COMPOSITIONS OF PROTEINS WITH DIPEPTIDES AS STABILISING AGENTS

Abstract

This invention relates pharmaceutical compositions comprising a protein as the active ingredient together with one or more dipeptides as stabilising agents, and optionally an enzyme inhibitor. The compositions are particularly useful for administration to the intestinal tract.

Claims

1. A pharmaceutical composition which comprises as active ingredient a protein, one or more di-peptides and optionally an enzyme inhibitor.

2. A pharmaceutical composition as claimed in claim 1 wherein the di-peptide is carnosine and/or diglycine.

3. A pharmaceutical composition as claimed in claim 1 wherein the enzyme inhibitor is aprotinin, or a fragment, or an analogue thereof.

4. A pharmaceutical composition as claimed in claim 1 wherein the protein is an antibody or fragment thereof.

5. A pharmaceutical composition as claimed in claim 1 in a liquid, solid or semi-solid form suitable for oral or rectal administration.

6. A pharmaceutical composition as claimed in claim 5, in a solid form suitable for oral administration, said composition having an enteric coating, preferably adapted for selective release of the protein in the lower gastro-intestinal tract.

7. A pharmaceutical composition as claimed in claim 1 comprising a solid dosage form with a core and a coating for the core, the core comprising the active ingredient, a first stabilising agent and optionally at least one second stabilising agent; and the coating comprising a mixture of a digestible polysaccharide and a film-forming material which has a solubility threshold at pH 6.0 or above.

8. A pharmaceutical composition as claimed in claim 7, 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;

9. A pharmaceutical composition as claimed in claim 7, in which the film-forming material is an acrylate polymer, a cellulose polymer or a polyvinyl-based polymer, preferably cellulose acetate phthalate; cellulose acetate trimellitate; hydropropylmethylcellulose acetate succinate; and polyvinyl acetate phthalate.

10. An orally administrable pharmaceutical composition comprising as active ingredient a protein, one or more di-peptides and optionally an enzyme inhibitor.

11. A solid dosage form for oral administration comprising a core comprising as active ingredient a protein, one or more di-peptides and optionally an enzyme inhibitor, and a delayed release coating for the core.

12. A rectally administrable pharmaceutical composition comprising as active ingredient a protein, one or more di-peptides and optionally an enzyme inhibitor.

13. An enema formulation comprising as active ingredient a protein.

14. A method of treating or preventing a disease or condition in a subject which comprises administering to the subject via the ileum and/or the colon a pharmaceutical composition which comprises as active ingredient a protein together with one or more di-peptides and optionally an enzyme inhibitor.

15. A method as claimed in claim 14, wherein the one or more di-peptides of the pharmaceutical composition is carnosine and/or diglycine.

16. A method as claimed in claim 14, wherein the disease or condition is inflammatory bowel disease; irritable bowel syndrome; constipation; diarrhoea; infection; autoimmune disease or cancer.

17. (canceled)

18. A method of stabilising a protein in the presence of intestinal fluid, which comprises contacting the protein with one or more di-peptides and optionally an enzyme inhibitor.

19. (canceled)

20. The method as claimed in claim 14, wherein the enzyme inhibitor of the pharmaceutical composition is aprotinin, or a fragment, or an analogue thereof.

21. The method as claimed in claim 14, wherein the protein of the pharmaceutical composition is an antibody or fragment thereof.

Description

[0092] FIG. 1 shows the level of the antibody in tissues following administration via an enteric coated capsule with and without the stabilising agents, or intravenously.

[0093] FIG. 2 shows the plasma level of the antibody following administration via an enteric coated capsule with and without the stabilising agents, or intravenously.

MATERIALS AND METHODS

[0094] Human Colon Model

[0095] A human colonic model based on a mixed faecal 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.

[0096] Antibody Incubation Studies

[0097] Antibody stock solution (vedolizumab, ruplizumab, infliximab, ustekinumab, infliximab Fab fragment or combination of infliximab and ustekinumab) was prepared in PBS at 2 mg/ml and added to 20% human or rat faecal slurry to obtain an incubation concentration of 1 mg/ml and 10% w/w faecal slurry. Samples were withdrawn at appropriate time points and added to a protease inhibitor cocktail (Sigma-Aldrich, P2714) in a ratio of 1:3. The samples were centrifuged at 9.6 g for 10 mins and the supernatant was analysed by size exclusion-HPLC (SE-HPLC).

[0098] SE-HPLC

[0099] Sample analysis 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). A 600×7.8-mm Biosep™ 5 μm SEC-s3000 290 Å (Phenomenex, Torrance, Calif.) size exclusion (SE) chromatography column was used for sample separation using phosphate buffer saline (pH 7.3) prepared in sterile HPLC grade water as the mobile phase for elution, at a flow rate of 1 ml/min. The analysis was operated at room temperature and UV detection wavelength was set at 280 nm. Each sample was run for 40 minutes to allow complete elution of the sample proteins and reduce run-over. The retention time for IgG1 antibody, F(ab′).sub.2 and Fab/Fc fragments was 17, 18.2 and 20.3 minutes, respectively.

[0100] In-vivo dosing of infliximab capsules with and without stabilizing agents to Wistar Rats Lyophilized antibody (Infliximab) was formulated into rat capsules (5 mm×2.6 mm) at a dose of 0.7 mg/kg co-formulated with mannitol or stabilizing agents (aprotinin, L-carnosine and diglycine combination). The capsules were coated with an enteric coating that consisted of a mixture of pH sensitive polymer and intestinal bacteria digestible polysaccharide. Healthy male Wistar rats (Charles River Laboratories) weighing 350-400 g were housed in groups of 4 per cage and acclimatized for at least 5 days prior to entering the study. The rats were dosed with the capsules after overnight fasting and post dosing until sacrificed. The animals had free access to water throughout the study. The rats were sacrificed after 7 hrs post dosing and the entire GI tract, plasma and feces were collected, and antibody levels were analyzed by ELISA.

[0101] A parallel arm involving IV injection of the same dose of antibody was also tested in rats to compare the tissue and plasma concentration of antibody.

[0102] ELISA

[0103] Each well of the 96 well plate was coated with 100 μL of 1 μg/mL antigen (recombinant Human TNF-α Protein) diluted in PBS and incubated overnight at 4° C. The wells were washed 3 times with 200 μL/well washing buffer and blocked for 1 hr at 37° C. Following washing, the test samples were added and incubated for 1 hr at 37° C. After three washing cycles, the secondary antibody solution was added (goat anti-human IgG Fc secondary antibody HRP-conjugated) and incubated for 45 min at 37° C. After washing steps, the wells were incubated with 150 μL/well of K-BLUE substrate and incubate for 10 min at room temperature in the dark. After 10 minutes, 50 μL of RED STOP solution was added to each well and the plate was read at 650 nm.

Example 1 Stability of Monoclonal Antibody in the Human Colon Model in the Presence of L-Carnosine, Diglycine (Gly-Gly) and Optionally Aprotinin

[0104] Colon stability was assessed using the Human Colon model with the amount of intact antibody (vedolizumab, ruplizumab, infliximab or ustekinumab) remaining at each time point assessed by SE-HPLC as described in the Methods section. The experiment was carried out in the absence of a dipeptide, aprotinin or an excipient, and then in the presence of 50 mM or 100 mM L-carnosine. The experiment was also carried out in the presence of L-carnosine together with 0.5 mg/mL aprotinin (Sigma-Aldrich).

[0105] The results are shown in the following Table.

TABLE-US-00002 % Antibody % Antibody % Antibody remaining remaining remaining Concentration after 0 hours after 4 hours after 6 hours Formulation of excipient (T = 0 h) (T = 4 h) (T = 6 h) Infliximab alone  1 mg/ml 100 0 0 Ustekinumab alone  1 mg/ml 100 2.12 ± 0.20 0 Infliximab + L-carnosine  10 mM 100  1.1 ± 0.02 0 Infliximab + L-carnosine  50 mM 100 6.27 ± 0.55 1.76 ± 0.31 Infliximab + L-carnosine 100 mM 100 17.57 ± 1.23  7.84 ± 0.74 Infliximab + diglycine 100 mM 100 23.25 ± 0.82  — Infliximab + aprotinin 0.5 mg/ml 100 20.2 ± 0.16 0 Infliximab + aprotinin 0.1 mg/ml 100 3.05 — Infliximab + aprotinin + 0.1 mg/ml + 100 73.4 ± 0.05 — L-carnosine 50 mM Infliximab + aprotinin + 0.1 mg/ml + 100 66.24 ± 1.09  — diglycine 50 mM Infliximab + aprotinin + 0.1 mg/ml + 100 76.86 ± 1.39  — L-carnosine + diglycine 50 mM + 50 mM Infliximab + aprotinin + 0.5 mg/ml + 100 81.60 ± 3.74  — L-carnosine + diglycine 50 mM + 50 mM Infliximab + aprotinin + 0.5 mg/ml + 100 60.69 ± 3.61  35.57 ± 2.15  L-carnosine + diglycine 25 mM + 50 mM Infliximab + aprotinin + 0.5 mg/ml + 100 55.82 ± 2.86  27.35 ± 1.76  L-carnosine + diglycine 10 mM + 50 mM Ustekinumab + aprotinin + 0.5 mg/ml + 100 41.21 ± 1.94  14.19 ± 1.11  L-carnosine + diglycine 50 mM + 50 mM

[0106] The results show that infliximab, vedolizumab and ruplizumab were degraded by the human colonic microbiota in the absence of the tested stabilizer molecules. The antibody+aprotinin (0.5 mg/ml) control was observed to result in less degradation of the antibody. In the presence of 100 mM L-carnosine and 100 mM diglycine, a significant stabilising effect of the antibodies was obtained. This stabilisation effect of L-carnosine was significantly enhanced by including 0.1 and 0.5 mg/mL aprotinin in the test sample.

Example 2 Stability of Combination of Monoclonal Antibodies Infliximab and Ustekinumab in the Human Colon Model in the Presence of Aprotinin, L-Carnosine and Diglycine (Gly-Gly)

[0107] Colon stability was assessed using the Human Colon model with the amount of intact antibody (infliximab, ustekinumab or combination of infliximab and ustekinumab) remaining at each time point assessed by SE-HPLC as described in the Methods section. The experiment was carried out in the absence of a dipeptide, aprotinin or an excipient, and then in the presence of L-carnosine, diglycine and aprotinin (Sigma-Aldrich).

[0108] The results are shown in the following Table.

TABLE-US-00003 % Antibody % Antibody % Antibody remaining remaining remaining Concentration after 0 hours after 4 hours after 6 hours Formulation of excipient (T = 0 h) (T = 4 h) (T = 6 h) Infliximab alone 1 mg/ml 100 12.34 ± 0.83  2.45 ± 0.66 Ustekinumab alone 1 mg/ml 100 2.12 ± 0.20 0 Infliximab + Ustekinumab 0.5 mg/ml + 100 6.48 ± 1.01 0 0.5 mg/ml Infliximab + aprotinin + 0.5 mg/ml + 100 81.60 ± 3.74  — L-carnosine + diglycine 50 mM + 50 mM Ustekinumab + aprotinin + 0.5 mg/ml + 100 41.21 ± 1.94  14.19 ± 1.11  L-carnosine + diglycine 50 mM + 50 mM Infliximab + Ustekinumab + 0.5 mg/ml + 100 42.43 ± 1.99  17.56 ± 0.44  aprotinin + L-carnosine + 50 mM + diglycine 50 mM

Example 3 In-Vivo PK of Antibody in Wistar Rats after Oral Dosing in Capsule

[0109] Intestinal tissue concentration of antibody (infliximab) was measured after oral capsule dosing with and without stabilization agents (aprotinin, L-carnosine and diglycine combination) and post IV injection at a dose of 0.7 mg/kg.

[0110] The results are shown in the FIGS. 1 and 2.

[0111] The results demonstrated superior antibody concentration in the intestinal tissue (ileo-colonic and colonic) after formulating with stabilization agents in enteric coated capsule, compared to formulating without stabilization agents and IV injection of the same dose of antibody. No plasma exposure of antibody was observed after capsule dosing (with and without stabilization agents), while the IV injection dose showed high concentration of antibody floating in the systemic circulation.