Population Pharmacodynamics for Monitoring Epoetin in Athletes

PHARMACODYNAMICS Clin. Drug Invest. 1997 Sep; 14 (3): 233-242 11 73-2563/97/(XX)9-{)233/S0S.OJ1O

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Population Pharmacodynamics for Monitoring Epoetin in Athletes Franfoise Bressolle,l Michel Audran,2 Raynald Gareau,3 Roy D. Baynes,4 Claudette Guidicelli2 and Roberto Gomenis

Laboratoire de Pharrnacocinetique, Faculte de Pharmacie, Universite Montpellier 1, France 2 Departement de Biophysique, Faculte de Pharmacie, Universite Montpellier I, France 3 Service d 'hematologie, Universite de Quebec, Trois Rivieres, Canada 4 Department of Internal Medicine, Division of Hematology, University of Kansas Medical Center, Kansas City,

Kansas, USA 5 Bestfit, Luxembourg

Summary Epoetin (recombinant human erythropoietin) is used by some endurance athletes to increase oxygen transport and aerobic power in an attempt to improve endurance capacity and recovery during training and competition. Although currently on the list of banned substances issued by the medical commission of the International Olympic Committee, the use of epoetin as an ergogenic agent remains uncontrollable using classical analytical techniques. In the present paper, after subcutaneous administration of repeated high doses (200 units/kg), the pharmacokinetics of epoetin were evaluated in 18 athletes. The mean elimination half-life was 42.0 hours. The total c1earance/bioavailability (F) and the volume of di stribution/F averaged 0.05 Llh/kg and 2.95 Llkg, respectively. Significant changes in ferritin (fr), soluble transferrin receptor (sTtR) and ratio (sTfRlfr) were observed after administration. A population sigmoidal asymptotic maximum expected effect concentration (emax) model has been developed to assess and quantify the relationship between the changes in sTtR, fr and the ratio sTtR/fr secondary to the repeated administration of epoetin. The mean population parameters were as follows: emax: 12.6 mg/L, 44.9 J..lg/L and 2313.5; the concentration producing 50% of the emax(C50): 18.3,18.1 and 37.7 lUlL; the sigmoidicity factor (gamma): 2.96,4.17 and 5.15 ; and ke9 (rate constant of transfer between central and effect compartment): 1.29 x 10- , 7.14 x 10-3 and 3.47 x 1O-3/h for the 3 markers, respectively. Moreover, in this paper, we propose an appropriate statistical methodology based on the measurement of sTtR, fr and sTfRlfr from blood samples to decide if the observed values of these markers could be related, at a given probability risk, to the administration of epoetin. Values greater than 10 mg/L for sTtR and 403 for sTtR/fr indicate a probable intake of epoetin. Because of the large interindividual variability of fr, it has not been possible to define a threshold value for this parameter. The sTtR seems to be the most effective marker in doping control. An increased haematocrit with concomitant changes in sTtR and sTfRlfr values seems to supply a valuable diagnostic tool that avoids falsepositive doping and allows the identification of epoetin abusers. Blood samples could be collected using capillary blood from the fingertip or earflap. These controls could be carried out during training because they are easy to perform and do not add substantially to total screening costs . At present, this is the only tool available for detecting epoetin abusers during competition.

234

Erythropoietin (Epo) is a glycoprotein hormone involved in the regulation of erythropoiesis. Epoetin (recombinant human erythropoietin) is used by some endurance athletes to increase oxygen transport and aerobic power in an attempt to improve their endurance capacity and recovery during training and competition,lI-3] Like endogenous Epo, epoetin stimulates the proliferation and differentiation of erythroid progenitor cells in bone marrow and their evolution into mature erythrocytes,l4,5]

Secondarily, stimulated erythropoiesis induces redistribution of storage iron into erythroid elements. Although on the list of banned substances issued by the medical commission of the International Olympic Committee, the use of epoetin as an ergogenic agent currently remains uncontrollable using classical analytical techniques.

In a study by Gareau et aU6], soluble transferrin receptor (sTfR) as an index of both tissue iron deficiency and expanded erythroid progenitor mass was expressed in relation to serum ferritin (fr) as a measure of body iron store, thereby generating the serum sTfR/fr ratio. The authors concluded that concomitant changes in haematocrit and in the ratio sTfR/fr was indicative of epoetin abuse.

In a recent paper,17] the pharmacokinetic profile of epoetin after repeated subcutaneous injections was investigated and a pharmacodynamic model developed to relate the effect of epoetin on reticulocytes.

The purpose of the present study was to evaluate a new methodology based on the use of population pharmacodynamic data modelling and statistical

Table I. Patient characteristics. Data are presented as mean ± SD

Bressolle et al.

decisional criteria to evaluate the probability of the abuse of doping agents by athletes. The primary objective was to define a population pharmacodynamic model suitable to assess and quantify the relationship between the changes in sTfR, fr and the ratio sTfRlfr secondary to the repeated administration of epoetin. The secondary objective of this paper was to evaluate an appropriate statistical methodology to decide ifthe observed values ofthe markers (sTfR, fr and sTfR/fr) could be related to the intake of epoetin at a given probability.

Subjects and Methods

Study Participants

Twenty-nine healthy male athletes ranging in age from 19 to 26 years were included in the study after a full physical examination. A complete blood cell count and serum chemistry analyses including total serum iron, transferrin, percentage saturation of the total iron-binding capacity, ferritin, vitamin BI2 and folic acid were performed at baseline.

None of the subjects were taking part in official competition during this period. They were randomly assigned to either active (19 subjects, treated group) or placebo (10 subjects, control group) treatment.

Comparison of the demographic characteristics summarised in table I did not reveal any differences between the two groups. The inclusion criteria were age ranging from 19 to 30 years and the absence of signs or symptoms of disease. Exclusion criteria were age « 18 years); hypertension (blood pressure: systolic> 160mm Hg, diastolic > 95mm Hg); cardiovascular disease; thrombosis;

Age (y)

Weight (kg)

Height (cm)

Haematocrit Serum iron Transferrin (gIL)

Ferritin (~g/L)

Saturation of the total iron-binding capacity (%)

(%) (~mol/L)

Controls (n = 10)

Mean 21.1 70.2 176.6 42.9 19.0 2.87 71.8 27.1

SD 1.46 9.62 5.49 1.92 2.89 0.40 36.6 6.3

Treated (n = 19)

Mean 21.8 72.3 173.6 42.9 23.9 3.01 55.2 32.2

SD 1.73 7.53 6.14 1.72 7.65 0.36 28.8 11.4

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Pharmacodynamic Model for Epoetin Monitoring

anaemia; iron, vitamin B 12 or folic acid deficiency; and thrombocytosis.

Written informed consent was obtained from all participants. The study protocol was approved by the Ethics Committee of the Assistance Publique des H6pitaux de Paris, France.

To determine the reference mean baseline values of the markers (fr, sTfR, sTfR/fr) in the largest possible population of subjects, 15 additional athletes (after a stay of 8 days at 1800 to 2000m in order to enhance erythropoiesis), were included in this study. Furthermore, to check the potential influence of gender on the variability of the markers, we analysed the values of fr, sTfR and sTfR/fr in 20 female nontraining healthy subjects during the follicular and luteal periods. These subjects were enrolled in the study according to the same inclusion and exclusion criteria as the 29 subjects included in the population study.

Study Design

Recombinant human Epo alpha (Eprex®) was administered subcutaneously at a dose of 200 units/kg each morning on days 0, 2, 4, 7 and 10. The drug was supplied by Cilag AG, France, stored protected from light at 4°C, and warmed to room temperature before administration.

The control group received isotonic saline solution according to the same experimental design.

Sampling Procedures

Blood samples were drawn from a forearm vein before each injection (days 0, 2, 4, 7 and 10) and on days 14, 17, 21 and 24. To exclude possible diurnal influences on the concentrations determined, all specimens were taken at the same time for each subject. The blood samples were left to clot at room temperature and serum was separated before storing (-20°C). At the same time, full haemograms were determined on cell counter apparatus from blood samples drawn into EDTAcoated tubes. The treatment was stopped if an increase in haematocrit greater than 5% of the initial value was observed.

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235

Analytical Procedures

A specific immunoradiometric assay based on a sandwich technique using two monoclonal anti-Epo antibodies (mouse) raised against Epo (1 25I-EPO Coatria® kit, BioMerieux, Lyon, France) was used to quantify erythropoietin)7) ELISA techniques were used to assay sTfR(8) and fr serum concentrations. (9)

Pharmacokinetic-Pharmacodynamic Analysis

A preliminary independent pharmacokinetic analysis was performed on the plasma concentrations of erythropoietin in order to estimate the individual pharmacokinetic parameters. The serum concentration-time curves were modelled according to a one-compartment open model with a zeroorder input rate constant)7] The pharmacodynamic data analysis was then performed using the changes in sTfR, fr and sTfR/fr ratio as an index of the therapeutic effect of epoetin. The individual pharmacokinetic parameters required in the calculations of the pharmacodynamic model were fixed to the ones previously estimated. The effect was modelled as an additional compartment linked to the central one by first-order kinetics.

A hysteresis plot of the serum erythropoietin concentrations against the effect, using concentrations predicted by the pharmacokinetic model for the central compartment, was constructed to assess the equilibration delay between the serum concentration and the effect site. Using the EM-fit computer program,[JO] two dynamic models were used to correlate the drug concentrations at the effect site with the effect: the sigmoidal asymptomatic maximum expected effect concentration (emax) and the exponential models. The model that provided the lowest Akaike criterion and the best residual distribution was retained.

The population pharmacodynamic parameters were estimated together with the posterior individual parameter estimates using a Bayesian methodology supplied by the EM computational approach. Preliminary analysis reve~led that the probability

Clin. Drug Invest. 1997 Sep; 14 (3)

236

distribution of the fixed effect parameters was better described by log normal than by normal distribution, and that the distribution of the residuals showed that the error variance was better described by an heteroscedastic (proportional to the squared value of the predictions) model.

Statistical Analysis

Throughout the presentation of the results, mean (± SD) data are given. The t-test was used to compare the demographic and biological characteristics of subjects between the control group and the group treated on day O. Statistical comparisons between effect parameters over the entire observation period in the control group were performed using analysis of variance.

Mean baseline (± SD) sTfR, fr and sTfR/fr values with the corresponding 99.9% standard confidence intervals (CI) were computed from basal values measured in the control and treated groups (29 subjects), and from marker values measured in the group of 15 athletes training at altitude. A criterion based on the use of t-statistics was defined to assess whether the value of a marker measured in a new individual has to be considered outside the expected baseline reference range. As these parameters are not normally distributed, this statistical test was performed after log-transformation of the data.

The criterion is based on the calculation of the minimal difference between the computed mean baseline and a new measurement value. The use of t-statistics provides a decisional criterion to assess if such a difference has to be considered as an acceptable random fluctuation around the baseline value or not, at a given probability level of 0.01.

In the case of a value statistically different from the baseline, a second stage independent statistical test was proposed to verify the consistency of the results obtained by the previous test. This second methodology was based on the measurement of the marker values in a second sample taken from the same individual 7 days after the first one. The principle of this new test was to evaluate if the change in the markers reflected the expected intra-individ-

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Bressolle et al.

ual variability or if the change was larger, due to the presence of an external agent.

An estimate of the intra-individual variability of each effect parameter in the placebo-treated subjects was determined by computing (for each subject) a mean value for sTfR, fr and sTfR/fr over the entire observation period, then the difference between this mean value and the value of the parameter at each time of measure. From these differences, a mean ± SD value with the 99.9% confidence intervals was computed for each parameter. A t-test on the log transformed data was used to determine the minimum difference that was significantly different to the mean difference value.

Results

Subjects

The pharmacokinetic-pharmacodynamic study was carried out in 18 subjects. One athlete had, at day 7, an increase in haematocrit higher than 5% of the initial value and consequently did not receive the final dose of epoetin. The demographic and biological characteristics of the subjects were not significantly different between the two groups (control vs treated). Analysis of variance indicated that no significant change (p > 0.05) occurred in serum sTfR, fr and sTfRlfr over the entire observation period in the control group. Moreover, for these parameters determined at day 0, no significant difference was observed between the control group and the treated group. Mean effect parameters at each sampling time are given in table II.

In the athletes training at altitude, after a period of 8 days at 1800 to 2000m, mean ± SD fr, sTfR and sTfRlfr values were 70.8 ± 31.6 J..Lg/L (range: 30 to 147), 6.64 ± 1.43 mg/L (range: 4.26 to 8.2) and 109.7 ± 46.9 (range: 41 to 169), respectively.

In the female non training population of subjects, during the follicular and luteal periods, the values of fr, sTfR and sTfRlfr were 61.4 ± 37.1 and 55.9 ± 33.2 J..Lg/L for fr, 4.26 ± 1.31 and 3.83 ± 1.44 mg/L for sTfR, and 95 ± 64 and 96 ± 66 for the ratio, respectively; no significant difference was seen between the two study periods.

Clin. Drug Invest. 1997 Sep; 14 (3)

Pharmacodynamic Model for Epoetin Monitoring 237

Pharmacokinetic Study ... "-CD co r-.: ... -i

The pharmacokinetic parameters of epoetin .,.; L() C'".i C') "-C') 0 +1 N N '+'I +1 +1 0 +1 +1 0 have been previously publishedJ7] The serum

CD 0> CD <6 0> C') 0 "- .,.; L() 0 r-.: ""

L() erythropoietin concentration-time profile L() L() -i L() 0> N was compatible with a one-compartment open model

N and zero-order input rate. The mean elimination 0 .,.; L() N C'".i "- ;:::. 0 .,.; "<I: L() .,.; N half-life (± SD) was 42.0 ± 34.2 hours. Total cIear-C') +1 N +1 +1 +1 C') +1 +1 "<I: ance and the volume of distribution, uncorrected ... C') N g N L()

N 0 C'".i C') 0 0 for bioavailability, were 0.05 ± 0.01 Llh/kg and L() L() -i ... C')

co 2.95 ± 2.13 Llkg, respectively. Serum erythro-C') N poietin concentrations returned to baseline 7 days L() <6 0 C') N

<6 0> ... C') "- co 0 +l <Xi N +1 after the last administration.

+l +l +1 +1 ... C'".i co "- en L() C') N

0 C'".i -i ~ <6 N 0 Pharmacodynamics ... ...

... The concentration-effect relationship was best C') CD <6

0 L() 0> C') 0; described using the sigmoid emax model; the fol-0 C\! en <6 N +1 N C') +1 +1 "-+1 +1 +1 co "" C'".i lowing models were used: ... ... ... 0 CD en N r-.: ;:::: 0

C') ~ C') ... -i 0>

• for data modelling of sTfR and sTfRlfr: C')

0 co 0> .,.;

N CD 0 <6 L() C'".i r-.: N CD emax x CeY C') 0 C') +1 +1 +1 +1 +1 +1 C') 0 0 y = eo +

CeY + CsoY 0 CD CD ... -i ~ -i ... 0 0> .,.; 0> 0 N CD C'".i "- 0>

... CD for data modelling of fr: <Xi • 0 ... ~ ~ C') r-.: L() <Xi en ... C') 0 ... +1 +1 +1 +1 +1 +1 0> N C') emax x CeY co "- ... C') <6 C') -i

CD C'".i N 0 <Xi ... Y = eo -CeY + CsoY ~ CD -i 0> CD

N

~ ... co 0 co C'".i r-.: ~ N

-i ... 0 N Q) ... 0 ... +l +1 +1 () where Ce is the serum erythropoietin concentration Q) ~

E +1 +1 +l 0> co C\! :;:; C') co r-.: ... C') c: in the effect compartment; eo is the effect concen-r-.: C'".i .,.; ·c Cl CD -i N co

~ .5 0> CD co N tration at time 0 (before epoetin administration); a. c: E N C') III ~ emax is the asymptotic maximum expected effect '" C\! co C') r-.: ~ (/) L()

~ co ~ .c ;;:; 0 N concentration; Cso is the concentration producing +1 +1 +1 J:J () ~

'" +l +1 +1 co 0 L() (5 Q) 0> ... N C'".i 0 r-.: (/) 50% of the emax ; and y is the sigmoidicity factor ., co .,.; C') r-.: ... .,.; ~ II

~ ... "- -i "- (reflecting the steepness of the curve). a:

Q) j:: Q; (/) Population pharmacodynamic parameters are E N C') "- e ~ C') "- ... .,.; co .,.; given in table III. The posterior individual fitting '" <6 co N N 0 0> :e 0. N 0 ... +1 +1 +1 2 U +1 +1 +1 CD co for each subject entering the study is presented in Q) ~ 0> 0> .,.; ~ .,.; E := r-.: L() -i L() .,.; C') 2 figure 1 for fr, figure 2 for sTfR and figure 3 for Q) 0 L() -i 0> Q)

0 (/)

sTfRlfr. An individual fitting is shown in figure 4. C/) II +1 S co .:: c: ~ c,; '" II ~ Statistical Results Q) :J' :J' 2 ~ .:. 0, .:. 0, - € "2 :J' .s

~ "tI :J' .s .~

QI 0, .!! 0, "" ~ The statistical analysis is presented in table IV. :c Q) C a: ., a: a: E ..:!, ..:!, .Q

0 j:: j:: QI j:: j:: .Q {!! F () .:: (/) (/) ~ .:: (/) (/) « The maximal acceptable difference between a

© Adis International Umited. All rights reserved. Clin. Drug Invest. 1997 Sep: 14 (3)

238 Bressolle et al.

Table III. Population pharmacodynamic parameters. Values are means (percentage coefficient of variation)

keo (h- ') eo a emax a gamma C50 (lUlL)

sTIR 1.29 . 10.3 (18.6) 5.00 (5.67) 12.6 (14.0) 2.96 (15.1) 18.3 (10.0%)

18.1 (9.59%)

37.7 (7.69%)

fr 7.14. 10.3 (13.2) 56.7 (8.38) 44.9 (9.06) 4.17 (34.6)

Ratio 3.47.10.3 (15.6) 154.3 (117.2) 2313.5 (10.6) 5.15 (12.3)

a Units: soluble transferrin receptor (sTfR), mg/L; serum ferritin (fr), ~g/L.

Abbreviations: C50 = concentration producing 50% of the emax; emax = asymptotic maximum expected effect concentration; eo = effect concentration at time 0 (before epoetin administration); gamma = sigmoidicity factor (reflecting steepness of the curve); keo = rate constant of transfer between central and effect compartment.

140

120

100

::::J a, .:; 80 §

~ E 60 :J Q;

(J)

40

20

0 0 100

o

200 300

Time (h)

400

o Subjects 1, 9, 17 o Subjects 2, 10, 18 '1 Subjects 3, 11 f:,. Subjects 4, 12 <> Subjects 5, 13 X Subjects 6, 14 )IE Subjects 7, 15 + Subjects 8, 16

500 600

Fig. 1. Pharmacokineticlpharmacodynamic analysis of serum ferritin as an index of the therapeutic effect of epoetin.

marker and its baseline value has been defined using the t-test. The marker having values lower than 10 mg/L for sTfR and 403 for sTfR/fr was considered as not different from the normal baseline. However, a larger value indicates the probable intake of epoetin. It has not been possible to define a threshold value for fr because of the great interand intra-individual variability of this parameter.

© Adis International Umited. All rights reserved.

From the results obtained in this study where athletes received only 5 doses of 200 units/kg, inducing a slight increase of haematocrit, we can see that the sTfR levels were higher than 10 mg/L on day 4 for 16 of 18 subjects, on day 7 for 17 of 18 subjects, on day 11 for 10 of 18 subjects, and on day 14 for 8 of 18 subjects after the last drug intake. No subjects had sTfR values higher than 10 mg/L

Clin. Drug Invest. 1997 Sep: 14 (3)

Pharmacodynamic Model for Epoetin Monitoring 239

20 0

[J

:::J 16 0, 0

.s '" 2 c. Q) <> 12 ~ .§ 2 '" c: ~ 8 Q)

:0 :::l "0 (f)

4

O+---_r--~--_,----~--.---~--._--~--_r--~--_,----~--._~

o 100 200 300 Time (h)

400 500 600

Fig. 2. Pharmacokineticlpharmacodynamic analysis of the soluble transferrin receptors as an index of the therapeutic effect of epoetin. See figure 1 for a key to the symbols.

.2 iii a:

4000

3200

2400

1600

800

o

o 100 200 300 Time (h)

6-o

400 500 600

Fig. 3. Pharmacokinetic/pharmacodynamic analysis of the soluble transferrin receptor/serum ferritin ratio values as an index of the therapeutic effect of epoetin. See figure 1 for a key to the symbols.

© Adis International Umited. All rights reserved. Clin. Drug Invest. 1997 Sep: 14 (3)

240

140

120

o Erythropoietin plasma concentration (lUl L) 6. Serum ferritin (IIg1L) • Soluble transferrin-receptors Ix 10] (mglL)

Bressolle et al.

'" Soluble transferrin-receptors/serum ferritin ratio [x to ']

• / ~ - - - -'- '---

100 - 1/

80

60

'T 40 , ,

•

20 '.---", -"-' ~:- ~ .. __ .. /J. ..•• _ . .. /J. •. - __ _ .. _ .... .• . _ .. - ' A'--'

/J.

o o

o 100 200 300

nme (h)

400 500 600

Fig. 4. Individual pharmacokineticlpharmacodynamic analysis.

Table IV. Statistical analysis. Data are presented as mean ± SO. Values between brackets are the 0.1% confidence intervals

Baseline values' (n = 43)

Mean intra-individual variabilities (n = 10)

fr (lig/L)

59.7 ± 28.1 [45.6 - 73.8]

7.73 ± 9.52 [4.33-11.1] (number of values: 85)

sTfR (mg/L)

5.52 ± 1.35 [4.84-6.20]

0.48 ± 0.42 [0.33-0.63] (number of values: 88)

sTlR/fr

117.2 ± 71.5 [81.3-153.1]

16.5 ± 5.34 [14.6-18.4] (number of values: 85)

a Computed from 43 subjects: 28 subjects (18 treated and 10 placebo) and 15 additional athletes training at altitude.

Abbreviations: fr = serum ferritin ; n = number of subjects; sTfR = soluble transferrin receptor.

at baseline. Similarly, the ratio levels were higher than 403 on day 4 for 18 of 18 subjects, on day 7 for 17 of 18 subjects, on day 11 for 5 of 18 subjects and on day 14 for 3 of 18 subjects, after the last drug intake.

As the aim of the doping was to significantly increase the haematocrit value, the acceleration of erythropoiesis should be higher, and one can speculate that the magnitude of marker increase should be followed for a longer time.


The maximal acceptable differences between two measurements of each parameter have been estimated at 1.6 mg/L for sTfR and 30 for sTfR/fr. These values were used in the second stage statistical approach to confirm the results found by the first test. Between days 7 and 14 after the last drug intake, in 14 of 18 subjects the change of sTfR was > 1.6 mg/L and for all subjects the change of the ratio was> 30. Such variations were not observed in the control group.

Clln. Drug Invest. 1997 Sep; 14 (3)

Pharmacodynamic Model for Epoetin Monitoring

Discussion

In the present paper, a population pharmacodynamic model is described to assess and quantify the relationship between the changes in sTfR, fr and in the ratio sTfRlfr secondary to the repeated administration of epoetin. The good agreement found between the predicted values of the markers and the fluctuations of the plasma erythropoietin concentrations indicated that the pharmacodynamic relationship based on a sigmoidal emax

model can be satisfactorily used to relate the changes observed in the markers to epoetin administration.

In addition, the use of the present modelling approach allows one to forecast expected kinetic profiles of the markers given any predefined epoetin dosage regimen [for example: daily administration of low dosages (50 mg/kg/ day) of the recombinant hormone].

Appropriate statistical methodology has been proposed as decisional criteria to assess the possible dependency between the observed values of the markers (sTfR, fr and sTfRlfr) and the intake of epoetin. In order to determine the reference mean baseline values of these markers in the largest possible population of subjects, we decided to consider a reference population constituted by the values measured in the athletes entered in the present study together with those obtained in 15 athletes training at altitude in order to enhance erythropoiesis.

Threshold values of 10 mg/L for sTfR and of 403 for sTfR/fr have been defined using t-test statistics. Values of the markers greater than these reference limits indicate a probable intake of epoetin. Because of the great interindividual variability in fr, it was not possible to define a threshold value for this parameter. From these results, sTfR seems to be the most effective marker.

Although on the list of banned substances issued by the medical commission of the International Olympic Committee, the nonmedical use of epoetin currently remains uncontrollable with nonreliable analytical techniques available to detect its use as an ergogenic agent. Thus, in order to verify


241

the consistency of the results, in the case of a value statistically different from the baseline, a second test based on the measurement of the marker values in a second sample taken from the same individual was performed. Taking into account the mean elimination half-life of epoetin (42 hours) and the slow decrease of the markers, the interval of 7 days seems reasonable. This new test evaluates if the difference in the markers reflects just the intraindividual variability or if the difference is larger than the expected value, due to the presence of an external agent.

To check the potential influence of gender on the variability of the markers, we analysed the baseline values of fr, sTfR and sTfRlfr in a female nontraining population of healthy subjects during the follicular and luteal periods. No significant difference occurred between the two study periods.

Another source of variability in the marker concentrations measured in athletes could be the type of physical exercise. However, it does not seem to be associated with increased sTfR/fr serum values per se,[111 thus precluding a false-positive epoetin conviction from blood sampled at the competition site.

In the absence of erythropoietin, plasma concentrations greater than the baseline value or in the absence of detectable epoetin with the appropriate analytical method,[l21 the decisional rule discussed above requires the initial measurement of the sTfR and the sTfR/fr ratio from a blood sample. Three possible situations can occur: • The marker values are within the reference con

fidence intervals. In this case we do not have evidence of the presence of doping factors.

• The marker values are outside the reference confidence intervals but lower than the threshold values. In this case we can consider these values as highly improbable « 111000) in ~ normal population of athletes . Consequently, a second blood sample should be collected I week later to proceed to the second stage statistical check. If the changes of the marker values in the two samples are greater than the reference


242

thresholds the presence of doping factors is highly probable.

• The marker values are greater than the reference thresholds; in this case we can presume with a low probability of error (lower than 1 %) the presence of a doping factor. However, to confirm this finding we recommend a second blood sample be obtained I week later and then to proceed to the second stage statistical verification. These blood samples could be collected using capillary blood from the fingertip or earflap.

Conclusion

In conclusion, the purpose of the present study was to mimic a situation of epoetin abuse in healthy athletes. The first objective was to find a 'reasonable' model suitable to describe changes in the sTfR, fr and sTfRlfr ratio consequent to the intake of epoetin. The second objective was to propose a statistical methodology to detect epoetin misuse. An increased haematocrit with concomitant changes in sTfR and sTfR/fr values seems to provide a valuable diagnostic tool that avoids falsepositive doping and allows the identification of epoetin abusers.[6]

These controls could be carried out during training because they are easy to perform and do not add substantially to total screening costs. At present, it constitutes the only available tool to detect epoetin abusers during competition.

The main clinical implication of this study is the provision of a tool to identify risk factors in individuals during training and competition due to the nonmedical use of epoetin. Indeed, athletes are particularly susceptible to the effects of hyperviscosity due to the biological effects that last for the life of the RBC (up to 120 days). Effects may be exacerbated during prolonged endurance events when dehydration increases the hyperviscosity. The athletes may also experience a thromboem-

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Bresso/le et al.

bolic hypoxic event because of 'sludging' of the blood.

Acknowledgements This work was supported by a grant from the French

Ministry of Sport. The authors gratefully acknowledge Janssen-Ortho Inc. for providing epoetin. The authors thank Professor J .P. Escande, president of the French antidoping commission , for his confidence and his support, and Dr A. Duvallet for the realisation of the clinical part of the study.

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4. Koury MJ, Bondurant Me. The molecular mechanism of erythropoietin action. Eur J Biochem 1992; 210: 649-63

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6. Gareau R, Audran M, Baynes RD. et al. Erythropoietin abuse in athletes . Nature 1996; 380: 113

7. Souillard A. Audran M, Bressolle F, et al. Recombinant human erythropoietin and pharmacodynamic parameters in athletes. Interest of blood sampling for doping control. Br J Clin Pharmacol 1996; 42: 355-60

8. Flowers CH, Skikne BS. Covell AM, et al. The clinical measurement of serum transferrin receptor. J Lab Clin Med 1989; 114: 368-77

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Correspondence and reprints: Prof. F. Bressolle, Laboratoire de Pharmacocinetique, Faculte de Pharmacie, 15 Ave Ch. Flahault, Universite Montpellier I, 34060 Montpellier Cedex 01, France.


Population Pharmacodynamics for Monitoring Epoetin in Athletes

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