Nilesh K. Wagh

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

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.

177Lu-labeled HPMA copolymers utilizing cathepsin B and S cleavable linkers: Synthesis, characterization and preliminary in vivo investigation in a pancreatic cancer model

INTRODUCTION A major barrier to the advancement of therapeutic nanomedicines has been the non-target toxicity caused by the accumulation of the drug delivery systems in organs associated with the reticuloendothelial system, particularly the liver and spleen. Herein, we report the development of peptide based metabolically active linkers (MALs) that are enzymatically cleaved by cysteine cathepsin B and S, two proteases highly expressed in the liver and spleen. The overall goal of this approach is to utilize the MALs to lower the non-target retention and toxicity of radiolabeled drug delivery systems, thus resulting in higher diagnostic and radiotherapeutic efficacy. METHODS In this study three MALs (MAL0, MAL1 and MAL2) were investigated. MAL1 and MAL2 are composed of known substrates of cathepsin B and S, respectively, while MAL0 is a non-cleavable control. Both MAL1 and MAL2 were shown to undergo enzymatic cleavage with the appropriate cathepsin protease. Subsequent to conjugation to the HPMA copolymer and radiolabeling with (177)Lu, the peptide-polymer conjugates were renamed (177)Lu-metabolically active copolymers ((177)Lu-MACs) with the corresponding designations: (177)Lu-MAC0, (177)Lu-MAC1 and (177)Lu-MAC2. RESULTS In vivo evaluation of the (177)Lu-MACs was performed in an HPAC human pancreatic cancer xenograft mouse model. (177)Lu-MAC1 and (177)Lu-MAC2 demonstrated 3.1 and 2.1 fold lower liver retention, respectively, compared to control ((177)Lu-MAC0) at 72h post-injection. With regard to spleen retention, (177)Lu-MAC1 and (177)Lu-MAC2 each exhibited a nearly fourfold lower retention, relative to control, at the 72h time point. However, the tumor accumulation of the (177)Lu-MAC0 was two to three times greater than (177)Lu-MAC1 and (177)Lu-MAC2 at the same time point. The MAL approach demonstrated the capability of substantially reducing the non-target retention of the (177)Lu-labeled HPMA copolymers. CONCLUSIONS While further studies are needed to optimize the pharmacokinetics of the (177)Lu-MACs design, the ability of the MAL to significantly decrease non-target retention establishes the potential this avenue of research may have for the improvement of diagnostic and radiotherapeutic drug delivery systems.

Development of hypoxia enhanced 111In-labeled Bombesin conjugates: design, synthesis, and in vitro evaluation in PC-3 human prostate cancer.

The gastrin-releasing peptide receptor (BB2r) has shown great promise for tumor targeting due to the increase of the receptor expression in a variety of human cancers including prostate, breast, small-cell lung, and pancreatic cancer. From clinical investigations, prostate cancer has been shown to be among the most hypoxic of the cancers investigated. Many solid tumors contain regions of hypoxia due to poor organization and efficiency of the vasculature. However, hypoxia is typically not present in normal tissue. Nitroimidazoles, a thoroughly investigated class of hypoxia selective drugs, have been shown to be highly retained in hypoxic tissues. The purpose of this study is to determine if the incorporation of hypoxia trapping moieties into the structural paradigm of BB2r-targeted peptides will increase the retention time of the agents in prostate cancer tumors. The present work involves the design, syntheses, purification, and in vitro investigation of hypoxia enhanced (111)In-BB2r-targeted radioconjugates. A total of four BB2r-targeted conjugates (1-4) were synthesized and coupled with increasing numbers of 2-nitroimidazoles, a hypoxia trapping moiety. Conjugates were radiolabeled with (111)In and purified by HPLC prior to in vitro studies. Receptor saturation assays under both normoxic and hypoxic conditions showed that the BB2r receptor expression on the PC-3 human prostate cancer cell line was not significantly affected by oxygen levels. Competitive binding assays revealed that incorporation of 2-nitroimidazoles had a detrimental effect to BB2r binding when adequate spacer groups, between the hypoxia trapping agent and the pharmacophore, were not employed. All of the 2-nitroimidazole containing BB2r-targeted agents exhibited significantly higher longitudinal retention in PC-3 cells under hypoxic conditions compared to the analogous normoxic studies. Protein association analysis revealed a 3-fold increase in binding of a 2-nitroimidazole containing BB2r-targeted agent under hypoxic relative to normoxic conditions. The positive nature of 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.

Cycloart-23-ene-3β, 25-diol stimulates GLP-1 (7–36) amide secretion in streptozotocin–nicotinamide induced diabetic Sprague Dawley rats: A mechanistic approach

In previous study, we have reported cycloart-23-ene-3β, 25-diol is an active antidiabetic constituent isolated from stem bark of Pongamia pinnata (Linn.) Pierre. The objective of the present investigation was to evaluate cycloart-23-ene-3β, 25-diol stimulates glucagon like peptide-1 (GLP-1) (7-36) amide secretion in streptozotocin-nicotinamide induced diabetic Sprague Dawley rats. Molecular docking studies were performed to elucidate the molecular basis for GLP-1 receptor agonistic activity. Type 2 diabetes was induced in overnight fasted Sprague Dawley rats pre-treated with nicotinamide (100mg/kg, i.p.) followed by administration of streptozotocin (55 mg/kg, i.p.) 20 min after. The rats were divided into following groups; I- non-diabetic, II- diabetic control, III- sitagliptin (5mg/kg, p.o.), IV- cycloart-23-ene-3β, 25-diol (1mg/kg, p.o.). The cycloart-23-ene-3β, 25-diol and sitagliptin treatment was 8 week. Plasma glucose was estimated every week (week 0 to week 8). Body weight, food and water intake were recorded daily. Glycosylated haemoglobin, lipid profile, plasma and colonic active (GLP-1) (7-36) amide, mRNA expression of proglucagnon GLP-1, plasma and pancreatic insulin, histology of pancreata as well as biomarkers of oxidative stress (superoxidase dismutase, reduced glutathione, malondialdehyde, glutathione peroxidase, glutathione S transferase) were measured after 8th week treatment. In acute study, active GLP-1 (7-36) amide release, plasma glucose and insulin were measured during oral glucose tolerance test. The docking data clearly indicated cycloart-23-ene-3β, 25-diol bind to the GLP-1 receptor. It decreased plasma glucose level, increased plasma and pancreatic insulin level as well as increased plasma and colonic active GLP-1 (7-36) amide secretion in streptozotocin-nicotinamide induced diabetic Sprague Dawley rats.

Evaluation of DOTA-chelated neurotensin analogs with spacer-enhanced biological performance for neurotensin-receptor-1-positive tumor targeting

INTRODUCTION Neurotensin receptor 1 (NTR1) is overexpressed in many cancer types. Neurotensin (NT), a 13 amino acid peptide, is the native ligand for NTR1 and exhibits high (nM) affinity to the receptor. Many laboratories have been investigating the development of diagnostic and therapeutic radiopharmaceuticals for NTR1-positive cancers based on the NT peptide. To improve the biological performance for targeting NTR1, we proposed NT analogs with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelation system and different lengths of spacers. METHODS We synthesized four NTR1-targeted conjugates with spacer lengths from 0 to 9 atoms (null (N0), β-Ala-OH (N1), 5-Ava-OH (N2), and 8-Aoc-OH (N3)) between the DOTA and the pharmacophore. In vitro competitive binding, internalization and efflux studies were performed on all four NT analogs. Based on these findings, metabolism studies were carried out on our best performing conjugate, (177)Lu-N1. Lastly, in vivo biodistribution and SPECT/CT imaging studies were performed using (177)Lu-N1 in an HT-29 xenograft mouse model. RESULTS As shown in the competitive binding assays, the NT analogs with different spacers (N1, N2 and N3) exhibited lower IC50 values than the NT analog without a spacer (N0). Furthermore, N1 revealed higher retention in HT-29 cells with more rapid internalization and slower efflux than the other NT analogs. In vivo biodistribution and SPECT/CT imaging studies of (177)Lu-N1 demonstrated excellent accumulation (3.1 ± 0.4%ID/g) in the NTR1-positive tumors at 4h post-administration. CONCLUSIONS The DOTA chelation system demonstrated some modest steric inhibition of the pharmacophore. However, the insertion of a 4-atom hydrocarbon spacer group restored optimal binding affinity of the analog. The in vivo assays indicated that (177)Lu-N1 could be used for imaging and radiotherapy of NTR1-positive tumors.

Antidiabetic Activity of Allium Sativum

Allium sativum , commonly known as garlic, belongs to the family Liliaceae. The allicin compound derived from garlic has been found to possess a significant blood sugar-lowering action. Clinical studies have suggested that active compounds from garlic lower blood glucose levels by competing with insulin sites in the liver, which results in an increase in free insulin. Research on garlic has shown that animals and humans with diabetes experienced a decline in blood sugar while taking garlic. Interestingly, if blood sugar is normal, garlic does not promote this lowering effect. Several animal and human studies have shown that garlic may be a very valuable therapeutic agent for diabetes. Studies conducted by medical doctors in India revealed that garlic is a completely natural and harmless substance, which is effective in the treatment of diabetes. The effect of garlic juice was studied in artificial diabetic rats, which showed a significant reduction in blood sugar levels. It is considered that some of the sulfur-containing compounds of garlic have special capabilities in regulating sugar metabolism.

Abstract LB-259: Pb203-AR-RMX conjugates for image-guided TAT of neuroendocrine tumors (NETs)

Objective: The peptide receptor radionuclide therapy (PPRT) for somatostatin receptor positive (SSTR) neuroendocrine tumors (NETs) has emerged more than 15 years ago. The beta-emitter-PRRT has shown to induce objective response in 30-45% of metastatic NETs patients with hematologic/renal toxicity reduced by dose fractionation. The complete response to therapy is rare due to the heterogeneity of NETs; advanced stage of disease at the time of diagnosis; and patient resistance to nonradioactive octreotide and 90Y/177Lu PRRT developed during the therapy. The targeted alpha-emitter therapy of NETs can overcome these limitations. It can enhance the therapeutic response of patients and decrease side-effect and overcome patient resistance of beta-emitter PRRT without significant acute and mid-term toxicity. The RadioMedix and AREVA Med teams together have recently developed several novel 203Pb-peptide derivatives targeting SSTR(+) cancer cells, 203Pb-AR-RMX. The 203Pb is a gamma emitter (279 keV) with t1/2=51.9 h, suitable for single-photon emission computed tomography (SPECT) imaging. The 203Pb is an ideal surrogate for 212Pb α-particle therapy because both isotopes share identical chemical properties. The objective of these studies were: (1) to evaluate the SSTR targeting properties of novel conjugates 203Pb-AR-RMX; and (2) to determine their PK and biodistribution in vivo in SSTR overexpressing xenographs; and (3) to select lead candidate for further pre-IND clinical studies. Methods: AR-RMX was manufactured under GMP by Macrocylics Inc. The 203Pb-radiolabeling of AR-RMX was carried out under mild conditions. The SSTR targeting properties of 203Pb-AR-RMX were determined in the cellular uptake/competition studies in AR-42J cancer cells and in vivo in SSTR(+) AR42J xenograph mice. Results:203Pb-AR-RMX-15 shows exceptionally high tumor-specific accumulation and retention in SSTR(+) AR42J xenograph mice. Tumor uptake of 203Pb-AR-RMX-15 was >14.4 %ID/g at 1h post injection and it remained at this level at least for 24h. The kidneys accumulation of 203Pb-AR-RMX-15 was >13 %ID/g at 1h and varies in different strains of mice but decreased progressively over the 24h. The kidney uptake of agent is similar to previously observed for octreotate labeled with other isotopes. The tumor uptake is significantly higher possible as a result of the change of charge of the 203Pb-AR-RMX-15. The acute hematotoxicity and chronic kidney toxicity is known to limit the doses of 90Y/177Lu PRRT. Preliminary studies of 203Pb-AR-RMX-15 showed that its kidney retention can be reduced by >32% by co-injection of our proprietary AminoMedixTM or Gelofusine-Lysine composition. 203Pb-AR-RMX-15 has shown >98% radio/chemical stability up 7 days post-formulation; no bone marrow uptake of agent was observed in bioD studies of 203Pb AR-RMX-15 done up to 24h post injection. These results allow us to hypothesize that the therapeutic dose of 203Pb/212Pb-AR-RMX-15 is expected to be significantly higher than the dose limiting activity. Conclusions:203Pb-AR-RMX-15 showed promising results in vitro and in vivo studies and will be further investigated as a 203Pb/212Pb-labeled theranostics agent. Our “theranostics” approach using 203Pb/212Pb-PRRT has a potential to advance image-guided radionuclide therapy that can detect and deliver therapeutic radiation dose precisely to SSTR(+) NETs. Citation Format: Izabela Tworowska, Tania Stallons, Amal Saidi, Nilesh Wagh, Federico Rojas-Quijano, Paul Jurek, Garry Kiefer, Julien Torgue, Ebrahim Delpassand. Pb203-AR-RMX conjugates for image-guided TAT of neuroendocrine tumors (NETs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-259. doi:10.1158/1538-7445.AM2017-LB-259