Patent Application
20230339757
The present invention relates to ion substituted calcium phosphate particles, to methods of producing such particles and uses thereof.
Dentin hypersensitivity is a clinical condition that can cause significant oral discomfort and pain, triggered by e.g., thermal, mechanical or evaporative stimuli. The underlying cause for the condition is that the dentin tubules have become exposed due to gingival recession or loss of enamel, in turn caused by e.g., demineralization, abrasion or erosion, excessive tooth brushing or flossing, pocket reduction surgery or as a secondary reaction to periodontal disease.
Demineralization of dentin and enamel may be countered by remineralization, in which calcium and phosphate ions present in saliva can deposit to form new mineral. This requires a local supersaturation of ions and preferably a pH above neutral to form hydroxyapatite. In order to regain mineral and tip the scale towards remineralization, additional calcium and phosphate ions are required.
WO 2021/086252 discloses spherical and hollow calcium magnesium phosphate particles and compositions comprising such particles.
U.S. Pat. No. 9,205,035 B2 discloses a method for formation of spherical particles of ion substituted calcium phosphate. The method is based on precipitation of particles from a buffered solution under static, stirring, or hydrothermal conditions.
There is still a need for improved calcium phosphate particles that could be used to prevent or treat the above-mentioned dental disorders.
The present invention aims to solve the problems of the prior art by providing hollow calcium phosphate particles.
A first aspect of the invention relates to hollow calcium phosphate particles having a mean diameter ranging from 400 nm to 1.5 μm. The hollow calcium phosphate particles comprise a respective shell comprising calcium, phosphate, water, magnesium, and strontium. The Ca:P atomic ratio is 0.9-1.3, the Sr2+ content is 1-6 wt %, the Mg2+ content is 5-10 wt % and the Ca2+ content is 15-25 wt %.
In one embodiment of the invention, the Ca:P atomic ratio in the respective shells is 0.9-1.3, the Sr2+ content in the respective shells is 1-6 wt %, the Mg2+ content in the respective shells is 5-10 wt %, and the Ca2+ content in the respective shells is 15-25 wt %.
In one embodiment of the invention, the particles are X-ray amorphous.
In one embodiment of the invention, the mean diameter of the particles ranges from 700 nm to 1.5 μm.
In one embodiment of the invention, the shell has a mean thickness ranging from 20 to 300 nm, preferably around 250 nm.
In one embodiment of the invention, the particles are substantially spherical.
In one embodiment of the invention, the water is bound water.
An aspect of the invention relates to a composition comprising a paste-forming compound and hollow calcium phosphate particles.
In one embodiment of the invention, the paste-forming compound is selected from the group consisting of glycerol, triglyceride, polyethylene glycol, propylene glycol, polypropylene glycol, polyvinyl alcohol, mineral oil, liquid paraffin, and any mixture thereof, preferably glycerol.
A third aspect of the invention relates to a method of manufacturing hollow calcium phosphate particles. The method comprises forming a first salt solution comprising 0.5-1.5 mM calcium, 0.2-1.0 mM strontium, and 0.2-1.0 mM magnesium, and a second salt solution comprising 5.0-15 mM phosphate. The method also comprises mixing the first and the second salt solutions to form a mixed solution at 15-25° C., and heating the mixed solution to equal to or above 70° C. at a rate of 1-20° C./min forming a heated solution. The method further comprising precipitating particles from the heated solution at 70-100° C. for a duration of 1 min to 24 h.
In one embodiment of the invention, the method additionally comprises retrieving and washing the particles.
In one embodiment of the invention, heating the mixed solution comprises heating the mixed solution to a temperature selected within a range of from 70 to 100° C.
In one embodiment of the invention, heating the mixed solution comprises heating the mixed solution to a temperature selected within a range of from 70 to 100° C. for a duration of 5 min to 3 h.
In one embodiment of the invention, the first salt solution comprises 0.9 mM calcium, 0.6 mM strontium and 0.5 mM magnesium.
In one embodiment of the invention, the second salt solution comprises 10 mM phosphate.
In one embodiment of the invention, the ratio between Ca:P in the mixed solution is around 0.09.
A fourth aspect of the invention relates to a method of treating or preventing caries in a subject. The method comprises administering hollow calcium phosphate particles or a composition to a surface of at least one tooth of the subject.
A fifth aspect of the invention relates to a method of treating or preventing dentin hypersensitivity in a subject. The method comprises administering hollow calcium phosphate particles or a composition to a surface of at least one tooth of the subject.
A sixth aspect of the invention relates to a method of treating or preventing enamel demineralization or decalcification defects, such as white spot lesions, in a subject. The method comprises administering hollow calcium phosphate particles or a composition to a surface of at least one tooth of the subject.
A seventh aspect of the invention relates to a dental product comprising hollow calcium phosphate particles or a composition. The dental product selected from the group consisting of a toothpaste, a dentifrice, a dental varnish, a desensitizing gel, a mineralizing gel, a tooth whitening gel, a tooth whitening strip, a dental prophy paste, a mouthwash, a tablet, a chewing gum, a dental sealant, a dental filling material, a dental cement, and a dental pulp capping material.
In the following, the invention will be described in more detail, by way of example only, with regard to non-limiting embodiments thereof, reference being made to the accompanying drawings.
‘Dentin hypersensitivity’—refers to dental pain arising from exposed dentin surfaces on teeth in response to a stimuli, e.g., thermal stimuli. Dentin hypersensitivity is also referred to as ‘sensitive teeth’;
‘bound water’—refers to water of hydration associated with the amorphous calcium phosphate particles according to the invention, based on the generalized chemical formula of amorphous calcium phosphate as CaxHy(PO4)z·nH2O, n=3-4.5;
‘X-ray amorphous’—refers to a material or particles that lacks or lack long range crystalline order. The crystallinity or X-ray or XRD amorphous state of the particles is determined by powder X-ray diffraction (XRD). A crystalline material reflects the X-rays according to the arrangement of its crystallographic planes and generates an identifiable pattern of sharp peaks, whereas an X-ray amorphous material only generates a single broad diffuse peak. Herein, the particles are, thus, classified as X-ray amorphous if the generated pattern lacks identifiable sharp peaks and is only characterized by a broad diffuse peak;
‘ACP’—is short for amorphous calcium phosphate;
‘wt %’—refers to weight percent of the ingredient in relation to the total weight of the particles or the composition; and
‘HA’—refers to hydroxyapatite, chemical formula Ca5(PO4)3(OH) but usually written as Ca10(PO4)6(OH)2.
Different calcium phosphate technologies and particles have been introduced in oral care products to promote remineralization of enamel as well as occlusion of dentin tubules for reduction of dentin hypersensitivity. One of the most effective approaches is to use amorphous calcium phosphate (ACP), which, with its high aqueous solubility, can supply a high concentration of calcium and phosphate ions, making it highly bioactive. The metastable nature of ACP also means that it is easily transformed into more stable calcium phosphate phases, such as HA. In fact, ACP is considered a precursor of natural apatite in teeth and therefore plays an important role in endogenous mineralization.
A challenge in utilizing ACP in biomedical applications, such as dentin and enamel mineralization and tubule occlusion, is to stabilize it in production and product formulations. In this regard, stability refers to stability of the amorphous state in order to prevent the ACP particles from crystallizing. Aqueous formulations with ACP and long-term storage of ACP formulations tend to crystallize the material, making it less bioactive. A way of stabilizing ACP is to substitute part of the calcium with magnesium, which, with its smaller ionic radius, will disrupt the active crystallite growth sites. This has been shown to reduce the crystallization rate of ACP. The formed particles may be referred to as ion substituted calcium phosphate particles.
Strontium is chemically closely related to both calcium and magnesium. The effect of strontium in bone formation and dental applications has been widely studied.
The present invention provides ion substituted calcium phosphate particles comprising magnesium and strontium, as well as a method for producing the particles. The particles are stable upon storage, in the sense that they at least partly maintain their morphology. The as-synthesized particles are stable during storage for at least a time period of a month. The particles may be applied in oral care products, such as toothpastes, dentifrices, fluoride varnish or desensitizing/mineralizing gels to prevent or treat caries, dentin hypersensitivity and/or white spot lesions.
A first aspect of the invention relates to hollow calcium phosphate particles having a mean diameter ranging from 400 nm to 1.5 μm. The particles comprise a respective shell comprising calcium, phosphate, water, magnesium, and strontium. The Ca2+, Mg2+ and Sr2+ concentrations in the shells are 15-25 wt %, 5-10 wt % and 1-6 wt %, respectively, and the Ca:P atomic ratio is 0.9-1.3
As mentioned above, the hollow calcium phosphate particles have a mean diameter ranging from 400 nm to 1.5 μm. This means that the mean or average diameter of individual hollow calcium phosphate particles is within the range of from 400 nm up to 1.5 μm. Accordingly, a collection of a plurality of such hollow calcium phosphate particles will have mean diameters ranging from 400 nm to 1.5 μm.
In one embodiment, the hollow calcium phosphate particles have a mean diameter ranging from 700 nm to 1.5 μm.
In one embodiment, the particles are substantially spherical. It is an advantage with the invention that the particles are well suited in size and shape to penetrate exposed dentin tubules.
In one embodiment, the particles are X-ray amorphous. Without being bound by theory, the amorphous character of the particles is believed to be caused by ion substitution and the conditions during particle precipitation. Ion substitution refers to the incorporation of Mg2+ and Sr2+ in the structure where Ca2+ otherwise would have been arranged. The ion substitution is believed to stabilize the amorphous phase of the calcium phosphate particles and thereby prolong the lifetime of the particles.
Without being bound by any theory it is possible that the ionic radii of the substituting ion influence the stability of the calcium phosphate particles. It might therefore not be possible to use any type of ion as a substitute in order to stabilize calcium phosphate particles.
In one embodiment, the water in the composition is bound water, i.e., water that is part of the chemical composition. Bound water can also be referred to as water that is incorporated within the structure of calcium phosphate particles.
A second aspect of the invention relates a composition comprising a paste-forming compound and hollow calcium phosphate particles according to the invention.
To obtain a stable composition, particles according to the invention are mixed with a paste-forming compound, for instance glycerol, then dried to remove any excess water. In one embodiment, the paste-forming compound is essentially water-free, such as <10 wt % water, preferably <5 wt % water and more preferably <1 wt % water.
It is an advantage with a composition according to the invention that the particles can be homogenously dispersed in the composition. It is further advantageous that the composition has good flowability and/or viscosity and can easily be mixed with other ingredients to prepare, for example, a toothpaste. Furthermore, by introducing the particles into a composition without a preceding drying step a narrow size distribution can be maintained by not letting the particles agglomerate into larger clusters.
In one embodiment, the paste-forming compound is selected from the group consisting of glycerol, triglyceride, polyethylene glycol, propylene glycol, polypropylene glycol, polyvinyl alcohol, mineral oil, liquid paraffin, and any mixture thereof.
Particles according to the invention can also be delivered as a slurry, i.e., in the form of a mixture of a solvent, or mixture of solvents, and particles. The solvent can, for example, be ethyl alcohol, isopropyl alcohol, or a mixture of those.
A third aspect of the invention relates to a method of manufacturing calcium phosphate particles that are ion substituted with Sr2+ and Mg2+. In short, two salt solutions, one containing the constituent cations (Ca2+, Mg2+, and Sr2+) and one containing the anions (H2PO4−/HPO42−) are mixed forming a mixed solution at or slightly less than room temperature (20-25° C.). The mixed solution is heated to above 70° C., after which precipitates start to form in the heated solution. The precipitates are collected after 1 min or up to 24 hours by for example vacuum filtration, and thereafter optionally washed with e.g., deionized water and/or ethanol.
In other words, a method for manufacturing particles 100 according to the invention comprises, see
In one embodiment, the method 100 also comprises retrieving and washing, at step 105, the particles.
In one embodiment, the mixed solution 102a is clear, hence no or only a minor amount of precipitates are formed in the solution until it is heated to 70° C.
In one embodiment, the temperature in step 103 during heating of the mixed solution is 70-100° C.
In one embodiment, the mixed solution 102a is heated in step 103 to 70-100° C. for a duration of 5 mM to 3 h.
In one embodiment, the first salt solution 101a comprises 0.9 mM calcium, 0.6 mM strontium and 0.5 mM magnesium. In one embodiment the second salt solution 101b comprises 10 mM phosphate.
In one embodiment, the ratio between Ca:P in the mixed solution 102a is around 0.09.
Without being bound by any theory, the formation of core-shell calcium phosphate particles comprising Sr and Mg can be the result of a simultaneous formation of primary NPs of calcium phosphate particles and gas bubbles of O2, and/or N2, and/or CO2. The gas bubbles may be ultrafine. It can be explained by the instability of gas bubbles driving adsorption of the NPs onto the bubble surface and the gas bubbles may therefore function as soft templates in the synthesis.
Furthermore, without being bound by any theory, the solubility of the gas bubbles discussed above are temperature dependent. It is therefore possible that at too low synthesis temperatures there is a decreased or no formation of core-shell shaped calcium phosphate particles.
In one embodiment, magnesium and strontium substituted calcium phosphate particles are formed at a temperature above 70° C.
A further aspect of the invention relates to the particles or a composition according to the present invention for use as a medicament. The present invention also relates to the particles or a composition according to the invention for use in prevention or treatment of caries, dentin hypersensitivity or enamel demineralization/decalcification defects, such as white spot lesions.
‘Treatment’ of caries, dentin hypersensitivity or white spot lesions as used herein does not necessarily mean curative treatment of caries, dentin hypersensitivity or white spot lesions but also encompass inhibition or reduction of the short- and long-term symptoms of the caries, dentin hypersensitivity or white spot lesions. Hence, ‘treatment’ also encompasses delaying onset of the caries, dentin hypersensitivity or white spot lesions, including delaying, preventing onset of symptoms or resolving established pathologies associated with caries, dentin hypersensitivity or white spot lesions, or any other demineralization or decalcification defect.
The present invention also relates to a method of prevention or treatment of caries, dentin hypersensitivity and/or white spot lesions. The method comprises administering hollow calcium phosphate particles or a composition of the invention to a subject to a surface of at least one tooth of the subject.
The subject is a mammalian subject, and preferably a human subject. The particles or composition is administered locally in the oral cavity of to the subject, and in particular to the teeth of the subject, in the form of a toothpaste, dentifrice, whitening gel, dental varnish or similar product. Particles according to the present invention can be added as an ingredient in a toothpaste, a dentifrice, a dental varnish, a desensitizing gel, a mineralizing gel, a tooth whitening gel or strip, a dental prophy paste, a mouthwash, a tablet, a chewing gum, a dental sealant, a dental filling material, a dental cement, a dental pulp capping material.
An aspect of the invention relates to a dental product selected from the group consisting of a toothpaste, a dentifrice, a dental varnish, a desensitizing gel, a mineralizing gel, a tooth whitening gel or strip, a dental prophy paste, a mouthwash, a tablet, a chewing gum, a dental sealant, a dental filling material, a dental cement, a dental pulp capping material. The dental product comprises hollow calcium phosphate particles or a composition of the invention.
All embodiments disclosed herein relate to all aspects of the present invention and all embodiments may be combined unless stated otherwise.
CaCl2·2H2O, MgCl2·6H2O, Sr(NO3)2, Na2HPO4, and KH2PO4 were used as received from the manufacturer without any purification.
Synthesis of the core-shell particles of calcium phosphate was performed based on previously published methods for synthesis using precipitation reactions in aqueous solutions (Berg et al. ‘Ion substitution induced formation of spherical ceramic particles’ Ceramics International (2019) 45: 10385-10393, and Xia et al. ‘Synthesis and release of trace elements from hollow and porous hydroxyapatite spheres’ Nanotechnology (2011) 22: 1-10). In short, two salt solutions, one containing the constituent cations (Ca2+, Mg2+ and Sr2+) were mixed with a solution containing the anions (H2PO4−/HPO42−), forming a clear solution. The reaction solutions were heated to 60-100° C. Precipitates were collected by vacuum filtration after 5 min-24 hours of heating, followed by washing once with deionized (DI) water and three times with ethanol to remove any salt residues. The reaction conditions are listed in Table 1.
Scanning electron microscopy (SEM; Zeiss Leo 1550/1530) was used for evaluation of the morphology of the primary NPs and core-shell particles. An acceleration voltage of 5 kV and secondary electrons was used for imaging. Samples were sputtered with a conductive Au/Pt layer to allow for imaging and to avoid charging the samples.
The crystal structure and the phase evolution of the samples were analyzed with X-ray Diffraction (XRD; Bruker, D8 Advanced) using Cu Kα-radiation (X=1.5418 Å). Samples were prepared by dispersing dried precipitates in ethanol, dropping the dispersion onto zero-background silicon sample holders where it was left to dry prior to analysis.
Different reaction temperatures between 60-100° C. were evaluated to investigate how it influenced the precipitation of calcium phosphate and the formation of core-shell particles. As shown in
The formation and morphological evolution of core-shell particles synthesized at 100° C., using both Sr2+ and Mg2+, was followed by collecting precipitates at different time points during the reaction as indicated in Table 1. In the early stages of the reaction, after 5 min, it was clear that the core-shell particles, having diameters of 700 nm-1.5 μm, were constructed of primary NPs with diameters of ˜20-40 nm, see
Cross-sections of particles collected after 24 hours were prepared by embedding the particles in resin followed by polishing. As can be seen in
To determine the specific role of the ions, the effect of Sr2+ and Mg2+ was compared separately to investigate how the ionic radius and the concentration were reflected in the characteristics of the primary NPs and the resulting properties of the core-shell particles synthesized at 100° C.
Without substituting ions flake-like structures formed with a morphology resembling that of HA (Ca10(PO4)6(OH)2) (
The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations, and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. The scope of the present invention is, however, defined by the appended claims.
| Number | Date | Country | |
|---|---|---|---|
| 63329981 | Apr 2022 | US |
