Characterization of site I on human serum albumin: concept about the structure of a drug binding site.

Human serum albumin (HSA) possesses at least three sites or areas for high-affinity binding of drugs. Of these sites, site I was investigated by series of ultrafiltration and equilibrium dialysis experiments. Three ligands, acenocoumarol, dansyl-L-asparagine (DNSA) and n-butyl p-aminobenzoate (n-butyl p-ABE) were employed as marker ligands. Each ligand binds to a single high-affinity site on HSA, and binding studies with different pairs of the ligands revealed independent high-affinity binding. Preliminary displacement studies performed with the typical site I binding drugs warfarin, phenylbutazone and iodipamide showed different displacement patterns of the three marker ligands. These studies were followed by stringent competition experiments involving all possible combinations of the three test ligands themselves and of these and the three marker ligands. On the basis of the results obtained it seems that the acenocoumarol and DNSA binding regions correspond to the warfarin and azapropazone binding regions, respectively, of site I reported by others (Fehske, Schläfer, Wollert and Müller (1982) Mol. Pharmacol. 21, 387-393). The new binding region, represented by n-butyl p-ABE, is probably located adjacent to the acenocoumarol binding region but apart from that of DNSA. We have elaborated a model for binding site I in which we propose novel nomenclatures, region Ia, Ib, and Ic for the acenocoumarol, DNSA and n-butyl p-ABE binding regions, respectively. Furthermore, the relation between these regions and the high-affinity binding sites for other drugs have been discussed.

Radiation inactivation of multispecific transport systems for bile acids and xenobiotics in basolateral rat liver plasma membrane vesicles.

The functional molecular mass of the cholate, phallotoxin, iodipamide, and ouabain transport proteins in isolated basolateral plasma membrane vesicles was determined by radiation inactivation. Purified basolateral plasma membrane vesicles were irradiated (-90 to -120 degrees C) with high energy electrons from a 10-MeV linear accelerator at doses from 0 to 30 megarads. After each dose, the initial uptake, the equilibrium binding, and the binding of the substrates at 4 degrees C were checked. The size of the transporting function was, for cholate, 107 +/- 8.9 kDa; for phallotoxin, 104 +/- 7 kDa; and for ouabain, 120 +/- 4.7 kDa. The target size for the binding proteins was 56 +/- 4.2, 57 +/- 5, and 47.2 +/- 1.95 kDa for cholate, phallotoxin, and taurocholate, respectively. In the case of iodipamide, the functional molecular mass for both the transport and binding proteins was 54 +/- 4.8 kDa.

New substrates of the multispecific bile acid transporter in liver cells: interference of some linear renin inhibiting peptides with transport protein(s) for bile acids.

Interactions between some stable linear peptides with renin inhibitory activity and a multispecific transport system in the basolateral plasma membrane of liver cells was studied on cell suspensions. The peptides used in our experiments were taken up by liver cells and subsequently eliminated without any biotransformation (e.g., proteolysis). No degradation products could be detected in the extracellular medium by thin-layer chromatography. All peptides tested inhibited the uptake of physiological and of some foreign substrates of the multispecific bile acid transporter (MT). The phalloidin response of liver cells was also inhibited to a similar degree in a concentration-dependent manner. The potency of inhibition did not correlate with the lipophilic properties of the peptides. On the other hand a tight correlation could be documented between the inhibition of cholate transport and that of the phalloidin response. Transport inhibition of typical substrates of the MT by the above renin inhibitors was competitive. In contrast, the transport of a typical substrate of the bilirubin carrier (rifampicin), of amino acids (alpha-aminoisobutyric acid), long chain fatty acids (oleic acid) and cationic compounds (thiamin hydrochloride) was not inhibited by the same renin inhibitors. These results indicate that linear renin inhibiting peptides are taken up into liver cells by carrier proteins related to the MT.

[Pharmacokinetic research on bilignost (a mathematical physiological model)]. / Issledovanie farmakokinetiki bilignosta (fiziologicheskaia matematicheskaia model').

The physiologically based mathematical model has been used to describe the pharmacokinetics of bilignost in albino rats. There is a good agreement between experimental and modeling data.

Iodipamide uptake by rat liver plasma membrane vesicles enriched in the sinusoidal fraction: evidence for a carrier-mediated transport dependent on membrane potential.

Iodipamide, a cholecystographic agent, is known to be taken up by isolated hepatocytes by a mechanism similar or identical with the inward transport of bile salts (Petzinger, E., Joppen, C. and Frimmer, M. (1983) Naunyn-Schmiedeberg's Arch. Pharmacol. 322, 174-179). To elucidate its mode of transport, uptake of iodipamide was studied by rapid-filtration techniques on plasma membrane vesicles enriched in the sinusoidal fraction. Uptake was found to be dependent upon the temperature, the intravesicular volume, a gradient of monovalent cations (Na+, K+ or Li+) and the substrate concentration (saturation kinetics with respect to iodipamide: apparent Km = 70 microM, Vmax = 0.31 nmol per mg protein per min at 100 mM NaCl and 25 degrees C). Countertransport and transstimulation in tracer exchange experiments indicate that in vesicles, iodipamide uptake rather than binding occurs. Na+ could be replaced by K+ or Li+ in our system without any effect. However, in the presence of choline chloride a slight, but distinct reduction occurred. Iodipamide uptake was inhibited by cholate, phalloidin, 4,4'-diisothiocyanato-1,2-diphenylethane-2,2'-disulfonic acid and by bromosulfophthalein with inhibition being competitive in the case of cholate and non-competitive in the case of bromosulfophthalein. Alteration of the membrane potential by addition of NO3-, SCN- or SO4(2-) modified the uptake rate for iodipamide. The above results support our earlier hypothesis that the hepatocellular uptake of iodipamide is due to a carrier-mediated transport, probably similar to that of bile acids. However, translocation of iodipamide is assumed to be driven by the membrane potential only and not by Na+ contransport.

Properties of iodipamide uptake by isolated rat hepatocytes.

Exposure of isolated rat hepatocytes to iodipamide resulted in its time dependent accumulation in the cells. No accumulation was observed with rat AS-30D hepatoma cells and isolated jejunal and ileal cells from guinea pig. At concentrations below 75 microM, the iodipamide uptake into the liver cells showed saturation kinetics with a Km of 55 microM and Vmax of 555 pmol/mg cell protein X min. At higher concentrations, a nonsaturable component with a permeability coefficient (P) of 1.02 X 10(-5) cm/s is superimposed on the hepatoselective iodipamide uptake. Uptake in liver cells was partially inhibited by DIDS, an irreversible inhibitor of bile acid and phalloidin uptake in liver cells. Iodipamide uptake was found to be dependent upon Cl- and was slightly reduced in the absence of Na+. Both SCN- and NO3- decreased iodipamide accumulation in liver cells whereas SO4(2-) enhanced the accumulation. As with bile acid and phalloidin uptake, monensin, valinomycin and gramicidin A markedly reduced iodipamide uptake in rat hepatocytes. The results support the hypothesis that the organotropic excretion of iodipamide is partially performed by an energy dependent carrier which is the bile acid transporter of hepatocytes.

[Pharmacokinetics of bilignost in the normal liver and in experimental pathology]. / Farmakokinetika bilignosta v norme i pri éksperimental'noi patologii pecheni.

In the first approximation the process of distribution of the radiographic contrast agent bilignost may be formalized with the aid of a linear two-compartmental model, while generally, with the aid of a non-linear many-compartmental perfusion model of the pharmacokinetics. Pharmacokinetic analysis of experimental data has shown that the decreased content of bilignost in the liver during acute carbon tetrachloride poisoning is largely determined by the impairment of radiographic contrast agent absorption by the liver.

Common properties of hepatocellular uptake of cholate, iodipamide and antamanide, as distinct from the uptake of bromosulfophthalein.

The uptake of iodipamide and of the cyclopeptide antamanide by isolated hepatocytes was reduced reversibly in the absence of oxygen as recently shown for the transport of cholate. Oligomycin, antimycin A and carbonylcyano-chlorophenylhydrazone (CCCP) completely blocked the uptake of iodipamide and antamanide whereas the uptake of cholate was only partially decreased. Reduction of ATP in hepatocytes following replacement of glucose by fructose inhibited the uptake of iodipamide, of antamanide, and also of cholate. In contrast, the penetration of bromosulfophthalein remained unaffected under the above conditions. Arrhenius paralysis yielded high apparent activation energies for the uptake of cholate, iodipamide, and antamanide being 89, 77 and 55 kJ/mol respectively but only 22 kJ/mol for bromosulfophthalein. Mutual transport inhibition was found for iodipamide, antamanide and cholate as well as for bromosulfophthalein. Cholate inhibited the uptake of iodipamide and antamanide competitively. In contrast, bromosulfophthalein inhibited iodipamide uptake in a mixed order fashion. The results suggest a common uptake mechanism for cholate, iodipamide and antamanide different from that of bromosulfophthalein.

The liver-like anion transport system in the rabbit uvea does not eliminate iodipamide from the eye.

Iodipamide is known to be actively taken up in vitro by the rabbit iris-ciliary process preparation. This uptake is partly resistant to high concentrations of hippurate and the resistant part has been called the 'liver-like' system. In vivo iodipamide is eliminated from the rabbit eye after injection into the vitreous by a saturable process. This process is hippurate-sensitive and no role for any hippurate-resistant system was found. Two explanations for the discrepancy between the results in vitro and in vivo are offered: (1) Iodipamide may be a less than perfect model substance for physiological compounds that normally are transported by a liver-like system from the vitreous cavity and the posterior aqueous humour to the blood. (2) Iodipamide is a model for compounds that are taken up by the non-pigmented epithelium of the ciliary processes by a liver-like system and transported to the pigmented epithelium for metabolic modification.

[A method for the investigation of cholegraphic contrast media. Experiments using isolated perfused rat livers (author's transl)]. / Zur Methodik der Cholegraphie mit Röntgenkontrastmitteln. Untersuchungen am Modell der isoliert perfundierten Rattenleber.

Isolated perfused rat livers were used for investigating possible interactions between two simultaneously injected contrast media, and which technique, using parenteral application of cholegraphic media, is optimal. The results show that excretion of a parenteral contrast medium is reduced by giving an oral contrast medium at the same time. Simultaneous administration of two different contrast media therefore does not result in improved diagnostic information. The effect depends on the dose, and a sufficiently long interval should be observed between giving an oral and a parenteral contrast medium. A comparison of excretion values following injection of a bolus and prolonged infusion shows higher biliary contrast concentration and increased excretion after a single injection. Comparing only the period after the infusion, no difference was found between these two methods of administration. The single injection offers pharmacokinetic advantages, but an infusion is better tolerated and has fewer side effects. A rapid infusion of 10 to 15 minutes is therefore recommended as the optimal means of administration.

Intravenous cholangiography by bolus injection of meglumine iotroxamate and meglumine iodoxamate: a comparative trial of two new contrast media.

The meglumine salts of iodoxamic and iotroxamic acids are recently developed intravenous cholangiographic media. In several studies these two media have been shown to be significantly better than meglumine iodipamide and meglumine ioglycamate for opacification of the biliary tree and incidence of adverse effects. As part of a multi-centre double-blind trial 100 patients were given iodoxamate or iotroxamate. Comparisons of opacification, side effects and renal excretion of contrast were made. The results showed no statistically significant difference in biliary tree opacification; more frequent renal excretion of contrast with iodoxamate; and contrary to previous reports a slightly higher incidence of side effects with iotroxamate.

The potential of iosulamide meglumine as a contrast material for intravenous cholangiography: an experimental study in dogs.

Because of relatively low acute toxicity, iosulamide meglumine has been recommended as an improved contrast material for intravenous cholangiography. It has been postulated that high doses of the compound could be given safely with the expectation of achieving greater biliary excretion and improved opacification of the biliary tree. Experiments performed in dogs show that higher rates of infusion of iosulamide result in greater urinary elimination without additional biliary excretion. Consequently, iosulamide is unlikely to have any special advantage as a contrast agent.

[Reaction between x-ray contrast media and dyes and hepatocyte plasma membranes]. / Vzaimodeistvie rentgenokontrastnykh sredstv i krasitelei s plazmaticheskimi membranami gepatotsitov.

It has been shown in experiments in vitro that the hepatotrophic organic anions, the radiographic contrast agent (RCA) bilignost used in cholecystography and Bengal pink, have an affinity, unlike the urographic RCA triombrin and renotrophic dye indigo-carmine, for the plasmatic membranes (PM) of liver cells. Hydrophobic interaction has been ascertained to be of primary importance in the course of Bengal pink binding to PM of hepatocytes. PM has a higher capacity for binding Bengal pink as compared to that of lysosomal and microsomal membranes of liver cells. The binding capacity of uterine and renal PM is about 2.5 and 3 times less, respectively than that of liver PM, that is accounted for by differences in the number of binding sites on the membranes with a similar affinity for the dye. The results obtained indicate that PM of liver cells carry specific binding sites responsible for recognition and selective absorption of the hepatotrophic organic anions.

Biliary iodipamide and iodoxamate excretion as function of basal bile flow in normal, common bile duct obstructed and liver-damaged dogs.

The investigation was performed in 6 cholecystectomized chronic bile fistula dogs in which, except in complete common bile duct obstruction, the bile was diverted and replaced with a constant taurocholate infusion of 0.3 mumoles per min. per kg. Iodipamide and iodoxamate were i.v. infused at a rate of 6.7 mumoles per minute per kg for 30 minutes. Different degrees of extrahepatic obstruction were simulated by producing different intrabiliary pressure conditions. Progressive hepatic parenchymal disease was induced by oral administration of dimethylnitrosamine. In both conditions basal (precontrast) bile flow, maximum biliary excretion rate and bile concentration of the contrast agents decreased with increasing hepatic dysfunction. This investigation suggests that, regardless of the underlying mechanism, the bile iodine concentration required for radiographic visualization of the biliary system is no longer attained in intravenous cholangiography when the basal bile flow decreases below 2 microliter per min per kg in the presence of a physiologic bile salt plasma pool. In hepatic dysfunction alkaline phosphatase correlated better with the maximum biliary excretion rate and concentration of the contrast agents than SGPT, SGOT, and serum bilirubin and therefore seems to be the best parameter to predict the chance of a successful intravenous cholangiography.

Biodistribution of a particulate hepatolienographic CT contrast agent: a study of iodipamide ethyl ester in the rat.

Particulate contrast agents have been shown to be efficacious in computerized tomographic detection of liver tumors. This article quantitatively defines the biodistribution of the experimental particulate agent iodipamide ethyl ester in the rat as a function of time after intravenous infusion. The contrast agent is actively accumulated in the liver and spleen with high selectivity compared to iodine concentrations in the blood or other organs. The contrast material remains at high concentration in the liver for more than 2 hours and then is slowly cleared from the organism in approximately two days. The high selectivity and retention of contrast in the liver are indicative of the value of particulate agents in contrast-enhanced computerized tomography.