Secondary plant substances in various extracts of the leaves, fruits, stem and bark of Caraipa densifolia Mart.

Thirty secondary plant substances were detected in various extracts of the leaves, fruits, stem and bark of Caraipadensifolia Mart. Phenolic compounds were preliminarily identified and quantitated by HPLC-ESI-MS and the structures of the compounds, purified by semi-preparative HPLC, were further characterized by nano-ESI-MS-MS. The presence of gallic acid, 3,4-dihydroxybenzoic acid, neochlorogenic acid, chlorogenic acid, methyl gallate, p-coumaric acid quinate, epicatechin, procaynidin dimer B(2), procyanidin trimer C(1), syringic acid, 1,2,3,6-tetragallate glucoside, 1,3,4,6-tetragallate glucoside, corilagin, ellagic acid, methyl ellagic acid rhamnoside, quercetin-3-O-rhamnoside, two apigenin-C-glycosides (vitexin and isovitexin) and two luteolin-C-glycosides (orientin and isoorientin) are reported in this species for the first time. In addition, the previously reported following terpenoids, lupeol, lupenone, betulinic acid, betulin, friedelin and a previously non-characterized terpenoid in this species, friedelinol were identified and quantitated by GC-MS. A previously identified sterol was beta-sitosterol along with stigmasterol in this species for the first time. The vitamins alpha-tocopherol and gamma-tocopherol were also identified in extracts of the leaves of Caraipa species for the first time. The data shows that the botanical parts of C. densifolia Mart. has a much richer spectrum of secondary plant substances than previously reported.

Polyphenol composition and antioxidant potential of Hibiscus esculentus L. fruit cultivated in Nigeria.

Consumption of certain fruits and vegetables is now widely associated with chemoprevention of degenerative diseases like cancer and cardiovacsular disorders because of their antioxidant components. Polyphenols, a heterogeneous group of compounds, are one of these constituents. Hibiscus esculentus L. (Family Malvaceae), commonly referred to as okro, okra, or lady's finger, is an important component of diet in Nigeria and other countries in sub-Saharan Africa. In this article, we describe the polyphenol composition and antioxidant potential of H. esculentus of Nigerian origin. Quercetin glucoside (quercetrin) and an unidentified flavonoid were detected. In vitro antioxidant assay of methanol extract of the fruits showed potent antioxidant/radical scavenging activities with 50% inhibitory concentration values of 25 and 43 microL when analyzed by the xanthine oxidase and 2-deoxyguanosine methods, respectively. These data suggest that H. esculentus, popular especially during the rainy season in Nigeria and many tropical West, Central, and Eastern African countries, is a good contributor to the antioxidant status and disease chemoprevention of people in these countries.

Evaluation of the polyphenol composition and antioxidant activity of African variety of Dacryodes edulis (G.Don) H.J Lam fruit.

Polyphenols are abundant micronutrients in our diet that have been credited with chemoprevention of diseases associated with oxidative stress. In this study, we investigated the whole ripened fruit of Dacryodes edulis (G.Don) H.J Lam, a multipurpose tree growing in West and Central Africa and other countries bordering the Gulf of Guinea, for polyphenol content as well as its antioxidant/radical scavenging capacity. Analysis of the methanol extract of the fruit by high-performance liquid chromatography coupled to an ultraviolet dual-array detector and mass-selective detector revealed the presence of catechol (9.27 mg/kg), gallate (10.40 mg/kg), methylgallate (0.88 mg/kg), ellagic acid (3.10 mg/kg), quercetin (0.21 mg/kg), and quercetin rhamnoside (0.76 mg/kg). The extract showed very high antioxidant potential (50% inhibitory concentration [IC(50)] = 14 microL), but a rather weak radical scavenging activity (IC(50) = 357 microL), when tested in vitro with the xanthine oxidase and 2-deoxyguanosine assay model systems, respectively. These results suggest that consumption of D. edulis could contribute to prevention of diseases that are related to oxidative stress.

Comparison of in vitro and in vivo properties of [99mTc]cRGD peptides labeled using different novel Tc-cores.

AIM: The alfa(v)beta(3) integrin is involved in angiogenesis and tumor metastasis. Arginine-glycine-aspartic acid (RGD)-peptides bind with high affinity to this integrin. This study compares the influence of (99m)Tc-labeling applying novel Technetium-cores on imaging characteristics of the radiolabeled peptide. METHODS: Different peptide conjugates based on the cyclic pentapeptide c(RGDyK) (cRGD) were prepared and characterized (HYNIC-, Cys-, L2- and Pz1-cRGD). Radiolabeling experiments using different coligands for HYNIC-cRGD, the (99m)Tc(CO)(3) metal fragment for PZ-1-cRGD (pyrazolyl-derivative), the Tc-nitrido-core using a phosphine-coligand (PNP) for Cys-cRGD and an isonitrile-conjugate (L2-cRGD) together with a NS(3)-coligand (4+1 concept) were performed and showed labeling yields >90% at high specific activities. RESULTS: A high in vitro stability was observed, plasma protein binding and lipophilicity varied considerably between different radiolabeled cRGD conjugates. Experiments on biological activity of the radiolabeled peptides using alfa(v)beta(3) positive (M21) and negative (M21L) tumor cells did show specific uptake of various conjugates. Studies in tumor bearing animals revealed significant differences between different conjugates concerning pharmacokinetic behavior (predominant renal excretion to considerable hepatobiliary clearance) as well as tumor uptake (0.2-2.7%ID/g). Highest specific tumor uptake and tumor/background values were found for [(99m)Tc]EDDA/HYNIC-c(RGDyK), [(99m)Tc]Nitrido-PNP-Cys-c(RGDyK) and [(99m)Tc(CO)(3)]-Pz1-c(RGDyK). CONCLUSIONS: Using novel Tc-cores such as the (99m)Tc(CO)(3) metal fragment, Tc-nitrido- and the 4+1 concept peptides could be labeled with [(99m)Tc]technetium at high specific activities resulting in complexes with high stability, but binding moieties have to be optimized especially concerning hydrophilicity resulting in renal rather than hepatobiliary excretion. This comparative study underlines that peptide labeling strategies using (99m)Tc have to be properly selected and optimized. Different in vitro assays are necessary to predict targeting properties in vivo.

Isolation, purification and identification of ellagic acid derivatives, catechins, and procyanidins from the root bark of Anisophyllea dichostyla R. Br.

The root bark of Anisophyllea dichostyla R. Br. is traditionally used in the Democratic Republic Congo for the treatment of several conditions such as anorexia, fatigue and intestinal infections. We have identified and quantitated several polyphenol antioxidants in the methanol extract of the root bark (120g). The polyphenol content (3.32g/kg) was predominantly ellagitannins (25%) and polyhydroxyflavan-3-ols (catechins and procyanidins, 75%) with 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside and (-)-epicatechin as the major species in each class. These two compounds and the following species were identified unequivocally by NMR spectroscopy: (+)-catechin, (-)-epicatechin 3-O-gallate, 3-O-methyl ellagic acid, 3,3'-di-O-methyl ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside, and 3'-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. The following additional compounds were purified by semi-preparative HPLC and tentatively identified on the basis of UV spectra, HPLC-ESI-MS and nano-ESI-MS-MS: (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), epicatechin-(4beta-->8)-epicatechin (procyanidin B(2)), an (epi)catechin trimer, 3-O-methyl ellagic acid 4-O-beta-d-glucopyranoside, (-)-epicatechin 3-O-vanillate, 3,4-methylenedioxo ellagic acid 4'-O- beta-d-glucopyranoside, and 3,3'-di-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. Fractionation of the raw extract by column chromatography on silicic acid yielded 10 fractions. In the hypoxanthine/xanthine oxidase antioxidant assay system, CC-9 which contained a range of polyphenols dominated by (-)-epicatechin-O-gallate proved to be the most potent antioxidant fraction (IC(50)=52 micro g/mL) in terms of ROS scavenging. In terms of XO inhibition CC-8, dominated by (epi)catechin trimer and which also contained appreciable amounts of 3'-O-methyl ellagic acid 4'-O-beta-d-xylopyranoside, as well as the catechins (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), and (-)-epicatechin 3-O-gallate, proved to be the most potent (IC(50)=36 micro g/mL).

Characterization of alkyl phenols in cashew (Anacardium occidentale) products and assay of their antioxidant capacity.

In this study the content of anacardic acids, cardanols and cardols in cashew apple, nut (raw and roasted) and cashew nut shell liquid (CNSL) were analysed. The higher amounts (353.6 g/kg) of the major alkyl phenols, anacardic acids were detected in CNSL followed by cashew fibre 6.1 g/kg) while the lowest (0.65 g/kg) amounts were detected in roasted cashew nut. Cashew apple and fibre contained anacardic acids exclusively, whereas CNSL also contained an abundance of cardanols and cardols. Cashew nut (raw and roasted) also contained low amounts of hydroxy alkyl phenols. Cashew nut shell liquid was used for a basic fractionation of the alkyl phenol classes and the individual anacardic acids, major cardanols and cardols were purified to homogeneity from these fractions by semi-preparative HPLC and definitively identified by nano-ESI-MS-MS, GC-MS and NMR analyses. The hexane extracts (10 mg/ml) of all cashew products tested plus CNSL, displayed significant antioxidant capacity. Cashew nut shell liquid was the more efficient (inhibition=100%) followed by the hexane extract of cashew fibre (94%) and apple (53%). The antioxidant capacity correlated significantly (P<0.05) with the concentration of alkyl phenols in the extracts. A mixture of anacardic acids (10.0 mg/ml) showed the higher antioxidant capacity (IC50=0.60 mM) compared to cardols and cardanols (IC50>4.0 mM). The data shows that of these substances, anacardic-1 was by far the more potent antioxidant (IC50=0.27 mM) compared to cardol-1 (IC50=1.71 mM) and cardanol-1 (IC50>4.0 mM). The antioxidant capacity of anacardic acid-1 is more related to inhibition of superoxide generation (IC50=0.04 mM) and xanthine oxidase (IC50=0.30 mM) than to scavenging of hydroxyl radicals. At present a substantial amount of cashew fibre is mostly used in formulations of animal or poultry feeds. The data presented in this study, indicates that this waste product along with CNSL, both of which contain high contents of anacardic acids, could be better utilized in functional food formulations and may represent a cheap source of cancer chemopreventive agents.

Isolation and structure elucidation of phenolic antioxidants from Tamarind (Tamarindus indica L.) seeds and pericarp.

Although it is already known that Tamarind (Tamarindus indica L.) seeds contain phenolic substances, the individual components of the seeds have not been fully identified and quantitated, and in the case of Tamarind pericarp not reported. Therefore, major polyphenolic compounds were extracted using organic solvents and the metabolites were isolated by semi-preparative high performance liquid chromatography. Their structures were elucidated by liquid chromatography-electrospray-ionisation-mass spectrometry (LC-ESI-MS), nano-electrospray-ionisation mass spectrometry (ESI-MS), and where possible by gas chromatography-mass spectrometry (GC-MS) and 1H and 13C NMR. Quantitative analysis of polyphenolic compounds in Tamarind seeds and pericarp was conducted by analytical high performance liquid chromatography (HPLC), calculated against standard curves of authentic compounds. The yields of total phenolic compounds after Soxhlet extraction with methanol were 6.54 and 2.82 g/kg (dry weight) in the seeds and pericarp respectively. The profile (%) of polyphenolics in Tamarind pericarp was dominated by proanthcyanidins (73.4) in various forms (+)-catechin (2.0), procyanidin B2 (8.2), (-)-epicatechin (9.4), procyanidin trimer (11.3), procyanidin tetramer (22.2), procyanidin pentamer (11.6), procyanidin hexamer (12.8) along with taxifolin (7.4), apigenin (2.0), eriodictyol (6.9), luteolin (5.0) and naringenin (1.4) of total phenols, respectively. The content of Tamarind seeds comprised only procyanidins, represented (%) mainly by oligomeric procyanidin tetramer (30.2), procyanidin hexamer (23.8), procyanidin trimer (18.1), procyanidin pentamer (17.6) with lower amounts of procyanidin B2 (5.5) and (-)-epicatechin (4.8). Extraction of Tamarind pericarp and seeds using acetone:methanol:acetic acid gave only procyanidin oligomers, but in much higher yield and variety. The antioxidant capacities of the Soxhlet methanolic extracts were determined, and indicates that Tamarind may be an important source of cancer chemopreventive natural products in tropical regions.

Olives and olive oil in cancer prevention.

Epidemiologic studies conducted in the latter part of the twentieth century demonstrate fairly conclusively that the people of the Mediterranean basin enjoy a healthy lifestyle with decreased incidence of degenerative diseases. The data show that populations within Europe that consume the so-called 'Mediterranean diet' have lower incidences of major illnesses such as cancer and cardiovascular disease. Studies have suggested that the health-conferring benefits of the Mediterranean diet are due mainly to a high consumption of fibre, fish, fruits and vegetables. More recent research has focused on other important factors such as olives and olive oil. Obviously fibre (especially wholegrain-derived products), fruits and vegetables supply an important source of dietary antioxidants. What is the contribution from olives and olive oil? Apparently the potential is extremely high but epidemiologic studies rarely investigate consumption of these very important products in-depth, perhaps due to a lack of exact information on the types and amounts of antioxidants present. Recent studies have shown that olives and olive oil contain antioxidants in abundance. Olives (especially those that have not been subjected to the Spanish brining process) contain up to 16 g/kg typified by acteosides, hydroxytyrosol, tyrosol and phenyl propionic acids. Olive oil, especially extra virgin, contains smaller amounts of hydroxytyrosol and tyrosol, but also contains secoiridoids and lignans in abundance. Both olives and olive oil contain substantial amounts of other compounds deemed to be anticancer agents (e.g. squalene and terpenoids) as well as the peroxidation-resistant lipid oleic acid. It seems probable that olive and olive oil consumption in southern Europe represents an important contribution to the beneficial effects on health of the Mediterranean diet.

Targeting of gelatinase activity with a radiolabeled cyclic HWGF peptide.

Matrix metalloproteinases (MMPs) are a family of proteinases that play an important role in cancer as well as in numerous diseases. In this article, we describe the labeling of a phage display selected cyclic decapeptide containing the HWGF (histidine-tryptophane-glycine-phenylalanine) sequence to target MMP-2 and MMP-9. To evaluate the ability of this labeled peptide to monitor non invasively MMP-2 and MMP-9 activity, in vitro studies, biodistribution, competition studies and plasma metabolites analyses in Lewis Lung cancer tumor bearing mice were performed.

Synthesis and biological evaluation of a (99m)Tc-labelled cyclic RGD peptide for imaging the alphavbeta3 expression.

AIM: The alphavbeta3 integrin is involved in tumour induced angiogenesis and tumour metastasis. We describe the synthesis and evaluation of a (99m)Tc-labelled RGD analogue for the visualisation of alphavbeta3 integrin expression. METHODS: The linear peptides were assembled on a solid support. Cyclisation was performed under high dilution conditions. For conjugation with the chelator peptide, a water soluble carbodiimide was used. Radiolabelling was carried out due to standard procedures with high radiochemical yield and radiochemical purity. For in vivo evaluation, nude mice bearing alphavbeta3-positive human melanoma M21 and alphav-negative human melanoma M21-L or Balb/c mice bearing alphav-positive murine osteosarcoma were used. RESULTS: Activity accumulation of (99m)Tc-DKCK-RGD 240 min p. i. was 1.1% ID/g in the alphavbeta3-positive melanoma and 0.3% ID/g in the negative control tumour. In the osteosarcoma model 2.2% ID/g was found 240 min p. i. Planar gamma camera images allowed contrasting visualisation of alphavbeta3-positive tumours 240 min p. i. Blocking of the tumour using the alphavbeta3-selective pentapeptide cyclo(-Arg-Gly-Asp-D-Phe-Val-) reduces activity accumulation in the tumour to background level. However, 240 min p. i. highest activity concentration was found in kidneys resulting in low tumour/kidney ratios. Metabolite analysis 240 min p. i. showed approximately 60% intact tracer in kidneys and 80% in the tumour. Only 24% intact tracer was found in blood 30 min p. i. CONCLUSION: (99m)Tc-DKCK-RGD allows imaging of alphavbeta3-positive tumours in mice. However, pharmacokinetics as well as metabolic stability of the tracer have to be improved for potential clinical application.

Isolation and structure elucidation of the major individual polyphenols in carob fibre.

Although it is already known that carob fibre contains several classes of polyphenolic substances, a comprehensive analysis of these has not been conducted to date. Therefore, the major polyphenolic compounds were extracted with organic solvents, and, following fractionation by normal-phase column chromatography on silicic acid, their structures were elucidated by liquid-chromatography electrospray-ionisation mass spectrometry (LC-ESI), nano-electrospray-ionisation mass spectrometry (ESI-MS), and gas-chromatography mass spectrometry (GC-MS). In addition, complete 1H and 13C NMR assignments were obtained for the isolated gallotannins 1,6-di-, 1,2,6-tri- and 1,2,3,6-tetra-O-galloyl-beta-D-glucose. Carob fibre was found to contain a rich variety of phenolic antioxidants. A total of 24 polyphenol compounds were identified with a yield of 3.94 g/kg (dry weight). The profile was dominated by gallic acid in various forms: free gallic acid (42% of polyphenols by weight), gallotannins (29%), and methyl gallate (1%), while simple phenols, mainly cinnamic acid, made up about 2% of the total. Flavonoids represented 26% of the polyphenols, and the major components were identified as the glycosides myricetin- and quercetin-3-O-alpha-L-rhamnoside (ca. 9% and 10%, respectively). These data indicate that carob fibre is rich in both amount and variety of phenolic antioxidant substances, and its inclusion in the diet may have chemopreventive properties.

Radiotracer-based strategies to image angiogenesis.

Tumour-induced angiogenesis plays an important role in tumour progression. Great efforts are made to develop therapeutic strategies to interfere with this process resulting in the starvation of the tumour. However, strategies to monitor conventional therapies seems to be inappropriate to control these approaches. Thus, there is a keen interest in developing methods supplying information about the corresponding therapeutical effects. Several radiotracer-based approaches focused on different targets in the angiogenic process are currently investigated. One class of tracers is based on matrix metalloproteinases inhibitors. These compounds show promising results in in vitro assays. However, initial data from in vivo studies using murine tumour models could not confirm successful non-invasive monitoring of MMP activity yet. Another strategy uses a radiolabelled single chain fragment against the ED-B domain of fibronectin, an extracellular matrix protein. Promising results demonstrated selective accumulation of the tracer in the tumour vasculature of a murine tumour model. Most of the studies are concentrated on the development of radiolabelled antagonists of the integrin alpha(v)beta(3). This heterodimeric transmembrane glycoprotein is involved in the migration of activated endothelial cells during formation of new vessels. Different compounds have been labelled with (18F), (111)In, (99m)Tc, (90)Y and several iodine isotopes. In in vitro assays most of them revealed high alpha(v)beta(3) affinity and selectivity. Moreover, in different murine tumour models successful non-invasive determination of alpha(v)beta(3) expression has been shown. Some of these approaches indicate that tumour-induced angiogenesis can be monitored in animal studies. Nevertheless, translation of these approaches into clinical settings allowing visualisation of tumour-induced angiogenesis in patients needs still to be demonstrated.

Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes.

Because olives represent an important component of the Mediterranean diet, it is necessary to establish unequivocal identification and quantitation of the major potential antioxidant phenolic compounds they contain. The major phenolic antioxidants in two types of brined olives were isolated and purified by semi-preparative high performance liquid chromatography. Structural analysis was conducted using UV spectrophotometry, mass spectrometry and nuclear magnetic resonance spectroscopy. In particular, completely assigned 1H and 13C NMR data are presented and errors in literature data are corrected. The data show that tyrosol, hydroxytyrosol, 3-(3, 4-dihydroxyphenyl) propanoic acid (dihydrocaffeic acid), dihydro-p-coumaric acid (phloretic acid), the phenylpropanoid glucosides acteoside (verbascoside) and isoacteoside, along with the flavonoids luteolin and apigenin are major components of the phenolic fraction of brined black olives. Brined green olives contain only hydroxytyrosol and traces of other minor phenolics. Brined olives contain even higher concentrations of phenolic antioxidants than olive oil and may, therefore, be more important modulators of cancer chemopreventive activity.

Tumor angiogenesis targeting using imaging agents.

The inhibition of tumor induced angiogenesis is an emerging therapeutic strategy in clinical oncology aimed at halting cancer progression by suppressing tumor blood supply. As anti-angiogenic therapy is primarily cytostatic and not cytotoxic, the established criteria for assessing tumor response to chemo- and radiotherapy cannot be applied to anti-angiogenic therapy. Therefore, functional and molecular parameters for imaging of tumor angiogenesis are being intensively studied. Computed tomography, magnetic resonance imaging, ultrasound and scintigraphic techniques can assess changes in vascular permeability and tumor blood flow during anti-angiogenic therapy. Scintigraphic techniques, especially positron emission tomography (PET), may be used to monitor the consequences of anti-angiogenic therapy on tumor cell metabolism, proliferation and apoptosis. The high sensitivity of PET which allows measurements of tracer concentrations in the picomolar range is promising for the visualization of specific molecular targets prior to therapy thus identifying patients most likely benefit from a particular form of anti-angiogenic therapy.

Comparison of [18F]FHPG and [124/125I]FIAU for imaging herpes simplex virus type 1 thymidine kinase gene expression.

Various radiotracers based on uracil nucleosides (e.g. [124I]2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil, [124I]FIAU) and acycloguanosine derivatives (e.g. [18F]9-[(3-fluoro-1-hydroxy-2-propoxy) methyl] guanine, [18F]FHPG) have been proposed for the non-invasive imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression. However, these radiotracers have been evaluated in different in vitro and in vivo models, precluding a direct comparison. Therefore, we directly compared [18F]FHPG and radioiodinated FIAU to assess their potential for PET imaging of transgene expression. The uptake of [125I]FIAU, [18F]FHPG and [3H]acyclovir was determined in vitro using four different HSV1-tk expressing cell lines and their respective negative controls. The in vitro tracer uptake was generally low in non-transduced parental cell lines. In HSV1-tk expressing cells, [3H]acyclovir showed approximately a twofold higher tracer accumulation, the [18F]FHPG uptake increased by about sixfold and the [125I]FIAU accumulation increased by about 28-fold after 120-min incubation of T1115 human glioblastoma cells. Similar results were found in the other cell lines. In addition, biodistribution and positron emission tomography (PET) studies with [18F]FHPG and [124/125I]FIAU were carried out in tumour-bearing BALB/c mice. Significantly higher specific accumulation of radioactivity was found for [125I]FIAU compared with [18F]FHPG. The ratio of specific tracer accumulation between [125I]FIAU and [18F]FHPG increased from 21 (30 min p.i.) to 119 (4 h p.i.). PET imaging, using [124I]FIAU, clearly visualised and delineated HSV1-tk expressing tumours, whereas only a negligible uptake of [18F]FHPG was observed. This study demonstrated that in vitro and in vivo, the radioiodinated uracil nucleoside FIAU has a significantly higher specific accumulation than the acycloguanosine derivative [18F]FHPG. This suggests that [124I]FIAU should be the preferred reporter probe for PET imaging of HSV1-tk gene expression. Thus, further attempts to develop suitable PET tracers for the assessment of HSV1-tk gene expression should also focus on 18F-labelled uracil derivatives.

Noninvasive imaging of alpha(v)beta3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography.

The alpha(v)beta3 integrin is an important cell adhesion receptor involved in tumor-induced angiogenesis and tumor metastasis. Here we describe the 18F-labeling of the RGD-containing glycopeptide cyclo(-Arg-Gly-Asp-D-Phe-Lys(sugar amino acid)-) with 4-nitrophenyl 2-[18F]fluoropropionate and the evaluation of this compound in vitro and in tumor mouse models. Binding assays with isolated immobilized alpha(v)beta3, alpha(v)beta5, and alpha(IIb)beta3 as well as in vivo studies using alpha(v)beta3-positive and -negative murine and xenotransplanted human tumors demonstrated receptor-specific binding of the radiolabeled glycopeptide yielding high tumor:background ratios (e.g., 120 min postinjection: tumor:blood, 27.5; tumor:muscle, 10.2). First imaging results using a small animal positron emission tomograph suggest that this compound is suitable for noninvasive determination of the alpha(v)beta3 integrin status and therapy monitoring.

Usefulness of intracoronary brachytherapy for in-stent restenosis with a 188Re liquid-filled balloon.

The objective of this randomized pilot trial with 21 patients was to evaluate the effectiveness of a rhenium-188 liquid-filled balloon system to prevent recurrent restenosis after percutaneous transluminal coronary angioplasty for in-stent restenosis. A significant benefit from brachytherapy was seen at 6-month repeat angiography, as well as during the clinical follow-up of 12 months.

Glycosylated RGD-containing peptides: tracer for tumor targeting and angiogenesis imaging with improved biokinetics.

UNLABELLED: The alpha(v)beta3 integrin plays an important role in metastasis and tumor-induced angiogenesis. Targeting with radiolabeled ligands of the alpha(v)beta3 integrin may provide information about the receptor status and enable specific therapeutic planning. Previous studies from our group resulted in tracers that showed alpha(v)beta3-selective tumor uptake. However, these first-generation compounds predominantly revealed hepatobiliary excretion with high radioactivity found in the liver. In this report, the synthesis and biological evaluation of the first glycosylated RGD-containing peptide (RGD-peptide) for the noninvasive imaging of alpha(v)beta3 expression are described. METHODS: Peptides were assembled on a solid support using fluorenylmethoxycarbonyl-coupling protocols. The precursor cyclo(-Arg-Gly-Asp-D-Tyr-Lys(SAA)-) GP1 was synthesized by coupling 3-acetamido-2,6-anhydro-4,5,7-tri-O-benzyl-3-deoxy-beta-D-glycero-D-gulo-heptonic acid (SAA(Bn3)) with cyclo(-Arg(Mtr)-Gly-Asp(OtBu)-D-Tyr(tBu)-Lys-) and subsequent removal of the protection groups. Iodine labeling was performed by the Iodo-Gen method (radiochemical yield > 50%). The in vitro binding assays were performed using purified immobilized alpha(IIb)beta3, alpha(v)beta5, and alpha(v)beta3 integrins. For in vivo experiments, nude mice bearing xenotransplanted melanomas and mice with osteosarcomas were used. RESULTS: The glycosylated peptide 3-iodo-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Lys(SAA)-) GP2 showed high affinity and selectivity for alpha(v)beta3 in vitro (50% inhibitory concentration = 40 nmol/L). Pretreatment studies indicate specific binding of [125I]GP2 on alpha(v)beta3-expressing tumors in vivo. Comparison of the pharmacokinetics of [125I]GP2 and [125I]-3-iodo-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) [125I]P2 revealed for [125I]GP2 an increased activity concentration in the blood (e.g., 3.59 +/- 0.35 percentage injected dose [%ID]/g vs. 1.72 +/- 0.44 %ID/g at 10 min postinjection) and a significantly reduced uptake in the liver (e.g., 2.59 +/- 0.24 %ID/g vs. 21.96 +/- 2.78 %ID/g at 10 min postinjection). Furthermore, a clearly increased activity accumulation in the tumor was found (e.g., 3.05 +/- 0.31 %ID/g vs. 0.92 +/- 0.16 %ID/g at 240 min postinjection), which remained almost constant between 60 and 240 min postinjection. This resulted in good tumor-to-organ ratios for the glycosylated tracer (e.g., 240-min postinjection osteosarcoma model: tumor-to-blood = 16; tumor-to-muscle = 7; tumor-to-liver = 2.5), which were confirmed by the first gamma-camera images of osteosarcoma-bearing mice at 240 min postinjection. CONCLUSION: This study demonstrates that the introduction of a sugar moiety improves the pharmakokinetic behavior of a hydrophobic peptide-based tracer. Additionally, this alpha(v)beta3-selective glycosylated radioiodinated second-generation tracer GP2 shows high tumor uptake and good tumor-to-organ ratios that allow noninvasive visualization of alpha(v)beta3-expressing tumors and monitoring therapy with alpha(v)beta3 antagonists. Finally, the favorable biokinetics make the glycosylated RGD-peptide a promising lead structure for tracers to quantify the alpha(v)beta3 expression using PET.

Uptake of radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil in cardiac cells after adenoviral transfer of the herpesvirus thymidine kinase gene: the cellular basis for cardiac gene imaging.

BACKGROUND: Gene therapy is a promising approach for the treatment of cardiac diseases. Coexpression of therapeutic genes with a suitable marker gene would allow for the noninvasive imaging of successful gene transfer and expression via radiolabeled marker substrates. In the present study, such an approach was first applied to cardiac tissue. METHODS AND RESULTS: The combination of the herpesvirus thymidine kinase reporter gene (HSV1-tk) and radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil (FIAU) was evaluated. H9c2 rat cardiomyoblasts were infected in vitro with a replication-defective HSV1-tk-containing adenovirus and a negative control virus. The intracellular uptake of [(14)C]FIAU increased with increasing multiplicity of infection and with time after infection. Uptake in negative controls remained <15% of positive controls. Additionally, vectors were applied intramyocardially in Wistar rats. The marker substrate [(125)I]FIAU was injected intravenously 3 days later, and animals were killed after 24 hours. Autoradiographically, regional transgene expression was clearly identified in animals receiving the adenovirus containing HSV1-tk (3. 4+/-2.2-fold increase of radioactivity at vector administration site compared with remote myocardium), whereas nonspecific uptake in negative controls was low (<10% of positive controls). CONCLUSIONS: Using an adenoviral vector, HSV1-tk can be successfully expressed in cardiac cells in vitro and in vivo, yielding high uptake of radiolabeled FIAU. The results suggest that imaging transgene expression in the heart is feasible and may be used to monitor gene therapy noninvasively.