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.

[Radiolabelled peptides in the diagnosis and therapy of tumours]. / Radioaktivne znacené peptidy v diagnostice a terapii nádoru.

In the last two decades, radiolabelled receptor-specific peptides have profiled themselves as excellent tools that have expressively increased the diagnostic and therapeutical possibilities of tumours predominantly of neuroendocrine origin. The aim of this review paper is to transparently inform the Czech scientific fraternity about the significance and limitations of the use of this group of radiopharmaceuticals in clinical practice.

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.

On the interaction of diazepam with human, rat and mouse plasma proteins and erythrocytes.

Binding of diazepam to the blood fractions (erythrocytes and plasma proteins) in man, rat and mouse was studied. Only little dependence of binding on total drug concentration was found. The main binding fraction of plasma in studied species is albumin, but interspecies differences are both in the amount of diazepam bound to albumin and in that bound to other components of plasma. The determination of the binding of diazepam with erythrocytic mass and with blood plasma demonstrates the proportion of these bonds in total distribution of the drug under study in blood and the importance of thus experimentally followed interspecies comparison.

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.

Pharmacokinetic examination of p-aminobenzoic acid passage through the placenta and the small intestine in rats.

In the present study the permeability of the rat small intestine and the placenta to p-aminobenzoic acid (PABA) and antipyrine (AP) was investigated. Perfusion of the rat term placenta was used to determine the materno-fetal transfer of both compounds. PABA appeared in the fetal compartment faster than AP (ktransfer = 0.064 and 0.046 min-1, respectively). The rate of equilibration between the maternal and fetal compartments and placental clearance were lower for PABA than for AP (kequilibration = 0.011 and 0.020 min-1; Clp = 0.22 and 0.33 ml/min, respectively); the feto-maternal concentration ratios at equilibrium (FMCReq) were, however, mutually comparable. Similarly, PABA proved to be absorbed from the small intestine significantly faster than AP (ka = 0.824 min-1 and 0.479 min-1; tmax = 3.1 min and 8.9 min, respectively). The apparent volume of distribution (Vd) of AP in non-pregnant animals showed that the drug is distributed into the whole body water as expected (Vd = 0.66 l/kg); however, Vd of AP in pregnant animals was estimated to be 1.81 l/kg. Vd of PABA in non-pregnant animals showed its partially limited distribution, which was only slightly increased in the pregnant animals. Our results confirmed a faster penetration of hydrophilic PABA across the placenta and the small intestine than that of lipophilic AP. The mechanism of transplacental passage of PABA, however, remains to be determined.

Different transfers of N-acetyl-p-aminobenzoic acid and p-aminobenzoic acid across the placenta and the small intestine in rats.

The aim of the present study was to evaluate the transfer of N-acetyl-p-aminobenzoic acid (AcPABA) across the rat term placenta and the rat small intestine and to compare it with that of its parent drug p-aminobenzoic acid (PABA). Umbilical perfusion of the rat term placenta was used to determine the materno-fetal transfer. AcPABA appeared in the fetal compartment significantly more slowly than PABA (k transfer = 0.023 and 0.064 min(-1), respectively). The rate of equilibration between the maternal and fetal compartments was slightly lower for AcPABA than for the parent drug (k eqilibration = 0.0082 and 0.011 min(-1), respectively). Similarly, AcPABA was shown to be absorbed from the small intestine significantly more slowly than PABA (ka = 0.052 and 0.82 min(-); tmax = 37 and 3.1 min, respectively). Our results showed that both investigated compounds which are structurally related and very similar in their physical-chemical characteristics crossed both the placental and small intestinal barrier with a different kinetics. AcPABA was transported across both barriers significantly more slowly than its parent compound, which might indicate a possible equipment of the placenta with a carrier for PABA, a similar one to that previously found in the rat small intestine.

Influence of P-glycoprotein on the transplacental passage of cyclosporine.

The transfer kinetics of cyclosporine across the dually perfused rat placenta in the maternal to fetal direction and a possible involvement of P-glycoprotein were investigated. The transplacental clearance of cyclosporine in the materno-fetal direction was found to be dependent on the maternal inflow concentration of cyclosporine. Coadministration of cyclosporine with an excess of quinidine or chlorpromazine into the maternal compartment revealed 1.7- and 1.9-fold increase in cyclosporine concentration in the fetal compartment. In the experiments where quinidine was present both in the maternal and fetal compartments, cyclosporine appeared in the fetal compartment significantly faster, and its amount was three times higher when compared with controls. Conversely, quinidine or chlorpromazine did not affect the transplacental passage of L-[(3)H]-glucose. The interference of quinidine with the metabolism of cyclosporine in the placenta was excluded because only traces of M-1 and M-17 metabolites were found in the fetal solutions. Sodium azide, a mitochondrial respiratory inhibitor, was found to double the rate of cyclosporine, but not L-[(3)H]-glucose, passage across the placenta. Our findings indicate that P-glycoprotein pumps cyclosporine out of the trophoblast cells of the rat placenta in the ATP-dependent manner and restricts the passage of cyclosporine across the placental barrier.

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.

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.

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.

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 clearance-lipophilicity relationships of some organic acids in rabbits, rats and mice.

The effect of lipophilicity on the renal clearance for a group of weak organic acids (benzoic, phenylacetic and hippuric acid derivatives) was studied in rabbits, rats and mice. These compounds are eliminated in the kidney by glomerular filtration and undergo both tubular secretion and tubular reabsorption. For quantification of the effect of lipophilicity, an equation [formula: see text] was employed, where CLR represents renal clearance of the parent drug, ERPF is effective renal plasma flow, D is the partition coefficient of the acids between octanol and water, and a and b are constants. In interspecies comparison, the values of parameters a and b are similar indicating no significant interspecies differences in this route of elimination.

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.

o- and M-iodohippurate binding to plasma proteins as a model drug transport mechanism.

The pharmacokinetics of two isomers, o- and m-iodohippurate, were determined in rabbits and rats and the effect of protein binding on their elimination is demonstrated. Both isomers are rapidly eliminated by transport systems in the kidney and their clearance by the kidney approaches the renal plasma flow regardless of protein binding, m-Iodohippurate is more highly bound to plasma proteins than o-iodohippurate and its rate of elimination is enhanced in comparison with o-iodohippurate. In the case of these two isomers, the binding to plasma proteins should be considered as a transport mechanism and not as a storage depot.

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.

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.

Distribution of catalase, ribonuclease and superoxide dismutase modified by monomethoxy (polyethylene glycol) into rat central lymph and lymphatic nodes.

The plasma-lymphatic distribution of ribonuclease (RNase), superoxide dismutase (SODase), and catalase (CTase) modified by monomethoxy (polyethylene glycol) (mPEG) was studied in rats. The lymphatic bioavailability (FL) of individual enzymes administered intravenously was determined on the basis of plasmatic and lymphatic concentration curves. It was concluded that FL values depend on enzyme-adduct molecular weight (m.w.). The highest FL value was found in mPEG-RNase (the lowest m.w.), medium value in mPEG-SODase (intermediate m.w.), and the lowest one in mPEG-CTase (the highest m.w.). The binding of these enzymes in the lymphatic tissue of iliac, intestinal, brachial and neck nodes was also proportional to their molecular weight. The lymphatic binding was dependent on the node localization, higher concentrations being found in the iliac and neck nodes in contrast to the other nodes (intestinal, brachial).