Biopolymer formulations for oral administration

Abstract

The present invention particularly relates to a method for preparing a pharmaceutical composition that can be provided to a subject in an oral form, the pharmaceutical composition comprising the following as active ingredients: a protein with a mass of more than 10 kilodaltons, a nucleic acid molecule consisting of more than 15 nucleotides, or a combination thereof. The one or more active ingredients are dispersed in particles to promote intestinal absorption of the particles and release of the active ingredients in the bloodstream. The method for preparing such a pharmaceutical composition comprises: preparing an aqueous composition comprising one or more active ingredients; monosaccharides or disaccharides; one or more other compounds and chitosan; and spraying the prepared aqueous composition to an anti-solvent and / or a supercritical fluid, wherein the water fraction of the composition is soluble or miscible and the solute of the composition is insoluble, so that the solute precipitates and produces particles with an average diameter of 50 nanometers (nm) to 20 micrometers (μm); collecting the particles; and preparing a pharmaceutical composition comprising the particles. Also provided is a particle assembly having an average diameter of 50nm to 20μm containing uniformly distributed components. Also provided is a pharmaceutical preparation in an oral dosage form comprising such a particle assembly for treating a disease.

Description

[0001] The present invention relates to pharmaceutical formulations for gastrointestinal delivery of biopolymers such as proteins and nucleic acid molecules. The present invention also relates to methods for preparing such formulations.

[0002] The absorption of particles containing biomacromolecules delivered via the gastrointestinal system and via the oral route is hampered by a series of barriers, such as: low regional pH, digestive enzymes and the presence of a mucus layer that prevents passive absorption (Lundquist P., Artusson P. Adv Drug Del Rev 106:256-276, 2016 and Muheem A. et al., Saudi Pharm J 24(4):379-506, 2016).

[0003] The digestive tract contains several systems that facilitate the absorption of biomolecules. Most of these systems are specific for a particular substance. Recently, it has been shown that particles can also cross the intestinal barrier. The exact path taken by the particles is still unknown and may vary depending on the type of particle. Some studies have shown that some particles migrate via M cells and the so-called Peyer's Patches.

[0004] Methods for passing through the harsh environment of the stomach are readily available. Such methods include, but are not limited to, enteric coatings of tablets or pills, delayed release formulations resistant to acidic environments, and capsules. Such delivery vehicles preferably contain particles that are released after passing through the stomach. This stomach delivers the particles substantially intact to the environment after the stomach through the delivery vehicle. It is preferably the small intestine and / or colon. The factors that are conducive to promoting release in the small intestine can be pH, the time that the carrier can be degraded after a certain period of time, such as in slow-release or delayed-release formulations, and / or the presence of digestive enzymes. The factors that are conducive to promoting release in the colon can be time, such as in delayed-release or slow-release formulations. Delayed-release formulations usually release their contents immediately after a certain period of time. Sustained-release formulations usually release continuously over a long period of time, and the release can be triggered at a certain time point, or triggered by changes in the environment or other signals, and the release can be, but not necessarily, constant during the period of time. After protection against the low pH of the stomach, the rest of the digestive tract contains systems such as absorptive enterocytes in microvilli. For the absorption of particles, passive and active transport mechanisms on the intestinal wall can be targeted. Loading the particles with biomolecules can facilitate the release of the biomolecules from the particles into the bloodstream.

[0005] To date, oral delivery of biologically active macromolecules (biologics) has been a challenge, with only small biologics being somewhat successful. Millotti et al. (2011) describe the preparation of chitosan-6-mercaptonicotinic acid particles for oral delivery of human insulin. Insulin, which is less than 6 kDa in size, cannot pass through the intestinal wall by itself, but when combined with particles containing thiolated chitosan, it can pass through the intestinal wall, but with low bioavailability (Millotti et al., 2011 Drug Delivery 18 (3) 190-197).

[0006] Proteins and oligonucleotides in the range of 10–500 kDa are currently delivered only by intravenous or subcutaneous injection. The low bioavailability of peptides and proteins in oral formulations (<10%) has triggered extensive research into better means and methods. Such research has particularly involved the use of penetration enhancers, but with mixed results (see Whitehead K. et al., Pharm Res 25(8):1782-1788, 2007; Yewale C. et al., Clin Rev Therap Drug Carrier systems 32(5):236-387, 2015). These methods have not yet developed into common practice.

[0007] The present invention provides particles and methods of making particles by which the particles can effectively cross the intestinal barrier and effectively make the entrapped protein or nucleic acid molecules bioavailable once entering the bloodstream. Summary of the invention

[0008] The present invention provides a method for preparing a pharmaceutical composition, which contains a protein with a mass of more than 10 kilodaltons, a nucleic acid molecule composed of more than 15 nucleotides, or a combination thereof as an active ingredient, the method comprising: preparing an aqueous composition, which contains one or more active ingredients; monosaccharides or disaccharides; one or more other compounds and chitosan; and spraying the prepared aqueous composition into an anti-solvent and / or a supercritical fluid, in which the water fraction of the composition is soluble or miscible and the solute of the composition is insoluble, so that the solute precipitates and produces particles with an average diameter of 50 nanometers (nm) to 20 micrometers (μm); collecting the particles; and preparing a pharmaceutical composition including the particles. Also provided is a particle assembly with an average diameter of 50nm to 20μm, which contains the components uniformly distributed. The components are described in the claims and include active ingredients (proteins with a mass of more than 10 kilodaltons, nucleic acid molecules composed of more than 15 nucleotides), chitosan and other components shown. Also provided is a pharmaceutical preparation in an oral dosage form, which includes such a particle assembly for treating a disease.

[0009] In one embodiment, there is provided a method for preparing a pharmaceutical composition, the method comprising:

[0010] Prepare an aqueous composition comprising

[0011] - a protein with a mass of 10 kilodaltons or more, a nucleic acid molecule consisting of 15 or more nucleotides, or a combination thereof, as an active ingredient, in an amount of 0.02% to 25% of the dry weight of the composition;

[0012] - monosaccharides or disaccharides in an amount of 2% to 25% by dry weight of the composition;

[0013] - one or more other compounds in an amount of 0-10% by dry weight of the composition;

[0014] - chitosan in an amount of at least 48.3% by dry weight of the composition;

[0015] spraying the prepared aqueous composition into an antisolvent and / or a supercritical fluid in which the aqueous fraction of the composition is soluble or miscible and the solute of the composition is insoluble, thereby precipitating the solute and producing particles having an average diameter of 50 nanometers (nm) to 20 micrometers (μm);

[0016] collecting the particles; and preparing a pharmaceutical composition comprising the particles.

[0017] Also provided is a method of preparing a pharmaceutical composition for oral delivery of a systemic active ingredient, the method comprising:

[0018] Prepare an aqueous composition comprising

[0019] - a protein with a mass of 10 kilodaltons or more, a nucleic acid molecule consisting of 15 or more nucleotides, or a combination thereof, as a systemically active ingredient, in an amount of 0.02% to 25% of the dry weight of the composition;

[0020] - monosaccharides or disaccharides in an amount of 2% to 25% by dry weight of the composition;

[0021] - one or more other compounds in an amount of 0-10% by dry weight of the composition;

[0022] - chitosan in an amount of at least 48.3% by dry weight of the composition;

[0023] spraying the prepared aqueous composition into an antisolvent and / or a supercritical fluid in which the aqueous fraction of the composition is soluble or miscible and the solute of the composition is insoluble, thereby precipitating the solute and producing particles having an average diameter of 50 nanometers (nm) to 20 micrometers (μm);

[0024] collecting the particles; and preparing the pharmaceutical composition for oral delivery, which comprises the particles containing the systemically active ingredient.

[0025] The prepared aqueous composition preferably comprises one or more other compounds in an amount of 0-8%, preferably 0-6%, preferably 0-4%, preferably 0-2%, preferably 0-1.7% by dry weight of the composition.

[0026] In one embodiment, the present invention provides a method for preparing a pharmaceutical composition, the method comprising:

[0027] Prepare an aqueous composition comprising

[0028] - a protein with a mass of 10 kilodaltons or more, a nucleic acid molecule consisting of 15 or more nucleotides, or a combination thereof, as an active ingredient, and its content accounts for 0.02% to 25% of the dry weight of the composition;

[0029] - monosaccharides or disaccharides in an amount of 2% to 25% by dry weight of the composition;

[0030] - one or more other compounds in an amount of 0-1.7% by dry weight of the composition;

[0031] - chitosan in an amount of at least 48.3% by dry weight of the composition;

[0032] spraying the prepared aqueous composition into an antisolvent and / or a supercritical fluid in which the aqueous fraction of the composition is soluble or miscible and the solute of the composition is insoluble, thereby precipitating the solute and producing particles having an average diameter of 50 nanometers (nm) to 20 micrometers (μm);

[0033] collecting the particles; and preparing a pharmaceutical composition comprising the particles.

[0034] The present invention provides a method for preparing a pharmaceutical composition, the method comprising:

[0035] - preparing an aqueous composition comprising

[0036] o a protein with a mass of 10 daltons or more, a nucleic acid molecule consisting of 15 or more nucleotides, or a combination thereof, as an active ingredient, in an amount of 0.001% to 1.5% (by weight) of the final aqueous composition;

[0037] ○ monosaccharide or disaccharide, the amount of which accounts for 0.1% to 1.5% (by weight) of the final aqueous composition;

[0038] o one or more other compounds, in an amount of 0.0-0.5%, preferably 0.1% by weight of the final aqueous composition;

[0039] Chitosan in an amount of at least 0.5% by weight of the final aqueous composition;

[0040] - spraying the prepared aqueous composition into an antisolvent in which the aqueous fraction of the composition is soluble or miscible and the solute of the composition is insoluble;

[0041] - causing the solute to precipitate, thereby producing particles having an average diameter of 50 nanometers (nm) to 20 micrometers (μm);

[0042] - collecting the particles; and

[0043] A pharmaceutical composition comprising the particles is prepared.

[0044] The prepared aqueous composition preferably comprises one or more other compounds in an amount of 0.0-0.4%, preferably 0.0-0.3%, preferably 0.0-0.2%, and preferably 0.0-0.1% by weight of the final aqueous composition.

[0045] The present invention also provides a particle assembly having an average diameter of 50 nm to 20 μm obtainable by the method described herein.

[0046] The method for preparing a pharmaceutical composition as described herein preferably further comprises: preparing an oral dosage form comprising the prepared particles or aggregates of particles.

[0047] The pharmaceutical composition prepared is preferably a pharmaceutical composition for oral delivery of systemic active ingredients.

[0048] Also provided is a particle assembly having an average diameter of 50 nm to 20 μm, comprising uniformly distributed:

[0049] - a protein with a mass of 10 kilodaltons or more, a nucleic acid molecule consisting of 15 or more nucleotides, or a combination thereof, as an active ingredient, in an amount of 0.02% to 25% of the dry weight of the granules;

[0050] - mono- or disaccharides in an amount of 2% to 25% by dry weight of the granules;

[0051] - one or more other compounds in an amount of 0.0-10%, preferably 0.0-8%, preferably 0.0-6%, preferably 0.0-4%, preferably 0.0-2%, preferably 1.7% by dry weight of the granules;

[0052] - Chitosan in an amount of at least 48.3% by dry weight of the particles.

[0053] Also provided is a method for delivering a protein of 10 kDa or more, or a nucleic acid molecule consisting of 15 or more nucleotides, or a combination thereof as an active ingredient to the blood of a subject in need thereof in a therapeutically relevant amount, the method comprising preparing a pharmaceutical composition comprising the protein or nucleic acid molecule according to the method described herein, and orally administering the pharmaceutical composition to the subject.

[0054] Also provided is a pharmaceutical preparation in oral dosage form, which comprises the particle assembly described herein for use in treating a disease. The preparation is preferably a solid dosage form.

[0055] Also provided is a particle assembly as described herein, which preferably comprises an antibody, such as a protein with a molecular mass of 10 kilodaltons. A method for delivering a protein of 10 kDa or more or a nucleic acid molecule consisting of 15 or more nucleotides as an active ingredient to the blood of a subject in need thereof in a therapeutically relevant amount, the method comprising: preparing a pharmaceutical composition comprising the protein or nucleic acid molecule according to any one of claims 1 to 8, and orally administering the pharmaceutical composition to the subject.

[0056] Detailed description of the invention

[0057] An active ingredient is an ingredient that has a biological effect when provided to a permissive biological system. It is generally an ingredient that is expected to have a therapeutic effect after administration to an individual in need thereof. It may have a therapeutic effect alone or in combination with one or more other molecules and / or cells. The other molecules and / or cells may be present in the same pharmaceutical composition or in a different pharmaceutical composition, or may already be present in the individual.

[0058] An active ingredient is a component that has a biological effect when administered or provided to an individual's circulation. Such components are also referred to as systemic active ingredients. Without being bound by theory, it is believed that the particles, particle assemblies, compositions and pharmaceutical compositions of the present invention and other means comprising the particles described herein can encapsulate components, provide stability in the gastrointestinal tract for the components and promote the transport of the components from the gastrointestinal tract to the blood. It is further believed that the particles promote the slow release of the encapsulated components in the subject's blood. This results in an increased bioavailability compared to intravenous injection of the components, particularly for proteins with a molecular weight of about 100-300 kDa, such as antibodies. Active ingredients may also be referred to as drugs.

[0059] The active ingredient used in the present invention is preferably soluble in water. The term "soluble in water" or "water-soluble" means that the water solubility of the active ingredient is generally not less than about 1 g per 100 ml of water at 20°C, preferably not less than about 3 g, and more preferably not less than about 5 g. Preferably, the active ingredient is readily soluble in water. The term "readily soluble in water" means that the water solubility of the physiologically active substance is not less than about 5 g per 100 ml of water at 20°C, preferably not less than about 10 g.

[0060] The active ingredient can be a compound or a biomolecule. The active ingredient is preferably a protein with a mass of more than 10 kilodaltons, a nucleic acid molecule consisting of more than 15 nucleotides, or a combination thereof. The pharmaceutical composition may contain one, two, three or even more active ingredients. The total amount of all these active ingredients (with a mass of more than 10 kilodaltons in the case of proteins, with more than 15 nucleotides in the case of nucleic acid molecules, or a combination thereof) accounts for 0.001%-1.5% of the weight of the aqueous composition, preferably 0.01%-1.5%; more preferably 0.05%-1.5%; more preferably 0.1%-0.5% of the weight of the aqueous composition. An active ingredient of a protein or nucleic acid molecule of non-specified mass or number of nucleotides (length) may be present in the particles, in which case its weight is provided as below "one or more other compounds" relative to the final weight of the aqueous composition. The upper limit of the amount of active ingredient in the aqueous composition may be 1.5%.

[0061] The active ingredient is preferably a protein consisting of more than 60 amino acid residues. Proteins include proteins synthesized by cellular mechanisms, proteins modified post-translationally, polypeptides, and their derivatives. The active ingredient can be a compound with a peptide-like structure. The molecular weight of the protein is preferably about 10 kilodaltons or more, preferably 15 kilodaltons or more, more preferably 20, 30, 50, 75, 100, 125 or 150 kilodaltons or more. In a preferred embodiment, the molecular weight of the protein is 100 to 20,000 kilodaltons. The present invention is particularly useful for proteins with a molecular weight greater than 100 kilodaltons. In some embodiments, the protein is an antibody. The mass of the antibody is generally between 100-300 kilodaltons. Therefore, the molecular weight of the protein is preferably 100-300 kilodaltons, preferably about 100-200 kilodaltons. The present invention is particularly suitable for proteins or nucleic acid molecules that need to be repeatedly administered over a long period of time. Many proteins and nucleic acid molecules are administered in a more or less invasive manner by injection needles. This often requires that the drug is usually administered by qualified personnel in a hospital. The pharmaceutical composition of the present invention can be administered orally. The pharmaceutical composition of the present invention helps to transport a large amount of active ingredients from the intestinal cavity to the bloodstream, and then the bloodstream transports nutrients from the gastrointestinal tract to the rest of the body. Most but not all of the blood from the gastrointestinal tract passes through the liver before being distributed to other parts of the body. Usually more than 20% of the active ingredients are transported. In a preferred embodiment, at least 30%, preferably at least 40%, more preferably at least 50%, 60%, 70% are transported to the bloodstream.

[0062] The terms "mass", "molecular mass" or "molecular weight", "size" or "molecular size" are used interchangeably with respect to the protein in the particles described herein and / or with respect to the active ingredients as described herein. For example, the mass of an antibody, or the molecular mass of an antibody or the molecular weight of an antibody is typically between 100-300 kilodaltons.

[0063] The disaccharide is preferably a biocompatible and biodegradable disaccharide. A disaccharide has two monosaccharides connected via a glycosidic bond. Non-limiting examples are lactose, sucrose, cellobiose, trehalose, and maltose. In a preferred embodiment, at least one monosaccharide is glucose. In a preferred embodiment, the glycosidic bond is a glucosidic bond. In a preferred embodiment, the disaccharide has the formula C 12 H 22 O 11 In a particularly preferred embodiment, the disaccharide is sucrose.

[0064] The monosaccharide or disaccharide is preferably present in an amount of 0.01% to 1.5% by weight of the final aqueous composition. Preferably, it is present in an amount of 0.01% to 0.25% by weight of the final aqueous composition. The monosaccharide or disaccharide is preferably present in an amount of 0.1% to 1.5% by weight of the final aqueous composition. Preferably, it is present in an amount of 0.1% to 0.25% by weight of the final aqueous composition. The upper limit of the amount of monosaccharide or disaccharide in the aqueous composition may be 2%.

[0065] Chitosan is a linear polysaccharide consisting of randomly distributed β-(1>4) linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetyl units). It can be produced by deacetylation of chitin, a structural element in the exoskeleton of crustaceans (such as crabs and shrimps) and in the cell walls of fungi. The degree of deacetylation (%DD) can be determined by NMR spectroscopy. The %DD degree in the chitosan of the present invention ranges from 60% to 100%. On average, the molecular weight of the chitosan of the present invention is between 3800 and 20,000 Daltons. Methods for producing chitosan are known in the art. A common method for synthesizing chitosan is to deacetylate chitosan using an excess of sodium hydroxide as a reagent and water as a solvent. Nowadays, chitosan can also be chemically synthesized. The chitosan used in the present invention is generally a weak base and is insoluble in water and organic solvents. However, it is soluble in dilute acidic aqueous solutions (pH < 6.5), which enables the conversion of the glucosamine units into soluble forms of R-NH3+ (Kumar, Muzzarelli, Muzzarelli, Sashiwa and Domb, 2004). The chitosan used in the present invention is soluble in water at room temperature and atmospheric pressure in an amount of less than 10 mg / ml, preferably less than 5 mg / ml.

[0066] The chitosan is preferably present in an amount of at least 1%, preferably 2%, preferably 4%, more preferably 8%, more preferably at least 16% by weight of the final aqueous composition. The chitosan is preferably present in an amount of 1-10% by weight of the final aqueous composition.

[0067] Particles are produced by spraying the aqueous composition into an antisolvent and / or by spraying the aqueous composition into a supercritical fluid. In a preferred embodiment, the prepared aqueous composition is sprayed into an antisolvent, wherein the water fraction of the composition is soluble or miscible and the solute of the composition is insoluble. The water fraction of the composition can be dissolved in the antisolvent and / or the supercritical fluid. The solvent and / or droplets or particles in the aqueous composition are generally substantially insoluble in the antisolvent and / or the supercritical fluid. Droplets can appear in the environment of emulsions, colloids and / or dispersions. Of course, it is conceivable that the aqueous solution contains solutes, particles or droplets that can be quickly dissolved in the antisolvent and / or the supercritical fluid. In this case, the solute, particle or droplet will not generally contribute significantly to the particles formed in the antisolvent and / or the supercritical fluid. In a preferred embodiment, the antisolvent is a supercritical fluid.

[0068] The average diameter of the particles produced by the method of the present invention is preferably 50nm to 20μm, preferably 50nm to 10μm, preferably 50nm to 5μm, preferably 50nm to 1μm. The most commonly used measurement when describing the particle size distribution is the D value. D values ​​such as D10, D50 and D90 are intercepts of the cumulative amount of 10%, 50% and 90%. Typical examples of particles produced:

[0069] D10(nm) D50(nm) D90(nm) 104 285 854

[0071] The D10 value represents the diameter at which the mass of particles with smaller diameters accounts for 10% of the total mass of all particles in the collection. Similarly, the D50 value represents the diameter at which the mass of particles with smaller diameters accounts for 50% of the total mass of all particles in the collection. The D50 value and diameter distribution of a collection of particles can be affected by the following spray variables:

[0072] Temperature, nozzle size and shape, pressure, CO2 flow rate, solution flow rate, composition of the fluid to be sprayed.

[0073] When referring to the average diameter of the particles herein, it is meant the D50 value of the collection. In a preferred embodiment, the average diameter of the particles is 50-500 nm.

[0074] WO2005 / 004838 describes a formulation for intranasal delivery of proteins comprising chitosan derivatives. The formulation comprises a blend of protein particles and soluble chitosan. Soluble chitosan is a derivative of chitosan that dissolves in water in an amount of at least 10 mg / ml at room temperature and atmospheric pressure. The formulation does not contain particles in which at least protein and chitosan are uniformly distributed. Kashid et al. point out that the particle size and morphology of the drug particles constitute important characteristics of nasal granular drug products. Particle size and morphology are related to drug solubility and should be controlled to obtain suitable drug solubility characteristics in the nostrils. In vitro dissolution in suitable simulated liquids should be considered. Particle size and morphology are also important for reducing the feeling of sand and possible irritation to the nasal cavity. Too fine particles less than five microns may be inhaled into the lungs and should be avoided for nasal products. Typically, particles in the 5-10 micron range are deposited in the nostrils (Kashid et al. (2016), World Journal of Pharmaceutical Research 5.891-912. DOI 10.20959 / wjpr20166-6427.

[0075] The supercritical fluid preferably has a temperature of 30-55 degrees Celsius. Examples of suitable supercritical fluids are ethane, propane, nitrous oxide (N2O) or carbon dioxide (CO2). The supercritical fluid is preferably supercritical CO2. The pressure of the supercritical fluid is of course high enough to ensure the supercritical state of the supercritical fluid. For CO2, the pressure is preferably at least 73Bar. Preferably, the pressure is at least 100Bar, more preferably, the pressure is 100-180Bar.

[0076] The flow rates of CO2 and the aqueous composition are typical parameters known to the skilled person that can be adjusted to fine tune the system. Suitable rates are 150-350 kg / hr for CO2 and 2-8 ml / min for the aqueous composition. Similarly, the skilled person knows that the size and shape of the nozzle through which the aqueous composition is sprayed can be changed as desired.

[0077] One or more other compounds can be typical pharmaceutical excipients, such as but not limited to one or more amino acids, anti-adhesive agents, adhesives, coating agents, pigments, disintegrants, flavoring agents, glidants, lubricants, preservatives, adsorbents, sweeteners, etc., or combinations thereof. One or more other compounds can also include active ingredients that are not proteins with a mass of more than 10 Daltons, nucleic acid molecules consisting of more than 15 nucleotides, or combinations thereof.

[0078] The one or more other compounds may be present in an amount of 0-10% by weight of the final aqueous composition, preferably 0.000-0.5%, more preferably 0.005%-0.5%, preferably 0.05%-0.5%, preferably 0.0-0.1%, preferably 0.005%-0.1%, preferably 0.01%-0.1%, preferably 0.05%-0.1% by weight of the final aqueous composition.

[0079] In one embodiment, the prepared aqueous composition comprises an amino acid in an amount of 0.005% to 0.015% by weight of the final aqueous composition, the lower limit of the range being preferably 0.005%, more preferably 0.01%. The upper limit is preferably 0.015%, more preferably 0.01%. Any lower range limit may be used in combination with any higher range limit. The range is preferably 0.001% to 0.5% by weight of the final aqueous composition; more preferably 0.01% to 0.25%, more preferably 0.02% to 0.1%.

[0080] The amino acid is preferably histidine, lysine, arginine, asparagine or glutamine. The amino acid is preferably histidine, lysine or arginine. Preferably, the amino acid is histidine. When there are two or more amino acids, at least one of them is histidine, lysine, arginine, asparagine or glutamine. In a preferred embodiment, two or more of them are selected from histidine, lysine, arginine, asparagine and glutamine. Preferably, at least one of them is histidine.

[0081] The prepared aqueous composition preferably contains one or more penetration enhancers, solubilizers, emulsifiers or combinations thereof. Based on their safety and effectiveness, the chemical substances that promote penetration are but not limited to zwitterionic surfactants, or having nitrogen-containing rings, or anionic surfactants, which are preferred additives for treating nanoparticles containing macromolecules.

[0082] The solubility of poorly water-soluble drugs in water can be increased by the action of solubilizers. Solubilizers can increase the bioavailability of active ingredients. Well-known solubilizers are cyclodextrins. The aqueous composition can contain polysorbate 20 to polysorbate 80 and / or sorbitan stearate.

[0083] The amount of water in the final aqueous composition can vary. A more concentrated or dilute composition can be used by the skilled artisan as desired. The dry weight to water ratio can be 1-20; 1-30; 1-40; 1-50; 1-60; 1-70; 1-80; 1-90; 1-100; 1-120; 1-140; 1-160; 1-180; 1-200 or less. In a preferred embodiment, the dry weight to water ratio in the final aqueous composition ranges from 1-25 to 1-200; preferably from 1-30 to 1-160; preferably from 1-50 to 1-100; preferably from 1:27 to 1:166.

[0084] The active ingredient may include an oligonucleotide, a cytokine, an enzyme, a soluble protein ligand of a cell receptor, a coagulation system factor, a fusion protein and / or an antibody. In the case of two or more active ingredients, all of the ingredients may be an oligonucleotide, a protein or a combination thereof. The combination of active ingredients is not limited in principle.

[0085] The nucleic acid molecule is preferably an oligonucleotide. The oligonucleotide may be double-stranded or single-stranded. The oligonucleotide is preferably single-stranded. In one embodiment, the oligonucleotide is an antisense oligonucleotide. At present, oligonucleotides are sought for reasons of manipulating the levels of certain transcripts in cells. They are also used as guides for Crispr / Cas type genome editing methods. They are also used as replicative and non-replicative gene expression boxes (cassettes), such as but not limited to nucleic acid vaccines and gene therapy vectors. In the case of two or more active ingredients, it is preferred to have two or more oligonucleotides, preferably two or more oligonucleotides or 15 or more nucleotides. The two or more oligonucleotides may be directed to different targets, the same target at different target positions, or a combination thereof. The number of more than 15 nucleotides in the nucleic acid molecule referred to herein refers to the length of the nucleic acid molecule. For example, a double-stranded oligonucleotide having two complementary single-stranded oligonucleotides hybridized to each other each consisting of 15 nucleotides is referred to as a nucleic acid molecule or oligonucleotide having a length of 15 nucleotides. The length of the nucleic acid molecule is preferably 15-10000 nucleotides, preferably 15-5000, more preferably 15-2000 nucleotides. The oligonucleotide preferably has a length that can be synthesized in vitro. Typically, the length of such oligonucleotides is 15-60 nucleotides, more preferably 15-40 nucleotides, more preferably 15-25 nucleotides. In a preferred embodiment, the oligonucleotide is single-stranded. The oligonucleotide may sometimes be less than 15 nucleotides. The oligonucleotide may have an artificial backbone. Such an artificial backbone includes, but is not limited to, locked nucleic acid (LNA); 2'-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE); 2'-O-methoxyethyl (MOE); 2'-O-methyl (OMe); morpholino; peptide nucleic acid (PNA); phosphorothioate (PS) or a combination thereof.

[0086] Cytokines are a broad class of proteins that are important in cell signaling. Their release has an effect on the behavior of the cells around them. It can be said that cytokines participate in autocrine signaling, paracrine signaling, and endocrine signaling as immunomodulators. Cytokines can include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.

[0087] Enzymes are protein catalysts. Enzymes speed up chemical reactions. The molecules on which enzymes can act are called substrates, and the enzymes convert substrates into different molecules called products. Almost all metabolic processes in cells require enzyme catalysis in order to occur at a fast enough rate to sustain life. Various enzymes are the subject of so-called enzyme replacement therapy. Replacement therapy aims to replace insufficient or deficient enzymes in the body. Today, this is done by giving patients an intravenous (IV) infusion of a solution containing the enzyme or by subcutaneous injection of an enzyme depot that shields the natural immune system. Replacement therapy can be used for certain lysosomal storage diseases: Gaucher's disease, Fabry disease, MPS I, MPS II (Hunter syndrome), MPS VI and Pompe disease. Adenosine deaminase and proline peptidase are other non-limiting examples of enzyme replacement therapy. Using the particles of the present invention, the enzyme can now be administered orally.

[0088] Many cells can accept molecules attached to cell receptors. Such molecules can be steroids and other small molecules. Typically, such receptors are receptors for protein ligands above 10 kDa. The protein molecule is typically a signaling protein. Such a signaling protein can be a growth factor such as, but not limited to, EGF, a WNT protein such as, but not limited to, WNT3, a bone morphogenetic protein. The protein ligand can be soluble or can be made soluble. For example, for cell membrane-associated proteins, by replacing the transmembrane region and the cytoplasmic region (if any) with an Fc tail.

[0089] Active ingredient can be the factor of coagulation system. The coagulation system (coagulation) involves two components, cells (platelets) and proteins (coagulation factors). Coagulation is caused by a cascade reaction of proteins that interact with coagulation and stimulate coagulation. This is called coagulation cascade. It includes tissue factor pathway (so-called extrinsic pathway), contact activation pathway (intrinsic pathway) and common pathway. A variety of factors have been confirmed. They are called by different names. Usually, they are simply referred to by numbers, i.e., factor I to factor XIII. Other factors are, for example, von Willebrand factor, fibronectin, antithrombin III, protein C, protein S, protein Z, plasminogen, etc.

[0090] The active ingredient may be a fusion protein. A fusion protein may be prepared for solubilization of the extracellular portion of a membrane protein. It may also be a fusion for other reasons.

[0091] In a preferred embodiment, the active ingredient is an antibody. Antibodies are very useful in many cases. One of the disadvantages of antibodies is that they must be administered usually within a certain time period of the day, and must be administered by injection needles in the form of intravenous injections, drip solutions or intramuscular injections. One of the advantages of embodiments of the present invention is that larger proteins such as antibodies (wherein IgG generally has an average size of 120-180kDa, depending on additives such as toxins, labels and other binding units, etc.) can now be effectively absorbed in a substantially unmodified form when administered orally. The antibody can be any isotype. It is preferably IgG, and IgA, IgM or a combination thereof. It can be a monoclonal antibody. It can be a bispecific antibody. Various other forms have also been produced today, such as trispecific or more specific antibodies.

[0092] In a preferred embodiment, the method includes providing two or more active ingredients. In this embodiment, it is preferred that the active ingredient comprises two or more different antibodies. The antibodies can be directed against different epitopes on a target molecule and the same target molecule. Typically, they are directed against different epitopes on different target molecules. Preferably one or more anti-cancer antibodies. Other preferred antibodies are antibodies against pathogens in vaccines, or antibodies against one or more proinflammatory cytokines in, for example, autoimmune diseases, or antibodies in the treatment of urticaria, allergies, asthma and allergic asthma.

[0093] In another preferred embodiment, the active ingredient is an antigen-binding portion of an antibody, or a derivative or analog thereof. Suitable portions are FAB fragments, single-chain Fv fragments and other forms. Nanobodies and monoclonal antibodies can be used, and camelid antibodies, such as single heavy chain antibodies, can be used. Binding peptides can be used. Suitable peptides are, for example, so-called bicyclic peptides, which are essentially conformationally restricted peptides. Derivatives and analogs of antibody portions are peptides having the same general structure (e.g., immunoglobulin folding) but having different amino acid sequences. Analogs are proteins that have different structures from antibodies but have similar binding properties, such as soluble T cell receptors.

[0094] Antibodies or parts, derivatives and / or analogs thereof can also be drug conjugates. Non-limiting examples of such drug conjugates are antibody drug conjugates (ADCs), nanobody drug conjugates (NDCs), single-chain Fv drug conjugates.

[0095] In a preferred embodiment, the active ingredient is an interferon, erythropoietin, an antibody, factor VIII, factor IX, von Willebrand factor, tumor necrosis factor (TNF), growth hormone (GH), etanercept or proline peptidase. In a particularly preferred embodiment, the antibody is omalizumab.

[0096] The present invention also provides a particle assembly having an average diameter of 50 nm to 20 μm obtainable by the method of the present invention.

[0097] Also provided is a particle assembly having an average diameter of 50 nm to 20 μm, comprising uniformly distributed:

[0098] - a protein with a mass of 10 kilodaltons or more, a nucleic acid molecule consisting of 15 or more nucleotides, or a combination thereof, as an active ingredient, in an amount of 0.02% to 25% of the dry weight of the granules;

[0099] - mono- or disaccharides in an amount of 2% to 25% by dry weight of the granules;

[0100] - one or more other compounds in an amount of 0-10%, preferably 0-8%, preferably 0-6%, preferably 0-4%, preferably 0-2%, preferably 0.0-1.7% by dry weight of the granules;

[0101] - Chitosan in an amount of at least 48.3%, preferably at least 50%, by dry weight of the granules.

[0102] The active ingredient, mono- or disaccharide, one or more other compounds, chitosan are the same as described for the method of preparing the granules. In a preferred embodiment, the active ingredient of the granules is an antibody, preferably the antibody omalizumab.

[0103] In the granule assembly described herein, the active ingredient is preferably present in an amount of 0.02%-25% of the dry weight of the granule. Preferably, the active ingredient is present in an amount of 0.2% to 20% of the dry weight of the granule, preferably 0.4% to 15% of the dry weight of the granule, preferably 1.0% to 10% of the dry weight of the granule. When more than one active ingredient is present, the range applies to the addition of all active ingredients. The monosaccharide or disaccharide is preferably present in an amount of 2%-25% of the dry weight of the granule, preferably 1%-15%, preferably 1%-12%, preferably 2%-10% of the dry weight of the granule, more preferably 3%-8%, preferably 4%-6%. The preferred lower limit of the monosaccharide or disaccharide within these ranges is 2%. One or more other compounds are preferably present in an amount of 0-12%, preferably 0-10% of the dry weight of the granule; preferably, one or more other compounds are present in an amount of 0-5%, preferably 0.1%-5%, preferably 0.5%-5%, preferably 2%-4% of the dry weight of the granule. The one or more other compounds are preferably present in an amount of 0.0-1.7% of the dry weight of the particles; preferably 0.1%-1.7%; more preferably 0.2%-1%; more preferably 0.4%-0.8%. Chitosan is preferably present in an amount of at least 48.3% of the dry weight of the particles, preferably at least 50%, more preferably at least 60%. In one embodiment, chitosan is present in an amount of at least 70% of the dry weight of the particles. In the case where the weight of the listed particle components in the assembly described herein does not total 100%, the remaining weight is generally composed of one or more suitable pharmaceutical excipients. Typically, in the assembly, the weight of chitosan is selected so that the other ingredients together with the weight of chitosan constitute 100% of the dry weight of the particles. In other words, the percentage of the compound is selected, and the remainder is composed of chitosan to reach 100%.

[0104] The moisture content of the particle assembly as described herein may be 0-10% of the total weight of the particles; preferably 1%-10%, preferably 1%-8%, more preferably 1%-6%, more preferably 1%-4% of the total weight of the particles.

[0105] The particles in the assembly generally have a uniform composition. The drug may include two or more different particle assemblies, each of which has a uniform composition different from the composition of other particle assemblies in the drug. Preferably, but not necessarily, the one or more other assemblies are also assemblies of the present invention. The embodiment of the multi-assembly can be used for the administration of two or more active ingredients, especially when two or more active ingredients require different release curves. In the case where the drug has two or more different particle assemblies, at least one of the assemblies is composed of the following particles: having a uniform distribution of the ingredients specified in the claims and containing active ingredients (proteins with a mass of more than 10 kilodaltons, nucleic acid molecules consisting of more than 15 nucleotides) and chitosan. In a preferred embodiment, the two or more different particle assemblies are each composed of the following particles: having a uniform distribution of the ingredients specified in the claims and containing active ingredients (proteins with a molecular weight of more than 10 kilodaltons, nucleic acid molecules consisting of more than 15 nucleotides) and chitosan.

[0106] In a preferred embodiment, the assembly of particles comprises:

[0107] - 0.02%-25%, preferably 0.2%-20%, preferably 0.4%-15%, or preferably 1.0%-10% of one or more active ingredients by dry weight of the granules;

[0108] - 2% to 25%, 2.5% to 15%, 2.5% to 12%, 2.5% to 10%, more preferably 3% to 8%, preferably 4% to 6% sucrose by dry weight of the granules;

[0109] - 0.2% to 1% of polysorbate 20 to polysorbate 80 by dry weight of the granules;

[0110] - 0.1% to 0.3% of the dry weight of the granules of one or more amino acids;

[0111] - at least 48.7%, preferably at least 40%, more preferably at least 50% chitosan by dry weight of the granules.

[0112] In a preferred embodiment, the assembly of particles comprises:

[0113] - 5%-25% omalizumab by dry weight of the particles;

[0114] - 2% to 25% sucrose by dry weight of the granules;

[0115] - 0.2% to 1% of polysorbate 20 to polysorbate 80 by dry weight of the granules;

[0116] - 0.1% to 0.3% of the dry weight of the granules of one or more amino acids;

[0117] - at least 48.7%, preferably 50%, more preferably at least 60% chitosan by dry weight of the granules.

[0118] The weight of water in the particle assembly can be as described above, for example but not limited to 1%-10% of the total weight of the particles, preferably about 5%-10%.

[0119] The particles and particle assemblies produced by the method of the present invention and the pharmaceutical composition comprising the particles are all so-called solid particles, which have a solid interior and preferably have a water (moisture) content of less than 20%, preferably less than 10%. Based on the total weight of the particles, the moisture content is usually 1%-10%. The various components of the particles containing the active ingredient are usually evenly distributed in the particles. The particles or particle assemblies can of course be coated or incorporated into other materials.

[0120] The present invention further provides a sustained release composition comprising the particle assembly of the present invention. The sustained release composition can release the encapsulated active ingredient when present in the bloodstream.

[0121] The present invention further provides a method for delivering a protein of 10 kDa or more or a nucleic acid molecule consisting of 15 or more nucleotides as an active ingredient to the blood of a subject in need, preferably in a therapeutically relevant amount, the method comprising: preparing a pharmaceutical composition comprising the protein or nucleic acid molecule according to the method of the present invention, and orally administering the pharmaceutical composition to the subject. Also provided is a pharmaceutical preparation in an oral dosage form, preferably a solid oral dosage form, which includes the particle assembly of the present invention for the treatment of a disease. The solid dosage form is preferably a tablet, a hard or soft capsule, a caplet, a lozenge, a pill, a mini-tablet, a pellet or a powder. Non-limiting examples of non-solid dosage forms are gels, capsules containing fluids or gels, etc.

[0122] Also provided is a pharmaceutical composition comprising the particle assembly described herein, which is used to deliver a protein of 10 kDa or more or a nucleic acid molecule consisting of 15 or more nucleotides as an active ingredient to the blood of a subject in need thereof, preferably in a therapeutically relevant amount, comprising preparing a solid oral dosage form comprising such a particle assembly, wherein the particle assembly contains the protein of 10 kDa or more or the nucleic acid molecule consisting of 15 or more nucleotides as the active ingredient described herein, wherein the oral dosage form is orally administered to the subject.

[0123] Also provided is a pharmaceutical composition comprising the particle assembly described herein, which is used to deliver a protein of 10 kDa or more or a nucleic acid molecule consisting of 15 or more nucleotides as an active ingredient to the blood of a subject in need thereof, preferably in a therapeutically relevant amount, comprising preparing a solid oral dosage form comprising such a particle assembly, wherein the particle assembly contains the protein of 10 kDa or more or the nucleic acid molecule consisting of 15 or more nucleotides as the active ingredient described herein, wherein the oral dosage form is orally administered to the subject.

[0124] Also provided is a pharmaceutical composition in an oral dosage form comprising the particles or particle assemblies described herein, comprising a protein of 10 kDa or more or a nucleic acid molecule consisting of 15 or more nucleotides as an active ingredient, preferably in a therapeutically relevant amount, for systemic treatment of a disease. The disease is preferably cancer, an immune disease or an infection. Preferably, it is an infection of a microorganism such as a virus, mycobacterium or a parasite.

[0125] The present invention also provides the use of particles or particle aggregates containing a protein of 10 kDa or more or a nucleic acid molecule consisting of 15 or more nucleotides as an active ingredient in the manufacture of a drug for oral delivery of the active ingredient. The present invention also provides the use of particles or particle aggregates containing a protein of 10 kDa or more or a nucleic acid molecule consisting of 15 or more nucleotides as an active ingredient in the manufacture of a drug in an oral dosage form.

[0126] The particles of the present invention are preferably packaged into a system suitable for oral delivery of drugs to the intestine. Such methods include, but are not limited to, enteric coatings of tablets or pills, extended release formulations and capsules that are resistant to acidic environments. After protection from the low pH of the stomach, the rest of the digestive tract contains systems such as absorptive enterocytes in microvilli. For absorption of the particles, it is possible to target active transport mechanisms on the intestinal wall. The particles of the present invention promote the release of biomolecules from the particles into the bloodstream. Without being bound by theory, it is believed that the particles migrate at least partially intact through the intestinal wall.

[0127] The diameter of a nearly spherical particle can be determined by determining the longest axis through the particle and selecting the perpendicular cross section with the largest surface area and selecting the distance of the longest straight line through two points on the edge of that surface.

[0128] Preferred methods of measuring particle diameter are by TEM (Transmission Electron Microscopy) or by SEM (Scanning Electron Microscopy).

[0129] As used herein, the term "moisture" refers to the water in the granules. This water is usually not present in free form, but is combined with other dry materials in the granules.

[0130] For purposes of clarity and concise description, features are described herein as part of the same or different embodiments, but it is to be understood that the scope of the invention may include embodiments having a combination of all or part of the described features. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 . Schematic diagram of an example of a supercritical setup.

[0132] Figure 2 .Chitosan particles. Chitosan contains 1% IFN.

[0133] Figure 3 . Release of IFN from chitosan measured by HPLC.

[0134] Figure 4 .Stability of interferon chitosan particles after 6 and 12 months at room temperature.

[0135] Figure 5 .SDS-PAGE showed that omalizumab remained remarkably intact.

[0136] Figure 6 .Scanning electron microscopy showed spherical particles.

[0137] Figure 7 . Serum omalizumab levels in rats after intraduodenal administration.

[0138] Figure 8 .Overlay of HPLC chromatograms of reference omalizumab and released omalizumab.

[0139] Fig. 9 .CD Analysis

[0140] Fig.10 . Melting curve analysis

[0141] Fig.11 .N-glycan % for batch analysis

[0142] Fig.12 .Continuous sedimentation coefficient distribution of sample 1 (left) and sample 2 (right)

[0143] Fig.13 SPR interaction analysis between injected Xolair IgG (t=0) and amine-coupled IgE surface in Lane 5 (containing 2993 RU of protein)

[0144] Fig.14 SPR interaction analysis between injected BP001 DS IgG sample (t=0) and amine-coupled IgE surface in lane 5 (containing 2993 RU of protein)

[0145] Fig.15 SPR interaction between injected omalizumab (t=6 months) and amine-coupled IgE surface in lane 6 (containing 2494 RU of protein)

[0146] Fig.16 . SPR interaction between injected BP001 "DPinPBS" sample and amine-coupled IgE surface in lane 6 (containing 2494 RU of protein).

[0147] Fig.17 : Circular dichroism spectroscopy analysis, combined spectrum of sample 1 (4485A) and sample 2 (4485B)

[0148] Fig.18 : Melting curve analysis. Combined spectra of sample 1 (4485A) and sample 2 (4485B).

[0149] Fig.19 : Sedimentation velocity analysis. Superposition of continuous sedimentation coefficient distributions for sample 1 (4485A) and sample 2 (4485B).

[0150] Fig. 20 : Kinetic titration experiments measuring the binding of sample 1 and sample 2 to fixed human IgE. In each replicate experiment, 4 (increasing) IgG concentrates were injected in a single cycle. Replicates of binding experiments are shown in each panel. The black line overlap indicates that the data are overall fitted to a 1:1 interaction model.

[0151] Fig.21 :In Fig. 20 The binding data of were fitted to the kinetic and affinity parameters derived from a 1:1 binding model. The lines represent sample 1 (orange), sample 2 (blue), and Reference (red).

[0152] Fig. 22 : Serum levels of omalizumab in minipigs after oral and subcutaneous administration. Example

[0153] Example 1

[0154] After protection from the low pH of the stomach by means of, for example, enteric coating of the granules, tablets with said granules passing through the stomach, or capsules containing these granules; the rest of the digestive tract contains systems such as absorptive enterocytes in microvilli, which for selective molecules, active transport mechanisms on the intestinal wall, systems may be targeted for absorption of the granules and subsequently release the biologic present in the granules to release it into the blood stream.

[0155] The particles of the present invention may have the following properties: Almost no protein is released in the first part of the intestine (pH 8). The particles can be transported through the intestinal wall. Once in the blood circulation, the active ingredient is released (pH 7.4). Preferably, the release profile of the active ingredient in the blood is continuous. This means that the active ingredient is released over a prolonged period of time.

[0156] To date, oral delivery of biologically active large molecules (biologics) has been a challenge and has only achieved limited success for small biologics such as insulin with a molecular size less than 6 kDa, which achieved only low bioavailability.

[0157] Table 1 shows non-limiting examples of macromolecules that may be advantageous for oral formulation and their kDa.

[0158] Table 1.

[0159]

[0160] Biologicals in the range of 10-500 kDa are currently administered only by intravenous or subcutaneous injection, whereas the oral route is highly preferred for chronic therapy.

[0161] The bioavailability of peptides and proteins from oral formulations is low (<10%) and penetration enhancers have been used with mixed results.

[0162] Materials and methods

[0163] The technology used in this application for encapsulating complex macromolecules is based on supercritical carbon dioxide technology and is an alternative method for producing solid dosage forms of biologics.

[0164] The technology is based on the premise that, at elevated temperatures and pressures (30°C / 73 Bar), CO2 becomes supercritical, i.e. “in a supercritical state.” Under these conditions, CO2 combines the high diffusivity of a gas with the solvating power of a liquid.

[0165] Specialized, dedicated equipment used in this technique is capable of safely withstanding high pressures and of varying several parameters while spraying the aqueous composition into a pressure chamber in which the CO2 is in a supercritical state.

[0166] Figure 1

[0167] By this method, solid particles can be obtained by spraying an aqueous composition comprising protein and excipients through a nozzle. Subsequently, the scCO2 effectively extracts the solvent and a completely solvent-free powder with a narrow diameter distribution is obtained. The spent CO2 contains a water fraction.

[0168] The diameters of these particles can range from nanometers (nm) to micrometers (μm).

[0169] The relatively mild conditions of the antisolvent method allow for the production of dried protein particle preparations with high retention of biological activity.

[0170] Powder properties can be adjusted by controlling various parameters (pressure, temperature, nozzle size, solution flow rate, CO2 flow rate, protein concentration, and the nature and concentration of excipients).

[0171] Table 2 Adjustable equipment settings for preparing particles

[0172] CO2 Pressure 100–180 Bar CO2 Temperature 40–50℃ CO2 flow rate 150–350 kg / hr Solution flow rate 2-8ml / min

[0173] Table 3 Variables in the composition of the fluid to be sprayed

[0174] Chitosan, deacetylation degree 60%-90%; viscosity 20-500CPS 1％–10％ Amino Acids 0.001％–0.005％ sucrose 0.01％–0.25％ Polysorbate PS 20-80 0.001％–0.005％ Sorbitan Stearate 0.001％–0.005％ Bioactive molecules 0.01％–0.5％

[0175] Before spraying, the chitosan was dissolved in water acidified with acetic acid (pH 3). The protein was subsequently dissolved in this solution just before spraying.

[0176] The dried powder encapsulates were trapped on a filter at the bottom of the container and collected manually at the end of the experiment. The dried powder was stored in a refrigerator (2-8°C) until further characterization.

[0177] Protein release assay

[0178] Powder (20 mg for BCA, or 200 mg for HPLC test) is weighed into a test tube. A certain volume (1 ml for BCA test, or 10 ml for HPLC test) of release medium is added to each test tube. The release medium is PBS pH 7.4 (it is a buffer system for simulating blood pH) or phosphate buffer pH 8 (it is a buffer system for simulating intestinal pH).

[0179] The tubes with the particle suspension were kept at 37°C on a roller bank. Sampling was performed at 0.5; 1; 1.5; 2; 4; 7; 17 and 24 hours (unless otherwise stated). At each sample point, 50% of the release medium was removed from the tube (followed by brief centrifugation to pellet the particles) and 50% of fresh medium was added to the suspension for the continued release test.

[0180] Determine protein levels using the QuantiPro BCA assay kit or HPLC.

[0181] result

[0182] Cytokine interferon-α-2a (IFN)

[0183] A study of a commercially available biosimilar interferon alpha-2a (IFN) encapsulated in particles.

[0184] The chitosan aqueous solution was sprayed into supercritical CO2. The chitosan was dissolved in water acidified with acetic acid (pH 3). Immediately before spraying, the IFN was dissolved.

[0185] Composition of spray fluid

[0186] Chitosan, degree of deacetylation 77%; 3％ Acetic acid 0.5％ sucrose 0.1 Polysorbate PS 20 0.001 Bioactive molecules: IFN 0.2％

[0187] Equipment setup for pellet preparation

[0188]

[0189]

[0190] The material was characterized for particle size (SEM; DLS), IFN loading (HPLC) and IFN release. Structural integrity was tested using SDS-PAGE.

[0191] Spray drying in scCO2

[0192] The spray experiments were performed on a scale of 1 to 2 grams of solids. The polymer to protein (IFN) ratio was 99% to 1%. The solids were dissolved in 60 grams of water.

[0193] In these small-scale spray experiments, powder yields (amount of solids recovered) were typically between 40% and 80%.

[0194] Powder Characterization (SEM) Figure 2

[0195] Scanning electron microscopy (SEM) characterization of the powders showed that the chitosan-based powders produced spherical particles with diameters varying between 1 and 10 micrometers.

[0196] Protein loaded in particles.

[0197] For chitosan based particles, it has been demonstrated that it is possible to dissolve the chitosan by incubating the particles in acetate at pH 4 and obtain a solution injectable into HPLC for determination of IFN in the particles.

[0198] Two different batches of 1% IFN in chitosan particles were dissolved in triplicate. The results are shown in Table 4.

[0199]

[0200] Protein release assay

[0201] Protein release experiments were performed using the BCA protein assay. The particles produced showed up to 80% release of IFN within 24 hours at pH 7.4 using the BCA assay. Figure 3

[0202] The stability of IFN chitosan particles over a one-year period was demonstrated. After one year of storage, the particles maintained a consistent release profile. Figure 4

[0203] in conclusion

[0204] Particles with encapsulated interferon α-a can be produced using scCO2 technology with good reproducibility in terms of release profiles and loading efficiency.

[0205] Monoclonal antibody Omalizumab

[0206] Study on Omalizumab-encapsulated Particles

[0207] The chitosan aqueous solution was sprayed into supercritical CO2. The chitosan was dissolved in water acidified with acetic acid (pH 3). Immediately before spraying, the antibody (omalizumab) was dissolved.

[0208] Table 5 Composition of spray fluid

[0209] Chitosan, degree of deacetylation 77%; 8％ Amino Acids 0.015％ sucrose 1％ Polysorbate PS 20 0.01％ Bioactive molecule: Omalizumab 0.9％

[0210] Table 6 Equipment settings for preparing particles

[0211] CO2 Pressure 150Bar CO2 Temperature 50℃ CO2 flow rate 300kg / hr Solution flow rate 2.5ml / min

[0212] The materials were characterized for particle size (SEM; DLS), loading of omalizumab (HPLC) and release of omalizumab. Structural integrity was tested using ELISA and SDS-PAGE.

[0213] Loading of omalizumab

[0214] 5 mg of particles were dissolved in 1.5 ml of acetate at pH 4 by rotation in an oven at 37°C overnight or longer. After dissolution, the solution was filtered through a 0.45 μm filter to remove any sub visible particles and then analyzed by HPLC.

[0215] Release of omalizumab

[0216] 10 mg of the particles were suspended in PBS (to simulate release in the blood) or in phosphate buffer at pH 8 to simulate release in the intestine. The suspension was kept rotating at 37° C. At given time intervals, the clear buffer was removed from the top of the particles and filtered through 0.45 μm before HPLC analysis.

[0217] Elisa

[0218] ELISA was performed using a commercially available kit. Based on the concentration determined by HPLC, samples were diluted to one-fourth the highest calibrator concentration.

[0219] SDS-PAGE

[0220] Approximately 2 μg of protein was loaded into the loading chamber of a 4-20% Tris HCl gel. The gel was run at 90 V for 5 minutes, and then the voltage was applied for 180 minutes. The total run time was 45 to 60 minutes. The gel was stained with Coomassie Brilliant Blue.

[0221] result

[0222] Particle loading

[0223] In initial experiments the particle loading was set at 16% chitosan and it was shown that in practice an actual loading of 14% could be measured.

[0224] ELISA

[0225] Based on the ELISA results, its biological integrity appeared to be intact after encapsulation and subsequent release.

[0226] SDS-PAGE

[0227] Omalizumab (TM0192) remained remarkably intact through this experiment. Figure 5

[0228] Particle size and SEM

[0229] Particle size was determined using dynamic light scattering (DLS).

[0230] For most of the particles produced, the particle size was approximately 500 nm.

[0231] Figure 6 The SEM shows the particles

[0232] Bioavailability in rats

[0233] To evaluate the bioavailability of encapsulated omalizumab in rats after intraduodenal administration, rats were administered intraduodenal administration.

[0234] 18 rats were divided into three groups. Group 1 was administered 0.2 ml of omalizumab solution (5 mg / ml). Groups 2 and 3 were administered with 200 μl of a suspension (10 mg / ml) of omalizumab particles suspended in a buffer (Group 2) and a buffer with a penetration enhancer (Group 3). At the end of the experiment, the rats were killed and serum was collected. Serum was collected after 1; 4 and 24 hours (in duplicate for each time point). The serum level of omalizumab was determined using an ELISA kit. Figure 7 The results showed that, with the current particle size, the encapsulated protein was absorbed by the intestine and subsequently released in the blood.

[0235] The possible effect of penetration enhancers may be masked by the large basal absorption. No significant effect of penetration enhancers was observed.

[0236] This could be explained by the high bioavailability of the antibodies contained in the chitosan particles. Chitosan itself is known to be a penetration enhancer.

[0237] in conclusion

[0238] Particles with encapsulated omalizumab could be produced using scCO2 technology with good reproducibility in terms of release profiles and loading efficiency.

[0239] Furthermore, based on HPLC and ELISA data, the chemical and biological integrity of the omalizumab molecule appeared to be intact.

[0240] In addition, a preliminary rat study showed that the levels of omalizumab in the blood were detectable in the group that received omalizumab-encapsulated particles, but undetectable in the group that received unencapsulated omalizumab.

[0241] The results obtained in this project demonstrated that scCO2-assisted chitosan encapsulation technology can be used to develop stable formulations of macromolecules to be used in oral dosage forms.

[0242] Example 2

[0243] Evaluation of the BiOraliX' production process on the protein structure and function of omalizumab

[0244] introduce

[0245] Before and after applying the process, a series of tests were performed to evaluate the structure, composition and function of omalizumab, namely:

[0246] -Structure: circular dichroism, N-glycans, peptide maps, intact mass, disulfide bridges

[0247] -Analysis (aggregation / particle size): SV-AUC

[0248] - Functionality: SPR binding kinetics and potency (based on ELISA)

[0249] sample

[0250] 1. The antibody used for this purpose is Omalizumab. The monoclonal antibody is produced in a fermentation process using Canine Hamster ovary cells and purified to obtain a purity of >95%. The drug substance (DS) is kept frozen (-20°C) until further processing.

[0251] ○ Sample 1 is the DS sample as described above

[0252] ○ Sample 2 is the sample after the above DS was exposed to the present invention and further processed according to the following steps:

[0253] ■ Release the obtained particles in PBS buffer and freeze before shipment

[0254] ■Samples are sent to external laboratories for characterization and comparison

[0255] Figure 8 It is the original research type of sample 2 and omalizumab The HPLC scan lines were compared and showed that the identities of the two active ingredients in the samples were identical.

[0256] A summary of the activities (functional, binding and analytical properties) of the base material (represented by Sample 1) and the material represented by Sample 2 is provided in the following table and Figure 9-Figure 16 middle.

[0257] The two samples were analyzed on two separate occasions, but the same reference sample was used on both occasions. Analyses were performed to bridge the data and make comparisons.

[0258]

[0259]

[0260]

[0261] in conclusion

[0262] The data collected for Sample 2 (untreated protein processed into particles and released in PBS) were very similar to the data collected for Sample 1 (untreated protein).

[0263] Analytical, structural and functional tests demonstrated that the molecule is intact and functional.

[0264] Example 3

[0265] Evaluation of the BiOraliX' production process on the protein structure and function of omalizumab

[0266] introduce

[0267] Before and after application of the described process, a series of tests were performed to evaluate the structure, composition and function of omalizumab, namely:

[0268] -Structure: circular dichroism, peptide map, intact mass, disulfide bridges

[0269] -Analysis (aggregation / particle size): SV-AUC

[0270] - Functionality: SPR binding kinetics and potency (based on ELISA)

[0271] Different from Example 2, the results of each test were obtained from the same analytical occasion. This means that the results of the two samples were obtained, for example, in the same analytical run or sequence, and / or analyzed on the same day, by the same technician.

[0272] sample

[0273] The antibody used for this purpose was Omalizumab. This monoclonal antibody was a lyophilized product produced at Halix (lot number B3010640). The Omalizumab drug product was kept frozen (-20°C) until further processing.

[0274] ○ Sample 1 is the omalizumab drug product as described above. This sample comprises:

[0275] Omalizumab (59%)

[0276] ■L-Histidine hydrochloride monohydrate (0.77%)

[0277] ■L-Histidine (0.49%)

[0278] Sucrose (39.8%)

[0279] ■Polysorbate (0.14%)

[0280] ○ Sample 2 is the omalizumab drug product after exposure to the present invention. This sample contains:

[0281] Omalizumab (16%)

[0282] ■Sucrose (10%)

[0283] ■Chitosan (74%)

[0284] PS20 (0.03%)

[0285] ■Histidine (0.3%)

[0286] The particles of Sample 2 were released and frozen in PBS buffer and then shipped to an outside laboratory for characterization and comparison with Sample 1.

[0287] A summary of the activities (functional, binding and analytical properties) of the base material (represented by Sample 1) and the test material (represented by Sample 2) is provided in the following table and Figure 17-Figure 21 middle.

[0288]

[0289]

[0290]

[0291] in conclusion

[0292] The data collected for Sample 2 (untreated protein processed into particles and released in PBS) were very similar to the data collected for Sample 1 (untreated protein).

[0293] Analytical, structural and functional tests demonstrated that the molecule is intact and functional.

[0294] Example 4

[0295] Pharmacokinetics and safety of omalizumab in miniature pigs following oral and subcutaneous administration

[0296] Materials and methods

[0297] Materials used:

[0298]

[0299] Composition of the spray solution:

[0300] WFI contained 1% of a mixture including 10% sucrose, 73.7% chitosan, 0.03% PS20, 0.3% histidine, and 16% omalizumab.

[0301] Preparation of granules

[0302] The particles were prepared by spraying a solution containing the coating material, omalizumab and excipients into supercritical carbon dioxide.

[0303] Supercritical spray conditions:

[0304] Carbon dioxide (CO2) 300kh / h, nozzle 1650, solution 2.5ml / min.

[0305] Obtained particle size distribution: D 10 0.75μm, D 50 1.49 μm, D 90 3.11μm (laser diffraction particle size analysis)

[0306] The loading amount of omalizumab in the particles was determined by HPLC to be 16.4% by weight.

[0307] Preparation of capsules for oral (po) administration

[0308] 156 mg of granules (25 mg of omalizumab) were weighed into each capsule (type 0). Each capsule was further filled with 70 to 80 mg of an excipient mixture consisting of Avicell PH101 and 1% fumed silica.

[0309] The capsules were enteric coated by dip coating in a solution containing Eudragit L100-55. Each capsule was dipped 5 times, which resulted in an average weight gain of about 38 mg per capsule. All capsules were tested for coating quality and proved to be resistant to hydrochloric acid at pH 2 for two hours. The capsules dissolved in phosphate buffer (pH 7.2) between 5 and 65 minutes.

[0310] Preparation of solution for subcutaneous (sc) administration

[0311] Omalizumab solution was dissolved in water for injection (WFI) at a concentration of 50 mg / ml.

[0312] Animal studies

[0313] 18 male miniature pigs aged 4-6 years and weighing between 40 and 55 kg were given 25, 50 or 75 mg of omalizumab by lateral subcutaneous injection or orally using a bolus injector for capsules. For subcutaneous injection, an omalizumab solution dissolved in water for injection (WFI) was used. Oral administration was performed with capsules containing particles. Blood was collected from the jugular vein into standard Falcon tubes with K2-EDTA at 0 (before administration); 1; 3; 5; 24; 48; 120; 240; 480; 720 and 960 hours after administration. Subsequently, the blood was processed into serum by allowing it to coagulate for about 30min-1h. After centrifugation at 3800rpm for 15 minutes, the serum was collected in an Eppendorf tube. The Eppendorf tube was then centrifuged at 8000rpm for 2 minutes to remove residual red blood cells.

[0314] Serum samples were kept frozen (-20°C) until further analysis of omalizumab levels using enzyme-linked immunosorbent assay (ELISA).

[0315] Detection of Omalizumab serum levels using ELISA

[0316] The level of omalizumab in serum samples was determined using the Shikari Q-Oma ELISA kit produced by Matriks Biotek. The kit is a sandwich principle based on the binding of omalizumab present in human plasma or serum to IgE at the bottom of the well. A horseradish peroxidase conjugate was subsequently added, which binds to omalizumab. After washing the wells, the activity of the bound enzyme was detected by adding a chromogen-substrate. The color development is proportional to the amount of omalizumab in the sample or standard.

[0317] Results were determined using a standard curve. The assay was designed to detect omalizumab in minipig serum rather than human serum.

[0318] Quantification was performed using the calibration samples provided with the kit (10–1000 ng / ml). Serum samples were diluted 10-fold before incubation.

[0319] Safety parameters

[0320] Blood and serum samples were collected at baseline t=0 and 24, 48, 120, 240, 480, 720 and 960 hours post-dose for hematology and chemistry assessments.

[0321] The following hematological parameters were measured: white blood cell count, red blood cell count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, reticulocyte count, reticulocyte count, red cell distribution width, neutrophils, lymphocytes, monocytes, eosinophils, basophils, and unstained large cells.

[0322] For serum chemistry, we determined: sodium, potassium, chloride, total protein by protein gel, alkaline phosphatase, cholesterol, phosphorus, aspartate aminotransferase, alanine aminotransferase, albumin, albumin / globulin ratio, blood urea nitrogen, glucose, gamma-glutamyl transpeptidase, calcium, and creatinine.

[0323] result

[0324] Animal Health and Safety:

[0325] No animals showed abnormal behavior or signs of pain or other adverse effects.

[0326] The animals were weighed two weeks before the start of the study and one week after dosing. No clear trend in weight gain or loss was observed, which positively confirmed the health of the animals during the study.

[0327] Omalizumab serum levels

[0328] All types of dosing showed an initial dose-dependent increase in serum omalizumab that was gradually eliminated over time (see Fig. 22 ).

[0329] Compared with subcutaneous administration, oral administration showed higher bioavailability. When injected subcutaneously, the maximum serum level reached was 12,000; 8,000 or 4,000 ng / ml when 25, 50 or 75 mg of omalizumab was administered, respectively. When 25, 50 or 75 mg of omalizumab was administered orally, the maximum serum level reached was 27,000, 11,000 or 5,000 ng / ml, respectively.

[0330] Compared with subcutaneous injection, oral administration has a longer T1 / 2 value. The relative bioavailability of oral administration is 2.3–3.1 times that of subcutaneous injection.

[0331] Compared with the subcutaneous injection group, the Tmax values ​​(i.e., the time required to reach the maximum concentration) of all oral administration groups were significantly prolonged.

[0332] Security:

[0333] All parameters showed no significant changes compared with the pre-drug values.

[0334] in conclusion:

[0335] The study demonstrated that oral administration of omalizumab achieved excellent, dose-dependent, high bioavailability in minipig serum using gastroresistant capsules filled with omalizumab formulated as microparticles. The microparticle formulation was shown to enable transport of omalizumab across the intestinal epithelium into the bloodstream without toxic effects as measured across a range of parameters in blood and serum.

[0336] Apparently, higher bioavailability in humans can be achieved when omalizumab is administered orally using microparticles when compared to the conventional clinical subcutaneous route of administration of omalizumab.

[0337] Biostatistical analysis of the sc group showed similar results with those based on literature on sc dosing in a clinical setting (Rivière et al., J. Bioequiv. Availab 2011, 3; 6) regarding Tmax (96 vs 120 hours) and T. (385 vs 580 hours).

Claims

1. A method for preparing a pharmaceutical composition for oral delivery of a systemic active ingredient, comprising: Prepare an aqueous composition comprising - proteins with a mass of more than 10 kilodaltons, as systemically active ingredients, in an amount of 0.02% to 25% of the dry weight of the composition; - sucrose in an amount of 2% to 25% by dry weight of the composition; - one or more other compounds in an amount of 0-10% by dry weight of the composition, wherein the one or more other compounds include polysorbate 20 to polysorbate 80; - chitosan in an amount of at least 48.3% by dry weight of the composition; spraying the prepared aqueous composition into an antisolvent and / or a supercritical fluid in which the aqueous fraction of the composition is soluble or miscible and the solute of the composition is insoluble, thereby precipitating the solute and producing particles having an average diameter of 50 nanometers (nm) to 20 micrometers (μm); collecting the particles; and preparing a pharmaceutical composition for oral delivery, the pharmaceutical composition comprising the particles containing the systemically active ingredient.

2. The method according to claim 1, wherein: The anti-solvent is supercritical CO2.

3. The method according to claim 2, wherein: The supercritical CO 2 has a temperature of 30-55 degrees Celsius and / or a pressure of at least 73 bar.

4. The method according to any one of claims 1 to 3, wherein: The prepared aqueous composition comprises amino acids in an amount of 0.001% to 0.5% of the final dry weight of the aqueous composition.

5. The method according to any one of claims 1 to 3, wherein: The prepared aqueous composition comprises one or more penetration enhancers, solubilizers; emulsifiers or a combination thereof.

6. The method according to claim 4, wherein: The prepared aqueous composition comprises one or more penetration enhancers, solubilizers; emulsifiers or a combination thereof.

7. The method according to any one of claims 1 to 3 and 6, wherein The active ingredients include cytokines, enzymes, soluble protein ligands of cell receptors, factors of the coagulation system, fusion proteins and / or antibodies.

8. The method according to claim 4, wherein: The active ingredients include cytokines, enzymes, soluble protein ligands of cell receptors, factors of the coagulation system, fusion proteins and / or antibodies.

9. The method according to claim 5, wherein: The active ingredients include cytokines, enzymes, soluble protein ligands of cell receptors, factors of the coagulation system, fusion proteins and / or antibodies.

10. The method according to any one of claims 1 to 3 and 6, wherein The active ingredients include interferon, erythropoietin, antibodies, factor VIII, factor IX, von Willebrand factor, tumor necrosis factor (TNF), growth hormone (GH), etanercept or proline peptidase.

11. The method according to claim 4, wherein: The active ingredients include interferon, erythropoietin, antibodies, factor VIII, factor IX, von Willebrand factor, tumor necrosis factor (TNF), growth hormone (GH), etanercept or proline peptidase.

12. The method according to claim 5, wherein: The active ingredients include interferon, erythropoietin, antibodies, factor VIII, factor IX, von Willebrand factor, tumor necrosis factor (TNF), growth hormone (GH), etanercept or proline peptidase.

13. An assembly of particles having an average diameter of 50 nm to 20 μm obtained by the method according to any one of claims 1 to 12.

14. A particle assembly having an average diameter of 50 nm to 20 μm, comprising uniformly distributed: - a protein having a mass of 10 kilodaltons or more, as an active ingredient, in an amount of 0.02% of the dry weight of the granules -25％； - sucrose in an amount of 2% to 25% by dry weight of the granules; - one or more other compounds in an amount of 0-10% by dry weight of the particles, wherein the one or more other compounds include polysorbate 20 to polysorbate 80; - Chitosan in an amount of at least 48.3% by dry weight of said particles.

15. The particle assembly according to claim 13 or claim 14, wherein: The active ingredients include antibodies.

16. The particle assembly according to claim 15, wherein: The antibody is omalizumab.

17. The particle assembly according to claim 16, comprising - 5% to 25% omalizumab by dry weight of the particles; - 2.5% to 20% sucrose by dry weight of the granules; - 0.025%-0.2% polysorbate 20 to polysorbate 80 by dry weight of the granules; - 2.5% to 10% amino acids by dry weight of the particles; - at least 48.3% chitosan by dry weight of the particles.

18. A pharmaceutical preparation in oral dosage form, comprising the particle assembly according to any one of claims 13 to 17, for treating a disease.

19. The pharmaceutical preparation according to claim 18, wherein The dosage form is a tablet, hard or soft capsule, caplet, lozenge, pill, or powder.

20. The pharmaceutical preparation according to claim 18, wherein The dosage form is a mini-tablet or a mini-pill.

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