Development of 99mTc(CO)₃-dendrimer-FITC for cancer imaging

Study of fluorophore and technetium labeling of poly(amido)-amine (PAMAM) generation 4 (G4) dendrimer and its evaluation as potential molecular imaging agent in both normal and melanoma-bearing mice, are described. Dendrimers were first conjugated with FITC (fluorescein isothiocyanate). Dendrimer-FITC was then incubated with the intermediate [(99m)Tc(CO)(3)(H(2)O)(3)](+) and purified by gel filtration. Biodistribution and scintigraphy images were performed administrating (99m)Tc(CO)(3)-dendrimer-FITC to normal mice (NM) or melanoma-bearing mice (MBM). Cryostat tissue sections from MBM mice were analyzed by confocal microscopy. Radiolabeling yield of dendrimer was approx. 90%. The (99m)Tc(CO)(3)-dendrimer-FITC complex was stable for at least 24h. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with high tumor uptake that allowed tumor imaging. Confocal microscopy analysis showed cytoplasmic distribution of (99m)Tc(CO)(3)-dendrimer-FITC.     

Synthesis and biological evaluation of 99mTc(CO)3(His–CB) as a tumor imaging agent

The chlorambucil l-histidine conjugate was synthesized and radiolabeled with [^99mTc(CO)₃]⁺ core to form the ^99mTc(CO)₃(His–CB) complex. The radiochemical purity of the complex was over 90%. It had good hydrophilicity and was stable at room temperature. The high initial tumor uptake with certain retention, fast clearance from background, good tumor/non-tumor ratios and satisfactory scintigraphic images highlighted the potential of ^99mTc(CO)₃(His–CB) as a tumor imaging agent.     

99mTc bone scanning agents—V. Influence of experimental conditions on the labeling efficiency and gel chromatography of 99mTc(Sn)HMDP

The preparation of ^99mTc(Sn)HMDP was investigated as a function of pH, Sn(II) and ligand concentration. HMDP could be labeled efficiently from pH 2–9. The Sn(II) and the ligand concentrations had a beneficial influence.The composition of the radiopharmaceutical under various experimental conditions was studied by means of gel chromatography on Biogel P-4. Six different complexes were found. A preparation consisted of maximally three major complexes. The presence of a particular complex was mainly determined by pH and ligand concentration. The Sn(II) concentration had little influence.     

Maillard Glycation of Peptides Containing the (N αHis)Ac Chelator for 99mTc(CO)3 Labeling

The retro-N ^α-carboxymethyl histidine moiety, short (N ^αHis)Ac, functions as an efficient chelator for the ^99mTc(CO)₃ core which allows the labeling of the peptides with a very high specific activity. However as a general consequence of the neutral [^99mTc(CO)₃] (N ^αHis)Ac-complex, undesired accumulation in kidney and liver may be high. In order to improve the pharmacokinetic properties of the radiolabeled peptides containing this chelate, attempts have been made to conjugate a carbohydrate using the Maillard reaction. Since the (N ^αHis)Ac moiety has an unusually reactive N ^α, various chemical strategies have been explored to perform the Maillard reaction followed by the Amadori rearrangement on peptides containing this chelator. This indicated that a selective protection of the secondary nitrogen in the chelator is necessary.Australian Peptide Conference Issue.     

Preparation of classical Re/99mTc(CO)3+ and novel 99mTc(CO)2(NO)2+ cores complexed with flavonol derivatives and their binding characteristics for Aβ(1–40) aggregates

Classical ^99mTc(CO)₃⁺ and novel ^99mTc(CO)₂(NO)²⁺ cores complexed with flavonol derivatives were prepared. Autoradiography of postmortem AD transgenic mice (Tg C57, APP, PS1 12-month-old) brain section confirmed the binding property of [^99mTc(CO)₃⁺-3-OH-flavone]⁰ to Aβ_(1–40) aggregates, while the novel ^99mTc(CO)₂(NO)²⁺ labeled compounds showed no binding sites in AD transgenic mice sections. Intravenous administration of [^99mTc(CO)₃⁺-3-OH-flavone]⁰ resulted in moderate brain uptake (0.48 ± 0.05%ID/g) at 5 min post-injection and slow clearance from the brain issues in 2 h post-injection (120 min: 0.39 ± 0.08%ID/g). Then an Aβ_(1–40)-receptor-targeted Re(CO)₃⁺-3-OH-flavone, was prepared to identify the structure of the technetium complex. UV–vis absorption and fluorescence emission properties have been studied at room temperature in order to determine the natures of the lowest electronically excited states of Re(CO)₃⁺-3-OH-flavone and the ligand. The fluorescent rhenium complex Re(CO)₃⁺-3-OH-flavone showed high affinity for Aβ_(1–40) aggregates in vitro by fluorescence spectra (dissociation constant (K_d) = 11.16 nM). In conclusion, the results suggested that ^99mTc(CO)₃⁺-3-OH-flavone should be a suitable candidate as Aβ plaque SPECT imaging agent for AD.     

Radioactive synthesis and biodistribution study of β-elemene–99mTc(CO)3 conjugates

β-Elemene, (5S,7R,10S)-(−)-(1-methyl-1-vinyl-2,4-diisopropenylcyclohexane), is an anticancer agent from traditional Chinese herbal medicine. Three novel ^99mTc(CO)₃–β-elemene conjugates were synthesized successfully, and compared with β-elemene exhibited improved water solubility. A biodistribution and micro single photon emission computed tomography image study showed there is a visible accumulation in Lewis lung cancer tumors. Y. Sun and Y. Ren contributed equally to this paper.     

Evaluation of a DMSA kit for instant preparation of 99mTc(V)—DMSA for tumour and metastasis scintigraphy

A kit has been developed to instantly prepare ^99mTc(V)—DMSA. The freeze-dried kit consisting of DMSA, stannous chloride and ascorbic acid in appropriate proportions, produces quality ^99mTc(V)—DMSA when mixed with 0.2 mL of 3.5% NaHCO₃ solution and 2–4 mL of [^99mTc] pertechnetate. The radiopharmaceutical characterized by chromatography with ITLC-SG in 0.9% saline and horizontal paper electrophoresis in 50 mM vernol buffer, pH 8.6, at a potential gradient of 15 V/cm showed a different mobility with respect to ^99mTc(III)-DMSA, a known agent for kidney imaging. The new agent exhibited less plasma protein binding as compared to that of ^99mTc(III)-DMSA. Biodistribution of the pentavalent DMSA in mouse demonstrated greater uptake in bone and muscle and lower uptake in liver and kidney with respect to trivalent DMSA. The soft tissue tumour specificity and its suitability for tumour scintigraphy was apparent from the scintigrams of mammary carcinoma in a C₃H Jax mouse and medullary carcinoma in a patient. Brain metastatic lesions were also visible in a breast carcinoma patient after administering him with the agent.     

Technetium-99m labeled renal function and imaging agents: I. Clinical evaluation of 99mTc CO2-DADS-A (99mTc N,N′-Bis-(Mercaptoacetyl)-2,3-Diaminopropanoate)

Animal experiments and preliminary clinical results showed that the N₂S₂-complex ^99mTc CO₂-DADS-A, which was claimed to be a potential replacement for o-I-hippurate as a renal function agent, had a lower affinity for the tubular transport system than o-1-hippurate.In order to evaluate if this finding offered the possibility of detecting decreases in tubular function with more sensitivity, or at earlier times, 6 patients in the early post-transplantation period were subjected to 53 simultaneous scintigraphic investigations with ¹³¹I o-I-hippurate and ^99mTc CO₂-DADS-A. The comparison of the renograms obtained with the respective agents showed that in almost all cases of acute graft rejection only o-I-hippurate yielded the typical, diagnostically useful accumulation curve which results from its high retention in the kidney parenchyma. ^99mTc CO₂-DADS-A did not reveal this effect.Additionally the plasma clearance of each agent was measured simultaneously under steady state conditions in nine patients. Although it was reported that relative to o-I-hippurate the analog images obtained with ^99mTc CO₂-DADS-A gave higher kidney-to-background ratios and the amount excreted in the urine at 30 min was slightly less, the clearance values obtained for ^99mTc CO₂-DADS-A were on average only 36% of those for o-I-hippurate.It is concluded that ^99mTc CO₂-DADS-A is not suitable as a substitute for o-I-hippurate.     

Multicultural society and everyday cultural racism: second generation of Ethiopian Jews in Israel's ‘crisis of modernization’

Abstract

The core of this article sets out to examine the extent to which a multicultural society can prevent cultural racism, which, like multiculturalism, is by definition based on a culture of diversity and separation. The ‘first modernity’ was organized along national lines, with a centralist state that opted to create an essentialist and uncontested national identity. Immigrants, especially those who came from ‘third world’ countries, were expected to undergo a process of assimilation, and to integrate into the dominant culture by relinquishing their particular past and tradition. Multiculturalism, which emerged historically as a criticism of that perspective, aims at creating a kaleidoscope of associations and cultural communities, which inevitably presents a challenge to the one ‘truth’ of the nation-state with the argument that this ‘truth’ favours some groups over others. Within the multicultural model, identity politics of various groups is perceived as a means to achieve recognition, acceptance, respect and even public affirmation of differences. However, do multicultural society and identity-related differences provide a solution to cultural racism as well?

Investigating the second generation of the Ethiopian Jews, who migrated to Israel during its transformation from ethno-national republicanism to a neo-liberal, multicultural society, can help answer this question. By presenting their patterns of association, character of protest activities and the newly formed hybrid identity that Ethiopian youth have developed as a means to liberate themselves from a discriminating reality, and by examining the Others’ reaction to that challenge, this article uncovers certain mechanisms and methods of action through which a multicultural society, having a thin and mild version of multiculturalism, does not diminish cultural racism, particularly its everyday non-institutional version, but in fact augments it.     

Partial correction of defective generation of lymphokine-activated killer cells in patients with chronic myelogenous leukaemia after in vivo treatment with interferon-α (wellferon)

Summary Patients with chronic myelogenous leukaemia (CML) in untreated chronic phase are deficient in their ability to generate lymphokine-activated killer (LAK) cells from peripheral blood mononuclear cells although they posses essentially normal levels of CD16⁺ and Leu19⁺ lymphocytes, which do not seem to be actively suppressed by tumour cells. Attempts to enhance LAK cell generation in these patients are reported here. Combining the lymphokines interleukins-2, with -4 and -5 (IL-2, IL-4, IL-5), was not successful; in fact, IL-4 depressed LAK cell induction in both normal donors and CML patients. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also failed to enhance cytotoxicity of normal donors or patients, and indomethacin was similarly without effect. The only agent found to enhance LAK cell induction by IL-2 in normal donors was interferon- (but not IFN-) and even this modest effect was not seen with the cells of CML patients. Increasing concentrations of IL-2 and/or culture duration also failed to improve LAK cell generation by patients. The only improvement in LAK cell generation was observed in CML patients treated for one or more months with IFN-, where a steady increase of LAK activity with time after initiation of therapy was noted. These results show that the blockade of LAK cell induction in chronic-phase myelogenous leukaemia patients is difficult to lift pharmacologically in vitro but possibly susceptible to biological response modifiers in vivo.     

Melanoma targeting with α-melanocyte stimulating hormone analogs labeled with fac-[99mTc(CO)3]+: effect of cyclization on tumor-seeking properties

Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled α-melanocyte stimulating hormone (α-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized α-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of α-MSH (Ac-Nle-cyclo[Asp-His-dPhe-Arg-Trp-Lys]-NH₂]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of α-MSH analogs by comparing the pharmacokinetics profile of the ^99mTc-labeled cyclic peptide βAla-Nle-cyclo[Asp-His-d-Phe-Arg-Trp-Lys]-NH₂ with that of the linear analog βAla-Nle-Asp-His-dPhe-Arg-Trp-Lys-NH₂ in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of βAla, and the resulting pz–peptide conjugates were reacted with the fac-[^99mTc(CO)₃]⁺ moiety. The ^99mTc(CO)₃-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic ^99mTc(CO)₃-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 °C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 ± 0.83 and 11.31 ± 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear ^99mTc(CO)₃-pz–peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle⁴,dPhe⁷)-αMSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h after injection for cyclic and linear radioconjugates, respectively).     

Rhodamine-based ‘turn-on’ fluorescent probe for Cu(II) and its fluorescence imaging in living cells

A novel rhodamine spirolactam derivative 3′,6′-Bis(diethylamino)-2-(2-hydroxyethylamino) spiro[isoindoline-1,9′-xanthen]-3-one (RO1) was synthesized, and characterized by high-resolution mass spectrometry (HRMS), X-ray crystallography, Infrared spectroscopy (IR), and ¹H NMR and ¹³C NMR spectroscopy. RO1 exhibited highly sensitive and exclusively selective fluorescence response toward Cu²⁺ over other metal ions with a detection limit of 0.56 ppb in mixed aqueous solution. The fluorescence was pH-independent in the wide range pH 3.1–11.6. The turn-on fluorescence enhancement of the probe is based on Cu²⁺ induced ring-opening mechanism of the rhodamine spirolactam. Moreover, by means of fluorescence microscopy experiments, it was demonstrated that RO1 could monitor trace Cu²⁺ changes by live cell imaging.     

Derivatization of (±) dihydrotetrabenazine for copper-64 labeling towards long-lived radiotracers for PET imaging of the vesicular monoamine transporter 2

Dihydrotetrabenazine (DTBZ) derivatized from (+) Tetrabenazine (TBZ) has been used for imaging the expression of VMAT2 when labeled with ¹¹C (t_1/2 = 20.3 min) or ¹⁸F (t_1/2 = 110 min) in neurodegenerative diseases or pancreatic beta-cell. Because ¹¹C or ¹⁸F radiolabels are only available in the proximity of a biomedical cyclotron facility, here we report our work of derivatizing (+) and (−) DTBZ using a ⁶⁴Cu-specific bifunctional chelator scaffold (⁶⁴Cu: t_1/2 = 12.7 h) for the preparation of long-lived VMAT2 targeted radiotracers, ⁶⁴Cu-CB-TE2A-(+)-DTBZ and ⁶⁴Cu-CB-TE2A-(−)-DTBZ. The specific VMAT2 binding affinity of ⁶⁴Cu-CB-TE2A-(+)-DTBZ measured using rat brain homogenate or porcine islets was not compromised by our chemical modifications while that of its (−) counterpart remained low as in ¹¹C or ¹⁸F labeled (±) DTBZ.     

Natural fermentation of soybean (glycine max) with added salt for ‘soy-daddawa’ production

Microbial growth, pH and titratable acidity decreased with increasing NaCl concentration during the fermentation of soybeans. NaCl at 1% (w/w) in the fermenting mash improved the organoleptic quality of soy-daddawa.The author is with the Department of Microbiology, Obafemi Awolowo University, lle-lfe, Nigeria.     

A caspase-3 ‘death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical ‘death-switch'' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [¹⁸F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The ‘death-switch'' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of ‘death-switched'' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [¹⁸F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [¹⁸F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.     

Application of 2,4,5-tris(2-pyridyl)imidazole as ‘turn-off’ fluorescence sensor for Cu(II) and Hg(II) ions and in vitro cell imaging

The 2,4,5-tris(2-pyridyl)imidazole ( L ) molecule has been evaluated as a probe for dual sensing of Hg²⁺ and Cu²⁺ ions in EtOH/HEPES buffer medium (5 mM, pH = 7.34, 1:1, v/v). Probe L shows a good sensitive and selective turn-off response in the presence of both Hg²⁺ and Cu²⁺ ions, which is comprehensible under long UV light. The probe can detect Cu²⁺ ion in the pH range 3–11 and Hg²⁺ ion in pH 6–8. The limit of detection for Cu²⁺ (0.77 μM) is well under the allowable limit prescribed by the United States Environmental Protection Agency. Two metal (Cu²⁺/Hg²⁺) ions are needed per L for complete fluorescence quenching. The probe shows marked reversibility on treatment with Na₂EDTA, making the protocol more economical for practical purposes. Paper strip coated with the L solution of EtOH can detect the presence of Cu²⁺ and Hg²⁺ ions in the sample using visible quenching of the fluorescence intensity. Density functional theory–time-dependent density functional theory (DFT–TDDFT) calculations support experimental observations, and d-orbitals of Cu²⁺/Hg²⁺ provide a nonradiative decay pathway. Cell imaging study using HDF and MDA-MB-231 cells also supported the viability of L in detecting Cu²⁺ and Hg²⁺ ions in living cells.     

Experiences with the use of a starter culture in the fermentation of maize for ‘kenkey’ production in Ghana

Controlled fermentation of maize was carried out using six strains of Lactobacillus fermentum and one strain of yeast, Saccharomyces cerevisiae, isolated from traditionally fermented maize dough as starter cultures for inoculum enrichement. The fermentations were monitored by pH, acidity, microbiological analysis and taste panel evaluation of two products, kenkey and koko, prepared from the fermented doughs. The strains of L. fermentum used as starter culture dominated the microflora during fermentation and in most inoculated doughs the required pH was attained by 24 h instead of 48 h of dough fermentation. Higher contents of lactic acid bacteria and yeasts were observed in inoculated doughs at the initial stages of fermentation but the spontaneously fermented doughs attained similar lactic acid bacteria and yeasts counts by 24 h of dough fermentation. The organoleptic quality of kenkey and koko prepared from doughs fermented with starter culture for 48 h was not significantly different from the traditional products. Kenkey prepared from doughs fermented for 24 h with starter culture were found to be unacceptable by the taste panel although similarly produced koko was acceptable.The authors are with the Food Research Institute, Council for Scientific and Industrial Research, P.O Box M 20. Accra, Ghana.     

Multimeric system of 99mTc-labeled gold nanoparticles conjugated to c[RGDfK(C)] for molecular imaging of tumor α(v)β(3) expression

Integrin α(V)β(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic RGD peptides can be used for noninvasive imaging of α(V)β(3) expression. The aim of this research was to prepare a multimeric system of technetium-99m-labeled gold nanoparticles conjugated to c[RGDfK(C)] and to evaluate its biological behavior as a potential radiopharmaceutical for molecular imaging of tumor angiogenesis. Hydrazinonicotinamide-GGC (HYNIC-GGC) and c[RGDfK(C)] peptides were synthesized and conjugated to gold nanoparticles (AuNP, 20 nm) by means of spontaneous reaction of the thiol groups of cysteine. The nanoconjugate was characterized by TEM, FT-IR, UV-vis, XPS, and Raman spectroscopy. To obtain (99m)Tc-HYNIC-GGC-AuNP-c[RGDfK(C)] ((99m)Tc-AuNP-RGD), the (99m)Tc-HYNIC-GGC radiopeptide was first prepared and added to 1.5 mL of AuNP solution (1 nM) followed by c[RGDfK(C)] (10 μL, 50 μM) at 18 °C with stirring for 15 min. Radiochemical purity (RP) was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in α(V)β(3) receptor-positive C6 glioma cancer cells. Biodistribution studies were accomplished in athymic mice with C6-induced tumors with blocked and nonblocked receptors, and images were obtained using a micro-SPECT/CT. TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with peptides. RP was 96 ± 2% without postlabeling purification. (99m)Tc-AuNP-RGD showed specific recognition for α(V)β(3) integrins expressed in C6 cells, and 3 h after i.p. administration in mice, the tumor uptake was 8.18 ± 0.57% ID/g. Micro-SPECT/CT images showed evident tumor uptake. (99m)Tc-AuNP-RGD demonstrates properties suitable for use as a target-specific agent for molecular imaging of tumor α(V)β(3) expression.     

The mechanism of apoptosis,cell membrane lipid peroxidation and a novel in vivo function for antioxidant vitamin E (α-tocopherol)

A critical novel function for the antioxidant vitamin E (α-tocopherol) may be its involvement in the control of apoptosis in which it acts as a ‘gate keeper’ in the regulation of membrane lipid peroxidation. The biochemical and biophysical antioxidant properties of the molecule are discussed together with recent evidence for its involvement in a possible model of the apoptotic mechanism. The original observation that led to the discovery of vitamin E (foetal resorption in pregnant rats fed on tocopherol-deficient diets) is considered as an unrecognised example of apoptosis.