WO2004052341A1

WO2004052341A1 - Adjuvant compositions containing a sugar in crystalline form and aminoalkyl glucosaminide-4-phosphate

Info

Publication number: WO2004052341A1
Application number: PCT/GB2003/005354
Authority: WO
Inventors: Julian Alexander Blair; Glen Patrick Martyn; Lei Mao
Original assignee: Quadrant Drug Delivery Limited
Priority date: 2002-12-09
Filing date: 2003-12-09
Publication date: 2004-06-24
Also published as: AU2003288441A1; GB0228689D0

Abstract

Adjuvant compositions are prepared by spray drying a sugar and an adjuvant. The compositions comprise a crystalline matrix and the adjuvant dispersed within the matrix.

Description

ADJUVANT COMPOSITIONS CONTAINING A SUGAR IN CRYSTALLINE FORM AND AMINOALKYL

GLUCOSAMINIDE-4 - PHOS PHATE

Field of the Invention

This invention relates to sugar compositions for the stabilisation of therapeutic agents, in particular liposomal and micellar products. 5 Background of the Invention

Spray-drying technology is now widely used for the production of particles that comprise a therapeutic agent. In particular, there is now a wide range of literature that demonstrates that spray-drying can be useful to produce microparticles that are suitable for inhalation delivery, e.g. via an inhaler device.

10 Typically, spray-drying is carried out by first producing a solution of the therapeutic agent with an appropriate material, e.g. a sugar, and spray-drying the solution at an appropriate temperature.

Trehalose is a well known sugar that is used for the stabilisation of labile molecules in the solid state. When prepared as very small particles (for example

15 by spray-drying), amorphous trehalose is formed. In some circumstances, the amorphous trehalose can be unstable, reverting quickly in relative humidity above 30% (at ambient temperature) to the crystalline dihydrate form. If a labile molecule is incorporated into an amorphous trehalose matrix which subsequently reverts to its thermodynamically more favourable crystalline state, a

20 corresponding loss in activity can also be seen.

Accordingly, there is a need for further compositions for stabilising a therapeutic product, especially when the products are to be prepared by a spray- drying process. Summary of the Invention

25 The present invention is based, in part, on a surprising finding that crystalline sugars can be used to stabilise therapeutic products or adjuvants.

According to a first aspect of the invention, a composition comprises a crystalline matrix formed from a carbohydrate sugar and, dispersed within the matrix, a molecule which is or comprises an acylated sugar.

30 It has been found that spray-drying an acylated sugar with a sugar that forms a crystalline structure, provides powders that are chemically stable and disperse well in dry powder delivery devices. Description of the Invention

The compositions of the invention are typically microparticles for therapeutic application.

The microparticles will be formed typically by spray-drying a solution of the acylated sugar and the carbohydrate sugar.

An acylated sugar is any carbohydrate sugar moiety which has one or more fatty acids attached. Suitable sugars are disclosed in WO-A-02/03961 , the content of which is incorporated herein by reference. Preferred acylated sugars include aminoalkyl glucosaminide-4-phosphate (AGP). Glycolipids are suitable acylated sugars.

The acylated sugar is preferably intended to be used as, or as part of, an adjuvant, to help stimulate an immune response to an antigen.

In addition, any suitable therapeutic product/agent may be present in the formulation. Therapeutic agents which may be used include, for example, proteins, peptides, nucleic acids and small organic molecules. The reference to therapeutic agents is intended to also include prophylactic agents, including vaccines. The vaccines may comprise any suitable antigenic component. Preferably the vaccine is a nucleic acid-based antigen, as described in WO-A- 0203961 , the content of which is incorporated herein. In a particularly preferred embodiment, the therapeutic agent is a phospholipid, micellar or liposomal product. In a further preferred embodiment, the phospholipid, micellar or liposomal product is complexed with, or encapsulates, a peptide, protein or polynucleotide antigen.

Other particularly preferred therapeutic agents include: human growth hormone, bovine somatotropin, porcine somatotropin, growth hormone releasing peptide, granulocyte-colony-stimulating factor, granulocyte-macrophage-colony- stimulating factor, erythropoietin, interferon, monoclonal antibody, TNF, interleukin, tumour-denaturing factor, insulin, insulin-like growth factor, epidermal growth factor, tissue plasminogen activator and urokinase. As stated previously, antigens (immunogens) are particularly preferred.

The antigens may be for the prophylaxis of any bacterial or viral disease. For example, the antigen may be for the prevention of meningococcal disease (meningitis, septicaemia, meningoccaemia and pneumonia). In this embodiment, the antigen may be used to prevent infection of meningococci of any of groups A, B, C, Y, W135, X and Z.

Other suitable antigens for use in the practice of the invention include those used to prepare vaccines against anthrax e.g. (Protective Antigen), plague, small pox, tularaemia, meliodosis, Q fever, botulism, typhus, cholera, yellow fever, brucellosis, encephalitis, ricin, salmonella and staphylococcal

Enterotoxin B.

Viral particles useful in the preparation of vaccines are known and are applicable to the invention. The invention may be used for the prophylaxis of HIV, HepB, CMV and TB.

In one embodiment, the antigen is a protein. The protein may be in the form of a fusion protein or may be conjugated (complexed) with a different molecule. Any sugar that is capable of forming a crystalline morphology can be used in the present invention.

Particularly preferred sugars are mannitol and erythritol. The compositions may be formed from a mixture of polyols (sugars), provided that the crystalline morphology is obtained. The compositions of the present invention may be formulated using techniques known to those skilled in the art. Suitable methods include, but are not limited to, spray-drying, freeze drying, spray-freeze drying, air drying, vacuum drying, fluidised-bed drying, milling and super-critical fluid evaporation. Spray-drying is a particularly preferred method. In the case of spray-drying, the components are first dissolved or suspended in a suitable solvent at a suitable concentration. The solution or suspension is then fed into the atomisation chamber of a spray-dryer at suitable feed rates and at a suitable temperature. The resulting microparticles are collected and stored in airtight containers.

The moisture content of the dried products is preferably less than 4%, more preferably less than 2%.

Microparticles according to the invention may be adapted for any suitable route of administration, i.e. oral, ocular, rectal, vaginal etc. For example, a composition comprising the microparticles may be prepared for delivery via injection, including subcutaneous injection, transdermal injection, intramuscular injection or using ballistic/needle-free injection systems. In a .preferred embodiment, the microparticles are to be delivered via inhalation, i.e. nasal or pulmonary administration.

The microparticles are generally of a size less than 100 μm ih diameter, depending on the route of administration. For delivery via oral or nasal inhalation, the microparticles will typically be of a size of about 0.1 μm to 50 μm in diameter. Preferably, the microparticles will be of a size of 0.1 μm to 5 μm in diameter. More preferably, the microparticles will be of a size of about 2 μm in diameter in order for the microparticles, when inhaled, to reach the alveoli of the lungs.

It will be appreciated by the skilled person that the therapeutic agents are to be formulated in pharmacologically effective amounts. That is, when delivered in a unit dosage form, there should be sufficient amount of therapeutic to achieve the desired response.

If the microparticles are intended for delivery as dried powders in an inhalation device, it will be appreciated that unit dose comprises a predefined amount of microparticles delivered to the patient in one inspiratory effort. In a preferred embodiment, the microparticles are prepared as single unit dosage forms for inclusion in dry powder inhalers. In this embodiment, a single unit dose will be approximately 0.1 to 15 mg, preferably between 0.5 to 10 mg.

The composition delivered to a patient may also comprise other components, e.g. carbohydrates or other glass-forming substances as stabilisers of excipients. Additional components may be desirable to modify the characteristics of the microparticles. For example, it may be desirable to add further components to improve the particle rigidity or release profile.

The invention will now be described further in the following Examples, with reference to the accompanying figures. Example 1

The adjuvant RC-529 is an aminoalkyl glucosaminide-4-phosphate and was supplied as an aqueous formulation in which RC-529-triethylamine was solubilised by DPPC micelles. HPLC assay of this material gave an RC-529 content of 0.842mg/ml (expressed as the triethylamine salt).

Three formulations were prepared by spray-drying from water (Table 1 ). Two mannitol-based batches were prepared in order to demonstrate reproducibility. Powder recovery yields of 70-80% w/w were obtained from 1.5g total solids. The RC-529 raw material was used as received, therefore, all spray- dried batches contained RC-529 and DPPC at the nominal concentrations given in Table 1. Table 1 RC-529 Formulations

* As triethylamine salt. The water content of the isolated powders (as determined by Karl Fischer titration) ranged from 0.6% to 2.2% w/w (Table 2). The mannitol formulations contained less moisture compared to the trehalose formulation. Differential Scanning Calorimetry (DSC) was performed in order to determine particle crystallinity. The aerodynamic particle size of each batch was determined by Time of Flight Aerosol Beam Spectroscopy (TOFABS) using an aerosizer. All batches were found to have Mass Median Aerodynamic Diameter (MMAD) values of approximately 2μm. Table 2

The full aerodynamic particle size distributions for the formulations are shown in Figure 1.

Effect of Processing on the Stability of RC-529 Formulations

Spray-dried formulations were assayed for RC-529 content (Table 3). Table 3 RC-529 Content of Spray-Dried Formulations

Stability of the formulations was assessed by comparing the RC-529 contents before and after storage in sealed glass vials in a 25°C/60% relative humidity (RH) environment for 14 days (Table 4). Table 4 RC-529 Content of Formulations during Stability Assessment

An insignificant change in adjuvant content was measured in the mannitol formulations after storage. However, interestingly an apparent reduction in adjuvant content was measured with the trehalose-based formulation. Example 2

An experiment was carried out to determine whether erythritol could be used to produce crystalline spray dried powders. The following conditions were used: Formulation 1

Erythritol 500 mg

Water 25 ml

Sprav dry conditions

Inlet temperature 100°C Outlet temperature 50°C

Feed rate 90 %

Atomisation pressure 4 bar

Dry air flow 1 bar

The spray dried powders were tested to establish whether they had a crystalline or glass morphology. The results are shown in Figure 2. A crystalline morphology was observed.

Formulation 2

Erythritol 500 mg

DPPC micelles in water 0.06 w/v 25 ml Sprav dry conditions

Inlet temperature 100°C

Outlet temperature 50 °C

Feed rate 90 %

Atomisation pressure 4 bar Dry air flow 1 bar

The morphology results are shown in Figure 3. A crystalline morphology was observed.

Claims

1. A composition comprising a crystalline matrix formed from a carbohydrate sugar and, dispersed within the matrix, a molecule that is or comprises an acylated sugar.

2. A composition according to claim 1 or claim 2, wherein the adjuvant is or comprises aminoalkyl glucosaminide-4-phosphate.

3. A composition according to any preceding claim, wherein the carbohydrate sugar is mannitol, erythritol or a mixture of polyols comprising mannitol or erythritol.

4. A composition according to any preceding claim, in the form of spray dried powder.

5. A composition according to any preceding claim, further comprising an antigen.

6. A composition according to claims 5, wherein antigen is a liposomal product encapsulating a therapeutic product.

7. A composition according to claim 6, wherein the therapeutic product is a peptide, protein or polynucleotide.

8. A composition according to any preceding claim, for therapeutic use.

9. A device comprising a composition according to any preceding claim.