Compositions for inhalation, method for priparing the same, using the same and inhaler

The invention relates to the field of medicine and relates to compositions and methods for introducing peptides and proteins of medical value.

Although the discovery of recombinant DNA technology has led to a rapid expansion of peptide-based drugs, the main disadvantage of peptide-based therapies has been the great impediment to realizing the full potential of the field: in general, active doses of peptide-based drugs before they reach the bloodstream. If the polypeptide of interest cannot be altered to become relatively resistant to these enzymes, then the parenteral route, such as intravenous, intramuscular or subcutaneous administration, is the only practical practical route of administration.

Drug administration is known, for example, by absorption through nasal membranes, using a composition containing ergopeptide alkaloids dissolved in aqueous ethanol and used as an aerosol (see Swiss Patent No. 636011).

It is known that smaller polypeptides can be effectively absorbed through the nasal mucosa (see L. Illume, Archiv for Pharmaci og Chemi, Vol. 94 (1987), pp. 127-135).

However, larger sized polypeptides are only partially absorbed through the nose and this process is unstable.

It has been observed that when the peptide or protein (collectively referred to as polypeptides) is mixed with an appropriate absorption enhancer and pulverized into the lungs, it reaches the pulmonary circulation readily through a layer of epithelial cells of the lower respiratory tract, with a certain particle size. This is conveniently accomplished by inhaling powder through an inhaler that delivers the correct doses of the polypeptide / promoter in powder form with particle sizes that provide maximum deposition in the lower airways rather than in the mouth or throat. (For simplicity of reference, the polypeptide and promoter will hereinafter be referred to collectively as "active compounds"). As much active compound as possible should be comprised of particles having a radius of about 10 µm (e.g., 0.01 to 10 µm, ideally between i and 6 µm) to deliver most of it to the lungs. In more preferred embodiments, at least 50% (preferably at least 60%, more preferably at least 70%, more preferably at least 80% and preferably at least 90%) of the total weight of the active compounds in the inhaler consists of particles having a diameter within the desired range.

Thus, the present invention encompasses a pharmaceutical composition comprising the active compounds (A) a pharmaceutically active polypeptide and (B) a stimulating compound that improves the systemic absorption of the polypeptide in a patient's lower respiratory tract (preferably lungs) when in dry powder inhalable form and at least 50 % of the total weight of the active compounds (A) and (B) consists of primary particles having a diameter of less than or equal to 10 microns. The primary particles may be disposed as such or may form agglomerates which are then substantially disintegrated before they enter the patient's respiratory tract. Of course, the composition may, if necessary, also contain other ingredients, including other pharmacologically active substances, other excipients and pharmacologically acceptable excipients such as diluents or carriers. Thus, the therapeutic preparation according to the present invention may contain only said active compounds or it may contain other substances, such as a pharmaceutically acceptable carrier. The bulk of this carrier may be comprised of particles less than about 10 microns in diameter to form at least 50% of the powder as a whole, optionally agglomerated primary particles less than about 10 microns in diameter; on the other hand, the bulk of the carrier can be made up of much larger particles (& quot; coarse particles & quot;), leading to the formation of a & quot; neat mixture & quot; between the active compounds and said carrier. In the ordered mixture, otherwise in the interaction or adhesive mixture, the fine drug particles (the active compounds of the present invention) are distributed fairly uniformly over the surface of the larger excipient particles (the pharmaceutically acceptable carrier of the present invention). Preferably, in this case, the active compounds do not form agglomerates prior to forming the ordered mixture. Coarse particles can have diameters above 20 microns, for example 60 microns. Above these thresholds, the diameter of the coarse particles is not very important, so coarse particles can be used in varying sizes, as required by the practical requirements of a particular recipe. Although there is no requirement for the coarse particles in the ordered mixture to have a uniform size, it is more useful when the coarse particles in the ordered mixture are similar in size. More preferably, the coarse particles have a diameter of 60-800 microns.

The polypeptide can be any peptide or protein of medical or diagnostic utility (including analogues and modified or truncated derivatives thereof) of small or medium size, i.e. up to 40 kD molecular weight (MS), which requires systemic administration. The mechanism of improved polypeptide absorption according to the present invention can and should be generally applied to all of these polypeptides, although the degree to which their absorption is improved may vary depending on the MS and the physical and chemical properties of the polypeptide and the particular promoter employed. Polypeptides having a molecular weight of up to 30 kD are expected to be most useful in the present invention, such as polypeptides having a molecular weight of up to 25 kD or up to 20 kD, and in particular up to 15 kD or 10 kD. Any desired polypeptide can be readily tested using the present invention with a particular promoter in in vitro and in vivo assays as described below. Further, any reference to a polypeptide, peptide or protein, or any specific polypeptide, peptide or protein, shall be understood to include any analogue or modified or truncated derivative thereof.

The stimulant compound used in the compositions of the present invention may be any substance that improves the absorption of the polypeptide through the lower respiratory tract epithelium into the major circulatory system. The words "improved absorption" means that the amount of polypeptide absorbed into the circulation is greater in the presence of the promoter than in the presence of the promoter. More preferably, the amount of polypeptide absorbed is significantly higher (p <0.05) in the presence of a promoter. Whether a particular potential promoter is suitable for use in the present invention is readily determined using in vivo and in vitro assays as described herein.

Preferably, the amount of polypeptide absorbed in the present invention is at least 150% of the amount absorbed in the absence of a promoter. In more preferred embodiments, the absorption of the polypeptide is at least doubled, more tripled, and preferably increased fourfold in the presence of a promoter, as compared to the absence of a promoter.

Preferably, the promoter is a surfactant such as a fatty acid salt, a bile salt, a bile salt derivative, an alkyl glycoside, a cyclodextrin or a phospholipid. The promoter may be, for example, a sodium, potassium or organic amine salt of a fatty acid, and preferably when the fatty acid is capric acid or another fatty acid of 10 to 14 carbon atoms. Preferably, the promoter is sodium caprate. Preferably, the ratio of polypeptide to promoter ranges from about 9: 1 to about 1: 1. While ratios greater than 1: 1 are expected to improve absorption as well as, or even more so, lower ratios, it is believed that the amount of promoter used should not exceed what is necessary to achieve the desired level of improvement, since excess promoter can cause undesirable side effects, e.g. , local irritation.

The invention also relates to a systemic method of administering a pharmaceutically active polypeptide which comprises inhaling to a patient a pharmaceutical composition of the invention comprising at least 50% of the total weight of the active compounds before they enter the patient's respiratory tract consisting of particles less than or equal to about 10 microns. This is preferably done using an inhaler through which the patient inhales the powder. If the powder formulation is composed of primary particulate agglomerates, it is preferable that the inhaler is designed to disintegrate a significant portion of the agglomerates by inhaling the powder from the inhaler to disintegrate most agglomerates into particles less than or equal to about 10 microns before entering the powder. . This fragmentation should occur inside the device and is usually induced by air turbulence caused by the inspiratory force in the device. In general, it is preferable that agglomerates do not form in the ordered mixture. In the case of a neat mixture, preferably when the active compounds are released from the large particles by inhalation, either by mechanical means in the inhaler, or simply by inhalation or otherwise, the active compounds are deposited in the lower respiratory tract and the carrier particles in the mouth.

Preferably, the inhaler is a single dose dry powder inhaler, but may also be a multiple dose dry powder inhaler.

The invention also encompasses a process for preparing a pharmaceutical composition suitable for administration by inhalation. In one such process, a solution is prepared that dissolves (a) a pharmaceutically active polypeptide and (b) a stimulating compound that improves the systemic absorption of the polypeptide in the patient's lower respiratory tract. The solvent is then removed from the solution to give a dry solid mass consisting of the polypeptide and promoter and the dry mass is triturated to give a powder. In a second such process, dry (a) the pharmaceutically active polypeptide and (b) the stimulating compound are mixed and crushed the resulting mixture to micron particles. In a further third such process, first, a micronized particulate preparation consisting of a polypeptide and, secondly, a micronized particulate preparation consisting of a stimulating compound are prepared, and the two micronized particles are mixed together. When a carrier not intended to be formulated is added, it can be added to the solution or to a dry mixture of the pharmaceutically active polypeptide, or the particulate carrier may be mixed dry with the other micronized particles prior to comminution. The micronized polypeptide and adjuvant are mixed with a suitable carrier to form a fine-grained mixture.

Brief description of the drawings

FIG. 1. A graph illustrating the effect of different concentrations of promoter, sodium caprate, on the transport of labeled compound (mannitol) via monolayer epithelial cell culture.

FIG. 2. Graph illustrating the effect of different concentrations of promoter, sodium caprate, on the transport of labeled compound (mannitol) via monolayer epithelial cell culture in the presence of polypeptide (sodium caprate: polypeptide 1: 3 by weight).

FIG. Plasma concentration of polypeptide as a function of time after inhalation of polypeptide alone, polypeptide with sodium caprate ratio 90:10 and polypeptide with sodium caprate ratio 75:25.

Below are general descriptions of some more preferred embodiments of the invention. A polypeptide

Preferably, the polypeptide is a peptide hormone other than insulin such as vasopressin, vasopressin analogs, desmopressin, glucagon, corticotropin (ACTH), gonadotropin (luteinizing hormone, or LHRH), calcitonin, human hormone Cpeptide, parathyroid hormone (parathyroid hormone). (hGH), growth hormone (GH), growth hormone releasing hormone (GHRH), oxytocin, corticotropin releasing hormone (CRH), somatostatin analogues, gonadotropin agonist analogs (GnRHa), atrial natriuretic hormone (hANP), thyroxine releasing hormone (TRHrh), follicle stimulating hormone (FSH), or prolactin.

Other possible polypeptides include growth factor, interleukins, polypeptide vaccines, enzymes, endorphins, glycoproteins, lipoproteins, or polypeptides involved in the blood coagulation chain, which exert systemic pharmacological effects. It is expected that most, if not all, polypeptides having a small to medium size, relatively good water solubility, and an isoelectric point between about pH3 and pH8 can be efficiently introduced by the methods of the present invention.

Promoter

The absorption enhancer is of particular importance since one polypeptide is very poorly absorbed by the lungs. Any compound that enhances absorption into adjacent pulmonary vascular vessels through the epithelial cell layer surrounding the airways may be used as a promoter. The promoter can do this in several possible ways:

(1) Improving the intracellular conductance of a polypeptide by inducing structural changes in solid junctions between epithelial cells.

(2) Improving the intracellular conductivity of a polypeptide by interacting with membrane proteins or lipids or by extracting membrane proteins or lipids, thereby affecting membrane integrity.

(3) An interaction between a promoter and a polypeptide that increases the solubility of the polypeptide in aqueous solution. This can be accomplished by preventing the formation of insulin assemblies (dimers, trimers, hexamers) or by dissolving the polypeptide molecules in the promoter micelles.

(4) Reduction or dissolution of the viscous barrier lining the alveoli and knees to the lungs, thereby directing the epithelial surface to directly absorb the polypeptide.

The promoter may act either by one of the mechanisms above or by two or more. A promoter that acts by multiple mechanisms is more likely to promote efficient absorption of a polypeptide than a promoter that uses only one or two mechanisms.

For example, surfactants are a class of promoters that are believed to act by all four mechanisms above. Surfactants are amphiphilic molecules having both a lipophilic and a hydrophilic side, with a variable balance of the two characteristics. If the molecule is highly lipophilic, its low water solubility may limit its usefulness. However, if the hydrophilic side is predominant, the surface-active properties of the molecule may be minimal. Thus, in order to be effective, a surfactant must strike a certain balance between sufficient solubility and sufficient surfactant.

Another property of the surfactant that may be important is the net charge of the surfactant at the pH of the lungs (approximately 7.4). At pH

7.4 some polypeptides have a negative net charge. This can cause electrostatic repulsion between molecules, which in turn prevents aggregation and thus increases solubility. If the surfactant also has a negative charge, it can interact with the polypeptide, for example hydrophobically, and thus cause an additional push between the molecules of the polypeptide. In this case, the anionic surfactant will have the additional advantage (as compared to those that are neutral or have a positive net charge at physiological pH) of improving absorption, helping to stabilize the polypeptide in the monomeric state.

A variety of different compounds potentially useful as promoters in the methods of the present invention were tested on mice as described in Example 2 below. Other substances with known absorption enhancers or physical properties that make them suitable candidates for the methods of the present invention can readily be tested by one of ordinary skill in the art in the in vivo assay or, alternatively, the in vitro assay described in Example 1.

It is possible that a combination of two or more stimulants will also give satisfactory results. The invention is intended to include the use of such combinations in the methods of the present invention.

The promoter useful in the methods of the present invention must combine an effective enhancement of the absorption of the polypeptide with (1) the absence of toxicity at the concentrations used and (2) the good powder properties, i.e., the absence of an adhesive or waxy solid state. The toxicity of the substance may be tested by standard methods such as MTT assays such as those described in Int. J. Pharm., 65 (1990), 249-259. The powder properties of a given substance may be determined from published material data or empirically.

One of the most promising promoters is the fatty acid salt. Sodium salt has been found to work well with saturated fatty acids having carbon chain lengths of 10 (i.e., sodium caprate), 12 (sodium laurate) and 14 (sodium myristate). The potassium and lysine salts of capric acid have also been found to be effective in the process of the present invention. If the carbon chain length is less than about 10, the surfactant may be too low for the surfactant, and if the chain length is greater than about 14, the reduced water solubility of the fatty acid limits its usefulness.

Sodium caprate is the most suitable material for improving the absorption of the polypeptide in the lower respiratory tract.

Different opposing charge ions can alter the water solubility of saturated fatty acid salts so that it may appear that a promoter with a carbon chain other than 10 to 14 lengths is even superior to the promoters specifically mentioned above. Unsaturated fatty acid salts may also be useful in the present invention since they are more soluble in water than saturated fatty acid salts and may therefore have a longer chain than the latter and still retain the solubility necessary to successfully improve the absorption of the polypeptide.

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

Phospholipids have also been screened as promoters. Single-chain phospholipid (lysophosphatidylcholine) was found to be a good promoter, whereas double-chain phospholipids (dioctanoylphosphatidylcholine and didecanoyl phosphatidylcholine) were not good promoters. This could be explained by the fact that double chain phospholipids are much less water soluble than their single chain counterparts; however, it is to be expected that double chain phospholipids with a shorter chain, more soluble in water than their longer chain counterparts, may be used as promoters in the present invention, so that both single and double chain phospholipids can be used.

One glycoside, octylglucopyranoside, has been tested as a promoter of the present invention and has been found to have some such stimulating properties. Other alkyl glycosides, such as thioglycopyranosides and maltopyranosides, may be expected to also exhibit absorption enhancing properties in the methods of the present invention.

Cyclodextrins and their derivatives efficiently improve nasal absorption and could act similarly in the lungs. Dimethyl-β-cyclodextrin has been tested and found to have absorption-enhancing properties.

Other potentially useful surfactants include sodium salicylate, sodium 5-methoxysalicylate, and naturally occurring surfactants such as glyceryl acidic salts, saponin glycosides, and acyl carnitines.

For ionic promoters (such as the anionic surfactants described above), the origin of the opposite charge ion may be important. The specific ion of the opposite charge may affect the powder properties, solubility, stability, hygroscopicity, and local / systemic toxicity of the promoter or any formulation containing the promoter. It may also affect the stability and / or solubility of the polypeptide with which it is combined. In general, one would expect that monovalent metal cations, such as sodium, potassium, lithium, rubidium, and cesium, would be useful as opposing charge ions to anionic promoters. Ammonia and organic amines form another class of cations which are expected to be used with anionic promoters having a carboxylic acid group. Such organic amines include ethanolamine, diethanolamine, triethanolamine, 2-aminomethylethylamine, betaines, ethylenediamine, Ν, Ν-dibenzylethylenetetraamine, arginine, hexamethylenetetraamine, histidine, N-methylpiperidine, lysine, piperazine, spermidine, spermidine, spermidine.

Because effective enhancement of polypeptide absorption in the lungs has been found with many of the promoters tested, it is to be expected that more will be discovered that act in similar ways. Starch microspheres effectively improve the availability of a polypeptide introduced through the nasal membrane and have been tested as a promoter in the methods of the present invention. Although they have proven to be of little use in the pulmonary route in the animal model used here, it is to be assumed that this is mainly due to technical difficulties and may be successful in overcoming the pulmonary route.

Chelates are a class of promoters that are thought to work by binding calcium ions. Because calcium ions help maintain cell-to-cell size and decrease solubility of the polypeptide, binding of these ions should, in theory, increase both the solubility of the polypeptide and the permeability of the parabolic polypeptide. Although one of the chelates tested, ethylenediaminetetraacetic acid sodium (EDTA), has been shown to be ineffective in improving insulin absorption in a tested rat model, other calcium-binding chelates may be more valuable.

Proportion of polypeptide to promoter

The relative proportions of the polypeptide and the promoter may be varied as necessary. There must be sufficient amount of promoter to absorb the inhaled polypeptide; however, the amount of the promoter should be kept as low as possible to minimize the risk of adverse effects caused by the promoter. While each polypeptide / promoter combination must be tested to determine the optimal proportions, it should be expected that more than 10% of the polypeptide / promoter mixture should be a promoter to achieve an acceptable level of absorption of the polypeptide; for most types of promoters, the promoter share should be greater than 15% or more than 20%, and preferably between 25% and 50%. A more acceptable polypeptide / promoter ratio (or polypeptide / promoter / diluent) can be readily determined by one of ordinary skill in the art of pharmacology using standard techniques based on criteria such as sufficient and constant administration of an optimal dose, minimization of side effects, and acceptable absorption rate.

No ingredients are needed for the preparations to work, but they can be turned on if needed. Additional materials may be incorporated into the formulation, for example, to reconstitute the powder to an amount that can be administered using the particular powder inhaler desired; to facilitate processing of the preparation; improve the powder properties of the preparation; increase the stability of the preparation, for example, by the use of antioxidants or pH-adjusting compounds; or add flavor to the preparation. Any of these additives must not adversely affect the stability or absorption of the insulin or adversely affect the absorption of the insulin. It should be stable, non-hygroscopic, have good powder properties, and should not adversely affect the respiratory tract. Examples of potentially useful additives are mono-, di- and polysaccharides, sugar alcohols and other polyols, such as lactose, glucose, raffinose, melezitose, lactitol, maltitol, trechalose, sucrose, mannitol and starch. As reducing sugars, lactose and glucose tend to form complexes with proteins, non-reducing sugars such as raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol and starch may be more acceptable additives to the present invention. Depending on the inhaler used, the total amount of such accessories may vary within wide limits. In some circumstances, little or no additives are needed, whereas inhalers requiring a high powder volume can contain any part, from 0% (ie without additives) to close to 100% of the total formulation.

In a more preferred embodiment, the present invention provides a therapeutic formulation of a pharmaceutically active polypeptide and a substance which improves the absorption of said polypeptide in the lower respiratory tract, in dry powder form suitable for inhalation and comprising at least 50% by weight of less than about 10 microns, or (b) agglomerates of said particles; in another more preferred embodiment, the invention provides a therapeutic formulation comprising a pharmaceutically active polypeptide, a substance that improves the absorption of said polypeptide in the lower respiratory tract, and a pharmaceutically acceptable carrier, and wherein the formulation comprises at least 50% by weight of particles (a) about 10 microns, or (b) agglomerates of said particles; and in still more preferred embodiments, the invention provides a therapeutic preparation comprising the active compounds of (A) a pharmaceutically active polypeptide and (B) a substance that improves the absorption of said polypeptide in the lower respiratory tract and at least 50% of the total active compounds. (B), the mass consisting of particles having a diameter of less than about 10 microns and a pharmaceutically acceptable carrier, and the preparation is in the form of a dry powder for inhalation, which may form a neat mixture between the active compounds and a pharmaceutically acceptable carrier.

The powder formulation described may be prepared in several ways using standard techniques. In most cases, the purified polypeptide may be obtained from commercial sources. Alternatively, the polypeptide of interest may be purified from naturally occurring sources using conventional biochemical techniques, or may be obtained by expressing prokaryotic or eukaryotic cells that have been engineered with a nucleotide sequence encoding the polypeptide and having correspondingly linked expression control sequences ( animals capable of producing the desired peptide or protein, for example in their milk). These are standard techniques in the art (e.g., see Sambrook et al., Molecular 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 readily synthesized using known chemical techniques.

Absorption enhancers described above can also generally be obtained from commercial sources or prepared using known techniques. In the case of ionic promoters, the ion to which it is bonded with the opposite charge can be replaced, if necessary, by standard ion exchange techniques.

In the preparation of the powder formulations described, it is simply necessary at some stage in the process to grind the powder to micron particles using suitable mills, such as a jet mill, to produce primary particles within the size range of j which provide maximum deposition in the lower respiratory tract. 10 pm). For example, it is possible to mix the dry polypeptide and promoter powder and then combine both materials to form micron particles; otherwise, the materials may be crushed individually and then blended. When mixed compounds have different physical properties, such as strength and brittleness, micron particle resistance varies, and different pressures may need to be applied to decompose to the appropriate particle size. Therefore, the particle sizes of one of the components may not be satisfactory when crushed together. In such cases, it would be better to grind the different components separately and then mix them.

It is also possible to first dissolve the ingredients in a suitable solvent such as water to mix at the molecular level. This procedure also allows the pH to be adjusted at the desired level, for example to improve the absorption of the polypeptide. Pharmaceutically acceptable pH ranges for inhaled products, pH 3.0 to 8.5, should be kept in mind, since products with pH outside these ranges would cause respiratory tract irritation and contraction. To obtain a powder, the solvent must be removed so as to maintain the biological activity of the polypeptide. Suitable drying techniques include vacuum evaporation, open drying, spray drying and freeze drying. Generally, temperatures above 40 ° C for more than a few minutes should be avoided as some polypeptides may degrade. After the drying step, the solid can be crushed to a coarse powder, if necessary, then crushed to micron particles if necessary.

If necessary, the micronized powders may be processed to improve flow properties, such as dry granulation, to form spherical agglomerates with improved operational properties prior to incorporation into the intended inhaler. In such cases, the inhaler should be designed to ensure that most agglomerates are disintegrated before leaving the device so that the majority of particles entering the respiratory tract are within the desired size range. Where a neat mixture is required, the active compound can be treated, e.g., chopped to micron particles, to produce particles having sizes within certain limits, if necessary. The carrier can also be treated, for example, to obtain the desired particle size and required surface properties, such as a specific surface to weight ratio or a certain roughness, to provide optimum adhesive forces in the cured mixture. Such physical requirements for an ordered mixture are well known because there are a variety of means to obtain an ordered mixture ii satisfying the above requirements and can be readily determined by the skilled person in the light of the particular circumstances.

A more acceptable inhaler should have the following design features: It must protect the powder from moisture and ensure that no accidental overdose is given; in addition, as many of the following properties as possible are desirable: protecting the powder from light; have a large inhalable fraction and a large proportion should settle in the lungs over a wide range of flow rates; slight deviation in dose and inhalation fraction; keeping a low powder content in the mouthpiece - this is particularly true in multi-dose inhalers where the polypeptide retained in the mouthpiece can degrade and then be inhaled at the next dose; have low absorption on the surface of the inhaler; flexibility in dose selection; and low resistance to inspiration. Preferably, the inhaler is a single dose, although multiple dose inhalers, such as a multiple dose, reusable, dry powder inhaler, may also be used. It is preferable to use a single dose inhalation dry powder disposable inhaler.

A variety of dry powder formulations including polypeptide and various promoters have been prepared and tested in vivo as described below. In vitro assays useful for testing polypeptide / promoter combinations are also described.

Example: Determination of a Specific Polypeptide Value of the Invention by In Vitro.

A standard in vitro assay with an epithelial cell line, CaCo-2 (available from the American Type Culture Collection (ATCC), Rockville, MD, USA) was developed to evaluate the ability of various promoter compounds to promote labeled substance transport through epithelial cells. a monolayer, which is a model of a layer of epithelial cells in the lungs that separates the alveoli from the pulmonary blood vessels.

In this assay, various proportions and / or concentrations of promoter and polypeptide or other labeled substance are dissolved in aqueous solution and applied to the apical side of the cell monolayer. After incubation for 60 min at 37 ° C and 95% SD (relative humidity), the amount of labeled material at the basolateral end of the cells is determined, e.g., using a radioactive labeled material.

For sodium caprate, the amount of labeled substance (mannitol, MS 360) that appears on the basolateral side depends on the amount of promoter used, at least up to 16 mM sodium caprate (Fig. 1). This is true even if the promoter / mannitol mixture is supplemented with polypeptide, insulin, (1: 3 caprateunsulin, w / w) (Figs. 2). It was also observed that this concentration of sodium caprate (16 mM) promotes the absorption of two low molecular weight peptides, insulin (MS 5734) and vasopressin (MS 1208), through the cell monolayer. The amount of insulin that passes through the monolayer is doubled when 16 mM sodium caprate is added compared to the amount without any stimulant; the amount of vasopressin that passes through the monolayer is increased 10 to 15-fold compared to the amount without any promoter.

In contrast, no significant increase in the transport rate of large proteins such as cytochrome C (MS 12300), carbon anhydrase (MS 30000) and albumin (MS 69000) was observed when tested with sodium caprate up to 16 mM. It is to be expected that at higher concentrations of sodium caprate, cell permeability will be further increased and will carry higher polypeptides; however, the potential cytotoxicity of sodium caprate may prevent the use of much higher concentrations of this particular promoter.

Other promoters may promote transport of larger polypeptides; they can also be tested with this in vitro model of epithelial cell permeability, which can be used as a rapid screening tool to test whether the desired polypeptide / promoter combination is suitable for the method of this invention.

2. Example: A method of selecting promoters suitable for the present invention.

Each compound in Table I was tested for its ability to improve polypeptide (insulin) uptake in a rat model. The results obtained with insulin are considered to be the promoter's potential ability to improve the absorption of other polypeptides.

Different forms of insulin have been used in different tests: human recombinant, human or bovine semisynthetic. Each preparation was prepared as described above by drying and processing an insulin / promoter or insulin / promoter / lactose solution to provide a powder suitable for inhalation. The powder was administered to rats by inhalation and blood glucose levels were further monitored in rats as a measure of insulin uptake. These levels were compared with the corresponding values ​​obtained in rats that had inhaled insulin preparations.

The same in vivo model system can be used to test the suitability of any peptide or protein in the methods of the present invention by administering a formulation containing the desired peptide or protein in admixture with promoters and monitoring the concentration of the desired peptide or protein in the major circulation of the test animals. such as standard immunoassays or biochemicals specific to a given peptide or protein).

TABLE I

<td>Material</td><td>Promoter: Insulin: Lactose</td><td>Effect</td>

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

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

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

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

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

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

<td>Didecanoylphosphatidylcholine</td><td> 4:4:94</td><td></td>

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

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

<td>Sodium Caprate</td><td> 10:90:0</td><td> (+)</td>

<td>Sodium Caprate</td><td> 17,5:82,5:0</td><td> (+)</td>

<td>Sodium Caprate</td><td> 25:75:0</td><td> +</td>

<td>Sodium Caprate</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>Myristate of sodium</td><td> 30:70:0</td><td> +</td>

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

+ effect, meaning that the promoter produces a significant drop in blood glucose, or no (+) effect, not as significant as +

Example: A therapeutic preparation according to the invention

Human growth hormone (hGH, MS 22kD, source Humatrope from Lilly, 3 parts) was mixed with sodium caprate (1 part). The mixture was ground in a Retsch mechanical mill to particles having a median diameter of 6.7 µm.

The resulting powder was injected into the rat trachea and the uptake of hGH was compared to that of a powder having a median diameter of 9.6 µm and consisting of hGH and mannitol in the same proportions and prepared in the same manner as described above.

The results showed a better uptake of hGH when sodium caprate was present, compared to a non-promoter.

Example: A preparation containing insulin polypeptide

Insulin is used herein as a budding agent of other polypeptides of the present invention.

Biosynthesized human insulin (53 g) was pulverized to micron particles in an Airfilco jet grinder (Brand name, Airfilco Process Plant Limited) with compressed nitrogen (feed pressure 7 bar, vessel pressure 5 bar) until a median diameter of 2.4 micrometers was achieved.

Sodium caprate (170 g) was pulverized to micron particles in an Airfilco Jet Grinder (TM) with pressurized nitrogen (feed pressure 5 bar, pressure 3 bar) until a median diameter of 1.6 micrometer was reached.

Micronized biosynthesized human insulin (45 g) and sodium caprate (14.26 g) were mixed dry according to this procedure. Half of the insulin was placed in a mixing unit consisting of a mixing cylinder having a volume of 4.4 liters, divided into a sieve having a width of 1 mm and containing a metal ring for mixing each. Sodium caprate is added and finally the remaining insulin. The mixing cylinder is closed, rotated 180 degrees and placed on a shaker with motor. The engine is started and shaken for approximately two minutes until all insulin and sodium caprate have passed through the sieve. The engine is switched off and the mixing cylinder is rotated 180 degrees, pushed back on the shaker, and shaken again until all powder has passed through a sieve. This procedure was repeated eight more times, stirring for about 20 minutes.

The preparation thus obtained was administered by inhalation in 5 dogs at a dose of 1 U / kg and plasma insulin levels were determined at various time points after administration.

The results obtained were compared with plasma insulin levels obtained when biosynthesized insulin pulverized to a median diameter of 2.4 micrometres, as described above, was administered to five dogs in the same manner and at the same dose, and plasma insulin levels obtained when therapeutic insulin and sodium caprate, 90:10, was administered to five dogs in the same manner and at the same dose as above. In this case, the therapeutic preparation was prepared as follows. Gel filtration with human semi-synthetic insulin was performed to reduce zinc from 0.52% to 0.01% relative to insulin. Insulin (4.5 g) and sodium caprate (0.5 g) were dissolved in water (232 mL). The solution was stirred by rotation until it became clear and the pH was brought to 7.0. The solution was concentrated by evaporation at 37 ° C for approximately two days. The resulting solid precipitate was crushed and sieved through a 0.5 mm sieve, and the resulting powder was crushed in a jet grater to a particle diameter of 3.1 microns.

The results of these comparisons are shown in Figs. 3 (p = 0.0147 for difference between 75:25 and 100: 0). The results showed some improvement in insulin uptake at 90:10 and a very marked improvement in insulin uptake at 75:25 with sodium caprate compared to one insulin.