Compositions for inhalation

This invention relates to compositions for distributing medically useful peptides and proteins.

Background of the Invention

Although the emergence of technology for DNA recombinant has resulted in a large increase in peptide-based drugs, the failure of treatment with peptide-based lymph nodes has really hampered that this range has been fully developed: in general, peptide-based drugs can not be administered by mouth to effective doses, as the enzymes in the gastrointestinal tract break them rapidly downstream into the bloodstream. If this polypeptide can not be modified to render it immune to such enzymes, the only practical method of distribution of these lyija is probably to be administered by the gastrointestinal tract, such as intravenous, intramuscular or subcutaneous injection. Subcutaneous administration of other gastrointestinal joints (eg through nasal, oral or rectal membranes or lung membranes) has limited performance.

WO-A-9116038 discloses microorganisms of interferon or human interleukin which are generally unstable in aqueous formulation which can be administered through the upper lower respiratory tract. The fine particles of the active ingredient as solidified by lyophilization of the aqueous solution of the bioactive peptide and then by milling (cautiously) are administered either in the form of an aerosol-free solution of a dry powder inhaler.

WO-A-8809163 discloses a pharmaceutical composition comprising a multiplicity of microorganisms for nasal administration. Each particle should be attached to a surfactant with the ability to increase the absorption of the active substance.

US-A-5011678 discusses drug-based compositions, mainly for the absorption of nasal drugs. Blends contain polypeptides and steroids such as sodium tetrahydro-24,25-dihydrousidate in liquid form liquid suspension. The purpose of the steroid is to increase the absorption of the drugs through the mucosa surface, usually by the nose.

P. Ruin, Sydsvenska (Dagbladet), Monday, 12 June 1989, pointed out in the article "Diabetics may not need their Insulin Shots", the possibility that insulin and unintended "detergents" may be dispensed with inhalation device.

US-A-5006343 discloses a lung dispersion of liposomes containing a physiologically active agent; as liposomes merge balloon-surfactant protein to increase liposomes uptake.

US-A-4994439 discloses formulations for drug distribution through mucous membranes consisting of a drug, one or more non-purified cleansers, and one or more bile salts or fusidates or derivatives thereof, primarily for nasal administration. The product may be prepared as a dry pad in a suspension.

It has been found that when a peptide or protein (referred to as a joint as a polypeptide) is mixed with a video capture source and is introduced into a pulmonary pulmonary fluid form, it easily penetrates into the lung circulation through uptake through the lower part of the body respiratory tract. This is advantageously accomplished by inhaling the powder from an inhaler which distributes the right dose of powdered particle peptide / particle size, which results in maximum lower respiratory tract lower than the mouth and throat. (For simplicity of reference, references to the polypeptide and the enhancer are commonly referred to as "active compounds"). In order to achieve this desired pulmonary distribution, as much as possible of the active compounds should consist of particles having a diameter of less than about 10 μm (e.g. between 0.01-10 μg and most preferably between 1-6 μαί). Preferred embodiments comprise at least 50% (at least 60%, preferably at least 70%, still at least 80%, and at least 90% total) of the total amount of active compounds contained in an inhaler of particles with a diameter within the desired range.

The invention thus comprises a pharmaceutical composition comprising a mixture of active compounds (A) a pharmaceutically acceptable polypeptide and (B) enhancer compound which enhances the systemic absorption of the polypeptide in the lower respiratory lung (patient) lung, the mixture is in the form of an inhalation dry powder, wherein at least 50% of the total amount of active compounds (A) and (B) consist of primary particles with a diameter less than or equal to 10 μη. The source data may be packaged in the same way as otherwise they may be placed in clusters, which are then severely broken while entering the patient's respiratory tract. The mixture may, of course, contain other ingredients as necessary, including other pharmaceutically active agents, adjuvant and pharmaceutically acceptable binders such as dilute such carriers. The pharmaceutical composition of this invention may contain only the active compounds as it may contain other substances such as a pharmacologically acceptable carrier. This charge can generally consist of particles with a diameter of less than 10 μτη so at least

50% of the powder formed consists of a total of possible aggregates of primary particles with a diameter of less than 10 μια ·, in addition, the buidirinnide can generally consist of much larger particles ("coarse particles"), so that a "mixer" can form between active compounds and the carrier. In a row mixture, also referred to as an interactive lime mixture, finger drug data (in this invention, the active compounds) are distributed relatively evenly over the coarse volume of the volumes (in this invention, a pharmacologically acceptable carrier). Preferably, in such cases, the active compounds are not in the form of clusters for the formation of the mixture. The coarse particles can exceed 20 μτη in diameter, such as over 60 μτη. Above this lower limit, the diameter of the coarse particles does not matter, so that different grades of coarse particles can be used if desired in accordance with the practical requirements of each case. There is no requirement for the coarse particles in the series mixture to be of the same degree, but economically, the coarse particles may be of similar strength within the mix. Preferably, the diameter of the coarse particles is 60800 μτη.

Multipeptide may be used as a peptide for any protein, which is a novel medicine for diagnosis, a small amount of medal, ie with a molecular weight (MW) of up to 40 kD, which is sought in a kerosene distribution.

The efficacy of enhanced polypeptide uptake of this invention is generally video-relevant and should apply to all such polypeptides, although the marked increase in absorption may vary widely depending on the molecular weight and physicochemical properties of the fepeptidid and particular enhancement noted. It is expected that a polypeptide with a molecular weight of up to 30 kD is best utilized in this invention, such as polypeptides of molecular weight up to 25 kD and up to 20 kD and especially up to 15 kD above 10 kD. Any desired polypeptide may be audibly tested for use in this invention with particular enhancement with in vivo in vitro assays as described herein.

The compound of formula used in the compositions of this invention may comprise any compound which enhances the uptake of the polypeptide through the ventral cells of the respiratory tract and into the cardiovascular cycle.

By "enhancing recording" refers to the amount of polypeptide that succeeds in the cyclical cycle in the presence of an increase, which is greater than when no enhancer is present. Preferably, the amount of polypeptide that is effective is significantly greater (p <0.05) in the presence of an increase. The suitability of any enhancements that may be used by this invention can be evaluated audibly by in vivo in vitro assays as described herein.

The amount of polypeptide produced according to the present invention is preferably at least 150% of the amount which is dissolved without any activity. In preferred embodiments, at least doubles the uptake of polypeptide, more preferably triple and, most preferably, fourfold in the presence of the additive, when compared to non-present.

The enhancer is preferably a surfactant such as fatty acid salt, bile salt, bile salt derivative, alkyl glycoside, cyclodextrin, eda phospholipid. For example, the enhancer may have sodium, potassium or ethyl alcohol of the fatty acid and the fatty acid is fermented with hydrochloric acid or other 10-14 carbon atoms of fatty acids. The most preferred enhancer is sodium caprate. The proportion of polypeptide and magnitude of epiphytic activity ranges from about that interval

9: 1 and about 1: 1 Although the ratio of magnitude greater than 1: 1 would probably increase the recording as well, but the lower ratios are considered as the amount of magnitude used should not exceed the level required to reach the level Increased increase, as excessive levels of eukaryism can cause unwanted side effects, such as local irritation.

Administration of the pharmaceutical composition according to the invention is carried out using an inhalation device which the patient uses for the preparation of the mixture. When the powdery mixture is in the form of clustering of primary particles, a device is designed to induce significant deterioration of the clusters when the patient breathes the powder from the device, so that the majority of the clusters break into particles with a diameter less than equal to 10 μτη prior to dust enters the patient's respiratory system. This disruption would enter a device into the device and is usually induced by the intake of air formed by the device by the inhalation force. Clusters are generally not formed in the series mix.

In the case of a radomic mixture, the active compounds should be released from the large particles by inhalation, whether by mechanical means in the inhaler simply by inhalation itself, in a disorderly manner, the active substances were thus present in the lower respiratory tract and the burden data in the mouth .

Inhalation device is a single dose dry breath inhaler, but may alternatively be a multi-dose dry powder inhaler.

Various forms of preparation of the preparation of the composition according to the invention are suitable for inhalation. In one such behavior, a solution is first solved (a) a pharmacologically active polypeptide and (b) an enhancer compound that increases the selective uptake of the polypeptide in the lower respiratory tract of the patient. The solvent is then removed from the solution to give a dry solid containing the polypeptide and the solid and dry solid. is crushed into powder. Another such method involves the dry-mixing of (a) a pharmaceutically acceptable polypeptide and (b) a compound compound and annealing of the resulting mixture. The third possible method comprises the steps of providing a first micronized mixture containing polypeptide and other micromeric mixture containing an enhancer compound, and mixing the microtitrated mixtures together. When carrier is included and is not intended to provide a series of compositions, it may be added to the solution or in the dry mixture of the pharmacologically active polypeptide for microtitration, or the micromeric carrier may be dry mixed with the micronized components. When preparing a series of preparations, micronized polypeptides and enhancers are mixed with suitable vehicles.

A brief list of the drawings

Figure 1 is a graph illustrating the effects of different concentrations of sodium caprate glyc on the transfer of marker compounds (mannitol) through a single layer of cultured epithelial cells.

Figure 2 is a graph showing the effect of different concentrations of sodium caprate glyc on the transfer of marker compounds (mannitol) through a single culture. epithelial cells in the presence of polypeptide (sodium caprate: polypeptide 1: 3 by weight).

Figure 3 is a graph showing the plasma polypeptide concentration as a function of time after the inhalation of the polypeptide alone, the polypeptide with sodium caprate at a ratio of 90:10 and the polypeptide with sodium caprate at a ratio of 75:25.

Accurate description

Some of the preferred embodiments of the invention are described generally in the following.

The polypeptide

The polypeptide is called peptide hormone other than insulin such as vasopressin, vasopressin analogues, desmopressin, glucagon, corticotropin (ACTH), gonadotropin (gulic-inducing hormone, or LHRH), calcitonin, C-peptide insulin, parathyroid hormone (FTH), human growth hormone (hGH) growth hormone (HG), growth hormone (GHRH), oxytocin, corticotrophin (CRH), growth hormone analogues, gonadotropin agonist analogues (GnRHa), atrial fibrillation peptide (hANP), tyroxin (TRHrh), follicle control hormone (FSH) and prolactin.

Other possible polypeptides include growth factors, interleukin, polypeptide vaccine, enzymes, endorphins, glycoproteins, lipoproteins and polypeptides involved in the blood clotting chain reaction, which utilize the pharmacological effects of the core drug. It is expected that most, if not all small to medium-sized polypeptides, with relatively high water solubility and with an equilibrium loading point between about pH 3 and pH 8, can be effectively dispersed by the methods of the invention.

The eagle

The use of a source of absorption is very important, as the polypeptide alone acts poorly through the lungs. The enhancer used may be any of a variety of compounds that act to promote absorption through the membrane compartment covering the lower respiratory tract, into the adjoining vessel lung. The owner can do this according to one of several possible ways:

(1) Enhancement of the mediated epiphytic permeability of polypeptides by the occurrence of structural changes in the narrow genome between the epithelial cells.

(2) Increasing the genomic transmission of polypeptides by interacting with or reducing the protein or lipid components of the membrane, thus interfering with the integrity of the membrane.

(3) Intermediate link between enhancer and polypeptide that enhances solubility of the polypeptide in aqueous solution. This can happen by preventing the formation of polypeptide clusters (the third, third, hexamera) or by dissolving polypeptide molecules in the eukaryotic genes.

(4) Reduce the viscosity of the mucous membrane that covers the pulmonary artery and lungs or dissolves, so that the surface of the epidermis is exposed to direct absorption of the polypeptide.

Echoes can work on only one of the aforementioned routes, or after two or more. An activator that works in several ways is more likely to promote the effective uptake of polypeptide, but you only use one or two.

For example, surfactants are a category of echoes that are considered to be effective in all four ways mentioned above. Surfactants are double-stranded molecules with both lipophilic and hydrophobic components, and with a variable balance between these two properties. If the molecule is very lipophilic, the low solubility of the substance in water may limit its usefulness. However, if the hydrophobic component is superimposed, the surfactant properties of the molecule can be reversed. In order to effect, the surfactant will be obtained to achieve an appropriate balance between sufficient solubility and sufficient surface activity.

Another feature of surfactant which may be important is the net surfactant loading at the pH of the lung (approximately 7.4). At pH 7.4 there are some polypeptides with a negative net charge. This results in electrostatic forces between molecules, which then prevent clusters, thus increasing the solubility. If the surfactant is also negatively charged, it may interact with the polypeptide with such a water-dependent relationship, and even greater shear force will occur among the polypeptide molecules. In such cases, anionic surfactant has the additional cost (compared to those with neutral or net positive charge at physiological pH) to increase uptake by assisting in stabilizing the polypeptide in monomer form.

A number of different compounds which are potentially useful as enhancers in the methods of the invention were tested in rats as described in Example 2 below. Other substances with known properties in terms of enhancement of absorption, or with physical symptoms that make them likely to be used in the method of the invention, can be easily tested by one of ordinary skill in the in vivo assay or alternatively with the in vitro assay described herein 1.

It is possible that the combination of two or more enhancers can provide satisfactory results. The use of such compositions in the method according to the invention is considered to be within the invention.

A beneficial agent in the methods according to the invention will combine an effective increase in polypeptide uptake in (1) the lack of toxicity in the concentrations used and (2) good powder properties, i.e. lack of solid or waxy solids solids. The toxicity of a particular substance can be tested by standard methods, such as by analysis, for example, as described in Int. J. Pharm., 65 (1990), 249-259. You can access the thumbnails of a certain content in the published data or in the experiment.

One type of egg that promises very good is salt fitusyrro. It has been found that the sodium salt of saturated fatty acids with the carbon chain lengths 10 (i.e., sodium caprate), 12 (sodium chloride) and 14 (sodium mylrate) function well in the method according to the invention. Potassium and lysine salts of capric acid have also been shown to be effective in the method according to the invention. If the carbon chain length is less than about 10, the surfactant of the surfactant may be too low, and if the chain length is longer than about 14, the reduced solubility of the fatty acid salt in water will limit its usefulness.

Preferably, according to this invention, the substance that increases the uptake of polypeptide in the lower respiratory tract is sodium caprate.

Different metions can change the solubility of saturated fatty acid salts in water, so that a carbon footprint other than 10 -14 would be even more advantageous than the ones specifically mentioned above. Salts of unsaturated fatty acids can also prove useful in this invention as they dissolve more in water than salts of saturated fatty acids and may therefore have longer chains than the latter and still possess the solubility necessary for the effective propagation of polypeptide uptake.

All bile salts and derivatives of bile salts tested (sodium salts of urethoxycolate, tearocholate, glycocolate and tributrophidus fusidate) effectively enhance polypeptide uptake in the lung.

Phospholipids were also tested as enhancers. It was found that single chain phospholipid (lysophosphatidylcholine) was an effective enhancer, but two double chain phospholipids (dioctanoylphosphatidylcholine and didecanoylphosphatidylcholine) were not. This can be explained by the fact that the bi-chain phospholipids dissolve much worse in water than their monoclonal analogues; However, it may be expected that short chain phospholipids with shorter chains that dissolve more in water than their longer chain analogs may be used as enhancers of this invention so that both mono- and di-chain phospholipids can be used.

One glycoside, octylgluopyranoside, was tested as enhancer in this invention and found to have some absorption enhancing properties. Therefore, other alkyl glycosides such as thioglucopyranoside and maltopyranoside can also be expected to exhibit properties that enhance the absorption in the methods of this invention.

Cyclodextrin and their derivatives effectively enhance nasal absorption and may have similar activity in the lung. Dimethyl-B-cyclodextrin has been tested and proved to have a recording-enhancing effect.

Other potentially useful topically active agents include sodium salicylate, sodium 5-methoxysalylate and natural topical active substances such as glycyrrhizin salts, saponin glycosides and asyl carnitines.

In the case of ionic ions (for example, the anionic active substances described above), it may be important to distinguish the potential. The selections selected may affect the duodenal properties, solubility, stability, fluidity, and local / synthetic toxicity of any of the ingredients containing the enhancer. She can also affect the stability and / or solubility of the polypeptide to which it relates. Generally, monovalent metallic cations such as sodium, potassium, lithium, rhbidium and sesium can be useful as useful agents for the anionics. Ammoniaf and organic amine form another category of cation which is suitable for use with anionic acids with carboxylic acid moieties. Examples of such organic amine are medalamines, diethanolamine, triethanolamine, 2-amino-2-methylethylamine, betaine, ethylenediamine, Ν,

Since many of the tested subjects showed an effective increase in the absorption of polypeptide in the lung, one can expect many more to be found to function as well. Sterilizer spheres effectively enhance the bioavailability of the polypeptide distributed across nasal membranes and were tested to enhance the behavior of the invention. Although they did not make much use of the distribution of the lung in the experimental animal used here, it is believed that this is mostly due to technical difficulties and, if avoided, it could lead to effective distribution of the lungs.

Engines are a category of ects that are currently active by linking calcium ions. Since the calcium ions adstoda by maintaining the dimension of the space between cells and thereby reducing the solubility of polypeptides, the association of these ions would both theoretically increase the solubility of polypeptides and increase the polypeptide polypeptide. Although one tested test substance, the sodium salt of the ethylenediamine tetraacetic acid (EDTA), was not effective in increasing the absorption of the insulin test test tested, other calcium ions-related adhesives could prove useful.

Fipeptide and Efficacy Ratio

Internal proportions of polypeptide and enhancement may be altered wildly. A sufficient amount of enhancement must be available to allow the effective absorption of polypeptide inhaled; In spite of that, the amount of oxygen should be kept as small as possible to minimize the risk of adverse effects caused by the eukaryth. While testing for any polypeptide / enhancer composition to determine the desired proportions, it is expected that in order to achieve acceptable uptake of the polypeptide more than 10% of the polypeptide / enhancer mixture may be increased; For most types of ewes, the rate of increase should be over 15% or over 20% and is between 25% and 50%. The preferred ratio for each polypeptide / enhancer (or polypeptide / enhancer / dilute) mixture can be determined by general pharmacology based on standardized methods based on certain criteria,

No additional components for the operation of the mixture, but may be requested. For example, an increase in the amount of powder that forms a single dose of a certain combination of polypeptides / surfactants (Ld for use in an inhalation device that requires a high dose of powder for each injection) is required by diluting the powder with pharmaceutically acceptable blisters. Additional additives may be used to facilitate the processing or improvement of the powder properties or stability of the mixture. Flavors could be added so that the part of the powder that will inevitably remain in the mouth and throat will indicate to the patient with positive retroactivity that a dose has been delivered from the inhaler. Any such additive should have the following characteristics: (a) to be stable and do not adversely affect the stability of the polypeptide and the enhancer; (b) that it does not cause undesirable disturbances on the uptake of the polypeptide; (c) that it has good duodenal properties, as defined in Pharmacy; (d) it is not liquid; and (e) does not adversely affect the respiratory tract in the concentrations used. Useful types of such additives are medal or mono-, di- and polysaccharides, sugar alcohol, and other polyols: for example, lactose, glucose, raffinose, melesitose, lactitol, maltitol, trehalose, sucrose, mannitol and starch. Since reducing sugars such as lactose and glucose tend to form complex with proteins, sugars that do not have decreasing effects such as raffinose, melesitose, lactitol, maltitol, trehalose, sucrose, mannitol and starch may be a preferred additive for use in this invention . Such additives can range from 0% (ie.

In a preferred embodiment, this invention provides a therapeutic formulation of a pharmacologically active polypeptide and a substance that enhances the uptake of the polypeptide in the lower respiratory tract, a mixture in the form of a dry powder mixture suitable for inhalation and wherein at least 50% of its mass consists of

(a) particles of less than about 10 μια in diameter; (b) clusters of particulate matter; In another preferred embodiment, the invention provides a therapeutic composition comprising a pharmacologically active polypeptide, a substance that enhances the absorption of polypeptide in the lower respiratory tract and a pharmaceutically acceptable carrier, wherein the mixture is in the form of a suitable inhaled dry powder and wherein at least 50% of mass they consist of (a) particles less than 10 μg / cm², or (b) clusters of particulate matter; and in yet a more preferred embodiment, this invention provides a therapeutic composition comprising active compound (A) pharmacologically active polypeptide and (Ð) a substance that enhances the uptake of the polypeptide in the lower respiratory tract,

The powder mixture described herein can be produced in several ways with conventional behaviors. In many cases, purified polypeptides can be obtained on a generalized basis. Ad ddrum is a potentially cleansing of polypeptide that is used by natural donors by using standardized behavioral behaviors, resulting in the expression of prodrug-arytic cells which, for the study of inheritance control, contain kimarod encoding polypeptide and has video-rich control the expression related thereto (including genetically modified animals that manage to produce the peptides of the desired proteins, for example in their milk). Such behavior is present in the glove (see, for example, Sambrook et al., Molecular Cloning:

A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Peptide (eg polypeptides with fewer amino acid residues) is readily synthesized by known chemical-dependent behaviors.

Recorders as described above are also generally available in the general market, or can be produced using published methods. In the case of ionic ions, you can change the benefit associated with enhancer by conventional ion exchange treatments, upon request.

For the preparation of the powder mixture described herein, it is usually necessary to emit the powder into a suitable compartment, such as a pneumatic membrane, at some point of the manufacturing process, to obtain primates within the levels that ensure peak respiratory distress in the lower respiratory tract (ie less than 10 μτη). For example, you can dry mix polypeptides and egg yolks, then mix the ingredients together, otherwise the ingredients can be micronized separately and then mixed together. In those cases, the composite compounds have different chemical properties such as hardness and leakage, their resistance to micronization varies and they may require varying pressures to break down into suitable sized particles. When they are identical, the particle size of another component that is obtained may be unacceptable.

It is also possible to dissolve the components first in a suitable solvent, for example

water, to obtain a mixture at the molecular level. This mode also makes it possible to adjust the pH to a desired level, for example to improve the uptake of the polypeptide. The pharmacologically acceptable levels of pH 3.0 to pH 8.5 for inhalation products should be considered, as products with pH beyond these limits can cause irritation and respiratory irritation. In order to obtain powder, the solvent must be removed by a method of living the biological activity of the polypeptide. Suitable drying methods include pressure relief, open drying, spray drying and freeze drying. Temperatures above 40 ° C for more than a few minutes should generally be avoided, as decomposition of certain polypeptides may occur. After the drying step, if necessary, grind solids into a coarse powder which, if necessary, is micro-malleable.

On request, the micro-powder can be treated to make up the 35's, for example, with a dry coma to form spherical colonies with superior treatment characteristics prior to insertion into the inhaler intended to be used. In this case the device would be equipped to ensure that the clusters are severely disrupted before leaving the device, so that the particles entering the patient's respiratory tract are largely within the desired size range. When looking for a series of compositions, the active compound can be treated, for example by microwell, to obtain, on request, particles within a certain size range. The carrier may also be treated, for example, to obtain a desired size and preferably surface properties, such as a certain ratio between surface and weight, or certain roughness, and to ensure maximum adhesion forces in the mixer. Such a condition of a series of compositions are well known as well as various methods of obtaining a series of compositions that meet the conditions, and they can easily be determined by a skilled person, as appropriate.

Optimal inhalation equipment has the following design characteristics: protection of the powder from moisture and no risk of excessive dosage being achieved; In addition, there are desirable as many of the following symptoms as possible: protection of the powder from light; a high proportion of breathable breathable particles and a high rate of pulmonary embolism at a wide velocity rate; small dose variations and proportion of breathable particles; the mouthpiece is low, this is especially important for multiple dose inhaler, as the polypeptide remaining in the mouthpiece may break down and then breathe in with the next dose; low absorption in the surface of the inhaler; dose flexibility; and low resistance to inhalation. The inhaler is a single dose inhaler, although a multi-dose inhaler may also be used, such as multi-dose multi-dose dry breath inhaler activated by breathing. Heist is a single-use unit dose dry breath inhaler that is activated by breathing.

Many dry powder mixtures containing polypeptide and various enhancers have been prepared and tested in an in vivo assay and are described below. An in vitro assay is also described which is useful in the testing of polypeptide / enhancer combinations.

Example 1: In vitro method for determining utility of particular polypeptides of this invention,

Standardized in vitro groining using cellular cell lines, CaCo-2 (available from

The American Type Culture Collection (ATCC), Rockville, MD, USA) has been developed to evaluate the ability of various compounds to promote the transfer of markers across the epithelial cell line used as models for the cellular body that function in the lung to separate the pulmonary arteries and blood vessels Iungnanna.

In this analysis, the enhancer and polypeptide or other label are dissolved in aqueous solutions of different proportions and / or concentrations, and placed on the top of the cell line. After 60 minutes microbial culture in a warming cup at 37 ° C and 95%

HR (relative humidity), the amount of the marker on the base and lateral sides of the cells is determined, for example by using radio-labeled labels.

Regarding the test substance, sodium caprate, the amount of the label (mannitol, MW 360) shown on the base and lateral side of the strength of the agent used is at least up to 16 mM sodium caprate (Figure 1). This is true even when the polypeptide insulin is added to the eukaryotic / mannitol mixture (1: 3 sodium caprate: insulin, weight) (Figure 2).

It was also found that this concentration of sodium caprate (16 mM) facilitated the uptake of two low molecular weight insulin peptides (MW 5734) and urinary tumors (MW1208) across the cytokine. The amount of insulin that crossed the monolayer doubled in the presence of 16 mM sodium caprate, compared with the amount when no enhancer was present; The amount of urine vacancy that exceeded the monolayer became 10 to 15 times more compared to the amount when no enhancer was present.

In contrast, no increase in transfer rates for larger proteins, such as cytochrome C (MW 12,300), carbonic anhydrase (MW 30,000) and albumin (MW 69,000) were detected when tested for up to 16 mM sodium caprate. It is expected that when the sodium caprate concentration is higher, the permeability of cells is expected to further increase transmission of larger polypeptides; However, potential cytotoxicity of sodium caprate may inhibit the use of this particular enhancement when its concentration is considerably higher.

Other enhancers can make the transfer of larger polypeptides possible; they can be tested in this in vitro model of epithelial cell proliferation, which can be used as a screening device for rapid testing of all combinations of polypeptides / enhancements that may be useful in the methods of the invention.

EXAMPLE 2: A process for the selection of compounds present in this invention.

All of the compounds listed in Table I were tested to evaluate their ability to increase the uptake of polypeptide (insulin) in the experimental session. Nine sites with insulin are used as evidence of the ability of the enhancer to increase the absorption of other polypeptides.

Various types of insulin were used in different experiments: human recombinant human, semi-man or cattle. Each mix was prepared as before, by wiping and working the insulin / enhancer or insulin / enhancer / lactose solution to produce breathable breaths. The powder was given by inhalation rats and then the blood sugar levels of the rats were monitored as a measure of insulin uptake. This amount was compared to the corresponding values ​​obtained from rats who had been inhaled without insulin.

The same in vitro model system could be used to test any peptides or proteins useful in the methods of the invention by dispersing with the same inhalation method a mixture containing the desired peptide or protein together with the eukaryotic, and detecting the concentration of the desired peptide or the protein in the core-cycle cycle of the experimental animal (eg, standard immunosuppression or biochemistry assays suitable for any peptide or protein).

TABLE I

<td>content</td><td>Eykir: Insulin, lactose</td><td>effects</td>

<td>octylglucopyranoside</td><td>4: 4: 92</td><td>(+)</td>

<td>Natríumúrsódeoxýkólat</td><td>4: 4: 92</td><td>+</td>

<td>glycoside</td><td>4: 4: 92</td><td>+</td>

<td>Sodium</td><td>4: 4: 92</td><td>+</td>

<td>lysophospatidylcholine</td><td>4: 4: 92</td><td>+</td>

<td>Dfoktanóýlfosfatidýlkólín</td><td>2: 4: 94</td><td>(+)</td>

<td>Dídekanóýlfosfatidýlkólín</td><td>4: 4: 94</td><td>-</td>

<td>Sodium taurodihydrofusidate</td><td>2: 4: 94</td><td>+</td>

<td>Sodium caprylate</td><td>25: 75: 0</td><td>-</td>

<td>sodium</td><td>10: 90: 0</td><td>(+)</td>

<td>sodium</td><td>17.5: 82.5: 0</td><td>(+)</td>

<td>sodium</td><td>25: 75: 0</td><td>+</td>

<td>sodium</td><td>4: 4: 92</td><td>+</td>

<td>sodium laurate</td><td>25: 75: 0</td><td>(+)</td>

<td>Potassium oleate</td><td>4: 4: 92</td><td>+</td>

<td>Potassium caprate</td><td>27: 73: 0</td><td>+</td>

<td>Lysine caprate</td><td>35: 65: 0</td><td>+</td>

<td>Sodium myristate</td><td>30: 70: 0</td><td>+</td>

<td>Dimethyl-B-cyclodextrin</td><td>75: 25: 0</td><td>+</td>

+ effect, ie enhancer causes a significant decrease in blood glucose levels

- no or very small effect (+) effect, not like those indicated by "+"

Example 3: A pharmaceutical composition according to the invention

Human growth hormone (hGH, MW 22kD, Lactated Humatrope from Lilly, 3 parts) was mixed with sodium caprate (one part). The mixture was milled in a Retsch engine glue to a particle size of 6.7 μη in the median mass diameter (MMD).

The powder that was formed was administered to rats within the bark and the uptake of hGH was compared to the absorption of powder, 9.6 μm MMD, and consists of hGH and mannitol in the same proportions as prepared in the same manner as before.

The results indicated an increase in the incorporation of hGH into the mixture, including sodium caprate, as compared to the absorption of the mixture without anxiety.

Example 4: A mixture containing the polypeptide insulin

Insulin is herein used as an indicator of other polypeptides of this invention.

Human biochemical insulin (53g) was microfilmide in Airfilco Jet Mill (trademark Airfilco Process Plant Limited), with nitrogen under pressure (7 bar pressure, 5 bar pressure) to 2.4 micrometer median mass.

Sodium caprate (170g) was air filtrate jet mill (TM), pressurized nitrogen (dose pressure 5 bar, 3 bar pressure), 1.6 micrometer mid-mass mass.

The micronized bioavailable human insulin (45g) and sodium caprate (14.26g) were dry-blended according to the following procedure: half of the insulin was placed in a mixer comprising a mixing tank with

4.4 liters volume divided by sew of the width of two compartments, with a metal ring in each compartment to assist in mixing and stirring. Sodium caprate and eventually the rest of the insulin were added.

The mixing cylinder was closed, turned 180 degrees and fitted to a mechanical shaker. The machine was started and shaken for about two minutes until the whole insulin and the sodium caprate had passed through the filter. The engine was switched off and the mixing cylinder turned 180 degrees, reattached to the shaker and shaken again until all the powder had passed through the sift. This process was repeated eight more times so that the total mixing time was approximately 20 minutes.

The mixture obtained in this manner was administered to 5 dogs by inhalation, at 1 U./kg dose range, and plasma insulin levels reported at different times after administration.

The results obtained were compared to the amount of insulin in plasma obtained when bioavailable insulin, microwaved as before 2,4 micrometre median mean mass, was administered to five dogs in the same dose as in the amount of insulin in plasma already obtained A cure of insulin and sodium caprate in the 90:10 proportions were given to five dogs in the same manner as in the same dose as before. In this case, the remedy was prepared as follows: a semi-man-made insulin was gelatinized to reduce the zinc content from 0.52% to 0.01% relative to the insulin content. Insulin (4.5g) and sodium caprate (0.5g) were dissolved in water (232ml). The solution was stirred until clear and the pH adjusted to 7.0. The solution was concentrated by evaporation at 37 ° C for about two days.

The results of these comparisons are shown in Figure 3 (p = 0.0147 for the difference between 75:25 and 100: 0). The results show some increase in insulin bioavailability with the 90:10 blend and a dramatic increase in the bioavailability of the insulin with the 75:25 mixture, including sodium caprate, relative to insulin alone.