5-AMINOLEVULINIC ACID FOR THE LOCAL TREATMENT OF INFLAMMATORY BOWEL DISEASE

Abstract

The present invention relates to the therapeutic use of compositions containing 5-aminolevulinic acid for the local treatment of inflammatory bowel disease, including but not restricted to ulcerative colitis and Crohn's disease.

Claims

1. A pharmaceutical composition comprising 5-aminolevulinic acid (5-ALA) or a pharmaceutically acceptable salt thereof, adapted for topical administration to the lower gastrointestinal tract of a human or animal.

2. A pharmaceutical composition as claimed in claim 1, adapted for rectal administration.

3. A pharmaceutical composition as claimed in claim 1, adapted for delayed release oral administration.

4. A pharmaceutical composition as claimed in claim 3, comprising an enteric coating.

5. A pharmaceutical composition as claimed in claim 3, comprising a solid oral dosage form with a core and a coating for the core, the core comprising 5-ALA or a pharmaceutically acceptable salt thereof, and the coating comprising a mixture of a digestible polysaccharide and a film-forming material which has a pH threshold at pH 5.0 or above,

6. A pharmaceutical composition as claimed in claim 5 wherein the digestible polysaccharide is selected from the group consisting of starch; amylose; amylopectin; chitosan; chondroitin sulfate; cyclodextrin; dextran; pullulan; carrageenan; scleroglucan; chitin; curdulan and levan.

7. A pharmaceutical composition as claimed in claim 5, in which the polysaccharide is starch, amylose or amylopectin.

8. A pharmaceutical composition as claimed in either claim 5 or claim 6, in which the film-forming is an acrylate polymer, a cellulose polymer or a polyvinyl-based polymer.

9. A pharmaceutical composition as claimed in claim 7, in which the film-forming is selected from cellulose acetate phthalate; cellulose acetate trimellitate; hydropropylmethylcellulose acetate succinate; and polyvinyl acetate phthalate.

10. A pharmaceutical composition as claimed in claim 8, in which the film-forming is a co-polymer of a (meth)acrylic acid and a (meth)acrylic acid C.sub.1-4 alkyl ester.

11. A pharmaceutical composition as claimed in any preceding claim, for use in the prophylaxis or treatment of inflammatory bowel disease, irritable bowel disease, autoimmune disease, constipation, diarrhoea, infection, or cancer.

12. A pharmaceutical composition for use of claim 11, wherein the inflammatory bowel disease is Ulcerative colitis or Crohn's disease.

13. A method of treating inflammatory bowel disease, irritable bowel disease, autoimmune disease, constipation, diarrhoea, infection, or cancer comprising topically administering 5-aminolevulinic acid (5-ALA) or a pharmaceutically acceptable salt thereof, to the lower gastrointestinal tract of a human or animal.

14. A method of claim 13 comprising administering a pharmaceutical composition as defined in any one of claims 1 to 12.

15. A method of claim 13 or claim 14 wherein the inflammatory bowel disease is Ulcerative colitis or Crohn's disease.

Description

[0058] The following Examples illustrate the invention. The Examples refer to the following figures:

[0059] FIG. 1 shows the stability of 5-ALA and formation of PpIX in human colon. A) Stability of 5-ALA in human colon fluid. B) Formation of PpIX after incubation of 5-ALA+SFC in human colon fluid.

[0060] FIG. 2 shows the stability of 5-ALA and formation of PpIX in mouse colon. A) Stability of 5-ALA in mouse colon fluid. B) Formation of PpIX after incubation of 5-ALA+SFC in mouse colon fluid.

[0061] FIG. 3 shows the 5-ALA levels in the apical, tissue and basal compartments after exposure to human colon tissue.

[0062] FIG. 4 shows the 5-ALA levels in the apical, tissue and basal compartments after exposure to C57BL6 mouse colon tissue.

[0063] FIG. 5 shows TNF-α levels in mouse colon tissue at 10 days following treatment.

[0064] FIG. 6 shows IL-6 levels in levels in mouse colon tissue at 10 days following treatment.

[0065] FIG. 7. IL-1β levels in mouse colon tissue at 10 days following treatment.

[0066] FIG. 8 shows TNF-α levels in mouse colon tissue at 10 days following treatment.

[0067] FIG. 9 shows IL-6 levels in mouse colon tissue at 10 days following treatment.

[0068] FIG. 10 shows IL-1δ levels in mouse colon tissue at 10 days following treatment.

[0069] FIG. 11 shows PpIX levels in the plasma at day 10 after 5-ALA dosing orally and intra-rectally at 10 mg/kg and 100 mg/kg doses.

[0070] FIG. 12 the levels of metabolites in the tissue and plasma. A) Bilirubin levels in the colon tissue and plasma of mice at day 10 following treatment. B) Biliverdin levels in mouse colon tissue and plasma in all groups at day 10 following treatment.

MATERIALS AND METHODS

[0071] Mouse Colon Model

[0072] A mouse colonic model based on a mixed fecal inoculum was used to mimic the luminal environment of the mouse large intestine. An anaerobic workstation (Electrotek 500TG™ workstation, Electrotek, West Yorkshire, UK) maintained at 37° C. and 70% relative air humidity was used to set up the model. Three healthy male C57BL6 mice were sacrificed and the fecal contents were collected. The fecal material was transferred in the anaerobic workstation and diluted with freshly prepared basal medium to obtain 20% w/w slurry by homogenization. The basal media provides nutrients and growth factors to the microbiota allowing viability for upto 24 hours. The homogenized bacterial media was sieved through an open mesh fabric (SefarNitex™, pore size 350 μm) to remove any nonhomogeneous fibrous material.

[0073] Human Colon Model

[0074] A human colonic model based on a mixed fecal inoculum was used to mimic the luminal environment of the human large intestine. An anaerobic workstation (Electrotek 500TG™ workstation, Electrotek, West Yorkshire, UK) maintained at 37° C. and 70% relative air humidity was used to set up the model. The fecal material was transferred in the anaerobic workstation and diluted with freshly prepared basal medium to obtain 20% w/w slurry by homogenization. The basal media provides nutrients and growth factors to the microbiota allowing viability for up to 24 hours. The homogenized bacterial media was sieved through an open mesh fabric (SefarNitex™, pore size 350 μm) to remove any nonhomogeneous fibrous material. The pH was maintained at approximately 7 to mimic the colonic luminal pH of the human.

[0075] 5-ALA+SFC Incubation Studies and Processing of Solution for Analysis of 5-ALA and PpIX

[0076] 5-ALA and SFC stock solution was prepared in PBS at 12 mg/ml and 2 mg/ml respectively. The stock was added to 20% human or mouse faecal slurry to obtain an incubation concentration of 6 mg/ml 5-ALA and 1 mg/ml SFC, and 10% w/w faecal slurry. Samples were withdrawn at appropriate time points and added to 0.5% TFA in a ratio of 1:2. The samples were centrifuged at 9.6 g for 10 mins and the supernatant was prepared for detection of 5-ALA and PpIX by HPLC-FLD.

[0077] For detection of 5-ALA, the solution containing 5-ALA was added to 0.1% fluorescamine solution and borate buffer solution in the ratio of 1:1 and 1:3 respectively. The solution was vortexed and incubated for 10 minutes at room temperature before added into the HPLC for analysis.

[0078] PpIX solution was prepared by adding the supernatant in an extraction solvent consisting of 100 parts of N,N-dimethylformamide and 1 part of 2-proponol. The mixture was vortexed and transferred for analysis by HPLC-FLD.

[0079] HPLC-FLD for 5-ALA and PpIX

[0080] Sample analysis for detecting of 5-ALA and PpIX was performed using a high performance liquid chromatography (HPLC) system (Agilent Technologies, 1260 Infinity II Series™) equipped with a pump (model G1311C), autosampler (model G1329B) and a diode-array UV detector (model G1314B).

[0081] For 5-ALA analysis, a 150×4.6-mm Jupiter 5 μm 300 Å (Phenomenex, Torrance, Calif.) C18 column was used using 70% water (0.1% TFA) and 30% acetonitrile (0.1% TFA) as the mobile phase for elution, at a flow rate of 1 ml/min. The analysis was operated at room temperature and fluorescence detection wavelength was set at 395/480 nm excitation/emission. The injection volume of each sample was 10 μl.

[0082] For PpIX analysis, a 150×4.6-mm Aeris 3.6 μm 100 Å (Phenomenex, Torrance, Calif.) C18 column was used using 70% acetonitrile and 30% 10 mM TBA solution (pH 7.5) as the mobile phase at a flow rate of 1 ml/min. The analysis was operated at room temperature and fluorescence detection wavelength was set at 400/630 nm excitation/emission. The injection volume of each sample was 50 μl.

[0083] Ussing Chamber System

[0084] A NaviCyte vertical ussing system (Harvard Apparatus, Cambridge, UK) was used to measure transport across epithelial membranes which are polar structures possessing an apical (mucosal) and basolateral (serosal) side. The chambers are made of solid acrylic and supports the tissue membrane in such a way that each side of the membrane is isolated and faces a different chamber representing the luminal (apical) and blood (basal) compartments. The working system consists of a unit to fit a maximum of six vertical chambers, a gas manifold for carbogen purging (95% O.sub.2, 5% CO.sub.2) and a heater block to maintain the temperature of the chambers at 37° C. during the experiments with the use of a circulating water bath. The chambers are two-piece assemblies held together by a high spring-tension retaining ring to ensure leak-free operation during the experiments.

[0085] The EVOM™ voltohmmeter (World Precision Instruments, Inc., Hertfordshire, UK) and Ag/AgCl electrodes (Harvard Apparatus, Cambridge, UK) were used to measure the trans-epithelial electrical resistance (TEER) of the tissue samples. TEER monitors the presence of functional tight junctions, which are responsible for the barrier function and which limit Paracellular permeation of water and solutes. TEER value of 200 Ω/cm.sup.2 was set as the lower limit to confirm the tissue viability and tight junction integrity.

[0086] For the tissue penetration studies, the freshly excised colon of human subject or C57BL6 mice was collected and transferred to an ice-cold solution of Krebs-Bicarbonate Ringer solution (KBr) of pH 7.4. The tissue was cut open transversally and was washed with KBr solution to remove the luminal contents and was then mounted in the Ussing chambers. The mucosal surface of the colon tissue was facing the apical chamber, and the endothelial surface of the tissue was facing the basolateral chamber. The exposed tissue area on each side of the chamber was 0.29 cm.sup.2 and the tissue mounting region was 4×8 mm (FIG. 3.6). The volume of KBr in apical and basolateral chamber was 5 ml and the pH was maintained at 7.4. The tissue was allowed to incubate with KBr for 20 minutes before addition of the drug. 5-ALA and SFC concentrations tested during the penetration experiments was 6 mg/ml and 1 mg/ml respectively. The penetration of 5-ALA and formation of metabolites biliverdin and bilirubin in the tissue was tested for 3 hours and in a minimum of 3 mice. The tissue without drug was incubated in parallel for the same time which acted as the negative control. The chambers were purged with carbogen and kept at 37° C. by water jackets during incubation. The TEER was continuously monitored during the experiment to confirm the viability and integrity of the tissue. Tissues with TEER value below 200 were not used for the experiments.

[0087] Tissue Homogenization and 5-ALA, Biliverdin and Bilirubin Extraction for Quantification by HPLC-FLD

[0088] Freshly excised colon tissues following completion of the Using chamber permeation study for 3 hrs were weighed and the appropriate amount of extraction buffer was added in the ratio 20 mg: 1 ml. The tissue was homogenized, and the homogenate was incubated for 2 hrs at 4° C. After centrifugation, the supernatant was analyzed for 5-ALA levels as per the protocol described above.

[0089] For detection of biliverdin and bilirubin, the samples were added into a dilution buffer (methanol, ammonia solution and water in the ratio of 50:1:49) in a ratio of 1:10. The solution was mixed in the dark and then added to HPLC-FLD for analysis.

[0090] HPLC-FLD Analysis of Biliverdin and Bilirubin

[0091] A 10×4.6-mm Aeris 3.6 μm 100 Å (Phenomenex, Torrance, Calif.) C18 column was used using 70% acetonitrile and 30% 10 mM TBA solution (pH 7.5) as the mobile phase at a flow rate of 1 ml/min. The analysis was operated at room temperature and fluorescence detection wavelength was set at 400/630 nm excitation/emission. The injection volume of each sample was 50 μl.

[0092] In-Vivo Anti-Inflammatory Efficacy of 5-ALA with SFC in DSS Colitis Mouse Model

[0093] ALA with SFC, the positive controls 5-aminosalicylic acid (5-ASA) or Cyclosporin A were dosed prophylactically either orally or intra-rectally once daily in C57BL/6 mice (male, 9 weeks old) that received treatment of 2.5% DSS in their drinking water. Animals were kept on 2.5% DSS for 7 days and then switched to normal water for 3 days before being sacrificed.

[0094] The different groups in the study were as follows:

TABLE-US-00001 Treatment Test Article Test Article Group Description Dose (mg/kg) Conc. (mg/ml) N 1 Vehicle (PBS) N/a N/a 7 2 2.5% DSS + N/a N/a 7 Vehicle 3 2.5% DSS + 70  7 7 Cyclosporin A 4 2.5% DSS + 50 15 7 5-ASA 5 2.5% DSS + 10 mg/kg ALA + 3 mg/kg ALA & 7 ALA & SFC 1.5 mg/kg SFC 0.45 mg/kg SFC 6 2.5% DSS + 100 mg/kg ALA + 30 mg/kg ALA + 7 ALA & SFC 15.7 mg/kg SFC 4.7 mg/kg SFC 7 Vehicle (PBS) N/a N/a 7 8 2.5% DSS + 70  7 7 Cyclosporin A 9 2.5% DSS + 50 15 7 5-ASA 10 2.5% DSS + 10 mg/kg ALA + 3 mg/kg ALA & 7 ALA & SFC 1.5 mg/kg SFC 0.45 mg/ks SFC 11 2.5% DSS + 100 mg/kg ALA + 30 mg/kg ALA + 7 ALA & SFC 15.7 mg/kg SFC 4.7 mg/kg SFC

[0095] Groups 1-6 were dosed orally via gavage, while groups 7-10 were dosed intra-rectally directly into the colon.

[0096] All groups were evaluated for changes in body weight, stool consistency, stool blood and colon weight/length ration compared to naïve tap-water drinking animals. Colon samples were also collected and subject to histopathology evaluation, cytokine/chemokine and metabolite analysis, and neutrophil myeloperoxidase activity. The study was terminated on Day 10, and necropsy was performed with tissue collection. Blood was collected by terminal cardiac puncture and plasma (>0.2 ml) was isolated by centrifugation, aliquoted into 50 μL aliquots, and stored at −80° C. Plasma was then subjected to chemokine/cytokine analysis of HO-1, TNF-α, IL-1β, IL-6, IL-10, and IL-2 levels as well as analysis of bilirubin and biliverdin levels. In the PK groups, plasma from 2 hours post-dose was analyzed for levels the metabolite PpIX.

Example 1. Stability of 5-ALA with SFC and Formation of PpIX in Human and Mouse Colon Model

[0097] Colon stability was assessed using the human and mouse colon model with the amount of intact 5-ALA remaining and subsequent formation of PpIX at each time point assessed by HPLC-FLD as described in the Methods section. The stability of 5-ALA and formation of PpIX in human and mouse colon are shown in FIGS. 1 and 2 respectively. The results show that 5-ALA is completely stable in both human and mouse colon fluid for up to 24 hrs. However, no conversion of 5-ALA in to PpIX was observed in the presence of SFC in both human and mouse colon.

Example 2. Human and Mouse Colon Tissue Penetration and Permeability of 5-ALA with SFC and Formation of PpIX, Biliverdin and Bilirubin

[0098] Colon tissue penetration and permeability of 5-ALA in the presence of SFC was assessed using healthy human and mouse colon tissue and formation of metabolites PpIX, biliverdin and bilirubin was analysed by HPLC-FLD as described in the Methods section. FIGS. 3 and 4 shows the penetration and permeability of 5-ALA in human and mouse colon tissue respectively. Formation of PpIX, biliverdin and bilirubin are shown in table below:

TABLE-US-00002 PpIX (μg/ml) Biliverdin (μg/ml) Bilirubin (μg/ml) Human 0 0 0 Mouse 0 0 0

[0099] The tissue conc. of 5-ALA was higher in human colon compared to mouse colon, while higher permeability of 5-ALA across the tissue was detected in mouse colon compared to human colon.

Example 3. In-Vivo Anti-Inflammatory Efficacy of 5-ALA with SFC in DSS Colitis Mouse Model

[0100] The ability of 5-ALA to reduce inflammation was investigated in a DSS induced colitis mouse model at 10 and 100 mg/kg dose. The drug was administered orally via gavage and intra-rectally directly into the colon. Expression of key inflammatory cytokines were measured locally in the colon tissue and plasma (FIGS. 5-10). To further understand the differences in the anti-inflammatory mechanisms of oral vs intra-colonic administration of 5-ALA, the formation of active metabolites biliverdin and bilirubin along with PpIX were measured in the tissue and plasma (FIGS. 11 and 12). 5-ALA was able to reduce the expression of inflammatory cytokines TNF-α, IL-1β and IL-6 in the colon tissue and plasma significantly better than after oral administration. The reduction in inflammatory cytokines was also superior to 5-ASA given both orally and intra-colonic. The expression of 5-ALA metabolite PpIX was higher in the plasma when given orally compared to intra-colonic, however, no difference in biliverdin and bilirubin expression was measured in the colon tissue and plasma after oral and intra-colonic dosing of 5-ALA. The data in the present invention suggests that the superior anti-inflammatory effect of intra-colonic administration of 5-ALA compared to oral administration is independent of SFC and anti-inflammatory metabolites formation (biliverdin and bilirubin) and is a direct anti-inflammatory effect of 5-ALA when administered locally in the inflamed colon. Local delivery of 5-ALA offers a unique therapeutic option for the treatment of inflammatory bowel disease at much lower doses and hence frequency of administration than 5-ASA which is the current standard first line therapy.