Methods and compositions for delivering biotin to mitochondria

Abstract

Certain exemplary embodiments relate to a composition of biologically active material (and uses thereof) configured for targeted delivery of biotin to mitochondria, the composition comprising a first D-biotin conjugated to a water-soluble, cell-permeable peptide sequence, wherein the peptide sequence is selected from the group of polypeptides having alternating aromatic-cationic motifs.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and is incorporated herein by reference in its entirety by reference to pending U.S. Provisional Patent Application 63 / 075,996 filed on September 9, 2020 (Attorney Docket No. 1255-003) and U.S. Provisional Patent Application 63 / 076,022 filed on September 9, 2020 (Attorney Docket No. 1255-004). BRIEF DESCRIPTION OF THE DRAWINGS

[0004] A variety of potential, feasible and / or useful embodiments will be more readily understood through the non-limiting, non-exhaustive description of certain exemplary embodiments provided herein, with reference to the accompanying exemplary drawings, in which:

[0005] Figure 1A , Figure 1B and Figure 1C is a chemical structure diagram showing how biotin can be conjugated to a mitochondrial targeting peptide sequence.

[0006] Figure 2 are fluorescence microscopy images associated with cellular uptake of certain exemplary compounds.

[0007] Figure 3 is a graph showing exemplary cellular uptake.

[0008] Figure 4 are fluorescence microscopy images associated with cellular uptake of certain exemplary compounds.

[0009] Figure 5 is a graph showing exemplary cellular uptake.

[0010] Figure 6 are fluorescence microscopy images associated with targeting of certain exemplary compounds to mitochondria.

[0011] Figure 7 is a graph showing exemplary cell viability.

[0012] Figure 8 are microscopic images showing exemplary cell numbers.

[0013] Fig. 9 is a graph showing exemplary cell viability.

[0014] Fig.10 is a graph showing exemplary cellular ATP levels.

[0015] Fig.11 is a graph showing exemplary cellular ATP levels.

[0016] Fig.12is a graph showing exemplary cellular ATP levels.

[0017] Fig.13 is a fluorescence microscopy image showing mitochondrial potential.

[0018] Fig.14 is a fluorescence microscopy image showing exemplary mitochondrial potential.

[0019] Fig.15 are microscopic images of scratched areas associated with certain exemplary compounds.

[0020] Fig.16 is a graph showing exemplary scratch areas.

[0021] Fig.17 is a graph showing exemplary scratch areas.

[0022] Fig.18 are microscopic images of scratched areas associated with certain exemplary compounds.

[0023] Fig.19 is a graph showing exemplary scratch areas.

[0024] Fig. 20 are microscopic images associated with cell proliferation for certain exemplary compounds.

[0025] Fig.21 is a graph showing exemplary cell proliferation.

[0026] Fig. 22 are microscopic images associated with cell proliferation for certain exemplary compounds.

[0027] Fig.23 is a graph showing exemplary cell proliferation.

[0028] Fig.24 are microscopic images associated with mitochondrial potential of certain exemplary compounds.

[0029] Fig.25 is a graph showing exemplary mitochondrial potentials.

[0030] Fig.26 are fluorescence microscopy images associated with retinal uptake of certain exemplary compounds.

[0031] Fig. 27 are fluorescence microscopy images associated with retinal uptake of certain exemplary compounds.

[0032] Details

[0033] Certain exemplary embodiments described herein may relate to methods and compositions for enhancing cellular and / or mitochondrial uptake of biotin. Targeted delivery of biotin to mitochondria may improve efficacy and avoid the use of high doses of biotin.

[0034] Certain exemplary embodiments described herein may provide a method for delivering biotin to mitochondria using a water-soluble, cell-permeable, mitochondrial-targeted peptide sequence.

[0035] Certain exemplary embodiments described herein relate to short water-soluble peptide sequences selected from the group of polypeptides having a general aromatic-cationic motif, meaning that the amino acids of the polypeptide group can be arranged, for example, as [aromatic-cationic-aromatic-cationic] or [cationic-aromatic-cationic-aromatic].

[0036] Certain exemplary embodiments described herein may include polypeptides comprising naturally occurring amino acids.

[0037] Some exemplary embodiments described herein can include D amino acids, which can help make peptides more tolerant to the hydrolysis of peptidases. Some exemplary embodiments described herein can include polypeptides comprising one or more non-natural amino acids. Non-natural amino acids are those amino acids that are not synthesized in the normal metabolic process in living organisms, and are not naturally present in proteins. Non-natural amino acids can include naturally occurring amino acid derivatives.

[0038] The peptides useful in the present invention may contain one or more non-naturally occurring amino acids. The non-naturally occurring amino acids may be L-amino acids, dextrorotatory (D) amino acids, or mixtures thereof. Optimally, the peptides do not have naturally occurring amino acids.

[0039] Non-naturally occurring amino acids are those amino acids that do not naturally occur in proteins. In addition, the non-naturally occurring amino acids that can be used in the present invention are preferably not recognized by common proteases.

[0040] The non-naturally occurring amino acid can be present at any position of the peptide. For example, the non-naturally occurring amino acid can be at the N-terminus, at the C-terminus, or at any position between the N-terminus and the C-terminus.

[0041] The non-natural amino acid may, for example, comprise an alkyl, aryl or alkylaryl group. Some examples of alkyl amino acids include α-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid and ε-aminocaproic acid. Some examples of aryl amino acids include o-aminobenzoic acid, m-aminobenzoic acid and p-aminobenzoic acid. Some examples of alkylaryl amino acids include o-aminophenylacetic acid, m-aminophenylacetic acid and p-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.

[0042] Non-naturally occurring amino acids also include derivatives of naturally occurring amino acids. Derivatives of naturally occurring amino acids may, for example, include the addition of one or more chemical groups to a naturally occurring amino acid.

[0043] For example, one or more chemical groups can be added to the 2', 3', 4', 5' or 6' position of the aromatic ring of phenylalanine or tyrosine residues, or one or more of the 4', 5', 6' or 7' positions of the benzo ring of tryptophan residues. The group can be any chemical group that can be added to an aromatic ring. Some examples of such groups include branched or unbranched C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl or tert-butyl, C1-C4 alkoxy (i.e., alkoxy), amino, C1-C4 alkylamino and C1-C4 dialkylamino (e.g., methylamino, dimethylamino), nitro, hydroxyl, halogenated (e.g., fluoro, chloro, bromo or iodo). Some specific examples of non-naturally occurring derivatives of naturally occurring amino acids include norvaline (Nva), norleucine (Nle) and hydroxyproline (Hyp).

[0044] Another example of amino acid modification in a peptide that can be used in the method of the invention is the derivatization of the carboxyl group of the aspartic acid or glutamic acid residue of the peptide. An example of derivatization is amidation with ammonia or with a primary or secondary amine (e.g., methylamine, ethylamine, dimethylamine, or diethylamine). Another example of derivatization includes esterification with, for example, methanol or ethanol.

[0045] Another such modification comprises the derivatization of the amino group of lysine, arginine or histidine residues.For example, such an amino group can be acylated.Some suitable acyl groups comprise, for example, a benzoyl group or an alkanoyl group comprising the C1-C4 alkyl group mentioned above, such as an acetyl or propionyl group.Some exemplary embodiments described herein comprise the amidation of the C-terminal of the peptide sequence, which can help make the peptide more tolerant to the hydrolysis of carboxypeptidase.

[0046] The carboxyl group, in particular the terminal carboxyl group of the C-terminal amino acid, can be amidated with, for example, ammonia to form a C-terminal amide. Alternatively, the terminal carboxyl group of the C-terminal amino acid can be amidated with any primary or secondary amine. The primary or secondary amine can be, for example, an alkylamine, in particular a branched or unbranched C1-C4 alkylamine or an arylamine. Therefore, the amino acid at the C-terminal of the peptide can be converted into an acylamino, N-methylamido, N-ethylamido, N,N-dimethylamido, N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido or N-phenyl-N-ethylamido group.

[0047] The free carboxylic acid groups of asparagine, glutamine, aspartic acid and glutamic acid residues not present at the C-terminus of the aromatic-cationic peptides of the invention can also be amidated, wherever they are present within the peptide. Amidation at these internal positions can be carried out with ammonia or any primary or secondary amine described above.

[0048] Certain exemplary embodiments described herein may include polypeptides comprising a minimum of four amino acids.

[0049] Certain exemplary embodiments described herein may include polypeptides comprising no more than six amino acids.

[0050] Certain exemplary embodiments described herein relate to water-soluble peptide sequences selected from polypeptides comprising 4-6 amino acids (in the D or L configuration) having alternating aromatic-cationic motifs.

[0051] Certain exemplary embodiments described herein relate to water-soluble peptide sequences, including but not limited to:

[0052] D-Arg-L-(2'6'-dimethylTyr)-L-Lys-L-Phe-NH2(SS-31);

[0053] D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys-NH2 (SPN02);

[0054] D-Trp-D-Arg-D-Trp-D-Lys-OH (SPN07);

[0055] L-Trp-L-Arg-L-Trp-L-Lys-NH2 (SPN10);

[0056] D-Trp-D-Arg-D-Trp-D-Lys-NH2 (SPN13); and

[0057] L-Trp-L-Arg-L-Trp-L-Lys-OH (SPN14).

[0058] Certain exemplary embodiments described herein may provide methods for conjugating D-biotin to a short (ie, 4 to 6 amino acids), water-soluble, cell-permeable, mitochondrial-targeted peptide sequence for targeted delivery of biotin to mitochondria.

[0059] Certain exemplary peptide sequences described herein are defined by an N-terminus and a C-terminus and may include an α-amine at the N-terminus. Certain exemplary embodiments described herein may provide for conjugating D-biotin to the N-terminal α-amine of a peptide sequence targeted to mitochondria.

[0060] Certain exemplary peptide sequences described herein may include a lysine residue having an ε-amine at its C-terminus. Certain exemplary embodiments described herein may provide methods for conjugating D-biotin to the ε-amine of a lysine residue at the C-terminus of a peptide sequence targeting mitochondria (ε-N-[d-biotinyl]-L-lysine).

[0061] Certain exemplary embodiments described herein may provide methods for conjugating D-biotin to the N-terminal α-amine and the ε-amine on the lysine residue at the C-terminus of a mitochondrial targeting peptide sequence.

[0062] Certain exemplary embodiments described herein are directed to at least one therapeutically effective composition of matter and / or methods for making and / or using such a composition and / or one or more components thereof, the composition comprising:

[0063] One or more compounds selected from the group consisting of:

[0064] D-biotin-D-Arg-L-(2'6'-dimethylTyr)-L-Lys-L-Phe-NH2(SPN05);

[0065] D-biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH (SPN08);

[0066] D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH (SPN09);

[0067] L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2 (SPN11);

[0068] D-biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2 (SPN12);

[0069] D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2 (SPN15); and

[0070] D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2(SPN16).

[0071] One or more exemplary compositions described herein may include one or more peptide-conjugated biotin molecules suitable for stimulating cellular ATP production.Exemplary compositions may include vitamins and / or amino acids and / or metabolic supplements that promote ATP synthesis at effective concentrations.

[0072] One or more exemplary compositions described herein can include one or more peptide-conjugated biotin molecules suitable for maintaining mitochondrial potential.

[0073] Certain exemplary embodiments described herein relate to novel compositions and / or methods suitable for promoting cell survival when culturing cells in vitro in the absence of serum. One or more exemplary compositions described herein may include one or more peptide-conjugated biotin molecules suitable for stimulating mitochondrial function (such as promoting cell proliferation). One or more exemplary compositions described herein may include vitamins and / or amino acids and / or metabolic supplements that exhibit additive or synergistic activity in maintaining cell growth at effective concentrations.

[0074] Due to the small number of stem cells, in order to perform cell therapy, a large amount of ex vivo expansion is required to obtain a sufficient number of cells for treating clinical indications. Culture medium containing fetal bovine serum (FBS) provides a supportive environment for the separation and expansion of mesenchymal stem cells. FBS provides attachment factors, growth factors and many other nutrients. However, in addition to the inherent variability and limited global supply of FBS, serum products may also be a source of pathogens and / or contain serum proteins with the potential to induce immune responses in human recipients.

[0075] Serum depletion causes cell cycle arrest and apoptosis, which severely limits the yield of stem cells or primary cells for clinical use. Multiple growth factors must be added to serum-free media to allow stem cell expansion, and this often involves the use of human-derived supplements that may be contaminated with human pathogens. Certain exemplary media described herein include chemically defined but serum-free and xeno-free components that support the growth and attachment of human primary cells and subcultures to allow large-scale production of primary mammalian cells for clinical use.

[0076] Large-scale production of primary mammalian cells may be important for laboratory-produced meat as a replacement for traditional livestock-derived meat. Cultivation of animal myoblasts with FBS is not sustainable. Certain exemplary culture media described herein contain a chemically defined serum-free component that supports the proliferation of animal myoblasts for large-scale production of laboratory-grown meat.

[0077] Certain exemplary embodiments described herein relate to novel compositions and / or methods suitable for promoting cell or tissue survival for transplantation in humans. One or more exemplary compositions described herein may include one or more peptide-conjugated biotin molecules suitable for stimulating mitochondrial cell ATP production to promote cell survival. One or more exemplary compositions described herein may include vitamins and / or amino acids and / or metabolic supplements that exhibit additive activity or synergistic activity in maintaining organ survival at effective concentrations.

[0078] Islet transplantation is a method of beta cell replacement therapy for type 1 diabetes. Addition of one or more peptide-conjugated biotin molecules that can improve cell viability throughout the harvesting and purification procedures can greatly improve islet yields, and / or increase the success rate of glycemic control in recipients.

[0079] Ischemia and hypoxia are inevitable events during organ preservation before transplantation. After the organ is deprived of normal blood supply, the depletion of mitochondrial ATP synthesis will lead to cell death. The storage duration of the heart and lungs varies from 4 hours to 6 hours, and the storage duration for the kidney is up to 36 hours. Adding one or more peptide-conjugated biotin molecules that can improve cell viability to the organ preservation solution can extend the survival time of the organ to allow for wider distribution to matching recipients. Improvements in the quality of transplanted organs can also reduce delayed graft function and graft failure.

[0080] Certain exemplary embodiments described herein relate to novel compositions and / or methods suitable for promoting tissue health and / or preventing tissue damage. Certain exemplary methods described herein provide systemic administration of exemplary compositions to mammals. Compositions may include one or more peptide-conjugated biotin molecules suitable for stimulating mitochondrial cell ATP production to promote cell function. Compositions may include vitamins and / or amino acids and / or metabolic supplements that exhibit additive or synergistic activity in maintaining tissue health at effective concentrations.

[0081] Aging is associated with a reduced proliferative capacity of many cell types, including skin and epithelial membranes. The intestinal epithelium completely renews itself within 5 days, while the lung epithelium may take up to 6 months to renew. Aging reduces the ability of the epithelium to regenerate and leads to progressive epithelial damage.

[0082] As we age, most tissues exhibit a progressive decrease in regenerative capacity, which leads to tissue degeneration, dysfunction, and pathology. Certain methods described herein can promote tissue health during aging and can prevent and / or reduce many of these age-related dysfunctions.

[0083] Exemplary methods described herein relate to novel compositions and / or methods suitable for promoting tissue health that can promote the in vivo proliferation of resident stem / progenitor cells in aged and / or damaged tissues.

[0084] Certain exemplary embodiments described herein relate to novel compositions and / or methods suitable for promoting tissue repair and regeneration. Certain exemplary methods described herein provide systemic administration of exemplary compositions to mammals after tissue damage. Compositions may include one or more peptide-conjugated biotin molecules suitable for stimulating mitochondrial cell ATP production to promote cell proliferation and / or tissue regeneration. Compositions may include vitamins and / or amino acids and / or metabolic supplements that exhibit additive activity or synergistic activity in maintaining tissue health at effective concentrations.

[0085] Tissue damage may include damage to the skin, and / or soft tissue (e.g., muscle, tendon, ligament, nerve, blood vessel), and / or hard tissue (e.g., bone, tooth), and / or solid organ (e.g., heart, kidney, lung, liver, spleen, intestine, etc.) Causes of tissue damage may include, for example, trauma, hypoxia (i.e., low oxygen supply), ischemia (i.e., low blood flow), infectious agents, drugs, chemicals, and / or toxins, etc.

[0086] Certain exemplary embodiments described herein relate to novel compositions and / or methods suitable for treating wounds and / or tissue damage and / or promoting tissue regeneration and faster wound repair. Certain exemplary methods described herein provide direct application of compositions to wounds, such as stimulating (i.e., promoting) neighboring cell proliferation (i.e., increasing the number of cells) around the wound to promote and / or achieve wound closure. The composition may include one or more peptide-conjugated biotin molecules suitable for stimulating mitochondrial cell ATP production to promote cell proliferation and / or migration (i.e., cell movement). The composition may include vitamins and / or supplements and / or metabolic supplements that exhibit additive activity or synergistic activity in terms of stimulating wound healing (such as postoperative wounds and / or non-healing chronic wounds for patients, such as pressure sores and / or diabetic ulcers and / or venous ulcers) at effective concentrations.

[0087] Exemplary methods described herein relate to novel compositions and / or methods suitable for treating tissue damage that can promote the in vivo proliferation of resident stem / progenitor cells in damaged tissue.

[0088] Certain exemplary embodiments may provide a pharmaceutical composition useful for promoting tissue repair and / or regeneration, the pharmaceutical composition comprising:

[0089] (a) one or more compounds that promote ATP production and cell proliferation;

[0090] (b) at least one vitamin; and / or

[0091] (c) at least one amino acid; and / or

[0092] (d) at least one metabolic supplement.

[0093] Certain exemplary embodiments described herein may provide a method of enhancing mitochondrial ATP production in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a compound having the formula:

[0094] D-biotin-D-Arg-L-(2'6'-dimethylTyr)-L-Lys-L-Phe-NH2(SPN05);

[0095] D-biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH (SPN08);

[0096] D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH (SPN09);

[0097] L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2 (SPN11);

[0098] D-biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2 (SPN12);

[0099] D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2 (SPN15);

[0100] and / or

[0101] D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2(SPN16).

[0102] Wherein the compound is administered to the mammal as a composition comprising a pharmaceutically acceptable carrier.

[0103] Certain exemplary embodiments described herein can provide a composition configured for increasing cell proliferation of primary cells cultured ex vivo in the absence of serum, the composition comprising an active ingredient comprising one or more peptide-conjugated biotin molecules that stimulate mitochondrial ATP production and cell proliferation, and a second ingredient comprising at least one vitamin and / or amino acid and / or metabolic supplement that enhances ATP production and cell proliferation.

[0104] Certain exemplary embodiments described herein can provide a composition configured for increasing the survival of cells and organs ex vivo, the composition comprising an active ingredient comprising one or more peptide-conjugated biotin molecules that stimulate mitochondrial ATP production and cell proliferation, and a second ingredient comprising at least one vitamin and / or amino acid and / or metabolic supplement that enhances ATP production and cell proliferation.

[0105] Certain exemplary embodiments described herein may provide a composition configured for increasing cell proliferation (such as in a wound area), the composition comprising an active ingredient comprising one or more peptide-conjugated biotin molecules, and / or a second ingredient comprising a mixture of at least one vitamin and / or an amino acid and / or a metabolic supplement that enhances cell proliferation and migration.

[0106] Examples of amino acids that can be used in certain exemplary embodiments described herein include the L-isomers of all natural amino acids, including the essential and non-essential amino acids (isoleucine, leucine, alanine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, histidine), as well as taurine, which is naturally derived from cysteine.

[0107] Examples of metabolic supplements that can be used in certain exemplary embodiments described herein include pyruvate, carnitine, acetylcarnitine, creatine, alpha-ketoglutarate, alpha-lipoic acid, coenzyme Q 10 , nicotinamide riboside, and nicotinamide mononucleotide.

[0108] Certain exemplary embodiments described herein may provide methods of enhancing the survival and proliferation of primary mammalian cells in a serum-free, chemically defined culture medium by adding to the culture medium a formulation comprising an effective amount of a mixture of one or more peptide-conjugated biotin molecules, and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement.

[0109] In certain exemplary embodiments of one or more methods described herein, primary cells may include bone marrow stem cells or mesenchymal stem cells for autologous, allogeneic, or xenogeneic regenerative medicine applications.

[0110] In certain exemplary embodiments of one or more of the methods described herein, the primary cells can be stem cells obtained from placenta or umbilical cord blood for allogeneic transplantation.

[0111] In certain exemplary embodiments of one or more methods described herein, the primary cells can be hematopoietic cells, such as T cells, for chimeric antigen receptor (CAR) T cell therapy.

[0112] In certain exemplary embodiments of one or more methods described herein, the primary cells can be mammalian cells cultured for the production of therapeutic proteins (such as monoclonal antibodies) and / or biopharmaceuticals, and / or for the development and / or production of viral vaccines.

[0113] In certain exemplary embodiments of one or more of the methods described herein, the primary cells may be animal cells, such as myoblasts, cultured for in vitro production of laboratory-grown non-slaughtered meat.

[0114] In certain exemplary embodiments of one or more of the methods described herein, the primary cells can be pancreatic islet cells harvested from a donor pancreas and purified in the laboratory prior to transplantation.

[0115] Certain exemplary embodiments described herein may provide methods for optimizing and / or improving stem cell transplantation and / or mitochondrial transfer for tissue regeneration by treating the stem cells and / or mitochondria prior to transplantation with a solution comprising a therapeutically effective amount of a mixture of one or more peptide-conjugated biotin molecules, and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement.

[0116] In certain exemplary embodiments of one or more methods described herein, the cells can be resident stem / progenitor cells in damaged tissue, and a mixture of one or more peptide-conjugated biotin molecules, and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement can be administered systemically to the subject.

[0117] In certain exemplary embodiments of one or more methods described herein, one or more peptide-conjugated biotin molecules and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement may be systemically administered to the subject following mitochondrial transplantation to optimize mitochondrial survival and function.

[0118] In certain exemplary embodiments of one or more methods described herein, one or more peptide-conjugated biotin molecules, and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement can be added to serum-free culture medium for culturing laboratory-grown meat to replace meat from livestock sources.

[0119] Certain exemplary embodiments described herein may provide a method of enhancing an organ preservation solution by adding to the preservation solution a formulation comprising an effective amount of one or more peptide-conjugated biotin molecules and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement.

[0120] In certain exemplary embodiments of one or more methods described herein, the organ can include a kidney, liver, heart, lung, pancreas, skin, intestine, cornea, trachea, and / or blood vessel.

[0121] In certain exemplary embodiments of one or more methods described herein, effective amounts of one or more peptide-conjugated biotin molecules and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement may be systemically administered to a transplant recipient to enhance early graft function and enhance graft survival.

[0122] Certain exemplary embodiments described herein may provide methods of enhancing tissue health in a subject by administering a micronutrient formulation comprising an effective amount of a mixture of one or more peptide-conjugated biotin molecules, and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement.

[0123] Certain exemplary embodiments described herein may provide methods of enhancing tissue health in a subject by administering a micronutrient formulation comprising an effective amount of a mixture of one or more peptide-conjugated biotin molecules, and acetylcarnitine and / or alpha-ketoglutarate.

[0124] Certain exemplary embodiments described herein may provide methods of enhancing tissue health in a subject by administering a micronutrient formulation comprising an effective amount of a mixture of one or more peptide-conjugated biotin molecules, and acetylcarnitine, alpha-ketoglutarate, and / or taurine.

[0125] In certain exemplary embodiments of one or more of the methods described herein, tissue health can include the health of keratinous tissue (e.g., skin, hair, and / or nails), and / or muscles, and / or joints, and / or bones, and / or heart, and / or lungs, and / or kidneys, and / or brain, and / or vision and / or hearing.

[0126] In certain exemplary embodiments of one or more of the methods described herein, tissue damage can be caused by aging. As we age, all cells can undergo changes. Many cells can lose their ability to function, waste products can accumulate in cells, connective tissue can become stiff, and many tissues can lose mass.

[0127] Certain exemplary embodiments described herein can provide methods of reducing, alleviating and / or reversing age-related damage in a subject by administering a composition comprising an effective amount of one or more peptide-conjugated biotin molecules and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement, the administration being performed orally, sublingually and / or subcutaneously, etc. Pharmaceutical preparations for oral administration can be solutions, suspensions or solid forms such as tablets, capsules and powders, etc. Pharmaceutical preparations for sublingual administration or for subcutaneous injection can be prepared by mixing such compositions with non-toxic, therapeutically inert and / or liquid carriers commonly used for sublingual and / or subcutaneous formulations.

[0128] Certain exemplary embodiments described herein may provide methods of enhancing skin wound healing in a subject by applying a topical solution comprising a therapeutically effective amount of one or more peptide-conjugated biotin molecules and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement directly to the wound area.

[0129] Certain exemplary embodiments described herein may provide methods of enhancing skin wound healing in a subject by administering a topical solution comprising a therapeutically effective amount of one or more peptide-conjugated biotin molecules and taurine directly to the wound area.

[0130] In certain exemplary embodiments of one or more methods described herein, the wound can be a pressure wound, a surgical wound, a burn wound, a trauma, and / or a wound that has been exposed to one or more chemicals and / or therapeutic radiation, among others.

[0131] For topical application to the skin, certain exemplary compositions described herein can be prepared as sprays, ointments, creams and / or gels. Pharmaceutical preparations for topical application to the skin can be prepared by mixing such compositions with non-toxic, therapeutically inert, solid and / or liquid carriers commonly used for topical pharmaceutical preparations.

[0132] Certain exemplary embodiments described herein may provide methods of enhancing gingival and / or periodontal healing in a subject by directly applying to the wound area a topical solution comprising a therapeutically effective amount of one or more peptide-conjugated biotin molecules and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement.

[0133] Certain exemplary embodiments described herein may provide methods of enhancing gingival and / or periodontal healing in a subject by administering a topical solution comprising a therapeutically effective amount of one or more peptide-conjugated biotin molecules and taurine.

[0134] For topical administration to the oral mucosa, certain exemplary compositions described herein can be prepared as sprays, ointments, gels, mouthwashes and / or toothpastes, etc. Pharmaceutical preparations for topical administration to mucosa can be prepared by mixing such compositions with non-toxic, therapeutically inert and / or liquid carriers commonly used for topical pharmaceutical preparations.

[0135] Certain exemplary embodiments described herein may provide a method of enhancing repair of ocular damage in a subject by directly administering to the eye a topical solution comprising a therapeutically effective amount of one or more peptide-conjugated biotin molecules and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement.

[0136] Certain exemplary embodiments described herein may provide a method of enhancing repair of ocular damage in a subject by administering a topical solution comprising a therapeutically effective amount of one or more peptide-conjugated biotin molecules and taurine.

[0137] In certain exemplary embodiments of one or more of the methods described herein, the injury to the eye can be one or more of the following: for example, acute corneal abrasion, subconjunctival hemorrhage, and / or retinal detachment, and / or chronic eye disease, including, for example, age-related macular degeneration, diabetic retinopathy, glaucoma, and / or dry eye.

[0138] Certain exemplary embodiments may provide methods of enhancing bone and soft tissue healing in a subject by administering directly to the wound area a solution comprising a therapeutically effective amount of one or more peptide-conjugated biotin molecules and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement.

[0139] In certain exemplary embodiments of one or more methods described herein, the injury can be acute and / or chronic, such as trauma, arthritis, tendinitis, one or more ligament tears, and / or nerve compression, and the like.

[0140] For direct application to soft tissue, certain exemplary compositions described herein can be prepared as sterile solutions for injection into one or more joints, tendons, muscles and / or nerves, etc. Pharmaceutical preparations for injection can be prepared by mixing such compositions with non-toxic, therapeutically inert and / or liquid carriers commonly used for such preparations, such as polyethylene glycol and hyaluronic acid.

[0141] Certain exemplary embodiments described herein can provide methods for enhancing organ repair in a subject by administering (such as intravenously, intramuscularly, subcutaneously and / or orally, etc.) a solution comprising a therapeutically effective amount of one or more peptide-conjugated biotin molecules and at least one vitamin and / or at least one amino acid and / or at least one metabolic supplement. Pharmaceutical products for injection can be prepared by mixing such compositions with a non-toxic, therapeutically inert and / or liquid carrier commonly used for injectable pharmaceutical products. Pharmaceutical products for oral administration can be administered, for example, in solutions, suspensions and / or solid forms (such as one or more tablets, capsules and / or powders), etc.

[0142] In certain exemplary embodiments of one or more methods described herein, tissue damage can be caused by an acute condition, such as trauma, reduced blood flow, reduced oxygen supply, infectious agents, drugs, and / or toxins to one or more organs, structures, and / or systems (such as the heart, brain, kidneys, liver, intestines, and / or limbs), and the like.

[0143] In certain exemplary embodiments of one or more of the methods described herein, the tissue damage can be caused by a chronic disease, such as heart failure, chronic kidney disease, inflammatory bowel disease, diabetic complications, stroke, macular degeneration, and / or neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, chronic traumatic encephalopathy, and / or Alzheimer's disease, among others.

[0144] In certain exemplary embodiments of one or more methods described herein, a mixture of one or more peptide-conjugated biotin molecules may be administered to a subject suffering from progressive multiple sclerosis, frontotemporal dementia, Parkinson's disease, and / or Alzheimer's disease.

[0145] In certain exemplary embodiments of one or more methods described herein, a mixture of one or more peptide-conjugated biotin molecules and thiamine can be administered to a subject suffering from a biotin-thiamine responsive basal ganglia disease.

[0146] In certain exemplary embodiments of one or more methods described herein, a mixture of one or more peptide-conjugated biotin molecules can be administered to a subject having inflammatory bowel disease.

[0147] Example 1 - Short aromatic-cationic peptide sequences increase cellular uptake of biotin

[0148] Certain peptide sequences described herein may be water-soluble polypeptides comprising 4 to 6 amino acids, which may have a general aromatic-cationic motif, meaning that the peptide sequence may be, for example, [aromatic-cationic-aromatic-cationic] or [cationic-aromatic-cationic-aromatic].

[0149] Certain "short" peptide sequences described herein may be polypeptides comprising a minimum of four amino acids and a maximum of six amino acids.

[0150] Amino acid can be naturally occurring. Naturally occurring amino acids include the 20 most common amino acids commonly found in proteins, i.e. alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). Aromatic amino acids can include Phe, Tyr and Trp. Cationic amino acids can include Lys, Arg and His. Amino acids can include natural amino acids in D- configuration. In some peptides, the carboxyl terminus can be amidated.

[0151] Amino acids can be non-naturally occurring. Non-naturally occurring amino acids are those amino acids that are not typically synthesized during normal metabolism in living organisms and do not occur naturally in proteins. Non-naturally occurring amino acids can include derivatives of naturally occurring amino acids in the L-configuration or the D-configuration.

[0152] Certain exemplary embodiments may provide for administering a composition for therapeutic purposes to a subject. In certain exemplary therapeutic applications, a composition and / or drug may be administered to a subject suspected of suffering from or already suffering from such a disease and / or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease and / or condition, including its complications and intermediate pathological phenotypes in the development of the disease and / or condition.

[0153] In certain exemplary embodiments, the therapeutic method comprises administering the composition together with one or more active agents.In certain exemplary embodiments, the peptide administration is chronic.

[0154] In certain exemplary embodiments, the peptides may be administered with one or more thrombolytic agents. In certain exemplary embodiments, the one or more thrombolytic agents may be selected from the group consisting of tissue plasminogen activator, urokinase, prourokinase, streptokinase, acylated forms of plasminogen, acylated forms of plasmin, and acylated streptokinase-plasminogen complex.

[0155] In certain exemplary embodiments, the therapeutic method may include administering the composition together with one or more antihypertensive agents. In certain exemplary embodiments, one or more antihypertensive agents may include diuretics, adrenergic receptor antagonists, calcium channel blockers, renin inhibitors, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, aldosterone antagonists, vasodilators and / or alpha-2 agonists.

[0156] In certain exemplary embodiments, the diuretic may include a loop diuretic, a thiazide diuretic, a thiazide-like diuretic, and / or a potassium-sparing diuretic. In certain exemplary embodiments, the diuretic may include bumetanide, ethacrynic acid, furosemide, torsemide, epithiazide, hydrochlorothiazide, chlorothiazide, bendroflumethiazide, indapamide, chlorthalidone, metolazone, amiloride, triamterene, and / or spironolactone.

[0157] In certain exemplary embodiments, adrenergic receptor antagonists may include beta blockers, alpha blockers, or mixed alpha and beta blockers. In certain exemplary embodiments, adrenergic receptor antagonists may include atenolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, timolol, doxazosin, phentolamine, indolamine, phenoxybenzamine, prazosin, terazosin, tolazoline, bucindolol, carvedilol and / or labetalol.

[0158] In certain exemplary embodiments, the calcium channel blocker may include dihydropyridines and / or non-dihydropyridines. In certain exemplary embodiments, the calcium channel blocker may include amlodipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, nitrendipine, diltiazem and / or verapamil.

[0159] In certain exemplary embodiments, a renin inhibitor may include

[0160] In certain exemplary embodiments, angiotensin converting enzyme (ACE) inhibitors may include captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, trandolapril, and / or benazepril.

[0161] In certain exemplary embodiments, angiotensin II receptor antagonists may include

[0162] In certain exemplary embodiments, the aldosterone antagonist may include eplerenone and / or spironolactone.

[0163] In certain exemplary embodiments, the vasodilator may include sodium nitroprusside and / or hydralazine.

[0164] In certain exemplary embodiments, the alpha-2 agonist may include clonidine, guanabenz, methyldopa, moxonidine, guanethidine, and / or reserpine.

[0165] In certain exemplary embodiments, any method known to those skilled in the art for contacting cells, organs and / or tissues with peptides can be used. Suitable methods may include in vitro, ex vivo and / or in vivo methods. When used for treatment in vivo, the composition can be administered to the subject in an effective amount (i.e., an amount with a desired therapeutic effect). Dosage and dosage regimen can depend on the degree of injury in the subject, the characteristics of the particular composition used, such as its therapeutic index, the subject and / or the subject's medical history.

[0166] The effective amount can be determined during preclinical and / or clinical trials by methods familiar to physicians and / or clinicians. An effective amount of a peptide useful in the method can be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The peptide can be administered systemically and / or topically.

[0167] The peptides can be formulated as pharmaceutically acceptable salts. Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. When the peptide contains both a basic moiety, such as an amine, pyridine or imidazole, and an acidic moiety, such as a carboxylic acid or tetrazole, zwitterions can be formed and are included in the term "salt" as used herein. Salts derived from pharmaceutically acceptable inorganic bases include ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts and zinc salts, etc. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, and the like, such as arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, D-glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. Salts derived from pharmaceutically acceptable inorganic acids include salts of boric acid, carbonic acid, hydrohalic acid (hydrobromic acid, hydrochloric acid, hydrofluoric acid or hydroiodic acid), nitric acid, phosphoric acid, sulfamic acid and sulfuric acid. Salts derived from pharmaceutically acceptable organic acids include salts of aliphatic hydroxy acids (e.g., citric acid, gluconic acid, glycolic acid, lactic acid, lactobionic acid, malic acid, and tartaric acid), aliphatic monocarboxylic acids (e.g., acetic acid, butyric acid, formic acid, propionic acid, and trifluoroacetic acid), amino acids (e.g., aspartic acid and glutamic acid), aromatic carboxylic acids (e.g., benzoic acid, p-chlorobenzoic acid, diphenylacetic acid, gentisic acid, hippuric acid, and triphenylacetic acid), aromatic hydroxy acids (e.g., o-hydroxybenzoic acid, p-hydroxybenzoic acid, Formic acid, 1-hydroxynaphthalene-2-carboxylic acid and 3-hydroxynaphthalene-2-carboxylic acid), ascorbic acid, dicarboxylic acids (e.g., fumaric acid, maleic acid, oxalic acid and succinic acid), glucuronic acid, mandelic acid, mucic acid, nicotinic acid, orotic acid, pamoic acid, pantothenic acid, sulfonic acids (e.g., benzenesulfonic acid, camphorsulfonic acid, ethanesulfonic acid (edisylic), ethanesulfonic acid, isethionic acid, methanesulfonic acid, naphthalenesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2,6-disulfonic acid and p-toluenesulfonic acid), xinafoic acid, etc. In some embodiments, the salt is acetate or trifluoroacetate.

[0168] The compositions described herein, or pharmaceutically acceptable salts thereof, such as acetate or trifluoroacetate, can be incorporated into pharmaceutical compositions for administration to a subject alone or in combination to treat and / or prevent the disorders described herein. Such compositions can include an active agent and a pharmaceutically acceptable carrier, which can include one or more of saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into certain exemplary compositions.

[0169] Pharmaceutical compositions can be formulated to be compatible with their intended routes of administration. Exemplary routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal, or subcutaneous), oral, sublingual, nasal, inhalation, transdermal (surface), intraocular, iontophoresis, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous applications may include any one or more of the following components: sterile diluents, such as water for injection, saline solutions, nonvolatile oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents such as sodium chloride or dextrose for adjusting tension. In certain exemplary embodiments, pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral products can be encapsulated in ampoules, disposable syringes, and / or multidose vials made of glass and / or plastic. For the convenience of the patient and / or treating physician, the administration formulation can be provided in a kit containing any or all necessary equipment (e.g., drug vials, diluent vials, syringes and / or needles, etc.) for a course of treatment (e.g., 7 days of treatment).

[0170] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) and / or dispersions and / or sterile powders for the extemporaneous preparation of sterile injectable solutions and / or dispersions. For intravenous administration, suitable carriers may include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) and / or phosphate buffered saline (PBS). Compositions for parenteral administration can be sterile and / or can be fluids that are easy to inject. Certain exemplary compositions can be stable and / or can prevent contaminating effects of microorganisms such as bacteria and / or fungi under preparation and / or storage conditions.

[0171] Composition components can include carriers, and carriers can be solvents or dispersion media, which include, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and / or liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained as follows, for example, by using coatings such as lecithin, by maintaining required particle size in the case of dispersions and / or by using surfactants. By various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, etc., it is possible to achieve the effect of preventing microorganisms. Glutathione and / or other antioxidants can be included to prevent oxidation. Some exemplary compositions can include isotonic agents, for example sugars and / or polyols, such as mannitol, sorbitol and / or sodium chloride. The absorption of the prolongation of injectable compositions can be achieved by including an agent such as aluminum monostearate and / or gelatin in the compositions that delays absorption.

[0172] Sterile injectable solutions can be prepared by: the required amount of active compound is mixed in a suitable solvent as required with one or combination of the above-mentioned ingredients, and then filtered for sterilization. Dispersions can be prepared by mixing the active compound into a sterile vehicle comprising an alkaline dispersion medium and other desired ingredients (such as one or more of those listed above). In the case of sterile powders for the preparation of sterile injectable solutions, exemplary preparation methods include vacuum drying and / or freeze drying, which can produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0173] Oral compositions may include inert diluents and / or edible carriers. For the purpose of oral therapeutic administration, the active compound may be incorporated with an excipient and / or used in the form of a tablet, lozenge or capsule (e.g., gelatin capsule) and / or a powder soluble in a diluent such as water. Oral compositions may be prepared using a fluid carrier used as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Exemplary tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth gum, and / or gelatin; excipients, such as starch and / or lactose, disintegrants, such as alginic acid, Primogel, and / or corn starch; lubricants, such as magnesium stearate and / or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose and / or saccharin; or flavorings, such as mint, methyl salicylate, and / or citrus flavoring; and the like.

[0174] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container and / or dispenser containing a suitable propellant (eg, a gas such as carbon dioxide) and / or a nebulizer.

[0175] Systemic administration of therapeutic compounds as described herein can be by transmucosal and / or transdermal means. For transmucosal or transdermal administration, penetrants suitable for the barrier to be penetrated can be used in the formulation. Such penetrants can include, for example, detergents, bile salts and / or fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays. For transdermal administration, the active compound can be formulated as an ointment, salves, gels and / or creams. In certain exemplary embodiments, transdermal administration can be performed by iontophoresis, microneedles and / or electroporation.

[0176] Therapeutic proteins and / or peptides can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome and / or a phospholipid bilayer vehicle. In certain exemplary embodiments, the therapeutic peptide can be encapsulated in a liposome while maintaining peptide integrity. The activating agent can be loaded into a particle prepared from pharmaceutically acceptable ingredients, including but not limited to soluble, insoluble, permeable, impermeable, biodegradable and / or gastric retention polymers and / or liposomes. Such particles can include nanoparticles, biodegradable nanoparticles, micron particles, biodegradable micron particles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and / or viral vector systems.

[0177] The carrier can be a polymer, for example, a biodegradable and / or biocompatible polymer matrix. In certain exemplary embodiments, the therapeutic peptide can be embedded in a polymer matrix while maintaining protein integrity. The polymer can be natural, such as a polypeptide, protein or polysaccharide, or synthetic, such as poly-alpha-hydroxy acid. Examples include carriers made of, for example, collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharides, fibrin, gelatin and combinations thereof. In certain exemplary embodiments, the polymer can be polylactic acid (PLA) and / or copolylactic acid / glycolic acid (PGLA). The polymer matrix can be prepared and / or separated in various forms and / or sizes, including microspheres and nanospheres. Polymer formulations can result in prolonged duration of therapeutic effect.

[0178] In certain exemplary embodiments, the therapeutic compound can be prepared with a carrier that will prevent the therapeutic compound from being rapidly eliminated from the body, such as a controlled release formulation, including implants and microcapsule delivery systems. Biodegradable and / or biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and / or polylactic acid can be used. Such preparations can be prepared using known techniques. Materials can also be obtained commercially from, for example, Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting specific cells with monoclonal antibodies to cell-specific antigens) can be used as pharmaceutically acceptable carriers.

[0179] Therapeutic compounds can be formulated to enhance intracellular delivery. For example, liposome delivery systems and / or fusogenic liposomes can be used to deliver proteins to cells in vivo and / or in vitro.

[0180] The dosage, toxicity and therapeutic efficacy of therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures and / or experimental animals, for example, for determining LD50 (a dose that is lethal to 50% of a population) and / or ED50 (a dose that is therapeutically effective to 50% of a population). The dose ratio between toxicity and therapeutic effect, called the "therapeutic index," can be expressed as the LD50 / ED50 ratio. Compounds that exhibit high therapeutic indexes may be preferred. Although compounds that exhibit toxic side effects can be used, delivery systems that target such compounds to affected tissue sites can be carefully designed to minimize potential damage to uninfected cells, thereby reducing side effects.

[0181] The data obtained from cell culture assays and / or animal studies can be used to formulate a dosage range for humans. The dosage of such a compound can be within the range of circulating concentrations that include ED50 and are almost non-toxic or non-toxic. The dosage can vary within this range depending on the dosage form used and / or the route of administration utilized. For some exemplary compounds, the therapeutically effective dose can be estimated initially from cell culture assays. In animal models, the dosage can be formulated into a circulating plasma concentration range that reaches the IC50 (i.e., the concentration of the test compound that reaches the half-maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine a useful dosage in humans. The level in plasma can be measured by, for example, high performance liquid chromatography.

[0182] The effective amount range of the composition sufficient to achieve a therapeutic or preventive effect can be about 0.000001 mg per kilogram of body weight per day to about 10,000 mg per kilogram of body weight per day. The dosage range can be about 0.0001 mg per kilogram of body weight per day to about 100 mg per kilogram of body weight per day. For example, the dosage can be about 1 mg / kg body weight or about 10 mg / kg body weight per day, every two days or every three days, or in the range of about 1 mg / kg to about 10 mg / kg per week, every two weeks or every three weeks. In certain exemplary embodiments, the single dose range of the peptide can be about 0.1 micrograms to about 10,000 micrograms per kilogram of body weight. In certain exemplary embodiments, the composition concentration range in the carrier can be about 0.2 micrograms to about 2000 micrograms per milliliter of delivery. Exemplary treatment regimens may require administration once a day or once a week. In certain therapeutic applications, relatively high doses at relatively short intervals may be required until the progression of the disease is reduced and / or terminated, and / or until the subject shows partial or complete improvement of disease symptoms. Thereafter, the patient may be administered a preventive regimen.

[0183] In certain exemplary embodiments, a therapeutically effective amount of the composition may be defined as about 10 -12 M to about 10 -6 M, for example, about 10 -7 The peptide concentration of M. This concentration can be administered at a systemic dose of about 0.01 mg / kg to about 100 mg / kg, or an equivalent dose calculated by body surface area. The dosage schedule can be optimized to maintain therapeutic concentrations at the target tissue, such as by a single daily or weekly administration, but also includes continuous administration (e.g., parenteral infusion and / or transdermal application).

[0184] In certain exemplary embodiments, the dosage of the composition can be provided at a "low", "medium" or "high" dosage level. In certain exemplary embodiments, a low dose of about 0.01 mg / kg / h to about 0.5 mg / kg / h, such as about 0.0001 mg / kg / h to about 0.1 mg / kg / h, can be provided. In certain exemplary embodiments, a medium dose of about 0.001 mg / kg / h to about 1.0 mg / kg / h, such as about 0.01 mg / kg / h to about 0.5 mg / kg / h, can be provided. In certain exemplary embodiments, a high dose of about 0.005 mg / kg / h to about 10 mg / kg / h, such as about 0.01 mg / kg / h to about 2 mg / kg / h, can be provided.

[0185] In certain exemplary embodiments, certain factors may influence the dosage and / or time required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and / or the presence of other diseases. In addition, treatment of a subject with a therapeutically effective amount of a therapeutic composition described herein may include a single treatment or a series of treatments.

[0186] The mammal treated according to certain exemplary methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cattle and horses; pet animals, such as dogs and cats; and / or laboratory animals, such as rats, mice and rabbits. In certain exemplary embodiments, the mammal can be a human.

[0187] Certain exemplary embodiments provide a bioactive material composition comprising a first D-biotin conjugated to a lysine located at the C-terminus of a water-soluble, cell-permeable, mitochondrial-targeted peptide sequence, wherein:

[0188] The peptide sequence targeting mitochondria contains a minimum of four amino acids and a maximum of six amino acids;

[0189] The peptide sequences that target mitochondria have a universal alternating aromatic-cationic motif;

[0190] The peptide sequence targeting mitochondria is selected from:

[0191] D-Arg-L-(2'6'-dimethylTyr)-L-Lys-L-Phe-NH2;

[0192] D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys-NH2;

[0193] D-Trp-D-Arg-D-Trp-D-Lys-OH;

[0194] L-Trp-L-Arg-L-Trp-L-Lys-NH2;

[0195] D-Trp-D-Arg-D-Trp-D-Lys-NH2; and / or

[0196] L-Trp-L-Arg-L-Trp-L-Lys-OH;

[0197] a second D-biotin conjugated to the N-terminal α-amine of the mitochondrial targeting peptide sequence;

[0198] The composition comprises one or more of the following:

[0199] D-biotin-D-Arg-L-(2'6'-dimethylTyr)-L-Lys-L-Phe-NH2; and

[0200] D-biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH;

[0201] First, D-biotin is conjugated to the ε-amine of the lysine at the C-terminus of the mitochondrial targeting peptide sequence;

[0202] The composition comprises one or more of the following:

[0203] D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH;

[0204] L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2; and

[0205] D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2;

[0206] A first D-biotin is conjugated to the ε-amine of a lysine at the C-terminus of the mitochondrial targeting peptide sequence, and a second D-biotin is conjugated to the N-terminal α-amine; and / or

[0207] The composition comprises one or more of the following:

[0208] D-biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2; and

[0209] D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2.

[0210] Certain exemplary embodiments provide a bioactive material composition comprising a first D-biotin conjugated to a lysine located at the N-terminus of a water-soluble, cell-permeable, mitochondrial-targeted peptide sequence, wherein the mitochondrial-targeted peptide sequence:

[0211] contains a minimum of four amino acids and a maximum of six amino acids; and

[0212] It has a common alternating aromatic-cationic motif.

[0213] Certain exemplary embodiments provide a composition of matter comprising:

[0214] Therapeutically effective preparations include:

[0215] One or more biologically active, water-soluble, cell-permeable, mitochondrial-targeted compounds selected from the group consisting of biotinylated peptides;

[0216] a pharmaceutically acceptable carrier for each of the one or more biologically active, water-soluble, cell-permeable, mitochondrial-targeted compounds;

[0217] One or more vitamins selected from the following:

[0218] Vitamin B1 (thiamine);

[0219] Vitamin B2 (riboflavin);

[0220] Vitamin B3 (niacin, niacinamide);

[0221] Vitamin B5 (pantothenic acid);

[0222] Vitamin B6 (pyridoxine);

[0223] Vitamin B7 (biotin);

[0224] Vitamin B9 (folic acid);

[0225] Vitamin B12 (cyanocobalamin); and

[0226] Vitamin C (ascorbic acid);

[0227] One or more metabolic supplements selected from the following:

[0228] Pyruvate;

[0229] Carnitine;

[0230] Acetylcarnitine;

[0231] Creatine;

[0232] α-ketoglutarate;

[0233] Alpha-lipoic acid;

[0234] Coenzyme Q;

[0235] Nicotinamide riboside; and

[0236] Nicotinamide mononucleotide; and / or

[0237] One or more amino acids selected from the group consisting of:

[0238] Leucine;

[0239] Isoleucine;

[0240] Valine;

[0241] Glutamine;

[0242] Serine;

[0243] Arginine;

[0244] Methionine;

[0245] Tryptophan;

[0246] Glycine;

[0247] Trimethylglycine;

[0248] β-Hydroxy-β-methylbutyrate; and

[0249] Taurine;

[0250] in:

[0251] Each biologically active, water-soluble, cell-permeable, mitochondrial-targeted compound in the biotinylated peptide group is defined as:

[0252] More than one amino acid arranged with a common alternating aromatic-cationic motif;

[0253] A minimum of four amino acids and a maximum of six amino acids;

[0254] a first D-biotin conjugated to a lysine located at the C-terminus or N-terminus of the biotinylated polypeptide;

[0255] The biotinylated polypeptide group consists of the following:

[0256] D-biotin-D-Arg-L-(2'6'-dimethylTyr)-L-Lys-L-Phe-NH2;

[0257] D-biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH;

[0258] D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH;

[0259] L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2;

[0260] D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2;

[0261] D-biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2; and

[0262] D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2.

[0263] Table 1 identifies certain exemplary short, water-soluble, alternating aromatic-cationic peptide sequences.

[0264] Table 1

[0265]

[0266] The alternating aromatic-cationic peptide sequences useful in certain exemplary methods described herein can be chemically synthesized by any of the methods described in the following U.S. patent documents, each of which is incorporated herein by reference in its entirety and / or for the portions thereof that describe or relate to the synthesis of peptides and / or the use of synthetic peptides:

[0267] U.S. Patent 4,749,742;

[0268] U.S. Patent 5,026,773;

[0269] U.S. Patent 7,576,061;

[0270] U.S. Patent 9,388,212;

[0271] U.S. Patent 9,695,214;

[0272] U.S. Patent 10,125,163;

[0273] U.S. Patent Application Publication No. 2019 / 0202861;

[0274] U.S. Patent Application Publication No. 2019 / 0015521; and

[0275] U.S. Patent Application Publication No. 2012 / 0149868.

[0276] Certain exemplary alternating aromatic-cationic peptide sequences listed in Table 1 are water soluble.

[0277] Certain exemplary alternating aromatic-cationic peptide sequences listed in Table 1 are water soluble, yet permeable to cell membranes.

[0278] Certain exemplary alternating aromatic-cationic peptide sequences listed in Table 1 are water soluble but can permeate the outer mitochondrial membrane.

[0279] Biotin( Figure 1A ) can be combined with alternating aromatic-cationic peptide sequences at the N-terminal α-amine ( Figure 1B ) or at the ε-amino group of the C-terminal lysine ( Figure 1C ) conjugation.

[0280] For certain exemplary embodiments, biotin may be conjugated to a polypeptide via chemical synthesis using one or more of the methods described in any of the following U.S. Patent Publications, each of which is incorporated herein by reference in its entirety and for its teachings of such methods:

[0281] U.S. Patent 5,391,711;

[0282] U.S. Patent 5,416,016; and

[0283] U.S. Patent Application Publication No.: 2006 / 0149035.

[0284] Certain exemplary embodiments may preferably use biotin to label the N-terminal α-amino group in the peptide. In certain exemplary embodiments, biotinylation can be easily achieved by activating the carboxyl group of biotin so that it reacts with the free amino group of the peptide. Certain exemplary embodiments may use biotinylation reagents such as D-biotin-N-hydroxy-succinimide ester or biotinyl-p-nitrophenyl ester. The activated ester can react with the amino group under mild conditions to incorporate the biotin residue into the desired molecule. Certain exemplary embodiments may use D-biotin-N-hydroxy-succinimide ester to biotinylate macromolecules and / or may use biotinyl-ε-nitrophenyl ester as a biotinylation reagent to biotinylate exogenous molecules. Certain exemplary embodiments may also use other reagents such as D-biotinyl-ε-aminocaproic acid N-hydroxysuccinimide ester, in which ε-aminocaproic acid is used as a spacer to connect to reduce steric hindrance.

[0285] For certain exemplary embodiments, biotinylation can be performed at the lysine ε-amino group to form biocytin. In certain exemplary embodiments, a biocytin-containing peptide can be achieved via solid phase synthesis using Fmoc-Lys (ε-biotinyl) at the C-terminal position, which can produce Lys (biotinyl) or biocytin after undergoing acid cleavage of the resin. For example, to produce biocytin, certain exemplary embodiments can utilize one or more methods described in U.S. Pat. No. 2,720,527, which is incorporated herein by reference in its entirety and its teachings on such methods.

[0286] Certain exemplary peptide-conjugated biotin molecules and the corresponding mitochondrial-targeted peptide sequences are listed in Table 2.

[0287] Table 2

[0288]

[0289] Cellular uptake and localization of peptide-conjugated biotin molecules were determined in HK-2 human renal epithelial cells and ARPE-19 human retinal pigment epithelial cells (ATCC, Manassas, VA). HK-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 1 g / L glucose and 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin. ARPE-19 cells were cultured in DMEM / F12 medium containing 1 g / L glucose and 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin. Cells were incubated at 37°C in a humidified incubator containing 5% CO2. HK-2 cells and ARPE-19 cells were cultured at 5×10 4 The initial density of cells was seeded in 35 mm culture dishes. FBS was removed from the culture medium for 3 days to deplete endogenous biotin in the cells. The cells were then incubated in serum-free medium containing biotin or peptide-conjugated biotin molecules for 12 hours (HK-2 cells) or 1 hour (ARPE-19 cells). All compounds were used at 1 μM.

[0290] Biotin uptake was determined using streptavidin binding. Streptavidin has a high affinity for biotin. By conjugating streptavidin with Alexa Fluor 594, biotin uptake can be visualized using fluorescence microscopy. Cells were fixed with 4% PFA for 10 minutes at RT, permeabilized in 0.1% triton X-100 / PBS for 10 minutes at RT, and incubated with 3.2 μg / ml streptavidin-Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA) and 5 μg / ml Hoechst 33342 (Novus Biologicals, Centennial, CO) for 30 minutes at RT. Hoechst 33342 is a fluorescent stain for labeling DNA, and it is used for nuclear staining. Live cell imaging buffer was added, and Alexa-594 fluorescence (Ex / Em=591 / 614nm) and Hoechst fluorescence (Ex / Em=490 / 461nm) were observed using a Nikon Eclipse Ti2 fluorescence microscope (100× oil immersion objective). Streptavidin fluorescence was quantified from 10 random fields of view for each sample by NIS-Elements imaging software (Nikon) and normalized to Hoechst fluorescence to account for the number of cells per field of view.

[0291] Figure 2Representative microscopic images of HK-2 human kidney epithelial cells after incubation for 12 hours with 1 μM free biotin or peptide-conjugated biotin molecules selected from Table 2 are shown. Intracellular biotin was visualized with streptavidin-Alexafluor 594 (red fluorescence). Cell nuclei were visualized with Hoechst 33342 dye (blue fluorescence) (1000× magnification). Minimal intracellular streptavidin fluorescence was observed when HK-2 cells were incubated with 1 μM biotin alone. In contrast, all peptide-conjugated biotin compounds (SPN05, SPN08, SPN09, SPN11, and SPN12) showed strong red fluorescence, indicating significant cellular uptake of biotin.

[0292] Figure 3 It is a graph quantifying the biotin uptake of HK-2 human kidney epithelial cells after incubation for 12 hours with 1 μM biotin or peptide-conjugated biotin molecules selected from Table 2. In HK-2 cells, peptide-conjugated biotin molecules deliver significantly higher intracellular biotin content compared to free biotin. Total streptavidin fluorescence normalized to cell number (Hoechst fluorescence) was calculated from 10 random fields of view and averaged for each treatment. All data were then normalized to free biotin treatment, with free biotin arbitrarily set to 1.0. Compared to free biotin, all peptide-conjugated biotin molecules resulted in significantly higher intracellular biotin (*P<0.05, ***P<0.001). The uptake of peptide-conjugated biotin molecules was 2 to 2.5 times higher than that of free biotin.

[0293] Figure 4 Representative microscopic images of ARPE-19 human retinal pigment epithelial cells after incubation for 1 hour with 1 μM biotin or peptide-conjugated biotin molecules selected from Table 2 are shown. Intracellular biotin was visualized with streptavidin-Alexafluor 594 (red fluorescence). Cell nuclei were visualized with Hoechst 33342 dye (blue fluorescence) (1000× magnification). All peptide-conjugated biotin compounds (SPN08, SPN09, SPN11, SPN12, SPN15, and SPN16) showed intense red staining with perinuclear distribution.

[0294] Figure 5is a graph showing biotin uptake by ARPE-19 human retinal pigment epithelial cells after incubation for 1 hour with 1 μM biotin or peptide-conjugated biotin molecules selected from Table 2. In ARPE-19 cells, peptide-conjugated biotin molecules delivered significantly higher intracellular biotin content compared to free biotin. The ratio of streptavidin fluorescence to Hoechst fluorescence was calculated from 10 random fields of view and averaged for each treatment. All data were then normalized for free biotin treatment, with free biotin arbitrarily set to 1.0. All peptide-conjugated biotin molecules resulted in significantly higher intracellular biotin staining compared to free biotin (***P<0.001). The uptake of peptide-conjugated biotin molecules was 1.5 to 2 times higher than that of free biotin.

[0295] These peptides enter the cells by simple diffusion and, after entering the cells, are observed exclusively in mitochondria. Figure 2 as well as Figure 4 Confirmed. For mitochondria, the distribution pattern shown by these peptides (a filamentous network starting from around the nucleus) is very obvious. Note that the nucleus is not stained, and the staining is not everywhere in the cell. This greatly reduces the chance of side effects of the compounds acting on other organelles.

[0296] These results demonstrate that conjugation of biotin to exemplary short, water-soluble, aromatic-cationic peptide sequences can significantly increase the cellular uptake of biotin. Figure 2 as well as Figure 4 The microscopic images in show that conjugation of biotin to SS-31, SPN02, SPN07, and SPN10 can greatly enhance the uptake of biotin by two different cell lines (kidney and retinal epithelial cells). All four short peptides follow an alternating aromatic-cationic motif, with SS-31 and SPN02 having a "cationic-aromatic-cationic-aromatic" sequence order, while SPN07 and SPN10 have an "aromatic-cationic-aromatic-cationic" sequence order. All exemplary peptides are water-soluble. SPN02 shows that a pentapeptide can act like a tetrapeptide and is water-soluble. SS-31 shows that non-naturally occurring amino acids (2'6'-dimethyl Tyr) can replace naturally occurring amino acids. These peptides also support the use of Tyr, Phe, or Trp as aromatic amino acids, and the use of Arg or Lys as cationic amino acids. The illustrative examples show that amino acids can be in either D- or L-configuration and that amidation of the C-terminus has no effect on mitochondrial delivery but can improve the stability of the peptide against degradation by carboxypeptidases in vivo.

[0297] Universal peptides of 4-6 amino acids, with alternating aromatic-cationic motifs composed of naturally occurring or non-naturally occurring amino acids in the D-configuration or L-configuration, with or without C-terminal amidation, can be used as water-soluble delivery vehicles to enhance the cellular uptake of biotin by mammalian cells.

[0298] Example 2 - Short aromatic-cationic peptide sequences selectively deliver biotin to the inner mitochondrial membrane

[0299] Certain exemplary alternating aromatic-cationic peptide sequences listed in Table 1 can be selectively targeted and localized to the inner mitochondrial membrane.

[0300] The perinuclear distribution pattern of all peptide-conjugated biotin molecules ( Figure 2 as well as Figure 4 ) indicate that they are localized to the filamentous mitochondrial network.

[0301] To show the mitochondrial localization of peptide-conjugated biotin molecules, ARPE-19 human retinal pigment epithelial cells were incubated with 1 μM SPN05, SPN12 or SPN15 in serum-free DMEM / F12 medium for 2 hours. The cells were fixed with 4% PFA at RT for 10 minutes, and then permeabilized with 0.1% Triton X-100 at RT for 10 minutes. The cells were blocked with 2% BSA (bovine serum albumin) at RT for 30 minutes, and then incubated with a primary antibody against cytochrome c oxidase subunit 4 (COX4), the major protein complex expressed on the inner membrane of the mitochondria. Rabbit polyclonal COX4 antibody (Invitrogen PA5-29992, Waltham, Ma) was used, and the dilution was 1:500 in 2% BSA at 4°C overnight. The cells were then incubated with secondary antibodies (goat anti-rabbit Alexa Fluor 488, 1:500 dilution, Invitrogen A-11008) and streptavidin-Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, Pa.) and 5 μg / ml Hoechst 33342 (Novus Biologicals, Centennial, Colo.) at RT for 30 min. Live cell imaging buffer was added, and Alexa Fluor-594 fluorescence, Alexa Fluor-488 fluorescence, and Hoechst fluorescence were observed using a Nikon Eclipse Ti2 fluorescence microscope (100× oil immersion objective).

[0302] Figure 6Co-localization of peptide-conjugated biotin molecules with cytochrome c oxidase is shown. The upper panel is a representative microscopic image of SPN15 (stained in red), cytochrome c oxidase (COX) (stained in green), and a merged image (yellow / orange). The lower panel shows images of SPN05 and SPN12 merged with COX staining (yellow / orange). All images are shown at 600× magnification.

[0303] These results show that short aromatic-cationic peptide sequences can be used as delivery vectors for biotin targeting to mitochondria.COX4 is a subunit of cytochrome c oxidase, which is the terminal complex (complex IV) of the electron transport chain.COX complex is the main regulatory site for oxidative phosphorylation of the inner mitochondrial membrane.The colocalization of biotin staining from SPN05, SPN12 and SPN15 and COX4 staining shows that these short aromatic-cationic peptide sequences can be used as sequences targeting mitochondria to deliver biotin to the inner mitochondrial membrane.

[0304] Example 3 - Peptide-conjugated biotin molecules can promote cell growth under prolonged serum starvation

[0305] Serum removal can cause a decrease in cellular ATP, cell cycle arrest and / or apoptosis. Serum deprivation can inhibit the ability of cultured cells to proliferate. Incubation with certain exemplary peptide-conjugated biotin molecules can promote cell viability under serum-free conditions.

[0306] In the first model, HK-2 cells were cultured in 96-well plates for 11 days in serum-free DMEM alone or in serum-free DMEM containing 10 nM SPN11 or SPN12. The medium was changed every 3 days.

[0307] Cell viability was measured using the Alamar Blue cell proliferation assay (Bio-Rad) according to the manufacturer's protocol. Alamar Blue uses resazurin to measure the reducing power of living cells. When reduced by cell metabolism, weakly fluorescent resazurin is reduced to highly fluorescent resazurin. In brief, alamar blue reagent (10 μl) was added to the culture medium and incubated at 37°C for 1 h. Fluorescence (Ex / Em530 / 590nm) from resazurin was detected using a microplate reader (SpectraMax iD3, Molecular Devices).

[0308] Figure 7 is a graph showing cell viability of HK-2 cells after 11 days of serum starvation. In the absence of serum, treatment with only 10 nM of SPN11 or SPN12 significantly increased cell viability by 35% (**P<0.005).

[0309] In the second model, ARPE-19 cells were grown in serum-free DMEM for 30 days in the absence or presence of peptide-conjugated biotin molecules. Serum-free medium supplemented with 10 nM of SPN12, SPN15 or SPN16 compounds (all at 10 nM) was replaced every 3 days. The medium was replaced every 3 days. Figure 8 are representative microscopic images showing that all three SPN compounds increased cell numbers after prolonged serum deprivation.

[0310] Fig. 9 It is shown that treatment with SPN12, SPN15 and SPN16 significantly doubled cell viability after 30 days of serum deprivation compared to control.Data represent mean±SEM of 6 samples from each treatment (**P<005; ***P<0.001 compared to control).

[0311] These results indicate that the addition of only 10 nM of peptide-conjugated biotin molecules can significantly double cell survival in serum-free conditions. These results show that exemplary mitochondrial-targeted biotin molecules are biologically active (ie, they alter cell biology), protecting cell survival in at least two different mammalian cell systems.

[0312] Example 4 - Peptide-conjugated biotin molecules increase cellular ATP content in serum-free and nutrient-deprived cultures

[0313] Serum deprivation can cause a decrease in cellular ATP. Certain exemplary peptide-conjugated biotin molecules can increase cellular ATP levels under serum-deprived and / or nutrient-deprived conditions.

[0314] Human kidney epithelial cells (HK-2) were cultured in DMEM containing 1 g / L glucose and 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin. The cells were incubated at 37°C in a humidified incubator containing 5% CO2. HK-2 cells were cultured at 5×10 3 The initial density of cells was seeded in 96-well culture plates. FBS was removed from the culture medium, and the cells were incubated for 7 days in serum-free DMEM alone (control group) or serum-free DMEM containing 10 nM biotin or peptide-conjugated biotin molecules. In each experiment, all treatments were performed with N=6. The culture medium was changed every 3 days.

[0315] Human retinal pigment epithelial cells (ARPE-19) were cultured in DMEM / F12 containing 1 g / L glucose and 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin. The cells were incubated at 37°C in a humidified incubator containing 5% CO2. ARPE-19 cells were plated at 5×103 The initial density of cells was seeded in 96-well culture plates. FBS was removed from the culture medium and the cells were incubated for 7 days in serum-free DMEM / F12 alone (control group) or DMEM / F12 containing 10 nM biotin or different peptide-conjugated biotin molecules. In each experiment, all treatments were performed with N=5-6. The culture medium was changed every 3 days.

[0316] According to the manufacturer's scheme, ATP is measured using ApoSENSOR ATP bioluminescence assay kit (BioVision). The kit utilizes luciferase catalysis to form light from ATP and luciferin. In brief, cells are accompanied by 100 μl nuclear release buffer and shaken gently at RT for 5 min. 10 μl ATP monitoring enzymes are added to the cell lysate, and luminescence is measured using a microplate reader (SpectraMax iD3, Molecular Devices). ATP levels are normalized for serum-free DMEM control group (arbitrarily set to 1.0).

[0317] Compared with free biotin, all peptide-conjugated biotin molecules resulted in higher ATP concentrations in HK-2 cells after 7 days under serum-free conditions ( Fig.10 ). Data shown are mean ± SEM from 6 samples for each treatment. SPN08, SPN09, SPN11, SPN12, SPN15 and SPN16 increased ATP levels by 35%-100% under serum-free conditions compared to free biotin (**P<0.005; ***P<0.001).

[0318] Peptide-conjugated biotin molecules also significantly increased ATP concentrations in ARPE-19 cells after 7 days of serum starvation. The data shown are the mean ± SEM of 6 samples from each treatment. Compared with free biotin, peptide-conjugated biotin molecules increased ATP levels by 60%-75% (***P<0.001) ( Fig.11 ).

[0319] The second model used an extreme starvation model, where HK-2 cells were cultured in 96-well plates in serum-free 5% DMEM in PBS (phosphate buffered saline) for 3 days. Cells were treated with 10 nM free biotin or peptide-conjugated biotin molecules. In each experiment, all treatments were performed with N=6.

[0320] All peptide-conjugated biotin molecules resulted in significantly higher ATP concentrations under extreme starvation conditions compared to free biotin. Data shown are mean ± SEM of 6 samples from each treatment. (*P<0.05; **P<0.01; ***P<0.001), with SPN11 and SPN12 doubling ATP content ( Fig.12 ).

[0321] These results show that exemplary mitochondria-targeted biotin molecules are biologically active, promoting mitochondrial ATP synthesis in at least two different mammalian cell systems under serum and nutrient deprivation conditions.

[0322] Example 5 - Peptide-conjugated biotin molecules can restore mitochondrial potential and prevent mitochondrial fragmentation caused by serum starvation

[0323] Serum deprivation can induce mitochondrial depolarization, followed by a decrease in ATP synthesis. Certain exemplary peptide-conjugated biotin molecules can restore mitochondrial potential in cells cultured in serum-free medium.

[0324] Electron transfer along the electron transport chain on the inner mitochondrial membrane results in the pumping of protons from the mitochondrial matrix to the intermembrane space. This creates a potential across the inner mitochondrial membrane, and the proton gradient is used to drive ATP synthase (complex V) to produce ATP from ADP. Withdrawal of serum or nutrients results in a drop in mitochondrial potential and reduced ATP production.

[0325] Human retinal pigment epithelial cells (ARPE-19) were cultured in DMEM / F12 medium containing 1 g / L glucose and 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin. The cells were incubated at 37°C in a humidified incubator containing 5% CO2. ARPE-19 cells were plated at 5×10 4 The cells were seeded at an initial density of 10 cells / mL in a 35 mm glass plate. FBS was removed from the culture medium for 3 days to deplete endogenous biotin. The cells were then incubated for 2 hours in serum-free DMEM / F12 alone (control group) or DMEM / F12 containing 1 μM SPN12 or SPN15.

[0326] To determine mitochondrial potential, live cells were incubated with 5nM tetramethylrhodamine methyl ester (TMRM, #70017, Biotium, Fremont, CA) in DMEM without phenol red. TMRM is a potential-dependent fluorescent dye (Ex / Em=548 / 573). TMRM accumulates in negatively charged polarized mitochondria and can be detected as red fluorescence. Total mitochondria were imaged with a mitochondrial potential-independent fluorescent dye (100nM MitoView Green; Ex / Em=490 / 523; #70054, Biotium, Fremont CA). Hoechst 33342 (10 μg / ml, Novus Biologicals, Centennial, CO) was added to stain the nucleus. Live cells were covered with phosphate buffer and imaged using a Nikon Eclipse Ti fluorescence microscope using a 60× water immersion objective.

[0327] Fig.13 (Top) Representative fluorescence microscopy images of ARPE-19 cells cultured in DMEM / F12 containing 10% FBS for 3 days. Fig.13 (Bottom) Representative images of ARPE-19 cells cultured in serum-free DMEM / F12 for 3 days. TMRM (red) shows a significant loss of mitochondrial potential under serum-free conditions compared to serum controls. Mitogreen (green) staining also shows that serum-free conditions cause mitochondrial fragmentation and aggregation in a perinuclear pattern without a clear filamentous network. When the images are merged, mitochondrial depolarization can be observed in all cells under serum-free conditions.

[0328] Fig.14 Representative fluorescence microscopy images of ARPE-19 cells cultured for 3 days in 10% FBS medium (serum control) or in serum-free medium alone (serum-free) or after incubation for 2 hours with 1 μM SPN12 or SPN15. The images shown are merged images of TMRM (red) and MitoGreen (green). Incubation for 2 hours with SPN compounds was sufficient to restore the mitochondrial potential of all cells and restore the filamentous mitochondrial network in ARPE-19 cells after 3 days of serum starvation.

[0329] These results show that after 3 days of serum starvation, mitochondria are completely depolarized and the mitochondrial network in cells is fragmented. In serum starvation, peptide-conjugated biotin molecules (SPN12 and SPN15) can rescue mitochondrial potential in about 2 hours. These results confirm that biotin conjugated to aromatic-cationic peptide sequences targets the mitochondrial inner membrane and is biologically active in protecting the transmitochondrial inner membrane potential.

[0330] Example 6 - Peptide-conjugated biotin molecules are more effective than free biotin in wound repair

[0331] Certain exemplary peptide-conjugated biotin molecules can accelerate wound healing in cell culture. The in vitro scratch assay is a simple and well-established method for measuring cell proliferation and migration in an in vitro "wound area". The assay involves creating a "scratch" in a cell monolayer with a pipette tip, and then examining the rate at which cells proliferate and migrate to close the scratch.

[0332] HK-2 cells were cultured in DMEM (1 g / L glucose) containing 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin. 5 Cells were plated in DMEM in 6-well plates at 37°C in a humidified incubator containing 5% CO2. To simulate the microenvironment of wounds in vitro, serum-free medium was used in the scratch assay. On the day of the scratch assay, the medium was changed to serum-free DMEM and a p1000 pipette tip was used to streak across the cell monolayer. The cells were washed to remove cell debris and replaced with DMEM alone (control) or DMEM containing 10 nM biotin or peptide-conjugated biotin molecules.

[0333] Immediately (day 0), the scratch area was examined using a Nikon Eclipse Ti2 fluorescent microscope. For each sample, six different visual fields were captured using a 4× objective lens. The scratch area (cell-free area) was calculated using ImageJ software (an open source Java image processing program inspired by NIHImage (National Institutes of Health)). The scratch area was checked again after 24 hours (day 1), and the area was normalized to the area determined for the same sample at day 0. All results were calculated as changes from untreated controls, and six visual fields were averaged.

[0334] Fig.15 24 hours after mechanical scratching, treatment with 10 nM SPN11 or SPN12 greatly accelerated the closure of HK-2 cells. For each treatment group, the scratch area determined after 24 hours was normalized to the area determined immediately after the scratch was applied (day 0). Biotin had a small effect on reducing the scratch area, while SPN11 and SPN12 almost completely closed the scratch area within 24 hours.

[0335] Fig.16The effects of biotin, SPN11 and SPN12 on the reduction of scratch area 24 hours after mechanical scratching of HK-2 cells are summarized. All treatments significantly accelerated the closure of the scratch area (***P<0.001 compared to the control). Compared with biotin, biotin-conjugated peptides (SPN11 and SPN12) were twice as efficient in reducing the scratch area (P<0.001).

[0336] Fig.17 It was shown that in another experiment in HK-2 cells, other peptide-conjugated biotin molecules SPN15 and SPN16 also significantly accelerated the closure of the scratch area after 24 hours (***P<0.001) and were more effective (three-fold) than free biotin (P<0.001).

[0337] Peptide-conjugated biotin molecules (SPN12, SPN15, and SPN16) also accelerated wound healing in ARPE-19 cells ( Fig.18 ). Fig.19 is a graph summarizing the effect of peptide-conjugated biotin molecules on scratch area (**P<0.01; ***P<0.001). Peptide-conjugated biotin molecules were significantly more effective (1.5-fold to 2-fold) compared to free biotin (P<0.001).

[0338] These results show that peptide-conjugated biotin molecules are not only biologically active, they are also superior to free biotin in promoting wound healing in vitro.

[0339] Example 7 - Peptide-conjugated biotin molecules can promote cell proliferation in the wound area

[0340] Re-epithelialization of the wound area requires cell proliferation at the wound edge, which may be impaired in most wounds due to the lack of blood flow to deliver nutrients and / or one or more growth factors that may be necessary for cell proliferation. Cell proliferation in the wound edge can be monitored by proliferating cell nuclear antigen (PCNA) staining. In order to simulate the microenvironment of the wound in vitro, serum-free medium was used for the scratch assay of cell culture.

[0341] According to the standard procedure proposed by IHC World, proliferating cells were identified by immunostaining with antibodies against proliferating cell nuclear antigen (PCNA). After determining the scratch area, PCNA staining was performed using the same cell plate. HK-2 cells were fixed with ethanol:methanol (1:1) at -20 ° C for 30 min, permeabilized with 0.1% riton X-100 / PBS at RT for 10 min, blocked with 2% goat serum at RT for 30 min, and incubated with mouse anti-PCNA primary antibody (Dako Agilent, Santa Clara, CA) and goat anti-mouse IgG-BI secondary antibody at RT for 30 min. The cells were then incubated with streptavidin-Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA) and Hoechst (Novus Biologicals, Centennial, CO) and imaged with a Nikon Eclipse Ti2 fluorescence microscope (20× objective). The nuclei of all cells were stained blue with Hoechst, but the nuclei of proliferating cells were stained red. The number of proliferating cells per field was quantified by the intensity of red nuclear staining and normalized to the intensity of blue nuclear staining (determined by Nikon NIS-Elements imaging software).

[0342] Fig. 20 Representative microscopic images (×40 magnification) of HK-2 human kidney epithelial cell monolayers in serum-free medium alone (control) or serum-free medium containing exemplary SPN compounds (10 nM) for 24 hours after mechanical scratching are shown. Cells positive for proliferating cell nuclear antigen are shown in red. All other cell nuclei are shown in blue. SPN11 and SPN12 increased the number of proliferating cells at the scratch edge compared to free biotin.

[0343] Fig.21 Figure 1 is a graph summarizing the number of proliferating cells in HK-2 human kidney epithelial cell monolayers in serum-free medium alone (control) or in serum-free medium supplemented with 10 nM biotin or peptide-conjugated biotin molecules for 24 hours after mechanical scratching. The number of proliferating cells was normalized to all nuclei. The effect of biotin was small and did not reach statistical significance. Incubation with SPN11 and SPN12 doubled the number of proliferating cells at the edge of the scratch compared to control or biotin (***P<0.001).

[0344] Fig. 22Representative microscopic images (40× magnification) of ARPE-19 human retinal pigment epithelial cell monolayers in serum-free medium alone (control) or in serum-free medium supplemented with 10 nM free biotin or peptide-conjugated biotin molecules for 24 hours after mechanical scratching are shown. Cells positive for proliferating cell nuclear antigen are shown in red. All other cell nuclei are shown in blue. Incubation with SPN08, SPN09, SPN11 or SPN12 increased the number of proliferating cells at the edge of the scratch, while 10 nM biotin had no effect on cell proliferation.

[0345] Fig.23 Figure 1 is a graph summarizing the number of proliferating cells in ARPE-19 human retinal pigment epithelial cell monolayers in serum-free medium alone (control) or in serum-free medium containing 10 nM biotin or peptide-conjugated biotin molecules for 24 hours after mechanical scratching. The number of proliferating cells was normalized to all nuclei. Incubation with each of SPN09, SPN11, SPN12, SPN15, and SPN16 significantly increased the percentage of proliferating cells at the edge of the scratch, but biotin itself had no effect (*P<0.05; **P<0.01; ***P<0.001 compared to control).

[0346] These results suggest that peptide-conjugated biotin molecules are more effective than biotin alone in promoting cell proliferation in a wound setting and this may be due to increased delivery of biotin to mitochondria.

[0347] Example 8 - Peptide-conjugated biotin molecules protect mitochondrial potential of cells at the wound edge

[0348] Rapidly dividing cells may need ATP to support the synthesis of important building blocks. Tissue damage can cause rapid depolarization of mitochondria, thereby impairing the production of ATP and / or leading to cell death. The loss of ATP may further impair cell proliferation and / or tissue repair. The mitochondrial potential of cells at the edge of the scratch area can be monitored using the cell-permeable potential-dependent dye TMRM (tetramethylrhodamine methyl ester). TMRM can accumulate in negatively charged polarized mitochondria and can be detected as red fluorescence.

[0349] In order to simulate the microenvironment of trauma in vitro, serum-free medium was used in the scratch assay. On the day of the scratch assay, the medium was replaced with serum-free DMEM, and a p1000 pipette tip was used to streak across the HK-2 cell monolayer. The cells were washed to remove debris and replaced with DMEM alone (control) or DMEM supplemented with 10 nM free biotin or peptide-conjugated biotin molecules for 24 hours. HK-2 cells were then incubated with 5 nM TMRM in DMEM without phenol red and incubated for 30 min. The cells were then covered with live cell imaging buffer and imaged using a Nikon Eclipse Ti2 fluorescence microscope (20× objective).

[0350] Fig.24 Representative microscopic images (200× magnification) of mitochondrial potential of HK-2 cells in serum-free medium alone (control) or serum-free medium containing 10 nM biotin, SPN11 or SPN12 for 24 hours after mechanical scratching are shown. Mitochondrial potential was detected with TMRM, which was stained red, while the nucleus was stained blue with Hoechst. Under control conditions, a few cells at the edge of the scratch showed TMRM staining, indicating that many cells underwent mitochondrial depolarization. Adding 10 nM of SPN11 or SPN12 increased the number of cells with good mitochondrial potential, while biotin had no effect.

[0351] Fig.25 It is a graph summarizing the ratio of mitochondrial potential (red fluorescence) normalized to the number of nuclei (blue fluorescence) of HK-2 cells in serum-free medium only (control) or in serum-free medium containing 10 nM biotin or peptide-conjugated biotin molecules for 24 hours after mechanical scratching. Incubation with SPN11 or SPN12 significantly increased the mitochondrial potential of cells at the scratch edge (***P<0.001 compared to control). The effect of free biotin did not reach statistical significance.

[0352] These results show that biotin-conjugated peptides (SPN11 and SPN12) are superior to free biotin in maintaining mitochondrial potential in a traumatic environment due to their targeted delivery to the inner mitochondrial membrane. This can translate into better ATP production and increased cell proliferation.

[0353] Example 9 - Uptake and distribution of SPN15 in the mouse retina after intraperitoneal administration

[0354] To show that peptide-conjugated biotin molecules can be administered in vivo, we determined the uptake and distribution of SPN15 in mice following intraperitoneal (ip) administration.

[0355] SPN15 (30 mg / kg) was administered ip to 13-month-old male mice (C57BL / 6J strain). At 2 hours after administration, mice were anesthetized with an overdose of ketamine / xylazine and the eyes were removed. After removal of the cornea and lens, the eye cups were fixed in 4% paraformaldehyde in 0.1 M Tris buffer for 1.25 hours. The eye cups were then rinsed 3 times in Tris buffer for 10 minutes each, and then cryoprotected in 10%, 20%, and 30% sucrose and then sectioned at 30 μm using a Leica 3050 cryostat. Sections were stored at -20°C until use.

[0356] To label the biotin on SPN15, sections were incubated with streptavidin-conjugated AlexaFluor 594 (1:500) from Jackson ImmunoResearch, West Grove, PA. The nuclei of the cells were stained using ToPro 3 (nuclear stain, 1:1000, Molecular Probes). Mitochondrial localization was determined using antibodies against Cox IV (cytochrome c oxidase subunit 4) (PA 529992, 1:300, Invitrogen) and Mitotracker Red (1:1500 in Tris, Invitrogen). All incubations were performed in a microwave (150 W), except for Cox IV and Mitotracker labeling, which was achieved by the following: primary antibody overnight at 4° C., followed by secondary antibody (AF488-conjugated donkey anti-rabbit, Jackson Immunoresearch). Sections were imaged at a resolution of 1024 × 1024 using an Olympus Fluoview 300 with helium, argon, and neon lasers using a 40× oil immersion lens.

[0357] Fig.26 (Left) Cryosections from control mice not administered peptide show nuclei labeled with TOPRO (blue) in different layers of the retina, where OS = outer segment; IS = inner segment; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; RGC = retinal ganglion cell.

[0358] Fig.26(Right) is a cryosection from a control mouse stained for COX4 (COX4 is a protein expressed on the inner membrane of mitochondria) (green), Mitotracker Red (which is a fluorescent dye that labels mitochondria) (red), and TOPRO (which labels the nucleus) (blue). COX4 staining for mitochondria is clearly visible in the photoreceptor IS, and INL, IPL, and RGCs, which have abundant mitochondria. Mitotracker Red staining in the photoreceptor OS was surprising because mitochondria are not present in OS.

[0359] Fig.26 (Middle figure) is a frozen section of a mouse 2 hours after SPN15 administration. Visualized by streptavidin Alexa Fluor 594 (red) staining, it is clear that SPN15 is taken up in different layers of the retina. SPN15 staining is concentrated in the inner plexiform layer and outer plexiform layer (IPL and OPL), i.e., the synapses between bipolar cells and amacrine cells in the ganglion, inner nuclear layer (INL) and photoreceptors. Streptavidin staining is co-localized with COX4 staining in RGC, IPL, INL and photoreceptor IS. The exception is the strong streptavidin staining in the photoreceptor OS where COX4 staining is not present. It is not clear why SPN15 is so heavily distributed in the OS containing densely-packed disks responsible for phototransduction.

[0360] Fig. 27 This is a magnified image of the photoreceptors IS and OS. The retinal pigment epithelium (RPE) is a single layer of cells above the OS. SPN15 staining can be clearly observed in the RPE cells.

[0361] These results indicate that SPN15 is rapidly absorbed after ip administration to live mice and is widely distributed throughout the retina. There are two sources of blood supply to the mammalian retina: the central retinal artery and the choroidal vessels. The central retinal artery begins at the optic nerve to supply the inner retina. Choroidal blood flow is essential for maintaining the outer retina (photoreceptors and retinal pigment epithelium (RPE)). Streptavidin staining showed that SPN15 is distributed through two vascular systems.

[0362] SPN15 staining is concentrated in the inner and outer plexiform layers (IPL and OPL). The outer nuclear layer (ONL) contains the cell bodies of rods and cones, and the inner nuclear layer (INL) contains the cell bodies of ganglion cells, horizontal cells, and amacrine cells. The OPL is where synapses between rods and cones and vertical bipolar cells and horizontal directional cells occur. The IPL acts as a relay station for bipolar cells and ganglion cells, and this is where information about visual images is transmitted to the brain along the optic nerve.

[0363] SPN15 is also concentrated in the inner segment (IS) which contains a cluster of very long thin mitochondria to support the metabolism of rod and cone photoreceptors. High concentrations of SPN15 in the IS were expected due to the density of mitochondria. These mitochondria play a role in the biosynthesis of many lipid discs in the outer segment (OS) which contains the visual pigment molecule (rhodopsin) used for visual transduction. The very high concentration of SPN15 in the OS was unexpected because mitochondria were completely absent. This was confirmed by the lack of COX4 staining in the OS. Mitotracker staining of the OS has been reported, but the reason is unclear and further study will be required. The concentration of SPN15 in the OS may be important for maintaining disc stability.

[0364] The OS disc is subject to light-induced oxidative damage and is normally engulfed by the retinal pigment epithelium (RPE) and degraded via lysosomal degradation, allowing the recycling of rhodopsin and the production of ketone bodies, which can be used as metabolic fuel by photoreceptors. The RPE is a single layer of cells between the OS and the choroid that also forms the blood-retinal barrier. The distribution of SPN15 in the RPE layer is highlighted in Fig. 27 middle.

[0365] These findings confirm that SPN15 can be distributed to areas of high mitochondrial density in the retina after systemic administration. The inventors believe that SPN15 and other peptide-conjugated biotin molecules can be beneficial to many ophthalmic diseases. The targeting of RPE shows that SPN15 can improve mitochondrial bioenergetics in aging and combat age-related macular degeneration and diabetic retinopathy. This is supported by the results of SPN compounds maintaining mitochondrial potential, increasing ATP synthesis, improving cell viability and increasing cell proliferation in ARPE-19 cells (human retinal pigment epithelial cell line). SPN15 can also increase the viability of retinal ganglion cells when the pressure from glaucoma increases and causes optic nerve damage.

[0366] definition

[0367] Where the following terms are used materially herein, the accompanying definitions apply. These terms and definitions are set forth without prejudice to the present application, and, consistent with the present application, the right to redefine these terms by amendment during the prosecution of the present application or any application claiming priority hereto is reserved. For purposes of interpreting the claims of any patent claiming priority hereto, every definition in that patent operates as an express disclaimer of subject matter outside that definition.

[0368] A (a) – at least one / one kind.

[0369] Activity – an action, movement, step and / or process or part thereof.

[0370] Adapt – to design, prepare, set up, arrange, shape, configure and / or make suitable and / or appropriate for a particular purpose, function, use and / or situation.

[0371] Add – to join and / or combine (something) with other things in order to increase size, amount, effect, and / or scope.

[0372] Administer – to give and / or apply.

[0373] Alcohol – Any of a class of compounds having the general formula ROH, in which R represents an alkyl group and –OH represents a hydroxyl group, as in methanol, CH3OH, or ethanol, C2H5OH.

[0374] Alternating – specifying or relating to every other one in a series.

[0375] Amine - Any of a group of nitrogen organic compounds, such as ethylamine C2H5NH2, which can be considered a derivative of ammonia in which one or more hydrogen atoms are replaced by a hydrocarbon group.

[0376] Amino acid – a compound in which at least one amino group and at least one carboxyl group are bound to the same carbon skeleton and the nitrogen atom of the amino group may form part of a ring, such compounds including the L-isomers and D-isomers of natural amino acids.

[0377] and – together with…

[0378] and / or – together with or optionally with.

[0379] Antibiotic – A substance, such as penicillin or erythromycin, produced by and / or derived from certain microorganisms (including fungi and bacteria) that can destroy or inhibit the growth of other microorganisms (especially bacteria). Antibiotics are widely used to prevent and treat infectious diseases.

[0380] Anticonvulsant – Any of a class of drugs used to prevent or eliminate seizures.

[0381] Antioxidant – a substance that reduces the production of reactive oxygen species, inhibits the oxidation of other substances, slows the oxidative deterioration of other substances, and / or is a scavenger of free radicals, reactive oxygen species, hydroxyl radicals, oxidized lipids, and / or lipid peroxidation products.

[0382] Any – one, some, every, and / or all, without specifying.

[0383] Instrument – ​​An implement or device used for a specific purpose.

[0384] About - approximately and / or nearly the same as, including every value in a series of two or more values; within an acceptable error for a particular value as determined by one of ordinary skill in the art, depending on how the particular value is measured or determined; within one standard deviation; when no specific margin of error is stated (e.g., the standard deviation of the mean given in a data graph or table), the range encompasses the stated value and the range that would be included by rounding the stated value up or down to take into account significant figures; and / or within ±20%, 15%, 10% or 5% of a specified value.

[0385] Aromatic – Organic compounds having unsaturated rings containing alternating double and single bonds, including those having benzene rings.

[0386] Arrange – To set in a specific order.

[0387] Associate / Relate – Connect, join together and / or relate.

[0388] at – in, on and / or near.

[0389] At least – not less than, and possibly more than, this applies to each value in any series of values ​​that follows the word “at least”.

[0390] Yes – actually exists.

[0391] Biologically active – formulated to alter cell biology.

[0392] Biotin – A crystalline, water-soluble vitamin of the vitamin B complex, C10H16O3N2S, sometimes called vitamin B7 and / or vitamin H.

[0393] Biotinidase – An enzyme that in humans is encoded by the BTD gene that readily cleaves and / or degrades biotinamide, releasing free biotin and an amine, and whose primary substrate is biocytin or biotin linked to lysine; biotinase is also able to degrade biotin esters.

[0394] Biotinylated - A polypeptide having a biotin molecule located at one or more of its termini.

[0395] C-terminus - (also called carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminus or COOH-terminus) the end of an amino acid chain (protein or polypeptide) terminating in a free carboxyl group (-COOH).

[0396] May - be able to, in at least some embodiments.

[0397] Carrier - a substance to which an active ingredient and / or agent is added as a means of applying and / or transferring the active ingredient and / or agent.

[0398] Cation – An ion or group of ions having a positive charge and characteristically moving toward the cathode in electrolysis.

[0399] Cause – bring about, inspire, contribute to, produce, initiate, be the cause of, lead to and / or affect.

[0400] Caused by —— caused by

[0401] Cell – The smallest unit of an organism capable of independent function, consisting of cytoplasm, usually a nucleus, and various other organelles, all surrounded by a semipermeable cell membrane.

[0402] Cell-permeable – able to move freely in and out of cells passively.

[0403] Cellular – of, relating to, or composed of cells.

[0404] Chemically-defined medium – Those media in which all components are identified at precise concentrations and in which the media do not contain added animal or human sera, growth factors, hormones, etc.

[0405] Composition of matter – a composition, reaction product, compound, mixture, preparation, material, and / or complex formed by humans and / or automation from two or more substances and / or elements.

[0406] Compound – A pure, macroscopically homogeneous substance composed of atoms or ions of two or more different elements in definite proportions that cannot be separated by physical means. Compounds usually have properties that differ from those of their constituent elements.

[0407] Comprising – including but not limited to.

[0408] Conceive – to imagine, conceptualize, form and / or develop in the mind.

[0409] Configure – to design, arrange, set up, shape and / or make suitable and / or appropriate for a particular purpose, function, use and / or situation.

[0410] Conjugation - The joining of two compounds together via a covalent bond.

[0411] Conservative substitutions - substitutions of amino acids in a polypeptide with functionally, structurally and / or chemically similar natural or non-natural amino acids, such as the following groups each contain natural amino acids that are conservative substitutions for one another:

[0412] 1) Glycine (Gly / G), Alanine (Ala / A);

[0413] 2) Isoleucine (Ile / I), leucine (Leu / L), methionine (Met / M), valine (Val / V);

[0414] 3) Phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W);

[0415] 4) Serine (Ser / S), threonine (Thr / T), cysteine ​​(Cys / C);

[0416] 5) Asparagine (Asn / N), glutamine (Gln / Q);

[0417] 6) Aspartic acid (Asp / D), Glutamic acid (Glu / E); and / or

[0418] 7) Arginine (Arg / R), lysine (Lys / K), and histidine (His / H).

[0419] And / or the following groups, each of which comprises natural amino acids that are conservative substitutions for each other:

[0420] 1) Non-polar: Ala, Val, Leu, Ile, Met, Pro (proline / P), Phe, Trp;

[0421] 2) Hydrophobicity: Val, Leu, Ile, Phe, Trp;

[0422] 3) Aliphatic: Ala, Val, Leu, Ile;

[0423] 4) Aromatic: Phe, Tyr, Trp, His;

[0424] 5) Uncharged polar or hydrophilic: Gly, Ala, Ser, Thr, Cys, Asn, Gln, Tyr;

[0425] 6) Containing aliphatic hydroxyl or sulfhydryl groups: Ser, Thr, Cys;

[0426] 7) Containing amide: Asn, Gln;

[0427] 8) Acidic: Asp, Glu;

[0428] 9) Basic: Lys, Arg, His; and / or

[0429] 10) Small: Gly, Ala, Ser, Cys.

[0430] and / or the following groups:

[0431] 1) Hydrophobicity: Val, Leu, Ile, Met, Phe, Trp;

[0432] 2) Aromatic: Phe, Tyr, Trp, His;

[0433] 3) Neutral hydrophilicity: Gly, Ala, Ser, Thr, Cys, Asn, Gln;

[0434] 4) Acidic: Asp, Glu;

[0435] 5) Basic: Lys, Arg, His; and / or

[0436] 6) Residues that affect backbone orientation: Pro, Gly.

[0437] Consists of – relates to a closed group (and its legal equivalent) that does not include anything not listed.

[0438] Take – eat and / or drink; take into the body by mouth for digestion and / or absorption.

[0439] Contains / Includes (including) but not limited to.

[0440] Transform – to change, adapt and / or alter.

[0441] Corresponding – related, associated, concomitant, similar in purpose and / or position, identical in every respect, and / or equal and / or identical in amount, quantity, order of magnitude, quality and / or degree.

[0442] Produce – make real.

[0443] Medium – A nutrient substance, such as agar gel or liquid medium, in which cultures of bacteria, fungi, animal cells, or plant cells are grown and / or cultured for scientific purposes.

[0444] Deficiency – a state and / or condition that deviates from a desired state and / or condition.

[0445] Define – establish meaning, relationships, outline, form, and / or structure; and / or describe and / or specify precisely and / or clearly.

[0446] Delivery – the act of transmitting and / or transferring.

[0447] Source – received, obtained and / or generated from a source and / or origin.

[0448] Determine – To discover, obtain, calculate, decide, infer, ascertain, and / or draw a conclusion, usually by investigation, reasoning, and / or calculation.

[0449] Apparatus – a machine, an article of manufacture and / or a collection thereof.

[0450] Each – Each one in the group is considered individually.

[0451] Effective – sufficient to arouse, inspire, provoke, and / or cause.

[0452] Implementation – a way of implementing, manifestation, and / or specific representation.

[0453] Estimate – (noun) a calculated value close to an actual value; (verb) to calculate and / or determine approximately and / or tentatively.

[0454] Excessive – exceeding normal, usual, reasonable and / or appropriate limits.

[0455] Exemplary—serving as an example, instance, and / or illustration, but not necessarily preferred or advantageous over other embodiments or features.

[0456] First – The initially referenced element in the collection.

[0457] Formulation – A pharmaceutical preparation for administration in a specific form such as a tablet, electuary, ointment or injection.

[0458] Derived from – used to indicate its source, origin and / or location.

[0459] Universal alternation - having a sequence of a logo or its inverse, for example aromatic-cationic or cationic-aromatic, such as a peptide sequence having ACACAC or CACACA residues (where A represents aromatic and C represents cationic).

[0460] generate – create, produce, give rise to, and / or cause to exist.

[0461] Greater than —— At least

[0462] group – (noun) a number of individuals and / or things considered together because of one or more similarities; (verb) to associate a number of individuals or things so that they are considered together and / or to associate a number of individuals or things together because of having similar characteristics.

[0463] Having – including but not limited to.

[0464] Health – The general condition of a living being at a given time; soundness, especially of body and / or mind; the absence of disease, injury, disorder and / or abnormality.

[0465] Improve – change to a better state and / or condition.

[0466] inadequate —— insufficient

[0467] Includes – has but is not limited to the following.

[0468] Initialize – prepare something for use and / or some future event.

[0469] Damage – damage and / or injury done to and / or sustained by persons and / or things.

[0470] Install – Connect or set in place and ready for use.

[0471] Ingestion – the act and / or instance of absorbing, ingesting, and / or receiving.

[0472] Intestinal Malabsorption – Defective and / or inadequate absorption of nutrients from the intestines.

[0473] Intramuscular – within a muscle.

[0474] Intranasal – inside the nose.

[0475] Intravenous – inside a vein.

[0476] Yes – actually exists.

[0477] Lysine – A crystalline, basic, essential amino acid produced primarily by hydrolysis of many proteins, H2N(CH2)4CH(NH2)COOH.

[0478] Sustain – Activities involving restoration and / or maintenance of program and / or system performance.

[0479] mammal – Any of various warm-blooded vertebrates of the class Mammalia, including humans, characterized by having hair covering the skin and, in females, mammary glands that secrete milk for nourishment of the young.

[0480] Mammalian – of and / or relating to mammals.

[0481] maximum – has the largest value.

[0482] may - in at least some embodiments, allowed and / or permitted.

[0483] Medical Condition – A physical and / or mental illness, injury, disorder and / or abnormality.

[0484] Drug - a substance suitable for alleviating at least one symptom of a medical condition and / or curing a medical condition.

[0485] Metabolic – of and / or relating to metabolism.

[0486] Metabolic Supplements – Naturally occurring compounds that enhance energy production.

[0487] Metabolism – Chemical processes that occur within living cells or organisms and are necessary for the maintenance of life. In metabolism, some substances are broken down to produce energy for life processes, while other substances necessary for life are synthesized.

[0488] Method – one or more actions performed on a subject to be transformed into a different state or thing and / or one or more actions associated with a specific instrument, which is not a basic principle and does not take precedence over all uses of the basic principle.

[0489] Minimum – has the lowest value.

[0490] Alleviate – make something less severe, serious, or painful.

[0491] Mitochondria – A spherical or elongated organelle in the cytoplasm of nearly all eukaryotic cells that contains genetic material and many enzymes important for cellular metabolism, including those responsible for converting food into usable energy.

[0492] Mitochondrial targeted - means that the indicated substance (eg, biotin) is transported to the mitochondria, such that upon entry into the cell, the substance will be preferentially localized to the mitochondria with substantially no distribution to other organelles or membranes.

[0493] More – A quantifier meaning greater in size, amount, scope, and / or degree.

[0494] Motif – a recurring pattern in a molecular sequence (usually nucleotides or amino acids in proteins) or in a molecular structure (usually corresponding to a specific biological activity).

[0495] N-terminus – (also called amino terminus, NH2-terminus, N-terminal end or amine terminus) is the beginning of a protein or polypeptide and refers to the free amine group (-NH2) at the end of a polypeptide. In peptides, the amine group is bonded to another carboxyl group in the protein to form a chain, but since the terminal amino acid of a protein is only attached at the carboxyl end, the remaining free amine group is called the N-terminus.

[0496] No – absence and / or lack of anything.

[0497] One – being and / or representing a single unit, individual, and / or entire thing, item, and / or object.

[0498] Operable – feasible and / or suitable, ready and / or fit for its intended use and / or effect.

[0499] Or – a conjunction used to indicate optional items, usually only before the last item in a group of optional items.

[0500] Oral – of and / or relating to the mouth.

[0501] Organ – A differentiated part of an organism that performs a specific function, such as an eye, wing, or leaf.

[0502] Patient – ​​A mammalian subject, such as a human subject.

[0503] Peptide - any of various natural or synthetic compounds comprising two or more amino acids covalently linked by at least one peptide bond and / or amide bond linking the carboxyl group of one amino acid to the amino group of another amino acid (including amino acid salts, such as pharmaceutically acceptable salts).

[0504] per – every one, every kind, and / or by…way.

[0505] Pharmaceutically acceptable - a substance (e.g., an active ingredient or an excipient) that is suitable for use in contact with the tissues and organs of a subject without undue irritation, allergic response, immunogenicity and / or toxicity, commensurate with a reasonable benefit / risk ratio, effective for its intended use, and / or compatible with the other ingredients of any composition in which it is contained; that is useful for preparing pharmaceutical compositions and is generally safe, nontoxic, and neither biologically nor otherwise undesirable, and includes being acceptable for veterinary use as well as human pharmaceutical use; and / or approved and / or approvable by regulatory agencies of the Federal and / or state governments, and / or equivalent agencies in countries other than the United States, and / or listed in the U.S. Pharmacopeia and / or other generally recognized pharmacopeias for use in animals and, more particularly, in humans.

[0506] Plural – The state of being plural and / or more than one.

[0507] Polypeptide – A chain of amino acids linked together by peptide bonds and having a molecular weight of up to about 10,000.

[0508] Polypeptides – generally peptides and proteins, where the left-hand end of the polypeptide sequence is called the "amino (N)-terminus" and the right-hand end of the sequence is called the "carboxyl (C)-terminus".

[0509] Part – a portion, component, section, percentage, proportion, and / or amount that is smaller than a larger whole.

[0510] Pre- – a prefix before an activity that occurs beforehand and / or in advance.

[0511] Predetermine – to determine, decide, and / or establish in advance.

[0512] Save – Store safely for later use.

[0513] Prevent – ​​to stop, avoid, resist, hinder, stop and / or prevent from happening.

[0514] Probability – A quantitative expression of the likelihood of an event occurring.

[0515] Product – Something that results from human and / or mechanical work.

[0516] Project – calculate, estimate, or predict.

[0517] Promote – to facilitate the progress and / or growth of; to further, advance, promote and / or market.

[0518] Protein – A linked sequence of amino acid residues consisting of 50 or more amino acid residues.

[0519] Provide – supply, furnish, give and / or make available.

[0520] Range – The degree of a set of values, and / or a measure of the amount and / or degree of variation.

[0521] Ratio – A relationship between two quantities expressed as the quotient of one quantity divided by the other.

[0522] Receive – to obtain, gather, acquire, and / or obtain as a signal.

[0523] Recommend – to suggest, praise, approve and / or approve.

[0524] Reduce – make less and / or smaller, and / or become less and / or smaller.

[0525] Regeneration – regrowth of lost and / or destroyed parts and / or organs.

[0526] Remove – eliminate, remove and / or lack, and / or remove from a place or position occupied.

[0527] Repair – restore to a desired condition.

[0528] Repeat – to do again and / or perform again.

[0529] Repeatedly – ​​over and over again; repeatedly.

[0530] Request – express a desire and / or request for…

[0531] result – (noun) the result and / or consequence of a particular action, operation and / or process; (verb) to cause the result and / or consequence of a particular action, operation and / or process.

[0532] Said - when used in a system or apparatus claim, an article indicating that a subsequent claim term has been previously introduced.

[0533] Second – The referenced element in the collection that follows the initial element.

[0534] Select – to make a choice or selection from among available options.

[0535] Selected – chosen from more than one possible option.

[0536] Sequence – An ordered collection.

[0537] Serum-free - lacking the clear, yellowish fluid obtained when whole blood is allowed to clot and separate into its solid and liquid components.

[0538] Collection – more than one related thing.

[0539] Solution – A homogeneous mixture of two or more substances which may be solids, liquids, gases or a combination of these.

[0540] Species – A class of individuals and / or objects grouped according to their common properties and given a common name; a division under a genus.

[0541] storage – The act of storing or the state of being stored.

[0542] Store – set aside, save, preserve, ensure and / or put away for future use.

[0543] Subcutaneous – Slightly beneath the skin.

[0544] Subject - an animal, including, but not limited to, a mammal, such as a primate (e.g., a human, chimpanzee, or monkey), a rodent (e.g., a rat, a mouse, a guinea pig, a gerbil, or a hamster), a lagomorph (e.g., a rabbit), a bovine (e.g., a cow), a porcine (e.g., a pig), an ovine (e.g., a sheep), an equine (e.g., a horse), a canine (e.g., a dog), and / or a feline (e.g., a cat).

[0545] Sublingual – Located under the tongue and / or on the lower surface of the tongue.

[0546] Substantially – to a great extent and / or degree.

[0547] suffer – feel pain or suffering; sustain injury and / or harm; endure, develop and / or become ill; be accurately diagnosed with.

[0548] Supplement – ​​(noun) a product containing one or more vitamins, herbs, enzymes, amino acids, and / or other ingredients that is taken to supplement an individual's diet by providing missing nutrients.

[0549] System – A mechanism, apparatus, machine, article of manufacture, method, composition of matter, data, and / or set of instructions designed to perform one or more specific functions.

[0550] Target – To interact with.

[0551] Treatment – ​​of or relating to the medical treatment of an illness, injury, disorder and / or abnormality.

[0552] Therapeutically effective amount – an amount of a substance that, when administered to a subject, is sufficient to prevent, reduce the risk of developing, delay the onset of, slow the progression of, and / or cause regression of the medical condition being treated, and / or alleviate some of the severity of the medical condition and / or one or more symptoms and / or complications of the condition, and / or elicit the biological and / or medical response of a cell, tissue, organ, system, animal, and / or human being that is being sought by a researcher, veterinarian, medical doctor, and / or clinician, at least in a portion of the subjects taking the substance.

[0553] Tissue – An aggregation of morphologically similar cells and associated intercellular materials that work together to carry out one or more specific functions in the body.

[0554] Tissue regeneration – The regrowth of tissue through cell proliferation, completely restoring a damaged section of tissue to its normal state.

[0555] Tissue repair - the restoration of tissue structure and function following injury, including tissue regeneration and tissue replacement.

[0556] Tissue replacement – ​​those types of healing that repair damaged tissue by building connective tissue or scar tissue.

[0557] Topical – The surface area of ​​the body, and usually the skin.

[0558] Transdermal – across or through the skin.

[0559] Transformation – A measurable change in: form, appearance, nature and / or characteristics.

[0560] Transmit – to send, provide, supply and / or furnish as a signal.

[0561] Transshipment – ​​To convey and / or move from one place to another.

[0562] Treat – To alleviate, ameliorate, inhibit the progression of, reverse, prevent and / or eliminate a medical condition and / or one or more causes, symptoms and / or complications associated with such condition; handle and / or deal with an individual and / or something.

[0563] Ingest – take in, take and / or use.

[0564] Use —— put into effect.

[0565] Used – adopted to achieve something.

[0566] via – through and / or by means of.

[0567] Vitamin – Any of various fat-soluble or water-soluble organic substances necessary in trace amounts for normal growth and activity of living organisms, such substances being synthesized by bacteria and / or plants and / or being obtained primarily by animals in their diet; and / or being an essential component, other than protein, carbohydrates, or fat, of the food of the animal to which the vitamin is administered, e.g., the B vitamins, including B1 (thiamine), B2 (riboflavin), B3 (niacin, niacinamide), B5 (pantothenic acid), B6 ​​(pyridoxine), B7 (biotin), B9 (folic acid), and B12 (cyanocobalamin), vitamin C (ascorbic acid), vitamin D, and vitamin K.

[0568] Water-soluble – capable of dissolving in water.

[0569] Weight – The force with which an entity is attracted by the Earth or other celestial body, equal to the product of the object’s mass and its gravitational acceleration; and / or a factor and / or value assigned to a number in a calculation (such as determining an average) so that the number’s effect on the calculation reflects its importance, significance, preference, influence, etc.

[0570] among – in this regard; and; and / or in addition to this.

[0571] with – accompanied by.

[0572] Region – an area and / or volume having at least one predetermined boundary.

[0573] Notice

[0574] Various substantial and specific practical and useful exemplary embodiments of the claimed subject matter are described herein in text and / or pictorial form, including the best mode (if any) known to the inventors, for one of ordinary skill in the art to implement the claimed subject matter. References herein to "in one embodiment," "in an embodiment," etc. do not necessarily refer to the same embodiment.

[0575] When reading this document, for a person of ordinary skill in the art, relying on his / her expertise and / or understanding of the entire field and without undue experimentation, any of the many possible changes (e.g., modification, expansion, modification, refinement and / or enhancement, etc.), details (e.g., types, aspects, nuances and / or fine processing, etc.) and / or equivalents (e.g., substitution, replacement, combination and / or substitution, etc.) of one or more embodiments described herein may become apparent. The inventors expect that any person of ordinary skill in the art, after obtaining authorization from the inventors, will appropriately implement such changes, details and / or equivalents, and the inventors therefore expect to implement the claimed subject matter differently from the specific description herein. Therefore, where permitted by law, the claimed subject matter includes and covers all changes, details and equivalents of the claimed subject matter. In addition, where permitted by law, every combination of the features, functions, activities, substances and / or structural elements described herein, and all possible changes, details and equivalents thereof, are included in the claimed subject matter, unless otherwise expressly indicated, expressly and specifically denied herein, or otherwise clearly inappropriate, inoperable or contradictory to the context.

[0576] Unless otherwise specified, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate one or more embodiments and does not limit the scope of any claimed subject matter. No language herein should be construed to indicate that any non-claimed subject matter is essential to the practice of the claimed subject matter.

[0577] Therefore, regardless of the contents of any part of this document (e.g., title, field, background, overview, description, abstract, drawings, etc.), unless expressly specified to the contrary (such as by an explicit definition, assertion or argument, etc.) or clearly contradicted by the context, with respect to any claims, whether claims of this document and / or any claims of any document claiming priority thereof, and whether originally filed or otherwise:

[0578] It is not required to include any specifically described features, functions, activities, materials or structural elements, any specific sequence of activities, any specific combination of materials, or any specific relationship between elements;

[0579] None of the features, functions, activities, materials, or structural elements described are “essential”; and

[0580] Within, between and in any of the embodiments described:

[0581] Any two or more of the substances described may be mixed, combined, reacted, separated, and / or isolated;

[0582] Any features, functions, activities, substances, components, and / or structural elements described, or any combination thereof, may be specifically included, duplicated, excluded, combined, reordered, reconfigured, integrated, and / or separated;

[0583] Any interrelationships, sequences and / or dependencies described between any features, functions, activities, substances, components and / or structural elements described may be omitted, altered, varied and / or reordered;

[0584] Any of the activities described can be performed manually, semi-automatically, and / or automatically;

[0585] Any of the activities described may be repeated, conducted by more than one entity, and / or carried out in more than one jurisdiction.

[0586] The terms "a," "an," "said," "the," and / or similar referents used in the context of describing various embodiments (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0587] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (ie, meaning "including, but not limited to,") unless otherwise noted.

[0588] When describing any quantity or range herein, unless otherwise expressly stated, the quantity or range is approximate. Unless otherwise indicated herein, the description of the range of values ​​herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range, and each individual value and each individual sub-range limited by such individual values ​​are incorporated into this specification as if they were individually stated herein. For example, if a range of 1 to 10 is described, the range includes all values ​​therebetween, such as, for example, 1.1, 2.5, 3.335, 5, 6.179, 8.9999, etc., and includes all sub-ranges therebetween, such as, for example, 1 to 3.65, 2.8 to 8.14, 1.93 to 9, etc., even if these specific values ​​or specific sub-ranges are not expressly stated.

[0589] When any phrase (ie, one or more words) appearing in a claim is followed by a drawing element number, that drawing element number is illustrative and does not limit the scope of the claim.

[0590] No claim or claim element in this document is intended to invoke 35 USC 112(f) unless the specific words "means for" are followed by a gerund.

[0591] Any information in any material (e.g., U.S. patents, U.S. patent applications, books, articles, web pages, etc.) incorporated herein by reference in its entirety is incorporated herein by reference in its entirety to the maximum extent permitted by law, but only to the extent that there is no conflict between such information and other definitions, statements, and / or drawings set forth herein. If such a conflict arises, including a conflict that would invalidate any claim herein or seek priority, any such conflicting information in such material is expressly not incorporated herein by reference. Any specific information in any part of any material incorporated herein by reference that identifies, criticizes, or compares any prior art is not incorporated herein by reference.

[0592] It is the applicant's intention that each and every claim issued herein and at any time during the prosecution of this application, and in any application claiming priority thereto, define a distinct patentable invention, and that if and as the scope of such claims changes during the prosecution thereof, the scope of such invention must change accordingly. Accordingly, any references in this document, and during the prosecution of any patent application to which this document relates, to any claimed subject matter are intended to reference only the exact language of the then-pending claimed subject matter at that particular point in time.

[0593] Accordingly, each section of this document (e.g., title, field, background, overview, description, abstract, drawings, etc.) should be regarded as illustrative in nature and not restrictive, except for any limitations provided by the claims themselves and the words used therein. The scope of subject matter protected by any claim of any patent issuing based on this document is defined and limited solely by the exact language of such claim (and all legal equivalents thereof) and any limitations provided by any words used in such claim, as informed by the context of this document when reasonably interpreted by one of ordinary skill in the relevant art.

Claims

1. A bioactive substance composition, comprising a first D-biotin, wherein the first D-biotin is conjugated to a water-soluble, cell-permeable, mitochondrial-targeted peptide sequence to form a biotinylated polypeptide, wherein the first D-biotin is conjugated to the ε-amine of the C-terminal lysine residue of the mitochondrial-targeted peptide sequence via an amide bond, wherein the mitochondrial-targeted peptide sequence is selected from the group consisting of: D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys-NH2; D-Trp-D-Arg-D-Trp-D-Lys-OH; L-Trp-L-Arg-L-Trp-L-Lys-NH2 (SEQ ID NO: 1); D-Trp-D-Arg-D-Trp-D-Lys-NH2; and L-Trp-L-Arg-L-Trp-L-Lys-OH (SEQ ID NO: 2).

2. The composition according to claim 1, wherein the biotinylated polypeptide is selected from the group consisting of: D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH; L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2; D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2; and L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-OH.

3. The composition of claim 1, wherein the second D-biotin is conjugated to the N-terminal α-amine of the mitochondrial targeting peptide sequence.

4. The composition according to claim 1, wherein the biotinylated polypeptide is selected from the group consisting of: D-biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH2; and D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH2, The biotinyl group in the above structural formula is the first D-biotin; the D-biotin in the above structural formula is the second D-biotin.

5. The composition according to claim 1, further comprising: A pharmaceutically acceptable carrier for the biotinylated polypeptide.

6. The composition according to claim 1, further comprising: One or more vitamins selected from the group consisting of: Vitamin B1; Vitamin B2; Vitamin B3; Vitamin B5; Vitamin B6; Vitamin B7; Vitamin B9; Vitamin B12; and Vitamin C.

7. The composition according to claim 1, further comprising: One or more metabolic supplements selected from the group consisting of: Pyruvate; Carnitine; Acetylcarnitine; Creatine; α-ketoglutarate; Alpha-lipoic acid; Coenzyme Q; Nicotinamide riboside; and Nicotinamide mononucleotide.

8. The composition according to claim 1, further comprising: One or more components selected from the following: Leucine; Isoleucine; Valine; Glutamine; Serine; Arginine; Methionine; Tryptophan; Glycine; Trimethylglycine; as well as Taurine.

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