The present invention relates to the use carbamylated erythropoietin for the treatment of Friedreich's Ataxia.

BACKGROUND OF THE INVENTION

Friedreich's ataxia (FRDA) is the most frequent of the inherited ataxias with an incidence of about 1 in 50,000 people. FRDA is an autosomal, recessive disorder charac- terized by degeneration within the cerebellum and upper medulla and leads progressive gait and limb ataxia. Also, other organs such as the heart and pancreas become affected as reflected by hypertrophic cardiomyopathy and diabetes. The disease is considered to arise from a mutation, an expansion of a GAA repeat, within the first in- tron of the frataxin gene, attenuating the expression and levels of frataxin. Reduced levels of the mitochondrial protein frataxin hampers synthesis of iron-sulfur clusters which in turn leads to a decreased cellular respiration and a build up of iron storage within the mitochondria, as well as enhanced production of free oxygen radicals. Reduced capacity for mitochondrial oxidative phosphorylation and associated oxidative stress may play a role in the disease mechanism, indirectly supported by the observa- tions that the organs affected the most are those of high energy-turnover, such as brain and heart.

Disease onset is mainly within the adolescents, in some reports about 75% are diagnosed under 18 years of age (Wilson et al. (2007). Eur J Neurol 24; 14:1040-7), but also children are diagnosed.

The current treatments approaches for FRDA includes coenzyme Qi ₀ , vitamin E, Ide- benone and L-carnitine, however none are of these treatments have proven effective or are widely used.

WO 2006/050819 discloses EPO for treatment of FRDA. Furthermore, in a previous published pilot study with EPO in FRDA, the patients were dosed with 5,0001 U (5,0001 U EPO corresponding to 40μg protein), 3 times per week for eight weeks (Boesch et al. Ann Neurol. 2007;62(5):521 -4). The treatment was well tolerated apart from the expected haematological side effects. EPO is believed to act through the classical EPO-receptor, however, the present invention shows that the effect on the frataxin levels are not mediated through this receptor, because frataxin levels also increases in cells which do not express EPO receptor (THP-1 cells). More importantly, the inventors have found that CEPO, which is known not to bind the classical EPO-receptor, have the same dosage dependent increase in frataxin levels. CEPO has the advantage of not stimulating the erythropoiesis and is thus a more attractive alternative in long term treatment regimes, such as FRDA. Moreover, the inventors of the present invention have found that by administering car- bamylated erythropoietin to FRDA patients in a dose ranging from about 300 μg to about 400 μg, preferably in a dose of 325 μg, a safe and efficacious treatment is obtained.

SUMMARY OF THE INVENTION

The invention relates to carbamylated erythropoietin for use in the treatment of Frie- drich's ataxia. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 and 2: Effect of recombinant EPO on frataxin-expression in K562 and THP-1 cells:

K562 cells (Fig 1 ) or THP-1 cells (Fig 2) were incubated with increasing concentration of recombinant EPO for 24 hours. Frataxin expression in cell lysates was detected by an electrochemical luminescence assay . Shown are the meansiSEM (n=3).

Figure 3 and 4: Effect of carbamylated erythropoietin on frataxin-expression in K562 and THP-1 cells: K562 cells (Fig 3) or THP-1 cells (Fig 4) were incubated with increasing concentration of carbamylated erythropoietin (CEPO) for 24 hours. Frataxin- expression in cell lysates was detected by an electrochemical luminescence assay . Shown are the meansiSEM (n=3).

Figure 5 and 6: Effect of carbamylated erythropoietin on frataxin-expression in primary lymphocytes from control and Friedreich's ataxia patients: Primary lymphocytes from control (Fig 5) or Friedreich's ataxia (Fig 6) patients were incubated with increasing concentration of carbamylated erythropoietin (CEPO) for 24 hours. Frataxin expression in cell lysates was detected by an electrochemical luminescence assay. Shown are the meansiSEM (n=3). Figure 7: Effect of EPO and CEPO in proliferating human SH - SY5Y neuroblastoma cells. A.Western blot analysis of frataxin level in proliferating SH - SY5Y cells incubated with EPO or CEPOfor 24h. B. Densitometric analysis of frataxin expression from three independent experiments. Density of the frataxin band of the untreated SH - SY5Y in the absence of either EPO or CEPO wassetat 100% of frataxin expres- sion. Differences were examined for statistical significance using the Stu- dent'st - test. ^* p<0,05 ^** p<0,005.

Figure 8: Effect of EPO and CEPO in differentiated human SH - SY5Y neuroblastoma cells. A. Western blot analysis of frataxin level in differentiated SH - SY5Y cells incu- bated with EPO or CEPO for 24h. B. Densitometric analysis of frataxin expression from three independent experiments. Density of the frataxin band of the untreated SH - SY5Y in the absence of either EPO or CEPO wassetat 100% of frataxin expression. Differences were examined for statistical significance using the Student's t- test. ^* p<0,05 ^** p<0,005

Figure 9: Effect of EPO and CEPO in human olfactory mucosa stem cells from healthy subject and a FDRA patient. A. Western blot analysis of frataxin level in cells treated with EPO or CEPO for 24h. B. Densitometric analysis of frataxin expression from three independent experiments. Density of the frataxin band of the untreated cells from healthy donors wassetat 100% of frataxin expression. Differences were examined for statistical significance using the Student's t-test. ^* p<0,05 ^** p<0,005.

DETAILED DESCRIPTION OF THE INVENTION

Frederick's ataxia (FRDA) is an autosomal-recessive disorder caused by mutations in the gene encoding the mitochondrial protein frataxin. The mutations cause a dramatic reduction in the expression of frataxin leading to iron accumulation, production of free radicals and respiratory chain dysfunction. Over the years the FRDA-patients experiences a progressive gait and limb ataxia, dysarthria, lower-limb areflexia, decreased vibration sense and muscular weakness in the legs. Non-neurological signs include hypertrophic cardiomyopathy and diabetes mellitus.

It has been shown that recombinant human erythropoietin (EPO) can increase frataxin expression in lymphocytes from patients with FRDA (Boesch et al. Ann Neurol. 2007;62(5):521 -4) and the treatment has been shown to give a favourable outcome with regards to the frataxin levels and oxidative stress biomarkers.

Additionally, it has been discovered that EPO exerts a protective effect in response to insults in various tissues, including the brain cells (such as e.g. neurons and astro- cytes) (Masuda et al. J Biol Chem. 1993;268(15):1 1208-16; Konishi et al. Brain Res. 1993;609(1 -2):29-35; Diaz et al. J Neurochem. 2005;93(2):392-402; Sakanaka et al. Proc Natl Acad Sci USA.1998;95(8):4635-40). However, a major drawback by using EPO for treatment in non-anaemic patients is its effects on the haematopoietic system, where EPO increases endothelial activation, platelet count, platelet reactivity and leu- kocyte count even after a few doses. This effect gives an increased risk of thromboembolic events to the patient (Stohlawetz et al. Blood. 2000;95(9):2983-9; Homoncik et al. Aliment Pharmacol Ther. 2004;20(4):437-43; Kirkeby et al. Thromb Haemost. 2008;99(4):720-8). EPO is believed to act through the classical EPO-receptor, how- ever, the present invention shows that the effect on the frataxin levels is not mediated through this receptor, because frataxin levels also increases in cells which do not express EPO-receptors (THP-1 cells, Example 5).

CEPO is a novel cytoprotective compound currently in development for acute ischemic stroke. It is chemically modified EPO by carbamylation of lysine residues (Leist et al. Science. 2004;305(5681 ):239-42, hereby incorporated by reference in its entirety) and does not bind to the EPO-receptor and therefore avoids the haematopoietic side- effects associated with EPO treatment. The inventors of the present invention have found that CEPO have the same dosage dependent increase in frataxin levels as EPO (Example 5), and CEPO is thus a more attractive alternative for the treatment of FRDA than EPO due to its safety profile.

Furthermore, as shown in e.g. figure 7, 8 and 9, the present inventors have found that CEPO

significantly increase frataxin expression both in human neuron - like cells and in olfactory mucosa stem cell from FRDA patients in a more effective way than EPO. The lack of severe side effects of CEPO and its ability to raise frataxin levels may be advantageous for clinical use. In the present invention, the term CEPO is intended to include any variant or derivative of carbamylated EPO (e.g described in US 2004157293 or Science, Vol. 35, pp 239- 242 or WO 2006/050819 herby incorporated by reference in its entirety), that is a variant or derivative of EPO in which at least one of the primary-amino groups (the lysines and the N-terminal group) of the protein is carbamylated. In particular, the invention relates to CEPO with an amino acid sequence as depicted below in table 1 (SEQ ID NO 2) or comprising an additional arginine in the C-terminal end (SEQ ID NO 1 ), or a sequence which is 95%, 98% or 99% identical to SEQ ID NO 1 or 2. Table 1

PPRLI CDSRVLERYLLEA

21 EAE N ITTGCAE HCS LN EN IT

41 VP DTKVN FYAWKRMEVGQQA

61 VEVWQG LALLSEAVLRGQAL

81 LVNSSQPWEPLQLHVD AVS

101 G LRSLTTLLRALGAQKEAIS

121 PPDAASAAPLRTITADTFR

141 LFRVYSN FLRG L LYTG EA

161 C RT G D

Table 1: Potential carbamylation sites are shown in bold cursive and conventional amino acids in arial font.

There are nine potential carbamylation sites as shown in table 1 : Alanine at position 1 and Lysine at positions 20, 45, 52, 97, 116, 140, 152 and 154. Accordingly, the invention relates to CEPO in which at least one or more of the of the amino acids selected from the group comprising alanine at position 1 and lysine at positions 20, 45, 52, 97, 116, 140, 152 and 154 (as shown in table 1) are carbamylated.

CEPO may be produced by carbamylating EPO e.g. as disclosed in WO2006/002646, hereby incorporated by reference in its entirety. Briefly, purified human EPO (or alter- natively recombinant human EPO or biologically or chemically modified human EPO) can be mixed with an approximately equal volume of 1M potassium cyanate/0.25M potassium tetraborate, pH about 9.0, and incubating at about 29°C for about 24-48 hours. The reaction can be stopped by cooling to room temperature, adding 3M ammonium sulphate/150m M Tris-HCI, pH 7.5 and hydrophobic interaction chromatography.

In it broadest concept the invention relates to CEPO for use in the treatment of FRDA. CEPO may be given in a dose ranging from about 300 μg to about 400 μg, preferably in a dose of 325 μg, given parenterally, such as e.g. subcutaneous, intramuscular, intrathecal, intravenous and intradermal routes, as a single active ingredient or in combi- nation with other active ingredients, such as e.g. coenzyme Qi ₀ , vitamin E, Idebenone and L-carnitine. CEPO may be administered at least 1 time per week, such as e.g. 2 times per week, 3 times per week or 4 times per week, for at least one week or more, such as e.g. for at least two weeks or more. Alternatively, the dose of CEPO used may be expressed in terms of μg per kg. As shown in Example 4, the dose of CEPO will range between about 0.5 and 50 μg kg body weight. However, it is preferred that the dose of CEPO is in the range of about 2 and about 10 μg kg body weight, preferably about 4 to about 6 μg kg body weight or about 5 μg kg body weight.

It is envisaged that the administration of CEPO may be combined with one or more agents used in the treatment of FRDA such as e.g. coenzyme Q10, vitamin E, Idebe- none, L-aetylcarnitine and/or L-carnitine. CEPO may be comprised in a pharmaceutical composition. The pharmaceutical compositions of the invention may comprise a therapeutically effective amount of CEPO and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized for- eign pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solu- tion is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from so- dium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of CEPO, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Other components that may be present in such compositions include water, alcohols, polyols, glycerine and vegetable oils, for example. Compositions adapted for parenteral administration may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, e.g., sterile saline solution for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. In one embodiment, an autoinjector comprising an injectable solution of a compound of the invention may be provided for emergency use by ambulances, emergency rooms.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically-sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile saline can be provided so that the ingredients may be mixed prior to administration.

The pharmaceutical compositions may be specifically formulated for administration by any suitable route such as parenteral, including subcutaneous, intramuscular, intrathecal, intravenous and intradermal routes. It will be appreciated that the route will depend on the general condition and age of the subject to be treated, the nature of the condi- tion to be treated and the active ingredient.

In a preferred embodiment the pharmaceutical composition is for intravenous administration and comprises CEPO, a buffer such as citric acid, an isoosmotic modifier such as sodium chloride and/or sodium citrate, a pH modifier such as sodium hydroxide and a solvent such as sterile water.

EXAMPLES

Example 1

Safety

CEPO has been administered to both rats and cynomolgus monkeys in 4-week toxicology studies at doses between 0 and 500C^g/kg per day. The kinetics showed some differences both between species and sex.

In rat no major sex-related difference was observed in C _max and AUC0-24h at 200 and 1000 μg kg per day. However, at 5000μg kg per day, C _max and AUC0-24h were 2 to 3 times higher in male rats than in female rats. In monkeys no major sex-related difference was observed in C _max or AUCs at 200μg kg per day. At 1000μg kg per day, females were 2 to 10 times more exposed than males, while at 5000μg kg per day, males were 5 to 13 times more exposed than females, irrespective of the occasion. Some ac- cumulation of CEPO was evident in both species after the 4-week administration period. The increase in systemic exposure was more than dose-proportional in males and less than dose-proportional in females.

Based on preclinical safety studies CEPO appears a safe compound with no clinically significant adverse effects. The central nervous, respiratory, and cardiovascular system studies performed demonstrated that CEPO were well tolerated in rats and cynomolgus monkeys. Similarly, the repeated-dose toxicity studies performed with CEPO demonstrated that CEPO was well tolerated at all doses tested in rats and cynomolgus monkeys. Treatment with CEPO did not result in any meaningful treatment-related changes in the parameters investigated: mortality, clinical signs, ECG (cynomolgus monkeys only), ophthalmoscopy, weight and food consumption, haematology, coagulation and serum clinical chemistry, urinary parameters, organ weight changes, macroscopic and histological findings. CEPO was recognized as a foreign protein in both rats and cynomolgus monkeys and the antibody formation was an expected finding with little predictive value for the potential immunogenicity of CEPO in humans. Therefore, a relative immunogenicity evaluation was performed in lymphocytes from 50 human donors. Results from this study showed that CEPO was less immunogenic than EPO, and safe. Example 2

Safety in humans

CEPO was tested in humans in a single dose, dose-escalation study of the safety, tolerability and pharmacokinetics of CEPO in acute ischemic stroke. The study investi- gated i.v. doses of CEPO in the range of 0.005-5C^g/kg in 5 cohorts and the total number of completed patients was 16 (1 1 CEPO, 5 placebo). The assessments performed included neurological examination, vital signs, ECG, AE monitoring, blood biochemis- try/haematology, blood sampling for explorative biomarkers, drug analysis and antibodies.

No safety concerns were found and there were no reports of Serious Adverse Events considered related to CEPO. Furthermore, no antibodies towards CEPO were detected. Example 3

Pharmacokinetics

Pharmacokinetic analysis from 3 doses (0.5, 5, and 50μg kg CEPO) in a single dose, dose-escalation study in patients with acute ischemic stroke, showed that all patients dosed with CEPO had a plasma concentration peak appearing 0.25 to 1.5h after the start of infusion. The data indicated a proportional increase in mean exposure with increasing dose from 0.5 to 50μg kg CEPO. All patients had low clearances and small volumes of distribution. The terminal elimination half-life was estimated to 9 to 14h for all patients. The pharmacokinetics of CEPO appears to be linear over the dose interval tested.

Example 4

Ongoing Clinical Phase Ha Study

In a previous published pilot study with EPO in FRDA, the patients were dosed with 5,000IU, 3 times per week for eight weeks (Boesch et al. Ann Neurol. 2007;62(5):521 - 4). The treatment was well tolerated apart from the expected haematological side effects, which CEPO does not induce.

A dose of 325μg CEPO was selected for this study which corresponds to the interim dose level of 5μg kg used in Example 3. Also, it is 8 fold higher than that of EPO ad- ministered to FRDA patients (5,0001 U EPO corresponding to 40μg protein) (Boesch et al. Ann Neurol. 2007;62(5):521 -4). In vitro experiments on frataxin levels suggest that EPO and CEPO have approximately the same molar potency. Haematological side- effects are not associated with CEPO which was safe and well tolerated in stroke pa- tients at doses up 50μg kg. Assuming an average adult weight of 70kg the 50μg kg CEPO dose corresponds to a 3,500μg CEPO dose. The 325μg CEPO dose in the present study is 10 fold below the maximum dose used in the Example 3.

Primary objective is to evaluate the safety and tolerability of 2 weeks treatment with CEPO in patients with Friedreich's Ataxia.

The first 3 patients will be enrolled sequentially with at least 1 week between the randomizations. The patients will receive i.v. injections of 325μg CEPO or placebo dosed 3 times per week for two weeks. After the recruitment of 6 patients (4 active and 2 pla- cebo) the recruitment will be stopped, and the safety and tolerability evaluated. The patient enrolled in the study after the treatment phase be followed up after 4 weeks for safety assessments.

The following efficacy and safety parameters will be evaluated: Frataxin, 8-OHdG, per- oxides, MDA, ataxia scales (SARA, FARS), CGI-I/S, adverse events, clinical safety laboratory tests, vital signs, physical and neurological examinations and antibodies. For pharmacokinetic and pharmacodynamic evaluations samples from CEPO population will be obtained during the study, and estimates of the average plasma concentrations, exposure and clearance will be calculated.

Example 5

Carbamylated erythropoietin increases frataxin expression in vitro

Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by decreased expression of the mitochondrial protein frataxin, described to be an iron chaperone for the

assembly of iron-sulphur clusters in the mitochondria, causing iron accumulation in mitochondria, oxidative stress and cell damage. Materials and Methods: In the experiments EPO receptor expressing human erythro- leukaemic K562 cells, and human monocytes THP-1 cells, a cell line which des not express EPO-R, as well as isolated primary lymphocytes from control and FRDA patients were incubated with different concentrations of recombinant EPO or CEPO which does not bind to the classical EPO receptor. Frataxin-expression was detected by a electrochemical luminescence assay (based on the principle of an ELISA) and real time RT- PCR for frataxin-mRNA.

Results: It was shown that recombinant EPO increases frataxin-expression in K562 cells expressing EPO receptor as well as in the cell line THP1 , which does not express EPO receptor (Figure 1 and 2). These results were confirmed by the finding that car- bamylated erythropoietin (Figure 3 and 4), which cannot bind to the classical EPO receptor increased frataxin expression in the same concentration range as recombinant EPO. In addition we show that recombinant EPO as well as CEPO significantly increases frataxin-expression in primary lymphocytes from control and Friedreich's ataxia patients (Figure 5 and 6).