Andrzej Czerwinski

Identification of the structural elements of amphotericin B and other polyene macrolide antibiotics of the hepteane group influencing the ionic selectivity of the permeability pathways formed in the red cell membrane

The selectivity of the transmembrane permeability induced by polyene antibiotics was studied in human erythrocytes and related to the hemolytic potency of the drugs. The selectivity induced was differently, dependent on the antibiotic structure in aromatic (vacidin A, gedamycin) and nonaromatic heptaenes (amphotericin B, candidin). Aromatic heptaenes were more effective than nonaromatic in inducing permeability to K+. For both groups of antibiotics, permeability to K+ was not affected by substitution at the carboxyl group but important differences in the induction of permeability to H+, OH- and Cl- were found. The strongly hemolytic aromatic heptaenes vacidin A and gedamycin exhibited much higher protonophoric activity than the nonaromatic ones: amphotericin B, and candidin. The protonophoric properties of aromatic heptaenes were related to the presence of a free carboxyl group in the antibiotic molecule. Indeed the esterification or amidation of the carboxyl group of vacidin A or gedamycin eliminated the ability of the antibiotic to increase H+ conductance and consequently diminished their hemolytic activity to an important extent. Both groups of antibiotics differed also in the efficiency of anion permeability induction. Only unsubstituted aromatic heptaenes, at high concentration, induced Cl-/OH- exchange and conductive flux of Cl- in a concentration-dependent manner. Substitution at the carboxyl group of vacidin A or gedamycin eliminated this property. Amphotericin B as well as its carboxyl-substituted derivatives formed a pathway characterized by low K+ over Cl- selectivity, whatever the concentration. The hemolytic activity, related to K+ permeability increased by heptaenes was dependent on simultaneous increase of the permeability to anions, and net KCl influx. Carboxyl-substituted derivatives of aromatic heptaenes presenting a remarkably high selectivity for K+, had consequently a very poor hemolytic activity.

FITC-Conjugated Cyclic RGD Peptides as Fluorescent Probes for Staining Integrin αvβ3/αvβ5 in Tumor Tissues

This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against 125I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 106 tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1–0.3 g. Tumors were harvested for integrin αvβ3/αvβ5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvβ3/αvβ5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvβ3/αvβ5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin β3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of 99mTc-3P-RGD2 (an integrin αvβ3/αvβ5-targeted radiotracer) and the relative integrin αvβ3/αvβ5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvβ3/αvβ5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvβ3/αvβ5 expression levels in tumor tissues.

Thermal behaviour and stability of amphotericin B

Abstract Thermoanalytical methods (DTA, TG, DTG) and other techniques (UV-VIS spectroscopy, thin-layer chromatography, biological assay) have been employed to examine the thermal behaviour of the polyene antibiotic amphotericin B. Heated to 410 K, in either argon or self-generating atmospheres, amphotericin B does not undergo chemical changes and does not lose biological activity. However, the compound decomposes completely on heating up to 720 K and this process is accompanied by a partial volatilization of light products. The thermal decomposition pattern is markedly affected by the nature of the gaseous phase.

Synthesis, Chemical Characterization and Multiscale Biological Evaluation of a Dimeric-cRGD Peptide for Targeted Imaging of α V β 3 Integrin Activity

Cyclic peptides containing the Arg-Gly-Asp (RGD) sequence have been shown to specifically bind the angiogenesis biomarker αVβ3 integrin. We report the synthesis, chemical characterization, and biological evaluation of two novel dimeric cyclic RGD-based molecular probes for the targeted imaging of αVβ3 activity (a radiolabeled version, 64Cu-NOTA-PEG4-cRGD2, for PET imaging, and a fluorescent version, FITC-PEG4-cRGD2, for in vitro work). We investigated the performance of this probe at the receptor, cell, organ, and whole-body levels, including its use to detect diabetes associated impairment of ischemia-induced myocardial angiogenesis. Both versions of the probe were found to be stable, demonstrated fast receptor association constants, and showed high specificity for αVβ3 in HUVECs (Kd ~ 35 nM). Dynamic PET-CT imaging indicated rapid blood clearance via kidney filtration, and accumulation within αVβ3-positive infarcted myocardium. 64Cu-NOTA-PEG4-cRGD2 demonstrated a favorable biodistribution, slow washout, and excellent performance with respect to the quality of the PET-CT images obtained. Importantly, the ratio of probe uptake in infarcted heart tissue compared to normal tissue was significantly higher in non-diabetic rats than in diabetic ones. Overall, our probes are promising agents for non-invasive quantitative imaging of αVβ3 expression, both in vitro and in vivo.