Compositions for inhalation

 

(57) Abstract:

The composition contains a pharmaceutically active polypeptide in the mixture with the connection-amplifier. Connection-amplifier increases the absorption of the polypeptide in the lower respiratory tract of the patient. The amplifier is a surface-active substance. Its amount is more than 10% of the total mass of the polypeptide and the connection of the amplifier. The mixture is a dry powder. At least 50% of the total mass of the powder consists of particles with diameter less than or equal to 10 microns, or agglomerates of these particles. The composition is administered by inhalation, provides systemic delivery of the drug into the lungs of peptides and proteins, and improved absorption of these drug compounds. 5 S. and 26 C. p. F.-ly, 3 ill., table 1.

The present invention relates to methods and compositions for drug delivery of useful peptides and proteins.

The background to the invention.

Although the advent of genetic engineering has led to the rapid expansion of the list of drugs based on peptides, however, the main obstacle therapy on the basis of the peptides is significantly Stradina realization of the full potential of this area: in General, lo collapse under the action of enzymes in the gastrointestinal tract before how can they reach the bloodstream. If desired polypeptide cannot be changed to make it relatively resistant to these enzymes, the only practical way to deliver drugs is probably parenteral, e.g. intravenous, intramuscular or subcutaneous injection. The introduction of other parenteral routes (e.g., by absorption through the nasal, buccal or rectal sheath or through the lungs) were less successful.

A brief statement of the substance of the invention

It was found that, when the peptide or protein ( hereinafter collectively referred to as polypeptides) combined with a suitable amplifier, absorption and injected into the lungs in the form of a powder with a suitable particle size, it easily enters the pulmonary circulation by absorption through the layer of epithelial cells in the lower respiratory tract. This is conveniently performed by inhalation of powder from the device is an inhaler that releases the exact dose of powdered combinations of polypeptide/amplifier with particle size, which maximizes deposition in the lower respiratory tract in contrast to the oral cavity and throat. (To facilitate reference polypeptide and the amplifier further Sobornosti more active compounds should consist of particles, having a diameter less than about 10 microns (e.g., between 0.01-10 μm, ideally between 1-6 μm). In preferred embodiments, at least 50% (preferably at least 60%, more preferably at least 70%, even more preferably at least 80% and most preferably at least 90%) of the total mass of active compounds that are found in the device-inhaler consists of particles with the desired size limit diameters.

The invention thus includes a pharmaceutical composition comprising a mixture of active compounds: (a) a pharmaceutically active polypeptide and (B) connection of the amplifier, which increases systemic absorption of the polypeptide in the lower respiratory system (preferably light) of the patient, the mixture is in a form suitable for inhalation as a dry powder in which at least 50% of the total mass of active compounds (a) and (b) consists of the initial particles having diameter less than or equal to about 10 microns. The initial particles can be Packed as such or can be converted into agglomerates, which are then mainly disagglomerated prior to the introduction into the respiratory tract of the patient. The composition may, of course, containing matemticas acceptable excipients, for example, diluents or carriers. Therefore, therapeutic drug of the present invention may contain only these active compounds or it may contain other substances, for example, pharmaceutically acceptable carrier. This carrier may preferably be composed of particles having a diameter of less than about 10 microns, so that at least 50% of the resulting powder consists of possible initial agglomerated particles having a diameter of less than about 10 microns; alternatively, the carrier may mainly consist of much larger particles (coarse particles), so that "ready-to-use mixture can be formed of the active compounds and the media. In an ordered mixture, alternatively known as interactive or adhesive mixture, the smallest particles of a drug (in the present invention the active compounds) are fairly evenly distributed on the surface of the coarse filler particles (in the present invention pharmaceutically acceptable carrier). In this case, the active compounds are preferably not in the form of agglomerates to the formation of the ready to use mixture. Coarse particles can have a diameter of more than 20 a MIC so if you want you can use the coarse particles of different sizes in accordance with the practical requirements of the finished formulation. You do not want the coarse particles in the assigned mixture were the same size, but they may have a similar size within the size of the assigned mixture with the preferred diameter equal 60-800 microns.

The polypeptide can be any drug or diagnostically suitable peptide or protein with small-to-medium, i.e., molecular weight (MW) to about 40 kDa, which is desirable systemic delivery. For all of these peptides in General are suitable and can be applied mechanisms of increased polypeptide absorption in accordance with the present invention, although the extent to which increases their adsorption may vary in accordance with the MW and physico-chemical properties of the polypeptide and specific applied by the amplifier. It is expected that this invention will be most suitable polypeptides having a molecular weight of 30 kDa, for example, polypeptides having a molecular mass of 25 kDa or 20 kDa, especially up to 15 kDa or 10 kDa. Any desired polypeptide can be easily tested for use in the present invention with a specific amplifier through analyses of in vivo or in vitro, as described here.

Connection-amplifier used in the compositions nastoj respiratory tract and the systemic circulation. The term "enhances absorption" means that the amount of polypeptide that is absorbed into the systemic circulation in the presence of amp, more than in its absence. Preferably the number of carbon polypeptide is significantly higher (p < 0.05) in the presence of amp. The suitability of any potential amplifier for use in the present invention can be easily determined by tests in vivo or in vitro, as described here.

The amount of absorbed polypeptide in accordance with the present invention is preferably at least 150% of the amount absorbed in the absence of the amplifier. In preferred embodiments, the absorption of the polypeptide is at least doubled, more preferably tripled and most preferably whateverelse in the presence of amp compared to its absorption in the absence of the amplifier.

The amplifier is preferably a surface-active substance, such as salt of fatty acid, salt of the bile acid derivative salts of bile acids, Alkylglucoside, a cyclodextrin or a phospholipid. The amplifier may be, for example, a salt of sodium, potassium, or organic amine fatty acids, fatty key is titanium amplifier is capret sodium. The ratio of the polypeptide to the amplifier preferably will vary from 9:1 to 1:1. Although the relative content of the amplifier is higher than 1: 1 should, apparently, to enhance absorption as well or better than a lower relative content, consider that the number of amplifier should not be higher than necessary to achieve the desired gain level, because the excess of the amplifier may cause unwanted side effects such as local irritation.

In the scope of the invention is also a method of system introduction pharmaceutically active polypeptide by assigning the patient inhalation, the pharmaceutical compositions of the present invention, in which at least 50% of the total mass of active compounds at the site of entrance into the respiratory tract of a patient consists of particles having diameter less than or equal to about 10 microns. This is preferably carried out using a device-inhaler from which the patient inhales the powder. When the powdered composition is in the form of agglomerates of the initial particles, the device preferably has such a shape that induces significant desagglomeration agglomerates inhalation powder patient from the device, the n, prior to entering the powder in the respiratory system of the patient. This desagglomeration must occur inside the unit, it usually is induced by air turbulence created in the device by the power of breathing. In the ordered mixture of agglomerates in General preferably not formed. In the case of an ordered mixture of active compounds must be released from large particles, preferably by inhalation, or by using mechanical means your inhaler, or only under the action of inhalation, or by other means, and an active connection then Deposit in the lower respiratory tract, and the particles of media forms in the mouth.

The device is an inhaler is preferably inhaler single dose dry powder, but may be an alternative inhaler with many doses of a dry powder.

The invention also includes methods of making a pharmaceutical composition suitable for administration by inhalation. One such method is to first prepare a solution, which is dissolved (a) a pharmaceutically active polypeptide and (b) the connection of the amplifier, which increases systemic absorption of the polypeptide in the lower respiratory tract of the patient. Rastogi the solid residue of finely pulverized to obtain a powder. The second method involves dry mixing (a) a pharmaceutically active polypeptide and (b) connection of the amplifier and fine grinding the resulting mixture. However, the third one suitable method includes the stage of receiving the first finely ground preparation containing the polypeptide, and the second finely ground product containing the compound amplifier, and the mixing together of two finely powdered drugs. When you want to include the media, except when it is desirable to have an ordered mixture, it can be added in solution or in a dry mixture of the pharmaceutically active polypeptide to fine grinding or finely chopped medium can be mixed with other finely ground components. When receiving an ordered mixture of finely ground polypeptide and the amplifier is mixed with a suitable carrier.

Brief description of drawings

Fig. 1 is a graph illustrating the effect of different concentrations of the amplifier, which is used capret sodium, to transfer the connection-token (mannitol) through a monolayer of cultured epithelial cells.

Fig. 2 is a graph illustrating the effect of different concentrations of the amplifier, as the cat is liannah cells in the presence of the polypeptide (mass ratio caprata sodium and polypeptide 1:3).

Fig. 3 is a graph of the concentration of the polypeptide of the plasma as a function of time after inhalation only polypeptide, the polypeptide with caprata sodium at a ratio of 90:10 and polypeptide and caprata sodium in the ratio of 75:25.

Detailed description

Some preferred embodiments of the invention in General are described below.

Polypeptide

The polypeptide preferably is a peptide hormone, with the exception of insulin, for example, a vasopressin analogue of vasopressin, desmopressin, glucagon, corticotropin (ACTH), gonadotropin (luteinizing hormone or LHPH), calcitonin, c-peptide of insulin, parathyroid hormone (PTH), human growth hormone (hGH), growth hormone (HG), releasing hormone growth hormone (GHRH), oxytocin, releasing hormone corticotropin (CRH), somatostatin analogue, analogue agonist gonadotropin (GhRHa), atrial natriuretic peptide (hANP), releasing hormone thyroxine (TRHrh), follicle-stimulating hormone (FSH) and prolactin.

Other possible polypeptides include growth factors, interleukins, polypeptide vaccines, enzymes, endorphins, glycoproteins, lipoproteins, and polypeptides included in the system smartyvalidate size small to medium, relatively high water solubility and isoelectric point between about pH3 and pH8 can be effectively delivered by the methods of the invention.

Amplifier

The use of amplifier absorption is critical, because the polypeptide itself (one) is poorly absorbed through the lungs. Applicable amplifier may be any of a number of compounds that act, increasing absorption through the layer of epithelial cells lining the lower respiratory tract, and in the adjacent pulmonary vasculature. The amplifier can perform this action by any of several possible mechanisms. These mechanisms shall include:

(1) Strengthening paracellular permeability of the polypeptide by inducing structural changes in place tight connection of epithelial cells.

(2) Strengthening paracellular permeability of the polypeptide by interacting with protein or lipid components of the membrane or extraction through them and this breach of membrane integrity.

(3) the Interaction between the amplifier and the polypeptide that increases the solubility of the polypeptide in aqueous solution. This can be done by preventing the formation of aggregates of insulin (DNIe viscosity or dissolution of the mucosal barrier, lining the alveoli and passages of the lungs, resulting in epithelial surface becomes capable of direct absorption of the polypeptide.

The amplifiers can operate only in one of the above mechanisms, or two or more. Power, acting through several mechanisms, is more suitable for promotion of effective absorption of the polypeptide than the amplifier, which uses only one or two mechanisms.

Surfactants are, for example, a class of amplifiers, which are believed to act through any of the above mechanisms. Surfactants are amphiphilic molecules having both lipophilic and hydrophilic part, with the changing balance between these two characteristics. If the molecule is very lipophilic, low solubility of substances in water may limit its suitability. However, if the hydrophilic part of the overwhelmingly dominant, then the surface-active properties of the molecule can be minimal. Therefore, in order to be effective, surface-active agent should provide a suitable balance between sufficient solubility and sufficient surface Aktobe charge surfactants at pH values in the lungs (approximately 7,4). At pH 7.4 for some polypeptides have a negative charge. This can lead to electrostatic repulsion between the molecules, which, in turn, can prevent aggregation and thus increase the solubility. If a surfactant is also negatively charged, it can interact with the polypeptide, for example, by hydrophobic interactions, and can be additional repulsion among the polypeptide molecules. In this case, the anionic surfactant will have an additional advantage in comparison with surface-active substances having a neutral or positive charge) in enhancing the absorption, contributing to stabilization of the polypeptide in Monomeric state.

A number of different compounds potentially useful as amplifiers in the methods of the invention was tested on rats, as described in the following example 2. Other substances with known properties to enhance the absorption or physical characteristics that make them likely candidates for use in the method of the present invention, can easily be tested by an expert ordinary skill analysis in viv-amplifiers also gives satisfactory results. It is believed that the use of such a combination in the method of the present invention is within the scope of the invention.

The amplifier is suitable for methods of the present invention may combine the effective strengthening of the absorption of the polypeptide with (1) lack of toxicity when applied concentrations and (2) good powder properties, i.e. no sticky or waxy consistency in the solid state. The toxicity of a given substance can be checked by standard methods, such as the MTT-test, for example, as described in Int. J. Pharm., 65 (1990), 249-259. Powder properties of this substance you can see from the published data for this substance or empirically.

One very promising type of amplifier is a salt of fatty acid. It is found that the sodium salt of saturated fatty acids with carbon chain of 10 (i.e., capret sodium), 12 (sodium laurate) and 14 (monistat sodium) atoms are well suited for the method of the invention. Also found that potassium salts and lysine capric acid is effective in the method of the present invention. If the carbon chain consists of less than 10 atoms, the surface activity of surfactants may be too low and, if carbon is bullying; appropriate.

Most preferred in the present invention is a substance which enhances the absorption of the polypeptide in the lower respiratory tract, is capret sodium.

Various counterions can change the solubility of the salt of saturated fatty acid in the water, so the amplifier, the carbon chain of which contains from 10 to 14 atoms, may be even more desirable than the amplifiers specifically listed above. Salts of unsaturated fatty acids can also be used in the present invention because they are more water soluble than salts of saturated fatty acids, and may have a longer carbon chain than the last, and at the same time maintain the solubility necessary for a good amplifier absorption of the polypeptide.

All of the tested salts of bile acids and derivatives of bile salts (sodium salt ursodeoxycholate, taurocholate, glycocholate and eurodiversity) effectively increase the absorption of the polypeptide in the lungs.

As amplifiers experienced the phospholipids. It was found that single-chain phospholipid (lysophosphatidylcholine) is an effective amplifier, whereas double-stranded phospholipids (dictyochophyceae and dedica fosfolipidy much less soluble in water, than their single counterparts; however, it is reasonable to assume that double-stranded phospholipids with a shorter chain having greater solubility in water than their counterparts with longer chain, it is possible to apply the present invention as amplifiers, so you can use both single-stranded and double-stranded phospholipids.

One glycoside, octylglucoside, experienced in the present invention as an amplifier. It was found that he has some ability to enhance absorption. It should also be assumed that other Alkylglucoside, such as thioglucopyranoside and multipurposed have the ability to enhance the absorption in the methods of the present invention.

Cyclodextrins and their derivatives effectively enhance nasal absorption and similarly can function in the lungs. Experienced dimethyl-cyclodextrin and found that it has enhance the absorption effect.

Other potentially suitable surfactants are sodium salicylate, 5-methoxysilyl sodium and naturally occurring surfactants such as salts of glycyrrhizic acid, spanisheasily and acylcarnitine.

Since the effective strengthening of the absorption of the polypeptide in the lungs were observed for a number of the amplifiers tested, it is assumed that you can find a lot more substances that function in the same way. Microspheres KRA is experienced as an amplifier in the methods of the present invention. Although they were not suitable for delivery of the polypeptide through the pulmonary path in the animal model used in the invention, it is believed that this was mainly due to technical difficulties, the overcoming of which can lead to successful delivery of the polypeptide via the pulmonary route.

Hepatoblastoma tools are a class of amplifiers, which are believed to act by binding calcium ions. Because calcium ions help to maintain the distance between the cells and further reduces the solubility of polypeptides, binding of these ions should in theory how to increase solubility of the polypeptide, or to increase paracellular permeability of polypeptides. Although experienced one chelate forming agent, sodium salt of ethylenediaminetetraacetic acid (EDTA), which was found ineffective in enhancing the absorption of insulin in the tested rat model, other binding calcium ions, hepatoblastoma agents may be more suitable.

The ratio of the polypeptide and amplifier

The relative ratio of the polypeptide and amplifier, you can optionally change. To ensure effective absorption of inhaled polypeptide has presti low, to minimize the risk of negative effects caused by the amplifier. Although you should test each specific combination polypeptide/power to determine the optimum ratio of components of the combination, it is expected that to achieve adequate absorption of the polypeptide content of the amplifier must be more than 10% of a mixture of polypeptide/amplifier. For most types of amplifiers contents of the amplifier should be more than 15% or more than 20%, preferably between 25% and 50%. The preferred ratio for each combination of the polypeptide/amplifier (or polypeptide/amplifier/diluent) can be easily determined by the expert of ordinary skill in the field of pharmacology, standard techniques based on criteria such as effective joint delivery of optimal dose to minimize side effects and acceptable rate of absorption.

For the action of the drug does not require additional components, but if desired, you can enter them. For example, the amount of powder, which is a single dose of the combination of the polypeptide/surfactant, can be increased (for example, for use in the device of the inhaler, which by design require boltenia processing or improving powder properties or stability of the drug can include other additives. You can add corrigent (flavoring agent), to the amount of powder that inevitably settles in the mouth and throat, serve to receive the patient instructions (positive feedback) that the dose from the device-inhaler delivered. Any such Supplement shall have the following properties: (a) it is stable and has no adverse effect on the stability of the polypeptide and amplifier, (b) does not prevent the absorption of the polypeptide, (c) it has good powder properties, as this term is understood in pharmacy, (d) it is not hygroscopic and (e) when she applied concentrations has no negative effect in the respiratory tract. Suitable types of such additives include mono-, di - and polysaccharides, alcohols of a number of sugars and other polyols, for example lactose, glucose, raffinose, melezitose, lactic, ▫ maltitol, trehalose, sucrose, mannitol and starch. As reducing sugars, such as lactose and glucose, have a tendency to form complexes with proteins, non sugar, such as raffinose, melezitose, lactic, ▫ maltitol, trehalose, sucrose, mannitol and starch, can be the preferred additives for use in the present invention. Such additives in all cases, m shall invention provides a therapeutic preparation of pharmaceutically active polypeptide and substance, which enhances the absorption of the polypeptide in the lower respiratory tract. This drug has the form of a dry powder suitable for inhalation. At least 50 wt.% this powder consists of (a) particles having a diameter of less than about 10 microns, or (b) agglomerates of these particles. In another preferred embodiment, the invention provides a therapeutic preparation containing pharmaceutically active polypeptide, a substance which enhances the absorption of the polypeptide in the lower respiratory tract, and pharmaceutically applicable carrier. The drug has the form of a dry powder suitable for inhalation. At least 50 wt. % this powder consists of (a) particles having a diameter of less than about 10 microns, or (b) agglomerates of these particles. In the following a preferred embodiment, the invention provides a therapeutic preparation containing active compounds: (A) A pharmaceutically active polypeptide and (B) a substance which enhances the absorption of the polypeptide in the lower respiratory tract, and at least 50% of the total mass of active compounds (A) and (B) consists of particles having a diameter of less than about 10 microns, and a pharmaceutically acceptable carrier. The drug has the form of a dry powder suitable for inhalational powdered drug can be manufactured in several ways, using conventional techniques. In many cases, the purified polypeptide can be obtained from commercial sources. Conversely, the desired polypeptide can be cleared from a natural source using standard biochemical methods or can be obtained by expression of prokaryotic or eukaryotic cells, genetically engineered to contain a nucleotide sequence that encodes the polypeptide and has United with the appropriate sequence of control expression (including transgenic animal capable of expression of the desired peptide or protein, such as milk). Such methods are standard in the art (see, for example, Sambrook et. al., Molekular Cloning: A Laboratory Manual: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Peptides (i.e., polypeptides having 30 or less amino acid residues) can be easily synthesize known chemical methods.

Amplifiers absorption, described above, are also commonly available from commercial sources or can be obtained by using published techniques. In the case of ionic amplifiers the counterion associated with the amplifier, can be replaced, if desired, to another using standard methods at some stage method finely chop in a suitable mill, for example, a jet mill, to obtain primary particles with a size limit of particles suitable for the maximum deposition in the lower respiratory tract (i.e., less than 10 microns). For example, you can spend the dry mixing of powders polypeptide and amplifier and then substances finely chop together. Alternative materials can be finely chop separately and then mix. When mixed compounds have different physical properties, such as hardness and brittleness, resistance to fine grinding them varies and grinding into particles of suitable size may require different pressures. Therefore, when the substance is finely milled together, the size of the resulting particles of one component may be unsatisfactory. In this case, it may be advantageous for various components finely chop separately and then mix them.

You can also dissolve the components in a suitable solvent, for example water, to obtain mixing at the molecular level. This technique also allows you to set the desired pH, for example, to improve the absorption of the polypeptide. You need to take into account the pharmaceutically acceptable pH range from 3.0 to 8.5 DL the respiratory tract. To obtain a powder solvent should be removed in a manner that allows to preserve the biological activity of the polypeptide. Suitable drying methods include vacuum concentration, open air drying, spray drying and drying by freezing. You should generally avoid temperatures above 40oC for more than a few minutes, because there may be some destruction of certain polypeptides. After the stage of drying the solid material can, if necessary, grind to obtain large powder and then, if necessary, finely chop.

If desired, finely ground powder can be processed to improve the rheological properties, for example, a dry granulation for the formation of spherical agglomerates with the best performance before introducing him to intended for his device is an inhaler. In this case, the device must be of the form, providing a significant desagglomeration agglomerates to the exit of the powder from the device, so that the particles included in the respiratory tract of a patient, generally have a size within the desired size. In those cases where you need to have an ordered mixture, the active compound can be the lah specific particle size. The media also can be processed, for example, to obtain particles of the desired size and the desired surface properties, such as specific relationships of surface to mass, or achieve a certain strength and optimum strength of adhesion in an ordered mixture. Such physical requirements for an ordered mixture is well known, as well as different ways of obtaining an ordered mixture that meets these requirements, they can easily determine the specialist of this field in accordance with the specific details.

The preferred inhalation device must have structural features that protect the powder from moisture and preventing the risk of accidental large doses. In addition, it is desirable that the device would satisfy as many as possible of the following conditions: protect the powder from light; provided easily respirable fraction and significant deposition in the lungs with a wide range of volumetric flow rates with low deviation dose and respirable fractions, low retention of powder in the tip - this is especially important for parenteral multi-dose inhaler, where the polypeptide that remains in the tip may break down and then inhaled along with posleduyushe. Preferred inhaler single dose, although you can also use the inhaler with multiple doses, for example, driven by the inhalation nebulizer reusable containing multiple dose dry powder. Preferred is driven by the inhalation nebulizer disposable, containing a single dose dry powder.

Was obtained and tested in vivo analyses described below, the number of dry powder ready-made forms, containing polypeptide and a variety of amplifiers. Also described in vitro assays suitable for testing combinations of polypeptide/power.

Example 1: Method for determining in vivo the suitability of specific polypeptides to the present invention

Standard analysis of in vivo using epithelial cell lines, SASO-2 (available from American Type Culture Collection (ATCC), Bockville, MD, USA) was developed to measure the ability of various compounds amplifiers promote the transfer of tokens through the monolayer of epithelial cells as a model of the epithelial cell layer, which functions in the lungs for the Department of the alveoli from the pulmonary circulation.

In this analysis the amplifier and the polypeptide or other marker is dissolved in water R is OYA. After incubation for 60 min at 37oC and 95% RH (relative humidity) was determined, for example, with the use of marker-labeled radioactive isotope, the number of marker basolateral side of the cell.

To test the amplifier, caprate sodium, the amount of marker (mannitol, molecular weight 360), which appears on basolateral side, depends on the concentration of the used amplifier, which is at least up to 16 mm caprata sodium (Fig. 1). This is true even if the mixture amplifier/mannitol add polypeptide insulin (mass ratio caprata sodium and insulin 1:3) (Fig. 2). Also found that this concentration of caprata sodium (16 mm) promotiom absorption through the cell monolayer two low molecular weight peptides, insulin (MW 5734) and vasopressin (MW1208). The amount of insulin that has passed through the monolayer in the presence of 16 mm caprata sodium doubled compared with the number in the absence of any amplifier, the number of vasopressin, which was absorbed through the monolayer, increased 10-15 times compared with the number in the absence of any amplifier.

On the contrary, no increase in the speed of transfer is not observed for more than you is the use of up to 16 mm caprata sodium. It is expected that at higher concentrations of caprata sodium permeability of the cells should be increased, which will allow the transfer of more high molecular weight polypeptides; however, the possible cytotoxicity of caprata sodium can prevent the application of significantly higher concentrations of this amplifier.

Other amplifiers may allow the transfer of more high molecular weight polypeptides; they can also be tested in this in vivo model of epithelial cell permeability, which can be used as a tool for screening in order to quickly test any desired combination of the polypeptide/power to determine its suitability in the methods of the present invention.

Example 2: the Method of selection of amplifiers suitable for the present invention.

Each of the compounds listed in the table, was tested for its ability to enhance the absorption of the polypeptide (insulin) in a rat model. Results insulin taken as a criterion of the capacity of the amplifier to increase the absorption of other polypeptides.

In different trials used different forms of insulin recombinant human semisynthetic human or bovine. Each is silical/lactose to obtain the inhaled powder. The powder was administered to rats by inhalation and the glucose content in the blood of the rats were then monitored to measure the absorption of insulin. These values were compared with the corresponding values obtained in rats that inhaled form of insulin without the amplifier.

The same model system in vivo can be used to test any given peptide or protein useful in the methods of the invention by delivering the same way inhalation form containing the desired peptide or protein in combination with the amplifier, and the quantitative determination of the concentration of the desired peptide or protein in a large range of blood test animal (e.g., standard immunological or biochemical analyses that are appropriate for a given peptide or protein).

Example 3: Therapeutic drug in accordance with the invention.

The human growth hormone (hGH, MW 22 kDa, the source of Humatrope Lilly, 3 parts) was mixed with caprata sodium (1 part). The mixture was ground in a mechanical mill Retsch to obtain particles with an average diameter of 6.7 μm.

The obtained powder was administered to rats vnutritrahealno and absorption of hGH compared with the absorption of the powder, the MMD of 9.6 μm, containing hGH and Mannlichen absorption of hGH in the finished form, including capret sodium, compared to the absorption in the case of ready-made forms without an amplifier.

Example 4. The preparation containing the polypeptide insulin

Insulin is here used as a criterion for the other peptides in accordance with the present invention.

Biosynthetic human insulin (53 g) was subjected to fine grinding in a jet mill Airfilco (trademark, Airfilco Process Plant Limited) with compressed nitrogen (pressure load 7105PA, the pressure in the chamber 5105PA) to obtain particles with an average size of 2.4 μm.

Capret sodium (170 g) was subjected to fine grinding in a jet mill Airfilco (TM) compressed nitrogen (pressure load 5105PA, the pressure in the chamber 3105PA) to obtain particles with an average diameter of 1.6 μm.

Finely ground biosynthetic insulin (45 g) and capret sodium (14,26 g) were mixed in dry condition in accordance with the following procedure: half of the insulin was added to the mixing device containing cylinder for mixing displacement 4.4 l, separated by a sieve with an aperture of 1 mm in two compartments, with a metal ring in each Department that helps mixing and stirring. Added capret sodium and the remaining conclusion is consistent apparatus for shaking. Motor included and shaking continued for approximately two minutes until all the powder is passed through a sieve. The motor was off and the cylinder for mixing turned 180 degrees, again installed in the machine to shake and shake again until until all the powder is passed through a sieve. This procedure was repeated eight times with a total mixing time of approximately 20 minutes.

Thus obtained the drug was administered 5 dogs by inhalation at a dose of 1 U/kg insulin Levels in plasma were determined at different time points after drug administration.

The results were compared with the results of the insulin level in the plasma was obtained when biosynthetic insulin, finely chopped, as described above, to obtain particles with an average diameter of 2.4 μm was administered to five dogs in the same manner and to the same doses and results insulin levels obtained when therapeutic preparation of insulin and caprata sodium in the ratio of 90:10 was administered to five dogs in the same way and in the same doses as above. In this case, therapeutic preparation was prepared as follows. Human synthetic insulin gel-filtered to reduce oral in water (232 ml). The solution was stirred until clarification (transparency) and the pH was set to 7.0. The solution was concentrated by evaporation at 37oC for a period of about two days. The obtained solid cake was grinded and sieved through a sieve with openings of 0.5 mm and formed the powder is finely crushed in a jet mill into particles with an average diameter of 3.1 μm.

The results of these comparisons are presented in Fig. 3. (p = 0,0147 for ratios of from 75:25 to 100:0). The results show some improvement in the bioavailability of insulin in the case of ready-made forms with a ratio of 90: 10 and dramatic improvement in bioavailability of insulin in the case of the preparation with a ratio of 75:25, including capret sodium, compared with the introduction of only insulin.

1. Pharmaceutical composition suitable for administration by inhalation, containing a pharmaceutically active polypeptide, characterized in that it contains in a mixture with a pharmaceutically active polypeptide connection-amplifier, which increases the absorption of the polypeptide in the lower respiratory tract of the patient, and the surface-active substance, taken in an amount more than 10% of the total mass of the polypeptide and the connection of the amplifier, and wikepedia and connect the amplifier consists of particles, having a diameter less than or equal to 10 microns, or agglomerates of the above-mentioned particles.

2. The composition according to p. 1, characterized in that it further comprises a pharmaceutically acceptable carrier, which contains particles having a diameter less than 10 microns, and at least 50% of the resulting powder consists of particles having diameter less than 10 microns, or agglomerates of the above-mentioned particles.

3. The composition according to p. 1, characterized in that it further comprises a pharmaceutically acceptable carrier, which consists of coarse particles having a diameter greater than 20 μm, with ready-to-use mixture is formed between the polypeptide and the compound-amplifier and the specified media.

4. The composition according to p. 1, wherein the polypeptide is a polypeptide hormone.

5. The composition according to p. 4, characterized in that the above-mentioned hormone is vasopressin analogue of vasopressin, desmopressina, glucagon, corticotropin (ACTH), gonadotropin (luteinizing hormone, or LTRH), calcitonin, C-peptide of insulin, parathyroid hormone (PTH), human growth hormone (hGH), growth hormone (HG), releasing hormone growth hormone (GHRH), oxytocin releasing hormone corticotroph is I (hANP), releasing hormone thyroxine (TRHrh), follicle-stimulating hormone (FSH) or prolactin.

6. Composition under item 1 or 2, wherein the polypeptide is a growth factor, an interleukin, a polypeptide vaccine, enzyme, endorphins, a glycoprotein, a lipoprotein, or a polypeptide, which may be included in the blood clotting system and has a pharmacological effect.

7. Composition according to any one of paragraphs.1 - 6, characterized in that the above polypeptide has a molecular mass of less than 30 kDa.

8. Composition according to any one of paragraphs.1 - 6, characterized in that the above polypeptide has a molecular mass of less than 25 kDa.

9. Composition according to any one of paragraphs.1 - 6, characterized in that the above polypeptide has a molecular weight less than 20 kDa.

10. Composition according to any one of paragraphs.1 - 6, characterized in that the above polypeptide has a molecular mass of less than 15 kDa.

11. Composition according to any one of paragraphs.1 - 6, characterized in that the above polypeptide has a molecular weight less than 10 kDa.

12. The composition according to p. 1, wherein the surfactant is a salt of a fatty acid.

13. Composition under item 1, differing Alkylglucoside, the cyclodextrin or its derivative, or a phospholipid.

14. The composition according to p. 1, wherein the surfactant is a salt of bile acid.

15. The composition according to p. 14, characterized in that the salt of the bile acid is the sodium salt ursodeoxycholate, taurocholate, glycocholate or eurodiversity.

16. The composition according to p. 15, characterized in that the salt of the bile acid is taurocholate sodium.

17. The composition according to p. 1, characterized in that the surfactant is acylcarnitines.

18. Composition according to any one of paragraphs.2 to 17, characterized in that the pharmaceutically acceptable carrier is chosen from mono-, di - and polysaccharides, alcohols of a number of sugars and other polyols.

19. The composition according to p. 18, characterized in that the carrier is predetermin sugar.

20. The composition according to p. 18, characterized in that the carrier is raffinati, melezitose, lactitol, ▫ maltitol, trehalose, sucrose, mannitol or starch.

21. The composition according to p. 1, characterized in that the above-mentioned connection-power is present in the amount of more than 15% of the total amount of the polypeptide and amplifier.

, is leaving more than 20% of the total amount of the polypeptide and amplifier.

23. The composition according to p. 22, characterized in that the above-mentioned connection-power is present in the amount of 25 - 50% of the total amount of the polypeptide and amplifier.

24. Composition according to any one of paragraphs.2 to 23, characterized in that it is inhalator device in the form of the above-mentioned agglomerates, while inhalation device facilitates the grinding of the above-mentioned agglomerates to particles having a diameter less than or equal to 10 μm, inhalation of the above-mentioned agglomerates of these devices.

25. Composition according to any one of paragraphs.2 to 23, characterized in that it is inhalator device, disposable, driven by inhalation, and is a single dose dry powder.

26. Composition according to any one of paragraphs.2 to 23, characterized in that it is inhalator device with multiple applications, driven by inhalation, and is a multiple dose dry powder.

27. The method of introduction pharmaceutically active polypeptide, characterized in that it provides for a composition in the form of a dry powder,OE enhances the absorption of the polypeptide in the lower respiratory tract of the patient and the surface-active substance, taken in an amount of more than 10% of the total number of polypeptide and amplifier; and inhalation by the patient of the above-mentioned composition; provided that the diameter of the particles of the active compounds at the point of entry into the respiratory tract of the patient is less than or equal to 10 microns.

28. The method according to p. 27, characterized in that the above composition inhale from the device is an inhaler containing powder in the form of agglomerates of the aforementioned particles, and the above-mentioned agglomerates mainly disagglomerated entrance to the respiratory tract of the patient.

29. A method of obtaining a pharmaceutical composition suitable for administration by inhalation, characterized in that it includes obtaining a solution by dissolving the pharmaceutically active polypeptide and the connection of the amplifier, which increases the absorption of the polypeptide in the lower respiratory tract of the patient and the surface-active substance, taken in an amount of more than 10% of the total number of polypeptide & amp; removing the solvent from the above solution to obtain a dry solid mass containing polypeptide and the above-mentioned connection of the amplifier; and grinding the dry solid mass to obtain a powder containing, by m the tion 10 μm, or agglomerates of the above particles.

30. A method of obtaining a pharmaceutical composition suitable for administration by inhalation, characterized in that it comprises dry mixing the pharmaceutically active polypeptide and the connection of the amplifier, which increases the absorption of the polypeptide in the lower respiratory tract of the patient and the surface-active substance, taken in an amount of more than 10% of the total number of polypeptide and amplifier; and grinding the aforementioned mixture to obtain a powder containing at least 50% of the total mass of active compounds in the form of particles having a diameter less than or equal to 10 microns, or agglomerates of the above particles.

31. A method of obtaining a pharmaceutical composition suitable for administration by inhalation, characterized in that it includes obtaining powdered pharmaceutically active polypeptide, and then give the ground connection of the amplifier, which increases the absorption of the polypeptide in the lower respiratory tract of the patient and the surface-active substance, taken in an amount of more than 10% of the total number of polypeptide and amplifier; and mixing them to obtain a powder, aderrasi or equal to 10 μm, or agglomerates of the above-mentioned particles.

 

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