JP2023113737A - Products and methods for assessing and increasing klotho protein levels - Google Patents

Abstract

To provide 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.SOLUTION: A method of stabilizing Klotho protein in a mammalian blood sample comprises: 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 preservative or anti-coagulant is mixed with the capillary blood in the container. The preservative or anti-coagulant stabilizes the soluble Klotho protein for at least 24 hours at room temperature or without freezing. The preservative or anti-coagulant comprises one or more of heparin, lithium heparin, EDTA, and K2 EDTA.SELECTED DRAWING: Figure 1

Description

BACKGROUND This disclosure relates to the assessment and increase of Klotho protein levels in patients. Specifically, the present disclosure provides for monitoring, detecting, and/or qualifying levels of endogenous and/or exogenous Klotho protein, and Klotho protein deficiency in human or non-human animal (e.g., mammalian) subjects. and the production, purification, and administration of pharmaceutical or nutraceutical compositions to increase Klotho protein levels in a subject.

2. Related Art Klotho (or alpha-klotho, α-klotho, etc.) is a recently characterized protein encoded by the KL (or klotho) gene located on human chromosome 13. Two transcripts have been identified that arise from a single Klotho gene through alternative RNA splicing. See Figures 1 and 2. The first transcript has a short cytoplasmic tail (human residues 1003-1012) and a transmembrane (TM) domain (human residues 982-1002) with 20%-40% amino acid sequence homology to β-glucosidase, respectively. two (mostly) homologous (internal repeat) domains, KL1 (human residues 56-506, which are 450 residues long) and KL2, which share the same sex but may lack similar levels of glucosidase catalytic activity. (referred to as human residues 515-953, which is 438 residues long), an extracellular region or domain (human residues 1-981) and a signal sequence (SS) domain (human residues 1-33 It is predicted to encode Klotho isoform 1, a single-pass transmembrane protein of 1,012 amino acids in length, containing the SS, KL1, and KL2 domains-containing extracellular region (human residues 1-981). ) can be enzymatically cleaved by α/β-secretase and released into circulation as a 130 kDa circulating protein termed soluble klotho (or s-klotho, s-klotho, alpha-soluble klotho, etc.). It can also be cleaved into separate 68 kDa proteins (KL1+SS) and 64 kDa proteins (KL2).

A second transcript, a splicing variant of the alpha-Klotho mRNA, encodes a second isoform of the Klotho protein that primarily corresponds to the KL1 domain. An internal splice donor site is believed to be located in exon 3 of the Klotho gene. The resulting alternatively spliced transcript contains a 50 base pair insertion after exon 3 with an in-frame translational stop codon at its end (Fig. 1, grey). The expressed protein product is secreted into the circulation and is called secreted Klotho (or Klotho isoform 2), which differs from the canonical sequence of isoform 1 at amino acid residues 535-549 by DTTLSQFTDLNVYLW→SQLTKPISSLTKPYH, amino acid residues Lacking 550-1012.

Therefore, at any given time, there may be several different Klotho proteins in circulation, depending on gene expression, RNA splicing, and enzymatic cleavage. Despite the presence of various forms of alpha-Klotho protein, only the full-length, membrane-bound isoform 1 complexes with fibroblast growth factor (FGF) receptors and induces urinary phosphate excretion. and is known to function as an essential co-receptor for FGF23, a bone-derived hormone with roles in regulating _Pi and vitamin D metabolism.

Klotho is highly expressed in kidney, brain, and to a lesser extent in other organs, and can also be found in mammalian blood, cerebrospinal fluid, and urine. Circulating levels of soluble Klotho protein in mammals, including humans, are thought to decrease with age. In addition, Klotho-deficient mice exhibit an accelerated aging phenotype, while overexpression of Klotho in mice has been shown to extend lifespan. In addition, Klotho has been implicated in several cellular processes associated with aging. In view of the foregoing, it has been hypothesized that soluble Klotho may function as an anti-aging compound in the human body.

Aging is an inevitable and progressive biological process that leads to the dysfunction and destruction of almost all tissues and organs, and ultimately to death. For example, aging of the human body is associated with a decline in cell function that can lead to the development of various diseases. Aging is thought to be caused by a tightly regulated and complex interplay between genetic and acquired factors, leading to increased aging, quantitative and qualitative decline in stem cells, and changes in stem cells at the tissue level. Abnormal structure is a typical feature.

The elderly (eg, 60-65 year old) population is rapidly increasing worldwide as the so-called "baby boomer" generation ages. The increasing demand for health care for this elderly population is a significant economic burden on any health care system. Recombinant Klotho protein may provide a promising therapeutic agent to combat age-related health conditions. To date, all relevant therapeutic data related to Klotho are preclinical research studies in animal models. (i) production and/or purification (e.g., to substantially homogeneity) of recombinant s-Klotho protein or protein variants, and (ii) to subjects, particularly humans, and/or the increasing elderly population. The development of strategies and health intervention products and methods based on the administration of recombinant s-Klotho protein or protein variants and/or s-Klotho protein level-increasing health supplements in the population can help improve this situation.

Recombinant soluble human alpha-Klotho protein or protein variants currently suitable for use in humans, especially current good manufacturing practices (cGMP) as determined and enforced by the US Food and Drug Administration (FDA). ) There is no product or method for providing exogenous forms of human Klotho protein, such as regulatory compliant proteins, either alone or in combination with one or more additional active ingredients.

Similarly, currently products or methods for (naturally) increasing endogenous Klotho protein levels or protein production in a safe and effective manner, particularly in compliance with the Dietary Supplement Health Education Act of 1994 (DSHEA). A product or product that complies with current good manufacturing practice (cGMP) regulations (as determined and enforced by the U.S. Food and Drug Administration (FDA)) may contain, either alone or with one or more additional active ingredients, drugs, pharmaceuticals, or Not even in combination with dietary supplements.

Moreover, currently products (e.g., kits or systems) for efficiently, reliably, reproducibly, practically and/or (commercially or economically) viable monitoring of Klotho protein levels or Methods, specifically endogenous and/or exogenous Klotho protein levels, and more specifically endogenous and/or exogenous soluble alpha Klotho protein or protein variant levels, on-demand and/or real-time monitoring and /or There are no products or methods for quantifying or diagnosing Klotho protein deficiency, specifically serum soluble Klotho protein deficiency, in humans and non-human animals.

It is now possible to efficiently, reliably, reproducibly, practically and/or produce pharmaceutical grade recombinant Klotho proteins, including recombinant Klotho proteins, specifically recombinant Klotho protein fragments and recombinant Klotho fusion proteins. There are also no suitable large-scale methods for viable (commercially or economically) production and purification.

Embodiments of the present disclosure solve one or more of the foregoing or other problems in the art with products and methods for assessing and increasing Klotho protein levels in patients. Some embodiments are directed to (1) compositions and methods for stabilizing Klotho protein in the blood or serum of a human or non-human animal (e.g., mammalian), (2) Klotho protein level(s) , specifically compositions and methods for monitoring, detecting, and/or quantifying endogenous and/or exogenous soluble alpha Klotho protein level(s) and/or human or non-human animals (e.g., compositions and methods for diagnosing Klotho protein deficiency in a mammalian subject, and products useful for their practice; (3) pharmaceutical or nutraceutical compositions for increasing Klotho protein levels in a subject; include. Some embodiments use Klotho protein(s), or fragment(s) or variant(s) thereof, and more specifically cGMP grade human (or non-human) recombinants, as therapeutic agents. Soluble Alpha Klotho protein(s), or fragment(s) or variant(s) thereof, or (pharmaceutical) compositions comprising them. Some embodiments provide products for producing (recombinant) Klotho protein(s), or fragment(s) or variant(s) thereof, and administering them to human or non-human subjects and and/or methods, and specifically products and/or methods for increasing serum soluble Klotho protein levels in a subject. Some embodiments comprise a nutritional or health supplement composition for increasing serum soluble Klotho protein levels in a subject. Specifically, some embodiments are products that, when administered to a subject, cause an increase in Klotho protein (specifically, endogenous soluble alpha Klotho protein) level(s), expression, or production in a subject. and (4) methods of purifying recombinant Klotho proteins, specifically pharmaceutical grade recombinant Klotho proteins, recombinant Klotho protein fragments, and recombinant Klotho fusion proteins.

Various embodiments include compositions. The composition comprises (i) one or more recombinant human Klotho proteins, protein fragments, and/or protein variants, expression nucleic acid constructs and/or vectors, cell lines and/or cell suspension cultures, and/or ( ii) when administered to a subject, cause an increase in Klotho protein level(s), expression or production in the subject, specifically endogenous soluble alpha Klotho protein level(s), expression or production, or It may contain one or more ingredients that induce. Certain embodiments include methods of making a composition of the disclosure, purifying it, and administering it to a subject (human or non-human animal). Some embodiments include methods of increasing Klotho protein (specifically (exogenous and/or endogenous) soluble alpha Klotho protein) level(s), expression, or production in a subject. Some embodiments provide a Products (eg, diagnostic kits) and/or methods and/or products and/or methods for diagnosing Klotho protein deficiency in a subject.

Some embodiments of the present disclosure provide recombinant human alpha Klotho protein, protein fragments, and/or protein variants (e.g., cGMP grade human recombinant soluble alpha Klotho proteins, protein fragments, and/or protein variants). compositions comprising recombinant human alpha soluble Klotho protein, protein fragments, and/or protein variants (e.g., therapeutic compositions, pharmaceutical compositions, medicaments, formulations, etc.); recombinant human alpha soluble Klotho protein and ( a composition comprising a pharmaceutically acceptable) vehicle (e.g., carrier or excipient); a composition comprising recombinant human alpha-soluble klotho protein and at least one additional (active) ingredient; human recombinant alpha-soluble klotho a nucleic acid construct or vector encoding the protein; (i) a cell line containing a nucleic acid construct or vector encoding a recombinant human alpha soluble Klotho protein and/or (ii) expressing a recombinant human alpha soluble Klotho protein; each) a cell suspension culture of one or more cells (i) containing a nucleic acid construct or vector encoding a recombinant human alpha soluble Klotho protein and/or (ii) expressing a recombinant human alpha soluble Klotho protein; a method of producing and optionally purifying a recombinant human alpha soluble Klotho protein; a method of producing a pharmaceutical (or therapeutic composition, i.e., formulation) of a recombinant human alpha soluble Klotho protein; a (human or non-human animal) subject diagnostic method for determining Klotho protein deficiency in a subject; method for diagnosing Klotho protein deficiency in a subject; administration requires receiving recombinant human alpha soluble Klotho protein methods of evaluating the efficacy and/or determining an effective dosage of a protein for a subject in need thereof; certain medical conditions in humans or non-human animals (e.g., non-human mammals) Recombinant human alpha soluble Klotho protein for use in the treatment of or other medical conditions; Use of recombinant human alpha soluble Klotho protein for the treatment of certain medical conditions or other conditions in humans or non-human animals; or use of a composition comprising recombinant human alpha-soluble Klotho protein for the treatment of certain medical conditions or other conditions in non-human animals; and/or certain medical conditions or other conditions in humans or non-human animals. use of the recombinant human alpha-soluble Klotho protein in the manufacture of a medicament for the treatment of conditions of

Some embodiments may comprise a recombinant Klotho protein, wherein at least a portion of the protein is SEQ ID NOs: 2-70, or SEQ ID NOs: 107-120, or SEQ ID NOs: 125-128, or SEQ ID NOs: 133, or at least a portion of one of SEQ ID NO:152. Some embodiments are one of SEQ ID NO:2-70, or SEQ ID NO:107-120, or SEQ ID NO:125-128, or SEQ ID NO:133, or SEQ ID NO:152, preferably at least a portion of one of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125-128, SEQ ID NO: 133, and SEQ ID NO: 152, and optionally with or without any linker sequence therebetween a Klotho protein sequence having at least 80% amino acid sequence identity with at least a portion of an immunoglobulin Fc domain sequence, a human serum albumin sequence, a His (eg, 6His) tag sequence, and/or a Twin-Strep tag sequence. , may contain the recombinant protein. Some embodiments may contain more than one recombinant Klotho protein.

Some embodiments may include a pharmaceutical composition comprising a pharmaceutically effective amount of a recombinant Klotho protein described herein and a pharmaceutically acceptable carrier. Some embodiments are a pharmaceutically effective amount of a recombinant soluble Klotho protein, wherein at least a portion of the protein comprises amino acid residues 1-981, 29-981, 34 of human alpha Klotho isoform 1 or isoform 2 -981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or at least a subset of 131-549, or SEQ ID NOs: 2-70, or SEQ ID NOs: 107- A recombinant soluble Klotho protein having at least 80% amino acid sequence identity with at least a portion of one of SEQ ID NOs: 120, or SEQ ID NOs: 125-128, or SEQ ID NO: 133, or SEQ ID NO: 152, and pharmaceutically A pharmaceutical composition comprising an acceptable carrier may be included. Some embodiments include a pharmaceutically acceptable carrier or excipient and a or at least part of one of SEQ ID NO: 152, preferably one of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125-128, SEQ ID NO: 133 and SEQ ID NO: 152; and at least a portion and at least 80% of an immunoglobulin Fc domain sequence, a human serum albumin sequence, a His (eg, 6His) tag sequence, and/or a Twin-Strep tag sequence, optionally with or without any linker sequence therebetween A pharmaceutical composition comprising an effective amount of a recombinant protein comprising a Klotho protein sequence having an amino acid sequence identity of . Some embodiments may include compositions comprising pharmaceutically effective amounts of two or more recombinant Klotho proteins.

Some embodiments are compositions comprising a therapeutic Klotho protein and at least one other active component such as a drug, antibody, hormone, human cell, tissue, cell product or tissue system product (HCT/Ps) and/or methods of administering them to human or non-human subjects. Combinatorial compositions and methods can be useful for treating subjects with age-related disorders or conditions, clinical (eg, metabolic) disorders, chronic diseases, acute injuries, and the like. Prophylactic administration of the combination therapy to subjects who do not have overt conditions or disorders may also be useful in delaying or preventing the specific conditions or disorders described herein.

Some embodiments may include nucleic acids or nucleic acid constructs. For example, embodiments can include expression vectors or nucleic acids. A nucleic acid can encode a recombinant human alpha soluble Klotho protein, protein fragment, or protein variant. Nucleic acids can encode natural or non-natural signaling sequences. For example, a nucleic acid can encode a non-native signaling sequence upstream (or N-terminal) to the encoded Klotho protein sequence. Some embodiments are recombinant proteins of preferably at least a portion of one of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125-128, SEQ ID NO: 133, and SEQ ID NO: 152, and optionally anything in between at least 80% amino acid sequence identity with at least a portion of the immunoglobulin Fc domain sequence, human serum albumin sequence, His (e.g., 6His) tag sequence, and/or Twin-Strep tag sequence, with or without the linker sequence of A nucleic acid construct comprising a nucleic acid sequence encoding a recombinant protein, including a Klotho protein sequence, having a Klotho protein sequence.

Some embodiments may involve cell lines. Cell lines may comprise Chinese Hamster Ovary (CHO) cell(s), HEK-293 cells, or other suitable (protein-expressing) cell lines. In some embodiments, the cells can be dihydrofolate reductase (DHFR)-deficient CHO cells, such as CHO-S cells, or glutamine synthetase (GS)-deficient CHO cells, such as GS-/- CHO cells. the cell is one of SEQ ID NO: 2 to SEQ ID NO: 70, or SEQ ID NO: 107 to SEQ ID NO: 120, or SEQ ID NO: 125 to SEQ ID NO: 128, or SEQ ID NO: 133, or SEQ ID NO: 152, preferably SEQ ID NO: 52; encoding a polypeptide having at least 80% amino acid sequence identity with at least a portion of one of SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125-128, SEQ ID NO: 133, and SEQ ID NO: 152 ( It may contain (copies of) one or more exogenous nucleic acids (including transgenes or cDNAs). The polypeptide may comprise human recombinant alpha soluble Klotho protein. The exogenous nucleic acid may comprise a transgene or cDNA, preferably having at least 80% nucleic acid sequence identity with one of SEQ ID NO:76-SEQ ID NO:106 or SEQ ID NO:121-124. In some embodiments, the nucleic acid is (also) a promoter (associated with the transgene), and/or any may contain or encode an (exogenous) enzyme of

At least one embodiment preferably contains a carbon source, a nitrogen source, and/or one or more vitamins, minerals, salts, amino acids, supplements, or additives so that the cells express the polypeptide encoded by the nucleic acid. Includes suspension cell cultures, including cell lines grown in liquid media such as Liquid media may be serum-free (human, bovine, fetal bovine, or otherwise) and/or free of animal (or animal-derived) protein (components). For example, liquid media can be free of bovine serum albumin, human serum albumin, and the like. In some embodiments, the suspension cell culture is a liquid medium, preferably a serum-free and/or animal protein component-free liquid medium, preferably containing a carbon source, a nitrogen source, and one or more A liquid medium comprising vitamins, minerals, salts, amino acids, supplements or additives, and more preferably lacking hypoxanthine, thymidine, and/or glutamine, and cells such that they express the polypeptide encoded by the nucleic acid. A cell line that grows in a liquid medium, wherein the polypeptide comprises the recombinant Klotho protein.

Some embodiments include extracts of or from suspension cell cultures of cells, liquid media, or both, wherein the extract comprises SEQ ID NO: 2-SEQ ID NO: 70, or one of SEQ ID NO: 107 to SEQ ID NO: 120, or SEQ ID NO: 125 to SEQ ID NO: 128, or SEQ ID NO: 133, or SEQ ID NO: 152, preferably SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125 to Contains a recombinant protein having at least 80% amino acid sequence identity with at least a portion of one of SEQ ID NO:128, SEQ ID NO:133, and SEQ ID NO:152. Certain embodiments include human recombinant alpha soluble Klotho protein-containing extracts of or from cells, liquid media, or both (eg, suspension cell cultures). At least one embodiment comprises at least a portion of one of SEQ ID NOs:2-70, or SEQ ID NOs:107-120, or SEQ ID NOs:125-128, or SEQ ID NO:133, or SEQ ID NO:152 and at least Contains isolated recombinant proteins with 80% amino acid sequence identity.

Some embodiments may include methods of producing recombinant Klotho protein (eg, recombinant human alpha soluble Klotho protein). Exemplary methods include, for example, in Chinese Hamster Ovary (CHO) cells HEK-293, preferably in dihydrofolate reductase (DHFR) deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS) cells. producing a recombinant Klotho protein in deficient CHO cells, more preferably in GS-/- CHO cells, wherein the protein is preferably SEQ ID NO: 2 to SEQ ID NO: 70, or SEQ ID NO: 107 to SEQ ID NO: 120 , or SEQ ID NO: 125-128, or SEQ ID NO: 133, or SEQ ID NO: 152.

In some embodiments, the manufacturing method comprises growing Chinese Hamster Ovary (CHO) cells in liquid medium, producing recombinant soluble Klotho protein in the cells, and/or purifying the recombinant soluble Klotho protein-containing extracts from both The extract comprises at least about 90%, preferably at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% dry weight of recombinant soluble Klotho protein, and/or It may contain less than about 1-100 ppm host cell protein (HCP). The cells can be HEK-293 cells, or dihydrofolate reductase (DHFR) deficient CHO cells such as CHO-S cells, or glutamine synthetase (GS) deficient CHO cells such as GS-/- CHO cells. The produced (expressed) protein may be released (eg, secreted) from the cell into the liquid medium and/or may have one or more glycans attached thereto.

The cell may contain one or more exogenous nucleic acids that encode proteins and optionally functional enzymes such as dihydrofolate reductase, glutamine synthetase (GS) enzymes. An exogenous nucleic acid can contain a promoter (eg, a strong promoter, a weak promoter, etc.), such as promoters customary or typical for expression of exogenous proteins in cells. The exogenous nucleic acid is preferably one of SEQ ID NOs: 76-106 or SEQ ID NOs: 121-124, or any other suitable nucleic acid sequence encoding a Klotho protein as described herein. (eg, an S-Klotho variant) that has at least 80% nucleic acid sequence identity with a transgene or cDNA (eg, under the control of a promoter).

A method can include introducing exogenous nucleic acid into a cell, such as by transfection. The method may involve growing the cells in liquid media, such as serum-free (human, bovine, fetal bovine, or other) and/or animal (or animal-derived) protein (component)-free media. The medium preferably contains a carbon source, a nitrogen source, and/or one or more vitamins, minerals, salts, amino acids, supplements, or additives, preferably within the bioreactor. Depending on the particular cell line, methods may include introducing an effective amount of methotrexate (MTX), methionine sulfoximine (MSX), or other agent into the liquid medium and/or (e.g., cell subcloning, limiting dilution selection of suspension cultures of viable cells growing in liquid medium by methods, fluorescence-activated cell sorting (FACS), etc.).

In some embodiments, selection and/or gene amplification is performed by culturing the transfected cells in a selective medium, such as medium lacking hypoxanthine and/or thymidine (e.g., -HT medium), glutamine, etc. can be performed by In at least one embodiment, low concentration(s) of MTX is added or used to amplify the transfected nucleic acid (or gene(s) thereof) thereby (e.g., transfected with the DHFR transgene). DHFR-deficient cells that are transfected) can be selected for increased protein expression. Alternatively (or in addition), selection and/or gene amplification may be performed by adding MSX ((endogenous) glutamine synthetase (GS ) by adding an inhibitor of ).

The method can include subculturing viable cells or cultures (eg, MTX-resistant and/or MSX-resistant cells or cultures). Selected suspension cultures and/or selected cells may exhibit increased protein production (e.g., by the cells), increased protein concentration (e.g., in the liquid medium), and/or e.g. It may have or exhibit an increased copy number (eg, per cell) of the exogenous nucleic acid (as compared to the culture or cells).

In some embodiments, the liquid medium may also contain effective amounts of MTX and/or MSX. Suspension cultures (or cells thereof) exhibit increased production of proteins (e.g., by cells), exhibit increased concentrations of proteins (e.g., in liquid media), and exhibit increased concentrations of proteins (e.g., in liquid media). and/or preferably have an increased copy number (eg, per cell) of the exogenous nucleic acid compared to unselected suspension cultures. A protein may have one or more glycans attached to it.

Some embodiments of the present disclosure relate to compositions configured or formulated to enhance, improve, or increase natural soluble Alpha Klotho protein production. Some embodiments include compositions or methods for increasing (endogenous) Klotho protein levels in a subject. Some embodiments include compositions (nutraceutical or health supplement) for increasing Klotho protein levels, particularly serum soluble Klotho protein levels, in a subject. Some embodiments reduce the level(s) of Klotho protein level(s), expression or production in the subject, specifically endogenous soluble alpha Klotho protein level(s), expression or production in the subject. Including products that cause an increase.

Some embodiments of the present disclosure relate to compositions configured or formulated to attenuate damage or degradation of Klotho protein. Some embodiments of the present disclosure relate to methods of making compositions of the present disclosure. Some embodiments of the present disclosure relate to the use of the disclosed compositions or methods of using them to treat human or non-human animal subjects. Some embodiments of the disclosure relate to therapeutic methods involving the compositions of the disclosure, including administration thereof to human or non-human animal subjects. Such uses and treatment methods may increase endogenous Klotho protein levels, such as by increasing natural soluble alpha Klotho protein production and/or attenuating Klotho protein damage or degradation.

Certain embodiments include health supplements that, when administered to a human or non-human animal (e.g., mammalian) subject, increase the level of endogenous soluble alpha Klotho protein in the human or non-human animal subject. including compositions. The composition or health supplement may contain (synthetic) chemical and/or natural constituents or ingredients. In other words, the constituents or ingredients of the composition or health supplement may be of or derived from (synthetic) chemical and/or natural sources. Exemplary compositions are adapted (or chemically configured) to elevate serum soluble Klotho protein levels by increasing or enhancing the endogenous production of soluble Klotho protein by a mammalian subject or such may include multiple components that are adapted (or chemically configured) to

Exemplary compositions preferably contain, for example, vitamin D3, vitamin E, vitamin C, vitamin K, 3,5,4′-trihydroxy-trans-stilbene (resveratrol), pterostilbene, N-acetylcysteine (NAC), Troglitazone, Rosiglitazone, Notopterygium incisum Ting, Gentian (root, extract), Cordyceps (Cordyceps Sinensis (CS) mycelium), Isoflavones (soybean), Genistein, Daidzein, Quercetin (dihydrate, dill, bay leaf) , Oregano), Docosahexaenoic Acid (DHA), Astaxanthin, Semen Sesamin Nigrum (black), Sesame (seed extract), Rosmarinic Acid (RA, (Marjoram)), Tetradecylthioacetic Acid (TTA), Diphosphate Calcium (Anhydrous), Microcrystalline Cellulose (MCC), Croscarmellose Sodium (Primerose), Stearing Acid, Magnesium Stearate, Alpha Lipoic Acid, Conjugated (Alpha) Linoleic Acid (CLA), Intestinal Beneficial Bacteria , activated carbon, and others known in the art.

Exemplary compositions preferably contain suitable nutritional supplement concentrations and preferably include, for example, 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 intestinal beneficial bacteria It may contain one or more selected components. The one or more intestinal beneficial bacteria is an effective amount of Bifidobacterium species and/or strains Bifidobacterium lactis BL-04, Bifidobacterium bifidum/lactis BB-02, and Bifidobacterium longum BL-05, and Lacto bacillus species and/or strains Lactobacillus acidophilus LA-14, Lactobacillus
rhamnosus LR-32, and Lactobacillus paracasei LPC-37.

Certain ingredients or components may have positive benefits and very low side effects in treating or affecting one or more conditions associated with cell and tissue dysfunction that occurs with age. Certain ingredients or components can increase (directly or indirectly) circulating plasma levels of soluble alpha klotho (s-klotho) levels in a human or non-human animal subject following its administration.

In at least one embodiment, the compositions of the present disclosure can be marketed as an over-the-counter drug, marketed, and/or sold direct to consumer (DTC) or purchased without a prescription/physician's direction. can be or include a nutritional supplement that complies with the Dietary Supplement Health Education Act of 1994 (DSHEA). In some embodiments, a composition of the present disclosure can be or comprise a US Food and Drug Administration (FDA) approved drug.

In some embodiments, the components may be co-administered, preferably via at least some co-formulations. In some embodiments, the components may be co-administered in separate formulations. For example, in some embodiments, the ingredients may be co-administered in a multi-pill, multi-capsule, or dosage pack. Some ingredients may be provided in solid, granular, powdered, liquid, or other form.

As described herein, some embodiments may include one or more (additional) ingredients or components. For example, some embodiments may comprise one or more recombinant (eg, human or mammalian) Klotho proteins, protein fragments, and/or protein variants, as described herein. Certain embodiments include expression nucleic acid constructs and/or vectors, cell lines and/or cell suspension cultures, and one or more recombinant (e.g., human or mammalian) Klotho proteins, protein fragments, and/or protein variants. It can include methods of manufacturing the body, purifying it, and administering it to a subject (human or non-human animal). Some embodiments include pharmaceutical or prescription drugs (e.g. ARBs (e.g. losartan, valsartan), testosterone, vitamin D receptor agonists (e.g. calcitriol, paricalcitol), PPAR (gamma) agonists (e.g. thiazolidine dione, troglitazone, rosiglitazone), and/or others described in the patent references incorporated above).

Some embodiments comprise a pharmaceutically effective amount of one or more recombinant Klotho proteins and an effective amount of a composition formulated to increase endogenous Klotho protein levels or production (e.g., in a mammalian subject) can include compositions that include substances (eg, health supplements). In some embodiments, the recombinant Klotho protein may be co-administered with the composition. In some embodiments, the recombinant Klotho protein can be combined with a pharmaceutically acceptable carrier. In some embodiments, the administered recombinant Klotho protein can elevate serum soluble Klotho protein levels by providing exogenous Klotho protein, while the composition reduces the amount of Klotho protein (naturally occurring) by the patient or subject. ) can raise serum soluble Klotho protein levels by increasing or enhancing production.

Some embodiments may include therapeutic methods. The method may comprise administering a (pharmaceutically) effective amount of a composition of the present disclosure to a subject in need thereof. The composition comprises (i) a pharmaceutically effective amount of one or more recombinant soluble Klotho proteins or protein variants of the present disclosure, and/or (ii) endogenous Klotho protein levels or production (e.g., in a mammalian subject) may include a pharmaceutically effective amount of the composition (eg, health supplement) formulated to increase the

Some embodiments may include methods of treating Klotho deficiency (eg, in a human or non-human animal (eg, mammalian) patient or subject). The method may optionally or preferably comprise administering the composition to the mammalian subject after diagnosing the mammalian subject with Klotho deficiency. The composition comprises, for example, a pharmaceutically acceptable carrier or excipient and a pharmaceutically effective amount of a recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion disposed in the carrier or excipient and nutrition, comprising a plurality of components adapted to increase serum soluble Klotho protein levels by increasing or enhancing the endogenous production of soluble Klotho protein by a mammalian subject. It may contain one or more of the auxiliary compositions.

Some embodiments may include methods of treating age-related or other conditions, diseases, or disorders. Some embodiments may include methods of treating and/or preventing acute kidney injury (AKI), chronic kidney disease (CKD), or other conditions. The subject to whom the composition is administered can suffer from or be at risk of various conditions (eg, disorders, diseases, injuries, illnesses, etc.). For example, some embodiments treat one or more chronic diseases and/or age-related conditions, such as physical, intellectual, neurological, or other conditions associated with (human) aging. including how to Some embodiments may promote healing, recovery, longevity, and/or other beneficial outcomes through one or more mechanisms or actions. Administration of the composition(s) of the invention can have a positive therapeutic effect on, and characterization of, the course and outcome of disease states, including chronic and/or age-related diseases and longevity in human subjects. .

A pharmaceutically effective amount of the composition(s) raises the serum soluble Klotho protein concentration in a subject to a predetermined level such as greater than, equal to, or between about 50 and 3000 picograms per milliliter of serum soluble Klotho protein. It may be sufficient to raise or increase. The amount may also, or alternatively, be sufficient to maintain the subject's serum soluble Klotho protein concentration at or above a predetermined threshold for a predetermined period of time. Embodiments may also include administering the composition(s) to a subject in need thereof to maintain the subject's serum soluble Klotho protein concentration at or above a predetermined threshold for a predetermined period of time.

Embodiments may also include determining the subject's serum soluble Klotho protein concentration. Embodiments may also include calculating a pharmaceutically effective amount of a composition of the disclosure. Embodiments may also include determining the rate of decline of soluble Klotho protein in the serum of the subject. Embodiments may also include calculating a subsequent dosing time at which the subject's serum soluble Klotho protein concentration is at or below a second predetermined level based on the determined rate. Embodiments also include calculating subsequent dosing times and/or amounts of compositions sufficient to raise the subject's serum soluble Klotho protein concentration from a second predetermined level to a first predetermined level. obtain. Embodiments can also include administering subsequent dosages of the composition to the subject. Certain embodiments may involve prescribing a regular (eg, daily) dose or dosage form of the composition to a subject (eg, based on the determined level of the subject's serum soluble Klotho protein concentration).

In certain embodiments, the composition (eg, protein or supplement) or elevated soluble Klotho protein levels can modulate (can be effective to modulate) the IGF-1 and/or Wnt signaling pathways, can exhibit β-glucuronidase and/or sialidase activity, can inhibit the p53/p21 signaling pathway, and/or reduce H ₂ O ₂ -induced cellular senescence and apoptosis, preferably through inhibition of the p53/p21 signaling pathway can. The protein or elevated protein level preferably exhibits pleiotropic activity and/or preferably oxidative stress, growth factor signaling, ion homeostasis and/or one or more ion channel proteins and/or insulin/insulin It may function as a humoral factor or may be functional in modulating the activity of glycoproteins on the cell surface such as growth factor receptors such as the like growth factor-1 receptor.

In certain embodiments, the composition (e.g., protein or supplement) or elevated soluble Klotho protein levels are also associated with frailty, decreased or decreased bone mineral density, weight loss, muscle atrophy or wasting, decreased muscle mass, decreased muscle strength. , decreased grip strength, leg strength or physical strength, movement, freedom of movement, quality of life assessment, ejection fraction, decreased motor capacity, decreased learning ability, ability to learn, memory, or intelligence quotient, cognitive decline or amnesia, cognition Treating (therapeutically effective) one or more age-related conditions (or (human) age-related conditions) such as reduced capacity or function, reduced synaptic plasticity or synaptic function, and cellular senescence. could be.

In certain embodiments, the composition (eg, protein or supplement) or elevated soluble Klotho protein levels are associated with Alzheimer's disease, Parkinson's disease, dementia or vascular dementia, amyotrophic lateral sclerosis (ALS) or motor neuron disease (MND), atrial fibrillation, chronic obstructive pulmonary disease (COPD), fibromyalgia, adult-onset diabetes, arthritis or rheumatoid arthritis, osteoarthritis, osteoporosis, glaucoma, cataracts, macular degeneration and others Treating one or more age-related conditions (or (human) age-related conditions) such as eye diseases/disorders, multiple sclerosis (MS), lupus, and/or ulcerative colitis. (be therapeutically effective).

In certain embodiments, the composition (eg, protein or supplement) or elevated soluble Klotho protein levels may treat (and be therapeutically effective) one or more other diseases or conditions. For example, some embodiments of the present disclosure are directed to treating cancer, lowering serum phosphate levels in a patient, diabetes or a diabetes-related condition (e.g., type 1 diabetes, etc.) in a subject in need of treatment. ), treating cardiac conditions in a subject (e.g., cardiovascular disease, left ventricular hypertrophy (LVH), pathological LVH, and/or congestive heart failure, etc.), (e.g., using nanoparticles treating acute lung injury in a subject; protecting a patient's lungs against oxidative injury; detecting early acute kidney injury in critically ill patients; attenuating vascular calcification in a subject; improving strength, treating renal and/or hepatic ischemia, modulating the stress response of (human) senescent endothelial cells, acute and/or chronic renal injury, disease, or disease progression, and /or prophylactically and/or therapeutically treating, preventing, reducing, inhibiting and/or reversing uremic cardiomyopathy, reversing or attenuating age-related resistance to therapy of melanoma, apoptosis of senescent cells and preferably thereby restoring tissue homeostasis, as well as in one or more of a variety of other indications.

Accordingly, embodiments may also include compositions for use in treating one or more age-related or other conditions. An exemplary method of treating age-related or other conditions, diseases, or disorders comprises administering an effective amount of a composition of the present disclosure to a subject in need thereof. The composition comprises (i) a supplement as described herein, and/or (ii) (eg, SEQ ID NOs: 2-70, or SEQ ID NOs: 107-120, or SEQ ID NOs: 125-128). or having at least 80% amino acid sequence identity with at least a portion of one of SEQ ID NO: 133, or SEQ ID NO: 152). Optionally, the composition may contain a pharmaceutically acceptable carrier or excipient.

Some embodiments include methods of detecting and/or quantifying endogenous and/or exogenous Klotho protein, and more specifically endogenous and/or exogenous soluble alpha Klotho protein. Some embodiments include methods of diagnosing Klotho protein deficiency in a subject. Some embodiments include products (eg, systems or kits) for detecting and quantifying Klotho protein levels, and more particularly endogenous and/or exogenous soluble Alpha Klotho protein levels. Some embodiments include methods of diagnosing Klotho protein deficiency in a subject. Some embodiments may include methods of treating Klotho deficiency in a subject.

Certain embodiments may comprise a (mammalian) subject's blood or serum sample, preferably capillary blood and/or blood obtained from a finger prick or other non-phlebotomy (non-phlebotomy) blood collection technique, or available. However, it will be understood that phlebotomy (phlebotomy) and other suitable blood collection techniques are also contemplated herein.

Some embodiments include kits. Embodiments can include, for example, sample collection vessels. The container may have an opening configured to receive capillary blood. In some embodiments, capillary blood may contain or be suspected of containing an amount of soluble Klotho protein. Embodiments may include sample preservatives or anticoagulants, preferably including one or more of EDTA and heparin. In some embodiments, a sample preservative or anticoagulant may be disposed within the sample collection compartment of the container. The solution can be configured to stabilize the soluble Klotho protein for at least 24 hours and up to 7 days or more at room temperature or without freezing. Embodiments may optionally include a capillary blood sampling device, preferably comprising a lancing device or lancet.

Some embodiments include methods. For example, some embodiments may include methods of stabilizing Klotho protein in mammalian blood samples. Embodiments can include, for example, drawing or obtaining capillary blood from a mammalian subject and storing the capillary blood in a container at room temperature or without freezing for at least 24 hours. The container may have a preservative or anticoagulant disposed therein such that the preservative or anticoagulant mixes with the capillary blood within the container. The preservative or anticoagulant can stabilize the soluble Klotho protein for at least 24 hours at room temperature or without freezing. The preservative or anticoagulant can be or include one or more of heparin, lithium heparin, EDTA, and _K2EDTA .

Embodiments may also include collecting capillary blood into a container and/or mixing capillary blood with a preservative or anticoagulant to form a mixture. In some embodiments, drawing capillary blood may comprise lancing the skin of the mammal. In some embodiments, collecting capillary blood into the container may comprise collecting at least or about 10-1000 μl, preferably about 50-200 μl of capillary blood into the container. Some embodiments may include storing the mixture or allowing storage of the mixture at room temperature or without freezing, wherein the preservative or anticoagulant is stored at room temperature or without freezing. Stabilize the soluble Klotho protein for at least 3 days.

Some embodiments may optionally include assaying the mixture for the presence of soluble Klotho protein. Assaying the mixture for the presence of soluble Klotho protein may comprise detecting soluble Klotho protein and/or quantifying the amount of soluble Klotho protein. Detecting soluble Klotho protein and/or quantifying the amount of soluble Klotho protein can comprise contacting the mixture with an antibody configured to bind to soluble Klotho protein. Detecting soluble Klotho protein and/or quantifying the amount of soluble Klotho protein comprises performing an ELISA on the mixture using an antibody configured to bind to soluble Klotho protein. can contain. Other detection methods include mass spectrometry or multi-analyte profiling (xMAP), Luminex technology (e.g., miniaturized liquid array bioassays, miniature lasers, light emitting diodes (LEDs), digital signal processors, photodetectors, and/or charge combined element imaging) and the like.

Some embodiments include methods of diagnosing Klotho deficiency in a mammalian subject or methods of treating Klotho deficiency in a mammalian subject. Embodiments can include, for example, providing or obtaining a fluid biological sample. The sample may comprise mammalian serum, preferably in the form of capillary blood. The method can optionally include mixing the mammalian serum with a preservative or anticoagulant to form a mixture. The sample may also, or alternatively, comprise a mixture comprising mammalian serum, preferably in the form of capillary blood, and a preservative or anticoagulant. The method may comprise assaying the mixture for the presence of soluble Klotho protein. The step of assaying the mixture for the presence of soluble Klotho protein comprises storing the Klotho-stabilized mixture at room temperature or without freezing for a period of time and then detecting the presence, if any, of soluble Klotho protein in the mixture. and/or quantifying the amount of soluble Klotho protein in the mixture, if present. The method can comprise diagnosing the mammalian subject with Klotho deficiency if no soluble Klotho protein is detected in the mixture or if the amount of soluble Klotho protein quantified in the mixture is less than a predetermined amount. . In some embodiments, diagnosing a mammalian subject with Klotho deficiency comprises displaying the determination or diagnosis of Klotho deficiency on a user interface of a computer system and/or displaying the determination or diagnosis of Klotho deficiency; It may include generating a file or report in physical or electronic form.

In some embodiments, providing or obtaining a fluid biological sample may comprise drawing capillary blood from a mammalian subject and/or drawing capillary blood into a container. In some embodiments, assaying the mixture for the presence of soluble Klotho protein may comprise performing an ELISA on the mixture using an antibody configured to bind to soluble Klotho protein. Some embodiments administer the composition to a mammalian subject if no soluble Klotho protein is detected in the mixture or if the amount of soluble Klotho protein quantified in the mixture is less than a predetermined amount can include Mass spectrometry may also or alternatively be performed.

The disclosed strategies, products, and/or health intervention methods specifically configured to increase circulating and/or endogenous levels of soluble klotho are useful in situations and problems associated with decreased levels of klotho in patients. can help improve.

Some embodiments may include any of the features, options, and/or possibilities described elsewhere in this disclosure, including other aspects or embodiments of this disclosure. Note also that each of the foregoing, following, and/or other features described herein also represents a separate embodiment of the present disclosure. Moreover, combinations of any two or more of such characteristics represent separate embodiments of this disclosure. Such features or embodiments may also be combined in any suitable combination and/or order without departing from the scope of the present disclosure. Accordingly, each of the features described herein may be combined with any one or more of the other features described herein in any suitable combination and/or order. Accordingly, the disclosure is not limited to the specific combinations of exemplary embodiments detailed herein.

Additional features and advantages of exemplary embodiments of the disclosure will be set forth in the description that follows, and in part will be apparent from the description, or may be learned by practice of such exemplary embodiments. The properties and advantages of such embodiments may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other characteristics will become more fully apparent from the following description and appended claims, or may be learned by practice of such exemplary embodiments as described below.

A diagram illustrating alternative RNA splicing of the Klotho gene. A diagram illustrating alternative RNA splicing of the Klotho gene. Ditto. Figures illustrating the surprising and unexpected stability of Klotho protein in human serum over long periods of time at room temperature and other temperatures. Purity and integrity of purified proteins analyzed by 4-20% SDS-PAGE (reducing and non-reducing) stained with GelCode Blue reagent. A dose-response curve is shown. A dose-response curve is shown. A dose-response curve is shown. Purity and integrity of purified antibodies analyzed by 4-20% SDS-PAGE (reducing and non-reducing) stained with GelCode Blue reagent. Purity and integrity of purified antibodies analyzed by 4-20% SDS-PAGE (reducing and non-reducing) stained with GelCode Blue reagent. Raw absorbance data at 450 nm and corresponding antibody titration curve. Raw absorbance data at 450 nm and corresponding antibody titration curve. It shows that there is a relatively weak signal for the IgY/HRP-IgY pair. It shows that there is a relatively weak signal for the IgY/HRP-IgY pair. Graphical representation of (daily) changes in circulating Klotho protein levels in human subjects. Further measurements of Klotho protein are shown. Further measurements of Klotho protein are shown. Klotho levels at different incubation times at room temperature are shown. Ditto. 3 illustrates an exemplary report graph or chart; A chromatograph of Klotho-Fc protein is shown. Exemplary Western blot analysis using anti-Fc antibody-(SEQ ID NOS:52 and 54) or Strep-Tactin-(SEQ ID NO:66) HRP conjugate-based detection. Exemplary Western blot analysis using anti-Fc antibody-(SEQ ID NOS:52 and 54) or Strep-Tactin-(SEQ ID NO:66) HRP conjugate-based detection. Exemplary Western blot analysis using anti-Fc antibody-(SEQ ID NOS:52 and 54) or Strep-Tactin-(SEQ ID NO:66) HRP conjugate-based detection. Exemplary Western blot analysis using anti-Fc antibody-(SEQ ID NOS:52 and 54) or Strep-Tactin-(SEQ ID NO:66) HRP conjugate-based detection. A gel of the various indicated recombinant Klotho protein constructs is shown. Shown is a series of gels of each indicated recombinant Klotho protein construct. Ditto. Ditto. Ditto. Ditto. Density data evaluated from top 10 clones. Viability data assessed from top 10 clones. Productivity data evaluated from top 10 clones. Productivity data evaluated from top 10 clones. Data for the top four clones. Data for the top four clones. Data for the top four clones. Data for the top four clones. Shows copy number stability. The stability of 5 of the clones is shown. The stability of 5 of the clones is shown. The stability of 5 of the clones is shown. The stability of 5 of the clones is shown. The stability of 5 of the clones is shown. Purified Klotho protein observed on the gel after ELISA is shown. Measured Klotho protein concentrations and spike recoveries observed in each sample are shown. Measured Klotho protein concentrations and spike recoveries observed in each sample are shown. Measured Klotho protein concentrations and spike recoveries observed in each sample are shown. Exhaustive glycan preliminary analysis. Exhaustive glycan preliminary analysis. Growth of each cell line is shown. Viability of each cell line is shown. A titration curve for each cell line is shown. Growth of each cell line is shown. Viability of each cell line is shown. A titration curve for each cell line is shown. Growth of each cell line is shown. Viability of each cell line is shown. A titration curve for each cell line is shown. Western blot analysis on expressed protein pools. Western blot analysis on expressed protein pools. Elution of Klotho protein is shown. Ditto. Elution of Klotho protein is shown. Elution of Klotho protein is shown. Analysis of K _on (association), K _off (dissociation) and K _D (Koff/Kon) are shown. FIG. 1 shows that purified Klotho proteins, both native His and Fc-Klotho, stimulated cell proliferation in a dose-dependent manner. FIG. 1 shows that purified Klotho proteins, both native His and Fc-Klotho, stimulated cell proliferation in a dose-dependent manner. FIG. 1 shows that purified Klotho proteins, both native His and Fc-Klotho, stimulated cell proliferation in a dose-dependent manner. FIG. 1 shows that purified Klotho proteins, both native His and Fc-Klotho, stimulated cell proliferation in a dose-dependent manner. FIG. 1 shows that purified Klotho proteins, both native His and Fc-Klotho, stimulated cell proliferation in a dose-dependent manner. Graph showing the mean (mean) body weight (±standard error of the mean, SEM bars) of rats in each group over the course of the 12-day study (daily).

Embodiments of the present disclosure provide compositions and methods for increasing serum soluble Klotho protein levels in a human or non-human animal (or mammalian) subject and Klotho protein level(s), specifically endogenous and and/or compositions and methods for monitoring, detecting, and/or quantifying exogenous soluble alpha Klotho protein level(s) and/or for diagnosing and/or treating Klotho protein deficiency in a subject. . Some embodiments include (i) recombinant human Klotho proteins, protein fragments, and/or protein variants, expression nucleic acid constructs and/or vectors, cell lines and/or cell suspension cultures, and/or (ii) ) that, when administered to a subject, cause an increase in Klotho protein level(s), expression or production, specifically endogenous soluble alpha Klotho protein level(s), expression or production (e.g. supplements), including one or more compositions. Certain embodiments are directed to methods of manufacturing, purifying, and administering a composition of the present disclosure to a subject (human or non-human animal), and/or Klotho protein level(s), expression, Alternatively, it may comprise methods for increasing production, specifically increasing (endogenous and/or exogenous) soluble alpha Klotho protein level(s), expression, or production. Some embodiments provide a and/or methods for diagnosing Klotho protein deficiency in a subject.

Before describing various embodiments of the present disclosure in detail, the present disclosure is intended only to describe specific parameters, language, and specific exemplary systems, methods, and/or articles of manufacture that may vary with each embodiment. It is understood that it is not limited to Accordingly, certain embodiments of the present disclosure have been described in detail with reference to particular characteristics (eg, configurations, parameters, characteristics, steps, components, components, members, elements, parts, and/or portions, etc.). The description, however, is exemplary and should not be construed as limiting the scope of the present disclosure and/or the claimed invention. Additionally, the terminology used herein is for the purpose of describing embodiments and is not necessarily intended to limit the scope of the present disclosure and/or the claimed invention.

Although the detailed description is divided into paragraphs, the paragraph headings and content within each paragraph are for organizational purposes only and are intended to stand alone descriptions and embodiments or otherwise limit the scope of the description or claims. not intended to Rather, the content of each paragraph in the Detailed Description should be read and understood as a collective whole in which elements of one paragraph may relate to and/or be characteristic of other paragraphs. intended to Accordingly, embodiments specifically disclosed in one paragraph may also be incorporated into additional and/or alternative embodiments within another paragraph having the same and/or similar products, methods, and/or terms. may be related and/or functional.

To facilitate understanding, where possible, like references (i.e., like components and/or like names for elements) are used to denote like elements common to different embodiments of the present disclosure. ing. Similarly, similar components, or components having similar functions, will be provided with similar reference designations where possible. Certain language will be used herein to describe the exemplary embodiments. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Rather, the language used to describe the exemplary embodiments is exemplary only and limits the scope of the disclosure (unless it is expressly stated herein that such language is essential). It is understood that it is not to be construed as a thing.

For brevity, this disclosure may recite numerical lists or ranges. However, such a list or range of numerical values (e.g., greater than a particular value, less than a particular value, up to a particular value, at least a particular value, and/or about a particular value, and/or two recited values) are disclosed or recited, any particular value or range of values within a disclosed value or list, or range of values, is also specifically disclosed and contemplated herein. It will be understood that As an illustrative example, disclosure of a particular ingredient or component "up to 1,000 mg" includes (i) 0.01 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, 750 mg, 990 mg, and 1,000 mg any value greater than zero and less than or equal to 1,000 milligrams, including, but not limited to, and/or (ii) 0.01 to 1,000 mg, 1 mg to 990 mg, 5 mg to 750 mg, 10 mg to 500 mg, and Includes specific disclosure of ranges of values greater than zero and less than or equal to 1,000 milligrams, or any value therebetween, including, but not limited to, 50 mg to 100 mg. Similarly, a disclosure of "at least 80% amino acid sequence identity" or "80% to 100% amino acid sequence identity" includes (i) 81%, 82%, 83%, 84%, 85%, 86% , 80% to 100%, including 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% any whole percentage value of %, as well as any fractional percentage value therebetween, and/or (ii) as non-limiting examples, 81% to 100%, 82% to 100%, 83% to 100%, 84%-100%, 85%-100%, 86%-100%, 87%-100%, 88%-100%, 89%-100%, 90%-100%, 91%-100%, 92% 80, including ~100%, 93%-100%, 94%-100%, 95%-100%, 96%-100%, 97%-100%, 98%-100%, and 99%-100% Includes specific disclosure of any percentage value range from % to 100%.

SUMMARY LIST OF DEFINED TERMS To assist in understanding the scope and content of the foregoing as well as the description and claims set forth below, some selected terms are defined below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
Various aspects of this disclosure, including systems, methods, and/or articles of manufacture, can be illustrated with reference to one or more embodiments that are exemplary in nature. As used herein, the term “embodiment” means “serving as an example, instance, or illustration,” and is not necessarily preferred over other aspects disclosed herein; or should not be construed as advantageous. In addition, references to the disclosure or to "embodiments" of the invention are intended to provide illustrative examples, rather than to limit the scope of the invention, which is indicated by the appended claims.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" each refer to both singular and plural referents unless the context clearly dictates otherwise. It contemplates, includes, and specifically discloses the For example, reference to "a protein" contemplates and specifically discloses one and more (eg, two or more, three or more, etc.) proteins. Similarly, the use of plural referents does not necessarily require plural such referents, but contemplates and includes support for singular and plural such referents unless the context clearly dictates otherwise. , specifically discloses and/or provides.

As used throughout this disclosure, the words "can" and "may" do not have a compulsory meaning (i.e., meaning must), but rather Used in a permissive sense (ie meaning having the potential). In addition, the terms "including," "having," "involving," "containing," "characterized by," variations thereof (e.g., Similar terms used herein, including “includes,” “has,” and “involves,” “contains,” etc.) and claims, include , shall be inclusive and/or open-ended and shall illustratively have the same meaning as the word "comprising" and variations thereof (e.g., "comprise" and "comprises") , and does not exclude additional, unlisted elements or method steps.

It is understood that all numerical values in this description that indicate amounts of materials or reaction conditions and/or use, except in the examples or where otherwise explicitly indicated, are modified by the word "about." It is what is done. As used herein, with respect to values, the term "about" means +/- 10% of the stated value or amount represented by it. For example, throughout this disclosure, the term "about" refers to the percent concentration or composition of a component or component (e.g., in mixtures such as fluid or liquid mixtures, aqueous mixtures, solutions, etc., optionally or preferably w/w percent, w/ (measured as v percent, v/v percent, etc.). In such cases, the term “about” and/or the term “+/-10%” refer to +/-10% of the stated numerical value, as opposed to +/-10 percentage points of the recited percentage. means and/or includes; As an example, if 20% (w/w) of a component or ingredient reflects 20 g of a component or ingredient per 100 mL of total mixture, then the term "about" and/or the term "+/- 10%" would mean 10 meaning a recited range of 18g to 22g (i.e., 18% (w/w) to 22% (w/w)) rather than a range of % (w/w) to 30% (w/w); and/or include. So-called “about” values and/or +/-10% alternatives are +/-1%, +/-2%, +/-3%, +/-4%, +/-5% of the stated value. %, +/- 6%, +/- 7%, +/- 8%, or +/- 9%, each of which is used herein for the term "about" or +/- 10% It is considered as a suitable alternative or substitute.

As used herein, the terms "approximately" and "substantially" refer to the amounts recited (e.g., still perform the desired function or achieve the (desired or expected) result). Denotes or means near (or any) quantity. For example, the terms "about" and "substantially" refer to amounts within or less than 10%, 5%, 1%, 0.1%, 0.01%, or other percentages of the stated amount. can point to As used herein, the term "substantially free" is generally regarded by those skilled in the art to reflect (1) an undetectable or unquantifiable amount, (2) a detectable or quantifiable amount and/or (3) an amount less than or less than an amount generally considered by those skilled in the art to be functional or capable of achieving a (desired or anticipated) result (eg, less than 10%, 5%, 1%, 0.1%, 0.01%, or other percentage).

A "sufficient amount" (also referred to as a "qs" or "qs") means a sufficient amount. Thus, a “100% dosage,” “provided in 100% dosage,” or “up to 100% dosage” component or ingredient refers to a component or ingredient that completes the composition or (listed provided or included in an amount sufficient to bring the totality of all components) to 100%, whether or not they are included. However, a "100% dosing", "provided at 100% dosing", or "dosing up to 100%" (final) component or ingredient means that the mixture is added immediately before the "100% dosing" component. It should be noted that it does not imply that it consists of, consists essentially of, or contains only the listed or enumerated components. In other words, "100% dosage" and similar terms are intended to be open-ended expressions indicating the source of whatever that remainder is.

As used herein, the term "product(s)" includes compositions, formulations, and mixtures, as well as devices, instruments, assemblies, kits, systems, and the like. Similarly, the term "method" includes processes, procedures, steps, and the like.

As used herein, "characteristics" or "aspects" of the present disclosure or of the embodiments disclosed herein refer to characteristics, characteristics, components, components, elements, members, parts, or components of the subject matter at hand. , part, (method) step, or other aspect.

As used herein, the word “or” generally refers to any one member of a particular list, but also includes any combination of two or more members of that list. Similarly, the term "and" can be interchangeable with the term "or", either of which can be understood to mean "and/or."

The term "comprising" is synonymous with "including," "having," "containing," or "characterized by." These terms are inclusive and open-ended and do not exclude additional, unquoted elements or method steps.

The phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim rather than immediately following the preamble, it limits only the element listed in that clause and does not exclude other elements from the claim as a whole.

The phrase “consisting essentially of” limits the scope of a claim to those that, in addition to the specified materials or steps, do not materially affect the basic and novel feature(s) of the claimed subject matter. do.

The terms “comprising,” “consisting of, and “consisting essentially of” may be used alternatively. Where one of these three terms is used, the presently disclosed and claimed subject matter may include use of either of the other two terms.

The terms "plurality" and "at least two" are used interchangeably.
The term "condition" refers to any disorder, disease, injury, or illness that manifests or is expected in a patient, as understood by those of ordinary skill in the art. Such pathological manifestations may be early, intermediate, or late stage manifestations known in the art, including pre-pathological symptoms, precursors, or markers. Anticipation of such conditions is predicted, expected, imagined, estimated, assumed and/or speculated, whether found on scientific or medical grounds, risk assessment, or simply fear or fear. can be, or include, certain occurrences.

As used herein, the term “patient” is synonymous with the term “subject” and specifically (i) (physicians, nurses, With respect to non-human animals such as humans (in the care of medical assistants, or medical volunteers) and (ii) non-human mammals (in the care of veterinarians or other veterinary professionals, assistants, or volunteers), generally: Refers to any animal under the care of a medical professional.

As used herein, the term "medical professional" generally refers to any individual or institution responsible for providing medical care to humans and animals, including non-human animals such as non-human mammals, Emphasis on qualified or unqualified providers (such as assistants, technicians, and/or volunteers), specifically those covered by (inclusive) licensing or provider insurance . The term may include, where contextually appropriate, oncologists, surgeons or physician assistants, nurses, phlebotomists, veterinarians, and the like.

The term "cancer" refers to abnormal, typically uncontrolled cell growth. As used herein, "cancer cells" include malignant cells with abnormal, typically uncontrolled growth. As such, the term cancer is an umbrella term encompassing a number of different and distinct diseases that are typically characterized by malignant cells that grow in an uncontrolled manner.

As used herein, the term "diagnosis" or "diagnosing" and similar terms are not required or regulated by the U.S. Food and Drug Administration (FDA) or any other governmental or non-governmental agency, organization, or agency. It is not intended to convey or require (in any way) an accredited and compliant medical diagnosis. Rather, as used herein, "diagnosis" or "diagnosing" and like terms relate to providing information indicative of and/or useful in making decisions related to a disease state. .

The term "co-administration" and like terms refer to parallel, sequential, and/or concomitant administration of two or more components. For example, the two components can be co-administered by concurrent, simultaneous, or sequential administration (eg, separate administrations separated by a period of time) in separate dosages of each component. The time period can be very short (e.g., substantially immediately after the first administration) or longer (e.g., 1-60 seconds, 1-60 minutes, 1-24 hours, 1-7 days, 1-7 4 weeks, 1-12 months, etc., or any value or range of values therebetween). Parallel or simultaneous administration can include overlapping time frames of administration of two or more components, or administration of a combination product comprising a mixture of two or more components.

As used herein, “room temperature” is above the freezing point (0° C., or other (equivalent) freezing temperature depending on the presence of freezing point adjusting components) and normal human body temperature (37° C. , or other (equivalent) boiling temperature depending on the presence of the boiling point modifying component), preferably greater than about 4°C and less than about 35°C, more preferably about 5°C to about 32°C, about 8 ° C to about 30 ° C, about 10 ° C to about 30 ° C, about 10 ° C to about 25 ° C, about 10 ° C to about 20 ° C, about 10 ° C to about 15 ° C, about 15 ° C to about 30 ° C, about 15 ° C to about 25° C., about 15° C. to about 20° C., about 20° C. to about 30° C., about 20° C. to about 25° C., about 20° C. to about 22° C., or any value or range of values therebetween can refer to the temperature of

As used herein, “refrigeration” means above freezing point (0° C.) and below about 32° C., preferably from about 1° C. to about 30° C., from about 1° C. to about 25° C., from about 1° C. to about 20° C., about 1° C. to about 15° C., about 2° C. to about 20° C., about 2° C. to about 15° C., about 3° C. to about 20° C., about 3° C. to about 15° C., about 4° C. to about 20° C. C., from about 4.degree. C. to about 15.degree. C., or any value or range of values therebetween.

As used herein, "nucleic acid" and like terms means DNA or RNA or DNA-RNA hybrids, single- or double-stranded, either sense or antisense, naturally occurring or Or refers to a synthetic oligonucleotide or polynucleotide. Specifically, nucleic acids can include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof. Nucleic acids of the present disclosure also include nucleotides or nucleic acid analogs known in the art (e.g., BrdU), and non-phosphodiester (internucleoside) linkages or backbones (e.g., peptide nucleic acid (PNA) or thiodiester linkages). can be included.

As used herein, the term "peptide" or "protein" refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. Peptides are made up of consecutive amino acids. The term "peptide" or "protein" encompasses naturally occurring or synthetic molecules.

As used herein, the term "standard amino acids" includes alanine-ala-A; arginine-Arg-R; asparagine-asn-N; gln-Q; glutamic acid-glu-E; glycine-gly-G; histidine-his-H; isoleucine-ile-I; leucine-leu-L; threonine-thr-T; tryptophan-trp-W; tyrosine-tyr-Y; and valine-val-V.

As used herein, "codon-optimized" or "codon-optimized" means that the codons within a nucleotide sequence are preferred to the codon usage pattern in the organism used for expression of that molecule, or Refers to the process of modifying or changing codons to more closely match. Thus, for use in particular organisms where expression is desired based on known codon usage in the organism, in order to enhance the efficiency of expression of nucleic acids, e.g., to achieve faster translation rates and greater accuracy. codons can be optimized for Codon usage in certain organisms is known.

Encoding nucleic acid molecules may be modified wild-type, or codon-expressed in particular host cells, e.g., in mammalian cells such as CHO cells or 293 cells, or in yeast, or in plant cells, in eukaryotic cells, and the like. can be a codon-optimized sequence, optimized for

In certain instances, nucleic acid sequences can be codon-optimized, eg, to increase the level of expression of the encoded sequences. Particular codon usage depends on the host organism in which the modified polypeptide is expressed. Those skilled in the art are familiar with the method of intramammalian or human cells, eg, E. We are familiar with the optimal codons for expression in bacteria, including E. coli or Saccharomyces cerevisiae, or in yeast. For example, the codon usage information is kazusa. or. jp. available from the Codon Usage Database available at codon (for a description of the database, see e.g. Richmond (2000) Genome
See Biology, 1:241. See also Forsburg (2004) Yeast, 10:1045-1047, Brown et al. (1991) Nucleic Acids Research, 19:4298, Sharp et al. (1988) Nucleic Acids Res. , 12:8207-8211; Sharp et al. (1991) Yeast, 657-78).

The first definition of an acronym or other abbreviation applies mutatis mutandis to all subsequent uses of the same abbreviation and applies mutatis mutandis to the ordinary grammatical variations of the first defined abbreviation, unless expressly stated to the contrary. Measurements of properties are determined by the same techniques referred to earlier or later for the same properties.

Exemplary Embodiments Exemplary embodiments of the present disclosure include compositions, compositions of matter, articles of manufacture, articles of manufacture, formulations, combination products, co-administration, co-formulations, dosages, processes, methods, treatment protocols, Including, but not limited to, diagnostic methods, kits, and the like.

Products and Methods for Stabilizing Blood Klotho Protein The stability of Klotho protein (e.g., soluble Klotho) in blood or serum, particularly at room temperature, and more particularly for (extended) periods of time, has been previously reported. It has not been. Figure 3 illustrates the surprising and unexpected stability of Klotho protein in human serum over long periods of time at room temperature and other temperatures.

Without being bound by any theory, a typical blood or serum protein diagnostic involves intravenous blood sampling to obtain a suitable amount of blood for protein (level) monitoring, detection, and/or quantification. need. In addition, drawn blood samples are typically processed immediately after sampling (e.g., the same day, within minutes or hours of sampling, etc.) or refrigerated for preservation of protein and sample stability. (below room temperature). However, in some embodiments of the present disclosure, the inventors have found that blood Klotho protein can be stable at room temperature for up to 7, 9, 14, or more days. In some embodiments, the Klotho protein is treated at room temperature for at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, e.g., by adding EDTA and/or heparin to the sample. 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days , 27 days, 28 days, 29 days, or 30 days (data not shown). As a non-limiting example, Klotho levels in blood samples taken from four donors were relatively constant over 7 days (and 14 days) at room temperature when EDTA and/or heparin were added. (See Figure 3). In addition, both anticoagulants tested, EDTA and heparin, showed no significant difference with respect to Klotho concentration. This was surprising and unexpected to the inventors.

Storage at room temperature can be important for commercial diagnostic applications where refrigeration of hundreds, thousands, tens of thousands, etc. of samples may not be commercially practical (logically and/or financially). Commercial diagnostic testing of Klotho blood or serum levels can be accomplished using the preferred room temperature stabilization methods described herein.

In view of the foregoing, embodiments of the present disclosure may include methods and/or products for combining EDTA and/or heparin with a biological sample (eg, blood or serum) containing Klotho protein. Some exemplary products may include sample collection kits that include sample collection containers and sample storage compositions.

Some embodiments include methods of stabilizing Klotho protein in mammalian blood samples. A method can include obtaining capillary blood from a mammalian subject. Capillary blood may contain (or be suspected of containing) a certain amount of soluble Klotho protein. The method preferably comprises soaking capillary blood (eg, in a container) for at least 24 hours (or preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) at room temperature. , 14, 18, 21, 28, or 30 days, or more). Capillary blood samples may be mixed with preservatives or anticoagulants. For example, it may have a preservative or anticoagulant disposed therein or added thereto such that the preservative or anticoagulant is mixed with the capillary blood in the container. The preservative or anticoagulant can be or include one or more of heparin, lithium heparin, EDTA, and _K2EDTA . The preservative or anticoagulant may stabilize the soluble Klotho protein for at least 24 hours (or more), preferably at room temperature. However, it will be understood that refrigeration and/or freezing are also contemplated herein.

In addition, the inventors have found that Klotho protein levels measured by the finger stick method (about 10-1000 μl, preferably about 50-200 μl of blood) are consistent with Klotho protein levels measured from venous blood draws of the same subjects. found to do. Therefore, Klotho protein can be measured accurately, reproducibly and reliably in relatively small commercially practical blood volumes. This was surprising and unexpected to the inventors. Specifically, the inventors expected that blood volumes collected in standard vials might be required to accurately, reproducibly, and reliably measure blood Klotho protein. .

Without being bound by any theory, venous blood draws (eg, FDA approved or other diagnostic) typically require a professional phlebotomist. Venous blood draws are also inconvenient and painful for the patient. The finger stick method, on the other hand, is simple and inexpensive. Commercially, direct-to-consumer applications (eg, diagnostic kits) may require at-home blood sampling. In such cases, venous blood draws may be undesirable and/or impractical. Embodiments of the present disclosure may enable capillary blood sampling by a simple and convenient finger prick (or other means of sampling capillary blood). The amount of capillary blood to be sampled is, for example, about 10-1000 μl, preferably about 20-800 μl, more preferably about 30-600 μl, even more preferably about 40-400 μl, even more preferably about 50-200 μl. It can be capillary blood.

In view of the foregoing, some embodiments of the present disclosure may include products and methods for capillary blood (non-venous, non-phlebotomy, etc.) sampling of blood or serum. Some embodiments may include a capillary blood sampling device. Illustratively, the capillary blood sampling device may be or include a lancing device and/or may include (blood) lancets). In some embodiments, the blood sampling or lancing device may be or include a finger stick device such as a (spring-loaded) finger prick. However, it will be appreciated that the present disclosure is not limited to fingers and quick finger prick devices. In some embodiments, the lancing device or its lancet may be disposable and/or replaceable. Embodiments may also include written instructions for using the kit to collect blood samples.

An exemplary method of stabilizing Klotho protein in a mammalian blood sample is obtaining capillary blood from a mammalian subject, wherein the capillary blood contains an amount of soluble Klotho protein. and storing the capillary blood in a container at room temperature for at least 24 hours, the container having a preservative or anticoagulant disposed therein, the preservative or anticoagulant is mixed with capillary blood in a container, and a preservative or anticoagulant stabilizes the soluble Klotho protein for at least 24 hours at room temperature, and the preservative or anticoagulant is heparin, lithium heparin, EDTA. , and storing, including one or more of K ₂ EDTA. In some embodiments, the capillary blood mixed with a preservative or anticoagulant is treated at room temperature for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 18, 21, 28, or 30 days, wherein the preservative or anticoagulant reduces soluble Klotho protein at room temperature to at least 2, 3, 4, 5, 6, 7, 8, Stabilize or store for 9, 10, 11, 12, 13, 14, 18, 21, 28, or 30 days. In certain embodiments, samples of the mixture may also be refrigerated.

In certain embodiments, a small amount of blood (1-4 drops, about 10-1000 μl, preferably about 50-200 μl) can be drawn (eg, at home) and unfrozen, unrefrigerated, or unchilled (eg, (ambient or room temperature) via normal shipping methods to a diagnostic facility. This provides a substantial advantage over existing methods or products. Thus, private, home, or other non-clinical based tests or diagnostics (eg, lateral flow with quantification/metrics, home ELISA kits, etc.) are contemplated herein. However, it will be understood that any clinical or clinically based test or diagnosis is also contemplated herein.

Products and Methods for Measuring Klotho Protein Levels Some embodiments detect and/or endogenous and/or exogenous Klotho protein, and more specifically endogenous and/or exogenous soluble alpha Klotho protein. Including how to quantify. Some embodiments include methods of diagnosing Klotho protein deficiency in a subject. Some embodiments include products (eg, systems or kits) for detecting and quantifying Klotho protein levels, and more particularly endogenous and/or exogenous soluble Alpha Klotho protein levels. Some embodiments include methods of diagnosing Klotho protein deficiency in a subject.

Levels of circulating soluble Klotho protein in mammalian serum are hypothesized to decrease with age. However, prior to the present disclosure, it was unclear at what age this hypothesized decrease in serum Klotho levels could begin to occur, and what is the normal level (or range) of naturally occurring soluble circulating Klotho protein in humans and non-human animals. reliable and reproducible as to whether there is, what is the rate of decline of circulating Klotho in humans and non-human animals, and to what extent the level (or extent) of naturally occurring soluble Klotho protein in humans and non-human animals is reduced. Good data are not available or widely accepted. Commercially available antibodies provide reliable, reproducible and/or It may not be suitable for cost-effective detection. Accordingly, suitable antibodies (eg, for blood sample ELISA detection) were developed according to the following procedure.

Phase 1: Klotho protein expression: various sizes of (mouse and human) alpha Klotho proteins (e.g. , in humans, amino acid residues 34-981, 34-549, etc.) were prepared. Gene constructs corresponding to the above proteins were synthesized and cloned into commercially available expression vectors, which were then transfected into HEK293 or CHO cells for Klotho protein production. The expressed Klotho protein was purified by methods known and described in this disclosure. Illustratively, using IMAC and IE protein chromatography, a protein sample of 0.78 mg/mL (measured by UV absorbance at 280 nm) in 330 mM NaCl, 20 mM Na phosphate buffer, pH 8.0 got Similarly, Protein A chromatography as described herein followed by size exclusion chromatography yielded active protein in monomeric and multimeric (eg, dimer and tetramer) forms.

Quality control: Purity and integrity of purified proteins were analyzed by 4-20% SDS-PAGE (reducing and non-reducing) stained with GelCode Blue reagent. For example, see FIG.

Lane 1: Klotho ECD, lot number 170918A, 2 μg/lane, reduced.
Lane M: protein molecular weight markers.
Lane 2: Klotho ECD, lot number 170918A, 2 μg/lane, non-reduced.

Lane 3: BSA standard, 2 μg/lane, reduced The calculated theoretical molecular weight of the expressed protein was 109.97 kDa.
Phase 2: Chicken Polyclonal (IgY) and Mouse Monoclonal (Hybridoma) Development: The expressed proteins were used as antigens to immunize mice and chickens to develop monoclonal and polyclonal antibodies. Mouse monoclonals are preferred for some embodiments of ELISA antibodies. After the fusion process (spleen cells with myeloma cells), two rounds of hybridoma selection were performed, initially screening approximately 3,000 clones and finally narrowing down to 10 clones based on activity. is. These 10 clones were pooled, cultured and purified to yield approximately 5 mg of purified antibody from each.

The table below shows exemplary mouse serum IgG titration using target antigen by indirect ELISA (standard ELISA protocol, TMB 5 minutes).
Antigen 1-0.5 μg/mL 6xHis-tagged Klotho in 50 mM Na bicarbonate buffer.

Antigen 2-Klotho-Fc 0.5 μg/mL in 50 mM Na bicarbonate buffer.

Summary of Table 1: Mice M3 and M5 were selected for spleen harvesting.
Primary screening of Klotho hybridomas: Splenocytes from mice #M3 and #M5 were harvested and fused with myeloma cells according to standard protocols.

Hybridoma supernatants were evaluated by indirect ELISA using target antigen (standard indirect ELISA protocol, TMB 10 minutes).
Antigen, Klotho protein (Lot #170918A, 0.78 mg/mL) 0.5 μg/mL in 50 mM Na bicarbonate, 100 μl/well, +4° C. overnight.

Anti-mouse HRP, Peroxidase AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3), Fcγ fragment specific, 1:4000 in SEDB (Jackson Lab., Catalog No. 115-035-164), 100 μL/well, 1 hour, room temperature.

Selected cut-off OD450nm>0.5.
The table below shows the primary Klotho hybridoma screen. Clones with OD450 nm between 0.5 and <2 are highlighted in yellow (light grey). Clones with OD450nm>2 are highlighted in orange (dark grey).

Summary of Table 2: 2,976 clones were isolated and analyzed. 66 target-specific clones expanded for confirmatory screening.
Secondary screening of Klotho hybridomas: Hybridoma supernatants were evaluated by indirect ELISA using target antigen (standard indirect ELISA protocol, TMB 10 minutes).

Antigen, (1) Klotho-6xHis protein (lot number 170918A, 0.78 mg/mL) in 50 mM Na bicarbonate 0.5 μg/mL, 100 μl/well, +4° C. overnight. (2) Klotho-Fc protein (Vista, 0.1 mg/mL) in 50 mM Na bicarbonate) 0.5 μg/mL, 100 μl/well, +4° C. overnight.

Detection antibody: anti-mouse HRP, peroxidase AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3), Fcγ fragment specific, 1:5000 in SEDB (Jackson Lab., catalog number 115-035-164), 100 μL/well, 1 hour, room temperature .

Summary of Table 3: 28 Klotho-6xHis specific clones were confirmed (OD450 cutoff: >0.5, Klotho-6xHis plate) - 1F6, 2E1, 2E12, 3D2, 4H4, 4H5, 5D12, 7A12, 7F9 , 8G11, 10A8, 10D1, 10G3, 11C9, 11F7, 11H3, 12G4, 12H3, 14B2, 14B7, 14B12, 15E9, 16H1, 18C2, 18G5, 21G12, 26G3, 28F11.

First array of 9 Klotho-6xHis specific clones recommended for native (untagged) Klotho ELISA development (OD450>2, Klotho-6xHis plate) - 2E12, 4H4, 4H5, 5D12, 11C9, 11F7 , 14B12, 26G3, 28F11.

Second array of 11 Klotho-6xHis specific clones recommended for native (untagged) Klotho ELISA development (OD450>1 and <2, Klotho-6xHis plate) - 1F6, 2E1, 3D2, 7A12, 7F9 , 8G11, 10G3, 11H3, 14B7, 18C2, 18G5.

A first array of 10 Klotho-6xHis and Klotho-Fc specific clones (OD450>1.7, Klotho-6xHis and Klotho-Fc plates)-1F6, 2E12, 4H4, 4H5, 8G11, 11C9, 11F7, 14B12, 26G3, 28F11.

A second array of two Klotho-6xHis and Klotho-Fc specific clones (OD450>1.2 and <1.7, Klotho-6xHis and Klotho-Fc plates)-5D12, 14B7.

mIgG Isotyping: mIgG Isotyping of Klotho Hybridoma Selected Clones: Antibody Isotyping from Selected Hybridoma Clone Culture Supernatants.
A sample of hybridoma culture medium was used to determine antibody isotype using a rapid ELISA mouse mAb isotyping kit (ThermoFisher, catalog number 37503) according to the manufacturer's instructions.

The table below shows the results of mIgG isotyping of Klotho hybridoma selected clones.

Summary of Table 4: mIgG isotype determination:
1F6, 2E12, 4H4, 4H5, 8G11, and 11F7: IgG1
11C9, 14B12, 26G3, and 28F11: IgG2b
Quality Control: An indirect ELISA was performed for quality control: ELISA plates were treated with 0.5 μg/ml unconjugated 6xHis-tagged Klotho protein or Klotho-Fc protein in 50 mM Na bicarbonate buffer at +4°C. Coated overnight. Purified 1F6, 2E12, 4H4, 4H5, 8G11, 11C9, 11F7, 14B12, 26G3, and 28F11 antibodies were applied in 5-fold serial dilutions starting at 500 ng/mL and the secondary antibody, goat anti-mouse HRP. , applied at 0.16 μg/ml. The reaction was allowed to occur for 7 minutes. Raw absorbance data at 450 nm and corresponding antibody titration curve.

See also dose-response curves illustrated in Figures 5A, 5B, and 6, respectively.
Quality Control: The purity and integrity of the purified antibody was analyzed by 4-20% SDS-PAGE (reducing and non-reducing) stained with GelCode Blue reagent. See Figures 7A and 7B, respectively.

Polyclonal IgY Antibodies: Polyclonal IgY antibodies were also developed. The above design was adapted as needed for the production of polyclonal IgY antibodies in chickens.
Chicken serum IgY titration using target antigen by indirect ELISA (TMB: 5 minutes). Read at 450 nm Antigen: Klotho 1 μg/mL (0.78 mg/mL, lot number 170918A) in 50 mM Na bicarbonate.

Sample: Chicken #1, #2, room temperature, 1 hour (5% milk in PBS).

ELISA plates were coated with 0.5 μg/ml 6xHis-tagged Klotho protein or Klotho-Fc protein in 50 mM Na bicarbonate buffer at +4° C. overnight. Purified anti-Klotho IgY antibody (Lot No. 180327A) was applied in 5-fold serial dilutions starting at 1000 ng/mL and secondary antibody goat anti-chicken HRP was applied at 0.16 μg/ml. The reaction was allowed to occur for 5 minutes. Raw absorbance data at 450 nm and corresponding antibody titration curve. See also Figures 8A-8B.

Summary: Both HRP and biotin anti-Klotho IgY show significant signals in indirect ELISA. Bio-IgY has a stronger signal than HRP-IgY.
As illustrated in Figures 9A and 9B, there is a relatively weak signal for the IgY/HRP-IgY pair. IgY/bio-IgY shows relatively high background even at low concentrations (0.25 μg/mL). The IgY/bio-4H5 pair appears to perform better than the IgY/labeled-IgY pair.

Levels of circulating soluble Klotho protein in mammalian serum are hypothesized to decrease with age. Baseline measurements of naturally occurring circulating soluble Klotho protein in human subjects have been reported in various publications. A thorough literature review yielded the following average range(s) of circulating soluble Klotho protein in the indicated human subjects (see Tables 8-9).

In the table above, CKD: chronic kidney disease, GH: growth hormone, T1D: type 1 diabetes, T2D: type 2 diabetes, CAD: coronary artery disease, COPD: chronic obstructive pulmonary disease, MS: multiple sclerosis.

In view of the variability observed in the literature, more robust, more consistent and/or more accurate means of measuring serum Klotho levels are needed. Embodiments of the present disclosure include Klotho diagnostic kits and methods of using same, described in further detail below.

Table 10 shows baseline measurements of naturally occurring circulating soluble Klotho protein in human subjects (before receiving any treatment, eg, health supplements, pharmaceuticals, therapeutic proteins, etc.). Figure 10 is a graphical representation of the (daily) changes in circulating Klotho protein levels in human subjects.

Blood samples were taken from human subjects once an hour beginning at 06:50 and Klotho protein levels were measured by ELISA.
A quantitative ELISA according to embodiments of the present disclosure detects (1) native (e.g., endogenous) Klotho, (2) recombinant Klotho-Fc fusions, and (3) recombinant 6xHis Klotho in human plasma samples be able to.

Additional measurements of Klotho protein are illustrated and shown in Figures 11A-11B and Table 11 below.

Klotho Stability in Human Plasma Using ELISA Kit Venous and capillary (“finger”) blood samples were taken from 4 donors (A, B, C, D) and ii) stored in heparin-containing tubes; A set of venous and capillary blood samples taken from Donor A was spiked with 1.5 ng/mL of Klotho (Lot #170918A—Human Soluble Alpha Klotho). Collected blood (blood samples) was stored at room temperature for 0, 1, 4, 16, 24, 48, 72, 96, or 168 hours and then sedimented. Plasma from each sample was stored at -80°C prior to ELISA testing. ELISA plate maps, raw data, standard curve data, and concentrations are shown in Tables 12-21 below.

Figure 12 illustrates Klotho levels at different incubation times at room temperature.
Table 22 below shows the mean Klotho protein levels (ng/mL) in plasma:

Table 23 below shows recovery (%) of spiked Klotho

Klotho Stability Test at Various Temperatures to Emulate Transport Stress The phlebotomist(s) collected over 12 mL of blood (samples) from each of the 4 donors into heparinized blood tubes. Technician(s) immediately dispensed 300-400 μL of blood from each donor's collected sample into separate heparinized microfuge tubes (BD). Tubes were stored and processed according to the study design (see table below). Blood was processed or centrifuged at each time point and stored (plasma) at -80°C. All samples were tested at approximately 17 days.

In this study, 120 data points representing 6 conditions over 14 days were collected (see below).

The plate layout (see Table 25) was the same for all four donors (see Tables 26-29 below).

The ELISA protocol was performed according to the instructions of the Soluble Klotho ELISA Kit (Human), IBL, Catalog No. 27998). The raw data are presented in Tables 26-29 below.

Data analysis is shown in Tables 30-41 below:

In view of the foregoing, at least one embodiment includes a Klotho detection kit and/or assay. Kits and/or assays of the invention may be suitable for CLIA laboratory testing of clinical samples. The kit monitors, detects and/or monitors one or more proteins selected from (1) native (e.g., endogenous) Klotho, (2) recombinant Klotho-Fc fusions, and (3) recombinant 6xHis Klotho. or can be configured for use in quantifying or diagnosing deficiencies thereof. Klotho protein can be detected in a biological sample. In some embodiments, the sample may be (substantially) unpurified. For example, the sample can be or include mammalian (eg, human) blood and/or plasma.

In at least one embodiment, the kit comprises one or more lancets adapted, configured, or suitable for a finger prick, adapted, configured, or suitable for drawing blood from a finger prick. A sample collection container, one or more disinfecting elements (such as disposable (e.g., alcohol) wipe(s)), one or more adhesive bandages, a foil envelope or other biodegradable packaging, and a transport container (such as an envelope). ).

Some embodiments may include methods of diagnosing Klotho deficiency in a mammalian subject. (i) mammalian serum in capillary form, optionally in the form of capillary blood, mixed with a preservative or anticoagulant comprising one or more of heparin, lithium heparin, EDTA, and K2EDTA; and (ii) mammalian serum optionally in the form of capillary blood, said mammalian serum having an amount of soluble Klotho protein. obtaining a fluid sample mixture selected from the group, the method mixing mammalian serum with a preservative or anticoagulant comprising one or more of heparin, lithium heparin, EDTA, and K2EDTA can further include: The method can also include storing the mixture at room temperature for at least 24 hours or longer, wherein the preservative or anticoagulant stabilizes the soluble Klotho protein at room temperature for at least 24 hours or longer. After the storing step, the method can include quantifying the amount of soluble Klotho protein in the mixture. The method can also include diagnosing the mammalian subject with Klotho deficiency when the quantified amount of soluble Klotho protein in the mixture is below a predetermined threshold amount.

As described above, the fluid sample mixture comprises about 10-1000 μl, preferably about 50-200 μl of capillary blood from the mammalian subject, or obtaining the fluid sample mixture comprises about 10 μl of capillary blood from the mammalian subject. Obtaining ˜1000 μl, preferably about 50-200 μl of capillary blood. Quantifying the amount of soluble Klotho protein in the mixture comprises detecting soluble Klotho protein using a first antibody that binds to a portion of the soluble Klotho protein and optionally or performing an enzyme-linked immunosorbent assay (ELISA) to detect and optionally quantify soluble Klotho protein. Mass spectrometry can also be used for protein detection.

In some embodiments, diagnostic or informational reports may be provided. The report may include an indication or display of the measured Klotho level(s) of one or more Klotho proteins. In some embodiments, the report includes comparative charts or graphs depicting measured levels overlaid on age-based graphs exemplifying mean or median Klotho levels for various ages, as well as a number of levels for various ages. Display 25th percentile and/or 75th percentile indications. FIG. 13 illustrates an example report graph or chart. Thus, in some embodiments, diagnosing a mammalian subject with Klotho deficiency comprises displaying the determination, indication, or diagnosis of Klotho deficiency on a user interface of a computer system, and/or determining Klotho deficiency; Determining, indicating, or diagnosing Klotho deficiency includes generating a file or report in physical or electronic form displaying an indication or diagnosis, optionally a quantified amount of soluble Klotho protein and/or including a predetermined threshold amount.

As used herein, the term “diagnosis” or “diagnosing” and like terms are required or It will be understood that it is not intended to convey or require (in any way) a regulated, certified and compliant medical diagnosis. Rather, "diagnosis" or "diagnosing" and like terms relate to providing information indicative of and/or useful in making decisions related to a disease state.

In some embodiments, one or more additional analytes can be monitored, detected and/or quantified. Illustratively, additional analytes may be associated with renal disease, fibrotic disease, and/or aging. In some embodiments, the one or more additional analytes are FGF, PTH, non-oxidized PTH, vitamin D, vitamin E, KIM-1, NGAL, interleukins and other inflammatory biomarkers, testosterone, estrogen and the like.

Therapeutic Proteins Embodiments of the present disclosure may comprise one or more therapeutic and/or recombinant human alpha soluble Klotho proteins, protein fragments, and/or protein variants.

The protein comprises amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549 of human alpha Klotho isoform 1 or 2 , 36-549 or 131-549, or a subset thereof. The protein comprises amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549 of human alpha Klotho isoform 1 or 2 , 36-549 or 131-549, or a subset thereof, and at least and/or about 80% of the amino acid sequence. For example, at least a portion of the protein is at least a portion of one of SEQ ID NOS:2-70, or SEQ ID NOS:107-120, or SEQ ID NOS:125-128, or SEQ ID NO:133, or SEQ ID NO:152 , or a combination of two or more thereof, such as at least 80% amino acid sequence identity. Other portions or fragments of the protein sequences described in this application are also contemplated herein. For example, some embodiments have at least a portion that has an amino acid sequence identity, such as at least 80%, with any suitable portion of one of SEQ ID NOS: 1-75, or a combination of two or more thereof May contain protein.

Some embodiments may include proteins with one or more amino acid divergences compared to human alpha Klotho isoform 1. Illustratively, the protein may include a human C370 variant. For example, the protein may contain a C370S modification, thereby containing S370. In some embodiments, proteins may include other than human F352, or F352V. In at least one embodiment, the protein comprises S370, which may comprise a C370S modification, thereby excluding the F352V variant and preferably comprising F352. Proteins may include H193, or other than H193R variants. All other canonical amino acid substitutions at amino acid residues (or positions) 193, 352, and/or 370 of human alpha Klotho isoform 1 are contemplated and expressly disclosed herein.

Some embodiments may include a diversity at amino acid residue 45 of human alpha Klotho isoform 1. At position 45, the residue can be valine (Val; V), phenylalanine (Phe; F), or another amino acid.

A protein may also include (attached to) one or more glycans. For example, native human alpha Klotho isoform 1 can have glycans attached (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612 and/or 694. Thus, a protein of the disclosure or its Klotho protein sequence has one or more of the same (or similar) glycans attached thereto (via glycosylation) (e.g., at the same amino acid position(s)). can have In a preferred embodiment, the protein comprises all of the same or similar (naturally occurring) glycans attached thereto at the same amino acid position(s).

In some embodiments, a protein may include a signal peptide or signaling sequence. For example, the protein may contain the native Klotho signaling sequence. Proteins may contain non-natural or synthetic signaling sequences. In some embodiments, the signaling sequence can be upstream (or N-terminal) to the N-terminal signaling sequence and/or the Klotho protein sequence. In other embodiments, the signaling sequence may be C-terminal or otherwise positioned. Preferably, the signaling sequence is, comprises, or has at least 80 % amino acid sequence identity.

In some embodiments, proteins may include amino acid tags. The tag can be downstream (or C-terminal) of the C-terminal tag and/or the Klotho protein sequence. In other embodiments, the tag can be N-terminal or otherwise positioned. The tag may be or include an Fc peptide (or Fc fusion tag, Fc domain, etc.). For example, the tag can be or include an IgG1-Fc protein sequence. Preferably, the tag is, comprises, or has at least 80% amino acid sequence identity with SEQ ID NO:74.

A tag can also, or alternatively, be or include a peptide that includes a Twin-Strep protein sequence, such as a Twin-Strep tag or protein sequence (eg, as known in the art). Preferably, the signaling sequence is, comprises, or has at least 80% amino acid sequence identity with SEQ ID NO:75, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104.

The tag may also, or alternatively, be or include polysialic acid (PSA). In at least one embodiment, the PSA tag can render the conjugated (Klotho) protein resistant to one or more proteases. In some embodiments, maintaining an amount (e.g., even a relatively small amount) of sialic acid in the protein conjugate reduces protease attack and subsequent targeting, degradation, and clearance of the molecule (e.g., through the liver) can be prevented.

In at least one embodiment, the tag can be cleaved from the protein. In other embodiments, tags may be retained as part of the protein. In some embodiments, the tag may enhance protein solubility and/or (serum) half-life. In some embodiments, tags may be utilized during protein purification (eg, as part of a purification mechanism).

In some embodiments, the protein may include a linker (eg, amino acid linker) placed between the Klotho protein sequence and the amino acid tag. Illustratively, the linker may comprise 1-40 amino acids, preferably 5-20 amino acids, more preferably 6-12 amino acids, and most preferably about 8-10 amino acids. In some embodiments, the linker may be or include a GS linker (eg, (G ₄ S) ₂ linker) or other linker (eg, GGENLYFQ linker). Preferably, the linker is, comprises, or has at least 80% amino acid sequence identity with SEQ ID NO:73 or SEQ ID NO:105.

In at least one embodiment, the protein may comply with cGMP regulations as determined and enforced by the US Food and Drug Administration (FDA). For example, the Klotho protein can be at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% pure by dry weight. In some embodiments, the Klotho protein sample is less than about 1 to 100 parts per million (ppm), about 100 to less than 1000 parts per billion (ppb), or about 1 to less than 100 ppb, or anywhere in between CHO host cell proteins (HCPs), nucleic acids, and/or other cellular components of any value or range of values provided.

Klotho protein variants Therapeutic S-Klotho proteins of various lengths (eg, S-Klotho, isoforms 1 or 2, 1-981, 29-981, 34-981, 36-981, 131-981, 1- 549, 29-549, 34-549, 36-549, 131-549, etc.) can be modified in various ways to achieve various beneficial effects and/or results that the native Klotho protein does not exhibit. Illustratively, the QuickChange XL site-directed mutagenesis kit (Stratagene) can be used to modify the nucleic acid sequences of various S-Klotho constructs. Other mutagenesis methods and kits known in the art can also be used. For example, various subcloning methods and kits are known in the art and commercially available. Amino acid changes can affect (eg, improve, enhance, decrease, etc.) protein expression, protein stability, protein solubility, protein binding, protein specificity, protein activity, protein function, immunogenicity, toxicity, and the like.

In at least one embodiment of the present disclosure, proteins are modified with one or more C-terminal and/or N-terminal tags. Such tags may function (in one or more therapeutic or other settings) to extend the protein's serum and/or soluble half-life. Tags may also be used for the presence or diagnostic localization of proteins, isolation or removal of proteins, delivery or transport of proteins, targeting one or more of proteins (e.g., proteins, nucleic acids, organelles, cellular structural components, etc.). It can be useful as a marker for binding to, enzymatic processing or cleavage, or the like. In at least one embodiment, the C-terminus of the protein is a TEV-Twin-Strep tag or sequence, an immunoglobulin (IgG1) Fc domain tag or sequence, or a protein known in the art and/or further described herein. It can be tagged with other tags or sequences. Additional description can be found in the article "Fusion Proteins for
Half-Life Extension of Biologies as a Strategy to Make Biobetters,""What is the
future of PEGylated therapies? ”, and “Strategies for extended serum half-life of protein therapeutics”. In certain embodiments, a linker or linker peptide may be inserted and/or positioned between the (naturally occurring or variant) Klotho protein sequence and the tag.

In some embodiments, a modified Klotho protein may include an alternative (eg, natural, non-natural, and/or synthetic) signal peptide. For example, in some embodiments the native signal peptide sequence may be replaced and/or supplemented with an alternative signal peptide or signaling sequence (SS). In some embodiments, the native methionine residue of the Klotho protein can be removed and a methionine residue can be included at the N-terminus of the SS. Additional description can be found in the article "Generation of high expressing CHO cell lines for the production of recombinant antibodies using
Optimized signal peptides and a novel ER stress based selection system". In certain embodiments, a linker or linker peptide may be inserted and/or positioned between the (naturally occurring or variant) Klotho protein sequence and the alternate SS.

Some embodiments may contain one or more amino acid variants. The present disclosure contemplates diversification of any one or more of any of the naturally occurring amino acids of any of the disclosed Klotho proteins to any other amino acid, whether natural, synthetic, or otherwise constructed. will be understood.

S-Klotho V45F Protein Variants The inventors have found that substitution of phenylalanine for valine at (natural) position 45 of human alpha Klotho (V45F) has surprising and unexpected benefits. For example, V45F soluble Klotho, and fragments or fusion proteins thereof, are expressed at higher levels in CHO and HEK-293 cells than are expressed with native wild-type V45 protein. Commercially, it may be desirable to achieve higher levels of soluble protein expression. Accordingly, some embodiments of the present disclosure include Klotho proteins with V45F substitutions.

S-Klotho C370S Protein Variant In humans, the Klotho gene is located on chromosome 13q12. A variant known as KL-VS is present in approximately 15-25% of Caucasians. This variant consists of six single nucleotide polymorphisms (SNPs), two of which cause amino acid substitutions (ie, F352V and C370S-phenylalanine 352 changes to valine, cysteine 370 to serine). In vitro transfection assays show that Klotho secretion levels are reduced 6-fold in the V352 variant, while increased nearly 3-fold in the S370 form. However, these two variants in the human Klotho gene segregate together and form a KL-VS haplotype that increases Klotho secretion by a range of 1.6-fold. For example, screening of over 300 individuals from geographically and/or ethnically distinct cohorts did not find a single individual carrying only one of the V352 or S370 variants. reportedly not.

One embodiment of the present disclosure includes a recombinant S-Klotho protein with a C370S homovariant (ie, absent (or with deletion) of the F352V variant). The C370S variant can be produced and/or expressed in the context of any of the protein constructs described herein. For example, the C370S variant is S-Klotho, isoforms 1 or 2, 1-981, 29- 981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

An embodiment involves producing an S370 heterozygous or homozygous variant construct, transfecting the resulting construct encoding the S-Klotho C370S protein (e.g., via transfection) into an appropriate expression system (e.g., CHO cells) and/or transiently expressing the S-Klotho S370 protein. The S370 Klotho protein can be expressed at higher levels than the F352V/C370S protein and/or the wild-type F352/C370 protein. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments may comprise administering a therapeutic or therapeutically effective amount of S-Klotho C370S protein to a subject in need thereof. A subject may, for example, carry or express a KL-VS variant. Alternatively, the subject may be of other mutants or variants of the wild type. Administration of recombinant S-Klotho C370S protein can lead to beneficial increases in blood S-Klotho levels. Thus, circulating concentrations of S-Klotho in subjects administered therapeutic recombinant S-Klotho C370S protein may not suffer the dilution effect observed when the F352V variant is present.

Nucleic Acids and Expression Vectors Some embodiments may include nucleic acids or nucleic acid constructs. For example, an embodiment can include an expression vector or nucleic acid construct. A nucleic acid can encode a recombinant human alpha soluble Klotho protein, protein fragment, or protein variant, as described herein. In at least one embodiment, the nucleic acid is a Klotho protein sequence, any (natural or non-natural) signaling sequence (e.g., at the N-terminal(s) of the Klotho protein sequence), as described herein; Optional linker sequences (eg, GS linker) and/or amino acid tags (eg, IgG1-Fc or TEV-Twin-Strep) may be encoded.

In some embodiments, the nucleic acid comprises amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549 of human alpha Klotho isoform 1 or 2, Proteins comprising all or a subset of 29-549, 34-549, 36-549 or 131-549 can be expressed. At least a portion of the protein comprises amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29- of human alpha Klotho (isoform 1 or 2) It can have at least or about 80% amino acid sequence identity with all or a subset of 549, 34-549, 36-549, or 131-549. For example, at least a portion of the protein can have at least and/or about 80% amino acid sequence identity with all or part of one of SEQ ID NOs: 1-75, or a combination of two or more thereof. In at least one embodiment, the protein is one of SEQ ID NO:2-70, or SEQ ID NO:107-120, or SEQ ID NO:125-128, or SEQ ID NO:133, or SEQ ID NO:152 It can have at least and/or about 80% amino acid sequence identity with all or a portion.

In some embodiments, at least a portion of the nucleic acid has at least and/or about 80% (e.g., at least about 82%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least a portion of a nucleic acid that may have 99% or 100% nucleotide sequence identity, hi preferred embodiments, the nucleic acid is one of SEQ ID NO:76-SEQ ID NO:106 or SEQ ID NO:121-124 may have at least and/or about 80% nucleotide sequence identity with one of

Nucleic acid sequences of the present disclosure can also include stop codons known in the art (eg, TGA, TAG, TAA).
Cell Lines and Methods of Manufacture An embodiment of the present disclosure may include cell lines. Cell lines may comprise any suitable cell type such as CHO cells, HEK cells, HL-60 cells, or other cell lines known in the art. Illustratively, a cell line can comprise CHO cells (eg, multiple CHO cells). For brevity, references to CHO cells may include HEK cells (e.g., HEK-293 cells), HL-60 cells, and/or any other (protein-expressing) cell(s) known in the art. ) or cell line(s). In some embodiments, the cells may contain (each) an exogenous nucleic acid (one or more copies). The nucleic acid has at least and/or about 80% amino acid sequence identity with one of SEQ ID NO: 1 to SEQ ID NO: 75, or a combination of two or more thereof, preferably one of SEQ ID NO: 2 to SEQ ID NO: 70 can encode a polypeptide having a specific property.

A nucleic acid can include at least one transgene or cDNA. In some embodiments, at least a portion of the nucleic acid is one of SEQ ID NOs:76-106 or SEQ ID NOs:121-124, or a combination of two or more thereof, preferably SEQ ID NOs:76-106 or sequences It can have at least and/or about 80% nucleic acid sequence identity with one of Nos. 121-124. The nucleic acid may be or include a plasmid or other (structural) form of nucleic acid.

In some embodiments, exogenous nucleic acids can encode functional enzymes such as dihydrofolate reductase (DHFR) and/or glutamine synthetase (GS). In at least one embodiment, the cells can be or comprise dihydrofolate reductase (DHFR) deficient CHO cells, such as CHO-S or CHO-M cells. A nucleic acid can include a promoter (eg, from a weak promoter to a maximally strong promoter, as understood by those of skill in the art). For example, in some embodiments, the nucleic acid has a One may include a (strong) promoter associated with the transgene having such as at least 80% nucleic acid sequence identity. Thus, the transgene can be under the control of a promoter.

For convenience, reference is made to CHO cells and/or cell lines throughout this disclosure. However, it should be noted that other cells, cell lines and/or host cells (besides CHO cells) are also contemplated within the scope of this disclosure. References to CHO cells and/or cell lines therefore also refer to other known cells, cell lines and/or host cells (e.g., HEK cells such as HEK-293, HL-60 cells, etc.) and / Or use is also contemplated.

Transfection Methods of producing cell lines may involve introducing exogenous nucleic acid into cells, preferably via transfection or other techniques known in the art. In at least one embodiment, using a serum-free growth optimized cell suspension of the cell line as the host cell line, the promoter, SEQ ID NO: 2-SEQ ID NO: 70, or SEQ ID NO: 107-SEQ ID NO: 120, or SEQ ID NO: 125 to SEQ ID NO: 128, or SEQ ID NO: 133, or SEQ ID NO: 152, and optionally a human alpha S-Klotho transgene that encodes a polypeptide having at least 80% amino acid sequence identity with at least a portion of one of SEQ ID NO: 152; A nucleic acid (plasmid) containing a (enzymatic) marker was inserted. The transgenes encode amino acids 1-981, 29-981, or 34-981 of human alpha soluble Klotho, respectively. In certain embodiments, the transgene has a sequence portion(s) corresponding to one or more of SEQ ID NOs:76-106 or SEQ ID NOs:121-124 (or SEQ ID NOs:76-124). having at least 80% nucleic acid sequence identity with SEQ ID NO: 106 or one of SEQ ID NOs: 121-124). In DHFR-deficient CHO cell lines (such as the CHO-S cell line), the selectable (enzymatic) marker was exogenous DHFR. In other cell lines the selectable (enzymatic) marker was exogenous GS.

Growth, Selection, and/or Gene Amplification Some embodiments use serum-free and/or animal (or animal-derived growing cells (eg, transfected cells) in medium lacking protein (components of ). For example, cells can be plated on solid growth medium for a period of time. Cells may also, or alternatively, be grown in suspension culture and/or in liquid media. Liquid media preferably contain a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives. In some embodiments, the medium may also lack hypoxanthine and thymidine (HT), glutamine, and the like.

In at least one embodiment, after a specified period of time (e.g., 48 hours post-transfection), cells are harvested (e.g., detached), optionally centrifuged (e.g., 100 xg for 5 minutes), and /or can be seeded (eg, at approximately 2000 cells/well), such as in 96-well adherent culture plates (eg, containing serum-supplemented -HT and/or -glutamine medium). In certain embodiments, the medium may also contain MTX and/or MSX. Non-transfected cells can die within 7-14 days after selection (eg, exposure to MTX and/or MSX in -HT and/or -glutamine medium).

In certain embodiments, the cells contain at least about 2-10 copies, at least about 10-20 copies, at least about 20-30 copies, or at least about 30-50 copies of the exogenous nucleic acid (eg, per cell). obtain (can be selected to contain). Thus, methods select cells that contain at least about 2-10 copies, at least about 10-20 copies, at least about 20-30 copies, or at least about 30-50 copies (eg, per cell) of the exogenous nucleic acid. can include For example, MTX and/or MSX (at serially increasing levels) can be administered to cells (eg, at a concentration of about 1 nM-1 μM, about 10-100 nM, etc.).

Dihydrofolate reductase (DHFR) gene amplification in DHFR-deficient CHO cells (such as the CHO-S cell line) transfected with exogenous DHFR continuously increases levels of methotrexate (MTX) in the growth medium. achieved by The plasmid contains DHFR, allowing amplification of the S-Klotho gene (fragment) in host cells upon exposure to MTX (10-100 nM). The GS gene expression system was also used to amplify cells transfected with exogenous GS (eg, upon exposure to MSX). Alternatively, a GS-/- host cell line was used to also eliminate the need for MSX. These steps resulted in the production of multiple copies of the S-Klotho gene (eg, 10-30 copies of the gene per cell) and the high levels of expression of the S-Klotho protein obtained within the transgenic cell lines.

In some embodiments, in suspension cultures, proteins can be secreted from the cells into the liquid medium. For example, certain cells and/or cell lines of the present disclosure can produce up to 200-500 mg protein per liter of liquid medium (without protein concentration), 500-2000 mg protein per liter of liquid medium, 1 liter of 2000-5000 mg protein per liquid medium, or any value or range of values therebetween (or may be selected to secrete). In at least one embodiment, the concentration of human recombinant alpha soluble Klotho protein in the medium (of the selected suspension culture(s) of the selected cell line) is at least 200 mg without concentrating the protein. /L, preferably at least 500 mg/L, more preferably at least 1000 mg/L, even more preferably at least 2000 mg/L, even more preferably at least 5000 mg/L. ) can be selected.

Subcloning of the resulting high S-Klotho-producing transgene-containing colonies by cell limiting dilution was performed to further generate S-Klotho secreting cell lines secreting S-Klotho in the range of 500-2000 mg/L into cell-conditioned media. produced. All cell constructs were restriction digested and sequence verified.

In some embodiments, the cells are at least 10 liters, preferably at least 25 liters, more preferably at least 50 liters, even more preferably at least 100 liters, even more preferably at least 250 liters, even more preferably at least 500 liters , even more preferably at least 1,000 liters, even more preferably at least 2,000 liters, even more preferably at least 2,500 liters, even more preferably at least 5,000 liters, even more preferably at least 10,000 liters can be grown in a bioreactor having a volume or working volume of

Cell line maintenance For high-producing S-Klotho cell lines that have been amplified (e.g., produced by the DHFR/MTX or GS/MSX systems) and subsequently cell-subcloned, during scale-up and until final bioreactor operation , was performed in a serum-free and animal protein component-free basal medium, using judiciously concentrated media stocks administered for cell line production.

Scale-up of high-yielding cell lines involves expansion of the cell inoculum in shake flasks or in cell suspension in the Wave Bag system, followed by production in 100 L and then 500 L volume bioreactors. cells were serially inoculated. Cell viability is greater than 85% viable cells throughout the growth cycle in shake flasks, wavebags, or bioreactors, followed by 80% viable cell numbers in the bioreactor during the plateau phase of CHO cell growth. A maximum of 1-3 g/L of S-Klotho is maintained and produced concomitantly (referred to as "high productivity").

Protein Production Certain embodiments employ recombinant DNA strategies that utilize strong promoter sequences and/or high copy number plasmids for the production of therapeutic amounts of Klotho protein in mammalian (e.g., CHO) cells. can be adopted. In at least one embodiment, for example, dihydrofolate reductase (DHFR) gene amplification in DHFR-deficient CHO cells provides methotrexate (MTX) and/or the use of (continuously increasing levels of) MTX can include Similarly, exogenous glutamine synthetase (GS) gene-containing cells can be treated with methionine sulfoximine (MSX).

A Klotho protein may also include (attached to) one or more glycans. For example, native human alpha Klotho isoform 1 (SEQ ID NO: 1) can have glycans attached (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612 and/or 694. A Klotho protein of the disclosure may have one or more of the same (or similar) glycans attached thereto (via glycosylation) (eg, to the same amino acid(s)).

In at least one embodiment, the protein may comply with cGMP regulations as determined and enforced by the US Food and Drug Administration (FDA). For example, the Klotho protein can be at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% pure by dry weight. In some embodiments, the Klotho protein sample is less than about 1 to 100 parts per million (ppm), about 100 to less than 1000 parts per billion (ppb), or about 1 to less than 100 ppb, or anywhere in between CHO host cell proteins (HCPs), nucleic acids, and/or other cellular components of any value or range of values provided.

The glycan structures present on the produced S-Klotho protein are similar compared to those on native S-Klotho protein isolated from human body fluids (i.e., blood, serum, urine, cerebrospinal fluid). or can be identical. In at least one embodiment, confirmation of native-like glycans can ensure that the correct native post-translational modifications (PTMs) are generated and stably maintained in the cell-produced S-Klotho protein.

Klotho Protein Solubility and/or Half-Life Extension Methods and compositions for extending the half-life and increasing the solubility of human S-Klotho protein are disclosed. Purification and characterization of the protein constructs so produced to achieve these results are also the subject of this disclosure. Nucleic acid changes made within the sequence of the Klotho gene or nucleic acid construct (see SEQ ID NOS: 76-96) and/or changes in the amino acid sequence of the Klotho protein (see SEQ ID NOS: 1-70) or chemical Modifications and/or relevant information regarding the addition or removal of chemical groups, peptides, or proteins to the amino acid sequence of the Klotho protein can be obtained naturally occurring in biological matrices such as blood, cerebrospinal fluid, urine, or various human tissues. The present disclosure teaches to obtain resulting human Klotho variant proteins (novel compositions) that have increased biological half-lives or increased solubility over that of the Klotho molecule. These novel compositions can be made through the methods described herein for modification of S-Klotho protein.

Illustratively, fusion protein constructs combine the S-Klotho protein with antibodies (IgG), albumins such as human serum albumin (HSA), transferrin such as human transferrin (TF), and/or proprietary antibodies such as XTEN®. by combining with the Fc domain of a recombinant polypeptide of Novel proteins can also be produced by conjugating (or modifying) the S-Klotho protein with post-translational modifications such as polysialic acid (PSA) or pegylation. Additionally, novel S-Klotho proteins can be produced via pegylation. These and other (half-life extension) methodologies have been shown to increase S-Klotho dosing intervals, provide superior patient convenience and compliance potential, reduce dosing frequency requirements, and result in less drug use in aggregate. reduce the cost of the drug; lower the drug amount at the same dosing interval as the parent protein; simplify the dosing formulation and allow subcutaneous formulation; Using S-Klotho to provide higher drug levels, resulting in longer drug exposure and potentially better efficacy, and reducing the immunogenicity of S-Klotho. It can improve protein performance.

Production of S-Klotho Fc Domain Fusion Protein Constructs Antibody Fc domains, human serum albumin (HSA), and polysialic acid (PSA) were tested for their effectiveness in extending half-life and increasing solubility of human S-Klotho protein. did. Fc fusions include fusion of a peptide, protein, or receptor exodomain to the Fc portion of an antibody. Both Fc- and albumin-fusions not only achieve half-life extension by increasing the size of the peptide drug, but both are also effective in the body through binding of the elongation protein to the neonatal Fc receptor, FcRn. A natural recirculation mechanism is also utilized. After binding of the elongation protein to the FcRn receptor, degradation of the fusion protein is prevented in the endosomes of the cell. Fusions based on the addition of Fc or albumin can result in biological half-lives in the range of 3-16 days, much longer than those reported for typical pegylated or lipidated peptides. For the use of protein fusion technologies such as Fc fusion proteins, fusions to human serum albumin, fusions to carboxy-terminal peptides, and other polypeptide fusion approaches to create biobetter drugs with more desirable pharmacokinetic profiles. For the commentary cited, see Strohl WR. Fusion Proteins for Half-Life Extension of Biologies as a Strategy to Make Biobetters. Biodrugs. 2015;29(4):215-239.

An Fc domain was added in this way to the parent protein (S-Klotho) to increase its binding affinity to the Fc receptor (FcRn). FcRn resides inside lysosomes within the endothelial cells that line blood vessels and functions to rescue antibodies from degradation that makes most proteins short-lived in circulation. As a result of the interaction with FcRn, the protein has a half-life ranging from days to weeks, making the extended form of the protein drug more attractive than biologics without this newly produced composition of matter. allows the drug to be administered less frequently.

The dimeric nature of Fc versus the monomeric structure of HSA may lead to the existence of Fc fusion peptides as dimers or monomers, in contrast to HSA. The dimeric nature of peptide Fc fusions has avidity effects when the target receptors for S-Klotho are sufficiently closely spaced or they themselves are dimeric in specific human target organs. can generate This may or may not be desirable, depending on the target. Fusion of S-Klotho protein to antibody Fc is also taught in this disclosure to improve the solubility and stability of S-Klotho. Surprisingly, Fc-fused Klotho proteins appear to have improved binding, avidity and/or immunogenicity. Addition of the Fc domain to S-Klotho also allows the fusion protein to be less immunogenic upon administration to human subjects.

Conjugation of S-Klotho Protein to Human Serum Albumin (HSA) The 66.5 kDa protein HSA, similar to human IgG, has a long mean half-life within 19 days. At concentrations of about 50 mg/mL (about 600 μM), HSA is the most abundant protein in human plasma and performs several functions, including maintaining plasma pH, metabolite and fatty acid transport, and a role in maintaining blood pressure. have. HSA, which is the upper size limit for glomerular filtration of proteins by the kidney, is also strongly anionic and helps to further delay filtration through the kidney. Similar to IgG, HSA also binds to FcRn in a pH-dependent manner and is recycled like IgG, albeit at a site distinct from IgG binding and via a mechanism distinct from that of IgG binding. , resulting in a prolongation of its half-life. HSA also tends to accumulate in tumors and inflamed tissues, suggesting that fusion or conjugation to albumin could potentially help target proteins or peptides to those sites. there is

The fusion of peptides or proteins with inherently short half-life properties to HSA has been extensively investigated since the early 1990's in order to extend the serum half-life of these molecules. Since then, dozens of different peptides and small proteins have been fused to HSA as both innovative and potential biobetter molecules. The first HSA-peptide or protein fusion product to be commercially approved was Tanzeum® (marketed as Eperzan® in the European Union), discovered at Human Genome Sciences, developed and marketed by GlaxoSmithKline ) was a DPP-4 resistant GLP-1-HSA fusion protein. Tanzeum® (albiglutide) was approved by the European Medicines Agency (EMA) and FDA in March and April 2014, respectively. Thus, HSA improves the half-life of pharmacologically active GLP-1 from 1-2 minutes to 4-7 days for native GLP-1, allowing once weekly dosing. Seven other known HSA fusion protein product candidates are currently in development or have been in recent years. Novozyme is also developing modified versions of recombinant HSA with improved FcRn binding for the construction of "next generation" HSA protein fusions that may have even longer half-life properties. This was based on the use of the K573P mutant of HSA, which was found to have a 12-fold higher affinity for FcRn, giving HSA a longer half-life than the wild-type molecule in both mice and cynomolgus monkeys. . It is anticipated that these longer half-life HSA variants may further be used as fusion proteins to improve the half-life of fusion proteins.

Thus, the inventors provide strategic therapeutic benefits such as superior patient convenience and compliance potential, reduced dosing frequency resulting in overall lower drug usage, and/or reduced cost of goods. To this end, the Klotho protein is fused to wild-type HSA or a mutant form of HSA to produce a Klotho fusion molecule with a significantly prolonged half-life in human blood, cerebrospinal fluid, and other human biological matrices ( (more than one) are disclosed herein. Also, lowering the drug amount at the same dosing interval as the parent protein may simplify the dosing formulation, allow for subcutaneous formulation, or may allow for reduced immunogenicity of S-Klotho. Surprisingly, HAS fusion Klotho proteins appear to have improved binding, avidity and/or immunogenicity.

Conjugation of S-Klotho Protein to Human Transferrin (TF) Transferrin is a highly abundant serum glycoprotein, found in serum at 3-4 mg/mL, which strongly and reversibly binds iron, Functions to carry iron to tissues. Transferrin has 679 amino acid residues, is approximately 80 kDa in size, and has two high-affinity Fe3+ binding sites, one in the N-terminal domain and the other in the C-terminal domain. Human transferrin has a reported half-life of 7-10 days, or 10-12 days. The aglycosylated form of human transferrin, which accounts for approximately 2-8% of the total transferrin pool, has a slightly longer half-life of 14-17 days. The prolonged persistence of transferrin in human serum is due to a mechanism mediated by the clathrin-dependent transferrin receptor, which recycles receptor-bound transferrin back into circulation.

Peptide and protein fusions have been made to the N- and C-termini of human transferrin and to the centrally located hinge region that links together the two major lobes of transferrin. The N-terminus of transferrin is free and can be directly fused. Because the C-terminus is more buried and constrained by a nearby disulfide bond, flexible linkers are typically used when proteins are fused to the C-terminus. This ability has led to the generation of libraries of peptides against specific targets, and then aglyco-transferrin (N-terminal, C-terminal, loop, or linker region).

Biotechnology company BioRexis Technologies, Inc. was founded in 2002 to develop a transferrin fusion protein platform, which it termed the “Trans Body” platform as a therapeutic platform. Their lead molecule, BRX-0585, was a transferrin-GLP-1 fusion protein for the treatment of type 2 diabetes mellitus (T2DM). Fusion of GLP-1 to transferrin was demonstrated to significantly enhance the half-life of GLP-1. BioRexis was acquired by Pfizer in March 2007. As far as can be ascertained, there are currently no BioRexis-derived fusion proteins in hospitals. Klotho protein can be fused to human transferrin to produce Klotho fusion molecules, which are clinically administered to significantly extend half-life, or stability in human biological matrices in vivo. obtain.

Conjugation of S-Klotho Protein to Amunix's XTEN® XTEN® is a unique recombinant polypeptide that extends the in vivo half-life of therapeutic payloads. XTEN consists of naturally occurring hydrophilic amino acids and is biodegradable. Pharmaceuticals such as proteins, peptides, and synthetic compounds can be XTENylated through chemical conjugation or gene fusion. XTEN proteins lack secondary and tertiary structure and their solution behavior resembles chemically prepared polymers with very large hydrodynamic radii. By size exclusion chromatography, XTEN protein polymers appear much larger than typical globular proteins of similar molecular weight. The bulking effect of XTEN greatly reduces the renal clearance of bound molecules, thus greatly increasing the in vivo half-life. In the present invention, the length of the XTEN polymer attached to the Klotho protein will be specified to optimize the pharmacokinetics as well as the biodistribution of the bound Klotho protein payload.

Thus, XTEN can be recombinantly fused to our S-Klotho protein S(s) to increase the in vivo half-life of the molecule. One benefit is that the genetic S-Klotho-XTEN fusion construct produces a molecule with the convenience of single molecule expression, purification, and characterization containing both therapeutic and bulking moieties. Recombinant fusion allows attachment of multiple XTEN chains per protein at precisely defined locations and has been successfully used by therapeutic drug manufacturers to produce XTENylated growth hormone (Somavaratan from Versartis) and It provides best-in-class pharmacokinetics, as exemplified by FVIII-XTEN (Biogen). For example, pharmacokinetic studies conducted in children receiving different doses of Somavaratan showed that XTENylation resulted in best-in-class half-life due to reduced receptor-mediated elimination in addition to renal clearance. there is

XTEN protein polymers can be produced as free intermediates for chemical conjugation to peptides, peptidomimetics, and other synthetic molecules. Reactive groups (thiols, amines) are inserted at precisely defined positions via the introduction of cysteine or lysine residues into the gene encoding XTEN. Amunix has developed XTEN containing 1-9 thiol groups at various intervals, which can be provided to partners. Thus, in the present invention, orthogonal conjugation to amino and thiol groups in XTEN facilitates production of our Klotho-XTEN molecules.

Protein Purification Klotho protein can be extracted from a cell suspension culture of cells (eg, of a CHO cell line). The cells can produce and optionally secrete (eg, into the liquid medium) the Klotho protein. Secretion of S-Klotho into the spent medium was also observed.

Purification of recombinant proteins of the present disclosure may be accomplished by any conventional method, including any suitable method known in the art or described herein, such as extraction, precipitation, chromatography, and/or electrophoresis. It can be performed by procedures. Additional purification procedures that can be used to purify the protein include affinity chromatography using monoclonal antibodies that bind the target protein(s). Some embodiments may include IgG-tagged proteins, Fc-tagged proteins, His-tagged proteins, HSA-tagged proteins, GST-tagged proteins, etc., which may be purified by affinity chromatography. For example, some embodiments may comprise at least a portion of the Fc domain of an IgG, such as IgG1. Generally, a crude preparation containing recombinant protein is passed over a column to which a suitable monoclonal antibody has been immobilized. For other tags, the corresponding tag-binding affinity entity can be placed on the column. After washing the column, proteins are eluted from the gel by changing pH or ionic strength. For example, spent medium from a CHO-S high-yielding cell line was concentrated via tangential flow filtration and the S-Klotho protein purified by affinity chromatography followed by ion exchange cartridge or column chromatography. Size exclusion chromatography can also be used for protein purification. It will be appreciated that various sequences of chromatography types may be applied. Any suitable combination of chromatographic steps or columns is contemplated herein.

In an alternative protocol, one or more pre- or post-affinity purification steps were performed. Such steps include, for example, (ultra)centrifugation, dialysis, membrane and/or tangential flow filtration, chromatographic separations such as ion exchange, liquid-liquid extractions such as (aqueous) two-phase extraction methods, or other Known purification steps may be included. In certain embodiments, one or more post-purification processing steps were performed. Such post-purification steps include, for example, tandem anion/cation flow-through chromatography (as opposed to bind and elute chromatography), membrane filtration (e.g., 0.2 micron, 0.1 micron, etc.). , or removal of viruses and/or bacteria by other means known to those skilled in the art.

Certain embodiments include methods of purifying recombinant Klotho protein. The method may comprise expressing the recombinant Klotho protein in suspension cell culture. Methods may include harvesting the Klotho protein, such as by harvesting suspension culture media and/or cells. In at least one embodiment, the method can include a first purification step. It is understood that the "first" purification step need not occur first, before any other purification step(s), or after any other purification step(s). Will. The first purification step may comprise, involve or involve chromatography such as high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), ion exchange chromatography, affinity chromatography. can be used. The first purification step may comprise, involve or use affinity chromatography. Affinity chromatography can be based on Klotho protein tags or other protein fusion entities. For example, His-tagged proteins can be purified by His-tag affinity chromatography (eg, HisTrap or Nickel column chromatography). Similarly, GST-conjugated proteins can be purified by GST affinity chromatography. Depending on the particular tag(s), a corresponding tag-binding affinity entity can be deployed for use in protein purification.

In at least one embodiment, the first purification step is, for example, Protein A or Protein A conjugated particles or beads (e.g., Protein A Sepharose 4 fast flow column (0.75 ml ) (0.5 cm×3 cm)). A column can be equilibrated in a suitable solution. The solution may contain a suitable concentration of buffer (eg, 10 mM Tris-HCl, pH 8.0). A suitable amount of protein-containing sample (e.g., suspension cell culture supernatant optionally adjusted to pH 8.0 with 1.5 M Tris-HCl, pH 8.0) is added (e.g., at a flow rate of 0.75 ml/min). ) column and optionally washed with a suitable wash buffer (eg, 10 mM Tris/arginine-HCl, pH 8.0) and a suitable flow rate (eg, about 0.50-0.25 ml/min or equivalent) and/or with a suitable elution buffer (eg, 4 M MgCl ₂ , pH 3 (ie, low pH, high salinity)) to produce a concentrated sample.

In at least one embodiment, the elution buffer can have a pH of about 3. In some embodiments, the elution buffer has a pH of about 2 to about pH 6, preferably about pH 2 to about pH 5, more preferably about pH 2 to about pH 4, even more preferably about pH 2.5. ~ pH about 3.5.

In at least one embodiment, the elution buffer may contain at least one salt. Illustratively, the salt may be or include MgCl ₂ , preferably at a concentration of about 4M. In some embodiments, the elution buffer is about 1 M to about 6 M, preferably about 2 M to about 5 M, more preferably about 3 M to about 5 M, even more preferably about 3.5 M to about 4.5 M salt. concentration.

Additional purification step(s) may include chromatography such as high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), ion exchange chromatography, affinity chromatography, size exclusion (or gel filtration) chromatography. may include, involve, or use the In at least one embodiment, the method can include a second purification step. It will be appreciated that the "second" purification step need not occur second, before or after any other purification step(s). A second purification step may include or involve size exclusion chromatography (and a suitable mobile phase buffer) to fractionate, resolve, clarify, or purify the protein-containing sample(s). or you can use it. For example, in an exemplary second purification step, the protein-containing elution sample(s) (from the first purification step) are optionally buffer exchanged and subjected to size exclusion chromatography in a suitable mobile phase buffer. (eg, using a Superdex 200 (1 cm x 30 cm) column). In at least one embodiment, the mobile phase buffer comprises a suitable buffering agent (eg, 100 mM Tris-HCl) at a suitable pH (eg, pH about 8) and one or more optional reducing agents (eg, 5 mM L-methionine and/or 0.6% sodium thioglycolate). In at least one embodiment, the buffer can be other than a phosphate-containing buffer (eg, Tris, citrate, etc.). In at least one embodiment, the reducing agent may be or include L-methionine and/or sodium thioglycolate, preferably in effective reducing concentration(s). Alternative reducing agents include acetylcysteine (ie, N-acetyl-L-cysteine, N-acetyl-D-cysteine, and racemic N-acetylcysteine, or N-acetyl-L-cysteine and N-acetyl-D-cysteine). N-acetylcysteine (NAC), including (racemic) mixtures with cysteine), ascorbic acid, dithionite, erythiorbate, cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, dierythritol, Resin supported thiol, resin supported phosphine, vitamin E and/or trolox or salts thereof, sodium citrate, potassium citrate, potassium iodide, ammonium chloride, guaifenesin (or guaifenesin), tolubalsam , vasaca, ambroxol, carbocisteine, erdosteine, mecysteine, dornase alfa, and the like.

In at least one embodiment, the mobile phase buffer can have a pH of about 8. In some embodiments, the elution buffer has a pH of about 6 to about pH 10, preferably about pH 7 to about pH 9, more preferably about pH 7.2 to about pH 8.8, even more preferably pH It can have a pH of about 7.5 to about pH 8.5.

In at least one embodiment, the eluted protein may be or include a monomeric Klotho protein. In at least one embodiment, the eluted protein may be or include a multimeric Klotho protein. In some embodiments, the eluted protein may be or include a dimeric Klotho protein. In some embodiments, the eluted protein may be or include a tetrameric Klotho protein. In some embodiments, the eluted protein may be or include a hexameric Klotho protein. In some embodiments, the eluted protein may be or include an octameric Klotho protein. In at least one embodiment, the eluted protein can be or include both monomeric and multimeric Klotho proteins. In at least one embodiment, the eluted protein can be or comprise a single species, either a monomeric Klotho protein or a multimeric Klotho protein. In at least one embodiment, the eluted protein may be or comprise about 80% or more of a single species of either monomeric or multimeric Klotho protein. In at least one embodiment, the eluted protein is about 82%, 85%, 88%, 90%, 92%, 95%, 96% of a single species of either monomeric or multimeric Klotho protein. %, 97%, 98%, or 99% or more.

Additional purification step(s) may include chromatography such as high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), ion exchange chromatography, affinity chromatography, size exclusion (or gel filtration) chromatography. may include, involve, or use the

Analysis of Klotho Protein Protein purity can be demonstrated by SDS-PAGE or other assays or means known in the art. For example, in at least one embodiment, a Klotho protein sample (50 μg) was fractionated on a pre-cast SDS-PAGE gel (4-15%, 10-well, Catalog No. 456-1083, BioRad) and stained with Coomassie blue dye. All samples were either separated by empty lanes or placed on separate gels to avoid contamination between samples. Greater than 98% of S-Klotho was isolated from the CHO S-conditioned medium as determined by Coomassie blue dye and densitometry transmission, or by silver staining visualization, or by HPLC or RP-HPLC. was shown. To obtain sequence information, proteins (after reduction and S-carboxymethylation) can be cleaved with cyanogen bromide, trypsin, and/or proteinase K, and peptides separated by HPLC, according to known methods of protein chemistry. . Samples so prepared were then run on an automated vapor-phase microsequencing instrument (Applied Biosystems model 470A) equipped with an on-line automated HPLC PTH amino acid analyzer (Applied Biosystems model 120, ABI, see above) connected to a power outlet. , ABI, Foster City, Calif., USA).

Proteins were also analyzed via mass spectrometry. For sample preparation for mass spectrometry, only the 75-150 kDa gel band was clipped for analysis in order to limit the analysis to the correct S-Klotho protein. The gel fraction was softened with a sterile blade and subjected to in-gel digestion. Gel fractions were destained by washing three times with 80 μL of 50% acetonitrile (ACN)/50 mM ammonium bicarbonate and washed with 100% ACN. The alkylation step was omitted given the absence of cysteine residues from the target α-Klotho peptide. Trypsin digestion was performed overnight at 37° C. in 50 mM ammonium bicarbonate (0.005 μg/μL) using 60 μL of trypsin (sequencing grade modification, catalog number V511A, Promega). This process yielded 25 μL, of which 5 μL (1 μL for S-Klotho) was subjected to liquid chromatography-electrophoresis on an Orbitrap nano-ESI Q-Exactive mass spectrometer (Thermo Scientific) attached to a nanoLC (Dionex Ultimate 3000 UHPLC). Subjected to spray ionization tandem mass spectrometry (MS/MS) and PRM analysis. In MS/MS analysis, human recombinant alpha-S-klotho produced by embodiments of the present disclosure is substantially similar to that found in human blood, serum, urine or cerebrospinal fluid (e.g., corresponding are identical in the amino acid sequences that correspond to each other).

Using the purification method described above, the level of contaminating CHO host cell protein (HCP) was determined to be acceptable in the purified S-Klotho protein. HCP was removed from 1 to less than 100 ppm in the final S-Klotho product. S-Klotho protein products of at least 90% and up to 98% purity were isolated from spent CHO S-producing cell lines (cells and/or broth). Specifically, cGMP grade human alpha-S-Klotho was produced and purified with an analytical profile suitable for clinical administration in human subjects. For example, an analytical profile of human recombinant alpha S-Klotho can be found at http://proteomecentral.com. proteome exchange. org/cgi/GetDataset? Reference number PXD002775 in the ProteomeXchange Database, which can be found at ID=PXD002775. The NIH's complete S-Klotho protein dataset is at http://www[dot]ncbi[dot]nlm[dot]nih[dot]gov/protein/Q9UEF7.

Analytical profiles of S-Klotho suitable for clinical administration, and analytical profiles obtained with embodiments of the present disclosure, contained endotoxin levels of less than 0.1 ng per μg of S-Klotho (1 EU/μg). In addition, the purified human recombinant S-Klotho was also shown to have a purity of over 98% by SDS PAGE.

The glycan structure present in S-Klotho produced in CHO S cells is identical compared to that of native S-Klotho isolated from human body fluids (i.e., blood, serum, urine, and cerebrospinal fluid). Met. This ensured that the same native post-translational modifications (PTMs) were generated and stably maintained in the cell-produced S-Klotho protein. Thus, using the production and purification methods described herein, the inventors have successfully produced cGMP grade human S-Klotho with analytical profiles suitable for clinical administration to human subjects. .

Therapeutic Compositions Some embodiments of the present disclosure may include pharmaceutical compositions, such as therapeutic compositions. The pharmaceutical compositions of this disclosure generally comprise a therapeutically effective amount of a recombinant soluble alpha Klotho protein and one or more additional components in a (pharmaceutically acceptable) vehicle, carrier, or excipient can include admixtures with Klotho protein in any suitable amount, such as about 0.001-1000 mg, about 0.01-100 mg, about 0.1-10 mg, about 1-5 mg, or any amount or range of amounts therebetween. can be included in In some embodiments, the composition may comprise a pharmaceutically or therapeutically effective amount of Klotho protein (e.g., to raise serum Klotho levels to a predetermined level in a subject to whom the composition is administered).

Components may include one or more aggregation inhibitors, buffers, tonicity agents, and additional excipients. The primary solvent in the carrier may be either aqueous or non-aqueous in nature. A composition can be prepared by combining the purified Klotho protein of this disclosure with a pharmaceutically acceptable carrier.

The combination of the various components included in the composition may be done in any suitable order, i.e. the buffer may be added first, middle or last and the tonicity agent may also be added first. , in the middle, or at the end. Those skilled in the art will also appreciate that some of these chemicals may be incompatible in certain combinations and are therefore readily substituted by different chemicals having similar properties but compatible with the relevant mixtures. It is what is done.

Aggregation inhibitors reduce the tendency of a polypeptide to associate into inappropriate or undesirable ternary or quaternary complexes. The amino acids L-arginine and/or L-cysteine can act to reduce aggregation of Fc domain-containing polypeptides in formulations for long periods of time, eg, two years or more. The concentration of aggregation inhibitor in the formulation is preferably about 1 mM to 1 M, more preferably about 10 mM to about 200 mM, more preferably about 10 mM to about 100 mM, even more preferably about 15 mM to about 75 mM, and even more preferably about 25 mM. These compounds are available from commercial suppliers.

Compositions of the disclosure may include a buffering agent. Buffers keep the pH within a desired range. Various buffers suitable for use in the pharmaceutical compositions of the present disclosure include histidine, potassium phosphate, alkaline salts, sodium or potassium phosphate or their hydrogen or dihydrogen salts, sodium citrate or Potassium citrate/citric acid, sodium acetate/acetic acid, maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris), various forms of acetate and diethanolamine, and solutions known in the art. Any other pharmaceutically acceptable pH buffering agent to maintain pH within the desired range is included. Mixtures of these buffers can also be used.

The amount of buffering agent useful in the composition is highly dependent on the particular buffer used and the pH of the solution. For example, acetate is a more efficient buffer at pH 5 than at pH 6, so less acetate can be used in the solution at pH 5 than at pH 6. The preferred pH of the preferred formulations is in the range of 4.0 to 5.0, and pH adjusters such as hydrochloric acid, citric acid, sodium hydroxide, or salts thereof are also included to obtain the desired pH. can be

One preferred buffer is sodium phosphate, as its buffering capacity is at or near pH 6.2. However, it will be appreciated that other buffers may be selected to achieve any desired pH buffering. The concentration of buffer in the formulation is preferably from about 1 mM to about 1 M, more preferably from about 10 mM to about 200 mM. Buffers are well known in the art, are manufactured by known methods, and are available from commercial suppliers.

Setting the pH of the pharmaceutical composition at or near physiological levels maximizes patient comfort during administration. Specifically, the pH is preferably within the range of about pH 5.8 to 8.4, preferably about 6.2 to 7.4, however, the pH may be adjusted as necessary to It is within the scope of this disclosure that the stability and solubility of a polypeptide in a particular formulation can be maximized and thus tolerated by patients even at pHs outside the physiological range. It is understood that

Formulations of the present disclosure may further include one or more tonicity agents (eg, to make the solution isotonic with the patient's blood for injection). A tonicity agent is understood to be a molecule that contributes to the osmolality of a solution. The osmolality of the pharmaceutical composition is preferably adjusted to maximize stability of the active ingredient and also minimize patient discomfort upon administration. If the serum is approximately 300+/-50 milliosmoles per kilogram. In general, it is preferred that the pharmaceutical composition be isotonic with serum, ie, have the same or similar osmolality achieved by the addition of a tonicity agent, thus having an osmolality of about 180 to about 420. It is contemplated that the osmolality can be in the milliosmolarity, but it is understood that the osmolality may be either higher or lower as required under particular conditions.

Typical tonicity agents are well known in the art and include, but are not limited to, various salts, amino acids, or polysaccharides. Non-limiting examples of suitable amino acids include glycine. Non-limiting examples of suitable polysaccharides include sucrose, mannitol, and sorbitol. It is understood that more than one tonicity agent can be used at once, for example sorbitol and glycine can be used in combination to modify the tonicity of the formulation.

Additional examples of tonicity agents suitable for modifying osmolality include amino acids (eg, arginine, cysteine, histidine, and glycine), salts (eg, sodium chloride, potassium chloride, and sodium citrate), and /or sugars (eg, sucrose, glucose, and mannitol), including but not limited to; The concentration of the tonicity agent in the formulation is preferably about 1 mM to 1 M, more preferably about 10 mM to about 200 mM. Tonicity agents are well known in the art, are manufactured by known methods, and are available from commercial suppliers.

Excipients, also called chemical additives, co-solutes, or co-solvents, that stabilize the polypeptide in solution (even in dried or frozen form) can also be added to the pharmaceutical composition. An excipient is defined herein as a non-therapeutic agent added to a pharmaceutical composition to provide a desired effect, eg, stabilization, isotonicity. Common attributes of desirable excipients are water solubility, non-toxicity, non-reactivity, rapid clearance from the body, and absence of immunogenicity. In addition, excipients should be capable of stabilizing the native conformation of the protein to maintain drug efficacy and safety during processing, storage, and administration to patients. . Examples include sugars/polyols such as sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose; serum albumin (bovine serum albumin (BSA), human SA, or recombinant HA), dextran, polymers such as PVA, hydroxypropylmethylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); polyhydric alcohols such as PEG, ethylene glycol, and glycerol, dimethylsulfoxide (DMSO), and non-aqueous solvents such as dimethylformamide (DMF); amino acids such as proline, L-serine, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, and gamma-aminobutyric acid; Tween-80™ (polysorbate 80), Surfactants such as Tween-20™ (polysorbate 20), SDS, polysorbates, polyoxyethylene copolymers; as well as potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions. (e.g., zinc, copper, calcium, manganese, and magnesium), CHAPS, monolaurate, 2-O-beta-mannoglycerate, or any combination of the above. include but are not limited to: As a caution, note that the highly toxic veratrum album can be mistaken for gentian.

The concentration of one or more excipients in the formulations of the present disclosure is preferably about 0.001-5 weight percent, more preferably about 0.1-2 weight percent. Excipients are well known in the art, are manufactured by known methods, and are available from commercial suppliers.

In one exemplary embodiment, the formulations of the present disclosure contain about 150 mM NaCl buffered with HEPES, MES, or Tris-HCl to a pH of about 7.3-7.4, and optionally as described herein. may include one or more additional components of

In one exemplary embodiment, the formulations of the present disclosure contain a (therapeutically effective) amount of Klotho protein, about 10 mM to about 50 mM sodium phosphate, about 0.75% to about 1.25% sucrose, and/or about 50 mM to about 150 mM NaCl. In another embodiment, L-arginine can be replaced with L-cysteine (from about 1 to about 500 micromolar) in the formulation. In yet another embodiment, the pH can be about pH 7.0. In another specific embodiment, the formulation of the present disclosure contains a (therapeutically effective) amount of Klotho protein in 25 mM L-arginine, about 25 mM sodium phosphate, about 98 mM sodium chloride, at a pH of about 6.2. , and/or about 1% sucrose.

In another embodiment, the formulation of the present disclosure comprises a (therapeutically effective) amount of Klotho protein in about 10 mM to about 100 mM L-arginine, about 10 mM to about 50 mM phosphate, at about pH 6 to about pH 7. sodium, about 0.75% to 1.25% sucrose, about 50 mM to about 150 mM NaCl. In certain embodiments, the formulations of the present disclosure contain a (therapeutically effective) amount of Klotho protein in about 25 mM sodium phosphate, about 98 mM sodium chloride, and/or about 1% May contain sucrose.

In yet another embodiment, a formulation of the present disclosure comprises, at a pH of about 6-7, a therapeutically effective amount of an Fc domain-containing Klotho fusion protein, about 10 mM to about 100 mM L-arginine, about 10 mM to about 50 mM sodium phosphate. , about 0-5% mannitol, and/or 0-0.2% Tween-20™ (polysorbate 20). In another embodiment, a formulation of the present disclosure comprises an effective amount of Klotho protein, about 25 mM L-arginine, about 25 mM sodium phosphate, about 4% mannitol, about 0.02% Tween-20™ (polysorbate 20) and/or about pH 6.0.

In yet another embodiment, the present disclosure provides a method of treating a mammal comprising administering a therapeutically effective amount of a pharmaceutical composition described herein, wherein the mammal has Methods are provided having diseases or disorders that can be beneficially treated with Fc domain-containing polypeptides. In yet another embodiment, the Fc domain-containing polypeptide is from the same species as the mammal that will be treated with the composition. In certain embodiments, the mammal is a human patient in need of treatment. Examples of diseases or disorders that may be treated when the Fc domain-containing polypeptide of the composition is Klotho:Fc include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease (granulomatosis), Crohn's disease ( or inflammatory bowel disease), chronic obstructive pulmonary disease (COPD), hepatitis C, endometriosis, asthma, cachexia, psoriasis, and atopic dermatitis, or mutations in one or more of the Klotho genes Including, but not limited to, persons with genetic disorders. Additional diseases or disorders that can be treated with Klotho:Fc include those described in WO00/62790, WO01/62272, and US Patent Application No. 2001/0021380, each of which is incorporated herein by reference in its entirety. included.

In yet another embodiment, the present disclosure provides a method for accelerated stability testing of Fc domain-containing polypeptide stability in pharmaceutical compositions of the present disclosure, wherein prior to storage, i.e., at initiation, the present Testing the activity of a polypeptide formulated according to the disclosure, storing the composition at 37° C. for 1 month, measuring the stability of the polypeptide and comparing the stability forms from the beginning to the 1 month time point. A method is provided, comprising: This information helps in early elimination of batches or lots that appear to have good stability initially but do not store well in the long term.

Additionally, the pharmaceutical composition provides for long-term storage such that the active ingredients, eg, Fc domain-containing polypeptides, are stable over the course of storage, either in liquid or frozen state. As used herein, the phrase "long-term" storage is understood to mean that the pharmaceutical composition can be stored for 3 months or longer, 6 months or longer, 1 year or longer, and preferably 2 years or longer. . Long-term storage also means that the pharmaceutical composition is either stored as a liquid at 2-8°C or frozen, for example at -20°C or below (eg -20°C or -80°C). Then understood. It is also contemplated that compositions can be frozen and thawed more than once. The term "stable" with respect to long-term storage means that the active polypeptide of the pharmaceutical composition is 20%, or more preferably 15%, or even more preferably 10% compared to the activity of the composition at the start of storage. , and most preferably not losing more than 5% of its activity.

One or more antioxidants may be included in the formulations of the present disclosure. Antioxidants contemplated for use in preparing the formulations include amino acids such as glycine and lysine, chelating agents such as EDTA and DTPA, and free radical scavengers such as sorbitol and mannitol.

(oral or parenteral) modified release formulations (e.g. coating, encapsulation, sustained release, controlled release, extended release, delayed release, sustained release, sequential release, depot, etc.), inhalation mists, orally active formulations, suppositories, and other effective dosage forms such as implants or implantable medical devices are also contemplated herein. Thus, the formulation also includes bulk erosion polymers such as poly(lactic-co-glycolic acid) (PLGA) copolymers, PLGA polymer blends, block copolymers of PEG, and lactic and glycolic acid, microparticle preparations of polymeric compounds such as poly(cyanoacrylates)); surface eroding polymers (e.g., poly(anhydrides), and poly(orthoesters)); hydrogel esters (e.g., pluronic polyols, poly(vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, cellulose, hyaluronic acid derivatives, alginates, collagen, gelatin, albumin, and starch and dextran), and compositions thereof based microparticle preparations, or liposomes or microspheres. can include preparations of Such formulations can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the subject proteins and derivatives. The optimal pharmaceutical formulation for the desired protein can be determined by one skilled in the art depending on the route of administration and desired dosage. Exemplary pharmaceutical formulations are described in Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing Co.; , Easton, Pa. 18042, pages 1435-1712, the disclosure of which is incorporated herein by reference.

Biological activity (preliminarily) to assess the efficacy of human recombinant alpha-soluble Klotho and/or to determine effective doses (for patients, subjects, or individuals presenting with age-related or clinical (e.g., metabolic) disorders) A method of can include conducting a bio-based assay (eg, using a mammal, such as a mouse, rat, primate, or some other non-human). Klotho protein can be administered to the organism once or according to a regimen (regular or irregular). For example, the protein may be administered at any suitable number of times (eg, once, twice, etc.) for a given period of time (eg, monthly, semi-monthly, weekly, semi-weekly, daily, etc.). Biological parameters (eg, age-related parameters) can then be assessed. A Klotho protein of interest may effect or result in a change in a parameter compared to a reference (eg, a parameter of a control organism). Other parameters (eg, related to toxicity, clearance, and pharmacokinetics) can also be evaluated.

The Klotho proteins of the present disclosure are useful in animals (models) having or exhibiting a particular disorder or condition, such as an age-related or age-related disorder or condition, a clinical (e.g., metabolic) disorder or condition, can be evaluated using Such disorders and conditions can also provide a sensitizing system in which the physiological effects of proteins can be observed. Exemplary disorders include, e.g., denervation, disuse atrophy, clinical (e.g., metabolic) disorders (e.g., disorders in obese and/or diabetic animals such as db/db mice, ob/ob mice), brain liver ischemia (kidney idschemia) or other liver damage, cisplatin/taxol/vincristine models, renal ischemia/reperfusion models, various tissue (xenograft) transplantation, transgenic bone models, pain syndromes (e.g. inflammation and neuropathy), paraquat, genotoxicity, oxidative stress models, and tumor (I) models.

To evaluate the S-Klotho protein of the present disclosure, the protein can be administered to a suitable animal (over a suitable treatment period) and the animal's parameters are evaluated (eg, 10-60 minutes, 1-24 hours, 1-24 hours, after a suitable period of time such as 30 days, 1-12 months, 1-5 years, or any value or range of values therebetween). Animals can be fed ad libitum or normally (eg, not under caloric restriction, although some parameters can be assessed under such conditions). Typically a cohort of such animals is used in the assay. In general, if the test polypeptide influences parameters in the direction of phenotype in similar animals subjected to caloric restriction, the test polypeptide may be shown to favorably alter lifespan control in the animal. Such test polypeptides can cause at least some lifespan-regulating effect due to caloric restriction (eg, a subset of such effects) without removing the organism's caloric intake.

The parameter(s) tested can be age-related or disease-related parameter(s) (eg, symptoms of disorders associated with animal models). Advantageously indicated test proteins may cause amelioration of symptoms compared to similar reference animals not treated with the polypeptide. Other parameters related to disability or aging include antioxidant levels (e.g., antioxidant enzyme levels or activity), stress tolerance (e.g., paraquat tolerance), core body temperature, glucose levels, insulin levels, thyroid stimulating hormone levels, Prolactin levels, and luteinizing hormone levels may be included.

Animals with reduced Klotho expression (eg, mice with mutant or Klotho gene defects) can be used to measure the efficacy of the S-Klotho proteins of the present disclosure for treating age-related disorders. For example, test proteins may be administered to mutant mice and age-related parameters monitored. Advantageously indicated test proteins may cause symptomatic improvement compared to similar reference animals not treated with the protein.

Parameters related to clinical (e.g., metabolic) disorders or aging are assessed by measuring body weight, testing for fertility gain, measuring blood glucose levels, observing lifespan, observing skin, and observing motor functions such as walking. obtain. Evaluation can also be performed by measuring thymus weight, observing the size of calcified nodules formed on the lining of the thoracic cavity, and the like. In addition, quantitative determination of the Klotho gene or Klotho protein mRNA may also be useful for evaluation.

Still other (in vivo) models and biological assays include evaluating animals for clinical (eg, metabolic) parameters, eg, parameters associated with insulin disorders, type II diabetes. Exemplary metabolic parameters include glucose concentration, insulin concentration, and insulin sensitivity.

Several age-related parameters or biomarkers can be monitored or evaluated in assessing whether a test protein can alter lifespan regulation. Exemplary age-related parameters include (i) the lifespan of a cell or organism, (ii) the presence or abundance of a gene transcript or gene product in a cell or organism having a biological age-dependent expression pattern; (iii) resistance of the cell or organism to stress, (iv) one or more metabolic parameters of the cell or organism (exemplary parameters include circulating insulin levels, blood glucose levels, fat content, core body temperature, etc.). (v) the ability of a cell or set of cells present in the organism to proliferate, and (vi) the physical appearance or behavior of the cell or organism.

The term "life expectancy" refers to the average age at death of a cohort of organisms. In some cases, "life expectancy" is assessed using cohorts of genetically identical organisms under controlled environmental conditions. Accidental deaths are not counted. If life expectancy cannot be determined under controlled environmental conditions (e.g., in humans), robust statistical information (e.g., from mathematical tables) for sufficiently large populations can be used as life expectancy.

Characterization of molecular differences between two such organisms, e.g., one reference organism and one organism treated with S-Klotho protein, can reveal differences in the physiological state of the organisms. . The reference organism and the treated organism are typically of the same (or substantially the same) chronological age and/or gender. As used herein, the term "chronological age" refers to the time elapsed since conception, the defined prenatal or fetal period, or more preferably, a preselected event such as birth. Various criteria can be used to determine whether organisms are of the "same" chronological age for comparative analysis.

Typically, the degree of accuracy required correlates with the life expectancy of wild-type organisms. For example, the C. elegans C. elegans, whose laboratory wild-type strain N2 survives on average about 16 days under some controlled conditions. For elegans, organisms of the same age can live for the same number of days. Organisms of the same age can live for the same number of weeks or months in the case of mice, the same number of years (ie within 2, 3 or 5 years) in the case of primates or humans, and so on. Generally, organisms of the same chronological age can live for an amount of time within 15, 10, 5, 3, 2, or 1% of the life expectancy of wild-type organisms of that species. Preferably, the organism is an adult (eg, the organism has lived for at least some amount of time for an average wild-type organism to mature to reproductive age).

Biological screening assays can be performed before the organism exhibits the overt physical characteristics of aging. For example, an organism may be an adult that has survived only 10, 30, 40, 50, 60, or 70% of the life expectancy of a wild-type organism of the same species. Age-related changes in metabolism, immune competence, and chromosomal structure have been reported. Any of these changes can occur in a test subject (e.g., an organism-based assay) or (e.g., in a (human or mammalian) patient) before, during, or after treatment with a therapeutic agent described herein. It can be evaluated either later.

Markers associated with caloric restriction can also be evaluated in the subject organism (or treated subject) of the screening assay. These markers may not be age-related, but may indicate physiological conditions that are altered when Klotho or Klotho-related pathways are modulated. Markers can be mRNAs or proteins that are highly altered in calorie-restricted animals. Cell models derived from cells of the animals described herein, or analogs of the animal models described herein, can be used for cell-based assays.

Models for evaluating the effect of a test protein on muscle atrophy include: 1) medial gastrocnemius muscle mass loss in rats resulting from denervation, e.g., by cutting the right sciatic nerve in the mid-thigh; 2) (e.g., 90 3) medial gastrocnemius mass loss in rats resulting from immobilization, 4) the cachectic cytokine interleukin-1 ( Skeletal muscle atrophy resulting from treatment with IL-1) and 5) skeletal muscle atrophy resulting from treatment with glucocorticoids, dexamethasone.

Products, compositions, formulations, supplements, etc. Some embodiments of the present disclosure are configured or formulated to increase production of naturally soluble Alpha Klotho protein and/or attenuate damage or degradation of Klotho protein. and methods, kits, formulations, combination products, co-administration, co-formulations, dosages, processes, methods, treatment protocols, and diagnostic methods associated therewith. Some embodiments of the present disclosure relate to methods of making compositions of the present disclosure. Some embodiments of the present disclosure relate to the use of the disclosed compositions or methods of using them to treat human or non-human animal subjects. Some embodiments of the disclosure relate to therapeutic methods involving the compositions of the disclosure, including administration thereof to human or non-human animal subjects. Such uses and treatment methods may increase endogenous Klotho protein levels, such as by increasing natural soluble alpha Klotho protein production and/or attenuating Klotho protein damage or degradation.

Certain embodiments include health supplements that, when administered to a human or non-human animal (e.g., mammalian) subject, increase the level of endogenous soluble alpha Klotho protein in the human or non-human animal subject. including compositions. The composition or health supplement may contain (synthetic) chemical and/or natural constituents or ingredients. In other words, the constituents or ingredients of the composition or health supplement may be of or derived from (synthetic) chemical and/or natural sources.

Exemplary compositions preferably contain, for example, vitamin D3, vitamin E, vitamin C, vitamin K, 3,5,4′-trihydroxy-trans-stilbene (resveratrol), pterostilbene, N-acetylcysteine (NAC), Troglitazone, Rosiglitazone, Notopterygium incisum Ting, Gentian (root, extract), Cordyceps (Cordyceps Sinensis (CS) mycelium), Isoflavones (soybean), Genistein, Daidzein, Quercetin (dihydrate, dill, bay leaf) , Oregano), Docosahexaenoic Acid (DHA), Astaxanthin, Semen Sesamin Nigrum (black), Sesame (seed extract), Rosmarinic Acid (RA, (Marjoram)), Tetradecylthioacetic Acid (TTA), Diphosphate Calcium (Anhydrous), Microcrystalline Cellulose (MCC), Croscarmellose Sodium (Primerose), Steering Acid, Magnesium Stearate, Alpha Lipoic Acid, Conjugated (Alpha) Linoleic Acid (CLA), Enteric Beneficial Bacteria, Activated Charcoal, and It may contain one or more components selected from the group consisting of others known in the art.

Certain ingredients or components may have positive benefits and very low side effects in treating or affecting one or more conditions associated with cell and tissue dysfunction that occurs with age. Certain ingredients or components can increase (directly or indirectly) circulating plasma levels of soluble alpha klotho (s-klotho) levels in a human or non-human animal subject following its administration.

The following examples are illustrative only. The following examples illustrate various optional ingredients, components, method steps, etc. that may be useful in practicing embodiments of the present disclosure.
Individually, embodiments of the present disclosure address, treat, affect, and/or combat one or more conditions associated with cellular and tissue dysfunction and/or that may occur with aging. , include formulations (compositions, co-administration, etc.) of several components (or ingredients) that may have positive benefits and/or very low side effect potential.

Without being bound by any theory, some components or ingredients of the disclosure may activate the promoter of the Klotho gene (e.g., in the human HEK293/kl cell line) or , and reverse the age-related decline in Klotho expression in other tissues. Some of the components may attenuate, reduce, inhibit, and/or prevent oxidative or other damage to the Klotho protein or Klotho gene.

Without being bound by any theory, some of the components address one or more conditions associated with cellular and tissue dysfunction and/or that may occur with aging. have been shown by one or more animal and human clinical trials to have positive benefit and/or very low potential for side effects in treatment, impact and/or opposition. The present disclosure utilizes these various components in compositions and methods for increasing endogenous Klotho protein levels. Specifically, the present disclosure provides these various components in compositions constructed or formulated to increase the production of native soluble Alpha Klotho protein and/or attenuate damage or degradation of Klotho protein. take advantage of

Without being bound by any theory, certain individual components of some embodiments may increase circulating plasma levels of soluble alpha-klotho in mammalian (eg, human or non-human) animals. Examples of ingredients include the antioxidant N-acetylcysteine (NAC), D vitamins such as vitamin D3 (1,25-dihydroxyvitamin D3 [1,25(OH)2D3]), and/or chronic kidney disease. increased Klotho expression, increased phosphaturia, corrected hyperphosphatemia, decreased serum fibroblast growth factor-23 (FGF-23) and aortic calcification in mice suffering from vitamin D receptor agonists (e.g., calcitriol, paricalcitol), which are thought to cause an associated decrease in Vitamin C and/or vitamin E may be included, but are not limited to.

Further exemplary ingredients include, for example, docosahexaenoic acid (DHA), linoleic acid, conjugated linoleic acid (CLA), isoflavones (e.g., genistein, daidzein, quercetin), rosmarinic acid, sesamin and astaxanthin (food-derived antioxidants). ), as well as non-formulation ingredients that may supplement Alpha Klotho's anti-aging properties such as, but not limited to, tetradecylthioacetic acid (TTA). Without being bound by any theory, the aging brain is particularly prone to inflammatory and oxidative alterations, which may underlie the decline in learning and memory, and omega-3 polyvalent depletions such as DHA. Saturated fatty acids (PUFAs) protect the aging brain from developing neurodegenerative diseases. Thus, specific formulas may include alpha-linoleic acid or conjugated alpha-linoleic acid, as alpha-linoleic acid may serve as a precursor molecule used by the human body in the production of DHA.

In at least one embodiment, one or more DHA precursors or omega-3 fatty acids (such as LA or CLA) may be omitted from the formulation. Without being bound by any theory, omega-3 fatty acids such as LA and CLA may increase the effectiveness of anticoagulant drugs and increase the risk of bleeding. Examples of drugs include, but are not limited to, warfarin (Coumadin), clopidogrel (Plavix), and aspirin, among others. In some embodiments, DHA may be included in the formulation. Without being bound by any theory, DHA does not directly cause anticoagulation.

Without being bound by any theory, mitochondrial damage may be the cause and consequence of cell injury resulting from various toxic insults, hypoxia, or trauma. Increased mitochondrial biogenesis after renal proximal tubule cell (RPTC) injury may accelerate recovery of mitochondrial and cellular function after such injury. Related to these observations, isoflavones such as daidzein and genistein increase peroxisome proliferator-activated receptor gamma-coactivator (PGC)-1alpha expression, ATP synthase beta and NADH:ubiquinone oxidoreductase core subfolders. It may result in mitochondrial biogenesis indicated by increased expression of unit 6 (ND6), which may result in a 1.5-fold increase in respiration and ATP in RPTCs, which may benefit recovery of injured RPTCs. Without being bound by any theory, daidzein can be metabolized in the intestine, specifically by the intestinal flora, to produce the bioactive compound equol. Equol may also be included in certain embodiments. The isoflavones, quercetin, on the other hand, play a major role in maintaining redox balance by recognizing and removing oxidatively modified or damaged proteins Large protein complexes involved in the proper regulation of cellular protein load may counter the deleterious effects of aging by increasing oxidative stress resistance in the proteasome, which is

Rosmarinic acid may also be added as an ingredient in some formulations. Rosmarinic acid (RA) is a naturally occurring phenolic compound that can contribute to the beneficial health-promoting effects of herbs, spices, and medicinal plants. Administration of RA at a dose of 200 mg/kg once daily for 30 days in aged mice produced superoxide dismutase (SOD) catalase (CAT) and glutathione peroxidase (GSH- Px) activity can be significantly increased and malondialdehyde (MDA) decreased. Thus, RA can promote significant antioxidant enzyme activity in liver and kidney tissues of aged mice.

Some embodiments may also contain food-derived antioxidants such as astaxanthin (A) and/or sesamin (S). Without being bound by any theory, these components (astaxanthin is the red carotenoid found in salmon, shrimp, crab, and microalgae, while sesamin is the major lignan found in sesame extracts). ) in combination (i.e., AS) and administered daily to subjects with mild cognitive impairment (MCI), significant improvements in psychomotor and processing speed were observed in the AS group compared to the placebo group. was done. Therefore, daily supplementation of AS may improve cognitive functions related to the ability to comprehend and perform complex tasks quickly and accurately.

One or more peroxisome proliferator-activated receptor (PPAR-γ) agonists such as tetradecylthioacetic acid (TTA) may also be added in certain embodiments. PPAR-γ agonists may have beneficial effects on the myocardium through regulation of amino acid metabolism and L-carnitine biosynthesis.

In addition to the purified chemical compounds described above, some embodiments may contain one or more (eg, several) natural ingredients or extracts. These include, but are not limited to, gentian root extract, Cordyceps mushroom, and Notopterygium incisum Ting.

Certain embodiments include Lactobacillus species (e.g., acidophilus, brevis, bulgaricus, casei, gasseri, johnsonii, lactis, plantarum, paracasei, rhamnosus, salivarius, sake, etc.), Bifidobacterium species (e.g. bifidum, breve, infantis, lactis, longum, etc.), Streptococcus spp. (e.g., thermophiles, etc.), Bacillus spp. (e.g., laterosporus, etc.), Pediococcus spp. may contain one or more (selective) strains of endophytic bacteria). Without being bound by any theory, intestinal beneficial bacteria may mitigate age-related oxidative stress (eg, in mice) and improve the expression of biomarkers of aging. Additional information regarding intestinal beneficial bacteria that may be included in certain embodiments of the present disclosure is provided in Int J Environ Res Public Health, 2014 May;11(5):4745, which is herein incorporated by specific reference in its entirety. 4767, in the article "Microorganisms with Claimed Probiotic Properties: An Overview of Recent Literature" by Sabina Fijan.

Table 42 provides a list of ingredients that can be formulated into a particular embodiment (denoted KSF101).

As a non-limiting example, the KSF101 formulation can be or include an orally administered DSHEA-compliant formulation or composition. Thus, in some cases, KSF101 supplements may be marketed and/or sold directly to the consumer (DTC) and/or without a prescription/physician's direction. However, it will be appreciated that KSF101 may be formulated in other dosage forms and/or as an FDA-approved drug without departing from the scope of this disclosure. Concentrations of components in KSF101 are based on a standard adult reference of a 150 lb (68 kg) person. Alternative dosage(s) are also contemplated herein, as will be appreciated by those skilled in the art.

Table 43 lists potential (eg, preferred and optional) ingredients that can be formulated into another embodiment of the present disclosure.

Table 44 lists potential (eg, preferred and optional) ingredients that can be formulated into another embodiment of the present disclosure.

The ingredients in Tables 42-44 may be combined in any suitable combination. As a non-limiting example, the alternative formulation(s) may be or include an orally administered DSHEA-compliant formulation or composition. However, it will be appreciated that alternative formulation(s) may be formulated in other dosage forms and/or as FDA approved drugs without departing from the scope of this disclosure. The concentrations of each of the ingredients in the alternative formulation(s) are based on a standard adult reference for a 150 pound (68 kilogram) person. Alternative dosage(s) are also contemplated herein, as will be appreciated by those skilled in the art.

Exemplary compositions (eg, nutraceutical compositions) include vitamin C, vitamin D3, vitamin E, N-acetylcysteine (NAC), quercetin (dihydrate), rosmarinic acid (RA), pterostilbene, docosahexaene two or more selected from the group consisting of acid (DHA), nicotinamide riboside (NR), nicotinamide adenine dinucleotide (NAD+), nicotinamide mononucleotide (NMN), and one or more intestinal beneficial bacteria may contain components of The one or more intestinal beneficial bacteria comprises an effective amount of (i) Bifidobacterium lactis BL-04, Bifidobacterium bifidum/lactis BB-02, and Bifidobacterium longum
one or more Bifidobacterium species and/or strains selected from BL-05 and/or one or more Laact selected from Lactobacillus acidophilus LA-14, Lactobacillus rhamnosus LR-32, and Lactobacillus paracasei L-37 obacillus species and /or may include one or more of the strains.

Illustratively, the composition (or formulation) comprises up to, at least, and/or about 50 billion CFU (per daily dose) of intestinal beneficial bacteria components. Alternative embodiments are up to, at least between, and/or about 20 billion CFU (per daily dose), 30 billion CFU (per daily dose), 40 billion CFU (per daily dose), 600 billion CFU (per daily dose), 70 billion CFU (per daily dose), and/or 80 billion CFU (per daily dose) of intestinal beneficial bacteria components. Illustratively, the gut beneficial bacteria component is a single gut beneficial (bacterial) species or strain, or up to, at least, and/or between 2, 3, 4, 5, 6, 7, 8 , 9, and 10 intestinal beneficial (bacterial) species or strains. In at least one embodiment, the enteric beneficial bacteria component is the following enteric beneficial (bacterial) species or strains, Bifidobacterium species, and/or Bifidobacterium lactis BL-04, Bifidobacterium bifidum/lactis BB-02, and Bifidobacterium longu m Includes 30 billion CFU/day for each strain of BL-05. However, certain embodiments contain one or two of the aforementioned Bifidobacterium species and/or strains, and/or up to, at least, between, and/or about 10 billion CFU (per daily dose), It is understood that amounts of 20 billion CFU (per daily dose), 30 billion CFU (per daily dose), 40 billion CFU (per daily dose), and/or 50 billion CFU (per daily dose) may be included. will be done. In at least one embodiment, the enteric beneficial bacteria component is the following enteric beneficial (bacterial) species or strains, Lactobacillus species and/or strains Lactobacillus acidophilus LA-14, Lactobacillus rhamnosus LR-32, and Lactobacillus paracasei Includes 20 billion CFU/day for each strain of LPC-37. However, certain embodiments include one or two of the aforementioned Lactobacillus species and/or strains, and/or up to, at least, between, and/or about 5 billion CFU (per daily dose), It is understood that amounts of 10 billion CFU (per daily dose), 20 billion CFU (per daily dose), 30 billion CFU (per daily dose), and/or 40 billion CFU (per daily dose) may be included. will be done.

In at least one embodiment, vitamin C is about 500 mg (per day or per dosage), or about 100-2000 mg, preferably about 200-1500 mg, more preferably about 250-1200 mg, even more preferably about 400 mg. A daily dose and/or concentration of ˜1000 mg, even more preferably about 500-1000 mg, even more preferably about 500-750 mg. In at least one embodiment, vitamin C may be included at a daily dose and/or concentration of at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg (per day or per dosage) . In at least one embodiment, vitamin C may be included at a daily dose and/or concentration of no more than about 2500 mg, 2000 mg, 1500 mg, 1000 mg, or 750 mg (per day or per dosage).

In at least one embodiment, vitamin D3 is about 1000 mg (per day or per dosage), or about 100-2500 mg, preferably 200-2000 mg, more preferably about 250-1800 mg, even more preferably about 400-1800 mg. It may be included in a daily dose and/or concentration of 1500 mg, even more preferably about 500-1500 mg, even more preferably about 750-1250 mg. and/or concentration. In at least one embodiment, vitamin D3 may be included at a daily dose and/or concentration of about 3000 mg, 2500 mg, 2000 mg, or 1500 mg or less (per day or per dosage).

In at least one embodiment, vitamin E is about 400 mg (per day or per dosage), or about 100-2000 mg, preferably about 150-1500 mg, more preferably about 200-1200 mg, even more preferably about 250 mg. daily doses and/or concentrations of -1000 mg, even more preferably about 250-750 mg, even more preferably about 250-500 mg, even more preferably about 300-750 mg, even more preferably about 300-500 mg . In at least one embodiment, vitamin E may be included at a daily dose and/or concentration of at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg (per day or per dosage) . In at least one embodiment, vitamin E may be included at a daily dose and/or concentration of no more than about 2000 mg, 1500 mg, 1000 mg, 750 mg, or 500 mg (per day or per dosage).

In at least one embodiment, N-acetylcysteine (NAC) is about 600 mg (per day or per dosage), or about 100-2000 mg, preferably about 150-1500 mg, more preferably about 200-1200 mg, and More preferably, it may be included in a daily dose and/or concentration of about 250-1000 mg, even more preferably about 250-750 mg, even more preferably about 500-750 mg. In at least one embodiment, N-acetylcysteine (NAC) is at a daily dose of at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg (per day or per dosage) and/or concentration. In at least one embodiment, N-acetylcysteine (NAC) can be included at a daily dose and/or concentration of no more than about 2000 mg, 1500 mg, 1000 mg, or 750 mg (per day or per dosage).

In at least one embodiment, quercetin (dihydrate) is about 150 mg (per day or per dosage), or about 10-1000 mg, preferably about 25-750 mg, more preferably about 50-500 mg, and More preferably, it may be included in a daily dose and/or concentration of about 100-250 mg, even more preferably about 100-200 mg. In at least one embodiment, quercetin (dihydrate) is at least about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, or 250 mg (per day or per dosage) It may be included in daily doses and/or concentrations. In at least one embodiment, quercetin (dihydrate) is administered at a daily dose and/or concentration of less than or equal to about 1000 mg, 750 mg, 500 mg, 400 mg, 300 mg, 250 mg, or 200 mg (per day or per dosage) can be included.

In at least one embodiment, rosmarinic acid (RA) is about 500 mg (per day or per dosage), or about 100-2000 mg, preferably about 200-1500 mg, more preferably about 250-1200 mg, even more preferably may be included in a daily dose and/or concentration of about 400-1000 mg, even more preferably about 500-1000 mg, even more preferably about 500-750 mg. In at least one embodiment, rosmarinic acid (RA) is at a daily dose and/or concentration of at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg (per day or per dosage) can be included. In at least one embodiment, rosmarinic acid (RA) can be included at a daily dose and/or concentration of no more than about 2500 mg, 2000 mg, 1500 mg, 1000 mg, or 750 mg (per day or per dosage).

In at least one embodiment, the pterostilbene is about 50 mg (per day or per dosage), or about 10-200 mg, preferably about 20-150 mg, more preferably about 25-120 mg, even more preferably about 25 mg. A daily dose and/or concentration of ˜120 mg, even more preferably about 40-100 mg, even more preferably about 50-100 mg, even more preferably about 50-75 mg. In at least one embodiment, pterostilbene may be included in a daily dose and/or concentration of at least about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, or 100 mg (per day or per dosage) . In at least one embodiment, pterostilbene may be included at a daily dose and/or concentration of about 250 mg, 200 mg, 150 mg, 100 mg, or 75 mg (per day or per dosage).

In at least one embodiment, DHA (preferably vegetarian DHA) is about 200 mg (per day or per dosage), or about 10-1000 mg, preferably about 25-750 mg, more preferably about 50-500 mg, Even more preferably, it may be included in a daily dose and/or concentration of about 100-250 mg, even more preferably about 150-250 mg, even more preferably about 100-200 mg. In at least one embodiment, quercetin (dihydrate) is at least about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg (per day or per dosage) , 400 mg, or 500 mg daily doses and/or concentrations. In at least one embodiment, quercetin (dihydrate) is at a daily dose of no more than about 2000 mg, 1500 mg, 1000 mg, 750 mg, 500 mg, 400 mg, 300 mg, or 250 mg (per day or per dosage) and/or concentration.

In at least one embodiment, nicotinamide riboside (NR) is about 500 mg (per day or per dosage), or about 100-2000 mg, preferably about 200-1500 mg, more preferably about 250-1200 mg, and More preferably, it may be included in a daily dose and/or concentration of about 400-1000 mg, even more preferably about 500-1000 mg, even more preferably about 500-750 mg. In at least one embodiment, nicotinamide riboside (NR) is at a daily dose of at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg (per day or per dosage) and/or concentration. In at least one embodiment, nicotinamide riboside (NR) may be included at a daily dose and/or concentration of no more than about 2500 mg, 2000 mg, 1500 mg, 1000 mg, or 750 mg (per day or per dosage).

In at least one embodiment, nicotinamide adenine dinucleotide (NAD+) is about 300 mg (per day or per dosage), or about 50-2000 mg, preferably about 100-1000 mg, more preferably about 150-750 mg; more preferably about 200-600 mg, even more preferably about 200-500 mg, even more preferably about 200-400 mg, even more preferably about 250-500 mg, even more preferably about 250-400 mg, even more preferably about 300 It may be included in a daily dose and/or concentration of -500 mg, even more preferably about 300-400 mg. In at least one embodiment, the nicotinamide adenine dinucleotide (NAD+) is at a daily dose of at least about 50 mg, 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg (per day or per dosage) and/or concentrations. In at least one embodiment, the nicotinamide adenine dinucleotide (NAD+) is at a daily dose and/or concentration of less than or equal to about 2000 mg, 1500 mg, 1000 mg, 750 mg, 500 mg, 400 mg, or 350 mg (per day or per dosage) can be included in

In at least one embodiment, nicotinamide mononucleotide (NMN) is about 200 mg (per day or per dosage), or about 10-1000 mg, preferably about 25-750 mg, more preferably about 50-500 mg, and More preferably, it may be included in a daily dose and/or concentration of about 100-250 mg, even more preferably about 150-250 mg, even more preferably about 100-200 mg. In at least one embodiment, nicotinamide mononucleotide (NMN) is at least about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg (per day or per dosage) , 400 mg, or 500 mg daily doses and/or concentrations. In at least one embodiment, nicotinamide mononucleotide (NMN) at a daily dose and/or concentration of no more than about 2000 mg, 1500 mg, 1000 mg, 500 mg, 400 mg, 300 mg, or 250 mg (per day or per dosage) can be included.

Without being bound by any theory, in embodiments comprising NAD+ and/or NAD+ precursor(s) (NR and/or NMN), NAD+ and/or NAD+ precursor(s) are ( For example, it may be in the form of lozenges for sublingual application (to enter the bloodstream, bypass the liver first, and extend the time until the liver converts them to NAMs). Other components may also be administered separately or co-administered. In some embodiments, the components may be co-administered, preferably via at least some co-formulations, or may be formulated as multi-dosage compositions. For example, some embodiments may be, include, or be provided as a multi-pill, multi-capsule, or other dosage kit or pack. Embodiments may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dosage components such as pills, capsules, and the like. Some ingredients may be provided in solid, granular, powdered, liquid, or other form.

Table 45 shows exemplary formulations according to embodiments of the present disclosure.

The ingredients in Table 45 can optionally be combined in any suitable combination. As a non-limiting example, the alternative formulation(s) may be or include an orally administered DSHEA-compliant formulation or composition. However, it will be appreciated that alternative formulation(s) may be formulated in other dosage forms and/or as FDA approved drugs without departing from the scope of this disclosure. The concentrations of each of the ingredients in the alternative formulation(s) are based on a standard adult reference for a 150 pound (68 kilogram) person. Alternative dosage(s) are also contemplated herein, as will be appreciated by those skilled in the art.

Some embodiments provide an effective amount of a composition described herein (e.g., health supplements). A pharmaceutically effective amount of the composition(s) will increase the subject's serum soluble Klotho protein concentration (e.g., about 50-3000 picograms per milliliter of serum, greater than, equal to, or between a predetermined amount of soluble Klotho protein, etc.). to the level of ). A pharmaceutically effective amount of the composition(s) may also, or alternatively, be sufficient to maintain a subject's serum soluble Klotho protein concentration at or above a predetermined threshold for a predetermined period of time.

In certain embodiments, a sufficient or suitable increase in Klotho protein levels may modulate (can be effective to modulate) IGF-1 and/or Wnt signaling pathways, β-glucuronidase and/or sialidase may exhibit activity, may inhibit the p53/p21 signaling pathway, and/or may reduce _{H2O2 -} induced cellular senescence and apoptosis, preferably through inhibition of the p53/p21 signaling pathway. The (elevated) protein preferably exhibits pleiotropic activity and/or preferably oxidative stress, growth factor signaling, regulation of ion homeostasis and/or one or more ion channel proteins and/or insulin/insulin-like It may function as a humoral factor or may be functional in modulating the activity of glycoproteins on the cell surface such as growth factor receptors such as growth factor-1 receptor.

A formulated composition (e.g., a health supplement) of the present disclosure increases or enhances the (natural) production of Klotho protein by a patient or subject, or causes oxidative or other damage or damage to Klotho protein or Klotho gene. Attenuating (eg, reducing, inhibiting, and/or preventing) degradation can increase serum soluble Klotho protein levels.

Some embodiments of the present disclosure may include pharmaceutical compositions such as therapeutic compositions or drugs. For example, some embodiments include pharmaceutical or prescription drugs (e.g. ARBs (e.g. losartan, valsartan), testosterone, vitamin D receptor agonists (e.g. calcitriol, paricalcitol), PPAR (gamma) agonists (e.g. , thiazolidinediones, troglitazone, rosiglitazone), or others). Some embodiments include formulations such as angiotension receptor blockers (ARBs) (i.e. losartan, valsartan), or vitamin D receptor agonists (VDRAs) (i.e. calcitriol or paricalcitol) In combination with drugs may include formulations or co-formulations. Other prescription drugs that can be administered with the formulation include troglitazone (200 mg/kg/day) and rosiglitazone (20 mg/kg/day), both peroxidase proliferator-activated receptor (PPAR-γ) agonists. . Without being bound by any theory, Klotho is believed to be a target gene of PPAR-γ in cultured human kidney cells and in mouse kidneys in vivo. In addition, Klotho protein expression can be enhanced (or upregulated) by treatment with a PPAR-γ agonist (eg, at the oral dosages described).

Without being bound by any theory, losartan can (significantly) increase circulating α-klotho levels in human type 2 diabetic patients by an average of 23% (from 542 pg/ml to 668 pg/ml, p = 0.001). Linear regression analysis also showed that an increase in plasma albumin-Klotho achieved an enhancement of healthy renal function seen by an associated decrease in urinary albumin/creatinine ratio (β=−0.263, p=0.029). suggests that Similarly, valsartan can significantly increase α-Klotho levels (from 432.7±179 to 506.4±226.8 pg/ml)).

Also, with respect to the above, in mice with chronic kidney disease (CKD) induced by calcitriol or paricalcitrol (at VDRA doses extrapolated from human subjects currently approved for the treatment of secondary hyperparathyroidism) Treatment is believed to result in half the amount of aortic calcification compared to no VDRA. Specifically, mice given calcitriol or paricalcitol intraperitoneally 3 times a week for 3 weeks (dosing: 30 ng/kg calcitriol (C30), or 100 ng/kg paricalcitol (P100) or 300 ng/kg paricalcitol (P300)) can exhibit increased serum and urinary Klotho levels, increased phosphaturia, correction of hyperphosphatemia, and decreased serum fibroblast growth factor-23. . Klotho and osteopontin can also be upregulated by VDRA therapy independently of changes in serum parathyroid hormone and calcium.

Some embodiments, or pharmaceutical components thereof, may comprise one or more recombinant (eg, human or mammalian) Klotho proteins, protein fragments, and/or protein variants. Such active ingredients or pharmaceutical components may contribute to increased levels of endogenous Klotho protein (production) in a patient or subject. Additional descriptions of (various and/or additional) active ingredients such as therapeutic agents, recombinant proteins, pharmaceuticals, and/or prescription drugs can be found in the patent references incorporated above (i.e., ( i) PCT/US2017/035755 filed June 2, 2017; (ii) PCT/US2017/063149 filed November 22, 2017; and (iii) filed December 6, 2017. (U.S. Provisional Patent Application No. 62/595,567). Such active ingredients may contribute to increased levels of endogenous Klotho protein (production) in a patient or subject.

A pharmaceutical composition or component, regardless of type, generally comprises a drug component optionally admixed with a (pharmaceutically acceptable) vehicle, carrier, or excipient consisting of one or more additional ingredients. can contain. Components may include one or more aggregation inhibitors, buffers, tonicity agents, and additional excipients. The primary solvent in the carrier may be either aqueous or non-aqueous in nature.

Some embodiments may include a combination product, composition, or formulation comprising recombinant Klotho protein and one or more additional active ingredients. The one or more additional active ingredients may be selected from among the components, drugs, substances, therapeutic compositions, etc. described herein or others known in the art. For example, the Klotho protein and one or more additional active ingredients may be injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulations. can be co-formulated in Alternatively, combination therapy or co-administration may involve combining the Klotho protein and one or more additional active ingredients into a combination product, composition, or formulation, or Treatment or administration of these additional active ingredients may be included. For example, the Klotho protein and one or more additional active ingredients may be separately injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other or may be in such a formulation.

Some embodiments provide an effective amount of a composition described herein (e.g., health supplement), and optionally a composition comprising an effective (eg, pharmaceutically effective) amount of the recombinant Klotho protein and/or pharmaceutical or prescription drug. In some embodiments, a recombinant Klotho protein and/or a pharmaceutical or prescription drug may be co-administered with the composition. In some embodiments, the recombinant Klotho protein and/or the pharmaceutical or prescription drug may be combined with a pharmaceutically acceptable carrier. In some embodiments, any optionally administered recombinant Klotho protein can increase serum soluble Klotho protein levels by providing exogenous Klotho protein, while any optionally administered pharmaceutical agent or The prescribed drug may increase or enhance the (natural) production of Klotho protein by a patient or subject, or attenuate (e.g., reduce, inhibit, and/or prevent oxidative or other damage or degradation of Klotho protein or Klotho gene). ) can increase serum soluble Klotho protein levels.

It is also understood that co-administration can include simultaneous administration of two or more components or separate administration of two or more components, the separate administrations preferably being separated by a period of time. Will. In some embodiments, the period of time may be very short. For example, the second component can be administered substantially immediately after administration of the first component. Alternatively, the first and second administrations are 1-60 seconds, 1-60 minutes, 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, etc., or any value or interval between may be separated by a period of time, such as a range of values for . Similarly, co-administration can involve overlapping dosing windows of two or more components.

Any of the foregoing or other compositions, formulations, or co-administrations may have additive or synergistic effects as compared to any of the individual components alone. For example, co-administration of various health supplement components or health supplement compositions with active components (e.g., recombinant Klotho proteins, pharmaceuticals, etc.) may result in individual outcomes of administering similar concentrations of individual components alone. It can result in a therapeutic outcome that is greater than the sum. In addition, synergistic effects can include therapeutic results similar to those of the individual results of administering the single components at lower concentrations. A synergistic effect can also be an increase in the maximum effective dose of one or more of the components, a reduction in toxicity of one or more of the components, or any other than merely additive effects of the individual therapeutic outcomes. can include beneficial results of Additionally, the additive effect of individual therapeutic outcomes can include one or more additive effects. Such additive/synergistic effects may not be expected or expected given the nature and understanding of the individual components.

Certain embodiments include expression nucleic acid constructs and/or vectors, cell lines and/or cell suspension cultures, and one or more recombinant (e.g., human or mammalian) Klotho proteins, protein fragments, and/or protein variants. It can include methods of manufacturing the body, purifying it, and administering it to a subject (human or non-human animal).

It will be appreciated that any of the foregoing, including health supplements, formulations, co-formulations, co-administrations, combination products, etc., can be referred to as a "composition" or "composition of the invention."
Administration of Exogenous S-Klotho (to increase and/or maintain ) S-Klotho formulations, clinical dosages, and administration.

Aspects or embodiments of the present disclosure include, for example, administering human recombinant alpha soluble Klotho protein (cGMP and/or clinical grade) or protein fragments (of isoform 1) to a (human) subject in need thereof. including. Embodiments may also include measuring (eg, by mass spectrometry (MS) or ELISA) serum S-Klotho levels or concentrations (in a (human) subject). Such measurements can occur before, after, and/or during S-Klotho administration, and, optionally, serum S-Klotho levels and/or metabolism, degradation, or reduction of serum S-Klotho levels. It can be repeated to determine velocity. MS is a technique well known in the art. MS can be used to identify and quantify the levels of one or more (natural and/or recombinant) Klotho proteins in a subject's serum.

One or more additional proteins can also be measured in the subject's serum. For example, one or more Klotho-related and/or aging-related proteins (e.g., FGF21, GDF-11, TIMP2, NAD+, CCL11, hormones testosterone, estrogen, etc.), and/or renal function proteins (e.g., KIM-1, cystatin-C, creatinine, BUN, creatinine, NGAL, etc.) can be measured separately or in combination with measuring Klotho in serum.

In at least one embodiment, a serum sample, such as a blood sample, is obtained. Samples may be obtained by drawing blood, as is known in the art. In preferred embodiments, a finger prick or other less invasive means of obtaining a blood sample may be used. Blood samples are therefore taken more frequently (eg, throughout the day and/or every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours). ) can be harvested. Measure total Klotho protein serum concentration and serum concentration of various alpha Klotho protein species such as native Klotho species (e.g., soluble Klotho, cleaved Klotho, secreted Klotho, etc.) and/or one or more Klotho proteins of the present disclosure For this purpose, MS can be used. In some embodiments, the Klotho level is 0.5, 1, 2, 3, 4, 5, 6, 7, 0.5, 1, 2, 3, 4, 5, 6, 7, 0.5, 1, 2, 3, 4, 5, 6, 7, before and again throughout the day and/or after administration of the therapeutic recombinant Klotho protein. It can be measured every 8, 9, 10, 11, or 12 hours (or one or more of these). Alternatively or additionally, the Klotho level is It can be measured every 21, 25, 28, 30, 36, 40, 45, 50, 60, or 90 days (or weeks) (or one or more of these).

Embodiments also determine such rates and/or maintain S-Klotho protein concentrations in the serum of such subjects within the range of normal juvenile human S-Klotho serum concentrations. for example, including frequency, amount, and/or duration of (subsequent) administration(s) of S-Klotho. In at least one embodiment, the concentration of S-Klotho can be maintained at approximately 1000 picograms of (S-Klotho) protein per milliliter of serum (pg/mL).

In at least one embodiment, the S-Klotho dosing strategy (in humans) may include measuring one or more pharmacokinetic parameters of S-Klotho. For example, the resulting in vivo changes in S-Klotho levels in serum, urine, and cerebrospinal fluid in response to S-Klotho administration can be measured. Some embodiments may include measuring the efficacy of S-Klotho administration on one or more clinical indicators. Clinical indicators of various conditions, diseases, and disorders are known in the art and are further described herein.

Embodiments also provide any (eg, before and after administration of S-Klotho)), (baseline) S-Klotho level measurement(s) may be taken.

Embodiments may also include determining a suitable frequency, amount, and/or duration of S-Klotho administration. For example, in a subject with low S-Klotho serum levels (eg, as measured by MS or ELISA immunoassay quantification), the subject's serum S-Klotho level can be increased to a first predetermined level (eg, about 1000 pg/mL) A first dose of Klotho configured and/or adapted to provide (e.g., via any suitable route of administration) may be given (e.g., via any suitable route of administration), the subject's serum S-Klotho concentration (the resulting change in ) was measured (by MS or ELISA), the level (after the first dose) and/or rate of metabolism, degradation or reduction of serum S-Klotho levels were measured, and the half-life of administered S-Klotho was calculated. and/or determine the frequency and/or time frame at which a second subsequent dose of S-Klotho should be given (eg, to maintain serum S-Klotho levels above a second predetermined level) can do. In at least one embodiment, the second predetermined level is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% of the first predetermined level, It can be 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% and/or 5% and/or in between.

Additional administrations are provided over a suitable time frame to produce (long-term) in the subject S-Klotho serum levels equivalent to serum maintenance levels (e.g., approximately 1000 pg/ml) in normal young people of the same sex. can be The total duration of S-Klotho administration (to a (human) subject) can range from 1 day to 5 years or more. Measurements and/or determinations of a subject's frailty based on the use of clinical frailty scores and other measures can also be made over a time frame.

Embodiments of the present disclosure include 5%, 10%, 15%, 20%, 25%, 30%, 35%, above the normal range (eg, illustratively at least about 500-1000 pg/ml serum). 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more to maintain S-Klotho level elevation , further comprising increasing the S-Klotho dosage.

Certain embodiments include administering S-Klotho protein. Non-limiting examples of suitable routes of administration include injection (e.g., bolus, gradual, intravenous, intradermal, intraperitoneal, intramuscular, intradermal, and/or subcutaneous injection), oral or oral-related administration ( oral, buccal, and/or sublingual), topical, transdermal, rectal, vaginal, inhalation, and the like.

Exemplary embodiments can include administering the S-Klotho protein or composition as a single bolus injection or a chronic (IV) injection (eg, an infusion over an extended period of time). In at least one embodiment, 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 2.75, 3, 3.5, per kg body weight of the subject, 4, 4.5, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or more micrograms of S-Klotho can be administered per treatment. Suitable dosages can be calculated through one or more methods known in the art. One such method is the allometric scaling method. For example, in rat studies, 0.01 mg S-Klotho/kg body weight or 10 μg/kg body weight per dose is used. Exemplarily, using 0.16, which is the equivalent human relative growth rate, Human Equivalent Dose (HED) = 10 μg/kg x 0.16 = 1.6 μg/kg. Thus, illustratively, a 70 kg human individual would need 70 kg×1.6 μg/kg=112 μg, and a 60 kg human would need 60 kg×1.6=96 μg. Table 46 below demonstrates the conversion of various animal doses to Human Equivalent Dose (HED) based on body surface area. Transformation assumes a 60 kg human.

HED was established using body surface area normalization, a process described by Reagan-Shaw (2008), which is incorporated herein by reference. This process, termed relative growth rate, corrects for underlying differences in metabolic rates between different species and may be preferable to simple dosage extrapolation. Illustratively, if the animal species dose is known, HED can be calculated as HED = animal dose in mg/kg times the relative growth factor for the appropriate animal species. The actual dose administered to the patient can then be calculated by multiplying the HED by the patient's weight.

Amount and/or Bioavailability of S-Klotho in Humans (Before and/or After Recombinant Protein Administration), Total Amount and/or Concentration of S-Klotho Administered, and/or Recombinant Protein Administration Multiple factors may be considered or taken into account in determining, measuring and/or estimating the subsequent serum level response (over time). For example, such factors include diluent composition, route of administration, site of administration, distribution to tissues and organs of interest, metabolic or other rates of interest, pharmacokinetics (PK), pharmacodynamics (PD), toxicological Tox, etc. may be included.

In at least one embodiment, the (normal) concentration of S-Klotho (eg, in healthy young (18-30 year old) human adults) may be approximately 1000 pg/ml in serum. A typical adult can have approximately 5 liters of blood volume, with women generally having less blood volume than men. Approximately 55% of human blood can be made up of serum. Therefore, (5 liters of blood/adult) x (0.55 serum/liter of blood) = 2.75 liters (2750 ml) of serum/adult. Assuming no endogenous serum S-Klotho, to achieve a final concentration of 1000 pg/ml in total serum, 2750 ml x 1000 pg/ml = 2,750,000 pg (or 2750 ng or 2.750 pg/ml) per adult subject. 75 μg) of exogenous S-Klotho may be administered.

To increase soluble Klotho by 50% over typical healthy levels (eg, 1500 pg/ml of serum), a dose of 4.125 micrograms/subject may be administered. To increase soluble klotho 100% above typical healthy levels (eg, 2000 pg/ml serum), a dose of 5.5 micrograms/subject may be administered, and so on.

per milliliter of serum to increase the serum soluble Klotho protein concentration in a subject to any suitable level , 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 11,000, 12,000, 13,000, 14,000, 15 >,000, 20,000, 25,000, 30,000 40,000, 50,000, 75,000, 100,000 picograms of soluble Klotho protein, equal to or between, or soluble Klotho protein in serum about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150% , 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1200%, 1500%, 2000%, 2500%, 3000%, 4000%, 5000 Greater than, equal to, or between pharmaceutically effective and/or sufficient amounts of purified recombinant S-Klotho protein may be administered.

In some embodiments, the subject is administered about 0.01 mg/day in a suitable amount of Klotho's buffer (e.g., 150 mM NaCl and 10 mM HEPES pH 7.4) or other pharmaceutically acceptable carrier. One or more (bolus) intravenous, intradermal, intraperitoneal, intramuscular, intradermal, subcutaneous, and/or other doses of recombinant human S-Klotho protein greater than, equal to, or between kg body weight injection can be administered. Thus, a subject weighing 160 pounds (i.e., weighing 72.57 kg) should receive approximately 0.73 mg S-Klotho per dose (based on a calculation of 0.01 mg S-Klotho/kg x 72.57 kg body weight). (bolus) injections can be administered. Similarly, a 170 pound person could receive 0.77 mg of S-Klotho per dose. The total number and frequency of administration can be determined, for example, based on achieving and maintaining a concentration in serum of 1000 pg/ml S-Klotho (equivalent to 0.000001 mg/ml serum). The latter can be measured and confirmed by MS or by the human S-Klotho ELISA assay.

In other embodiments, the dosage is greater than about 0.0001-10 mg/kg body weight, 0.0001-10 μg/kg body weight, 0.0001-10 ng/kg body weight, 0.0001-10 pg/kg body weight, can be equal to or any value or range of values therebetween. Urine and/or blood may be used for recombinant Klotho protein in medical procedures or procedures (e.g., to measure, test, and/or determine changes in serum S-Klotho levels and dose response and/or response over time). About 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours after administration (dose) of 1 , 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 2.5 days, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 4 weeks, 1 month , 2 months, 3 months, less than, equal to, and one or more time points therebetween.

One or more embodiments involve the manufacture and/or (subsequent) administration of unique formulations of S-Klotho active drug products and/or combinations with pharmaceutically acceptable carriers. Carriers may be suitable for IV and/or bolus injection. Embodiments also include S-Klotho and/or pharmacologically It may involve the manufacture and/or (subsequent) administration of unique inactive prodrug formulations in combination with acceptable carriers, Such prodrug formulations may include coatings or sequential sustained release formulations.

Methods of Treatment, Uses, Etc. Some embodiments include methods or methods of treatment (eg, including one or more method steps). Some embodiments may comprise administering one or more compositions described herein to a subject (eg, a subject in need thereof). Administration of the composition(s) of the invention raises a subject's serum soluble Klotho protein concentration above a predetermined threshold or maintains a subject's serum soluble Klotho protein concentration above a predetermined threshold for a period of time. can be sufficient for

The composition may comprise, for example, (i) a therapeutic recombinant Klotho protein or fragment or fusion protein thereof described in the present disclosure, and/or (ii) endogenous Klotho protein production and/or levels in a patient to whom the product has been administered ( (iii) optionally with one or more additional (active or inactive) ingredients or compositions. One of ordinary skill in the art will appreciate that each of the therapeutic effects, indications, and/or treatments outlined herein with respect to recombinant therapeutic Klotho proteins and products, compositions, and methods associated therewith are associated with endogenous Klotho protein production and It will be appreciated that this may be achieved by (naturally) increasing, enhancing, augmenting and/or supporting the level(s). One skilled in the art will also appreciate the therapeutic effects, indications and It will be understood that each of the/or treatments can be accomplished by administering recombinant therapeutic Klotho proteins and products, compositions, and through methods associated therewith. Accordingly, for the sake of brevity, the present disclosure provides the disclosed therapeutic effects for both (i) administration of a recombinant therapeutic Klotho protein and (ii) administration of a health supplement or nutraceutical composition of the present disclosure. , indications, and/or treatments need not be elaborated upon, as each is expressly contemplated herein.

Certain embodiments may comprise the compositions described herein for use in treating one or more diseases, disorders, injuries and/or (medical) conditions. Similarly, embodiments may include methods of treating (or alleviating, addressing, preventing, inhibiting, etc.) one or more diseases, disorders, injuries, and/or (medical) conditions. An exemplary therapeutic method comprises administering to a subject in need thereof an effective amount of a composition of the present disclosure. Without being bound by any theory, administration of an effective amount of the composition to a subject in need thereof causes or results in an increase (or elevation) of serum soluble Klotho protein concentration in the subject. obtain. Thus, an “effective amount” of a composition of the present disclosure increases or elevates a subject's serum soluble Klotho protein concentration above a predetermined threshold, or increases a subject's serum soluble Klotho protein concentration above a predetermined threshold over a predetermined period of time. can be an amount effective to maintain the Alternatively, or additionally, an "effective amount" of a composition of the present disclosure can be an amount effective to achieve a beneficial therapeutic effect in a patient to whom the composition is administered.

The method, composition or (elevated) protein may also be effective in treating one or more diseases, disorders, injuries and/or (medical) conditions. Illustratively, the condition is frailty, low or low bone mineral density, weight loss, muscle atrophy or wasting, low muscle mass, decreased muscle strength, grip strength, leg strength or strength, movement, freedom of movement, quality of life assessment, such as reduced ejection fraction, decreased motor capacity, decreased learning power, learning ability, memory, or intelligence quotient, cognitive deterioration or amnesia, decreased cognitive ability or function, decreased synaptic plasticity or synaptic function, and cellular senescence. , an age-related or aging-related condition (or a condition associated with (human) aging). However, the condition may also, or alternatively, be any physical, intellectual, emotional condition in which increased serum Klotho levels may provide beneficial therapeutic effects, including those described in this disclosure and known to those of ordinary skill in the art. It will be understood that it may be or include a target, or other deficiency, need, or desire.

The method, composition, or (elevated) protein is also a composition (e.g., protein or supplement) or elevated soluble Klotho protein levels are associated with Alzheimer's disease, Parkinson's disease, dementia or vascular dementia, amyotrophic Lateral sclerosis (ALS) or motor neuron disease (MND), atrial fibrillation, chronic obstructive pulmonary disease (COPD), fibromyalgia, adult-onset diabetes, arthritis or rheumatoid arthritis, osteoarthritis, osteoporosis, glaucoma , cataracts, macular degeneration and other eye diseases/disorders, multiple sclerosis (MS), lupus, and/or ulcerative colitis (or (human) aging) can be effective in the treatment of conditions associated with

Some embodiments of the present disclosure are for treating cancer, lowering serum phosphate levels in a patient, treating diabetes or diabetes-related conditions (e.g., type 1 diabetes, etc.) in a subject in need thereof. treating cardiac conditions in a subject (e.g., cardiovascular disease, left ventricular hypertrophy (LVH), pathological LVH and/or congestive heart failure, etc.); treating acute lung injury in a subject (e.g., using nanoparticles), protecting the lungs of patients against oxidative injury, detecting early acute renal injury in critically ill patients, reducing vascular calcification in subjects, improving cognition, treating renal and/or hepatic ischemia, modulating the stress response of (human) senescent endothelial cells, acute and/or chronic renal injury, disease, or disease progression, and/or uremic myocardium prophylactically and/or therapeutically treating, preventing, reducing, inhibiting and/or reversing disease, reversing or reducing age-related treatment resistance of melanoma and other cancers and muscular dystrophies (including DMD); It may be useful in targeting apoptosis of senescent cells, preferably thereby restoring tissue homeostasis, and in one or more of a variety of other indications.

Some embodiments of the present disclosure may be useful in treating one or more other conditions. Such conditions may be described or disclosed herein. Such conditions may be known to those skilled in the art to be associated with Klotho serum levels.

As briefly noted above, the present disclosure includes and contemplates various dosage forms and/or methods of administration of the disclosed compositions. Some embodiments may be dosed in an oral dosage form and/or administered orally (eg, in one or more convenient forms including tinctures, tablets, capsules, powders, teas, etc.). For example, tinctures consisting of alcoholic or glycerin-based extracts of herbs or natural ingredients can be packaged in glass dropper bottles. Glycerin-based tinctures may provide a sweeter taste and make the formulation easier to swallow. Tinctures can also be administered by dropper. Tinctures may include a labeled dosage range (eg, 20-40 drops per adult dose).

Capsules and tablets are also common forms for oral administration of supplements. For example, dried herbs may be powdered and compressed into gelatin capsules. Supplements may be supplied as capsules and/or tablets. The supplement may be supplied as a powder in a bottle or envelope with the dosage to be used listed on the container. A suitable amount of powder per dose may also be obtained by opening a dosage capsule and sprinkling the contents onto a drink or food.

Tea (eg, herbal decoction) is a common form of herbal remedy and a sedative method of taking herbs. An herbal decoction can be made by placing a small amount of herb in a cup, filling the cup with boiling water, and steeping. In the form of tea, the potency of the herbal components of the supplement may increase with prolonged steeping time, with the tea best steeped for at least 10 minutes. Covering the cup (a small saucer works well) keeps the steam and keeps the tea hot longer.

Some embodiments are methods of treating age-related or other conditions, diseases, or disorders comprising administering to a subject in need thereof an effective amount of a composition of the present disclosure , may include methods. Some embodiments are a method of treating and/or preventing a condition, disease, or disorder, preferably an age-related condition, disease, or disorder, in a mammalian subject, comprising an effective amount of A method can include administering the composition to a subject in need thereof. Some embodiments are methods of treating or preventing acute kidney injury (AKI), chronic kidney disease (CKD), or other conditions comprising an effective amount of a composition of the present disclosure in a subject in need thereof can include a method comprising administering a Some embodiments are methods of treating and/or preventing renal injury in a mammalian subject, wherein prior to a medical procedure or administration of nephrotoxin and/or after a medical procedure or administration of nephrotoxin, the subject and optionally prophylactically, comprising administering an effective amount of a composition of the present disclosure. Some embodiments may include a method of administering a composition of this disclosure to a human or non-human animal (eg, mammalian) subject in need thereof. The subject to whom the composition is administered can suffer from or be at risk of various conditions (eg, disorders, diseases, injuries, illnesses, etc.). For example, some embodiments treat one or more chronic diseases and/or age-related conditions, such as physical, intellectual, neurological, or other conditions associated with (human) aging. including how to Some embodiments may promote healing, recovery, longevity, and/or other beneficial outcomes through one or more mechanisms or actions.

A pharmaceutically effective amount of the composition(s) will increase the subject's serum soluble Klotho protein concentration (e.g., about 50-3000 picograms per milliliter of serum, greater than, equal to, or between a predetermined amount of soluble Klotho protein, etc.). to the level of ). A pharmaceutically effective amount of the composition(s) may also, or alternatively, be sufficient to maintain a subject's serum soluble Klotho protein concentration at or above a predetermined threshold for a predetermined period of time.

In certain embodiments, a suitable increase in protein levels may modulate (can be effective to modulate) IGF-1 and/or Wnt signaling pathways and may exhibit β-glucuronidase and/or sialidase activity. , may inhibit the p53/p21 signaling pathway and/or may reduce H ₂ O ₂ -induced cellular senescence and apoptosis, preferably through inhibition of the p53/p21 signaling pathway. The (elevated) protein preferably exhibits pleiotropic activity and/or preferably oxidative stress, growth factor signaling, regulation of ion homeostasis and/or one or more ion channel proteins and/or insulin/insulin-like It may function as a humoral factor or may be functional in modulating the activity of glycoproteins on the cell surface such as growth factor receptors such as growth factor-1 receptor.

Certain embodiments include: determining serum soluble Klotho protein concentration in a subject; calculating a pharmaceutically effective amount; determining the rate of decline of soluble Klotho protein in the serum of a subject; calculating the subsequent dosing time at which will be at or below a second predetermined level based on the measured rate; reducing the subject's serum soluble Klotho protein concentration from the second predetermined level to the first predetermined level; This can include calculating subsequent dosages of the composition sufficient to raise the level and/or administering subsequent dosages of the composition to the subject. Certain embodiments may involve prescribing a regular (eg, daily) dose or dosage form of the composition to a subject (eg, based on the determined level of the subject's serum soluble Klotho protein concentration).

Exemplary embodiments include (treatment) methods. Treatment methods can mimic and/or model testing of therapeutic efficacy or health benefits of various compositions of this disclosure. The results of administration of various compositions are monitored in human subjects. After subjects voluntarily agree to participate in the study and sign a patient consent form, a physical examination and blood and saliva tests are performed. Upon meeting the study protocol's inclusion criteria, subjects are enrolled in the study. Illustratively, a study can include 100 healthy individuals who are not currently receiving vitamin D supplementation and do not live in a sunny environment. Subjects with KLVS variants (Klotho overexpression factors) and/or other genetic variants of the Klotho gene(s) may be excluded from the study. After enrollment, subjects will receive a 3-month supply of the Klotho supplement formulation of the present disclosure, or placebo in a double-blind study. Subjects receive a complimentary bottle of unlabeled supplement formulation or placebo. Subjects take this supplement daily for 3 months.

At the end of this 3-month period, blood is drawn for fasting blood tests, the subject's weight, blood pressure is determined, and the Alpha Klotho (α-Klotho) Human Soluble ELISA Assay Kit (eg, IBL America, Minneapolis, Minn., USA) is administered. (Catalog No. 27998)).

The test/design procedure for human subjects in this study is provided below.
Study design/procedure:
Up to 100 patients Duration: 3 months to first endpoint, crossover, second endpoint at 6 months including placebo patients who elected to take replacement medications.

Baseline questionnaire to include: current medications (statins, ARBs), nutritional supplements, blood pressure, exercise frequency, alcohol consumption.
Before registration:
Blood sample and cheek swab collection (PI office or mobile phlebotomist)
KLVS Variant (Klotho Overexpressor) Determination—Buccal Swabs Send to Laboratory to Determine KLVS Variant (Klotho Overexpressor) Status Lab Analysis of Klotho Levels, Vitamins D and E, and Pre-Treatment Baseline Other Analysis of Value Replenishment: Shipped to participants (three months supply). (active supplement and placebo control, equal number of active and placebo controls)
Outcome Measurements: Participants download a mobile application to monitor compliance and questionnaires (exercise, blood pressure) Follow-up blood sampling at 3 and 6 months Ingredients in supplements pose risks composed of very low nutrient constituents. If a subject experiences any discomfort or adverse reaction to the formulation, the subject will be instructed to stop taking the product and contact the study physician.

Any significant side effects will be reported to the study's IRCM Institutional Review Board (IRB) within 48 hours.
Exemplary Conditions In some embodiments, conditions, diseases, or disorders to be treated with the disclosed composition(s) and/or method(s) are preferably, e.g., frailty, loss of bone mineral density, , Bone mineral loss, Weight loss, Muscle atrophy, Muscle atrophy, Decreased muscle mass, Decreased muscle strength, Decreased grip strength, Decreased leg strength, Decreased physical strength, Decreased movement, Decreased freedom of movement, Decreased quality of life evaluation, Ejection Decreased rate, Decreased motor performance, Decreased learning ability, Decreased learning ability, Decreased memory, Decreased intelligence quotient, Cognitive deterioration, Amnesia, Decreased cognitive ability, Decreased cognitive function, Decreased synaptic plasticity, Decline in synaptic function, cellular senescence, chronic kidney disease (CKD), bone mineral disorders associated with chronic kidney disease (CKD-MBD), polycystic kidney disease (PKD), autosomal dominant polycystic kidney disease (ADPKD), acute kidney injury (AKI), acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), glomerulonephritis, renal disease, renal failure, Alport syndrome, non-oliguric renal failure, alcoholism, hyperphosphatemia Hememia, muscular dystrophy (MS), type 1 diabetes, type 2 diabetes, cardiovascular disease (CVD), cardiovascular calcification, cerebrovascular insufficiency, vascular calcification, ischemic heart disease, heart failure, left ventricular hypertrophy, uremic myocardium hypertension, hypertension, salt-sensitive hypertension, tissue calcification, calcified atherosclerotic plaque burden, calcification, familial neoplastic calcification, cancer, one or more tumors, myelin-related disease, demyelinating disease , neurodegenerative disease, neurovascular disease, progressive supranuclear palsy (PSP), Pompe disease, Niemann-Pick disease, microgliosis, Farber disease (FD), bone mass disease, osteoporosis, osteopenia, osteopenia (specifically (specifically decreased cortical BMD), emphysema, pulmonary fibrosis, cystic fibrosis, idiopathic (i.e. unexplained) pulmonary fibrosis, radiation-induced lung injury, cirrhosis, biliary atresia, atrial fibrosis , endocardial myocardial fibrosis, (old) myocardial infarction, glial scar, arteriosclerosis, joint fibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, kidney systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, adhesive capsulitis, skin atrophy, thymic atrophy, accumulation of renal interstitial matrix, glomerulosclerosis, anemia , albuminuria, proteinuria, infertility, Alzheimer's disease, Parkinson's disease, dementia, vascular dementia, amyotrophic lateral sclerosis (ALS), motor neuron disease (MND), atrial fibrillation, chronic obstruction COPD, fibromyalgia, adult-onset diabetes, arthritis, rheumatoid arthritis, osteoarthritis, glaucoma, cataracts, macular degeneration, multiple sclerosis (MS), lupus, ulcerative colitis, cachexia obesity, vitamin D-related conditions, bone disease, bone remodeling-mediated bone disease, bowel disease and related conditions, stem cell depletion, seasickness, space maladaptation syndrome (SAS), nausea, vertigo, It may be selected from the group consisting of nonalcoholic steatohepatitis (NASH), cirrhosis, and alcoholic steatohepatitis.

Klotho serum levels have also been reported to be associated with muscle recovery, including muscle healing after injury. Specifically, and without being bound by any theory, age-related decline in α-Klotho may cause mitochondrial dysfunction in progenitor cells and impaired muscle regeneration. Clinically, this could lead to the treatment of older people who have suffered muscle injury or have undergone surgery to damage the muscle. Embodiments of the present disclosure may include administering the compositions described herein prophylactically and/or post-operatively. Administration at the appropriate time(s) may enhance or support muscle regeneration, leading to more complete recovery. Serum Klotho protein levels can also enhance or support muscle regeneration in athletes and other patients. Accordingly, embodiments of the present disclosure may include administering the compositions described herein prophylactically (pre-workout) and/or post-exercise.

Administration of Compositions to Treat Age-Related Frailty in Humans and Non-Human Mammals and administering the composition of s-Klotho rescues myogenic stem cells, improves muscle repair, and/or inhibits fibrosis in animal models of human disease. Therefore, s-Klotho may be a promising approach to combat muscle wasting in elderly human subjects showing signs of frailty.

For example, the present disclosure maintains maintenance serum concentrations of s-Klotho in subjects within the range of normal (e.g., without any chronic conditions) and/or young persons (e.g., 18-30 years old) s-Klotho level-increasing compositions, formulations and dosages, and their administration to vulnerable individuals and/or elderly individuals (eg, 60-95 years old) for the purpose.

Such Klotho treatment over an extended period of time can reverse and/or ameliorate one or more age-related indicators or conditions in the elderly, infirm, or other physiologically aged individuals.
Administration of Compositions to Treat (Reduce) Muscle Atrophy in Humans and Non-Human Mammals , skeletal muscle tissue mass, and, preferably, the composition of the present disclosure for treating (e.g., reducing (the rate of)) muscle atrophy in humans, as measured by the simultaneous use of the above compositions and molecular beacons. Regarding dosing.

Muscle wasting can include numerous neuromuscular, metabolic, immune, and neurological disorders and diseases, as well as starvation, nutritional deficiencies, metabolic stress, diabetes, aging, muscular dystrophy, or myopathy. Muscle atrophy can occur during the aging process. Muscle atrophy can also result from reduced use or disuse of muscles. Symptoms include decreased skeletal muscle tissue mass. In human males, muscle mass declines by a third between the ages of 50 and 80. Some molecular features of muscle atrophy may include upregulation of ubiquitin ligases and reduction of myofibrillar proteins. Degradation of these proteins can be monitored, for example, in specific muscle myosins (eg, by measuring 3-methyl-histidine production, a specific component of actin). Release of creatine kinase (a cell damage marker) can also be an indicator.

Administration of Compositions to Treat (Improve) Muscle Repair or Recovery in Humans and Non-Human Mammals Klotho serum levels have also been reported to be associated with muscle recovery, such as muscle healing after injury. there is Specifically, and without being bound by any theory, age-related decline in α-Klotho may cause mitochondrial dysfunction in progenitor cells and impaired muscle regeneration. Clinically, this could lead to the treatment of older people who have suffered muscle injury or have undergone surgery to damage the muscle. Embodiments of the present disclosure may include administering the compositions described herein prophylactically and/or post-operatively. Administration at the appropriate time(s) may enhance or support muscle regeneration, leading to more complete recovery. Serum Klotho protein levels can also enhance or support muscle regeneration in athletes and other patients. Accordingly, embodiments of the present disclosure may include administering the compositions described herein prophylactically (pre-workout) and/or post-exercise.

Administration of Compositions to Treat Intellectual and/or Cognitive Decline throughout Old Age and/or Human Lifespan administration of the compositions of the present disclosure for the improvement and/or inhibition of At the time of this disclosure, it was unknown whether administration of the disclosed compositions could prevent cognitive decline in humans. However, transgenic mice with systemic overexpression of Klotho performed better than controls in multiple tests of learning and memory. Klotho elevation in mice also enhanced long-term synergy, a form of synaptic plasticity, and increased synaptic activity, an N-methyl-D-aspartate receptor (NMDAR) subunit that has important functions in learning and memory. GluN2B was concentrated. GluN2B blockade abolished the Klotho-mediated effect.

Klotho-regulated pathways may be involved in slowing the progression of Alzheimer's disease and other forms of dementia. Brain scans of more than 400 healthy men and women over the age of 53 found that those who carried a single copy of a particular Klotho gene variant had larger brain regions involved in planning and decision-making. It was found that Further testing on this group found that people with enlarged right dorsolateral prefrontal cortex (rDLPFC) performed better on a range of intellectual tasks.

About 1 in 5 people inherit a single copy of a genetic variant or allele known as KL-VS that improves heart and kidney function and increases human lifespan by about 3 years on average. However, in terms of brain function, the improvement in intelligence test scores in people with a larger rDLPFC only accounts for 12%, whereas the editors note that carrying one copy of the KL-VS allele This appears to provide 10 years of resilience against the expected decline in rDLPFC structure and function. Thus, KL-VS heterozygosity appears to be associated with a larger volume of the right dorsolateral prefrontal cortex (rDLPFC).

Since the rDLPFC is important for executive function, the researchers also analyzed individuals' working memory and processing speed. KL-VS heterozygosity may be associated with enhanced executive function over the life span tested. In sum, the results suggest that Klotho gene diversity may be associated with greater brain volume and better function.

Exemplary embodiments of the present disclosure improve in vivo Klotho protein levels and/or (cellular, molecular, and/or downstream) (e.g., to enhance cognition and reduce cognitive deficits throughout the human lifespan). It relates to administration of the compositions of the present disclosure to enhance efficacy. Administration of s-Klotho level-increasing compositions can preserve and/or improve cognitive function (eg, in humans).

Administration of Compositions to Treat (Prolong) Human Longevity and/or Lifespan to improve life expectancy. Life expectancy results obtained with composition administration (experimental group of human subjects) are combined with statistical information (e.g., from mathematical tables) for individuals not receiving the composition (control group) and/or for sufficiently large populations. can be reliably compared.

Administration of Compositions to Treat Other Clinical Indications Exemplary embodiments of the present disclosure are directed to administering compositions of the invention to treat any age-related or other non-age-related condition. Regarding the administration of , pathologies include human frailty (increase), longevity (decrease), cell senescence (decrease), muscle strength (decrease), bone loss or density (decrease), cognitive ability (decrease ), (decreased) muscle mass, (decreased) physical strength, (decreased) grip strength, (decreased) leg strength, etc., but are not limited thereto. The disclosure also provides increased bone mineral density (BMD) (e.g., in women but not men), increased BMD that is reduced after menopause (e.g., in older women), regeneration of (degenerate) skeletal muscle or its degeneration of the present disclosure for reduction in gait, improvement in spatial learning and memory, movement, freedom of movement, quality of life assessment, improvement in ejection fraction (or reduction in decline), change in movement, improvement in movement, etc. Regarding administration of the composition. The disclosure further provides a reduction in cognitive deterioration or amnesia, an increase in cognitive performance, an improvement in cognitive function and synaptic plasticity, a reduction in learning ability, learning ability, or decline in IQ, an improvement in learning ability, learning ability, or IQ. administration of the compositions of the present disclosure for, and the like.

Administration of Compositions to Treat Acute Kidney Injury (AKI) Acute Kidney Injury (AKI), formerly called Acute Renal Failure (ARF), often ranges from mild functional loss to dysfunctional Defined as the sudden onset of renal dysfunction. AKI is a common clinical complication occurring in approximately 4% to 7% of hospitalized patients each year and can have a poor prognosis. Mortality rates associated with AKI range from 5% to 35%. Renal Klotho expression has been shown to be suppressed following AKI. Adenoviral gene transfer of Klotho may be cytoprotective in AKI.

Acute kidney injury (AKI) is reported in approximately 4.9% to 7% of hospitalized patients each year. Rates of AKI can be as high as 60% in the (hospitalized) elderly and 20-30% in the elderly or critically ill. AKI is also associated with increased mortality, length of stay (LOS), 30-day readmission rate, and hospital costs.

AKI includes kidney transplantation or other surgery, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis (eg, glomerulonephritis), nephrotoxicity (eg, drug-induced nephrotoxicity), It may result, at least in part, from hypotension, sepsis or a septic condition, or other contributing factor(s). Kidney transplantation and other surgeries cause acute damage or injury to the kidneys and can lead to kidney disease and/or kidney failure. Nephrotoxicity can contribute to AKI, ATN, AAIN, nephritis, and the like. Drugs (eg, clinically administered, prescription, illicit, or other drugs) have been reported to be associated with 15% to 25% of all cases of AKI. Contrast agents alone account for 10% of all causes of hospital-acquired acute renal failure (e.g., contrast agent-induced acute kidney injury CIAKI), deterioration of renal function during hospitalization after decreased renal perfusion and postoperative renal failure. is the third major cause of

In some cases, drug-induced AKI can be or include antimicrobial-induced nephrotoxicity (resulting from antimicrobial treatment). For example, certain (Gram-negative) bacterial infections can be treated with one or more aminoglycosides such as paromomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin. Aminoglycosides have been shown to be nephrotoxic. Table 47 shows treatment data collected for adult patients receiving aminoglycosides. For example, as illustrated in Figure 47, nearly 23% of patients treated with the common aminoglycoside amikacin developed acute kidney disease, and more than 17% of amikacin-treated patients died before discharge. are doing. Other aminoglycosides, including gentamicin and tobramycin, also induced (or were associated with or contributed to) renal disease.

Over 1.2 million adult patients of various age groups were treated with aminoglycosides in 2010, as illustrated in Figures 13A and 13B. Other antimicrobial agents include, for example, penicillins, ampicillins, cephalosporins, sulfonamides, ciprofloxacin, vancomycin, macrolides, tetracyclines, rifampins, and the like.

Drug-induced nephrotoxicity can also be caused by one or more of the following: aspirin (acetylsalicylic acid), celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, tolmetin. It can result from treatment with non-steroidal anti-inflammatory drugs (NSAIDs).

Drug-induced nephrotoxicity can also be caused by one or more cyclooxygenase-2 (COX-2) inhibitors (e.g. valdecoxib, rofecoxib, celecoxib, etc.), proton pump inhibitors (e.g., omeprazole, lansoprazole, etc.), anticonvulsants ( phenytoin, valproic acid, etc.), histamine H2 receptor antagonists (e.g., nizatidine, ranitidine, famotidine, cimetidine, etc.), diuretics (e.g., carbonic anhydrase inhibitors, loop diuretics (e.g., bumetanide, ethacrynic acid, torasemide, furosemide, etc.), potassium-sparing diuretics (e.g., triamterene, spironolactone, amiloride, etc.), thiazide diuretics (e.g., indapamide, chlorthalidone, metolazone, methiclothiazide, hydrochlorothiazide, chlorothiazide, bendroflumethiazide, polythiazide, hydro Flumethiazide, etc.), or other diuretics such as pamabrom, mannitol, etc.

Drug-induced nephrotoxicity also results from treatment with lithium, which can affect the flux of sodium through nerve and muscle cells in the body, often resulting in hyperactivity, urgent speech, and poor judgment. , may be used to treat manic episodes of bipolar disorder characterized by reduced sleep desire, aggression, and anger. Lithium can also help prevent or reduce the intensity of manic episodes. Drug-induced nephrotoxicity can also result from treatment with or exposure to gold, mercury, copper, or other elemental substances.

Drug-induced nephrotoxicity is also associated with scleroderma, Wilson's disease (by accumulating and binding copper for removal through urine), cysteinuria (by binding cysteine to produce mixed disulfides that are more soluble than cysteine). (D-) penicillamine, relaxants acting directly on smooth muscle (e.g. hydralazine), antispasmodics (e.g. carisoprodol, cyclobenzaprine , metaxalone, methocarbamol, diazepam, benzodiazepines such as clonidine and other imidazoline compounds, tizanidine, baclofen, hydantoin derivatives, dantrolene, and the like.

Other forms of drug-induced nephrotoxicity include, for example, contrast agent-induced nephrotoxicity (e.g., after exposure to (iodinated) contrast agents, also known as radiocontrast-induced nephropathy (CIN)). narcotic (opioid)-induced nephrotoxicity (e.g., after use or abuse of certain narcotics (e.g., opioids) such as cocaine, heroin); chemotherapy-induced nephrotoxicity (e.g., cisplatin; carboplatin; oxaliplatin). alkylating agents such as bendamustine, cyclophosphamide, ifosfamide, nitrosourea, temozolomide, melphalan; antitumor antibiotics such as mitomycin C, bleomycin, anthracyclines, and related agents; capecitabine, hydroxyurea, methotrexate, pemetrexed , pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, cytarabine, and other antimetabolites; vinca alkaloid; topotecan; etoposide; taxane; irinotecan; lenalidomide; after treatment). In fact, a wide variety of nephrotoxic drugs can induce nephrotoxicity and lead to AKI. Drug-induced nephrotoxicity (and other forms of AKI) can be life-threatening if untreated and can result in enormous treatment costs (to patients, hospitals, and insurance companies).

Embodiments of the present disclosure may include methods of treating or preventing acute kidney injury (AKI), chronic kidney disease (CKD), or other conditions (e.g., prophylactically or in response to at least one event). . A method can comprise administering a composition of the present disclosure to a subject in need thereof. For example, a method requires an effective amount of a composition of the present disclosure (e.g., to raise and/or maintain a subject's serum soluble Klotho protein concentration above a predetermined threshold for a predetermined period of time). administering to a subject. The composition increases, enhances (i) a therapeutic recombinant Klotho protein or fragment or fusion protein thereof and/or (ii) endogenous Klotho protein production and/or level(s) in a patient to whom the product is administered. , augmenting, and/or supporting products, optionally with one or more additional ingredients or compositions.

In some embodiments, the condition is (i) acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, and/or nephrotoxicity, or (ii) sepsis, AKI resulting at least in part from kidney transplant or other surgery or medical procedure, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, nephrotoxicity, or hypotension can contain. Conditions can include drug-induced (eg, aminoglycoside-induced) nephrotoxicity. Proteins can be administered prophylactically, for example, prior to kidney transplantation, nephrotoxin administration, or other activities, treatments, or events known or expected to cause or contribute to AKI. . Alternatively, or in addition, the protein may be used for kidney transplantation or other surgery, aminoglycoside or other nephrotoxin administration, or other activities known or expected to cause or contribute to AKI. , treatment, or after an event, in response to AKI.

In some embodiments, the nephrotoxin or other drug is, for example, one or more aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, etc.), one or more antifungal agents (e.g., amphotericin B, flucytosine, etc.), one or more contrast agents (e.g., (iodinated) radiocontrast agents, hyperosmotic contrast agents (HOCM) with an iodine to molecule ratio of about 1.5:1, about 3:1 hypotonic non-ionic contrast media (LOCM) with an iodine to molecule ratio of about 6:1, iso-osmotic (iso-osmotic) contrast media (IOCM) with an iodine to molecule ratio of about 6:1), one antiretroviral agents (e.g., adefovir, cidofovir, tenofovir, foscarnet, etc.), one or more cancer (or chemotherapy) therapeutic agents (e.g., cisplatin, carboplatin, oxaliplatin, alkylating agents (e.g., bendamustine) , cyclophosphamide, ifosfamide, nitrosourea, temozolomide, melphalan, etc.), antitumor antibiotics (mitomycin C, bleomycin, anthracyclines, and related substances, etc.), antimetabolites (capecitabine, hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, cytarabine, etc.), vinca alkaloids, topotecan, etoposide, taxanes, irinotecan, lenalidomide, eribulin, arsenic trioxide, ixazomib, etc.), one or more bisphosphonates, or derivatives thereof (e.g., zoledronate/zoledronic acid, ibandronate, alendronate, alendronate/cholecalciferol, etidronate, risedronate (optionally with calcium carbonate), pamidronate, tiludronate, etc.) and/or cocaine, heroin, etc. It can be or include one or more narcotics (eg, opioids).

Embodiments of the disclosure may include methods of administering compositions of the disclosure. A method can comprise administering a composition of the present disclosure to a human or non-human subject to (prophylactically) treat or prevent AKI or one or more conditions associated with AKI. The method comprises determining the level of serum soluble klotho levels in a subject, administering a first dosage of a composition of the present disclosure sufficient to raise the serum soluble klotho levels in the subject to a predetermined level or percent of normal levels. administering a first dosage to the subject, such as by calculating, orally, bolus, or incremental administration; determining the rate of soluble klotho reduction in the serum of the subject, such as after administration of the first dosage calculating subsequent dosing times and amounts, and/or administering subsequent doses of the protein to the subject.

Administration of Compositions to Treat Chronic Kidney Disease (CKD) Without being bound by any theory, Klotho may be involved in human chronic kidney disease. Circulating soluble klotho begins to decline early in chronic kidney disease (CKD) stage 2, and urinary klotho begins to decline in CKD stage 1 probably earlier. Therefore, soluble Klotho may serve as an early and sensitive marker of renal decline. Furthermore, preclinical animal data support that Klotho deficiency is not just a biomarker, but an etiology for CKD progression and extrarenal CKD complications, including cardiovascular disease and mineral metabolism disturbances. Prevention of Klotho decline, reactivation of endogenous Klotho production, or exogenous Klotho supplementation all attenuate renal fibrosis, slow progression of CKD, improve mineral metabolism, ameliorate cardiomyopathy, and vascular calcification in CKD. can contribute to the mitigation of

CKD is characterized by progressive deterioration of renal function with a high risk of ESRD. CKD risk increases with age, occurring in approximately half of the cases of CKD stage 3 or higher in subjects aged 70 years and older. CKD can be considered a condition of accelerated aging. The relative risk of cardiovascular mortality in dialysis patients aged 25-34 years is similar to non-CKD patients aged 75 years and older. Cardiovascular disease is the leading cause of death in CKD and ESRD patients. CKD and ESRD patients have low renal Klotho expression and low levels of circulating Klotho. Renal Klotho deficiency in the early stages of CKD may be primarily due to suppression of Klotho expression rather than loss of viable renal tubules. In addition, some dialysis patients still have detectable circulating Klotho, which indicates that renal Klotho expression is not completely suppressed and whose origin has not previously been clarified, but is of extrarenal sources. suggesting that it may be derived from(s). It is of utmost importance to establish extrarenal sources of Klotho and to characterize how these can be upregulated when renal production fails.

Administration of the compositions of the present disclosure can help prevent, delay, and reduce the burden of comorbidities in CKD.
Administration of Compositions to Treat Bowel Diseases and Related Conditions Without being bound by any theory, intercalated Cajal cells (ICCs) are other cells in the gastrointestinal tract whose function is to control peristalsis. interstitial cells. The (main) function of the ICC is to create electrical signals that control rhythmic smooth muscle contractions in the intestine (different regions of the gastrointestinal system have different frequencies of contractions called peristalsis). ICC can induce migration of mesenchymal stem cells within the intestine (and many other organs).

In the intestine, ICCs are produced and/or activated by Klotho, which may lead to optimized regeneration processes. In Klotho knockout mice, the ICC is reduced, leading to impaired peristaltic processes, mimicking the intestinal disturbances that occur in aged humans.

Administration of the compositions of the present disclosure can help prevent, slow, and reduce disturbances in peristaltic processes and/or at least increase, support, and/or optimize regenerative processes in the intestine. These effects may further support gut health and/or prevent, delay, and reduce the incidence and/or severity of bowel disease and related conditions.

Therapeutic Treatment of Age-Related Conditions Investigators have discovered associations between Klotho and biological parameters that are generally accepted as indicators of clinical status in hospitalized elderly patients. Investigators genotyped the single nucleotide polymorphisms (SNPs) rs9536314, rs1207568, and rs564481 at the KL locus in 594 hospitalized elderly patients (ages 65-99) who attended the geriatric ward consecutively, Covariance and genetic risk score model analyzes were used to test the association of these KL variants with bioquantitative traits. A significant association of rs9536314 with serum levels of hemoglobin, albumin, and high-density lipoprotein cholesterol (HDL-C), and of rs564481 with serum levels of hemoglobin, fasting insulin, and fasting glucose was found. observed. Gender analysis confirmed these associations, and while the associations between KL genotype and HDL-C, fasting glucose, and fasting insulin levels may be driven by female gender, serum levels of hemoglobin and suggest that the association between An association between KL genotype and creatine levels was found in females, while an association between insulin-like growth factor-1 (IGF-1) and lymphocyte count (LC) was found in males. A genetic risk score (GRS) model further confirmed significant associations between KL SNPs and hemoglobin, total cholesterol, and HDL-C. Gender analysis using the GRS-tagged approach confirmed associations with HDL-C, fasting glucose levels, and fasting insulin levels in females, and hemoglobin and LC in males. This finding suggests that the KL locus can affect quantitative traits such as serum levels of lipids, fasting glucose, albumin, and hemoglobin in hospitalized elderly patients, with some gender differences in creatine, IGF-1 levels, and LC. has been suggested, thus suggesting that one of the genetic factors may contribute to age-related diseases and longevity.

Embodiments of the present disclosure provide compositions of the present disclosure to a subject (e.g., an individual or patient, optionally elderly, and/or age-related conditions, low endogenous S-Klotho protein expression) in need thereof. , and/or individuals or patients suffering from age-related conditions or symptoms of decreased longevity). Administration of the composition(s) of the present disclosure can lead to beneficial increases in blood s-Klotho levels. The administration reverses or inhibits the adverse effects of age-related conditions and/or in a positive therapeutic manner (e.g., in hospitalized and/or elderly patients) total cholesterol, HDL-C, fasting glucose, fasting Quantitative traits such as serum levels of insulin, albumin, creatine, IGF-1, hemoglobin, and lymphocyte counts can be affected.

Prophylactic Composition Administration In addition to the foregoing, embodiments of the present disclosure provide for administration of a composition of the present disclosure to an individual or subject in need thereof for prophylactic purposes and/or maintenance of particular health attributes. can include doing For example, administration of certain compositions of the present disclosure can help maintain youthfulness in any aging patient not yet suffering from a diagnosed age-related condition. Thus, while certain embodiments of the present disclosure may relate to and/or involve treatment of a condition in a patient, other embodiments may prevent, inhibit the development of, and treat one or more conditions. /or may relate to and/or include prophylactic measures.

Administration of Exogenous S-Klotho to Treat Genetic Defects Exemplary embodiments of the present disclosure relate to administration of exogenous S-Klotho to treat (eg, correct) known human genetic defects . For example, a 13-year-old girl with familial neoplastic calcification and the Klotho variant has been reported. Familial neoplastic calcification is an autosomal recessive metabolic disease characterized by ectopic calcification and hyperphosphatemia due to inactivating mutations in FGF23 or GALNT3. FGF23 is a hormone required for renal excretion of phosphate, while GALNT is an enzyme that contributes to the maturation and secretion of FGF23. A homozygous mutation in the Klotho gene has been identified in this 13-year-old girl. Klotho encodes a secreted protein that is required for transduction of the signal released by FGF23 towards its receptor. Administration of exogenous human recombinant S-Klotho constitutes a highly targeted and effective therapy to address the dysfunctions and symptoms associated with familial neoplastic calcification.

Compositions and Treatments Comprising S-Klotho and/or Supplements in Combination with Other Component(s) Alternate soluble alpha klotho (S-klotho) protein and natural supplement compositions that increase endogenous klotho production or one or more components thereof may be included. Klotho also has other compounds and/or constituents, whether endogenous or exogenous (recombinant), that affect one or more aspects of human health and well-being. can act in synergistic or synergistic substances. For example, a therapeutic human recombinant soluble alpha klotho (S-klotho) protein and/or a natural supplement composition that increases endogenous klotho production in combination with and/or concurrently with one or more additional active components. Treatments involving may benefit human patients. Such treatment may be prophylactic or responsive to any human condition in which Klotho protein and/or other component(s) may have a therapeutic effect. Such conditions can include, for example, age-related conditions, clinical (eg, metabolic) conditions, chronic or acute conditions, and the like. Certain non-limiting examples of certain conditions are disclosed herein.

S-Klotho may be present in the human body along with other blood-borne anti-aging compounds such as growth/differentiation factor 11 (GDF-11). Thus, in certain embodiments, therapeutic S-Klotho may be co-administered (eg, simultaneously, sequentially, and/or in combination) with therapeutic GDF-11. In some embodiments, such administration may have additive or synergistic anti-aging or other effects. Similarly, co-administration of S-Klotho to human subjects with (neutralizing) antibodies to CCL11, or inhibitors thereof, may act in concert to combat aging or other disease states (CCL11 (eotaxin -1) is understood to be a negative regulator of stem cell rejuvenation). S-Klotho may also, or alternatively, be co-administered with other eotaxins such as eotaxin-2 (CCL24) and/or eotaxin-3 (CCL26).

In some embodiments, S-Klotho may be co-administered with an inhibitor or antibody of transforming growth factor beta-1 (TGF-β1). S-Klotho administration can counteract the effects of the TGF-β1 signaling pathway, which is involved in endogenous anti-cell epithelial-mesenchymal transition (anti-EMT) leading to renal fibrosis and fibrosis of other tissues. Anti-EMT is also associated with cancer cells, and it is understood that inhibition of EMT may confer metastatic potential on cancer cells, and this latter process is countered by Klotho. Therefore, co-administration of S-Klotho with inhibitors or antibodies of transforming growth factor beta-1 (TGF-β1) may have synergistic or additive effects.

In some embodiments, S-Klotho may be co-administered with an antibody or inhibitor of insulin growth factor-1 (IGF-1). Klotho is a hormone that inhibits the intracellular insulin/IGF-1 signaling cascade, and this inhibition increases resistance to oxidative stress at the cellular and organismal level in mammals and has been evolved to extend lifespan. It is understood to be a mechanism that is considered conserved. Therefore, co-administration of S-Klotho with inhibitors or antibodies of insulin growth factor-1 (IGF-1) may have synergistic or additive effects.

Numerous studies have demonstrated the mutually regulated positive/negative feedback effects of Klotho alpha-K1, FGF23, and 1,25(OH) ₂ D, and/or Klotho β-K1, regulating bile acid/cholesterol metabolism. Since a comprehensive regulatory scheme of mineral homeostasis has been revealed involving a similar regulatory network composed of FGF15/human FGF19, and bile acids, in some embodiments S-Klotho may be added to vitamin D (e.g., vitamin D3), or in combination with 1,25-dihydroxyvitamin D ₃ [1,25(OH) ₂ D ₃ ], FGF-15, FGF-19, and/or Klotho-β. Such co-administration can have synergistic or additive effects on numerous disease states and/or processes in the body. In some embodiments, S-Klotho may be administered in combination with FGF-21
In some embodiments, S-Klotho may be co-administered with a carbonic anhydrase inhibitor such as acetazolamide, methazolamide, dichlorphenamide, dorzolamide, brinzolamide, and/or topiramate. Such combinatorial administration can be useful in treating ankylosing spondylitis (AS), rheumatoid arthritis (RA), and various other conditions. Various studies have shown that increased bone resorption is a hallmark of AS and RA, and that carbonic anhydrase inhibitors play an anti-arthritic role by inhibiting bone resorption. At the bone level, S-Klotho stimulates bone resorption and phosphate release through distinct mechanisms by acting on TRPV5, a recently identified regulator of osteoclast function. Elevated levels of 1,25(OH) ₂ D ₃ caused by S-Klotho administration also stimulate osteoclast differentiation and bone resorption, thereby releasing phosphate. Therefore, co-administration of S-Klotho with a carbonic anhydrase inhibitor may have additive or synergistic effects, particularly in AS and RA, particularly in promoting bone health.

For the treatment of severe active rheumatoid arthritis, S-Klotho may be administered in combination with one or more disease-modifying antirheumatic drugs (DMARDs).
S-Klotho may be administered in combination with cyclosporine because cyclosporine decreases klotho mRNA and protein and increases oxidative stress leading to cyclosporin-induced renal injury (CsA). Associated decreases in Klotho mRNA and protein and increases in oxidative stress can be counteracted by exogenous co-administration of S-Klotho.

In some embodiments, S-Klotho may be co-administered with losartan and/or cyclosporine. Treatment with the angiotensin type II 1 (AT1) receptor blocker losartan reversed the decrease in Klotho expression seen with cyclosporine. Losartan also produced concomitant improvement in renal tissue (with losartan reducing tubulointerstitial fibrosis caused by cyclosporine).

In some embodiments, S-Klotho may be co-administered with one or more aminoglycosides such as amikacin, gentamicin, tobramycin. When aminoglycosides are used to treat infections with (Gram-negative) pathogens, which could greatly expand the use of aminoglycosides to treat infections, such treatment may result in nephrotoxicity and/or acute kidney injury (AKI). ) can be useful in preventing Administering S-Klotho with verapamil and/or diltiazem, which have been used to block AKI, may be therapeutic in treating and/or preventing renal impairment from AKI.

In some embodiments, S-Klotho may be co-administered with a testosterone or androgen receptor (AR) upregulating compound. Recent reports indicate that no beneficial effects of testosterone treatment in men on personality, psychological well-being, or mood are observed. In addition, prescribing testosterone supplementation for low T as cardiovascular health, sexual function, physical function, mood, or cognitive function was not seen as supported by randomized clinical trials. However, while testosterone supplementation was consistently found to increase muscle strength, no beneficial effects on physical function were found. S-Klotho administration in combination with testosterone and/or androgen receptor (AR) upregulating compounds exceeds any effect that testosterone or S-Klotho may have on these treatment groups alone, in the elderly, It can significantly increase muscle strength and/or physical function in frail or low T men.

In some embodiments, S-Klotho may be co-administered with estrogen or estrogenic hormones (eg, estradiol, estriol, estrone, etc.). Such co-administration can improve female health indicators (e.g., menstruation, menopause, or women transitioning to menopause) and/or infertility, polycystic ovary disease or disorder, obesity, hormonal imbalance, Balance and related medical conditions and/or other female health conditions may be treated.

In some embodiments, S-Klotho may be co-administered with one or more nootropics, also called smart drugs or cognitive enhancers. Nootropics, supplements, and/or other substances may improve cognitive function, particularly executive performance, memory, creativity, motivation, task saliency (motivation to perform tasks), (especially high effort). It can improve performance (required laborious tasks) and can be useful in treating cognitive or motor impairments that contribute to disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and ADHD. The most commonly used class of drugs known to improve some aspect of cognition are stimulants, particularly dopamine receptor D1, adrenergic receptor A2, or both, in the prefrontal cortex. It is a class of stimulants that exhibit cognitive-enhancing effects in humans by acting as direct or indirect agonists of their receptors. Stimulants include, for example, amphetamines (e.g., amphetamine, dextroamphetamine, lisdexamphetamine), dimethylamylamines (DMAA) such as 1,3-dimethylamylamine, which can improve physical performance, alertness, reaction time, etc., methylphenidate—a range of cognitive functions (e.g., working memory, Substituted phenethylamines that can improve episodic memory and inhibitory control, attentional aspects, and planning latency, especially in sleep-deprived individuals, increasing alertness and facilitating reasoning and problem-solving; eugeroics (e.g., armodafinil, modafinil, etc.) that can function as wakefulness-promoting agents that can treat narcolepsy, shift-work sleep disorders, and daytime sleepiness that remains after treatment for sleep apnea, etc.; Xanthines (eg, caffeine, etc.), nicotine, etc., which may enhance alertness, performance, and/or memory.

In some embodiments, S-Klotho may be co-administered with one or more osteoporosis and/or osteopenia drugs, as known in the art. Klotho may play a role in regulating bone mineral density, and the absence of Klotho can lead to reduced bone mineral density in animals. For example, Klotho knockout mice exhibit decreased bone mineral content over time. Klotho expression can rescue bone defects such as reduced bone mineral content that Klotho knockout mice exhibit over time. Epidemiologic studies have shown associations between various Klotho gene variants and alterations in bone mineral content and prevalence of osteoarthritis of the hands.

S-Klotho may be administered in combination with one or more anticancer treatments and/or preventions, such as chemotherapy. In lung cancers such as non-small cell lung cancer (NSCLC), for example, S-Klotho administration can affect resistance of lung cancer cells to cisplatin and/or other chemotherapy. In addition, S-Klotho may function as a potential tumor suppressor in lung cancer, gastric cancer, pancreatic cancer (adenocarcinoma), and other forms of cancer. S-Klotho may be administered in combination with sorafenib chemotherapy for the treatment of hepatocellular carcinoma (HCC). Overexpression of Klotho and treatment with soluble Klotho protein can reduce hepatoma cell growth in vitro and in vivo. Other cancer types that can be treated with S-Klotho co-administration include hepatocellular carcinoma (HCC), central nervous system (CNS) cancers such as brain (eg, glioma, craniopharyngioma, medulloblastoma, and meningioma), spinal cord and other tumors, lymphoma, etc.), (metastatic) colon cancer, etc.

S-Klotho can also be administered in combination with chemotherapeutic agent(s) to treat chemotherapy-induced frailty in cancer patients. S-Klotho can also be administered after other known treatments to treat cancer-induced weakness in cancer patients.

S-Klotho may be administered in combination with renal dialysis or other procedures. Since frailty is associated with poor prognosis in dialysis patients, S-Klotho can be administered to treat frailty in dialysis patients.

S-Klotho may be used in combination with one or more Alzheimer's disease treatments or prevention, or in combination with brain-derived neurotrophic factor (BDNF), because adequate amounts of BDNF can help develop and maintain normal neuronal circuits in the brain ) in combination with

S-Klotho increases the ability of Klotho to cross the blood-brain barrier to increase the ability of Klotho to enter the central nervous system (CNS) to treat or prevent CNS-related conditions. It can be administered in combination with molecules. For example, neither S-Klotho nor BDNF are known to cross the blood-brain barrier. Embodiments of the present disclosure administer S-Klotho and/or S-Klotho with BDNF to the CNS to treat Alzheimer's disease and/or to improve cognition in individuals not suffering from Alzheimer's disease and utilizing blood-brain barrier delivery technology to.

S-Klotho positively regulates 5′ adenosine monophosphate-activated protein kinase (AMPK) or AMPK-activating drugs, or signaling pathways that replenish the cellular ATP supply, including fatty acid oxidation and autophagy, or sugars. It can be administered in combination with components that negatively regulate ATP-consuming biosynthetic processes, including neogenesis, lipid, and protein synthesis.

S-Klotho can be administered in combination with one or more antidiabetic agents, such as insulin, phlorizin, or the antioxidant tiron, and these combined treatments are beneficial in preventing renal damage due to oxidative stress that occurs in diabetic disorders. can have Co-administration of S-Klotho with other antidiabetic drugs for type 1 diabetes inhibits β-cell apoptosis through activation of the integrin β1-FAK/Akt pathway, leading to inhibition of caspase 3 cleavage, thereby inhibiting β- - can protect cells;

S-Klotho can be administered in combination with one or more type 2 antidiabetic agents, such as metformin, to improve glycemic control and vascular function in overweight and obese diabetic subjects.
S-Klotho may be administered in combination with one or more blood pressure medications, calcium control agents, or treatment or prevention of chronic kidney disease (CKD). For example, soft tissue calcification is a hallmark of CKD, and Klotho ameliorates vascular calcification by enhancing phosphaturia, maintaining glomerular filtration rate, and directly inhibiting phosphate uptake by vascular smooth muscle. obtain.

S-Klotho is thought to extend the lifespan of Klotho-deficient (kl/kl) mice while slowing the aging process and preserving organ structure and function. of PAI-1 inhibitors (plasminogen activator 1).

S-Klotho can be administered in combination with Sirtuin 1 (SIRT1) or a SIRT1 activating compound (STAC) such as resveratrol. SIRT1, a type III protein deacetylase, is believed to be a novel anti-aging protein involved in the regulation of cellular senescence/aging and inflammation. SIRT1 levels and activity decrease during lung inflammation caused by oxidative stress. SIRT1-mediated protective mechanisms against inflammation are associated with regulation of inflammation, premature aging, telomere shortening, secretory phenotypes associated with aging, and the DNA damage response. Various dietary polyphenols and pharmacological activators have been shown to regulate SIRT1 to intervene in the progression of chronic obstructive pulmonary disease associated with type 2 diabetes, cancer, cardiovascular disease, and inflammation. Therefore, some or all of the health benefits of SIRT-1 may be augmented by co-administration of SIRT1 and/or SIRTI-activating compounds administered with S-Klotho.

S-Klotho can be administered in combination with one or more FDA-approved human cell, tissue, and cell and tissue-based products (HCT/P). Such products include, for example, bone (including demineralized bone, ligaments, tendons, fascia, cartilage), ocular tissue (cornea and sclera), skin, vascular grafts (veins and arteries), optionally ( or otherwise) preserved umbilical vein, pericardium, amniotic membrane (when used alone (no cells added) for eye repair), dura mater, heart valve allograft, peripheral blood or umbilical cord One or more of blood, semen, oocytes, or embryo-derived hematopoietic stem cells may be included. In at least one embodiment, the HCT/P can be or include one or more stem cells. Stem cell therapy of damaged body tissues and organs is becoming popular. Administration of therapeutic recombinant Klotho protein in combination with stem cells has provided surprising, unexpected and even synergistic results for subjects in need thereof.

Embodiments of the present disclosure further include combination products comprising therapeutic recombinant Klotho protein in combination with human stem cells. Compositions can also include a pharmaceutically acceptable carrier as described herein. Such compositions may be included or classified as regenerative medicines approved by the FDA for the treatment, modification, reversal, or cure of serious or life-threatening diseases or conditions. . Preliminary clinical evidence indicates that the composition (drug) has the potential to address a medical need not matched to such disease or condition.

Illustratively, stem cells can be or include mesenchymal stem cells (MSCs), such as from human umbilical cord or placenta. In at least one embodiment, compositions comprising huMSCs and therapeutic recombinant Klotho proteins of the present disclosure attenuate the inflammatory and oxidative stress responses that occur in AKI and/or decrease the expression of proteins and microRNAs associated with aging. reduced.

S-Klotho can be administered in combination with one or more anti-aging agents. For example, Klotho protein may be combined with Pin1-FOXM1 and/or other anti-aging agents to enhance results in patients undergoing such treatment.

S-Klotho is a Klotho stimulant (e.g. Vitamin D, Losartan, Testosterone), GDF-11, Trichostatin A antifungal (GDF-11 stimulator), TIMP-2, CCL-11 inhibitor/antibody, Dasatinib , nicotinamide riboside (e.g. NAD+), nicotinamide mononucleotide (NMN) (e.g. NDA+), AMPK stimulants (e.g. resveratrol, aspirin, salicylates, phytochemicals, DR), C60 fullerene, rapamycin, FGF It may be administered in combination with one or more of inhibitors, senolytic agents/compounds such as FOXO4-p53 interfering peptides (eg, FOXO4-DRI), inhibitors of the anti-apoptotic proteins BCL-2 and BCL-xL.

Any of the foregoing or other treatments or co-administration may have additive or synergistic effects over either treatment alone. For example, co-administration of a Klotho protein with one or more of the foregoing may produce therapeutic results greater than the sum of the individual results of administering the individual components at similar concentrations. In addition, synergistic effects can include therapeutic results similar to those of the individual results of administering the single components at lower concentrations. A synergistic effect can also be an increase in the maximal effective dosage of one or more of the components, a reduction in toxicity of one or more of the components, or any more than merely additive effects of the individual therapeutic outcomes. It may contain other beneficial results. Additionally, the additive effect of individual therapeutic outcomes can include one or more synergistic effects. Such additive/synergistic effects may not be expected or expected given the nature and understanding of the individual components.

As used herein, combination therapy or co-administration can include treatment or administration of a combination product, composition, or formulation comprising the Klotho protein and one or more additional active ingredients. The one or more additional active ingredients may be selected from among the components, drugs, substances, therapeutic compositions, etc. described herein or others known in the art. For example, the Klotho protein and one or more additional active ingredients may be administered in an injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulation. can be co-formulated in

Alternatively, combination therapy or co-administration may involve combining the Klotho protein and one or more additional active ingredients into a combination product, composition, or formulation, or Treatment or administration of these additional active ingredients may be included. For example, the Klotho protein and one or more additional active ingredients may be separately injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other or may be in such a formulation.

It is also understood that co-administration may comprise simultaneous administration of two or more components, or separate administration of two or more components, the separate administrations preferably being separated by a period of time. deaf. In some embodiments, the period of time may be very short. For example, administration of a first component of Klotho protein and one or more additional active ingredients, followed by administration of a second component of Klotho protein and one or more additional active ingredients substantially immediately after administration (e.g., injection). Alternatively, the first and second administrations are 1-60 seconds, 1-60 minutes, 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, etc., or any value or interval between may be separated by a period of time, such as a range of values for . Similarly, co-administration can involve overlapping dosing windows of two or more components.

Therapeutic treatment of hyperphosphatemic familial neoplastic calcification (HFTC) In affected human individuals, HFTC is transformed from histidine (H) at amino acid (AA) position 193 of S-Klotho-rs121908423. Caused by mutations to arginine (R). Without being bound by any theory, it is believed that the H193R mutation in HFTC individuals impairs the ability of S-Klotho to form a ternary complex with FGF23 and FGFR1c, which impairs KL-dependent FGF23 signaling. It is As a result, afflicted subjects exhibit a severe metabolic disorder manifested by hyperphosphatemia and massive calcium deposits in the skin and subcutaneous tissue. Some patients present with recurrent, transient, painful swelling of long bones associated with periosteal reaction and radiological findings of cortical osteophysis and absence of skin involvement.

One embodiment of the disclosure includes an S-Klotho protein with H193. H193 proteins can be produced or expressed in the context of any of the protein constructs described herein. For example, H193 variants may be S-Klotho, isoforms 1 or 2, 1-981, 29-981, 34 with or without a tag (eg, Fc tag, TEV-Twin-Strep tag, TEV sequence, etc.). ~981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be in the corresponding configuration.

Embodiments include producing H193 heterozygous or homozygous variant constructs, transfecting the resulting constructs encoding the S-Klotho H193 protein (eg, via transfection) into an appropriate expression system (eg, CHO cells) and/or transiently expressing the S-Klotho H193 protein. The H193 Klotho protein can also be expressed at higher levels than the R193 (or H193R) protein. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments provide a therapeutic or therapeutically effective amount of S-Klotho H193 protein to a subject in need thereof (e.g., an HFTC individual, an individual diagnosed with HFTC, or carrying H193R (rs121908423) or other variant patient). Alternatively, the subject may be of other mutant or variant wild type. Administration of recombinant S-Klotho H193 protein can lead to beneficial increases in blood S-Klotho levels. Administration can reverse or suppress the deleterious effects of the R193 variant that is transcribed and circulated in HFTC individuals as a result of the H- to -R193 point mutation found in the human Klotho gene in individuals with HFTC. Therefore, circulating concentrations of S-Klotho H193 may help counteract the effects observed in H193R or HFTC individuals.

Therapeutic treatment in CC genotype patients with end-stage renal disease (ESRD) Approximately 350,000 patients with end-stage renal disease (ESRD) suffer a very high mortality rate in their first year of chronic hemodialysis. I'm in. Both vitamin D and fibroblast growth factor (FGF)-23 levels correlate with survival in these patients. Without being bound by any theory, Klotho is a protein in the vitamin D/FGF-23 signaling pathway that has been associated with accelerated aging and premature death in animal models. It has been hypothesized that genetic diversity in the Klotho gene may be associated with survival of subjects with ESRD. The researchers found an association between 12 single nucleotide polymorphisms (SNPs) in the Klotho gene and mortality in a cohort of ESRD patients (n=1307, Caucasian and Asian) during the first year of hemodialysis. was tested. One tagging SNP, the CC genotype at rs577912 (a common HapMap variant with a minor allele frequency MAF within the Klotho gene sequence located in intron 1)>0.05, and an increased risk of 1-year mortality ( A significant association was found between RR, 1.76; 95% CI, 1.19-2.59; p=0.003). Among patients who were not treated with active vitamin D supplements (HR, 2.51; 95% CI, 1.18-5.34; p=0.005), this increase in individuals with the CC genotype The effect was even more pronounced. In lymphoblastoid cell lines from HapMap subjects, the CC genotype was associated with 16-21% lower Klotho expression compared to the AA or AC genotypes. However, none of the rs577912 SNP nucleotide changes described above result in amino acid changes in the Klotho protein. Therefore, this functional SNP (rs577912) can quantitatively affect Klotho gene expression at the mRNA level.

One embodiment of the present disclosure includes S-Klotho protein expressed from AA or AC genotypes. Proteins can be produced or expressed in the context of any of the protein constructs described herein. For example, the protein may be S-Klotho, isoform 1 or 2 tagged or untagged 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34- 549, 36-549, 131-549, etc. (eg, Fc tag, TEV-Twin-Strep tag, TEV sequences, etc.). Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

Embodiments include producing AA or AC heterozygous or homozygous constructs, transfecting the resulting constructs encoding S-Klotho protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells). ) and/or transiently expressing the S-Klotho protein. Klotho protein expressed in AA or AC heterozygous or homozygous cells may be expressed at higher levels than in CC cells. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments provide a therapeutic or therapeutically effective amount of S-Klotho protein to a subject in need thereof (e.g., low endogenous S-Klotho protein expression, carrying a CC mutation of one tagging SNP, rs577912, and /or to an individual or patient with end stage renal disease (ESRD). Alternatively, the subject may be of other mutant or variant wild type. Administration of recombinant S-Klotho protein can lead to beneficial increases in blood S-Klotho levels. Administration can reverse or suppress the deleterious effects of CC mutations that are transcribed and circulated in the individual as a result of the point mutation(s) found in the human Klotho gene of the affected individual. Thus, the circulating concentration of S-Klotho is associated with the effects observed in CC individuals, particularly those with end-stage renal disease (ESRD), i. can help combat

Therapeutic Treatment of Radiographic Hand Osteoarthritis (OA) and Osteophytes Osteoarthritis (OA) is a common and complex disease with a strong hereditary component. Researchers tested the association between four putative functional genetic variants of the Klotho gene and OA of the hands in a large population of Caucasian women. Investigators found a significant association between SNP G-395A and the presence/absence of radiographic hand OA and osteophyte formation. Allele G was 1.44 (P=0.008, 95% confidence interval (CI) 1.09-1.91) and 1.36 (P=0.006, 95% CI 1.09-1.91), respectively. 70) significantly increased the risk of radiographic hand OA and osteophytes. From logistic regression modeling, genotype GG was associated with radiographic hand OA (OR=3.10, 95% CI 1.10-8.76) and osteophyte (OR=3.10) when compared with genotype AA. , 95% CI 1.10-8.75) showed a more than 3-fold increase in both risks. After adjusting for age, the OR for genotype GG was 4.39 (P=0.006, 95% CI 1.51-12.74) for radiographic hand OA and 4.47 for osteophyte (P=0. 005, 95% CI 1.56-12.77). The researchers also suggested that one variant in the Klotho gene (SNP G-395A) is associated with susceptibility to OA of the hand and appears to act through osteophyte formation rather than cartilage damage.

One embodiment of the present disclosure includes S-Klotho protein expressed from a construct with SNP G395A. The resulting protein can be produced or expressed in the context of any of the protein constructs described herein. For example, A395 variants may be S-Klotho, isoforms 1 or 2, 1-981, 29-981, 34 with or without tags (eg, Fc-tag, TEV-Twin-Strep-tag, TEV sequences, etc.) ~981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

Embodiments include producing a G395A heterozygous or homozygous construct, transferring the resulting construct encoding the S-Klotho protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells). introducing and/or transiently expressing the S-Klotho protein. Klotho protein expressed in A395 heterozygous or homozygous cells may be expressed at higher levels than in G396 cells. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments provide a therapeutic or therapeutically effective amount of S-Klotho protein to a subject in need thereof (e.g., the G395 SNP, and/or radiographic osteoarthritis (OA) of the hand and/or osteophyte). administration to individuals or patients carrying (at risk of developing them). Alternatively, the subject may be of other mutant or variant wild type. Administration of recombinant S-Klotho protein can lead to beneficial increases in blood S-Klotho levels. Administration may reverse or suppress the adverse effects of the G395 SNP that is transcribed and circulated in afflicted individuals. Thus, circulating concentrations of S-Klotho may help counter the effects observed in G395 individuals, particularly those at risk of developing radiographic hand osteoarthritis (OA) and/or osteophytes. . Thus, administration of the G-395A S-Klotho protein may reduce the risk of radiographic hand osteoarthritis (OA) and osteophyte formation in patients (eg, patients carrying the G395 SNP).

Therapeutic Treatment of Metabolic Syndrome The risk and/or incidence of metabolic syndrome (MetS), a cluster of cardiometabolic risk factors including abdominal obesity, hyperglycemia, dyslipidemia, and hypertension, increases with age. In older adults, MetS not only increases the risk of cardiovascular disease and type 2 diabetes, but is also associated with cognitive decline and disability. Current evidence suggests that MetS is partly heritable and that genetic factors play a greater role than environmental factors on the incidence of MetS. Researchers have discovered an association between the G-395A polymorphism and metabolic syndrome (MetS) in the Chinese 90s and 100s population. Subjects were from the Longevity and Aging Project of Dujiangyan City (PLAD). Genotyping of G-395A (rs1207568) in the promoter region of the Klotho gene was performed using the TaqMan allelic discrimination assay. MetS was diagnosed according to International Diabetes Federation criteria. 695 subjects aged 93.5±3.2 years were included. The allele frequencies of G and A were 0.852 and 0.148, respectively. In the population as a whole, the frequency of MetS was 10.8% and 5.9% in the GG and GA+AA genotype groups, respectively (p=0.004). −395A allele carriers in the overall population (odds ratio [OR] 0.50, 95% confidence interval [CI] 0.25-0.98) and in women (OR 0.51, 95% CI 0.24 ∼0.97), but in males (OR 0.42, 95% CI 0.05-3.85). Across the population and in women, the relationship between the Klotho G-395A SNP and MetS was associated with hypertension (OR 0.48, 95% CI 0.34-0.67; OR 0.47, 95% CI 0.31-0.67, respectively). 71), and possibly due to its effect on hypertriglyceridemia (OR 0.66, 95% CI 0.39-0.95; OR 0.54, 95% CI 0.31-0.98, respectively). In men, this relationship may be due to its effect on hypertension (OR 0.47, 95% CI 0.25-0.90) and low HDL-C (OR 0.69, 95% CI 0.27-0.93) was there. The researchers concluded that carriers of the -395A allele of the Klotho gene correlated with a lower risk of MetS during the 90s and 100s in China, especially among women.

One embodiment of the present disclosure includes S-Klotho protein expressed from a construct having the -395A allele. The resulting protein can be produced or expressed in the context of any of the protein constructs described herein. For example, the A395 allele, with or without a tag (eg, Fc tag, TEV-Twin-Strep tag, TEV sequences, etc.), S-Klotho, isoform 1 or 2, 1-981, 29-981, 34 ~981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

An embodiment is to produce a -395A heterozygous or homozygous construct, inserting the resulting construct encoding the S-Klotho protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells). and/or transiently expressing the S-Klotho protein. Klotho protein expressed in A395 heterozygous or homozygous cells may be expressed at higher levels than in G396 cells. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments provide a therapeutic or therapeutically effective amount of S-Klotho protein to a subject in need thereof (e.g., the G395 SNP, and/or having (at risk of developing) metabolic syndrome (MetS) Administration of recombinant S-Klotho protein may comprise administering to an individual or patient.Alternatively, the subject may be of other mutants or variants of wild-type.Administration of recombinant S-Klotho protein may increase blood S-Klotho levels Administration can reverse or suppress the adverse effects of G395 SNPs that are transcribed and circulated in afflicted individuals.Thus, circulating concentrations of S-Klotho are associated with G395 individuals, particularly those with metabolic syndrome ( MetS), administration of the G-395A S-Klotho protein may help counteract the effects observed in individuals at risk of developing G395 SNP. female patients) risk of metabolic syndrome (MetS).

Therapeutic Treatment of Cancer S-Klotho is thought to inhibit basal Wnt signaling activity, thereby functioning as a tumor suppressor in colorectal cancer (CRC). In addition, Klotho gene variants associated with lifespan differences may suppress butyrate-mediated Wnt hyperactivation, thus increasing the risk of CRC. In this manner, it has been hypothesized that the type of Klotho variant present and its relative expression interact with dietary-derived butyrate levels and may modify CRC risk. In addition, mTOR signaling is also associated with human aging, and cross-talk between Wnt and mTOR signaling may affect colon tumorigenesis.

KL-VS variants or other constructs may be used to determine which SNPs (eg, within KL-VS) affect S-Klotho to reduce basal Wnt signaling and/or butyrate-mediated Wnt hyperactivity. It can serve as a vehicle to study whether S-Klotho is involved in the generation of suppression of cytotoxicity, the latter activity associated with Wnts associated with S-Klotho tumor suppression. Embodiments include modifying appropriate amino acids (eg, KL-VS stretch of S-Klotho) that have been shown to affect the tumor suppressor action of KL-VS variants. One embodiment of the present disclosure includes a recombinant S-Klotho protein with one or more amino acid alterations in the KL-VS stretch of 6 SNPs. Proteins can be produced and/or expressed in the context of any of the protein constructs described herein. For example, proteins may be S-Klotho, isoforms 1 or 2, 1-981, 29-981, 34-981, with or without tags (eg, Fc-tag, TEV-Twin-Strep-tag, TEV sequences, etc.). , 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

An embodiment is to produce a heterozygous or homozygous variant construct, inserting the resulting construct encoding the S-Klotho protein (eg, via transfection) into a suitable expression system (eg, CHO cells). and/or transiently expressing the S-Klotho protein. Klotho proteins can be expressed at higher levels than other Klotho proteins, including wild-type. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments administer a therapeutic or therapeutically effective amount of S-Klotho protein to a subject in need thereof (e.g., a patient having or at risk of developing colorectal cancer (CRC) or another tumor) can include A subject may, for example, carry or express a Klotho variant with reduced Wnt inhibitory activity. Alternatively, the subject may be of other mutant or variant wild type. Administration of recombinant S-Klotho protein can lead to beneficial increases in blood S-Klotho levels.

Therapeutic Treatment of Non-Human Mammals In addition to the above, one or more non-human mammalian Klotho proteins or protein sequences (or portion(s) thereof) are useful in practicing certain embodiments of the present disclosure. obtain. SEQ ID NOs: 121-124 represent DNA sequences for encoding various non-human mammalian Klotho proteins. SEQ ID NOS: 111-120 represent amino acid sequences of various non-human mammals. These Klotho protein sequences (or portion(s) thereof) can be expressed, purified, formulated into compositions, and/or administered to animals in the same manner as described above. Therapeutic administration of recombinant Klotho (fusion) proteins can be effective in treating non-human mammalian versions of the medical and other conditions outlined herein. Specifically, embodiments of the present disclosure may include veterinary therapeutic methods, proteins, and compositions, as understood by those skilled in the art.

Example 1
Table 48 illustrates the results of transient expression and purification of the listed Klotho variants in HEK and/or CHO cell lines. For the results provided in Table 48 below, the following simplified protocol was followed.

For Fc fusion proteins, the protein expression vector was transformed into HEK293. sus or CHO. Briefly, cells were grown for 7 days and harvested. Cell numbers are given in the notes column. Supernatant pH was adjusted with 1M Hepes pH 7.4 and sodium azide was added. Proteins were captured using KanCap A resin. The resin was washed with PBS. The resin was added to PBS and washed with 1M NaCl. The resin was washed with PBS. Proteins were eluted with 50 mM citrate pH 3.5, 100 mM NaCl. Proteins were immediately neutralized with 1 M Tris pH 8, 0.5 M Arginine. SDS PAGE gel samples were removed at this stage. Proteins were buffer exchanged into PBS. Proteins were quantified by OD280 and calculated extinction coefficients were used to determine quantity and concentration. Purity and molecular weight were determined using reducing and non-reducing SDS-PAGE (Biorad standard Tris/glycine/SDS, 4-20%). Aggregation status was determined by HPLC with detection at 280 nm using a Sepax Zenix-C SEC-300, 3 um, 300 A, 4.6 x 150 mm size exclusion column and PBS buffer run. After filter sterilization and quick freezing in liquid nitrogen, proteins were shipped in aliquots. Protein reduction was observed for some Fc proteins during desalting into PBS as judged from samples run on SDS-PAGE before and after purification. These proteins were expressed from HPLC but encountered problems during desalting or assay on silica HPLC columns in PBS. Therefore, buffer selection was optimized to reduce protein loss.

For Strep-tagged proteins, the protein expression vector was transformed into HEK293. sus or CHO. Briefly, cells were grown for 7 days and harvested. Supernatant pH was adjusted with 1M Hepes pH 7.4 and sodium azide was added. BioLock Biotin Sequestration Reagent was added. Proteins were captured using StrepTactin superflow resin. The resin was washed with 100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA. Proteins were eluted with 100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, and 2.5 mM desthiobin. Proteins were quantified by OD280 and calculated extinction coefficients were used to determine quantity and concentration. Purity and molecular weight were determined using reducing and non-reducing SDS-PAGE (Biorad standard Tris/glycine/SDS, 4-20%). Aggregation status was determined by HPLC with detection at 280 nm using a Sepax Zenix-C SEC-300, 3 um, 300 A, 4.6 x 150 mm size exclusion column and PBS buffer run. After filter sterilization and quick freezing in liquid nitrogen, proteins were shipped in aliquots. Note that for Strep-tagged proteins, samples were assayed in elution buffer. This elution buffer is very "neutral" and non-toxic to proteins. Also, since the running buffer of the SEC column was PBS, the protein underwent a buffer exchange during the assay. Absorbances at times greater than 7 minutes are small molecules.

In certain embodiments, EDTA and/or other metal chelator(s) were removed from (or not included in) one or more (eg, all) of the purification steps and/or buffers. . In some embodiments, EDTA and/or other metal chelator(s) may contribute to or cause inactivation of Klotho protein.

In a follow-up study, transiently expressed Fc-Klotho protein was analyzed on an Aquity UPLC BEH200 SEC analytical column: 1.7 μm, 4.6×150 mm; mobile phase: 100 mM sodium phosphate, 200 mM sodium chloride, pH 6.5. Run time: 9 minutes; Detection: A280, MALS&RI detector; ambient sample compartment, 30C column compartment; 100 μL sample injection (37 μg) was used for purification. FIG. 14 illustrates the chromatograph of Klotho-Fc protein. Most of the Fc Klotho protein appears to overlap the first peak of the MW marker near the void volume. This corresponds to the very high molecular weight seen in the main peak (in megadaltons), much higher than native klotho. By SDS-PAGE (non-reducing) the peak (fraction) contained monomeric sized protein, suggesting non-covalent binding in solution. A small shoulder is observed at the tail of the main Klotho peak (labeled with a dashed oval), which, compared to the MW marker, indicates a larger MW than IgG due to elution time on the SEC column and ~700 kDa due to light scattering. (LS signals are very low and may be overestimated due to trailing aggregates), less than 1% of the total Klotho peak area. This peak corresponds to multimers, possibly dimers or tetramers.

Various alternative purification schemes involving different columns, buffers, conditions, etc. were used to address the problem of protein aggregation and multimerization. However, in at least one embodiment, multimeric (dimeric, tetrameric, etc.) Klotho proteins can be purified.

Example 2
_{SEQ . _} S) ₂ Linker_Human IgG1 Fc-C′), and stable binding of the (human) Klotho-derived protein represented by SEQ ID NO: 66 (N′-Human Alpha Klotho 34-981 Isoform 1_Twin-Strep cleavage site residues) Expression was performed in CHO cells. Each of the three Klotho protein constructs was expressed with a non-naturally occurring N-terminal signal sequence, which was subsequently cleaved during the stable expression/purification process to obtain the N-terminal (Klotho protein) amino acids. At the C-terminus of the Klotho protein sequences of SEQ ID NOs:52 and 54 is a GS linker (GGGGSGGGGS) and an Fc fusion tag (human IgG1 Fc domain), which are (or appear to be) retained in the expressed protein. is). The C-terminus of the Klotho protein sequence of SEQ ID NO: 52 is a TEV-Twin-Strep tag (with a GS linker) (GGENLYFQ/SSAWSHPQFEK-GGGSGGGSGGS-SAWSHPQFEK), which is a TEV-Twin-Strep tag (i.e., GGENLYFQ). Cleaved after glutamine 8 (Q8), releasing the Twin-Strep tag portion (SSAWSHPQFEK-GGGSGGGSGGS-SAWSHPQFEK) and retaining at least part of the TEV (protease recognition or matching) sequence (GGENLYFQ-C').

The coding sequence was codon optimized using DNA2.0's proprietary algorithm. The gene was synthesized and assembled into a Pd3600 transposon scaffold-based expression construct.
A GS knockout CHOK1 derivative host cell line was used to express human Klotho variants. Cells were co-transfected with Leap-In transposon-based expression plasmids (ATUM) encoding three different hKlotho designs and a Leap-In transposase (ATUM). Transfection was performed by electroporation using the Neon apparatus (Thermo). Transfected cells were selected under glutamine-free conditions and the resulting stable pools were cryopreserved. Cells from established stable pools were inoculated into shake flasks and fed-batch production trials were performed using ATUM's small-scale established feed and cell culture conditions. Clarified harvest samples were collected and subjected to Western blot analysis using anti-Fc antibody-(SEQ ID NOS:52 and 54) or Strep-Tactin-(SEQ ID NO:66) HRP conjugate-based detection. Exemplary Western blots are shown in Figures 15A-16B.

Stable pool 291645 expressing the FL-ECD(34-981)-Fc Klotho derivative (SEQ ID NO: 52) mainly produces protein of the expected size (see Figure 15A). The predicted size of the full-length extracellular domain-Fc fusion protein monomer is 139 kDa. The predominant anti-Fc positive band is of the expected ˜140 kDa size (arrow), indicating that expression construct 291645 expresses the correct size fusion protein. Proteins dimerize under non-native and non-reducing conditions (see Figure 15B). Under non-reducing conditions, full-length extracellular domain-Fc fusion proteins can form homodimers. The size of the predominant band detected by the anti-Fc antibody in the non-reduced Western blot (arrow) is consistent with the expected homodimer formation.

The FL-ECD(34-981)-TEV-Twin-Strep molecule expressed by stable pool 291647 (SEQ ID NO: 66) also showed the correct size in Western blots and remained monomeric under non-native and non-reducing conditions. (see FIGS. 16A-16B). The expected size isoform-2-Fc variant represents the secreted human Klotho protein. The predicted molecular weight of the mature full-length ECD(34-981)-twin-strep tagged Klotho protein (SEQ ID NO:66) is approximately 109 kDa. Non-reducing Western blot analysis demonstrates successful expression of the correct size protein from CHO cells encoded by expression construct 291647 (see Figure 16A). Non-reducing denaturing Western blot analysis indicates that the full-length extracellular domain of hKlotho does not form covalent homodimers when secreted from CHO cells (see Figure 16B). Other protein constructs represented in the attached sequence listing were also prepared. FIG. 17 is a gel of various indicated recombinant Klotho protein constructs. Figure 18 is a series of gels of each of the indicated recombinant Klotho protein constructs.

In some embodiments, in addition to one or more other Klotho variants disclosed herein but not shown in Table 48, disclosed in Table 48 and/or It should be understood that the expression and/or purification of the Klotho variants shown in 18 provide advantages over the expression and/or purification of native Klotho. For example, expressing and/or purifying a Klotho variant may reduce the number and/or variety of alternative products. Additionally or alternatively, the expression level of the desired Klotho variant may be increased compared to the expression level of native Klotho. Additionally or alternatively, the desired Klotho variant is expressed and/or purified in a purer form under comparable conditions and methods (e.g., the concentration of the desired Klotho variant is expressed and/or or with a concomitant decrease in purified by-products). Preliminary results indicated that the designed recombinant fusion proteins were expressed and purified within suitable titer ranges and were soluble in acceptable buffers, carriers and excipients.

Additional cell lines were developed and evaluated for density, viability, and production. Data from the top 10 clones are illustrated in Figures 19-22. Data for the top four clones are illustrated in Figures 23-26. Based on feeding rank, the top two clones appear to be 138D9 and 145G8. Based on the cumulative productivity of sham perfusion, 138D9 appears to have the best performance. The 140F6 reached the highest VCD. Copy number stability was also assessed, as illustrated in FIG. Total RNA was extracted and RNA isolation was performed using the RNeasy Mini Kit (Qiagen, catalog number 74104). After RNA isolation, template amplification was performed using the Omniscript RT kit (Qiagen, catalog number 205111). All cDNA sequences were as expected.

Five of the clones were further tested for stability, as illustrated in Figures 28-32. These clones were grown in growth medium with or without glutamine and were shown to have higher growth rates in the presence of 4 mM glutamine compared to conditions without glutamine. No change in growth rate was observed during more than 60 population doublings in either set. Viable cell densities and clonal viability over a 7 day time course are illustrated in Figures 30-31. Figure 32 illustrates the volumetric productivity of clones in fed-batch culture at time 0, 60 PD for 7 days in the presence (+) and absence (-) of 4 mM glutamine. Volumetric productivity without T0 and PD60 glutamine cells is comparable. The volumetric productivity of PD60+glutamine was similar or somewhat lower, but never lower than about 70% of the T0 value.

Cells were adapted to 100% adaptation in EX-CELL, ActiPro, and CD OptiCHO media, and nearly 100% adaptation to CD CHO, CD FortiCHO, BalanCD CHO Growth A, and Select CD1000 media.

Example 3
As shown in Figure 33, purified Klotho protein was observed on the gel after ELISA. Table 49 shows the ELISA plate map key (top two segments) and the heatmap labeled ELISA 450 nm data (bottom two segments). Table 50 shows ELISA data for Klotho protein from cell culture supernatant and human serum samples. Table 51 shows the mean concentration of Klotho in each sample group.

Table 52 shows the ELISA plate map key of human serum Klotho levels before (top two segments) and after (bottom two segments) recombinant Klotho protein injection. Table 53 shows heatmap labeled ELISA 450 nm data of Klotho protein levels in human serum samples before (top two segments) and after (bottom two segments) injection of recombinant Klotho protein as measured by ELISA. show. Human serum samples were diluted 2-fold and cell culture supernatants were diluted 5,000-fold using Klotho ELISA assay sample dilution buffer. Serum B1 and cell culture supernatant were included on both ELISA plates as controls. TMB 30 minutes. Table 54 shows a summary of the average concentrations of Klotho protein in the indicated samples.

Table 55 shows the concentration and monoclonality of replicate Klotho protein samples.

An exemplary method for estimating concentrations involved quantification (quantification) by PAGE against BSA standards. An exemplary method for estimating concentration involved measurement against a purified Klotho standard.

One interpretation of the data is that the native-like recombinant (TwinStrep-TEV-tagged) Klotho protein expresses better, purifies cleaner, and/or has more activity than the recombinant Fc-tagged Klotho protein. It can be that holding This was surprising and unexpected. An alternative interpretation is that neither protein, or the Fc-fusion protein more than the native-like protein, showed a higher level of Klotho protein response to ELISA antibodies than the native-like protein. Therefore, we have found that the expression, purification, storage, and/or administration of native-like recombinant Klotho proteins and Fc-fusion Klotho proteins is not as simple as expected and that standard techniques have not worked well. may have identified. Proceeding based on the later interpretation, the procedure was modified to obtain more active proteins while continuing to process the data for further interpretation.

Three commercially available ELISA detection kits for Klotho protein were also tested for suitability according to certain embodiments of the present disclosure. See Tables 57 and 58 below. The IBL kit exhibits low background, linear dilution of serum samples, and acceptable spike recovery after 1:4 dilution. The Cloud-clone kit showed high background, linear dilution of serum samples, and no acceptable spike recovery. The EIAab kit showed low background, very low Klotho concentration in serum samples, no linear dilution of serum samples, and acceptable spike recovery.

A total of eight human serum samples were diluted 1:2, 1:4, 1:8 with each kit's dilution buffer. Four of these eight serum samples were spiked with the highest concentration standard from each kit at a 1:20 dilution. The protocol for each kit followed the instructions provided.

Figures 34A-34C illustrate the measured Klotho protein concentrations and observed spike recovery in each sample.

Example 4
A global glycan preliminary analysis was also performed, as illustrated in Figures 35A-35B. Experimental conditions and results for glycan analysis are shown in Table 59.

Tables 60 and 61 below show the data illustrated in FIGS. 35A-35B, respectively.

An additional pool of nine clones (see Table 62 below) was evaluated for suitability.

The growth, viability, and titers of each cell line are shown in Table 63 below.

Figures 36A-36C respectively illustrate growth, viability, and titer curves for each cell line.
An additional pool of three clones (see Table 64 below) was evaluated for suitability.

The growth, viability, and production of each cell line are shown in Table 65 below.

Figures 37A-37C respectively illustrate growth, viability, and titer curves for each cell line.
One of the promising clones (see Table 66 below) was re-evaluated for growth, viability and production.

Figures 38A-38C illustrate cell line growth, viability, and titer curves, respectively.
Example 5
Western blot analysis was performed on the expressed protein pools as illustrated in Figures 39A-39B. Figure 39A illustrates the Klotho ECD (34-981) 6xHis protein and Figure 39B illustrates the Klotho proteins shown. Unexpectedly, the Klotho protein appears to aggregate into higher molecular weight multimers.

A 5 L bioreactor expression run was performed followed by an adapter purification protocol. In the first purification step, a Protein A Sepharose 4 fast flow (0.75 ml) (0.5 cm x 3 cm) column was equilibrated with 10 mM Tris-HCl, pH 8.0. 100 ml of cell supernatant adjusted to pH 8.0 with protein sample (1.5 M Tris-HCl, pH 8.0 (approximately 1 ml)) was loaded at a flow rate of 0.75 ml/min and 10 mM Tris/arginine- A concentrated sample is generated by washing with HCl, pH 8.0 buffer and eluting with 4M MgCl ₂ , pH 3 (ie, low pH, high salt) at 0.50-0.25 ml/min. See Figure 40A. The column was re-equilibrated with 10 mM Tris-HCl, pH 8.0 (to prevent salt precipitation) and sanitized with 0.1 M NaOH: 0.5-0.3 ml/ml to avoid overpressure. . Minimal Klotho protein eluted during re-equilibration and sanitization.

Peak #1 and Peak #2 from Protein A elution were analyzed separately, buffer exchanged, 100 mM Tris-HCl/5 mM L-methionine/0.6% sodium thioglycolate, pH 8.0, migration. Fractionation was performed using a Superdex 200 (1 cm x 30 cm) size exclusion chromatography column with phases (0.5 mL/min flow rate). See Figure 40B. The mobile phase was buffered using Tris instead of phosphate-containing buffers to prevent magnesium salt precipitation. A reducing agent (L-methionine) was used to prevent oxidation of Klotho-Fc. Sodium thioglycolate was used to prevent scrambling of the free sulfhydrylcysteine groups of Klotho-Fc. The buffer components are FDA listed GRAS.

Superdex200 fractions from single separated peak #1 and peak #2 each represent a different MW species of Klotho-Fc protein. See Figures 41A and 41B. In summary, analysis of the harvested protein (in culture medium) by SDS-PAGE suggests that Klotho-Fc exists in various aggregation states. Purification produced two populations of multimeric Klotho-Fc corresponding to dimers (300 kDa or less) and tetramers (600 kDa or less). The bind/elution protocol from protein A shows a much better product elution profile and produces minimal protein after sanitization. Since the formulation has thioglycolic acid as reducing agent, the product separates as monomer after heating and as monomer and dimer without heating in gel electrophoresis. These new purification procedures resulted in non-aggregated Fc-Klotho protein.

Example 6
A bioassay was developed to test the activity of both native and Fc-Klotho proteins, and specifically to test the binding affinity of Klotho proteins to FGF23 using ForteBio's BLITZ. First, the surface plasmon resonance (SPR) binding method was used to assess the binding affinity of recombinant mouse Klotho to mouse FGF-23. SPR is an optical technique used to detect molecular interactions between a mobile molecule, an analyte, and an immobilized molecule, a ligand, bound to a sensor, which changes the refractive index of the sensor upon interaction. In this study, we compared the affinity of six concentrations of recombinant mouse Klotho (2-fold serial dilutions from 400 μg/mL to 12.5 μg/mL) antibody to mouse FGF-23. An anti-HIS (HIS2) sensor is used to bind mouse FGF-23 to the sensor and these sensors are then used subsequently to generate binding curves for six different concentrations of mouse Klotho. A dissociation constant (K _d ) value for the antibody is determined from the six association and dissociation curves generated for the antibody. Assays were performed and data validated (data not shown).

For human recombinant Klotho protein, the HIS-tag biosensor chip was hydrated in PBS for 10 minutes at room temperature. 300 μg/ml of HIS-tagged FGF23 (ProSpec, Catalog No. CYT-374) in PBS was allowed to bind to the biosensor for 2 minutes. A baseline was established on the BLITZ instrument for 30 seconds. The binding of five two-fold serial dilutions (from 400 μg/ml to 12.5 μg/ml) of native and Fc-s Klotho proteins to immobilized FGF23 was then tested in PBD for 2 minutes followed by PBS. Dissociation was allowed for 2 minutes. K _on (association), K _off (dissociation), and K _D (K off/Kon) were analyzed using global analysis. See Figure 42.

Cell-based functional assays were used to test the biological activity of the native and Fc-Klotho proteins, and specifically by measuring their effect on stimulating NIH/3T3 cell proliferation. Further assays were performed to test both biological activities of Based on the biology of Klotho, the rationale for developing this assay is to test the effect of Klotho on stimulating the proliferation of FGFR-expressing NIH-3T3 cells.

Cells in growth phase were seeded in culture medium (10% FBS-DMEM) at a density of 5000 cells/well/96-well plate and cultured overnight before changing the medium to 0.5% FBS. , cultured overnight. Cells were treated with 10 doses of each Klotho protein (2-fold serial dilutions, each dose in triplicate) starting at 30 μg/ml and incubated for 48 hours. Cell proliferation was measured using the Cell Titer-Glu Assay Kit and data were analyzed in GraphPad Prism using a four parameter fit curve. The data show that purified Klotho proteins, both native His and Fc-Klotho, stimulated cell proliferation in a dose-dependent manner. See Figures 43A-43E.

Example 7
A study was conducted to determine the acutely tolerated dose (ATD) of alpha human native Klotho and alpha human Fc fusion Klotho proteins following intravenous administration to male Sprague-Dawley rats. In an acute tolerable dose study, the safety of test article alpha human native Klotho protein and alpha human Fc-fused Klotho protein was evaluated in male Sprague-Dawley rats. Twenty-one animals were subcutaneously implanted with transponders prior to dose administration. On study day 1, animals were randomized into seven groups of three animals based on body weight. Alpha human native Klotho protein was tested at three dose concentrations of 10 μg/kg, 30 μg/kg, and 100 μg/kg (based on individual body weight) in vehicle (PBS, pH 7.2) on study day 0. A dose of 1 ml/kg (based on individual body weight) was administered intravenously.

Alpha human Fc fusion Klotho protein was also tested at three dose levels of 3 μg/kg, 10 μg/kg, and 30 μg/kg (based on individual body weight) and treated with vehicle (PBS, pH 7.2) on study day 0. ) in a volume of 1 ml/kg (based on individual body weight). Dosing was initiated on study day 0 in the vehicle only (PBS, pH 7.2) group, the 10 μg/kg alpha human native Klotho protein group, and the 3 μg/kg alpha human Fc fusion Klotho protein group, with each test article dose Level groups were staggered by at least 24 hours. All dosing was given once on each initiation day. Animals were observed for any adverse effects up to 14 days after treatment.

Lifetime measures included twice-daily checks for morbidity and mortality, and clinical observations were recorded daily. Body weight measurements were recorded daily. After completion of the observation phase, all animals were euthanized by cardiac exsanguination. Final measurements included blood hematology, serum biochemistry, and coagulation. Gross pathological changes were recorded for all animals at necropsy and the weights of 10 organs (adrenal gland, brain, epididymis, heart, kidney, liver, spleen, testis, thyroid/parathyroid, and thymus) were determined. Taken.

Table 67 shows a summary of the animals tested.

Table 68 shows a summary of the study design.

Group assignment: For MatchedPairDistribution, an optimized distribution method was performed using an algorithm that matches individual measurements to the mean of all selected animals. The method first takes matched pairs close to the mean of all selected animals and then distributes them into groups that are matched (or as close as possible) to the mean of all selected animals. The animals are distributed so that the mean of each group's measurements is as close as possible to the mean of all selected animals.

For statistical analysis, descriptive statistics were generated from trial data. Data were evaluated to determine whether parametric or nonparametric analysis was appropriate. For parametric data, analysis of variance (ANOVA) followed by post hoc testing was performed to determine significant differences between treatments, time points and/or groups. For nonparametric data, appropriate statistical analyzes were performed (eg, Kaplan-Meier survival, Kruskal-Wallis one-way ANOVA, Mann-Whitney, or Wilcoxon rank sums, etc.).

Table 69 summarizes the sampling procedure.

Table 70 summarizes the sampling tasks.

Any animal that weighed less than 85% of initial body weight or showed severe, long-term adverse clinical signs was euthanized. Animals were euthanized by cardiac bleed for collection of plasma, serum and whole blood as described above.

Preliminary results indicated that rats tolerated relatively high doses of recombinant Klotho protein administered to them.
Example 8
A study was conducted to determine the pharmacokinetics of alpha human native Klotho and alpha human Fc-fused Klotho proteins following IV administration to male Sprague-Dawley rats. Prior to study day 0, animals were subcutaneously implanted with transponders. Body weights were obtained on study day 1 and animals were randomized based on body weight. On study day 0, animals in groups 5, 6, and 7 (10 animals per group) were treated at 3, 10, and 30 μg/kg, respectively, in 1 ml/kg vehicle (PBS, pH 7.2). , received a single dose of alpha human Fc-fusion Klotho protein intravenously via the tail vein (see detailed dosing schedule below). On study day 1, animals in groups 2, 3, and 4 (10 animals per group) were treated at 10, 30, and 100 μg/kg, respectively, in 1 ml/kg vehicle (PBS, pH 7.2). , received a single dose of alpha human native Klotho protein intravenously via the tail vein (see detailed dosing schedule below). Animals in vehicle group 1 (3 animals) were also dosed with vehicle (PBS, pH 7.2) without protein at 1 ml/kg on study day 1. The volume of dosing solution administered to each animal was calculated and adjusted based on individual body weight measurements obtained on Study Day-1.

Three blood samples were obtained from each group (2-7) for all time points corresponding to 3 blood samples per rat (see detailed blood collection schedule below). Blood samples were taken at pre-dose, 3 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours post-dose, as shown in Table 71 below. (Groups 2, 3, and 4).

Blood samples were obtained at pre-dose, 3 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 7 days post-dose, as shown in Table 72 below. (Groups 5, 6, and 7). Additional bleeds were obtained in Groups 5, 6, and 7 on Day 14 post-dose.

Animals in vehicle group 1 were terminally bled one hour after dosing, as shown in Table 73 below.

Obtained serum samples were analyzed with the Alpha Klotho human soluble ELISA kit.
Table 74 shows a summary of the animals tested.

Table 75 shows a summary of the study design.

Group assignments: For stratified sampling, a non-optimized randomization method using "binning" of animals with similarly sized measures was used. By this method, pools of animals are stratified by sorting them in ascending order by the measurement of interest. Animals are then sorted into "bins" or hierarchies, depending on the size of the measurements. The number of strata is determined by the number of animals assigned per group. For example, if there are 3 animals per group, 3 bins will be created: small, medium and large. One animal from each size bin is randomly assigned to each group. Stratified sampling ensures that each group receives one animal at random from each size bin, and the mean for each group will remain similar to the other groups. This method balances the difference between the group mean and standard deviation to control for pool order bias.

For statistical analysis, descriptive statistics were generated from test data. Data were evaluated to determine whether parametric or nonparametric analysis was appropriate. For parametric data, analysis of variance (ANOVA) followed by post hoc testing was performed to determine significant differences between treatments, time points and/or groups. For nonparametric data, appropriate statistical analyzes were performed (eg, Kaplan-Meier survival, Kruskal-Wallis one-way ANOVA, Mann-Whitney, or Wilcoxon rank sums, etc.).

Table 76 summarizes the sampling procedure.

Table 77 summarizes the sampling tasks.

Any animal that weighed less than 85% of initial body weight or showed severe, long-term adverse clinical signs was euthanized (data not shown). Animals were euthanized by cardiac bleed for collection of plasma, serum and whole blood as described above.

Example 9
Dose dependence of alpha human Fc-Klotho and alpha human native Klotho on I/R-induced AKI and renal biomarkers (by measuring renal I/R-induced increases in plasma renal injury biomarkers) in Wistar rats. A study was conducted to determine efficacy. Specifically, the purpose of this study was to assess the dose-dependent effects of alpha human Fc-Klotho and alpha human native Klotho on kidney I/R-induced increases in plasma renal injury biomarkers in rats. This study consisted of bilateral renal ischemia-reperfusion (I/R) injury (30 min ischemic period) surgery (n=63) versus sham surgery (n=4), compound dosing (one intravenous injection/rat). ), serial urine collection via metabolic cage farm (3 collections/rat, days 1, 2, and 7), clinical chemistry analysis of serial urine specimens for creatinine and protein (days 1, 2 serial bleeds (3 bleeds/rat, days 1, 2 and 7), clinical chemistry analysis of serial plasma samples for creatinine and BUN (days 1, 2) Day 7, and Day 7), designed to provide data from or related to daily weight and health observations post-surgery to endpoint, and tissue collection and processing at endpoint. are presented in tabular and graphical form.

In addition, analysis of serial urine specimens for Kim-1 via species-specific ELISA (days 1, 2, and 7), analysis of serial urine specimens for NGAL via species-specific ELISA (1 day, 2, and 7), left and/or right kidney mRNA expression analysis (1-2 kidneys/animal: n=67 samples, 10 analytes, 3 normal performed using the Affymetrix Quantigene Plex 2.0 platform), MCP-1, TGF-β, α-SMA, Col1A1, Col3A1, Fn-1, CTGF), and specific staining and ED-1+ macrophages. Optionally left and/or right renal cortex immunohistochemistry and quantification of macrophage infiltration via staining were also performed.

Table 78 shows the test parameters.

Table 79 shows a summary of the study design.

Sixty-seven male Wistar rats weighing approximately 151-175 g were obtained, fed standard chow (Harlan 8640), group-housed under standard conditions, and acclimated for at least 7 days prior to study enrollment. Prior to study initiation, test rats were placed into weight-matched treatment groups. Animals were enrolled in a balanced design so that approximately equal numbers of animals were enrolled per group on each surgical day, with the heaviest animals enrolled first to minimize differences in animal age. enrolled in the study. When not in metabolic cage housing, rats were group-housed in static cage housing under standard conditions.

At t-0.5 hours, test rats were anesthetized with isoflurane anesthesia over nosecone prepared for surgery to allow core temperature to equilibrate prior to the renal I/R procedure. To minimize fluid volume fluctuations, rats were administered 10 ml/kg of warm sterile saline (subcutaneously; 25% pre- and 75% post-surgery). Core temperature was maintained normative (37±0.2° C.) throughout the ischemic period. At t-0.5 hours, animals in Groups 7 and 8 also received intravenous compound injections as needed.

A laparotomy was then performed. At t0, rats were subjected to sham (group 1, n=4) or bilateral renal artery ischemia (groups 2-8, n=9) using heat-sterilized instruments, vasoocclusive clamps, and sterile surgical technique. / group). Following a 30 minute continuous ischemia period (t+0.5), the occlusive clamp was removed, the kidney was reperfused, and the abdominal wound was closed with sterile silk sutures. Rats were given 0.01 mg/kg buprenorphine as an analgesic after surgery and allowed to recover briefly in a clean heated cage. At t+1 hours, animals in groups 1-6 were briefly restrained and received intravenous injections of vehicle or Klotho compounds in vehicle as needed. Groups 7 and 8 rats were also briefly restrained (but not injected) to control stress response. Rats were then immediately placed in metabolic cages.

Twenty-four hours after reperfusion (D1), urine volume was determined and sampled in all animals. All animals were then bled (500 μl) into Li-heparin by direct tail vein puncture. Rats were given 500 μl of warm saline immediately after bleeding. Urine and blood collection was repeated again on D2 (48 hours after reperfusion) and D7 (168 hours after reperfusion). On D2-D5, all rats were housed in static cages under standard conditions, weighed and health observations were made daily. Whole blood (500 μl) was appropriately processed for plasma production and processed as described.

Clinical observations were performed once daily after the start of dosing. With respect to collected/calculated data/tissues, animal health was observed daily after the surgical procedure and kidney mass (weight of left and right kidneys) was indexed against tibia length and body weight. Urine was collected on days 1, 2, and 7 (n=3 samples/animal, targeting 500 μl/sample). At least one sample was subjected to clinical chemistry analysis (creatinine, protein) and at least one sample was optionally used for Kim-1 and NGAL analysis. Plasma was collected on Li-heparin on days 1, 2, and 7 (n=2 samples/animal, 125 μl/sample targeted). At least one sample was submitted for clinical chemistry analysis (creatinine, BUN).

On day D7, rats were anesthetized with isoflurane immediately after bleeding and tissues were harvested. Both left and right kidneys were immediately placed in 0.9% ice-cold NaCl, unencapsulated and weighed. After tissue collection (for histological and other analyses), all remaining inner cortex/outer medulla (i.e., , S3/THAL-enriched) were snap frozen in appropriately labeled tubes and stored at −80° C. for potential future biochemical analysis. Prior to performing the analysis, cortical tissue was optionally cryogenically powdered and mixed to allow better homogeneity of tissue samples across biochemical assays, as well as the introduction of sampling bias that can occur in cortical biopsies. restricted. At least one mid-transverse section of each sham and ischemic kidney (left and right from each animal) was randomly taken and immersion fixed in 10% neutral buffered formalin for histological analysis. Rats were then sacrificed.

Example 10
Test rats were weighed to test the effect of Klotho protein administration on the subject. Table 80 shows a summary of the animals tested.

FIG. 44 graphically illustrates the mean (mean) body weight (±standard error of the mean, SEM bars) of rats in each group over the course of the 12-day study (daily). Table 81 below shows the corresponding data for mean body weights illustrated in Figure 44, Table 82 below shows the corresponding SEMs illustrated in Figure 44, and Table 83 below shows the 12 day study ( Shows the percent change in mean body weight of rats in each group over the course of each day.

Example 11
To test the therapeutic effect of Klotho administration on acute kidney injury (AKI) after renal ischemia-reperfusion injury (IRI), Sprague-Dawley rats were randomized into sham or AKI groups and animals in each group were randomized. Animals were randomly assigned to vehicle or Klotho treatment. In humans, AKI is a formidable clinical problem resulting primarily from ischemia or nephrotoxins, with outcomes ranging from complete recovery to development of dialysis-dependent chronic kidney disease, or death. Animals in the Klotho group received a single bolus i.p. (150 mM NaCl and 10 mM HEPES, pH 7.4). Twenty-four hour urine and blood were collected on days 1, 2, and 7 after surgery. This study showed that a single bolus injection of mouse S-Klotho 30 minutes after injury was effective at attenuation (of AKI) in an animal model of human pathology.

Example 12
To determine whether Klotho protects the lungs against oxidative injury, Sprague-Dawley rats (weighing approximately 300 g) were either normoxic (N: 21% inspired O) or hyperoxic (H: 90%). O) for 3 days, during which time three preparations: Dulbecco's Modified Eagle's Medium (DMEM), or Klotho (Klotho CM, approximately 60 pmol) or empty vector (Control CM) (n=6-8 each) animals) received an intraperitoneal injection of one of the CHO cell-derived conditioned medium (CM) overexpressing Injections were given 12 hours prior and repeated at 24 and 48 hours during exposure.

3LL cells were also implanted subcutaneously into the flanks of athymic mice (2×10 ⁶ cells per mouse) and treated with Klotho protein (0.01 mg/kg, ip) or vehicle every other day for 10 days. . Lungs were harvested 21 days after transplantation and the number of metastatic nodules was counted.

Conclusion Existing Klotho protein measurements can be prohibitively expensive and/or technically difficult for home collection and/or quantitative diagnosis. Embodiments of the present disclosure provide methods for measuring Klotho protein level(s), specifically endogenous and/or exogenous soluble Alpha Klotho protein level(s), and more specifically soluble Alpha Klotho protein level(s) in a subject. methods for monitoring, detecting and/or quantifying Klotho protein deficiency, diagnosing Klotho protein deficiency, and/or increasing Klotho protein level(s), expression, or production, and treating Klotho protein deficiency Articles of manufacture useful in those practices are provided, including diagnostic kits and compositions for.

Additionally, although gene therapy can be effective in increasing certain protein levels in animal models or trials, the safety of gene therapy, particularly for human treatment, remains questionable. Compared to viral delivery of the Klotho gene to animals (cells), administration of exogenous and/or recombinant Klotho proteins in humans is safer, easier and more efficient for restoring (endocrine) Klotho levels. It can be in a direct fashion. Preclinical data support the therapeutic potential of soluble Klotho protein for age-related disorders and diseases associated with Klotho deficiency. Epidemiological data indicate that soluble klotho is lower in older adults than in young adults, and levels of soluble klotho are inversely correlated with age, indicating that aging may be associated with decreased soluble klotho. . However, in general, the production and administration of exogenous and/or recombinant proteins to humans is strictly regulated by governments and other agencies. Due to the time and expense associated with regulatory compliance, the cost of such treatments can be prohibitive for developers, manufacturers, and patients alike. However, the compositions and methods of the present disclosure are a viable and effective option for increasing endogenous Klotho protein levels in humans and non-human animals without one or more of the drawbacks associated with other solutions. can provide Specifically, some embodiments of the present disclosure provide recombinant human Klotho, such as human recombinant soluble alpha-Klotho protein (of current good manufacturing practice (cGMP) grade), protein fragments, and/or protein variants. Products, compositions, and/or methods of making and/or using proteins are included. In addition, some embodiments of the present disclosure provide for (naturally) increasing (endogenous and/or serum soluble) Klotho protein level(s), expression, or production in a patient or subject (nutrition or health supplement) products, compositions and/or methods.

One of ordinary skill in the art will appreciate that each of the therapeutic effects, indications, and/or treatments outlined herein with respect to recombinant therapeutic Klotho proteins and products, compositions, and methods associated therewith are associated with endogenous Klotho protein production and It will be appreciated that this may be achieved by (naturally) increasing, enhancing, augmenting and/or supporting the level(s). One skilled in the art will also appreciate the therapeutic effects, indications and It will be understood that each of the/or treatments can be accomplished by administering recombinant therapeutic Klotho proteins and products, compositions, and through methods associated therewith. Accordingly, for the sake of brevity, the present disclosure provides the disclosed therapeutic effects for both (i) administration of a recombinant therapeutic Klotho protein and (ii) administration of a health supplement or nutraceutical composition of the present disclosure. , indications, and/or treatments need not be elaborated upon, and such are expressly contemplated herein.

Although the foregoing detailed description refers to specific exemplary embodiments, the disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the described embodiments are to be considered in all respects only as illustrative and not restrictive. For example, various substitutions, alterations, and/or modifications of the features of the invention described and/or exemplified herein, and the /or additional applications of the exemplary principles may be made to the described and/or illustrated embodiments without departing from the spirit and scope of the present disclosure as defined by the appended claims. Such substitutions, alterations, and/or modifications are intended to be within the scope of this disclosure.

The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. Limitations recited in the claims are to be interpreted broadly based on the language employed in the claims and are not limited to the specific examples set forth in the foregoing Detailed Description, rather than to the specific examples set forth in the foregoing Detailed Description. The examples are to be construed as non-exclusive and non-limiting. All changes that come within the range and meaning of the claims are to be embraced within their scope.

It will also be understood that various features of certain embodiments may be compatible with, combined with, included in, and/or incorporated in other embodiments of the present disclosure. For example, systems, methods, and/or articles of manufacture according to certain embodiments of the present disclosure may include, incorporate, or incorporate features described in other embodiments disclosed and/or described herein. may be included in other ways. Therefore, disclosure of particular features in relation to particular embodiments of the present disclosure should not be construed as limiting the application or inclusion of those features to particular embodiments.

Additionally, unless a property is stated as being required in a particular embodiment, features described in various embodiments are optional and may not be included in other embodiments of the present disclosure. . Further, any feature herein may be combined with any other feature of the same or a different embodiment disclosed herein, unless the feature is stated as requiring another feature in combination with it. can be It will be appreciated that in certain embodiments, features may be optional, but when features are included in such embodiments, they may be required to have specific configurations described in this disclosure. be.

Similarly, any steps recited in any method or process described herein and/or recited in the claims may be performed in any suitable order, unless otherwise specified (whether express or implied). The order in which they are described and/or listed is not necessarily limited unless stated explicitly). However, such steps may also be required to be performed in a particular order or any suitable order in certain embodiments of the present disclosure.

Moreover, various well-known aspects of example systems, methods, articles of manufacture, etc. are not described in detail herein to avoid obscuring aspects of the example embodiments. However, such aspects are also contemplated herein.

Claims (21)

A method of stabilizing a Klotho protein in a mammalian blood sample, comprising:
obtaining capillary blood from a mammalian subject, said capillary blood containing an amount of soluble Klotho protein;
storing said capillary blood in a container at room temperature or without freezing for at least 24 hours, said container having a preservative or anticoagulant disposed therein; An agent or anticoagulant is mixed with the capillary blood in the container, wherein the preservative or anticoagulant stabilizes the soluble Klotho protein for at least 24 hours at room temperature or without freezing. , storing, wherein said preservative or anticoagulant comprises one or more of heparin, lithium heparin, EDTA, and _K2EDTA . storing said capillary blood mixed with said preservative or anticoagulant at room temperature or without freezing for at least 3 days, wherein said preservative or anticoagulant is at room temperature or 2. The method of claim 1, further comprising the step of storing, stabilizing the soluble Klotho protein for at least 3 days without freezing. storing said capillary blood mixed with said preservative or anticoagulant at room temperature or without freezing for at least 7 days, wherein said preservative or anticoagulant is at room temperature or 2. The method of claim 1, further comprising the step of storing, stabilizing the soluble Klotho protein for at least 7 days without freezing. 2. The method of claim 1, further comprising quantifying the amount of the soluble Klotho protein or assaying for the presence of the soluble Klotho protein after the storing step. quantifying the amount of said soluble Klotho protein comprises: determining said soluble Klotho protein using mass spectroscopy, multi-analyte profiling (xMAP), and/or a first antibody that binds to a portion of said soluble Klotho protein; 5. The method of claim 4, comprising detecting. quantifying the amount of said soluble Klotho protein comprises detecting said soluble Klotho protein using a detection antibody that binds to a portion of said first antibody or performing an enzyme-linked immunosorbent assay (ELISA) to detect and optionally quantify the soluble Klotho protein. 2. The method of claim 1, wherein obtaining the capillary blood comprises one or more of lancing the skin of the mammal and collecting the capillary blood into the container. Method. Said capillary blood is about 10-1000 μl, preferably about 50-200 μl of said capillary blood, or obtaining said capillary blood comprises about 10-1000 μl, preferably about 50-200 μl of said 2. The method of claim 1, comprising obtaining capillary blood. A method of diagnosing Klotho deficiency in a mammalian subject, comprising:
obtaining a fluid sample mixture, the fluid sample mixture comprising:
mammalian serum, optionally in the form of capillary blood, mixed with a preservative or anticoagulant comprising one or more of heparin, lithium heparin, EDTA, and K ₂ EDTA, wherein an amount of soluble a mammalian serum comprising Klotho protein, and a mammalian serum optionally in the form of capillary blood, having an amount of soluble Klotho protein, said method comprising adding said mammalian serum to heparin, lithium heparin, mammalian serum, further comprising mixing with a preservative or anticoagulant comprising one or more of EDTA, and K ₂ EDTA to form said mixture. a step;
storing the mixture at room temperature or without freezing for at least 24 hours, wherein the preservative or anticoagulant stabilizes the soluble Klotho protein for at least 24 hours at room temperature or without freezing. causing and storing;
quantifying the amount of the soluble Klotho protein in the mixture after the storing step;
diagnosing said mammalian subject as having Klotho deficiency when the quantified amount of said soluble Klotho protein in said mixture is below a predetermined threshold amount. Said fluid sample mixture comprises about 10-1000 μl, preferably about 50-200 μl of capillary blood from said mammalian subject, or said step of obtaining said fluid sample mixture comprises about 10 μl of capillary blood from said mammalian subject. 10. A method according to claim 9, comprising obtaining ˜1000 μl, preferably about 50-200 μl of capillary blood. Said step of quantifying the amount of said soluble Klotho protein in said mixture comprises mass spectroscopy, multi-analyte profiling (xMAP), and/or a first antibody that binds to a portion of said soluble Klotho protein and optionally said first antibody. detecting said soluble Klotho protein using a detection antibody that binds to a portion of the antibody of 1, or performing an enzyme-linked immunosorbent assay (ELISA) to detect and optionally quantify said soluble Klotho protein. 10. The method of claim 9, comprising: The step of diagnosing the mammalian subject with Klotho deficiency comprises displaying the determination or diagnosis of Klotho deficiency on a user interface of a computer system and/or displaying the determination or diagnosis of Klotho deficiency, either physically or electronically. 10. The method of claim 9, comprising generating a formatted file or report, wherein the determination or diagnosis of Klotho deficiency optionally comprises the quantified amount and/or the predetermined threshold amount of the soluble Klotho protein. the method of. A method of treating Klotho deficiency in a mammalian subject, comprising:
obtaining serum or capillary blood from a mammalian subject, said serum or capillary blood having an amount of soluble Klotho protein;
storing the serum or capillary blood in a container at room temperature or without freezing for at least 24 hours, the container being placed therein and containing the serum or capillary blood to form a mixture; a preservative or anticoagulant mixed with blood, said preservative or anticoagulant stabilizing said soluble Klotho protein for at least 24 hours at room temperature or without freezing, said preservative or storing, wherein the anticoagulant comprises one or more of heparin, lithium heparin, EDTA, and K ₂ EDTA;
quantifying the amount of the soluble Klotho protein in the mixture after the storing step;
diagnosing the mammalian subject as Klotho-deficient when the quantified amount of the soluble Klotho protein in the mixture is less than a predetermined threshold amount;
After diagnosing the mammalian subject with Klotho deficiency, administering a composition to the mammalian subject, the 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 said carrier or excipient. , one or more pharmaceutical compositions,
one or more nutritional supplement compositions comprising a plurality of components adapted to increase serum soluble Klotho protein levels by increasing or enhancing endogenous production of soluble Klotho protein by said mammalian subject; Pharmaceuticals, Angiotensin II Receptor Blockers (ARBs), Angiotensin Converting Enzyme (ACE) Inhibitors, Losartan, Valsartan, Telmisartan, AT1 Receptor Antagonists or Blockers, Testosterone, Growth Hormone, Growth Hormone Releasing Peptides, Sermorelin, Prescription or Prescription dose optionally selected from the group consisting of vitamin D, 1,25-dihydroxycholecalciferol, calcitrol, paricalcitrol, indoxyl sulfate binders, Kremezin, AST-120, statins, PPAR agonists, troglitazone, and rosiglitazone and one or more pharmaceutical compositions that increase endogenous klotho levels. The administering step comprises
oral or oral-related administration optionally selected from the group consisting of ingestion, buccal administration, and sublingual administration, optionally in modified release form;
14. A modified release form comprising one or more bolus or incremental injections optionally selected from intravenous, intradermal, intraperitoneal, intramuscular, intradermal, and subcutaneous injections, according to claim 13. Method. obtaining the serum or capillary blood,
lancing the mammalian subject's skin with a lancet;
obtaining about 10-1000 μl, preferably about 50-200 μl of said capillary blood;
collecting said capillary blood into a container having a preservative or anticoagulant placed therein or added thereto, wherein said capillary blood optionally contains about 10-1000 μl, preferably about 14. The method of claim 13, comprising one or more of collecting 50-200 μl of capillary blood. wherein said pharmaceutical composition is with at least a portion of one of SEQ ID NOs: 2-70, or SEQ ID NOs: 107-120, or SEQ ID NOs: 125-128, or SEQ ID NOs: 133, or SEQ ID NOs: 152; 14. The method of claim 13, comprising 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. said pharmaceutical composition 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. 17. The method of claim 16, comprising a recombinant Klotho fusion protein. The nutraceutical composition contains 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 intestinal beneficial bacteria. described method. wherein the one or more intestinal beneficial bacteria is an effective amount of Bifidobacterium species and/or strains Bifidobacterium lactis BL-04, Bifidobacterium bifidum/lactis BB-02, and Bifidobacterium longum BL-05, and Lacto bacillus species and/or strains Lactobacillus acidophilus LA-14, Lactobacillus rhamnosus LR-32, and Lactobacillus paracasei LPC-37. A method of purifying a pharmaceutical grade recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein, comprising:
obtaining a suspension cell culture fluid comprising (i) a recombinant Klotho protein, a recombinant Klotho protein fragment, or a recombinant Klotho fusion protein, and (ii) one or more contaminants;
performing a purification step using affinity chromatography, wherein said recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein is purified from protein A or other Bound to the affinity entity, optionally washed, and eluted from Protein A with an elution buffer having a pH of about pH 2 to about pH 6 and a salt concentration of about 1 M to about 6 M, said salt optionally being dissolved in MgCl _{2 .} performing steps comprising
performing a purification step using size exclusion chromatography, wherein the eluted recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein further eluted in a buffer, wherein said mobile phase buffer comprises a buffer and one or more optional reducing agents, said buffer optionally other than a phosphate-containing buffer, said reducing agent comprising optionally comprising L-methionine and/or sodium thioglycolate, wherein said mobile phase buffer optionally has a pH of from about pH 6 to about pH 10;
The method wherein at least some of said further eluted recombinant Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho fusion protein is optionally in multimeric form. at least a portion of one of said recombinant Klotho proteins, SEQ ID NOs:2-70, or SEQ ID NOs:107-120, or SEQ ID NOs:125-128, or SEQ ID NO:133, or SEQ ID NO:152 and at least 22. The method of claim 21, having 80% amino acid sequence identity.