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Novel Structural Modification Based on Evans Blue Dye to Improve Pharmacokinetics of a Somastostatin Receptor Based Theranostic Agent Nilantha Bandara, Orit Jacobson, Cedric Mpoy, Xiaoyuan Chen, and Buck Edward Rogers Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00341 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 23, 2018

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Bioconjugate Chemistry

Novel Structural Modification Based on Evans Blue Dye to Improve Pharmacokinetics of a Somastostatin Receptor Based Theranostic Agent

Nilantha Bandara,† Orit Jacobson,§ Cedric Mpoy,† Xiaoyuan Chen,*,§ Buck E. Rogers,*,†

†

Department of Radiation Oncology, Washington University School of Medicine,

St. Louis, MO 63108

§

Laboratory of Molecular Imaging and Nanomedicine, National Institute of

Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA

Corresponding authors *

E-mail: [email protected]. Phone: +1 3143629787. Fax: +1 3143629790.

*

E-mail: [email protected]. Phone: +1 3014514246.

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Development of somastatin (SS) peptide analogues for detection and treatment of neuroendocrine tumors has been successful with the recent FDA approval of 68Ga-DOTA-TATE and 177Lu-DOTA-TATE. The structure of these peptide constructs contains the peptide binding motif that binds to the receptor with high affinity, a chelator to complex the radioactive metal, and a linker between the peptide and chelator. However, these constructs suffer from rapid blood clearance, which limit their tumor uptake. In this study, this design has been further improved by incorporating a modification to control the in vivo pharmacokinetics. Adding a truncated Evans Blue (EB) dye molecule into the construct provides a prolonged half-life in blood due to its low micromolar affinity to albumin. We compared 177Lu-DOTA-TATE to the modified 177Lu Evans Blue compound (177Lu-DMEB-TATE), in vitro and in vivo in mice bearing A427-7 xenografts. The tumor uptake of 177Lu-DMEB-TATE was significantly greater than the uptake of 177Lu-DOTA-TATE in biodistribution and SPECT imaging studies. The therapeutic effect of 177Lu-DMEB-TATE construct was superior to the 177Lu-DOTA-TATE construct at the doses evaluated. ____________________________________________________________________________________


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INTRODUCTION The somatostatin receptor subtype 2 (SSTR2) is overexpressed on neuroendocrine tumors

and has been the target for somatostatin-based imaging and therapy1-6. The native somatostatin peptide hormone binds with high affinity to all five SSTR subtypes and has two biologically active forms that are fourteen or twenty-eight amino acids long with a short biological half-life, on the order of minutes5. Eight amino acid peptide analogues were developed that maintained high affinity to SSTR2 and extended the biological half-life by incorporating D-amino acids into the peptide7, 8. These analogues (octreotide) have been evaluated as therapeutic agents themselves but have also been modified for carrying radionuclides for nuclear imaging and targeted radiotherapy1-6. In this regard, 111In-DTPA-octreotide (Octreoscan) was approved by the U.S. Food and Drug Administration (FDA) in 1994 for SPECT imaging of neuroendocrine tumors9. While this agent proved useful in the detection and staging of neuroendocrine tumors, much research has been performed on the development of octreotide analogues (octreotate (TATE), tyrosine-3-octreotate, SSTR2 antagonists (sst2-ANT)) radiolabeled with PET radionuclides to improve the sensitivity of tumor detection10-13. This research culminated in the FDA approval of 68Ga-DOTA-octreotate (DOTA-TATE) (Netspot) for PET imaging of patients with neuroendocrine tumors in 2016. In addition to imaging, octreotide analogues have been radiolabeled with therapeutic radionuclides for targeted radiation therapy and evaluated extensively preclinically and clinically14-17. A recent publication of a Phase 3 trial using 177Lu-DOTA-TATE for treatment of midgut neuroendocrine tumors showed a 65% progression free survival response rate compared to 11% in the control group with an interim analysis of overall survival also being greater with 177

Lu-DOTA-TATE compared to control (P = 0.004) 6. This 177Lu-DOTA-TATE (LutaThera)



was recently approved by the FDA. Although 177Lu-DOTA-TATE is effective in treating patients with neuroendocrine tumors, the therapy is limited by relatively low doses of radiation (23-29 Gy) being delivered to the tumor18-20 due to the rapid clearance of the peptide from the blood. Therefore, we sought to improve tumor uptake by decreasing the blood clearance of the radiolabeled peptide. One method of accomplishing this is through the addition of an albumin binding domain to the peptide. In this regard, we have used a Evan’s Blue (EB) derivative to decrease the blood clearance of a radiolabeled RGD peptide21, while others have used 4-(piodophenyl)butyric acid to accomplish something similar in a variety of systems22, 23. EB has been used in clinical practice to determine patient plasma volume because of its affinity for serum albumin24, 25. In these studies, we synthesized a DOTA-TATE analogue in which a truncated Evan’s Blue (EB) molecule was conjugated to the peptide (DMEB-TATE, Figure 1) in order to modify the pharmacokinetics and increase tumor uptake. We compared 177Lu-DMEBTATE to 177Lu-DOTA-TATE in vitro and in vivo. In vivo studies consisted of biodistribution, SPECT imaging, and therapy. These studies show a dramatic increase in the tumor uptake and therapeutic efficacy of 177Lu-DMEB-TATE compared to 177Lu-DOTA-TATE at the doses evaluated. Further evaluation of 177Lu-DMEB-TATE is warranted to determine potential toxicities associated with this agent and to determine if an increase in the therapeutic index has been achieved when compared to 177Lu-DOTA-TATE.


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Figure 1. The structure of DMEB-TATE. This shows the DOTA chelator (1,4,7,10tetraazacyclotetradodecane-1,4,7,10-tetraacetic acid), the truncated Evans blue modification (blue circle), and SSTR2-binding octreotate peptide (green circle).

RESULTS Radiolabeling. The radiochemical purity was > 99% as observed for both 177Lu-DOTA-

TATE and 177Lu-DMEB-TATE as determined by ITLC (Figures 2a and 2b, respectively). The specific activity was obtained as 3.7 ± 0.5 MBq/µg (100.0 ± 1.3 µCi/µg) for both DMEB-TATE and DOTA-TATE.



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Figure 2. ITLC (mobile phase 50 mM DTPA) chromatographic profiles from 177Lu radiolabeling of (a) DOTA-TATE and (b) DMEB-TATE. Internalization into A427-7 is shown in (c) as the percentage internalized based on the total activity used. Blocking with cold compounds was used to demonstrate specificity and the data listed for 30, 60, 120, 120 and 1440 min time points.

Internalization Assay. Cell internalization was evaluated using A427-7 cells by performing an acid wash to remove cell surface radioactivity, and then lysing and collecting the cells to determine the amount of internalized radioactivity (Figure 2c). These cells were used due to our experience with them as well as their high expression of SSTR226. This shows that internalization of both 177Lu-DOTA-TATE and 177Lu-DMEB-TATE was specific as they were both significantly inhibited by excess of unlabeled peptide at all time points.

177

Lu-DOTA-

TATE rapidly internalized at 30 minutes (13.7 ± 0.4%) compared to 177Lu-DMEB-TATE which


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only had 2.2 ± 0.1% at this time point. Both peptides reached a maximum at 4 h with 177LuDOTA-TATE having 26.5 ± 1.1% and 177Lu-DMEB-TATE having 17.4 ± 0.5%. There was still good uptake and retention at 24 h with 177Lu-DOTA-TATE having 21.9 ± 1.1% and 177LuDMEB-TATE having 15.4 ± 1.2%.

Biodistribution Studies. In vivo biodistribution experiments were conducted to investigate the pharmacokinetics of 177Lu-DOTA-TATE (Figure 3a, Table S1 and Figure S2) and 177LuDMEB-TATE (Figure 3b, Table S1 and Figure S2). This shows that the tumor uptake was significantly higher (p < 0.004) for 177Lu-DMEB-TATE at all time points compared to 177LuDOTA-TATE. As anticipated due to the albumin binding of DMEB, 177Lu-DMEB-TATE also had significantly (p < 0.007) greater amount of radioactivity in the blood (Figure S3) at all time points compared to 177Lu-DOTA-TATE. The tumor uptake reached a maximum of 78.8 ± 4.1% ID/g for 177Lu-DMEB-TATE at 24 h compared to a maximum of 9.3 ± 0.8% ID/g for 177LuDOTA-TATE at 4 h. Due to the longer retention in the blood for 177Lu-DMEB-TATE its uptake was significantly greater than the uptake of 177Lu-DOTA-TATE in the normal tissues examined at all time points.



Figure 3. Results from the in vivo biodistribution studies (%ID/g) in athymic nude mice with A427-7 xenografts, a) 177Lu-DOTA-TATE and b) 177Lu-DMEB-TATE at 4, 24, 48, 72 and 144 h p.i.


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Figure 4. Representative coronal SPECT images of 177Lu-DMEB- TATE and 177Lu-DOTATATE injected in athymic nude mice with A427-7 xenografts. (a) to (d) 177Lu-DMEB-TATE nonblock with 1, 24, 72 and 144 h p.i., and (h) 24 h p.i. block with 125 µg DMEB-TATE coinjected with the dose. (e) and (f) 177Lu-DOTA-TATE nonblock with 1 and 24 h p.i. and (g) 24 h p.i. block with 125 µg DOTA-TATE.



Nano-SPECT/CT Imaging Studies. The representative images from SPECT imaging are shown in Figure 4. In order to establish the in vivo specificity of the labeled analogues, blocking studies were performed as described in the experimental section. In the presence of unlabeled DMEB-TATE tumor uptake of 177Lu-DMEB-TATE was reduced at 24 h (Figure 4b vs. 4h). However, with 177Lu-DOTA-TATE, both non-block (Figure 4f) and blocked (Figure 4g) images looked similar as the tumor uptake of 177Lu-DOTA-TATE was low to begin with.

Therapy Studies. The effect of a single administration of 177Lu-DOTA-TATE or 177LuDMEB-TATE on tumor growth is shown in Figure 5. The tumor volume of mice treated with 7.4 MBq of 177Lu-DOTA-TATE was similar to the volume of control mice. When 18.5 MBq of 177

Lu-DOTA-TATE was administered, tumor growth inhibition was observed with the tumor

volumes being significantly smaller only from days 18-22 (p < 0.02, Figure S4). However, these tumors continued to grow and were not significantly different by the end of the study. For mice treated with 177Lu-DMEB-TATE, the 18.5 MBq group demonstrated a complete regression of tumors in 4 out of 5 mice, while the 7.4 MBq group maintained a tumor volume that was similar to the starting volume. Interestingly, the 3.7 MBq group had a tumor growth profile that was similar to the 177Lu-DOTA-TATE group that was administered 18.5 MBq.


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Figure 5. 177Lu tumor therapy. Athymic nude mice with A427-7 xenografts, (a) control / saline, (b) 7.4 MBq (200 µCi) 177Lu-DOTA-TATE, (c) 18.5 MBq (500 µCi) 177Lu-DOTA-TATE, (d) 3.7 MBq (100 µCi) 177Lu-DMEB-TATE, (e) 7.4 MBq (200 µCi) 177Lu-DMEB-TATE, and (f) 18.5 MBq (500 µCi) 177Lu-DMEB-TATE injected iv and tumor growth monitored after that.

DISCUSSION

The most common type of malignant gastrointestinal neuroendocrine tumors are ones of the midgut and have 5-year survival rates less than 50% for patients with metastatic disease4, 6. Standard of care for these patients consists of the use of somatostatin-based inhibitors to control tumor growth and hormone secretion27, 28. However, no standard second-line treatment options were available until recently29, 30. In this regard, research on the use of radiolabeled somatostatin analogues to deliver targeted radiation to these tumors began in preclinical mouse models more than twenty-five years ago31. Some of the early clinical studies used the Auger-emitting, 111InDTPA-TOC at high doses (max of 75 GBq (2 Ci) per patient)1. It was subsequently found that



either 90Y-DOTA-TOC or 177Lu-DOTA-TATE were more efficacious than 111In-DTPA-TOC presumably due to the longer path lengths of their beta emissions14, 18, 32. The recent phase 3 Neuroendocrine Tumors Therapy (NETTER-1) trial evaluated the safety and efficacy of 177LuDOTA-TATE compared to high-dose octreotide in patients with advanced, progressive, somatostatin-receptor-positive midgut neuroendocrine tumors6. This showed significantly greater progression free and overall survival that helped gain 177Lu-DOTA-TATE FDA approval. Despite this, relatively low doses of radiation were delivered to the tumor and we hypothesized that this could be improved by increasing the blood circulation time of 177Lu-DOTA-TATE. In order to achieve this, we chose to conjugate an albumin binding domain to the DOTA-TATE peptide. Müller et al. used 4-(p-iodophenyl)butyric acid conjugated to folic acid and DOTA for targeting 177Lu to folate receptor-positive tumors in mice23. These studies showed good tumor uptake up to 7 days with increased blood circulation compared to the construct without the albumin binding domain. This group has also investigated different radionuclides, other targets such as PSMA, and a variety of albumin binding moieties33-35. In our laboratory, we have utilized a truncated form of the Evan’s Blue dye that binds with micromolar affinity to albumin21. Conjugation of this to an RGD peptide and NOTA for PET imaging with 64Cu or DOTA for therapy with 90Y, demonstrated that increased blood retention was observed which led to better PET imaging in mice bearing U87MG xenografts compared to 64Cu-NOTA-RGD without the EB moiety21. Similarly, a greater therapeutic effect was observed when using 90YDMEB-RGD. We have recently conjugated EB to DOTA-Octreotate for evaluation after radiolabeling with 86/90Y in preclinical studies36 or with 177Lu for initial clinical studies37, 38. These clinical studies showed that 177Lu-DMEB-TATE was safe, with high NET uptake, and potentially efficacious. It was demonstrated that DMEB-TATE had similar binding to SSTR2


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when compared to DOTA-TATE (9.2 nM vs. 3.5 nM) and that DMEB-TATE bound to serum albumin with an affinity of 4.8 µM36. In the preclinical studies, we demonstrated that 86YDMEB-TATE had approximately 35-fold higher uptake in HCT116/SSTR2+ tumor xenografts compared to 86Y-DOTA-TATE. In addition, 90Y-DMEB-TATE resulted in tumor regression for both HCT116/SSTR2+ and AR42J xenografts at single doses of 7.4 MBq, while 7.4 MBq of 90YDOTA-TATE resulted in minimal inhibition of tumor growth in each of these models. Since 177

Lu-DOTA-TATE was recently approved by the FDA for treating neuroendocrine tumors, we

sought to evaluate 177Lu-DMEB-TATE in comparison to 177Lu-DOTA-TATE with regard to tumor uptake and therapeutic response. Both DMEB-TATE and DOTA-TATE were radiolabeled with 177Lu at high specific activity and radiochemical purity, which is needed for subsequent therapy studies. We utilized the A427-7 non-small cell lung cancer cells as a model system in these studies due to their high expression of SSTR226. In addition, these cells are relatively radioresistant39, which allow us to determine differences between 177Lu-DMEB-TATE and 177Lu-DOTA-TATE in therapy studies. Internalization studies showed that both 177Lu-DMEB-TATE and 177Lu-DOTA-TATE were highly internalized (> 17%) at 4 h, which is greater than the 9% reported for the 86Y-labeled compounds in HCT116/SSTR2 cells 36. Internalization of 177Lu-DMEB-TATE was significantly less (p < 0.05) than that of 177Lu-DOTA-TATE at all time points, which differs from the 86Y studies where both compounds show similar levels of internalization. It is not clear why these differences exist in the current studies. Tumor uptake of 177Lu-DMEB-TATE was 60-80% ID/g at 24-48 h (Figure 3), which is greater than observed for 86Y-DMEB-TATE in the HCT116/SSTR2 model (30-35% ID/g), but similar to 86Y-DMEB-TATE uptake in the AR42J model (60-65% ID/g)36. This is particularly interesting as the AR42J cells have been reported to



express less SSTR2 (~2,500 fmol/mg) compared to HCT116/SSTR2 (~4,500 fmol/mg), while the A427-7 cells express (~7,000 fmol/mg)26, 40, 41. It is possible that this expression profile changes when the cells are grown as xenografts, which may account for the biodistribution differences. Similar to the biodistribution studies, the tumor uptake of 177Lu-DMEB-TATE is the highest at 24 h and clearly visualized by SPECT imaging with decreased tumor uptake at 72 and 144 h (Figure 4). This decrease is likely due to normal clearance of the radioactivity from the tumor, but could also be the result of a therapeutic effect since a 37 MBq dose was needed for adequate imaging results. Therapy studies were conducted based on our previous studies using 90Y at doses of 3.7 and 7.4 MBq as well as a high dose of 18.5 MBq. The 7.4 MBq dose of 177Lu-DMEB-TATE resulted in a tumor volume that remained constant over the course of the study. This is different than the same dose of 90Y-DMEB-TATE, which resulted in tumor regressions for both the HCT116/SSTR2 and AR42J tumors. As mentioned earlier, this could be due to the radioresistance of the A427-7 tumors or the differences in energy deposition between 90Y and 177

Lu. The 18.5 MBq dose of 177Lu-DMEB-TATE, however, resulted in tumor regression and

this dose is still an order of magnitude less than the clinical accumulative dose. Importantly, the therapeutic effect is significantly greater using 177Lu-DMEB-TATE when compared to 177LuDOTA-TATE. However, further studies must be performed to compare both agents near their maximally tolerated dose to confirm an increase in the therapeutic index.

CONCLUSIONS

The 177Lu-DMEB-TATE showed prolonged circulation half-life and enhanced tumor accumulation compared to the 177Lu-DOTA-TATE. This lead to significantly greater tumor


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growth inhibition when using the 177Lu-DMEB-TATE compared to 177Lu-DOTA-TATE at the same doses. However, the prolonged circulation time and increased uptake in normal tissues for 177

Lu-DMEB-TATE compared to 177Lu-DOTA-TATE may lead to toxicity preventing an

increase in the therapeutic index. If the therapeutic index between the two compounds is similar, there may still be advantages to the use of 177Lu-DMEB-TATE in terms of using less 177Lu, which will reduce costs and lower radiation exposure while handling the product.

EXPERIMETAL SECTION General methods. All solvents and reagents were ultra-pure or trace metal grade, obtained

from Sigma-Aldrich (St. Louis, MO) and used as received unless stated otherwise. All solutions and buffers were prepared using water purified from a Millipore Integral 5 Milli-Q water system (18 MΩ·cm resistivity, Billerica, MA). All solvents were treated with Chelex overnight and filtered through a 0.22 µm nylon filter to remove trace amounts of metal ions. Radioactivity was counted with a Beckman Gamma 8000 counter containing a NaI crystal (Beckman Instruments, Inc., Irvine, CA). DMEB-TATE and DOTA-TATE were synthesized and characterized as previously described36.

Cell culture. A427-7 is human lung carcinoma cell line that was modified to overexpress the somatostatin receptor subtype 2 (SSTR2) as previously described26. The cells were cultured in 90% DMEM with 10% heat inactivated Fetal Bovine Serum (FBS). Media components were acquired from ThermoFisher scientific (Grand Island, NY).



Radiolabeling. 177Lu was obtained from the University of Missouri Research Reactor (MURR), Columbia, MO. A stock solution of 177LuCl3 generally contained about 740 MBq (20 mCi) of activity in 20 µL 0.1 M HCl and used after dilution with 0.4 M NH4OAc pH = 5.5 for radiolabeling. Labeling of DMEB-TATE or DOTA-TATE with 177Lu was achieved by adding 100 µg of compounds (50 µL by volume) to 370 MBq (10 mCi) of 177LuCl3 in 150 µL of 0.4 M NH4OAc (pH = 5.5). The reactions were incubated on a thermomixer with 800 rpm agitation at 37 ºC for 1 h. Radiolabeled complexes were analyzed by thin layer chromatography (TLC) with a mobile phase of 50 mM DTPA with ITLC-SG paper. The free 177Lu elutes with the mobile phase while the complex remains in the origin. A radiochemical yield of greater than 95% was achieved for most labelling. When purification was needed, a light C18 Sep-Pak was used with 70% EtOH to elute the product.

Internalization of 177Lu-DMEB-TATE and 177Lu-DOTA-TATE into A-427-7 cells. A427-7 cells were plated in 6-well plates and 24 h later the 177Lu-DMEB-TATE or 177Lu-DOTATATE was added (1 nM) in 1 mL of internalization media (DMEM, 30 mM HEPES, 2 mM Lglutamine, 1 mM sodium pyruvate, 1% BSA) and incubated at 37 ºC for 30, 60, 120, 240 and 1440 min. To three of the six wells per plate, 20 µg of unlabeled conjugates were added to act as a block. The cells were rinsed with PBS and then with Hank’s balanced salt solution (HBSS) containing 20 mM sodium acetate (pH 4.0) to remove surface bound radioactivity. Then to each well, 1 ml of 10 mM sodium borate + 1% SDS was added to facilitate cell lysis. The lysates were then collected to determine the internalized radioactivity and counted in a Packard II gamma counter.


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Biodistribution studies. All animal experiments were performed in compliance with the Guidelines for Care and Use of Research Animals established by the Division of Comparative Medicine and the Animal Studies Committee of Washington University School of Medicine. Biodistribution studies were conducted in female 5-6 weeks old athymic nude mice (Charles River Laboratories). Tumors were implanted subcutaneously on the right flank with 1 × 107 A427-7 cells with a volume of 100 µl. The tumors were allowed to grow for 2-3 weeks to approximately 150 mg. Animals were injected with 0.37-0.55 MBq (10-15 µCi) of 177Lu-DMEBTATE or 177Lu-DOTA-TATE while anesthetized with 2% isoflurane. Blocking studies were conducted using i.v. injections of 125 µg of DMEB-TATE or DOTA-TATE immediately prior to injection of radioactivity and evaluated at 24 h p.i. Tumor, pancreas, liver, kidney, and other organs of interest were harvested 4, 24, 48, 72 and 144 h p.i (n=5). The amount of radioactivity in each organ was determined by gamma counting and percent injected dose per gram of tissue (%ID/g) calculated. Samples were calibrated against a known standard. Quantitative data were processed by Prism 6 (GraphPad Software, v 6.03, La Jolla, CA) and expressed as Mean ± SEM. Statistical analysis performed using one-way analysis of variance and Student’s t test. Differences at the 95% confidence level (p < 0.05) were considered statistically significant.

Nano-SPECT/CT Imaging Studies. Small animal SPECT/CT imaging studies were conducted using the model above, but the tumor cells implanted in the axillary thorax. Approximately 37.0 MBq (1.0 mCi) of 177Lu-DMEB-TATE or 177Lu-DOTA-TATE were administrated to mice via tail vein injection. Blocking studies were conducted using i.v. injections of 125 µg of DMEB-TATE or DOTA-TATE diluted in saline. Mice were anesthetized with 1-2% isofluorane/oxygen and whole-body SPECT images were obtained at 1, 24, 72 and



144 h after injection using a Nano-SPECT/CT Imager (Bioscan Inc.) fitted with a 2-mm pinhole collimator in helical scan mode. A 45 keV helical scan was obtained first, and then SPECT was performed at 24 projections and 60 s per projection, for a total time about 40 min per mouse. Tomographic data were reconstructed iteratively with the manufacturer-supplied InVivoScope and HiSPECT software (noise suppression: middle, voxel size (mm): 0.40, and reconstruction settings: fast) for CT and SPECT, respectively.

Therapy Studies. The same tumor model as described in the biodistribution were used in the therapy studies.

Mice were weighed, and tumors were measured 2-3 times weekly using

electronic callipers. Tumor volume (mm3) was calculated as length (mm) x width (mm) x width (mm)/2. Mice were randomized into 6 groups of n = 5, with similar sized tumors (mean tumor volume ~91 mm3) on day 22 after implant. The 6 groups were (1) control / saline, (2) 7.4 MBq (200 µCi) 177Lu-DOTA-TATE, (3) 18.5 MBq (500 µCi) 177Lu-DOTA-TATE, (4) 3.7 MBq (100 µCi)

177

µCi)

177

Lu-DMEB-TATE, (5) 7.4 MBq (200 µCi)

177

Lu-DMEB-TATE, and (6) 18.5 MBq (500

Lu-DMEB-TATE. All treatments were given intravenously by tail vein injection. The

mice were euthanized once tumors reached a volume in excess of 4000 mm3 or when the tumors began to ulcerate.

Statistical Analysis. Data were processed by Prism 7 (GraphPad Software, v 7.00, La Jolla, CA) and expressed as Mean ± SEM. Statistical analysis performed using one-way analysis of variance and Student’s t test. Differences at the 95% confidence level (p < 0.05) were considered statistically significant. For the therapy study, a 2-way ANOVA was performed with a Tukey’s multiple comparisons test to calculate significance for each therapy group for each day.


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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

We would like to acknowledge the Department of Radiation Oncology for the financial support for these studies. The authors would also like to thank the small animal imaging facility at Washington University School of Medicine for technical assistance. We would also like to acknowledge the University of Missouri Research Reactor (MURR) for production of 177Lu. We acknowledge Jalen Scott, Eric Tint, and Diana Tran, for their assistance in imaging and therapy studies. We are grateful for Dr. Marquiza C. Sablon (Research Scholar, Havana, Cuba)42 for her assistance in this work during her stay in Rogers lab.

Supporting information is available on the ACS Publications website.

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Abstract Figure


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