Biomed& Pharmocother(1992)46,37-43 0 Elsevier, Paris
37
riginal article Serum erythropoietin levels in hemodialysed patients after administration of recombinant human erythro V PavloviC-Kentera’,
GK Clemons2, L Bil’anoviC-PaunoviC’, V LeiaiC4, M Pokrajac 4, Lj Djukanovid4
D MarisavljeviC3,
‘Instituie for Medical Research, PO Box 721, JJO01 Belgrade; 2JLawrenceBerkeley Laboratory, Berkeley, CA. USA; 31nstituteof Hematology and 41nstitute of Nephrology. University Clinical Center, &igrade; ‘Department of Pharmacokinetics, Faculty of Pharmacy, Belgrade. Yugoslavia (Received 23 August 1991; accepted 16 December 1991)
Summary - In anemic patients on regular hemodiaiysis (HD), correction of anemia with recombinant human erythropoietin @HuEpo) administe~d intravenously (iv} or subcutaneously (SC) was followed over a 2-month period. Monitoring serum Epo post-dose concentrations after the first iv rHuEpo injection and following another regular injection after 2 months of therapy with rHuEpo iv in 9 patients showed that the Epo elimination half-life was rednced from 7.48 h to 4.68 h. In the same patients the initially low percentage of e~~r~las~ and mature erythroid progenitors increased during 2 months of rHuEpo therapy. Because Bpo molecules bound to Epo receptors are intemakzd in target cells we suggest that the expansicn of the Bpo responsive cell pool could explain the shorted Epo elimination time afier 2 months of rHtQo treatment. By rno~~to~ng serum Epo concentration following SCrHuEpo injection in 7 HD patients it was found that the modest increase in serum Epo levels (30-40 mU/ml~ was sufficient to correct anemia. serum erythropoietin tevel I chronic renal failure I rHuEpo therapy Msumi: - Taux d’~rythropo~~tine sbrique chez les maladef h~m~~alys~s aprb administration d’~r~h~~ffine ktomaine r~om~inant~ La correction dune ankmie a et& men&epar ~~thropo~~tine humaine reco~~~nte {rHuEpo] ituraveineuse ou sous-cutant!e et suivie sur une pkiode de deux tnois chez des patfents en h~rn~~~~se. Le monitoring des concenrrfftion~ d’Epo skique aprts une premi&e injection IV de rHuEpo et apr& une autre dew: mois plus tard cbez ne& patients a montrt! un rac~our~Jssement de la demi-vie ~~lirni~tion de f’Epo de ?h4B ci 4h6B. Chet ces mgmes malades. les pou~enta~es ~~~throblastes et de prog~niteurs ~~thro~des matures, initialement bas, ant augment&au eours des deux mois de traitement par Ia rHuEJw. En raison du fait que les mokdes d’Epo fm&es sur les r&cepteurs correspondants se trouvent ci l’ext&ieur des ~eJ~ules-~ibJes,ies auteurs dmettent I’hypothcise que l’expansion du pool de celiuJes r&ondant ir I’Epo pourrait expliquer la rkductioa du temps ~~~irni~tion de I’Epo apr&s deux mois de traitement par I’rHuEpo. En surveillant fes concentrations d’Epo s&ique apr& injection sous-cutantfe de rHuEpo chez sept ht?madialysPs, an aurait &aiement trot& que la l&&e augmentation due aux d’Epo skique (30 d 60 mUlm1) &ait st@sante pow corriger PanZmie. taux d*&ythropoMine sbique / ddfaillance r&tale rhronique I traitement par rHuEpo
Introduction The efficacy of recombinant human erythropoietin (rHuEpo) therapy for anemia in patients on regular hemodialysis (HD) was demonstrated in clinical trials [21]. The correction of anemia in HD patients by rHuEpo indicates that studies of the effects of Epo in those patients in vivo could
provide
valuable
information
concerning
the bio-
logy of this hormone. During a 2-month period, correction of anemia in HD patients treated with rHuEpo intravenously (iv) or subcutaneously (SC) was followed. Serum Epo levels were monitored after the first injection at the beginning of therapy and following another regular injection after 2 months of therapy, in order to obtain more infor-
38 mation regarding the dependence of serum Epo levels on the size of the Epo responsive cell pool and the serum Epo levels anemia in ND patients.
required
to counteract
Patients and methods Patiettts Two groups of HD patients with anemia were investigated. The study was approved by the Ethics Committee of the University Clinical Center. Written informed consent was obtained From each patient. Nine patients were treated with rHuEpo iv and 7 SC. Patients were dialysed 3 times a week for 4 h nsing cuprophan membrane dialyzers. rHuEpo was administered at the end of dialysis. The starting dose was 50 U/kg body weight (bw) for iv and 30 U/kg bw for SC, after each dialysis, ie 3 times a week. The dose was increased by 50 percent of the starting dose after every 4 weeks whenever target hemoglobin level was not achieved. Iron supplementation was given to patients with normal or low serum iron concentration or ferritin levels < lOO/pg/l. A complete blood count was performed at the outset of the study and then once a week for 8 weeks. Methods Bone marrow specimens were obtained during the morning on the day preceding rHuEpo injection. Samples of 1-2 ml of bone marrow were aspirated from the iliac crest. Samples for bone marrow cuitures were collected in preservative-free sterile heparin (50 U/ml). Bone marrow smears were made on glass slides and analyzed after May-Griinwald-Giemsa and Prussian blue staining. Four hundred elements were differentiated on coded glass slides. To obtain the erythroblast maturation curve, the percemage of different erythroblast maturation stages was determined by differentiation of up to 200 erythroblasts. Iron content in the bone marrow was evaluated by the percentage of sideroblasts as well as the presence of extracellular iron. For the CFU-E assay, mononuclear cells from bone marrow samples were separated by Lymphoprep gradient centrifugation before plating. The methylcellulose culture technique described by Iscove et al [5] was used. Cells were cultured in duplicate in 35-mm tissue culture dishes at lO’/ml in Dulbecco’s medium containing 30% fetal calf serum, 0.8% methylcellulose, 0.1% bovine serum albumin, fi-mercaptoethanol 2 x 10m5 M, and 1.O U of rHuEpo (kindly donated by W Jelkman, Liibeck, Germany). Erythroid colonies were identified after 7 days incubation at 37°C in a humidified 5% CO2 atmosphere. Epo levels were determined at the Lawrence Berkeley Laboratory by radioimmunoassay [4]. Serum
samples for basal value of Epo were obtained just prior to the first rHuEpo injection and then 30 min, 2 and 6 h after iv and 2, S, 14, 24 and 36 h after SC injection. The same schedule in obtaining serum samples for Epo determinations after one iv or SC injection was used again after 2 months of rHuEpo therapy. Pharmacokinetic
calculations
The elimination rate constant (&I) for rHuEpo after one bolus iv injection was estimated from the slope of the curve after 2 h, as the distribution phase was terminated in all patients at that time. The kmenc parameter eiimination half-life (tin) was estimated using the standard pharmacokinetic equation, assuming first order elimination. Both mathematical and graphic methods of calculation gave similar results.
Results The c!inical characteristics of the patients studied are presented in table I. These patients were anemic prior to rHuEpo therapy with hematocrit values below 20%. As can be seen in table II, during 2 months of treatment with rHuEpo, anemia improved with both iv and SC routes of administration, although the target hemoglobin value of 10.0 g/d1 was not reached in that time. The values of 2 patients whose anemia was not corrected during 2 months of rHuEpo treatment were not included in table II. Their hematocrit values were respectively 19 and 26 before and 20 and 21 after 2 months’ treatment. The first patient was bleeding from a papilloma in the stomach and the second had to undergo surgery. The bone marrow differential count revealed low percentages of erythroblasts before Epo. Values increased after 2 months in the patients who received therapy. The rate of increase differed from patient to patient, while bone marrow cellularity did not change significantly (data not shown). It is particularly important that the increase in the percentage of erythroblasts was
Table
I. Clinical characteristics of the patients studied.
Route of Epo administration iv SC
No of patients M
F
4 2
5 5
Age (yr)
BW (kg)
Duration of hemodialysis (months)
37-64 36-64
40-67 40-75
6-216 13-107
39 Table II. Measures of red cell concentration in hemodialysis patients treated with rHuEpo intravenously (iv) or subcutaneotts~y (xc).
iv rHuEpo therapy RBC x IO’=/1
Hb gldl
1.86 + 0.21
5.64 f 0.58
Before therapy After 2 months of therapy
SCrHuEpa therapy PCV l/l
RBC x Io’211
Hb gldl
PCV III
2.06 zk 0.46
6.23 + 1.16
19.0 f 3.65
17.29 z!z1.80
2.70*** f 0.45 9.06*** f 1.44 26.29, + 4.89
2.89* f 0.64 9.19** f 2.12 26.57. + 6.11
The values are mean + SD for II = 7 in each group. Significance cf the differences when compared to the values before therapy: *P < 0.05, **P c 0.01;
***P
c 0.001. RBC: red blood cells; Hb: hemoglobin; PCV: packed cell volume.
Table III. Bone rnarrow smear analyses prior to (A) and after 2 months (B) of rHuEpo therapy. Patient
225 A B 226 A B 223 A B 252 A B 227 A B 231 A B
CFU-El16
79 172 58 226 82 238 66 201 136 656 205 229
Erbf W)
3 40 11 26 2 10 16 21 31 36 4.5 8
Erythroblast&’ Pro @)
Baso (%)
0
0
2 0 0 0 0
10 1 13 0 9
1 1.5 3 0 0
6 6 7 1 10
Poly m)
Ortho (a)
35 52 37 58 30 49
65 35 62 29 70 42
26
67 51.5 60 55 38
41 40 45 52
Sideroblasts iw 80 46 64 80 0 30 40 58 80 20 70 34
Extrace~hdaf iron
4+
2+ 4+ 4+ 1-k l+ 2+ 3+ 5+
l+ 3+ 2+
a Percent of erythroblasts in bone marrow differential; b percentageof erythroblast maturation stagesobtained by differentiating 100 to 2M) erythroblasls:pro-proerythroblasts;baso4asophilic; poly-polychromatophilic;ortho-orthochromatic;’ extracellularim: normal value +2 to +3.
due to an increase in basophilic and polychromatophilic erythroblasu (table III) while orthochromatic erythroblasts decreased to near normal values. In the same patients the percentage of sideroblasts in the bone marrow decreased after 2 months of therapy. In all the patients studied, the number of CFUE before therapy was significantly lower in comparison to normal controls: the mean value for a group of 6 patients was 104 + 56. Values for normal controls were obtained from 13 non hematologic patients in whom bone marrow was aspirated for diagnostic purposes. The mean value for normal controls was 409 + 191/105 cells mainly
seeded. After 2 months of rHuEpo therapy, the number of CFU-E increased in 5 patients and in one remained at the same level as before therapy (table III). It should be emphasized that in this patient, the number of CFU-E before therapy was the highest in this group of patients. The mean value for the group of 6 patients investigated after 2 months of therapy was 287 f 182 CFU-E/IO’ cells plated. This means that although the number of CFU-E increased during therapy, it was not normalized. In all but one patient CFU-E increased to about 50% of the normal value. Serum immunoreactive Epo levels prior to rHuEpo therapy in HD patients were 14.5 f 5.18
40 (mean f SD). In figure 1 serum Epo concentration time profiles for patients treated with rHuEpo iv are portrayed: i) after the first iv bolus injection of 50 U/kg rHuEpo; and ii), after 2 months of rHuEpo therapy following iv bolus injection of 75 U/kg rHuEpo. Serum Epo levels increased 30 min after injection in all the patients but the increase varied very much. From these highest values of serum Epo the levels also decreased at 6 h to variable values. The calculated rHuEpo elimination half-life after the first iv injection was 7.48 + 4.07 h (range 3.43 +- 15.40 h). After 2 months of therapy it had decreased to 4.68 f 1.57 h (range 1.38 f 6.47 h). In 2 healthy volunteers rHuEpo elimination half-life after iv injection of 50 U/kg (time profiles in fig 2) was 5.54 h. As can be seen from figure 3 signilicant negative correlation of calculated rHuEpo elimination half-life and number of CFU-E in the bone marro\r. ..; treated patients was found. The correlation of rHuEpo elimination half-life and percentage of erythroblasts was not significant (r = -0.303). Changes in serum Epo concentrations following SC rHuEpo administration of 30 U/kg at the beginning of therapy and 45 U/kg after 2 months of therapy can be seen in figure 4. When rHuEpo was given SC, peak concentrations were achieved between 8-14 h later and maintained for another
20 h. Time profiles of serum Epo concentrations after 2 months of therapy were similar to those found after the first SC rHuEpo injection. However, Epo levels were somewhat higher at that time, but the rHuEpo doses administered were higher as well. Contrary to the moderately in-
1
2
3
hours atlrr
4
5
6
rHu Epo
Fig 2. Serum Epo concentration time profile following an iv rHuEpo dose of 50 U/kg Lo 2
normai volunteers.
. 600
i
6 hour8
l2
aftor
rbtuLpo
Zb i.r.
Fig 1. Serum Epo concentration time profile prior to rHuEpo therapy following the first iv rHuEpo dose of 50 U/kg (0) and 2 months of therapy following an rHuEpo dose of 75 U/kg Values are mean 3~ SE for 9 patients.
Fig 3. Linear regression and correlation of rHuEpo elimination half-life after one bolus iv injection and number of CFU-E derived colonies in patients treated with rHuEpo iv.
41 lOO228
50-
./ /’ .*-
,7+
,a--------3-a
‘X1
40,
,___-l_----O-___~*, 301
:p--o-o
I
0 hcmts
6 uflcr
12
rHu
18
24
30
36
, 0
c 6
12
16
24
30
36
Epo S.C.
Fig 4. Serum Epo concentration time profile prior to rHuEpo therapy following the first sc rHuEpo dose (--) and after 2 months of therapy (- - -). Patient numbers are given with each curve. Numbers in parentheses denote weekly rHuEpo dose given.
creased serum Epo levels (30-50 mu/ml) found in 6 patients following SC rHuEpo injection, in one patient the serum Epo level increased from 12.2 to 107.2 mu/ml 2 h following the first rHuEpo injection and to 73 mu/ml after 2 months of therapy. Serum Epo levels were not monitored for a sufficiently long period after SC dosing to estimate a terminal t1/2. Discussion In the patients on HD described here, correction of anemia was achieved by both iv and SC application of rHuEpo. The rHuEpo iv starting dose used after each dialysis session was the dose presently widely accepted (50-80 U/kg). This dose is sufficient to induce a slow increase in hematocrit in most patients [19]. The anemia was corrected by SC rHuEpo administration over 2 months, although the doses were 40 percent lower than the iv doses (table II). This is in agreement with the findings of other authors [l].
Anuric HD patients were chosen for the study in order to ercta3e any possible influence of urinary Epo excretion on serum Epo levels following rHuEpo administration. Serum Epo concentration time profiles following iv rHuEpo administration differ from those described by others in the wide variations of peak levels at 30 min after the same doses (fig 1). Technical artefacts are not likely to cause those variations because of the precautions taken during rHuEpo iv application. It is possible, however, that the peak serum Epo level has been missed in some patients by measurements at only one point, -30 min after iv injection. Elimination half-life of rHuEpo injected iv (7.48 + 4.07) in our HD patients corresponds to the values reported for predialysis patients [lo] and patients with different degrees of renal failure [8], while it is slightly shorter than that reported for HD patients [3, 13J. The values we obtained (fig 3) in normal subjects are the same as reported [12, 181.
42 The shortening of rHuEpo half life observed during repeated administration for 2 months was first described by Egrie et al after 7 injections [3]. It was also observed after 8 weeks 1101 of rHuEpo therapy of predialysis patients. Interestingly enough, monitoring serum Epo post-dose concentrations in healthy volunteers revealed shortening of the serum half-life[l2]. The reasons for this have not yet been determined [lo]. We investigated mature erythroid progenitors (CFU-E) and erythroid precursor cells in the bone marrow of HD patients knowing that both CFU-E and erythroblasts have Epo receptors while for other hemopoietic cells this has not yet been clarified [9]. The difference between the concentration of CFU-E and erythroblasts (particularly pro- and basophilic) prior to rHuEpo therapy and following repeated rHuEpo dosing for 2 months shows an expansion of the cell pool of erythroid progenitors and precursors under rHuEpo therapy. Further on, expansion of erythroid progenitors negatively correlated with Epo elimination halflife (fig 3). Knowing that Epo mclecnles bound to Epo receptors are internalized in target cells, we would suggest that expansion of erythroid progenitors and the precursor cell pool could explain the faster Epo elimination after 2 months of rHuEpo treatment in the patients studied. This would be in line with Epo catabolism by utilization as first proposed by Stohlman [20], and in consensus with our earlier findings [ 161 (cited by Nathan and Sytkowski in the Editorial) [14] that serum Epo levels are inversely correlated with marrow activity. In support of our suggestion is the work of Jelkmann and Wiedemann [7], demonstrating that nonrenal anemic patients with erythrocytic hypoplasia have higher levels of serum Epo at any given hemoglobin concentration than do patients with hyperactive erythroid marrows. However, a different argument has recently been published work of Piroso ef al [17]. In their discussion, the authors have stated that because erythroid progenitors contain relatively few receptors, the consumption of Epo by erythroid progenitors should not measurably change the number of circulating Epo molecules. Their conclusion that it is unlikely that erythroid activity determines Epo lifespan and catabolism is based on the findings of similar half-lives found in rats regardless of decreased or increased bone marrow activity. Included in our suggestion is the presumption that rHuEpo penetration into the extracellular space does not change from the first rHuEpo iv injection to the
one given after 2 months of therapy. However, it should be emphasized that the metabolic fate of Epo is still not explained and that other unknown mechanisms could have induced shortening of elimination half-life of rHuEpo in our patients. SC rHuEpo administration resulted in a lower hut more constant serum Epo level compared to iv administration, as found by others [ 1, 111. Time profiles of serum concentrations of Epo following SC administration of rHuEpo (fig 4) are similar to those portrayed by Salmonson et al [ 181 for healthy subjects. It can be seen from the results presented that the patients required different doses of rHuEpo SC to achieve similar correction of anemia but that their serum Epo levels were in the same range. We have found that the modest increase of serum Epo over basal values after SC rHuEpo effectively stimulated the patients erythropoiesis. This indicates that Epo levels kept between 30-60 mu/ml between 2 injections were optimal for correcting anemia in our HD patients. We have described earlier a group of HD patients with acquired cystic transformation in the kidney, whose anemia improved markedly during HD. Serum Epo levels in those patients were moderately increased too [15]. Similar serum Epo levels were reported in transfusion independent HD [Q] and in nonanemic patients with polycystic kidney disease [2]. The small number of patients we have followed does not allow us to make firm conclusions but our results can be integrdted with those of others to provide the data on the serum Epo levels required for HD patients to maintain target red cell parameters that correspond to moderate anemia (a 12.0 g/d1 hemoglobin and PCV FJ 30%).
Acknowledgments This work was supported by a grand from the Serbian Research Foundation and also supported in part by Grant No HL 22469 from the National Heart, Lung and Blood Institutes, USA.
References 1 Bommer J, Ritz E, Weinreich T, Bommer G, Ziegler T (1988) Subcutaneous erythropoietin. Lancer ii, 406 2 Chandra M, Miller ME, Garcia JF, Mossey RT, McVicar M (1985) Serum immunoreactive erythropoietin levels in patients with polycystic kidney dis-
43
3
4
5
6
7
8
9 10
11
12
ease as compared with other hemodialysis patients. Nephron 39, 26 Egrie JC. Eschbach JW, McGuire T, Adamson JW (1988) Pharrnacokinetics of recombinant human erythropoietin (rHuEpo) administered to hemodialysis patients. Kidrtey Znt 33, 262 Garcia JF, Ebbe SN, Hollander L, Cutting HO, Milier ME, Cronkite EP (1982) Radioimmunoassay of erythropoietin: circulating levels in normal and polycystemic human beings. J Lab Chin Med 99, 624 Iscove N, Sieber F, Winterhalter K (1974) Erythroid colony formation in cultures of mouse and human bone marrow: analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose-concanavalin A. J Cell Physiol 83, 309 De Klerk G, Wilmink JM, Rosengarten PCJ, Vet RJWM, GG:ldsmit R (1982) Serum erythropoietin (ESF) titers in anemia of chronic renal failure. J Lab Cli?; Me2 100. 720 Jelkmann W, Wiedemann G (1990) Serum erythropoierin level: Relationships to blood hemoglobin concentration and erythrocytic activity of the bone marrow. Klin Wochenscbr 68, 403 Kindler J, Eckardt KU, Ahmer B, Jandeleit K, Kurtz A, Schreiber A, Scigalla P” Siberth HG (1989) Single dose pharmacokinetics of recombinant human erythropoietin in patients with various degrees of renal failure. Nephrol Dial Tranqvlant 4, 345 Krantz SB (1991) Erythroplaietin. Blood 77, 419 Lim VS, DeGowin RL, Zavala D, Kirchner PT, Abels R, Perry P, Fangman J (1989) Recombinant human erythropoietin treaunent in pre-dialysis patients. Ann Znt Med 110, II08 Macdougall IC, Roberts DE, Neubert P, Dharmsena AD, Coles GA, Williams JD l(1989) Pharmacokinetits of recombinant human eqrthropoietin in patients on continuous ambulatory peritoneal dialysis. Lancer 8635, 425 McMahon FG, Vargas R, Ryan M, Jain AK, Abels RJ, Ferry B, Smith Ii (1990) Pharmacokinetics and
effects of recombinant human erythropoietin after intravenous and subcutaneous injections in healthy volunteers. Blood 76, 1718 13 Muirhead N, Keown PA, Slaughter D, Mazaherir N, Jevnikar AM, Hollomby DJ, Hodsman AB, Cordy PE, Lindsay RM, Clark WF, Faye WP (1988) Recombinant human erythropoietin in the anaemia of chronic renal failure: a pharmacokinetic study. Nephrol Dial Transplant 3, 499 14 Nathan DC, Sytkowski A (1983) Editorial retrospective: Erythropoietin and the regulation of erythropoiesis. N Engl J Med 306, 520 12,.Tavlovid-Kentera V. Ciemons GK. Diukanovid L (1989) Pathophysioiogy of eryihropoidsis in renal diseases. In: Erythropoietin (Jelkmsnn W, Gross AJ, eds), Springer-Verlag. Berlin p 92 16 Pavlovid-Kentera V, Milenkovit P, RuvidiC R. Jovanovik V, Biljanovi&Paunovib L (1979) Erythropoietin in aplastic anemia. Blur 39, 345 17 Piroso E, Erslev AI, Flaharty KK, Caro J (1991) Erythropoietin life span in rats with hypoplastic and hyperplastic bone marrows. Am J Hematol 36, 105 18 Salmonson T, Danielson BG, Wikstriim B (1990) The pharmacokinetics of recombinant human erythropoietin after intravenous and subcutaneous administration to healthy subjects. Br J Clin Pharmacol 29, 709 19 Samtleben W, lvanovich P, Gurland HJ (1989) Recombinant human erythropoietin in nephrology. Biomed Pharmacother 43, 607 20 Stohlman F (1959) Observations on the physiology of erythropoietin and its role in the regulation of red cell production. Ann NY Acad Sci 77; 710 21 Sundal E, Kaeser U (1989) Correction of anemia of chronic renal failure with recombinant human crythropoietin: safety and efficacy of one year’s treatment in a European multicentre study of 150 haemodialysis-dependent patients. Nephrol Dial Transplant 4, 979
37
riginal article Serum erythropoietin levels in hemodialysed patients after administration of recombinant human erythro V PavloviC-Kentera’,
GK Clemons2, L Bil’anoviC-PaunoviC’, V LeiaiC4, M Pokrajac 4, Lj Djukanovid4
D MarisavljeviC3,
‘Instituie for Medical Research, PO Box 721, JJO01 Belgrade; 2JLawrenceBerkeley Laboratory, Berkeley, CA. USA; 31nstituteof Hematology and 41nstitute of Nephrology. University Clinical Center, &igrade; ‘Department of Pharmacokinetics, Faculty of Pharmacy, Belgrade. Yugoslavia (Received 23 August 1991; accepted 16 December 1991)
Summary - In anemic patients on regular hemodiaiysis (HD), correction of anemia with recombinant human erythropoietin @HuEpo) administe~d intravenously (iv} or subcutaneously (SC) was followed over a 2-month period. Monitoring serum Epo post-dose concentrations after the first iv rHuEpo injection and following another regular injection after 2 months of therapy with rHuEpo iv in 9 patients showed that the Epo elimination half-life was rednced from 7.48 h to 4.68 h. In the same patients the initially low percentage of e~~r~las~ and mature erythroid progenitors increased during 2 months of rHuEpo therapy. Because Bpo molecules bound to Epo receptors are intemakzd in target cells we suggest that the expansicn of the Bpo responsive cell pool could explain the shorted Epo elimination time afier 2 months of rHtQo treatment. By rno~~to~ng serum Epo concentration following SCrHuEpo injection in 7 HD patients it was found that the modest increase in serum Epo levels (30-40 mU/ml~ was sufficient to correct anemia. serum erythropoietin tevel I chronic renal failure I rHuEpo therapy Msumi: - Taux d’~rythropo~~tine sbrique chez les maladef h~m~~alys~s aprb administration d’~r~h~~ffine ktomaine r~om~inant~ La correction dune ankmie a et& men&epar ~~thropo~~tine humaine reco~~~nte {rHuEpo] ituraveineuse ou sous-cutant!e et suivie sur une pkiode de deux tnois chez des patfents en h~rn~~~~se. Le monitoring des concenrrfftion~ d’Epo skique aprts une premi&e injection IV de rHuEpo et apr& une autre dew: mois plus tard cbez ne& patients a montrt! un rac~our~Jssement de la demi-vie ~~lirni~tion de f’Epo de ?h4B ci 4h6B. Chet ces mgmes malades. les pou~enta~es ~~~throblastes et de prog~niteurs ~~thro~des matures, initialement bas, ant augment&au eours des deux mois de traitement par Ia rHuEJw. En raison du fait que les mokdes d’Epo fm&es sur les r&cepteurs correspondants se trouvent ci l’ext&ieur des ~eJ~ules-~ibJes,ies auteurs dmettent I’hypothcise que l’expansion du pool de celiuJes r&ondant ir I’Epo pourrait expliquer la rkductioa du temps ~~~irni~tion de I’Epo apr&s deux mois de traitement par I’rHuEpo. En surveillant fes concentrations d’Epo s&ique apr& injection sous-cutantfe de rHuEpo chez sept ht?madialysPs, an aurait &aiement trot& que la l&&e augmentation due aux d’Epo skique (30 d 60 mUlm1) &ait st@sante pow corriger PanZmie. taux d*&ythropoMine sbique / ddfaillance r&tale rhronique I traitement par rHuEpo
Introduction The efficacy of recombinant human erythropoietin (rHuEpo) therapy for anemia in patients on regular hemodialysis (HD) was demonstrated in clinical trials [21]. The correction of anemia in HD patients by rHuEpo indicates that studies of the effects of Epo in those patients in vivo could
provide
valuable
information
concerning
the bio-
logy of this hormone. During a 2-month period, correction of anemia in HD patients treated with rHuEpo intravenously (iv) or subcutaneously (SC) was followed. Serum Epo levels were monitored after the first injection at the beginning of therapy and following another regular injection after 2 months of therapy, in order to obtain more infor-
38 mation regarding the dependence of serum Epo levels on the size of the Epo responsive cell pool and the serum Epo levels anemia in ND patients.
required
to counteract
Patients and methods Patiettts Two groups of HD patients with anemia were investigated. The study was approved by the Ethics Committee of the University Clinical Center. Written informed consent was obtained From each patient. Nine patients were treated with rHuEpo iv and 7 SC. Patients were dialysed 3 times a week for 4 h nsing cuprophan membrane dialyzers. rHuEpo was administered at the end of dialysis. The starting dose was 50 U/kg body weight (bw) for iv and 30 U/kg bw for SC, after each dialysis, ie 3 times a week. The dose was increased by 50 percent of the starting dose after every 4 weeks whenever target hemoglobin level was not achieved. Iron supplementation was given to patients with normal or low serum iron concentration or ferritin levels < lOO/pg/l. A complete blood count was performed at the outset of the study and then once a week for 8 weeks. Methods Bone marrow specimens were obtained during the morning on the day preceding rHuEpo injection. Samples of 1-2 ml of bone marrow were aspirated from the iliac crest. Samples for bone marrow cuitures were collected in preservative-free sterile heparin (50 U/ml). Bone marrow smears were made on glass slides and analyzed after May-Griinwald-Giemsa and Prussian blue staining. Four hundred elements were differentiated on coded glass slides. To obtain the erythroblast maturation curve, the percemage of different erythroblast maturation stages was determined by differentiation of up to 200 erythroblasts. Iron content in the bone marrow was evaluated by the percentage of sideroblasts as well as the presence of extracellular iron. For the CFU-E assay, mononuclear cells from bone marrow samples were separated by Lymphoprep gradient centrifugation before plating. The methylcellulose culture technique described by Iscove et al [5] was used. Cells were cultured in duplicate in 35-mm tissue culture dishes at lO’/ml in Dulbecco’s medium containing 30% fetal calf serum, 0.8% methylcellulose, 0.1% bovine serum albumin, fi-mercaptoethanol 2 x 10m5 M, and 1.O U of rHuEpo (kindly donated by W Jelkman, Liibeck, Germany). Erythroid colonies were identified after 7 days incubation at 37°C in a humidified 5% CO2 atmosphere. Epo levels were determined at the Lawrence Berkeley Laboratory by radioimmunoassay [4]. Serum
samples for basal value of Epo were obtained just prior to the first rHuEpo injection and then 30 min, 2 and 6 h after iv and 2, S, 14, 24 and 36 h after SC injection. The same schedule in obtaining serum samples for Epo determinations after one iv or SC injection was used again after 2 months of rHuEpo therapy. Pharmacokinetic
calculations
The elimination rate constant (&I) for rHuEpo after one bolus iv injection was estimated from the slope of the curve after 2 h, as the distribution phase was terminated in all patients at that time. The kmenc parameter eiimination half-life (tin) was estimated using the standard pharmacokinetic equation, assuming first order elimination. Both mathematical and graphic methods of calculation gave similar results.
Results The c!inical characteristics of the patients studied are presented in table I. These patients were anemic prior to rHuEpo therapy with hematocrit values below 20%. As can be seen in table II, during 2 months of treatment with rHuEpo, anemia improved with both iv and SC routes of administration, although the target hemoglobin value of 10.0 g/d1 was not reached in that time. The values of 2 patients whose anemia was not corrected during 2 months of rHuEpo treatment were not included in table II. Their hematocrit values were respectively 19 and 26 before and 20 and 21 after 2 months’ treatment. The first patient was bleeding from a papilloma in the stomach and the second had to undergo surgery. The bone marrow differential count revealed low percentages of erythroblasts before Epo. Values increased after 2 months in the patients who received therapy. The rate of increase differed from patient to patient, while bone marrow cellularity did not change significantly (data not shown). It is particularly important that the increase in the percentage of erythroblasts was
Table
I. Clinical characteristics of the patients studied.
Route of Epo administration iv SC
No of patients M
F
4 2
5 5
Age (yr)
BW (kg)
Duration of hemodialysis (months)
37-64 36-64
40-67 40-75
6-216 13-107
39 Table II. Measures of red cell concentration in hemodialysis patients treated with rHuEpo intravenously (iv) or subcutaneotts~y (xc).
iv rHuEpo therapy RBC x IO’=/1
Hb gldl
1.86 + 0.21
5.64 f 0.58
Before therapy After 2 months of therapy
SCrHuEpa therapy PCV l/l
RBC x Io’211
Hb gldl
PCV III
2.06 zk 0.46
6.23 + 1.16
19.0 f 3.65
17.29 z!z1.80
2.70*** f 0.45 9.06*** f 1.44 26.29, + 4.89
2.89* f 0.64 9.19** f 2.12 26.57. + 6.11
The values are mean + SD for II = 7 in each group. Significance cf the differences when compared to the values before therapy: *P < 0.05, **P c 0.01;
***P
c 0.001. RBC: red blood cells; Hb: hemoglobin; PCV: packed cell volume.
Table III. Bone rnarrow smear analyses prior to (A) and after 2 months (B) of rHuEpo therapy. Patient
225 A B 226 A B 223 A B 252 A B 227 A B 231 A B
CFU-El16
79 172 58 226 82 238 66 201 136 656 205 229
Erbf W)
3 40 11 26 2 10 16 21 31 36 4.5 8
Erythroblast&’ Pro @)
Baso (%)
0
0
2 0 0 0 0
10 1 13 0 9
1 1.5 3 0 0
6 6 7 1 10
Poly m)
Ortho (a)
35 52 37 58 30 49
65 35 62 29 70 42
26
67 51.5 60 55 38
41 40 45 52
Sideroblasts iw 80 46 64 80 0 30 40 58 80 20 70 34
Extrace~hdaf iron
4+
2+ 4+ 4+ 1-k l+ 2+ 3+ 5+
l+ 3+ 2+
a Percent of erythroblasts in bone marrow differential; b percentageof erythroblast maturation stagesobtained by differentiating 100 to 2M) erythroblasls:pro-proerythroblasts;baso4asophilic; poly-polychromatophilic;ortho-orthochromatic;’ extracellularim: normal value +2 to +3.
due to an increase in basophilic and polychromatophilic erythroblasu (table III) while orthochromatic erythroblasts decreased to near normal values. In the same patients the percentage of sideroblasts in the bone marrow decreased after 2 months of therapy. In all the patients studied, the number of CFUE before therapy was significantly lower in comparison to normal controls: the mean value for a group of 6 patients was 104 + 56. Values for normal controls were obtained from 13 non hematologic patients in whom bone marrow was aspirated for diagnostic purposes. The mean value for normal controls was 409 + 191/105 cells mainly
seeded. After 2 months of rHuEpo therapy, the number of CFU-E increased in 5 patients and in one remained at the same level as before therapy (table III). It should be emphasized that in this patient, the number of CFU-E before therapy was the highest in this group of patients. The mean value for the group of 6 patients investigated after 2 months of therapy was 287 f 182 CFU-E/IO’ cells plated. This means that although the number of CFU-E increased during therapy, it was not normalized. In all but one patient CFU-E increased to about 50% of the normal value. Serum immunoreactive Epo levels prior to rHuEpo therapy in HD patients were 14.5 f 5.18
40 (mean f SD). In figure 1 serum Epo concentration time profiles for patients treated with rHuEpo iv are portrayed: i) after the first iv bolus injection of 50 U/kg rHuEpo; and ii), after 2 months of rHuEpo therapy following iv bolus injection of 75 U/kg rHuEpo. Serum Epo levels increased 30 min after injection in all the patients but the increase varied very much. From these highest values of serum Epo the levels also decreased at 6 h to variable values. The calculated rHuEpo elimination half-life after the first iv injection was 7.48 + 4.07 h (range 3.43 +- 15.40 h). After 2 months of therapy it had decreased to 4.68 f 1.57 h (range 1.38 f 6.47 h). In 2 healthy volunteers rHuEpo elimination half-life after iv injection of 50 U/kg (time profiles in fig 2) was 5.54 h. As can be seen from figure 3 signilicant negative correlation of calculated rHuEpo elimination half-life and number of CFU-E in the bone marro\r. ..; treated patients was found. The correlation of rHuEpo elimination half-life and percentage of erythroblasts was not significant (r = -0.303). Changes in serum Epo concentrations following SC rHuEpo administration of 30 U/kg at the beginning of therapy and 45 U/kg after 2 months of therapy can be seen in figure 4. When rHuEpo was given SC, peak concentrations were achieved between 8-14 h later and maintained for another
20 h. Time profiles of serum Epo concentrations after 2 months of therapy were similar to those found after the first SC rHuEpo injection. However, Epo levels were somewhat higher at that time, but the rHuEpo doses administered were higher as well. Contrary to the moderately in-
1
2
3
hours atlrr
4
5
6
rHu Epo
Fig 2. Serum Epo concentration time profile following an iv rHuEpo dose of 50 U/kg Lo 2
normai volunteers.
. 600
i
6 hour8
l2
aftor
rbtuLpo
Zb i.r.
Fig 1. Serum Epo concentration time profile prior to rHuEpo therapy following the first iv rHuEpo dose of 50 U/kg (0) and 2 months of therapy following an rHuEpo dose of 75 U/kg Values are mean 3~ SE for 9 patients.
Fig 3. Linear regression and correlation of rHuEpo elimination half-life after one bolus iv injection and number of CFU-E derived colonies in patients treated with rHuEpo iv.
41 lOO228
50-
./ /’ .*-
,7+
,a--------3-a
‘X1
40,
,___-l_----O-___~*, 301
:p--o-o
I
0 hcmts
6 uflcr
12
rHu
18
24
30
36
, 0
c 6
12
16
24
30
36
Epo S.C.
Fig 4. Serum Epo concentration time profile prior to rHuEpo therapy following the first sc rHuEpo dose (--) and after 2 months of therapy (- - -). Patient numbers are given with each curve. Numbers in parentheses denote weekly rHuEpo dose given.
creased serum Epo levels (30-50 mu/ml) found in 6 patients following SC rHuEpo injection, in one patient the serum Epo level increased from 12.2 to 107.2 mu/ml 2 h following the first rHuEpo injection and to 73 mu/ml after 2 months of therapy. Serum Epo levels were not monitored for a sufficiently long period after SC dosing to estimate a terminal t1/2. Discussion In the patients on HD described here, correction of anemia was achieved by both iv and SC application of rHuEpo. The rHuEpo iv starting dose used after each dialysis session was the dose presently widely accepted (50-80 U/kg). This dose is sufficient to induce a slow increase in hematocrit in most patients [19]. The anemia was corrected by SC rHuEpo administration over 2 months, although the doses were 40 percent lower than the iv doses (table II). This is in agreement with the findings of other authors [l].
Anuric HD patients were chosen for the study in order to ercta3e any possible influence of urinary Epo excretion on serum Epo levels following rHuEpo administration. Serum Epo concentration time profiles following iv rHuEpo administration differ from those described by others in the wide variations of peak levels at 30 min after the same doses (fig 1). Technical artefacts are not likely to cause those variations because of the precautions taken during rHuEpo iv application. It is possible, however, that the peak serum Epo level has been missed in some patients by measurements at only one point, -30 min after iv injection. Elimination half-life of rHuEpo injected iv (7.48 + 4.07) in our HD patients corresponds to the values reported for predialysis patients [lo] and patients with different degrees of renal failure [8], while it is slightly shorter than that reported for HD patients [3, 13J. The values we obtained (fig 3) in normal subjects are the same as reported [12, 181.
42 The shortening of rHuEpo half life observed during repeated administration for 2 months was first described by Egrie et al after 7 injections [3]. It was also observed after 8 weeks 1101 of rHuEpo therapy of predialysis patients. Interestingly enough, monitoring serum Epo post-dose concentrations in healthy volunteers revealed shortening of the serum half-life[l2]. The reasons for this have not yet been determined [lo]. We investigated mature erythroid progenitors (CFU-E) and erythroid precursor cells in the bone marrow of HD patients knowing that both CFU-E and erythroblasts have Epo receptors while for other hemopoietic cells this has not yet been clarified [9]. The difference between the concentration of CFU-E and erythroblasts (particularly pro- and basophilic) prior to rHuEpo therapy and following repeated rHuEpo dosing for 2 months shows an expansion of the cell pool of erythroid progenitors and precursors under rHuEpo therapy. Further on, expansion of erythroid progenitors negatively correlated with Epo elimination halflife (fig 3). Knowing that Epo mclecnles bound to Epo receptors are internalized in target cells, we would suggest that expansion of erythroid progenitors and the precursor cell pool could explain the faster Epo elimination after 2 months of rHuEpo treatment in the patients studied. This would be in line with Epo catabolism by utilization as first proposed by Stohlman [20], and in consensus with our earlier findings [ 161 (cited by Nathan and Sytkowski in the Editorial) [14] that serum Epo levels are inversely correlated with marrow activity. In support of our suggestion is the work of Jelkmann and Wiedemann [7], demonstrating that nonrenal anemic patients with erythrocytic hypoplasia have higher levels of serum Epo at any given hemoglobin concentration than do patients with hyperactive erythroid marrows. However, a different argument has recently been published work of Piroso ef al [17]. In their discussion, the authors have stated that because erythroid progenitors contain relatively few receptors, the consumption of Epo by erythroid progenitors should not measurably change the number of circulating Epo molecules. Their conclusion that it is unlikely that erythroid activity determines Epo lifespan and catabolism is based on the findings of similar half-lives found in rats regardless of decreased or increased bone marrow activity. Included in our suggestion is the presumption that rHuEpo penetration into the extracellular space does not change from the first rHuEpo iv injection to the
one given after 2 months of therapy. However, it should be emphasized that the metabolic fate of Epo is still not explained and that other unknown mechanisms could have induced shortening of elimination half-life of rHuEpo in our patients. SC rHuEpo administration resulted in a lower hut more constant serum Epo level compared to iv administration, as found by others [ 1, 111. Time profiles of serum concentrations of Epo following SC administration of rHuEpo (fig 4) are similar to those portrayed by Salmonson et al [ 181 for healthy subjects. It can be seen from the results presented that the patients required different doses of rHuEpo SC to achieve similar correction of anemia but that their serum Epo levels were in the same range. We have found that the modest increase of serum Epo over basal values after SC rHuEpo effectively stimulated the patients erythropoiesis. This indicates that Epo levels kept between 30-60 mu/ml between 2 injections were optimal for correcting anemia in our HD patients. We have described earlier a group of HD patients with acquired cystic transformation in the kidney, whose anemia improved markedly during HD. Serum Epo levels in those patients were moderately increased too [15]. Similar serum Epo levels were reported in transfusion independent HD [Q] and in nonanemic patients with polycystic kidney disease [2]. The small number of patients we have followed does not allow us to make firm conclusions but our results can be integrdted with those of others to provide the data on the serum Epo levels required for HD patients to maintain target red cell parameters that correspond to moderate anemia (a 12.0 g/d1 hemoglobin and PCV FJ 30%).
Acknowledgments This work was supported by a grand from the Serbian Research Foundation and also supported in part by Grant No HL 22469 from the National Heart, Lung and Blood Institutes, USA.
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