WO2006090127A1

WO2006090127A1 - Pharmaceutical formulation

Info

Publication number: WO2006090127A1
Application number: PCT/GB2006/000587
Authority: WO
Inventors: Mark Seymour; Ingrid Vereyken; Lorraine Jordan
Original assignee: Syngenta Limited; Octoplus Development B.V.
Priority date: 2005-02-22
Filing date: 2006-02-20
Publication date: 2006-08-31
Also published as: GB0503640D0

Abstract

The present invention relates to pharmaceutical compositions. In particular, the present invention relates to a pharmaceutical composition comprising a pharmaceutically active polypeptide comprising at least one cysteine residue having a redox-active thiol group in a reduced state and at least one saccharide or saccharide derivative capable stabilising the redox-active thiol. Preferably, the polypeptide is a thioredoxin. The present invention further relates to a process for manufacturing the pharmaceutical composition and uses of the composition.

Description

STABLE PHARMACEUTICAL FORMULATION COMPRISING THIOREDOXIN

The present invention relates, inter alia, to the provision of a pharmaceutical composition comprising a pharmaceutically active polypeptide that comprises at least one cysteine residue having at least one redox-active thiol group in a reduced state, for example thioredoxin. Such compositions are useful in the treatment of various diseases including, for example, inflammatory diseases and pulmonary diseases.

Reduced thioredoxin is inherently unstable, insofar as it is able to oxidise in the presence of oxygen in the atmosphere. For example, the -SH groups associated with the cysteines at the thioredoxin active site (CyS₁ -Gly-Pro-Cys₂) can oxidise to form a, di-sulphide bridge. Additionally, where the thioredoxin comprises cysteines other than at the active site, these may oxidise to form thioredoxin oligomers.

A need exists therefore to provide a pharmaceutical composition that is able to maintain the polypeptide in a reduced state for prolonged periods of time.

Surprisingly, it has been shown that certain saccharides or saccharide derivatives are able to stabilise thioredoxin to such an extent that the thioredoxin-formulated material is maintained in a reduced state for several months, even when stored at elevated temperature.

Thus, according to the present invention there is provided a pharmaceutical composition comprising: a. a pharmaceutically active polypeptide comprising at least one cysteine residue haying at least one redox-active thiol group in a reduced state; and b. at least one saccharide or saccharide derivative capable of stabilising the redox-active thiol group.

By redox-active thiol group it is meant a thiol group that may exist in either a reduced state (-SH) or an oxidised state (-S-S-). The term "stabilising" includes, for example, reducing the rate of oxidation of the redox-active thiol group in a reduced state when the polypeptide is present in a pharmaceutical composition with the saccharide or saccharide derivative relative to a composition in which the saccharide or saccharide derivative is omitted. Preferably, the polypeptide referred to in (a) above is a thioredoxin. By thioredoxin it is meant any polypeptide that comprises a thioredoxin active site i.e Cysi-Gly-Pro-Cys₂. Thioredoxin is a small (10-14 kDa), ubiquitous protein that is an important component of the cellular redox regulation system. Suitable thioredoxins for use in the pharmaceutical compositions of the present invention include, for example, thioredoxin from (1) a prokaryote e.g E. coli, (2) a plant e.g Arabidopsis and (3) animal e.g human.

Thioredoxin can exist in a reduced state (wherein the two cysteines at the active site provide a dithiol) and an oxidised state (wherein there is a disulphide bridge present between the two cysteines at the active site). Under physiological conditions both redox states can exist. In respect of the pharmaceutical compositions of the present invention thioredoxin containing a reduced active site is preferred including, for example, a thioredoxin population comprising a substantial number of thioredoxin polypeptides containing a reduced active site. Furthermore, it is known that certain thioredoxins can exist in multimeric forms. For example, it is known that human thioredoxin can form dimers wherein a disulphide bridge exists between Cys-73. In respect of the pharmaceuti cal compositions of the present invention thioredoxin in substantially monomelic state is preferred. A preferred molecule for use in the compositions of the present invention is the human thioredoxin depicted in SEQ ID NO. 1 or SEQ ID NO. 2 — since this thioredoxin is an endogenous human protein, and is therefore unlikely to cause either adverse effects, or an immune response when administered to patients. Particularly preferred is a pharmaceutical composition comprising reduced, monomelic human recombinant thioredoxin (rhTRX).

By "saccharide" it is meant any mono-, di-, oligo- or poly-saccharide. Examples of saccharides are glucose, fructose, sucrose, lactose, maltose, galactose, raffinose, inulin, dextran and trehalose. By saccharide derivative it is meant a compound that structurally resembles the saccharide from which it is derived. For example, sucralose, which is a chlorinated sucrose, would be considered a saccharide derivative of sucrose. Further derivatives include, for example, alditol derivatives for example mannitol and xylitol. Preferred compositions of the present invention comprise non-reducing saccharides, for example raffinose, trehalose, stachyose and particularly sucrose. More preferably the pharmaceutical composition comprises recombinant human thioredoxin in a substantially reduced, monomelic state and sucrose. Preferred human thioredoxin has the polypeptide sequence depicted in SEQ ID NO. 1 or SEQ ID NO. 2.

The pharmaceutical composition of the present invention may further comprise other pharmaceutically acceptable excipients, for example stabilisers and tonicity adjusters. An example of such an excipient is EDTA.

Furthermore, it is preferred that the pharmaceutical composition of the present invention is a dry (or substantially dry) composition, for example a powder. This may be achieved, for example, by freeze-drying the pharmaceutical composition of the present invention. Preferably, the composition is provided and/or stored under an inert gas, for example nitrogen or argon.

In a preferred embodiment, the composition is manufactured as an aqueous composition which is then lyopbilised. For example, the aqueous composition may comprise:-

a. 20 to 100 mg/ml recombinant human thioredoxin; b. 10 to 100 mg/ml sucrose; c. 0.1 to 1.0 mg/ml EDTA

The present invention further provides a kit comprising:

a. a first component consisting of a pharmaceutical composition of the present invention; b. a second component suitable for the reconstitution of the first component, preferably sterile water.

The present invention further relates to the use of the pharmaceutical composition of the present invention in the manufacture of a medicament for therapeutic or prophylactic use. In particular, the present invention further relates to the use of the pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of a pulmonary disease. Preferably, the pulmonary disease is selected from the group consisting of acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, infant respiratory distress syndrome, interstitial lung disease and asthma. Most preferably, the pulmonary disease is cystic fibrosis.

The present invention further relates to a process of treating a pulmonary disease comprising administering to a patient diagnosed with a pulmonary disease the pharmaceutical composition of the present invention. The pharmaceutical composition may be administered to the patient via any suitable means, for example by use of a nebulizer or a breath-activated device.

The present invention further relates to a process for the manufacture of a pharmaceutical composition of the present invention comprising:

a. obtaining the pharmaceutically active polypeptide comprising at least one cysteine residue having at least one redox-active thiol group in a reduced state of interest; and b. at least one saccharide or saccharide derivative capable of stabilising the redox-active thiol group.

If the composition of the present invention is manufactured via an aqueous solution then the process may further comprise a lyophilisation step (c) after said contacting step (b).

Furthermore, the pharmaceutical composition of the present invention may further comprise an additional active ingredient. By additional active ingredient it is meant an ingredient that also has a pharmacological effect, which may be either additive of synergistic with thioredoxin. Examples of additional active ingredients include those having deoxyribonuclease activity. The pharmaceutical compositions of the present invention could further include other ingredients such anti-oxidants (e.g glutathione, vitamin A, vitamin E).

The present invention further relates to the use of a saccharide or saccharide derivative as a cyro-protectant and/or antioxidant and/or osmotic adjuster in the manufacture of a thioredoxin-containing medicament for the treatment of a pulmonary disorder. List of Figures:

WFI = Water For Injection. FD = Freeze Drying

Figure 1. Effect of freeze-drying on thioredoxin (TRX) + sucrose compositions. Results indicate that the amount of dimer in the composition remains significantly unchanged following the freeze-drying process.

Figure 2. Effect of freeze-drying on thioredoxin (TRX) + mannitol compositions. Results indicate that the amount of dimer in the composition increases following the freeze-drying process, indicating that mannitol-containing compositions are less stable than sucrose- containing compositions.

Figure 3. Results of monitoring freeze-dried thioredoxin-containing compositions for a period of two months stored at 25°C. Results indicate that the percentage of reduced monomeric thioredoxin remains constant in compositions comprising sucrose, but does not remain constant in compositions comprising mannitol.

Figure 4. Results of monitoring freeze-dried thioredoxin-containing compositions for a period of two months stored at 25°C. Results show the increase in oxidized TRX monomer and suggest that the percentage of reduced thioredoxin remains approximately constant in compositions comprising sucrose, whereas the percentage of oxidized monomer increases in compositions comprising mannitol.

Figure 5. Results of monitoring freeze-dried thioredoxin-containing compositions for a period of two months stored at 25°C. Results indicate that oligomerisation is a less prominent degradation route in respect of both sucrose and mannitol compositions.

Figure 6. Results of monitoring freeze-dried thioredoxin-containing compositions for a period of two months stored at 5°C. Results indicate that the percentage of reduced monomeric thioredoxin remains constant in compositions comprising sucrose, but did not remain constant in compositions comprising mannitol. Figure 7. Results of monitoring freeze-dried thioredoxin-containing compositions for a period of two months stored at various temperatures. Results indicate that the percentage of oxidized monomer remained unchanged in compositions comprising sucrose, even in compositions stored at 4O⁰C, whereas an increase in oxidized monomer is observed in compositions comprising mannitol,

Figure 8. Results of monitoring freeze-dried thioredoxin-containing compositions (further comprising EDTA) for a period of two months stored at various temperatures. Results indicate that the percentage of oxidized monomer remains unchanged in compositions comprising sucrose, even in compositions stored at 40⁰C, whereas an increase in oxidized monomer is observed in compositions comprising mannitol.

Figure 9. Stability comparison of thioredoxin-containing compositions comprising sucrose. The results indicate that the stability of the sucrose formulations is comparable.

Figure 10. Overview of the percentage of rhTRX retained for each sugar formulation following 6 weeks incubation at 4O⁰C (the glucose formulation is not included in this overview). The graph indicates that non-reducing sugars such as sucrose, trehalose and rafϊinose are particularly effective in providing stabilised thioredoxin formulations.

Figure 11. Overview of the percentage of hTRX oligomers present in respect of each sugar formulation following 6 weeks incubation at 40⁰C. The graph indicates that the non- reducing sugars sucrose, trehalose, and rafflnose and also lactose were more effective in preventing oligomerisation of the TRX polypeptides.

Figure 12. Overview of stability of freeze-dried rhTRX when stored under nitrogen at various temperatures in the absence of any saccharide or saccharide derivative over a 25- week period. Graph shows a steady increase in the observed concentration of oxidised monomer when the material is stored at +5°C or room temperature (RT). SEQUENCES

SEQ ID NO. 1 - Human thioredoxin.

SEQ ID NO. 2 - Human thioredoxin (minus N-terminal Met).

Examples :

Example 1

Formulations are prepared according to the table below.

Lyophilised tbioredoxin-containing compositions are prepared as follows.

1. Prepare formulation according to above table (where acetate buffer is not present sterile water is used). Sufficient saccharide and/or saccharide derivative is provided in order to establish an isotonic solution.

2. Add recombinant human thioredoxin (rhTRX) in a substantially reduced state (40 mg/ml).

3. Sterile filter.

4. Add 2.5 ml of filtered solution to vial.

5. Freeze dry.

Freeze dried compositions were stored under nitrogen and samples taken at various time points in order to assess the changes that had occurred in respect the reduced material.

Claims

1. A pharmaceutical composition comprising: a. a pharmaceutically active polypeptide comprising at least one cysteine residue having a redox-active thiol group in a reduced state; b. at least one saccharide or saccharide derivative capable of stabilising the redox-active thiol.

2. A pharmaceutical composition according to claim 1, wherein the polypeptide is a thioredoxin.

3. A pharmaceutical composition according to claim 1 or claim 2, wherein the saccharide or saccharide derivative is selected from the group consisting of sucrose,

. sucralose, lactose, trehalose, maltose, galactose and raffinose.

4. A pharmaceutical composition according to any one of the previous claims, wherein the saccharide is a non-reducing sugar.

5. A pharmaceutical composition according to claim 4, wherein the non-reducing sugar is selected from the group consisting of sucrose, trehalose and raffinose.

6. A pharmaceutical composition according to claim 5, wherein the saccharide or saccharide derivative is sucrose.

7. A pharmaceutical composition according to any one of the preceding claims, wherein the thioredoxin is recombinant human thioredoxin.

8. A pharmaceutical composition according to claim 7, wherein the human thioredoxin has the sequence depicted in SEQ ID ISfO. 1 or SEQ ID NO. 2.

9. A pharmaceutical composition according to any one of the previous claims, further comprising EDTA.

10. A pharmaceutical composition according to any one of the previous claims, comprising: a. 20 to 100 mg/ml recombinant human thioredoxin; b. 20 to 100 mg/ml sucrose; c. 0.1 % to 1.0% mg/mJ EDTA

11. A pharmaceutical composition according to any of the preceding claims, which does not comprise an alditol derivative of a saccharide.

12. A pharmaceutical composition according to claim 11 , wherein the alditol derivative of a saccharide is mannitol.

13. A pharmaceutical composition according to any one of the previous claims, wherein said composition is a substantially dry composition.

14. A pharmaceutical composition according to any one of the previous claims, wherein said composition is provided under an inert gas.

15. A kit comprising:

a. a first component consisting of a pharmaceutical composition according to claim 13 or claim 14;

b. a second component suitable for the reconstitution of the first component.

16. A kit according to claim 15, wherein the second component is sterile water.

17. A pharmaceutical composition according to any one of claims 1 to 14 fox therapeutic or prophylactic use.

18. The use of a pharmaceutical composition according to any one of claims 1 to 14, in the manufacture of a medicament for the treatment of a pulmonary disease.

19. The use according to claim 18, wherein said pulmonary disease is cystic fibrosis.

20. A process of treating a pulmonary disease comprising administering to a patient diagnosed with a pulmonary disease a pharmaceutical composition according to any one of claims 1 to 14.

21. A process according to claim 20, wherein said phaπnaceutical composition is administered to the patient by means of a breath activated device.

22. A process for the manufacture of a pharmaceutical composition according to claim 1 comprising:

a. obtaining the thioredoxin of interest in a substantially reduced state; b. contacting the thioredoxin of step (a) with a saccharide or saccharide derivative capable of stabilising the redox-active thiol.

23. A process according to claim 22, wherein the at least one saccharide or saccharide derivative is selected from the group consisting of sucrose, sucralose, lactose, trehalose, maltose, galactose and raffinose.

24. A process according to claim 23, wherein the saccharide is sucrose.

25. A process according to arty one of claims 22 to 24, further comprising a lyophilisation procedure a.fter said contacting step.

26. A pharmaceutical composition according to any one of claims 1 to 14, further comprising an additional active ingredient.

27. A pharmaceutical composition according to claim 26, wherein said additional active ingredient has deoxyribonuclease activity.

28. The use of a saccharide or saccharide derivative as a cyro-protectant and/or antioxidant and/or osmotic adjuster in the manufacture of a thioredoxin-containing medicament for the treatment of a pulmonary disease.