Description ALUMINIUM PHOSPHATE NANOPARTICLES

FIELD OF INVENTION

The present invention relates to aluminium phosphate nanoparticles and method of preparation thereof.

BACKGROUND OF THE INVENTION

Adjuvants are defined as substances used in combination with a specific antigen to enhance the immune response. An effective vaccine usually requires an adjuvant to stimulate the immune response. The only adjuvants approved for use in currently licensed vaccines are the aluminium-containing adjuvants. Aluminium hydroxide and aluminium phosphate are the most commonly used adjuvants. In 1926, the adjuvant effect of aluminium compounds was described by Glenny et. al. (Glenny AT, Pope CG, Waddington H, Wallace U. Immunological Notes XVII to XXIV. J. Pathol. 29, 31-40, 1926). Scholtz et. al. in 1984 demonstrated that pure aluminium phosphate can be prepared using equimolar amounts of aluminium chloride and trisodium phosphate.

Although such adjuvants are widely used today, aluminium phosphate adjuvants can be difficult to manufacture with reproducible properties as the experimental conditions like temperature, concentration and even rate of addition of chemicals reagents strongly influence the properties of the gel prepared. The important properties that are affected are particle size distribution, electric charge and hydrated colloid nature of the precipitate formed. These properties then affect the adsorption of antigen and thus the immunogenicity of the antigen.

Subcutaneous administration of vaccines is considered as the most appropriate route for delivering an antigen to secondary lymphoid organs such as lymph nodes where adaptive immunity is initiated. It is known that the fate of the particles following subcutaneous administration depends on the size of the particles. Large particles, having a size greater than 1 micron are unable to access and drain into the lymphatics and may not be phagocytosed readily and they are retained at the injection site until they degrade to a sufficiently small size. On the contrary, small particles having a size less than 1 micron enter lymphatic capillaries, which form a one-way drainage system connected to the lymphatic vessels, lymph nodes and lymphatic ducts, respectively. Particles with a diameter less than 100 nm enter the lymphatic capillaries through gaps between the lymphatic endothelial cells while particles having diameter between 100 nm to 1 micron are phagocytosed by antigen representing cells such as dendritic cells which subsequently passage into the lymphatic capillaries.

Nanoparticles of adjuvants are considered to be the most promising system for delivery of antigen to the draining lymph nodes following subcutaneous administration. Nanoparticles are defined as particles with diameter ranging from 1 to 1000 nm.

Nanometer size particles were used as carrier particles in the prior art. See 5178882, 5219577, 5306508, 5334394, 5460830, 5462750 and 5464634. The paricles described herein are in the range of 10 to 200 nm size range and are difficult to make with a degree of consistency.

Calcium phosphate particles have been under investigation as an alternative to aluminium-containing adjuvants in parenteral vaccines. Calcium Phosphate particles (>1000nm) as vaccine adjuvants are unsuitable due to inferior adjuvanting activity and possess a rough and oblong morphology. (Goto et. al., Vaccine, vol.15, no. 12-13, 1364-1371, 1997).

US 6355271 discusses a method for preparing one or more particles of calcium phosphate having diameters between about 300 nm to about 4000 nm, comprising reacting a soluble calcium salt with a soluble phosphate salt, wherein the reaction comprises: (a) mixing an aqueous solution of calcium chloride with an aqueous solution of sodium citrate to form a mixture, (b) adding an aqueous solution of sodium phosphate to the mixture to form a solution, (c) stirring the solution until particles of the desired size and comprising calcium phosphate are obtained and co-crystallized with antigenic material. The particles of size more than 1000 nm may not be phagocytosed and therefore are not suitable as adjuvants.

WO 2007/098186 discloses a composition prepared by combining one or more oils, one or more non-ionic surfactants, optionally one or more sterols, aluminium salt and an aqueous phase. The aluminium phosphate particles prepared therein range from 50 nm to 5000 nm and are stabilized by the surfactants added therein.

WO 2008/009592 discloses a method of preparing nanoparticles of metal phosphates with desired particle size, size distribution and purity wherein the method involves atomization of the solution in a reactor in excess of 100 deg C wherein the said temperature is selected in such a way that the organic acid and the water evaporate from the solution to produce nanoscale particles. Such process seems to be hazardous and teadious.

The instant invention overcomes the drawbacks of the prior art and provides a method for production of nanoparticles of aluminium phosphate with particle diameter less than 1000 nm, preferably 10 to 600 nm.

SUMMARY OF THE INVENTION

The instant invention relates to stable nanoparticles of Aluminium Phosphate and a process for preparation of thereof.

The instant invention provides nanoparticles of aluminium phosphate with particle diameter in the range of 10 to 600 nanometer (nm).

The instant invention also provides a process for preparation of aluminium phosphate nanoparticles comprising

1. a. adjusting pH of the aluminium phosphate gel to a pH above 6, preferably

between 6.5 to 10

1. b. passing the solution through high pressure homogenizer to get desired particle size

1. c. optional stabilization of the nanoparticles at a pH in the range of 6 to 8, preferably at pH 7.0 The instant invention has following advantages over prior art:

1. 1. Stability of the nanoparticles

1. 2. Simple, robust and repeatable process

1. 3. Process does not involve use of chemicals or high temperature

1. 4. No additional stabilizer like surfactants, glycols or oils required

1. 5. The robust nanoparticles afforded are stored at physiological pH hence can be used as adjuvant without additional processing

DESCRIPTION OF FIGURES

Figure 1 : Particle size statistics report by intensity of nanoparticles immediately after preparation

Figure 2: Particle size statistics report by intensity of nanoparticles one month after preparation

DETAILED DESCRIPTION OF THE INVENTION

The first embodiment of the instant invention is the stable nanoparticles of aluminium phosphate with particle diameter less than 1000 nm.

The second embodiment of the instant invention is the process for preparation of the stable nanoparticles of aluminium phosphate with particle diameter less than 1000 nm.

Specifically, the stable nanoparticles of aluminium phosphate are of diameter less than 600 nm, preferably between 10 to 600 nm. The particle size or mean particle size of the nanoparticles can be controlled and the particle size distribution can be restricted to a desired level. The process of preparation of aluminium phosphate nanoparticles is a simple and robust process which produces the particles of desired diameter. The process produced nanoparticles with high degree of consistency in terms of properties of the particles.

The aluminium phosphate gel used for preparation of the nanoparticles can be prepared in situ or aluminium phosphate powder can be dissolved in suitable buffer before size reduction.

The nanoparticles of the instant invention can be prepared from aluminium phosphate gel or precipitate. The pH of the solution can be adjusted to a desired pH in the range of 6 to 10. The preferred pH is around 7.0. The particles can be prepared by treating the gel with either a homogenizer or a sonicator. Appropriate technical parameters e.g. pressure, frequency, speed or amplitude of the homogenizer or sonicator can be adjusted to produce particles of desired size. The particles thus obtained can be stabilized in a suitable buffer. The preferred buffer is phosphate buffer of pH 7.0. The buffer can be the buffer used to formulate the final vaccine.

The following Examples further illustrate the essential features of the present invention. However, it will be apparent to those skilled in the art that the specific chemicals, apparatus, instruments, conditions and methods used in the Examples do not limit the scope of the present invention.

EXAMPLES

Example 1

Preparation of Aluminium Phosphate Gel

Slowly add 5 L solution of trisodium phosphate (16.9%) with 5 L solution of aluminium chloride (10.74%) with continuous stirring. Stirred the above mixture, for approximately 2 hours, to allow gel or precipitate formation. Diluted the suspension to around 50 L using water for injection (WFI). Allowed the aluminium phosphate gel to settle. Decanted the supernatant and replaced with WFI. The step was repeated till the supernatant is free of chloride ions.

Example 2

Preparation of Aluminium Phosphate Nanoparticles by homogenization

Aluminium phosphate gel suspension, either from example 1, prepared from aluminum phosphate powder or prepared in situ, was adjusted to pH 7.0 using trisodium phosphate. The gel was subjected to high pressure homogenization at 40000 psi for 15 cycles. The average particle diameter was found to be -150 nm. The nanoparticles thus obtained were stabilized using 10 mM phosphate buffer with pH 7.0. The gel was kept at 2 to 8 degrees temperature. Stability of the nanoparticles was checked after one month. The average particle size (diameter) along with the particle size distribution was substantially stable.

Example 3

Preparation of Aluminium Phosphate Nanoparticles by sonication

Aluminium phosphate gel suspension, either from example 1, prepared from aluminum phosphate powder or prepared in situ, was adjusted to pH 7.0 using trisodium phosphate. The gel was subjected to sonication using at 40 amplitude with 3 second pulse rate for 1 hour. The nanoparticles thus obtained were stabilized using 10 mM phosphate buffer with pH 7.0. The gel was kept at 2 to 8 degrees temperature. The average particle diameter was found to be 250 nm.