May Q. Wong

Kit-like 18F-labeling of RGD-19F-arytrifluroborate in high yield and at extraordinarily high specific activity with preliminary in vivo tumor imaging.

INTRODUCTION Positron Emission Tomography (PET) is a rapidly expanding, cutting edge technology for preclinical evaluation, cancer diagnosis and staging, and patient management. A one-step aqueous (18)F-labeling method, which can be applied to peptides to provide functional in vivo images, has been a long-standing challenge in PET imaging. Over the past few years, we have sought a rapid and mild radiolabeling method based on the aqueous radiosynthesis of in vivo stable aryltrifluoroborate (ArBF(3)(-)) conjugates. Recent access to production levels of (18)F-Fluoride led to a fluorescent-(18)F-ArBF(3)(-) at unprecedentedly high specific activities of 15Ci/μmol. However, extending this method to labeling peptides as imaging agents has not been explored. METHODS In order to extend these results to a peptide of clinical interest in the context of production-level radiosynthesis, we applied this new technology for labeling RGD, measured its specific activity by standard curve analysis, and carried out a preliminary evaluation of its imaging properties. RESULTS RGD was labeled in excellent radiochemical yields at exceptionally high specific activity (~14Ci/μmol) (n = 3). Preliminary tumor-specific images corroborated by ex vivo biodistribution data with blocking controls show statistically significant albeit relatively low tumor uptake along with reasonably high tumor:blood ratios (n = 3). CONCLUSIONS Isotope exchange on a clinically useful (18)F-ArBF(3)(-) radiotracer leads to excellent radiochemical yields and exceptionally high specific activities while the anionic nature of the aryltrifluoroborate prosthetic results in very rapid clearance. Since rapid clearance of the radioactive tracer is generally desirable for tracer development, these results suggest new directions for varying linker arm composition to slightly retard clearance rather than enhancing it. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE This work is the first to use production levels of (18)F-activity to directly label RGD at specific activities that are an order of magnitude higher than most reports and thereby increases the distribution window for radiotracer production and delivery.

68Ga Small Peptide Imaging: Comparison of NOTA and PCTA

In this study, a bifunctional version of the chelate PCTA was compared to the analogous NOTA derivative for peptide conjugation, (68)Ga radiolabeling, and small peptide imaging. Both p-SCN-Bn-PCTA and p-SCN-Bn-NOTA were conjugated to cyclo-RGDyK. The resulting conjugates, PCTA-RGD and NOTA-RGD, retained their affinity for the peptide target, the α(v)β(3) receptor. Both PCTA-RGD and NOTA-RGD could be radiolabeled with (68)Ga in >95% radiochemical yield (RCY) at room temperature within 5 min. For PCTA-RGD, higher effective specific activities, up to 55 MBq/nmol, could be achieved in 95% RCY with gentle heating at 40 °C. The (68)Ga-radiolabeled conjugates were >90% stable in serum and in the presence of excess apo-transferrin over 4 h; (68)Ga-PCTA-RGD did have slightly lower stability than (68)Ga-NOTA-RGD, 93 ± 2% compared to 98 ± 1%, at the 4 h time point. Finally, the tumor and nontarget organ uptake and clearance of (68)Ga-radiolabeled PCTA-RGD and NOTA-RGD was compared in mice bearing HT-29 colorectal tumor xenografts. Activity cleared quickly from the blood and muscle tissue with >90% and >70% of the initial activity cleared within the first 40 min, respectively. The majority of activity was observed in the kidney, liver, and tumor tissue. The observed tumor uptake was specific with up to 75% of the tumor uptake blocked when the mice were preinjected with 160 nmol (100 μg) of unlabeled peptide. Uptake observed in the blocked tumors was not significantly different than the background activity observed in muscle tissue. The only significant difference between the two (68)Ga-radiolabeled bioconjugates in vivo was the kidney uptake. (68)Ga-radiolabeled PCTA-RGD had significantly lower (p < 0.05) kidney uptake (1.1 ± 0.5%) at 2 h postinjection compared to (68)Ga-radiolabeled NOTA-RGD (2.7 ± 1.3%). Overall, (68)Ga-radiolabeled PCTA-RGD and NOTA-RGD performed similarly, but the lower kidney uptake for (68)Ga-radiolabeled PCTA-RGD may be advantageous in some imaging applications.

Dual Mode Fluorescent 18F-PET Tracers: Efficient Modular Synthesis of Rhodamine-[cRGD]2-[18F]-Organotrifluoroborate, Rapid, and High Yielding One-Step 18F-Labeling at High Specific Activity, and Correlated in Vivo PET Imaging and ex Vivo Fluorescence

The design of dual mode fluorescent-PET peptidic tracers that can be labeled with [(18)F]fluoride at high specific activity and high yield has been challenged by the short half-life of (18)F and its aqueous indolence toward nucleophilic displacement, that often necessitates multistep reactions that start with punctiliously dry conditions. Here we present a modular approach to constructing a fluorescent dimeric peptide with a pendant radioprosthesis that is labeled in water with [(18)F]fluoride ion in a single, user-friendly step. The modular approach starts with grafting a new zwitterionic organotrifluoroborate radioprosthesis onto a pentaerythritol core with three pendent alkynes that enable successive grafting of a bright fluorophore (rhodamine) followed by two peptides (cylcoRGD). The construct is labeled with [(18)F]fluoride via isotope exchange within 20 min in a single step at high specific activity (>3 Ci/μmol) and in good yield to provide 275 mCi and high radiochemical purity. Neither drying of the [(18)F]fluoride ion solution nor HPLC purification of the labeled tracer is required. Facile chemical synthesis of this dual mode tracer along with a user-friendly one-step radiolabeling method affords very high specific activity. In vivo PET images of the dual mode tracer are acquired at both high and low specific activities. At very high specific activity, i.e., 3.5 Ci/μmol, tumor uptake is relatively high (5.5%ID/g), yet the associated mass is below the limits of fluorescent detection. At low specific activity, i.e., 0.01 Ci/μmol, tumor uptake in the PET image is reduced by approximately 50% (2.9%ID/g), but the greater associated mass enables fluorescence detection in the tumor. These data highlight a facile production of a dual mode fluorescent-PET tracer which is validated with in vivo and ex vivo images. These data also define critical limitations for the use of dual mode tracers in small animals.

Single step 18F-labeling of dimeric cycloRGD for functional PET imaging of tumors in mice

INTRODUCTION Arylboronates afford rapid aqueous (18)F-labeling via the creation of a highly polar (18)F-aryltrifluoroborate anion ((18)F-ArBF3(-)). HYPOTHESIS Radiosynthesis of an (18)F-ArBF3(-) can be successfully applied to a clinically relevant peptide. To test this hypothesis, we labeled dimeric-cylcoRGD, [c(RGDfK)]2E because a) it is molecularly complex and provides a challenging substrate to test the application of this technique, and b) [c(RGDfK)]2E has already been labeled via several (18)F-labeling methods which provide for a preliminary comparison. GOAL To validate this labeling method in the context of a complex and clinically relevant tracer to show tumor-specific uptake ex vivo with representative PET images in vivo. METHODS An arylborimidine was conjugated to [c(RGDfK)]2E to give the precursor [c(RGDfK)]2E-ArB(dan), which was aliquoted and stored at -20 °C. Aliquots of 10 or 25 nmol, containing only micrograms of precursor, were labeled using relatively low levels of (18)F-activity. Following purification eight mice (pre-blocked/unblocked) with U87M xenograft tumors were injected with [c(RGDfK)]2E-(18)F-ArBF3(-) (n = 4) for ex vivo tissue dissection. Two sets of mice (pre-blocked/unblocked) were also imaged with PET-CT (n = 2). RESULTS The [c(RGDfK)]2E-ArB(dan) is converted within 15 min to [c(RGDfK)]2E-(18)F-ArBF3(-) in isolated radiochemical yields of ~10% (n = 3) at a minimum effective specific activity of 0.3 Ci/μmol. Biodistribution shows rapid clearance to the bladder via the kidney resulting in high tumor-to-blood and tumor-to-muscle ratios of >9 and >6 respectively while pre-blocking with [c(RGDfK)]2E showed high tumor specificity. PET imaging showed good contrast between tumor and non-target tissues confirming the biodistribution data. CONCLUSION An arylborimidine-RGD peptide is rapidly (18)F-labeled in one step, in good yield, at useful specific activity. Biodistribution studies with blocking controls show tumor specificity, which is corroborated by PET images. Advances in Knowledge and Implications for patient Care: Despite many antecedent examples of labeled RGD tracers, this work is the first to show direct aqueous labeling of bisRGD with an (18)F-ArBF3(-). Labeling occurs in near record rapidity (45 min) at useful effective specific activities and competitive yields for high contrast tumor specific images. As bisRGD has been imaged in humans with several prosthetics, this work suggests potential clinical applications of tracers appended with an (18)F-ArBF3(-). More generally, the ability to label a molecularly complex tracer suggests that this method could be useful to label many other peptides. Furthermore, these results portend the development of kits that use only microgram quantities of lyophilized precursor for on demand labeling. The ability to perform one-step aqueous labeling in under an hour to provide tracers with high T:NT ratios has important implications for developing radiotracers for use in fundamental research and in preclinical tracer studies.

A new 18F-heteroaryltrifluoroborate radio-prosthetic with greatly enhanced stability that is labelled by 18F–19F-isotope exchange in good yield at high specific activity

An 18F-heteroaryltrifluoroborate was designed as a novel radioprosthetic for PET imaging. The stability of the new prosthetic was confirmed using 19F-NMR spectroscopy: the solvolytic half-life was measured to be >16 000 min. Conjugation to RGD gave a stable radiotracer precursor for kit-like labeling by 18F–19F isotope exchange in 15 min. The total radiosynthetic procedure including HPLC purification requires <45 min. A plasma stability assay showed negligible defluoridation following 150 min and stability is corroborated by in vivo imaging showing minimal bone uptake. With Curie levels of 18F–fluoride, yields of 20% and specific activities as high as 3 Ci μmol−1 (110 GBq μmol−1) are achieved (n = 3).

Irinophore C™, a lipid nanoparticulate formulation of irinotecan, improves vascular function, increases the delivery of sequentially administered 5-FU in HT-29 tumors, and controls tumor growth in patient derived xenografts of colon cancer

PURPOSE A liposomal formulation of irinotecan, Irinophore C™ (IrC™) is efficacious in a panel of tumor models, normalizes tumor vasculature, and increases the accumulation of a second drug in the same tumor. We now show that Irinophore C™ is also effective against patient derived xenografts (PDX) of colon cancer, and examine the kinetics of vascular normalization in the HT-29 tumor model and assess how these changes might be used with 5-FU sequentially. MATERIALS AND METHODS Rag2M mice bearing HT-29 tumors were treated with IrC™ (25mg/kg; Q7D×3) for up to three weeks. Groups of tumors were harvested for analysis at 7, 14 and 21days after the start of treatment. Drug and lipid levels in the tumor were evaluated using HPLC and scintillation counts, respectively. Changes in tumor morphology (H&E), vasculature (CD31), perfusion (Hoechst 33342) and apoptosis (TUNEL) were quantified using microscopy. The accumulation of a second drug ([(14)C]-5-FU, 40mg/kg) given 3h before sacrifice was determined using liquid scintillation. The efficacy of IrC™ (Q7D×3) followed by 5-FU treatment (Q7D×3) was assessed in mice bearing established HT-29 tumors. The efficacy of IrC™ was also evaluated in primary human colorectal tumors grown orthotopically in NOD-SCID mice. RESULTS Following a single dose of IrC™ the active lactone forms of irinotecan and its metabolite SN-38 were measurable in HT-29 tumors after 7days. The treatment reduced tumor cell density and increased apoptosis. Hoechst 33342 perfusion and accumulation of [(14)C]-5-FU in the treated tumors increased significantly on days 7 and 14. This was accompanied by an increase in the number of endothelial cells relative to total nuclei in the tumor sections. Pre-treatment with IrC™ (Q7D×3) followed by 5-FU (Q7D×3) delayed the time taken for tumors to reach 1cm(3) by 9days (p<0.05). IrC™ was just as effective as free irinotecan when used on patient derived xenografts of colorectal cancer. CONCLUSIONS Treatment with IrC™ reduces tumor cell viability and appears to normalize the vascular function of the tumor after a single treatment cycle. A concomitant increase in the accumulation of a second drug (5-FU) in the tumor was observed in tumors from IrC™ treated animals and this was correlated with changes in vascular structure consistent with normalization. The treatment effects of sequential 5-FU dosing following IrC™ are additive with no additional toxicity in contrast to previous studies where concurrent 5-FU and IrC™ treatment exacerbated 5-FU toxicity. The studies with PDX tumors also indicate that IrC™ is just as effective as free irinotecan on PDX tumors even though the delivered dose is halved.

The differential effects of metronomic gemcitabine and antiangiogenic treatment in patient-derived xenografts of pancreatic cancer: treatment effects on metabolism, vascular function, cell proliferation, and tumor growth

BackgroundMetronomic chemotherapy has shown promising activity against solid tumors and is believed to act in an antiangiogenic manner. The current study describes and quantifies the therapeutic efficacy, and mode of activity, of metronomic gemcitabine and a dedicated antiangiogenic agent (DC101) in patient-derived xenografts of pancreatic cancer.MethodsTwo primary human pancreatic cancer xenograft lines were dosed metronomically with gemcitabine or DC101 weekly. Changes in tumor growth, vascular function, and metabolism over time were measured with magnetic resonance imaging, positron emission tomography, and immunofluorescence microscopy to determine the anti-tumor effects of the respective treatments.ResultsTumors treated with metronomic gemcitabine were 10-fold smaller than those in the control and DC101 groups. Metronomic gemcitabine, but not DC101, reduced the tumors’ avidity for glucose, proliferation, and apoptosis. Metronomic gemcitabine-treated tumors had higher perfusion rates and uniformly distributed blood flow within the tumor, whereas perfusion rates in DC101-treated tumors were lower and confined to the periphery. DC101 treatment reduced the tumor’s vascular density, but did not change their function. In contrast, metronomic gemcitabine increased vessel density, improved tumor perfusion transiently, and decreased hypoxia.ConclusionThe aggregate data suggest that metronomic gemcitabine treatment affects both tumor vasculature and tumor cells continuously, and the overall effect is to significantly slow tumor growth. The observed increase in tumor perfusion induced by metronomic gemcitabine may be used as a therapeutic window for the administration of a second drug or radiation therapy. Non-invasive imaging could be used to detect early changes in tumor physiology before reductions in tumor volume were evident.

Abstract 5261: Irinophore C, a liposomal formulation of irinotecan, has anti-vascular effects in primary tumors of colorectal cancer grown orthotopically in mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Colorectal cancer accounts for ∼10% of cancer deaths in North America. Our group has developed a series of primary tumors from human colorectal cancer tissue obtained during surgery. These tumors are passaged orthotopically in mice and maintain the complexity and heterogeneity of the original patient sample. We have used these tumors to examine the cytotoxic and anti-vascular effects of Irinophore CTM, a liposomal form of irinotecan, which is more efficacious and less toxic than the parent drug. Materials and Methods: Primary tumor tissues from colorectal cancer patients, were validated by a reference pathologist and implanted subcutaneously in SCID mice. Tumors that grew successfully were then passaged orthotopically on the ascending colon of new mice. When these tumors reached ∼200mm3, groups of mice were treated with saline, irinotecan (50mg/kg), or IrinophoreCTM (25mg/kg) once a week for 6 weeks. Separate groups of tumors, A, B and C were harvested on days 3, 21 and 42 after treatment started, respectively. Magnetic resonance imaging (MRI) was used to assess tumor perfusion in mice from group B. Treatment effects on tumor metabolism were assessed with 18F -fluorodeoxyglucose and positron emission tomography (FDG-PET) for groups A and C mice. Immunofluorescence staining was carried out on tumors from all treatment groups to determine levels of cell proliferation, apoptosis, hypoxia, and vessel density. Results: 4 of 14 samples were successfully propagated and maintain their original morphology. Irinophore CTM treatment reduced tumor volume by 54% to 92% compared to the untreated controls depending on the tumor line. No toxic effects were seen with Irinophore CTM. The aggregate data for cell proliferation (Ki67), necrosis (HE however, Irinophore CTM treatment did reduce vascular density in the tumors. The volume transfer coefficient, Ktrans, derived from MRI, decreased when tumors were treated with irinotecan, but increased with Irinophore CTM treatment. Differences in the metabolic activity of the tumors were also seen. Conclusion: Orthotopic models of colorectal cancer propagated from patient tumors were successfully developed. These models retain the characteristics of the original patient sample and are a good alternative to xenograft models grown from immortalized cell-lines. The anti-tumor activity of Irinophore CTM at lower doses is greater than irinotecans, and with fewer side effects. Treatment with Irinophore CTM also reduces tumor metabolism and appears to improve vascular function. The data imply that Irinophore CTM has sustained anti-tumor activity and multiple mechanisms of action compared to irinotecan. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5261. doi:1538-7445.AM2012-5261

Abstract B12: The effects of Irinophore C™ on the microenvironment and vasculature of a primary orthotopic model of colorectal cancer.

Introduction: Colorectal cancer is a common cancer that accounts for ∼10% of cancer cell deaths in North America. There are numerous in vivo xenograft models for colorectal cancer available, but in general, these are derived from immortalized cell lines and fail to capture the complexities and heterogeneity of tumors seen in the clinic. Our group has developed a series of orthotopic, primary tumors from samples of human colorectal cancer tissue obtained during surgical resection that maintain the characteristics of the original sample. The effects of Irinophore C™, (a liposomal formulation of irinotecan that is more efficacious and less toxic than the parent drug) on the microenvironment and vascular function of these primary colorectal tumors were examined in the present study. Materials and Methods: Primary tumor tissues, obtained from colorectal cancer patients, were validated by a reference pathologist and implanted subcutaneously in SCID mice. Small pieces of subcutaneous tumors that grew successfully were then passaged orthotopically on the ascending colon of new mice. When these tumors reached ∼200mm3, groups of mice were treated with vehicle control (0.9% saline), irinotecan (50mg/kg), or Irinophore C™ (25mg/kg) once a week for 6 weeks. Separate groups of tumors were harvested on days 3 and 42 after treatment started. Immunofluorescence staining was performed on tumor cryosections followed by computerized image analysis to determine levels of cell proliferation, apoptosis, hypoxia, and vessel density. Results: 4 of 14 samples were successfully propagated orthotopically and were characterized by a reference pathologist. The tumors maintain their original morphology, and one is a highly mucinous adenocarcinoma whereas the others are typical colorectal adenocarcinomas. Treatment with irinotecan and Irinophore C™ reduced tumor volumes by 54% and 92%, respectively, compared to the vehicle control. No toxic effects were seen with Irinophore C™. Immunostaining data showed that the distance between blood vessels remained the same for Irinotecan when compared to control, but increased with Irinophore C™ treatment (+30%; P Conclusion: An orthotopic model of colorectal cancer was successfully developed using patient tumor materials that retained the morphology of the primary tumor. Irinophore C™ was more effective in controlling tumor growth than irinotecan despite being delivered at a lower dose. In addition, treatment with Irinophore C™ appeared to improve overall vascular function in the tumor. Based on these data, it is clear that Irinophore C™ has different mechanisms of action and is more efficacious than irinotecan against primary models of colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B12.

Abstract 1422: Cancer-associated fibroblasts influence the sensitivity of pancreatic cancer cells to gemcitabine in pancreatic ductal adenocarcinoma

Introduction: Pancreatic ductal adenocarcinoma is chemo- and radio-resistant, and has the worst survival rates of all cancers. Gemcitabine improves quality of life, but only provides modest survival benefits. Notably, pancreatic cancer is characterized by a strong “reactive” stroma in association with extensive fibroblast proliferation. Our hypothesis is that the large population of cancer-associated fibroblasts (CAFs) in pancreatic tumors produces a plethora of growth factors and cytokines which in turn, influence the sensitivity of pancreatic cancer cells to gemcitabine. Methods: Fourteen primary pancreatic cancer-associated fibroblast (CAF) lines were established in culture using freshly resected pancreatic tumor tissues from 14 different patients. To assess whether these CAFs influence chemosensitivity, MiaPaCa-2 pancreatic cancer cells were injected alone or co-injected with one of the CAF lines CAF11 subcutaneously into SCID mice. When tumors reached ∼200 mm 3 , mice bearing MiaPaCa-2 tumors or MiaPaCa-2+CAF11 tumors were treated with the vehicle control (0.9% saline) or gemcitabine (120 mg/kg, q7d, i.p.). Tumor volume was measured weekly. Concurrently, Affymetrix U133 plus 2.0 arrays were used to determine the differentially expressed genes in pancreatic CAFs vs. normal fibroblasts. The genes were then analyzed with Ingenuity Systems to identify network and canonical pathways that are altered in CAFs, and which may mediate chemosensitivity. Selected genes were further validated by RT-PCR, western blotting, or ELISA. Results: Gemcitabine was found to suppress the growth of MiaPaCa-2 tumors but not that of MiaPaCa-2+CAF11 tumors, suggesting that the presence of CAF11 renders MiaPaCa-2 cells more resistant to the drug. Affymetrix results showed that 1704 probe sets representing 1183 unique genes were differentially expressed by 2-fold or greater (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1422.