Platelet-activating factor (PAF) receptor binding antagonists from Alpinia officinarum

The bioassay-guided purification of ether extracts of Alpinia officinarum led to the isolation of two new compounds 6-hydroxy-1,7-diphenyl-4-en-3-heptanone (1) and 6-(2-hydroxy-phenyl)-4-methoxy-2-pyrone (4) as well as three known compounds 1,7-diphenyl-4-en-3-heptanone (2), 1,7-diphenyl-5-methoxy-3-heptanone (3), and apigenin (5). Their structures were established on the basis of spectral methods. All three diarylheptanoids 1, 2, and 3 exhibited potent PAF receptor binding inhibitory activities with an IC₅₀ of 1.3, 5.0, and 1.6 μM, respectively. These studies have identified diarylheptanoids as a novel class of potent PAF antagonists.     

Vanadium treatment of type 2 diabetes: A view to the future

3-Hydroxy-2-methyl-4-pyrone and 2-ethyl-3-hydroxy-4-pyrone (maltol and ethyl maltol, respectively) have proven especially suitable as ligands for vanadyl ions, in potential insulin enhancing agents for diabetes mellitus. Both bis(maltolato)oxovanadium(IV) (BMOV), and the ethylmaltol analog, bis(ethylmaltolato)oxovanadium(IV) (BEOV), have the desired intermediate stability for pro-drug use, and have undergone extensive pre-clinical testing for safety and efficacy. Pharmacokinetic evaluation indicates a pattern of biodistribution consistent with fairly rapid dissociation and uptake, binding to serum transferrin for systemic circulation and transport to tissues, with preferential uptake in bone. These bis-ligand oxovanadium(IV) (VOL₂) compounds have a clear advantage over inorganic vanadyl sulfate in terms of bioavailability and pharmaceutical efficacy. BEOV has now completed Phase I and has advanced to Phase II clinical trials. In the Phase I trial, a range of doses from 10 mg to 90 mg BEOV, given orally to non-diabetic volunteers, resulted in no adverse effects; all biochemical parameters remained within normal limits. In the Phase IIa trial, BEOV (AKP-020), 20 mg, daily for 28 days, per os, in seven type 2 diabetic subjects, was associated with reductions in fasting blood glucose and %HbA1c; improved responses to oral glucose tolerance testing, versus the observed worsening of diabetic symptoms in the two placebo controls.     

Molecular Cloning and Construction of agp Gene Deletion-mutant in Cyanobacterium Synechocystis sp. PCC 6803

Nine compounds were isolated from Elsholtzia blanda (Benth.) Benth. Their　structures were identified with spectral and chemical methods as follows: 5,6-dihydro-6-styry-2-pyrone (1), friedelin (2), 4-hydroxy-3-methoxystyrene (3), 5,2′-dimethoxy-6,7-methylene dioxyflavanone (4), 5-hydroxy-7-methoxy-6-O-［α- L -rhamnopyranosyl(1→2)-β- D -fucopyranosyl］ flavone glycoside (5), 5,5′-dihydroxy-7-acetoxyl-6,8,3″,3″-tetramethylpyran　(3′,4′)　flavone (6), 5,5′-dihydroxy-7-(α-methyl) butyroxyl-6,8,3″,3″-tetramethylpyran (3′,4′) flavone (7), 5,5′-dihydroxy-6,7-methylenedioxy-8,3″,3″-trimethylpyran　(3′,4′)　flavone (8), glucosyringic acid (9). Among them, 6, 7 and 8 are new compounds, named as sifanghaoine Ⅰ,Ⅱ and Ⅲ, respectively.     

Similar binding sites for unsaturated fatty acids and alkyl 2-pyrone inhibitors of human sputum elastase

Evidence is presented to support the hypothesis that oleic acid and 3-(1'-oxo-7'-carboxyheptyl)-4-hydroxy-6-octyl-2-pyrone (and other 3,6-dialkyl-2-pyrones) occupy the same binding region on human sputum elastase. The mechanism of inhibition is strongly dependent on the substitution pattern of the 2-pyrone, and these mechanisms correlate with those of oleic acid and 11-undecenoic acid ("half oleic acid"). Based on the assumption that the 2-pyrone moiety and the double bond of the fatty acids bind to the same region of elastase (subsite S3), we believe that the alkyl chain points towards the S1 subsite, with the carboxylate anion fragment pointing away and probably associated with positively charged Arg217. (subsite S4 or S5).     

Structure of a geranyl-alpha-pyrone from Mimulus aurantiacus leaf resin

A previously reported but misidentified geranyl-alpha-pyrone, in addition to six known compounds, was isolated from the leaf resin of Mimulus aurantiacus. HMBC NMR analyses of the geranyl-alpha-pyrone resolved uncertainties in the site of attachment of the two side chains and necessitated a revision of the previously reported structure. This compound is shown to be 3-geranyl-4-hydroxy-6-(2-hydroxypropyl)-2-pyrone.     

Effect of fasting on thyroid hormone levels, and TR(alpha) and TR(beta) mRNA accumulation in late-stage embryo and juvenile rainbow trout, Oncorhynchus mykiss

The accumulation of mRNA encoding for hepatic and intestinal T3-receptor (TR) and body and liver masses were measured in fed and 3-week fasted juvenile and swim up stage rainbow trout embryos. Plasma and total body thyroid hormone (TH) levels were measured for juvenile and swim up stages, respectively. Fasted juveniles exhibited a lower hepatosomatic index (HSI), liver mass and plasma T4 and T3 concentrations than fed animals, but there were no changes in body mass or the accumulation of mRNA encoding for either of the TR(alpha) or TR(beta) isoforms in liver or intestine. TR(beta) mRNA accumulation was greater than TR(alpha) mRNA accumulation in both tissues. Fasted embryos had lower whole body TH levels and body, liver and intestinal tract masses, in addition to a lower intestinosomatic index. However, there was no change in HSI. Fasting did not affect whole body or hepatic TR(alpha) and TR(beta) mRNA accumulation, although intestinal tract TR(alpha) and TR(beta) mRNA accumulation was lower in the fasted embryos. The HSI and body mass changes in fasted juvenile and embryo stages, respectively, indicated that both developmental stages were impacted by fasting. Both stages also showed evidence of decreased TH production. The lower TR gene expression in the intestinal tract of fasted embryos may suggest a role for THs in the transitional stage of intestinal development during this period of development.     

Aspergillus oryzae CsyB Catalyzes the Condensation of Two β-Ketoacyl-CoAs to Form 3-Acetyl-4-hydroxy-6-alkyl-α-pyrone

The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C₉–C₁₇ in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs.     

Thyroid hormone receptor α and regulation of type 3 deiodinase

Mice deficient in thyroid hormone receptor α (TRα) display hypersensitivity to thyroid hormone (TH), with normal serum TSH but diminished serum T(4). Our aim was to determine whether altered TH metabolism played a role in this hypersensitivity. TRα knockout (KO) mice have lower levels of rT(3), and lower rT(3)/T(4) ratios compared with wild-type (WT) mice. These alterations could be due to increased type 1 deiodinase (D1) or decreased type 3 deiodinase (D3). No differences in D1 mRNA expression and enzymatic activity were found between WT and TRαKO mice. We observed that T(3) treatment increased D3 mRNA in mouse embryonic fibroblasts obtained from WT or TRβKO mice, but not in those from TRαKO mice. T(3) stimulated the promoter activity of 1.5 kb 5'-flanking region of the human (h) DIO3 promoter in GH3 cells after cotransfection with hTRα but not with hTRβ. Moreover, treatment of GH3 cells with T(3) increased D3 mRNA after overexpression of TRα. The region necessary for the T(3)-TRα stimulation of the hD3 promoter (region -1200 to -1369) was identified by transfection studies in Neuro2A cells that stably overexpress either TRα or TRβ. These results indicate that TRα mediates the up-regulation of D3 by TH in vitro. TRαKO mice display impairment in the regulation of D3 by TH in both brain and pituitary and have reduced clearance rate of TH as a consequence of D3 deregulation. We conclude that the absence of TRα results in decreased clearance of TH by D3 and contributes to the TH hypersensitivity.     

Characterization of alternative cytosolic forms and cellular targets of mouse mitochondrial thioredoxin reductase

Thioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains cysteine residues in numerous proteins in the reduced state. Both cytosolic (TR1 and Trx1) and mitochondrial (TR3 and Trx2) enzymes are essential in mammals, but the function of the mitochondrial system is less understood. In this study, we characterized subcellular localization of three TR3 forms that are generated by alternative first exon splicing and that differ in their N-terminal sequences. Only one of these forms resides in mitochondria, whereas the two other isoforms are cytosolic. Consistent with this finding, TR3 did not have catalytic preferences for mitochondrial Trx2 versus cytosolic Trx1, both of which could serve as TR3 substrates. Similarly, TR1 was equally active with Trx1, Trx2, or a bacterial Trx. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc, but not by calcium or cobalt ions. We further developed a proteomic method for identification of targets of TRs in mammalian cells utilizing affinity columns containing recombinant TR3 forms differing in C-terminal sequences. Using this procedure, we found that Trx1 was the major target of TR3 in both rat and mouse liver cytosol. The truncated form of TR3 lacking selenocysteine was particularly efficient in binding Trx1, consistent with the previously observed role of truncated TR1 in apoptosis. Overall, these data establish that the function of TR3 is not limited to its role in Trx2 reduction.     

Effects and interaction studies of triflusal and other salicylic derivatives on cyclooxygenase in rats

Triflusal (TR) is a new salicylic acid derivative used clinically as an antiplatelet drug. Both aspirin (ASA) and TR inhibit platelet cyclooxygenase but the effects of these drugs are different. TR (0.5-2 mM) strongly inhibited platelet aggregation and malondialdehyde formation induced by arachidonic acid. The IC50 was 0.8 mM for TR and less than 0.1 mM for ASA. Deacetylated compounds, salicylic acid (SA) and HTB (the main metabolite of TR) were apparently competitive and reversible inhibitors of cyclooxygenase and HTB was 15 times more potent than SA. They did, however, partially prevent the inhibitory effects of ASA and TR in vitro. A similar effect was observed ex vivo in rats treated with HTB (100 mg/k i.p.) before TR or ASA (20 and 5 mg/kg i.v., respectively). Moreover, TR at 10 and 20 mg/kg i.v., inhibited thromboxane production by more than 50% while its effect on vascular cyclooxygenase was negligible. These findings indicated that TR is a weaker inhibitor of cyclooxygenase than ASA, and that HTB interferes with the effect of TR and ASA, despite the fact that HTB is a more potent reversible inhibitor than SA with probably a higher affinity for this enzyme.     

The goitrogen 3-hydroxy-4(1H)-pyridone, a ruminal metabolite from Leucaena leucocephala: effects in mice and rats.

Mice fed a diet containing 1% (w/w) 3-hydroxy-4(1H)-pyridone (DHP) developed goitre even with a diet high in iodine whereas mimosine (0.5% w/w) did not produce goitre even with a low-iodine diet. Thyroid enlargement was apparent (measured morphometrically) by the 7th week and was advanced by the 11th week. Histologically the goitre was hyperplastic in type. No marked histological changes were found in other organs of mice fed DHP or any organs of mice fed mimosine, except for some atrophy of hair follicles. A single intragastric dose of DHP inhibited the uptake of 125I by the thyroid in the rat but an equivalent dose of mimosine did not. Evidence is presented that the inhibition occurs at the iodine binding step, as with methyl thiouracil, rather than at the iodide trapping step, as with thiocyanate. Chronic treatment of mice with DHP, as with 6-methyl thiouracil, increased the avidity of the thyroid in taking up 125I. The major conjugated form of DHP in mammals, DHP-3-O-glucuronide, was almost as effective a goitrogen as the unconjugated compound when given by mouth but considerably less active than the free form in the blood stream. It was concluded that DHP is a potent antithyroid compound of the thiouracil type with low general toxicity, since mammals can tolerate a level of intake sufficient to produce goitre in spite of iodine supplementation.     

Polyketide derivatives active against Botrytis cinerea in Gerbera hybrida

A previously isolated cDNA molecule from Gerbera hybrida (Asteraceae) codes for a new chalcone synthase-like polyketide synthase, 2-pyrone synthase (2PS). 2PS is able to synthesise 4-hydroxy-6-methyl-2-pyrone (triacetolactone), a putative precursor for gerberin and parasorboside, two abundant glucosides in gerbera. In this study, we show that gerbera plants transformed with the gene for 2PS in an antisense orientation and unable to synthesise gerberin and parasorboside are susceptible to Botrytis cinerea infection. In addition to the preformed glucosides, the transgenic plants also lack several compounds that are induced in control plants when infected with the mould. Some of these induced substances are effective in inhibiting fungal growth both in vitro and in vivo. Two of the phytoalexins were identified as the aglycones of gerberin and trans-parasorboside. The third phytoalexin is a rare coumarin, 4-hydroxy-5-methylcoumarin; however, it is typical of many plants of the sunflower family Asteraceae. The coumarin cannot be structurally derived from either gerberin or parasorboside, but may be derived from a related polyketide intermediate.     

Characterization and evaluation of pyrone and tropolone chelators for use in metalloprotein inhibitors

The tetrahedral zinc and cobalt complexes [(Tp^Ph,Me)ZnOH] (Tp^Ph,Me = hydrotris(3,5-phenylmethylpyrazolyl)borate) and [(Tp^Ph,Me)CoCl] were combined with 3-hydroxy-2H-pyran-2-one (3,2-pyrone), 3-hydroxy-4H-pyran-4-one (3,4-pyrone), and tropolone to form the corresponding [(Tp^Ph,Me)M(L)] complexes (L = bidentate ligand, M = Zn²⁺, Co²⁺). X-ray crystal structures of these complexes were obtained to determine the mode of binding for each chelator and the coordination geometry of each complex. The complexes [(Tp^Ph,Me)M(3,2-pyrone)] (M = Zn²⁺, Co²⁺) are the first structurally characterized metal complexes with this chelator. These complexes with the various chelators show that the cobalt(II) complexes are generally isostructural with their zinc(II) counterparts. In addition to structural characterization, inhibition data for each ligand against two different zinc(II) metalloproteins, matrix metalloproteinase-3 (MMP-3) and anthrax lethal factor (LF), were obtained. Examination of these chelators in the MMP-3 active site demonstrates the possible mode of inhibition.     

Dietary oxidized n-3 PUFA induce oxidative stress and inflammation: role of intestinal absorption of 4-HHE and reactivity in intestinal cells

Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to -19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.     

Hispidin biosynthesis in cultures of Polyporus hispidus

Cultures of Polyporus hispidus grown on a liquid medium with glucose as the principal carbon source produced the yellow pigment hispidin (4-hydroxy-6-(3,4-dihydroxystyryl)-2-pyrone) (II). Tracer studies showed that DL-phenylalanine, DL-tyrosine, cinnamate, p-coumarate and caffeate were incorporated into the hispidin molecule without scrambling of the label. Good incorporation of acetate and roalonate into the pyrone portion of the molecule was observed. The related styrylpyrone, bis-noryangonin (4-hydroxy-6-(4-hydroxystyryl)-2-pyrone) (1) was also detected in extracts of cultured mycelium and a cell-free enzyme preparation was obtained which catalyzed the hydroxylation of bis-noryangonin to hispidin.     

Pyrone and pyridone compounds in the liquid culture of Physisporinus sanguinolentus

Chromatographic separation of the liquid culture filtrate of the basidiomycete fungus Physisporinus sanguinolentus has yielded three new compounds viz., 2-methyl-4-pyrone, 2-methyl-5,6-dihydro-4-pyrone and the pyridone form of 4-hydroxy-2-methylpyridine, together with the known triacetic acid lactone, the sesquiterpene dialdehyde merulidial and a derivative of merulidial. Their structures were elucidated by spectroscopic analysis and by comparison to literature data and a synthetic sample. One of the compounds, merulidial, was shown to inhibit the germination of spores and the hyphal growth of the wood-rotting basidiomycete Heterobasidion annosum and the saprophytic mould Cladosporium cucumerinum.     

Colon cancer and solar ultraviolet B radiation and prevention and treatment of colon cancer in mice with vitamin D and its Gemini analogs

It has been recognized that people who live at higher latitudes and who are vitamin D deficient are at higher risk of dying from many common cancers including colon cancer. To evaluate the role of vitamin D deficiency on colon tumor growth, Balb/c adult male mice were fed either a vitamin D sufficient or vitamin D deficient diet for 10 weeks. Mice were arranged into groups of six and each animal received subcutaneously 10(4) MC-26 cells in the posterior trunk. The tumor size was recorded daily. By day 9 there was a significant difference in tumor volume between the vitamin D sufficient and vitamin D deficient mice. By day 18 the vitamin D deficient animals had a tumor size that was 56% larger compared to the animals that were vitamin D sufficient. To determine whether treatment with active vitamin D analogs could further decrease colon tumor growth in a vitamin D sufficient state, groups of mice were treated with the novel 19-nor-Gemini compounds. The mice were fed a low calcium diet. Twenty-four hours after tumor implantation, the mice received, three times weekly, one of the vitamin D analogs or the vehicle. The group that received Gemini 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-19-nor-20S-cholecalciferol (3) showed a dose-dependent decrease in tumor volume. On day 19, at the dose level of 0.02microg molar equivalents (E), the tumor volume was reduced by 41% when compared to the control group. At the same time point, the hexadeuterated analog 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-26,27-hexadeutero-19-nor-20S-cholecalciferol (4), administered at the 10-fold lower dose of 0.002microgE, showed a 52% reduction in tumor volume (p<0.05), compared to the control group. Animals that received 1,25(OH)(2)D(3) at 0.002 and 0.02microg showed a trend in tumor volume reduction at the highest dose but the changes were not statistically significant. An evaluation of serum calcium concentrations revealed that the calcium levels were normal in all groups, except the group receiving 0.02microgE of 4. The results from these studies demonstrate that vitamin D deficiency may accelerate colon cancer growth and that novel Gemini analogs of 1,25(OH)(2)D(3) may be an effective new approach for colon cancer treatment.     

A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors

Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3-dependent manner. A 9-amino-acid peptide derived from full-length TR3 with polyarginine tail (TR3-r8) recapitulates TR3's binding specificity, displaying high affinity for Bcl-B. TR3-r8 peptide was used to screen for small molecule Bcl-B inhibitors. A fluorescence polarization assay (FPA) employing fluorescein isothiocyanate (FITC)-labeled TR3-r8 peptide (FITC-TR3-r8) and Bcl-B protein was optimized, with nonfluorescent TR3-r8 serving to demonstrate reversible, competitive binding. Approximately 50,000 compounds were screened at 3.75 mg/L, yielding 145 reproducible hits with >/=50% FITC-TR3-r8 displacement (a confirmed hit rate of 0.29%). After dose-response analyses and counterscreening with an unrelated FITC-based FPA, 6 candidate compounds remained. Nuclear magnetic resonance (NMR) showed that 2 of these compounds bound Bcl-B, but not glutathione S-transferase (GST) control protein. One Bcl-B-binding compound was unable to displace FITClabeled BH3 peptides from Bcl-B, confirming a unique binding mechanism compared with traditional antagonists of antiapoptotic Bcl-2-family proteins. This compound bound Bcl-B with K(d) 1.94 +/- 0.38 microM, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrated that this compound induced Bcl-B-dependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins.     

Studies on the Reduction of 2,6-Dichlorophenolindophenol by 6-Substituted 3-Hydroxy-4-Pyrones,Using a Stopped-flow Method

The reduction of 2,6-dichlorophenolindophenol (DCIP) by 6-substituted 3-hydroxy-4-pyrones was studied in the pH range of 3.0 to 7.8, using a stopped-flow apparatus.

1) Kinetic characteristics of 6-substituted 3-hydroxy-4-pyrones are shown as the apparent first-order rate constant, K_app, or the second-order rate constant, k. The plot of log k against pH was different from those obtained for the reactions of L-ascorbic acid and triose reductone with DCIP.

2) The log (k/k_H) of 6-substituted 3-hydroxy-4-pyrone solutions at acidic pHs have been correlated with Δδ_C?3 values for 3-hydroxy-4-pyrone.     

Synthesis, chelating properties towards gallium and biological evaluation of two N-substituted 3-hydroxy-4-pyridinones

Two N-substituted 3-hydroxy-4-pyridinones (1-(3'-aminopropyl)-3-hydroxy-2-methyl-4-pyridinone (L1) and 1-(2'-carboxyethyl)-3-hydroxy-2-methyl-4-pyridinone (L2)) were prepared through one- and three-step reactions, respectively. The pKa values of the ligands and the stability constants of their Ga(III) complexes have been determined. Both the complexes are strongly coordinated to three (O,O) hydroxypyridonate moieties. There is a clear effect of the N-substituents in the lipophilic-hydrophilic balance and in the Ga(III) binding interaction; the acid derivative (L2) has lower lipophilicity but higher chelating strength than the amine derivative (L1). Both chelators are shown to interfere in the typical biological behavior of 67Ga-citrate in mice: L1 enhanced the urinary excretion leading to an increased 67Ga removal from the soft tissue, while L2 induced a lower blood clearance with a pronounced bone uptake mainly at 48 h after injection, thus suggesting that the 67Ga-L2 complex could have potential interest as a bone imaging agent.