Pharmacolunetics of recombinant human superoxide dismutase in healthy volunteers We studied the pharrnacokinetics and effects of recombinant human superoxide dismutase (rhSOD) in 3 2 normal human volunteers after intravenous bolus doses from 1 mglkg to 4 5 mglkg in a single-blind, placebo-controlled, crossover design. The drug was well tolerated. Neither cardiovascular nor renal function, such as the echocardiographically determined cardiac index, inulin or para-aminohippurate clearances, or the urinary excretion of P,-microglobulin or N-acetylglucosaminidase, was affected. Pharmacokinetic analysis by use of noncompartmental methods showed an overall half-life of rhSOD to be about 4 hours for doses from 3 mgikg to 4 5 mgikg. The peak concentrations ranged from 2 4 to 8 3 7 mg&, and urinary excretion increased from 3% to 57% of total dose after single intravenous bolus administrations of the drug from 1 mg/kg to 4 5 mg/kg. The mean renal clearance of rhSOD initially increased with dose then plateaued at the highest dose, whereas the nonrenal clearance decreased with dose to a plateau; total clearance remained essentially constant. The progressive increase in renal clearance may be explained by saturation of the tubular reabsorption and degradation of the protein, a mechTHER1991;50:713-20.) anism previously described in animal models. (CLINPHARMACOL
Cecillia Tsao, MD, Paula Greene, BSN, Bo Odlind, MD, and D. Craig Brater, MD Indianapolis, Ind.
The relationship of oxygen-derived free radicals, or superoxides, to cell injury has been an area of active research with considerable early evidence for releSuperoxide vance to a number of disease dismutase (SOD), an enzyme naturally present in many tissues of humans and other organisms, catalyzes the conversion of superoxides to less reactive oxygen species, thereby protecting the cell. This protective role has been demonstrated in various animal models of ischemia and reperfusion injury such as myocardial infarction, cardioplegia, and renal transplantation.4-11Before the availability of human SOD, clinical studies of SOD in humans were limited to the use of bovine SOD.'^.^^ Human SOD has recently become available by recombinant technology with use of a yeast vector (rhSOD; Chiron-Pharmacia Partnership, Piscataway, N.J.); clinical investigation of its use in humans by the intravenous route is currently underway in the area of renal tran~plantation.'~ Although
From the Division of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine. Received for publication April 11, 1991; accepted Sept. 4, 1991. Supported by a grant from Chiron-Pharmacia Partnership. Reprint requests: D. Craig Brater, MD, Rm. 320 WOP Bldg., LOO1 W. loth St., Indianapolis, IN 46202. 1311133584
the effects and pharmacokinetics of rhSOD after intravenous administration have been studied in animal models, these characteristics of the protein in humans have not been reported.I6-I* The purpose of this study is to establish the pharmacokinetic parameters and to monitor the renal and cardiovascular effects of rhSOD in normal volunteers after intravenous bolus doses of rhSOD from 1 mgikg to 45 mglkg in a single-blind (to volunteers), placebo-controlled, crossover study design. Use of increasing doses allowed the unique observation of increasing renal clearance to a plateau level that likely represents saturation of renal tubular degradation of filtered SOD.
METHODS We studied 32 male volunteers between the ages of 20 and 40 years who weighed between 60.1 and 94.2 kg. All subjects were deemed to be normal on the basis of screening medical history, physical examination, and laboratory parameters including clinical chemistries, hematology, urinalysis, and electrocardiogram. Subjects were divided into five dose groups: 1 mgikg and 3 mglkg each with four subjects and 7.5 mgikg, 15 mgikg, and 45 mgikg each with eight subjects. Each subject was randomly assigned to initially receive either rhSOD or sucrose placebo; the alternate treatment followed after a washout period of at least 7
CLIN PHAIt'vIACOL THER
714 Tsao etal. days. Subjects were excluded if they had any previous drug allergies; they took no medications or alcohol during the study. Protocol. Subjects followed a no-added-salt diet at least 5 days before the study and fasted overnight before the date of study. Intravenous catheters were placed in each forearm, one for infusion of study drug and marker compounds (inulin and para-aminohippurate [PAH]) and the other for serial blood sampling. Inulin at a loading dose of 50 mgikg and PAH at a loading dose of 10 mgikg were administered intravenously followed by continuous infusion of the two compounds to maintain serum concentrations at about 20 mgidl for inulin and 1.5 mgldl for PAH. To ensure adequate urine output, a water load of 20 mllkg distilled water was ingested by the subject during the 45minute equilibration period, which was followed by three 30-minute urine collections with midpoint blood samples for baseline inulin and PAH clearance measurements. Urine f uid loss was replaced with intravenous 5% dextrose solution during the control and experimental collection periods. At the end of the third control urine collection, two-dimensional echocardiographic examination with Doppler evaluation was performed. The study drug, either rhSOD or placebo, was administered intravenously as a bolus over either 1 minute for doses up to 7.5 mgikg or over 5 minutes for doses greater than 7.5 mgikg. Immediately afterward and approximately 2 hours after drug administration (at the end of the experimental period), repeat echocardiography was performed. The subject was placed on continuous electrocardiographic monitoring and had vital signs taken every 5 minutes for 1 hour after drug injection. Four additional 30-minute urine collections with midpoint blood samples (experimental periods) were carried out for marker compound and drug clearance studies. Serial blood samples for drug concentrations of rhSOD were collected at 1 , 2, 5, 10, 15, 30, 45, and 60 minutes and then every hour until 8 hours and every 2 hours until 12 hours after drug injection. Clinical laboratory parameters were checked at 30 minutes, 2 hours, and 24 hours after drug injection. Timed urine collections were continued for the first 24 hours for measurement of drug excretion. Follow-up blood samples were examined at 2, 4, and 6 weeks after drug administration for possible development of antibodies to rhSOD. No antibodies were detected. Data analysis. Plasma portions of the blood Samples and urine aliquots were obtained for drug concentrations and frozen at -20" C before analysis. Con-
centrations of rhSOD in plasma and urine samples were measured by use of a previously described douIn brief, plasma ble antibody radioimm~noassay.'~.'~ or urine samples to be analyzed were diluted with phosphate buffer to concentrations in the mid-range of the assay (range, 2 to 200 mg/L). The sample was mixed with 125~-labeled rhSOD tracer and incubated at room temperature with antibodies specific for rhSOD raised in rabbits. Sheep anti-rabbit antibodies coupled onto micro-Sepharose (Pharmacia, Piscataway, N.J.) were added to the mixture, which was then centrifuged and the ' 2 5 ~activity of the sediment measured. The concentrations of rhSOD in the unknown samples were determined by comparison to a standard curve generated with known concentrations of rhSOD. The coefficients of variation for control samples were 14.3%, 6.0%, and 4.4% at concentrations of 6, 30, and 150 mgiL, respectively. Samples were measured in duplicate. The mean pretreatment plasma concentration of rhSOD (0.34 0.09 mg/L for subjects 25 to 32) was not significantly different from the mean value after placebo treatments (0.42 i 0.13 mg/L for all subjects). SOD either was not detected or was found only in trace amounts (