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1. WO2009010107 - USE OF EPO RECEPTOR ACTIVATION OR STIMULATION FOR THE IMPROVEMENT OF THE EDSS SCORE IN PATIENTS WITH MULTIPLE SCLEROSIS

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[ EN ]

Use of EPO receptor activation or stimulation for the
improvement of the EDSS score in patients with multiple
sclerosis

The present invention relates to a method for the improvement of the EDSS score achieved by mammals which are affected by multiple sclerosis and also the use of substances effecting increased and/or prolonged activation and/or stimulation of the erythropoietin receptor for this purpose and also for the production of a drug for this purpose.

With approx. 80 to 110 cases of multiple sclerosis (MS) per 100,000 persons, multiple sclerosis is the most frequent chronic disease of the central nervous system. A third of patients thereby shows a primary or secondary progressive course of the multiple sclerosis, for which no therapy has been available to date. Understanding and treatment of the
progressive phase of multiple sclerosis (MS) , which is
characterized by the steady accumulation of neurological disability, is far from being satisfactory. Progression is most likely driven by the high prevalence of neurodegenerative compared with inflammatory pathological changes, explaining the limited long-term efficacy of current anti-inflammatory and immunosuppressive treatment strategies . It seems that once the cascade of events leading to neuronal and axonal loss is established, even an effective suppression of inflammation fails to protect from clinical disease progression. The
development of add-on treatments targeting axonal repair and remyelination and / or the slowing of disease progression through neuroprotection / neuroregeneration remains therefore the most important challenge and goal in clinical management of chronic progressive MS.

Disability of patients affected by multiple sclerosis can be determined based on different parameters. One of the
parameters - mainly determining motoric ability - is the
Expanded Disability Status Scale (EDSS) . This parameter is described in Kurtzke J. F., Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS), Neurology 1983; 33: 1444-52. At present, no therapy is
available which would improve the EDSS score achieved by patients.

In the following and throughout this application, the term "achieved by patients" shall have the same or corresponding meaning as "achievable by patients" .

Erythropoietin (EPO) is a glycoprotein produced naturally in the body, having a molecular weight of 34,000 D. It is an essential growth factor for the production of erythrocytes and was isolated for the first time in 1977. It binds to and stimulates the endogenous erythropoietin receptor.

Erythropoietin has been in frequent clinical use for many years in patients with renal anaemia, in the case of nephrodialysis, in order to obtain fairly large quantities of autologous blood before planned operations, and it also appeared in press headlines as a doping agent.

Erythropoietin thereby proved to be exceptionally well
tolerated with only minor or no side effects at all.
Intolerance reactions or anaphylactic reactions are rarities with erythropoietin.

There are a plethora of substances, known and produced, which all are considered to increase or prolong the activation and/or increase or prolong stimulation of the erythropoietin receptor (e.g. as shown in the following paragraph) . Besides
erythropoietin itself, whether native or recombinant, whether in native sequence or even after sequence changes or sequence shortening, erythropoietin analogues, erythropoietin fragments or erythropoietin agonists were developed. As examples for recombinant erythropoietins, Epoetin a (Epogen by Amgen Inc., Procrit by OrthoBiotech Inc., Johnson and Johnson Inc.),
Epoetin β (Neorecormon™ by F. Hoffmann-La Roche AG) and Epoetin ω (Epomax™) , Epoetin δ (DynEpo™, Shire Pharmaceuticals Group PLC) with variing glycosilation and sialysation are known.
Genetically modified EPO are known as Darbepoetin α™ (by Amgen Inc.) and CERA™ (by F. Hoffmann-La Roche AG) . Further,
erythropoietin receptor activating antibody (e.g. by Abbott), fusion proteins like Epo-Fc and carbamylated EPO (CEPO™, by H. Lundbeck A/S) are known. Endogenous erythropoietin stimulating substances, substances increasing the release and/or activity of endogenous erythropoietin (e.g. HIF-stabilisers, e.g. by FibroGen Inc. or Torrent Pharmaceuticals Ltd.) are also known. Furthermore, erythropoietin analogues or mimetics are known, e.g. SEP (Synthetic Erythropoiese Protein, by Gryphon
Therapeutics), Hematide™ (by Affymax Inc.) and others known as EPO-analogues or EPO-mimetics by AplaGen. It is even known to transfer the gene coding for erythropoietin into a patient in order to express erythropoietin.

All of these substances are known to increase and/or prolong the activation and/or stimulation of the erythropoietin receptor besides their antiapoptotic, tissueprotective effect. Some useable EPO variants are published for example in the following publications:

Leist et al . , Science 2004, Vol. 305, pp. 239 - 242, WO
86/03520, WO 85/02610, WO 90/11354, WO 91/06667, WO 91/09955, WO 93/09222, WO 94/12650, WO 95/31560, WO 95/05465. An overview of known EPO variants, analogues, mimetics and equivalents which can also be used in their entirety in the present invention and also of known fields of use thereof appears in Brines and Cerami, Nature Reviews, Neuroscience, June 2005, Vol. 6, pages 484 - 494.

WO 00/61164 discloses the use of EPO for the protection of neuronal tissue, in particular also of the central nervous system. It is mentioned in passing in this document that multiple sclerosis might also be treated with EPO.

WO 00/61164 in fact discloses the use of EPO for the treatment of multiple sclerosis but indicates no experimental data or treatment regime at all. It is not disclosed therein, in what respect (cognitive, motoric function or others) patients suffering from multiple sclerosis might benefit from treatment with EPO.

It is therefore the object of the present invention to develop a method for the safe improvement of the expanded disability status scale (EDSS) score achieved by mammals and the use of substances effecting increased and/or prolonged activation and/or stimulation of the erythropoietin receptor for the safe improvement of the expanded disability status scale (EDSS) score achieved by mammals affected by multiple sclerosis.

This object is achieved by the method according to claim 1 and also the use according to claim 14. Advantageous developments of the method according to the invention and of the use
according to the invention are given in the respective
dependent claims.

According to the invention, in the method of the present invention in mammals, in particular in humans, a substance effecting increased and/or prolonged activation and/or
increased and/or prolonged stimulation of the erythropoietin receptor is administered to the mammal, e.g. to a human patient affected by multiple sclerosis.

As substance effecting increased and/or prolonged activation and/or stimulation of the erythropoietin receptor and substance as described above may be used, e.g. erythropoietin, native or recombinant, variants or derivatives of erythropoietin,
analogues, mimetics, agonists of erythropoietin or substances for genetically altering the patient in order to achieve said activation/stimulation of the erythropoietin receptor.

The present invention for the first time shows, that a
treatment or use according to the present invention improves not only cognitive parameters but effect a significant improvement of the EDSS score in patients suffering from multiple sclerosis. This improvement has not been known before and could not have been foreseen considering the purely
speculative nature of the above cited documents generally relating to the use of erythropoietin in treatment of multiple sclerosis. It is of great importance that the proposed method and proposed use is safe with no or only minor side effects.

The method can be used with patents suffering from any kind of multiple sclerosis, e.g. primary progressive multiple sclerosis (PPMS) , secondary progressive multiple sclerosis (SPMS) , relapsing/remitting multiple sclerosis (RRMS) or progressive relapsing multiple sclerosis (PRMS) .

In particular, an improvement in the symptoms was observed not only in the case of chronic-progressive multiple sclerosis but also in the case of relapsing-remitting multiple sclerosis. In particular in the case of (chronic) progressive multiple sclerosis, a deterioration in the symptoms would have been expected during the treatment-free interval. However
stabilisation also occurred here.

Advantageously, the erythropoietin is applied intermittently. This means that, as an advantageous development of the present invention, an interval treatment is proposed. It is thereby particularly advantageous if the treatment comprises a sequence of periods of time with application of EPO (application period) and periods of time without application of EPO (application-free periods) .

The individual periods of time thereby comprise several weeks . A sequence has emerged as particularly advantageous in which each application period lasts 12 to 48 weeks, advantageously 18 to 36 weeks, advantageously 24 to 28 weeks, whilst the
application-free periods last 8 to 53 weeks, advantageously 16 to 28 weeks. Within the application periods, the dosage can be varied, for example firstly a period of time with a weekly application and a subsequent period of time with a two-weekly application.

The dosage is thereby respectively in the values described in the claims, particularly advantageously in a dosage range of 5,000 IU to 100,000 IU (international units), advantageously in a dosage range of 30.000 IU to 60.000 IU, advantageouly in a dosage range of 40000 IU to 50000 IU, per week or per administration, including or excluding the respective range limits. In the alternative, doses equivalent to said doses given in the preceding sentence, which lead to comparable erythropoietin levels or comparable erythropoietin receptor activating or stimulating biological activity may be used as dosage.

With the mentioned interval dosage schemes, the result
surprisingly is a constant improvement in the clinical symptoms during treatment. The improved level is maintained
astonishingly in the interval and the second cycle produces a further improvement .

The interval treatment according to the invention is an
innovative improvement to the entire concept of neuroprotection which, in the case of EPO with multiple sclerosis, exploits in addition the fact that the result with half-yearly
erythropoietin treatment is a latent, desired lack of iron.
Since lack of iron can be advantageous in addition for the known neuroprotective EPO effect in chronic inflammatory diseases such as multiple sclerosis, advantageously iron is substituted neither in the treatment nor in the treatment-free phase. The treatment-free phase serves therefore also for slow replenishment of the depleted iron stores, as a result of balanced nutrition.

In particular in the case of use of EPO with haematopoietic effect, the neuroprotective effect is consequently supplemented by the latent lack of iron produced by the EPO treatment .

The effect according to the invention is also achieved however already by using EPO derivatives or variants without
haematopoietic effect. It turned out, that is not of
particular relevance in the present invention whether the EPO analogues or erythropoietin fragments which are used have a haematopoietic effect (e.g. Epogen™) or not (e.g. CEPO™) .

The above explanation relates to the method but the invention is not only directed to the therapeutic method but also to the use of the above mentioned substances in a method of this type and also the use of the above mentioned substances for the production/manufacturing of a drug for use in a method of this type.

In the following, an explanatory study is described which provides experimental proof for the invention. This example contains four tables (table 1, supplementary table 1,
supplementary table 2, supplementary table 3) as part of the description and is further explained on the basis of 5 figures. Therein

figure 1 shows the design of the EPO MS exploratory study. Low-dose EPO treatment was only performed until week 12;

figure 2 shows changes in blood cell counts and iron parameters during and after EPO treatment. The mean of two baseline values of each patient was set to 100% for each of the laboratory parameters and used for calculating individual change over time. Mean change of all patients within each group during follow-up upon treatment or during the treatment-free period is expressed in % baseline. Low-dose EPO MS patients and Parkinson patients were only followed until week 12. Filled circles: high-dose EPO MS patients (N=5) ; open circles: low-dose EPO MS patients (N=3) ; gray triangles: high-dose EPO Parkinson patients (N=2) ;

figure 3 shows changes in parameters of motor function upon EPO treatment, (a) The mean of all available baseline values of maximum walking distance of each patient obtained during the whole lead-in period was set to 100% and used for calculating individual change over time. Mean change of all patients within each group during follow-up upon treatment or during the treatment-free period is expressed in % baseline, (b) Follow-up of maximum walking distance of one high-dose EPO MS patient over a total of 60 weeks, including two EPO treatment periods, is presented as raw data for every test time-point. The trend line illustrates the improvement over time, (c) For presenting the course of EDSS scores during the study, mean individual baseline was set to 0 and subsequent values denote change of EDSS score during follow-up of individual MS patients (left panel) or of mean EDSS score of the groups (right panel) . For determining individual EDSS change over time, the mean value of consecutive ratings over six week follow-up periods was used.

(d) Intra-individual course of central motor conduction time

(Tibialis MEP, original data; MS high-dose patients N=4 , due to methodological problems in one patient during baseline measurement) .
Low-dose EPO MS patients and Parkinson patients were only followed until week 12. Filled circles: high-dose EPO MS patients (N=5) ; open circles: low-dose EPO MS patients (N=3);

gray triangles: high-dose EPO Parkinson patients (N=2) . Significance values refer to the high-dose EPO MS patients only and denote changes of the respective parameter from baseline to the end of each treatment period, including all testing time-points. Statistical analysis: Friedman test (SPSS 14.0 for Windows) ;

figure 4 shows changes in parameters of fine motor coordination upon EPO treatment. (a) Change of performance in MacQuarrie Tapping test (MacQuarrie TW. MacQuarrie Test for Mechanical Ability. Monterey, CA: CTB/McGraw-Hill , 1925, 1953), (b) MacQuarrie Dotting test (MacQuarrie TW. MacQuarrie Test for Mechanical Ability. Monterey, CA: CTB/McGraw-Hill, 1925, 1953), and (c) 9-hole peg test (Cutter GR, Baier ML, Rudick RA, Cookfair DL, Fischer JS, Petkau J, et al . Development of a multiple sclerosis functional composite as a clinical trial outcome measure. Brain 1999; 122 ( Pt 5): 871-82), is expressed as % individual baseline. The mean of all available baseline values of each patient was set to 100% for each of these parameters and used for calculating individual change over time. Mean change of all patients within each group during follow-up upon treatment or during the treatment-free period is expressed in % baseline. Low-dose EPO MS patients and Parkinson patients were only followed until week 12. Filled circles: high-dose EPO MS patients (N=5) ; open circles: low-dose EPO MS patients (N=3); gray triangles: high-dose EPO Parkinson patients (N=2) . Significance values refer to the high-dose EPO MS patients only and denote changes of the respective parameter from baseline to the end of each treatment period, including all testing time-points. Statistical analysis: Friedman test; and figure 5 shows changes in cognitive function tests upon EPO treatment, (a) Decrease of reaction time in Trail Making - Part B. (b) Improvement of performance on WMS-R Letter Number
Sequencing, (c-d) Reduction of reaction time in TAP subtests Visual Scanning - critical trials and Working Memory in the high-dose EPO MS group (N=5) . Low-dose EPO MS and Parkinson patients fail to show improvement. The mean of two baseline values of each patient was set to 100% for each of the
cognitive parameters and used for calculating individual change over time. Mean change of all patients within each group during follow-up upon treatment or during the treatment-free period is expressed in % baseline. Low-dose EPO MS patients and Parkinson patients were only followed until week 12. Filled circles:
high-dose EPO MS patients (N=5) ; open circles: low-dose EPO MS patients (N=3); gray triangles: high-dose EPO Parkinson
patients (N=2) . Significance values refer to the high-dose EPO MS patients only and denote changes of the respective parameter from baseline to the end of each treatment period, including all testing time-points. Statistical analysis: Friedman test.

Detailed description of the present study and its results

The main objectives of the present study were: (1) to evaluate safety of long-term high-dose EPO treatment in chronic
progressive MS, and (2) to collect first evidence of potential efficacy with respect to a variety of clinical parameters, in particular Expanded Disability Status Scale (EDSS) , but also walking distance, fine motor function and cognition. A careful and comprehensive individual follow-up of a small number of patients during a 6-week lead-in phase, a 12-24 week treatment phase, and a 24-week post-treatment phase delivered information about dosing of EPO, necessary duration of treatment in order to see improvement, and effect of EPO treatment-free periods on maintaining status. Finally, effects found in chronic
progressive MS could be compared to effects observed in drug-naϊve patients suffering from another degenerative disease, Morbus Parkinson.

Materials and methods

Patients and procedures

Following announcement of the present exploratory study to the local ethical committee, a total of eight patients suffering from chronic progressive MS (primary or secondary) , who had previously failed on disease-modifying drugs, and, as disease control, two patients with Morbus Parkinson were included into this study (for overview see Table 1) . Written informed consent was obtained after comprehensive and repeated information of the respective patient in the absence and in the presence of selected relatives or close advisors (mostly physicians) .
Patients were fully aware that they participated in an
experimental study, set up to evaluate safety and efficacy of EPO in chronic progressive MS with respect to EDSS score.
The study was designed as an exploratory open- label study employing two different doses of recombinant human (rh) EPO.

The high dose (48000 IU of EPOα, ERYPOR, Janssen-Cilag,
Germany) was selected as presumably effective dose, based on previous trials in stroke and in schizophrenia patients, whereas the low dose (8000 IU of EPOα) lies in the upper dose range of anemia treatment (e.g. Eschbach JW, Egrie JC, Downing MR, Browne JK, Adamson JW. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin.
Results of a combined phase I and II clinical trial. N Engl J Med 1987; 316: 73-8) and was explored for the first time in a neurological indication. Of the eight MS patients, five
received high-dose rhEPO, whereas three received low-dose rhEPO. Dosing was randomly assigned. Patients were unaware of the dose being "high" or "low". The two drug-naϊve Parkinson patients received high-dose rhEPO (48000 IU of EPOα) .

An overview of the study design including tests and follow-up parameters is presented in Figure 1. After baseline examination and confirmation of inclusion / exclusion criteria, patients entered a lead-in phase of six weeks duration, where they were asked to regularly score or test their performance in a number of items, either specifically reflecting their individual handicaps or important for general well-being and health. For that, every patient received an individually tailored
questionnaire, which should provide insight into the
longitudinally evaluated baseline performance as well as, later, the treatment and post-treatment follow-up period.

Following the lead-in period, the treatment period was
initiated. It started with a one-week inpatient setting, which allowed a comprehensive examination of the patient including magnetic resonance tomography (MRI) of brain and spinal cord, neurological, neuropsychological, electrophysiological,
urological, ophthalmological examination, and routine
laboratory analyses (including EPO-antibody testing) . Patients were then started on high-dose prednisolone (1000 mg / 100 ml over 30 min intravenously) in order to create a comparable immunosuppression as a starting point of neuroprotective therapy. One day later, a second infusion of prednisolone was given, followed by the first infusion of EPO (48000 or 8000 IU of EPOα, respectively, in 50 ml of 0.9% sodium chloride over 15 min intravenously) . The third treatment day included
prednisolone followed by EPO. Parkinson patients did not receive prednisolone, but were otherwise treated identically. One day later, patients were discharged and asked to return weekly to the clinic for application of the study drug,
documentation of clinical state, performance rating, monitoring of adverse events and safety, including measurement of blood pressure and routine laboratory workup. Blood letting (350-450 ml) was performed if the hematocrit exceeded 50% in male or 48% in female patients on two consecutive weeks. During the one-week inpatient phase, the 12-24 week outpatient treatment phase, as well as the 24-week post-treatment phase, patients were asked to continue regular (daily / weekly) self-rating using their individually tailored questionnaire.

Neuropsychological baseline and follow-up testing of patients included the Mehrfachwahl-Wortschatz-Intelligenztest (Lehrl S. Mehrfachwahl-Wortschatz-Intelligenztest . MWT-B. Erlangen:
Straube, 1999) , four subtests (Information, Similarities,
Picture Completion, Block Design) of the revised German version of the Wechsler Adult Intelligence Scale (Tewes U. Hamburg- Wechsler Intelligenztest fur Erwachsene. Revision 1991. Bern: Huber, 1991) , the Repeatable Battery for the Assessment of Neuropsychological Status (Randolph C. RBANS Manual -Repeatable Battery for the Assessment of Neuropsychological Status. Harcourt, Texas: Psychological Corporation, 1998), subtests Visual Scanning, Working Memory and Alertness of the computer-assisted battery for attention testing (Zimmermann P, Fimm B. Testbatterie zur Aufmerksamkeitsprufung (TAP).
Herzogenrath: PsyTest, 1995), subtest Letter Number Sequencing of the Wechsler Memory Scale - Revised (Wechsler D. Wechsler Memory Scale - 3rd edition (WMS-III). Harcourt, Texas:
Psychological Corporation, 1998) , Wisconsin Card Sorting Test -64 Card Version (Kongs SK, Thompson LL, Iverson GL, Heaton RK. WCST-64: Wisconsin Card Sorting Test - 64 Card Version. Odessa, FL: Psychological Assessment Resources, 2000) and the Trail

Making Test (Reitan RM. Validity of the Trail Making test as an indicator of organic brain damage. Percept Motor Skills 1958; 8: 271-276) .

MRI of brain and spinal cord was conducted on a Siemens 1.5T scanner (scans before and after gadolinium; for cranial imaging transverse, coronal and sagittal Tl-weighted sequences, T2-weighted TSE- and TIRM-sequences; for spinal imaging sagittal and transverse Tl-weighted TIRM- and TSE- sequences, T2 -weighted TSE-sequences; slice thickness: 6mm (19 slices, cranial), 3mm (15 slices, cervical) ; 4mm (11 slices, thoracic) ; semi-automated volumetrical analyses carried out, in a blinded fashion, with Centricity Radiology RA 1000, General Electrics, in T2 -weighted images) . Electrophysiology wherever technically possible (motor-evoked potentials: MEP), maximum walking distance, fine motor assessment using the 9-hole peg test
(Cutter GR, Baier ML, Rudick RA, Cookfair DL, Fischer JS, Petkau J, et al . Development of a multiple sclerosis functional composite as a clinical trial outcome measure. Brain 1999; 122 ( Pt 5) : 871-82; Rudick RA, Cutter G, Baier M, Fisher E,
Dougherty D, Weinstock-Guttman B, et al . Use of the Multiple Sclerosis Functional Composite to predict disability in
relapsing MS. Neurology 2001; 56: 1324-30), as well as
MacQuarrie Tapping and Dotting tests (MacQuarrie TW. MacQuarrie Test for Mechanical Ability. Monterey, CA: CTB/McGraw-Hill, 1925, 1953) were performed.

Inclusion and exclusion criteria

An overview of all eight MS patients and the two Parkinson patients is provided in Table 1. MS patients were in a chronic progressive state (either primary or secondary) of their disease. They were free of other severe psychiatric or neurological disorders. A minimum of measurable walking
distance was required. Patients were not allowed to smoke or to take sex steroid hormones to avoid a potential additional vascular risk on top of their relative immobility. Two patients quit smoking several weeks before the lead-in phase, another patient stopped taking sex steroid medication (in accordance with her gynecologist) . All medication was documented. Start of any MS-related novel medication during the study was not allowed nor was any kind of iron substitution. Previous
medication in all MS patients included corticosteroids,
intrathecal prednisolone, beta interferon, Copaxone,
mitoxantrone, cyclophosphamide, iv-immunoglobulins,
deoxyspergualin and riluzole. Treatment was terminated at least half a year before EPO treatment due to lack of efficacy. All patients had shown distinct progression of their disease in the past year.

"Consilium" : Decision on study continuation after 12 weeks

After 12 weeks of weekly EPO treatment a so-called consilium took place, integrating all test results and observations of all participating parties, the patient, potential relatives or family members, an independent MS specialist, members of the clinical neuroscience team, a clinical neurophysiologist, a neuroradiologist, and a physiotherapist. Continuation of treatment was only recommended if a patient had clearly
improved performance in at least three independent items, previously identified to be affected in this particular
individual, e.g. walking distance, cognition, and bladder function.

Statistical analysis

All numerical results are presented as mean + SD. For analysis, individual mean baseline performance in each of the items of interest was set to 100% in order to reach comparable baseline values among patients. Individual change during follow-up upon treatment or during the treatment-free period was expressed in % individual baseline. This way, patients can be compared and intra-group comparisons investigating the course of various variables in the high-dose EPO MS group, including all testing time-points from baseline on, can be performed using the
Friedman test of SPSS 14.0 for Windows. This special
nonparametric procedure for comparing repeated measures data with small sample sizes can be used for metric as well as ordered categorical data. Supplementary Tables 2 and 3
additionally provide significance values based on analyses of raw scores. Inter- and intragroup comparisons of MRI data were carried out using the nonparametric Mann-Whitney-U-test
(independent and dependant) . Statistical significance was set to 0.05 for all analyses.

Results

Study participation

All ten patients participated in the study until the consilium took place after 12 weeks of weekly EPO treatment. At that time-point, only the five high-dose MS patients met criteria for continuation, whereas all three low-dose EPO MS patients as well as the two high-dose EPO Parkinson patients did not show sufficient improvement that would have justified continuation according to our consilium criteria for beneficial treatment response. This, however, does not prejudice effectiveness of EPO treatment with low-doses. The five high-dose EPO MS patients stayed on continuous treatment for another 12 weeks to receive intravenous high-dose EPO now once every other week. After this second treatment period, there was a 24 -week
treatment- free follow-up period for all high-dose EPO MS patients. One of these patients could be followed over another EPO treatment cycle.

Safety

There were no adverse events reported or observed in any of the patients at any time. One Parkinson patient complained about being tired for approximately two to three days after each EPO infusion. All other patients (high-dose as well as low-dose EPO MS and Parkinson) reported on feeling physically stronger, less tired, more optimistic, more enduring. They did not report that their sleep was in any way affected. Quality of life self-rating during that time stayed stable (data not shown) . No relapses were observed in any of the MS patients during the study period. Mean changes in blood cell counts and iron parameters during and after EPO treatment are illustrated in Figure 2. Original data (mean + SD) of high-dose and low-dose MS patients and of Parkinson patients are presented in
Supplementary Table 1. The response of hemoglobin, hematocrit, and erythrocytes to EPO in MS patients was surprisingly low. Of the high-dose EPO MS group, a total of only five blood-lettings were necessary during the whole treatment phase (three times in one patient and once in two patients; see Supplementary Table 1) . In the low-dose EPO group, no blood-letting had to be performed. The two Parkinson patients responded stronger to EPO and both required blood-letting. MCV and MCH declined similarly strongly in all high-dose patients. The iron parameters showed the expected pattern, more pronounced upon high-dose and less upon low-dose EPO treatment: Distinct decrease in serum
ferritin levels, paralleled by increases in serum transferrin and, particularly, in soluble transferrin receptor (Figure 2; Supplementary Table 1) . Platelet counts increased in all patients at approximately the same rate but stayed essentially within the normal range. Whereas there was no measurable change in c-reactive protein (CRP) , erythrocyte sedimentation rate tended to decrease in all patients over time of EPO treatment. All patients were EPO-antibody negative at baseline and none of the patients had developed EPO-antibodies by the end of the treatment period. No appreciable change in blood pressure upon EPO treatment was observed in any of the patients (data not shown) .

Motor function

All patients in the high-dose EPO MS group showed a significant improvement in their maximum walking distance over time as compared to baseline, which became first apparent after three weeks, gradually improved during the treatment phase to reach a plateau at around eight weeks and still persisted after
cessation of EPO treatment (Figure 3a, b; Supplementary Table 2) . The increase in maximum walking distance in high-dose EPO

MS patients resulted in a reduction of the EDSS (Figure 3c) and was paralleled in patients, who could be followed
electrophysiological^, by a reduction in the central motor conduction time (left Tibialis MEP; Figure 3d, Supplementary Table 2) . In contrast, neither low-dose EPO MS patients nor Parkinson patients, according to the Unified Parkinson's
Disease Rating Score (Movement Disorder Society Task Force on Rating Scales for Parkinson's Disease, 2003; Fahn S, Elton RL. Unified Parkinson's Disease Rating Scale. In: Fahn S, Marsden CD, Calne DB and Goldstein M, editors. Recent Developments in Parkinson's Disease. Florham Park, NJ: Macmillan Health Care Information, 1987) , displayed any measurable beneficial effect on walking distance/gait. Figure 3b illustrates the course of the maximum walking distance, both supervised and self-measurements, over more than one year in one of the high-dose EPO MS patients who underwent two EPO treatment periods. The trend- line from lead-in to the end of the observation period clearly directs upwards, with no change after switch to biweekly application. The EPO treatment break did not provoke any loss of the gained function, underlining that the effect of EPO is long lasting. Also, results of supervised ratings and self-ratings were quite consistent.

Fine motor performance in MacQuarrie Tapping and Dotting tests also showed improvement in high-dose EPO MS patients only
(Figure 4a, b), whereas in the 9-hole peg test, there was no significant beneficial effect of EPO treatment in any group and no group difference (Figure 4c ; Supplementary Table 2) .
Medianus MEP stayed stable over time (Supplementary Table 2) . Bladder function was initially reported to be affected in three of the five high-dose and all three low-dose EPO MS patients. Subjective rating by the patients yielded distinct improvement only upon high-dose EPO treatment (data not shown) . One MS patient with respiratory insufficiency due to muscle weakness showed improvement in lung function (vital capacity and forced vital capacity) upon high-dose EPO treatment.

Cognitive functions

Premorbid intelligence as measured at baseline with the MWT-B was almost identical in all MS patients (Table 1) . In contrast, estimation of the current intelligence using HAWIE-R showed a higher variability (high-dose EPO MS: 132.8±15.4; low-dose EPO MS: 119.3±14.7). High-dose EPO MS patients displayed a clear improvement in cognitive tests related to executive
functioning, i.e. Trail Making - Part B, WMS-R Letter Number Sequencing, Visual Scanning, RBANS Coding and Working Memory, and psychomotor speed (Trail Making - Part A) , which was absent both in low-dose MS and Parkinson patients (Figure 5,
Supplementary Table 3) . This improvement remained stable even during the EPO treatment break. In contrast, parameters of learning and memory (see Supplementary Table 3 for the most relevant items) remained essentially unchanged on a high performance level, consistent with a ceiling effect.

Other outcome and follow-up parameters

Analysis of MRI data did not uncover changes upon EPO
treatment. Volumetrical analysis of total brain (excluding cerebellum, brain stem and ventricles) as well as of ventricles did not yield differences among high and low-dose MS patients upon study entry (1195.8±65.6ml versus 1013.6+189.7ml, p=0.4; and 48.3+17.6ml versus 46.4±12.8ml, p=0.857). Follow-up of the high-dose patients after three months (3.6±1.1 months) showed no change in total brain or ventricle volume compared to baseline (baseline versus 3 months: 1195.8±65.6ml versus
1152.4±47.7ml, p=0.317; and 48.3±17.6ml versus 47.9±17.8ml, p=1.0) . Serum levels of the glial damage marker SlOOB did not reveal consistent changes in any of the patients upon treatment (Supplementary Table 1) .

Conclusion

The results presented above demonstrate safety and potential beneficial effects of long-term EPO treatment in chronic progressive MS. In this study, there were no adverse events, no safety concerns, and an astonishingly low need of blood lettings . Using an intra-individual follow-up design, a significant clinical and a tendency of electrophysiological improvement of motor function in chronic progressive MS at least upon high-dose EPO treatment was found as reflected by a reduction in EDSS. Improvement of this score has to our knowledge not yet been observed in any chronic progressive MS trial.

The demonstrated beneficial effects on motor function and cognition cannot simply explained by improved mood since an improvement in general well-being and mood was observed in all patients, independent of the dose, and became evident already after the first infusion of EPO. In contrast to mood and general well-being, no clear beneficial effect of low-dose EPO treatment in MS patients on any of the other parameters tested could be demonstrated at present. Further, there was no measurable effect of high-dose EPO treatment in the two drug-naϊve Parkinson patients. These latter findings may also exclude a pure placebo effect to explain the improvement found in EPO MS patients.

The improvement was kept during EPO reduction and even after complete cessation of EPO treatment over a follow-up time of 24 weeks, pointing to a regenerative effect mediated by EPO rather than a temporary and short-lived action. These results may be tested using an interval treatment as described in DE 10 2006 004 008.2 and in PCT/EP2007/000640. The teaching of this document with respect to the beneficial effects of EPO in patients suffering from multiple sclerosis using an interval treatment is fully included into the present document by reference. It remains to be determined, however, whether continuation of EPO treatment in MS patients after a treatment-free interval will lead to further improvement, or at least contribute to maintaining the improved status and to slowing of progression. The observations in one of our patients who could be followed over more than one year and two EPO treatment cycles, supports the latter assumption.

Taken together, we were able to provide first evidence that EPO may show an effect on the clinical course, as demonstrated by the EDSS score, of chronic progressive MS, acting via as yet undetermined mechanisms that improve function, enhance regeneration and / or slow deterioration. Although there are reports of beneficial effects of EPO in rodent studies of EAE, EPO is the first compound that apparently demonstrated to effect an improvement in EDSS and a tendency of improvement in central motor conduction time in a cohort of chronic progressive MS patients.

Of note is the fact that all MS patients, high-dose as well as low-dose EPO received the same three-day high-dose corticosteroid infusion to create an immunologically comparable starting-point of longterm EPO therapy. Although no persisting beneficial effects of steroid treatment per se would be expected (but rather a potential rebound effect, i.e. a relapse of the disease after discontinuation of therapy - Agnello D, Bigini P, Villa P, Mennini T, Cerami A, Brines ML, et al . Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis. Brain Res 2002; 952: 128-34), the lasting clinical improvement in the high-dose EPO MS group makes a steroid effect explaining this improvement very unlikely.

Regarding the mechanism of action of EPO on motor and cognitive performance in chronic progressive MS, the observed gradual improvement, first visible after a latency of several weeks, and, in particular, its stability over several months of EPO reduction and EPO treatment-free interval, suggests a morphological rather than a purely functional and transient effect. This is further supported by the observed improvement of the central motor conduction time. This improvement is particularly striking and difficult to reconcile with the known spectrum of EPO functions, as remyelination by EPO has not yet been demonstrated.

Whereas the obvious failure of low-dose EPO to lead to an appreciable improvement in MS patients might be explained by an insufficient concentration of EPO achieved in the central nervous system, it is unclear why the Parkinson patients did not have any measurable benefit. EPO penetrates through a blood-brain-barrier at least upon high-dose peripheral application (Banks et al . , 2004; Brines et al . , 2000; Ehrenreich et al . , 2004b; Xenocostas et al . , 2005) and should therefore have reached the brain also in the two Parkinson patients in amounts sufficient to exert neurotrophic effects. In this disease, however, symptoms become usually overt when 70% of neurons in the substantia nigra are degenerated (Roller WC. When does Parkinson's disease begin? Neurology 1992; 42: 27-31; discussion 41-8) . Neurotrophic effects on the remaining neuronal population may not lead to rapid clinical improvement or require longer time periods of treatment and follow-up.

The infrequent requirement of blood lettings in MS patients, consistent with a relative hyporesponsiveness of the hematopoietic system to EPO (Kwack C, Balakrishnan VS. Managing erythropoietin hyporesponsiveness. Semin Dial 2006; 19: 146-51) , might be due to a systemic latent inflammatory condition altering cytokine patterns that modulate the bone marrow response to EPO. This proposed latent inflammatory condition cannot be easily diagnosed with routine laboratory parameters of inflammation. Here, the response of the hematopoietic system to EPO might even serve in future studies of MS as an indicator of occult inflammation, and thus requirement of additional immunosuppression. Moreover, the hyporesponsiveness of the hematopoietic system, in contrast to the nervous system, to EPO in MS supports the notion that the observed therapeutic efficacy in MS is not simply due to improved oxygen supply via increased red blood cell mass.

EPO treatment leads to temporary shifts in iron stores as delineated here by several determinants of iron metabolism. Accelerated and intensified integration of iron into new red blood cells and thereby withdrawal of iron from its stores, leads to a picture similar to that of true iron deficiency. This picture is corrected after termination of EPO treatment without extra iron substitution. In the absence of appreciable blood loss and upon normal nutrition, iron deficiency will not occur. Binding more and more of the freely available iron, on the other hand, might additionally reduce inflammatory processes in MS and thereby contribute to the beneficial effect of high-dose long-term EPO treatment. In fact, iron chelators have been proposed for treatment of MS (e.g. desferrioxamine;

Lynch SG, Fonseca T, Levine SM. A multiple course trial of desferrioxamine in chronic progressive multiple sclerosis. Cell

MoI Biol (Noisy-le-grand) 2000; 46: 865-9)). Interestingly, disturbed iron metabolism has been described in MS patients

(Sfagos C, Makis AC, Chaidos A, Hatzimichael EC, Dalamaga A,

Kosma K, et al . Serum ferritin, transferrin and soluble transferrin receptor levels in multiple sclerosis patients.

Mult Scler 2005; 11: 272-5) .

To summarize, in the presented example, eight MS patients, five randomly assigned to high-dose (48000 IU) , three to low-dose (8000 IU) rhEPO treatment, and, as disease controls, two drug-naϊve Parkinson patients (receiving 48000 IU) were followed over up to 48 weeks: A 6-week lead-in phase, a 12-week treatment phase with weekly EPO, another 12 -week treatment phase with bi-weekly EPO, and a 24-week post- treatment phase. Not only an improvement of cognitive performance or the first time but also a reduction in EDSS was observed upon high-dose EPO treatment in MS patients, persisting for three to six months after cessation of EPO application. In contrast, low-dose EPO MS patients and drug-naϊve Parkinson patients did not improve in any of the parameters tested in the present study. There were no adverse events, no safety concerns and a surprisingly low need of blood-lettings.

Table 1 : Patient Characteristics
Treatment Patient Age Sex Education Premorbid Disease Disease Optic nerve Leading symptoms EDSS Walking distanc group IO (years) total intelligence duration subtype upon upon
(years) quotient' (years) inclusion inclusion
34 female 19 5 118 92 PPMS tetraspastic (mainly legs/right side), slight ataxia 5 5 88m
2 42 male 22 112 97 SPMS none ataxia, dysarthria, urinary dysfunction, mild cognitive 6 0 140m
deficits, fatigue, inadequate affect (with crutch)

MS
3 53 male 20 5 136 19 3 PPMS bilateral tetraspastic (mainly legs/left side), urinary/bowel 6 5 25m
high-dose dysfunction, slight fatigue (with walker)

((N=S)
4 44 male 20 5 112 12 1 PPMS bilateral tetraspastic (mainly arms/nght side) ataxia, respiratory 5 5 110m
insufficiency (muscle weakness)
5 45 female 20 143 14 8 SPMS none tetraspastic ataxia of upper limbs, urinary/bowel 6 0 77m
dysfunction fatigue dysthymia (intermittent assistant;
43.6 m/f- 20.5 124.2 13.0 3x PPMS 5.9 88.0
(6.8) 3/2 (0.9) (14.4) (4 1) 2x SPMS (0.4) (42.7)
6 38 male 20 130 9 5 PPMS unilateral ataxia, paraparesis, severe sensory dysfunction, urinary 4 5 381m
dysfunction slight dysarthria
MS
7 42 male 23 107 18 3 SPMS bilateral tetraspastic (mainly legs), urinary dysfunction, cognitive 6 5 54m
low-dose
deficits INO, dysarthria (with walker)

K* (N=3)
fi 63 female 23 136 10 4 SPMS none paresis (mainly legs/right side), muscle cramps, urinary 6 0 118m
dysfunction dysarthria I"1*1 =""!
47.7 m/f: 22.0 124.3 12.7 1 x PPMS 57 184.3
(13.4) 2/1 (1.7) (15.3) (48) 2X SPMS (1.0) (173.3)
Treatment Patient Age Sex Education Premorbid Disease Disease Leading symptoms UPDRS
group ID (years) total intelligence duration subtype upon - (years) quotient* (years) inclusion
9 43 male 24 145 1 8 idiopathic tremor (mainly right side), rigidity, bradykinesia 30
Parkinson
micrography, mild cognitive deficits, dysarthria
high-dose
10 73 female 18 118 0 7 idiopathic tremor (mainly legs), bradykinesia, impaired fine motor 20 - (N=2)
function, fatigue, cognitive dysfunction
Means (SD) presented for treatment groups "MS high dose" and "MS low-dose" PPMS primary progressive multiple sclerosis SPMS secondary progressive multiple sclerosis EDSS Expanded Disabilit Status Scale, UPDRS Unified Parkinson's Disease Rating Scale INO internuclear opthalmoplegia aPremorbιd intelligence quotient based on results of the Mehrfachwahl-Wortschatz-lntelligenztest (MWT

B) bState of ophthalmological examination and VEPs (visual evoked potentials) upon study entry which remained unchanged during follow-up

Supplementary Table 1 : Routine Laboratory Data - Part 1
BaseEPO weekly EPO bi-weekly EPO-free
line period
Mean Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week
baseline 1 2 3 4 5 6 7 8 9 10 11 12 14 16 18 20 22 24 36 Ref
Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Rang
(SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD)
Hemoglobin (g/dlj
MS high-dose 15 06 13 98 14 62 1490 15 38 15 18 15 34 15 34 1460 14 30 14 54 1470 14 36 14 06 14 16 1396 14 14 13 98 1343 13 98 malt

(N=5) (0 87) (1 19) (072) (046) (1 05) (1 48) (067) (1 82) (1 65) (1 30) (0 81) (1 25) (1 15) (1 22) (1 26) (1 50) (1 53) (1 15) (094) (0 67) 13 5 1

MS low-dose 14 22 14 90 15 OO 14 83 14 57 15 63 15 00 15 10 1523 15 13 15 50 15 13 1567
(N-3) (1 18) (0 80) (070) (081 (0 81) (0 91) (1 06) (098) (0 81) (070) (1 14) (067) (0 59) tema

Parkinson high-dose 14 23 13 65 14 30 1460 15 35 15 55 15 45 15 95 1595 15 75 15 75 1660 15 95
(N=2) (2 93) (1 4B) (1 70) (099) (2 33) (2 33) (064) (1 06) (0 49) (078) (0 78) (1 98) (1 34)
Hematocrit (%)
MS high-dose 44 82 41 94 4460 45 50 48 38 46 521 47 78 47 042 4572' 44 721 4526 45 96 45 14 44 10 44 96 4424 4524 44 18 4265 42 16 mal

(N=5) (2 48) (3 09) (1 50) (1 37) (249) (5 02) (098) (566) (4 54) (422) (262) (3 68) (2 55) (3 12) (397) (4 28) (5 12) (3 30) (3 19) (1 84) 39-5

MS low-dose 4272 4547 44 73 44 73 43 6 4723 44 77 45 80 4587 45 70 46 73 45 97 48 10
(N=3) (4 19) (249) (2 22) (386) (2 34) (2 48) (340) (3 66) (2 05) (245) (3 61) (2 41) (2 03) ' ' " ' ' tema
35-4

Parkinson high-dose 41 58 40 30 42 55 43 00 47 40 47 351 45 90 49 50 4875 47 65 48 55 52 951 50 201
(N=2) (7 60) (368) (5 30) (3 11) (6 93) (6 15) (028) (4 67) (0 78) (262) (049) (6 86) (4 38)
O
Erythrocytes (Mio/μl)
MS high-dose S OO 464 4 87 504 541 5 20 538 5 37 525 522 5 32 546 5 46 544 561 5 50 565 548 522 4 93 mal

(N=S) (0 26) (040) (026) (0 19) (0 09) (0 55) (028) (069) (0 51) (041) (0 20) (047) (0 22) (033) (041) (0 54) (062) (042) (040) (0 31) 44 5

MS low-dose 4 78 504 4 87 4 92 4 82 5 18 492 5 03 5 06 507 5 20 5 31 5 33
(N=3) (0 39) (0 15) (0 29) (035) (0 18) (0 24) (031) (0 34) (0 11) (0 19) (0 30) (0 25) (0 17) fema

Parkinson high-dose 4 72 4 53 4 81 485 5 33 5 33 524 566 560 5 50 572 6 30 5 96
(N=2) (1 11) (068) (0 83) (057) (0 97) (0 92) (026) (0 83) (0 16) (045) (0 27) (0 98) (0 77
Reticulocytes (%o)
MS high-dose 1330 2060 2940 2340 15 25 1480 11 80 1400 1300 11 20 11 40 10 00 7 80 600 700 6 00 740 8 20 875 11 60 <2£

(N=5) (3 85) (1 67) (7 09) (792) (5 32) (3 27) (4 82) (570) (5 70) (4 97) (3 51) (339) (1 92) (224) (3 16) (2 12) (270) (2 28) (275) (3 36)
MS low-dose 1000 19 33 17 00 12 67 1267 1267 10 67 10 33 1067 10 00 9 33 767 8 00
(N=3) (1 32) (1 15) (6 08) (321) (0 58) (3 51) (1 53) (1 53) (2 52) (2 65) (3 21) (1 15) (1 00) ' " " ' " '
Parkinson high-dose 1575 24 50 42 00 20 50 20 00 1400 20 50 15 50 1600 9 00 10 50 12 50 9 00
(N=2) (5 30) (2 12) (2 83) (2 12) (5 66) (0 00) (120) (0 71) (1 41) (566) (2 12) (2 12) (2 83)
MCV (ϋ)
MS high-dose 8970 90 60 90 20 90 20 89 50 8960 88 60 87 80 8700 85 74 84 80 84 20 8260 81 20 80 20 8060 80 20 80 80 81 75 85 60 81 -S

(W=5J (3 91) (2 97) (3 27) (349) (3 87) (4 04) (288) (2 59) (3 39) (3 54) (2 17) (342) (3 65) (327) (3 11) (2 30) (268) (2 59) (222) (2 61)
MS low-dose 8950 90 17 90 33 91 00 9067 91 33 91 00 91 00 90 33 90 00 90 00 9067 9033
(N=3) (1 50) (2 36) (1 15) (1 73) (1 53) (0 58) (1 73) (1 00) (2 08) (1 73) (1 73) (231) (2 08)
Parkinson high-dose 8875 89 50 89 00 89 00 89 50 8900 88 00 88 00 8700 86 50 85 00 84 50 84 50
(N=2) (4 60) (4 95) (4 24) (424) (3 54) (4 24) (424) (4 24) (4 24) (2 12) (2 83) (2 12) (3 54)
SD= standard deviation, ref range= reference range of laboratory
1 2 denotes number of blood lettiπgs necessary in the respective week (blood letting if hematocπt > 50% in male, > 48% in female patients)

Supplementary Table 1: Routine Laboratory Data - Part 2
BaseEPO weekly EPO bi-weekly EPO-free
line period
Mean Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week
baseline 1 2 3 4 5 6 7 8 9 10 11 12 14 16 18 20 22 24 36 Re
Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Ran
(SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD)

MCH (Pg)
MS high-dose 30 14 30 14 29 52 29 52 29 23 29 22 28 54 2868 27 80 2742 27 32 2694 26 30 25 88 2530 25 40 25 04 25 58 25 75 28 34 26- (N=S) (0 79) (1 17) (1 51) (0 69) (0 85) (1 27) (1 35) (1 43) (1 03) (048) (0 99) (1 58) (1 89) (1 50) (1 29) (1 59) (1 03) (1 10) (0 95) (070)

MS low-dose 2970 2960 30 17 3020 30 20 30 17 3047 30 00 30 10 29 87 29 67 2983 29 40
(N=S) (0 44) (1 04) (0 83) (0 82) (066) (0 76) (083) (0 26) (1 11) (045) (0 93) (0 61) (098) '
Parkinson high-dose 30 25 30 25 29 90 3020 28 80 29 30 2S 60 28 35 28 55 28 60 27 80 2640 26 90
(N=2) (0 92) (1 34) (1 56) (1 56) (0 85) (0 71) (028) (2 19) (0 07) (099) (042) (0 99) (1 27)
Thrombocytes(Tsd/μl)
MS high-dose 259 6 231 8 267 8 2630 326 3 321 4 3108 2756 293 6 3096 3164 315 6 303 6 273 2 281 4 255 4 270 4 2850 287 3 254 4 150- (N=5) (190) (55 8) (257) (51 8) (756) (436) (41 3) (15 7) (42 1) (61 8) (23 1) (33 3) (403) (406) (33 5) (529) (34 9) (51 3) (21 3) (42 8)

MS low-dose 282 8 307 7 301 7 3240 3227 381 0 331 7 328 7 307 7 341 7 338 0 3227 323 3
(N=3) (626) (54 5) (21 5) (57 7) (59 0) (460) (31 6) (40 4) (606) (41 0) (43 5) (48 9) (66 5) " ' ' '
Parkinson high-dose 245 8 244 0 277 0 2280 2505 272 5 294 5 2820 275 0 261 0 290 0 3085 292 0
w (N=2) (58 3) (73 5) (750) (25 5) (61 5) (72 8) (78 5) (21 2) (50 9) (43 8) (93 3) (74 2) (877)
Iron (μmol/l)
MS high-dose 2393 860 8 S4 29 88 12 82 1086 1060 9 50 30 08 25 82 30 38 34 74 24 90 2863 21 92 mε

(N=S) (49) (3 1) (3 3) (7 6) (3 9) (42) (46) (1 4) (17 3) (2 5) (7 1) (187) (6 7) (57) (84) 12- MS low-dose 1470 19 53 14 10 17 20 1743 19 50 17 97
(N=3) (28) (2 5) (36) (2 7) (67) (3 3) (7 5) " ' " fem

Parkinson high-dose 2395 1075 10 10 17 20 13 30 1245 1275 9 55
(N=2) (89) (0 9) (06) (3 0) (9 8) (62) (5 0) (2 5)
Transferrin (μmol/l)
MS high-dose 269 6 246 0 264 4 299 0 301 4 3254 337 0 339 2 333 2 3274 310 8 321 8 3106 305 3 2926 168- (N=S) (44 3) (26 8) (287) (207) (22 4) (55 0) (25 8) (327) (30 8) (34 5) (434) (39 3) (159) (35 7) (25 2)

MS low-dose 232 0 255 0 250 3 2467 2557 2507 267 3 275 3
(N=3) (21 8) (4 2) (21 4) (3 5) (4 7) (85) (6 5) (30 0)
Parkinson high-dose 262 0 251 0 260 5 285 0 301 0 319 0 314 5 312 5
(N=2) (32 5) (62 2) (530) (55 2) (679) (58 0) (60 1) (247)
Transferrin
saturation (%)
MS high-dose 3607 13 96 13 30 39 82 17 31 1368 12 72 11 25 3543 31 83 4022 43 14 31 97 3668 3066 16- (N=5) (8 1) (4 9) (4 2) (104) (6 5) (59) (5 6) (2 0) (19 9) (4 9) (13 2) (23 9) (8 6) (33) (14 8)

MS low-dose 2503 3048 31 28 22 72 26 82 2761 26 94 2543
(N=3) (30) (10 2) (5 6) (5 6) (4 5) (10 4) (37) (7 7)
Parkinson high-dose 3774 1740 1567 24 07 16 56 1651 17 06 12 33
(N=2) (18 5) (2 9) (23) (0 5) (9 2) (10 7) (9 6) (4 1)
SD= standard deviation ref range= reference range of laboratory

Supplementary Table 1 : Routine Laboratory Data - Part 3
BaseEPO weekly EPO bi-weekly EPO-fre
line period
Mean Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week
baseline 1 2 3 4 5 6 7 8 9 10 11 12 14 16 18 20 22 24 36
Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean
(SD) (SD) (SD) (SD) (SD) (SD] (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD)

Fembn (μg/l)
MS high-dose 118 6 97 0 530 39 0 174 14 6 11 0 94 206 19 4 21 8 29 8 31 0 33 5 40 2

(N=S) (55 9) (534) (285) (17 1) (8 1) (96) (3 0) (2 5) (9 3) (8 S) (10 0) (15 1) (153) (20 1) (25 0)

MS low-dose 162 3 146 0 126 7 84 3 75 3 79 7 567 547
(88 3) (834) (769) (56 3) (474) (70 9) (41 1) (35 1) '
Parkinson high-dose 177 8 159 0 775 42 5 20 5 13 5 14 0 120
(N=2) (7 4) (O D) (106) (2 1) (4 9) (2 1) (0 0) (1 4)
Soluble transferrin
receptor (mg/l)
MS high-dose 3 36 6 54 11 01 11 41 775 6 70 7 58 3 80

(N=5) (0 59) (2 11) (248) (2 59) (2 18) (1 65) (094) (0 71)

MS low-dose 2 29 3 66 402 4 17 4 11
(N=3) (0 14) (0 74) (056) (0 79) (1 16) "
Parkinson high-dose 260 673 994 9 95 11 09
κ> (N=2) (082) (247) (1 32) (0 42) (1 19)
CRP (mg/l)
MS high-dose 2 15 2 12 676 2 33 200 2 34 2 00 2 14 2 34 2 00 2 06 2 02 3 34 2 00 260

(N=S) (0 34) (0 27) (863) (047) (000) (0 56) (0 00) (0 31) (0 76) (000) (0 13) (0 04) (300) (000) (093)

MS low-dose 3 83 2 95 473 6 70 427 1693 2 80 3 33
(N=3) (263) (1 34) (473) (7 54) (393) (25 9) (1 39) (231) '
Parkinson high-dose 200 2 50 255 200 200 2 00 2 00 2 00
(N=Z) (0 00) (0 71) (078) (0 00) (000) (0 00) (0 00) (0 00)
Erythrocyte sedimentation rate (mmHh)
MS high-dose 5 50 675 3 60 2 40 2 80 220 6 80 375 2 60 4 00 1 67 3 60 1 40 2 20 2 00 320 5 00 560

(N=5) (268) (802) (2 07) (1 52) (1 79) (1 64) (7 26) (550) (1 82) (5 20) (1 15) (1 67) (055) (1 10) (1 22) (335) (3 37) (2 07)

MS low-dose 9 83 500 2 50 2 00 6 67 667 7 67 733 4 33 6 33 5 00
(N=3) (7 08) (529) (2 12) (1 00) (3 79) (208) (8 96) (4 16) (2 31) (6 81) (4 36)
Parkinson low-dose 12 00 10 50 17 00 5 00 2 00 3 50 3 00 1 50 4 50 3 50 3 00
(N=2) (9 19) (778) (22 6) (4 24) (1 41) (2 12) (2 83) (071) (0 71) (0 71) (2 83)
S100B (μgll)
WIS high-dose 0 07 0 06 005 0 05 0 07 0 08 0 07 007 0 07 007 0 06 0 07 0 07 0 07 007 0 07 0 10 008 0 06 0 09

(N=5) (0 04) (0 02) (003) (005) (005) (0 04) (0 04) (004) (0 03) (002) (0 04) (0 03) (003) (002) (003) (0 03) (0 03) (003) (0 04) (004)

MS low-dose 0 06 005 0 06 0 07 0 06 0 06 007 0 05 006 0 06 0 05 0 04
(N=3) (001) ' (001) (0 02) (0 02) (0 01) (0 02) (002) (0 04) (002) (0 01) (0 02) (002)
Parkinson low-dose 008 0 08 008 0 09 0 11 0 07 0 06 0 10 0 07 0 10 0 07 0 09 008
(N=2) (0 00) (0 00) (002) (0 03) (0 01) (0 01) (0 01) (004) (0 02) (002) (0 00) (0 04) (002)
SD= standard deviation, ref range= reference range of laboratory



P2 denotes significance from baseline to week 36 Statistical analysis Friedman test (performed in high-dose EPO MS patients only)

Week Week Week Week
Mean Mean Mean

problems during baseline measurement

Supplementary Table 3: Neuropsychological Test Data (original) - Part 1


Ul



RBANS Repeatable Battery for the Assessment of Neuropsychological Status
PT denotes significance from baseline to week 24, Pi denotes significance from baseline to week 36 Statistical analysis Friedman test (performed in high-dose EPO MS patients only)

Su lementar Table 3: Neuro s cholo ical Test Data ori inal - Part 2


RBAN Repeatable Battery for the Assessment of Neuropsychological Status, WCST-64 Wisconsin Card Sorting Test - 64 Card Version
Pt denotes significance from baseline to week 24, P2 denotes significance from baseline to week 36, Statistical analysis Friedman test (performed in high-dose EPO MS patients only)