Method of treating relapsing-remitting multiple sclerosis using arsenic trioxide

Abstract

The present invention relates to a method for preventing or treating multiple sclerosis, particularly relapsing-remitting multiple sclerosis using arsenic trioxide.

Claims

1. A method of treating relapsing-remitting multiple sclerosis in a patient, comprising administering from 0.05 to 0.5 mg of arsenic trioxide per kilogram of bodyweight per day to the patient.

2. The method of claim 1, wherein the arsenic trioxide is administered intravenously.

3. The method of claim 1, wherein the arsenic trioxide is administered orally.

4. The method of claim 1, wherein administration of arsenic trioxide to the patient delays new attacks of relapsing-remitting multiple sclerosis in the patient.

5. The method of claim 1, wherein administration of arsenic trioxide to the patient reduces the severity of relapsing-remitting multiple sclerosis in the patient.

Description

(1) Other subjects and advantages of the invention will emerge on reading the detailed description illustrated by means of the drawings in which:

(2) FIG. 1 shows EAE severity (mean clinical score) vs time.

(3) FIG. 2 shows change in body weight vs time.

(4) FIG. 3 shows IL-6 concentration (average of triplicates) in spleen cultures after 72 hours of incubation with various concentrations of MOG35-55 (0, 0.7, 2.2, 6.7 and 20.0 g/mL).

EXAMPLES

Example 1

(5) Summary

(6) The study objective was to evaluate the effects of arsenic trioxide (1 mg/ml), administered i.v., QD, starting 7 days before EAE induction, on EAE development in the MOG35-55-induced prophylactic/therapeutic EAE model, known as the more robust and direct animal model to trigger and observe the autoimmune cascade involving the central nervous system and ensuing demyelination.

(7) The inventors' goal was to verify the compound efficacy on the inflammatory processes active in early MS development (RRMS), thus focusing on the early manifestations of the RRMS form of the disease, since the progressive form is now thought to involve neurodegenerative mechanisms more than inflammatory or strictly autoimmune mechanisms.

(8) EAE was induced by MOG35-55/CFA immunization and pertussis toxin injection in C57BL/6 mice.

(9) There were 2 experimental groups with 12 mice per group. Mice were treated prophylactically.

(10) Arsenic trioxide was efficacious at postponing EAE onset and reducing disease severity in this study. In addition, the efficacy was maintained 7 days after the treatment was stopped.

(11) Material and Methods

(12) Model Background

(13) Experimental autoimmune encephalomyelitis (EAE) is the most commonly used mouse model of human multiple sclerosis (MS). Because of its many similarities to MS, EAE is used to study pathogenesis of autoimmunity, CNS inflammation, demyelination, cell trafficking and tolerance induction.

(14) EAE is characterized by paralysis, CNS inflammation and demyelination. EAE is mediated by myelin-specific CD4+ T cells, but CD8+ cells and B cells may also play a role in some models of EAE.

(15) EAE is induced in C57BL/6 mice by immunization with MOG35-55 or MOG1-125 in CFA emulsion followed by administration of PTX in PBS. The emulsion provides antigen which initiates expansion and differentiation of MOG-specific autoimmune T cells.

(16) PTX enhances EAE development by providing additional adjuvant and facilitating entrance of autoimmune T cells into the CNS.

(17) FTY720 is the most commonly used positive control in this model.

(18) Chronic EAE develops in C57BL/6 mice after immunization with an emulsion of MOG35-55/CFA or MOG1-125/CFA followed by injection of pertussis toxin. This model is used to test the potential of compounds to prevent or mitigate EAE disease. It can be run with the compound dosed from the time of immunization (prophylactic treatment), or with the aim of reversing the course of disease and facilitating recovery by dosing the compound from the time of EAE onset (therapeutic treatment).

(19) The MOG1-125 antigen is used for testing therapeutics which specifically target B-cells; EAE development after immunization with MOG1-125 is reported to be impaired in B-cell deficient mice.

(20) The model uses female C57BL/6 mice of age 10 to 14 weeks at the start of the study. Typically, EAE develops 8 to 18 days after immunization. EAE development is usually followed for 4 weeks (28 days) after immunization.

(21) Stress reduces mouse susceptibility to EAE. Aside from any compound effects, the administration of treatment during the disease induction period (0-10 days after immunization) postpones disease onset and reduces disease severity. This is due to the stress of compound administration and the effects of the vehicle on the mice. The more frequent the administration and the less tolerated the vehicle, the greater the impact on disease development.

(22) The stress of treatment and administration of vehicle has less effect on disease development after clinical signs of EAE have appeared.

(23) EAE Induction

(24) Mice were acclimated to our facility for 1 week prior to the start of the study.

(25) The study used a total of 24 female C57BL/6 mice (Taconic Farms, 10 weeks old).

(26) EAE was induced in all mice as follows:

(27) Day 0, Hour 0Immunization with MOG.sub.35-55/CFA

(28) Day 0, Hour 2Injection of pertussis toxin

(29) Day 1, Hour 02.sup.nd injection of pertussis toxin (24 hours after initial immunization)

(30) Mice were injected subcutaneously at two sites in the back with the emulsion component (containing MOG.sub.35-55) of Hooke Kit MOG.sub.1-125/CFA Emulsion PTX, catalog number EK-2110 (Hooke Laboratories, Lawrence Mass.). One site of injection was in the area of upper back, approximately 1 cm caudal of the neck line. The second site was in the area of lower back, approximately 2 cm cranial of the base of the tail. The injection volume was 0.1 mL at each site.

(31) Within 2 hours of the injection of emulsion, and then again 24 hours after the injection of emulsion, the pertussis toxin component of the kit was administered intraperitoneally. The volume of each injection was 0.1 mL.

(32) To optimize disease severity for this particular study, the pertussis toxin was administered at 165 ng/dose for the first injection and 154 ng/dose for the second injection.

(33) Groups and Treatment

(34) There were 2 groups of 12 mice each.

(35) Mice were assigned to groups 7 days before immunization (Day 7), balanced to achieve similar weight at the start of the study.

(36) Treatment was according to Table 1 below.

(37) TABLE-US-00001 TABLE 1 Groups and treatment Group Treatment Route Dose Volume Freq. Purpose 1 Vehicle i.v. 10 mL/kg QD Negative control 2 arsenic i.v. To Day 1: 10 mL/kg QD Test trioxide 2 mg/kg compound To Day 27: 3 mg/kg

(38) Dosing of all mice was at the same time (+/1 hour) on each day.

(39) Dosing of all mice started on Day 7 (7 days before EAE induction) and continued until Day 27.

(40) Group 2 was dosed at 2 mg/kg from Day 7 to Day 1, and at 3 mg/kg until Day 27.

(41) Mice were scored for EAE and weighed until Day 35, the last day of the study.

(42) Scoring and Readout

(43) Readouts were EAE scores and body weight at the end of the study.

(44) Mice were scored daily from Day 7 after immunization until the end of the study, and body weight was measured three times/week (Monday, Wednesday and Friday), starting on Day 7.

(45) Scoring was performed blind, by a person unaware of both treatment and of previous scores for each mouse.

(46) EAE Scoring

(47) EAE was scored on scale 0 to 5: 0 No obvious changes in motor functions of the mouse in comparison to non-immunized mice. When picked up by the tail, the tail has tension and is erect. Hind legs are usually spread apart. When the mouse is walking, there is no gait or head tilting. 1 Limp tail. When the mouse is picked up by the tail, instead of being erect, the whole tail drapes over finger. 2 Limp tail and weakness of hind legs. When mouse is picked up by tail, legs are not spread apart, but held closer together. When the mouse is observed walking, it has a clearly apparent wobbly walk. 3 Limp tail and complete paralysis of hind legs (most common). OR Limp tail with paralysis of one front and one hind leg. OR ALL of: Severe head tilting, Walking only along the edges of the cage, Pushing against the cage wall, Spinning when picked up by the tail. 4 Limp tail, complete hind leg and partial front leg paralysis. Mouse is minimally moving around the cage but appears alert and feeding. Usually, euthanasia is recommended after the mouse scores level 4 for 2 days. When the mouse is euthanized because of severe paralysis, score of 5 is entered for that mouse for the rest of the experiment. 5 Complete hind and complete front leg paralysis, no movement around the cage. OR Mouse is spontaneously rolling in the cage. OR Mouse is found dead due to paralysis.

(48) In-between scores were assigned when the clinical signs fell between two above defined scores.

(49) Statistical Analysis

(50) Hooke's standard statistical analysis for EAE studies was applied, according to Table 2 below.

(51) TABLE-US-00002 TABLE 2 Statistical analysis performed Readout Statistical analysis EAE incidence Chi-square test Mean day of EAE onset (sick mice) 2-tailed Student's t-test Median day of EAE onset (all mice) Wilcoxon's survival test Average clinical score Plotted only Average end clinical score Wilcoxon's non-parametric test Mean maximum score (MMS) Wilcoxon's non-parametric test Average weight gain/loss 2-tailed Student's t-test, plotted End weight gain/loss 2-tailed Student's t-test
Results and Interpretation

(52) EAE development was evaluated by comparing clinical EAE readouts between the vehicle group and all other groups (Tables 3-6, FIGS. 1 and 2.)

(53) TABLE-US-00003 TABLE 3a Summary of results to Day 28 (end of treatment) EAE Median day of Mean day of incidence onset onset +/ SEM Day 28 Day 27% body Group Treatment (%) (all mice) (sick mice) MMS +/ SD score +/ SD weight +/ SD 1 Vehicle, i.v., QD 100.0% 12.0 11.4 +/ 0.5 3.68 +/ 0.46 3.23 +/ 0.79 83.7 +/ 8.3 2 arsenic trioxide, 3 100.0% 17.0 17.5 +/ 1.1 3.41 +/ 0.30 2.00 +/ 0.74 97.3 +/ 5.1 mg/kg, i.v., QD

(54) TABLE-US-00004 TABLE 3b Summary of results to Day 35 (end of study) End End % body score +/ weight +/ Group Treatment SD SD 1 Vehicle, i.v., QD 3.36 +/ 0.67 82.8 +/ 8.1 2 arsenic trioxide, 2.27 +/ 0.75 93.2 +/ 6.4 3 mg/kg, i.v., QD
Group 1: Vehicle, i.v., OD (Negative Control)

(55) Clinical results for the vehicle group (Group 1, negative control) are shown in Tables 4 and 6, and in FIGS. 1 and 2.

(56) TABLE-US-00005 TABLE 4a Vehicle group (Group 1, negative control) to Day 28 (end of treatment) EAE Median day of Mean day of incidence onset onset +/ SEM Day 28 Day 27% body Treatment (%) (all mice) (sick mice) MMS +/ SD score +/ SD weight +/ SD Vehicle, i.v., QD 100.0% 12.0 11.4 +/ 0.5 3.68 +/ 0.46 3.23 +/ 0.79 83.7 +/ 8.3

(57) TABLE-US-00006 TABLE 4b Vehicle group (Group 1, negative control) to Day 35 (end of study) End End % body score +/ weight +/ Treatment SD SD Vehicle, i.v, QD 3.36 +/ 0.67 82.8 +/ 8.1

(58) EAE development in this group was as expected for this model, with maximum scores ranging from 3.5 to 5.0.

(59) All mice in this group lost weight during this study, as expected (Tables 3 and 5, FIG. 2).

(60) Two mice died in this group. One mouse died due to severe EAE while the other death resulted from complications of intravenous dosing (mouse suffocated in the restrainer while being dosed).

(61) Group 2: Arsenic Trioxide, 3 mg/kg, i.v., QD (Test Compound)

(62) Clinical results for this group compared to the vehicle group (Group 1, negative control) are shown in Tables 5 and 6, and in FIGS. 1 and 2.

(63) TABLE-US-00007 TABLE 5a Arsenic trioxide group (Group 2, test compound) compared to the vehicle group (Group 1, negative control) to Day 28 (end of treatment) EAE Median day of Mean day of incidence onset onset +/ SEM Day 28 Day 27% body Treatment (%) (all mice) (sick mice) MMS +/ SD score +/ SD weight +/ SD Vehicle, i.v., QD 100.0% 12.0 11.4 +/ 0.5 3.68 +/ 0.46 3.23 +/ 0.79 83.7 +/ 8.3 arsenic trioxide 100.0% 17.0 17.5 +/ 1.1 3.41 +/ 0.30 2.00 +/ 0.74 97.3 +/ 5.1 3 mg/kg, i.v., QD

(64) TABLE-US-00008 TABLE 5b Arsenic trioxide group (Group 2, test compound) compared to the vehicle group (Group 1, negative control) to Day 35 (end of study) End End % body score +/ p weight +/ p Treatment SD value SD Value Vehicle, i.v., QD 3.36 +/ 0.67 82.8 +/ 8.1 arsenic trioxide 2.27 +/ 0.75 0.0016 93.2 +/ 6.4 0.0035 3 mg/kg, i.v., QD

(65) While 100% of mice in arsenic trioxide group developed EAE (same as in the vehicle group), disease onset was postponed and overall severity was lower in this group compared to the vehicle group (Tables 4 and 5, FIG. 1).

(66) The clinical scores at the end of dosing period (Day 28), as well as at the end of the study, which was 7 days after the treatment was stopped (Day 35), were both significantly lower in the arsenic trioxide treated mice than in the vehicle group.

(67) Consistent with improved clinical scores, arsenic trioxide treated mice had higher body weight compared to the vehicle treated controls (Tables 4 and 5, FIG. 2).

(68) One mouse died in this group. The death did not appear to have resulted from EAE and did not result from complications of i.v. dosing.

(69) Treatment with arsenic trioxide was efficacious at postponing EAE onset and reducing disease severity in this study.

Example 2

(70) The objective of this study was to determine the effects of arsenic trioxide on cytokine production by T lymphocytes from draining lymph nodes and spleen after immunization with MOG.sub.35-55/CFA.

(71) There were 2 experimental groups with 6 mice/group.

(72) Treatment started on Day 16.

(73) Mice were immunized with MOG.sub.35-55/CFA on Day 0.

(74) Eleven days after immunization, mice were euthanized, spleens and lymph nodes collected and cell suspensions prepared. Cell suspensions were cultured for 3 days in the presence of multiple concentrations of MOG.sub.35-55. The culture supernatants were collected at the end of this 3-day culture period. The concentrations of 7 cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNF, and IFN-) were determined in the culture supernatants using Th1/Th2/Th17 cytokine bead assay (CBA) kit from Becton Dickinson.

(75) In the spleen cell cultures there was a significant reduction of IL-6 concentration in the arsenic trioxide group at all concentrations of MOG.sub.35-55 (FIG. 3). This reduction may be large enough to be the mechanism of action for arsenic trioxide in reducing EAE severity. IL-6 is known to be critical in EAE development; this finding may be relevant to arsenic trioxide's mechanism of action in inhibiting EAE development.