Transport of biguanides by human organic cation transporter OCT2

Biguanides have the severe side effect of lactic acidosis. Although both metformin and phenformin are biguanide derivatives, there is a difference in the frequency at which they induce lactic acidosis. However, the reasons for the difference are not clear. Metformin has been reported to be mainly excreted into urine by human organic cation transporter 2 (hOCT2). The present study was designed to investigate the renal transport of metformin and phenformin, focusing on hOCT2, using hOCT2–expressing oocytes. Both biguanides were found to be good substrates for hOCT2. However, phenformin exhibited a higher affinity and transport activity than metformin. The K_m values for metformin and phenformin were 235 and 37.4 μM, with CL_int (V_max/K_m) values of 71.9 × 10⁻³ μL/min per oocyte and 209 × 10⁻³ μL/min per oocyte, respectively. This is the first report that has compared the transport profiles of these biguanides in hOCT2–expressing oocytes. The results suggest that plasma concentration of phenformin in subjects carrying hOCT2 variant may be higher compared to reference subjects, as reported in metformin. In addition, the relationship between plasma concentration of these biguanides and blood lactate level as well as the possible reasons for the difference in the associated frequency of occurrence of lactic acidosis are discussed.     

Rapid hepatic clearance of 99mTc‐TMEOP: a new candidate for myocardial perfusion imaging

Background

^99mTc labeled radiotracers used in clinical practice lack the perfect characteristics for myocardial perfusion imaging. In particular, the high liver uptake can interfere in the interpretation of the inferior myocardial wall. Within the tricarbonyl approach, we used tris(pyrazolyl)methane ^99mTc organometallic complexes as a lead structure. Herein we present the production, in vivo and in vitro metabolic studies in rats and the first in vivo biodistribution in rats for tri‐methoxy‐tris‐pyrazolyl‐^99mTc‐(CO)₃ (^99mTc‐TMEOP), compared with ^99mTc‐sestamibi and ^99mTc‐tetrofosmin.

Methods

The chemical identity of ^99mTc‐TMEOP was characterized by RP‐HPLC. The octanol–water partition coefficient was determined under physiological conditions. In vitro stability and protein binding were determined using RP‐HPLC. In vivo stability was determined in blood, heart, liver and kidney homogenates, intestine and urine using RP‐HPLC. In vivo biodistribution was determined using dynamic planar acquisitions. Pinhole gated SPECT images were performed in other animals. Cardiac, liver and lung uptake were expressed as differential uptake ratios by drawing regions of interest in the organs of interest and around the total body. Heart–liver and heart–lung ratios were derived. Cardiac uptake was also expressed as percentage of injected activity. SPECT images were processed to determine the heart–liver ratio on SPECT images, to compare functional parameters between different tracers and to visualize homogeneous intracardiac tracer distribution.

Results

^99mTc‐TMEOP is a moderately lipophilic cation, is stable and does not undergo any transformation in vitro. ^99mTc‐TMEOP also shows a high in vivo stability. In vivo imaging shows liver kinetics faster than those of ^99mTc‐sestamibi and ^99mTc‐tetrofosmin. Cardiac uptake and functional analysis of pinhole gated SPECT data are comparable to those of ^99mTc‐sestamibi and ^99mTc‐tetrofosmin.

Conclusion

Although ^99mTc‐TMEOP shows a cardiac uptake between those of ^99mTc‐sestamibi and ^99mTc‐tetrofosmin, a better heart–liver ratio is obtained due to the faster liver washout. These results suggest possible faster cardiac perfusion imaging using ^99mTc‐TMEOP without liver activity interference. Copyright © 2010 John Wiley & Sons, Ltd.     

Regulatory mechanisms of C4b‐binding protein (C4BP)α and β expression in rat hepatocytes by lipopolysaccharide and interleukin‐6

Summary. Background: C4b‐binding protein (C4BP), a multimeric protein structurally composed of α chains (C4BPα) and a β chain (C4BPβ), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)‐6. However, it is not fully understood how lipopolysaccharide (LPS) and IL‐6 affect the plasma C4BP antigen level and C4BPα and C4BPβ expression in hepatocytes. Objectives: To assess the effect of LPS and IL‐6 on plasma C4BP, PS–C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. Results: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg^?1) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL‐6 (10 μg kg^?1) injection, and then gradually decreased up to 24 h after IL‐6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPα and C4BPβ in rat hepatocytes, and this effect was inhibited by NFκB and MEK/ERK inhibitors. IL‐6 mediated increase in C4BPβ expression in rat hepatocytes, which leads to increased plasma PS–C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT‐3. Conclusion: LPS decreases both C4BPα and C4BPβ expression via the NFκB and MEK/ERK pathways, whereas IL‐6 specifically increases C4BPβ expression via the STAT‐3 pathway, causing an increase in plasma PS–C4BP complex, and thus decreasing the anticoagulant activity of PS.     

Specific E‐selectin targeting with a superparamagnetic MRI contrast agent

Targeting of the endothelial inflammatory adhesion molecule E‐selectin by magnetic resonance imaging (MRI) was performed with a superparamagnetic contrast agent in the context of in vitro and in vivo models of inflammation. The specific contrast agent was obtained by grafting a synthetic mimetic of sialyl Lewis^x (sLe^x), a natural ligand of E‐selectin expressed on leukocytes, on the dextran coating of ultrasmall particles of iron oxide (USPIO). This new contrast agent, called USPIO‐g‐sLe^x, was tested, in vitro, on cultured human umbilical vein endothelial cells (HUVECs) stimulated to express inflammatory adhesion molecules, and in vivo, on a mouse model of hepatitis. In vitro, HUVECs were stimulated with the pro‐inflammatory cytokine tumor necrosis factor alpha (TNF‐α) and were then incubated with USPIO‐g‐sLe^x or ungrafted USPIO. In vivo, hepatitis was induced on NMRI mice by injection of concanavalin A (Con A). USPIO‐g‐sLe^x and ungrafted USPIO were injected intravenously. In vitro results showed an extensive retention of USPIO‐g‐sLe^x on TNF‐α stimulated HUVECs. Image intensity and R₂ measurements performed on T₂‐weighted MR images demonstrated a significantly higher binding of USPIO‐g‐sLe^x on stimulated HUVECs. In vivo, USPIO are known to pass through the fenestrae of the liver and to be captured by Kupffer cells, inducing a loss of signal intensity on T₂‐weighted MR images. Unexpectedly, when injected to Con A‐treated mice, USPIO‐g‐sLe^x induced a significantly lower attenuation of liver signal intensity than USPIO or USPIO‐g‐sLe^x injected to healthy mice, or USPIO injected to Con A‐treated mice, suggesting that the specific contrast media is retained extracellularly by an interaction with E‐selectin overexpressed on the vascular endothelium. Both in vitro and in vivo results therefore indicate that USPIO‐g‐sLe^x is recognizing endothelial E‐selectin. USPIO‐g‐sLe^x is thus well suited for the MRI diagnosis of inflammation and for the in vitro evaluation of endothelial cells activation. Copyright © 2006 John Wiley & Sons, Ltd.     

Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies

Summary. Background: Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late‐stage clinical development for the prevention and treatment of thromboembolic diseases. Objective: We evaluated the in vitro properties of apixaban and its in vivo activities in rabbit models of thrombosis and hemostasis. Methods: Studies were conducted in arteriovenous‐shunt thrombosis (AVST), venous thrombosis (VT), electrically mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. Results: In vitro, apixaban is potent and selective, with a K_i of 0.08 nm for human FXa. It exhibited species difference in FXa inhibition [FXa K_i (nm ): 0.16, rabbit; 1.3, rat; 1.7, dog] and anticoagulation [EC_2× (μm , concentration required to double the prothrombin time): 3.6, human; 2.3, rabbit; 7.9, rat; 6.7, dog]. Apixaban at 10 μm did not alter human and rabbit platelet aggregation to ADP, γ‐thrombin, and collagen. In vivo, the values for antithrombotic ED₅₀ (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT and the values for BT ED_3× (dose that increased BT by 3‐fold) were 0.27 ± 0.03, 0.11 ± 0.03, 0.07 ± 0.02 and > 3 mg kg^?1 h^?1 i.v. for apixaban, 0.05 ± 0.01, 0.05 ± 0.01, 0.27 ± 0.08 and > 3 mg kg^?1 h^?1 i.v. for the indirect FXa inhibitor fondaparinux, and 0.53 ± 0.04, 0.27 ± 0.01, 0.08 ± 0.01 and 0.70 ± 0.07 mg kg^?1 day^?1 p.o. for the oral anticoagulant warfarin, respectively. Conclusions: In summary, apixaban was effective in the prevention of experimental thrombosis at doses that preserve hemostasis in rabbits.     

Role of K ATP channels in cephalic vasodilatation induced by calcitonin gene-related peptide, nitric oxide, and transcranial electrical stimulation in the rat

Objective.— The objective of this study was to explore the role of K_ATP channels in vasodilatation induced by calcitonin gene‐related peptide (CGRP), nitric oxide (NO), and transcranial electrical stimulation (TES) in intracranial arteries of rat. Background.— Dilatation of cerebral and dural arteries causes a throbbing, migraine‐like pain. Both CGRP and NO are potent vasodilators that can induce migraine. Their antagonists are effective in the treatment of migraine attacks. K_ATP channel openers cause headache in the majority of healthy subjects suggesting a role for K_ATP channels in migraine pathogenesis. We hypothesized that vasodilatation induced by CGRP and the NO donor glyceryltrinitrate (GTN) is mediated via K_ATP channels. Methods.— We examined the effects of the K_ATP channel inhibitor glibenclamide on dural and pial vasodilatation induced by CGRP, NO, and endogenously released CGRP by TES. A rat genuine closed cranial window model was used for in vivo studies and myograph baths for studying the effect in vitro. In the closed cranial window model the diameter of dural vessels was measured directly in anesthetized animals to investigate the vascular effects of infused CGRP, NO, and endogenous CGRP after electrical stimulation. Also diameter changes of pial arteries, mean arterial blood pressure and local cerebral blood flow by Laser Doppler flowmetry (LCBF_Flux) were measured. Results.— CGRP, NO, and TES caused dilatation of the 2 arteries in vivo and in vitro. In anesthetized rats glibenclamide significantly attenuated CGRP induced dural and TES induced dural/pial artery dilatation (P = .001; P = .001; P = .005), but had no effect on dural/pial vasodilatation induced by GTN. In vitro glibenclamide failed to significantly inhibit CGRP‐ and GTN‐induced vasodilatation. Conclusions.— These results show that a K_ATP channel blocker in vivo but not in vitro inhibits CGRP, but not GTN‐induced dilatation of dural and pial arteries, a mechanism thought to be important in migraine.     

Reversible biotinylated oligosaccharides: a new approach for a better management of anticoagulant therapy

Summary. Objective: In order to obtain a neutralizable antithrombotic, a chimeric molecule (SSR126517E) containing the sequence of a long‐lasting antithrombin (AT)‐dependent anti‐factor Xa pentasaccharide, idraparinux, linked to a biotin molecule was synthesized and tested for anticoagulant and antithrombotic activity. Methods: SSR126517E was tested in several models in vitro and in vivo for its pharmacological properties as well as its ability to be neutralized by avidin. Results: SSR126517E displayed exactly the same properties as idraparinux. In vitro, SSR126517E had a very high affinity for AT (K_d < 1 nm ) and showed a potent anti‐FXa effect and inhibition of thrombin generation with IC₅₀ values similar to those of idraparinux. Ex vivo, after intravenous administration to rats, SSR126517E produced a potent and long‐lasting anti‐FXa effect comparable to that obtained with idraparinux; as with idraparinux, the subcutaneous bioavailability was 100%. In vivo, SSR126517E was a potent antithrombotic in rat and mouse venous and arterial thrombosis models. Direct comparison in rats showed that SSR126517E was as active as idraparinux, when administered at the same molar dose. Furthermore, injection of avidin triggered the immediate elimination of SSR126517E from the bloodstream, resulting in complete neutralization of the antithrombotic activity of SSR126517E. Conclusions: These results show for the first time that coupling an oligosaccharide with biotin has no effect on the former’s pharmacokinetic and pharmacologic properties and renders neutralization easy by injection of avidin.     

Use of prostaglandin E1 during preparation of platelet concentrates

Summary. A paired study in 10 autologous volunteer donors was undertaken to investigate the efficacy of adding prostaglandin E₁ (PGE₁) in vitro during routine platelet concentrate (PC) production. After 5 days storage, PCs prepared with PGE₁ were compared with control PCs. In vivo platelet recovery, survival and biodistribution were determined following autologous infusion of indium-111 labelled platelets into volunteers, together with the in vitro evaluation of platelet function and biochemistry. PGE₁ facilitated easier and faster platelet resuspension following centrifugation. After storage there were few significant in vitro differences between PCs prepared with PGE₁ and control PCs. The artifactual leucocyte concentration was significantly lower in the presence of PGE₁, suggesting less platelet aggregates had been formed during storage and β-thromboglobulin release was significantly reduced by PGE₁, 14.0±6.0 μg per 10⁹platelets compared with 22.3±9.8μg per 10⁹platelets in control PCs, (P < 0.01), indicating PGE₁ reduced both platelet aggregation and activation probably at the initial preparation stage, known to produce the greatest trauma. Initial in vivo platelet recovery for PCs prepared with PGE₁ was similar to that of control PCs, 41.1 ± 12.5% vs. 44.4±80%, respectively, and there were no differences in organ distribution at 24h. However, in vivo multiple hit survival was reduced in the presence of PGE₁, 5.8 ± 1.6 days compared with 6.9 ± 1.4 days in control PCs (P < 0.05). Despite the ability of PGE₁ to facilitate platelet resuspension and inhibit platelet aggregation and activation during preparation of the PCs, the reduced in vivo survival time may preclude the use of PGE₁ during routine PC preparation.     

Blood Clearance Kinetics,Biodistribution, and Radiation Dosimetry of a Kit-Formulated Integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript>-Selective Radiotracer <Superscript>99m</Superscript>Tc-3PRGD<Subscript>2</Subscript> in Non-Human Primates

Purpose  

^99mTc-3PRGD₂ is a ^99mTc-labeled dimeric cyclic RGD peptide with increased receptor binding affinity and improved kinetics for in vivo imaging of integrin α_vβ₃ expression in nude mouse model. To accelerate its clinical translation, we reported here the evaluation of the kit-formulated ^99mTc-3PRGD₂ in healthy cynomolgus primates for its blood clearance kinetics, biodistribution, and radiation dosimetry.     

Oxygen consumption is depressed in patients with lactic acidosis due to biguanide intoxication

Introduction  

Lactic acidosis can develop during biguanide (metformin and phenformin) intoxication, possibly as a consequence of mitochondrial dysfunction. To verify this hypothesis, we investigated whether body oxygen consumption (VO₂), that primarily depends on mitochondrial respiration, is depressed in patients with biguanide intoxication.     

Kinetic analysis of impaired work-cost performance in jaundiced rabbit liver

Impairment of energy metabolism was studied in jaundiced rabbit liver by kinetic analysis of energy transfer function. Free cytosolic ADP (ADP_f), as calculated from the measured components of the glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase/lactate dehydrogenase reactions, decreased from the control value of 48.1 to 37.0 μM at 24 h after bile duct ligation. The maximal velocity (V_max) of ATP synthesis, as measured by state 3 respiration of isolated mitochondria, decreased from the control value of 62.1 to 38.3 nmol ATP synthesized per min per mg mitochondrial protein, while the Michaelis constant for ADP (K _m) decreased from the control value of 19.2 to 12.8 μM. ATP synthesis velocity in vivo {v:V_max/[1+(K _m/[ADP_f])]}, as calculated by V_max,K _m and ADP_f, decreased from the control value of 44.4 to 28.5 nmol ATP synthesized per min per mg mitochondrial protein. Δv/ΔADP_f (Δv/ΔADP_f: V_max·K _m/(K _m+[ADP_f])²), which indicates work-cost performance of the liver, decreased from the control value of 0.263 to 0.198. Biochemical output of the liver, as measured by hippurate synthesis from benzoate, decreased from the control value of 98.4 to 32.7 mg/h. These results indicate that synergistic decreases in ADP_f, V_max, v and Δv/ΔADP_f take place in the course of deterioration of mitochondrial ATP synthesis and work output in jaundiced liver     

An Efficient Automated Radiosynthesis and Bioactivity Confirmation of VMAT2 Tracer [18F]FP-(+)-DTBZ

Purpose

The aim of this study was to optimize the radiolabeling method of [¹⁸F]fluoropropyl-(+)-dihydrotetrabenazine ([¹⁸F]FP-(+)-DTBZ) to fulfill the demand of preclinical and clinical application.

Procedures

Optimized labeling conditions were performed by altering the molar ratio of precursor to base (P/B), base species, solvents, reaction temperature, reaction time, and precursor concentration through manual radiosynthesis of [¹⁸F]FP-(+)-DTBZ. The conditions with the highest radiochemical yield (RCY) were applied to automated radiosynthesis, and the crude product was purified with a Sep-Pak Plus C18 cartridge. Quality control and stability of [¹⁸F]FP-(+)-DTBZ were carried out by HPLC. In vitro cellular uptake and blocking assays were conducted in human neuroblastoma cell line SH-SY5Y. In vivo imaging with small animal positron emission tomography (microPET) was performed with Sprague–Dawley rats.

Results

Under the optimized conditions (P/K₂CO₃?=?1:8, heating at 120 °C for 3 min in dimethyl sulfoxide), an RCY of 88.7 % was obtained with 1.0 mg precursor. The optimized reaction conditions were successfully applied to an automated module and gave a high activity yield (AY) of 30–55 % in about 40 min with a >?99.0 % radiochemical purity (RCP) and a >?44.4 GBq/μmol molar activity (A_m). Stability test displayed that the RCP retained >?98.0 % in 8 h in saline and in phosphate buffer saline (PBS, pH 7.4). In vitro cellular uptake assay showed accumulation of [¹⁸F]FP-(+)-DTBZ in SH-SY5Y cells, which could be significantly inhibited by vesicular monoamine transporter 2 (VMAT2) inhibitor DTBZ. MicroPET images of rat brain displayed that the striatum showed the highest uptake with a standardized uptake value (SUV) of 3.91?±?0.30 at ~?70 min. Co-injection with DTBZ (1.0 mg/kg) resulted in a 75 % decrease of the striatal SUV, confirming the specificity of [¹⁸F]FP-(+)-DTBZ to VMAT2.

Conclusions

We obtained an optimized radiolabeling method of [¹⁸F]FP-(+)-DTBZ and successfully applied it to a commercial available module. The automated synthesis gave a high AY and RCP of [¹⁸F]FP-(+)-DTBZ with high and specific binding to VMAT2, facilitating its routine application for VMAT2 tracing.

     

Venous Admixture During Exercise in Sitting Position

Abstract

Introduction. The aim of this study was to establish a radio synthesis of ^99mTc-HYNIC-lactadherin for in vivo studies and to perform biodistribution analysis studies in mice, comparing ^99mTc-HYNIC-lactadherin to ^99mTc-HYNIC-annexin V. Methods. The radiochemical purity of ^99mTc-HYNIC-lactadherin was optimized by varying the amount of SnCl₂ in the synthesis. Furthermore, the need for bovine serum albumin (BSA) as a stabilizing agent was evaluated by following the stability by radiochemical purity measurement with and without the addition of BSA. A total of 24 mice were assigned to groups of 15 and nine mice, respectively. The animals were sacrificed at different time points; 10 min, 60 min, and 180 min. Results. The synthesis of ^99mTc-HYNIC-lactadherin for in vivo studies has been optimized to give a stable product without addition of BSA and with a radiochemical purity of more than 95%. Approximately 60% of the injected dose of ^99mTc-HYNIC-lactadherin was found in the liver and 4–5% could be assigned to kidneys. In contrast, ^99mTc-HYNIC-annexin V distributes with around 13% and 45% of the injected dose in liver and kidneys, respectively. Over the experimental period (10–180 min) only small distributional changes were observed for both probes. Conclusion. In conclusion, the biodistribution of ^99mTc-HYNIC-lactadherin, a potential new tracer for in vivo quantification of apoptosis, was evaluated. The small renal uptake of ^99mTc-HYNIC-lactadherin makes it possible to image apoptosis in the kidneys, but the high liver clearance may be a disadvantage during myocardial perfusion.     

Manganese cell labeling of murine hepatocytes using manganese(III)‐transferrin

Manganese(III)‐transferrin [Mn(III)–Tf] was investigated as a way to accomplish manganese‐labeling of murine hepatocytes for MRI contrast. It is postulated that Mn(III)–Tf can exploit the same transferrin‐receptor‐dependent and ‐independent metabolic pathways used by hepatocytes to transport the iron analog Fe(III)–Tf. More specifically, it was investigated whether manganese delivered by transferrin could give MRI contrast in hepatocytes. Comparison of the T₁ and T₂ relaxation times of Mn(III)–Tf and Fe(III)–Tf over the same concentration range showed that the r₁ relaxivities of the two metalloproteins are the same in vitro, with little contribution from paramagnetic enhancement. The degree of manganese cell labeling following incubation for 2–7 h in 31.5 µm Mn(III)–Tf was comparable to that of hepatocytes incubated in 500 µm Mn²⁺ for 1 h. The intrinsic manganese tissue relaxivity between Mn(III)–Tf‐labeled and Mn²⁺‐labeled cells was found to be the same, consistent with Mn(III) being released from transferrin and reduced to Mn²⁺. For both treatment regimens, manganese uptake by hepatocytes appeared to saturate in the first 1–2 h of the incubation period and may explain why the efficiency of hepatocyte cell labeling by the two methods appeared to be comparable in spite of the ～16‐fold difference in effective manganese concentration. Hepatocytes continuously released manganese, as detected by MRI, and this was the same for both Mn²⁺‐ and Mn(III)–Tf‐labeled cells. Manganese release may be the result of normal hepatocyte function, much in the same way that hepatocytes excrete manganese into the bile in vivo. This approach exploits a biological process—namely receptor binding, endocytosis and endosomal acidification—to initiate the release of an MRI contrast agent, potentially conferring more specificity to the labeling process. The ubiquitous expression of transferrin receptors by eukaryotic cells should make Mn(III)–Tf particularly useful for manganese labeling of a wide variety of cells both in culture and in vivo. Published in 2008 by John Wiley & Sons, Ltd.     

Targeting of lanthanide(III) chelates of DOTA‐type glycoconjugates to the hepatic asyaloglycoprotein receptor: cell internalization and animal imaging studies

The characterization of a new class of hydrophilic liver‐targeted agents for γ‐scintigraphy and MRI, consisting, respectively, of [¹⁵³Sm]³⁺ or Gd³⁺ complexes of DOTA monoamide or bisamide linked glycoconjugates (DOTA = 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid), is reported. In vitro studies show high uptake of radiolabeled [¹⁵³Sm]‐DOTAGal₂ by the human hepatocyte carcinoma cell line Hep G2 containing the asialoglycoprotein receptor (ASGP‐R), which is decreased to less than 50% by the presence of its high‐affinity ligand asialofetuin (ASF). In vivo biodistribution, γ‐imaging and pharmacokinetic studies on Wistar rats using the [¹⁵³Sm]³⁺‐labeled glycoconjugates show a high uptake in the receptor‐rich organ liver of the radiolabeled compounds containing terminal galactosyl groups, but very little uptake for those compounds with terminal glycosyl groups. Blocking the receptor in vivo reduced liver uptake by 90%, strongly suggesting that the liver uptake of these compounds is mediated by their binding to the asyaloglycoprotein receptor (ASGP‐R). This study also demonstrated that the valency increase improves the targeting capability of the glycoconjugates, which is also affected by their topology. However despite the specific liver uptake of the radiolabeled galactose‐bearing multivalent compounds, the animal MRI assessment of the corresponding Gd³⁺ chelates shows liver‐to‐kidney contrast effects which are not significantly better than those shown by GdDTPA. This probably results from the quick wash‐out from the liver of these highly hydrophilic complexes, before they can be sufficiently concentrated within the hepatocytes via receptor‐mediated endocytosis. Copyright © 2006 John Wiley & Sons, Ltd.     

Hepatoprotective and antioxidant potential of Asphodeline lutea (L.) Rchb. roots extract in experimental models in vitro/in vivo

The aim of this study was to investigate the effect of Asphodeline lutea (L.) Rchb. dry root extract (ALE) administered alone and against carbon tetrachloride (CCl₄)-induced liver injury in vitro/in vivo. The dried roots of A. lutea were extracted with 70% ethanol and was characterized with HPLC-UV. Hepatoprotective potential was investigated by in vivo/in vitro assays in Wistar rats as well as antioxidant properties. At concentrations ranging from 10 to 200 μg/mL of ALE significant cytotoxic effects on isolated hepatocytes were found. ALE showed some toxicity in Wistar rats discerned by increased ALT (Alanine transaminase), ALP (Alkaline phosphatase) activities and MDA (malondialdehyde) quantity, decreased GSH (reduced glutathione) levels without affecting the activity of the antioxidant enzymes (GPx (Gluthatione peroxidase), GR (Glutathione reductase) and GST (Glutathione-S-transferase activity)). The antioxidant and hepatoprotective potential of ALE was also observed in vitro/in vivo against CCl₄-induced liver injury, where ALE normalizes all the examined parameters perturbated by CCl₄ administration. In addition, ALE preserved the decreased cytochrome P450 level and EMND (Ethylmorphine-N-Demethylase) activity without affecting AH (Aniline 4-Hydroxylase) activity. ALE is rich in anthraquinones, naphthalenes and caffeic acid. The pro-oxidant effects of ALE could be due to naphthalene and anthraquinone bioactivation pathways involving toxic metabolites.     

Resveratrol inhibits fatty acid and triacylglycerol synthesis in rat hepatocytes

Background The putative role of resveratrol, a polyphenol present in grapes and other plants, in modulating dislypidemia, thus preventing cardiovascular diseases, is generally based on proliferating cell lines and in vivo studies in different pathological conditions. The aim of the present study was to investigate whether resveratrol plays a role on lipid biosynthesis in rat hepatocytes. Materials and methods The effect of resveratrol on total rate of fatty acid, cholesterol and complex lipid synthesis, assayed by the incorporation of [1‐¹⁴C]acetate into these lipid fractions, was investigated in rat hepatocyte suspensions. Enzyme activities of acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS) as well as 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase (HMG‐CoA‐R), pace‐setting steps of de novo fatty acid and cholesterol synthesis, respectively, were in situ measured in digitonin‐permeabilized hepatocytes. Results Resveratrol‐treated hepatocytes exhibited a short‐term (30 min) inhibition (IC₅₀ ~25 µm ) of total fatty acid synthesis from [1‐¹⁴C]acetate. Among neosynthesized fatty acids, palmitic acid formation was mainly reduced, thus suggesting that enzymatic step(s) of de novo fatty acid synthesis was affected by resveratrol. In digitonin‐permeabilized hepatocytes, only ACC activity was noticeably reduced, while no change in FAS activity was observed. A noticeable resveratrol‐induced reduction of label incorporation into triacylglycerols was also detected. Conversely, cholesterol synthesis and HMG‐CoA‐R activity were unaffected by resveratrol. Conclusion Results here reported show that in isolated hepatocytes from normal rats a resveratrol‐induced short‐term inhibition of fatty acid and triacylglycerol synthesis occurs. This finding may represent a potential mechanism contributing to the reported hypolipidemic effect of resveratrol.     

Purification and some properties of the enzyme which conjugates sulfobromophthalein to glutathione in rat liver

The enzyme in rat liver which conjugates sulfobromophthalein (BSP) with glutathione (GSH) has been purified 100-fold by ammonium sulfate fractionation, gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose.The purified enzyme follows Michaelis-Menten kinetics. The apparent K_m's for BSP and GSH were 2.7 × 10^?4 M and 1 × 10^?2 M, respectively. The enzyme showed a pH optimum between 8.0 and 8.2 and the enzyme activity was proportional to enzyme concentration over a broad range. Inhibition of activity occurred at concentrations of both BSP and GSH at or above their K_m values. Crude enzyme present in acetone powder extracts of liver was stable for 2 months, but all activity was lost in 48 h at 4° by enzyme purified 100-fold.Enzymatic conjugation of GSH to three organic “acceptor” molecules in addition to BSP was observed in acetone powder extracts of rat liver. However, only one acceptor molecule, i.e., ethylmethanesulfonate, in addition to BSP, was conjugated in enzyme preparations purified 100-fold.     

Functionalized single‐walled carbon nanotubes containing traces of iron as new negative MRI contrast agents for in vivo imaging

Single‐walled carbon nanotubes (SWCNTs) containing traces of iron oxide were functionalized by noncovalent lipid‐PEG or covalent carboxylic acid function to supply new efficient MRI contrast agents for in vitro and in vivo applications. Longitudinal (r₁) and transversal (r₂) water proton relaxivities were measured at 300 MHz, showing a stronger T₂ feature as an MRI contrast agent (r₂/r₁ = 190 for CO₂H functionalisation). The r₂ relaxivity was demonstrated to be correlated to the presence of iron oxide in the SWNT‐carboxylic function COOH, in comparison to iron‐free ones. Biodistribution studies on mice after a systemic injection showed a negative MRI contrast in liver, suggesting the presence of the nanotubes in this organ until 48 h after i.v. injection. The presence of carbon nanotubes in liver was confirmed after ex vivo carbon extraction. Finally, cytotoxicity studies showed no apparent effect owing to the presence of the carbon nanotubes. The functionalized carbon nanotubes were well tolerated by the animals at the dose of 10 µg g^?1 body weight. Copyright © 2012 John Wiley & Sons, Ltd.     

FLT-PET Imaging of Radiation Responses in Murine Tumors

Background  3′-[F-18]Fluoro-3′-deoxythymidine (FLT) traces thymidine phosphorylation catalyzed by thymidine kinase during cell proliferation. Knowing the rate of cell proliferation during cancer treatment, such as radiation therapy, would be valuable in assessing whether tumor recurrence is likely and might indicate the need for additional treatments. However, the relationship between FLT kinetics and the effects of radiation is not well-understood. Nor has the method for optimal quantification of FLT uptake within the irradiated tumor microenvironment been extensively examined. Materials and Methods  We performed dynamic FLT-positron emission tomography (PET) studies (60 min) on 22 mice implanted subcutaneously with syngeneic mammary MCaK tumors bilaterally in the shoulder area. A day before the FLT-PET imaging, the tumor on the right side was irradiated with a single dose (0, 2.5, 5, 10, or 20 Gy) or with fractionated exposures (4 × 2.5 Gy given in 12 h intervals). Standardized uptake value (SUVs) of FLT on tumors at 10 and 60 min post injection were calculated; model fitting was used to estimate the kinetic parameters. Significant radiation-induced changes were shown by comparing the irradiated tumor with the control tumor in the same animal and by comparing it to nonirradiated mice. The effect of radiation on MCaK cell cycle parameters and FLT uptake was also examined in vitro. Results   In vivo FLT kinetics were sensitive to radiation doses of 5 Gy and higher (administered 1 day earlier), as judged by SUV semiquantitative measures and by modeling. Single irradiation with 10 Gy had greater impact on SUVs and kinetic parameters than fractionated exposures. Overall, the uptake constant K _i appeared to be the best marker for these radiation effects. FLT uptake by irradiated cells in vitro at various doses gave similar findings, and the in vitro FLT uptake correlated well with K _i . Radiation-induced G2/M arrest appeared to influence FLT uptake, and this was more pronounced after single than fractionated doses. Conclusion  The kinetics of FLT uptake into murine mammary tumors was altered 1 day after radiation treatment. The dose-dependent response correlated well with in vitro FLT cellular uptake. Parameters (e.g., K _i ) derived from FLT kinetics are expected to be useful for assessing the efficacy of irradiation treatment of tumors.