Acta Neurologica Belgica

, Volume 114, Issue 4, pp 273–278

Efficacy of combination therapy with erythropoietin and methylprednisolone in clinical recovery of severe relapse in multiple sclerosis

Authors

  • Fatemeh Najmi Varzaneh
    • Sina MS Research Center, Neuroscience InstituteSina Hospital, Tehran University of Medical Sciences
  • Farnaz Najmi Varzaneh
    • Sina MS Research Center, Neuroscience InstituteSina Hospital, Tehran University of Medical Sciences
    • Research Center for ImmunodeficienciesChildren’s Medical Center, Tehran University of Medical Sciences
  • Amir Reza Azimi
    • Sina MS Research Center, Neuroscience InstituteSina Hospital, Tehran University of Medical Sciences
  • Nima Rezaei
    • Research Center for ImmunodeficienciesChildren’s Medical Center, Tehran University of Medical Sciences
    • Molecular Immunology Research CenterSchool of Medicine, Tehran University of Medical Sciences
    • Department of ImmunologySchool of Medicine, Tehran University of Medical Sciences
    • Sina MS Research Center, Neuroscience InstituteSina Hospital, Tehran University of Medical Sciences
Original Article

DOI: 10.1007/s13760-014-0286-y

Cite this article as:
Najmi Varzaneh, F., Najmi Varzaneh, F., Azimi, A.R. et al. Acta Neurol Belg (2014) 114: 273. doi:10.1007/s13760-014-0286-y

Abstract

Multiple sclerosis (MS) is a multifaceted disease in which genetic and environmental factors are involved. Although neurodegeneration aspect of MS has major influence in patients’ disability, none of the available treatments have been shown to obviously reduce neurodegeneration. Recently, the role of Erythropoietin (EPO) as a neuroprotective and anti-inflammatory agent has been attracted tremendous interest. In the present randomized double-blind pilot study, we combined EPO with methylprednisolone (MPred) in severe motor relapsing–remitting MS (RR-MS) patients to target both inflammatory and neurodegenerative aspects of disease. Twenty patients with RR-MS in relapse phase were randomized into two groups. The case group (10 patients) received intravenous MPred (1,000 mg/24 h) and intravenous EPO (20,000 U/24 h) for five consecutive days, and the control group (10 patients) received just MPred at the same dose as the case group, and a placebo. Both groups were followed for 3 months by ambulatory index (AI), Expanded Disability Status Scale (EDSS) and by magnetic resonance imaging (MRI) parameters. Improvement in maximal distance walking, reflected by reduction in AI and EDSS, was observed in EPO group after second month and continued after 3 months. Furthermore, MRI data analysis showed significant reduction in the number of T2WI lesions in EPO group without any significant change in contrast enhancing and black hole lesions. There was no major side effect in EPO group. The results of this first therapeutic pilot trial in RR-MS patients are promising, but need to be validated in larger trials.

Keywords

Multiple sclerosisErythropoietinMethylprednisoloneTreatment

Introduction

Multiple sclerosis (MS) is an autoimmune central nervous system (CNS) disease, characterized by inflammation, demyelination, axonal and neuronal degeneration. It has been increasingly apparent that neurodegenerative aspect of MS has the most important impact on developing the irreversible neurological deficits and long-term disability in MS patient [15].

Steroid as a standard therapy for acute relapse in MS has been shown to have negative effect on neuronal survival and apoptotic properties in axons [6, 7].

In addition, current MS therapies in chronic phase of the disease have been shown to be efficacious in terms of modifying the clinical disease course by their anti-inflammatory, immunomodulatory and immunosuppressive properties; however, any obvious neuroprotective properties have been identified for none of them [8, 9].

Methylprednisolone (MPred), as a steroid which promotes neuronal cell death by inhibition of mitogen-activated protein kinase (MAPK) phosphorylation, an enzyme in endogenous neuroprotective pathway, has shown to produce rapid improvement in MS [7]. Among potential agents for neuroprotection in MS, erythropoietin (EPO) seems to be a very promising compound [10].

EPO, a hematopoietic growth factor, has shown neuroprotective properties in both in vitro and in vivo models of brain diseases [1115]. EPO which contributes in several pathophysiologic mechanisms involved in MS [10] has been demonstrated to promote axonal repair and neurogenesis [1621]. In addition, EPO diminishes inflammatory cell infiltration and demyelination [22, 23] and alters proinflammatory cytokine expression [2426]. Furthermore, it has been shown that EPO enhances oligodendrocyte progenitor cell proliferation and prevents from brain atrophy [19, 27].

In the present pilot study, we investigated whether combination of MPred and EPO acts synergistically during attacks of MS patients and thereby prevents axonal degeneration. The main study aims were: (1) to investigate safety of short-term high-dose EPO treatment in relapsing–remitting MS (RR-MS) as primary endpoint and (2) to accumulate first evidence of EPO potential efficacy with regard to a variety of clinical and radiologic parameters, including Ambulatory index (AI), Expanded Disability Status Scale (EDSS) [28], T2, contrast enhancing and black hole lesions in magnetic resonance imaging (MRI) as secondary endpoint. Furthermore, this pilot should deliver information in determining outcome parameters and estimating the number of patients necessary for a future phase II trial.

Patients and methods

A total of twenty patients suffering from RR-MS were investigated in this clinical trial pilot study (trial registration number: IRCT2013081914407N1) after approval of the study by the Ethics Committee of Tehran University of Medical Sciences. After comprehensive information to patients, written informed consent was obtained. The inclusion criteria were: age between 18 and 45 years, definite diagnosis of RR-MS, according to the McDonald criteria by a qualified neurologist [29], duration of symptoms between 24 and 72 h, motor force 3/5 or less at presentation. The study population was restricted to patients with severe motor relapse because motor components could be measured more precisely than sensory or cerebellar attacks and also due to little evidence about safety and tolerability of EPO in MS patients, only severe motor relapse patients have been selected based on the ethical committee advised.

Exclusion criteria were: pregnancy, breastfeeding, history of vasculitis or collagen vascular disease, history of diabetes mellitus, hypertension, hyperlipidemia, heavy cigarette smoking, ischemic heart disease, myocardial infarction, cerebrovascular accident (CVA), thromboembolic events, history of any cancer (treated or untreated), presence of polycythemia (hematocrit above 50 % in males and above 45 % in females), presence of chronic renal failure (CRF), use of contraceptive medication and/or iron substitution, family history of ischemic heart disease and/or CVA before 45 years in first-degree relatives.

The selected patients were randomly divided into two different treatment groups. Patients, EDSS rater, and statisticians were all unaware of random allocation and treatment arms. A brain MRI was performed, using a 1.5-T unit (Philips Gyroscan Intera, Amsterdam, and The Netherlands). The MR sequences included axial and coronal (FSE) T2-weighted (TR/TE: 3631-4000/100-117), axial FLAIR (TR/TE/TI: 6000-6660/100-117/1200-2000), pre- and post-contrast axial, coronal and sagittal (SE) T1-weighted (TR/TE: 495-500/15-20). Section thickness was 5 mm. Gadolinium-based contrasts were used in first and/or follow-up MR and post-contrast axial, coronal, and sagittal T1 images were obtained. The interval between contrast injection and imaging was 10 min.

The treatment regimen included intravenous (IV) MPred for both case and control groups, IV EPO for the treatment group, and IV placebo for the control group. All participants received infusions of 1,000 mg IVMP in 500 ml normal saline over 5 h every day for 5 consecutive days. The treatment group received 20,000 units of recombinant human EPO (Eprex, parsdarou, Iran) intravenously in 200 ml of saline solution over 1 h every day for 5 consecutive days (simultaneously with IVMP). In the control group, 200 ml saline solution, as a placebo, was infused according to the same schedule as in the treatment group.

The attending physicians monitored probable adverse effects and safety of EPO, including measurement of blood pressure and routine laboratory workup. Complete blood count (CBC), blood glucose, blood urea nitrogen (BUN), creatinine and serum electrolytes were checked daily to screen for any undesirable changes. All patients were followed by the neurologist (new episodes, blood pressure, and laboratory data) 5 days after administration of drug and at the end of the months 1, 2 and 3.

Follow-up brain and cervical MRI with contrast were carried out in month 3. The primary endpoint was evaluating EDSS and AI. The secondary endpoints were number of hyperintense lesions on T2WI, number of contrast enhancing lesions, and number of black holes in MRI.

Inter- and intra-group comparisons of data were carried out using the repeated measure analysis of variance (Greenhouse–Geisser correction), nonparametric Mann–Whitney U test. A probability value of less than 0.05 was considered significant for all statistical tests. Statistical analyses were performed using the Friedman test of SPSS 19.0.

Results

Study groups

The treatment group consisted of 10 patients (9 women, 1 man) with a mean age of 30.5 ± 2.71 years. The control group consisted of 10 patients (9 women, 1 man) with a mean age of 29.7 ± 2.9 years. There were no significant differences between groups in mean age, gender distribution, type of MS and severity of attacks (p > 0.05).

As Table 1 shows, there were no significant differences in EDSS and AI and the number of hyperintense lesions in T2WI, number of contrast enhancing lesions, or number of black holes between groups before the intervention (p > 0.05).
Table 1

Baseline mean of EDSS, AI, number of hyperintense lesions in T2-weighted imaging (T2WI), number of enhancing lesions, and number of black holes in patients treated with erythropoietin (EPO) and non-treated controls (non-EPO)

Parameters

Non-EPO mean (min/max, median)

EPO mean (min/max, median)

p value

EDSS

6.60 (6/7, 7)

6.60 (6/7, 7)

1

AI

6.10 (5/8, 6)

6.20 (5/8, 6)

0.85

No. of T2-WI hyperintense lesions

15.30

15.70

0.81

No. of enhancing lesions

0.90

0.80

0.71

No. of black holes

0.70

0.70

1

EDSS expanded disability status scale, AI ambulatory index

Safety

All patients adhered to the study and tolerated the intervention. There was no significant difference between the frequency of polycythemia and hypertension, the two most worrying adverse effects of EPO.

One patient in the EPO group developed headache associated with nausea. No other adverse effects occurred during the study period in either group. No motor relapse was observed in any of the MS patients during the study period; however, a sensory relapse in one MS patient in control group has been detected.

Mean changes in red blood cell (RBC) counts during and after EPO treatments are shown in Table 2 in the EPO group, and no phlebotomy had to be performed. As Table 2 shows, there was no substantial difference in blood pressure upon EPO treatment.
Table 2

Mean of mean blood pressure (BP) and red blood cell (RBC) mass in patients treated with erythropoietin (EPO) and non-treated controls (non-EPO)

Parameters

Non-EPO mean (min/max, median)

EPO mean (min/max, median)

p value

Mean BP (mmHg)

 Day 5

88.10 (83–94, 87.5)

88.00 (84–94, 86.5)

0.47

 Month 1

87.80 (83–93, 88)

87.70 (84–93, 86)

0.47

 Month 3

87.70 (83–92, 87.5)

88.00 (84–93, 86)

0.47

RBC mass (106)

 Baseline

4.40 (4.1–4.7, 4.4)

4.42 (4.1–4.7, 4.5)

0.7

 Day 5

4.42 (4.1–4.7, 4.35)

4.41 (4.1–4.7, 4.5)

0.7

 Month 3

4.40 (4.1–4.7, 4.35)

4.42 (4.1–4.7, 4.45)

0.7

Motor function

All the patients in the EPO MS group showed a significant improvement in AI over time, as compared to Non-EPO group, which became apparent after 2 months and continued to improve during the third month. The increase in AI in EPO MS patients resulted in an increase of the EDSS (Table 3).
Table 3

Mean of expanded disability status scale (EDSS) and ambulatory index (AI) in patients treated with erythropoietin (EPO) and non-treated controls (non-EPO)

Parameters

Non-EPO mean (min/max, median)

EPO mean (min/max, median)

p value

EDSS

 Month 1

5.30 (4/6, 5.5)

5.30 (4/6, 5.5)

1

 Month 2

4.50 (3/6, 4)

3.00 (2/4, 3)

0.003

 Month 3

3.90 (2/6, 4)

1.40 (1/2, 1)

<0.001

AI

 Month 1

4.00 (3/5, 4)

4.00 (3/5, 4)

1

 Month 2

3.70 (3/5, 3)

1.40 (1/3, 1)

<0.001

 Month 3

3.10 (1/6, 3)

0.60 (0/2, 0.5)

<0.001

MRI outcome

Analysis of MRI data after 3 months revealed considerable changes in T2 number lesions upon EPO treatment, but no significant difference in contrast enhancing and black hole lesions among EPO and placebo group was detected (Table 4).
Table 4

Mean number of hyperintense lesions in T2-weighted imaging (T2WI), number of enhancing lesions, and number of black holes in patients treated with erythropoietin (EPO) and non-treated controls (non-EPO)

Parameters

Non-EPO mean

EPO mean

p value

No. of T2-WI hyperintense lesions month 3

17.40

12.40

0.014

No. of enhancing lesions month 3

0.10

0.10

1

No. of black holes month 3

0.70

0.70

1

Discussion

This study was designed as a pilot study to evaluate safety and short-term efficacy of EPO treatment in relapse phase of RR-MS patients. Heretofore, MPred was observed to inhibit endogenous neuroprotective pathway leading to adverse effects on neuronal survival during CNS chronic inflammatory autoimmune disease such as MS [7]. In contrast, it was demonstrated that EPO increases survival of neurons [30] and acts as a neurotrophic factor which influences the maintenance and regeneration of neurons [16].

Furthermore, it has been shown that EPO decreases demyelination and permeability of blood brain barrier (BBB) [22]. Under the theory that protective effects of EPO and anti-inflammatory effect of MPred might be complementary to each other if both substances are combined, we examined different treatment protocols to compare the efficacy of combination therapies of EPO and MPred with the standard method of IV MPred alone in promoting recovery of moderate to severe acute relapses in MS patients.

At serial short-term follow-up, we found significant clinical motor function improvement in relapsing–remitting MS patients treated with combined therapy in which reflected by a reduction in EDSS. We also conducted brain and cervical MRI to evaluate any change in the number of T2, contrast enhancing and black hole lesions to see the effect of this combination therapy on MRI measures.

T2-weighted MRI lesions represent accumulative MS lesion load, while contrast enhancing and black hole lesions are indicative of blood–brain barrier leakage and permanent axonal loss, respectively [31].

A reduction in number of T2 lesions was observed in combined therapy in MS patients; however, there was no significant difference in number of contrast enhancing and black hole lesions.

The findings of the current study are consistent with those of Ehrenreich et al. [10] who showed effectiveness of high-dose EPO in clinical course of chronic progressive MS patients according to reduction in EDSS and improvement of cognitive performance.

In line with our findings, a beneficial superior efficacy of combination therapy of EPO and MPred has been demonstrated in a rat model of experimental autoimmune encephalomyelitis (EAE) [32].

In the mentioned study, Diem and coworkers [32] revealed that axon and neuron protection was more effective in combination therapy of EPO and MPred in comparison to monotherapy with MPred in both functional and histopathologic data.

In the current study, gradual recovery in motor function of MS patients and decrease in number of T2 lesions first became detectable after 2 months of EPO administration and continued after 3 months. These findings advocate the regenerative and permanent effects of EPO rather than a temporary one. These findings are in agreement with Ehrenreich et al.’s [10] findings which insisted on permanent effect of EPO in chronic progressive MS patients. This result also accords with previous observations, which showed that EPO has impact in neurogenesis, axonal repair and stimulating oligodendrocyte proliferation which are all structural and long-lasting changes in CNS rather than transient one [18, 21, 27].

Since, previous evidence has been confirmed beneficial effects of EPO including reduction of axonal damage and promoting oligodendrocyte proliferation in rodent studies of EAE [2225, 32, 33], we expected significant difference in the number of black hole and T2 lesions in treatment group. However, using conventional MRI, we could not find any significant changes in the number of black hole lesions which are surrogate markers of axonal damage [34, 35]. A possible explanation for this might be due to small number of patients in this trial. Another possible explanation for this could be due to short observation of follow-up. It remains to be determined whether continuation of follow-up time in MS patients will lead to further decline in the number of black hole lesions.

Recent studies have shown that EPO decreases inflammatory cell infiltration and BBB leakage. Furthermore, Agnello et al. showed that EPO exerts its anti-inflammatory effects by a mechanism different from steroid, emphasizing the potential synergistic effects of two drugs [24, 25, 36]. Since contrast enhancing lesions are considered markers of inflammation related to the permeability of the BBB [33]; we therefore hypothesized that combination of EPO with MPred may be a novel mechanism contributing to decreased inflammation in relapse phase of MS patients. Nonetheless, unexpectedly in our study, there was no clear cut difference in number of contrast enhancing lesions between two groups.

One explanation for such discrepancy could be due to antagonize effect of MPred with EPO which was previously revealed by Gorio et al. in a spinal cord injury model which showed that co-administration of MPred antagonizes the beneficial effects of EPO; however, the clearly different pathologies underlying MS versus spinal cord injury, have to be considered [10, 34]. Another explanation for discrepancies in results might be attributed to small number of patients in current trial.

The most important shortcomings of our study were the small sample size and short duration of follow-up of MS patients. In addition, since OCP and EPO both increase thrombotic state, and female patients using contraceptive medication were excluded, and because generally majority of MS patients are female; the applicability of EPO would be so limited.

Taken together, to our knowledge, we were able to provide first evidence that EPO may show an effect on the clinical course of relapsing–remitting MS patients in attack phase. We believe that the encouraging results of combination therapy of EPO and MPred in motor function of relapsing–remitting MS patients deliver some pivotal information for designing a phase II trial. More research for applying other measures such as 25 feet walk and nine hole peg test and also measure 25FWT and 9HP test to obtain precise results regarding the efficacy is needed.

Acknowledgments

The authors would like to thank research development office of Sina hospital for their assistance in the preparation of the manuscript and Cinnagen Company for the unrestricted research grant to perform this study.

Conflict of interest

The authors declare that they have no conflict of interest.

Copyright information

© Belgian Neurological Society 2014