Pharmacokinetics Of Cefotaxime Research Articles

Therapeutic considerations in using combinations of newer β-lactam antibiotics

The in vitro activity, pharmacokinetic interactions, and clinical efficacy of newer beta-lactam antibiotic combinations are reviewed. Combinations of beta-lactam antibiotics offer an antimicrobial spectrum similar to that of aminoglycoside-beta-lactam combinations without the renal or eighth cranial nerve toxicity of aminoglycosides. Synergistic activity with beta-lactam combinations is demonstrable in vitro against a wide variety of aerobic gram-negative bacilli, but the frequency, with which it is found is substantially less than for aminoglycoside-beta-lactam combinations. Also, in vitro antagonism can be demonstrated, particularly with combinations containing an agent capable of inducing beta lactamase. Substantial alterations in the pharmacokinetics of cefotaxime and desacetylcefotaxime have been demonstrated by the concomitant administration of mezlocillin or azlocillin. In addition, the clearance of moxalactam has been shown to be reduced by concomitant administration of piperacillin, and the clearance of oxacillin is reduced by concomitant mezlocillin therapy. Dosage reductions of these drugs may be appropriate in certain situations. Several clinical trials comparing therapy with beta-lactam combinations versus aminoglycoside-containing regimens in neutropenic patients have shown no difference in overall efficacy between the two regimens, with the possible exception of infections in persistently granulocytopenic patients and perhaps in patients with Pseudomonas aeruginosa infections. beta-lactam combinations are generally less nephrotoxic, but potentially more costly when newer compounds are included, than amino-glycoside-containing regimens. These beta-lactam combinations should be reserved for use in patients at high risk for aminoglycoside toxicity.

Pharmacokinetics of cephalosporins in normal and septicemic rabbits.

The differences in the pharmacokinetics of cefotaxime, moxalactam, and CPW 86-363, a new expanded-spectrum cephalosporin, were studied in healthy rabbits and in rabbits infected intravenously with Streptococcus pneumoniae. The pharmacokinetic analysis of concentration-time courses in the sera of infected animals according to a two compartment-model evidenced a clear decrease of drug fractions in the central compartment but enhanced drug fractions in the peripheral compartment. The shift was more pronounced in animals which received CPW 86-363 (60%; P less than 0.05) than in those which received cefotaxime (20%) or moxalactam (5%). Corresponding increases in drug concentration were observed in soft tissue interstitial fluid; therefore, the areas under the curve and mean residence times in the soft tissue interstitial fluid of infected rabbits were prolonged. The shift of drug fractions from the central compartment to other body fluid compartments during infection was thought to be due to cardiovascular changes associated with fever. No changes in serum binding of the three drugs were found during the course of the infection. The quantitative differences in the extent of altered distribution properties of the drugs might be due to variations in the physicochemical properties of the drugs.

The human pharmacokinetics of cefotaxime and its metabolites and the role of renal tubular secretion on their elimination.

The pharmacokinetics of cefotaxime were investigated in human volunteers given constant intravenous infusions, intravenous bolus, and intramuscular doses of the drug. After intravenous dosing, the plasma levels of cefotaxime declined in a biphasic manner with a terminal half-life varying between 0.92 and 1.65 hr. Moreover, the pharmacokinetics were linear up to at least a 2.0 g dose for volume of distribution based on area (23.3-31.3 l), plasma clearance (249-2.88 ml/min), and renal clearance (151-177 ml/min). Renal tubular secretion of intact cefotaxime and each of its metabolites was demonstrated by its interaction with probenecid, although the ratio of drug to metabolites ultimately excreted in urine after probenecid was similar to that seen normally (54 +/- 6, 19 +/- 4, 6.5 +/- 0.7 and 5.5 +/- 0.7% for cefotaxime, DACM, M2, and M3, respectively, when calculated as a percentage of the dose). The observed half-lives of DACM, M2, and M3 were 2.3 +/- 0.4, 2.2 +/- 0.1 and 2.2 hr, respectively. However, when the true half-life of DACM was calculated (0.83 +/- 0.23 hr) it was not only significantly shorter than that observed but also shorter than that for intact cefotaxime. The plasma clearance of DACM (744 +/- 226 ml/min) was much higher than that of cefotaxime while the volume of distribution was of a similar order (56 +/- 24 l). When administered intramuscularly, there was good absorption of cefotaxime from the site of injection (92-94%) giving maximum plasma levels of the drug of between 30 and 35 mg/l at approximately 40 min after dosing. Thereafter, the plasma levels of cefotaxime declined in a monophasic manner with a half-life (1.0-1.2 hr) similar to that of the terminal half-life seen after intravenous administration. Lidocaine had no significant effect on either its absorption or elimination kinetics.

Effect of probenecid on the pharmacokinetics of cefotaxime in sheep.

The effect of probenecid given by intravenous (i.v.), intramuscular (i.m.) and subcutaneous (s.c.) injection on the pharmacokinetics of cefotaxime was studied in six Merino ewes. When given intravenously, probenecid increased significantly (P less than 0.05) the plasma half-life of cefotaxime three-fold (to 0.94 +/- 0.32 h) and the area under the curve (AUC) approximately two-fold (to 41.1 +/- 16.8 micrograms.h/ml), and decreased plasma cefotaxime clearance (ClB) 45% (to 0.648 +/- 0.191 l/h/kg). When given with probenecid intravenously, renal clearance (ClR), volume of the central compartment (VC), volume of distribution steady state (Vd(ss], and the amount excreted in urine unchanged did not alter significantly. When given by i.m. injection, probenecid and cefotaxime were well tolerated and cefotaxime was well absorbed (101 +/- 45%). When given by s.c. injection, only 40 +/- 25% cefotaxime was absorbed. When given intramuscularly or subcutaneously, probenecid appeared to reduce the ClB and ClR of cefotaxime, probably because plasma probenecid concentrations are prolonged. Probenecid did not appear to affect the distribution of cefotaxime.

The pharmacokinetics of cefotaxime and ceftriaxone in renal and hepatic dysfunction

The pharmacokinetics of cefotaxime, its desacetyl metabolite and ceftriaxone were studied in 72 patients with various degrees of renal and hepatic failure. Patients with severe renal failure (creatinine clearance 3 to 10 ml/min) had a cefotaxime serum half-life of 2.6 h, a desacetyl cefotaxime serum half-life of 10 h and ceftriaxone serum half-life of 17 h. In the case of ceftriaxone, this serum half-life could be very variable--increasing to over 50 h, possibly due to co-existing hepatic dysfunction. Increases in serum half-life were found for cefotaxime in the presence of liver disease.

Pharmacokinetics of Intravenous and Intraperitoneal Cefotaxime in Patients Undergoing CAPD

We investigated the pharmacokinetics of non-metabolized cefotaxime in 10 patients undergoing CAPD. The elimination half-life after IV administration of I g cefotaxime was 3.1 ± 1.3 hr, i.e. two to three times longer than in individuals with normal renal function but similar to patients with severe renal insufficiency. An average of 2.18% of the dose was recovered in the effluent. The halflife of I g cefotaxime administered in the dialysis solutions was 1.4 ± 0.8 hr. This difference between the half-lives after intraperitoneal and intravenous administration indicates a faster transport through the peritoneal membrane. Intraperitoneally administered cefotaxime -250 mg four times daily, was effective in the treatment of peritonitis in three CAPD patients. Since its introduction in 1976, CAPD has become an effective therapy for end-stage renal disease. The most serious complication is peritonitis and effective treatment is essential. Cefotaxime, a new broad-spectrurn cephalosporin, is active against most gramnegative and gram-positive organisms. It possesses no nephrotoxicity and may be useful in the treatment of peritonitis and other infections in patients on CAPD. This study was done to evaluate the pharmacokinetics of cefotaxime administered intravenously and intraperitoneally during CAPD.

Rapid chromatographic determination of cefotaxime and its metabolite in biological fluids

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of cefotaxime and its metabolite desacetylcefotaxime in plasma and urine was developed. Plasma was deproteinized with small amounts of acetonitrile. After separation of the proteins the supernatant was extracted with a mixture of chloroform and 1-butanol. A phase separation was obtained leaving the cephalosporin and its metabolite in the aqueous part and extracting most of the interfering endogenous material. The aqueous phase was injected directly into the chromatograph. As part of the plasma water was dissolved in the acetonitrile—1-butanol—chloroform layer, the concentration of the cephalosporin in the aqueous phase was significantly higher than in the original plasma sample. Therefore, the usual diluting effect of the deproteinization could be avoided. In a similar way the assay was applicable to measure cefotaxime and its metabolite in urine. Calibration curves were set up and were linear up to 25 μg/ml for desacetylcefotaxime and 250 μg/ml for cefotaxime. The assay was applied to study the pharmacokinetics of cefotaxime and its metabolite in a healthy volunteer. In a similar way this deproteinization and extraction method was also applied to assay for ceftazidime, cephalexin, cephazolin and cefoxitin.

Pharmacokinetics of cefotaxime and probenecid in sheep with normal and reduced renal function.

The pharmacokinetics of cefotaxime and probenecid, given by intravenous injection, were determined in six Merino ewes which had been subjected to a 75% reduction in renal mass. These results were compared with results previously determined in sheep with normal renal function. In the sheep with reduced renal mass, the following significant changes in parameter values for cefotaxime were observed. The elimination rate constant (kel) decreased by 47%, the apparent volume of the central compartment (Vc) decreased by 59%, the steady state volume (Vss) decreased by 50%, and the total body clearance (ClB) decreased by 78%. The rate constant for distribution of drug into tissues (k12) increased 6.9 times, the rate constant for distribution out of tissues (k21) increased 3.7 times, and the area under the plasma concentration-time curve (AUC) increased by a factor of 4.9. The parameter values, determined in sheep with reduced renal mass, for probenecid plasma half-life, Vss and the rate constants k12, k21, and kel were not significantly different from the values obtained previously in sheep with normal renal mass. However, the rate constant for renal excretion of probenecid (ke), renal clearance (ClR), ClB and Vc decreased by 79, 90, 54 and 36%, respectively. The results indicate that reduced renal mass increased the plasma half-life for cefotaxime as well as increasing its diffusion into tissue. In the case of probenecid the overall distribution and elimination kinetics were not altered by reduced renal mass; however, the rate of urinary excretion of the drug was reduced.

Pharmacokinetics of cefotaxime in neonates.

The effects of gestational age on the pharmacokinetics of cefotaxime and its desacetyl metabolite during the first days of life was investigated in a group of four full-term infants and 12 preterm infants of less than 35 weeks gestation. Half of the preterm infants had received betamethasone, a drug known to facilitate hepatic microsomal drug metabolism, whilst the others had not. No significant differences in the pharmacokinetics of cefotaxime were observed between the various groups, with elimination half-life (T 1/2 beta) of cefotaxime ranging from 4.04 +/- 1.52 to 4.56 +/- 1.31 h. The desacetyl metabolite of cefotaxime was present in all post-dose serum samples, irrespective of the gestational age of the baby. Its formation was apparently unaffected by prior exposure to betamethasone. The elimination half-life of cefotaxime is significantly longer in newborn infants than in older children or adults, this increase probably results from decreased renal excretion of the drug, rather than from immaturity in its metabolism. A dose of 50 mg/kg of cefotaxime given every 12 h is appropriate for infants of less than seven days old.

Pharmacokinetics of cefotaxime in patients undergoing haemodialysis and continuous ambulatory peritoneal dialysis.

The pharmacokinetics of cefotaxime were studied in 13 patients on haemodialysis or continuous ambulatory peritoneal dialysis (CAPD). One gram of cefotaxime was administered intravenously or intraperitoneally. In the haemodialysis patients, the serum concentration after 4h was 9.4 mg/l and the T1/2 was 1.31 h. The 6-h value and T1/2 in the CAPD (intravenous) patients were 4.5 mg/l and 1.59 hours, while the peak value in the dialysate was 11.6 mg/l. In the CAPD (intraperitoneal) patients, the cefotaxime concentration in the dialysate was 157 mg/l after 6 h and the peak value in the serum was 9.1 mg/l. Since cefotaxime is easily removed from the blood by both the haemodialysis and CAPD methods, accumulation of the drug in patients undergoing dialysis is unlikely to occur.

Pharmacokinetics of cefotaxime and desacetyl-cefotaxime in cirrhosis of the liver.

In 9 patients with advanced hepatic cirrhosis and a normal serum creatinine concentration, the pharmacokinetics of cefotaxime (CTX) and its desacetyl metabolite (DACM) were examined in serum and urine after intravenous administration of 2.0 g CTX. The peak serum levels were 130.3 +/- 33.9 and 8.5 +/- 4.6 mg/l for CTX and DACM, respectively. T50% beta was calculated to be 138.1 min (69.3-245.8 min) for CTX. The approximation of the terminal half-life of DACM revealed a prolongation which exceeded 10 h. Accordingly, the AUC amounted to 255.3 +/- 92.0 and 151.9 h X mg/l, respectively. Within 24 h, 62.9% was eliminated in urine as CTX and 19.4% as DACM. The total clearance of CTX was reduced to 164.3 ml/min (58.3-296.6 ml/min), but the renal clearance was only moderately decreased to 90.0 ml/min (24.1-169.3 ml/min). These altered kinetics can be explained by altered metabolic function of the liver as well as by cirrhosis-linked alterations of renal elimination mechanisms.

Cerebrospinal fluid penetration after combined administration of cefotaxime and ampicillin to rabbits with staphylococcal meningitis.

The pharmacokinetics of cefotaxime and ampicillin in the cerebrospinal fluid (CSF) were studied by a HPLC method after administering each of the antibiotics singly or as a mixture to rabbits with experimental meningitis caused by Staphylococcus aureus. The half-lives of cefotaxime and desacetyl-cefotaxime in the serum and of ampicillin in the CSF were found to be significantly shorter in the mixture group than in the respective single administration groups. No other statistically significant differences were observed between the mixture and single administration groups with respect to the maximum concentrations (Cmax) of the antibiotics in the serum and the CSF, to the relative percentage of the Cmax of each antibiotic in the CSF to the Cmax in the serum, and to the relative percentage of the AUC of each antibiotic in the CSF to that in the serum. These results differ from earlier findings that the concentration of ampicillin in the CSF of rabbits was significantly lower in animals concomitantly dosed with latamoxef (moxalactam) and ampicillin than in a group dosed only with ampicillin.

Elimination kinetics of cefotaxime and desacetyl cefotaxime in patients with renal insufficiency and during hemodialysis.

The pharmacokinetics of cefotaxime, a new semi-synthetic cephalosporin for injection, was studied in 30 subjects with various degrees of renal function after a single 1-gram intramuscular injection. Serum and urinary concentrations of cefotaxime and desacetyl cefotaxime were determined by high pressure liquid chromatography. The pharmacokinetic parameters of cefotaxime were obtained using a one-compartment open model. The mean serum half-life of the parent compound (cefotaxime), 0.87 h in normal subjects, was prolonged to 2.35 h in hemodialysis patients. There was a significant linear correlation between the elimination rate constant of cefotaxime and creatinine clearance. The mean cumulative urinary recovery of the administered dose in the 24-hour urine was 51.7% as cefotaxime and 25.6% as desacetyl cefotaxime in normal subjects.

Comparative Multiple-Dose Pharmacokinetics of Cefotaxime, Moxalactam, and Ceftazidime

The pharmacokinetics of cefotaxime, moxalactam, and ceftazidime were investigated in six human volunteers who received in a crossover fashion doses of 0.5, 1.0, and 2.0 g of each drug by a 5-min infusion. Doses of 1.0 g were repeated after the administration of probenecid. Serum and urine concentrations were assayed with an agar diffusion method. Serum concentrations of moxalactam exceeded those of ceftazidime at all times and were distinctly higher than those of cefotaxime. The normalized area under the concentration time curve (defined as the ratio of the area under the curve per dose) reflects this relationship: compared with cefotaxime the normalized area under the curve of moxalactam was 3 to 4 times higher, and that of ceftazidime was 2 to 3 times higher. By intra-individual comparisons, the area under the curve of moxalactam was 44% larger than that of ceftazidime. With increasing doses, cefotaxime exhibited a nonlinear increase of the area under the curve. The half-lives of moxalactam, ceftazidime, and cefotaxime were 2.34, 1.95, and 1.16 h, respectively. The volume of distribution averaged 0.20 ± 0.03, 0.23 ± 0.02, and 0.25 ± 0.04 liters per kg, and the mean total body clearance was 84, 131, and 328 ml/min for moxalactam, ceftazidime, and cefotaxime, respectively. The 24-h urinary recovery was highest for moxalactam (75 ± 4%) followed by ceftazidime (68 ± 11%) and cefotaxime (53 ± 6%). The influence of probenecid on serum concentrations, half-life, area under the curve, and clearance was most apparent with cefotaxime, whereas the pharmacokinetics of moxalactam and ceftazidime were only slightly affected. After the 0.5− and 2.0− g doses of cefotaxime, desacetyl-cefotaxime activity (determined by high-pressure liquid chromatography) reached a peak of 2.7 and 9.9 μg/ml and declined with a half-life of 1.9 and 1.4 h. The ratio of the R(−) and S(−) epimers of moxalactam, which could be differentiated by high-pressure liquid chromatography, fell rapidly from 0.81 at 0.17 h to 0.5 at 5 h, indicating the presence of twice as much of the microbiologically less active S(−) epimer. From a pharmacokinetic standpoint it appears reasonable to conclude that moxalactam and possibly ceftazidime could be administered twice daily and that cefotaxime could be administered three or even four times daily.

The pharmacokinetics of cefotaxime and its metabolites in subjects with normal and impaired renal function.

The pharmacokinetics of single doses (15 mg/kg) of cefotaxime and each of its metabolites were examined in six normal subjects and 24 patients who showed various degrees of renal insufficiency. An additional nine patients with renal failure were entered into a multiple-dose study in which cefotaxime (1 g) was given twice daily for five to seven days. After intravenous administration, the levels of cefotaxime in serum declined in two phases. The half-life for the terminal phase was 1.10 +/- 0.78 hr. Cefotaxime administered intramuscularly was well absorbed from the injection site (91% +/- 19%) and produced maximal levels of 25.5 +/- 3.5 microgram/ml in serum at approximately 0.5 hr after injection. The levels of the desacetyl metabolite in serum were comparatively low, and the half-life of this metabolite was 1.3 hr. Microbiologic measurements of cefotaxime showed that the half-life of the drug was lower in normal subjects (1.71 +/- 0.90 hr) than in patients with severe renal impairment (half-life, 10.20 +/- 3.43 hr). However, this difference was due primarily to desacetylcefotaxime. Specific measurement by high-performance liquid chromatography indicated only a slight increase in the half-life of cefotaxime, whereas the increase in half-life of desacetylcefotaxime was greater. The most pronounced increases in half-life of metabolites occurred when values for creatinine clearance in patients were below about 5 ml/min per 1.73 m2. When cefotaxime was administered repetitively, the ratio of maximal serum levels after the last dose to those after the first dose demonstrated minimal accumulation of intact drug. The average accumulation of desacetylcefotaxime in this group of patients was about twofold, and that for metabolites M2 and M3 was approximately fourfold. This accumulation of metabolites was observed only in those patients with severe renal impairment and was not excessive. Hemodialysis caused a significant reduction in the half-life of both cefotaxime and desacetylcefotaxime. Recommendations are made for the adjustment of dosing schedules in patients with renal failure.

Clinical pharmacokinetics of cefotaxime in patients with normal and reduced renal function.

Two analytic methods for measuring cefotaxime, microbiologic and high-pressure liquid chromatography (HPLC), were compared in normal healthy subjects and in patients with impaired renal function. Results of studies showed that in healthy subjects the levels of desacetylcefotaxime are low relative to those of cefotaxime and that either a bioassay or HPLC is adequately specific for determination of pharmacokinetic parameters. However, in patients with decreased renal function, desacetylcefotaxime reaches much higher levels than in healthy subjects. Thus, the less specific bioassay produces misleading data on serum and urine concentrations in these patients, and the HPLC assay of cefotaxime is recommended for estimating pharmacokinetic parameters in patients with decreased renal function. In healthy subjects and in patients with a creatinine clearance greater than 20 ml/min, neither cefotaxime nor its metabolites accumulates after multiple dosing. If creatinine clearance is less than 20 ml/min, metabolites begin to accumulate. It is recommended that until further data are obtained the dose of cefotaxime given to patients with an estimated creatinine clearance of less than 20 ml/min be half that given to patients with values greater than 20 ml/min.

Pharmacokinetics of cefotaxime in newborn infants.

The pharmacokinetics of cefotaxime were determined in newborn infants who were 1 to 7 days of age. Mean peak serum concentrations of 116 and 132 micrograms/ml were observed at completion of a 10-min intravenous infusion of 50 mg of cefotaxime per kg in low and average birth weight infants, respectively. The mean elimination half-lives were 4.6 and 3.4 h and rates of clearance from serum were 23 and 44 ml/min per 1.73 m2, respectively. A dosage schedule for cefotaxime in newborn infants is presented.

Cefotaxime kinetics after multiple-dose intramuscular study in healthy volunteers.

The pharmacokinetics of cefotaxime after repeated doses of intramuscular injections were studied in 8 healthy volunteers receiving 1 g cefotaxime every 8 h for 6 days. Mean peak serum level after the first 1 g intramuscular dose was 26.8 +/- 6.4 micrograms/ml and the peak serum concentration was reached within 75 min. The half-life of cefotaxime was 1.35 +/- 0.33 h and the total apparent clearance 263 +/- 63 ml/min. After the last injection (day 6), the mean peak serum level reached 19.1 +/- 3.2 micrograms/ml and the areas under the curve were significantly lower than on day 1. No body accumulation of the drug could be detected. Serum levels of cefotaxime monitored throughout the study 5 min prior to and 30 min after the intramuscular injection were found above the minimum inhibitory concentration for most aerobic gram-positive and gram-negative sensitive organisms.

Cefotaxime pharmacokinetics following a single intravenous dose to patients with varying renal function.

The pharmacokinetics of cefotaxime (HR 756) were investigated in two groups of patients (Groups I and II) with creatinine clearances of greater than or equal to 60 and < 60 ml/min per 1.73 m2 respectively. Each of the 24 patients included in the study received 0.5 g or 1.0 g cefotaxime intravenously as a bolus injection. The decline of serum concentrations was biphasic in all patients, and the data were fitted to the pharmacokinetic two-compartment model. The mean distribution and elimination half-lives of cefotaxime in individuals in Group I were 0.2 and 1.2 hours respectively. In the uremic individuals, the pharmacokinetic parametes did not differ markedly from those in normal subjects, except when renal function was markedly reduced. In severe renal impairment, the elimination half-life increased to 8.3 hours. Cumulative 24-hour urinary excretion accounted for a mean of 59% of the dose in normal individuals, and from 0.3 to 36% of the dose in uremic individuals. Incomplete recovery of intact drug in urine of normal individuals reflects excretion by an extrarenal pathway, possibly as desacetyl cefotaxime. Urine levels were greater than the minimum inhibitory concentrations for susceptible organisms for at least eight hours after dosing in all individuals who produced urine. Because of the relatively small effect of renal impairment on the pharmacokinetics of cefotaxime, dose reduction is necessary only in cases of severe renal impairment.

Pharmacokinetics of cefotaxime in normal human volunteers.

The pharmacokinetic parameters of cefotaxime after either intramuscular (Lm.) or intravenous (i.v.) administration were determined in several studies involving about 200 healthy volunteers. In single-dose studies, the pharmacokinetics of cefotaxime appeared to be linear and dose-independent for doses up to 2 g. The mean peak concentration in serum after i.m. injection of 1 g of cefotaxime was ≃22 μg/ml at 0.5 h, whereas the level at 6 h was ≃1.5 μg. After i.v. bolus injection of 1 g of cefotaxime, the mean peak level was ≃105 μg, with an apparent volume of distribution of ≃25 1. After i.v. infusion serum levels ranged from ≃15 μg to ≃40μg, depending on the duration of infusion. Cefotaxime is rapidly eliminated: serum half-life ranged from 0.92 to 1.35 h after 1 g i.m. injection, and from 0.84 to 1.25 h after i.v. injection (terminal phase), the majority of the dose being excreted unchanged in the urine (≃51% within 8 h). Protein binding of cefotaxime ranged from 35 to 45 %. Thus the pharmacokinetic profile of cefotaxime was similar to that reported for cefoxitin, cefamandole and cefuroxime. Co-administration of lignocaine had no significant effect on the absorption and elimination kinetics of cefotaxime while probenecid increased and sustained plasma levels, acting primarily by inhibition of renal tubular secretion. In multiple-dose studies, cefotaxime was administered i.m. and i.v. at different doses, dosing intervals and for periods of 6 to 14 days. There was no evidence that the administration of cefotaxime up to 1 g 6-hourly for 14 days caused any significant accumulation in healthy volunteers.