CZ20002417A3 - Medicament intended for administration by inhalation - Google Patents

Abstract

The present invention relates to the use of AspB28-human insulin for preparation medicaments suitable for administration by inhalation.

Description

The present invention relates generally to methods of treating a human suffering from diabetes mellitus. More particularly, the present invention relates to pulmonary administration of monomeric insulin analogs for systemic lung absorption to significantly reduce or eliminate the need for administration of monomeric insulin analogs by injection.

BACKGROUND OF THE INVENTION

Since the introduction of insulin after 1920, constant attempts have been made to improve the treatment of diabetes mellitus. The most important progress has been made in terms of insulin purity and availability, and various preparations with different duration of action have been developed. Non-injectable forms of insulin are desirable to increase patient compliance with intensive insulin therapy and reduce the risk of complications.

Diabetes mellitus is a disease affecting approximately 6% of the world's population. In addition, the population of most countries is aging and diabetes is especially common in aging populations. It is often this part of the population that has difficulty or reluctance to inject insulin alone. In the United States, approximately 5% of the population has diabetes, and approximately one third of these diabetics self-administer one or more doses of insulin daily by subcutaneous injection. This type of intensive therapy is necessary to lower blood glucose levels. High blood glucose, resulting from low levels or absence of endogenous insulin, alters the normal chemistry of the body and can lead to microvascular system failure in many organs. Untreated diabetics often undergo amputation and have blindness and kidney failure. It is estimated that treating side effects of diabetes and loss of productivity due to inadequate diabetes treatment lead to total annual losses of $ 40 billion in the United States alone.

A nine-year-long Diabetes Control and Complications Trial (DCCT) experiment involving 1441 patients with type I diabetes showed that maintaining blood glucose with close tolerance reduces the frequency and severity of diabetic complications. Conventional insulin therapy involves only two injections of insulin per day. Intensive insulin therapy in the DCCT study included three or more injections of insulin each day. In this study, the incidence of diabetes side effects was dramatically reduced. For example, in patients receiving intensive therapy, retinopathy was reduced by 50-76%, nephropathy by 35-56% and neuropathy by 60%.

Unfortunately, many diabetics do not wish to receive intensive therapy due to the discomfort associated with the many injections required to keep glucose levels within a narrow range. This type of therapy can be painful both psychologically and physically. When administered orally, insulin degrades rapidly in the gastrointestinal tract and is not absorbed into the bloodstream. Therefore, many researchers have investigated alternative routes of insulin administration such as the oral, rectal, transdermal and nasal routes. However, these routes of administration have not yet resulted in effective insulin absorption.

For many years, it has been known that some proteins can be absorbed in the lungs. Administration of insulin in the form of an inhalation aerosol to the lungs was first described by Gaensslen in 1925. Although numerous animal and human studies have shown that some insulin preparations may be absorbed by the lungs, pulmonary administration has not been widely accepted as a means of effectively treating diabetes. This is due in part to the fact that only a small part of the insulin administered is absorbed. In addition, researchers have observed great variability in the amount of insulin absorbed by pulmonary administration of different insulin preparations and even doses of the same formulation administered at different times.

There is therefore a need to provide an efficient and reliable method of administering insulin by pulmonary means. This need is particularly evident in patients undergoing aggressive treatment protocols using fast-acting human monomeric insulin analogs. Effective pulmonary administration of fast-acting human monomeric insulin analogs would have the effect of rapidly reducing blood glucose concentrations when this need arises, such as after a meal or for a longer period without insulin therapy.

Obviously, not all proteins can be effectively absorbed in the lungs. There are numerous factors that affect whether a protein can be effectively administered through the lungs. Lung absorption is largely dependent on the physical properties of the particular therapeutic protein to be administered. Therefore, although pulmonary delivery of regular human insulin has been observed, physical differences between regular human insulin and fast-acting monomeric insulin analogs make it unclear whether these analogs can be effectively administered by the pulmonary route.

Effective pulmonary protein delivery is dependent on the protein's ability to enter the deep pulmonary alveolar epithelium. Proteins deposited in the upper airway epithelium are not absorbed significantly. This is because the mucus covering the tissue, which has a thickness of approximately 30-40 μιη, acts as a barrier to absorption. In addition, proteins deposited on this epithelium are removed by mucociliary transport to the airways and then removed into the gastrointestinal tract. This mechanism also contributes substantially to low absorption of some protein particles. The extent to which proteins are not absorbed and are instead removed in this manner depends on their solubility, size, and other, less explored properties.

It is difficult to predict whether the therapeutic protein can be rapidly transferred from the lung to the blood, even if the protein can be successfully delivered to the deep pulmonary alveolar epithelium. Absorption values were calculated for some proteins delivered by the lungs and range from 15 minutes for parathyroid hormone (fragment 1-34) to 48 hours for glycosylated α-antitrypsin. Due to the wide spectrum of peptidases that exist in the lungs, longer absorption times increase the possibility that the protein will be significantly degraded or removed by mucociliary transport earlier

- 5 · fc · fcfc · fcfc · fcfc «fc before absorption.

Insulin is a peptide hormone with a molecular weight of approximately 5,800 Daltons. In the presence of zinc, human insulin self-associates to a more stable hexameric form. Dissociation of stable hexamers is believed to be a step limiting the rate of insulin absorption from the subcutaneous injection site into the blood stream. However, fast-acting insulin analogues do not form readily stable hexamers. These analogs are known as monomeric insulin analogs because they are less prone to self-association to stable higher order complexes. This lack of self-association is due to modifications in the amino acid sequence of human insulin that reduce association by disrupting dimer formation. Unfortunately, modifications of insulin that make these analogs monomeric also lead to non-specific aggregation of monomers. This non-specific aggregation can cause the analogs to be insoluble and unstable.

Due to the intrinsic instability of monomeric insulin analogs and the possibility of forming insoluble precipitates of insulin analogs, physical differences between insulin and monomeric insulin analogs, and due to the high degree of variability in absorption of regular human insulin administered by the lungs, it is surprising that aerosolized formulations containing monomeric insulin analogs and efficiently delivered by the lungs. Most advantageous and least anticipated is the discovery that, unlike the data obtained for regular human insulin, the change in inhaled volume does not lead to detectable variations in the pharmacokinetics or pharmacodynamics of monomeric insulin analogs, especially Lys ^B28 Pro ^B29 -human

· · 4 t t 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 insulin. In addition, it was surprising that Lys ^B28 Pro ^{B29 -human} insulin is absorbed at least as fast as the lungs after subcutaneous administration.

SUMMARY OF THE INVENTION

The present invention relates to a method of administering a monomeric insulin analog which comprises administering an effective amount of a monomeric insulin analog to a patient in need thereof by the pulmonary route. The present invention also relates to a method of treating diabetes, comprising administering an effective amount of a monomeric insulin analog to a patient in need thereof by the pulmonary route. Another object of the present invention relates to a method of treating hyperglycemia, comprising administering an effective amount of a monomeric insulin analog to a patient in need thereof by the pulmonary route. Preferably, the monomeric insulin analogs are administered by inhalation and into the lower airways of the patient.

The monomeric insulin analogs can be administered in a carrier, as a solution or suspension, or as a dry powder using a variety of devices suitable for administration by inhalation. Preferably, the monomeric insulin analogs are administered with a particle size effective to achieve lower airway in the lung. The preferred particle size of the monomeric insulin analogs is less than 10 microns. An even more preferred particle size of the monomeric insulin analogs is between 1 and 5 microns.

Figure 1 shows the mean glucose response in beagle dogs versus time after aerosol administration of Lys ^B28 Pro ^B29 human insulin.

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9 9 9 · 9 9

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 99 99 99

The term insulin as used herein refers to a mammalian insulin such as bovine, porcine or human insulin, the sequence and structure of which are known to those skilled in the art. The amino acid sequence and spatial structure of human insulin are well known. Human insulin consists of a chain of 21 amino acids and a chain of 30 amino acids which are crosslinked by disulfide bonds. Properly cross-linked human insulin contains three disulfide bridges: one between position 7 of chain A and position 7 of chain B, the other between position 20 of chain A and position 19 of chain B, and the third between position β and position 11 of chain A.

The term insulin analog means proteins having chain A and chain B that have substantially the same amino acid sequence as chain A and chain B of human insulin but differ from chain A and chain B of human insulin by one or more amino acid deletions, one or more amino acid substitutions and / or one or more amino acid insertions that do not interfere with the insulin activity of the insulin analog.

One type of insulin analog, monomeric insulin analog, is well known to those skilled in the art. These human insulin analogs are fast-acting and include, for example, monomeric insulin analogs in which:

(a) the aminoacyl residue at position B28 is substituted with Asp, Lys, Leu, Val or Ala and the aminoacyl residue at position B29 is Lys or Pro;

(b) aminoacyl residues at positions B28, B29 and B30 are omitted; or • 4 4 4 4 4 • 4 • 44 · 4

(c) the aminoacyl residue at position B21 is omitted.

A preferred insulin monomer analog is Asp ^B28 . An even more preferred monomeric insulin analog is Lys ^B28 Pro ^B29 .

Monomeric insulin analogs are described in Chance, et al., U.S. Pat. U.S. Patent No. 5,514,646; Chance et al. Patent Application No. 08 / 255,297; Brems et al., Protein

Engineering, 5: 527-533 published Application No. 1987); and Brange et al., Biology, 1: 934-940 (1991).

included as an insulin reference.

(1992); Brange et al., EPO 214,826 (published March 18

Current Opinion in Structural These disclosures are herein explicitly describing monomeric analogs

Insulin analogues may also have amidated amino acid replacements by acidic forms. For example, Asn may be replaced by Asp or Glu. Similarly, Gln can be replaced by Asp or Glu. In particular, Asn (A18), Asn (A21) or Asp (B3) or any combination of these residues may be replaced by Asp or Glu. Also Gln (A15) or Gln (B4) or both can be replaced by Asp or Glu.

The term preservative refers to a compound added to a pharmaceutical composition to act as an antimicrobial agent. The parenteral formulation must meet the requirements for preservative efficacy to be able to be used as a multipurpose commercial product. Preservatives known to those skilled in the art to be effective and acceptable for parenteral formulations include benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, o-cresol, p-cresol;

Chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid and various mixtures thereof. See, for example, Wallhausser, K.-H., Develop. Biol. Standard, 24: 9-28 (Basel, S. Krager, 1974). Certain phenol preservatives, such as phenol and m-cresol, are known to bind to insulin-type molecules, thereby inducing conformational changes that enhance either physical or chemical stability or both [Birnbaum, et al., Pharmac. Res. 14: 25-36 (1997); Rahuel-Clermont, et al., Biochemistry 36: 5837-5845 (1997)]. M-cresol and phenol are preferred preservatives in the protein preparations of the monomeric insulin analogs for use in the present invention.

The term buffer or pharmaceutically acceptable buffer refers to a compound known to be safe when used in insulin preparations and which has the effect of maintaining the pH of the formulation at the pH required for the formulation. Pharmaceutically acceptable buffers for maintaining the pH at a slightly acidic pH to a slightly basic pH include, for example, compounds such as phosphates, acetates, citrates, TRIS, arginine, or histidine.

The term isotonicity agent refers to a compound that is physiologically tolerated and provides appropriate tonicity of the formulation to prevent water flow through cell membranes. Compounds such as glycerin at known concentrations are commonly used for this purpose. Other acceptable acceptable isotonic agents include salts such as NaCl, dextrose, mannitol, and lactose. Glycerol at a concentration of 12-25 mg / ml is a preferred isotonicity agent.

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Administration of monomeric insulin analogs

The monomeric insulin analogs are administered by inhalation at a dose corresponding to an effective amount to increase the level of insulin protein in the bloodstream and / or reduce the level of glucose in the circulation. Such administration may be effective for treating disorders such as diabetes or hyperglycemia. Achieving an effective dose of monomeric insulin analogs requires administration of an inhaled dose of greater than about 0.5 μς / kg to about 50 μς / kg of the monomeric insulin protein analog, preferably about 3 μς / kg to about 20 μς / kg, and most preferably about 7 μς / kg to approximately 14 μς / kg. A therapeutically effective amount can be determined by the skilled practitioner, considering factors including insulin protein level, blood glucose level, patient's physical condition, patient's lung condition, and the like.

According to the present invention, monomeric insulin analogs are administered by inhalation to achieve rapid absorption of these analogs. Administration by inhalation may lead to pharmacokinetics comparable to the subcutaneous administration of insulin. Inhalation of monomeric insulin analogues results in a rapid increase in circulating insulin levels, followed by a rapid decrease in blood glucose levels. Different inhalation devices typically provide similar pharmacokinetics when similar particle sizes and lung delivery levels are compared.

According to the present invention, the monomeric insulin analogs may be administered by any of a number of inhalation devices known to those skilled in the art for administration of the therapeutic agent by inhalation. These devices include metered dose inhalers, nebulizers, generators, and three dry powder, sprays, and the like. Preferably, the monomeric insulin analogs are administered in a dry powder inhalation device or spray. An inhalation device for administering monomeric insulin analogs should have some desirable features. For example, administration by an inhaler device is preferably reliable, reproducible and accurate. The inhalation device should deliver small particles, for example less than about 10 µm, preferably about 1-5 µm, for good respiration. Some specific examples of commercially available inhalation devices suitable for the method of the present invention are Turbohaler ™ (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros ™ Inhaler (Dera), devices supplied by Inhaler Therapeutics, AERx ™ ( Aradigm), Ultravent ® (Mallinckrodt), Acorn II ® (Marquest Medical Products), Ventolin ® MDI (Glaxo), Spinhaler ® powder inhaler (Visons) and the like.

As will be appreciated by those skilled in the art, the formulation comprising the monomeric insulin analog, the amount of formulation administered, and the duration of administration of a single dose depend on the type of inhaler used. For some delivery systems, such as aerosol dispensers, the frequency of administration and the time the system is activated depends mainly on the concentration of the monomeric insulin analog in the aerosol. For example, shorter administration periods can be used at higher concentrations of the monomeric insulin analog in the nebulized solution. Devices such as metered dose inhalers produce higher aerosol concentrations and can work for a shorter time to deliver the desired amount of monomeric insulin analog. Apparatus such as powder inhalers deliver • 4 44 «4 • 4 49 44» · • · «« 44 4 4 4 4 • 4 444 ·· ····

4 4 44 444 44 4 4 4 4 4 4 4

4444 · 44 «4 49 4 · active substance until the dose of reagent used is expelled from the device. In this type of inhaler, the amount of monomeric insulin analog in a given amount of powder determines the dose administered in a single administration.

The particle size of the monomeric insulin analog in the formulation administered by the inhaler is critical for the ability of the protein to enter the lungs and preferably the lower airways or alveoli. The monomeric insulin analog is preferably used in such a composition that at least 10% of the administered monomeric insulin analog is deposited in the lung, preferably 10% to 20% or more. It is known that the maximum lung deposition efficiency of a breathing human mouth is achieved at a particle size of about 2 µm to about 3 µm. If the particle size is greater than about 5 µm, deposition in the lungs decreases substantially. A particle size of less than about 1 µm causes a decrease in lung deposition and becomes difficult to deliver particles in sufficient quantity to be therapeutically effective. Thus, the monomeric insulin analog particles administered by inhalation preferably have a particle size of less than about 10 µm, more preferably in the range of about 1 µm to about 5 µm, and most preferably in the range of about 2 µm to about 3 µm. The monomeric insulin analog formulation is selected to provide the desired particle size for the selected inhaler device.

Administration of monomeric insulin analogs by a dry powder inhaler

For administration as a dry powder, the monomeric insulin analog is prepared with a particle size of less than about 10

9

99 • 9 9

9 * 9

99 · 9 9

9 9 9

99 μιη, preferably about 1 to about 5 μιη, and most preferably about 2 μτη to about 3 μπι. A preferred particle size is effective for delivery to the alveoli of the patient. The dry powder preferably consists of particles formed so that most of the particles have a size within the desired range. Preferably, at least 50% of the dry powder is represented by particles having a diameter of less than about 10 µπι. Such formulations may be obtained by spray drying, milling or condensation at a critical point in a solution of the monomeric insulin analog and other desired components. Other methods are also known in the art and are also suitable for forming particles useful in the present invention.

The particles are usually separated from the formulation in the container and are then transported to the patient's lungs by a carrier air stream. Typically, in conventional dry powder inhalers, the force to inhale solids develops exclusively by patient inhalation. One suitable dry powder inhaler is Turbohaler ™, manufactured by Astra (Sodertalje, Sweden). In another type of inhaler, the air flow generated by the patient's inhalation activates the propeller motor, which deagglomerates the monomeric insulin analog particles. The Dôra Spiros ™ inhaler is such a device.

Preparations of monomeric insulin analogs for administration by a dry powder inhaler typically comprise a finely divided dry powder containing the monomeric insulin analog, but the powder may also contain a bulking agent, carrier, excipient, other additives and the like. The additives may be contained in a dry powdered monomeric insulin analog preparation, for example, to dilute the powder to a value of »4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

Required for administration from a particular powder inhaler, to facilitate processing of the powder formulation, to provide advantageous properties of the powder in the formulation, to facilitate dispersion of the powder from the inhaler, to stabilize the formulation (e.g., antioxidants or buffers) alike. Preferably, the additives do not adversely affect the patient's airways. The monomeric insulin analogs may be mixed with the additive at the molecular level, or the solid preparation may comprise particles of the monomeric insulin analog mixed with the additive or deposited on the additive particles. Typical additives include mono-, di- and polysaccharides; sugar alcohols and other polyols such as, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch or combinations thereof; surfactants such as sorbitols, diphosphatidylcholine or lecithin; etc. Typically, an additive such as a bulking agent is present in an amount effective for the above-described purposes, often about 50% to about 90% by weight. preparation. Additional agents known in the art of making protein preparations, such as proteins represented by insulin analogs, may also be included in the formulation.

Administration of the Humalog® dry powder formulation, which is Lys ^B28 Pro ^B29 human insulin, by inhalation is a preferred method of treating diabetes.

Administering the monomeric insulin analogues in the form of a spray A spray containing the monomeric insulin analogue may be formed by forcing a suspension or solution of the monomeric insulin analogue through a nozzle under pressure. Nozzle size and its size

000 0 · ♦ 00 «0 00 0 000 000» 0 0 0 0 000 0 0 0 0 0 0 00 00 000 00 0 „0 0 00000000 - 15 - ♦ ·· ♦ ·· ·· ·· ·· configuration, the applied pressure and liquid feed rate can be selected to achieve the desired outlet and particle size. The electrospray may be formed, for example, by an electric field together with a capillary or nozzle. Preferably, the monomeric insulin analog particles, administered in the form of a spray, have a particle size of less than about 10 µm, preferably in the range of about 1 µm to about 5 µm, and most preferably from about 2 µm to about 3 µm.

Monomeric insulin analog preparations suitable for use in a spray apparatus typically contain the monomeric insulin analog in an aqueous solution at a concentration of about 1 mg to about 20 mg monomeric insulin analog per ml of solution. The composition may contain agents such as an excipient, a buffer, an isotonic agent, a preservative, a surfactant, and preferably zinc. The composition may also contain an excipient or agent for stabilizing the monomeric insulin analog, such as a buffer, a reducing agent, a bulk protein, or a hydrocarbon. Bulk proteins useful for making preparations containing monomeric insulin analogs include albumin, protamine, and the like. Typical hydrocarbons useful in formulating insulin analog preparations include sucrose, trehalose, glucose, and the like. The monomeric insulin analog formulation may also contain a surfactant that reduces or prevents surface-induced aggregation of the monomeric insulin analog caused by spraying the solution to form an aerosol. Various conventional surfactants such as polyoxyethylene fatty acid esters and alcohols and alcohols, polyoxyethylene sorbitol fatty acid esters may be used.

containing monomeric mannitol, lactose, • 4,444

4 · 44 ··

4 4 4 4 4 · 4 44444 4

Ί £ 4 44444 ~ J_ Ό ~ 4444 4 44 44 44

The amount is generally between 0.001 and 4% by weight. preparation. Particularly preferred surfactants for use herein are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Additional agents known in the art of protein preparations, such as insulin analogs, may also be included in the formulation.

Administration of monomeric insulin analogs by nebulizer

The monomeric insulin analog can be administered by a nebulizer, such as a jet nebulizer or an ultrasonic nebulizer. Typically, a compressed air source is used in the jet sprayer to produce a high velocity air jet through the nozzle. As the gas expands beyond the nozzle, a low air pressure region is formed which entrains the monomeric insulin analog solution by a capillary attached to the liquid reservoir. Upon exiting the capillary, the liquid stream from the capillary bursts into unstable fibers and droplets to form an aerosol. A variety of configurations, flow rates, and nozzle types can be used to achieve the desired performance characteristics of a given atomizer. In an ultrasonic atomizer, high-frequency electrical energy is used to generate vibrational mechanical energy, typically using a piezoelectric transducer. This energy is supplied to the monomeric insulin analog preparation either directly or via a binding fluid, thereby forming an aerosol containing the monomeric insulin analog. Preferably, the atomic insulin analog monomer particles have a particle size of less than about 10 µm, preferably in the range of about 1 µm to about 5 µm, and most preferably about 2 µm to about 3 µm.

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Monomeric insulin analog preparations suitable for use in a nebulizer, whether jet or ultrasonic, typically contain the monomeric insulin analog in an aqueous solution at a concentration of about 1 mg to about 20 mg of the monomeric insulin analog per ml of solution. The composition may contain agents such as an excipient, a buffer, an isotonic agent, a preservative, a surfactant, and preferably zinc. The composition may also contain an excipient or agent for stabilizing the monomeric insulin analog, such as a buffer, a reducing agent, an agent, a bulk protein or a hydrocarbon. Bulk proteins useful for making preparations containing monomeric insulin analogs include albumin, protamine, and the like. Typical hydrocarbons useful in formulating compositions containing monomeric insulin analogs include sucrose, mannitol, lactose, trehalose, glucose, and the like. The monomeric insulin analog formulation may also contain a surfactant that reduces or prevents surface-induced aggregation of the monomeric insulin analog caused by spraying the solution to form an aerosol. The surfactants, fatty acid esters can be used in various customary forms such as polyoxyethylene and alcohols and alcohols, polyoxyethylene sorbitol fatty acid esters. The amount is generally between 0.001 and 4% by weight. preparation. Particularly preferred surfactants for use herein are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Additional agents known in the art of protein preparations, such as insulin analogs, may also be included in the formulation.

fcfc fcfcfcfc • fc • fc • fcfcfc • fcfc fcfc fcfc • fc

Administration of monomeric insulin analogs by a metered dose inhaler

In a metered dose inhaler (MDI), the propellant, the insulin monomer analog and any excipients or other additives are contained in the container as a mixture containing liquefied compressed gas. Lowering the metering valve releases the composition as an aerosol, preferably containing particles in a size range of less than about 10 µm, preferably about 1 µm to about 5 µm, and most preferably about 2 µm to about 3 µm. The desired aerosol particle size can be achieved by using a monomeric insulin analog formulation prepared by methods known to those skilled in the art, such as jet milling, spray drying, critical point condensation, and the like. Preferred metered-dose inhalers include those manufactured by 3M or Glaxo and employ a fluorocarbon propellant.

The monomeric insulin analog formulations for use in a metered dose inhaler device generally comprise a fine powder comprising the monomeric insulin analogue in suspension in a non-aqueous medium, for example suspended in a propellant using a surfactant. The propellant may be any conventional material used for this purpose, such as chlorinated and fluorinated carbon compounds, and chlorinated and fluorinated hydrocarbons or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227) and the like. Preferably, the propellant is a fluorocarbon. The surfactant may be used to stabilize the monomeric insulin analogue in the form of a suspension in the propellant

9 99 99 99 • 9 9 9 9999

9,999,999 · 9,999 • 9,999,999,999 9

9 9999 9999 • 9999 99 99 99 99 gas, for protecting the active agent against chemical degradation and the like. Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid and the like. In some cases, aerosol solutions using solvents such as ethanol are preferred. Additional agents known in the art of protein preparations, such as insulin analogs, may also be used in the formulation.

One of ordinary skill in the art will recognize that the methods of the present invention can be performed by pulmonary administration of monomeric insulin analogs using devices not described herein.

Pharmaceutical preparations containing monomeric insulin analogues

The present invention also relates to pharmaceutical compositions or compositions comprising a monomeric insulin analog and suitable for administration by inhalation. According to the present invention, the monomeric insulin analog can be used for the manufacture of a formulation or medicament for administration by inhalation. The present invention also relates to methods of making compositions comprising a monomeric insulin analog in a form suitable for administration by inhalation. For example, a dry powder formulation can be manufactured in a number of ways using conventional techniques. Particles in a range of sizes suitable for maximum deposition in the lower parts of the respiratory tract can be formed by micronization, grinding, spray drying, and the like. The liquid formulation may be made by dissolving the monomeric insulin analog in a suitable solvent such as water, at a suitable pH, containing buffers or other excipients.

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One particular pharmaceutical composition comprising a particular monomeric insulin analog for administration by the pulmonary route is Humalog®. Humalog® have been described

DeVelippis, U.S. Pat. U.S. Patent No. 5,474,578; and 5,504,188. These discoveries

5,461,031; Bakaysa et al. U.S. Pat. Baker et al. U.S. Pat. U.S. Patent No. 3, are expressly incorporated by reference, describing various compositions comprising monomeric insulin analogs. Other formulations include sterile water solutions alone and aqueous solutions containing low concentrations of surfactants and / or preservatives and / or stabilizers and / or buffers. Other suitable formulations containing monomeric zinc insulin analogs are known to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be better understood with reference to the following examples. These examples are intended to represent specific embodiments of the present invention and are not intended to limit the scope of the invention.

Example 1

Serum pharmacokinetics of Lys ^B28 Pro ^B29 human insulin in Beagle dogs following pulmonary administration of a single aerosol dose of Lys ^B28 Pro ^B29 -human insulin (Lys ^B28 Pro ^{B29 -hI} ) formed from Lys ^B28 Pro ^{B29 -hI} solutions. in sterile water, were administered to anesthetized dogs via the pulmonary route through the endotracheal tube via an ultrasonic nebulizer. Immunoreactive serum concentration

Lys ^B28 For ^{B29 -hI} was determined by recognized radioimmunological methods.

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Six beagle dogs (3 males and 3 females) were used in this experiment. Animals were housed either in a cage or individually in a stainless steel cage with suspended mesh floor. Initially, dogs were fed approximately 450 g of Purina Certified Canine Diet 5007 food each day. The animals were fasted approximately eight hours prior to dosing. After recovery from anesthesia, food and water were provided ad libitum up to the final dose after 48 hours. The initial daily diet regimen started with the final dose at 48 hours. At the start of the study, the weight of the animals was between 12.5 and 11.6 kg.

Blood samples were taken at various times post-dose to determine the plasma concentrations of Lys ^B28 Pro ^{B29 -hI} and the bioavailability of the inhaled material was determined. The dogs were chosen because they are large animals with particle deposition in a human-like respiratory tract.

Pulmonary administration of Lys ^B28 Pro ^{B29 -hI} resulted in systemic exposure as evidenced by increased serum concentrations of immunoreactive Lys ^B28 Pro ^{B29 -hI} in all dogs.

Table 1: Serum concentrations of Lys ^B28 For ^{B29 -hI} (ng / ml) versus time after pulmonary administration are shown in Table 1:

Time (h ^a ) 0 O, 08 / 0, 17 0.33 0.5 0.75 1 1.5 2 3 4 6 Dog no. (sex) 26754 (M) 0.35 O, 76 0, 67 0.84 0, 81 0.59 0, 96 0, 48 0, 98 0.81 0, 66 0.57 28536 (F) 0.82 3, 22nd 3.16 2, 99 1.33 2.01 1.59 0.40 2.30 0.52 0, 77 0.29 26852 (M) 0, 61 2, 61 2.40 3, 98 2.35 2.17 2.17 1.12 0.35 0, 61 2, 71 0.34

4 · ························· · · · ······· · · β · ** · *

28910 (F) 0, 83 2, 61 2.14 2.27 1, 67 1, 90 1.79 0, 59 0.53 0.28 0.30 BLQ 27258 NS ^b 1.70 2.24 2.36 1, 85 1, 02 0.87 0.59 0.36 0.32 0.46 0.37 29244 (F) 0, 60 6.01 5.34 3.81 3.21 2.32 1.44 1,25 0, 68 0.27 0.35 0.33 Ν 5 6 6 6 6 6 6 6 6 6 6 6 Diameter 0, 64 2.82 2, 66 2.71 1.87 1, 67 1.47 0.74 0, 87 0.47 0.88 0, 32 SD 0.20 1, 78 1.54 1.16 0.83 0.70 0,0, 5 0.36 0, 74 0.22 0, 92 0, 18 PULL 0, 09 0.73 0, 63 0.47 0.34 0.28 0.20 0.15 0, 30 0.09 0.37 0, 07

• · · · • • • 4 · · · ^and Abbreviations used:

h, hour;

M, male;

F, female;

N, number of animals included in the calculation;

SD, standard deviation;

SEM, standard error of the mean;

BLQ, below the measurement threshold (40.25 ng / ml).

For calculation purposes, the BLQ was set equal to zero.

^b NS = No Sample. A serum sample was not collected from dog 27258 prior to dosing (0 h).

Pulmonary administration resulted in a rapid increase in immunoreactive insulin with peak concentrations (T _max ) occurring in most dogs approximately 5 to 20 minutes after aerosol exposure.

Table 2:

Pharmacokinetic parameters of pulmonary Lys ^B28 Pro ^{B29 -hI} .

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···· * *

Exposed Overall exposed

Gender Dog Weight kg Dose gg / kg Dose gg / kg Cmax ng / ml tax h AUC ^t ' _o ' β h “ ¹ tl / 2 h ng * h / m h M 28536 13.1 3.76 49.3 3.22 0,083 4.75 3.0 2,2394 0.31 M 28911 13.5 7.62 102, 9 2, 61 0,083 2.54 1.5 0.8607 0.81 M 29245 13, 9 8.71 121.1 6.01 0,083 4.89 3.0 0.9158 0.76 F 26754 11.1 6.69 74.3 0.98 2 4.32 6.0 0.1977 3.51 F 26852 11, 9 7.08 84.3 2.36 0, 33 2.17 6.0 0.8341 0, 83 F 27258 9.7 23, 45 227 3, 98 0.33 8.89 2,0 1,8245 0.38 Center (M) 13.5 6.70 91.1 3.95 0.08 4.06 2.5 1,3386 0.52 SD 0.4 2, 60 37.3 1.81 - 1.32 0.9 0.7805 % cv 3, 0 38, 8 41, 0 45, 9 - 32.5 35 58.3 N 3 3 3 3 3 3 3 3 3 Center (F) 10, 9 12.41 129 2.44 0.89 5.13 4.7 0.9521 0.73 SD 1.1 9.57 85.7 1.50 0, 96 3.43 2.3 0.8198 % CV 10.2 77.1 66, 6 61.5 109 67.0 49 86.1 N 3 3 3 3 3 3 3 3 3 Center (M + F) 12.2 9.6 109, 9 3.19 0, 48 4.59 3.6 1,1454 0, 61 SD 1.6 7.0 62.6 1.70 0.75 2.40 2,0 0.7466 % CV 13.2 73, 4 57.0 53.3 155 52.2 55 65.2 N 6 6 6 6 6 6 6 6 6 All dogs except 27258 Center 12.7 6.8 86.3 3.04 0.52 3.73 3.9 1,0095 0.69 SD 1,2 1,8 27.4 1.85 0, 84 1.29 2,0 0.7472 % CV 9.2 27, 3 31.7 61.1 162 34.4 52 74.0 N 5 5 5 5 5 5 5 5 5

• · · ·

Used shortcuts:

kg, kilogram;

pg, microgram;

ng, nanogram;

ml, milliliters;

h, hour;

Cmax, maximum serum concentration;

T _max , time to maximum serum concentration;

AUC 40, the area under the dose-response curve to return to baseline; návrat, return to basic value;

S, closing velocity constant; t 1/2 half-life;

M, male;

F, female;

SD, standard deviation;

% CV, percentage change coefficient;

N, number of animals used in the calculation.

The data show that pulmonary administration of aerosolized Lys ^B28 Pro ^{B29 -hI} results in detectable concentrations of immunoreactive Lys ^B28 Pro ^{B29 -hI} in the serum of beagle dogs. Lys ^B28 For ^{B29 -hI} was rapidly absorbed with mean peak concentrations achieved in less than 30 minutes. Serum concentrations of immunoreactive Lys ^B28 Pro ^{B29 -hI} decreased with a mean half-life of about 40 minutes. No gender differences in the deposition and release of Lys ^B28 Pro ^B29 hl. Blood glucose values showed a decrease to approximately 55% of control values in fasted animals following inhalation of Lys ^B28 Pro ^{B29 -hI} (Fig. 1). The mean lung dose required to produce these effects was approximately 7 pg / kg, as determined by Technitium gamma detection, which was used as a radiolabel for aerosol droplets. The time required for the decrease in blood glucose values was slightly less for the Lys Pro -hl to be compared to that observed with subcutaneous injection.

Example 2

Absorption of Asp ^B28 -human insulin in beagle dogs after pulmonary administration

Asp ^B28 -human insulin was administered to conscious beagle dogs in the form of an aerosol using a head bag. Blood samples were taken to determine blood glucose levels and serum Asp ^B28 human insulin levels after exposure to the test substance. The dogs were chosen because they are large animals with particle deposition in a human-like respiratory tract.

Six female beagle dogs were used in the study. Animals were kept either in a cage or individually in stainless steel cages with suspended grid floors. All animals were fed approximately 450 grams of Hill's Science Diet daily. Animals were fasted approximately 12 hours prior to dosing. At the start of the study, the weight of the animals was between 10.8 and 14.1 kg.

All animals were exposed to aerosols containing Asp ^B28 human insulin while standing in the harness. One layer of 0.03 inch latex film was placed around the neck of the animal to form a non-restrictive airtight coating. For the purpose of the experiment, the manufactured 11 liter bag was placed over the animal's head and attached to the harness. The total flow through the bag was approximately 11-15 liters per minute. Aerosols were formed using a nebulizer with an inlet of approximately 7.5 liters per minute. 6 ml of Asp ^B28- human insulin at a concentration of 2.4 mg / ml sterile water plus 100-500 pCi of ⁹⁹ Tc were charged to the generator. The generator output was directed directly to the bag. One gravimetric blood sample was taken at 11 / min during each exposure. for 15 minutes. The target pulmonary dose was 10 mg / kg. The actual delivered pulmonary dose was determined by gamma imaging immediately after exposure.

Blood samples were taken 0 (pre), 0.08, 0.25, 0.5,

0.75, 1, 1.5, 2, 3, and 4 hours after exposure to the blood glucose and Asp ^B28 human insulin levels. The mean concentration for each dog exposed to Asp ^B28 human insulin ranged from 17.9 to 30.5 pg / L. The mean (± standard deviation) concentration for all animals was 22.4 ± 4.3 pg / L. The mean dose of Asp ^B28 -human insulin delivered to the lungs of 6 dogs was calculated to be 10 ± 5 pg / kg (mean ± standard deviation). The median inhaled dose was 287 ± 107 µg / kg (mean ± standard deviation). Individual animal data is shown in Table 3 below.

Table 3 Animal Target Inhaled Estimated C. dose dose dose delivered to (pg / kg) delivered to lung (pg / kg) lung (pg / kg)

• 9 9 9 9

Μ · · · ·

• · 9 · 9 · 9

27682 10 230 8.48 27684 10 210 8.15 27685 10 230 8.47 27686 10 320 4.94 27687 10 240 11.88 27689 10 490 19.60 Pulmonary administration Asp ^B28 ~ human insulin led to systemic exposure , how shows increase concentration

immunoreactive Asp ^B28 -human insulin in the serum of all dogs. Serum Asp ^B28 -human insulin concentrations versus time following pulmonary administration were determined with the Coat-ACount Insulin RIA Kit (Diagnostic Products, Los Angeles, California).

4 4 4

9

- 29 -;

«· 3 4 · · ·

4 4 · · 4 · · · 4 4 · · 4 ··

4 44 44 * 4 * • 4 · ·

4 * 4 · 4

Table 4: Serum Asp ^B28 human insulin concentrations (ng / ml) versus time after pulmonary administration.

Aerosol Dog 0 hrs 0,083 throw. 0, 25 throw. 0.5 throw. 0.75 throw. 1 hour 276821 0.68 1.55 1.51 1.65 1.31 1.06 276841 1, 64 3.18 1, 98 1.75 1,23 1.71 276851 0, 37 1.70 2.11 1.18 0.93 0.93 276861 0.39 2.92 4.15 2.63 1.88 1.20 276871 0.80 2.98 2.78 2.64 2.29 1.81 276891 0.39 4.15 3.12 2.83 1, 69 1.47

Aerosol Dog 1.5 throw. 2 hrs 3 hours 4 hours 276821 0.87 0, 54 0.52 0, 69 276841 1.15 0.70 1.09 1.08 276851 0.89 0.80 0.71 0.71 276861 1.10 0, 90 1.01 1.87 276871 1,21 1.07 1.01 0.66 276891 1,23 1.08 0.48 0.40

Changes in blood glucose were also measured after pulmonary administration of Asp ^B28- human insulin (Table 5). Serum glucose was determined using the Hitachi Chemistry System (Boehringer Mannheim Corp., Indianapolis, IN). The maximum percentage decrease in blood glucose compared to control values was observed 1 hour after exposure following pulmonary administration. The decreases of approximately 40% and 70% were · 0000

0 0 «

Observed at a dose delivered to the lungs of approximately 10 pg / kg.

Table 5:

Time Percent change in level Percent change in level (minutes) serum glucose serum glucose after inhalation after subcutaneous administration Asp ^B28 -human insulin Asp ^B28 -human insulin (mean ± std.) (mean ± std.) 0 100 + 0 100 + 0 5 105 ± 6 98 ± 7 15 Dec 87 ± 8 72 ± 13 30 65 ± 8 44 ± 7 45 65 ± 8 47 ± 6 60 61 ± 8 33 ± 4 90 64 + 9 38 ± 4 120 7 8 ± 8 45 ± 6 180 10 6 ± 6 72 ± 7 240 112 ± 7 91 ± 9

Pulmonary administration of Asp ^B28 -human insulin reached measurable levels of test substance above pre-administration levels. Comparisons of pharmacokinetic parameters after subcutaneous and pulmonary administration are shown in Table 6.

Table 6:

Method of administration AUC (ng.h / ml) C _max (ng / ml) Tmax (h)

·· ·· ♦ ♦ ·

9 9 ·· «· · · · ·

(middle + SEM) (middle + SEM) (mean ± SEM) subcutaneous 10.30 ± 0.47 5.57 ± 0.35 0.42 ± 0.05 pulmonal 5.21 ± 0.47 3.04 ± 0.42 0.21 ± 0.07

For pulmonary administration, the AUC was determined from a time interval of -0.25 to 2 hours (the time when blood levels approximately returned to baseline); for subcutaneous administration, AUC was determined from 0 to 4 hours (the time when blood levels approximately returned to baseline). These AUC values were then adjusted by subtracting the baseline endogenous canine insulin level. After deducting the contribution of endogenous insulin, the bioavailability of the pulmonary route was estimated to be approximately 26% compared to the subcutaneous injection. For comparison purposes, the bioavailability of the pulmonary route is estimated to be approximately 38% compared to a subcutaneous injection for human insulin.

Claims (8)

Use of Asp ^B28 human insulin for the preparation of a medicament suitable for administration by inhalation. Use according to claim 1, wherein the medicament is in the form of a solution or an aqueous medium or suspension or an anhydrous medium. Use according to claim 1, wherein the medicament is in the form of an aerosol. Use according to claim 1, wherein the medicament is in the form of a dry powder. The use of claim 1, wherein the monomeric insulin analog has a particle size of less than about 10 microns. The use of claim 1, wherein the monomeric insulin analog has a particle size having a particle size of from about 1 to about 5 microns. The use of claim 1, wherein the monomeric insulin analog has a particle size of from about 2 to about 3 microns. 8. Use according to any of claims 1-7, when Asp ^B28 -human insulin with served in batches in range approximately 3 pg / kg to about 2C i pg / kg. 9. Use according to any of claims 1-7, when Asp ^B28 -human insulin with served in batches in range approximately 7 μg / kg to about 14 pg / kg.