JP2021508037A - Products and methods for assessing and improving Klotho protein levels - Google Patents

Abstract

Products and methods for monitoring Klothoprotein levels and stabilizing Klothoproteins in mammalian blood samples over a period of time, especially at room temperature or without freezing, are disclosed. Methods for Detecting and Quantifying Croto Protein Levels, Specifically Intrinsic and / or Extrinsic Soluble Alpha Croto Protein Levels, Diagnosing Croto Protein Defects, and Increasing Croto Protein Levels or Production, Specifically Useful for their practice, including diagnostic kits and compositions for treating endogenous and / or exogenous soluble alpha croto protein levels (s), expression, or production, and croto protein deficiency in mammalian subjects. The product is disclosed. The composition is configured or formulated to increase natural soluble alpha croto protein production, attenuate damage or degradation of croto protein, and / or supplement croto protein levels with exogenous recombinant protein. Treatment methods and uses include administration of the composition to human or non-human mammalian subjects.

Description

Background The present disclosure relates to the evaluation and increase of Klotho protein levels in patients. Specifically, the present disclosure is for monitoring, detecting, and / or certifying levels of endogenous and / or extrinsic Crotoproteins, and Crotoprotein deficiency in human or non-human animal (eg, mammal) subjects. With respect to products and methods for diagnosing, and the production, purification, and administration of pharmaceutical or nutritional supplemental compositions for increasing levels of Cloto protein in a subject.

2. 2. Related Techniques Croto (or alpha-croto, α-croto, etc.) is a recently characterized protein encoded by the KL (or Croto) gene located on human chromosome 13. Through alternative RNA splicing, two transcripts originating from a single Croto gene have been identified. See FIGS. 1 and 2. The first transcript consists of a short cytoplasmic tail (human residues 1003-1012), a transmembrane (TM) domain (human residues 982-1002), and 20% -40% amino acid sequence homology with β-glucosidase, respectively. Two (mostly) homologous (internal repeat) domains (KL1 (human residues 56-506 with a length of 450 residues) and KL2) that share sex but may lack similar levels of glucosidase catalytic activity. An extracellular region or domain (human residues 1-981) containing (referred to as human residues 515-953 having a length of 438 residues) and a signal sequence (SS) domain (human residues 1-33). ), Which is a single transmembrane protein with a total length of 1,012 amino acids. It is predicted to encode Klothoisoform 1. SS, KL1, and KL2 domain-containing extracellular regions (human residues 1 to 981). ) Can be enzymatically cleaved by an α / β-secretase and released into the circulating stream as a 130 kDa circulating protein called a soluble croto (or s croto, s-croto, alpha-soluble croto, etc.). It can also be cleaved into separate 68 kDa proteins (KL1 + SS) and 64 kDa proteins (KL2).

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

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

Klotho is highly expressed in the kidneys, brain, to a lesser extent in other organs, and can also be found in mammalian blood, cerebrospinal fluid, and urine. Circulatory levels of soluble Klotho protein in mammals, including humans, are believed to decrease with age. In addition, Croto-deficient mice exhibit accelerated aging phenotype, while overexpression of Croto in mice has been shown to prolong lifespan. In addition, Klotho is associated with several age-related cellular processes. From the above standpoint, it has been hypothesized that soluble crotho can function as an anti-aging compound in the human body.

Aging is an unavoidable and progressive biological process that results in dysfunction and destruction of almost all tissues and organs, and ultimately death. For example, aging of the human body is associated with decreased cellular function that can lead to the development of various diseases. Aging is thought to be caused by tightly controlled and complex interactions between genetic and acquired factors, including increased aging, quantitative and qualitative loss of stem cells, and at the tissue level. Anomalous structure is a typical feature.

As the so-called "baby boomer" generation grows older, the elderly (eg, 60-65) population is growing rapidly worldwide. This increase in demand for health care for the elderly population is a significant financial burden on all health care systems. Recombinant Croto protein may provide a promising therapeutic agent against age-related health conditions. To date, all relevant therapeutic data associated with Klotho are preclinical studies in animal models. (I) Production and / or purification (eg, until substantially homogenous) of recombinant s-croto protein or protein manifold, and (ii) to subjects, especially humans, and / or increasing elderly Development of strategies and health intervention products and methods based on administration of recombinant s-croto protein or protein manifolds and / or s-croto protein level-increasing health supplements in the population may help improve this situation.

Currently suitable for human use, recombinant soluble human alpha-croto proteins or protein variants, in particular Good Manufacturing Practice (cGMP) determined and enforced by the US Food and Drug Administration (FDA). ) No product or method for providing an exogenous form of human Croto protein, such as a regulatory compliant protein, either alone or in combination with one or more additional active constituents.

Similarly, it now complies with products or methods for (naturally) increasing endogenous Klotho protein levels or protein production in a safe and effective manner, especially the 1994 Dietary Supplement Health Education Act (DSHEA). The product or product that complies with Good Manufacturing Practices (cGMP) regulations (as determined and enforced by the US Food and Drug Administration (FDA)) alone or with one or more additional active ingredients, drugs, drugs, or Not present, even in combination with dietary supplements.

In addition, products (eg, kits or systems) for efficiently, reliably, reproducibly, practically, and / or (commercially or economically) viablely monitoring Klotho protein levels are currently available. Methods, specifically endogenous and / or extrinsic crotho protein levels, and more specifically endogenous and / or extrinsic soluble alpha crotho proteins or protein diversity levels are monitored on demand and / or in real time. / Or there is no product or method for quantification, or for diagnosing Klothoprotein deficiency in humans and non-human animals, specifically serum-soluble Klothoprotein deficiency.

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

Embodiments of the present disclosure solve one or more of the aforementioned or other problems in the art with products and methods for assessing and increasing Croto protein levels in patients. Some embodiments include (1) compositions and methods for stabilizing crotoproteins in the blood or serum of human or non-human animals (eg, mammals), (2) crotoprotein levels (s). Compositions and methods for monitoring, detecting, and / or quantifying endogenous and / or extrinsic soluble alphacroto protein levels (s), and / or human or non-human animals (eg, eg,). Mammals) Compositions and methods for diagnosing Crotoprotein deficiency in subjects, and products useful in their practice, (3) Pharmaceutical or nutritional supplement compositions for increasing Crotoprotein levels in subjects. Including. In some embodiments, the therapeutic agent is a croto protein (s), or a fragment (s) or manifold thereof (s), and more specifically cGMP grade human (or non-human) recombination. Includes soluble alpha croto protein (s), or fragments (s) or manifolds (s) thereof, or (pharmaceutical) compositions containing them. Some embodiments are products and products for producing (recombinant) seroproteins (s), or fragments (s) or manifolds thereof (s), which are administered to human or non-human subjects. Includes / or methods, and specifically products and / or methods for increasing serum-soluble Klotho protein levels in a subject. Some embodiments include dietary supplement compositions or health supplement compositions 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 the level (s), expression, or production of the Cloto protein (specifically, an endogenous soluble alpha Cloto protein) in the subject. , And (4) methods of purifying recombinant croto proteins, specifically pharmaceutical grade recombinant croto proteins, recombinant croto protein fragments, and recombinant croto fusion proteins.

Various embodiments include compositions. The compositions include (i) one or more recombinant human croto proteins, protein fragments, and / or protein variants, expressed nucleic acid constructs and / or vectors, cell lines and / or cell suspension cultures, and / or ( ii) When administered to a subject, it causes an increase in clot protein level (s), expression, or production in the subject, specifically endogenous soluble alpha clot protein levels (s), expression, or production. It may contain one or more components that induce. Certain embodiments include methods of producing the compositions of the present disclosure, purifying them, and administering them to a subject (human or non-human animal). Some embodiments include methods of increasing the level (s), expression, or production of a Klotho protein (specifically (exogenous and / or endogenous) soluble alpha Klotho protein) in a subject. Some embodiments are for monitoring, detecting, and / or quantifying croto protein levels (s), specifically (endogenous and / or extrinsic) soluble alpha croto protein levels (s). Includes 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 include recombinant human alpha-soluble croto proteins, protein fragments, and / or protein variants (eg, cGMP grade human recombinant soluble alpha croto proteins, protein fragments, and / or protein varieties). Compositions comprising recombinant human alpha-soluble croto proteins, protein fragments, and / or protein variants (eg, therapeutic compositions, pharmaceutical compositions, pharmaceuticals, formulations, etc.); recombinant human alpha-soluble croto proteins and ( Composition containing a pharmaceutically acceptable vehicle (eg, carrier or excipient); composition containing recombinant human alpha-soluble croto protein and at least one additional (active) ingredient; human recombinant alpha-soluble croto A protein-encoding nucleic acid construct or vector; (i) a cell line containing a nucleic acid construct or vector encoding a recombinant human alpha-soluble Croto protein and / or (ii) expressing a recombinant human alpha-soluble Croto protein; A cell suspension culture of one or more cells containing (i) a nucleic acid construct or vector encoding a recombinant human alpha-soluble croto protein and / or (ii) expressing a recombinant human alpha-soluble croto protein. Methods of Producing and Optionally Purifying Recombinant Human Alpha-Soluble Croto Protein; Methods of Producing Pharmaceuticals (or Therapeutic Compositions, That Is, Formulations) of Recombinant Human Alpha-soluble Croto Protein; (Human or Non-Human Animal) Subjects Method of administering recombinant human alpha-soluble croto protein to a subject; diagnostic method for determining croto protein deficiency in a subject; method of diagnosing croto protein deficiency in a subject; And how to diagnose a subject; how to assess the effectiveness of a protein and / or determine the effective dosage for a subject who needs it; certain medical disorders in humans or non-human animals (eg, non-human mammals) Or recombinant human alpha-soluble croto protein for use in the treatment of other pathologies; use of recombinant human alpha-soluble croto protein for the treatment of specific medical or other pathologies in human or non-human animals; human Or recombinant humans for the treatment of certain medical or other pathologies in non-human animals Use of compositions containing alpha-soluble crotho protein; and / or of the use of recombinant human alpha-soluble crotho protein in the manufacture of pharmaceuticals for the treatment of certain medical or other pathologies in humans or non-human animals. It may include one or more.

Some embodiments may comprise a recombinant Croto protein, wherein at least a portion of the protein is SEQ ID NO: 2-SEQ ID NO: 70, or SEQ ID NO: 107-SEQ ID NO: 120, or SEQ ID NO: 125-SEQ ID NO: 128, or SEQ ID NO: It has at least 80% amino acid sequence identity with at least one portion of 133, or SEQ ID NO: 152. Some embodiments are 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. With or without at least a portion of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125-SEQ ID NO: 128, SEQ ID NO: 133, and SEQ ID NO: 152, and optionally any linker sequence in between. Contains 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 include recombinant proteins. Some embodiments may include two or more recombinant croto proteins.

Some embodiments may comprise a pharmaceutically effective amount of the recombinant Croto protein described herein and a pharmaceutically acceptable carrier. Some embodiments are pharmaceutically effective amounts of recombinant soluble croto protein in which at least a portion of the protein is amino acid residues 1-981, 29-981, 34 of human alpha croto 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-SEQ ID NO: 70, or SEQ ID NOs: 107-SEQ ID NOs. A recombinant soluble Klotho protein having at least 80% amino acid sequence identity with at least one portion of No. 120, or SEQ ID NO: 125 to SEQ ID NO: 128, or SEQ ID NO: 133, or SEQ ID NO: 152, and pharmaceutically soluble. It may comprise a pharmaceutical composition comprising an acceptable carrier. Some embodiments include pharmaceutically acceptable carriers or excipients, as well as SEQ ID NOs: 2 to SEQ ID NO: 70, or SEQ ID NOs: 107 to 120, or SEQ ID NOs: 125 to SEQ ID NO: 128, or SEQ ID NO: 133. , Or at least one of SEQ ID NO: 52, preferably SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125-SEQ ID NO: 128, SEQ ID NO: 133, and SEQ ID NO: 152. And at least 80% with at least a portion of the immunoglobulin Fc domain sequence, human serum albumin sequence, His (eg, 6His) tag sequence, and / or Twin-Strep tag sequence, with or without any linker sequence in between. Can comprise a pharmaceutical composition comprising an effective amount of recombinant protein, comprising the Croto protein sequence having the same amino acid sequence of. Some embodiments may comprise a composition comprising a pharmaceutically effective amount of two or more recombinant croto proteins.

Some embodiments include a Therapeutic Klotho protein and at least one other active constituent such as a drug, antibody, hormone, human cell, tissue, cell product or tissue system product (HCT / Ps). It may include objects and / or methods of administering them to human or non-human subjects. Combinatorial compositions and methods may be useful in treating subjects with age-related disorders or conditions, clinical (eg, metabolic) disorders, chronic diseases, and acute injuries. Prophylactic administration of combination therapy to a subject who does not have an overt pathology or disorder may also be useful in delaying or preventing the particular pathology or disorder described herein.

Some embodiments may include nucleic acids or nucleic acid constructs. For example, embodiments may include expression vectors or nucleic acids. The nucleic acid may encode a recombinant human alpha-soluble croto protein, protein fragment, or protein manifold. Nucleic acids can encode natural or non-natural signaling sequences. For example, the nucleic acid may encode an unnatural signaling sequence upstream (or N-terminus) of the encoded Croto protein sequence. Some embodiments are recombinant proteins of SEQ ID NO: 2-SEQ ID NO: 70, or SEQ ID NO: 107-SEQ ID NO: 120, or SEQ ID NO: 125-SEQ ID NO: 128, or SEQ ID NO: 133, or SEQ ID NO: 152. At least one of, preferably at least one of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125-SEQ ID NO: 128, SEQ ID NO: 133, and SEQ ID NO: 152, and optionally any in between. At least 80% amino acid sequence identity with at least a portion of immunoglobulin Fc domain sequences, human serum albumin sequences, His (eg, 6His) tag sequences, and / or Twin-Strep tag sequences, with or without linker sequences. It may comprise a nucleic acid construct comprising a nucleic acid sequence encoding a recombinant protein, including a Croto protein sequence having.

Some embodiments may include cell lines. The cell line may include (s) Chinese Hamster Ovary (CHO) cells (s), HEK-293 cells, or other suitable (protein expression) cell lines. In some embodiments, the cell can be a dihydrofolate reductase (DHFR) deficient CHO cell, such as a CHO-S cell, or a glutamine synthetase (GS) deficient CHO cell, such as a GS-/-CHO cell. 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. Encodes a polypeptide having at least 80% amino acid sequence identity with at least one portion of SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125-SEQ ID NO: 128, SEQ ID NO: 133, and SEQ ID NO: 152. It may contain (a copy of) one or more exogenous nucleic acids (including the transgene or cDNA). The polypeptide may include a human recombinant alpha-soluble croto protein. The extrinsic nucleic acid may preferably comprise a transgene or cDNA having at least 80% nucleic acid sequence identity with one of SEQ ID NOs: 76-106 or SEQ ID NOs: 121-102. In some embodiments, the nucleic acid is (also) a promoter (related to the transgene) and / or any of the (functional) dihydrofolate reductase (DHFR) enzyme, (functional) glutamine synthetase (GS) enzyme, and the like. (Extrinsic) enzymes can be included or encoded.

The at least one embodiment preferably comprises a carbon source, a nitrogen source, and / or one or more vitamins, minerals, salts, amino acids, supplements, or additives in which the cell expresses a polypeptide encoded by the nucleic acid. Includes suspended cell cultures, including cell lines that grow in such liquid media. The liquid medium can be serum-free (human, bovine, fetal bovine, or other) and / or free of animal (or animal-derived) proteins (components). For example, the liquid medium may be free of bovine serum albumin, human serum albumin, and the like. In some embodiments, the suspended cell culture is a liquid medium, preferably a serum-free and / or liquid medium free of animal protein constituents, preferably a carbon source, a nitrogen source, and one or more. Liquid medium and cells expressing nucleic acid-encoded polypeptides, including vitamins, minerals, salts, amino acids, supplements, or additives, more preferably lacking hypoxanthin, thymidine, and / or glutamine. A cell line that grows in a liquid medium, wherein the polypeptide may include a cell line containing a recombinant croto protein.

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

Some embodiments may include methods of producing recombinant human alpha-soluble croto proteins (eg, recombinant human alpha-soluble croto proteins). An exemplary method is, 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). It may comprise producing recombinant croto protein in deficient CHO cells, more preferably in GS − / − CHO cells, the proteins preferably SEQ ID NOs: 2 to SEQ ID NO: 70, or SEQ ID NOs: 107 to 120. , Or SEQ ID NO: 125-SEQ ID NO: 128, or SEQ ID NO: 133, or at least one portion of SEQ ID NO: 152 and at least 80% amino acid sequence identity.

In some embodiments, the production method is to grow Chinese hamster ovary (CHO) cells in liquid medium, to produce recombinant soluble croto protein intracellularly, and / or cells, liquid medium, or them. It may include purifying a recombinant soluble Klotho protein-containing extract from both of the above. The extract contains at least about 90%, preferably at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% dry weight of recombinant soluble croto protein, and / or It may contain less than about 1-100 ppm of 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 can be released (eg, secreted) from the cell into a liquid medium and / or have one or more glycans bound to it.

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

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

In some embodiments, selective and / or gene amplification involves culturing the transfected cells in a selective medium such as a medium lacking hypoxanthine and / or thymidine (eg, -HT medium), glutamine, and the like. Can be carried out by. In at least one embodiment, a low concentration (s) of MTX is added or used to amplify the transfected nucleic acid (or its gene (s)) thereby (eg, transfecting with a DHFR transgene). Increased protein expression (in infected DHFR-deficient cells) can be selected. Alternatively (or in addition), selection and / or gene amplification is performed on suspension cultures of cells carrying at least one (extrinsic) glutamine synthetase (GS) transgene in MSX ((endogenous) glutamine synthetase (GS). ) Inhibitor) can be added.

The method may include subculturing viable cells or cultures (eg, MTX-resistant and / or MSX-resistant cells or cultures). Selective suspension cultures and / or selected cells include increased production of protein (eg, by cells), increased concentration of protein (eg, in liquid medium), and / or, eg, non-selected suspension. It may have or present an increase in the number of copies (eg, per cell) of the exogenous nucleic acid (eg, per cell) compared to the culture or cells.

In some embodiments, the liquid medium may also contain effective amounts of MTX and / or MSX. Suspended cultures (or their cells) exhibit increased production of protein (eg, by cells), increased concentration of protein (eg, in liquid medium), and protein (eg, in liquid medium). And / or preferably have an increase in the number of copies of the exogenous nucleic acid (eg, per cell) as compared to a suspension culture that was not selected. A protein may have one or more glycans bound to it.

Some embodiments of the present disclosure relate to compositions configured or formulated to increase, improve, or increase the production of naturally soluble alpha croto proteins. Some embodiments include compositions or methods for increasing (endogenous) Klotho protein levels in a subject. Some embodiments include compositions (nutritional supplements or health supplements) to increase Klothoprotein levels in the subject, particularly serum-soluble Klothoprotein levels. Some embodiments, when administered to a subject, have a clot protein level (s), expression, or production in the subject, specifically an endogenous soluble alpha clot protein level (s), expression, or production. Includes products that cause an increase.

Some embodiments of the present disclosure relate to compositions configured or formulated to attenuate damage or degradation of the Klotho protein. Some embodiments of the present disclosure relate to methods of producing the compositions of the present disclosure. Some embodiments of the present disclosure relate to the use of the compositions of the present disclosure, or methods of using it to treat human or non-human animal subjects. Some embodiments of the present disclosure relate to therapeutic methods involved in the compositions of the present disclosure, including their administration to human or non-human animal subjects. Such uses and therapeutic methods can increase endogenous croto protein levels, such as by increasing the production of naturally soluble alpha croto protein and / or diminishing damage or degradation of croto protein.

Certain embodiments of a substance comprising a health supplement that increases the level of endogenous soluble alpha human protein in a human or non-human animal subject when administered to a human or non-human animal (eg, mammal) subject. Contains the composition. 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 (synthetic) chemical and / or natural sources or may be derived from them. The exemplary composition is adapted (or chemically constructed) to increase serum-soluble Crotoprotein levels by increasing or enhancing the endogenous production of soluble Crotoprotein by a mammalian subject, or as such. May contain multiple components adapted (or chemically composed) to.

The exemplary composition is preferably, for example, vitamin D3, vitamin E, vitamin C, vitamin K, 3,5,4'-trihydroxy-trans-stilbene (resveratrol), pterostilbene, N-acetylcysteine. (NAC), troglycazone, rosmarintazone, Notopterygium incisum Ting, gentiana (root, extract), Cordyceps (Cordiceps Sinensis (CS) mycelium), isoflavone (soybean), genistein, daizein, quercetin (dihydrate, dihydrate, leaf) , Oregano), docosahexaenoic acid (DHA), astaxanthin, sesamine (Semen Sesamin Nigrum (black)), sesame (seed extract), rosmarinic acid (RA, (majoram)), 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 one or more constituents selected from the group consisting of others known in the art.

The exemplary composition is preferably at a suitable dietary supplement concentration, and preferably, for example, vitamin C, vitamin D3, vitamin E, N-acetylcysteine (NAC), quercetin (dihydrate), rosmarinic acid ( From the group consisting of RA), pterostilben, docosahexaenoic acid (DHA), nicotinamide riboside (NR), nicotinamide adenine dinucleotide (NAD +), nicotinamide mononucleotide (NMN), and one or more enteric beneficial bacteria. It may contain one or more constituents of choice. One or more intestinal beneficial bacteria are effective amounts of Bifidobacterium species and / or strains Bifidobacterium lactis BL-04, Bifidobacterium bactisum / lactis BB-02, and Bifidobacterium strains Lactobacillus-Lactobacillus-Lactobacillus BL-04. Includes certain Lactobacillus acidofilus LA-14, Lactobacillus rhamnosus LR-32, and Lactobacillus paracasei LPC-37.

Certain components or constituents may have positive benefits and very low side effects in the treatment or effect of one or more pathologies associated with age-related cell and tissue dysfunction. Certain components or constituents may (directly or indirectly) increase the circulating plasma levels of soluble alpha human (s-croto) levels in human or non-human animal subjects after their administration.

In at least one embodiment, the compositions of the present disclosure can be sold as over-the-counter medicines, marketed and / or sold directly to consumers (DTC), or purchased without prescription / physician instructions. It can be, or can include, a dietary supplement that complies with the Dietary Supplement Health and Education Act of 1994 (DSHEA). In some embodiments, the compositions of the present disclosure may or may be pharmaceuticals approved by the US Food and Drug Administration (FDA).

In some embodiments, the ingredients can be co-administered, preferably via at least some co-formulations. In some embodiments, the ingredients can be co-administered in separate formulations. For example, in some embodiments, the ingredients may be co-administered in multipills, multicapsules, or dosing packs. Some ingredients may be provided in solids, granules, powders, liquids, or other forms.

As described herein, some embodiments may include one or more (additional) components or constituents. For example, some embodiments may comprise one or more recombinant (eg, human or mammalian) croto proteins, protein fragments, and / or protein manifolds, as described herein. Specific embodiments include expressed nucleic acid constructs and / or vectors, cell lines and / or cell suspension cultures, and one or more recombinant (eg, human or mammalian) croto proteins, protein fragments, and / or protein diversity. It may include a method of producing a body, purifying it and administering it to a subject (human or non-human animal). Some embodiments include pharmaceutical or prescription pharmaceuticals (eg, ARB (eg, rosaltan, balsartan), testosterone, vitamin D receptor agonists (eg, calcitriol, paricalcitol), PPAR (gamma) agonists (eg, thiazolidine). It may contain one or more active ingredients such as dione, troglitazone, rosiglitazone), and / or others described in the patent references incorporated above).

In some embodiments, a pharmaceutically effective amount of one or more recombinant croto proteins, and an effective amount of composition formulated to increase the level or production of an endogenous croto protein (eg, in a mammalian subject). It may include a composition comprising a substance (eg, a health supplement). In some embodiments, the recombinant Klotho protein can be co-administered with the composition. In some embodiments, the recombinant Croto protein can be combined with a pharmaceutically acceptable carrier. In some embodiments, the administered recombinant croto protein can increase serum soluble croto protein levels by providing an exogenous croto protein, while the composition is a (natural) croto protein by the patient or subject. ) Serum-soluble Klotho protein levels can be increased by increasing or enhancing production.

Some embodiments may include therapeutic methods. The method may include administering a (pharmacological) effective amount of the 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 croto proteins or protein manifolds of the present disclosure and / or (ii) endogenous croto protein levels or production (eg, in a mammalian subject). May include a pharmaceutically effective amount of the composition (eg, a health supplement) formulated to increase.

Some embodiments may include methods of treating croto deficiency (eg, in human or non-human animal (eg, mammalian) patients or subjects). The method may optionally or preferably include administering the composition to the mammalian subject after diagnosing the mammalian subject as Croto deficiency. The composition is, for example, a pharmaceutically acceptable carrier or excipient and a pharmaceutically effective amount of recombinant croto protein, recombinant croto protein fragment, or recombinant croto fusion in the carrier or placed in the excipient. A pharmaceutical composition comprising a protein; as well as a nutritional composition comprising a plurality of components adapted to increase serum soluble croto protein levels by increasing or enhancing the endogenous production of soluble croto 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 pathologies, disorders, 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 may suffer from or be at risk for a variety of conditions (eg, disorders, disorders, injuries, illnesses, etc.). For example, some embodiments treat one or more chronic diseases and / or age-related conditions, such as (human) age-related physical, intelligent, neurological, or other conditions. Including how to do it. Some embodiments may promote healing, recovery, longevity, and / or other beneficial outcomes through one or more mechanisms or actions. Administration of the compositions of the invention (s) may have a positive therapeutic effect on the course and consequences of the condition, including chronic and / or age-related diseases and longevity, as well as their characterization in human subjects. ..

A pharmaceutically effective amount of the composition (s) will increase the concentration of the serum soluble croto protein of interest to a predetermined level, such as soluble croto protein, greater than, about 50 to 3000 picograms per milliliter of serum, equivalent, or in between. It can be sufficient to rise or increase. The amount may also, or alternative, be sufficient to maintain the serum soluble croto protein concentration of the subject above a predetermined threshold over a predetermined period of time. Embodiments may also include administering the composition (s) to a subject in need thereof so as to maintain the serum soluble croto protein concentration of the subject above a predetermined threshold over a predetermined period of time.

Embodiments may also include determining the serum soluble croto protein concentration of the subject. Embodiments may also include calculating pharmaceutically effective amounts of the compositions of the present disclosure. Embodiments may also include determining the rate of decrease of soluble croto protein in the serum of interest. The embodiment may also include calculating the subsequent dosing time at which the serum soluble croto protein concentration of the subject falls below a second predetermined level based on the determined rate. The embodiment also comprises calculating the subsequent dosing time and / or the amount of composition sufficient to raise the serum soluble croto protein concentration of the subject from a second predetermined level to a first predetermined level. obtain. Embodiments may also include administering to the subject a subsequent dosage of the composition. Certain embodiments may include prescribing a composition in a normal (eg, daily) dose or dosage form to a subject (eg, based on a determined level of serum-soluble Klotho protein concentration in the subject).

In certain embodiments, the composition (eg, protein or supplement) or elevated soluble apoptosis protein levels may (and may be effective for) regulating the IGF-1 and / or Wnt signaling pathway. obtained exhibited β- glucuronidase and / or sialidase activity, obtained by suppressing the p53 / p21 signaling pathway, and / or through preferably inhibition of p53 / p21 signaling _pathway, reduce the H 2 _{O 2} induced cell senescence and apoptosis Can be. Proteins or elevated protein levels preferably exhibit polymorphic expression activity and / or preferably oxidative stress, growth factor signaling, maintenance of ion homeostasis, and / or one or more ion channel proteins and / or insulin / insulin. Like a growth factor-1 receptor can function or be functional as a humoral factor in the regulation of the activity of glycoproteins on cell surfaces such as growth factor receptors.

In certain embodiments, the composition (eg, protein or replacement) or elevated soluble croto protein levels are also frailty, bone density loss or bone mineral loss, weight loss, muscle atrophy or degeneration, muscle loss, strength. , Grip, leg or physical weakness, movement, freedom of movement, quality of life evaluation, ejection rate, decreased athletic ability, learning ability, learning ability, memory ability, or decreased intelligence index, cognitive deterioration or oblivion, cognition Treat (or treat) one or more age-related conditions (or (human) age-related conditions), such as decreased capacity or function, decreased synaptic plasticity or synaptic function, and cellular senescence. possible.

In certain embodiments, the composition (eg, protein or replacement) or elevated soluble Klotho protein levels are Alzheimer's disease, Parkinson's disease, dementia or vascular dementia, muscular atrophic 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, luteal degeneration and other Treat one or more age-related conditions (or (human) age-related conditions) such as eye disease / disorder, multiple sclerosis (MS), rheumatoid arthritis, and / or ulcerative arthritis. Can be (effective for treatment).

In certain embodiments, the composition (eg, protein or replacement) or elevated soluble croto protein levels may treat (therapeutically be effective) one or more other diseases or conditions. For example, some embodiments of the disclosure include treating cancer, lowering serum phosphate levels in a patient, diabetes in a subject in need of treatment, or a condition associated with diabetes (eg, type 1 diabetes, etc.). ), To treat the pathology of the subject's heart (eg, cardiovascular disease, left ventricular hypertrophy (LVH), pathological LVH, and / or congestive heart failure), (eg, using nanoparticles). Treatment of subject's acute lung injury, protection of patient's lungs against oxidative injury, detection of early acute renal injury in critically ill patients, attenuation of subject's vascular calcification, cognition Improving force, treating renal and / or hepatic ischemia, regulating the stress response of (human) aging endothelial cells, acute and / or chronic renal injury, disease, or progression of disease, and / Or prophylactically and / or therapeutically treat, prevent, alleviate, prevent, and / or reverse urotoxic cardiomyopathy, reverse or diminish age-related treatment resistance of melanoma, apoptosis of aging cells Can be useful in targeting and thereby restoring tissue homeostasis, as well as in one or more of a variety of other indications.

Thus, embodiments may also include compositions for use in the treatment of one or more age-related pathologies or other pathologies. An exemplary method of treating an age-related or other pathology, disease, or disorder comprises administering an effective amount of the composition of the present disclosure to a subject in need thereof. The compositions include (i) the replenishers described herein and / or (ii) (eg, SEQ ID NOs: 2-SEQ ID NO: 70, or SEQ ID NOs: 107-SEQ ID NOs: 120, or SEQ ID NOs: 125-SEQ ID NO: 128). , Or a recombinant soluble croto protein (having at least 80% amino acid sequence identity with at least one portion of SEQ ID NO: 133, or SEQ ID NO: 152). Optionally, the composition may comprise a pharmaceutically acceptable carrier or excipient.

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

Certain embodiments may include blood or serum samples of (mammalian) subjects, preferably blood obtained from capillary blood and / or fingertips or other non-phlebotomy (non-venous incision) blood sampling techniques, or Can be used. However, it will be understood herein that phlebotomy (venous cutdown) and other suitable blood sampling techniques are also contemplated.

Some embodiments include kits. Embodiments may include, for example, a sampling container. The vessel may have an opening configured to receive capillary blood. In some embodiments, capillary blood may or is suspected of containing a certain amount of soluble croto protein. Embodiments may include a sample preservative or anticoagulant, preferably comprising one or more EDTA and heparin. In some embodiments, the preservative or anticoagulant may be placed within the sampling compartment of the container. The solution can be configured to stabilize the soluble croto 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 lancet incision device or lancet.

Some embodiments include methods. For example, some embodiments may include methods of stabilizing the Klotho protein in a mammalian blood sample. Embodiments may include, for example, collecting or obtaining capillary blood from a mammalian subject and storing the capillary blood in a container for at least 24 hours at room temperature or without freezing. The container may have a preservative or anticoagulant placed therein such that the preservative or anticoagulant is mixed with the capillary blood in the container. Preservatives or anticoagulants can stabilize soluble Klotho protein for at least 24 hours at room temperature or without freezing. The preservative or anticoagulant may be, or may include, one or more of heparin, lithium heparin, EDTA, and K _{2 EDTA.}

Embodiments may also include collecting capillary blood in a container and / or mixing the capillary blood with a preservative or anticoagulant to form a mixture. In some embodiments, blood collection of capillary blood may include incising the skin of a mammal with a lancet. In some embodiments, the step of collecting capillary blood in a container may comprise collecting at least or about 10 to 1000 μl, preferably about 50 to 200 μl of capillary blood in a container. Some embodiments may include storing the mixture at room temperature or without freezing, or allowing storage of the mixture, and preservatives or anticoagulants may be stored at room temperature or frozen. Without, the soluble Klotho protein is stabilized for at least 3 days.

Some embodiments may optionally include assaying the mixture for the presence of soluble Klotho protein. The step of assaying the mixture for the presence of soluble croto protein can include detecting soluble croto protein and / or quantifying the amount of soluble croto protein. Detecting the soluble croto protein and / or quantifying the amount of the soluble croto protein can include contacting the mixture with an antibody configured to bind the soluble croto protein. Detecting soluble croto protein and / or quantifying the amount of soluble croto protein can be performed by performing an ELISA on the mixture using an antibody configured to bind to the soluble croto protein. Can include. Other detection methods include mass spectrometry or multianalyte profiling (xMAP), Luminex technology (eg, miniaturized liquid array bioassays, small lasers, light emitting diodes (LEDs), digital signal processors, photodetectors, and / or charges. Coupling element imaging) and the like may be included.

Some embodiments include a method of diagnosing a croto deficiency in a mammalian subject or a method of treating a croto deficiency in a mammalian subject. Embodiments may include, for example, providing or obtaining a fluid biological sample. The sample may preferably contain mammalian serum in the form of capillary blood. The method may optionally include mixing mammalian serum with a preservative or anticoagulant to form a mixture. The sample may also, or optionally, contain a mixture containing mammalian serum, preferably in the form of capillary blood, and a preservative or anticoagulant. The method may include assaying the mixture for the presence of soluble Klotho protein. The step of assaying the mixture for the presence of soluble croto protein is to detect the presence of soluble croto protein in the mixture, if present, after storing the croto-stabilized mixture for a period of time at room temperature or without freezing. , And / or if present, may include quantifying the amount of soluble croto protein in the mixture. The method may include diagnosing a mammalian subject as croto deficiency if no soluble croto protein is detected in the mixture, or if the amount of quantified soluble croto protein in the mixture is less than a predetermined amount. .. In some embodiments, diagnosing a mammalian subject as a Croto deficiency displays a Croto deficiency determination or diagnosis on the user interface of a computer system and / or displays a Croto deficiency determination or diagnosis. It may include generating files or reports in physical or electronic format.

In some embodiments, providing or obtaining a fluid biological sample may include collecting capillary blood from a mammalian subject and / or collecting capillary blood in a vessel. In some embodiments, the step of assaying the mixture for the presence of a soluble croto protein may include performing an ELISA against the mixture using an antibody configured to bind the soluble croto protein. In some embodiments, the composition is administered to the mammalian subject if no soluble croto protein is detected in the mixture, or if the amount of quantified soluble croto protein in the mixture is less than a predetermined amount. Can include that. Mass spectrometry can also be performed, or alternative.

Disclosure strategies, products, and / or health intervention methods specifically configured to increase the circulation and / or intrinsic levels of soluble croto are of situations and problems associated with reduced croto levels in patients. Can help improve.

Some embodiments may include any of the properties, options, and / or possibilities described elsewhere in the disclosure, including other aspects or embodiments of the disclosure. It should also be noted that each of the aforementioned and / or other properties described herein, as described above, also represents a separate embodiment of the present disclosure. Moreover, any combination of any two or more of such properties represents a separate embodiment of the present disclosure. Such properties or embodiments may also be combined in any suitable combination and / or order without departing from the scope of the present disclosure. Thus, each of the properties described herein can be combined with any one or more other properties described herein in any suitable combination and / or order. Accordingly, the present disclosure is not limited to the particular combination of exemplary embodiments described in detail herein.

Additional properties and benefits of the exemplary embodiments of the present disclosure are set forth in the following description, some of which may be apparent from the description or may be learned by implementing such exemplary embodiments. The properties and advantages of such embodiments may be realized and obtained by the equipment and combinations specifically pointed out in the appended claims. These and other properties may become more fully apparent from the claims below and in the appended claims, or may be learned by practicing such exemplary embodiments as described below.

The figure explaining the alternative RNA splicing of the Klotho gene. The figure explaining the alternative RNA splicing of the Klotho gene. Same as above. The figure which illustrates the surprising and unexpected stability of the Croto protein in human serum over a long period of time at room temperature and other temperatures. The purity and integrity of the purified protein analyzed with 4-20% SDS-PAGE (reduced and non-reduced) stained with GelCode Blue reagent. The dose responsiveness curve is shown. The dose responsiveness curve is shown. The dose responsiveness curve is shown. The purity and integrity of the purified antibody analyzed with 4-20% SDS-PAGE (reduced and non-reduced) stained with GelCode Blue reagent. Purity and integrity of purified antibody analyzed with 4-20% SDS-PAGE (reduced and non-reduced) stained with GelCode Blue reagent. Raw absorbance data at 450 nm and the corresponding antibody titration curve. Raw absorbance data at 450 nm and the corresponding antibody titration curve. We show that there is a relatively weak signal in the IgY / HRP-IgY pair. We show that there is a relatively weak signal in the IgY / HRP-IgY pair. Graph representation of (daily) changes in circulating human protein levels in human subjects. Further measurements of Klotho protein are shown. Further measurements of Klotho protein are shown. It shows the Croto level at different incubation times at room temperature. Same as above. Shows a graph or chart of an exemplary report. The chromatograph of Klotho-Fc protein is shown. An exemplary Western blot analysis using anti-Fc antibody- (SEQ ID NOs: 52 and 54) or Strept-tactin- (SEQ ID NO: 66) HRP conjugate-based detection. An exemplary Western blot analysis using anti-Fc antibody- (SEQ ID NOs: 52 and 54) or Strept-tactin- (SEQ ID NO: 66) HRP conjugate-based detection. An exemplary Western blot analysis using anti-Fc antibody- (SEQ ID NOs: 52 and 54) or Strept-tactin- (SEQ ID NO: 66) HRP conjugate-based detection. An exemplary Western blot analysis using anti-Fc antibody- (SEQ ID NOs: 52 and 54) or Strept-tactin- (SEQ ID NO: 66) HRP conjugate-based detection. Gels of various recombinant Croto protein constructs shown are shown. A series of gels of each of the indicated recombinant Croto protein constructs is shown. Same as above. Same as above. Same as above. Same as above. Density data evaluated from the top 10 clones. Survival data evaluated from the top 10 clones. Productivity data evaluated from the top 10 clones. Productivity data evaluated from the top 10 clones. Data of the top 4 clones. Data of the top 4 clones. Data of the top 4 clones. Data of the top 4 clones. Shows copy number stability. It shows the stability of 5 of the clones. It shows the stability of 5 of the clones. It shows the stability of 5 of the clones. It shows the stability of 5 of the clones. It shows the stability of 5 of the clones. The purified Klotho protein observed on the gel after ELISA is shown. The measured Klotho protein concentration and the spike recovery observed in each sample are shown. The measured Klotho protein concentration and the spike recovery observed in each sample are shown. The measured Klotho protein concentration and the spike recovery observed in each sample are shown. Comprehensive glycan preliminary analysis. Comprehensive glycan preliminary analysis. Shows the growth of each cell line. The survival rate of each cell line is shown. The titer curve of each cell line is shown. Shows the growth of each cell line. The survival rate of each cell line is shown. The titer curve of each cell line is shown. Shows the growth of each cell line. The survival rate of each cell line is shown. The titer curve of each cell line is shown. Western blot analysis for the expressed protein pool. Western blot analysis for the expressed protein pool. Indicates the elution of Klotho protein. Same as above. Indicates the elution of Klotho protein. Indicates the elution of Klotho protein. K _{on (association) shows} an analysis of _{K off} (dissociation), and _K D (Koff / Kon). The figure which shows that the purified Klotho protein of both natural His and Fc-Klotho stimulated cell proliferation in a dose-dependent manner. The figure which shows that the purified Klotho protein of both natural His and Fc-Klotho stimulated cell proliferation in a dose-dependent manner. The figure which shows that the purified Klotho protein of both natural His and Fc-Klotho stimulated cell proliferation in a dose-dependent manner. The figure which shows that the purified Klotho protein of both natural His and Fc-Klotho stimulated cell proliferation in a dose-dependent manner. The figure which shows that the purified Klotho protein of both natural His and Fc-Klotho stimulated cell proliferation in a dose-dependent manner. Graph showing the mean (mean) body weight (mean ± standard error, SEM bar) of rats in each group over the course of a 12-day study (daily).

Embodiments of the present disclosure include compositions and methods for increasing serum-soluble Klothoprotein levels in human or non-human animal (or mammal) subjects, and Klothoprotein levels (s), specifically endogenous and. / Or compositions and methods for monitoring, detecting, and / or quantifying exogenous soluble alpha chromatoprotein levels (s) and / or for diagnosing and / or treating a chromatoprotein deficiency in a subject. .. Some embodiments include (i) recombinant human croto proteins, protein fragments, and / or protein variants, expressed nucleic acid constructs and / or vectors, cell lines and / or cell suspension cultures, and / or (ii). ) A product (eg,) that, when administered to a subject, causes an increase in crotoprotein level (s), expression, or production, specifically endogenous soluble alpha crotoprotein level (s), expression, or production. Contains one or more compositions, including supplements). A particular embodiment is a method of producing the composition of the present disclosure, purifying it and administering it to a subject (human or non-human animal), and / or the Croto protein level (s), expression, in the subject. Alternatively, it may include methods for increasing production, specifically (endogenous and / or extrinsic) soluble alpha human protein levels (s), expression, or increased production. Some embodiments are for monitoring, detecting, and / or quantifying croto protein levels (s), specifically (endogenous and / or extrinsic) soluble alpha croto protein levels (s). Methods and / or methods for diagnosing Klotho protein deficiency in a subject.

Prior to elaborating on the various embodiments of the present disclosure, the present disclosure only describes specific parameters, phrases, and specific exemplified systems, methods, and / or products that may vary in each embodiment. It is understood that it is not limited to. Accordingly, certain embodiments of the present disclosure are described in detail with reference to specific properties (eg, configurations, parameters, properties, steps, components, components, components, elements, parts, and / or parts, etc.). However, the description is exemplary and is not construed as limiting the scope of the disclosed and / or claimed inventions. In addition, the terms used herein are for purposes of describing embodiments and are not necessarily intended to limit the scope of the disclosed and / or claimed inventions.

The detailed description is divided into paragraphs, but the paragraph headings and content within each paragraph are for constitutive purposes only and are independent description and embodiments, or limit the scope of the description or claims. Not intended to be. Rather, the content of each paragraph in the detailed description is read and understood as a collective whole, in which the elements of one paragraph relate to and / or characterize another paragraph. Intended. Thus, embodiments specifically disclosed within one paragraph may also be added and / or alternative embodiments within another paragraph having the same and / or similar products, methods, and / or terms. Can be involved and / or can function.

To facilitate understanding, where possible, similar references (ie, components and / or similar names of elements) are used to describe similar elements that are common to the different embodiments of the present disclosure. ing. Similarly, similar components, or components with similar functionality, will be provided with similar reference notation, if possible. Specific languages will be used herein to illustrate exemplary embodiments. Nevertheless, it will be understood that it is not intended to limit the scope of this disclosure. Rather, the language used to illustrate the exemplary embodiments is merely exemplary (unless it is clearly stated herein that such a language is essential) and limits the scope of this disclosure. It is understood that it is not interpreted as a thing.

For brevity, the present disclosure may enumerate a list or range of numbers. However, such a list or range of numbers (eg, above a certain value, less than a certain value, a maximum specific value, at least a specific value, and / or about a specific value, and / or two listed. Where (between values) is disclosed or enumerated, the disclosed values or lists, or any particular value or range of values within a range of values, are similarly disclosed and contemplated herein. It will be understood that it will be done. By way of example, disclosure of a particular component or component of "up to 1,000 mg" is as follows: (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, but not limited to, greater than and less than 1,000 milligrams, and / or (ii) 0.01-1,000 mg, 1 mg-990 mg, 5 mg-750 mg, 10 mg-500 mg, and. Includes specific disclosures of greater than zero and less than or equal to 1,000 milligrams, or any range of values in between, including, but not limited to, 50 mg to 100 mg. Similarly, disclosures of "at least 80% amino acid sequence identity" or "80% to 100% amino acid sequence identity" are (i) 81%, 82%, 83%, 84%, 85%, 86%. , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%, 80% to 100 Any whole percentage value of%, and any fractional part of the percentage value in between, and / or (ii) as a non-limiting example, 81% -100%, 82% -100%, 83% -100%, 84% -100%, 85% -100%, 86% -100%, 87% -100%, 88% -100%, 89% -100%, 90% -100%, 91% -100%, 92% Includes -100%, 93% -100%, 94% -100%, 95% -100%, 96% -100%, 97% -100%, 98% -100%, and 99% -100%, 80 Includes specific disclosures in the range of any percentage value from% to 100%.

Outline List of Defined Terms To help you understand the scope and content of the above and the description and claims set forth below, some selected terms are defined below.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure relates.
Various aspects of the disclosure, including systems, methods, and / or products, may be exemplified with reference to one or more embodiments that are essentially exemplary. As used herein, the term "embodiment" means "acts as an example, case, or example," and is not necessarily preferred over other aspects disclosed herein. Or it should not be interpreted as advantageous. In addition, references to the present disclosure or "embodiments" of the invention are intended to provide exemplary examples without limiting the scope of the invention as indicated by the appended claims.

As used herein and in the appended claims, the singular forms "a", "an", and "the" are both singular and multiple referents, respectively, unless expressly indicated in the context. Is intended, included, and specifically disclosed. For example, references to "proteins" contemplate and specifically disclose one and more than one (eg, two or more, three or more, etc.) proteins. Similarly, the use of multiple referents does not necessarily require multiple such referents, but intends and includes the support of one or more such referents, unless explicitly indicated by context. , Specifically disclose and / or provide.

As used throughout the present disclosure, the words "can" and "may" do not have a compulsory meaning (ie, mean must), but rather. Used in an acceptable sense (ie, meaning having potential). In addition, the terms "inclusion", "having", "involving", "contining", "characterized by", and variations thereof (eg, "characterized by"). Similar terms used herein include "includes," "has," and "involves," "contines," etc.), as well as claims. , Comprehensive and / or unlimited, and, exemplary, shall have the same meaning as the word "comprising" and its variants (eg, "comprise" and "comprises"). , And do not exclude additional unlisted elements or method steps.

It is understood that all numerical values in this description, which indicate a material or reaction condition and / or amount of use, are modified by the word "about" except in the examples or unless otherwise explicitly indicated. Is to be done. As used herein, with respect to a value, the term "about" means +/- 10% of the value stated or the amount represented therein. For example, throughout the disclosure, the term "about" refers to the percent concentration or composition of a component or component (eg, in a mixture such as a fluid or liquid mixture, an aqueous mixture, a solution, etc., optionally or preferably w / w percent, w /. Used for (measured as v percent, v / v percent, etc.). In such cases, the term "about" and / or the term "+/- 10%" will add +/- 10% of the stated number, as opposed to the listed percentages +/- 10 percentage points. Means and / or includes. As an example, if 20% (w / w) of a component or component reflects 20 g of component or component per 100 mL of the total mixture, the term "about" and / or the term "+/- 10%" is 10 Means the listed range of 18 g to 22 g (ie, 18% (w / w) to 22% (w / w)) rather than the range of% (w / w) to 30% (w / w). And / or include. Substitutes for so-called "approx." Values and / or +/- 10% are +/- 1%, +/- 2%, +/- 3%, +/- 4%, +/- 5 of the stated values. %, +/- 6%, +/- 7%, +/- 8%, or +/- 9%, each of which uses the term "about" or +/- 10% herein. Considered as a suitable alternative or alternative.

As used herein, the terms "approximately" and "substantially" are used in the amounts described (eg, still performing the desired function or achieving the (desired or expected) result). Represents or means a close (or arbitrary) quantity. For example, the terms "approximately" and "substantially" are in the range of 10%, 5%, 1%, 0.1%, 0.01%, or other percent of the stated amount or less. Can point to. As used herein, the term "substantially free" is generally considered by one of ordinary skill in the art to reflect (1) an undetectable or unquantifiable amount, and (2) a detectable or quantifiable amount. Less than or less than or less than or less than or less than (3) an amount generally considered by one of ordinary skill in the art to be functional or to achieve (desired or expected) results. (For example, 10%, 5%, 1%, 0.1%, 0.01%, or less than any other percentage).

"Appropriate amount" (also referred to as "appropriate amount (qs)" or "appropriate amount (qs)") means a sufficient amount. Thus, a component or component "in the right amount of 100%", "provided in the right amount of 100%", or "up to 100% in the right amount" is used by the component or component to complete the composition, or (listed). Indicates that it is provided or contained in an amount sufficient to bring the whole (of all components) to 100%, whether or not it is. However, the "appropriate amount 100%", "provided in the appropriate amount 100%", or "up to 100% appropriate amount" (final) component or component is such that the mixture immediately precedes the "appropriate amount 100%" component. Note that it does not indicate that it consists of, is essentially made up of, or contains only those components listed or enumerated. In other words, the terms "appropriate amount 100%" and similar terms are intended to be an unlimited expression indicating the source of the rest, whatever the rest.

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

As used herein, the "characteristics" or "modes" of this disclosure or the embodiments disclosed herein are the characteristics, properties, components, components, elements, members, parts of the immediate subject matter. , Part, (method) step, or other face.

As used herein, the word "or" generally means any one member of a particular list, but also includes any combination of two or more members of that list. Similarly, the terms "and" can be compatible with the term "or" and both can be understood to mean "and / or".

The term "comprising" is synonymous with "inclusion," "having," "contining," or "characterized by." These terms are comprehensive and unlimited and do not exclude additional, uncited elements or method steps.

The phrase "consisting of" excludes elements, steps, or components that are not specified in the claims. If this phrase appears in a clause of the body of the claim rather than immediately after the preamble, it limits only the elements described in that clause and the other elements are not excluded from the entire claim.

The phrase "becomes essential" limits the scope of the claims to those that, in addition to the particular material or step, do not substantially affect the basic and novel features (s) of the claimed subject matter. To do.

The terms "comprising", "consisting of", and "consisting of essentially" can be used alternatives. If one of these three terms is used, the currently disclosed and claimed subject matter may include the use of any of the other two terms.

The terms "plurality" and "at least two" are used interchangeably.
The term "pathology" refers to any disorder, disease, injury, or disease that appears or is expected in a patient, as understood by those skilled in the art. The manifestation of such a condition can be an early, middle, or late stage manifestation known in the art, including pre-symptoms, precursors, or markers. Predictions of such conditions are predicted, predicted, imagined, estimated, assumed, and / or speculated, whether found on scientific or medical basis, risk assessment, or mere anxiety or fear. Can occur or include them.

As used herein, the term "patient" is synonymous with the term "subject" and, as the term is defined herein, specifically (i) (doctor, nurse, With respect to humans (under the protection of a medical assistant or medical volunteer) and non-human mammals (ii) such as non-human mammals (under the protection of a veterinarian or other veterinary specialist, assistant, or volunteer), generally, Refers to any animal under the care of a medical professional.

As used herein, the term "healthcare 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. Focuses on qualified or unqualified healthcare providers (such as assistants, technicians, and / or volunteers), specifically those covered by a (comprehensive) license or healthcare provider's insurance .. The term may include oncologists, surgeons, or physician assistants, nurses, phlebotomists, veterinarians, and the like, where appropriate in context.

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

As used herein, the terms "diagnosing" or "diagnosing" and similar terms are required or regulated by the US Food and Drug Administration (FDA) or any other government or non-governmental agency, organization, or agency. Is not intended to convey or require (in any case) a certified and regulated medical diagnosis. Rather, as used herein, "diagnosing" or "diagnosing" and similar terms relate to providing information that indicates a condition and / or can help make a determination related to the condition. ..

The term "co-administration" and similar terms refer to parallel, continuous, and / or concomitant administration of two or more components. For example, the two components may be co-administered by administering each component in separate dosages in parallel, simultaneously, or consecutively (eg, separate administrations separated by a period of time). The period may be very short (eg, substantially immediately after the first dose) but longer (eg, 1-60 seconds, 1-60 minutes, 1-24 hours, 1-7 days, 1- It may be 4 weeks, 1-12 months, etc., or any value or range of values between them). Parallel or co-administration can include overlapping dosing time frames for two or more components, or administration of a combination product containing a mixture of two or more components.

As used herein, "room temperature" exceeds the freezing point (0 ° C., or other (equivalent) freezing temperature depending on the presence of freezing point adjusting components) and is normal human body temperature (37 ° C.). , Or other (equivalent) boiling temperature), preferably above about 4 ° C and less than about 35 ° C, more preferably from about 5 ° C to about 32 ° C, about 8 depending on the presence of boiling adjustment components. ° 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 Approximately 25 ° C, approximately 15 ° C to approximately 20 ° C, approximately 20 ° C to approximately 30 ° C, approximately 20 ° C to approximately 25 ° C, approximately 20 ° C to approximately 22 ° C, or any value or range of values between them. Can refer to the temperature of.

As used herein, "refrigerate" is above the freezing point (0 ° C) and below about 32 ° C, preferably about 1 ° C to about 30 ° C, about 1 ° C to about 25 ° C, 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 It can refer to a temperature of ° C., about 4 ° C. to about 15 ° C., or any value or range of values in between.

As used herein, "nucleic acid" and similar terms are naturally occurring, either DNA or RNA or DNA-RNA hybrids, single-stranded or double-stranded, sense or antisense. Alternatively, it 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. The nucleic acids of the present disclosure also include nucleotide or nucleic acid analogs known in the art (eg, BrdU), and non-phosphodiester (inter-nucleoside) or backbone (eg, peptide nucleic acid (PNA) or thiodiester bonds). 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 composed of contiguous amino acids. The term "peptide" or "protein" includes naturally occurring or synthetic molecules.

As used herein, the term "standard amino acid" includes alanin-ala-A; arginine-Arg-R; aspartin-asn-N; aspartic acid-asp-D; cysteine-cys-C; glutamine-. gln-Q; glutamic acid-glu-E; glycine-gly-G; histidine-his-H; isoleucine-ile-I; leucine-leu-L; lysine-lys-K; methionine-met-M; phenylalanine-phe- F; proline-pro-P; serine-ser-S; threonin-thr-T; tryptophan-trp-W; tyrosine-tyr-Y; and valine-val-V are included.

As used herein, "codon-optimized" or "codon-optimized" means that a codon in a nucleotide sequence is preferred for the codon usage pattern in the organism used for the expression of its molecule, or Refers to the process of modifying or altering a more closely matched codon. Thus, for use in specific organisms where expression is desired based on known codon usage in the organism to enhance the effectiveness of nucleic acid expression, eg, to achieve faster translation rates and higher accuracy. Therefore, codons can be optimized. The use of codons in certain organisms is known.

The coding nucleic acid molecule is such that the modified wild form, or codon, is expressed in a particular host cell, eg, in a mammalian cell such as a CHO cell or 293 cell, or in a yeast, or in a plant cell, eukaryotic cell, etc. Can be a codon-optimized sequence optimized for.

In certain examples, the nucleic acid sequence can be codon-optimized, for example, to increase the expression level of the encoded sequence. Specific codon usage depends on the host organism on which the modified polypeptide is expressed. One of ordinary skill in the art can use in mammalian or human cells, eg, E. Familiarity with codons optimal for expression in bacteria or yeast, including coli or Saccharomyces cerevisiae. For example, codon usage information can be found in kazusa. or. jp. It is available from the Codon Usage Database, available at codon (see, for example, Richmond (2000) Genome Biology, 1: 241 for a database description, and Forceburg (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 to all subsequent uses of the same abbreviation and applies mutatis mutandis to the usual grammatical variety of the first defined abbreviation, unless explicitly stated otherwise. Measurement of properties is determined by the same technique as previously or later mentioned for the same properties.

Exemplary Embodiments The exemplary embodiments of the present disclosure include compositions, composition of substances, products, products, formulations, combination products, co-administration, co-formulations, dosages, processes, methods, therapeutic protocols, Includes, but is not limited to, diagnostic methods, kits, etc.

Products and Methods for Stabilizing Blood Croto Proteins Stability of blood or serum croto proteins (eg, soluble croto), especially at room temperature and for a more particularly (extended) period, has been previously reported. It has not been. FIG. 3 illustrates the surprising and unexpected stability of Klotho protein in human serum over time at room temperature and other temperatures.

Without being bound by any theory, typical blood or serum protein diagnostics are intravenous blood draws to draw a suitable amount of blood for monitoring, detecting, and / or quantifying proteins (levels). Needs. In addition, the collected blood sample is typically processed immediately after sampling (eg, same day, within minutes or hours of sampling) or refrigerated to preserve protein and sample stability. (Less than room temperature). However, in some embodiments of the present disclosure, the inventors have found that the blood crotho protein can be stable at room temperature for up to 7, 9, 14 or more days. In some embodiments, at room temperature, for example, by adding EDTA and / or heparin to the sample, the Croto protein is applied for at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 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 can be stabilized (data not shown). As a non-limiting example, the level of croto in blood samples taken from 4 donors was relatively constant over 7 days (and 14 days) at room temperature with the addition of EDTA and / or heparin. (See Figure 3). In addition, both anticoagulants tested, EDTA and heparin, showed no significant difference in terms of croto concentration. This was surprising and unexpected for the inventor.

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

In view of the above, embodiments of the present disclosure may include methods and / or products of combining EDTA and / or heparin with a biological sample (eg, blood or serum) containing a Klotho protein. Some exemplary products may include a sampling container and a sampling kit containing a sample storage composition.

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

In addition, we found that the Klotho protein level measured by the finger stick method (about 10 to 1000 μl, preferably about 50 to 200 μl of blood) is consistent with the Klotho protein level measured from intravenous blood draws of the same subject. I found out to do. Therefore, Klotho protein can be measured accurately, reproducibly and reliably with a relatively small amount of commercially practical blood. This was surprising and unexpected for the inventor. Specifically, the inventors predicted that the amount of blood collected in a standard vial could be required to accurately, reproducibly, and reliably measure blood croto protein. ..

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

From the above standpoint, some embodiments of the present disclosure may include products and methods for sampling blood or serum capillaries (non-venous, non-phlebotomy, etc.). Some embodiments may include a capillary blood sampling device. Illustratively, the capillary blood sampling device can be or include a lancet incision device and / or may include a (blood) lancet). In some embodiments, the blood sampling or lancet incision device can be or include a finger stick device such as a (spring-loaded) finger puncture device. However, it will be appreciated that the present disclosure is not limited to finger and rapid finger puncture devices. In some embodiments, the lancet incision device or lancet thereof may be disposable and / or replaceable. Embodiments may also include written instructions for taking a blood sample using the kit.

An exemplary method of stabilizing crotoprotein in a mammalian blood sample is to obtain capillary blood from a mammalian subject, the capillary blood containing an amount of soluble crotoprotein. That is, at room temperature, the capillary blood is stored in the container for at least 24 hours, wherein the container has a preservative or anticoagulant placed therein and is a preservative or anticoagulant. However, the preservative or anticoagulant stabilizes the soluble croto protein for at least 24 hours at room temperature, and the preservative or anticoagulant is heparin, lithium heparin, EDTA. , And storage, including one or more of _{K 2 EDTA.} In some embodiments, capillary blood mixed with a preservative or anticoagulant is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 at room temperature. , 14, 18, 21, 28, or 30 days, where the preservative or anticoagulant will at least 2, 3, 4, 5, 6, 7, 8, soluble croto protein at room temperature. Stabilize and store for 9, 10, 11, 12, 13, 14, 18, 21, 28, or 30 days. In certain embodiments, the sample of the mixture may also be refrigerated.

In certain embodiments, small amounts of blood (1-4 drops, about 10-1000 μl, preferably about 50-200 μl) can be collected (eg, at home) and are unfrozen, unrefrigerated, or uncooled (eg, eg, at home). It can be delivered via the usual method of transport to a diagnostic facility (ambient temperature or room temperature). This provides substantial benefits compared to existing methods or products. Therefore, personal, home, or other non-clinical based trials or diagnoses (eg, lateral flow with quantification / measurement indicators, home ELISA kits, etc.) are contemplated herein. However, it will be understood herein that clinical or clinically based trials or diagnoses are also contemplated.

Products and Methods for Measuring Croto Protein Levels Some embodiments detect endogenous and / or extrinsic croto proteins, and more specifically endogenous and / or exogenous soluble alpha croto proteins. Includes methods for quantification. Some embodiments include a method of diagnosing a Croto protein deficiency in a subject. Some embodiments include products (eg, systems or kits) for detecting and quantifying Klotho protein levels, and more specifically endogenous and / or extrinsic soluble alpha Klotho protein levels. Some embodiments include a method of diagnosing a Croto protein deficiency in a subject.

It is hypothesized that the level of circulating soluble croto protein in mammalian serum decreases with age. However, prior to this disclosure, at what age this assumed reduction in serum humanity levels can begin to occur, what is the normal level (or range) of naturally occurring soluble circulating humanity proteins in humans and non-human animals. Reliable and reproducible of what is, what is the rate of decline of circulating humans in humans and non-human animals, and how low the level (or range) of naturally occurring soluble human protein is in humans and non-human animals. Good data is not available or widely accepted. Over-the-counter antibodies are reliable, reproducible, and / or of (both endogenous and extrinsic) Klotho proteins in mammalian blood or serum samples (crude and / or substantially unpurified). It may not be suitable for cost-effective detection. Therefore, suitable antibodies (eg, for detecting blood sample ELISA) were developed according to the following procedure.

Phase 1: Crotoprotein expression: Alpha croto proteins of various sizes (eg, mouse and human) with (C-terminal) 6xHis tag, (C-terminal) Fc tag, or (N-terminal or C-terminal) HAS tag. , In the case of humans, amino acid residues 34-981, 34-549, etc.) were produced. The gene construct corresponding to the above protein was synthesized, cloned into a commercially available expression vector, and then transfected into HEK293 cells or CHO cells for Klotho protein production. The expressed Klotho protein was purified by a known and disclosed method. Illustratively, using IMAC and IE protein chromatography, a 0.78 mg / mL protein sample (measured by UV absorbance at 280 nm) in 330 mM NaCl, 20 mM Na phosphate buffer, pH 8.0. Got Similarly, the protein A chromatography described herein, followed by size exclusion chromatography, gave active proteins in the form of monomers and multimers (eg, dimers and tetramers).

Quality Control: Purity and completeness of the purified protein was analyzed with 4-20% SDS-PAGE (reduced and non-reduced) stained with GelCode Blue reagent. See, for example, FIG.

Lane 1: Croto ECD, lot number 170918A, 2 μg / lane, reduction.
Lane M: Protein molecular weight marker.
Lane 2: Croto ECD, lot number 170918A, 2 μg / lane, non-reduced.

Lane 3: BSA standard, 2 μg / lane, reduction The theoretical molecular weight calculation of the expressed protein was 109.97 kDa.
Phase 2: Development of chicken polyclonal (IgY) and mouse monoclonal (hybridoma): Using the expressed protein as an antigen, mice and chickens were immunized to develop monoclonal and polyclonal antibodies. In some embodiments of the ELISA antibody, a mouse monoclonal is preferred. After the fusion process (spleen cells with myeloma cells), two rounds of hybridoma selection were performed, approximately 3,000 clones were screened first, and finally narrowed down to 10 clones based on activity. That's right. These 10 clones were stored, cultured and purified to produce about 5 mg of purified antibody from each.

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

Croto-Fc 0.5 μg / mL in Na bicarbonate buffer of antigen 2-50 mM.

Summary of Table 1: Mice M3 and M5 were selected for spleen harvesting.
Primary screening of croto hybridomas: Mouse number M3 and number M5 splenocytes were harvested and fused with myeloma cells according to standard protocols.

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

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

Selective cutoff OD 450nm> 0.5.
The table below shows the primary Klotho hybridoma screening. Clones with an OD of 450 nm between 0.5 and <2 are highlighted in yellow (light gray). Clones with an OD of 450 nm> 2 are highlighted in orange (dark gray).

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

Antigen, (1) Croto-6xHis protein in 50 mM sodium bicarbonate (lot number 170918A, 0.78 mg / mL) 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.

Detected antibodies: anti-mouse HRP, peroxidase AffiniPure goat anti-mouse IgG (subclass 1 + 2a + 2b + 3), Fcγ fragment specific, SEDB 1: 5000 (Jackson Lab., Catalog No. 115-035-164), 100 μL / well, 1 hour, room temperature ..

Summary of Table 3: Twenty-eight Croto-6xHis-specific clones were identified (OD450 cutoff:> 0.5, Croto-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 Nine Croto-6xHis Specific Clone-2E12, 4H4, 4H5, 5D12, 11C9, 11F7 Recommended for Natural (Untagged) Croto ELISA Development (OD450> 2, Croto-6xHis Plate) , 14B12, 26G3, 28F11.

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

A first array of 10 Croto-6xHis and Croto-Fc specific clones (OD450> 1.7, recommended for the development of an ELISA capable of measuring both untagged and Croto-Fc proteins. Clone-6xHis and Clone-Fc Plates) -1F6, 2E12, 4H4, 4H5, 8G11, 11C9, 11F7, 14B12, 26G3, 28F11.

A second array of two Croto-6xHis and Croto-Fc specific clones (OD450> 1.2 and recommended for the development of an ELISA capable of measuring both untagged and Croto-Fc proteins. <1.7, Clone-6xHis and Clone-Fc Plate) -5D12, 14B7.

mIgG Isotyping: mIgG Isotyping of Croto-Hybridoma Selective Clone: Antibody Isotyping from Selective Hybridoma Clone Culture supernatant.
Using a sample of hybridoma culture medium, a rapid ELISA mouse mAb isotyping kit (ThermoFisher, Catalog No. 37503) was used according to the manufacturer's instructions to determine antibody isotypes.

The table below shows the results of mIgG isotyping of Croto hybridoma selective clones.

Summary of Table 4: mIgG Isotype Determination:
1F6, 2E12, 4H4, 4H5, 8G11, and 11F7: IgG1
11C9, 14B12, 26G3, and 28F11: IgG2b
Quality Control: Indirect ELISA was performed for quality control: ELISA plate in 0.5 μg / ml non-conjugated 6xHis tagged Croto protein or Croto-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 to give the secondary antibody goat anti-mouse HRP. , 0.16 μg / ml. The reaction was allowed to occur for 7 minutes. Raw absorbance data at 450 nm and the corresponding antibody titration curve.

See also the dose responsiveness curves exemplified in FIGS. 5A, 5B, and 6, respectively.
Quality Control: Purity and completeness of the purified antibody was analyzed on 4-20% SDS-PAGE (reduced and non-reduced) stained with GelCode Blue reagent. See FIGS. 7A and 7B, respectively.

Polyclonal IgY antibody: Polyporous IgY antibody has also been developed. The above design was adapted as needed for the production of polyclonal IgY antibodies in chickens.
Evaluation of chicken serum IgY titer using target antigen by indirect ELISA (TMB: 5 minutes). Reading at 450 nm Antigen: Croto 1 μg / mL (0.78 mg / mL, lot number 170918A) in 50 mM sodium bicarbonate.

Samples: Chicken No. 1, No. 2, room temperature, 1 hour (5% milk in PBS).

ELISA plates were coated with 0.5 μg / ml 6xHis tagged Croto protein or Croto-Fc protein in 50 mM Na bicarbonate buffer overnight at + 4 ° C. Purified anti-Klotho IgY antibody (lot number 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 the corresponding antibody titration curve. See also FIGS. 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 FIGS. 9A and 9B, there is a relatively weak signal in the IgY / HRP-IgY pair. IgY / Bio-IgY shows a relatively high background even at low concentrations (0.25 μg / mL). The IgY / Bio-4H5 pair appears to work better than the IgY / labeled-IgY pair.

It is hypothesized that the level of circulating soluble croto protein in mammalian serum decreases with age. Reference measurements of naturally occurring circulating soluble croto proteins in human subjects have been reported in various publications. A thorough review of the literature gave the following average range (s) of circulating soluble croto proteins in the human subjects shown (see Tables 8-9).

In the above table, 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.

From the point of view of volatility observed in the literature, a more robust, more consistent and / or more accurate means of measuring serum crotho levels is needed. Embodiments of the present disclosure include a Klotho diagnostic kit and methods of using it, which are described in more detail below.

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

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

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

Crotho Stability in Human Plasma Using ELISA Kits Samples of venous and capillary (“finger”) blood were taken from 4 donors (A, B, C, D) and (i) EDTA or (i) EDTA or ( ii) Stored in a heparin-containing tube. A 1.5 ng / mL croto (lot number 170918A-human soluble alpha croto) was spiked into a set of venous and capillary blood samples taken from donor A. The collected blood (blood sample) was stored at room temperature for 0, 1, 4, 16, 24, 48, 72, 96, or 168 hours and then settled. Plasma from each sample was stored at −80 ° C. prior to the ELISA test. ELISA plate maps, raw data, standard curve data, and concentrations are shown in Tables 12-21 below.

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

Table 23 below shows the recovery rate (%) of spiked Croto.

Croto Stability Tests at Various Temperatures Emulating Transport Stress Blood technologists (s) collected over 12 mL of blood (sample) from each of the 4 donors into heparinized blood tubes. The technician (s) immediately dispensed 300-400 μL of blood from each donor's sample into a separate heparinized microtube (manufactured by BD). Tubes were stored and processed according to the test design (see table below). Blood was treated at each time point or centrifuged and stored (plasma) at −80 ° C. All samples were tested approximately on day 17.

In this test, 120 data points representing 6 conditions over a 14-day period 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 in the soluble Klotho ELISA kit (human), IBL, Catalog No. 27998). The raw data are shown in Tables 26-29 below.

The data analysis is shown in Tables 30-41 below:

From the above standpoint, at least one embodiment comprises a Klotho detection kit and / or assay. The kits and / or assays of the present invention may be suitable for CLIA laboratory testing of clinical samples. The kit includes monitoring, detection, and / or monitoring of one or more proteins selected from (1) native (eg, endogenous) croto, (2) recombinant croto-Fc fusion, and (3) recombinant 6xHis croto. Alternatively, it may be configured for use in quantification or diagnosis of its deficiency. Klotho protein can be detected in biological samples. In some embodiments, the sample can be (substantially) unpurified. For example, the sample can be or contain mammalian (eg, human) blood and / or plasma.

In at least one embodiment, the kit is adapted, configured, or suitable for one or more lancets, adapted, configured, or suitable for drawing blood from a fingertip. Sampling container, one or more disinfecting elements (such as disposable (eg, alcohol) wipes (s)), one or more adhesive bandages, foil envelopes or other biodegradable packages, and shipping containers (such as envelopes). ) Can be included.

Some embodiments may include methods of diagnosing croto deficiency in a mammalian subject. The method is (i) mammalian serum in the form of capillary blood, optionally mixed with a preservative or anticoagulant containing one or more of heparin, lithium heparin, EDTA, and K2EDTA. Consists of a mammalian serum having a certain amount of soluble croto protein, and (ii) a mammalian serum optionally in the form of heparinous blood, having a certain amount of soluble croto protein. The method may include obtaining a fluid sample mixture selected from the group, the method mixing the mammalian serum with a preservative or anticoagulant containing one or more of heparin, lithium heparin, EDTA, and K2EDTA. Can further include that. The method may also include storing the mixture at room temperature for at least 24 hours or longer, with the preservative or anticoagulant stabilizing the soluble croto protein at room temperature for at least 24 hours or longer. After the storage step, the method may include quantifying the amount of soluble croto protein in the mixture. The method may also include diagnosing a mammalian subject as a Croto deficiency when the quantified amount of soluble Croto protein in the mixture is less than a predetermined threshold amount.

As mentioned above, the fluid sample mixture comprises from about 10 to 1000 μl, preferably about 50 to 200 μl of capillary blood from the mammalian subject, or the step of obtaining the fluid sample mixture is from about 10 from the mammalian subject. Includes obtaining ~ 1000 μl, preferably about 50-200 μl of capillary blood. The steps to quantify the amount of soluble croto protein in the mixture are to detect the soluble croto protein using a first antibody that binds to a portion of the soluble croto protein, and optionally a portion of the first antibody. Includes detecting soluble Klotho protein using a detection antibody that binds to, or performing enzyme-bound immunoadsorption assay (ELISA) to detect and optionally quantify soluble Klotho protein. Mass spectrometry can also be used to detect proteins.

In some embodiments, diagnostic reports or information reports may be provided. The report may include indications or indications of the measured croto level (s) of one or more croto proteins. In some embodiments, the report overlays an age-based graph exemplifying mean or median Klotho levels of various ages, as well as comparative charts or graphs depicting measured levels, as well as percentiles of various ages. Display instructions for the 25th and / or 75th percentile. FIG. 13 illustrates a graph or chart of an exemplary report. Thus, in some embodiments, diagnosing a mammalian subject as a Croto deficiency is displaying a determination, instruction, or diagnosis of a Croto deficiency on the user interface of a computer system, and / or determining a Croto deficiency. Determining, instructing, or diagnosing croto deficiency, including generating files or reports in physical or electronic form that display instructions or diagnoses, optionally quantified amounts of soluble croto protein and / or Contains a predetermined threshold amount.

As used herein, the terms "diagnosing" or "diagnosing" and similar terms are required or by the US Food and Drug Administration (FDA) or any other government or non-governmental agency, organization, or agency. It will be understood that it is not intended to convey or require (in any case) a regulated, accredited and regulated medical diagnosis. Rather, "diagnosis" or "diagnosing" and similar terms relate to providing information that indicates a condition and / or can be useful in making a determination related to the condition.

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, one or more additional analytes include FGF, PTH, non-oxidized PTH, vitamin D, vitamin E, KIM-1, NGAL, interleukin and other inflammatory biomarkers, testosterone, estrogens. It can be selected from the group consisting of.

Therapeutic Proteins The embodiments of the present disclosure may comprise one or more therapeutic and / or recombinant human alpha-soluble croto proteins, protein fragments, and / or protein manifolds.

The proteins are amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549 of human alpha crotoisoform 1 or 2. , 36-549 or 131-549, all or a subset. The proteins are amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549 of human alpha crotoisoform 1 or 2. , 36-549 or 131-549 and all or a subset and at least and / or about 80% of the amino acid sequence. For example, at least a portion of the protein is at least one portion 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. , Or a combination thereof, and may have at least 80% amino acid sequence identity. Other parts or fragments of the protein sequences described in this application are also contemplated herein. For example, some embodiments have any suitable portion of any one of SEQ ID NOs: 1-75, or a combination of two or more thereof, and at least a portion having an amino acid sequence identity, such as at least 80%. May contain proteins.

Some embodiments may comprise a protein having one or more amino acid varieties as compared to human alpha chloride isoform 1. Illustratively, the protein may comprise a human C370 manifold. For example, the protein may include a C370S modification, thereby comprising S370. In some embodiments, the protein may include something other than human F352, or F352V. In at least one embodiment, the protein comprises S370, which may comprise a C370S modification, thereby containing no F352V manifold and preferably containing F352. The protein may include anything other than the H193, or H193R manifold. At amino acid residues (or positions) 193, 352, and / or 370 of human alphacrotoisoform 1, all other standard amino acid substitutions are contemplated and expressly disclosed herein.

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

The protein can also contain one or more glycans (bound to it). For example, native human alpha crotoisoform 1 may have glycans bound (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612 and / or 694. Thus, the proteins of the present disclosure or their Klotho protein sequences contain one or more of the same (or similar) glycans attached to it (eg, at the same amino acid position (s)) (via glycosylation). Can have. In a preferred embodiment, the protein comprises all of the same or similar (natural) glycans attached thereto at the same amino acid position (s).

In some embodiments, the protein may comprise a signal peptide or signaling sequence. For example, the protein may contain a native Croto signal transduction sequence. Proteins can include non-natural or synthetic signaling sequences. In some embodiments, the signaling sequence can be upstream (or N-terminal) of the N-terminal signaling sequence and / or the Croto protein sequence. In other embodiments, the signaling sequence can be C-terminal or otherwise arranged. Preferably, the signaling sequence is, or comprises, or at least 80 of the native human alpha crotoisoform 1 signaling sequence, the native human alpha croto isoform 2 signaling sequence, SEQ ID NO: 71 or SEQ ID NO: 72. % May have amino acid sequence identity.

In some embodiments, the protein may comprise an amino acid tag. The tag can be the C-terminal tag and / or downstream (or C-terminal) of the Croto protein sequence. In other embodiments, the tag can be N-terminal or otherwise placed. The tag can be or may include an Fc peptide (or Fc fusion tag, Fc domain, etc.). For example, the tag can be or contain an IgG1-Fc protein sequence. Preferably, the tag is or contains SEQ ID NO: 74, or can have at least 80% amino acid sequence identity with it.

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

The tag can also, or alternative, be polysialic acid (PSA) or include it. In at least one embodiment, the PSA tag can make a conjugate (croto) protein resistant to one or more proteases. In some embodiments, maintaining an amount (eg, even a relatively small amount) of sialic acid in the protein conjugate is a protease attack, as well as targeting, degradation, and clearance of subsequent molecules (eg, for example). (Through the liver) can be prevented.

In at least one embodiment, the tag can be cleaved from the protein. In other embodiments, the tag 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, the tag can be utilized during protein purification (eg, as part of a purification mechanism).

In some embodiments, the protein may comprise a linker (eg, an 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, most preferably about 8-10 amino acids. In some embodiments, the linker, GS linker _{(e.g., (G} 4 _{S) 2} linkers) or other linkers (e.g., GGENLYFQ linker) or can be a, or include it. Preferably, the linker is or contains SEQ ID NO: 73 or SEQ ID NO: 105, or can have at least 80% amino acid sequence identity with it.

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, Klotho protein can be at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% pure by dry weight. In some embodiments, the Croto protein sample is placed at or between parts per million (ppm) less than about 1-100 and one billion parts (ppb) less than about 100-1000, or about 1-100 ppb. It may contain any value or range of CHO host cell proteins (HCPs), nucleic acids, and / or other cellular constituents.

Croto protein manifolds Various lengths of therapeutic S-croto proteins (eg, S-croto, isoforms 1 or 2, 1-2, 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 not exhibited by the native Croto protein. Illustratively, the QuickChangeXL site-specific mutagenesis kit (Stratagene) can be used to modify the nucleic acid sequences of various S-Cloteau constructs. Other mutagenesis methods and kits known in the art may also be used. For example, various subcloning methods and kits are known in the art and are commercially available. Amino acid changes can affect protein expression, protein stability, protein solubility, protein binding, protein specificity, protein activity, protein function, immunogenicity, toxicity, etc. (eg, improvement, enhancement, reduction, etc.).

In at least one embodiment of the disclosure, the protein is modified with one or more C-terminal tags and / or N-terminal tags. Such tags can function to prolong the serum and / or soluble half-life of proteins (in one or more treatments or other environments). Tags are also the presence or diagnostic localization of proteins, the isolation or removal of proteins, the delivery or transport of proteins, one or more targets of proteins (eg, proteins, nucleic acids, organelles, cellular structural components, etc.). It can be useful as a marker for binding to, enzymatic treatment or cleavage, etc. 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 known and / or further described herein in the art. It can be tagged with other tags or arrays. Additional descriptions are incorporated herein by reference in their entirety, the references "Fusion Proteins for Half-Life Extension of Biopharmacy as a Strategy to Make Biobetters", "What a PEG. And can be found in "Strategies for extended serum half-life of protein therapeutics". In certain embodiments, the linker or linker peptide can be inserted and / or placed between the (natural or manifold) Croto protein sequence and the tag.

In some embodiments, the modified Klotho protein may comprise an alternative (eg, natural, non-natural, and / or synthetic) signal peptide. For example, in some embodiments, the native signal peptide sequence can 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 the N-terminus of the SS can contain a methionine residue. Additional explanations are incorporated in its entirety by a particular reference, the article "Generation of high pressing CHO cell lines for production of recombinant antibodies system". In certain embodiments, the linker or linker peptide can be inserted and / or placed between the (natural or manifold) Croto protein sequence and the alternative SS.

Some embodiments may include one or more amino acid manifolds. The present disclosure contemplates diversifying any one or more of the native amino acids of any of the disclosed Klotho proteins to any other amino acid, whether natural, synthetic, or other composition. Will be understood.

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

S-Klotho C370S protein manifold In humans, the Klotho gene is located on chromosome 13q12. The manifold known as KL-VS is present in approximately 15-25% of Caucasians. This manifold is composed of six single nucleotide polymorphisms (SNPs), two of which cause amino acid substitutions (ie, F352V and C370S-phenylalanine 352 are converted to valine, cysteine 370 is converted to serine). In vitro transfection assays have shown that levels of Klotho secretion are reduced 6-fold in the V352 manifold, while increased almost 3-fold in the S370 form. However, these two manifolds in the human croto gene separate together to form the KL-VS haplotype, which increases croto secretion by a factor of 1.6. For example, screening of more than 300 individuals from geographically and / or ethnically distinct cohorts found no individual carrying only one of the V352 or S370 manifolds. It is reported that it has not.

One embodiment of the present disclosure comprises a recombinant S-croto protein having a C370S homogene (ie, in the absence of (or with a deletion thereof) of the F352V manifold). The C370S manifold can be produced and / or expressed in the context of any of the protein constructs described herein. For example, C370S manifolds, with or without tags (eg, (IgG) Fc tags, TEV-Twin-Strep tags, TEV sequences, etc.), S-croto, isoforms 1 or 2, 1-981, 29- It can be produced or expressed in contexts such as 981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549. Therefore, the nucleic acid construct or cDNA in which the protein is expressed can be of corresponding length.

In an embodiment, the S370 heterozygous or homozygous complex construct is produced, and the resulting construct encoding the S-Clotho C370S protein is expressed in a suitable expression system (eg, via transfection) (eg, CHO). It may include introduction into cells) and / or transient expression of the S-Crotho S370 protein. The S370 croto protein can be expressed at higher levels than the F352V / C370S protein and / or the wild-type F352 / C370 protein. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control tests). Embodiments may include administering a therapeutic or therapeutically effective amount of S-Cloteau C370S protein to a subject in need thereof. The subject may carry or express, for example, the KL-VS manifold. Alternatively, the subject can be of the wild type of another mutant or manifold. Administration of recombinant S-Klotho C370S protein can lead to a beneficial increase in blood S-Klotho levels. Therefore, the circulating concentration of S-croto in subjects administered with the therapeutic recombinant S-croto C370S protein may not be subject to the dilution effect observed in the presence of the F352V manifold.

Nucleic Acids and Expression Vectors Some embodiments may include nucleic acids or nucleic acid constructs. For example, embodiments may include expression vectors or nucleic acid constructs. Nucleic acids can encode recombinant human alpha-soluble croto proteins, protein fragments, or protein manifolds, as described herein. In at least one embodiment, the nucleic acid is a croto protein sequence, any (natural or non-natural) signaling sequence (eg, at the N-terminus of the croto protein sequence), as described herein. Any linker sequence (eg, GS linker) and / or amino acid tag (eg, IgG1-Fc or TEV-Twin-Strep) can 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 chloride isoform 1 or 2. A protein can be expressed that includes all or a subset of 29-549, 34-549, 36-549 or 131-549. At least a portion of the protein is amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29- of human alpha croto (isoform 1 or 2). It may 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 a protein may have at least and / or about 80% amino acid sequence identity with all or part of one of SEQ ID NOs: 1 to SEQ ID NO: 75, or a combination of two or more thereof. In at least one embodiment, the protein is one of SEQ ID NO: 2-SEQ ID NO: 70, or SEQ ID NO: 107-SEQ ID NO: 120, or SEQ ID NO: 125-SEQ ID NO: 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 part of it.

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

The nucleic acid sequences of the present disclosure may also contain stop codons known in the art (eg, TGA, TAG, TAA).
Cell Lines and Production Methods One embodiment of the present disclosure may include a cell line. The cell line may include 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 may include CHO cells (eg, multiple CHO cells). For brevity, references to CHO cells include HEK cells (eg, HEK-293 cells), HL-60 cells, and / or any other (protein-expressing) cell known in the art. ) Or a specific reference to a cell line (s). In some embodiments, the cell 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 NOs: 1 to SEQ ID NO: 75, or a combination thereof, preferably one of SEQ ID NOs: 2 to SEQ ID NO: 70. It can encode a polypeptide having sex.

The nucleic acid may comprise 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-SEQ ID NOs: 106 or sequences. It may have at least and / or about 80% nucleic acid sequence identity with one of numbers 121-SEQ ID NO: 124. The nucleic acid can be a plasmid or other (structural) form of nucleic acid, or can include it.

In some embodiments, the exogenous nucleic acid may encode a functional enzyme such as dihydrofolate reductase (DHFR) and / or glutamine synthetase (GS). In at least one embodiment, the cell can be or contain dihydrofolate reductase (DHFR) deficient CHO cells such as CHO-S cells or CHO-M cells. Nucleic acids can include promoters (eg, from weak promoters to the strongest promoters, as will be appreciated by those skilled in the art). For example, in some embodiments, the nucleic acid is of SEQ ID NO: 2-SEQ ID NO: 70, or SEQ ID NO: 107-SEQ ID NO: 120, or SEQ ID NO: 125-SEQ ID NO: 128, or SEQ ID NO: 133, or SEQ ID NO: 152. One may include a (strong) promoter associated with the transgene having at least 80% nucleic acid sequence identity. Therefore, the transgene can be under the control of a promoter.

For convenience, CHO cells and / or cell lines are referred to throughout the disclosure. However, it should be noted that other cells, cell lines and / or host cells (in addition to CHO cells) are also contemplated within the scope of this disclosure. Therefore, references to CHO cells and / or cell lines also refer to other known cells, cell lines and / or host cells (eg, HEK cells such as HEK-293, HL-60 cells, etc.) and / Or also intend to use.

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

Growth, Selection, and / or Gene Amplification Some embodiments are on solid medium and / or in liquid medium (eg, in suspension cell culture), preferably serum-free and / or animal (or animal-derived). It may include growing cells (eg, transfected cells) in a medium that is free of proteins (components). For example, cells can be plated on solid growth medium over a period of time. Cells can also, or alternative, grow in suspension cultures and / or in liquid media. The liquid medium preferably contains 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 specific period of time (eg, 48 hours after transfection), cells are collected (eg, isolated), optionally centrifuged (eg, 5 minutes 100 xg), and / Or 96 (eg, containing serum-supplemented -HT and / or -glutamine medium) well-adhesive culture plates can be seeded (eg, with approximately 2000 cells / well). In certain embodiments, the medium may also contain MTX and / or MSX. Non-transfected cells can die within 7-14 days after selection (eg, after exposure to MTX and / or MSX in -HT and / or-glutamine medium.

In certain embodiments, the cell contains 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 exogenous nucleic acid (eg, per cell). Obtain (can be selected to contain). Thus, the method selects cells containing at least about 2-10 copies (eg, per cell), at least about 10-20 copies, at least about 20-30 copies, or at least about 30-50 copies of exogenous nucleic acid. Can include that. For example, MTX and / or MSX (at continuously increasing levels) can be administered to cells (eg, at concentrations such as about 1 nM to 1 μM, about 10 to 100 nM).

Dihydrofolate reductase (DHFR) gene amplification in DHFR-deficient CHO cells transfected with exogenous DHFR (such as CHO-S cell lines) continuously increases the level of methotrexate (MTX) in the growth medium. Achieved by. Since the plasmid contains DHFR, the S-croto gene (fragment) in the host cell can be amplified when exposed to MTX (10 to 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 rule out 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 high levels of expression of the S-Klotho protein obtained in transgenic cell lines.

In some embodiments, in suspension culture, the protein can be secreted from the cells into the liquid medium. For example, the particular cells and / or cell lines of the present disclosure may include up to 200-500 mg protein per liter of liquid medium (without enriching the protein), 500-2000 mg protein per liter of liquid medium, 1 liter. 2000-5000 mg of protein per liquid medium, or any value or concentration in the range of values between them can be secreted (or can be selected to be secreted). In at least one embodiment, the concentration of human recombinant alpha-soluble croto protein in medium (of selected suspension cultures (s) of selected cell lines) is at least 200 mg without enriching the protein. High-producing cell lines (or suspension cultures) such that / 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 obtained high S-Crotho transgene-containing colony by cell limiting dilution was performed to further subclon the S-Crotho-secreting cell line that secretes S-Clotho in the range of 500 to 2000 mg / L in the cell conditioned medium. Produced. All cell constructs were restricted digested and sequenced.

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 (eg, produced by DHFR / MTX or GS / MSX systems) Amplified and subsequently cell subcloned high-producing S-Klotho cell lines during scale-up and until final bioreactor operation Carefully used concentrate raw materials administered for cell line production, performed in serum-free and animal protein-free basal media.

Scale-up of high-yielding cell lines is produced in a bioreactor with a volume of 100 L and then 500 L of cell inoculum material expanded in a cell suspension in a shaking flask or in a Wave Bag system. This was done by continuous inoculation of the cells. Cell viability is greater than 85% of viable cells throughout the growth cycle in a shaking flask, wavebag, or bioreactor, followed by 80% of viable cells in the bioreactor during the plateau phase of CHO cell growth. The S-Klotho maintained and produced as described above is concomitantly produced at a maximum of 1 to 3 g / L (referred to as "high productivity").

Protein Production Certain embodiments include recombinant DNA strategies that utilize strong promoter sequences and / or high copy count plasmids for the production of therapeutic amounts of Croto protein in mammalian (eg, 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 use of MTX (at continuously increasing levels). May include. Similarly, exogenous glutamine synthetase (GS) gene-containing cells can be treated with methionine sulfoxymin (MSX).

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

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, Klotho protein can be at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% pure by dry weight. In some embodiments, the Croto protein sample is placed at or between parts per million (ppm) less than about 1-100 and one billion parts (ppb) less than about 100-1000, or about 1-100 ppb. It may contain any value or range of CHO host cell proteins (HCPs), nucleic acids, and / or other cellular constituents.

The glycan structure present on the produced S-croto protein is similar compared to the structure on the native S-croto protein isolated from human body fluids (ie, blood, serum, urine, cerebrospinal fluid). Or it can be the same. In at least one embodiment, confirmation of native-like glycans can ensure that the correct natural post-translational modification (PTM) is produced and stably maintained in the cell-produced S-croto protein.

Dissolution and / or Half-Life Extension of Klotho Protein Disclosed methods and compositions for extending the half-life and increasing solubility of human S-Crotho protein. Purification and characterization of protein constructs so produced to achieve these results is also the subject of the present 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 or chemicals within the amino acid sequence of the Klotho protein (see SEQ ID NOs: 1-70). Relevant information regarding modification and / or addition or removal of a chemical group, peptide, or protein to the amino acid sequence of the Klotho protein is naturally available in biological matrices such as blood, cerebrospinal fluid, urine, or various human tissues. It is taught in the present disclosure to obtain the resulting human Klotho hybrid protein (a novel composition) having an increased biological half-life or increased solubility over that of the Klotho molecule. These novel compositions can be made through the methods described herein for modification of the S-croto protein.

Illustratively, fusion protein constructs are unique such as antibody (IgG), albumin such as human serum albumin (HSA), transferrin such as human transferrin (TF), and / or XTEN®. Can be made by combining with the Fc domain of a recombinant polypeptide of. New proteins can also be produced by conjugating (or modifying) the S-croto protein with post-translational modifications such as polysialic acid (PSA) or pegation. In addition, novel S-croto proteins can be produced via pegging. The aforementioned and other (half-life extension) methodologies increase the S-Crotho dosing interval, provide superior patient convenience and compliance, reduce dosing frequency requirements, and total less drug use. Bringing the amount, reducing the cost of the drug, lowering the drug amount at the same dosing interval as the parent protein, simplifying the dosing formulation and enabling subcutaneous formulation, the same dose and dosing interval as the parent protein S-Crotho by one or more of using to provide higher drug levels, resulting in longer drug exposure and potentially better efficacy, and reducing the immunogenicity of S-Crotho. It can improve protein performance.

Production of Fc Domain Fusion Protein Constructs of S-Crotho Antibody Fc Domain, Human Serum Albumin (HSA), and Polysialic Acid (PSA) have been tested for their effectiveness in prolonging their half-life and increasing the solubility of human S-Crotho protein. did. Fc fusions include fusion of a peptide, protein, or receptor exodomain to the Fc portion of an antibody. Not only do both Fc and albumin fusions achieve an extended half-life by increasing the size of the peptide drug, but both also through the binding of extended proteins to the neonatal Fc receptor FcRn. The natural recirculation mechanism is also used. After binding of the elongated protein to the FcRn receptor, degradation of the fusion protein in the endosome of the cell is prevented. Fusions based on the addition of Fc or albumin can result in a biological half-life in the range of 3-16 days, much longer than that reported for typical PEGylated or lipidified peptides. For the use of protein fusion techniques such as Fc fusion proteins, fusion to human serum albumin, fusion to carboxy-terminal peptides, and other polypeptide fusion approaches to create biobetter drugs with a more desirable pharmacokinetic profile. The comments described are incorporated herein by reference in their entirety, Strohhl WR. Fusion Proteins for Half-Life Extension of Biopharmacy as a Strategy to Make Biobetters. Biodrugs. 2015; 29 (4): 215-239.

The Fc domain was thus added to the parent protein (S-croto) to increase its binding affinity to the Fc receptor (FcRn). FcRn resides inside lysosomes in the endothelial cells that line blood vessels and functions to rescue antibodies from degradation that shortens most proteins in the circulation. As a result of the interaction with FcRn, the protein has a half-life ranging from days to weeks, and the extended form of the protein drug is more than a biologic without the composition of this newly produced substance. Also allows for less frequent dosing.

The dimeric nature of Fc versus the monomeric structure of HSA can lead to the presence of the Fc fusion peptide as a dimer or monomer, as opposed to HSA. The dimerity of the peptide Fc fusion is a binding activity effect when the target receptors for S-croto are sufficiently closely spaced or they themselves are dimers in a particular human target organ. Can be generated. This may or may not be desirable, depending on the target. Fusion of S-croto protein to antibody Fc is also taught herein to improve the solubility and stability of S-croto. Surprisingly, the Fc-fused Klotho protein appears to have improved binding, binding activity, and / or immunogenicity. Addition of the Fc domain to S-croto also allows a reduction in the immunogenicity of the fusion protein upon administration to human subjects.

Conjugation of S-Crotho Protein with Human Serum Albumin (HSA) A 66.5 kDa protein HSA similar to human IgG has a long average half-life within the 19-day range. At a concentration of about 50 mg / mL (about 600 μM), HSA is the most abundant protein in human plasma and has several functions, including its role in maintaining plasma pH, metabolite and fatty acid transport, and maintaining blood pressure. Have. HSA, which is the upper limit of size for renal glomerular filtration of proteins, is also strongly anionic and helps further delay filtration through the kidney. Like IgG, HSA also binds to FcRn in a pH-dependent manner and is recirculated like IgG, even at a site different from IgG binding and through a mechanism separate from that of IgG binding. , Its half-life is extended. HSAs also tend to accumulate in tumors and inflamed tissues, suggesting that fusion or binding to albumin can potentially help target proteins or peptides to their sites. There is.

Fusion of peptides or proteins with essentially short half-life properties to HSA for the extension of serum half-life of these molecules has been extensively studied since the early 1990s. Since then, dozens of different peptides and small proteins have been fused to HSA as both innovative and potential biobetter molecules. The first commercially approved HSA-peptide or protein fusion product was discovered at Human Genome Sciences, developed and marketed by GlaxoSmithKline, and marketed as Tanzeum® (Registered Trademark in the European Union). ) It 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. Therefore, HSA improves the half-life of pharmacologically active GLP-1 from 1-2 minutes to 4-7 days for native GLP-1, which allows weekly dosing. Seven other known HSA fusion protein product candidates are currently under development or are under development in recent years. Novozymes is also developing a modified version 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 wild-type molecules in both mice and cynomolgus monkeys. .. It is expected that these longer half-life HSA variants could be further used as fusion proteins to improve the half-life of fusion proteins.

Therefore, we provide tactical therapeutic benefits such as superior patient convenience and compliance, reduced dosing frequency resulting in lower drug usage in total, and / or reduced product costs. To be used, a Klotho fusion molecule with a significantly extended half-life in human blood, cerebrospinal fluid, and other human biological matrices by fusing the Klotho protein into a wild-type HSA or mutant form of HSA ( It is disclosed herein to produce (s). Also, by reducing the drug dose at the same dosing interval as the parent protein, the dosing formulation may be simplified and allowed for subcutaneous formulation, or the immunogenicity of S-crotoe may be reduced. Surprisingly, the HAS-fused Klotho protein appears to have improved binding, binding activity, and / or immunogenicity.

Conjugation of S-Crotho protein with human transferrin (TF) Transferrin is a highly abundant serum glycoprotein found in serum at 3-4 mg / mL, which strongly and reversibly binds to iron. It functions to carry iron to the tissue. Transferrin has 679 amino acid residues, is about 80 kDa in size, 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 half-life reported to be 7-10 days, or 10-12 days. The aglycosylated form of human transferrin, which accounts for about 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 clathrin-dependent transferrin receptors (receptor-bound transferrin returned to circulation and recirculated).

Peptide and protein fusions are made at the N-terminus and C-terminus of human transferrin, and in the centrally located hinge region connecting the two major protrusions of transferrin together. The N-terminus of transferrin is vacant and can be fused directly. Flexible linkers are typically used when the protein is fused to the C-terminus because the C-terminus is more buried and constrained by nearby disulfide bonds. This ability creates a library of peptides for specific targets and then develops binders from those libraries into therapeutic fusion proteins with an extended half-life aglyco-transferrin (N-terminus, It was expanded by fusing to the C-terminus, loop, or linker region).

Biotechnology company BioRexis Technologies, Inc. Was founded in 2002 to develop a transferrin fusion protein platform, which was referred to as the "TransBody" platform as a therapeutic platform. Their reed 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 has been demonstrated to significantly enhance the half-life of GLP-1. BioRexis was acquired by Pfizer in March 2007. As far as it can be confirmed, there is currently no bioRexis-derived fusion protein in the hospital. The Klotho protein can be fused to human transferrin to produce a Klotho fusion molecule, which is administered clinically to significantly prolong the half-life, or stability in the human biological matrix in vivo. obtain.

The conjugate of the S-Crotho protein Amunix with 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 XTEN-ized via chemical conjugates or gene fusions. 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 to be much larger than typical globular proteins of similar molecular weight. The bulking effect of XTEN significantly reduces renal clearance of bound molecules and thus significantly increases in vivo half-life. In the present invention, the length of the XTEN polymer added to the Klotho protein will be specified to optimize the pharmacokinetics as well as the biodistribution of the bound Klotho protein payload.

In this way, XTEN can be recombinantly fused to our S-Clotho 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 that has the convenience of expression, purification, and characterization of a single molecule, including both therapeutic and bulking moieties. Recombinant fusion allows the binding of multiple XTEN chains for each protein at precisely defined positions, which therapeutic drug manufacturers have successfully used to produce XTEN growth hormone (Versartis Somavaratan) 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 XTEN formation 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 peptides, peptide mimetics, and chemical conjugation to 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 is developing XTENs containing 1-9 thiol groups at various intervals, which may be offered to partners. Therefore, in the present invention, orthogonal conjugation to amino and thiol groups in XTEN promotes the production of our Croto-XTEN molecule.

Purification of Protein Croto protein can be extracted from cell suspension cultures of cells (eg, of CHO cell lines). Cells can produce Croto protein and optionally secrete it (eg, in a liquid medium). Secretion of S-croto to the consumed medium was also observed.

Purification of the recombinant proteins of the present disclosure can be any conventional method known in the art or comprising any suitable method described herein, eg, extraction, precipitation, chromatography, and / or electrophoresis. Can be performed by procedure. Further purification procedures that can be used to purify the protein include affinity chromatography using a monoclonal antibody that binds to the target protein (s). Some embodiments may include IgG-tagged proteins, Fc-tagged proteins, His-tagged proteins, HSA-tagged proteins, GST-tagged proteins, and the like, which can be purified by affinity chromatography. For example, some embodiments may include at least a portion of the Fc domain of an IgG, such as IgG1. In general, crude preparations containing recombinant proteins are passed through a column on which a suitable monoclonal antibody is immobilized. For other tags, the corresponding tag binding affinity entity can be placed on the column. After washing the column, the protein is eluted from the gel by varying the pH or ionic strength. For example, consumed medium from a CHO-S high-yielding cell line was concentrated via tangential filtration and the S-croto protein was purified by affinity chromatography followed by ion exchange cartridge or column chromatography. Size exclusion chromatography can also be used to purify proteins. It will be appreciated that different sequences of chromatographic types can be applied. As used herein, any suitable combination of chromatography steps or columns is contemplated.

In the alternative protocol, one or more affinity pre-purification or post-purification steps were performed. Such steps include, for example, (ultra) centrifugation, dialysis, membrane and / or tangential filtration, chromatographic separation such as ion exchange, liquid-liquid extraction such as (aqueous) two-phase extraction, or other. Known purification steps may be included. In certain embodiments, one or more post-purification treatment steps have been performed. Such post-purification treatment steps include, for example, tandem anion / cation transit fraction chromatography (as opposed to binding and elution chromatography), membrane filtration (eg, 0.2 micron, 0.1 micron, etc.). , Or the removal of viruses and / or bacteria by other means known to those skilled in the art.

Certain embodiments include methods of purifying recombinant Croto protein. The method may include expressing recombinant Klotho protein in suspended cell culture. The method may include harvesting the Croto protein, such as by harvesting suspension culture medium and / or cells. In at least one embodiment, the method may include a first purification step. It is understood that the "first" purification step does not have to occur first or before any other purification step (s) or after any other purification step (s). Will. The first purification step may include or be involved in chromatography such as high performance liquid chromatography (HPLC), high performance protein liquid chromatography (FPLC), ion exchange chromatography, affinity chromatography, or the like. Can be used. The first purification step may include affinity chromatography, may be involved in it, or may use it. Affinity chromatography can be based on the tag of the Klotho protein or other protein fusion entity. 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), the corresponding tag-binding affinity entity may be placed 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 for purification of Ig-tagged or Fc-fused chromatoproteins (eg, protein ASEpharose 4 fast flow column (0.75 ml)). ) (0.5 cm x 3 cm)) can be included, involved in, or used. The column can be equilibrated in a suitable solution. The solution may contain a buffer of suitable concentration (eg, 10 mM Tris-HCl, pH 8.0). A suitable amount of protein-containing sample (eg, optionally 1.5 M Tris-HCl, suspension cell culture supernatant adjusted to pH 8.0 at pH 8.0) is provided (eg, at a flow rate of 0.75 ml / min). ) Columns are loaded and optionally washed with a suitable wash buffer (eg, 10 mM Tris / arginine-HCl, pH 8.0) and at a suitable flow rate (eg, about 0.50 to 0.25 ml / min or with it). Equivalent) and / or can be eluted from the column with a suitable elution buffer (eg, 4M MgCl ₂ , pH 3 (ie, low pH, high salt)) 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 is pH about 2 to pH 6, preferably pH about 2 to pH 5, more preferably pH about 2 to pH about 4, even more preferably pH about 2.5. It can have a pH of ~ pH about 3.5.

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

Additional purification steps (s) include chromatography such as high performance liquid chromatography (HPLC), high performance protein liquid chromatography (FPLC), ion exchange chromatography, affinity chromatography, size exclusion (or gel filtration) chromatography. Can be included, can be involved in it, or can be used. In at least one embodiment, the method may include a second purification step. It will be appreciated that the "second" purification step does not need to occur, secondly, before or after any other purification step (s). The second purification step may include or be involved in size exclusion chromatography (and suitable mobile phase buffer) for fractionation, degradation, clarification, or purification of the protein-containing sample (s). Or you can use it. For example, in an exemplary second purification step, the protein-containing eluted sample (s) (s) are optionally buffer exchanged and size exclusion chromatography in a suitable mobile phase buffer. (For example, a Superdex 200 (1 cm × 30 cm) column) can be used for purification. In at least one embodiment, the mobile phase buffer is a suitable buffer (eg, 100 mM Tris-HCl) of a suitable pH (eg, pH about 8) and one or more optional reducing agents (eg, eg, pH). It may contain 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 can, or may contain, preferably at an effective reducing concentration (s) of L-methionine and / or sodium thioglycolate. Alternative reducing agents are acetylcysteine (ie, N-acetyl-L-cysteine, N-acetyl-D-cysteine, and racemic N-acetylcysteine, or N-acetyl-L-cysteine and N-acetyl-D- N-Acetylcysteine (NAC)), ascorbic acid, subdithionate, erythiorbate, cysteine, glutathione, dithiotreitol, 2-mercaptoethanol, dierythritol, including (racemi) mixture with cysteine, 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), tolbalsum , Basaka, Ambroxol, Carbocysteine, Eldostain, Mecysteine, Dornase Alpha, etc.

In at least one embodiment, the mobile phase buffer can have a pH of about 8. In some embodiments, the elution buffer is pH about 6 to pH 10, preferably pH about 7 to pH 9, more preferably pH about 7.2 to pH about 8.8, even more preferably pH. It can have a pH of about 7.5-5 and a pH of about 8.5.

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

Additional purification steps (s) include chromatography such as high performance liquid chromatography (HPLC), high performance protein liquid chromatography (FPLC), ion exchange chromatography, affinity chromatography, size exclusion (or gel filtration) chromatography. Can be included, can be involved in it, or can be used.

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 Croto protein sample (50 μg) was fractionated on a precast SDS-PAGE gel (4-15%, 10 wells, Catalog No. 456-1083, BioRad) and stained with Kumasy blue dye. All samples were placed with empty lanes in between or on separate gels to avoid contamination between samples. More than 98% of S-crotoes were isolated from CHOS conditioned medium as determined by fluoroscopy of Kumasy blue dye and densitometry, or by visualization of silver staining, 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. .. The sample thus prepared is then subjected to an automated vapor phase microsequencing instrument (Applied Biosystems model 470A) equipped with an online automated HPLC PTH amino acid analyzer (Applied Biosystems model 120, ABI, see above) connected to an outlet. , ABI, Foster City, California, USA).

Proteins were also analyzed through mass spectrometry. For sample preparation for mass spectrometry, only the 75-150 kDa gel band was cut out for analysis in order to limit the analysis to the correct S-Croto protein. The gel fraction was softened with a sterile blade and subjected to in-gel digestion. The gel fraction was decolorized by washing 3 times with 80 μL of 50% acetonitrile (ACN) / 50 mM ammonium bicarbonate and washed with 100% ACN. The alkylation step was omitted due to the absence of cysteine residues from the target α-crotopeptide. Trypsin digestion was performed overnight at 37 ° C. in 50 mM ammonium hydrogencarbonate (0.005 μg / μL) using 60 μL of trypsin (sequencing grade modification, Catalog No. V511A, Promega). This process yielded 25 μL, of which 5 μL (1 μL for S-Croto) was liquid chromatographed on an Orbitrap nano-ESI Q-Exactive mass spectrometer (Thermo Electro-Scientific) attached to nanoLC (Dionex Ultimate 3000 UHPLC). It was subjected to spray ionization tandem mass spectrometry (MS / MS) and PRM analysis. In MS / MS analysis, the human recombinant alpha-S-croto produced by the embodiments of the present disclosure is substantially similar to that found in human blood, serum, urine or cerebrospinal fluid (eg, correspondence). It was confirmed that they are the same in the amino acid sequence.

Using the above purification method, the level of contaminated CHO host cell protein (HCP) was determined to be acceptable in the purified S-croto protein. In the final S-Crotho product, HCP was removed to less than 1-100 ppm. S-Croto protein products with a purity of at least 90% and up to 98% were isolated from consumed CHOS-producing cell lines (cells and / or liquid medium). Specifically, cGMP grade human alpha-S-crotoe having an analytical profile suitable for clinical administration in human subjects was produced and purified. For example, the analytical profile of human recombinant alpha S-croto is described at http: // proteomecentral. proteomexchange. org / cgi / GetDatet? It is described in the reference number PXD002775 of the ProtocolXchangeDatabase, which can be found at ID = PXD002775. The complete NIH S-Crotho protein dataset is located at http: // www [dot] ncbi [dot] nlm [dot] nih [dot] gov / protein / Q9UEF7.

The analytical profile of S-Crotho suitable for clinical administration, and the analytical profile obtained in the embodiments of the present disclosure, contained endotoxin levels of less than 0.1 ng (1 EU / μg) per 1 μg of S-Crotho. In addition, purified human recombinant S-croto was also shown to have a purity of 98% or higher by SDS PAGE.

The glycan structure present in S-crotoes produced in CHO S cells is identical compared to the structure of native S-crotoes isolated from human body fluids (ie, blood, serum, urine, and cerebrospinal fluid). Met. This ensured that the same naturally occurring post-translational modification (PTM) was produced and stably maintained in the S-Clotho protein produced in the cells. Therefore, using the production and purification methods described herein, we have succeeded in producing cGMP grade human S-croto with an analytical profile 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 the present disclosure generally consist of a (pharmaceutically acceptable) vehicle, carrier, or excipient consisting of a therapeutically effective amount of recombinant soluble alpha croto protein and one or more additional components. May include admixtures with. Klotho protein is in any suitable amount, such as about 0.001 to 1000 mg, about 0.01 to 100 mg, about 0.1 to 10 mg, about 1 to 5 mg, or any amount or range of amounts in between. Can be included in. In some embodiments, the composition may comprise a pharmaceutically effective or therapeutically effective amount of Croto protein (eg, to raise serum Croto levels to a predetermined level in the subject to which the composition is administered).

The components may include one or more aggregation inhibitors, buffers, tonicity agents, and additional excipients. The main solvent in the carrier may be essentially aqueous or non-aqueous. The composition can be prepared by combining the purified Klotho protein of the present disclosure with a pharmaceutically acceptable carrier.

The combination of the various constituents contained in the composition can be made in any suitable order, i.e. the buffer can be added first, intermediate or last, and the tonicity agent is also first. It will be appreciated by those skilled in the art that it can be added in the middle, or at the end. It is also understood by those skilled in the art that some of these chemicals can be incompatible in certain combinations and are therefore easily replaced by different chemicals that have similar properties but are compatible with the relevant mixture. Is to be done.

Aggregation inhibitors reduce the tendency of polypeptides to associate with inappropriate or undesired tri- or quaternary complexes. The amino acids L-arginine and / or L-cysteine may act to reduce the aggregation of Fc domain-containing polypeptides in the formulation over a long period of time, eg, 2 years or more. The concentration of the aggregation inhibitor in the formulation is preferably from about 1 mM to 1 M, more preferably from about 10 mM to about 200 mM, more preferably from about 10 mM to about 100 mM, even more preferably from about 15 mM to about 75 mM, and even more preferably. It is about 25 mM. These compounds are available from commercial suppliers.

The compositions of the present disclosure may include a buffer. The buffer keeps the pH within the desired range. Various buffers suitable for use in the pharmaceutical compositions of the present disclosure include histidine, potassium phosphate, alkali salts, sodium phosphate or potassium phosphate or hydrogen salts or dihydrogen salts thereof, sodium citrate or Potassium citrate / citric acid, sodium / acetate, maleic acid, ammonium acetate, tris- (hydroxymethyl) -aminomethane (tris), various forms of acetate and diethanolamine, and solutions known in the art. Included are any other pharmaceutically acceptable pH buffers for keeping the pH within the desired range. Mixtures of these buffers can also be used.

The amount of buffer useful in the composition largely depends on the pH of the particular buffer and solution used. For example, less acetate can be used in solution at pH 5 than pH 6 because acetate is a more efficient buffer at pH 5 than pH 6. The preferred pH of the preferred formulation is in the range 4.0-5.0 and also includes pH regulators such as hydrochloric acid, citric acid, sodium hydroxide, or salts thereof to obtain the desired pH. It can be.

One preferred buffer is sodium phosphate, because 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 buffer. 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 to or near physiological levels maximizes patient comfort during administration. Specifically, the pH is preferably in the range of about 5.8 to 8.4, preferably about 6.2 to 7.4, however, the pH is adjusted as necessary. It is within the scope of the present disclosure that the stability and solubility of the polypeptide in a particular formulation can be maximized and thus is acceptable to the patient even at pH outside the physiological range. Is understood.

The formulations of the present disclosure may further comprise one or more isotonic agents (eg, to make the solution isotonic with the patient's blood for injection). The tonicity agent is understood to be a molecule that contributes to the weight osmolality of the solution. The weight osmolal concentration of the pharmaceutical composition is preferably adjusted to maximize the stability of the active ingredient and also to minimize patient discomfort upon administration. When the serum is approximately 300 +/- 50 milliosmol per kilogram. In general, it is preferred that the pharmaceutical composition be isotonic with serum, i.e. having the same or similar weight osmolality achieved by the addition of an isotonic agent, thus having a weight osmolality of about 180-about 420 Although it is intended to be myriosmoles, it is understood that the weight osmolality may be either higher or lower, as required under certain conditions.

Typical isotonic 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 isotonic agent can be used at a time and, for example, sorbitol and glycine can be used in combination to modify the tonicity of the formulation.

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

Excipients, also referred to as chemical additives, co-solubilizers, or co-solvents that stabilize the polypeptide in solution (even in dry or frozen form), can also be added to the pharmaceutical composition. Excipients are defined herein as non-therapeutic agents added to a pharmaceutical composition to provide the 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 able to stabilize the natural conformation of the protein in order to maintain the efficacy and safety of the drug during processing, storage, and administration to the patient. .. 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 (eg, PEG, ethyleneglycol, and glycerol) dimethylsulfoxide (DMSO), and Non-aqueous solvents such as dimethylformamide (DMF); amino acids such as proline, L-serine, sodium glutamate, alanine, glycerin, lysine hydrochloride, sarcosin, and gamma-aminobutyric acid; Tween-80 ™ (polysorbate 80), Surfactants such as Tween-20 ™ (Polysorbate 20), SDS, polysorbate, polyoxyethylene copolymer; as well as potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions Includes various excipients (eg, zinc, copper, calcium, manganese, and magnesium), CHAPS, monolaurate, 2-O-beta-mannoglycerate, or any combination of the above. However, it is not limited to these. 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 from about 0.001 to 5 weight percent, more preferably from about 0.1 to 2 weight percent. Excipients are well known in the art, manufactured by known methods and available from commercial suppliers.

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

In one exemplary embodiment, the formulations of the present disclosure have a (therapeutically effective) amount of sucrose protein in L-arginine at a pH of about 6.0 to about 7.0 and about 10 mM to about 100 mM. It may contain from 10 mM to about 50 mM sodium phosphate, from about 0.75% to about 1.25% sucrose, and / or from about 50 mM to about 150 mM NaCl. In another embodiment, L-arginine can be replaced with L-cysteine (about 1 to about 500 micromoles) in the formulation. In yet another embodiment, the pH can be about pH 7.0. In another particular embodiment, the formulations of the present disclosure have a pH of about 6.2, a (therapeutically effective) amount of sucrose protein in 25 mM L-arginine, about 25 mM sodium phosphate, about 98 mM sodium chloride. , And / or may contain about 1% sucrose.

In another embodiment, the formulations of the present disclosure are a (therapeutically effective) amount of sucrose protein in L-arginine at about 10 mM to about 100 mM, at a pH of about 6 to about 7, and about 10 mM to about 50 mM phosphate. It may contain 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 have a pH of about 6.2, a (therapeutically effective) amount of sucrose protein in about 25 mM sodium phosphate, about 98 mM sodium chloride, and / or about 1%. May include sucrose.

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

In yet another embodiment, the disclosure is a method of treating a mammal, comprising administering a therapeutically effective amount of the pharmaceutical composition described herein, wherein the mammal is in the composition. Provided are methods of having a disease or disorder that can be beneficially treated with an Fc domain-containing polypeptide. In yet another embodiment, the Fc domain-containing polypeptide is derived 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. When the Fc domain-containing polypeptide of the composition is Croto: Fc, examples of diseases or disorders that can be treated include rheumatoid arthritis, psoriatic arthritis, tonic spondylitis, Wegener's disease (granulomatosis), Crohn's disease ( Or inflammatory bowel disease), chronic obstructive pulmonary disease (COPD), hepatitis C, endometriosis, asthma, malaise, psoriasis, and atopic dermatitis, or mutations in one or more Croto genes Includes, but is not limited to, persons with hereditary disorders. Klotho: Additional diseases or disorders that can be treated with Fc include those described in WO00 / 62790, WO01 / 62272, and US Patent Application 2001/0021380, each of which is incorporated herein by reference in its entirety. included.

In yet another embodiment, the present disclosure is a method for accelerating stability testing of Fc domain-containing polypeptide stability in the pharmaceutical compositions of the present disclosure, the present invention before storage, i.e. at the start. The activity of the polypeptide formulated according to the disclosure is tested, the composition is stored at 37 ° C. for 1 month, and the step of measuring the stability of the polypeptide is compared with the stability morphology from start to 1 month. Provide methods, including steps. This information is useful for early removal of batches or lots that initially appear to have good stability but are not well stored for long periods of time.

In addition, the pharmaceutical composition provides long-term storage such that the active ingredient, eg, an Fc domain-containing polypeptide, is stable over the storage process, either in a 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 at, for example, −20 ° C. or lower (eg, −20 ° C. or −80 ° C.). Then it is understood. It is also contemplated that the composition 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 more than 5% is understood to mean not losing its activity.

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

Modified release formulations (eg, coating, encapsulation, sustained release, controlled release, prolonged release, delayed release, sustained release, sequential continuous release, depot, etc.), inhaled mist, oral active preparation, suppository, And other effective dosage forms such as implants or implantable medical devices are also contemplated herein. As such, the formulations are also bulk erosion polymers (eg, poly (lactic acid-glycolic acid copolymers) (PLGA) copolymers, PLGA polymer blends, PEG block copolymers, and lactic acid and glycolic acids, Fine particle preparations of polymer compounds such as poly (cyanoacrylate); surface eroding polymers (eg, poly (anhydrous), and poly (orthoesters)); hydrogel esters (eg, pluronic polyols, poly (vinyl alcohol), Poly (vinylpyrrolidone), maleic anhydride-alkylvinyl ether copolymers, cellulose, hyaluronic acid derivatives, alginates, collagen, gelatin, albumin, and starch and dextran), and fine particle preparations of their composition systems, or liposomes or microspheres. Can include preparations of. Such formulations may affect the physical condition, stability, in vivo release rate, and in vivo clearance rate of the protein and derivatives. The optimal pharmaceutical formulation for the desired protein can be determined by one of ordinary skill in the art, depending on the route of administration and the desired dosage. An exemplary pharmaceutical formulation is Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mac Publishing Co., Ltd. , Easton, Pa. 18042, pp. 1435-1712, which disclosure is incorporated herein by reference.

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

The Klotho protein of the present disclosure refers to an animal (model) that has or presents with a particular disorder or condition, such as an age-related or age-related disorder or condition, a clinical (eg, metabolic) disorder or condition. Can be evaluated using. Such disorders and conditions may also provide a sensitizing system in which the physiological effects of proteins can be observed. Exemplary disorders include, for example, neuropathy, disuse atrophy, clinical (eg, metabolic) disorders (eg, disorders of obese and / or diabetic animals such as db / db mice, ob / ob mice), brain. Disorders, hepatic ischemia (kidney idschemia) or other hepatic disorders, cisplatin / taxol / bincristine model, renal ischemia / reperfusion model, various tissue (heterologous transplant) transplants, transgenic bone model, pain syndrome (eg, inflammation Sexual and neuropathy), paracoat, genetic toxicity, inflammatory stress models, and tumor (I) models are included.

To assess the S-Crotho protein of the present disclosure, the protein can be administered to a suitable animal (over a suitable treatment period) and the animal parameters (eg, 10-60 minutes, 1-24 hours, 1- It is evaluated (after a suitable period such as 30 days, 1-12 months, 1-5 years, or any value or range of values between them). Animals can be nourished as appropriate or as usual (eg, not under calorie restriction, but some parameters can be evaluated under such conditions). Typically, a cohort of such animals is used in the assay. In general, if the test polypeptide affects the parameters in the direction of a similar animal phenotype subject to calorie restriction, the test polypeptide may be indicated to favorably alter animal lifespan control. Such test polypeptides can cause at least some lifespan control effect by calorie restriction (eg, a subset of such effects) without eliminating the organism's caloric intake.

The parameter (s) tested can be an age-related or disease-related parameter (s) (eg, symptoms of a disorder associated with an animal model). The test protein shown favorably can cause amelioration of symptoms as compared to similar reference animals not treated with the polypeptide. Other parameters related to disability or aging include antioxidant levels (eg, antioxidant enzyme levels or activity), stress tolerance (eg, paracoat resistance), core body temperature, glucose levels, insulin levels, thyroid stimulating hormone levels, Prolactin levels, and luteinizing hormone levels may be included.

Animals with reduced croto expression (eg, mice with mutants or deficiencies in the croto gene) can be used to measure the effectiveness of the S-croto proteins of the present disclosure for treating age-related disorders. For example, the test protein may be administered to mutant mice and age-related parameters may be monitored. The test protein shown favorably can cause amelioration of symptoms compared to similar reference animals that have not been treated with the protein.

Parameters related to clinical (eg, metabolic) disorders or aging are evaluated by measuring body weight, testing fertility, measuring blood glucose levels, observing lifespan, observing skin, observing motor functions such as walking, etc. obtain. Evaluation can also be done by measuring thymus weight, observing the size of calcified nodules formed on the inner surface 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.

Yet other (in vivo) models and bioassays include assessing animals for clinical (eg, metabolic) parameters, such as those 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 the test protein can alter lifespan control. Illustrative age-related parameters include (i) lifespan of cells or organisms, (ii) presence or abundance of gene transcriptions or products in cells or organisms with biological age-dependent expression patterns. (Iii) Resistance of cells or organisms to stress, (iv) one or more metabolic parameters of cells or organisms (exemplary parameters include circulating insulin levels, blood glucose levels, fat content, core body temperature, etc.) Includes, (v) the ability of a cell or cell set to proliferate in an organism, and (vi) the physical appearance or behavior of a cell or organism.

The term "life expectancy" refers to the average age of death in a cohort of organisms. In some cases, "life expectancy" is assessed using a cohort of genetically identical organisms under controlled environmental conditions. Deaths from accidents are not counted. If life expectancy cannot be determined under controlled environmental conditions (eg, in the case of humans), reliable statistical information for a sufficiently large population (eg, from a mathematical table) can be used as life expectancy.

Differences in the physiological state of an organism can be revealed by characterizing the molecular differences between two such organisms, eg, one reference organism and one organism treated with the S-croto protein. .. The reference organism and the treated organism are typically of the same (or substantially the same) real age and / or gender. As used herein, the term "actual age" refers to the time elapsed from a preselected event, such as conception, defined fetal or fetal period, or more preferably birth. Various criteria can be used to determine if an organism is of "same" real age for comparative analysis.

Typically, the degree of accuracy required correlates with the life expectancy of wild-type organisms. For example, C. elegans C. elegans, in which the laboratory wild-type strain N2 survives for an average of about 16 days under some controlled conditions. In the case of elegans, organisms of the same age can survive for the same number of days. In the case of mice, organisms of the same age can survive for the same number of weeks or months, for the same number of years (ie, within 2, 3 or 5 years) in the case of primates or humans. In general, organisms of the same real age can survive for hours 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 survives for at least a certain amount of time that the average wild-type organism matures to a fertile age).

A biological screening assay can be performed before the organism exhibits the apparent physical properties of aging. For example, the organism may be an adult that survives 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 capacity, and chromosomal structure have been reported. Any of these changes can be performed in a test subject (eg, a biological assay) or before, during, or treated with a therapeutic agent described herein (eg, in a (human or mammalian) patient). It can be evaluated in any of the latter.

Markers associated with calorie restriction can also be evaluated in the subject organism (or treated subject) of the screening assay. Although these markers may not be age-related, they may indicate a physiologic condition that is altered by the regulation of croto or croto-related pathways. The marker can be mRNA or protein that changes significantly in calorie-restricted animals. Cell models derived from the cells of the animals described herein, or analogs of the animal models described herein, can be used in cell-based assays.

Models for assessing the effect of test proteins on muscular atrophy include 1) reduction of rat medial gastrocnemius muscle mass resulting from denervation, eg, by cutting the right sciatic nerve in the central thigh, 2) (eg, 90). Rat medial gastrocnemius muscle atrophy resulting from immobilization (by immobilizing the right ankle joint with degree flexion) 3) Rat medial gastrocnemius muscle atrophy resulting from hind limb atrophy 4) Interleukin-1 (a humoral cytokine) Includes skeletal muscular atrophy resulting from treatment with IL-1) and 5) skeletal muscular atrophy resulting from treatment with glucocorticoids, dexamethasone.

Products, Compositions, Formulations, Supplements, etc. Some embodiments of the present disclosure are configured or formulated to increase the production of naturally soluble alpha Croto protein and / or diminish damage or degradation of Croto protein. And related methods, kits, formulations, combination products, co-administration, co-formulations, dosages, processes, methods, therapeutic protocols, and diagnostic methods. Some embodiments of the present disclosure relate to methods of producing the compositions of the present disclosure. Some embodiments of the present disclosure relate to the use of the compositions of the present disclosure, or methods of using it to treat human or non-human animal subjects. Some embodiments of the present disclosure relate to therapeutic methods involved in the compositions of the present disclosure, including their administration to human or non-human animal subjects. Such uses and therapeutic methods can increase endogenous croto protein levels, such as by increasing the production of naturally soluble alpha croto protein and / or diminishing damage or degradation of croto protein.

Certain embodiments of a substance comprising a health supplement that increases the level of endogenous soluble alpha human protein in a human or non-human animal subject when administered to a human or non-human animal (eg, mammal) subject. Contains the composition. 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 (synthetic) chemical and / or natural sources or may be derived from them.

The exemplary composition is preferably, for example, vitamin D3, vitamin E, vitamin C, vitamin K, 3,5,4'-trihydroxy-trans-stilbene (resveratrol), pterostilbene, N-acetylcysteine. (NAC), troglycazone, rosmarintazone, Notopterygium incisum Ting, gentiana (root, extract), Cordyceps (Cordiceps Sinensis (CS) mycelium), isoflavone (soybean), genistein, dizein, quercetin (dihydrate, dihydrate, leaf) , Oregano), docosahexaenoic acid (DHA), astaxanthin, sesamine (Semen Sesamin Nigrum (black)), sesame (seed extract), rosmarinic acid (RA, (majoram)), 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 components or constituents may have positive benefits and very low side effects in the treatment or effect of one or more pathologies associated with age-related cell and tissue dysfunction. Certain components or constituents may (directly or indirectly) increase the circulating plasma levels of soluble alpha human (s-croto) levels in human or non-human animal subjects after their administration.

The following examples are merely examples. The following examples exemplify various optional components, components, method steps, and the like that may be useful in implementing the embodiments of the present disclosure.
The embodiments of the present disclosure individually address, treat, affect, and / or counteract one or more pathologies that are associated with cell and tissue dysfunction and / or may occur with age. Includes formulations (compositions, co-administration, etc.) of several constituents (or ingredients) that may have positive benefits and / or potential for very low side effects.

Without being bound by any theory, some of the constituents or components of the present disclosure may activate the promoter of the Croto gene (eg, within a human HEK293 / kl cell line) or the liver, kidney. , And age-related declines in the expression of the promoter in other tissues can be reversed. Some of the constituents 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 constituents are associated with cell and tissue dysfunction and / or coping with one or more pathologies that can occur with age. It has been shown by one or more animal and human clinical trials to have a positive benefit and / or a very low potential for side effects in treatment, effects, and / or countermeasures. The present disclosure utilizes these various components in compositions and methods for increasing endogenous Klotho protein levels. Specifically, the present disclosure comprises these various components in compositions configured or formulated to increase the production of naturally soluble alpha croto proteins and / or diminish damage or degradation of croto proteins. To use.

Without being bound by any theory, certain individual components of some embodiments may increase circulating plasma levels of soluble alpha-croto in mammalian (eg, human or non-human) animals. Examples of ingredients include the antioxidants N-acetylcysteine (NAC), D vitamins such as vitamin D3 (1,25-dihydroxyvitamin D3 [1,25 (OH) 2D3]), and / or chronic renal disease. Increases Klotho expression, increases phosphate urine, corrects hyperphosphatemia, decreases serum fibroblast growth factor-23 (FGF-23), and aortic calcification in mice suffering from Vitamin D receptor agonists (eg, calcitriol, paricalcitol) that are thought to result in the associated reduction of, and age-related renal damage in salt urine and proximal tubules are thought to improve. Vitamin C and / or Vitamin E may be included, but not limited to these.

Further exemplary ingredients include, for example, docosahexaenoic acid (DHA), linoleic acid, conjugated linoleic acid (CLA), isoflavones (eg genistein, daidzein, quercetin), rosmarinic acid, sesamine and astaxanthin (food-derived antioxidants). ), As well as non-prescription ingredients that can supplement the antioxidant properties of alphacrotoe, 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 can underlie learning and memory loss and are omega-3 polyunsaturated fatty acids such as DHA. Saturated fatty acids (PUFAs) protect the aging brain from the development of neurodegenerative diseases. Thus, certain formulas may include alpha linoleic acid or conjugated alpha linoleic acid, as alpha linoleic acid can function as a precursor molecule for use in 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 can increase the effectiveness of anticoagulants and increase the risk of bleeding. Examples of drugs include, but are not limited to, warfarin, clopidogrel, and aspirin, in particular. In some embodiments, DHA may be included in the formulation. Without being bound by any theory, DHA does not directly cause anticoagulant.

Without being bound by any theory, mitochondrial damage can be the cause and consequence of cytotoxicity resulting from various toxic attacks, hypoxia, or trauma. Increased mitochondrial biosynthesis after renal proximal tubular cell (RPTC) injury can accelerate the recovery of mitochondrial and cellular function after such injury. In connection with these observations, isoflavones such as daidzein and genistein increase peroxysome growth factor-activating receptor gamma-co-activating factor (PGC) -1alpha expression, ATP synthase beta and NADH: ubiquinone oxide reductase core subunit. It can result in mitochondrial biosynthesis indicated by increased expression of unit 6 (ND6), which can result in a 1.5-fold increase in respiration and ATP in RPTC, which can benefit the recovery of injured RPTC. 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 can also be included in certain embodiments. On the other hand, isoflavones and quercetin are large protein complexes involved in the proper control of cellular protein loading, which plays a major role in maintaining redox balance by recognizing and removing oxidatively modified or damaged proteins. By increasing the resistance to oxidative stress in the proteasome, the adverse effects of aging can be countered.

Rosmarinic acid can 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 200 mg / kg of RA per kg once daily for 30 days to aged mice resulted in superoxide dismutase (SOD) catalase (CAT) and glutathione peroxidase (GSH-) compared to aging controls that did not receive RA. The activity of Px) can be significantly increased and malondialdehyde (MDA) can be decreased. Therefore, RA can promote significant antioxidant enzyme activity in the 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 extract. When given daily in combination with both (ie, AS) and subjects with mild cognitive dysfunction (MCI), significant improvements in psychomotor and processing rates were observed in the AS group compared to the placebo group. Was done. Therefore, daily supplementation of AS can improve cognitive function associated with the ability to quickly and accurately understand and perform complex tasks.

One or more peroxisome proliferator-activated receptor (PPAR-γ) agonists, such as tetradecylthioacetic acid (TTA), can also be added to 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, some) natural ingredients or extracts. These include, but are not limited to, gentian root extract, Cordyceps notopterygium, and Cordyceps incisum Ting.

Specific embodiments include Lactobacillus species (eg, acidofilus, brevis, bulgaricus, casei, gasseri, jhonsiii, lactis, platinum, paracassei, rhamnosus, salivarius, savibi, etc.) Lactis, streptococcus (eg, lactobacillus, etc.), Streptococcus (eg, thermophiles, etc.), Bifidobacterium (eg, lactobacillus, etc.), Pediococcus (eg, acidilactici, etc.), and others known in the art (intestines). It may contain one or more (selective) strains of (internally beneficial) bacteria. Without being bound by any theory, enteric beneficial bacteria can relieve age-related oxidative stress (eg, in mice) and improve the expression of age-related biomarkers. Additional information regarding enteric beneficial bacteria that may be included in a particular embodiment of the present disclosure is incorporated herein by reference in its entirety, Int J. Literature Res Public Health, 2014 May; 11 (5): 4745. It can be found in the document "Microorganisms with Classed Probiotic Properties: An Overview of Retent Literature" by Sabina Fijan, published in -4767.

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

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

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

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

The components of Tables 42-44 can be combined in any suitable combination. As a non-limiting example, the alternative formulation (s) may or may be a DSHEA-compliant formulation or composition administered orally. However, it will be appreciated that alternative formulations (s) can be formulated in other dosage forms and / or as FDA-approved drugs without departing from the scope of the present disclosure. The concentration of each ingredient in the alternative formulation (s) is based on a standard adult reference of 150 pounds (68 kilograms) of a person. As will be appreciated by those of skill in the art, alternative dosages (s) are also contemplated herein.

Exemplary compositions (eg, dietary supplement compositions) include vitamin C, vitamin D3, vitamin E, N-acetylcysteine (NAC), quercetin (dihydrate), rosmarinic acid (RA), pterostilben, 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 enteric beneficial bacteria. May contain the constituents of. One or more intestinal beneficial bacteria are selected from effective amounts of (i) Bifidobacterium lactis BL-04, Bifidobacterium bactisum / lactis BB-02, and Bifidobacterium longum BL-05 or one or more Bifido strains. And / or include one or more of one or more Lactobacillus species and / or strains selected from Lactobacillus acidofilus LA-14, Lactobacillus rhamnosus LR-32, and Lactobacillus paracasei LPC-37.

Illustratively, the composition (or formulation) comprises a maximum, at least, and / or about 50 billion CFU (per daily dose) of enteric beneficial bacterial components. Alternative embodiments are up to, at least between them, and / or about 20 billion CFU (per daily dose), 30 billion CFU (per daily dose), 40 billion CFU (per daily dose), 600. It may contain 100 billion CFU (per daily dose), 70 billion CFU (per daily dose), and / or 80 billion CFU (per daily dose) of enteric beneficial bacterial components. Illustratively, the intestinal beneficial bacterial component is a single intestinal beneficial bacterial (bacterial) species or strain, or maximum, at least, and / or between 2, 3, 4, 5, 6, 7, 8 , 9, and 10 intestinal beneficial bacterial (bacterial) species or strains may be included or included. In at least one embodiment, the intestinal beneficial bacterial components are the following intestinal beneficial bacterial (bacterial) species or strains, Bifidobacterium species, and / or Bifidobacterium lactis BL-04, Bifidobacterium bifidum / lactis BB-02, and Bifidobacterium. Includes 30 billion CFU / day for each strain of BL-05. However, certain embodiments include one or two of the Bifidobacterium species and / or strains described above, and / or maximum, at least between them, and / or about 10 billion CFU (per daily dose). Understood that it can contain 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). Will be done. In at least one embodiment, the intestinal beneficial bacterial components are the following intestinal beneficial bacterial (bacterial) species or strains, Lactobacillus species and / or strains Lactobacillus acidofilus LA-14, Lactobacillus rhamnosus LR-32, and Lactobacillus. Includes 20 billion CFU / day for each strain of LPC-37. However, certain embodiments include one or two of the Lactobacillus species and / or strains described above, and / or maximum, at least between them, and / or about 5 billion CFU (per daily dose). Understood that it can contain 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). 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. It may be included in 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 about 2500 mg, 2000 mg, 1500 mg, 1000 mg, or 750 mg or less (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-. 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 to 1250 mg. In at least one embodiment, vitamin D3 is at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, 1000 mg, 1250 mg, 1500 mg, or 2000 mg daily doses and / or 2000 mg. May be included in 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. It may be included in a daily dose and / or concentration 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 about 2000 mg, 1500 mg, 1000 mg, 750 mg, or 500 mg or less (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 further. It may be included in a daily dose and / or concentration of more preferably 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 least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg daily dose and / or 1000 mg (per day or per dosage). Can be included in concentration. In at least one embodiment, N-acetylcysteine (NAC) may be included at a daily dose and / or concentration of about 2000 mg, 1500 mg, 1000 mg, or 750 mg or less (per day or per dosage).

In at least one embodiment, quercetin (dihydrate) is about 150 mg (per day or per dosage), or about 10 to 1000 mg, preferably about 25 to 750 mg, more preferably about 50 to 500 mg, and further. It may be included in a daily dose and / or concentration of more preferably 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). May be included in daily dose and / or concentration. In at least one embodiment, quercetin (dihydrate) is at a daily dose and / or concentration of about 1000 mg, 750 mg, 500 mg, 400 mg, 300 mg, 250 mg, or 200 mg or less (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. Can be included in daily doses and / or concentrations 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) may be included at a daily dose and / or concentration of about 2500 mg, 2000 mg, 1500 mg, 1000 mg, or 750 mg or less (per day or per dosage).

In at least one embodiment, 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. It may be included in 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 at 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 edible DHA) is about 200 mg (per day or per dosage), or about 10 to 1000 mg, preferably about 25 to 750 mg, more preferably about 50 to 500 mg. It can be contained in even more preferably about 100-250 mg, even more preferably about 150-250 mg, even more preferably about 100-200 mg daily dose and / or concentration. 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 about 2000 mg (per day or per dosage) at a daily dose of about 2000 mg, 1500 mg, 1000 mg, 750 mg, 500 mg, 400 mg, 300 mg, or 250 mg or less and / or May be included in 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 further. It may be included in a daily dose and / or concentration of more preferably 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 least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg daily dose and / or 1000 mg (per day or per dosage). Can be included in concentration. In at least one embodiment, nicotinamide riboside (NR) may be included at a daily dose and / or concentration of about 2500 mg, 2000 mg, 1500 mg, 1000 mg, or 750 mg or less (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, nicotinamide adenine dinucleotide (NAD +) is 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 may be included in concentration. In at least one embodiment, nicotinamide adenine dinucleotide (NAD +) is a daily dose and / or concentration of about 2000 mg, 1500 mg, 1000 mg, 750 mg, 500 mg, 400 mg, or 350 mg or less (per day or per dosage). Can be included in.

In at least one embodiment, the nicotinamide mononucleotide (NMN) is about 200 mg (per day or per dosage), or about 10 to 1000 mg, preferably about 25 to 750 mg, more preferably about 50 to 500 mg, and further. It may be included in a daily dose and / or concentration of more preferably 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, the nicotinamide mononucleotide (NMN) is at a daily dose and / or concentration of about 2000 mg, 1500 mg, 1000 mg, 500 mg, 400 mg, 300 mg, or 250 mg or less (per day or per dosage). Can be included.

Without being bound by any theory, in embodiments comprising NAD + and / or NAD + precursors (s) (NR and / or NMN), NAD + and / or NAD + precursors (s) are (s). For example, it can be in the form of lozenges for sublingual application (to extend the time it takes for the liver to enter the bloodstream, bypass the liver first, and convert them to NAM). Other components can also be administered separately or co-administered. In some embodiments, the ingredients can preferably be co-administered via at least some co-formulations or formulated as multiple dose compositions. For example, some embodiments may be multi-pills, multi-capsules, or other dosing kits or packs, may include them, or may be provided as such. 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 solids, granules, powders, liquids, or other forms.

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 or may be a DSHEA-compliant formulation or composition administered orally. However, it will be appreciated that alternative formulations (s) can be formulated in other dosage forms and / or as FDA-approved drugs without departing from the scope of the present disclosure. The concentration of each ingredient in the alternative formulation (s) is based on a standard adult reference of 150 pounds (68 kilograms) of a person. As will be appreciated by those of skill in the art, alternative dosages (s) are also contemplated herein.

Some embodiments (eg, in mammalian subjects) are formulated or co-prepared to increase endogenous Klotho protein levels or production, as described herein in an effective amount of composition (eg, in a mammalian subject). Can include compositions containing (health supplements). A pharmaceutically effective amount of the composition (s) may increase the concentration of the serum soluble croto protein of interest, such as, for example, a soluble croto protein of greater than, or equal to, about 50-3000 picograms per milliliter of serum. Can be sufficient to increase or increase (up to the level of). A pharmaceutically effective amount of the composition (s) may also, or alternative, be sufficient to maintain a serum-soluble Klotho protein concentration of interest above a predetermined threshold over a predetermined period of time.

In certain embodiments, a sufficient or preferred increase in apoptotic protein levels can regulate (can be effective in regulating) the IGF-1 and / or Wnt signaling pathway, β-glucuronidase and / or sialidase. obtained exhibit activity, resulting suppresses p53 / p21 signaling pathway, and / or through preferably inhibition of p53 / p21 signaling _pathway, may reduce the H 2 _{O 2} induced cell senescence and apoptosis. The (elevated) protein preferably exhibits multifaceted 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 or be functional as a humoral factor in the regulation of the activity of glycoproteins on cell surfaces such as growth factor receptors such as growth factor-1 receptors.

The formulated compositions of the present disclosure (eg, health supplements) increase or enhance (natural) production of seroprotein by a patient or subject, or oxidative or other damage to seroprotein or serogene. Serum-soluble Klotho protein levels can be increased by attenuating (eg, reducing, inhibiting, and / or preventing) degradation.

Some embodiments of the present disclosure may include therapeutic compositions or pharmaceutical compositions such as drugs. For example, some embodiments include pharmaceutical or prescription drugs (eg, ARB (eg, rosaltan, balsartan), testosterone, vitamin D receptor agonists (eg, calcitriol, paricalsitol), PPAR (gamma) agonists (eg, eg). , Thiazolidinedione, troglitazone, rosiglitazone), or others) may contain one or more active ingredients. Some embodiments include formulations such as angiotension receptor blocker (ARB) (ie, losartan, valsartan), or vitamin D receptor agonist (VRRA) (ie, calcitriol or paricalcitol). May include formulations or co-formulations in combination with the drug. Other prescription drugs that can be administered with the formulation include troglitazone (200 mg / kg / day) and rosiglitazone (20 mg / kg / day), both of which are peroxidase growth factor-activated receptor (PPAR-γ) agonists. .. Without being bound by any theory, Klotho is believed to be the target gene for 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 PPAR-γ agonists (eg, at the oral dosages described).

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

Also, with respect to the above, mice with chronic kidney disease (CKD) due to calcitriol or paricalcitolol (at a VSRA dose extrapolated from a human subject currently approved for the treatment of secondary hyperparathyroidism) Treatment is believed to result in half the amount of aortic calcification compared to without VFRA. Specifically, mice given intraperitoneally calcitriol or paricalcitol three times a week for three weeks (medication: 30 ng / kg calcitriol (C30), or 100 ng / kg paricalcitol (P100) or 300 ng / kg paricalcitol (P300)) may exhibit increased serum and urinary crotho levels, increased phosphate urine, correction of hyperphosphatemia, and decreased serum fibroblast growth factor-23. .. Croto and osteopontin can also be upregulated by VRRA therapy independently of changes in serum parathyroid hormone and calcium.

Some embodiments, or pharmaceutical constituents thereof, may comprise one or more recombinant (eg, human or mammalian) croto proteins, protein fragments, and / or protein manifolds. Such active or pharmaceutical constituents 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 are described in the patent references incorporated above (ie, (ie). i) PCT / US2017 / 0355755 submitted on June 2, 2017, (ii) PCT / US2017 / 063149 submitted on November 22, 2017, and (iii) submitted on December 6, 2017. US Patent Provisional Application No. 62 / 595,567). Such active ingredients may contribute to increased levels of endogenous Klotho protein (production) in the patient or subject.

Pharmaceutical compositions or components, regardless of type, generally consist of a drug component optionally mixed with a (pharmaceutically acceptable) vehicle, carrier, or excipient consisting of one or more additional components. Can include. The components may include one or more aggregation inhibitors, buffers, tonicity agents, and additional excipients. The main solvent in the carrier may be essentially aqueous or non-aqueous.

Some embodiments may comprise a combination product, composition, or formulation comprising recombinant croto protein and one or more additional active ingredients. One or more additional active ingredients may be selected from the constituents, drugs, substances, therapeutic compositions, etc. described herein, or others known in the art. For example, Klotho protein and one or more additional active ingredients are injectable (eg, intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulation. Can be co-injected into. Alternatively, in combination therapy or co-administration, the Croto protein and one or more additional active ingredients are combined with the combination product, composition, or formulation, or without being formulated. It may include treatment or administration of the above additional active ingredients. For example, the Klotho protein and one or more additional active ingredients can be injected separately (eg, intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other. Each contains or may be in such a formulation.

Some embodiments (eg, in mammalian subjects) are formulated or co-prepared to increase endogenous Klotho protein levels or production, as described herein in an effective amount of the composition (eg, in a mammalian subject). It may include a composition comprising (health supplement) and optionally an effective (eg, pharmaceutically effective) amount of recombinant Klotho protein and / or pharmaceutical or prescription drug. In some embodiments, the recombinant Klotho protein and / or pharmaceutical or prescription drug can be co-administered with the composition. In some embodiments, the recombinant Croto protein and / or pharmaceutical or prescription drug can be combined with a pharmaceutically acceptable carrier. In some embodiments, any recombinant croto protein that is optionally administered can increase serum-soluble croto protein levels by providing an extrinsic croto protein, while any drug or optionally administered. Prescription drugs increase or enhance (natural) production of seroprotein by the patient or subject, or attenuate (eg, reduce, inhibit, and / or prevent) oxidative or other damage or degradation of seroprotein or sero gene. ) Can increase serum-soluble Klotho protein levels.

It is also understood that co-administration can include co-administration of two or more components, or separate administration of two or more components, with separate administrations preferably separated by a period of time. Will. In some embodiments, the period 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 doses may be 1 to 60 seconds, 1 to 60 minutes, 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, etc., or any value or in between. Can be separated by a period, such as a range of values for. Similarly, co-administration can include overlapping dosing time frames for two or more components.

Any of the aforementioned or other compositions, formulations, or co-administrations may have additive or synergistic effects as compared to those by any of the individual components alone. For example, co-administration of various health supplement components or health supplement compositions with active components (eg, recombinant crotoproteins, pharmaceuticals, etc.) results in the administration of individual single components at similar concentrations. Can result in greater than total treatment results. In addition, synergistic effects may include therapeutic outcomes similar to those of individual outcomes of administering a single component at lower concentrations. Synergistic effects are also any other than increasing the maximum effective dose of one or more of the constituents, reducing the toxicity of one or more of the constituents, or merely additive effects of individual treatment outcomes. May include beneficial results of. In addition, the additive effects of individual treatment outcomes may include one or more additive effects. Such additive / synergistic effects may be predicted or unpredictable given the nature and understanding of the individual constituents.

Specific embodiments include expressed nucleic acid constructs and / or vectors, cell lines and / or cell suspension cultures, and one or more recombinant (eg, human or mammalian) croto proteins, protein fragments, and / or protein diversity. It may include a method of producing a body, purifying it and administering it to a subject (human or non-human animal).

It will be appreciated that any of the above, including health supplements, formulations, co-formulations, co-administration, combination products, etc., may be referred to as "compositions" or "compositions of the invention".
Administration of Extrinsic S-Crotho This disclosure discloses serum levels of S-Crotho within the range of young (eg, 18-30 years old) persons (eg, normal and / or (eg, without chronic pathology)). With respect to S-Clotho preparations, clinical dosages, and administration) to increase and / or maintain.

Aspects or embodiments of the present disclosure include, for example, administering a (cGMP and / or clinical grade) human recombinant alpha-soluble croto protein or protein fragment (of isoform 1) to a (human) subject in need thereof. including. Embodiments may also include measuring serum S-crotoe levels or concentrations (in (human) subjects) (eg, by mass spectrometry (MS) or ELISA). Such measurements can occur before, after, and / or during S-Crotho administration, and optionally for metabolic, degradation, or reduction of serum S-Clotho levels and / or serum S-Cloteau levels. Can be repeated to determine speed. MS is a well-known technique in the art. MS can be used to identify and further quantify the level of one or more (natural and / or recombinant) croto proteins in the serum of a subject.

One or more additional proteins can also be measured in the serum of interest. For example, one or more croto-related and / or age-related proteins (eg, FGF21, GDF-11, TIMP2, NAD +, CCL11, hormone testosterone, estrogen, etc.), and / or kidney function proteins (eg, eg, estrogen). KIM-1, cystatin-C, creatinine, BUN, creatinine, NGAL, etc.) can be measured separately from or in combination with the measurement of croto in serum.

In at least one embodiment, a serum sample, such as a blood sample, is obtained. Samples can be obtained by blood sampling, as is known in the art. In a preferred embodiment, a finger stick to obtain a blood sample, or other less invasive means may be used. Therefore, blood samples are collected more frequently (eg, throughout the day and / or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or every 12 hours. ) Can be collected. Measure total Croto protein serum concentration and serum concentration of various Alpha Croto protein species such as native Croto species (eg, soluble Croto, truncated Croto, secretory Croto, etc.) and / or one or more Croto proteins of the present disclosure. For this, MS can be used. In some embodiments, the croto level is 0.5, 1, 2, 3, 4, 5, 6, 7, before and again throughout the day and / or after administration of the therapeutic recombinant croto protein. It can be measured every 8, 9, 10, 11 or 12 hours (or one or more of these). Alternatively or in addition, the crotch levels are 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 18, 20, 20, It can be measured every 21, 25, 28, 30, 36, 40, 45, 50, 60, or 90 days (or weekly) (or one or more of these).

Embodiments also determine such rates and / or maintain the serum concentration of S-croto protein in the serum of such subjects within the serum concentration range of S-croto in normal young individuals. It may include calculating the treatment protocol for (eg, including the frequency, amount, and / or duration of administration (s) of S-Cloteau (successive)). In at least one embodiment, the concentration of S-crotoe can be maintained with approximately 1000 picograms (pg / mL) of (S-crotoe) protein per milliliter of serum.

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

The embodiments are also optional (eg, at the start (before administration of any extrinsic S-crotoe) and / or at different times prior to the treatment protocol and / or throughout the treatment protocol, in the (human) subject (s) To illustrate the circadian rhythm effect of (eg, before and after administration of S-croto), (reference value) S-croto level measurements (s) may be included.

Embodiments may also include measuring the preferred frequency, amount, and / or duration of S-Cloteau administration. For example, for subjects with low S-Crotho serum levels (eg, measured by MS or ELISA immunoassay quantification), the serum S-Cloteau level of the subject should be set to a first predetermined level (eg, about 1000 pg / mL). A first dose of Klotho configured and / or adapted to be given (eg, via any suitable dosing route) may be given (eg, changes obtained in the serum S-Klotho concentration of interest). ) Is measured (by MS or ELISA), the rate of metabolism, degradation or reduction of levels (after the first dose) and / or serum S-croto level is measured, and the half-life of the administered S-croto is calculated. And / or determine the frequency and / or time frame in which a second subsequent dose of S-Klotho should be given (eg, to maintain serum S-Crotho 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.

Further administration provides S-Klotho serum levels equivalent to normal homosexual young person serum maintenance levels (eg, approximately 1000 pg / ml) over a time frame suitable for (long-term) production in the subject. Can be. The total duration of S-Clotho administration (to (human) subjects) can range from 1 day to 5 years or more. Measurements and / or determinations of a subject's frailty based on the use of a clinical frailty score and other measurements can also be performed over a time frame.

Embodiments of the present disclosure are 5%, 10%, 15%, 20%, 25%, 30%, 35%, beyond the normal range (eg, at least about 500-1000 pg / ml serum, for example). To maintain elevated S-Crotho levels of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more. Further includes increasing the S-Crotho dosage.

Certain embodiments include administering the S-Crotho protein. Non-limiting examples of suitable routes of administration include injection (eg, bolus, gradual, intravenous, intradermal, intraperitoneal, intramuscular, intradermal, and / or subcutaneous injection), oral or oral related administration (eg, intradermal, intraperitoneal, intramuscular, intradermal, and / or subcutaneous injection). For example, oral ingestion, buccal administration and / or sublingual administration), topical administration, transdermal administration, rectal administration, intravaginal administration, inhalation and the like.

An exemplary embodiment may include administering the S-Clotho protein or composition in a single bolus injection or a long-term (IV) injection (eg, a long-term infusion). 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 micrograms or more of S-Crotho can be administered per treatment. Suitable doses can be calculated through one or more methods known in the art. One such method is the allometric scaling method. For example, in rat experiments, 0.01 mg of S-croto / kg body weight or 10 μg / kg is used per dose. Illustratively, using 0.16, which is equivalent to the human relative growth rate, the human equivalent dose (HED) = 10 μg / kg x 0.16 = 1.6 μg / kg. Thus, exemplary, a 70 kg human individual would require 70 kg x 1.6 μg / kg = 112 μg, and a 60 kg human would require 60 kg x 1.6 = 96 μg. Table 46 below demonstrates the conversion of various animal doses to human equivalent (HED) based on body surface area. The conversion assumes a 60 kg human.

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

The amount and / or bioavailability of S-croto in humans (before and / or after recombinant protein administration), the total amount and / or concentration of S-croto administered, and / or recombinant protein administration. Multiple factors may or may be considered in the subsequent determination, measurement, and / or estimation of 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, metabolism or other rates of subject, pharmacokinetics (PK), pharmacodynamics (PD), toxins. It may include studies (Tox) and the like.

In at least one embodiment, the (normal) concentration of S-crotoe (eg, in a healthy, young (18-30 year old) human adult) can be approximately 1000 pg / ml in serum. A typical adult can have a blood volume of approximately 5 liters, and women generally have a lower blood volume than men. Approximately 55% of human blood can be composed of serum. Therefore, (5 liters of blood / adult) x (0.55 serum / liter of blood) = 2.75 liters (2750 ml) of serum / adult. Assuming the absence of endogenous serum S-croto, 2750 ml x 1000 pg / ml = 2,750,000 pg (or 2750 ng or 2.) per adult subject to achieve a final concentration of 1000 pg / ml in total serum. 75 μg) of extrinsic S-croto can be administered.

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

Approximately 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750 per milliliter of serum to raise the serum-soluble Klotho protein concentration of interest 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 Soluble croto protein in excess of, equal to, or in between 000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, 100,000 picograms, or soluble croto protein in serum Typical health levels of 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 A pharmaceutically effective and / or sufficient amount of purified recombinant S-Klotho protein can be administered in excess of, equivalent to, or in between.

In some embodiments, the subject is about 0.01 mg / in a suitable amount of Croto's buffer (eg, 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-Crotho protein above kg by weight, equivalent, or in between. Injections can be administered. Therefore, for a 160 lb (ie, 72.57 kg body weight) subject, based on a calculation of approximately 0.73 mg S-croto (0.01 mg S-croto / kg x 72.57 kg body weight) per dose. (Borus) injections can be administered. Similarly, a 170 lb person can receive 0.77 mg of S-Crotho per dose. The total number and frequency of administrations can be determined, for example, on the basis of achieving and maintaining a concentration of 1000 pg / ml S-croto (equivalent to 0.000001 mg / ml serum) in serum. The latter can be measured and confirmed by MS or by the human S-Croto 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, they Can be equivalent to, or any value or range of values between them. Urine and / or blood (eg, to measure, test, and / or determine changes in serum S-crotoe levels and dose and / or response over time) are medical procedures or procedures for recombinant croto protein. Approximately 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 Can be harvested at one or more time points between them, greater than, less than, equal to, greater than, 2 months, and 3 months.

One or more embodiments include the manufacture and / or (subsequent) administration of a unique formulation of an S-Clotho active drug product and / or a combination with a pharmaceutically acceptable carrier. The carrier may be suitable for IV and / or bolus injection. The embodiments also include S-Crotho and / or pharmaceutically such that the Inactive S-Clotho can be activated in vivo to release a biologically effective S-Cloteau in an animal or human subject. It may include the manufacture and / or (subsequent) administration of a unique Inactive Prodrug formulation in combination with an acceptable carrier. Such a Prodrug formulation may include a coating or sequential continuous release formulation.

Therapeutic methods, uses, etc. Some embodiments include methods (eg, including one or more method steps) or therapeutic methods. Some embodiments may include administering to a subject (eg, a subject in need thereof) one or more of the compositions described herein. Administration of the composition of the invention (s) raises the serum-soluble croto protein concentration of the subject above a predetermined threshold, or maintains the serum-soluble croto protein concentration of the subject above a predetermined threshold over a period of time. Can be enough.

The composition comprises, for example, (i) the therapeutic recombinant croto protein or fragments or fusion proteins thereof described in the present disclosure, and / or (ii) endogenous croto protein production and / or level in a patient to whom the product has been administered Products that increase, enhance, increase, and / or support (s) may optionally include (iii) with one or more additional (active or inactive) ingredients or compositions. Those skilled in the art will appreciate that each of the therapeutic effects, indications, and / or treatments outlined herein with respect to recombinant therapeutic Crotoproteins and products, compositions, and methods associated thereto is endogenous Clotoprotein production and. You will understand that / or levels (s) can be achieved by (naturally) increasing, enhancing, increasing, and / or supporting. Those skilled in the art will also (naturally) increase, enhance, increase, and / or support endogenous Klotho protein production and / or levels (s) of the therapeutic effects, indications, and indications outlined herein. It will be appreciated that each of / or treatments can be achieved by administering recombinant therapeutic Klotho proteins and products, compositions, and through methods associated with them. Therefore, for brevity, the present disclosure relates to both (i) administration of recombinant therapeutic Croto protein and (ii) administration of health supplements or dietary supplement compositions of the present disclosure. , Indications, and / or treatments are expressly contemplated herein and need not be elaborated.

Certain embodiments may include the compositions described herein for use in the treatment of one or more diseases, disorders, injuries, and / or (medical) pathologies. Similarly, embodiments may include methods of treating (or alleviating, coping, preventing, inhibiting, etc.) one or more diseases, disorders, injuries, and / or (medical) conditions. An exemplary treatment method comprises administering an effective amount of the composition of the present disclosure to a subject in need thereof. 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 increase) in serum-soluble Klotho protein concentration in the subject. obtain. Thus, an "effective amount" of the compositions of the present disclosure increases or raises the serum-soluble croto protein concentration of a subject above or above a predetermined threshold, or increases the serum-soluble croto protein concentration of a subject above a predetermined threshold over a predetermined period of time. It can be an effective amount to maintain. Alternatively, or in addition, an "effective amount" of the composition of the present disclosure can be an amount effective in achieving a beneficial therapeutic effect in the patient to whom the composition is administered.

The method, composition, or (elevated) protein can also be effective in treating one or more diseases, disorders, injuries, and / or (medical) pathologies. Illustratively, the pathology is weakness, bone density loss or bone mineral loss, weight loss, muscle atrophy or degeneration, muscle loss, muscle strength, grip strength, leg strength or weakness, movement, freedom of movement, quality of life assessment, Decreased ejection rate, decreased motor capacity, learning ability, learning ability, memory, or intellectual index, cognitive deterioration or oblivion, cognitive or functional deterioration, synaptic plasticity or hyposynaptic function, and cellular senescence , May be or include age-related or age-related conditions (or (human) age-related conditions). However, the pathology, or alternative, can provide beneficial therapeutic effects, including those described in the present disclosure and those known to those of skill in the art, with increased serum crotch levels, any physical, intelligent, emotional. It will be understood that it can be, or include, a target or other deficiency, need, or desire.

Methods, compositions, or (elevated) proteins are also compositions (eg, proteins or supplements) or elevated soluble Klotho protein levels, Alzheimer's disease, Parkinson's disease, dementia or vascular dementia, muscular atrophy. 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 One or more age-related conditions (or (human) aging, such as, cataracts, luteal degeneration and other eye diseases / disorders, multiple sclerosis (MS), cysts, and / or ulcerative arthritis It may be effective in the treatment of pathological conditions related to.

Some embodiments of the disclosure include treating cancer, lowering serum phosphate levels in a patient, and treating diabetes or a diabetes-related condition in a subject in need of treatment (eg, type 1 diabetes). That, treating the condition of the subject's heart (eg, cardiovascular disease, left ventricular hypertrophy (LVH), pathological LVH and / or congestive heart failure, etc.), treating the subject's acute lung injury (eg, eg). (Using nanoparticles), protecting the patient's lungs against oxidative injury, detecting early acute renal injury in critically ill patients, reducing vascular calcification in the subject, improving cognitive ability, Treating renal and / or hepatic ischemia, regulating the stress response of (human) aging endothelial cells, acute and / or chronic renal injury, disease, or progression of disease, and / or urotoxic myocardium Prophylactically and / or therapeutically treating, preventing, alleviating, blocking and / or reversing the disease, reversing or reducing age-related resistance to melanoma and other cancers and muscular dystrophy (including DMD), It may be useful in targeting the apoptosis of senile 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 pathologies. Such conditions may be described or disclosed herein. Such conditions may be known to those of skill in the art to be associated with Klotho serum levels.

As briefly mentioned above, the present disclosure includes and contemplates various dosage forms and / or methods of administration of the compositions of the present disclosure. Some embodiments may be administered in oral dosage form and / or orally (eg, in one or more convenient forms including tinctures, tablets, capsules, powders, tea, etc.). For example, a tincture consisting of a herbal or natural ingredient alcohol or glycerin-based extract can be packaged in a glass drip bottle. Glycerin-based tinctures can provide a sweet taste and make the formulation easier to swallow. The tincture can also be administered by an infusion device. The tincture may include the labeled dosage range (eg, 20-40 drops per adult dose).

Capsules and tablets are also common forms for oral administration of replacements. For example, dried herbs may be pulverized and compressed into gelatin capsules. The replenisher may be supplied as capsules and / or tablets. The replenisher may be delivered as a powder in a bottle or envelope in which the dosage used is listed in a container. The appropriate amount of powder per dose can also be obtained by opening the dosage capsule and sprinkling the contents on the drink or food.

Tea (eg, herbal decoction) is a common form of herbal remedy and is a sedative method of taking herbs. Herb decoction can be made by placing a small amount of herb in a cup, filling the cup with boiling water and immersing it. In the form of tea, the potency of the herbaceous component of the replenisher can increase with increasing soaking time, and the tea is best soaked for at least 10 minutes. Covering the cup (a small saucer works well) keeps the steam and keeps the tea hot for a longer period of time.

Some embodiments are methods of treating age-related or other pathologies, diseases, or disorders, comprising administering an effective amount of the composition of the present disclosure to a subject in need thereof. , May include methods. Some embodiments are methods of treating and / or preventing a condition, disease, or disorder, preferably an age-related condition, disease, or disorder in a mammalian subject, in an effective amount of the present disclosure. Methods may be included that 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, the composition of the present disclosure in an effective amount for a subject in need thereof. May include methods, including administration of. Some embodiments are methods of treating and / or preventing renal injury in a mammalian subject, subject to prior to medical procedure or administration of nephrotoxicity and / or after administration of medical procedure or nephrotoxicity. May include methods, optionally prophylactically, comprising administering an effective amount of the composition of the present disclosure. Some embodiments may include a method of administering the composition of the present disclosure to a human or non-human animal (eg, mammal) subject in need thereof. The subject to whom the composition is administered may suffer from or be at risk for a variety of conditions (eg, disorders, disorders, injuries, illnesses, etc.). For example, some embodiments treat one or more chronic diseases and / or age-related conditions, such as (human) age-related physical, intelligent, neurological, or other conditions. Including how to do it. 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) may increase the concentration of the serum soluble croto protein of interest, such as, for example, a soluble croto protein of greater than, or equal to, about 50-3000 picograms per milliliter of serum. Can be sufficient to increase or increase (up to the level of). A pharmaceutically effective amount of the composition (s) may also, or alternative, be sufficient to maintain a serum-soluble Klotho protein concentration of interest above a predetermined threshold over a predetermined period of time.

In certain embodiments, a preferred increase in protein levels may modulate (can be effective in regulating) the IGF-1 and / or Wnt signaling pathway and exhibit β-glucuronidase and / or sialidase activity. , obtained by suppressing the p53 / p21 signaling pathway, and / or through preferably inhibition of p53 / p21 signaling _pathway, may reduce the H 2 _{O 2} induced cell senescence and apoptosis. The (elevated) protein preferably exhibits multifaceted 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 or be functional as a humoral factor in the regulation of the activity of glycoproteins on cell surfaces such as growth factor receptors such as growth factor-1 receptors.

Specific embodiments include determining the serum-soluble croto protein concentration of a subject, calculating a pharmaceutically effective amount, determining the rate of decrease of the soluble croto protein in the subject's serum, and determining the serum-soluble croto protein concentration of the subject. To calculate the subsequent dosing time, which will be below the second predetermined level based on the measured rate, the serum soluble croto protein concentration of the subject from the second predetermined level to the first predetermined level. It may include calculating a subsequent dosage of the composition sufficient to raise to a level and / or administering the composition of the subsequent dosage to the subject. Certain embodiments may include prescribing a composition in a normal (eg, daily) dose or dosage form to a subject (eg, based on a determined level of serum-soluble Klotho protein concentration in the subject).

Exemplary embodiments include (therapeutic) methods. The method of treatment may be similar to and / or modeled on testing the therapeutic effects or health benefits of the various compositions of the present disclosure. The results of administration of the various compositions are monitored in human subjects. After the subject voluntarily agrees to participate in the study and signs the patient consent form, a physical examination and blood and saliva tests will be performed. If the inclusion criteria of the test protocol are met, the subject is enrolled in the test. Illustratively, the study may include 100 healthy individuals who are not currently supplemented with vitamin D and do not live in a sunny environment. Subjects with KLVS variants (Klotho overexpression factors) and / or other gene variants of the Klotho gene (s) may be excluded from the study. After enrollment, subjects will receive the Klotho supplement formulation of the present disclosure, or a three-month supply of placebo in a double-blind study. Subjects receive an unlabeled replacement formulation or a bottle of placebo free of charge. Subjects take this supplement daily for 3 months.

At the end of this 3-month period, blood was drawn for a fasting blood test, the subject's body weight and blood pressure were measured, and an alpha-croto (α-croto) human soluble ELISA assay kit (eg, IBL America, Minneapolis, MN, USA). Record with the concentration of plasma alpha-soluble crotho as determined by (Cat. No. 27998).

The test / design procedure for human subjects in this test is shown below.
Test design / procedure:
Up to 100 patients Duration: 3 months to the first endpoint, 2nd endpoint at 6 months, including placebo patients who chose to take crossovers and replacements.

Criteria to include Questionnaire: Current medications (statins, ARBs), nutritional supplements, blood pressure, exercise frequency, alcohol consumption.
Before registration:
Blood sample and cheek swab collection (PI office or mobile blood technician)
Judgment of KLVS Manifold (Klotho Overexpression Factor) -Send a cheek swab to the laboratory to determine KLVS manifold (Klotho Overexpression Factor) status Croto levels, laboratory analysis of vitamins D and E, and pretreatment criteria Other analysis of values Supplement: Transport to participants (3 months supply). (Activity supplement and placebo control, equal number of activity and placebo control)
Result measurement: Participants download a mobile application to monitor compliance and questionnaires (exercise, blood pressure) Follow-up blood sampling after 3 and 6 months Ingredients in supplements carry risks Is composed of very low nutritional components. If the subject experiences any discomfort or adverse reaction to the formulation, the subject will be instructed to stop taking the product and contact the investigator.

Any significant side effects will be reported to the IRCM Institutional Review Board (IRB) of the study within 48 hours.
Illustrative Pathology In some embodiments, the pathology, disease, or disorder treated with the compositions (s) and / or methods (s) of the present disclosure preferably, for example, is frail, reduced bone density. , Bone mineral loss, weight loss, muscle atrophy, muscle degeneration, muscle loss, muscle weakness, grip weakness, leg strength weakness, physical weakness, movement loss, movement freedom, loss of quality of life evaluation, drive out Decreased rate, decreased motor ability, decreased learning ability, decreased learning ability, decreased memory ability, decreased intelligence index, decreased cognitive ability, forgetfulness, decreased cognitive ability, decreased cognitive function, decreased synaptic plasticity, Decreased synaptic function, cell aging, chronic renal disease (CKD), bone-inorganic disorder associated with chronic renal disease (CKD-MBD), polycystic kidney disease (PKD), autosomal dominant polycystic kidney (ADPKD), acute kidney injury (AKI), acute tubule necrosis (ATN), acute allergic interstitial nephritis (AAIN), glomerular nephritis, renal disease, renal failure, Alport syndrome, non-poor urinary renal failure, alcohol dependence, hyperphosphate Hememia, muscular dystrophy (MS), type 1 diabetes, type 2 diabetes, cardiovascular disease (CVD), cardiovascular calcification, cerebrovascular failure, vascular calcification, ischemic heart disease, heart failure, left ventricular hypertrophy, urinary myocardium Disease, abnormal blood pressure, salt-sensitive hypertension, tissue calcification, calcification atherosclerotic plaque loading, calcification, familial neoplastic calcification, cancer, one or more tumors, myelin-related disease, demyelination disease , Neurodegenerative disease, neurovascular disease, progressive nuclear palsy (PSP), Pompe disease, Niemannpic disease, microgliosis, Farber's disease (FD), bone mass disease, osteoporosis, bone loss, bone loss (specifically) (Reduction of BMD in cortical bone), pulmonary emphysema, pulmonary fibrosis, cystic fibrosis, idiopathic (ie, unknown cause) pulmonary fibrosis, radiation-induced lung injury, cirrhosis, biliary atresia, atrial fibrosis , Endocardial myocardial fibrosis, (old-fashioned) myocardial infarction, collagen scar, arteriosclerosis, joint fibrosis, Crohn's disease, Dupuytran contraction, Keroid, mediastinal fibrosis, myeloid fibrosis, Pelony's disease Systemic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, adhesive arthritis, skin atrophy, thoracic atrophy, renal interstitial matrix accumulation, glomerular sclerosis, anemia , Albuminuria, proteinuria, infertility, Alzheimer's disease, Parkinson's disease, dementia, vascular dementia, muscular atrophic lateral sclerosis (ALS), motor neuron disease (MND), atrial fibrillation, chronic obstruction Sexual lung disease (COPD), fibromyalgia, adult-onset diabetes, arthritis, rheumatoid arthritis, degenerative joints Disease, glaucoma, cataract, cirrhosis, multiple sclerosis (MS), ulcerative colitis, ulcerative colitis, nausea, obesity, vitamin D-related pathology, bone disease, bone disease through bone remodeling, intestinal disease And related conditions, stem cell depletion, sea sickness, space maladaptive syndrome (SAS), nausea, rotatable dizziness, nonalcoholic steatohepatitis (NASH), cirrhosis, and alcoholic steatohepatitis. obtain.

Croto serum levels have also been reported to be associated with muscle recovery, such as muscle healing after injury. Specifically, and not bound by any theory, age-related decline in α-crotoe can lead to impaired mitochondrial dysfunction and muscle regeneration of progenitor cells. Clinically, this can lead to the treatment of elderly people who have had muscle injuries or have undergone surgery to damage muscles. Embodiments of the present disclosure may include administering the compositions described herein prophylactically and / or after surgery. Administration at the appropriate time point (s) can enhance or support muscle regeneration and lead to more complete recovery. Serum Croto protein levels can also enhance or support muscle regeneration in athletes and other patients. Thus, embodiments of the present disclosure may include administering the compositions described herein prophylactically (before training) and / or after exercise.

Administration of Compositions for Treating Age-Related Weaknesses in Humans and Non-Human Mammals An exemplary embodiment of the present disclosure is for treating age-related weaknesses (eg, in humans or non-human animals). With respect to administration of the composition of. s-Crotho rescues myoprototype stem cells, improves muscle repair and / or suppresses fibrosis in animal models of human disease. Therefore, s-crotoe may be a promising approach to combat muscle degeneration in older human subjects showing signs of weakness.

For example, the disclosure maintains maintenance serum levels of s-croto in subjects within the range of normal and / or young people (eg, 18-30 years) (eg, without any chronic pathology). For s-croto level increasing compositions, formulations, and dosages, and their administration, to vulnerable individuals and / or the elderly (eg, 60-95 years).

Such long-term Klotho treatment may ameliorate and / or ameliorate one or more age-related indicators or conditions in the elderly, frail, or other physiologically aged.
Administration of Compositions for Treating (Reducing) Muscle Atrophy in Humans and Non-Human Mammalians The exemplary embodiments of the present disclosure provide guidance on the effect of administration of Croto against muscle atrophy. , Skeletal muscle tissue mass, and preferably the compositions of the present disclosure for treating (eg, reducing) muscle atrophy in humans, as measured by the simultaneous use of the above compositions and molecular indicators. Regarding administration.

Muscle atrophy can include a number of neuromuscular, metabolic, immune, and neuropathy 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 or disused muscle use. Symptoms include a decrease in skeletal muscle tissue mass. In human males, muscle mass is reduced by a third between the ages of 50-80. Some molecular properties of muscular atrophy may include upregulation of ubiquitin ligase and a decrease in myofibrillar proteins. Degradation of these proteins can be monitored, for example, in specific muscle myosin (eg, by measuring the production of 3-methyl-histidine, a specific constituent of actin). Release of creatine kinase (cell damage marker) can also be an indicator.

Administration of compositions to treat (improve) muscle repair or recovery in humans and non-human mammals Croto serum levels have also been reported to be associated with muscle recovery such as muscle healing after injury. There is. Specifically, and not bound by any theory, age-related decline in α-crotoe can lead to impaired mitochondrial dysfunction and muscle regeneration of progenitor cells. Clinically, this can lead to the treatment of elderly people who have had muscle injuries or have undergone surgery to damage muscles. Embodiments of the present disclosure may include administering the compositions described herein prophylactically and / or after surgery. Administration at the appropriate time point (s) can enhance or support muscle regeneration and lead to more complete recovery. Serum Croto protein levels can also enhance or support muscle regeneration in athletes and other patients. Thus, embodiments of the present disclosure may include administering the compositions described herein prophylactically (before training) and / or after exercise.

Administration of Compositions for Treating Intellectual and / or Cognitive Decreased Throughout Old Age and / or Human Life The exemplary embodiments of the present disclosure include impaired cognitive function (s) (s). With respect to administration of the compositions of the present disclosure to improve and / or suppress. At the time of this disclosure, it was unclear whether administration of the compositions of the present disclosure could suppress cognitive decline in humans. However, transgenic mice with systemic overexpression of Klotho performed better than controls in multiple tests of learning and memory. Mouse crotho elevation is also a synaptic form of synaptic plasticity, a synapse that is an N-methyl-D-aspartate receptor (NMDA) subunit that enhances long-term synergies and has important functions in learning and memory. GluN2B was concentrated. Due to the blocking of GluN2B, the effect of croto intervening was lost.

Klotho-regulated pathways may be associated with slowing the progression of Alzheimer's disease and other forms of dementia. In brain scans of more than 400 healthy men and women over the age of 53, those who had a single copy of a particular Croto gene manifold had a larger area of brain to handle planning and decision making. Was found. Further studies in this group found that people with enlarged right dorsolateral prefrontal cortex (rDLPFC) were better at a series of intelligent tasks.

Approximately 1 in 5 inherits a single copy of the gene manifold or allele known as KL-VS, which improves heart and kidney function, extending human lifespan by an average of approximately 3 years. However, with respect to brain function, the improvement in people's intelligent test scores by having a larger rDLPFC accounts for only 12%, while the editors point out that they carry one copy of the KL-VS allele. This appears to provide 10 years of resilience to the expected degradation of rDLPFC structure and function. Therefore, KL-VS heterozygotes appear to be associated with a larger volume of the right dorsolateral prefrontal cortex (rDLPFC).

Since rDLPFC is important for executive function, researchers also analyzed the working memory and processing speed of individuals. KL-VS heterozygotes may be associated with enhanced executive function over the lifespan tested. In short, the results suggest that diversity of the Klotho gene can be associated with larger brain volume and better functioning.

Exemplary embodiments of the present disclosure include in vivo human protein levels (eg, to enhance cognition and suppress cognitive deficiency throughout human life) and / or (cells, molecules, and / or downstream). With respect to administration of the compositions of the present disclosure to increase efficacy. Administration of the s-croto level increasing composition can preserve and / or improve cognitive function (eg, in humans).

Administration of Compositions for Treating (Extending) Human Longevity and / or Lifespan Exemplary embodiments are compositions of the present disclosure for human subjects of real age (eg, age from birth) and of the same sex. Is administered to improve life expectancy. Life expectancy results obtained with composition administration (experimental group of human subjects) include statistical information (eg, from a mathematical table) for individuals not receiving the composition (control group) and / or a sufficiently large population. Can be compared reliably.

Administration of Compositions for Treating Other Clinical Indications The exemplary embodiments of the present disclosure are compositions of the invention for treating pathological conditions associated with or not associated with any age. Regarding the administration of, the pathological conditions include human weakness (increase), longevity (decrease), cellular senescence (decrease), muscle strength (decrease), bone loss or density (decrease), and cognitive ability (decrease). ), Muscle mass (decrease), physical strength (decrease), grip strength (decrease), leg strength (decrease), etc., but are not limited thereto. The disclosure also includes increased bone mineral content (BMD) (eg, in women rather than men), decreased BMD (eg, in older women) after menopause, regeneration of (degenerated) skeletal muscle or its degeneration. The present disclosure for reduction of walking, improvement of gait, spatial learning ability and memory, movement, freedom of movement, quality evaluation of life, improvement of ejection fraction (or reduction of decrease), change of movement, improvement of movement, etc. With respect to administration of the composition. The present disclosure further reduces cognitive deterioration or amnesia, increases cognitive ability, improves cognitive function and synaptic plasticity, reduces learning ability, learning ability, or IQ decline, learning ability, learning ability, or IQ improvement. With respect to administration of the compositions of the present disclosure for such.

Administration of Compositions to Treat Acute Kidney Injury (AKI) Acute Kidney Injury (AKI), formerly known as Acute Kidney Injury (ARF), often ranges from mild to dysfunction. It is defined as the sudden onset of renal dysfunction. AKI is a common clinical complication that affects 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%. Expression of renal croto has been shown to be suppressed following AKI. Croto adenovirus gene transfer can be cytoprotective in AKI.

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

AKI includes kidney transplantation or other surgery, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephrotoxicity (eg glomerulonephritis), nephrotoxicity (eg drug-induced nephrotoxicity), It can result at least partially from hypotension, septic or nephrotoxic pathology, or other contributors (s). Kidney transplantation and other surgery can cause acute damage or injury to the kidney, leading to renal disease and / or renal failure. Nephrotoxicity can contribute to AKI, ATN, AAIN, nephritis and the like. Drugs (eg, clinically administered drugs, prescription drugs, illicit drugs, or other drugs) have been reported to be associated with 15% to 25% of all cases of AKI. Contrast alone accounts for 10% of all causes of in-hospital infected acute renal failure (eg, contrast-induced acute renal injury CIAKI), deterioration of renal function during hospitalization and postoperative renal failure after diminished renal perfusion. Is the third major cause of.

In some cases, drug-induced AKI may or may be antibacterial-induced (resulting from antimicrobial treatment) nephrotoxicity. 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 FIG. 47, nearly 23% of patients treated with the common aminoglycoside amikacin develop acute renal disease and more than 17% of patients treated with amikacin die before discharge. doing. Other aminoglycosides, including gentamicin and tobramycin, also induced (or were associated with or contributed to) renal disease.

As illustrated in FIGS. 13A and 13B, more than 1.2 million adult patients of various age groups were treated with aminoglycosides in 2010. Other antibacterial agents include, for example, penicillin, ampicillin, cephalosporins, sulfonamides, ciprofloxacin, vancomycin, macrolides, tetracyclines, riffampins and the like.

Drug-induced nephropathy is also one or more of aspirin (acetylsalicylic acid), selecoxib, diclofenac, diflunisal, etdrac, ibuprofen, indomethacin, ketoprofen, ketoprofen, nabumetone, naproxen, oxaprozin, pyroxicum, salsarate, sulindac, torumetin. It can result from treatment with non-steroidal anti-inflammatory drugs (NSAIDs).

Drug-induced nephropathy also includes one or more cyclooxygenase-2 (COX-2) inhibitors (eg, valdecoxib, lofecoxib, selecoxib, etc.), proton pump inhibitors (eg, omeprazole, lansoprazole, etc.), anticonvulsants (eg, omeprazole, lansoprazole, etc.). For example, phenitoin, valproic acid, etc.), histamine H2 receptor antagonists (eg, nizatidine, lanitidine, famotidine, simethidin, etc.), diuretics (eg, carbonate dehydration enzyme inhibitors, loop diuretics (eg, thiazide, etaclinic acid, etc.), Trasemide, Frosemia, etc.), potassium-retaining diuretics (eg, triamterene, spironolactone, amylolide, etc.), thiazide diuretics (eg, indapamide, chlortalidone, metrasone, methicrotiazide, hydrochlorothiazide, chlorothiazide, bendroflumethiazide, polythiazide, hydro It can result from treatment with other diuretics such as flumethiazide) or other diuretics such as pamablom, mannitol.

Drug-induced nephropathy also results from treatment with lithium, which can affect the flow of sodium through nerve and muscle cells in the body, often with hyperactivity, imminent discourse, and diminished judgment. Can be used to treat manic episodes of bipolar disorder, characterized by reduced sleep cravings, aggression, and anger. Lithium can also help prevent or reduce the intensity of manic episodes. Drug-induced nephrotoxicity can also result from treatment with gold, mercury, copper, or other elemental substances or exposure to them.

Drug-induced nephropathy also results in penicillamine, Wilson's disease (due to the accumulation and binding of copper for removal through the urine), cysteineuria (binding to cysteine to produce a mixed disulfide that is more soluble than cysteine). Penicillamine (D-), a chelating agent class drug that can be used to treat (depending on), relaxants that act directly on smooth muscles (eg, hydralazine), antispasmodics (eg, carisoprodol, cyclobenzaprine) , Metaxalon, methocarbamol, diazepam, chronidine and other imidazoline compounds such as benzodiazepine, tizanidine, baclofen, hydantin derivatives, dantrolene and the like.

Other forms of drug-induced nephrotoxicity are also known, for example, after exposure to contrast agent-induced nephrotoxicity (eg, (iodination) contrast agent, radiocontrast-induced nephrotoxicity (CIN)). ); Drug (opioid) -induced nephrotoxicity (eg, after use or abuse of certain drugs (eg, opioid) such as cocaine, heroine); Chemotherapy-induced nephrotoxicity (eg, cisplatin; carboplatin; oxaliplatin) Alkylating agents such as bendamstin, cyclophosphamide, iposphamide, nitrosourea, temozolomid, melphalan; antitumor antibiotics such as mitomycin C, bleomycin, anthracylin, and related agents; capecitabin, hydroxyurea, methotrexate, pemetrexed Antimetabolites such as pralatrexate, pentostatin, fludalabine, cladribine, gemcitabine, citarabin; vinca alkaloids; topotecan; etopocid; taxan; irinotecan; lenalidomide; elibrin; arsenic trioxide; ixazomib; After treatment) etc. may be included. 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 left untreated and can result in enormous treatment costs (for 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 (eg, prophylactically or in response to at least one event). .. The method may include administering the composition of the present disclosure to a subject in need thereof. For example, the method requires an effective amount of the composition of the present disclosure (eg, to increase and / or maintain a serum-soluble Croto protein concentration of interest above a predetermined threshold over a predetermined period of time). It may include administration to the subject. The composition increases and enhances (s) the production and / or level (s) of endogenous croto protein in patients receiving (i) therapeutic recombinant croto protein or fragments or fusion proteins thereof, and / or (ii) product. , Augmentation, and / or supporting products may optionally be included with one or more additional ingredients or compositions.

In some embodiments, the pathology is (i) acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, and / or nephritis, or (ii) septicemia, AKI resulting from kidney transplantation or other surgical or medical procedures, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, nephritis, or low blood pressure Can include. Pathology can include drug-induced (eg, aminoglycoside-induced) nephrotoxicity. The protein can be administered prophylactically prior to kidney transplantation, nephrotoxicity administration, or other activity, treatment, or event that is known or expected to cause or contribute to AKI, for example. .. Alternatively, or in addition, the protein is known or expected to cause or contribute to AKI, kidney transplantation or other surgery, aminoglycoside or other nephrotoxic administration, or other activity. , Treatment, or after an event, may be administered in response to AKI.

In some embodiments, the nephrotoxin or other drug is, for example, one or more aminoglycosides (eg, paromomycin, tobramycin, gentamycin, amicacin, canamycin, neomycin, etc.) or one or more antifungal agents (eg, amphotericin). B, flucitosine, etc.) One or more contrast agents (eg, (iodinated) radioactive contrast agents, high osmotic contrast agents with an iodine-to-molecular ratio of about 1.5: 1 (HOCM), about 3: 1 Low osmotic nonionic contrast agent (LOCM) with iodine to molecular ratio, isotonic (isopermeable) contrast agent (IOCM) with iodine to molecular ratio of about 6: 1), one These antiretroviral agents (eg, adehovir, sidohovir, tenhovir, foscalnet, etc.) and one or more cancer (or chemotherapy) therapeutic agents (eg, cisplatin, carboplatin, oxaliplatin, alkylating agents (eg, bendamstin) , Cyclophosphamide, iphosphamide, nitrosourea, temozoledronic acid, melphalan, etc.), antitumor antibiotics (mitomycin C, bleomycin, anthracylin, and related substances, etc.) Pralatrexate, pentostatin, fludalabine, cladribine, gemcitabine, citalabin, etc.), binca alkaloids, topotecan, etopocid, taxan, irinotecan, lenalidomide, elibrin, arsenic trioxide, ixazomib, etc.), or one or more bisphosphonates, or derivatives thereof. (For example, zoledronic acid / zoledronic acid, ibandronate, alendronate, alendronate / cholecalciferol, etidronate, lysedronate (optionally with calcium carbonate), pamidronate, chilldronate, etc.) and / or cocaine, heroine, etc. It can be or include one or more drugs (eg, opioids).

Embodiments of the present disclosure may include methods of administering the compositions of the present disclosure. The method may include administering the compositions of the present disclosure to a human or non-human subject to (prophylactically) treat or prevent AKI or one or more pathologies associated with AKI. The method is to determine the level of serum-soluble crotho in a subject, the composition of the present disclosure in a first dosage sufficient to raise the serum-soluble crotho level in the subject to a predetermined level or a percentage of normal levels. Determining the rate of decrease of soluble croto in serum of a subject, such as by calculating, oral, bolus, or gradual administration, to administer a first dosage to the subject, after administration of the first dosage, etc. That, calculating the subsequent dosage time and amount, and / or administering the subsequent dosage of protein to the subject.

Administration of Compositions for the Treatment of Chronic Kidney Disease (CKD) Without being bound by any theory, Klotho may be involved in human chronic kidney disease. Soluble crotho in the circulation begins to decline early in stage 2 of chronic kidney disease (CKD), and urinary crotho begins to decline to CKD stage 1 probably earlier. Therefore, soluble croto can function as an early and sensitive marker of renal dysfunction. In addition, preclinical animal data support that Croto deficiency is not just a biomarker, but a cause of CKD progression and extrarenal CKD complications, including cardiovascular disease and disruption of inorganic metabolism. Prevention of lowering of crotho, reactivation of endogenous crotho production, or replacement of extrinsic crotho are all attenuated in renal fibrosis, delayed CKD progression, improved inorganic metabolism, improved cardiomyopathy, and vascular calcification in CKD. Can contribute to the mitigation of.

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

Administration of the compositions of the present disclosure may help prevent, delay, and reduce the burden of comorbidities in CKD.
Administration of Compositions for the Treatment of Enteropathy and Related Pathologies Although not bound by any theory, interstitial cells of Kahar (ICC) are other cells in the gastrointestinal tract where the function of controlling peristalsis is ongoing. The stromal cells in between. The (main) function of the ICC is to create electrical signals that control rhythmic smooth muscle contraction in the intestine (different regions of the gastrointestinal system have contractions of different frequencies called peristalsis). The ICC can induce the migration of mesenchymal stem cells within the intestine (and many other organs).

In the intestine, ICC is produced and / or activated by Klotho, which can lead to an optimized regeneration process. In Klotho knockout mice, ICC is reduced, which leads to impaired peristaltic processes, mimicking intestinal disorders that occur in older humans.

Administration of the compositions of the present disclosure may help prevent, delay, and reduce peristaltic process disorders, and / or at least increase, support, and / or optimize the intestinal regeneration process. These effects may further support bowel health and / or prevent, delay, and reduce the onset and / or severity of bowel disease and related conditions.

Therapeutic Treatment of Age-Related Pathologies Researchers have found a link between Croto and biological parameters that are generally accepted as indicators of the clinical status of hospitalized elderly patients. The researchers genotyped the KL locus single nucleotide polymorphisms (SNPs) rs95363314, rs1207568, and rs564481 in 594 hospitalized elderly patients (65-99 years old) and went to the geriatric ward in succession. The association of these KL variants with bioquantitative traits was tested using analysis of covariant and genotype risk score models. A significant association between rs9536314 and serum levels of hemoglobin, albumin, and high-density lipoprotein cholesterol (HDL-C), and a significant association between rs564481 and serum levels of hemoglobin, fasting insulin, and fasting glucose. It was observed. Gender analysis confirms these associations, with serum levels of hemoglobin and serum levels, while associations between KL genotype and HDL-C, fasting glucose, and fasting insulin levels can be triggered by female gender. It suggests that the association of insulin can be caused by the gender of the male. An association between KL genotype and creatine levels was found in women, while an association between insulin-like growth factor-1 (IGF-1) and lymphocyte count (LC) was found in men. A genetic risk score (GRS) model further confirmed the significant association between KL SNP and hemoglobin, total cholesterol, and HDL-C. Gender analysis using the GRS-tagged approach confirmed the association of HDL-C, fasting glucose and fasting insulin levels in women and hemoglobin and LC in men. 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. Is suggested, thus suggesting that one of the genetic factors may contribute to age-related illness and longevity.

Embodiments of the present disclosure use the compositions of the present disclosure for subjects requiring them (eg, individuals or patients, optionally the elderly, and / or age-related pathologies, low endogenous S-croto protein expression. And / or administration to an individual or patient suffering from age-related pathology or symptoms of reduced longevity). Administration of the compositions (s) of the present disclosure can lead to a beneficial increase in blood s-crotoe levels. Administration reverses or suppresses the adverse effects of age-related conditions and / or is a positive treatment regimen (eg, in hospitalized and / or elderly patients) total cholesterol, HDL-C, fasting glucose, fasting. When it can affect quantitative traits such as insulin, albumin, creatin, IGF-1, hemoglobin, and serum levels of lymphocyte count.

Prophylactic composition administration In addition to the above, embodiments of the present disclosure administer the composition of the present disclosure to an individual or subject in need thereof for prophylactic purposes and / or maintenance of specific health attributes. May include doing. For example, administration of the particular composition of the present disclosure may help maintain youth in optionally aging patients who have not yet suffered from a diagnosed age-related condition. Thus, while certain embodiments of the present disclosure may relate to and / or include treatment of a condition in a patient, other embodiments may prevent, inhibit the onset, and / or condition of one or more conditions. / Or may be involved in and / or include prophylactic coping.

Administration of Extrinsic S-Crotho to Treat Genetic Defects An exemplary embodiment of the present disclosure relates to administration of exogenous S-crotoes to treat (eg, correct) a known human genetic defect. .. For example, a 13-year-old girl with familial neoplastic calcification and a Croto 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 secretory protein required for the transmission of signals that FGF23 releases towards its receptor. Administration of exogenous human recombinant S-Crotho constitutes a highly targeted and effective treatment to address the dysfunction and symptoms associated with familial neoplastic calcification.

Compositions and Treatments Containing S-Crotho and / or Supplements in Combination with Other Components (s) In at least one embodiment, the composition, dosage, dosage, or therapeutic treatment is a therapeutic human set. Recombinant soluble alpha croto (S-croto) proteins, and natural supplement compositions that increase endogenous croto production or one or more components thereof may be included. Klotho is also additive with other compounds and / or constituents that affect one or more aspects of human health and well-being, whether endogenous or extrinsic (recombinant). Can act in target or synergistic substances. For example, a Therapeutic Human Recombinant Soluble Alpha Croto (S-Croto) Protein and / or a Natural Supplementary Composition that Increases Endogenous Croto Production in Combination with and / or Simultaneously with One or More Additional Active Components Treatments that include may benefit human patients. Such treatment may be prophylactic or responsive to any human condition in which the Klotho protein and / or other components (s) may have a therapeutic effect. Such conditions may include, for example, age-related conditions, clinical (eg, metabolic) conditions, chronic or acute conditions, and the like. Specific non-limiting examples of specific pathologies are disclosed herein.

S-Crotho may be present in the human body along with other blood-carried anti-aging compounds such as growth / differentiation factor 11 (GDF-11). Thus, in certain embodiments, the Therapeutic S-Clotho can be co-administered with the Therapeutic GDF-11 (eg, simultaneously, continuously and / or in combination). In some embodiments, such administration may have additive or synergistic anti-aging or other effects. Similarly, co-administration of S-Clotho to human subjects with a (neutralizing) antibody against CCL11, or an inhibitor thereof, may act synchronously to combat aging or other pathologies (CCL11 (eotaxin). (Also known as -1) is understood to be a negative regulator of stem cell rejuvenation). S-Crotho can also be co-administered with other eotaxins such as eotaxin-2 (CCL24) and / or eotaxin-3 (CCL26), or alternatives.

In some embodiments, S-Clotho can be co-administered with an inhibitor or antibody of transforming growth factor β-1 (TGF-β1). S-Crotho administration can counteract the effects of the TGF-β1 signaling pathway involved in endogenous anticellular 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 can confer metastatic potential on cancer cells, the latter process being countered by Klotho. Therefore, co-administration of S-Crotho with a transforming growth factor β-1 (TGF-β1) inhibitor or antibody can have synergistic or additive effects.

In some embodiments, S-croto can be co-administered with an antibody or suppressor of insulin growth factor-1 (IGF-1). Klotho is a hormone that inhibits the intracellular insulin / IGF-1 signaling cascade, which evolutionarily increases resistance to oxidative stress at the cellular and biological levels of mammals and prolongs lifespan. It is understood to be a mechanism that is considered conserved. Therefore, co-administration of S-croto with an inhibitor or antibody of insulin growth factor-1 (IGF-1) can have synergistic or additive effects.

The number of tests, Klotho alpha -K1, FGF23, and 1,25 _{(OH) 2} D regulated positive / negative feedback interact, as well as adjusting the / or bile acid / cholesterol metabolism Klotho beta-K1, In some embodiments, S-croto is vitamin D (eg, vitamin D) because a comprehensive control scheme for mineral homeostasis has been revealed that includes a similar control network consisting of FGF15 / human FGF19, and bile acids. It can be administered in combination with D3), or 1,25-dihydroxyvitamin D ₃ [1,25 (OH) ₂ D ₃ ], FGF-15, FGF-19, and / or Croto β. Such co-administration can have synergistic or additive effects on numerous pathologies and / or processes in the body. In some embodiments, S-Clotho can be administered in combination with FGF-21.
In some embodiments, S-croto can be co-administered with a carbonic anhydrase inhibitor such as acetazolamide, metazolamide, dichlorphenamide, dorzolamide, brinzolamide, and / or topiramate. Such combinatorial administration may be useful in the treatment of ankylosing spondylitis (AS), rheumatoid arthritis (RA), and various other pathologies. Various studies have shown that increased bone resorption is a hallmark of AS and RA, and that carbonic anhydrase inhibitors play an anti-arthritis role by inhibiting bone resorption. At the bone level, S-crotoe stimulates bone resorption and phosphate release through different mechanisms by action on TRPV5, a recently identified osteoclast function regulator. The rise of 1,25 _(OH) 2 _{D 3} levels caused by S- Klotho administration also stimulates osteoclast differentiation and bone resorption, thereby releasing the phosphate. Therefore, co-administration of S-Crotho with a carbonic anhydrase inhibitor can have additive or synergistic effects, especially in AS and RA, specifically in promoting bone health.

For the treatment of severe active rheumatoid arthritis, S-croto can be administered in combination with one or more disease-modifying antirheumatic drugs (DMARD).
S-Clotho can be administered in combination with cyclosporine because cyclosporine reduces croto mRNA and protein and increases oxidative stress leading to cyclosporin-induced renal injury (CsA). Decreased associated Klotho mRNA and protein and increased oxidative stress can be countered by exogenous co-administration of S-Crotho.

In some embodiments, S-croto can be co-administered with losartan and / or cyclosporine. Treatment with losartan, an angiotensin type II1 (AT1) receptor blocker, reversed the reduction in croto expression seen with cyclosporine. Losartan also resulted in simultaneous improvement of renal tissue (using losartan, which reduces tubulointerstitial fibrosis caused by cyclosporine).

In some embodiments, S-croto can be co-administered with one or more aminoglycosides such as amikacin, gentamicin, tobramycin. The use of aminoglycosides in the treatment of infections can be significantly expanded (gram-negative) When aminoglycosides are used to treat pathogen infections, such treatments are nephrotoxic and / or acute kidney injury (AKI). ) Can be useful in preventing. Administration of S-croto with verapamil and / or diltiazem, which has been used to block AKI, may be therapeutic in the treatment and / or prevention of renal dysfunction from AKI.

In some embodiments, S-Clotho can be co-administered with testosterone or an androgen receptor (AR) upregulatory compound. Recent reports have shown that no beneficial effects have been observed with testosterone treatment in men for personality, psychological well-being, or mood. In addition, prescribing testosterone supplementation for low T as cardiovascular health, sexual function, physical function, mood, or cognitive function was considered unsupported by randomized clinical trials. However, although testosterone supplementation was consistently found to increase muscle strength, no beneficial effect on physical function was found. Administration of S-Crotho in combination with testosterone and / or androgen receptor (AR) upregulatory compounds exceeds any effect that testosterone or S-Clotoe alone can have on these treatment groups in the elderly. It can significantly increase muscle strength and / or physical function in frail or low T men.

In some embodiments, S-croto can be co-administered with estrogen or estrogen hormones (eg, estradiol, estriol, estrone, etc.). Such co-administration can improve women's health indicators (eg, women who are menstruating, menopause, or transitioning to menopause) and / or infertility, polycystic ovary disease or disorder, obesity, hormonal deficiency. It can treat equilibrium and related conditions, and / or other women's health conditions.

In some embodiments, S-croto can be co-administered with one or more nutropics, also called smart drugs or cognitive enhancers. Nutropic drugs, supplements, and / or other substances have cognitive function, especially ability to perform, memory, creativity, motivation, work salience (motivation to perform work), (especially with high effort). It can improve performance (of the tedious work required) and can be useful in the treatment of cognitive or motor dysfunction that causes 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 aspects of cognition are agonists, especially dopamine receptors D1 in the prefrontal cortex, adrenergic receptors A2, or both. A class of stimulants that exert a cognitive-enhancing effect on humans by acting as a direct or indirect agonist of the receptor. Stimulants include amphetamines (eg, amphetamines, dextroamphetamines, etc.) that can be beneficial, for example, to a range of cognitive functions (eg, in terms of inhibitory control, episode memory, work memory, and attention), especially in individuals with ADHD. Lisdexamfetamine, etc.), dimethylamylamine (DMAA), such as 1,3-dimethylamylamine, which can improve physical fitness, alertness, reaction time, etc., methylphenidate-range of cognitive function (eg, work memory, etc.) Substitute amphetamines, which can improve episode memory and suppression control, attention, and planning rate, increase alertness, especially in sleep-deficient individuals, facilitate logical thinking and problem solving, Eugeroics (eg, armodafinil, modafinil, etc.), which can act as arousal agents that can treat narcolepsy, shift-based sleep disorders, and daytime sleepiness that remains after treatment for sleep apnea, etc. Includes xanthin (eg, caffeine), nicotine, etc., which can enhance alertness, performance, and / or memory.

In some embodiments, S-Crotho can be co-administered with one or more osteoporosis and / or osteopenia drugs, as is known in the art. Klotho can play a role in regulating bone mineral content, and the absence of Klotho can lead to reduced bone mineral content in animals. For example, Croto knockout mice show a decrease in bone mineral content over time. Klotho expression can relieve bone defects such as reduced bone mineral content that Klotho knockout mice exhibit over time. Epidemiological studies have shown an association between various Croto gene manifolds and changes in bone mineral content and the prevalence of osteoarthritis of the hand.

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

S-Clotho can also be administered in combination with chemotherapeutic agents (s) to treat chemotherapeutic-induced weakness in cancer patients. S-Crotho can also be administered to treat cancer-induced weakness in cancer patients after other known treatments.

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

Brain-derived neurotrophic factor (BDNF) can be used in combination with one or more treatments or prevention of Alzheimer's disease, because the appropriate amount of BDNF can help develop and maintain normal neuronal circuits in the brain. ) Can be administered in combination.

To increase the ability of Klotho to enter the central nervous system (CNS) to treat or prevent conditions related to CNS, S-Klotho is one or more that increase the ability of Klotho to cross the blood-brain barrier. It can be administered in combination with the molecule. For example, neither S-Klotho nor BDNF is known to cross the blood-brain barrier. In the embodiments of the present disclosure, S-croto and / or S-croto with BDNF is administered to the CNS to treat Alzheimer's disease and / or to improve the cognitive ability of individuals not suffering from Alzheimer's disease. Includes utilizing blood-brain barrier delivery techniques for

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

S-Clotho can be administered in combination with one or more antidiabetic agents such as insulin, phlorizin, or the antioxidant Tyrone, and these combination treatments benefit in preventing renal damage due to oxidative stress caused by diabetic disorders. Can have. Co-administration of S-crotoe with other antidiabetic agents for type 1 diabetes inhibits β-cell apoptosis through activation of the integrin β1-FAK / Akt pathway, leading to inhibition of caspase 3 cleavage. -Can protect cells.

S-Crotho 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-Clotho can be administered in combination with one or more blood pressure medications, calcium regulators, or treatment or prevention of chronic kidney disease (CKD). For example, soft tissue calcification is a prominent property of CKD, and Croto improves vascular calcification by improving phosphate urine, maintaining glomerular filtration rate, and directly inhibiting phosphate uptake by vascular smooth muscle. obtain.

S-Clotho is TM5441 or others, as PAI-1 inhibition or deficiency is thought to delay the progression of aging and prolong the lifespan of Croto-deficient (kl / kl) mice while preserving organ structure and function. Can be administered in combination with a PAI-1 inhibitor (plasminogen activator 1).

S-Cloteau can be administered in combination with a SIRT1 activating compound (STAC) such as sirtuin 1 (SIRT1) or 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 are reduced during lung inflammation caused by oxidative stress. SIRT1-mediated protective mechanisms against inflammation are associated with inflammation, premature aging, telomere shortening, aging-related secretory phenotypes, and regulation of DNA damage responses. 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 can be enhanced by co-administration of SIRT1 and / or SIRTI-activating compounds administered with S-Crotho.

S-Crotho can be administered in combination with one or more FDA-approved human cells, tissues, and cell and tissue products (HCT / P). Such products include, for example, bone (including decalcified bone, ligaments, tendons, myocardium, cartilage), eye tissue (corneal and strong membranes), skin, vascular implants (venes and arteries), and optionally ( Or other) Conserved umbilical vein, pericardium, sheep membrane (when used alone for eye repair (without cells added)), hard membrane, heart valve allogeneic implant, peripheral blood or cord blood It can include one or more of blood, semen, umbilical vein cells, or embryo-derived hematopoietic stem cells. In at least one embodiment, the HCT / P can be or include one or more stem cells. Stem cell therapy for damaged body tissues and organs is becoming more common. Administration of therapeutic recombinant Klotho protein in combination with stem cells provided surprising, unexpected, and even synergistic results for subjects in need of it.

The embodiments of the present disclosure further comprise a combination product comprising a Therapeutic Recombinant Crotoprotein in combination with human stem cells. The composition may also include the pharmaceutically acceptable carriers described herein. Such compositions may be included as a regenerative agent for treating, modifying, reversing, or curing a serious or life-threatening disease or condition approved by the FDA, or may be classified as a regenerative agent. .. Preliminary clinical evidence indicates that the composition (drug) has the potential to address a medical need that is incompatible with such a disease or condition.

Illustratively, stem cells can be or contain mesenchymal stem cells (MSCs), such as from the human umbilical cord or placenta. In at least one embodiment, the compositions comprising the huMSC and therapeutic recombinant croto proteins of the present disclosure diminish the inflammatory and oxidative stress responses that occur in AKI and / or express aging-related proteins and microRNAs. Was reduced.

S-Crotho can be administered in combination with one or more aging inhibitors. For example, the Klotho protein can be combined with Pin1-FOXM1 and / or other aging inhibitors to enhance results in patients receiving such treatment.

S-Klotho is a Klotho stimulant (eg, vitamin D, rosaltan, testosterone), GDF-11, tricostatin A antifungal agent (GDF-11 stimulant), TIMP-2, CCL-11 inhibitor / antibody, dasatinib. , Nicotinamide riboside (eg NAD +), nicotinamide mononucleotide (NMN) (eg NDA +), AMPK stimulants (eg resveratrol, aspirin, salicylate, phytochemicals, DR), C60 fullerene, rapamycin, FGF It can be administered in combination with an inhibitor, a senolytic agent / compound such as a FOXO4-p53 interfering peptide (eg, FOXO4-DRI), an inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL.

Either the aforementioned or other treatments or co-administration may have additive or synergistic effects over those of either treatment alone. For example, co-administration of Klotho protein with one or more of the aforementioned may result in greater therapeutic outcome than the sum of the individual outcomes of administering the single component at similar concentrations. In addition, synergistic effects may include therapeutic outcomes similar to those of individual outcomes of administering a single component at lower concentrations. The synergistic effect is also any more than an increase in the maximum active dosage of one or more of the constituents, a reduction in toxicity of one or more of the constituents, or a mere additive effect of the individual treatment outcome. It may include other beneficial results. In addition, the additive effects of individual treatment outcomes may include one or more synergistic effects. Such additive / synergistic effects may be predicted or unpredictable given the nature and understanding of the individual constituents.

As used herein, combination therapy or co-administration may include treatment or administration of a combination product, composition, or formulation containing a Croto protein and one or more additional active ingredients. One or more additional active ingredients may be selected from the constituents, drugs, substances, therapeutic compositions, etc. described herein, or others known in the art. For example, Klotho protein and one or more additional active ingredients are injectable (eg, intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulation. Can be co-injected into.

Alternatively, in combination therapy or co-administration, the Croto protein and one or more additional active ingredients are combined with the combination product, composition, or formulation, or without being formulated. It may include treatment or administration of the above additional active ingredients. For example, the Klotho protein and one or more additional active ingredients can be injected separately (eg, intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other. Each contains or may be in such a formulation.

It is also understood that co-administration may include co-administration of two or more components, or separate administration of two or more components, with separate administrations preferably separated by a period of time. Let's do it. In some embodiments, the period may be very short. For example, immediately after administration of the first component of the Klotho protein and one or more additional active ingredients, the second component of the Klotho protein and one or more additional active ingredients is substantially administered (eg, eg). Can be injected). Alternatively, the first and second doses may be 1 to 60 seconds, 1 to 60 minutes, 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, etc., or any value or in between. Can be separated by a period, such as a range of values for. Similarly, co-administration can include overlapping dosing time frames for two or more components.

Therapeutic Treatment of Hyperphosphatemia Familial Neoplastic Calcification (HFTC) In affected human individuals, HFTC is derived from histidine (H) at amino acid (AA) 193 of S-croto-rs121908423. Caused by a mutation to arginine (R). Without being bound by any theory, it is believed that the H193R mutation in HFTC individuals impairs the ability of S-croto to form a three-component complex with FGF23 and FGFR1c, which impairs KL-dependent FGF23 signaling. Has been done. As a result, affected subjects exhibit severe metabolic disorders with hyperphosphatemia and heavy calcification in the skin and subcutaneous tissue. In some patients, recurrent, transient, painful swelling of the long bones appears, which is associated with periosteal reactions and radiological findings of cortical osteoproliferative disease and the absence of skin infiltration.

One embodiment of the present disclosure comprises an S-croto protein having H193. The H193 protein can be produced or expressed in the context of any of the protein constructs described herein. For example, the H193 manifold, with or without tags (eg, Fc tags, TEV-Twin-Strep tags, TEV sequences, etc.), S-croto, isoforms 1 or 2, 1 to 981, 29 to 981, 34. It can be produced or expressed in contexts such as ~ 981, 36 ~ 981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549. Thus, the nucleic acid construct or cDNA in which the protein is expressed can be the corresponding configuration.

In an embodiment, the H193 heterozygous or homozygous complex construct is produced, and the resulting construct encoding the S-Croto H193 protein is expressed in a suitable expression system (eg, via transfection) (eg, CHO). It may include introduction into cells) and / or transient expression of the S-Clotho H193 protein. The H193 Croto protein can also be expressed at higher levels than the R193 (or H193R) protein. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control tests). Embodiments carry a therapeutic or therapeutically effective amount of S-Crotho H193 protein in a subject requiring it (eg, an HFTC individual, an individual diagnosed with HFTC, or H193R (rs121908423) or other manifold. May include administration to the patient). Alternatively, the subject can be of the wild type of another mutant or manifold. Administration of recombinant S-Klotho H193 protein can lead to a beneficial increase in blood S-Klotho levels. Administration can reverse or suppress the adverse effects of the R193 mutant transcribed and circulated in HFTC individuals as a result of the H- to -R193 point mutations found in the human Croto gene in individuals suffering from HFTC. Therefore, the circulating concentration of S-Crotho H193 may help counteract the effects observed in H193R or HFTC individuals.

Therapeutic Treatment in CC Genotype Patients with End-End Renal Disease (ESRD) Approximately 350,000 patients with End-Term Renal Disease (ESRD) suffer from very high mortality in the first year of habitual hemodialysis I'm out. 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 is associated with accelerated aging and premature mortality in animal models. It has been hypothesized that the genetic diversity of the Klotho gene may be associated with the survival of subjects with ESRD. Researchers found an association between the 12 single nucleotide polymorphisms (SNPs) of the Klotho gene and the mortality rate of the ESRD patient cohort (n = 1307, Caucasian and Asian) during the first year of hemodialysis. Was tested. CC genotype of one tag SNP, rs577912 (common HapMap project with minor allele frequency MAF in the Croto gene sequence located in intron 1)> 0.05 and increased risk of 1-year mortality ( A significant association was found between RR, 1.76; 95% CI, 1.19-2.59; p = 0.003). This in individuals with the CC genotype among patients not treated with the active vitamin D replacement (HR, 2.51; 95% CI, 1.18 to 5.34; p = 0.005). The effect was even more pronounced. In lymphoblast-like cell lines from HapMap subjects, CC genotype was associated with 16-21% lower Cloto expression compared to AA or AC genotype. 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 comprises an S-croto protein expressed from an AA or AC genotype. The protein can be produced or expressed in the context of any of the protein constructs described herein. For example, the proteins are S-Clotho, Isoform 1 or 2 tagged or untagged 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34- It can be produced or expressed in the context of 549, 36-549, 131-549 and the like (eg, Fc tag, TEV-Twin-Strep tag, TEV sequence, etc.). Therefore, the nucleic acid construct or cDNA in which the protein is expressed can be of corresponding length.

In an embodiment, an AA or AC heterozygous or homozygous construct is produced, the resulting construct encoding the S-croto protein is expressed in a suitable expression system (eg, via transfection) (eg, CHO cells). ) And / or transient expression of the S-croto protein. Croto proteins expressed in AA or AC heterozygous or homozygous cells can be expressed at higher levels than in CC cells. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control tests). The embodiment carries a therapeutic or therapeutically effective amount of S-Clotho protein in a subject requiring it (eg, a CC mutation in one tag SNP, rs577912, with low endogenous S-Clotho protein expression, and / Or administration to an individual or patient with end-stage renal disease (ESRD) may be included. Alternatively, the subject can be of the wild type of another mutant or manifold. Administration of recombinant S-croto protein can lead to a beneficial increase in blood S-croto levels. Administration can reverse or suppress the adverse effects of CC mutations transcribed and circulated in an individual as a result of point mutations (s) found in the human Croto gene of the affected individual. Therefore, the circulating concentration of S-croto is the effect observed in CC individuals, especially in patients with end-stage renal disease (ESRD), namely the mortality rate of ESRD patients undergoing habitual hemodialysis during the first year. Can help counter.

Therapeutic Treatment of Hand Osteoarthritis (OA) and Osteophytes by Roentgen Photography Osteoarthritis (OA) is a common complex disease with strong hereditary components. The researchers tested the association between four putative functional gene manifolds of the Croto gene and osteoarthritis of the hand OA in a large population of Caucasian women. The researchers found a significant association between SNP G-395A and the presence / absence of hand OA and osteophyte formation on roentgen photography. Alleles G are 1.44 (P = 0.008, 95% confidence interval (CI) 1.09 to 1.91) and 1.36 (P = 0.006, 95% CI 1.09 to 1.91), respectively. The odds ratio (OR) of 70) significantly increased the risk of hand OA and osteophytes by roentgen photography. From logistic regression modeling, genotype GG was roentgen-photographed hand OA (OR = 3.10, 95% CI 1.10 to 8.76) and osteophytes (OR = 3.10) when compared to genotype AA. , 95% CI 1.10 to 8.75) both showed a more than triple increase in risk. After age adjustment, the OR of genotype GG was 4.39 (P = 0.006, 95% CI 1.51-12.74) for hand OA by roentgen photography and 4.47 (P = 0.) for osteophytes. 005, 95% CI 1.56 to 12.77) was further increased. Researchers also suggested that one manifold of the Klotho gene (SNP G-395A) is associated with susceptibility to OA in the hand and appears to act through osteophyte formation rather than cartilage damage.

One embodiment of the present disclosure comprises an S-croto protein expressed from a construct having SNP G395A. The resulting protein can be produced or expressed in the context of any of the protein constructs described herein. For example, the A395 manifold can be S-croto, isoforms 1 or 2, 1-981, 29-981, 34 with or without tags (eg, Fc tags, TEV-Twin-Strep tags, TEV sequences, etc.). It can be produced or expressed in contexts such as ~ 981, 36 ~ 981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549. Therefore, the nucleic acid construct or cDNA in which the protein is expressed can be of corresponding length.

The embodiment is to produce a G395A heterozygous or homozygous construct, transfer the resulting construct encoding the S-croto protein to a suitable expression system (eg, via transfection). It may include introduction and / or transient expression of the S-croto protein. Croto proteins expressed in A395 heterozygous or homozygous cells can be expressed at higher levels than in G396 cells. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control tests). In embodiments, a therapeutic or therapeutically effective amount of S-Crotho protein is applied to a subject in need of it (eg, G395 SNP, and / or roentgen-photographed hand osteoarthritis (OA) and / or osteophytes. May include administration to individuals or patients who carry (at risk of developing these). Alternatively, the subject can be of the wild type of another mutant or manifold. Administration of recombinant S-croto protein can lead to a beneficial increase in blood S-croto levels. Administration can reverse or suppress the adverse effects of G395 SNPs transcribed and circulated in affected individuals. Therefore, circulating concentrations of S-crotoe may help counteract the effects observed in G395 individuals, especially those at risk of developing hand osteoarthritis (OA) and / or osteophytes on roentgen photography. .. Therefore, administration of G-395A S-croto protein may reduce the risk of hand osteoarthritis (OA) and osteophyte formation by roentgen photography in patients (eg, patients with G395 SNPs).

Therapeutic Treatment of Metabolic Syndrome The risk and / or incidence of metabolic syndrome (MetS), a population of cardiovascular 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 partially hereditary and that genetic factors play a greater role than environmental factors in the incidence of MetS. Researchers have found an association between G-395A polymorphism and metabolic syndrome (METS) in Chinese populations in their 90s and 100s. The subject was from the Dujiangyan City (PLAD) longevity and aging project. Genotyping of G-395A (rs1207568) in the promoter region of the Klotho gene was performed using the TaqMan allele discrimination assay. MetS was diagnosed according to the standards of the International Diabetes Federation. 695 subjects aged 93.5 ± 3.2 years were included. The allele frequencies of G and A were 0.852 and 0.148, respectively. Throughout the population, 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 were found throughout the population (odds ratio [OR] 0.50, 95% confidence interval [CI] 0.25 to 0.98) and in women (OR 0.51, 95% CI 0.24). It had a significantly lower risk of MetS from ~ 0.97), but was (OR 0.42, 95% CI 0.05 to 3.85) in men. In the entire population and in women, the relationship between Klotho G-395A SNP and MetS was hypertension (OR 0.48, 95% CI 0.34 to 0.67; OR 0.47, 95% CI 0.31 to 0, respectively. 71), and its effects on hypertriglyceridemia (OR 0.66, 95% CI 0.39 to 0.95; OR 0.54, 95% CI 0.31 to 0.98, respectively). In men, this relationship may be due to its effect on hypertension (OR 0.47, 95% CI 0.25 to 0.90) and low HDL-C (OR 0.69, 95% CI 0.27 to 0.93). was there. The researchers concluded that the -395A allele carrier of the Klotho gene correlates with a lower risk of MetS, especially among women in their 90s and 100s.

One embodiment of the present disclosure comprises an S-croto protein expressed from a construct carrying 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 is S-croto, isoforms 1 or 2, 1-981, 29-981, 34, with or without tags (eg, Fc tags, TEV-Twin-Strep tags, TEV sequences, etc.). It can be produced or expressed in contexts such as ~ 981, 36 ~ 981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549. Therefore, the nucleic acid construct or cDNA in which the protein is expressed can be of corresponding length.

The embodiment is to produce a -395A heterozygous or homozygous construct, to express the resulting construct encoding the S-croto protein (eg, via transfection) in a suitable expression system (eg, CHO cells). And / or transient expression of the S-croto protein can be included. Croto proteins expressed in A395 heterozygous or homozygous cells can be expressed at higher levels than in G396 cells. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control tests). Embodiments carry a therapeutic or therapeutically effective amount of S-Crotho protein in a subject requiring it (eg, G395 SNP and / or metabolic syndrome (MetS) (at risk of developing these)). It may include administration to an individual or patient. Alternatively, the subject may be of the wild form of another variant or variant. Administration of recombinant S-croto protein is at blood S-croto levels. Administration can reverse or suppress the adverse effects of G395 SNP transcribed and circulated in affected individuals. Therefore, circulating concentrations of S-Crotho can be found in G395 individuals, especially metabolic syndrome (). It can help counteract the effects observed in individuals at risk of developing MetS). Therefore, administration of G-395A S-Klotho protein may help patients (eg, older humans with G395 SNPs and / or). It can reduce the risk of metabolic syndrome (METS) in female patients).

Therapeutic Treatment of Cancer S-Crotho is thought to inhibit basal Wnt signaling activity and thereby function as a tumor suppressor for colorectal cancer (CRC). In addition, lifespan-related Klotho gene manifolds can suppress butyrate-mediated Wnt overactivation and thus increase the risk of CRC. In this way, it has been hypothesized that the types of Klotho manifolds present and their relative expression can interact with dietary butyrate levels and modify CRC risk. In addition, mTOR signaling is also associated with human aging, and crosstalk between Wnt and mTOR signaling can affect colon tumorigenesis.

For KL-VS manifolds or other constructs, which SNP (eg, within KL-VS) affects S-crotoe to reduce basal Wnt signaling and / or overactivity of Wnt mediated by butyrate. It can serve as a vehicle for studying whether it is involved in the generation of inhibition of morphogenesis, the latter being a Wnt-related activity associated with S-Clotho tumor inhibition. Embodiments include modifying appropriate amino acids that have been shown to affect the tumor suppressive effects of KL-VS manifolds (eg, KL-VS stretch of S-Klotho). One embodiment of the present disclosure comprises a recombinant S-croto protein having one or more amino acid modifications in a KL-VS stretch of six SNPs. Proteins can be produced and / or expressed in the context of any protein construct described herein. For example, the protein may be tagged (eg, Fc tag, TEV-Twin-Strep tag, TEV sequence, etc.) with or without S-croto, isoforms 1 or 2, 1-981, 29-981, 34-981. , 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549 and the like. Therefore, the nucleic acid construct or cDNA in which the protein is expressed can be of corresponding length.

In an embodiment, a heterozygous or homozygous complex construct is produced, the resulting construct encoding the S-croto protein is expressed in a suitable expression system (eg, via transfection) (eg, CHO cells). And / or transient expression of the S-croto protein can be included. Klotho proteins can be expressed at higher levels than other Klotho proteins, including wild-type. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control tests). In the embodiment, a therapeutic or therapeutically effective amount of S-Klotho protein is administered to a subject in need of it (eg, a patient who has or is at risk of developing colorectal cancer (CRC) or another tumor). Can include that. The subject may carry or express, for example, a Klotho manifold with reduced Wnt inhibitory activity. Alternatively, the subject can be of the wild type of another mutant or manifold. Administration of recombinant S-croto protein can lead to a beneficial increase in blood S-croto levels.

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

Example 1
Table 48 illustrates the results of transient expression and purification of the enumerated Klotho manifolds within HEK and / or CHO cell lines. The results provided in Table 48 below followed the simplified protocol below.

For Fc fusion proteins, use standard methods to formulate the protein expression vector at HEK293. Transfected to sus or CHO. In summary, cells were grown for 7 days and harvested. The cell number is stated in the comment section. The supernatant pH was adjusted to 1 M Hepes pH 7.4 and sodium azide was added. Protein was 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. The protein was eluted with 50 mM citrate pH 3.5, 100 mM NaCl. The protein was immediately neutralized with 1 M Tris pH 8 and 0.5 M arginine. SDS PAGE gel samples were removed at this stage. The protein was buffer exchanged for PBS. The protein was quantified by OD280 and the calculated extinction coefficient was used to determine the amount and concentration. Reduced and non-reduced SDS-PAGE (Bio-Rad Laboratories / Glycine / SDS, 4-20%) was used to determine purity and molecular weight. Aggregation status was determined by HPLC with detection at 280 nm using Sepax Zenix-C SEC-300, 3um, 300A, 4.6 x 150 mm size exclusion column and PBS buffer operation. After filter sterilization and quick freezing in liquid nitrogen, the protein was transported as an aliquot. Judging from samples run on SDS-PAGE before and after purification, protein reduction was observed in some Fc proteins during desalting to PBS. Although these proteins were expressed from HPLC, problems arose during desalting or assaying on a silica HPLC column in PBS. Therefore, buffer selection was optimized to reduce protein loss.

For Strept-tagged proteins, use standard methods to formulate the protein expression vector to HEK293. Transfected to sus or CHO. Briefly, cells were grown for 7 days and harvested. The supernatant pH was adjusted to 1 M Hepes pH 7.4 and sodium azide was added. BioLock biotin isolation 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. The protein was eluted with 100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, and 2.5 mM desthiobin. The protein was quantified by OD280 and the calculated extinction coefficient was used to determine the amount and concentration. Reduced and non-reduced SDS-PAGE (Bio-Rad Laboratories / Glycine / SDS, 4-20%) was used to determine purity and molecular weight. Aggregation status was determined by HPLC with detection at 280 nm using Sepax Zenix-C SEC-300, 3um, 300A, 4.6 x 150 mm size exclusion column and PBS buffer operation. After filter sterilization and quick freezing in liquid nitrogen, the protein was transported as an aliquot. It should be noted that for Strept-tagged proteins, samples were assayed in elution buffer. This elution buffer is very "neutral" and is not harmful to proteins. Also, since the operating buffer of the SEC column was PBS, the protein underwent buffer exchange during the assay. Absorbance over a time greater than 7 minutes is a small molecule.

In certain embodiments, EDTA and / or other metal chelating agents (s) have been removed (or not included) from one or more (eg, all) of the purification step and / or buffer. .. In some embodiments, EDTA and / or other metal chelating agents (s) can contribute to or cause the inactivation of the Klotho protein.

In the follow-up test, the transiently expressed Fc-Crotho protein was expressed in the AQuity UPLC BEH200 SEC analysis column: 1.7 μm, 4.6 × 150 mm; mobile phase: 100 mM sodium phosphate, 200 mM sodium chloride, pH 6. 8; Flow rate: 0.35 mL / min; Operating time: 9 min; Detection: A280, MALS & RI detector; Peripheral sample compartment, 30C column compartment; Purification using injection of 100 μL sample (37 μg). FIG. 14 illustrates a chromatograph of Klotho-Fc protein. Most of the Fc croto protein appears to overlap the first peak of the MW marker near the void volume. This corresponds to the very high molecular weight found in the main peak (at Mega Dalton) and is much higher than the natural croto. By SDS-PAGE (non-reduction), the peak (fraction) contains a protein of monomeric size, which suggests non-covalent binding in solution. A small shoulder was observed at the tail of the main crotch peak (marked with a dashed oval), which was greater than IgG by elution time on the SEC column compared to the MW marker, and about 700 kDa by light scattering. Less than 1% of the total Croto peak area with (the LS signal is very low and the aggregates are trailing and can be overestimated). This peak corresponds to a multimer, perhaps a dimer or tetramer.

Various alternative purification schemes, including different columns, buffers, conditions, etc., were used to address the problem of protein aggregation and multimer formation. However, in at least one embodiment, multimeric (dimer, tetramer, etc.) croto proteins can be purified.

Example 2
SEQ ID NO: 52 (N'human alpha Klotho 34-981 isoform 1_ _(G 4 _{S) 2} linker _ human IgG1Fc-C '), SEQ ID NO: 54 (N'human alpha Klotho 34-549 isoform 2_ _{(G 4 S) 2} linker _ human IgG1 Fc-C '), and SEQ ID NO: 66 (N'human alpha Klotho 34-981 represented by (human) Klotho stable protein derived isoform 1_Twin-Strep cleavage site residue) 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 then cleaved during a stable expression / purification process to give the N-terminal (Klotho protein) amino acid. The C-terminus of the Croto protein sequences of SEQ ID NOs: 52 and 54 is the GS linker (GGGGSGGGGS) and Fc fusion tag (human IgG1 Fc domain), which are retained (or appear to be retained) in the expressed protein. Is). The C-terminus of the Croto protein sequence of SEQ ID NO: 52 is a TEV-Twin-Strep tag (having a GS linker) (GGENLYFQ / SSAWSHPQFEK-GGGSGGGSGGS-SAWSHPQFEK), which is a TEV-Twin-Strep tag (ie, GGEN). It is cleaved after glutamine 8 (Q8) to release the Twin-Strep tag portion (SSAWSHPQFEK-GGGSGGGGSGGS-SAWSHPQFEK) and retain at least a portion of the TEV (protease recognition or match) sequence (GGENLYFQ-C').

The coding sequence was codon-optimized using a DNA 2.0 proprietary algorithm. Genes were synthesized to construct a Pd3600 transposon skeleton-based expression construct.
A GS knockout CHOK1 derivative host cell line was used to express the human Croto variety. Cells were co-transfected with a Leap-In transposon-based expression plasmid (ATUM) encoding three different h-crotow designs and Leap-In transfections (ATUM). Transfection was performed by electroporation using a Neon device (Thermo). Transfected cells were selected under glutamine-free conditions and the resulting stable pool was cryopreserved. Cells from an established stable pool were inoculated into shake flasks and fed-batch production tests were performed using a small established supply of ATUM and cell culture conditions. Clarified collection samples were taken and subjected to Western blot analysis using anti-Fc antibody- (SEQ ID NOs: 52 and 54) or Strept-tactin- (SEQ ID NO: 66) HRP conjugate-based detection. An exemplary Western blot is shown in Figures 15A-16B.

A stable pool of 291645 expressing the FL-ECD (34-981) -Fc croto derivative (SEQ ID NO: 52) produces predominantly expected sized proteins (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 the predicted approximately 140 kDa size (arrow), indicating that expression construct 291645 expresses the correct size fusion protein. Proteins dimerize under non-natural and non-reducing conditions (see Figure 15B). Under non-reducing conditions, the full-length extracellular domain-Fc fusion protein can form a homodimer. The size of the predominant band detected by the anti-Fc antibody on non-reducing Western blots (arrows) is consistent with the predicted homodimer formation.

The FL-ECD (34-981) -TEV-Twin-Strep molecule expressed by the stable pool 291647 (SEQ ID NO: 66) also showed the correct size on Western blots and was monomeric under non-natural and non-reducing conditions. It remains (see FIGS. 16A-16B). The expected size isoform-2-Fc manifold represents a secreted human croto 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 denatured Western blot analysis shows that the full-length extracellular domain of h croto does not form a covalent homodimer 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 shown recombinant Croto protein constructs. FIG. 18 is a series of gels of each of the indicated recombinant Croto protein constructs.

In some embodiments, in addition to one or more other Klotho manifolds disclosed herein but not shown in Table 48, they are disclosed in Table 48 and / or from FIG. 15A. It should be understood that the expression and / or purification of the Klotho manifold shown in 18 provides an advantage over the expression and / or purification of the native Klotho. For example, expressing and / or purifying a Klotho manifold can reduce the number and / or type of alternative products. In addition, or alternative, the expression level of the desired croto manifold can be increased compared to the expression level of the native croto. In addition, or alternatively, the desired Crotoe manifold is expressed and / or purified in a purer form under equivalent conditions and methods (eg, the concentration of the desired Crotoy Manifold is expressed and / / Or increase with the accompanying reduction of purified by-products). Preliminary results showed that the designed recombinant fusion protein was expressed and purified within a suitable titer range and was 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 FIGS. 19-22. The data of the top four clones are illustrated in FIGS. 23-26. Based on the fed-batch order, the top two clones appear to be 138D9 and 145G8. Based on the cumulative productivity of quasi-perfusion, 138D9 seems to have the best performance. 140F6 has reached the highest VCD. Copy number stability was also evaluated, as illustrated in FIG. Total RNA was extracted and RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Catalog No. 74104). After RNA isolation, template amplification was performed using the Omniscript RT kit (Qiagen, Catalog No. 205111). All cDNA sequences were as expected.

The stability of 5 of the clones was further tested, as illustrated in FIGS. 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 population doubling greater than 60 in either group. The viable cell density and the viability of the clones over a period of 7 days are illustrated in FIGS. 30-31. Figure 32 illustrates the volume productivity of clones in a 7-day fed-batch culture at time 0,60 PD in the presence (+) and in the absence (-) of 4 mM glutamine. The volume productivity of T0 and PD60 without glutamine cells is comparable. The volume productivity of PD60 + glutamine was comparable or somewhat lower, but never lower than about 70% of the T0 value.

In EX-CELL, ActiPro, and CD OptiPro media, cells were 100% adapted and almost 100% adapted to CD CHO, CD FortiCHO, BalanCD CHO Growth A, and Select CD1000 media.

Example 3
Purified Klotho protein was observed on the gel after ELISA, as shown in FIG. Table 49 shows the ELISA plate map keys (top two segments) and heatmap labeled ELISA 450 nm data (bottom two segments). Table 50 shows Elisa data for Klotho protein from cell culture supernatants and human serum samples. Table 51 shows the average concentration of Klotho in each sample group.

Table 52 shows the Elisa plate map keys for human serum croto level before (top two segments) and after (bottom two segments) recombinant croto protein injection. Table 53 shows 450 nm data of heatmap labeled ELISA of croto protein levels in human serum samples before (top two segments) and after (bottom two segments) recombinant croto protein injection as measured by ELISA. Shown. Human serum samples were diluted 2-fold and cell culture supernatants were diluted 5,000-fold using Croto 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 concentration of Croto protein in the samples shown.

Table 55 shows the concentration and monoclonality of the replicated Croto protein sample.

An exemplary method for estimating concentrations involved quantification by PAGE against BSA standards. An exemplary method for estimating the concentration involved measurements against the purified Croto standard.

One interpretation of the data is that the native-like recombinant (TwinStrep-TEV-tagged) croto protein expresses better, is cleanly purified, and / or has more activity than the recombinant Fc-tagged croto protein. Can be to hold. This was surprising and unexpected. An alternative interpretation is that none of the proteins, or Fc fusion proteins, than native-like proteins, showed a higher level of Klotho protein response to ELISA antibodies than native-like proteins. Therefore, we found that the expression, purification, storage, and / or administration of native-like recombinant croto proteins and Fc fusion croto proteins was not as simple as expected, and that standard techniques did not work well. May have been identified. Proceeding on the basis of later interpretations, the procedure was modified to obtain more active proteins while continuing the process for further interpretation of the data.

According to the specific embodiments of the present disclosure, three commercially available Elisa detection kits for Klotho protein have also been tested for suitability. See Tables 57 and 58 below. The IBL kit shows low background, linear dilution of serum samples, and acceptable spike recovery after 1: 4 dilution. The Clone-clone kit showed high background, no linear dilution of serum samples, and no acceptable spike recovery. The EIAab kit showed low background, very low Crotoe concentrations in serum samples, no linear dilution of serum samples, and no acceptable spike recovery.

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

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

Example 4
A comprehensive glycan preliminary analysis was also performed, as illustrated in FIGS. 35A-35B. The experimental conditions and results of the glycan analysis are shown in Table 59.

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

An additional pool of 9 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 illustrate the growth, viability, and titer curves of each cell line, respectively.
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 illustrate the growth, viability, and titer curves of each cell line, respectively.
One of the promising clones (see Table 66 below) was reassessed 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 FIGS. 39A-39B. FIG. 39A illustrates the Klotho ECD (34-981) 6xHis protein and FIG. 39B illustrates the Klotho protein shown. Unexpectedly, the Klotho protein appears to aggregate into higher molecular weight multimers.

A 5 L bioreactor expression operation was performed, followed by an adapter purification protocol. In the first purification step, a protein ASEpharose 4 fast flow (0.75 ml) (0.5 cm × 3 cm) column was equilibrated with 10 mM Tris-HCl, pH 8.0. 100 ml of cell supernatant adjusted to pH 8.0 with a protein sample (1.5 M Tris-HCl, pH 8.0 (about 1 ml)) was filled with a flow rate of 0.75 ml / min and 10 mM Tris / arginine-. Wash with HCl, pH 8.0 buffer _{and elute with 4M MgCl 2} , pH 3 (ie, low pH, high salt) at 0.50 to 0.25 ml / min to produce a concentrated sample. See FIG. 40A. The column was rebalanced 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 was eluted during reequilibrium and hygiene.

Peak numbers 1 and 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, transfer. Fractionation was performed using a Superdex 200 (1 cm × 30 cm) size exclusion chromatography column having a phase (flow rate of 0.5 mL / min). See FIG. 40B. Tris was used in place of the phosphate-containing buffer to buffer the mobile phase and prevent magnesium salt precipitation. A reducing agent (L-methionine) was used to prevent the oxidation of Croto-Fc. Sodium thioglycolate was used to prevent scrambling of the free sulfhydrylcysteine group of Croto-Fc. The buffer component is GRAS listed by the FDA.

The Superdex 200 fractions from a single isolated peak number 1 and peak number 2 each represent a different MW species of Croto-Fc protein. See FIGS. 41A and 41B. In summary, analysis of harvested proteins (in medium) by SDS-PAGE suggests that Croto-Fc is present in various aggregated states. Purification produced two populations of multimeric Croto-Fc corresponding to a dimer (300 kDa or less) and a tetramer (600 kDa or less). The binding / elution protocol from Protein A shows a much better product elution profile and produces minimal protein after hygiene. Since the formulation has thioglycolic acid as a reducing agent, the product separates as a monomer after heating and as a monomer and a dimer without heating on a gel electrophoresis. These new purification procedures resulted in non-aggregating Fc-croto 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 for FGF23 using ForteBio's BLITZ. First, the surface plasmon resonance (SPR) binding method was used to assess the binding affinity of recombinant mouse croto for mouse FGF-23. SPR is an optical technique used to detect a molecular interaction between an analyte, which is a mobile molecule, and a ligand, which is an immobilized molecule bound to a sensor, and the refractive index of the sensor changes during the interaction. In this test, we compared the affinity of 6 concentrations of recombinant mouse croto (2-fold serial dilution from 400 μg / mL to 12.5 μg / mL) antibody against mouse FGF-23. An anti-HIS (HIS2) sensor is used to bind mouse FGF-23 to the sensor, and then these sensors are subsequently used to generate binding curves for six different concentrations of mouse croto. _{The dissociation constant (K d} ) value of an antibody is determined from the six associations and dissociation curves generated for the antibody. Assays were performed and the data were validated (data not shown).

For human recombinant croto protein, HIS tag biosensor chips were hydrated in PBS for 10 minutes at room temperature. 300 μg / ml HIS-tagged FGF23 (ProSpec, Catalog No. CYT-374) in PBS was bound to the biosensor for 2 minutes. The baseline was established on a BLITZ device for 30 seconds. The binding of five 2-fold serial dilutions (from 400 μg / ml to 12.5 μg / ml) of native and Fc-s croto proteins to immobilized FGF23 was then tested in PBD for 2 minutes, followed by in PBS. It was dissociated for 2 minutes. K _{on (association), K off} (dissociation), and _K D a (Koff / Kon), and analyzed using an exhaustive analysis. See FIG. 42.

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

Growth phase cells were seeded in culture medium (10% FBS-DMEM) at a density of 5000 cells / well / 96 well plates, cultured overnight, and then the medium was replaced with 0.5% FBS. , Incubated overnight. Cells were treated with 10 doses of each Croto protein (2-fold serial dilutions, 3 doses each) starting at 30 μg / ml and incubated for 48 hours. Cell proliferation was measured using the Cell Titter-Glu assay kit and data were analyzed in GraphPad Prism using a four parameter fit curve. The data show that both native His and Fc-Klotho purified Klotho proteins stimulated cell proliferation in a dose-dependent manner. See FIGS. 43A-43E.

Example 7
A test was conducted to determine the acute tolerability (ATD) of alpha-human native croto and alpha-human Fc-fused croto proteins after intravenous administration to male Prague dolly rats. In the acute tolerated dose study, the safety of the test substances alpha-human natural croto protein and alpha-human Fc fusion croto protein was evaluated in male Prague dolly rats. Twenty-one animals were subcutaneously transplanted with transponders prior to dose administration. On day 1 of the test, animals were randomly divided into 7 groups of 3 animals based on body weight. Alphahuman natural croto protein was tested at three dose concentrations of 10 μg / kg, 30 μg / kg, and 100 μg / kg (based on individual body weight) and in vehicle (PBS, pH 7.2) on day 0 of the test. It was administered intravenously at a dose of 1 ml / kg (based on individual body weight).

Alphahuman Fc fusion crotho protein was also tested at three dose concentrations of 3 μg / kg, 10 μg / kg, and 30 μg / kg (based on individual body weight) and vehicle (PBS, pH 7.2) on day 0 of the test. ) At a dose of 1 ml / kg (based on individual body weight). On day 0 of the study, dosing was started in the vehicle-only (PBS, pH 7.2) group, 10 μg / kg alpha human native crotho protein group, and 3 μg / kg alpha human Fc fusion clot protein group, and the dose of each test substance was started. The levels were staggered for at least 24 hours. All medications were given once on each start date. Animals were observed for any adverse effects for up to 14 days after treatment.

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

Table 67 shows a summary of the test animals.

Table 68 shows a summary of the test design.

Group Allocation: For MatchedPairDistribution, an optimized distribution method was performed using an algorithm that matched individual measurements with the mean of all selected animals. The method first takes matching pairs that are close to the average of all selected animals and then distributes them to groups that match (or are as close to) the average of all selected animals. Animals are distributed so that the average of the measurements in each group is as close as possible to the average of all selected animals.

For statistical analysis, descriptive statistics were generated from test data. The data were evaluated to determine whether parametric or nonparametric analysis was appropriate. For parametric data, analysis of variance (ANOVA) was followed by a post-hoc study to determine treatment, time point, and / or significant differences between groups. Appropriate statistical analysis was performed on nonparametric data (eg, Kaplan-Meyer survival, Klaskar-Wallis one-way ANOVA, Mann-Whitney, or Wilcoxon rank sum).

Table 69 summarizes the sampling procedure.

Table 70 summarizes the sampling tasks.

All animals that weighed less than 85% of their initial body weight or showed severe, long-term adverse clinical signs were euthanized. As mentioned above, animals were euthanized by cardiac hemorrhage for plasma, serum, and whole blood collection.

Preliminary results showed that rats were resistant to relatively high doses of recombinant Klotho protein administered to it.
Example 8
A study was conducted to determine the pharmacokinetics of alpha-human native croto and alpha-human Fc-fused croto proteins after IV administration to male Prague dolly rats. Before the 0th day of the test, the transponder was subcutaneously transplanted into the animals. Weight was gained on day 1 of the study and animals were randomized based on body weight. On day 0 of the study, animals in groups 5, 6 and 7 (10 animals per group) were at 3, 10, and 30 μg / kg in 1 ml / kg vehicle (PBS, pH 7.2), respectively. , Received a single dose of alpha-human Fc-fusion crotho protein intravenously via the tail vein (see detailed dosing schedule below). On day 1 of the study, groups 2, 3 and 4 animals (10 animals per group) were at 10, 30, and 100 μg / kg in 1 ml / kg vehicle (PBS, pH 7.2), respectively. , Received a single dose of alpha human native Cloto protein intravenously via the tail vein (see detailed dosing schedule below). Animals in vehicle group 1 (3 animals) were also dosed with protein-free vehicle (PBS, pH 7.2) at 1 ml / kg on day 1 of the test. The volume of dosing solution administered to each animal was calculated and adjusted based on individual body weight measurements taken on day 1 of the test.

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

Blood samples were taken before, 3 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 7 days after administration, as shown in Table 72 below. (Groups 5, 6, and 7). Additional blood draws 14 days after dosing were taken in groups 5, 6 and 7.

As shown in Table 73 below, animals in vehicle group 1 were terminally bleeding 1 hour after dosing.

The resulting serum samples were analyzed with an Alpha Klotho human soluble ELISA kit.
Table 74 shows a summary of the test animals.

Table 75 shows a summary of the test design.

Group Allocation: For stratified sampling, a non-optimized randomization method using animal "binning" with similarly sized measurements was used. In this way, pools of animals are stratified by classifying them in ascending order according to the measurements of interest. The animals are then classified into "bins" or hierarchies, depending on the size of the measurements. The number of hierarchies is determined by the number of animals assigned to each group. For example, if there are 3 animals per group, 3 small, medium and large bottles will be created. One animal from each size bin is randomly assigned to each group. Stratified sampling will ensure that each group receives one animal at random from a bottle of each size, and the average for each group will remain similar to the other groups. This method balances the difference between the group mean and the standard deviation and controls the bias of pool orders.

Descriptive statistics were generated from test data for statistical analysis. The data were evaluated to determine whether parametric or nonparametric analysis was appropriate. For parametric data, analysis of variance (ANOVA) was followed by a post-hoc study to determine treatment, time point, and / or significant differences between groups. Appropriate statistical analysis was performed on nonparametric data (eg, Kaplan-Meyer survival, Klaskar-Wallis one-way ANOVA, Mann-Whitney, or Wilcoxon rank sum).

Table 76 summarizes the sampling procedure.

Table 77 summarizes the sampling tasks.

All animals that weighed less than 85% of their initial body weight or showed severe, long-term adverse clinical signs were euthanized (data not shown). As mentioned above, animals were euthanized by cardiac hemorrhage for plasma, serum, and whole blood collection.

Example 9
Dose-dependent of alpha-human Fc-croto and alpha-human native croto in I / R-induced AKI and renal biomarkers (by measuring renal I / R-induced increase in plasma renal injury biomarkers) in Wistar rats A test was conducted to determine the effect. Specifically, the purpose of this study was to evaluate the dose-dependent effects of alpha-human Fc-croto and alpha-human native croto on renal I / R-induced increase in plasma renal injury biomarkers in rats. This study included bilateral renal ischemia-reperfusion (I / R) injury (30-minute ischemia) surgery (n = 63) vs. sham surgery (n = 4), compound medication (single intravenous injection / rat). ), Continuous urine collection via metabolic cage breeding (3 collections / rat, 1st, 2nd, and 7th days), clinical chemistry analysis of creatinine and protein continuous urine specimens (1st, 2nd) (Days and 7s), continuous blood draws (3 blood draws / rats, 1st, 2nd, and 7th days), clinical chemistry analysis of continuous plasma samples of creatinine and BUN (1st, 2nd) Day and 7), designed and data to provide daily weight and health observations from postoperative to endpoint, and data from or related to tissue collection and processing at the endpoint. Is shown in table and graph format.

In addition, analysis of Kim-1 continuous urine samples via species-specific ELISA (Days 1, 2, and 7) and analysis of NGAL continuous urine samples via species-specific ELISA (1). Day, 2, and 7), left and / or right kidney mRNA expression analysis (1-2 kidneys / animals: n = 67 samples, 10 analyzes, 3 normals Includes immunohistochemical genes; performed using the Affymetrix Quantine Plex 2.0 platform), MCP-1, TGF-β, α-SMA, Col1A1, Col3A1, Fn-1, CTGF), and specific staining and ED-1 + macrophages. Immunohistochemistry and quantification of the left and / or right renal cortex of macrophage infiltration via staining was also optionally performed.

Table 78 shows the test parameters.

Table 79 provides a summary of the test design.

67 male Wistar rats weighing approximately 151-175 g were fed a standard solid diet (Harlan 8640), group-reared under standard conditions and acclimatized for at least 7 days prior to enrollment in the study. Prior to the start of the study, the study rats were placed in a weight-matched treatment group. Use a balanced design so that approximately the same number of animals are enrolled per group per day of surgery and the heaviest animals are enrolled first to minimize age differences between animals. Registered for the exam. When not in metabolic cage farms, rats were group-contained in static cages under standard conditions.

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

After that, laparotomy was performed. At t0, rats were subjected to pseudo (group 1, n = 4) or bilateral renal artery ischemia (groups 2-8, n = 9) using heat-sterilized equipment, vascular occlusion clamps, and aseptic surgical techniques. / Group). Following a continuous ischemic time of 30 minutes (t + 0.5), the occlusion clamp was removed, the kidney was reperfused, and the abdominal wound was closed with sterile silk suture. Rats were administered 0.01 mg / kg buprenorphine as a post-surgical analgesic and recovered briefly in a clean heating cage. At t + 1 hours, animals in groups 1-6 were briefly detained and received a vehicle or intravenous injection of Croto compound in the vehicle as needed. Rats in groups 7 and 8 were also briefly restrained (but not injected) to control stress response. The rats were then immediately placed in a metabolic cage.

Twenty-four hours after reperfusion (D1), urine output from all animals was determined and sampled. Then, all animals were collected (500 μl) into Li-heparin by direct tail vein puncture. Immediately after bleeding, rats were given 500 μl of warm saline. Urine and blood sampling were repeated again at D2 (48 hours after reperfusion) and D7 (168 hours after reperfusion). In D2 to D5, all rats were placed in static cages and weighed under standard conditions and daily health observations were performed. Whole blood (500 μl) was appropriately treated for plasma production and treated as described.

After the start of administration, clinical observation was performed once a day. For 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 tibial length and body weight. Urine was collected on days 1, 2, and 7 (targeting n = 3 samples / animal, 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 (targeting n = 2 samples / animal, 125 μl / sample). At least one sample was subjected to clinical chemistry analysis (creatinine, BUN).

On day D7, the rats were anesthetized with isoflurane immediately after bleeding and tissue was collected. 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 medial cortex / lateral medulla (ie, left and right kidneys from each animal, 1 tube / kidney / animal) from each pseudo and ischemic kidney. , S3 / THAL enriched) was rapidly frozen in a properly labeled tube and stored at −80 ° C. for potential biochemical analysis in the future. Prior to performing the analysis, the cortical tissue is optionally cryopolized and mixed to allow for better homogeneity of the tissue sample throughout the biochemical assay, as well as the introduction of possible sampling bias in cortical biopsy. Limited. At least one central cross-sectional section of each pseudo and ischemic kidney (left and right from each animal) was optionally taken and fixed in 10% neutral buffered formalin for histological analysis. The rats were then sacrificed.

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

FIG. 44 graphically illustrates the mean (mean) body weight (mean ± standard error, SEM bar) of rats in each group over the course of a 12-day study (daily). Table 81 below shows the corresponding data of average body weight exemplified in FIG. 44, Table 82 below shows the corresponding SEM exemplified in FIG. 44, and Table 83 below shows the 12-day study ( The percentage change in average body weight of rats in each group over the course of daily) is shown.

Example 11
To test the therapeutic effect of Croto administration on acute kidney injury (AKI) after renal ischemia-reperfusion injury (IRI), Sprague dolly rats were randomly divided into sham or AKI groups and animals in each group were absent. Artificially assigned to vehicle or Croto treatment. In humans, AKI is a terrifying clinical problem that results primarily from ischemia or nephrotoxicity, with outcomes ranging from complete recovery to the development or death of dialysis-dependent chronic renal disease. Animals in the chloride group received a single bolus intraperitoneal injection of recombinant mouse chloride protein (0.01 mg / kg body weight) 30 or 60 minutes after reperfusion, and rats in the vehicle group received the same volume of chloride buffer. Received (150 mM NaCl and 10 mM HEPES, pH 7.4). Urine and blood were collected for 24 hours on days 1, 2, and 7 after surgery. This study showed that a single bolus injection of mouse S-Crotho 30 minutes after injury was effective in attenuating (AKI) in animal models of human pathology.

Example 12
To determine whether Klotho protects the lungs against oxidative damage, Sprague Dolly rats (body weight about 300 g) are subjected to normal oxygen (N: 21% inspiratory O) or hyperoxygen (H: 90%). 3 days of exposure to O), during which time 3 preparations, Dulveco modified Eagle's medium (DMEM), or Croto (Klotho CM, about 60 pmol) or empty vector (control CM) (n = 6-8, respectively). Animal) received an intraperitoneal injection of one of the CHO cell-derived conditioned medium (CM) overexpressing. Injections were given 12 hours prior and repeated 24 and 48 hours during exposure.

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

CONCLUSIONS: Existing Croto protein measurements can be exorbitantly expensive and / or technically difficult for home harvesting and / or quantitative diagnosis. Embodiments of the present disclosure include clotau protein levels (s) in a subject, specifically endogenous and / or exogenous soluble alpha clot protein levels (s), and more specifically soluble alpha clot protein levels. To monitor, detect, and / or quantify, to diagnose croto protein deficiency, and / or to increase clot protein level (s), expression, or production, and to treat croto protein deficiency. Provide useful products for their practice, including diagnostic kits and compositions for.

In addition, gene therapy may be effective in increasing certain protein levels in animal models or trials, but the safety of gene therapy, especially for the treatment of humans, remains questionable. Administration of exogenous and / or recombinant cloprotein in humans is safer, easier, and more effective in restoring (endocrine) croto level, as compared to viral delivery of the croto gene to animals (cells). It can be a direct style. Preclinical data support the potential of soluble Klotho protein treatment for age-related disorders and diseases associated with Klotho deficiency. Epidemiological data show that soluble croto is lower in older adults than in young adults, and that levels of soluble croto are inversely correlated with age, indicating that aging may be associated with decreased soluble croto. .. However, in general, the production and administration of exogenous and / or recombinant proteins to humans is strictly regulated by government and other agencies. Due to the time and cost associated with regulatory compliance, the cost of such treatment can be exorbitantly high for developers, manufacturers, and patients as well. However, the compositions and methods of the present disclosure do not have one or more of the drawbacks associated with other solutions and are viable and effective options for increasing endogenous human protein levels in humans and non-human animals. Can be provided. Specifically, some embodiments of the disclosure are recombinant human croto, such as human recombinant soluble alpha-croto protein (of Good Manufacturing Practices (cGMP) grade), protein fragments, and / or protein manifolds. Includes products, compositions, and / or methods of making and / or using proteins. In addition, some embodiments of the disclosure are (nutritional supplements) to (naturally) increase (s), expression, or production of (s) croto protein levels (s) in a patient or subject. Or health supplements) include products, compositions, and / or methods.

Those skilled in the art will appreciate that each of the therapeutic effects, indications, and / or treatments outlined herein with respect to recombinant therapeutic Crotoproteins and products, compositions, and methods associated thereto is endogenous Clotoprotein production and. You will understand that / or levels (s) can be achieved by (naturally) increasing, enhancing, increasing, and / or supporting. Those skilled in the art will also (naturally) increase, enhance, increase, and / or support endogenous Klotho protein production and / or levels (s) of the therapeutic effects, indications, and indications outlined herein. It will be appreciated that each of / or treatments can be achieved by administering recombinant therapeutic Klotho proteins and products, compositions, and through methods associated with them. Therefore, for brevity, the present disclosure relates to both (i) administration of recombinant therapeutic Croto protein and (ii) administration of health supplements or dietary supplement compositions of the present disclosure. , Indications, and / or treatments need not be elaborated, and such are expressly contemplated herein.

Although the detailed description described above refers to a particular exemplary embodiment, the present disclosure may be embodied in other particular embodiments without departing from its spirit or essential features. Therefore, the described embodiments should be considered in all respects as merely exemplary and not limiting. For example, various substitutions, modifications, and / or modifications of the properties of the invention described and / or exemplified herein that may be conceivable to those skilled in the art and the owners of the present disclosure, as well as described and described herein. / Or additional application of the exemplary principles may be made to the described and / or exemplary embodiments without departing from the spirit and scope of the present disclosure as defined by the appended claims. Such substitutions, modifications, and / or modifications are considered to be within the scope of this disclosure.

Therefore, the scope of the present invention is shown not by the above description but by the appended claims. The limitations listed in the claims are broadly interpreted based on the language adopted in the claims and are not limited to the particular examples described in the detailed description above. The examples are to be interpreted as non-exclusive and non-limiting. All changes that fall within the scope and meaning of the claims are included within that scope.

It will also be appreciated that the various properties of a particular embodiment are compatible with, and may be combined with, and / or incorporated with, other embodiments of the present disclosure. For example, a system, method, and / or product according to a particular embodiment of the present disclosure may include, incorporate, or incorporate the properties described in other embodiments disclosed and / or described herein. It can be included in other ways. Therefore, disclosure of a particular property in connection with a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of that property in a particular embodiment.

In addition, the properties described in the various embodiments are optional and may not be included in the other embodiments of the present disclosure, unless certain properties are stated to be required in a particular embodiment. .. In addition, any property herein is combined with any other property of the same or different embodiments disclosed herein, unless the property is described as requiring another property in combination with it. Can be done. In certain embodiments, the properties can be arbitrary, but it is understood that when such embodiments include properties, they may be required to have the particular configuration described in this disclosure. There will be.

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

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

Claims (21)

A method of stabilizing Klotho protein in mammalian blood samples.
A step of obtaining capillary blood from a mammalian subject, wherein the capillary blood contains a certain amount of soluble croto protein.
The step of storing the capillary blood in a container for at least 24 hours at room temperature or without freezing, wherein the container has a preservative or anticoagulant placed therein and said storage. The agent or anticoagulant is mixed with the capillary blood in the container and the preservative or anticoagulant stabilizes the soluble croto protein for at least 24 hours at room temperature or without freezing. , A method comprising a storage step, wherein the preservative or anticoagulant comprises one or more of _{heparin, lithium heparin, EDTA, and K 2 EDTA.} The step of storing the capillary blood mixed with the preservative or anticoagulant at room temperature or without freezing for at least 3 days, wherein the preservative or anticoagulant is at room temperature or The method of claim 1, further comprising the step of storing, stabilizing the soluble cloto protein for at least 3 days without freezing. The step of storing the capillary blood mixed with the preservative or anticoagulant at room temperature or without freezing for at least 7 days, wherein the preservative or anticoagulant is at room temperature or The method of claim 1, further comprising the step of storing, stabilizing the soluble croto protein for at least 7 days without freezing. The method of claim 1, further comprising a step of quantifying the amount of the soluble croto protein or an assay for the presence of the soluble croto protein after the storage step. The step of quantifying the amount of the soluble croto protein is to use mass spectrometry, multianalyte profiling (xMAP), and / or a first antibody that binds to a portion of the soluble croto protein. The method of claim 4, comprising detecting. The step of quantifying the amount of the soluble croto protein is to detect the soluble croto protein using a detection antibody that binds to a portion of the first antibody, or to carry out an enzyme-linked immunosorbent assay (ELISA). 5. The method of claim 5, further comprising detecting and optionally quantifying the soluble Klotho protein. The first aspect of claim 1, wherein the step of obtaining the capillary blood comprises one or more of incising the mammalian skin with a lancet and collecting the capillary blood in the container. Method. The capillary blood is about 10 to 1000 μl, preferably about 50 to 200 μl of the capillary blood, or the step of obtaining the capillary blood is about 10 to 1000 μl, preferably about 50 to 200 μl of the said. The method of claim 1, comprising obtaining capillary blood. A method for diagnosing Klotho deficiency in mammalian subjects.
A step of obtaining a fluid sample mixture, wherein the fluid sample mixture is
Heparin, lithium heparin, EDTA, and mixed with preservatives or anticoagulants include one or more of K ₂ EDTA, a mammal serum in the form of capillary blood optionally soluble a quantity Mammalian serum, optionally in the form of capillary blood, having a certain amount of soluble Klotho protein, the method comprising the mammalian serum in heparin, lithium heparin,. Obtained, selected from the group consisting of mammalian serum, further comprising forming said mixture by mixing with EDTA, and _{a preservative or anti-heparinic agent comprising one or more of K 2 EDTA.} Steps and
The step of storing the mixture for at least 24 hours at room temperature or without freezing, wherein the preservative or anticoagulant stabilizes the soluble croto protein for at least 24 hours at room temperature or without freezing. Let, store steps,
After the storage step, a step of quantifying the amount of the soluble croto protein in the mixture, and
A method comprising the step of diagnosing the mammalian subject as croto deficiency when the quantified amount of the soluble croto protein in the mixture is less than a predetermined threshold amount. The fluid sample mixture comprises about 10 to 1000 μl, preferably about 50 to 200 μl of capillary blood from the mammalian subject, or the step of obtaining the fluid sample mixture is about 10 from the mammalian subject. 9. The method of claim 9, comprising obtaining ~ 1000 μl, preferably about 50-200 μl of capillary blood. The step of quantifying the amount of the soluble croto protein in the mixture is mass analysis, multi-analyte profiling (xMAP), and / or a first antibody that binds to a portion of the soluble croto protein and optionally the first. The soluble croto protein is detected and optionally quantified by detecting the soluble croto protein using a detection antibody that binds to a portion of the antibody of 1 or by performing an enzyme-bound immunoadsorption assay (ELISA). 9. The method of claim 9, comprising: The step of diagnosing a croto deficiency in a mammalian subject is to display the croto deficiency determination or diagnosis on the user interface of a computer system and / or display the croto deficiency determination or diagnosis, physically or electronically. 9. A claim comprising generating a file or report in the form, wherein the determination or diagnosis of the crotho deficiency optionally comprises the quantified amount and / or the predetermined threshold amount of the soluble crotho protein. the method of. A method of treating Klotho deficiency in mammalian subjects
A step of obtaining serum or capillary blood from a mammalian subject, wherein the serum or capillary blood has a certain amount of soluble croto protein.
The step of storing the serum or capillary blood in a container for at least 24 hours at room temperature or without freezing, the serum or capillary so that the container is placed therein and forms a mixture. Having a preservative or anticoagulant mixed with blood, the preservative or anticoagulant stabilizes the soluble croto protein for at least 24 hours at room temperature or without freezing, said preservative. Or a storage step, wherein the anticoagulant comprises one or more of heparin, lithium heparin, EDTA, and K _{2 EDTA.}
After the storage step, a step of quantifying the amount of the soluble croto protein in the mixture, and
A step of diagnosing the mammalian subject as Croto deficiency when the quantified amount of the soluble Klotho protein in the mixture is less than a predetermined threshold amount.
A step of administering a composition to the mammalian subject after diagnosing the mammalian subject as Croto-deficient.
Includes a pharmaceutically acceptable carrier or excipient and a pharmaceutically effective amount of recombinant Croto protein, recombinant Croto protein fragment, or recombinant Croto fusion protein placed 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 Clotoprotein levels by increasing or enhancing the endogenous production of soluble Crotoprotein by said mammalian subject, as well as blood pressure. Pharmaceuticals, angiotensin II receptor blockers (ARBs), angiotensin converting enzyme (ACE) inhibitors, rosartans, balsartans, thermisartans, AT1 receptor antagonists or blockers, testosterone, growth hormones, growth hormone-releasing peptides, cellmorelin, prescriptions or Prescription Dosage Optional selection from the group consisting of vitamin D, 1,25-dihydroxycholecalciferol, calcitrol, paricalcitrol, indoxyl sulfate binder, Kremezin, AST-120, statin, PPAR agonist, troglycazone, and rosiglitazone. A method comprising a step of administration, comprising one or more of one or more pharmaceutical compositions that increase endogenous crotho levels. The administration step
Oral or oral related administration, optionally selected from the group consisting of ingestion, buccal administration, and sublingual administration, in optionally modified release forms.
13. The thirteenth claim, comprising one or more bolus or gradual injections of a modified release form, optionally selected from intravenous, intradermal, intraperitoneal, intramuscular, intradermal, and subcutaneous injections. Method. The step of obtaining the serum or capillary blood is
Making an incision in the skin of the mammalian subject with a lancet
To obtain about 10 to 1000 μl, preferably about 50 to 200 μl of the capillary blood,
The capillary blood is collected in a container having a preservative or anticoagulant placed therein or added thereto, wherein the capillary blood is optionally about 10 to 1000 μl, preferably about. 13. The method of claim 13, comprising collecting 50-200 μl of capillary blood and one or more of the following. The pharmaceutical composition comprises at least one portion 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. 13. The method of claim 13, comprising a pharmaceutically effective amount of recombinant croto protein, recombinant croto protein fragment, or recombinant croto fusion protein having at least 80% amino acid sequence identity. A pharmaceutically effective amount of 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 between them. 16. The method of claim 16, comprising a recombinant Klotho fusion protein. The dietary supplement composition includes vitamin C, vitamin D3, vitamin E, N-acetylcysteine (NAC), quercetin dihydrate, rosmarinic acid (RA), pterostilben, docosahexaenoic acid (DHA), nicotinamide riboside ( NR), nicotinamide adenine dinucleotide (NAD +), nicotinamide mononucleotide (NMN), and two or more constituents selected from the group consisting of one or more enteric beneficial bacteria, according to claim 13. The method described. The one or more intestinal beneficial bacteria are effective amounts of Bifidobacterium species and / or strains Bifidobacterium lactis BL-04, Bifidobacterium bifidom / lactis BB-02, and Bifidobacterium and / or strains Lactobacillus lactobacillus-Lactobacillus BL-04. The method of claim 18, comprising Lactobacillus acidofilus LA-14, Lactobacillus rhamnosus LR-32, and Lactobacillus paracasei LPC-37. A method of purifying a pharmaceutical grade recombinant Cloto protein, recombinant Croto protein fragment, or recombinant Croto fusion protein.
To obtain a suspended cell culture fluid comprising (i) a recombinant croto protein, a recombinant croto protein fragment, or a recombinant croto fusion protein, and (ii) one or more contaminants.
A step of performing a purification step using affinity chromatography, wherein the recombinant croto protein, recombinant croto protein fragment, or recombinant croto fusion protein is protein A or other. It is bound to an affinity entity, optionally washed, eluted from protein A with an elution buffer having a pH of about 2 to pH 6 and a salt concentration of about 1 M to about 6 M, and the salt is optionally MgCl ₂ Steps to carry out, including
A step of performing a purification step using size exclusion chromatography, wherein the eluted recombinant croto protein, recombinant croto protein fragment, or recombinant croto fusion protein is the mobile phase. Further eluted in the buffer, the mobile phase buffer has a buffer and one or more optional reducing agents, the buffer is optionally other than a phosphate-containing buffer, and the reducing agent is The mobile phase buffer, optionally containing L-methionine and / or sodium thioglycolate, optionally has a pH of about 6 to about 10 pH, comprising:
A method, wherein at least some of the further eluted recombinant Cloto proteins, recombinant Croto protein fragments, or recombinant Croto fusion proteins are optionally in multimeric form. At least a portion and at least one of the recombinant Croto protein, SEQ ID NO: 2-SEQ ID NO: 70, or SEQ ID NO: 107-SEQ ID NO: 120, or SEQ ID NO: 125-SEQ ID NO: 128, or SEQ ID NO: 133, or SEQ ID NO: 152. 21. The method of claim 21, which has 80% amino acid sequence identity.