Relative Contribution of Different Mitochondrial Oxidative Phosphorylation Components to the Retinal Pigment Epithelium Barrier Function: Implications for RPE-Related Retinal Diseases.

Disruption of retinal pigment epithelial (RPE) barrier integrity is involved in the pathology of several blinding retinal diseases including age-related macular degeneration (AMD) and diabetic retinopathy (DR), but the underlying causes and pathophysiology are not completely well-defined. Mitochondria dysfunction has often been considered as a potential candidate implicated in such a process. In this study, we aimed to dissect the role of different mitochondrial components; specifically, those of oxidative phosphorylation (OxPhos), in maintaining the barrier functionality of RPE. Electric cell-substrate impedance sensing (ECIS) technology was used to collect multi-frequency electrical impedance data to assess in real-time the barrier formation of the RPE cells. For this purpose, the human retinal pigment epithelial cell line-ARPE-19-was used and treated with varying concentrations of specific mitochondrial inhibitors that target different steps in OxPhos: Rotenone for complex I (the largest protein complex in the electron transport chain (ETC)); oligomycin for ATP synthase; and carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) for uncoupling ATP synthesis from the accompanying ETC. Furthermore, data were modeled using the ECIS-Zθ software to investigate in depth the effects of these inhibitors on three separate barrier parameters: cell-cell interactions (Rb), cell-matrix interactions (α), and the cell membrane capacitance (Cm). The viability of ARPE-19 cells was determined by lactate dehydrogenase (LDH) Cytotoxicity Assay. The ECIS program's modeling demonstrated that FCCP and thus OxPhos uncoupling disrupt the barrier function in the ARPE-19 cells across all three components of the total resistance (Rb, α, and Cm) in a dose-dependent manner. On the other hand, oligomycin and thus ATP synthase inhibition mostly affects the ARPE-19 cells' attachment to their substrate evident by a significant decrease in α resistance in a dose-dependent manner, both at the end and throughout the duration of the experiment. On the contrary, rotenone and complex I inhibition mostly affect the ARPE-19 paracellular resistance Rb in a dose-dependent manner compared to basolateral resistance α or Cm. Our results clearly demonstrate differential roles for different mitochondrial components in maintaining RPE cell functionality in which uncoupling of OxPhos is a major contributing factor to the disruption barrier function. Such differences can be used in investigating gene expression as well as for screening of selective agents that improve the OxPhos coupling efficiency to be used in the therapeutic approach for treating RPE-related retinal diseases.

Using Rotenone to Model Parkinson's Disease in Mice: A Review of the Role of Pharmacokinetics.

Rotenone is a naturally occurring toxin that inhibits complex I of the mitochondrial electron transport chain. Several epidemiological studies have shown an increased risk of Parkinson's disease (PD) in individuals exposed chronically to rotenone, and it has received great attention for its ability to reproduce many critical features of PD in animal models. Laboratory studies of rotenone have repeatedly shown that it induces in vivo substantia nigra dopaminergic cell loss, a hallmark of PD neuropathology. Additionally, rotenone induces in vivo aggregation of α-synuclein, the major component of Lewy bodies and Lewy neurites found in the brain of PD patients and another hallmark of PD neuropathology. Some in vivo rotenone models also reproduce peripheral signs of PD, such as reduced intestinal motility and peripheral α-synuclein aggregation, both of which are thought to precede classical signs of PD in humans, such as cogwheel rigidity, bradykinesia, and resting tremor. Nevertheless, variability has been noted in cohorts of animals exposed to the same rotenone exposure regimen and also between cohorts exposed to similar doses of rotenone. Low doses, administered chronically, may reproduce PD symptoms and neuropathology more faithfully than excessively high doses, but overlap between toxicity and parkinsonian motor phenotypes makes it difficult to separate if behavior is examined in isolation. Rotenone degrades when exposed to light or water, and choice of vehicle may affect outcome. Rotenone is metabolized extensively in vivo, and choice of route of exposure influences greatly the dose used. However, male rodents may be capable of greater metabolism of rotenone, which could therefore reduce their total body exposure when compared with female rodents. The pharmacokinetics of rotenone has been studied extensively, over many decades. Here, we review these pharmacokinetics and models of PD using this important piscicide.

Characterization of the four isomers of (123)I-CMICE-013: a potential SPECT myocardial perfusion imaging agent.

UNLABELLED: Myocardial perfusion imaging (MPI) with single photon emission computed tomography (SPECT) is widely used in the assessment of coronary artery disease (CAD). We have developed (123)I-CMICE-013 based on rotenone, a mitochondrial complex I (MC-1) inhibitor, as a promising new MPI agent. Our synthesis results in a mixture of four species of (123)I-CMICE-013 A, B, C, D. In this study, we separated the four species and evaluated their biodistribution and imaging properties. The cold analogs (127)I-CMICE-013 A, B, C, D were isolated and characterized and their chemical structures proposed. METHODS: (123)I-CMICE-013 was synthesized by radiolabeling rotenone with Na(123)I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60°C for 45min, and the four species were separated by RP-HPLC. The cold analogs (127)I-CMICE-013 A, B, C and D were isolated with a similar procedure and characterized by NMR and mass spectrometry. Biodistribution and microSPECT imaging studies were carried out on normal rats. RESULTS: We propose the mechanism of the rotenone iodination and the structures of the four species. First, I(+) forms an intermediate three-membered ring with 6' and 7' carbons. Second, the lone electron pair of the water molecule attacks the 6' or 7'-carbon, following by the formation of 6'-OH, and 7'-I bonds as in major products C and D, or 6'-I and 7'-OH bonds as in minor products A and B. The weaker 6'-I bond in the intermediate prompts the nucleophilic attachment of water at the favorable 6'-carbon to generate C and D. MicroSPECT images of (123)I-CMICE-013 A, B, C, D in rats showed clear visualization of myocardium and little interference from lung and liver. The imaging time activity curves and biodistribution data showed complex profiles for the four isomers, which is not expected from the structure activity relationship theory. CONCLUSION: (123/127)I-CMICE-013 A and B are constitutional isomers with C and D, while A and C are diastereomers of B and D, respectively. Overall, the biological characteristics of the four species are not correlated perfectly with their molecular structures.

Delivering instilled hydrophobic drug to the bladder by a cationic nanoparticle and thermo-sensitive hydrogel composite system.

Some bladder disease therapies can benefit from intravesical drug delivery, which involves direct instillation of drug into the bladder via a catheter, to attain high local concentrations of the drug with minimal systemic effects. Deguelin is a potential anticancer agent, however, its poor water solubility and neurotoxicity restrict its clinical application. To address these challenges, we investigated the promising application of deguelin in the intravesical therapy of bladder cancer by designing a novel intravesical drug delivery system for deguelin. It was found that deguelin could efficiently kill bladder cancer cells and inhibit angiogenesis. Intravesically administrated deguelin had better tolerance than systemically applied deguelin. Encapsulation of deguelin in cationic DOTAP and monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) hybrid nanoparticles (DMP) created the deguelin loaded DMP nanoparticles (D/DMP). They had a mean particle size of 35 nm and zeta potential of 21 mV, rendering deguelin completely dispersible in aqueous media. Encapsulation of deguelin in cationic DMP nanoparticles enhanced the anticancer activity of deguelin in vitro. In addition, D/DMP nanoparticles were incorporated into a thermo-sensitive Pluronic F127 hydrogel, forming a novel D/DMP-F system, which remained in a flowing liquid state at lower than 25 °C, but underwent gelation at higher temperatures. The DMP nanoparticles in the F127 hydrogel system (DMP-F) could significantly extend the hydrophobic drug residence time and increase the drug concentration within the bladder. These results suggested that DMP-F was a good intravesical drug delivery system and D/DMP-F may have promising applications in intravesical therapy of bladder cancer.

Myocardial uptake of 7'-(Z)-[(123)I]iodorotenone during vasodilator stress in dogs with critical coronary stenoses.

BACKGROUND: There is a well-recognized need for a new generation of single photon emission computed tomography (SPECT) perfusion tracers with improved myocardial extraction over a wide flow range. Radiotracers that target complex I of the mitochondrial electron transport chain have been proposed as a new class of myocardial perfusion imaging agents. 7-(Z)-[(125)I]iodorotenone ((125)I-ZIROT) has demonstrated superior myocardial extraction and retention characteristics in rats and in isolated perfused rabbit hearts. We sought to fully characterize the biodistribution and myocardial extraction versus flow relationship of (123)I-ZIROT in an intact large-animal model. METHODS AND RESULTS: The (123)I-ZIROT was administered during adenosine A(2A) agonist-induced hyperemia in 5 anesthetized dogs with critical left anterior descending (LAD) stenoses. When left circumflex (LCx) flow was maximal, (123)I-ZIROT and microspheres were coinjected and the dogs were euthanized 5 minutes later. (123)I-ZIROT biodistribution was evaluated in 2 additional dogs by in vivo planar imaging. At (123)I-ZIROT injection, transmural LAD flow was unchanged from baseline (mean±SEM, 0.90±0.22 versus 0.87±0.11 mL/[min · g]; P=0.92), whereas LCx zone flow increased significantly (mean±SEM, 3.25±0.51 versus 1.00±0.17 mL/[min · g]; P<0.05). Myocardial (123)I-ZIROT extraction tracked regional myocardial flow better than either thallium-201 or (99m)Tc-sestamibi from previous studies using a similar model. Furthermore, the (123)I-ZIROT LAD/LCx activity ratios by ex vivo imaging or well counting (mean±SEM, 0.42±0.08 and 0.45±0.1, respectively) only slightly underestimated the LAD/LCx microsphere flow ratio (0.32±0.09). CONCLUSIONS: The ability of (123)I-ZIROT to more linearly track blood flow over a wide range makes it a promising new SPECT myocardial perfusion imaging agent with potential for improved coronary artery disease detection and better quantitative estimation of the severity of flow impairment.

Liposomal encapsulation of deguelin: evidence for enhanced antitumor activity in tobacco carcinogen-induced and oncogenic K-ras-induced lung tumorigenesis.

Deguelin has shown promising chemopreventive and therapeutic activities in diverse types of cancers. However, the potential side effect of deguelin over a certain dose could be the substantial hurdle in the practical application of the drug. One of the successful strategies for the use of deguelin in clinical trials could be lung-specific delivery of the drug. The present study evaluates the efficacy of liposome-encapsulated deguelin with a dose of 0.4 mg/kg, which is 10 times less than the dose (4 mg/kg) for preventive and therapeutic activities validated in previous in vivo studies. Liposomal deguelin revealed cytotoxic activity in vitro in premalignant and malignant human bronchial epithelial cells and non-small cell lung cancer cells through the same mechanistic pathway previously reported for deguelin (i.e., suppression of the heat shock protein 90 chaperone function and induction of apoptosis). Delivery of liposomal deguelin at a dose of 0.4 mg/kg by intranasal instillation resulted in markedly increased drug partitioning to the lungs compared with that of 4 mg/kg deguelin or 0.4 mg/kg liposomal deguelin administered by oral gavage. Lung-specific delivery of deguelin (0.4 mg/kg) via nasal or intratracheal instillation in a liposomal formulation also showed significant chemopreventive and therapeutic activities in 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone/benzo(a)pyrene-treated A/J mice and K-rasLAC57Bl6/129/sv F1 mice with no detectable toxicity. Our findings support the potential use of deguelin in a liposomal formulation via lung-specific delivery to improve efficacy and to reduce the potential side effects of the agent.

Mitochondrial avid radioprobes. Preparation and evaluation of 7'(Z)-[125I]iodorotenone and 7'(Z)-[125I]iodorotenol.

The loss of mitochondrial function has been implicated in a number of maladies such as Huntington's disease, Parkinson's disease (PD), cancer and cardiovascular disease. The objective of this research was to develop a radiolabeled mitochondrial probe. Two tracers, 7'-Z-iodorotenol and 7'-Z-iodorotenone, analogs of rotenone a natural product that inhibits Complex I of the mitochondrial electron transport chain, have been labeled with iodine-125 in 45-85% yield in a single step from the corresponding tributylstannyl precursor. In vivo distribution in adult male Sprague-Dawley rats for both compounds showed high accumulation in the heart (1.7-3.7 %ID/g at 1 h), a tissue with high mitochondrial content. Z-Iodorotenol did not washout of most tissues between 1 and 2 h postinjection, whereas Z-iodorotenone showed moderate washout (7-26%) over the same period. By 24 h, there was significant loss of both compounds from most tissues including the heart. Heart-to-blood, -lung and -liver ratios for Z-iodorotenone of 28.9, 10.7 and 2.4, respectively, were two- to fourfold higher than the Z-iodorotenol ratios. Compared to the current clinical perfusion tracers, 99mTc-sestamibi and 99mTc-tetrofosmin, Z-iodorotenone demonstrates similar 1 h heart accumulation and significantly higher heart-to-lung ratio (P<.001). Z-Iodorotenone heart-to-liver ratio is equivalent to 99mTc-sestamibi. 7'-Z-Iodorotenone possesses distribution characteristics of an improved tracer for SPECT perfusion studies.

Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson's disease.

Parkinson's disease (PD) has been linked to mitochondrial dysfunction and pesticide exposure. The pesticide rotenone (ROT) inhibits complex I and reproduces features of PD in animal models, suggesting that environmental agents that inhibit complex I may contribute to PD. We have previously demonstrated that ROT toxicity is dependent upon complex I inhibition and that oxidative stress is the primary mechanism of toxicity. In this study, we examined the in vitro toxicity and mechanism of action of several putative complex I inhibitors that are commonly used as pesticides. The rank order of toxicity of pesticides to neuroblastoma cells was pyridaben > rotenone > fenpyroximate > fenazaquin > tebunfenpyrad. A similar order of potency was observed for reduction of ATP levels and competition for (3)H-dihydrorotenone (DHR) binding to complex I, with the exception of pyridaben (PYR). Neuroblastoma cells stably expressing the ROT-insensitive NADH dehydrogenase of Saccharomyces cerevisiae (NDI1) were resistant to these pesticides, demonstrating the requirement of complex I inhibition for toxicity. We further found that PYR was a more potent inhibitor of mitochondrial respiration and caused more oxidative damage than ROT. The oxidative damage could be attenuated by NDI1 or by the antioxidants alpha-tocopherol and coenzyme Q(10). PYR was also highly toxic to midbrain organotypic slices. These data demonstrate that, in addition to ROT, several commercially used pesticides directly inhibit complex I, cause oxidative damage, and suggest that further study is warranted into environmental agents that inhibit complex I for their potential role in PD.

Synergism of rotenone by piperonyl butoxide in Haemonchus contortus and Trichostrongylus colubriformis in vitro: potential for drug-synergism through inhibition of nematode oxidative detoxification pathways.

The anthelmintic properties of rotenone and its activity in combination with the cytochrome P450 inhibitor piperonyl butoxide, were examined in in vitro assays with adults and larvae of Haemonchus contortus and larvae of Trichostrongylus colubriformis. Rotenone was toxic to larvae of both species, with LC(50) values in larval development assays of 0.54 and 0.64 microg/ml for H. contortus and T. colubriformis, respectively. The compound also caused complete cessation of movement in adult H. contortus after 72 h at a concentration of 20 microg/ml. Toxicity of rotenone towards the larvae of both species was increased in the presence of piperonyl butoxide (synergism ratios of 3-4-fold at the LC(50)) and the activity against adult H. contortus was also significantly enhanced following pre-treatment with piperonyl butoxide. This significant synergism suggests that these nematode species are able to utilize a cytochrome P450 enzyme system to detoxify rotenone and indicates that a role may exist for cytochrome P450 inhibitors to act as synergists for other anthelmintics which are susceptible to oxidative metabolism within the nematode.

A delivery strategy for rotenone microspheres in an animal model of Parkinson's disease.

In order to study the pathogenesis of Parkinson's disease (PD), and explore therapeutic drug or approaches, the accurate animal model of PD with inexpensive, biocompatible and convenient administration was necessary. The aim of the present work was to investigate a delivery strategy for rotenone microspheres in an animal model of PD. The rotenone microspheres were prepared by solvent evaporation technique. The rotenone microspheres showed high entrapment efficiency (97.4+/-2.2%) with particle size about 100 microm. In vitro release of rotenone microspheres demonstrated different profiles from medium with different pH or concentration of isopropyl alcohol. The most consistent medium with in vivo rotenone levels in rat plasma was PBS (pH 5.8) with 20% isopropyl alcohol, and the cumulated release amount of rotenone over 30 days was 95.4% in it. The rotenone microspheres (9 mg/kg) produced typical PD symptoms in rats, for example, the cataleptic behavior test demonstrated a obviously prolonged descent latency compared with control animals after administration, and the tyrosine hydroxylase (TH) immunohistochemistry tests showed typical histological evidence of selective degeneration of the nigrostriatal dopaminergic system (striatum and substantia nigra) in rotenone microspheres-treated rats. In addition, this delivery system for rotenone model showed many noticeable advantages such as inexpensive, biocompatible and expedient administration by direct subcutaneous injection. This information suggested that rotenone microspheres as a delivery strategy for setting up an ideal animal model of PD was feasible.

Kinetic analysis of 18F-fluorodihydrorotenone as a deposited myocardial flow tracer: comparison to 201Tl.

UNLABELLED: The goals of this investigation were to assess the accuracy of (18)F-fluorodihydrorotenone ((18)F-FDHR) as a new deposited myocardial flow tracer and to compare the results to those for (201)Tl. METHODS: The kinetics of these flow tracers in 22 isolated, erythrocyte- and albumin-perfused rabbit hearts were evaluated over a flow range encountered in patients. The 2 flow tracers plus a vascular reference tracer ((131)I-albumin) were introduced as a bolus through a port just above the aortic cannula. Myocardial extraction, retention, washout, and uptake parameters were computed from the venous outflow curves with the multiple-indicator dilution technique and spectral analysis. RESULTS: The mean +/- SD initial extraction fractions for (18)F-FDHR (0.85 +/- 0.07) and (201)Tl (0.87 +/- 0.05) were not significantly different, although the initial extraction fraction for (18)F-FDHR declined with flow (P < 0.0001), whereas the initial extraction fraction for (201)Tl did not. The washout of (201)Tl was faster (P < 0.001) and more affected by flow (P < 0.05) than was the washout of (18)F-FDHR. Except for the initial extraction fraction, (18)F-FDHR retention was higher (P < 0.001) and less affected by flow (P < 0.05) than was (201)Tl retention. Reflecting its superior retention, the net uptake of (18)F-FDHR was better correlated with flow than was that of (201)Tl at both 1 and 15 min after tracer introduction (P < 0.0001 for both comparisons). CONCLUSION: The superior correlation of (18)F-FDHR uptake with flow indicates that it is a better flow tracer than (201)Tl in the isolated rabbit heart. Compared with the other currently available positron-emitting flow tracers ((82)Rb, (13)N-ammonia, and (15)O-water), (18)F-FDHR has the potential of providing excellent image resolution without the need for an on-site cyclotron.

Rotenone, deguelin, their metabolites, and the rat model of Parkinson's disease.

Rotenone and deguelin are the major active ingredients and principal components of cuberesin from Lonchocarpus utilis used as a botanical insecticide and piscicide. They are also potent complex I (NADH:ubiquinone oxidoreductase) inhibitors. Rotenone was known earlier, and deguelin is shown here to induce a Parkinson's disease (PD)-like syndrome after subcutaneous treatment of rats by osmotic minipump. Rotenone at 3 mg/kg/day or deguelin at 6 but not 3 mg/kg/day induces degeneration of the nigrostriatal dopaminergic pathway, as shown by reduced tyrosine hydroxylase immunoreactivity with treatments for 5 or 6 days. The neuropathological lesions are associated with a brain level of parent rotenoid of 0.4-1.3 ppm but not with the much smaller brain level of 12abeta-hydroxyrotenoids or other metabolites analyzed by HPLC and LC/MS. We previously established that the hydroxylated metabolites and derivatives of rotenone and deguelin are all less active (i.e., detoxified) as complex I inhibitors relative to the parent rotenoids. The PD-like syndrome induced in rats by rotenone and deguelin is therefore due to the parent compounds rather than metabolites. Deguelin is about half as active as rotenone in inducing the PD-like syndrome in rats and in acute ip LD50 in mice. Rotenone and deguelin are metabolized by human recombinant 3A4 and 2C19 but not five other P450 enzymes. 2C19 is more selective than 3A4 in forming the 12abeta-hydroxyrotenoids. Identified sites of metabolic attack individually or in combination are as follows: 12abeta hydroxylation and 2-O-demethylation of both compounds, oxidation of the rotenone isopropenyl substituent to mono and diol derivatives, and probable oxidation of the deguelin dimethylchromene double bond. These toxicological features must be considered in using rotenone, deguelin, and their analogues as pesticides, candidate radioimaging and cancer chemopreventive agents, and models of PD.

Pharmacokinetics of deguelin, a cancer chemopreventive agent in rats.

PURPOSE: To study the pharmacokinetics of deguelin, a naturally occurring potential cancer chemopreventive agent, in rats. METHODS: [3H]Deguelin was administered intravenously (i.v.) under anesthesia, and blood samples were collected over 24 h. [3H]Deguelin and metabolites were extracted from plasma with ethyl acetate, and quantified by HPLC. Data were analyzed with the WinNolin pharmacokinetic software package to determine pharmacokinetic parameters. A three-compartment first-order elimination model was used to fit the plasma concentration-time curve. In addition, deguelin concentrations in tissues after i.v. and intragastric (i.g.) administration were determined by HPLC, and excretion (feces and urine) was evaluated over a 5-day period after i.g. administration. RESULTS: Deguelin exhibited a mean residence time (MRT) of 6.98 h and terminal half-life (t1/2(gamma)) of 9.26 h. The area under the curve (AUC) and total clearance (Cl) were 57.3 ng.h/ml and 4.37 l/h per kg, respectively, with an apparent volume of distribution (V) and volume of distribution at steady-state (Vss) of 3.421 l/kg and 30.46 l/kg, respectively. Following i.v. administration, the relative levels of tissue distribution were as follows: heart > fat > mammary gland > colon > liver > kidney > brain > lung. Following i.g. administration, the relative levels of tissue distribution were as follows: perirenal fat > heart > mammary gland > colon > kidney > liver > lung > brain > skin. Within 5 days of i.g. administration, about 58.1% of the [3H]deguelin was eliminated via the feces and 14.4% via the urine. Approximately 1.7% of unchanged deguelin was found in the feces, and 0.4% in the urine. CONCLUSIONS: An initial pharmacokinetic investigation of deguelin showed that this rotenoid has a relatively long MRT and half-life in plasma in the rat. The compound distributed in the tissues and excreted as metabolites, mainly via the feces.

Kinetic analysis of 125I-iodorotenone as a deposited myocardial flow tracer: comparison with 99mTc-sestamibi.

UNLABELLED: The goal of this investigation was to assess the accuracy of 7'-Z-[125I]iodorotenone (125I-iodorotenone) as a new deposited myocardial flow tracer and compare the results with those for 99mTc-sestamibi. METHODS: The kinetics of these two flow tracers were evaluated in 25 isolated, erythrocyte- and albumin-perfused rabbit hearts over a flow range relevant to patients. The two flow tracers and a vascular reference tracer (131I-albumin) were introduced simultaneously as a compact bolus through a port just above the aortic cannula in the absence of tracer recirculation. Myocardial extraction, retention, washout, and uptake parameters were computed from the venous outflow curves using the multiple-indicator dilution technique and spectral analysis. RESULTS: The extraction of 125I-iodorotenone was much higher than the extraction of 99mTc-sestamibi (0.84 +/- 0.05 vs. 0.48 +/- 0.10, respectively, P < 0.001). 125I-iodorotenone extraction was also less affected by flow than was 99mTc-sestamibi (P < 0.001). Net retention of 125I-iodorotenone was significantly greater than 99mTc-sestamibi net retention at 1 min (0.77 +/- 0.08 vs. 0.41 +/- 0.11, respectively, P < 0.001) and 26 min (0.46 +/- 0.13 vs. 0.27 +/- 0.11, respectively, P < 0.001) after tracer injection. Flow had less effect on 125I-iodorotenone net retention than on 99mTc-sestamibi net retention 1 min after tracer injection (P < 0.04). However, at 26 min, flow had an equivalent effect on the retention of both flow tracers (P < 0.4). The relationship between 125I-iodorotenone and 99mTc-sestamibi washout was complex and depended on elapsed time after isotope introduction and perfusion rate. Reflecting the favorable extraction and retention characteristics of 125I-iodorotenone, both its maximum myocardial uptake and its 26-min uptake were more closely related to flow than were those of 99mTc-sestamibi (P < 0.001 for both comparisons). CONCLUSION: The extraction and retention of 125I-iodorotenone were greater than those of 99mTc-sestamibi, making 125I-iodorotenone the superior flow tracer in the isolated rabbit heart.

In vivo labeling of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone.

Defects in mitochondrial energy metabolism have been implicated in several neurodegenerative disorders. Defective complex I (NADH:ubiquinone oxidoreductase) activity plays a key role in Leber's hereditary optic neuropathy and, possibly, Parkinson's disease, but there is no way to assess this enzyme in the living brain. We previously described an in vitro quantitative autoradiographic assay using [(3)H]dihydrorotenone ([(3)H]DHR) binding to complex I. We have now developed an in vivo autoradiographic assay for complex I using [(3)H]DHR binding after intravenous administration. In vivo [(3)H]DHR binding was regionally heterogeneous, and brain uptake was rapid. Binding was enriched in neurons compared with glia, and white matter had the lowest levels of binding. In vivo [(3)H]DHR binding was markedly reduced by local and systemic infusion of rotenone and was enhanced by local NADH administration. There was an excellent correlation between regional levels of in vivo [(3)H]DHR binding and the in vitro activities of complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase), suggesting that the stoichiometry of these components of the electron transport chain is relatively constant across brain regions. The ability to assay complex I in vivo should provide a valuable tool to investigate the status of this mitochondrial enzyme in the living brain and suggests potential imaging techniques for complex I in humans.

Synthesis and biological evaluation in mice of (2-[11C]methoxy)-6',7'-dihydrorotenol, a second generation rotenoid for marking mitochondrial complex I activity.

Evidence has accumulated suggesting that impairment of the function of the complexes of the mitochondrial respiratory chain might be involved in the pathology of neurological diseases including Parkinson's and Huntington's diseases. Recently we reported the synthesis of (2-[11C]methoxy)rotenone ([11C]ROT) as a tool for in vivo studies of complex I. In an effort to develop a complex I imaging radiotracer which might be easier to synthesize and less likely to be metabolized, we prepared (2-[11C]methoxy)-6',7'-dihydrorotenol ([11C]DHROT). The radiotracer was synthesized by [11C]methylation of 2-O-desmethyl-6',7'-dihydrorotenol under basic [11C]alkylation conditions. (2-[11C]Methoxy)-6',7'-dihydrorotenol was produced in 30-35% radiochemical yields (decay corrected), with synthesis times shorter than 35 min. Radiochemical purities were over 95% and specific activities averaged 1000 Ci/mmol. The brain distributions of [11C]ROT and [11C]DHROT were investigated in mice after intravenous injections. For both radiotracers, distribution of radioactivity was similar in all brain regions examined. However, significantly higher uptake was observed with [11C]DHROT than with [11C]ROT, indicating that the alterations introduced in the structure of rotenone during the design of [11C]DHROT resulted in a tracer with greater brain barrier permeability.