Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induce Post-Translational Modifications of AKAP121, DRP1, and OPA1 That Promote Mitochondrial Fission.

RATIONALE: Cardiac lipotoxicity, characterized by increased uptake, oxidation, and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes mellitus. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. OBJECTIVE: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. METHODS AND RESULTS: Using a transgenic mouse model of cardiac lipotoxicity overexpressing ACSL1 (long-chain acyl-CoA synthetase 1) in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes. Palmitate exposure to neonatal rat ventricular cardiomyocytes initially activates mitochondrial respiration, coupled with increased mitochondrial polarization and ATP synthesis. However, long-term exposure to palmitate (>8 hours) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of AKAP121 (A-kinase anchor protein 121) leading to reduced phosphorylation of DRP1 (dynamin-related protein 1) at Ser637 and altered proteolytic processing of OPA1 (optic atrophy 1). Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. CONCLUSIONS: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mechanism for mitochondrial dysfunction in lipotoxic cardiomyopathy.

Synthesis and evaluation of 15-(4-(2-[¹8F]Fluoroethoxy)phenyl)pentadecanoic acid: a potential PET tracer for studying myocardial fatty acid metabolism.

15-(4-(2-[¹8F]fluoroethoxy)phenyl)pentadecanoic acid ([¹8F]7) was synthesized as a PET probe for assessing myocardial fatty acid metabolism. The radiosynthesis of [¹8F]7 was accomplished using a two-step reaction, starting with the corresponding tosylate ester, methyl 15-(4-(2-(tosyloxy)ethoxy)phenyl)pentadecanoate (5), and gave the radiolabeled fatty acid, [¹8F]7 in a radiolabeling yield of 55-60% and a specific activity of >2000 Ci/mmol (decay corrected to EOB). The biological evaluation of [¹8F]7 in rats displayed high uptake in heart (1.94%ID/g at 5 min), which was higher than the uptake (%ID/g) in blood, lung, muscle, pancreas, and brain. MicroPET studies of [¹8F]7 in Sprague-Dawley rats demonstrated excellent images of the myocardium when compared with [¹¹C]palmitate images in the same animal. Moreover, the tracer kinetics of [¹8F]7 paralleled those seen with [¹¹C]palmitate, with an early peak followed by biphasic washout. When compared to [¹¹C]palmitate, [¹8F]7 exhibited a slower early clearance (0.17 ± 0.01 vs 0.30 ± 0.02, P < 0.0001) and a significantly higher late clearance (0.0030 ± 0.0005 vs 0.0006 ± 0.00013, P < 0.01). These initial studies suggest that [¹8F]7 could be a potentially useful clinical PET tracer to assess abnormal myocardial fatty acid metabolism.

Assessment of myocardial triglyceride oxidation with PET and 11C-palmitate.

BACKGROUND: The goal of this study was to test whether myocardial triglyceride (TG) turnover including oxidation of TG-derived fatty acids (FA) could be assessed with PET and (11)C-palmitate. METHODS AND RESULTS: A total of 26 dogs were studied fasted (FAST), during Intralipid infusion (IL), during a hyperinsulinemic-euglycemic clamp without (HIEG), or with Intralipid infusion (HIEG + IL). (11)C-palmitate was injected, and 45 minutes were allowed for labeling of myocardial TG pool. 3D PET data were then acquired for 60 minutes, with first 15 minutes at baseline followed by 45 minutes during cardiac work stimulated with constant infusion of either phenylephrine (FAST, n = 6; IL, n = 6; HIEG + IL, n = 6) or dobutamine (FAST, n = 4; HIEG, n = 4). Myocardial (11)C washout during adrenergic stimulation (AS) was fitted to a mono-exponential function (Km(PET)). To determine the source of this (11)C clearance, Km(PET) was compared to direct coronary sinus-arterial measurements of total (11)C activity, (11)C-palmitate, and (11)CO(2). Before AS, PET curves in all groups were flat indicating absence of net clearance of (11)C activity from heart. In both FAST groups, AS resulted in negligible net (11)C activity and (11)CO(2) production higher than net (11)C-palmitate uptake. AS with phenylephrine resulted in net myocardial uptake of total (11)C activity and (11)C-palmitate in IL and HIEG + IL, and (11)CO(2) production lower than (11)C-palmitate uptake. In contrast, AS with dobutamine in HIEG resulted in net clearance of all (11)C metabolites (total (11)C activity, (11)C-palmitate and (11)CO(2)) with (11)CO(2) contributing 66% to endogenous FA oxidation. The AS resulted in significant Km(PET) in all the groups, except HIEG + IL. However, positive correlation between Km(PET) and (11)CO(2) was observed only in HIEG (R (2) = 0.83, P = .09). CONCLUSIONS: This is the first study to demonstrate that using PET and pre-labeling of intracardiac TG pool with (11)C-palmitate, noninvasive assessment of myocardial TG use is feasible under metabolic conditions that favor endogenous TG use such as increased metabolic demand (beta-adrenergic stimulation of cardiac work) with limited availability of exogenous substrate (HIEG).

7alpha-18F-fluoromethyl-dihydrotestosterone and 7alpha-18F-fluoromethyl-nortestosterone: ligands to determine the role of sex hormone-binding globulin for steroidal radiopharmaceuticals.

UNLABELLED: Sex hormone-binding globulin (SHBG) is believed to play a key role in steroidal radiopharmaceutical delivery to target tissues in humans. To better understand the action of SHBG, we have synthesized and tested in vivo 2 novel 18F-labeled androgens: 7alpha-18F-fluoromethyl-dihydrotestosterone (7alpha-18F-FM-DHT) and 7alpha-18F-fluoromethyl-nortestosterone (7alpha-18F-FM-norT). Both 7alpha-18F-FM-DHT and 7alpha-18F-FM-norT have high affinity for the androgen receptor (AR); however, 7alpha-18F-FM-DHT has a high affinity for SHBG, whereas 7alpha-18F-FM-norT has a relatively low affinity. METHODS: We developed an efficient radiochemical synthesis for both 7alpha-18F-FM-DHT and 7alpha-18F-FM-norT, producing them in good radiochemical yield and high specific activity. Biodistribution studies of both compounds were done on diethylstilbestrol-pretreated and DHT-blocked Sprague-Dawley male rats. Metabolism studies were done to determine the amount of intact ligand in the prostate. RESULTS: We obtained 7alpha-18F-FM-DHT and 7alpha-18F-FM-norT in radiochemical yields of about 30% and radiochemical purities of greater than 99%. Rat biodistribution studies showed selective AR-mediated uptake in the prostate for both compounds. Both compounds showed relatively little defluorination, but the norT analog was more metabolically stable than the DHT analog. CONCLUSION: These studies show that 7alpha-18F-FM-DHT and 7alpha-18F-FM-norT have potential for use in human clinical imaging trials to evaluate more definitively the role of SHBG in radiotracer delivery of steroidal systems to target tissues.

Evaluation of a bromine-76-labeled progestin 16alpha,17alpha-dioxolane for breast tumor imaging and radiotherapy: in vivo biodistribution and metabolic stability studies.

INTRODUCTION: Progesterone receptors (PRs) are present in many breast tumors, and their levels are increased by certain endocrine therapies. They can be used as targets for diagnostic imaging and radiotherapy. METHOD: 16alpha,17alpha-[(R)-1'-alpha-(5-[(76)Br]Bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione ([(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione (3)), a PR ligand with relative binding affinity (RBA)=65 and log P(o/w)=5.09+/-0.84, was synthesized via a two-step reaction, and its tissue biodistribution and metabolic stability were evaluated in estrogen-primed immature female Sprague-Dawley rats. RESULTS: [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 was synthesized in 5% overall yield with specific activity being 200-1250 Ci/mmol. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 demonstrated high PR-mediated uptake in the target tissue uterus (8.72+/-1.84 %ID/g at 1 h) that was reduced by a blocking dose of unlabeled progestin R5020, but the nonspecific uptake in blood and muscle (2.11+/-0.14 and 0.89+/-0.16 %ID/g at 1 h, respectively) was relatively high. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 was stable in whole rat blood in vitro, but it was not stable in vivo due to the fast metabolism that occurred in the liver, resulting in the formation of a more polar radioactive metabolite and free [(76)Br]bromide. The level of free [(76)Br]bromide in blood remained high during the experiment (2.11+/-0.14 %ID/g at 1 h and 1.52+/-0.24 %ID/g at 24 h). The tissue distribution of [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 at 1 and 3 h was compared with that of the (18)F analogs, [(18)F]FFNP fluoro furanyl norprogesterone (FFNP) 1 and ketal 2. CONCLUSION: [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 may have potential for imaging PR-positive breast tumors at early time points, but it is not suitable for imaging at later times or for radiotherapy.

Synthesis and biological evaluation of [18F]bicalutamide, 4-[76Br]bromobicalutamide, and 4-[76Br]bromo-thiobicalutamide as non-steroidal androgens for prostate cancer imaging.

Androgen receptors (AR) are overexpressed in most primary and metastatic prostate cancers. To develop a nonsteroidal AR-mediated imaging agent, we synthesized and radiolabeled several analogs of the potent antiandrogen bicalutamide: [18F]bicalutamide, 4-[76Br]bromobicalutamide, and [76Br]bromo-thiobicalutamide. Two of these analogs, 4-[76Br]bromobicalutamide and [76Br]bromo-thiobicalutamide, were found to have a substantially increased affinity for the androgen receptor (AR) compared to that of bicalutamide. The synthesis of [18F]bicalutamide utilized a pseudocarrier approach to effect addition of a carbanion generated from tracer-level amounts of a radiolabeled precursor to an unlabeled carbonyl precursor. 4-[76Br]Bromobicalutamide and [76Br]bromo-thiobicalutamide were labeled through electrophilic bromination of a tributylstannane precursor. The former could be prepared in high specific activity, and its tissue distribution was tested in vivo. Androgen target tissue uptake was evident in castrated adult male rats; however, in DES-treated, AR-positive, tumor-bearing male mice, tumor uptake was low.

Transgenic expression of fatty acid transport protein 1 in the heart causes lipotoxic cardiomyopathy.

Evidence is emerging that systemic metabolic disturbances contribute to cardiac myocyte dysfunction and clinically apparent heart failure, independent of associated coronary artery disease. To test the hypothesis that perturbation of lipid homeostasis in cardiomyocytes contributes to cardiac dysfunction, we engineered transgenic mice with cardiac-specific overexpression of fatty acid transport protein 1 (FATP1) using the alpha-myosin heavy chain gene promoter. Two independent transgenic lines demonstrate 4-fold increased myocardial free fatty acid (FFA) uptake that is consistent with the known function of FATP1. Increased FFA uptake in this model likely contributes to early cardiomyocyte FFA accumulation (2-fold increased) and subsequent increased cardiac FFA metabolism (2-fold). By 3 months of age, transgenic mice have echocardiographic evidence of impaired left ventricular filling and biatrial enlargement, but preserved systolic function. Doppler tissue imaging and hemodynamic studies confirm that these mice have predominantly diastolic dysfunction. Furthermore, ambulatory ECG monitoring reveals prolonged QT(c) intervals, reflecting reductions in the densities of repolarizing, voltage-gated K+ currents in ventricular myocytes. Our results show that in the absence of systemic metabolic disturbances, such as diabetes or hyperlipidemia, perturbation of cardiomyocyte lipid homeostasis leads to cardiac dysfunction with pathophysiological findings similar to those in diabetic cardiomyopathy. Moreover, the MHC-FATP model supports a role for FATPs in FFA import into the heart in vivo.

Assessment of myocardial metabolism in diabetic rats using small-animal PET: a feasibility study.

UNLABELLED: This feasibility study was undertaken to determine whether kinetic modeling in conjunction with small-animal PET could noninvasively quantify alterations in myocardial perfusion and substrate metabolism in rats. METHODS: All small-animal PET was performed on either of 2 tomographs. Myocardial blood flow and substrate metabolism were measured in 10 male Zucker diabetic fatty rats (ZDF, fa/fa) and 10 lean littermates (Lean, Fa/+) using (15)O-water, 1-(11)C-glucose, 1-(11)C-acetate, and 1-(11)C-palmitate. Animals were 12.0 +/- 1.4-wk old. RESULTS: Consistent with a type 2 diabetic phenotype, the ZDF animals showed higher plasma hemoglobin A(1c), insulin, glucose, and free fatty acid (FFA) levels than their lean controls. Myocardial glucose uptake (mL/g/min) was not significantly different between the 2 groups. However, higher glucose plasma levels in the ZDF rats resulted in higher myocardial glucose utilization (nmol/g/min) (Lean, 629 +/- 785, vs. ZDF, 1,737 +/- 1,406; P = 0.06). Similarly, myocardial FFA uptake (mL/g/min) was not significantly different between the 2 groups, (Lean, 0.51 +/- 28, vs. ZDF, 0.72 +/- 0.19; P = not significant) However, due to higher FFA plasma levels, utilization and oxidation (nmol/g/min) were significantly higher in the ZDF group (Lean, 519 +/- 462, vs. ZDF, 1,623 +/- 712, P < .001; and Lean, 453 +/- 478, vs. ZDF, 1,636 +/- 730, P < .01). CONCLUSION: Noninvasive measurements of myocardial substrate metabolism in ZDF rats using small-animal PET are consistent with the expected early metabolic abnormalities that occur in this well-characterized model of type 2 diabetes mellitus. Thus, small-animal PET demonstrates significant promise in providing a means to link the myocardial metabolic abnormalities that occur in rat of disease with the human condition.

Assessment of myocardial blood flow using 15O-water and 1-11C-acetate in rats with small-animal PET.

UNLABELLED: This feasibility study was undertaken to determine whether myocardial blood flow (MBF, mL/g/min) could be quantified noninvasively in small rodents using microPET and 15O-water or 1-11C-acetate. METHODS: MBF was measured in 18 healthy rats using PET and 15O-water (MBF-W) under different interventions and compared with direct measurements obtained with microspheres (MBF-M). Subsequently, MBF was estimated in 24 rats at rest using 1-11C-acetate (MBF-Ace) and compared with measurements obtained with 15O-water. Using factor analysis, images were processed to obtain 1 blood and 1 myocardial time-activity curve per tracer per study. MBF-W was calculated using a well-validated 1-compartment kinetic model. MBF-Ace was estimated using a simple 1-compartment model to estimate net tracer uptake, K1 (K1 (mL/g/min) = MBF.E; E = first-pass myocardial extraction of 1-11C-acetate) and washout (k2 (min(-1))) along with F(BM) (spillover correction) after fixing F(MM) (partial-volume correction) to values obtained from 15O-water modeling. K1 values were converted to MBF values using a first-pass myocardial extraction/flow relationship measured in rats (E = 1.0-0.74.exp(-1.13/MBF)). RESULTS: In the first study, MBF-W correlated well with MBF-M (y = 0.74x + 0.96; n = 18, r = 0.91, P < 0.0001). However, the slope was different than unity, P < 0.05). Refitting of the data after forcing the intercept to be zero resulted in a nonbias correlation between MBF-W and MBF-M (y = 0.95x + 0.0; n = 18, r = 0.86, P < 0.0001) demonstrating that the underestimation of the slope could be attributed to the overestimation of MBF-W for 2 MBF-M values lower than 1.50 mL/g/min. In the second study, MBF-Ace values correlated well with MBF-W with no underestimation of MBF (y = 0.91x + 0.35; n = 24, r = 0.87, P < 0.0001). CONCLUSION: MBF can be quantified by PET using (15)O-water or 1-11C-acetate in healthy rats. Future studies are needed to determine the accuracy of the methods in low-flow states and to develop an approach for a partial-volume correction when 1-11C-acetate is used.

Synthesis and biological evaluation of a fluorine-18-labeled nonsteroidal androgen receptor antagonist, N-(3-[18F]fluoro-4-nitronaphthyl)-cis-5-norbornene-endo-2,3-dicarboxylic imide.

INTRODUCTION: Androgen receptor (AR), which is overexpressed in most prostate cancers, is the target of androgen ablation and antiandrogen therapies: it is also the target for the receptor-mediated imaging of AR-positive prostate cancer using radiolabeled ligands. Previous AR imaging agents were based on a steroidal core labeled with fluorine. To develop a novel class of nonsteroidal imaging agents, with binding and pharmacological characteristics that are more similar to those of clinically used AR antagonists, we synthesized N-(3-fluoro-4-nitronaphthyl)-cis-5-norbornene-endo-2,3-dicarboxylic imide (3-F-NNDI), an analog of recently reported AR antagonist ligands. METHODS: 3-F-NNDI was synthesized in six steps starting with 1-nitronaphthalene, with fluorine incorporation as the final step. The labeling of 3-F-NNDI with fluorine-18 was achieved through a novel, extremely mild, S(N)Ar displacement reaction of an o-nitro-activated arene trimethylammonium salt, and 3-[(18)F]F-NNDI was prepared in high specific activity. RESULTS AND DISCUSSION: 3-F-NNDI was found to have an AR-binding affinity similar to that of its parent compound. In vitro assays demonstrated high stability of the labeled compound under physiological conditions in buffer and in the blood. Androgen target tissue uptake in diethylstilbestrol-pretreated male rats, however, was minimal, probably because of extensive metabolic defluorination the radiolabeled ligand. CONCLUSIONS: This study is part of our first look at a novel class of nonsteroidal AR antagonists as positron emission tomography (PET) imaging agents that are alternatives to steroidal AR agonist-based imaging agents. Although 3-[(18)F]F-NNDI has significant affinity for AR, it showed limited promise as a PET imaging agent because of its poor target tissue distribution properties.

Synthesis of an estrogen receptor beta-selective radioligand: 5-[18F]fluoro-(2R,3S)-2,3-bis(4-hydroxyphenyl)pentanenitrile and comparison of in vivo distribution with 16alpha-[18F]fluoro-17beta-estradiol.

Estrogen receptor beta (ERbeta), a less active ER subtype that appears to have a restraining effect on the more active ERalpha, could be a factor that determines the level of estrogen action in certain estrogen target tissues. ERbeta is found in breast cancer, and its levels relative to ERalpha decline with disease progression. Thus, the independent quantification of ERalpha and ERbeta levels in breast cancer by imaging might be predictive of responses to different hormone therapies. To develop an imaging agent for ERbeta, we synthesized a fluoroethyl analogue of DPN (2,3-bis(4-hydroxyphenyl)propanonitrile), a known ERbeta-selective ligand. This analogue, FEDPN (5-fluoro-(2R,3S)-2,3-bis(4-hydroxyphenyl)pentanenitrile), has an 8.3-fold absolute affinity preference for ERbeta. [18F]Fluoride-labeled FEDPN was prepared from a toluenesulfonate precursor, which provided [18F]FEDPN with a specific activity greater than 3100 Ci/mmol after HPLC purification. Biodistribution studies in immature female rats using estradiol as a blocking agent revealed specific uptake of [18F]FEDPN in the uterus and ovaries. Experiments using ERalpha- and ERbeta-knockout mice demonstrated the expected ERalpha-subtype dependence in the tissue uptake of the known 16alpha-[18F]fluoro-17beta-estradiol ([18F]FES), which has a 6.3-fold preference for ERalpha. The tissue uptake of [18F]FEDPN in the ER knockout mice showed some evidence of mediation by ERbeta, but the levels of specific uptake of this agent were relatively modest. Based on our results, imaging of ERalpha can be done effectively with [18F]FES, but imaging of ERbeta will likely require agents with more optimized ERbeta binding affinity and selectivity than [18F]FEDNP.

Techniques necessary for multiple tracer quantitative small-animal imaging studies.

INTRODUCTION: An increasing number and variety of studies on rodent models are being conducted using small-animal positron emission tomography scanners. We aimed to determine if animal handling techniques could be developed to perform routine animal imaging in a timely and efficient manner and with minimal effect on animal physiology. These techniques need to be reproducible in the same animal while maintaining hemodynamic and physiological stability. METHODS: The necessary techniques include (a) the use of inhalant anesthesia, (b) arterial and venous cannulation for multiple tracer administrations and blood sampling, (c) development of small-volume analytic columns and techniques and (d) measurement of the physiological environment during the imaging session. RESULTS: We provide an example of a cardiac imaging study using four radiotracers (15O-water, 1-[11C]-acetate, 1-[11C]-palmitate and 1-[11C]-glucose) injected into normal rats. Plasma substrates, CO2 production and total metabolites were measured. The animals remained anesthetized over the entire imaging session, and their physiological state was maintained. CONCLUSION: The intrastudy stability of the physiological measurements and substrate levels and interstudy reproducibility of the measurements are reported.

Measurement of input functions in rodents: challenges and solutions.

INTRODUCTION: Tracer kinetic modeling used in conjunction with positron emission tomography (PET) is an excellent tool for the noninvasive quantification of physiological, biological and molecular processes and their alterations due to disease. Currently, complex multi-compartment modeling approaches are being applied in a variety of clinical studies to determine myocardial perfusion, viability and glucose utilization as well as fatty acid metabolism and oxidation in the normal and diseased heart. These kinetic models require two key measurements of tracer activity over time, tracer activity in arterial blood (input function) and its corresponding activity in the organ of interest. The alteration in the time course of tracer activity as it travels from blood to the organ of interest describes the kinetics of the tracer. To be able to implement these approaches in rodent models of disease using small-animal PET (microPET), it is imperative that the input function is measured accurately. METHODS: The blood input functions in rodent experiments were obtained by (1) direct blood sampling, (2) direct measurement of blood activity by a beta-detecting probe that counts the activity in the blood, (3) an arterial-venous bypass (A/V shunt), (4) factor analysis of dynamic structures from dynamic PET images and (5) measurement from region-of-interest (ROI) analysis of dynamic PET images. Direct blood sampling was used as the reference standard to which the results of the other techniques were compared. RESULTS: Beta probes are difficult to operate and may not provide accurate blood input functions unless they are used intravenously, which requires complicated microsurgery. A similar limitation applies to the A/V shunt. Factor analysis successfully extracts the blood input function for mice and rats. The ROI-based method is less accurate due to limited image resolution of the PET system, which results in severe partial volume effect and spillover from myocardium. CONCLUSION: The current reference standard, direct blood sampling, is more invasive and has limited temporal resolution. With current imaging technology, image-based extraction of blood input functions is possible by factor analysis, while forthcoming technological developments are likely to allow extraction of input function directly from the images. These techniques will reduce the level of complexity and invasiveness for animal experiments and are likely to be used more widely in the future.

Longitudinal noninvasive imaging of progesterone receptor as a predictive biomarker of tumor responsiveness to estrogen deprivation therapy.

PURPOSE: To investigate whether longitudinal functional PET imaging of mammary tumors using the radiopharmaceuticals [(18)F]FDG (to measure glucose uptake), [(18)F]FES [to measure estrogen receptor (ER) levels], or [(18)F]FFNP [to measure progesterone receptor (PgR) levels] is predictive of response to estrogen-deprivation therapy. EXPERIMENTAL DESIGN: [(18)F]FDG, [(18)F]FES, and [(18)F]FFNP uptake in endocrine-sensitive and -resistant mammary tumors was quantified serially by PET before ovariectomy or estrogen withdrawal in mice, and on days 3 and 4 after estrogen-deprivation therapy. Specificity of [(18)F]FFNP uptake in ERα(+) mammary tumors was determined by competition assay using unlabeled ligands for PgR or glucocorticoid receptor (GR). PgR expression was also assayed by immunohistochemistry (IHC). RESULTS: The levels of [(18)F]FES and [(18)F]FDG tumor uptake remained unchanged in endocrine-sensitive tumors after estrogen-deprivation therapy compared with those at pretreatment. In contrast, estrogen-deprivation therapy led to a reduction in PgR expression and [(18)F]FFNP uptake in endocrine-sensitive tumors, but not in endocrine-resistant tumors, as early as 3 days after treatment; the changes in PgR levels were confirmed by IHC. Unlabeled PgR ligand R5020 but not GR ligand dexamethasone blocked [(18)F]FFNP tumor uptake, indicating that [(18)F]FFNP bound specifically to PgR. Therefore, a reduction in FFNP tumor to muscle ratio in mammary tumors predicts sensitivity to estrogen-deprivation therapy. CONCLUSIONS: Monitoring the acute changes in ERα activity by measuring [(18)F]FFNP uptake in mammary tumors predicts tumor response to estrogen-deprivation therapy. Longitudinal noninvasive PET imaging using [(18)F]FFNP is a robust and effective approach to predict tumor responsiveness to endocrine treatment.

Comparison of 1-(11)C-glucose and (18)F-FDG for quantifying myocardial glucose use with PET.

UNLABELLED: In this study, we compared the accuracy of the rate of myocardial glucose use (rMGU) measured using PET and 1-(11)C-glucose with the rate measured using PET and the more conventional tracer (18)F-FDG. METHODS: PET measurements of myocardial tracer uptake (K, in mL/g/min) and rMGU (in nmol/g/min) were obtained with 1-(11)C-glucose and (18)F-FDG in 21 dogs using kinetic modeling and the Patlak graphical method, respectively. Eighteen dogs were studied during hyperinsulinemic-euglycemic clamp performed either at rest or combined with phenylephrine, dobutamine, intralipid infusion, or intralipid infusion and dobutamine. Three dogs were studied during intralipid infusion alone under resting conditions. Arterial-coronary sinus sampling was performed to measure the K of both tracers (n = 14) and rMGU by the Fick method (n = 21). RESULTS: PET-derived values for K from either 1-(11)C-glucose or (18)F-FDG correlated closely with directly measured tracer K values (glucose: y = 0.98x + 0.01, r = 0.79, P < 0.001; (18)F-FDG: y = 0.74x + 0.03, r = 0.77, P < 0.001). In contrast, correlation with K values of unlabeled glucose measured directly was better for 1-(11)C-glucose (y = 0.92x + 0.02, r = 0.96, P < 0.0001) than for (18)F-FDG (y = 0.66x + 0.05, r = 0.72, P < 0.01) (P < 0.001 for comparison of correlation coefficients). As a consequence, PET-derived values for rMGU correlated more closely with Fick-derived measurements of unlabeled glucose using 1-(11)C-glucose (y = 0.82x + 168, r = 0.97, P < 0.0001) than with (18)F-FDG (y = 0.81x + 278, r = 0.79, P < 0.001) (P < 0.001 for comparison of correlation coefficients). CONCLUSION: Over a wide range of conditions, PET-derived measurements of rMGU are obtained more accurately with 1-(11)C-glucose than with (18)F-FDG.

Delineation of hypoxia in canine myocardium using PET and copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone).

UNLABELLED: Copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone) (copper-ATSM) is a hypoxia-avid tracer for the selective identification of hypoxic tissue. Using canine models of hypoxic myocardium, we report our findings on *Cu-ATSM PET (*Cu is defined as either (60)Cu, (61)Cu, or (64)Cu) for the delineation of ischemic and hypoxic myocardium. METHODS: In protocol I, myocardial hypoxia was induced by global hypoxia (n = 3). In protocol II, myocardial ischemia was generated by occlusion of the left anterior descending coronary artery (n = 9). In protocol III, coronary artery stenosis was induced by a stenosis in the left anterior descending coronary artery (n = 4). PET dynamic data were acquired immediately after tracer injection. Tracer retention kinetics were analyzed using either monoexponential analysis (1/k(mono)) or a simple 2-compartment model (1/k(4)). RESULTS: In protocol I, tracer retention in hypoxic myocardium was 2-fold greater than in normal myocardium, despite a 7-fold increase in blood flow (normal, 0.70 +/- 0.42 mL.min(-1).g(-1); hypoxic, 4.94 +/- 3.00 mL.min(-1).g(-1) [P < 0.005]). In protocol II, approximately 3 h after occlusion, retention of *Cu-ATSM within 20 min was greater in ischemic regions (myocardial blood flow, 0.28 +/- 0.26 mL.min(-1).g(-1)) than in normal tissue (myocardial blood flow, 0.52 +/- 0.19 mL.min(-1).g(-1)) (1/k(mono), 40.72 +/- 39.0 min vs. 26.69 +/- 22.29 min [P < 0.05]; 1/k(4), 6.85 +/- 4.90 min vs. 3.51 +/- 1.97 min [P < 0.05]). In selected dogs, tracer retention decreased at 24 h, suggesting the development of necrosis with no subsequent retention of *Cu-ATSM. In protocol III, dobutamine infusion after stenosis placement resulted in increased tracer retention consistent with hypoxia in the damaged regions. CONCLUSION: *Cu-ATSM PET has shown quantitative selective uptake in hypoxic myocardium within 20 min of tracer administration in 3 canine models of hypoxia.

Impact of reversible trapping of tracer and the presence of blood metabolites on measurements of myocardial glucose utilization performed by PET and 18F-fluorodeoxyglucose using the Patlak method.

In this study we demonstrated that significant egress of FDG from myocardium occurs within the first hour after tracer injection leading to nonlinear Patlak plots. There are also significant amounts of acidic FDG metabolites present in the blood. However, the impact of these metabolites on the estimates of myocardial glucose utilization (MGU) is negligible. Although further studies will be required to elucidate the reason for the egress of tracer from myocardium, not accounting for it will result in erroneous estimates of MGU.

MicroPET assessment of androgenic control of glucose and acetate uptake in the rat prostate and a prostate cancer tumor model.

PET has been used to monitor changes in tumor metabolism in breast cancer following hormonal therapy. This study was undertaken to determine whether PET imaging could evaluate early metabolic changes in prostate tumor following androgen ablation therapy. Studies were performed comparing two positron-emitting tracers, 18F-FDG and 11C-acetate, in Sprague-Dawley male rats to monitor metabolic changes in normal prostate tissue. Additional studies were performed in nude mice bearing the CWR22 androgen-dependent human prostate tumor to evaluate metabolic changes in prostate tumor. In rats, for the androgen ablation pretreatment, 1 mg diethylstilbestrol (DES) was injected subcutaneously 3 and 24 hours before tracer injection. For androgen pretreatment, 500 microg dihydrotestosterone (DHT) was injected intraperitoneally 2 and 6 hours before tracer injection. The rats were divided into three groups, Group A (no-DES, no-DHT, n = 18), Group B (DES, no-DHT, n = 18) and Group C (DES, DHT, n = 18). In each group, 10 animals received 18F-FDG, whereas the remaining eight animals were administered 11C-acetate. Rats were sacrificed at 120 min post-injection of 18F-FDG or 30 min post-injection of 11C-acetate. Pretreatment of the mouse model using DHT (200 microg of DHT in 0.1 mL of sunflower seed oil) or DES (200 microg of DES in 0.1 mL of sunflower seed oil) was conducted every 2 days for one week. Mice were imaged with both tracers in the microPET scanner (Concorde Microsystems Inc.). DES treatment caused a decrease in acetate and glucose metabolism in the rat prostate. Co-treatment with DHT maintained the glucose metabolism levels at baseline values. In the tumor bearing mice, similar effects were seen in 18F-FDG study, while there was no significant difference in 11C-acetate uptake. These results indicate that changes in serum testosterone levels influence 18F-FDG uptake in the prostate gland, which is closely tied to glucose metabolism, within 24 hours of treatment and in the prostate tumor within 1 week. These early metabolic changes could enable monitoring metabolic changes in prostate tumor following treatment by imaging using 18F-FDG PET. Further studies are needed to clarify the reason for the insensitivity of 11C-acetate for measuring metabolic change in prostate tumor.

Preparation of 66Ga- and 68Ga-labeled Ga(III)-deferoxamine-folate as potential folate-receptor-targeted PET radiopharmaceuticals.

A folate-receptor-targeting radiopharmaceutical, Ga(III)-deferoxamine-folate (Ga-DF-Folate), was radiolabeled with two positron-emitting isotopes of gallium, cyclotron-produced (66)Ga (9.5 hour half-life) and generator-produced (68)Ga (68 minute half-life). The [(66)Ga]Ga-DF-Folate was administered to athymic mice with folate-receptor-positive human KB cell tumor xenografts to demonstrate that microPET mouse tumor imaging is feasible with (66)Ga, despite the relatively high positron energy of this radionuclide. Using the athymic mouse KB tumor xenograft model, dual-isotope autoradiography was also performed following i.v. co-administration of [(18)F]-FDG, a marker of regional metabolic activity, and folate-receptor-targeted [(111)In]In-DTPA-Folate. The autoradiographic images of 1 mm tumor sections demonstrate the gross heterogeneity of the KB cell tumor xenograft, as well as subtle disparity in the regional accumulation of the two radiotracers.

PET imaging of chemokine receptors in vascular injury-accelerated atherosclerosis.

UNLABELLED: Atherosclerosis is the pathophysiologic process behind lethal cardiovascular diseases. It is a chronic inflammatory progression. Chemokines can strongly affect the initiation and progression of atherosclerosis by controlling the trafficking of inflammatory cells in vivo through interaction with their receptors. Some chemokine receptors have been reported to play an important role in plaque development and stability. However, the diagnostic potential of chemokine receptors has not yet been explored. The purpose of this study was to develop a positron emitter-radiolabeled probe to image the upregulation of chemokine receptor in a wire-injury-accelerated apolipoprotein E knockout (ApoE(-/-)) mouse model of atherosclerosis. METHODS: A viral macrophage inflammatory protein II (vMIP-II) was used to image the upregulation of multiple chemokine receptors through conjugation with DOTA for (64)Cu radiolabeling and PET. Imaging studies were performed at 2 and 4 wk after injury in both wire-injured ApoE(-/-) and wild-type C57BL/6 mice. Competitive PET blocking studies with nonradiolabeled vMIP-II were performed to confirm the imaging specificity. Specific PET blocking with individual chemokine receptor antagonists was also performed to verify the upregulation of a particular chemokine receptor. In contrast, (18)F-FDG PET imaging was performed in both models to evaluate tracer uptake. Immunohistochemistry on the injury and sham tissues was performed to assess the upregulation of chemokine receptors. RESULTS: (15)O-CO PET showed decreased blood volume in the femoral artery after the injury. (64)Cu-DOTA-vMIP-II exhibited fast in vivo pharmacokinetics with major renal clearance. PET images showed specific accumulation around the injury site, with consistent expression during the study period. Quantitative analysis of tracer uptake at the injury lesion in the ApoE(-/-) model showed a 3-fold increase over the sham-operated site and the sites in the injured wild-type mouse. (18)F-FDG PET showed significantly less tracer accumulation than (64)Cu-DOTA-vMIP-II, with no difference observed between injury and sham sites. PET blocking studies identified chemokine receptor-mediated (64)Cu-DOTA-vMIP-II uptake and verified the presence of 8 chemokine receptors, and this finding was confirmed by immunohistochemistry. CONCLUSION: (64)Cu-DOTA-vMIP-II was proven a sensitive and useful PET imaging probe for the detection of 8 up-regulated chemokine receptors in a model of injury-accelerated atherosclerosis.