Methods and compositions for pulmonary delivery of insulin

 

(57) Abstract:

The invention relates to the field of medicine. Systemic delivery of insulin to the mammal host occurs by inhalation of a dry powder insulin through the mouth. Dry insulin powders quickly absorbed through the alveolar region of the lungs. 5 C. and 14 C.p. f-crystals, 16 ill., 5 table.

BACKGROUND OF THE INVENTION

1. The technical field

This invention relates in General to methods and compositions for pulmonary delivery of insulin to patients with diabetes. In particular this invention relates to pulmonary delivery of drugs dry powder insulin 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 cells of the pancreas in normal (neadiabaticheskikh) individuals. Insulin is necessary for the regulation of carbohydrate metabolism by lowering levels of glucose in the blood, and systemic deficiency causes diabetes. Survival of diabetic patients depends on the frequency and duration of insulin, which maintains an acceptable level of glucose in the blood.

The more insulin is administered glucose levels in the blood, it is often necessary to inject insulin at least once or twice a day, moreover, when necessary, impose additional injections of fast-acting insulin. Invasive method of treatment of diabetes may require even more frequent injections, when the patient is carefully controlled levels of blood glucose, using at-home diagnostic kits. The invention in particular relates to the introduction of fast-acting insulin, which is able to provide peaks of serum insulin within one hour and fall of glucose within 90 minutes.

Administration of insulin by injection is undesirable for several reasons. First, many patients find it difficult and cumbersome to be injected yourself as often as necessary to maintain acceptable levels of glucose in the blood. This dislike can lead to failure of the regimen of medicines that in the most severe cases can be life threatening. In addition, systemic absorption of insulin after subcutaneous injection is relatively slow, often requires 45 to 90 minutes, even when using fast-acting formulations of insulin. Thus, there has long been a challenge in the development of alternative formulations of insulin and routes of its introduction, which would allow isbilen.

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

Although these techniques provide an opportunity to avoid the discomfort and poor compliance associated with subcutaneous injections, each of them suffers from its own limitations. Intrarectal and intravaginal routes uncomfortable, very uncomfortable, and the latter is not available for the entire population of diabetics. Intranasal delivery may be convenient and possibly less unpleasant than the injection does not require the use of potentially toxic "penetration enhancers", which have an impact on the passage of insulin through the nasal mucosa, which is characterized by a thick epithelial layer, resistant to the passage of macromolecules. Of particular interest to this invention is pulmonary delivery when the patient makes the inhaled formulations of insulin and when there is systemic absorption through the thin layer of epithelial cells in the alveolar regions of the lung. Apparently, such pulmonary delivery of insulin provides faster system availability than does subcutaneous injection, and it avoids using avco, in most cases, carried out by spraying the liquid formulations of insulin, requiring the use of heavy liquid sprayers. In addition, the aerosols formed by using such nozzles have a very low concentration of insulin, inevitably leading to a large number of inhalations to provide an adequate dose. The insulin concentration is limited due to the low solubility of insulin in the corresponding aqueous solutions. In some cases, up to 80 or more breaths may be required to achieve adequate dose that leads to the inception of from 10 to 20 minutes or more.

It is desirable to develop improved methods and compositions for pulmonary delivery of insulin. Particularly desirable if such methods and compositions would be quite easy to implement injecting themselves even outside the home and will be able to deliver the desired total dose with a relatively small number of breaths, preferably less than ten. Such methods and compositions should also provide for rapid systemic absorption of insulin, preferably reaching peak serum within 45 minutes or less and get a drop of glucose within about one hour or less. Such Bitola treatment, when the injection of insulin intermediate steps and long-term steps can be reduced or eliminated. The compositions of this invention must be, in addition, stable, preferably consisting of a concentrated dry powder formulations.

2. Prior art

Respiratory aerosol delivery of aqueous solutions of insulin are described in several 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; Overlying 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 powder drugs, such as insulin, in the filler with the carrier in the form of large particles, is described in U.S. Pat. USA N 5254330. Inhaler with measuring dose (DHS, DI) for delivery of crystalline insulin, suspended in propellant (liquid media aerosol), described Lee and Sciura (1976) J. Pharm. Sei, 65:567-572. DHS (DI) for insulin delivery in the spacer for regulating the speed of inhalation flow, described in U.S. Pat. USA N 5320094. Intrabronchial introduction of recombinant human insulin briefly described Schluter e is Dov, including insulin, in the presence of the amplifier described in U.S. Pat.USA N 5011678 and Nagai et al. (1984) J. Contr.rel.1:15-22. Intranasal delivery of insulin in the presence of enhancers and/or contained in the formulations of controlled release, described in U.S. Pat. USA N 5204108; 4294829; 4153689; PCT Applications WO 93/02712, WO 91/02545, WO 90309780, and WO 88/04556; Patent UK 1527605; Ryden and Edman(1992) Int. J. Pharm. 83: 1-10; and Bjork and We (1988) Int. J. Pharm. 47:233-238. Obtaining and stability of amorphous insulin described Rigsbee and Pikal at the American Association of Pharmaceutical Sciences (AAPS), November 14-18, 1993, Lake Buena Vista, Florida. Methods spray drying polypeptide, polynucleotide and other labile drugs in the media, which forms an amorphous structure, which stabilizes the drug described in the application on Heb.Pat. 520748.

The invention

According to the invention methods and compositions for aerosolization and systemic delivery of insulin to the mammal host, in particular the patient-person suffering from diabetes, provide for rapid absorption into the bloodstream, while eliminating subcutaneous injection. In particular, the methods of the present invention is based on the pulmonary delivery of insulin in the form of a dry powder. Unexpectedly found that respirable dry powders insulin Osada into the bloodstream. Thus, pulmonary delivery of insulin powders can be an effective alternative to introduction via subcutaneous injection.

In the first aspect of the present invention, insulin is provided in the form of a dry powder, usually, but not necessarily, mainly amorphous state, and is dispersed in the air or other physiologically acceptable gas stream to form an aerosol. The aerosol is in the camera with the tip of the mouth, where it is suitable for subsequent inhalation by the patient. Not necessarily dry powder insulin is combined with a pharmaceutically acceptable dry powder carrier, as described in more detail below. Powder insulin preferably includes particles having a diameter less than 10 microns, more preferably less than 7.5 μm, and most preferably below 5 μm, and usually it is in the range from 0.1 μm to 5 μm. Suddenly discovered that the dry powder insulin compositions of the present invention are absorbed in the lung without the use of penetration enhancers, such as amplifiers required for absorption through the mucous membrane of the nose and upper respiratory tract.

In the second aspect, the invention measures the particles less than 10 microns, which can be connected with the dry powder pharmaceutical carriers. Insulin composition is preferably free from penetration enhancers and includes particles having diameter less than 10 microns, preferably less than 7.5 μm, and most preferably below 5 μm, and typically in the range from 0.1 μm to 5 μm. Usually the content of dry powder insulin 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 salts, amino acid, peptide or protein, as described in more detail in the future.

In the third aspect of the present invention, insulin dry powder obtained by dissolving insulin in aqueous buffer to form a solution and spray drying the solution to obtain mainly amorphous particles having a particle size of less than 10 microns, preferably less than 7.5 μm, and most preferably below 5 μm, and usually their size is in the range from 0.1 μm to 5 μm. Arbitrarily, the pharmaceutical carrier is also dissolved in the buffer, with the formation of a homogeneous solution in which the spray drying of the solution gives the individual particles, including Inam is a carbohydrate, organic salt, an amino acid, peptide, or protein, which provides mainly amorphous structure after spray drying. Amorphous media can be either glassy or koutsokoumnis, and it improves the stability of insulin during storage. Mostly, such stable formulations, in addition, are able to effectively deliver insulin into the bloodstream when inhaled into the alveolar region of the lungs.

Additional understanding of the nature and advantages of this invention become apparent from consideration of the following sections of the description and drawings.

Brief description of drawings

Fig. 1 is a schematic illustration of a system for aerosolization dose of insulin according to the method of the present invention.

Fig. 2 is a schematic illustration of how is inhaled by the patient 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 receiving glucose response after administration of three different aerosol of dry powder formulations. Each point represents the average value for three different CR min and 20 minutes For powders 87% insulin/citrate, 20% insulin-mannitol/citrate and 20% of insulin-raffinose/citrate, respectively. Animals are locked at night.

Fig. 4A and 4B are graphs illustrating characteristics of the average serum concentration of insulin and glucose in time, respectively, when comparing aerosol and subcutaneous injection cynomolgus monkeys. For aerosol group is given the average value of the three monkeys, for a group subcutaneous injection is given the average value for the four monkeys.

Fig. 5A is a graph illustrating the mean concentration of insulin in time for subcutaneous injection (a) and inhalation of three clicks (), in humans.

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

Fig. 6A is a graph illustrating serum insulin concentration over time as a result of subcutaneous injection (a) and three taps with the introduction of the aerosol (), in humans.

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

Fig. 7A and 7B represent the comparison of variation from subject to subject levels of serum insulin (7A) and the HPLC) chromatogram of human insulin. Fig. 8A represents the chromatogram of the insulin standard, balanced (Stressed) in 10 mm HCl at 25oC, demonstrating human insulin, eluting through 23, 87 minutes, desamethasone, eluting through 30,47 minutes. In Fig. 8B presents a similar chromatogram of standard human insulin. Fig. 8C is similar to the chromatogram of formulation reconstructed, spray dried insulin obtained according to this invention.

In Fig. 9 shows the UV spectrum of insulin compositions before and after spray drying. In the visible part of the spectrum is not observed scattering light that indicates that the insulin is not aggregates during the process of spray drying.

Detailed description of the specific variants of embodiment of the invention

According to this invention is provided insulin in the form of a dry powder. The term "dry powder" realize that the moisture content in the powder below about 10% by weight, usually below about 5% by weight, and preferably below about 3% by weight. The term "powder" understand that insulin includes free flowing (macro)particles having a size selected to provide PR is e than 7.5 μm, and most preferably less than 5 μm, and usually it is in the range from 0.1 μm to 5 μm in diameter.

This invention is based, at least in part, on the surprising observation that the dry powder insulin quickly and easily absorbed through the lungs of the host. It was unexpectedly found that the dry insulin powders can reach the alveolar region of the lung, whereas 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, we should expect that when the particles pass through the Airways of the lung (which has a relative humidity of over 99% at 37oC), the individual particles must have a tendency to absorb water and grow up to the effective size of the particles larger than 10 μm, the upper limit of this invention. If a significant fraction of the insulin particles larger than the size of the target area, it should be expected that the particles will be deposited in the Central Airways of the lungs rather than inside the alveolar region, thus limiting the delivery and subsequent systemic absorption. In addition, the liquid layer above epitheli the m way prior to this invention it was unpredictable whether dry insulin particles to dissolve in the deposition within the alveolar regions of the lungs. Suddenly the dry powders of insulin were able to penetrate into the alveolar region of the lung, and to dissolve immediately after their deposition within the alveolar region of the lung. Dissolved insulin is then able to pass through the epithelial cells into the circulation.

Now consider that the effective absorption of insulin is the result of rapid dissolution in ultrathin (< 0.1 ám) liquid layer of the alveolar content. Particles of this invention thus have an average size from 10 to 50 times larger than the thickness of lung liquid layer, making unexpected that the particles dissolve and insulin quickly systemically absorbed. Indeed, as shown in the Experimental section below, the dry insulin formulations of the present invention can provide faster peaks of serum insulin and fall of glucose, than this is achieved by subcutaneous injection, which today is the most common form of insulin administration. Understanding the exact mechanism, however, does not atimeline compositions according to the invention, mostly free from penetration enhancers. "Penetration enhancers" are surface-active compounds that promote the penetration of insulin (or other medicinal means) through the membrane of the mucosa or content, and are intended for use in intranasal, intrarectal, intravaginal the formulations of the drug. Examples of penetration enhancers are salts of bile acids, for example taurocholate, glycocholate, and dezoksiholatom; fusidate, such as tradewikipedia; and biocompatible detergents, such as Twins, Laureth-9, etc., the Use of penetration enhancers in the formulations to the lungs, however, is generally undesirable because such surfactants can have a harmful effect on the epithelial blood barrier in the lung. Suddenly discovered that the dry powder insulin compositions of the present invention is easily absorbed in the lungs without the use of penetration enhancers.

Insulin dry powder, suitable for use in this invention include amorphous insulin, crystalline insulin and a mixture of both amorphous and kristallovich, which lead to a mainly amorphous powder, having a particle size within videosteenage range. Alternatively, amorphous insulin can be obtained by freeze-drying (drying-freezing), vacuum drying or evaporative drying of a suitable solution of insulin in terms of providing an amorphous structure. Amorphous insulin, thus obtained, can then be chopped or ground to obtain a particle size within the desired range. Crystalline dry powder insulin can be obtained by crushing or grinding in a jet mill mass of crystalline insulin. The preferred method of obtaining insulin powders, including (macro) particles in the desired size range, is spray drying, where the net 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 from about 2 to 9. Insulin was dissolved at a concentration of from 0.01% by weight to 1% by weight, usually from 0.1% to 0.2%. Then the solutions can be subjected to spray drying in a standard equipment for spray drying from commercial suppliers, such as Bu is linovia powders can basically, consist of particles of insulin within the range of the desired size and can be mostly free from any other biologically active ingredients, pharmaceutical carriers, etc., Such "pure" formulations may include minor amounts of components such as preservatives present in small quantities, typically less than 10% by weight and usually less than 5% by weight. Using such pure formulations, the number of inhalations required to even high doses can be significantly reduced, often to only a single breath.

Insulin powders of this invention may not necessarily be either with pharmaceutical carriers or excipients, which are suitable for respiratory and pulmonary administration. Such media can serve just as filling means, when it is required to reduce the concentration of insulin in the powder, which will be introduced to the patient, but may also serve to increase the stability of the insulin compositions and to improve the dispersive ability of the pigment powder inside the device for dispersion of the powder in order to obtain more efficient and reproducible delivery of insulin and to improve the characteristics of manual manip CLASS="ptx2">

Suitable substances-carriers may be in the form of amorphous powder, 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, etc., disaccharides such as lactose, trehalose, cellobiose, and etc., cyclodextrins, such as 2-hydroxypropyl--cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, etc., (b) amino acids such as glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine, and so forth; (C) organic salts derived from organic acids and bases, such as sodium citrate, sodium ascorbate, gluconate, magnesium gluconate, sodium, tromethamine hydrochloride, etc.; (d) peptides and proteins, such as aspartame, human serum albumin, gelatin, etc.; (e) alditol, such as mannitol, xylitol, etc., the Preferred group of carriers include lactose, trehalose, raffinose, maltodextrins, glycine, sodium citrate, tromethamine hydrochloride, human serum albumin and mannitol.

Such substances-carriers can be shifted with insulin before spray drying, i.e., by adding carrier-ventureone with particles of insulin and as part of the insulin particles. Typically, when the carrier is produced by spray drying together with insulin, insulin will be present in each individual particle at a weight percentage ranging from 5% to 95%, preferably from 20% to 80%. The remaining particles will be mainly to provide substance-media (and typically ranges from 5% to 95%, usually 20% to 80% by weight), but it will also include a buffer(s) and may include other above-described components. The presence of substance-media particles that are delivered to the alveolar region of the lung (i.e., particles in the desired size range below 10 microns), as set slightly prevents systemic absorption of insulin.

Alternatively, the carriers can be obtained separately in the form of a dry powder and combine with dry powder insulin by mixing. Separately obtained powdery carriers usually must be crystalline (to avoid absorption of water), but can, in some cases, be amorphous or mixtures of crystalline and amorphous powders. The particle size of the carrier should be chosen so as to improve the fluidity of the powder of insulin, typically the particle size is in the range from 25 μm to 100 μm. Particles n is from insulin delivery device prior to inhalation. Thus, the particles, which penetrate into the alveolar region of the lung, should, in the main, consist of insulin and buffer. The preferred substance carrier is a crystalline mannitol having a size in videoustanovok range.

Dry insulin powders of this invention can also be mixed with other active ingredients. For example, to improve the treatment of diabetes, it is desirable to combine a small amount of Amylin or active analogs of Amylin to insulin powders. Amylin is a hormone that is secreted together with insulin from pancreatic-cells in normal (non-diabetic) individuals. Believe that Amylin modulates the activity of insulin ib vivo, and suggest that the simultaneous introduction of Amylin to insulin may improve control of blood glucose. The combination of dry powdered Amylin to insulin in the compositions of the present invention provides a particularly convenient product to achieve the simultaneous introduction. Amylin can be combined with insulin at a content of 0.1% by weight to 10% by weight (calculated on the total weight of the insulin dose), preferably from 0.5% by weight to 2.5% by weight. Amylin available from commercial postava, for example, Amylin can be dissolved in water or other suitable solutions together with insulin, and, optionally, together with the media, and then the solution is spray dried, obtaining a powdery product.

Dry powder insulin compositions of the present invention is preferably subjected to the aerosolization by dispersion in the flow of air or other physiologically acceptable gas flow in the normal way. One system suitable for such dispersion, described in the concurrently pending application, U.S. Ser. N 07/910 048, which published as WO 93/00951, a full description of which is here shown as prior art. With regard to Fig. 1, the 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. Receiving chamber 10 includes a tip for the mouth 12 at the opposite end from the point of entrance of the air dispersion powder. Volume of the chamber 10 is large enough to enclose the desired dose and the camera can have guides screens and/or one-way valves to ensure deterrence, after the dose of insulin powder is enclosed in a chamber 10, the patient P (Fig. 2) does ingal the Yu, the incoming air is introduced through the tangentially located inlet port 14 for air, and the air flows generally in the form of a whirlwind, carrying aerosol insulin from the camera into the lungs of the patient. Volume of the chamber and aerosol dose such that the patient is able to make a fully inhalation of the dose of aerosol insulin with the amount of air sufficient to ensure the delivery of insulin in the lower alveolar region of the lung.

Such aerosol powders insulin is particularly useful instead of subcutaneous injections of fast-acting insulin used to treat diabetes and related insincerities. Unexpectedly found that the aerosol introduction of dry powder insulin leads to much more rapid absorption of insulin and glucose response than this is achieved by subcutaneous injection. Thus, the methods and compositions of this invention should be particularly valuable in the treatment protocols, where the patient often controls the levels of glucose in the blood and enters, as necessary insulin to maintain target serum concentration, but, in addition, they are useful in cases when you want, systemic injection of insulin. The patient may receive trebuia delivery of insulin using the just described method, usually is in the range from about 15% to about 30%, with individual doses (inhalation), typically range from about 0.5 mg to 10 mg Usually the total dose required insulin within one respiratory injection is in the range from about 0.5 to 15 mg. Thus, the desired dose may be effective for the patient, making 1 inhalation to 4 breaths.

The following examples are for the purpose of illustration and not as limitations.

EXPERIMENTAL PART

Materials and methods

Substances

Human crystalline zinc insulin, 26,3 Units/mg, (Lilly Lot # CC) receive from Elililly and Company, Indianapolis IN, as installed, has a purity of > 99% defined using offrig (rpHPLC). FSSA (USP) mannitol get from Roguette Corporation (Gurnee, IL). The raffinose get y Pfanstiehe Laboratories (Waukegan, IL). Sodium citrate dihydrate, USP, ACS and citric acid monohydrate USP receive from I. T. Baker (Phillipsburg, NI).

Obtaining powder

Powders insulin is produced by dissolving mass of crystalline insulin in sodium citrate buffer containing the excipient (mannitol, or raffinose or without filler) to obtain the final concentration of solids 7.5 mg/ml and pH 6,70,3. Spray sushila 5 ml/min, that led to the exit temperature of 70-80oC. Then the solution is filtered through 0.22 μm filter and subjected to spray drying in a Buchi spray drier with the formation of a fine white amorphous powder. The obtained powders stored in tightly closed containers in a dry atmosphere (< 10% RH).

Analyses of the powder

The distribution of powder particles size was determined using the liquid sedimentation by centrifugation in particle size analyzer Horiba CAPA-700 Particbe Size Analyzer after dispersion of powders in Sedisperse A-11 (Micromeritics, Norcross, GA). The moisture content of the powders was determined by Karl Fisher, using moisture meter Mitsubishi CA-06 Moisture Meter.

Safety of insulin before and after the formation of the powder was determined by comparing with standard human insulin, by re-dissolving the weighed powder in distilled water and comparing the re-dissolved solution with the initial solution, passed through a spray dryer. Retention time and peak area for affric (rpHPLC) was used to determine modified whether chemically or degraded during the processing of the insulin molecule. UV absorption is used for opreme, determine the pH of the original and reconstructed solutions. The amorphous nature of the insulin powder was confirmed with the aid of polarized light microscopy.

The impact of aerosol on rats

Experiments on rats were performed in the chamber for aerosol exposure. The female rats (280-300 g) were not fed during the night. Animals (21 - 24/experiment) were placed in a Plexiglas tube and installed in 48 port the camera, nose - only, for aerosol exposure (In-Tox Products, Albuquetque, NM). The air flow in the area of inhalation was established when a 7.2-9.8 liters/min, and air was removed by vacuum so as to create a weak negative pressure (about 1.5 cm H2O) in the chamber defined by the magnagelic meter. Times of aerosol exposure was between 5-20 minutes, depending on how much powder was introduced into the chamber. The powders were injected manually into the small Venturi (Venturi) nozzle, which was dispersional the powder particles to form a thin aerosol cloud. Venturi (Venturi) nozzle worked at a gauge pressure of more than 1,055 kg/cm2(15 psig), and air duct installed from 7.2 l/min up to 9.8 l/min Venturi (Venturi) nozzle inserted in the bottom of a transparent Plexiglas chamber of dispositio.

Calibration aerosol chamber for rats

The concentration of powder in the area of inhalation was measured by repeated timed sampling filter in the area of inhalation in In-Toch filter holder on the way vacuumised at the rate of 2 liters/min flow. The camera was calibrated with animals and no animals. The weight of the powder was determined gravimetrically. The particle size of the powders in the area of inhalation was determined using a cascade impactor (In-Tox Products) placed in the hole for breathing and working in the mode of flow of 2 liters/min. Powder mass at each stage was determined gravimetrically

In each test with the powder used 21-24 rats and aerosol exposure lasted 5-20 minutes. Three rats were scored at time 0 and then at CA. 7, 15, 30, 60, 90, 120, 180 and 240 minutes after exposure aerosol. Animals were anestesiology, opened the abdominal cavity and selected a large sample of blood from the abdominal aorta. Then, animals were scored by cervical dislocation.

Blood was allowed to clot at room temperature for 30 minutes and then centrifuged for 20 minutes at 3500 rpm in test tubes for separation of serum. Serum was either analyzed immediately, regulation 3 rats were scored, took their blood and their lungs were washed in phosphate-saline buffer solution (FBI) six times in 5 ml. Quantity of insulin in the final summative wash sample was used as aerosol dose for rats in the calculation of bioavailability.

Exposure system for primates

For investigations of aerosol impacts on primates (3-4 animals/group) used young wild male cynomolgus monkeys, line Macaca fascicularis (2-5 kg) (Charles River Primates, Inc.). Animals were either injected subcutaneously Humulin (Humulin) (Eli Lilly, Indianopolis, Indiana), or were subjected to powder aerosol insulin. Each animal was placed head - only section for exposure, to ensure a fresh supply of the test atmosphere at an adequate flow rate (7 l/min), providing a minimum oxygen demand animal. Animals kept in the device type chairs, where they were placed in pramoedya position (sitting with legs up). Helmets (hoods) are transparent, allowing animals to fully look around. A permanent catheter was placed in the leg so that blood samples could be taken at any time. The monkey was awake during the entire procedure, and looked calm. The blood of primacy was treated with t the AET monitor for inhalation, which allows you to measure the amount of air, inhalation of each monkey. This value, multiplied by measuring the concentration of insulin in the air we breathe, allows you to accurately calculate how much insulin is inhaled each animal.

Trials in humans

Insulin administered 24 normal human subjects subcutaneously, or by inhalation of an aerosol of dry powder insulin. Each subcutaneous injection consists of 10,4 E Humulin (Humulin R), 100 u/ml (Eli Litly, Indianapolis, Indiana). Dry powders of amorphous insulin, and was obtained by spray drying, as described above, with 20% by weight of mannitol as filler. Dose (5 mg) insulin dry powder was dispersible in high-speed air stream to obtain fine aerosol, which was in the camera. Each subject did inhalation aerosol powder through the implementation of slow, deep breaths each spherical aerosol mass (bolus), or pressing. The powder was administered by three strokes for dose 31,9 E). Serum levels of insulin and glucose were determined over a certain period of time, as described below.

Assays of serum

Serum levels of insulin in rats, primates and humans were determined using Coat-Artie samples constructed standard curves. The assay sensitivity was approximately 43 PG/ml Variance within assay (%CV) was < 5%. Tests for glucose was carried out using Colifornia Veterinary Diagnostics, Inc.in West Sacramentc. CA, using strips reagent Gluose IHK Reagent Pack System analyzer Bcehringer Mannheim/ Hitachi 747 Analyzer. Variance within assay (%CV) was < 3%.

In experiments on rats relative bioavailability of the aerosol was calculated by comparing the area under the curve (CPD) for corrected dose immunoreactive insulin (IRI) profile of concentration - time area under the curve, obtained by subcutaneous injection. In rats, all washed a lot of insulin used in the form of an aerosol dose. A certain amount of insulin absorbed to the lungs, can be washed, so the dose that was determined using this technique, probably a bit low (undervalued) relative to the total deposited dose. Any fixes for this alleged loss was not done.

In experiments on monkeys relative bioavailability was calculated similarly to the above calculation for rats, except that instead of using insulin, washed from the lung as an aerosol dose used oby in the nasal passages and throat, was included in the dose assessment. In monkeys the whole insulin, which was received by the animal, was included in the assessment of the doses.

The results of absorption of insulin in rats

All insulin powders used in animal studies, had particle sizes (mass-average diameters in the range of 1-3 microns and a moisture content of 3%. The purity of insulin powders measured using offrig (rpHPLC) was > 97%. A typical chromatogram of 20% of the insulin formulations is shown in Fig. 8S. Powders gave a clear solution in the reconstruction of clean water absorption < 0,01 in the ultraviolet region at 400 nm and a pH of 6.7 to 0.3. In Fig. 9 shows a typical ultraviolet (UV) spectrum for 20% of the insulin formulation.

The following three formulations of insulin powder were tested on rats in the form of aerosols into the in Toch 48 port the camera for exposure.

1. 87,9% insulin; 11.5% of sodium citrate; 0,6% citric acid.

2. 20% insulin; 66% mannitol; 12.4% of sodium citrate; 0,6% citric acid.

3. 20% insulin; 66% raffinose; 12.4% of sodium citrate; 0,6% citric acid.

Table 1 lists the key dimensions for three different research exposure in rats, including characteristics of the aerosol in the zone ª rats (34-67%) due to losses in the walls due to adhesion (collisions) and incomplete dispersive ability of the pigment powder during the powder supply. The size of aerosol particles in the zone of breathing, however, was perfect for pulmonary deposition (1,3-1,9 µm) and was slightly less than the particle size of the original formulation (2.0 and 2.8 μm) due to selective loss of particles of larger size in the camera for exposure of animals.

Table 2 shows the results on serum insulin and glucose of the three aerosol research and one subcutaneous studies in rats. In Fig. 3A and 3B presents the concentration profiles of serum immunoreactive insulin (IRI) in time and concentration profiles of serum glucose over time for the three formulations (formulations), entered using aerosol. Table 3 presents tmaxfor insulin and tminfor glucose from three different studies, as well as the relative bioavailability of aerosol in comparison with subcutaneous (SC) injection.

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

The results in primates

Dose identity is yanam, to get the PC results for comparison with the results of the application of the aerosol (Fig. 4A and 4B). Table 4 presents the effects on monkeys aerosol. Table 5 shows the mean levels of serum insulin and glucose for aerosol exposure aerosol and subcutaneous studies. Aerosol dose gives a strong insulin and glucose response (high dose). Fig. 4 illustrates the comparison of the average profiles of serum insulin for two aerosol and one PC research. From PPK (AUCS) of these profiles relative bioavailability of aerosolized insulin, as calculated, is 12%.

The results on the people

Comparative results between the delivery of insulin by respiratory and using subcutaneous injections are presented in table 5. Respiratory aerosol delivery leads to more rapid absorption (peak 20 minutes) than injection (peak after 60 minutes), with a more rapid glucose response (drop after 60 minutes) than in the case of injection (drop after 90 minutes). Reproducibility was good, if not better in the case of aerosol than in the case of injections, as for insulin and glucose response. Injected dose was carefully adjusted by weight, aerosol dose not. Bioa 28-36%. Bioavailability of aerosolized insulin, based on the area under the curve for insulin, compared with injection, 22.8, for a group with 3 clicks.

The results of the tests on humans is shown in Fig. 5A-5B. Fig. Figure 5A shows the average levels of serum insulin in time with the introduction by subcutaneous injection (a), inhalation (3 hits) (). Similarly to Fig. 5B presents the average levels of serum glucose. Spiking insulin and fall of glucose is shown in Fig. 6A and 6B, respectively, while the variation between subjects in the definitions of serum insulin and glucose is shown in Fig. 7A and 7B, respectively.

In addition, shallow inhalations (tidal maelstrom breath) monkeys during aerosol exposures do not represent the optimal procedure breath deep Deposit (deposits) in the lungs. Higher bioavailability observed in humans (table 5), as expected in the case when using the optimal procedure is breathing and when using aerosol bolus for oral inhalation, not by nasal inhalation.

Although the foregoing invention has been described in some detail by way of illustration and example, for the purpose of making the clarity is going beyond the scope of the following claims.

1. The method of aerosolization dose of insulin, namely, that the dry powder insulin is dispersed in the gas stream with the receipt of an aerosol particle size of insulin from 0.1 to 10 μm, which is placed in the camera with the tip of the mouth, for subsequent inhalation by the patient.

2. The method according to p. 1, in which the insulin is present in a dry powder carrier in a weight concentration ranging from about 5 to 99%.

3. The method according to p. 2, in which the insulin is present in a dry powder carrier in a weight concentration in the range from 20 to 80%.

4. The method according to p. 2 or 3, in which powdered media includes carbohydrate, organic salt, an amino acid, peptide or protein.

5. The method according to p. 1, in which the dry insulin powder includes particles having an average size less than 10 microns.

6. The method according to p. 1, in which the dry insulin powder includes particles with a size in the range from 0.1 to 10 μm.

7. The method according to p. 1, in which the dry powder comprises individual particles, including both insulin and the substance of the media.

8. Improved method for respiratory delivery of insulin, in which this improvement includes the provision of insulin in the form of the pharmaceutical carrier, the dispersion of insulin in the air flow and the delivery of insulin through the mouth into the lungs in the form of a dry powder having an average particle size in the range from 0.1 to 10 μm, in which the peak insulin and fall of glucose is achieved faster than with subcutaneous injection of an equivalent amount of insulin.

9. The improved method according to p. 8, in which the insulin is present in a dry powder carrier in a weight concentration ranging from about 10 to 99%.

10. The improved method according to p. 9, in which powdered media includes carbohydrate, organic salt, an amino acid, peptide or protein.

11. The improved method according to p. 8, in which the dry powder comprises individual particles, including both insulin and the substance of the media.

12. The method of obtaining a stable dry powder insulin compositions, including the dissolution of insulin and a pharmaceutical carrier in an aqueous buffer, where insulin composition (insulin and media) is from 0.01 to 1% by weight of the solution, and the insulin is from 20 to 80% by weight of the total insulin composition (insulin and a pharmaceutical carrier in the solution, and spray drying the solution to obtain amorphous particles, including the tion of moisture is less than 10%.

13. The method according to p. 12, in which the pharmaceutical carrier is a carbohydrate, an organic salt, an amino acid, peptide or protein, which gives the powder after spray drying.

14. The method according to p. 12, in which the carbohydrate is selected from the group consisting of mannitol, raffinose, lactose, maltodextrin and trehalose.

15. The method according to p. 13, in which the organic salt is selected from the group consisting of sodium citrate, sodium acetate and sodium ascorbate.

16. Oral insulin composition for pulmonary delivery, containing the dry powder of the individual particles, which include the insulin in an amount of from 20 to 80% by weight of a pharmaceutical carrier and have a size in the range from 0.1 μm to 10 μm.

17. Oral insulin composition on p. 16, in which the pharmaceutical carrier substance comprises a carbohydrate selected from the group consisting of mannitol, raffinose, lactose, maltodextrin and trehalose.

18. Oral insulin composition on p. 16, in which the pharmaceutical carrier is a substance includes an organic salt selected from the group consisting of sodium citrate, sodium gluconate and sodium ascorbate.

19. Oral insulin composition obtained SPO is

 

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