# US 2019/0169593 A1 · Products and methods for assessing and increasing klotho protein levels

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- Number: US 2019/0169593 A1
- Document ID: US20190169593A1
- Status: Published application
- Kind: A1
- Application number: 16/208,552
- Filed: 2018-12-03
- Published: 2019-06-06
- Earliest claimed: 2016-06-02
- Assignees: Klotho Therapeutics, Inc.
- Applicants: Klotho Therapeutics, Inc.
- Inventors: Joseph F. Tarsio; Dinesh Raturi; William Ian Ramage; James R. Plante
- Web page: https://patentgrep.com/patent/US20190169593A1

## CPC

- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- A23L 33/13: Nucleic acids or derivatives thereof (A23L33/145 takes precedence)
- A23L 33/135: Bacteria or derivatives thereof, e.g. probiotics
- A23L 33/15: Vitamins
- A23L 33/155: Vitamins A or D
- A23L 33/17: Amino acids, peptides or proteins
- B01D 15/1871: {placed in series}
- B01D 15/34: Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
- B01D 15/3804: {Affinity chromatography}
- C07K 14/765: Serum albumin, e.g. HSA
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12Y 302/01031: Beta-glucuronidase (3.2.1.31)
- G01N 33/573: for enzymes or isoenzymes
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- G01N 2333/924: acting on glycosyl compounds (3.2)
- G01N 2800/7042: Aging, e.g. cellular aging
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Disclosed are products and methods for monitoring Klotho protein levels and for stabilizing Klotho protein in a mammalian blood sample, especially at room temperature or without freezing, for a period of time. Methods of detecting and quantifying Klotho protein levels, particularly endogenous and/or exogenous soluble alpha Klotho protein levels, methods of diagnosing Klotho protein deficiency, and methods of increasing Klotho protein levels or production, particularly endogenous and/or exogenous soluble alpha Klotho protein level(s), expression, or production, in a mammalian subject, and products useful in performing the same, including diagnostic kits and compositions for treating Klotho protein deficiency, are disclosed. Compositions are configured or formulated to augment natural soluble alpha Klotho protein production, attenuate Klotho protein damage or degradation, and/or supplement Klotho protein levels with exogenous, recombinant protein. Treatment methods and uses include administration of the compositions to human or non-human mammalian subjects.

## Claims

### Claim 1

A method of stabilizing Klotho protein in a mammalian blood sample, the method comprising:
obtaining capillary blood from a mammalian subject, the capillary blood containing an amount of soluble Klotho protein; and
storing the capillary blood in a container for at least 24 hours at room temperature or without freezing, the container having a preservative or anti-coagulant disposed therein, the preservative or anti-coagulant being mixed with the capillary blood in the container, the preservative or anti-coagulant stabilizing the soluble Klotho protein for at least 24 hours at room temperature or without freezing, the preservative or anti-coagulant comprising one or more of heparin, lithium heparin, EDTA, and K2 EDTA.

### Claim 2 (depends on 1)

The method of claim 1, further comprising storing the capillary blood mixed with the preservative or anti-coagulant for at least 3 days at room temperature or without freezing, the preservative or anti-coagulant stabilizing the soluble Klotho protein for at least 3 days at room temperature or without freezing.

### Claim 3 (depends on 1)

The method of claim 1, further comprising storing the capillary blood mixed with the preservative or anti-coagulant for at least 7 days at room temperature or without freezing, the preservative or anti-coagulant stabilizing the soluble Klotho protein for at least 7 days at room temperature or without freezing.

### Claim 4 (depends on 1)

The method of claim 1, further comprising, after the storing step, quantifying the amount of soluble Klotho protein or assaying for the presence of the soluble Klotho protein.

### Claim 5 (depends on 4)

The method of claim 4, wherein the step of quantifying the amount of soluble Klotho protein comprises detecting the soluble Klotho protein using mass spectrometry, Multi-Analyte Profiling (xMAP), and/or a first antibody that binds to a portion of the soluble Klotho protein.

### Claim 6 (depends on 5)

The method of claim 5, wherein the step of quantifying the amount of soluble Klotho protein further comprises detecting the soluble Klotho protein using a detection antibody that binds to a portion of the first antibody or performing an enzyme-linked immunosorbent assay (ELISA) to detect and optionally quantify the soluble Klotho protein.

### Claim 7 (depends on 1)

The method of claim 1, wherein the step of obtaining capillary blood comprises one or more of lancing skin of the mammal with a lancet and collecting the capillary blood in the container.

### Claim 8 (depends on 1)

The method of claim 1, wherein the capillary blood is between about 10-1000 ul, preferably about 50-200 ul of the capillary blood, or wherein the step of obtaining capillary blood includes obtaining between about 10-1000 ul, preferably about 50-200 ul of the capillary blood.

### Claim 9

A method of diagnosing Klotho deficiency in a mammalian subject, the method comprising:
obtaining a fluid sample mixture selected from the group consisting of:
mammalian serum, optionally in the form of capillary blood, mixed with a preservative or anti-coagulant comprising one or more of heparin, lithium heparin, EDTA, and K2 EDTA, the mammalian serum having an amount of soluble Klotho protein; and
mammalian serum, optionally in the form of capillary blood, the mammalian serum having an amount of soluble Klotho protein, the method further comprising mixing the mammalian serum with a preservative or anti-coagulant comprising one or more of heparin, lithium heparin, EDTA, and K2 EDTA to form the mixture;
storing the mixture for at least 24 hours at room temperature or without freezing, the preservative or anti-coagulant stabilizing the soluble Klotho protein for at least 24 hours at room temperature or without freezing;
after the storing step, quantifying the amount of the soluble Klotho protein in the mixture; and
diagnosing the mammalian subject with Klotho deficiency when the quantified amount of soluble Klotho protein in the mixture is less than a predetermined threshold amount.

### Claim 10 (depends on 9)

The method of claim 9, wherein the fluid sample mixture comprises about 10-1000 ul, preferably about 50-200 ul of capillary blood from the mammalian subject, or wherein the step of obtaining the fluid sample mixture comprises obtaining about 10-1000 ul, preferably about 50-200 ul of capillary blood from the mammalian subject.

### Claim 11 (depends on 9)

The method of claim 9, wherein the step of quantifying the amount of the soluble Klotho protein in the mixture comprises detecting the soluble Klotho protein using mass spectrometry, Multi-Analyte Profiling (xMAP), and/or a first antibody that binds to a portion of the soluble Klotho protein and, optionally, a detection antibody that binds to a portion of the first antibody, or performing an enzyme-linked immunosorbent assay (ELISA) to detect and optionally quantify the soluble Klotho protein.

### Claim 12 (depends on 9)

The method of claim 9, wherein diagnosing the mammalian subject with Klotho deficiency comprises displaying a Klotho deficiency determination or diagnosis on a user interface of a computer system and/or producing a file or report, in physical or electronic form, that displays the Klotho deficiency determination or diagnosis, the Klotho deficiency determination or diagnosis optionally including the quantified amount of the soluble Klotho protein and/or the predetermined threshold amount.

### Claim 13

A method of treating Klotho deficiency in a mammalian subject, the method comprising:
obtaining serum or capillary blood, from a mammalian subject, the serum or capillary blood having an amount of soluble Klotho protein;
storing the serum or capillary blood in a container for at least 24 hours at room temperature or without freezing, the container having a preservative or anti-coagulant disposed therein and mixed with the serum or capillary blood to form a mixture, the preservative or anti-coagulant stabilizing the soluble Klotho protein for at least 24 hours at room temperature or without freezing, the preservative or anti-coagulant comprising one or more of heparin, lithium heparin, EDTA, and K2 EDTA;
after the storing step, quantifying the amount of the soluble Klotho protein in the mixture;
diagnosing the mammalian subject with Klotho deficiency when the quantified amount of soluble Klotho protein in the mixture is less than a predetermined threshold amount; and
administering a composition to the mammalian subject after diagnosing the mammalian subject with Klotho deficiency, the composition comprising one or more of:
one or more pharmaceutical composition comprising a pharmaceutically-acceptable carrier or excipient and a pharmaceutically effective amount of a recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein disposed in the carrier or excipient;
one or more nutraceutical composition comprising a plurality of components adapted to raise serum soluble Klotho protein levels by increasing or enhancing endogenous production of soluble Klotho protein by the mammalian subject; and
one or more pharmaceutical composition that increases endogenous Klotho levels, optionally selected from the group consisting of a blood pressure medication, an Angiotensin II receptor blocker (ARB), an Angiotensin-converting enzyme (ACE) inhibitor, Losartan, Valsartan, Telmisartan, an AT1 receptor antagonist or blocker, testosterone, growth hormone, a growth hormone releasing peptide, sermorelin, prescription or prescription-strength vitamin D, 1,25-dihydroxycholecalciferol, calcitrol, paricalcitrol, an Indoxyl sulphate binder, Kremezin, AST-120, a statins, a PPAR agonists, troglitazone, and rosiglitazone.

### Claim 14 (depends on 13)

The method of claim 13, wherein the administering step comprises:
an oral or oral-related administration, optionally selected from the group consisting of ingestion, buccal administration, and sublingual administration, optionally in modified-release form;
one or more bolus or gradual injection, optionally selected from intravenous, intradermal, intraperitoneal, intramuscular, intracutaneous, and subcutaneous injection, optionally in modified-release form.

### Claim 15 (depends on 13)

The method of claim 13, wherein the step of obtaining serum or capillary blood comprises one or more of:
lancing skin of the mammalian subject with a lancet;
obtaining between about 10-1000 ul, preferably about 50-200 ul of the capillary blood; and
collecting the capillary blood in a container having a preservative or anti-coagulant disposed therein or added thereto, the capillary blood optionally being between about 10-1000 ul, preferably about 50-200 ul of capillary blood.

### Claim 16 (depends on 13)

The method of claim 13, wherein the pharmaceutical composition comprises a pharmaceutically-effective amount of a recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein having at least 80% amino acid sequence identity to at least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70 or SEQ ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO: 125 through SEQ ID NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152.

### Claim 17 (depends on 16)

The method of claim 16, wherein the pharmaceutical composition comprises a pharmaceutically-effective amount of a recombinant Klotho fusion protein comprising at least a portion of an immunoglobulin Fc domain sequence or human serum albumin sequence fused to at least a portion of a soluble Klotho protein sequence, with or without a linker sequence therebetween.

### Claim 18 (depends on 13)

The method of claim 13, wherein the nutraceutical composition comprises two or more components selected from the group consisting of vitamin C, vitamin D3, vitamin E, N-acetylcysteine (NAC), quercetin dihydrate, rosmarinic acid (RA), pterostilbene, docosahexaenoic acid (DHA), nicotinamide riboside (NR), nicotinamide adenine dinucleotide (NAD+), nicotinamide mononucleotide (NMN), and one or more probiotic.

### Claim 19 (depends on 18)

The method of claim 18, wherein the one or more probiotic comprises an effective amount of the Bifidobacterium species and/or strains Bifidobacterium lactis BL-04, Bifidobacterium bifidum/lactis BB-02, and Bifidobacterium longum BL-05, and the Lactobacillus species and/or strains Lactobacillus acidophilus LA-14, Lactobacillus rhamnosus LR-32, and Lactobacillus paracasei LPC-37.

### Claim 20

A method of purifying a pharmaceutical grade recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein, the method comprising:
obtaining a suspension cell culture fluid comprising (i) recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein and (ii) one or more contaminants;
performing a purification step using affinity chromatography in which the recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein is bound to Protein A or other affinity entity, optionally washed, and eluted from Protein A with and elution buffer having a pH between about pH 2 and about pH 6 and a salt concentration between about 1 M and about 6 M, the salt optionally comprising MgCl2;
performing a purification step using size exclusion chromatography in which the eluted recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein is further eluted in a mobile phase buffer, the mobile phase buffer having a buffering agent and one or more optional reducing agent, the buffering agent optionally being other than a phosphate-containing buffer, the reducing agent optionally comprising L-Methionine and/or Sodium Thioglycolate, the mobile phase buffer optionally having a pH between about pH 6 and about pH 10,
wherein at least some of the further eluted recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein is optionally in multimeric form.

### Claim 21 (depends on 21)

The method of claim 21, wherein the recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein has at least 80% amino acid sequence identity to at least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70 or SEQ ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO: 125 through SEQ ID NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152.