Method of production, use and composition of minor spherical particles produced in controlled phase separation

FIELD: process engineering.

SUBSTANCE: invention relates to production of minor spherical particles of active agent in sole liquid phase solution. Sole liquid phase comprises active agent, agent facilitating phase separation and first thinner. Phase separation is induced at controller rate in solution to cause separation active agent into "fluid-solid" and to form liquid and solid phases. Note here that inducing comprises solution cooling. Solid phase contains minor spherical particles of active agent. Liquid phase comprises agent facilitating phase separation and thinner. Minor spherical particles feature particle size varying from 0.01 mcm to about 200 mcm.

EFFECT: minor spherical particles of active agent in sole liquid phase solution.

77 cl, 49 dwg, 4 tbl, 36 ex

 

CROSS-REFERENCE TO RELATED APPLICATION:

This application claims priority based on provisional application U.S. 60/488712 registered on July 18, 2003, which is incorporated in its entirety by reference and is a part of this application.

FEDERAL SPONSORSHIP of RESEARCH OR DEVELOPMENT

Was not carried out

The prior art INVENTIONS

Technical area

The invention relates to methods of manufacture, methods of use and compositions of small spherical particles of the active agent. In accordance with the production method of the active agent is dissolved in water or miscible with water, the solvent containing dissolved reinforcing phase separation agent (UVR)to form a solution in a single liquid phase. The solution is then subjected to phase separation, liquid-solid, and available active agent contained in the solid phase, and UV and solvent contained in the liquid phase. Phase separation of liquid-solid can be caused by a number of ways, for example, by changing the solution temperature below the phase transition temperature of the system. The method is most suitable for the formation of small spherical particles of therapeutic agents, which can then be delivered to the object that needs therapeutic agent. JV the property is most suitable for the formation of a solid, small spherical particles of macromolecules, in particular, macromolecules, which are thermally unstable, for example, proteins.

Prior art

Several technologies have been used in the past for manufacturing a biopolymer nano - and microparticles. Conventional technologies for education particles include spray drying and grinding, and can be used to produce a particle size of 5 microns or less.

In patents U.S. 5654010 and U.S. 5667808 described the production of solid forms of recombinant human growth hormone, HGH, through complexation with zinc in order to create an amorphous complex, which is then finely milled, passing through an ultrasonic nozzle, and sprayed into liquid nitrogen to freeze the droplets. Liquid nitrogen then allow to evaporate at a temperature of -80°C, and the resulting material is dried by sublimation.

Microparticles, microspheres and microcapsules are solid or semi-solid particles with a diameter less than one millimeter, more preferably less than 100 microns, and most preferably less than 10 μm, which can be formed from various materials, including proteins, synthetic polymers, polysaccharides, and combinations thereof. Microspheres have been used for many different purposes, mainly for the separation, detection and delivery Lek is rstv.

The most well-known examples of microspheres used in separation technologies are microspheres formed by polymers of synthetic or natural origin, such as polyacrylamide, hydroxyapatite or agarose. In the field of drug controlled delivery of molecules are often combined or encapsulated in a small spherical particles or combined in a monolithic matrix for subsequent release. A number of different technologies are regularly used to obtain these microspheres made of synthetic polymers, natural polymers, proteins and polysaccharides, including phase separation, solvent evaporation, coacervation, emulsification and spray drying. Typically, the polymers form the structure of the media data of the microspheres, and the desired drug is in the polymer structure.

Currently available particles prepared using lipids to encapsulate the target drugs. Liposomes are spherical particles consisting of one or multiple phospholipid and/or cholesterol bilayers. Liposomes have a size of 100 nanometers or more, and can carry a lot of water-soluble or lipitorhistory drugs. For example, lipids organized in duhl inye membrane, surrounding the many water departments to form particles, can be used to encapsulate water-soluble drugs for subsequent delivery as described in U.S. patent 5422120, Sinil Kim.

Spherical beads commercially available as a tool for biochemists for many years. For example, antibodies, United with beads, creating a relatively large particles, which are specifically associated with specific ligands. Antibodies used regularly for binding to receptors on the cell surface to cell activation, binds to the solid phase to form particles coated with antibodies, for immunoaffinity cleaning, and can be used to deliver therapeutic agents, which are slowly excreted over time, using tissue or tumor-specific antibodies attached to the particles, to deliver the agent to the desired site.

Currently, there is an urgent need to develop new methods for producing particles, especially those that can be adapted for use in the fields of drug delivery preparation, separation and diagnostics. The most desirable particle from the point of view of usage, would be a small spherical particles with the following characteristics: limited granulometric the cue structure, essentially, spherical, essentially consisting only of active agent, the preservation of biochemical integrity and biological activity of the active agent. Particles should provide a suitable hardness, which provides additional stabilization of the particles in the coating or microencapsulation. Furthermore, the method of production of small spherical particles should possess the following desirable characteristics: ease of fabrication, essentially water-way, high output and do not require further screening.

The INVENTION

The present invention relates to methods of production and usage of small spherical particles of the active agent. In accordance with the method, the active agent is dissolved in a solvent containing a reinforcing phase separation agent to form a solution, which is a single liquid phase. The solvent is preferably water or miscible with water, the solvent. The solution is then subjected to phase separation, liquid-solid, and available active agent contained in the solid phase, and UV and solvent contained in the liquid phase. Phase separation of liquid-solid can be caused by a number of ways, for example, by changing the solution temperature below the temperature of the phase is about the transition of the solution.

In a preferred embodiment, the present invention uses a method of phase separation of the solution of the fluid-solid by cooling the solution below the phase transition temperature of the active agent in solution. The temperature may be above or below the freezing temperature of the solution. For solutions in which the freezing temperature above the phase transition temperature, the solution may include an agent that lowers the freezing point, for example, polyethylene glycol or polypropylenglycol to lower the freezing point of the solution and allow to happen to a phase separation in the solution without freezing solution.

Reinforcing phase separation agent of the present invention enhances or induces phase separation of liquid-solid active agent in the solution when the solution is subjected to phase, phase change, in which the active agent hardens, forming a suspension of small spherical particles as a discrete phase, while reinforcing phase separation agent remains dissolved in the continuous phase. That is, the reinforcing phase separation agent does not change phase, while the active agent changes the phase.

A method of producing particles according to the present invention may also include additional phase control of the phase separation of the particles of the LM is bone-hard, to adjust the size and shape of the formed particles. Ways to control the phase separation include the regulation of ionic strength, pH, concentration-enhancing phase separation agent, the concentration of the active agent in solution or regulation of the rate of change of temperature of the solution, and the regulation of these parameters is carried out or before phase separation or change one or more of them in order to cause phase separation.

In a preferred embodiment of the present invention a small spherical particles are separated from the UV in the continuous phase after the formation of the particles. In another preferred embodiment, the method of separation is a washing solution containing particles, the liquid in which the active agent is not soluble, while reinforcing phase separation agent is soluble in the liquid. Wash liquid may contain an agent that reduces the solubility of the active agent in the liquid. Rinsing fluid may also contain one or more filler. The filler can act as a stabilizer for a small spherical particle or the active agent or carrier agent. The filler may also be given active agent or particle additional features, such as controlling is my release of active agent from the particles, or a modified penetration of the active agent through biological tissue.

In another preferred embodiment, despite the fact that small particles do not include UV, they can be formed in the presence of phase UVR for subsequent processing steps before the separation from the phase of UVR.

In another preferred embodiment, the solution is an aqueous solution containing water or miscible with water, the solvent.

The active agent of the present invention is, preferably, pharmaceutically active agent, which can be a therapeutic agent, diagnostic agent, a cosmetic substance, food additive or pesticide. In a preferred embodiment of the present invention the active agent is a macromolecule such as a protein, polypeptide, carbohydrate, polynucleotide or nucleic acid. In another preferred embodiment, the particles containing the active agent, are suitable for delivery in vivo agent to the desired object appropriate means, such as parenteral injection, tapicerki, orally, rectally, through the lungs, vaginally, through the cheek, under the tongue, subcutaneously, through the mucous membranes of the eye, inside the eye or through the ear.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 represents a two-dimensional phase diagram of dependence of the concentration of the active component from the temperature.

Figure 2 is a profile of decreasing temperature, illustrating the influence of solution temperature and cooling rate on the phase change of insulin in buffer solution of the polymer. At temperatures above 60°C the insulin remains in solution (region A). The region is a square optimal formation of small spherical particles bounded by the highest and the lowest rate of temperature change observed in the heat exchanger. Large cooling rate (area) lead to the formation of very small non-spherical particles, while low cooling rate (region D) lead to a mixture of small spherical particles of various sizes, along with particles of irregular shape and loose sediment.

Figa represents the image of the source material to insulin, resulting scanning electron micrographs (SAM).

Fig.3b is an image (SEM) of a small spherical particles of insulin (example 4).

Figure 4 is a HPLC analysis showing the preservation of the chemical stability of insulin in the preparation of small spherical particles, illustrating the chemical stability during the method of manufacturing micrope the insulin.

HPLC analysis showed no increase in high molecular weight compounds associated with the method, and the increase (relative to the feedstock of insulin) % dimer, % A21 of desamethasone, % late eluruumid peaks and % of other compounds within the USP.

Figure 5 is a schematic demonstration of reproducibility from portion to portion. The figure shows the distribution of small spherical particles of insulin (time-of-flight data Aerosizer). For all six parties received more than 96% of the particles were between 0,86 and 2.9 µm, with more than 60% fall between 1.6 and 2.5 μm. Less than 1.1% of small spherical particles were beyond the dimensions covered by the chart.

6 is a schematic demonstration of reproducibility from portion to portion. The figure shows the distribution of small spherical particles of insulin using a cascade impactor Andersen. Data are averages (means+/-TO (standard deviation)) results for the six batches of small spherical particles of insulin, resulting in the device Cyclohaler at 60 LM. EPD for steps 1, 2, 3 and 4 was 4.4; 3,3; 2.0 and 1.1 µm, respectively.

7 is a schematic diagram of the continuous flow method of producing small spherical particles of insulin in example 3.

Fig represents the image(scanning electron micrograph (at 10 KV and H increase)) small spherical particles of insulin, obtained in the continuous flow method of example 3.

Fig.9 represents the HPLC chromatogram of dissolved small spherical particles of insulin, resulting in method with a continuous stream of example 3.

Figa demonstrates the influence of sodium chloride (2.5 mg/ml) on the solubility of insulin in the volume of the tube (NaCl versus temperature). The figure presents the following data (elevation 60°C):

% NaCl0,10,30,50,70,91,11,31,51,7
Dissolution, °C777369676665636362
Residenee, °C58555042373429 2222

Fig.10b demonstrates the influence of sodium chloride (5 mg/ml) on the solubility of insulin in the volume of the tube (NaCl versus temperature). The figure presents the following data (elevation 60°C):

% NaCl0,81,21,62,02,42,83,23,64,0
Dissolution, °C777472656464636262
Residenee, °C484336302622<22<20<20

Figs demonstrates the influence of sodium chloride (10 mg/ml) on the solubility of insulin in the volume of the tube (NaCl against the temperature). The figure presents the following data (elevation 40°C):

% NaCl1,02,03,04,05,02,22,42,62,8
Dissolution, °C858374706874747473
Residenee, °C674633232039373735

% NaCl3,03,23,43,6the 3.84,0
Dissolution, the 717070707069
Residenee, °C313027262422

Fig.10d demonstrates the influence of sodium chloride (20 mg/ml) on the solubility of insulin in the volume of the tube (NaCl versus temperature). The figure presents the following data (elevation 40°C):

% NaCl1,02,03,04,05,0
Dissolution, °C8883807774
Residenee, °C6760494027

Five-10h illustrate the impact of salts on the solubility of the insulin.

Fig is a Raman spectrum si the th material of insulin, insulin isolated from small spherical particles, and insulin small spherical particles. See CR spectrum in the region of amide band I for the raw powder of insulin and powder of small spherical particles, and the corresponding solutions.

11 represents the results obtained at the cascade impactor Andersen for radiochango insulin of example 10. Results from a cascade impactor Andersen for99mTC radiochango powder insulin show a strong linkThTC with insulin before the first dog was injected dose, and after the last animal was delivered dose.

Fig represents the histogram of the P/I for example 8, showing the average value of the ratio P/I, equal 0,93 for the five tested dogs.

Fig is a scintigraphic appearance of light from example 8, where99mTC radiometry insulin was gomogenizirovannom distributed on the periphery of the lung. There is no visual confirmation deposition in the center of the lungs. It supports the unimodal size distribution of small spherical particles of insulin after administration to dogs.

Figa is a graph showing the circular dichroism (CD) for alpha-1-antitrypsin (AAT).

Fig.14b is a graph showing the dependence of activity on time storage when it is matney temperature in example 17.

Fig with is a graph showing the dependence of activity on the time of storage at 4°C in example 17.

Fig is a graph showing DSC, showing the first heating thermogram of 50 mg/ml rat sample in the BDS solution against BDS as standard.

Fig is a graph showing DSC, showing the first cooling thermogram of 50 mg/ml rat sample in the BDS solution against BDS as standard.

Fig is a graph showing DSC, showing a second heating thermogram of 50 mg/ml rat sample in the BDS solution against BDS as standard.

Fig is a graph showing DSC, showing a second cooling thermogram of 50 mg/ml rat sample in the BDS solution against BDS as standard.

Fig is a graph showing DSC, showing a second heating thermogram 45 mg/ml rat sample in acetate solution against acetate buffer as a reference.

Fig is a graph showing DSC, showing a second cooling thermogram 45 mg/ml rat sample in acetate solution against acetate buffer as a reference.

Fig is a graph showing DSC, showing a second heating thermogram of 1 mg/ml rat sample in the BDS solution against BDS buffer as reference. raat sample obtained by dissolving 3 small spherical particles rat in the BDS.

Fig is a graph showing DSC, showing the second thermogram cooling 1 mg/ml rat sample in BD solution against BDS buffer as a reference, raat sample obtained by dissolving 3 small spherical particles rat in the BDS.

Fig is a graph showing DSC, showing the first cooling thermogram small spherical particles made of the party.

Fig is a graph showing DSC, showing the first heating thermogram small spherical particles made of the party.

Figa is a graph showing DSC, showing a second cooling thermogram small spherical particles obtained party.

Fig.25b is a graph showing DSC, showing a second heating thermogram small spherical particles made of the party.

Fig is a graph showing data on particle size (TSI Corporation Aerosizer).

Fig is a SAM small spherical particles of human growth hormone (HGH).

Fig is a graph showing that the insulin in the form of microspheres is stable after storage in gas-propellant HFA 134a.

Fig is a graph comparing the aerodynamic qualities of insulin using three devices for inhalation, where the aerodynamic qualities of a small spherical particles of insulin were compared using three different devices for inhalation: IRD, Cyclohaler COI, Disphaler COI.

Fig is a graph showing data on the stability of small sphere the institutions particles of insulin in the initial insulin, when stored at 25°C, showing that when stored at 25°C percentage of education A21-desamethasone from the source material much more in comparison with the formation of small spherical particles of insulin. This result shows that a small spherical particles of insulin significantly more resistant to chemical attack than the original material, without the addition of stabilizing fillers.

Fig is a graph showing data on the stability of small spherical particles of insulin in the initial insulin stored at 37°C, showing that when stored at a temperature of 37°C, the percentage of education A21-desamethasone from the source material much more in comparison with the formation of small spherical particles of insulin. This result shows that a small spherical particles of insulin significantly more resistant to chemical attack than the original material, without the addition of stabilizing fillers.

Fig is a graph showing data on the stability of small spherical particles of insulin in the initial insulin stored at 25°C showing that when stored at 25°C percentage of formation of dimer and oligomer of insulin from the source material much more in comparison with the formation of bolshih spherical particles of insulin. This result shows that a small spherical particles of insulin significantly more resistant to chemical attack than the original material, without the addition of stabilizing fillers.

Fig is a graph showing data on the stability of small spherical particles of insulin in the initial insulin stored at 37°C, showing that when stored at a temperature of 37°C. the percentage of formation of dimer and oligomer of insulin from the source material much more in comparison with the formation of small spherical particles of insulin. This result shows that a small spherical particles of insulin significantly more resistant to chemical attack than the original material, without the addition of stabilizing fillers.

Fig is a graph showing data on the stability of small spherical particles of insulin in the initial insulin stored at 25°C showing that when stored at 25°C percentage of education total connections, related to insulin, from the source material much more in comparison with the formation of small spherical particles of insulin. This result shows that a small spherical particles of insulin significantly more resistant to chemical attack than the original material, without time to relax is of stabilizing fillers.

Fig is a graph showing data on the stability of small spherical particles of insulin in the initial insulin stored at 37°C, showing that when stored at a temperature of 37°C, the percentage of education total connections, related to insulin, from the source material much more in comparison with the formation of small spherical particles of insulin. This result shows that a small spherical particles of insulin significantly more resistant to chemical attack than the original material, without the addition of stabilizing fillers.

Fig represents the histogram of the aerodynamic stability of small spherical particles of insulin using dry powder inhaler (COI) Cyclohaler in the cascade impactor Andersen.

Fig is a light micrograph of a small spherical particles Gnkazy (light microscope Nikon, 100x oil immersion lens).

Fig is a graph showing the enzymatic activity Gnkazy.

Fig is a light micrograph of a small spherical particles SOD (light microscope Nikon, 100x oil immersion lens, a sample of dry powder).

Fig is a graph showing the enzymatic data for small spherical particles SOD (method for determining the activity described Worthington Biochemical Catalogue).

Figa-b are schematic illustrations of the reactor continuous emulsification, where figa is a schematic illustration of a reactor of the continuous emulsification, when surface-active compound is added to the continuous phase or the dispersed phase before emulsification, and FIGU is a schematic illustration of a reactor of the continuous emulsification, when surface-active compound is added after emulsification.

Fig illustrates the effect of PEG on the IVR profile PLLA-encapsulated particles HSA (example 32).

Fig illustrates the IVR profile of PLGA-encapsulated small spherical particles LDS (example 33).

Fig illustrates the effect of pH of the continuous phase on the IVR profile of PLGA-encapsulated small spherical particles of insulin (example 31).

Fig illustrates the IVR profile of PLGA-encapsulated small spherical particles of HGH (example 34).

Fig illustrates the influence of variables microencapsulation (pH of the continuous phase and the matrix material) to form dimers Ann in encapsulated Isms (example 35).

Fig illustrates the influence of variables microencapsulation (pH of the continuous phase and the matrix material) on the formation of VM particles encapsulated in INSs (example 35).

Fig illustrates in vivo selection of recombinant insulin, human is the ka of not encapsulated and encapsulated pre-manufactured small spherical particles of insulin in rats (example 36).

Fig is a SEM at 10 kV particles of example 27.

DETAILED description of the INVENTION

The present invention includes embodiments of many different forms. Preferred embodiments of the invention are stated with the understanding that this statement should be viewed as a series of examples of the principle of the invention without intending to limit the broad aspects of the invention are illustrated variants of implementation.

The present invention relates to methods of manufacture, methods of use and compositions of small spherical particles of the active agent. In accordance with the method of manufacture of an active agent dissolved in a solvent containing dissolved agent, a reinforcing phase separation to form a solution, which is a single continuous liquid phase. The solvent preferably is a water or miscible with water, the solvent. The solution is further subjected to a phase change, for example, by lowering the solution temperature below the phase transition temperature of the active agent, the active agent passes through the phase separation of liquid-solid with formation of a suspension of small spherical particles that constitute a discrete phase, while reinforcing phase separation agent about what remains in the continuous phase.

Phase

Continuous phase

Method of preparation of small spherical particles of an active agent according to the present invention starts with a solution having an active agent and a reinforcing phase separation agent, dissolved in the first solvent in a single liquid phase. The solution may be an organic system containing an organic solvent or a mixture of miscible organic solvents. The solution can also be a solution, based on the water containing the aquatic environment or miscible with water, an organic solvent, or a mixture of miscible with water, organic solvents, or combinations thereof. The aqueous medium may be water, salt solution or buffer solution, the buffer solution of the salt, and the like. Suitable miscible with water and organic solvents include, but are not limited to, N-methyl-2-pyrrolidinone (N-methyl-2-pyrrolidone), 2-pyrrolidinone (2-pyrrolidone), 1,3-dimethyl-2-imidazolidinone (SIAS), dimethylsulfoxide, dimethylacetamide, acetic acid, lactic acid, acetone, methyl ethyl ketone, acetonitrile, methanol, ethanol, isopropanol, 3-pentanol, n-propanol, benzyl alcohol, glycerol, tetrahydrofuran (THF), polyethylene glycol (PEG), PEG-4, PEG-8, PEG-9, PEG-12, PEG-14, PEG-16, PEG-120, PEG-75, PEG-150, esters of polyethylene glycol, PEG-4 dilaurate, P The G-20 dilaurate, PEG-6 isostearate, PEG-8 palmitostearate, PEG-150 palmitostearate, polietilenglikolsuktsinata, PEG-20 serbianization, monoalkyl ethers of polyethylene glycol, PEG-3 dimethyl ether, PEG-4 dimethyl ether, polypropyleneglycol (BCP), polypropyleneimine, GPR-10 butanediol, GPR-10 methyl glucose ether, GPR-20 methyl glucose ether, GPR-15 stearyl ether, propylene glycol dicaprylate/dicaprate, propilenglikolstearat and glycoluril (tetrahydrofurfuryl alcohol ether of propylene glycol), alkanes, including propane, butane, pentane, hexane, heptane, octane, Noonan, Dean, or their combination.

The only continuous phase can be obtained by first preparing a solution reinforcing phase separation agent, which is either soluble or miscible with the first solvent. This is followed by adding a solution of the active agent. The active agent can be added directly to the solution or the active agent may be first dissolved in the second solvent and then added to the solution. The second solvent can be the same solvent, the first solvent, or it may be a different solvent, selected from those listed above, and which is mixed with a solution. Preferably, the agent was added to the solution at ambient temperature or below, which is especially important for those who olabilmek molecules, such as some proteins. Temperature "environment" means a temperature of about room temperature, from about 20°to about 40°C. However, the system can also be heated to increase the solubility of the active agent in the system as long as the heat does not cause a significant reduction in the activity of the agent.

Reinforcing phase separation agent

Reinforcing phase separation agent (UVR) of the present invention enhances or induces phase separation of liquid-solid active agent from the solution when the solution is in the process of phase separation, in which the active agent is a solid or almost solid with formation of a suspension of small spherical particles as a discrete phase, while reinforcing phase separation agent remains dissolved in the continuous phase. Reinforcing phase separation agent reduces the solubility of the active agent, when the solution is in terms of phase separation. Suitable reinforcing phase separation agents include, but are not limited to, polymers or mixture of polymers that are soluble or are mixed with a solution. Examples of suitable polymers include linear or branched polymers. These polymers may be soluble, partially soluble in water, mixing the situations with water or insoluble.

In the preferred form of the invention the reinforcing phase separation agent is a water-soluble or miscible with water. The types of polymers which can be used include polymers based on carbohydrates, paleoliticheskie alcohols, poly(vinyl)polymers, polyacrylic acid, paliggenesia acid, polyaminoamide, copolymers and block copolymers (for example, poloxamer, such as Pluronics F127 or F68) tert-polymers, polyesters, polymers found in nature, polyimides, surfactants, polyesters, branched and cyclic polymers and polyallelic.

Preferred polymers are polymers that are acceptable as pharmaceutical additives for the intended route of administration of the particles of active agent. Preferred polymers are pharmaceutically acceptable additives such as polyethylene glycol (PEG) with different molecular weight, such as PEG 200, PEG 300, PEG 3350, PEG 8000, PEG 10000, PEG 20000, etc. and poloxamer, such as Pluronics F127 or Pluronics F68. Another preferred polymer is a polyvinylpyrrolidone (PVP). Another preferred polymer is gidroxiatilkrahmal. Other amphiphilic polymers can also be used individually or in combinations. Reinforcing phase separation agent may is not to be a polymer, for example, a mixture of propylene glycol and ethanol.

Phase separation of liquid-solid

Phase separation of liquid-solid active agent in the solution may be caused by any method known in this field, for example, temperature change, pressure change, change in pH, change in ionic strength of the solution, changing the concentration of the active agent, changes in the concentration of the reinforcing phase separation agent, the change of osmotic pressure of a solution, the combination of these factors, and the like.

In a preferred embodiment of the present invention, the phase change is a temperature induced phase change by lowering the temperature below the phase transition temperature of the active agent in solution.

Figure 1 represents a two-dimensional phase diagram 10 for a solution, solvent, UV and active agent. Chart depicts the dependence of the concentration of the active agent from the solution temperature. The concentration of UVR constant support.

The chart contains saturation curve 12, the curve glut 14; metastable area 16 between them; the first area 18 below the saturation curve, where the system is a homogeneous single liquid phase, where all components are in the liquid phase; and a second area 20 above the curve re is asimenia, where the system is a two-phase system, having a solid phase active agent and a liquid phase UV and solvent. The phase diagram is useful for determining the temperature of the system and the relative concentration of the components in the pure liquid phase, in-phase liquid-solid and the transition between these two phases.

As here set forth, preparing small spherical particles of an active agent principally involves the freezing of the unsaturated solution (point a') to achieve saturation at point a, where the solution is in equilibrium with any solid phase that may be present. When additional cooling is achieved when a solution contains more active agent than it corresponds to the equilibrium solubility at the given temperature; the solution thus becomes supersaturated. The spontaneous formation of the solid phase does not occur until the point is reached Century. This point represents the boundary of the metastable zone. The width of the metastable zone may be expressed or maximum achievable supercooling ∆Tmax=T2-T1or oversaturation ∆max=*2-C*1.These two expressions are thermodynamically equivalent:

Path A'-a-b represents on termicheski way to obtain metastable solution. In the isothermal method, the starting point would be. Increasing concentration at constant temperature, the saturation is again reached at point A. Isothermal increase in the concentration (for example, by evaporation of the solvent or the introduction of seed/plus active agent) to point C will cause a shift of the solution in the metastable state, while the metastable limit is again reached. When the metastable limit is exceeded, the solution becomes unstable, and immediately there is a spontaneous formation of a hard phase.

The value of (∆Cmax)T=*3-C*2received isothermal, may be different from the corresponding values of ∆Tmax=T3-T2received polythermal. When approaching the boundary of the metastable zone, decreases the time required for the formation of solid particles, while the metastable limit is reached.

In polythermal method of cooling is carried out at a controlled rate in order to adjust the size and shape of the particles. Under controlled velocity to understand the rate of approximately 0.2°C./minute to about 50°C./minute and, more preferably, from 0.2°C./minute to 30°C/minute. The rate of change may be constant or linear velocity, nonlinear speed, uneven or programmable speed (with cash is the chii many phase cycles).

Particles can be separated from the UV in solution and purified by washing, as will be discussed below.

In the present invention examined the regulation of the concentration of the active agent, the concentration of UV, temperature, or any combination of these parameters in order to cause a phase change in which the active agent passes from a liquid state to the solid state, while the UV and solvent do not pass through the phase change and remain liquid. Also consider changing pH, ionic strength, osmotic pressure and the like to enhance, promote, control or suppress the phase change. For solutions in which the freezing temperature is relatively high or freezing temperature above the phase transition temperature, the solution may include an agent that lowers the freezing point, for example, propylene glycol, sucrose, ethylene glycol, alcohols (e.g. ethanol, methanol) or water a mixture of agents that lower the freezing temperature, to lower the freezing point of the system and to make it possible phase change system without freezing the system. The method can also be carried out in such a way that the temperature is reduced below the freezing temperature of the system. The method described here is particularly suitable for molecules that are Ermoupoli (for example, proteins).

Possible fillers

Particles of the present invention may include one or more filler. The filler can give active agent or the additional particle characteristics, such as increased stability of the particles of active agent or carrier agents, controlled release of active agent from the particles or the modification of the penetration of the active agent through biological tissue. Suitable fillers include, but are not limited to, carbohydrates (e.g., trehalose, sucrose, mannitol), cations (e.g., Zn2+, Mg2+Ca2+), anions (e.g., SO42-), amino acids (e.g. glycine), lipids, phospholipids, fatty acids, surfactants, triglycerides, bile acids or their salts (for example, Holt or its salts, such as Holt sodium; desoxycholate acid or its salts), esters of fatty acids and polymers that are present at levels below their functioning as a UVR. When using the filler, the filler does not impact significantly on the phase diagram of a solution.

Separation and washing of the particles

In a preferred embodiment of the present invention a small spherical particles collecting, separating them from reinforcing phase separation agent in the solution. Another pre is respectful embodiment, the method of separation is a washing solution, containing small spherical particles and liquid medium in which the active agent is not soluble, while reinforcing phase separation agent is soluble. Some ways of washing may be diafiltrate or by centrifugation. The liquid medium can be an aqueous medium or an organic solvent. For active agents with low solubility in water liquid medium can be an aqueous medium or an aqueous medium containing agents, which reduce the solubility of the active agent, such as divalent cations. For active agents with high solubility in water, such as many proteins can be used organic solvents or aqueous solvents containing agent, precipitating the protein, such as ammonium sulfate.

Examples of suitable organic solvents for use as the liquid medium include organic solvents described above as suitable for the continuous phase, and a more preferred methylene chloride, chloroform, acetonitrile, ethyl acetate, methanol, ethanol, pentane and the like.

Also considered is the use of mixtures of any of these solvents. One preferred mixture represents a methylene chloride or a 1:1 mixture of methylene chloride and acetone. Preferably, the liquid medium had a low key is to be placed for easy removal, for example, lyophilization, evaporation or drying.

Liquid medium can also be a supercritical fluid, for example, liquid carbon dioxide, or a fluid near its critical point. Supercritical fluid may be suitable solvents for enhancing phase separation agents, in particular, some of the polymers, but are not solvents for protein particles. Supercritical fluids can be used alone or with a co-solvent. Can be used following supercritical fluid: liquid CO2, ethane or xenon. Potential co-solvents can be acetonitrile, dichloromethane, ethanol, methanol, water or 2-propanol.

The liquid medium used for the separation of small spherical particles from UVR described herein can contain an agent that lowers the solubility of the active agent in a liquid medium. Most preferably, the particles showed a minimum solubility in the liquid medium, to maximize the yield of particles. For some proteins, such as insulin or human growth hormone, decrease in solubility can be achieved by the addition of divalent cations, such as Zn2+for protein. Other ions that may be used for the formation of complexes include, but are not limited to, Ca2+, Cu+ , Fe2+, Fe3+and similar.

The solubility of complexes of insulin-Zn or growth hormone-Zn quite low, which makes it possible to diafiltration complex in aqueous solutions.

The liquid medium may also contain one or more filler, which can provide an active agent or a particle of additional properties such as increased stability of the particles and/or active or carrier agents, controlled release of active agent from the particles, or the modification of the penetration of the active agent through biological tissue, as discussed earlier.

In other forms of the invention the small spherical particles are not separated from the solution containing the UV.

The method, based on the aqueous system

In another preferred embodiment, the method of obtaining this system is an aqueous system comprising water or miscible with water, the solvent. Examples of suitable mixed with water solvents include, but are not limited to, those identified above for the continuous phase. The advantage of using based on the water system of the method is that the solution can be buffered and may contain fillers, which provide biochemical stabilization to protect active agents such as proteins.

The act is wny agent

The active agent of the present invention, preferably is a pharmaceutically active agent, which can be a therapeutic agent, diagnostic agent, cosmetic, food additive or pesticide.

therapeutic agent can be a biological substance, which includes, but not limited to, proteins, polypeptides, carbohydrates, polynucleotides and nucleic acids. Protein can be an antibody, which may be polyclonal or monoclonal. therapeutic agent may be a low molecular weight. In addition, therapeutic agents can be selected from a variety of known pharmaceutical substances, but are not limited to: analgesics, anesthetics, analeptics, adrenergic agents, adrenergic blocking agents, adrenolytic, adrenocorticoid, agonists, anticholinergics agents, anticholinesterases, anticonvulsants, alkylating agents, alkaloids, allosteric inhibitors, anabolic steroids, anorexiant, antacids, agents against diarrhea, antidotes, antipolice, antipyretics, Antirheumatic agents, psychotherapeutic agents, neural blocking agents, anti-inflammatory agents, anthelmintic, antiarrhythmic agents, antibiotics, anticoagulants, antidepressants is, antidiabetic agents, antiepileptic, antifungal agents, antihistamines, protivogipertonicheskoe agents, agents antimuskarinovoe act occurs, antimycobacterial agents, antimalarial agents, antiseptics, antineoplastics agents, Antiprotozoal agents, immunosuppressants, Immunostimulants, antithyroid agents, antiviral agents, tranquilizers, astringency, beta-adrenoceptor blocking agents, contrast media, corticosteroids, protivokashlevy agent, diagnostic agents, diagnostic reproducing agents, diuretics, dopaminergic, hemostatic, hematological agents, modifiers of hemoglobin, hormones, hypnotics, immunologicheskie agents, antihyperlipidemic and other agents that regulate lipid, muscarinic, muscle relaxers, parasympathomimetics, parathyroid hormone, calcitonin, prostaglandins, radiopharmaceutical agents, sedative agents, sex hormones, anti-allergic agents, stimulants, sympathomimetics, thyroid agents, vasodilators, agents, vaccines, vitamins and xantina. Antineoplastic or anticancer agents include, but are not limited to, paclitaxel and derivatives connections, and other antineoplastic selected from the group consisting of alkaloids, antimetabolites, inhibitors of enzymes, alquiler the participating agents and antibiotics.

Cosmetic agent is any active ingredient, is able to have a cosmetic activity. Examples of these active ingredients can be, for example, softening agents, moisturizers, agents that inhibit free radicals, anti-inflammatory agents, vitamins, depigmentation agents, protivougrevoe agents, agents against seborrhea, keratolytic, agents for weight loss, coloring the skin agents and sunscreen agents, and, in particular, linoleic acid, retinol, retinova acid, complex alkalemia esters of ascorbic acid, polyunsaturated fatty acid, nicotinic esters, tocopherylacetate, unsaponifiable residues of rice, soy or tree oil, ceramide, hydroxy acids such as glycolic acid, derivatives of selenium, antioxidants, beta-carotene, gamma-oryzanol and sterilisers. Cosmetics commercially available and/or can be prepared by technology, well known to specialists in this field.

Examples of food additives considered for use in practice of the present invention, include, but are not limited to, proteins, carbohydrates, water-soluble vitamins (such as vitamin C, b-complex vitamins, and the like), fat-soluble vitamins (such as vitamins A, D, E, K and the like) and herbal extracts, Food additives are commercially available and/or can be prepared by technology, well-known specialists in this field.

The term "pesticide" is understood in the aggregate, herbicides, insecticides, acaricides, nematicides, ectoparasiticide and fungicides. Examples of classes of compounds can belong to the pesticide in the present invention include urea, triazine, triazole, carbamates, esters of phosphoric acid, dinitroanilines, morpholines, acylalanines, pyrethroids, esters benzyl acid, diphenyl ethers, and polycyclic halogenated hydrocarbons. Specific examples of pesticides each of these classes are registered in the Pesticide Manual, 9th Edition, British Crop Protection Council. Pesticides are commercially available and/or can be prepared by technology, well known to specialists in this field.

In a preferred embodiment of the present invention the active agent is a macromolecule such as a protein, polypeptide, carbohydrate, polynucleotide, virus, or nucleic acid. Nucleic acids include DNA, oligonucleotides, anticysticeral oligonucleotides, optimera, RNA and SiRNAs. The macromolecule may be natural or synthetic. Protein can be an antibody that can be monoclonal or Polikanov. The protein may also be any known therapeutic protein isolated from natural sources is of IKI or received or synthetic recombination methods. Examples of therapeutic proteins include, but are not limited to, proteins of the cascade of blood clotting (for example, Factor VII, Factor VIII, Factor IX and other), subtilisin, egg albumin, alpha-1-antitripsin (AAT), Tnkase, peroxide dismutase (UNDER), lysozyme, ribonuclease, hyaluronidase, collagenase, growth hormone, erythropoietin, insulin-like growth factors or their analogues, interferons, glatiramer, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, antibodies, Paglierani proteins, glycosylated or sverkhvinoslivie proteins, desmopressin, LHRH agonists, such as leuprolide, goserelin, nafarelin, buserelin; LHRH antagonists, vasopressin, cyclosporine, calcitonin, the hormone of the parathyroid gland, a peptide hormone of the parathyroid gland and insulin. Preferred therapeutic proteins are insulin, alpha-1-antitripsin, LHRH antagonists, and growth hormones.

Examples of therapeutic molecules with a low molecular weight include, but are not limited to, steroids, beta-agonists, antimicrobial agents, antifungal agents, taxanes (antimitoticescoy and antimicrotubule agents), amino acids, aliphatic compounds, aromatic compounds and urea.

In a preferred embodiment, the active Agay is t is a therapeutic agent for the treatment of pulmonary disorders. Examples of such agents include, but are not limited to, steroids, beta-agonists, antifungal agents, antimicrobial compounds, bronchial dialator, Antiasthmatic agents, non-steroidal anti-inflammatory agents (NCPAG), alpha-1-antitripsin and agents for the treatment of cystic fibrosis. Examples of steroids include, but are not limited to, beclomethasone (including beclomethasone), fluticasone (including fluticasonet), budesonide, estradiol, fludrocortisone, fluocinonide, triamcinolone (including triaminobenzene) and flunisolide. Examples of beta-agonists include, but are not limited to, salmeterol, xinafoate, formaterror, left-albuterol, bambuterol and tulobuterol.

Examples of antifungal agents include, but are not limited to, Itraconazole, fluconazole and amphotericin Century

Diagnostic agents include an agent that reproduces the x-ray exposure, and contrast media. Examples of agents reproducing x-ray images include WIN-8883 (ethyl-3,5-diacetamido-2,4,6-triiodobenzoate), also known as complex ethyl ester diatrizoic acid (AEDC), WIN 67722, ie (6 ethoxy-6-oxohexyl-3,5-bis(acetamido)-2,4,6-triiodobenzoate); ethyl-2-(3,5-bis(acetamido)-2,4,6-triiodobenzoate)butyrate (WIN 16318); ethyldiethanolamine is at (WIN 12901); ethyl-2-(3,5-bis(acetamido)-2,4,6-triiodobenzoate)propionate (WIN 16923); N-ethyl-2-(3,5-bis(acetamido)-2,4,6-triiodobenzoate)ndimethylacetamide (WIN 65312); isopropyl-2-(3,5-bis(acetamido)-2,4,6-triiodobenzoate)ndimethylacetamide (WIN 12855); diethyl-2-(3,5-bis(acetamido)-2,4,6-triiodobenzoate)malonate (WIN 67721); ethyl-2-(3,5-bis(acetamido)-2,4,6-triiodobenzoate)phenylacetate (WIN 67585); perpendicularly acid, [[3,5-bis(acetylamino)-2,4,5-triiodobenzoic]oxy]bis(1-methyl)ester (WIN 68165) and benzoic acid, 3,5-bis(atsetamino)-2,4,6-triiodo-4-(ethyl-3-ethoxy-2-butenoate)ester (WIN 68209). Preferred contrast agents include those that dissolve relatively quickly in the body, thus minimizing any associated with particles of an inflammatory response. The decomposition can be the result of enzymatic hydrolysis, dissolution of carboxylic acids at physiological pH, or other mechanisms. Thus, it may be preferable iodirovannoye carboxylic acids, such as iodipamide, diacrisia acid and matricola acid together with hydrolytically labile iodirovannoye representatives, such as WIN 67721, WIN 12901, WIN 68165 and WIN 68209 or other.

The number of combinations of active agents may be desirable, including, for example, a combination of steroid and beta-agonist, for example, fluticasonet and salmeterol on the list budezonida and formet the Rola etc.

Examples of carbohydrates are dextrans, GetAttachment, cyclodextrins, alginates, chitosans, chondroitin, heparin and the like.

Small spherical particles

Particles and small spherical particles of the present invention, preferably, have an average geometric particle size of from about 0.01 µm to about 200 µm, more preferably from 0.1 μm to 10 μm and even more preferably from about 0.5 microns to about 5 microns, and most preferably from about 0.5 μm to about 3 μm, as measured by means of dynamic light scattering (for example, photocorrelation spectroscopy, laser diffraction laser diffraction small-angle scattering (LDAR), laser diffraction with an average angle of scattering (LDAR), ways of shading devices (for example, a method analysis of Coulter) or in other ways, such as rheology, or microscopy (light or electron). Particles for delivery to the lungs will have the aerodynamic particle size, determined by measuring the time of flight (for example, Aeroespacial) or measurements using cascade impactor Andersen.

Small spherical particles are essentially spherical. "Essentially spherical" means that the ratio of the length of the longest to the shortest perpendicular to the axis of the cross section of the part is s less than or equal to about 1.5. Essentially, the sphericity does not require the symmetry axis. In addition, the particles can have a surface texture, such as lines or dimples or bumps that are small in scale when compared to the full size of the particles, and still are, essentially spherical. More preferably, when the ratio of the lengths of the longest and the shortest axes of the particles are less than or equal to about 1.33. Most preferably, when the ratio of the lengths of the longest and the shortest axes of the particles are less than or equal to approximately 1.25. Contact surface minimum in the microspheres, which are essentially spherical, which minimizes undesirable agglomeration of the particles during storage. Many crystals or flakes have a flat surface that can lead to a large area of surface contact, where agglomeration may occur ionic or non-ionic interaction. The field allows you to communicate on much smaller area.

Particles are also preferably the same size particles. Particles having a wide size distribution, where there are relatively large, and small particles allow smaller particles to fill the interstices between large particles, thus creating new contact surface. A wide size distribution can lead to the larger areas with a large number of possibilities of contact for linking agglomeration. This invention creates spherical particles with a narrow distribution in size, thus minimizing opportunities for contact agglomeration. "Narrow size distribution" means that the preferred distribution of particle size will have a volumetric ratio of diameter 90thpercentile of the small spherical particles to the volume diameter of 10thpercentile less than or equal to 5. More preferably, when the ratio of the volume diameter of the 90thpercentile of the small spherical particles to the volume diameter of 10thpercentile less than or equal to 3. Most preferably, when the distribution of particle size will have a volumetric ratio of diameter 90thpercentile of the small spherical particles to the volume diameter of 10thpercentile less than or equal to 2.

Geometric standard deviation (GSO) can also be used to characterize a narrow distribution in size. GSO estimates include the determination of the effective marginal diameter (EPD) the accumulation of less than 15.9% and 84,1%. GSO is equal to the square root of the ratio EPD smaller than 84,17% EPD less than 15.9 per cent. GSO has a narrow size distribution, when GSO<a 2.5, more preferably less than 1.8.

In a preferred embodiment of the invention the active agent is a small spherical particles is a semi-crystalline or non-crystalline substance.

Typically, when a small spherical particles obtained by the method of the invention are essentially non-porous and have a density greater than 0.5 g/cm3more preferably, greater than 0.75 g/cm3and most preferably greater than 0.85 grams/cm3. The preferred range of density is from about 0.5 to about 2.0 g/cm3more preferably from about 0.75 g/cm3up to around 1.75 g/cm3and most preferably from about 0.85 grams/cm3to about 1.5 g/cm3.

Particles of this invention can exhibit a high content of the active agent. There are no requirements significant quantities of bulk agents or similar additives that are required in many other ways of making particles. For example, small spherical particles of insulin consist of equal to or greater than 95 wt.%. the number of particles. However, particles can be enabled volumetric agents or fillers. Preferably, when the active agent is from 0.1% to more than 95% wt. particles, more preferably from about 30% to about 100 wt.%, even more preferably from about 50% to about 100 wt.%, even more preferably from about 75% to about 100 wt.%, and most preferably more than 90 wt.%. The establishment here of these intervals about the mean, they include any range or combination of them.

An additional aspect of the present invention is that a small spherical particles retain biochemical purity and biological activity of the active agent with or without the addition of fillers.

Delivery of particles in vivo

The particles containing the active agent of the present invention, suitable for in vivo delivery of the agent to the desired object suitable means, such as injection, tapicerki, orally, rectally, through the nose, through the lungs, vaginally, through the cheek, under the tongue, subcutaneously, through the mucous membranes, through the ears, inside the eye, through the eye. The particles can be delivered as a stable liquid suspension or receptionby as a solid dose form, such as tablets, pills in the form of capsules, capsules, etc. Preferred way of delivery is an injection, which includes intravenous, intramuscular, subcutaneous, intraperitoneal, intrathecal, epidural, intra-arterial, intra-articular, and the like. Another preferred way of delivery is a pulmonary inhalation. In this way the delivery of the particles can be deposited deep in the lungs, upper respiratory tract or somewhere in the respiratory tract. The particles can be delivered as a dry powder using a nebulizer with the steamboat powder or they may be delivered using a metered dose inhaler or nebulizer.

Drugs for systemic effects, such as insulin, it is desirable to precipitate in the alveoli, which has a very large surface area available for absorption in the bloodstream. If the goal is the deposition of the drug on specific areas inside the lungs, the aerodynamic diameter of the particles can be adjusted to an optimum interval by changing the fundamental physical characteristics of particles, such as shape, density and particle size.

Acceptable respirable fraction of particles of a drug by inhalation are often achieved by adding filler to the recipe or the introduction into the composition of the particles, or in the form of a mixture with particles of a drug. For example, to improve the dispersion of the crushed particles of the drug (about 5 μm) is affected by the mixing with large (30-90 μm) particles of inert carrier, such as trehalose, lactose or maltodextrin. Large particles of filler improves the flow properties of the powder, which correlates with the pharmacodynamic effect. With additional improvements to the fillers are injected directly into small spherical particles, which affects the quality of the aerosol, and also potentially increases the stability of protein drugs. In General, the fillers are chosen so that they were tentative the but FDA approved for inhalation, such as lactose or organic molecules endogenous to the lungs, such as albumin and dipalmitoyl-DL-α-phosphatidylcholine (DPPC). Other fillers such as poly(lactic acid-co-glycolic acid) (PMHC) used to create particles with the desired physical and chemical characteristics. However, extensive experience inhalation fillers approved by the FDA, had with Antiasthmatic drugs with large aerodynamic size of the particles to precipitate them in the tracheobronchial area, and which do not penetrate significantly deep into the lungs. About inhaled protein or peptide, a therapeutically delivered deep into the lungs, there is a concern that may appear undesirable long-term side effects such as inflammation or irritation that can be caused by an immunological response or fillers when they are delivered in the alveolar area.

In order to minimize the potentially harmful side effects from the deep lung inhalation of therapeutic agents, it may be appropriate to produce particles for inhalation, which essentially consist only of the delivered drug. This strategy can minimize alveolar impact fillers and reduce the total weight of the particles, the ex is made on the alveolar surfaces with each dose, if possible, minimizing irritation in chronic use of inhaled therapeutics. Small spherical particles with an aerodynamic properties, suitable for deposition deep in the lung, which essentially consist completely of a therapeutic protein or peptide, can be particularly useful for individual study the effects of chronic therapeutic dosing the alveolar membrane of the lung. The influence of the systematic delivery of protein or peptide in the form of small spherical particles in inhalation can then be studied without complicating factors introduced by appropriate fillers.

Requirements for delivery of particles deep into the lungs during inhalation consist in the fact that the particles had a small median aerodynamic diameter of 0.5 to 10 μm and a narrow size distribution. The invention also considers the mixing together of different batches of particles having different intervals of particle size. The method according to the present invention enables to produce small spherical particles with the above characteristics.

There are two main approaches to the formation of particles with an aerodynamic diameter of from 0.5 to 3 μm. The first approach is to obtain relatively large, but very porous (or perforated) microca the tics. Since there is a relationship between aerodynamic diameter (Daerodynamic) and a geometric diameter (Dgeometric), Daerodynamicequal Dgeometricmultiplied by the square root of the particle density, particles with very low bulk density (about 0.1 g/cm3) may have slight aerodynamic diameter (0.5 to 3 microns), while relatively large geometric diameter (5 to 10 µm).

An alternative approach is the obtaining particles with a relatively low porosity, in the case of the present invention the particles have a density set forth in interval above, and usually it is close to 1 g/cm3. Thus, the aerodynamic diameter of such non-porous dense particles close to their geometric diameter.

This method of particle formation, described above, relates to the formation of particles with fillers or no fillers.

The production of small spherical particles of protein from the protein without supplements provides great advantages when used in pulmonary delivery, because it offers choice for large payloads drug, increases safety and reduces the number of inhalations.

Microencapsulation made earlier small spherical ASTIC

Small spherical particles of the present invention or small particles, prepared in other ways (including microparticles, microspheres, nanospheres, nanoparticles, etc.), may optionally be encapsulated in the matrix of the material forming the wall, for education microencapsulating particles. Microencapsulation can be carried out by any method known in this field. In a preferred embodiment, the microencapsulation small spherical particles of the present invention or any other small particles perform ways emulsification/solvent extraction, as described below. The matrix may transfer the property to a prolonged release of the active agent, the resulting rate allocation that persist from minutes to hours, days or weeks in accordance with the desired therapeutic use. Microencapsulation particles can also delay the allocation formulas previously obtained a small spherical particles. In the preferred embodiment, pre-fabricated small spherical particles are particles of macromolecules. In another preferred embodiment, the macromolecule is a protein or polypeptide.

In the way emulgin the tion/extraction solvent emulsification is conducted by mixing two immiscible phases, continuous phase and discrete phase (also known as the dispersed phase), for the formation of the emulsion. In a preferred embodiment, the continuous phase is an aqueous phase (water phase), and the discrete phase is an organic phase or an oil phase to form the emulsion, oil-in-water (M/V). Discrete phase may further contain a dispersion of solid particles or as a fine suspension, or as a fine dispersion, forming a solid phase-in-oil (T/M). The organic phase is preferably not miscible with water or partially miscible with water, an organic solvent. The mass ratio of the organic phase and the aqueous phase is in the range from about 1:99 to about 99:1, more preferably from 1:99 to about 40:60, and most preferably from about 2:98 to about 1:3, or any range or combination of these intervals. In a preferred embodiment, the ratio of organic and aqueous phase is approximately 1:3. The present invention further consider the use of inverted emulsions or emulsions water-in-oil (W/M), where the oil phase forms the continuous phase and the aqueous phase forms a discrete phase. The present invention further consider used the e emulsions, having more than two phases such as emulsion oil-in-water-in-oil (M//M) or emulsion water-in-oil-in-water (W/M/In).

In a preferred embodiment, the method microencapsulation using the method of emulsification/solvent extraction, begins with the preparation of pre-fabricated small spherical particles by the methods described previously, and the organic phase containing the forming wall material. Pre-manufactured small spherical particles dispersed in the organic phase from forming wall material to form a solid phase-in-oil (T/M)containing a dispersion of pre-fabricated small spherical particles in the oil phase. In a preferred embodiment, the dispersion is complete homogenization of the mixture of small spherical particles and the organic phase. Water forms the continuous phase. In this case, the emulsion system is formed by emulsification T/M phase with a water phase is an emulsion system solid-in-oil-in-water (T/M/B).

Forming wall material refers to materials capable of forming the structural nature of the matrix individually or in combination. Biorazlagaemykh forming wall materials are preferred, particularly for injectable applications. Examples so the x materials include, but not limited to, family polylactide/polyglycolide polymers (PLGP), polyethylene glycol conjugate PLGP (PLGP-PEG) and triglycerides. In the embodiment, in which the use PGP or PGP-PEG, PLGP preferably have a ratio of polylactide to polyglycolides from 100:0 to 0:100, more preferably from about 90:10 to about 15:85, and most preferably about 50:50. In General, the larger the ratio of polyglycolides to the polylactide in the polymer, the more hydrophilic are microencapsulation particles, resulting in faster hydration and to more rapid decomposition. Can also be used PLGP with different molecular weight. In General, when the same ratio polyglycolides and polylactide in the polymer, the greater the molecular weight PLGP, the slower the release of active agent and the wider size distribution microencapsulating particles.

Organic solvent in the organic phase (oil phase) oil-in-water (M/C) or emulsion of a solid-in-oil-in-water (T/M/In) may not be miscible with water or partially miscible with water. The term is not miscible with water solvent" is understood as such solvents, which form the boundary of the meniscus when combined with an aqueous solution in a ratio of 1:1 (M/B). Appropriate is not miscible with the ode solvents include, but not limited to, substituted or unsubstituted, linear, branched or cyclic alkanes with the number of carbon atoms between 5 and higher, substituted or unsubstituted, linear, branched or cyclic alkenes with carbon atoms 5 and above, substituted or unsubstituted, linear, branched or cyclic alkynes with the number of carbon atoms between 5 and higher aromatics, fully or partially halogenated hydrocarbons, ethers, esters, ketones, mono-, di - or triglycerides, natural oils, alcohols, aldehydes, acids, amines, linear or cyclic silicones, hexamethyldisiloxane or any combination of these solvents. Halogenated hydrocarbons include, but are not limited to, carbon tetrachloride, methylene chloride, chloroform, tetrachloroethylene, trichloroethylene, trichlorethane, fluorocarbons, chlorinated benzene (mono, di, three), Trichlorofluoromethane. Particularly suitable solvents are methylene chloride, chloroform, diethyl ether, toluene, xylene and ethyl acetate. The term "partially miscible with water solvents" refers to those solvents that do not mix with water at the same concentration and mixed with water at the other lower concentration. Examples of partially miscible with water solvents are tetrahydrofur the na (THF), propylene carbonate, benzyl alcohol and ethyl acetate.

Can be added surface-active compound, for example, to increase the wetting properties of the organic phase. Surface-active compound can be added prior to emulsification to the aqueous phase to the organic phase, to both and to the aquatic environment and to an organic solution or emulsion after emulsification. The use of surfactants can reduce the number of unencapsulated public or partially encapsulated small spherical particles, resulting in reduction of the initial release of the active agent at the time of selection. Surface-active compound can be added to the organic phase or the aqueous phase, or both - and the organic and aqueous phases, depending on the solubility of the compounds.

The term "surface-active compounds" refer to compounds such as anionic, cationic, amphoteric, non-ionic surfactants or biological surface-active molecules. Surface-active compound can be present in an amount by weight of the aqueous phase or the organic phase or the emulsion depending on the application, less than about 0.01% to about 30%, more preferably from about 0.01% to about 10%, or any combination of these interval is.

Suitable anionic surfactants include, but are not limited to, potassium laurate, sodium lauryl sulfate, sodium dodecylsulfate, alkylpolyoxyethylene, sodium alginate, dioctylsulfosuccinate sodium, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, fosfatidov acid and its salts, glycerol esters, sodium carbometalation, haliewa acid and other bile acids (for example, haliewa acid, desoxycholate acid, glycocholate acid, taurocholate acid, glikogenofiksiruta acid) and their salts (e.g. sodium deoxycholate and so on).

Suitable cationic surfactants include, but are not limited to: Quaternary ammonium compounds such as benzalkonium chloride, bromide, cetyltrimethylammonium, chloride of lauryldimethylamine, hydrochloride of acylcarnitine or halides alkylpyridine. As anionic surfactants can be used phospholipids. Suitable phospholipids include, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, fosfatidov acid, lysophospholipid, phospholipids eggs or soybeans, or their combination. Phospholipids can be salt or salt-free, hydrogenated sludge is partially hydrogenated, or natural, semi-synthetic or synthetic.

Suitable nonionic surfactants include esters polyoxyethylene fatty alcohol (Macrogol and Brij), polyoxyethylene esters sorbitan and fatty acids (Polysorbates), esters polyoxyethylene fatty acids (Myrj), esters sorbitan (Span), glycerol monostearate, polyethylene glycols, polypropylenglycol, cetyl alcohol, cetosteatil alcohol, stearyl alcohol, arylalkylamine alcohols, copolymers of polyoxyethylene-polyoxypropylene (poloxamer), poloxamine, polyvinyl alcohol, polyvinylpyrrolidone and polysaccharides including starch and starch derivatives, such as gidroxiatilkrahmal (BSE), methylcellulose, hydrocellulose, hydroxypropylcellulose, hypromellose and non-crystalline cellulose). In the preferred form of the invention the nonionic surfactant is a copolymer of polyoxyethylene and polyoxypropylene and preferably block copolymers of polypropylenglycol and ethylene glycol. Such polymers are sold under the trade name POLOXAMER also sometimes refer to PLURONIC®, and are sold by several vendors, such as Spectrum Chemical and Ruger. Among the esters polyoxyethylene fatty acids are those that have a short alkyl chain. One is m an example of such a surfactant is SOLUTOL® HS 15, polyethylene-660-hydroxystearate produced by BASF Aktiengesellschaft.

Surface-active biological molecules include molecules like albumin, casein, heparin, hirudin, GetAttachment and other suitable biochemical agents.

In a preferred embodiment of the invention the aqueous phase includes protein as a surface-active compounds. The preferred protein is albumin. Protein can also function as a filler. In the variants of implementation, in which the protein is not surface-active compound, other fillers may be incorporated in the emulsion by adding either before or after emulsification. Suitable fillers include, but are not limited to, saccharides, disaccharides and sugar alcohols. The preferred disaccharide sucrose is the preferred sugar alcohol is mannitol.

In addition, the use canalobre agents such as polyethylene glycol (PEG), can increase the rate of permeability of the final product, which leads to a change of the initial kinetics of excretion of the active agent from the matrix, as well as to changes in the decay rate of the matrix and depend on the decomposition kinetics of selection when changing the speed of hydration. The use of PEG as a channel-forming agent during encapsul the simulation can be advantageous in the sense of excluding part of the washing process during the production of small spherical particles, where PEG is used as the reinforcing phase separation agent. In addition, changes in pH of the continuous phase with the use of buffers can significantly increase the wetting between the surface of the particles and an organic phase, therefore, the result is a significant reduction in the initial release of the encapsulated therapeutic agent from the matrix microencapsulating particles. The properties of the continuous phase can also be modified, for example, increasing its salinity by adding a salt such as NaCl, which reduces the Miscibility of the two phases.

After dispersion of small spherical particles in the organic phase (oil phase) continuous phase of the water environment (water phase) is vigorously stirred, for example, by homogenization or ultrasonic treatment, with discrete phase organic phase to form an emulsion containing emulsified droplets embryonic microencapsulating particles. The continuous aqueous phase may be saturated with an organic solvent used in the organic phase prior to mixing the aqueous phase and organic phase in order to minimize the quick selection of the organic solvent from the emulsified droplets. The emulsification can be carried out at any temperature at which the mixture can maintain its fluid properties. Stabiles the ü emulsion is a function of the concentration of surface-active compounds in the organic phase or in the aqueous phase, or in emulsion, if the surface-active compound is added to the emulsion after emulsification. This is one of the factors that determines the size of the droplets in the emulsion system (embryonic microencapsulation particles) and the size and size distribution microencapsulating particles. Other factors affecting the size distribution microencapsulating particles represent the viscosity of the continuous phase, the viscosity of the discrete phase, effect of shear during emulsification, the type and concentration of surface-active compounds and the ratio of oil/water.

After emulsification, the emulsion is then transferred to the environment hardening. Wednesday solidification solvent extracts of discrete phases of embryonic microencapsulating particles, resulting in formation of a solid microencapsulating particles having a solid polymeric matrix around pre-fabricated small spherical particles in the vicinity of emulsified droplets. In the embodiment, systems M/b or T/M/In the environment of solidification is an aqueous medium which may contain surface-active compounds or agents that create volume, or other fillers. Microencapsulation particles are preferably spherical with a particle size of from about 0.6 to about 300 MK is, and more preferably, from about 0.8 to about 60 microns. In addition, microencapsulation particles preferably have a narrow size distribution. To reduce the extraction time is discrete phase to the environment hardening can be applied heating or reduced pressure. The speed of extraction of discrete phases of embryonic microencapsulating particles is an important factor in the degree of porosity of the final microencapsulating particles, because of the rapid destruction of, for example, by evaporation (the effect of boiling), discrete phase leads to the destruction of the integrity of the matrix.

In a preferred embodiment, the emulsification is conducted in a continuous way, and not periodic. Fig depicts the design of the reactor continuous emulsification.

In another preferred embodiment, the hardened, forming a wall of a polymeric matrix that encapsulates a small spherical particles of an active agent, optionally collected by centrifugation and/or filtration (including diafiltration) and washed with water. The remaining liquid phase can optionally be removed in such a way as lyophilization or evaporation.

A.Small spherical particles of insulin

Example 1

A common way to obtain small spherical particles of insulin

Prepared buffer solution with a pH 5,65 (buffer, 0,M sodium acetate)containing 16,67% PEG 3350. Was added to this solution with stirring, a concentrated suspension of crystalline insulin zinc. The insulin concentration in the final solution was made 0.83 mg/ml Solution was heated to about 85 to 90°C. the insulin Crystals were completely dissolved in this temperature interval within five minutes. Small spherical particles of insulin was started to be formed at a temperature of about 60°C. when the temperature of the solution was reduced at a controlled rate. The yield increased with increasing concentration of PEG. In this way received a small spherical particles with different distribution in size, with an average size of 1.4 microns.

Received a small spherical particles of insulin was separated from the PEG lavage microparticles diafiltrate under conditions in which small spherical particles are not dissolved. Small spherical particles of insulin washed from the suspension using an aqueous solution containing Zn2+. Zn2+ion lowers the solubility of insulin and prevents dissolution, which reduces yield and causes the agglomeration of small spherical particles.

Example 2

Periodic process without stirring to obtain small spherical particles of insulin

202 mg of crystalline insulin zinc suspended in 1 ml of deionized water at room temperature. Insulin was added 50 microliters 0,5h. HCl. 1 ml of deionized water was added to form a solution of 10 mg/ml zinc crystalline insulin. 12.5 g of polyethylene glycol 3350 (Sigma) and 12.5 g of polyvinylpyrrolidone (Sigma) was dissolved in 50 ml of 100 millimolar buffer of sodium acetate, pH of 5.7. The volume of polymer solution was brought to 100 ml of a buffer of sodium acetate. To 800 μl of the polymer solution in an Eppendorf tube was added 400 μl of a solution of insulin 10 mg/ml Solution of insulin/polymer mixing became opaque. A control experiment was prepared using water instead of the polymer solution. The Eppendorf tubes were heated in a water bath at 90°C for 30 minutes without agitation or stirring, then was removed and placed in ice for 10 minutes. A solution of insulin/polymer became transparent when removed from the water bath at 90°C, but started to thicken when cooled. Supervisory experience without polymer remained transparent the entire experiment. Particles insulin/polymer was isolated by centrifugation tubes, followed by washing twice for removal of the polymer. The last suspension in water was dried freeze drying to obtain a dry powder. SAM analysis of freeze dried drying particles insulin/polymer of the tubes showed a homogeneous distribution of small spherical particles about 1 μm in diameter. Analysis of light scattered is I Coulter showed a narrow distribution of particle size with an average particle size, equal 1,413 µm, 95% confidence interval 0,941-of 1.88 μm and a standard deviation equal to 0,241 μm. Insulin control experience without polymer and the washing steps, but otherwise processed and the last freeze drying in the same way, showed only cereal (no particles) analysis SAM, similar in appearance to that usually obtained after freeze-drying of proteins.

Example 3

Continuous flow method of obtaining the small spherical particles of insulin

of 36.5 mg of insulin was weighed and suspended in 3 ml of deionized water. Added 30 μl of 1N. HCl to dissolve the insulin. The final volume of solution was brought to the 3.65 ml of deionized water. 7.3 ml of a solution of PEG/PVP (25% PEG/PVP pH of 5.6 in 100 mm buffer from the NaO) was added to a solution of insulin to a final total volume of a solution of insulin, equal 10,95 ml of the Solution was then shaken for the formation of a homogeneous suspension of insulin and PEG/PVP.

Suspension of insulin was connected to a peristaltic pump BioRad, Sipper with a speed of 0.4 ml/min through a Teflon® tube (TFE 1/32" inner diameter flexible tubing). The tube after the pump was immersed in a water bath, supported at 90°C, before entering into the collecting pipe is lowered into the ice. Small spherical particles of insulin was formed when the temperature of a solution of insulin was decreased from 90°C in Wodan the second bath to approximately 4°C in the tube. 7 is a schematic diagram of this method. The total time of the process was 35 minutes for a volume equal 10,95 Jr. After collecting a small spherical particles capped tube was centrifuged at 3000 rpm for 20 minutes in a centrifuge Beckman J6B. Secondary leaching water was finished and the balls are small spherical particles was centrifuged at 2600 rpm for 15 minutes. After the last wash water was centrifuged at 1500 rpm for 15 minutes. An aliquot was taken for analysis of particle size. Small spherical particles were frozen at -80°C and freeze dried drying for 2 days.

The particle size, defined by volume, was equal 1,397 microns, surface area of 1,119 μm and the number 0,691 μm, as determined using a particle counter Beckman Coulter LS 230. Scanning electronic micrograph showed the equality of the dimensions and the absence of agglomeration of small spherical particles of insulin (Fig).

Using the continuous flow method in which a solution of insulin kept at 90°C for a short time, gave the opportunity to obtain small spherical particles. This method led to the final composition, which contains 90% protein, as determined by high performance liquid chromatography (HPLC) (Fig.9). HPLC analysis of t is the train showed that dissolved a small spherical particles of insulin had an elution time of about 4,74 minutes, differs only slightly from the time of standard insulin or source material of natural insulin, thus showing the conservation of biochemical purity of insulin after manufacture in the form of small spherical particles.

Example 4

Periodic way to obtain small spherical particles of insulin in the heat exchanger

Crystalline insulin zinc human suspended in a minimal amount of deionized water with ultrasonic agitation to ensure complete dispersion. Suspension of insulin was added to a mix containing buffer solution of polymer (pH 5,65 at 25°C), pre-heated to 77°C., so that the final concentration of dissolved has made 0.83% crystalline insulin zinc, 18.5% polyethylene glycol 3350, 0.7% of sodium chloride in 0.1 m buffer solution of sodium acetate. Opaque first mixture brightened up within three minutes when dissolved crystalline insulin. Immediately after his enlightenment, the solution was transferred into a glass, water-jacketed chromatography column, which was used as the heat exchanger (column with an internal diameter: 25 mm, length: 600 mm; Ace Glass Incorporated, Vineland, NJ). A glass column was positioned vertically, and the coolant centuries the Dili in the water jacket from the bottom of the column and taken out from the top. In order to document the heat transfer properties of the system, thermocouples (Type J, Cole Parmer) was placed in the center of insulin liquid drug from the top and bottom of the column, and the profile of the lower temperature was obtained during preliminary tests. thermocouple was removed during six cycles of conducting this experiment, in order not to introduce extraneous variables surface.

The heat exchanger is pre-heated to 65°C and a solution of insulin-polymer buffer moved in such a way that the solution temperature did not fall below 65°C and air bubbles do not appear in the solution. Then the clear solution was left at four minutes to achieve equilibrium at 65°C in the heat exchanger, filed coolant temperature, adjustable from 65°C to 15°C. the Preparation of insulin in the heat exchanger was left at 15°C for twenty minutes to achieve equilibrium. Small spherical particles of insulin was formed, when the temperature dropped from 60 to 55°C with the formation of a homogeneous stable creamy white suspension.

Small spherical particles of insulin was separated from the glycol by diafiltrate (cartridge for UF A/G Technologies, 750000 MWCO) with five volumes of 0.16% sodium acetate was 0.026% of zinc chloride as a buffer, pH 7.0, followed by concentration to one-fifth the original volume. The suspension is slightly the x spherical particles of insulin was additionally washed with diafiltrate with five volumes of deionized water, followed by lyophilization to remove water. Measures have been taken, preventing agglomeration of small spherical particles during diafiltration (from polarizing seal of particles on the membrane surface) and during lyophilization (from deposition of small spherical particles prior to freezing). Dried small spherical particles circulate freely and were ready for use without the need of deagglomeration and sifting.

Small spherical particles of insulin

The above described method of obtaining spherical particles of the same size of crystalline insulin zinc without added fillers. Small spherical particles prepared by this method have excellent aerodynamic properties, as determined by measuring time-of-flight (AerosizerTM) and measurements using cascade impactor Andersen, with high respirable fractions, indicating delivery deep into the lungs, with the delivery of a simple, widely used dry powder inhaler (CyclohalerTM). When using insulin as a model protein, the authors were also able to study the influence of the method on the chemical purity of the protein, using the U.S.P. ways.

Small spherical particles of a dry powder insulin reflected by microscopy in polarized St is the (Leica EPISTAR®, Buffalo, NY) and with a scanning electron microscope (AMRAY 1000, Bedford, MA). Analysis of particle size was performed using a system of measuring the size of particles Aerosizer® Model 3292, which includes Aero-Disperser® Dry Powder Disperser Model 3230 for introducing powder into the device (TSI Incorporated, St. Paul, MN). Individual particle size was confirmed by comparing the results obtained using the Aerosizer, with the results of electron micrographs.

Chemical purity of insulin before and after the implementation of the method was determined by HPLC according to the USP monograph Insulin human body (USP 26). Insulin and protein of high molecular weight was measured using isocratic method SEC HPLC with UV detection at 276 nm. For measurement of insulin, A-21 desamethasone and other related insulin substances, the sample was analyzed using gradient HPLC with reversed phase. Insulin was measured using a UV detector at 214 nm. High molecular weight protein, desamethasone and other related insulin substances, conducted a quantitative assessment of any chemical decomposition caused by the method.

Aerodynamic characteristics of small spherical particles of insulin was tested using the device Aerosizer®. Measurement of the distribution on the size of the dry powder insulin were and the use of fixtures AeroDisperser with a small shearing force, the average feed speed and normal deagglomerate. The instrument software transforms the time-of-flight data in the size and positions them on a logarithmic scale. The number of particles, determined for each set of dimensions used for statistical analysis, as well as the total volume of particles identified in each set of dimensions. The spatial distribution allocates large particles better than the numeric distribution, and, therefore, is more sensitive for the determination of the agglomerates is not dispersed particles, and large particles.

Set cascade impactor Andersen consists of a pre-separator, nine levels, eight plates of the collection and a backup filter. Stages are numbered -1, 0, 1, 2, 3, 4, 5, 6 and F. Step -1 represents only the nozzle. Step F includes a plate Assembly for a level 6 and a backup filter. Plates for collection of stainless steel covered with a thin layer of food grade silicone, to prevent "rebound" of the particles. A simple regulator air flow 60 LM through the sample used for analysis. Accurately weighed sample of about 10 mg was weighed in each starch capsule (Vendor) of powder delivered as an aerosol from a Cyclohaler within four seconds. The amount of powder insulin deposited on each plate were determined using HPLC with reversed the phase detection at 214 nm, according to USP 26 analysis for human insulin.

Mean mass aerodynamic diameter (Smad) was calculated by the computer program Sigma plot, using probit selection when the accumulation is smaller than the mass percentage against the effective marginal diameter (EPD). Emitted dose (ED) was defined as the total observed mass of insulin deposited in the cascade impactor. This is reflected as a percentage of the mass of small spherical particles of insulin loaded in a capsule Cyclohaler.

The results show that with careful control of the parameters of the method in connection with the phase change formulation can be obtained: 1) predominantly spherical particles with a diameter of about 2 microns; 2) a narrow size distribution; 3) reproducible aerodynamic properties from batch to batch, and 4) a small spherical particles consisting of more than 95% of the active drug (insulin human), excluding residual moisture. The inventors have determined that the solubility of crystalline insulin zinc can be regulated by the temperature of the solution, pH, polymer concentration and ionic strength. The inventors have also found that the regulation of cooling rate during the period of the phase change represents an important parameter, which gives the possibility of formation of predominantly spherical particles with a narrow interval is of size.

Figure 2 is a profile of lowering the temperature for the method corresponding to this example. The profile was measured using a chromatographic column with a water jacket located vertically, and the heat exchange fluid within the water jacket from the bottom of the column and out the top. Two thermocouples were placed in the column in contact with the solution. One thermocouple was located on the top of the column and the second from the bottom of the column. Temperature curves divide the graph of the time-temperature in individual areas in which preliminary experiments to optimize, due to the induced phase change above or below the optimal rate of change of temperature, lead to the broadening of the interval size of the particles and non-spherical form. At temperatures above 60°C the insulin remains soluble in a buffer solution of polymer (area A; figure 2). When the temperature decreases at speeds from approximately 8,6°C/minute to 26.5°C/minute, it is beneficial for optimal formation of the same size spherical particles (region V; figure 2). If the recipe is to apply the rate of temperature drop of more than 26.5°C/min, there is a tendency to the formation of very small (less than 0.5 µm) of nonspherical particles insulin, easily forming the agglomerate (area C; figure 2). The cooling rate is lower than the 8.6°C/min lead to user is the distribution according to size of a small spherical particles of insulin, as well as non-spherical shape and amorphous flocculent precipitate (area D; figure 2).

When the temperature of the insulin-superstarcase of the polymer solution within the heat exchanger falls within the region In figure 2, is a phase change, resulting in a milk white stable suspension of small spherical particles of insulin. Phase separation, showing the formation of microspheres, begins when the temperature falls below 60°C, and ends when the temperature reaches 40°C. No additional changes to the suspension was not observed when the formulation was cooled to 15°C before washing with diafiltration to remove polymer PEG.

While SAM feedstock crystalline insulin zinc human showed heterogeneity of size and crystalline form with a particle size of from about 5 to 40 μm, SAM pattern obtained for one of the parties of this example showed a spherical shape and the same size of a small spherical particles of insulin (pigv). The shape and size of the particles, shown SAM, are characteristic of the other five parties obtained in this example.

After diafiltration to separate from the polymer containing buffer, and the subsequent washing and lyophilization of the suspension with deionized water dry powder small spherical particles of insulin was relatively freedoms what about the fluid and easily weighed and processed. The moisture content in small spherical particles of insulin ranged from 2.1 to 4.4%, compared with 12% in the feedstock crystalline insulin zinc. Chemical analysis of small spherical particles of insulin by HPLC showed a very slight degradation of insulin in the method (figure 4) with no increase of compounds with high molecular weight. Although there was an increase (over the raw material of insulin) in % dimer, % A21 of desamethasone, % late eluruumiks peaks and % of other compounds, the results for all six batches were within USP limits. The retention efficiency of insulin ranged from 28,3 to 29.9 IU/mg, compared to 28.7 IU/mg for feedstock. Residual levels of polymer used in the method (polyethylene glycol)were below 0,13% and up to netdetective level, showing that the polymer is not a significant component of the small spherical particles of insulin.

The reproducibility of the aerodynamic properties in the comparison of different batches of small spherical particles of insulin

Observed excellent reproducibility of the aerodynamic properties when comparing the six batches received a small spherical particles of insulin, as demonstrated by the data obtained using the Aerosizer and cascade impactor Andersen. For all six desks is th Aerosizer data showed that more than 99.5% of the particles have a size in the range of 0.63 to 3.4 μm, at least 60% of small spherical particles of insulin have a size in the narrow interval from 1.6 to 2.5 microns (figure 5). Statistically, the data show that with probability 95%, at least 99% of small spherical particles in the obtained quantities of insulin are at least 96,52% of particles with sizes in the range of 0.63 to 3.4 μm (from 68.5 per cent to 70 per cent with a diameter of about 2 microns).

Data cascade impactor Andersen a good match to the data Aerosizer, except that, on average, 17.6% of the dose delivered from Cuclohaler, was deposited in the inlet and in the pre-separator/the neck of the instrument (6). Data show that the efficiency of dispersion of the powder with the use of the device Aerosizer higher than with the use of the device Cyclohaler. However, the average emitted dose for six parties amounted to 71.4 per cent of the Cyclohaler, with 72.8% of the emitted dose deposited in stage 3 of the impactor. If it is determined that respirable fraction for deep lung delivery should be the faction with the EPD between the 1.1 and 3.3 μm, average 60.1% of small spherical particles inhaled insulin may be available for deep lung delivery and subsequent systemic absorption. Excellent reproducibility of the method is demonstrated in table 1 where the values of standard deviation for Smad and GSO average for six separate parties, is the tsya extremely low. This shows that the variables of the method are under strict control, leading to the same aerodynamic properties from batch to batch.

Table 1
The aerodynamic properties of small spherical particles of insulin
Smad (µm)GSO (µm)% step 2-F (EPD of 3.3 μm)% step 3-F (EPD 2,0 µm)Emitted dose (%)
Average2,481,51and 88.872,871,4
TO0,1000,0644,584,07lower than the 5.37

Table 1 presents the aerodynamic properties of small spherical particles of insulin. Results (mean±CO) was calculated from the analysis of individual batches of small spherical particles of insulin (N=6) at the cascade impactor Andersen. Very good reproducibility of the method is demonstrated e the lowest values of the square deviations for Smad and GSO.

Small spherical particles of insulin obtained by cooling method, showed a slight tendency to agglomeration, which can be seen from the aerodynamic data of table 1.

Example 5

The way to obtain small spherical particles of insulin in the mix vessel

2880 ml of buffer solution of polymer (18.5% polyethylene glycol 3350, 0.7% of sodium chloride in 0.1 M sodium acetate as a buffer with a pH of 5,65 at 2°C) were loaded into a 3-liter glass vessel with jacket filled with water, with stirring and pre-heated to 75°C. 2.4 grams of crystalline insulin zinc human suspended in 80 ml of buffer solution of polymer with ultrasound to ensure complete dispersion. Suspension of insulin was added to the mix, pre-heated buffer to the polymer solution and stirred for another 5 minutes. The mixture became transparent during this time, showing that crystalline insulin zinc dissolved. Water from the refrigerator set at 10°C was pumped through the jacket of the vessel, while the insulin in solution of the polymer is cooled to 15-20°C. the resulting suspension was diafiltrate with five volumes of buffer solution of 0.16% sodium acetate was 0.026% of zinc chloride, pH 7.0, followed by five volumes of deionized water and subsequent lyophilization to remove water. Seminalis of liofilizirovannogo powder showed homogeneous small spherical particles with an average aerodynamic diameter, equal 1,433 μm, using the time-of-flight analysis TSI Aerosizer. Analysis using a cascade impactor Andersen showed 73% of the emitted dose deposited on the filter stage 3, Smad of 2.2 μm and GSO 1.6 ám; everything points to excellent aerodynamic properties of the powder.

Example 6

The reduction in the formation of degradation products of insulin in the regulation of the ionic strength of the composition to obtain small spherical particles

Insulin can also be dissolved in solution at lower initial temperatures, for example 75°C, without increasing the time and / or acidification of the environment, but it results in significant aggregation when added to the NaCl solution.

Superior way to obtain small spherical particles was completed using the following methods. A concentrated suspension of crystalline insulin zinc (at room temperature) was added (with stirring) to 16.7 percent solution of polyethylene glycol in 0.1 m sodium acetate, pH 5,65, pre-heated to about 85-90°C. the insulin Crystals were completely dissolved in this temperature interval within five minutes. Small spherical particles of insulin was formed by lowering the temperature of the solution.

Significant education And21desamethasone and dimers of insulin as a result of chemical reactions about what went down when raising the temperature from the initial temperature, equal to 85-90°C. However, at 75°C, it takes a significant period of time. For a long time also leads to a significant degradation of insulin. Pre-dissolving insulin in the acidic environment also causes undesirable conversion of a large percentage of insulin in the product decomposition And21desamethasone.

In the experiment, the sodium chloride was added to the reaction mixture of buffer polymer to chemical means to reduce the formation of dimers of insulin. Although the added sodium chloride was slightly reduced the formation of desamino or dimers of insulin, which is the decomposition product, adding sodium chloride significantly reduced the formation of oligomers (molecular produce insulin) (table 2).

Table 2
NaCl added to a suspension of insulin-water
Description sample% dimer% VM% dazamide% other related compounds
Control without NaCl0,940,230,781,52
NaCl, 0.7% of the final concentration0,830,050,821,43
NaCl added to the polymer solution
Description sample% dimer% VM% dazamide% other related compounds
NaCl, 0.7% of the final concentration0,850,070,931,47

In addition, a crystalline insulin-Zn dissolves much faster in the presence of NaCl than the control without NaCl. This leads to the assumption that the addition of sodium chloride increases the dissolution rate of insulin and makes it possible to reduce a temperature of the dissolution of crystals of insulin-zinc. This hypothesis was confirmed in the experiment, which demonstrated that the addition of 0.7% NaCl to the composition allows raw crystalline insulin zinc dissolved at 75°C for five minutes, significantly lower temperature than 87°C required previously without added NaCl. At 75°C in the absence of NaCl insulin in 13 minutes dissolved completely.

Series the experience is imento demonstrate the increase in NaCl concentration (2.5 mg/ml, 5.0 mg/ml, 10 ml/ml and 20 mg/ml) in addition lowers the temperature at which the dissolved crystals of insulin and also lowers the temperature at which begin to form small spherical particles (figa-d). Additionally, it was determined that increasing the concentration of NaCl in the formulation of fast dissolving high concentrations of crystalline insulin-Zn. Thus, it was confirmed that the solubility of insulin at a given temperature can be closely controlled by regulating the level of sodium chloride in the initial continuous phase. This allows for a method at temperatures that are less favorable for the formation of decomposition products.

In order to determine whether the sodium chloride unique chemical properties that make it possible to reduce the temperature of the dissolution of insulin, equimolar concentrations of ammonium chloride and sodium sulfate were compared to control with sodium chloride. And NH4Cl, and Na2SO4equally lowered the temperature required for dissolving the raw material is crystalline insulin zinc. Apparently, the increase in ionic strength increases the solubility of insulin in the composition to obtain microspheres without affecting the ability to form small spherical particles at lower the temperature of the solution.

Example 7

Study of the effect of concentration of PEG on the yield and concentration of insulin and the size of a small spherical particles of insulin

Data for the titration of polyethylene glycol (3350) showed that increasing the PEG-3350 also increases the release of small spherical particles. However, when the concentration of PEG is too high, the particles lose their spherical shape, which negates a small increase in output.

Data on concentrations of insulin show the direction opposite to the PEG, in which the increase in the concentration of insulin leads to a decrease in the output of small spherical particles.

The authors see a General trend that higher concentrations of insulin lead to the formation of small spherical particles of larger diameter. In this experiment, higher concentrations also lead to mixtures of nonspherical particles and small spherical particles.

Example 8

The study of small spherical particles of insulin in dogs

The aim of this experimental study was both quantitative and visual experiment with deposition of aerosol powder of insulin in the lungs of hounds.99mTc labeled particles insulin were obtained by methods described herein. The deposition of aerosol insulin in the lungs was assessed using gamma-ray topography.

Five with the tank used in this study, and each dog was introduced aerosol99mTc radiolabelled particles of insulin. Identification number of the dogs were 101, 102, 103, 104 and 105.

Before the introduction of the aerosol animals were subjected to anesthesia propofol through the line infusion for anesthesia, and each animal was placed endotracheal tube for delivery of the aerosol.

Each dog was placed in a cell “Spangler box” for inhalation radiolabelled aerosol. Immediately after administration of radiolabelled aerosol, received a computer image of the gamma-camera for the front and rear chest. Two in-vitro collection cascade impactor were evaluated by one before the introduction of the aerosol first animal (101) and the next after exposure to the last animal (105)to determine the stability of the99mTc radiolabelled powder insulin.

The results are presented in figure 11. Fees cascade impactor in both cases showed a unimodal distribution.

Fig shows the results of a calculation of the ratio P/I for all animals. The ratio of P/I is a measure of the ratio99mTc powder insulin that ass in the peripheral parts of the lungs, that is deep in the lungs. A typical ratio of P/I is probably about 0.7. The ratio of P/I is larger than 0.7 indicate significant deposition in perifericheskoi part of the lung compared with the Central part of the lung or bronchial region.

Scintigraphic image on Fig shows deposits of insulin in the respiratory system and is compatible with the data P/I (Fig). Scintigraphic image of the dog 101 is typical for all five dogs in this study.

Scintigraphic image for dogs 101 shows a small tracheal or bronchial deposits with the obvious increase in deposits in the peripheral parts of the lungs. Radioactivity outside the lungs caused by rapid absorption99mTc of the deposition of aerosol powder deep into the lungs.

The ratio of P/I and the data on the image show that insulin, radiolabelled99mTc, settles mostly deep in the lungs. The amount of radiolabelled insulin settled on the periphery of the lung, indicated low levels of agglomeration of particles.

Example 9

Diafiltrate with zinc-containing buffer to remove polymer from small spherical particles of insulin

For small spherical particles of insulin from the UV solution was desirable to remove all of UVR from the suspension prior to lyophilization. Even a few percent remaining of UVR can act as a binder, forming Naviglio small agglomerates of spherical particles. These agglomerates can adversely N. the emitted dose and aerodynamic properties of the powder, supplied from the ISP device. In addition, in the lung tissues under repeated impacts doses of UVR may increase Toxicological consequences.

Three methods were considered for the separation of small spherical particles from the UV to lyophilization. Filtering could be used to collect small amounts of particles. However, the large number of small spherical particles quickly block the pores of the filtration medium, making washing and returning more than a few milligrams impractical.

Centrifugation to collect the particles, followed by several washing cycles, including re-suspension in an aqueous solvent and recentrifuging was successfully applied for the removal of UVR. Deionized water was used as the aqueous solvent as small spherical particles dissolve poorly, and UVR remains in solution. One disadvantage centrifugation was that small spherical particles were tightly compressed into balls high accelerations required for the deposition of particle rotation. With each subsequent washing was becoming more and more difficult to resuspendable balls into discrete particles. Agglomeration of particles of insulin was often an unwanted side effect of the method of centrifugation.

Diafiltration using hollow in the fibrous ampoules used as an alternative to centrifugation for washing small spherical particles of insulin. In a typical installation with equipment for diafiltration buffer suspension of UV/particles insulin was placed in a sealed container, and the suspension was recycled through the fiber with sufficient pressure to cause the filtrate to pass through the voids of the fibrous membrane. The rate of recycling and the back pressure optimized, in order to prevent clogging (polarization) of the pores of the membrane. The amount of filtrate removed from the suspension, continuously replenished siteniravam solvent for washing stir in a sealed container. During the process of diafiltration concentration of UVR in suspension gradually decreased, and the suspension of small spherical particles of insulin was essentially free of UVR after five-, seven-time exchange of the original volume of the suspension with the solvent for washing within a period of hours or about.

Though the way of diafiltration very effective for the removal of the polymer and is very suitable for increase in commercial quantities, small spherical particles of insulin was slowly dissolved in deionized water, originally used as the solvent for washing. The experiments showed that insulin was gradually lost in the filtrate, and the particles of insulin were completely dissolved after an exchange of deionized water in an amount equivalent to dvadtsat the times the original volume of the suspension. Although it was found that the small spherical particles of insulin poorly soluble in deionized water, due to the high efficiency of the method of diafiltration permanently remove dissolved insulin, and probably zinc ions in the slurry. Therefore, the equilibrium between dissolved and the concentration of dissolved insulin in the given volume of deionized water is not installed when diafiltration, a condition that favors the dissolution of the insulin.

Table 3 shows the different solutions that were evaluated as potential leaching environment. Ten milligrams of dry small spherical particles of insulin suspended in 1 ml of each solution and weakly was stirred for 48 hours at room temperature. The percentage of dissolved insulin was measured after 24 and 48 hours. It was found that the insulin is poorly soluble in deionized water with the balance achieved when 1% of the total mass of dissolved insulin, less than 24 hours. However, as noted earlier, high efficiency diafiltration continuously removes dissolved insulin (and zinc), so this equilibrium is never achieved, and a small spherical particles of insulin continuously dissolve. Therefore, the solubility of insulin in the ideal washing solution should be lower than in water. Because insulin least of all races is work near its isoelectric point, checked dumesny acetate buffer and pH 5,65. It was found that the solubility of insulin depends on polyarnosti buffer and compared with the solubility in water at low polyarnosti. Ethanol significantly reduces the solubility of insulin, but only near anhydrous concentrations. The actual increase in the solubility of insulin when blending ethanol aqueous solution was used in the suspensions of UV/small spherical particles of insulin in the early stages of diafiltration.

Table 3
The solubility of small spherical particles of insulin in different leaching solutions
The washing liquor% dissolved insulin after 24 hours% dissolved insulin after 48 hours
Deionized water0,910,80
0.1 M sodium acetate, pH 5,652,482,92
0.001 M sodium acetate, pH 5,650,540,80
0,16% sodium acetate -0,016% ZnO, pH of 5.3 0,140,11
0,16% sodium acetate -0,027% ZnCl2, pH 7.00,090,06
50% ethanol/deionized water (about./about.)for 9.479,86
100% anhydrous ethanol0,050,04

Buffer solutions used in commercial suspensions for injection containing crystalline insulin zinc, also contained zinc in solution. Two of these solutions were tested with a small spherical particles of insulin, and found that they significantly reduce the solubility of insulin compared to deionized water. According to the literature, crystalline insulin zinc should be from 2 to 4 Zn ions associated with each hexameron insulin. Crystalline insulin zinc obtained with different number of zinc ions on hexamer in the interval from a to 2.46 1.93 and used as raw material for the production of small spherical particles of insulin. This corresponds to from 0.36 to 0.46% of zinc on the quantity of crude crystalline insulin zinc. After the formation of small spherical particles of insulin and diafiltration with deionized water from 58% to 74% zinc were lost during processing. Loss of zinc is C particles of insulin causes an increase in the solubility of insulin and loss during diafiltration.

Diafiltrate small spherical particles of insulin from 0.16% sodium acetate - 0,027% ZnCl2, pH 7.0 actually eliminate the loss of insulin in the filtrate. Unexpectedly, however, the content of zinc in the small, spherical particles of insulin increased to almost 2%, which is significantly higher than 0.46%of that measured for feedstock crystalline insulin zinc. Another unexpected result of diafiltration with zinc-containing buffer resulted in a substantial improvement in emitted dose, observed from a Cyclohaler COI equipment (68% diafiltration with deionized water compared to 84-90% after diafiltration with zinc buffer) and a decrease in the number of particles of insulin, pending at the mouth of cascade impactor Andersen. Diafiltrate with zinc buffer improves the ability of small spherical particles of the dry powder insulin to the dispersion and reduces agglomeration of the particles, resulting in reduction of Smad and greater deposition at low stages of the impactor. This led to the assumption that diafiltrate with zinc buffer and a higher content of zinc in the small, spherical particles of insulin can improve the percentage of doses, delayed deep into the lungs.

When suspendirovanie in the gas propellant GFA(hydrofluroalkane)-134a without adding fillers for use in I & d (inhaler measured dose) the application was not idivi irreversible agglomeration washed zinc buffer a small spherical particles of insulin. Particles of insulin, indeed, rolled flakes of suspension less than a minute, but easy to move in suspension by shaking before use. The shaking of the I & d of the container immediately prior to use is a normal part of the instructions issued for the use of any of I & d product. In fact, the loss dropped the cereal particles, which are located at the bottom of the I & d of the container, may actually inhibit long-term agglomeration of particles of insulin (in addition to the minimum contact because of their spherical shape), because the particles are not arranged in close-Packed layer at the bottom of the I & d of the container under pressure. Therefore, the properties acquired by small spherical particles of insulin in diafiltration with zinc buffer, can improve the long term storage and dispersion ability of the I & d of insulin and other related zinc compounds.

As was found with the analysis of powder x-ray diffraction, small spherical particles of insulin are noncrystalline associated zinc is not associated with the coordination of the zinc ion with insulin monomers for the formation of hexamer. Therefore, the nonspecific binding of ions and the resulting potential benefits can be extended to ions other than zinc. According to CNAE proteins, which does not bind zinc can bind other ions that will reduce the solubility in the way of diafiltration and to provide similar beneficial effects.

Small spherical particles suspended in gas-displacer hydrofluroalkane (GFA) 134a concentration equal to 10 mg/ml Chemical stability of insulin after storage in GFA 134a evaluated at time 0 and after one month. The data presented in Fig show the preservation of the microspheres of insulin on the insulin monomer, dimer of insulin, insulin oligomers, the main peak of insulin and A21-desamethasone.

In the following study of small spherical particles of insulin, obtained according to the methods of example 4, were compared for their quality in three different devices for inhalation using the method of cascade impactor Andersen. The device Cyclohaler is a commercial dry powder inhaler, Disphaler is another dry powder inhaler and inhaler measured doses (IRD) is a device in which the microspheres are suspended in GFA 134a, as described in this example, and push through a 100-Microlitre or other size of the measuring valve. The results on Fig clearly showed that the small spherical particles, the past stage device cascade impactor Andersen, otkladyvat is conducted on stages 3 and 4. This indicates the high reproducibility of the quality of small spherical particles, regardless of the device used as an inhaler. The only main difference between the COI and IRD devices is substantially larger number of small spherical particles, suspended in the mouth of cascade impactor Andersen, when using the IRD. The high speed with which the IRD device pushes a small spherical particles through the mouth of the Andersen impactor, explains a higher proportion of deposits microspheres of insulin compared with the ISP device. Specialists in this field can assume that the IRD device with reduced or modified by the exit velocity can be used to reduce the number of small spherical particles, suspended in the neck. For additional measurements can be used in the separation device at the end of the IRD.

Small spherical particles of insulin (party room YQ010302) made of liofilizirovannogo source material of insulin according to the methods described in this example. The storage stability during the year, small spherical particles of insulin compared with liofilizovane source material of insulin at 25°C and 37°C. the Stability of insulin were compared in the study all related to insulin with the joining, dimers of insulin, insulin oligomers and A21-desamethasone.

Fig. 30-35 show that after a period of one year, small spherical particles of insulin showed significantly smaller amounts of dimers of insulin, insulin oligomers, A21-desamethasone and all related insulin compounds when compared with the original material of insulin stored in the same conditions. This indicates that the insulin in the form of microspheres is significantly more resistant to chemical changes than the original material.

Small spherical particles of insulin was tested using a cascade impactor Andersen at time 0, and 10 months after manufacturing. The device Cyclohaler COI used to determine the aerodynamic stability after long-term storage. Fig shows that the aerodynamic performance remains highly constant after 10 months of storage.

Research using spectroscopy CU was undertaken to explain the structural differences between the raw sample of insulin and insulin small spherical particles prepared in this example. It was shown that the insulin small spherical particles has a significantly higher content of β-sheet, and subsequently lower content of α-helix than the outcome of the initial raw sample of insulin. Discovery data coincide with the formation of aggregates microfiber structures in small spherical particles. However, when dissolved in water spectra show substantially identical structures of proteins obtained or untreated microspheres, or insulin, indicating that any structural changes in the microspheres are fully reversible when dissolved.

Two batches of insulin were tested using spectroscopy CU: (A) raw USP insulin (Intergen, Cat no.4502-10, Lot# XDH 1350110) and (B) the insulin small spherical particles (JKPL072502-2 NB 32: P.64). Samples in the form of a powder or solution of insulin (about 15 mg/ml in 0,01M HCl) was placed in a standard glass capillaries and thermostatically at 12°C for analysis of CU. Usually 2-15 μl aliquot was enough to fill the sample portion of the capillary exposed to laser light. The spectra were excited at 514.5 nm argon laser (Coherent Innova 70-4 Argon Ion Laser, Coherent Inc., Santa Clara, CA) and recorded on the scan dual spectrometer (Ramalog V/VI, Spex Industries, Edison, NJ) with fotona-counting detector (Model R928P, Hamamatsu, Middlesex, NJ). The data in intervals of 1.0 cm-1collected with integration time of 1.5 s and a spectral slit width of 8 cm-1. The specimens were scanned again, and individual images were reproduced and studied before averaging. Usually sobi is Ali, at least 4 images of each sample. The spectrometer was calibrated using indene and carbon tetrachloride. The spectra were compared different numerical methods, using the SpectraCalc and GRAMS/AI Version 7 software (Thermo Galactic, Salem, NH). The spectra were corrected taking into account the solvent (if any) and the background. Spectra of the solutions were adjusted received a range of 0,01M HCl under identical conditions and customized to a series of five overlapping functions Gauss-LJ for falling background [S.-D. Yeo, P.G. Debenedetti, S.Y.Patro, T.M.Przybycien, J. Pharm. Sci., 1994, 83, 1651-1656]. The fitting was carried out in the area of 1500-1800 cm-1.

The Raman spectra were obtained for powder samples of insulin, and their respective solutions (Fig). The spectrum of the untreated sample corresponds very well with the previously described spectra of samples of commercial insulin [S.-D. Yeo, P.G. Debenedetti, S.Y. Krom besides patr-o, T.M. Przybycien, J. Pharm. Sci., 1994, 83, 1651-1656; J.L. Lippert, D. Tuminski, P.J. Desmueles, J. Amer. Chem. Soc., 1976, 98, 7076-7080]. A small sample of spherical particles showed a significant shift (from approximately +10 to +15 cm-1to amide I, pointing to significant changes in the secondary structure of the protein. It is noteworthy, however, that the range of commercial powder and a range of small spherical particles were virtually identical by dissolving the samples in the aquatic environment, showing that changes in the secondary structure when education is Otke were fully reversible.

Secondary structural parameters were determined using computer algorithms, which include smoothing, subtraction fluorescence and aromatic background, and the deconvolution of the amide bands I. Exponential decay of fluorescence was subtracted substantially as described elsewhere [S.-D. Yeo, P.G. Debenedetti, S.Y. Krom besides patr-o, T.M. Przybycien, J. Pharm. Sci., 1994, 83, 1651-1656]. Installed structural parameters are collected in table 4.

Table 4
Structural parameters of the samples of insulin set of spectra KR
SampleThe total content of α-helix, %The total content of β-Plast, %β-reverse coil, %Random tangle, %
The raw powder44311411
Untreated insulin in solution44281117
Small spherical particles, the powder is OK 1167157
Small spherical particles in solution44301115

Example 10

Preparation of small spherical particles of human insulin isothermal method

The USP human insulin (Intergen) was dispersible in NaCl and PEG (3350 MM, range Lot# RP0741) c education final concentration of insulin, equal 0,86 mg/ml, and concentrations of NaCl and PEG, equal to 0.7% wt. and 8.3 wt.%. the pH was brought to 5.65 addition of minute quantities of a solution of glacial acetic acid and 1M NaOH. After heating up to T1=77°C was obtained transparent solution of the protein to a final concentration of insulin Ceq. Then the solutions were cooled with a certain predetermined rate to a temperature T2=37°C. At T2observed precipitation of protein. Precipitation was removed by centrifugation (13000×g, 3 min) at 37°C, and insulin concentrations (*in the obtained supernatant is determined bicinchoninic analysis protein was 0.45 mg/ml of thus Obtained solution of insulin, which was kept at 37°C, identified a solution of A.

The solution was prepared by dissolving insulin persons the century of 0.7% wt. NaCl/8,3% wt. PEG (pH brought to approximately 2.1 by adding HCl) with the final concentration of insulin, equal to 2 mg/ml Solution was kept at 37°C With stirring for 7 h followed by sonication for 2 min Aliquots of the resulting solution was added to the solution And getting the total insulin concentration 1 mg/ml of the resulting mixture was vigorously stirred at 37°C during the night, getting the precipitate of insulin, which was carefully removed from the liquid using a membrane filter (effective pore diameter of 22 μm). The obtained microparticles protein then quickly frozen with liquid nitrogen and subjected to lyophilization.

CenturySmall spherical particles of alpha-1-antitrypsin (AAT)

The present invention can also be used to obtain small spherical particles of the AAT, which is practically convenient for delivery into the lungs.

Example 11

Preparation of small spherical particles AAT periodic manner in a column with the shirt (scale 10-300 mg)

Buffer solution at a pH of 6.0 with 10 mm ammonium acetate containing 16% PEG 3350 and 0.02% Pluronic F-68, was stirred with a magnetic stirrer in a beaker with a shirt and was heated to 30°C. The temperature in the glass was controlled using a water circulation water bath. A concentrated solution of recombinant AAT (rat) was added to this p is the target with stirring and brought the pH to 6.0. Concentration rat in the final solution was 2 mg/ml. raat completely dissolved at the same temperature in this composition solution. All contents of the vessel were transferred to a column with a shirt and was heated to 25-30°C. Circulating water bath for columns were installed to reduce the temperature to -5°C. the Column and its contents were cooled to about 1°C/min to a temperature of about 4°C. a Small spherical particles raat formed during the cooling phase. A suspension of microparticles was suspended in a glass mold and subjected to lyophilization to remove water and buffer.

In order to extract the PEG of small spherical particles of protein after lyophilization, PEG/protein precipitate was washed with methylene chloride (Cl2). Others used the washing liquid was a methylene chloride:acetone 1:1 methylene chloride:pentane 1:1. The wash procedure was repeated a total of 3 times the original volume of leaching. The obtained granules resuspendable in a small volume of acetone or pentane and dried or directly in gaseous nitrogen, or by rotary evaporation.

Example 12

Preparation of small spherical particles AAT periodic manner in a column with the shirt (scale 200-2000 mg)

This type of cooking was carried out using the same comp is the position of the formulation, as the column with the shirt, but it is adapted for large volumes and is more suitable for increasing the scale of production. In this case, the formulation was mixed at 75 rpm using A-shaped impeller vane-type vessel with a shirt, usually 500-1000 ml, and heated to 30°C. the Temperature in the vessel was regulated using a circulating water bath. The solution was left in the same vessel, and switched the source of the water bath to 30°C in the bath to 2°C in the bath. The vessel and contents were cooled at about 1°C./min to a temperature of 4°C. a Small spherical particles raat formed during the cooling phase. The temperature was monitored using thermocouples, and when the suspension reached 4°C, it was held near this temperature for another 30 minutes After the stage of keeping a small spherical particle suspension was concentrated by diafiltration at about 4°C to remove approximately 75% of the polymer and volume. The suspension remaining small spherical particles were frozen in a thin layer in a pre-chilled prior to lyophilization to remove water and the remaining buffer.

Small spherical particles of protein was separated from the rest of the dry polymer or by centrifugation with organic solvents (as described in example 10), or extraction supercritical fluid (NWC). For the extraction of N Is W the dried material was transferred into the extraction chamber of a high pressure, which was raised to 2500 psi (at room temperature) using a CO2. When he reached the operating pressure in the inlet fluid stream injected ethanol as a mixture of 70:30 CO2:ethanol. This NWC was dissolved polymer, leaving a small spherical particles. At the end of the way the system was rinsed with ethanol and slowly decompressional.

Example 13

Output method - % conversion of rat in small spherical particles

Small spherical particles were made as described in examples 10 and 11. After completion of the cooling process, a small aliquot of the suspension was removed and filtered through 0.2 μm syringe filter to remove small solid spherical particles. The optical density of the filtrate, which represented raat remaining in the solution was determined at 280 nm using UV spectrophotometer. The concentration of rat was then calculated from the standard curve. % conversion was calculated as:

(initial concentration rat-concentration raat filtrate)

-----------------------------------------------------------*100%=%conversion

the initial concentration raat

Scale% conversion in a small spherical particles
100-200 mg (n=9, column)91,7±4,4
300 mg (n=4, column)for 93.4±1,6
2 g (n=5, receptacle)to 90.4±1,8

As shown in the table above, a high percentage of AAT protein turned into small spherical particles regardless of the scale method.

Example 14

The particle size distributions AAT at different scales method

Aerosizer data

A sample of a small spherical particles of the final dry powder AAT were analyzed TSI Aerosizer 3225, which measures the size of particles using time-of-flight measurements. From these measurements was calculated for various volumetric ratio of diameters to demonstrate the size distribution of small spherical particles AAT and used for comparison with the particles obtained by methods other than the method of the present invention.

Scaled90/d10(volume)d80/d20(volume)(d90-d10)/d50 (volume)
5-10 mg (n=12, column)1,88±0,201,49±0,100,67±0,14
100-200 mg (n=5, column)1,83±0,051,41±0,05 0,66±0,05
300 mg (n=3, column)2,05±0,171,61±0,110,77±0,06
1-2 g (n=4, container)2,21±0,301,60±0,110,86±0,19

Andersen data

5-10 mg of sample was weighed in a gel capsule and introduced into the cascade impactor Andersen, using Cyclohaler inhaler dry powder at a flow rate of 60 liters per minute (LFM). Small spherical particles were collected from all stages of the impactor, was dissolved in 0.2m Tris-Hcl buffer at pH 8.0 and quantitatively analyzed using HPLC with reversed phase. Data were analyzed and calculated geometric standard deviation (GSO), as described in United States Pharmacopeia (USP). The results showed a narrow size distribution.

ScaleGEO
100-200 mg(n=5, column)1,74±0,22
300 mg(n=3, column)1,77±0,40
2 g(n=5, receptacle)1,70±0,09

All the parameters of the distribution, shown above, has demonstrated excellent distribution of the size of the RA particles, what is the result of the retrieval method of the present invention.

Example 15

Preservation of bioactivity AAT

To determine the specific activity of small spherical particles of rat was dissolved in 0.2m Tris-HCl pH 8.0 at room temperature. The resulting solution was analyzed by a method that measures the ability rat to inhibit the ability of porcine pancreatic elastase (PPA) to hydrolyze synthetic peptides that contain p-nitroaniline group on their end. The same solution small spherical particles raat then analyzed for protein concentration using the method bicinchoninic acid (BCC). The control solution raat source material was also analyzed by both methods. As the activity analysis was developed to determine the activity on the basis of a concentration of 1 mg/ml protein in a sample, the activity value was adjusted based on the actual protein concentration determined using BCC, receiving the value of the specific activity:

the activity value sample

------------------------------------- = specific activity of the sample is

the actual concentration of protein

Inhibition of porcine pancreatic elastase rat
ScaleIU/mg small spherical particlesIU/mg control
100-300 mg (n=12, column)64,19±5,0164,34±4,95
200-300 mg (n=8, receptacle)62,53±of 5.2965,87±0,98

Thus, the specific activity demonstrates the preservation of bioactivity after production of AAT in the form of small spherical particles.

Example 16

Maintaining structural purity AAT

One of the main differences in the technology of controlled phase separation (EQF) is a particle formation under mild conditions, using the water system during the formation of particles and avoiding other causing stress conditions such as increased temperature, shear, etc. In the field of particle technology, the main problems are the stability of proteins during the manufacture and storage stability. I believe that the main route of degradation, such as oxidation, deliciousa and, especially, the aggregation of proteins responsible for the side effects of protein formulations, including immunogenicity. Therefore, the problems of regulation require exceptionally low levels of the decomposition products in the final formulations of the particles. To determine the actual happens if modification of the protein during the formation, used HPLC, physico-chemical characteristics, such as CD or DSK.

Circular dichroism (CD) is the most commonly used method to assess the structural changes in the protein subjected to disturbance, or comparison of the structure of the produced protein from the parental protein. The way KD determines the conformation of the protein secondary and tertiary structure of the protein.

Secondary structure can be determined by CD spectroscopy in the far-UV" spectral region (190-250 nm). At these wavelengths the chromophore represents a peptide bond, when it is in normal conformational environment. Alpha-helix, beta-sheet and random coiled structure, each, give an increase of the characteristic shape and intensity of the CD spectrum. Approximate proportion of each type of secondary structure, which is present in any protein can be, therefore, determined by analysis of the far UV CD spectrum as a sum of the relative contributions of these reference spectra for each structural type.

The CD spectrum of the protein in the "near-UV" spectral region (250-350 nm) can be sensitive to certain aspects of the Quaternary structure. At these wavelengths the chromophores are aromatic amino acids and disulfide bonds, and CD signals they produce, feeling timeline entire Quaternary structure of the protein. Signals in the region from 250 to 270 nm can be attributed to phenylalanine residues, signals from 270 to 290 nm can be attributed to the tyrosine and the signals from 280 to 300 nm can be attributed to tryptophan. Disulfide bonds give a weak broad signal around the middle UV spectrum.

The far UV CD spectra of the original solution raat and AAT isolated from small spherical particles in phosphate buffer (pH 7.4, T=25°C, the protein concentration of 0.05 mg/ml), shown in Fig. Each spectrum is averaged over 10 scans.

The far UV CD spectra are indistinguishable, demonstrating that the production of AAT in small spherical particles and their subsequent isolation leads to the AAT molecules with a structure identical to that of the source material AAT.

RP-HPLC

Small spherical particles was dissolved in 0.2m Tris-HCl at pH 8.0 and analyzed using HPLC with reversed phase. When compared with the control solution of the original rat protein noticeable difference in the chromatograms was not found.

HPLC system:

HPLC column - Pheomenex Jupiter, 5 μm, C4, 300A, 250×4.6 mm

Waters Alliance 2965 pump/automatic sampler

Wavelength of 280 nm

The volume of a sample of 75 ál

Concentration gradient:

The mobile phase 1: 0,1% THF in water

The mobile phase 2: 0,085% THF in 90% (c/v) acetonitrile in water

Experience time - 60 min

Volumetric flow rate - 1.0 ml/min

DSK

Received graphics DSK. Cm. Fig. 15-25b.

Example 17

Stability store small spherical particles AAT regarding the storage stability of the source material AAT

Small spherical particles were analyzed on the preservation of bioactivity (using the analysis described in example 15) after storage at room temperature and 4°C for 1 week, 1 month, 2 months, 3 months, 6 months and 12 months (Fig. 14b and 14C.) The solid material is an original solution raat, which was Valitova and then liofilizovane. For each point in time and storage conditions were duplicate samples, which were analyzed in parallel.

C. Small spherical particles of HGH (human growth hormone)

The present invention can also be used for making small spherical particles of HGH.

Example 18

Preparation of small spherical particles of HGH

in vitro periodic way (scale from 20 to 50 mg)

Buffer solution with a pH of 5.6 (50 mm ammonium acetate/50 mm ammonium bicarbonate)containing 18% PEG 3350, with the final concentration of HGH in solution 1 mg/ml was mixed in a 50-ml conical tube and heated in a stationary water bath to 58°C. HGH was dissolved in this solution under these conditions. The tube was removed from the water bath and cooled in ice is Noah bath until a solution of 10°C. The cooling rate was maintained from 4 to 6°C/min, Small spherical particles of HGH protein was formed during the cooling phase. Small spherical particles begin to form when the temperature of the solution reached approximately 40°C. After the formation of particles of small spherical particles of HGH protein was separated from the PEG in one of two ways, which are described below.

Rinsing with an organic solvent requires that after the stage of cooling and the formation of particles of a suspension of small spherical particles were quickly frozen with liquid nitrogen and was liofilizovane to remove water and buffer. To separate small spherical particles from PEG after lyophilization, the briquette PEG/protein suspended in methylene chloride (Cl2). PEG is soluble in Cl2while small spherical particles of insoluble protein. The suspension was stirred at room temperature for 5 minutes. As the density of small spherical particles of HGH is close to the density Cl2(d=1,335 g/ml)a second solvent to reduce the density of the liquid for successful centrifugation. Acetone, which is mixed with Cl2added in a volume equal to the volume Cl2. A suspension of small spherical particles and then centrifuged at 3300 rpm for 5 minutes at room temperature. The supernatant LM is bone decantation, and granules resuspendable in Cl2and the mixture was stirred for 5 minutes at room temperature. This washing procedure is repeated for 5 washes. After the last wash, the pellet re-suspended in a small volume Cl2and dried by rotary evaporation, getting the final powder small spherical particles of HGH.

Flushing zinc buffer demanded that after the stage of cooling and the formation of particles of a suspension of small spherical particles was centrifuged at 4000 rpm for 10 minutes at 4°C to separate the small spherical particles from the PEG. The supernatant was removed and pellets resuspendable in cold buffer containing 50 mm zinc acetate, in an amount equal to the amount of removed the supernatant. Ion Zn2+reduced solubility of HGH and prevent dissolution during leaching. Wash buffer and kept on ice. The suspension is then immediately centrifuged at 3000 rpm for 5 minutes at 4°C. the Supernatant was removed, and the leaching of zinc buffer a fully repeated 3 times. After 3-pay leaching zinc buffer, the pellets were washed 2 times with water and centrifuged at 3000 rpm for 5 minutes at 4°C to remove excess zinc. After the final water rinse granules resuspendable in a little water and quickly Zamora is supported, using liquid nitrogen. Frozen pellets were then liofilizovane to remove the water, getting the final powder small spherical particles of HGH.

Example 19

Preparation of small spherical particles of HGH in the vessel with jacket periodic way (100 mg scale)

This method of cooking is carried out using a composition similar to example 18, but can accommodate large volumes and is more suitable for larger scale.

Buffer solution with a pH of 6.1 (80 mm ammonium acetate/10 mm ammonium bicarbonate)containing 18% PEG 3350 and 0.02% Pluronic F-68, was stirred in a beaker with a shirt through the top of the impeller and was heated to 58°C. the Temperature of the mixture was controlled by means of a water bath with circulation. A concentrated solution of HGH was added to this solution with stirring. The final concentration of HGH in solution was 1 mg/ml HGH is completely soluble at a given temperature in the composition of this solution. The vessel and contents were then cooled at a rate of 8°C/min to a temperature of approximately 10°C. a Small spherical particles of HGH were formed during the cooling phase. Small spherical particles began to form around 40°C, and this process continued with the further cooling of the suspension. After the stage of cooling of a small spherical particles were separated from the PEG-ODN is m of two ways, described in example 20A.

Example 20

Preserving the purity of HGH

Protein purity of HGH in small spherical particles were evaluated in the following stages of the method: after the formation of the particles after extraction PEG and after removal of the solvent or after drying. Measurement of the chemical purity of HGH after manufacture in the form of small spherical particles was determined using HPLC analysis (size exclusion chromatography (RAH), reversed phase (RP)) for the quantitative analysis of products agglomeration and decomposition. The results showed no significant accumulation of agglomerates or other related substances during the development process of the formulation of small spherical particles.

A. Rinsing with an organic solvent

Agglomeration of HGH using size exclusion: an increase in the agglomeration compared with the original material

Substances related HGH, using reversed phase: increase decomposition in comparison with the source material

Step of the way% increase in the dimer% increase in VM particles
After the formation of particles1,170
After the PEG extraction and dryingto 2.670,43
Step of the way% increase in early discharge particles% increase in dazamide% increase in later washed out particles
After the formation of particles0,220,660
After the PEG extraction and drying1,292,930

b. Flushing zinc buffer

Agglomeration of HGH using size exclusion: an increase in the agglomeration compared with the original material

Step of the way% increase in the dimer% increase in VM particles
After the formation of particles0,880
After the PEG extraction2,250
After vissian the I particles of 2.510

Substances related HGH, using reversed phase: increase decomposition in comparison with the source material

Step of the way% increase in early eluruumid
particles
% increase in dazamide% increase in late eluruumid particles
After the formation of particles0,381,910,26
After the PEG extraction0,19of 1.340,26
After drying of the particles0,341,580,37

Example 21

The particle size distributions of small spherical particles of HGH

The removal characteristics of the size distribution of small particles of spherical particles was performed using aerodynamic time-of-flight measurements using a TSI Aerosizer (Fig) and scanning electron microscopy (FEG).

Example 22

The kinetics of dissolution of small spherical particles of HGH

CPA is aligned kinetics of dissolution of small spherical particles of HGH, subjected to two different extraction procedures.

Small spherical particles of HGH, washed with an organic solvent, was dissolved directly in the aqueous environment similar to the source material HGH.

When a small spherical particles of HGH washed zinc buffer, the solubility decreased (Fig). Dissolution of small spherical particles of HGH was carried out in 10 mm Tris, 154 mm NaCl, 0,05% Brij 35, pH 7.5 at 37°C. a More complete selection of protein was achieved in other environments in vitro. The kinetics of dissolution showed that approximately 30% of the total HGH stood out in the first 15 minutes and approximately 50% was allocated in the first 24 hours. The selection of the protein was fully completed within 1 month. The fact that the dissolution of small spherical particles was biphasic manner, may result in some delay of excretion in vivo.

D.Small spherical particles of lysozyme

Example 23

Preparation of small spherical particles of lysozyme

Solution: 1.6 mg/ml lysozyme, 13.2% PEG 3350, 55 mm ammonium acetate, pH 9,5, 53 mm ammonium sulfate, 263 mm NaCl, 26 mm of calcium chloride.

PEG and buffer were heated to 40°C (pH of 9.55). The resulting suspension was rapidly frozen in liquid nitrogen and were liofilizovane in multiple liofilizadora. Received a small spherical particles.

E.Small spherical particles is desoxyribonuclease (Gnkazy)

Example 24

Preparation of small spherical particles Gnkazy

Example formulation: solution: 0.18 mg/ml Gnkazy (from raw materials 1 mg/ml), and 18.2% PEG 3350 (from raw materials 25%), 9 mm ammonium acetate, pH of 5.15 (from raw materials 1M).

This suspension was cooled in the freezer at -80°C and after cooling was liofilizovane in multiple liofilizadora, and then washed by centrifugation with MeCl2/acetone.

Tested the initial concentration was equal to 0.1 mg/ml Gnkazy and 20% PEG 3350. But after trying the cooling from 37°C to 0°C and absence of sediment added another number Gnkazy to get a higher concentration. This solution was chilled in the freezer at -80°C and after freezing was liofilizovane in multiple liofilizadora. Washed by centrifugation with MeCl2/acetone (Fig. 37, 38).

Activity (analysis on Tenkasu-I using the DNA-methyl green, obtained from Sigma).

theoretical activity of the source material is 775 CI/mg protein. In the solution of raw materials identified 0,145 mg/ml protein. This concentration was diluted to 5 ml for a final concentration of 0,0199 mg/ml. Activity should be 775 CI/mg * 0,0199 mg/ml = 15,46 CI/ml

CI/ml=-0,0004×40×1/-0,0011=14,55 CI/ml

Comparison with theoretical:

small spherical particles/theoretical*100%=% of the activity

14,55 CI/ml / 15,46 To the/ml * 100% = 94,1%

F.Small spherical particles of superoxide dismutase

Example 25

Preparation of small spherical particles of superoxide dismutase

A solution of 0.68 mg/ml UNDER (from raw materials 5 mg/ml), 24,15% PEG 3350 (from raw materials of 31.25%), 9.1 mm ammonium acetate (from raw materials 1M), final pH 4,99 established by ammonium hydroxide and acetic acid. The solution was cooled from 40°C to 0°C for 50 minutes (~0,8°C/min), and the deposition was started about 25°C. the Suspension was rapidly frozen in liquid nitrogen and were liofilizovane in multiple liofilizadora, and then washed by centrifugation with MeCl2/acetone (Fig. 39, 40).

Cooled from 40°C to 0°C for 50 minutes (~0,8°C/min). Deposition began about 25°C. was Rapidly frozen in liquid nitrogen and were liofilizovane in multiple liofilizadora. Washed by centrifugation with MeCl2/acetone. Formed small spherical particles, and most of the acetone was kept.

G.Small spherical particles of subtilisin

Example 26

Small spherical particles of subtilisin using non-polymeric reinforcing phase separation agents

The continuous phase of the original system may not contain reinforcing polymer phase separation agent to cause phase separation of protein during cooling. Small spherical particles of subtilisin could the t to be formed according to the present invention using a mixture of propylene glycol and ethanol without the use of any other polymers. Propylene glycol acts as an agent lowering the freezing temperature, and the ethanol serves as a reinforcing phase separation agent in the system. Propylene glycol also contributes to the formation of the spherical shape of small spherical particles.

Prepared 20 mg/ml solution of subtilisin 35% propylene glycol - 10% of the formate - 0,02% CaCl2. A solution of 35% propylene glycol-subtilisin then brought in 67% ethanol with stirring. The solution remained transparent at room temperature. However, when cooled to -20°C for one hour to form a suspension of particles. After centrifugation to collect the particles and washing 90% ethanol was performed Coulter analysis of particle size with absolute ethanol as suspendida fluid. Particles, giving Coulter results, consist of discrete particles having an average diameter of 2.2 μm, and 95% of these particles were from 0.46 to 3,94 μm. Evaluation of optical microscopy confirmed these results, showing the substantially spherical particles. SAM particle analysis confirmed Coulter results.

The preservation of the enzymatic activity of subtilisin after the formation of small spherical particles

The preservation of the enzymatic activity after transformation of subtilisin in solution in a small spherical particles of subtilisin was confirmed by colorimetric analysis. Theoretical summary the units of activity of small spherical particles was calculated by subtracting the total units, found in the supernatant (after separation of the particles subtilisin), from the total units subtilisin found in the ethanol-subtilisin-propilenglikolem solution before cooling. The actual total units found for small spherical particles subtilisin, divided into theoretical units and expressed as a percentage, reflects the persistence of the activity of subtilisin after the formation of the particles. According to this calculation 107% of theoretical activity of subtilisin remained after the formation of small spherical particles of subtilisin.

N.Small spherical particles of the carbohydrate

Example 27

The formation of small spherical particles of the carbohydrate

The present invention can be used for making small spherical particles of carbohydrate. It is possible to cause phase separation between the phase of the PEG and the phase of dextran in the cooling system. You can use the dextrans with different molecular weight, for example, 5K, 40K, C and 500K. A mixture of 5 mg/ml dextran 40K in 30% PEG 300 was brought to equilibrium at 35°C, then the mixture was cooled to 0°C and was liofilizovane. Particles were collected by washing the mixture with methylene chloride:acetone (1:1) and centrifugation. As can be seen from Fig formed small spherical particles. Other carbohydrates, such as starch, gidroxiatilkrahmal, trehalose, La the Tosa, mannitol, sorbitol, gelosa, dextran sulfate, etc. can be obtained in the form of small spherical particles using this method.

I.Microencapsulation pre-manufactured small spherical particles

Example 28

Preparation of PLGA-encapsulated small spherical particles are pre-manufactured insulin

a) 20% (wt./about.) the polymer solution (8 ml) was prepared by dissolving 1600 mg polylactide-with-glycolide (PMGC, MM 35k) in methylene chloride. To this solution was added 100 mg of a small spherical particles of insulin (Isms) and obtained homogeneous suspension by vigorous stirring of the environment using a rotor/stator homogenizer at 11k rpm Continuous phase consisted of 0.02% aqueous solution of methylcellulose (24 ml), saturated with methylene chloride. The continuous phase was stirred at 11k rpm using the same homogenizer, and the suspension was gradually introduced into the environment to generate embryonic microencapsulating particles of the organic phase. This emulsion had a ratio M/1:3. Emulsification was continued for 5 minutes. Then the emulsion is immediately transferred to the environment hardening, consisting of 150 ml of deionized (DI) water, and the medium was stirred at 400 rpm Organic solvents were removed within one hour under reduced pressure of-0.7 bar. For verdeschi microencapsulation particles were collected by filtration and washed with water. Washed microencapsulation particles were liofilizovane to remove excess water. Received microencapsulation particles had an average particle size of about 30 microns, with most of the particles was less than 90 microns, and contained 5,7% (wt./wt.) the insulin.

b) 30% (wt./about.) the polymer solution (4 ml) was prepared by dissolving 1200 mg of 50:50 polylactic-with-glycolide (PMGC, MM 35k) in methylene chloride. Then a suspension of 100 mg Isms in a solution of the desired polymer was prepared using a homogenizer. This suspension was used to generate M/emulsion in 12 ml of a 0.02% aqueous solution of methylcellulose as described in example 28 (b/M ratio = 1:3). The same procedures as in example 28 was used for the preparation of the final microencapsulating particles. Received microencapsulation particles had an average particle size of 25 microns, varying from 0.8 to 60 μm. The content of insulin in the data microencapsulating particles was equal to 8.8% (wt./wt.).

Alternatively, 10% (wt./about.) the polymer solution used for implementing the method of microencapsulation described in the same terms. This method led to microencapsulating particles with an average particle size of about 12 microns, with the majority of particles less than 50 microns and the insulin content of 21.1% (wt./wt.).

The method of in vitro selection

In vitro selection (IVC) of insulin from microencapsulation is the R of the particles was achieved by adding 10 ml of release buffer (10 mm Tris, of 0.05% Brij 35, 0.9% NaCl, pH of 7.4) in a glass ampoule containing 3 mg of equivalent encapsulated insulin was maintained at 37°C. at designated time intervals 400 ál of IVC medium was transferred into a tube microcentrifuge and centrifuged for 2 min at 13k rpm Top 300 µl of the supernatant was removed and kept at -80°C until analysis. Taken volume was replaced with 300 μl of fresh medium, which was used to recreate the solid residue with the remaining supernatant fluid (100 μl). The suspension is transferred back to the corresponding in vitro-emitting medium.

Example 29

Method for microencapsulating pre-manufactured small spherical particles of insulin in the matrix system alloy PMGC/JMC

30% (wt./about.) the solution PMGC/PMC alloy was prepared in methylene chloride (4 ml). The alloy consisted of 50:50 PMHC (MM 35k), D,L-polylactic acid (PLA, MM 19k) and poly L-PLA (PLLA, MM 180k) 40, 54 and 6% (0,48, and 0.07 to 0.68 g), respectively. For the preparation of the final microencapsulating particles followed the same procedures as in example 28b. Examples microencapsulating particles had a size range of particles from 0.8 to 120 μm, average at 40 μm, with the highest number of particles smaller than 90 microns.

Example 30

Methodology microencapsulation pre-manufactured small spherical particles of insulin in PM is To the matrix system using PEG and in the continuous and discrete phases

A solution of 4 ml of 10% 50:50 PMHC (0.4 g) and 25% polyethylene glycol (PEG, MM 8k) was prepared in methylene chloride. Using a rotor/stator homogenizer, 100 mg Insms suspended in this solution at 11k rpm Continuous phase consisted of an aqueous solution (12 ml) of 0.02% (wt./about.) methylcellulose and 25% PEG (MM 8k), saturated with methylene chloride. The continuous phase was stirred at 11k rpm, using the same homogenizer, and the suspension was gradually introduced into the environment to generate embryonic microencapsulating particles of the organic phase. This emulsion had a ratio M/1:3. Emulsification was carried out for 5 minutes. Then the emulsion is immediately transferred to the environment hardening, consisting of 150 ml of DI-water, and the medium was stirred at 400 rpm Organic solvent was removed within one hour under reduced pressure of-0.7 bar. Hardened microencapsulation particles were collected by filtration and washed with water. Washed microencapsulation particles were liofilizovane to remove excess water. Microencapsulation particles of this example had an average particle size of 30 μm in the range from 2 to 90 μm, with most of the particles was less than 70 μm. The insulin content in the microspheres was equal to 16.0% (wt./wt.).

Example 31

Method for microencapsulating pre-manufactured n is large spherical particles of insulin in PMGC the matrix system at different pH of the continuous phase using phosphate buffer

A solution of 4 ml of 20% to 50:50 35 D PMHC (0.8 g) was prepared in methylene chloride. Using a homogenizer of the rotor/stator, 100 mg Insms was suspensively in this solution at 11k rpm Continuous phase consisted of an aqueous solution of 0.1% (wt./about.) methylcellulose and 50 mm phosphate buffer at pH of 2.5 to 5.4 and 7.8. Microencapsulation was performed using continuous installation (figa). The continuous phase was stirred at 11k rpm and served in the chamber of emulsification at 12 ml/min Dispergirovannoyj phase was introduced into the chamber at 2.7 ml/min to generate embryonic microencapsulating particles. The resulting emulsion was removed from the chamber and transferred to a bath of solidification in a continuous manner. Wednesday solidification was stirred at 400 rpm Organic solvent was removed within one hour under reduced pressure -0,4 bar. Hardened microencapsulation particles were collected by filtration and washed with water. Washed microencapsulation particles were liofilizovane to remove excess water.

The content of insulin in the received microencapsulating particles prepared at pH of 2.5 to 5.4 and 7.8, was assessed as a 12.5, and 11.5 and 10.9, respectively. The results of the analysis of the size distribution of microencapsulating particles are summarized in table 5.

Table
The size distribution microencapsulating particles of insulin containing PMHC made at different pH of the continuous phase
Particle size (µm)
the pH of the continuous phaseRangeAverage95% lower5% below
2,51,4-542435,913,8
of 5.40,9-4623to 33.811,8
7,80,8-251116,0the 5.7

The method of in vitro selection

In vitro insulin secretion from microencapsulating particles was achieved by adding 10 ml of release buffer (10 mm Tris, 0.05% of Brij 35, 0.9% NaCl, pH of 7.4) in a glass ampoule containing 3 mg of equivalent encapsulated insulin was maintained at 37°C. at designated time intervals 400 ál of IVC medium was transferred into a tube microcentrifuge and centrifuged who for 2 min at 13k rpm Top 300 µl of the supernatant was removed and kept at -80°C until analysis. Taken volume was replaced with 300 μl of fresh medium, which was used to recreate the solid residue with the remaining supernatant fluid (100 μm). The suspension is transferred back to the corresponding in vitro-emitting medium.

Results in vitro selection (IVC) above preparations shown in Fig and demonstrate a significant effect of the pH of the continuous phase on the kinetics of insulin release from the formulations.

Example 32

Methodology microencapsulation pre-manufactured small spherical particles of albumin human serum in the matrix system PLLA or PLLA/PEG

A solution of 2 ml of 25% (wt./about., 500 mg) PEG (MM 3k or 8k) was prepared in methylene chloride. The PEG solution or 2 ml of methylene chloride was used to form a suspension of 50 mg pre-manufactured small spherical particles serum albumin human (SAC) using a rotor/stator homogenizer at 11k rpm To this suspension was added 2 ml of 4% PLLA (80 mg, MM 180k) in methylene chloride, and the environment homogenized at 11-27k rpm with obtaining the organic phase. The continuous phase consisted of 12 ml of a 0.02% aqueous solution of methyl cellulose saturated with methylene chloride. Emulsification initiated by vigorous mixing of the continuous phase at 11k rpm, followed by tapenum the introduction of the organic phase. Wednesday was emulsiable within 5 minutes, and then the emulsion was transferred into a 150 ml DI-water, stirred at 400 rpm All described procedures were performed at 4°C. Environment solidification then moved to room temperature, and the organic solvent was removed within one hour under reduced pressure of-0.7 bar. Hardened microencapsulation particles were collected by filtration and washed with water. Washed microencapsulation particles were liofilizovane to remove excess water. Channel-forming effect of PEG on IVC SAC of the above formulations shown in Fig.

The method of in vitro selection

In vitro selection (IVC) SAC of the encapsulated microencapsulating particles was achieved by adding 15 ml of release buffer (20 mm HEPES, 0.01% of Tween-80, 0,1M NaCl, 1 mm CaCl2, pH 7,4) in 15-ml polypropylene tube for centrifugation, containing 2.5 mg equivalent encapsulated SAC, maintained at 37°C. the sampling Procedure described in example 31.

Example 33

Preparation of PLGA-encapsulated pre-manufactured small spherical particles leuprolide/dextran sulfate

30% (wt./about.) the polymer solution (4 ml) was prepared by dissolving 1200 mg of 50:50 polylactic-with-glycolide (PMGC, MM 35K) in methylene chloride. Then 65.9 mg pre-manufactured small spherical particles leuprolide/sulfate de the country (LSD), containing 50 mg of leuprolide, suspended in the described polymer solution using a homogenizer. This suspension was used to generate M/emulsion in 12 ml of a 0.02% aqueous solution of methylcellulose as described in example 28 (b/M ratio=1:3). The same procedures as in example 28b, used to prepare the final microencapsulating particles.

Microencapsulation particles had an average particle size of 20 microns with the majority of particles less than 50 microns. The results of the IVC leuprolide of microencapsulating particles shown in Fig.

The method of in vitro selection

In vitro selection (IVC) leuprolide of microencapsulating particles was achieved by adding 15 ml of release buffer (10 mm Na-phosphate buffer, 0.01% of Tween-80, 0.9% NaCl, 0.04% Of NaN3pH 7,4) in 15-ml polypropylene centrifuge tubes containing 2.5 mg equivalent encapsulated leuprolide, maintained at 37°C. the sampling Procedure described in example 28.

Example 34

Preparation of PLGA-encapsulated pre-manufactured small spherical particles of recombinant human growth hormone

10% (wt./about.) the polymer solution (4 ml) was prepared by dissolving 0.4 g PLGA-PEG in methylene chloride. Then 100 mg previously obtained a small spherical particles of recombinant human growth hormone (HGH) suspended in the description is " a polymer solution, using the homogenizer. The continuous phase consisted of an aqueous solution of 0.1% (wt./about.) methylcellulose and 50 mm phosphate buffer at pH 7.0. Microencapsulation was performed using continuous installation (figa), as described in example 31. The average size of the data microencapsulating particles was 25 microns with a range of 1 to 60 μm. IVC profile of HGH from the polymer matrix shown in Fig.

The method of in vitro selection

IVC HGH from microencapsulating particles was achieved as described in example 28.

Example 35

Determination of purity microencapsulating pre-manufactured small spherical particles of insulin

To assess the influence of process microencapsulation purity inkapsulirovannykh pre-manufactured small spherical particles of insulin, polymer microencapsulation particles containing pre-made Incms, laid out using the method of two-phase dual extraction. The weighted sample is encapsulated Insms suspended in methylene chloride and gently stirred to dissolve the polymer matrix. For the extraction of protein was added 0,01H. HCl, and the two phases were mixed, obtaining an emulsion. Then the two phases were separated, the aqueous phase was removed and replaced with the same solution, and the extraction process repeated. The purity of extracted is of Sulina were determined using size exclusion chromatography (RAH). This method identifies the content of the monomer, dimer and high molecular weight (VM) particles ins extracted in the environment. The appropriate test was used to identify the effect of decomposition on the purity of the Ann. The results showed no significant influence of this process on the purity of the Ann.

Encapsulated Insms contained from 97,5 to 98.94% of monomers of the protein depending on the conditions and process microencapsulation compared to 99,13% of the content of the monomer in the original Isms (unencapsulated public). The particle content of the dimer in encapsulated Insms changed from 1.04% to 1.99% compared to 0.85% in the original Insns. The contents of the VM in encapsulated Insms varied from 0.02% to 0.06% vs 0.02% in the original Insns. The results are summarized in table 6. The influence of the polymer matrix is shown in Fig. 46 and 47.

Table 6
The influence of process microencapsulation purity encapsulated prior small spherical particles of insulin.
The monomer(%)Dimer(%)VM(%)
Unencapsulated public Insms 99,130,850,02
Encapsulated
Insms
97,5-98,941,04-1,990,02-0,06

Example 36

In vivo insulin secretion from microencapsulating pre-manufactured small spherical particles of insulin

In vivo insulin secretion from microencapsulating pre-manufactured small spherical particles of insulin examined at Spragu Dawley (SD) rats. Animals received initial subcutaneous dose of 1 IU/kg unencapsulated public or encapsulated, pre-fabricated small spherical particles of insulin. ELISA was used to determine levels of recombinant serum human insulin (rcis) in the collected samples. The results are shown in Fig.

Although there have been illustrated and described specific embodiments of, can be proposed numerous modifications without violating the spirit of the invention and the scope of protections are limited only by the framework of the accompanying claims.

1. Method of preparation of small spherical particles of an active agent, including
obtaining a solution in a single liquid phase containing the active agent, a reinforcing phase separation agent and per the first solvent, and
the induction of phase change variable speed in this solution for the occurrence of phase separation liquid-solid active agent with the formation of the solid phase and the liquid phase and the solid phase comprises a solid, small spherical particles of an active agent, and the liquid phase contains a reinforcing phase separation agent and a first solvent, a small, essentially spherical particles, and the specified induction includes a cooling solution.

2. The method according to claim 1, wherein the solution has a transition temperature, the first temperature and the second temperature, and this solution is cooled from the first temperature to the second temperature, the first temperature is above the specified temperature phase transition of the solution, and the second temperature is below the specified temperature phase transition of the solution.

3. The method according to claim 1 or 2, additionally comprising a step selected from the group consisting of regulation of the concentration of the active agent, regulating the concentration of the reinforcing phase separation agent, the regulation of the ionic strength of the solution, pH regulation and regulation of osmolality of the solution.

4. The method according to claim 1 or 2, further comprising changing the concentration of the active agent.

5. The method according to claim 1 or 2, additionally containing a change con is entrale reinforcing phase separation agent.

6. The method according to claim 2, in which the second temperature is above the freezing temperature of the solution.

7. The method according to claim 2, in which the second temperature is below the freezing temperature of the solution.

8. The method according to claim 1 or 2, wherein the step of cooling is carried out with a variable speed component from 0.2 to 50°C/min

9. The method according to claim 8, in which the adjustable speed is in the range from about 0.2 to about 30°C/min

10. The method according to claim 1 or 2, wherein the step of obtaining the solution involves the dissolution of the reinforcing phase separation agent in the first solvent to form a mixture; and adding the active agent to the mixture to form a solution.

11. The method according to claim 10 further comprises the step of dissolving the active agent in the first solvent or the second solvent which is mixed with the first solvent before adding the active agent to the mixture.

12. The method according to claim 6, in which the solution further comprises lowering the temperature of the freezing agent to reduce the freezing temperature of the solution.

13. The method according to item 12, which lowers the freezing temperature of the agent chosen from the group of polyethylene glycol and propylene glycol.

14. The method according to claim 1 or 2, in which the reinforcing phase separation agent is a water-soluble or miscible with water agent.

15. The method according to claim 1 or 2, in which the ω-enhancing phase separation agent selected from the group consisting of linear or branched polymers, polymers based on carbohydrates, paleoliticheskikh alcohols, polyvinyl polymers, polyacrylic acids, polyorganic acids, polyaminoacid, copolymers and block copolymers, tert-polymers, polyethers, natural polymers, polyimides, surfactants, polyesters, branched and cyclopolymer, polyarteritis, starches, substituted starches, polyethylene glycol, polyvinylpyrrolidone, poloxamers, ethanol, acetone and isopropanol.

16. The method according to claim 1 or 2, in which the reinforcing phase separation agent is a polyethylene glycol (PEG).

17. The method according to claim 1 or 2, in which a small spherical particles additionally contain a filler to increase the stability of small spherical particles, to provide a regulated selection of an active agent from a small spherical particles, or to enhance penetration of the active agent through biological tissue.

18. The method according to 17, in which the filler is selected from the group consisting of carbohydrates, cations, anions, amino acids, lipids, fatty acids, surfactants, triglycerides, bile acids or their salts, esters of fatty acids and polymers.

19. The method according to p, in which the cation is chosen from the group consisting of Zn2+Mg 2+and CA2+.

20. The method according to p, in which the bile acid is a cholic acid or its salt.

21. The method according to claim 1 or 2, further containing the step of collecting small spherical particles.

22. The method according to item 21, in which the step of collecting small spherical particles carried out by washing the particles with a liquid medium at a temperature at which the active agent is not soluble in this liquid medium, and a reinforcing phase separation agent is soluble in this liquid medium.

23. The method according to item 22, in which the step of washing is carried diafiltrate or by centrifugation.

24. The method according to item 22, in which the liquid medium is aqueous or organic.

25. The method according to item 22, in which the fluid is a supercritical fluid or a mixture of supercritical fluid and solvent, miscible with the supercritical fluid.

26. The method according to paragraph 24, in which the organic liquid medium selected from the group consisting of methylene chloride, chloroform, acetonitrile, ethyl acetate, ethanol and pentane.

27. The method according to item 22, in which the liquid medium further comprises an agent that reduces the solubility of the active agent in a liquid medium.

28. The method according to item 27, in which the agent that reduces the solubility of the active agent in the liquid medium contains a complexing ion.

29. The method according to p in which complexo brazowy agent is a cation, selected from the group consisting of: Zn2+, CA2+, Fe2+, Mg2+, Mn2+, Na+and NH4+.

30. The method according to item 22 additionally comprising the step of removing the liquid medium.

31. The method according to item 30, in which the step of removing the liquid medium is performed by lyophilization, drying or evaporation.

32. The method according to item 22, in which the liquid medium further comprises a filler.

33. The method according to p, in which the filler increases the stability of small spherical particles, provides controlled release of active agent from a small spherical particles, or increases the penetration of the active agent through biological tissue.

34. The method according to p, in which the filler is selected from the group consisting of carbohydrates, cations, anions, amino acids, lipids, fatty acids, surfactants, triglycerides, bile acids or their salts, esters of fatty acids and polymers.

35. The method according to clause 34, in which the cation is chosen from the group consisting of Zn2+, Mg2+and CA2+.

36. The method according to clause 34, in which the filler is a cholic acid or its salt.

37. The method according to p, in which the reinforcing phase separation agent selected from the group consisting of poloxamers, glycols and mixtures thereof.

38. The method according to claim 1 or 2, wherein the solution contains water or mixes the present with the water solvent.

39. The method according to § 38, which is miscible with water, the solvent is chosen from the group consisting of N-methyl-2-pyrrolidinone (N-methyl-2-pyrrolidone), 2-pyrrolidinone (2-pyrrolidone), 1,3-dimethyl-2-imidazolidinone (SIAS), dimethyl sulfoxide, dimethylacetamide, acetic acid, lactic acid, methanol, ethanol, isopropanol, 3-pentanol, n-propanol, benzyl alcohol, glycerol, polyethylene glycol (PEG), PEG-4, PEG-8, PEG-9, PEG-12, PEG-14, PEG-16, PEG-120, PEG-75, PEG-150, esters of polyethylene glycol, PEG-4 dilaurate, PEG-20 dilaurate, PEG-6 isostearate, PEG-8 palmitostearate, PEG-150 palmitostearate, polietilenglikolmonostearat, PEG-20 servicemetadata, monoalkyl esters of polyethylene glycol, PEG-3 dimethyl ether, PEG-4 dimethyl ether, polypropyleneglycol (BCP), polypropyleneimine, GPR-10 butanediol, GPR-10 methyl glucose ether, GPR-20 methyl glucose ether, GPR-15 stearyl ether, dicaprylate/dicaprate propylene glycol, laurate propylene glycol and glycoluril (ether tetrahydrofurfuryl alcohol and polyethylene glycol), or combinations thereof.

40. The method according to claim 1 or 2, in which the active agent is a pharmaceutically active agent.

41. The method according to p, in which the pharmaceutically active compound selected from the group consisting of therapeutic agents, diagnostic agents, cosmetics / products the definition of funds food additives and pesticides.

42. The method according to claim 1 or 2, in which the active agent is a macromolecule.

43. The method according to § 42, in which the macromolecule selected from the group consisting of proteins, polypeptides, carbohydrates, polynucleotides, viruses and nucleic acids.

44. The method according to item 43, in which the protein is chosen from the group consisting of protein clotting of blood flow, factor VII, factor VIII, factor IX, subtilisin, ovalbumin, alpha-1-antitrypsin, Gnkazy, superoxide dismutase, lysozyme, ribonuclease, hyaluronidase, collagenase, growth hormone, erythropoietin, insulin-like growth factors or their analogues, interferons, glatiramer, factor stimulation of colonies of granulocytes/macrophages, factor stimulation of granulocyte colony, antibodies, monoclonal antibodies, polyclonal antibodies, Fab fragments, single-chain antibodies, Paglierani proteins, glycosylated or hyperglycosylated proteins, desmopressina, LHRH agonists, such as leuprolide, goserelin, nafarelin, buserelin, LHRH antagonists, vasopressin, cyclosporine, calcitonin, the hormone of the parathyroid gland, a peptide hormone of the parathyroid gland and insulin.

45. The method according to claim 1 or 2, in which the particles are suitable for in vivo delivery to the object in need of the active agent.

46. The method according to item 45, in which way Dostuk is selected from the group consisting of methods, injection, inhalation, parenteral, local, oral, rectal, nasal, pulmonary, vaginal, buccal, through the skin, through the mucosa, sublingual, ear, eye, and intraocular ophthalmic.

47. The method according to item 46, in which the method of delivery is a delivery into the lungs.

48. The method according to p in which particles suitable for deposition in the Central or peripheral region of the lungs.

49. The method according to p, in which the particles are delivered by a device selected from the group consisting of a dry powder inhaler, nebulizer measured dose and spray.

50. The method according to item 45, wherein the particles are delivered in the form of a stable liquid suspension.

51. The method according to claim 1 or 2, in which the particles have essentially the same particle size.

52. The method according to claim 1 or 2, in which the particles have an average particle size of from about 0.01 to about 200 microns.

53. The method according to claim 1 or 2, in which the particles have an average particle size of from about 0.5 to about 10 microns.

54. The method according to claim 1 or 2, in which the active agent is from about 0.1 to about 100% of particles by mass.

55. The method according to claim 1 or 2, in which the active agent is from about 75 to about 100% of particles by mass.

56. The method according to claim 1 or 2, in which the content of active agent is equal to or exceeds 90% of the particles by mass.

57. The method according to claim 1 or 2, wherein the small spherical particles have a narrow size distribution.

58. The method according to § 57, in which the ratio of the volume diameter of the 90th percentile of the small spherical particles to the volume diameter of the 10th percentile less than or equal to about 5.

59. The method according to claim 1 or 2, wherein the small spherical particles are semi-crystalline or not crystalline.

60. Method of preparation of small spherical particles of an active agent, and the method comprises the steps:
dissolution of the active agent and the reinforcing phase separation agent in water or miscible with water, the solvent to form solution a single continuous phase, and
induce a phase change, whereby the active agent is undergoing phase separation of liquid-solid with the formation of a solid phase containing the solid, small spherical particles of an active agent, and the liquid phase enhances the phase separation agent, and the specified induction includes a cooling solution with variable speed.

61. The method according to p, in which the solution has a temperature of phase transformation, the first temperature and the second temperature, and a step of bringing a solution to the phase change is performed by cooling the solution from the first temperature to the second temperature, g is e, the first temperature is above the temperature of phase transformation solution and the second temperature is below the temperature of phase transformation solution.

62. The method according to p, in which the adjustable speed is in the range from about 0.2 to about 50°C/min

63. The method according to claim 1, in which the indicated induction includes a cooling solution with an adjustable rate to a temperature above the freezing temperature of the specified solution.

64. The method according to p in which the specified adjustable speed above 25,6°C/min

65. The method according to claim 1, wherein said reinforcing phase separation agent comprises a mixture of poloxamer and polyethylene glycol.

66. The method according to claim 1, wherein the first solvent is water.

67. The method according to claim 1, in which the percentage of conversion of the active agent from the source solution into particles is at least 90%.

68. The method according to claim 1, wherein the active agent is released from the particles, retains the biological activity of the active agent in the initial solution, which is determined on the basis of its special properties.

69. The method according to claim 1, wherein the active agent is released from the particles, retains the structural purity of the active agent in the initial solution, which is determined by spectroscopy circular dichroism.

70. The method according to claim 1, wherein the small spherical particles have a geometric standard deviation of less than 2.5.

71. The method according to item 70, in which the geometric standard deviation of less than 1.8 and the ratio of the volume diameter of the 90th percentile of these particles to the volume diameter of the 10th percentile of these particles is less than 3.

72. The method of preparation of the solid, small spherical particles of an active agent, including:
obtaining an aqueous solution in a single liquid phase, and containing an active agent and a reinforcing phase separation agent;
cooling the aqueous solution with an adjustable rate to a temperature below the phase transition of the active agent in solution;
obtaining thus a suspension containing small solid particles of the active agent suspended in the liquid phase, containing the reinforcing phase separation agent and water, and a small spherical particles are essentially spherical.

73. The method according to item 72, in which the cooling of the aqueous solution is carried out with a constant or linear velocity, nonlinear speed, uneven or programmable speed.

74. The method according to item 72, in which the cooling of the aqueous solution is conducted at speeds of more than 0.2°C/min

75. The method according to item 72, in which the aqueous solution further comprises an agent that lowers the freezing point.

76. The method according to item 72, in which the indicated cooling includes cooling the solution to a temperature above the freezing temperature of the solution.

77. The method according to item 75, in which the agent that lowers the freezing temperature, contains polyethylene glycol.



 

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1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: group of invention covers the microcapsules consisting of at least one core which contains the rubber supplement, and the first polymeric coat of melamine-formaldehyde or phenol-formaldehyde resin; the second polymeric coat is applied upon the microcapsule and its content differs from that of the first one and consists of the low-molecular inorganic or organic compound as a sliding and wear layer to reduce the static friction.

EFFECT: method of preparation microcapsules and use in rubber vulcanisation.

20 cl, 14 ex, 2 tbl

FIELD: derivatives of chitosan.

SUBSTANCE: invention relates to preparing biologically active chitosan substances and their derivatives. Invention describes a modified chitosan substance showing pH-neutral reaction and plastic structure of chitosan particles as fractal chitosan particles of size of nanofractals from 1 nm, not less, and to 5000 nm, not above, or as cross-linked net-shaped polymer having multiple cavities of size from 1 nm, not less, to 50 nm, not above. Invention describes methods for their preparing. Invention provides high transdermal penetration of chitosan substance and enhanced capacity for administration of medicinal or biologically active substances into chitosan substance. Invention can be used in manufacturing cosmetic, curative-cosmetic, pharmacological preparations, biologically active food supplements and foodstuffs.

EFFECT: improved and valuable properties of chitosan substances.

14 cl, 4 tbl, 7 dwg, 9 ex

FIELD: food and pharmaceutical industries.

SUBSTANCE: object of invention is production of freely flowing alcohol-containing encapsulated products. Food fat and/or wax are melted in heated reactor at 55-65°C, after which required food additives or additive complexes are added, resulting mixture is stirred to achieve homogenous mass and alcoholic produce with alcohol content from 5 to 96.6% is added in amount 5 to 60 wt parts per 100 wt parts of the reactor charge. Emulsion obtained is supplied to atomization turbine wherein arising drops are solidified in air and collected in receiving device. Thus obtained powder is screened and tightly packaged. Granule size is controlled by turbine rotation speed and varied within a range of 0.1 to 1 mm.

EFFECT: enabled creation of fundamentally novel formulations and production technologies, improved quality, reduced expenses, and prolonged shelf time of produce.

3 cl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmacology and medicine, namely to obtaining orally decomposing powder of cilostazol. For this purpose into pharmaceutical preparation included are from 10% wt to 20% wt of cilostazol and from 70% to 79.5% wt of mannitol.

EFFECT: creation of cilostazol preparation, easily decomposing in oral cavity and not requiring taking water with it.

13 cl, 3 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmacology, in particular to medication for treatment of diabetic ulcers of foot or ski9n chronic ischemic wounds of lower extremities in patients with diabetes and for prevention of amputation of said extremities. Application of pharmaceutical composition, which contains microspheres of epidermal growth factor, of definite diameter, for treatment of diabetic ulcers of foot or skin chronic ischemic wounds of lower extremities in patients with diabetes and for prevention of amputation of said extremities.

EFFECT: composition makes it possible to reduce frequency of introduction of medications during treatment and ensures quicker healing of lesions.

8 cl, 8 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of medicine and chemical-pharmaceutical industry, in particular, to solid therapeutic preparative form, which includes granulated materials, containing as therapeutically active compound, ospemifene, also known as (deaminohydroxy)toremifene, or its geometric isomer, stereoisomer, pharmaceutically acceptable salt, ester or metabolite, in combination with one or more intragranular excipients.

EFFECT: obtaining required medications.

2 cl, 1 dwg, 1 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of medicine. Composition includes inert core, onto which evenly layered is active substance, which in its turn is covered with envelope including hardening and lipophilic agents. Also described is method of obtaining said pharmaceutical composition.

EFFECT: obtaining pharmaceutical composition with controlled rate of venlafaxine hydrochloride release.

15 cl, 2 dwg, 5 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compositions of particle-like sulfoalkyl ether of cyclodextrin SAE-CD. The SAE-CD composition has a primary combination of physical properties which are not found out in common solid forms of SAE-CD.

EFFECT: SAE-CD compositions under the invention have improved fluidity, crushing resistance, tableting ease and improved water dissolution rate.

21 cl, 4 dwg, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to chemical-pharmaceutical industry, and can be applied in technology of pill production. After granulation in pseudoliquefied layer in dispersion carried out is calibration of granulated material on vertical conical calibrator with following process parametres: rotor speed - 2-5 rot/sec; gap between grid and rotor - 1-3 mm; diametre of grid cell - 1-2 mm. Active pharmaceutical substance is selected from group, which includes enalapril, captopril, methionine. Binding agent is selected from group, which includes polyvinylpyrrolydone, methylcellulose, gelatin, starch, copolymer of vinylpyrrolydone with vinylacetate, sucrose, vegetable extracts.

EFFECT: method of obtaining pills makes it possible to increase dosing accuracy by prevention of stratification of multi-component pilled masses, due to improvement of components and their mixtures friability and ensuring evenness of their supply to matrix of pill machine.

7 cl, 3 ex, 12 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of pharmaceutics, in particular deals with composition with prolonged release of active substance, representing iron source, containing at least one coated granule, said coated granule consisting of particle, which contains said active substance, covered with at least two coats, containing combination of excipients, as well as to method of obtaining it. Claimed compositions can be used for obtaining medication, intended for treatment and/or prevention of iron deficiency and anemia, induced by iron deficiency.

EFFECT: invention provides composition with prolonged release of active substance, possessing high stability and ensuring protection of active substance from oxidation.

30 cl, 2 ex, 3 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmacology and medicine, and represents a solid and stable oral dispersion of water-soluble Vinca alkaloid derivative, particularly vinorelbine in at least one polyethylene glycol of molecular weight within 1000 and 6000, wherein a mass ratio of water-soluble vinorelbine and more specifically, firstly vinorelbine ditartrate and, secondly polyethylene glycol is within 1.5:1 and 1:10 and, preferentially within 1:3 and 1:6.

EFFECT: invention provides higher stability of Vinca alkaloid derivatives, and especially vinorelbine salts, particularly vinorelbine ditartrate.

14 cl, 3 ex, 2 tbl, 2 dwg

FIELD: medicine.

SUBSTANCE: vitamin dosage form contains a combination of biologically active substances chosen, first of all, from a group of vitamins, mineral substances and microelements, and also their mixtures and at least partially included or introduced in a swellable matrix providing controlled or time shift release of biologically active substances after intake of such composition.

EFFECT: higher bioavailability of the biologically active substances, optimised absorbtion in a gastrointestinal tract, preferentially with avoided time overdose of the relevant biologically active substances and prevented excessive load on involved absorption systems.

22 cl, 3 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of medicine. Composition containing microparticles of glucagons-like peptide 1 (GLP-1) in combination with diketopiperazine (DKP) is stable in vitro and in vivo. Composition may be used as pharmaceutical preparation to treat diseases or conditions, including, but not limited to, diabetes, cancer and obesity.

EFFECT: invention provides for minimum risk in case of intrapulmonic administration.

24 cl, 31 dwg, 20 ex

FIELD: medicine, in particular composition for quick-disposable in buccal cavern tablets.

SUBSTANCE: claimed composition contains granulated product of fine dispersed long releasing particles, comprising drug and fillers selected from group including sugars and sugar alcohols together with binder, wherein content of non-granulated fine dispersed long releasing particles is 0-15 %. Method for production of such tablets is also disclosed.

EFFECT: pharmaceutical composition with accelerated degradation.

24 cl, 9 ex, 3 dwg

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