Previously, daptomycin was shown to reduce tobramycin nephrotoxicity in vivo (D. Beauchamp, M. Pellerin, P. Gourde, M. Pettigrew, and M. G. Bergeron, Antimicrob. Agents Chemother. 34:139-147, 1990; C. A. Wood, H. C. Finkbeiner, S. J. Kohlhepp, P. W. Kohnen, and D. C. Gilbert, Antimicrob. Agents Chemother. 33:1280-1285, 1989). Female Sprague-Dawley rats were treated with saline (NaCl, 0.9%), daptomycin (10 mg/kg of body weight every 12 h, subcutaneously), gentamicin (30 mg/kg/12 h, intraperitoneally) or with a combination of daptomycin plus gentamicin over a 10-day period. Animals were killed 4, 10, and 20 days after the end of treatment. Four days after the end of drug administration, gentamicin and daptomycin levels in the renal cortices of animals treated with the combination of daptomycin and gentamicin were significantly higher than in those of rats given gentamicin or daptomycin alone (P < 0.01). Despite the higher cortical concentrations of gentamicin, rats given the combination of gentamicin and daptomycin had less reduction in renal cortex sphingomyelinase activity, less evidence of regeneration of cellular cortical cells ([3H]thymidine incorporation into cortex DNA), lower creatinine concentration in serum, and less histopathologic evidence of injury than rats given gentamicin alone. By immunogold technique, both daptomycin and gentamicin were localized to the lysosomes of proximal tubular cells, regardless of whether animals received the drugs alone or in combination. Interestingly, myeloid body formation occurred in both those animals given gentamicin alone and those given daptomycin plus gentamicin. No significant changes were observed for all groups between 10 and 20 days after the end of therapy, suggesting that the toxicity of gentamicin was not delayed by the concomitant injection of daptomycin. The results confirm that daptomycin can attenuate experimental gentamicin nephrotoxicity.

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The lipopeptidic antibiotic daptomycin is reported to reduce experimental tobramycin nephrotoxicity (D. Beauchamp, M. Pellerin, P. Gourde, M. Pettigrew and M. G. Bergeron, Antimicrob. Agents Chemother. 34:139-147, 1990; C. A. Wood, H. C. Finkbeiner, S. J. Kohlhepp, P. W. Kohnen, and D. C. Gilbert, Antimicrob. Agents Chemother. 33:1280-1285, 1989). In an attempt to explain these results, the in vivo and in vitro interactions between daptomycin and tobramycin were studied. Tobramycin alone and preincubated with negatively charged phospholipid bilayers (liposomes) was dialyzed against increasing concentrations of daptomycin in buffer at pH 5.4. A significant drop in the concentration of tobramycin was observed when daptomycin was added to the opposite half cells. Furthermore, daptomycin induced a concentration-dependent release of lipid-bound tobramycin. Gold labeling experiments showed that daptomycin could be incorporated into phospholipid layers. Female Sprague-Dawley rats were treated with daptomycin alone, with tobramycin alone, or with the combination over 2 to 10 days. Levels of daptomycin and tobramycin in serum were similar in all groups. The levels of tobramycin in the renal cortex increased significantly with time and, on day 10, reached values of 654 +/- 122 and 844 +/- 298 micrograms/g of tissue (mean +/- standard deviation; not significant) in animals treated with tobramycin and the combination of daptomycin-tobramycin, respectively. No significant difference was observed in the levels of tobramycin in the kidneys between animals treated with tobramycin or the daptomycin-tobramycin combination at any time. By contrast, daptomycin levels were significantly higher in the renal cortexes of animals treated with daptomycin-tobramycin in comparison with those in the renal cortexes of animals treated with daptomycin alone on days 6,8, and 10 (P < 0.01). For immunogold labeling studies, animals were killed 4 h after a single injection of daptomycin alone or daptomycin in combination with tobramycin. Daptomycin was found throughout the matrixes of the lysosomes of proximal tubular cells of animals treated with daptomycin alone. In animals treated with the combination of daptomycin and tobramycin, daptomycin was associated with intralysosomal myeloid bodies. Our results suggest that daptomycin might attenuate experimental aminoglycoside nephrotoxicity by interacting with the aminoglycoside, perhaps electrostatically, and thereby protecting intracellular targets of toxicity.

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Previous studies in experimental animals showed that daptomycin, a lipopeptide antibiotic, protects against aminoglycoside nephrotoxicity (C. A. Wood, H. C. Finkbeiner, S. J. Kohlhepp, P. W. Kohnen, and D. N. Gilbert, Antimicrob. Agents Chemother. 33:1280-1285, 1989; D. Beauchamp, M. Pellerin, P. Gourde, M. Pettigrew, and M. G. Bergeron, Antimicrob. Agents Chemother. 34:139-147, 1990). In order to better understand the mechanism involved in this protective effect, the subcellular distribution of daptomycin was investigated in the proximal tubular cells of animals treated with daptomycin alone or in combination with tobramycin. A first group of female Sprague-Dawley rats received a single intravenous injection of daptomycin at a dose of 100 mg/kg of body weight and were killed at 10 min, 1 h, or 24 h after the injection. Other groups of rats were treated during 10 days with saline (NaCl, 0.9%), tobramycin at dosages of 20 mg/kg/12 h, daptomycin at dosages of 10 mg/kg/12 h, or the combination tobramycin-daptomycin at the same dosages. At the time of sacrifice, the renal cortex of the right kidney of each animal was dissected, and small blocks of tissue were fixed, dehydrated, and embedded in Araldite 502 epoxy resin. The subcellular distribution of daptomycin and tobramycin was determined on ultrathin sections by immunogold labeling. Ten minutes after the injection of daptomycin alone, gold particles were seen over the brush border membrane and on the membranes of the endocytic vacuoles of proximal tubular cells. One hour after the injection, a similar distribution was seen and numerous gold particles were found over the lysosomes of proximal tubular cells. The results suggest that daptomycin might protect against aminoglycoside nephrotoxicity by interfering with the interaction between the aminoglycoside and phospholipids inside the lysosomes of proximal tubular cells.

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The differential tissue distributions of aztreonam and ceftazidime within fibrin clots infected with Pseudomonas aeruginosa, Enterobacter cloacae, and Serratia marcescens, their efficacies, and the in vivo bacterial morphological changes induced by these drugs were evaluated. Rabbits were given intravenously a single dose of 100 mg of either agents/kg of body weight. In the cores of the clots, the peak levels of both drugs were much lower than those observed in the peripheries and in serum. Aztreonam's half-lives within the peripheries and in the cores of the fibrin clots were up to six times higher than observed in serum, while ceftazidime's half-lives in clots were twice that observed in serum. This resulted in a much greater penetration ratio for aztreonam than for ceftazidime. Both drugs controlled the growth of P. aeruginosa in vivo, but E. cloacae and S. marcescens responded better to ceftazidime. Morphological changes were more abundant in the peripheries than in the cores of the clots. In the control group, P. aeruginosa's morphology in the cores was different than that in the peripheries of the clots. Against P. aeruginosa, aztreonam did induce morphological changes in the cores while ceftazidime did not. Electron microscopic studies revealed that morphological changes associated with aztreonam seemed different than those of ceftazidime. Along with elongation of bacteria, more bow tie and herniated bacteria were observed with aztreonam. Though both agents selectively affect PBP 3, as manifested by elongated bacteria, they induce in the peripheries of the clots thickening, breaks, and detachment in bacterial cell walls, alterations which are generally associated with antibiotics affecting PBP 1a and 1b.

The effect of timing of gentamicin dosing relative to food access periods was evaluated in experimental animals. Female Sprague-Dawley rats were treated for 4 and 10 days with gentamicin (40 mg/kg of body weight/day) intraperitoneally at either 0700, 1300, 1900, or 0100 h according to three food presentation schedules: food was available from 0800 to 1600 h in the first group, from 1600 to 0000 h in the second group, and from 0000 to 0800 h in the last group. Animals were thus subjected to a restricted feeding period. Results indicate that time-restricted feeding schedules displace the peak and the trough of gentamicin-induced renal toxicity, as evaluated by changes in the inhibition of sphingomyelinase activity, cellular regeneration (incorporation of [3H]thymidine into DNA of renal cortex), and blood urea nitrogen and serum creatinine levels, as well as histopathological lesions observed after 10 days of treatment. In fact, the toxicity was minimal when gentamicin was injected during the feeding period, while the maximal toxicity was found when gentamicin was administered during the fasting period. It is concluded that the feeding period can modulate aminoglycoside nephrotoxicity. The time of dosing of gentamicin relative to the time of feeding seems to be a more important modulator of gentamicin nephrotoxicity than the light-dark cycle.

The effect of fleroxacin on gentamicin-induced nephrotoxicity was evaluated with female Sprague-Dawley rats. Animals were injected during 4 or 10 days with saline (NaCl; 0.9%), gentamicin alone at doses of 10 and 40 mg/kg of body weight/12 h (subcutaneously), fleroxacin alone at a dose of 25 mg/kg/12 h (intraperitoneally), or the combination gentamicin-fleroxacin in the same regimen. Gentamicin induced a dose- and time-dependent renal toxicity as evaluated by gentamicin cortical levels, sphingomyelinase activity in the renal cortex, histopathologic and morphometric analysis, blood urea nitrogen and serum creatinine levels, and cellular regeneration ([3H]thymidine incorporation into DNA of cortical cells). The extent of these changes was significantly reduced when gentamicin was given in combination with fleroxacin. Although the mechanisms by which fleroxacin reduces the nephrotoxic potential of gentamicin are unknown, we propose that the fleroxacin-gentamicin combination enhances exocytosis activity in proximal tubular cells, as suggested by the higher excretion of urinary enzymes and lower cortical levels of gentamicin observed in animals treated with the combination fleroxacin-gentamicin compared with those treated with gentamicin alone. The protective effect of fleroxacin on gentamicin nephrotoxicity should be investigated further.

Recent findings suggest that nitric oxide (NO) is an important biologic mediator which exerts a wide variety of effects on numerous physiological and pathophysiological processes. L-Arginine is oxidized to L-citrulline with concomitant NO production; as a result, nitrate and nitrite accumulates. This study was conducted to determine the potential NO production by proximal tubular cells (PTC) in response to bacterial lipopolysac-charides (LPS) and cytokines and to evaluate the cytotoxic effect associated with NO release. After a 7-day stimulation with LPS (100 micrograms/ml), interleukin-1 beta (IL-1 beta) (10 ng/ml), and tumor necrosis factor alpha (TNF-alpha) (10 ng/ml), the nitrate and nitrite levels were determined by a spectrophotometric method based on the Griess reaction. Moreover, alpha-methylglucopyranoside phosphate and lactate dehydrogenase release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay served as indicators of sodium-dependent hexose transport integrity and cell death, respectively. IL-1 beta and TNF-alpha used alone or together or combined with LPS led to a significant generation of NO by PTC. Our results also demonstrate that NO induced by LPS and cytokines could inhibit sodium-dependent transport and could induce PTC damage.

Evidence for temporal variations in the nephrotoxicity of low doses of aminoglycosides were recently shown by using specific and sensitive parameters of renal toxicity. The aim of the present study was to evaluate the effect of a short period of fasting on the temporal variations in the renal toxicity of gentamicin. Twenty-eight normally fed (i.e., food and water were available ad libitum throughout the experiment) female Sprague-Dawley rats (weight, 175 to 220 g) and 28 fasted rats (i.e., only water was available during a 12-h fast before and a 24-h fast after gentamicin injection) were used. The animals were synchronized on a 14-h light, 10-h dark cycle (lights on at 0600 h) for 1 week before gentamicin administration. In July 1993, each group of animals was treated with a single intraperitoneal injection of saline (NaCl, 0.9%) or gentamicin (150 mg/kg of body weight) at either the peak (1400 h) or the trough (0200 h) of the previously determined toxicity. On day 1, the 24-h urinary excretion of beta-galactosidase, N-acetyl-beta-D-glucosaminidase, and gamma-glutamyltransferase was significantly higher in normally fed animals treated with gentamicin at 1400 h than in their time-matched controls and in normally fed animals treated at 0200 h (P < 0.01), which had normal levels of these enzymes. By contrast, the urinary excretion of these enzymes was significantly higher in both groups of gentamicin-treated, fasted rats than in their time-matched control groups (P < 0.01), reaching levels similar to those measured in normally fed rats treated at 1400 h. The accumulation of gentamicin was significantly lower in the renal cortex of normally fed rats treated at 0200 h than in rats treated at 1400 h (P < 0.05), but this time-dependent difference was not found in fasted rats treated at 0200 and 1400 h. Immunogold labeling done on ultrathin sections and observed by electron microscopy showed a similar subcellular localization of gentamicin in normally fed and fasted rats treated at either 1400 or 0200 h. These results suggest that the feeding period is of crucial importance in the temporal variations of the nephrotoxicity of gentamicin in rats.

The temporal variation in the nephrotoxicity of low doses of isepamicin was studied in male Sprague-Dawley rats treated with a single daily intraperitoneal injection of saline (NaCl, 0.9%) or isepamicin (80 mg/kg of body weight) at either 0800, 1400, 2000, or 0200 h for 4 and 10 days. On day 10, the cellular regeneration (incorporation of [3H] thymidine into DNA of renal cortex) and cortical accumulation of isepamicin were significantly higher in animals treated at 1400 h than at 0200 h (P < 0.01). Immunogold labeling studies showed that isepamicin was essentially localized in the lysosomes of proximal tubular cells in all treated groups, but the density of the gold particles over the lysosomes was higher in animals treated at 1400 than at 0200 h. The results of the present study show that the renal toxicity of isepamicin was maximal at 1400 h (midlight period) and minimal at 0200 h (middark period).

The pharmacokinetics and distribution in tissue of 2',3'-dideoxyinosine (ddI) encapsulated in sterically stabilized liposomes have been evaluated in rats. Most of the sterically stabilized liposomes concentrated in the spleen with a peak level at 24 h after their intravenous injection. An extended half-life in plasma was observed for sterically stabilized liposomes (14.5 h) compared with that of conventional liposomes (3.9 h). The systemic clearance of ddI incorporated in sterically stabilized liposomes was 180 times lower than that of the free drug. The levels of in vitro and in vivo protein binding on both conventional and sterically stabilized liposomes were also evaluated. Results suggest that the amount of proteins associated with liposomes might not be the only factor involved in the in vivo clearance of liposomes, as this process may also be influenced by the nature of the bound blood proteins.

A possible explanation for the difficulties encountered in curing deep fibrin-embedded infections is that antibiotic diffusion inside the infected fibrin matrix is not homogeneous and is insufficient to neutralize the pathogen. To evaluate this conjecture, the differential pharmacodynamics of daptomycin in fibrin clots infected with methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis was estimated. Daptomycin (20 or 50 mg/kg of body weight) was infused over 30 min. Fibrin clots and blood samples were evaluated from 0.5 to 42 h after the injections. The half-lives of daptomycin in serum and fibrin clot were close to identical after the two doses and averaged 5.4 and 22 h, respectively. The mean areas under the concentration-time curves from 0 to 42 h (AUC0-infinity) for daptomycin concentrations in serum and infected clots were 575 +/- 36.7 and 215 +/- 6.2 micrograms/g/h after administration of 20 mg/kg and 1,089 +/- 39.9 and 326 +/- 16.8 micrograms/g/h after administration of 50 mg/kg. A concentration gradient from the periphery to the core of the clots was observed in many clots up to 18 h after treatment. Mean peak concentrations in the core of the clots reached 60% of the peripheral values (P < 0.05) and were delayed for at least 3 h compared with the peripheral peak concentrations. AUC0-42 h of daptomycin concentration in the periphery and the core of clots were significantly different (P < 0.01). Survival of microorganisms was better in the core than in the periphery, with as much as a 3 log10 CFU/g difference between the center and the surface of the clot. Bacterial examination by transmission electron microscopy also showed noticeable differences in ultrastructural changes between those in the periphery and those in the core of the clots. In conclusion, the pharmacokinetics of daptomycin are significantly different at the periphery and within the core of fibrin clots, which may have led to the higher bacterial survival in the core of clots. Limited diffusion of daptomycin in fibrin, an essential component of the vegetation in bacterial endocarditis, could explain at least in part some of the treatment failures.

Hypocalcemia and an increase in creatinine level are the most important serious effects associated with foscarnet (PFA) therapy. In an animal model, we have explored the potential protective role of liposome-encapsulated foscarnet (LE-PFA) on these metabolic abnormalities. PFA administered as one bolus injection (0.5 or 1.0 g/kg) caused significant rapid decreases (approximately 20%) in the levels of calcium and phosphorus in serum within a few minutes and up to 30 min after injection. LE-PFA did not induce any of these changes, while peak levels in serum and the half-life of this formulation were much higher than those of the free drug. PFA administered for 2 weeks (340 or 500 mg/kg/day) resulted in no changes in creatinine or blood urea nitrogen levels in serum at the low-dosage level, but at the higher-dosage level, the creatinine level in serum increased by day 5 posttreatment. Furthermore, there was no increase in the creatinine or blood urea nitrogen level after 2 weeks of treatment with LE-PFA at a dosage of 35 mg/kg/day. When the pharmacokinetics of both free PFA and LE-PFA were compared, the plasma half-life of the encapsulated drug was approximately four times longer than that of the free drug. In addition, the systemic clearance of LE-PFA was approximately one-fifth of that of the free drug. In conclusion, free PFA causes hypocalcemia and hypophosphatemia and increases the creatinine level in serum, whereas the LE form of this drug seems to protect against the abnormal changes in calcium and phosphorus levels caused by the free drug. By preventing hypocalcemia and increasing its half-life, LE-PFA can be used at lower doses and at longer intervals. Clinical investigations of these formulations may be worthwhile.

In this study, the relationship between leukotrienes, peritubular cell infiltration with polymorphonuclear cells (PMNs) and renal tubular damage was investigated in a rat model of acute ascending pyelonephritis. Infection was induced by the injection of 10(5) CFU of Escherichia coli into the bladder and occlusion of the left ureter for 24 h. Treatment of infected animals was started 24 h after the induction of pyelonephritis with either hydrocortisone (25 mg/kg of body weight per day), the leukotriene inhibitor L-651,392 (10 mg/kg/day), or the vehicle of L-651,392 and was maintained for 5 days. At the end of treatment, the animals were killed, serum was collected, and both kidneys were removed for colony counts and histopathology. Renal function was evaluated by the measurement of blood urea nitrogen levels and creatinine clearance. The numbers of PMNs and mononuclear cells (MNs) in the cortex and medulla were recorded for all groups on plastic sections done from the left kidney. Infection alone (vehicle of L-651,392) resulted in intensive interstitial infiltration and a severe tubular destruction in the cortex. Treatment with hydrocortisone did not prevent PMN migration and tissue damage. By contrast, treatment with L-651,392 resulted in a significant reduction in PMNs (P < 0.001 in comparisons with all other groups) and greater preservation of the tubular structure despite identical bacterial counts than in the group receiving hydrocortisone. We conclude that L-651,392 prevents inflammatory cells from reaching the site of infection and protects the kidney from tubular damage associated with inflammation during pyelonephritis. Inhibitors of leukotrienes should be further investigated for their potential benefit as adjuvants to antibiotherapy in the treatment of pyelonephritis.

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The effect of ceftriaxone on tobramycin-induced nephrotoxicity was investigated. Female Sprague-Dawley rats were treated during 4 and 10 days with saline (NaCl, 0.9%), ceftriaxone at a dose of 100 mg/kg of body weight/12 h subcutaneously, tobramycin at doses of 40 and 60 mg/kg/12 h intraperitoneally, or the combination ceftriaxone-tobramycin. Creatinine levels in serum were significantly higher in animals treated with tobramycin alone given at 60 mg/kg/12 h during 10 days, compared with control animals (P < 0.01) or animals receiving the combination tobramycin-ceftriaxone (P < 0.01). After 10 days of treatment, ceftriaxone did not accumulate in renal tissue but did reduce the renal intracortical accumulation of tobramycin (P < 0.05). Tobramycin given alone at either 40 or 60 mg/kg/12 h induced a significant inhibition of sphingomyelinase activity compared with control animals (P < 0.05). However, this enzyme activity was significantly less inhibited when tobramycin was injected in combination with ceftriaxone (P < 0.05). Ceftriaxone alone had no effect on the activity of this enzyme. The [3H]thymidine incorporation into the DNA of renal cortex was also significantly lower in animals treated with tobramycin-ceftriaxone compared with animals receiving tobramycin alone (P < 0.05). The 24-h urinary excretion of beta-galactosidase was significantly reduced in animals treated with the combination tobramycin-ceftriaxone compared with the administration of tobramycin alone at 40 and 60 mg/kg/12 h after 5 and 10 days (P < 0.05). Histologically, ceftriazone induced very few cellular alterations and reduced considerably the presence of typical signs of tobramycin nephrotoxicity. This investigation demonstrated that ceftriaxone protects animals against tobramycin-induced nephrotoxicity.

The present study was designed to determine the temporal changes in tobramycin nephrotoxicity during the dark and the light periods of the day and to look for the mechanisms of such changes. Female Sprague-Dawley rats (9 to 11 weeks old) were housed in a 14-h-light-10-h-dark cycle (lights on 0600 to 2000 h). A bolus of tobramycin (60 mg/kg of body weight) was intravenously injected into a first group of 15 rats, at either 1400 or 0200 h. Six blood samples were taken from each rat, 30 to 210 min after the bolus injection. The total clearance of the drug was reduced during the rest period (1400 h) of rats compared with the activity period (0200 h) (P = 0.0007). Another group of 99 rats was given intraperitoneally a single dose of tobramycin (40 mg/kg), and renal cortices were collected 2 to 222 h after injection. The cortical drug levels were always higher in animals injected at 1400 h than in those injected at 0200 h. A last group of 32 rats was used in the studies of tobramycin (30 mg/kg/day, once daily for 10 days, intraperitoneally) nephrotoxicity and subcellular distribution. Weight gain in the rats receiving tobramycin (both 1400 and 0200 h) was significantly (P = 0.028) less than that in the controls. Nephrotoxicity, indicated by the incorporation of [3H]thymidine into cortical DNA and urinary excretion of N-acetyl-beta-D-glucosaminidase, was significantly higher in animals treated at 1400 h than in those treated at 0200 h. No difference in the subcellular distribution of tobramycin was observed. The data indicate that the reduction in the clearance of tobramycin during the rest period is in part responsible for the higher nephrotoxicity in rats.

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This report describes the effects of endotoxin and a thromboxane receptor antagonist, L-655,240, on kidney function and the intrarenal pharmacokinetics of aminoglycosides. The rationale for these studies was that thromboxane antagonists may eventually be used in combination with aminoglycosides in patients with gram-negative sepsis and endotoxemia. As aminoglycosides are nephrotoxic and endotoxin has already been shown to increase the renal uptake of gentamicin, we investigated the possibility that thromboxane antagonists might interfere with the nephrotoxic potential of both substances. A decrease in the volume of distribution and an increase in the intracortical concentration of gentamicin were observed in animals given endotoxin. Compared with animals given endotoxin alone, those which received endotoxin plus L-655,240 had significant accumulation of gentamicin in the renal cortex and medulla, as determined by the area under the concentration-time curve, and a significant reduction in the total clearance of the antibiotic (P < 0.05). This difference in uptake could not be attributed to hypotension or changes in the glomerular filtration rate or renal plasma flow. L-655,240 alone did not modify gentamicin pharmacokinetics but did decrease p-aminohippuric acid secretion. Thromboxane antagonists in the context of endotoxemia increase intrarenal uptake of aminoglycosides. If these compounds are to be used as therapeutic agents when endotoxin is present, their influence on renal handling of nephrotoxic drugs needs to be considered. Multiple-dosing regimens deserve investigation.

The fibrin clot penetration and in vivo bactericidal activity of RP 59500, a new semisynthetic streptogramin, for two Staphylococcus aureus strains (one methicillin resistant and the other methicillin susceptible), two Staphylococcus epidermidis strains (one methicillin resistant and the other methicillin susceptible), and one Enterococcus faecalis strain were evaluated. The clots, inserted subcutaneously, were infected with a mean of 10(8) CFU of the pathogen per g. For each strain, groups of four rabbits received a single intravenous injection of 50 mg of RP 59500 per kg of body weight over 30 min. The mean peak level of RP 59500 in serum in the infected rabbits was 61.9 +/- 6.3 micrograms/ml. The drug was detectable in serum at a level of 0.8 micrograms/ml up to 4 h after administration. The mean peak fibrin clot drug level at 1 h was 3.3 +/- 0.1 micrograms/g. At 6 h, the level in clots was 1.2 +/- 0.1 micrograms/g. The mean half-life in serum in infected rabbits was 0.34 +/- 0.01 h, while in clots the drug exhibited a longer half-life of 3.8 +/- 0.4 h. In vivo, this new streptogramin sterilized the clots infected with the two S. aureus strains studied in less than 1 h and induced a marked reduction in colony counts of the two S. epidermidis strains studied for up to 24 h. The activity of the streptogramin against E. faecalis was limited. These results suggest that RP 59500 should be further evaluated for the treatment of infection with methicillin-resistant staphylococci.

The subcellular localization of tobramycin and vancomycin in the renal cortices of rats was determined with ultrathin sections by immunogold labeling. Four groups of four rats each were treated for 10 days with saline (NaCl, 0.9%), tobramycin at dosages of 20 mg/kg of body weight per 12 h intraperitoneally, vancomycin at dosages of 25 mg/kg/12 h subcutaneously, or the combination tobramycin-vancomycin. On day 11, the animals were killed, and cubes of renal cortex were fixed overnight in phosphate-buffered glutaraldehyde (0.5%), dehydrated in ethanol, and embedded in Araldite 502 resin. Ultrathin sections were made and incubated with sheep antitobramycin antibody followed by protein A-gold (15-nm diameter) complex or rabbit antivancomycin antibody followed by gold (30-nm diameter)-labeled goat anti-rabbit antibody. For the double labeling, incubations were made on opposite sides of the grid. Tobramycin was detected over the lysosomes of proximal tubular cells, but the labeling was concentrated into small areas in the matrix of the lysosomes. Vancomycin was seen over the lysosomes of proximal tubular cells and was distributed uniformly throughout the matrix of the lysosomes. In rats treated with tobramycin-vancomycin, both drugs were still detected in lysosomes of proximal tubular cells. It is concluded that tobramycin and vancomycin accumulate in lysosomes of proximal tubular cells throughout 10 days of treatment and that vancomycin has no effect on the subcellular distribution of tobramycin.

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A study recently conducted across Canada showed that 64 of 2,503 clinical isolates of Haemophilus influenzae were resistant to beta-lactams without production of a beta-lactamase (L. D. Tremblay, J. L'Ecuyer, P. Provencher, M. G. Bergeron, and Canadian Study Group, Can. Med. Assoc. J. 143:895-900, 1990). The beta-lactamase-negative strains formed three distinct groups, with ampicillin MICs of 0.5 to 1, 2 to 4, and greater than or equal to 8 micrograms/ml for groups I, II, and III, respectively. We have investigated the mechanisms of resistance for eight strains originating from different infections and geographic areas. These strains were representative of groups I to III. Five strains were nontypeable, two were type B, and one was non-B. Chromosomal DNA extracted from each strain was used to transform the laboratory strain Rd. Transformants were selected on beta-lactam-containing plates and showed the same level of resistance to ampicillin as the donor strains. Differences in outer membrane proteins, porins, and lipopolysaccharide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) did not change with resistance. Functional analyses of purified porins in artificial lipid bilayer experiments did not explain resistance. Peptidoglycan synthesis was measured by incorporation of [14C]alanine into trichloroacetic acid-insoluble cell wall material in the presence of chloramphenicol. The growth rate and the rate of peptidoglycan synthesis observed for the transformants of the isogenic set did not correlate with resistance. Whole-cell labeling with 125I-penicillin revealed modifications in penicillin-binding proteins (PBPs) among the transformants. In particular, PBPs 3A and 3B (65 and 63 kDa, respectively) showed a decrease in affinity for beta-lactams in all transformants (groups I, II, and III) and correlated with an increased MIC except in the transformant of group III, which showed higher levels of resistance. Partial purification and proteolytic digestion of 125I-penicillin-labeled PBP 3B led to two types of CnBr peptide profiles on SDS-PAGE, the profiles of the transformed strains from groups I and II being different from those of the control group and group III. Finally, electron microscopy revealed a distinct cell filamentation for the group III transformants. These data clearly indicate that changes in PBPs are a common mechanism that results in a significant level of non-beta-lactamase-mediated beta-lactam resistance in H. influenzae despite serotype, origin of isolation, or geographic distribution.

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The subcellular distribution of gentamicin in rat renal proximal tubular cells was evaluated by immunogold labeling. The distribution of the drug was monitored from 10 min to 10 days following single (40 mg/kg of body weight) and multiple (5 and 20 mg/kg/12 h) injections of gentamicin. Animals were killed on day 11, and cubes of renal cortex tissue were fixed overnight in cold phosphate-buffered glutaraldehyde (0.5%), dehydrated in ethanol, and embedded in Araldite 502 epoxy resin. Ultrathin sections were made and incubated with sheep antigentamicin and then with protein A-gold (15 nm) complex. At 10 min after a single injection, the labeling was found over the brush border membrane and over the membranes of endocytic apical vesicles of proximal tubular cells. After 1 h, a similar distribution was observed and the labeling was also seen over small lysosomes located close to the brush border membrane. At 24 h, gold particles were found over large lysosomes of proximal tubular cells. Following 10 days of treatment, lysosomes of proximal tubular cells were densely labeled with gold particles. The labeling was distributed uniformly over the lysosomes, although a lower density of labeling was observed over the myeloid bodies inside the lysosomes. Necrotic proximal tubular cells showed labeling over intact lysosomes and also in the cytoplasms of the cells, in the mitochondria, and in the nucleoli. The various control experiments demonstrated the high specificity of these results. The present immunocytochemical study better documents the subcellular disposition of gentamicin in proximal tubular cells, as previously evaluated by subcellular fractionation and autoradiography. This technique will be useful for better understanding the relationship between drug disposition and drug-induced toxicity.

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Aminoglycoside-induced renal damage is enhanced in animals with Escherichia coli pyelonephritis. Bacterial endotoxin is liberated during antibiotic therapy. The toxic effect of endotoxin and tobramycin, alone or in combination, was investigated in primary cultures of rabbit proximal tubular cells grown to confluence in serum-free medium. Sodium-dependent uptakes of Pi and alpha-methylglucopyranoside (MGP) and enzymatic activities (lactate dehydrogenase [LDH] released as a marker of cell necrosis and gamma-glutamyltransferase [GGT] and N-acetyl-beta-D-glucosaminidase [NAG] present in the homogenate as markers of brush border membrane and lysosome integrity) were measured. Cells were exposed to (i) endotoxin (20 mg/liter), tobramycin (1 mM), or endotoxin plus tobramycin for 48 h, or (ii) endotoxin (100 mg/liter), tobramycin (4 mM), or endotoxin plus tobramycin for 72 h. Endotoxin alone did not alter Pi uptake, but tobramycin inhibited Pi uptake through a decrease in Vmax. The effect was not enhanced by the combination of endotoxin and tobramycin. Endotoxin and tobramycin alone exerted no significant effect upon MGP uptake, but strong inhibition of the Vmax was observed after exposure to a combination of endotoxin plus tobramycin, without alteration of the Km. Endotoxin decreased residual GGT activity in the cell homogenate. Tobramycin increased LDH release in the medium and NAG activity in the homogenate. Endotoxin plus tobramycin resulted in an additive effect upon LDH and NAG activities. In conclusion, by disturbing apical membrane integrity, endotoxin increased tobramycin toxicity in vitro in the absence of serum hormonal mediator.

The concentrations of teicoplanin in sera and heart tissues of 49 patients undergoing coronary bypass were measured. Each patient received a 6- or 12-mg/kg dose of teicoplanin administered in a slow intravenous bolus injection over 3 to 5 min beginning at the time of induction of anesthesia. Mean +/- standard error of the mean concentrations in serum were, for the two doses, respectively, 58.1 +/- 1.7 and 123.3 +/- 7.4 micrograms/ml 5 min after administration and 22.2 +/- 0.7 and 56.5 +/- 2.8 micrograms/ml at the time of removal of atrial appendages. Mean +/- standard error of the mean concentrations in tissue were 70.6 +/- 1.7 and 139.8 +/- 2.2 micrograms/g, respectively, giving mean tissue/serum ratios of 3.7 +/- 0.3 and 2.8 +/- 0.2, respectively. Teicoplanin penetrates heart tissue readily and reaches levels in the serum far in excess of the MICs for most pathogens that have been found to cause infections following open heart surgery.

The aim of this study was to evaluate the role of chronic endotoxemia in the nephrotoxicity of gentamicin (GM). Saline or Escherichia coli lipopolysaccharide (LPS) was administered to conscious rats by continuous intravenous perfusion (1 mg/kg per day for 7 days) from a subcutaneously implanted osmotic pump. Twenty-four hours after surgery (day zero), treatment with saline or GM (15 mg/kg; intraperitoneally, twice a day) was started for 5 days. Levels of LPS in plasma measured by Limulus amoebocyte lysate activity decreased significantly from days 1 through 8. At days 5 and 8, the cortical concentrations of GM were higher in the LPS-perfused and GM-treated group (LPS plus GM) than they were in the saline-perfused and GM-treated group (saline plus GM) (P less than 0.05). Blood urea nitrogen and serum creatinine remained at normal levels throughout the experiment. A significant increase of cortical tubular cell regeneration was observed in the LPS plus GM animals as compared with regeneration observed in the other groups (saline plus saline, LPS plus saline, and saline plus GM), as measured by [3H]thymidine incorporation into DNA. Moreover, histopathological nephrotoxicity scores showed a synergistic toxic effect between LPS and GM. These results demonstrate that chronic perfusion of low doses of LPS potentiates the nephrotoxicity of GM.

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The mechanism by which endotoxin (lipopolysaccharide [LPS]) modifies the intrarenal distribution and the nephrotoxic potential of gentamicin is unknown. We studied the influence of LPS on the intracortical accumulation kinetics of gentamicin in rats infused intravenously for 6 h, during which time steady-state levels of the antibiotic in serum were achieved. We compared gentamicin accumulation rates (V) in normal rats and in rats receiving LPS (0.5 and 5 mg/kg) as levels in serum (S) varied from 0.5 to 130 micrograms/ml. The pharmacokinetic parameters of gentamicin were previously measured in the three groups of rats that were studied in order to reach and maintain in each rat the desired levels of antibiotic in serum during the 6 h of infusion. Two hours before the infusion of gentamicin, LPS was injected intravenously over a period of 15 min. In normal rats, the increase in S was associated with a nonlinear increase in V. The Michaelis-Menten kinetics, which was the best-fitting function, gave an apparent Vmax (maximal capacity of uptake) of 195.03 +/- 9.75 micrograms/g per h and an apparent Km (concentration in serum at Vmax/2, an index of affinity) of 34.91 +/- 4.45 micrograms/ml (linear transformation of the experimental data by the Hanes-Woolf plot: r = 0.93, n = 85). In the rats that received LPS, the increase in S was associated with a linear increase of V: for LPS at 0.5 mg/kg, V = 27.00 + 1.50 S (r = 0.94, n = 80); for LPS at 5 mg/kg, V = 22.72 + 1.48 S (r = 0.94, n = 75). We conclude that endotoxin modifies the accumulation kinetics of gentamicin in the kidney cortices of rats.

Daptomycin is a new biosynthetic antibiotic which belongs to a new class of drugs known as lipopeptides. The objective of this study was to evaluate the effects of daptomycin and vancomycin on tobramycin-induced nephrotoxicity. Female Sprague-Dawley rats were treated during 4 and 10 days with either saline (NaCl, 0.9%) or tobramycin at doses of 4 and 40 mg/kg per day (given every 12 h [q12h] intraperitoneally). Each treatment was combined with saline, daptomycin at a dose of 20 mg/kg per day (given q12h subcutaneously), and ancomycin at a dose of 50 mg/kg per day (given q12h subcutaneously). Daptomycin and vancomycin had no effect on the intracortical accumulation of tobramycin. Daptomycin did not accumulate in renal tissue even after 10 days of treatment. Tobramycin given at a dose of 40 mg/kg per day during 10 days induced a significant inhibition of sphingomyelinase activity in the renal cortex (P less than 0.01) and increased cellular regeneration (P less than 0.01), as measured by the incorporation of [3H]thymidine into DNA of the renal cortex. These changes were minimal when daptomycin was combined with tobramycin. Histologically, signs of tobramycin toxicity were also less severe in the presence of daptomycin. The intracortical accumulation of vancomycin was not modified by tobramycin. The sphingomyelinase activity was significantly more inhibited (P less than 0.01) when vancomycin was associated with tobramycin (4 and 40 mg/kg) without affecting the rate of [3H]thymidine incorporation into DNA. Histologically, signs of tobramycin toxicity were not affected by vancomuycin, but the cellular vacuolizations which were also observed in vancomycin-treated animals were still present in the proximal tubular cells of animals that were treated with the combination vancomycin-tobramycin. This study strongly suggests that daptomycin protects animals from tobramycin-induced nephrotoxicity but that vancomycin may enhance the effect of tobramycin. We conclude that daptomycin is safe and protects kidney cells from tobramycin-induced nephrotoxicity.

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