WO2014059536A1 - Combinaison d'une insuline et de la kallikréine 1 tissulaire - Google Patents

Combinaison d'une insuline et de la kallikréine 1 tissulaire Download PDF

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Publication number
WO2014059536A1
WO2014059536A1 PCT/CA2013/050755 CA2013050755W WO2014059536A1 WO 2014059536 A1 WO2014059536 A1 WO 2014059536A1 CA 2013050755 W CA2013050755 W CA 2013050755W WO 2014059536 A1 WO2014059536 A1 WO 2014059536A1
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WIPO (PCT)
Prior art keywords
insulin
klkl
composition
diabetes
human
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PCT/CA2013/050755
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English (en)
Inventor
Dietrich John PAULS
Tadeusz Mitchell KOLODKA
Mark Williams
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Diamedica Inc.
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Publication of WO2014059536A1 publication Critical patent/WO2014059536A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4853—Kallikrein (3.4.21.34 or 3.4.21.35)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21035—Tissue kallikrein (3.4.21.35)

Definitions

  • Diabetes mellitus or simply diabetes, is a group of metabolic diseases in which a person has high blood sugar, either because the pancreas does not produce enough insulin, or because cells do not respond to the insulin that is produced. Around 366 million people worldwide suffer from diabetes mellitus. Untreated, diabetes can cause many
  • Acute complications include diabetic ketoacidosis and nonketotic hyperosmolar coma.
  • Serious long-term complications include cardiovascular disease, chronic renal failure, diabetic retinopathy, and diabetic neuropathy. The adequate treatment of diabetes is thus important and there is a need for improved therapies for the treatment of diabetes.
  • the present invention includes a composition having as one component at least one insulin and as a second component a tissue kallikrein-1 (KLK1) polypeptide, or variant or fragment thereof.
  • the at least one insulin includes human insulin, or an analog, derivative, or metabolite thereof.
  • the at least one insulin is a rapid-acting insulin, a short-acting insulin, an intermediate- acting insulin, a long-acting insulin, or a mixture thereof.
  • the at least one insulin is Gly(A21)-Arg(B31)-Arg(B32) human insulin, Lys B2S Pro B29 human insulin, B28 Asp human insulin, and B29Lys(s ⁇ tetradecanoyl), or desB30 human insulin.
  • the insulin is long acting insulin. In some aspects, the long acting insulin is Gly(A21)- Arg(B31)-Arg(B32) human insulin. In some aspects, the insulin is short acting insulin. In some aspects, the KLKl is recombinant KLKl (rKLKl). In some aspects, the KLKl is human KLKl (hKLKl). In some aspects, the KLKl is recombinant human KLKl (rhKLKl). In some aspects, the KLKl includes an effective dosage of about 0.025 to about 250 IU kg or about 0.1 to about 1000 g/kg. In some aspects, a composition of the present invention includes a total day's effective dosage of insulin. In some aspects, a composition of the present invention further includes a pharmaceutically acceptable carrier.
  • the present invention includes a kit including a composition of the present invention.
  • the present invention includes a kit including two compositions, a
  • the at least one insulin includes human insulin, or an analog, derivative, or metabolite thereof.
  • the at least one insulin is a rapid- acting insulin, a short-acting insulin, an intermediate-acting insulin, a long-acting insulin, or a mixture thereof.
  • the at least one insulin is Gly(A21)-Arg(B31)- Arg(B32) human insulin, Lys B28 Pro B29 human insulin, B28 Asp human insulin, and B29Lys(8-tetradecanoyl), or desB30 human insulin.
  • the insulin is long acting insulin.
  • the long acting insulin is Gly(A21)-Arg(B31)-Arg(B32) human insulin.
  • the insulin is short acting insulin.
  • the KLKl is recombinant KLKl (rKLKl).
  • the KLKl is human KLKl (hKLKl).
  • the KLKl is recombinant human KLKl (rhKLKl).
  • the KLKl includes an effective dosage of about 0.025 to about 250 IU/kg or about 0.1 to about 1000 g kg.
  • the insulin includes a total day's effective dosage.
  • the present invention includes a device including a composition as described above, wherein the device is suitable for delivering the composition parenterally.
  • parenteral delivery includes subcutaneous or intravenous delivery.
  • the device includes a syringe.
  • the syringe further includes a hypodermic needle assembly attached to the syringe.
  • the syringe further includes a protective cover around the needle assembly.
  • the needle is about 1 ⁇ 2 inch to about 5/8 of an inch in length and has a gauge of about 25 to about 31.
  • the present invention includes a device including a pharmaceutical composition including KLKl and a pharmaceutical composition including at least one insulin, wherein the device is suitable for the combined delivery of both compositions.
  • deliver includes parenteral delivery.
  • parenteral delivery includes subcutaneous delivery or intravenous delivery.
  • the device includes a syringe.
  • the syringe further includes a hypodermic needle assembly attached to the syringe.
  • the syringe further includes a protective cover around the needle assembly.
  • the needle is about 1 ⁇ 2 inch to about 5/8 of an inch in length and has a gauge of about 25 to about 31.
  • the at least one insulin includes human insulin, or an analog, derivative, or metabolite thereof. In some aspects, the at least one insulin is a rapid-acting insulin, a short-acting insulin, an intermediate-acting insulin, a long-acting insulin, or a mixture thereof. In some aspects, the at least one insulin is Gly(A21)-Arg(B31 )-Arg(B32) human insulin, Lys B28 Pro B29 human insulm, B28 Asp human insulin, and B29Lys(s-tetradecanoyl), or desB30 human insulin. In some aspects, the insulin is long acting insulin. In some aspects, the long acting insulm is Gly(A21)-Arg(B31)-Arg(B32) human insulin. In some aspects, the insulin is short acting insulin. In some aspects, the KLKl is recombinant KLKl
  • the KLKl is human KLKl (hKLKl). In some aspects, the KLKl is recombinant human KLKl (rhKLKl).
  • the present invention includes a method for treating a disease or condition that benefits from treatment with insulin in a patient in need thereof, the method including administering to said patient a therapeutically effective amount of a composition of the present invention.
  • the present invention includes a method for treating for treating a disease or condition that benefits from treatment with insulin in a patient in need thereof, the method including administering to said patient a therapeutically effective amount of a co- formulation including a tissue kallikrein-1 (KLKl) polypeptide and a therapeutically effective amount of at least one insulin.
  • the at least one insulin includes human insulin, or an analog, derivative, or metabolite thereof.
  • the at least one insulin is a rapid-acting insulin, a short-acting insulin, an intermediate-acting insulin, a long-acting insulin, or a mixture thereof.
  • the at least one msulin is Gly(A21)-Arg(B31)-Arg(B32) human insulin, Lys B28 Pro B29 human insulin, B28 Asp human insulin, and B29Lys(s-tetradecanoyl), or desB30 human insulin.
  • the insulin is long acting insulin.
  • the long acting insulin is Gly(A21)-Arg(B31)-Arg(B32) human insulin.
  • the insulin is short acting insulin.
  • the KLKl is recombinant KLKl (rKLKl). In some aspects, the KLKl is human KLKl (hKLKl). In some aspects, the KLKl is recombinant human KLKl (rhKLKl).
  • the present invention includes method for treating a disease or condition that benefits from treatment with insulin in a patient in need thereof, the method including coadministering or sequentially administering to said patient a therapeutically effective amount of a formulation including a tissue kallikrein-1 (KLKl) polypeptide and a therapeutically effective amount of a formulation including at least one insulin.
  • the at least one insulin includes human msulin, or an analog, derivative, or metabolite thereof.
  • the at least one insulin is a rapid-acting insulin, a short-acting insulin, an intermediate-acting insulin, a long-acting insulin, or a mixture thereof.
  • the at least one insulin is Gly(A21)-Arg(B3 l)-Arg(B32) human insulin, Lys B28 Pro B29 human insulin, B28 Asp human insulin, and B29Lys(s- tetradecanoyl), or desB30 human insulin.
  • the insulin is long acting insulin.
  • the long acting insulin is Gly(A21)-Arg(B31)-Arg(B32) human insulin.
  • the insulin is short acting insulin.
  • the KLKl is recombinant KLKl (rKLKl).
  • the KLKl is human KLKl (hKLKl).
  • the KLKl is recombinant human KLKl (rhKLKl).
  • said patient is treated for adjusting the fasting blood glucose levels, postprandial and/or postabsorptive blood glucose levels, improving insulin tolerances, reducing insulin resistance, improving glucose tolerance, and/or lowering the risk of hypoglycemia.
  • the disease or condition is a disease or condition that benefits from treatment of insulin, including, type 1 diabetes (T1D), pre-diabetes, metabolic syndrome, impaired fasting blood glucose, late stage type 2 diabetes mellitus, insufficient glycemic control, gestational diabetes, new onset diabetes after transplantation (NODAT), post-transplant metabolic syndrome (PTMS), or type 2 diabetes (T2D).
  • T1D type 1 diabetes
  • pre-diabetes metabolic syndrome
  • impaired fasting blood glucose including late stage type 2 diabetes mellitus, insufficient glycemic control
  • gestational diabetes new onset diabetes after transplantation (NODAT), post-transplant metabolic syndrome (PTMS), or type 2 diabetes (T2D).
  • NODAT new onset diabetes after transplantation
  • PTMS post-transplant metabolic syndrome
  • T2D type 2 diabetes
  • the at least one insulin includes human insulin, or an analog, derivative, or metabolite thereof. In some aspects, the at least one insulin is a rapid- acting insulin, a short-acting insulin, an intermediate-acting insulin, a long-acting insulin, or a mixture thereof. In some aspects, the at least one insulin is Gly(A21)-Arg(B31)- Arg(B32) human insulm, Lys B2S Pro B29 human insulin, B28 Asp human insulin, and
  • the insulin is long acting insulin.
  • the long acting insulm is Gly(A21)-Arg(B31)-Arg(B32) human insulin.
  • the insulin is short acting insulin.
  • Figure 1 Effect of insulin glargine, rhKLKl and a GLP-1 analogue separately on fasting blood glucose levels in the diabetic db/db mouse.
  • Figure 2 Effect of an insulin glargine and rhKLKl in combination on fasting blood glucose levels in the diabetic db/db mouse.
  • Figure 3 Effect of insulin glargine, rhKLKl and a GLP-1 analogue separately in the oral glucose tolerance test in the diabetic db/db mouse.
  • Figure 4 Effect of insulin glargine and rhKLKl in combination in the oral glucose tolerance test in the diabetic db/db mouse.
  • the invention relates to co-formulations of insulin and tissue kallikrein-1 (KLKl) polypeptide for use in methods of treating diabetes and devices and methods that deliver such co-formulations to a subject in need thereof. Further, the present invention relates to compositions, devices, and methods that provided for the coordinated co-administration of a formulation with insulin and a fonnulation with a KLKl polypeptide to a patient for the treatment of a disease or condition that benefits from the administration of insulin. According to the invention, there are provided methods for treating a patient with regard to diseases and conditions benefit from the administration of insulin, the methods including the administration of KLK1 and an insulin, including, for example,
  • diseases and conditions include, but are not limited to, diabetes mellitus, type 1 diabetes (T1D), prediabetes, metabolic syndrome, impaired fasting blood glucose, late stage type 2 diabetes mellitus, insufficient glycemic control, gestational diabetes, new onset diabetes after transplantation (NOD AT), post- transplant metabolic syndrome (PTMS), and type 2 diabetes (T2D).
  • T1D type 1 diabetes
  • prediabetes prediabetes
  • metabolic syndrome impaired fasting blood glucose
  • late stage type 2 diabetes mellitus insufficient glycemic control
  • gestational diabetes new onset diabetes after transplantation (NOD AT), post- transplant metabolic syndrome (PTMS), and type 2 diabetes (T2D).
  • NOD AT new onset diabetes after transplantation
  • PTMS post- transplant metabolic syndrome
  • T2D type 2 diabetes
  • the administration of KLK1 provides for the need to administer less insulin in the treatment of the patient, is safer, and/or is more effective for the patient.
  • the formulations, devices and methods of the present invention are particularly suited for the treatment of patients or individuals that require insulin injections.
  • the patient or individual has type 2 diabetes (T2D).
  • the patient or individual has type 2 diabetes (T2D) and still has residual endogenous insulin production.
  • the patient or individual has type 1 diabetes (T1D).
  • patient or individual is insulin resistant or has insufficient glycemic control.
  • An insulin of the present invention includes insulin and insulin analogs being used in the therapy of patients, in particular humans, and includes, but is not limited to, normal insulin, human insulin, insulin derivatives, zinc insulin and insulin analogues, including formulations thereof with modified release profiles, in particular as used in the therapy of humans.
  • An insulin of the present invention includes, for example, a rapid-acting insulin, a short-acting insulin, an intermediate-acting insulin, a long-acting insulin, and mixtures thereof (for example, mixtures of short- or rapid-acting insulin with long-acting insulin).
  • An insulin of the present invention includes insulin which is administered to the patient via injection, via infusion, including pumps, via inhalation, via oral, via transdermal or other routes of administration.
  • a rapid-acting insulin usually starts lowering the blood glucose within about 5 to 15 minutes and is effective for about 3 to 4 hours.
  • rapid-acting insulin include, but are not limited to, insulin aspart, insulin lispro and insulin glulisine.
  • Insulin lispro is marketed under the trade name HUMALOGTM and LIP OLOGTM.
  • Insulin aspart is marketed under the trade names NOVOLOGTM and NOVORAPIDTM.
  • Insulin glulisine is marketed under the trade name APIDRATM.
  • a short-acting insulin usually starts lowering the blood glucose within about 30 minutes and is effective about 5 to 8 hours.
  • An example is regular insulin or human insulin.
  • An intermediate-acting insulin usually starts lowering the blood glucose within about 1 to 3 hours and is effective for about 16 to 24 hours.
  • NPH insulin also known as HUMULINTM N, NOVOLINTM N, NOVOLINTM NPH
  • isophane insulin and a lente insulin, such as, for example, SEMLENTETM or MONOTARDTM
  • a long-acting insulin usually start lowering the blood glucose within 1 to 6 hours and are effective for up to about 24 hours or even up to or beyond 32 hours.
  • Long-acting insulin usually provides a continuous level of insulin activity (for up to 24-36 hours) and usually operates at a maximum strength (with flat action profile) after about 8-12 hours, sometimes longer. Long-acting insulin is usually administered in the morning or before bed.
  • a synergistic effect of the administration of a combination of insulin and a KLKl polypeptide is observed.
  • it has been found that the synergistic effects of the combination of insulin and a KLKl polypeptide on the concentration of glucose in the blood plasma occur in a concentration range of human KLKl of nearly one order of magnitude (factor 10).
  • a synergistic concentration range of KLKl allows a pharmaceutical composition of the invention that contains a defined proportion of at least one msulin to KLKl to cover a therapeutic range of msulin doses simultaneously with the associated, synergistic amount of KLKl .
  • the proportion can be selected such that every desired insulin dose has its corresponding dose of KLKl, which is situated within the desired range, e.g., the synergistic range.
  • the dose of KLKl at the desired insulin dose of a composition is outside (generally above) the desired dosage range of KLKl
  • the next composition e.g., the second composition
  • a further composition with a greater proportion of the at least one insulin to KLKl is selected for use, in which the amount of KL 1 at the desired insulin dose lies within the desired range.
  • the proportions of the first, the second, and, where used, further therapeutic agents may be further chosen such that the ranges of the insulin dosages which correspond to the desired dosages of KL 1 border one another and/or overlap one another. Preferably the ranges overlap.
  • Overlapping means more particularly that it is possible to select at least two compositions which, at the desired dose of the at least one insulin, each contain an amount of KLK1 which lies within the desired dosage range.
  • long acting insulin is injected once a day to provide a basal insulin level (to control fasting blood glucose levels) and perhaps short-acting or rapid acting insulin is injected two or three times a day just before meals to control postprandial or postabsorptive hyperglycemia.
  • basal insulin level to control fasting blood glucose levels
  • rapid acting insulin is injected two or three times a day just before meals to control postprandial or postabsorptive hyperglycemia.
  • short/rapid acting insulin may cause dangerous hypoglycemia if sufficient calories are not ingested. For example, if a meal is late at a restaurant or the patient feels ill and does not eat after the administration of insulin.
  • the formulations, devices and methods of the present invention provide for administering insulin and KLK1.
  • KL 1 may function as an insulin sensitizer and a glucose dependent insulin secretagogue, to sensitize the patient to the injected and/or endogenously produced insulin.
  • Administration of KLK1 to a patient with a disease or condition that benefits from treatment of insulin may reduce insulin resistance, thus requiring less insulin to be administered to the patient.
  • the insulin that is administered to the patient will be more effective in controlling blood glucose levels.
  • the combination of KLK1 and insulin will also reduce the risk of hypoglycemia.
  • the KLKl also allows endogenously produced insulin to control postprandial or postabsorptive hyperglycemia, decreasing or eliminating the need for short acting insulin.
  • the formulations, devices and methods of the present invention may be safer and easier to use because the patient can eat whenever they wish and not worry about counting calories and multiple daily short acting insulin injections, as KLKl and long acting insulin can be administered once a day.
  • KLKl may decrease insulin resistance (decrease HOMA-IR) and thus allow lower doses of long acting insulin or short/rapid acting insulin to the administered to a patient to achieve the desired blood glucose control.
  • the type 1 diabetic patient may require lower doses of short or rapid acting insulin prior to a meal.
  • the type 1 diabetic patient may require lower doses of long acting insulin to achieve the desired level of blood glucose control.
  • a change in HOMA-IR following KLKl administration may be calculated by methods known in the medical community.
  • a decrease in the amount of insulin required for the treatment of a disease or condition that benefits from the administration of insulin will be self-evident. Since most patients self-monitor their blood glucose levels and will administer insulin "to effect," the patients will administer less insulin if their bodies are more responsive to the insulin. Alternatively, the patient together with a medical professional may determine that the patient should administer less inulin by detecting hypoglycemia in the patient.
  • the present invention provides an improved dosing strategy, using KLKl along with insulin in a synergistic manner to control blood glucose levels. And, the present invention provides for an improved method of managing blood glucose levels in a patient with advanced type 2 diabetes, by treating or preventing insulin resistance.
  • the improved glucose control achieved by KLKl and insulin may result in improved glucose control in the patient, and thus decrease or delay the development of complications of diabetes.
  • Such conditions include, but are not limited to, treating, preventing, reducing the risk of, slowing progression of, and/or delaying metabolic disorder selected from the group consisting of type 1 diabetes mellitus, type 2 diabetes mellitus, impaired glucose tolerance (IGT), impaired fasting blood glucose (IFG), hyperglycemia, postprandial hyperglycemia, postabsorptive hyperglycemia, overweight, obesity, metabolic syndrome and gestational diabetes; or improving glycemic control and/or for reducing of fasting plasma glucose, of postprandial plasma glucose and/or of glycosylated hemoglobin HbAlc; or preventing, slowing, delaying or reversing progression from impaired glucose tolerance (IGT), impaired fasting blood glucose (IFG), insulin resistance and/or from metabolic syndrome to type 2 diabetes mellitus; or preventing, slowing the progression of, delaying or treating of a condition or disorder selected from the group consisting of complications of diabetes mellitus such as cataracts and micro- and macrovascular diseases, such as
  • ectopic fat or maintaining and/or improving the insulin sensitivity and/or for treating or preventing hyperinsulinemia and/or insulin resistance; preventing, slowing progression of, delaying, or treating new onset diabetes after transplantation (NOD AT) and/or post-transplant metabolic syndrome (PTMS); preventing, delaying, or reducing NODAT and/or PTMS associated complications including micro- and macrovascular diseases and events, graft rejection, infection, and death; treating diabetes associated with cystic fibrosis treating hyperuricemia and hyperuricemia associated conditions; treating or prevention kidney stones; treating hyponatremia; in a patient in need thereof characterized in that KL 1 is administered, for example in combination or alternation, with an insulin.
  • NOD AT new onset diabetes after transplantation
  • PTMS post-transplant metabolic syndrome
  • Tissue kallikreins are members of a gene super family of serine proteases comprising at least 15 separate and distinct proteins (named tissue kallikrein 1 through 15) (Yousef et al., 2001, Endocrine Rev; 22:184-204). Tissue kallikrein-1 is produced predominantly in the pancreas, hence the origin of the name from the Greek term
  • Tissue kallikrein-1 is also known as KLK1, pancreatic/renal kallikrein, glandular kallikrein 1, kallikrein serine protease 1, kallikrein 1, renal kallikrein, renal/pancreas/salivary kallikrein, kidney/pancreas/salivary gland kallikrein.
  • tissue kallikrein-1 and KLK1 are synonymous.
  • Tissue kallikrein-1 is a trypsin-like serine protease.
  • tissue kallikrein-1 cleaves kininogen into lysyl-bradykinin (also known as kallidin), a decapeptide kinin having physiologic effects similar to those of bradykinin.
  • lysyl-bradykinin also known as kallidin
  • Bradykinin is a peptide that causes blood vessels to dilate and therefore causes blood pressure to lower.
  • the KLKl gene encodes a single pre-pro- enzyme that is 262 amino acid residues in length and that includes the "pre-" sequence (residues 1-18) and the "pro-" sequence (residues 19-24), which is activated by trypsin-like enzymes.
  • the mature and active form human KLKl is a glycoprotein of 238 amino acid residues (residues 25-262) with a molecular weight of 26 kDa and a theoretical pi of 4.6.
  • KLKl has five disulphide bonds in its tertiary structure that are believed to be responsible for the protein's high stability, both against trypsin digestion and heat inactivation.
  • tissue kallikrein-1 The amino acid sequence of tissue kallikrein-1 is available for a wide variety of species, including, but not limited to, human (SEQ ID NO:l and SEQ ID NO:2), mouse (see, for example, GenBank: AAA39349.1, February 1, 1994); domestic cat (see, for example, NCBI Reference Sequence: XP_003997527.1, November 6, 2012); gorilla (see, for example, NCBI Reference Sequence: XP_004061305.1, December 3, 2012); cattle (see, for example, GenBank: AAI51559.1, August 2, 2007); dog (see, for example, CBI Reference Sequence: NP_001003262.1, February 22, 2013); rat (see, for example, GenBank: CAE51906.1, April 25, 2006); and olive baboon (see, for example, NCBI Reference Sequence: XP_003916022.1, September 4, 2012).
  • human SEQ ID NO:l and SEQ ID NO:2
  • mouse see, for example, GenBank: AAA
  • KLKl is functionally conserved across species in its capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen.
  • a tissue kallikrein-1 polypeptide of the present invention may have any of the known amino acid sequences for KLKl, or a fragment or variant thereof.
  • tissue kallikrein-1 polypeptide is a human tissue kallikrein-1 (hKLKl), including, but not limited to, a hKLKl polypeptide represented by SEQ ID NO:l or SEQ ID NO:2.
  • hKLKl may be represented by the amino acid sequence of
  • GenBank Ref. NP_002248.1 having the complete KLKl preproprotein amino acid sequence shown below: MWFLVLCLALSLGGTGAAPPIQSRIVGGWECEQHSQPWQAALYHFSTFQC 50
  • amino acids 1 to 18 of SEQ ID NO:l represent the signal peptide
  • amino acids 19 to 24 represent propeptide sequences
  • amino acids 25 to 262 represent the mature peptide.
  • the preproprotien includes a presumptive 17-amino acid signal peptide, a 7-amino acid proenzyme fragment and a 238-amino acid mature KLK1 protein.
  • a second amino acid sequence for human KLK1 is represented by SEQ ID NO:2, shown below:
  • VKWIEDTIAENS SEQ ID NO: 2
  • amino acids 1 to 18 of SEQ ID NO:2 represent the signal peptide
  • amino acids 19 to 24 represent propeptide sequences
  • amino acids 25 to 262 represent the mature peptide.
  • the preproprotien includes a presumptive 17-amino acid signal peptide, a 7-amino acid proenzyme fragment and a 238-amino acid mature KLK1 protein.
  • SEQ ID NO:l A comparison between SEQ ID NO:l and SEQ ID NO:2 shows two amino acid differences between the two hKL l amino acid sequences.
  • Single-nucleotide polymorphism (SNP's) between the two individuals within a species account for an E to Q substitution at amino acid residue 145 of 262 and an A to V substitution at position 188 of 262.
  • SEQ ID NO:l has an E (glutamic acid) at position 145 and an A (alanine) at position 188
  • SEQ ID NO:2 has a Q (glutamine) at position 145 and a V (valine) at position 188.
  • a KLK1 polypeptide of the present invention may have an E at position 145; may have a Q at position 145; may have an A at position 188; may have an A at position 188; may have an E at position 145 and an A at position 188; may have a Q at position 145 and a V at position 188; may have an Q at position 145 and an A at position 188; or may have an E at position 145 and a V at position 188.
  • tissue kallikrein-1 polypeptide may include residues 1- 262, residues 19-262, or residues 25-262 of a kallikrein preproprotein sequence, including, but not limited to human KLK1 having SEQ ID NO:l or SEQ ID NO:2, and fragments and variants thereof.
  • a "variant" of a starting or reference polypeptide is a polypeptide that has an amino acid sequence different from that of the starting or reference polypeptide. Such variants include, for example, deletions from, insertions into, and/or substitutions of residues within the amino acid sequence of the polypeptide of interest.
  • a variant amino acid in this context, refers to an amino acid different from the amino acid at the corresponding position in a starting or reference polypeptide sequence. Any combination of deletion, insertion, and substitution may be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics.
  • the amino acid changes also may alter post-translational processes of the polypeptide, such as changing the number or position of glycosylation sites.
  • a polypeptide variant may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity with a reference sequence, such as, for example, an amino acid sequence described herein.
  • a KLK1 polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to SEQ ID NO: 1 , or to a fragment of SEQ ID NO: 1 , such as for example, residues 25-262 or residues 78-141 of SEQ ID NO: 1.
  • Such a KLKl polypeptide may have an E or a Q at amino acid residue 145, and/or an A or a V at position 188.
  • a KLK1 polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 1%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to SEQ ID NO:2, or to a fragment of SEQ ID NO:2, such as for example, residues 25-262 or residues 78- 141 of SEQ ID NO:2.
  • Such a KL 1 polypeptide may have an E or a Q at amino acid residue 145, and/or an A or a V at position 188.
  • Percent (%) amino acid sequence identity with respect to a polypeptide is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B can be calculated as follows:
  • Variants may also include sequences added to the reference polypeptide to facilitate purification, to improve metabolic half-life or to make the polypeptide easier to identify, for example, an Fc region, a His-tag, and/or a PEGylation sequence.
  • fragment includes smaller portions of a KLK1 polypeptide that retain the activity of a KL 1 polypeptide. Fragments includes, for example, a KLK1 polypeptide fragment that ranges in size from about 20 to about 50, about 20 to about 100, about 20 to about 150, about 20 to about 200, or about 20 to about 250 amino acids in length. In other embodiments, a KLK1 polypeptide fragment ranges in size from about 50 to about 100, about 50 to about 150, about 50 to about 200, or about 50 to about 250 amino acids in length.
  • a KLK1 polypeptide fragment ranges in size from about 100 to about 150, about 100 to about 200, about 100 to about 250, about 150 to about 175, about 150 to about 200, or about 150 to about 250 amino acids in length. In other illustrative embodiments, a KLK1 polypeptide fragment ranges in size from about 200 to about 250 amino acids in length. Certain embodiments comprise a polypeptide fragment of a full-length KLK1 of about, up to about, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more contiguous amino acid residues. In some embodiments, a fragment may have residues 25-262 or residues 78-141 of a preproprotein sequence. In some embodiments, a fragment may be any such fragment size, as described above, of SEQ D NO:l or SEQ ID NO:2.
  • a fragments or variant of a L 1 polypeptide may retain the enzymatic capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen.
  • an active variant or fragment retains serine protease activity of a KLK1 polypeptide that releases kallidin from a higher molecular weight precursor such as kininogen, or that cleaves a substrate similar to kininogen such as D-val- leu-arg-7 amido-4-trifluoromethylcoumarin to release a colorimetric or fluorometric fragment.
  • a “wild type” or “reference” sequence or the sequence of a "wild type” or “reference” protein/polypeptide may be the reference sequence from which variant polypeptides are derived through the introduction of changes.
  • the "wild type” amino acid sequence for a given protein is the sequence that is most common in nature.
  • a “wild type” gene sequence is the polynucleotide sequence for that gene which is most commonly found in nature. Mutations may be introduced into a "wild type” gene (and thus the protein it encodes) either through natural processes or through human induced means.
  • human KLK may be used.
  • hKLKl may be, for example, isolated from human urine.
  • the KLKl may be porcine KLKl that has been isolated from the pig pancreas.
  • porcine KLKl preparations may have contamination with other porcine proteins, and have been formulated and administered orally in humans.
  • porcine KLKl has 67% amino acid homology with human KLKl, and administration of porcine KLKl into a human patient risks eliciting an uirmune reaction against porcine KLKl .
  • KLKl polypeptides described herein may be prepared by any suitable procedure known to those of skill in the art, including recombinant techniques.
  • KLKl may be isolated and/or purified from a source, such as, for example, urine.
  • the KLKl is not KLKl isolated from urine.
  • KLKl may be prepared by a procedure including one or more of the steps of: preparing a construct comprising a polynucleotide sequence that encodes a rhKLKl and that is operably linked to a regulatory element; introducing the construct into a host cell; culturing the host cell to express the rhKLKl; and isolating the rhKLKl from the host cell.
  • the construct and expression system may be such that the mature or active rhKLKl is expressed from the host cell.
  • the rhKLKl may be expressed in an inactive form, such as a propeptide, and the rhKLKl serine protease activity may be activated (for example, by removing the "pro" sequence) after the rhKLKl is isolated form the host cell.
  • a nucleotide sequence encoding the polypeptide, or a functional equivalent may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of expression vector/host systems are known and may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • plant cell systems transformed with virus expression vectors e.g
  • a non- mammalian cell expression system such as bacteria
  • a process would need to be used to add glycan groups to the rhKLKl, such as genetically engineered cells that express the enzymes required for mammalian style glycosylation.
  • a number of viral-based expression systems are generally available.
  • sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan and Shenk, 1984, PNAS USA; 81 :3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RS V) enhancer, may be used to increase expression in mammalian host cells.
  • RS V Rous sarcoma virus
  • Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells sub-cloned for growth in suspension culture, Graham et al., 1977, J Gen Virol; 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, 1980, BiolReprod; 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (Wl 38, ATCC
  • Hep G2 human liver cells
  • MMT 060562 mouse mammary tumor
  • TR1 cells (Mather et al, 1982, Annals NY Acad Sci; 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., 1980, PNAS USA; 77:4216)); and myeloma cell lines such as NSO and Sp2/0.
  • KLKl polypeptide products of cell culture expression in vertebrate (e.g., mammalian and avian) cells may be further characterized by freedom from association with human proteins or other contaminants, which may be associated with KLKl in its natural mammalian cellular environment or in extracellular fluids such as plasma or urine.
  • Polypeptides of the invention may also include an initial methionine amino acid residue (at position-1).
  • Certain embodiments therefore include host cells (e.g., eukaryotic host cells such as CHO cells, 293 cells) that comprise a recombinant or introduced polynucleotide that encodes a KLKl polypeptide described herein, such as the polypeptide of SEQ ID NO:l or SEQ ID NO:2. Also included are host cells that comprise a polynucleotide that encodes recombinant (e.g., non-naturally occurring) KLK-1 polypeptide described herein, such as the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • host cells e.g., eukaryotic host cells such as CHO cells, 293 cells
  • host cells that comprise a polynucleotide that encodes recombinant (e.g., non-naturally occurring) KLK-1 polypeptide described herein, such as the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • the cell culture expressed KLKl polypeptides of the present invention may be isolated and purified by using, e.g., chromatographic separations or immunological separations involving monoclonal and/or polyclonal antibody preparations, or using inhibitors or substrates of serine proteases for affinity chromatography.
  • the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO:2 list the sequence for pre-pro KLKl. If the gene coding for either of these sequences is expressed in mammalian cells, the 17-amino acid signal peptide (residues 1- 18) should result in the KLKl polypeptide to be secreted by the cell, and the signal peptide removed by the cell.
  • a gene encoding KLKl may be generated in which the signal sequence is omitted or replaced with another sequence.
  • the 7 amino acid pro- sequence (residues 19-24) will inhibit the serine protease activity of the KLKl and may be removed to allow activity of the mature KLKl polypeptide.
  • the pro- sequence may be removed after the KLKl polypeptide is isolated, for example by exposing the pro-KLKl to trypsin under conditions that will allow cleavage of the pro- sequence, or by generating a gene encoding KLKl in which the pro-sequence omitted or replaced with another sequence.
  • KLKl polypeptides described herein may be "labeled" by covalent association with a detectable marker substance (such as, for example, radiolabels such as I 125 or P 32 and nonisotopic labels such as biotin) to provide reagents useful in detection and quantification of KLKl in solid tissue and fluid samples such as blood or urine.
  • a detectable marker substance such as, for example, radiolabels such as I 125 or P 32 and nonisotopic labels such as biotin
  • hKLKl polypeptides may be produced by direct peptide synthesis using solid-phase techniques (see, for example, Merrifield, 1963, J Am Chem Soc; 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • various fragments may be chemically synthesized separately and combined using chemical methods to produce the desired polypeptide.
  • cell-free expression of proteins typically utilize purified R A polymerase, ribosomes, tR A and ribonucleotides; these reagents may be produced by extraction from cells or from a cell-based expression system. Purity. Determinations of the purity of a composition of the present invention may include, but are not limited to, determination so endotoxin, host cell protein, host cell DNA, and/or percentage single peak purity by SEC HPLC.
  • HCPs host cell proteins
  • the host cells used for recombinant expression may range from bacteria and yeast to cell lines derived from mammalian or insect species.
  • the cells contain hundreds to thousands of host cell proteins (HCPs) and other biomolecules that could contaminate the final product.
  • the HCP may be secreted along with the protein of interest, or released by accidental lysing of the cells, and may contaminate the protein of interest.
  • Two types of immunological methods may be applied to HCP analysis: Western blotting (WB) and immunoassay (IA), which includes techniques such as ELISA and sandwich immunoassay or similar methods using radioactive, luminescent, or fluorescent reporting labels.
  • Compositions of the present invention may include host cell protein of less than about 500, less than about 400, less than about 300, less than about 200, less than about 100 or less than about 50 ng/mg total protein.
  • compositions of the present invention may include host cell deoxyribonucleic acid (DNA) of less than about 100, less than about 90, less than about 80, less than about 70, less than about 60, less than about 50, less than about 40, less than about 30, less than about 20, or less than about 10 pg/mg total protein.
  • DNA host cell deoxyribonucleic acid
  • Endotoxin testing Endotoxin is extremely potent, is heat stable, passes sterilizing membrane filters and is present everywhere bacteria are or have been present.
  • An Endotoxin Unit (EU) is a unit of biological activity of the USP Reference Endotoxin Standard.
  • the bacterial endotoxins test is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate (white blood cells) from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). Limulus amebocyte lysate (LAL) reagent, FDA approved, is used for all USP endotoxin tests.
  • Method A the gel-clot technique, which is based on gel formation
  • Method B the turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate
  • Method C the chromogenic technique, based on the development of color after cleavage of a synthetic peptide- chromogen complex.
  • Photometric tests require a spectrophotometer, endotoxin-specific software and printout capability.
  • the simplest photometric system is a handheld unit employing a single-use LAL cartridge that contains dried, pre-calibrated reagents; there is no need for liquid reagents or standards.
  • the FDA-approved unit is marketed under the name of ENDOSAFE®- PTSTM The device requires about 15 minutes to analyze small amounts of sample, a 25 ⁇ , aliquot from CSP diluted in a sterile tube, and to print out results.
  • gel-clot methods require a dry-heat block, calibrated pipettes and thermometer, vortex mixer, freeze-dried LAL reagents, LAL Reagent Water (LRW) for hydrating reagents and depyrogenated glassware.
  • diluted sample and liquid reagents require about an hour for sample and positive- control preparation and an hour's incubation in a heat block; results are recorded manually.
  • LRW LAL Reagent Water
  • SEC Size-exclusion chromatography
  • the "purity" of a KLK1 polypeptide in a composition may be specifically defined.
  • certain compositions may include a hKLKl polypeptide that is at least about 80, at least about 85, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, or 100% pure, including all decimals in between, as measured, for example and by no means limiting, by high pressure liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.
  • HPLC high pressure liquid chromatography
  • compositions are also substantially free of aggregates (greater than about 95% appearing as a single peak by SEC HPLC). Certain embodiments are free of aggregates with greater than about 96%, about 97%, about 98%, or about 99%, appearing as a single peak by SEC HPLC.
  • a KLKl composition for parenteral administration is substantially pure, as determined by one or more of the following determinations of purity: less than about 1 EU endotoxin/mg protein, less that about 100 ng host cell protem/mg protein, less than about 10 pg host cell DNA/mg protein, and/or greater than about 95% single peak purity by SEC HPLC.
  • Naturally occurring KLKl has three potential N-linked glycosylation sites at position 78, 84 and 144 (relative to SEQ ID NO. 1). However, the N-linked glycosylation sites at position 78, 84 and 144 (relative to SEQ ID NO. 1). However, the N-linked glycosylation sites at position 78, 84 and 144 (relative to SEQ ID NO. 1). However, the N-linked glycosylation sites are three potential N-linked glycosylation sites at position 78, 84 and 144 (relative to SEQ ID NO. 1). However, the N-linked
  • glycosylation site at position 144 is only glycosylated on 60%» of the KLKl polypeptides. Therefore, naturally occurring KLKl (isolated from urine) is a 40:60 mixture of double to triple glycosylated KLKl (see, for example, WO/1989/000192, Lu et al., Protein
  • the KLKl is a composition as described in more detail in International Application No. PCT/CA2013/050425, which is herein incorporated by reference in its entirety.
  • the KLKl is a composition comprising a triple glycosylated isoform of a KLKl polypeptide and a double glycosylated isoform of a KLKl polypeptide, wherein the triple
  • glycosylated isoform of the KLKl polypeptide and the double glycosylated isoform of the KLKl polypeptide are present in the composition in a ratio ranging from about 45:55 to about 55:45.
  • the triple glycosylated isoform includes N-linked glycans at amino acid residues 78, 84, and 141 of KLKl, as defined by SEQ ID NO:l .
  • the double glycosylated isoform includes N-linked glycans at amino acid residues 78 and 84, but not at amino acid residue 141 of KLKl, as defined by SEQ ID NO:l.
  • the triple glycosylated isoform and the double glycosylated isoform of KLKl are present in the composition in a ratio of about 60:40 to about 40:60. In some aspects, the triple glycosylated isoform and the double glycosylated isoform of KLKl are present in the composition in a ratio of about 50:50. In some aspects of a composition of the present invention, one or more KLKl polypeptide(s) is a human tissue kallikrein-l (hKLKl) polypeptide.
  • hKLKl human tissue kallikrein-l
  • one or more KLKl polypeptide(s) includes amino acid residues 25-262 of SEQ ID NO:l or amino acid residues 25-262 of SEQ ID NO:2.
  • KLKl fusion protein includes a KLKl polypeptide or polypeptide fragment linked to either another KLKl -polypeptide (e.g., to create multiple fragments), to a non- KLK1 polypeptide, or to both.
  • a "non-KLKl polypeptide” refers to a "heterologous polypeptide” having an amino acid sequence corresponding to a protein which is different from KLKl protein, and which is derived from the same or a different organism.
  • the KLKl polypeptide of the fusion protein can correspond to all or a portion of a
  • a KLKl fusion protein includes at least one (or two) biologically active portion of a KLKl protein.
  • the polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N- terminus to N-terminus, or N-terminus to C-terrninus.
  • the polypeptides of the fusion protein can be in any order.
  • the fusion partner may be designed and included for essentially any desired purpose provided they do not adversely affect the therapeutic activity of the KLKl polypeptide.
  • a fusion partner may comprise a sequence that assists in expressing the KLKl protein (an expression enhancer) at higher yields than the native recombinant protein.
  • Other fusion partners may be selected so as to increase the solubility of the KLKl protein or to enable the protein to be targeted to desired tissues.
  • the disclosure provides nucleic acids encoding a KLK1 polypeptide.
  • DNA sequences encoding a human KLK1 polypeptide have been isolated and characterized.
  • human DNA may be utilized in eukaryotic and prokaryotic expression systems to provide isolatable quantities of KLK1 protein having biological and immunological properties of naturally-occurring KLK1 as well as in vivo and in vitro biological activities, in particular therapeutic activity or serine protease activity, of naturally-occurring KLK1.
  • an isolated nucleic acid codes for a KLK1 polypeptide that has serine protease activity and has at least about 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% amino acid sequence identity with a reference sequence, such as the full length, propeptide or mature KLK1 having a sequence of SEQ ID NO:2 s or which optionally retains a SNP that encodes an El 45 substitution, an A188 substitution, or both, relative to SEQ ID NO: 1.
  • Illustrative of the present invention are cloned DNA sequences of human species origins and polypeptides suitably deduced therefrom which represent, respectively, the primary structural conformation of KL 1 of human species origins.
  • Insulin is a polypeptide composed of 51 amino acids which are divided over 2 amino acid chains: the A chain, with 21 amino acids, and the B chain, with 30 amino acids. The chains are linked together by 2 disulfide bridges.
  • insulin preparations may use not only a naturally occurring insulin, but also, insulin derivatives and insulin analogs.
  • an insulin includes not only unmodified insulins but also insulin analogs, insulin derivatives, and insulin metabolites.
  • the compositions of the invention comprise one or more independently selected from the group consisting of insulins (e.g., unmodified insulins), insulin analogs, insulin derivatives, and insulin metabolites, and any desired combinations thereof.
  • the at least one insulin may independently be selected from the group consisting of bovine insulins, analogs, derivatives, and metabolites thereof, porcine insulins, analogs, derivatives, and metabolites thereof, and human insulins, analogs, derivatives, and metabolites thereof.
  • the at least one insulin is independently selected from human insulins, analogs, derivatives, and metabolites thereof.
  • an insulin of the invention may be selected independently from unmodified insulins, more particularly from bovine insulins, porcine insulins, and human insulins.
  • Insulin derivatives of the invention are derivatives of a naturally occurring insulin and/or an insulin analog, which are obtained by chemical modification.
  • the chemical modification may consist, for example, in the addition of one or more defined chemical groups onto one or more amino acids.
  • Insulin analogs are analogs of naturally occurring insulins, namely human insulin or animal insulins, which differ by replacement of at least one naturally occurring amino acid residue by other amino acids and/or addition/deletion of at least one amino acid residue from the corresponding, otherwise identical, naturally occurring insulin.
  • the amino acids in question may also be amino acids which do not occur naturally.
  • Insulin derivatives are derivatives of naturally occurring insulin or an insulin analog which are obtained by chemical modification.
  • the chemical modification may consist, for example, in the addition of one or more defined chemical groups and to one or more amino acids.
  • the activity of insulin derivatives and insulin analogs is somewhat altered as compared with human insulin.
  • Insulin analogs with an accelerated onset of action include any of those that are described in EP 0 214 826, EP 0 375 437, EP 0 678 522, EP 0 885 961, EP 0 419 504, WO 92/00321, German patent applications 10 2008 003 568.8 and 10 2003 003 566.1, and EP-A 0 368 187 may be part of the compositions of the invention.
  • EP 0 214 826 relates, among other things, to replacements of B27 and B28.
  • EP 0 678 522 describes insulin analogs which have different amino acids in position 1329, preferably proline, but not glutamic acid.
  • EP 0 375 437 encompasses insulin analogs with lysine or arginine in 1328, which may optionally also be modified in B3 and/or A21. An accelerated activity is also exhibited by the insulin analogs described in EP-A-0 885 961.
  • EP 0 419 504 discloses insulin analogs which are protected from chemical modifications by modification of asparagine in B3 and of at least one further amino acid in positions A5, Al 5, Al 8 and/or A21.
  • WO 92/00321 describes insulin analogs in which at least one amino acid in positions B1-B6 has been replaced by lysine or arginine. Such insulins, according to WO 92/00321, have an extended activity. A delayed activity is also exhibited by insulin analogs described in EP-A 0 368 187 and by the insulin analogs described in German patent applications 10 2008 003 568.8 and 10 2008 003 566.1.
  • the insulin preparations of naturally occurring insulins for insulin replacement that are on the market differ in the origin of the insulin (e.g., bovine, porcine, human insulm) and also in their composition, whereby the profile of action can be influenced (onset of action and duration of action).
  • the profile of action can be influenced (onset of action and duration of action).
  • an insulin of the present invention is insulin glargine (Gly(A21)-Arg(B31)-Arg(B32) human insulin), with an extended duration of action.
  • Insulin glargine is injected as an acidic, clear solution and, on account of its solution properties in the physiological pH range of the subcutaneous tissue, is precipitated as a stable hexamer associate.
  • Insulin glargine (LANTUSTM) is injected once daily and is notable over other long-activity insulins for its flat serum profile and the associated reduction in the risk of nocturnal hypoglycemias.
  • the specific preparation of insulin glargine that leads to a prolonged duration of action is characterized by a clear solution with an acidic pH.
  • Insulin glargine marketed by Sanofi-Aventis under the name Lantus, is a long- acting basal insulin analogue, given once daily to help control the blood sugar level of those with diabetes. It consists of microcrystals that slowly release insulm, giving a long duration of action of 18 to 26 hours, with a "peakless" profile (according to the Lantus package insert). Pharmacokinetically, it resembles basal insulin secretion of non-diabetic pancreatic beta cells. Sometimes, in type 2 diabetes and in combination with a short acting sulfonylurea (drugs which stimulate the pancreas to make more insulin), it can offer moderate control of serum glucose levels. In the absence of endogenous insulin— type 1 diabetes, depleted type 2 (in some cases) or latent autoimmune diabetes of adults in late stage— Lantus needs the support of fast acting insulin taken with food to reduce the effect of prandially derived glucose.
  • Insulin glargine have substitution of glycine for asparagine at A21 and two arginines added to the carboxy terminal of B chain.
  • the arginine amino acids shift the isoelectric point from a pH of 5.4 to 6.7, making the molecule more soluble at an acidic H, allowing for the subcutaneous injection of a clear solution.
  • the asparagine substitution prevents deamidization of the acid-sensitive glycine at acidic pH. In the neutral subcutaneous space, higher-order aggregates form, resulting in a slow, peakless dissolution and absorption of insulin from the site of injection. It can achieve a peakless level for at least 24 hours.
  • An insulin analog of the invention may be Gly(A21)-Arg(B31)-Arg(B32) human insulin (insulin glargine, Lantus); Arg(A0)-His(A8)-Glu(A15)-Asp(A18)-Gly(A21)- Arg(B31)-Arg(B32) human insulin amide, Lys(B3)-Glu(B29) human (insulin;
  • Lys Pro human insulin insulin lyspro
  • B28 Asp human insulin insulin aspart
  • AlaB26 human insulin des(B28-B30) human insulin
  • des(B30) human insulin insulin detemir
  • An insulin derivative of the invention may be B29-N-myristoyl-des(B30) human insulin, B29-N-palmitoyl-des(B30) human insulin, B29-N-myristoyl human insulin, B29- N-palmitoyl human insulin, B28-N-myristoyl Lys B28 Pro B29 human insulin, B28-N- palmitoyl-Lys Pro human insulin, B30-N-myristoyl-ThrB29LysB30 human insulin, B30-N-palmitoyl-Thr B29 Lys B3 ° human insulin.
  • B29-N-(N-palmitoyl-Y-glutamyl)- des(B30) human insulin B29-N-(N-lithocholyl-Y-glutamyl)-des(B30) human insulin, B29-N-(E-carboxyheptadecanoyl)-des(B30) human insulin, and ⁇ 29- ⁇ -( ⁇ - carboxyheptadecanoyl) human insulin.
  • An insulin derivative of the invention may be Gly(A21)-Arg(B31)-Arg(B32) human insulin, LysB28ProB29 human insulin (insulin lyspro), B28 Asp human insulin (insulin aspart), B29Lys(s-tetradecanoyl), desB30 human insulin (insulin detemir).
  • One embodiment of the invention is a medicament as described herein comprising at least one insulin independently selected from LysB28ProB29 human insulin (insulin lyspro), B28 Asp human insulin (insulin aspart), B29Lys(s-tetradecanoyl), desB30 human insulin (insulin detemir), and insulin glargine (Gly(A21)-Arg(B31)-Arg(B32) human insulin), and comprising KLK1.
  • a further particularly preferred subject is a medicament as described herein comprising insulin glargine (Gly(A21)-Arg(B31)- Arg(B32) human insulin) and KLK1.
  • compositions of the invention may contain about 60 to about 6000 nmol/ml, preferably about 240 to about 3000 nmol/ml of an insulin. Depending on the insulin used, a concentration of 240 to 3000 nmol ml corresponds approximately to a
  • a formulation of the present invention may include one or more physiologically acceptable carriers.
  • Such carriers include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., Edition 21 (2005).
  • physiologically-acceptable or “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • aqueous composition that contains a L 1 protein as an active ingredient is well understood in the art.
  • injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparations can also be emulsified.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disacchaiides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; and/or salt-forming counterions such as sodium.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • Type 2 diabetes contrasts with type 1 diabetes in that there is not always a deficiency of insulin, but in a large number of cases, especially at the advanced stage, treatment with insulin, where appropriate in combination with an oral antidiabetic, is considered the most advantageous form of therapy.
  • an improved therapy of diabetes must be aimed primarily at keeping blood glucose as closely as possible within the physiological range.
  • intensified insulin therapy this is to be achieved by means of injections, several times a day, of fast-acting and slow-acting insulm preparations.
  • Fast- acting formulations are given at mealtimes in order to compensate the postprandial rise in blood glucose.
  • Long-acting basal insulins are intended to ensure the basic supply of insulin especially during the night, without leading to hypoglycemia.
  • fast acting insulin is injected prior to every meal, and thus requires multiple injections per day.
  • the threat of hypoglycemia is always present, especially if a meal is missed after administering the fast acting insulin, (for example, a person in need of glucose control administers fast acting insulin but misses a meal).
  • the person in need of glucose control may accidently inject too much fast acting insulin, or not ingest sufficient calories for the amount of inj ected insulin, resulting in the person becoming
  • hypoglycemic Alternatively, there is also a threat of hyperglycemia.
  • the person may miss an injection prior to a meal, such as forgetting to inject the fast acting insulin, forgetting to bring their fast acting insulin, or not injecting sufficient amount of insulin for the meal.
  • the present invention overcomes the above described limitations to
  • the present invention improves control of blood glucose levels in diabetic patients, especially after a meal or under fasting conditions, lowers the risk of hypoglycemia, and/or decreases the need for injections of fast acting insulin before every meal.
  • the amount of insulin to be administered is adjusted to the individual requirement of the individual diabetes patients. Individual patients generally need different amounts of insulin and/ KL 1.
  • the predetermined dose is administered by administering a defined amount of a composition having a defined concentration.
  • a therapeutically effective amount of long-acting insulin is administered to a patient in need of glucose control.
  • the therapeutically effective amount of insulin may be calculated using various algorithms known to persons skilled in the art, and specifically described in package inserts for approved long-acting insulins. Insulin regimens should be designed taking lifestyle and meal schedule into account. The algorithms can only provide basic guidelines for initiation and adjustment of insulin.
  • a therapeutically effective amount of KLK1 includes an amount that lowers fasting glucose or that increases glucose tolerance, or other indicator.
  • a therapeutically effective amount of L 1 includes an amount that lowers fasting glucose or that increases glucose tolerance, or other indicator.
  • an effective amount of KLK1 administered parenterally per dose includes about 0.025 IU/kg (about 0.1 ⁇ g/kg) to about 250 lU/kg (about 1000 ⁇ g/kg) of patient body weight, although, as noted above, this is subject to therapeutic discretion.
  • a dose may be about 0.2 IU/kg to about 25 IU/kg (about 0.8 ⁇ g/kg to about 100 ⁇ g/kg), or about 0.2 IU/kg to about 10 IU/kg (about 0.8 ⁇ g kg to about 40 ⁇ g/kg) of patient body weight.
  • the conversion from units to micrograms assumes a specific activity of KLK1 of 250 IU/mg.
  • the specific activity of KLK1 may differ depending on various parameters.
  • the specific activity of KLK1 may be determined as described herein below.
  • the dose of KLK1 may be about 0.1 ⁇ g/kg to about 100 ⁇ g/kg; about 0.5 ⁇ g/kg to about 75 ⁇ g kg; about 1 ⁇ g kg to about 50 ⁇ g kg; about 1 ⁇ g kg to about 25 ⁇ g kg; about 5 ⁇ g kg to about 50 ⁇ g kg; about 5 ⁇ /kg to about 25 ⁇ g/kg; about 10 ⁇ g/kg to about 50 ⁇ g/kg.
  • the dose of KLK1 may be about 0.1 ⁇ g/kg; about 0.2 ⁇ g/kg; about 0.3 ⁇ g/kg; about 0.4 ⁇ g kg; about 0.5 ⁇ g/kg; about 0.6 ⁇ g/kg; about 0.7 ⁇ g/kg; about 0.8 ⁇ g kg; about 0.9 ⁇ g kg; about 1 ⁇ g/kg; about 1.5 ⁇ g/kg about 2 ⁇ g/kg; about 3 ⁇ g/kg; about 4 ⁇ g/kg; about 5 ⁇ g/kg; about 6 g kg; about 7 ⁇ g/kg; about 8 ⁇ g/kg; about 9 ⁇ g/kg; about 10 ⁇ g/kg; about 11 ⁇ g/kg; about 12 ⁇ g/kg; about 13 ⁇ g/kg; about 14 ⁇ g kg; about 15 ⁇ g k ; about 16 ⁇ g/kg; about 17 ⁇ g/kg; about 18 ⁇ g/kg; about 19 ⁇ g/kg; about 20 ⁇ g/kg; about 25
  • the dose of KLK1 may be increased if a patient's blood glucose is elevated above a predetermine levels (for example, greater than about 180 mg/dl or greater than about 200 mg/dl) immediately after meal or during a meal tolerance test or an OGTT or the KLK1 dose may be decreased if postabsorptive or fasting blood glucose levels are too low (for example, below approximately 80 mg/dl or below approximately 60 mg/dl).
  • a predetermine levels for example, greater than about 180 mg/dl or greater than about 200 mg/dl
  • postabsorptive or fasting blood glucose levels are too low (for example, below approximately 80 mg/dl or below approximately 60 mg/dl).
  • the KLK1 dosage amount can be increased, merely by way of example, by about l.lx, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, lOx, 1 x, 20x or more, relative to the previous dosage.
  • the dosage frequency can be increased, merely by way of illustration, by about 1, 2, 3, 4, 5 or more dosages per day, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dosages per week, relative to the previous dosing schedule.
  • the dose of KLKl may also be decreased by the amounts indicated above, such as a 0.95x, 0.90, 0.85, 0.80, 0.75, 0.70, 0.65, 0.60, etc.
  • the dosage amount can be increased or decreased separately or in combination with the dosage frequency, and vice versa, optionally until a desired level or range of glucose levels or other treatment indicators are achieved.
  • Appendix D Converting animal doses to human equivalent doses.
  • a human equivalent dose is 1/12 the mouse dose.
  • a mouse dose 0.08 IU of KLKl per mouse is described here.
  • the mice used in the instant study weighted approximately 30 grams, equating to a mouse dose of 2.7 IU/kg.
  • the human equivalent dose (1/12) would be approximately 0.22 IU/kg.
  • a mouse dose of about 0.4 IU (about 16 ⁇ g) of KLKl per mouse equates to approximately 13.3 IU/kg (53.2 ⁇ g kg) in mice.
  • the human equivalent dose would be approximately 1.11 IU/kg (4.4 pg/kg).
  • a mouse dose 2 IU (8 ⁇ g) of KLKl per mouse equates to approximately 66. 7 IU/kg (267 ⁇ g/kg) in mice.
  • the human equivalent dose would be approximately 5.55 IU kg (22.2 pg/kg).
  • KLKl may also be administered daily, or may be administered less frequently than once a day.
  • KLKl may be administered every 2 days, every three days, every four days, or every five days.
  • KLKl may be administered every 1-7 days, every 1-5 days, every 2-7 days, every 2-5 days, every 2-4 days, every 2-3 days, every 3-4 days, every 3-5 days, or every 3-7 days.
  • the frequency of dosing of KLKl will depend on maintaining KLKl in the therapeutic range or therapeutic concentration (below the minimum toxic concentration and above the minimum effective concentration). This may require dosing KLKl every second day, every third day, every fourth day, every fifth day, every sixth day, or once a week.
  • a result of this is that a composition which comprises insulin and KLKl at the same time allows the administration of only one particular proportion of insulin and KLKl .
  • the active compounds may be formulated in a composition and provided in a device, as for example in a prefilled syringe.
  • a system of this kind does allow the dosing of the combination, but only in a fixed portion of the active compounds, as is present in the composition.
  • this is a disadvantage for the combination of an insulin with KLK1, since different amounts of the insulins and LK1 have to be administered, according to the therapeutic requirement.
  • two active compounds can be administered in two separate formulations, each comprising one of the two active compounds, which are injected independently of one another each with one device (e.g., prefilled syringes).
  • an injection therapy such as the injection of insulin
  • patient compliance is a key prerequisite for the success of the therapy.
  • pain, needle-phobia, and the carrying facility for the injection apparatus are a problem, which can lead to reduced compliance. If the patient is to use two separate devices for injection, then these problems multiply.
  • U.S. Pat. No. 4,689,042, U.S. Pat. No. 5,478,323, U.S. Pat. No. 5,253,785, and WO 01/02039 describe devices for the simultaneous administration of two or more injectable products to a patient. These devices comprise two containers each containing one composition. In these devices the two compositions are injected via a needle. This does make it possible to overcome the disadvantages produced by the use of two separate devices. As a result of the mixing process, there is a dilution in the concentrations of the two active compounds. This may impact adversely on the pharmacokinetics.
  • the pharmacokinetics of insulin is influenced by the dilution of the insulin in the administered composition.
  • the concentration of insulin ought to be kept constant as for as possible. Dosing ought to take place essentially via the volume of the insulin composition administered. This is also true for the administration of a combination of insulin and KL 1.
  • this proviso can only be met if both substances are dosed in a fixed proportion to one another in one composition.
  • both substances are provided in separate compositions and are mixed for injection in a suitable device (e.g., from WO 01/02039), then a constant concentration of insulin can be realized only if the insulin composition is not substantially diluted by the composition of KLKl. This imposes limits on the possibility of independent dosing of insulin and of KLKl .
  • One conceivable solution would be to provide human KLKl in such a high concentration that the dosed addition of human KLKl produces not significant dilution of the insulin composition (e.g., not more than 10%).
  • Polypeptides such as insulins (e.g., insulin glargine, LANTUSTM) or human KLKl cannot be concentrated ad infinitum.
  • insulins e.g., insulin glargine, LANTUSTM
  • human KLKl cannot be concentrated ad infinitum.
  • the solubility of proteins is limited, and high concentrations of protein may alter the flow characteristics of the solution.
  • the most important problem for the use of solutions with a high concentration of active compound is the dosing accuracy. At high
  • the invention provides a medicament comprising at least one insulin and KLKl, the medicament being formulated and/or compounded in such a way that it comprises the insulin and KL 1 each in a predetermined amount and can be administered in a dose adapted to the individual requirement of a patient.
  • the medicament of the invention may be used in particular for the treatment of a disease or condition that benefits from the administration of insulin in a patient in need thereof, such a disease or condition includes, but is not limited to, diabetes mellitus, more particularly type 1 or type 2 diabetes.
  • the medicament of the invention allows the blood glucose concentration to be adapted more effectively to normoglycemic levels in the case of patients with diabetes, more particularly type 1 or type 2 diabetes.
  • the medicament is used preferably to adjust the fasting, postprandial and/or postabsorptive blood glucose concentration of patients with diabetes, more particularly patients with type 1 or type 2 diabetes. More preferably the medicament of the invention is used to adjust the postprandial and/or postabsorptive blood glucose concentration of patients with diabetes, more particularly patients with type 1 or type 2 diabetes.
  • Adjustment in this context means that normoglycemic blood glucose concentrations are substantially achieved or at least an approximation thereto is obtained.
  • normoglycemic levels are meant more particularly blood glucose concentrations in the normal range (breadth of fluctuation 60-140 mg/di, corresponding to 3.3 to 7.8 mmol/1). This range of fluctuation encompasses blood glucose concentrations under fasting conditions, postprandial conditions, and postabsorptive conditions.
  • Postprandial and postabsorptive are terms familiar to the person skilled in the field of diabetology, postprandial is used herein to refer more particularly to the phase after a meal and/or after glucose loading in an experiment. This phase is characterized more particularly in a healthy individual by an increase and fall again in the concentration of glucose in the blood.
  • Postabsorptive, or postabsorptive phase is used herein to refer more particularly to the phase which follows the postprandial phase.
  • the postprandial phase typically ends up to 4 h after the meal and/or glucose loading.
  • the postabsorptive phase lasts typically for up to 8 to 16 h.
  • the medicament of the invention is also used preferably for improving glucose tolerance in the treatment of a patient with diabetes, more particularly with a type 1 or type 2 diabetes. Improving the glucose tolerance means that the medicament of the invention lowers the postprandial blood glucose concentration. Improving the glucose tolerance is also taken to mean that the medicament of the invention lowers the postabsorptive blood glucose concentration. Lowering means more particularly that the blood glucose concentration substantially reaches normoglycemic values or at least is approximated thereto.
  • the medicament of the invention is able to lower the risk of hypoglycemia, which may occur, for example, in the postabsorptive phase.
  • the medicament of the invention is used preferably for lowering the risk of hypoglycemia in the treatment of a patient with diabetes, more particularly with a type 1 or type 2 diabetes, it being possible for the hypoglycemia to occur more particular in the postabsorptive phase.
  • the medicament of the invention may maintain the function of the pancreatic ⁇ - cells.
  • the medicament of the invention is used preferably for preventing a loss of function of the pancreatic ⁇ -cells in a patient with diabetes, more particularly with a type 1 or type 2 diabetes.
  • the loss of function of the ⁇ -cells may be caused more particularly by apoptosis.
  • the medicament of the invention can be used in order to treat more than one of the preferred indications described therein in a patient with a disease or condition that benefits form the administration of insulin, more particularly with a type 1 or type 2 diabetes. Accordingly the present invention encompasses not only the individual preferred indications but also arbitrary combinations of the indications.
  • the medicament of the invention can therefore be used to treat one or more of the herein- described indications in patients with diabetes, more particularly of patients with type 1 or type 2 diabetes, for the purpose, for example, of adjusting the fasting, postprandial and/or postabsorptive blood glucose concentration, for improving glucose tolerance, for preventing hypoglycemia, and for preventing a loss of function of the pancreatic ⁇ -cells. Preference is given to the adjustment of fasting, postprandial and/or postabsorptive blood glucose concentration, the improvement of glucose tolerance and/or the prevention of hypoglycemia.
  • the medicament of the invention can also be used for producing a medicinal product for treating one or more of the herein-described indications, as, for example, for adjusting the fasting, postprandial and/or postabsorptive blood glucose concentration, for improving glucose tolerance, for preventing hypoglycemia, and for preventing a loss of function of the pancreatic ⁇ -cells.
  • the at least one insulin and LK1 a variant or active fragment thereof may also be used for producing a medicinal product for treating one or more of the herein- described indications, as for example for adjusting the fasting, postprandial and/or postabsorptive blood glucose concentration, for improving glucose tolerance, for preventing hypoglycemia, and for preventing a loss of function of the pancreatic ⁇ -cells.
  • a formulation of the present invention may include one or more additional therapeutic agents.
  • the dose of insulin for an individual patient to a level selected from the range from about 15 to about 80 units of insulin and at the same time to dose KLK1 with an amount within the range from about 0.1 to about 100 IU/kg (about 0.4 g/k to about 400 ⁇ g/kg3.
  • substantially equal weight fractions of an active compound in two compositions means that one of the two compositions contains the active compound in a weight fraction which is, for example, not more than 10%, not more than 5%, not more than 1% or not more than 0.1%» higher than its weight fraction in the other composition.
  • the combination of KLK1 and an insulin according to the present invention significantly improves the glycemic control, in particular in patients as described herein, compared with a monotherapy using either KLK1 or an insulin alone, for example with a monotherapy of a long-acting insulin, such as insulin glargine.
  • the combination of KLK1 and insulin according to this invention may allow for a reduction of the dose of the insulin compared with a monotherapy of said insulin, for example with a monotherapy of a long-acting insulin, such as insulin glargine. With a reduction of the dose of the insulin any side effects associated with the therapy using said insulin may be prevented or attenuated.
  • a dose reduction is beneficial for patients which otherwise would potentially suffer from side effects in a therapy using a higher dose of one or more of the active ingredients, in particular with regard to side effect caused by the insulin. Therefore, the pharmaceutical composition and methods according to the present invention show reduced side effects, thereby making the therapy more tolerable and improving the patient's compliance with the treatment.
  • the efficacy of the insulin for example in a basal insulin therapy with a long acting insulin or with a short- or rapid acting insulin, including human insulin, via infusion with a pump, may be prolonged by a combined treatment with KLKl . Therefore the time interval between two applications, for example subcutaneous injections or infusions via a pump, of the insulin may be prolonged.
  • the dose of the long acting insulin, the dose of KLKl, the time interval between two applications of the long acting insulin and the time interval between the application of the long acting insulin and KLKl are chosen such that a good glycemic control is provided to the patient for a given time period, in particular for 24 hours.
  • this invention refers to patients requiring treatment or prevention, it relates primarily to treatment and prevention in humans, but the pharmaceutical composition may also be used accordingly in veterinary medicine in mammals.
  • the term "patient” covers adult humans (age of 18 years or older), adolescent humans (age 10 to 17 years) and children (age 6-9 years).
  • the methods and uses according to the present invention are advantageously applicable in those patients who show one, two or more of the following conditions: type 1 diabetes mellitus; need for treatment with insulin; and/or latent autoimmune diabetes with onset in adults (LAD A).
  • the methods and uses according to the present invention are advantageously applicable in those patients who are or shall be treated with an insulin, for example with insulin glargine or detemir insulin, in particular in patients diagnosed with type 1 diabetes mellitus, and show one, two or more of the following conditions, including the risk to develop such conditions: nocturnal and/or early morning
  • hypoglycemia hypoglycemic episodes; hyperglycemic episodes; cardiac or cerebral complications; retinopathy, in particular proliferative retinopathy; and/or injection site reactions, for example skin or subcutaneous tissue disorders.
  • ITT impaired glucose tolerance
  • IGF impaired fasting blood glucose
  • a method for improving glycemic control in patients, in particular in adult patients, with type 1 or type 2 diabetes mellitus as an adjunct to diet and exercise is provided.
  • the invention additionally provides a kit comprising a medicament of the invention.
  • the kit of the invention may be intended for use by medical staff or by persons without specialist medical training, more particularly by the patients themselves or helpers such as relatives.
  • the individual pharmaceutical compositions comprising the medicament of the invention are assembled in separate packs, and so the patient is able to select the composition appropriate to the current requirement and to administer an amount in line with that requirement.
  • the kit of the invention comprises, for example, the medicament of the invention in the form of a set of syringes, glass ampoules and/or pens which comprise a composition of the invention.
  • the medicament of the invention can be administered parenterally.
  • the medicament may be injected, with the possible use of injection systems with or without needles.
  • the medicament may be administered by inhalation.
  • liquid compositions may be inhaled, or the compositions can be inhaled in the form of powder.
  • the medicament of the invention may be administered as a spray, more particularly as a nasal spray.
  • the medicament of the invention may be administered by a transdermal system. The skilled worker is aware of these methods of administration and is able to formulate the medicament of the invention in such a way that it can be effectively administered by one of these methods of
  • compositions of the medicament of the invention are preferably liquid. It is preferred, furthermore, for the medicament of the invention to be
  • the present invention further provides a device for administering the medicament of the invention.
  • This device comprises the pharmaceutical compositions which are encompassed by the medicament of the invention, in separate containers, and allows the pharmaceutical compositions to be dosed independently of one another.
  • the device of the invention may be a device for parenteral administration.
  • the device of the invention may be a device for injection with or without needles.
  • the device may be a device for inhalation, in which case liquid compositions are inhaled, or the compositions can be inhaled in the form of powder.
  • the device may be a device for administering a spray, more particularly a nasal spray.
  • the device may be a transdermal administration system. It is preferred for the device of the invention to be a device for parenteral administration, more particularly an injection device.
  • Compounding is a term which is known to the skilled worker and which in pharmacology identifies the finishing treatment, such as portioning and packing, for example, of medicaments for use by the end user.
  • finishing treatment such as portioning and packing, for example, of medicaments for use by the end user.
  • compositions of the invention are packaged in a suitable way in a therapeutically effective amount to allow the herein-described selection of at least one of the
  • a parenteral administration preferably an injection, more preferably subcutaneous injection.
  • Suitable packaging is, for example, a syringe or a glass vessel with a suitable closure, from which, as required, individual therapeutically active doses can be taken.
  • injection pens for the administration of insulin comprising a container (e.g., a cartridge) which contains a pharmaceutical composition of the invention.
  • Formulating or “formulation M is a term which is known to the skilled worker and which, in the field of pharmacology, refers to the production of medicaments and medicament compositions, and their preparation with excipients. In the present specification “formulating” or “formulation” means more particularly that the
  • composition of the invention is provided in a suitable form which allows administration of a therapeutically effective amount of the active compounds. More particularly a formulation is intended for parenteral administration, preferably for injection, more preferably for subcutaneous injection.
  • KLKl The dosing of KLKl will depend on various factors, including the type and dose of insulin, the disease to be treated, other medications that the patient is taking, etc.
  • KLKl is also dependent on the specific activity of the KLKl protein. Dosages of KLKl are administered based on the number of units, which are converted into mg of protein.
  • KLKl is a serine protease which cleaves low-molecular- weight kininogen resulting in the release of kallidin (lys-bradykmin). This activity of KLKl may be measured in an enzyme activity assay by measuring either the cleavage of low-molecular- weight kininogen, or the generation of lys-bradykinin. Assays include examples wherein a labelled substrate is reacted with KLKl , and the release of a labelled fragment may be detected.
  • D-val- leu-arg-7 amido-4-trifluoromethylcoumarin D-VLR-AFC, FW 597.6 (Sigma, Cat # V2888 or Ana Spec Inc Cat # 24137.)
  • D-VLR-AFC D-val- leu-arg-7 amido-4-trifluoromethylcoumarin
  • fluorometric detection excitation 400 nm, emission 505 nm
  • Other methods and substrates may also be used to measure KLKl proteolytic activity.
  • KLKl activity measured in Units or Units/ml, may be determined by comparing the relative activity of a KLKl sample to the Kininogenase, Porcine standard acquired from the National Institute for Biological Standards and Control (NIBSC Product No. 78/543).
  • the assigned potency is 22.5 international units (IU) per 20 ⁇ g ampoule of porcine pancreatic kininogenase.
  • serial dilutions are made of the standard, and the activity in an unknown sample of KLKl is compared to the standard.
  • the rhKLKl has a specific activity of approximately 250 to 300 IU/mg, though specific activities of certain lots may be outside this range. However, the specific activity of rhKLKl may vary from lot to lot, and thus would need to be checked to determine the dosage in mg/kg of total mg of rhKLKl to administer.
  • the KLKl polypeptide is subcutaneously administered in an individual dose of at least about 20 pg/kg (0.02 mg/kg of 5 IU/kg), or in range of about 8 pg kg to about 400 pg/kg (0.008 to .40 mg/kg or 2 to 100 U/kg). If KLKl is required at a dose of 200 pg kg into a 90 kg patient, a total of 18.0 mg of rhKLKl is required. If the KLKl is formulated at 5 mg/mL, then a total of 3.6 mL would need to be injected, which is a large volume and could cause discomfort if injected subcutaneously. However, if the KLKl were formulated at 25 mg/mL, the total injection volume is 0.72 mL, which is within the recommended injection volume for subcutaneous delivery of 1.0 to 1.5 mL.
  • a dose of 500 pg/kg to a 90 kg individual equates to about 45 mg of KLKl .
  • the injection volume is 1.8 mL, which is above the recommended volume for subcutaneous injection.
  • the KLKl is formulated at 50 mg/mL, the injection volume is 0.9 mL or within the tolerable limit for subcutaneous injection into a human.
  • compositions of the present invention is that of liquid compositions suitable in particular for parenteral
  • composition of the present invention is suitable for injection once daily.
  • composition of the invention may comprise a suitable preservative.
  • suitable preservatives include phenol, m-cresol, benzyl alcohol and/or p' esters.
  • composition of the invention may further comprise a suitable buffer.
  • Buffer substances which can be used, particularly for setting a pH level between about 4.0 and 8.5, include, for example, sodium acetate, sodium citrate, sodium phosphate, etc.
  • physiologically unobjectionable dilute acids typically HC1
  • alkalis typically NaOH
  • concentrations of the buffers and also of corresponding salts are in the range of 5-250 mM, more preferably in the range of 10-100 mM.
  • a composition of the invention may comprise zinc ions. The concentration of the zinc ions is preferably in the range from 0 ⁇ g /ml to 500 ug/ml s more preferably from 5 ⁇ g to 200 ⁇ g of zinc/ml.
  • a composition of the invention may further comprise suitable isotonicity agents.
  • suitable examples include glycerol, dextrose, lactose, sorbitol, mannitol, glucose, NaCl, calcium compounds or magnesium compounds such as CaCl 2 , etc.
  • Glycerol, dextrose, lactose, sorbitol, mannitol, and glucose are typically in the range of 100-250 mM, NaCl in a concentration of up to 150 mM.
  • composition of the invention may further comprise other additives such as, for example, salts, which retard the release of at least one insulin.
  • the invention further provides a method of treating a patient with a kit or medicament of the invention as described herein.
  • body mass index or "BMI” of a human patient may be defined as the weight in kilograms divided by the square of the height in meters, such that BMI has units of kg/m 2 .
  • overweight may be defined as the condition wherein the individual has a BMI greater than or 25 kg/m 2 and less than 30 kg/m 2 .
  • overweight and pre- obese may be used interchangeably.
  • obesity may be defined as the condition wherein the individual has a BMI equal to or greater than 30 kg/m 2 .
  • the term obesity may be categorized as follows: class I obesity is the condition wherein the BMI is equal to or greater than 30 kg/m 2 but lower than 35 kg/m 2 ; class II obesity is the condition wherein the BMI is equal to or greater than 35 kg/m but lower than 40 kg/m ; class III obesity is the condition wherein the BMI is equal to or greater than 40 kg/m 2 .
  • normoglycemia may be defined as the condition in which a subject has a fasting blood glucose concentration within the normal range, greater than 70 mg/dL (3.89 mmol/L) and less than 100 mg/dL (5.6 mmol/L).
  • fasting has the usual meaning as a medical term.
  • hypoglycemia may be defined as the condition in which a subject has a fasting blood glucose concentration above the normal range, greater than 100 mg/dL (5.6 mmol/L).
  • hypoglycemia may be defined as the condition in which a subject has a blood glucose concentration below the normal range, in particular below 70 mg/dL (3.89 mmol/L) or even below 60 mg/dl.
  • postprandial hyperglycemia may be defined as the condition in which a subject has a 2 hour postprandial blood glucose or serum glucose concentration greater than 200 mg/dL (11.1 mmol/L).
  • IGF paired fasting blood glucose
  • a subject with "normal fasting glucose” has a fasting glucose concentration smaller than 100 mg/dl, i.e. smaller than 5.6 mmol/1.
  • ITT paired glucose tolerance
  • the abnormal glucose tolerance for example, the 2 hour postprandial blood glucose or serum glucose concentration, can be measured as the blood sugar level in mg of glucose per dL of plasma 2 hours after taking 75 g of glucose after a fast.
  • a subject with normal glucose tolerance has a 2 hour postprandial blood glucose or serum glucose concentration smaller than 140 mg/dl (7.8 mmol/L).
  • hyperinsulinemia may be defined as the condition in which a subject with insulin resistance, with or without euglycemia, has fasting or postprandial serum or plasma insulin concentration elevated above that of normal, lean individuals without insulin resistance, having a waist-to-hip ratio ⁇ 1.0 (for men) or ⁇ 0.8 (for women).
  • insulation-sensitizing As used herein, the terms “insulin-sensitizing,” “insulin resistance-improving,” or “insulin resistance-lowering” are synonymous and may be used interchangeably.
  • insulin resistance may be defined as a state in which circulating insulin levels in excess of the normal response to a glucose load are required to maintain the euglycemic state (Ford et al., 2002, JAMA: 287:356-9).
  • Methods of determining insulin resistance include, for example, the euglycaernic-hyperinsulinaemic clamp test.
  • the ratio of insulin to glucose may be determined within the scope of a combined insulin-glucose infusion technique. There is found to be insulin resistance if the glucose absorption is below the 25th percentile of the background population investigated (WHO definition). Rather less laborious than the clamp test are so called minimal models in which, during an intravenous glucose tolerance test, the insulin and glucose
  • the response of a patient with insulin resistance to therapy, insulin sensitivity, and/or hyperinsulinemia may be quantified by, for example, assessing the "homeostasis model assessment to insulin resistance (HOMA- IR)" score, a reliable indicator of insulin resistance ( atsuki et al., 2001, Diabetes Care; 24: 362-5). Further reference is made to methods for the determination of the HOMA- index for insulin sensitivity (Matthews et al., 1985, Diabetologia; 28: 412-19), of the ratio of intact proinsulin to insulin (Forst et al., 2003, Diabetes; 52(Suppl.
  • HOMA-IR score may be calculated with the formula (Galvin et al., 1992, Diabet eJ;9:921-8):
  • HOMA-IR [fasting serum insulin (uU/mL)] X [fasting plasma
  • the patient's triglyceride concentration is used, for example, as increased triglyceride levels correlate significantly with the presence of insulin resistance.
  • Patients with a predisposition for the development of IGT or IFG or type 2 diabetes are those having euglycemia with hyperinsulinemia and are by definition, insulin resistant.
  • a typical patient with insulin resistance is usually overweight or obese, but this is not always the case. If insulin resistance can be detected, this is a particularly strong indication of the presence of pre-diabetes.
  • a person have, for example, 2-3 times as high endogenous insulin production as a healthy person, without this resulting in any clinical symptoms.
  • the methods to investigate the function of pancreatic beta-cells are similar to the above methods with regard to insulin sensitivity, hyperinsulinemia or insulin resistance.
  • beta-cell function can be measured for example by determining a HOMA-index for beta-cell function (Matthews et al., 1985, Diabetologia; 28: 412-19), the ratio of intact proinsulin to insulin (Forst et al., 2003, Diabetes; 52(SuppLl): A459), the insulin/C -peptide secretion after an oral glucose tolerance test or a meal tolerance test, or by employing a hyperglycemic clamp study and/or minimal modeling after a frequently sampled intravenous glucose tolerance test (Stumvoll et al. ; 2001, Eur J Clin Invest; 31 : 380-81).
  • Pre-diabetes is the condition wherein an individual is pre-disposed to the development of type 2 diabetes.
  • Pre-diabetes extends the definition of impaired glucose tolerance to include individuals with a fasting blood glucose within the high normal range 100 mg/dL (Meigs, et al., 2003, Diabetes 52:1475-1484) and fasting hyperinsulinemia (elevated plasma insulin concentration).
  • the scientific and medical basis for identifying pre-diabetes as a serious health threat is laid out in a Position Statement entitled "The Prevention or Delay of Type 2 Diabetes” issued jointly by the American Diabetes Association and the National Institute of Diabetes and Digestive and Kidney Diseases (Diabetes Care 2002; 25:742-749).
  • insulin resistance is defined as the clinical condition in which an individual has a HOMA-IR score >4.0 or a HOMA-IR score above the upper limit of normal as defined for the laboratory performing the glucose and insulin assays.
  • treatment of patients who are insulin resistance (have an elevated HOMA-IR) with KLK1 may decrease the patient's insulin resistance (as measured by decreased HOMA-IR). Decreasing the HOMA-IR or decreasing insulin resistance in the patients may allow the patients to be more effectively treated with insulin.
  • Type 1 diabetes includes the condition in which a subject has, in the presence of autoimmunity towards the pancreatic beta-cell or insulin, a fasting blood glucose or serum glucose concentration greater than 125 mg/dL (6.94 mmol/L).
  • the blood sugar level of a diabetic will be in excess of 200 mg of glucose per dl_ ( 11.1 mmol/I) of plasma 2 hours after 75 g of glucose have been taken on an empty stomach, in the presence of autoimmunity towards the pancreatic beta cell or insulin.
  • 75 g of glucose are administered orally to the patient being tested after 10-12 hours of fasting and the blood sugar level is recorded immediately before taking the glucose and 1 and 2 hours after taking it.
  • the presence of autoimmunity towards the pancreatic beta-cell may be observed by detection of circulating islet cell autoantibodies ["type 1 A diabetes mellitus”], for example, at least one of: GAD65 [glutamic acid decarboxylase-65], ICA [islet-cell cytoplasm], IA-2
  • a genetic predisposition is present (for example, HLA, INS VNTR and PTPN22).
  • Type 2 diabetes includes the condition in which a subject has a fasting blood glucose or serum glucose concentration greater than about 125 mg/dL (6.94 mmol/L).
  • the measurement of blood glucose values may be, for example, as is a standard procedure in routine medical analysis. If a glucose tolerance test is carried out, the blood sugar level of a diabetic will be in excess of about 200 mg of glucose per dL (11.1 mmol/1) of plasma 2 hours after 75 g of glucose having been taken on an empty stomach. In a glucose tolerance test 75 g of glucose are administered orally to the patient being tested after 10-12 hours of fasting and the blood sugar level is recorded immediately before taking the glucose and 1 and 2 hours after taking it.
  • the blood sugar level before taking the glucose will be between about 60 and about 110 mg per dL of plasma, less than about 200 mg per dL 1 hour after taking the glucose and less than about 140 mg per dL after 2 hours. If after 2 hours the value is between about 140 and about 200 mg, this is regarded as abnormal glucose tolerance.
  • Late stage type 2 diabetes mellitus includes patients with a secondary drug failure, indication for insulin therapy and progression to micro- and macrovascular complications e.g. diabetic nephropathy, or coronary heart disease (CHD).
  • CHD coronary heart disease
  • HbAlc refers to the product of a non-enzymatic glycation of the haemoglobin B chain. Its determination is well known to one skilled in the art. In monitoring the treatment of diabetes mellitus the HbAlc value is of exceptional importance. As its production depends essentially on the blood sugar level and the life of the erythrocytes, the HbAlc in the sense of a "blood sugar memory” reflects the average blood sugar levels of the preceding 4-6 weeks. Diabetic patients whose HbAlc value is consistently well adjusted by intensive diabetes treatment (for example, less than about 6.5% of the total haemoglobin in the sample), are significantly better protected against diabetic microangiopathy.
  • metformin on its own achieves an average improvement in the Hb A 1 c value in the diabetic of the order of 1.0- 1.5 % .
  • This reduction of the HbAlc value is not sufficient in all diabetics to achieve the desired target range of ⁇ 6.5% and preferably ⁇ 6% HbAlc.
  • insufficient glycemic control or inadequate glycemic control include a condition wherein patients show HbAlc values above about 6.5%, above about 7.0%, above about 7.5%, or above about 8%.
  • metabolic syndrome also called “syndrome X” (when used in the context of a metabolic disorder), also called the dysmetabolic syndrome) is a syndrome complex with the cardinal feature being insulin resistance (Laaksonen et al, ⁇ 2002, Am J Epidemiol; 156:1070-7). According to the ATP III/NCEP guidelines (Executive
  • Abdominal obesity defined as waist circumference >40 inches or 102 cm in men, and >35 inches or 94 cm in women; or with regard to a Japanese ethnicity or Japanese patients defined as waist circumference >85 cm in men and >90 cm in women;
  • Triglycerides >150 mg/dL; 3. HDL-cholesterol ⁇ 40 mg/dL in men;
  • Triglycerides and HDL cholesterol in the blood can also be determined by standard methods in medical analysis and are described for example in Thomas L (Editor): “Labor and Diagnose", TH-Books Verlagsgesellschaft mbH, FrarJcfurt/Main, 2000.
  • Hypertension may be diagnosed, for example, if the systolic blood pressure (SBP) exceeds a value of 140 mm Hg and diastolic blood pressure (DBP) exceeds a value of 90 mm Hg. If a patient is suffering from manifest diabetes it is currently recommended that the systolic blood pressure be reduced to a level below 130 mm Hg and the diastolic blood pressure be lowered to below 80 mm Hg.
  • SBP systolic blood pressure
  • DBP diastolic blood pressure
  • NOD AT new onset diabetes after transplantation
  • PTMS PTMS
  • NOD AT and/or PTMS are associated with an increased risk of micro- and macrovascular disease and events, graft rejection, infection, and death.
  • a number of predictors have been identified as potential risk factors related to NOD AT and/or PTMS including a higher age at transplant, male gender, the pre- transplant body mass index, pre-transplant diabetes, and immunosuppression.
  • gestational diabetes denotes a form of the diabetes which develops during pregnancy and usually ceases again immediately after the birth.
  • Gestational diabetes may be diagnosed by a screening test which is carried out between the 24th and 28th weeks of pregnancy. It is a simple test in which the blood sugar level is measured one hour after the administration of 50 g of glucose solution. If this 1 h level is above 140 mg/dl, gestational diabetes is suspected. Final confirmation may be obtained by a standard glucose tolerance test, for example with 75 g of glucose.
  • the present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
  • the following two examples show the beneficial effect on glycemic control of the combination of a KLK1 (rhKLKl) and an insulin (insulin glargine) as compared to rhKLKl or insulin glargine alone.
  • Test compounds Insulin glargine (LANTUSTM - 5 IU/kg subcutaneous); GLP-1 analog (AVE10 (Pro 36 exendin-4(l-3 )-Lys 6 -NH 2 - 10 ⁇ g kg subcutaneous); and KL 1 (Human recombinant KLKl derived from CHO cell line).
  • mice 10 female 10- weeks old db/db mice in each group.
  • test compound(s) at the dosages described above, and continue blood glucose measurements in 30 min. intervals for the next 4 hours after injection.
  • OGTT the diabetic db/db animals from Example 1 were allowed to recover from the fasting blood glucose test for 6 days. The experiment was carried out in accordance with the protocol described in Example 1. Female db/db mouse, 12 weeks
  • n 10, raean.+-.Sem
  • GLP-1 analogue (10 ⁇ g/kg sc)
  • the animals were divided into groups and fasted overnight (8 hours), and in the morning started blood glucose monitoring.
  • test compound(s) at the same as before dosages
  • Table 3 is the mean blood glucose value +/- Standard error of the mean for fasting blood glucose levels following administration of DM-199 and/or insulin.
  • Table 4 is the mean blood glucose value +/- Standard error of the mean for blood glucose levels during an OGTT following administration of DM- 199 and/or insulin.
  • the following example will show the beneficial effect on glycemic control of a KL 1 (rhKLKl) sequentially added to a rapidly acting insulin (insulin lispro) as compared to rhKLKl or insulin alone.
  • the objectives of this study are to determine rhKLKl administration in a Streptozotocin-induced Type 1 diabetic rat model, has beneficial effects on blood glucose levels and sensitivity to 'rapid-acting' insulin (insulin lispro). Insulin tolerance tests and glucose tolerance tests will be conducted with a low and a high dose of insulin lispro.
  • the study is designed to test for additive effects of rhKLKl and rapid acting insulin in STZ-induced diabetic rats.
  • the rats will be treated with three doses of rhKLKl (100 g/kg) administered over a seven day period or a vehicle control.
  • the rhKLKl will persist in the rats for approximately 72 hrs, and the three doses are meant to load the rats with rhKLKl .
  • animals from the vehicle and rhKLKl treatment groups will be administered insulin tolerance tests with single doses of OU kg, lU/kg and 3 U/kg of insulin lispro.
  • Housing individually per cage at 22-25°C and 30-70% relative humidity
  • Photoperiod Diurnal; 12 hours light, 12 hours dark Acclimation: Animals will be acclimated for a period of at least 7 days. During the acclimation period, the animals will be observed for signs of abnormal behavior or disease. Induction of Diabetes: Following acclimation, non-fasting rats will be injected with streptozotocin (Day 0, 55 mg/kg, i.v.) and blood glucose levels will be checked 72 hours later. Only animals with non-fasting blood glucose level >350 mg/dL will be considered diabetic and will be randomized based on body weight glucose level and entered into the study (Day 3).
  • Animals will be injected subcutaneously with either the vehicle or DM- 199 (100 ⁇ g/kg) at the same time of day on days 3, 6 and 9.
  • Animals will be observed daily for general health. Body weights and non-fasted blood glucose levels will be measured daily. On day 8, 50 nL blood will be collected for measurement of plasma insulin levels, and non-fasting blood glucose will be measured. Equal numbers of animals from each treatment group will then be assigned to two sub-groups. Animals will be administered an insulin tolerance test by subcutaneous injection of either 0 U/kg, 1 U kg or 3 U/kg of 'rapid-acting' insulin aspart. Blood glucose levels will be tested at 15, 30, 45, 60 and 120 mins, followed by collection of 50 u blood for measurement of insulin. The animals are not fasted before the ITT test, but will be fasted for 2-3 hours while testing. As such, there may be some decline in blood glucose as a result of the fast. The blood glucose lowering effects of the rapid acting insulin should dissipate after approximately 4 hours.
  • the Area Under the Curve (AUC) glucose can be calculated. It is expected that rats treated with the combination of rhKLKl and insulin will have a more pronounced decrease in blood glucose levels than animals treated with insulin or rhKLKl alone. This may include a lower AUC, a greater decrease in blood glucose levels, and/or blood glucose levels being decrease for a longer period of time (e.g. decreased for 3 hours rather than 2 hours) compared to animals treated with insulin or rhKLKl alone.
  • the following example will show the beneficial effect on glycemic control of a KLKl (rhKLKl) sequentially added to a rapid acting insulin (insulin aspart) as compared to the insulin alone in an Oral Glucose Tolerance Test.
  • rhKLKl KLKl
  • insulin aspart rapid acting insulin
  • diabetes will be induced in rats with STZ.
  • the rats will be randomized into 5 groups, with rats in group 3, 4 and 5 being treated with 100 ⁇ g kg KLKl on days 1, 3 and 7. On day 8, following an overnight fast, the rats will be administered 0 U/kg, 1 U/kg or 3 U/kg of 'rapid-acting' insulin.
  • each rat will be given a single 1.5 ml dose of glucose solution (2 g/kg body weight of D-(+)-glucose (G7528, Sigma) solubilized in deionized water) administered by oral gavage.
  • the blood glucose levels will then be measured by glucose meter at 15, 30, 60, 90, 120 and 180 minutes to observe the rate of glucose clearance from the blood over time.
  • glucose meter At each time point of an OGTT
  • AUC glucose levels can be calculated.
  • diabetic rats administered KLK1 together with insulin will have lower blood glucose levels, and lower AUC glucose compared to rats treated with insulin or rhKLKl alone.
  • the following example will show the beneficial effect on glycemic control of a KLK1 (rhKLKl) sequentially added to an insulin (insulin glargine) as compared to the insulin alone.
  • Rats will be treated with STZ as described in Example 3 above to induce diabetes.
  • the rats will be randomized into 5 groups, with rats in group 3, 4 and 5 being treated with 100 ⁇ g/kg KLK1 on days 1, 3 and 7.
  • the rats will be administered 1 U kg (groups 1 and 3) or 3 U/kg (groups 2 and 4) of 'long- acting' insulin glargine.
  • each rat will be given a single 1.5 ml dose of glucose solution (2 g/kg body weight of D-(+)-glucose (G7528, Sigma) solubilized in deionized water) administered by oral gavage.
  • the blood glucose levels will then be measured by glucose meter at 15, 30, 60, 90, 120 and 180 minutes to observe the rate of glucose clearance from the blood over time.
  • glucose meter At each time point of an OGTT
  • AUC glucose levels can be calculated.
  • Treating patients with type 2 diabetes with the pharmaceutical composition according to the invention is expected, in addition to producing an acute improvement in the glucose metabolic situation, to also contribute to a sustainable well metabolic situation in the long term. This may be observed in patients being treated for a longer period, e.g. 3 months to 1 year or even 1 to 6 years, with the pharmaceutical composition according to the invention and compared with patients who are treated with insulin alone. There is evidence of therapeutic success compared with patients treated with insulin alone if no increase in the fasting glucose and/or HbAlc value is observed but where a reduction in hypoglycaemia event rate, glucose excursions or insulin requirement is seen.
  • Treating patients with type 1 diabetes with the pharmaceutical composition according to the invention is expected to, in addition to producing an acute improvement in the glucose metabolic situation, to also contribute to a sustainable well metabolic situation in the long term. This may be observed in patients being treated for a longer period, e.g. 3 months to 1 year or even 1 to 6 years, with the pharmaceutical composition according to the invention and compared with patients who are treated with insulin alone. There is evidence of therapeutic success compared with patients treated with insulin alone if no increase in the fasting glucose and/or HbAlc value is observed but where a reduction in hypoglycaemia event rate, glucose excursions or insulin requirement is seen.

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Abstract

L'invention concerne des compositions et des méthodes de traitement d'une maladie ou d'un état qui bénéficie de l'administration d'insuline, le diabète, comprenant l'administration d'une quantité thérapeutiquement efficace de KLK1, ou d'un variant ou d'un fragment actif de celle-ci, et d'une quantité thérapeutiquement efficace d'au moins une insuline.
PCT/CA2013/050755 2012-10-15 2013-10-08 Combinaison d'une insuline et de la kallikréine 1 tissulaire WO2014059536A1 (fr)

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US9364521B2 (en) 2012-06-04 2016-06-14 Diamedica Inc. Human tissue kallikrein 1 glycosylation isoforms
US9616015B2 (en) 2012-05-25 2017-04-11 Diamedica Inc. Formulations of human tissue kallikrein-1 for parenteral delivery and related methods
WO2018165551A1 (fr) * 2017-03-09 2018-09-13 Diamedica Inc. Formes posologiques de kallicréine tissulaire 1

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Cited By (8)

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Publication number Priority date Publication date Assignee Title
US9616015B2 (en) 2012-05-25 2017-04-11 Diamedica Inc. Formulations of human tissue kallikrein-1 for parenteral delivery and related methods
US9364521B2 (en) 2012-06-04 2016-06-14 Diamedica Inc. Human tissue kallikrein 1 glycosylation isoforms
US9839678B2 (en) 2012-06-04 2017-12-12 Diamedica Inc. Human tissue kallikrein 1 glycosylation isoforms
WO2018165551A1 (fr) * 2017-03-09 2018-09-13 Diamedica Inc. Formes posologiques de kallicréine tissulaire 1
CN110446501A (zh) * 2017-03-09 2019-11-12 代阿麦迪卡股份有限公司 组织激肽释放酶1的剂型
JP2020510023A (ja) * 2017-03-09 2020-04-02 ダイアメディカ， インコーポレイテッド 組織カリクレイン１の剤形
EP3592377A4 (fr) * 2017-03-09 2021-02-17 Diamedica Inc. Formes posologiques de kallicréine tissulaire 1
US11857608B2 (en) 2017-03-09 2024-01-02 Diamedica Inc. Dosage forms of tissue kallikrein 1

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