Determination and prediction of tissue binding of prednisolone in the rabbit

Binding of prednisolone to rabbit tissue was determined experimentally and by computation to assess factors and proteins responsible for tissue distribution of this corticosteroid. Binding in tissue slices ranged from 36% in the lung to 83% in the liver. The results obtained were compared with steady-state tissue distribution data generated in vivo in the rabbit. Linear uptake with good agreement between the two sets of data was observed for the skeletal muscle and kidney, but not for the liver and lung. In vivo uptake of prednisolone in the liver is severely affected by metabolism and biliary excretion, while in vitro distribution into liver slices showed a pattern typical of saturation of a low-capacity site followed by linear nonspecific binding at higher concentrations. Specific saturable binding (K1 = 1.95 X 10(7) M-1) accounted for 74% of drug bound at a steroid concentration in the liver of 0.3 X 10(-6) M, but its contribution decreased to 5% at a concentration of 20 X 10(-6) M. Expected prednisolone binding in rabbit tissue was calculated based on various effective protein concentrations in tissues and binding parameters of prednisolone to rabbit plasma. The use of total Lowry protein concentrations markedly overestimates, while albumin (and transcortin) concentrations severely underestimate the tissue binding of prednisolone. The thiopental effective protein fraction best approximates the in vivo and tissue slice binding of prednisolone. Metabolism and saturable binding in liver complicate the otherwise relatively linear uptake of prednisolone into various tissues where macromolecules other than albumin dominate in tissue binding of the steroid.     

Strong binding of ditrisarubicin B to DNA

DNA binding characteristics of ditrisarubicin B were studied by the fluorescence titration technique. Ditrisarubicin B bound to calf thymus DNA with an affinity higher than any we have ever seen among anthracyclines. The apparent association constant (Kapp) of ditrisarubicin B was 2.36 X 10(8) M-1, which is 22.7 times larger than that of doxorubicin. The apparent number of binding sites (napp) of ditrisarubicin B per nucleotide of DNA was 0.164, and this value is identical with that of doxorubicin. Betaclamycin A, which has a trisaccharide chain at C-7 but no carbohydrate at C-10 in the aglycone, interacted with DNA to give a Kapp of 5.92 X 10(6) M-1 and napp of 0.178. These results suggest to us that the high affinity of ditrisarubicin B for DNA is caused by the existence of a glycosidic chain at C-10.     

Erythrosin B inhibits high affinity ouabain binding in guinea-pig heart Na+-K+-ATPase without influence on cardiac glycoside induced contractility.

Binding of [3H]-ouabain to guinea-pig heart membranes enriched in Na+-K+-ATPase revealed two different cardiac glycoside binding sites. High affinity binding was obtained at a KD = 2.2 X 10(-7) mol 1(-1) (Bmax = 16.8 pmol ouabain mg-1 protein) whereas low affinity ouabain binding occurred at a KD much greater than 10(-6) mol 1(-1). To discover whether the two ouabain binding sites are functional in guinea-pig heart muscle, erythrosin B, an inhibitor of the high affinity ouabain binding in rat brain tissue, was tested in guinea-pig isolated heart muscle preparations. Erythrosin B proved to be a potent inhibitor of the Mg2+ (Na+)-dependent-, as well as Na+-K+-activated ATPase (ID50 = 9 X 10(-6) mol 1(-1). Contractility of guinea-pig isolated papillary muscles, however, was not influenced by erythrosin B in concentrations up to 1 X 10(-5) mol 1(-1). Only very high concentrations (4 X 10(-4) mol 1(-1) resulted in a slightly negative inotropic effect (about 20%). Erythrosin B dose-dependently inhibited [3H]-ouabain binding to the Na+-K+-ATPase (KD = - 3.6 X 10(-6) mol 1(-1). In a concentration of 1 X 10(-5) mol 1(-1) the dye abolish high affinity [3H]-ouabain binding without affecting the low affinity binding sites. In contrast, in guinea-pig isolated atria, no functional antagonism between erythrosin B (5 X 10(-5) mol 1(-1) and ouabain was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binedaline binding to plasma proteins and red blood cells in humans

Serum binding of binedaline, a new antidepressant drug, was studied in vitro by equilibrium dialysis. The percent of binding in serum is high, 99.2%, and remains constant within the range of therapeutic concentrations; no saturation to the binding sites was seen. Investigations performed on isolated proteins with a wide range of concentrations showed one site with a high affinity constant (Ka = 2 X 10(6) M-1) for alpha 1-acid glycoprotein and two sites with a low affinity constant (Ka = 3 X 10(4) M-1) for human serum albumin. Binding to lipoproteins was nonsaturable, with a total affinity constant of 1.25 X 10(5) less than nKa less than 2.79 X 10(6) M-1. Over the range of therapeutic concentrations, the ratio of binedaline concentrations in serum and red blood cells remained constant (1%) and was shown to be dependent on the free fraction of binedaline in serum.     

Nature of steroid hormone binding to mitochondria

Binding of 3H-corticosterone, 3H-estradiol and 3H-testosterone to the rat liver mitochondria was studied. One type of specific binding sites for corticosterone with association constant (3.25 +/- 0.51) X 10(10) M-1 and the number of binding sites (1.79 +/- +/- 0.21) X 10(-13) mol/mg of protein was found. Adrenalectomy did not change significantly corticosterone affinity to mitochondria but increased binding capacity by 70%. Binding of estradiol and testosterone was of non-specific, hydrophobic character.     

Binding of antiarrhythmic drugs to purified human alpha 1-acid glycoprotein

The binding of lidocaine, verapamil, propafenone and propranolol to isolated, purified human alpha 1-acid glycoprotein was studied using equilibrium dialysis. Lidocaine and verapamil bound to a single class of binding sites which was characterized by high affinity (kd1 for lidocaine was 5.79 x 10(-6)M-1 and for verapamil 3.43 X 10(-6)M-1) and low capacity (n = 0.40 for lidocaine and 0.62 for verapamil). The binding of propafenone revealed two classes of binding sites, both with high affinity (kd1 was 7.62 X 10(-6)M-1 and kd2 was 6.00 X 10(-8)M-1) and low capacity (n1 = 0.79 and n2 = 0.20). Propranolol bound to at least two classes of binding sites (kd1 was 2.56 X 10(-6)M-1; n1 = 0.58). Complete characterization of the binding parameters of the second site was not possible due to failure to achieve saturation.     

Diclofenac binding to albumin and lipoproteins in human serum

The binding of diclofenac to human serum albumin (HSA) and to lipoproteins was studied in vitro by equilibrium dialysis. Binding to HSA is characterized by two classes of sites with one site each (K1 = 5 X 10(5) M-1 and K2 = 0.6 X 10(5) M-1). The binding to lipoproteins was shown to be saturable with a larger number of binding sites and low association constants. The evidence of two specific binding sites on HSA was confirmed by circular dichroism data. In addition, an identification of those sites was performed by displacement of fluorescent probes. The data show that the high affinity site (K1 = 5 X 10(5) M-1) is likely to be shared by benzodiazepines while the second one (K2 = 0.6 X 10(5) M-1) is common with the warfarin site.     

Release of specific proteins from nuclei of HL-60 and MOLT-4 cells by antitumor drugs having affinity to nucleic acids

The binding sites for mitoxantrone (MIT), Ametantrone (AMT), doxorubicin (DOX), actinomycin D (AMD) and ethidium bromide (EB) in nuclei from exponentially growing and differentiating human promyelocytic HL-60 and lymphocytic leukemic MOLT-4 cells were studied by gel electrophoresis of proteins selectively released during titration of these nuclei with the drugs. Each drug at different drug: DNA binding ratios resulted in a characteristic pattern of protein elution and/or retention. For example, in nuclei from exponentially growing HL-60 cells, MIT affected 44 nuclear proteins that were different from those affected by EB; of these 29 were progressively released at increasing MIT:DNA ratios, 11 were transiently released (i.e. only at a low MIT:DNA ratio) and 4 entrapped. Patterns of proteins displaced from nuclei of exponentially growing HL-60 cells differed from those of cells undergoing myeloid differentiation as well as from those of exponentially growing MOLT-4 cells. The first effects were seen at a binding density of approximately one drug molecule per 10-50 base pairs of DNA. The observed selective displacement of proteins may reflect drug-altered affinity of the binding sites for those proteins, for example due to a change of nucleic acid or protein conformation upon binding the ligand. The data show that the binding site(s) for each of the ligands studied is different and the differences correlate with variability in chemical structure between the ligands. The nature of the drug-affected proteins may provide clues regarding antitumor or cytotoxic mechanisms of drug action.     

The binding of the anthelmintic pyrvinium cation to deoxyribonucleic acid in vitro

Pyrvinium pamoate, an anthelmintic drug with mutagenic activity, binds to duplex DNA in vitro. Binding markedly enhances the intrinsic fluorescence of the drug, permitting a characterization of the binding reaction to be performed directly through fluorimetric analysis. The monopyrvinium salt, pyrvinium iodide, binds cooperatively to native calf thymus DNA with an intrinsic binding constant of 1.1 X 10(4) M-1 as determined fluorimetrically and 1.4 X 10(4) M-1 as determined by equilibrium dialysis. The respective binding site was estimated to be 2.5 base pairs. A change in the absorption of bound drug as a function of the binding ratio identifies secondary, nonfluorescent binding which is essentially ionic in character. The binding of pyrvinium removes negative supercoils from PM2-ccc-DNA with an experimental efficiency greater than that observed for the intercalating drug chloroquine. Electron microscopy shows that molecules of pBR322 DNA treated with pyrvinium increase in length by as much as 18% over controls, consistent with an increase in DNA base pair separation upon binding. The evidence indicates that the primary interaction of pyrvinium with DNA involves intercalation.     

The distribution of beta-adrenoceptors in dog kidney: an autoradiographic analysis

The distribution of beta-adrenoceptors in slide-mounted dog kidney sections was determined using the radioligand (-)-[125I]cyanopindolol ((-)-[125I]CYP) and autoradiography. Using conditions designed to prevent (-)-[125I]CYP binding to non-beta-adrenoceptor sites, biochemical studies revealed that (-)-[125I]CYP binding equilibrated within 150 min (K1 = 3.2 X 10(8) M-1 min-1), was saturable (KD = 30.72 +/- 2.96 pM; Bmax = 0.57 +/- 0.03 fmol/section, n = 4) and stereoselective with respect to the stereoisomers of propranolol and pindolol. Delineation of beta-adrenoceptor subtypes with the selective beta 1-adrenoceptor antagonist betaxolol and beta 2-adrenoceptor antagonist ICI 118,551 demonstrated that the proportions of beta 1-: beta 2-adrenoceptors was between 1:6 and 1:11. Autoradiographic studies showed that beta 1-adrenoceptors were localized on the juxtaglomerular apparatus and glomeruli, while beta 2-adrenoceptors were localized on medullary rays. The distribution of beta-adrenoceptors with respect to renal function in the dog kidney is discussed.     

Disposition, distribution, plasma protein binding and biliary excretion of pinazepam after i.p. administration to rat

The pharmacokinetics, distribution, plasma protein binding and the biliary excretion of pinazepam were studied in the rat. The drug was administered (5 mg/kg) by i.p. injection. The chemical analysis of pinazepam and its metabolites was carried out by a gas-chromatographic method. The parent compound was rapidly absorbed, accumulated into the tissues and converted into N-desmethyldiazepam. The highest plasma levels of the parent compound (367 +/- 13 ng/ml) were found 3 min after administration. The volume of distribution and the clearance of the drug were 1315 ml and 7.23 ml/min respectively. The metabolite was detected in the plasma and tissues 3 min after administration. At this sampling time its concentrations were 76 +/- 16 ng/ml in the plasma and 1081 +/- 68 ng/g in the liver. The decay curve of both pinazepam and N-desmethyldiazepam in the plasma, liver, lung, heart, kidney, brain, and gastrochemius muscle were characterized for their Kel, t 1/2, and AUC values. The tissue AUC to plasma AUC ratios indicated a preferential accumulation of pinazepam over its metabolite in the tissues. The plasma protein binding of pinazepam was measured by dialysis at the equilibrium. Rat plasma proteins bound 89.17 +/- 0.20 percent of the drug. The association constant was 2.60 X 10(3) l/mole and the number of sites 0.44 X 10(-6) sites/g. The biliary excretion of pinazepam and N-desmethyldiazepam was poor.     

Buffer and pH effects on propranolol binding by human albumin and alpha 1-acid glycoprotein

Propranolol binding to isolated human alpha-1-acid glycoprotein (AGP) and human albumin (HSA) was studied by equilibrium dialysis at 37 degrees C. With AGP (0.067%) and HSA (4%), total propranolol concentration was varied from 0.7 to 93,000 ng mL-1. Over this concentration range the percentage drug bound to HSA declined from 49 to 39% while that to AGP declined from 68 to 4%. Two classes of sites were identified on AGP with n1k1 = 8.50 X 10(4) M-1 and n2k2 = 3.12 X 10(4) M-1. With a pH 7.4 phosphate buffer, propranolol binding to AGP was greatest when the protein was initially dissolved in pH 7.4 water compared with pH 7.2 water or the phosphate buffer. Thus, the method of AGP solution preparation affected propranolol binding by this protein. For both AGP and HSA, greater drug binding was noted with phosphate buffers in comparison with a physiological buffer. With phosphate buffers, decreasing pH from 7.4 to 7.0 decreased propranolol binding by AGP, while decreasing pH from 7.7 to 7.4 had little effect. With HSA, the percent propranolol bound consistently decreased on lowering pH from 7.7 to 7.0.     

Studies on the molecular pharmacology of GR63178A. A novel pentacyclic pyrolloquinone anticancer drug.

GR63178A (NSC D611615) is the second pentacyclic pyrolloquinone to be evaluated clinically as an anticancer drug. Its mechanism of action is unknown but may be related either to its quinone group or planar ring system. In this report we have investigated the ability of GR63178A to bind non-covalently to DNA, inhibit topoisomerase II and undergo reduction to reactive free radical species. Using two DNA duplexes, a 12-mer oligonucleotide which is a preferred sequence for minor groove binders and a hexamer which is a preferred sequence for intercalators, no evidence of significant binding with GR63178A was found. Neither GR63178A nor GR54374X (its 9-hydroxy metabolite) inhibited purified human topoisomerase II in a decatenation assay. Free radical chemistry was studied by both pulse radiolysis and ESR spectroscopy as well as by in vitro drug incubations with NADPH-fortified rat liver microsomes and purified cytochrome P450 reductase. The one-electron reduction potential of GR63178A was -207 mV +/- 10 which is much more positive than other quinone-containing anticancer drugs such as doxorubicin, mitomycin C and mitozantrone. GR63178A underwent enzyme-catalysed quinone reduction more readily than doxorubicin but produced significantly fewer reactive oxygen species. No evidence was detected of drug-induced, radical-mediated DNA damage in vitro using pBR322 plasmid DNA. Disproportionation of the GR63178A semi-quinone free radical proceeded with a rate constant of 1 x 10(9) M-1 sec-1 under anaerobic conditions, one order of magnitude faster than doxorubicin. The preferential disproportionation of the semi-quinone may explain our inability to detect a free radical signal by ESR. The hydroquinone of GR63178A was stable and exhibited strong visible absorption with a bathochromic shift of 120 nm over the parent drug. These unusual properties may be due to the hydroquinone undergoing a form of keto-enol tautomerization. Thus, GR63178A free radical formation does not appear to result in significant drug activation. In conclusion, GR63178A is unlikely to mediate its antitumour activity by DNA binding, topoisomerase II inhibition or free radical formation in direct contrast to similar anthracycline- and anthraquinone-based anticancer drugs.     

Factors affecting quinidine protein binding in humans

The free (unbound) concentration of drug in plasma is often an important determinant of pharmacological and toxicological effects. Unfortunately, studies examining the factors influencing the free fraction of quinidine in plasma have yielded inconsistent results. It is probable that differences in the type of blood collection tubes utilized and the analytical procedure employed biased some of these estimates of quinidine binding. The present study was executed in a manner free of factors now known to introduce artifacts into estimates of the free fraction of quinidine. In healthy volunteers, the free fraction of quinidine (1.0 microgram/mL) was 0.129 +/- 0.019 (mean +/- SD) and was constant throughout the therapeutic range. A high-affinity, low-capacity binding site (K = 1.17 X 10(5) M-1; nP = 3.49 X 10(-5) M) and a low-affinity, high-capacity binding site (K = 1.33 X 10(3) M-1; nP = 3.14 X 10(-3) M) were identified. The characteristics of quinidine binding in a 4.5-g/dL solution of human serum albumin (K = 3.05 X 10(3) M-1; nP = 1.36 X 10(-3) M) suggested that the low-affinity, high-capacity binding site was on this quinidine free fraction increased from 0.114 to 0.231. A lidocaine concentration of 250 micrograms/mL caused a similar increase. Patients suffering traumatic injury had a significant increase in alpha 1-acid glycoprotein concentration (197 mg/dL) and a decreased quinidine free fraction (0.075 +/- 0.019). Patients with hyperlipidemia had free fractions similar to those observed in healthy individuals (0.118 +/- 0.019).(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of the antitumor antibiotic chartreusin to poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC), calf thymus DNA, transfer RNA, and ribosomal RNA

Chartreusin binds cooperatively to poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Both the site-exclusion model and the specific site model yield cooperative binding constants of about 5 X 10(5) M-1 and 3 X 10(5) M-1 for the AT and GC polymers, respectively, and the same stoichiometry and intrinsic binding constant for both polymers of 5 nucleotides per binding site and 3.1 X 10(4) M-1. The Scatchard plot for calf thymus DNA is curved in the opposite sense from that of cooperative binding. These binding data did not fit the site-exclusion model with the cooperative binding parameter as a variable nor the specific site, negative-cooperative binding model. The site-exclusion model with a cooperative binding parameter of unity yielded a binding constant of about 4 X 10(4) M-1 and a stoichiometry of about 5 nucleotides per binding site. The same model for transfer and ribosomal RNA yielded binding constants of 5 X 10(3) M-1 and 7 X 10(3) M-1 and stoichiometries of about 13 and 6 nucleotides per binding site, respectively.     

Drug-protein interaction: plasma protein binding of furocoumarins.

The binding of six furocoumarins (angelicin, psoralen, 8-methoxypsoralen, 5-methoxypsoralen, 8-methylpsoralen, 4,5'8-trimethylpsoralen) to human serum and human serum albumin was studied by equilibrium dialysis using tritium labelled compounds. The results indicated that in serum all furocoumarins are bound mostly by albumin, the extent of binding being related to the structure of the furocoumarins; at any rate, high values of the bound drug, ranging from 84 to 97% were observed. The percentage of binding is strictly related to the water solubility of the compounds. A limited number of binding sites, n = 1-2.4, were detected in the albumin molecule, indicating a high specificity in the binding process. The association constants of the furocoumarins to albumin. Ka, ranged from 1.2 X 10(4) M-1 (8-methoxypsoralen) to 1.9 X 10(5) M-1 (4,5'8-trimethylpsoralen).     

Determinants of carbamazepine and carbamazepine 10,11-epoxide binding to serum protein, albumin and alpha 1-acid glycoprotein

The binding of carbamazepine and carbamazepine 10,11-epoxide to serum, albumin and alpha 1-acid glycoprotein (AAG) was determined and compared at drug concentrations ranging from 0.5 to 400 mg/l using equilibrium dialysis and liquid chromatography. The total binding of carbamazepine in serum was determined primarily by albumin and to a lesser extent (20-30%) by AAG. Modified Scatchard plots for carbamazepine binding in serum were biphasic, suggesting the presence of two binding sites on serum protein. Association constants characterizing the first (k1 = 2.4 X 10(4) l/mol) and second (k2 = 4.6 X 10(2) l/mol) binding sites agreed with those measured for AAG and albumin respectively. Modified Scatchard plots for carbamazepine 10,11-epoxide binding in serum were linear and serum binding was largely accounted for by binding to albumin. The epoxide metabolite did not bind to AAG. Carbamazepine binding to AAG was drug concentration-dependent over the concentration range considered to be therapeutic, while the percent binding values for carbamazepine and epoxide binding to albumin and serum from a normal individual were constant over this range. Computer simulations showed that physiological extremes in AAG and albumin concentrations can result in a range of carbamazepine unbound fractions of 0.17 to 0.47. These data suggest that normal variations in concentrations of both proteins may be the principal cause of interpatient variability in serum protein binding of carbamazepine.     

In vitro protein binding of diclofenac sodium in plasma and synovial fluid

The in vitro protein binding behavior of diclofenac sodium (sodium[o-(2,6-dichloroanilino)phenyl]acetate) in plasma and synovial fluid was investigated by equilibrium dialysis. The drug was highly protein bound (approximately 99.5%) and the extent of binding remained constant for drug concentrations of 2-10 micrograms/mL. Comparable results were obtained with human serum albumin solution (45 g/L) indicating that albumin is probably the responsible protein. The extent of binding remained relatively constant for drug concentrations of 0.25-10 micrograms/mL when albumin concentrations were greater than 25 g/L. For albumin concentrations less than 10 g/L, the extent of binding tended to decrease with increased drug concentration. This concentration (10 g/L) is substantially lower than that usually observed in plasma or synovial fluid of arthritic patients. Curvature of the Scatchard plot indicated the existence of two classes of sites. Excellent results were obtained from fitting of the data according to two classes of sites (r2 greater than 0.999). Parameter estimates (SEM) of the number of binding sites, n1 and n2, and the corresponding association constants, k1 and k2, were 2.26 (0.55), 10.20 (0.69), and 1.32 (0.54) X 10(5) M-1, and 3.71 (1.11) X 10(3) M-1, respectively. Simultaneous samples obtained from arthritic patients indicate considerably higher total protein and albumin concentrations in plasma compared with synovial fluid, but the albumin:total protein ratios were essentially the same. There was very little difference in plasma binding in arthritic patients compared with normal subjects. The extent of binding in synovial fluid samples was consistently lower than that for plasma samples (mean +/- SD of 99.5 +/- 0.2% versus 99.7 +/- 0.1%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetic parameters influencing tissue specificity in chemical carcinogenesis.

Highly and specifically (3H)-labeled trans-4-dimethylaminostilbene was orally administered to female Wistar rats in doses ranging from 5 X 10(-10) to 2.5 X 10(-4) mol/kg. The following parameters were determined: tissue index in several tissues, binding index of liver proteins, rRNA, DNA, plasma proteins and haemoglobin and stomach proteins. Up to a dose of 2.5 X 10(-6) mol/kg all parameters remained constant, i.e. binding increased proportionately with the dose. It is concluded that simple and linear kinetics prevail and that a pharmacokinetic threshold does not exist in this dose range. At higher doses deviations from linearity were observed, however, the binding index decreased rather than increased with the dose. The results indicate that reactive metabolities are involved in the acute toxic effects observed in the rat stomach and that proximate or ultimate activation products are distributed in the circulation. Their concentration in the target tissue appears not be be specifically high to account for the tissue susceptibility.     

Mode and kinetics of DNA binding of the anti-tumour agent bisantrene

Viscometric measurements using covalently closed-circular DNA and sonicated rod-like fragments of calf thymus DNA show that the DNA-binding anti-cancer drug bisantrene intercalates with a helix unwinding angle of 14 degrees, and causes an increase in DNA contour length of 2.8A per bound drug molecule. Measurements of DNA affinity using the ethidium displacement method indicate binding constants of 7-8 X 10(7) M-1 for both natural and synthetic DNAs in buffer of ionic strength 0.1. The bimolecular rate constant for association with calf thymus DNA is greater than 1 X 10(7)M-1 s-1 at 20 degrees C and ionic strength 0.1, as determined by stopped-flow spectrophotometry. Surfactant sequestration studies of the dissociation of bisantrene from both calf thymus and synthetic DNAs reveal that the mechanism is complex, involving at least three distinguishable processes in all cases. The kinetic profiles are found to be essentially independent of DNA type, with average reciprocal time constants in the range 1-3 s-1 at a binding ratio of one drug molecule per 20 base pairs in buffer of ionic strength 0.1 at 20 degrees C. These results are discussed with respect to the mechanism of binding of bisantrene to DNA.