Mb. Carlier

Inhibition of lysosomal phospholipases by aminoglycoside antibiotics: in vitro comparative studies.

Aminoglycoside antibiotics induce an early and characteristic lysosomal phospholipidosis in cultured fibroblasts and in kidney tubular cells. We have recently demonstrated an inhibition of lysosomal phospholipases A1 and A2 by gentamicin and amikacin in vitro. In vivo, gentamicin decreases the activity of phospholipase A1 (Laurent et al., Biochem. Pharmacol. 31:3861-3870, 1982). In the present study, we examined 14 aminoglycosides for in vitro inhibition of phospholipases. To mimic the situation prevailing in lysosomes, the enzymatic activities were assayed with phospholipid vesicles (liposomes) with a composition similar to that of lysosomal phospholipids (phosphatidylcholine, sphingomyelin, phosphatidylinositol, cholesterol; 4:4:3:5.5, molar ratio). We measured the hydrolysis of 1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine contained in the liposomes by a soluble fraction of highly purified lysosomes isolated from rat liver. Similar IC50S (concentrations causing 50% inhibition of enzymatic activity) were observed for dibekacin, gentamicin (with no major difference between C1, C1a, or C2), netilmicin, tobramycin, and kanamycin B. Sisomicin was slightly more inhibitory. Kanamycin A, N1-(L-4-amino-2-hydroxy-1-oxobutyl)dibekacin, and amikacin showed increasing IC50S. Streptomycin caused the least inhibition. Octa- and tetramethylkanamycin A are much less inhibitory than the parent drug. These results point to the number, the nature, and the respective positions of the cationic groups as essential determinants in causing inhibition of phospholipid breakdown. The binding of three aminoglycosides (gentamicin, amikacin, streptomycin) to the liposomes at pH 5.4 was also measured by gel permeation and was found to be related to the respective inhibitory potency of each drug. Insofar as lysosomal phospholipidosis is an early sign of intoxication by aminoglycosides, these results may serve as a basis for the development or screening of less toxic compounds in this class of antimicrobial agents.

Increased renal DNA synthesis in vivo after administration of low doses of gentamicin to rats.

Kidney cortex DNA synthesis was studied in female rats treated with a low dose of gentamicin (10 mg/kg) up to 14 days. Synthesis was measured by incorporation of [3H]thymidine into DNA 1 h after intraperitoneal injection of the labeled precursor (200 muCi per animal). Gentamicin given in one injection per day resulted in a greater incorporation of [3H]thymidine into DNA after both 7 and 14 days of treatment as compared with control animals. When the daily dose was divided into three equal injections given at 8-h intervals, a statistically significant increase in thymidine incorporation was observed as early as 4 days after starting gentamicin administration. Excellent agreement was found between DNA specific radioactivity and kidney cortex nuclear labeling, as measured by histoautoradiography. The greatest amount of [3H]thymidine incorporation occurred within proximal tubular cells and interstitial cells. We conclude that a finite duration of gentamicin treatment at low dosage induces an increased DNA synthesis in vivo in rat kidney cortex. We suggest that this reaction results from cellular proliferation and could reflect a regenerative process after focal necrosis induced by gentamicin at low doses. The demonstrated early increase in DNA synthesis could be a useful tool to measure kidney cortex alterations caused by various aminoglycosides at low, therapeutic doses. Images

Interaction of Streptomycin and Streptomycylamine Derivatives With Negatively Charged Lipid Layers - Correlation Between Binding, Conformation of Complexes and Inhibition of Lysosomal Phospholipase Activities

Aminoglycoside antibiotics induce a lysosomal phospholipidosis in kidney proximal tubules after conventional therapy in animals and man. We have previously demonstrated that these drugs bind to negatively charged phospholipid bilayers at acid pH and inhibit the activity of lysosomal acid phospholipases in vitro and in vivo. A combined biochemical and conformational study [Brasseur et al., Biochem. Pharmac. 33, 629 (1984)] showed major and consistent differences between 6 aminoglycosides in current clinical use with respect to the stability of the complexes they form with phosphatidylinositol, their inhibitory potency towards the activity of lysosomal phospholipases and their current toxicity ranking (e.g. gentamicin greater than amikacin greater than streptomycin). In the present study we have extended this approach to experimental derivatives of streptomycin. The derivatives examined were: dihydrostreptomycin, dideguanyldihydrostreptomycin, streptomycylamine, dideguanylstreptomycylamine, N-butyl- and N-benzyl-dideguanylstreptomycylamine. These compounds were examined for (i) their binding to negatively charged liposomes, measured by gel permeation on Sepharose 4B; (ii) their interactions with phosphatidylinositol assessed by semi-empirical conformational analysis and (iii) their inhibitory effect on the activities of lysosomal phospholipases towards phosphatidylcholine present in negatively charged liposomes. Streptomycin and gentamicin were also used as reference compounds with low and high affinity (and inhibitory potency), respectively. Our observations can be summarized as follows: (i) the replacement of the aldehyde in the streptose ring by a methylamino group strikingly changes the conformation of the molecule, allowing a better interaction with phosphatidylinositol. Thus, streptomycylamine binds much more tightly to phospholipid bilayers and shows a higher inhibitory potency towards phospholipase activity, as compared to streptomycin. The conformational analysis shows, however, that this effect is only partially due to the additional cationic charge carried by streptomycylamine. Other modifications of the streptomycin molecule, such as the replacement of the guanidinium groups by aminogroups or the addition of hydrophobic moieties (butyl or benzyl groups) to the streptose do not markedly further strengthen the interactions of the molecule with phosphatidylinositol. (ii) Even though some derivatives (e.g. dideguanylstreptomycylamine) bind as tightly to phospholipids as gentamicin, they remain much less inhibitory towards lysosomal phospholipases.(ABSTRACT TRUNCATED AT 400 WORDS)

In Vitro Inhibition of Lysosomal Phospholipases by Aminoglycoside Antibiotics: A Comparative Study

Aminoglycosides induce an early and characteristic phospholipidosis in the lysosomes of kidney tubular cells. Inhibition of lysosomal phospholipase activity by gentamicin has been demonstrated in vivo and in vitro. Ten aminoglycosides have been examined for their potency in inhibiting phospholipases A1 and A2. Similar IC50s and IC90s (concentrations causing 50 and 90% inhibition) were found for sisomicin, dibekacin, gentamicin, tobramycin, kanamycin B and netilmicin. Kanamycin A, HABA-dibekacin and amikacin showed increasingly higher ICs. Thus, both the number and the position of amino groups are important. Streptomycin had little effect on phosphatidylcholine hydrolysis. Kinetic studies with gentamicin suggest a competitive type of inhibition. This approach may help for the in vitro screening of less toxic aminoglycosides.

Early Toxic Events in Kidney of Rat and Man Following Administration of Gentamicin at Low Doses

Kidney cortex of rats and humans treated with low doses of Gentamicin have been examined both morphologically and biochemically. Specific alterations of the lysosomes (loss of activity of phospholipases; accumulation of phospholipid-like material) was detected as early as from 2-4 days of treatment. This finding is consistent with the concept that Gentamicin exerts its toxic action on kidney through lysosomal dysfunction.

In Vitro Study and Conformational Analysis of 1-N-Aminohydroxybutyryl Derivatives of Aminoglycosides in Correlation with Their Inhibitory Potency Towards Lysosomal Phospholipases

Aminoglycoside nephrotoxicity involves the development of a lysosomal phospholipidosis in proximal tubular cells (Tulkens 1986). In vitro, aminoglycosides bind to negatively charged phospholipid bilayers and inhibit the activities of lysosomal phospholipases (Laurent et al. 1982; Mingeot-Leclercq et al. 1987). By computer-aided conformational analysis, it was observed that amikacin, a semisynthetic derivative of kanamycin A in which the N1 aminofunction is substituted by a 4-amino-2-hydroxy-1-oxobutyl (aminohydroxybutyryl, AHBA) moiety and which is reported to be less nephrotoxic than gentamicin and tobramycin (Hottendorf and Gordon 1980; Holm et al. 1983), displays a markedly different mode of insertion and a lower energy of interaction, binds less tightly to negatively charged lipid layers, and is a less potent inhibitor of lysosomal phospholipases than most other 2-deoxystreptamine-containing aminoglycosides (Carlier et al. 1983). This difference, however, could be related to the lower number of cationic charges of amikacin (4) or to the presence of the AHBA side chain. Therefore other experimental aminoglycosides carrying a 1-N-aminohydroxybutyryl side chain and patterned after dibekacin, tobramycin, and kanamycin B, which all possess five amino groups (Fig. 1), have been examined for their ability to bind to negatively charged lipids and inhibit lysosomal phospholipase activity.

Tubular Regeneration in Rat Kidney Cortex During Treatment with Gentamicin at a Low Dose

Tissue injury induced in rat kidney cortex by gentamicin at a low dose (10 mg/kg) has been studied by measuring subsequent regeneration after treatment for 4, 7, and 14 days. Cell proliferation was demonstrated by the incorporation of [3H]-thymidine into kidney cortex DNA. In treated animals, the specific radioactivity of DNA increased up to 4.6 times the mean value found in the controls. Autoradiography showed labelling of nuclei in proximal tubular cells. Electron microscopy showed that a subpopulation of tubular cells, devoid of myeloid bodies, which are characteristic of gentamicin intoxication, is also poorer in peroxisomes, and therefore may belong to less differentiated, regenerating cells. It is concluded that a low dose of gentamicin induces measurable tissue regeneration.

Renal Phospholipidosis in Rats after Gentamicin and Pefloxacin Coadministration at Low Doses

Fluoroquinolones constitute a new class of highly active, wide spectrum antimicrobials (see Wolfson & Hooper, 1985; and Hooper & Wolfson, 1985 for review), among which pefloxacin is one of the first to have reached wide clinical usage. Since up to 60% of the administered dose of pefloxacin is eliminated through the kidney, it appears important to examine its potential nephrotoxicity. Moreover, pefloxacin can be administered in combination with an aminoglycoside in order to broaden the spectrum covered by the antiinfective therapy and/or to decrease the risk of emergence of bacterial resistance to either of these drugs (Pechere et al., 1987). Combination of other antibiotics with an aminoglycoside may increase (viz. vancomycin; Wood et al., 1986; Francq-Dufief et al., 1987), or decrease (viz. fosfomycin or piperacillin; Laurent et al., 1985; Carlier et al., 1987) aminoglycoside toxicity. In this connection, we have examined and report here on the influence of pefloxacin on the development of lysosomal phospholipidosis, an early, sensitive and predictive index of aminoglycoside-induced nephrotoxicity in animals and man (see Tulkens, 1986, for review).

Comparative Uptake and Lysosomal Phospholipidosis Induced by Gentamicin Components C1, C1a, and C2

Aminoglycosides are nephrotoxic and this adverse effect has triggered many efforts towards the design and/or the screening of less toxic derivatives (see Price, 1986 for a recent review). Yet, the first broad-spectrum and still widely used aminoglycoside, gentamicin, is not a pure substance and is actually commercialized as a mixture of three main components, C1, C1a and C2, which differ by the methylation of the N6 and C6 atoms in the 2’,6’ diaminosugar moiety. Surprisingly enough, little information is available concerning the relative nephrotoxicities of these components. Whereas some reports suggest that gentamicin C1 induces less nephrotoxicity than gentamicin complex in humans (see e.g., Mossegaard et al., 1975) , others failed to substantiate such difference (e.g., Forrey et al., 1978). Kohlepp et al. (1984) showed in a comparative study in rats that gentamicin C2 and gentamicin C1a are more nephrotoxic than gentamicin C1 at an equivalent, high dosage (40 mg/kg). The uptake of gentamicin complex by rat kidney cortex, however, is saturable, with an apparent Km in a 10–20 mg/k serum concentration range (Gauliano et al., 1986).