TW576750B - Pharmaceutical composition for respiratory/pulmonary delivery of insulin and preparation method thereof - Google Patents

Abstract

Systemic delivery of insulin to a mammalian host is accomplished by inhalation of a dry powder of insulin. It has been found that dry insulin powders are rapidly absorbed through the alveolar regions of the lungs.

Description

576750 A7 B7 V. Description of the invention () Background of the invention 1. The invention of the chrysanthemum field The present invention is probably related to the method and composition for the administration of insulin to the respiratory tract of diabetic patients. In particular, the present invention relates to the pulmonary administration of dry powdered insulin, and the preparation of this insulin is intended for rapid systemic collection via the lungs. Insulin is a 50-amino acid polypeptide hormone with a molecular weight of approximately 6000, which is produced by the / 3-cells of pancreas in normal individuals (non-diabetics). Insulin is required to regulate carbohydrate metabolism by lowering blood glucose levels, and a lack of uniformity that leads to diabetes. Survival of diabetes depends on frequent and long-term insulin administration to maintain acceptable blood glucose levels . Printed by the Consumers' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs • (Please read the notes on the back before filling out this page)

Insulin is most commonly administered by subcutaneous injection, typically into the abdomen or upper thigh. In order to maintain an acceptable blood glucose level, it is often necessary to inject insulin at least once or twice a day, and if necessary, a rapid injection of supplemental insulin. Further treatment of diabetes requires even higher frequency injections, where the patient uses a home test kit to closely check blood glucose levels. The present invention does not involve the administration of fast-acting membranin, which can provide a local peak of membranin within one hour and glucose spreading within 90 minutes. Insulin administration by injection is undesirable in many ways. First, many patients find it difficult and annoying to frequently inject themselves whenever an acceptable blood glucose level is needed. Such a low willingness will lead to patient disobedience, which may have life in severe cases ~ 4-This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 576750 A7 B7 V. Description of the invention () Danger. Furthermore, the systemic absorption of insulin obtained from subcutaneous injections is quite slow. Even insulin formulas that use rapid absorption often take 45 to 90 minutes. Therefore, it has been a long-term goal to provide another insulin formulation and a route of administration that does not require self-injection and can provide fast and systematic insulin. Other forms of insulin administration have been proposed, including intranasal, rectal, and intravaginal. These techniques avoid the pain and disobedience of subcutaneous injections, each of which has its own limitations. Rectal and vaginal administration is inconvenient and uncomfortable, and the latter is not effective for all diabetic populations. Intranasal administration is more convenient and may be less objectionable than injection, but the use of a potentially toxic " penetration enhancer " is required to complete the passage of insulin through the nasal mucosa because a thick epithelial layer resists the passage of macromolecules. Pending. It is particularly interesting that the invention administers insulin to the lungs. A patient inhales an insulin formula and absorbs it in a thin layer of epithelial cells in the alveolar region of the lungs. These lung insulin administrations have shown that they can provide faster and more systematic access than subcutaneous injections and avoid the need for needles. However, pulmonary administration will eventually become widely accepted. Lung administration has so far been most commonly achieved by spraying with a liquid insulin formulation, requiring the use of a nasty liquid sprayer. Also, the spray formed by this sprayer has a very low insulin concentration, and a large amount of inhalation is necessary to provide a suitable dose. Insulin concentration is limited by the low solubility of insulin in suitable aqueous solvents. For example, it may take 80 or more breaths to reach a suitable dose, which can take 10 to 20 minutes, or longer. -5-I paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) _ (Please read the precautions on the back before filling in this page)

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 576750 A7 B7___ V. Description of the Invention () Provide improved methods and components for insulin lung administration. It is hoped that these methods and compositions allow sufficient self-administration even when away from home, and can give a desired total dose with a relatively small number of breaths, preferably less than ten times . These methods and compositions can also provide rapid, systematic absorption of insulin, preferably reaching a serum peak in 45 minutes or less and causing the "glucose to spread throughout about an hour or less" . These fast-acting formulations are preferred for use in further courses of treatment in which mid- or long-term insulin can be reduced or omitted. The composition of the present invention is also stable, preferably a formula containing a concentrated dry powder. 2 * Background The respiratory administration of sprayed aqueous insulin formulations according to the instructions are discussed in many references, including Gnsslen (1925) Klin. Wochenschr. 4: 71 and Laube et al, (1993) JAMA 269: 2106-21- 9 ;

Elliottet al. (1987) Aust. Paediatr. J. 22_ * 293-297; Vigley et al. (1971) Diabetes 2A: 552-556 · Corthorpe et al. (1992) Pharm. Res. 9_ · 764-768; Govinda (1959) Indian J. Physiol. Pharmacol. 3_ ♦ 161-167; Hastings et al. (1992) J. AppI. Physiol. 73_: 1310-1316; Liu et al. (1993) JAMA 269: 2106-2109; Naganoet a 1 · (1985) Jikeikai Med. J. 22. * 503-506; Sakr (1992) Int. J. Phar · S_6_: 1-7; and Yoshida at al · (1987) Clin · Res · 35: 160- 166 · Pulmonary administration of dry powdered agents, such as insulin, in a large-particle carrier, as described in US Patent No. 5,254,330-6-This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) ~ ί-^ ------ 2 ^^-• (Please read the notes on the back before filling out this page) "， 1T printed by the Consumer Cooperatives of the Central Standards Bureau, Ministry of Economic Affairs ~ 5〇A7 、, _B7 __ 5. Description of the invention () It is beneficially discussed. The administered fixed dose inhaler (MDI) as crystalline insulin suspended in the propellant is in Lee and Sciara (1976) J. Pharm. Sc i. Si: 567-572 has been discussed. An MD I used to administer insulin to a space for adjusting the inhalation flow rate is discussed in U.S. Patent No. 5,320,094. The bronchial administration of recombinant insulin is described in Schl liter et a 1, (Abstract) (1984) Diabetes 33: 75A and Kohler et al. (1984) Ateraw. Lungenkrkh. 1_3_: 230-232. Different peptides, including insulin, are in a booster Intranasal and respiratory administration in the presence of acetone is discussed in U.S. Patent No. 5,01,678 and 卩 383 丨 ^ 3 1. (1984) J. C ο ntr ♦ R e 1. L: 15-22. Intranasal administration of insulin in the presence of a booster and / or in a formulation containing controlled release has been issued in U.S. Patent Nos. 5,204,108; 4,294,829 and 4,153,689; PCT Nos.W0 93/02712, W0 91/02545, W0 90/09780 W0 Application No. 88/04556; British Patent No. 1,527,605; Rydgri and Edman (1992) Int. J. Phar in. 8i: 1-10; and Bjork and Edman (1988) Int. J. Pharm. 4Z · 233 -238 discussed. The preparation and stability of amorphous insulin is discussed in Riggs bee and Pika a 1 in the America Association of Pharmaceutical Sciences (AAPS), November 14-18, 1993, Lake Buena Vista, Florida. The method of spraying dry polypeptides, polynucleotides, and unstable drugs in a carrier that can form an amorphous structure and stabilize drugs is discussed in European Patent Application No. 520 748. Summary of the invention According to the present invention, for a mammalian host (especially a person with diabetes) The paper size applies the Chinese National Standard (CNS) 8-4 specification (210X297 mm) (Please read the precautions on the back before filling this page)

576750 A7 B7__ V. Description of the invention () Patients with diseases) Method and composition for insulin spray and systematic administration, which provides rapid absorption into circulating blood without subcutaneous injection. In particular, the method of the present invention is lung administration for insulin in a dry powder form. Surprisingly, it has been found that inhaled dry insulin powder will adhere to the alveolar region of the lungs and be absorbed by epithelial cells through the alveolar region and enter the blood circulation. Therefore, pulmonary administration of insulin powder is another effective method of administration in addition to subcutaneous injection. First, insulin in the present invention is provided in the form of a dry powder, usually but not necessarily in a substantially amorphous state, and dispersed in an air or other physiologically acceptable air stream to form a spray. This spray is enclosed in a medicine chamber with a mouthpiece, which is then obtained by inhalation by a patient. Alternatively, the dry powdered insulin can be combined with a pharmaceutically acceptable dry powder carrier, as described in more detail below. The insulin powder preferably contains particles having a radius of less than 10 μm, more preferably less than 7.5 μm, and most preferably less than 5 Mm, usually in the range of 0.1 μm to 5 μm. Surprisingly, it has been found that the dry powdered insulin composition of the present invention is absorbed in the lungs without the use of penetration enhancers such as those required for their absorption through the intranasal mucosa and upper respiratory tract. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs f — " ^ ------ ^ 衣 II-(Please read the precautions on the back before filling this page)

In a second aspect, the present invention provides an insulin composition comprising a dry powdered insulin, which has an average particle size of less than 10 Mm and can be combined with a dry powdered pharmaceutical carrier. The insulin composition preferably contains no penetration enhancer and contains particles having a radius of less than 10 μιη, more preferably less than 7. 5 μιη, most preferably less than 5 μιη, usually in the range of 0.1 μm m to 5 μm. In general, the dry powder of insulin is in the composition-8-This paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 576750 A7 B7 5. Description of the invention () There are 5 Up to 99 vt% of insulin, more usually from 15 to 80%, and in a suitable pharmaceutical carrier, usually a class, an organic salt, an amino acid, a peptide or a protein, as described in more detail below Discussion. In the third aspect of the present invention, the preparation of dried insulin powder is performed by dissolving insulin in an aqueous buffer solution to form a solution, and spray-drying the solution to substantially produce amorphous particles having a radius of less than 10 μm. The best is less than 7.5 M m, the best is less than 5 μm, usually in the range of 0.1 μm to 5 μm. Optionally, the pharmaceutical carrier is also dissolved in the buffer to form a homogeneous solution, wherein the spray-drying of the solution produces an individual that contains insulin, the carrier buffer, and any other ingredients present in the solution. Particles. Preferably, the carrier is a type, organic salt, amino acid, peptide or protein, which produces a substantially amorphous structure when spray-dried. The amorphous carrier can be glassy or rubbery and can enhance the stability of insulin during storage. Beneficially, such stable formulations can also be inhaled into the alveolar region of the lungs, effectively injecting insulin into the bloodstream. By referring to the following sections and illustrations of the instructions, the characteristics and advantages of the present invention can be further improved Understanding of the Figures Brief Description of the Figures Figure 1 is a schematic diagram of the structure of a method for spraying a dose of insulin according to the method of the present invention. Figure 2 is a schematic diagram of a patient inhaling an insulin spray dose from the system of Figure 1. Figures 3A and 3B show the absorption of recombinant human insulin in rats-9-This paper size is applicable to China National Standard (CNS) 8-4 (210X297 mm) (Please read the precautions on the back before filling this page)

576750 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 ___ V. Description of the invention () and three different dry powdered formulas caused by the ® glucose reaction after spraying. Each point represents the average value obtained from three different rats. At zero, the dry powder spray maker is activated. The sprays of 87¾ insulin / citric acid, 20¾ insulin-mannitol / citric acid, and 20¾ insulin-melitabose / citric acid powder were completed at 5 minutes, 14 minutes, and 20 minutes, respectively. Animals need to be fasted overnight. Sections 4A and 4B are plots of insulin and glucose illustrating average time-concentration in serum, and the results of spraying and subcutaneous administration in cynomolgus monkeys are compared, respectively. The spray group shows the average of three monkeys, and the subcutaneous injection group shows the average of four monkeys. Figure 5A is a graphical illustration of the results of the mean insulin concentration versus time when administered subcutaneously (0) and three times inhaled (♦) in humans. Figure 5B shows the average glucose concentration relative to the insulin concentration in Figure 5A. Figure 6A is a graphical illustration of the results of the mean serum insulin concentration versus time when humans were administered subcutaneously (0) and three inhaled sprays (·). Figure 6B shows the serum® glucose level relative to the insulin level of Figure 6A. Figures 7A and 7B provide a comparison of the changes in insulin concentration (7A) and glucose level (7B) between subcutaneous injections (0) and three inhalations (·). Figures 8A, 8B, and 8C show the rpHPLC chromatograms of a human insulin. Figure 8 A is a paper size of one of the standard pressurized insulin in 10 mM HC1 at 25 ° C. Applicable to China National Standard (CNS) A4 specification (210X297 mm) · (Please read the precautions on the back before filling (This page)

576750 Α7 Β7 V. Description of the invention () Chromatography 圔 shows that human insulin was pumped out at 23.87 minutes and deamidine insulin was pumped out at 30.47 minutes. Panel 8B 圔 shows a chromatogram similar to one of the human insulin standards. Figure 8C shows a similar chromatogram of one of the recombinant, spray-dried insulin formulations prepared according to the present invention. Figure 9 shows an ultraviolet spectrum of an insulin formula before and after spray drying. The absence of light scattering in the visible light pattern indicates that the insulin did not coagulate during the spray drying process. Detailed Description of Specific Embodiments In accordance with the present invention, insulin is provided as a dry powder. Dry powder means that the moisture content of the powder is less than about 10 wU, usually about 5 wt% or less, and preferably less than about 3 w «. "Powder" means that the insulin contains free-flowing particles with a size that allows penetration into the alveoli of the lungs, preferably with a radius of less than 10 μm, more preferably less than 7.5 μm, and most preferably less than 5 μ m, whose radius is usually in the range of 0.1 μm to 5 μm. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ▼-(Please read the precautions on the back before filling out this page)

The present invention has, at least in part, an unexpected result, that is, dry powder insulin can be easily and quickly absorbed through the lungs of a host. Dry powdered insulin can reach the alveolar region of the lungs, as water-soluble drugs (such as insulin granules) are known to be hygroscopic. See Byron, ed., Respi-ratory Drug Delivery, CRC Press, Boca Raton (1990), P. 150. Therefore, it has been expected that the particles will pass through the respiratory tract of the lungs (the relative humidity at 37 ° C is more than 99¾), the individual particles will have a tendency to absorb water, and will grow to an effective particle size of one of 10 Mm which is greater than the upper limit of the present invention. If a substantial part of the insulin particle is larger than the locked size -11-This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 576750 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Β7 _____ V. Description of the invention () Range, it is expected that the particles will fall in the lungs of the respiratory tract and not in the alveolar region, thus limiting the administration and subsequent systemic absorption. What's more, the fluid layer on the lung epithelial cells is very thin, and usually a part of the radius of the insulin powder is sent by lotus. Therefore, the previous case of the present invention cannot predict whether the dried insulin particles will dissolve after being located in the alveolar region of the lungs. Amazingly, this dry insulin powder clearly penetrates the alveolar region of the lungs and dissolves once it sits in the alveolar region of the lungs. This dissolved insulin can then pass through the epithelial cells and enter the circulation. It is currently believed that the effective absorption of insulin results from its ultra-thin (< K 1 μιη) flow layer which dissolves rapidly on the inner surface of the alveoli. The particles of the invention therefore have an average particle size from 10 to 50 times the flow layer of the lung So that the dissolution of the granules and the rapid and systematic absorption of insulin become unpredictable. Indeed, as shown in the following experiments, the dry insulin formula of the present invention can provide a better than subcutaneous injection (the most common administration Method) Producer has a faster serum insulin peak and M glucose spread. However, the understanding of its correct mechanism need not be explained in the present invention. According to the present invention, its preferred composition does not substantially contain penetration enhancement Agents. ”Penetration enhancers are surface-active compounds that help insulin (or other drugs) penetrate through a mucosal surface or inner layer, and they are formulated for intranasal, rectal, and vaginal drug formulations. Typical Osmotic enhancers include bile salts, such as taurocholic acid, glycine cholate, and deoxycholate; brown mycolates, such as taurine dehydrofumonate; and Biocompatible cleaners such as Twee η, Laurel-9 and similar. However, the use of penetration enhancers in lung-administered formulations is often undesirable because the lung-12-this paper size Applicable to China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 576750 A7 B7 5. The epithelial blood disorder in the description of the invention () can be adversely affected by these surface-active compounds. Surprisingly, it has been found that the dry powder insulin composition of the present invention can be easily absorbed in the lungs without using a penetration enhancer '. Suitable dry insulin powder for use in the present invention includes amorphous insulin, crystalline insulin, and a mixture of amorphous and crystalline insulin. 3 Dry powdered insulin is preferably spray-dried to produce a substantial amount of insulin. In the case of an amorphous powder, the powder has a particle size within the above range. In addition, amorphous insulin can be prepared by freeze-drying, vacuum drying or evaporation drying under the conditions used to produce an amorphous structure using a suitable insulin solution. The amorphous insulin so produced can then be mashed or ground to produce particles in a desired size range. Crystalline dry powder insulin can be formed by mashing or jet milling of crystalline insulin in bulk. The preferred method of forming insulin powder with particles in the desired size range includes spray drying. Pure block insulin (usually in a crystalline form) is first dissolved in a physiologically acceptable aqueous buffer, typically A citric acid buffer with a pH in the range of about 2 to 9. The insulin is dissolved at a concentration of 0.1 to 1 wt%, usually from 0.1 to 0.2%. The solution can then be spray-dried in a conventional spray-drying device obtained from a supplier, such as Buch i, N Bu and the like, and finally a substantially amorphous particulate product is obtained. The dried insulin powder may contain Insulin granules within the required size range as well as ingredients that do not substantially have any other biological activity 'pharmaceutical carriers, and the like. This * pure pure 〃 formula may contain less important ingredients, such as preservatives in small amounts, which are typically less than 10 -13-This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm) * ( (Please read the precautions on the back before filling out this page)

576750 A7 B7 V. Description of the invention () It is usually lower than 5. When using this pure formula, even if a high dose is required, the number of inhalations can be substantially reduced, often requiring only a single breath. The insulin powder of the present invention can be selectively combined with a pharmaceutical carrier or suitable for the respiratory tract and lung Additive combination for administration. When the insulin concentration in a powder administered to a patient is desired to be reduced, the carrier can be used simply as a block, but it can also be used to enhance the stability of the insulin composition, and to improve the powder distribution in a powder. Dispersibility in the device to provide more effective and repeatable insulin administration, and to improve the handling properties of insulin, such as fluidity and solidity that aids manufacturing and powder calming. Suitable carrier materials may be in the form of an amorphous powder, a crystalline powder, or a combination of amorphous and crystalline powder. Suitable substances include (a) carbohydrates, such as monosaccharides, such as sugar, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, trehalose, cellobiose and the like Similar; cyclodextrin, such as 2-hydroxypropyl-cyclodextrin; and polysaccharides, such as melibiose, maltodextrin, dextrin and similar; printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs ( (Please read the notes on the back before filling in this page) (b) Amino acids such as glycine, spermine, aspartic acid, glutamic acid, cysteine, lysine and the like (C) Organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, and the like; Peptides and proteins, such as aspartame methyl phenylalanine, human serum albumin, animal glue and the like; (e) aldols, such as mannitol, xylitol and the like. A preferred carrier contains lactose, trehalose, melibiose, maltodextrin, glycine, sodium citrate, tromethamine hydrochloride, and human serum white-14-this paper applies Chinese National Standard (CNS) A4 Specifications (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 576750 A7 B7___ 5. Description of the invention () Protein and mannitol. The carrier substance can be combined with insulin before spray drying, i.e. by adding the carrier substance to a buffer solution for spray drying. In this way, the carrier substance will be simultaneously formed as part of the insulin particle. Typically, when the carrier is formed with insulin by spray drying, the insulin will be present in the range of 5% to 95%, preferably 20% to 80%, of the individual particles. The remaining particles are mainly carrier material (typically 5% to 95%, usually 20 to 80 wt%), but also contain buffers and may contain other ingredients as described above. It is known that the presence of a carrier substance in the granules of the alveolar region of the lung (i.e., in a size range below the required 10 μm) does not significantly affect the systematic absorption of insulin. In addition, the carrier can be prepared separately in the form of a dry powder and combined with the dry powdered insulin by a stirrer. The separately prepared powder carrier is usually crystalline (to avoid water absorption), but in some cases may be amorphous or a mixture of crystalline and amorphous. The size of the carrier particles can be selected to improve the fluidity of the insulin powder, typically in the range of 25 μm to 100 μm. Carrier granules in this range will generally not penetrate into the alveolar region of the lungs, and are usually separated from insulin in the device being administered prior to inhalation. Therefore, the particles that will penetrate the alveolar region of the lungs contain insulin and buffer. A preferred carrier material is mannitol which is crystalline and has a size within the above range. The dry insulin powder of the present invention can also be combined with other active ingredients. For example, it can be combined with a small amount of starchy cellulose or starchy cellulose analog in insulin powder to improve the treatment of diabetes. Starch fiber quality is -15-This paper size applies Chinese National Standard (CNS) A4 specification (210 × 297 mm) • (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 576750 A7 ___. _ B7____ V. Description of the invention () A hormone, which is secreted by the / 3 cells of the pancreas together with insulin in normal individuals (non-diabetic). It is believed that starch fibrin regulates insulin activity in vivo, and the simultaneous administration of starch fibrous and insulin can improve blood. The control of glucose has been proposed. The combination of dry powdered starch fibroin and insulin in the composition of the present invention will provide a unique convenient product that can be administered at the same time. Starch fibrous substance can be combined with insulin from 0.1 w «to 10 wt% (based on the total weight of insulin in one dose), preferably from 0.5 to 2.5 wt%. Starch fibrous material is available from suppliers such as Amylin Corporation, San Diego, California, and can be conveniently formulated into the composition of the present invention. For example, amylocellulose can be dissolved in an aqueous or other suitable solution with insulin and optionally a carrier, and the solution can be spray-dried to produce a powder product. The dry powdery insulin composition of the present invention is preferably sprayed in a convenient form by dispersing it in moving air or other physiologically acceptable airflow. A system suitable for such decentralization has been discussed in another application numbered 07 / 910,048 (which has been published as WO 93/00951), all of which is incorporated herein by reference. Referring to FIG. 1 herein, the dry, free-flowing insulin powder is introduced into a high-speed air or gas stream, and the resulting dispersion is introduced into a container 10. The container 10 includes a mouthpiece 12 at an inlet dispersed with respect to the air powder. The skeletal volume of the container 10 is adequate to contain a desired dose, and may optionally have a one-way valve for barrier and / or enhanced containment. After a dose of the insulin powder is contained in the container 10, a patient P (picture 2) inhales at the mouthpiece 12-16-This paper size applies to the Chinese National Standard (CNS) A4 (210X297 mm)） Please (Read the notes on the back before filling out this page)

576750 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 ___B7 ___ V. Description of the invention () To introduce the spray dispersion into its lungs. When the patient p inhales, the generated air is introduced through the air inlet 14 in all directions, whereby the air current blows the sprayed insulin from the container into the patient's lungs in an approximately vertical direction. The volume of the container and the spray dose are such that a patient can fully inhale the entire spray insulin dose after inhaling sufficient air to determine that the insulin will reach the low alveolar region of the lung. These spray insulin powders are used in diabetes and related insulin In the treatment of deficiency, it is particularly useful after subcutaneous injection of fast-acting insulin. Surprisingly, it has been found that spray administration of this dry powder insulin can result in significantly faster insulin absorption and glucose response than subcutaneous injection. Therefore, when a patient frequently monitors the blood glucose level during the course of treatment, and when necessary, insulin is administered to maintain a locked serum concentration, the method and composition of the present invention will appear to be of special value, but even in It is also useful when systematic insulin administration is needed. As discussed above, the patient can achieve a desired dose by injecting an appropriate amount of insulin. Through the above method, the efficacy of systematic insulin administration will typically be about 15% to 30% at the dose of each individual (based on each inhalation), that is, typically in the range of about 0.5 mg to 10 mg. Within the range of%. Generally, during a single respiratory administration, the total dose of the desired insulin will be in the range of approximately 0,5 mg to 15 mg. Therefore, the desired dose can be effective by the patient taking 1 to 4 breaths. The examples provided below are for the purpose of description, not for limiting. -17 A paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm)-(Please read the precautions on the back before filling this page. )

576750 V. Description of the invention () Printed A7 B7 experimental materials and methods printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economics. Crystalline human zinc insulin (26.3 Dnits / fflg) (Lilly Lot it784KK2) was obtained from Eli Lillyand Company, Indianapolis, IN And was found to have a purity of > 99% by rpHPLC. USP mannitol was obtained from Roquette Corporation (Gurnee, IL). Triose was purchased from Pfanstiehl Laboratories (Waukegan, IL). Sodium citrate dihydrate, DSP, ACS and citric acid monohydrate USP are prepared from Baker (Phillipsburg, NJ) ° powder. Insulin powder is prepared by dissolving a block of crystalline insulin in an additive (mannitol). 7 or 0.3。 or triose, or without) sodium citrate buffer to obtain a final solids concentration of 7.5 mg / ml and a pH of 6.7 ± 0.3. The spray dryer is operated with an inlet temperature between 11 ° C and 120 ° F and a liquid injection rate of 5 m 1 / mi η, resulting in an outlet temperature between 70 ° C and 80 υ. Then the solution It was filtered through a 0.22 mm filter and spray-dried in a Bushi spray dryer to form a fine white amorphous powder. The obtained powder was dried in a dry environment (< 10% RH ) Is stored in a tight container. Analysis of powder -18-This paper size applies to China National Standard (CNS) 8-4 (210X297 mm) 乂 Please read the precautions on the back before filling this page)

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 576750 A7 B7 V. Description of the Invention () The particle size distribution of the powder was dispersed in Sedisperse A -11 (Micromeritics, Norcross, GA) and dispersed in a Hor i ba CAPA-700 particle size analyzer is calculated by liquid centrifugal sedimentation. The moisture content of the powder was measured by Karl Fischer technology using a M i t s u b i s h i C A-06 hygrometer. The integrity of the insulin before and after powder processing is calculated with respect to a reference standard for human insulin, which can be achieved by re-dissolving the weighed portion of the powder in distilled water and comparing the re-dissolved solution to the The original solution in a spray dryer. With rpHPLC, its residence time and peak area are used to determine whether the vicin molecule has been modified or degraded in the process. UV absorption was used to determine the concentration of insulin (at 278 nm) and whether insoluble coagulation occurred (at 400 rim). In addition, its initial and redissolved pH values were also measured. The amorphous nature of the insulin powder was confirmed by a polarizing microscope. Exposure of rats to sprays Experiments of rats were performed in a spray exposure container. Female rats (280-300 gm) were fasted overnight. Animals (21-24 / experiment) were placed in Plexiglas tubes and filled into a 48-mouth, nose-only spray exposure container (In-Tox product, Albuquerque, NM). The air flow to the breathing area is maintained at 7.2-9.8 liters / minute and removed by vacuum, so that when measured with a magnetic spiral meter, a slightly negative pressure (about 1.5 cm Η 2 〇). The spray exposure time is between 5-20 minutes, depending on the amount of powder being injected into the container. -19-This paper size is in accordance with Chinese National Standard (CNS) Α4 size (21〇 × 297mm) · (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 576750 A7 __ ^ _ _B7___ V. Description of the invention () The powder is injected into a small Venturi nozzle (Venturi η ο zz 1 e) by hand, which applies the powder The particles dispersed to form a fine spray cloud. Venturi-type nozzles are operated at a pressure in excess of 15 psig and the air flow is set at 7.2 I / min to 9.8 I / min. A Venturi-type nozzle was fitted into the bottom of a plexix glass dispersion container (750 ml), which directly delivered the spray to an nasal exposure container. Calibration of Rat Spray Containers The powder concentration in the snore interval was measured by collecting several, timely filter samples with a 2 l / min vacuum flow in the breathing zone with an In-Tox filter container. The container was corrected with and without animals. The powder quality is determined by weight. The particle size of the powder in the breathing area was measured by operating a cascade punch (In Tox product) placed in a breath hole at a flow rate of 2 liters / mi η. The powder quality at each stage was determined by gravimetry. Each powder test used 21-24 rats, and the spray exposure was continued for 5-20 minutes. Three rats were killed at time 0 o'clock after the end of the spray exposure, and then at about 7, 15, 30, 60, 90, 120, 180 and 240 minutes. The animal was anesthetized, its abdomen was opened, and a large blood sample was drained from its ventral aorta by oil. The animal was then killed by a broken neck. The blood was allowed to clot at room temperature for 30 minutes and then centrifuged in a serum separation tube at 3500 rpm for 20 minutes. Serum can be analyzed immediately or frozen at -80 ° C until analysis. Three rats were killed as soon as possible (0-7 min) after the end of the spray application, and their blood was drawn out and six times 5 ml of phosphoric acid were used. Mm) • (Please read the precautions on the back before filling out this page)

576750 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention () Wash the lungs with saline (PBS). In the final rinse sample collected, the amount of insulin was used as the amount of spray in calculating the biological gain of rats. Exposed strips of primates Young, wild-captured male M a c a c a fascicularis lion monkey cynomolgus monkey strips (2-5 kg) (Charles R i ver Primates,

Inc.) was used for primate spray studies (3-4 animals per group). The animal was subcutaneously injected with Humul in (El Lily, Indianapol is, Indiana) or exposed to a powder spray of insulin. Each animal was placed in a head-only exposure device to provide a fresh supply of test environment at an appropriate flow rate (7 L / m in) that provided the animal with the lowest oxygen requirements. The animal was imprisoned in a chair-like device to maintain a straight sitting position. The fume hood is clean, allowing the animal to fully see its environment. An internal catheter is placed in the leg to allow blood samples to be collected at any time. During the entire process, the monkey was completely awake and appeared calm. The treatment of primate blood is the same as in rats (see above). The primate spray exposure strip includes a breathing monitor that allows the amount of air inhaled by each monkey to be counted. This value, together with the insulin concentration measured in the inhaled air, can reliably calculate the amount of insulin inhaled by each animal. Human test Insulin was administered to 24 normal persons by subcutaneous injection and inhalation by spraying dry insulin powder. Each subcutaneous injection contains 10.4U of Humtilin -21-This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) · (Please read the »Notes on the back before filling this page) Winter · Central Standard of the Ministry of Economic Affairs Printed by the Bureau ’s Consumer Cooperatives 576750 A7 _____B7__ 5. Description of the invention () R, 100 U / ffll (Eli Lilly, Indianapolis, Indiana). The dried insulin powder was amorphous and was prepared by the above-mentioned spray drying. The dosage of the dried insulin powder (5 mg) with 20% by weight of mannitol additive was dispersed in a high-speed air stream to produce A fine spray that can be contained in a container. The spray powder is inhaled by taking a slow, deep breath with each spray filling or "puff" each inhalation. The powder was administered in three sprays (for a dose of 3L · 9U). Serum insulin and glucose levels were measured over time as described below. Serum insulin levels in rats, primates, and humans were determined using Coat-A-Count proportional immunoassay kits (Diagnostic Products Corporation, Los Angeles, CA) for human insulin. The standard curve is Made with each batch of samples. The sensitivity of this analysis is approximately 43 pg / ml. The change value (UCV) in the analysis was <5¾. Glucose analysis was performed by California Veterinary Diagnostics, Inc., West Sacramento, CA, using a glucose / ringK reagent strip package from Boehringer Mannheim / Hitachi 7 47 An a 1 y z e r. The change value (% CV) in the analysis was &lt; 3¾. In experiments in rats, the relative bioavailability of the spray was calculated by comparing the area of the insulin (IR I) with the immune response under the curve of concentration vs. time versus the dose obtained by adjusting the dose with those obtained from subcutaneous injection It was. In rats, this total lavage insulin mass was used as a spray dose. Some insulin is absorbed before the lungs can be lavaged, so the dose estimated by this technique may be a slightly lower estimate of the total input dose. The amount of loss assumed in this regard is not corrected. This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) · (Please read the »Notes on the back before filling this page)

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 576750 A7 B7 V. Description of the invention () In the monkey experiment, the calculation of the relative biological gain is similar to that of the above rats, except that it is based on the total amount of insulin inhaled. Instead of lavaged lung insulin in rats as a spray dose. In the rat's dose estimation, only substances falling in the lungs are included, and insulin falling in the nasal passages and throat is not included. In monkeys, all insulin entering the animal is included in its dose estimates. Results of Insulin Absorption in Rats All insulin powders used in animal studies have a particle size (mass intermediate particle size) in the range of 1-3 μm and a moisture content of <3¾. The powder had an insulin purity of &gt; 97% as measured by rpHPLC. The representative chromatogram for this 20% insulin formula is not shown in Figure 8C. Once the powder was reconstituted with pure water, a clear solution having an ultraviolet light absorption value <0.01 at 400 nm and a pH value of 6 · 7 ± 0 · 3 was generated. A representative ultraviolet (UV) spectrum of the 20¾ insulin formula is shown in Figure 9. In a 48-well In-Tox exposure container, the following three groups of insulin powder formulations were tested as sprays in rats. 1. 87.9% insulin; 11.5¾ sodium citrate; 0.6¾ citric acid. 2. 20% insulin; 66% mannitol: 12. «Sodium citrate: 0.6% citric acid. 3. 20% insulin; 66% melibiose; 12 · «sodium citrate; 0.6¾ citric acid. Table 1 lists the main measurements in three different groups of rats exposure studies, including the characteristics of the spray in the breathing zone and container operating conditions. Part of the powder fed into the venturi nozzle will reach the breath of the rat-23-This paper size applies to China National Standard (CNS) A4 (210X297 mm) 乂 Please read the precautions on the back before filling in this Page) clothing. Order 576750

A B Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. The area of the invention () (3 «-67%), due to the loss in the wall, is due to the compaction and dispersion of the powder during the feeding of the powder. The particle size of the spray in the breathing area is ideal for pulmonary administration (1 · 3-1 · 9 µm), and because the large particles are selectively lost in the animal exposure container, the size of the pupae is somewhat larger than the original The formulation is smaller (2.0-2.8 μκι). Table 1. Measured values of spray exposure for rats 88¾ Insulin 201 Insulin mannitol 202 Insulin honey Tricholine flow rate 7.2 L / min 9.6 L / min 9.8 L / min Powder mass median particle size (HMD) 2 · 2 μπι 2.8 μπ \ 2.0 μχη Powder fed into the container 70 mgs 255 mgs 260 mgs Powder feeding time 5 min 14 min 20 min Powder in the breathing zone 40 mgs 171 mgs 88 mgs Insulin in the breathing zone 35 mgs 34 mgs 18 mqs Fly Percentage of total powder in the open area 57% 67% 34% Mass intermediate aerodynamic particle size (MAD) 1.1 mg / L 1.3 mg / L 0.45 mg / L Particle size in the breathing area 1 · 4 μιη 1.9 μπι 1 · 3 μτα Insulin recovered during lavage 30 · 7 ± S. 2 pg 12 · 7 ± 6 · 9 / jg 31 · 6 ± 12.9 pg serum insulin AUC (ng fflin / m1) 104 201 150 9 ^ 17 _ to | Drafts | Table 2 shows the results of serum insulin and glucose of rats obtained by the three groups of sprays and one group of subcutaneous injection studies. Figures 3A and 3B show the concentration-time diagrams of the serum immune response insulin (IR I) and the serum glucose concentration-time diagrams of the three groups of formulations administered by spray. Table 3 shows the comparison with SC injection. Insulin and glucose obtained from different studies and the relative bioavailability of the spray η This paper size applies the Chinese National Standard (CNS) A4 specification (2 丨 〇X 297 mm) '(Please read the precautions on the back before filling (This page) &quot;

I 576750 Β V. Description of the invention (Table 2) Serum insulin obtained from rats and the formula printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (time) 1 Insulin (pg / mlil SD ·) i = 3rats / ti also epi serum 1 grape (mg / dl + l SD) n = 3rats / t iaept β 0 / Douliang plum 0 230 ± 184 106 ± 12 _ Q 0 / 〇0 / ¾ 1¾ rrt spray 12 1020 soil 312 114 ± 10 ”(spray exposure is completed in 5 minutes) 21 165 ± 768 81 ± 10 An average dose = Q 1 μ cf / ~ h · ^ Serving 36 876 ± 764 66 soil 7 W 66 684 ± 416 62 ± 15 96 568 ± 128 65 ± 10 126 564 ± 260 73 ± 11-186 712 ± 140 93 ± 5 20 ¾ Insulin-mannitol _-(spray exposure completed at -14 minutes) _ average dose =-13 rats Λ 0 476 soil 56 165 ± 18 k 22 14761428 117 ± 15 35 2480 ± 892 101 ± 19 fog 57 1204 ± 64 64 ± 13 87 1084 ± 396 63 ± 17 117 664 ± 180 105 ± 38 agent 147 1228 ± 416 108 ± 22 207 676 100 100 119 ± 33 -20¾ Insulin-meltotriose- _ (spray exposure completed at 20 minutes)-Flat horse dose = one "32 Kg / rat_ 0 426 ± 97 157 ± 37 spray 27 2948 soil 2816 139 ± 46 42 1504 ± 592 181 ± 11 fog 57 1272 ± 496 124 ± 45 87 852 ± 164 128 ± 17 * η1 117 604 ± 156 124 ± 9 147 532 ± 172 172 ± 12 207 556 ± 100 218 ± 34, 2η insulin-mannitol_ Dose =: 3 Q μ g nf- «£ a / i-^-t 〇360 ± 140 107 ± 5 k 15 14200 ± 3160 53 ± 2 Under the wind 30 10160 ± 720 24 ± 5 60 11000 ± 1080 28 ± 6 沣 90 2440 ± 1160 25 ± 7 LL 120 3520 ± 840 49 ± 3 shot 180 1280 ± S00 40 ± 17 240 400 * 260 77 ± 34 \ ^: Please read the precautions on the back before filling in this page.) Clothing,,? Τ This paper size applies Chinese National Standard (CNS) Α4 specification (21〇xp7_2 576750 printed by A7 B7, a consumer cooperative of the Central Standards Bureau of the Ministry of Economic Affairs) Description of the invention () Table 4. Exposure data of monkey spray ID Srav. I filter mass (®g) average spray concentration 0g / L) volume inhaled (L) estimated inhaled spray mist mass 0g) estimated inhaled Insulin mass and weight (Kg) Estimated insulin thinness 0 g / kg) AUC (ng jnin / ml) # 1,23-46 1.07 178 8.96 1597 320 3.92% \ 6 34 7 # 2, 23-48 1.01 168 19.98 3363 673 3.81 176.6 1196 # 3, 122-55 0.97 162 14.68 2373 475 4.1 115.7 739 489 ± 178 The results of the holy humans will be compared between the airway administration and subcutaneous injection. As mentioned in Table 5. Respiratory spray administration (peaking at 20 minutes) provides faster absorption than injection (peaking at 60 minutes), which has a faster glucose response (in 60%) than injection (minimizing at 90 minutes). Down to minutes). In both the insulin and glucose reactions, the reproducibility of the spray is not as good as that of the injector. The dose at the time of injection must be carefully adjusted to its weight, but not the spray. The biological activity of the spray insulin is based on the «glucose reaction», which is 28-36¾ relative to the injection. The bioavailability of the aerosol insulin is based on the area under the insulin curve, which is 22.8¾ per three blows relative to the injector. Table 5. Serum insulin and glucose insulin obtained in humans

The dose / injected by the experimented subject * The maximum serum insulin is based on the time of the increase in the insulin AUC or the dose in the foam. The relative μϋ / ml biocapacity 1-24 (SC injection) 10.4U 10.4U 5.8 -20.9 60 min 100,0% 7-24 (3 sprays :) 76,0U 31 * 91] 6.1 -28.5 20 min 22 · 8% _ 27-This paper size applies to China National Standard (CNS) Α4 specification (210 × 297 (Mm) Please read the notes on the back before filling in this page)

576750 A7 B7 V. Description of the invention (). The time when glucose was lowered by the experimental tfs average serum glucose mg / dl mg / dl minimum value 1 SC The relative biological acquisition based on the decrease of glucose 1-24 (SC injection) 93.6 -64.9 28.7 90 min 100 1 100% 7-24 (3 sprays) 91.8-67.6 24.2 60 min 84.3 ^ 27.4% Printed design efficiency of employee consumer cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs = 42% Shown in Figures 5A-5B. Figure 5A shows the mean serum insulin over time as a result of subcutaneous injection (0), inhalation (three sprays, ·). The average serum glucose level is similar to that shown in Figure 5B. Peak insulin and minimum glucose are shown in Figures 6A and 6B, and changes in serum insulin and glucose in the subject are shown in Figures 7A and 7B, respectively. In addition, the monkey's superficial breathing (inhalation) during spray exposure did not perform optimal breathing methods that fell deep in the lungs. As expected, a higher bioavailability was seen in humans when the most appropriate breathing method was used and the spray was inhaled through the mouth rather than through the nose (Table 5 :). Although for the purpose of clear understanding of the foregoing invention, certain details have been discussed by way of description and examples, it is obvious that it may still fall into this patent when making certain changes or modifications. Within the range of _. · (Please M-read the notes on the back before filling this page) · # 衣. Order

-28-This paper size applies to China National Standard (CNS) A4 (210X 297mm)

Claims (1)

576750 576750 Member of the Intellectual Property Office of the Ministry of Economic Affairs of the People's Republic of China, X Consumer Co., Ltd. Application Scope: κ is an upper amorphous particle; and where spray-dried produces a particle having a weight of 20 to 90. / 〇 Dry powder of insulin present in individual particles. 7. The method according to claim 6 of the patent application scope, further comprising dissolving the tontoxin and a certain II carrier in an aqueous buffer solution, wherein a 20% to 90% by weight A dry powder of insulin present in individual particles. For example, the method of claim 7 in the patent application range, wherein the pharmaceutically acceptable carrier is a carbon xide, an organic salt, an amino acid, a peptide or a protein, which is spray-dried to produce a powder. For example, the method of claim 8 wherein the carbohydrate is selected from the group consisting of mannitol, melibiose, lactose, maltodextrin and trehalose. 10 · = The method of claim 8 in which the organic salt is selected from the group consisting of sodium arsenate, sodium acetate, and sodium ascorbate. A compound of tenisin in lung and medicine comprising individual particles containing 5% to 99% by weight of tensin in a pharmaceutical carrier substance and having a size of less than 10 μΓη, And this composition = contains penetration enhancer. Non-claimed patent No. 11 item of the tensilin composition, wherein the pharmaceutical carrier material package s-is selected from the group consisting of mannitol, melibiose, lactose, maltodextrin and trehalose Carbohydrate. 13. The insulin composition according to item u of the patent scope, wherein the pharmaceutical carrier ^ 3 is an organic salt selected from the group consisting of sodium citrate, sodium acetate, and sodium ascorbate. 8. 9. 11 . 12 Correction Supplementary V #