Patent Application
20230002457
This disclosure relates to novel glucoregulatory peptides and their use for increasing glucose uptake and decreasing hepatic glucose production. The disclosure also relates to use of the peptides for treating diabetes.
Type 2 diabetes (T2D) is a complex multifactorial disorder resulting from insulin resistance in peripheral tissues such as skeletal muscle, and pancreatic β-cell dysfunction (Stumvol et al., 2005). According to a recent report from the International Diabetes Federation, in 2000, 151 million people aged between 18 to 99 years had T2D. In 2017, 425 million people were suffering from T2D (International Diabetes Federation, 2017). This disease is growing at a fast rate (Wild et al., 2004).
Salmon Protein Hydrolysate (SPH) has been tested in in vitro studies. SPHs may have effects on glucose uptake (Chevrier et al., 2015, Roblet et al., 2016) and hepatic glucose production (Chevrier et al., 2015). These bioactivities may be caused by the presence of low molecular (<1 kDa) bioactive peptides (BPs) in the SPHs (Chevrier et al., 2015, Roblet et al., 2016). Nevertheless, the identification of these BPs has never been done.
In this context, the inventors aimed to generate bioactive fractions useful for the treatment of T2D and to identify potential peptide sequences responsible for this bioactivity.
Provided herein are glucoregulatory peptides, compositions and combinations, and methods and uses thereof.
Accordingly an aspect of the present disclosure includes a peptide comprising (i) an amino acid sequence as shown in SEQ ID NO: 1 (IPVE); or (ii) a peptide comprising at least 50 or 75% sequence identity with the amino acid sequence as shown in SEQ ID NO: 1 that increases glucose uptake.
A further aspect includes a peptide comprising (i) an amino acid sequence as shown in any one of SEQ ID NO: 2 (IEGTL), SEQ ID NO: 3 (IVDI), or SEQ ID NO: 4 (VAPEEHPTL), or (ii) a peptide comprising at least 33, 40, 50, 67, 75, 80, or 90% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 2-4 that decreases hepatic glucose production.
In an embodiment, the peptide consists of the amino acid sequence of any one of SEQ ID NOs: 1-4.
In an embodiment, the peptide further comprises additional amino acids and is at least: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In an embodiment, the peptide is less than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids in length and comprises an amino acid sequence encoding a peptide that increases glucose uptake or decreases hepatic glucose production as described herein.
In an embodiment, the peptide is modified for cell permeability, stability or bioavailability.
Also provided is a composition comprising a peptide described herein and a carrier.
Further provided is a composition or combination comprising (i) at least two peptides described herein and optionally (ii) at least two, at least three or four peptides of any one of SEQ ID NOs: 1-4 and a carrier.
Yet a further aspect includes a method of increasing glucose uptake in a subject in need thereof, the method comprising administering to the subject a peptide, composition, or combination described herein.
Also provided is a method of decreasing hepatic glucose production in a subject in need thereof, the method comprising administering to the subject a peptide, composition, or combination described herein.
A further aspect includes a method of regulating glucose levels in a subject in need thereof, the method comprising administering to the subject a peptide, composition, or combination described herein.
Yet a further aspect includes a method of treating diabetes, optionally type 1 or type 2 diabetes, in a subject in need thereof, the method comprising administering to the subject a peptide, composition, or combination described herein.
In an embodiment, the subject is a diabetic subject.
In an embodiment, the subject is a mammal, optionally a dog, cat, horse, or human. In one embodiment, the subject is a human.
In an embodiment, the peptide, composition, or combination is administered or is for use orally or intravenously.
Also provided is a method of obtaining the peptides described herein, the method comprising:
Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
Embodiments are described below in relation to the drawings in which:
Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.
Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.
Terms of degree such as “about”, “substantially”, and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies. All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other.
A “therapeutically effective amount” is intended to mean that amount of a compound that is sufficient to treat, prevent or inhibit a disease or condition such as T2D and/or hyperglycemia. The amount of a given compound of the present disclosure that will correspond to such an amount will vary depending upon various factors, such as the given compound, the composition, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. In one embodiment, a “therapeutically effective amount” is an amount sufficient to have a desired effect on a subject, such as reducing hyperglycemia, increasing cellular glucose uptake and/or decreasing hepatic glucose production.
The disclosure provides peptides that have effects, such as to increase glucose uptake or decrease hepatic glucose production. The peptides described herein can increase glucose uptake or decrease hepatic glucose production in vitro or in vivo.
Glucose uptake can typically occur in one of two ways: passively (such as by facilitated diffusion) or actively (such as by secondary active transport).
An increase in glucose uptake by a cell refers to the increase in the amount, whether active or passive, of glucose that is taken up by the cell. Thus, reducing glucose uptake of a cell includes the reduction of uptake of glucose by the cell from the extracellular environment, e.g., from blood vessels or surrounding environment. Reducing glucose uptake includes a reduction or decrease in the uptake of glucose by at least some cells of a subject. The terms higher or increase refer to any increase above normal homeostatic levels. For example, control levels are in vitro, ex vivo, or in vivo levels prior to, or in the absence of, addition of an agent. Thus, the increase can be at least: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of increase in between as compared to native or control levels.
Peptides provided by the present disclosure are set out in Table 1 (SEQ ID NOs: 1-4).
As used herein, the term “peptide” refers to two or more amino acids linked by a peptide bond, and includes synthetic and natural peptides as well as peptides that are modified. Various lengths of peptides are contemplated herein.
The peptide can for example be 4-50 amino acids in length as amino acids may be added to the peptides in Table 1, optionally 7-30 amino acids in length or at least 25 or 30 amino acids in length. The peptide can for example be any number of amino acids between 4 and 30.
Accordingly, in one embodiment, the peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-4, or a conservatively substituted variant thereof.
Also provided is a peptide that is a part of a sequence described herein, optionally a part of any one of SEQ ID NOs: 1-4, that retains all or part of the biological activity.
The term “part” with reference to amino acids over 4 amino acids long means at least 4 contiguous amino acids of the reference sequence. The reference sequence can for example by any one of SEQ ID NOs: 1-4, or a conservatively substituted variant thereof.
In another embodiment, the peptide consists essentially of, or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 1-4, or a conservatively substituted variant thereof.
In another embodiment, the peptide comprises an amino acid sequence with at least: 25, 30, 33, 40, 50, 60, 67, 70, 75, 80, 90, 95 or 99% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 1-4 or a part thereof. In another embodiment, the peptide comprises or consists of an amino acid sequence comprising at least 4, 5, 6, 7 or 8 contiguous amino acids of SEQ ID NOs: 1-4.
In particular, described herein is the peptide “IPVE” comprising the amino acid sequence set out in SEQ ID NO: 1, or a conservatively substituted variant thereof, wherein the peptide increases glucose uptake.
Also described herein is the peptide “IEGTL” comprising the amino acid sequence set out in SEQ ID NO: 2, or a conservatively substituted variant thereof, wherein the peptide decreases hepatic glucose production.
Also described herein is the peptide “IVDI” comprising the amino acid sequence set out in SEQ ID NO: 3, or a conservatively substituted variant thereof, wherein the peptide decreases hepatic glucose production.
Also described herein is the peptide “VAPEEHPTL” comprising the amino acid sequence set out in SEQ ID NO: 4, or a conservatively substituted variant thereof, wherein the peptide decreases hepatic glucose production.
The peptide comprising any one of SEQ ID NOs: 1-4 may further comprise additional amino acids and be at least: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In an embodiment, the peptide is less than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids in length and comprises an amino acid sequence encoding a peptide that increases glucose uptake or decreases hepatic glucose production as described herein, such as any one of SEQ ID NOs: 1-4.
In one embodiment, the disclosure provides a peptide that has at least: 25, 30, 33, 40, 50, 60, 67, 70, 75, 80, 90, 95 or 99% sequence identity with any one of SEQ ID NOs: 1-4.
Sequence identity can be calculated according to methods known in the art. Sequence identity is optionally assessed by the algorithm of BLAST version 2.1 advanced search. BLAST is a series of programs that are available, for example, online from the National Institutes of Health. The advanced blast search is set to default parameters. (ie Matrix BLOSUM62; Gap existence cost 11; Per residue gap cost 1; Lambda ratio 0.85 default). References to BLAST searches are: Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403410; Gish, W. & States, D. J. (1993) “Identification of protein coding regions by database similarity search.” Nature Genet. 3:266272; Madden, T. L., Tatusov, R. L. & Zhang, J. (1996) “Applications of network BLAST server” Meth. Enzymol. 266:131_141; Altschul, S. F., Madden, T. L., Schiffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI_BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:33893402; Zhang, J. & Madden, T. L. (1997) “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation.” Genome Res. 7:649656. In addition, percent identity between two sequences may be determined by comparing a position in the first sequence with a corresponding position in the second sequence. When the compared positions are occupied by the same nucleotide or amino acid, as the case may be, the two sequences are conserved at that position. The degree of conservation between two sequences is often expressed, as it is here, as a percentage representing the ratio of the number of matching positions in the two sequences to the total number of positions compared.
As used herein, the term “conservatively substituted variant” refers to a variant with at least one conservative amino acid substitution. A “conservative amino acid substitution” as used herein, refers to the substitution of an amino acid with similar hydrophobicity, polarity, and R-chain length for one another. In a conservative amino acid substitution, one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Without the intention of being limited thereby, in one embodiment, the substitutions of amino acids are made that preserve the structure responsible for the ability of the peptide to increase glucose uptake or decrease hepatic glucose production as disclosed herein. Examples of conservative amino acid substitutions include:
In one embodiment, the peptides described herein are optionally modified for cell permeability, improved stability, and/or better bioavailability. These modifications include, without limitation, peptide conjugation, peptide cyclization, peptide end modification (e.g. N-acetylation or C-amidation, side chain modifications including the incorporation of non-coded amino acids or non-natural amino acids, N-amide nitrogen alkylation, chirality changes (incorporation of or replacement of L-amino acids with D-amino acids), generation of pseudopeptides (e.g. amide bond surrogates), or peptoids, or azapeptides or azatides). In one embodiment, the peptides described herein are modified by the addition of a lipophilic moiety.
The peptides described above may be prepared using recombinant DNA methods. These peptides may be purified and/or isolated to various degrees using techniques known in the art. Accordingly, nucleic acid molecules having a sequence which encodes a peptide of the disclosure may be incorporated according to procedures known in the art into an appropriate expression vector which ensures good expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used. The expression “vectors suitable for transformation of a host cell”, means that the expression vectors contain a nucleic acid molecule encoding a peptide of the disclosure and regulatory sequences, selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. “Operatively linked” is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.
The peptides may be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).
In one embodiment, the peptides may be modified with a detectable label. For example, in one embodiment the peptide is fluorescently, radioactively or immunologically labeled.
The peptides may also be modified with an enhancer moiety. Accordingly, another aspect provides a compound comprising a peptide described herein and an enhancer moiety. In one embodiment, the peptide is conjugated directly or indirectly to the enhancer moiety. As used herein, an enhancer moiety can increase or enhance the activity of the peptide. For example, the enhancer may be a permeability enhancer, a stability enhancer or a bioavailability enhancer. The enhancer moiety is optionally selected from a protein carrier, or a polymer carrier. In one embodiment, the enhancer moiety is a carrier protein, thereby forming a fusion protein. In another embodiment, the enhancer moiety is a PEG moiety.
The peptides may also be modified with a cell-penetrating moiety. As used herein, the term “cell-penetrating moiety” refers to a moiety that promotes cellular uptake of the peptide upon delivery to a target cell. Examples of cell-penetrating moieties include cell-penetrating peptides that translocate across the plasma membrane of eukaryotic cells at higher levels than passive diffusion. In one embodiment, the cell-penetrating peptide can translocate the nuclear membrane of a cell to enter the nucleus. In another embodiment, the cell-penetrating peptide can enter the nucleolus.
In one embodiment, the cell-penetrating peptide is an amphipathic peptide comprising both a hydrophilic (polar) domain and a hydrophobic (non-polar) domain. Cell-penetrating peptides can include sequences from membrane-interacting proteins such as signal peptides, transmembrane domains and antimicrobial peptides.
The peptides described herein can also be conjugated to a carrier protein, thereby forming a fusion protein.
The disclosure also includes nucleic acids that encode the peptides described herein. As used herein, the term “nucleic acids” includes isolated nucleic acids. In one embodiment, the disclosure provides nucleic acids that encode a peptide comprising or consisting of any one of SEQ ID NOs: 1-4 or any peptide described herein.
In another embodiment the disclosure provides a nucleic acid having at least 50, 60, 67, 70, 80, 90, 95 or 99% sequence identity with a nucleic acid that encodes a peptide comprising or consisting of any one of SEQ ID NOs: 1-4, a nucleic acid that hybridizes to a nucleic acid that encodes a peptide comprising or consisting of any one of SEQ ID NOs: 1-4 or any peptide described herein under at least moderately stringent hybridization or stringent hybridization conditions.
By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log 10 (Na+))+0.41(% (G+C)−600/l), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5× sodium chloride/sodium citrate (SSC)/5×Denhardt's solution/1.0% SDS at Tm (based on the above equation) −5° C., followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3×SSC at 42° C. It is understood however that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in Ausubel, 1989 and in Sambrook et al., 1989.
The disclosure further contemplates a vector comprising a nucleic acid described herein, optionally a recombinant expression vector containing a nucleic acid molecule that encodes a peptide of the disclosure and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. In an embodiment, the vector is a viral vector such as a retroviral, lentiviral, adenoviral or adeno-associated viral vector.
Recombinant expression vectors can be introduced into host cells to produce a transformed host cell for the purpose of producing the peptides described herein. The term “transformed host cell” is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the disclosure. The terms “transformed with”, “transfected with”, “transformation” and “transfection” are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
Also provided in another aspect is a recombinant cell expressing a peptide, nucleic acid, vector or compound described herein. In an embodiment, the cell is a bacterial cell, yeast cell, a mammalian cell, or a plant cell.
The disclosure also provides a composition comprising one or more of the peptides described herein. Also provided is a combination of two or more peptides described herein.
In one aspect, the composition comprises a peptide described herein and a carrier. In another embodiment, the composition or combination comprises at least two peptides described herein, optionally at least two, at least three or at least four peptides of SEQ ID NOs: 1-4 and a carrier.
In one embodiment, the carrier is a carrier acceptable for administration to humans.
As used herein, the term “acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Optional examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions and dextrose solution.
In one embodiment, a composition or combination described herein is formulated to be compatible with its intended route of administration. Examples of routes of administration include oral and parenteral, e.g. intravenous, intradermal, subcutaneous.
For example, in one embodiment, the active ingredient such as a peptide described herein is prepared with a carrier that will protect it against rapid elimination from the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
In one embodiment, oral or parenteral compositions or combinations are formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required carrier. The specification for the dosage unit forms are dictated by and directly dependent on the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and the limitations inherent in the art of preparing such an active ingredient for the treatment of individuals.
In one embodiment, the compositions described herein comprise an agent that enhances its function, such as, for example, insulin, other diabetes medication(s), omega 3, and/or polyphenols. The composition can also contain other active ingredients as necessary or beneficial for the particular indication being treated, optionally those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
The disclosure also provides uses and methods relating to the peptides, compositions, and combinations described herein.
Some of the peptides disclosed herein increase glucose uptake by cells, while others decrease hepatic glucose production. Accordingly, the peptides, compositions, and combinations of the present disclosure are useful for regulating blood glucose levels in a subject and optionally for treating diabetes in a subject. In one embodiment, the peptides described herein are useful for reducing hyperglycemia in a subject, optionally in a subject with T2D.
In one embodiment, the methods and uses include the administration to a subject or use in a subject of a peptide, composition or combination as described herein. In one embodiment, the subject is a diabetic subject. In one embodiment, the subject is a mammal, optionally a dog, cat, horse, or human. In one embodiment, the mammal is a human. In one embodiment, the peptide, composition, or combination is administered orally or intravenously. In another embodiment, the peptide, composition, or combination is for use orally or intravenously.
The disclosure provides a method of increasing glucose uptake in a subject in need thereof, the method comprising administering to the subject a peptide, composition, or combination described herein. Also provided is use of a peptide, composition, or combination disclosed herein to increase glucose uptake. In another embodiment, a peptide, composition, or combination disclosed herein is used in the manufacture of a medicament to increase glucose uptake. In yet another embodiment, a peptide, composition, or combination disclosed herein is for use in treating hyperglycemia.
As used herein, the term “hyperglycemia” refers to higher than normal fasting blood glucose concentration, optionally at least 125 mg/dL.
The disclosure further provides a method of decreasing hepatic glucose production in a subject in need thereof, the method comprising administering to the subject a peptide, composition, or combination described herein. Also provided is use of a peptide, composition, or combination disclosed herein to decrease hepatic glucose production. In another embodiment, a peptide, composition, or combination disclosed herein is used in the manufacture of a medicament to decrease hepatic glucose production. In yet another embodiment, a peptide, composition, or combination disclosed herein is for use in treating hepatic hyperglycemia.
The disclosure further provides a method of regulating glucose levels in a subject in need thereof, the method comprising administering to the subject a peptide, composition, or combination described herein. Also provided is use of a peptide, composition, or combination disclosed herein to regulate glucose levels. In another embodiment, a peptide, composition, or combination disclosed herein is used in the manufacture of a medicament to regulate glucose levels. In yet another embodiment, a peptide, composition, or combination disclosed herein is for use in regulating glucose levels.
Regulating glucose levels comprises the lowering of hyperglycemic glucose levels to a normoglycemic range. Optionally a normoglycemic range is 70-130 mg/dL. Optionally the glucose levels are maintained substantially in that normoglycemic, for example for at least: 30, 60, 90, 120, 180 or 240 minutes. For example, 30-60, 30-120, or 30-240 minutes.
The disclosure further provides a method of treating prediabetes in a subject in need thereof, the method comprising administering to the subject a peptide, composition, or combination described herein. Also provided is use of a peptide, composition, or combination disclosed herein to treat prediabetes. In another embodiment, a peptide, composition, or combination disclosed herein is used in the manufacture of a medicament to treat prediabetes. In yet another embodiment, a peptide, composition, or combination disclosed herein is for use in treating prediabetes.
Prediabetes is also referred to as “impaired glucose tolerance” or “impaired fasting glucose” and refers to blood glucose levels that are higher than a normal fasting blood glucose concentration, but are not high enough to be classified as type-2 diabetes. For example, from 100 to 125 mg/dL.
The disclosure further provides a method of treating diabetes, optionally type 1 or type 2 diabetes, in a subject in need thereof, the method comprising administering to the subject a peptide, composition, or combination described herein. Also provided is use of a peptide, composition, or combination disclosed herein to treat diabetes, optionally type 1 or type 2 diabetes. In another embodiment, a peptide, composition, or combination disclosed herein is used in the manufacture of a medicament to treat diabetes, optionally type 1 or type 2 diabetes. In yet another embodiment, a peptide, composition, or combination disclosed herein is for use in treating diabetes, optionally type 1 or type 2 diabetes.
The disclosure provides a method of treating metabolic syndrome in a subject in need thereof by reducing one or more of hyperglycemia and hypertension, the method comprising administering to the subject a peptide, composition, or combination described herein. Also provided is use of a peptide, composition, or combination disclosed herein to treat metabolic syndrome. In another embodiment, a peptide, composition, or combination disclosed herein is used in the manufacture of a medicament to treat metabolic syndrome. In yet another embodiment, a peptide, composition, or combination disclosed herein is for use in treating metabolic syndrome.
The disclosure further provides a method of obtaining the peptides disclosed herein. In one embodiment the method comprises providing a homogenized salmon frame or fraction, precipitating proteins from the homogenized fraction, hydrolyzing the precipitated proteins to form a hydrolyzed solution, filtering the hydrolyzed solution using an ultrafiltration membrane to generate a filtrate, and isolating the peptides from the filtrate, optionally isolating peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 into separate fractions.
In an embodiment, precipitating the proteins is performed by isoelectric precipitation at pH 4.5.
Hydrolysis of precipitated proteins may be carried out with a variety of enzymes known to a person skilled in the art.
In an embodiment, hydrolyzing the peptides precipitated proteins is performed using trypsin, chymotrypsin, pepsin, or any combination thereof.
Ultrafiltration may comprise several techniques known to a skilled person. In an embodiment, ultrafiltration comprises-pressure driven ultrafiltration. In another embodiment ultrafiltration comprises electrodialysis with an ultrafiltration membrane.
Ultrafiltration membranes comprise pores that may be, for example, 0.1 to 0.001 μm.
In an embodiment, the ultrafiltration membrane has a molecular weight cutoff of 1 kDa.
Peptide isolation may be performed using a variety of methods known to a skilled person and may include various chromatography methods such as size-exclusion, affinity purification, and ion exchange.
In an embodiment, isolating the peptides is performed using reverse-phase liquid chromatography.
Also provided is a method of producing a peptide as described herein comprising culturing a host cell that expresses a nucleic acid encoding the peptide, such as a peptide selected from SEQ ID NO: 1-4, and optionally isolating the peptide.
The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the disclosure. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
The following non-limiting examples are illustrative of the present disclosure:
Salmon Protein Hydrolysate (SPH) was produced according to the procedure described previously by Chevrier et al, (2015). Briefly, salmon frames were thawed, mechanically deboned and homogenized in a 1.0 M NaOH solution. Then, fish proteins were isoelectrically precipitated, recovered and a sequential hydrolysis was carried out with pepsin, then trypsin and chymotrypsin. Once hydrolysis was complete, the supernatant was filtered through a 5 μm pore size paper filter to remove any insoluble fat or protein. Finally, the filtrate was subsequently ultrafiltered using a Prep/Scale Tangential Flow Filtration (TFF) 2.5 ft2 cartridge with 1 kDa exclusion limit (Millipore Corporation, Bedford, Mass., USA). Permeates containing peptides with molecular weights <1 kDa were collected, demineralized by electrodialysis and freeze-dried.
One ultrafiltration membrane made of polyether sulfone (PES) with a molecular weight exclusion limit of 50 kDa, was purchased from Synder filtration (Vacaville, Calif., USA). Food grade Neosepta™ CMX-SB cationic membranes and Neosepta AMX-SB anionic membranes were obtained from Astom (Tokyo, Japan).
The electrodialysis cell used for the experiment was an MP type cell with an effective surface area of 100 cm2, manufactured by ElectroCell Systems AB Company (Täby, Sweden). The cell was composed of one anion-exchange membrane (AEM), one cation-exchange membranes (CEM), one ultrafiltration membrane (UFMs) with MWCO 50 kDa as illustrated in
First configuration: The first EDUF cell configuration, shown in
Second configuration: In a second configuration (
The spray dried SPH was diluted with demineralized water at a final protein concentration of 0.7% (w/v) and the EDUF fractionation was performed for 4 h. EDUF experiments were performed in batches for both cell configurations using constant electrical field strength of 6 V/cm (corresponding to a current density varying between 0.005 and 0.008 A/cm2 during the treatment). The system was run at controlled temperature (˜16° C.) to prevent growth of microorganisms (Suwal, Roblet, Amiot, & Bazinet, 2015). The pH of SPH and recovery (KCl) solutions were adjusted to pH 6 before each run with 0.1 N NaOH and/or 0.1N HCl and maintained constant thereafter (Roblet et al., 2016). For each treatment 10 mL-sample of SPH and recovery solutions were collected every hour before applying voltage and during the treatment to determine the peptide migration rate and their kinetics of migration. The electrical conductivity of the SPH feedstock and recovery solutions was maintained at a constant level by adding KCl, following the recommendations of Suwal et al, (2015) (Suwal et al., 2015). The current intensity, electrical potential differences of the AEM, CEM and UFMs were recorded every 30 min during EDUF treatment for both configurations. Finally, 3 replicates of each condition were performed. At the end of each replicate, a cleaning-in-place was performed according to the membrane manufacturer's instructions and the cell was dismantled before being reassembled.
The peptide concentration in recovered compartments of both configurations, during and after 4 h of EDUF separation were determined using micro bicinchoninic acid (μBCA) protein assay reagents (Pierce, Rockford, Ill., USA). Assays were conducted on microplates by mixing 150 μL of the sample with 150 μL of the working reagent followed by incubation at 37° C. during 2 h. The microplate was then cooled to room temperature and the absorbance was read at 562 nm on a microplate reader (Thermomax, Molecular devices, Sunnyvale, Calif.). Concentration was determined with a standard curve of bovine serum albumin (BSA) following the manufacturer's indications.
RP-UPLC analyses were performed using a 1290 Infinity™ II UPLC (Agilent Technologies, Santa Clara, Calif., USA). The equipment consisted of a binary pump (G7120A), a multisampler (G7167B), an in-line degasser and a variable wavelength detector (VWD G7114B) adjusted to 214 nm. Peptides were diluted to 0.5 mg/mL and filtered through 0.22 μm PVDF filter into a glass vial. The sample was loaded (5 μL) onto an Acquity UPLC CSH 130 1.7 μm C18 column (2.1 mm i.d.×150 mm) (Waters Corporation, Milford, Mass., USA). The column was operated at a flow rate of 400 μL/min at 45° C. A linear gradient consisting of solvent A (LC-MS grade water with 0.1% formic acid) and solvent B (LC-MS grade ACN with 0.1% formic acid) was applied with solvent B going from 2% to 25% in 50 min holding until 53 min, after, ramping to 90% and holding until 57 min, then back to initial conditions. Each sample was run in triplicate for statistical evaluation of technical reproducibility.
A hybrid ion mobility quadrupole TOF mass spectrometer (6560 high definition mass spectrometry (IM-Q-TOF), Agilent, Santa Clara, USA) was used to identify and quantify the relative abundances of the peptides. All LC-MS/MS experiments were acquired using Q-TOF. Signals were recorded in positive mode at Extended Dynamic Range, 2 Ghz, 3200 m/z with a scan range between 100-3200 m/z. Nitrogen was used as the drying gas at 13.0 L/min and 150° C., and as nebulizer gas at 30 psig. The capillary voltage was set at 3500 V. The nozzle voltage was set at 300 V and the fragmentor at 400 V. The instrument was calibrated using an ESI-L low concentration tuning mix (G1969-85000, Agilent Technologies, Santa Clara, Calif., USA). Data acquisition and analysis were done using the Agilent Mass Hunter™ Software package (LC/MS Data Acquisition, Version B.07.00 and Qualitative Analysis for IM-MS, Version B.07.00 with BioConfirm Software). Additional search was done using the Spectrum Mill MS Proteomics Workbench Rev B.05.00.180.
Evolutions of peptide concentration and relative abundance were subjected to a one way analysis of variance (ANOVA) using SAS software version 9.1 (SAS institute Inc., Cary, N.C., USA) with Tukey's post hoc tests at a significant P values of 0.05 for acceptance. In vitro glucose uptake assays were subjected to a one way ANOVA using SAS software version 9.1 (SAS institute Inc., Cary, N.C., USA) with Dunnett's post hoc test at a significant P values of 0.05 for acceptance. The relative energy consumption was compared by Student's t-test (P<0.05 as probability level for acceptance).
The evolution of peptide separation and concentration as a function of time in KCL compartments of both cationic and anionic EDUF configurations measured by micro-BCA method is represented in
As shown in
Without being bound by theory, the differences in abundance observed between both compartments (feed and recovery compartments) may have been principally due to the selectivity of the EDUF process. Inventors demonstrated significant differences in terms of abundances and total peaks between the feed and recovery compartments after 360 minutes of EDUF separation. Moreover, a number of compounds were not able to cross the ultrafiltration membrane (those marked with one asterisk) while, some compounds were concentrated in the recovery compartments (those marked with two asterisks). For configuration 1, forty compounds were concentrated in KCL. According to the potential anti-diabetic peptides previously identified (Henaux et al. 2019), three of these peptides were concentrated in the KCL−. For configuration 2, three compounds were recovered in peak 5 and thirteen in peak 6, among which two compounds (743.36 and 724.36 Da) were observed only in KCL+. For peak 5, three compounds were identified (598.37, 587.32 and 491.24 Da) but none were only found in the KCL+. Nevertheless, a compound (587.32 Da) was found only in the feed compartment (in both AFFC and AFFC2 fractions). Therefore, the increase in peak 5 area for KCL+ was due to the concentration by EDUF of compounds 598.37 and/or 491.24 Da.
Peptide synthesis and purification was performed. Peptides were synthesized by standard Fmoc solid-phase synthesis using 2-Cl-Trt resin [GB Fields, R. Hammami]. Briefly, the Fmoc protecting group was removed from the resin by two 10 min treatments with 20% piperidine in dimethylformamide (DMF, v/v) and amino acid coupling was performed with Fmoc-XaaOH (3 equivalents), 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU, 3 equivalents) and N-methylmorpholine (12 equivalents) in dimethylformamide (DMF, 2×30 min). The synthesized peptides were released by treating the resin with 20% hexafluoro-2-propanol (HFIP) in dichloromethane (DCM) for 30 min [206]. Side chain deprotection was achieved by treating the peptides with TFA/Triisopropylsilane (TIPS)/H2O (95:2.5:2.5, v/v/v) for 3 h. The resulting peptides were precipitated with cold ether and purified by RP-HPLC with a Shimadzu Prominence instrument (Columbia, Md., USA) on a Vydac 218 MS column (22.0×250 mm, 300 Å, 10 μm, C18) using 0.1% TFA/H2O (solvent A) and 0.1% TFA/CH3CN (solvent B) with a linear gradient of 10-100% solvent B for 20 min at 10 mL/min and UV detection at 220 nm and 254 nm. After freeze-drying, the purified peptides were characterized by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF) on an AB SCIEX 4800 Plus MALDI-TOF/TOF instrument using alpha-cyano-4-hydroxycinnamic acid as matrix.
Glucose uptake experiments were conducted as described by Roblet 2013. L6 skeletal muscle cells were grown in an α-minimum essential medium (α-MEM) containing 2% (v/v) fetal bovine serum (GBS) in an atmosphere of 5% CO2 at 37° C. [Tremblay 2001]. Cells were plated at 600,000 cells/plate in 24-well plates to obtain about 25,000 cells/mL. The cells were incubated 7 days, to reach their complete differentiation to myotubes (7 days post-plating). L6 myotubes were deprived of GBS for 3 h, with a α-MEM containing 0% of GBS. Then, the cells were incubated for 75 minutes, with 10 μl of EDUF fractions at a concentration of 1 μg/mL and 1 ng/mL. Finally, insulin was added (10 μl at 1.10-5M) for 45 min. Experiments were repeated 9 times, and each repetition was run in triplicate. After experimental treatments, cells were rinsed once with 37° C. HEPES-buffered solution (20 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, and 1 mM CaCl2)) and were subsequently incubated in HEPES-buffered solution containing 10 μM 2-deoxyglucose and 0.3 μCi/mL 2-deoxy-[3H] glucose for 8 minutes. Then, the cells were rinsed three times with 0.9% NaCl solution at 4° C. and then frozen. The next day, the cells were disrupted by adding 500 μl of a 50 mM NaOH solution. The radioactivity was determined by scintillation.
Hepatic glucose production experiments were conducted as described by Chevrier et al, (2015). Briefly, FAO rat hepatocytes were grown and maintained in monolayer culture in Roswell Park Memorial Institute medium (RPMI) containing 10% FBS in an atmosphere of 5% CO2 at 37° C. Cells were plated at 4.106 cells/plate. FAO cells were deprived with 1 mL/well of RPMI without FBS, and the EDUF's fractions were added at 10 μl/well with or without insulin at 1 nmol. Cells were washed three times with PBS, then incubated for 5 h (in an atmosphere of 5% CO2 at 37° C.) with the peptide fractions in the presence or absence of insulin at 1 nmol in a hepatic glucose production medium (glucose-free DMEM containing sodium bicarbonate at 3.7 g/L, 2 mmol sodium pyruvate, and 20 mmol sodium L-lactate. Glucose production was measured in the medium by using the Amplex Red Glucose/Glucose Oxidase Assay kit (Invitrogen). Results shown are the mean response of at least 6 independent experiments realized in triplicate.
IPVE was synthetized and its capacity to increase the glucose uptake was tested in basal and insulin stimulation conditions (
Next, the dose-response effect to IPVE was tested (
The capacity of IVDI, IEGTL, and VAPEEHPTL to regulate the hepatic glucose production (“HGP”) was investigated and results are presented in
The effect of the peptides, compositions, and combinations described herein on glucose regulation is shown in human patients with type-2 diabetes. Patients are divided into two groups: treatment and control. Both patient groups are administered the same type and quantity of food, wherein the food has a glycemic index value of 56 or higher. After the food is administered, patients in the treatment group are given a composition comprising one or more peptides of the amino acid sequence of any one of SEQ ID NOs: 1-4, while patients in the control group are given a placebo composition that does not have an effect on glucose uptake, production, or regulation. Following administration of the placebo or the treatment composition, the blood glucose levels of the patients in both groups is measured. On average, patients in the treatment group are observed have a lower blood glucose level than the patients in the control group.
While the present application has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
a calculated at pH 6.00
This disclosure claims the benefit of U.S. provisional application No. 62/944,794, filed Dec. 6, 2019, the contents of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/051665 | 12/4/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62944794 | Dec 2019 | US |
