LT3445B - Therapeutic preparation for inhalation - Google Patents

Abstract

respiratory alleviation preparations. SUBSTANCE: composition in the form of dry powder contains pharmaceutically-active polypeptide associated with surfactant-type promoter. The latter increases absorption of polypeptide in lower respiratory pathways. Its amount represents more than 10% of the total weight of polypeptide and promoter. At least 50% of powder consists of particles with diameter lesser than or equal to 10 mcm, or agglomerates of these particles. Inhaled composition provides system transportation of therapeutically-active peptides and proteins into lungs and absorption of drugs. EFFECT: enhanced treatment efficiency. 31 cl, 3 dwg, 1 tbl, 4 ex

Description

The present invention relates to a therapeutic insulin preparation for inhalation.

Insulin plays a key role in the metabolism of carbohydrates, fats and proteins in the body. Diabetes mellitus (diabetes, diabetes mellitus) (commonly referred to as diabetes mellitus) is a disease characterized by deregulation of metabolism, especially glucose metabolism. In normal individuals, elevated blood glucose levels (such as levels immediately after a meal) stimulate pancreatic beta cells to secrete insulin, a peptide hormone, into the blood. Insulin binds to insulin receptors on many cell types, especially muscle cells, thereby signaling to cells that they need to increase glucose uptake into cells. When blood glucose returns to pre-meal levels, insulin levels in the blood also fall. In the absence of insulin, your blood sugar level may rise to dangerous levels (conditions known as hyperglycaemia), which can lead to death. Excessive insulin levels can cause an abnormal decrease in blood glucose (hypoglycaemia), which is also dangerous and can be fatal. The existence of feedback circuits in normal individuals that regulate insulin secretion and elimination from the major circulatory system prevents both hyperglycemia and hypoglycemia.

Diabetes is a disease that affects about 3% of the Swedish population. Of these 3%, approximately 20% suffer from type 1 diabetes and the rest from type 2 diabetes.

Type I diabetes or insulin dependent diabetes mellitus (IDDM) usually begins in childhood. It is characterized by atrophy of the pancreatic beta cells, which results in a reduction or stopping of insulin production, and the patient's life depends solely on exogenous insulin.

More common type 2 diabetes or non-insulin dependent diabetes mellitus (NIDDM) usually develops in patients over 40 years of age. These patients may have, at least initially, normal or even higher levels of insulin in their blood, but may have abnormally low glucose uptake into cells in response to insulin. Although type II diabetes can often be cured by regulating the patient's diet, administration of exogenous insulin as a supplement to the secreted patient's beta cells may also prove necessary.

Active doses of insulin cannot be administered orally as it is rapidly degraded by gastrointestinal and low gastric enzymes before it reaches the bloodstream. The standard route of administration is the subcutaneous injection of isotonic insulin solution by the patient. The need for injections causes many difficulties and inconveniences to most patients and may cause local reactions at the injection sites. In addition, insulin administration produces an abnormal plasma concentration profile. This abnormal plasma concentration profile is undesirable and increases the risk of side effects associated with long-term treatment of diabetes mellitus.

These disadvantages make it necessary to look for a form of insulin that can be administered otherwise than by injection. In an attempt to discover this form of insulin, various suggestions have been made. For example, products have been proposed for nasal, oral, or rectal administration, with particular emphasis on nasal products. However, nasal administration is problematic and yields only very low bioavailability. In recent years, interest in the systemic administration of active pulmonary drugs has intensified, including some studies on pulmonary insulin delivery. Daygums of these were intended to be administered via pulmonary solutions or suspensions, such as nebulizers and metered dose inhalers, but all had limited success.

We have discovered that insulin can also be incorporated into a dry powder formulation for inhalation, as well as incorporation of a substance that accelerates the absorption of insulin into the lungs, and from this formulation insulin can be absorbed at a therapeutically acceptable rate and amount. The term accelerated absorption means that the amount of insulin absorbed into the major circulatory system in the presence of a promoter is greater than the amount absorbed in the absence of the promoter.

Thus, the present invention provides a therapeutic preparation comprising the active compounds (A) insulin and (B) a substance which improves insulin absorption in the lower respiratory tract and is in the form of a dry powder suitable for inhalation containing at least 50% of the total weight of the active compounds comprising (a) primary particles having a diameter of less than about 10 microns, e.g. between 0.01 and 10 microns, preferably between 1 and 6 microns, or (b) agglomerates of said particles.

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 so that at least 50% of the powder as a whole is optionally composed of agglomerated primary particles less than about 10 microns in diameter; on the other hand, the bulk of the carrier may be composed of much larger particles (coarse particles), which may result in the formation of an ordered mixture 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 a diameter of more than 20 microns, for example 60 microns. Above these thresholds, the diameter of the coarse particles is not critical, and coarse particles of various sizes may be used, as required by the practical requirements of the particular recipe.

Although there is no requirement for the coarse particles in the ordered mixture to have the same size, it is preferable for the coarse particles in the ordered mixture to have a similar size. More preferably, the coarse particles have a diameter of 60-800 microns.

Thus, in a separate embodiment, the present invention provides a therapeutic formulation of insulin and a substance which improves lower respiratory tract insulin delivery in a dry powder form suitable for inhalation and comprising at least 50% by weight of particles having a diameter of less than about 10%. micron, or (b) agglomerates of said particles; in another particular embodiment, the invention provides a therapeutic preparation consisting of insulin, a substance which improves the absorption of insulin in the lower respiratory tract, and a pharmaceutically acceptable carrier, the preparation being in the form of a dry powder for inhalation and at least 50% by weight having a diameter of less than about 10 microns to about 3445 B; or (b) agglomerates of said particles; and in still another particular embodiment, the present invention provides a therapeutic formulation comprising the active compounds of (A) insulin and (B) which improves insulin absorption in the lower respiratory tract, and comprising at least 50% by weight of the total active compounds (A) and (B). 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 fine mixture between the active compounds and a pharmaceutically acceptable carrier.

In more preferred embodiments, at least 60%, such as at least 70%, or at least 80%, and more preferably at least 90%, of the total weight of the active compounds, (A) and (B), consisting of particles having a diameter of less than about 10 microns; such particulate agglomerates, and wherein the dry powder formulation contains a carrier not intended to be incorporated into the admixed mixture, preferably at least 60%, such as at least 70%, or at least 80% and more preferably at least 90% by weight of the total dry powder Diameters less than or equal to about 10 microns or from agglomerates of such particles.

Although dry powder for inhalation, with or without a pharmaceutically acceptable carrier, may contain particulate agglomerates as mentioned above, any agglomerates must be substantially disintegrated during inhalation to form a powder having a particle size of less than about 50% 10 microns. Agglomerates can be formed by a controlled agglomeration process or simply by close contact between the powder particles. In any case, it is important that the agglomerates are able to disintegrate, for example by mechanical inhalation or otherwise, into the above-mentioned particles. In general, it is preferable that agglomerates do not form in the ordered mixtures. In the case of a neat mixture, the active compounds should be released from the large particles by inhalation or mechanical means in the inhaler or other means, then the active compounds are deposited in the lower respiratory tract and the carrier particles in the mouth.

Various forms or derivatives of insulin can be used in the present invention. For example, bovine, porcine, or biosynthetic or equilibrium human insulin may be used, or a biologically active human insulin derivative (modified insulin), for example, substituted with certain amino acids as set forth in Brage et al. Diabetes care 13: 923, 1990. Modified insulin is developed to improve various properties, such as improved stability or better pharmacokinetic form (i.e., better absorption through the epithelial membrane). Insulin must be low in zinc because zinc lowers the solubility of insulin, probably reducing the rate of insulin absorption, and also because zinc can form an insoluble precipitate with some of the excipients that can be used in the present invention. In addition, the insulin should be in the form of a dry powder, which may rapidly dissolve in aqueous solution.

Substances that improve pulmonary insulin absorption, hereinafter referred to as enhancers, may be any of a series of compounds that enhance absorption through the lower respiratory tract epithelial cell layer 1 adjacent pulmonary circulation vessels. The promoter can do this in several possible ways:

(1) Improvement of intracellular insulin conductance by inducing structural changes in solid junctions between epithelial cells.

(2) Improving extracellular insulin permeability by interacting with membrane proteins or lipids or by extracting membrane proteins or lipids, thereby affecting membrane integrity.

(3) Interaction between promoter and insulin, which increases the solubility of insulin in aqueous solution. This can be accomplished by preventing the formation of insulin units (dimers, trimers, hexamers) or by dissolving the insulin molecules in the promoter micelles.

(4) Reduction or dissolution of the viscous barrier lining the alveoli and the lungs, thereby removing the surface of the epithelium to directly absorb insulin.

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 thought 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, to be effective, the 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). The isoelectric pH of the insulin is 5.5. Insulin has a negative net charge at pH 7.4. This can cause electrostatic repulsion between insulin molecules, which in turn prevents aggregation and thus increases solubility. If the surfactant also has a negative charge, it can interact with insulin, for example, hydrophobically, causing an additional push between insulin molecules. Thus, 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 insulin in the monomeric state.

A variety of different compounds potentially useful as promoters in the methods of this invention were tested on rats as described in Example 5 below. Other substances with known absorption enhancers or physical properties that make them suitable candidates for the methods of the present invention can be readily tested by one of ordinary skill in the art for such in vivo assays or, alternatively, in vitro assays as described in Example 6.

A combination of two or more stimulants may 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 the effective improvement of insulin absorption with (1) the absence of toxicity at the concentrations used and (2) the good properties of the powder, e.g. lack of adhesive or waxy consistency in solid state. The toxicity of the material may be tested by standard methods such as MTT assays, such as those described in Int. J. Pharmac., 65 (1990), 249-259. The powder properties of a given substance can be determined from published material data or empirically.

One of the most promising promoters is the fatty acid salt. Sodium salts of saturated fatty acids having carbon chain lengths of 10 (i.e., sodium caprate), 12 (sodium laurate), and 14 (sodium myristate) have been found to work well in the process of the present invention. 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, its reduced water solubility limits its usefulness.

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

Thus, in a particularly preferred embodiment of the invention, the present invention provides a pharmaceutical composition containing the active compounds, (A) insulin and (B) sodium caprate, in the form of a dry powder for inhalation containing at least 50% of the total active compounds, (A) and (B). the masses comprising (a) primary particles having a diameter of less than about 10 microns, for example between 0.01 and 10 microns or better between 1 and 6 microns, or (b) agglomerates of these particles; a particularly particularly preferred embodiment of the present invention provides:

a therapeutic preparation of insulin and sodium caprate in the form of a dry powder for inhalation, comprising at least 50% by weight of (a) primary particles of less than 10 microns in diameter, or (b) agglomerates of these particles;

a therapeutic preparation consisting of insulin, sodium caprate, and a pharmaceutically acceptable carrier, in the form of a dry powder for inhalation, containing at least 50% by weight of a particle of diameter less than about 10 microns or (b) ) agglomerates of said particles; and a therapeutic preparation comprising the active compounds, (A) insulin and (B) sodium caprate, and wherein the preparation comprises at least 50% by weight of the total active compounds, (A) and (B), of particles having a diameter of less than about 10. microns, and a pharmaceutically acceptable carrier, the preparation is in the form of a dry powder suitable for inhalation, which may form a fine mixture between the active compounds and the pharmaceutically acceptable carrier.

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 insulin absorption.

Bile salts and bile salt derivatives have been tested as promoters in the process of the present invention. All tested (ursodeoxycholate, taurocholate, glycocholate, and taurodihydrofuzidate sodium) are effective in improving pulmonary insulin absorption.

dual water in water for

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

A single glycoside, octylglucopyranoside, has been tested as a promoter of the present invention and has been found to have some such promoter 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 effectively improve the absorption of insulin in the nose and could have similar effects in the lungs. Dimethyl-β-cyclodextrin was tested in the process of the present invention and had an absorption-enhancing effect.

Other potentially useful surfactants are 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 / total toxicity of the promoter or any formulation containing the promoter. It may also affect the stability and / or solubility of the insulin to 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-amino-2-methylamine, betaines, ethylenediamine, N, N-dibenzylethylenetetramine, arginine, hexamethylenetetramine, histidine, N-methylpiperidine, lysine, piperazine, spermidine, spermidine, ) aminomethane.

was

Although

Because effective enhancement of insulin absorption in the lungs has been found with many of the promoters tested, more can be expected to work in similar ways. The starch microspheres are effective in improving the bioavailability of insulin delivered through the nasal membrane and, when tested as a promoter in the methods of the present invention, have proven to be of little use in the lung administration of the animal model used herein; once overcome, pulmonary delivery may be successful. Chelates are a class of promoters that are thought to work by binding calcium ions. Because calcium ions help to maintain between ¹ : between cellular measurements and the additive, reducing the solubility of insulin, binding of these ions should, in theory, both increase insulin and increase cellular solubility and permeability. Although one investigated chelate, insulin ethylene LT 3445 B diamintetraacetic acid sodium salt (EDTA), has been shown to be ineffective in improving insulin absorption in a tested rat model, other calcium-binding chelating agents may prove to be more valuable.

In general, the ratio of insulin to stimulant should be kept as low as possible within the range where rapid and effective insulin absorption is achieved. This is important in order to minimize the adverse effects of the promoter, both locally and systematically. The optimal ratio of insulin to stimulant can be determined by testing different proportions of each given stimulus in in vivo models as described herein. For example, insulin was combined with sodium caprate in the following weight / weight ratios: 50/50, 75/25, 82.5 / 17.5, and 90/10. Significant improvements in insulin absorption were obtained with 50% and 25% sodium caprate; 10% gave a slight increase in absorption and 17.5% gave intermediate results. This indicates that the lowest effective concentration of sodium caprate, when used in the methods of the present invention, is approximately 15-25% and possibly 20-25%. Other promoters may have higher or lower optimum concentrations relative to insulin, and therefore each individual promoter must be tested individually. However, based on the results described above, it should be expected that the optimum proportions of the promoter, which is a surfactant, will generally be between 10 and 50% of the insulin / promoter mixture, such as between 15 and 50% as between 25% and 50%. It should be noted that the proportions given above represent proportions related solely to insulin and do not include any carrier or other additive which may be added, for example, to improve the powder properties of the preparation.

The amount of insulin absorbed according to the present invention may be significantly higher than the amount absorbed without the stimulant.

Example 4 shows that the therapeutic preparation according to the present invention exhibits a bioavailability much greater than three times the inhaled preparation from insulin alone.

More preferably, the amount of insulin absorbed according to the present invention is reliably greater (p <0.05) than the amount absorbed without the enhancer.

As stated above, additional substances commonly used in therapeutic preparations, such as pharmaceutically acceptable carriers, may be incorporated into a therapeutic preparation according to the present invention. Further, for example, the powder is reconstituted to an amount that can be administered using a particular powder to facilitate the preparation of the desired powder inhaler; processing; improved powder properties of the preparation; increase the stability of the preparation, for example, by the use of antioxidants or pH-adjusting compounds; or give the preparation a taste. Any of these additives must not adversely affect the stability of the insulin or the absorption enhancer or adversely affect the absorption of the insulin. It must be stable, non-hygroscopic, have good powder properties, and do not adversely affect the respiratory tract. Among the 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, trechalose, sucrose, mannitol, and starch may be more inventive. Depending on the total amount of such accessories, the accessories for this inhaler can vary within wide limits. There is little or no need for additives in some circumstances, whereas an inhaler that requires a large amount of powder additives can make a very large amount of additives necessary for a person skilled in the art to determine.

volume, the portion of the preparation can be readily inhaled with the aid of a generally powdered inhaler

A convenient way to deliver the powder to the patient's lungs is to use a dry powder inhaler. Many such devices designed to deliver anti-asthmatic and anti-inflammatory drugs to the respiratory system are already on the market. A more acceptable dry powder inhaler must 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; keep small in mouthpiece; must have low adsorption to surfaces; should have flexibility in dose selection; and low resistance to inspiration. Preferably, the inhaler is a single dose, although multiple dose inhalers, such as, preferably, a multiple dose, reusable, dry powder inhaler, may also be used. It is preferable to use a single dose, single use, dry powder inhaler, started by inhalation.

The powder formulation described may be prepared in several ways using standard techniques. At some stage in the process, the active compounds and, where appropriate (i.e., no neat mixture is anticipated), any carrier may need to be crushed to micron particles using suitable mills, such as a jet mill, to provide primary particles in the range of size. maximum deposition in the lower airways (ie less than 10 µm). For example, it is possible to mix dry insulin and promoter powders and, where appropriate, carrier, and then to comminute the materials to micron particles; otherwise, the materials may be crushed individually and then blended. When mixed compounds have different physical properties, such as strength and brittleness, the micron particle resistance varies, and different pressures may be required to decompose them to the appropriate particle size. Therefore, the particle size 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 dissolve the first constituents, including those which are not intended to be an ordered mixture, in a suitable solvent, such as water, to mix at the molecular level. This procedure also allows you to adjust the pH value at the desired level. Nasal absorption of insulin is known to depend on the pH of the preparation and increases with up or down movement from the insulin isoelectric point of about 5.5. However, insulin may be less stable at pHs much higher or lower than

5.5, and furthermore, pharmaceutically acceptable limits, from pH 3.0 to 8.5 for inhaled products, should be kept in mind, since products with pH outside these limits may cause respiratory irritation and contraction. To obtain a powder, the solvent must be removed in such a way as to maintain the biological activity of the insulin. Suitable drying techniques include vacuum evaporation, open drying, spray drying and freeze drying. In general, temLT 3445 B temperatures above 40 ° C should be avoided for more than a few minutes as insulin may degrade to some extent. 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 most of the particles entering the patient's 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 may 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 degree of roughness, to provide optimum adhesive forces in the cured mixture. Such physical requirements for neat mixtures are well known, as there are a variety of means to obtain arranged mixtures that meet the above requirements, and can be readily determined by one skilled in the art in light of the particular circumstances.

The invention will now be described by the following examples, which are intended to illustrate but not limit the scope of the invention.

EXAMPLES

COMPARATIVE EXAMPLE

Insulin therapeutic therapy without promoter

Semi-synthetic human insulin (Diosynth, 0.8 g) and water (150 ml) were placed in a beaker. The pH was lowered with 1 M HCl to pH 3, 4 and then raised with 1 M NaOH to pH 7.4 to dissolve the insulin.

Lactose (commercially available, 9.2 g) was added and the pH was again adjusted to 7.4. The solution was stirred until it became clear and slightly discolored and dried 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 pulverized into a micron particle having a diameter of about 2 microns.

EXAMPLE

Therapeutic preparations of insulin and sodium caprate;

ratio 75:25

Semi-synthetic human insulin (9.75 g) and water (250 mL) were placed in a beaker. The pH was lowered with 1 M HCl to pH 3.4 and then raised with 1 M NaOH to pH 7.4 to dissolve the insulin.

Sodium caprate (Sigma, 3.25 g) was added and the pH was again adjusted to 7.4. The solution was stirred until it became clear and slightly discolored and dried 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 pulverized into a micron particle having a diameter of about 2 microns.

EXAMPLE

Therapeutic preparations of insulin and sodium caprate with lactose; ratio 50:25:25

Semi-synthetic human insulin (7.5 g) was dissolved in water (150 mL) as in Example 1. Sodium caprate (3.75 g) and lactose (3.75 g) were added and the procedure of Example 1 was performed to obtain a powder consisting mainly of particles with a diameter of about a micron.

EXAMPLE

Therapeutic preparations of insulin and sodium caprate with lactose; ratio 4: 4: 92

Example 2 was performed with 0.5 g of equilibrium human insulin, 150 ml of water, 0.5 g of sodium caprate and 11.5 g of lactose.

INHALATION TESTS study sample preparation was used in inhalation studies in two dogs. The formulation was filled into a Wright Dust Feed inhaler and administered to dogs. The dose was 1 U./ kg (1 U. = one unit of human insulin = 35 µg of human insulin, 100%). Blood glucose and plasma insulin values were measured at various time intervals and the results are summarized in Tables 1 and 2 below.

TABLE

Blood sampling time after exposure (minutes) Blood glucose (mmol / L) Insulin concentration j (pU / ml) against 3 <) 6.70 0.5 3.6 120.66 5 2.8 194.47 30th 2.6 195.39 20th n.d 139.74 22.5 1.6 n.d 31st 2.0 73.42 45 1.7 47.49 59.5 1.7 36.21 89.5 2.3 19.28 120 3.0 14.58 240 45 _

Π TABLE

Blood sampling time after exposure (minutes) Blood glucose (mmol / L) Insulin concentration (pU / tnl) against 3.9 44.84 3 4.2 165.10 6th 4.3 158.28 12th 3.9 n.d 14th n.d 180.72 19th 3.0 133.75 30th 2.7 143.71 45 2.5 91.62 I 60 2.4 66.70 90 2.7 38.58 122 3.7 29.15 241 4.1 n.d 242.5 n.d 19.76 j

n. d = not set

The tables show that insulin sodium caprate significantly increases insulin levels in plasma and lowers blood glucose. Peak plasma insulin levels and trough blood glucose values were obtained after approximately 15 and 60 minutes, respectively.

study

Comparative and Example 1 preparations were each administered to four or five dogs using the Wright Dust Feed Inhaler at a constant dose of 1 U./kg. The effect of each preparation on plasma insulin and blood glucose levels was measured at various time intervals and the results are shown in Figs. 1 and 2. It was observed that the control without promoter did not substantially alter plasma insulin levels, whereas preparations containing both insulin and promoter increased plasma insulin levels from approximately 20 μυ / ml at zero time to 80 UU / ml after 15 mins after powder inhalation. Similarly, control animals had a maximum fall in blood glucose of 0.5 mmol / l after non-promoter insulin inhalation, whereas animals that inhaled insulin with a promoter had a transient fall of about 1.7 mmol / l, from approximately 4.0 mmol / l. 1 to about 2.3 mmol / l. Thus, insulin, in combination with promoter, sodium caprate, is rapidly absorbed and cleared from the major circulatory system, with a brief reduction in blood glucose levels. In contrast, the absorption of carrier insulin (lactose) but without the promoter was very low (p = 0.0002 insulin / caprate versus insulin / lactose).

study

Preparations 1-3 were tested at various doses in two dogs. The formulations were administered using a Wright Dust Feed Inhaler. Plasma insulin and blood glucose levels were measured at various time intervals after inhalation. The results are shown in Figs. 3-6 and shows that insulin adjusted to sodium caprate in various proportions is rapidly absorbed and peak value is reached after 20-30 minutes followed by a corresponding decrease in blood glucose levels. The results also indicate that inhalation of the insulin powder may result in a plasma profile that is more similar to the natural physiological profile than the profile obtained after subcutaneous injection of insulin.

EXAMPLE

Insulin and Sodium Caprate, 75:25; mixing of powdered particles to micron particles

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

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 micrometers was reached.

Micronized biosynthesized human insulin (45 g) and sodium caprate (14.26 g) were mixed dry according to this procedure. Half the insulin was fed into a mixing unit consisting of a mixing cylinder having a volume of 4.4 liters, divided by a sieve of 1 mm width into two parts and each containing a metal ring for mixing. 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 for a total of about 20 minutes.

The preparation thus obtained was administered by inhalation to 5 dogs using a Wright Dust Feed inhaler 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 with therapeutic insulin and sodium caprate, 90:10, was administered to five dogs in the same manner and in the same doses as above. In this case, the therapeutic preparation was prepared as follows. Gel filtration with human equilibrium 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 37C 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 grinder to particles having a median diameter

3.1 microns.

The results of these comparisons are shown in Figs. 9. The results showed some improvement in insulin uptake for the 90:10 formulation and a very marked improvement in the insulin uptake for the 75:25 formulation of the present invention compared to one insulin. (p = 0.0147 for a difference of 75:25 and 100: 0).

EXAMPLE

Selection of promoters

Each compound in Table III was tested for its ability to improve insulin uptake and thereby affect blood glucose levels in a rat model. Various forms of insulin have been used: human recombinant or human equilibrium or bovine. Each formulation was prepared as described in Examples 1-3 above by drying and treating the insulin / promoter solution to give 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 from rats inhaled insulin preparations.

TABLE III

Material Promoter: Insulin: Lactose Effect Octylglucopyranoside 4: 4: 92 (+) Sodium ursodeoxycholate 4: 4: 92 + Sodium taurocholate 4: 4: 92 + Sodium glycocholate 4: 4: 92 + Lysophosphatidylcholine 4: 4: 92 -1- Dioctanoylphosphatidylcholine 2: 4: 94 (+) Didecanoylphosphatidylcholine 4: 4: 94 - Sodium taurodihydrofuzidate 2: 4: 94 + Sodium caprylate 25: 75: 0 - Sodium caprate 10: 90: 0 (+) Sodium caprate 17.5: 82.5: 0 (+) Sodium caprate 25: 75: 0 + Sodium caprate 4: 4: 92 + Sodium laurate 25: 75: 0 (+) Potassium oleate 4: 4: 92 + Potassium Caprate 27: 73: 0 + Lysine caprate 35: 65: 0 + Myristate of sodium 30: 70: 0 + Dimethyl ^ -cyclodextrin 75: 25: 0 +

+ effect, i.e. the promoter produces a significant drop in blood glucose or no (+) effect, not as significant as +

EXAMPLE

Selection of promoters

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 the epithelial cell layer in the lungs that separates the alveoli from the pulmonary blood vessels. In this assay, various proportions and / or concentrations of promoter and insulin or other labeled substance are dissolved in aqueous solution and applied to the apical side of the cell monolayer. After 60 min. incubation at 37 ° C and 95% SD (relative humidity) determines the amount of labeled material on the basolateral side of the cells, e.g., using a radioactive labeled material.

When testing a particular promoter (sodium caprate) in the experiments shown in Figs. 5 and 6, the amount of labeled material (mannitol, MS 360) that appears on the basolateral side depends on the concentration of promoter used, at least up to 16 mM sodium caprate (Fig. (Fig.7). 7). This is true even if insulin is added to the promoter / mannitol mixture (1: 3 sodium caprate: insulin, by weight) (Fig. 8). It was also observed that this concentration of sodium caprate (16 mM) promotes the absorption of insulin through the cell monolayer. The amount of insulin passing through the monolayer is doubled when 16 mM sodium caprate is added compared to the amount without any diffuser. At higher concentrations of sodium caprate, cell permeability is expected to increase further; however, the potential cytotoxicity of sodium caprate may prevent the use of significantly higher concentrations of this particular promoter.

This in vitro model of epithelial cell permeability can be used as a rapid screening tool to test the desired promoter for the methods of the present invention.

Claims (5)

DESCRIPTION OF THE INVENTION A therapeutic preparation comprising a mixture of the active compounds (A) insulin and (B) which improves the absorption of insulin in the patient's lower airways in the form of a dry powder suitable for inhalation comprising at least 50% by weight of the total active compounds. from (a) primary particles up to 10 microns in diameter, or (b) agglomerates of such particles. Therapeutic preparation according to claim 1, characterized in that it comprises active compounds. different from the above 3. A therapeutic preparation according to claim 1, wherein the dry powder additionally comprises a pharmaceutically acceptable carrier without said active compounds. -4 · 'Οη rrj cJ f \ j {1 / jotu}} azo ^ nrfg ofirer ^ T o i v a c «n pVąnnzic.iios oradŽiOS fmin) Effect of insulin + promoter inhalation on dog plasma insulin 1 U / kg. Exposure 5 minutes (n = 7) (N vS r- tn oh & ΐβ M όό Pu '- / οβ -u A4 N O O 3 J S z c / 5 vi rt a δ tn n a s after exposure starts (min) H I I I I I I I I | I i I I 1 I 1T1 | ~ ri I ll'll I I In Γ ι'ΊΤΤΓΠ 1 | I T I l'TI I I I In I I T i i i I I I j o q o C \, o oo Ό * 4- r \ | () ui / nrt) scnunsur souizeįj Λ S .g t / i § s · —M 0-, 250 η 200 - r < i 1 150 J 1 1 1 - r 100- / A I2 1 * * V * 50- r 0- r t- r- i * Insulin levels in dog plasma following inhalation with ins + Na Kaprat + lact 157 study, Dolly. Dose (! U / kg) -fns + Na- Kap + Lac (502525) 1.9 ......... Ins + Na-Kap + Lac (kk92) 1.8 --- Ins + Na-Kap (75:25) 2.1 250 c S 200 ςΛ 150 liters O Ί r-l l i Γ 1 — I — I — I— | —I— | 20 LO 60 80 100 120 1L0 160 180 200 220 2L0 260 Time after exposure starts (min) Insulin levels in dog plasma after inhalation with ins + Na Kaprat + lact 157 study, Lady. δ .g ίΛ § s 50100 --- Ins + Na-Kap (75-25) I 'Γ> -r I'm not I i j t- i i r T-p Dose (! U / kg} Ins + Na-Kap + Lcc (50: 2525) 22 Ins + Na-Kap + Lac (L-4 92) 1.9 0 ^Δ 0 20 LO 60 80 100 120 1L0 160 180 200 220 2L0 260 Time after exposure starts ( ^mtn -) Effect of Insulin + Na Corporal + Lactose on Blood Glucose, 15 7; Silence, Dollv. 4. A therapeutic preparation according to claim 3, wherein said carrier is comprised of particles up to 10 microns in diameter, such that at least 50% of said dry powder is comprised of (a) particles up to 10 microns in diameter, or (b) agglomerates of such particles. 5. A therapeutic preparation according to claim 3, wherein said carrier is composed of coarse particles so that an ordered mixture can be formed between said active compounds and the carrier. 6. A therapeutic preparation according to claim 4, wherein at least 50% of the dry powder consists of (a) particles having a diameter between 1 and 6 microns, or (b) agglomerates of such particles. Ί. Therapeutic preparation according to claim 1 or 5, characterized in that at least 50% by weight of the total active compounds (A) and (B) consist of particles having a diameter between 1 and 6 microns. 8. A therapeutic preparation according to claim 1, wherein the insulin is bovine, porcine, biosynthetic or equilibrium human insulin, or a biologically active derivative of human insulin. 9. A therapeutic preparation according to claim 8, wherein the insulin is an equilibrium human insulin. 10. The therapeutic preparation according to claim 8, wherein the insulin is biosynthetic human insulin. 11. The therapeutic preparation of insulin according to claim 1, wherein the substance which improves insulin absorption in the lower respiratory tract is the substance which stimulates the absorption of insulin through the epithelial cell layer in the lower airways into adjacent pulmonary blood vessels. 12. A therapeutic preparation according to claim 11, wherein said agent which improves insulin absorption in the lower respiratory tract is a surfactant. 13. A therapeutic preparation according to claim 11, wherein said substance which improves insulin absorption in the lower respiratory tract is an anionic surfactant. 14. A therapeutic preparation according to claim 11, wherein said substance which enhances lower respiratory absorption of insulin is bile salt or bile salt derivative. 15. A therapeutic preparation according to claim 11, wherein said substance which enhances lower respiratory insulin absorption is a phospholipid. 16. A therapeutic preparation according to claim 11, wherein said substance which improves insulin absorption in the lower respiratory tract is an alkyl glycoside. 17. A therapeutic preparation according to claim 11, wherein said substance which improves insulin absorption in the lower respiratory tract is cyclodextrin or a derivative thereof. 18. A therapeutic preparation according to claim 11, wherein said substance which improves insulin absorption in the lower respiratory tract is a fatty acid salt. 19. A therapeutic preparation according to claim 11, wherein said substance which improves insulin absorption in the lower respiratory tract is capric acid salt. 20. The therapeutic preparation of claim 11, wherein. the aforesaid substance which improves the absorption of the well ii ..in the lower airways, y a sodium caprate. 21. A therapeutic preparation comprising the active compounds (A) insulin and (B) sodium caprate in a dry powder form suitable for inhalation comprising at least 50% by weight of the active compounds (A) and (B). of (a) primary particles having a diameter of less than 10 microns, or (b) agglomerates of such particles. 22. A therapeutic preparation according to claim 21, characterized in that it comprises only said active compounds. 23. A therapeutic preparation according to claim 21, wherein the dry powder further comprises a pharmaceutically acceptable carrier in addition to said active compounds. 24. A therapeutic preparation comprising an insulin, sodium caprate and a pharmaceutically acceptable carrier in a dry powder form suitable for inhalation comprising at least 50% by weight of particles having a diameter of less than 10 microns, or (b) agglomerates of said particles. 25. A therapeutic preparation comprising the active compounds (A) insulin and (B) sodium caprate, wherein at least 50% of the total weight of the active compounds (A) and (B) is comprised of particles having a diameter of less than 10. microns, and a pharmaceutically acceptable carrier in the form of a dry powder suitable for inhalation, in which the admixed composition may be formulated between the active compounds and a pharmaceutically acceptable carrier. 26. The therapeutic preparation according to claim 1 or 21, wherein the ratio of insulin to promoter in said preparation is between 9: 1 and 1: 1. 27. A therapeutic preparation according to claim 1 or claim 21 wherein said ratio is in the range of 5: 1 to 2: 1. 28. A therapeutic preparation according to claim 1 or claim 21 wherein said ratio is in the range of 4: 1 to 3: 1. 29. A therapeutic preparation according to claim 3 or 23, wherein said additive is selected from mono-, di- and polysaccharides, sugar alcohols and other polyols. 30. A therapeutic preparation according to claim 3 or 23, wherein said additive is a non-reducing sugar. 31. A therapeutic preparation according to claim 3 or 23, wherein said additive is raffinose, melezitose, lactitol, maltitol, trechalose, sucrose, mannitol or starch. Use of a therapeutic preparation according to claim 1 or 21 in an inhaler. 33. Use according to claim 32, wherein the inhaler protects the powder for inhalation from moisture and has a minimal risk of overdose. 34. The use of claim 32, wherein the inhaler is a single dose, single use, dry powder inhaler that is actuated by inhalation. Use according to claim 32, characterized in that the inhaler is a multi-dose, multiple-use, dry powder inhaler actuated by inhalation. 36. A dry powder inhaler, characterized in that it contains a therapeutic preparation according to claim 1 or 21. 37. A single dose, single use, dry powder inhaler, which is administered by inhalation, comprising a therapeutic formulation for inhalation consisting of the active compounds (A) insulin and (B) sodium caprate, and containing at least one dry powder. 50% of the total weight of the active compounds, (A) and (B), consists of particles up to 10 microns in diameter. 38. A process for the preparation of a therapeutic insulin preparation, comprising the step of preparing a solution of insulin and a substance which improves insulin absorption in the lower respiratory tract by evaporating or otherwise removing the solvent to obtain a solid mass and optionally grinding and / or mixing said solid mass to obtain a powder having at least 50% particles up to 10 microns in diameter. 39. The method of claim 38, further comprising adding a pharmaceutically acceptable carrier in addition to said substance which improves absorption in the lower airways. 40. A method of manufacturing a therapeutic insulin preparation comprising mixing dry insulin with a substance that improves insulin absorption in the lower respiratory tract and optionally grinding and / or mixing said solid mass to form a powder having at least 50% particulate matter. having a diameter of up to 10 microns. 41. The method of claim 40, wherein the dry pharmaceutically acceptable carrier is mixed with insulin and a substance that improves insulin absorption in the lower respiratory tract. 42. Method by 38 or 40 score points s k i - r i duck in that additionally grinds to of micron particles. 43. Buddha by 39 or 41 score points s k i - characterized in that it further admixes the blend of said powder with a pharmaceutically acceptable carrier. Use of a promoter in the preparation of an insulin inhaled dry powder formulation with improved systemic insulin absorption in the lower respiratory tract, wherein at least 50% of the total weight of the insulin and the promoter consists of (1) particles having a diameter of 10 microns or less; agglomerates. 45. The use of claim 44, wherein the promoter is a surfactant. 46. The use of claim 44, wherein the promoter is a fatty acid salt. 47. The use of claim 44, wherein the promoter is sodium caprate. Insulin + promoter inhalation ve C4 5.5 n 0 20 U) 60 80 JOG 120 KO 160 180 200 220 2U) 260 Time after exposure starts (mm) Time after start of exposure (min) o_ control <- 10 m M 13mM -λ— 16m M control 10mM 13mM 16mM Mannitol transport via Caeo-2 monolayer in Na caprate (10-16 mM) A. Well Caprate B. Well caprate / insulin (1: 3 w / w) Insulin concentration 5 min after inhalation 1 U / kg. Mean values ± s e m (n = 5)