Influence of Treatment with Continuous Erythropoietin Receptor Activator on Markers Of

Abstract

Aim of the study: We investigated in a prospective, non randomized, controlled study a possible direct effect of C.E.R.A. on markers of endothelial dysfunction, inflammation and malnutrition in chronic kidney disease

(CKD) patients.

Materials and Methods: Routine parameters and markers of inflammation and endothelial dysfunction were dosed before (T0), after three (T1) and six months (T2) of treatment with C.E.R.A. (mean dose 98,33±4,6 mcg month), and again after three (T3) and six (T4) months of α-darboepoetin (mean dose 27,30±3,60

µg/week) in 60 stage 3 and 4 CKD patients on stable anemia correction previously achieved with α- darboepoetin.

Results: At 3 (T1) and 6 (T2) months we observed a significant reduction of sHcy (p=0,0011 for T0 vs T1 and

0,001 for T0 vs T2) and hs-CRP (p<0,0001 for both T0 vs T1 and T0 vsT2). At 6 months (T2) a significant decrease of fibrinogen (p=0,0066) also occurred. After three-months of retreatment with α-darboepoetin sHcy, hs-CRP and fibrinogen returned comparable to baseline (p=non significant). Follow up was thus interrupted at three months (T3).Hb remained stable during the whole investigation period. Other markers tested were not significantly affected by Erythropoiesis Stimulating Agents (E.S.A.s) therapy.

Conclusions: Our results suggest that treatment with C.E.R.A. may influence some markers of inflammation and endothelial dysfunction usually related to cardiovascular risk in renal patients, with a mechanism that seems to be unrelated to anemia correction.

Key-Words: Renal Anemia- Continuous erythropoietin receptor activator-Inflammatory markers-

Homocysteine- Chronic kidney disease.

1 Running title: C.E.R.A. and cardiovascular risk markers in CKD patients

2 Introduction

In patients with chronic renal failure anemia is a multifactorial disorder primarily related to erythropoietin deficiency. Iron deficiency, malnutrition and a chronic inflammatory state are often present as contributing causes and may be responsible for the resistance to treatment with recombinant human erythropoietin that is present in some cases. A recent investigation showed that monthly administration of metoxy-polyethilen- glycol-β-epoetin (Continuous Erythropoietin Receptors Activator, C.E.R.A.) is able to achieve and maintain target plasma Hb better than monthly α-darboepoetin [1].

C.E.R.A may thus provide stable correction of anemia and stable maintenance of target Hb at extended administration intervals in uremic patients.

A reduction of oxidative stress, endothelial dysfunction and chronic inflammation related to the correction of anemia has been reported in chronic kidney disease (CKD) patients after administration of Erythropoiesis

Stimulating Agents (E.S.A.s) [2].

Aim of our study was otherwise to detect a possible direct effect of C.E.R.A. on markers of endothelial dysfunction, inflammation and malnutrition in CKD patients under stable treatment with E.S.A.s, independently from the correction of anemia.

Materials and Methods

Study design and population.

It this prospective, non randomized, controlled trial study, conducted from January 2009 to January 2010, target population was CKD (stage III-IV, according to K-DOQI guidelines) [3] adult outpatients attending our clinic, treated since at least six consecutive months with subcutaneous α-darboepoetin for anemia and with stable hemoglobin (12±0.5 g/dl) since three months at least. Exclusion criteria were: blood transfusions in the previous six months, acute or chronic bleeding, severe liver diseases, neoplasia, haemolytic disease, hemoglobinopathies, ferritin <90 ng/ml, either myocardial infarction, stroke or unstable coronary artery disease in the previous three months, congestive hearth failure (NYHA class IV), gastrointestinal malabsorption, chronic inflammatory diseases, acute kidney injury (AKI). Patients could be withdrawn at the discretion of the investigators for concurrent illness, adverse events or violation of protocol.

In all patients E.S.A. therapy was switched from α-darboepoetin to C.E.R.A. for six months and then switched again to α-darboepoetin.

At the beginning of the study all patients received a single dose of subcutaneous C.E.R.A. (≤150 mcg/month), repeated monthly for six months. After six months (T2), all patients returned to their previous treatment with subcutaneous α-darboepoetin (≤30 mcg/week).

3 Thus, in our case-control study patients were used as controls of themselves.

High-sensitive C-Reactive Protein (Hs-CRP), α1-acid-glycoprotein, serum homocysteine (sHcy), total, HDL and LDL-cholesterol, Hb, albumin and pre-albumin, fibrinogen, serum iron and ferritin were dosed at baseline

(T0), after three (T1) and six (T2) months of treatment with C.E.R.A. and again after three months of retreatment with α-darboepoetin (T3).

Adverse events (AEs) were recorded throughout the study, including hypertension and any patients complaint, whether related to E.S.A.s therapy or not.

Only patients previously treated with a single kind of E.S.A. (α–darboepoetin) were enrolled into the study in order avoid potential confounding factors in the analysis of results. The correct doses of C.E.R.A were calculated based on the α-darboepoetin dosages, according to a conversion table previously described [4].

The study protocol complied with the declaration of Helsinki and was appointed by the Ethical Committee of our institution. A written fully-informed consent was obtained by all participants before enrollment into the study.

Laboratory Parameters

Hs-CRP, α1-acid-glycoprotein, sHcy, total, HDL and LDL-cholesterol, Hb, albumin and pre-albumin, fibrinogen, serum iron and ferritin, were then dosed before (T0) and after three (T1) and six (T2) months of therapy with monthly subcutaneous C.E.R.A and again after three months of re-treatment with α- darboepoetin (T3).

Blood samples were obtained from an antecubital vein. For sHcy determinations, blood samples were collected into K3EDTA vacutainer tubes as well, put on ice and immediately centrifuged and stored at -20 °C until analysis. Plasma total sHcy was assayed by a fully automated HPLC method using reversed-phase separation and fluorescence detection as reported previously [5].

Red blood cell count (RBCC) and plasma Hb were determined with an automated haematology analyzer

Sysmex XE-2100 (Japan). Fibrinogen was quantified by a phototurbidimetric method (Ca 7000 Sysmex,

Japan).

All routine laboratory measurements (serum creatinine, albumin, total, HDL and LDL-cholesterol, albumin, serum iron and ferritin) were determined by an automated method using the Modular P800-Roche apparatus.

Supplementary analysis on clinical and inflammation markers (e.g. α1-acid-glycoprotein, β-2-microglobulin, pre-albumin, hs-CRP) were performed using a nephelometric method (BN IITM BNHTM nephelometer,

Siemens Healthcare Diagnostics, Milano, Italy).

4 Baseline Body Mass Index (BMI) was calculated as the fasting weight to squared-height ratio. Statistical analysis.

All results are expressed as mean±SD. Two-tailed paired sample T test was employed for analysis of results; p-values<0.05 were considered statistically significant. Data were elaborated through the MedCalc Statistical

Software (MedCalc Software, 9030 Mariakerke, Belgium).

Results

Twenty-two out of the initial 82 eligible patients were not enrolled into the study, due to the following exclusion criteria: iron deficiency (n=11), recent change in α-darboepoetin dose (n=5), lack of consent (n=1), acute or chronic bleeding (n=2) and recent myocardial infarction (n=3). Sixty patients (31 male, 29 female) represented the study population. All patients were Caucasian. Demographic and clinical features at baseline

(T0) are summarized in Table 1.

The mean dose of α-darboepoetin and C.E.R.A. were 27,30±3,60 mcg weekly and 98,33±4,6 mcg monthly, respectively.

Both E.S.A.s (α-darboepoetin and C.E.R.A.) treatments resulted in the maintenance of RBCC.

In particular, plasma hemoglobin remained stable between 11 and 12 g/dl during the whole investigation period with no statistical differences between the two treatments (Table 2 and Figure 1). Also serum iron, ferritin and transferrin remained constant during the entire study, confirming effective control of renal anemia.

Among markers of endothelial dysfunction, inflammation and malnutrition evaluated, sHcy and hs-CRP showed a significant reduction following treatment with C.E.R.A. (T0 27,46±8,42, T1 25,09±8,12 and T2

23,52±8,09 μmol/L; p=0,0011 T0 vs. T1 and 0,001 T0 vs. T2, T0 10,28±12,99, T1 6,48±7,99 and T2

4.07±4.47 mg/dl; p<0,0001 T0 vs. T1 and T0 vs. T2 for sHcy and hs-CRP, respectively). Fibrinogen significantly decreased at T2 only (T0 413,83±114,30 and T2 354,50±97,48 mg/dl, p=0,0066).

Remarkably, switching again patients to α-darboepoetin resulted in prompt return to baseline (T0) values of either sHcy, hs-CRP and fibrinogen at T3 (Table 2 and Figure 1). Follow up was thus interrupted at three months. Other investigated inflammatory and nutrition markers were not significantly affected by both treatments (Table 2).

No AEs were observed during the whole study period.

Discussion

Chronic kidney disease is a well established cardiovascular risk factor: mortality rate for cardiovascular disease is 10 to 20-fold greater in uremic patients compared to the general population, even after correction to age, sex, race and the presence of diabetes.

5 Non conventional risk factors may contribute to this bad outcome such as increased oxidative stress, ADMA, chronic inflammatory state and hyperhomocysteinemia, among others [6].

Uraemia has been considered a chronic inflammatory state for the last few years. Thus, even in the absence of overt inflammation or infection, many patients with renal failure show increased levels of acute phase proteins, such as CRP, ferritin and fibrinogen. Consequently, CKD patients showed low serum albumin, enhanced lipid peroxidation and increased oxidative stress. CKD patients with high CRP or low serum albumin show a significantly poor outcome compared to those with normal levels of inflammatory parameters

[7-9].

It has been recognized that about 30-50% of predialysis, hemodialysis (HD) and peritoneal dialysis (PD) patients show serologic evidence of an activated inflammatory response with elevated serum CRP (i.e.>8-

10 mg/L) [10]. Among non conventional cardiovascular risk factors, hyperhomocysteinemia has been associated with atherosclerosis, arterial thrombosis and vascular endothelial dysfunction [5,11]. Hyper- homocysteinemia is observed in about 90% of dialysis patients.

Recombinant human EPO (rHuEPO) is well known to correct anaemia in patients with chronic renal failure

[12]. However, several studies showed the existence of non-haematopoietic effects of EPO, that may be related to the present of EPO-receptor far from the bone-marrow, such as in cardiomyocites, renal and neuronal cells [13]. E.S.A.s have been reported to be cytoprotective for these cells [14]. EPO showed also pleiotropic protective effects in non renal animal models of ischemia-reperfusion injury [15-21]. In animal models EPO can reduce renal dysfunction and injury caused by oxidative stress, hypoxia and haemorrhagic shock, generally by reducing caspase activation and apoptosis [22-29]. Moreover, EPO significantly reduced the lung inflammatory cell infiltration, and capillary endothelial cell injury in hyperoxic lung injury in newborn rats. The mechanism may be related to the inhibition of MCP-1 and CINC-1 gene expression by EPO [30].

Moreover, EPO inhibits apoptosis in a variety of tissues and promotes endothelial progenitor cell proliferation and differentiation. Finally, it is now well known that EPO has renal and cardioprotective effects

[31].Additionally, an increased in cardiac systolic function as been observed in patients with chronic hearth failure treated with EPO [12].

The long-acting methoxy-polyethylene-glycol-derivate, C.E.R.A., has been introduced only recently in the treatment of CKD associated anemia. This drug is characterized by a slower association and a faster dissociation from the cellular epoetin receptor and thus by a longer half-life compared to other E.S.A.s.

Hence C.E.R.A. allows monthly administration [32]. C.E.R.A. differs from native human erythropoietin and other E.S.A.s for the integration of a large polymeric chain that is linked via amide bonds between amino-

6 groups and methoxy-polyethylene glycol-succinimidil butanoic acid. The resulting molecule has a weight of approximately 60.000D [33].

Our results suggest that treatment with C.E.R.A. may interfere with some markers of inflammation (i.e. hs-

CRP and fibrinogen) and of endothelial dysfunction (Hcy) that are usually associated with an increased cardiovascular risk in CKD patient, through mechanisms that appear unrelated to the correction of anemia, as plasma Hb and iron status remained stable for the whole duration of the study. The significant decrease in fibrinogen observed only after six months of treatment, may indicate the need of a prolonged administration of C.E.R.A. in order to obtain the anti-inflammatory effect. This possible enhanced anti-inflammatory effect of

C.E.R.A. compared to other E.S.A.s might be related to the very particular secondary and tertiary configuration of the molecule associated to the involvement of epoetin receptors far from the bone marrow

[34]. This is the first study that evaluates the possible effect of a conversion from α-darboepoetin to C.E.R.A. on inflammatory and endothelial dysfunction parameters in non dialysis CKD patients.

Our results suggest that treatment with monthly doses of C.E.R.A. may determine an improvement in some laboratory parameters related to cardiovascular risk.

Finally, our present investigation may prepare a hopeful ground for larger clinical studies aimed to determine whether the improvement of inflammatory and endothelial dysfunction has an impact on clinical outcome in renal patients.

Acknowledgments.

The Authors are indebted with Prof. Massimo Taccone-Gallucci for his skilful suggestions

Disclosure.

All the authors declared no competing interests.

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Gender (male/female) 31 / 29

Age (years) 74,6 ± 5,6*

Weight (kg) 74,0 ± 19,4*

Height (cm) 162,5 ± 8,3* 10 Body Mass Index 30,0 ± 8,5*

GFR [CKD – EPI formula] (ml/min) 23,1 ± 12,8*

Hypertension, n (%) 39 (65%)

Diabetes, n (%) 15 (25%)

Smoke, n (%) 19 (31,6%)

Dyslipidemia, n (%) 45 (75%)

Cause of Chronic Kidney Disease, n (%) Nephroangiosclerosis 35 (58,4%) Glomerulonephritis 20 (33,3%) Polycystic kidney disease 3 (5,0%) Interstizial nephritis - pyelonephritis 2 (3,3%)

Table 1. Demographic and clinical characteristic of study patients at baseline (n=60). *Data are expressed as mean ± standard deviation.

11 T0 T1 T2 T3

mean±SD mean±SD mean±SD mean±SD Hb (g/dl) 11,09±1,18 11,19±1,23 11,03±1,05 11,08±1,06 Serum Iron(mg/dl) 63,22±24,56 68,06±28,72 59,11±17,21 62,03±18,05 Ferritin (ng/ml) 96,16±84,20 102,87±76,85 83,28±66,25 86,18±69,31 Transferrin(mg/dl) 215,8±15,2 216,7±18,1 217,8±17,1 219,9±18,3 Hcy (mmol/l) 27,46±8,42 25,09±8,12* 23,52±8,09* 26,8±8,35 Hs-CRP (mg/dl) 10,28±12,99 6,48±7,99** 4,07±4,47** 8,45±7,91 α1-acid glicoprotein (g/l) 1,12±0,32 1,12±0,31 1,50±2,03 1,13±0,31 Albumin (g/l) 4,16±0,40 4,11±0,53 4,11±0,40 4,13±0,38 Pre-albumin (g/l) 0,27±0,07 0,26±0,08 0,26±0,08 0,27±0,05 Fibrinogen (mg/dl) 413,83±114,30 390,61±100,30 354,50±97,48a* 391,12±103,45 Total Cholesterol (mg/dl) 166,44±54,90 167,72±38,36 169,00±33,89 169,45±35,85 HDL-Cholesterol (mg/dl) 45,17±11,53 47,00±10,41 47,11±12,13 47,12±12,15 LDL-Cholesterol (mg/dl) 94,83±49,98 95,72±33,61 98,94±7,23 99,01±7,51

Table 2. Laboratory findings at baseline (T0), after three (T1) and six months (T2) of treatment with subcutaneous C.E.R.A. and after returning from three months (T3) to therapy with α-darbepoetin. *p<0,05 as compared with baseline. **p<0,001 as compared with baseline.

12 Table 2. Laboratory findings at baseline (T0), after three (T1) and six months (T2) of treatment with subcutaneous C.E.R.A. and after returning from three months (T3) to therapy with α-darbepoetin. *p<0,05 as compared with baseline. **p<0,001 as compared with baseline.

Figure 1. Graphical representation of the values, expressed in logarithmic scale, of Hcy, hs-CRP and Fibrinogen at baseline (T0), after three (T1) and six months (T2) of treatment with subcutaneous C.E.R.A. and after returning from three months (T3) to therapy with α-darbepoetin. (This figure is drawn from the data shown in Table 2). *p<0,05 as compared with baseline. **p<0,001 as compared with baseline.