Cristina Müller

Short‐term dietary restriction and fasting precondition against ischemia reperfusion injury in mice

Dietary restriction (DR) extends lifespan and increases resistance to multiple forms of stress, including ischemia reperfusion injury to the brain and heart in rodents. While maximal effects on lifespan require long‐term restriction, the kinetics of onset of benefits against acute stress is not known. Here, we show that 2–4 weeks of 30% DR improved survival and kidney function following renal ischemia reperfusion injury in mice. Brief periods of water‐only fasting were similarly effective at protecting against ischemic damage. Significant protection occurred within 1 day, persisted for several days beyond the fasting period and extended to another organ, the liver. Protection by both short‐term DR and fasting correlated with improved insulin sensitivity, increased expression of markers of antioxidant defense and reduced expression of markers of inflammation and insulin/insulin‐like growth factor‐1 signaling. Unbiased transcriptional profiling of kidneys from mice subject to short‐term DR or fasting revealed a significant enrichment of signature genes of long‐term DR. These data demonstrate that brief periods of reduced food intake, including short‐term daily restriction and fasting, can increase resistance to ischemia reperfusion injury in rodents and suggest a rapid onset of benefits of DR in mammals.

In vivo anti-tumor activity of the organometallic ruthenium(II)-arene complex [Ru(η6-p-cymene)Cl2(pta)] (RAPTA-C) in human ovarian and colorectal carcinomas

Based on the clinical success of platinum-based anti-cancer drugs such as cisplatin, carboplatin and oxaliplatin, a variety of other metal-based anti-cancer compounds are being investigated. In particular, a number of ruthenium-based compounds have been identified which exhibit unique biochemical properties and reduced toxicity profiles compared to the clinically used platinum-based drugs. We have developed a series of organometallic ruthenium(II)-arene complexes that were shown to exert anti-metastatic activity with relatively minor activity on primary tumor growth. Here, we show that the prototype compound, [Ru(η6-p-cymene)Cl2(pta)], where pta = 1,3,5-triaza-7-phosphaadamantane (RAPTA-C), reduces the growth of primary tumors in preclinical models for ovarian and colorectal carcinomas. When administered daily at relatively low doses (0.2 mg kg−1), RAPTA-C was shown to significantly reduce the growth of the A2780 ovarian carcinoma transplanted onto the chicken chorioallantoic membrane model. Similar activity was observed in LS174T colorectal carcinoma in athymic mice, albeit at a higher dose. In both models, a clear inhibition of microvessel density was observed, confirming the previously discovered anti-angiogenic mechanism of RAPTA-C. Biodistribution studies with radiolabeled (103Ru) RAPTA-C indicate that the compound is rapidly cleared from the organs and the bloodstream through excretion by the kidneys. As such, RAPTA-C is a promising compound for translation to clinical evaluation.

Folic Acid Conjugates for Nuclear Imaging of Folate Receptor–Positive Cancer

The folate receptor (FR) is overexpressed on a variety of tumor types, whereas its distribution in normal tissues and organs is highly limited. Exploration of the utility of the FR revealed its promising potential for targeting with folate-based radiopharmaceuticals. Herein, we report the principle of the FR-targeting strategy and summarize the development of several folic acid radioconjugates useful for SPECT and PET of cancer diseases. The potential applicability of folate radiopharmaceuticals for FR-targeted radionuclide therapy is also discussed.

A “Click Chemistry” Approach to the Efficient Synthesis of Multiple Imaging Probes Derived from a Single Precursor

Different imaging modalities can provide complementary information on biological processes at the cellular or molecular level in vitro and in vivo. However, specific molecular probes suitable for a comparison of different imaging modalities are often not readily accessible because their preparation is usually accomplished by individually developed and optimized syntheses. Herein, we present a general, modular synthetic approach that provides access to multiple probes derived from a single precursor by application of the same, efficient functionalization strategy, the Cu(I)-catalyzed cycloaddition of terminal alkynes and azides (click chemistry). To demonstrate the viability and efficiency of this approach, folic acid (FA) was selected as a targeting vector because the preparation of FA-based imaging probes used for SPECT, PET, MRI, and NIRF by reported synthetic strategies is usually difficult to achieve and often results in low overall yields. We prepared a versatile γ-azido-FA precursor as well as a set of alkyne functionalized probes and precursors including ligand systems suitable for the chelation of various (radio)metals, an NIR dye and (18)F- and (19)F-derivatives, which enabled the parallel development of new FA-imaging probes. The Cu(I)-mediated coupling of the alkynes with the γ-azido-FA precursor was accomplished in high yields and with minimal use of protective groups. The various probes were fully characterized spectroscopically as well as in vitro and in vivo. In vitro, all new FA-derivatives exhibited high affinity toward the folic acid receptor (FR) and/or were specifically internalized into FR-overexpressing KB cells. In vivo experiments with nude mice showed that all probes (except the MRI probes which have not been tested yet) accumulated specifically in FR-positive organs and human KB-cell xenografts. However, in vivo imaging revealed significant differences between the various FA-derivatives with respect to unspecific, off-target localization. In general, the comparison of different probes proved the superiority of the more hydrophilic, radiometal-based imaging agents, a result which will guide future efforts for the development of FA-based imaging probes and therapeutic agents. In addition, the strategy presented herein should be readily applicable to other molecules of interest for imaging and therapeutic purposes and thus represents a valuable alternative to other synthetic approaches.

SPECT Study of Folate Receptor-Positive Malignant and Normal Tissues in Mice Using a Novel 99mTc-Radiofolate

The folate receptor (FR) is overexpressed on epithelial cancers (FR-α) and on activated, but not resting, macrophages (FR-β) involved in a variety of inflammatory and autoimmune diseases. Therefore, folate-based radiopharmaceuticals have the potential to be used as imaging agents of FR-positive tumor and inflammatory cells. In this study SPECT/CT of FR-positive malignant and normal tissues and organs in mice was performed using an improved organometallic 99mTc-radiofolate. Methods: The 99mTc radiolabeling of the histidine-folate was performed using the tricarbonyl technique described earlier for preparation of other organometallic radiofolates. Nude male mice with FR-positive KB tumor xenografts were used. Biodistribution studies were performed 1, 4, and 24 h after injection of the 99mTc-His-folate (1.5 MBq/mouse). Images were acquired with a dedicated small-animal SPECT/CT camera 24 h after injection of the radiofolate (500 MBq/mouse). Ex vivo autoradiography was performed on tumors and FR-positive normal tissues. Adjacent sections were used for in vitro autoradiography of FRs after decay of the injected radioactivity. Results: The SPECT/CT studies revealed accumulation of the radiotracer in FR-positive KB tumor xenografts and kidneys as reported previously. At the same time, specific uptake of the radiofolate in normal tissues—that is, salivary glands and choroid plexus—could be visualized with SPECT. FR-specific accumulation in these tissues and organs was confirmed by coinjection of excess folic acid, which resulted in a complete blockade of radiofolate uptake. In addition, ex vivo and in vitro autoradiography of these organs and tissues confirmed FR expression and displayed radioactivity distribution patterns almost identical to those found on SPECT images. In biodistribution studies we found a high tumor uptake (4.29 ± 0.67 %ID/g [percentage of the injected dose per gram of tissue], 4 h after injection) that was almost completely retained over time (3.51 ± 0.37 %ID/g, 24 h after injection). Conclusion: The novel 99mTc-histidine-folate showed improved in vivo characteristics compared with other organometallic radiofolates that allowed imaging of FR-positive malignant (KB tumor xenografts) and kidneys. For the first time, to our knowledge, specific tracer uptake in salivary glands and the choroid plexus could be visualized using a high-resolution animal SPECT/CT camera.

DOTA Conjugate with an Albumin-Binding Entity Enables the First Folic Acid–Targeted 177Lu-Radionuclide Tumor Therapy in Mice

The folate receptor (FR) has proven a valuable target for nuclear imaging using folic acid radioconjugates. However, using folate-based radiopharmaceuticals for therapy has long been regarded as an unattainable goal because of their considerable renal accumulation. Herein, we present a novel strategy in which a DOTA–folate conjugate with an albumin-binding entity (cm09) was designed with the aim of prolonging circulation in the blood and therewith potentially improving tumor-to-kidney ratios. Methods: The folate conjugate cm09 was radiolabeled with 177LuCl3, and stability experiments were performed in plasma. Cell uptake studies were performed on FR-positive KB tumor cells, and an ultrafiltration assay was used to determine the plasma protein–binding properties of 177Lu-cm09. In vivo, 177Lu-cm09 was tested in KB tumor–bearing mice using SPECT/CT. The therapeutic anticancer effect of 177Lu-cm09 (20 MBq) applied as a single injection or as fractionated injections was investigated in different groups of mice (n = 5) by monitoring tumor size and the survival time of treated mice, compared with untreated controls. Results: Compound cm09 was radiolabeled at a specific activity of 40 MBq/nmol, a radiochemical yield of more than 98%, and a stability of more than 99% over 5 d in plasma. Ultrafiltration revealed significant binding of 177Lu-cm09 to serum proteins (∼91%) in plasma, compared with folate radioconjugate without an albumin-binding entity. Cell uptake and internalization of 177Lu-cm09 was FR-specific and comparable to other folate radioconjugates. In vivo studies resulted in high tumor uptake (17.56 percentage injected dose per gram [%ID/g] at 4 h after injection), which was almost completely retained for at least 72 h. Renal accumulation was significantly reduced (28 %ID/g at 4 h after injection), compared with folate conjugates that lack an albumin-binding entity (∼70 %ID/g at 4 h after injection). These circumstances enabled SPECT imaging of excellent quality. Radionuclide therapy (1 × 20 MBq) revealed complete remission of tumors in 4 of 5 cases and a significantly prolonged survival time, compared with untreated controls. Conclusion: The modification of a folate radioconjugate with an albumin-binding entity resulted in a significant increase of the tumor-to-kidney ratio of radioactivity, enabling for the first time, to our knowledge, the preclinical application of folic acid–targeted radionuclide therapy in mice.

[18F]fluoro-deoxy-glucose folate: a novel PET radiotracer with improved in vivo properties for folate receptor targeting.

The folate receptor (FR) is upregulated in various cancer types (FR-α isoform) and in activated macrophages (FR-β isoform) which are involved in inflammatory and autoimmune diseases, but its expression in healthy tissues and organs is highly restricted to only a few sites (e.g kidneys). Therefore, the FR is a promising target for imaging and therapy of cancer and inflammation using folate-based radiopharmaceuticals. Herein, we report the synthesis and evaluation of a novel folic acid conjugate with improved properties suitable for positron emission tomography (PET). [(18)F]-fluoro-deoxy-glucose folate ([(18)F]3) was synthesized based on the click chemistry approach using 2-deoxy-2-[(18)F]fluoroglucopyranosyl azide and a folate alkyne derivative. The novel radiotracer [(18)F]3 was produced in good radiochemical yields (25% d.c.) and high specific radioactivity (90 GBq/μmol). Compared to previously published (18)F-folic acid derivatives, an increase in hydrophilicity was achieved by using a glucose entity as a prosthetic group. Biodistribution and PET imaging studies in KB tumor-bearing mice showed a high and specific uptake of the radiotracer in FR-positive tumors (10.03 ± 1.12%ID/g, 60 min p.i.) and kidneys (42.94 ± 2.04%ID/g, 60 min p.i.). FR-unspecific accumulation of radioactivity was only found in the liver (9.49 ± 1.13%ID/g, 60 min p.i.) and gallbladder (17.59 ± 7.22%ID/g, 60 min p.i.). No radiometabolites were detected in blood, urine, and liver tissue up to 30 min after injection of [(18)F]3. [(18)F]-fluoro-deoxy-glucose-folate ([(18)F]3) is thus a promising PET radioligand for imaging FR-positive tumors.

Promises of Cyclotron-Produced 44Sc as a Diagnostic Match for Trivalent β−-Emitters: In Vitro and In Vivo Study of a 44Sc-DOTA-Folate Conjugate

In recent years, implementation of 68Ga-radiometalated peptides for PET imaging of cancer has attracted the attention of clinicians. Herein, we propose the use of 44Sc (half-life = 3.97 h, average β+ energy [Eβ+av] = 632 keV) as a valuable alternative to 68Ga (half-life = 68 min, Eβ+av = 830 keV) for imaging and dosimetry before 177Lu-based radionuclide therapy. The aim of the study was the preclinical evaluation of a folate conjugate labeled with cyclotron-produced 44Sc and its in vitro and in vivo comparison with the 177Lu-labeled pendant. Methods: 44Sc was produced via the 44Ca(p,n)44Sc nuclear reaction at a cyclotron (17.6 ± 1.8 MeV, 50 μA, 30 min) using an enriched 44Ca target (10 mg 44CaCO3, 97.00%). Separation from the target material was performed by a semiautomated process using extraction chromatography and cation exchange chromatography. Radiolabeling of a DOTA-folate conjugate (cm09) was performed at 95°C within 10 min. The stability of 44Sc-cm09 was tested in human plasma. 44Sc-cm09 was investigated in vitro using folate receptor–positive KB tumor cells and in vivo by PET/CT imaging of tumor-bearing mice Results: Under the given irradiation conditions, 44Sc was obtained in a maximum yield of 350 MBq at high radionuclide purity (>99%). Semiautomated isolation of 44Sc from 44Ca targets allowed formulation of up to 300 MBq of 44Sc in a volume of 200–400 μL of ammonium acetate/HCl solution (1 M, pH 3.5–4.0) within 10 min. Radiolabeling of cm09 was achieved with a radiochemical yield of greater than 96% at a specific activity of 5.2 MBq/nmol. In vitro, 44Sc-cm09 was stable in human plasma over the whole time of investigation and showed folate receptor–specific binding to KB tumor cells. PET/CT images of mice injected with 44Sc-cm09 allowed excellent visualization of tumor xenografts. Comparison of cm09 labeled with 44Sc and 177Lu revealed almost identical pharmacokinetics. Conclusion: This study presents a high-yield production and efficient separation method of 44Sc at a quality suitable for radiolabeling of DOTA-functionalized biomolecules. An in vivo proof-of-concept study using a DOTA-folate conjugate demonstrated the excellent features of 44Sc for PET imaging. Thus, 44Sc is a valid alternative to 68Ga for imaging and dosimetry before 177Lu-radionuclide tumor therapy.

Pemetrexed Improves Tumor Selectivity of 111In-DTPA-Folate in Mice with Folate Receptor–Positive Ovarian Cancer

Folate-based radiopharmaceuticals can be used as imaging agents and for potential radiotherapy of folate receptor (FR)–positive malignant tissue (e.g., ovarian carcinomas). However, substantial FR expression in the kidneys results in undesired renal retention of radioactivity. Recently, we found that the preinjection of an antifolate significantly improved tumor selectivity of organometallic 99mTc-radiofolates in mice. The aim of this study was to corroborate the effect of pemetrexed with the clinically tested 111In-DTPA-folate (DTPA is diethylenetriaminepentaacetic acid) in a human ovarian cancer xenografted mouse model. Methods: In vivo studies were performed in female athymic nude mice bearing subcutaneous FR-positive ovarian tumors (IGROV-1 and SKOV-3) or metastases (after intraperitoneal SKOV-3 cell inoculation). Biodistribution studies were performed 1, 4, and 24 h after administration of 111In-DTPA-folate (0.7 MBq/mouse, 0.35 μg) with or without preinjection of pemetrexed (PMX, 400 μg) 1 h before the radiofolate. Images were acquired with a high-resolution, high-sensitivity SPECT/CT camera, 4 and 24 h after injection of the radiotracer (30–50 MBq/mouse, 4.5–10 μg). Results: In biodistribution studies the tumor uptake of 111In-DTPA-folate (IGROV-1: 9.79 ± 3.21 %ID/g [percentage injected dose per gram]; SKOV-3: 7.57 ± 0.61 %ID/g, 4 h after injection) was high and retained over the time of investigation. However, considerable retention of radioactivity was found in kidneys (85–105 %ID/g, 4 h after injection), resulting in unfavorably low tumor-to-kidney ratios (∼0.10). Preinjection of PMX resulted in a significant reduction of renal uptake (20%–30% of control values, P < 0.03) at all time points after injection of 111In-DTPA-folate, whereas the tumor uptake was retained. Thus, the tumor-to-kidney ratio was significantly increased to ∼0.50. SPECT/CT images confirmed the superior tumor-to-background ratio in mice injected with PMX. These findings were particularly evident in mice with SKOV-3 metastases that could be visualized only when 111In-DTPA-folate was administered in combination with PMX. Conclusion: The application of PMX resulted in a significant reduction of undesired radioactivity accumulation in kidneys, whereas the tumor uptake remained unaffected. These observations suggest a general validity of the reducing effect of PMX on the uptake of radiofolates in kidneys. Our findings will lead the way toward the development of folate-based radiotherapy.

Folate Receptor–Targeted Single-Photon Emission Computed Tomography/Computed Tomography to Detect Activated Macrophages in Atherosclerosis: Can It Distinguish Vulnerable from Stable Atherosclerotic Plaques?

The need for noninvasive imaging to distinguish stable from vulnerable atherosclerotic plaques is evident. Activated macrophages play a role in atherosclerosis and express folate receptor folate receptor β (FR-β). The feasibility of folate targeting to detect atherosclerosis was demonstrated in human and mouse plaques, and it was suggested that molecular imaging of FR-β through folate conjugates might be a specific marker for plaque vulnerability. However, these studies did not allow differentiation between stable and vulnerable atherosclerotic plaques. We investigated the feasibility of a folate-based radiopharmaceutical (111)In-EC0800) with high-resolution animal single-photon emission computed tomography/computed tomography (SPECT/CT) to differentiate between stable and vulnerable atherosclerotic plaques in apolipoprotein E(−/−) mice in which we can induce plaques with the characteristics of stable and vulnerable plaques by placing a flow-modifying cast around the common carotid artery. Both plaques showed (111)In-EC0800 uptake, with higher uptake in the vulnerable plaque. However, the vulnerable plaque was larger than the stable plaque. Therefore, we determined tracer uptake per plaque volume and demonstrated higher accumulation of (111)In-EC0800 in the stable plaque normalized to plaque volume. Our data show that (111)In-EC0800 is not a clear-cut marker for the detection of vulnerable plaques but detects both stable and vulnerable atherosclerotic plaques in a mouse model of atherosclerosis.The need for noninvasive imaging to distinguish stable from vulnerable atherosclerotic plaques is evident. Activated macrophages play a role in atherosclerosis and express folate receptor folate receptor β (FR-β). The feasibility of folate targeting to detect atherosclerosis was demonstrated in human and mouse plaques, and it was suggested that molecular imaging of FR-β through folate conjugates might be a specific marker for plaque vulnerability. However, these studies did not allow differentiation between stable and vulnerable atherosclerotic plaques. We investigated the feasibility of a folate-based radiopharmaceutical (111In-EC0800) with high-resolution animal single-photon emission computed tomography/computed tomography (SPECT/CT) to differentiate between stable and vulnerable atherosclerotic plaques in apolipoprotein E 7 mice in which we can induce plaques with the characteristics of stable and vulnerable plaques by placing a flow-modifying cast around the common carotid artery. Both plaques showed 111In-EC0800 uptake, with higher uptake in the vulnerable plaque. However, the vulnerable plaque was larger than the stable plaque. Therefore, we determined tracer uptake per plaque volume and demonstrated higher accumulation of 111In-EC0800 in the stable plaque normalized to plaque volume. Our data show that 111In-EC0800 is not a clear-cut marker for the detection of vulnerable plaques but detects both stable and vulnerable atherosclerotic plaques in a mouse model of atherosclerosis.