Martin Behe

Stability of siRNA polyplexes from poly(ethylenimine) and poly(ethylenimine)-g-poly(ethylene glycol) under in vivo conditions: Effects on pharmacokinetics and biodistribution measured by Fluorescence Fluctuation Spectroscopy and Single Photon Emission Computed Tomography (SPECT) imaging

In search of optimizing siRNA delivery systems for systemic application, one critical parameter remains their stability in blood circulation. In this study, we have traced pharmacokinetics and biodistribution of each component of siRNA polyplexes formed with polyethylenimine 25 kDa (PEI) or PEGylated PEIs by in vivo real-time gamma camera recording, SPECT imaging, and scintillation counting of blood samples and dissected organs. In vivo behavior of siRNA and polymers were compared and interpreted in the context of in vivo stability of the polyplexes which had been measured by fluorescence fluctuation spectroscopy (FFS). Both pharmacokinetics and biodistribution of polymer-complexed siRNA were dominated by the polymer. PEGylated polymers and their siRNA polyplexes showed significantly less uptake into liver (13.6-19.7% ID of PEGylated polymer and 9.5-10.2% ID of siRNA) and spleen compared to PEI 25 kDa (liver deposition: 36.2% ID of polymer and 14.6% ID of siRNA). With non-invasive imaging methods we were able to predict both kinetics and deposition in living animals allowing the investigation of organ distribution in real time and at different time points. FFS measurements proved stability of the applied polyplexes under in vivo conditions which explained the different behavior of complexed from free siRNA. Despite their stability in circulation, we observed that polyplexes dissociated upon liver passage. Therefore, siRNA/(PEG-)PEI delivery systems are not suitable for systemic administration, but instead may be useful when the first-pass effect is circumvented, which is the case in local application.

Exendin-4–Based Radiopharmaceuticals for Glucagonlike Peptide-1 Receptor PET/CT and SPECT/CT

Strong overexpression of glucagonlike peptide-1 (GLP-1) receptors in human insulinoma provides an attractive target for imaging. The first clinical trials demonstrated that GLP-1 receptor SPECT/CT using [Lys40(Ahx [6-aminohexanoic acid]-DOTA-111In)NH2]-exendin-4 can localize hardly detectable insulinomas. However, [Lys40(Ahx-DOTA-111In)NH2]-exendin-4 imaging has drawbacks related to the use of 111In in that it is costly and carries a relatively high radiation burden for the patient. The aim of this study was the preclinical evaluation of [Lys40(Ahx-DOTA-68Ga)NH2]-exendin-4 for PET/CT and [Lys40(Ahx-hydrazinonicotinamide [HYNIC]-99mTc)NH2]-exendin-4 for SPECT/CT. Methods: Internalization, biodistribution, dosimetry, and imaging studies were performed in the Rip1Tag2 mouse model of pancreatic β-cell carcinogenesis and compared with our gold standard [Lys40(Ahx-DOTA-111In)NH2]-exendin-4. Poly-glutamic acid and Gelofusine, a gelatin-based plasma expander, were used for renal uptake reduction studies. Results: The tumor uptake of [Lys40(Ahx-DOTA-68Ga)NH2]-exendin-4 was 205 ± 59 percentage injected activity per gram of tissue at 4 h. Other GLP-1 receptor–positive organs showed more than 4.8 times lower radioactivity uptake. [Lys40(Ahx-HYNIC-99mTc/ethylenediaminediacetic acid [EDDA])NH2]-exendin-4, compared with its 111In- and 68Ga-labeled sister compounds, showed significantly less tumor and organ uptake. The significantly lower tumor and organ uptake of [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4 did not result in inferior tumor-to-organ ratios or reduced image quality. All radiopeptides tested showed a high tumor-to-background ratio, resulting in the visualization of small tumors (maximum diameter between 1.0 and 3.2 mm) by SPECT and PET. The only exception was the kidneys, which also showed high uptake. This uptake could be reduced by 49%−78% using poly-glutamic acid, Gelofusine, or a combination of the 2. The estimated effective radiation dose was 3.7 μSv/MBq for [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4, which was 8 times less than that for [Lys40(Ahx-DOTA-68Ga)NH2]-exendin-4 and 43 times less than that for [Lys40(Ahx-DOTA-111In)NH2]-exendin-4. Conclusion: These promising pharmacokinetic and imaging data show that [Lys40(Ahx-DOTA-68Ga)NH2]-exendin-4 and [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4 are suitable candidates for clinical GLP-1 receptor imaging studies.

Radioimmunotherapy of small-volume disease of metastatic colorectal cancer.

Whereas radioimmunotherapy (RIT) has shown disappointing results in bulky, solid tumors, preclinical results in small‐volume disease and in an adjuvant setting are promising. In a previous Phase I study, the authors had encouraging results with the iodine‐131 (131I)–labeled humanized anti–carcinoembryonic antigen (anti‐CEA) antibody (MAb) hMN‐14 in small‐volume disease of colorectal cancer. The aim of this study was to evaluate, in a subsequent Phase II trial, the therapeutic efficacy of this 131I‐labeled humanized anti‐CEA antibody in colorectal cancer patients with small‐volume disease or in an adjuvant setting.

Pre-clinical comparison of [DTPA0] octreotide, [DTPA0,Tyr3] octreotide and [DOTA0,Tyr3] octreotide as carriers for somatostatin receptor-targeted scintigraphy and radionuclide therapy

We have evaluated the potential usefulness of radiolabelled [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide as radiopharmaceuticals for somatostatin receptor–targeted scintigraphy and radiotherapy. In vitro somatostatin receptor binding and in vivo metabolism in rats of the compounds were investigated in comparison with [111In‐DTPA0] octreotide. Comparing different peptide–chelator constructs, [DTPA0,Tyr3]octreotide and [DOTA0, Tyr3]octreotide were found to have a higher affinity than [DTPA0]octreotide for subtype 2 somatostatin receptors (sst2) in mouse AtT20 pituitary tumour cell membranes (all IC50 values obtained were in the low nanomolar range). In vivo studies in CA20948 tumor‐bearing Lewis rats revealed a significantly higher uptake of both 111In‐labelled [DOTA0,Tyr3]octreotide and [DTPA0,Tyr3]octreotide in sst2‐expressing tissues than after injection of [111In‐DTPA0]octreotide, showing that substitution of Tyr for Phe at position 3 in octreotide results in an increased affinity for its receptor and in a higher target tissue uptake. Uptake of 111In‐labelled [DTPA0]octreotide, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide in pituitary, pancreas, adrenals and tumour was decreased to less than 7% of control by pre‐treatment with 0.5 mg unlabelled octreotide/rat, indicating specific binding to sst2. Comparing different radionuclides, [90Y‐DOTA0,Tyr3]octreotide had the highest uptake in sst2‐positive organs, followed by the [111In‐DOTA0,Tyr3]octreotide, whereas [DOTA0, 125I‐Try3]octreotide uptake was low compared to that of the other radiopharmaceuticals, when measured 24 hr after injection. Renal uptake of 111In‐labelled [DTPA0]octreotide, [DTPA0, Tyr3]octreotide and [DOTA0,Tyr3]octreotide was reduced over 50% by an i.v. injection of 400 mg/kg d‐lysine, whereas radioactivity in blood, pancreas and adrenals was not affected. Int. J. Cancer 75:406–411, 1998.

[Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 is a highly efficient radiotherapeutic for glucagon-like peptide-1 receptor-targeted therapy for insulinoma.

Purpose: Although metabolic changes make diagnosis of insulinoma relatively easy, surgical removal is hampered by difficulties in locating it, and there is no efficient treatment for malignant insulinoma. We have previously shown that the high density of glucagon-like peptide-1 receptors (GLP-1R) in human insulinoma cells provides an attractive target for molecular imaging and internal radiotherapy. In this study, we investigated the therapeutic potential of [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4, an 111In-labeled agonist of GLP-1, in a transgenic mouse model of human insulinoma. Experimental Design: [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 was assessed in the Rip1Tag2 mouse model of pancreatic β-cell carcinogenesis, which exhibits a GLP-1R expression comparable with human insulinoma. Mice were injected with 1.1, 5.6, or 28 MBq of the radiopeptide and sacrificed 7 days after injection. Tumor uptake and response, the mechanism of action of the radiopeptide, and therapy toxicity were investigated. Results: Tumor uptake was >200% injected activity per gram, with a dose deposition of 3 Gy/MBq at 40 pmol [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4. Other GLP-1R–positive organs showed ≥30 times lower dose deposition. A single injection of [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 resulted in a reduction of the tumor volume by up to 94% in a dose-dependent manner without significant acute organ toxicity. The therapeutic effect was due to increased tumor cell apoptosis and necrosis and decreased proliferation. Conclusions: The results suggest that [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 is a promising radiopeptide capable of selectively targeting insulinoma. Furthermore, Auger-emitting radiopharmaceuticals such as 111In are able to produce a marked therapeutic effect if a high tumor uptake is achieved.

First Clinical Evidence That Imaging with Somatostatin Receptor Antagonists Is Feasible

Preclinical studies have indicated that somatostatin receptor (sst)–expressing tumors demonstrate higher uptake of radiolabeled sst antagonists than of sst agonists. In this study, we evaluated whether imaging with sst antagonists was feasible in patients. Methods: Biodistribution and tumor uptake of the sst antagonist 111In-DOTA-pNO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)DTyrNH2 (111In-DOTA-BASS) were studied in 5 patients with metastatic thyroid carcinoma or neuroendocrine tumors. Findings were compared with 111In-pentetreotid (111In-DTPA-octreotide) scan. Results: No adverse effects of 111In-DOTA-BASS (20 μg) were observed. 111In-DOTA-BASS detected 25 of 28 lesions, whereas 111In-DTPA-octreotide detected only 17 of 28 lesions. In the same patient, 111In-DOTA-BASS showed higher tumor and lower renal uptake than 111In-DTPA-octreotide (3.5 ± 2.8 percentage injected activity [%IA] vs. 1.0 ± 0.99%IA and 1.5 ± 0.3 %IA vs. 2.3 ± 0.7 %IA) at 4 h after injection. Conclusion: Imaging of neuroendocrine tumors with sst antagonists is clinically feasible. The favorable human biodistribution data suggest that sst antagonists could significantly affect peptide receptor–mediated imaging and therapy.

In vivo SPECT and real-time gamma camera imaging of biodistribution and pharmacokinetics of siRNA delivery using an optimized radiolabeling and purification procedure.

Single photon emission computed tomography (SPECT) imaging provides a three-dimensional method for exactly locating gamma emitters in a noninvasive procedure under in vivo conditions. For characterization of siRNA delivery systems, molecular imaging techniques are extremely helpful to follow biodistribution under in experimental animal studies. Quantification of biodistribution of siRNA and nonviral delivery systems using this technique requires efficient methods to stably label siRNA with a gamma emitter (e.g., 111In or 99mTc) and to purify labeled material from excesses of radiolabel or linkers. In the following study, we have optimized labeling and purification of siRNA, which was then applied as free siRNA or after complexation with polyethylenimine (PEI) 25 kDa for in vivo real-time gamma camera and SPECT imaging. Quantification of scintillation counts in regions of interest(ROIs) was compared to conventional scintillation counting of dissected organs, and the data acquired by imaging was shown to corroborate that of scintillation counting. This optimization and proof of principle study demonstrates that biodistribution and pharmacokinetics of siRNA and the corresponding polyplexes can be determined using SPECT, leading to comparable results as conventional methodology.

Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100

Acute graft-versus-host disease (GvHD) is a complex process involving endothelial damage and neovascularization. Better understanding of the pathophysiology of neovascularization during GvHD could help to target this process while leaving T-cell function intact. Under ischemic conditions, neovascularization is regulated by different micro RNAs (miRs), which potentially play a role in inflamed hypoxic GvHD target organs. We observed strong neovascularization in the murine inflamed intestinal tract (IT) during GvHD. Positron emission tomography imaging demonstrated abundant αvβ3 integrin expression within intestinal neovascularization areas. To interfere with neovascularization, we targeted αv integrin-expressing endothelial cells, which blocked their accumulation in the IT and reduced GvHD severity independent of immune reconstitution and graft-versus-tumor effects. Additionally, enhanced neovascularization and αv integrin expression correlated with GvHD severity in humans. Expression analysis of miRs in the inflamed IT of mice developing GvHD identified miR-100 as significantly downregulated. Inactivation of miR-100 enhanced GvHD indicating a protective role for miR-100 via blocking inflammatory neovascularization. Our data from the mouse model and patients indicate that inflammatory neovascularization is a central event during intestinal GvHD that can be inhibited by targeting αv integrin. We identify negative regulation of GvHD-related neovascularization by miR-100, which indicates common pathomechanistic features of GvHD and ischemia.

18F-FDG PET, somatostatin receptor scintigraphy, and CT in metastatic medullary thyroid carcinoma: a clinical study and an analysis of the literature.

AimTo determine the clinical potential of 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) in patients with medullary thyroid carcinoma (MTC), we compared it to computed tomography (CT), and somatostatin receptor scintigraphy (SRS). Patients and methodsBlinded evaluation of PET, CT and SRS images obtained from 26 patients with histologically proven metastatic MTC was done by nuclear medicine and radiology specialists. Sites of tumour involvement were classified as ‘sure’ or ‘suspicious’. The data were analysed in comparison to two different standards. Either those sites classified as ‘sure’ by at least one of the methods were defined as the standard or those sites of involvement which were classified as ‘sure’ by at least two methods. ResultsDependent on the type of data analysis performed, PET was able to demonstrate 56.8%/80.6% of the tumour sites, CT showed 64.5%/79.6%, and SRS showed 47.5%/69.9% of the tumour sites. ConclusionOverall, CT is similar or better than PET in our patients (dependent on the standard) while SRS is inferior to both other techniques. Our data are in agreement with publications that consider CT superior to PET in the diagnosis of metastatic MTC while other studies show superiority of PET. However, a combination of CT and PET seems to be the most appropriate non-invasive diagnostic approach in patients with MTC.

Efficacy of 99mTc pertechnetate and 131I radioisotope therapy in sodium/iodide symporter (NIS)-expressing neuroendocrine tumors in vivo

PurposeThere is growing interest in the human sodium/iodide symporter (NIS) gene both as a molecular imaging reporter gene and as a therapeutic gene. Here, we show the feasibility of radioisotope therapy of neuroendocrine tumors. As a separate application of NIS gene transfer, we image NIS-expressing tumors with pinhole SPECT in living subjects.MethodsBiodistribution studies and in vivo therapy experiments were performed in nude mice carrying stably NIS-expressing neuroendocrine tumor xenografts following i.v. injection of 131I and 99mTc pertechnetate. To show the usefulness of NIS as an imaging reporter gene, 99mTc pertechnetate uptake was imaged in vivo using a clinical gamma camera in combination with a custom-made single pinhole collimator, followed by SPECT/small animal MRI data coregistration.ResultsNIS-expressing neuroendocrine tumors strongly accumulated 131I and 99mTc pertechnetate, as did thyroid, stomach, and salivary gland. The volume of NIS-expressing neuroendocrine tumors decreased significantly after therapeutic administration of 131I or 99mTc pertechnetate, whereas control tumors continued to grow. NIS-mediated uptake of 99mTc pertechnetate could be imaged in vivo at high resolution with a clinical gamma camera equipped with a custom-made single pinhole collimator. High-resolution functional and morphologic information could be combined in a single three-dimensional data set by coregistration of SPECT and small animal MRI data. Lastly, we demonstrated a therapeutic effect of 99mTc pertechnetate on NIS-expressing neuroendocrine tumors in cell culture and, for the first time, in vivo, thought to be due to emitted Auger and conversion electrons.ConclusionsNIS-expressing neuroendocrine tumors efficiently concentrate radioisotopes, allowing for in vivo high-resolution small animal SPECT imaging as well as rendering possible successful radioisotope therapy of neuroendocrine tumors.