Treatment of disease with relaxin

Abstract

The present invention relates to relaxin or a homologue or variant thereof for use in a method of treating an inflammatory condition, for example an autoimmune inflammatory condition. The invention also relates to the treatment of an inflammatory condition, for example an autoimmune inflammatory condition comprising administering to a subject in need thereof a relaxin, or a homologue or variant thereof. Such conditions may be CNS conditions or be organ specific.

Claims

1. A method of treating traumatic spinal cord injury comprising administering an effective amount of relaxin selected from human relaxin 1 and relaxin 2, a homologue selected from prorelaxin and preprorelaxin or variant thereof to a subject in need thereof, wherein the relaxin, the homologue or the variant thereof is a polypeptide, wherein the variant thereof has at least 95% similarity or identity to the relaxin or the homologue, and wherein administration of the relaxin, the homologue or the variant thereof reduces the levels of IL-6 and IL-17 in the spinal cord and brain, reduces demyelination, preserves neurofilaments, protects neurons, and stops or reverses gliosis.

2. The method according to claim 1, wherein the method further comprises the administration of estrogen.

3. The method according to claim 1, wherein the variant thereof has at least 98% similarity or identity to the relaxin or the homologue.

Description

(1) The present invention will now be further described with reference to the following figures which show:

(2) FIG. 1a shows the improvement in clinical score in EAE model animals receiving RLX, as compared to PBS control. Improvement in clinical scores; 0=normal, 1=tail paralysis, 2=tail paralysis and strong hind limb weakness, 3=complete hind limb paralysis, 4=hind limb and forelimb paralysis, 5=found dead;

(3) FIG. 1b shows similar results to 1a—Animals receiving RLN treatment (n=8) showed greater improvement in clinical scores (2.0->0.5) compared to the placebo treated group (n=8, 2.0-1.5). Using a scale of 0-5 (0=normal, 1=tail paralysis, 2=tail paralysis and strong hind limb weakness, 3=complete hind limb paralysis, 4=hind limb and forelimb paralysis, 5=found dead). (*=p<0.05).

(4) FIG. 2a shows the reductions in chemokine receptors in relative units normalised to β-actin following RLX treatment, as compared to PBS control. The pro-inflammatory chemokine receptors CCR2, CCR5 and CCR7 were decreased in relaxin treated mice

(5) FIG. 2b shows similar results to 2a—spinal cord expression of chemokine receptors in relative units normalized to β-actin. The pro-inflammatory chemokine receptors CCR2, CCR5 and CCR7 were decreased in relaxin treated mice (n=8) when compared to the placebo group (n=8). (*=p<0.05)

(6) FIG. 3 shows spinal cord expression of cytokines in relative units normalised to β-actin spinal cord tissue. The cytokines IL-6, IL-10 and IL-17 were decreased and MIR2 was increased in relaxin treated mice.

(7) FIG. 4 shows spinal cord expression of cytokines in relative units normalised to β-actin spinal cord tissue. The cytokines IL-6 and IL-17 were significantly decreased in the relaxin treated group (n=8) when compared to the placebo group (n=8). (*=p<0.05)

(8) FIG. 5 shows cross sections of spinal cord where myelin is stained blue. The decrease in blue colour indicates demyelination in untreated mice (1a). Whereas, mice treated with RLN (2b) have normal appearing myelin which stains an intense blue. Images are enlarged 40×.

(9) FIG. 6 shows a decrease in inflammation and Wallerian degeneration of the spinal cord using H&E staining, following treatment with Relaxin (b) as compared to untreated spinal cord (a).

(10) FIG. 7 shows decreased macrophage infiltration of the spinal cord of RLN treated mice (7b) when compared to untreated mice (7a). Tissue samples were embedded in paraffin and stained for CD68. Images are magnified 200×.

(11) FIG. 8 indicates a greater loss of neurofilament in the spinal cords of untreated mice (8a) compared to mice treated with RLN (8b). Indicating a loss of neurons in the untreated mice as compared to those treated with RLN. Images are magnified 200×; and

(12) FIG. 9 shows spinal cord slices stained for the presence of GFAP. The untreated sample (9a) has large amounts of GFAP present indicating extensive gliosis. In contrast, the RLN treated samples did not stain positively for GFAP. Magnification 200×.

(13) EXAMPLES SECTION

(14) Materials and Methods

(15) Animals and Treatment

(16) Female C57BL/6 mice were obtained and housed at Biomedical Research Models (Worcester, Mass.)

(17) At 7-8 weeks of age, mice were immunized with MOG33-35/CFA emulsion (Hooke Laboratories #EK-2110 provided pre-formulated). Mice received pertussis toxin (Hooke Laboratory, Cat #EK-2110 provided lyophilized) reconstituted in phosphate buffered saline (PBS) at the time of immunization (day 0; 75 ng) and on day 2 (200 ng). Animals were scored daily for disease severity: 0=normal, 1=tail paralysis, 2=tail paralysis and strong hind limb weakness, 3=complete hind limb paralysis, 4=hind limb and forelimb paralysis, 5=found dead.

(18) At the onset of EAE (the first day with a score of 1 or greater), subcutaneous alzet pumps (DURECT Corporation, Cupertino, Calif.) were inserted and the animals were randomly assigned to receive either porcine relaxin (SKY BioHealth, Inc., Eden Prairie, Minn., 66.0 μg/day) or placebo treatment (1 μ/Hr PBS), either recombinant porcine relaxin (pRLN, n=8) or placebo (PBS, n=8) at a rate of 0.110/hr. Relaxin (SKY BioHealth, Inc., Eden Prairie, Minn/) was administered at a concentration of 0.25 mg/ml for the remainder of the study. This delivered a standard dose of 0.66 μg/day of pRLN, which is equivalent to the identified effective dose of 30 μg/KG body weight/day (Hasse et al, 2014). Statistical analysis was done using the t-test and P values <0.05 were considered to be statistically significant.

(19) Spinal Cord

(20) Spinal cords and brains were collected for RT-PCR and histopathology. Total RNA was isolated from spinal cord and brain tissue using the RNeasy mini-kit protocol (Qiagen; Valencia, Calif.) and converted to cDNA using oligo(dT), random hexamers, and Superscript RT II (Invitrogen; Grand Island, N.Y.). RT-PCR for CCR2, CCR5, CCR7, IL-6, IL-10, IL-17 and TNF-α was run on a Prism 7000 sequence detection system (Applied Biosystems; Foster City, Calif.) using Taqman PCR master mix (Qiagen) and primers from Applied Biosystems. Levels of expression were normalized to

(21) β-actin. Alternatively RT-PCR for the chemokine receptors (CCR); CCR2, CCR5, CCR7, the interleukins (IL-6 and IL-17) were analyzed using quantitect primers (table 2) and SYBR green based RT-PCR kits (Qiagen, Valencia, Calif.). For histology, spinal cords sections were stained using luxol fast blue-periodic acid shiff for semi-quantitative analysis of infiltration and demyelination.

(22) For alternative histology, spinal cords were formalin fixed and paraffin embedded (FFPE) as described by Canene-Adams [Canene-Adams, 2013]. Briefly, Spinal cords were rinsed in PBS to remove blood and fixed in 10% formalin for 48 hours at room temperature. Tissues were then transferred to 70% ethanol (ETOH) for 1 hour, 95% ETOH (95% ETOH/5% methanol) for 1 hour, then incubated in four absolute ETOH baths first for 1 hour, then 2 for 1½ hours, followed by a fourth for 2 hours. Tissues were then cleared with two xylene baths for 1 hour each. Tissues were then incubated in two paraffin baths at 58° C. for 1 hour each. Tissues were then removed and placed in cassettes.

(23) The FFPE tissues were sectioned into 5 μM slices on a microtome. Slices were transferred to glass slides and warmed to 65° C. for 20 minutes to bond the tissue to the glass.

(24) Sections were stained with Kiernan's eriochrome cyanin for myelin [Kiernan J A, 1999], cluster of differentiation 68 (CD68) for macrophages [Holness and Simmons, 1993], neurofilaments [Alberts, B. 2002] and glial fibrillary acidic protein (GFAP) [Jacque C M, et al, 1978].

(25) Kiernan's stain for myelin was done as described. First tissues were placed in a solution containing eriochrome cyanine R, ferric chloride and sulphuric acid in water for 20 minutes. Rinsed briefly under tap water and differentiated with aqueous ferric chloride (5.6%). Tissues were then dried with ETOH.

(26) Staining with Hematoxylin and eosin (H&E) stain was also carried out according to standard protocols.

(27) Standard immunohistochemistry [Ramos-vara and Miller, 2014] was used to identify the presence of CD68, neurofilaments and GFAP in the FFPE tissues. Essentially the appropriate antibody directed against the antigen of choice was incubated with the tissue and allowed to bind. The tissue was then washed and incubated with the secondary antibody which was conjugated to the dye. The tissues were then washed and the conjugated dye developed for visualization.

(28) Results

(29) Disease Severity

(30) Clinical onset of EAE was detected on day 14 of the study. The alzet pumps were inserted in the mice and continuous infusion of either RLX or PBS was started. Animals receiving RLX treatment showed a marked improvement in clinical score within 3 days of commencing treatment (FIG. 1a). In further studies at the end of treatment the group receiving RLN treatment had a score of >0.5 while the placebo group had a score 1.5. The difference in scores between the RLN and placebo treated group were significant on days 19, 20, & 21 (p=<0.05), see FIG. 1b).

(31) Real-Time PCR

(32) Levels of mRNA for cytokines and chemokine receptors were analysed in the spinal cord samples.

(33) Further studies show that in comparison to the placebo group (n=8) RNA in the spinal cords of the relaxin treated group (n=8) showed a significant decreases in the chemokine receptors CCR2 (0.13 vs 0.044, p<0.05) and CCR7 (0.003 vs 0.014, p<0.05). There was also an observed decrease in CCR5 (0.0035 vs 0.0046) although this was not significant (see FIG. 2b). There was also a reduction in the cytokines IL-6 (0.0004 vs 0.0009, p<0.05) and IL-17 (0.000078 vs 0.00026, p<0.05, see FIG. 4).

(34) RNA expression in the spinal cords of relaxin treated mice showed significant reductions in the chemokine receptors CCR2, CCR5 and CCR7 (see FIG. 2a). The cytokines IL-6, IL-10 and IL-17 were also decreased in the RLX treated mice (see FIG. 3).

(35) Histology

(36) Relaxin treatment reduced lesion load and size which indicates a decrease in cellular infiltration into the spinal cord compared to control animals (data not shown).

(37) Kiernan's Eriochrome Cyanin for Myelin and H&E Staining

(38) The spinal cords of mice with EAE which were untreated showed inflammation, Wallerian degeneration and a loss of myelin. While mice treated with RLN had normal appearing myelin (see FIGS. 5 and 6).

(39) CD68

(40) Tissue samples stained for the presence of CD68 showed greater infiltration of macrophages in the untreated samples than in those treated with RLN (see FIG. 6).

(41) Neurofilaments

(42) The spinal cords from untreated mice shoed a relatively greater loss of neurofilaments compared to mice receiving RLN therapy (see FIG. 7).

(43) GFAP

(44) The spinal cords for RLN treated mice were negative for GFAP while the untreated mice stained positive for GFAP, indicating the presence of gliosis (see FIG. 8).

(45) Discussion

(46) Inflammatory autoimmune diseases of the CNS which include MS and EAE may differ in onset and etiology, but the goals of treatment are the same; control inflammation, regulate autoimmunity and enhance neural protection. Despite the development of agents capable of modulating inflammation and autoimmunity, treatment remains problematic. Low efficacy and severe side effects being the most limiting factors in the use of these agents (Costello 2008).

(47) The discovery that estrogen treatment could reduce the relapse rate and symptoms in MS (Sicotte 2002) provided a novel approach to treatment based on a naturally occurring peptide. But estrogen is not without unwanted side effects, especially with prolonged use (Barrett-Connor 2001). The challenge of using sex hormone-based therapy for these diseases is to maximize the effects of this treatment on the immune system and minimize the effects on the reproductive system. By studying the actions of molecules produced downstream from estrogen may identify one which has many of the immune-modulating effects with minimum or no effect of the reproductive system. In this study we investigated the ability of one such molecule, RLX in the treatment of EAE. The results of which showed that like estrogen, it down regulated the secretion of pro-inflammatory cytokines and chemokine receptors, reduced cellular infiltration and demyelination in EAE. And increased the expression of the MIR2, which can downregulate a wide range of cell surface proteins (Anania 2011) including the major histocompatability complex 1 (MHC-1, Thakur 2011).

(48) In addition, treatment with RLN down regulated the secretion of pro-inflammatory chemokine receptors CCR2 and CCR5. These results are significant since CCR2 has been identified as a key driver of encephalitogenic Th17 cell recruitment into the CNS in MS and EAE. Molecules capable of modulating CCR2 have been identified as possible novel therapies in MS and EAE (Kara et al, 2015). Similarly, CCR5+ Th-1 cells are increased in the CSF and brain lesions of active demyelinating MS patients. Glatiramer acetate which is widely used for treatment of MS, acts via decreasing the expression of CCR5 and related Th-1 cells (Cheng and Chen, 2014). Th-1 and Th-17 cells are also modulated by CCR7, the presence of which is required for the development of EAE (Kuwabara, 2009).

(49) Modulation of the cytokines IL-6 and IL-17 by RLN also has implication for novel therapeutic strategies in MS and EAE. Therapies that reduce IL-6 levels in EAE reduce symptoms and disease progression (Erta, 2016). And the pro-inflammatory cytokine IL-17 is a crucial effector cytokine. Therapies designed to block or reduce levels of IL-17 has been shown to be effective treatments in EAE (Hofstetter et al, 2005). Novel therapies targeted at blacking or reducing IL-6 and IL-17 are currently undergoing clinical trials for the treatment of MS.

(50) The results of histology and immunohistochemistry showed a reduction of infiltrating macrophages in the RLN treated group. There was evidence of neuroprotective effects from RLN treatment as it reduced demyelination, preserved neurofilaments, protected neurons and stopped or reversed gliosis.

(51) RLX treatment can ameliorate the symptoms of EAE, reduce lesion load and modulate the expression of cytokines and chemokine receptors. It is a naturally occurring peptide capable of modulating the ER's, GCR and PPARγ; pathways known to modulate the symptoms of MS. And in limited clinical trials there have been no severe side effects reported with its use (Smith 2006;). These results taken together with ability of RLX to modulate some of the pathways currently targeted in the treatment of MS, support further investigation of RLX as a novel therapeutic pathway in treating MS and other inflammatory, neurodegenerative and inflammatory auto-immune diseases and conditions.

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