WO2008084051A1

WO2008084051A1 - Mixtures of pegylated insulin and fast acting insulin for pulmonary administration

Info

Publication number: WO2008084051A1
Application number: PCT/EP2008/050156
Authority: WO
Inventors: Peter Madsen; Thomas Børglum KJELDSEN
Original assignee: Novo Nordisk A/S
Priority date: 2007-01-12
Filing date: 2008-01-09
Publication date: 2008-07-17

Abstract

A pharmaceutical formulation is obtained comprising a mixture of long-acting insulin and fast-acting insulin, wherein the long-acting insulin is PEGylated insulin or a PEGylated insulin analogue and wherein the fast-acting insulin is human insulin or an insulin analogue. Particular embodiments of the invention include a formulation in a solid form or as a solution for pulmonary delivery. Further described is the use of a formulation according to the invention for the manufacture of a medicament for the treatment of a mammal having reduced ability to produce serum insulin compared to a normal mammal and a method of treating diabetes in a patient in need of such treatment, comprising administering to a patient a therapeutically effective amount of a pharmaceutical formulation according to the invention.

Description

Mixtures of PEGylated Insulin and Fast Acting Insulin for Pulmonary Administration

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The inherited physical and chemical stability of the insulin molecule is a basic condition for insulin therapy of diabetes mellitus. These basic properties are fundamental for insulin formulation and for applicable insulin administration methods, as well as for shelf-life and storage conditions of pharmaceutical preparations. Use of solutions in administration of insulin exposes the molecule to a combina- tion of factors, e.g., elevated temperature, variable air-liquid-solid interphases as well as shear forces, which may result in irreversible conformation changes, e.g., fibrillation.

Unfortunately, many diabetics are unwilling to undertake intensive therapy due to the discomfort associated with the many injections required to maintain close control of glucose levels. This type of therapy can be both psychologically and physically painful. Upon oral administration, insu- lin is rapidly degraded in the gastro intestinal tract and is not absorbed into the blood stream. Therefore, many investigators have studied alternate routes for administering insulin, such as oral, rectal, transdermal, and nasal routes. Thus far, however, these routes of administration have not resulted in effective insulin absorption.

Efficient pulmonary delivery of a protein is dependent on the ability to deliver the protein to the deep lung alveolar epithelium. Proteins that are deposited in the upper airway epithelium are not absorbed to a significant extent. This is due to the overlying mucus which is approximately 30- 40 μm thick and acts as a barrier to absorption. In addition, proteins deposited on this epithelium are cleared by mucociliary transport up the airways and then eliminated via the gastrointestinal tract. This mechanism also contributes substantially to the low absorption of some protein particles. The extent to which proteins are not absorbed and instead eliminated by these routes depends on their solubility, their size, as well as other less understood characteristics.

It is, however, well recognised that the properties of peptides can be enhanced by grafting organic chain-like molecules onto them. Such grafting can improve pharmaceutical properties such as half life in serum, stability against proteolytical degradation and reduced immunogenicity. The organic chain-like molecules often used to enhance properties are polyethylene glycol- based chains, i.e., chains that are based on the repeating unit -CH₂CH₂O-. Hereinafter, the abbreviation "PEG" is used for polyethyleneglycol.

Classical PEG technology takes advantage of providing polypeptides with increased size (Stoke radius) by attaching a soluble organic molecule to the polypeptide (Kochendoerfer, G., et al., Science (299) 884 et seq., 2003). This technology leads to reduced clearance in man and animals of a hormone polypeptide compared to the native polypeptide. However, this technique is often hampered by reduced potency of the hormone polypeptides subjected to this technique (Hinds, K., et al., Biocon- jugate Chem. (1 1 ), 195 - 201 , 2000).

Insulin formulations for pulmonary administration comprising a conjugate of two-chain insulin covalently coupled to one or more molecules of non-naturally hydrophilic polymers including polyalkyl- ene glycols and methods for their preparation are disclosed in WO 02/094200, WO 94/20069 and WO 02/092147.

WO 03/094951 discloses pharmaceutical formulations containing insulin aspart and insulin detemir.

It has traditionally been difficult to obtain mixtures of insulins having a prolonged profile of action with insulins having a fast action onset where the two insulin components act as or act substantially as they would have acted if they had been the only insulin components present.

There is still a need for mixtures of insulins having a more prolonged profile of action with fast acting insulins. Furthermore, there is need for such insulin formulations which are well suited for pulmonary application.

SUMMARY OF THE INVENTION

In one embodiment of the invention a pharmaceutical formulation is obtained comprising a mixture of long-acting insulin and fast-acting insulin.

In another embodiment a pharmaceutical formulation for pulmonal delivery is obtained comprising a mixture of long-acting insulin and fast-acting insulin. In yet another embodiment a pharmaceutical formulation is obtained comprising a mixture of long-acting insulin and fast-acting insulin, wherein the long-acting insulin is PEGylated insulin or a PEGylated insulin analogue and wherein the fast-acting insulin is human insulin or an insulin analogue

In yet another embodiment a formulation is obtained comprising a mixture of long-acting insulin and fast-acting insulin, wherein the long-acting insulin is human insulin via a linker conjugated with PEG in one or more positions or an insulin analogue via a linker conjugated with PEG in one or more positions; and wherein the fast-acting insulin is human insulin or an insulin analogue.

A formulation according to the invention may in one embodiment be in a solid form or in the form of a solution.

Also comprised by the invention is the use of a fast-acting insulin in an amount in the range from 10 % to 90 % of the total amount of insulin component calculated on a unit to unit basis to prepare a solution or a solid formulation having both a fast-acting and a long-acting insulin component and the use of a formulation according to the invention for the manufacture of a medicament for the treatment of a mammal having reduced ability to produce serum insulin compared to a normal mammal. Further comprised is a method of treating diabetes in a patient in need of such treatment, comprising administering to a patient a therapeutically effective amount of a pharmaceutical formulation according to the invention. DESCRIPTION OF THE INVENTION

Insulin is a polypeptide hormone secreted by β-cells of the pancreas and consists of two polypeptide chains designated the A and B chains which are linked together by two inter-chain disulphide bridges. The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids in the configuration: prepeptide-B-Arg-Arg-C-Lys-Arg-A, in which C is a connecting peptide of 31 amino acids, and A and B are the A and B chains, respectively, of insulin. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide between the A and B chains to form the two-chain insulin mole- cule. Insulin is essential in maintaining normal metabolic regulation.

In embodiment 1 of the invention a pharmaceutical formulation is obtained comprising a mixture of a long-acting insulin and a fast-acting insulin

Embodiment 2. Formulation according to embodiment 1 in a form suitable for pulmonary administration Embodiment 3. Formulation according to any one of the preceding embodiments wherein the long-acting insulin is PEGylated insulin or a PEGylated insulin analogue and wherein the fast-acting insulin is human insulin or an insulin analogue

Embodiment 4. Formulation according to any one of the preceding embodiments comprising a mixture of long-acting insulin and fast-acting insulin wherein the long-acting insulin is selected from a group consisting of:

• human insulin conjugated with PEG in one or more positions; and

• an insulin analogue conjugated with PEG in one or more positions; and wherein the fast-acting insulin is selected from a group consisting of:

• human insulin; and • an insulin analogue wherein one or more amino acid residues of insulin is substituted and/or deleted and/or one or more amino acid residues is inserted into and/or added to the insulin.

Embodiment 5. Formulation according to any one of the preceding embodiments comprising a mixture of long-acting insulin and fast-acting insulin wherein the long-acting insulin is selected from a group consisting of

• human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 , and B29; and

• an insulin analogue conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 , B3, B28 and B29; and wherein the fast-acting insulin is selected from a group consisting of

• human insulin; • an insulin analogue wherein the amino acid residue in position B28 of insulin is Pro, Asp,

Lys, Leu, VaI, or Ala and the amino acid residue in position B29 is Lys or Pro and optionally the amino acid residue in position B30 is deleted;

• des(B28-B30) human insulin, des(B27) human insulin or des(B30) human insulin; and • an insulin analogue wherein the amino acid residue in position B3 is Lys and the amino acid residue in position B29 is GIu or Asp.

Embodiment 6. Formulation according to any one of the preceding embodiments comprising a mixture of long-acting insulin and fast-acting insulin wherein the long-acting insulin is selected from a group consisting of • human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29;

• DesB30 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29;

• AspB28 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29;

• AspB28,DesB30 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29;

• LysB3,GluB29 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B3; and • LysB28,ProB29 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B28; and wherein the fast-acting insulin is selected from a group consisting of

• human insulin;

• DesB30 human insulin; • AspB28 human insulin ;

• AspB28,DesB30 human insulin;

• LysB3,GluB29 human insulin; and

• LysB28,ProB29 human insulin.

Embodiment 7. Formulation according to any one of the previous embodiments wherein the nominal average molecular weight of a PEG covalently coupled to insulin is in the range from about 200 to about 20,000 daltons, from about 500 to about 5000 daltons, from about 500 to about 2000 daltons, from about 750 to about 2000 daltons.

Embodiment 8. Formulation according to embodiment 7 herein the nominal average molecular weight of a PEG covalently coupled to insulin is selected from the group consisting of about 750, about 2000, about 5000 and about 20000 daltons.

Embodiment 9. Formulation according to embodiment 8 herein the nominal average molecular weight of a PEG covalently coupled to insulin is about 750 daltons. Embodiment 10. Formulation according to embodiment 8 herein the nominal average molecular weight of a PEG covalently coupled to insulin is about 2000 daltons.

Embodiment 1 1. Formulation according to any one of the embodiments 1-6 wherein a PEG residue via a linker covalently coupled to insulin is monodisperse. Embodiment 12. Formulation according to embodiment 1 1 wherein a PEG covalently coupled to insulin is elected from the group consisting of mdPEGi₂, mdPEG₂4, mdPEGi₂- CH2CH2NHCOCH2CH2OCH2)3CNHCOCH2CH2(OCH2CH2)4NHCOCH2CH₂CO- or mdPEGi2- CH₂CH₂NHCOCH₂CH₂OCH₂)₃CNHCOCH₂CH₂(OCH₂CH₂)₄NHCOCH₂CH₂CH₂CO-.

Embodiment 13. Formulation according to any one of the previous embodiments wherein the long-acting insulin is a mixture of one, two or three PEG residues via linkers covalently coupled to insulin.

Embodiment 14. Formulation according to embodiment 13 wherein the long-acting insulin comprises three conjugates of PEG residues via linkers covalently coupled to insulin.

Embodiment 15. Formulation according to embodiment 13 wherein the long-acting insulin comprises two conjugates of PEG residues via linkers covalently coupled to insulin.

Embodiment 16. Formulation according to embodiment 13 wherein the long-acting insulin comprises one conjugate of PEG residue via a linker covalently coupled to insulin.

Embodiment 17. Formulation according to any one of the previous embodiments wherein the molar ratio between the long-acting insulin and the fast-acting insulin is in a range from 10:90 to 90:10. Embodiment 18. Formulation according to embodiment 17 wherein the molar ratio between the long-acting insulin and the fast-acting insulin is selected from the group consisting of 90:10, 85:15, 80:20, 75:25, 70:30, 67:33, 50:50, 30:70, 33:67, 25:75, 20:80, 15:85 and 10:90.

Embodiment 19. Formulation according to any one of the previous embodiments further comprising one or more agents selected from the group consisting of an excipient, an isotonicity agent, a preservative, a buffer, a surfactant, a stabilizer, a chelating agent, a reducing agent, a bulk protein, a carbohydrate and a suitable zinc salt.

Embodiment 20. Formulation according to any one of the previous embodiments, wherein the formulation is in the form of a solution.

Embodiment 21. Formulation according to embodiment 20 wherein the concentration of fast- acting insulin is in the range from 3 - 20 mM. Embodiment 22. Formulation according to embodiment 21 wherein the concentration of fast-acting insulin is in the range from 5 - 15 mM.

Embodiment 23. Formulation according to embodiment 22 wherein the concentration of fast- acting insulin is 9 mM.

Embodiment 24. Formulation according to embodiment 22 wherein the concentration of fast- acting insulin is 12 mM.

Embodiment 25. Formulation according to embodiment 20 wherein the concentration of long- acting insulin is in the range from 3 - 20 mM.

Embodiment 26. Formulation according to embodiment 25 wherein the concentration of long- acting insulin is in the range from 5 - 15 mM. Embodiment 27. Formulation according to embodiment 26 wherein the concentration of long- acting insulin is 9 mM.

Embodiment 28. Formulation according embodiment 26 wherein the concentration of long- acting insulin is 12 mM. Embodiment 29. Formulation according to any one of the embodiments 20-28, wherein the pH value is in the range from 6.5 to 8.5.

Embodiment 30. Formulation according to embodiment 29 wherein the pH value is in the range from 7.4 to 7.9.

Embodiment 31. Formulation according to any one of the embodiments 20-30, wherein the preservative is phenol.

Embodiment 32. Formulation according to any one of the embodiments 20-31 wherein the pH-buffer is a physiologically acceptable buffer selected from the group consisting of diglycine buffer, phosphate buffer, TRIS buffer, acetate buffer, carbonate buffer and mixtures thereof.

Embodiment 33. Formulation according to any one of the embodiments 20-32, wherein the pH-buffer is a physiologically acceptable buffer in a concentration in the range from 3 mM to 20 mM.

Embodiment 34. Formulation according to any one of the embodiment 20-33 wherein the pH-buffer is a physiologically acceptable buffer in a concentration in the range from 5 mM to 15 mM. Embodiment 35. Formulation according to any one of the embodiments 1-19, wherein the formulation is in a solid form. Embodiment 36. Formulation according to embodiment 35 wherein the formulation is in a particulate form.

Embodiment 37. Formulation according embodiment 36 wherein the formulation is in a particulate form with a particle size of less than about 10 μm.

Embodiment 38. Formulation according to embodiment 37 wherein the formulation is in a particulate form with a particle size of 1 to 5 μm.

Embodiment 39. Formulation according to any one of the embodiments 35-38 further comprising one or more agents selected from the group consisting of a bulking agent, a carrier and an ex- cipient.

Embodiment 40. The use of a fast-acting insulin in an amount in the range from 10 % to 90 %, of the total amount of insulin component calculated on a unit to unit basis to prepare a solution or a solid powder having both a fast-acting and a long-acting insulin component.

Embodiment 41. Use of a formulation comprising a mixture of long-acting insulin and fast- acting insulin wherein said long-acting insulin and said fast-acting insulin are provided in a molar ratio of between 90:10 to 10:90 for the manufacture of a medicament for the treatment of a mammal having reduced ability to produce serum insulin compared to a normal mammal.

Embodiment 42. A method of treating diabetes in a patient in need of such treatment, comprising administering to a patient a therapeutically effective amount of a pharmaceutical formulation according to any one of the embodiments 1-39. In one embodiment of the invention a pharmaceutical formulation is obtained comprising a mixture of a long-acting insulin and a fast-acting insulin.

Herein, the term insulin covers natural occurring insulins, e.g., human insulin, as well as insulin analogues thereof. With fast-acting insulin is meant an insulin having a similar or faster onset of action than normal or regular human insulin. With long-acting insulin is meant an insulin having a longer duration of action than normal or regular human insulin, i.e. a protracted activity.

It has been found that formulations of the invention may be obtained wherein the two insulin components act as or act substantially as they would have acted if they had been the only insulin components present. I.e. the protracted activity of the long-acting insulin is retained or substantially retained and the fast onset of the fast-acting insulin is retained or substantially retained. In one embodiment a formulation for pulmonary delivery comprising a mixture of long-acting insulin and fast- acting insulin is obtained. In a further embodiment a formulation for pulmonary delivery comprising a mixture of long-acting insulin and fast-acting insulin is obtained wherein the two insulin components act as or act substantially as they would have acted if they had been the only insulin components pre- sent.

In one embodiment, the present invention is based upon formulations of long-acting and fast- acting insulin for administration to the systemic circulation via the deep lung. In a further embodiment the formulations of the invention comprise a conjugate of insulin covalently coupled to one or more molecules of a non-naturally occurring hydrophilic polymer and human insulin or an insulin analogue. In yet a further embodiment, the non-naturally occurring, hydrophilic polymer covalently coupled to insulin is a polyalkylene glycol such as polyethylene glycol (PEG).

By insulin analogue as used herein is meant a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, for example that of human insulin, by deleting and/or substituting at least one amino acid residue occurring in the natural insulin and/or by adding or inserting at least one amino acid residue. For example between 1-10 amino acid residues, 1-5 amino acid residues, 4 amino acid residues, 3 amino acid residues 2 amino acid residues or 1 amino acid residue may be substituted, deleted, added and/or inserted to a naturally occurring insulin. The added, inserted and/or substituted amino acid residues are codable.

The ratio between the long-acting insulin and the fast-acting insulin can be determined by a person skilled in the art. In one embodiment the ratio is 90:10, 85:15, 80:20, 75:25, 70:30, 67:33, 50:50, 30:70, 33:67, 25:75, 20:80, 15:85 or 10:90.

In one embodiment the long-acting insulin is a conjugate of insulin or an insulin analogue covalently coupled to one or more non-naturally occurring hydrophilic polymer(s). The long-acting insulin can be a conjugate of insulin or an insulin analogue coupled to one or more polyalkylene glycols such as polyethylene glycol (PEG), herein also referred to as a "PEGinsulin", "PEGinsulin conjugate" or "PEGylated insulin". For example between 1-3 PEG molecules, 1-2 or 1 PEG molecule(s) may be coupled to insulin or an insulin analogue according to the invention.

With "PEG" or polyethylene glycol, as used herein is meant any water soluble poly(alkylene oxide). The expression PEG will comprise the structure -CI-I₂CI-I₂O(CI-I₂CI-I₂O)_nCH₂CH₂O-, where n is an integer from 2 to about 1000. A commonly used PEG is end-capped PEG, wherein one end of the PEG termini is end-capped with a relatively inactive group such as alkoxy, while the other end is a hy- droxyl group that may be further modified by linker moieties. An often used capping group is methoxy and the corresponding end-capped PEG is often denoted mPEG. Hence, mPEG is CI-I₃O(CI-I₂CI-I₂O)_nCI-I₂CI-I₂-O-, where n is an integer from 2 to about 1000 sufficient to give the average molecular weight indicated for the whole PEG moiety, e.g., for mPEG Mw 2,000, n is approximately 44 (a number that is subject for batch-to-batch variation). The notion PEG is often used instead of mPEG.

Specific PEG forms of this invention are branched, linear or multi-armed PEGs, and the like. In one embodiment, a PEG-insulin conjugate may comprise two mono-functionallyderivatized insulin molecules interconnected by a di-activated polyethylene glycol (insulin-PEG-insulin). An insulin molecule within this "dumbell" architecture may be further modified by additional PEGs. In another embodiment, a PEG-insulin conjugate of the invention comprises a forked polyethylene glycol having a branching moiety at one end of the polymer chain and two free reactive groups (or a multiple of two) linked to the branching moiety for covalent attachment to insulin. In this embodiment of the invention, the branched architecture of polyethylene glycol allows attachment of the polymer chain to two or more molecules of insulin. The PEG groups are typically polydisperse, i.e. a mixture of various lengths (or molecular weights) of the PEG polymer, possessing a low polydispersity index of less than about 1.05. The PEG moieties present in a PEGinsulin conjugate will for a given molecular weight typically consist of a range of ethyleneglycol (or ethyleneoxide) monomers. For example, a PEG moiety of molecular weight 2000 will typically consist of 44 ± 10 monomers, the average being around 44 monomers. The molecular weight (and number of monomers) will typically be subject to some batch-to- batch variation.

Other specific PEG forms are monodisperse that can be branched, linear, forked, or dumb- bell shaped as well. Being monodisperse means that the length (or molecular weight) of the PEG polymer is specifically defined and is not a mixture of various lengths (or molecular weights). Herein the notion mdPEG is used to indicate that the mPEG moiety is monodisperse, using "d" for "discrete". The number in subscript after mdPEG, for example mdPEG_i2, the number (12) indicates the number of ethyleneglycol monomers within the monodisperse polymer (oligomer). Herein the term linker covers a chemical moiety which connects an -HN- group of the insulin with the -O- group of the PEG moiety. The linker does not have any influence on the desired action of the final PEGylated insulin, especially it does not have any adverse influence.

The term PEGylation covers modification of insulin by attachment of one or more PEG moieties via a linker. The PEG moiety can either be attached by nucleophilic substitution (acylation) on N- terminal alpha-amino groups or on lysine residue(s) on the gamma-positions, e.g., with OSu-activated esters, or PEG moieties can be attached by reductive alkylation - also on amino groups present in the insulin molecule - using PEG-aldehyde reagents and a reducing agent, such as sodium cyanoboro- hydride. Terms like A1 , A2, A3 etc. indicates the position 1 , 2 and 3, respectively, in the A chain of insulin (counted from the N-terminal end). Similarly, terms like B1 , B2, B3 etc. indicates the position 1 , 2 and 3, respectively, in the B chain of insulin (counted from the N-terminal end). Using the one letter codes for amino acids, terms like A21A, A21 G and A21 Q designates that the amino acid in the A21 position is A, G and Q, respectively. Using the three letter codes for amino acids, the corresponding expressions are AlaA21 , GlyA21 and GlnA21 , respectively. Terms like desB29 and desB30 indicate an insulin analogue lacking the B29 or B30 amino acid residue, respectively.

In one embodiment, a long-acting insulin of the formulation of the invention is a conjugate wherein insulin is covalently coupled to PEG at one or more of its amino sites. Long-acting insulin con- tained within a formulation of the invention may be mono-substituted, di-substituted, or tri-substituted (i. e., having one, two, or three PEG moieties covalently coupled thereto). Particular PEG-insulin conjugates in accordance with the invention possess a polyethylene glycol moiety covalently attached to one, two or three position(s) on the insulin molecule selected from the group consisting of PheB1 , GIyAI , and LysB3, LysB28 or LysB29. In one embodiment PEG-insulin conjugates in accordance with the invention possess a polyethylene glycol moiety covalently attached to one position on the insulin molecule selected from the group consisting of PheB1 , GIyAI and LysB3, LysB28 or LysB29.

In a preferred embodiment, the PEG moiety is covalently attached at the PheB1 site of insulin. In a one embodiment, at least about 75% of the PheB1 sites on insulin are covalently coupled to PEG. In another embodiment, at least about 90% of the PheB1 sites on insulin are covalently coupled to PEG.

In a preferred embodiment, the PEG moiety is covalently attached to one of the positions LysB3, LysB28 or LysB29 of insulin or an insulin analogue. In a one embodiment, at least about 75% at one of the positions LysB3, LysB28 or LysB29 on insulin or an insulin analogue are covalently coupled to PEG. In another embodiment, at least about 90% at one of the positions LysB3, LysB28 or LysB29 on insulin or an insulin analogue are covalently coupled to PEG.

In one embodiment a long-acting insulin of the invention is human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29; desB30 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29; AspB28 human insulin (Insulin Aspart) conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29; AspB28, desB30 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29; LysB3,GluB29 human insulin (Insulin Glulisine) conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B3; and/or LysB28,ProB29 human insulin (Insulin Lispro) conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B28. In general, an insulin-polymer conjugate of the invention can exhibit pharmacokinetic and pharmacodynamic properties improved over native insulin, particularly when administered to the lung. In one embodiment, the PEG-insulin conjugates provided herein exhibit good absolute bioavailabilities when administered to the lung and deep lung. In a particular embodiment, a PEG-insulin conjugate of the invention is characterized by an absolute pulmonary bioavailability that is greater than that of na- tive insulin. Preferably, a PEG-insulin conjugate of the invention is characterized by having an absolute pulmonary bioavailability that is at least 1.5-2.0 times greater than that of native insulin. In a more preferred embodiment, a PEG-insulin conjugate in accordance with the invention is characterized by an absolute pulmonary bioavailability that is greater than about 15%, even more preferably greater than about 20% or most preferably greater than about 30%.

In yet another embodiment, a PEG-insulin conjugate of the invention, when administered pulmonarily, exhibits a Tmax (the amount of time required to reach maximum concentration) that is at least 1 .5 times that of native insulin, or more preferably is at least 2 or 3 times, or even more preferably that is at least five times that of native insulin. In yet another embodiment, a PEG-insulin conjugate of the invention, when administered pulmonarily, exhibits a t<_/2 (the amount of time required for the concentration to be halved) that is at least 1 .5 times that of native insulin, or alternatively that is at least 2, 3, 4 or 5, or also alternatively that is at least 10 times that of native insulin.

The polyethylene glycol-portion of an insulin conjugate of the invention may optionally con- tain one or more degradable linkages.

Typically, insulin is covalently coupled to PEG via a linking moiety positioned at a terminus of the PEG. Preferred linking moieties for use in the invention include those suitable for coupling with reactive insulin amino groups such as N-hydroxysuccinimide active esters, active carbonates, aldehydes, and acetals. In one embodiment, a PEG covalently coupled to insulin in a conjugate of the invention will comprise from about 2 to about 300 subunits of (OCH₂CH₂), preferably from about 4 to 200 subunits, and more preferably from about 10 to about 100 subunits, and even more preferably from about 10 to about 50 subunits.

In one embodiment, a PEG covalently coupled to insulin will possess a nominal average mo- lecular weight of from about 200 to about 20,000 daltons. In a preferred embodiment, the PEG will possess a nominal average molecular weight from about 200 to about 5000 daltons. In yet a more preferred embodiment, the PEG will possess a nominal average molecular weight from about 200 to about 2000 daltons or from about 200 to about 1000 daltons.

The following examples are offered by way of illustration, not by limitation. In the following list, selected PEGylation reagents are listed as activated Λ/-hydroxy- succinimide esters (OSu). Obviously, other active esters may be employed, such as 4-nitrophenoxy and many other active esters known to those skilled in the art. The PEG (or mPEG) moiety, CH₃O- (CH₂CH₂O)_n-, can be of any size up to Mw 40.000 Da, e.g., 750 Da, 2000 Da, 5000 Da, 20.000 Da and 40.000 Da. The mPEG moiety can be polydisperse but also monodisperse consisting of mPEG's with well defined chain lengths (and, thus, molecular weights) of, e.g., 12 or 24 repeating ethylene glycol units - denoted mdPEG_x for m: methyl/methoxy end-capped, d: discrete and x for the number of repeating ethylene glycol residues, e.g., 12 or 24. The PEG moiety can be either straight chain or branched. The structure/sequence of the PEG-residue on the insulin can formally be obtained by replacing the leaving group (e.g., "-OSu") from the various PEGylation reagents with "NH-insulin", where the insulin is PEGylated either in an epsilon position in a lysine residue or in the alpha-amino position in the A- or B-chain (or both): mPEG-COCH₂CH₂CO-OSu, mPEG-COCH₂CH₂CH₂CO-OSu, mPEG-CHzCO-OSu, mPEG-CHzCHzCO-OSu, mPEG-CHzCHzCHzCO-OSu, mPEG-CHzCHzCHzCHzCO-OSu, mPEG-CHzCHzCHzCHzCHzCO-OSu, mPEG-CH₂CH(CH₃)CO-OSu,

FTIPEG-CH₂CH₂CH(CH₃)CO-OSU, mPEG-CH₂CH₂NH-COCH₂CH₂CO-OSu, mPEG-CH₂CH₂CH₂NH-COCH₂CH₂CH₂CO-OSu, mPEG-CH₂CH₂CH₂NH-COCH₂CH₂CO-OSu, mPEG-CH₂CH₂NH-COCH₂CH₂CH₂CO-OSu, mPEG-CO-(4-nitrophenoxy),

(mdPEG₁₂-CH₂CH₂NHCOCH₂CH₂OCH₂)₃CNHCOCH₂CH₂(OCH₂CH₂)₄NHCOCH₂CH₂CO- OSu (or, in short: (mdPEG₁₂)₃-dPEG₄-OSu),

(mdPEGi₂-CH₂CH₂NHCOCH₂CH₂OCH₂)₃CNHCOCH₂CH₂(OCH₂CH₂)₄NH- COCH₂CH₂CH₂CO-OSU (or, in short: (mdPEGi₂)₃-dPEG₄-OSu), mdPEG_x-COCH₂CH₂CO-OSu, mdPEG_x-COCH₂CH₂CH₂CO-OSu, mdPEG_x-CH₂CO-OSu, mdPEG_x-CH₂CH₂CO-OSu, mdPEG_x-CH₂CH₂CH₂CO-OSu, mdPEG_x-CH₂CH₂CH₂CH₂CO-OSu, mdPEG_x-CH₂CH₂CH₂CH₂CH₂CO-OSu, mdPEG_x-CH₂CH(CH₃)CO-OSu, mdPEG_x-CH₂CH₂CH(CH₃)CO-OSu, mdPEG_x-CH₂CH₂NH-COCH₂CH₂CO-OSu, mdPEG_x-CH₂CH₂CH₂NH-COCH₂CH₂CH₂CO-OSu, mdPEG_x-CH₂CH₂CH₂NH-COCH₂CH₂CO-OSu, mdPEG_x-CH₂CH₂NH-COCH₂CH₂CH₂CO-OSu or mdPEG_x-CO-(4-nitrophenoxy) wherein x is an integer in the range from about 6 to about 48, e.g. ,12 or 24.

In a particular embodiment, the long-acting insulin is PEGylated human insulin.

In yet another embodiment, the conjugate of the formulation of the invention possesses a purity of greater than about 90% (i. e., of the conjugate portion of the formulation, 90% or more by weight comprises one or more PEG-insulins). That is to say, formulations of the invention may be character- ized by a high degree of purity of conjugated insulin component, i.e., the formulation is absent detectable amounts of free polyethylene glycol species and other PEG-related impurities.

In an alternative embodiment, a PEG insulin conjugate in accordance with the invention is characterized by a rate of proteolysis that is reduced relative to non-pegylated or native insulin. A fast-acting insulin of the invention may be human insulin or such wherein the amino acid residue in position B28 of insulin is substituted with Pro, Asp, Lys, Leu, VaI, or Ala; the amino acid residue in position B29 is Lys or Pro; and optionally the amino acid residue in position B30 is deleted (i.e. the des(B30) analogue), des(B28-B30) human insulin, des(B27) human insulin or des(B30) human insulin, and an analogue wherein the amino acid residue in position B3 is Lys and the amino acid residue in position B29 is GIu or Asp.

In one embodiment a fast-acting insulin of the invention is human insulin; desB30 human insulin; AspB28 human insulin (Insulin Aspart); AspB28,desB30 human insulin; LysB3,GluB29 human insulin (Insulin Glulisine); and/or LysB28,ProB29 human insulin (Insulin Lispro).

In one embodiment a formulation is provided comprising a mixture of long-acting insulin and fast-acting insulin wherein the long-acting insulin is PEGylated insulin or a PEGylated insulin analogue and wherein the fast-acting insulin is human insulin or an insulin analogue.

In a further embodiment a formulation is provided comprising a mixture of long-acting insulin and fast-acting insulin wherein the long-acting insulin is selected from a group consisting of:

• human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29; and

• an insulin analogue conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 , B3 and B29; and wherein the fast-acting insulin is selected from a group consisting of:

• des(B28-B30) human insulin, des(B27) human insulin or des(B30) human insulin; and

• an insulin analogue wherein the amino acid residue in position B3 is Lys and the amino acid residue in position B29 is GIu or Asp.

Formulations of the invention may comprise a long acting insulin and a short-acting insulin where the long-acting insulin comprises a mixture of mono-conjugated and di-conjugated PEG insulin and optionally tri-conjugated, the PEG insulin having any one or more of the features described above.

Also provided herein is a mixture of a bioactive polyethylene glycol-insulin conjugate and human insulin or an insulin analogue suitable for administration by inhalation to the deep lung.

In yet another embodiment, the invention provides a method for delivering a formulation comprising a PEGinsulin conjugate and human insulin or an insulin analogue to a mammalian subject in need thereof by administering by inhalation a formulation as previously described in aerosolized form.

The invention also provides in another embodiment, a method for providing a substantially non-immunogenic insulin formulation for administration to the lung. The method includes the steps of covalently coupling insulin to one or more molecules of a non-naturally occurring hydrophilic polymer conjugate as described herein for obtaining a long-acting insulin, substituting an insulin or using human insulin for obtaining a fast-acting insulin, mixing the long-acting and the fast acting insulins, and administering the formulation to the lung of a subject by inhalation, whereby as a result, the mixture of long-acting and fast-acting insulins passes through the lung and enters into the blood circulation. In another embodiment, provided is a method for providing an insulin formulation for administration to the lung of a human subject whereby an immediate reaction is obtained followed by a protracted effect. The method includes covalently coupling insulin to one or more PEG molecules to provide a long-acting insulin, substituting insulin or using human insulin to provide a fast-acting insulin, mixing the long-acting and the fast acting insulins, and administering the formulation to the lung of the subject by inhalation. Upon the administering step, the long-acting and the fast-acting insulins pass through the lung and enter into the blood circulation, and an immediate elevation of blood levels of insulin is obtained followed by elevated blood levels of insulin sustained for at least 8 hours post administration.

The formulation of this invention may also be used on combination treatment together with an antidiabetic agent.

Antidiabetic agents will include insulin, GLP-1 (1 -37) (glucagon like peptide-1 ) described in WO 98/08871 , WO 99/43706, US 5424286 and WO 00/09666, GLP-2, exendin-4(1-39), insulinotropic fragments thereof, insulinotropic analogues thereof and insulinotropic derivatives thereof. Insulinotropic fragments of GLP-1 (1-37) are insulinotropic peptides for which the entire sequence can be found in the sequence of GLP-1 (1-37) and where at least one terminal amino acid has been deleted.

The formulation of this invention may also be used on combination treatment together with an oral antidiabetic such as a thiazolidindione, metformin and other type 2 diabetic pharmaceutical preparation for oral treatment.

Furthermore, the pharmaceutical formulation of this invention may be administered in combi- nation with one or more antiobesity agents or appetite regulating agents.

A formulation of the invention, when aerosolized and administered via inhalation, is useful in the treatment of diabetes mellitus (DM).

Herein the term parent insulin means human insulin or insulin analogue without appended PEG moieties. The parent insulins are produced by expressing a DNA sequence encoding the insulin in question in a suitable host cell by well known technique as disclosed in, e.g., US patent No. 6500645. The parent insulin is either expressed directly or as a precursor molecule which has an N-terminal extension on the B-chain. This N-terminal extension may have the function of increasing the yield of the directly expressed product and may be of up to 15 amino acid residues long. The N-terminal extension is to be cleaved of in vitro after isolation from the culture broth and will therefore have a cleavage site next to B1. N-terminal extensions of the type suitable in this invention are disclosed in U.S. Patent No. 5,395,922, and European Patent No. 765.395A.

The polynucleotide sequence coding for the parent insulin may be prepared synthetically by established standard methods, e.g., the phosphoamidite method described by Beaucage et al. (1981 ) Tetrahedron Letters 22:1859-1869, or the method described by Matthes et al. (1984) EMBO Journal 3: 801 -805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, duplexed and ligated to form the synthetic DNA construct. A currently preferred way of preparing the DNA construct is by polymerase chain reaction (PCR). The polynucleotide sequences may also be of mixed genomic, cDNA, and synthetic origin.

For example, a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the A and B chains, after which the DNA sequence may be modified at a site by inserting synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures or preferably generating the desired se- quence by PCR using suitable oligonucleotides.

The recombinant method will typically make use of a vector which is capable of replicating in the selected microorganism or host cell and which carries a polynucleotide sequence encoding the parent insulin. The recombinant vector may be an autonomously replicating vector, i.e., a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replica- tion, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. The vector may be linear or closed circular plasmids and will preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

The recombinant expression vector is capable of replicating in yeast. Examples of sequences which enable the vector to replicate in yeast are the yeast plasmid 2 μm replication genes REP 1-3 and origin of replication.

The vector may contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (ornithine carbamoyltransferase), pyrG (orotidine-5'-phosphate decarboxylase) and trpC (anthranilate synthase. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3. A well suited selectable marker for yeast is the Schizosaccharomyces pompe TPI gene (Russell (1985) Gene 40:125-130).

In the vector, the polynucleotide sequence is operably connected to a suitable promoter sequence. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extra-cellular or intra-cellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene {dagA), Bacillus subtilis levansucrase gene {sacB), Bacillus licheniformis alpha-amylase gene {amyL), Bacillus stearothermophilus maltogenic amylase gene {amyM), Bacillus amyloliquefaciens alpha-amylase gene {amyQ), and Bacillus licheniformis penicillinase gene {penP). Examples of suitable promoters for directing the transcription in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, and Aspergillus niger acid stable alpha-amylase. In a yeast host, useful promoters are the Saccharomyces cerevisiae Ma1 , TPI, ADH or PGK promoters.

The polynucleotide sequence encoding the parent insulin will also typically be operably connected to a suitable terminator. In yeast a suitable terminator is the TPI terminator (Alber et al. (1982) J. MoI. Appl. Genet. 1 :419-434). The procedures used to ligate the polynucleotide sequence encoding the parent insulin, the promoter and the terminator, respectively, and to insert them into a suitable vector containing the information necessary for replication in the selected host, are well known to persons skilled in the art. It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire DNA sequence encoding the insulins of this invention, and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for the individual elements (such as the signal, pro-peptide, connecting peptide, A and B chains) followed by ligation.

The vector comprising the polynucleotide sequence encoding the parent insulin is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating ex- tra-chromosomal vector. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, Streptomyces cell, or gram negative bacteria such as E. coli and Pseudomonas sp. Eu- karyote cells may be mammalian, insect, plant, or fungal cells. In one embodiment, the host cell is a yeast cell. The yeast organism may be any suitable yeast organism which, on cultivation, produces large amounts of the insulin of the invention. Examples of suitable yeast organisms are strains selected from the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pas- toris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida ca- caoi, Geotrichum sp., and Geotrichum fermentans.

The transformation of the yeast cells may for instance be effected by protoplast formation followed by transformation in a manner known per se. The medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms. The secreted insulin, a significant proportion of which will be present in the medium in correctly processed form, may be recovered from the medium by conventional procedures including separating the yeast cells from the medium by cen- trifugation, filtration or catching the insulin precursor by an ion exchange matrix or by a reverse phase absorption matrix, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g., ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g., ion exchange chromatography, affinity chromatography, or the like.

PHARMACEUTICAL FORMULATIONS

The formulations according to the invention may be biphasic.

Formulations of the invention may be dissolved or suspended in liquid or in dry form, and may additionally comprise a pharmaceutically acceptable excipient. Formulations in dry form typically contain less than 20% moisture, alternatively less than 10% moisture.

The pharmaceutical formulation of this invention may be administered subcutaneously, orally, or pulmonary.

In one embodiment, the long-acting and the fast-acting insulins are administered as a mixed pharmaceutical preparation. In another embodiment, the long-acting and the fast-acting insulins are administered separately either simultaneously or sequentially.

For subcutaneous administration, the mixture of long-acting and fast-acting insulins of this invention are formulated analogously with the formulation of known insulins. Furthermore, for subcutaneous administration, the mixture of long-acting and fast-acting insulins of this invention are adminis- tered analogously with the administration of known insulins and, generally, the physicians are familiar with this procedure.

Mixtures of long-acting and fast-acting insulins of this invention may be administered by inhalation in a dose effective to increase circulating insulin levels and/or to lower circulating glucose levels. Such administration can be effective for treating disorders such as diabetes or hyperglycemia. Achieving effective doses of insulin requires administration of an inhaled dose of more than about 0.5 μg/kg to about 50 μg/kg of a mixture of long-acting and fast-acting insulins of this invention. A therapeutically effective amount can be determined by a knowledgeable practitioner, who will take into account factors including insulin level, blood glucose levels, the physical condition of the patient, the patient's pulmonary status, or the like. The mixture of long-acting and fast-acting insulins of this invention may be delivered by inhalation to achieve slow absorption and/or reduced systemical clearance of the long-acting insulin as well as fast absorption and/or fast clearance of the fast-acting insulin. Different inhalation devices typi- cally provide similar pharmacokinetics when similar particle sizes and similar levels of lung deposition are compared.

The mixture of long-acting and fast-acting insulins of this invention may be delivered by any of a variety of inhalation devices known in the art for administration of a therapeutic agent by inhala- tion. These devices include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Preferably, the mixture of long-acting and fast-acting insulins of this are delivered by a dry powder inhaler or a sprayer. There are a several desirable features of an inhalation device for administering mixture of long-acting and fast-acting insulins of this invention. For example, delivery by the inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device should deliver small particles or aerosols, e.g., less than about 10 μm, for example about 1-5 μm, for good respirability. Some specific examples of commercially available inhalation devices suitable for the practice of this invention are Turbohaler™ (Astra), Rotahaler^® (Glaxo), Diskus^® (Glaxo), Spiros™ inhaler (Dura), devices marketed by Inhale Therapeutics, AERx™ (Aradigm), the Ultravent^® nebulizer (Mallinckrodt), the Acorn II^® nebulizer (Marquest Medical Products), the Ventolin^® metered dose in- haler (Glaxo), the Spinhaler^® powder inhaler (Fisons), or the like.

As those skilled in the art will recognize, the formulation of mixtures of long-acting and fast- acting insulins of this invention, the quantity of the formulation delivered and the duration of administration of a single dose depend on the type of inhalation device employed. For some aerosol delivery systems, such as nebulizers, the frequency of administration and length of time for which the system is activated will depend mainly on the concentration of respectively the long-acting and the fast-acting insulins in the aerosol. For example, shorter periods of administration can be used at higher concentrations of the mixture of long-acting and fast-acting insulins in the nebulizer solution. Devices such as metered dose inhalers can produce higher aerosol concentrations, and can be operated for shorter periods to deliver the desired amount of the mixture of long-acting and fast-acting insulins. Devices such as powder inhalers deliver active agent until a given charge of agent is expelled from the device. In this type of inhaler, the amount of mixture of long-acting and fast-acting insulins of this invention in a given quantity of the powder determines the dose delivered in a single administration.

The particle size of a mixture of long-acting and fast-acting insulins of this invention in the formulation delivered by the inhalation device is critical with respect to the ability of insulin to make it into the lungs, and preferably into the lower airways or alveoli. Preferably, the mixture of long-acting and fast-acting insulins of this invention is formulated so that at least about 10% of the mixture of long- acting and fast-acting insulins delivered is deposited in the lung, preferably about 10 to about 20%, or more. It is known that the maximum efficiency of pulmonary deposition for mouth breathing humans is obtained with particle sizes of about 2 μm to about 3 μm. When particle sizes are above about 5 μm, pulmonary deposition decreases substantially. Particle sizes below about 1 μm cause pulmonary deposition to decrease, and it becomes difficult to deliver particles with sufficient mass to be therapeutically effective. Thus, particles of the mixture of long-acting and fast-acting insulins delivered by inhalation have a particle size preferably less than about 10 μm, more preferably in the range of about 1 μm to about 5 μm. The formulation of the mixture of long-acting and fast-acting insulins is selected to yield the desired particle size in the chosen inhalation device.

Advantageously for administration as a dry powder a mixture of long-acting and fast-acting insulins of this invention is prepared in a particulate form with a particle size of less than about 10 μm, preferably about 1 to about 5 μm.

In one embodiment a dry powder may be prepared by mixing dry powder long-acting insulin and dry powder fast-acting insulin and optionally further mixing with dry powder pharmaceutical carriers), bulking agent, excipient and/or other additive(s). In another embodiment a dry powder is prepared by mixing a dry powder comprising a long-acting insulin and pharmaceutical ingredients such as carrier(s), bulking agent and/or additive(s) with a dry powder comprising a fast-acting insulin and pharmaceutical ingredients such as carrier(s), bulking agent and/or additive(s). Usually, the dry powder will have from 5% to 99% by weight mixture of long-acting and fast-acting insulin in the formulation, more usually from 15% to 80%, in a suitable pharmaceutical carrier, usually a carbohydrate, an organic salt, an amino acid, peptide, or protein. In another embodiment of the present invention, dry powders are prepared by dissolving the mixture of long-acting and fast-acting insulin in an aqueous buffer to form a solution and spray drying the solution to produce substantially amorphous particles. Optionally, the pharmaceutical carrier is also dissolved in the buffer, to form a homogeneous solution, wherein spray drying of the solution produces individual particles comprising long-acting insulin, fast-acting insulin, carrier buffer, and any other components which were present in the solution. Preferably the carrier is a carbohydrate, organic salt, amino acid, peptide, or protein which produces a substantially amorphous structure upon spray drying. The amorphous carrier may be either glassy or rubbery and enhances stability of the insulin during storage.

The preferred particle size is effective for delivery to the alveoli of the patient's lung. Advan- tageously, such stabilized formulations are also able to effectively deliver long-acting and fast-acting insulin to the blood stream upon inhalation to the alveolar regions of the lungs. Preferably, the dry powder is largely composed of particles produced so that a majority of the particles have a size in the desired range. Advantageously, at least about 50% of the dry powder is made of particles having a diameter less than about 10 μm. Such formulations can be achieved by spray drying, milling, or critical point condensation of a solution containing the mixture of long-acting and fast-acting insulins of this invention and other desired ingredients. Other methods also suitable for generating particles useful in the current invention are known in the art.

The particles are usually separated from a dry powder formulation in a container and then transported into the lung of a patient via a carrier air stream. Typically, in current dry powder inhalers, the force for breaking up the solid is provided solely by the patient's inhalation. In another type of inhaler, air flow generated by the patient's inhalation activates an impeller motor which deagglomerates the particles.

Formulations of mixtures of long-acting and fast-acting insulins of this invention for administration from a dry powder inhaler typically include a finely divided dry powder containing the deriva- tive, but the powder can also include a bulking agent, carrier, excipient, another additive, or the like. Additives can be included in a dry powder formulation of a mixture of long-acting and fast-acting insulins, e.g., to dilute the powder as required for delivery from the particular powder inhaler, to facilitate processing of the formulation, to provide advantageous powder properties to the formulation, to facili- tate dispersion of the powder from the inhalation device, to stabilize the formulation (for example, antioxidants or buffers), to provide taste to the formulation, or the like. Advantageously, the additive does not adversely affect the patient's airways. The mixture of long-acting and fast-acting insulins can be further mixed with an additive at a molecular level or the solid formulation can include particles of the mixture of long-acting and fast-acting insulins further mixed with or coated on particles of the additive. Typical additives include mono-, di-, and polysaccharides; sugar alcohols and other polyols, such as, e.g., lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or the like. Typically an additive, such as a bulking agent, is present in an amount effective for a purpose described above, often at about 50% to about 90% by weight of the formulation. Additional agents known in the art for formulation of a protein such as insulin analogue protein can also be included in the formulation.

A spray including the mixture of long-acting and fast-acting insulins of this invention can be produced by forcing a suspension or solution of the mixture of long-acting and fast-acting insulins through a nozzle under pressure. The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size. An electrospray can be produced, e.g., by an electric field in connection with a capillary or nozzle feed. Advantageously, particles of insulin conjugate delivered by a sprayer have a particle size less than about 10 μm, preferably in the range of about 1 μm to about 5 μm.

Formulations of mixture of long-acting and fast-acting insulins of this invention suitable for use with a sprayer will typically include the mixture of long-acting and fast-acting insulins in an aqueous solution at a concentration of from about 1 mg to about 20 mg of each of the long-acting and the fast-acting insulins per ml of solution. The formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc. The formulation can also include an excipient or agent for stabilization of the long-acting and the fast-acting insulins, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating insulin conjugates include albumin, protamine, or the like. Typical carbohydrates useful in formulating the mixture of long-acting and fast-acting insulins include sucrose, mannitol, lactose, trehalose, glucose, or the like. The mixture of long-acting and fast-acting insulins formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the insulin conjugate caused by atomiza- tion of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation.

Pharmaceutical formulations containing a mixture of long-acting and fast-acting insulins of this invention may also be administered parenterally to patients in need of such a treatment. Par- enteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump.

Formulations of the mixture of long-acting and fast-acting insulins of this invention can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product. Thus, according to one procedure, a mixture of long-acting and fast-acting insulins is dissolved in an amount of water which is somewhat less than the final volume of the formulation to be prepared. Zinc, an isotonic agent, a preservative and/or a buffer is/are added as required and the pH value of the solution is adjusted - if nec- essary - using an acid, e.g., hydrochloric acid, or a base, e.g., aqueous sodium hydroxide as needed. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients. According to another procedure a long-acting insulin solution is prepared comprising zinc, an isotonic agent, a preservative and/or a buffer as required and the pH value of the long-acting insulin solution is adjusted - if necessary - using an acid, e.g., hydrochloric acid, or a base, e.g., aqueous so- dium hydroxide as needed; a fast-acting insulin solution is prepared comprising zinc, an isotonic agent, a preservative and/or a buffer as required and the pH value of the fast-acting insulin solution is adjusted - if necessary - using an acid, e.g., hydrochloric acid, or a base, e.g., aqueous sodium hydroxide as needed; and the long-acting and the fast-acting insulin solutions are mixed and the pH value of the mixture of long-acting and fast-acting insulins solution is adjusted - if necessary - to give the desired pharmaceutical formulation of a mixture of long-acting and fast-acting insulins.

In a further embodiment of this invention the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)- aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of this invention.

In a further embodiment of this invention the formulation further comprises a pharmaceutically acceptable preservative which may be selected from the group consisting of phenol, o-cresol, tricresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p- hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, ben- zethonium chloride, chlorphenesine (3-(4-chlorophenoxy)-1 ,2-propanediol) or mixtures thereof. In a further embodiment of this invention the preservative is present in a concentration from about 0.1 mg/ml to 20 mg/ml. In a further embodiment of this invention the preservative is present in a concen- tration from about 0.1 mg/ml to 5 mg/ml. In a further embodiment of this invention the preservative is present in a concentration from about 5 mg/ml to 10 mg/ml. In a further embodiment of this invention the preservative is present in a concentration from about 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of this invention. The use of a preserva- tive in pharmaceutical formulations is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995.

In a further embodiment of this invention, the formulation further comprises an isotonic agent which may be selected from the group consisting of a salt ( e.g., sodium chloride), a sugar or sugar alcohol, an amino acid (for example, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), an alditol (e.g. glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3- propanediol or 1 ,3-butanediol), polyethyleneglycol (e.g., PEG400) or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodex- trin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In one embodiment the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group and includes, e.g., mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodiment the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of this invention. In one embodiment, the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 1 mg/ml to 50 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 1 mg/ml to 7 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 8 mg/ml to 24 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of this invention. The use of an isotonic agent in pharmaceutical formulations is well-known to the skilled person. For convenience reference is made to Remington: The Sci- ence and Practice of Pharmacy, 19^th edition, 1995.

Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfone and glycerol and typical preservatives are phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are diglycine buffer, phosphate buffer, TRIS buffer, acetate buffer, carbonate buffer, glycylglycine and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

A formulation for nasal administration of a mixture of long-acting and fast-acting insulins of this invention may, e.g., be prepared as described in European Patent No. 272097.

Formulations containing mixture of long-acting and fast-acting insulins of this invention can be used in the treatment of states which are sensitive to insulin. Thus, they can be used in the treat- ment of type 1 diabetes, type 2 diabetes and hyperglycaemia for example as sometimes seen in seriously injured persons and persons who have undergone major surgery. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific insulin derivative employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the state to be treated. It is recommended that the daily dosage of the mixture of long-acting and fast-acting insulins of this invention be determined for each individual patient by those skilled in the art in a similar way as for known insulin formulations.

In one embodiment, the two active components, long-acting insulin and fast-acting insulin, are administered as a mixed pharmaceutical preparation. In another embodiment, the two components are administered separately either simultaneously or sequentially.

EXAMPLES

General procedure (A) for preparation of insulins PEGylated at ε-positions in lysine residues (positions B3, B28 or B29) according to the invention:

Human insulin or analogue thereof (125 mg, 22 μmol) was dissolved in 0.1 M Na₂CO₃ (2.8 ml). PEGylation reagent as Λ/-hydroxysuccinimide ester (e.g., mPEG-SPA 2000) (25 μmol) dissolved in acetonitrile (1.25 ml) was added. If necessary, pH is adjusted from up to 10.5 with 0.1 N NaOH. If necessary more mPEG-SPA 2000 (25 mg) dissolved in acetonitrile (1.25 ml) is added after 45 min following HPLC analysis of the reaction mixture. After slow stirring for 80 min, water (4.5 ml) was added and pH was adjusted to 5 with 1 N HCI. The mixture is lyophilized. The PEGylated insulin is obtained by preparative HPLC purification followed by lyophilisation. The insulin is analysed by HPLC, MALDI-TOF, HPLC-ES/MS, and/or other methods

General procedure (B) for preparation of insulins PEGylated at ε-positions in lysine residues (positions B3, B28 or B29) according to the invention:

Human insulin or analogue thereof (200 mg, 35 μmol) is added to 0.05 M boric acid (10 ml) and pH is adjusted to 10.2 by addition of 1 N NaOH whereby the insulin is dissolved. PEGylation reagent as Λ/-hydroxysuccinimide ester (e.g., mPEG-SPA 2000) (70 μmol) dissolved in acetonitrile (6 ml) is added and the mixture is diluted with water (14 ml). If necessary, pH is adjusted from up to 10.5 with 0.1 N NaOH. After slow stirring for 80 min, pH was adjusted to 5 with 1 N HCI. The mixture is lyophilized. The PEGylated insulin is obtained by preparative HPLC purification followed by lyophilisation. The insulin is analysed by HPLC, MALDI-TOF, HPLC-ES/MS, and/or other methods

Alternatively, PEGylated insulins can be prepared by published methods, e.g., as described in WO 02/094200, Hinds et al., Bioconjugate Chem., 1 1 (2), 195-201 (2000), or Hinds & Kim, Advanced Drug Deliv. Rev., 54, 505-530 (2002)

The following insulins were prepared according to one of the above general procedures: In the following, PEGylated insulin analogues with polydisperse PEG residues have been drawn as they contain a specific number of PEG residues. For example, in example 1 the insulin contains a polydisperse PEG moiety with average molecular weight 20000 Da. It has been drawn as it contains exactly 453 ethyleneglycol residues. Correctly, it contains a number of PEG residues of different lengths with an average length of approximately 453, which is a number that is subject to con- siderable batch-to-batch variation. Example 1, B29K(N^εmPEG20000-Propionyl), desB30 human insulin

MALDI-TOF MS: m/z = 27000 (broad)

Example 2, B29K(N^εmPEG2000-Pmpionyl), desB30 human insulin

MALDI-TOF MS: m/z = 7900 (broad)

Example 3, B29K(N^εmPEG5000-Pmpionyl), desB30 human insulin

MALDI-TOF MS: m/z = 1 1000 (broad) Example 4, B29K(N^ε(mPEG750-Carbamoyl)propionyl) desB30 human insulin

H G I VEQ

MALDI-TOF MS: m/z = 6500 (broad)

Example 5, B29K(N^ε-(4-[2-(2-{2-[2-(2-Tri-(2-ethoxy-N-(mdPEG₁₂)carbamoyl)methyl)methyl- carbamoylethoxy)ethoxy]ethoxy}ethoxy)ethylcarbamoyl]butanoyl), desB30 human insulin (B29K(t*f(mdPEG₁₂)₃-dPEG₄-yl), desB30 human insulin)

MALDI-TOF MS: m/z = 8012

Example 6: A1G-, B1F- & BΣΘ^^mPEGTSO-Carbamoylfpropionyl) desB30 human insulin, randomly PEGylated

This insulin was prepared as described in WO 02/094200

MALDI-TOF MS: m/z = 6600 (broad) (mono-PEG), 7500 (broad) (di-PEG).

Example 7, B28D, B29K(N^εmPEG2000-Propionyl) human insulin

MALDI-TOF MS: m/z = 7700 (broad)

Example 8, B28D, BΣΘK^mPEGΣOOO-Propionyl), desB30 human insulin

H-G I VEQ

H-F V N Q H

MALDI-TOF MS: m/z = 7900 (broad) Example 9, B28D, BΣΘK^mdPEG_∑rPropionyl), desB30 human insulin

H-G I VEQCCTS I CS LYQL

MALDI-TOF MS: m/z = 6935

Example 10, B28D, B29K(N^εmdPEG₂₄-Propionyl), desB30 human insulin

H-G I V E Q

H-F V N Q H

MALDI-TOF MS: m/z = 6398

Example 11, B28D, B29K(N^ε(mPEG750-Carbamoyl)propionyl) human insulin

MALDI-TOF MS: m/z = 6644 (broad) Other preferred PEGylated insulins of the invention includes:

Randomly PEGylated (PEG750 at B29, B1 and/or A1 ) human insulin,

Randomly PEGylated (PEG2000 at B29, B1 and/or A1 ) human insulin, Randomly PEGylated (PEG5000 at B29, B1 and/or A1 ) human insulin,

PEGylated (PEG750 at B29) human insulin,

PEGylated (PEG2000 at B29) human insulin,

PEGylated (PEG5000 at B29) human insulin,

Randomly PEGylated (PEG750 at B29, B1 and/or A1 ) insulin aspart, Randomly PEGylated (PEG2000 at B29, B1 and/or A1 ) insulin aspart,

Randomly PEGylated (PEG5000 at B29, B1 and/or A1 ) insulin aspart,

PEGylated (PEG750 at B29) insulin aspart,

PEGylated (PEG2000 at B29) insulin aspart,

PEGylated (PEG5000 at B29) insulin aspart, B29K(Λ/^εmdPEG₂₄-propionyl), desB30 human insulin,

B29K(Λ/^εmdPEG₁₂-propionyl), desB30 human insulin,

B29K(Λ/^ε(mPEG750-carbamoyl)propionyl) human insulin,

B29K(Λ/^εmPEG2000-propionyl) human insulin,

B29K(Λ/^εmPEG5000-propionyl) human insulin, B29K(Λ/^εmPEG20000-propionyl) human insulin,

B29K(Λ/^ε(mdPEGi₂)3-dPEG₄-yl) human insulin,

B29K(Λ/^εmdPEG₂₄-propionyl) human insulin,

B29K(Λ/^εmdPEGi₂-propionyl) human insulin,

A1 G-, B1 F- & B29K(Λ/^ε(mPEG750-carbamoyl)propionyl) human insulin, A1 G-, B1 F- & B29K(Λ/^ε(mPEG2000-carbamoyl)propionyl) human insulin,

A1 G-, B1 F- & B29K(Λ/^ε(mPEG2000-carbamoyl)propionyl) desB30 human insulin,

B28D, B29K(Λ/^εmPEG5000-propionyl) human insulin,

B28D, B29K(Λ/^εmPEG20000-propionyl) human insulin,

B28D, B29K(Λ/^ε(mdPEGi₂)₃-dPEG₄-yl) human insulin, B28D, B29K(Λ/^εmdPEG₂₄-propionyl) human insulin,

B28D, B29K(Λ/^εmdPEGi₂-propionyl) human insulin,

B28D, B29K(Λ/^ε(mPEG750-carbamoyl)propionyl), desB30 human insulin,

B28D, B29K(Λ/^εmPEG5000-propionyl), desB30 human insulin,

B28D, B29K(Λ/^εmPEG20000-propionyl), desB30 human insulin, B28D, B29K(Λ/^ε(mdPEGi₂)₃-dPEG₄-yl) desB30 human insulin,

B28K(Λ/^ε(mPEG750-carbamoyl)propionyl), B29P human insulin,

B28K(Λ/^εmPEG2000-propionyl), B29P human insulin, B28K(Λ/^εmPEG5000-propionyl), B29P human insulin, B28K(Λ/^εmPEG20000-propionyl), B29P human insulin, B28K(Λ/^ε(mdPEGi₂)₃-dPEG₄-yl), B29P human insulin, B28K(Λ/^εmdPEG₂₄-prc)pionyl), B29P human insulin, B28K(Λ/^εmdPEG₁₂-propionyl), B29P human insulin,

BSK^mPEGyδO-carbamoyOpropionyl), B29E human insulin, B3K(Λ/^εmPEG2000-propionyl), B29E human insulin, BSK^mPEGδOOO-propionyl), B29E human insulin, B3K(Λ/^εmPEG20000-propionyl), B29E human insulin, B3K(W^ε(mdPEGi₂)3-dPEG₄-yl), B29E human insulin,

B3K(Λ/^εmdPEG₂₄-propionyl), B29E human insulin and BSK^mdPEG^-propionyl), B29E human insulin.

Preferred fast acting insulins of the invention includes: Human insulin, desB30 human insulin,

B28D human insulin (insulin aspart),

B28D, desB30 human insulin,

B3K, B29E human insulin (insulin Glulisine) and B28K, B29P human insulin (insulin Lispro).

Example 12: Insulin receptor binding of the insulins of this invention.

The affinity of the insulins of this invention for the human insulin receptor is determined by a SPA as- say (Scintillation Proximity Assay) microtiterplate antibody capture assay. SPA-PVT antibody-binding beads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) are mixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodium chloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mix for a single Packard Optiplate (Packard No. 6005190) is composed of 2.4 μl of a 1 :5000 diluted purified recombinant human insulin receptor (either with or without exon 1 1 ), an amount of a stock solution of A14Tyr[¹²⁵l]-human insulin corresponding to 5000 cpm per 100 μl of reagent mix, 12 μl of a 1 :1000 dilution of F12 antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml. A total of 100 μl reagent mix is then added to each well in the Packard Optiplate and a dilution series of the insulin derivative is made in the Optiplate from appropriate samples. The samples are then incubated for 16 hours while gently shaken. The phases are the then separated by centrifugation for 1 min and the plates counted in a Topcounter. The binding data were fitted using the nonlinear regression algorithm in the GraphPad Prism 2.01 (GraphPad Software, San Diego, CA). Example 13: Blood glucose lowering effect after Lv. bolus injection in rat of the insulins of this invention

Male Wistar rats, 200-300 g, fasted for 18 h, is anesthetized using either Hypnorm-Dormicum s.c. (1.25 mg/ml Dormicum, 2.5 mg/ml fluanisone, 0.079 mg/ml fentanyl citrate) 2 ml/kg as a priming dose (to timepoint -30 min prior to test substance dosing) and additional 1 ml /kg every 20 minutes.

The animals are dosed with an intravenous injection (tail vein), 1 ml/kg, of control and test compounds (usual dose range 0.125-20 nmol/kg). Blood samples for the determination of whole blood glucose concentration are collected in heparinized 10 μl glass tubes by puncture of the capillary vessels in the tail tip to time -20min and 0 min (before dosing), and to time 10, 20, 30, 40, 60, 80, 120, and 180 min after dosing. Blood glucose concentrations are measured after dilution in analysis buffer by the immobilized glucose oxidase method using an EBIO Plus autoanalyzer (Eppendorf, Germany). Mean plasma glucose concentrations courses (mean ± SEM) are made for each dose and each compound.

Example 14: Potency of the insulins of this invention relative to human insulin

Sprague Dawley male rats weighing 238-383 g on the experimental day are used for the clamp experiment. The rats have free access to feed under controlled ambient conditions and are fasted overnight (from 3 pm) prior to the clamp experiment.

Experimental Protocol:

The rats are acclimatized in the animal facilities for at least 1 week prior to the surgical procedure. Approximately 1 week prior to the clamp experiment, Tygon catheters are inserted under halothane anaesthesia into the jugular vein (for infusion) and the carotid artery (for blood sampling) and exteriorised and fixed on the back of the neck. The rats are given Streptocilin vet. (Boehringer Ingelheim; 0.15 ml/rat, i.m.) post-surgically and placed in an animal care unit (25 ⁰C) during the recovery period. In order to obtain analgesia, Anorphin (0.06 mg/rat, s.c.) is administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.) is administered after full recovery from the anaesthesia (2-3 h) and again once daily for 2 days.

At 7 am on the experimental day overnight fasted (from 3 pm the previous day) rats are weighed and connected to the sampling syringes and infusion system (Harvard 22 Basic pumps, Harvard, and Per- tectum Hypodermic glass syringe, Aldrich) and then placed into individual clamp cages where they rest for ca. 45 min before start of experiment. The rats are able to move freely on their usual bedding during the entire experiment and have free access to drinking water. After a 30 min basal period during which plasma glucose levels were measured at 10 min intervals, the insulin derivative to be tested and human insulin (one dose level per rat, n = 6-7 per dose level) are infused (i.v.) at a constant rate for 300 min. Plasma glucose levels are measured at 10 min intervals throughout and infusion of 20% aqueous glucose is adjusted accordingly in order to maintain euglyceamia. Samples of re-suspended erythrocytes are pooled from each rat and returned in about 14 ml volumes via the carotid catheter.

On each experimental day, samples of the solutions of the individual insulin derivatives to be tested and the human insulin solution are taken before and at the end of the clamp experiments and the concentrations of the peptides are confirmed by HPLC. Plasma concentrations of rat insulin and C-peptide as well as of the insulin derivative to be tested and human insulin are measured at relevant time points before and at the end of the studies. Rats are killed at the end of experiment using a pentobarbital overdose.

Example 15: Pulmonary delivery of insulins to rats

The test substance will be dosed pulmonary by the drop instillation method. In brief, male Wistar rats (app.250 g) are anaesthesized in app. 60 ml fentanyl/dehydrodenzperidol/-dormicum given as a 6.6 ml/kg sc priming dose and followed by 3 maintainance doses of 3.3 ml/kg sc with an interval of 30 min. Ten minutes after the induction of anaesthesia, basal samples are obtained from the tail vein (t = -20 min) followed by a basal sample immediately prior to the dosing of test substance (t=0). At t=0, the test substance is dosed intra tracheally into one lung. A special cannula with rounded ending is mounted on a syringe containing the 200 ul air and test substance (1 ml/kg). Via the orifice, the cannula is introduced into the trachea and is forwarded into one of the main bronchi - just passing the bifurcature. During the insertion, the neck is palpated from the exterior to assure intratracheal positioning. The content of the syringe is injected followed by 2 sec pause. Thereafter, the cannula is slowly drawn back. The rats are kept anaesthesized during the test (blood samples for up to 4 or 8 hrs) and are euthanized after the experiment.

Example 16, Mixtures of long-acting and fast-acting insulins of the invention:

Claims

1. A pharmaceutical formulation comprising a mixture of long-acting insulin and fast-acting insulin, wherein the long-acting insulin is PEGylated insulin or a PEGylated insulin analogue and wherein the fast-acting insulin is human insulin or an insulin analogue.

2. Formulation according to claim 1 in a form suitable for pulmonary administration.

3. Formulation according to claim 1 or 2 comprising a mixture of long-acting insulin and fast-acting insulin wherein the long-acting insulin is selected from a group consisting of: • human insulin conjugated with PEG in one or more positions;

• human insulin;

• an insulin analogue wherein one or more amino acid residues of insulin is substituted and/or deleted and/or one or more amino acid residues is inserted into and/or added to the insulin.

4. Formulation according to claim 3 comprising a mixture of long-acting insulin and fast-acting insulin wherein the long-acting insulin is selected from a group consisting of

• human insulin conjugated with PEG in one or more positions selected from the group con- sisting of A1 , B1 , and B29;

• an insulin analogue conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 , B3 and B29; and wherein the fast-acting insulin is selected from a group consisting of

• des(B28-B30) human insulin, des(B27) human insulin or des(B30) human insulin; and an insulin analogue wherein the amino acid residue in position B3 is Lys and the amino acid resi- due in position B29 is GIu or Asp.

5. Formulation according to claim 4 comprising a mixture of long-acting insulin and fast-acting insulin wherein the long-acting insulin is selected from a group consisting of

• human insulin conjugated with PEG in one or more positions selected from the group con- sisting of A1 , B1 and B29;

• DesB30 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29; • AspB28 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29;

• AspB28,DesB30 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29; • LysB3,GluB29 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29;

• LysB28,ProB29 human insulin conjugated with PEG in one or more positions selected from the group consisting of A1 , B1 and B29; and wherein the fast-acting insulin is selected from a group consisting of • human insulin;

• DesB30 human insulin;

• AspB28 human insulin ;

• AspB28,DesB30 human insulin;

• LysB3,GluB29 human insulin and • LysB28,ProB29 human insulin.

6. Formulation according to any one of the previous claims wherein the molar ratio between the long- acting insulin and the fast-acting insulin is in a range from 10:90 to 90:10.

7. Formulation according to any one of the previous claims, wherein the nominal average molecular weight of a PEG covalently coupled to insulin is in the range from 200 to 20,000 daltons.

8. Formulation according to any one of the previous claims, wherein the formulation is in a form of a dry powder.

9. Formulation according to any one of the claims 1-7, wherein the formulation is in the form of a solution.

10. The use of a fast-acting insulin in an amount in the range from 10 % to 90 %, of the total amount of insulin component calculated on a unit to unit basis to prepare a solution or a powder having both a fast-acting and a long-acting insulin component.

1 1. Use of a formulation comprising a mixture of long-acting insulin and fast-acting insulin wherein said long-acting insulin and said fast-acting insulin are provided in a molar ratio of between 10:90 to 90:10 for the manufacture of a medicament for the treatment of a mammal having reduced ability to produce serum insulin compared to a normal mammal.

12. A method of treating diabetes in a patient in need of such treatment, comprising administering to a patient a therapeutically effective amount of a pharmaceutical formulation according to any one of claims 1-9.

13. Any novel feature or combinations of features described herein.