitroimidazoles into the BB2r-targeted peptide significantly increases the in vitro retention of the agent in hypoxic prostate cancer cells. The study described herein represents our first investigation of the in vivo properties of these hypoxia-enhanced BB2r-targeted agents in a PC-3 xenograft mouse model. Methods: Four 111In-labeled BB2r-targeted conjugates—111In-1, 111In-2, 111In-3, and 111In-4, composed of 2-nitroimidazole moieties of 0, 1, 2, and 3, respectively—were synthesized, labeled, and purified. The BB2r binding affinities, externalization, and protein-association properties of these radioconjugates were assessed using the BB2r-positive PC-3 human prostate cancer cell line under hypoxic and normoxic environments. The in vivo biodistribution and micro-SPECT/CT imaging of the 111In-1, 111In-2, and 111In-4 radioconjugates were investigated in PC-3 tumor–bearing severely combined immunodeficient mice. Results: All conjugates and natIn-conjugates demonstrated nanomolar binding affinities. 111In-1, 111In-2, 111In-3, and 111In-4 demonstrated 41.4%, 60.7%, 69.1%, and 69.4% retention, correspondingly, of internalized radioactivity under hypoxic conditions relative to 34.8%, 35.3%, 33.2%, and 29.7% retention, respectively, under normoxic conditions. Protein-association studies showed significantly higher levels of association under hypoxic conditions for 2-nitroimidazole–containing BB2r-targeted radioconjugates than for controls. On the basis of the initial 1-h uptake in the PC-3 tumors, 111In-1, 111In-2, and 111In-4 demonstrated tumor retentions of 1.5%, 6.7%, and 21.0%, respectively, by 72 h after injection. Micro-SPECT/CT imaging studies of 111In-1, 111In-2, and 111In-4 radioconjugates resulted in clear delineation of the tumors. Conclusion: On the basis of the in vitro and in vivo studies, the BB2r-targeted agents that incorporated 2-nitroimidazole moieties demonstrated improved retention. These results indicate that further exploration into the potential of hypoxia-selective trapping agents for BB2r-targeted agents, as well as other targeted compounds, is warranted.

According to the American Cancer Society estimates, prostate cancer is the second leading cause of death and accounts for 29% of all new cancer cases for men in the United States (1). The gastrin-releasing peptide receptor (BB2r) has been thoroughly investigated as a diagnostic and therapeutic target for prostate and other cancers because of the high expression of the receptor on neoplastic relative to normal tissues (2,3). To date, a variety of BB2r-targeted agents have been developed using the BBN(7-14)NH2 sequence (Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) (4–7). The developed BB2r-targeted agents, as with most low-molecular-weight, receptor-targeted drugs, demonstrate rapid targeting of receptor-positive tumors and swift clearance from nontargeted tissues. However, the disadvantage of many of these agents is low retention at the tumor site because of intrinsically high diffusion and efflux rates. The lower tumor retention can substantially reduce the diagnostic and therapeutic efficacy of the agent and its potential for clinical translation.

Tissue hypoxia is the result of an inadequate supply of oxygen. In most solid cancers, hypoxic regions commonly exist because of a chaotic vascular architecture, which impedes delivery of oxygen and other nutrients. A recent clinical investigation found that 63% (median, n = 247) of prostate tumors gave pO2 measurements of less than 1.3 kPa (10 mm Hg; tissues less than this are generally defined as hypoxic) (8). The extent of hypoxia in tumors appears to be strongly associated with the aggressiveness of the tumor phenotype, therapeutic resistance, and patient prognosis (9). Because hypoxia is not present in most normal human tissues, a variety of bioreductive, hypoxia-selective prodrugs has been developed for the purpose of diagnostic and therapeutic applications for cancer. Nitroimidazoles have been used extensively in basic and clinical investigation as diagnostic imaging agents (10,11). In hypoxic environments, nitroimidazoles undergo a series of enzymatic reductions—mediated by nitroreductase enzymes—leading to the formation of strong electrophiles, which can irreversibly bind to intracellular nucleophiles, thereby trapping the agent in the hypoxic tissue (12). Recently, radiohalogenated nitroimidazoles, such as 18F-fluoromisonidazole, 18F-1-α-d-(2-deoxy-2-fluoroarabinofuranosyl)-2-nitroimidazole, and 123I-iodoazomycin arabinoside, have been used clinically to detect hypoxia in tumors (13–15).

One focus of our laboratory is the development of tumor-selective chemical moieties to increase the retention of receptor-targeted agents. We have begun investigating the inclusion of 2-nitroimidazoles into the structure of BB2r-targeted peptides. We have previously demonstrated that these hypoxia-enhanced BB2r-targeted peptides significantly increase retention in hypoxic PC-3 human prostate cancer cells (16). From these studies, it was determined that the proximity of the 2-nitroimidazole relative to the pharmacophore had a substantial impact on BB2r binding affinity. Herein, we present the synthesis and in vitro properties of hypoxia-enhanced BB2r-targeted radioconjugates with extended linking groups to improve BB2r binding affinity. Additionally, using biodistribution and micro-SPECT/CT imaging studies, we report the first, to our knowledge, in vivo investigation of these agents in a PC-3 xenograft mouse model.

MATERIALS AND METHODS

Full details regarding the chemicals, equipment, and methodology used in this manuscript are presented in the supplemental materials (available at http://jnm.snmjournals.org).

Cell Lines and Xenograft Models

Prostate cancer (PC-3) cell lines were obtained from American Type Culture Collection and cultured under vendor-recommended conditions. All animal experiments were conducted in accordance with the Principles of Animal Care outlined by National Institutes of Health and approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. Four-week-old Institute of Cancer Research severely combined immunodeficient (SCID) mice were obtained from Charles River Laboratories. Food and water were given ad libitum. Each animal was kept in individual cages equipped with a HEPA air filter cover in a light- and temperature-controlled environment. Bilateral PC-3 tumors were induced by subcutaneous injection of 5.0 × 106 cells in Matrigel (BD Biosciences). The tumors were allowed to grow to 0.1–1 g (4–6 wk after inoculation) before the mice were used in pharmacokinetic studies.

Synthesis and Radiolabeling of 2-Nitroimidazole Acetic Acid (2-NIAA) Bombesin Conjugates

Peptides 1, 2*, 3*, and 4* were synthesized using an automated solid-phase peptide synthesizer (CEM) with traditional Fmoc chemistry using a Rink Amide resin (Nova Biochem). Electrospray mass spectrometry was used to determine the molecular mass of the prepared peptides. All conjugates were peak-purified to 95% or greater purity and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) before in vitro and in vivo investigations. The 2-NIAA was synthesized as previously described (16). The 2-NIAA was manually coupled to the ε-amino group of the lysine residue for peptides 2*, 3*, and 4* using standard amidation chemistry, peak-purified by RP-HPLC, and characterized by mass spectrometry. For the convenient characterization of the 111In-bombesin conjugates, naturally abundant natIn was used to substitute for 111In in the electrospray mass spectrometry and in vitro binding studies. Isolated yields were 57.6%, 28.4%, 56.8%, and 40.4% for natIn-1, natIn-2, natIn-3, and natIn-4, respectively. All natIn-conjugates were of 95% or greater purity before mass spectrometric characterization. Radiolabeling was performed on all conjugates by mixing 100-μg samples with 37 MBq of 111InCl3 in ammonium acetate buffer (1 M, 200 μL, pH 5.5). The solution was heated for 60 min at 90°C. The resulting specific radioactivities were 0.64, 0.71, 0.78, and 0.86 MBq/nmol for 111In-1, 111In-2, 111In-3, and 111In-4, respectively. To separate radiolabeled peptides from unlabeled peptides on HPLC, 4–5 mg of CoCl2 were then added and incubated for 5 min at 90°C to increase the hydrophobicity of unlabeled conjugates. The solutions were allowed to cool to room temperature, peak-purified using RP-HPLC (≥95%), and concentrated using a C18 extraction disk (3M Empore). The specific activities for all peak-purified 111In-conjugates are essentially the theoretic maximum of 1,725 MBq/nmol. Radiolabeling yields for 111In-1, 111In-2, 111In-3, and 111In-4 were 86.3%, 45.2%, 71.1%, and 58.8%, respectively.

In Vitro Studies

The inhibitory concentration (IC50) for all conjugates and natIn-conjugates was determined using the PC-3 human prostate cancer cell line. natIn-conjugates were used as substitutes for the corresponding 111In-radioconjugates. The cell-associated activity was measured using a γ-counter (LTI). Efflux studies were performed using PC-3 cells, which were incubated in 6-well plates (0.5 × 106/well) under hypoxic conditions (94.9% N2, 0.1% O2, 5% CO2) overnight. On the day of the experiment, the medium was replaced with fresh normoxic or hypoxic medium and incubated for 2 h under normoxic (95% air, 5% CO2) and hypoxic conditions, respectively. The cells were preincubated for 2 h at 37°C in the presence of 100,000 cpm of each 111In-radioconjugate. At time points 0, 2, 4, and 8 h, the radioactivity of the effluxed, surface-bound, and internalized fractions for each radioconjugate was collected and determined using a γ-counter. For the cellular fractionation studies, normoxic and hypoxic PC-3 cells (2.5 × 105/well) were prepared as outlined in the efflux studies above. At time points 2, 4, and 8 h, the cells were lysed and transferred to an Amicon Ultracel 30-kDa filter with extra phosphate-buffered saline (1 mL). The samples were centrifuged at 4,000g for 10 min and washed with phosphate-buffered saline (1 mL × 2). The radioactivity associated with the molecular-weight fractions was collected and determined using a γ-counter.

Biodistribution and Small-Animal SPECT/CT Imaging Studies

Biodistribution studies were performed using PC-3 tumor–bearing SCID mice. Each mouse (average weight, 20 g) received an intravenous bolus injection via the tail of 277.5 kBq (7.5 μCi) of the radio–RP-HPLC peak-purified 111In-radioconjugate (111In-1, 111In-2, or 111In-4) in 100 μL of saline. The mice were sacrificed, and their tissues were excised at 1, 4, 24, 48, and 72 h after injection. The excised tissues were weighed, the radioactivity measured, and the percentage injected dose (ID) or percentage ID per gram (%ID/g) calculated for each tissue. Blocking studies were also investigated on 111In-4 by coinjection with 300 μg of unlabeled conjugate 4 (n = 3). The mice for SPECT/CT imaging studies were administered 4–11 MBq (0.108–0.300 mCi) of the desired BB2r-targeted peptide in 100–200 μL of saline via tail vein injection. At 1, 24, 48, and 72 h after injection, mice were anesthetized with 1%–1.5% isoflurane delivered in a 2:1 mixture of nitrous oxide:oxygen. Images were acquired using a FLEX Triumph CT/SPECT system (SPECT/CT) and software (Gamma Medica, Inc.) fitted with a 5-pinhole (1.0 mm/pinhole) collimator. SPECT projections (30- to 90-min acquisition time per mouse based on the amount of activity) were acquired and reconstructed using SpectReconstructionApp (Gamma Medica, Inc.), followed by CT scans that were acquired and reconstructed using Triumph X-O 4.1 (Gamma Medica, Inc.).

Statistical Analysis

IC50 values were determined by nonlinear regression using the 1-binding-site model of GraphPad PRISM 5 (GraphPad Software, Inc). Comparisons of the 2 groups for efflux studies, cellular protein analysis studies, and biodistribution studies were analyzed by the 2-tailed Student t test, and P values of less than 0.05 were considered statistically significant.

RESULTS

Conjugate Synthesis and Radiolabeling

Four radioconjugates were synthesized using the DOTA-X-8-AOC-BBN(7-14)NH2 paradigm (Fig. 1). The yields of conjugates 1, 2*, 3*, and 4* ranged from 16.67% to 20.44% as determined by RP-HPLC. The 2-NIAA coupling of conjugates 2* and 4* was performed by O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU) conjugation, whereas conjugate 3* was coupled using N,N′-dicyclohexylcarbodiimide. All attempts to conjugate 3* with 2-NIAA using HBTU resulted in poor yields (<1%). The products were purified by RP-HPLC and isolated with yields of 17.4%, 26.7%, and 19.0% for conjugates 2, 3, and 4, respectively. RP-HPLC retention time and mass spectrometric identification of the conjugates are listed in Table 1.

FIGURE 1.
FIGURE 1.

Hypoxia-enhanced 111In-BB2r–targeted conjugates.

View this table:
TABLE 1

Mass Spectrometric and RP-HPLC Characterization of Conjugates

In Vitro Competitive Cell-Binding Studies

The BB2r binding affinity of the conjugates and natIn-conjugates were investigated by competitive binding studies using the BB2r-positive, PC-3 cell line. All conjugates and natIn-conjugates demonstrated nanomolar binding affinities. The natIn-labeled conjugates had IC50 values of 7.1 ± 1.1, 7.3 ± 1.1, 5.8 ± 1.1, and 6.9 ± 1.2 nM for natIn-1, natIn-2, natIn-3, and natIn-4, respectively. Slightly lower binding affinities were observed for unlabeled conjugates than for natIn-labeled.

In Vitro Internalization and Efflux Studies

We have previously reported that the surface expression of the BB2r remains essentially unchanged under the hypoxic conditions used in our studies (16). In these studies, 111In-1, which does not have a 2-nitroimidazole incorporated, is the control to compare the relative effectiveness of the hypoxia-trapping conjugates (111In-2–4). The PC-3 cells were first incubated in the presence of the radioconjugates for 2 h before the start of the efflux studies. During this incubation period, all of the radioconjugates investigated under both normoxic and hypoxic conditions demonstrated similar levels of internalization, ranging from 18% to 22% of the total radioactivity added.

The efflux of the radioconjugates over time given as a percentage of the initial internalized activity is depicted in Figure 2. Within the first 2 h of the experiment, the 111In radioconjugates 1–4 under hypoxic conditions demonstrated a lower efflux rate relative to normoxic conditions. At 8 h after incubation, 41.4%, 60.7%, 69.1%, and 69.4% of the initially internalized radioactivity for 111In-1, 111In-2, 111In-3, and 111In-4, respectively, was retained under hypoxic conditions, compared with only 34.8%, 35.3%, 33.2%, and 29.7% retained under normoxic conditions. 111In-1 also exhibited a significant decrease in clearance rate after 2 h under hypoxic conditions. However, the increased retention showed by 111In-1 is limited relative to 111In-2–4, which have 2-nitroimidazoles incorporated into the structure of the radioconjugate. The radioconjugates 111In-2–4 demonstrated significantly higher retention under hypoxic conditions than normoxic conditions (P < 0.0001).

FIGURE 2.
FIGURE 2.

Efflux assays depicted as percentage of initial internalized activity for 111In-radioconjugates in PC-3 cells. Values are mean ± SD (n = 5).

The internalized activity of the radioconjugate was compared as an additional means of evaluating the retention effect under normoxic and hypoxic conditions. Hypoxia enhancement factor (HEF) is defined as the ratio of the amount of activity remaining in the hypoxic cells versus the normoxic PC-3 cells of radioconjugates internalized. The HEF for each radioconjugate at each time point is depicted in Figure 3. At the initial time point, all of the radioconjugates demonstrated a similar accumulation under both normoxic and hypoxic conditions where the HEF is approximately equal to 1. At the 2-h time point, all of the radioconjugates incorporated with 2-nitroimidazole started to display significantly higher retention in hypoxic than normoxic cells (i.e., HEF > 1). By the 8-h time point, the 111In-1, 111In-2, 111In-3, and 111In-4 demonstrated an average HEF of 1.17 ± 0.12, 1.95 ± 0.28, 2.72 ± 0.35, and 3.29 ± 0.25, respectively. The radioconjugates 111In-2–4 exhibited significantly higher average HEF ratios than the control. The strong positive linear relationship between the HEF and the number of 2-nitroimidazoles incorporated was confirmed by linear regression analysis (R > 0.96).

FIGURE 3.
FIGURE 3.

HEF ratios for efflux studies of 111In-BB2r–targeted radioconjugates under normoxic (95% air, 5% CO2) and hypoxic (94.9% N2, 0.1% O2, 5% CO2) conditions. Values are mean ± SD (n = 5).

Cellular Protein Analysis

It is well established that 2-nitroimidazoles are reductively activated in a hypoxic environment. This activation leads to the irreversible conjugation of the reduced 2-nitroimidazole moiety with intracellular nucleophiles (e.g., thiols), including those contained in proteins, to form adducts (11,17,18). To better elucidate the mechanism of the observed increase in retention of the 2-nitroimidazole–containing BB2r-targeted agents under hypoxic conditions, the protein-association properties of the conjugates were evaluated under hypoxic and normoxic environments. At the 2-, 4-, and 8-h postincubation time points, the PC-3 cells were lysed and centrifuged. The supernatant was then filtered using a 30-kDa centrifugal filter. The ratio of protein-associated radioactivity as a percentage of total intracellular radioactivity under hypoxic conditions over the percentage of protein-associated radioactivity under a normoxic environment is depicted in Figure 4. The control radioconjugate 111In-1 demonstrated similar ratios, from 1.08 to 1.38, during the timespan of the experiment. The 2-nitroimidazole–containing BB2r-targeted conjugates demonstrated at least 1-fold-higher protein association under hypoxic conditions than that observed under normoxic conditions. For 111In-4, at the 4- and 8-h time points, up to 3-fold-higher hypoxic/normoxic protein-association ratios were observed relative to the control. These results strongly suggest that 2-nitroimidazoles are partially responsible for this enhancement.

FIGURE 4.
FIGURE 4.

Protein-association studies under normoxic (95% air, 5% CO2) and hypoxic (94.9% N2, 0.1% O2, 5% CO2) conditions in PC-3 cells. Values are mean ± SD (n = 5).

Biodistribution Studies

The in vivo biodistributions of the 111In-1, 111In-2, and 111In-4 radioconjugates were investigated in PC-3 tumor–bearing SCID mice. Because of the similar efflux and protein-association properties of 111In-3 and 111In-4, 111In-3 was not investigated in vivo. The results obtained from pharmacokinetic studies of 111In-1, 111In-2, and 111In-4 at 1, 4, 24, 48, and 72 h after injection are summarized in Table 2. All of the investigated 111In-radioconjugates demonstrated rapid blood clearance at 1 h after injection. Clearance of the radioconjugates proceeded largely through the renal or urinary system. At 1 h after injection, the highest accumulation was found in the pancreas for radioconjugates 111In-1, 111In-2, and 111In-4 (70.96 ± 15.88, 33.70 ± 27.11, and 33.04 ± 19.50 %ID/g, respectively). These results are due to the high expression of BB2r in rodent pancreas and are consistent with previous reports (5,19). The tumor retention of radioconjugate 111In-4 (2.80 ± 1.18 %ID/g) at 1 h after injection is substantially lower than 111In-1 (5.82 ± 2.63 %ID/g) and 111In-2 (6.06 ± 3.35 %ID/g). However, by the 72-h postinjection time point, 1.5%, 6.7%, and 21.0% (corresponding to radioconjugates 111In-1, 111In-2, and 111In-4, respectively) of the initial 1-h uptake was retained in the tumor tissue (Fig. 5). The tumor retention observed for both 111In-2 (0.41 ± 0.07 %ID/g, P < 0.01) and 111In-4 (0.60 ± 0.40 %ID/g, P < 0.05) was found to be significantly higher than for the control 111In-1 (0.09 ± 0.10 %ID/g). With the exception of the kidneys, the addition of the 2-nitroimidazoles did not increase the nontarget retention of the BB2r-targeted agents. The initial kidney uptake for all of the radioconjugates investigated was approximately 15 %ID/g at the 1-h time point. By 72 h after injection, the conjugates 111In-2 (2.66 ± 0.73 %ID/g) and 111In-4 (8.83 ± 5.69 %ID/g), as compared with 111In-1 (0.76 ± 0.67 %ID/g), demonstrated significant retention (P < 0.05) in the kidneys. In this study, the kidney retention correlated with an increase in the 2-nitroimidazole moieties of the BB2r-targeted agent. The coinjection of an excess of unlabeled conjugate 4 along with 111In-4 resulted in significantly reduced radioactivity in the pancreas (1.16 ± 0.66 %ID/g), kidney (12.37 ± 7.92 %ID/g), and tumor (0.44 ± 0.34 %ID/g) at 4 h after injection (P < 0.05, 1-tailed).

View this table:
TABLE 2

Biodistribution Studies in PC-3 Tumor–Bearing SCID Mice

FIGURE 5.
FIGURE 5.

Percentage tumor retention of 111In-1, 111In-2, and 111In-4 in PC-3 tumor–bearing SCID mice. Values are mean ± SEM (n = 4).

Small-Animal SPECT/CT Imaging Studies

Small-animal SPECT/CT imaging studies were performed in PC-3 tumor–bearing SCID mice using the 111In-1, 111In-2, and 111In-4 radioconjugates. The whole-body images and the respective axial slices of the PC-3 tumors at 1, 24, 48, and 72 h after injection are depicted in Figure 6. At 1 h after injection, significant abdominal uptake was observed in all cases due to the accumulation of radioactivity in the gastrointestinal tract and pancreas, as previously demonstrated in the biodistribution studies. Axial slices of the PC-3 tumor for all the radioconjugates investigated exhibited substantial accumulation of radioactivity in the tumor tissue after the rapid clearance of the radioconjugates through the renal or urinary system. For 111In-4 radioconjugates, conspicuous kidney retention was observed, echoing the biodistribution studies.

FIGURE 6.
FIGURE 6.

Fused micro-SPECT/CT and axial images of 111In-1, 111In-2, and 111In-4 in PC-3 tumor–bearing mice at 1, 24, 48, and 72 h after injection. Tumors and kidneys are indicated by red and green arrows, respectively. Max = maximum; Min = minimum.

DISCUSSION

To determine whether the incorporation of 2-nitroimidazoles would increase the retention of the radioconjugate in hypoxic PC-3 cells, we have previously synthesized 4 BB2r-targeted agents, which included 2-nitroimidazole moieties (16). In vitro studies showed improved longitudinal retention of the 2-nitroimidazole–containing BB2r-targeted agents in hypoxic relative to normoxic PC-3 cells. However, it was determined that the steric interference of the 2-nitroimidazole with the BB2r-targeting vector resulted in poor binding affinities, severely impeding internalization of these conjugates. In this study, an extended linker (8-AOC) was incorporated between the 2-nitroimidazole-amino-acid residue and the pharmacophore. The BB2r affinities of (1–4) natural indium–labeled and –unlabeled conjugates versus [125I-Tyr4] BBN were performed for the gastrin-releasing peptide receptor using the PC-3 cell line. All natIn-BBN conjugates demonstrated nanomolar binding affinity. On the basis of these results, the incorporation of the 8-AOC linker has eliminated the detrimental impact of a 2-NIAA side chain on pharmacophore binding.

Internalization and efflux studies demonstrated that the clearance rate of the radioconjugates containing 2-nitroimidazole was substantially lower relative to the control under hypoxic conditions. Specifically, at the 8-h time point for 111In-1, 111In-2, 111In-3, and 111In-4, 6.6%, 25.4%, 35.9%, and 39.7%, respectively, more retention was observed under hypoxic conditions than normoxic conditions. 111In-3 and 111In-4, which have more than one 2-nitroimidazole (2 and 3, respectively), exhibited a higher retention effect than 111In-2, which has only one 2-nitroimidazole. Inclusion of more than one hypoxia-trapping moiety may increase the chances for the 2-nitroimidazole–containing radioconjugates to form protein adducts, thus enhancing the long-term retention of the radioconjugate in the cell. Ultimately, further investigation into the identification and quantification of the protein adduct is needed to obtain a clearer understanding of the mechanism involved in this process. Also, 111In-1 exhibited a slightly lower clearance rate under hypoxic conditions, which could be due to the decreased metabolic rate under hypoxic conditions (9). With respect to the HEF, significantly higher retention in hypoxic than normoxic cells was observed for 111In-2–4 at the 2-h time point. The HEF continued to increase for 111In-1 and 111In-2 throughout the experiments but remained constant for 111In-3 and 111In-4 after the 4-h time point. A strong positive linear relationship between the HEF and the number of 2-nitroimidazoles incorporated was confirmed for the 8-h time points.

Cellular protein analysis of the control radioconjugate 111In-1 demonstrated minimal hypoxic-to-normoxic cellular protein-association ratios, which are likely due to reversible, nonspecific binding. For the 2-nitroimidazole–containing BB2r-targeted conjugates, up to a 2-fold increase was observed under hypoxic conditions. These results suggest that the significantly higher protein-association ratio of 2-nitroimidazole–containing radioconjugates is due, at least in part, to the irreversible binding to intracellular proteins, which is consistent with the known trapping mechanism of 2-nitroimidazole.

The in vivo biodistribution of each radioconjugate was investigated in PC-3 tumor–bearing SCID mice, except for 111In-3 because this radioconjugate had efflux and protein-association properties similar to 111In-4. At 1 h after injection, 111In-1 and 111In-2 share comparable tumor uptake, 5.82 ± 2.63 and 6.06 ± 3.35 %ID/g, respectively. However, a substantially lower tumor uptake (2.80 ± 1.18 %ID/g) was observed for 111In-4, which has three 2-nitroimidazoles incorporated. Given the similar uptake of 111In-2 and 111In-4 in the BB2r-positive pancreas, the reason behind the reduced tumor uptake is unclear. At 4 h after injection, most of the radioconjugates were cleared through the renal or urinary system (80–82 %ID), which is consistent with other investigations of BB2r-targeted radioconjugates (19). By the 72-h postinjection time point, the radioconjugates were largely cleared from most tissues, including the pancreas (20). Significant tumor retention enhancement was observed at 72 h after injection for radioconjugates 111In-2 and 111In-4. Specifically, 6.7% and 21.0% of the initial 1-h uptake in tumor was retained for 111In-2 and 111In-4, compared with only 1.5% remaining for the control 111In-1. However, the hypoxia burden of each tumor is unknown, limiting the ability to fully interpret the relationship between tumor retention effect and incorporation of the hypoxia-trapping moiety. The hypoxia burden in tumor xenograft mouse models has been shown to increase (modest correlation) with an increase in tumor size, but this trend is highly dependent on the cell line (21). Linear regression analysis of the %ID/g retention of our radioconjugates in PC-3 tumors versus tumor weight revealed no correlation between retention and tumor size. Interestingly, in the clinic, the extent of hypoxia is independent of tumor size in a variety of human cancers, including head and neck, cervical, and lung (22–24). Further study to correlate the tumor hypoxia burden with radioconjugate retention is ongoing. Significantly lower tumor and pancreas uptake caused by the coinjection of an excess of unlabeled conjugate 4 indicates that accumulation of 111In-4 is largely mediated by the BB2r.

It is interesting to note the unusually high kidney retention for 2-nitroimidazole–containing conjugates relative to the control conjugate. This is particularly true of 111In-4, with a kidney retention of 8.83 ± 5.69 %ID/g at 72 h after injection. This renal retention value is higher, to the best of our knowledge, than any reported research related to bombesin or 2-nitroimidazole–based radioconjugates (5,19,25,26). Renal retention of radiolabeled targeting peptide has long been addressed as one of the dose-limiting factors in radionuclide therapy, and various mechanisms have been demonstrated to be involved in high renal uptake (27). Cationic peptides are preferentially reabsorbed by the proximal tubules because of the anionic binding sites on the brush border membrane (28,29). Megalin and cubilin are known to be associated with the proximal tubular reabsorption of structurally different proteins, peptides, and drugs (30). Moreover, low or very low tissue oxygen tensions exist under physiologic conditions in the kidneys, facilitating urine concentration (31). Coinfusion of competitive inhibitors such as lysine, arginine, and succinylated gelatin can reduce the reabsorption by endocytosis or transporters (32–34). In preliminary coinjection blocking studies (data not shown), both EF5 (a 2-nitroimidazole–based hypoxia marker) and lysine coinjection with 111In-4 can reduce the retention of radioconjugates in the kidneys. Further studies are needed to fully understand the mechanism of kidney retention of hypoxia-trapping enhanced bombesin conjugates to develop specific methods to reduce the renal toxicity.

Micro-SPECT/CT images of all radioconjugates using PC-3 tumor–bearing mice at 1 h after injection (Fig. 6), showing significant abdominal uptake, are consistent with the data obtained from biodistribution studies. The PC-3 tumor xenografts in all mice are easily visualized. For the 111In-4 radioconjugates, containing three 2-nitroimidazoles, significant activity in the kidneys was observed, strongly agreeing with previously established biodistribution data.

CONCLUSION

We have synthesized 3 BB2r-targeted radioconjugates with 2-nitroimidazole hypoxia-trapping moieties incorporated to enhance the retention in hypoxic cancer cells. In vitro competitive binding studies indicate that inclusion of extended linker 8-AOC eliminated the detrimental effect on binding affinity that was determined in our previous report. The 2-nitroimidazole–trapping moieties containing BB2r-targeted agents demonstrated significantly higher retention and protein-association properties in hypoxic than in normoxic PC-3 cells. In vivo biodistribution studies revealed great potential of incorporated trapping moieties to increase the residence time of BB2r-targeted agents in a PC-3 xenograft tumor. Further works are needed to clarify the mechanisms of increased retention effects at the molecular level and to correlate the tumor hypoxia burden with the retention efficacy.

DISCLOSURE

The costs of publication of this article were defrayed in part by the payment of page charges. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. This study was supported by the National Cancer Institute (5 R00 CA137147) and the National Center for Research Resources (8 P20 GM103480). No other potential conflict of interest relevant to this article was reported.

Acknowledgments

We thank Dr. Katherine Estes for assistance in SPECT/CT imaging and the Bioimaging Core at UNMC.

Footnotes

  • Published online Jul. 29, 2013.

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  • Received for publication November 30, 2012.
  • Accepted for publication March 27, 2013.
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