Dietary supplements, compositions, and uses thereof for increasing telomerase activity and extending telomere length

Abstract

Disclosed herein are dietary supplements, and nutraceutical compositions thereof, comprising a polypeptide having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO 1 and 2; and methods of increasing telomerase activity and extending the length of telomeres using a composition comprising a polypeptide having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO 1 or 2. Also disclosed is an isolated polypeptide according to SEQ ID NO 2.Disclosed herein are dietary supplements, and nutraceutical compositions thereof, comprising a polypeptide having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO 1 and 2; and methods of increasing telomerase activity and extending the length of telomeres using a composition comprising a polypeptide having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO 1 or 2. Also disclosed is an isolated polypeptide according to SEQ ID NO 2.

Description

DIETARY SUPPLEMENTS, COMPOSITIONS, AND USES THEREOF FOR INCREASINGTELOMERASE ACTIVITY AND EXTENDING TELOMERE LENGTHRELATED APPLICATIONThis Application claims priority to and the benefit of U.S. Provisional Patent ApplicationNo. 63 / 571,675, filed on March 29, 2024, the contents of which are incorporated by reference.BACKGROUNDIt is widely known that dietary and nutritional supplements can play a role in prevention,alleviation, or delay of onset of many diseases or symptoms, can help boost the immunesystem, and may even play a pivotal role in the treatment of some diseases. Dietary andnutritional supplements may also play a role in helping to delay or prevent aging and the signsof aging.As we age, we amass a series of maladies and conditions that affect our day to daylives. Age-related diseases are illnesses and conditions that occur more frequently in olderpeople. Age is a significant risk factor in such conditions. Some of most common agingassociateddiseases are atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, and Alzheimer's disease. About 60% of the olderpopulations suffer from obesity which leads to type 2 diabetes. Many also suffer from metabolicsyndrome which increases risk factor for developing cardiovascular diseases. Out of the150,000 people that die each day across the globe, about two thirds die of age-related causes.During ageing, the functionality of cells declines, and various types of cell-intrinsic damage andalterations in the niche and the circulating blood have been demonstrated to contribute to thisage-related decline. Telomere, a region of repetitive nucleotide sequences (TTAGGG)associated with specialized proteins at the ends of linear chromosomes, length shortens withage. Progressive shortening of telomeres leads to senescence, apoptosis, or oncogenictransformation of somatic cells, affecting the health and lifespan of an individual.Shorter telomeres have been associated with increased incidence of age-related diseases,maladies, and mortality. Therefore, a telomeres length is an important factor in maintaining thelife span of cells in various tissues. The introduction of "youthful" progenitor cells into the humanbody may help rejuvenate existing cells, increase telomere length, and allow the body to agemore gracefully, and even reverse some effects of the aging process.Imagine Pharma's U.S. Patent No. 10,548,941 to Thai et al. ("Thai") discloses andclaims pharmaceutical compositions including a 40s ribosomal protein S2 (with 293 amino acids(see Sequence ID No. 1) and referred to as "IMG-1") useful for the treatment of hypertensionand hyperglycemia using a therapeutically effective amount of IMG-1. Thai identifies oralformulations that include 50-150 IJg / kg of IMG-1, which for a 70-kg human amounts to 3.5 mg to10.5 mg. Thai does not disclose use of IMG-1 for increasing telomerase activity and / orincreasing telomere length.SUMMARYTelomerase is an enzyme protein that catalyzes the addition of a telomere repeatsequence to the 3' end of a telomere and serves to restore telomere loss due to cellularsenescence. Telomerase is not expressed in most normal adult somatic cells, and telomerelength is gradually decreased in cell replication.In specific diseases, the telomere's end is abnormally rapidly lost and leads to aging ordegeneration of immature cells. In the case of a human, cells expressing human telomerase(hTERT, human-telomerase reverse transcriptase) are found to bypass a normal cellularsenescence pathway. When the expression of telomerase is induced in aged cells having ashort telomere, a telomere length is increased, and phenotypes similar to younger cells arerestored.Since the loss of the telomeric repeat sequence is reduced by an increase in telomeraseactivity, it is expected that the induction of telomerase activity having an effect of adding atelomeric repeat sequence at the end of the telomere restarts the replication and division ofsomatic cells in a senescent or death phase and induces restoration of damaged tissue in whicha large quantity of the senescent or death-phase cells are distributed. Since telomere length hasa role in many aging-related diseases, increasing telomere length could provide therapeuticbenefits to alleviate, or reverse, age-related diseases.In one aspect of the disclosure, a composition is disclosed comprising a polypeptidehaving at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acidsequence set for the in SEQ ID NO(s) 1 -2 (or fragments thereof), when administered tosomatic cells in culture (in vitro; ex vivo) and to a mammal (in vivo) causes an increase intelomerase activity in somatic cells, and a corresponding increase in telomere length.In one aspect, the peptide disclosed herein may include one or more peptides having atleast 80%, 85%, 90, 95%, 96%, 97%, 98% or 99% sequence homology to SEQ ID No(s) 1 -2.In another aspect, the peptide disclosed herein may include a peptide having adifference in 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or moreamino acids, 5 or more amino acids, 6 or more amino acids, or 7 or more amino acids from thepeptide of SEQ ID No(s) 1 - 2or fragments thereof.In yet another aspect, the disclosure provides a composition comprising a polypeptidewith an amino acid sequence having at least 80% sequence homology with the amino acidsequence according to SEQ ID No(s) 1 - 2, or a fragment thereof together with one or morenutritional additives, that when administered to a human causes an increase in telomeraseactivity and increase in length of telomeres.A dietary supplement comprising an amino acid sequence according to SEQ ID No(s) 1or 2 is provided. The dietary supplement may be formulated in a dosage form selected from thegroup consisting of a capsule, a cachet, a pill, a tablet, a powder, a granule, a pellet, a bead, aparticle, a troche, a lozenge, a gel, a liquid, a suspension, a solution, an elixir, and a syrup. Thedietary supplement formulation may further include one or more additional components,including but not limited to a carrier, an excipient, a binder, an antioxidant, a colorant, a flavoringagent, a preservative, a buffer, a diluent, a second food additive or supplement, and / orcombinations thereof. DETAILED DESCRIPTION OF THE DRAWINGSThe compositions and methods disclosed herein are further described by theaccompanying figures shown in the Appendix, attached hereto.FIG. 1 shows use of SEQ ID 1 causes an increase in telomerase activity in endothelial cells invitro. Endothelial cells were treated with IMG-1 (20ug / mL, SEQ ID 1) for 72hrs and thentelomerase products per cell equivalent was measured by TeloTAGGG™ Telomerase PCRELISAPLUS (Roche). EC376 refers to and EC 382 refers to two primary endothelial cellpopulations. Stem cells with established telomerase activity were used as a control. Telomeraseactivity was determined by telomerase products per cell equivalent.FIG. 2 shows use of SEQ ID 1 causes an increase in telomere length in endothelial cells in vitro.Using the same cells under the same conditions as described in Figure 1, Telomere length wasalso measured 72hrs post-treatment. Telomere length was determined using the AbsoluteHuman Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories) asper manufacturer's instructions.FIG. 3 shows use of SEQ ID 1 causes an increase in telomere length in vivo as seen by theanalysis of blood samples taken from eight adult human subjects administered an oral dose(200 ug I daily) of the composition for 12-weeks. Telomere activity was determined byTeloTAGGG™ Telomerase PCR ELISAPLUS (Roche).FIG. 4 shows use of SEQ ID 1 causes an increase in telomere length in vivo- as seen by theanalysis of blood samples taken from adult human subjects pre and post administration of anoral dose in a range of 200 mglml of the composition. Telomere length was quantified as Kb perdiploid genome and as base pairs.FIG. 5 shows use of SEQ ID 1 causes an increase in telomere length in vivo as seen by theanalysis of blood samples taken from eight adult human subjects administered an oral dose(200 ug I daily) of the composition for 12-weeks. The average length in base pairs was8963.875 (pre) and 10425 (post dosing of SEQ ID 1 ).FIG. 6 shows use of SEQ ID 1 causes an increase in telomere length in vivo as seen by theanalysis of blood samples taken from eight adult human subjects administered an oral dose(200 ug I daily) of the composition for 12-weeks. The average length in base pairs was 8250(pre) and 8700 (post).FIG. 7 shows test results in the observational study. Data shown is the aggregate average ofparticipants pre and post dosing of SEQ ID 1 over 12 weeks. Cholesterol, Triglycerides, LDLand glucose are measured as mgldl; Hs-CRP is measured as mgll; and HgA 1 c is measuredas percentage.FIG. 8 shows the markers of liver function in study participants taking an oral dose of SEQ ID 1,pre and post study (data shown is the aggregate average of the participants, pre and postdosing of SEQ ID 1 ). Alkaline phosphatase, AL T and AST are measured as Unitsll; andBilirubin, BUN and Creatinine are measured as mgldl.FIG. 9 shows that use of SEQ ID 1 causes an increase in telomerase activity in vivo as seen bythe analysis of blood samples taken from six adult human subjects administered an oral dose(200 ug I daily) of the composition for 12-weeks. The average increase in telomerase activity(measured as Relative Telomerase Activity) was 0.494.DETAILED DESCRIPTIONThe following terms are used in this disclosure to describe different embodiments. Theseterms are used for explanation purposes only and are not intended to limit the scope for anyaspect of the subject matter claimed herein.As used herein "GRAS" means an acronym for the phrase Generally Recognized AsSafe, under sections 201 (s) and 409 of the Federal Food, Drug, and Cosmetic Act (the Act).As used herein, "food" means a product containing a peptide, amino acid, protein,carbohydrate and I or fat and is intended for consumption and includes a source of nourishmentand nutritional value. For example, the food may include, but is not limited to, a hot or coldcereal, a baking product, a beverage, pasta, bread, snack products, dairy products, and the like.As used herein, "food product" means any ingredient, component or composition thatprovides nutritional value to a mammal, including humans.As used herein, "isolated" or "purified" refers to a polypeptide that has been removedfrom at least one naturally associated component. For example, the polypeptide may be at least1% pure, such as at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, atleast 60% pure, at least 80% pure, Pure, at least 90% pure, or at least 95% pure.As used herein a "nutraceutical" is any functional food (including beverages) thatprovides an additional benefit other than its nutritional benefit.As used herein "SEQ ID NO 1 or SEQ ID NO 2, etc." refers to a protein, polypeptide,peptide fragment, or analogue thereof, and including any modification thereto, having an aminoacid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acidsequence according to SEQ ID No(s) 1 and / or 2 (See Table 1). Also contemplated is a peptidefragment, or analogue thereof, and including any modification thereto, having an amino acidsequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acidsequence according to Table 1. For example, a fragment of SEQ. ID NO 1 may include Thai'ssequence ID numbers 2-5, where Thai's subject matter is disclosed herein by reference.Likewise, a fragment of SEQ. ID NO 2 may include Thai's sequence ID numbers 2-5 that includea histidine tag comprised of 2-10 histidine moieties (e.g., 4-10 histidine moieties, 6-10 histidinemoieties, or 6 histidine moieties) located at theN- or C-terminus of the particular peptide.It has been demonstrated by in vitro studies using polypeptides according to SEQ ID NO1 and SEQ ID NO 2 that treatment of myriad cell types (keratinocytes, enterocytes, islet,endothelial and pneumocyte cells) with polypeptides according to SEQ ID NO 1 or SEQ ID NO 2added to cell culture medium causes stimulation and increase in cell growth, resulting in viableprogenitor cells, as measured by the percent of CD133 positive cells in culture. It has also beenshown that treatment of cells with a peptide according to SEQ ID NO 1 or SEQ ID NO 2 causesan increase in the anti-aging-related genes Sirts 1 ,2 and 6, as well as a dramatic increase inmTor expression. The cells treated with a peptide according to SEQ ID 1 or 2 can beregenerated and propagated into the billions.As used herein, the term "sequence identity" refers to the identity between two or moreamino acid sequences expressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; the higher the percentage,the more identical the sequences are. The percentage identity is calculated over the entirelength of the sequence. Homologs or orthologs of amino acid sequences possess a relativelyhigh degree of sequence identity when aligned using standard methods. This homology is moresignificant when the orthologous proteins are derived from species which are more closelyrelated (e.g., human and mouse sequences), compared to species more distantly related (e.g.,human and C. elegans sequences). Methods of alignment of sequences for comparison are wellknown in the art. Various programs and alignment algorithms are described in: Smith &Waterman; Needleman & Wunsch, J. Mol. Bioi. 48:443, 1970; Pearson & Lipman, Proc. Nat.Acad Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237 44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Carpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al.Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Bioi. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations. The level of sequence identity may bedetermined using the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol.Bioi. 215:403-10, 1990), which is available from several sources, including the National Centerfor Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805,Bethesda, Md. 20894, US) and on the Internet.As used herein, "supplement" or "dietary supplement" (used interchangeably) means acomposition that is intended to supplement the diet by providing certain nutrients or othermolecules in contrast to a large number of calories, and as further defined by DietarySupplement Health and Education Act (often referred to as DSHEA). Supplements may be insolid, semi-solid, or liquid form (e.g., including tablets, capsules, soft gels, gel caps, powders,bars, gummies, and liquids) to provide the polypeptides of the present invention. In addition tothe polypeptides of the present invention, the supplements may contain any one or more of thefollowing: vitamins, minerals, herbs, amino acids, essential fatty acids and other substances thatare traditionally supposed to be supplements or which may be used in supplements or others.It will be understood that a numerical value may be associated with a certain amount ofexperimental error. Thus, recitation of the qualifier "about" (or "approximately") prior to anumerical error is meant to embody the experimental error that may be associated with therecited numerical value. To the extent that a numerical value obtained experimentally is notpreceded by the expression "about" (or "approximately") does not mean that the numericalvalue is not associated with a certain amount of experimental error.

Claims

 1.A method of treating a patient at risk for developing atherosclerotic cardiovascular disease (ASCVD) comprising administering to the patient a therapeutically effective dose of an anti-interleukin-6 (anti-IL-6) antibody or antibody fragment having the variable heavy (VH) CDRs as defined in SEQ ID NOs 2, 3 and 4, and the variable light (VL) CDRs as defined in SEQ ID NOs 8, 9 and 10, wherein the therapeutically effective dose is about 15, about 25, or about 50 mg.   2.The method of claim 1, wherein the treatment is administered to ASCVD high-risk primary prevention patients of 50 years or older, hs-CRP ≥3 mg / L, and with the diagnosis of diabetes or 2 or more of the following risk factors: tobacco use, hyperlipidemia, or hypertension, excluding patients with a concomitant diagnosis of inflammatory or infectious disease.  3.The method of claim 1, wherein, the treatment is administered to ASCVD secondary prevention patients of 50 years or older and with hs-CRP ≥2 mg / L, excluding secondary prevention patients with a concomitant diagnosis of inflammatory or infectious disease.   4.The method of claim 1, wherein the method comprises following the patient for safety for 365 days, preferable at 240 days, 300 days and 365 days.   5.The method of claim 1-4, wherein the antibody or antibody fragment comprises a heavy chain polypeptide comprising a polypeptide having at least 98% identity to SEQ ID NO: 1 and a light chain polypeptide comprising a polypeptide having at least 98% identity to SEQ ID NO: 7.   6.The method of claim 5, wherein the antibody or antibody fragment comprises a heavy chain polypeptide having the sequence of SEQ ID NO: 1 and a light chain polypeptide having the sequence of SEQ ID NO: 7.    7.The method of any one of claims 1-6, further comprising:a) administering a loading dose of the anti-IL-6 antibody or antibody fragment to the patient for at least the first two doses during a loading regimen; andb) thereafter administering a maintenance dose of the anti-IL-6 antibody or antibody fragment to the patient during a maintenance regimen.   8.The method of claim 7, wherein the loading regimen comprises administering the loading dose every 1 week, every 2 weeks, or every 4 weeks.    9.The method of claim 7, wherein the maintenance regimen comprises administering the maintenance dose every 4 weeks, every 8 weeks, every 12 weeks, or every 24 weeks.   10.The method of claim 7, further comprising(a) administering a loading dose of the anti-IL-6 antibody or antibody fragment to the patient every 4 weeks for the first two doses during a loading regimen; and (b) thereafter administering a maintenance dose of the anti-IL-6 antibody or antibody fragment to the patient every 8 or 12 weeks during a maintenance regimen.    11.The method of any one of claims 7-10, wherein the loading dose is greater than or equal to the maintenance dose.   12.The method of any one of claims 1-11, wherein the patient has inflammation.    13.The method of claim 12, wherein the patient has IL-6 mediated inflammation.     14.The method of claim 12 or 13, wherein the treatment is sufficient to reduce the inflammation without causing immune suppression.   15.The method of claim 12, wherein the immune suppression is measured by absolute neutrophil count (ANC).    16.The method of claim 15, wherein a post-treatment ANC is at least 500, 1000, 1500, or 2000 cells / pL.   17.The method of claim 16, wherein the post-treatment ANC is decreased by no more than 50%, 40%, 30%, 20% or 10% as compared to a pre-treatment level.   18.The method of claim 16, wherein the ANC is not decreased as compared to a pre-treatment level.   19.The method of any one of claims 12-18, wherein the inflammation is measured by high-sensitivity C-reactive protein (hsCRP) levels.    20.The method of claim 19, wherein the patient has a pre-treatment hsCRP level of greater than 2 mg / L.     21.The method of claim 20, wherein the patient has a pre-treatment hsCRP level of 2 mg / L or less.    22.The method of claim 20, wherein the patient has a pre-treatment hsCRP level of 1 mg / L or less.    23.The method of claim 20, wherein the treatment is sufficient to reduce the hsCRP level to 2 mg / L or less.    24.The method of claim 20, wherein the treatment is sufficient to reduce the hsCRP level to 1 mg / L or less.   25.The method of any one of claims 19-24, wherein the treatment is sufficient to reduce the hsCRP level by at least 50%, 60%, 70%, 80%, or 90% as compared to the pre-treatment level.    26.The method of claim 25, wherein the treatment results in hsCRP reduction within 4, 8 or 12 weeks of treatment.   27.The method of claim 25, wherein the treatment is sufficient to sustain the hsCRP reduction for at least 24 or 48 weeks.   28.The method of claim 23 or 25, wherein the treatment results in hsCRP reduction after about 180 days of treatment.    29.The method of any one of claims 1-28, wherein the treatment is sufficient to reduce fibrinogen, haptoglobin, serum amyloid A (SAA), secretory phospholipase A2 (sPLA2), lipoprotein (a), or neutrophil-to-lymphocyte ratio (NLR) by at least 15%, 30%, or 50%. 

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