Patent Grant
10376555
The present disclosure relates to cyclic peptide agonists of human galanin receptor 2 (GalR2) and galanin receptor 3 (GalR3) and methods of preparation and use in the treatment of spexin-deficient disorders thereof. More particularly, present disclosure provides cyclic peptide agonists of GalR2 and GalR3, which are useful in for the treatment of GalR2-related, GalR3-related, and/or spexin-deficient disorders, such as obesity.
Galanin receptors (GalRs) are G-protein-coupled receptors (GPCRs) that mediate signaling of galanin and other neuropeptides in the galanin family. So far, three subtypes of GalRs have been identified, namely GalR1, GalR2 and GalR3. With relatively low sequence similarity between one and other. However, their sequences are highly conserved among different vertebrate species.
Widely expressed in the brain and peripheral tissues, GalRs are involved in the regulation of a number of biological functions, including nociception, cognition, mood control, neuroendocrine function, reproduction, feeding control, energy and osmotic homeostasis, and metabolism. They have also been implicated in many human diseases, including neurological and metabolic disorders, inflammation, and cancers. However, understanding the roles of GalRs in health and disease, and clinical translation have been hindered by the lack of metabolically stable and receptor-specific probes.
Spexin (SPX) is a newly identified, GalR-cognate neuropeptide. Expressed centrally and peripherally, SPX can regulate gastrointestinal motility, adrenocortical cell proliferation, cardiovascular and renal function, nociception, reproduction, and feeding. It has also been implicated in a number of human disorders, including obesity, type-II diabetes, nonalcoholic fatty liver disease, and constipation.
Containing 14 amino acids and amidated at C-terminal, SPX is similar to the N-terminal sequence of galanin. Nevertheless, receptor recognition profiles of these two neuropeptides are different: galanin activates GalR1, GalR2 and GalR3. Whereas, SPX only activates GalR2 and GalR3. For GalR2, SPX and galanin have comparable activity; while for GalR3, SPX has much better activity than galanin.
Elucidating the biological role of GalRs in health and disease, and clinical translation of this knowledge to new methods of medical treatment has been hindered by the lack of metabolically stable and receptor-specific probes. Thus, there is a need to develop new agonists receptor selective probes of GalR2 and GalR3 with improved metabolic properties.
Accordingly, it is an object of the present disclosure to provide peptide agonists of GalR2 and GalR3 and methods of use thereof in the treatment of GalR2- and GalR3-related and spexin-deficient disorders, such as obesity.
In a first aspect of the present disclosure, there is provided herein is a peptide comprising a sequence having at least 92.8% homology with SEQ ID NO: 23 or SEQ ID NO: 24; or having at least 93.7% homology with SEQ ID NO: 25 or a therapeutically effective salt thereof, wherein R is hydrogen, alkyl, or acyl with the proviso that SEQ ID NO: 23 must contain cysteine at position one and position thirteen of SEQ ID NO: 23; SEQ ID NO: 24 must contain cysteine at position four and position thirteen of SEQ ID NO: 25; SEQ ID NO: 25 must contain cysteines at position one and position thirteen of SEQ ID NO: 25.
In a first embodiment of the first aspect, provided herein is the peptide of the first aspect, wherein R is hydrogen, (C1-C6)alkyl, or (C1-C6)acyl.
In a second embodiment of the first aspect, provided herein is the peptide of the first aspect, wherein R is hydrogen or (C1-C3)acyl.
In a third embodiment of the first aspect, provided herein is the peptide of the first aspect, wherein R is hydrogen or acetyl.
In a fourth embodiment of the first aspect, provided herein is the peptide of the first aspect, wherein the peptide comprises SEQ ID NO: 23; SEQ ID NO: 24; or SEQ ID NO: 25.
In a fifth embodiment of the first aspect, provided herein is the peptide of the fourth embodiment of the first aspect, wherein R is hydrogen, (C1-C6)alkyl, or (C1-C6)acyl.
In a sixth embodiment of the first aspect, provided herein is the peptide of the fourth embodiment of the first aspect, wherein R is hydrogen or (C1-C3)acyl,
In a seventh embodiment of the first aspect, provided herein is the peptide of the fourth embodiment of the first aspect, wherein R is hydrogen or acetyl.
In a second aspect of the present disclosure, provided herein is a peptide consisting of SEQ ID NO: 23; SEQ ID NO: 24; or SEQ ID NO: 25 or a therapeutically effective salt thereof, wherein R is hydrogen, alkyl, or acyl.
In a first embodiment of the second aspect, provided herein is the peptide of the second aspect, wherein R is hydrogen, (C1-C6)alkyl, or (C1-C6)acyl.
In a second embodiment of the second aspect, provided herein is the peptide of the second aspect, wherein the peptide is SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; or SEQ ID NO: 11.
In a third aspect of the present disclosure, provided herein is a pharmaceutical composition comprising a peptide of the first aspect and at least one pharmaceutically acceptable excipient.
In a fourth aspect of the present disclosure, provided herein is a method of treating a spexin-deficient disorder in a patient in need thereof comprising the step of administering a therapeutically effective amount of a peptide of the first aspect to the patient.
In a first embodiment of the fourth aspect, provided herein is the method of the fourth aspect, wherein the spexin-deficient disorder is obesity, type-II diabetes, nonalcoholic fatty liver disease or constipation.
In a second embodiment of the fourth aspect, provided herein is the method of the fourth aspect, wherein the peptide comprises SEQ ID NO: 23; SEQ ID NO: 24; or SEQ ID NO: 25; or a therapeutically acceptable salt thereof.
In a third embodiment of the fourth aspect, provided herein is the method of the fourth aspect, wherein the peptide is SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; or SEQ ID NO: 11; or a therapeutically acceptable salt thereof.
In a fourth embodiment of the fourth aspect, provided herein is the method of the fourth aspect, wherein the spexin-deficient disorder is obesity.
In a fifth embodiment of the fourth aspect, provided herein is the method of the first embodiment of the fourth aspect, wherein the peptide comprises SEQ ID NO: 23; SEQ ID NO: 24; or SEQ ID NO: 25; or a therapeutically acceptable salt thereof.
In a sixth embodiment of the fourth aspect, provided herein is the method of the first embodiment of the fourth aspect, wherein the peptide is SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; or SEQ ID NO: 11; or a therapeutically acceptable salt thereof.
In a seventh embodiment of the fourth aspect, provided herein is the method of the fourth aspect, wherein the peptide is administered to the patient intraperitoneally.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively, and any and all combinations or any two or more of the steps or features.
The term “amino acid” refers to naturally occurring and non-natural amino acids. Naturally occurring amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.
An “amino terminus modification group” refers to any molecule that can be attached to the amino terminus of a polypeptide. Similarly, a “carboxy terminus modification group” refers to any molecule that can be attached to the carboxy terminus of a polypeptide. Terminus modification groups include, but are not limited to, various water soluble polymers (such as polyethylene glycol (PEG)), peptides or proteins such as serum albumin, albumin binding domain or other moieties that increase serum half-life of peptides.
As used herein, the term “variant” refers to a polynucleotide or nucleic acid differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide.
A variant can, for example, comprise the amino acid sequence of the parent polypeptide sequence with at least one conservative amino acid substitution. Alternatively or additionally, the variant can comprise the amino acid sequence of the parent polypeptide sequence with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the variant, such that the biological activity of the variant is increased as compared to the parent polypeptide.
Amino acid substitutions of the described polypeptides can be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic/negatively charged polar amino acid substituted for another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino acid (e.g. Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., Ile, Thr, and Val), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), an amino acid with a side chain of a certain size with another amino acid with a side chain of similar size, etc.
The terms “percentage homology” and “percentage sequence identity”, when used in reference to a polypeptide or polynucleotide sequence, are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680; Higgins et al. 1996, Methods Enzymol. 266:383-402; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Altschul et al., 1993, Nature Genetics 3:266-272). In certain embodiments, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”) which is well known in the art (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402).
The term “alkyl” is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term “alkyl” refers to unsubstituted alkyl groups that do not contain heteroatoms. In certain embodiments, a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and alternatively, about 20 or fewer, about 10 or fewer, about 9 or fewer, about 8 or fewer, about 7 or fewer, about 6 or fewer, about 5 or fewer, about 4 or fewer, about 3 or fewer, or about 2 or fewer carbon atoms. Thus, the term includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The term also includes branched chain isomers of straight chain alkyl groups, including but not limited to, the following which are provided by way of example: —CH(CH3)2, —CH(CH3)(CH2CH3), —CH(CH2CH3)2, —C(CH3)3, —C(CH2CH3)3, —CH2CH(CH3)2, —CH2CH(CH3)(CH2CH3), —CH2CH(CH2CH3)2, —CH2C(CH3)3, —CH2C(CH2CH3)3, —CH(CH3)CH(CH3)(CH2CH3), —CH2CH2CH(CH3)2, —CH2CH2CH(CH3)(CH2CH3), —CH2CH2CH(CH2CH3)2, —CH2CH2C(CH3)3, —CH2CH2C(CH2CH3)3, —CH(CH3)CH2CH(CH3)2, —CH(CH3)CH(CH3)CH(CH3)2, —CH(CH2CH3)CH(CH3)CH(CH3)(CH2CH3), and others. Likewise, cycloalkyls have from about 3 to about 10, about 3 to about 6, or about 3 to about 5 carbon atoms in their ring structure, and alternatively about 3, 4, 5, 6, or 7 carbons in the ring structure. Thus, the term also includes cyclic alkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and such rings substituted with straight and branched chain alkyl groups as defined above.
The term “acyl” is art-recognized and refers to a group represented by the general formula:
wherein alkyl is as defined above. As used herein, “Ac” represents acetyl (i.e., CH3(C═O)—).
As used herein, the term “therapeutically effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or disorder or one or more signs or symptoms associated with a disease or disorder. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. A therapeutically effective amount can be given in one or more administrations. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder.
As used herein, an “isolated” or “purified” polypeptide or peptide is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the agent is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. For example, an isolated peptide would be free of materials that would interfere with diagnostic or therapeutic uses of the agent. Such interfering materials may include enzymes, hormones and other proteinaceous and nonproteinaceous solutes.
As used herein, the terms “treat”, “treating”, “treatment”, and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. In certain embodiments, treatment includes prevention of a disorder or condition, and/or symptoms associated therewith. The term “prevention” or “prevent” as used herein refers to any action that inhibits or at least delays the development of a disorder, condition, or symptoms associated therewith. Prevention can include primary, secondary and tertiary prevention levels, wherein: a) primary prevention avoids the development of a disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing opportunities for interventions to prevent progression of the disease and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established disease by restoring function and reducing disease-related complications.
As used herein the term “patient” refers to a human or non-human subject (rodent, non-human primate, canine, bovine, ovine, equine, feline, etc) to be treated.
Cysteine residues having side chains forming disulfide linkages present in the polypeptides described herein can be represented by C.
Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
Furthermore, throughout the specification and claims, unless the context requires otherwise, the word “include” or variations such as “includes” or “including”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.
Other aspects and advantages of the invention will be apparent to those skilled in the art from a review of the ensuing description.
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which:
The present disclosure is not to be limited in scope by any of the specific embodiments described herein. The following embodiments are presented for exemplification only.
The solution structure of human spexin (hSPX) was investigated by an approach that combines computational and biophysical analysis, which revealed an unexpected β-turn-helix-β-turn βαβ conformation of hSPX that, is different from the previously reported α-helix conformation of goldfish spexin (gSPX). In this conformation, two β turns are connected by a short, nascent a helix, and the N- and C-terminal residues are in extended form and close to each other. Following this finding, cyclic SPX analogs (CSAs) were designed and synthesized, which were expected to adopt the same βαβ conformation as demonstrated by MD simulation and NMR analysis and bear the disulfide bond between the N- and C-terminal residues. These CSAs were proven to be potent activators of GalR2 and GalR3 in serum response element-luciferase (SRE-Luc) assay.
The invention suggested that GalR2 and GalR3 recognize SPX in this conformation. In short, guided by conformational analysis, a novel class of cyclic peptide agonists of GalR2 and GalR3 with improved serum stability and in vivo efficacy were identified.
The investigations of galanin receptors (GalRs) function and the translation of these functions into clinically relevant applications have been hindered by lack of metabolically stable and receptor-specific agonists. Spexin (SPX) is a newly identified neuropeptide which selectively activates GalR2 and GalR3.
In a first aspect of the present disclosure there is provided a cyclic peptide agonist of galanin receptor 2 and galanin receptor 3 comprising a sequence having at least 92.8% homology with SEQ ID NO: 23 or SEQ ID NO: 24; or having at least 93.7% homology with SEQ ID NO: 25 or a therapeutically effective salt thereof, wherein R is hydrogen, alkyl, or acyl with the proviso that SEQ ID NO: 23 must contain cysteine at position one and position thirteen of SEQ ID NO: 23; SEQ ID NO: 24 must contain cysteine at position four and position thirteen of SEQ ID NO: 25; and SEQ ID NO: 25 must contain cysteines at position one and position thirteen of SEQ ID NO: 25.
In certain embodiments the sequence having at least 92.8% homology with SEQ ID NO: 23 or SEQ ID NO: 24; or having at least 93.7% homology with SEQ ID NO: 25 includes a single non-conservative amino acid substitution or a single conservative amino acid substitution.
In certain embodiments, the N-terminal amine of peptides described herein can be represented by the following structure:
Wherein R1 is an amino acid side chain; —NHR represents the N-terminal amine; and R is hydrogen, alkyl, or acyl.
In certain embodiments, the alkyl can be (C1-C12)alkyl or (C3-C6)cyclcoalkyl. In other embodiments, the alkyl group can be (C1-C12)alkyl, (C1-C11)alkyl, (C1-C10)alkyl, (C1-C9)alkyl, (C1-C8)alkyl, (C1-C7)alkyl, (C1-C6)alkyl, (C1-C5)alkyl, (C1-C4)alkyl, (C1-C3)alkyl, (C3-C5)cyclcoalkyl, or (C3-C4)cyclcoalkyl.
In certain embodiments, the acyl can be (C1-C12)alkyl(C═O)— or (C3-C6)cyclcoalkyl(C═O)—. In other embodiments, the alkyl group can be (C1-C12)alkyl(C═O)—, (C1-C11)alkyl(C═O)—, (C1-C10)alkyl(C═O)—, (C1-C9)alkyl(C═O)—, (C1-C8)alkyl(C═O)—, (C1-C7)alkyl(C═O)—, (C1-C6)alkyl(C═O)—, (C1-C5)alkyl(C═O)—, (C1-C4)alkyl(C═O)—, (C1-C3)alkyl(C═O)—, (C3-C5)cyclcoalkyl(C═O)—, or (C3-C4)cyclcoalkyl(C═O)—.
R1 can be the side chain of any naturally occurring amino acid.
In instances where R is hydrogen, the N-terminal amino acid can be covalently attached to an amino terminus modification group, such as PEG (e.g., PEG having a molecular weight of about 1,000, about 3,000, about 5,000, about 10,000, or about 20,000 amu), human serum albumin, or an albumin binding domain.
In a first embodiment of the first aspect of the present disclosure there is provided the peptide wherein said compound is LH101 (SEQ ID NO: 6), LH101 (Ac) (SEQ ID NO: 8), LH102 (SEQ ID NO: 7), LH201 (SEQ ID NO: 10), or LH201 (Ac) (SEQ ID NO: 11) or a therapeutically effective salt thereof.
In a second embodiment of the first aspect of the present invention this is provided the cyclic peptide wherein said compound is comprising of compound LH101 (SEQ ID NO: 6) of molecule formula C73H111N19O18S3 or compound LH101 (Ac) (SEQ ID NO: 8) of molecule formula C75H113N19O19S3 or compound LH102 (SEQ ID NO: 7) of molecule formula C72H110N20O19S3 or compound LH201 of molecule formula C76H113N21O20S2 or compound LH201 (Ac) (SEQ ID NO: 11) of molecule formula C78H115N21O21S2.
The pharmaceutically acceptable salts of the peptides described herein include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
In other cases, the peptides described herein may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically-acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.)
Pharmaceutical compositions comprising the peptides disclosed herein may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray).
Pharmaceutical compositions comprising the peptides disclosed herein can comprise a pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
For parenteral administration, the pharmaceutical composition is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
Generally, the formulations are prepared by contacting the components of the pharmaceutical composition uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as poly-sorbates, poloxamers, or PEG.
The components of the pharmaceutical composition to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic components of the pharmaceutical composition generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
The components of the pharmaceutical composition ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound(s) using bacteriostatic Water-for-Injection.
The investigations of galanin receptors (GalRs) function and the translation of these functions into clinically relevant applications have been hindered by lack of metabolically stable and receptor-specific agonists. Spexin (SPX) is a newly identified neuropeptide which selectively activates GalR2 and GalR3. To gain insights into structural basis of how spexin is recognized by GalR2 and GalR3 and to develop novel agonists, human spexin solution structure was investigated using a computational approach. All-atom, unbiased, and microsecond molecular dynamics (MD) simulations captured the α-helix conformation, but surprisingly, an unknown β-hairpin conformation was also observed. The β-fold was more stable in water, while α-fold was more profound in hydrophobic solutions. To verify if β-hairpin is biologically active, a series of cyclic peptides were designed, synthesized, and tested. Cysteine-scanning mutagenesis suggested that residue 1, 4, and 13 in spexin were candidate sites for disulfide bonding (
Spexin-deficient disorders include those diseases and/or disorders that can be treated by agonizing GalR2 and/or GalR3.
Diseases and/or disorders that can be treated by agonizing GalR2 and/or GalR3 include, but are not limited to attention deficit hyperactivity disorder (ADHD), bipolar disorder, body dysmorphic disorder, bulimia nervosa eating disorder, cataplexy, dysthymia, anxiety disorders, irritable bowel syndrome (IBS), impulse control disorders, pathological gambling problem, kleptomania, migraines, major depressive disorder, narcolepsy, obsessive-compulsive disorder (OCD), chronic pain, drug abuse, addiction, Alzheimer's disease, pain, seizures, learning and memory, anxiety, overeating, and obesity and the like. Additional diseases and/or disorders that can be treated by agonizing GalR2 and/or GalR3 are recited in U.S. Pat. No. 9,057,726, which is incorporated herein by reference
Results
Folding Simulations of hSPX Captured α-Helix and β-Hairpin Conformation
To understand the folding process of hSPX, 10-μs MD simulations were performed in pure water and water-TFE (50:50) mixture solutions, which mimics the Nuclear Magnetic Resonance (NMR) solution conditions as well as cell membrane environment. With 14 amino acids, this short peptide was extremely flexible in solution. However, conformation clustering results indicated that it folded into several major conformations. Similar with goldfish spexin solved by NMR, the largest conformation cluster of hSPX in 50% TFE solution was α-helix (
MD Simulations and NMR Analysis Suggested a βαβ hSPX Conformation
Experimental and theoretical studies have indicated that α-helix is the primary conformation of hGAL N-terminal fragment. The structure of goldfish spexin (gSPX) in solution has been solved by NMR, where the α-helix spans the distance from Q5 to Q14. Circular dichroism (CD) experiments have indicated that gSPX in water has α-helix, and that increasing the 2,2,2-trifluoroethanol (TFE) concentration also increases the α-helix content. hSPX is expected to have the same α-helix conformation, which may be stabilized by TFE. To verify this, three independent, all-atom MD simulations of hSPX (10 μs each) in pure water or in TFE: water (50:50) solutions at 298 K were performed.
With 14 amino acids, hSPX was flexible in both water and TFE:water (50:50) during MD simulations (
It was expected that the α-helix conformation of gSPX was of the high ranking (more populated) clusters; however, this is not the case for hSPX, neither in water nor in TFE:water (50:50). The α-helix conformation was hardly found in water; while in TFE:water (50:50), it was ranked at 11, 10 and 6 (
NMR analysis was then performed for hSPX in water at 298 K. With 2D 1H—1H Total Correlated SpectroscopY (TOCSY) experiments, the hydrogen atom chemical shifts of most hSPX residues (except N1) were successfully assigned (
1H assignment and chemical shifts for
The overall main-chain atom root-mean-square deviation (RMSD) between this new conformation and gSPX α-helix conformation was 6.53 Å, which share local structural similarity from T3 to Y9 (
1The 13th residue in hSPX is Ala, while it is Thr in gSPX.
1The 13th residue in hSPX is Ala, while it is Thr in gSPX.
The folding kinetics of hSPX α and βαβ conformations were also investigated using MD simulation data (
It is concluded that hSPX commonly adopts the βαβ conformation in solution, but the question remained as to whether the conformation is the biologically active conformation recognized by GalR2 and GalR3? To answer this crucial question, cyclic peptides stapled by a disulfide bond were prepared. The flexibility of hSPX could be reduced by cyclic arrangement, and it will be easier to understand the conformation-activity relationships.
Cyclic Analogs of hSPX Retain the Activation Profile on GalR2 and GalR3
It was the aim to make a cyclic form of hSPX that did not lose bioactivity with disulfide bonding constraints. To do this, it was important to know which sites can tolerate cysteine substitutions. Cysteine-scanning mutagenesis was performed from position 1 to position 14 for hSPX. Fourteen hSPX mutants were designed and synthesized. The bioactivities of these mutants against GalR2 and GalR3 were measured by SRE-Luc assay at single dose (1 μM, 6 hours) treatment in HEK293 cells transiently transfected with GalRs and Gqi plasmids, while hGAL and hSPX were utilized as positive controls (
In certain embodiments, variants of SEQ ID NO: 23 or SEQ ID NO: 24 having at least 92.8% homology do not include a cysteine at any of positions 2, 3, 5, 6, 9, 10, 12, and 14.
CWTPQAMLYLK
CWTLNSAGYLL
1Relative to hGAL Emax;
2Relative to hSPX Emax;
Based on the βαβ conformation suggested by MD and NMR, as well as the results of cysteine-scanning experiment, N1/A13 or P4/A13 were replaced in hSPX with cysteines and connected by disulfide bonds, resulting in two cyclic peptides: LH101 (CWTPQAMLYLKGCQ-NH2 (SEQ ID NO: 6)) (
Thus, provided herein are peptides capable of agonizing both hGalR2 and hGalR3. In certain embodiments, the peptides provided herein have little (e.g., EC50≥5 μM or ≥10 μM) or substantially no hGalR1 agonist activity.
In other embodiments, the peptides provided herein are capable of selectively agonizing one of hGalR2 or hGalR3. In certain embodiments, the peptide provided herein is a selective agonist of hGalR2 having about 1.5×, about 2×, about 3×, about 4×, about 5×, about 6×, about 8×, about 9×, about 10×, about 20×, about 30×, about 40×, or lower EC50 for hGalR2 than hGalR3. In certain embodiments, the peptide provided herein is a selective agonist of hGalR3 having about 1.5×, about 2×, about 3×, about 4×, about 5×, about 6×, about 8×, about 9×, about 10×, about 20×, about 30×, about 40×, or lower EC50 for hGalR3 than hGalR2.
In certain embodiments, the peptides provided herein have little (e.g., EC50≥10 μM) or substantially no hGalR3 agonist activity.
Previous studies have suggested that the hGAL N-terminal fragment, hGAL (1-16) (
The in vitro bioactivity data suggested that the CSAs (LH101 and LH101 (Ac)), can potently activate GalR2 and GalR3, and that the same cyclic form could also be adopted by hGAL(1-16). It was interesting that in LH101 and LH101(Ac), the N- and C-terminal residues were linked by disulfide bond and close to each other—which was consistent with the hSPX βαβ conformation.
LH101(Ac) Adopts the βαβ Conformation in Water
MD simulation and NMR analysis were used to elucidate the structure of LH101 (Ac) in water. The Ca atom RMSF of LH101 (Ac) in MD simulations were much reduced (
1H—1H 2D-TOCSY and NOESY experiments for LH101 (Ac) were conducted. Most hydrogen atom chemical shifts of all 14 residues in LH101 (Ac) could be assigned (
1H assignment and chemical shift for LH101
1The number in bracket represented the minor chemical shifts of that residue in 1H assignment.
The key structural features of hSPX βαβ conformation, including the two β turns characterized by backbone hydrogen bonds T3.O-A6.H and A6.O—Y9.H, were well preserved in LH101(Ac) (
1The 1st residue in hSPX is Asn, while it is Cys in LH101 (Ac).
2The 13th residue in hSPX is Ala, while it is Cys in LH101 (Ac).
1The 13th residue in hSPX is Ala, while it is Cys in LH101 (Ac).
The folding trajectory suggests that LH101 (Ac) rarely adopts the α-helix conformation in water (
Improved Serum Stability of the Cyclic Analogs of hSPX
The serum stability of CSAs was tested using an in vitro assay followed by reverse-phase high-performance liquid chromatography (RP-HPLC) analysis (
The intact peptides of hSPX degraded quickly in serum (t1/2=66.5 min), and almost vanished after 8 h incubation (
LH101 Suppressed Body Weight Gain in High-Fat-Diet Induced Obesity Mice
Previous studies suggested that down-regulation of spexin was associated with several disorders, such as obesity, type-II diabetes, nonalcoholic fatty liver disease, and constipation. To demonstrate the therapeutic value of this hSPX cyclic analog, the effect of LH101 on body weight gain in male mice (C57BL/6J) with diet-induced obesity (DIO) was investigated.
Male mice (C57BL/6J) weighing about 22-25 g were purchased from the Laboratory Animal Services Center, The Chinese University of Hong Kong, Hong Kong. The animals were fed with a high fat diet (Research Diets, New Brunswick, N.J.) ad libitum with free access to water and were housed in rooms maintained at 22±1° C. with a 12 h light/dark cycle (lights on 6:00-18:00). Animals were constantly acclimated to the facility until the body weight reached 45-50 g. Then all the diet induced obese mice were divided into 2 groups randomly for the drug treatment. The Animal Ethics Committees of Hong Kong Baptist University, approved all experimental protocols, in accordance with “Institutional Guidelines and Animal Ordinance” from Department of Health, Hong Kong Special Administrative Region.
In the result, LH101 at 25 μg/kg by intraperitoneal injection (i.p.) suppressed mice body weight gain over the course of 14-days-treatment while controls receiving PBS continued to gain weight on the high fat diet (
LH101(Ac) Demonstrated Better Body Weight Control Efficacy than hSPX
Previous studies have suggested that down-regulation of SPX is associated with several disorders, including obesity. To demonstrate the potential therapeutic value of CSAs, the effect of LH101 (Ac) on body weight gain in C57BL/6J mice with high-fat-diet (HFD) induced obesity was tested.
Female and male C57BL/6J mice weighing 22-25 g were purchased from the Laboratory Animal Services Center, The Chinese University of Hong Kong, Hong Kong. The animals were fed with rodent diet with 60 kcal % fat (Research Diets, New Brunswick, N.J.) ad libitum with free access to water and were housed in rooms maintained at 22±1° C. with a 12 h light/dark cycle (lights on 6:00-18:00). Animals were constantly acclimated to the facility until the body weight reached no less than 30 g. Then all the diet-induced obese mice were divided into 3 groups randomly for the drug treatment. Mice body weight was daily monitored, and the food intake amount was recorded every 3 days. The Animal Ethics Committee of Hong Kong Baptist University approved all experimental protocols in accordance with “Institutional Guidelines and Animal Ordinance” from Department of Health, Hong Kong Special Administrative Region.
During an 18 day treatment study, LH101 (Ac) at 25 μg/kg by i.p. injection significantly suppressed body weight gain compared with negative controls, which received phosphate-buffered saline (PBS) (
Since it was discovered three decades ago, the solution structures of GAL have drawn substantial research interest. Initially, the solution structure of rat GAL (identical to hGAL in the first 15 amino acids) was demonstrated to be primarily helical in TFE and disordered in water. However, CD and NMR detected a nascent helix spanning from Thr3 to Leu11 in hGAL, in aqueous solution and the first 12 amino acids of GAL kept helical conformation in the chimeric peptide transportan, in neutral phospholipid bicelles. Furthermore, Gly1 was thought to be crucial to stabilize the N-terminal helix of GAL, which was in turn believed to be important to the activation of GalR1. The α-helix conformation of GAL was thought to be biologically active, being recognized by GalRs, at least for GalR1. In the proposed complex model by Kask et al., GAL made interactions with GalR1 via the N-terminal fragment in α-helix conformation. In line with all these observations, the α-helix conformation was also observed in gSPX in 60% TFE solution, suggesting that the helical structure of gSPX can be formed in close proximity to plasma membrane of target cells. This α-conformation has been a well-established model to explain ligand-GalR recognition, used for years. In line with the observation in galanin, the α-helix conformation of goldfish spexin was observed in 60% TFE solution. However, in the present invention, MD simulations suggested an unexpected low-energy conformation of hSPX in aqueous solution, β-hairpin, which was not observed in NMR study of goldfish spexin. If the β conformer were true, hSPX could have two distinct low-energy conformations, but how is hSPX recognized by its cognate receptors?
Proposed herein is a new βαβ conformation of hSPX in the recognition of GalR2 and GalR3, which is supported by a combination of computational simulation, biophysical, and molecular biology studies. Moreover, the results of cyclic analogs of hGAL (1-16) suggest that they are also applicable to other GAL family members, such as hGAL, in activation of GalR2 and GalR3.
By using cysteine-scanning, three sites (1, 4, 13) were identified as candidate sites for incorporation of cysteine residues and disulfide formation. Cyclic peptide LH101 (CWTPQAMLYLKGCQ-NH2 (SEQ ID NO: 6)), wherein a disulfide bond is present between C1 and C13, was demonstrated to be a potent activator of both hGalR2 and hGalR3. The results described herein support the hypothesis that hSPX activates cognate receptors via a possible β conformation. The results of the cyclic analog of human galanin (1-16) support the conclusion that human galanin (1-16) may activate GalR2 and GalR3 by utilizing a similar β conformation as hSPX.
With a hydrophobic core in the middle, the βαβ conformer may be inserted into the receptor binding cavity, leaving both N- and C-terminals pointing to the extracellular space. In this binding orientation, it is expected that modifications to the N- and C-terminus could yield analogs with similar properties. This proposed binding mode may explain why chemical modifications with large functional groups on the SPX N-terminal does not affect its binding to GalR2/GalR3. It may also explain why the galanin-like peptide (GALP)—a neuropeptide containing 60 amino acids with large flanking extensions at both N- and C-terminals compared with GAL—has preferential binding affinity to GalR2.
The βαβ model can be used to design a new generation of GalR agonists/antagonists. Provided herein is a novel class of GalR2 and GalR3 agonists, e.g., the cyclic spexin analogs (CSAs). The CSAs exhibit sub-micromolar bioactivity, superior serum stability, and improved anti-obesity efficacy. Peptide-based therapeutics oftentimes suffer from poor stability and/or bioavailability. However, exhibit improved stability and bioavailability.
Cyclic peptides containing a disulfide bond, e.g., LH101, LH102, and LH101 (Ac) as analogs of hSPX, and LH201 and LH201 (Ac) as analog of human galanin (1-16) have been discovered. Compared with wild type hSPX, the concentration of one agonist to induce 50% maximum activation of its receptor (EC50) of LH101 was slightly increased, but can be recovered by N-terminal acetylation (in the case of LH101(Ac)). For LH201, the EC50 on hGalR2 was indeed better than human galanin (1-16). Another advantage of cyclic peptides is better serum stability. LH101 and LH101 (Ac) were both more stable in serum than hSPX. Given these advantages, LH101, LH102, LH101 (Ac), LH201 and LH201 (Ac) can be further developed as intravenous administration pharmaceuticals, like the pioneering works on somatostatin. It is also possible that orally administrated spexin or galanin analogs could be designed based on LH101, LH102, LH101 (Ac), LH201 and LH201 (Ac) scaffolds.
The galanin receptors (GalR1, GalR2 and GalR3) are close to somatostain receptors (SSTR1, SSTR2, SSTR3, SSTR4 and SSTR5) in polygenetic tree, and both of them belong to the γ-subfamily of GPCR. It seemed to be naturally that these receptors recognize their cognate ligands in a similar way.
The present disclosure also paves the way to clinical application of spexin or galanin signaling modulators. With good bioactivity and plasma stability, cyclic peptides LH101, LH102, LH101 (Ac), LH201 and LH201 (Ac) have potential to be applied in the treatment of GalR2 and GalR3-related diseases. LH101, LH102, and LH101(Ac) can be regarded as hSPX analogs with prolonged action, and can be used in treatment of a number of human disorders with spexin-deficiency, such as obesity, type-II diabetes, nonalcoholic fatty liver disease, constipation, and other diseases related with cardiovascular and renal function, nociception, reproduction. In particular, spexin was the most down-regulated gene in obese human fat, and spexin treatment reduced caloric intake with weight loss in rats. The data provided herein demonstrates that LH101 does inhibit body weight gain in high-fat-diet induced obesity mice, which supports the therapeutic value of LH101.
MD Simulations
Initial structure in fully extended conformation was generated by cpptraj within AMBER 14. It was solvated into a cubic box containing TIP3P water or water-TFE mixture (percentage of TFE are 0% and 50%, respectively) with buffer distance 12 Å. Charge was neutralized by counter ions. An AMBERff99SB force field was used for protein parameterization, while parameters for TFE were adopted from published work. Prepared systems were minimized and equilibrated by sander via three stages: (1) heating from 100 K to 300 K in 20 ps; (2) adjusting solvent density in 80 ps with constant pressure and constant temperature (NPT); (3) further equilibrating in 2,000 ps with NPT. Finally, all-atoms, unbiased, 10-μs, constant volume and constant temperature (NVT) simulations were performed in Amber 14 with CUDA (Compute Unified Device Architecture) accelerated PMEMD (algorithm for accelerated computation). 2 fs time step and SHAKE-enabled (algorithm for accelerated computation with fixed hydrogen atoms) setting were used for all equilibration and production stages, and a Berendsen thermostat was used for temperature control. Simulation frame was saved every 250 ps. Resulted trajectories were further analyzed with cpptraj. Conformational clusters of hSPX were calculated using Ca RMSD of hSPX (2-13) with 2.5 Å as threshold. Representative conformation of each cluster was used to define secondary structural components. Among representative conformers, the one belonged to the largest β cluster was used as reference structure of β conformer. Each frame of hSPX was compared with hSPX α-helix conformer solved by NMR and hSPX β-hairpin conformer predicted by MD simulation in terms of Ca RMSD of Trp2 to Lys11. A folding event of α-helix or β-hairpin was reached if Ca RMSD of Trp2 to Lys11 was less than 2.5 Å. In folding trajectory analysis, each frame was compared with gSPX α-helix conformer and the hSPX βαβ conformer in terms of main-chain atom RMSD of hSPX (2-13). A folding event of a or βαβ conformation was reached if the RMSD was less than 2.5 Å compared with reference structures. Folding time was defined as how long it took from end of last folding event to start of current folding event, while unfolding time was defined as how long it stayed in current folding event.
NMR Analysis
NMR experiments were performed in H2O:D2O (90:10) with 5 mM sample concentration solvent at 298° K. For TOCSY experiments, the mixing time was 70 ms; while for NOESY, it was 500 ms. Data was acquired with Avance 600 MHz NMR Spectrometry (Bruker, Germany) in Shanghai Jiao Tong University. Data analysis were done with CcpNmr Analysis (version 2.4.2).
Peptide Synthesis and Characterization
All peptides used in this study were synthesized by Scilight Biotechnology LLC (Beijing, China) with standard solid-phase peptide synthesis procedures. Cyclic peptides (<1 mg/mL) were generated by oxidation in 20% dimethyl sulfoxide (DMSO) with overnight incubation. All peptides were characterized by MALDI-MS spectrum in Department of Chemistry, Hong Kong Baptist University.
Receptor Activation Assay
The hGalR1, hGalR2, hGalR3, and SRE-Luc plasmids were provided by Yingrun Biotechnology Co., Ltd (Changsha, China), while Gqi plasmids were gifts from Prof. Jae Young Seong. HEK293 cells were cultured regularly in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and seeded into 24-well plates with 50,000 cells per well. After being cultured for 24 h, the seeded cells were transiently transfected with equal amount of hGalR-, Gqi-, and SRE-Luc plasmids by using Lipofectamine 3000 (Thermo Fisher Scientific, USA). Eighteen hours ahead of drug treatment, the medium was changed to serum-free medium. Cells were lysed after 6 h drug treatment, and the cell lysates were centrifuged at 13,000 rpm for 10 mins. 20 μL supernatant was subject to luciferase assay using Dual-Luciferase® Reporter Assay System (Promega, Madison, Wis., USA).
Serum Stability Assay
750 μL RPMI medium 1640 (Thermo Fisher Scientific) was mixed with 250 pt rat serum, and incubated at 37° C. for 15 min before adding peptide. The peptide dissolved in water was added into the reaction buffer to make a final concentration of 100 μg/mL. At each time point (5, 15, 30, 60, 120, 240, and 480 min), 100 μL reaction solution were taken, and mixed with 200 μL 6% trichloroacetic acid (TCA, Sigma-Aldrich). The resulting cloudy solution was cooled at 4° C. for 15 min, and centrifuged for 10 mins at 13,000 rpm. The supernatant was then analyzed by RP-HPLC. The extent of intact peptide was calculated based on the peak area and was normalized using a control sample.
If desired, the different functions discussed herein may be performed in a different order and/or concurrently with each other. Furthermore, if desired, one or more of the above-described functions may be optional or may be combined.
While the foregoing invention has been described with respect to various embodiments and examples, it is understood that other embodiments are within the scope of the present invention as expressed in the following claims and their equivalents. Moreover, the above specific examples are to be construed as merely illustrative, and not limitative of the reminder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.
Provided herein are cyclic peptide agonists toward human galanin receptor 2 (GalR2) and galanin receptor 3 (GalR3) useful in the treatment of GalR2 and GalR3 related and spexin-deficient disorders. In particular, the cyclic peptides and derivatives thereof are potent spexin and hGAL analogs with prolonged action, which are novel therapies for GalR2, GalR3 spexin-deficient disorders, such as obesity.
This application claims priority from U.S. Provisional Patent Application No. 62/482,751 filed on Apr. 7, 2017 and U.S. Provisional Patent Application No. 62/506,654 filed on May 16, 2017, the disclosures of which are hereby incorporated by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 8435940 | Bulaj | May 2013 | B2 |
| 9057726 | Leroy | Jun 2015 | B2 |
| 20040234537 | Bougueleret | Nov 2004 | A1 |
| 20090281031 | Bulaj | Nov 2009 | A1 |
| 20130196348 | Leroy | Aug 2013 | A1 |
| Number | Date | Country |
|---|---|---|
| 101448513 | Jun 2009 | CN |
| 103391784 | Nov 2013 | CN |
| 2016146513 | Sep 2016 | WO |
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| Number | Date | Country | |
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| 20180289766 A1 | Oct 2018 | US |
| Number | Date | Country | |
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| 62482751 | Apr 2017 | US | |
| 62506654 | May 2017 | US |
