PROTEASE STABILIZED, ACYLATED INSULIN ANALOGUES
FIELD OF THIS INVENTION
The present invention relates to novel acylated insulin analogues exhibiting resistance towards proteases, a method for the preparation of such insulin analogues, insulin preparations containing the insulin analogues of the invention and a method of treating diabetes mellitus using these insulin analogues.
BACKGROUND OF THIS INVENTION
Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost. About 5% of all people suffer from diabetes and the disorder approaches epidemic proportions. Since the introduction of insulin in the 1920's, continuous efforts have been made to improve the treatment of diabetes mellitus. Since people suffering from diabetes are subject to chronic treatment over several decades, there is a major need for safe, convenient and life quality improving insulin formulations.
The oral route is by far the most widely used route for drug administration and is in general very well accepted by patients, especially for chronic therapies. Administration of therapeutic peptides or proteins is however often limited to parenteral routes rather than the preferred oral administration due to several barriers such as enzymatic degradation in the gastrointestinal (Gl) tract and intestinal mucosa, drug efflux pumps, insufficient and variable absorption from the intestinal mucosa, as well as first pass metabolism in the liver.
Normally, insulin formulations are administered by subcutaneous injection. However, administration by other routes, e.g., orally or pulmonary, would be advantageous due to patient compliance, safety and convenience. Some of the commercial available insulin formulations are characterized by a fast onset of action and other formulations have a relatively slow onset but show a more or less prolonged action. It is vary important for diabetic patients that there is, on the market, a big variety of insulins with different durations of actions (profiles of actions). Briefly, insulins can be classified as being short-, intermediate- or long-acting.
WO 2008/034881 relates to certain insulin analogues wherein at least two hydrophobic amino acids have been substituted with hydrophilic amino acids which insulin analogues are not acylated.
EP 2008/060733 and EP 2008/060733 relate to certain acylated insulin analogues wherein the insulin analogue comprises an elongation with an amino acid or a peptide residue connected C ter-mianly to the A21 amino acid.
EP 2008/060734 relates to certain acylated insulins wherein an acyl moiety is attached to the parent insulin and wherein said acyl moiety comprises repeating units of alkylene glycol containing amino acids.
ASPECTS OF THIS INVENTION
An aspect of this invention relates to the furnishing of insulin analogues which, when administered orally, can give a satisfactory control of the blood glucose level.
Another aspect of this invention relates to furnishing of insulin analogues which, when administered orally, can give a prolonged lowering of the glucose level
Another aspect of this invention relates to furnishing of basal insulin analogues which, when administered orally, can give a prolonged lowering of the glucose level
Another aspect of this invention relates to furnishing of basal insulin analogues which, when administered orally, can give a satisfactory control of the blood glucose level following thrice daily administration.
Another aspect of this invention relates to furnishing of basal insulin analogues which, when administered orally, can give a satisfactory control of the blood glucose level following twice daily administration.
Another aspect of this invention relates to furnishing of basal insulin analogues which, when administered orally, can give a satisfactory control of the blood glucose level following once daily administration.
Another aspect of this invention relates to furnishing of basal insulin analogues which are hydrophilic.
Another aspect of this invention relates to furnishing of basal insulin analogues which are more hydrophilic than human insulin.
Another aspect of this invention relates to furnishing of basal insulin analogues which are less hydrophobic than human insulin, as measured by the relative hydrophobicity (k'rel) as described herein.
Another aspect of this invention relates to furnishing of basal insulin analogues which are less hydrophobic than of similar non-protease stabilised parent insulins acylated with the same acyl moiety, as measured by the relative hydrophobicity (k'rel) as described herein. K'rel of the basal insulin analogues of the invention are preferably less than 5, more preferably less than 3, more preferably less than 2, more preferably less than 1, more preferably less than 0.8, more preferably less than 0.6, more preferably less than 0.5, more preferably less than 0.4, more preferably less than 0.3, more preferably less than 0.2, more preferably less than 0.1.
Another aspect of this invention relates to furnishing of basal insulin analogues which, when administered orally, have satisfactory bioavailabilities. Compared with the bioavailabilities of similar acylated insulins without the protease stabilising mutations given in similar doses, the bioavailability of preferred compounds of this invention is at least 10% higher, preferably 20% higher, preferably 25% higher, preferably 30% higher, preferably 35% higher, preferably 40% higher, preferably 45% higher, preferably 50% higher, preferably 55% higher, preferably 60% higher, preferably 65% higher, preferably 70% higher, preferably 80% higher, preferably 90% higher, preferably 100% higher, preferably more than 100% higher than that of the non-protease stabilised comparator.
Another aspect of this invention relates to furnishing of basal insulin analogues which, when
administered orally, have satisfactory bioavailabilities. Bioavailabilities of preferred compounds of this invention (relative to i.v. administration) are at least 0.3%, preferebly >0.5%, preferebly >1%, preferably >1.5%, preferebly >2%, preferebly >2.5%, preferebly >3%, preferebly >3.5%, preferebly >4%, preferebly >5%, preferebly >6%, preferebly >7%, preferebly >8%, preferebly >9%, preferebly >10%.
Another aspect of this invention relates to furnishing of basal insulin analogues which, when administered by intravenous infusion, have satisfactory potencies. Compared with the potency of human insulin, potencies of preferred protease stabilised insulin analogues of the invention are preferably >5%, preferably >10%, preferably >20%, preferably >30%, preferably >40%, preferably >50%, preferably >75% and preferably > 100% .
Another aspect of this invention relates to the furnishing of insulin analogues which, when administered pulmonarily, can give a satisfactory control of the blood glucose level.
Another aspect of this invention relates to the furnishing of insulin analogues which, when administered pulmonarily, can give a satisfactory control of the blood glucose level with a relatively slow onset of action and/or a more or less prolonged action.
Another aspect of this invention relates to the furnishing of insulin analogues having a satisfactory prolonged action following pulmonary administration. Compared with similar acylated insulin without protease stabilising mutations given in similar doses, the duration of action of preferred compounds of this invention is at least 10% longer, preferably 20% longer, preferably 25% longer, preferably 30% longer, preferably 35% longer, preferably 40% longer, preferably 45% longer, preferably 50% longer, preferably 55% longer, preferably 60% longer, preferably 65% longer, preferably 70% longer, preferably 80% longer, preferably 90% longer, preferably 100% longer, preferably more than 100% longer than that of the comparator. Duration of action can be measured by the time that blood glucose is suppressed, or by measuring relevant pharmacokinetic properties, for example t½ or MRT (mean residence time).
Another aspect of this invention relates to the furnishing of insulin analogues having a satisfactory pulmonary bioavailability. Compared with the bioavailability of human insulin or compared with similar acylated insulin without protease stabilising mutations given in similar doses, the bioavailability of preferred compounds of this invention is at least 10% higher, preferably 20% higher, preferably 25% higher, preferably 30% higher, preferably 35% higher, preferably 40% higher, preferably 45% higher, preferably 50% higher, preferably 55% higher, preferably 60% higher, preferably 65% higher, preferably 70% higher, preferably 80% higher, preferably 90% higher, preferably 100% higher, preferably more than 100% higher than that of the comparator.
Another aspect of this invention relates to the furnishing of insulin analogues having increased apparent in vivo potency.
Another aspect of this invention relates to the furnishing of prolonged acting insulins with oral bioavailability.
Another aspect of this invention relates to the furnishing of insulin analogues having an increased proteolytical stability compared to the stability of human insulin. Compared with human insulin, the proteolytical stability of preferred compounds of this invention is at least 2 fold more stable,
preferably 3 fold more stable, preferably 4 fold more stable, preferably 5 fold more stable, preferably 6 fold more stable, preferably 7 fold more stable, preferably 8 fold more stable, preferably 9 fold more stable, preferably 10 fold more stable, preferably 12 fold more stable, preferably 14 fold more stable, preferably 16 fold more stable, preferably 18 fold more stable, preferably 20 fold more stable, preferably 25 fold more stable, preferably more than 25 fold more stable than that of the comparator. Prote-olytical stability can be measured by exposing the insulins to (a mixture of) proteolytic enzymes, e.g. an extract of gut enzymes as described herein.
The object of this invention is to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
DEFINITIONS
Herein, the term insulin covers natural occurring insulins, e.g., human insulin, as well as insulin analogues thereof. Human insulin consists of two polypeptide chains, the so-called A and B chains which contain 21 and 30 amino acid residues, respectively, and which are interconnected by two cystine disulphide bridges.
Herein, the term amino acid residue covers an amino acid from which a hydrogen atom has been removed from an amino group and/or a hydroxy group has been removed from a carboxy group and/or a hydrogen atom has been removed from a mercapto group. Imprecise, an amino acid residue may be designated an amino acid.
Herein, hydrophobic amino acids are to be understood as the naturally occurring amino acids tryptophan (Trp, W), phenylalanine (Phe, F), valine (Val, V), isoleucine (lie, I), leucine (Leu, L) and tyrosine (Tyr, Y) (with the three-letter and the one-letter abbreviation in brackets).
Herein, hydrophilic amino acids are to be understood as natural amino acids that are not hydrophobic amino acids according to the definition above. In one embodiment hydrophilic acids according to the invention are selected from the group consisting of: Glutamic acid (Glu, E), aspartic acid (Asp, D), histidine (His, H), glutamine (Gin, Q), asparagine (Asn, N), serine (Ser, S), threonine (Thr, T), proline (Pro, P), glycine (Gly, G), lysine (Lys, K) and arginine (Arg, R). In a further embodiment hydrophilic amino acids according to the invention are selected from the group consisting of: Glutamic acid (Glu, E), aspartic acid (Asp, D), histidine (His, H), glutamine (Gin, Q), asparagine (Asn, N), lysine (Lys, K) and arginine (Arg, R).
Herein, the term insulin analogue covers a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, e.g., human insulin, by deleting and/or substituting (replacing) one or more amino acid residue occurring in the natural insulin and/or by adding one or more amino acid residue. The added and/or substituted amino acid residues can either be codable amino acid residues or other naturally occurring amino acid residues or purely synthetic amino acid residues. In a preferred embodiment, the insulin analogue has two or more mutations compared to human insulin.
Herein, the term protease stabilised insulin means the insulin without an appended acyl moiety. Said protease stabilised insulins have an improved stability against degradation from proteases.
Herein, the term parent insulin means the insulin without an appended acyl moiety and without mutations to improve stability against degradation from proteases. Said parent insulins have optionally mutations relative to human insulin. Parent insulins are thus also insulin analogues as defined above. Herein, the terms parent insulin and non-protease stabilised insulin covers the same compounds.
Herein, the term mutation covers any change in amino acid sequence (substitutions and insertions with codable amino acids as well as deletions).
Herein, the term analogues of the A chain and analogues of the B chains of human insulin covers A and B chains of human insulin, respectively, having one or more substitutions, deletions and or extensions (additions) of the A and B amino acid chains, respectively, relative to the A and B chains, respectively, of human insulin.
Herein, terms like A1, A2, A3 etc. indicate 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, A21G and A21Q 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.
Herein, the terms A(0) or B(0) indicate the positions N-terminally neighbouring the A1 or B1 positions, respectively, in the A or B chains, respectively. The terms A(-1) or B(-1) indicate the positions of the first amino acids N-terminally to A(0) or B(0), respectively. Thus A(-2) and B(-2) indicate positions N-terminally to A(-1) and B(-1), respectively, A(-3) and B(-3) indicate positions N-terminally to A(-2) and B(-2), respectively, and so forth.
Herein, terms like desB29 and desB30 indicate an insulin analogue lacking the B29 or B30 amino acid residue, respectively.
Herein, the term "fast acting insulin" covers an insulin having a faster onset of action than normal or regular human insulin.
Herein, the term "long acting insulin" or the term "basal insulin" covers an insulin having a longer duration of action than normal or regular human insulin. Preferably, the time-action is more than 5, or 8 hours, in particularly of at least 9 hours. Preferably, the basal insulin has a time-action of at least 10 hours. The basal insulin may thus have a time-action in the range from about 8 to 24 hours, preferably in the range from about 9 to about 15 hours.
The numbering of the positions in insulin analogues, insulins and A and B chains is done so that the parent compound is human insulin with the numbering used for it.
Herein, the term "acylated insulin" covers modification of insulin by attachment of one or more acyl moieties via a linker to the protease stabilised insulin.
By acylated insulin having insulin activity is meant an acylated insulin with either the ability to lower the blood glucose in mammalians as measured in a suitable animal model, which may, e.g., be
a rat, rabbit, or pig model, after suitable administration, e.g., by intravenous or subcutaneous administration, or an insulin receptor binding affinity.
Herein, the term alkyl covers a saturated, branched or straight hydrocarbon group.
Herein, the term alkoxy covers the radical "alkyl-O-". Representative examples are methoxy, ethoxy, propoxy (e.g., 1-propoxy and 2-propoxy), butoxy (e.g., 1-butoxy, 2-butoxy and 2-methyl-2-propoxy), pentoxy (1-pentoxy and 2-pentoxy), hexoxy (1-hexoxy and 3-hexoxy), and the like.
Herein, the term alkylene covers a saturated, branched or straight bivalent hydrocarbon group having from 1 to 12 carbon atoms. Representative examples include, but are not limited to, methylene; 1,2-ethylene; 1,3-propylene; 1,2-propylene; 1,3-butylene; 1,4-butylene; 1,4-pentylene; 1,5-pentylene; 1,5-hexylene; 1,6-hexylene; and the like.
Herein, the term "neutral linear amino acid" covers . Non limiting examples of neutral linear amino acids are.
Herein, the term "cyclic amino acid" covers . Non limiting examples of cyclic amino acids are .
Herein, the term "acidic amino acid" covers . Non limiting examples of acidic amino acids are .
Herein, the term "fatty acid" covers a linear or branched, aliphatic carboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non limiting examples of fatty acids are myristic acid, palmitic acid, and stearic acid.
Herein, the term "fatty diacid" covers a linear or branched, aliphatic dicarboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non limiting examples of fatty diacids are succinic acid, hexanedioic acid, octanedioic acid, decanedioic acid, dodecanedioic acid, tetradec-anedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, and eicosanedioic acid.
Herein, the naming of the insulins is done according to the following principles: The names are given as mutations and modifications (acylations) relative to human insulin. For the naming of the acyl moiety, the naming is done according to IUPAC nomenclature and in other cases as peptide nomenclature. For example, naming the acyl moiety:
(Formula Removed)
can for example be "octadecanedioyl-γGlu-OEG-OEG", or "17-carboxyheptadecanoyl-γGlu-OEG-OEG", wherein
OEG is short hand notation for the amino acid NH2(CH2)2O(CH2)2OCH2CO2H, γGlu is short hand notation for the amino acid gamma glutamic acid. Other short hand notations for amino acids are, for example: PEG3 is NH2((CH2)2O)4CH2CH2CO2H PEG7 is NH2((CH2)2O)8CH2CH2CO2H
For example, the insulin of example 9 (with the sequence/structure given below) is named "A14E, B25H, B29K(NεOctadecanedioyh'Glu-OEG-OEG), desB30 human insulin" to indicate that the amino acid in position A14, Y in human insulin, has been mutated to E, the amino acid in position B25, F in human insulin, has been mutated to H, the amino acid in position B29, K as in human insulin, has been modified by acylation on the epsilon nitrogen in the lysine residue of B29, denoted Nε, by the residue octadecanedioyl-γGlu-OEG-OEG, and the amino acid in position B30, T in human insulin, has been deleted. Asterisks in the formula below indicate that the residue in question is different (i.e. mutated) as compared to human insulin. Throughout this application both formulas and names of preferred insulins of the invention are given
(Formula Removed)
Herein, the term "chemical stability" and "high chemical stability", means that chemically, the insulins of the invention are sufficiently stable in the desired formulation. That is that chemical degradation products are only formed in amounts that do not compromise shelf life of the final drug product. Chemical degradation products includes deamidation products, iso-aspartate formation, dimer formation, racemisation products, products resulting from dehydration processes etcetera. Chemical stability may be measured by HPLC analyses of aged samples or formulations.
Herein, the term "high physical stability" covers a tendency to fibrillation being less than 50% of that of human insulin. Fibrillation may be described by the lag time before fibril formation is initiated at a given conditions.
A polypeptide with insulin receptor and IGF-1 receptor affinity is a polypeptide which is capable of interacting with an insulin receptor and a human IGF-1 receptor in a suitable binding assay. Such receptor assays are well-know within the field and are further described in the examples. The present acylated insulin will not bind to the IGF-1 receptor or will have a rather low affinity to said re-
ceptor. More precisely, the acylated insulins of this invention will have an affinity towards the IGF-1 receptor of substantially the same magnitude or less as that of human insulin
The term "pharmaceutically acceptable" as used herein means suited for normal pharmaceutical applications, i.e., giving rise to no serious adverse events in patients etc.
The terms treatment and treating as used herein 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 term treatment of a disease as used herein means the management and care of a patient having developed the disease, condition or disorder. The purpose of treatment is to combat the disease, condition or disorder. Treatment includes the administration of the active compounds to eliminate or control the disease, condition or disorder as well as to alleviate the symptoms or complications associated with the disease, condition or disorder.
The term prevention of a disease as used herein is defined as the management and care of an individual at risk of developing the disease prior to the clinical onset of the disease. The purpose of prevention is to combat the development of the disease, condition or disorder, and includes the administration of the active compounds to prevent or delay the onset of the symptoms or complications and to prevent or delay the development of related diseases, conditions or disorders.
The term effective amount as used herein means a dosage which is sufficient in order for the treatment of the patient to be effective compared with no treatment.
POT is the Schizosaccharomyces pombe those phosphate isomerase gene, and TPI1 is the S. cerevisiae triose phosphate isomerase gene.
By a leader is meant an amino acid sequence consisting of a pre-peptide (the signal peptide) and a pro-peptide.
The term signal peptide is understood to mean a pre-peptide which is present as an N-terminal sequence on the precursor form of a protein. The function of the signal peptide is to allow the heterologous protein to facilitate translocation into the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the yeast organism producing the protein. A number of signal peptides which may be used with the DNA construct of this invention including yeast aspartic protease 3 (YAP3) signal peptide or
any functional analog (Egel-Mitani et al. (1990) YEAST 6:127-137 and US 5,726,038) and the α-factor
signal of the MFα1 gene (Thorner (1981) in The Molecular Biology of the Yeast Saccharomyces cerevisiae, Strathern et al., eds., pp 143-180, Cold Spring Harbor Laboratory, NY and US 4,870,00.
Herein, the term "pro-peptide" covers a polypeptide sequence whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast
cell). The pro-peptide may be the yeast α-factor pro-peptide, vide US 4,546,082 and 4,870,008. Alternatively, the pro-peptide may be a synthetic pro-peptide, which is to say a pro-peptide not found in nature. Suitable synthetic pro-peptides are those disclosed in US 5,395,922; 5,795,746; 5,162,498 and WO 98/32867. The pro-peptide will preferably contain an endopeptidase processing site at the C-terminal end, such as a Lys-Arg sequence or any functional analogue thereof.
Unless indicated explicitly, the amino acids mentioned herein are L-amino acids. Further, the left and right ends of an amino acid sequence of a peptide are, respectively, the N- and C-termini, unless otherwise specified.
SUMMARY OF THE INVENTION
It has been discovered that insulins that are stabilised towards proteolytic degradation (by specific mutations) and acylated at the B29-lysine are efficacious and protracted and possess high potential as protracted insulins that can be administered pulmonary or orally. The acylation confers binding to serum albumin, and, consequently, protraction. In addition, the acylated insulins of the invention display substantial reduction of insulin receptor affinity, compared to similar acylated insulins that are not stabilised towards proteolytic degradation. This reduction in insulin receptor affinity of albumin-bound insulins of the invention contributes to the protraction of the acylated insulin in circulation, since insulin is internalised and degraded upon receptor activation. Hence, clearance of the insulins of the invention is reduced. The reduction of insulin receptor affinity does probably not cause a loss of potency, e.g., as measured in the hyperinsulinaemic euglycaemic clamp as described herein. The combination of high albumin binding affinity and low insulin receptor affinity is, thus, beneficial for obtaining long duration of action of the insulins (basal insulins). Furthermore, after oral administration, these acylated insulins have a higher degree of bioavailability than similar known acylated insulins, that are not stabilised towards proteolytic degradation. Hence, these acylated insulin analogues are valuable for oral administration. Similarly, after pulmonary administration, these acylated protease stabilised insulins displays higher apparent potency and/or bioavailability than similar known acylated insulins, that are not stabilised towards proteolytic degradation. Furthermore, these acylated protease stabilised insulins displays protracted time-action profiles when administered pulmonary to mammals. Hence, these acylated insulin analogues are valuable for pulmonary administration.
The above-mentioned insulins that are stabilised towards proteolytic degradation are herein designated protease stabilised insulins.
The protease stabilised insulin molecule has a limited number of the naturally occurring amino acid residues substituted with other amino acid residues relative to human insulin as explained in the detailed part of the specification.
In one embodiment, this invention relates to an acylated insulin, wherein the protease stabilised insulin analogue deviates from human insulin in one or more of the following deletions or substitutions: Q in position A18, A, G or Q in position A21, G or Q in position B1 or no amino acid residue in position B1,
Q, S or T in position B3 or no amino acid residue in position B3, Q in position B13, no amino acid residue in position B27, D, E or R in position B28 and no amino acid in position B30.
In still a further aspect, this invention relates to pharmaceutical preparations comprising the acylated insulin of this invention and suitable adjuvants and additives such as one or more agents suitable for stabilization, preservation or isotoni, e.g., zinc ions, phenol, cresol, a parabene, sodium chloride, glycerol or mannitol. The zinc content of the present formulations may be between 0 and about 6 zinc atoms per 6 molecules of insulin. The pH value of the pharmaceutical preparation may be between about 4 and about 8.5, between about 4 and about 5 or between about 6.5 and about 7.5.
In a further embodiment, this invention is related to the use of the acylated insulin as a pharmaceutical for the reducing of blood glucose levels in mammalians, in particularly for the treatment of diabetes.
In a further aspect, this invention is related to the use of the acylated insulin for the preparation of a pharmaceutical preparation for the reducing of blood glucose level in mammalians, in particularly for the treatment of diabetes.
In a further embodiment, this invention is related to a method of reducing the blood glucose level in mammalians by administrating a therapeutically active dose of an acylated insulin of this invention to a patient in need of such treatment.
In a further aspect of this invention, the acylated insulins are administered in combination with one or more further active substances in any suitable ratios. Such further active agents may be selected from human insulin, fast acting insulin analogues, antidiabetic agents, antihyperlipidemic agents, antiobesity agents, antihypertensive agents and agents for the treatment of complications resulting from or associated with diabetes.
In one embodiment, the two active components are administered as a mixed pharmaceutical preparation. In another embodiment, the two components are administered separately either simultaneously or sequentially.
In one embodiment, the acylated insulins of this invention may be administered together with fast acting human insulin or human insulin analogues. Such fast acting insulin analogue may be such wherein the amino acid residue in position B28 is Asp, Lys, Leu, Val, or Ala and the amino acid residue in position B29 is Lys or Pro, 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 Glu or Asp. The acylated insulin of this invention and the rapid acting human insulin or human insulin analogue can be mixed in a ratio from about 90% of the acylated insulin to about 10% of the rapid acting human insulin or human insulin analogue; preferably from about 70% of the acylated insulin to about 30% of the rapid acting human insulin or human insulin analogue, and even more preferred from about 50 % of the acylated insulin to about 50% of the rapid acting human insulin or human insulin analogue (% being weight percentage).
The acylated insulins 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, WO 00/09666, WO 2006/097537, PCT/EP2008/061755 and PCT/EP2008/061830, 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 acylated insulins 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 acylated insulin of this invention may be administered in combination with one or more antiobesity agents or appetite regulating agents.
In one embodiment this invention is related to a pulmonal pharmaceutical preparation comprising the acylated insulin of this invention and suitable adjuvants and additives such as one or more agents suitable for stabilization, preservation or isotoni, e.g., zinc ions, phenol, cresol, a parabene, sodium chloride, glycerol, propyleneglycol or mannitol.
It should be understood that any suitable combination of the acylated insulins with diet and/or exercise, one or more of the above-mentioned compounds and optionally one or more other active substances are considered to be within the scope of this invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The stability and solubility properties of insulin are important underlying aspects for current insulin therapy. This invention is addressed to these issues by providing stable, acylated insulin analogues wherein the acylation decreases molecular flexibility and concomitantly reduce the fibrillation propensity and limit or modify the pH precipitation zone.
The acylated insulins of this invention are in particularly intended for pulmonary or oral administration due to their relatively high bioavailability compared to, e.g., human insulin and acylated human insulin. Furthermore, the acylated insulins will have a protracted insulin activity.
As mentioned above, insulins that are stabilised towards proteolytic degradation are herein designated protease stabilised insulins. The acylated insulins of this invention are said protease stabilised insulins which have been acylated as described herein.
Said protease stabilised insulins are derived from insulin compounds which herein are designated parent insulins or non-protease stabilised insulins.
In one embodiment a parent insulin is selected from the group consisting of a) human insulin; b) an insulin analogue of human insulin wherein the amino acid residue in position B28 of is Pro, Asp, Lys, Leu, Val, 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; c) an insulin analogue which is des(B28-B30) human insulin, des(B27) human insulin or des(B30) human insulin; d) an insulin analogue of human insulin wherein
the amino acid residue in position B3 is Lys and the amino acid residue in position B29 is Glu or Asp; e) an insulin analogue of human insulin wherein the amino acid residue in position A21 is Gly and wherein the insulin analogue is further extended in the C-terminal with two arginine residues; f) an insulin derivative wherein the amino acid residue in position B30 is substituted with a threonine methyl ester; and g) an insulin derivative wherein to the Nε position of lysine in the position B29 of des(B30) human insulin a tetradecanoyl chain is attached. Each of these groups is a specific embodiment.
In another embodiment, a parent insulin is selected from the group consisting of human insulin; desB30 human insulin; AspB28 human insulin; AspB28,DesB30 human insulin; LysB3,GluB29 human insulin; LysB28,ProB29 human insulin; GlyA21, ArgB31, ArgB32 human insulin; and desB30, ArgB31, ArgB32 human insulin.
More specifically, the protease stabilised insulin is an insulin molecule having two or more mutations of the A and/or B chain relative to the parent insulin. Surprisingly, it has been found that by substituting two or more hydrophobic amino acids within or in close proximity to two or more protease sites on an insulin with hydrophilic amino acids, an insulin analogue (i.e., a protease stabilised insulin) is obtained which is proteolytically more stable compared to the parent insulin. In a broad aspect, a protease stabilised insulin is an insulin analogue wherein at least two hydrophobic amino acids have been substituted with hydrophilic amino acids relative to the parent insulin, wherein the substitutions are within or in close proximity to two or more protease cleavage sites of the parent insulin and wherein such insulin analogue optionally further comprises one or more additional mutations.
In another embodiment, a protease stabilised insulin is an insulin analogue wherein
• the amino acid in position A12 is Glu or Asp and/or the amino acid in position A13 is His, Asn, Glu or Asp and/or the amino acid in position A14 is Asn, Gln, Glu, Arg, Asp, Gly or His and/or the amino acid in position A15 is Glu or Asp; and
• the amino acid in position B24 is His and/or the amino acid in position B25 is His and/or the amino acid in position B26 is His, Gly, Asp or Thr and/or the amino acid in position B27 is His, Glu, Gly or Arg and/or the amino acid in position B28 is His, Gly or Asp; and
which optionally further comprises one or more additional mutations.
In another embodiment a protease stabilised insulin is an analogue comprising the B25H or B25N mutations in combination with mutations in B27, optionally in combination with other mutations.
In another embodiment a protease stabilised insulin is an analogue comprising the B25H or B25N mutations in combination with mutations in B27, optionally in combination with other mutations. The mutations in position B27 can, for example, be Glu or Asp.
These protease stabilised acyated insulin analogues comprising both the B25 and B27 mutations have advantageous properties.
In another embodiment, a protease stabilised insulin is an insulin analogue comprising an A-chain amino acid sequence of formula 1:

(Formula Removed)
Formula (1) (SEQ ID No:1) and a B-chain amino acid sequence of formula 2:
(Formula Removed)
Formula (2) (SEQ ID No:2)
wherein XaaA(-2) is absent or Gly; XaaA(-1) is absent or Pro; XaaA0 is absent or Pro;
XaaA8 is independently selected from Thr and His; XaaA12 is independently selected from Ser, Asp and Glu;
XaaA13 is independently selected from Leu, Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser and Glu; XaaA14 is independently selected from Tyr, Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser and Glu; XaaA15 is independently selected from Gln, Asp and Glu; XaaA18 is independently selected from Asn, Lys and Gln; XaaA21 is independently selected from Asn and Gln; XaaB(-2) is absent or Gly; XaaB(-1) is absent or Pro; XaaB0 is absent or Pro;
XaaB1 is absent or independently selected from Phe and Glu; XaaB2 is absent or Val;
XaaB3 is absent or independently selected from Asn and Gln; XaaB4 is independently selected from Gin and Glu; XaaB10 is independently selected from His, Asp, Pro and Glu; XaaB16 is independently selected from Tyr, Asp, Gln, His, Arg, and Glu; XaaB24 is independently selected from Phe and His; XaaB25 is independently selected from Asn, Phe and His; XaaB26 is absent or independently selected from Tyr, His, Thr, Gly and Asp; XaaB27 is absent or independently selected from Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser and Glu;
XaaB28 is absent or independently selected from Pro, His, Gly and Asp;
XaaB29 is absent or independently selected from Lys, Arg and Gln; and, preferably, XaaB29 is absent or independently selected from Lys and Gln; XaaB30 is absent or Thr;
XaaB31 is absent or Leu; XaaB32 is absent or Glu;
the C-terminal may optionally be derivatized as an amide;
wherein the A-chain amino acid sequence and the B-chain amino acid sequence are connected by disulphide bridges between the cysteines in position 7 of the A-chain and the cysteine in position 7 of the B-chain, and between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain and wherein the cysteines in position 6 and 11 of the A-chain are connected by a disulphide bridge.
In another embodiment, a protease stabilised insulin is an insulin analogue comprising an A-chain amino acid sequence of formula 3:
(Formula Removed)
Formula (3) (SEQ ID No:3) and a B-chain amino acid sequence of formula 4:
(Formula Removed)
Formula (4) (SEQ ID No:4)
wherein XaaA8 is independently selected from Thr and His; XaaA12 is independently selected from Ser, Asp and Glu;
XaaA13 is independently selected from Leu, Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser and Glu; XaaA14 is independently selected from Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser and Glu; XaaA15 is independently selected from Gln, Asp and Glu; XaaA18 is independently selected from Asn, Lys and Gln; XaaA21 is independently selected from Asn, and Gln; XaaB1 is independently selected from Phe and Glu; XaaB3 is independently selected from Asn and Gln; XaaB4 is independently selected from Gin and Glu; XaaB10 is independently selected from His, Asp, Pro and Glu; XaaB16 is independently selected from Tyr, Asp, Gln, His, Arg, and Glu; XaaB24 is independently selected from Phe and His; XaaB26 is absent or independently selected from Tyr, His, Thr, Gly and Asp; XaaB27 is absent or independently selected from Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser and Glu;
XaaB28 is absent or independently selected from Pro, His, Gly and Asp;
XaaB29 is absent or independently selected from Lys, Arg and Gln; and, preferably, XaaB29 is absent or
independently selected from Lys and Gln;
XaaB30 is absent or Thr;
the C-terminal may optionally be derivatized as an amide; wherein the A-chain amino acid sequence and the B-chain amino acid sequence are connected by disulphide bridges between the cysteines in position 7 of the A-chain and the cysteine in position 7 of the B-chain, and between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain and wherein the cysteines in position 6 and 11 of the A-chain are connected by a disulphide bridge.
In another embodiment, a protease stabilised insulin is an insulin analogue wherein
XaaA8 is independently selected from Thr and His; XaaA12 is independently selected from Ser and Glu;
XaaA13 is independently selected from Leu, Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, Pro, Ser and Glu; XaaA14 is independently selected from Asp, His, and Glu; XaaA15 is independently selected from Gin and Glu; XaaA18 is independently selected from Asn, Lys and Gln; XaaA21 is independently selected from Asn, and Gln; XaaB1 is independently selected from Phe and Glu; XaaB3 is independently selected from Asn and Gln; XaaB4 is independently selected from Gin and Glu; XaaB10 is independently selected from His, Asp, Pro and Glu; XaaB16 is independently selected from Tyr, Asp, Gln, His, Arg, and Glu; XaaB24 is independently selected from Phe and His; XaaB25 is independently selected from Phe, Asn and His; XaaB25 is independently selected from Tyr, Thr, Gly and Asp;
XaaB27 is independently selected from Thr, Asn, Asp, Gln, His, Lys, Gly, Arg, and Glu; XaaB28 is independently selected from Pro, Gly and Asp; XaaB29 is independently selected from Lys and Gln; XaaB30 is absent or Thr;
the C-terminal may optionally be derivatized as an amide; wherein the A-chain amino acid sequence and the B-chain amino acid sequence are connected by disulphide bridges between the cysteines in position 7 of the A-chain and the cysteine in position 7 of the B-chain, and between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain and wherein the cysteines in position 6 and 11 of the A-chain are connected by a disulphide bridge.
Other embodiments of protease stabilised insulins are mentioned below.
A "protease" or a "protease enzyme" is a digestive enzyme which degrades proteins and peptides and which is found in various tissues of the human body such as e.g. the stomach (pepsin), the intestinal lumen (chymotrypsin, trypsin, elastase, carboxypeptidases, etc.) or mucosal surfaces of the Gl tract (aminopeptidases, carboxypeptidases, enteropeptidases, dipeptidyl peptidases, endopep-tidases, etc.), the liver (Insulin degrading enzyme, cathepsin D etc), and in other tissues.
A proteolytically stable insulin analogue (also designated a protease stabilised insulin) is herein to be understood as an insulin analogue, which is subjected to slower degradation by one or more proteases relative to human insulin. In one embodiment, a protease stabilised insulin is subjected to slower degradation by one or more proteases relative to the parent insulin. In a further embodiment, a protease stabilised insulin is stabilized against degradation by one or more enzymes selected from the group consisting of: pepsin (such as, e.g., the isoforms pepsin A, pepsin B, pepsin C and/or pepsin F), chymotrypsin (such as, e.g., the isoforms chymotrypsin A, chymotrypsin B and/or chymotrypsin C), trypsin, Insulin-Degrading Enzyme (IDE), elastase (such as, e.g., the isoforms pancreatic elastase I and/or II), carboxypeptidase (e.g., the isoforms carboxypeptidase A, carboxypepti-dase A2 and/or carboxypeptidase B), aminopeptidase, cathepsin D and other enzymes present in intestinal extracts derived from rat, pig or human.
In one embodiment, a protease stabilised insulin is stabilized against degradation by one or more enzymes selected from the group consisting of: chymotrypsin, trypsin, Insulin-Degrading Enzyme (IDE), elastase, carboxypeptidases, aminopeptidases and cathepsin D. In a further embodiment, a protease stabilised insulin is stabilized against degradation by one or more enzymes selected from the group consisting of. chymotrypsin, carboxypeptidases and IDE. In a yet further embodiment, a protease stabilised insulin is stabilized against degradation by one or more enzymes selected from: chymotrypsin and carboxypeptidases.
T½ may be determined as described in the Examples as a measure of the proteolytical stability of a protease stabilised insulin towards protease enzymes such as chymotrypsin, pepsin and/or carboxypeptidase A. In one embodiment of the invention, T½ is increased relative to human insulin. In a further embodiment, T½ is increased relative to the parent insulin. In a yet further embodiment, T½ is increased at least 2-fold relative to the parent insulin. In a yet further embodiment, T½ is increased at least 3-fold relative to the parent insulin. In a yet further embodiment, T½ is increased at least 4-fold relative to the parent insulin. In a yet further embodiment, T½ is increased at least 5-fold relative to the parent insulin. In a yet further embodiment, T½ is increased at least 10-fold relative to the parent insulin.
An alternative way of measuring proteolytical stability is to measure the relative stability towards a comparator, e.g., human insulin. The relative stability is defines as T½/T½(comparator), where T½ and T½ (compatator) are the half-lives of the analogue and the comparator, respectively, in the degradation assay. In the examples section, the relative stability of selected insulins of the invention towards an enzyme mixture extracted from duodemum from rats is given (relative to human insulin as well as relative to a protease-resistant insulin without acylation).
Protease cleavage sites (herein also mentioned as protease sites) are to be understood as amino acid residues that are recognized by proteases and/or amino acid residues whose peptide bond is cleaved by proteases. Protease cleavage sites may be determined by determining cleavage "hot-spots" by HPLC, MS or LC-MS analyses and/or by prediction based on enzyme specificity of the protease enzyme for which the protease cleavage site is to be determined. A skilled person in the art will know how to determine protease cleavage sites for example based on enzyme specificities as for example described in Handbook of Proteolytical Enzymes, 2nd ed., Barrett, A.J., Rawlings, N.D., Woes-ner, J.F. editors, Elsevier Academic Press 2004. For example chymotrypsin is predicted to cleave peptide bonds C-terminal to aromatic residues (Trp, Tyr, Phe or Leu), that are not followed by Pro. Similarly, trypsin is predicted to cleave peptide bonds C-terminal to basic residues Lys or Arg, that are not followed by Pro, elastase is predicted to cleave residues C-terminal to Ala, Val, Gly or Ser and car-boxypeptidase A will remove any C-terminal amino acid, but not Arg, Lys or Pro. Insulin-degrading enzyme (IDE) is predicted to cleave the following positions of human insulin B9-10, B10-11, B13-14, B14-15, B24-25, B25-26, A13-14 and A14-15.
The term substituting (an) amino acid "within or in close proximity" to a protease cleavage site is herein used to indicate the substitution of an amino acid within or in close proximity to a position of the parent insulin which has been determined to be a protease cleavage site. In one embodiment, two or more hydrophobic amino acids within or in close proximity to two or more protease sites on an insulin are substituted, wherein said hydrophobic amino acids are substituted with hydrophilic amino acids. In a further embodiment, two or more hydrophobic amino acids within two or more protease sites on an insulin are substituted with hydrophilic amino acids. In a yet further embodiment, two or more hydrophobic amino acids situated next to two or more protease sites on an insulin are substituted with hydrophilic amino acids. In a still further embodiment, two or more hydrophobic amino acids situated two amino acids away from to two or more protease sites on an insulin are substituted with hydrophilic amino acids. In a yet further embodiment, two or more hydrophobic amino acids situated three amino acids away from two or more protease sites on an insulin are substituted with hydrophilic amino acids. In a still further embodiment, two or more hydrophobic amino acids situated up to four amino acids away from two or more protease sites on an insulin are substituted with hydrophilic amino acids. In a yet further embodiment two or more hydrophobic amino acids situated one, two or three amino acids away from or within two or more protease sites on an insulin are substituted with hydrophilic amino acids. In a still further embodiment, two or more hydrophobic amino acids situated one or two amino acids away from or within two or more protease sites on an insulin are substituted with hydrophilic amino acids. In a yet further embodiment, two or more hydrophobic amino acids situated next to or within two or more protease sites on an insulin are substituted with hydrophilic amino acids.
A protease stabilised insulin may have a net charge which is different than the net charge of the parent insulin. In one embodiment, the net charge of a protease stabilised insulin is more positive than the net charge of the parent insulin. In one embodiment, the net charge of a protease stabilised insulin is more negative than the net charge of the parent insulin. In one embodiment, the average positive net charge of a protease stabilised insulin is between 0.5 and 5 as measured in an aqueous
solution. In one embodiment, the average positive net charge of a protease stabilised insulin is between 1 and 5. In one embodiment, the average positive net charge of a protease stabilised insulin is between 1 and 4. In one embodiment, the average positive net charge of a protease stabilised insulin is between 1 and 3. In one embodiment, the average positive net charge of a protease stabilised insulin is between 2 and 3. In one embodiment, the average negative net charge of a protease stabilised insulin is between -0.5 and -5 as measured in an aqueous solution. In one embodiment, the average negative net charge of a protease stabilised insulin is between -1 and -5. In one embodiment, the average negative net charge of a protease stabilised insulin is between -1 and -4. In one embodiment, the average negative net charge of a protease stabilised insulin is between -1 and -3. In one embodiment, the average negative net charge of a protease stabilised insulin is between -2 and -3.
In one embodiment, a protease stabilised insulin may have increased solubility relative to human insulin. In a further embodiment, a protease stabilised insulin has increased solubility relative to human insulin at pH 3-9. In a yet further embodiment, a protease stabilised insulin has increased solubility relative to human insulin at pH 4-8.5. In a still further embodiment, a protease stabilised insulin has increased solubility relative to human insulin at pH 4-8. In a yet further embodiment, a protease stabilised insulin has increased solubility relative to human insulin at pH 4.5-8. In a further embodiment, a protease stabilised insulin has increased solubility relative to human insulin at pH 5-8. In a yet further embodiment, a protease stabilised insulin has increased solubility relative to human insulin at pH 5.5-8. In a further embodiment, a protease stabilised insulin has increased solubility relative to human insulin at pH 6-8.
In one embodiment, a protease stabilised insulin has increased solubility relative to human insulin at pH 2-4.
In one embodiment, a protease stabilised insulin may have increased solubility relative to the parent insulin. In a further embodiment, a protease stabilised insulin has increased solubility relative to the parent insulin at pH 3-9. In a yet further embodiment a protease stabilised insulin has increased solubility relative to parent insulin at pH 4-8.5. In a still further embodiment, a protease stabilised insulin has increased solubility relative to parent insulin at pH 4-8. In a yet further embodiment, a protease stabilised insulin has increased solubility relative to parent insulin at pH 4.5-8. In a still further embodiment, a protease stabilised insulin has increased solubility relative to parent insulin at pH 5-8. In a yet further embodiment, a protease stabilised insulin has increased solubility relative to parent insulin at pH 5.5-8. In a further embodiment, a protease stabilised insulin has increased solubility relative to parent insulin at pH 6-8.
In one embodiment, a protease stabilised insulin has increased solubility relative to parent insulin at pH 2-4.
By "increased solubility at a given pH" is meant that a larger concentration of a protease stabilised insulin dissolves in an aqueous or buffer solution at the pH of the solution relative to the parent insulin. Methods for determining whether the insulin contained in a solution is dissolved are known in the art.
In one embodiment, the solution may be subjected to centrifugation for 20 minutes at 30,000 g and then the insulin concentration in the supernatant may be determined by RP-HPLC. If this concentration is equal within experimental error to the insulin concentration originally used to make the composition, then the insulin is fully soluble in the composition of the invention. In another embodiment, the solubility of the insulin in a composition of the invention can simply be determined by examining by eye the container in which the composition is contained. The insulin is soluble if the solution is clear to the eye and no particulate matter is either suspended or precipitated on the sides/bottom of the container.
A protease stabilised insulin may have increased apparent potency and/or bioavalability relative to the parent insulin when compared upon measurement.
Standard assays for measuring insulin in vitro potency are known to the person skilled in the art and include inter alia (1) insulin radioreceptorassays, in which the relative potency of an insulin is defined as the ratio of insulin to insulin analogue required to displace 50% of 125l-insulin specifically bound to insulin receptors present on cell membranes, e.g., a rat liver plasma membrane fraction; (2) lipogenesis assays, performed, e.g., with rat adipocytes, in which relative insulin potency is defined as the ratio of insulin to insulin analogue required to achieve 50% of the maximum conversion of [3-3H] glucose into organic-extractable material (i.e. lipids); (3) glucose oxidation assays in isolated fat cells in which the relative potency of the insulin analogue is defined as the ratio of insulin to insulin analogue to achieve 50% of the maximum conversion of glucose-1-[14C] into [14C02]; (4) insulin radioimmunoassays which can determine the immunogenicity of insulin analogues by measuring the effectiveness by which insulin or an insulin analogue competes with 125l-insulin in binding to specific anti-insulin antibodies; and (5) other assays which measure the binding of insulin or an insulin analogue to antibodies in animal blood plasma samples, such as ELISA assays possessing specific insulin antibodies.
Increased apparent in vivo potency can be estimated/visualised by comparison of blood glucose vs. time profiles of the insulin in question with a similar insulin without protease stabilising mutations given in similar doses. The insulin of the invention will have increased blood glucose lowering effect relative to the comparator.
Standard assays for measuring insulin bioavailability are known to the person skilled in the art and include inter alia measurement of the relative areas under the curve (AUC) for the concentration of the insulin in question administered pulmonary or orally and intra venously (i.v.) in the same species. Quantitation of insulin concentrations in blood (plasma) samples can be done using for example antibody assays (ELISA) or by mass spectrometry. Pulmonary administration can be performed by several means. For example, insulins can be dosed to rats by drop instillation, or to pigs by dry powder insufflation.
Protease stabilised insulin may optionally be analyzed for further protease sites which may be subject to further substitutions of one or more hydrophobic amino acids with hydrophilic amino acids. A protease stabilised insulin may be an insulin analogue which has at least two hydrophilic acids in protease sites compared to the parent insulin, the first modified insulin, and which has further at
least one amino acid substitution in a new protease site of the first modified insulin wherein at least one hydrophobic amino acid has been substituted with at least one hydrophilic amino acid.
For the sake of convenience, here follows the names of codable, natural amino acids with the usual three letter codes & one letter codes in parenthesis: Glycine (Gly & G), proline (Pro & P), alanine (Ala & A), valine (Val & V), leucine (Leu & L), isoleucine (lle & I), methionine (Met & M), cysteine (Cys & C), phenylalanine (Phe & F), tyrosine (Tyr & Y), tryptophan (Trp & W), histidine (His & H), lysine (Lys & K), arginine (Arg & R), glutamine (Gin & Q), asparagine (Asn & N), glutamic acid (Glu & E), aspartic acid (Asp & D), serine (Ser & S) and threonine (Thr & T). If, due to typing errors, there are deviations from the commonly used codes, the commonly used codes apply. The amino acids present in the insulins of this invention are, preferably, amino acids which can be coded for by a nucleic acid. In one embodiment insulin or an insulin analogue is substituted by Gly, Glu, Asp, His, Gln, Asn, Ser, Thr, Lys, Arg and/or Pro and/or Gly, Glu, Asp, His, Gln, Asn, Ser, Thr, Lys, Arg and/or Pro is added to insulin or an insulin analogue. In one embodiment insulin or an insulin analogue is substituted by Glu, Asp, His, Gln, Asn, Lys and/or Arg and/or Glu, Asp, His, Gln, Asn, Lys and/or Arg is added to insulin or an insulin analogue.
In one embodiment, a protease stabilised insulin is selected from the group consisting of the following compounds: A14E, B25H, desB30 human insulin; A14H, B25H, desB30 human insulin; A14E, B1E, B25H, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; A14E, B25H, B28D, desB30 human insulin; A14E, B25H, B27E, desB30 human insulin; A14E, B1E, B25H, B27E, desB30 human insulin; A14E, B1E, B16E, B25H, B27E, desB30 human insulin; A8H, A14E, B25H, desB30 human insulin; A8H, A14E, B25H, B27E, desB30 human insulin; A8H, A14E, B1E, B25H, desB30 human insulin; A8H, A14E, B1E, B25H, B27E, desB30 human insulin; A8H, A14E, B1E, B16E, B25H, B27E, desB30 human insulin; A8H, A14E, B16E, B25H, desB30 human insulin; A14E, B25H, B26D, desB30 human insulin; A14E, B1E, B27E, desB30 human insulin; A14E, B27E, desB30 human insulin; A14E, B28D, desB30 human insulin; A14E, B28E, desB30 human insulin; A14E, B1E, B28E, desB30 human insulin; A14E, B1E, B27E, B28E, desB30 human insulin; A14E, B1E, B25H, B28E, desB30 human insulin; A14E, B1E, B25H, B27E, B28E, desB30 human insulin; A14D, B25H, desB30 human insulin; B25N, B27E, desB30 human insulin; A8H, B25N, B27E, desB30 human insulin; A14E, B27E, B28E, desB30 human insulin; A14E, B25H, B28E, desB30 human insulin; B25H, B27E, desB30 human insulin; B1E, B25H, B27E, desb30 human insulin; A8H, B1E, B25H, B27E, desB30 human insulin; A8H, B25H, B27E, desB30 human insulin; B25N, B27D, desB30 human insulin; A8H, B25N, B27D, desB30 human insulin; B25H, B27D, desB309 human insulin; A8H, B25H, B27D, desB30 human insulin; A(-1)P, A(0)P, A14E, B25H, desB30 human insulin; A14E, B(-1)P, B(0)P, B25H, desB30 human insulin; A(-1)P, A(0)P, A14E, B(-1)P, B(0)P, B25H, desB30 human insulin; A14E, B25H, B30T, B31L, B32E human insulin; A14E, B25H human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B10P, B25H, desB30 human insulin; A14E, B10E, B25H, desB30 human insulin; A14E, B4E, B25H, desB30 human insulin; A14H, B16H, B25H, desB30 human insulin; A14H, B10E, B25H, desB30 human insulin; A13H, A14E, B10E, B25H, desB30 human insulin; A13H, A14E, B25H, desB30 human insulin; A14E, A18Q, B3Q, B25H, desB30 human insulin; A14E, B24H,
B25H, desB30 human insulin; A14E, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29R, desB30 human insulin; A14E, A21G, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, A21G, B25H, B26G, B27G, B28G, B29R, desB30 human insulin; A14E, A18Q, A21Q, B3Q, B25H, desB30 human insulin; A14E, A18Q, A21Q, B3Q, B25H, B27E, desB30 human insulin; A14E, A18Q, B3Q, B25H, desB30 human insulin; A13H, A14E, B1E, B25H, desB30 human insulin; A13N, A14E, B25H, desB30 human insulin; A13N, A14E, B1E, B25H, desB30 human insulin; A(-2)G, A(-1)P, A(0)P, A14E, B25H, desB30 human insulin; A14E, B(-2)G, B(-1)P, B(0)P, B25H, desB30 human insulin; A(-2)G, A(-1)P, A(0)P, A14E, B(-2)G, B(-1)P, B(0)P, B25H, desB30 human insulin; A14E, B27R, B28D, B29K, desB30 human insulin; A14E, B25H, B27R, B28D, B29K, desB30 human insulin; A14E, B25H, B26T, B27R, B28D, B29K, desB30 human insulin; A14E, B25H, B27R, desB30 human insulin; A14E, B25H, B27H, desB30 human insulin; A14E, A18Q, B3Q, B25H, desB30 human insulin; A13E, A14E, B25H, desB30 human insulin; A12E, A14E, B25H, desB30 human insulin; A15E, A14E, B25H, desB30 human insulin; A13E, B25H, desB30 human insulin; A12E, B25H, desB30 human insulin; A15E, B25H, desB30 human insulin; A14E, B25H, desB27, desB30 human insulin; A14E, B25H, B26D, B27E, desB30 human insulin; A14E, B25H, B27R, desB30 human insulin; A14E, B25H, B27N, desB30 human insulin; A14E, B25H, B27D, desB30 human insulin; A14E, B25H, B27Q, desB30 human insulin; A14E, B25H, B27E, desB30 human insulin; A14E, B25H, B27G, desB30 human insulin; A14E, B25H, B27H, desB30 human insulin; A14E, B25H, B27K, desB30 human insulin; A14E, B25H, B27P, desB30 human insulin; A14E, B25H, B27S, desB30 human insulin; A14E, B25H, B27T, desB30 human insulin; A13R, A14E, B25H, desB30 human insulin; A13N, A14E, B25H, desB30 human insulin; A13D, A14E, B25H, desB30 human insulin; A13Q, A14E, B25H, desB30 human insulin; A13E, A14E, B25H, desB30 human insulin; A13G, A14E, B25H, desB30 human insulin; A13H, A14E, B25H, desB30 human insulin; A13K, A14E, B25H, desB30 human insulin; A13P, A14E, B25H, desB30 human insulin; A13S, A14E, B25H, desB30 human insulin; A13T, A14E, B25H, desB30 human insulin; A14E, B16R, B25H, desB30 human insulin; A14E, B16D, B25H, desB30 human insulin; A14E, B16Q, B25H, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14R, B25H, desB30 human insulin; A14N, B25H, desB30 human insulin; A14D, B25H, desB30 human insulin; A14Q, B25H, desB30 human insulin; A14E, B25H, desB30 human insulin; AUG, B25H, desB30 human insulin; A14H, B25H, desB30 human insulin; A8H, B10D, B25H human insulin; and A8H, A14E, B10E, B25H, desB30 human insulin and this embodiment may, optionally, comprise A14E, B25H, B29R, desB30 human insulin; B25H, desB30 human insulin; and B25N, desB30 human insulin.
In a preferred embodiment, a protease stabilised insulin is selected from the group consisting of the following compounds: A14E, B25H, desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; A14E, B25H, B29R, desB30 human insulin; A14E, B25H, B26G, B27G, B28G, desB30 human insulin; B25H, desB30 human insulin and A14E, B25H, desB27, desB30 human insulin.
In a preferred embodiment, a protease stabilised insulin is selected from any of the groups above that, in addition, are containing the desB27 mutation.
In a preferred embodiment, a protease stabilised insulin is selected from the group consisting of the following compounds: A14E, B25H, desB27, desB30 human insulin; A14E, B16H, B25H, desB27, desB30 human insulin; A14E, B16E, B25H, desB27, desB30 human insulin; A14E, B25H, desB27, B29R, desB30 human insulin and B25H, desB27, desB30 human insulin.
In one embodiment, a protease stabilised insulin is selected from any of the groups above that, in addition, are containing the following mutations in position A21 and/or B3 to improve chemical stability: A21G, desA21, B3Q, orB3G.
In a preferred embodiment, a protease stabilised insulin is selected from the following protease stabilised insulins: A14E, A21G, B25H, desB30 human insulin; A14E, A21G, B16H, B25H, desB30 human insulin; A14E, A21G, B16E, B25H, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, A21G, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, A21G, B25H, B26G, B27G, B28G, B29R, desB30 human insulin; A21G, B25H, desB30 human insulin and A21G, B25N, desB30 human insulin, and, preferably, it is selected from the following protease stabilised insulins: A14E, A21G, B25H, desB30 human insulin; A14E, A21G, B16H, B25H, desB30 human insulin; A14E, A21G, B16E, B25H, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A21G, B25H, desB30 human insulin and A21G, B25N, desB30 human insulin.
In a preferred embodiment, a protease stabilised insulin is acylated in the B29 position, at the epsilon nitrogen position of B29K.
In a preferred embodiment, a protease stabilised insulin is acylated in the A1 position, at the alpha nitrogen position of A1.
In a preferred embodiment, a protease stabilised insulin is acylated in the A1 position, at the alpha nitrogen position of A1, and the protease stabilized insulin is comprising the B29R mutation.
The protease stabilised 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. 6,500,645. The protease stabilised 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 protease stabilised 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 at. (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 sequence 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 protease stabilised 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 replication, 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 protease stabilised insulin will also typically be op-erably connected to a suitable terminator. In yeast a suitable terminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1:419-434).
The procedures used to ligate the polynucleotide sequence encoding the protease stabilised 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 protease stabilised insulin is introduced into a host ceil so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-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 eu-karyote. 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 Pseudomo-nas sp. Eukaryote 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 single chain 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 polymor-pha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, 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 perse. 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.
Preferably, the acylated insulins of this invention are mono-substituted having only one acyla-tion group attached to a lysine amino acid residue in the protease stabilised insulin molecule.
In one embodiment, the acyl moiety attached to the protease stabilised insulin has the general formula:
(Formula Removed)
wherein n is 0 or an integer in the range from 1 to 3; m is 0 or an integer in the range from 1 to 10; p is 0 or an integer in the range from 1 to 10; Acy is a fatty acid or a fatty diacid comprising from about 8 to about 24 carbon atoms; AA1 is a neutral linear or cyclic amino acid residue; AA2 is an acidic amino acid residue; AA3 is a neutral, alkyleneglycol-containing amino acid residue; the order by which AA1, AA2 and AA3 appears in the formula can be interchanged independently; AA2 can occur several times along the formula (e.g., Acy-AA2-AA32-AA2-); AA2 can occur independently (= being different) several times along the formula (e.g., Acy-AA2-AA32-AA2-); the connections between Acy, AA1, AA2 and/or AA3 are amide (peptide) bonds which, formally, can be obtained by removal of a hydrogen atom or a hydroxyl group (water) from each of Acy, AA1, AA2 and AA3; and attachment to the protease stabilised insulin can be from the C-terminal end of a AA1, AA2, or AA3 residue in the acyl moiety of the formula (I) or from one of the side chain(s) of an AA2 residue present in the moiety of formula (I).
In another embodiment, the acyl moiety attached to the protease stabilised insulin has the general formula Acy-AA1n-AA2m-AA3p- (I), wherein AA1 is selected from Gly, D- or L-Ala, ßAla, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, D- or L-Glu-α-amide, D- or L-Glu-γ-amide, D- or L-Asp-α-amide, D- or L-Asp-ß-amide, or a group of one of the formula:
(Formula Removed)
from which a hydrogen atom and/or a hydroxyl group has been removed and wherein q is 0, 1, 2, 3 or 4 and, in this embodiment, AA1 may, alternatively, be 7-aminoheptanoic acid or 8-aminooctanoic acid.
In another embodiment, the acyl moiety attached to the protease stabilised insulin has the general formula Acy-AA1n-AA2m-AA3p- (I), wherein AA1 is as defined above and AA2 is selected from L- or D-Glu, L- or D-Asp, L- or D-homoGlu or any of the following:
(Formula Removed)
from which a hydrogen atom and/or a hydroxyl group has been removed and wherein the arrows indicate the attachment point to the amino group of AA1, AA2, AA3, or to the amino group of the protease stabilised insulin.
In one aspect, the neutral cyclic amino acid residue designated AA1 is an amino acid containing a saturated 6-membered carbocyclic ring, optionally containing a nitrogen hetero atom, and preferably the ring is a cyclohexane ring or a piperidine ring. Preferably, the molecular weight of this neutral cyclic amino acid is in the range from about 100 to about 200 Da.
The acidic amino acid residue designated AA2 is an amino acid with a molecular weight of up to about 200 Da comprising two carboxylic acid groups and one primary or secondary amino group. Alternatively, acidic amino acid residue designated AA2 is an amino acid with a molecular weight of up to about 250 Da comprising one carboxylic acid group and one primary or secondary sulphonamide group.
The neutral, alkyleneglycol-containing amino acid residue designated AA3 is an alkyleneglycol moiety, optionally an oligo- or polyalkyleneglycol moiety containing a carboxylic acid functionality at one end and a amino group functionality at the other end.
Herein, the term alkyleneglycol moiety covers mono-alkyleneglycol moieties as well as oligo-alkyleneglycol moieties. Mono- and oligoalkyleneglycols comprises mono- and oligoethyleneglycol based, mono- and oligopropyleneglycol based and mono- and oligobutyleneglycol based chains, i.e., chains that are based on the repeating unit -CH2CH20-, -CH2CH2CH20- or -CH2CH2CH2CH20-. The alkyleneglycol moiety is monodisperse (with well defined length / molecular weight). Monoalkyleneglycol moieties comprise -OCH2CH20-, -OCH2CH2CH20- or -OCH2CH2CH2CH20- containing different groups at each end.
As mentioned herein, the order by which AA1, AA2 and AA3 appears in the acyl moiety with the formula (I) (Acy-AA1n-AA2m-AA3p-) can be interchanged independently. Consequently, the formula Acy-AA1n-AA2m-AA3p- also covers moieties like, e.g., the formula Acy-AA2m-AA1n-AA3p-, the formula Acy-AA2-AA3n-AA2-, and the formula Acy-AA3p-AA2m-AA1n-, wherein Acy, AA1, AA2, AA3, n, m and p are as defined herein.
As mentioned herein, the connections between the moieties Acy, AA1, AA2 and/or AA3 are formally obtained by amide bond (peptide bond) formation (-CONH-) by removal of water from the parent compounds from which they formally are build. This means that in order to get the complete formula for the acyl moiety with the formula (I) (Acy-AA1n-AA2rn-AA3p-, wherein Acy, AA1, AA2, AA3, n, m and p are as defined herein), one has, formally, to take the compounds given for the terms Acy, AA1, AA2 and AA3 and remove a hydrogen and/or hydroxyl from them and, formally, to connect the building blocks so obtained at the free ends so obtained.
Non-limiting, specific examples of the acyl moieties of the formula Acy-AA1n-AA2m-AA3p- which may be present in the acylated insulin analogues of this invention are the following:
(Formula with Table Removed)
Any of the above non-limiting specific examples of acyl moieties of the formula Acy-AA1n-AA2m-AA3p-can be attached to an epsilon amino group of a lysine residue present in any of the above non-limiting specific examples of insulin analogues thereby giving further specific examples of acylated insulin analogues of this invention.
Any of the above non-limiting specific examples of acyl moieties of the formula Acy-AA1n-AA2m-AA3p-can be attached to an alpha amino group of a A1 residue present in any of the above non-limiting specific examples of insulin analogues thereby giving further specific examples of acylated insulin analogues of this invention.
The protease stabilized insulins can be converted into the acylated protease stabilized insulins of this invention by introducing of the desired group of the formula Acy-AA1n-AA2m-AA3p- in the lysine residue or in a N-terminal position in the insulin analogue. The desired group of the formula Acy-AA1n-AA2m-AA3p- can be introduced by any convenient method and many methods are disclosed in the prior art for such reactions. More details appear from the examples herein.
In an embodiment, the present invention does not relate to compounds described in EP 07114387.9, i.e., acylated insulins wherein an acyl moiety is attached to the parent insulin and wherein said acyl moiety comprises repeating units of alkylene glycol containing amino acids and wherein there is only one lysine residue (K & Lys) in the parent insulin.
PHARMACEUTICAL COMPOSITIONS
The acylated insulins of this invention may be administered subcutaneously, nasally, orally, or pulmonary.
For subcutaneous administration, the acylated insulins of this invention are formulated analogously with the formulation of known insulins. Furthermore, for subcutaneous administration, the acylated insulins of this invention are administered analogously with the administration of known insulins and, generally, the physicians are familiar with this procedure.
Acylated 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 ug/kg to about 50 ug/kg of acylated 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 acylated insulins of this invention may be delivered by inhalation to achieve slow absorption and/or reduced systemical clearance thereof. Different inhalation devices typically provide similar pharmacokinetics when similar particle sizes and similar levels of lung deposition are compared.
The acylated 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 inhalation. These devices include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Preferably, the acylated 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 acylated 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 inhaler (Glaxo), the Spinhaler® powder inhaler (Fisons), or the like.
As those skilled in the art will recognize, the formulation of acylated 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 acylated insulins in the aerosol. For example, shorter periods of administration can be used at higher concentrations of acylated 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 acylated 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 insulin acylated insulins of this invention in a given quantity of the powder determines the dose delivered in a single administration.
The particle size of acylated 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 acylated insulins of this invention ion is formulated so that at least about 10% of the acylated 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 acylated 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 acylated insulins is selected to yield the desired particle size in the chosen inhalation device.
Advantageously for administration as a dry powder an acylated insulin 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. The preferred particle size is effective for delivery to the alveoli of the patient's lung. 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 acylated insulin 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 acylated insulins of this invention for administration from a dry powder inhaler typically include a finely divided dry powder containing the derivative, 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 acylated insulin, 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 facilitate 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 acylated insulin can be mixed with an additive at a molecular level or the solid formulation can include particles of the acylated insulin 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 acylated insulins of this invention can be produced by forcing a suspension or solution of the acylated insulin 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 acylated insulins of this invention suitable for use with a sprayer will typically include the acylated insulins in an aqueous solution at a concentration of from about 1 mg to about 500 mg of the acylated insulin per ml of solution. Depending on the acylated insulin chosen and other factors known to the medical advisor, the upper limit may be lower, e.g., 450, 400, 350, 300, 250, 200, 150, 120, 100 or 50 mg of the acylated insulin 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 acylated insulin, 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 acylated insulin include sucrose, mannitol, lactose, trehalose, glucose, or the like. The acylated insulins formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the insulin conjugate caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxy-ethylene sorbitol fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation.
Pharmaceutical compositions containing an acylated insulin of this invention may also be administered parenterally to patients in need of such a treatment. Parenteral 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.
Injectable compositions of the acylated 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, an acylated insulin is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared. Zink, an isotonic agent, a preservative and/or a buffer is/are added as required and the pH value of the solution is adjusted - if necessary - 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.
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 dihy-drogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)amino-methan, 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, m-cresol, 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 concentration 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 preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th 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 compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th 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 sodium acetate, glycylglycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodium phosphate.
A composition for nasal administration of an acylated insulins of this invention may, e.g., be prepared as described in European Patent No. 272,097.
Oral preparations containing an acylated protease stabilised insulin of this inventions can be prepared in a manner known perse. One way of making preparations containing an acylated protease stabilised insulin of this invention which can conveniently be administered orally is by using a procedure which is analagous to the process described in WO 2008/145728.
Another way of preparing oral preparations containing an acylated protease stabilised insulin of this invention is to prepare a water-free liquid or semisolid pharmaceutical compositions comprising an acylated protease stabilised insulin of this invention (a), at least one polar organic solvent (b) for the acylated protease stabilised insulin, at least one lipophilic component (c), and optionally a surfactant (d) and/or at least one solid hydrophilic component (e). This could be in the form of an oily solution. Alternatively, the at least one solid hydrophilic component (d) is at least one solid hydrophilic polymer. Alterantively, the pharmaceutical composition comprising at least one solid hydrophilic component is free of surfactant, wherein said surfactant has an HLB value which is at least 8, i.e. there is no surfactant, which has an HLB value which is at least 8, present in the composition.
For example, a pharmaceutical composition contining an acylated protease stabilised insulin may be a water-free oily solution and/or a SEDDS or SMEDDS pharmaceutical composition.
Alternatively said pharmaceutical composition is a self emulsifying drug delivery system (herein designated SEDDS).
It is believed that the high solubility of an acylated protease stabilised insulin in the polar organic solvent of the pharmaceutical composition resulting in the relatively low total amount of polar organic solvent needed in said pharmaceutical composition may improve compatibility of the pharmaceutical composition with capsule materials.
The pharmaceutical composition may contain a carrier that comprises a lipophilic component, a surfactant and a polar organic solvent and optionally a solid hydrophilic component (e). If there is a solid hydrophilic component present, at least one of the components selected from the group consisting of a lipophilic component and a surfactant is liquid or semi-solid. If there is a liquid hydrophilic component (e) present, both the lipophilic component and the surfactant may be solid. For example, the surfactant is liquid or semisolid. In one aspect, a solid hydrophilic component is present.
As used herein, the term "carrier" refers to the pharmaceutically acceptable vehicle that transports the therapeutically active water-soluble polypeptide across the biological membrane or within a biological fluid. The carrier comprises a lipophilic component and a polar organic solvent, and optionally a solid hydrophilic component and/or a surfactant. The carrier is capable of spontaneously producing an emulsion or colloidal structures, when brought in contact, dispersed, or diluted, with an aqueous medium, e.g., water, fluids containing water, or in vivo media in mammals, such as the gastric juices of the gastrointestinal tract. The colloidal structures can be solid or liquid particles including domains, droplets, micelles, mixed micelles, vesicles and nanoparticles.
For example, when the pharmaceutical composition is brought into contact with an aqueous medium, an emulsion, such as a microemulsion, spontaneously forms. In particular, an emulsion or microemulsion forms in the digestive tract of a mammal when the delivery system is orally ingested. In addition to the aforementioned components, the spontaneously dispersible preconcentrate can also
optionally contain other excipients, such as buffers, pH adjusters, stabilizers and other adjuvants recognized by one of ordinary skill in the art to be appropriate for such a pharmaceutical use.
The term "water-free" as used herein refers to a composition to which no water is added during preparation of the pharmaceutical composition. The acylated protease stabilised insulin and/or one or more of the excipients in the pharmaceutical composition may have small amounts of water bound to it before preparing a pharmaceutical composition. Fore example, a water-free pharmaceutical composition comprises less than 10% w/w water, for example, less than 5% w/w water, for example, less than 4% w/w water, for example, less than 3% w/w water, for example, less than 2% w/w water, for example, less than 1% w/w water.
As used herein, the term "microemulsion preconcentrate" means a composition, which spontaneously forms a microemulsion, e.g., an oil-in-water microemulsion, in an aqueous medium, e.g., in water or in the gastrointestinal fluids after oral application. The composition self-emulsifies upon dilution in an aqueous medium for example in a dilution of 1:5, 1:10, 1:50, 1:100 or higher.
Due to the high solubility of the acylated protease stabilised insulin, the total amount of polar organic solvent in the SEDDS can be kept low which on the one hand improves compatibility of the formulation with capsule materials and on the other hand gives more design space for the composition.
The pharmaceutical composition comprises a lipophilic component, and an organic polar component. The components of the drug delivery system can be present in any relative amounts. For example, the drug delivery system can comprises up to 40% polar organic component by weight of the composition of the carrier, e.g., less than 30%, 20%, 15% or 10%. In another aspect, the drug delivery system comprises from 5% to 40% by weight polar organic solvent of the total composition of the carrier. In yet a further aspect, the drug delivery system comprises from 10% to 30 % by weight polar organic solvent of the total composition of the carrier.
The pharmaceutical composition may be in the form of a non-powder composition, i.e. in a semi-solid or liquid form.
As used herein, the term "liquid" means a component or composition that is in a liquid state at room temperature ("RT"), and having a melting point of, for example, below 20°C. As used herein room temperature (RT) means approximately 20-25°C.
As used herein, the term "semi-solid" relates to a component or composition which is not liquid at room temperature, e.g., having a melting point between room temperature and about 40°C. A semisolid can have the qualities and/or attributes of both the solid and liquid states of matter. As used-herein, the term "solidify" means to make solid or semi-solid.
Examples of semi-solid or liquid compositions are pharmaceutical compositions in the form of, e.g., oils, solutions, liquid or semisolid SMEDDS and liquid or semisolid SEDDS.
"SMEDDS" (being an abbreviation for self-micro-emulsifying drug delivery systems) are herein defined as isotropic mixtures of a hydrophilic component, a surfactant, optionally a cosurfactant and a drug that rapidly form an oil in water microemulsion when exposed to aqueous media under conditions of gentle agitation or digestive motility that would be encountered in the Gl tract.
"SEDDS" (being an abbreviation for self emulsifying drug delivery systems) are herein defined as mixtures of a hydrophilic component, a surfactant, optionally a cosurfactant and a drug that forms spontaneously a fine oil in water emulsion when exposed to aqueous media under conditions of gentle agitation or digestive motility that would be encountered in the Gl tract.
As used herein, the term "microemulsion" refers to a clear or translucent, slightly opaque, opalescent, non-opaque or substantially non-opaque colloidal dispersion that is formed spontaneously or substantially spontaneously when its components are brought into contact with an aqueous medium.
As used herein, the term "emulsion" refers to a slightly opaque, opalescent or opague colloidal dispersion that is formed spontaneously or substantially spontaneously when its components are brought into contact with an aqueous medium.
A microemulsion is thermodynamically stable and contains homogenously dispersed particles or domains, for example of a solid or liquid state (e.g., liquid lipid particles or droplets), of a mean diameter of less than about 500 nm, e.g., less than about 400 nm or less than 300 nm, less than 200 nm, less than 100 nm, and greater than about 2-4 nm as measured by standard light scattering techniques, e.g., using a MALVERN ZETASIZER Nano ZS. The term "domain size" as used herein refers to repetitive scattering units and can be measured by, e.g., small angle X-ray. In one aspect, the domain size is smaller than 400 nm, in another aspect, smaller than 300 nm and in yet another aspect, smaller than 200 nm.
As used herein the term "spontaneously dispersible" when referring to a pre-concentrate refers to a composition that is capable of producing colloidal structures such as microemulsions, emulsions and other colloidal systems, when diluted with an aqueous medium when the components of the composition are brought into contact with an aqueous medium, e.g. , by simple shaking by hand for a short period of time, for example for ten seconds. In one aspect a spontaneously dispersible concentrate according to the invention is a SEDDS or SMEDDS.
As used herein, the term "lipophilic component" refers to a substance, material or ingredient that is more compatible with oil than with water. A material with lipophilic properties is insoluble or almost insoluble in water but is easily soluble in oil or other nonpolar solvents. The term "lipophilic component" can comprise one or more lipophilic substances. Multiple lipophilic components may constitute the lipophilic phase of the spontaneously dispersible preconcentrate and form the oil aspect, e.g., in an oil-in-water emulsion or microemulsion. At room temperature, the lipophilic component and lipophilic phase of the spontaneously dispersible preconcentrate can be solid, semisolid or liquid. For example, a solid lipophilic component can exist as a paste, granular form, powder or flake. If more than one excipient comprises the lipophilic component, the lipophilic component can be a mixture of liquids, solids, or both.
In one aspect, the lipophilic component is present in the pharmaceutical composition in an amount of at least 20% w/w. In a further aspect, the lipophilic component is present in an amount of at least 30%, at least 50%, at least 80% or at least 90% w/w. For example, the lipophilic component may be present from about 5% to about 90 % by weight of the composition, e.g., from about 15% to about
60%, e.g., from about 20% to about 40%. Examples of solid lipophilic components, i.e., lipophilic components which are solid or semisolid at room temperature, include, but are not limited to, the following:
1. mixtures of mono-, di- and triglycerides, such as hydrogenated coco-glycerides (melting point (m.p.) of about 33.5°C to about 37°C], commercially-available as WITEPSOL HI5 from Sasol Germany (Witten, Germany); Examples of fatty acid triglycerides e.g., C10-C22 fatty acid triglycerides include natural and hydrogenated oils, such as vegetable oils;
2. esters, such as propylene glycol (PG) stearate, commercially available as MONOSTEOL (m.p. of about 33°C to about 36°C) from Gattefosse Corp. (Paramus, NJ); diethylene glycol palmito stearate, commercially available as HYDRINE (m.p. of about 44.5°C to about 48.5°C) from Gattefosse Corp.;

What is claimed is:
1. An acylated protease stabilised insulin wherein the protease stabilised insulin, formally, consists of a non-protease stabilised insulin (parent insulin) wherein at least one hydrophobic amino acid has been substituted with hydrophilic amino acids, and wherein said substitution is within or in close proximity to one or more protease cleavage sites of the non-protease stabilised insulin (parent insulin) and wherein such protease stabilised insulin optionally further comprises one or more additional mutations with the proviso that there is only one lysine residue in the stabilized insulin, and wherein the acyl moiety is attached to the lysine residue or to a N-terminal position in the protease stabilized insulin.
2. An acylated protease stabilized insulin according to the preceding claim, wherein at least two hydrophobic amino acids in the non-protease stabilized insulin have been substituted with hydrophilic amino acids.
3. An acylated protease stabilized insulin according to any of the preceding claims, wherein the acyl moiety is attached to the lysine residue in the protease stabilized insulin.
4. An acylated protease stabilized insulin according to any of the preceding claims, wherein the acyl moiety is attached to the amino group of the A-chain N-terminal residue in the protease stabilized insulin.
5. An acylated protease stabilized insulin according to any of the preceding, possible claims wherein the protease stabilized insulin is selected from the group consisting of A8H, B25N, B27E, desB30 human insulin; A14E, A18L, B25H, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, B1E, B25H, B27E, B28E, desB30 human insulin; A14E, B1E, B25H, B28E, desB30 human insulin; A14E, B1E, B27E, B28E, desB30 human insulin; A14E, B1E, B28E, desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B25H, desB30 human insulin; A14E, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, B25H, B27E, desB30 human insulin; A14E, B25H, desB27, desB30 human insulin; A14E, B25H, B29R, desB30 human insulin; A14E, B28D, desB30 human insulin; A14E, B28E, desB30 human insulin; B25N, B27E, desB30 human insulin; A14E, A21G, B16H, B25H, desB30 human insulin; A14E, A21G, B25H, B26G, B27G, B28G, desB30 human insulin; B25H, desB30 human insulin; A21G, B25H, desB30 human insulin; A14E, A21G, B25H, desB30 human insulin and A14E, A21G, B25H, desB27, desB30 human insulin,.
6. An acylated protease stabilized insulin according to any of the preceding claims, wherein the acyl moiety attached to the protease stabilised insulin has the general formula Acy-AA1n-AA2m-AA3p-
(I), wherein n is 0 or an integer in the range from 1 to 3; m is 0 or an integer in the range from 1 to 10; p is 0 or an integer in the range from 1 to 10; Acy is a fatty acid or a fatty diacid comprising from about 8 to about 24 carbon atoms; AA1 is a neutral linear or cyclic amino acid residue; AA2 is an acidic amino acid residue; AA3 is a neutral, alkyleneglycol-containing amino acid residue; the order by which AA1, AA2 and AA3 appears in the formula can be interchanged independently; AA2 can occur several times along the formula (e.g., Acy-AA2-AA32-AA2-); AA2 can occur independently (= being different) several times along the formula (e.g., Acy-AA2-AA32-AA2-); the connections between Acy, AA1, AA2 and/or AA3 are amide (peptide) bonds which, formally, can be obtained by removal of a hydrogen atom or a hydroxyl group (water) from each of Acy, AA1, AA2 and AA3; and attachment to the protease stabilised insulin can be from the C-terminal end of a AA1, AA2, or AA3 residue in the acyl moiety of the formula (I) or from one of the side chain(s) of an AA2 residue present in the moiety of formula (I).
7. An acylated protease stabilized insulin according to any of the preceding, posssible claims which is any one of the compounds mentioned specifically in the above specification.
8. An acylated protease stabilized insulin according to the preceding claim which is selected from the group consisting of A14E, B25H, B29K(Nε-hexadecandioyl), desB30 human insulin; A14E, B25H, B29K(Nεoctadecandioyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nε3-carboxy-5-octadecanedioylaminobenzoyl), desB30 human insulin; A14E, B25H, B29K(Nε-N-octadecandioyl-N-(2-carboxyethyl)glycyi), desB30 human insulin; A14E, B25H, B29K(Nε (N-octadecandioyl-N-carboxymethyl)-beta-alanyl), desB30 human insulin; A14E, B25H, B29K(Nε4-([4-({19-carboxynonadecanoylamino}methyl)trans-cyclohexanecarbonyl]-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεheptadecanedioyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nεmyristyl), desB30 human insulin; A14E, B25H, B29K-(Nεeicosanedioyl-γGlu-γGlu), desB30 human insulin; A14E, B25H, B29K(Nε4-([4-({19-carboxy-nonadecanoylamino}methyl)trans-cyclohexanecarbonyl]-γGlu-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεoctadecanedioyl-γGlu-γGlu), desB30 human insulin; A14E, B28D, B29K(Nεocta-decandioyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεoctadecandioyl-γGlu-PEG7), desB30 human insulin; A14E, B25H, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nεeicosanedioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}-ethoxy)propionyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεhexadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nεhexadecanedioyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεheptadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nεoctadecanedioyl-γGlu-γGlu-γGlu-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεeicosanedioyl-γGlu-γGlu-γGlu), desB30 human insulin; A14E, B25H, B27E, B29K(Nεocta-decanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B26G, B27G, B28G,
B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B16H, B25H, B29K-(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B16E, B25H, B29K(Nεocta-decanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(Nεhexadecane-dioyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεeicosanedioyl-γGlu-OEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K(Nεhexadecandioyl-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(Nεoctadecanedioyl-γGlu-γGlu-γGlu), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεhexadecandioyl-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(Nεoctadecanedioyl-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K-(N(eps)eicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nεocta-decanedioyl-OEG-γGlu-γGIu), desB30 human insulin; A14E, A18L, B25H, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, A18L, B25H, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B27E, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A1G(Nαoctadecandioyl-γGlu-OEG-OEG), A14E, B25H, B29R, desB30 human insulin; A14E, B1F(Nαoctadecandioyl-γGlu-OEG-OEG), B25H, B29R, desB30 human insulin; A1G(Nαhexadecandioyl-γGlu), A14E, B25H, B29R, desB30 human insulin; A14E, B25H, B29K(Nεoctadecanedioyl-γGlu-Abu-Abu-Abu-Abu), desB30 human insulin; A14E, B25H, B29K-(NPeicosanedioyl), desB30 human insulin; A14E, B25H, B29K(Nα4-[16-(1H-tetrazol-5-yl)hexa-decanoylsulfamoyl]butanoyl), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K-(Nεoctadecandioyl-γGlu), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K-(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεocta-decandioyl), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεeicosanedioyl), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nεdocosanedioyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεdocosanedioyl-γGlu-γGlu), desB30 human insulin; A14E, B25H, B29K-(Nεicosanedioyl-γGlu-OEG-OEG-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεoctadecane-dioyl-γGlu-OEG-OEG-γGlu), desB30 human insulin; A14E, B25H, B29K(Nε (N-icosanedioyl-N-carboxymethyl)-ßAla), desB30 human insulin; A14E, B25H, B29K(Nε3-[2-(2-{2-[2-(17-carboxy-heptadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nε3-[2-(2-{2-[2-(19-carboxynonadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]-propionyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεoctadecandioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl), desB30 human insulin; A14E, B25H, B29K(Nεocta-decandioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεicosanedioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}-ethoxy)propionyl), desB30 human insulin; A14E, B25H, B29K(Nε4-([4-({17-carboxynonadecanoyl-amino}methyl)trans-cyclohexanecarbonyl]-γGlu), desB30 human insulin; A14E, B25H, B29K(Nε4-([4-({17-carboxyheptadecanoylamino}methyl)trans-cyclohexanecarbonyl]-γGlu-γGlu), desB30 human insulin; A14E, B28D, B29K(Nεhexadecandioyl-γGlu), desB30 human insulin; A14E, B28D,
B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B28D, B29K(Nεoctadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B28D, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B28E, B29K(Nεhexadecandioyl-γGlu), desB30 human insulin; A14E, B28E, B29K(Nεoctadecandioyl-γGlu), desB30 human insulin; A14E, B28E, B29K(Nε eicosanedioyl-γGlu), desB30 human insulin; A14E, B28E, B29K(Nεoctadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B28E, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B28E, B29K(Nεhexadecandioyl-γGlu), desB30 human insulin; A14E, B1E, B28E, B29K(Nεoctadecandioyl-γGlu), desB30 human insulin; A14E, B1E, B28E, B29K-(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B1E, B28E, B29K(Nεhexadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B28E, B29K(Nεoctadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B28E, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B27E, B28E, B29K(Nεhexadecandioyl-γGlu), desB30 human insulin; A14E, B1E, B27E, B28E, B29K(Nεoctadecandioyl-γGlu), desB30 human insulin; A14E, B1E, B27E, B28E, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B1E, B27E, B28E, B29K(Nεhexadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B27E, B28E, B29K(Nεoctadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B27E, B28E, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B25H, B28E, B29K-(Nεhexadecandioyl-γGlu), desB30 human insulin; A14E, B1E, B25H, B28E, B29K(Nεoctadecan-dioyl-γGlu), desB30 human insulin; A14E, B1E, B25H, B28E, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B1E, B25H, B28E, B29K(Nεhexadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B25H, B28E, B29K(Nεoctadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B25H, B28E, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B25H, B27E, B28E, B29K(Nεhexadecandioyl-γGlu), desB30 human insulin; A14E, B1E, B25H, B27E, B28E, B29K(Nεoctadecandioyl-γGlu), desB30 human insulin; A14E, B1E, B25H, B27E, B28E, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B1E, B25H, B27E, B28E, B29K(Nεhexadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B25H, B27E, B28E, B29K(Nεoctadecandioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B1E, B25H, B27E, B28E, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B28D, B29K(Nεhexadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B28E, B29K(Nεhexadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25N, B27E, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25N, B27E, B29K(Nεocta-decanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25N, B27E, B29K(Nεhexadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25N, B27E, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; B25N, B27E, B29K(Nεoctadecanedioyl-γGlu), desB30 human insulin; B25N, B27E, B29K(Nεhexadecanedioyl-γGlu), desB30 human insulin; A8H, B25N, B27E, B29K(Nεeicosane-dioyl-γGlu-OEG-OEG), desB30 human insulin; A8H, B25N, B27E, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A8H, B25N, B27E, B29K(Nεhexadecanedioyl-γGlu-OEG-
OEG), desB30 human insulin; A8H, B25N, B27E, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A8H, B25N, B27E, B29K(Nεoctadecanedioyl-γGlu), desB30 human insulin; A8H, B25N, B27E, B29K(Nεhexadecanedioyl-γGlu), desB30 human insulin; A14E, B25H, B29K(Nε (N-icosane-dioyl-N-carboxymethyl)-ßAla-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nε (N-octa-decanedioyl-N-carboxymethyl)-pAla-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nε (N-hexadecanedioyl-N-carboxymethyl)-pAla-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nεoctadecanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)-methoxy]acetyl), desB30 human insulin; A14E, B25H, B29K(Nεeicosanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl), desB30 human insulin; A14E, B16H, B25H, B29K(Nεoctadecanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propyl-carbamoyl)methoxy]acetyl), desB30 human insulin; A14E, B16H, B25H, B29K(Nεeicosanedioyl-γGlu-2-[(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamoyl)methoxy]acetyl), desB30 human insulin; B25H, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25H, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25H, B29K(Nεoctadecanedioyl-γGlu), desB30 human insulin; B25H, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; B25H, B29K(Nεoctadecanedioyl), desB30 human insulin; B25H, B29K(Nεeicosanedioyl), desB30 human insulin; B25H, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25H, B29K-(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25H, B29K(Nεoctadecanedioyl-γGlu), desB30 human insulin; B25H, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; B25H, B29K-(Nεoctadecanedioyl), desB30 human insulin; B25H, B29K(Nεeicosanedioyl), desB30 human insulin; B25H, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25H, B29K-(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; B25H, B29K(Nεoctadecanedioyl-γGlu), desB30 human insulin; B25H, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B25H, desB27, B29K(Nεoctadecanedioyl), desB30 human insulin; A14E, B25H, desB27, B29K-(Nεeicosanedioyl), desB30 human insulin; A14E, B25H, desB27, B29K(Nεoctadecanedioyl-γGlu), desB30 human insulin; A14E, B25H, desB27, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B25H, desB27, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, desB27, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, A21G, B25H, desB27, B29K(Nεoctadecanedioyl), desB30 human insulin; A14E, A21G, B25H, desB27, B29K(Nεeicosanedioyl), desB30 human insulin; A14E, A21G, B25H, desB27, B29K-(Nεoctadecanedioyl-γGlu), desB30 human insulin; A14E, B25H, desB27, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, A21G, B25H, desB27, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, desB27, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A1G(Nαoctadecandioyl-γGlu-OEG-OEG), A14E, A21G, B25H, desB30 human insulin; A14E, B25H, B29K(Nεeicosanedioyl-OEG), desB30 human insulin; A14E, B25H, B27K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB28, desB29, desB30 human insulin; A14E, B25H, B29K(Nε(5-eicosanedioylaminoisophthalic acid)), desB30 human insulin; A14E, B25H,

B29K(Nεoctadecanedioyl), desB30 human insulin; A14E, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nεoctadecanedioyl-γGlu-OEG), desB30 human insulin; A14E, B25H, B29K(Nεeicosanedioyl-OEG-OEG), desB30 human insulin; A14E, B25H, B29K(Nεeicosanedioyl-Aoc), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεeicosanedioyl-γGlu-γGlu), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεeicosanedioyl-γGlu-γGlu), desB30 human insulin; A14E, B25H, B29K(Nεoctadecanedioyl-OEG), desB30 human insulin; A14E, B25H, desB27, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, B25H, B16H, B29K(Nεoctadecanedioyl-γGlu), desB30 human insulin; AIG(Nαoctadecanedioyl), A14E, B25H, B29R, desB30 human insulin; A14E, B16H, B25H, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B25H, B27K(Nεeicosanedioyl-γGlu), desB28, desB29, desB30 human insulin; A14E, B25H, B29K(Nεoctadecanedioyl-γGlu-γGlu-γGlu), desB30 human insulin; A21G, B25H, B29K(Nεoctadecanedioyl), desB30 human insulin; A14E, A21G, B25H, desB27, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, A21G, B25H, B29K(Nεoctadecanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, A21G, B25H, B29K(Nεeicosanedioyl-γGlu-OEG-OEG), desB30 human insulin; A14E, A21G, B25H, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, A21G, B25H, B29K(Nεeicosanedioyl), desB30 human insulin; A14E, A21G, B25H, B29K(Nεoctadecanedioyl-γGlu), desB30 human insulin; A14E, A21G, B25H, B29K(Nεoctadecanedioyl), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεoctadecanedioyl-γGlu), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεoctadecanedioyl), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεeicosanedioyl-γGlu), desB30 human insulin; A14E, B25H, B26G, B27G, B28G, B29K(Nεeicosanedioyl), desB30 human insulin; A1G(Nαoctadecandioyl-γGlu), A14E, B25H, B26G, B27G, B28G, desB30 human insulin; AIG(Nαeicosanedioyl-γGlu), A14E, B25H, B26G, B27G, B28G, desB30 human insulin; A1G(Nαoctadecandioyl-γGlu), A14E, B25H, B26G, B27G, B28G, B29R, desB30 human insulin; AIG(Nαeicosanedioyl-γGlu), A14E, B25H, B26G, B27G, B28G, B29R, desB30 human insulin; A1G(Nαoctadecandioyl), A14E, B25H, B26G, B27G, B28G, desB30 human insulin; A1G(Nαeicosanedioyl), A14E, B25H, B26G, B27G, B28G, desB30 human insulin; A1G(Nαoctadecandioyl), A14E, B25H, B26G, B27G, B28G, B29R, desB30 human insulin and AIG(Nαeicosanedioyl), A14E, B25H, B26G, B27G, B28G, B29R, desB30 human insulin.
9. An acylated protease stabilized insulin according to any of the above claims for use as medicament.
10. An acylated protease stabilized insulin according to any of the above claims for use in the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, syndrome X or dyslipidemia.
11. Use of a therapeutically effective amount of an protease stabilised insulin according to any one of the above claims for the preparation of a pharmaceutical formulation for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, syndrome X or dyslipidemia.
12. Any novel feature or combination of features described herein.