CZ20002417A3 - Drug for administration by inhalation - Google Patents

Abstract

The solution relates to the use of AspB28-human insulin for the preparation of a medicament suitable for administration by inhalation.

Description

The present invention generally relates 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 absorption through the lungs to significantly reduce or eliminate the need to administer monomeric insulin analogs by injection.

State of the art

Since the introduction of insulin in the 1920s, there have been continuous attempts to improve the treatment of diabetes mellitus. The most important advances have been made in the purity of insulin and its availability, and various preparations with different durations 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 particularly common in aging populations. It is often this segment of the population that has difficulty or is reluctant to self-inject insulin. 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 levels, which result from low levels or absence of endogenous insulin, alter the body's normal chemistry and can lead to microvascular failure in many organs. Untreated diabetics often undergo amputations and develop blindness and kidney failure. It is estimated that the treatment of diabetes side effects and the loss of productivity due to inadequate diabetes management result in a total annual loss of $40 billion in the United States alone.

The nine-year Diabetes Control and Complications Trial (DCCT), which included 1,441 patients with type 1 diabetes, demonstrated that maintaining blood glucose levels within a narrow tolerance reduces the frequency and severity of diabetic complications. Conventional insulin therapy involves only two insulin injections per day. Intensive insulin therapy in the DCCT study involved three or more insulin injections each day. In this study, the incidence of diabetic side effects was dramatically reduced. For example, in patients using intensive therapy, retinopathy was reduced by 50-76%, nephropathy by 35-56%, and neuropathy by 60%.

Unfortunately, many diabetics are reluctant to undergo intensive therapy due to the discomfort associated with the multiple injections required to maintain glucose levels within a narrow range. This type of therapy can be painful both psychologically and physically. When administered orally, insulin is rapidly degraded 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, to date, these routes of administration have not resulted in effective insulin absorption.

It has been known for many years that some proteins can be absorbed in the lungs. The administration of insulin by inhalation aerosol to the lungs was first described by Gaensslen in 1925. Although numerous animal and human studies have shown that some insulin preparations can be absorbed through the lungs, pulmonary administration has not been widely accepted as an effective means of treating diabetes. This is partly because only a small portion of the administered insulin is absorbed. In addition, investigators have observed great variability in the amount of insulin absorbed by the pulmonary administration of different insulin preparations and even doses of the same preparation given at different times.

There is therefore a need to provide an effective and reliable method of delivering insulin by pulmonary means. This need is particularly acute in patients undergoing aggressive treatment protocols using rapid-acting human monomeric insulin analogs. Effective pulmonary delivery of rapid-acting human monomeric insulin analogs would provide the effect of rapidly lowering blood glucose concentrations when the need arises, such as after a meal or after an extended period without insulin therapy.

It is clear that not all proteins can be effectively absorbed in the lungs. There are numerous factors that influence whether a protein can be effectively delivered via the lungs. Pulmonary absorption is dependent to a large extent on the physical properties of the particular therapeutic protein being administered. Therefore, although pulmonary delivery of regular human insulin has been observed, the physical differences between regular human insulin and rapid-acting monomeric insulin analogs make it unclear whether these analogs can be effectively delivered via the pulmonary route.

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

It is difficult to predict whether a therapeutic protein can be rapidly transported from the lung to the blood, even when the protein can be successfully delivered to the deep lung alveolar epithelium. Uptake values have been calculated for some proteins delivered through the lung and range from fifteen minutes for parathyroid hormone (fragment 1-34) to 48 hours for glycosylated α1-antitrypsin. Given the wide range of peptidases that exist in the lung, longer uptake 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 occurs.

Insulin is a peptide hormone with a molecular weight of approximately 5,800 Daltons. In the presence of zinc, human insulin self-associates into a more stable hexameric form. Dissociation of the stable hexamer is thought to be the rate-limiting step in the absorption of insulin from the subcutaneous injection site into the bloodstream. However, rapid-acting insulin analogs do not readily form stable hexamers. These analogs are known as monomeric insulin analogs because they are less likely to self-associate into 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, the modifications of insulin that make these analogs monomeric also lead to nonspecific aggregation of monomers. This nonspecific aggregation can render the analogs insoluble and unstable.

Due to the intrinsic instability of monomeric insulin analogs and the potential for the formation of insoluble precipitates of insulin analogs, the physical differences between insulin and monomeric insulin analogs, and the high degree of variability in the absorption of regular human insulin administered via the lungs, it is surprising that aerosolized formulations containing monomeric insulin analogs can be delivered via the lungs in a reproducible and effective manner. Most advantageous and least expected is the discovery that, in contrast to data obtained for regular human insulin, variation in inhaled volume does not lead to detectable differences in the pharmacokinetics or pharmacodynamics of monomeric insulin analogs, particularly Lys ^B28 Pro ^B29 -human

• · ·· ·· ·· • · · · « · · • · ♦ · · · > · * • · · · t 4» · < · • · · · · · · · • * · · ·» · · insulin. In addition, it was surprising that Lys ^B28 Pro ^B29 -human insulin is absorbed at least as quickly through the lungs after administration as it is after subcutaneous administration.

The essence of the invention

The present invention relates to a method of administering a monomeric insulin analog, which comprises administering an effective amount of the 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 the monomeric insulin analog to a patient in need thereof by the pulmonary route. Another aspect of the present invention relates to a method of treating hyperglycemia, comprising administering an effective amount of the 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 respiratory tract of the patient.

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 in a particle size effective to reach the lower respiratory tract in the lungs. The preferred particle size of the monomeric insulin analogs is less than 10 microns. 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 over time following 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 99 99 99

The term insulin, as used herein, refers to 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 an A chain of twenty-one amino acids and a B chain of thirty amino acids, which are cross-linked by disulfide bonds. Properly cross-linked human insulin contains three disulfide bridges: one between position 7 of the A chain and position 7 of the B chain, a second between position 20 of the A chain and position 19 of the B chain, and a third between position β and position 11 of the A chain.

The term insulin analog refers to proteins that have an A chain and a B chain that have essentially the same amino acid sequences as the A chain and B chain of human insulin, but differ from the A chain and B chain 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 impair the insulin activity of the insulin analog.

One type of insulin analog, a 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) the aminoacyl residues at positions B28, B29 and B30 are omitted; or • 4 4 4 4 4 • 4 • 44 · · « 4 4 •4 44 ·· • · « * · · · · 4 4 4

c) the aminoacyl residue at position B21 is omitted.

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

Monomeric insulin analogs are described in Chance, et al., U.S. Patent No. 5,514,646; Chance et al., U.S. Patent Application Serial 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 a reference, insulin.

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

Current Opinion in Structural These discoveries are here explicitly describing monomeric analogues

Insulin analogs may also have amidated amino acids replaced by acidic forms. For example, Asn may be replaced by Asp or Glu. Similarly, Gln may 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 may be replaced by Asp or Glu.

The term preservative refers to a compound added to a pharmaceutical preparation to act as an antimicrobial agent. A parenteral preparation must meet the requirements for preservative efficacy in order to be capable of being used as a multipurpose commercial product. Preservatives known to those skilled in the art to be effective and acceptable for parenteral preparations include benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, o-cresol, p-cresol, • 0 0 00 0

0 0 0 00 »000 00000 00 00 · · * · chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid and various mixtures thereof. See, for example, Wallháusser, K.-H., Develop. Biol. Standard, 24: 9-28 (Basel, S. Krager, 1974). Certain phenolic preservatives such as phenol and m-cresol are known to bind to insulin-like molecules and thereby induce conformational changes that increase 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 protein-containing preparations of monomeric insulin analogs for use in the present invention.

The term buffer or pharmaceutically acceptable buffer refers to a compound known to be safe for use in insulin preparations and which has the effect of maintaining the pH of the preparation at the pH value desired for the preparation. 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 isotonic agent refers to a compound that is physiologically tolerated and provides a suitable tonicity of the preparation to prevent the flow of water across cell membranes. Compounds such as glycerin are commonly used for this purpose at known concentrations. Other acceptable isotonic agents include salts, for example NaCl, dextrose, mannitol and lactose. Glycerol at a concentration of 12 to 25 mg/ml is a preferred isotonic agent.

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Administration of monomeric insulin analogues

Monomeric insulin analogs are administered by inhalation in a dose corresponding to an effective amount to increase the level of insulin protein in the bloodstream and/or to reduce the level of glucose in the bloodstream. Such administration may be effective for the treatment of 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 monomeric insulin protein analog, preferably about 3 μς/kg to about 20 μς/kg, and most preferably about 7 μς/kg to about 14 μς/kg. The therapeutically effective amount can be determined by a skilled physician who will consider factors including the level of insulin protein, the level of glucose in the blood, the physical condition of the patient, the condition of the patient's lungs, and the like.

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

According to the present invention, monomeric insulin analogs can be administered by any of a number of inhalation devices known to those skilled in the art for administering a therapeutic agent by inhalation. These devices include metered dose inhalers, nebulizers, dry powder generators, sprays, and the like. Preferably, monomeric insulin analogs are administered in a dry powder inhaler or spray. An inhalation device for administering monomeric insulin analogs should have certain desirable features. For example, administration by an inhalation device is preferably reliable, reproducible, and accurate. The inhalation device should deliver small particles, for example, less than about 10 pm, preferably about 1-5 pm, for good respirability. 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 (Důra), devices supplied by Inhaler Therapeutics, AERx™ (Aradigm), Ultravent® nebulizer (Mallinckrodt), Acorn II® nebulizer (Marquest Medical Products), Ventolin® MDI (Glaxo), Spinhaler® powder inhaler (Visons), and the like.

As will be appreciated by the skilled artisan, the composition of the monomeric insulin analogue, the amount of composition administered, and the duration of administration of a single dose depend on the type of inhalation device used. For some delivery systems, such as aerosol nebulizers, the frequency of administration and the length of time the system is activated depend primarily on the concentration of the monomeric insulin analogue in the aerosol. For example, shorter administration periods may be used with higher concentrations of the monomeric insulin analogue in the nebulized solution. Devices such as metered dose inhalers produce higher concentrations of aerosol and can operate for a shorter period of time to deliver the desired amount of monomeric insulin analogue. Devices such as dry 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 4

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

The particle size of the monomeric insulin analog in a formulation administered by an inhalation device is critical to 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 formulation that at least 10% of the administered monomeric insulin analog is deposited in the lungs, preferably 10% to 20% or more. It is known that maximum lung deposition efficiency in a mouth-breathing human is achieved at particle sizes of about 2 pm to about 3 pm. If the particle size is greater than about 5 pm, lung deposition is significantly reduced. Particle sizes of less than about 1 pm cause decreased lung deposition and it becomes difficult to deliver particles in sufficient quantities to be therapeutically effective. Thus, monomeric insulin analog particles administered by inhalation preferably have a particle size of less than about 10 pm, more preferably in the range of about 1 pm to about 5 pm, and most preferably in the range of about 2 pm to about 3 pm. The formulation containing the monomeric insulin analogue is selected to provide the desired particle size for the chosen inhalation device.

Administration of monomeric insulin analogues by dry powder inhaler

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

9 9 99 9

99 • 9 9

9 9*9

99 9 9

9 9 9 • 9 9 9

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

The particles are usually separated from the formulation in a container and are then transported to the patient's lungs by a carrier air stream. Typically, in conventional dry powder inhalers, the force for inhaling the solid is generated solely by the patient's inhalation. One suitable dry powder inhaler is the Turbohaler™, manufactured by Astra (Södertälje, Sweden). In another type of inhaler, the air stream generated by the patient's inhalation activates a propeller motor that deagglomerate the monomeric insulin analogue particles. The Důra Spiros™ inhaler is such a device.

Formulations of monomeric insulin analogs for administration by dry powder inhaler typically comprise a finely divided dry powder containing the monomeric insulin analog, but the powder may also comprise a bulking agent, carrier, excipient, other additives and the like. Additives may be included in the dry powder formulation of the monomeric insulin analog, for example, to dilute the powder to a value of ·« »«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 device, to stabilize the formulation (for example, antioxidants or buffers), to impart a flavor to the formulation, and the like. Preferably, the additives do not have an adverse effect on the patient's respiratory tract. Monomeric insulin analogs may be mixed with the additive at the molecular level, or the solid formulation may comprise particles of monomeric insulin analog mixed with or supported 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; and the like. Typically, an additive, such as a bulking agent, is present in an amount effective for the purposes described above, often from about 50% to about 90% by weight of the formulation. Additional agents known in the art of making protein formulations, such as proteins represented by insulin analogs, may also be included in the formulation.

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

Administration of monomeric insulin analogues in spray form A spray containing a monomeric insulin analogue can be produced by forcing a suspension or solution of the monomeric insulin analogue through a nozzle under pressure. The size of the nozzle and its

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

Formulations of monomeric insulin analog suitable for use in a spray device typically comprise monomeric insulin analog in aqueous solution at a concentration of about 1 mg to about 20 mg monomeric insulin analog per mL of solution. The formulation may contain agents such as an excipient, buffer, isotonicity agent, preservative, surfactant, and preferably zinc. The formulation may also contain an excipient or agent for stabilizing the monomeric insulin analog, such as a buffer, reducing agent, bulk protein, or carbohydrate. Bulk proteins useful for formulating formulations of monomeric insulin analogs include albumin, protamine, and the like. Typical carbohydrates useful for formulating formulations of insulin analogs include sucrose, trehalose, glucose, and the like. The formulation of monomeric insulin analog may also contain a surfactant that reduces or prevents surface-induced aggregation of the monomeric insulin analog caused by atomization of the solution during aerosol formation. Various conventional surfactants may be used, such as polyoxyethylene fatty acid esters and alcohols, polyoxyethylene sorbitol fatty acid esters.

containing monomers mannitol, lactose, •4 4444

4· 44 ··

4 4 4 4 4 · • 4 44444 4

Ί £ 4 4 44444 ~ J_ Ό ~ 4444 4 44 44 44

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

Administration of monomeric insulin analogues by nebulizer

The monomeric insulin analog can be administered by a nebulizer, such as a jet nebulizer or an ultrasonic nebulizer. Typically, a jet nebulizer uses a source of compressed air to create a high velocity air stream through a nozzle. As the gas expands past the nozzle, a low pressure region is created that entrains the monomeric insulin analog solution through a capillary connected to a liquid reservoir. Upon exiting the capillary, the liquid stream from the capillary breaks into unstable filaments and droplets, thereby producing an aerosol. A variety of configurations, flow rates, and nozzle types can be used to achieve the desired performance characteristics of a given jet nebulizer. An ultrasonic nebulizer uses high frequency electrical energy to create vibratory mechanical energy, typically using a piezoelectric transducer. This energy is delivered to the monomeric insulin analog formulation either directly or through a coupling fluid, thereby producing an aerosol containing the monomeric insulin analog. Preferably, the monomeric insulin analog particles formed by the nebulizer 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.

«· ···· · · 99 99 99

9 9 9 9 9 9 9 9 9

9 9 9 999 9 9 9 9 * 9 9 9 9 9 9 9 9 ·· · _ 1 7 — · · 9 99 9 9 99 9

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Formulations of monomeric insulin analog suitable for use in a nebulizer, whether jet or ultrasonic, typically comprise monomeric insulin analog in aqueous solution at a concentration of about 1 mg to about 20 mg monomeric insulin analog per mL of solution. The formulation may contain agents such as an excipient, buffer, isotonicity agent, preservative, surfactant, and preferably zinc. The formulation may also contain an excipient or agent for stabilizing the monomeric insulin analog, such as a buffer, reducing agent, bulking protein, or carbohydrate. Bulking proteins useful for forming formulations containing monomeric insulin analogs include albumin, protamine, and the like. Typical carbohydrates useful for forming formulations containing monomeric insulin analogs include sucrose, mannitol, lactose, trehalose, glucose, and the like. The composition containing the monomeric insulin analog may also contain a surfactant that reduces or prevents surface-induced aggregation of the monomeric insulin analog caused by spraying the solution during aerosol formation. Various conventional surfactants, such as polyoxyethylene and alcohols, polyoxyethylene sorbitol fatty acid esters, may be used. The amount is generally in the range of 0.001 and 4% by weight of the composition. Particularly preferred surfactants for use in the present invention 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 composition.

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

Administration of monomeric insulin analogues by metered dose inhaler

In a metered dose inhaler (MDI), the propellant, monomeric insulin analog, and any excipients or other additives are contained in a container as a mixture containing a liquefied compressed gas. Actuation of the metering valve releases the mixture 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 using fluorocarbon propellants.

Formulations containing a monomeric insulin analogue for use in a metered dose inhaler device generally comprise a fine powder containing the monomeric insulin analogue in the form of a suspension in an anhydrous medium, for example suspended in a propellant using a surfactant. The propellant may be any of the conventional materials 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 (hydrofluralkane-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 9 9 999 · 9 9 9 • 9 99 99 999 99 9

9 9999 9999 • 9999 99 99 99 99 gas, to protect the active agent against chemical degradation and the like. Suitable surfactants include sorbitan trioleate, soy 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.

It will be apparent to one of ordinary skill in the art that the methods of the present invention can be carried out by pulmonary administration of monomeric insulin analogs using devices not described herein.

Pharmaceutical preparations containing monomeric insulin analogue

The present invention also relates to pharmaceutical compositions or preparations comprising a monomeric insulin analogue and suitable for administration by inhalation. According to the present invention, a monomeric insulin analogue can be used to manufacture a preparation or medicament for administration by inhalation. The present invention also relates to methods of manufacturing preparations comprising a monomeric insulin analogue in a form suitable for administration by inhalation. For example, a dry powder preparation can be manufactured by a variety of methods using conventional techniques. Particles in a size range suitable for maximum deposition in the lower respiratory tract can be formed by micronization, milling, spray drying, and the like. A liquid preparation can be manufactured by dissolving the monomeric insulin analogue 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 pulmonary administration is Humalog®. Humalog® preparations have been described

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

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

Examples of embodiments of the invention

The present invention will be better understood by 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 after pulmonary administration of a single aerosol dose. Aerosols of Lys ^B28 Pro ^B29 -human insulin (Lys ^B28 Pro ^B29 -hI), prepared from solutions of Lys ^B28 Pro ^B29 -hI in sterile water, were administered to anesthetized dogs by the pulmonary route via an endotracheal tube using an ultrasonic nebulizer. Serum concentration of immunoreactive

Lys ^B28 Pro ^B29 -hI was determined by established radioimmunoassay methods.

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Six beagle dogs (3 males and 3 females) were used in this experiment. The animals were housed either in pairs per cage or individually in stainless steel cages with suspended mesh floors. Initially, the dogs were fed approximately 450 g of Purina Certified Canine Diet 5007 each day. The animals were fasted for approximately eight hours prior to dosing. After recovery from anesthesia, food and water were provided ad libitum until the final doses were given 48 hours later. The initial daily feeding regimen began with the final dose given 48 hours later. At the start of the study, the animals weighed between 12.5 and 11.6 kg.

Blood samples were collected at various time points after dosing to determine plasma concentrations of Lys ^B28 Pro ^B29 -hI and to determine the bioavailability of the inhaled material. Dogs were chosen because they are large animals with particle deposition in the respiratory tract similar to humans.

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

Table 1: Serum concentrations of Lys ^B28 Pro ^B29 -hI (ng/ml) as a function of time after pulmonary administration are shown in Table 1:

Time (h ^a ) 0 oh, 08 0.17 0.33 0.5 0.75 1 1.5 2 3 4 6 Dog No. (sex) 26754 (M) 0.35 oh, 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, 22 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.

28911 (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 (M) NS ^b 1.70 2.24 2.36 1.85 1.02 0.87 0.59 0.36 0.32 0.46 0.37 29245 (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 N 5 6 6 6 6 6 6 6 6 6 6 6 Average 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 • · · · ^a 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, 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 administered Lys ^B28 Pro ^B29 -hI.

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···· • *· • · · • ♦ ·

Exhibited Total exhibited

Sex Dog Weight kg Dose gg/kg Dose gg/kg Cmax ng/ml tax h AUC ^t ' _o ť βh" 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 Wednesday (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 Wednesday (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 Wednesday (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 outside 27258 Wednesday 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

• · · ·

Abbreviations used:

kg, kilogram;

pg, microgram;

ng, nanogram;

ml, milliliter;

h, hour;

Cmax, maximum serum concentration;

T _max , time to maximum serum concentration;

AUC, area under the dose-time curve to return to baseline; t, return to baseline;

S, final rate constant; ti/2z half-life;

M, male;

F, female;

SD, standard deviation;

%CV, percentage coefficient of variation;

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 Pro ^B29 -hI was rapidly absorbed with mean peak concentrations achieved in less than 30 minutes. Serum concentrations of immunoreactive Lys ^B28 Pro ^B29 -hI declined with a mean half-life of about 40 minutes. No gender differences were observed 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 after inhalation of Lys ^B28 Pro ^B29 -hI (Fig. 1). The median lung dose required to produce these effects was approximately 7 pg/kg, as determined by gamma detection of Technitium, which was used as a radioactive label for aerosol droplets. The time required for blood glucose levels to fall was slightly longer for inhaled Lys Pro-hl compared with 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 as an aerosol via a head-mounted bag. Blood samples were collected to determine blood glucose levels and serum Asp ^B28 -human insulin levels after exposure to the test substance. Dogs were chosen because they are large animals with similar respiratory tract deposition to humans.

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

All animals were exposed to aerosols containing Asp ^B28 human insulin while standing in a harness. A single layer of 0.03 inch thick latex film was placed around the animal's neck to create a non-restrictive, airtight envelope. A custom-made 11 liter bag was placed over the animal's head and secured to the harness. The total flow rate through the bag was approximately 11 to 15 liters per minute. Aerosols were generated using a nebulizer with an inlet of approximately 7.5 liters per minute. The generator was loaded with 6 ml of Asp ^B28 human insulin at a concentration of 2.4 mg/ml in sterile water plus 100 to 500 pCi ⁹⁹ Tc. The output from the generator was directed directly into the bag. During each exposure, one gravimetric blood sample was collected at a flow rate of 11/min. for 15 minutes. The target pulmonary dose was 10 mg/kg. The actual delivered lung dose was determined by gamma imaging immediately after exposure.

Blood samples were collected at 0 (before), 0.08, 0.25, 0.5,

0.75, 1, 1.5, 2, 3 and 4 hours after exposure to measure blood glucose and serum 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 the 6 dogs was calculated to be 10 ± 5 pg/kg (mean ± standard deviation). The mean inhaled dose was 287 ± 107 μg/kg (mean ± standard deviation). Individual animal data are shown in Table 3 below.

Table 3 Animal Target Inhaled Estimated No. dose dose dose delivered to (pg/kg) delivered to lung (pg/kg) lung (pg/kg)

• · · · · · • 9 9 9 9

M · · · • · ·

• · « 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

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

4 4 4

9

- 29 - ;

« · 4 4 • · ·

4 4 · · • 4 · · · • 4 4 · • 4 ··

4» 44 «4 * • 4 · ·

4 * 4 · ·· · « 4 ·«

Table 4: Serum concentrations of Asp ^B28 -human insulin (ng/ml) as a function of time after pulmonary administration.

Aerosol Dog 0 hours 0.083 hours 0.25 hours 0.5 hours 0.75 hours 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 hours 2 hours 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 levels were also measured after pulmonary administration of Asp ^B28 -human insulin (Table 5). Serum glucose was determined using a Hitachi Chemistry System (Boehringer Mannheim Corp., Indianapolis, IN). The maximum percentage decrease in blood glucose levels compared to control values was observed 1 hour after pulmonary exposure. Decreases of approximately 40% and 70% were ... 0000

0 0«

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

Table 5:

Time Percentage of level change Percentage of level change (minutes) serum glucose serum glucose after inhalation after subcutaneous administration Asp ^B28 -human insulin Asp ^B28 -human insulin (mean ± std. deviation) (mean ± std. deviation) 0 100+0 100+0 5 105±6 98±7 15 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 achieved measurable levels of the test substance above pre-administration values. Comparisons of pharmacokinetic parameters after subcutaneous and pulmonary administration are presented in Table 6.

Table 6:

Method of administration AUC (ng.h/ml) _Cmax (ng/ml) Tmax (hours)

·· ·· ♦ ♦ ·

9 9·· « · · · · ·

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

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

Claims (8)

1. Use of Asp ^B28 -human insulin for the preparation of a medicament suitable for administration by inhalation. 2. Use according to claim 1, wherein the medicament is in the form of a solution or aqueous medium or a suspension or non-aqueous medium. 3. The use according to claim 1, wherein the medicament is in the form of an aerosol. 4. The 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 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 from claims 1-7, when Asp ^B28 -human insulin is serves in doses in range approximately 3 pg/kg to approximately 2C i pg/kg. 9. Use according to any from claims 1-7, when Asp ^B28 -human insulin is serves in doses in range approximately 7 μg/kg to approximately 14 pg/kg.