Vamsidhar Akurathi

Intracoronary Delivery of Mitochondria to the Ischemic Heart for Cardioprotection

We have previously shown that transplantation of autologously derived, respiration-competent mitochondria by direct injection into the heart following transient ischemia and reperfusion enhances cell viability and contractile function. To increase the therapeutic potential of this approach, we investigated whether exogenous mitochondria can be effectively delivered through the coronary vasculature to protect the ischemic myocardium and studied the fate of these transplanted organelles in the heart. Langendorff-perfused rabbit hearts were subjected to 30 minutes of ischemia and then reperfused for 10 minutes. Mitochondria were labeled with 18F-rhodamine 6G and iron oxide nanoparticles. The labeled mitochondria were either directly injected into the ischemic region or delivered by vascular perfusion through the coronary arteries at the onset of reperfusion. These hearts were used for positron emission tomography, microcomputed tomography, and magnetic resonance imaging with subsequent microscopic analyses of tissue sections to confirm the uptake and distribution of exogenous mitochondria. Injected mitochondria were localized near the site of delivery; while, vascular perfusion of mitochondria resulted in rapid and extensive dispersal throughout the heart. Both injected and perfused mitochondria were observed in interstitial spaces and were associated with blood vessels and cardiomyocytes. To determine the efficacy of vascular perfusion of mitochondria, an additional group of rabbit hearts were subjected to 30 minutes of regional ischemia and reperfused for 120 minutes. Immediately following regional ischemia, the hearts received unlabeled, autologous mitochondria delivered through the coronary arteries. Autologous mitochondria perfused through the coronary vasculature significantly decreased infarct size and significantly enhanced post-ischemic myocardial function. In conclusion, the delivery of mitochondria through the coronary arteries resulted in their rapid integration and widespread distribution throughout the heart and provided cardioprotection from ischemia-reperfusion injury.

The ionic charge of copper-64 complexes conjugated to an engineered antibody affects biodistribution

The development of biomolecules as imaging probes requires radiolabeling methods that do not significantly influence their biodistribution. Sarcophagine (Sar) chelators form extremely stable complexes with copper and are therefore a promising option for labeling proteins with (64)Cu. However, initial studies using the first-generation sarcophagine bifunctional chelator SarAr to label the engineered antibody fragment ch14.18-ΔCH2 (MW 120 kDa) with (64)Cu showed high tracer retention in the kidneys, presumably because the high local positive charge on the Cu(II)-SarAr moiety resulted in increased binding of the labeled protein to the negatively charged basal cells of the glomerulus. To test this hypothesis, ch14.18-ΔCH2 was conjugated with a series of Sar derivatives of decreasing positive charge and three commonly used macrocyclic polyaza polycarboxylate (PAC) bifunctional chelators (BFC). The immunoconjugates were labeled with (64)Cu and injected into mice, and PET/CT images were obtained at 24 and 48 h postinjection (p.i.). At 48 h p.i., ex vivo biodistribution was assessed. In addition, to demonstrate the potential of metastasis detection using (64)Cu-labeled ch14.18-ΔCH2, a preclinical imaging study of intrahepatic neuroblastoma tumors was performed. Reducing the positive charge on the Sar chelators decreased kidney uptake of Cu-labeled ch14.18-ΔCH2 by more than 6-fold, from >45 to <6% ID/g, whereas the uptake in most other tissues, including liver, was relatively unchanged. However, despite this dramatic decrease, the renal uptake of the PAC BFCs was generally lower than that of the Sar derivatives, as was the liver uptake. Uptake of (64)Cu-labeled ch14.18-ΔCH2 in neuroblastoma hepatic metastases was detected using PET.

(18)F-labeled rhodamines as potential myocardial perfusion agents: comparison of pharmacokinetic properties of several rhodamines.

INTRODUCTION We recently reported the development of the [(18)F]fluorodiethylene glycol ester of rhodamine B as a potential positron emission tomography (PET) tracer for myocardial perfusion imaging (MPI). This compound was developed by optimizing the ester moiety on the rhodamine B core, and its pharmacokinetic properties were found to be superior to those of the prototype ethyl ester. The goal of the present study was to optimize the rhodamine core while retaining the fluorodiethyleneglycol ester prosthetic group. METHODS A series of different rhodamine cores (rhodamine 6G, rhodamine 101, and tetramethylrhodamine) were labeled with (18)F using the corresponding rhodamine lactones as the precursors and [(18)F]fluorodiethylene glycol ester as the prosthetic group. The compounds were purified by semipreparative HPLC, and their biodistribution was measured in rats. Additionally, the uptake of the compounds was evaluated in isolated rat cardiomyocytes. RESULTS As was the case with the different prosthetic groups, we found that the rhodamine core has a significant effect on the in vitro and in vivo properties of this series of compounds. Of the rhodamines evaluated to date, the pharmacologic properties of the (18)F-labeled diethylene glycol ester of rhodamine 6G are superior to those of the (18)F-labeled diethylene glycol esters of rhodamine B, rhodamine 101, and tetramethylrhodamine. As with (18)F-labeled rhodamine B, [(18)F]rhodamine 6G was observed to localize in the mitochondria of isolated rat cardiomyocytes. CONCLUSIONS Based on these results, the (18)F-labeled diethylene glycol ester of rhodamine 6G is the most promising potential PET MPI radiopharmaceutical of those that have evaluated to date, and we are now preparing to carry out first-in-human clinical studies with this compound.

Convenient synthesis of 18F-radiolabeled R-(−)-N-n-propyl-2-(3-fluoropropanoxy-11-hydroxynoraporphine

Aporphines are attractive candidates for imaging D2 receptor function because, as agonists rather than antagonists, they are selective for the receptor in the high affinity state. In contrast, D2 antagonists do not distinguish between the high and low affinity states, and in vitro data suggests that this distinction may be important in studying diseases characterized by D2 dysregulation, such as schizophrenia and Parkinsons disease. Accordingly, MCL-536 (R-(-)-N-n-propyl-2-(3-[(18)F]fluoropropanoxy-11-hydroxynoraporphine) was selected for labeling with (18)F based on in vitro data obtained for the non-radioactive ((19)F) compound. Fluorine-18-labeled MCL-536 was synthesized in 70% radiochemical yield, >99% radiochemical purity, and specific activity of 167 GBq/µmol (4.5 Ci/µmol) using p-toluenesulfonyl (tosyl) both as a novel protecting group for the phenol and a leaving group for the radiofluorination.

New chemical and radiochemical routes to [18F]Rho6G-DEG-F, a delocalized lipophilic cation for myocardial perfusion imaging with PET

New chemical and radiochemical syntheses are described for the preparation of [18F]Rho6G-DEG-F, an 18F-labeled analogue of the fluoresecent dye rhodamine 6G, which has shown promise as myocardidal perfusion imaging agent. Tosylated precursors of [18F]Rho6G-DEG-F amenable to 18F-labeling were obtained either through a two-step synthesis from rhodamine 6G lactone (33% yield), or in one step from rhodamine 575 (64% yield), then purified by preparative C18 chromatography. Manual synthesis of [18F]Rho6G-DEG-F was achieved in a single radiochemical step from either the tosylate salt or the tosylate/formate double salt in DMSO under standard nucleophillic aliphatic 18F-fluorination conditions (K[18F]F/K2CO3/Kryptofix 2.2.2.). Incorporation of the [18F]F- was found to be satisfactory (≥34% by TLC), despite the protic character of the precursor molecules. [18F]Rho6G-DEG-F was manually synthesized in final decay-corrected radiochemical yields of 11-26% (tosylate salt) and 9-21% (tosylate/formate double salt). The protocol was transferred to an automated synthesis unit, where the product was obtained in 3-9% radiochemical yield (n=3) decay corrected to start-of-synthesis, >99% radiochemical purity, and a molar activity of 122-267 GBq/μmol (3.3-7.2 Ci/μmol).