Milan Laznicek

Comparison of biological characteristics of EDTMP complexes with 99mTc, 111In and 153Sm in rats

The pharmacokinetics and elimination of EDTMP chelates with different radionuclides (99mTc, 111In and 153Sm) has been investigated in rats. The biodistribution of the complexes under study was similar and two main processes, namely bone uptake and the elimination of glomerular filtration, take part in their rapid blood clearance. A substantially slower blood clearance of 111In-EDTMP in comparison with the other complexes suggests partial indium exchange between the chelate and transferrin. All the complexes exhibited high affinity for bone and the radionuclide uptake into the skeleton was in the order 153Sm > 111In > 99mTc. No specific extra-skeletal uptake was found.

The effect of lipophilicity on the protein binding and blood cell uptake of some acidic drugs.

Quantitative relationships between lipophilicity (characterized by the octanol-water partition coefficient) and binding to both human plasma proteins and blood cells have been studied in a group of model anionic drugs (benzoic and phenylacetic acid derivatives). Protein binding in plasma and accumulation in blood cells in suspension increases with increasing lipophilicity. For quantitative evaluation, the equation log R = a + b log D has been employed, where R is the bound-to-free drug ratio, D is lipophilicity, and a and b are constants. Whereas the protein bound-to-free drug ratio is proportional to drug lipophilicity, the cell bound-to-free drug ratio correlates with lipophilicity to the power 0.685. Distribution in whole blood is affected by protein binding and also by cell accumulation. In blood, the free drug fraction and the fraction in blood cells decrease with increasing lipophilicity, whereas the protein-bound fraction correspondingly increases.

Protein interactions with HER-family receptors can have different characteristics depending on the hosting cell line

Cell lines are common model systems in the development of therapeutic proteins and in the research on cellular functions and dysfunctions. In this field, the protein interaction assay is a frequently used tool for assessing the adequacy of a protein for diagnostic and therapeutic purposes. In this study, we investigated the extent to which the interaction characteristics depend on the choice of cell line for HER-family receptors. The interaction characteristics of two therapeutic antibodies (trastuzumab and cetuximab) and one Affibody molecule (ZHER2:342), interacting with the intended receptor were characterized with high precision using an automated real-time interaction method, in different cell lines (HaCaT, A431, HEP-G2, SKOV3, PC3, DU-145). Clear differences in binding affinity and kinetics, up to one order of magnitude, were found for the interaction of the same protein binding to the same receptor on different cells for all three proteins. For HER-family receptors, it is therefore important to refer to the measured affinity for a protein-receptor interaction together with the hosting cell line. The ability to accurately measure affinity and kinetics of a protein-receptor interaction on cell lines of different origins may increase the understanding of underlying receptor biology, and impact the selection of candidates in the development of therapeutic or diagnostic agents.

Pharmacokinetics and plasma protein binding of two platinum cytostatics CHIP and CBDCA in rats.

SummaryPlasma protein binding and pharmacokinetic parameters of CHIP (cis-dichloro-trans-dihydroxy-bisisopropylamine platinum IV) and CBDCA (cis-diammine-1,1-cyclobutane dicarboxylate platinum II) were investigated in male Wistar rats. The plasma clearance of total and non-protein-bound platinum was determined and compared with that of 99mTc-DTPA. for binding experiments, a novel, simple, and quick method based on adsorption of non-protein-bound platinum species to charcoal was used. The clearance of total platinum after CHIP and CBDCA administration was markedly lower than the glomerular filtration rate (determined as the clearance of 99mTc-DTPA). The renal clearance of non-proteinbound platinum corresponded to 168% and 50% of the glomerular filtration rate for CHIP and CBDCA, respectively. These studies suggested that CHIP was excreted by the rat kidney.

2,3-Dihydro-1H-cyclopenta[b]quinoline Derivatives as Acetylcholinesterase Inhibitors—Synthesis, Radiolabeling and Biodistribution

In the present study we describe the synthesis and biological assessment of new tacrine analogs in the course of inhibition of acetylcholinesterase. The obtained molecules were synthesized in a condensation reaction between activated 6-BOC-hydrazinopyridine-3-carboxylic acid and 8-aminoalkyl derivatives of 2,3-dihydro-1H-cyclopenta[b]quinoline. Activities of the newly synthesized compounds were estimated by means of Ellman’s method. Compound 6h (IC50 = 3.65 nM) was found to be most active. All obtained novel compounds present comparable activity to that of tacrine towards acetylcholinesterase (AChE) and, simultaneously, lower activity towards butyrylcholinesterase (BChE). Apart from 6a, all synthesized compounds are characterized by a higher affinity for AChE and a lower affinity for BChE in comparison with tacrine. Among all obtained molecules, compound 6h presented the highest selectivity towards inhibition of acetylcholinesterase. Molecular modeling showed that all compounds demonstrated a similar binding mode with AChE and interacted with catalytic and peripheral sites of AChE. Also, a biodistribution study of compound 6a radiolabeled with 99mTc was performed.

Radiolabeling of PAMAM dendrimers conjugated to a pyridine-N-oxide DOTA analog with 111In: Optimization of reaction conditions and biodistribution

Polyamidoamine dendrimers (PAMAMs) of generations 1 (G1) and 4 (G4) were conjugated with a bifunctional pyridine-N-oxide DOTA analog, 10-[(4-carboxy-1-oxidopyridin-2-yl)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (H(4)do3a-py(NO-C)), through the pyridine-4-carboxylic acid group, and the conjugates were radiolabeled with indium-111. Reaction conditions for the radiolabelling were optimized. Both radiolabeled conjugates, G1-[(111)In(do3a-py(NO-C))] and G4-[(111)In(do3a-py(NO-C))], were kinetically stable for at least 48h after preparation; in the presence of competitive ligands, the radiochemical purity of the conjugates slightly decreased (4-7%) over the same time period. The preclinical pharmacokinetics of both agents were evaluated. Biodistribution and elimination in rats were more favorable for the G1-[(111)In(do3a-py(NO-C))] conjugate than G4-[(111)In(do3a-py(NO-C))] conjugate. However, the G1-[(111)In(do3a-py(NO-C))] conjugate was rapidly eliminated from the body, mainly through urine, while, significant and long-term radioactivity uptake in the liver and kidney was observed for the G4-[(111)In(do3a-py(NO-C))] conjugate.

Plasma protein binding-lipophilicity relationships: interspecies comparison of some organic acids

Relationships between plasma protein binding of 11 organic acids (benzoic and phenylacetic acid derivatives) and their lipophilicity were studied in man, rabbits, rats and mice. For description, the relationship fu = 1/(1 + aDb) was developed, where fu is the fraction of the unbound drug in plasma, D is the partition coefficient octanol/water and a and b are parameters. While the value of the parameter a is widely different in interspecies comparison, the value of the parameter b is very close in all species studied and is approximately equal to 1. The model used allows the simple calculation of the extent of plasma binding of structurally similar drugs from their lipophilicity, or conversion of the extent of plasma binding from one species to another.

Octreotide and Octreotate Derivatives Radiolabeled with Yttrium: Pharmacokinetics in Rats

Distribution profiles and elimination pathways in rats of two new octreotate derivatives radiolabeled with yttrium, namely Y-DOTAGA-tate and Y-DOTA-t-GA-tate, were compared with those of Y-DOTA-octreotide and Y-DOTA-Tyr(3)-octreotide. All synthetic somatostatin analogues under study were rapidly cleared from the blood and most organs of rats. The main elimination pathway for all peptides under study was urine excretion. High and long-term uptakes of radioactivity in the kidneys and also in organs with high density of somatostatin receptors (the adrenals and pancreas) were found. Radioactivity concentrations in these somatostatin receptor-rich organs were substantially higher for octreotate derivatives in comparison with octreotide analogues; the highest values for Y-DOTAGA-tate were determined. The octreotate derivatives under study appear to be specific ligands for treatment of somatostatin receptor-positive tumors if some mechanism to decrease their kidney retention is provided.

Preclinical Evaluation of 177Lu-Nimotuzumab: A Potential Tool for Radioimmunotherapy of Epidermal Growth Factor Receptor–Overexpressing Tumors

BACKGROUND The humanized monoclonal antibody Nimotuzumab (h-R3) has demonstrated an exceptional and better clinical profile than other monoclonal antibodies for immunotherapy of epidermal growth factor receptor-overexpressing tumors. This work deals with the preparation and radiolabeling optimization of (177)Lu-Nimotuzumab and their preclinical evaluation. METHODS Nimotuzumab was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), testing different molar ratios. The immunoconjugates were characterized. The radiolabeling with (177)Lu was optimized. Radioimmunoconjugates stability was tested in 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA) excess and human serum. In vitro studies were performed in tumor model cell lines. Receptor-specific binding was tested by competitive inhibition. (177)Lu-Nimotuzumab in vivo studies were conducted in healthy and xenograft animals. RESULTS Nimotuzumab conjugates were obtained with high purity. Radiolabeling yield and specific activities ranged from 63.6% to 94.5% and from 748 to 1142 MBq/mg, respectively. The stability in DTPA excess and human serum was 95.9% and 93.2% after 10 days, respectively. The radioimmunoconjugate showed specific receptor binding in tumor cell lines. Biodistribution in healthy animals showed the typical behavior of the immunoconjugates based on monoclonal antibodies. The study in xenografts mice demonstrated uptake of (177)Lu-Nimotuzumab in the tumor and reticuloendothelial organs. CONCLUSIONS (177)Lu-Nimotuzumab was obtained with high purity and specific activities under optimal conditions without significant loss in immunoreactivity and might be a potential radioimmunoconjugate for radioimmunotherapy of tumors with epidermal growth factor receptor overexpression.

The effect of molecular weight on the biodistribution of hyaluronic acid radiolabeled with111In after intravenous administration to rats

SummaryHyaluronic acid (HA), is a high molecular weight (HMW) glucosaminoglycan with significant acitivity, and which influences a number of physiological and pathological processes such as tumorogenesis, arthritis, etc. The aim of this study was to determine the difference in the biodistributional pathways of111in-labeled diethylenetriaminepentaacetic acid-hyaluronic acid (111In-DTPA-HA) molecule of three different MWs (10, 100 and 450 kDA) in a rat model, and to determine possible relationships between the biodistribution and the MW of the investigated agent for future medical applications.111In-DTPA-HA was prepared by mixing activated DTPA and activated HA, then adding111InCl3 to the previously prepared mixture at pH 5,5 in an acetic buffer. Biodistributional studies were performed using 36 male Wistar rats aged 2 months and weighing 280–350 g. The radioactivity in the samples was measured via a radiometer and the radioactivity in the different organs, blood, plasma and urine was determined. It was found that 50–54% for 10 and 100 kDa and 80% for 450 kDa of the administered dose of radiolabel was present in the liver after 5min. Other organs show no significant increase during the experimental period. The elimination of the radiolabel was mostly renal and in low molecular weight (LMW) form. Radioactivity remained in liver throughout the 72h experimental period. A difference in the biodistribution of 450 kDa and LMW radiolabeled molecules was found. Higher amounts of radiolabel were taken up by the liver when the 450 kDa molecule was used. LMW fractions were found in the urine, and could have been a product of non-enzymatic cleavage. The extended retention of radiolabel in the liver could be related to changes in the polarity of DTPA-HA molecules.