PHARMACOKINETICS, SAFETY AND IMMUNOMODULATORY EFFECTS OF HUMAN RECOMBINANT INTERLEUKIN-1 RECEPTOR ANTAGONIST IN HEALTHY HUMANS Eric V. Granowitz, Reuven Porat, James W. Mier, John P. Pribble, David M. Stiles, Duane C. Bloedow, Michael A. Catalano, Sheldon M. Wolff, Charles A. Dinarello A phase I study of human recombinant interleukin-1 receptor antagonist (IL-lra) was conducted in healthy males between the ages of 18 and 30. Twenty-five volunteers received a single, 3 h continuous intravenous infusion of doses ranging between 1 mg/kg and 10 mg/kg IL-lra. At 3 h into the infusion, plasma IL-lra levels were 3.1 &ml and 29 pg/ml for the 1 mg/kg and 10 mg/kg doses, respectively. Post-infusion plasma IL-lra levels declined rapidly, exhibiting an initial half-life of 21 min and a terminal half-life of 108 min. Clinical, hematological, biochemical, endocrinological and immunomodulatory effects were monitored over 72 h and compared to those of four subjects receiving a 3 h infusion of saline. There were no clinically significant differences between the drug and saline groups in symptoms, physical examinations, complete blood counts, mononuclear cell phenotypes, blood chemistry profiles, serum iron and serum cortisol levels. Peripheral blood mononuclear cells (PBMC) obtained after completion of the IL-lra infusion synthesized significantly less interleukin 6 ex vivo than PBMC from saline-injected controls. These data suggest that transient blockade of interleukin 1 receptors is safe and does not significantly affect homeostasis.

Interleukin la (IL-lo) and interleukin 1B (IL-lp) are two distinct geneproducts which have 25% amino acid similarity, recognize both IL-1 receptors (IL-lR), and exert similar biological effects. Elevated plasma levels of IL-1 are present in a variety of diseasesincluding septic shock.r-4 Consequently, IL-1 is considered a mediator of sepsis. 5 This theory is supported by the finding that infusing IL-l into humans induces fever, hypotension, leukocytosis and thrombocytosis.6,7 The IL-l receptor antagonist (IL-lra) is a naturally occurring 22 kDa glycoprotein which blocks the bind-

From the Department of Medicine, New England Medical Center Hospitals, and Tufts University School of Medicine, Boston, MA and Synergen, Inc., Boulder, CO, USA. Address correspondence and reprint requests to: C.A. Dinarello, M.D., Department of Medicine, New England Medical Center Hospitals, 750 Washington Street, Boston, MA 02111, USA. Received 16 March 1992; accepted for publication 1 May 1992 @ 1992 Academic Press Limited 1043-4666/92/050353+08 $08.00/O KEY

WORDS:

CYTOKINE,

human/IL-l/IL-lraipharmacokinetics

Vol.

4, No. 5 (September),

1992: pp 353-360

ing of IL-l to the IL-1R type I on T-cellssro and the IL-1R type II on polymorphonuclear leukocytes and B-cells.ri-13 IL-lra has 19% amino acid similarity with IL-lo and 26% amino acid similarity with ILlB.14 Despite this amino acid identity with IL-1 and similar binding affinity for the IL-1R type I, IL-lra has no agonist activity.9J5J6 In fact, IL-lra blocks in vitro IL-l-induced cytokine synthesis,17 fever,18 hypotension, leukopenia,lg and acute phase protein synthesis.20Ji In addition, IL-lra blocks Escherichia co&-induced shock** and lipopolysaccharide (LPS)induced mortality.19 In experimental endotoxemia in humans, plasma concentrations of endogenous IL-lra are loo-fold greater than those of IL-lp.23 Similar elevations in IL-lra are seen in sepsis.24 In this report we describe the first administration of intravenous human recombinant IL-lra to humans. In a Phase I trial performed in healthy male volunteers, we examined the safety of short term use of IL-lra. In addition, we found that peripheral blood mononuclear ceils (PBMC) from those volunteers who received IL-lra synthesized significantly less interleukin 6 (IL-6) in response to LPS ex vivo. 353

354 I Granowitz et al.

CYTOKINE,

RESULTS Pharmacokinetics Mean peak plasma IL-lra levels measured at the end of the infusion were 3.1 f 0.3 pg/ml for the 1 mg/kg dose group and 29 + 2 pg/ml for the 10 mg/kg dose group (Fig. 1). Pharmacokinetics were unaffected by dose over the dose range studied. Post-infusion plasma IL-lra levels declined quickly, exhibiting an initial half-life of 21 k 3 min and a terminal half-life of 108 + 18 min. IL-lra distributed into a steady-state distribution volume of 0.18 f 0.04 l/kg. Plasma clearance for IL-lra was 2.0 + 0.3 ml/min/kg. Less than 3.2% of the administered dose of IL-lra was detected in the urine. Plasma IL-lra clearance did not correlate with creatinine clearance.

Vol.

4, No.

5 (September

TABLE

1.

Schedule of studies in human volunteers

receiving

Evaluation

Plasma Cytokine Assays In all groups, baseline plasma levels of lL-lp, interleukin 2 (IL-2), IL-6, interleukin 8 (IL-g),

0

0.5

0.75

1

1.5

2

3

2

4

6

Figure IL-lra.

1.

Plasma

IL-lra

levels

s

Imgikg

-

2 mg/kg

-

3 mg/kg

-

5 mg/kg

-

7 mg/kg

--t

IO mg/kg

8

Time

during

IO

I2

(hours)

and after

a 3 h infusion

of

Volunteers were given 1 (N = 3), 2 (N = 6), 3 (N = 4), 5 (N = 4), 7 (N = 4) or 10 mgikg (N = 4) IL-lra as a 3 h continuous intravenous infusion beginning at time zero. Plasma IL-lra levels in the saline controls were < 1 ngiml.

IL-lra. Time (hours

0

353-360)

after the infusion liver function tests were within normal limits. A second volunteer had an asymptomatic, transient rise in ALT to 57 U/l 72 h after his 2 mg/kg infusion.

Clinical Evaluation Table 1 shows the various times of clinical evaluation. At all time points there were no clinically significant differences in symptoms, vital signs, complete blood counts (CBC), serum cortisols, blood chemistries, or urinalyses between any dose group and the saline-infused controls. One volunteer developed lightheadedness, diffuse pruritis, nausea, and vomited once 10 h after completion of his 2 mg/kg IL-lra infusion. There were no abnormalities detected on physical exam. Alanine aminotransferase (ALT) was elevated to 122 U/l. White blood cell count, total bilirubin, and amylase were within normal limits. Symptoms resolved within 2 h. Twenty-one hours after the infusion had ended ALT was 147 U/l and aspartate aminotransferase (AST) was 116 U/l. Two days after the infusion, ALT and AST were 84 U/l and 30 U/l, respectively. Thirteen days

1992:

3.25 3.5

after beginning 4

4.5

3 h infusion)

5

6

7

8

9

10

11

12

24

12

x

x

x

x

x

x

x

x

x

x x x x

x x x

Clinical evaluation Symptoms

and vital

signs

X

X

x

x

X

x

x

X X X X

X

x

x

X

x

X X

x x

x x

X X

x x

x X x x

Bloodwork CBC Blood Serum

chemistries cortisol

Plasma cytokines Immunological tests FACS analysis Stimulus-induced T-cell proliferation

cytokines

X X X

x x

X X X

X X X

X X X

X

X

X

Pharmacokinetics Plasma Urine

x

x

x

x

xxxxxxxxxxxxxxx

IL-lra

tumor necrosis factor (Y (TNF+), and granulocyte macrophage-colony stimulating factor (GM-CSF) were below the limits of detection of the radioimmunoassays (RIA). There were no significant increases in plasma levels of these cytokines during or after the infusion of saline or IL-lra. Mononuclear

Cell Phenotypes

During the course of the study there were no significant differences in mononuclear cell phenotypes between any dose group and the saline-injected controls. Stimulus-induced

in humans

/ 355

endotoxemia23 and septic shock.24 IL-lra disappeared rapidly from the plasma with an initial half-life of 21 min and a terminal half-life of 108 min. Comparisons between the groups receiving IL-lra and saline showed no clinically significant differences in clinical, hematological, biochemical or endocrinological parameters. The only reaction occurred in one volunteer who developed a mild, transient case of hepatitis following a 2 mg/kg IL-lra infusion. Although the reason for this response is unclear, the overall data indicate that transient blockade of IL-lra is safe. The data also confirmed in-vitro reports9,isJ6 that IL-lra has no agonist activity. In contrast, humans

Cytokine Synthesis

When stimulated ex vivo with LPS, PBMC isolated from saline-injected recipients at the completion, 3 h and 21 h after the infusion synthesized more IL-6 than at baseline (P < 0.05 at all three time points) (Fig. 2). However, this increase in LPS-induced IL-6 was not observed in PBMC obtained from subjects following an infusion of IL-lra. Three hours after the infusion IL-6 synthesis was significantly greater in the saline-injected recipients than in the 1,2,3,5,7 or 10 mg/kg IL-lra dose group (P < 0.05 at all IL-lra doses) (Fig. 2B). S’rmi 1ar results were obtained from PBMC isolated immediately after the infusion (Fig. 2A) as well as 21 h later (Fig. 2C).

250 A

-50

2

T-Cell Proliferation There were no significant differences in T-cell proliferation between any dose group and the salineinjected controls. Antibodies

infusion

F 1 c al ," 2 0 ,\"

DISCUiSION IL-lra is a cytokine which inhibits the biological activity of IL-l by blocking IL-l from binding to its receptors.%13 Consequently, IL-lra is potentially therapeutic in IL-l-mediated diseases. However, before administering IL-lra to seriously ill patients, it is imperative to show that the molecule is safe. This report describes the first intravenous administration of human recombinant IL-lra to humans. Twenty-five healthy male volunteers received a single, 3 h continuous intravenous infusion of doses ranging between 1 mg/kg and 10 mgikg IL-lra. The group receiving the 10 mg/kg dose achieved a mean peak plasma IL-lra level which was 4500-fold greater than the peak levels seen in experimental

I

2

3

5

7

lo

Saline

I

2

3

5

7

IO

2

3

5

7

IO

4

150

50

-50

to IL-1 ra

When evaluated between 2 and 6 weeks after the infusion, no subject had detectable antibody to IL-lra.

Saline

250

C

-T 50

-50 Saline

I

IL-I ra (mg/kg)

Figure 2. LPS-induced saline or IL-lra.

IL-6

in PBMC

of volunteers

receiving

either

PBMC were isolated from volunteers before and then again immediately upon completion (A), 3 h (B) or 21 h (C) after an infusion of saline or IL-lra. Cells were stimulated ex vivo with LPS (10 @kg) for 24 h. RIA were performed to determine the total concentration of IL-6. For each subject, IL-6 is expressed as a percentage of that subject’s IL-6 concentration at time zero. *P < 0.05 when comparing PBMC from saline-injected subjects to PBMC from subjects who received IL-lra.

356 I Granowitz

et al.

receiving 10 rig/kg intravenous IL-l experienced fever, headache, anorexia, myalgias and arthralgias.677 If ILlra had only one millionth the activity of IL-l, our volunteers would have exhibited some of these clinical signs and symptoms. However, the groups receiving IL-lra had no more complaints than the saline-injected controls. It is unknown whether binding of IL-l to its receptors is required to maintain homeostasis. In particular, it has been suggested that IL-1 might have an important constitutive role in the pituitary-adrenal axis. In support of such a concept are immunohistochemistry studies of pituitary glands of healthy rats showing the presence of IL-l. zs.Furthermore, administration of subpyrogenic doses of recombinant IL-ll3 to mice and rats increased blood levels of adrenocorticotropic hormone.26 In-vitro studies found that pituitary cells exposed to IL-l released adrenocorticotropic hormone.27 Therefore, it was suspected that IL-1 might be required for basal cortisol synthesis. Our finding that plasma IL-lra as high as 29 l.rg/ml did not affect serum cortisol levels argues against a role for IL-l in cortisol production in health. In addition, administration of intravenous IL-l to humans produced leukocytosis and thrombocytosis.6,7 In rats, exogenous IL-l caused a decrease in plasma glucose28 and iron. 29 In the present study white blood cell and platelet counts, glucose and iron were unaffected by IL-lra administration. However, IL-lra was not devoid of immunomodulatory effects. When stimulated ex vivo with LPS, PBMC obtained after completion of the saline infusion synthesized more IL-6 than PBMC obtained prior to the infusion. This observation suggests that the stress of the saline infusion resulted in the synthesis of IL-l which upregulated IL-6 production in PBMC subsequently exposed to LPS. Such a theory is supported by the findings that peritoneal macrophages from mice exposed to cold water stress synthesize more IL-130 and that in rats, psychological stress results in elevated plasma levels of IL-6.31 In this study, the increase in LPS-induced IL-6 was not observed in PBMC from subjects receiving IL-lra. We postulate that in humans IL-lra blocked stress-induced IL-l from priming PBMC for subsequent LPS-induced IL-6 synthesis. This inhibition of LPS-induced IL-6 is not likely to be a consequence of carry over of IL-lra from the plasma into the PBMC cultures. Although the plasma concentration of IL-lra reached 29 pg/ml, we estimate that processing of the PBMC resulted in a final in-vitro IL-lra concentration of less than 5 rig/ml. Concentrations of IL-lra as high as 1 pg/ml inhibit in-vitro LPS-induced IL-6 by only 25% .a2 In addition, if plasma IL-lra carried over into the PBMC cultures was responsible for the inhibition of stimulus-induced IL-6, there would have been concomitant reduction

CYTOKINE, Vol. 4, No. 5 (September1992:353-360)

of LPS-induced IL-l, IL-8 and TNF-a in these same subjects. Such changes were not observed. This example of differential regulation of cytokine synthesis is not a new concept. For example, in-vitro indomethacin enhances IL-l@induced TNF 01,downregulates IL-l@ induced IL-la and does not affect IL-@-induced IL-6 (unpublished observations). Animal studies have shown that IL-lra is effective therapy for septic shock, 19~2 rheumatoid arthritis,asJ4 colitis35 and graft-versus-host disease.36 Since, as shown in these studies, IL-lra can be given safely to humans, it is reasonable to study this agent in IL-l-mediated human diseases.

MATERIALS AND METHODS Volunteer Selection Protocol and consent forms were approved by The Human Investigation Review Committee of New England Medical Center and Tufts University Health Sciences. Twenty-nine healthy male volunteers between the ages of 18 and 30 entered the study. Eligibility requirements included no evidence of underlying disease on history or physical examination, adequate hematopoiesis (hemoglobin 13.2-17.3 gidl, white blood cell count 3.s10.4 x lOs/mms, platelets 150-400 X lOs/mms), normal serum electrolytes (sodium 13.5-145 mEq/l, potassium 3.5-5.0 mEq/l, chloride 97-106 mEq/l, calcium 8.5-10.5 mg/dl, phosphate 2.54.5 mgidl), normal renal function (creatinine 0.61.5 mg/dl, blood urea nitrogen 8-25 mg/dl), glucose 50-120 mg/dl, uric acid 3.5-7.3 mg/dl, normal hepatic function (total protein 6.0-8.5 gidl, albumin 3.5-5.1 g/dl, total bilirubin 0.1-1.7 mgidl, alkaline phosphatase &200 U/l, ALT l&38 U/l, AST l&40 U/l), normal macroscopic urinalysis, normal electrocardiogram, normal chest radiograph, and no antibodies to the human immunodeficiency virus (Bio-EnzaBead HIV-l ELISA, Organon Teknika, Durham, NC). All subjects gave informed consent for both the study and human immunodeficiency virus testing. Volunteers abstained from taking oral cyclooxygenase inhibitors during the two weeks preceding the infusion.37

Study Design Volunteers were admitted to the Clinical Study Unit of the New England Medical Center. The following day subjects received an intravenous infusion of human recombinant IL-lra (obtained by expressing the human IL-lra cDNA in E. coW4). IL-lra at a concentration of 200 mg/ml in 10 mM citrate buffer, 150 mM sodium chloride and 0.5 mM ethylenediaminetetraacetic acid (EDTA) was diluted in 460 ml 0.9% sterile sodium chloride (Abbott Laboratories, N. Chicago, IL) and was infused over 3 h. Controls received 460 ml of 0.9% sodium chloride over 3 h. Doses of IL-lra were 1 mg IL-lra/kg of body weight (three subjects), 2 mg/kg (six subjects), 3 mg/kg (four subjects), 5 mg/kg (four subjects), 7 mg/kg (four subjects) or 10 mg/kg (four subjects). Four volunteers received saline.

IL-lra infusion in humans / 357

Pharmacokinetics Blood was collected in sterile, vacuum blood collection tubes containing EDTA (Becton Dickinson, Rutherford, NJ)beforeand0.5,0.75,1,1.5,2,3,3.25,3.5,4,4.5,5,6,7, 8,9,10, 11 and 12 h after the infusion was begun. Blood was immediately centrifuged at 450 X g for 15 min and plasma was stored at -70°C. Urine was collected from 0 to 3, 3 to 6, and 6 to 24 h. Urine volumes were recorded, creatinines were determined and 5 ml aliquots were frozen at -70°C. Subsequently, plasma and urine samples were assayedfor ILlra using an enzyme-linked immunosorbent assay (ELISA). A polyclonal rabbit antibody raised against recombinant human IL-lra and purified by affinity chromatography was used as the primary capture antibody. An affinity purified monoclonal antibody was biotinylated and added as the secondary antibody. Horseradish peroxidase conjugated to avidin was used to visualize the captured protein. To control for any factors in plasma which might interfere with the detection of IL-lra, the IL-lra standards were diluted in plasma. The limit of detection of the ELISA was 1 ngiml. Volume of distribution at steady-state and plasma clearance of IL-lra were estimated for each plasma IL-lra concentration versus time curve using non-compartmental analysis based on statistical moment theory.38 IL-lra plasma half-lives were estimated by curve-fitting (RSTRIP, MicroMath Scientific Software, Salt Lake City, UT) the post-infusion IL-lra plasma disappearance.

Clinical Evaluation Vital signs (temperature, pulse, blood pressure, respirations) were monitored every hour beginning before the infusion and continuing for 12 h. Blood samples for CBC (hemoglobin, white blood cell count with differential, platelets) and serum cortisol were drawn immediately prior to the infusion, hourly for the next 6 h, and again 12 h after beginning the infusion. Blood for chemistries (sodium, potassium, chloride, calcium, phosphate, creatinine, blood urea nitrogen, glucose, uric acid, total protein, albumin, total bilirubin, alkaline phosphatase, ALT, AST, chollesterol, triglycerides, serum amyloid A, insulin, growth hormone, epinephrine, iron, transferrin, ferritin, fibrinogen, factor VIII, erythrocyte sedimentation rate), and urine for macroscopic analysis were obtained prior to the infusion and 6 h later. Twenty-four hours after beginning the infusion vital signs, CBC, serum cortisol and blood chemistries were repeated At 72 h after the start of the infusion, history, physical examination, CBC, serum cortisol and blood chemistries were again performed. Between 2 and 6 weeks after the infusion, a CBC was repeated.

Plasma CytolzineAssays Blood was collected in sterile vacuum blood collection tubes containing EDTA immediately before and 1, 2, 3, 4, 5, 6, 12 and 24 h after beginning the infusion. Samples were stored on ice for 3 h or less. Plasma was separated using the methods of Cannon et al.39 and then frozen at -70°C. All samples were assayed for IL-lB,aa IL-2, IL-6,40 IL-g,41 TNF(~42or GM-CSF.43 These RIA were performed without the addition of normal rabbit serum. The limit of detection for

each RIA was 80 pg/ml IL-lB, 40 pgiml IL-6,20 pg/ml IL-8, 160 pg/ml TNF-a and 20 pg/ml GM-CSF. Previous studies have shown no cross-reactivity between these RIA. Plasma IL-2 levels were determined using an ELISA (Intertest-2X, Genzyme, Cambridge, MA). The limit of detection for IL-2 was 1 @ml.

Isolation of PBMC Immediately before and 3, 6, and 24 h after the start of the infusion, blood was drawn into syringes containing heparin (20 units/ml final concentration, LymphoMed Inc., Rosemont, IL). PBMC were isolated by centrifugation through Ficoll (Sigma Chemical Co., St. Louis, MO) and Hypaque (90%, Winthrop Laboratories, New York, NY). Preparations of Ficoll-Hypaque used sterile, non-pyrogenic water (Abbott). PBMC were washed twice in sterile 0.9% sodium chloride (Abbott) before being suspended.

Fluorescent-activatedCell Sorting Analysis (FACS) of PBMC PBMC were resuspended in FACS buffer consisting of phosphate-buffered saline containing 0.1% bovine serum albumin (Fraction V, Sigma) and 0.1% sodium azide (Sigma). Then 5 X 10s cells were aliquoted into each of 4 tubes. Following centrifugation, supernatants were discarded and the pellets were mixed with 10 ~1of anti-CD3, anti-CD14, anti-CD19 or anti-CD56 mouse anti-human monoclonal antibody (Becton Dickinson). Afer a 30 min incubation at 4°C the cells were washed, then combined with goat anti-mouse fluorescein isothiocyanate-conjugated antibody (Becton Dickinson) for another 30 min. Finally, PBMC were washed, fixed in FACS buffer with 0.1% formalin, and analyzed with a fluorescent-activated cell sorter.

Stimulus-induced Cytokine Synthesis Human recombinant IL-1B was generously provided by Dr Aldo Tagliabue (Sclavo Research Centre, Siena: Italy). Toxic shock syndrome toxin-l (TSST-1) was kindly donated by Dr Takashi Ikejima (Tufts University School of Medicine, Boston, MA). LPS (from E. coli 055:BS) was purchased from Sigma. RPM1 1640 (Sigma) containing 10 mM L-glutamine, 24 mM NaHCO, (Mallinckrodt, Paris, KY), 10 mM HEPES (Sigma), 100 U/ml penicillin, and 100 pg/ml streptomycin (Irvine Scientific, Santa Ana, CA), pH 7.4, was ultrafiltered using polysulfone hollow fiber filters (F40, Fresenius AG, Bad Homburg, Germany) to remove substances capable of inducing cytokine production.4 Human AB serum collected aseptically from one donor was heated for 45 min at 56°C. PBMC were resuspended at a concentration of 5 x 106 cells/ml in RPM1 supplemented with 2% v/v heat-inactivated AB serum. Cells (750 ~1) were aliquoted into 5 ml polypropylene tubes (Falcon, Becton Dickinson, Lincoln Park, NJ) and 750 pl of either RPM1 or stimulus in RPM1 (200 rig/ml IL-lB, 20 ngiml LPS, or 200 @ml TSST-1) was added. PBMC were then incubated for 24 h ’ at 37°C in a humidified atmosphere containing 5% CO,. After incubation, cultures were frozen at -70°C. PBMC were subjected to three freeze-thaw cycles to

358 I Granowitz

CYTOKINE,

et al.

yield maximal recovery of total cytokines.45 IL-l-stimulated samples were assayed in duplicate for IL-1016 and TNF-a.42 Unstimulated, LPS- and TSST-1-stimulated samples were assayed for IL-lp,47 IL-6,40 IL-S,41 or TNF-a by RIA. The limit of detection for each RIA was 40 pg/ml IL-la, 80 pg/ml IL-lp, 40 pg/ml IL-6,40 pg/ml IL-8 and 80 pg/ml TNF-a.

T-cell Proliferation Phytohemagglutinin P (PHA) was purchased from Difco Laboratories (Detroit, MI). Concanavalin A (Con A) was obtained from Sigma. One hundred microliters of PBMC at 5 x 106 cells/ml in RPM1 and 2% AB serum were aliquoted into 9 wells of a 96-well polystyrene flat bottom cell culture plate (Costar Corporation, Cambridge, MA) and 250 ~1 of either RPM1 or stimulus in RPM1 (11.2 pg/ml PHA or 4.2 pg/ml Con A) was added. All three conditions were performed in triplicate. After a 24 h incubation at 37°C in 5% CO,, 10 ~1 of 100 &i/ml [sH]thymidine (New England Nuclear, Boston, MA) was added to each well. Eighteen hours later the plates were frozen at -70°C. All cultures were harvested on the same day by aspiration in water with the cellular debris trapped in glass-fiber paper. The filter paper was cut and P-radioactivity was counted in 2.5 ml of scintillation cocktail (Ready Safe, Beckman, Fullerton, CA). The standard deviation of counts per min of triplicate wells was consistently less than 15%.

Antibodies

to IL-lra

Before the IL-lra infusion and again between 2 and 6 weeks after drug administration, blood was drawn into sterile, vacuum blood collection tubes and allowed to clot. The samples were centrifuged at 450 x g for 15 min. Serum was removed and stored at -70°C. Subsequently, samples were analyzed for antibodies to IL-lra. Briefly, a 96-well flat-bottom plate (EIA/RIA, Costar) was coated with 1 kg/ml human recombinant IL-lra. Serial dilutions of serum (from l/20 to l/160) were added to the wells. The plates were washed and alkaline phosphatase-conjugated anti-human

IgG was added to each well. Following another wash, paranitrophenyl phosphatate was also added. Optical density at 405490 nm was measured (V,,, Kinetic Microplate Reader, Molecular Devices, Menlo Park, CA). A comparison of titration

curves

from

the pre-dose

and post-dose

samples showed the curves were superimposable, indicating no increase in antibodies to IL-lra.

Statistics Plasma half-lives, volume of distribution and clearance are expressed as the mean f one standard deviation. All other results are shown as the mean + standard error of the mean. Data were analyzed

using analysis of variance

with

Dunnett’s multiple comparisons. Acknowledgements We gratefully acknowledge the advice and help of Seymour Reichlin, Patricia M. Noga, Marion J.

Vol. 4, No. 5 (September 1992: 353-360)

Hancox, Anne-Marie Conway, Timothy Cummings and the staff of the Clinical Study Unit. We are also grateful for the assistance of Kevin Ash, Laura Bartle, Isabelle Bedrosian, Anita L. Beshore, Joseph G. Cannon, Michael V. Callahan, Brenda Crawford, David J. Dripps, Jeffrey A. Gelfand, Fiona H. Hill, Kathleen Kimball, Karl-Christian Koch, John LaBrecque, Elizabeth A. Lynch, Julie W. Morris, Robert P. Numerof, Scott F. Orencole, Deborah D. Poutsiaka, Leland Shapiro, Jean D. Sipe, Christopher G. Smith, Gloria Vachino, Edouard Vannier and Ke Ye. This work was supported by grants from the National Institutes of Health (AI-15614 and AI-07329)

and in part by the Sandoz Research Institute (R.P.).

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CYTOKINE, JWM, Cannon JG, human interleukin by human blood Lymphokine Res

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47. Lisi PJ, Chu CW, Koch GA, Endres S, Lonnemann G, Dinarello CA (1987) Development and use of a radioimmunoassay for human interleukin Q3. Lymphokine Res 6:229-244.