Binding Of Gentamicin Research Articles

Decreased distribution of gentamicin in rat kidney by complexation with dextran sulfate

The effect of dextran sulfate on aminoglycoside concentration in rat renal cortex was studied. Dextran sulfate has one class involving many binding sites for aminoglycosides. Gentamicin has a higher affinity for dextran sulfate than either amikacin or dibekacin. A complex of dextran sulfate and gentamicin had no influence on the elimination of gentamicin from the blood, but gentamicin concentration in the renal cortex showed a significant decrease. The binding of gentamicin to brush-border membrane vesicles was inhibited non-competitively by dextran sulfate. Substantial amounts of administered dextran sulfate were found in the urine, possibly indicating that dextran sulfate may inhibit the interaction between aminoglycoside and brush-border membranes at the lumen of the renal proximal tubule. There is thus the possibility that dextran sulfate may effectively inhibit the distribution of aminoglycosides on the reabsorption process and decrease the accumulation within the renal cortex.

Binding of propranolol and gentamicin to small unimellar phospholipid vesicles: Contribution of ionic and hydrophobic forces

Binding of propranolol and gentamicin to small unilamellar phospholipid vesicles having different surface charges was studied at pH 4.4 using an ultra-centrifugation method, and the results were analyzed by an equation describing the Langmuir adsorption isotherms. Gentamicin, a polycationic drug, bound to negatively-charged small unilamellar vesicles composed of 60% phosphatidylcholine and 40% of either phosphatidylinositol, phosphatidylglycerol or phosphatidylserine in a manner consistent with a single class of binding sites but did not bind at all to small unilamellar vesicles of phosphatidylcholine alone. In contrast, propranolol bound readily to both neutral and negatively-charged liposomes in a manner consistent with two types of binding sites. Based on the binding parameters calculated from replots, it is suggested that the high-affinity site is probably at the surface of the liposome and that ionic forces are primarily responsible for this binding. The low-affinity, high-capacity binding site for propranolol was demonstrated with both neutral and negatively-charged liposomes and appeared to be independent of the surface charge. Gentamicin, which is not hydrophobic, did not bind to the low-affinity site. It is hypothesized that hydrophobic interactions are the driving force for propranolol binding to the low-affinity site which may be the interior of the lipid bilayer.

Renal tubule fragments compared with renal cortical slices for the study of gentamicin binding and uptake In vitro

Renal tubule fragments compared with renal cortical slices for the study of gentamicin binding and uptake In vitro

A membrane model for studying mechanisms of nephrotoxicity

Isolated brush border membrane vesicles were prepared from rat kidney cortex for examining the primary interaction between the nephrotoxic aminoglyscoside, gentamicin, and the kidney cell. The uptake of [ 3H]gentamicin was studied kinetically and was found to involve a single class of binding sites (k d = 40.8 μ m and n max = 3.9 nmol/mg protein). Structure-activity relationships were examined by determining the effects of chemically related substances on the uptake. Polyamino acids showed the greatest inhibitory activity, while sugars, polyamines and mono-amino acids were relatively inactive. Inhibition of gentamicin binding was found to be a function of polymer size, but charge did not appear to constitute an important structure-activity requirement for binding to the renal receptor.

Reduction of gentamicin nephrotoxicity by the concomitant administration of poly-l-aspartic acid and poly-l-asparagine in rats.

Williams and Hottendorf (1985) recently reported that poly-l-aspartic acid (pAsp) and poly-l-asparagine (pAsn) inhibit gentamicin (G) binding to brush border membrane vesicles in vitro and protect from G-induced nephrotoxicity in vivo. A model of infused rats was used to check for the early tissue alterations induced by G in animals receiving either G alone or the combination G + pAsp or G + pAsn. The cortical tissue was analysed 2 h or 2 days after the end of a 12 h infusion for signs of i) lysosomal phospholipidosis due to interference of G on phospholipids catabolism assessed by both biochemical (measurement of sphingomyelinase activity and of the total phospholipids content in renal cortex) and morphological analysis and ii) tubular regeneration and peritubular cells infiltration subsequent to focal necroses. While no reduction of G cortical levels was detected, significant changes in these parameters showed that G + pAsp and G + pAsn caused less phospholipidosis than G alone. Thus pAsp and pAsn decrease the severity of the early renal alterations induces by G.

Inhibition of Renal Membrane Binding and Nephrotoxicity of Gentamicin by Polyasparagine and Polyaspartic Acid in the Rat

The effect of the polyamino acids, polylysine, polyasparagine and polyaspartic acid, on 3H-gentamicin binding to rat renal brush border membranes was observed. All three polyamino acids showed inhibition of 3H-gentamicin binding at 1X and 0.1X the molar amount of gentamicin. Polylysine, polyasparagine and polyaspartic acid were subsequently tested in vivo for potential nephroprotective activity vs. gentamicin in a 14-day subacute nephrotoxicity model at 3X, 0.5X and 0.3X the molar dose of gentamicin, respectively. While polylysine did not produce significant nephroprotection, polyasparagine and polyaspartic acid completely eliminated gentamicin-induced nephrotoxicity.

Mechanism of action of gentamicin components. Characteristics of their binding to Escherichia coli ribosomes.

The binding of gentamicin (Gm) to Escherichia coli ribosomes and ribosomal subunits has been studied. By means of equilibrium dialysis and of statistical interpretation of the data it was found that [3H]gentamicin C2 and 6'-N-[3H]methylgentamicin C1a interact with three classes of sites on tight-coupled 70-S species: a first class concerning the tight and non-cooperative interaction with one drug molecule (Kd = 0.6 microM), a second class in which about five Gm molecules bind cooperatively (mean Kd = 10 microM), and a third class of very high capacity in which up to 70 drug molecules may interact. The extreme cooperativity of the third class of sites induces such an increase in the affinity for Gm that it may allow the shift of molecules already bound from high-affinity sites towards lower-affinity sites. The alteration of a ribosomal protein, L6, in a gentamicin-resistant mutant of E. coli abolished the multiclass and the cooperative aspects of ribosomes--gentamicin interaction. The large ribosomal subunits from E. coli MRE 600 strain interact cooperatively with Gm, whereas 50-S particles from the resistant mutant bind the drug in a diffuse way with high capacity and low affinity. The small subunits from both strains behave identically towards Gm. A good correlation is observed in comparing the gentamicin concentrations capable of saturating the different ribosomal classes of sites with concentrations inducing its multiphasic effects on protein synthesis.

Interaction of gentamicin and spermine with bilayer membranes containing negatively charged phospholipids.

We measured the electrophoretic mobility of multilamellar phospholipid vesicles, the 31P NMR spectra of both sonicated and multilamellar vesicles, and the conductance of planar bilayer membranes to study the binding of spermine and gentamicin to membranes. Spermine and gentamicin do not bind significantly to the zwitterionic lipid phosphatidylcholine. We measured the concentrations of gentamicin and spermine that reverse the charge on vesicles formed from a mixture of phosphatidylcholine and either phosphatidylserine or phosphatidylinositol. From these measurements, we determined that the intrinsic association constants of the cations with these negative lipids are all about 10 M-1. This value is orders of magnitude lower than the apparent binding constants reported in the literature by other groups because the negative electrostatic surface potential of the membranes and the resultant accumulation of these cations in the aqueous diffuse double layer adjacent to the membranes have not been explicitly considered in previous studies. Our main conclusion is that the Gouy-Chapman-Stern theory of the aqueous diffuse double layer can describe surprisingly well the interaction of gentamicin and spermine with bilayer membranes formed in a 0.1 M NaCl solution if the negative phospholipids constitute less than 50% of the membrane. Thus, the theory should be useful for describing the interactions of these cations with the bilayer component of biological membranes, which typically contain less than 50% negative lipids. For example, our results support the suggestion of Sastrasinh et al. [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350-358] that phosphatidylinositol is the major binding site for gentamicin in renal brush border membranes.

Gentamicin binding to brush border and basolateral membranes isolated from rat kidney cortex.

The interaction of gentamicin with renal cortex of rats has been studied in vitro by means of a binding assay to brush border and basolateral membranes. Gentamicin specifically bound to plasma membrane fractions, compared to other subcellular fractions. Gentamicin binding to brush border and basolateral membranes was markedly inhibited by polycations such as spermine, and was slightly inhibited by high concentrations of tetraethylammonium. The treatment of phospholipase A2 to both types of membranes increased gentamicin binding, although the treatments by proteolytic enzymes and sulfhydryl reagent did not affect the binding. Gentamicin binding was increased in the brush border membranes treated with acidic phospholipids, whereas it was decreased in the membranes treated with calcium. Judging from the determination of membrane surface charge by metachromasy of cationic dye, basolateral membranes seemed to contain more anionic sites than brush border membranes. The alterations of gentamicin binding described above correlated with the changes of anionic charge on the membranes, indicating a charge interaction between gentamicin and anionic binding sites on the membranes. The addition of other aminoglycoside antibiotics to the incubation mixture induced significant reductions in the binding of gentamicin in the order of aminoglycosides according to their positive charge. The present results suggest that the characteristics of gentamicin binding to brush border and to basolateral membranes are essentially similar, and therefore the renal accumulation of gentamicin may be regulated by the transport of gentamicin across both plasma membranes.

Disposition of tobramycin in patients with cystic fibrosis: A prospective controlled study

The pharmacokinetics of tobramycin in adolescents or young adults with cystic fibrosis and in age-matched controls were prospectively compared. Patients with CF had a higher tobramycin total body clearance (121.2 +/- 14.2 ml/min/1.73 m2) than did controls (102.2 +/- 18.9 ml/min/1.73 m2, P less than 0.05). This was not associated with a higher glomerular filtration rate (iothalamate total body clearance 147.5 +/- 29.2 ml/min/1.73 m2 in patients vs 142.9 +/- 33.3 ml/min/1.73 m2 in controls) or a lower binding of gentamicin to serum proteins (14.3% +/- 2.6% in patients vs 17.4% +/- 3.8% in controls). Tobramycin renal clearance was not significantly different in the two groups (89.5 +/- 17.9 ml/min/1.73 m2 in patients vs 81.0 +/- 15.8 ml/min/1.73 m2 in controls). In the control group, tobramycin total body and renal clearances were highly correlated with iothalamate total body clearance (r = +0.95 and +0.88, P less than 0.01). In patients with cystic fibrosis, the correlation was not significant (r = +0.56, P greater than 0.05 for total body clearance, and r = 0.32, P greater than 0.1 for renal clearance). There was no significant difference in volume of distribution normalized to body surface area or in half-life of elimination. The higher tobramycin total body clearance without an increase in renal clearance, and the lower correlation with glomerular filtration rate indicate that an extrarenal clearance pathway might play a significant role in the elimination of tobramycin from the serum of patients with cystic fibrosis.

Gentamicin/Heparin Interactions

Heparin has been reported to have two different effects on gentamicin. One is the interference by heparin with the quantitation of gentamicin; the other is an increase in the bound fraction of gentamicin in the presence of serum. The reports conflict regarding what concentration of heparin causes these effects. The main objective of this study was to determine what effect heparin had on the determination of gentamicin using an enzyme immunoassay (EIA) or a fluorescence polarization immunoassay (FPIA). Heparin concentrations studied simulated the amounts that would be found in plasma when blood samples are collected with an evacuated heparinized tube (less than 30 U/ml), or the greater amounts that might be present when blood is collected from indwelling lines (100-200 U/ml). Heparin had no effect on the quantitation of gentamicin by FPIA. In measurements done by EIA, heparin had no effect at 28 U/ml but did significantly inhibit the reaction by 10-20% when its concentration was 100-200 U/ml. We studied the effects of heparin on the distribution equilibrium of gentamicin by measuring free gentamicin produced in an ultrafiltrate. It was found that the bound fraction increased as the heparin concentration increased. Our results with aqueous and serum-based samples suggest that the increased binding was the result of a direct binding of gentamicin to heparin, as well as a more complex interaction involving heparin, gentamicin, and proteins.

Molecular approach to the binding of the antibiotics gentamicin (GM) and amikac in (AK) to lipid bilayers containing phosphatidylinositol: A method to study the mechanism of aminoglycoside-induced phospholipidosis and nephrotoxicity

Molecular approach to the binding of the antibiotics gentamicin (GM) and amikac in (AK) to lipid bilayers containing phosphatidylinositol: A method to study the mechanism of aminoglycoside-induced phospholipidosis and nephrotoxicity

Gentamicin Binding to the Isolated Crista Ampullaris of the Guinea Pig

Isolated cristae ampullares of the guinea pig were incubated in vitro to study the kinetics of gentamicin binding. Binding of radiolabelled gentamicin reached an equilibrium at 10 min. Three binding sites were determined: Kd = 30 microM, n = 3.2 nmoles drug/mg protein; Kd = 317 microM, n = 26 nmoles; and Kd = 965 microM, n = 52 nmoles. Gentamicin remained bound to the crista after glutaraldehyde fixation and processing for electron microscopy. It is suggested that the isolated crista ampullaris may serve as a useful model for the study of aminoglycoside kinetics as related to ototoxicity.

Mechanism of aminoglycoside-induced lysosomal phospholipidosis: In vitro and in vivo studies with Gentamicin and Amikacin

Gentamicin, a widely used aminoglycoside antibiotic, is concentrated in lysosomes of proximal tubular cells of the kidney, and induces therein an accumulation of myelin-like material. We show that treatment of rats with Gentamicin (10 mg/kg, 7 days) induces a loss of activity of lysosomal sphingomyelinase and phospholipase A 1, associated with an increase in the amount of total lipid phosphorus in the kidney cortex. In vitro, Gentamicin is shown by gel permeation to bind to phospholipid bilayers (liposomes) under conditions which mimic the lysosomal environment (acid pH and presence of phosphatidylinositol). The reversal of this binding by an increase in the ionic strength (> 0.04) suggests electrostatic interaction between the hydrophilic, polycationic aminoglycoside and the negatively charged phospholipids. Binding of Gentamicin impairs the hydrolysis of phosphatidylcholine present in the bilayer, by lysosomal phospholipases A 1 and A 2 from the liver or kidney. We also show that lysosomal sphingomyelinase is readily and irreversibly inactivated by liposomes in the absence of detergent. The lysosomal phospholipidosis induced by Gentamicin in the kidney, as in cultured cells [Aubert Tulkens et al., Lab. Invest. 40, 481 (1979)] appears therefore to be a direct consequence of the lysosomotropic character of this drug and its ability to inhibit therein phospholipid breakdown. Amikacin, a semi-synthetic aminoglycoside, binds more loosely to phospholipid bilayers, induces less inhibition of phospholipases in vitro and is less taken up by tubular cells in vivo. Accordingly, Amikacin does not provoke significant lysosomal phospholipidosis or loss of sphingomyelinase and phospholipase A 1 activities in vivo at the doses and time investigated (0–40 mg/kg, 7 days). Inasmuch as Amikacin is reported to be less toxic to the kidney, we suggest that lysosomal alterations are an early and significant

Reduction of experimental gentamicin nephrotoxicity in rats by dietary calcium loading.

Since gentamicin accumulates in the renal cortex before renal failure and calcium reduces gentamicin binding to cell membranes, we examined the effect of dietary calcium loading on gentamicin toxicity in male F344 rats. Rats were fed either a normal (0.5%)- or a high (4%)-calcium-content chow. Calcium loading did not alter inulin clearance, urinary excretion of sodium or total osmoles, or serum ionized calcium. However, calcium loading caused elevation of urinary calcium excretion and lowered urinary cyclic AMP levels. The data suggest that calcium loading may have slowed the accumulation of gentamicin (40 mg/kg per day) by the renal cortex. Histological and functional evidence of nephrotoxicity was delayed in onset and attenuated in magnitude. The results indicate that dietary calcium loading reduces experimental gentamicin nephrotoxicity. Protection may be associated with slower renal cortical accumulation of gentamicin. Calcium-dependent membrane or intracellular events may mediate this effect.

The Tissue‐Disposition of Gentamicin in Rats

Abstract: The tissue disposition of 14C‐gentamicin was studied in young rats after a single‐dose administration by autoradiographic techniques. The autoradiograms showed a high accumulation of the substance in non‐parenchymateous tissues such as cartilages and connective tissues and a strong uptake in the kidney cortex. The in vivo bound substance was displaced from the cartilage at incubations in cation‐containing solutions, indicating an ionic binding of the polycationic gentamicin to negatively charged groups in the cartilage (probably carboxyl and sulphate groups of chondroitin sulphate). In the inner ear a localization of the substance was found in the membraneous walls of the chochlea and, at short survival intervals, in the perilymph, whereas the radioactivity in the endolymph was low. Studies in vitro showed an ionic binding of gentamicin to cartilage and in addition to several other tissues, such as the lymphomyeloid system, the gastrointestinal mucosa, the exocrine pancreas, salivary glands, and most prominent, the pigmented tissues. A strong in vitro binding of gentamicin to pigment from beef eyes was also shown. The potential binding sites in the latter tissues are localized intracellularly and are therefore apparently unattainable for the charged hydrophilic gentamicin molecule in vivo.

Gentamicin inactivation in purulent exudates: role of cell lysis.

Factors contributing to the binding and reversible inactivation of gentamicin by purulent exudates were studied in a simplified in vitro model consisting of purified human polymorphonuclear leukocytes (PMNLs). Whereas intact PMNLs (10(6)-10(8)/ml) bound almost no [14C]gentamicin, freeze-thawed PMNLs showed extensive [14C]gentamicin binding, expressed as antibiotic cosedimenting with particulate material from the lysed PMNLs. Antibiotic binding could be related to the concentration of lysed PMNLs and to the amount of [14C]gentamicin added. Binding of [14C]gentamicin by lysed PMNLs was highly sensitive to DNase I but was unaffected by RNase, Triton X-100, or protease. Purified chromatin or DNA from either purulent exudates or lysed PMNLs reproduced the [14C]gentamicin-binding pattern obtained with crude PMNL lysate. These results show that gentamicin inactivation in purulent exudates can be correlated with binding of the antibiotic to lysed PMNLs; PMNL chromatin DNA is identified as one of the major binding factors.

Binding of gentamicin to subcellular fractions of rabbit kidney: inhibition by spermine and other polyamines.

Binding of gentamicin to subcellular fractions of rabbit kidney: inhibition by spermine and other polyamines.

Gentamicin binding to serum and plasma proteins.

Gentamicin binding to serum proteins was studied by equilibrium dialysis at 37 degrees C and pH 7.4 in the presence of both physiologic and adjusted concentrations of ionized calcium and magnesium. The percentage of bound drug was inversely related to the concentration of these two divalent cations, raning from 27% bound with no calcium and magnesium present to 17% bound in the presence of four times physiologic concentrations. No significant difference in the extent of drug-protein binding was noted in a comparison of sera from healthy and uremic subjects. Heparin also was found to affect gentamicin binding. Increasing heparin concentration in serum increased apparent gentamicin-protein binding to 34% in the presence of physiologic amounts of calcium and magnesium. Buffered heparin solutions without plasma proteins bound up to 65% of total drug concentration. Gentamicin-protein binding may have implications regarding pharmacokinetics and renal cortical uptake.

Standardization of a preparative ultracentrifuge method for quantitative determination or protein binding of seven antibiotics.

A preparative ultracentrifuge method was standardized for determination of quantitative binding of cephalothin, cefamandole, cefazolin, cefaclor, erythromycin, gentamicin, and chloramphenicol to human serum proteins. At achievable in vivo concentrations, serum binding was 78.5% for cephalothin, 79.9% for cefamandole, 88.5% for cefazolin, 23.5% for cefaclor, 41.9% for erythromycin, 22.7% for gentamicin, and 59.5% for chloramphenicol. Techniques that use semipermeable cellophane or diaflow membranes, cross-linked dextran, inhibition of bacterial growth, protein precipitation, or liquid partitioning all have inherent problems with either the ligand or the antibiotic adversely interacting with the experimental apparatus. Ultracentrifugation provides a rapid, reproducible technique for protein-binding determinations of the classes of antibiotics described.