INTRODUCTION

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The major limitation to the clinical use of aminoglycosides continues to be concern for the development of nephrotoxicity. Evidence from studies with animals and humans has demonstrated a correlation between the nephrotoxic effects of aminoglycosides and the accumulation of these drugs in the cortex of the kidney (9, 23, 39). It is also evident that aminoglycoside accumulation in the kidney may be related to the dosing schedule; i.e., administration of larger doses less frequently may reduce the level of drug accumulation in the kidney cortex and thereby may reduce the nephrotoxic potentials of aminoglycosides.

Aminoglycoside nephrotoxicity is thought to be centered in the proximal renal tubular epithelial (PRTE) cells. Investigators have indicated that inhibition of phosphatidylinositol phospholipases A₁ and A₂ by aminoglycosides results in the formation of lamellar bodies which are postulated to cause cellular toxicity (37). The method of uptake into PRTE cells is closely linked to the rationale underlying the use of once-daily dosing to delay the onset of nephrotoxicity. The uptake of aminoglycosides is limited to the luminal border of the PRTE cell. It is likely that they bind to the cell surface and are taken up by pinocytosis, which is inherently limited to a maximal rate. Consequently, no matter how much aminoglycoside is present in the tubular lumen, only a maximal amount can be taken up per unit of time. The administration of larger doses less frequently allows high concentrations of drug to be present in the tubular lumen early in the dosing interval. Consequently, with high concentrations of aminoglycosides in the tubular lumen, when uptake is saturated, the rate of uptake is maximal and thus is not increased with higher concentrations. Therefore, much of the aminoglycoside bypasses the PRTE cell and is excreted. Smaller amounts of the drug are present for longer periods of time. The total amount of drug taken up by the cells per 24 h is smaller, as seen from the data of Verpooten et al. (39).

Traditionally, aminoglycosides have been administered in two to three divided doses per day by intermittent infusion. Numerous studies conducted with animals and humans have demonstrated equal efficacy (5, 11, 12, 14-16, 18, 20-22, 26-31, 34, 35, 38, 41) and nephrotoxicity (11, 12, 14-16, 20-22, 26, 27, 29, 31, 33-35, 38, 39, 41) with once-daily dosing of aminoglycosides compared with those with conventional dosing. All clinical studies to date have been unblinded. Moreover, many patients have received therapy for short periods of time. Such limited therapy may minimize toxicity and may thus obscure any difference between the effects of different dosing schedules. Recently, five different investigations by meta-analysis examined the data from the majority of published studies of once- versus multiple-daily dosing of aminoglycosides (1, 3, 4, 25, 36). The results of those studies indicated that the rates of toxicity with once-daily dosing were less than or equal to those with multiple-daily dosing. Furthermore, one of the analyses indicated a more favorable clinical outcome with once-daily dosing with dosed aminoglycosides compared with that from multiple-daily dosing (25). Meta-analysis may point the way to a correct hypothesis, but the ultimate proof of that hypothesis is still in the realm of the prospective randomized trial. We designed and carried out a prospective, randomized, double-blind study of once-daily aminoglycoside therapy compared to twice-daily therapy to examine toxicity in patients being treated for at least 72 h for suspected gram-negative bacterial infections.

	MATERIALS AND METHODS

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One hundred twenty-three patients admitted to the Detroit Receiving Hospital and University Health Center for bacteremia or sepsis, respiratory infections, skin and soft tissue infections, or urosepsis or pyelonephritis that were suspected to be due to gram-negative pathogens and for whom aminoglycoside therapy was required were enrolled in the study. Patients were identified for screening by physician order for an aminoglycoside. Of these patients, 123 were enrolled in the study on the basis of the following criteria; patients enrolled were greater than 18 years of age, had suspected infection for which an aminoglycoside was prescribed, had received no more than two aminoglycoside doses for the current infection, and were expected to receive therapy for at least 3 days. Patients were excluded if they had a serum creatinine concentration of >2.4 mg/dl, weighed 30% above their ideal body weight, had a history of hypersensitivity to any aminoglycoside, had tinnitus, deafness, or vestibular disturbances, or were pregnant, neutropenic (neutrophil count, <1,000/mm³), suspected of having meningitis, or in shock.

The protocol was approved by the Human Investigation Committee of Wayne State University, and informed consent was obtained from each patient or guardian. Patients were stratified by disease state (bacteremia or sepsis, respiratory infection, skin and soft tissue infection, or urosepsis or pyelonephritis) and were then randomly assigned by use of a random-numbers table to either dosing every 12 h as described below or dosing by once-daily administration of the entire daily dose of aminoglycoside, followed 12 h later by 100 ml of 5% dextrose in water as a placebo. Aminoglycoside doses were diluted in 100 ml of 5% dextrose in water. Drug and placebo doses were administered over 1 h. Both the patients and the physicians remained blinded to the treatment. Patients were also treated with beta-lactam or other antimicrobial agents as deemed necessary by their physicians.

Aminoglycoside dosing was individualized for both groups and was guided by targeted peak and trough concentrations in serum on the basis of the patient's individual pharmacokinetic parameters and standard equations (40). For the twice-daily dosing group, target peak concentrations in serum were 8 to 10 mg/liter for those with respiratory infections and 5 to 6 mg/liter for those with all other indications for gentamicin and tobramycin treatment, with trough concentrations of <2.0 mg/liter. For patients who received amikacin, peak concentrations in serum were 30 to 40 mg/liter for those with respiratory infections and 20 to 30 mg/liter for all other patients. Desired trough amikacin concentrations were <10.0 mg/liter. Target peak concentrations of gentamicin or tobramycin in serum concentrations for the once-daily dosing group were 16 to 20 mg/liter for those with respiratory infections and 10 to 12 mg/liter for patients with other infections. The desired peak serum amikacin concentrations were 60 to 80 mg/liter for patients with respiratory infections and 40 to 60 mg/liter for all others. The desired trough concentrations for once-daily dosing were targeted to be below 1.0 mg/liter. These target concentrations were based on doubling of the conventional target ranges for the Detroit Receiving Hospital and University Health Center (24). Initial dosing was based on population parameters in conjunction with the patient's calculated creatinine clearance and estimated ideal body weight. After serum aminoglycoside levels were obtained, dosing regimens were adjusted to maintain peak and trough concentrations in the targeted ranges. All patients were monitored daily by the unblinded pharmacokinetic consultation service. For patients in both groups three blood samples were drawn for determination of the aminoglycoside concentration after the administration of active doses (3rd, 7th, and 11th doses) and weekly thereafter. In addition, other determinations were made if deemed clinically necessary (e.g., for diminished renal function). Serum sampling times were 0.5, 8.0, and 10.5 h after the end of the 1-h infusion. Peak concentrations (end of infusion) and trough concentrations (at 12 and 24 h for groups who received aminoglycosides twice and once daily, respectively) were extrapolated by using a one-compartment equation with data for the serum samples obtained at the various times. Concentrations in serum were released only to unblinded pharmacists and were not reported in the patient's medical record.

Patient histories including the disease state that required aminoglycoside therapy, underlying medical conditions, and other drug therapies were obtained from each patient and the medical records. All patients were classified by the Acute Physiology and Chronic Health Evaluation (APACHE II) score (17). Creatinine clearance was calculated by the method of Cockroft and Gault (8). Lean body weight was used in the calculation of creatinine clearance for obese patients. Patients who received an aminoglycoside for at least 72 h and for whom plasma aminoglycoside concentration-time data were available were considered evaluable for nephrotoxicity. The serum creatinine concentration was obtained for each patient at the baseline (the lowest serum creatinine concentration within the period from 24 h before to 48 h after the initiation of therapy), on days 2, 4, 6, and 9, and then twice weekly or sooner if it was deemed clinically necessary. Nephrotoxicity was defined as an increase in the baseline serum creatinine concentration of 0.5 mg/dl or a 50% increase, whichever was greater, on two consecutive occasions any time during therapy or up to 1 week after the cessation of therapy (19). Nephrotoxicity was assessed by the same blinded investigator.

Audiologic testing was performed at the baseline (within 72 h of the start of therapy), weekly thereafter or sooner if it was deemed clinically necessary, and after the cessation of therapy. Pure-tone audiometry over the frequency range of 500 to 8,000 Hz (at the series of doubling frequencies and also at 3000 and 6,000 Hz) was performed for each ear by a certified audiologist in the audiologic laboratory or at the patient's bedside. Testing consisted of both air and bone conduction measurements and was performed with instruments calibrated to the American National Standards (ANSI S36-1969) specifications for audiometers. Vestibular function was not tested. Hearing impairment was evaluated by using the criteria of the American Academy of Otolaryngology Committee on Hearing and Equilibrium (2). All audiograms were interpreted by the same blinded investigator. Ototoxicity was defined as a decrease in auditory threshold of at least 15 dB at two adjacent tested frequencies in one or both ears.

Statistical analysis for patient characteristics and pharmacokinetic parameters was performed by the Mann-Whitney U rank sum test for unpaired data. The Fisher exact test was used for comparison of proportions. Data are reported as means ± standard deviations, and tests were performed at the 5% level of significance. Data were analyzed with Quattro Pro 5.0 for Windows (Borland International, Scotts Valley, Calif.) and SYSTAT 5.0 for Windows (SYSTAT Inc., Evanston, Ill.).

For the multivariate logistic regression analysis for nephrotoxicity, seven variables were examined. Days of therapy (DOT), area under the plasma concentration-time curve (AUC), and cumulative AUC (CumAUC; calculated as AUC · DOT) were treated as continuous variables. Site of infection (Site), Vancomycin use (Vanco), amphotericin B use (AmphoB), and schedule (Sched) were treated as categorical variables. Peak and trough concentrations were not evaluated separately. The hypothesis of this trial was that the dosing interval affects the probability of aminoglycoside toxicity. The peak concentration and the trough concentration are covariate with schedule (administration of the whole dose once daily will give higher peaks and lower troughs than administration of half of the dose every 12 h). We therefore decided to test the primary hypothesis that the dosing schedule is linked to the occurrence of nephrotoxicity.

The occurrence or absence of nephrotoxicity was coded as 1 or 0, respectively. The probability of occurrence of nephrotoxicity was modeled through the use of logistic regression with the logistic regression module of SYSTAT. Each covariate was examined univariately. Model building was approached in the following way: the covariate, which was most significant when tested univariately, was used as the base model. Other model covariates were tested for model expansion, one by one, in the order of the univariate significance. Because a maximum-likelihood estimator was used, the significance of model expansion was determined by the likelihood ratio test. Twice the likelihood difference for the competing models was referred to the ² distribution with the appropriate number of degrees of freedom for determination of significance.

We generated our hypothesis from data from previous studies with animals and humans, which demonstrated that schedule of administration influences the daily rate of uptake of aminoglycoside into the PRTE cell. This implies that once-daily administration will not protect from nephrotoxicity indefinitely but will delay the development of toxicity. We decided to test the hypothesis that schedule of drug administration also influenced the time to occurrence of the nephrotoxicity. Kaplan-Meier analysis was performed with stratification for dosing schedule. In addition, the same covariates detailed above were examined for their ability to influence the time to occurrence of nephrotoxicity with a Cox proportional hazards model. Model building proceeded as described above. In this analysis, schedule of administration was used as a stratification variable in all the analyses, and the effect of other covariates was examined by Cox modeling. Schedule of administration was tested for significance as a stratification variable by a Tarone-Ware test. In addition, a cumulative log hazards plot was examined to see whether schedule was most appropriately used as a stratification variable or as a covariate in the Cox proportional hazards model analysis.

	RESULTS

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Of a total of 123 patients enrolled in the study, 83 patients received therapy for at least 72 h; for 74 patients plasma aminoglycoside concentrations were available for analysis, and these patients formed the group evaluable for toxicity; 39 received doses twice daily and 35 received doses once daily. No patient discontinued therapy due to side effects attributed to an aminoglycoside. Of the patients not evaluable for toxicity, the majority were changed to some other antibiotic therapy before completing the minimum 72 h of aminoglycoside therapy: 14 of 23 (61%) were in the twice-daily dosing group and 17 of 26 (65%) were in the once-daily dosing group. Two patients in each group died before 72 h of treatment. One patient in the twice-daily dosing group was withdrawn because of an Ethics Committee decision to withdraw life support measures. Two patients who received aminoglycosides twice daily and one patient who received the drug once daily were receiving an aminoglycoside for prophylaxis treatment, and one patient in the once-daily dosing group was mistakenly enrolled after having received more than two prior doses of gentamicin. For the remaining patients plasma concentration-time data were not available.

Clinical characteristics for the 74 evaluable patients are summarized in Table 1. At the baseline the groups were comparable with respect to sex, age, weight, infectious disease diagnosis, serum creatinine clearance, and serum creatinine concentration. Two patients in the twice-daily dosing group and three patients in the once-daily dosing group were greater than 70 years of age. The majority of patients in both groups were treated for skin and soft tissue infections.

Peak and trough aminoglycoside concentrations for the evaluable patients are presented in Table 2. Data for patients who received either gentamicin or tobramycin were combined. For patients who received amikacin, the AUC was divided by 4, and the data were included in the logistic regression analysis. Average concentrations were derived by taking the average of the mean concentration for each patient. As expected, peak and trough concentrations in serum were significantly different (P < 0.0001) for the two dosing groups. Average daily doses were 4.3 ± 1.1 mg/kg of body weight with twice-daily dosing versus 6.1 ± 0.8 mg/kg with once-daily dosing for patients with respiratory infections and 3.6 ± 0.7 mg/kg with twice-daily dosing versus 3.9 ± 0.7 mg/kg with once-daily dosing for patients with all other infections combined. Three patients in the twice-daily dosing group experienced trough serum gentamicin or tobramycin concentrations above 2.0 mg/liter, whereas one patient in the once-daily dosing group experienced a trough concentration in serum above 2.0 mg/liter. One patient with twice-daily dosing (bacteremia) and three patients with once-daily dosing (pneumonia) never achieved the targeted peak concentrations in serum.

Thirty-four of the 74 (46%) evaluable patients had bacteriologically documented infections. A wide variety of organisms were cultured. Escherichia coli (n = 12) was the most commonly isolated organism in each group. Other major organisms included Acinetobacter baumannii (n = 4), Pseudomonas aeruginosa (n = 5), Klebsiella species (n = 4), Enterococcus species (n = 5), and Staphylococcus aureus (n = 2). Bacteremia was more common in the once-daily dosing group (seven versus three patients), and polymicrobial infections were more common in the twice-daily group (four patients versus one patient).

Patients in both groups received an average of one additional antibiotic during the study period. Most patients received a beta-lactam as concomitant therapy. The type of beta-lactam therapy was similar between the two groups and consisted of the following for the twice- and once-daily dosing groups: ampicillin, 10 and 11 patients, respectively; ampicillin-sulbactam, 1 and 2 patients, respectively; cefazolin, 7 and 5 patients, respectively; imipenem, 2 and 2 patients, respectively; nafcillin, 0 and 1 patients, respectively; piperacillin, 11 and 8 patients, respectively; and ticarcillin-clavulante, 1 and 2 patients, respectively. Eleven patients received concomitant vancomycin therapy (seven and four patients in the twice-daily and once-daily aminoglycopeptide dosing groups, respectively). One patient in each group received amphotericin B. No statistically significant differences were noted for the groups with respect to baseline APACHE II score, change in serum creatinine concentration, duration of therapy, cumulative dose, or mean total daily dose (Tables 1 and 3). Overall, the majority of evaluable patients defervesced during the study and were discharged to home or rehabilitation with oral antimicrobial therapy. One patient in the twice-daily dosing group died during the study period (post-72 h). Soon after enrollment in the study, this patient developed multi-organ system failure. Efficacy was not evaluated in this comparative study.

According to our definition, 6 of 39 patients (15.4%) in the twice-daily dosing group and 0 of 35 patients in the once-daily dosing group developed nephrotoxicity. This difference was significant when tested by the Fisher exact test (P = 0.026). Toxicity was observed to begin 8.8 ± 3.4 days after the start of treatment in the twice-daily dosing group.

The results of logistic regression analyses performed univariately are summarized in Table 4. Only the schedule of administration, vancomycin use, and daily AUC of aminoglycoside were significant in affecting the probability of nephrotoxicity. All were significant in the model-building process, as assessed by likelihood ratios. The final parameter values are displayed in Table 5.

The probability of nephrotoxicity as a function of AUC is displayed in Fig. 1A and B for once-daily and twice-daily administration, respectively, with and without vancomycin use.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   (A) Curve of probability of development of aminoglycoside nephrotoxicity for patients receiving the drug on a twice-daily basis as estimated by multivariate logistic regression analysis. The probability rises as a function of increasing daily exposure to aminoglycoside, as indexed to the AUC. Concurrent vancomycin use provides a marked increase in the probability of nephrotoxicity for equivalent exposure to aminoglycosides, as indexed to the daily AUC. (B) Once-daily administration shifts the curves of probability of nephrotoxicity as influenced by daily aminoglycoside AUC to the right.

A Kaplan-Meier analysis demonstrated that schedule of administration (primary hypothesis) significantly influenced time to nephrotoxicity (Log-rank test/mantel variant, P < 0.006; log-rank test/Breslow-Gehan variant, P < 0.026). Because schedule of administration influenced time to nephrotoxicity, this was retained as a stratification variable in all subsequent Cox proportional hazards modeling studies.

The parameters from the univariate Cox proportional hazards models (with stratification for schedule) are displayed in Table 6. Because the final model contained only vancomycin use with schedule of administration as a stratification variable, the final parameter value for vancomycin coadministration is presented in Table 6.

Twenty-two patients, 10 with twice-daily dosing and 12 with once-daily dosing, received two or more audiometric evaluations during and after treatment and were eligible for statistical analysis. Of these patients, one who received twice-daily aminoglycoside therapy met the criteria for ototoxicity.

	DISCUSSION

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Extensive data from animal models and clinical studies indicate that once-daily dosing of aminoglycosides results in lower accumulation rates per day in the PRTE cell. Whether this would result in lower rates of aminoglycoside-associated nephrotoxicity was the central question posed in this study. Several studies with patients have demonstrated no significant difference in efficacy (5, 11, 12, 14-16, 18, 20-22, 26-31, 34, 35, 38, 41) and nephrotoxicity (11, 12, 14-16, 20-22, 26, 27, 29, 31, 33-35, 38, 39, 41) between conventional and once-daily regimens of aminoglycoside dosing. These studies were, by and large, underpowered to test the hypothesis of lower nephrotoxicity as a function of once-daily dosing, and most importantly, all were unblinded. However, a meta-analysis investigation by Barza et al. (4) has demonstrated that examination of data from multiple studies demonstrates a difference in the rate of aminoglycoside nephrotoxicity in favor of the once-daily dosing group. Munckhof et al. (25), using a similar meta-analysis approach to data from published once-daily versus multiple-daily aminoglycoside dosing studies (2,881 patients), found equal rates of nephrotoxicity between the two regimens but demonstrated a more favorable clinical outcome (P = 0.027) with once-daily administration of these drugs. More recently, three additional meta-analyses have evaluated once-daily versus multiple-daily dosing of aminoglycosides. None of those studies found differences in efficacy, and all studies reported similar rates of toxicity between once-daily and multiple-daily aminoglycoside dosing (1, 3, 36).

Our study represents the first prospective, double-blind evaluation of this question. We found that once-daily dosing did, indeed, alter the risk of aminoglycoside nephrotoxicity. The factors identified in the logistic regression analysis as altering the probability of occurrence of nephrotoxicity and the factors that affect the time to nephrotoxicity, as identified in the Cox proportional hazards model, are concordant with each other and with the findings with animal models.

Examination of the data of terBraak et al. (35) demonstrates that once-daily dose administration and multiple-daily dose administration each will ultimately produce toxicity. What one purportedly gains from single-daily-dose administration is a lower daily intracellular accumulation rate, consistent with the clinical findings of Verpooten et al. (39) and the animal model data of Wood et al. (41). Therefore, the once-daily dosing schedule should provide a longer time of administration until the threshold for toxicity is met. This is precisely what is seen in our data, in which the AUC primarily drives the probability of nephrotoxicity but is modulated by the schedule of administration and the concurrent use of vancomycin. More explicitly, the time to nephrotoxicity was affected by both the schedule of administration and vancomycin use in the Cox model.

The concurrent use of vancomycin plus an aminoglycoside and the effect on the development of nephrotoxicity have been areas surrounded by controversy (7, 32). The use of vancomycin in this study was not controlled, and the effect of its concurrent use on nephrotoxicity was not the primary hypothesis being tested. As such, conclusions drawn from these data need to be viewed with caution. Nevertheless, both multivariate analyses indicated that concurrent vancomycin use significantly affects the patient's probability of developing nephrotoxicity and shortening the time to its occurrence. While this finding needs to be validated prospectively, clinicians would be prudent to monitor intensively the renal functions of patients who are receiving both agents.

Amphotericin B use was not linked to the occurrence of nephrotoxicity in any of the analyses. This is almost certainly because of the small number of patients who received this drug in our study. One of the patients in the twice-daily administration group received both vancomycin and amphotericin B and developed nephrotoxicity. Elimination of data for this patient from the data set changed none of the factors identified in either the logistic regression analysis or the Cox model as influencing the risk of nephrotoxicity.

Furthermore, all studies of once-daily dosing of aminoglycosides with patients to date have been unblinded. In contrast, this study randomized patients to either twice-daily therapy or once-daily therapy with a placebo infusion 12 h following the administration of each active dose. Concentrations in serum were not reported in the patient's medical report during the study period. This design allowed both the patient and the health care givers (physicians and nurses) to remain blinded to prevent investigator bias.

Nephrotoxicity developed in six patients in the twice-daily dosing group but in none of the patients in the once-daily dosing group. The serum creatinine concentrations for two of these patients in the twice-daily dosing group returned to the baseline before discharge from the hospital. Although the sample size is small, there was an equal distribution of males and females among the patients who experienced nephrotoxicity. To date, only one prospective study has reported a significant decrease in the incidence of nephrotoxicity in patients treated with aminoglycosides once daily compared to that in patients treated thrice daily. Prins et al. (29) observed a 5% (n = 40) incidence of nephrotoxicity in patients receiving once-daily therapy versus a 24% (n = 45) incidence in patients receiving an aminoglycoside three times daily. In their study, patients were also evaluable if they received 72 h of therapy. These data are quite consistent with the rates observed in the present study. Prins et al. (30) have also recently reported no difference in the rate of nephrotoxicity associated with once-daily gentamicin therapy (6.9%) versus that associated with once-daily netilmicin therapy (14.5%). In the absence of compelling data suggesting differences among aminoglycosides in their nephrotoxic potentials, we felt free to consider patients regardless of which aminoglycoside was used.

The use of the terminology "once-daily" or "single-daily" aminoglycoside therapy might be misleading. It is important to stress the concept of achieving high peak concentrations in serum rapidly and allowing concentrations to fall substantially below 2.0 mg/liter prior to administration of the next dose. Some patients in whom the aminoglycoside half-life is short will experience long periods during which subinhibitory concentrations go beyond the postantibiotic effect and may require dosing intervals shorter than 24 h, but this will require further study. For patients with slower elimination rates, it may not be appropriate to administer high doses of aminoglycosides on a once-daily basis. Studies have documented the need for larger initial doses of aminoglycosides in critically ill patients to achieve the desirable peak concentrations (40). This concern would be especially important with once-daily aminoglycoside dosing regimens.

The debate on whether once-daily aminoglycoside administration has value for the treatment of patients with infection continues to be voiced in the current literature, increasing the value of our investigation (6, 13). We have shown that, as predicted from data from animal models, single-daily-dose aminoglycoside administration, when applied to patients with a normal baseline renal function, is significantly less toxic than a more fractionated schedule. Although we intended to analyze the impact of dosing schedule on ototoxicity as well, the actual incidence in our study was too low to analyze the data in terms of ototoxicity. The potential for lowering the risk of ototoxicity from aminoglycosides via once-daily aminoglycoside administration is important and should be further investigated. As long as once-daily dosing of an aminoglycoside is at least as efficacious as conventional dosing, it clearly brings benefit to the patient in terms of toxicity avoidance and added convenience, especially for outpatient therapy. Furthermore, this therapeutic approach is more economical, since it produces less toxicity, which is highly costly (10), and requires less disposable equipment (e.g., syringes and intravenous supplies) and labor time for administration and fewer determinations of serum drug concentrations. On the basis of the results of this prospective study, for patients with normal renal function, we recommend that clinicians give serious consideration to the use of single-daily dosing for this class of anti-infective agents as a way of ameliorating the nephrotoxicity attendant to aminoglycoside administration.