Rise in RBC aggregability and concomitant decrease in blood pressure 10 days after injection of the long acting erythropoietin analogue methoxy polyethylene glycol-epoetin-β (MIRCERA ® )

1 Introduction

Erythropoietin, a hormone released by the kidney, is a major regulator of blood viscosity [30]. It experimentally increases hematocrit [19] and has an effect on red cell deformability and aggregation.

Its long lasting action analogue methoxy polyethylene glycol-epoetin-β (MIRCERA^®; F. Hoffmann-La Roche, Basel, Switzerland) is defined as a continuous erythropoietin receptor activator (C.E.R.A.) with a long half-life allowing once-monthly dosing [32]. Its efficacy and safety have been largely studied [2, 25–27].

MIRCERA^® is likely to be a candidate for modern sports doping. We took the opportunity of a study aiming at developing a detection of recent MIRCERA^® injection in the context of doping detection to assess the effects of this EPO analogue on RBC aggregation.

2 Subjects and methods

3 Results

Figure 1 shows the changes in the 2 aggregation indexes “M” and “M1”. Both increase (p < 0.01) and we can evidence a dramatic (+67%) increase in RBC aggregation index “M” on day 10 (from 9.49±1.01 to 17.66±1.8 p < 0.01).

Table 2 shows the main hemorheologic changes after injection. There was no significant changes in hematocrit. All other changes were significant with ANOVA (p < 0.01). Red cell aggregation “M” was higher at day 10. The rise was not significant with the red cell aggregation “M1” despite marked increases in most individuals due to a decrease in 2 subjects.

On the whole all measurements of blood pressure displayed a decrease over the 24 days of follow-up. Systolic blood pressure was lower on days 7 and 10 (p < 0.001). Diastolic blood pressure was lower on days 7, 10 and 17. Mean blood pressure was lower on days 4 (p < 0.02), 10 (p < 0.001), 17 (p < 0.005) and 24 (p < 0.05). Pulse blood pressure was lower on day 10 (p < 0.05). Parallel evolutions of RBC aggregation “M” and blood pressure are shown on Figs. 2 and 3.

Correlations between this decrease in blood pressure and “M” did not reach significance but pulse pressure was positively correlated to “M” (r = 0.743 p < 0.05), as shown on Fig. 4.

There were also changes that are not shown here and that will be presented in a separate paper. After injection a decrease in mean corpuscular volume at day 2 (p < 0.01) and day 10 (p < 0.02), a rise in reticulocyte count (p < 0.001) between day 4 and day 17 and a decrease in ferritin a day 5 (p < 0.05). Hemoglobin decreased at day 4 (p < 0.005).

4 Discussion

These data show that a single injection of the long acting erythropoietin analogue MIRCERA^® increases RBC aggregation parallel to a decrease in blood pressure, while it does not change systemic hematocrit. Correlations between this decrease in blood pressure and the increase in aggregation did not reach significance. The time course of these various changes is not the same. It can be seen on Figs. 2 and 3 that the decrease in mean, systolic and diastolic blood pressure becomes significant on day 4, before the rise in RBC aggregation, while pulse blood pressure decreases after the peak of RBC aggregation, on day 17. On day 10, during the peak of RBC aggregation, pulse blood pressure is not different from baseline but is positively correlated to RBC aggregation.

There is a large body of literature showing that long-term EPO administration increases blood pressure [33]. Several mechanisms have been described. Erythropoietin potentiates vascular responsiveness to norepinephrine [10] and decreases nitric oxide synthase expression in the endothelial cell [34]. EPO also increases vasoconstrictor tone via endothelin-1 and constrictor prostanoids [3]. There are few studies on long term effects of EPO but in normotensive subjects EPO-induced increases in Hct to approximately 49% were reported to be associated with a significant increase in resting mean arterial pressure of +6 mmHg when measured by intra-arterial catheter [17] and with a significantly increased response of blood pressure during exercise [1].

According to Krapf [12] EPO-induced increase in blood pressure is due to its vasoconstrictive effect, caused both by decreased systemic NO production and resistance to NO vasodilation. However this effect is modulated by an ET-1/NO-induced hypotensive effect mediated by inhibition of collecting duct sodium and water reabsorption. This latter effect can in turn be counteracted by EPO which induces sodium retention by a renin-angiotensin II-mediated mechanism at the kidney level [4]. For some authors however, EPO-induced decrease in nitric oxide synthase expression does not explain EPO-induced hypertension [7].

Interestingly, effects of EPO on blood pressure are probably not due to its effects on hematocrit, since a rise in blood pressure is observed after EPO even if hematocrit is kept constant [33] and is not correlated to changes in red blood cell mass and viscosity [12]. A striking demonstration of this dissociation of effects of EPO on blood pressure and hematocrit is given by the fact that a similar increase in hematocrit is associated with increased blood pressure if induced by EPO and decreased blood pressure if induced by transfusion [5, 17].

In contrast with the chronic effect of EPO which is an increase in blood pressure, a single intravenous administration of EPO even at a high-dose (400 and 5,000 U/kg), estimated to yield plasma concentrations comparable with those employed in vitro, does not increase blood pressure and does not modify the blood pressure response to angiotensin II [33].

EPO administration to top endurance athletes is one of the most popular (and apparently successful) doping procedure. In the Tour de France it has been employed on a regular basis by the most famous winning teams [24]. MIRCERA^® is a logic candidate for the same use. However, it is clear that the effects of MIRCERA^® are not totally similar to those of the native molecule rh-EPO. This may also apply to its hemorheologic effects.

The study of effects of rh-EPO on red cell deformability gave conflictual results. While Taylor reports no change in red cell deformability [31], Schmidt [29] reports a remarkable increase in RBC deformability which is also observed by Delamaire [8]. This fluidification of red cells prevented in part hyperviscosity despite the rise in hematocrit. The recent report that EPO activates nitric oxide synthase in erythrocytes [21] may provide an explanation of this effect. However Linde and coworkers [14] assessed erythrocyte fluidity at Hct 55% in a 7.4 saline-phosphate buffer at a shear rate of 1/s, in rh-EPO treated patients, and found a more complex picture. There was a decrease in this parameter between weeks 10 and 24 during the rapid production of erythrocytes, followed by a return to normal when the EPO doses were reduced after the target Hct was reached and the production rate diminished. They hypothesized that this EPO-related rigidification of red cells could play an important role in this blood pressure increase.

Concerning red cell aggregation, Delamaire [8] found that it was increased in hemodialyzed patients and EPO treatment resulted in a further increase. By contrast Schmidt [29] found no change in RBC aggregation after EPO.

Clearly, MIRCERA^® has a strong, transient effect on RBC aggregation which is markedly increased. In patients receiving erythropoietin analogues (ESAs), there has been reports of cardiovascular and thrombotic events such as myocardial ischaemia and infarction, cerebrovascular haemorrhage and infarction, transient ischaemic attacks, deep venous thrombosis, arterial thrombosis, pulmonary emboli, retinal thrombosis and haemodialysis graft occlusion [23]. Concerning blood pressure, it has been reported to rise in some patients during treatment of anemia with MIRCERA^® as withother ESAs.

In the French multicenter, longitudinal, observational study OCEANE adverse effects related to C.E.R.A. were observed in less than 5% of patients over 1 year of follow-up and led to modification or discontinuation of treatment in 2% [9]. Data of this series suggest therefore that this EPO analogue is relatively safe. In this respect it is interesting to notice that a single injection of MIRCERA^® does not increase hematocrit, while this increase is found withEPO [19].

As reminded above, the link between hemorheologic alterations and blood pressure after EPO is unclear. Moreover, classically, high blood pressure is associated with increased blood viscosity and in particular erythrocyte hyperaggregation [6, 35] and this study evidences the opposite picture.

5 Conclusions

In conclusion, this study shows two effects of a single injection of MIRCERA^®: a decrease in blood pressure and a rise in red cell aggregation. The association of these two effects is apparently paradoxical and further studies are needed in order to elucidate this mechanism.