ORIGINAL ARTICLES Pharmacokinetics and erythropoietic response to human recombinant erythropoietin in healthy men To assess the safety, pharmacokinetics, and erythropoietic responses to human recombinant erythropoietin (epoetin beta), single intravenous doses (10, 50, 150, and 500 IU / kg) were administered at monthly intervals to 16 healthy subjects in a two-panel, placebo-controlled, double-blind ascending-dose trial. A 1000 IU/ kg dose was subsequently administered in an open manner. Epoetin concentrations were determined in serum and urine by radioimmunoassay. Reticulocyte, hemoglobin, and hematocrit values were serially measured after each dose. Mean epoetin apparent half-lives ranged from 4.42 to 11.02 hours. The apparent volume of distribution was between 40 and 90 ml / kg, consistent with plasma water, and the apparent clearance values ranged from 4 to 15 ml/kg/hr, with both parameters having the highest values at the 10 IU/kg dose level. Clearance tended to decrease as a function of dose. Maximum reticulocyte counts were dose-dependent and occurred 3 to 4 days after the epoetin dose. Epoetin was well tolerated, and no antibodies were detected. (CLIN PHARMACOL THER 1990;47:557-64.)

Kristen K. Flaharty, PharmD, Jaime Caro, MD, Allan Erslev, MD, John J. Whalen, MD, Edward M. Morris, BSc, Thorir D. Bjornsson, MD, PhD, and Peter H. Vlasses, PharmD Philadelphia, Pa., and Princeton, NJ. Erythropoietin, a 36 kd glycoprotein, is the primary regulator of red blood cell production.' The main action of erythropoietin is to stimulate the differentiation of progenitor cells in the bone marrow toward functional erythroblasts. The subsequent maturation requires 5 to 9 days under normal physiologic conditions. Erythropoietin is produced in the kidney (90%) and the liver (10%).3 The specific site of production in the kidney is thought to be endothelial cells in close proximity to the proximal tubules.4'5 Endogenous erythropoietin production is regulated by renal oxygen sensing

From the Jefferson Medical College, Department of Medicine, Division of Clinical Pharmacology, and Cardeza Foundation, Philadelphia, and G.H. Besselaar Associates, Princeton. Supported by Chugai Pharmaceuticals, Tokyo, Japan, and grants HL04612, DK34642, A1-13231, and HL40791 from the National Institutes of Health, Bethesda, Md. Received for publication Oct. 11, 1989; accepted Jan. 15, 1990. Reprint requests: Kristen K. Flaharty, PharmD, Jefferson Medical College, Division of Clinical Pharmacology, 1100 Walnut St. (601 MOB), Philadelphia, PA 19107-5563.

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mechanisms. The anemia associated with chronic renal failure has been attributed primarily to an erythropoietin deficiency, inasmuch as patients with kidney disease are not able to produce adequate amounts. 6 Recent advances in biotechnology have allowed for the cloning and mass production of human recombinant erythropoietin (epoetin).7 Data from clinical trials with epoetin have demonstrated that the anemia associated with end stage renal disease can be corrected in most patients with epoetin therapy." Epoetin is currently under investigation for the treatment of anemia associated with rheumatoid arthritis, chemotherapy, and acquired immunodeficiency syndrome.'" In addition, it has been hypothesized to be of value in increasing the harvest in autologous transfusion programs.' The purpose of this study was to assess the in vivo disposition and erythropoietic response of increasing single doses of intravenous epoetin in normal volunteers. This is the first report of epoetin pharmacokinetics and pharmacodynamics in healthy men and will allow for further assessment of epoetin therapy in autologous transfusion programs.

557

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Flaharty et al. Group A

(n=8)

Placebo

(2)

Epoetin

(6)

10

Group C (n=6) Placebo (2) Epoetin (6) 150 1U/Kg

IU/Kg

Epoetin (6) 1000 1U/Kg .1,

Group B (n=8) Placebo (2) Epoetin (6) 50 1U/Kg

(2) (6) 1U/Kg

Placebo

Epoetin 500

Calendar Day:

29

15

1

43

81

67

Fig. 1. Two-panel double-blind study design (groups A and B). Group C was added in an open manner after preliminary results.

100000

10000

1000

100

10 0

10

20

30

40

TIME (Hr) Fig. 2. Average serum erythropoietin concentrationtime curves at 10 IU/kg (0), 50 IU/kg 150 IU/kg (E), 500 IU/kg (), and 1000 IU/kg (0) (n = 6 per dose).

MATERIAL AND METHODS Subjects. Sixteen healthy men ranging in age from 20 to 32 years and weighing 56 to 89 kg were enrolled in the study. Each subject was deemed to be healthy on the basis of normal history, physical examination, electrocardiogram, and laboratory tests (blood chemistry, complete blood count, iron studies, urinalysis, hepatitis B surface antigen, and human immunodeficiency virus antibody). Subjects were instructed not to consume alcohol or other medications for at least 7 days before the study and throughout the study period. Study design. This was a two-panel, double-blind randomized study to evaluate the phannacokinetics,

(),

pharmacodynamics, and renal excretion of single increasing doses of epoetin beta (Marogenrm, ChugaiUpjohn, Rosemont, Ill.). Study treatments are depicted in Fig. 1. All treatments were intravenous injections that were delivered over a 1-minute period. Group A consisted of eight subjects, six of whom received 10 IU/kg of epoetin first and two of whom received placebo first. Fifteen days later, subjects in Group B, another group of eight subjects, first received 50 IU/kg of epoetin (n = 6) or placebo (n = 2). Subjects in group A returned 15 days later and received 150 IU/kg of epoetin (n = 6) or placebo (n = 2). The placebo subjects remained the same. Subjects in group

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Fig. 3. Individual subject pharmacokinetic parameters for half-life (t2), clearance (CL), and volume of distribution (Vd). Panel A, Subjects (n = 4) received 10, 150, and 1000 IU /kg. Panel B, Subjects (n = 6) received 50 and 500 IU/kg. (Note only four subjects from group A received all three epoetin doses of 10, 150, 1000 IU/kg.) B returned 15 days later and received 500 IU /kg (n = 6) or placebo (n = 2); again, placebo subjects remained the same. Finally, 15 days later, six subjects from group A returned to receive 1000 IU / kg of epoetin in an open-label extension. Two of these subjects had

previously received placebo, whereas four of the subjects received all three doses of epoetin, that is, 10, 150, and 1000 IU/kg. These subjects were identified as group C. Subjects fasted from the night before each dose of epoetin or placebo and remained fasting for 3 hours after each dose. Otherwise, subjects maintained a normal diet throughout the study period. Samples of whole blood were collected just before and at ¼, ½, 1, 2, 4, 8, 12, 16, 24, and 48 hours after the intravenous administration of epoetin or placebo.

All blood sampling was timed from the start of the bolus administration. Blood samples were centrifuged and the sera transferred to plastic vials and frozen. Urine was collected before epoetin administration and quantitatively over the time intervals of 0 to 4, 4 to 8, 8 to 12, 12 to 24, and 24 to 48 hours after the intravenous dose. The urine volume was recorded, and an aliquot was transferred to a plastic vial and frozen.

Analytic methods. Determination of erythropoietin in plasma and urine was performed by radioimmunoassay. Labeled recombinant human erythropoietin (Genetics Institute, Boston, Mass.) was used as a tracer, as previously described.' The antibody was developed in rabbits against human urinary erythropoietin. This antibody has been tested against n-terminal and c-

CLIN PHARMACOL THER MAY 1990

560 Flaharty et al.

600

-5

C

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400

0 300

200

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10

20

30

40

50

60

Fraction Tube # Fig. 4. Sephadex column extraction of patient sera (closed squares) versus control epoetin (open squares). Control substances are not shown on this figure.

Table I. Mean apparent pharmacokinetic parameters Dose (1U/kg) 10

50 150 500 1000

C

(mUlml)

181 ± 35 1430 ± 162 4438 ± 440 16,257 ± 4,883 32,567 ± 13,226

C

D

(ml-')

18.1 ± 3.5*

28.6 ±_ 3.2t 29.6 ± 2.9t 32.5 ± 9.8t 32.6 ± 9.5t

AUC (mU hr

ml-')

731 ± 223 at 789

9,306 30,990 142,480 252,759

± 5,581 ± 35,683 ± 52,200

CL (ml/hr/kg)

± 3.94* ± 0.44t 4.98 ± 0.81t 3.70 ± 0.87f 4.08 J..- 0.94t

14.68 5.38

Data are mean values :L.- SD. C.., Maximum concentration; Cm,D, dose-adjusted C..; AUC, area under the plasma concentrationtime curve; CL, total clearance; Vss, steady-state volume of distribution; ty,, half-life; MRT, mean residence time; CLR, renal clearance; Ae(0-48 hr), urinary excretion of epoetin from 0 to 48 hours. *Values are not statistically different from each other at p < 0.05. tValues are not statistically different from each other at p < 0.05. Values are not statistically different from each other at p < 0.05.

terminal erythropoietin and found to be reactive, whereas other plasma glycoproteins are nonreactive. Anti-erythropoietin antibodies were determined before and 2 weeks after each epoetin or placebo exposure by a double-antibody radioactive binding assay as modified from Skolm et al.' Briefly, a 1:50 dilution of the patient serum was incubated with radiolabeled epoetin (approximately 10,000 to 15,000 cpm) for 24 hours. This was followed by precipitation of the erythropoietin-antibody complex with a goat antihuman immunoglobulin antibody. Results are expressed as percentage precipitation of the labeled material. Sam-

ples were considered positive for antibody presence if the percentage binding was more than three times the background radioactivity. Phannacokinetic calculations. Individual baseline serum concentrations of endogenous erythropoietin were subtracted from subsequent serum concentration values after the 10, 50, and 150 IU/kg doses of epoetin. Pharmacokinetic assessments with or without baseline values for the 500 and 1000 IU/ kg doses, however, were essentially identical because of the small contribution of endogenous concentrations obtained at these doses. All pharmacokinetic parameters are described as

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8 0

0>0

7 6

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4 3 cc

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50 150

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5 00

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DOSE (IU/KG) Fig. 5. Mean reticulocyte response by dose (n = 6 per dose). X, p *, p < 0.05 from placebo.

tk,(hr)

Vss (m11 kg)

89.05 39.50 49.20 46.47 62.72

± ± ± ± ±

23.57*

3.87t 8.12t 13.38t 22.77t

± ± ± ± 11.02 ± 4.42 5.34 6.10 8.49

1.18*

0.52* 0.88*

0.83t 0.031:

MRT (hr)

6.27 7.35 10.00 12.70 15.00

± ± ± ± ±

apparent values because the radioimmunoassay cannot distinguish between endogenous erythropoietin and exogenous epoetin. The area under the plasma concentration versus time curve (AUC) from zero to time infinity was calculated by Lagrange polynomial interpolation.' Apparent terminal half-life (42) was calculated by linear regression of the terminal phase of the semilogarithmic plasma concentrationtime curves. Total clearance, (CL) was calculated by division of the dose by the total AUC. The volume of distribution at steady state, (Vss) was calculated by use of model-independent analysis as follows: (Dose

AUMC)/(AUC)2

in which AUMC is the area under the first moment

0.23 0.06 0.06 0.01 1.49