RU2702690C2 - Method of producing products containing stabilized active substances and compositions containing thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to production of stabilized exenatide. Method of producing a product containing exenatide involves providing a coating liquid containing exenatide, a saccharide and a water-miscible solvent; providing particles containing one or more water-soluble gelling compounds. Particles are fluidised in the working chamber of the device such that the particles move in the chamber upward along the screw trajectory. Thereafter, the coating liquid is sprayed onto the particles to obtain coated particles and allowed to dry the coated particles. Also disclosed is a product obtained using said method and versions of using said method and product.

EFFECT: invention enables to obtain a convenient solid form for peroral delivery of exenatide and optimal suction in the oral cavity by destroying microcapsules and converting them into a microgel.

9 cl, 15 dwg, 5 ex

Description

Technical field

The present invention in a broad sense relates to a method for producing products containing active components, and in particular biological materials, where the active components are stabilized. The present invention further relates to compositions containing products, and in particular, to compositions containing therapeutic biological materials.

State of the art

Many biological materials, such as proteins and whole cells, which are useful in the treatment and prevention of diseases in humans and animals or as food additives, have a limited shelf life. The limited shelf life is believed to be the result of protein instability at storage temperature. While the shelf life of some proteins and cell cultures can be extended by storing them at low temperatures (i.e., from 4 ° C to 8 ° C), shelf life of less than eighteen months is common.

Biologically active proteins, as a rule, are folded into a complex three-dimensional structure, which is unique for each protein. Proteins, as a rule, have three levels of organization; with a primary structure consisting of a linear chain of covalently linked amino acid residues (peptide chain); a secondary structure in which the peptide chain folds into regular structures (such as α-helices and β-folded sheets); and a tertiary structure in which the folded chain further collapses onto itself to form a compact structure. In addition, some proteins consist of more than one polypeptide chain, tightly packed to form what is called the Quaternary structure. This tertiary and / or quaternary structure determines the final biological activity of the protein.

The final protein structure can be influenced by a number of environmental factors, for example, temperature, pH, the presence or absence of certain cofactors or metals, the presence of oxygen, enzymes, oxidizing agents or reducing agents, and the presence of water or moisture. If conditions are not optimal, the protein may not form properly or may denature, so that its biological function is lost or at least weakened.

Cells of animals, plants and microorganisms can be considered in the broadest sense complex protein materials, since they contain numerous proteins enclosed in the cell membrane and / or cell wall, and the membrane or internal bend of the wall represents additional proteins on the cell surface. As with proteins, cell viability depends on the environment in which it is located; for example, temperature, pH, the presence or absence of certain cofactors or metals, the presence or absence of certain nutrients, metabolic waste products, oxygen, enzymes, oxidizing agents or reducing agents, and the presence of water or the degree of humidity can act individually or in combination, affecting viability.

There are a number of protein stabilization techniques known in the art, some of which are briefly discussed below.

To obtain proteins used in vaccines and the like, freeze-drying (lyophilization) is usually used. Freeze-drying traditionally involves freezing a solution of a biological protein and removing ice crystals from there by converting them to water vapor under vacuum (sublimation). This method often results in damage to the native protein structure.

To help increase the stability of the biological protein obtained by freeze-drying, additives such as buffers or stabilizers can be used in the product. However, during freeze-drying, when the temperature of the solution slowly drops to minus 20 ° C over a period of several days, the additives may harden at various freezing points. As a result, the final product may be a finely divided puff like cake, actually consisting of different layers, each of which represents a separate component. In fact, the additives added to protect the biological protein can be physically and chemically separated from it, which makes them useless as protective agents.

An alternative technique commonly used in the food and dairy industries for the manufacture of dried fruit concentrates and powdered milk, for example, is spray drying using heat. This method involves spraying the solution in the form of fine mist down from the top of the tower with spray irrigation against the upward flow of hot air. Hot air removes water from the droplets before they reach the bottom of the tower. Spray drying is usually carried out at an inlet air temperature exceeding 190 ° C, and the product temperature can significantly exceed 60 ° C. In this working environment, most of the biological proteins or cells, such as bacterial cells, denature.

Another method for producing proteins known in the art is drying in a supercritical fluid. In this method, biological agents, such as peptides, proteins, and nucleic acids, are maintained in aqueous solution until particles are formed. The aqueous solvent is removed during particle formation using solvents capable of controlled hydrogen bonding, such as ethanol and isopropanol.

Fluidized bed spray drying is a modified heat spray spray drying technology. The method is usually used in the pharmaceutical and chemical industries to obtain tablets by granulation and / or for drying thermostable materials. The method includes spraying a solution in the form of a fine mist containing active substances down from the top of the spray head in the direction of the mass of dry fillers. At the same time, an upward flow of hot air passes through the mass of fillers with the formation of a fluidized bed. Hot air removes water from the fluidized wetted solids at the bottom of the fluidized bed.

Fluidized bed spray drying technology may be applicable to pharmaceutical proteins that are thermostable at approximately 50-60 ° C. However, the native structure of the protein may be disrupted, and, accordingly, the protein may lose all biological activity, or at least some of it.

Additional problems may be associated with the above described fluidized bed spray drying; for example, it is required that the spray nozzles located near the top of the working chamber have a substantial clearance above the surface of the fluidized bed of filler materials so that such materials do not block the spray nozzles; a significant amount of coating material or coating liquid containing the active ingredient (s) may block the filter system in the nozzles, resulting in processing losses; and such a spraying operation from above onto a fluidized bed can only be optimal for granulation purposes, but not spray coating. The method of spray drying in a fluidized bed also, as a rule, provides coarse and uneven surface granules with different shapes and sizes. Contact between the granules results in a grinding effect, which can result in protein denaturation.

Among the techniques that have found commercial application for the production of biological proteins and cells prior to the development of the present invention, the microencapsulation technique may be considered the most suitable. As a rule, basic equipment is not required, and the batch size can be in the range of 10 g-20 g, which, therefore, makes it suitable for the production of biological proteins, which may not be in excess. This method uses organic solvents to solubilize the biological protein, which is then encapsulated in polymer microspheres using a method using either a water-in-oil-in-water emulsion (w / o / w) or a solid-in-oil-in-water emulsion (s / o / w). Protein is captured in solid microspheres after water is removed by simple filtration and the solvent is evaporated.

Microencapsulation technology was used to make carbon or self-adhesive paper in the paper industry and, at least in Japan, food products such as artificial caviar and decorative products made using gelatin microcapsules to trap fish or meat flavors.

Although microencapsulation may be considered a favorable way to obtain biological proteins and whole cells for storage and use in the future, technology in the pharmaceutical and biotechnological industry is still under development. The technology has obvious difficulties in that the proteins are likely to denature under the influence of the solvents used and the necessary emulsification / homogenization methods. In addition, the quality of the product obtained according to this method may be considered undesirable due to the fact that trace amounts of solvent remain in the core of the microcapsules, which may hinder the commercialization of the product obtained using this technology.

The inventor of the present invention has developed an alternative method based on spray drying using heat and microencapsulation technology, in which active components, such as proteins, can be stabilized over long periods.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method for producing a product containing at least one active component, the method comprising:

(i) providing a coating fluid containing at least one active ingredient, a saccharide, and a water miscible solvent;

(ii) providing particles containing one or more water-soluble gelling agents;

(iii) fluidization of particles in the working chamber of the device so that the particles move in the chamber upward along a helical path;

(iv) spraying the coating liquid onto the particles to form coated particles;

(v) allowing coated particles to dry.

At least one active component may be unstable.

At least one active component may be unstable in the presence of light, heat, air and / or moisture.

At least one active component may be a biological material, for example, a protein, peptide, living cells, or a microorganism.

At least one active component may be unstable biological material.

At least one active component may be a biological material that is sensitive to light, heat, air and / or moisture.

At least one active component may be a cytokine.

At least one active component can be selected from the group consisting of interleukin, interferon, EPO, exenatide, G-CSF, human growth hormone and thymosin. In one embodiment, the at least one active component is exenatide.

At least one active component can be selected from the group consisting of an anticancer agent, an anti-inflammatory agent, a hemolytic agent, an antidiabetic agent, an antiallergic agent, and an antifungal agent.

The working chamber during the implementation of the method may have a temperature of from about 30 ° C to 45 ° C or at a temperature of from about 35 ° C to 40 ° C.

The coating fluid may be in the form of micro-droplets before contact with the particles.

The coating fluid can be solidified on the particles within one second after entering the working chamber.

Before step (iii), the particles can be fluidized in the working chamber and dried by applying heat to them.

The particles can be dried by heating to a temperature of from about 30 ° C to 80 ° C.

In step (iii), the particles can rotate.

Step (iii) can be carried out by introducing into the working chamber a moving upward rotating gas stream, which thus causes particles to move upward along a helical path.

Step (iii) can be carried out by drawing gas through a guide located on the bottom or adjacent to the bottom of the working chamber, where after leaving the guide the gas moves up the rotation path, which thus causes particles to move upward along the helical path.

The guide may have an inlet through which the gas is drawn in and an outlet through which the gas is released, wherein the guide comprises a gas guiding member acting on the gas passing between the inlet and the outlet, so that the gas released through the outlet moved up the rotation path.

The guide may comprise at least two functional units adapted to rotate about a common axis, where the first functional unit has an inlet through which gas can be drawn in, and where the second functional unit contains one or more gas-guiding elements that are designed outside the surface of the second functional link, where the second functional link is located above the first functional link and where the applied gas drawn through the inlet acts on one or not how many gas-guiding elements, causing the rotation of the second functional link so that the gas released from the guide moves upward along the rotation path.

The gas may be air or an inert gas, for example nitrogen.

The gas entering the working chamber may have a temperature of from about 30 ° C to 45 ° C or from about 35 ° C to 40 ° C.

Before starting the execution of step (iii) or step (iv), the air in the working chamber can be replaced with an inert gas, for example nitrogen.

Step (iv) may include spraying the coating liquid from the bottom of the working chamber in the direction of the particles.

Step (iv) may include spraying the coating fluid upward so that the coating fluid moves along the column formed by the particle stream.

Step (iv) may include spraying the coating liquid upward in the direction of the particles at an angle of from about 10 ° to 70 °, or at an angle of from about 20 ° to 60 °, or at an angle of from about 30 ° to 50 °, or an angle of from about 35 ° to 45 °, or at an angle of about 40 °.

Step (iv) may include spraying the coating fluid from several spray outlets in the working chamber.

In step (iv), the coating liquid may be at a temperature of from about 0 ° C to 50 ° C, or at a temperature of from about 10 ° C to 50 ° C, or at a temperature of from about 15 ° C to 45 ° C, or at at a temperature of from about 30 ° C to 45 ° C, or at a temperature of from about 35 ° C to 40 ° C. In one embodiment, the coating fluid may be at room temperature, for example, about 20 ° C.

Step (v) may include thermal drying of the coated particles.

Steps (iv) and (v) can be carried out under equilibrium conditions.

The method may further include one or more additional coating steps, wherein the particles are further coated with a coating selected from the group consisting of an enteric coating, a film coating, a water-repellent coating, and a taste-correcting coating.

The coating liquid may further contain additional components selected from the group consisting of amino acids such as lysine, arginine, cysteine and leucine, proteins such as human serum albumin, egg albumin, chelating agents such as EDTA and EDTA sodium, buffers such such as sodium phosphate buffer, citric acid / citrate buffer, Tris buffer, preservatives such as hydroxybenzoic acid and its derivatives, stabilizers, antioxidants such as vitamin E, ascorbic acid, and lubricants such as yes, silicon and silicates.

The water miscible solvent may be a glycol, for example glycerol or propylene glycol.

The saccharide may be a polymer of sugar.

The saccharide can be selected from the group consisting of fructose, glucose, invert sugar, lactitol, lactose, maltitol, maltose, mannitol, sorbitol, sucrose, trehalose, mannitol, and combinations thereof.

One or more water-soluble gel-forming compounds can be selected from the group consisting of acrylate and its derivatives, albumin, alginates, carbomers, carrageenan, cellulose and its derivatives, dextran, dextrose, dextrin, gelatin, polyvinylpyrrolidone, starch, pregelatinized starch and their combination.

The method can be carried out in a reversed fluidized bed reactor.

The working chamber of the device may be closed.

The working chamber of the device may be a sterile environment.

The working chamber of the device may be an inert environment.

Particles may be microparticles.

In a second aspect, the present invention provides a product containing at least one active ingredient, in all cases obtained by the method according to the first aspect.

At least one active component can be selected from the group consisting of interleukin, interferon, EPO, exenatide, G-CSF, human growth hormone and thymosin. In one embodiment, the at least one active component is exenatide.

At least one active component can be selected from the group consisting of an anticancer agent, an anti-inflammatory agent, a hemolytic agent, an antidiabetic agent, an antiallergic agent, and an antifungal agent.

In a third aspect, the present invention provides the use of a product according to a second aspect for delivering at least one active component to a subject. In one embodiment, the at least one active component is exenatide.

The subject may be a human or an animal.

The product may be suitable for injection, oral or nasal administration, sublingual administration, local administration, or vaginal or rectal administration.

The product may be in a dosage form selected from the group consisting of a composition intended for injection, tablets for sublingual use, tablets for oral use, sustained release tablets for sublingual use, microcapsules intended for filling capsules, microcapsules, pre-prepared food mixture pessary, a pre-formed solid dose intended for use in the form of a nasal spray or drops, water drops, eye lotion or drops l and a solution for washing the skin.

The product may be in a dosage form selected from the group consisting of a sustained release tablet or microcapsules.

In a fourth aspect, the present invention provides the use of the method according to the first aspect for stabilizing at least one active component. In one embodiment, the at least one active component is exenatide.

In a fifth aspect, the present invention provides the use of the method according to the first aspect for the preparation of sustained release dosage forms. In one embodiment, the at least one active component is exenatide

For the purposes of the second, third, fourth, and fifth aspects, at least one active component may be an active component, which is defined in the first aspect and which is described herein. In one embodiment, the at least one active component is exenatide

In a sixth aspect, the present invention provides an apparatus for producing a product comprising at least one active component comprising a working chamber, in which one or more outlets for introducing liquid into the working chamber are placed, an inlet located at the bottom of the working chamber, and an outlet thereby allowing the gas to move along the working chamber where the inlet is provided with the guide, so that when the gas is drawn in through the inlet, after exiting the direction The flue gas moves upward along the rotation path. In one embodiment, the at least one active component is exenatide

The guide may be a guide described herein.

In a seventh aspect, the present invention provides a method for producing an exenatide-containing product, the method comprising:

(i) providing a coating fluid containing exenatide, a saccharide and a water miscible solvent;

(ii) providing particles containing one or more water-soluble gelling agents;

(iii) fluidization of particles in the working chamber of the device so that the particles move in the chamber upward along a helical path;

(iv) spraying the coating liquid onto the particles to form coated particles;

(v) allowing coated particles to dry.

In an eighth aspect, the present invention provides a product obtained by the method defined in the seventh aspect.

In a ninth aspect, the present invention provides the use of the product defined in the eighth aspect for delivering exenatide to a subject.

In a tenth aspect, the present invention provides the use of the method defined in the seventh aspect for stabilizing exenatide.

In an eleventh aspect, the present invention provides the use of a method as defined in a seventh aspect for the preparation of sustained release dosage forms.

In a twelfth aspect, the present invention provides an apparatus for producing an exenatide-containing product comprising a working chamber in which one or more outlets for introducing liquid into the working chamber are placed, an inlet located at the bottom of the working chamber, and an outlet, thereby allowing the gas to move along the working chamber, where the inlet is provided with the guide, so that when the gas is drawn in through the inlet, after leaving the guide, the gas moves along ION up the rotation trajectory.

In a thirteenth aspect, the present invention provides a method for treating a metabolic syndrome or diabetes in a subject, the method comprising administering an effective amount of the product defined in the eighth aspect of the subject.

In a fourteenth aspect, the present invention provides a method for treating a metabolic syndrome or diabetes mellitus in a subject, the method comprising oromucous administration of an effective amount of exenatide to the subject.

The administration of exenatide may be sublingual or buccal administration.

In a fifteenth aspect, the present invention provides a method for preventing or treating hypoglycemia or hyperglycemia in a subject, the method comprising administering an effective amount of the product defined in the eighth aspect to the subject.

In a sixteenth aspect, the present invention provides a method for the prophylaxis or treatment of hypoglycemia or hyperglycemia in a subject, the method comprising oromucous administration of an effective amount of exenatide to the subject.

In a seventeenth aspect, the present invention provides the use of exenatide for the manufacture of a medicament for the treatment of diabetes mellitus or metabolic syndrome and / or for the regulation of blood glucose levels in which the medicament is formulated for oromucosal administration.

Brief Description of the Drawings

A preferred embodiment of the present invention will now be described only as an example with reference to the accompanying figures, where

1 shows fasting plasma glucose levels in a human subject given metformin and an exenatide-based product for oral (sublingual) use, obtained in accordance with the method of the present invention;

2 shows plasma glucose levels 2 hours after a meal in a human subject who was given metformin and an exenatide-based product for oral (sublingual) use, obtained in accordance with the method of the present invention.

the figure 3 shows the blood glucose levels in diabetic mice after administration of Exenatide -60 min followed by an intraperitoneal glucose load of 0 min. Mugs (top), delivered sublingually inert carrier; squares, 50 mcg exenatide sublingually; triangles base down; 100 mcg exenatide sublingually; diamonds, 150 mcg exenatide sublingually; triangles with the base up, 200 mcg exenatide sublingually; circles, 1 mcg exenatide subcutaneously (s / c).

Figure 4 shows blood glucose levels at 0 min (A) and 20 min (B), obtained from the results shown in Figure 1. ***, p <0.01 compared to an inert carrier.

figure 5A shows an analysis of the area under the curve (AUC) of blood glucose levels from 0 to 120 min, obtained from the results shown in figure 1. ***, p <0.01. Figure 5B shows the percentage change in AUC, calculated as (AUC [exenatide] - AUC [inert carrier]) / AUC [inert carrier].

6 shows blood glucose levels in diabetes mice after administration of exenatide in -60 min followed by an intraperitoneal glucose load of 0 min. Mugs delivered sublingually an inert carrier; squares, 2 mcg exenatide sublingually; triangles base down; 5 mcg exenatide sublingually; triangles with the base up, 10 mcg exenatide sublingually; diamonds, 25 mcg exenatide sublingually; crosses, 50 mcg exenatide sublingually; asterisks, 1 mcg exenatide subcutaneously (s / c).

Figure 7 shows blood glucose levels at 0 min (A) and 20 min (B), obtained from the results shown in Figure 4. ***, p <0.01 compared to an inert carrier.

figure 8A shows an analysis of the area under the curve (AUC) of blood glucose levels from 0 to 120 min, obtained from the results shown in figure 4. ***, p <0.01. Figure 8B shows the percentage change in AUC calculated as (AUC [exenatide] - AUC [inert carrier]) / AUC [inert carrier].

Figure 9 shows blood glucose levels in diabetes mice after administration of Exenatide in -60 min followed by an intraperitoneal glucose load of 0 min. Mugs delivered sublingually an inert carrier; squares, 10 mcg exenatide sublingually; triangles base down; 15 mcg exenatide sublingually; triangles with the base up, 20 mcg exenatide sublingually; diamonds, 25 mcg exenatide sublingually; asterisks, 1 mcg exenatide subcutaneously (s / c).

figure 10 shows blood glucose levels at 0 min (A) and 20 min (B), obtained from the results shown in figure 7. ***, p <0.01 compared with an inert carrier.

figure 11A shows an analysis of the area under the curve (AUC) of blood glucose levels from 0 to 120 min, obtained from the results shown in figure 7. ***, p <0.01. 11B shows the percent AUC change calculated as (AUC [exenatide] - AUC [inert carrier]) / AUC [inert carrier].

figure 12 shows the relative bioavailability of exenatide in monkeys. Squares, 20 μg of exenatide, administered sublingually, adjusted to t0 (average of four monkeys); triangles, 5 μg of exenatide, administered subcutaneously (average of three monkeys).

13 shows serum glucose levels in a human subject suffering from diabetes mellitus after administration of exenatide in -60 minutes followed by an intraperitoneal glucose load of 0 minutes. Diamonds, 100 mcg exenatide sublingually; squares, placebo sublingual; triangles, 5 mcg Exenatide (Byetta) subcutaneously.

Figure 14 shows blood glucose levels over a 12-week period in a person suffering from diabetes mellitus, to whom 50 μg of exenatide was administered sublingually for 12 weeks. Squares, glucose levels 2 hours after a meal; circles, fasting blood glucose levels.

the figure 15 shows postprandial (2 hours after a meal) plasma glucose levels for 11 days in a person suffering from diabetes mellitus, who were injected with 20 μg of exenatide together with 500 μg of metformin (squares) or 500 mg of metformin separately (diamonds )

Definitions

The following are some definitions that may be useful in understanding the description of the present invention. They are intended to be general definitions and will in no way limit the scope of the present invention to these expressions only, but are used to better understand the following description.

Throughout this description, unless the context otherwise requires, the word “comprise” or its variants, such as “comprises” or “comprising”, should be understood as implying the inclusion of a particular step, or element, or an integer or group of steps, or elements, or integers, but not the exclusion of any other step, or element, or integer, or group of elements or integers. Thus, with reference to this description, the expression "comprising" means "including predominantly, but not necessarily exclusively."

In relation to this description, the expression "approximately" is understood as referring to a range of numbers that a person skilled in the art would consider equivalent to the stated value in relation to achieving the same function or result.

In relation to this description, expressions denoting the singular form are used in this document, mentioning one or more than one (i.e., at least one) of the grammatical objects of the article. As an example, “element” means one element or more than one element.

For the purposes of this description, the expression "microparticles" is understood to include particles with a diameter of from about 50 to 800 microns, and preferably from about 200 to 400 microns.

In relation to this description, the expression "equilibrium conditions" is understood as meaning that the amount of moisture at the inlet is equivalent to the amount of moisture lost from the particles due to evaporation due to the input heat.

As applied to this description, the term “active component” should be interpreted in its broadest sense, and it includes, for example, drugs, living cells, including parts thereof, proteins, peptides, nucleic acids, viruses, viral particles, microorganisms, and the like. or any other substance or material that can be administered to a subject, such as a human or animal, for any purpose, including therapeutic purposes.

For the purposes of this description, the term “unstable” is understood to mean that the material to which it refers is susceptible to undesirable changes, whether physical or chemical, under certain conditions, for example, atmospheric conditions.

In the context of the present description, the term "effective amount" includes in its meaning a non-toxic, but sufficient amount of an agent or compound to provide a therapeutic effect. The exact amount needed will vary from subject to subject, depending on factors such as the type of subject to be treated, the age and general condition of the subject, the severity of the condition to be treated, the particular means and route of administration to be administered, and the like. Thus, it is not possible to establish the exact "effective amount". However, for a particular case, one skilled in the art will be able to determine the appropriate "effective amount" using exclusively routine experimentation.

As used herein, the terms “treating” and “treating” refer to any and all uses that treat a disease state or symptoms, prevent the development of a disease or otherwise prevent, impede, slow down or reverse the direction of disease progression or other undesirable symptoms whatever the way. Thus, “treatment” refers not only to treatment designed to treat or eliminate symptoms in an individual, but also to ongoing therapy to control and suppress the onset of symptoms. Treatment may last for a specified period of time or may be carried out on an ongoing basis depending on the specific circumstances of each individual.

DETAILED DESCRIPTION OF THE INVENTION

The present invention in a broad sense relates to a method for producing products containing the active component. Products obtained according to the present method may be referred to as "microcapsules." In certain embodiments, the present invention provides a method for stabilizing / preserving unstable materials, for example, materials sensitive to light, heat, air and / or moisture, and in particular biological materials, such as, for example, proteins, peptides, living cells and microorganisms . The method of the present invention finds particular application in the stabilization / preservation of proteins and peptides intended for administration to humans or animals. The stabilized products obtained according to the method of the present invention result in improved efficacy of their proteins or peptides when administered to subjects. The method of the present invention can be carried out at an operating temperature of only 30 ° C to 40 ° C, which means that it is compatible with many heat sensitive active components. In one embodiment, the at least one active component is exenatide.

In one aspect, the present invention provides a method for producing a product containing at least one active component, the method comprising:

(i) providing a coating fluid containing at least one active ingredient, a saccharide, and a water miscible solvent;

(ii) providing particles containing one or more water-soluble gelling agents;

(iii) fluidization of particles in the working chamber of the device so that the particles move in the chamber upward along a helical path;

(iv) spraying the coating liquid onto the particles to form coated particles;

(v) allowing coated particles to dry.

The method generally includes spraying a liquid containing the active component in combination with at least one saccharide and a water-miscible solvent onto an acceptable particulate filler material (particles, preferably microparticles) in an appropriate working chamber, where the particles, if they are covered, move along the camera up a helical path / helical path of movement. The movement of particles in the working chamber resembles the movement of air in a hurricane. Coating the particles results in microencapsulation and therefore stabilization / preservation of the active component. The helical path of the movement of particles in the working chamber provides a smooth, uniform coating with a coating fluid forming a hydrogel containing the active component and other water-soluble materials such as sugars and amino acids.

The nature of the coating fluid and particles is such that the products obtained according to the present method find particular application in applications related to delayed release delivery, such as mucosal delivery and sublingual delivery of therapeutically active components. In such applications, water-soluble materials dissolve upon contact with saliva, resulting in the formation of micropores on the surface of the coating, contributing to the destruction of microcapsules in a liquid gel. The gel covers the surface of the mucous membrane, thereby delivering the active component and optimizing absorption. Accordingly, the products obtained in accordance with the method of the present invention, are convenient solid forms for oral delivery of therapeutically active components, which may eliminate the need for injections.

The method of the present invention also provides for the successful preparation of dosage forms with a certain number of parts per billion active component evenly distributed therein. The in vivo action of cytokines is often catalytic, and therefore only quantities in nanograms or picograms are required to achieve the desired biological activity. The uniform distribution of such small amounts in the solid mass is particularly difficult and has no precedent in the pharmaceutical industry.

Using the method of the present invention, the active components can be encapsulated in a protected environment, excluding water, light, oxygen and microbes. In the case of biological materials, such as proteins and peptides, stability can be achieved for up to four years without cooling.

At least one active component may be unstable. In one embodiment, the at least one active component is sensitive to light, heat, air, and / or moisture. At least one active component may be any biological material, including without limitation proteins, peptides, living cells, microorganisms, and nucleic acids. Biological material may be unstable, for example, sensitive to light, heat, air and / or moisture. In one embodiment, the at least one active component is a cytokine, such as interleukin or interferon. In an alternative embodiment, the at least one active ingredient is an anticancer agent, an anti-inflammatory agent, a hemolytic agent, an anti-allergic agent, an antidiabetic agent, or an antifungal agent. At least one active component may be a peptide, for example, exenatide. In a specific embodiment, the at least one active component is exenatide. Accordingly, the present invention relates to a method for producing a product containing exenatide, the method comprising:

(i) providing a coating fluid containing exenatide, a saccharide and a water miscible solvent;

(ii) providing particles containing one or more water-soluble gelling agents;

(iii) fluidization of particles in the working chamber of the device so that the particles move in the chamber upward along a helical path;

(iv) spraying the coating liquid onto the particles to form coated particles;

(v) allowing coated particles to dry.

Those skilled in the art will recognize that the method of the present invention is also applicable to a wide range of other active components in addition to the aforementioned, for example to any material requiring stabilization / protection for any reason. In particular, the method of the present invention is applicable to any unstable therapeutic materials intended for administration to humans or animals.

The coating fluid contains at least one active ingredient, a saccharide, and a water miscible solvent. The saccharide can be, for example, mannitol, trehalose, isomalt, xylitol, dextrose, dextran, fructose, glucose, invert sugar, lactitol, lactose, maltitol, maltose, maltodextrin, sorbitol, sucrose, or any combination thereof. However, those skilled in the art will appreciate that alternative saccharides can be used depending, for example, on the nature of the active component contained in the solution and the intended use of the product. It should be understood that the expression “saccharide” as used herein includes sugar alcohols, so that the expression “saccharide” is synonymous with that of “saccharide or sugar alcohol”.

In some embodiments, the implementation of the present invention can be applied to several coatings containing various components. For example, in the preparation of microcapsules containing interferon, the first coating liquid to be applied may contain gelatin or ovalbumin in propylene glycol and water. The second (or active) coating may contain interferon, glycine, leucine, dextran, phosphate buffer, albumin, glucose, trehalose and maltodextrin.

Water-miscible solvents suitable for use in the method of the present invention include all water-miscible solvents considered to be safe for contact with tissues of the human or animal body based on an equitable risk / benefit ratio. Preferably, the water miscible solvent is a glycol, such as glycerol, or propylene glycol, or a mixture thereof. Other suitable water miscible solvents will be apparent to those skilled in the art given the intended use of the product.

The coating fluid may contain additional components, including without limitation additional proteins, amino acids, diluents, chelating agents, buffers, preservatives, stabilizers, antioxidants, lubricants and other additives that can act to complement the function or stabilize the specific active substance contained in it. Those skilled in the art will recognize that the nature of the additional components present will primarily depend on the active component and the intended use of the product. Additional examples of specific additional components include, without limitation, lysine, glycine, leucine, isoleucine, arginine and cysteine; human serum proteins, albumin, egg albumin and gelatin; sodium phosphate buffer, citric acid / citrate buffer, Tris buffer; derivatives of hydroxybenzoic acid; vitamin E, ascorbic acid; water miscible silicone / silicates; citric acid, EDTA and EGTA.

Those skilled in the art will appreciate that a variety of alternative proteins, amino acids, diluents, chelating agents, buffers, preservatives, stabilizers, antioxidants and lubricants may also be suitable for use in the present invention, again depending on the nature of the active component and the intended product application.

The particles contain water-soluble gelling agents, which can be either natural or synthetic polymers or monomers, which preferably do not become liquids or semi-solids in the presence of relatively high levels of moisture. Suitable water-soluble gelling agents include, without limitation, acrylate or its derivatives, albumin, alginates, carbomers, carrageenan, cellulose or its derivatives, dextran, dextrin, gelatin, polyvinylpyrrolidone, starch, or combinations thereof. In one embodiment, the water soluble gelling compound is gelatin, pregelatinized starch, carboxymethyl cellulose, or a combination thereof. A water-soluble gelling compound may form part of the core of the particles. Such a core may be referred to herein as a "hydrogel" core. The particles may additionally contain one or more simple sugars, which can serve to influence the dissolution rate of the product when exposed to an aqueous medium. The dissolution rate of the product can be increased by increasing the percentage of simple Sugars present in the core of the particles, or lower by lowering the percentage of simple Sugars present in the core of the particles. In addition, by incorporating one or more water-soluble gelling agents into the core and / or increasing the molecular weight of the water-soluble gelling agents, solid sustained release dosage forms can be formed.

The particles used in the method of the present invention are preferably microparticles with a diameter of from about 50 to 800 microns, or from about 100 to 600 microns, or from about 200 to 400 microns, however, it should be noted that particles with alternative sizes are also can be used depending on the active ingredient and the intended use of the product. Small particles dissolve faster than larger particles, since water is able to penetrate faster through the surface and into the core, which leads to the formation of a gel. In this regard, the use of a mixture of particles of various sizes can optimize the long-term effect of sustained release.

The composition of the particles used in the present method ensures that the coating fluid containing the active component effectively binds to the surface of the particles without significant agglomeration or loss.

The method of the present invention includes fluidization of particles in the working chamber of the device so that the particles move in the chamber in an upward direction along a helical path. Particles can also rotate simultaneously. The helical path of the movement of particles in the working chamber results in a smooth, uniform coating of the liquid for coating particles. A smooth, uniform coating is advantageous due to the fact that it minimizes the abrasive interactions that can occur between rough, unevenly coated particles. Such an abrasive effect may have a negative effect on the active component, especially in cases where the active component is a protein or peptide.

Helical movement of particles can be achieved by causing the movement of gas (which may include air or inert gas) in the working chamber along a helical path. This can be achieved by introducing into the working chamber a moving upward rotating gas stream. A rotating gas flow moving upward raises particles from the bottom of the working chamber, causing them to move upward along a helical path. In one embodiment, the helical movement of the particles is achieved by drawing gas through a guide located on the bottom or adjacent to the bottom of the working chamber. Gas can be drawn in through the guide by placing the working chamber under negative pressure conditions. The guide is adapted to cause the gas drawn in through it to move up the rotation path, which in turn lifts the particles, causing them to move upward along the helical path.

In one embodiment, the guide has an inlet through which gas is drawn in and an outlet through which gas is released, wherein the guide comprises a gas guiding element acting on a gas passing between the inlet and the outlet, so that gas released through the outlet, moved up the rotation path.

In an alternative embodiment, the guide comprises at least two functional units adapted to rotate about a common axis, where the first functional unit has an inlet through which gas can be drawn in, and where the second functional unit contains one or more gas guiding elements that are designed externally from the surface of the second functional unit, where the second functional unit is located above the first functional unit and where the gas used is drawn in through the inlet the hole acts on one or more gas-guiding elements, causing the second functional unit to rotate so that the gas released from the guide moves upward along the rotation path. In this embodiment, the first functional unit may be in the form of a disk, and the second functional unit may be in the form of a disk or cone-shaped. The second functional link during use can rise, become located at a distance from the first functional link and rotate so that the gas released from the guide moves upward along the rotation path. The inlet may be located in the center of the first functional link. One or more gas guiding elements may be located on the lower surface of the second functional unit oriented to the first functional unit. The gas guiding elements may be a series of curved blades. The diameter of the first functional unit may be larger than the diameter of the second functional unit. The guide may further comprise a third functional unit located between the first and second functional units, the third functional unit having an inlet communicating with the inlet of the first functional unit and further comprising one or more gas guiding elements that are designed outside the surface of the third functional unit the link where the applied gas drawn through each inlet acts on one or more gas guiding elements Causing rotation of the second and third functional units so that the gas released from the guide, moving upward along the rotation path. The second and third functional units during use can rise, so that the first, second and third functional units are spaced apart from each other and rotate so that the gas released from the guide moves upward along the rotation path. The gas guiding elements of the third functional link can be a series of curved blades. The third functional link may be in the form of a disk. The diameter of the third functional unit may be larger than the diameter of the second functional unit, and smaller than the diameter of the first functional unit. One or more gas guiding elements may be located on the lower surface of the third functional unit oriented to the first functional unit.

In an alternative embodiment, the guide comprises at least four functional units provided in the form of a first disk, a second disk, a third disk and a cone stacked on each other, rotated around a common axis, where each of the first, second and third disks has an inlet located at their center, through which gas can be drawn in, and where the surface of each of the second disk, third disk and cone include a series of gas-guiding elements that are designed outside of their surface, where The traction gas drawn through each inlet acts on one or more gas guiding elements, causing the second disk, third disk and cone to rotate so that the gas released from the guide moves upward along the rotation path. In this embodiment, the first disk may have a larger diameter than the second disk, the second disk may have a larger diameter than the third disk, and the third disk may have a larger diameter than the end surface of the cone, so that when the disks and the cone are folded together, the guide in general appearance it is conical, where the base is formed by the first disk, and the apex is formed by a cone. One or more gas guiding elements may be located on the lower surface of the second and third disks and on the surface of a cone oriented towards the third disk. The gas guiding elements may be a series of curved blades. The second disk, the third disk and the cone can rise during use, so that the first disk, the second disk, the third disk and the cone become separated and rotate so that the gas released from the guide moves upward along the rotation path.

In a further embodiment of the present invention, the guide comprises three circular functional links with central holes defining a path through them. Round functional links are adapted to rotate around a common axis and have decreasing diameters, starting from a functional link located at the lowest point of the working chamber. The diameter ratio of the round functional units is approximately 2: 1, 5: 1. On top of the round functional units is a solid cone-shaped functional unit adapted to rotate around the same axis as the round functional units. The cone-shaped functional unit has a diameter smaller than the diameter of the circular functional unit located directly below it. The edges of the two round functional units located directly below the cone-shaped functional unit are beveled so that when the two round functional units and the cone-shaped functional unit are placed together, the three sections form the shape of a large cone with a continuous flat surface. The lower sides of the circular functional links, with the exception of the circular functional link located at the lowest point of the working chamber, contain a series of generally curved blades extending from the outer edge to the central holes. The blades function by directing the flow of air to the holes. The applied negative pressure is applied in relation to the working chamber, as a result of which the gas is drawn upward through the round sections. In the presence of a continuous gas stream, the circular sections and the cone-shaped section rise, become spaced apart and rotate. The combined effect of spinning sections and curved blades causes gas to escape from the rail with an upward movement along the rotation path, or, in other words, along a path resembling air movement in a hurricane. A rotating gas flow moving upward raises particles from the bottom of the working chamber, causing them to move upward along a helical path.

The guide described in the previous paragraph may be referred to as “Rocket Drive” in this document.

The stage of spraying the coating liquid, as a rule, occurs from the bottom of the working chamber upward. Accordingly, the coating liquid is sprayed in the same general direction of motion as that for particles in the working chamber. In one embodiment, the coating fluid is sprayed upward so that the coating fluid moves along a column formed by a stream of particles in the working chamber. When particles move along a helical trajectory, a column form with an outer surface is formed in the working chamber. In this embodiment, the coating fluid is sprayed upward so that the fluid moves along the outer surface of the particle column. In an alternative embodiment, the coating liquid can be sprayed upward in the direction of the particles at an angle of from about 20 ° to 60 °, or at an angle of from about 30 ° to 50 °, or at an angle of from about 35 ° to 45 ° (relative to bottom of the working chamber). The exact angle at which the coating liquid is sprayed will depend on the size of the working chamber. For example, if you use a high, narrow working chamber, then the required angle is likely to be about 40 ° -50 °. Alternatively, if a wider, lower working chamber is used, then the required angle is likely to be less than 40 °. Specialists in this field will be able to determine the most suitable angle at which spray liquid for coating according to standard tests and experiments, given the size of the working chamber.

The coating fluid may have a temperature of from about 0 ° C to 50 ° C, or a temperature of from about 10 ° C to 50 ° C, or a temperature of from about 15 ° C to 45 ° C, or a temperature of from about 30 ° C to 45 ° C, or a temperature of from about 35 ° C to 40 ° C. Alternatively, the coating fluid at the inlet to the working chamber may have room temperature.

Depending on the expected batch size, the working chamber may contain one spray gun or a number of those from which a liquid is ejected for coating. When using multiple spray guns, different guns can be charged with different coatings. Spray guns can be activated at any time during the implementation of the method in combination, sequentially or one after another. This provides significant flexibility in the preparation of products having several coating layers and various active components. The method of the present invention allows to obtain a product with many active components in one solid dose, in contrast to the freeze-dried powder in a bottle. By encapsulating one active component at a time, and then mixing the resulting microcapsules, the method of the present invention makes it possible to combine chemically and / or physically incompatible components in a single dose.

The spray guns used in the method of the present invention can be adapted so that the coating liquid turns into microdroplets before contact with the particles. The size of the microdrops can be changed by controlling the rate of consumption of the sprayed material and the volume of gas entering the working chamber.

After spraying, the coating fluid is transformed into a continuous glassy film ("bioglass") enveloping the particles. The conversion of a liquid to a glassy solid is quick (in less than 1 second), so that the stability of the active component is not compromised. The active component will not be damaged by heat, as it dissipates due to the latent heat of evaporation of the water.

Optionally, additional coating steps can be carried out in order to obtain products having specific characteristics. For example, before or after the drying step, additional coatings may be applied to the resulting product. Those skilled in the art will recognize situations in which this may be advantageous, for example, when it is desired that the product be administered orally, enteric coatings may be used to protect the product from degradation in the stomach. Typically, such additional coating will be performed at a coating rate similar to that used to coat the particles with the starting coating liquid.

The method of the present invention is preferably carried out in a closed sterile environment or, in other words, in an environment that does not contain or practically does not contain pollutants. Typically, pollutants include microorganisms and the like, however, those skilled in the art will readily understand that it is also desirable to eliminate any other harmful pollutants in the process of the present invention.

The environment in which the method is carried out preferably does not contain or practically does not contain oxygen, so that the possibility of oxidation of the active components is minimized. Environmental conditions that do not contain oxygen or practically do not contain oxygen can be achieved by replacing the air contained in the working chamber with an inert gas, for example, nitrogen. However, it should be appreciated that alternative inert gases such as carbon dioxide or argon can be used.

The method of the present invention may include excess particle loading of the working chamber. In the normal mode in the fluidized bed, equipment manufacturers recommend not to exceed 50% of the mass / volume ratio of the working chamber capacity. For example, if the volume of the working chamber is 100 l, the weight of the processed material should not be more than 50 kg. However, the method of the present invention provides that the ratio of the weight of the processed material to the volume of the chamber is more than 100% mass / volume. Accordingly, the recyclable particle weight in any batch can be at least doubled or even tripled, which results in a significant improvement in production efficiency and significant cost savings compared to a conventional fluidized-bed coating system.

In order to minimize damage to the active component, the method can be performed under equilibrium conditions. In this regard, the amount of moisture at the inlet is equal to the amount of moisture lost from the particles due to evaporation due to the input heat. Accordingly, the method can be carried out in an environment that is neutral with respect to moisture and heat. In an "environment neutral to moisture and heat" conditions are such that the surface of the microparticles does not contain moisture. Moisture-free environment can be achieved by setting the spray rate for the material to be sprayed at a constant evaporation rate.

The method of the present invention can be performed in any suitable device for spraying on a fluidized bed. The following examples use Rocket Drive, designed by Vitapharm Technology Development Co. Hinge Engineering Ltd BDA, Beijing, China, housed in a CPU Driven Turbojet ™ fluidized bed coater manufactured by BWI Huttlin (Daimlerstrasse 7, D-79585, Steinen, Germany). Those skilled in the art will be familiar with the device from BWI Huttlin.

It should be borne in mind that in order to facilitate effective and efficient microencapsulation, certain modifications can be made to the device. For example, the device used in the examples described in this document can be modified in accordance with the requirements of the user as follows.

- The design of the spray nozzle has been redesigned so that the central part of the nozzle (which delivers the coating fluid to the working chamber) can be removed during operation of the device for cleaning or unlocking. This modification provides the possibility of continuous operation of the device.

- All contact surfaces were carefully polished to a high gloss to facilitate thermal sterilization after a standard CIP cycle.

- Replacing the air introduced into the working chamber with a recirculating inert gas, such as nitrogen, which, unlike air, can be used for fluidization. This modification can minimize the oxidation of susceptible active components and increase the resistance of anaerobic bacteria.

- The central air return column, available in the standard unit, has been replaced with the Rocket Drive guide described above.

In one embodiment, the method of the present invention (for a batch of 2 to 3 kg) can be performed as follows:

1. Solid hydrogel particles (microparticles) having a suitable composition are loaded under vacuum into a fluidized bed Huttlin Turbojet ™ device for coating and subjected to fluidization. Fluidization occurs at an air flow rate of 500-1000 m ³ / h.

2. The microparticles are heated to a temperature of from about 30 ° C to 80 ° C, more preferably to a temperature of about 60 ° C, for about one hour at a low speed of the working air (which enters the working chamber through the Rocket Drive), so that they are exposed delayed fluidization. Slow fluidization ensures that the fraction of microparticles inside is dry and minimizes friction between particles.

3. The temperature of the microparticles is then reduced to approximately 35 ° C-45 ° C, and the speed of the working air that enters the working chamber is increased until the microparticles move upward along a helical line of movement.

4. When the temperature of the microparticles reaches approximately 35 ° C-45 ° C, the working air is replaced with an inert gas, such as nitrogen. This step can be carried out for at least about 5 minutes in order to ensure that all the air present in the working chamber is replaced with an inert gas.

5. The active component is bound in a suitable coating liquid. The stock solution was heated to approximately 38 ° C to ensure complete dissolution of the solid. Immediately prior to spray coating, the active component is added to the stock solution (mixing at a speed of about 60 rpm) and mixed well.

6. The desired amount of coating liquid is then sprayed onto the fluidized microparticles, preferably at the highest possible speed, while the microparticles move upward along a helical line of movement. Coating occurs at a rate of approximately 20 grams to 40 grams of coating fluid per minute. The coating fluid is sprayed from the bottom of the working chamber at an angle of approximately 40 ° -50 ° (relative to the bottom of the working chamber) in the direction of the moving microparticles.

7. The coating speed is preferably from about 25 grams to 35 grams of coating fluid per minute when the microparticles do not contain excess moisture, which thus ensures that the microparticles will continuously perform helical movement. Accordingly, the coating liquid (containing the active component, such as a protein or peptide) is subjected to continuous dehydration in a nitrogen free environment. The product is usually dried to a water activity of less than 0.25.

It should be appreciated that the above steps and processing parameters can be changed to provide a variety of products or products containing various active substances. You can make changes, for example, in relation to the temperature of the inlet air, the temperature of the product, the volume of air used for fluidization, the speed of spraying the liquid, the temperature of the coating liquid, the viscosity of the coating liquid, the solids levels in the coating liquid, the total specific surface area of the core for microparticles, water solubility of the microparticle core, inlet air humidity, compressed air pressure for atomization, pore size of the device filter and frequency of automatic dust removal. If a change is made with respect to one parameter, those skilled in the art will be able to easily identify any appropriate adjustments that may be required with respect to another parameter in order to compensate for the initial change.

In the method of the present invention, the batch size may vary depending on the volume of the working chamber and whether overload is required. In the examples described herein, the batch size is typically 3 kg. In this regard, "batch size" refers to the total amount of solids used in the process, and includes solids contained in microparticles, coating liquids, and any additional coating liquids used. Accordingly, the percentages of specific components used in this document are expressed as a percentage of the total lot size.

The present invention further relates to products containing at least one active ingredient obtained according to the method of the present invention. The products preferably contain biological materials, however, those skilled in the art will appreciate that the products may contain any material, possibly sensitive to light, heat, moisture or air, or requiring stabilization / protection for any reason. The present invention further relates to the use of the method of the present invention for stabilizing at least one active component. In one embodiment, the at least one active component is exenatide

The present invention further relates to the use of a product according to a second aspect for the delivery of at least one active ingredient to a human or animal. In one embodiment, the at least one active component is exenatide. The products obtained in accordance with the method of the present invention find particular use in the delivery of active components, preferably biologically active components, to humans and animals. The product may be suitable for injection, oral or nasal administration, sublingual administration, local administration, vaginal administration or rectal administration. The product may be in a dosage form selected from the group consisting of a composition intended for injection, tablets for sublingual use, tablets for oral use, sustained release tablets for sublingual use, microcapsules intended for filling capsules, microcapsules, pre-prepared food mixture pessary, a pre-formed solid dose intended for use in the form of a nasal spray or drops, water drops, eye lotion or drops l and a solution for washing the skin. The products obtained in accordance with the present invention, due to the use of a water-soluble gelling compound, are highly effective in delivery with a slow release of the active components by the sublingual or mucosal route. Accordingly, a further aspect of the present invention includes the use of the method of the present invention for the preparation of sustained release dosage forms. In one embodiment, the delivery formulations of the present invention comprise proteins and peptides, preferably proteins and peptides, intended for therapeutic use, for example, cytokines such as interleukin and interferon.

The present invention also relates to a device for producing a product containing at least one active component, comprising a working chamber, in which one or more outlets for introducing liquid into the working chamber, an inlet located at the bottom of the working chamber, and an outlet are placed, which, thus, allows gas to move along the working chamber, where the inlet is provided with a guide, so that when the gas is drawn through the inlet after exiting the guide, the gas moves about direction up the rotation path. The device can be used to implement the method of the present invention. The guide used in the device according to the present invention may be the guide described herein with reference to the first aspect. An outlet may be located at the top of the cooking chamber. In one embodiment, the at least one active component is exenatide. Accordingly, the present invention provides an apparatus for producing a product containing at least one active component comprising a working chamber, in which one or more outlet openings for introducing liquid into the working chamber, an inlet located at the bottom of the working chamber, and an outlet are placed, thereby allowing gas to move along the working chamber where the inlet is provided with the guide, so that when the gas is drawn in through the inlet, then after exiting the direction boiling gas moves upward along the rotation path.

The present invention also provides a method for treating metabolic syndrome or diabetes in a subject, the method comprising oromucous administration of an effective amount of exenatide to the subject. The administration of exenatide may be sublingual or buccal administration.

The present invention also provides a method for preventing or treating hypoglycemia or hyperglycemia in a subject, the method comprising oromucous administration of an effective amount of exenatide to the subject.

The present invention also provides the use of exenatide for the manufacture of a medicament for the treatment of diabetes mellitus or metabolic syndrome and / or for the regulation of blood glucose levels, the medicament being formulated for oromucosal administration.

Examples

Example 1 - Interleukin-2 sustained release tablet containing 350 ng of recombinant human interleukin-2 (IL2) per 200 mg tablet

Lot size: 3 kg.

Container Size: 1.5 L

Coating temperature: 38 ° C ± 2 ° C.

Spray rate: 30 grams / minute.

Typical microparticle core size: 700 μm-425 μm (30%), 425 μm-180 μm (55%), <180 μm (15%).

The hydrogel core of microparticles: mannitol (70%), sodium SMS (2%), polyvinylpyrrolidone (2%), gelatin (13%) and starch (13%).

Coating liquid: mannitol (10%), propylene glycol (3.5%), succinyl gelatin (0.6%), glycine (0.3%), ascorbic acid (0.3%), albumin (0.05% ), standard sodium phosphate buffer up to pH 7 and water for injection up to 100%.

The method was carried out as follows.

1. Glycine, mannitol, succinylated gelatin, propylene glycol, ascorbic acid and a buffer were dissolved in water for injection and then filtered through a 0.2 micron membrane filter. Albumin was added to add water following injection.

2. The working chamber of the Huttlin Turbojet ™ Rocket Drive-equipped fluidized bed coater was sterilized by heat treatment according to the manufacturer's instructions for one hour.

3. The device was then switched to filtered nitrogen circulation.

4. The microparticles were loaded into the working chamber under vacuum, subjected to fluidization and then heated to a temperature of approximately 60 ° C for one hour.

5. The temperature of the microparticles was then lowered to 40 ° C.

6. The contents of the working chamber were then subjected to fluidization at an air flow rate of 300 m ³ / h, which, if necessary, was slowly increased to 900 m ³ / h. regarding air. This resulted in microparticles moving upward along a helical path.

7. Sterile IL2 (recombinant human IL2 (2.25 mg)) was then added to the coating liquid, which was then sprayed upward from the bottom of the working chamber under equilibrium conditions in the direction of the microparticles at a flow rate of 30 g / min.

8. The resulting product was dried to water levels at which its activity was less than 0.25%.

9. The product was then compressed according to standard techniques in the art into 200 mg tablets.

10. The tablets were then packaged in nitrogen-blown aluminum / aluminum foil bags and stored at a temperature not exceeding 25 ° C.

Method Validation Results

Two batches of tablets with 350 ng IL2 were processed on the same day using the same equipment, operating parameters and the same batch of active substances and filler materials. The batches received in the morning and in the afternoon were assigned batch codes BX20100831-1 and BX20100831-2, respectively. Tablets were also made using two batches of microcapsules with IL2. Each tablet had a weight of 200 mg. Tablet characteristics = 350 ng IL 2 ± 30%.

ELISA Validation and Biological Analysis Results

Two batches of tablets with 250 ng IL2 were processed on two separate days using the same equipment, operating parameters and the same batch of active substances and filler materials. The batches received on day 1 and on day 2 were assigned party codes BX20100414-1 and BX20100422-2, respectively. Tablets were also made using two batches of microcapsules with IL2. Each tablet had a weight of 200 mg. Tablet Characteristics = 250 ng IL2 ± 30%.

Example 2 - Exenatide Sustained Release Tablet Containing 20 Micrograms of Exenatide per 200 mg Tablet

Lot size: 3 kg

Container Size: 1.5 L

Coating Temperature: 38 ° C ± 2 ° C

Spray rate: 30 grams / minute

Typical microparticle core size: 700 μm-425 μm (30%), 425 μm-180 μm (55%), <180 μm (15%)

The hydrogel core of microparticles: mannitol (66%), sodium SMS (4%), polyvinylpyrrolidone (2%), gelatin (13%) and starch (15%).

Coating fluid: mannitol (10%), propylene glycol (3.5%), succinyl gelatin (0.6%), glycine (0.3%), dextran (0.3%), albumin (0.05%) , standard sodium phosphate buffer up to pH 7 and water for injection up to 100%.

The method was carried out as follows.

1. Glycine, mannitol, succinyl gelatin, propylene glycol, dextran and buffer were dissolved in water for injection and then filtered through a 0.2 micron membrane filter. Albumin was added to add water following injection.

2. The working chamber of the Huttlin Turbojet ™ Rocket Drive-equipped fluidized bed coater was sterilized by heat treatment according to the manufacturer's instructions for one hour.

3. The device was then switched to filtered nitrogen circulation.

4. The microparticles were loaded into the working chamber under vacuum, subjected to fluidization and then heated to a temperature of approximately 60 ° C for one hour.

5. The temperature of the microparticles was then lowered to 40 ° C.

6. The contents of the working chamber were then subjected to fluidization at an air flow rate of 300 m ³ / h, which, if necessary, was slowly increased to 900 m ³ / h with respect to air. This resulted in microparticles moving upward along a helical path.

7. Sterile exenatide (300 mg) was then added to the coating liquid, which was then sprayed upward from the bottom of the working chamber under equilibrium conditions in the direction of the microparticles at a flow rate of 30 g / min.

8. The resulting product was dried to water levels at which its activity was less than 0.25%.

9. The product was then compressed according to standard techniques in the art into 200 mg tablets.

10. The tablets were then packaged in nitrogen-blown aluminum / aluminum foil bags and stored at a temperature not exceeding 25 ° C.

Example 3 - Sublingual Exenatide Administration

A diabetes patient undergoing metformin treatment was transferred to a once-daily, sublingual tablet containing 20 μg of sustained release exenatide prepared in accordance with Example 2. Fasting glucose and plasma glucose were measured, and they are shown in figures 1 and 2. When the patient was taking metformin, the glucose level in his blood was fluctuating and difficult to control. The patient then began treatment with Exenatide tablets for sublingual use (under medical supervision), which provided a very stable blood glucose profile.

Those skilled in the art will appreciate that the present invention described herein is capable of variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications.

Example 4 - Sublingual Exenatide Diabetic Mice

200 mg standard placebo tablets containing sugars, amino acids, buffering and gelling agents were made as described in Examples 1 and 2. The tablets were dissolved in 1 ml of water and slightly homogenized until a uniform gel was obtained. This gel was used as a standard inert delivery vehicle, with or without exenatide (Vitapharm Technology Development Co. Ltd.).

Diabetic male mice (line C57 / BL6) were obtained at Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). For the experiments described below, mice weighing approximately 60 g were used. Mice were fasted for a six-hour period before administration of exenatide or placebo (-60 min). After 1 hour (0 min), a glucose load was administered and the blood glucose was measured at -60 min, 0 min, 30 min, 40 min, 60 min and 120 min. To administer 5 μg of a gel containing various doses of exenatide or placebo was placed under the tongue of mice using a micropipette.

In experiment 1, treatment was performed in six groups of five mice. In the four treatment groups, mice were injected with 50 μg, 100 μg, 150 μg or 200 μg of exenatide sublingually. They were compared with 1 μg subcutaneously administered exenatide and sublingually administered placebo. The results of experiment 1 are shown in figure 3. Blood glucose levels in each treatment group at 0 min and 20 min are shown in figure 4. Analysis of the area under the curve of blood glucose levels from 0 to 120 min (AUC _{0-120 min} ) for each treatment groups are shown in figure 5.

In experiment 2, treatment was performed in seven groups of five mice. In five treatment groups, mice were injected with 2 μg, 5 μg, 10 μg, 25 μg or 50 μg of exenatide sublingually. They were compared with 1 μg subcutaneously administered exenatide and sublingually administered placebo. Experiment 2 results are shown in Figure 6. Blood glucose levels in each treatment group at 0 min and 20 min are shown in Figure 7. The analysis of the area under the curve of blood glucose levels from 0 to 120 min (AUC _{0-120 min} ) for each treatment groups are shown in figure 8.

Experiment 3 included four treatment groups, including sublingual administration of 10 μg (five mice), 15 μg (ten mice), 20 μg (ten mice), or 25 μg (five mice) of exenatide. They were compared with 1 μg subcutaneously administered exenatide (five mice) and sublingually administered placebo (five mice). Experiment 3 results are shown in Figure 9. Blood glucose levels in each treatment group at 0 min and 20 min are shown in Figure 10. The analysis of the area under the curve of blood glucose levels from 0 to 120 min (AUC _{0-120 min} ) for each treatment groups are shown in figure 11.

The results of these experiments demonstrate that sublingual delivery of exenatide is feasible and effective. According to the analysis of the area under the curve, subcutaneously administered exenatide at a dose of 1 μg significantly reduced blood glucose levels compared to an inert carrier (placebo) from 0 to 120 minutes. Similarly, sublingually administered exenatide at a dose of 10 μg, 15 μg, 20 μg and 25 μg also significantly reduced blood glucose levels compared to an inert carrier (placebo) from 0 to 120 minutes, and the efficacy is comparable between doses based on statistical calculation. Sublingually administered exenatide at a dose of 10 μg, 15 μg, 20 μg and 25 μg significantly reduced fasting glucose levels (i.e., without glucose loading, 60 minutes after the administration of exenatide).

Based on the experiments described above, the bioavailability of sublingual exenatide produced using the gel matrix-based delivery system developed by the present inventor was 10%. Moreover, similar experiments revealed a bioavailability of approximately 20% using 5 μg of exenatide (data not shown). These values are very favorable (25-55 times greater) compared with the published bioavailability of only 0.37% for exenatide administered sublingually at a dose of 210 μg (Gedullin et al, 2008, Int J Pharm 356: 231-238 )

Example 5 - Sublingual Exenatide Administration to Primates

Monkey study

A study of the relative bioavailability of sublingual exenatide compared with subcutaneous administration was performed on monkeys. Exenatide was prepared as described in Example 4. Three monkeys were injected with 5 μg of exenatide subcutaneously and four monkeys were injected with 20 μg of exenatide sublingually and serum glucose levels were determined by ELISA at regular intervals up to 2 hours after administration. As shown in FIG. 12, sublingual administration leads to improved regulation of serum glucose levels (more even, less variable levels) compared to subcutaneously administered exenatide.

People study

Studies of the effects of sublingually administered Exenatide were then performed on human subjects. Studies were similar to studies conducted on mice (example 4). Briefly, subjects fasted overnight (11 hours) before exenatide or placebo was administered at 9:00 a.m. (-60 min). After 1 hour (0 min), a glucose load (75 g in 250 ml of water) was introduced and the blood glucose was measured at -60 min, 0 min, 30 min, 40 min, 60 min and 120 min. For administration, tablets containing 100 μg of exenatide (obtained using the same composition and in the same manner as the gel used in the mouse studies described above) or placebo were placed under the subjects tongue. For comparison, 5 μg of exenatide (Byetta) was administered subcutaneously. The study included nine healthy (non-diabetic) subjects and two diabetic subjects (referred to below as diabetic subjects No. 1 and No. 2).

Subjected sublingually, 100 μg exenatide did not have any adverse side effects on healthy subjects or diabetic subjects. Serum glucose levels over a 2-hour period after administration in healthy subjects who received exenatide administered sublingually fully correspond to levels in subjects who received a sublingual placebo, increasing 30 minutes after administration to a slow decline afterwards (data not shown). In contrast, in healthy subjects who received exenatide subcutaneously, serum glucose levels remained essentially unchanged (data not shown).

Serum glucose levels in subject No. 1 diabetic patient are shown in FIG. 13. It can be seen that, compared to levels in a subject who received placebo or subcutaneously administered exenatide, serum glucose levels in subject # 1 who received subexual exenatide received remained relatively constant over a 2-hour period after administration.

Subject 2 suffering from diabetes received 50 μg of exenatide sublingually for three months (12 weeks). As shown in figure 14, both fasting blood glucose levels and postprandial blood glucose levels remained fairly constant throughout the treatment period. At the beginning of treatment, the hemoglobin A1c (HbA1c) levels of subject No. 2 were borderline high, at 6.5, indicating poor blood sugar control. After 12 weeks of exenatide treatment, HbA1c levels dropped to 6.3, within the normal range of 4 to 6.5. Subject did not suffer from any side effects from exenatide treatment and reported that he felt significantly healthier and better after 12 weeks of treatment.

Postprandial (+ 2 hours) plasma glucose levels were also monitored for 11 days in a diabetic subject who received 20 µg subexual exenatide and 500 mg metformin compared to 500 mg metformin alone. As can be seen in FIG. 15, postprandial plasma glucose levels remained essentially constant for an 11-day period during treatment using sublingual administration of exenatide in conjunction with metformin, while levels were extremely variable during the day during treatment with metformin separately.

Claims (15)

1. A method of obtaining a product containing exenatide, including: (i) providing a coating fluid containing exenatide, a saccharide and a water miscible solvent; (ii) providing particles containing one or more water-soluble gelling agents; (iii) fluidization of particles in the working chamber of the device so that the particles move in the chamber upward along a helical path; (iv) spraying the coating liquid onto the particles to form coated particles; (v) allowing coated particles to dry, wherein the product is a sustained release dosage form for delivering exenatide to a subject. 2. The product obtained according to the method according to claim 1. 3. The application of the method according to claim 1 for stabilizing exenatide. 4. The use of the product according to claim 2 in the manufacture of a medicament for the treatment of diabetes. 5. The use of the product according to claim 2 in the manufacture of a medicinal product for the treatment of metabolic syndrome. 6. The use of the product according to claim 2 in the manufacture of a medicament for regulating blood glucose levels. 7. The use of the product according to claim 2 in the manufacture of a medicament for the prevention or treatment of hypoglycemia or hyperglycemia. 8. The use according to any one of paragraphs. 4-7, in which the drug is introduced into the composition for oromucous administration. 9. The use of claim 8, wherein the introduction is sublingual and / or buccal.

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