USE OF THE BASIC FORM OF RECOMBINANT HUMAN ERYTHROPOIETIN IN THE TREATMENT OF A PATIENTS WITH SPINOCEREBELLAR ATAXIA WITH CAG REPEAT MUTATIONS

Abstract

The present invention relates to use of pharmaceutical compositions that have as active ingredient the basic form of recombinant human erythropoietin (rhEPO) and that are administered nasally for the treatment of spinocerebellar ataxia (SCA) with mutations of CAG repeats type, particularly of SCA type 2. Another object of the present invention is a method for stratifying patients into responders and non-responders to treatment with the basic form of rhEPO.

Claims

1-8. (canceled)

9. A method for treating spinocerebellar ataxia (SCA) with mutations of CAG repeats type in a patient, the method comprising nasally administering to the patient a basic form of recombinant human erythropoietin (rhEPO).

10. The method according to claim 9, wherein the SCA is type 2.

11. A method of stratification of spinocerebellar ataxia (SCA) with mutations of CAG repeats type patients into responders or non-responders to treatment with a basic form of recombinant human erythropoietin (rhEPO) which involves administration of said rhEPO to patients in which: the number of CAG repeats correlates with severity of the SCA and/or, the number of CAG repeats correlates inversely with concentration of rhEPO in cerebrospinal fluid (CSF) of patients.

12. The method according to claim 11, wherein the non-responder patients show no improvement in Spinocerebellar ataxia Functional Index (SCAFI) scale after one year of treatment.

13. The method according to claim 11, wherein the basic form of the rhEPO is administered three times a week in a dose range from 0.1 mg/ml to 4 mg/ml.

14. The method according to claim 11, further comprising individually optimizing the basic form of rhEPO dosage regimen based on: a correlation between the number of CAG repeats with the severity of the disease and/or, an inverse correlation between the number of CAG repeats and the concentration of rhEPO in CSF of patients.

15. The method according to claim 13, wherein the basic form of rhEPO is administered for a time period from 6 to 12 months.

16. A method for treating SCA patients with mutations of the CAG repeats type wherein said treatment comprises stratifying patients into responders and non-responders to treatment by evaluating whether: said patient's number of CAG repeats correlates with the severity of the disease and/or, said patient's number of CAG repeats correlates inversely with the concentration of erythropoietin (EPO) in the cerebrospinal fluid (CSF) of said patient.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0131] FIG. 1. Qualitative analysis of the images of the Clasping Test showing the position taken by the animals of each group. As can be seen the position differed according to the estimate of the area between the four limbs and the distance from the axis of each animal.

[0132] FIG. 2. Comparison of survival of the different study groups. Statistically significant differences between the group of ataxic animals treated with placebo and the group of animals treated with the nasal formulation of the basic form of the hrEPO and healthy controls are observed, these differences become more evident as time goes by, no differences between the group of ataxic mice treated with hrEPO and the healthy control were observed. Different letters were used for statistically significant differences.

[0133] FIG. 3a. Decreased levels of EPO in the CSF of patients with SCA2, significant differences between SCA2 patients and healthy controls are shown.

[0134] FIG. 3b. Relationship between decreased EPO levels in the CSF with molecular damage. An inverse correlation is observed, that is, as the number of CAG repeats in SCA2 patients increases, EPO levels decrease in the CSF.

[0135] FIG. 4. EPO concentration measured by ELISA in the CSF of the patients analyzed.

EXAMPLES

Example 1

Demonstration of the Neuroprotective Effect of rhEPO in the Nasal Formulation of SCA Type-2 Ataxic Mice.

[0136] Twenty hybrid mice weaned at 21 days of age, descendants of parental males of the OF1 line and females of B6D2F1 line, homozygous dominant for SCA-2 gene were used. They were separated by sex into 2 groups, 10 females and 10 males, and these two groups further divided into two experimental groups. As healthy controls of the experiment five male mice of the OF1 line and five female mice of line B6D2F1 were used.

[0137] A 1 mg/ml of the basic form of rhEPO was administered nasally to one of the groups of ataxic female mice and one of the groups of ataxic males. The remaining two groups of ataxic animals were administered placebo. This administration was performed on alternate days, during morning time for 12 months. Healthy controls received no treatment.

[0138] The neurological behavior of the animals and clinical course of the disease was analyzed. The neurological behavior was studied by means of the Clasping Test, in order to perform this test the animals received training for a time period of 30 days prior to it. The protocol of the test consists of hanging the animal by the tail with his head down and analyzing the position adopted, taking into account the position of the limbs with relation to the axis (position of equilibrium or fetal), and a qualitative assessment by images. The test was performed every month to analyze the equilibrium position. The clinical course of the disease was analyzed by using the parameter survival.

[0139] For statistical processing of the results the Statgraphics Plus 5.1 software was used.

[0140] FIG. 1 shows images of the position taken by the animals in each group. Ataxic animals treated with placebo mostly adopted a position similar to fetal position showing reduced mobility; healthy controls adopted various unstable positions with spread legs, constantly seeking equilibrium by moving the legs; and the animals treated with the nasal formulation of rhEPO, showed higher variability of positions, going from fetal position to spread legs position, with a majority of mice adopting intermediate positions.

[0141] FIG. 2 shows survival in the different animal groups. As shown by the figure as times goes by the differences between the group of untreated ataxic animals and the other two groups significantly increase. The result obtained is obviously attributable to the described evolution of the disease and confirms the effectiveness of the treatment with rhEPO in modifying the clinical course of the disease.

Example 2

Quantification of the Number of CAG Repeats in Patients with SCA2.

[0142] DNA was isolated from 10 ml of peripheral blood of each patient. Amplification of the repetitive sequence of CAG in SCA2 locus was performed by PCR using for this purpose the DAN1 (5-cgtgcgagccggtgtatggg-3, C and 5 fluorescence) and UH10 (5-ggcgacgctagaaggccgct-3) primers.

[0143] For the determination of the exact number of CAG repeats an analysis of fragments was performed, for this purpose 4 l of products from the PCR were combined with 3 l of loading dye and 1 l of each ALFexpress Sizer of 100 and 300 base pairs (Amersham Biosciences).

[0144] This mixture was heated at 95 C. for three minutes before separation with 8% ReproGel gel electrophoresis using a gene sequencer (ALFexpress II, Amersham Biosciences). Size of CAG repeats was determined by comparison using an ALFexpress Sizer of 50-500 base pairs, which has a collection of ten kinds of fragments differentiated by increments of 10 base pairs as an external standard of molecular weight and an ALFexpress Sizer of 100 and 300 base pairs as internal standard of molecular weight. To control the electrophoretic run and data collection ALFwin Sequence Analyser 2.00 software was used and for data analysis and estimation of the allele size AlleleLinks 1.00 (Amersham Biosciences) software.

[0145] An average of 39.4 CAG repeats was obtained, the minimum number of repetitions was 35 and the maximum was 55.

Example 3

Decreased Levels of EPO in the CSF of Patients with SCA2.

[0146] EPO concentration in CSF was determined in a sample of 20 patients with SCA2 and in 20 healthy controls by Sandwich ELISA (EPO ELISA version 08, Roche). The results showed a significant decrease (mean: 0.12 mIU/mL), which represents 95% decrease of normal levels of EPO in the CSF as compared to the control group (mean: 2.65 mIU/mL) (FIG. 3a). Forty-five percent of the patients analyzed had values below the detection limit of the technique and in the rest the values had decreased as compared to healthy controls, the highest concentration value obtained in the group of patients with SCA2 was 0.396 mIU/mL. These biochemical changes are more evident in patients with greater polyglutamine expansions (FIG. 3b) and/or longer duration of disease.

Example 4

Evaluation of the Effect of Treatment with the Nasal Formulation of the Basic Form of rhEPO in Patients with SCA2.

[0147] A phase I-IIa, single-center, randomized, placebo-controlled, double-blind clinical trial, in patients with a clinical diagnosis of SCA2 and molecularly with a quantity higher or equal to 32 repetitions of the trinucleotide CAG, in early stages (I-II) of the disease was performed. In the trial 34 patients were randomly divided into two study groups; a group 17 patients received the basic form of rhEPO and the other 17 patients received placebo.

[0148] RhEPO was administered nasally to a concentration of 1 mg/mL three times a week, until six months of treatment were completed. A dose of 0.25 ml was applied first in each nostril and 15 minutes later a second dose of the same volume was again applied, for a final volume in each nostril of 0.5 mL and a total volume of 1 mL administered each day of treatment.

[0149] The primary endpoint to measure the effect of the product administered was the SCA Functional Index (SCAFI). Patients were evaluated before the beginning and end of the treatment stage and changes were determined by means of the SCAFI. The decrease in time of the average of the three scores (Z) that comprise it allows to monitor the progression of the disease.

[0150] The data analysis shows that at the end of treatment, in the group of patients treated with the basic form of rhEPO there is an increase in the values of the mean of the differences of the SCAFI, due to the decrease of values of this scale at end of treatment; contrary to what happens in the placebo group. The differences between the means of both groups are statistically significant (Table 1). The number of patients who completed the trial was different from baseline due to voluntary abandonment of the trial and in two cases due to treatment discontinuation caused by the progression of the disease, in both cases the patients who discontinued treatment coincided with the ones who had 52 and 55 CAG triplet repeats.

TABLE-US-00001 TABLE 1 Results of measurement of SCAFI parameter before and after completion of the trial. Mean of Variable Group n differences Significance SCAFI Treated with rhEPO 13 0.75 0.04 Placebo 16 0.41

[0151] EPO concentration in the CSF of the patients was determined by Sandwich ELISA (EPO ELISA version 08, Roche). FIG. 4 shows the increase of EPO concentration in patients treated with the basic form of rhEPO as compared to the levels they had before treatment and to those of patients who received the placebo.

[0152] These results indicate that in the group treated with the basic form of the rhEPO there is a significant clinical improvement associated with improvement of motor symptoms which evidences the influence of treatment on the most significant parameters that characterize the disease and consequently in the deceleration of disease progression.