COMPOSITIONS AND USES THEREOF

Abstract

The present invention relates to therapeutic compositions comprising a PDE10A inhibitor for use in the prevention, management and/or treatment of inflammatory bowel diseases, and particularly for use in individuals who are suffering from ulcerative colitis.

Claims

1. A composition for the prevention, management, and/or treatment of an inflammatory bowel disease in a subject in need thereof, the composition comprising an effective amount of a PDE10A inhibitor to treat, manage, and/or prevent the inflammatory bowel disease in the subject.

2. The composition according to claim 1, wherein the inflammatory bowel disease is ulcerative colitis.

3. The composition according to claim 1, wherein the PDE10A inhibitor is a selective inhibitor of PDE10A.

4. The composition according to claim 1, wherein the PDE10A inhibitor is selected from one or more of the following: PF-02545920, TAK-063, Papaverine, RG7203, JNJ-42314415, AMG-579, PQ-10, BMS-843496, PDM-042 and derivatives thereof.

5. The composition according to claim 1, wherein the PDE10A inhibitor is PF-02545920 or TAK-063.

6. The composition according to claim 1, wherein the PDE10A inhibitor selectively inhibits cGMP hydrolysis over cAMP hydrolysis.

7. The composition according to claim 1, wherein the management and/or treatment of inflammatory bowel diseases of the present invention involves increasing cGMP signaling in bowel tissue of a patient.

8. The composition according to claim 1, wherein the management and/or treatment of inflammatory bowel diseases of the present invention involves reducing levels of inflammatory cytokines in bowel tissue of a patient.

9-12. (canceled)

13. A method of prevention, management and/or treatment of inflammatory bowel diseases comprising the administration of a PDE10A inhibitor in an individual in need of such prevention, management and/or treatment.

14. The method according to claim 13, wherein the inflammatory bowel disease is ulcerative colitis.

15. The method according to claim 13, wherein the PDE10A inhibitor is a selective inhibitor of PDE10A.

16. The method according to claim 13, wherein the PDE10A inhibitor is selected from one or more of the following: PF-02545920, TAK-063, Papaverine, RG7203, JNJ-42314415, AMG-579, PQ-10, BMS-843496, PDM-042 and derivatives thereof.

17. The method according to claim 13, wherein the PDE10A inhibitor is selected from PF-02545920 or TAK-063.

18-21. (canceled)

Description

DETAILED DESCRIPTION OF THE INVENTION

[0406] Embodiments of the invention are described below, by way of example only, with reference to the accompanying figures in which:

[0407] FIG. 1 are plots showing RNA expression of PDE10A in normal tissue. The plots represent baseline gene expression of PDE10A and GUCY2C (guanylate cyclase 2C) in healthy samples based on GTEx data, where the X-axis represents tissue, y-axis represents log.sub.2 transformed expression;

[0408] FIG. 2 are volcano plots showing differential RNA expression of PDE10A and GUCY2C. The volcano plots show differential gene expression for a selected comparison, where the x-axis represents log fold change (FC) and y-axis represents log.sub.10 transformed adjusted p-value (FDR). Horizontal dotted line is FDR=0.05 threshold and values above the dotted line are considered significant. Values to the right of the central axis indicate upregulation, values to the left of the central axis indicate down regulation. The OmicSoft differential expression datasets used in the analysis were as follows: colonic mucosa -OmicSoft Project names: GSE14580, GSE16879, GSE36807, GSE59071, GSE65114, GSE73661; colon - OmicSoft Project names: GSE10191, GSE10616, GSE6731, GSE9686;

[0409] FIG. 3 is a graph showing the effect of a PDE10A inhibitor PF-02545920 on isolated human neutrophil activation in response to IL-8;

[0410] FIG. 4 are graphs showing PF-02545920 and TAK-063 inhibiting the release of inflammatory cytokines IL-6 and IL-8 in ex-vivo cultures of colon biopsy samples from UC patients. A. UC donor 1, B. UC donor 2 (n=2; Mean±SD), wherein Pred = prednisolone, Tofa = tofacitinib;

[0411] FIG. 5 are graphs showing PF-02545920 and TAK-063 inhibiting the release of inflammatory cytokines IL-6 and IL-8 in ex-vivo cultures of inflamed UC tissue. A. UC donor 1, B. UC donor 2 (n=5; Mean±SD; * p<0.05), wherein Pred = prednisolone, Tofa = tofacitinib ; and

[0412] FIG. 6 are graphs showing PF-02545920 (1 .Math.M) inhibiting release of the inflammatory cytokine TNFα in ex-vivo cultures of inflamed colon tissue obtained from surgical resection from treatment-refractory UC patients. A. UC donor 1, B. UC donor 2 (n=5; Mean±SD; * p<0.05).

Example 1

Assessing a PDE10A Inhibitor for Use in the Treatment of Ulcerative Colitis

[0413] To explore the role of PDE 10A in ulcerative colitis (UC) the Genotype-Tissue Expression (GTEx) database was used to look at PDE10A RNA expression in normal and diseased tissues. Alongside this expression levels of guanylate cyclase 2C (GUCY2C) were also assessed. GUCY2C is an enzyme which synthesises cGMP in response to the endogenous peptides guanylin and uroguanylin as well as E.coli heat-stable enterotoxin.

[0414] As previously described in the literature, in normal tissue PDE10A is expressed at low levels except in brain (as shown in FIG. 1). However in colonic mucosa and colon tissue from ulcerative colitis patients, PDE10A expression levels were significantly upregulated compared to normal controls (as shown in FIG. 2). This is a finding that has not been previously described in the literature and highlighting a potential undiscovered role for PDE10A in UC pathology.

[0415] GUCY2C was seen to be specifically expressed at high levels in colon and small intestine (as shown in FIG. 1) suggesting a role for this enzyme in normal gut homeostasis. In UC colonic mucosa and colon, GUCY2C was significantly downregulated (as shown in FIG. 2), a finding that has previously been described in the literature.

[0416] Guanylate cyclase-C and cGMP signalling is downregulated in ulcerative colitis (Brenna et al The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease Scand J Gastroenterol. 2015;50(10):1241-52) and decreases in expression of guanylate cyclase 2C, guanylin, and uroguanylin correlate with severity of disease. (Lan et al. Expression of guanylate cyclase-C, guanylin, and uroguanylin is downregulated proportionally to the ulcerative colitis disease activity index Sci Rep. 2016; 6: 25034. Published online 2016 Apr 29. doi: 10.1038/srep25034). This suggests that reduced cGMP signalling plays a role in UC pathology. cGMP in the GI tract has also been shown to play a role in fluid and electrolyte secretion, barrier function, inflammation and proliferation (Waldman et al, Guanylate cyclase-C as a therapeutic target in gastrointestinal disorders., Gut. 2018 67(8):1543-1552).

[0417] While less studied in inflammation than cAMP, reduced cGMP signalling has also been shown to increase inflammation in other systems (Ahluwalia et al, Antiinflammatory activity of soluble guanylate cyclase: cGMP-dependent down-regulation of P-selectin expression and leukocyte recruitment. Proc Natl Acad Sci U S A. 2004 101(5):1386-91; Rapôso et al, Role of iNOS-NO-cGMP signaling in modulation of inflammatory and myelination processes.Brain Res Bull. 2014 104:60-73)

[0418] Taken together, in UC colon and colonic mucosa, cGMP hydrolysing activity by PDE10A would be increased and cGMP synthesizing activity by guanylate cyclase 2C would be decreased resulting in a net decrease in cGMP levels and signalling.

[0419] Experiments were then conducted to assess whether inhibiting PDE10A with a small molecule inhibitor would help restore cGMP signalling to normal levels and therefore represent a useful treatment for ulcerative colitis.

[0420] A PDE10A selective tool compound PF-02545920 was assessed in an in vitro assay of IL-8 neutrophil activation. PF-02545920 dose dependently inhibited IL-8 induced neutrophil activation (as shown in FIG. 3). This was of interest as a role for PDE10A in neutrophil function had not been previously described and further suggested a role for PDE10A in modulating inflammation and that a PDE10A inhibitor would be suitable as a therapeutic for inflammatory bowel diseases, and ulcerative colitis in particular.

[0421] The therapeutic potential of inhibitors of PDE10A to treat ulcerative colitis was further assessed using tissue samples from inflamed colonic mucosa from ulcerative colitis patients.

[0422] The effect of selective PDE10 inhibition was tested on inflamed colonic mucosa from ulcerative colitis patients taken during routine endoscopy (Method 1 detailed below). These samples retain a disease phenotype in ex-vivo culture and secrete high basal levels of inflammatory cytokines. The effect of selective PDE10 inhibition on levels of the inflammatory cytokines IL-8 and IL-6 released from these tissue samples was measured. Both IL-6 and IL-8 are key regulators in ulcerative colitis pathology and their levels correlate with disease severity (Waldner MJ et al., Master regulator of intestinal disease: IL-6 in chronic inflammation and cancer development. Semin Immunol. 2014 26(1),75-9.; Bernardo D et al, IL-6 promotes immune responses in human ulcerative colitis and induces a skin-homing phenotype in the dendritic cells and T-cells they stimulate. Eur J Immunol. 2012, 42(5),1337-53.; Pearl DS, Cytokine mucosal expression in ulcerative colitis, the relationship between cytokine release and disease activity. J Crohns Colitis. 2013, 7(6), 481-9).

[0423] The structurally distinct PDE10A inhibitors PF-02545920 and TAK-063 were tested alongside 2 positive control compounds, the steroid prednisolone and the Janus kinase inhibitor tofacitinib, in colon biopsy samples from two ulcerative colitis patients. These colon biopsies retain an inflammatory phenotype and secrete high levels of inflammatory cytokines in ex-vivo culture. Selective PDE10A inhibition significantly reduced the secreted levels of IL-6 and IL-8 when compared to the DMSO vehicle (FIGS. 4 and 5) This reduction was comparable to that seen with the positive controls. PF-02545920 was tested at concentrations of 0.1 .Math.M and 1 .Math.M. The doses tested of each inhibitor will result in selective PDE10A inhibition over the other PDE family members.

[0424] In isolated enzyme biochemical assays, PF-02545920 has been shown to be a highly selective PDE10A inhibitor with an IC50 for PDE10A <5 nM and IC50s for other PDE family members >1 .Math.M (Grauer SM et.al. Phosphodiesterase 10A inhibitor activity in preclinical models of the positive, cognitive, and negative symptoms of schizophrenia. J Pharmacol Exp Ther. 2009 331(2), 574-90.) Therefore, at the test concentration of 0.1 .Math.M and 1 .Math.M in an ex-vivo tissue assay, PF-02545920 will selectively inhibit PDE10A.

[0425] In isolated enzyme biochemical assays, TAK-063 has been shown to be a highly selective PDE10A inhibitor with an IC50 for PDE10A of 0.3 nM and IC50s for other PDE family members >5 .Math.M (Kunitomo J et.al. Discovery of 1-[2-fluoro-4-(1 H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1 H-pyrazol-5-yl)pyridazin-4(1 H)-one (TAK-063), a highly potent, selective, and orally active phosphodiesterase 10A (PDE10A) inhibitor. J Med.Chem. 2014 57(22),9627-43.) Therefore, at the test concentration of 1 uM in an ex-vivo tissue assay, TAK-063 will selectively inhibit PDE10A.

[0426] The effect of selective PDE10A inhibition was also tested on inflamed colonic mucosa from pharmacotherapy treatment-refractory ulcerative colitis patients taken during colon resection surgery (Method 2 detailed below). The PDE10A inhibitor PF-02545920 (1 .Math.M) was tested in colon samples from two ulcerative colitis patients. The effect of selective PDE10A inhibition on levels of the inflammatory cytokine TNFα released from these tissue samples were measured. TNFα is a pro-inflammatory mediator that is expressed at high levels in the colonic mucosa of patients with UC and is the target of anti-TNFα biologics which have demonstrated efficacy in the treatment of UC (Pugliese D. et al, Anti TNF-α therapy for ulcerative colitis: current status and prospects for the future., Expert Rev Clin Immunol. 2017 13(3):223-233.). Selective PDE10A inhibition significantly reduced the secreted levels of TNFα compared to the DMSO vehicle (FIG. 6.)

[0427] The ability of selective PDE10A inhibition to significantly reduce levels of inflammatory cytokines in UC patient-derived colonic mucosa demonstrates the therapeutic utility of PDE10A inhibitors for the treatment of UC.

Method 1

[0428] Biopsy tissue was obtained from inflamed colonic mucosa from ulcerative colitis patients during routine endoscopy. Ex-vivo biopsy cultures for the analysis of inflammatory cytokine biomarkers were run as previously described (Vossenkamper A. et al. A CD3-specific antibody reduces cytokine production and alters phosphoprotein profiles in intestinal tissues from patients with inflammatory bowel disease. Gastroenterology, 2014, 147, 172-183). Biopsies were incubated in organ culture for 24 h with the addition of positive control compounds, or the specific PDE10A inhibitor PF-02545920. Supernatants collected at the end of the experiment were snap-frozen and stored at -70° C. For the measurement of cytokines, the frozen culture supernatants were thawed and analysed for levels of the inflammatory cytokines using Luminex cytokine assay kits (R&D Systems) and an R&D Systems MAGPIX® analyser. Mean values ± SDs were calculated for the levels of spontaneous cytokine production measured in biopsy culture supernatants from each treatment group.

Method 2

[0429] Ulcerative colitis donor samples were obtained with full ethical consent from patients undergoing therapeutic resection for ulcerative colitis. Tissues were placed apical (mucosal) side facing upwards on a Netwell filter. The biopsies were then be cultured in either control media or media containing the test compound in an incubator at 37° C. and high O.sub.2 atmospheric conditions. To try to minimize variation, the biopsies were also cultured in the presence of the inflammatory stimulant Staphylococcal Enterotoxin B (SEB) to help normalise cytokine levels. At approximately 18 hours post-culture start, media samples were collected, protease inhibitor added and samples stored at -80° C. Supernatants were subsequently subjected to ELISA analysis for cytokine measurement.

Example 2

Example Formulations and Treatments for Ulcerative Colitis

[0430] A number of example formulations are provided below along with suggested dosage regimes. It will be understood that these are for illustrative purposes and these would be optimized during further experimentation, which may include clinical trials. For simplicity, the formulations do not stipulate any non-active components (such as pharmaceutically acceptable carriers or excipients etc.)

TABLE-US-00001 TAK-063 - Oral Tablet for the Treatment of Ulcerative Colitis Active Ingredient Form mg Dose TAK-063 Oral Tablet 20 Once daily

TABLE-US-00002 TAK-063 - Oral Tablet for the Treatment of Ulcerative Colitis Active Ingredient Form mg Dose TAK-063 Oral Tablet 10 Once daily

TABLE-US-00003 Papaverine - Oral Tablet for the Treatment of Ulcerative Colitis Active Ingredient Form mg Dose Papaverine Oral Tablet 150 Twice daily

TABLE-US-00004 PF-02545920 - Oral Tablet for the Treatment of Ulcerative Colitis Active Ingredient Form mg Dose PF-02545920 Oral Tablet 20 Twice daily

TABLE-US-00005 PF-02545920 - Oral Tablet for the Treatment of Ulcerative Colitis Active Ingredient Form mg Dose PF-02545920 Oral Tablet 5 Twice daily

TABLE-US-00006 PQ-10 - Intravenous Injection for the Treatment of Ulcerative Colitis Active Ingredient Form mg/kg Dose PQ-10 Intravenous injection 0.1 Once daily

TABLE-US-00007 PQ-10 - Intravenous Injection for the Treatment of Ulcerative Colitis Active Ingredient Form mg/kg Dose PQ-10 Intravenous injection 3 Once daily

TABLE-US-00008 PDM-042 - Intravenous Injection for the Treatment of Ulcerative Colitis Active Ingredient Form mg/kg Dose PDM-042 Intravenous injection 0.1 Once daily

TABLE-US-00009 PDM-042 - Intravenous Injection for the Treatment of Ulcerative Colitis Active Ingredient Form mg/kg Dose PDM-042 Intravenous injection 3 Once daily

[0431] The skilled addressee will of course understand that alternative PDE10A inhibitors could also be employed in place of those outlined above. The therapeutically effective doses will of course depend on the activity and format of the chosen inhibitor.

[0432] The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.