HIGHLY STRUCTURED POROUS SILICA MATERIALS AND BIOLOGICAL USES THEREOF

Abstract

There is provided herein a porous silica particle characterized by having a hierarchical pore structure wherein at least about 20% of the pores of the silica particle are of a size in the range of from about 7.0 to about 13.0 nm, and at least about 10% of the pores of the silica particle are of a size in the range of from about 0.5 to about 5.0 nm, and by having a particle size of from about 0.1 μm to about 10.0 μm. There is also provided compositions comprising such silica particles, process for their preparation, and medical and non-medical uses thereof.

Claims

1. A porous silica particle characterized by having a hierarchical pore structure wherein: (a) at least about 20% of the pores of the silica particle are mesopores of a size in the range of from about 7.0 to about 13.0 nm; and (b) at least about 10% of the pores of the silica particle are lateral pores of a size in the range of from about 0.5 to about 5.0 nm, and by having a particle size of from about 0.1 μm to about 10.0 μm.

2. The porous silica particle of claim 1, wherein: (a) at least about 25% of the pores of the silica particle are mesopores of a size in the range of from about 9.0 to about 12.0 nm; and (b) at least about 10% of the pores of the silica particle are lateral pores of a size in the range of from about 0.5 to about 5.0 nm.

3. The porous silica particle of claim 1, wherein: (a) at least about 25% of the pores of the silica particle are mesopores of a size in the range of from about 9.0 to about 11.0 nm; and (b) at least about 15% of the pores of the silica particle are lateral pores of a size in the range of from about 0.5 to about 5.0 nm.

4. The porous silica particle of any one of claims 1 to 3, wherein the particle has a BET surface area of at least about 500 m.sup.2/g.

5. The porous silica particle of any one of claims 1 to 4, wherein the particle has a BET surface area of from about 500 to about 1500 m.sup.2/g.

6. The porous silica particle of any one of claims 1 to 5, wherein the particle has a BET surface area of from about 600 to about 1200 m.sup.2/g.

7. The porous silica particle of any one of claims 1 to 6, wherein the particle has a substantially non-spherical morphology (i.e. an aspect ratio of greater than 1:1, such as greater than 2:1).

8. The porous silica particle of any one of claims 1 to 7, wherein the particle has an essentially rod-shaped morphology.

9. The porous silica particle of any one of claims 1 to 8, wherein the particle has a particle size of from about 0.5 to about 5.0 μm.

10. The porous silica particle of any one of claims 1 to 9, wherein the average pore size of the pores in the range 5.0 to 50.0 nm is from about 7.0 to about 13.0 nm.

11. The porous silica particle of any one of claims 1 to 10, wherein the average pore size of the pores in range 5.0 to 50.0 nm is from about 9.0 to about 11.0 nm.

12. The porous silica particle of any one of claims 1 to 11, wherein the surface hydroxyl groups of the silica particle are unmodified (i.e. the silica is pristine silica).

13. The porous silica particle of any one of claims 1 to 12, wherein: (i) the surface hydroxyl (OH) groups are in an amount of from about 0.5 to about 7.0 per nm.sup.2; and (ii) the surface hydroxyl (OH) groups have two distinct pK.sub.a values being at a first pK.sub.a of from 3.5 to 4.2 and at a second pK.sub.a of from 8.0 to 8.7.

14. The porous silica particle of any one of claims 1 to 13, wherein the pores in the range 5.0 to 50.0 nm are structurally ordered.

15. A composition comprising a plurality of silica particles wherein at least 50% of the silica particles are as claimed in any one of claims 1 to 14.

16. A pharmaceutical composition comprising a plurality of silica particles wherein at least 50% of the silica particles are as claimed in any one of claims 1 to 14, and optionally one or more pharmaceutically acceptable excipient, colorant and/or flavouring.

17. A porous silica particle as claimed in any one of claims 1 to 14, or a composition as claimed in claim 15, for use in medicine.

18. The use of a porous silica particle as claimed in any one of claims 1 to 14, or a composition as claimed in claim 15, as a medical device.

19. A method for the treatment or prophylaxis of a metabolic disease or disorder, comprising administering to a patient in need thereof a therapeutically effective amount of a silica particle as claimed in any one of claims 1 to 14, a composition as claimed in claim 15, or a pharmaceutical composition as claimed in claim 16.

20. A silica particle as claimed in any one of claims 1 to 14, a composition as claimed in claim 15 or a pharmaceutical composition as claimed in claim 16 for use in the treatment or prophylaxis of a metabolic disease or disorder.

21. A method as claimed in claim 19, or a particle or composition for use as claimed in claim 20, wherein the treatment or prophylaxis of a metabolic disease or disorder is: (a) the reduction of metabolic and/or cardiovascular risk-factors of type 2 diabetes; (b) the treatment or prophylaxis of type 2 diabetes; (c) the treatment or prophylaxis of prediabetes; (d) the treatment or prophylaxis of metabolic syndrome; (e) the treatment or prophylaxis of obesity; (f) the lowering of, or prevention of increase in, body fat levels in the form of adipose tissue; (g) the lowering of, or prevention of increase in, triglyceride and/or cholesterol levels; and (h) the treatment or prophylaxis of dyslipidaemia.

22. A method or a composition for use as claimed in claim 21, wherein the metabolic risk-factors of type 2 diabetes are based on analysis of levels of one or more biomarkers selected from the group consisting of LDL, HDL, triglycerides, cholesterol, Apo A1, Apo B, the ratio between Apo A1 and Apo B and between LDL and HDL cholesterol, blood pressure, insulin resistance and glucose levels, and levels of HbA1c.

23. A method of lowering the efficiency of a food or drink item, comprising administering with said food or drink a silica particle as claimed in any one of claims 1 to 14, a composition as claimed in claim 15, or a pharmaceutical composition as claimed in claim 16.

24. The method of claim 23, wherein the lowering of the efficiency of a food or drink item comprises the lowering of the glycaemic response resulting from consumption of the food or drink item.

25. A method for separation of a fraction of (typically small) biomolecules, such as enzymes and metabolic products, including lipid complexes, carbohydrate and proteins, from undigested food in the digestive system, comprising administering a silica particle as claimed in any one of claims 1 to 14, a composition as claimed in claim 15, or a pharmaceutical composition as claimed in claim 16.

26. The use of a silica particle as claimed in any one of claims 1 to 14 or a composition as claimed in claim 15 as a dietary active ingredient.

27. A process for the preparation of porous silica particles as claimed in any one of claims 1 to 14 comprising the steps of: (i) forming a homogenous mixture of (a) an organic pore-forming material, and (b) a source of silica, in an aqueous solution at non-neutral pH, optionally at elevated temperature, such as at a temperature of from about 20° C. to about 60° C.; then (ii) curing the mixture formed in step (i) by maintaining at increased temperature, such as a temperature of from about 70° C. to about 150° C. for at least 3 hours; then (iii) removing the solid material from the cured mixture formed in step (ii) and washing the solid material to neutral pH; then optionally (iv) removing organic material.

28. The process as claimed in claim 27, wherein the organic pore-forming material is a diblock or triblock copolymer or a Pluronic.

29. The process as claimed in claim 27 or claim 28, wherein the silica source is selected from the group consisting of tetraethyl orthosilica (TEOS), tetramethyl orthosilicate (TMOS), tetrapropyl orthosilicate (TPOS) and sodium silicate or a combination of two or more thereof.

30. The process as claimed in any one of claims 27 to 29, wherein step (i) and/or step (ii) comprise stirring the mixture.

31. The process as claimed in any one of claims 27 to 30, wherein the aqueous solution at non-neutral pH comprises an acid, a base or a suitable buffer.

32. The process as claimed in any one of claims 27 to 31, wherein step (i) is performed at a temperature of about 40° C.

33. The process as claimed in any one of claims 27 to 32, wherein step (ii) is performed at a temperature of about 98° C.

34. Porous silica particles obtained or obtainable by the process as claimed in any one of claims 27 to 33.

Description

SUMMARY OF THE FIGURES

[0308] FIGS. 1A to 1F illustrate examples of the morphology and the physiochemical properties of the material prepared in Example 1, wherein:

[0309] FIG. 1A shows the N.sub.2 sorption isotherm of the calcined Silica material;

[0310] FIG. 1B shows pore size distribution measured via density functional theory (DFT);

[0311] FIG. 1C shows low-angle X-Ray diffraction (XRD) pattern showing fingerprints typically associated with 2-dimensional hexagonal pores structure. Peaks 110 and 200 are raised by a factor of 7 for clarity purposes;

[0312] FIG. 1D shows images of aggregates of rod-shaped silica material taken with scanning electron microscope (SEM);

[0313] FIG. 1E illustrates a model particle as described herein;

[0314] FIG. 1F shows a Bright Field image taken with transmission electron microscope (TEM), showing pores skeleton of silica particles in different orientations.

[0315] FIG. 2 is an illustration of the proposed mode of action of the invention. Upon ingestion, the porous silica particles mix with the food and the intestinal juices. In the digestive system, larger food molecules are broken down by enzymes into biomolecules small enough for the body to absorb. The intestinal content therefore contains both large and small molecules. The porous silica particle works as a molecular sieve by physically separating smaller molecules from larger ones through its tailored porosity. Only small molecules will diffuse into the mesopores as labelled. This physically separates a fraction of smaller biomolecules such as enzymes and metabolic products from larger undigested biomolecules. This separation is dependent on pore size and architecture (spatially ordered, cylindrical mesopores interconnected via narrowly elongated lateral pores) as well as the physiochemical properties of the material. Labels are as follows: A. Molecular Sieve, B. Physiochemical properties of material within particle, C. Narrowly elongated lateral pores, D. Spatially ordered, cylindrical mesopores, E. Silica particle, F. Small biomolecule (e.g. enzyme), G. Large molecule (e.g. undigested food particle), H. Water molecule.

[0316] FIG. 3A. Two different types of silica are compared in order to exemplify that a combination of correct pore size and physiochemical properties is needed for efficient molecular sieve entrapment. In this small panel, when comparing to reference silica material SM0002, only silica from example 1, labelled as SM0023 has the right properties (as described herein) to allow for efficient entrapment of biomolecules (here enzymes).

[0317] FIG. 3B illustrates the principle of how silica pore size affects the entrapment of biomolecules. i) Free biomolecules, here exemplified by enzymes (.square-solid.), can act unrestrained in the below enzyme in vitro assays. ii) When silica batch SM0002 is added to the reaction, the enzyme cannot enter due to pore size restriction. iii) Silica from example 1, herein also known as SM0023, has the right physiochemical properties for the enzymes to enter into the structure and stay in the structure. *see FIG. 3A and Table 1.

[0318] FIG. 3C. The Lipase assay illustrates how SM0023 with a pore size of 9.6 nm efficiently inhibits the Lipase enzyme (approx. dimensions 7×8×25 nm). In brief, the pH stat method was used by mixing freshly prepared Lipase extract (400 mg) in 1 ml tris-maleate buffer at pH 6.5 together with 8.3 mg of Silica batches SM0002 and SM0023. The samples were agitated and incubated at room temperature for 10 minutes before being centrifuged for 6 minutes at 1600 G. The supernatant was added to a digestion vessel containing the substrate tributyrin (6 g) dispersed in digestion buffer (9 mL). Throughout the digestion, NaOH (0.6 M) was titrated into the digestion vessel to maintain the pH at 6.5. The digestion was run for up to 10 mins and the volume of NaOH needed to keep the pH at 6.5 was measured (as the free fatty acids were released). The initial slope of NaOH (μmol) vs time (min) was normalized to the enzyme alone (white bar) tubes and expressed as percentage. Data is shown as means±standard deviation of 3 experiments. The method is also described in Phan, S. et al., Journal of Pharmaceutical Sciences, 104, 1311-1318 (2015).

[0319] FIG. 3D. The Amylase Absorption Assay exemplifies how SM0023 with a pore size of 9.6 nm efficiently entraps the α-Amylase enzyme (approx. dimensions 5×8×13 nm). In brief, a solution of silica (5 mg/mL) was mixed with an equal volume of porcine α-Amylase (0.1 mg/mL, PBS pH 5.4) and the mixture was incubated for 37° C. for 30 min with agitation. The samples were spun for 3 min and the supernatant was incubated for another 30 min together with soluble starch (2 mg/mL), following addition of 3,5-Dinitrosalicylic acid (DNS) and heating at 95 degrees for 15 min. DNS changes colour in the presence of reducing sugars (starch to maltose here) and is detected at 540 nm. The data is expressed as percentage, normalized to the enzyme alone (white bar) tubes. SM0002 and SM0023 are tested here in duplicate tubes, and data is shown as mean±standard error of the mean. The experiment has been repeated 3 times with identical results.

[0320] FIG. 3E. SM0023 depletes the biomolecules (enzyme in this context) from solution. Western blot staining with an anti-α-Amylase antibody shows that the supernatant in the above experiment (FIG. 3D) is depleted of α-Amylase (54 kDa) after treatment with SM0023. The silica with smaller (SM0002, 3.3 nm) pore sizes does not deplete the solution of the enzyme, since it does not have efficient physiochemical properties and cannot efficiently entrap the biomolecule.

[0321] FIG. 3F. SM0023 depletes the biomolecules (enzyme in this context) from solution. The QuantiPro BCA assay Kit (Sigma cat no. QPBCA) was utilized to measure the protein concentration of the supernatant in the above experiment (FIG. 3D) after treatment with SM0023. This data confirms the data in FIG. 3E that the amount of enzyme is substantially decreased. The silica with smaller (SM0002, 3.3 nm) pore sizes does not deplete the solution of the enzyme, since it does not have efficient physiochemical properties and cannot efficiently entrap the biomolecule. The data is expressed as protein concentration (μg/ml) after exposure to 1.25 mg/ml silica. SM0002 and SM0023 are tested here in duplicate tubes, and data is shown as mean±standard error of the mean.

[0322] FIG. 4. SM0023 delays the absorption and exposure of 14C triolein in rats. (4A) Kinetic profiles of postprandial uptake of 14C oleic acid measure in rat plasma after 14C triolein was spiked to 3.5% full fat milk with or without SM0023 and subsequently delivered to rats by gavage (n=4-5/group). Data is expressed as mean±standard error of the mean. (4B). Calculations from the data in FIG. 4A to illustrate the delayed time to maximum plasma concentration (Tmax) of oleic acid, mediated by SM0023 and (4C) the oleic acid exposure shown here as area-under-the-curve (AUC) again calculated from FIG. 4A. Data is expressed as mean±standard error of the mean. The student T-test (assuming Gaussian distribution) was used to compare the groups. ** in figure=p<0.01.

[0323] FIG. 5. SM0023 treatment changes long-term fat metabolism. In a metabolic mouse model, male C57BL/6 mice were fed high fat diet with or without 4% of SM0023 (n=7). (5A) Food intake was measured weekly. Total energy intake was calculated from food intake. (5B) Magnetic Resonance Imaging (MRI) measurements of lean body mass at the end of the study. (5C) MRI measurements also generate data on fat mass increase. (5D) Animals were weighed throughout the study and the data here show the mean increase±standard error of the mean after 7 weeks. (5E) Food efficiency over the 7 weeks of treatment, calculated as fat mass increase in gram multiplied by the energy content of 1 g fat (37 kJ/g) divided by kJ energy eaten, multiplied by 100 to obtain a percentage. Error bars represent standard error of the mean. The student T-test was used for statistical analysis.

[0324] FIGS. 6A and 6B illustrate the effect seen in a clinical trial performed, in which:

[0325] FIG. 6A provides a bar graph showing individual changes in HbA1c levels after treatment, wherein it can be seen that the silica of Example 1 delivered a significant reduction in HbA1c of almost 5% in average from baseline;

[0326] FIG. 6B provides a bar graph showing the individual changes in lipid levels (specifically, LDL) after treatment. A significant reduction in blood lipid levels was observed.

EXAMPLES

[0327] The present invention will be further described by reference to the following examples, which are not intended to limit the scope of the invention.

Example 1: Synthesis and Characterization of Porous Material

[0328] Manufacturing Process

[0329] All the chemicals used in the synthesis were purchased from Sigma Aldrich. The invention was manufactured in a stepwise manner as described below.

[0330] Step 1: 54 g of a poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) triblock copolymer with an average Molecular Weight≈5800, EO.sub.20PO.sub.70EO.sub.20, (commonly referred to as Pluronic P123) paste was dissolved in total of 1793 mL doubly distilled water. Dissolution was performed by stirring at 40° C. under mechanical stirring. Complete dissolution generates an opaque polymer solution. This dissolution was preformed either in a closed or open autoclavable system.

[0331] Step 2: An acid, acting as a catalyst, herein HCl (37%) 270 mL, was charged in the polymer solution to lower the pH (<0). Resulting mixture was then charged into a glass reactor anchored with a mechanical stirrer and temperature control unit. Temperature was controlled at 40±5° C.

[0332] Step 3: After reaching this temperature, a solution (108 mL) of silica source, TEOS (tetraethyl orthosilicate), was charged rapidly under 1-2 minutes. Resulting mixture was kept under stirring and same temperature for an additional 9±5 min.

[0333] Step 4: Stirring was stopped and gel was aged at the same temperature for an additional 20±4 hours. Gel was further cured without stirring at 98±2° C. measuring gel temperature for the course of 10±2 hours having set temperature of the control unit at 105±5° C. The gel was cooled to a temperature lower than 70° C. after 10-20 min vigorous stirring at 500±100 rpm, and transferred to a vacuum connected filtration system and drying system.

[0334] Step 5: Initial washing was performed using doubly distilled water until a pH of 5 or above was achieved. Further washing was performed with ethanol or acetone or combination of both ending with acetone.

[0335] Step 6: Finally, material dried at 50° C. for 24-48 hours with or without vacuum was subjected to calcination (at 550° C. in air) to remove the polymeric part from the hybrid material resulting in the formation of a porous silica network.

[0336] For the avoidance of doubt, silica material referred to herein may be identified by batch number. Using the above-mentioned method, several batches of silica were obtained, including batch SM0023.

[0337] A silica comparator was made according to modified protocols from the literature, which was characterized with the same techniques and methods. It is named herein as SM0002 (see Kim, S., Pauly, T. R. Pinnavaia, T. J., Chemical Communications, 1661-1662 (2000)) having pore size of 3.3 nm. The properties of this batch were analysed as described below.

[0338] Nitrogen Sorption Analysis

[0339] Brunauer-Emmett-Teller (BET) surface area, total pore volume and pore size were calculated from optimized synthesis. BET surface area was calculated from sorption isotherm at a relative pressure)(p/p°) of <0.2 (plots in figure BET surface area; FIG. 1A). Total pore volume for all the three studied silica candidates were recorded at a relative pressure)(p/p°)=0.99. Pore size was derived using the density functional theory (DFT) method assuming a cylindrical pore model. The pore size distribution data for the silica particles is presented in FIG. 1B. The nitrogen sorption isotherm of all the studied silica particles were in accordance with porous silica materials and can be classified as type IV according to the International Union of Pure and Applied Chemistry (IUPAC) nomenclature (Sing, K. S. W., Pure Appl. Chem. 57, 603-619 (1985)) (see FIG. 1A). The analysis was performed at liquid nitrogen temperature (−196° C.) using a TriStar II volumetric adsorption analyser (Micromeritics Instrument Corp., GA, USA).

[0340] Pore Structure

[0341] The low angle XRD pattern in FIG. 1C shows peaks that can be indexed on the basis of 2-dimensional hexagonal pore geometry. The unit cell parameter (α) was calculated using (2/√3) d.sub.100, calculated values are in accordance with previous reports for this class of silica porous particles. The unit cell parameter and d-spacing for all silica particles are provided in Table 1. Pore structure was characterized by low-angle X-Ray diffraction (XRD) on a powder PANalytical diffractometer (PANalytical, Karlsruhe, Germany) operated at 45 kV and 40 mA, with 0.02° step size and equipped with Cu Kα radiation source and by Transmission Electron Microscopy (TEM) using a JEOL JEM-2100F instrument (JEOL Ltd., Tokyo, Japan) equipped with Schottky-type field emission gun.

[0342] Particle Size and Morphology

[0343] Silica particle morphology was determined by SEM revealing large aggregates of several micrometers of individual rod-shaped particles (FIG. 1D). SEM micrographs were obtained using a JEOL JSM-7401F (JEOL Ltd., Tokyo, Japan) equipped with Schottky-type field emission gun was used to characterize the particle agglomerates and morphology. Furthermore, hydrodynamic particle size characterization was also performed using a nanosizer. Particles at a concentration of 0.38 (w/v) were dispersed in phosphate buffer of pH 7.3 or an aqueous solution of sodium chloride (0.2 M) and sonicated for 2-5 min to achieve a clear suspension. Particle size distribution was determined in a Malvern® Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK).

[0344] Tables 1 to 3: Material Characteristics

[0345] Various properties of synthesized silica material of batch SM0023 were systematically measured using above listed techniques with operational conditions. The features identified are described in Table 1 below.

TABLE-US-00001 TABLE I Parameter Measured Values Method Appearance White fine powder Ph Eur 7.3 01/2011:0434 Identification Test passed Ph Eur 7.3 01/2011:0434 (Structure) Average particle size 835 nm DLS and/or SEM % pores in 7.0-13.0 66% Gas Sorption Analysis nm .sup.(1) % Lateral pores 20% Gas Sorption Analysis (0.5-5.0 nm) .sup.(1) Specific Surface Area 687 m.sup.2/g Gas Sorption Analysis Pore Volume 0.87 cm.sup.3/g   Gas Sorption Analysis Assay ≥99%  Ph Eur 7.3 01/2011:0434 .sup.(1) {Pore Volume or surface area (at 0.5-5.0 nm or 7.0-13.0 nm)/Total Pore Volume (single point) or Total Surface Area (BET Method)} × 100

[0346] In further experiments wherein multiple bulk samples of the material were obtained, the following features were observed in the range indicated (which other features of the material being substantially in accordance with those indicated for the sample referred to in Table 1 above):

TABLE-US-00002 TABLE 2 Parameter Measured Values Method Appearance White or almost Ph Eur 7.3 white fine powder 01/2011:0434 Average particle size 539-1764 nm DLS and/or SEM % pores in 7.0-13.0 nm .sup.(1) 50-61% Gas Sorption Analysis % Lateral pores 22-31% Gas Sorption Analysis (0.5-5.0 nm) .sup.(1) Specific Surface Area 795-1094 m.sup.2/g Gas Sorption Analysis Pore Volume 0.79-1.27 cm.sup.3/g Gas Sorption Analysis .sup.(1) {Pore Volume or surface area (at 0.5-5.0 nm or 7.0-13.0 nm)/Total Pore Volume (single point) or Total Surface Area (BET Method)} × 100

[0347] It is contemplated that in other experiments the following features might be observed:

TABLE-US-00003 TABLE 3 Parameter Measured Values Method Appearance White fine powder Ph Eur 7.3 01/2011:0434 Identification (Structure) Test passed Ph Eur 7.3 01/2011:0434 Average particle size 835 nm DLS and/or SEM % pores in 7.0-13.0 nm .sup.(1) 49% Gas Sorption Analysis % Lateral pores 28% Gas Sorption Analysis (0.5-5.0 nm) .sup.(1) Specific Surface Area 687 m.sup.2/g Gas Sorption Analysis Pore Volume 0.87 cm.sup.3/g   Gas Sorption Analysis Assay ≥99%  Ph Eur 7.3 01/2011:0434 .sup.(1) {Pore Volume or surface area (at 0.5-5.0 nm or 7.0-13.0 nm)/Total Pore Volume (single point) or Total Surface Area (BET Method)} × 100

Example 2: Entrapment of Biomolecules by Silica In Vitro

[0348] The silica material according to Example 1 (batch SM0023) was tested on two biomolecules present in the human digestive system, pancreatic amylase and pancreatic lipase. The enzyme activity tests described herein are standard tests that can be found in the literature to measure the activity of digestive enzymes. In this example, the tests have been performed in vitro with the porcine versions of the enzymes. The example shows that interactions between the silica materials according to the invention entrap biomolecules such as digestive enzymes and consequently the remaining depleted solution has a lower enzyme activity (see FIG. 3).

[0349] α-Amylase entrapment by SM0023

[0350] The effect of silica particles on digestion of carbohydrates was studied by detecting the activity of α-Amylase (see FIG. 3D). It has been tested in principle under two different conditions: (a) by co-incubating the silica particles together with the two essential components, α-Amylase (the enzyme, which is a small biomolecule) and starch (the substrate which is a larger molecule) or (b) when α-Amylase has been incubated alone with the silica particles first, followed by removing the pelleted silica particles and detection of the activity in the remaining supernatant thereafter. When protocol (a) has been tested, in some instances inhibition of α-Amylase activity has been observed. When protocol (b) is used, if the α-Amylase has been pre-incubated with silica particles of a specific pore size (9.6 nm) and with the correct physiochemical properties, then the α-Amylase activity remaining in the supernatant is reduced (see FIG. 3D).

[0351] By detecting the specific α-Amylase protein band of 54 kDa in a conventional Western blot assay, it can be concluded that the enzyme was removed from the solution after binding to SM0023 (FIG. 3D). Using an alternative method (Bicinchoninic acid (BCA) assay)) to measure α-Amylase protein concentration in the solution confirm that the enzyme was removed after being exposed to SM0023 (FIG. 3F). Equally importantly, reference silica batch SM0002 with physiochemical properties falling outside of the ranges required by the present invention (as prepared using techniques known to those in the art) do not markedly affect the amount of α-Amylase present in the solutions.

[0352] The data in FIG. 3 can be seen as a model of how the silica acts as a molecular sieve by entrapping biomolecules, thus lowering the concentration of the free biomolecule and thereby lowering the overall enzymatic activity of the solution.

[0353] Lipase Entrapment—a Second Model Confirming the Mode of Action

[0354] The principle of biomolecule entrapment was confirmed with a second in vitro model system, namely that of pancreatic lipase degradation of a triglyceride substrate. In brief, a similar set-up was conducted by allowing the silica batches SM0002 and SM0023 to interact with lipase prior to a brief centrifugation and thereafter addition of a substrate in the form of tributyrin (FIG. 3C).

[0355] SM0023 inhibits the lipase activity in the remaining supernatant, whereas silica with other physiochemical properties does not achieve the same effect.

Example 3: Lowering the Immediate Energy Uptake In Vivo

[0356] Full fat milk (containing 3.5% fat) was spiked with 14C radiolabelled triolein and delivered to rats, followed by measuring the radioactivity in the plasma over 24 hours (FIG. 4). The results show a clear shift in the profile for the animals treated with SM0023 (FIG. 4A). Both a delay in early absorption of the biomolecule (FIG. 4B) as well as an overall lower exposure as illustrated by a significantly lower AUC can be seen (FIG. 4C). Treating the animals with SM0002 (with other physiochemical properties) in the same model does not show any effect, again underlining the importance of using silica particles with the correct physiochemical properties. This exemplifies that SM0023 has an effect detectable already at the level of absorption seen within 24 hours in a relevant model of lipid metabolism.

[0357] As for the protocol used, 1.5 mL of 3.5% full fat milk was spiked with 14-C radiolabelled triolein and sonicated (to incorporate the triolein into the fat droplets of the milk) followed by adding of 60 mg silica particles. Prior to the study the male Sprague Dawley rats were acclimatized for a minimum of 3 days. When the rats reached a weight between 250-300 grams, their right carotid artery was cannulised to allow for less stressful blood sampling. All rats were fasted for 8 hours prior to dose administration and 8 hours after dose administration.

Example 4: Lowering the Long-Term Adipose Tissue Formation and Food Efficacy In Vivo

[0358] SM0023 treatment changes long-term fat metabolism. A metabolic mouse model of male C57BL/6 mice were fed high fat diet with or without 4% of SM0023 (n=7). The total energy intake was similar between the groups, suggesting that the treatment does not in itself affect the appetite of the animals (FIG. 5A) and measurements of lean mass were also similar between the groups (FIG. 5B). Measurements of “fat mass increase” on the other hand show a significantly lower level in the SM0023 treated animals (FIG. 5C). Animals were also weighed throughout the study and the data strikingly shows a significantly lower weight gain in the animals treated with SM0023 (FIG. 5D). The fact that the weight differences were due to differences in fat mass and not in lean weight confirms that mice treated with silica particles grew normally, showed by normal weight increase on organ and muscle mass, while the adipose tissue did not increase in the same extent as for the control animals. This indicates that the mice were healthy during the treatment. Finally, these data can be used to calculate the “food efficiency”, defined as “fat mass increase” divided by “energy eaten” expressed as percentage (FIG. 5E). The “food efficiency” is also lower in the SM0023 treated animals, clearly showing that the treated animals have an altered energy metabolism leading to less fat being stored in their bodies as adipose tissue.

[0359] These data, combined with Examples 2 and 3, indicate that treatment with SM0023 lowers degradation of food and directly decreases biomolecule and energy uptake into the body, which secondarily lowers formation of adipose tissue in vivo.

[0360] As for the protocol details, nine weeks old male C57BL/6N mice (Scanbur, Sweden) were single caged and held under a 12 h:12 h light/dark cycle at thermo-neutral temperature, 30° C. with 50% humidity, at the animal facility at Stockholm University per standard protocol for animal husbandry. Mice had free access to their respective diets and water during the experiment. Food efficiency was calculated as fat weight gained in g multiplied by the energy content of 1 g fat (37 kJ/g) divided by the energy from food ingested, in kJ. This gives food efficiency as a unit-free rate, multiplying it by 100 gives the percentage of energy that gets stored as fat.

Example 5: Clinical Effects Seen of Silica Particles on Reduction in Metabolic and Cardiovascular Risk Factors

[0361] A pilot study in human subjects with both normal weight and obese male volunteers was performed using several batches of silica (each prepared according to the process set out in Example 1 and each conforming to the parameters as required by the present invention). The aim of the study was to evaluate safety, tolerability and feasibility of dosing regimen. 10 normal weight and 10 obese subjects were enrolled.

[0362] After signature on the written consent, medical examination, questions regarding eating habits, sleep patterns, living conditions and digestive health and blood and faces sampling, both the normal weight and obese subjects (Group A and Group B) received placebo capsules during study days 1-5 (five day run-in period). Thereafter all subjects received porous silica as set out in the schedule below.

[0363] Dosage of Porous Silica [0364] Study days 6-9 (4 days): 1 g before breakfast, lunch and dinner. [0365] Study days 10-14 (5 days): 2 g before breakfast, lunch and dinner. [0366] Study days 15-21 (7 days): 3 g before breakfast, lunch and dinner.

[0367] Upon successful completion of the initial dosing regimen, Group B continued with a dosing regimen of 3 g three times daily, a daily total intake of 9 grams. The treatment was continued for 10 additional weeks, i.e. in total 12 weeks treatment.

[0368] The adverse events reported were unrelated to the treatment. No changes in bowel and GI function was detected. In addition, blood levels of vitamins, trace elements and hormones were not affected. A decrease in cholesterol and blood lipids was observed but the main finding was the unexpected effect seen on HbA1c. HbA1c is glycated haemoglobin and this acts as a marker for long-term glucose exposure. HbA1c levels were measured using a Siemens DCA Vantage Analyser, using techniques known to those skilled in the art.

[0369] A statistically significant decrease of HbA1c levels were seen after 12 weeks of treatment with silica material of the present invention in obese male volunteers (see FIG. 6A). As neither fasting plasma glucose nor fasting insulin were statistically significantly altered it indicates that the reduction in HbA1c is due to reduction in postprandial levels.

[0370] No deaths, no study discontinuations and no serious adverse events were reported. Adverse events observed were mild and did not result in discontinuation. In summary, there were fewer related AEs in the obese compared to those of normal weight, and all were mild and transient.

[0371] Biochemistry (Summarized in Table 4)

[0372] In Normal weight/Group A the change from baseline was statistically significant for Cholesterol 4.5 to 4.1 mmol/L (p=0.04) and LDL 2.7 to 2.4 mmol/L (p=0.04) after 3 weeks of treatment.

[0373] In the Obese/Group B (see FIGS. 6A and 6B) the change from baseline was statistically significant for LDL from 2.6 to 2.3 mmol/L (p=0.001) after 3 weeks of treatment. The change from baseline was still statistically significant after 12-week treatment, with LDL to 2.2 mmol/L (p=0.03) (FIG. 6B), LDL/HDL from 2.2 to 1.8 (p=0.02) and HbA1c from 34.7 to 33.0 mmol/mol (p=0.04) (FIG. 6A) after 12-week treatment. FIG. 6 shows % reduction over time from baseline.

TABLE-US-00004 TABLE 4 The effects on blood lipids and HbA1c of silica particles as prepared according to Example 1 in human male volunteers Mean Baseline Week 3 P-value Week 12 P-value Cholesterol Normal 4.5 4.1 0.04 — — (mmol/L) Obese 4.3 4.2 0.24 3.9 0.13 LDL Normal 2.7 2.4 0.04 — — (mmol/L) Obese 2.6 2.3 0.001 2.2 0.03 LDL/HDL Normal 2.1 2.1 1.0 — — Obese 2.2 1.9 0.05 1.8 0.02 HbA1c Normal 31.9 32.1 0.62 — — (mmol/mol) Obese 34.7 34.1 0.14 33.0 0.04