Br. J. clin. Pharmac. (1990), 29, 709-713

The pharmacokinetics of recombinant human erythropoietin after intravenous and subcutaneous administration to healthy subjects T. SALMONSON', B. G. DANIELSON2 & B. WIKSTROM2 'National Board of Health and Welfare, Department of Drugs, Uppsala, and 2Department of Medicine, University Hospital, Uppsala, Sweden

1 The pharmacokinetics of recombinant erythropoietin, r-Epo, were evaluated after intravenous and subcutaneous administration of 50 u kg-' to six healthy male volunteers. 2 The calculated mean values (± s.d.) for volume of distribution at steady state and clearance after an i.v. dose were 76 (±33) ml kg-l and 12 (±3) ml h'- kg-l, respectively. 3 Serum concentrations of r-Epo peaked at 13 (±6) h after the s.c. dose and the bioavailability over 72 h was 36 (±23)%. The mean residence time and half-life of erythropoietin were 6.2 (± 1.0) and 4.5 (±0.9) h respectively after i.v. and 46 (± 18) and 25 (± 12) h after s.c. administration. 4 The results demonstrate the possibility of changing the profile of the concentrationtime curve by changing the mode of administration of r-Epo, with implications for the time-course of clinical response.

Keywords erythropoietin pharmacokinetics intravenous subcutaneous Introduction The development of recombinant DNA techniques has recently allowed the production of recombinant erythropoietin (r-Epo) in quantities sufficient for clinical trials. Several investigators have reported that intravenous administration of r-Epo successfully corrects anaemia in patients with chronic renal failure (CRF) (Eschbach et al., 1987; Sundal & Kaeser, 1989; Winearls et al., 1986). In these trials r-Epo has been given i.v. 1-3 times/week and the doses (15-500 u kg-') have been titrated in each patient to the desired response. The levels of endogenous erythropoietin in anaemic patients with CRF have been reported to be lower than in patients with anaemia due to other causes (Erslev et al., 1987; Naets et al., 1986; Rege et al., 1982). The reason for this is probably an inability to increase erythropoietin production, which mainly takes place in the kidney, in response to the anaemia. Pharmacokinetic studies of r-Epo in patients with CRF have shown that the half-life of r-Epo after i.v. administration is relatively short

(5-6 h), with respect to the dosing interval of 2-7 days (Salmonson et al., 1990). Stevens et al. (1989), Sinai-Trieman et al. (1989) and Salmonson etal. (1990) have presented data demonstrating that subcutaneous administration is effective in the treatment of anaemia in patients on peritoneal and haemodialysis (CAPD). The s.c. maintenance doses used in these studies were similar to the reported i.v. maintenance doses (Esbach et al., 1987; Sundal & Kaeser, 1989). In order to investigate if it is possible to change the concentration-time profile of r-Epo significantly by changing the route of administration, a study comparing intravenous and subcutaneous administration has been conducted. Methods

Subjects Six healthy male volunteers, aged 34 ± 12 years (mean ± s.d.) and weighing 79 ± 12 kg were

709

710

T. Salmonson, B. G. Danielson & B. Wikstrom

included in the study. Each subject gave their informed consent to participate prior to the study. Because there was no reported blood loss or anaemia prior to the study the endogenous Epo level was considered to be at steady state. The endo enous Epo concentration was 12 + 5 mu ml- prior to i.v. administration and 10 + 4 mu ml-' prior to s.c. administration. The clinical trial of r-Epo was approved by the Ethics Committee of the Medical Faculty, University of Uppsala and by the National Board of Health and Welfare, Department of Drugs. Drug administration Recombinant human erythropoietin (AMGEN, Thousand Oaks, California) was supplied by Cilag AG (Schaffhausen, Switzerland) as a sterile buffered solution with a specific activity of 4000 u ml-'. Both the intravenous (i.v.) and subcutaneous (s.c.) dose was 50 u kg-. The subcutaneous dose was injected in the upper arm.

Study design

Assay A radioimmunoassay was used to measure Epo concentrations. This method was previously validated for the measurement of both endogenous and recombinant erythropoietin (Egrie et al., 1987). The samples were analyzed in duplicate and the limit of assay was approximately 2 mu ml - 1. The interassay coefficient of variation at an rEpo concentration of 6 mu ml1 was 6%. serum

Pharmacokinetic analysis The baseline value was subtracted from the measured concentration and a poly-exponential equation was fitted to the concentration-time data by the non-linear regression program Siphar (Gomeni, 1984) using weighted least squares (weight = 1/y) analysis. The chosen model was used to characterize the absorption and to determine the terminal elimination halflife. The pharmacokinetic parameters were calculated using statistical moment theory. The calculation of the endogenous erythropoietin production was based on two assumptions: (a) the calculated clearance value was valid for endogenous Epo and (b) the measured baseline value was the steady state concentration of Epo.

Three blood samples were obtained from a forearm vein before each dose to determine the basal level of endogenous Epo. The volunteers first received r-Epo i.v. followed after a wash-out period of at least 1 week by the s.c. dose.

Results

Samples

l. v.

Venous blood samples were collected in plastic vials at 0.17, 0.33, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0, 8.0, 12.0, 24.0, 48.0 (s.c. only) and 72.0 (s.c. only) h after the dose. After sampling the serum was separated and kept frozen at -70° C, pend-

The serum concentration-time profiles after an i.v. dose of r-Epo followed a mono- (n = 5) or biexponential (n = 1) decline (Figure 1). The area under the serum concentration-time curve, AUC, and the calculated pharmacokinetic parameters (± s.d.) are presented in Table 1.

ing analysis.

Table 1 Pharmacokinetic parameters of r-Epo after i.v. administration of 50 u kg-' to six healthy volunteers

Epo produced t,/2z (u day-' kg) (h)

AUC (mu ml-' h)

CL (ml kg-' h)

(ml kg-')

Vss

MRT

(ml kg-)

1 2 3 4 5 6

4955 5401 2791 4190 4443 4657

10.1 9.3 17.9 12.0 11.3 10.7

60 48 145 73 64 89

55.6 53.8 141.5 70.0 60.5 74.7

5.5 5.8 7.9 5.8 5.4 7.0

4.1 2.3 2.8 2.2 2.4 3.7

4 4 6 4 4 6

Mean s.d.

4401 897

11.9 3.1

80 35

76.0 33.1

6.2 1.0

2.9 0.8

5 0

Vz

(h)

Pharmacokinetics of recombinant erythropoietin

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Discussion

1000.00, c

'r-

100.00

0

c

E

10.00

0-

11

nn,X 0.0

6.0

12.0

24.0

18.0

Time (h) Figure 1 Observed (x) and predicted (-) serum concentrations of r-Epo after an i.v. dose of 50 u kg-1 r-Epo (subject 1). 1000.001 c

0_

c

o-_E

0

100.00k 10.00

0

I.Unnor

6. 72.0 96.0 48.0 Time (h) Figure 2 Observed (x) and predicted (-) concentration of r-Epo after an s.c. dose of 50 u kg-' r-Epo (subject 1).

0.0

24.0

When uncorrected for weight the clearance was 15.6 ± 3.8 ml min-' and the volume of distribution at steady state was 5.9 ± 2.4 1. S.c.

The absorption of r-Epo in volunteers 2, 3 and 4 best described by a zero-order process ending at tmax. The data for the other subjects were best fitted by a first-order absorption function (Figure 2). The calculated lag time was 0.4 ± 0.5 h. The elimination after a s.c. dose was best described by a mono-exponential decline. The AUC and calculated pharmacokinetic parameters are presented in Table 2. was

The aim of this study was to estimate the pharmacokinetic parameters of r-Epo after an i.v. and a s.c. dose to healthy volunteers and to compare the results with those reported for patients with chronic renal failure (Salmonson et al., 1990). Erythropoietin is distributed in an apparent volume which is close to extracellular plasma volume. This is an expected finding considering the molecular size of the hormone and comparable volumes of distribution for similar proteins. It is also consistent with the detection of a distribution phase in only one of the six concentration-time profiles. Absorption after a subcutaneous r-Epo dose was slow, resulting in a maximum concentration 10-15 h after the dose. The bioavailability estimated from data collected over 72 h averaged 36%. The calculated pharmacokinetic parameters from this study can be compared with the results from a previous i.v. study in patients with chronic renal failure (Salmonson et al., 1990). The estimated values for clearance, volume of distribution, half-life and endogenous production in that study were (mean ± s.d.) 64 17 ml kg-,, 10 ± 6 ml kg-' h, 5 ± 1 h and 3.2 1.7 u day-' kg-'. The slightly, but not statistically significant, higher value for clearance in the healthy volunteers could be due to more efficient urinary excretion. Epo is excreted in the urine but the relative extent of this excretion in relation to the total elimination is unknown. The calculated rate of endogenous Epoproduction was about 3 u day-1 kg-1 in both groups. Production in the patients was inappropriately low in relation to the degree of anaemia. The measurement of endogenous Epo in anaemic non-uraemic patients (Erslev et al., 1987k) showed concentrations of up to 10000 mu ml- depending on the severity of the anaemia.

Table 2 Pharmacokinetic parameters of r-Epo after s.c. administration of 50 u kg-1 to six healthy volunteers

AUC (mu ml-' h)

tmax (h)

Cma

t*a

MRT

(%)

(mu ml-)

(h)

(h)

1 2 3 4 5 6

1599 1392 2232 986 794 1834

32 26 80 24 18 39

12 12 6 24 12 12

40 27 92 14 16 25

7 11 2 12 4 3

30 39 25 63 43 74

14 17 16 33 26 46

Mean s.d.

1473 534

36 23

13 6

36 29

6 4

46 18

25 12

F

t,/2z (h)

T. Salmonson, B. G. Danielson & B. Wikstrom

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1 000.O

°_750.0 E 500. 0

2500 0.0 48.0 96.0 144.0 192.0 240.0 288.0

Time (h) Figure 3 Simulated serum concentrations of r-Epo during repeated i.v. dosing of 50 u kg-1 r-Epo every

48h. 50.0 a

40.0

o

3 0.0. 200 100 00 0.0

0.0 48.0

96.0 144.0 192.0 240.0 288.0

Time (h) Figure 4 Simulated serum concentrations of r-Epo during repeated s.c. dosing of 50 u kg-1 r-Epo every

48h.

Uraemic patients cannot increase their levels of Epo adequately in response to decreased oxygen supply. The increased demand for erythropoietin resulting from a lowered oxygen supply is probably due to the decreased survival time of the red blood cells, inhibition of erythropoesis by

uraemic toxins, increased blood loss in the gastrointestinal tract and during haemodialysis treatment and impaired iron absorption in the gut. Using the calculated parameters from this study, Figures 3 and 4 show simulations of serum r-Epo cases during repeated i.v. and s.c. dosing. The chosen dosing interval was 48 h and the dose was 50 u kg-'. The peak serum concentrations of r-Epo reached after s.c. administration are about 5% of the values obtained after i.v. The lower bioavailability results in s.c. steady state concentrations after s.c. injection which are about 30% of those after i.v. administration. However, owing to the slow rate-limiting absorption after s.c. injection administration by this route, in contrast to i.v. injection, results in a detectable difference (15 mu ml-1) between the minimum concentration during a dosing interval and the baseline value of endogenous Epo. Our findings demonstrate that r-Epo is absorbed after s.c. administration and that the serum concentration-time profile is markedly different from that following an i.v. dose. Subcutaneous administration might be advantageous if smaller fluctuations and a sustained increase over baseline levels are desirable. Clinical trials in progress should indicate how changes in the profile of the serum concentration-time curve of r-Epo influences the pharmacodynamic response. The authors gratefully acknowledge the secretarial assistance of Ms Annika Lundin.

References Egrie, J. C., Cotes, P. M., Lane, J., Gaines Das, R. E. & Tam, R. C. (1987). Development of radioimmuno assays for human erythropoietin using recombinant erythropoietin as tracer and immunogen. J. immunol. Methods, 99, 235-241. Erslev, A. J., Wilson, J. & Caro, J. (1987). Erythropoietin titers in anemic, nonuremic patients. J. lab. clin. Med., 109, 429-433. Eschbach, J. W., Egrie, J. C., Downing, M. R., Browne, J. K. & Adamson, J. W. (1987). Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. New Engl. J. Med., 316, 73-78. Gomeni, R. (1984). Pharm-an interactive graphic program for individual and population pharmacokinetic parameter estimation. Comput. Biol. Med., 14, 25-34. Naets, J. P., Garcia, J. F., Tousaaint, Ch., Buset, M. & Waks, D. (1986). Radioimmunoassay of erythropoietin in chronic uraemia of anephric patients. Scand. J. Haematol., 37, 390-394.

Rege, A. B., Brookins, J. & Fisher, J. W. (1982). A radioimmunoassay for erythropoietin: serum levels in normal human subjects and patients with hemopoietic disorders. J. lab. clin. Med., 100, 829843. Salmonson, T., Danielson, B. G., Grahnen, A. & Wikstrom, B. (1990). Pharmacokinetics of intravenous recombinant human erythropoietin in patients with chronic renal failure. J. intern. Med., (in press). Sinai-Trieman, L., Salusky, I. B. & Fine, R. N. (1989). Use of subcutaneous recombinant human erythropoietin in children undergoing continuous cycling peritoneal dialysis. J. Pediatrics, 114, 550-554. Stevens, J. M., Strong, C. A., Oliver, D. O., Winearls, C. G. & Cotes, P. M. (1989). Subcutaneous erythropoietin and peritoneal dialysis. Lancet, i, 1388-1389. Sundal, E. & Kaeser, U. (1989). Correction of anaemia of chronic renal failure with recombinant human erythropoietin: results from a multicentre study in

Pharmacokinetics of recombinant erythropoietin 150 haemodialysis-dependent patients. Nephrol. Dial. Transpl., 4, 979-987. Winearls, C. G., Oliver, D. O., Pippard, M. J., Reid, C., Downing, M. R. & Cotes, P. M. (1986). Effect of human erythropoietin derived from recombinant

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DNA on the anaemia of patients maintained by chronic haemodialysis. Lancet, ii, 1175-1178.

(Received 19 June 1989, accepted 22 January 1990)

The pharmacokinetics of recombinant human erythropoietin after intravenous and subcutaneous administration to healthy subjects.

1. The pharmacokinetics of recombinant erythropoietin, r-Epo, were evaluated after intravenous and subcutaneous administration of 50 u kg-1 to six hea...
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