Synthesis and identification of products derived from the metabolism of the carcinostatic 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by rat liver microsomes.

Liver microsomal metabolism of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea in the presence of reduced nicotinamide adenine dinucleotide phosphate and O2 was shown to produce seven metabolites that included the parent urea. A cytochrome P-450-dependent monohydroxylation of the cyclohexyl ring occurred in 3 positions, cis-3, trans-3, and cis-4, and on the methyl group to form a trans-4-hydroxymethyl derivative. In addition, monohydroxylation of the 2-chloroethyl carbon attached to the N-1 urea nitrogen yielded an alpha-hydroxy metabolite. A ring-hydroxylated derivative remained unidentified while the structures of all other such derivatives were established by comparison with compound synthesized, purified by high-pressure liquid chromatography, and characterized by mass spectral and nuclear magnetic resonance analyses. It was tentatively concluded that some parent urea is formed by a cytochrome P-450 dependent reaction because of a requirement for reduced nicotinamide adenine dinucleotide phosphate and inhibition by CO. Microsomes from rats pretreated with phenobarbital showed about a 3-fold increase in hydroxylation rate while phenobarbital-treated mice microsomes were induced 8-fold. However, in both species, the induced hydroxylation rate was about 4 nmol/min/mg protein. When microsomes from phenobarbital-induced rats were used, a mixture of 80% CO:20% O2 decreased the rate of formation of all metabolites to 14% of that in 80% N2:20% O2.

Meropenem pharmacokinetics in a patient with multiorgan failure from Meningococcemia undergoing continuous venovenous hemodiafiltration.

Meropenem is a carbapenem antibiotic with a broad antibacterial spectrum of activity. Its main route of elimination is through the kidneys, with 63% of the drug excreted unchanged in the urine. Meropenem clearance is diminished in renal impairment; therefore, doses need to be adjusted in patients with varying degrees of renal function. An appropriate dose of meropenem for patients undergoing continuous venovenous hemodiafiltration (CVVHDF) is unknown. We evaluated the pharmacokinetics of meropenem in a patient with fulminant meningococcemia undergoing CVVHDF. Meropenem concentrations in serial venous, arterial, and ultrafiltrate samples after a 1 g intravenous dose were measured using high-performance liquid chromatography (HPLC). Meropenem clearance was found to be 129.36 mL/min and 141.29 mL/min for every 8- and 12-hour dosing, respectively. Trough levels were above the MIC90 for Neisseria meningitidis and most anaerobic pathogens. We recommend that meropenem 1 g intravenously every 12 hours be used as the initial dose in patients undergoing CVVHDF. Differences between meropenem clearance during CVVHDF and other forms of renal replacement therapy are discussed.

Zidovudine pharmacokinetics in five HIV seronegative patients undergoing continuous ambulatory peritoneal dialysis.

A few reports in the literature describe zidovudine (AZT) pharmacokinetics in patients undergoing hemodialysis; however, the effect of continuous ambulatory peritoneal dialysis (CAPD) on the drug's disposition has not been studied. The pharmacokinetics of AZT were evaluated in five patients, age 37-62 years, who were seronegative for the human immunodeficiency virus and were undergoing CAPD. Serial plasma, urine, and dialysate samples were collected after oral administration of AZT 200 mg. Samples were assayed using radioimmunoassay (RIA). Model-independent analysis was used to determine total plasma clearance, apparent volume of distribution, mean residence time, and half-life. Net peritoneal dialysis clearance was calculated to measure the effect of CAPD on AZT disposition. We found wide interpatient variability in AZT pharmacokinetics. Peak serum concentrations ranged from 0.3-47.8 microns and area under the curve from 0.5-26.1 mg x hour/L. These differences resulted in corresponding differences in clearance (range 66-3176 ml/min/1.73 m2) and volume of distribution (range 16-825 L). Interpatient variability in glucuronidation may partially account for this variability. Net peritoneal dialysis clearance of AZT was 5 ml/minute. Although the effect of peritoneal dialysis on AZT disposition was negligible, clinicians should be aware of the large differences in the way in which individual patients with renal dysfunction handle this drug.

New sodium hydroxide digestion method for measurement of renal tobramycin concentrations.

Because of the known propensity of the cationic aminoglycosides to interact with a variety of anionic tissue constituents, it was postulated that present elution methods fail to detect a substantial proportion of the aminoglycoside that accumulates in the kidney. After preliminary experiments documented the pH and temperature stability of tobramycin, a new sodium hydroxide (NaOH) tissue digestion method was applied to renal tissue collected from rats given tobramycin at 80 mg/kg (body weight) per day in two divided doses for 3, 7, 10, 14, and 17 days. Compared with elution with buffer from the renal homogenate, digestion with 1.0 N NaOH at 70 degrees C for 15 min significantly increased the amount of assayable tobramycin (P less than 0.001). The absolute increase varied between 37 and 96%, depending on the number of days of in vivo drug administration. A single trichloroacetic acid (TCA) treatment of the homogenate altered the amount of assayable tobramycin over a range which varied from a decrease of 6% to an increase of 32%. After TCA treatment, it was possible to increase the amount of measurable tobramycin by 17 to 22% by treatment of the TCA-induced precipitate with 1.0 N NaOH. Digestion of renal homogenates with 1.0 N NaOH significantly increases the amount of tobramycin detectable by immunoassay.

The influence of aminoglycoside antibiotics on the in vitro function of rat liver ribosomes.

There are few studies of the influence of aminoglycoside antibiotics on the ribosomes of higher eukaryotic organisms. To this end, cytoplasmic ribosomes were prepared from rat liver. In vitro, poly(U)-directed ribosome protein synthesis was studied in the presence and absence of selected aminoglycosides. Misreading of poly(U) was also assessed. Consistent with earlier studies using different sources of ribosomes, paromomycin inhibited cell-free protein synthesis and caused poly(U) misreading. In contrast to the findings of other studies in cell-free ribosomes of eukaryotic organisms, netilmicin, tobramycin, and neomycin were most active in inhibiting protein synthesis, and gentamicin C2 and neomycin caused appreciable misreading. Thus the previous suggestion that a paromamine fragment (found in paromomycin) might be a structural requirement for in vitro inhibition of protein synthesis and misreading is not substantiated by the results in rat liver ribosomes. Commercial gentamicin C is a mixture of gentamicins C1, C1a, and C2. Despite nearly identical chemical structures, the three constituents displayed greatly different propensities for inducing poly(U) misreading. C2 was the most active, followed by C1a. In summary, selected aminoglycoside antibiotics caused inhibition and mistranslation of poly(U) messenger in an in vitro ribosome system prepared from rat liver. These effects were not limited to paromamine-containing aminoglycoside antibiotics. Gentamicin C2 caused much more poly(U) misreading than the other two constituents of the gentamicin C complex.

Urinary metabolites of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea.

Urinary metabolites of ring 14C-labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (Methyl CCNU) from rats have been isolated and characterized by high-performance liquid chromatography and mass spectrometry. About 44% of the cyclohexyl moiety of CCNU was excreted in 24 hr and included approximately 10% of the excreted dose as free amines and 40% as conjugates that could be converted to amines by hydrolysis. Amine composition of free base plus hydrolyzable conjugates was 55% hydroxycyclohexylamines (3-trans, 3-cis, 4-cis, and 4-trans) and 30% cyclohexylamine. This strongly supports previous studies which indicated that CCNU is largely hydroxylated in vivo as well as in vitro. Rats pretreated with phenobarbital excreted high relative amounts of cis-4-hydroxy derivatives (41%), again showing a high degree of correlation between in vitro and in vivo results. Treatment of urine with beta-glucuronidase gave no apparent increase in free amines. However, sulfatase was about 25% as effective as alkaline hydrolysis for releasing free amines from whole urine. Major urinary metabolites were found to have m.w. of about 629, 413, 329, and 243 and represented 55%, 20%, 20%, and 5% of total excreted 14C, respectively. It was concluded that the higher m.w. metabolites may be conjugates of peptides possibly derived from active site-directed inactivation of specific enzymes. Previous work has shown that enzymes such as chymotrypsin and glutathione reductase are inhibited by isocyanates in this manner. Hydroxylated metabolites of Methyl CCNU had a pattern similar to that of CCNU. The major free (12%) and conjugated amine (54%) metabolites of Methyl CCNU in the urine in decreasing order of quantity present were cis-3-hydroxy-trans-4-methylcyclohexylamine, trans-4-methylcyclohexylamine, trans-4-hydroxymethylcyclohexylamine, and trans-3-hydroxy-trans-4-methyl-cyclohexylamine.

Influence of assay methodology on the measurement of free serum ceftriaxone concentrations.

The influence of assay methodology on the measurement of the active free fraction of ceftriaxone in plasma was determined. The free fraction was measured by three methods: agar diffusion bioassay, precipitation of plasma protein with methanol followed by high-performance liquid chromatography (HPLC) of the supernatant, and ultrafiltration of plasma followed by HPLC of the filtrate. In human serum, the free ceftriaxone levels were significantly lower (P = 0.03) when measured on ultrafiltrates compared to the other two methods. This difference disappeared when dolphin serum was studied. After ultrafiltration, human serum was shown, by Scatchard plot analysis, to have two ceftriaxone binding sites. Species differences were also demonstrated. Hence, in humans, determination of free plasma ceftriaxone varies with the assay method employed.

Pig kidney (LLC-PK1) cell membrane fluidity during exposure to gentamicin or tobramycin.

The surface membrane properties of LLC-PK1 cells grown with and without various amounts of gentamicin or tobramycin for various lengths of time were determined by measuring the diffusion coefficient of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L- alpha-phosphatidylethanolamine (NBD-PE) and the percentage of NBD-PE free to diffuse after photobleaching. One hour of exposure to tobramycin decreased the percentage that was free to diffuse. After 1 day or longer of exposure to either aminoglycoside the percentage that was free to diffuse returned to preexposure levels and the diffusion coefficient decreased.

Gentamicin does not chelate calcium.

The influence of increasing gentamicin concentrations on ionized calcium concentration was determined in pH-controlled, phosphate-buffered saline and normal human serum with an ion-specific calcium electrode. No evidence of calcium chelation was found.

Aminoglycoside-induced increase of intracellular calcium in LLC-PK1 cells due to an artifact caused by trypsin and EDTA.

Dietary calcium supplements attenuate experimental aminoglycoside nephrotoxicity. In cultured renal tubular cells, intracellular calcium levels have been reported to rise with aminoglycoside addition to the culture medium. In experiments designed to verify the in vitro influence of calcium on cultured kidney cells, we detected an unexpected artifact. When we resuspended cultured LLC-PK1 cells with trypsin and EDTA to measure intracellular calcium levels, our results correlated well with previously reported values. However, we saw no increase in intracellular calcium levels when we measured them by digital imaging video microscopy unless trypsin-EDTA exposure preceded aminoglycoside exposure. This apparent artifact should be considered in any study of the effects of various agents on intracellular calcium levels.

Investigation of bioequivalence and tolerability of intramuscular ceftriaxone injections by using 1% lidocaine, buffered lidocaine, and sterile water diluents.

The pharmacokinetics and tolerability of 1-g doses of ceftriaxone diluted in sterile water, 1% lidocaine, or buffered lidocaine were investigated. No difference in bioequivalence was noted between the three treatments. No difference in peak creatine kinase values was seen. By use of a quantitative pain scale, injection of ceftriaxone with the water diluent was significantly more painful than that with either of the other two diluents. No difference in injection pain was noted for lidocaine or buffered lidocaine.

Influence of daptomycin on staphylococcal abscesses and experimental tobramycin nephrotoxicity.

The antibacterial efficacies of daptomycin and vancomycin were compared in male Fischer rats with subcutaneous abscesses caused by either methicillin-susceptible Staphylococcus aureus (MSSA) or methicillin-resistant S. aureus (MRSA). The influence of daptomycin on tobramycin nephrotoxicity was also assessed. MSSA or MRSA abscesses were treated with subcutaneous daptomycin (10 mg/kg every 12 h), vancomycin (125 mg/kg every 12 h), or diluent (every 12 h) for 5 to 10 days. Rats in both antibiotic treatment groups had lower abscess bacterial counts than did controls at days 5 and 10 (P less than 0.0025). The daptomycin treatment groups had lower abscess bacterial counts than did the vancomycin treatment groups for MSSA at day 5 (P less than 0.0025) and day 10 (P less than 0.025) and for MRSA at day 10 (P less than 0.0025). Nephrotoxicity treatment groups included animals treated for 3, 7, 10, 14, and 17 days with subcutaneous diluent (every 12 h), daptomycin (20 mg/kg every 12 h), tobramycin (40 mg/kg every 12 h), and the combination of daptomycin and tobramycin. Compared with controls, animals treated with daptomycin alone exhibited no detectable nephrotoxicity. Rats given tobramycin alone developed functional and histopathologic abnormalities from days 7 through 17. Animals treated with daptomycin and tobramycin for 14 days had a lower mean concentration of creatinine in serum (P less than 0.005), higher mean creatinine clearance values (P less than 0.05), and less cortical tubular cell regeneration (P less than 0.05) than did rats treated with tobramycin alone. In rats with staphylococcal subcutaneous abscesses, daptomycin was superior to vancomycin in treating both MSSA and MRSA. Daptomycin alone caused no detectable renal injury, and in rats given daptomycin combined with tombramycin, there was less histologic and functional renal injury than in animals given tobramycin alone.

Nephrotoxicity of the constituents of the gentamicin complex.

Commercial gentamicin C is a mixture of gentamicin C1, C1a, and C2. The nephrotoxicity of each of these constituents was compared with that of the gentamicin complex. After seven days the mean creatinine level in serum was 0.8 mg/dl in rats given C2 and 0.5 mg/dl in rats given C1, C1a, or the gentamicin complex (P less than .001). Toxicity attributable to C1a was not detected until day 14, and only minimal toxicity was noted in C1-treated rats after 21 days. Nephrotoxicity caused by the gentamicin complex was similar to that caused by C2. By a new high-pressure liquid chromatographic method, the renal concentration of C1, C1a, and C2 was quantified in rats given the gentamicin complex. The results indicated an early, preferential renal accumulation of C2. Subsequently, the C2 content of 12 commercial lots of gentamicin C was measured. The C2 concentration ranged from 12.4 to 20.1 mg/ml. In short, experimental nephrotoxicity from gentamicin C is largely the result of the C2 constituent, and the concentration of this constituent in commercial preparations of gentamicin varies by as much as 7.7 mg/dl.

Duration of the protective effect of polyaspartic acid on experimental gentamicin nephrotoxicity.

It is known that daily polyaspartic acid (PAA) protects the kidney from gentamicin nephrotoxicity in a standardized rat model despite marked cortical accumulation of the aminoglycoside. The present experiments address the duration of PAA protection. When administered every other day, PAA provided functional and histologic protection against gentamicin-induced toxicity. A stepwise reduction in nephroprotection occurred as the dosage interval was prolonged.

Long-term protection of polyaspartic acid in experimental gentamicin nephrotoxicity.

Polyaspartic Acid (PAA) protects the kidney from experimental gentamicin nephrotoxicity despite large increases in renal cortical gentamicin content. In these experiments, prominent cytoplasmic vacuoles were noted in all animals that received PAA with or without gentamicin. The present study showed that there were no renal structural or functional consequences of PAA given alone or with gentamicin for up to 14 days, followed by a 16-week washout period. Creatinine clearance was similar to that of controls in animals that received gentamicin and in those that received PAA alone. Thus, complete functional protection was conferred by PAA and gentamicin, confirming previous reports from our laboratory. There was no protection by PAA from the nephrotoxic effects of mercuric chloride and cis-platinum.

Determinants of the in vitro interaction of polyaspartic acid and aminoglycoside antibiotics.

The in vitro interaction of polyaspartic acid (PAA) with aminoglycosides was evaluated using double diffusion in agar, dialysis chambers and changes in the optical density of test solutions. The results document a reversible, presumably electrostatic interaction that is optimized at a pH of approximately 5.0, by the absence of proteins over 800 Da, and at a 20:1 or 10:1 molar ratio of aminoglycoside to PAA. The PAA-aminoglycoside complex lost antibacterial activity and the ability to inhibit pronase E enzymatic activity. These results allow generation of a hypothesis as to the mechanism whereby PAA prevents aminoglycoside experimental nephrotoxicity.

Vancomycin enhancement of experimental tobramycin nephrotoxicity.

The influence of vancomycin on tobramycin nephrotoxicity was assessed in male Fischer rats. Treatment groups included controls receiving diluent and groups receiving vancomycin alone at a dosage of 200 mg/kg (body weight) per day, tobramycin alone at a dosage of 80 mg/kg per day, and a combination of vancomycin and tobramycin at the above dosages. All regimens were injected on a twice-a-day schedule. The animals were sacrificed on days 1, 3, 10, 14, 17, and 21. When compared with controls, animals receiving vancomycin alone exhibited no detectable renal toxicity. Compared with the case with controls, tobramycin alone was toxic, as manifested by lower mean animal weights, increased blood urea nitrogen concentrations on days 14 and 17 (P less than 0.005), increased serum creatinine concentrations on days 17 and 21 (P less than 0.005), and the presence of renal cortical tubular necrosis and regeneration. When compared with tobramycin alone, the combination of vancomycin and tobramycin caused earlier and more severe toxicity. By day 10, the magnitude of weight loss, the rise in blood urea nitrogen, and the increase in serum creatinine concentration were all greater in the rats given the combination of vancomycin plus tobramycin than in the animals given tobramycin alone (P less than 0.005). In addition, there was more proximal tubular necrosis and regeneration in rats given vancomycin plus tobramycin compared with those given tobramycin alone. In this animal model, vancomycin alone caused no detectable renal injury, tobramycin alone produced minimal proximal tubular damage, and the combination of vancomycin and tobramycin resulted in a greater degree of kidney injury than observed with tobramycin alone.

Pharmacologic limits of the protective effect of polyaspartic acid on experimental gentamicin nephrotoxicity.

Polyaspartic acid (PAA) ameliorates experimental gentamicin nephrotoxicity despite marked accumulation of gentamicin in the renal cortex. The experiments described here probe the extent of PAA's nephroprotective action when increasing doses of gentamicin, in excess of an established nephrotoxic dose (40 mg/kg of body weight per day), are administered. After 10 days, virtually complete nephroprotection was conferred by PAA coadministered to animals receiving three times the nephrotoxic dose (120 mg/kg/day) of gentamicin.

Amikacin nephrotoxicity in the rat.

When amikacin was administered to Fischer rats at a dose of 120 mg/kg/day for up to 14 days, renal proximal tubule cells became vacuolated, but BUN and creatinine remained normal. Renal cortical drug levels rose steadily throughout the treatment period. When, in a second trial of the same duration, the drug dose was tripled, focal proximal tubular necrosis, then regeneration, occurred and the animals became azotemic. Tissue drug concentrations peaked and began to decline during the treatment period, having reached levels more than three times higher than achieved at the lower dose. Ultrastructural changes were similar to those observed with other aminoglycosides. The results indicate that amikacin is less nephrotoxic than gentamicin and more toxin than tobramycin and netilmicin in the Fischer rat.

Polyaspartic acid prevents experimental aminoglycoside nephrotoxicity.

The influence of the polyamino acid polyaspartic acid (PAA) on experimental aminoglycoside nephrotoxicity was determined. PAA prevented all measured functional and pathologic evidence of gentamicin nephrotoxicity for less than or equal to 27 d of study. All the animals given PAA, either alone or with gentamicin, developed prominent cytoplasmic vacuoles in the cells of the renal proximal convoluted tubules; the vacuoles in rats given just PAA differed from those observed in rats given PAA plus gentamicin. Rats given PAA plus gentamicin accumulated roughly 10 times more renal aminoglycoside as did rats given gentamicin alone. Immunohistochemical localization studies confirmed the presence of increased amounts of gentamicin in the cytoplasm of the tubular cells of animals given gentamicin plus PAA. PAA did not alter the in vitro antimicrobial activity of gentamicin versus Escherichia coli or Pseudomonas aeruginosa. These studies demonstrate the ability of PAA to prevent experimental gentamicin nephrotoxicity.