Dosage forms of tissue kallikrein-1

Abstract

To provide dosage forms of tissue kallikrein-1.SOLUTION: Provided are dosage forms of one or more tissue kallikrein-1 (KLK1) polypeptides which have a total KLK1 polypeptide dosage of 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 conditions. Embodiments of the present disclosure relate to the unexpected discovery that formulations of tissue kallikrein-1 (KLK1) have an inverse dose curve, where up to a certain point the administration of lower-dosage formulations show an improved pharmacokinetic and / or activity profile relative to higher-dosage formulations.SELECTED DRAWING: 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 October 3, 2017, U.S. Provisional Patent Application No. 62 / 516,463, filed June 7, 2017, and U.S. Provisional Patent Application No. 62 / 469,385, filed March 9, 2017, each of which is incorporated by reference in its entirety.

[0002] Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file name containing the sequence listing is DIAM_037_03WO_ST25.txt. This text file is 9KB, was created on March 9, 2018, and has been submitted electronically 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, including subcutaneous and intravenous dosage forms, having a total KLK1 polypeptide dosage of about 0.1 μg / kg to about 10.0 μg / kg. Also provided are related devices and methods of use thereof, for example, to treat ischemic and hemorrhagic conditions. [Background technology]

[0004] 2. Description of Related Art All tissue kallikreins have protease activity with substrate specificity similar to that of trypsin or chymotrypsin. The best-characterized activity of KLK1 is its enzymatic cleavage of kininogens to generate bradykinin (BK)-like peptides, collectively called kinins, that directly or indirectly activate both subtypes of bradykinin receptors (BK-B1, BK-B2). Activation of BK receptors by kinins sets in motion a number of complex metabolic pathways in response to ischemia in the body, which may include improved blood flow (through vasodilation), anti-inflammatory responses, cellular repair through angiogenesis or vasculogenesis, and reduced apoptosis. There is a considerable amount of scientific research showing that tissue kallikrein-mediated release increases blood flow in various tissues, including the kidney and heart (see, for example, Stone et al., Arterioscler Thromb Vasc Biol.29:657-664,2009), which makes kallikrein therapy likely to be one way of addressing certain pathologies.KLK1 is therefore believed to have the potential to treat a wide range of clinical scenarios in which re-establishing blood flow and reducing inflammation in patients is essential to maintaining organ function, including brain function, kidney function, and heart function.However, there remains a need to identify optimal dosage forms and administration routes that achieve and maintain therapeutic levels of KLK1 in humans.The present 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 Summary of the Invention

[0006] Quick Overview Embodiments of the present disclosure relate to the unexpected discovery that formulations of tissue kallikrein-1 (KLK1) have an inverted dose curve, whereby administration of a low dose formulation up to a certain point exhibits an improved pharmacokinetic and / or activity profile compared to a high dose formulation.

[0007] Thus, certain embodiments include dosage forms comprising one or more tissue kallikrein (KLK1) polypeptides formulated at a total KLK1 polypeptide dosage of about 1.0 μg / kg to about 5.0 μg / kg or to about 10.0 μg / kg. In certain embodiments, the dosage form is suitable for subcutaneous or intravenous administration.

[0008] In some embodiments, the dosage form comprises about 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, 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 therebetween) of KLK1 polypeptide total dose.

[0009] Particular dosage forms include a total KLK1 polypeptide subcutaneous administration dosage form of about 1.0 μg / kg to about 4.0 μg / kg, or about 1.0 μg / kg to about 3.0 μg / kg, or about 1.0 μg / kg to about 2.0 μg / kg, or about 2.0 μg / kg to about 5.0 μg / kg, or about 2.0 μg / kg to about 4.0 μg / kg, or about 2.0 μg / kg to about 3.0 μg / kg, or about 3.0 μg / kg to about 5.0 μg / kg, or about 3.0 μg / kg to about 4.0 μg / kg, or about 2.5 μg / kg to about 3.5 μg / kg, or about 3 μg / kg.

[0010] Particular dosage forms include an intravenous dosage form of about 0.5 μg / kg to about 3.0 μg / kg, or about 0.5 μg / kg to about 2.0 μg / kg, or about 0.5 μg / kg to about 1.0 μg / kg, or about 0.5 μg / kg to about 0.8 μg / kg, or about 0.5 μg / kg to about 0.75 μg / kg, or about 0.75 μg / kg of total KLK1 polypeptide.

[0011] Some dosage forms include a mixture of KLK1 glycoforms, e.g., 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 about 45:55 to about 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 KLK1 polypeptide and the second KLK1 polypeptide are present in the dosage form in a ratio of about 50:50.

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

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

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

[0016] In some embodiments, the hKLK1 polypeptide comprises, consists of, or consists essentially of amino acid residues 25-262 of SEQ ID NO:1 or amino acid residues 25-262 of SEQ ID NO:2, or an active fragment thereof, or an active variant having at least about 90, 95, 96, 97, 98, or 99% sequence identity to amino acid residues 25-262 of SEQ ID NO:1 or amino acid residues 25-262 of 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 pharma- ceutically acceptable diluent, adjuvant, or carrier.In some embodiments, the dosage form is substantially free of other glycosylated isoforms (glycoforms) of KLK1.

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

[0020] In some embodiments, the dosage includes a second drug, such as an angiotensin receptor blocker, selected from one or more of edabalon, finelenone, and valdoxalone (including combinations thereof). In some embodiments, the angiotensin receptor blocker is selected from one or more of losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan (including combinations thereof).

[0021] Also included are methods of treating a subject in need thereof, comprising administering a dosage form described herein to the subject. Some embodiments include administering the dosage form subcutaneously or intravenously to the subject. Certain methods relate to treating an ischemic condition in a subject, optionally selected from one or more of cerebral ischemia (ischemic stroke), transient ischemic attack (TIA), cardiac ischemia (myocardial ischemia), ischemic colitis, limb ischemia, and cutaneous ischemia. Certain methods relate to treating vascular dementia. Some methods relate to treating a hemorrhagic condition in a subject, optionally hemorrhagic stroke, including intracerebral (inside the brain) hemorrhagic stroke and subarachnoid hemorrhagic stroke. Some methods relate to treating diabetes, e.g., type 2 diabetes (T2D). Certain embodiments relate to treating traumatic brain injury (TBI). Some embodiments relate to treating kidney disease, e.g., chronic kidney disease, diabetic kidney disease, or polycystic kidney disease. Also included are methods of treating systemic lupus erythematosus (SLE) and related conditions or complications such as lupus nephritis, pulmonary arterial hypertension (PAH), focal segmental glomerulosclerosis, and essential hypertension.

[0022] In some embodiments, subcutaneous administration of the dosage form achieves a therapeutically effective serum level of one or more KLK1 polypeptides in the subject, and optionally maintains a therapeutically effective serum level of one or more KLK1 polypeptides in the subject 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. In some embodiments, intravenous administration of the dosage form to the subject achieves a therapeutically effective serum level of one or more KLK1 polypeptides in the subject, optionally within about 0.5, 1, 2, 3, or 4 hours, or within less than about 0.5, 1, 2, 3, or 4 hours after intravenous administration. In some embodiments, the therapeutically effective serum level is about 1.0 to about 5.0 ng / ml, or about 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, 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, 8.1, 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, 10.0, 10.1, 10.2, 10.3, 10 8, 4.9, or 5.0 mg / ml (including all ranges therebetween), 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 compared to a high dose dosage form, for example, 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 an increase in serum half-life after a single subcutaneous administration, for example, 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 on a dosing schedule of about 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 per week. Certain embodiments include administering the dosage form to a subject on a dosing schedule of about once per day, for example, by subcutaneous administration, every three days.

[0025] Certain embodiments include administering one intravenous dosage form intravenously to a subject, followed by administering one or more subcutaneous dosage forms subcutaneously to a subject, e.g., on a dosing schedule of about 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 per week. In some embodiments, intravenous administration achieves a therapeutically effective serum level of one or more KLK1 polypeptides in a subject at 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] Certain methods include administering a second agent selected from one or more of the following angiotensin receptor blockers, edabalon, finelenone, and valdoxalone (including combinations thereof), 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 of the following angiotensin receptor blockers, including combinations thereof: losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan.

[0027] Also included are devices comprising the dosage forms described herein that are adapted or suitable for subcutaneous administration. In some embodiments, the device is a syringe. In some embodiments, the syringe comprises a hypodermic needle assembly attached to the syringe. In some embodiments, the syringe comprises 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 comprising one or more tissue kallikrein (KLK1) polypeptides formulated at a total dosage of about 0.5 μg / kg to about 10.0 μg / kg of KLK1 polypeptide. (Item 2) 2. The dosage form according to 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, 3. The dosage form of item 1 or 2, comprising a total administered amount 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) 4. The dosage form according to any one of items 1 to 3, comprising a total KLK1 polypeptide subcutaneous dosage form of about 1.0 μg / kg to about 5.0 μg / kg, or about 1.0 μg / kg to about 4.0 μg / kg, or about 1.0 μg / kg to about 3.0 μg / kg, or about 1.0 μg / kg to about 2.0 μg / kg, or about 2.0 μg / kg to about 5.0 μg / kg, or about 2.0 μg / kg to about 4.0 μg / kg, or about 2.0 μg / kg to about 3.0 μg / kg, or about 3.0 μg / kg to about 5.0 μg / kg, or about 3.0 μg / kg to about 4.0 μg / kg, or about 2.5 μg / kg to about 3.5 μg / kg, or about 3 μg / kg. (Item 5) 5. The dosage form according to any one of items 1 to 4, comprising an intravenous dosage form of about 0.5 μg / kg to about 3.0 μg / kg, or about 0.5 μg / kg to about 2.0 μg / kg, or about 0.5 μg / kg to about 1.0 μg / kg, or about 0.5 μg / kg to about 0.8 μg / kg, or about 0.5 μg / kg to about 0.75 μg / kg, or about 0.75 μg / kg of total KLK1 polypeptide. (Item 6) A first KLK1 polypeptide and a second 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; 6. 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 about 45:55 to about 55:45. (Item 7) 7. The dosage form of item 6, wherein one or more of the glycans is an N-linked glycan. (Item 8) 8. The dosage form of item 6 or 7, wherein one or more of the glycans are linked to amino acid residues 78, 84, or 141 of KLK1, as defined by SEQ 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) 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) 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 about 50:50. (Item 12) 6. The dosage form of any one of items 1 to 5, wherein the dosage form comprises a triple glycoform of a KLK1 polypeptide and a double glycoform of a KLK1 polypeptide, and the triple glycoform and the double glycoform are present in the dosage form in a ratio of about 45:55 to about 55:45. (Item 13) 13. The dosage form of item 12, wherein the triple glycoform comprises N-linked glycans at amino acid residues 78, 84, and 141 of KLK1 as defined by SEQ ID NO: 3 or 4. (Item 14) 14. The dosage form of item 12 or 13, wherein the dual glycoform comprises N-linked glycans at amino acid residues 78 and 84 of KLK1, but not at amino acid residue 141 of KLK1, as defined by SEQ ID NO: 3 or 4. (Item 15) 15. The dosage form according to any one of items 12 to 14, wherein the triple glycoform and the dual glycoform are present in the dosage form in a ratio of about 50:50. (Item 16) 16. The dosage form of any one of items 1 to 15, wherein the one or more KLK1 polypeptides are recombinant KLK1 polypeptides, mature KLK1 polypeptides, human KLK1 (hKLK1) polypeptides, or any combination thereof. (Item 17) 17. The dosage form of item 16, wherein the hKLK1 polypeptide comprises, consists of, or consists essentially of amino acid residues 78 to 141 of SEQ ID NO:1 or amino acid residues 78 to 141 of SEQ ID NO:2, or an active fragment thereof, or an active variant having at least about 90, 95, 96, 97, 98, or 99% sequence identity to amino acid residues 78 to 141 of SEQ ID NO:1 or amino acid residues 78 to 141 of SEQ ID NO:2. (Item 18) 17. The dosage form according to item 16, wherein the hKLK1 polypeptide comprises, consists of, or consists essentially of amino acid residues 25 to 262 of SEQ ID NO:1 or amino acid residues 25 to 262 of SEQ ID NO:2, or an active fragment thereof, or an active variant having at least about 90, 95, 96, 97, 98, or 99% sequence identity to amino acid residues 25 to 262 of SEQ ID NO:1 or amino acid residues 25 to 262 of SEQ ID NO:2. (Item 19) 17. The dosage form of item 16, wherein 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 to 262 of SEQ ID NO:2, and the KLK1 polypeptide comprises E145 and / or A188. (Item 20) 17. The dosage form of item 16, wherein 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 to 262 of SEQ ID NO:2, and the KLK1 polypeptide comprises Q145 and / or V188. (Item 21) 21. The dosage form according to any one of items 1 to 20, comprising a pharma- ceutically acceptable excipient, diluent, adjuvant, or carrier. (Item 22) 22. The dosage form according to any one of items 1 to 21, which is substantially free of other glycosylated isoforms (glycoforms) of KLK1. (Item 23) 23. The dosage form of any one of items 1 to 22, having an endotoxin level of less than about 1 EU per mg of protein, less than about 100 ng of host cells per mg of total protein, less than about 10 pg of host cell DNA per mg of total protein, and / or being substantially free of aggregates (greater than about 95% appearing as a single peak by SEC HPLC). (Item 24) 24. The dosage form of any one of items 1 to 23, further comprising a second agent selected from one or more of angiotensin receptor blockers, edabalon, finerenone, and valdoxalone (including combinations thereof). (Item 25) 25. The dosage form of item 24, wherein the angiotensin receptor blocker is selected from one or more of losartan, adisartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan (including combinations thereof). (Item 26) 26. A method for treating a subject in need thereof, comprising administering to said subject a dosage form according to any one of items 1 to 25. (Item 27) 28. The method of claim 26, comprising administering the dosage form to the subject subcutaneously or intravenously. 28. The method according to item 26 or 27, for treating an ischemic condition in the subject, optionally selected from one or more of cerebral ischemia (ischemic stroke), cardiac ischemia (myocardial ischemia), ischemic colitis, limb ischemia, and cutaneous ischemia. (Item 29) 28. The method according to item 26 or 27, for treating a hemorrhagic condition in said subject, optionally a hemorrhagic stroke, including intracerebral (inside the brain) hemorrhagic stroke and subarachnoid hemorrhagic stroke. (Item 30) 28. The method according to item 26 or 27, for treating a disease selected from one or more of vascular dementia, diabetes (optionally type 2 diabetes (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 arterial hypertension (PAH), focal segmental glomerulosclerosis, and essential hypertension. (Item 31) 31. The method of any one of items 26 to 30, comprising subcutaneously administering the dosage form to the subject, wherein subcutaneous administration of the dosage form achieves a therapeutically effective serum level of the 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more following 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 intravenous administration of the dosage form achieves a therapeutically effective serum level of the one or more KLK1 polypeptides in the subject, optionally at about 0.5, 1, 2, 3, or 4 hours or less after the intravenous administration. 33. The method of claim 31 or 32, wherein the therapeutically effective serum level is about 1.0 to about 5.0 ng / ml, or about 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 of 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, optionally 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. (Item 35) 35. The method of any one of items 26-34, comprising administering the dosage form to the subject as a dosing regimen of about 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 per week. (Item 36) 36. The method of claim 35, comprising administering the dosage form to the subject on a dosing regimen of about once a day every three days, optionally by subcutaneous administration. (Item 37) 37. The method of any one of items 26-36, comprising administering one intravenous dosage form intravenously to said subject, optionally followed by subcutaneous administration of one or more subcutaneous dosage forms to said subject on a dosing regimen of about 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, once or twice per week. (Item 38) 38. The method of item 37, wherein the intravenous administration achieves a therapeutically effective serum level of the one or more KLK1 polypeptides in the subject at or less than about 0.5, 1, 2, 3, or 4 hours after the intravenous administration, and the subcutaneous administration 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 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 of any one of items 26 to 38, optionally as part of the same or a different dosage form or composition, further comprising administering a second agent selected from one or more of angiotensin receptor blockers, edabalon, finerenone, and valdoxalone (including combinations thereof). (Item 40) 40. The method of claim 39, wherein the angiotensin receptor blocker is selected from one or more of losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, telmisartan, and valsartan (including combinations thereof). (Item 41) 26. A device comprising the dosage form according to any one of items 1 to 25, wherein the device is suitable for delivering said dosage form subcutaneously or intravenously. [Brief description of the drawings]

[0028] [Figure 1]Results of a Phase I / II preliminary study of KLK1 (DM199) in humans for the treatment of type 2 diabetes (T2D) are presented. Patients (n=37) were administered placebo or one of two doses of KLK1 (high dose of 15 μg / kg, or low dose of 3 μg / kg) every 3 days for 28 days, and fasting blood glucose (FBG) levels were measured. The high dose (15 μg / kg) showed no significant effect, while the low dose (3 μg / kg) showed a statistically significant (p<0.05) effect on FBG levels compared to baseline. Thus, the low dose of 3 μg / kg provides an unexpected improvement compared to the high dose of 15 μg / kg. [Figure 2A] We show that in healthy human subjects, subcutaneous administration of a low-dose formulation of mature human KLK1 glycoform (3 μg / kg, n=12) results in a significant increase in serum half-life compared to intravenous administration of a low-dose formulation (0.75 μg / kg, n=12). [Figure 2B] We show that in healthy human subjects, subcutaneous administration of a low-dose formulation of mature human KLK1 glycoform (3 μg / kg, n=12) results in a significant increase in serum half-life compared to intravenous administration of a low-dose formulation (0.75 μg / kg, n=12). [Diagram 3] Results from clinical trials show that a KLK1 (DM199) dose of 3 μg / kg maintained fairly stable drug concentrations with desired or therapeutically effective serum / plasma concentrations of approximately 3-5 ng / ml. In contrast, the higher doses of 15 / 25 μg / kg resulted in proportionally higher plasma levels with greater dose-to-dose variability. [Figure 4A] The results of a KLK1 administration study conducted in type 2 diabetes patients are shown. Figure 4A shows a summary, Figures 4B and 4D show the results of the placebo group, and Figure 4C shows the results of the (KLK1)DM199 group. The numbers are a derived measure of insulin resistance (HOMA2-IR), with higher numbers representing higher insulin resistance and more severe disease. [Figure 4B]The results of a KLK1 administration study conducted in type 2 diabetes patients are shown. Figure 4A shows a summary, Figures 4B and 4D show the results of the placebo group, and Figure 4C shows the results of the (KLK1)DM199 group. The numbers are a derived measure of insulin resistance (HOMA2-IR), with higher numbers representing higher insulin resistance and more severe disease. [Figure 4C] The results of a KLK1 administration study conducted in type 2 diabetes patients are shown. Figure 4A shows a summary, Figures 4B and 4D show the results of the placebo group, and Figure 4C shows the results of the (KLK1)DM199 group. The numbers are a derived measure of insulin resistance (HOMA2-IR), with higher numbers representing higher insulin resistance and more severe disease. [Figure 4D] The results of a KLK1 administration study conducted in type 2 diabetes patients are shown. Figure 4A shows a summary, Figures 4B and 4D show the results of the placebo group, and Figure 4C shows the results of the (KLK1)DM199 group. The numbers are a derived measure of insulin resistance (HOMA2-IR), with higher numbers representing higher insulin resistance and more severe disease. [Figure 5A] presents the results of a 28-day multiple-dose study in type 2 diabetes patients (n = 12-13 per group) on measures of renal function (creatinine, blood urea). The results showed that the low dose (3 μg / kg) produced significantly more effect than the high dose (15 μg / kg) on ​​improving measures of renal function. [Figure 5B] presents the results of a 28-day multiple-dose study in type 2 diabetes patients (n = 12-13 per group) on measures of renal function (creatinine, blood urea). The results showed that the low dose (3 μg / kg) produced significantly more effect than the high dose (15 μg / kg) on ​​improving measures of renal function. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

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

[0031] "About" means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0032] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of the stated step or element or group of steps or elements, but the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that no other elements may be present. "Consisting essentially of" means including the elements recited after this phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they materially affect the activity or action of the recited 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 mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis, as well as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine, and the like. Unnatural amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, which are known to those skilled in the art. Amino acid analogs include modified forms of natural and unnatural amino acids. Such modifications can include, for example, by substitution or replacement of chemical groups and moieties on the amino acid, or by derivatization of the amino acid. Amino acid mimetics include organic structures that exhibit functionally similar properties, such as, for example, charge and charge spacing characteristic of the reference amino acid. For example, an organic structure that mimics arginine (Arg or R) would have a positively charged moiety that is located in a similar molecular space and has the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures to maintain optimal spacing and charge interactions of 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 mimetics.

[0034] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to dosage forms, compositions, vehicles, devices, and / or vessels that contain at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects on a subject), preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain bacteria, usually gram-negative bacteria, although endotoxins can be found in gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), found in the outer membrane of various gram-negative bacteria, and represent a central pathogenic feature of the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can produce fever, decreased blood pressure, and activation of inflammation and coagulation, among other physiological side effects.

[0035] Therefore, in pharmaceutical manufacturing, it is often desirable to remove most or all traces of endotoxin from the drug and / or drug container, since even small amounts can cause side effects in humans. A depyrogenation oven can be used for this purpose, since temperatures above 300°C are usually required to break down most endotoxins. For example, based on primary packaging materials such as syringes or vials, a glass temperature of 250°C in combination with a holding time of 30 minutes is sufficient to achieve a 3-log reduction in endotoxin levels. Other methods of removing endotoxin are contemplated, including, for example, chromatography and filtration methods described herein and known in the art. Also included are methods of producing KLK1 polypeptides in eukaryotic cells, such as mammalian cells, and isolating them from eukaryotic cells to reduce, if not eliminate, the risk that endotoxins are present in the compositions of the invention. Preferred are methods of producing KLK1 polypeptides and isolating them from recombinant cells grown in a chemically defined serum-free medium.

[0036] Endotoxin can be detected using routine techniques known in the art. For example, the Limulus amoebocyte lysate assay, which utilizes horseshoe crab blood, is a highly sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the Limulus lysate due to a powerful enzyme cascade that amplifies this reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, the level of endotoxin can be less than about 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 10 EU per mg of protein or per ml. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1-10 EU.

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

[0038] The terms "modulate" and "modify" generally include "increase," "enhancement," or "stimulation," as well as "decrease" or "reduction," of a statistically significant or physiologically significant amount or degree relative to a control. An "increased," "stimulated," or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (all integers greater than 1 and decimal points therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) the amount or level produced by a control composition, sample, or test subject. A "reduction" or amount of "reduction" is usually a "statistically significant" amount and may include a 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% reduction in the amount or level of a resulting control composition, sample, or test subject. As one non-limiting example, a comparison may be made between the amount or level of a pharmacokinetic parameter / profile or biological / therapeutic response resulting from administration of a low dosage form (e.g., 1-10 μg / kg) of KLK1 versus administration of a high dosage form of KLK1. Other examples of comparisons and "statistically significant" amounts are described herein.

[0039] The terms "polypeptide", "protein" and "peptide" are used interchangeably and refer to a polymer of amino acids, not limited to a specific length. The term "enzyme" includes polypeptide or protein catalysts. The term includes modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of a signal sequence. The term "polypeptide" or "protein" refers 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 that are non-covalently linked and / or have the sequence of a native protein, i.e., a protein produced by a specific non-recombinant cell occurring in nature, or a genetically engineered or recombinant cell, and includes 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 the native sequence. In certain embodiments, a polypeptide is a "recombinant" polypeptide produced by a recombinant cell that contains one or more recombinant DNA molecules that are made of a heterologous polynucleotide sequence or combination of polynucleotide sequences not normally found in the cell.

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

[0041] A result is usually called "statistically significant" if it is unlikely to occur by chance. The significance level of a test or result is traditionally related to the amount of evidence needed to accept that an event is unlikely to occur by chance. In certain cases, statistical significance may be defined as the probability of making a decision to reject the null hypothesis when it is actually true (a decision known as a type I error, or "false positive decision"). 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 greater the result. Bayes factors can also be utilized to determine statistical significance (see Goodman, Ann Intern Med. 130:1005-13, 1999).

[0042] The term "solubility" refers to the property of a KLK1 polypeptide provided herein to dissolve in a liquid solvent to form a homogeneous solution. Solubility is usually expressed as a concentration by mass of solute per unit volume of solvent (g solute per kg of solvent, g per dL (100 mL), mg / ml, etc.), molar concentration, molar concentration, molar ratio, or other similar description of concentration. The maximum equilibrium amount of solute that can be dissolved per volume of solvent is the solubility of the solute in that solvent under specific conditions such as temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH or other pH, for example, 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 a physiological buffer such as PBS or NaCl (with or without NaP). In certain embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and a relatively high salt (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, the solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, about 21, about 22, about 23, about 24, or about 25°C) or about 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 nearly entirely or completely, e.g., 95%, 96%, 97%, 98%, 99% or more of a given amount.

[0044] As used herein, "treatment" or "treating" includes any desired 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 "treating" does not necessarily indicate complete eradication or cure of the disease or condition, or its associated symptoms. The subject receiving this treatment is the subject in need thereof. Exemplary 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" are the amount of an agent (e.g., a KLK1 polypeptide or a dosage form thereof) required to elicit a desired biological response following administration.

[0046] As used herein, "subject" includes animals that exhibit or are at risk of exhibiting a condition that can be treated with KLK1 polypeptide or its formulation.Suitable subjects (patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock, and domestic animals or pets (such as cats and dogs).Includes non-human primates, preferably human patients.

[0047] "Isolated" refers to material that is substantially or essentially free from components that normally accompany it in its native state. For example, "isolated peptide" or "isolated polypeptide" as used herein includes the in vitro isolation and / or purification of a peptide or polypeptide molecule from its natural cellular environment and from association with other components of the cell, i.e., not significantly associated with in vivo materials such as host cell proteins or nucleic acids.

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

[0049] Each embodiment in this specification is intended to apply to all other embodiments unless expressly stated otherwise.

[0050] Dosage form Embodiments of the present disclosure relate to dosage forms comprising one or more tissue kallikrein (KLK1) polypeptides formulated at a total KLK1 polypeptide dosage of about 0.1 μg / kg to about 5 μg / kg or about 10.0 μg / kg. In some cases, the dosage form is suitable (or adapted) for subcutaneous or intravenous administration to a subject, e.g., a human subject.

[0051] Particular dosage forms include those having about 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, 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, ​​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 therebetween).

[0052] For example, certain dosage forms may be from about 0.1 μg / kg to about 10.0 μg / kg, from about 0.1 μg / kg to about 9.0 μg / kg, from about 0.1 μg / kg to about 8.0 μg / kg, from about 0.1 μg / kg to about 7.0 μg / kg, from about 0.1 μg / kg to about 6.0 μg / kg, from about 0.1 μg / kg to about 5.0 μg / kg, or from about 0.1 μg / kg to about 4.0 μg / kg, or from about 0.1 μg / kg to about 3.0 μg / kg, or from about 0.1 μg / kg to about 2.0 μg / kg, or from about 0.01 μg / kg to about 1.0 μg / kg, or from about 0.5 μg / kg to about 10.0 μg / kg, about 0.5 μg / kg to about 9.0 μg / kg, about 0.5 μg / kg to about 8.0 μg / kg, about 0.5 μg / kg to about 7.0 μg / kg, about 0.5 μg / kg to about 6.0 μg / kg, about 0.5 μg / kg to about 5.0 μg / kg, or about 0.5 μg / kg to about 4.0 μg / kg, or about 0.5 μg / kg to about 3.0 μg / kg, or about 0.5 μg / kg to about 2.0 μg / kg, or about 0.5 μg / kg to about 1.0 μg / kg, or about 1.0 μg / kg to about 10.0 μg / kg, about 1.0 μg / kg to about 9.0 μg / kg, about 1.0 μg / kg to about 8.0 μg / kg, about 1.0 μg / kg to about 7.0 μg / kg, about 1.0 μg / kg to about 6.0 μg / kg, about 1.0 μg / kg to about 5.0 μg / kg, or about 1.0 μg / kg to about 4.0 μg / kg, or about 1.0 μg / kg to about 3.0 μg / kg, or about 1.0 μg / kg to about 2.0 μg / kg, or about 2.0 μg / kg to about 10.0 μg / kg, or about 2.0 μg / kg to about 9.0 μg / kg, or about 2.0 μg / kg to about 8.0 μg / kg, or about 2.0 μg / kg to about 7.0 μg / kg, or about 2.0 μg / kg to about 6.0 μg / kg g, or from about 2.0 μg / kg to about 5.0 μg / kg, or from about 2.0 μg / kg to about 4.0 μg / kg, or from about 2.0 μg / kg to about 3.0 μg / kg, or from about 3.0 μg / kg to about 10.0 μg / kg, or from about 3.0 μg / kg to about 9.0 μg / kg, or from about 3.0 μg / kg to about 8.0 μg / kg, or from about 3.0 μg / kg to about 7.0 μg / kg, or from about 3.0 μg / kg to about 6.0 μg / kg, or from about 3.0 μg / kg to about 5.0 μg / kg, or from about 3.0 μg / kg to about 4.0 μg / kg, or from about 4.0 μg / kg to about 10.0 μg / kg, or about 4.0 μg / kg to about 9.0 μg / kg, or about 4.0 μg / kg to about 8.0 μg / kg, or about 4.0 μg / kg to about 7.0 μg / kg, or about 4.0 μg / kg to about 6.0 μg / kg, or about 4.0 μg / kg to about 5.0 μg / kg, or about 5.0 μg / kg to about 10.0 μg / kg, or about 5.0 μg / kg to about 9.0 μg / kg, or about 5.0 μg / kg to about 8.0 μg / kg, or about 5.0 μg / kg to about 7.0 μg / kg, or about 5.0 μg / kg to about 6.0 μg / kg, or about 6.0 μg / kg to about 10.0 μg / kg, or or from about 6.0 μg / kg to about 9.0 μg / kg, or from about 6.0 μg / kg to about 8.0 μg / kg, or from about 6.0 μg / kg to about 7.0 μg / kg, or from about 7.0 μg / kg to about 10.0 μg / kg, or from about 7.0 μg / kg to about 9.0 μg / kg, or from about 7.0 μg / kg to about 8.0 μg / kg, or from about 8.0 μg / kg to about 10.0 μg / kg, or from about 8.0 μg / kg to about 9.0 μg / kg, or from about 9.0 μg / kg to about 10.0 μg / kg. Particular dosage forms include those having a total KLK polypeptide dosage of about 2.5 μg / kg to about 3.5 μg / kg, or about 3 μg / kg, suitable for intravenous administration. Particular dosage forms include those having a total KLK polypeptide dosage of about 0.5 μg / kg to about 1.0 μg / kg, or about 0.75 μg / kg, suitable for intravenous administration.

[0053] Tissue kallikrein-1 (KLK1) Polypeptides. As discussed above, certain dosage forms include one or more tissue kallikrein-1 or KLK1 polypeptides. Tissue kallikrein is a member of a genetic superfamily of serine proteases that includes at least 15 separate distinct proteins (designated 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), a decapeptide kinin with physiological effects similar to those of bradykinin. Bradykinin is a peptide that dilates blood vessels, thereby reducing blood pressure. Kallidin is identical to bradykinin, with an additional lysine residue added to the N-terminus, and signals through the bradykinin receptor.

[0054] The KLK1 gene encodes a single preproenzyme that is 262 amino acid residues in length and contains 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" form of human KLK1 is a glycoprotein 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 high stability of the protein against both trypsin digestion and heat inactivation, KLK1 has five disulfide bonds.

[0055] The amino acid sequences of tissue kallikrein-1 are available from human (SEQ ID NO: 1 and SEQ ID NO: 2), mouse (see, e.g., GenBank: AAA39349.1, February 1, 1994), domestic cat (see, e.g., NCBI Reference Sequence: XP_003997527.1, November 6, 2012), gorilla (see, e.g., NCBI Reference Sequence: XP_004061305.1, December 3, 2012), cow ( For example, see GenBank: AAI51559.1, August 2, 2007), dog (see, for example, CBI Reference Sequence: NP_001003262.1, February 22, 2013), rat (see, for example, GenBank: CAE51906.1, April 25, 2006), olive baboon (see, for example, NCBI Reference Sequence: XP_003916022.1, September 4, 2012). KLK1 is functionally conserved among species in 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 a fragment or variant 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, for example, a recombinant human KLK1 polypeptide, optionally 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), including homogenous rhKLK1 preparations, easier regulatory controls, and the option to vary 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 below in Table K1. [Table K1]

[0058] In certain embodiments, the KLK1 polypeptide comprises, consists of, or consists essentially of SEQ ID NOs:1-3 or SEQ ID NO:4, or residues 1-262, 19-262, or 25-262 of SEQ ID NO:1 or SEQ ID NO:2 (including fragments and variants thereof). 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 the predicted 17 amino acid signal peptide, the 7 amino acid proenzyme fragment, and the 238 amino acid mature KLK1 protein.

[0059] Comparison of SEQ ID NO:1 and SEQ ID NO:2 (or SEQ ID NO:3 and SEQ ID NO:4) shows two amino acid differences between the two hKLK1 amino acid sequences. Single nucleotide polymorphisms (SNPs) between two individuals within a species account for an E to Q substitution at amino acid residue 145 of 262 and an A to V substitution at position 188 of 262. SEQ ID NO:1 has an E (glutamic acid) at position 145 and an A (alanine) at position 188, and SEQ ID NO:2 has a Q (glutamine) at position 145 and a V (valine) at position 188. In some embodiments, the KLK1 polypeptide has an E at position 145, a Q at position 145, an A at position 188, an A at position 188, an E at position 145 and an A at position 188, a Q at position 145 and a V at position 188, a Q at position 145 and an A at position 188, or an E at position 145 and a V at position 188.

[0060] As mentioned above, certain embodiments include active variants and fragments of reference KLK1 polypeptide. A "variant" of a starting or reference polypeptide is a polypeptide that has an amino acid sequence that differs from the amino acid sequence of the starting or reference polypeptide. Such variants include, for example, deletion, insertion, and / or substitution of residues in the amino acid sequence of the target polypeptide. In this context, variant amino acid refers to an amino acid that differs from the amino acid at the corresponding position of the starting or reference polypeptide sequence. Any combination of deletion, insertion, and substitution can be performed to arrive at the final variant or variant construct, provided that the final construct possesses the desired functional properties. Amino acid changes can also change the post-translational process of the polypeptide, such as changing the number or position 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 to a reference sequence, such as, for example, an amino acid sequence described herein (e.g., SEQ ID NOs: 1-4).

[0062] In some embodiments, a KLK1 polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to SEQ ID NO: 1 or 3, or a fragment of SEQ ID NO: 1 or 3, such as, for example, residues 25-262 or residues 78-141 of SEQ ID NO: 1. Such a KLK1 polypeptide can have an E or Q at amino acid residue 145 and / or an A or V at position 188.

[0063] In some embodiments, a KLK1 polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% amino acid identity to SEQ ID NO:2 or 4, or a fragment of SEQ ID NO:2 or 4, such as, for example, residues 25-262 or residues 78-141 of SEQ ID NO:2. Such a KLK1 polypeptide can have an E or Q at amino acid residue 145 and / or an A or V at position 188.

[0064] "Percent (%) amino acid sequence identity" with respect to a polypeptide is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. The ALIGN-2 program is published by Genentech, Inc., South San Francisco, California.

[0065] For purposes herein, the % amino acid sequence identity of a given amino acid sequence A, or the % amino acid sequence identity to a given amino acid sequence B, (which can also be said that a given amino acid sequence A has and contains a particular amino acid sequence identity to, as well as to, a given amino acid sequence B), can be calculated as follows: 100 times X / Y, where X is the number of amino acid residues scored as identical matches by a program's sequence alignment program in that 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, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

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

[0067] The term "fragment" includes smaller portions of a KLK1 polypeptide (or variants thereof) that retain the activity of the KLK1 polypeptide. Fragments include, for example, KLK1 polypeptide fragments ranging in size from about 20 to about 50, about 20 to about 100, about 20 to about 150, about 20 to about 200, or about 20 to about 250 amino acids in length. In other embodiments, KLK1 polypeptide fragments range in size from about 50 to about 100, about 50 to about 150, about 50 to about 200, or about 50 to about 250 amino acids in length. In other embodiments, KLK1 polypeptide fragments range in size from about 100 to about 150, about 100 to about 200, about 100 to about 250, about 150 to about 175, about 150 to about 200, or about 150 to about 250 amino acids in length. In other exemplary embodiments, KLK1 polypeptide fragments range in size from about 200 to about 250 amino acids in length. Certain embodiments include polypeptide fragments of full-length KLK1 of about, up to about, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more (e.g., contiguous) amino acid residues. In some embodiments, the fragment can have residues 25-262 or residues 78-141 of the preproprotein sequence. In some embodiments, the fragment can be any such fragment size of SEQ ID NO:1 or SEQ ID NO:2, as described above.

[0068] In some cases, fragments and variants of KLK1 polypeptides retain the enzymatic ability to release the vasoactive peptide Lys-bradykinin from low molecular weight kininogens. In some embodiments, active variants or fragments retain the serine protease activity of KLK1 polypeptides, which cleave substrates similar to kininogens, such as D-val-leu-arg-7 amido-4-trifluoromethylcoumarin, which release kallidin from high molecular weight precursors such as kininogens, or release colorimetric or fluorometric fragments. The protease activity of KLK1 polypeptides can be measured in enzyme activity assays by measuring either the cleavage of low molecular weight kininogens or the production of lys-bradykinin. In one assay format, a labeled substrate reacts with KLK1 glycoforms and the release of labeled fragments is detected. An example of such a fluorogenic substrate suitable for measuring the activity of KLK1 is D-val-leu-arg-7 amido-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 (extinction 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, including dosage forms that contain defined ratios of doubly and triply 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, for example, relative to the mature amino acid sequence shown in SEQ ID NO: 3 or 4, as well as predicted O-linked glycosylation sites. However, O-linked glycosylation is not detected in native KLK1. KLK1 polypeptides glycosylated at all three positions (positions 78, 84, and 141) are detected as high molecular weight bands by SDS-PAGE analysis, and are referred to herein as high molecular weight triple glycosylated glycoforms of KLK1 (or "high glycoform" or "triple glycoform" KLK1). KLK1 polypeptides glycosylated at only two of the three available positions (positions 78 and 84) are detected as low molecular weight bands, and are referred to herein as low molecular weight double glycosylated glycoforms of KLK1 (or "low glycoform" or "double glycoform" KLK1).

[0071] Thus, certain dosage forms include a mixture of KLK1 glycoforms in a defined ratio, for example, including a first KLK1 polypeptide and a second KLK1 polypeptide, where the first KLK1 polypeptide has three glycans attached to three different positions available for glycosylation of the polypeptide, and the second KLK1 polypeptide has two glycans attached to only two of the three different positions available for glycosylation of the polypeptide. 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 therebetween). 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 and second KLK1 polypeptides is not about 60: 40. In some embodiments, the ratio of the first and second KLK1 polypeptides is not about 40: 60. In certain embodiments, the dosage form is free or substantially free of other glycosylated isoforms (glycoforms) of KLK1.

[0072] Some dosage forms include a triple glycoform of a KLK1 polypeptide and a double glycoform of a KLK1 polypeptide, where the triple glycoform and the double glycoform 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. In some embodiments, the triple glycoform and the double glycoform are present in the dosage form in a ratio of about 50:50. In some embodiments, the ratio of the triple glycoform and the double glycoform is not about 60:40. In some embodiments, the ratio of the triple glycoform and the double glycoform is not about 40:60. In certain embodiments, the dosage form is free or substantially free of other glycosylation isoforms (glycoforms) of KLK1.

[0073] The ratio of double and triple glycosylated isoforms of KLK1 can be detected and quantified by a variety of methods, such as reverse 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 KLK1 glycoform mixtures are described in U.S. Patent Application Serial No. 14 / 677,122, which is incorporated by reference in its entirety.

[0074] Additional drug. In certain embodiments, the dosage form comprises one more additional therapeutic agent, for example, a second therapeutic agent. In some embodiments, the additional drug is selected from one or more of the following angiotensin receptor blockers: edabalon, finelenone, and valdoxalone (including combinations thereof). Examples of angiotensin receptor blockers include losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, 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 protein, host cell DNA, endotoxin, and / or 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 KLK1 polypeptide compared to other components, for example, any one or more of the above.

[0076] In some embodiments, the purity of the dosage form is characterized with respect to or by the level or amount of host cell protein. Host cells used for recombinant expression can range from bacteria and yeast to cell lines derived from mammalian or insect species. 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 and may contaminate the protein of interest. Two types of immunological methods can be applied for 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 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 in the host cell genome. Residual host cell DNA is generally reported to be below a certain threshold level, but can also be quantified by rPCR methods. The compositions of the present invention can 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 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 and heat stable, passes through sterilizing membrane filters, and is present wherever bacteria is or has been.Endotoxin Unit (EU) is the unit of biological activity of the USP Reference Endotoxin Standard.

[0079] The bacterial endotoxin test (BET) is a test that uses amebocyte lysates (white blood cells) from horseshoe crabs (Limulus polyphemus or Tachypleus tridentatus) to detect or quantify endotoxins from gram-negative bacteria. The FDA-approved Limulus amebocyte lysate (LAL) reagent is used for all USP endotoxin tests. There are at least three methods for this test: Method A, a gel-clot technique based on gel formation; Method B, a turbidimetric method based on the development of turbidity after cleavage of an endogenous substrate; and Method C, a chromogenic method based on color development after cleavage of a synthetic peptide-chromogen complex.

[0080] At least two types of endotoxin tests are USP <85> It is described in the BET. Photometric testing requires a spectrophotometer, endotoxin-specific software, and printing capabilities. The simplest photometric system is a handheld unit that employs a disposable LAL cartridge containing dry, pre-calibrated reagent; no liquid reagents or standards are required. FDA-approved units are sold under the name Endosafe®-PTS™. The device takes approximately 15 minutes to analyze a small sample, a 25 μL aliquot of CSP diluted in a sterile tube and print out the results. In contrast, the gel clot method requires a dry heat block, calibrated pipettes and thermometer, a vortex mixer, lyophilized LAL reagent, LAL reagent water (LRW) to hydrate the reagent, and depyrogenated glassware. In this clot test, diluted samples and liquid reagents require approximately 1 hour for sample and positive control preparation, 1 hour of incubation in a heat block, and results are recorded manually. Thus, the simplicity and speed of the automated system makes it ideally suited for a pharmacy setting.

[0081] In some cases, the purity of the 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 structure of purified proteins. SEC is primarily used to analyze large molecules such as proteins or polymers. SEC works by trapping these small molecules in the pores of the particles. Larger molecules simply pass through the pores because they are too large to enter them. Thus, larger molecules flow through the column faster than smaller molecules, meaning that smaller molecules have longer retention times. Certain compositions are also substantially free of aggregates (greater than about 95% appear as a single peak by SEC HPLC). Certain embodiments are free of more than about 96%, about 97%, about 98%, or about 99% of aggregates 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 certain dosage form comprises 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 decimal points therebetween) pure, compared with other components of the dosage form.Purity can be measured, without limitation, for example, by high performance liquid chromatography (HPLC), which is a well-known form of column chromatography that is 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 about 1 EU endotoxin per mg protein, less than about 100 ng host cell protein per mg protein, less than about 10 pg host cell DNA per mg protein, and / or a single peak purity of greater than about 95% by SEC HPLC.

[0084] In some cases, the dosage form is formulated with pharma- ceutically acceptable excipients, diluents, adjuvants, or carriers, for example, to optimize stability and obtain isotonicity.In certain aspects, the pH of the dosage form is physiological pH or about pH 7.4, including 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 KLK1 polypeptide combined with a physiologically acceptable carrier.Such carriers include pharma-ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used.Formulation methods are well known in the art and are disclosed, for example, in Remington.The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., Edition 21 (2005).

[0085] The phrases "physiologically acceptable" or "pharmaceutical acceptable" refer to molecular entities and compositions that do not produce significant allergic or similar untoward reactions when administered to humans. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. Preparations can also be emulsified.

[0086] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

[0087] The dosage forms described herein can be formulated to be administered by various techniques, including, for example, subcutaneous and intravenous administration. Particular 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 just below 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 outer area of ​​the upper arm just above and below the waist, except for the area around the navel, which is a specific side (about a 2-inch circle), the top of the buttocks just behind the hip bone, and the front of the thigh on the way out, from about 4 inches below the top of the thigh to about 4 inches above the knee. These areas will vary depending on the size of the person. Varying the injection site also prevents lumps or small indentations, called lipoatrophy, from forming in the skin.

[0088] Subcutaneous injections are typically given into fatty tissue beneath the skin, and in certain cases, smaller, shorter needles can be used. In certain instances, needles with gauges of about 25 to about 31 and lengths of about ½ inch to about ⅝ inch are sufficient to administer the agent subcutaneously. As will be appreciated by those skilled in the art, these are general recommendations and SC injections can be administered with other sized 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 about 45 degrees to the skin.

[0089] Also included are methods of treating a subject in need thereof, comprising administering to the subject an effective amount of a dosage form described herein. For example, certain embodiments include methods of treating ischemic conditions, vascular dementia, hemorrhagic conditions, traumatic brain injury (TBI), diabetes, or kidney disease, among others.

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

[0091] In certain cases, administration of the dosage form achieves a therapeutically effective serum level of one or more KLK1 polypeptides in the subject.In some cases, administration of the dosage form achieves a therapeutically effective serum level of one or more KLK1 polypeptides at 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.In some cases, the dosage form is administered intravenously or subcutaneously. In some cases, a therapeutically effective serum level is about or at least about 1.0 to about or at least about 5.0 ng / ml, or about or at least about 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 therebetween).

[0092] In some cases, administration of the dosage form achieves and maintains a therapeutically effective serum level of one or more KLK1 polypeptides in the subject.For example, in some embodiments, administration of the dosage form achieves a therapeutically effective serum level of one or more KLK1 polypeptides at 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, and maintains a therapeutically effective serum level 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, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more after administration. In some cases, a therapeutically effective serum level is about or at least about 1.0 to about or at least about 5.0 ng / ml, or about or at least about 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 therebetween).

[0093] In some cases, administration of the dosage form achieves or results in an improved pharmacokinetic profile or biological (e.g., therapeutic) effect compared to a higher dose KLK1 polypeptide dosage form. For example, in some cases, subcutaneous administration of the dosage form achieves an improved pharmacokinetic profile or biological (e.g., therapeutic) effect compared to a 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 cases, the improved pharmacokinetic profile includes an increased serum half-life after a single subcutaneous or intravenous administration measured, for example, by 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 (e.g., a single subcutaneous dose).

[0094] Particular embodiments include dosing regimens in which one or more KLK1 dosage forms are administered at defined intervals over a period of time.For example, particular dosing regimens include administering KLK1 dosage forms once or twice a day, once or twice every 2 days (e.g., once every other day), once or twice every 3 days (e.g., once every 3 days after initial or initial administration), once or twice every 4 days, once or twice every 5 days, once or twice every 6 days, once or twice a week, once or twice every other week.Particular dosing regimens include administering KLK1 dosage forms once a day every 3 days (e.g., once a day every 3 days after initial or initial administration), and the dosage forms are administered subcutaneously.

[0095] Certain embodiments include administering at least one intravenous dosage form intravenously to a subject, followed by administering one or more subcutaneous dosage forms subcutaneously to a subject, e.g., on a dosing schedule of about 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 per week. In certain embodiments, the intravenous administration or dosage form achieves a therapeutically effective serum level of one or more KLK1 polypeptides in a subject at or less than about 0.5, 1, 2, 3, or 4 hours after intravenous administration, and the subcutaneous administration or dosage form 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.

[0096] Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject. Furthermore, for administration to humans, the preparation should meet sterility, pyrogenicity, and general safety and purity standards required by the FDA. In some cases, the preparation is substantially endotoxin-free or pyrogen-free, as described herein. According to the FDA Guidance for Industry, Estimation of the Maximum Safe Starting Dose for Early Clinical Trials of Therapeutics in Healthy Adult Volunteers (July 2005), Appendix D: Converting Animal Doses to Human Equivalent Doses. 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 with one or more additional therapeutic agents or therapeutic procedures. In some aspects, administration of the dosage forms allows for the effectiveness of lower doses of other therapeutic procedures and reduces toxicity observed with administration of higher doses of other procedures when compared to administration of the other therapeutic procedures alone. One or more additional therapeutic agents can be administered before, after, and / or simultaneously (e.g., together) with administration of the dosage forms described herein. The dosage forms and the additional therapeutic agents can be administered separately or as part of the same mixture or cocktail. As used herein, additional therapeutic agents include, for example, agents known to those skilled in the art for use in treating conditions (e.g., ischemic or hemorrhagic conditions). Examples of additional agents include angiotensin receptor blockers, edabalon, finerenone, and valdoxalone (including combinations thereof). Specific examples of angiotensin receptor blockers include losartan, azilsartan, candesartan, eprosartan, fimasartan, irbesartan, olmesartan, saprisartan, 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 hypodermic needle assembly, optionally with a protective cover around the needle assembly. In some embodiments, the needle may be about ½ inch to about ⅝ inch in length and have a gauge of about 25 to about 31. Thus, certain embodiments include devices that are attached 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 the vial or syringe is optionally attachable or attached to a hypodermic needle assembly. Also included are vials with a rubber cap, where a needle / syringe can be inserted into the vial through the rubber cap to withdraw 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 hypodermic 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 people from the needle before administration, and a second protective cover can be put into place (i.e. snapped on) after administration to safely dispose of the device.

[0100] The present invention is illustrated by the following examples, it being understood that the specific examples, materials, amounts and procedures are to be broadly interpreted 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 was evaluated in humans. The formulation consisted of a mixture of di- and tri-glycosylated 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 KLK1 formulations following a 30-minute IV infusion and a single subcutaneous injection. The IV dose was 0.75 μg / kg, and the subcutaneous dose was 3 μg / kg. Study groups consisted of 12 volunteers, 6 females and 6 males each.

[0103] The intravenous dose was 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 the weight and dose level of each study volunteer. The subcutaneous dose was delivered 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 injection volume was adjusted according to the weight of the study participant.

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

[0105] As shown in Figures 2A-2B, we demonstrate that in healthy human subjects, subcutaneous administration of a low-dose formulation of mature human KLK1 glycoforms (3 μg / kg, n=12) not only achieves effective plasma levels of KLK1, but also results in a significant prolongation of serum half-life compared to intravenous administration of a low-dose formulation (0.75 μg / kg, n=12). Effective plasma levels of KLK1 were rapidly achieved by intravenous administration of the low-dose formulation of mature human KLK1 glycoforms. 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. The points represent the mean values ​​obtained from six participants per group. The arrows represent the time of administration. The 3 μg / kg dose, considered as the target dose from the latest patent, maintained fairly stable drug levels with desirable or therapeutically effective serum / plasma concentrations of approximately 3-5 ng / ml. In contrast, higher doses caused proportionally higher levels with greater dose-to-dose variability.

[0107] A dosage test of KLK1 was performed in patients with type 2 diabetes. The results are shown in Figures 4A-4D. The points are the results of a meal tolerance test performed 2 hours after administration using HOMA2-IR measurements of insulin resistance 3 hours after administration. The numbers are a derived measure of insulin resistance, with higher numbers indicating higher insulin resistance and higher likelihood of disease. Figure 4D shows the results of the placebo group, and Figure 4C shows the results of the drug group (DM199). The test on day 1 was performed as a baseline, followed by a single dose of the specified dose level from the next day. Insulin resistance improved on day 5, when a low dose was tested, compared to the results on day 8, when a high dose was tested (Figure 4C), suggesting that the low dose was more effective.

[0108] A 28-day multiple-dose study in type 2 diabetes patients (n=12-13 per group) was also conducted. The results are shown in Figures 5A-5B. Bars represent the mean ± SEM change from baseline in serum creatinine and urea concentrations. These are typical measures of renal function, and when exceeded above normal, they indicate renal impairment. The results show that the low dose (3 μg / kg) produced a significantly greater effect than the higher dose on improving measures of renal function. P values ​​are based on comparisons between the 3 μg / kg group and placebo.

[0109] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0110] The complete disclosures of all patents, patent applications, and publications, and 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 regions in GenBank and RefSeq) are incorporated by reference. The above detailed description and examples are provided for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, and modifications obvious to one skilled in the art will be included within the invention as defined by the claims.

Claims

[Claim 1] The invention as depicted in the drawings.

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