Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/28—Insulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
  • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins

Definitions

• the present invention relates to insulin analogues having a fast onset of action.
• the invention also relates to compositions comprising such insulin analogues, and to compositions comprising a mixture of an insulin analogue having a fast onset of action and insulin having a protracted action.
• Human insulin is a 51 amino acid peptide hormone consisting of an A-chain and a B-chain having 21 and 30 amino acid residues respectively, interconnected by two cysteine bridges. Insulin may aggregate into hexamers, in which form the hormone is protected from chemical and physical degradation during synthesis and storage. The action of such insulin hexamers is delayed because the hexamers must diffuse and dissociate into dimmers and monomers.
• Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin to cover the basal requirement supplemented by bolus injections of a rapid acting insulin to cover the requirement related to meals.
• Protracted insulin compositions are well known in the art.
• one main type of protracted insulin compositions comprises injectable aqueous suspensions of insulin crystals or amorphous insulin.
• the insulin compounds utilized typically are protamine insulin, zinc insulin or protamine zinc insulin.
• Certain drawbacks are associated with the use of insulin suspensions, e.g. the need to suspend the insulin particles by gentle shaking before a defined volume of the suspension is withdrawn from a vial or expelled from a cartridge
• protracted insulin compositions are solutions having a pH value below physiological pH from which the insulin will precipitate because of the rise in the pH value when the solution is injected.
• a drawback with these solutions is that the particle size distribution of the precipitate formed in the tissue on injection, and thus the timing of the medication, depends on the blood flow at the injection site and other parameters in a somewhat unpredictable manner.
• the solid particles of the insulin may act as predictable manner.
• the solid particles of the insulin may act as a local irritant causing inflammation of the tissue at the site of injection.
• a further type of protracted insulin compositions is those in which the ⁇ -amino group of residue Lys B29 has been acylated with a long-chain fatty acid, see e.g. WO 95/07931 and WO 98/02460 (Novo Nordisk A/S).
• One such soluble insulin derivative is B29-N ⁇ -(N-lithocholyl- ⁇ - glutamyl)-des(B30) human insulin.
• the protracted action has been explained by a reversible binding to albumin in subcutis, blood and peripheral tissue (see e.g. Markussen, Diabetologia 39, 281-288, 1996).
• Rapid-acting insulin analogues are known, in which a mutation has been introduced with the aim of reducing the tendency to associate into higher molecular weight forms.
• Examples of such analogues are Asp B28 and Lys B28 Pro B29 human insulin.
• Some patients use insulin compositions having both a fast onset of action and a more prolonged action. This is effected by using an insulin composition comprising two types of insulin, where one has a fast onset of action and the other a more prolonged action.
• the two types of insulin may be present in different ratios.
• Such compositions may be prepared by the patients themselves prior to injection, or more conveniently the composition may be pre-mixed and ready for injection. In such pre-mixed compositions the exchange of insulin monomers between the rapid-acting insulin analogue and the prolonged-action insulin hexamer may take place, which may result in a inferior release profile of insulin.
• the invention provides analogues of human insulin wherein the amino acid in position B26 is Phe.
• the remaining amino acid sequence may be identical to that of human insulin or may contain substitutions or deletions.
• the invention also provides a pharmaceutical preparation comprising i. A derivative of human insulin ii. an analogue of human insulin having a fast onset of action, wherein Phe(B1) is deleted, or Tyr(B26) is replaced with Phe, or Phe(B1) is deleted and Tyr(B26) is replaced with Phe.
• the ratio of the derivative of human insulin i. to the analogue of human insulin having a fast onset of action ii. is between 1 :99 and 99:1, such as between 10:90 and 90:10, e.g. between 30:70 and 70:30.
• the derivative of human insulin i. may be selected from the group consisting of
• a pharmaceutical preparation which further comprises a phenolic preservative.
• Also provided by the invention is a method of treating type 1 or type 2 diabetes comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceu- tical preparation according of the invention, and the use of a preparation of the invention for the preparation of a medicament for treatment of type 1 or type 2 diabetes.
• insulin As used herein is intended to refer to not only human insulin as such, but also insulin analogues and derivatives. "Human insulin” is well known in the art, and is given below for convenience:
• analogue of human insulin refers to a polypeptide having the amino acid sequence of human insulin in which one or more amino acids have been deleted and/or replaced by other amino acids, including amino acids not encoded by the genetic code, or comprising additional amino acids, i.e. more than the 51 amino acids of human insulin.
• derivative of human insulin refers to human insulin or an analogue thereof in which at least one organic substituent is bound to one or more of the amino acids.
• Non-limiting examples of a “derivative of human insulin” are B29-N ⁇ -myristoyl-des(B30) human insulin, B29-N ⁇ -palmitoyl-des(B30) human insulin, B29-N ⁇ -myristoyl human insulin, B29- N ⁇ -palmitoyl human insulin, B28-N ⁇ -myristoyl Lys B28 Pro 629 human insulin, B28-N ⁇ -palmitoyl Lys B28 Pro 829 human insulin, B30-N ⁇ -myristoyl-Thr B29 Lys B30 human insulin, B30-N ⁇ -palmitoyl- Thr B29 Lys B30 human insulin, B29-N ⁇ -(N-palmitoyl- ⁇ -glutamyl)-des(B
• chemical stability refers to the tendency of an insulin composition to form stable hexamer structures.
• the unit "U" corresponds to 6 nmol.
• treatment means the management and care of a patient for the purpose of combating a disease, disorder or condition.
• the term is intended to include the delaying of the progression of the disease, disorder or condition, the alleviation or relief of symptoms and complications, and/or the cure or elimination of the disease, disorder or condition.
• the patient to be treated is preferably a mammal, in particular a human being.
• the present invention relates to rapid-acting analogues of human insulin wherein the amino acid in position B26 is Phe.
• Such analogues have been found to be particularly useful when used in premixed pharmaceutical preparations where another, long-acting insulin is also present.
• the monomer insulin molecules of the present invention do not interact with the hexamer formed by the long-acting insulin molecules to the same degree as known fast- acting analogues.
• the release profile of insulin after injection of the pre-mixed preparation shows an improved release profile, in that both an rapid initial release as well as a continuing, basal release is observed.
• the invention provides analogues of human insulin wherein the amino acid in position B26 is Phe and wherein the amino acid sequence is at least 80 % identical to that of human insulin.
• This and the following analogues wherein the amino acid in position B26 is Phe are termed Group A analogues.
• the invention provides an analogue of human insulin wherein the amino acid in position B26 is Phe and which has a half life of less than 3 hours measured in the disappearance assay described herein.
• A-Chain (contd.) 20 Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO:i; 13 14 15 16 17 18 19
• B-Chain (contd.) Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- 13 14 15 16 17 18 19 20 21 22 23 24
• Xa1 is Phe, Glu or -H
• Xa2 is Asn or Lys
• Xa3 is His or Asp
• Xa4 is Thr or Pro
• Xa5 is Pro, Thr, Lys, Asp or lie,
• Xa6 is Lys ,Thr, Pro or Glu
• Xa7 is Thr, Lys, Pro, Glu or -OH
• ment Xa5 s Lys In anothe embod ment Xa5 s Lys. In anothe embod ment Xa5 s Asp. In anothe embod ment Xa5 s Thr. In anothe embod ment Xa5 s lie. In anothei embod ment Xa6 s Lys. In another embodiment Xa6 is Thr. In another embodiment Xa6 is Pro. In another embodiment Xa6 is Glu. In another embodiment Xa7 is Thr. In another embodiment Xa7 is Lys. In another embodiment Xa7 is Pro. In another embodiment Xa7 is Glu. In another embodiment Xa7 is -OH.
• the invention provides a pharmaceutical preparation comprising i. A derivative of human insulin ii. an analogue of human insulin having a fast onset of action, wherein Phe(B1) is deleted, or Tyr(B26) is replaced with Phe, or Phe(B1) is deleted and Tyr(B26) is replaced with Phe.
• the invention provides a pharmaceutical preparation wherein the ratio of the derivative of human insulin i. to the analogue of human insulin having a fast onset of action ii. is between 1 :99 and 99:1.
• the ratio is between 10:90 and 90:10. In another embodiment the ratio is between 30:70 and 70:30.
• the invention provides a pharmaceutical preparation wherein the deriva- tive of human insulin i. is selected from the group consisting of
• the derivative of human insulin i. is B29-N ⁇ -(N-lithocholyl- ⁇ -glutamyl)- des(B30) human insulin.
• the derivative of human insulin i. is B29-N ⁇ -myristoyl-des(B30) human insulin.
• the invention provides a pharmaceutical preparation wherein the analogue of human insulin ii. is Des(B1) human insulin or Des(B1) Des(B30) human insulin. In another embodiment the analogue of human insulin ii. is Des(B1) human insulin.
• the invention provides a pharmaceutical preparation wherein the analogue of human insulin ii. is an analogue selected from Group A analogues.
• the invention provides a pharmaceutical preparation which further comprises a phenolic preservative.
• the invention furthermore provides a method of treating type 1 or type 2 diabetes comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical preparation of the invention.
• the invention provides the use of a preparation of the invention for the preparation of a medicament for treatment of type 1 or type 2 diabetes.
• the invention provides an analogue of human insulin wherein the amino acid in position B26 is Phe and wherein the amino acid sequence is at least 80 % identical to that of human insulin.
• the invention provides an analogue of human insulin wherein the amino acid in position B26 is Phe and which has a time of disappearance of less than 3 hours measured in the disappearance assay described herein.
• Xa1 is Phe, Glu or -H
• Xa2 is Asn or Lys
• Xa3 is Ala, Asp, Glu, Phe, Gly, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, or
• Xa4 is Thr or Pro
• Xa5 is Pro, Thr, Lys, Asp or lie
• Xa6 is Lys ,Thr, Pro or Glu
• Xa7 is Thr, Lys, Pro, Glu or -OH
• Xa2 s Lys In one embodiment Xa2 s Lys. In one embodiment Xa3 is Ala, Asp, Glu, Phe, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val,
• Trp Trp, or Tyr.
• Xa3 is Ala, Thr, Ser, Asn or Gin.
• Xa3 is Ala, Thr, or Ser. In one embodiment Xa3 is Ala.
• Xa4 is Thr.
• Xa4 is Lys.
• Xa5 is Pro, Lys or Asp.
• Xa5 is Pro. In one embodiment Xa5 is Lys.
• Xa5 is Asp.
• Xa5 is Thr.
• Xa5 is lie.
• Xa6 is Lys. In one embodiment Xa6 is Thr.
• Xa6 is Pro
• Xa6 is Glu
• Xa7 is Thr.
• Xa7 is Lys. In one embodiment Xa7 is Pro.
• Xa7 is Glu
• Xa7 is -OH.
• the invention provides a pharmaceutical preparation comprising i. A derivative of human insulin ii. an analogue of human insulin having a fast onset of action, wherein Phe(B1) is deleted, or Tyr(B26) is replaced with Phe, or Phe(B1) is deleted and Tyr(B26) is replaced with Phe.
• the ratio of the derivative of human insulin i. to the analogue of human insulin having a fast onset of action ii. is between 1 :99 and 99:1.
• the ratio is between 10:90 and 90:10.
• the ratio is between 30:70 and 70:30.
• the derivative of human insulin i. is selected from the group consisting of
• the derivative of human insulin i. is B29-N ⁇ ⁇ (N-lithocholyl- ⁇ -glutamyl)- des(B30) human insulin.
• the derivative of human insulin i. is B29-N ⁇ -myristoyl-des(B30) human insulin.
• the analogue of human insulin ii. is Des(B1) human insulin or Des(B1) Des(B30) human insulin.
• the analogue of human insulin ii. is Des(B1) human insulin. In one embodiment the analogue of human insulin ii. is an analogue according to any one of the embodiments above. In one embodiment the invention provides a pharmaceutical preparation according to any one of the embodiments above which further comprises a phenolic preservative. In one embodiment the invention provides a method of treating type 1 or type 2 diabetes comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical preparation according to any one of the embodiments above. In one embodiment the invention provides the use of a preparation according to any one of the embodiments above for the preparation of a medicament for treatment of type 1 or type 2 diabetes.
• Insulin preparations of the invention are usually administered from multi-dose containers where a preservative effect is desired.
• the preservative may be a phenolic molecules.
• the phenolic molecules in the insulin preparation may be selected from the group consisting of phenol, m-cresol, chloro-cresol, thymol, m-chlor-phenol, resorcinole, 7- hydroxyindole or any mixture thereof.
• 0.5 to 5.0 mg/ml of phenolic compound may be employed.
• inventi on 0.6 to 5.0 mg/ml of m-cresol may be employed, In another embodiment of the inventi on 0.5 to 5.0 mg/ml of phenol may be employed, In another embodiment of the inventi on 1.4 to 5.0 mg/ml of phenol may be employed. In another embodiment of the invention 0.5 to 5.0 mg/ml of a mixture of m-cresol or phenol may be employed.
• the pharmaceutical preparation may further comprise a buffer substance, such as a TRIS, phosphate, glycine or glycylglycine (or another zwitterionic substance) buffer, an isotonicity agent, such as NaCI, glycerol, mannitol and/or lactose
• a buffer substance such as a TRIS, phosphate, glycine or glycylglycine (or another zwitterionic substance) buffer
• an isotonicity agent such as NaCI, glycerol, mannitol and/or lactose
• the pharmaceutical preparation may further comprise physiologically acceptable agents that increase the viscosity of the pharmaceutical preparation.
• the pharmaceutical preparation according to the invention may furthermore comprise an agent which increases the viscosity, such as polyethylene glycol, polypropylene glycol, copolymers thereof, dextrans and/or polylactides.
• the insulin preparation of the invention comprises between 0.001 % by weight and 1 % by weight of a non-ionic surfactant, for example tween 20 or Poloxamer 188.
• a non-ionic surfactant for example tween 20 or Poloxamer 188.
• the insulin preparation of the present invention may have a pH value in the range of 3.5 to 8.5, more preferably 7.1 to 7.9.
• the invention furthermore relates to treatment of a patient in which the Group A insulin analogue of the invention and/or a pharmaceutical preparation of the invention, i.e. a preparation comprising both a Group A insulin analogue and a derivative of human insulin such as B29- N ⁇ -(N-lithocholyl- ⁇ -glutamyl)-des(B30) human insulin, is combined with another form of treat- ment.
• a pharmaceutical preparation of the invention i.e. a preparation comprising both a Group A insulin analogue and a derivative of human insulin such as B29- N ⁇ -(N-lithocholyl- ⁇ -glutamyl)-des(B30) human insulin
• treatment of a patient with the pharmaceutical preparation of the invention is combined with diet and/or exercise.
• the pharmaceutical preparation of the invention is administered in combination with one or more further active substances in any suitable ratios.
• Such further active substances may e.g. be selected from antiobesity agents, antidiabetics, anti- hypertensive agents, agents for the treatment of complications resulting from or associated with diabetes and agents for the treatment of complications and disorders resulting from or associated with obesity.
• the pharmaceutical preparation of the invention may be administered in combination with one or more antiobesity agents or appetite regulating agents.
• Such agents may be selected from the group consisting of CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, MC4 (melanocortin 4) agonists, MC3 (melanocortin 3) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, urocortin agonists, ⁇ 3 adrenergic agonists such as CL- 316243, AJ-9677, GW-0604, LY362884, LY377267 or AZ-40140, MSH (melanocyte- stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin) agonists, serotonin re-uptake inhibitors such as fluoxetine, seroxat or cita
• the antiobesity agent is leptin. In another embodiment the antiobesity agent is dexamphetamine or amphetamine. In another embodiment the antiobesity agent is fenfluramine or dexfenfluramine. In still another embodiment the antiobesity agent is sibutramine. In a further embodiment the antiobesity agent is orlistat.
• the antiobesity agent is rna ⁇ indol or phentermine. In still another embodiment the antiobesity agent is phendimetrazine, diethylpropion, fluoxetine, bupropion, topiramate or ecopipam.
• the orally active hypoglycemic agents comprise imidazolines, sulphonylureas, biguanides, meglitinides, oxadiazolidinediones, thiazolidinediones, insulin sensitizers, insulin secre- tagogues such as glimepride, ⁇ -glucosidase inhibitors, agents acting on the ATP-dependent potassium channel of the ⁇ -cells eg potassium channel openers such as those disclosed in WO 97/26265, WO 99/03861 and WO 00/37474 (Novo Nordisk A/S) which are incorporated herein by reference, or mitiglinide, or a potassium channel blocker, such as BTS-67582, nateglinide, glucagon antagonists such as those disclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporated herein by reference, GLP-1 agonists such as those disclosed in
• the pharmaceutical preparation of the invention is administered in combination with a sulphonylurea e.g. tolbutamide, chlorpropamide, to- lazamide, glibenclamide, glipizide, glimepiride, glicazide or glyburide.
• a sulphonylurea e.g. tolbutamide, chlorpropamide, to- lazamide, glibenclamide, glipizide, glimepiride, glicazide or glyburide.
• a biguanide e.g. metformin.
• the pharmaceutical preparation of the invention is administered in combination with a meglitinide eg repaglinide or nateglinide.
• the pharmaceutical preparation of the invention is administered in combination with a thiazolidinedione insulin sensitizer, e.g. troglitazone, cig ⁇ - tazone, pioglitazone, rosiglitazone, isaglitazone, darglitazone, englitazone, CS-011/CI-1037 or T 174 or the compounds disclosed in WO 97/41097, WO 97/41119, WO 97/41120, WO 00/41121 and WO 98/45292 (Dr.
• the pharmaceutical preparation of the invention may be administered in combination with an insulin sensitizer, e.g. such as Gl 262570, YM- 440, MCC-555, JTT-501 , AR-H039242, KRP-297, GW-409544, CRE-16336, AR-H049020, LY510929, BX-102, CLX-0940, GW-501516 or the compounds disclosed in WO 99/19313, WO 00/50414, WO 00/63191 , WO 00/63192, WO 00/63193 (Dr.
• an insulin sensitizer e.g. such as Gl 262570, YM- 440, MCC-555, JTT-501 , AR-H039242, KRP-297, GW-409544, CRE-16336, AR-H049020, LY510929, BX-102, CLX-0940, GW-501516 or the compounds disclosed in WO 99/19313, WO 00/504
• the pharmaceutical preparation of the invention is administered in combination with an ⁇ -glucosidase inhibitor, e.g. voglibose, emiglitate, migli- tol or acarbose.
• an ⁇ -glucosidase inhibitor e.g. voglibose, emiglitate, migli- tol or acarbose.
• the pharmaceutical preparation of the invention is administered in combination with an agent acting on the ATP-dependent potassium channel of the ⁇ -cells, e.g. tolbutamide, glibenclamide, glipizide, glicazide, BTS-67582 or repaglinide.
• an agent acting on the ATP-dependent potassium channel of the ⁇ -cells e.g. tolbutamide, glibenclamide, glipizide, glicazide, BTS-67582 or repaglinide.
• the pharmaceutical preparation of the invention may be administered in combination with nateglinide.
• the pharmaceutical preparation of the invention is administered in combination with an antilipidemic agent, e.g. cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyroxine.
• an antilipidemic agent e.g. cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyroxine.
• the pharmaceutical preparation of the invention is admin- istered in combination with more than one of the above-mentioned compounds, e.g.
• the pharmaceutical preparation of the invention may be administered in combi- nation with one or more antihypertensive agents.
• antihypertensive agents examples include ⁇ - blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (an- giotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, and ⁇ -blockers such as doxa- zosin, urapidil, prazosin and terazosin.
• the pharmaceutical preparation of the invention may also be combined with NEP inhibitors such as candoxatril.
• a Group A analogue of this invention is combined with with another form of treat- ment, this administration can be simulataneous or sequential, in a manner effective to result in their combined actions within the subject treated.
• a Group A analogue may be administered in combination with long-acting derivatives of human insulin as described above, either as a pre-mixed preparation, or by substantially simultaneous adminis- traton of two separate preparations, or by sequential administration, i.e. administrations may be separated in time.
• the agents would be provided in amounts effective and for periods of time effective to result in their combined presence and their combined actions.
• the administration of a Group A analogue of the invention may precede, or follow, the other form of treatment by, e.g., intervals ranging from minutes to weeks and months.
• compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, ophthalmiccaly, vaginally or via an implanted reservoir.
• the pharmaceutical compositions according to the invention may be used for parenteral administration, such as subcutaneous, intramuscular, intrathecal, intravenous, intradermal, intraspinal or intrasternal administration. It will be appreciated that the preferred route of administration will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient chosen.
• Suitable administration forms include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. Depot injectable preparations are also contemplated as being within the scope of the present invention.
• Suitable administration forms include ophthalmic preparations such as eye drops and eye ointments and topical preparations such as wound dressings.
• the preparations of the invention are used in connection with insulin pumps.
• the insulin pumps may be prefilled and disposable, or the insulin preparations may be supplied from a reservoir which is removable.
• Insulin pumps may be skin-mounted or car- ried, and the path of the insulin preparation from the storage compartment of the pump to the patient may be more or less tortuous.
• Non-lirniting examples of insulin pumps are disclosed in US 5,957,895, US 5,858,001 , US 4,468,221 , US 4,468,221 , US 5,957,895, US 5,858,001 , US 6,074,369, US 5,858,001 , US 5,527,288, and US 6,074,369.
• preparations of the invention are used in connection with pen-like injection devices, which may be prefilled and disposable, or the insulin preparations may be supplied from a reservoir which is removable.
• pen-like injection devices are FlexPen ® , InnoLet ® , InDuoTM, Innovo ® .
• preparations of the invention are used in connection with devices for pulmonary administration of aqueous insulin preparations, a non-limiting example of which is the AerX ® device.
• Rapid-acting analogues of human insulin of the invention were prepared by site-directed mutagenesis as described in Brange et al., Nature vol 333 page 679 - 682 (1988).
• Derivatives of human insulin having a protracted action were made by acylation of biosyn- thetic des(B30) human insulin in position Lys B29 N e by tetradecanoic acid and a series of cholic acid derivatives using conventional peptide chemistry as described in WO 99/21888.
• Insulin and insulin analogues for pharmacokinetic experiments were prepared as preparations containing 600 nmol of insulin per ml and 2 - 2.5 Zn 2+ per hexamer, 1,5 % glycerol and 0.3% phenol.
• Insulin preparations for pharmacokinetic experiments were labelled by 65 Zn 2+ or by iodinationation of Tyr ⁇ 14 of the appropriate analogues by 125 l 2 (see Jorgensen.KH, Lar- sen.UD: Homogeneous mono-125l-insulins. Preparation and characterization of Mono-1251- (Tyr A14)- and Mono-125l-(Tyr A19)-insulin. DIABETOLOGIA 19:546-554, 1980) and NHP insulin was labelled by Tyr A14 ( 125 l-human insulin).
• Formulated preparations of insulin analogues labelled in position Tyr A14 by 125 l or by 65 Zn 2+ were injected subcutaneously in pigs as previously described (Ribel, U., J ⁇ rgensen, K, Brange, J, and Henriksen, U. : The pig as a model for subcutaneous insulin absorption in man. Serrano-Rios, M and Lefebvre, P. J. 891-896. 1985. Amsterdam; New York;Qxford, Elsevier Science Publishers. 1985). The disappearance of the radioactive label from the site of subcutaneous injection was monitored using a modification of the traditional external gamma-counting method (Ribel, U.: Subcutaneous absorption of insulin analogues.
• Analogue 1 B10Asp B26Phe DesB30 human insulin.
• Analogue 2 B10Asp B26Phe B28Asp DesB30 human insulin.

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