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The ligands are generally bound in plasma to a significant extent by several transport proteins (both high and low affinity), irrespective of their endogenous or exogenous origin. The protein binding of endogenous compounds (such as hormones) exhibits higher affinity and specificity than those of exogenous compounds (such as drugs). For plasma proteins that bind the same ligand(s), structural similarities or a common genetic origin may be found, although this is not a general rule. Alterations in ligand binding may be due to modifications of either the structure or the level of the binding protein. These modifications may result from genetic make up, physiology or pathology. In some situations, plasma binding may impair the distribution of drugs to tissues, with drug distribution then mainly restricted to the distribution compartment of the drug-binding protein. In other instances, the plasma drug-binding is permissive, and does not limit drug distribution to tissues. A given drug-transport protein system may have either a permissive or a restrictive effect on the drug distribution, depending on the tissue. The physiological significance of the high-affinity transport proteins is not completely understood. These proteins may increase the plasma concentration of poorly hydrosoluble ligands, ensure a more uniform tissue distribution and increase the life of the ligands. The life of the protein may also be increased by ligand binding. High-affinity transport proteins are also involved in some specific carrier mediated transfer mechanisms. It is possible to demonstrate structure-binding relationships or binding selectivity for the plasma transport proteins, but these are quite independent of relationships observed at the receptor level.

The binding of tenoxicam to human serum albumin has been shown by affinity chromatography proton titration and equilibrium dialysis to be dependent on the neutral to basic conformational change of the protein. The influence of diazepam on the interaction was also investigated using the same techniques, suggesting that diazepam increases the association of tenoxicam to albumin. Affinity chromatography revealed that the reciprocal effect also occurs. Displacement studies indicated that diazepam causes a significant increase in the affinity of tenoxicam to its main binding site, albumin site I, which is different from the diazepam site (site II). Tenoxicam seemed to cause an allosteric change in the conformation of the protein during its own binding, as did warfarin. The mechanism of this effect was a pH-dependent conformational change of albumin induced by electrostatic forces within the protein. Diazepam induced a distant accommodation of the protein, an effect accompanied by an enhanced inhibition of the release of protons from albumin.

Using [3H]-vinorelbine, we demonstrated the presence of saturable and time-dependent high-affinity binding sites on human platelets and lymphocytes. The dissociation constant and binding-site values observed were 200 +/- 38 nM, 20.0 +/- 2.2 amol/platelet, and 155 +/- 20 amol/lymphocyte, respectively. Among other blood components, saturable low-affinity binding of vinorelbine to alpha 1-acid glycoprotein, serum albumin, and lipoproteins was observed. The binding to erythocytes was nonsaturable. Given the relative concentrations of these carriers, vinorelbine mainly distributes in the platelet compartment in blood (70%), and the amount of free vinorelbine in plasma relative to the total amount in blood is2%. It is suggested that because of the preferential retention of vinorelbine by platelets, variations in the platelet count are very likely to produce changes in the free blood fraction of vinorelbine.

The binding of nimesulide within human serum to isolated proteins and to erythrocytes was studied by equilibrium dialysis. Within the range of therapeutic concentrations, nimesulide was 99% bound to serum involving a nonsaturated process (NKA = 91). This binding was almost identical to binding of nimesulide to serum albumin (NKA = 95). Physiological concentrations of free fatty acids did not affect binding of nimesulide to serum albumin. The retention of nimesulide by erythrocytes suspended in buffer was moderate (67%), although in whole blood no erythrocyte binding was observed because of the greater affinity of this drug for serum. Over the range of therapeutic concentrations (2.5 to 63 mumol/L), the free fraction of nimesulide in serum remains constant. Serum binding was decreased in samples obtained from patients with renal failure or hepatic cirrhosis associated with hypoalbuminaemia and hyperbilirubinaemia, respectively. At therapeutic concentrations, the binding of nimesulide was unaffected by warfarin, cefoperazone, furosemide (frusemide), glibenclamide, tamoxifen or digitoxin. However, valproic acid and fenofibrate (80 mumol/L) may displace nimesulide. 4-Hydroxy-nimesulide, the principal metabolite, significantly increased the free fraction of nimesulide. Although methotrexate had no effect on the free fraction of nimesulide, the free fraction of methotrexate was significantly increased in the presence of nimesulide. The present study also demonstrated 2 distinct nimesulide binding sites (site I and site II) on serum albumin (10 mumol/L) with different affinities: site II KA = 3.57 x 10(5) L/mol and site I KA = 1.24 x 10(5) L/mol. Interaction studies using markers that bind specifically to site I (warfarin and azapropazone) and site II (diazepam and ibuprofen) indicated that nimesulide binds to site II with higher affinity and to a lesser extent to site I.

We investigated potential competition between L-tryptophan (TRP) and 6-fluoro-DL-tryptophan (6-F-TRP) for binding to albumin and for passage through the blood-brain barrier (BBB). In experiments based on equilibrium dialysis, albumin (600 microM) bound about 80% of TRP and 50% of 6-F-TRP with affinity constants (Ka) of 3.7 +/- 0.04 x 10(4) and 0.62 +/- 0.01 x 10(4) M-1, respectively. Competitive inhibition was assessed as the decrease in the apparent Ka (K' a) of TRP in the presence of 6-F-TRP, with no modification of the N value. Competition between TRP, 6-F-TRP and L-valine (VAL) for passage across the BBB was demonstrated using two approaches. When administered concomitantly with TRP or 6-F-TRP to rats, VAL decreased brain uptake of TRP and 6-F-TRP and reversed their action on serotonin. In Oldendorf's model, 6-F-TRP and VAL decreased the brain uptake of TRP.

The effects of several antidepressants, amitrityline, citalopram, desipramine, fluoxetine, maprotiline, mianserin, nialamide, nomifensine, tranylcypromine and viloxazine, on the accumulation of cyclic AMP and inositol monophosphates were studied in rat cerebral cortical slices. The two enzymatic systems were stimulated either by adrenergic agonists or by forskolin. Cyclic AMP and inositol monophosphates (IPs) formed were determined by a double label method. In vitro all drugs, except inhibitors of monoamine oxidase, nialamide and tranylcypromine, inhibited α,-agonist-mediated production but did not modify the cyclic AMP accumulation. Otherwise, chronic desipramine but not citalopram administration decreased the accumulation of cyclic AMP (−39%) elicited by β-adrenoceptor agonists; no change was observed in inositol phosphate metabolism after administration of these two drugs. These data support previous investigations showing a decrease in cyclic AMP production after chronic treatment with norepinephrine uptake blockers but do not confirm the hypothesis of a modification of α 1 -adrenoceptor-stimulated inositol phosphate metabolism.

Joint and muscle pain have been reported with quinolones; however, arthropathies induced by quinolones do not result in erosive changes in humans, although such changes have occurred in animal studies. We report an unusual case of destructive polyarthropathy in a 17-year-old boy after treatment with pefloxacin 800 mg/day for 3 months. Pefloxacin may have accentuated the cartilage damage in this case, even if an underlying joint disease could not be excluded.

Using a two-dimensional isoelectrofocusing (IEF)—electrophoresis technique, the pH titration curves of the three main desialylated variants (F1, S and A) of human α 1 -acid glycoprotein (AAG) were studied to assist in the development of a fractionation method for the AAG variants. For this purpose, different AAG samples, each corresponding to one of the three main phenotypes of the protein (F1S/A, F1/A and S/A), were first purified by chromatographic separation of individual human plasma samples on immobilized Cibacron Blue F3G-A. The purified AAG samples were then desialylated and their heterogeneity was checked by analytical IEF. The pH-mobility curves of the desialylated AAG samples were displayed in polyacrylamide gel slabs, under a constant set of experimental conditions, by carrying out electrophoresis of the protein samples perpendicularly to two stationary pH gradients: a large gradient (pH 3.5–9.5) and a narrow gradient (pH 5–8). The curves showed that all the desialylated variants of AAG exhibited small charge differences and moved closely together between about pH 3.5–5.5 and pH 7.5–9.5. However, the variants were found to show microheterogeneity in their total charge between about pH 5.5 and 7.5 due to the titrated ionizable groups involved along this pH zone. This microheterogeneity was assumed to be accounted for by the existence of differences between the titratable histidyl residues of the AAG variants. Consequently, the interactions of the variants with immobilized transition metal ions were studied at pH 7, using affinity chromatography on an iminodiacetate Sepharose—Cu(II) gel. It was found that the A variant was strongly bound by immobilized Cu(II) ions, whereas the F1 and S variants interacted non-specifically with the immobilized ligand. This finding allowed the development of a rapid and effective fractionation method for desialylated AAG into its A and F1 or S variants, depending on the AAG phenotype, by chromatography on an immobilized affinity Cu(II) adsorbent. The quantitative relationships between immobilized Cu(II) ions and desialylated AAG (the apparent association constant and gel protein-binding capacity) were also determined using a non-chromatographic equilibrium binding technique.

To provide an in vitro system for studying brain capillary functions, we have developed a process of co-culture that closely mimics the in vivo situation, by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In these conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the blood-brain barrier. To validate our in vitro blood-brain barrier model, we have compared the transport of ten different compounds across the in vitro model vs. transport across the blood-brain barrier in vivo. In vivo brain extraction (Eo) are measured according to the method of Oldendorf. The in vivo and in vitro Eo values show a strong correlation as indicated by the Spearman’s co-efficient (r = 0.88, p < 0.01). The in vitro blood-brain barrier permeability for glucose and leucine i.e. compounds that traverse the blood-brain barrier via carrier mediation is in the same range as the blood-brain barrier permeability in vivo indicating that in our model the glucose and leucine transporters are always present. The relative ease with which such co-cultures can be produced in large quantities would facilitate the investigations in delivery of nutriments across the blood-brain barrier, and the screening of new centrally acting drugs.

Binding of (125I)iodocyanopindolol (ICYP) and (3H)CGP-12177 to rat brain homogenates was characterized and compared. ICYP was shown to bind to both beta-adrenergic and serotonin1B (5HT1B) receptors whereas (3H)CGP-12177 only labelled the first ones. The addition of 10 microM serotonin (5HT) prevented ICYP binding to 5HT receptors and under these experimental conditions both ligands labelled a similar total number of beta-adrenoceptors in the different rat brain regions. ICYP displayed a higher affinity for cerebellar (mainly beta 2-subtype) than for cerebral cortex beta-adrenoceptors (mainly beta 1-subtype) suggesting a subtype selectivity. A multiple displacement binding approach using CGP-20712A, a beta 1-subtype ligand, as competitor revealed a 2.6 fold selectivity of ICYP for the beta 2-adrenoceptor subtype. On the other hand, (3H)CGP-12177 binds only to beta-adrenoceptors and is not subtype selective in the rat brain homogenate. Considering both its high specificity and its lack of subtype selectivity (3H)CGP-12177 seems to be a more suitable ligand than ICYP to non-selectively label beta-adrenoceptors in rat brain.

The binding interactions of a series of basic ligands with alpha 1-acid glycoprotein (AAG) were examined as a function of pH. The binding to AAG increased with increasing pH, and the binding data were satisfactorily fitted to a model that incorporates the effect of pH and discriminates the association constants of neutral (non-protonated) and protonated forms of ligands. It was shown that ligands in the neutral form have a markedly higher affinity for AAG than the protonated forms, resulting in a concomitant decrease in the pKa of bound ligands. The u.v.-visible difference spectra generated upon binding of a representative ligand to AAG also showed that there was a contribution to the binding arising from the deprotonation of the ligand. It is suggested that all tested ligands bind similarly to AAG and that hydrophobic interactions dominate high-affinity binding to AAG.

1. Determination of debrisoquine oxidation phenotype was carried out in 119 healthy subjects, 135 patients with chronic bronchitis and 153 patients with lung cancer, all of Caucasian origin. 2. A non-Gaussian distribution of the log D/HD ratio was observed in the three groups. 3. Assuming an antimode of 1.12, the proportion of PMs was found to be 6.7% in healthy subjects, 8.9% in chronic bronchitics and 6.5% in patients with lung cancer. These differences were not significant. 4. The presence of a lung tumour itself had no influence on phenotype in a group of 14 patients who were phenotyped before and after surgery. 5. We conclude that a link between debrisoquine phenotype and lung cancer is unlikely.

The review gives a critical evaluation of the different separation procedures used to study drug-protein interactions and describes their various fields of application. For pharmacological studies, the most widely used methods are dialysis and ultrafiltration, because they allow measurements with solutions of high protein concentrations, such as those found in therapeutic conditions. Both techniques use membrane devices, which may induce additional binding effects. Another drawback of these techniques is the need for radiolabelled compounds. Chromatographic methods, which now take advantage of the technology of high-performance liquid chromatography, are generally faster and do not use drug labelling because of the higher sensitivities of the detectors. Two different approaches are possible: either all the interacting species (protein and drug) are dissolved in the mobile phase, or one of them (protein or drug) is immobilized on the support. Several chromatographic methods are available for studies in solution that differ according to the sample injection mode (frontal or zonal elution) and the nature of the mobile phase used. They include quantitation of the drug-protein complex by zonal elution, the Hummel and Dreyer method, frontal elution, the vacancy peak method, and retention analysis by zonal elution. Frontal elution is the most rigorous method since all the species at equilibrium are present in the mobile phase with known and constant concentrations. The most promising one is the Hummel and Dreyer method, because of the very small amount of protein injected in the mobile phase containing the drug. Drug-protein interactions may be studied by affinity chromatography by immobilizing one of the interacting species on the support. Comparison of the constants obtained with methods when both the drug and the protein are in solution is questionable, since the immobilized species in affinity separations differ in their physical properties from those in solution. The main advantage with studies on immobilized proteins is the easy comparison of the binding properties of various drugs, especially when they are enantiomeric. The results of the binding constants measured by different separation methods are given for the albumin-phenylbutazone and albumin-warfarin systems. Good agreement is generally obtained, which proves the validity of using chromatography as a tool to study drug-protein interactions.

Part I of this article, which appeared in the previous issue of the Journal, discussed the implications of variations in plasma protein levels in a number of diseases: hepatic and renal disease, acute myocardial infarction, burns, cancer, diabetes mellitus, hyperlipidaemia and inflammatory diseases. In Part II the authors continue their review with a further range of disease states, and consider their import for drug dosages.

In order to better understand their binding and blood distribution, we have investigated the binding of retilliptine to blood components

The aim of our study was to answer the following questions: (1) is thiamine deficient in chronic excessive drinkers; and (2) is peripheral neuropathy associated with thiamine deficiency or with alcohol intake itself? We performed direct assays of blood concentrations of free thiamine and thiamine phosphate in excessive drinkers with or without peripheral neuropathy and in control subjects. We found no difference in free thiamine concentrations between excessive drinkers with.and without neuropathy, and no difference in free thiamine concentrations between the two groups of excessive drinkers and the control group. By contrast, a deficiency in thiamine phosphate was observed in each group of excessive drinkers compared to the control group. This was reflected in blood concentrations of total thiamine which were also lower in excessive drinkers than in controls. Finally, the thiamine phosphate: free thiamine ratio was slightly but significantly lower in the two groups of excessive drinkers than in the control group. Both groups of excessive drinkers showed typical moderate liver disease of alcoholic origin. In conclusion, the free thiamine fraction was not diminished in this group of alcoholic hospital inpatients. Thiamine deficiency would not therefore appear to play a determining role in the onset of peripheral neuropathy. In contrast, the phosphorylated fraction was slightly reduced, probably owing to the liver disease in these subjects. Contrary to studies using indirect assay techniques, our results suggest that thiamine deficiency is either slight or absent in chronic drinkers.

The effects of cyclosporin A (CSA) in low dosage (4 mg/kg/24 h i.v.) were studied in 17 patients awaiting heart transplantation. The lymphocyte subsets were typed, enumerated, and their proliferation measured before CSA perfusion, after infusion (over 24 h), and then again after two further 24 h intervals (T0 h, T24 h, T48 h, and T72 h). No significant change was found in T or B enumerations although lymphocyte function was markedly modified. For all 17 patients, there was a significant decrease in lymphocyte proliferation that was, however, re-established after 72 h for 14 of the patients, but not for the remaining 3. Inhibition of the proliferative response was found to occur rapidly, to be potent although rapidly reversible in most cases, while yet subject to wide interindividual variability. These four features suggest that (a) CSA in fixed doses may later the balance between helper and suppressor cells in varying degrees according to the patient, and that (b) CSA given immediately or shortly after heart transplantation could be beneficial.

1. The binding of tianeptine to human plasma, isolated plasma proteins, red blood cells and to plasma from patients with cirrhosis or renal failure was studied in vitro by equilibrium dialysis. 2. Tianeptine is highly bound to plasma (95%) at therapeutic concentrations (0.3-1 microM). No saturation of the binding sites was seen. 3. Human serum albumin (HSA) was shown to be mainly responsible for this binding (94%) with a saturable process characterized by one binding site with a moderate affinity (Ka = 4.2 x 10(4) M-1) and a non-saturable process with a low total affinity (nKa = 1.2 x 10(4) M-1). 4. Like many basic and amphoteric drugs, tianeptine showed a saturable binding to alpha 1-acid glycoprotein (AAG) with one site and a moderate affinity (Ka = 3.7 x 10(4) M-1). Its binding to lipoproteins and red blood cells (RBC) was weak and non-saturable. Over the range of therapeutic drug concentrations (0.3-1 microM), the unbound fraction in blood remains constant (4.5%). 5. Interactions were studied using non-esterified fatty acids (NEFA) at pathological concentrations; they altered tianeptine binding to plasma and to isolated HSA. Tianeptine seems to bind to a HSA site different from sites I (warfarin) and II (diazepam), but close to site II. It also shares the only basic-site on AAG. However, at therapeutic drug concentrations (0.3-1 microM), not all of these interactions occur. 6. The binding of tianeptine varied according to HSA, AAG and NEFA concentrations both in patients and healthy subjects. In patients with chronic renal failure having high NEFA concentrations the unbound fraction of tianeptine (fu) increased from 0.045 to 0.153 compared with normal (P less than 0.001). In cirrhotic patients, with relatively low HSA concentrations, the fu of tianeptine increased from 0.045 to 0.088 compared with normal (P less than 0.01). 7. Multiple regression analysis of all of the data indicated that the fu of tianeptine was related significantly to HSA, NEFA and AAG concentrations, with a particularly strong correlation with NEFA concentrations. Therefore, variation of HSA and NEFA concentrations in patients on maintenance therapy may cause an increase of tianeptine fu.

Sodium valproate (VPA) is a drug widely used in the treatment of epileptics often in association with benzodiazepines. Recent animal studies have shown that the addition of valproate increases diazepam levels in the cortex and the cerebellum (Hariton et al, 1985). The aim of our study was to determine the effect of VPA on the transfer of benzodiazepines through the blood-brain barrier. They were investigated using the intracarotid injection technique in rats as described by Oldendorf (1971). Our results show that the 14C-chlordiazepoxide brain extraction is significantly higher in rats on prolonged valproate treatment than in controls. With regard to plasma protein binding effects on chlordiazepoxide transport, our data indicate that a fraction of the protein-bound chlordiazepoxide could transfer from the intracapillary space to the brain tissue space because of enhanced drug dissociation from albumin in the brain microcirculation (Kd in vitro = 74.1 microM; Kd in vivo = 793.7 microM). Two distinct mechanisms can be deduced from this study: 1) chlordiazepoxide is displaced from HSA by valproate, 2) in addition, this fatty acid could increase drug permeation through the blood brain barrier (PS/F (chlordiazepoxide) = 0.60 in controls, PS/F (chlordiazepoxide) = 0.97 in treated rats). On the contrary, the washout of the benzodiazepine from the rat brain does not seem to be modified by the addition of valproate.

Summary— Early investigation of protein binding of a new drug is mandatory. The following questions have to be answered: is unbound fraction constant over tested concentrations? Which proteins are involved? What are the binding parameters? Can the drug compete with other therapeutic agents for the binding sites or in other words can drug displacements be predicted? What is the interindividual variability in protein binding? Is the binding stereoselective? All this information is necessary in predicting the pharmacokinetic behaviour of the drug and in assisting in the design of future pharmacokinetic protocols in phases II and III. The use of free drug concentration should also be considered when comparing the bioavailability of regular vs sustained release dosage forms of drugs exhibiting concentration-dependent binding and when studying concentration-effect relationships.

Ischemia reperfusion injury is still a leading cause of early graft dysfunction after transplantation. Trimetazidine (TMZ) has been postulated to be protective against renal damage from oxygen free radicals. The aim of this study was to assess the effect TMZ during cold storage (CS) and normothermic reperfusion in an isolated perfused pig kidney model. Three groups were studied: control group, immediately perfused (G0), 48 h CS in Euro-Collins solution (G 1), and 48 h CS in Euro-Collins solution plus TMZ (G 2). Glomerular filtration rate (GFR) and fractional sodium reabsorption (FRNa+) were calculated during reperfusion from urine and perfusate samples. Lipid peroxidation was determined by the renal tissue level of Schiff bases (SB) and malondialdehyde (MDA) after reperfusion. A histological evaluation was performed after reperfusion. Renal function was significantly improved and lipid peroxidation reduced after preservation in Euro-Collins solution plus TMZ. Functional data were closely related to histological damage. In conclusion, TMZ is a useful protective agent against renal damage induced by CS.

Serotonin increased the isoproterenol-induced cyclic AMP accumulation in rat brain cortical slices but did not itself stimulate the production of cyclic AMP in this area. This effect appeared to be inhibited by the 5-HT2 receptor antagonist, ritanserin. This potentiation was not observed in the hippocampus. These data reinforce the hypothesis of a coupling between the noradrenergic and serotonergic systems, which may be involved in the mechanism of action of antidepressant drugs.

The equilibrium saturation chromatographic method has been applied to the study of the influence of sodium dodecyl sulphate (SDS) on warfarin–human serum albumin binding. The ligand retention volume depends upon the association and gives text-decoration:overliner, the number of bound ligands per mole of macromolecule. Another method for evaluating the influence of SDS upon drug–protein binding is introduced.

Parameters of [3H]erythromycin binding to Legionella pneumophila were determined in vitro using both an equilibrium and a kinetic method. Different L. pneumophila serogroups, 1-3, and a virulent strain serogroup, 1, were tested. All strains of bacteria exhibited the same binding pattern, with a dissociation constant of 0.15 microM. Other macrolides, streptogramin B-types, and lincosamides competitively displaced bound erythromycin suggesting that these compounds share common binding sites on the bacteria. Minimum inhibitory concentration (MIC) values for macrolides, streptogramin B-types, and lincosamides were determined with buffered charcoal yeast extract (BCYE) medium. A good correlation (r = 0.994) was found between the corresponding inhibition constants of these antibiotics and their MIC. It was also noted that for lincosamides the microbiological inactivity was associated with a very low bacterium affinity. Thus, it is concluded that binding parameters of these antibiotics reflect their efficacy against L. pneumophila in vitro and may serve as a useful adjunct in developing new compounds.

In the Rat, after a single intravenous injection of 25 mg/kg of l4C labelled Ticlopidine, an anti-platelet compound, the decrease in plasma radioactivity follows a polyphasic curve. In all the tissues studied, no lasting accumulation of radioactivity occurred. Its elimination half-life is 1,54 ± 0.12 days. This radioactivity is eliminated essentially in feces ba](54 ± 1,7%) but also in urine (32 ±4,9%), since Ticlopidine undergoes a partial entero-hepatic cycle.

A study of the binding to human serum albumin (HSA) of caffeine and its deuterated isotopomers, 1-C2H3-,3-C2H3-, 1,7-(C2H3)2-, 3,7-(C2H3)2- and 1,3,7-(C2H3)3-caffeine, was performed by equilibrium dialysis. Free and bound fractions were measured by gas chromatography/mass spectrometry. Important and significant (Fischer and Student tests) isotope effects were observed on binding parameters: sites total concentration (N = 1732 microM for 1,3,7-(C2H3)3-caffeine versus 822 microM for caffeine; number of sites (n = 3 for 1,3,7-(C2H3)3-caffeine v. 1 for caffeine); and extent of binding (46% for 1,3,7-(C2H3)3-caffeine v. 27% for caffeine). A study of competition for HSA binding between caffeine and its 1,3,7-(C2H3)3- and 3,7-(C2H3)2-isotopomers confirmed the results obtained in direct binding studies. These isotope effects are discussed in terms of (a) tools for molecular pharmacology, (b) precautions to be taken when such labelled drugs are used in clinical pharmacology.

Summary: Using isotope techniques, equilibrium dialysis, and incubation experiments, we characterized the binding of nicardipine to isolated plasma proteins, human serum, blood cells, and platelets. Nicardipine was mainly bound to lipoproteins, orosomucoid, albumin, and erythrocytes in human blood. Nicardipine, pindolol, and imipramine were found to share the same site on orosomucoid. The determinants of nicardipine binding to lipoproteins were triglycerides, phospholipids, and cholesterol ester. Nicardipine partitioned into erythrocytes, showing a constant ratio of distribution between the intra- and extracellular compartments. Nicardipine partitioned less to erythrocytes when increasing amounts of binding plasma proteins were present in the extracellular compartment. In human blood, 12 to 18% of total nicardipine was present in erythrocytes. The overall binding of nicardipine in serum varied from 98 to 99.5% and correlated with serum orosomucoid and serum lipid concentrations.

A size exclusion chromatographic method for studying ligand-macromolecule binding parameters is described. This equilibrium saturation method allows the determination of the concentrations of constituents in equilibrium and is specially useful for characterizing ligand-protein binding under conditions that can be compared with physiological conditions. The method has been used for measuring warfarin-human serum albumin (HSA) binding and for studying the influence of free fatty acids (FFA) and sodium dodecyl sulphate on warfarin-HSA binding. Some comparisons with the Hummel and Dreyer method are given. The influence of the FFA is strongly dependent on their chain length, with an inversion of the effect for a 10-carbon chain.

An assay was developed to measure alizapride (a new antiemetic compound) in biological specimens. The method involved reversed-phase high-performance liquid chromatography and fluorescence detection. The detection limit was 5 ng/ml in plasma or urine samples. The value of the assay was demonstrated with a dose-dependent pharmacokinetic study. It showed a two-phase decrease in plasma concentrations, after intravenous injection, with half-lives of 7.5-min and 2.5-hr, respectively. From plasma and urine results, pharmacokinetic parameters remained constant in the dose range of 50-200 mg.

Tetracycline and erythromycin concentrate highly in pulmonary tissues in humans as well as in the rat. Their binding to the lung, whatever the species and the pathological state, is weak. Their intrapulmonary concentrations could be explained by a passive diffusion which depends on the pH variation between the intra- and extratissue compartments, the percentage of un-ionized form present, and their liposolubility. The importance of retention of tetracycline and erythromycin by plasma proteins is demonstrated by the decrease of their pulmonary index of penetration (IP, the intra- and extratissue concentrations ratio). The IP values are, respectively, 1.09 and 1.23 for tetracycline and erythromycin. These concentrations are in excess of their minimal inhibitory concentrations for bacteria responsible for pneumopathies. The lung homogenate binding of these antibiotics is weak (5% for erythromycin and 33% for tetracycline), corresponding to a nonsaturable binding to three main subcellular fractions (nucleus, mitochondria, and cytosol). Tetracycline has the same penetration in healthy or cancerous human lungs, whereas erythromycin presents a decreased IP in cancerous tissue. However, the binding of these antibiotics to healthy or cancerous lung homogenates is similar. So, the structure of cancerous cells is solely responsible for this modification of erythromycin penetration. The intrapulmonary concentration of tetracycline is increased in rat lungs infected by Legionella pneumophila. This modification is due to a great bacteria retention. In contrast, erythromycin possesses the same IP in healthy and infected rat lungs.

Moroxybrate, a drug obtained by the reaction of clofibric acid with moroxydine, a biguanide with practically no effect on carbohydrate metabolism, has been examined for its effects on blood lipids, fibrinogen, in-vitro platelet aggregation, and also on experimental atherosclerotic lesions obtained by feeding the rabbits a diet containing 1% cholesterol. At the same dose, moroxybrate has an effect at least equal to those of clofibrate and moroxydine alone, on the lipidic parameters and haemostasis in rat and rabbit. The combination of these actions together with an effect on the prevention of experimental atherosclerosis lesions in the rabbit suggests moroxybrate should be examined for its effects in ischaemic cardiovascular disease.

Summary [ 3 H] imipramine exhibits both saturable and high affinity binding sites in human lung with a maximal number of binding sites of 7.50 pmoles/mg protein and a dissociation constant of 1.74 nM. Displacement studies indicate that these sites can be considered as specific of imipramine, tricyclic compounds and also monoamine uptake inhibitors: fluoxetine and nisoxetine. Atypical antidepressants were inactive as ligands of main known receptors.

Four rates of rifampicin infusion ranging from 3.3 to 15 mg/min in 12 tuberculous patients were studied. Blood samples (n = 10) were drawn during infusion and 8 h later. Urine samples were collected in six fractions during a 24-h period. Rifampicin and desacetylrifampicin were measured by high-pressure liquid chromatography. Results show that the maximum plasma concentrations increase linearly for each dose with the rate of infusion, and that the amounts excreted in the urines are highly dependent on the administered dose. Simulation of plasma concentrations after different dosage regimens shows that a double rate of infusion--20 mg min-1 during 1 h and then 200 mg h-1--allows plasma concentrations to be quickly reached and maintained at a 20 mg L-1 level, far higher than the minimum inhibitory concentrations of most germs.

Using radiolabelled bepridil, equilibrium dialysis experiments and direct ligand-binding studies were conducted to characterize the binding of bepridil to some human serum proteins, and to blood cells and platelets, respectively. Bepridil was bound to serum albumin with K = 26 +/- 1.6 X 10(3) mol-1 X 1, n = 1.07 +/- 0.04; to alpha 1-acid glycoprotein with K = 442 +/- 68 X 10(3) mol-1 X 1, n = 0.90 +/- 0.04; and with a lesser extent to lipoproteins and gamma-globulins. Bepridil, propranolol, quinidine and erythromycin were found to share the same site on alpha 1-acid glycoprotein. The binding of bepridil to both RBC and WBC was nonspecific with the following parameters: NK = 10.55 +/- 0.10 per 5 X 10(6) RBC/mm3 and NK = 0.34 +/- 0.01 per 10(3) WBC/mm3, whereas the binding to platelets was saturable with N = 10.97 +/- 1.06 mumol/l per 108 +/- 10(3) platelets/mm3 and K = 40 +/- 8 X 10(3) mol-1 X 1. The binding parameters were used to simulate the distribution of bepridil between serum proteins, blood cells and platelets over the therapeutic range and showed that serum albumin, alpha 1-acid glycoprotein and RBC were the major binding components.

Polymerization of a commercial human serum albumin (Sigma A-1887) was investigated by two different techniques, high-performance liquid chromatography and gel electrophoresis. The chromatographic technique was based on the frontal analysis principle using a column which excludes polymers but retains monomers. The results allowed the determination of the monomer--polymer affinity constant, X = 526 +/- 100. The electrophoresis technique was performed with a polyacrylamide gel containing sodium dodecyl sulphate in order to separate the different polymer species according to their molecular weights. The two techniques gave results in good accordance and showed a concentration-dependent aggregation. The higher the human serum albumin concentration, the more the monomer proportion decreases.

Brain extraction of two antiepileptic compounds, progabide and its acid metabolite, SL 75102, was investigated using the carotid injection technique in the rat. The extent to which drug binding to plasma proteins could inhibit the brain extraction was measured. Equilibrium dialysis at 4 degrees showed that both drugs were highly bound to human serum proteins, mainly to serum albumin. Progabide is also bound to red blood cells and to lipoproteins. The free dialyzable drug fraction was inversely related to the protein concentration. Similarly, the brain extraction of the drugs in the presence of either albumin, or red blood cells for progabide was inversely related to their respective concentrations. However, the rat brain extraction of both drugs was higher than expected from the in vitro measurement of dialyzable fraction. Furthermore, despite a significant degree of progabide binding to lipoproteins, no significant reduction in the brain extraction of the drug was observed. These data indicate that the amount of circulating progabide or SL 75102 available for penetration in a peripheral tissue such as brain exceeds the dialyzable fraction of drug. However, the in vivo exchangeable drug fraction still parallels the dialyzable fraction, except if the drug is lipoprotein-bound.

Two selective and sensitive methods for the quantitative analysis of butofilol in human plasma and urine are discussed. The first method is a gas chromatographic assay with electron-capture detection using extraction with toluene, several clean-up procedures and derivatization. The second method is based on high-performance liquid chromatography and a single extraction with dichloromethane. The two assay methods were applied to the determination of the same human plasma samples after administration of a single, oral 200-mg dose of butofilolol. A good correlation between the results (inter-laboratory comparison) is obtained, validating both techniques.

Parameters of [ 3 H]-erythromycin binding to Streptococcus are determined in vivo using both equilibrium and kinetic methods. This binding is saturable, reversible and independent of energetic systems. Whatever the methods used, the binding parameters are identical as 14 nM for the dissociation constant of the complex erythromycin- Streptococcus and a density of binding sites of 11,865 molecules/ cell. Other macrolides, streptogramins and lincosamides competitively displaced bound [ 3 H]-erythromycin suggesting that these compounds share common binding sites on the bacteria. In parallel, the MIC values of these antibiotics against Streptococcus are determined by agar dilution method in Mueller-Hinton medium with 5% of horse blood in order to compare the binding and microbiological parameters. A strong correlation ( n = 0.863) has been found between the corresponding inhibition constants and MIC values. Such binding studies could be used in conjunction with microbiological assays for primary screening of active analogous or other compounds with interfere with [ 3 H]-erythromycin binding to the bacteria.

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.

The serum concentrations of alpha-1-acid glycoprotein (AAG), albumin (HSA), and non-esterified fatty acids (NEFA), and the serum binding of indapamide were measured in four groups of individuals: control (healthy) subjects (N = 24), patients with inflammatory syndrome (N = 28), with hepatic (N = 20) and renal (N = 27) insufficiency. Indapamide serum binding was increased in patients with inflammatory syndrome (82.2 +/- 3.4%, P less than .001), decreased in patients with hepatic insufficiency (72.3 +/- 5.9%, P less than .001) and unchanged in patients with renal insufficiency (77.7 +/- 2.8%) as compared with controls (78.2 +/- 3.1%). A multivariate analysis indicated that these changes were mainly related to concomitant changes in AAG concentration (that explained 63% of intersubject variability in bound/free binding ratio), and to a lesser extent to HSA (that explained only 4% of the variability in the binding). These data show that the free fraction of the acidic drug indapamide in serum is affected by pathologic conditions in which changes in AAG concentration occur and that, unexpectedly, HSA plays a negligible role in the binding.

A strong increase in the 315 nm induced ellipticity of the human serum albumin-warfarin complex has been observed in the presence of long-chain free fatty acids. Measurement of the binding properties of racemic warfarin and its enantiomers on human serum albumin by high-performance liquid chromatography shows only a slight enhancement of binding in the presence of the fatty acids concerned. Furthermore, the enhancement of the circular dichroic signal, observed at saturation of high-affinity sites, has been attributed to a modification in the conformation of the warfarin binding site on the protein caused by fatty acids.