Abstract
The pharmacodynamics (PD) of the reticulocyte response resulting from phlebotomy-induced erythropoietin (EPO) was investigated in adult sheep. The anemia caused by the controlled phlebotomy (Hb < 4 g/dl, t = 0) resulted in a rapid increase in EPO with peak concentrations from 200 to 1400 mU/ml at 0.5 to 3 days generating a delayed reticulocyte response with peak levels from 9.3 to 14.1% at 2.5 to 5.1 days. The PD EPO-reticulocyte relationship is well described by a simple kinetic model involving 3 relevant physiologic parameters: T1 = lag-time (0.73 ± 0.32 days, mean ± S.D.), T2 = reticulocyte maturation time (5.61 ± 1.41 days), andk = EPO efficacy coefficient (0.052 ± 0.048% g/dl mU/ml/day). Accordingly, 0.52% reticulocytes at 10 g/dl Hb level are generated per day at an EPO concentration of 100 mU/ml. The difference between the T2 parameter in this study and the maturation time reported for humans may be due to interspecies differences or different technique and experimental conditions. The PD transduction appears largely linear in the observed EPO concentration range, indicating a full utilization of EPO without any significant PD saturation. Also, the EPO concentration versus time profiles resulting from the phlebotomy were similar to exogenous EPO profiles resulting from s.c. therapeutic dosing. This study supports the hypothesis that s.c. EPO dosing is more efficacious than i.v. dosing.
Erythropoietin (EPO) is a 34-kDa glycoprotein that is the primary hormone regulator of erythrocyte production. Recombinant human EPO (rhEPO) has been widely used clinically as an effective treatment of anemic patients with insufficient EPO production, e.g., end-stage renal disease. rhEPO is also approved for anemic patients suffering from neoplastic disease and acquired immunodeficiency with azidothymidine treatment.
Several pharmacokinetics/pharmacodynamics (PK/PD) models for EPO's stimulating effect on erythropoiesis have been developed (Brockmoller et al., 1992; Uehlinger et al., 1992; Bressolle et al., 1997). The PD response variables considered in EPO PK/PD models have been Hb, hematocrit, reticulocyte count, and serum soluble transferrin receptors. However, none of these previous studies have been considered under “physiologic conditions”, i.e., investigating the PD of the response to the large endogenous EPO concentrations seen in hypoxemic episodes. Instead, the studies have been done using exogenous rhEPO under nonanemic conditions.
The goal of this investigation is to study the kinetic mechanisms underlying the PD of EPO through controlled physiologic experimentation, in which erythropoiesis was endogenously stimulated. A sheep model was employed to observe how the body naturally reacts to a large demand for hematopoiesis. The PD relationship between reticulocyte response and the endogenous EPO response was investigated under conditions of severe phlebotomy-induced anemia.
Materials and Methods
Study Animals.
All surgical and experimental procedures received prior approval by the local institutional animal care review committee. Five healthy normal young adult sheep were selected. They were 2 months old and weighed 21.1 (±3.5) kg (mean ± S.D.) at the beginning of the experiments. The animals were housed in an indoor, light- and temperature-controlled environment. All animals were in good health. Jugular venous catheters were placed under anesthesia using pentobarbital. Intravenous ampicillin (1 g) was administered daily for the first 3 days.
Study Protocol.
An increase in endogenous EPO was induced by controlled phlebotomy performed using the jugular venous catheter. Animals were bled until their Hb levels reached between 3.0 and 4.0 g/dl. To maintain a constant blood volume during the procedure, equal volumes of 0.9% NaCl solution was infused for each volume (∼2000 ml) of blood removed. Each animal underwent two such phlebotomies given 4 to 6 weeks apart. EPO concentrations, reticulocyte counts, and Hb were measured from one to three samples per day drawn before and during the study period. EPO concentration was measured in plasma using a modification of the double antibody radioimmunoassay procedure as previously described (Widness et al., 1992). The number of reticulocytes was determined by flow cytometry (FACScan; Becton-Dickinson, San Jose, CA) as described by Peters et al. (1996). Plasma iron concentration was also monitored. No extra iron supplementation other than through food was given to any subjects in this study.
PK/PD Modeling.
We applied a system analysis approach to analyze the PD (Cutler, 1978; Gillespie et al., 1988; Veng-Pedersen and Gillespie, 1988). In this approach we consider the rate of formation of reticulocyte, R, to depend on the EPO concentration,CEPO:
Data Analysis.
Equations 6 and 7 were fitted to the corresponding reticulocyte data using a Windows version of the general nonlinear regression program FUNFIT (Veng-Pedersen, 1977). A linear activation function was used to describe the formation rate of reticulocytes induced by EPO:
Results
EPO Concentrations and Reticulocyte Counts in Phlebotomized Sheep.
Baseline characteristics of EPO concentrations and relative reticulocyte counts were determined as 18.3 ± 3.6 mU/ml and 0.81 ± 0.57%, respectively. Figure1 illustrates the dynamic changes in the PD response variables after the phlebotomy. The anemia caused by phlebotomy (Hb < 4 g/dl) resulted in a rapid and immediate increase in endogenous EPO concentrations with peak concentrations ranging from 200 to 1400 mU/ml, from 0.5 to 3 days after the phlebotomy. The phlebotomy-induced EPO plasma concentrations returned to normal within 3 to 6 days after the start of the increase in EPO. The maximum relative percentage reticulocyte counts observed ranged between 9.3 and 14.1% at 2.5 to 5.1 days after the start of the phlebotomy. The increase in reticulocytes showed a delay with a lag time of 0.2 to 2.0 days from the commencement of phlebotomy. The elevated reticulocyte counts returned to baseline values at 10 to 15 days after the phlebotomy. The Hb levels lowered by phlebotomy recovered to the baseline by the time that the elevated reticulocytes returned to the normal range. None of the study animals showed significant iron deficiency, considering the observed ratio of plasma iron and total iron binding capacity throughout the experimental periods (0.37 ± 0.083; range, 0.31–0.50, n = 5).
The Fitted Model and Parameter Estimates.
The fitted model (eqs. 6 and 7) showed a good agreement with the observed data. Figure2 provides representative plots of such fittings, and Tables 1 and 2 summarize the parameter estimates of the fitted PK/PD model. In Fig. 2, two examples of the curve fitting are shown, one with the best correlation (r = 0.992) and the other with the worst (r = 0.853) among the 10 data sets. Owing partly to the parsimony in the model, the parameter estimates showed relatively little variability with coefficients of variation less than 100%. The three estimated parametersT1, T2, andk summarize the kinetics, i.e., the onset of EPO action, the systemic lifespan of EPO induced reticulocytes, and the efficacy coefficient, respectively. The estimated means ofT1, T2, andk are 0.47 days, 4.98 days, and 0.0099% · (mU/ml)−1 day−1with standard deviations of 0.28, 1.31, and 0.0077, respectively. Accordingly, newly generated reticulocytes are first observed 0.47 day on average after EPO's activation of the hematopoietic progenitor cells and can be observed circulating in the peripheral blood for 4.98 days before maturating to red blood cells. The efficacy coefficient k represents an efficacy measure of EPO for the rate of formation of new reticulocytes.
Fitting Using ARI Data.
There was no significant difference found in the overall shapes of reticulocyte response to EPO as shown in Fig. 3, when ARI was used as the dependent variable in the model instead of percentage reticulocyte count. This was observed consistently among all ten phlebotomies of the five study animals. The sampling was not frequent enough near the peaks to accurately determine the difference in the two different modeling situations, i.e., one with percentage of reticulocytes and the other using the ARI. Correspondingly, the parameter estimates using eq. 7with ARI also appeared to be similar to those from fitting eq. 6 to the data as shown in Tables 1 and2.
Discussion
Linearity in Pharmacodynamic Response of EPO.
In this study, the EPO concentrations resulting from severe phlebotomy-induced anemia are similar to those seen following s.c. EPO administration (Cheung et al., 1998). Within the endogenous range of EPO concentrations that the body could produce after this stimulus, the rate of reticulocyte formation was linearly related to the EPO concentration (eq. 8). This linearity was not expected, because most PD concentration-effect relationships are nonlinear and often well represented by anEmax or sigmoidEmax model. However, it is also recognized that a linear concentration range may exist as a part of nonlinear relationships, e.g., the sigmoid Emaxmodel. In this modeling approach, the Akaike information criterion (Akaike, 1974) did not justify the inclusion of a nonlinear concentration-effect relationship. A deviation from PD linearity would likely have been observed if the EPO plasma concentrations had been higher.
From a therapeutic point of view it is encouraging to see that, in the present study, the EPO concentrations from the phlebotomy appear to fall within the linear PD range indicating lack of saturation. Consequently, the EPO released is not “wasted” in a saturable process but is fully utilized. As previously noted, the EPO concentration profiles encountered in this study are similar to those seen in s.c. administration in humans. Several studies in humans indicated that s.c. administration of EPO is more efficacious than the same weekly dose given i.v. (Kaufman et al., 1998; Macdougall, 1999). Hence, our results in sheep are consistent with this finding in humans. The alternative i.v. administration of EPO will produce higher EPO concentrations than s.c. administration and possibly result in “saturation” in the PD effect and accordingly be less efficacious.
Saturation in receptor-mediated endocytosis is a possible mechanism for EPO's nonlinear PK (Kato et al., 1997; Veng-Pedersen et al., 1999). The majority of EPO receptors are located on progenitor cells in the bone marrow. EPO's elimination and PD response are accordingly related and not totally independent processes. The apparent lack of a nonlinear relationship between the EPO concentration and the rate of formation of new reticulocytes may be explained by the fact that in previous studies the exogenous EPO doses used to analyze the nonlinear elimination kinetics resulted in higher EPO concentrations, making a nonlinearity more easily detectable than in the present study (Veng-Pedersen et al., 1999).
However, one subject in this study showed some indication of nonlinearity in the PD. That subject experienced considerably higher EPO concentrations, i.e., 1400 mU/ml and 1128 mU/ml peak concentrations in the first and second phlebotomies, respectively, than the mean peak concentration of 624 mU/ml in the rest of subjects. In the analysis of the data from the subject showing the higher peak EPO concentrations, the inclusion of a nonlinear activation rate improved the fitting result, but not to the extent that it reached statistical significance according to the Akaike criterion.
The present analysis focused on the PD of EPO. It has been proposed that EPO's binding to EPO receptors on progenitor cells is a receptor-mediated process, which initiates the differentiation of these cells into reticulocytes with subsequent maturation to red blood cells. The consumption of EPO in this way appears to be an important component in EPO's nonlinear elimination (Kato et al., 1997; Veng-Pedersen et al., 1999). A nonlinear elimination is usually associated with a saturation type of elimination process. According to this receptor-based nonlinear elimination hypothesis, a single large i.v. dose creates saturation nonlinearity from high “spike” concentrations of EPO, whereas the lower concentrations seen as a result of phlebotomy and after a s.c. dose fall in the linear, more efficacious concentration range.
Model Parameters.
The proposed model provides information through the T1 andT2 parameters about the time course for the release of reticulocytes into the systemic circulation and the maturation of the reticulocytes into red blood cells. The transit time of human reticulocytes in blood is reported to be 1 to 2 days (Finch et al., 1977; Beutler et al., 1995), which was measured by injection of radiolabeled reticulocytes. The reported estimates of the circulating life span of reticulocytes are shorter than the correspondingT2 estimates of 5 days we observed in sheep as assessed by our model. The exact reason is unknown, but it may be partly due to different analysis methodologies for the determination and the fact that our study was performed under anemia induced by phlebotomy, contrary to other studies. Possible interspecies differences may also exist. When the data in our study are visually compared with those in a human study by Breymann et al. (1996), the reticulocytes remained elevated for a significantly longer period relative to our EPO profiles in sheep.
Our T2 estimates for individual sheep ranged from 3.0 to 6.7 days. According to the result from an ANOVA, the difference between the intrasubject variability and the intersubject variability in T2 (first and second phlebotomies) did not reach statistical significance. TheT2 parameter estimates in the second phlebotomy appeared to be smaller than those obtained in the first phlebotomy experimental units (P < .05). This indicates a possible change in the hematological system in the body. We speculate that the accelerated induction of new progenitor cells from the first phlebotomy may subsequently have produced reticulocytes predisposed for more rapid employment, i.e., having a faster conversion to red blood cells resulting in the smallerT2 value. Baseline blood parameters such as Hb and iron status did not show a significant difference between the first and second phlebotomies.
In contrary to the T2 estimates,T1 parameter estimates showed greater intrasubject variability, which was not statistically significant, either. The reason may be that the T1parameter is more difficult to estimate due to a gradual initial increase in reticulocyte responses and substantial fluctuation in the baseline reticulocytes level before the onset of the pharmacological effects.
The k parameter is a measure of EPO's efficacy in the reticulocyte production. It measures the rate of formation of reticulocytes normalized to the EPO concentration (units = rate/concentration). Physiologically, this can be interpreted as a reflection of the number of EPO receptors per progenitor cells and/or the number of progenitor cells. In terms of its variability, it showed the same trend as T2. The parameterk showed larger intersubject variability than intrasubject variability, but due to the small number of subject, it did not reach statistical significance.
Care should be taken in the interpretation of the kparameter. The estimate based on ARI is meaningful assuming that the size of the red cells and the amount of Hb per red cell remain constant. Accordingly, modeling with absolute reticulocyte counts will possibly provide a more meaningful k parameter estimate.
In summary, the proposed model employing a simple linear PD relationship can describe the reticulocyte response to phlebotomy-induced endogenous EPO and also provides a useful tool for determination of hematological parameters by PK means. The information obtained from this study offers a better understanding of the physiologic events after a large amount of blood loss. The linear relationship in EPO reticulocytes provides valuable information about the mechanisms for EPO's PD and supports the notion that s.c. administration of EPO is more efficacious than i.v. bolus injections.
Acknowledgments
The recombinant human EPO used in the EPO radioimmunoassay was a gift from Dr. H. Kinoshita of Chugai Pharmaceutical Company, Ltd. (Tokyo, Japan). The rabbit EPO antiserum used in the EPO radioimmunoassay was a generous gift from Gisela K. Clemens, Ph.D. The authors gratefully acknowledge the technical help of Lance S. Lowe.
Footnotes
-
Send reprint requests to: P. Veng-Pedersen, College of Pharmacy, University of Iowa, Iowa City, IA 52242. E-mail:veng{at}uiowa.edu
-
↵1 This work was supported by the United States Public Health Service National Institutes of Health (NIH) Grants PO1 HL46925 and GM57367 and by Grant RR000359 from the General Clinical Research Center Program, National Center for Research Resources, NIH.
- Abbreviations:
- EPO
- erythropoietin
- rhEPO
- recombinant human erythropoietin
- PD
- pharmacodynamics
- PK
- pharmacokinetics
- RRC
- relative reticulocyte count
- UIR
- unit impulse response
- ARI
- absolute reticulocyte index
- Received March 24, 2000.
- Accepted June 12, 2000.
- The American Society for Pharmacology and Experimental Therapeutics