Role of alpha-1 acid glycoprotein, albumin, and nonesterified fatty acids in serum binding of apazone and warfarin.

Altered concentrations of serum proteins and nonesterified fatty acids (NEFAs) often accompany malignant diseases. Free fractions (fu) of apazone and warfarin were measured by equilibrium dialysis in serum samples obtained from 31 patients with cancer and 18 control subjects. Mean fu values of both drugs were significantly higher in the patient group. Multivariate analysis showed albumin, NEFA, and AAG for apazone and albumin, NEFA, and age for warfarin accounted for 60% and 63%, respectively, of interpatient variation in bound/free drug concentration ratios in the group of patients with cancer. The interactions of apazone and warfarin with AAG were further characterized; the more avid site had association constants of 4.5 X 10(5) and 2.3 X 10(5) L/mol, respectively. Finally, it is strongly suggested that when hypoalbuminemia is present and a drug binds to AAG with an affinity constant comparable to or higher than that to albumin, then fu will become dependent on the concentration of AAG.

Drug binding in plasma. A summary of recent trends in the study of drug and hormone binding.

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.

Differential effects of zidovudine and zidovudine triphosphate on mitochondrial permeability transition and oxidative phosphorylation.

1. The effects of zidovudine (ZDV) and zidovudine triphosphate (ZDV-3P) on Ca2+-induced mitochondrial permeability transition (MPT), respiratory control ratio (RCR) and ATP synthesis have been investigated on isolated rat liver mitochondria. 2. ZDV slightly but significantly decreased RCR and ATP synthesis but was ineffective in inhibiting MPT. In contrast, ZDV-3P did not alter RCR and ATP synthesis but strongly inhibited MPT (IC50 = 3.0 +/- 0.9 microM). 3. The effect of ZDV-3P on mitochondrial swelling required a preincubation time. When incubated 10 min with mitochondria, ZDV-3P (8 microM) totally inhibited the rate of swelling. 4. ADP, ATP and atractyloside, which are agents known to interact with the mitochondrial adenine nucleotide carrier (ANC), antagonized the effect of ZDV-3P on mitochondrial swelling. Indeed, the IC50 value of ZDV-3P increased from 3.0 to 17.4, 93.6 and 66.5 microM, in the presence of 20 microM, ADP, ATP or atractyloside, respectively. 5. ZDV-3P did not displace [3H]-ATP from its mitochondrial binding site(s) whereas ADP and atractyloside did, suggesting that ZDV-3P and [3H]-ATP do not share the same binding sites. 6. ZDV-3P did not affect either mitochondrial respiration or ATP synthesis but inhibited Ca2+-dependent mitochondrial swelling. It was concluded that mitochondrial toxic effects observed during the chronic administration of ZDV cannot be related to its active metabolite (ZDV-3P).

Nimesulide binding to components within blood.

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.

Evidence for binding of certain acidic drugs to alpha 1-acid glycoprotein.

The binding of some acidic drugs to alpha 1-AGP was studied by equilibrium dialysis at 37 degrees, pH 7.4. Certain acidic drugs bound to alpha 1-AGP at one binding site with a high affinity. Though the alpha 1-AGP plasma concentration is far lower than the HSA concentration, the association constants of some acidic drugs with alpha 1-AGP are high enough to suggest that binding to alpha 1-AGP will contribute significantly to the total plasma binding of these drugs.

Evidence for isoxicam binding to site I as a primary site and to site II as a secondary site of human serum albumin.

Isoxicam binding to HSA was studied using equilibrium dialysis and fluorescence methods. It was shown that this drug binds to or near site I (warfarin or azapropazone site) and to site II (the diazepam site) as a secondary site, although it is generally considered that their respective drug structural requirements are often exclusive. The binding parameters were calculated with different mathematical models; a site oriented model with or without fixing the number of binding sites as integer values and a stoichiometric model. The relevant results are in good agreement under the selected experimental conditions. The stoichiometric method indicates that no positive cooperativity occurred during the binding process but other interactions between the two sites cannot be excluded.

Systemic antifungal agents. Drug interactions of clinical significance.

There are 3 main classes of systemic antifungals: the polyene macrolides (e.g. amphotericin B), the azoles (e.g. the imidazoles ketoconazole and miconazole and the triazoles itraconazole and fluconazole) and the allylamines (e.g. terbinafine). Other systemic antifungals include griseofulvin and flucytosine. Most drug-drug interactions involving systemic antifungals have negative consequences. The interactions of amphotericin B, flucytosine, griseofulvin, terbinafine and azole antifungals can be divided into the following categories: (i) additive dangerous interactions; (ii) modifications of antifungal kinetics by other drugs; and (iii) modifications of the kinetics of other drugs by antifungals. Amphotericin B and flucytosine mainly interact with other agents pharmacodynamically. Clinically important drug interactions with amphotericin B cause nephrotoxicity, hypokalaemia and blood dyscrasias. The most important drug interaction of flucytosine occurs with myelotoxic agents. Hypokalaemia can precipitate the long QT syndrome, as well as potentially lethal ventricular arrhythmias like torsade de pointes. Synergism is likely to occur when either QT interval-modifying drugs (e.g. terfenadine and astemizole) and drugs that induce hypokalaemia (e.g. amphotericin B) are coadministered. Induction and inhibition of cytochrome P450 enzymes at hepatic and extrahepatic sites are the mechanisms that underlie the most serious pharmacokinetic drug interactions of the azole antifungals. These agents have been shown to notably decrease the catabolism of numerous drugs: histamine H1 receptor antagonists, warfarin, cyclosporin, tacrolimus, digoxin, felodipine, lovastatin, midazolam, triazolam, methylprednisolone, glibenclamide (glyburide), phenytoin, rifabutin, ritonavir, saquinavir, nevirapine and nortriptyline. Non-antifungal drugs like carbamazepine, phenobarbital (phenobarbitone), phenytoin and rifampicin (rifampin) can induce the metabolism of azole antifungals. The bioavailability of ketoconazole and itraconazole is also reduced by drugs that increase gastric pH, such as H2 receptor antagonists, proton pump inhibitors, sucralfate and didanosine. Griseofulvin is an enzymatic inducer of coumarin-like drugs and estrogens, whereas terbinafine seems to have a low potential for drug interactions. Despite important advances in our understanding of the mechanisms underlying pharmacokinetic drug interactions during the 1990s, at this time they still remain difficult to predict in terms of magnitude in individual patients. This is because of the large interindividual and intraindividual variations in the catalytic activity of those metabolising enzymes that can either be induced or inhibited by various drugs. Notwithstanding these variations, increasing clinical experience is allowing pharmacokinetic interactions to be used to advantage in order to improve the tolerability of some drugs, as recently exemplified by the use of a fixed combination of ketoconazole and cyclosporin.

A case of destructive polyarthropathy in a 17-year-old youth following pefloxacin treatment.

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.

An international comparison of case definition of severe adverse cutaneous reactions to medicines.

There is substantial intercountry variation in the proportion of cases of toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) which are attributed to specific drugs. This study was undertaken to determine whether these differences might reflect biases in diagnosis of these conditions. A total of 138 reactions in 5 countries originally diagnosed as TEN or SJS were coded on to standardised forms. A single observer blind to the original diagnosis assessed each case according to specified criteria. This observer's diagnoses were compared with the original diagnoses. Overall, 111 of the 138 cases had information adequate for assessment. The blinded observer agreed with the diagnosis for 61% of cases where the original diagnosis was TEN and 58% of cases where the original diagnosis was SJS. There was no significant difference in rates of agreement when reactions attributed to sulphonamide antibiotics were compared with reactions attributed to other drugs. There were substantial and significant differences in percentage agreement between the blinded observer's diagnosis and the original diagnoses between countries. The lowest rates of agreement between the blinded observer and the original reports occurred in the US. Our results illustrate the difficulty in comparing reaction rates based on spontaneous reports between countries where the systems for gathering such reports vary. This illustrates the need for a minimum quantity of standard data and precise definitions of reactions if spontaneous reports of adverse reactions are to provide useful information about severe adverse skin reactions associated with drugs.

S15176 and S16950 interaction with Cyclosporin A antiproliferative effect on cultured human lymphocytes.

S15176 and S16950 are trimetazidine derivatives that antagonize more strongly than the parent drug mitochondrial toxicity, which leads to cellular hypoxia and nephrotoxicity in kidneys experimentally exposed to cyclosporin A. We have investigated whether every derivative might interact or not with the inhibitory effect of Cyclosporin A on the proliferation of cultured human lymphocytes. S15176 significantly increased the antilymphoproliferative effect of Cyclosporin A, whereas S15176 by itself neither displayed any antilymphoproliferative effect, nor did it induce any apoptotic process in cultured human lymphocytes. The effect of S16950 was not significant.

Lack of pharmacodynamic interaction between trimetazidine and cyclosporin A in human lymphoproliferative and mouse delayed hypersensitivity response models.

The hypothesis of an interaction between trimetazidine and the immunosuppressive effect of cyclosporin A was investigated in two models: a) ex vivo, the lymphoproliferative response of normal human lymphocytes to phytohemagglutinin and a murine monoclonal antibody against the CD3 T-lymphocyte membrane complex; b) in vivo, the delayed hypersensitivity response model in mouse. The uptake of methyl-3H-thymidine was measured in both models. For the lymphoproliferative response, statistical analysis showed that there was a significant inhibitory effect of cyclosporin A on cell proliferation (P < 0.001) and confidence intervals obtained by ANOVA showed the equivalence of the results when trimetazidine was combined with cyclosporin A (all CI95% < or = 10). In the delayed hypersensitivity model, cyclosporin A was also found to be very effective in inhibiting the immune response (P < 0.001), while trimetazidine did not interfere with cyclosporin A's effect. It was concluded that trimetazidine exerted neither an immunostimulatory nor an immunosuppressive effect in the two models, suggesting of the absence of interaction between trimetazidine and cyclosporin A's effectiveness when both drugs are given in combination.

How chronically administered valproate increases chlordiazepoxide transfer through the blood-brain barrier.

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.

Blood distribution of tenoxicam in humans: a particular HSA drug interaction.

Blood binding of tenoxicam was studied in vitro by equilibrium dialysis. Isolated human plasma proteins and blood cells were checked, and the distribution of the bound form was then calculated. The results showed that tenoxicam is mainly bound to HSA and that binding percentages are not different when measured in plasma (98.4%) and in an HSA solution at physiological concentration (704 microM, 98.15%). In these conditions, within the range of 1-150 microM, the tenoxicam binding percentage remained constant, evidence of a nonsaturable process. When a lower HSA concentration (10 microM) was used, the binding parameters of the tenoxicam interaction were calculated by using the same equilibrium dialysis data, by 3 methods of analysis- a stoichiometric method and site-oriented methods, fixing or not the number of HSA binding sites (n) as integer values. The best fit was observed with the first method, suggesting that two main interactions occurred. The site-oriented method gave lesser fits, the better being observed when n was not fixed. Its value, 1.77, suggest the possibility of two binding sites, one of them not preformed. The effects of known markers of site I, warfarin and apazone, of site II, diazepam and ibuprofen and of palmitic acid showed that tenoxicam is bound simultaneously to both sites I and II. The binding capacity of site I for tenoxicam is enhanced by diazepam: as this compound alone is bound to site II, this result suggests that the two HSA binding sites are not independent.

Blood binding and tissue uptake of drugs. Recent advances and perspectives.

The free drug hypothesis, which states that only the unbound moiety of drug in blood is available for tissue diffusion, is discussed according to recent investigations. In some experimental conditions, it must be assumed that part of the protein-bound drug in plasma is extracted during a single passage through the organ studied. The mechanisms underlying these observations are not unequivocal and remain hypothetical. In the liver, high-affinity binding sites for serum albumin have been demonstrated, and they would explain the high extraction by liver of endogenous and exogenous compounds. However, these experiments measure the unidirectional transfer of a drug from the vascular to the extravascular space in non-steady-state conditions. Hence, in steady-state conditions, the free drug hypothesis cannot be ruled out because it is supported by numerous pharmacokinetic studies.

Evidence for resveratrol-induced preservation of brain mitochondria functions after hypoxia-reoxygenation.

We have previously shown, as have other authors, that trans-resveratrol (E-resveratrol, 3,4,5-trihydroxy-E-stilbene) reduces reactive oxygen species (ROS) generation of mitochondria freshly isolated from healthy rat brains and that it also counteracts the effect of uncouplers (CCCP) on mitochondrial respiration and oxidative phosphorylation. Two main mechanisms have been shown: firstly, a scavenger effect toward O2- and secondly inhibition of complex III ROS generation. We now report on the effects of resveratrol in a pathological model that mimics the ischemia followed by the reperfusion process which may occur in the human brain. Isolated brain mitochondria were submitted first to hypoxia then to reoxygenation. The aim of this study was to determine the extent of mitochondrial damage induced by this experimental model, to demonstrate which mitochondrial functions were altered and to quantify the extent to which they were prevented by resveratrol. Resveratrol was either added to mitochondria freshly isolated from healthy rat brains or was injected by subcutaneous chronically implanted pumps (0.5, 2 and 10 mg/kg/day for 7 days). The rats were then sacrificed and mitochondria were extracted from brains. To evaluate the respective effects of hypoxia and reoxygenation on mitochondrial functions and the relevant effects of resveratrol, this drug was added (first protocol) either before the complete process (i.e., hypoxia and reoxygenation), or after anoxia before reoxygenation. We found that resveratrol prevented alterations of mitochondrial functions. This substance partly counteracted the decrease in respiratory control and the increase in ROS generation. It fully inhibited the alteration of membrane fluidity and the mitochondrial step of the apoptotic process (evidenced by cytochrome c release and membrane potential collapse). The effects of resveratrol were concentration-dependent (in vitro) or dose-dependent (ex vivo, second protocol). They were not significantly different when the drug was added before or after hypoxia, which suggests that in this model, reoxygenation was the most deleterious process and the stage at which resveratrol was most effective.

Potential interest of anti-ischemic agents for limiting cyclosporin A nephrotoxicity.

Chronic administration of cyclosporin A induces nephrotoxicity in humans. This is related to a cyclosporin A-induced constriction of afferent glomerular arterioles and mesangial cells, which leads to a decrease in filtration pressure and creatinine clearance. Afterwards, cellular lesions are observed involving mainly tubular atrophy and interstitial fibrosis, both of which are nonspecific. The initial mechanism of its toxicity is not clearly explained. The current pharmacological approach is symptomatic in order to counteract or minimize the consequences of a prime cause, which still remains to be defined. However, cyclosporin A has a deletereous effect on mitochondrial functions and mainly on ATP synthesis, which occurs when Ca2+ accumulates in matrix mitochondria. The effects of trimetazidine, an antischemic drug used in the treatment of angina pectoris, have been assessed. This drug is effective in experimental models of hypoxia induced by cyclosporin A: it restores ATP synthesis previously decreased by Ca2+ and cyclosporin A, and releases a part of Ca2+ excess accumulated by mitochondria at concentrations reached in humans at usual dosage regimens. At higher concentrations, it reverses the mitochondrial permeability transition previously generated (opened) by Ca2+ and a pro-oxidant such as terbutylperoxide (t-BH). It was also observed that trimetazidine does not modify the immunosuppressive effects of cyclosporin A in various models. These data suggest that nephrotoxicity of cyclosporin A is not irrevocably linked to its immunosuppressive effect but that it may be possible to counteract at least partly its nephrotoxic effects without altering its effectiveness in preventing graft rejection.

Clinical pharmacology of oxicams: new insights into the mechanisms of their dose-dependent toxicity.

Six oxicams, sudoxicam, isoxicam, piroxicam, tenoxicam, meloxicam and lornoxicam, were compared in an attempt to understand why, despite close chemical structures, two of them were associated with an increased risk of toxicity in patients. Different factors have been revealed which may explain these differences. A weak association constant to human serum albumin (HSA), together with a high plasma concentration, favours a rapid increase in unbound concentration (Cu) when total plasma concentration rises (peak of absorption). Pathological states may enhance this increase when both HSA plasma concentration is decreased and free fatty acid concentrations are increased. However, the main cause of toxicity may be the existence in some subjects of HSA natural mutants whose ability to bind oxicams is markedly lower than normal.

Methodological aspects of the evaluation of the binding of drugs to plasma and tissue proteins.

This paper describes the methodological aspects of the evaluation of the binding of drugs to plasma and tissue proteins. The relevance of the procedures employed to certain problems in clinical-pharmacology as well as the role of plasma binding in drug distribution and pharmacokinetics are discussed. The possibly important role of plasma protein binding of some drugs is emphasized. The incidence of plasma binding on drug distribution and pharmacokinetics are developed and the possible incidence of pathological states on drug plasma binding are considered.

A re-evaluation of the HSA-piroxicam interaction.

Piroxicam binding to HSA was studied using equilibrium dialysis and fluorescence methods. It was shown that this drug, like its analogs isoxicam and tenoxicam, binds to the apazone locus (site I area) and to a lesser extent to the diazepam site (site II). The piroxicam binding to HSA can be modulated by various specific ligands--apazone, warfarin, diazepam, ibuprofen--and these drug interactions have to be considered not only as potential displacement from the HSA binding sites but also in terms of induced allosteric effects.

[Pharmacologic and biologic basis of drug allergies]. / Bases pharmacologiques et biologiques des allergies médicamenteuses.

Drugs are able to activate the immune system, which may generate hypersensitivity states in individuals. This article first deals with the critical processes that are involved in drug sensitizations: what are the specific features of drugs as immunogens; how are drugs recognized as non-self by activated immune cells; what is the spontaneous outcome of an immune response to drugs in individuals; does a genetic predisposition regulate the immune response to drug antigens; how much are biotransformation processes involved in the immunogenicity of drugs? The second part is mostly devoted to the biological investigation of drug sensitizations: what are we looking for and why; are all the available methods equally suitable for routine diagnosis; what are the major methological problems that we have to deal with in investigating patients who have presented with symptoms which are clinically suspected to be of immuno-allergic origin?