RU2175556C2 - Methods and compositions for light delivery of insulin - Google Patents

Abstract

FIELD: medicine, endocrinology, pharmacy. SUBSTANCE: systemic delivery of insulin to mammalian-host is carried out by inhalation of dried insulin powder by oral route. Dried insulin powders show the more rapid absorption through alveolar zones in lung. EFFECT: improved method and composition. 19 cl, 5 tbl, 16 dwg

Description

BACKGROUND OF THE INVENTION
1. The technical field
The present invention relates generally to methods and compositions for the respiratory delivery of insulin to patients with diabetes. In particular, this invention relates to pulmonary delivery of dry powdered insulin preparations for rapid systemic absorption through the lungs.

 Insulin is a polypeptide hormone of 50 amino acids, having a molecular weight of about 6000, which is produced by pancreatic β-cells in normal (non-diabetic) individuals. Insulin is needed to regulate carbohydrate metabolism by lowering blood glucose levels, and its systemic deficiency causes diabetes. The survival of diabetic patients depends on the frequency and duration of insulin administration, which maintains an acceptable level of glucose in the blood.

 Insulin is most often administered by subcutaneous injection, usually in the abdomen or upper thighs. To maintain acceptable blood glucose levels, it is often necessary to inject insulin at least once or twice a day, with additional injections of fast-acting insulin being administered when necessary. An invasive diabetes treatment may require even more frequent injections when the patient carefully monitors blood glucose levels using at-home diagnostic kits. This invention, in particular, relates to the administration of fast-acting insulins which are capable of providing peaks of serum insulin within one hour and a drop in glucose within 90 minutes.

 The administration of insulin by injection is undesirable for a number of reasons. First, many patients find it difficult and cumbersome to inject themselves as often as necessary to maintain acceptable blood glucose levels. Such non-compliance may lead to non-compliance with the regimen of taking medications, which in the most serious cases can be life threatening. In addition, systemic absorption of insulin by subcutaneous injection is relatively slow, often requiring 45 to 90 minutes, even when fast-acting insulin formulations are used. Thus, there has long been a challenge in developing alternative formulations of insulin and routes for its administration, which would avoid the need to inject ourselves and which could provide fast systemic availability of insulin.

 A number of such alternative insulin routes have been proposed, including intranasal, intrectal and intravaginal.

 Although these techniques make it possible to avoid the discomfort and mild compliance associated with subcutaneous injection, each of them suffers from its own limitations. Intrarectal and intravaginal routes are uncomfortable, uncomfortable, and the latter is not accessible to the entire population of diabetics. Intranasal delivery can be convenient and perhaps less unpleasant than an injection, it does not require the use of potentially toxic “penetration enhancers” that affect the passage of insulin through the nasal mucosa, which is characterized by a thick epithelial layer resistant to passage of macromolecules. Of particular interest in this invention is pulmonary delivery when the patient is inhaled with the insulin formulation and when systemic absorption occurs through a thin layer of epithelial cells in the alveolar regions of the lung. Apparently, such pulmonary insulin delivery provides faster systemic access than subcutaneous injection does, and it avoids the use of a needle. Pulmonary insulin delivery, however, has yet to gain widespread acceptance. Until now, pulmonary delivery, in most cases, is accomplished by spraying liquid formulations of insulin requiring the use of heavy liquid nebulizers. In addition, aerosols generated by such nebulizers have a very low insulin concentration, which inevitably leads to a large number of inhalations in order to provide an adequate dose. The concentration of insulin is limited due to the low solubility of insulin in the corresponding aqueous solutions. In some cases, up to 80 or more inhalations may be required to achieve an adequate dose, which leads to an administration time of 10 to 20 minutes or more.

 It is desirable to develop improved methods and compositions for pulmonary insulin delivery. It is especially desirable if such methods and compositions are convenient enough to administer to themselves even outside the home and will be able to deliver the required total dose with a relatively small number of inhalations, preferably less than ten. Such methods and compositions should also include rapid systemic absorption of insulin, preferably reaching a serum peak within 45 minutes or less, and obtaining a drop in glucose within about one hour or less. Such high-speed formulations will preferably be suitable for use according to invasive treatment protocols when the injection of intermediate and long acting insulin can be reduced or eliminated. The compositions of this invention should also be stable, preferably consisting of concentrated formulations of dry powder.

2. The prior art
Respiratory delivery of aerosol insulin aqueous solutions is described in a number of references, starting with Gansslen (1925) Klin Wochensehr. 4:71 and including Laube et al. (1993) JAMA 269: 2106-21-9; Elliot et al. (1987) Aust Paediatr J. 23: 293-297; Wigley et al. (1971) Diabetes 20: 552-556. Corthorpe et al. (1992) Pharma Res 9: 764-768; Govinda (1959) Indian J. Physicl. Pharmacol 3: 161-167, Hastings et al. (1992). J. Appl. Physiol. 73: 1310-1316; Liu et al. (1993) JAMA 269: 2106-2109; Nagano et al. (1985) Jikeikai med.J. 32: 503-506; Sakr. (1992) Int. J. Phar. 86: 1-7; and Yoshida et al. (1987) Clin. Res. 35: 160-166. Pulmonary delivery of dry powdered drugs, such as insulin, in a vehicle with a carrier in the form of large particles, is described in US Pat. USA N 5254330. A metered-dose inhaler (MDI, MDI) for the delivery of crystalline insulin suspended in a propellant (liquid aerosol carrier) is described by Lee and Sciura (1976) J. Pharm. Sei 65: 567-572. MDI (MDI) for insulin delivery in a spacer for controlling the rate of inhalation flow is described in US Pat. USA N 5320094. The intrabronchial administration of recombinant insulin is briefly described by Schluter et al. B (Abstract) (1984) Diabetes 33: 75A and Kohler et al. (1987) Atemw. Lungenkrkh. 13: 230-232. Intranasal and respiratory delivery of a number of polypeptides, including insulin, in the presence of an enhancer, are described in US Pat. No. 5011678 and Nagai et al. (1984) J.Contr.rel. 1: 15-22. Intranasal delivery of insulin in the presence of enhancers and / or controlled release formulations is described in US Pat. U.S. N 5204108; 4,294,829; 4,153,689; PCT Applications WO 93/02712, WO 91/02545, WO 90309780, and WO 88/04556; British Patent 1,527,605; Ryden and Edman (1992) Int. J. Pharm. 83: 1-10; and Bjork and Esman (1988) Int. J.Pharm. 47: 233-238. The preparation and stability of amorphous insulin is described by Rigsbee and Pikal at the American Association of Pharmaceutical Sciences (AAPS), November 14-18, 1993, Lake Buena Vista, Florida. Methods of spray drying polypeptide, polynucleotide and other labile drugs in a carrier that forms an amorphous structure that stabilizes the drug are described in the application for Heb. Pat. 520748.

SUMMARY OF THE INVENTION
According to this invention, methods and compositions for aerosolization and systemic delivery of insulin to a mammalian host, in particular a human patient with diabetes, provide for rapid absorption into the bloodstream while eliminating subcutaneous injection. In particular, the methods of this invention are based on pulmonary delivery of insulin in the form of a dry powder. It was unexpectedly found that inhaled dry insulin powders precipitate in the alveolar regions of the lung and are rapidly absorbed through the alveolar epithelial cells into the bloodstream. Thus, pulmonary delivery of insulin powders can be an effective alternative to subcutaneous injection.

 In a first aspect of the invention, insulin is provided in the form of a dry powder, typically, but not necessarily, in a substantially amorphous state, and is dispersed in air or another physiologically acceptable gas stream to form an aerosol. The aerosol is contained in a chamber having a mouthpiece, from where it is suitable for subsequent inhalation to the patient. Not necessarily dry powdered insulin is combined with a pharmaceutically acceptable dry powdered carrier, as described in more detail below. The insulin powder preferably includes particles having a diameter of less than 10 microns, more preferably less than 7.5 microns, and most preferably below 5 microns, and usually it is in the range from 0.1 microns to 5 microns. It has surprisingly been found that the dry powdered insulin compositions of the present invention are absorbed in the lung without the use of penetration enhancers, such as those required for absorption through the nasal mucosa and upper respiratory tract.

 In a second aspect, the present invention provides insulin compositions consisting essentially of a dry insulin powder having an average particle size lower than 10 μm, which can be combined with dry powdered pharmaceutical carriers. The insulin composition is preferably free of penetration enhancers and includes particles having a diameter of less than 10 microns, preferably less than 7.5 microns, and most preferably below 5 microns, typically in the range of 0.1 microns to 5 microns. Typically, the dry content of insulin powder in the composition should be from 5% to 99% by weight, more preferably from 15% to 80%, in a suitable pharmaceutical carrier, usually a carbohydrate, organic salt, amino acid, peptide or protein, as described in more detail below. .

 In a third aspect of the invention, insulin dry powders are prepared by dissolving insulin in an aqueous buffer to form a solution and spray drying the solution to produce substantially amorphous particles having a particle size of less than 10 microns, preferably less than 7.5 microns, and most preferably below 5 microns, and usually their size is in the range from 0.1 microns to 5 microns. Optionally, the pharmaceutical carrier is also dissolved in a buffer to form a homogeneous solution in which spray drying the solution gives individual particles, including insulin, a carrier buffer, and any other components that were present in the solution. Preferably, the carrier is a carbohydrate, organic salt, amino acid, peptide, or protein, which provides a substantially amorphous structure after spray drying. The amorphous carrier can be either glassy or rubbery, and it increases the stability of insulin during storage. Advantageously, such stabilized formulations are also capable of efficiently delivering insulin to the bloodstream when inhaled into the alveolar regions of the lungs.

 A further understanding of the nature and advantages of this invention will become apparent from a consideration of the following description sections and drawings.

Brief Description of the Drawings
FIG. 1 is a schematic illustration of a system for aerosolizing a dose of insulin according to the method of the present invention.

 FIG. 2 is a schematic illustration of how a patient is inhaled with an aerosol dose of insulin from the system of FIG. 1.

 FIG. 3A and 3B are graphs illustrating the absorption of recombinant human insulin in rats and the glucose response after aerosol administration of three different dry powder formulations. Each point represents the average of three different rats. At time zero, a dry powder aerosol generator is turned on. Aerosolization is completed after 5 minutes, 14 minutes and 20 minutes. For powders, 87% insulin / citrate, 20% insulin-mannitol / citrate and 20% insulin-raffinose / citrate, respectively. Animals are locked at night.

 FIG. 4A and 4B are graphs illustrating the mean serum concentrations of insulin and glucose over time, respectively, when comparing aerosol and subcutaneous administration of cynomolgus to monkeys. For the aerosol group, the average value for three monkeys is given; for the subcutaneous administration group, the average value for four monkeys is given.

 FIG. 5A is a graph illustrating the average concentration of insulin over time for subcutaneous injection (o) and for inhalation of three taps (•) in humans.

 FIG. 5B shows the average glucose concentration corresponding to the insulin concentrations of FIG. 5A.

 FIG. 6A is a graph illustrating the serum insulin concentration over time as a result of subcutaneous injection (o) and three presses with aerosol (•), in humans.

 FIG. 6B is a graph illustrating serum glucose levels corresponding to the insulin levels in FIG. 6A.

FIG. 7A and 7B represent a comparison of the scatter from subject to subject, serum insulin (7A) and glucose (7B) levels for subcutaneous (o) and aerosol (•)
In FIG. 8A, 8B, and 8C show OFLCH (gHPLC) chromatograms of human insulin. FIG. 8A is a chromatogram of an insulin standard equilibrated (Stressed) in 10 mM HCl at 25 ^° C, showing human insulin eluting after 23, 87 minutes, desamidoinsulin eluting after 30.47 minutes. In FIG. 8B presents a similar chromatogram of human insulin standard. FIG. 8C is a similar chromatogram of reconstituted, spray-dried dried insulin formulations prepared according to this invention.

 In FIG. 9 shows the ultraviolet spectrum of the insulin composition before and after spray drying. No light scattering is observed in the visible part of the spectrum, which indicates that insulin does not aggregate during the spray drying process.

Detailed Description of Specific Embodiments of the Invention
According to the invention, insulin is provided in the form of a dry powder. By the term “dry powder” is meant that the moisture content of the powder is below about 10% by weight, usually below about 5% by weight, and preferably below about 3% by weight. The term "powder" means that insulin includes freely flowing (macro) particles having a size selected to allow penetration into the alveoli of the lungs, preferably less than 10 microns in diameter, preferably less than 7.5 microns, and most preferably less than 5 microns, and usually it is in the range from 0.1 microns to 5 microns in diameter.

This invention is based, at least in part, on the unexpected observation that dry powdered insulins are readily and rapidly absorbed through the lungs of the host. Surprisingly, dry insulin powders can reach the alveolar region of the lungs, while water-soluble drugs such as insulin particles are known to be hygroscopic. See, for example, Byron ed., Respiratory Drug Delivery, CRL Press, Boca Raton (1990), p. 150. Thus, it should be expected that when particles pass through the airways of the lung (which has a relative humidity of over 99% at 37 ^° C), individual particles should tend to absorb water and grow to an effective particle size greater than 10 microns of the upper limit of the present invention. If a significant fraction of the insulin particles is larger than the size of the target area, then it should be expected that the particles will precipitate within the central airways of the lungs rather than within the alveolar region, thereby limiting delivery and subsequent systemic absorption. In addition, the liquid layer above the lung epithelial cells is very thin, and a fraction of this diameter of insulin powder is usually delivered. Thus, prior to the present invention, it was unpredictable whether dry insulin particles would dissolve upon deposition within the alveolar regions of the lungs. Suddenly, powders of dry insulin were able to penetrate the alveolar regions of the lungs and dissolve immediately after their deposition inside the alveolar region of the lung. Dissolved insulin is then able to pass through the epithelial cells into the circulation.

 Currently, it is believed that effective absorption of insulin is the result of rapid dissolution in an ultrathin (<0.1 μm) liquid layer of alveolar contents. The particles of the present invention thus have an average size that is 10 to 50 times larger than the thickness of the pulmonary fluid layer, making it unexpected that the particles dissolve and insulin is rapidly absorbed systemically. Indeed, as shown in the Experimental section below, the dry insulin formulations of the present invention can provide even faster peaks of serum insulin and a drop in glucose than is achieved by subcutaneous injection, which is by far the most common form of insulin administration. An understanding of the exact mechanism, however, is not necessary for the practice of the present invention described herein.

 Preferred compositions of the invention are substantially free of penetration enhancers. "Penetration enhancers" are surface-active compounds that facilitate the penetration of insulin (or other drugs) through the membrane of the mucous membrane or contents, and are intended for use in intranasal, intrectal, and intravaginal formulations of the drug. Examples of penetration enhancers are bile salts, for example taurocholate, glycocholate, and deoxycholate; fusidates, for example taurodehydrofusidates; and biocompatible detergents, for example Twins, Laureth-9, and the like. The use of penetration enhancers in formulations for the lungs, however, is usually undesirable, since such surface-active compounds can have an adverse effect on the epithelial blood barrier in the lung. Surprisingly, dry powdered insulin compositions of this invention are readily absorbed in the lungs without the need for penetration enhancers.

 Dry insulin powders suitable for use in this invention include amorphous insulins, crystalline insulins, and mixtures of both amorphous and crystalline insulins. Dry powdered insulins are preferably obtained by spray drying under conditions that result in a substantially amorphous powder having a particle size within the above specified range. Alternatively, amorphous insulins can be obtained by lyophilization (freeze-drying), vacuum drying or evaporative drying a suitable insulin solution under conditions providing an amorphous structure. The amorphous insulin thus obtained can then be ground or ground to obtain particles of a size within the required range. Crystalline dry powdered insulin can be obtained by grinding or crushing a mass of crystalline insulin in a jet mill. A preferred method for producing insulin powders comprising (macro) particles in a desired size range is spray drying, where pure insulin in bulk (usually in crystalline form) is first dissolved in a physiologically acceptable aqueous buffer, usually a citrate buffer, having a pH in the range of about 2 up to 9. Insulin is dissolved at a concentration of from 0.01% by weight to 1% by weight, usually from 0.1% to 0.2%. The solutions can then be spray dried in standard spray drying equipment from commercial suppliers such as Buchi, Niro and the like, resulting in a substantially amorphous product in the form of (macro) particles.

 Dry insulin powders can mainly consist of insulin particles within the range of the desired size and can be mainly free of any other biologically active components, pharmaceutical carriers, etc. Such “pure” formulations may include minor amounts of components, such as preservatives, present in small amounts, typically below 10% by weight and usually below 5% by weight. Using such pure formulations, the number of inhalations required for even high doses can be significantly reduced, often to only a single breath.

 The insulin powders of the present invention may optionally be mixed with pharmaceutical carriers or excipients that are suitable for respiratory and pulmonary administration. Such carriers can serve simply as filling agents when required to reduce the concentration of insulin in the powder that will be administered to the patient, but can also serve to increase the stability of the insulin compositions and to improve the dispersibility of the powder inside the powder dispersion device in order to obtain a more effective and reproducible insulin delivery, and improve manual insulin handling characteristics, such as fluidity and consistency, facilitating production and packaging Patent Application insulin.

 Suitable carrier materials may be in the form of an amorphous powder, a crystalline powder, or a combination of amorphous and crystalline powders. Suitable substances are coal water, for example monosaccharides such as fructose, galactose, glucose, D-mannose, sorbose, and the like. disaccharides such as lactose, trehalose, cellobiose, and the like, cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin; and polysaccharides such as raffinose, maltodextrins, dextrans and the like (b) amino acids such as glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine and the like; (c) organic salts derived from organic acids and bases, such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, and the like; (d) peptides and proteins, such as aspartame, human serum albumin, gelatin and the like; (e) alditols, such as mannitol, xylitol and the like. A preferred carrier group includes lactose, trehalose, raffinose, maltodextrins, glycine, sodium citrate, tromethamine hydrochloride, human serum albumin and mannitol.

 Such carrier materials can be displaced with insulin prior to spray drying, i.e., by adding the carrier substance to the buffer solution that is obtained for spray drying. In this way, a carrier substance can be obtained simultaneously with insulin particles and as part of insulin particles. Typically, when the carrier is obtained by spray drying along with insulin, insulin will be present in each individual particle at a weight percentage in the range from 5% to 95%, preferably from 20% to 80%. The remainder of the particle will mainly be a carrier substance (typically typically 5% to 95%, typically 20% to 80% by weight), but it will also include buffer (s) and may include the other components described above. . The presence of a carrier substance in particles that are delivered to the alveolar region of the lung (i.e., particles in a desired size range below 10 microns) has been found to slightly interfere with systemic absorption of insulin.

 Alternatively, carriers can be prepared separately in the form of a dry powder and combined with a dry insulin powder by mixing. Separately obtained powdery carriers usually must be crystalline (to avoid absorption of water), but may, in some cases, be amorphous or mixtures of crystalline and amorphous powders. The particle size of the carrier should be selected so as to improve the fluidity of the insulin powder, typically the particle size is in the range from 25 μm to 100 μm. Carrier particles in this size range usually do not penetrate the alveolar region of the lung and can often be separated from insulin in the delivery device before inhalation. Thus, particles that penetrate the alveolar region of the lung should mainly consist of insulin and buffer. A preferred carrier material is crystalline mannitol having a size in the above range.

 The dry insulin powders of the present invention can also be mixed with other active ingredients. For example, to improve diabetes management, it is desirable to combine small amounts of amylin or active amylin analogs with insulin powders. Amylin is a hormone that is secreted together with insulin by pancreatic β-cells in normal (non-diabetic) individuals. Amylin is believed to modulate ib vivo insulin activity, and it is believed that the simultaneous administration of amylin with insulin can improve blood glucose control. The combination of dry powdered amylin with insulin in the compositions of this invention provides a particularly convenient product to achieve this simultaneous administration. Amylin can be combined with insulin at a content of from 0.1% by weight to 10% by weight (based on the total weight of insulin in a dose), preferably from 0.5% by weight to 2.5% by weight. Amylin is available from commercial suppliers such as Amylin Corporation, San Diego, California, and it can be easily formulated in the compositions of this invention, for example, amylin can be dissolved in aqueous or other suitable solutions together with insulin, and, optionally, together with carriers, and then the solution is spray dried to obtain a powdery product.

 The dry powdered insulin compositions of this invention are preferably aerosolized by dispersion in an air stream or other physiologically acceptable gas stream in the usual way. One system suitable for such dispersion is described in U.S. Ser. N 07/910 048, which is published as WO 93/00951, full descriptions of which are given here as a prior art. With reference to FIG. 1, a dry, free-flowing insulin powder is introduced into a high-speed stream of air or gas, and the resulting dispersion is introduced into the receiving chamber 10. The receiving chamber 10 includes a mouthpiece 12 at the opposite end from the entry point of the air dispersion of the powder. The volume of the chamber 10 is large enough to enclose the required dose, and the chamber may have guiding screens and / or one-way valves to provide containment, after the dose of insulin powder has been enclosed in chamber 10, patient P (Fig. 2) is inhaled with a tip 12 for the mouth by drawing the aerosol dispersion into the lungs. When patient P makes inhalation, the incoming air is introduced through a tangentially located air inlet port 14, the air usually flowing in the form of a vortex, taking with it aerosol insulin from the chamber to the patient's lungs. The volume of the chamber and the aerosol dose are such that the patient is able to completely inhale the entire dose of aerosol insulin with an amount of air sufficient to ensure the delivery of insulin to the lower alveolar regions of the lung.

 Such insulin aerosol powders are especially useful instead of subcutaneous injections of fast-acting insulin in the treatment of diabetes and related insulin deficiencies. It has been unexpectedly discovered that aerosol administration of dry powdered insulin leads to a significantly faster absorption of insulin and a glucose response than is achieved by subcutaneous injection. Thus, the methods and compositions of this invention should be especially valuable in treatment protocols, where the patient often monitors blood glucose levels and injects insulin as needed to maintain the target serum concentration, but they are also useful in cases when systemic administration of insulin is required. The patient can receive the required dose by inhalation of the appropriate amount of insulin, as just described. The efficacy of systemic insulin delivery using the method just described is usually in the range of about 15% to about 30%, with individual doses (based on inhalation) usually being in the range of about 0.5 mg to 10 mg. Typically, the total dose of insulin required during a single respiratory administration is in the range of about 0.5 to 15 mg. Thus, the required dose can be effective for a patient taking from 1 inhalation to 4 inhalations.

 The following examples are provided for purposes of illustration and not limitation.

EXPERIMENTAL PART
Substances and methods
Substances
Human crystalline zinc-insulin, 26.3 Units / mg, (Lilly Lot # 784KK2), obtained from Elililly and Company, Indianapolis IN, was found to have a purity> 99% as determined using rVHLC (rpHPLC). USP (USP) mannitol is obtained from Roguette Corporation (Gurnee, IL). Raffinose is acquired by y Pfanstiehe Laboratories (Waukegan, IL). Sodium citrate dihydrate, USP, ACS, and USP citric acid monohydrate are obtained from ITBaker (Phillipsburg, NI).

Powder production
Insulin powders are prepared by dissolving a mass of crystalline insulin in sodium citrate buffer containing an excipient (mannitol, or raffinose, or without an excipient) to obtain a final solids concentration of 7.5 mg / ml and a pH of 6.7 ± 0.3. The spray dryer was operated at an inlet temperature of 110 ^o C-120 ^o C and a liquid feed rate of 5 ml / min, which led to an outlet temperature of 70-80 ^o C. Then the solutions were filtered through a 0.22 μm filter and dried sprayed in a Buchi spray dryer to form a finely divided white amorphous powder. The resulting powders are stored in tightly closed containers in a dry atmosphere (<10% RH).

Powder Tests
The particle size distribution of the powders was determined by liquid sedimentation by centrifugation in a Horiba CAPA-700 Particbe Size Analyzer particle size analyzer after dispersing the powders in Sedisperse A-11 (Micromeritics, Norcross, GA). The moisture content of the powders was determined by the Karl Fischer method using a Mitsubishi CA-06 Moisture Meter.

 The preservation of insulin before and after the process of powder formation was determined by comparing with the standard of human insulin by re-dissolving the suspended portions of the powder in distilled water and comparing the re-dissolved solution with the initial solution passed through a spray dryer. The retention time and peak area by OFRFA (rpHPLC) was used to determine whether the insulin molecule is chemically modified or degraded during processing. UV absorption is used to determine the concentration of insulin (at 278 nm) and the presence or absence of insoluble aggregates (at 400 nm). In addition, the pH of the stock and reconstituted solutions is determined. The amorphous nature of the insulin powder is confirmed by polarizing light microscopy.

Aerosol effects on rats
Experiments on rats were performed in an aerosol exposure chamber. Female rats (280-300 g) were not fed overnight. Animals (21-24 / experiment) were placed in Plexiglass tubes and placed in a 48 port chamber, for the nose only for aerosol exposure (In-Tox Products, Albuquetque, NM). The airflow into the inhalation zone was set at 7.2-9.8 liters / minute, and the air was removed by evacuation so that a slight negative pressure (about 1.5 cm H ₂ O) was created in the chamber, determined using a magnagelic meter. Aerosol exposure times were between 5-20 minutes, depending on how much powder was injected into the chamber. The powders were manually injected into a small Venturi nozzle that dispersed the powder particles to form a thin aerosol cloud. The Venturi nozzle operated at an overpressure in excess of 1,055 kg / cm ² (15 psig), and the air flow was set from 7.2 L / min to 9.8 L / min. Venturi (Venturi) nozzle was inserted into the bottom of a transparent Plexiglass dispersion chamber (750 ml), which passed the aerosol directly into the chamber, for the nose - only for aerosol exposure.

Calibration of the aerosol chamber for rats
The concentration of the powder in the inhalation zone was measured by repeatedly chronometrical sampling on the filter in the inhalation zone in the In-Tox filter holders in the path of a 2-liter / min vacuum flow. The camera was calibrated both with animals and without animals. The mass of the powder was determined gravimetrically. The particle size of the powders in the inhalation zone was determined using a cascade impactor (In Tox Products) placed in the inhalation opening and operating in a flow rate of 2 liters / min. The powder mass at each stage was determined gravimetrically
In each powder test, 21-24 rats were used and aerosol exposures lasted 5-20 minutes. Three rats were killed at time 0 and then at approx. 7, 15, 30, 60, 90, 120, 180 and 240 minutes after the end of aerosol exposure. Animals were anesthetized, the abdominal cavity was opened, and a large blood sample was taken from the abdominal aorta. Then the animals were killed by cervical dislocation.

Blood was allowed to clot at room temperature for 30 minutes and then centrifuged for 20 minutes at 3500 rpm in serum separation tubes. Serum was either analyzed immediately or frozen at -80 ^° C until analysis. As soon as possible (0-7 minutes) after the end of aerosol dosing, 3 rats were sacrificed, their blood was taken and their lungs were washed with phosphate-buffered saline (FBI) six times 5 ml each. The amount of insulin in the final cumulative wash sample was used as an aerosol dose for rats in bioavailability calculations.

Exposition system for primates
To study the effects of aerosols on primates (3-4 animals / group), young wild male cynomolgus monkeys, Macaca fascicularis line (2-5 kg) (Charles River Primates, Inc.) were used. Animals were either injected subcutaneously with Humulin (Eli Lilly, Indianopolis, Indiana) or exposed to insulin powder aerosol. Each animal was placed with its head - only in the section for exposure, in order to ensure a fresh supply of the test atmosphere at an adequate flow rate (7 l / min), ensuring the minimum oxygen demand for the animal. Animals were kept in a chair-type device, where they were placed in a straight-sitting position (with their seated legs up). Helmets (hoods) were transparent, allowing the animals to fully look around. A permanent catheter was placed in the leg so that blood samples could be taken at any time. The monkeys were awake during the whole procedure and looked calm. Primate blood was treated in the same way as rat blood (see above).

 The primate aerosol exposure system includes an inhalation monitor that measures the amount of air inhaled by each monkey. This value, multiplied by measuring the concentration of insulin in the inhaled air, allows you to accurately calculate how much insulin is inhaled by each animal.

Human trials
Insulin is administered subcutaneously to 24 normal human subjects, as well as by inhalation of aerosolized dry insulin powders. Each subcutaneous injection consists of 10.4 E Humulin (Humulin R), 100 E / ml (Eli Litly, Indianapolis, Indiana). The dry insulin powders are amorphous and were prepared by spray drying as described above, with 20% by weight mannitol as a filler. Doses (5 mg) of insulin dry powder were dispersed in a high-speed stream of air to obtain a fine aerosol, which was enclosed in a chamber. Each subject was inhaled with aerosol powder by taking a slow, deep breath of each aerosol spherical mass (bolus) or pressing. The powder was administered by three presses (for a dose of 31.9 E). Serum insulin and glucose levels were determined over a period of time, as described below.

Serum Tests
Serum insulin levels in rats, primates, and humans were determined using Coat-A-Count radio-immunodiagnostic kits for human insulin (Diagnostic Products Corporation, Los Angeles, OA). Standard curves are plotted for each batch of samples. The sensitivity of the assay was approximately 43 pg / ml. The spread within the analysis (% CV) was <5%. Glucose assays were performed using Colifornia Veterinary Diagnostics, Inc. in West Sacramentc. CA using Gluсose IHK Reagent System Pack reagent strips for the Bcehringer Mannheim / Hitachi 747 Analyzer. The spread within the analysis (% CV) was <3%.

 In rat experiments, the relative bioavailability of the aerosol was calculated by comparing the area under the curve (PPC) for the adjusted dose of immunoreactive insulin (IRI) concentration-time profile with the area under the curve obtained by subcutaneous injection. In rats, all washed insulin was used as an aerosol dose. A certain amount of insulin absorbed to the lungs can be washed, so the dose determined using this technique is probably slightly underestimated (underestimated) relative to the total deposited dose. No corrections have been made for this alleged loss.

 In experiments on monkeys, the relative bioavailability was calculated similarly to the calculation described above for rats, except that instead of using insulin washed from the lung, the total amount of inhaled insulin was used as an aerosol dose. In rats, only the substance deposited in the lungs, but not the insulin deposited in the nasal passages and throat, was included in the dose estimate. In monkeys, all insulin that was given to animals was included in the dose estimate.

The results of the absorption of insulin in rats
All insulin powders used in animal studies had particle sizes (weight average diameters) in the range of 1-3 μm and a moisture content of 3%. The purity of insulin in powders, measured using OFVRH (rpHPLC) was> 97%. Typical chromatograms of 20% insulin formulations are shown in FIG. 8C. The powders gave a clear solution during reconstruction with pure water with absorption <0.01 in the ultraviolet region at 400 nm and a pH of 6.7 ± 0.3. In FIG. Figure 9 shows a typical ultraviolet (UV) spectrum for a 20% insulin formulation.

 The following three formulations of insulin powder were tested in rats as aerosols in an In-Tox 48 port exposure chamber.

 1. 87.9% insulin; 11.5% sodium citrate; 0.6% citric acid.

 2. 20% insulin; 66% mannitol; 12.4% sodium citrate; 0.6% citric acid.

 3. 20% insulin; 66% raffinose; 12.4% sodium citrate; 0.6% citric acid.

 Table 1 lists key measurements for three different exposure studies for rats, including inhalation aerosol characteristics and chamber operating conditions. The powder fraction supplied to the venturi nozzle reached the inhalation zones of rats (34-67%) due to wall losses due to adhesion (collision) and incomplete dispersibility of the powder during powder feeding. The size of the aerosol particles in the inhalation zone, however, was ideal for pulmonary deposition (1.3-1.9 μm) and was slightly smaller than the particle size of the original formulation (2.0-2.8 μm) due to the selective loss of larger particles in a chamber for animal exposure.

Table 2 shows the results for serum insulin and glucose from three aerosol studies and one subcutaneous study, for rats. In FIG. 3A and 3B show the concentration profiles of serum immunoreactive insulin (IRI) over time and the concentration profiles of serum glucose over time for the three formulations administered by aerosol. Table 3 presents t _max for insulin and t _min for glucose from three different studies, as well as the relative bioavailability of the aerosol compared to subcutaneous (PC) injection.

 All three formulations provide rapid absorption of insulin into the systemic circulation of rats (Fig. 3A and 3B). Bioavailability and glucose response were higher for 20% insulin / mannitol powder (table 3), although without many duplicate experiments it is not clear whether this difference is significant.

Results on primates
A dose identical to that used in the human test (0.2 E / kg, ~ 27 μg / monkey) is administered by injection to four monkeys to obtain PC results for comparison with aerosol results (FIGS. 4A and 4B). Table 4 presents the effects of aerosol exposure on monkeys. Table 5 shows the average serum insulin and glucose levels for aerosol aerosol exposure and subcutaneous studies. The aerosol dose gives a strong insulin and glucose response (high dose). FIG. 4 illustrates a comparison of mean serum insulin profiles for two aerosol and one PK study. Of the AUCSs of these profiles, the relative bioavailability of aerosol insulin was calculated to be 12%.

Results in humans
Comparative results between insulin delivery by respiratory and subcutaneous injection are presented in Table 5. Respiratory aerosol delivery leads to faster absorption (peak after 20 minutes) than injection (peak after 60 minutes), with a faster glucose response (drop after 60 minutes) than in the case of an injection (drop after 90 minutes). Reproducibility was good, if not better, in the case of aerosol than in the case of injection, both for insulin and for the glucose response. Injection doses were carefully controlled by weight, no aerosol doses. The biological activity of aerosol insulin, based on the glucose response, in relation to the injection was 28-36%. The bioavailability of aerosol insulin, based on the area under the curve for insulin, compared with injection, was 22.8 for a group with 3 taps.

 The human test results are shown in FIG. 5A-5B. FIG. 5A shows the mean serum insulin levels over time when administered by subcutaneous injection (o), inhalation (3 presses) (•). Similarly in FIG. 5B presents average serum glucose levels. Peaks of insulin and a drop in glucose are shown in FIG. 6A and 6B, respectively, while the scatter between subjects in the definitions of serum insulin and glucose is shown in FIG. 7A and 7B, respectively.

 In addition, shallow inhalations (tidal breathing) of monkeys during aerosol exposures do not represent the optimal breathing procedure for deep deposition (deposition) in the lungs. Higher bioavailability is observed in humans (Table 5), as expected when the optimal breathing procedure is used and when an aerosol bolus is used for oral inhalation rather than nasal inhalation.

 Although the aforementioned invention has been described in sufficient detail by way of illustration and example, in order to clarify its understanding, it should be obvious that some changes and modifications may be made thereto without departing from the scope of the following claims.

Claims (19)

 1. The method of aerosolization of a dose of insulin, which consists in the fact that the dry insulin powder is dispersed in a gas stream to obtain an aerosol with an insulin particle size of from 0.1 to 10 μm, which is placed in a chamber having a mouthpiece for subsequent inhalation by the patient.  2. The method according to claim 1, in which the insulin is present in a dry powder carrier at a weight concentration in the range from about 5 to 99%.  3. The method according to claim 2, in which the insulin is present in a dry powder carrier at a weight concentration in the range from 20 to 80%.  4. The method according to claim 2 or 3, in which the powder carrier comprises a carbohydrate, organic salt, amino acid, peptide or protein.  5. The method according to claim 1, in which the dry insulin powder comprises particles having an average size of less than 10 microns.  6. The method according to claim 1, in which the dry insulin powder comprises particles with a size in the range from 0.1 to 10 microns.  7. The method according to claim 1, in which the dry powder includes individual particles, including both insulin and a carrier substance.  8. An improved method for respiratory delivery of insulin, wherein said improvement comprises providing insulin in the form of a dry powder comprising individual particles of 5 to 99% by weight of insulin in a pharmaceutical carrier, dispersing insulin in an air stream and delivering insulin by mouth into the lungs in the form of a dry powder having an average particle size in the range from 0.1 to 10 microns, in which the peak of insulin and a drop in glucose are achieved faster than when subcutaneous administration of an equivalent amount of insulin .  9. The improved method of claim 8, wherein the insulin is present in a dry powder carrier at a weight concentration in the range of about 10 to 99%.  10. The improved method according to claim 9, in which the powder carrier includes a carbohydrate, organic salt, amino acid, peptide or protein.  11. The improved method of claim 8, in which the dry powder includes individual particles, including both insulin and a carrier substance.  12. A method of obtaining a stable dry powdered insulin composition, comprising dissolving insulin and a pharmaceutical carrier in an aqueous buffer, where the insulin composition (insulin and carrier) is from 0.01 to 1% by weight of the solution, and the actual insulin is from 20 to 80% by the total mass of the insulin composition (insulin and pharmaceutical carrier in solution), and spray drying of the solution to obtain amorphous particles, including both insulin and pharmaceutical carrier, the average size of which ranges from 0.1 about 10 microns, and a moisture content of less than 10%.  13. The method according to item 12, in which the pharmaceutical carrier is a carbohydrate, organic salt, amino acid, peptide or protein, which gives the powder after spray drying.  14. The method according to item 12, in which the carbohydrate is selected from the group consisting of mannitol, raffinose, lactose, maltodextrin and trehalose.  15. The method according to item 13, in which the organic salt is selected from the group consisting of sodium citrate, sodium acetate and sodium ascorbate.  16. An oral insulin composition for pulmonary delivery, containing a dry powder of individual particles, which include insulin in an amount of from 20 to 80% by weight in a pharmaceutical carrier and have a size in the range from 0.1 μm to 10 μm.  17. The oral insulin composition according to clause 16, in which the pharmaceutical carrier substance comprises a carbohydrate selected from the group consisting of mannitol, raffinose, lactose, maltodextrin and trehalose.  18. The oral insulin composition according to clause 16, in which the pharmaceutical carrier substance includes an organic salt selected from the group consisting of sodium citrate, sodium gluconate and sodium ascorbate.  19. The oral insulin composition obtained by the method according to item 12.

Images