WO1997023241A1

WO1997023241A1 - Particle compositions

Info

Publication number: WO1997023241A1
Application number: PCT/EP1996/005601
Authority: WO
Inventors: Mark John Berry; Paul James Davis; Philip Porter
Original assignee: Quest International B.V.
Priority date: 1995-12-21
Filing date: 1996-12-05
Publication date: 1997-07-03
Also published as: JP2000503000A; EP0868199A1; AU1194697A

Abstract

A composition for delivering an antimicrobially active protein, such as an enzyme, to a microbial cell target comprising, optionally in a vehicle, positively charged porous inorganic carrier particles having one or more antimicrobially active proteins adsorbed thereon.

Description

PARTICLE COMPOSITIONS

The present invention relates to compositions for the delivery of antimicrobially active proteins to a microbial cell target site. In particular, it relates to compositions in which the proteins are adsorbed on to an inorganic porous carrier particle by means of which they are delivered to the target site.

It is known to deliver a combination of enzymes, one of which produces a substrate for the other, conjugated through covalent bonds to a synthetic polymer such as polyethylene i ine, to a target site by means of an antibody or antibody fragment able to bind to the target site. Such an approach is described, for example, in EP-A-0450800 and EP-A-0451972 (Unilever) where preparations of particular use for attacking oral microflora are disclosed. Chemical coupling of the enzymes involves the use of reagents which may be toxic and^' it is necessary to take steps to ensure their removal in products intended for oral use.

EP-A-0566368 (Unilever) describes cosmetic compositions for the delivery of a cosmetically effective benefit agent to a target site on the skin and/or hair comprising particles including the benefit agent and having means to bind to an organic surface at the target location. Suitable particles disclosed are made of synthetic polymeric substances such as polycyanoacrylate or proteins such as albumin or gelatin. Preferred particles which are disclosed and exemplified are liposomes in which benefit agent is encapsulated.

It is known from O-A-93/05815 to target nucleic acids to cells by providing inorganic particles (particularly biodegradable metal oxides or salts) with a cell binding component, i.e. a targetting molecule for a particular cell type, and the nucleic acid. BE-A-844657 discloses porous inorganic particles, such as silica, coated with an aminated polysaccharide which reversibly bind various biologically active molecules. The particles can be used in chromatografic processes to purify such molecules.

A similar material is disclosed in JP-A-63031538 in which a cationic polymer such as polyethyleneimine is used as the coating. In this case the binding is said to be irreversible.

It has now been found that antimicrobially active proteins, adsorbed onto a positively charged inorganic porous carrier particle which is able to bind to a microbial cell target site, can efficiently be delivered to the target site with high affinity and specificity. Retention of the antimicrobially active proteins at the target site and prolonged action may thereby be achieved. Furthermore, by adsorbing the proteins onto the particle carrier, the need for chemical coupling reactions can be avoided, leading to a simple process of manufacture which can readily be scaled-up.

According to a first aspect, the present invention provides a composition comprising positively charged porous inorganic carrier particles having one or more antimicrobially active proteins adsorbed thereon.

In a second aspect, the invention provides a method for delivering antimicrobially active proteins to microbial cell targets comprising applying to the targets a composition according to the first aspect, and thereby inhibit or kill the microbial cell.

In a further aspect, the invention provides a method for the production of a composition according to the first aspect comprising providing the porous inorganic carrier particles with a positive electric charge and adsorbing one or more antimicrobially active proteins on the porous inorganic carrier particle.

Suitable porous inorganic particles for use according to the invention include silicas such as available from Crosfield, W-R Grace or Rhone Poulenc and hydrotalcites such as available commercially from Crosfield. The porous particles will conveniently have a pore size to suit the molecular size of the proteins. For example, if the protein is a large enzyme, the mean pore size should preferably be such that sufficient pores greater that 20nm, more preferably greater than 50nm are present. Pore sizes are preferably less than 200nm, but it is technically possible to use larger pores. It will be appreciated that other porous inorganic materials having properties analogous to those of silica may suitably be employed. An advantage of using carrier materials having a porous structure is that they have a large internal surface area able to accommodate a large payload of benefit agent .

Positively charged inorganic (e.g. silica) particles may be produced by physical adsorption of a positively charged ligand onto the surface of the negatively charged silica particle. Suitable positively charged ligands which may be employed include organic polymers such as polyethylene imine or polylysine and metal ions such as Al^3* and Mg²⁺. Porous hydrotalcites, such as those commercially available from

Crosfield under the trade name Macrosorb, naturally contain aluminium and magnesium ions and therefore carry an innate positive charge. Such carrier particles are therefore particularly useful in the present invention as they do not need to be further derivatised with a particularly charged ligand prior to use.

For the purposes of this invention the phrase "antimicrobially active proteins" refers to proteins which are either antimicrobially active themselves such that they directly act on the microorganism in such a way as to kill it or hinder its growth or multiplication, or to proteins which react with other compounds present in the microorganism or its environment thereby directly or indirectly producing compounds which have any of the above mentioned activities.

Thus, the antimicrobially active proteins to be adsorbed onto the carrier particles preferably include one or more enzymes which are antimicrobially active themselves or in combination with a suitable substrate produce antimicrobially active molecules. Examples are various oxidases and peroxidases, proteases, glycosidases, lipases etc. In particular, oxidases can function as cytotoxic agents. Oxidases such as glucose oxidase and galactose oxidase generate hydrogen peroxidase which is cytotoxic. Peroxidases can use the hydrogen peroxide as a substrate to form hypohalite which is even more cytotoxic. Commercially available peroxidases which may suitably be used in conjunction with these oxidases include horseradish peroxidase and lactoperoxidase. Other suitable peroxidases are chloroperoxidases . Both hydrogen peroxide and hypohalite are rapidly decomposed in vivo, but nevertheless active because they can effectively be delivered to the intended site of action by the present invention.

The compositions according to the invention may also include other enzymes which aid the antimicrobially active enzymes in their action e.g. by converting a substrate present near the microbial cell into a substrate which in turn can be used by the antimicrobially active enzymes, such other enzymes are preferably also absorbed on the carrier particles. An example of such other enzyme is invertase, as outlined below.

Compositions according to the invention may conveniently be used for the delivery of antimicrobial agents as oral care active agents. In particular, one application of the invention lies in the delivery of oral care active agents, particularly oxidative enzymes as discussed above against the microbial species in dental plaque. Suitable target microorganisms include Streptococcus mutans and S sanguis. The particles may carry some or all of the substrates for the enzymes or some or all of the enzyme substrates may be present at the target site. For example, where glucose oxidase is used and the intended target microorganism is in the mouth, dietary glucose may be relied upon as the enzyme substrate or the particles may incorporate glucose. Alternatively, the particles may incorporate invertase which converts sucrose to glucose.

Other enzymes may alter the pH of the environment, e.g. the dental plague, to a level which is not or less suitable for the microorganism to grow. Thus, certain enzymes are capable of raising the pH to a level unsuitable for growth of microorganisms in the dental plaque; an example is urease which converts ureum to ammonia and carbon dioxide.

In a further preferred embodiment of the invention, the target is microorganisms present on general household surfaces. Examples of such microorganisms include S . aureus, P . aeruginosa and E. coli . Suitable benefit agents for use in this application include enzymes such as horseradish peroxidase, lactoperoxidase or vanadium chloroperoxidase.

It will be appreciated that the invention is not limited to the various applications described above.

The compositions of the invention preferably include a vehicle to act as a diluant, dispersant or carrier for the particles so as to facilitate their application to and distribution at the site of application. It will be appreciated that the choice of vehicle will depend on the method and site of administration of the composition. For any oral application, for example, the vehicle must be acceptable for topical application in the mouth. Conventional cosmetically acceptable vehicles are well known in the art and can include water or substances such as liquid or solid emollients, solvents, humectants, thickeners and powders .

Where the composition is intended for oral (dental) application, the cosmetically acceptable vehicle will generally form from 10 to 99.9%, preferably form 50 to 99.9% by weight of the composition. Compositions according to the invention intended for oral use may conveniently be formulated as a mouthwash, toothpaste or lozenge. Alternatively, the compositions of the invention may be solid or semi-solid, for example sticks, creams or gels for use in conjunction with a suitable applicator.

The proportion of antimicrobially active protein in the compositions according to the invention varies depending upon the intended application. Typically the benefit agent provides from 0.01% to 10% by weight of the particles. Generally the benefit agent provides from 0.005% to 1.5% by weight of the composition.

The compositions according to the invention will generally contain various other components known in the art, depending on the intended application. In the case of tooth pastes such components will include the usual polishing agents. Many compositions will also include some sort of surface active or detergent active component. Tooth pastes and other compositions for oral care will also generally contain a suitable flavour, particularly a mint or menthol-like flavour.

EXAMPLES

The following examples are given by way of illustration.

I) r-erivatisation of silica with polyethyleneimine (PET)

Two samples of porous silica particles were obtained from Crosfield: SD1497 (= Gasil 23DP, particle size 5μm, mean pore diameter ca 22nm) and SD1498 (particle size 4μm, mean pore diameter ca 80nm, a wide pore silica prepared according to WO 94/11302) . Each sample was derivatised by rotating overnight in an 0.2% solution of polyethylene imine (PEI) (Sigma) at room temperature. Derivatised silica was separated by centrifugation and then washed three times in lOmM tris, pH8.

II) Adsorption of glucose oxidase enzyme onto particles

Six different particles were investigated: SD1497 and SD1498 derivatised with PEI, underivatised SD1497 and SD1498, Macrosorb CT100 (particle size 3.7μm, mean pore diameter 30nm) and Macrosorb CT2000M (particle size 4μm, mean pore diameter lOnm) . An 0.5 mis aliquot of a 12.5% slurry (total mass of particles = 62.5 mgs) of each of these particles was mixed with 4mgs of glucose oxidase enzyme made up in lOmM tris pH8. The total volume of the mixture was 2 mis. The mixture was gently rotated overnight at room temperature. Then the particles were separated by centrifugation. The amount of enzyme remaining in the supernatant was determined spectro-photometrically after filtering through an 0.2μm filter. This value was subtracted from the amount of enzyme that had been added to deduce the amount of enzyme that had been adsorbed onto the particles.

All the particles with positively charged surfaces bound glucose oxidase. SD1498 derivatised with PEI had the highest capacity (88mg of enzyme per gram of solid) . Neither of the underivatised silicas bound glucose oxidase (see Figure 1) . III) Adsorption of invertase and glucose oxidase onto

Macrosorb particles

A 12.5% slurry of Macrosorb (CT 100) was made up in lOmM tris pH8. 0.5 mis aliquots of this slurry (total mass of particles = 62.5 mgs) were added to glass vials. Then invertase (Sigma) was added from a stock solution (5mg/ml in lOmM tris, pH8) . Different amounts of invertase were added to each vial: 0, O.lmg, 0.2mg, 0.5mg, 1.0 mg and 5.0 mg; equivalent to 0, 1.6mg, 3.2mg, 8mg, 16mgs and 80mgs per gram of particles. Each vial was made up to 1.5mls with lOmM tris pH8. The vials were rotated slowly for 4 hours at room temperature. Then the particles were separated by centrifugation (2,000 r.p.m for 5 minutes) . The amount of invertase remaining in the supernatant was determined spectrophotometrically after filtering through an 0.2μm filter. This value was subtracted from the amount of enzyme that had been added to deduce the amount of enzyme that had been adsorbed onto the particles.

The invertase sensitised particles were resuspended in a 1.5mls volume of lOmM tris containing 2mgs of glucose oxidase (equivalent to 32mgs per gram of particles) . The tubes were rotated overnight at room temperature. The double-enzyme sensitised particles were separated by centrifugation and the amount of glucose oxidase that had been adsorbed was determined as previously. Particles were washed with and then stored in lOmM tris, pH8.

Macrosorb particles (CT 100) were successfully derivatised with both invertase and glucose oxidase. The ratio of invertase to glucose had been efficiently controlled by sensitising with different amounts of invertase and a fixed amount of glucose oxidase in a two-step reaction (see Figure 2) .

IV) Kill of Streptococci sά-tll Macrosorb particles derivatised with invertase and σlucose oxidase

An overnight broth culture of S. sanguis cells was washed in phosphate-buffered saline (PBS) pH7 by repeated centrifugation and finally resuspended in PBS pH6.5 such that the final reaction mixture (cells + particles + substrate) contained the original culture cell density. Enzyme-loaded Macrosorb samples obtained according to Example III were added, with thorough mixing, to the S . sanguis suspension, to a final concentrations of 0.5% solids and- finally enzyme substrate was added (9.6% sucrose, lOmM KI, lOug/ml lactoperoxidase) . Immediately following addition of substrate, a time zero sample was removed from the reaction mixture for viability measurement. Further samples were removed following 1, 5, 10, 20 and 30 minutes of incubation at 37°C. During incubation, settling of Macrosorb particles was prevented by periodic agitation. Samples were removed into quenching solution comprising 12 mg/ml cystein hydrochloride in PBS pH6.5 followed by enumeration of viable cells by the Miles, Misra and Irwin method (A A Miles, S S Misra and J 0 Irwin, Journal of Hygiene 1938 3_&, 737-48) .

In the presence of Macrosorb particles labelled with both invertase and glucose oxidase, together with the substrate system defined, loss of viability of suspended S . sanguis cells occurred rapidly: an 8log reduction in cell viability was observed in 5 to 10 minutes of substrate incubation (see Figure 3) . The particles given greatest invertase loading did not provide the most potent kill, indicating the desirability to balance the GOx: invertase loading ratio to achieve optimal potency.

Claims

1. A composition comprising positively charged porous inorganic carrier particles having one or more antimicrobially active proteins adsorbed thereon.

2. A composition according to claim 1 wherein the particles comprise silica or hydrotalcite.

3. A composition according to any one of claims 1 and 2 wherein the antimicrobially active proteins comprise one or more enzymes.

4. A composition according to claim 3 wherein the enzymes comprise an oxidase.

5. A composition according to claim 3 or 4 wherein the enzymes comprise a peroxidase.

6. A composition according to claims 3-5 wherein the enzymes also comprise an invertase.

7. A composition according to any one of claims 1-6 wherein the silica particles are treated with polyethylene imine.

8. A composition according to any one of claims 1 to 7 further comprising a vehicle.

9. A composition according to any one of claims 1 to 8 which is a composition for oral care.

10. A composition according to any one of claims 1 to 8 which is a composition for cleaning household surfaces.

11. A method for delivering an antimicrobially active protein to a microbial cell target comprising applying a composition according to any one of claims 1 to 8.

12. A method according to claim 11 wherein the microbial cell target is present in dental plaque.

13. A method according to claim 12 wherein the microbial cells are those of S. mutans or S. sanguis .

14. A method according to claim 11 wherein the microbial cell target is present on household surfaces.