Targeted Yttrium 89-Doxorubicin Drug-Eluting BeadâA Safety and

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Targeted Yttrium 89-Doxorubicin Drug-Eluting Bead – a Safety and Feasibility Pilot Study in a Rabbit Liver Cancer Model Johannes M Ludwig, Minzhi Xing, Yongkang Gai, Lingyi Sun, Dexing Zeng, and Hyun S Kim Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00336 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 15, 2017

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Molecular Pharmaceutics

Targeted Yttrium 89-Doxorubicin Drug-Eluting Bead – a Safety and Feasibility Pilot Study in a Rabbit Liver Cancer Model Johannes M. Ludwig, MD1,2, Minzhi Xing, MD1, Yongkang Gai, PhD2, Lingyi Sun, PhD2, Dexing Zeng, PhD2*, Hyun S. Kim, MD1,3*

1. Division of Interventional Radiology, Department of Radiology and Biomedical Imaging, Yale School of Medicine, 330 Cedar Street, New Haven, CT 06510, USA 2. Department of Diagnostic and Interventional Radiology and Neuroradiology, University Hospital Essen, University of Duisburg-Essen, Hufelandstr. 55, 45147 Essen, Germany. 3. Molecular Imaging Laboratory, Department of Radiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America, 100 Technology Drive, Pittsburgh, PA 15219, USA 4. Yale Cancer Center, Yale School of Medicine, New Haven, 330 Cedar Street, New Haven, CT 06510, USA *) Hyun S. Kim and Dexing Zeng are co-corresponding authors.

Corresponding Authors: Hyun S. Kim, MD Division of Interventional Radiology Department of Diagnostic Radiology Yale University School of Medicine Yale Cancer Center 330 Cedar Street, TE 2-224 New Haven, CT 06510 Phone: (203) 785-6938 Fax: (203) 785-3024 Email: [email protected] Dexing Zeng, Ph.D. Assistant Professor, Molecular Imaging Laboratory Department of Medicine, University of Pittsburgh

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Biomedical Science Tower #3, Suite 10015 3501 Fifth Avenue, Pittsburgh, PA 15260 Phone: 412-624-6874 Disclosures: All authors have no financial or other disclosures relationship with any commercial organization that may have a direct or indirect interest in this manuscript.


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Abstract: Purpose: To evaluate feasibility and safety of the cancer targeting (Radio)-Chemoembolization drug eluting bead (TRCE-DEB) concept drug SW43-DOX-L-NETA(89Y) DEB for the intraarterial treatment of VX2 rabbit liver tumors. The treatment compound comprises of the Sigma-2 receptor ligand SW43 for cancer targeting, Doxorubicin (DOX), and

89

Yttrium (89Y) as non-

radioactive surrogate for therapeutic (Yttrium-90, Lutetium-177) and imaging (Yttrium-86) radioisotopes via the chelator L-NETA. Material and Methods: Ten New Zealand white rabbits with VX2 tumor allografts were used. SW43-DOX-89Y was synthesized, loaded onto DEB (100µl; 100-300µm) and administered intraarterially in six rabbits at increasing doses (0.2-1.0 mg/kg). As controls, two rabbits each received either Doxorubicin IV (0.3 mg/kg) or no treatment. Consecutive serum analysis for safety and histopathological evaluation after sacrifice were performed. One-Way ANOVA incl. Bonferroni Post-Hoc test was performed to compare groups. Results: Targeted compound synthesis, loading onto DEB and intra-arterial administration was feasible and successful in all cases. Serum liver enzyme levels increased in a dose dependent manner within 24h and normalized within 3 days for 0.2/0.6 mg/kg SW43-DOX-89Y loaded onto DEB. The two rabbits treated with 1 mg/kg SW43-DOX-89Y had to be euthanized after 3/24h due to worsening general condition. Histopathological necrosis increased over time in a dose depended manner with 95-100% tumor necrosis 3-7 days post treatment (0.6 mg/kg). Conclusion: SW43-DOX-89Y loaded onto DEB can be formulated and safely administered at a concentration of 0.6 mg/kg. Loading with radioactive isotopes (e.g. 86

Yttrium/90Yttrium/177Lutetium) to synthesize the Targeted Radio-Chemoembolization Drug-

eluting Bead” (TRCE-DEB) concept drug is feasible.



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Keywords: Interventional Radiology, New Zealand White Rabbit, VX2, radiochemoembolization, Sigma-2 Receptor, SW43.


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Introduction: Primary liver cancer such as the hepatocellular carcinoma (HCC) was the 6th most common cancer type in 2012 with increasing incidence rates 1, 2. Despite ongoing efforts to improve survival rates of unresectable HCC with e.g. locoregional therapies such as drug-eluting bead transarterial chemoembolization (DEB-TACE) 3-6, HCC is still one of the most deadliest cancers with an overall 5-year survival rate improvement from 5% in 1987 to 18% in 2011 in the US 7. Despite its therapeutic benefits, DEB-TACE with e.g. Doxorubicin has some limitations including limited anticancer treatment efficacy 8, 9 and lack of cancer cell specificity 6, 10, 11. Moreover, conventional drug-eluting bead and Doxorubicin tumor distribution cannot be imaged appropriately in a clinical setting. Hence, the exploration of new and more effective treatment strategies is highly desirable. One of the potential cancer biomarkers suitable for cancer targeting and treatment is the Sigma-2 receptor which has been shown to be overexpressed in several rapidly growing tumor types including HCC, pancreatic cancer, colorectal carcinoma, breast cancer, lung cancer, melanoma and the VX2 rabbit tumor model 12-15. Sigma-2 receptor agonists such as SW43 have been described to induce a potent antiproliferative and proapoptotic effect in cancer cells with only very limited to no harm to non-tumorous tissue 14, 16, 17. Additionally, SW43 acts synergistically with antineoplastic drugs such as doxorubicin and, when attached to SW43, internalizes specifically and quickly into the tumor cells with increased intracellular accumulation and treatment efficacy making it an ideal molecule for developing a cancer targeting drug compound 14, 17-19

.

We therefore developed and synthesized SW43-DOX-L-NETA, a drug compound which targets cancer cells directly via the Sigma-2 receptor, contains the antineoplastic drug doxorubicin and


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the chelator L-NETA which can be loaded with radioactive isotopes like 90Yttrium and 177Lu (Lutetium-177) for treatment and/or imaging purposes. When loaded onto drug-eluting bead (DEB), the targeted compound could be delivered selectively to the tumor via injection into the tumor feeding artery. The concept of this “Targeted RadioChemoEmbolization” (TRCE) is outlined in figure 1. To test the concept for feasibility and safety of this drug compound in a preliminary exploratory dose escalation study in vivo, SW43-DOX-L-NETA chelated with the non-radioactive isotope 89

Y via the BFC (L-NTEA) to test for feasibility purposes of loading 90Y, and after being loaded

onto DEB, and it was used to treat New Zealand white rabbits bearing VX2 liver tumor allograft.

Material and Methods: All animal procedures were performed in accordance with the approval from the Institutional Animal Care and Use Committee (IACUC). Ten male New Zealand White rabbits (3.3 - 4.19kg) with VX2 carcinoma liver tumor allografts were used as study platform 20. Animals were randomly allocated to one of the treatment groups: I) untreated control group, II) intra-arterial injection of SW43-DOX-89Y loaded DEB or III) intravenous Doxorubicin treatment. Liver cancer model: VX2 cells were provided by H.S.K. VX2 tumor culturing and liver implantation were prepared similar to previous descriptions 20. Briefly, VX2 tumors for liver implantation were grown in the hind leg muscles of rabbits for ~2-3 weeks and, after euthanasia, viable tumor tissue was obtained for re-implantation. For liver implantation of VX2 tumor pieces, a small subxiphoidal skin incision was performed and the left liver lobe prepared. Thereafter, a 14-gauge venous access catheter was carefully inserted into the liver parenchyma and 4-6 viable tumor pieces with


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~1-2 mm each were gently pushed through the catheter into the liver. Liver injection site was cauterized to avoid extrahepatic tumor spillage. Two weeks later, animals underwent treatment. All anesthesia and euthanasia procedures were performed by trained animal facility personnel. For hind leg VX2 tumor implantation animals received 3 mg/kg Ketamine and 30-35mg/kg Xylazine via intramuscular injection. For tumor liver implantation, animals additionally were ventilated with Isoflurane (2-3%) during procedure and given Buprenorphine (0.02-0.01 mg/kg) at recovery subcutaneously. Euthanasia was performed injecting Pentobarbital (150 mg/kg) intravenously. Preparation of SW43-DOX-89Y and loading onto Drug-Eluting Bead: SW43-DOX-L-NETA synthesis and loading procedure onto Drug eluting beads is outlined in figure 5 and has been described in detail previously 14. Briefly, the Sigma-2 agonist SW43 (1R,3R,5S)-9-(10-aminodecyl)-9-azabicyclo[3.3.1]nonan-3-yl (2-methoxy-5methylphenyl)carbamate was synthesized and modified with a linker bearing azide and amino group followed by the coupling of an oxyamine group in order to attach Doxorubicin. The synthesized BFC L-NETA (2,2'-((6-amino-1-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1yl)hexan-2-yl)azanediyl)diacetic acid) was conjugated with SW43-DOX via copper free click chemistry, and the resulting SW43-DOX-L-NETA was chelated with 89Y3+: SW43-DOX-LNETA and YCl3 were mixed in DI water at molar ratio of 1:1, and the resulting mixture was incubated at 40oC for 30min. Then the pH was adjusted to 6.8 using 0.1M NaOH and further incubation was performed. During incubation, check the pH every 10mins and keep the pH at 6.8 until the pH is steady at 6.8. DEB Loading procedure: 100 µl of 100-300 µm DC Bead™ (DEBDOX™; Biocompatibles UK Ltd, Farnham, UK; 2 ml per vial) were washed three times with sterile water to remove the saline solution and mixed with 0.2-1.0 mg/kg SW43-DOX-89Y


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dissolved in 1 ml sterile water. Loading was performed directly prior to administration under gentle agitation and light protection at room temperature until full loading (20-60 min.) was achieved by visual inspection. Treatment - Exploratory dose escalation with Intra-arterial injection of loaded DEB: Rabbits were treated with increasing compound doses loaded onto DEB (number of rabbits; survival time after treatment): 0.2 mg/kg (n=1; 24h), 0.6 mg/kg (n=3; 1/3/7d) & 1 mg/kg (n=2; 3/24h). As treatment controls, 2 rabbits received intravenous Doxorubicin injection (0.3 mg/kg; 3d) and 2 rabbits were not treated. All intra-arterial procedures were performed by an experienced interventional radiologist (H. S. K.) under fluoroscopic guidance (GE Healthcare, OEC 9600 C-Arm) as similar to previously descriptions 20. Briefly, after achieving full anesthesia, the groin artery was cutdown under sterile conditions and accessed via a 3.0 French sheath (Cook Incorporated, Bloomington, IN, USA). The inserted 2.0 French JB1 catheter (Cook Incorporated, Bloomington, IN, USA) was then advanced into the aorta and the celiac axis. The left hepatic artery was then catheterized with the aid of a 0.010 inch guide wire (Seeker Lite®-10 Guidewire, Target Therapeutics, Fremont, CA, USA) and tumor growth was identified via inspection of local hypervascularity after contrast media injection. The loaded DEB were slowly injected into the left hepatic artery until the entire dose was administered. Here, complete tumor embolization was not an endpoint and not achieved. Subsequently, after successful injection, the catheter was removed and the femoral artery was ligated. Doxorubicin hydrochloride solution for injection (2 mg/ml; Pfizer Inc. New York, NY, USA) was diluted 1:10 with 0.9% sodium chloride solution and 0.3 mg/kg were injected into the ear vein over a 10-15 min. time period. Histopathological and Biochemical Serum Analysis: Animals were sacrificed after several time points as described above post treatment and


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untreated animals were sacrificed equivalent to 24h treatment. The abdominal and thoracic organs including liver, spleen, gastrointestinal tract, kidney, lung, heart, were inspected during gross necropsy for any pathological signs possibly related to treatment. Tissue from the tumor and the right liver lobe were obtained and fixed in 10% Formalin prior to paraffin embedding. Histopathological slide preparation, staining and evaluation was performed by institutional veterinary pathology services. Haematoxylin and Eosin (H&E) staining was used for tumor necrosis evaluation in % (no necrosis, ~10%, ~25%, ~50%, ~75%, ~90%, complete necrosis). Furthermore, tissues were evaluated for Hypoxia Induced Factor 1-alpha (HIF 1-α), Cytokeratin 19 (CK-19), TdT-mediated dUTP-biotin nick end labeling (TUNEL), Epithelial Growth Factor Receptor (EGFR), p53 and Ki-67. The relative staining of viable/ non-necrotic cells was graded as no stain (0), lightly stained (1), moderately stained (2), and strongly stained (3). Inhomogeneous staining patterns were recognized by giving the percentage of staining strength for each slide. An average score of overall staining was obtained by relativizing the percentage of staining expression for each staining degree (e.g. 50% of cells showed no staining (0) and 50 % showed slight staining (1) = average score of 0.5). In the serum, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), total bilirubin, albumin and creatinine were measured before and 3, 24 and up to 72h post treatment. Statistical Analysis: All data is represented as mean ± standard error (SE). Groups were compared using the OneWay ANOVA incl. Bonferroni Post-Hoc test. All calculations were performed with PRISM 6 (GraphPad Software Inc., La Jolla, CA, USA). P

Targeted Yttrium 89-Doxorubicin Drug-Eluting BeadâA Safety and

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