Dendrimers: Analytical characterization and applications.

This review focuses on analytical techniques used for separation and characterization of dendrimers and their derivatives. These macromolecules have been attractive material for a development of new drug carriers and imaging agents. They are also interesting for many biological and industrial applications. The review mentions a few of them.

The effect of molecular weight on the biodistribution of hyaluronic acid radiolabeled with 111In after intravenous administration to rats.

Hyaluronic 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 of 111In-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 adding 111InCl3 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 5 min. 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.

The involvement of selected membrane transport mechanisms in the cellular uptake of (177)Lu-labeled bombesin, somatostatin and gastrin analogues.

INTRODUCTION: Radiolabeled receptor-targeting peptides are a useful tool for the diagnostic imaging and radiotherapy of some malignancies. However, the retention of radioactivity in the kidney may result in renal radiotoxic injury. This study seeks to evaluate the role of endocytic receptor megalin, renal SLC influx transporters and fluid phase endocytosis (FPE) in the cellular accumulation of radiolabeled peptides. METHODS: In vitro transport cellular studies using megalin ligands (RAP, albumin), fluid phase endocytosis (FPE) inhibitor rottlerin and low temperature were employed to evaluate the transport mechanisms of the peptides. Cells transfected with hOAT1 or hOCT2 were used to analyze the role of these SLC transporters. Somatostatin ((177)Lu-DOTA-[Tyr(3)]octreotate, (177)Lu-DOTA-[1-Nal(3)]octreotide), gastrin ((177)Lu-DOTA-sargastrin) and bombesin ((177)Lu-DOTA-[Pro(1),Tyr(4)]bombesin, (177)Lu-DOTA-[Lys(3)]bombesin, (177)Lu-PCTA-[Lys(3)]bombesin) analogues were involved in the study. RESULTS: RAP, albumin and low temperature decreased the accumulation of all the studied peptides significantly. With one exception, rottlerin caused the concentration dependent inhibition of the cellular accumulation of the radiopeptides. No significant differences in the uptake of the peptides between the control cells and those transfected with hOAT1 or hOCT2 were observed. CONCLUSION: The study showed that active transport mechanisms are decisive for the cellular accumulation in all tested (177)Lu-labeled somatostatin, gastrin and bombesin analogues. Besides receptor-mediated endocytosis by megalin, FPE participates significantly in the uptake. The tested types of renal SLC transporters are not involved in this process.

Pharmacokinetics and renal handling of 99mTc-labeled peptides.

UNLABELLED: 99mTc-labeled peptides, particularly those of a lipophilic nature, are often excreted through the hepatobiliary system, and the subsequent accumulation in the intestine may obscure receptor-mediated uptake in tumor sites in the pelvis. We have therefore explored the route and rate of excretion of a small series of Tc-labeled peptides to shed some light on the mechanisms that influence the clearance of these agents. METHODS: Pharmacokinetic parameters, biodistribution, routes of elimination of 99mTc-complexes of 3 model tetrapeptides--namely, acetyl-N-Gly-Gly-Cys-Gly (AGGCG), acetyl-N-Ser-Ser-Cys-Gly (ASSCG), and acetyl-N-Gly-Gly-Cys-Lys (AGGCL)--were determined in rats in vivo. Renal handling of the complexes was studied in the perfused rat kidney. RESULTS: After intravenous injection, a relatively fast disappearance of the complexes from blood was found. Although the parameters of distribution in all 3 chelates were very similar, the elimination rate of 99mTc-AGGCG was higher than those of 99mTc-ASSCG and 99mTc-AGGCL. The Tc complexes under study were distributed mainly to the excretory organs (kidneys and liver), and no specific accumulation in other organs or tissues was found. Most of the radioactivity after intravenous administration of the chelates was rapidly eliminated through the urine, but a significant amount was also excreted through the feces, in the following order among the 3 chelates: 99mTc-AGGCL < 99mTc-ASSCG < 99mTc-AGGCG. Different proportions of glomerular filtration and secretion in renal tubules of the complexes were found in the perfused rat kidney. Elimination by glomerular filtration was dominant only in the case of 99mTc-AGGCL, whereas the rate of filtration of 99mTc-AGGCG was very low because of its high protein binding. Various rates of secretion into renal tubules were shown for all 3 agents. This renal excretion pathway was decisive in 99mTc-AGGCG and lowest in 99mTc-AGGCL. 99mTc-ASSCG was eliminated by both mechanisms at similar rates. CONCLUSION: These studies show that increasing the hydrophilic nature or reducing the negative charge of the peptides will reduce their hepatobiliary excretion, whereas the incorporation of suitable peptide sequences permits them to exploit efficient routes of renal excretion, such as tubular secretion, thereby optimizing the pattern of biodistribution of these radiopharmaceuticals.

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

Plasma protein binding and pharmacokinetic parameters of CHIP (cis-dichloro-trans-dihydroxy-bis-isopropylamine 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-protein-bound 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.

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.

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.

Effect of oxoplatinum and CBDCA on renal functions in rats.

Changes in renal function in rats after single-dose intravenous administration of CBDCA (cis-diammine-1,1-cyclobutane dicarboxylate platinum(II) and oxoplatinum [cis-dichlorodiammine-trans-dihydroxo-platinum(IV)] at a dose of 20 mg/kg were determined and compared. Renal function was monitored by several determinations of effective renal plasma flow (ERPF), glomerular filtration rate (GRF), plasma creatinine and urea and urine beta 2-microglobulin. A significant reduction of ERPF and GRF and a significant increase of plasma creatinine and urea concentration after oxoplatinum treatment were found. On the other hand, no significant changes in renal function parameters were determined after CBDCA. The increase in beta 2-microglobulin amount in rat urine and polyuria persisted until the 14th day after oxoplatinum administration. Histological examination of the kidneys in the experimental animals revealed marked nephrotoxicity changes in the distal tubules after the single-dose administration of oxoplatinum. Administration of CBDCA did not produce pathological renal changes.

Comparative pharmacokinetics of four platinum cytostatics in rats.

The time-course of plasma and red blood cells platinum concentration was investigated after the administration of cis-dichlorodiammineplatinum II (cisplatinum), cis-dichlorodiammine-trans-dihydroxyplatinum IV (oxoplatinum), cis-dichloro-trans-dihydroxy-bis-isopropylamine-platinum IV (CHIP) and cis-diammine-1,1-cyclobutanedicarboxylate-platinum II (CBDCA) to male Wistar rats. A physiologically based three-compartment open model was used for pharmacokinetic evaluation. This model provides an estimation of free and protein-bound platinum time-courses from the total platinum species decrease. The total plasma clearance of total platinum increased in the order cisplatinum, CHIP, oxoplatinum and CBDCA. Plasma half-live of gamma-phase, which probably represents the elimination of protein-bound platinum species is similar (51-72 h) in all drugs under study and is longer than that of 131I-HSA (33 h). The platinum concentration in red blood cells shows plateau for all complexes studied at long time intervals and its elimination is very slow.

99Tcm-DTPMP as a skeletal scintigraphy agent: distribution in rats in comparison with 99Tcm-MDP.

The distribution and elimination of 99Tcm-complexes with methylene-diphosphonate (MDP) and with the calcium salt of diethylene-triamine-penta(methylene phosphonate) (DTPMP) were compared in rats. Both compounds exhibited high bone uptake and long-term retention of radioactivity in the skeleton. No significant accumulation of the complexes in non-osseous tissues was found. The pharmacokinetics of both chelates were similar, small differences in their distribution and elimination probably being due to different binding to plasma proteins. Two processes, namely bone uptake and kidney elimination, contributed to the disappearance of the complexes from the blood. The higher protein binding of 99Tcm-MDP probably caused its slower rate of urine elimination and insignificantly higher bone uptake compared with 99Tcm-DTPMP. On the other hand, the more rapid reduction in blood and muscle radioactivity with 99Tcm-DTPMP resulted in accelerated non-osseous tissue clearance compared with 99Tcm-MDP. This suggests that the time between administration and imaging may be shorter for 99Tcm-DTPMP than for 99Tcm-MDP. Furthermore, the much greater stability of 99Tcm-DTPMP may also reduce degradation of the complex and 99Tcm liberation in the body. For a general evaluation of both compounds, it will be necessary to determine lesion-to-bone ratios.

Renal handling of iodobenzoates in rats.

Renal elimination pathways of three positional isomers of iodobenzoic acid (2-iodobenzoate, 3-iodobenzoate and 4-iodobenzoate radiolabelled with 125I) were compared using the perfused rat kidney in-situ. All agents were eliminated both in a parent form (involving all renal elimination mechanisms i.e. glomerular filtration, tubular secretion, and tubular reabsorption) and also metabolized to a large extent in the kidney. After 3-iodobenzoate and 4-iodobenzoate administration, the major fractions of radioactivity found in urine were in the form of their metabolites, whereas 2-iodobenzoate was eliminated into urine mostly as the parent compound. Proportions of the individual metabolites in the urine of the perfused rat kidney were similar to those in intact rats for all agents. The results suggest that the kidney is the major organ for both the excretion and metabolism of iodobenzoates in rats. The principal renal metabolic reaction for all compounds under study was conjugation with glycine to produce the corresponding hippuric acid derivatives.

Interspecies pharmacokinetic scaling of some iodinated organic acids.

We have investigated the possibility of interspecies scaling of relationships between the structure and total plasma clearance in a group of nine organic acids (iododerivatives of benzoic, phenylacetic and hippuric acids) in rabbits, rats and mice. The intercompound comparison established the dependence of total plasma clearance predominantly on the molecular structure in all the animals under study, but the dependence on drug lipophilicity was also meaningful. For interspecies scaling of total plasma clearance, the use of a biological clock with an effective renal plasma flow as the unit seemed most suitable and is probably connected with the principal role of the kidney in the elimination of the compounds under study.

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.

Kidney and liver contributions to salicylate metabolism in rats.

The pharmacokinetics and metabolism of radiolabelled salicylate were studied in rats and compared with that of the isolated perfused rat liver and the perfused rat kidney. Both parent compound and salicylate metabolites (mainly conjugates with glycine and glucuronic acid) were eliminated mostly into urine. The comparison of a relative proportion of metabolites eliminated in whole rats with that of the isolated perfused rat liver and the perfused rat kidney showed that both kidney and liver contributed to the salicylate metabolism. The glycine conjugate of salicylate was formed predominantly in the kidney whereas both kidney and liver participated in the formation of glucuronic acid derivatives.

Mono(pyridine-N-oxide) DOTA analog and its G1/G4-PAMAM dendrimer conjugates labeled with 177Lu: radiolabeling and biodistribution studies.

(177)Lu radiolabeling of the first (G1-) or fourth (G4-) generation polyaminoamide (PAMAM) dendrimer conjugates with DOTA-like bifunctional chelator with one methylenepyridine-N-oxide pendant arm (DO3A-py(NO-C)) stability of the radiolabeled species and their pharmacokinetic characteristics were evaluated in preclinical experiments. The results showed that the G1- and G4-dendrimer conjugates, modified in average with 7.5 or 57 DO3A-py(NO-C) chelating units, respectively, can also be labeled with (177)Lu with a high specific activity and radiochemical purity even at 37 °C. The radiolabeled species were stable for at least 24h. Distribution profile of G1-dendrimer conjugate in organs and tissues of rats was more favorable than that of G4 one. On the other hand, the later dendrimer conjugate bears a substantially higher number of metal chelators per molecule enabling binding of a considerably larger number of radiometals. Our results indicate that an employment of dendrimer-chelate conjugates with bound radiometals might represent a prospective way for radiolabeling of biologically active target-specific macromolecules to obtain markedly high specific activity.

Biodistribution of two octreotate analogs radiolabeled with indium and yttrium in rats.

BACKGROUND: In this study, two octreotate derivatives N-[4-carboxy-4-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoyl]-Tyr(3)-octreotate (DOTAGA-tate) and N-[[4,10-bis(carboxymethyl)-7-(1(1,3-dicarboxypropyl))-1,4,7,10-tetraaza-cyclododec-1-yl]acetyl]-Tyr(3)-octreotate (DOTA-t-GA-tate) were radio-labeled with (111)In or (88)Y and their biodistribution profiles together with their elimination characteristics in rats were compared. MATERIALS AND METHODS: Radiolabeling of the peptides with high radiochemical purity was carried out in an acetate buffer with gentisic acid as radioprotective compound. Biodistribution profiles of the radiolabeled peptides were determined in intact male Wistar rats after an intravenous dose of 1 microg/kg. For elimination pathways analysis, studies in intact rats in metabolic cages and perfused rat kidney and liver were carried out. RESULTS: Fast radioactivity clearance from rat tissues (excepting somatostatin receptor-rich organs and the kidney) was determined for all agents under study. Profound radioactivity uptake in organs with a high density of somatostatin receptors (namely the adrenals and pancreas as biomarkers of somatostatin receptor-positive tissue) was slightly higher for radiolabeled DOTAGA-tate when compared with DOTA-t-GA-tate. Significantly higher accumulation in kidney and somewhat lower urinary elimination of (111)In-labeled peptides in comparison with that of (88)Y-agents were determined. Perfused rat kidney experiments confirmed that glomerular filtration was the main elimination mechanism for the compounds under study; their bile clearances in the perfused rat liver were negligible. CONCLUSION: (111)In((88)Y)-DOTAGA-tates exhibited higher distribution into somatostatin receptor-rich organs when compared with the corresponding radiolabeled DOTA-t-GA-tates. Higher uptake of (111)In-labeled peptides in the kidney is attributed to its different coordination properties.

Preparation and the kinetic stability of hyaluronan radiolabeled with 111In, 125I and 14C.

Three different procedures for the labeling of hyaluronan (HA) with (111)In, (125)I and (14)C radionuclides were compared, and the kinetic stability of radiolabeled HA under different conditions (saline, artificial gastric juice and plasma) was established. Modification of HA structure with bifunctional chelating agents (DTPA) or with the prosthetic group (tyramine or tyrosine) was essential prior (111)In and (125)I labeling. These chemical labeling techniques were fast, simple and inexpensive, and labeled agents with a high specific activity were obtained. The only disadvantage of these methods was the occurrence of unknown functional groups in the HA molecule requiring further characterization of the compound. Conversely, HA labeling with (14)C by biotechnological synthesis was found to be rather expensive and time-consuming process. Although, the final product (14)C-HA was identical to natural HA its low specific activity presents certain limitation for its application in biological experiments. Stability studies showed that (14)C-HA and (125)I-Tm-HA were stable in all studied mediums. In the case of (125)I-Trs-HA, stability slightly decreased in rat plasma and in artificial gastric juice with increasing time. The least stable was (111)In-DTPA-HA, which degraded completely after 48h in artificial gastric juice. Kinetic stability studies may provide primary information concerning the properties of radiolabeled HA in vitro, which is essential for the use and explanation of its behavior in biological experiments.

[The role of the kidney and liver in the total metabolism of (125I) ortho-iodobenzoate in rats]. / Podíl ledvin a jater na celkovém metabolismu (125I)orto-jodbenzoanu u potkanu.

The contribution of the kidney and liver to the total metabolism of (125I)ortho-iodobenzoate (OIB) was studied in rats with the aim considering the suitability of the use of this radiopharmaceutical as a diagnostic agent of the conjugation function of the liver. For the analysis of OIB metabolism (formation of conjugates with glycine and glucuronic acid) in rats, the techniques of the perfused rat kidney "in situ" and the isolated perfused rat livers were used. A comparison of the relative representation of OIB metabolites in the urine of intact rats with the results obtained in the urine from the perfused kidney and the perfusion fluid from the isolated perfused liver gives evidence of preferential biotransformation of OIB in the kidney. As shown in a biodistributional study of OIB in rats, OIB concentration in the renal tissue and thus the supply for the metabolic processes in the kidney is higher by orders than it is in the liver tissue, which could explain the above-mentioned finding. Assuming that the biotransformational organ capacity for this drug in man is similar to that in the experimental rat, the use of OIB for quantification of the detoxicating function of the liver in man is debatable.

[Pharmacokinetics of salicylate in rabbits with acute renal damage]. / Farmakokinetika salicylanu u králíku s akutním poskozením ledvin.

Changes in the pharmacokinetics and metabolism of sodium salicylate were studied in rabbits with acute renal damage induced by intravenous administration of uranyl nitrate in the dose of 0.2 mg/kg. In the pathological group there occurs a marked decrease in elimination characteristics, the total plasma clearance of salicylate linearly decreasing with a decrease in the value of glomerular filtration rate. The plasma levels of creatinine and urea are suitable indices for the estimation of the changes in the plasma binding of salicylate. An increase in the fraction of free salicylate in the plasma of rabbits with acute impairment of the kidney results in a not very conclusive increase in the size of distributional volumes. In the control group mainly the unchanged drug is excreted into urine, whereas in rabbits with acute renal damage salicylate metabolites represent the majority of the eliminated amount.

Analysis of elimination mechanisms of some 99mTc-complexes.

The biological behaviour of complexes of 99mTc with aminopolycarboxylic and aminocarbohydroxamic ligands EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), EDTAH (ethylenediaminetetraacetohydroxamic acid) and HIDAmH (N-2-hydroxyethyl-N-carboxymethyl-aminoacetohydroxamic acid) was studied in rabbits. The pharmacokinetic parameters determined in intact rabbits were compared with the results obtained in the study of renal and hepatic clearance of the complexes under study. Hepatobiliary excretion, which in [99mTc]EDTA forms 20-30% of the total excreted amount, is of negligible magnitude in the other 99mTc-complexes studied (less than 2%). Their renal clearance is not influenced by the inhibition of tubular secretion with probenecid. Binding to plasma proteins increases in the order [99mTc]DTPA less than [99mTc]EDTA less than [99mTc]HIDAmH less than [99mTc]EDTAH and the elimination half-life increases in the same order. The value of renal clearance of the complexes studied related to inulin clearance correlates well with the fraction of the free drug in the plasma. In rabbits the complexes under study are excreted mainly by the mechanism of glomerular filtration in the kidney.