Compositions for inhalation

ES 2 162 865 T3

DESCRIPTION

Compositions for inhalation.

This invention relates to methods and compositions for the delivery of peptides and proteins useful in medicine.

Background of the invention

Although the advent of recombinant DNA technology has resulted in a rapidly expanding list of peptide-based drugs, a major disadvantage of baba therapy. Sada in peptides has severely hampered the realization of the full potential of this field: In general, peptide-based drugs cannot be administered orally in effective doses, since they are rapidly broken down by enzymes in the gastrointestinal tract before they can reach the bloodstream. Unless the polypeptide of interest can be altered to make it relatively resistant to said enzymes, it is likely that the only practical method of delivering the drug is a parenteral route, such as by intravenous, intramuscular or subcutaneous injection. Administration by other parenteral routes (eg, by absorption through the nasal, buccal, or rectal membranes, or through the lungs) has met with limited success.

International patent application document WO-A-9116038 is concerned with providing micronized particles (reduced to a size on the order of micrometer s) of human interferon or human interleukin, which are generally unstable in an aqueous solution, in a form that can be administered to the upper or lower respiratory tract. The fine particles of the active ingredient, obtained by lyophilization of an aqueous solution of the biologically active peptide, and then by grinding (taking care) are intended to be administered either from an aerosol formulation in solution or from a powder inhaler. dry.

WO-A-8809163 describes a drug delivery system, including a plurality of microsphere particles for intranasal administration. It is stated that each of the particles has associated with it a surfactant material which has the property of enhancing the ingestion of the active drug.

The US patent document USA 5,011,678 concerns compositions based on propellants, mainly for to the collection of drugs through the nasal vein. The compositions contain a polypeptide drug and a steroid such as sodium tauro-24,25-dihydro-fusidate suspended in a liquid propellant. The purpose of the steroid is to increase the absorption of the drug through a mucosal surface, generally intranasally.

P. Ruin, in the Sydsvenska newspaper (Dagbladet), of Monday June 12, 1989 reported in an article entitled "Diabetics may not need their insulin injections" about the possibility that insulin and a "detergent Unspecified can one day be delivered through an inhaler.

US-A-5,006,343 describes the pulmonary delivery of liposomes containing a pharmaceutically active substance; the liposomes having been combined with an alveolar surfactant protein to enhance the absorption of the liposomes.

US-A-4,994,439 d writes compositions for the transmucosal delivery of drugs, consisting of a drug, one or more non-ionic detergents and one or more bile salts or fusidates or their derivatives, mainly for intranasal administration. The drug can be formulated as an aerosol in the form of a dry powder suspended in a propellant.

It has been found that when a peptide or protein (hereinafter collectively referred to as polypeptides) is combined with an appropriate absorption enhancing agent and is introduced into the lungs in the form of a powder with an appropriate particle size, easily penetrates the pulmonary circulation by absorption through the layer of epithelial cells existing in the lower respiratory tract. This is conveniently accomplished by inhaling the powder from an inhaler device that delivers the correct dose of a powdery combination of a polypeptide and an Intensifier people in a particle size that maximizes stool in the lower respiratory tract, rather than in the mouth and throat. (For ease of reference, the polypeptide and enhancing agent are collectively referred to hereinafter as the "active compounds"). To achieve this preferential delivery to the lungs, as much of the active compounds as possible should consist of particles having a diameter less than about 10 pm (eg between 0.01 and 10 pm and ideally between 1 and 6 pm). In preferred embodiments, at least 50% (preferably at least 60%, more preferably at least 70%, still more preferably at least 80% and most preferably at least 90%) of the total mass of the active compounds, exiting the inhaler device , consists of particles that are within the desired range of diameters.

The invention therefore includes a a pharmaceutical composition containing a mixture of active compounds based on (A) a pharmaceutically active polypeptide and (B) an enhancer compound that enhances the systemic absorption of the polypeptide in the lower respiratory system (preferably in the lungs) of a patient, the mixture being in the form of a dry powder suitable for inhalation, in which at least 50% of the total mass of the active compounds (A) and (B) consists of primary particles having a diameter less than or equal to 10 pm (micrometers) . The primary particles may be packaged as is, or may optionally be formed into agglomerates, which are then substantially deagglomerated prior to entering the respiratory tract of the patient. Of course, the composition may contain other ingredients as needed, including other pharmaceutically active agents, other intensifying agents.

ES 2 cadores , and pharmaceutical excipients generally acceptable such as diluents or carriers. Therefore, the therapeutic preparation of the present invention may contain only such active compounds or it may contain other substances, such as a pharmaceutically acceptable carrier. This carrier can largely consist of particles having a diameter of less than 10 pm (micrometers) such that at least 50% of the resulting powder as a whole consists of optionally agglomerated primary particles having a diameter of less than 10 pm. (micrometers); alternatively, the carrier can largely consist of much larger particles ("coarse particles"), such that an "ordered mixture" can be formed between the active compounds and said carrier. In an ordered mixture, alternatively known as an interactive or adhesive mixture, the fine particles of the drugs (in this invention, the active compounds) are fairly evenly distributed over the surface. surface of the particles of a coarse excipient (in this invention, the pharmaceutically acceptable carrier). Preferably, in such a case the active compounds are not in the form of agglomerates prior to formation of the ordered mixture. Coarse particles can have a diameter greater than 20 pm (micrometers), for example one greater than 60 pm (micrometers). Above these lower limits, the diameter of the coarse particles is not critically important, so various sizes of coarse particles can be used, if desired in accordance with the practical requirements of the particular formulation. There is no requirement that the coarse particles in the ordered mix be of the same size, but the coarse particles can advantageously be of a similar size within the ordered mix. Preferably, the coarse particles have a diameter of 60-800 pm (micrometers).

The polypeptide can be any r (a) peptide or protein useful in medicine or for diagnosis with a size from small to intermediate, that is to say up to a molecular weight (MW) of about 40 kD, for which a systemic delivery is desired. The mechanisms of improved absorption of a polypeptide according to the present invention are widely applicable and should apply to all polypeptides of this type, although the degree to which their absorption is improved may vary depending on the MW and the properties. physicochemical properties of the polypeptide, as well as the particular enhancing agent used. The most useful in the present invention are expected to be polypeptides having a molecular weight of up to 30 kD, such as polypeptides having a molecular weight of up to 25 kD or up to 20 kD, and especially up to 15 kD or up to of 10 kD. Any desired polypeptide can be readily tested for use in the present invention with an enhancing agent. particular cator, by in vivo or in vitro analysis, such as those described in the present context.

The enhancer compound used in the compositions of the present invention can be any-

865 T3 4 wants a compound that enhances the absorption of the polypeptide through the epithelium of the lower respiratory tract, and into the systemic circulation. By the expression "enhances absorption" is meant that the amount of the polypeptide that has been absorbed into the systemic circulation in the presence of an enhancing agent is greater than in the absence of such an enhancing agent. Preferably, the amount of absorbed polypeptide is significantly higher (p <0.05) in the presence of an enhancing agent. The suitability of any potential enhancing agent for use in the present invention can be readily assessed by in vivo or in vitro analysis, as described herein.

The amount of polypeptide absorbed according to the present invention is preferably at least 150% of the amount absorbed in the absence of the enhancing agent. In preferred embodiments, the absorption of the polypeptide is at least doubled, more preferably tripled and most preferably quadrupled in the presence of the enhancing agent, when compared to its absence.

The enhancing agent is preferably a surface active agent such as a salt of a fatty acid, a bile salt, a derivative of a bile salt, an alkyl glycoside, a cyclodextrin or a phospholipid. The enhancing agent may be, for example, a sodium, potassium or organic amine salt of the fatty acid, and the fatty acid is preferably cypric acid or another fatty acid with 10-14 carbon atoms. The preferred enhancing agent is sodium caprate. The ratio of the polypeptide to the enhancing agent will vary and Between approximately 9: 1 and approximately 1: 1. Although enhancing agent ratios greater than 1: 1 could presumably enhance harvesting as well or better than other lower ratios, it is believed that the amount of enhancing agent used should not be greater than is necessary to achieve the desired level of enhancement, since that too much intensifying agent can trigger unwanted side effects, such as local irritation.

Also within the invention is a method of systemically administering a pharmaceutically active polypeptide, resulting in a patient inhaling the pharmaceutical composition of the invention, in which at least 50% of the total mass of the active compounds in the site of entry within the respiratory tract of the patient, consists of particles that have a diameter less than or equal to 10 pm (micrometers). This is preferably achieved by the use of a device ivo inhaler from which the patient inhales the powder. When the powdery composition is in the form of agglomerates of primary particles, the device is preferably configured to induce substantial deagglomeration of the agglomerates after inhalation of the powder from the device by the patient, such that most of the Agglomerates decompose to particles having a diameter less than or equal to 10 pm (micrometers) before the dust enters the respiratory system of the patient. Is

Deagglomeration should occur within the device, and is typically induced by air turbulence created in the device by inhalation force. Agglomerates do not generally form preferably in the ordered mixture. In the case of orderly mixing, the active compounds should be released from the large particles preferably after inhalation. either by mechanical means existing in the inhaler device or simply by the action of inhalation, or by other means, the active compounds then being deposited in the lower respiratory tract and the vehicle particles in the mouth.

The inhaler device is preferably a single dose dry powder inhaler, but may alternatively be a multi dose dry powder inhaler.

The invention also includes processes for the preparation of a pharmaceutical composition suitable for administration by inhalation. In one such method, a solution is first provided in which (a) a pharmaceutically active polypeptide and (b) an enhancer compound that enhance the systemic absorption of the polypeptide in the lower respiratory tract of a patient are dissolved. The solvent is then removed from the solution to provide a dry solid containing e The polypeptide and the enhancer compound, and the solid is pulverized to produce a powder. A second such procedure involves dry mixing (a) a pharmaceutically active polypeptide and (b) an enhancer compound, and micronizing (= reducing to a size on the order of micrometres) the mixture obtained. A still third appropriate procedure includes the steps of providing a first micronized preparation containing a polypeptide and a second micronized preparation containing an enhancer compound, and mixing the two micronized preparations together. When a carrier other than that used when orderly mixing is desired is to be included, it can be added to the solution, or to the dry mix of the pharmaceutically active polypeptide prior to micronization, or a micronized carrier can be dry mixed with the other micronized components. To produce an orderly mixture, the micronized polypeptide and the age The intensifier is mixed with an appropriate vehicle.

Brief description of the drawings

Figure 1 is a graph illustrating the effects of different concentrations of the enhancing agent sodium caprate on the transport of a marker compound (mannitol) through a monolayer of cultured epithelial cells.

Figure 2 is a graph illustrating the effects of different concentrations of the enhancing agent sodium caprate on the transport of a marker compound (mannitol) through a monolayer of cultured epithelial cells, in the presence of a polypeptide (ratio of caprate of sodium to polypeptide 1 : 3 by weight).

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

Detailed description

Some of the preferred embodiments of the invention are described generally below.

Polypeptide

The polypeptide is preferably a peptide hormone other than insulin, such as vasopressin, vasopressin-like compounds, desmopressin, glucagan, corticotropin (ACTH), gonadotropin (luteinizing hormone, or LHRH), calcitonin, the C-peptide of insulin, the hormone parathyroid (PTH), human growth hormone (hGH), growth hormone (HG), growth hormone releasing hormone (GHRH), oxytocin, corticotropin releasing hormone (CRH), Somatostatin-like compounds, gonadotropin agonist-like compounds (GnRHa), atrial natriuretic peptide (hANP), thyroxine-releasing hormone (TRHrh), follicle-stimulating hormone (FSH), and prolactin.

Other possible polypeptides include growth factors. ento, interleukins, polypeptide vaccines, enzymes, endorphins, glycoproteins, lipoproteins, and the polypeptides involved in the blood coagulation cascade, which exert their pharmacological effect through the systemic route. It is expected that most, if not all, polypeptides exhibiting a small to intermediate size, relatively high water solubility, and an isoelectric point between about pH 3 and pH 8 can be effectively delivered by the methods of the invention.

The enhancing agent

The use of an absorption enhancing agent is critically important, since the polypeptide alone is poorly absorbed through the lungs. The enhancing agent used can be any of a number of compounds that act to enhance absorption through the layer of epithelial cells lining the lower respiratory tract, and into the airway. adjacent pulmonary sculature. The enhancing agent can achieve this by any one of several possible mechanisms:

(1) Enhancement of the paracellular permeability of a polypeptide by inducing structural changes in the tight junctions (occlusion) between epithelial cells.

(2) Enhancement of the transcellular permeability of a polypeptide by interaction with, or extraction of, constituents of the protean type or tombstones of the membrane, and thus by disturbing the integrity of the membrane.

(3) Interaction between the enhancing agent and the polypeptide that increases the solubility of the polypeptide in an aqueous solution. This can be done by preventing the formation of polypeptide aggregations (dimers, tramers, hexamers) or by solubilizing polypeptide molecules in micelles of the enhancing agent.

(4) Decrease in viscosity, or dissolution, of the barrier of mucus that coats the al

ES 2 veoli and the lung passages, thereby exposing the epithelial surface for direct absorption of the polypeptide.

Enhancing agents can act only through one of the mechanisms discussed above, or through two or more of them. An enhancing agent that acts by several mechanisms is more likely to promote and activate an efficient absorption of a polypeptide than one that uses only one or two of these.

For example, surfactants are a class of enhancing agents that are believed to work by all of the four mechanisms listed above. Surfactants are amphiphilic molecules that have both a lipophilic and a hydrophilic moiety, with a variable balance between these two characteristics. If the molecule is highly lipephilic, the low solubility of the substance in water may limit its usefulness. If the hydrophilic part overwhelmingly dominates, however, the surface activity properties of the molecule may be minimal. To be effective, the surfactant must therefore strike a proper balance between sufficient solubility and sufficient surface activity.

Another property of surfactants, which may be of importance, is the net charge of the surfactant at the pH value that exists in the lungs (approximately 7.4). At pH 7.4, some polypeptides have a net negative charge. This will result in electrostatic repulsion between molecules, which in turn will prevent aggregation and thereby increase solubility. If the surfactant is also negatively charged, it can interact with the polypeptide, for example, through hydrophobic interactions, and further repulsion will occur between the polypeptide molecules. In such a case, an aniogenic surfactant will possess the added advantage tional (compared to those with a net neutral or positive charge at physiological pH) of enhancing absorption by helping to stabilize the polypeptide in the monomeric state.

A number of different compounds that are potentially useful as enhancing agents in the methods of the invention were tested in rats, as described in Example 2 below. Other substances with known absorption enhancing properties, or with physical characteristics that make them likely candidates for use in the method of the invention, can easily be tested by a person having ordinary experience in such analysis in vivo, or alternatively in the in vitro analysis described in Example 1.

A combination of two or more enhancers may also provide satisfactory results. The use of such a combination in the method of the invention is considered to be found within the invention.

An enhancing agent useful in the methods of the invention will combine effective enhancement of polypeptide uptake with (1) a lack of toxicity at the concentrations used and (2) good powdery properties, ie, the lack of a sticky or waxy consistency in the solid state. The toxicity of a given substance is

865 T3 8 can be assayed by classical standard means, such as by MTT analysis, for example as described in the citation of Int. J. Pharm., 65 (1990), 249-259 . The powdery properties of a given substance can be ascertained from published data about the substance, or empirically.

A very promising type of enhancing agent is the salt of a fatty acid. The sodium salt of saturated fatty acids having a carbon chain length of 10 (ie sodium caprate), 12 (sodium laurate), and 14 (sodium myristate) have been found to be I will behave well in the method of the invention. The potassium and lysine salts of caepric acid have also been found to be effective in the method of the invention. If the carbon chain length is shorter than about 10, the surface activity of the surfactant may be too low, and if the chain length is longer than about 14, the decreased solubility of the fatty acid salt in water limits. Its utility.

Most preferably, in the present invention, the substance that enhances the absorption of the polypeptide in the lower respiratory tract is sodium caprate.

Different ions of opposite sign can change the solubility of the salt of a saturated fatty acid in water, such that an enhancing agent having a carbon length other than 10-14 May prove to be even more advantageous than the specifically mentioned enhancing agents . in the above. The salts d Unsaturated fatty acids can also be useful in the present invention, since they are more soluble in water than salts of saturated fatty acids, and therefore they can have a longer chain length than the latter and still maintain the solubility that is necessary for a successful polypeptide absorption enhancing agent.

All of the bile salts and bile salt derivatives that were tested (sodium salts of ursodeoxycholate, taurocholate, glycocholate and taurodihydrofusidate) effectively enhance the absorption of polypeptides in the lungs.

Phospholipids were also tested as enhancing agents. A single-chain phospholipid (lysophosphatidylcholine) was found to be an effective enhancing agent, whereas the two-chain phospholipids (dioctanoel-phosphatidylcholine and didecanoel-phosphatidylcholine) were not. This can be explained by the fact that Two-chain phospholipids are much less soluble in water than their single-chain counterparts; However, it is reasonable to expect that shorter chain length two-chain phospholépids, having greater water solubility than their longer chain counterparts, will be useful as enhancing agents in the present invention, thus they can use both single chain and two chain phospholipids.

One glycoside, octyl glucopyranoside, was tested as an enhancing agent in the present invention and was found to have some absorption enhancing properties. It could be expected

ES 2 162 865 T3 rar also that other alkyl glycosides such as thioglucopyranosides and maltopyranosides exhibit absorption enhancing properties in the methods of the present invention.

Cyclodextrins and their derivatives effectively enhance absorption by v the nasal passage, and they can exert their function similarly in the lungs. Dimethyl-β-cyclodextrin has been tested and found to have an absorption enhancing effect.

Other potentially useful surfactants are sodium salicylate, sodium 5-methoxy salicylate, and naturally occurring surfactants such as glycyrrhizonic acid salts, saponin glycosides and acyl carnitines.

For ionic enhancing agents (eg the anionic surfactants described above) the nature of the ion with the opposite sign may be important. The particular counter sign ion that is selected can influence the powdery properties, solubility, stability, hygroscopicity, and local / systemic toxicity of the enhancing agent or any formulation containing the enhancing agent. It may also affect the stability and / or solubility of the polypeptide with which it is com bine. In general, certain monovalent metal cations, such as those of sodium, potassium, lithium, rubidium, and cesium, are expected to be useful as counter ions for anioonic enhancing agents. Ammonia and certain organic amines constitute another class of cations that are expected to be appropriate for use with anioonic enhancing agents having a carboxylic acid moiety. Examples of such organic amines include ethanolamine, diethanolamine, triethanolamine, (2-amino-2-methyl-ethyl) -amine, betaones, ethylene-diamine, N, N-dibenzyl-ethylene-tetraamine, arginine, hexamethylene- tetraamine, histidine, N-methyl-piperidine, lysine, piperazine, spermidine, spermine, and tris (hydroxymethyl) aminomethane.

Since effective enhancement of polypeptide uptake in the lungs was observed for a number of the enhancing agents tested, it is expected that many more of these also exert their potential. they worked this way. Starch-based microspheres effectively enhance the bioavailability of a polypeptide delivered through nasal membranes and were tested as an enhancing agent in the methods of the invention. Although these proved to be of little use in delivering via the pulmonary route in the one animal model used here, it is believed that this was mainly due to technical difficulties which, if overcome, could lead to a satisfactory delivery through the lungs. the pulmonary route.

Chelating agents are a class of enhancing agents that are believed to work by binding calcium ions. Since calcium ions help maintain the dimensions of the space between cells and further reduce the solubility of a polypeptide, fixation of these ions would theoretically lead to both an increase in the solubility of the polypeptides and an increase in the solubility of the polypeptides. the paracellular permeability of these polypeptides. Although one of the chelating agents tested, the sodium salt of ethylene diamine tetraacetic acid (EDTA), was found to be ineffective in enhancing insulin absorption in the rat model that was tested, other chelating agents Chelates that bind calcium ions may prove to be more useful.

Polypeptide and enhancing agent ratios

The relative proportions of the polypeptide and the enhancing agent can be varied as desired. Sufficient amount of the enhancing agent must be present to allow efficient absorption of the inhaled polypeptide; However, the amount of the enhancing agent should be kept as low as possible in order to minimize the risk of unfavorable effects caused by the enhancing agent. Although each of the particular combinations of a polypeptide and an agent int Ensifier should be tested to determine ooptimal proportions, it is expected that, to achieve acceptable absorption of the polypeptide, more than 10% of the mixture of a polypeptide and an enhancing agent should be the enhancing agent; For most types of enhancing agents, the proportion of enhancing agent should be greater than 15% or greater than 20%, and preferably it will be between 25% and 50%. The preferred ratio for each combination of a polypeptide and an enhancer agent (or a polypeptide, an enhancer agent, and a diluent) can easily be determined by a person having ordinary experience in the field of classical pharmacological technique, based on on criteria such as a consistent and efficient delivery of the optimal dosage, a minimization of side effects, and an acceptable absorption rate.

No additional ingredients are needed for the action of the preparation, but these can be included, if desired. For example, the amount of powder that makes up a single dose of a given combination of a polypeptide and a surfactant could be increased (eg, for use in an inhaler device that by design requires a greater volume of powder per each dosage) by diluting the powder with pharmacoeutically acceptable diluents. Other additives may be included to facilitate processing or to improve the powdery properties or stability of the preparation. A flavoring agent could be added such that the proportion of the powder inevitably settling in the mouth and throat would serve to give the patient positive information that a dose had been delivered from the inhaler device. Any such additive should have the following properties: (a) it should be stable and not adversely affect the stability of the polypeptide or the intensifying agent. icador; (b) it should not adversely interfere with the absorption of the polypeptide; (c) it must have good properties as powders, as this expression is understood in pharmaceutical techniques; (d) it must not be hygroscopic; and (e) it must not have adverse effects on the airways

ES 2 162 865 T3 (respiratory) in the concentrations used. Useful types of such additives include mono-, di-poly-saccharides, sugar alcohols, and other polyols: for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, and a starch. Since reducing sugars, such as lactose and glucose, have a tendency to complex with proteins, non-reducing sugars such as raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, and a starch may be preferred additives for use. in the present invention. Said additives can constitute any proportion between 0% (that is, without no additives) up to almost 100% of the total preparation.

In a preferred embodiment, this invention provides a therapeutic preparation of a pharmacoeutically active polypeptide and of a substance that enhances the absorption of said polypeptide in the lower respiratory tract, the preparation of which is in the form of a dry powder preparation that is appropriate for inhalation, of which at least 50% by mass consists of (a) particles having a diameter less than 10 µm (micrometers) or (b) agglomerates of said particles; In another preferred embodiment, the invention provides a therapeutic preparation comprising a pharmacoeutically active polypeptide, a substance that enhances the absorption of the polypeptide in the lower respiratory tract, and a pharmaceutically acceptable carrier, whose preparation is in the form of a dry powder suitable for inhalation in which at least 50% by mass consists of (a) particles that have a diameter of less than 10 pm (micrometers) or (b) agglomerates of said particles; and in a further preferred embodiment this invention provides a therapeutic preparation comprising as active compounds (A) a pharmaceutically active polypeptide and (B) a substance that enhances the absorption of said polypeptide in the lower respiratory tract, in which at least 50% of the total mass of the active compounds (A) and (B) consists of particles having a diameter of less than 10 pm (micrometers), and a pharmacoeutically acceptable vehicle, the preparation of which is in the form of a dry powder preparation that is suitable for inhalation, in which an ordered mixture can be formed between the active compounds and the pharmaceutically acceptable carrier.

The described powder preparation could be produced in various ways, using conventional techniques. In many cases, the purified polypeptide can be obtained from commercial sources. Alternatively, the polypeptide of interest can be purified from a naturally occurring source using classical biochemical techniques, or it can be obtained by expression of prokaryotic or eukaryotic cells treated by genetic engineering so that they contain a nucleotide sequence that encodes the polypeptide and has appropriate expression control sequences linked to it (including a transgenic animal treated by genetic engineering so that produce the desired peptide or proteon, for example in your milk).

These methods are classic in the field of technology (eg see Sambrook et al., Molecular Cloning: A Laboratory Manual (= molecular cloning, laboratory manual); Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Peptides (ie, polypeptides having 30 or fewer amino acid residues) can be synthesized easily by known chemical means.

The absorption enhancing agents described above are also generally available from commercial sources, or can be produced using published methods. For ion enhancing agents, the ion of the opposite sign associated with one enhancing agent can be replaced by another, if desired, using classical ion exchange techniques.

To produce the generally described powder preparation it would be necessary to micronize the powder in a suitable mill, eg ex. a jet mill, at some point during the process, in order to produce primary particles in a range of sizes that is appropriate for maximum deposition in the lower respiratory tract (ie, below 10 pm). For example, powders of the polypeptide and enhancing agent can be dry mixed and then micronized the substances together; alternatively, Substances can be micronized separately, and then mixed. When the compounds to be mixed have different phosphoric properties such as hardness and brittleness, resistance to varicose and micronization may require different pressures to disintegrate to appropriate particle sizes. When they are micronized together, therefore, the obtained particle size of one of the components can be unsatisfactory. In such a case it could be advantageous to micronize the different components separately and then mix them.

It is also possible to first dissolve the components in a suitable solvent, eg water, to obtain mixing at the molecular level. This procedure also makes it possible to adjust the pH value to a desired level, for example to improve the absorption of the polypeptide. The pharmacoeutically accepted limits of pH values ​​from 3.0 to 8.5 for inhalation products should be taken into account. Account, since products with a pH value outside these limits can induce irritation and constriction of the airways. To obtain a powder, the solvent must be removed by a procedure that retains the biological activity of the polypeptide. Suitable drying methods include concentrating in vacuo, drying in an open space, spray drying and freeze drying (= lyophilize). Temperatures above 40 ^or C for more than a few minutes should generally be avoided, as some degradation of certain polypeptides may occur. Following the drying operation, the solid material, if necessary, can be crushed to obtain a coarse powder and then, if necessary, it can be micronized.

If desired, the micronized powder can be treated to improve flow properties, eg.

ES 2 162 865 T3 by dry granulation for form spherical agglomerates that have superior handling characteristics, before this powder is incorporated into the intended inhaler device. In such a case, the device could be configured to ensure that the agglomerates are substantially deagglomerated before exiting the device, such that the particles entering the respiratory tract of the patient are well within the desired size range. When orderly mixing is desired, the active compound can be treated, for example by micronization, in order to obtain, if desired, particles within a particular range of sizes. The carrier can also be treated, for example, to obtain a desired size and desirable surface properties, such as a particular surface-to-weight ratio, or a certain roughness, and to ensure optimal adhesion forces in the ordered mix. Such phosphical requirements of a mixture or Nothing is well known, as are the various means of obtaining an orderly mixture that meets these requirements, and can be easily determined by a skilled person according to particular circumstances.

A preferred apparatus for inhalation should have the following design features: protection of dust from moisture and no risk of occasional large doses; in addition, the highest possible number of the following is desired: protection of dust from light; high respirable fraction and high stool in the lungs in a wide range of flow rates; low deviation of the dose and the respirable fraction; low retention of dust in the mouthpiece - this is particularly important for a multi-dose inhaler where the polypeptide retained in the mouthpiece could degrade and then be inhaled in conjunction with subsequent doses; low adsorption to the surfaces of the inhaler; a flexibility in the size of the doses; and a low resistance to inhalation. The inhaler is preferably a single-dose inhaler, although a multi-dose inhaler, such as a multi-dose, breath-actuated, dry powder inhaler may also be employed for multiple use. Preferably, the inhaler used is a unit dose, breath actuated dry powder inhaler for single use.

A number of dry powder formulations containing a polypeptide and various enhancing agents have been prepared and tested in in vivo analysis, and these are described below. Also described is an in vitro assay that is useful for testing combinations of a polypeptide and an enhancing agent.

Example 1

In vitro method of determining the utility of particular polypeptides for the present invention

An in vitro analysis or classical using an epithelial cell line, CaCo-2 (available through the American Type Culture Collection (ATCC), Rockville, MD, USA), has been developed to evaluate the ability of various agent compounds. enhancers to promote the transport of markers through a monolayer of epithelial cells, as a model for the epithelial coellular layer that functions in the lungs to separate the alvoeoli from the pulmonary blood supply.

In this analysis, the enhancing agent and the polypeptide or other marker are dissolved in an aqueous solution in different proportions and / or concentrations, and applied to the apical face of the monolayer of coellules. After incubation for 60 min to 37 ^or C and a RH (relative humidity) of 95%, the amount of the marker existing on the basolateral face of the cells is determined, for example, by using a marker that is marked diactively.

For the intensifying agent tested, sodium caprate, the amount of the marker (mannitol, MW 360) that appears on the basolateral side is dependent on the concentration of the enhancing agent used, at least up to 16 mM sodium caprate (Figure 1). . This is true even when the insulin polypeptide is added to the mixture of enhancer and mannitol (insulin sodium caprate 1: 3, by weight) (Figure 2). It was also found that this concentration of sodium caprate (16 mM) favors the absorption through the cell monolayer of two low molecular weight polypeptides, insulin (MW 5,734) and vasopressin (MW 1,208). The amount of insulin that had passed through the monolayer was doubled in the presence of 16 mM sodium caprate, compared to the amount that occurred in the absence of any enhancing agent; the amount of vasopressin that had been absorbed through the monolayer increased 10-15 times compared to rationed with the amount that was present in the absence of any enhancing agent.

In contrast, no increase in the transport rate was observed for larger proteins such as cytochrome C (MW 12,300), carbonic anhydrase (MW 30,000) and albumin (MW 69,000) when these were tested with up to 16 mM of sodium caprate . It is expected that with higher concentrations of sodium caprate, the permeability of the cells will be further increased, allowing the transport of larger polypeptides; However, the potential cytotoxicity of sodium caprate can preclude the use of substantially higher concentrations of this particular enhancing agent.

Other enhancing agents may allow the transport of larger polypeptides; These can also be tested in this in vitro model of epithelial cell permeability, which can be used as a screening tool for testing. Any desired combination of a polypeptide and an enhancing agent is readily available for utility in the methods of the invention.

Example 2 Methods for Selecting Enhancing Agents Useful for the Present Invention

Each of the compounds listed in Table I was tested for their ability to enhance the uptake of a polypeptide (insulin) in the rat model. The results obtained

ES 2 nests with insulin are taken as indicative of the potential of an enhancing agent to enhance the absorption of other polypeptides.

Various forms of insulin were used in the different tests: recombinant human, semi-synthetic human, or bovine. Each formulation was prepared as before by drying and treating the solution of a mixture of insulin and the enhancing agent or of a mixture of insulin, the enhancing agent and lactose to produce an inhalable powder. Dust it was administered to rats by inhalation, and subsequently the blood glucose levels of the rats were monitored as a measure of insulin uptake. These levels were compared to the corresponding values ​​obtained with rats that had inhaled formulations with insulin without any enhancing agent.

The same in vivo model system could be used to test any peptide or protein that is considered to be useful in the methods of the invention, providing by the same inhalation method a formulation containing the desired peptide or protein in combined with an enhancing agent, and by analysis of the desired peptide or protein concentration in the systemic circulation of the tested animal (eg. by classical immunoassays or biochemical analyzes, they are still appropriate for the peptide or protein under consideration).

TABLE I

<td> Substance &lt;PT XT&gt; Enhancing agent: insulin: lactose </td><td> Effect </td><td> Octylglucopyranoside </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 glycollate </td><td> 4:4:92</td><td> +</td>

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

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

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

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

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

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

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

+ there is an effect, that is, the intensifying agent 865 T3 16 dor provides a significant decrease in the level of glucose in the blood

- there is no effect or there is a very small one ( +) there is an effect,not as marked as or in + Example 3

Therapeutic preparation according to the invention

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

The resulting powder was administered intratracheally to rats and the collection of hGH was compared with that of a powder, MMD of 9.6 comprising hGH and mannitol in the same proportions and prepared in the same manner as above.

The results indicated an improvement in the collection of hGH in the formulation that included sodium caprate, compared to the collection of the formulation that lacked the enhancing agent.

Example 4 Preparation containing the polypeptide insulin

Insulin is used here as indicative of other polypeptides according to the present invention. nto.

A biosynthetic human insulin (53 g) was micronized in an Airfilco Jet Mill (Registered Trade Mark, Airfilco Process Plant Limited), with nitrogen put under pressure (supply pressure 7 bar, chamber pressure 5 bar) to a medium diameter in 2.4 micrometer mass.

Sodium caprate (170 g) was micronized in an Airfilco Jet Mill (MR) with nitrogen pressurized (feed pressure 5 bar, chamber pressure 3 bar) to a mass median diameter of 1.6 micrometers.

Micronized biosynthetic human insulin (45 g) and sodium caprate (14.26 g) were dry mixed according to the following procedure: Half of the insulin was added to a mixing device comprising a mixing cylinder with a volume of 4.4 liters divided, by a sieve with a width of 1 mm, in two compartments, with a metal ring in each compartment to aid in mixing and shaking. The sodium caprate and finally the rest of the insulin. The mixing cylinder was closed, rotated 180 degrees, and mounted on a motor-driven stirrer. The motor was turned on and stirring continued for approximately two minutes, until all of the insulin and sodium caprate had passed through the sieve. The motor was switched off and the mixing cylinder was rotated 180 degrees, mounted back on the agitator and agitation was again carried out until all of the powder had passed through the screen. This process was repeated eight more times to give a total mixing time of approximately 20 minutes.

The preparation thus obtained was administered to 5 dogs by inhalation, at a dosage level of 1 U / kg, and the plasma insulin level was

ES 2 162 865 T3 18 determined at various times after administration.

The results obtained were compared with the plasma insulin levels obtained when a biosynthetic insulin, micronized as above to a mass median diameter of 2.4 microns, was administered to five dogs in the same manner and at the same dosage levels, and with the plasma insulin levels that were obtained when five dogs were administered a therapeutic preparation of insulin and sodium caprate in a ratio of 90:10 in the same way and at the same dosage levels as before. In this case, the therapeutic preparation was produced as follows: A human semisyntotic insulin was filtered through a gel to reduce the zinc content from 0.52% to 0.01% relative to the insulin content. Insulin (4.5 g) and sodium caprate (0.5 g) were dissolved in water (232 ml). The solution was stirred until clear and the pH was adjusted to 7.0. The solution was concentrated by evaporation at 37 ^or C for a period d e 5 time of about two days. The solid cake obtained was crushed, and sieved through a 0.5 mm sieve, and the resulting powder was micronized through a jet mill to the form of particles with a mass median diameter of 3.1 micrometers.

The results of these comparisons are presented in Figure 3 (p = 0.0147 for the difference between 75:25 and 100: 0). The results demonstrate some improvement in insulin bioavailability with the 90:10 formulation, and a dramatic improvement in insulin bioavailability with the 75:25 preparation including sodium caprate, compared to insulin alone.