Tissue kallikrein 1 dosage form

Abstract

Dosage forms of one or more tissue kallikrein-1 (KLK1) polypeptides are provided, including subcutaneous and intravenous dosage forms, having a total dosage of about 0.1 μg / kg to about 10.0 μg / kg of KLK1 polypeptide. Related devices and methods of use thereof are also provided, for example, for treating ischemic and hemorrhagic conditions. [Selected Figure] Figure 2A

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 567,406, filed Oct. 3, 2017; U.S. Provisional Patent Application No. 62 / 516,463, filed Jun. 7, 2017; and U.S. Provisional Patent Application No. 62 / 469,385, filed Mar. 9, 2017, each of which is hereby incorporated by reference in its entirety.

[0002] Description of the Sequence Listing The sequence listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference in its entirety. The name of this text file containing the sequence listing is DIAM_037_03WO_ST25.txt. This text file is 9 KB, was created on Mar. 9, 2018, and was electronically submitted via EFS - Web.

[0003] Background Technical Field Embodiments of the present disclosure relate to dosage forms of one or more tissue kallikrein - 1 (KLK1) polypeptides having a total KLK1 polypeptide dosage of from about 0.1 μg / kg to about 10.0 μg / kg, including subcutaneous and intravenous dosage forms. Also provided are related devices and methods of use thereof, for example, for treating ischemic and hemorrhagic pathologies.

Background Art

[0004] Description of Related Art All tissue kallikreins possess protease activity with substrate specificity similar to that of trypsin or chymotrypsin. The most characteristic activity of KLK1 is its enzymatic cleavage of kininogen, which produces bradykinin (BK)-like peptides collectively called kinins, directly or indirectly activating subtypes of both bradykinin receptors (BK-B1, BK-B2). Activation of BK receptors by kinins triggers numerous complex metabolic pathways in response to ischemia in the body, which may include improved blood flow (by vasodilation), anti-inflammatory responses, cell repair by angiogenesis or angiogenesis, and reduced apoptosis. Tissue-mediated kallikrein release increases blood flow in various tissues, including the kidneys and heart (see, e.g., Stone et al., Arterioscler Thromb Vasc Biol. 29:657-664, 2009), and a considerable number of scientific studies suggest that this is likely one mode of addressing specific pathological conditions. Therefore, KLK1 is considered to have the potential to treat a wide range of clinical scenarios in which re-establishing blood flow and reducing inflammation in patients are essential for maintaining organ function, including brain, renal, and cardiac function. However, there remains a need to identify the optimal dosage form and route of administration to achieve and maintain therapeutic levels of KLK1 in humans. This disclosure addresses these and other needs. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Stone et al.,Arterioscler Thromb Vasc Biol.29:657-664,2009 [Overview of the project]

[0006] Brief Overview Embodiments of this disclosure relate to the unexpected finding that formulations of tissue kallikrein-1 (KLK1) have an inverse dose curve, where administration of the low-dose formulation up to a certain point in time shows an improved pharmacokinetic and / or activity profile compared to the high-dose formulation.

[0007] Therefore, certain embodiments include dosage forms comprising one or more tissue kallikrein (KLK1) polypeptides formulated in a total dose of KLK1 polypeptides ranging from about 1.0 μg / kg to about 5.0 μg / kg or about 10.0 μg / kg. In certain embodiments, the dosage forms are suitable for subcutaneous or intravenous administration.

[0008] In some embodiments, the dosage forms are approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, This includes total doses of KLK1 polypeptide of 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 μg / kg (including all ranges in between).

[0009] Specific dosage forms include subcutaneous KLK1 polypeptide dosage forms of approximately 1.0 μg / kg to approximately 4.0 μg / kg, or approximately 1.0 μg / kg to approximately 3.0 μg / kg, or approximately 1.0 μg / kg to approximately 2.0 μg / kg, or approximately 2.0 μg / kg to approximately 5.0 μg / kg, or approximately 2.0 μg / kg to approximately 4.0 μg / kg, or approximately 2.0 μg / kg to approximately 3.0 μg / kg, or approximately 3.0 μg / kg to approximately 5.0 μg / kg, or approximately 3.0 μg / kg to approximately 4.0 μg / kg, or approximately 2.5 μg / kg to approximately 3.5 μg / kg, or approximately 3 μg / kg.

[0010] Certain dosage forms include intravenous dosage forms of total KLK1 polypeptide ranging from approximately 0.5 μg / kg to approximately 3.0 μg / kg, or approximately 0.5 μg / kg to approximately 2.0 μg / kg, or approximately 0.5 μg / kg to approximately 1.0 μg / kg, or approximately 0.5 μg / kg to approximately 0.8 μg / kg, or approximately 0.5 μg / kg to approximately 0.75 μg / kg, or approximately 0.75 μg / kg.

[0011] Some dosage forms contain a mixture of KLK1 glycoforms, for example, a first KLK1 polypeptide and a second tissue KLK1 polypeptide. The first KLK1 polypeptide has three glycans attached at three different positions per polypeptide, and the second KLK1 polypeptide has two glycans attached at two different positions per polypeptide. The first KLK1 polypeptide and the second KLK1 polypeptide are present in the dosage form in a ratio of approximately 45:55 to approximately 55:45.

[0012] In some embodiments, one or more of the glycans are N-linked glycans. In some embodiments, one or more of the glycans are linked at amino acid residues 78, 84, or 141 of KLK1 as defined by SEQ ID NO: 3 or 4. In some embodiments, three glycans of the first KLK1 polypeptide are N-linked glycans at residues 78, 84, and 141. In some embodiments, two glycans of the second KLK1 polypeptide are N-linked glycans at residues 78 and 84, but not at residue 141. In some embodiments, the first and second KLK1 polypeptides are present in the dosage form in a ratio of approximately 50:50.

[0013] Some dosage forms contain a mixture of a triple glycoform and a double glycoform of the KLK1 polypeptide, with the triple and double glycoforms present in the dosage form in a ratio of approximately 45:55 to approximately 55:45. In some embodiments, the triple glycoform contains N-linked glycans at amino acid residues 78, 84, and 141 of KLK1, as defined by SEQ ID NO: 3 or 4. In some embodiments, the double glycoform contains N-linked glycans at amino acid residues 78 and 84 of KLK1, as defined by SEQ ID NO: 3 or 4, but not at amino acid residue 141 of KLK1. In some embodiments, the triple and double glycoforms are present in the dosage form in a ratio of approximately 50:50.

[0014] In certain embodiments, one or more KLK1 polypeptides are mature KLK1 polypeptides, human KLK1 (hKLK1) polypeptides, or mature hKLK1 polypeptides (including any combination thereof) (e.g., SEQ ID NO: 3 or 4 and its variants).

[0015] In some embodiments, the hKLK1 polypeptide comprises, consists of, or is essentially composed of, active variants having at least about 90, 95, 96, 97, 98, or 99% sequence identity with amino acid residues 78-141 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0016] In some embodiments, the hKLK1 polypeptide comprises, consists of, or is essentially composed of, active variants having at least about 90, 95, 96, 97, 98, or 99% sequence identity with amino acid residues 25-262 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] In some embodiments, the KLK1 polypeptide comprises an amino acid sequence having at least about 90, 95, 96, 97, 98, or 99% sequence identity to amino acid residues 25-262 of SEQ ID NO: 2, and the KLK1 polypeptide comprises E145 and / or A188. In some embodiments, the KLK1 polypeptide comprises an amino acid sequence having at least about 90, 95, 96, 97, 98, or 99% sequence identity to amino acid residues 25-262 of SEQ ID NO: 2, and the KLK1 polypeptide comprises Q145 and / or V188.

[0018] In some embodiments, the dosage form comprises a pharmaceutically acceptable diluent, adjuvant, or carrier. In some embodiments, the dosage form substantially does not contain other glycosylated isoforms (glycoforms) of KLK1.

[0019] In some embodiments, the dosage form has an endotoxin level of less than approximately 1 EU / mg of protein, host cells with total protein of less than approximately 100 ng / mg, host cell DNA with total protein of less than approximately 10 pg / mg, and / or is substantially free of aggregates (more than approximately 95% appear as a single peak by SEC HPLC).

[0020] In some embodiments, the dosage includes a second agent selected from one or more (including combinations thereof) angiotensin receptor blockers, edavalone, finelenone, and bardoxalon. In some embodiments, the angiotensin receptor blocker is selected from one or more (including combinations thereof) losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, suprisartan, telmisartan, and valsartan.

[0021] The invention also includes methods for treating subjects in need thereof, which involve administering the dosage forms described herein to the subjects. Some embodiments involve subcutaneous or intravenous administration of the dosage forms to the subjects. Certain methods relate to the treatment of ischemic conditions in subjects, which are optionally selected from one or more of the following: cerebral ischemia (ischemic stroke), transient ischemic attack (TIA), myocardial ischemia, ischemic colitis, limb ischemia, and cutaneous ischemia. Certain methods relate to the treatment of vascular dementia. Some methods relate to the treatment of hemorrhagic conditions in subjects, which optionally include hemorrhagic strokes, including intracerebral (internal brain) hemorrhagic stroke and subarachnoid hemorrhagic stroke. Some methods relate to the treatment of diabetes, for example, type 2 diabetes (T2D). Certain embodiments relate to the treatment of traumatic brain injury (TBI). Some embodiments relate to the treatment of kidney diseases, for example, chronic kidney disease, diabetic nephropathy, or polycystic kidney disease. This also includes methods for treating systemic lupus erythematosus (SLE) and related conditions or complications such as lupus nephritis, pulmonary hypertension (PAH), focal segmental glomerulosclerosis, and essential hypertension.

[0022] In some embodiments, subcutaneous administration of the dosage form achieves therapeutically effective serum levels of one or more KLK1 polypeptides in the subject, and, in some cases, maintains these levels for approximately or at least approximately 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or longer, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or longer after subcutaneous administration. In some embodiments, intravenous administration of the dosage form to the subject optionally achieves therapeutically effective serum levels of one or more KLK1 polypeptides in the subject approximately 0.5, 1, 2, 3, or 4 hours after intravenous administration, or less than approximately 0.5, 1, 2, 3, or 4 hours. In some embodiments, therapeutically effective serum levels are approximately 1.0 to approximately 5.0 ng / ml, or approximately 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4. 8, 4.9, or 5.0 mg / ml (including all ranges in between), for example, 1.0 to about 5.0 ng / ml, or about 1.0 to about 4.0 ng / ml, or about 1.0 to about 3.0 ng / ml, or about 1.0 to about 2.0 ng / ml, or about 2.0 to about 5.0 ng / ml, or about 2.0 to about 4.0 ng / ml, or about 2.0 to about 3.0 ng / ml, or about 3.0 to about 5.0 ng / ml, or about 3.0 to about 4.0 ng / ml.

[0023] In some embodiments, administration of the dosage form achieves an improved pharmacokinetic profile or biological effect as compared to a high-dose dosage form, e.g., a high-dose dosage form having a total KLK1 polypeptide dosage of at least about 15 μg / kg, or at least about 20 μg / kg, or at least about 50 μg / kg, or at least about 100 μg / kg, or at least about 400 μg / kg or more. In some embodiments, the improved pharmacokinetic profile includes, for example, an increase in serum half-life after a single subcutaneous administration, measured at about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more after subcutaneous administration.

[0024] Certain embodiments include administering the dosage form to a subject according to a dosing schedule of about once or twice a day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, once or twice every week. Certain embodiments include, for example, administering the dosage form to a subject according to a dosing schedule of about once a day every three days by subcutaneous administration.

[0025] Certain embodiments include administering an intravenous dosage form intravenously to a subject, followed by administering one or more subcutaneous dosage forms subcutaneously to the subject according to a dosing schedule of about once or twice a day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, once or twice every week. In some embodiments, intravenous administration achieves a therapeutically effective serum level of one or more KLK1 polypeptides in the subject at about 0.5, 1, 2, 3, or 4 hours, or less than about 0.5, 1, 2, 3, or 4 hours after intravenous administration, and subcutaneous administration maintains a therapeutically effective serum level for about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more after subcutaneous administration.

[0026] A particular method involves administering a second agent selected from one or more (including combinations thereof) of angiotensin receptor blockers, edavalone, finelenone, and bardoxalon, for example, as part of the same dosage form or as part of a different dosage form or composition. In some embodiments, the angiotensin receptor blocker is selected from one or more (including combinations thereof) of losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, suprisartan, telmisartan, and valsartan.

[0027] This specification also includes devices containing the dosage forms described herein, which are adapted or suitable for subcutaneous administration. In some embodiments, the device is a syringe. In some embodiments, the syringe includes a subcutaneous injection needle assembly attached to the syringe. In some embodiments, the syringe includes a protective cover around the needle assembly. In some embodiments, the syringe has a needle that is about 1 / 2 inch to about 5 / 8 inch in length and has a gauge of about 25 to about 31. In certain embodiments, for example, the following items are provided: (Item 1) A dosage form containing one or more tissue kallikrein (KLK1) polypeptides, formulated with a total KLK1 polypeptide dose ranging from approximately 0.5 μg / kg to approximately 10.0 μg / kg. (Item 2) The dosage form described in item 1, suitable for subcutaneous or intravenous administration. (Item 3) Approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2 .7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, The dosage forms described in item 1 or 2, containing a total dose of KLK1 polypeptide of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 μg / kg. (Item 4) Dosage forms according to any one of items 1 to 3, comprising a total KLK1 polypeptide subcutaneous dosage form in a dose of approximately 1.0 μg / kg to approximately 5.0 μg / kg, or approximately 1.0 μg / kg to approximately 4.0 μg / kg, or approximately 1.0 μg / kg to approximately 3.0 μg / kg, or approximately 1.0 μg / kg to approximately 2.0 μg / kg, or approximately 2.0 μg / kg to approximately 5.0 μg / kg, or approximately 2.0 μg / kg to approximately 4.0 μg / kg, or approximately 2.0 μg / kg to approximately 3.0 μg / kg, or approximately 3.0 μg / kg to approximately 5.0 μg / kg, or approximately 3.0 μg / kg to approximately 4.0 μg / kg, or approximately 2.5 μg / kg to approximately 3.5 μg / kg, or approximately 3 μg / kg. (Item 5) A dosage form according to any one of items 1 to 4, comprising an intravenous dosage form of total KLK1 polypeptide in an amount of approximately 0.5 μg / kg to approximately 3.0 μg / kg, or approximately 0.5 μg / kg to approximately 2.0 μg / kg, or approximately 0.5 μg / kg to approximately 1.0 μg / kg, or approximately 0.5 μg / kg to approximately 0.8 μg / kg, or approximately 0.5 μg / kg to approximately 0.75 μg / kg, or approximately 0.75 μg / kg. (Item 6) It comprises a first KLK1 polypeptide and a second KLK1 polypeptide, The first KLK1 polypeptide has three glycans attached to three different positions per polypeptide, and the second KLK1 polypeptide has two glycans attached to two different positions per polypeptide. The dosage form according to any one of items 1 to 5, wherein the first KLK1 polypeptide and the second KLK1 polypeptide are present in the dosage form in a ratio of approximately 45:55 to approximately 55:45. (Item 7) The dosage form described in item 6, wherein one or more of the glycans are N-linked glycans. (Item 8) The dosage form according to item 6 or 7, wherein one or more of the glycans are bound to amino acid residues 78, 84, or 141 of KLK1 as defined by Sequence ID No. 3 or 4. (Item 9) The dosage form according to any one of items 6 to 8, wherein the three glycans of the first KLK1 polypeptide are N-linked glycans at residues 78, 84, and 141. (Item 10) The dosage form according to any one of items 6 to 9, wherein the two glycans of the second KLK1 polypeptide are N-linked glycans at residues 78 and 84, but not at residue 141. (Item 11) The dosage form according to any one of items 1 to 10, wherein the first KLK1 polypeptide and the second KLK1 polypeptide are present in the dosage form in a ratio of approximately 50:50. (Item 12) The dosage form according to any one of items 1 to 5, wherein the dosage form comprises a triple glycoform of KLK1 polypeptide and a double glycoform of KLK1 polypeptide, and the triple glycoform and the double glycoform are present in the dosage form in a ratio of approximately 45:55 to approximately 55:45. (Item 13) The dosage form according to item 12, wherein the triple glycoform comprises an N-linked glycan at amino acid residues 78, 84, and 141 of KLK1 as defined by SEQ ID NO: 3 or 4. (Item 14) The dosage form according to item 12 or 13, wherein the biglyceride contains an N-linked glycan at amino acid residues 78 and 84 of KLK1, as defined by Sequence ID No. 3 or 4, but does not contain an N-linked glycan at amino acid residue 141 of KLK1. (Item 15) The dosage form according to any one of items 12 to 14, wherein the triple glycoform and the double glycoform are present in the dosage form in a ratio of approximately 50:50. (Item 16) The dosage form according to any one of items 1 to 15, wherein one or more KLK1 polypeptides are recombinant KLK polypeptides, mature KLK1 polypeptides, human KLK1 (hKLK1) polypeptides, or any combination thereof. (Item 17) The dosage form according to item 16, wherein the hKLK1 polypeptide comprises, consists of, or is essentially composed of, an active variant having at least about 90, 95, 96, 97, 98, or 99% sequence identity with amino acid residues 78-141 of SEQ ID NO: 1 or SEQ ID NO: 2. (Item 18) The dosage form according to item 16, wherein the hKLK1 polypeptide comprises, consists of, or is essentially composed of, an active variant having at least about 90, 95, 96, 97, 98, or 99% sequence identity with amino acid residues 25-262 of SEQ ID NO: 1 or SEQ ID NO: 2. (Item 19) The dosage form according to item 16, wherein the KLK1 polypeptide comprises an amino acid sequence having at least about 90, 95, 96, 97, 98, or 99% sequence identity with amino acid residues 25-262 of SEQ ID NO: 2, and the KLK1 polypeptide comprises E145 and / or A188. (Item 20) The dosage form according to item 16, wherein the KLK1 polypeptide comprises an amino acid sequence having at least about 90, 95, 96, 97, 98, or 99% sequence identity with amino acid residues 25-262 of SEQ ID NO: 2, and the KLK1 polypeptide comprises Q145 and / or V188. (Item 21) A dosage form according to any one of items 1 to 20, comprising a pharmaceutically acceptable excipient, diluent, adjuvant, or carrier. (Item 22) A dosage form according to any one of items 1 to 21, substantially free of other glycosylated isoforms (glycoforms) of KLK1. (Item 23) A dosage form according to any one of items 1 to 22, having an endotoxin level of less than approximately 1 EU per 1 mg of protein, host cells of less than approximately 100 ng per 1 mg of total protein, host cell DNA of less than approximately 10 pg per 1 mg of total protein, and / or being substantially free of aggregates (more than approximately 95% appearing as a single peak by SEC HPLC). (Item 24) A dosage form according to any one of items 1 to 23, further comprising a second agent selected from one or more (including combinations thereof) of angiotensin receptor blockers, edavalone, finelenone, and bardoxalon. (Item 25) The dosage form described in item 24, wherein the angiotensin receptor blocker is selected from one or more of losartan, azicilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, suprisartan, telmisartan, and valsartan (including combinations thereof). (Item 26) A method for treating a subject in need thereof, comprising administering to the subject a dosage form described in any one of items 1 to 25. (Item 27) The method according to item 26, comprising administering the dosage form subcutaneously or intravenously to the subject. (Item 28) The method according to item 26 or 27 for treating an ischemic condition in the subject, which is arbitrarily selected from one or more of the following: cerebral ischemia (ischemic stroke), myocardial ischemia (myocardial ischemia), ischemic colitis, limb ischemia, and skin ischemia. (Item 29) The method according to item 26 or 27 for treating hemorrhagic conditions in the subject, which are, optionally, hemorrhagic strokes including intracerebral (internal brain) hemorrhagic stroke and subarachnoid hemorrhagic stroke. (Item 30) The method according to item 26 or 27 for treating one or more of the following conditions: vascular dementia, diabetes mellitus (optionally type 2 diabetes mellitus (T2D)), traumatic brain injury (TBI), kidney disease (optionally chronic kidney disease, diabetic nephropathy, or polycystic kidney disease), systemic lupus erythematosus (SLE) and related conditions including lupus nephritis, pulmonary hypertension (PAH), focal segmental glomerulosclerosis, and essential hypertension. (Item 31) The method according to any one of items 26 to 30, comprising subcutaneously administering the dosage form to the subject, wherein the subcutaneous administration of the dosage form achieves a therapeutically effective serum level of one or more KLK1 polypeptides in the subject, and optionally maintains the therapeutically effective serum level for about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more after the subcutaneous administration. (Item 32) The method according to any one of items 26 to 30, comprising intravenously administering the dosage form to the subject, wherein the intravenous administration of the dosage form selectively achieves a therapeutically effective serum level of one or more KLK1 polypeptides in the subject about 0.5, 1, 2, 3, or 4 hours after the intravenous administration, or less than about 0.5, 1, 2, 3, or 4 hours after the intravenous administration. (Item 33) The method according to item 31 or 32, wherein the therapeutically effective serum level is approximately 1.0 to approximately 5.0 ng / ml, or approximately 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg / ml. (Item 34) The method according to any one of items 26 to 33, wherein administration of the dosage form achieves an improved pharmacokinetic profile or biological effect compared to a high-dose dosage form having a total dose of KLK1 polypeptide of at least about 15 μg / kg, or at least about 20 μg / kg, or at least about 50 μg / kg, or at least about 100 μg / kg, or at least about 400 μg / kg or more. (Item 35) The method according to any one of items 26 to 34, comprising administering the dosage form to the subject as a dosage schedule of approximately once or twice per day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, or once or twice every week. (Item 36) The method according to item 35, comprising administering the dosage form to the subject by subcutaneous administration as an optional measure, approximately once a day every three days. (Item 37) The method according to any one of items 26 to 36, comprising intravenously administering one intravenous dosage form to the subject, and then subcutaneously administering one or more subcutaneous dosage forms to the subject as an optional dosing schedule of approximately once or twice daily, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, or once or twice every week. (Item 38) The method according to item 37, wherein the intravenous administration achieves a therapeutically effective serum level of one or more KLK1 polypeptides in the subject approximately 0.5, 1, 2, 3, or 4 hours after the intravenous administration, or less than approximately 0.5, 1, 2, 3, or 4 hours after the intravenous administration, and the subcutaneous administration maintains the therapeutically effective serum level for approximately or at least approximately 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, or 96 hours or more, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more after the subcutaneous administration. (Item 39) The method according to any one of items 26 to 38, comprising administering a second agent selected from one or more (including combinations thereof) angiotensin receptor blockers, edavalone, finelenone, and bardoxalon as part of the same or different dosage form or composition, either optionally. (Item 40) The method according to item 39, wherein the angiotensin receptor blocker is selected from one or more of losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, supplementartan, telmisartan, and valsartan (including combinations thereof). (Item 41) A device comprising a dosage form described in any one of items 1 to 25, the device being suitable for delivering the dosage form subcutaneously or intravenously. [Brief explanation of the drawing]

[0028] [Figure 1]The results of a preliminary Phase I / II trial of KLK1 (DM199) in humans for the treatment of type 2 diabetes (T2D) are presented. Patients (n=37) received either placebo or one of two doses of KLK1 (high dose of 15 μg / kg or low dose of 3 μg / kg) every three days for 28 days, and fasting blood glucose (FBG) levels were measured. The high dose (15 μg / kg) did not show a significant effect, but the low dose (3 μg / kg) showed a statistically significant effect (p<0.05) on FBG levels compared to baseline. Therefore, the low dose of 3 μg / kg resulted in an unexpected improvement compared to the high dose of 15 μg / kg. [Figure 2A] This study demonstrates that subcutaneous administration of a low-dose mature human KLK1 glycoform (3 μg / kg, n=12) significantly prolongs the serum half-life compared to intravenous administration of a low-dose formulation (0.75 μg / kg, n=12) in healthy human subjects. [Figure 2B] This study demonstrates that subcutaneous administration of a low-dose mature human KLK1 glycoform (3 μg / kg, n=12) significantly prolongs the serum half-life compared to intravenous administration of a low-dose formulation (0.75 μg / kg, n=12) in healthy human subjects. [Figure 3] The results of clinical trials show that a 3 μg / kg dose of KLK1 (DM199) maintained fairly stable drug concentrations at desired or therapeutically effective serum / plasma concentrations of approximately 3–5 ng / ml. In contrast, higher doses of 15 / 25 μg / kg resulted in greater dose-to-dose variability and proportionally higher plasma levels. [Figure 4A] The results of a KLK1 administration trial conducted in patients with type 2 diabetes are shown. Figure 4A shows a summary, Figures 4B and 4D show the results for the placebo group, and Figure 4C shows the results for the (KLK1)DM199 group. The numerical values ​​are a derivative scale of insulin resistance (HOMA2-IR), with higher values ​​indicating greater insulin resistance and more severe disease. [Figure 4B]The results of a KLK1 administration trial conducted in patients with type 2 diabetes are shown. Figure 4A shows a summary, Figures 4B and 4D show the results for the placebo group, and Figure 4C shows the results for the (KLK1)DM199 group. The numerical values ​​are a derivative scale of insulin resistance (HOMA2-IR), with higher values ​​indicating greater insulin resistance and more severe disease. [Figure 4C] The results of a KLK1 administration trial conducted in patients with type 2 diabetes are shown. Figure 4A shows a summary, Figures 4B and 4D show the results for the placebo group, and Figure 4C shows the results for the (KLK1)DM199 group. The numerical values ​​are a derivative scale of insulin resistance (HOMA2-IR), with higher values ​​indicating greater insulin resistance and more severe disease. [Figure 4D] The results of a KLK1 administration trial conducted in patients with type 2 diabetes are shown. Figure 4A shows a summary, Figures 4B and 4D show the results for the placebo group, and Figure 4C shows the results for the (KLK1)DM199 group. The numerical values ​​are a derivative scale of insulin resistance (HOMA2-IR), with higher values ​​indicating greater insulin resistance and more severe disease. [Figure 5A] This shows the results of a 28-day multi-dose study in patients with type 2 diabetes (n=12-13 per group) regarding the measurement of renal function (creatinine, blood urea). The results showed that a low dose (3 μg / kg) had a significantly greater effect on improving renal function measurements than a high dose (15 μg / kg). [Figure 5B] This shows the results of a 28-day multi-dose study in patients with type 2 diabetes (n=12-13 per group) regarding the measurement of renal function (creatinine, blood urea). The results showed that a low dose (3 μg / kg) had a significantly greater effect on improving renal function measurements than a high dose (15 μg / kg). [Modes for carrying out the invention]

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, and preferred methods and materials are described below. For the purposes of the present invention, the following terms are defined below.

[0030] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "element" means one or more elements.

[0031] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that is 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a given quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0032] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply that they encompass the process or element or group of processes or elements described, but exclude any other process or element or group of processes or elements. “Consisting of” means that anything following the phrase “consisting of” is included and limited to them. Thus, the phrase “consisting of” indicates that the enumerated elements are necessary or essential, and that no other elements may be present. “Consisting essentially of” means that the elements enumerated after this phrase are included, and limited to other elements that do not interfere with or contribute to the activity or action identified in the disclosure of the enumerated elements. Thus, the phrase “consisting essentially of” indicates that the enumerated elements are necessary or essential, but other elements are optional and may or may not be present depending on whether they substantially affect the activity or action of the enumerated elements.

[0033] As used herein, the term “amino acid” is intended to mean both natural and unnatural amino acids, as well as amino acid analogs and mimics. Naturally occurring amino acids include 20(L)-amino acids utilized in protein biosynthesis, as well as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine, among others. Unnatural amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, and ethionine, which are known to those skilled in the art. Amino acid analogs include modified forms of natural and unnatural amino acids. Such modifications may include, for example, substitution or replacement of chemical groups and moieties on an amino acid, or derivatization of an amino acid. Amino acid mimics include, for example, organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristic of a reference amino acid. For example, an organic structure mimicking arginine (Arg or R) would be located in a similar molecular space and have a positively charged moiety with similar mobility to the e-amino group of the side chain of the naturally occurring Arg amino acid. The mimics also include constrained structures to maintain optimal spacing and charge interactions between amino acids or amino acid functional groups. Those skilled in the art know or can determine which structures constitute functionally equivalent amino acid analogs and amino acid mimics.

[0034] The terms “endotoxin-free” or “substantially endotoxin-free” generally refer to dosage forms, compositions, solvents, devices, and / or blood vessels containing at most trace amounts of endotoxin (e.g., amounts that do not cause clinically harmful physiological effects on the subject), preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain bacteria, usually Gram-negative bacteria, although endotoxins can also be found in Gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found on the outer membranes of various Gram-negative bacteria, representing a central pathogenic feature of these bacteria's ability to cause disease. Small amounts of endotoxin in humans can cause fever, hypotension, and activation of inflammation and coagulation, among other physiological side effects.

[0035] Therefore, in the manufacture of pharmaceuticals, it is often desirable to remove most or all traces of endotoxins from drugs and / or drug containers, as even small amounts can cause adverse effects in humans. Since temperatures above 300°C are usually required to decompose most endotoxins, a pyrogenic removal oven can be used for this purpose. For example, based on primary packaging materials such as syringes or vials, a combination of a glass temperature of 250°C and a holding time of 30 minutes is sufficient to achieve a 3-log reduction in endotoxin levels. Other methods for removing endotoxins are considered, including, for example, chromatography and filtration methods described herein and known in the art. Also included are methods for producing KLK1 polypeptides in eukaryotic cells such as mammalian cells and isolating them from eukaryotic cells in order to reduce, if not eliminate, the risk of endotoxins being present in the compositions of the present invention. Preferably, a method is used to produce KLK1 polypeptides and isolate them from recombinant cells grown in a chemically defined serum-free medium.

[0036] Endotoxins can be detected using techniques known and commonly practiced in the art. For example, the Limulus mimic cell lysate assay using horseshoe crab blood is a highly sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation of Limulus lysates due to a potent enzyme cascade that amplifies the reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). Because they are virtually endotoxin-free, endotoxin levels can be approximately 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or less than 10 EU per 1 mg or 1 ml of protein. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1 to 10 EU.

[0037] The “half-life” of a drug, such as a dosage form of KLK1 polypeptide, may refer to the time it takes for the drug to lose half of its pharmacological, physiological, or other activity compared to such activity at the time of administration to the serum or tissue of an organism, or to another defined time point. “Half-life” may also refer to the time it takes for the level of the drug to decrease by half of the initial dose administered to the serum or tissue of an organism, compared to such amount or concentration at the time of administration to the serum or tissue of an organism, or to another defined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0038] The terms “modify” and “alter” typically include an “increase,” “enhancement,” or “stimulation,” as well as a “decrease” or “reduction,” of a statistically significant or physiologically significant amount or degree compared to a control. An “increased,” “stimulated,” or “enhanced” amount is typically a “statistically significant” amount and may include increases of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (all integers greater than 1 and decimals between them, e.g., 1.5, 1.6, 1.7, 1.8, etc.) from the amount or level produced by the control composition, sample, or test subject. The “reduction” or amount of “reduction” is typically a “statistically significant” amount and may include reductions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the amount or level of the resulting control composition, sample, or test subject. As one non-limiting example, the comparison may be between the amount or level of pharmacokinetic parameters / profiles or biological / therapeutic responses resulting from the administration of a low-dose formulation (e.g., 1–10 μg / kg) of KLK1 compared to the administration of a high-dose formulation of KLK1. Other examples of comparisons and "statistically significant" quantities are described herein.

[0039] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably and refer to polymers of amino acids, not limited to any particular length. The term “enzyme” includes polypeptide or protein catalysts. This term includes modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The terms “polypeptide” or “protein” refer to one or more amino acid chains, each chain containing amino acids covalently linked by peptide bonds, and the polypeptide or protein may include multiple chains covalently linked together by peptide bonds having non-covalent and / or native proteins, i.e., sequences of proteins naturally occurring in specific non-recombinant cells, or proteins produced by genetically engineered or recombinant cells, and include molecules having the amino acid sequence of a native protein, or molecules having deletions, additions, and / or substitutions of one or more amino acids of a native sequence. In certain embodiments, a polypeptide is a “recombinant” polypeptide produced by a recombinant cell, which contains one or more recombinant DNA molecules consisting of heterogeneous polynucleotide sequences or combinations of polynucleotide sequences not normally found in cells.

[0040] The term "reference sequence" generally refers to a nucleic acid coding sequence or amino acid sequence that is being compared to another sequence. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those listed by name and those listed in tables and sequence listings.

[0041] Results are usually called "statistically significant" if they are unlikely to occur by chance. The significance level of a test or result traditionally relates to the amount of evidence needed to accept that an event is unlikely to occur by chance. In certain cases, statistical significance can be defined as the probability of making a decision that rejects the null hypothesis when the null hypothesis is actually true (a decision known as a Type I error, or a "false positive"). Often, this decision is made using a p-value. If the p-value is lower than the significance level, the null hypothesis is rejected. The smaller the p-value, the larger the result. Bayesian factors can also be used to determine statistical significance (see Goodman, Ann Intern Med. 130:1005-13, 1999).

[0042] The term "solubility" refers to the property of the KLK1 polypeptide provided herein to dissolve in a liquid solvent to form a homogeneous solution. Solubility is typically expressed as a concentration, either by the mass of solute per unit volume of solvent (e.g., g of solute per 1 kg of solvent, g per dL (100 mL), mg / ml), molar concentration, molar ratio, or any other similar description of concentration. The maximum equilibrium amount of solute that can dissolve per unit volume of solvent is the solubility of the solute in that solvent under specific conditions such as temperature, pressure, pH, and the properties of the solvent. In certain embodiments, solubility is measured at physiological pH or other pH levels, e.g., pH 6.0, pH 7.0, pH 7.4, pH 8.0, or pH 9.0. In certain embodiments, solubility is measured in water or in physiological buffers such as PBS or NaCl (with or without NaP). In certain embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt levels (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature may be near room temperature (e.g., about 20, about 21, about 22, about 23, about 24, or about 25°C) or near body temperature (37°C). In certain embodiments, the KLK1 polypeptide has a solubility of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, or at least about 60 mg / ml at room temperature or 37°C.

[0043] "Substantially" or "essentially" means almost entirely or completely, for example, 95%, 96%, 97%, 98%, 99% or more of a given quantity.

[0044] As used herein, “treatment” or “to treat” includes any desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include minimal change or reduction in one or more measurable markers of the disease or condition being treated. “Treatment” or “to treat” does not necessarily indicate the complete eradication or cure of the disease or condition or its associated symptoms. The subjects receiving this treatment are those who need it. Illustrative markers of clinical improvement will be apparent to those skilled in the art.

[0045] As used herein, the terms “therapeutically effective amount,” “therapeutic dose,” “prophylactically effective amount,” or “diagnostically effective amount” refer to the amount of the drug (e.g., KLK1 polypeptide or its dosage form) required to elicit a desired biological response after administration.

[0046] As used herein, “subjects” include animals exhibiting or at risk of exhibiting symptoms that can be treated with the KLK1 polypeptide or its dosage form. Suitable subjects (patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock, and domesticated animals or pets (such as cats and dogs). Non-human primates, preferably human patients, are also included.

[0047] "Isolated" means a substance that substantially or essentially does not contain the components normally associated with it in its original state. For example, as used herein, "isolated peptide" or "isolated polypeptide" includes in vitro isolation and / or purification of peptide or polypeptide molecules derived from their natural cellular environment and from association with other cellular components, i.e., not significantly related to in vivo substances such as host cell proteins or nucleic acids.

[0048] A "wild-type" or "reference" sequence, or a "wild-type" or "reference" protein / polypeptide sequence, can be a reference sequence from which a mutant polypeptide is induced by the introduction of a mutation. Generally, the "wild-type" amino acid sequence of a given protein is the most common sequence in its original form. Similarly, the "wild-type" gene sequence is the polynucleotide sequence of that gene most commonly found in nature. Mutations can be introduced into a "wild-type" gene (i.e., the protein it encodes) either through natural processes or artificial means.

[0049] Each embodiment described herein shall apply to all other embodiments unless otherwise specified.

[0050] Dosage form Embodiments of this disclosure relate to dosage forms comprising one or more tissue kallikrein (KLK1) polypeptides formulated in total doses of KLK1 polypeptides ranging from approximately 0.1 μg / kg to approximately 5 μg / kg or approximately 10.0 μg / kg. In some cases, the dosage forms are suitable (or adapted) for subcutaneous or intravenous administration to subjects, such as human subjects.

[0051] The specific dosage forms are approximately 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0. ,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0,5.1,5.2,5.3, Contains, consists of, essentially consists of, or comprises total doses of KLK1 polypeptides of 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 μg / kg (including all ranges in between).

[0052] For example, specific dosage forms include approximately 0.1 μg / kg to 10.0 μg / kg, approximately 0.1 μg / kg to 9.0 μg / kg, approximately 0.1 μg / kg to 8.0 μg / kg, approximately 0.1 μg / kg to 7.0 μg / kg, approximately 0.1 μg / kg to 6.0 μg / kg, approximately 0.1 μg / kg to 5.0 μg / kg, or approximately 0.1 μg / kg to 4.0 μg / kg, or approximately 0.1 μg / kg to 3.0 μg / kg, or approximately 0.1 μg / kg to 2.0 μg / kg, or approximately 0.01 μg / kg to 1.0 μg / kg, or approximately 0.5 μg / kg to 10.0 μg / kg. Approximately 0.5 μg / kg to approximately 9.0 μg / kg, approximately 0.5 μg / kg to approximately 8.0 μg / kg, approximately 0.5 μg / kg to approximately 7.0 μg / kg, approximately 0.5 μg / kg to approximately 6.0 μg / kg, approximately 0.5 μg / kg to approximately 5.0 μg / kg, or approximately 0.5 μg / kg to approximately 4.0 μg / kg, or approximately 0.5 μg / kg to approximately 3.0 μg / kg, or approximately 0.5 μg / kg to approximately 2.0 μg / kg, or approximately 0.5 μg / kg to approximately 1.0 μg / kg, or approximately 1.0 μg / kg to approximately 10.0 μg / kg, approximately 1.0 μg / kg to approximately 9.0 μg / kg, approximately 1.0 μg / kg to approximately 8.0 μg / kg, approximately 1.0 μg / kg to approximately 7.0 μg / kg, approximately 1.0 μg / kg to approximately 6.0 μg / kg, approximately 1.0 μg / kg to approximately 5.0 μg / kg, or approximately 1.0 μg / kg to approximately 4.0 μg / kg, or approximately 1.0 μg / kg to approximately 3.0 μg / kg, or approximately 1.0 μg / kg to approximately 2.0 μg / kg, or approximately 2.0 μg / kg to approximately 10.0 μg / kg, or approximately 2.0 μg / kg to approximately 9.0 μg / kg, or approximately 2.0 μg / kg to approximately 8.0 μg / kg, or approximately 2.0 μg / kg to approximately 7.0 μg / kg, or approximately 2.0 μg / kg to approximately 6.0 μg / kg g, or approximately 2.0 μg / kg to approximately 5.0 μg / kg, or approximately 2.0 μg / kg to approximately 4.0 μg / kg, or approximately 2.0 μg / kg to approximately 3.0 μg / kg, or approximately 3.0 μg / kg to approximately 10.0 μg / kg, or approximately 3.0 μg / kg to approximately 9.0 μg / kg, or approximately 3.0 μg / kg to approximately 8.0 μg / kg, or approximately 3.0 μg / kg to approximately 7.0 μg / kg, or approximately 3.0 μg / kg to approximately 6.0 μg / kg, or approximately 3.0 μg / kg to approximately 5.0 μg / kg, or approximately 3.0 μg / kg to approximately 4.0 μg / kg, or approximately 4.0 μg / kg to approximately 10.0 μg / kg, or approximately 4.0 μg / kg to approximately 9.0 μg / kg, or approximately 4.0 μg / kg to approximately 8.0 μg / kg, or approximately 4.0 μg / kg to approximately 7.0 μg / kg, or approximately 4.0 μg / kg to approximately 6.0 μg / kg, or approximately 4.0 μg / kg to approximately 5.0 μg / kg, or approximately 5.0 μg / kg to approximately 10.0 μg / kg, or approximately 5.0 μg / kg to approximately 9.0 μg / kg, or approximately 5.0 μg / kg to approximately 8.0 μg / kg, or approximately 5.0 μg / kg to approximately 7.0 μg / kg, or approximately 5.0 μg / kg to approximately 6.0 μg / kg, or approximately 6.0 μg / kg to approximately 10.0 μg / kg, The total dose of KLK1 polypeptides is approximately 6.0 μg / kg to 9.0 μg / kg, or approximately 6.0 μg / kg to 8.0 μg / kg, or approximately 6.0 μg / kg to 7.0 μg / kg, or approximately 7.0 μg / kg to 10.0 μg / kg, or approximately 7.0 μg / kg to 9.0 μg / kg, or approximately 7.0 μg / kg to 8.0 μg / kg, or approximately 8.0 μg / kg to 10.0 μg / kg, or approximately 8.0 μg / kg to 9.0 μg / kg, or approximately 9.0 μg / kg to 10.0 μg / kg. Certain dosage forms include those with a total KLK polypeptide dose of approximately 2.5 μg / kg to approximately 3.5 μg / kg, or approximately 3 μg / kg, and are suitable for intravenous administration. Other specific dosage forms include those with a total KLK polypeptide dose of approximately 0.5 μg / kg to approximately 1.0 μg / kg, or approximately 0.75 μg / kg, and are suitable for intravenous administration.

[0053] Tissue kallikrein-1 (KLK1) polypeptide. As mentioned above, certain dosage forms contain one or more tissue kallikrein-1 or KLK1 polypeptides. Tissue kallikreins are members of a gene superfamily of serine proteases containing at least 15 distinct proteins (named tissue kallikreins 1-15) (Yousef et al., 2001, Endocrine Rev; 22: 184-204). Tissue kallikrein-1 is a trypsin-like serine protease. In human and animal tissues, tissue kallikrein-1 cleaves kininogen into lysyl-bradykinin (also known as kallidin), and the decapeptide kinin has physiological effects similar to those of bradykinin. Bradykinin is a peptide that dilates blood vessels, thereby lowering blood pressure. Kallidin is identical to bradykinin, with an additional lysine residue added to the N-terminus, and signals via the bradykinin receptor.

[0054] The KLK1 gene is 262 amino acid residues long and encodes a single prepro enzyme containing a "pre" sequence (residues 1-18) and a "pro" sequence (residues 19-24), which is activated by a trypsin-like enzyme. The "mature" and "active" forms of human KLK1 are glycoproteins of approximately 238 amino acid residues (residues 25-262) with a molecular weight of 26 kDa and a theoretical pI value of 4.6. In the tertiary structure of KLK1, which is thought to be responsible for the protein's high stability against both trypsin digestion and thermal inactivation, KLK1 has five disulfide bonds.

[0055] The amino acid sequences of tissue kallikrein-1 are from humans (SEQ ID NOs. 1 and 2), mice (e.g., GenBank:AAA39349.1, see February 1, 1994), domestic cats (e.g., NCBI reference sequence:XP_003997527.1, see November 6, 2012), gorillas (e.g., NCBI reference sequence:XP_004061305.1, see December 3, 2012), and cattle ( For example, see GenBank:AAI51559.1, August 2, 2007), dogs (for example, see CBI reference sequence:NP_001003262.1, February 22, 2013), rats (for example, see GenBank:CAE51906.1, April 25, 2006), and olive baboons (for example, see NCBI reference sequence:XP_003916022.1, September 4, 2012). KLK1 is functionally conserved across species for its ability to release the vasoactive peptide Lys bradykinin from low molecular weight kininogen. The tissue kallikrein-1 polypeptide of the present invention may have any of the known amino acid sequences of KLK1, or fragments or variants thereof.

[0056] In certain embodiments, the KLK1 polypeptide is a “mature” KLK1 polypeptide. In certain embodiments, the KLK1 polypeptide is a human KLK1 polypeptide, optionally a mature human KLK1 polypeptide. In certain embodiments, the KLK1 polypeptide is a recombinant human polypeptide, e.g., optionally a recombinant human KLK1 polypeptide in a mature form. Recombinant human KLK1 (rhKLK1) can offer certain advantages over other KLK1 sources, such as urinary KLK1 (e.g., human KLK1 isolated from human urine), and include homogeneous rhKLK1 preparations, modulo for easier regulatory approval, and the option to alter the amino acid sequence or glycosylation pattern based on cell culture conditions.

[0057] Exemplary amino acid sequences of human tissue kallikrein-1 (hKLK1) polypeptides are provided in Table K1 below. [Table K1]

[0058] In certain embodiments, the KLK1 polypeptide comprises, consists of, or is essentially composed of residues 1-262, residues 19-262, or residues 25-262 (including fragments and variants thereof) of SEQ ID NOs: 1-3 or SEQ ID NO: 4, or SEQ ID NOs: 1 or 2. Amino acids 1-18 of SEQ ID NOs: 1 and 2 represent the signal peptide, amino acids 19-24 represent the propeptide sequence, and amino acids 25-262 represent the mature peptide. Thus, the preproprotein comprises a putative 17-amino acid signal peptide, a 7-amino acid proenzyme fragment, and a 238-amino acid mature KLK1 protein.

[0059] A comparison of SEQ ID NOs: 1 and 2 (or SEQ ID NOs: 3 and 4) shows a difference of two amino acids between two hKLK1 amino acid sequences. Single nucleotide polymorphisms (SNPs) between two individuals within the same species explain the E-to-Q substitution at position 145 of 262 amino acid residues and the A-to-V substitution at position 188 of 262. SEQ ID NOs: 1 has E (glutamic acid) at position 145 and A (alanine) at position 188, while SEQ ID NOs: 2 has Q (glutamine) at position 145 and V (valine) at position 188. In some embodiments, the KLK1 polypeptide has E at position 145, Q at position 145, A at position 188, A at position 188, E at position 145 and A at position 188, Q at position 145 and V at position 188, or E at position 145 and V at position 188.

[0060] As described above, certain embodiments include active variants and fragments of the reference KLK1 polypeptide. A “variant” of a starting or reference polypeptide is a polypeptide having an amino acid sequence different from the amino acid sequence of the starting or reference polypeptide. Such variants include, for example, deletions, insertions, and / or substitutions of residues in the amino acid sequence of the polypeptide of interest. In this context, a mutant amino acid refers to an amino acid different from the amino acid at the corresponding position in the starting or reference polypeptide sequence. Any combination of deletions, insertions, and substitutions can be performed to arrive at a final variant or mutant construct, provided that the final construct possesses the desired functional properties. Changes in amino acids can also alter the post-translational processes of the polypeptide, such as changes in the number or location of glycosylation sites.

[0061] In some embodiments, the KLK 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 with respect to a reference sequence such as the amino acid sequences described herein (e.g., SEQ ID NOs. 1-4).

[0062] In some embodiments, the 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 with respect to SEQ ID NO: 1 or 3, or fragments of SEQ ID NO: 1 such as residues 25-262 or residues 78-141. Such a KLK1 polypeptide may have E or Q at amino acid residue 145 and / or A or V at position 188.

[0063] In some embodiments, the 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 with respect to SEQ ID NO: 2 or 4, or fragments of SEQ ID NO: 2 such as residues 25-262 or residues 78-141. Such a KLK1 polypeptide may have E or Q at amino acid residue 145 and / or A or V at position 188.

[0064] The "amino acid sequence identity percentage (%)" for polypeptides is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference sequence after aligning the sequences and introducing gaps, achieving the maximum sequence identity percentage if necessary, and not considering conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to achieve the maximum alignment over the entire length of the sequences being compared. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California.

[0065] For the purposes of this specification, the amino acid sequence identity % of a given amino acid sequence A, or the amino acid sequence identity % of a given amino acid sequence B (which can also be rephrased as a given amino acid sequence A having and containing a specific amino acid sequence identity to a given amino acid sequence B) can be calculated as follows: X / Y 100 times, where X is the number of amino acid residues scored as identical by the program's sequence alignment program in the program's alignment of A and B, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A to B is not equal to the amino acid sequence identity % of B to A.

[0066] The variants may also include heterologous sequences or chemical modifications added to the reference KLK1 polypeptide to facilitate purification, improve metabolic half-life, or facilitate polypeptide identification. Examples include His tags, Fc regions, and / or affinity tags such as PEGylated sequences and PEG.

[0067] The term “fragment” includes smaller portions of the KLK1 polypeptide (or its variants) that retain the activity of the KLK1 polypeptide. Fragments include, for example, KLK1 polypeptide fragments with sizes ranging 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, KLK1 polypeptide fragments are in the range of about 50 to about 100, about 50 to about 150, about 50 to about 200, or about 50 to about 250 amino acid lengths. In other embodiments, KLK1 polypeptide fragments are in the range of 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 acid lengths. In other exemplary embodiments, the KLK1 polypeptide fragments are in the size range of about 200 to about 250 amino acid lengths. Certain embodiments include full-length KLK1 polypeptide fragments with about, at most 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 (e.g., consecutive) amino acid residues. In some embodiments, the fragments may have residues 25-262 or 78-141 of the preproprotein sequence. In some embodiments, the fragments may be any such fragment size as SEQ ID NO: 1 or SEQ ID NO: 2, as described above.

[0068] In some cases, fragments and variants of the KLK1 polypeptide retain the enzymatic ability to release the vasoactive peptide Lys-bradykinin from low molecular weight kininogen. In some embodiments, active variants or fragments retain the serine protease activity of the KLK1 polypeptide to cleave kininogen-like substrates such as D-val-leu-arg-7 amide-4-trifluoromethylcoumarin, releasing calidine from high molecular weight precursors such as kininogen, or releasing a colorimetric or fluorescently quantifiable fragment. The protease activity of the KLK1 polypeptide can be measured by enzyme activity assays by measuring either the cleavage of low molecular weight kininogen or the production of lys-bradykinin. In one assay configuration, a labeled substrate reacts with the KLK1 glycoform, and the release of the labeled fragment is detected. An example of such a fluorescence-generating substrate suitable for measuring KLK1 activity is D-val-leu-arg-7 amide-4-trifluoromethylcoumarin (D-VLR-AFC, FW597.6) (Sigma, Cat#V2888 or Ana Spec Inc Cat#24137). When D-VLR-AFC is hydrolyzed, the free AFC produced in the reaction can be quantified by fluorescence detection (excitation 400 nm, emission 505 nm) or spectrophotometric detection at 380 nm (absence coefficient = 12,600, pH 7.2). Other methods and substrates may also be used to measure KLK1 proteolytic activity.

[0069] Glycoforms and mixtures thereof. In certain embodiments, the dosage form comprises a mixture of one or more KLK1 polypeptide glycoforms, and includes dosage forms comprising specified proportions of bi- and tri-glycosylated KLK1 polypeptides (see U.S. Application No. 14 / 677,122, which is incorporated by reference in its entirety).

[0070] Human kallikrein has three potential Asn-linked (N-linked) glycosylation sites at residues 78, 84, and 141 relative to the mature amino acid sequence and presumed O-linked glycosylation site shown, for example, in SEQ ID NO: 3 or 4. However, O-linked glycosylation is not detected in natural KLK1. SDS-PAGE analysis detects KLK1 polypeptides glycosylated at all three positions (positions 78, 84, and 141) as a high molecular weight band and is referred to herein as the high molecular weight triply glycosylated glycoform of KLK1 (or "high glycoform" or "triply glycoform" KLK1). SDS-PAGE analysis detects KLK1 polypeptides glycosylated at only two of the three available positions (positions 78 and 84) as a low molecular weight band and is referred to as the low molecular weight biglycosylated glycoform of KLK1 (or "low glycoform" or "biglycosylated" KLK1).

[0071] Therefore, a particular dosage form comprises, for example, a mixture of KLK1 glycoforms in a defined ratio, comprising a first KLK1 polypeptide and a second KLK1 polypeptide, wherein the first KLK1 polypeptide has three glycans bonded to three different positions available for polypeptide glycosylation, and the second KLK1 polypeptide has two glycans bonded to only two of the three different positions available for polypeptide glycosylation. In certain embodiments, the first and second KLK1 polypeptides are present in the dosage form in a ratio of about 45:55 to about 55:45, including, for example, about 46:54, about 47:53, about 48:52, about 49:51, about 51:49, about 52:48, about 53:47, and about 54:46 (including all integers and decimals in between). In certain embodiments, the first and second KLK1 polypeptides are present in the dosage form in a ratio of about 50:50. In some embodiments, the ratio of the first to second KLK1 polypeptides is not approximately 60:40. In some embodiments, the ratio of the first to second KLK1 polypeptides is not approximately 40:60. In certain embodiments, the dosage form does not contain, or substantially does not contain, other glycosylated isoforms (glycoforms) of KLK1.

[0072] Some dosage forms contain a triple glycoform and a biglyceride of the KLK1 polypeptide, and the triple and biglycerides are present in the dosage form in ratios of approximately 45:55 to approximately 55:45, including, for example, approximately 46:54, approximately 47:53, approximately 48:52, approximately 49:51, approximately 51:49, approximately 52:48, approximately 53:47, and approximately 54:46. In some embodiments, the triple and biglycerides are present in the dosage form in a ratio of approximately 50:50. In some embodiments, the ratio of the triple and biglycerides is not approximately 60:40. In some embodiments, the ratio of the triple and biglycerides is not approximately 40:60. In certain embodiments, the dosage form does not contain, or substantially does not contain, other glycosylated isoforms (glycoforms) of KLK1.

[0073] The ratio of the bi and triglycosylated isoforms of KLK1 can be detected and quantified by various methods, including reversed-phase high-performance liquid chromatography (RP-HPLC), lectin affinity chromatography, and high-performance liquid chromatography (HPLC), which may include lectin affinity electrophoresis. The preparation and characterization of the KLK1 glycoform mixture are described in U.S. Patent Application No. 14 / 677,122, which is incorporated in its entirety by reference.

[0074] Additional agents. In certain embodiments, the dosage form includes another additional therapeutic agent, e.g., a second therapeutic agent. In some embodiments, the additional agent is selected from one or more angiotensin receptor blockers, edavalone, finelenone, and bardoxalon (including combinations thereof). Examples of angiotensin receptor blockers include losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, supplementsartan, telmisartan, and valsartan (including combinations thereof).

[0075] Purity. In some embodiments, the "purity" of a dosage form is characterized by, for example, the amount (e.g., total amount, relative amount, percentage) of host cell proteins, host cell DNA, or endotoxin, and / or the percentage of single-peak purity by SEC HPLC. In some cases, the purity of a dosage form is characterized by the amount (e.g., percentage) of other components, such as KLK1 polypeptide compared to one or more of the aforementioned.

[0076] In some embodiments, the purity of the dosage form is characterized in relation to, or by, the level or amount of host cell protein. The host cells used for recombinant expression can range from bacteria and yeasts to cell lines derived from mammalian or insect species. The cells contain hundreds to thousands of host cell proteins (HCPs) and other biomolecules that may contaminate the final product. HCPs can be secreted along with the protein of interest or released by accidental cell lysis, potentially contaminating the protein of interest. Two types of immunological methods can be applied to HCP analysis: Western blotting (WB) and immunoassay (IA), which include techniques such as ELISA and sandwich immunoassay, or similar methods using radioactive, luminescent, or fluorescent reporting labels. The compositions of the present invention may contain less than about 500 ng, less than about 400 ng, less than about 300 ng, less than about 200 ng, less than about 100 ng, or less than about 50 ng of host cell protein per 1 mg of total protein.

[0077] In some cases, purity is characterized in relation to or by the level or amount of host cell DNA. Detection of residual host cell DNA can be performed by polymerase chain reaction (PCR) using various primers for sequences within the host cell genome. Residual host cell DNA is generally reported to be below a certain threshold level, but it can also be quantified by rPCR. The compositions of the present invention may contain less than about 100 pg, less than about 90 pg, less than about 80 pg, less than about 70 pg, less than about 60 pg, less than about 50 pg, less than about 40 pg, less than about 30 pg, less than about 20 pg, or less than about 10 pg / mg of host cell deoxyribonucleic acid (DNA) per 1 mg of total protein.

[0078] In certain embodiments, purity is characterized in relation to or by the amount or level of endotoxin. As described herein, endotoxin is highly potent, thermally stable, passes through sterile membrane filters, and is present wherever bacteria are present or have been present. An endotoxin unit (EU) is a unit of biological activity in the USP Reference Endotoxin Standard.

[0079] The Bacterial Endotoxin Test (BET) is a test that uses horseshoe crab (Limulus polyfemus or Tachypleus tridentatus) amoeboid cell lysates (leukocytes) to detect or quantify endotoxins from Gram-negative bacteria. FDA-approved Limulus amoeba cell lysates (LAL) reagents are used in all USP endotoxin tests. There are at least three methods for this test: Method A, a gel coagulation technique based on gel formation; Method B, a turbidimetric method based on the development of turbidity after cleavage of the endogenous substrate; and Method C, a colorimetric method based on the development of color after cleavage of synthetic peptide-chromogen complexes.

[0080] At least two types of endotoxin tests are available in the USP <85> As described in the BET, photometric testing requires a spectrophotometer, endotoxin-specific software, and printing capabilities. The simplest photometric system is a handheld unit employing a disposable LAL cartridge containing dry, pre-calibrated reagents; no liquid reagents or standards are required. FDA-approved units are marketed under the name Endosafe®-PTS®. The device takes approximately 15 minutes to analyze small sample volumes, specifically 25 μL aliquots of diluted CSP in sterile tubing, and print out the results. In contrast, the gel coagulation method requires a dry heat block, calibrated pipettes and thermometer, vortex mixer, lyophilized LAL reagent, LAL reagent water (LRW) for hydrating the reagent, and pyrotherm removal glassware. In this coagulation test, the diluted sample and liquid reagent require approximately 1 hour for sample and positive control preparation, 1 hour incubation on the heat block, and the results are manually recorded. Thus, the simplicity and speed of automated systems make them ideally suited to pharmaceutical settings.

[0081] In some cases, the purity of a dosage form is characterized by the degree of aggregation. For example, the degree of aggregation of KLK1 can be measured by size exclusion chromatography (SEC), which separates particles based on size. This is a commonly accepted method for determining the tertiary and quaternary structures of purified proteins. SEC is primarily used for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of the particles. Larger molecules simply pass through the pores because they are too large to enter them. Therefore, larger molecules flow through the column faster than smaller molecules, meaning that smaller molecules have longer retention times. Certain compositions also contain virtually no aggregates (more than about 95% appear as a single peak by SEC HPLC). Certain embodiments contain more than 96%, 97%, 98%, or 99% aggregates that appear as a single peak by SEC HPLC.

[0082] In certain embodiments, the "purity" of the KLK1 polypeptide in the dosage form is specifically defined. For example, a particular dosage form contains one or more hKLK1 polypeptides that are 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% (including all decimals in between) purity compared to other components of the dosage form. Purity can be measured without limitation, for example, by high-performance liquid chromatography (HPLC), a well-known form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0083] In certain embodiments, the dosage form has one or more of the following purity determinations: less than approximately 1 EU of endotoxin per 1 mg of protein, less than approximately 100 ng of host cell protein per 1 mg of protein, less than approximately 10 pg of host cell DNA per 1 mg of protein, and / or a single peak purity of more than approximately 95% by SEC HPLC.

[0084] In some cases, the dosage form is formulated with pharmaceutically acceptable excipients, diluents, adjuvants, or carriers, for example, to optimize stability and obtain isotonicity. In certain embodiments, the pH of the dosage form is physiological pH or about pH 7.4 and includes about pH 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.5, or any range thereof. In some embodiments, the dosage form comprises a KLK1 polypeptide combined with a physiologically acceptable carrier. Such carriers include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the doses and concentrations used. Methods of formulation are well known in the art and are disclosed, for example, in Remington's *The Science and Practice of Pharmacy*, Mack Publishing Company, Easton, Pa., Edition 21 (2005).

[0085] The terms "physiologically acceptable" or "pharmaceutically acceptable" refer to molecular entities and compositions that, when administered to humans, do not produce significant allergic reactions or similar adverse reactions. Typically, such compositions are prepared as injections, either as liquid solutions or suspensions. Solid forms suitable for liquid solutions or suspensions before injection can also be prepared. Preparations can also be emulsified.

[0086] As used herein, “carrier” includes all solvents, dispersions, vehicles, coatings, diluents, isotonic agents, and absorption retarders, buffers, carrier solutions, suspensions, colloids, etc. It is intended for use in therapeutic compositions unless any conventional medium or agent is incompatible with the active ingredient.

[0087] The dosage forms described herein can be formulated for administration by various techniques, including, for example, subcutaneous and intravenous administration. Certain embodiments include administration by subcutaneous injection. In some cases, subcutaneous injections (abbreviated as SC, SQ, sub-cu, sub-Q, or subcut including SQ) are administered as a bolus into the subcutaneous tissue, which is the layer of skin directly beneath the dermis and epidermis, collectively referred to as the dermis. Exemplary locations on the body where a person can most easily inject SC include, but are not limited to, the lateral region of the upper arm just above and below the waist, excluding a specific side, the area around the navel (about a 2-inch circle), the upper part of the buttocks just behind the hip bone, and the anterior part of the thigh midway outward, from about 4 inches below the top of the thigh to about 4 inches above the knee. These areas vary depending on the person's size. Also, changing the injection site helps prevent the formation of lumps or small depressions in the skin, known as lipoatrophy.

[0088] Subcutaneous injections are typically administered into the fatty tissue beneath the skin, and in certain cases, smaller and shorter needles may be used. In certain instances, needles with a gauge of approximately 25 to 31 and a length of approximately 1 / 2 to 5 / 8 inches are sufficient for subcutaneous administration of medication. As those skilled in the art will understand, these are general recommendations, and SC injections can be administered with other sizes of needles. In some embodiments, SC administration is performed by pinching the tissue to prevent injection into the muscle and / or by inserting the needle at an angle of approximately 45 degrees to the skin.

[0089] Furthermore, methods for treating subjects in need thereof include administering effective doses of the dosage forms described herein to the subject. For example, certain embodiments include methods for treating ischemic conditions, vascular dementia, hemorrhagic conditions, traumatic brain injury (TBI), diabetes, or kidney disease.

[0090] Therefore, in some embodiments, the subjects have ischemic conditions. Non-limiting examples include cerebral ischemia (ischemic stroke), transient ischemic attack (TIA), myocardial ischemia, ischemic colitis, limb ischemia, and cutaneous ischemia. In some embodiments, the subjects have vascular dementia. In some embodiments, the subjects have hemorrhagic conditions, such as hemorrhagic strokes including intracerebral (internal brain) hemorrhagic stroke and subarachnoid hemorrhagic stroke. In some embodiments, the subjects have diabetes, such as type 2 diabetes (T2D). In certain embodiments, the subjects have traumatic brain injury (TBI). In some embodiments, the subjects have kidney disease, such as chronic kidney disease, diabetic nephropathy, or polycystic kidney disease. In some embodiments, the subjects have systemic lupus erythematosus (SLE), or related conditions or complications such as lupus nephritis. In certain embodiments, the subject has pulmonary hypertension (PAH), focal segmental glomerulosclerosis, or essential hypertension. These and related medical conditions can be diagnosed according to the conventional techniques of the art.

[0091] In certain cases, administration of the dosage form achieves therapeutically effective serum levels of one or more KLK1 polypeptides in the subject. In some cases, administration of the dosage form achieves therapeutically effective serum levels of one or more KLK1 polypeptides approximately or at least approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours after administration. In some cases, the dosage form is administered intravenously or subcutaneously. In some cases, a therapeutically effective serum level is approximately or at least approximately 1.0 to approximately or at least approximately 5.0 ng / ml, or approximately or at least approximately 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg / ml (including all ranges in between).

[0092] In some cases, the administration of the dosage form achieves and maintains therapeutically effective serum levels of one or more KLK1 polypeptides in the subject. For example, in some embodiments, the administration of the dosage form achieves therapeutically effective serum levels of one or more KLK1 polypeptides in about or at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours after administration, and maintains therapeutically effective serum levels of one or more KLK1 polypeptides in the subject for about or at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more after administration, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more. In some cases, a therapeutically effective serum level is approximately or at least approximately 1.0 to approximately or at least approximately 5.0 ng / ml, or approximately or at least approximately 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg / ml (including all ranges in between).

[0093] In some cases, administration of a dosage form achieves or produces an improved pharmacokinetic profile or biological (e.g., therapeutic) effect compared to higher-dose KLK1 polypeptide dosage forms. For example, in some cases, subcutaneous administration of a dosage form achieves an improved pharmacokinetic profile or biological (e.g., therapeutic) effect compared to dosage forms with a total KLK1 polypeptide dose of at least about 15 μg / kg, or at least about 20 μg / kg, or at least about 50 μg / kg, or at least about 100 μg / kg, or at least about 400 μg / kg or more. In some cases, the improved pharmacokinetic profile includes an increased serum half-life after a single subcutaneous or intravenous administration, measured at approximately or at least approximately 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more after subcutaneous administration (e.g., single subcutaneous administration), or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more.

[0094] Certain embodiments include a dosing plan in which one or more KLK1 dosage forms are administered at specified intervals over a period of time. For example, a particular dosing plan may include administering a KLK1 dosage form once or twice daily, once or twice every two days (e.g., once every other day), once or twice every three days (e.g., once every three days after the initial or initial dose), once or twice every four days, once or twice every five days, once or twice every six days, once or twice every week, or once or twice every other week. Another particular dosing plan may include administering a KLK1 dosage form once daily every three days (e.g., once daily every three days after the initial or initial dose), with the dosage form being administered subcutaneously.

[0095] Certain embodiments include intravenous administration of at least one intravenous dosage form followed by subcutaneous administration of one or more subcutaneous dosage forms as a dosing schedule, for example, once or twice per day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, or once or twice every week. In certain embodiments, intravenous administration or dosage forms achieve therapeutically effective serum levels of one or more KLK1 polypeptides in the subject approximately 0.5, 1, 2, 3, or 4 hours after intravenous administration, or less than approximately 0.5, 1, 2, 3, or 4 hours; subcutaneous administration or dosage forms maintain therapeutically effective serum levels for approximately or at least approximately 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, or 96 hours or longer after subcutaneous administration, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or longer.

[0096] Some variation in dosage is inevitable depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for each individual subject. Furthermore, for administration to humans, preparations should be sterile, pyrogenic, and meet the general safety and purity standards required by the FDA. In some cases, as described herein, preparations are substantially endotoxin-free or pyrogenic-free. According to FDA guidance for industry, Estimation of Maximum Safe Starting Dose in Early Clinical Trials of Therapeutics in Healthy Adult Volunteers (July 2005), Appendix D: Converting Animal Dose to Human Equivalent Dose. The human equivalent dose is 1 / 7 of the rat dose, and the human equivalent dose is 1 / 12 of the mouse dose.

[0097] In some embodiments, the dosage forms described herein are administered in conjunction with one or more additional therapeutic agents or treatment methods. In some embodiments, the administration of the dosage forms enables the efficacy of lower doses of other therapeutic methods compared to the administration of other therapeutic methods alone, and reduces the toxicity observed with higher doses of other therapeutic methods. One or more additional therapeutic agents may be administered before, after, and / or simultaneously (e.g., together) the administration of the dosage forms described herein. The dosage forms and additional therapeutic agents may be administered separately or as part of the same mixture or cocktail. When used herein, additional therapeutic agents include, for example, agents known to those skilled in the art for use in the treatment of medical conditions (e.g., ischemic or hemorrhagic conditions). Examples of additional agents include angiotensin receptor blockers, edavalone, finelenone, and bardoxalone (including combinations thereof). Specific examples of angiotensin receptor blockers include losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, supplementsartan, telmisartan, and valsartan (including combinations thereof).

[0098] Devices. Also included are devices containing the dosage forms described herein, including devices suitable for subcutaneous or intravenous delivery. In some embodiments, the device is a syringe. In some embodiments, the syringe is attached to a subcutaneous needle assembly and optionally includes a protective cover around the needle assembly. In some embodiments, the needle may be about 1 / 2 inch to about 5 / 8 inch in length and have a gauge of about 25 to about 31. Thus, certain embodiments include devices attached to or attachable to a needle assembly suitable for subcutaneous administration, containing the dosage forms described herein. For example, certain devices include a vial or syringe, where optionally the vial or syringe is attachable to or attached to the subcutaneous needle assembly. Also included are vials having a rubber cap, into which a needle / syringe can be inserted through the rubber cap to extract the dosage form for subcutaneous administration.

[0099] In certain embodiments, the device is a syringe that can be attached to or is attached to a subcutaneous injection needle and is packaged with one or more removable and / or permanent protective covers around the needle or needle assembly. For example, a first removable protective cover (removed during administration) can protect the user or other persons from the needle before administration, and a second protective cover can be placed in position (i.e., snapped) after administration for safe disposal of the device.

[0100] The present invention will be illustrated by the following examples. It should be understood that specific examples, substances, quantities, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as described herein. [Examples]

[0101] Example 1 Pharmacokinetics of low-dose KLK1 formulations in humans The pharmacokinetics of a low-dose KLK1 formulation were evaluated in humans. The formulation consists of a mixture of bi and triglycosylated isoforms of mature human KLK1, prepared as described in U.S. Patent Application No. 14 / 677,122 (incorporated by reference).

[0102] The single-dose comparative study was designed to establish the safety, tolerability, and pharmacokinetics of the KLK1 formulation after a 30-minute intravenous infusion and a single subcutaneous injection. The intravenous dose was 0.75 μg / kg, and the subcutaneous dose was 3 μg / kg. Each study group consisted of 12 volunteers: 6 women and 6 men.

[0103] Intravenous doses were delivered to the clinical site as a 104.4 mg / ml formulation dissolved in phosphate-buffered saline (pH=7.2). The amount of KLK1 polypeptide required for administration was calculated based on each study volunteer's body weight and dose level. Subcutaneous doses were provided as a 26.1 mg / ml solution in phosphate-buffered saline. Prior to administration, this dose was diluted to 2.61 mg / ml with saline, and the infusion volume was adjusted according to the study participant's body weight.

[0104] Plasma KLK1 concentrations were measured by ELISA using KLK1-specific antibodies. This method has been developed and validated for use in human clinical trials.

[0105] As shown in Figures 2A-2B, subcutaneous administration of a low-dose formulation of mature human KLK1 glycoform (3 μg / kg, n=12) not only achieves effective plasma levels of KLK1 but also significantly prolongs the serum half-life compared to intravenous administration of a low-dose formulation (0.75 μg / kg, n=12) in healthy human subjects. Intravenous administration of the low-dose formulation of mature human KLK1 glycoform rapidly achieved effective plasma levels of KLK1. Figure 2A shows serum levels 80 hours after administration, and Figure 2B shows serum levels 4 hours after administration.

[0106] The pharmacokinetic (PK) profiles of two KLK1 subcutaneous administration strategies (3 μg / kg and 15 / 25 μg / kg) in healthy volunteers were also evaluated in a Phase I clinical trial. The results are shown in Figure 3. Points represent the mean values ​​obtained from 6 participants per group. Arrows represent administration time. The 3 μg / kg dose, considered the target dose from the latest patent, maintained fairly stable drug levels at desirable or therapeutically effective serum / plasma concentrations of approximately 3–5 ng / ml. In contrast, higher doses showed greater dose-to-dose variability and resulted in proportionally higher levels.

[0107] A dose trial of KLK1 was conducted in patients with type 2 diabetes. The results are shown in Figures 4A-4D. The key point is the result of a food tolerance test conducted 2 hours after administration, using HOMA2-IR measurement of insulin resistance 3 hours after administration. The numerical values ​​are a derivative scale of insulin resistance, with higher values ​​indicating higher insulin resistance and a greater likelihood of disease. Figure 4D shows the results for the placebo group, and Figure 4C shows the results for the drug group (DM199). After the trial on day 1 was performed as baseline, single doses at the specified dose level were administered from the following day. Insulin resistance improved on day 5 when the low dose was tested compared to day 8 when the high dose was tested (Figure 4C), suggesting that the low dose was more effective.

[0108] A 28-day multi-dose trial was also conducted in patients with type 2 diabetes (n=12-13 per group). The results are shown in Figures 5A-5B. The bars represent the mean ± SEM change from baseline in serum creatinine and urea concentrations. These are typical measures of renal function, and values ​​exceeding normal indicate renal impairment. These results indicate that the low dose (3 μg / kg) had a significantly greater effect than the high dose on improving the measurement of renal function. The P-value is based on a comparison between the 3 μg / kg group and placebo.

[0109] Although the above invention has been described in some detail by examples and embodiments for the purpose of clarifying understanding, it will be readily apparent to those skilled in the art that, in view of the teachings of the present invention, certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0110] All patents, patent applications, and publications in their entirety, and all electronically available materials cited herein (including, for example, nucleotide sequence submissions in GenBank and RefSeq, amino acid sequence submissions in SwissProt, PIR, PRF, PDB, and translations from annotated coding areas in GenBank and RefSeq), are incorporated by reference. The above detailed description and examples are provided solely for clarity of understanding. No unnecessary limitations should be understood therefrom. The present invention is not limited to the exact details shown and described, and modifications that are obvious to those skilled in the art are included within the present invention as defined by the claims.

Claims

1. A subcutaneous formulation for use in a method of treating cerebral ischemia (ischemic stroke) in a subject, comprising a first human tissue kallikrein (hKLK1) polypeptide and a second hKLK1 polypeptide, formulated at a total hKLK1 polypeptide dose of approximately 2.0 μg / kg to approximately 5.0 μg / kg, The first and second hKLK1 polypeptides are mature recombinant hKLK1 polypeptides containing amino acid sequences having at least 90% sequence identity with SEQ ID NO: 3 or 4. The first hKLK1 polypeptide has three N-linked glycans bound at residues 78, 84, and 141 as defined by SEQ ID NO: 3 or 4, and the second hKLK1 polypeptide has two N-linked glycans bound at residues 78 and 84 as defined by SEQ ID NO: 3 or 4, but not bound at residue 141. A subcutaneous dosage form in which the first hKLK1 polypeptide and the second hKLK1 polypeptide are present in a ratio of 45:55 to 55:45, and the preparation is not used in combination with insulin.

2. The subcutaneous formulation according to Claim 1, comprising a total dose of hKLK1 polypeptide of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 μg / kg.

3. A subcutaneous formulation according to Claim 1, comprising a total subcutaneous dose of hKLK1 polypeptide of approximately 2.0 μg / kg to approximately 5.0 μg / kg, or approximately 2.0 μg / kg to approximately 4.0 μg / kg, or approximately 2.0 μg / kg to approximately 3.0 μg / kg, or approximately 3.0 μg / kg to approximately 5.0 μg / kg, or approximately 3.0 μg / kg to approximately 4.0 μg / kg, or approximately 2.5 μg / kg to approximately 3.5 μg / kg, or approximately 3 μg / kg.

4. The subcutaneous dosage form formulation according to any one of claims 1 to 3, wherein the first hKLK1 polypeptide and the second hKLK1 polypeptide are present in the formulation in a ratio of approximately 50:

50.

5. The subcutaneous formulation according to any one of claims 1 to 4, wherein the hKLK1 polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3 or 4.

6. The subcutaneous formulation according to claim 5, wherein the hKLK1 polypeptide comprises the amino acid sequence of SEQ ID NO:

4.

7. A subcutaneous dosage form formulation according to any one of claims 1 to 6, comprising a pharmaceutically acceptable excipient, diluent, adjuvant, or carrier.

8. A subcutaneous dosage form according to any one of claims 1 to 7, substantially free of a glycosylated isoform (glycoform) of hKLK1 different from that of the first hKLK1 polypeptide or the second hKLK1 polypeptide.

9. A subcutaneous formulation according to any one of claims 1 to 8, having an endotoxin level of less than about 1 EU per 1 mg of protein, less than about 100 ng of host cell protein per 1 mg of total protein, less than about 10 pg of host cell DNA per 1 mg of total protein, and / or being substantially free of aggregates (more than 95% appearing as a single peak by SEC HPLC).

10. A subcutaneous formulation according to any one of claims 1 to 9, further comprising a second agent selected from one or more (including combinations thereof) of angiotensin receptor blockers, edavalone, finelenone, and bardoxalon.

11. The subcutaneous formulation according to claim 10, wherein the angiotensin receptor blocker is selected from one or more of losartan, azicilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, supplementartan, telmisartan, and valsartan (including combinations thereof).

12. The subcutaneous dosage form formulation according to any one of claims 1 to 11, wherein subcutaneous administration of the subcutaneous dosage form formulation achieves a therapeutically effective serum level of the hKLK1 polypeptide in the subject, and optionally maintains the therapeutically effective serum level for about or at least about 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more after subcutaneous administration.

13. The subcutaneous formulation according to claim 12, wherein the therapeutically effective serum level is approximately 1.0 to approximately 5.0 ng / ml, or approximately 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg / ml.

14. The subcutaneous dosage form formulation according to any one of claims 1 to 13, wherein the administration of the subcutaneous dosage form formulation achieves an improved pharmacokinetic profile or biological effect compared to formulations formulated with a total dose of hKLK1 polypeptide of at least about 15 μg / kg, or at least about 20 μg / kg, or at least about 50 μg / kg, or at least about 100 μg / kg, or at least about 400 μg / kg or more.

15. The subcutaneous dosage form formulation according to any one of claims 1 to 14, wherein the method comprises administering the subcutaneous dosage form formulation to the subject as a dosage schedule of approximately once or twice per day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, and once or twice every week.

16. The subcutaneous dosage form formulation according to claim 15, wherein the method comprises administering the subcutaneous dosage form formulation to the subject as an administration plan of approximately once a day every three days.

17. The subcutaneous dosage form according to any one of claims 1 to 16, wherein the method comprises intravenously administering one intravenous dosage form to the subject, and subsequently subcutaneously administering one or more of the subcutaneous dosage form to the subject as an optional administration schedule of approximately once or twice per day, once or twice every two days, once or twice every three days, once or twice every four days, once or twice every five days, once or twice every six days, or once or twice every week, wherein the intravenous dosage form comprises the first hKLK1 polypeptide and the second hKLK1 polypeptide administered before the subcutaneous dosage form and formulated at a total dose of 0.5 to 1.0 μg / kg of hKLK1 polypeptide, and the first hKLK1 polypeptide and the second hKLK1 polypeptide are present in the intravenous dosage form in a ratio of 45:55 to 55:

45.

18. The subcutaneous formulation according to claim 17, wherein the intravenous administration achieves a therapeutically effective serum level of the hKLK1 polypeptide in the subject approximately 0.5, 1, 2, 3, or 4 hours after the intravenous administration, or less than approximately 0.5, 1, 2, 3, or 4 hours after the intravenous administration, and the subcutaneous administration maintains the therapeutically effective serum level for approximately or at least approximately 2, 4, 6, 8, 10, 12, 24, 23, 48, 60, 72, 84, 96 hours or more, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more after the subcutaneous administration.

19. The subcutaneous formulation according to any one of claims 1 to 18, wherein the method comprises administering a second agent selected from one or more (including combinations thereof) of angiotensin receptor blockers, edavalone, finelenone, and bardoxalon as part of the same or different formulation or composition.

20. The subcutaneous formulation according to claim 19, wherein the angiotensin receptor blocker is selected from one or more of losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, supplementartan, telmisartan, and valsartan (including combinations thereof).

21. A device comprising a subcutaneous dosage form formulation according to any one of claims 1 to 20, the device being suitable for subcutaneous delivery of the formulation.

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