WO2013151627A1 - Procédés de traitement de troubles du métabolisme du glucose - Google Patents

Procédés de traitement de troubles du métabolisme du glucose Download PDF

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Publication number
WO2013151627A1
WO2013151627A1 PCT/US2013/026879 US2013026879W WO2013151627A1 WO 2013151627 A1 WO2013151627 A1 WO 2013151627A1 US 2013026879 W US2013026879 W US 2013026879W WO 2013151627 A1 WO2013151627 A1 WO 2013151627A1
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peptide
antibody
amino acid
peptides
seq
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PCT/US2013/026879
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English (en)
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Daniel David KAPLAN
Maziyar SABERI
Beilin Zhang
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Ngm Biopharmaceuticals, Inc.
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Publication of WO2013151627A1 publication Critical patent/WO2013151627A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to, among other things, peptides and antibodies having glucose modulating activity, and associated methods and uses in treatment of diabetes and other metabolic-related disorders.
  • Patients who have a glucose metabolism disorder can suffer from hyperglycemia, hyperinsulinemia, and/or glucose intolerance.
  • An example of a disorder that is often associated with the aberrant levels of glucose and/or insulin is insulin resistance, in which liver, fat, and muscle cells lose their ability to respond to normal blood insulin levels.
  • the present disclosure contemplates the use of the agents described herein, and compositions thereof, to treat and/or prevent various diseases, disorders and conditions, and/or the symptoms thereof.
  • the diseases, disorders and conditions, and/or the symptoms thereof pertain to metabolic-related disorders, while in other embodiments they pertain to glucose metabolism disorders.
  • the agents, and compositions thereof can be used for the treatment and/or prevention of diabetes (e.g., Type 2 diabetes), insulin resistance and diseases, disorders and conditions characterized by insulin resistance, decreased insulin production, hyperglycemia, hypoinsulinemia, and metabolic syndrome.
  • diabetes e.g., Type 2 diabetes
  • the agents, and compositions thereof may also be useful in, for example, subjects who may be overweight or obese.
  • the agents of the present disclosure are referred to herein as "Modulators".
  • peptide subsequences derived from the Tor2a gene product have been identified. More particularly, ten murine peptides (ms pl-ms plO) and ten human peptides (hu pl-hu plO) have been identified (see Figure 2), and these peptides are distinct from the salusin peptides (i.e., neither the murine peptides nor the human peptides overlap with the murine and human amino acid sequences of the salusin peptides (salusin-a and salusin- ⁇ ; see, e.g., GenBank Accession No. NP 001238952). While an understanding of all of the characteristics of these murine peptides and these human peptides is not required, they are believed to be secreted peptides.
  • the present disclosure contemplates homologues, variants, fragments and other modified forms thereof.
  • the aforementioned human peptides and homologous and variants thereof are collectively referred to hereafter as the "Human Peptides”
  • the aforementioned murine peptides and homologous and variants thereof are collectively referred to hereafter as the “Murine Peptides”
  • the aforementioned human nucleic acids and homologous and variants thereof are collectively referred to hereafter as the "Human Nucleic Acid Molecules”
  • the “Human Peptides” the aforementioned murine peptides and homologous and variants thereof.
  • murine nucleic acids and homologous and variants thereof are collectively referred to as the "Murine Nucleic Acid Molecules".
  • “human” or “mouse” in connection with polypeptides, peptides and nucleic acid molecules of the present disclosure is not meant to be limiting with respect to the manner in which the peptide or nucleic is obtained or the source, but rather is only with reference to the sequence as it may correspond to a sequence of a naturally occurring human or mouse polypeptide or nucleic acid molecule.
  • the Human Peptides and the Murine Peptides may be collectively referred to as the "Peptides", while the Human Nucleic Acid Molecules and the Murine Nucleic Acid Molecules may be collectively referred to as the "Nucleic Acid Molecules”.
  • Tor2a - related peptides is meant to refer to both the Human Peptides and the Murine Peptides.
  • the present disclosure also contemplates Tor2a - related peptides, and the nucleic acid molecules which encode them, from other species (e.g., non-human primates (e.g., chimpanzees), dogs, rats, etc.).
  • Activators (as defined hereafter) of the Peptides.
  • the term “Inhibitors” refers to agents that, for example, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down-regulate the function or activity of one or more Peptides, such as, for example, antagonists including antibodies, small molecule antagonist compounds, and antagonistic peptides structurally distinguishable from the peptides disclosed herein; other examples of Inhibitors are described below.
  • Activators refers to agents that, for example, stimulate, increase, activate, facilitate, enhance activation, sensitize or up-regulate the function or activity of one or more Peptides, such as, for example, agonists including antibodies, small molecule agonist compounds, the peptides disclosed herein, and other agonistic peptides structurally distinguishable from the peptides disclosed herein; other examples of Activators are described below.
  • Inhibitors and Activators are collectively referred to as
  • compositions are provided that comprise one or more peptides or antibodies of the present disclosure, which compositions are useful in treating or preventing a metabolic disorder.
  • the compositions modulate aberrant glucose and/or insulin levels in a subject and may be used to treat or prevent diabetes mellitus (e.g., Type I and Type II diabetes and gestational diabetes).
  • diabetes mellitus e.g., Type I and Type II diabetes and gestational diabetes.
  • the compositions comprising the peptides described herein may also be used to treat, for example, insulin resistance,
  • hyperinsulinemia glucose intolerance, hyperglycemia or metabolic syndrome
  • various body weight - related disorders e.g., obesity
  • a use or method of treatment of a subject includes administering a Peptide to a subject, such as a subject having, or at risk of having, a disease or disorder treatable by a Peptide, in an amount effective for treating the disease or disorder.
  • a method includes administering a Peptide to a subject, such as a subject having a hyperglycemic condition, insulin resistance, hyperinsulinemia, glucose intolerance or metabolic syndrome, a Peptide, in an amount effective for treating the disease or disorder.
  • one embodiment of the present disclosure relates to a peptide comprising any one of: a subsequence of human isoform of Tor2a or a murine isoform of Tor2a as depicted in Figure 3, or a variant thereof; a subsequence of human Tor2a or a subsequence of murine Tor2a as depicted in Figure 4, Panel b, or a variant thereof; a human sequence of Figure 2 (hu pi - hu p2) or a murine sequence of Figure 2 (ms pi - ms plO), or a variant thereof; or the sequence ALVVKXiLKAFVRDPAPX 2 KPLVLSLHGWTGT (SEQ ID NO:4), where Xi and X 2 are semi-conserved residues.
  • Xi is A or S and X 2 is T or S.
  • the peptide comprises a carboxyl-terminal modification, e.g., instead of a carboxyl group at the carboxyl terminus, the peptide comprises a CONH 2 group (e.g., SEQ ID NO:5).
  • the peptide comprises any one of ms pi, ms p5, hu pl, or hu p5.
  • the peptide comprises a variant of a human peptide of
  • the variant of a human peptide comprises an amino acid sequence having at least 85% amino acid identity, at least 90% amino acid identity, at least 93% amino acid identity, at least 95% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to the amino acid sequence of a human peptide of Figure 2 (hu pi - hu p2) or the variant of a murine peptide of Figure 2 (ms pi - ms plO).
  • the peptide comprises the amino acid sequence PLVLSLHGWTGT (SEQ ID NO:6) and has fewer than 100 amino acid residues, fewer than 75 amino acid residues, fewer than 50 amino acid residues, fewer than 25 amino acid residues, or fewer than 20 amino acid residues.
  • the peptide comprises the amino acid sequence PLVLSLHGWTGT (SEQ ID NO:7) comprising a CONH2 group instead of a COOH group at the carboxyl terminus of the peptide.
  • the peptide may be isolated.
  • nucleic acid molecules encoding the aforementioned peptides.
  • a nucleic acid molecule is operably linked to an expression control element that confers expression of the nucleic acid molecule encoding the peptide in vitro, in a cell or in vivo.
  • a vector e.g., a viral vector
  • Some embodiments include transformed or host cells that express one or more of the aforementioned peptides.
  • one or more of the aforementioned peptides is formulated to yield a pharmaceutical composition, wherein the composition also includes one or more pharmaceutically acceptable diluents, carriers or excipients.
  • a pharmaceutical composition also includes at least one additional prophylactic or therapeutic agent.
  • Still further embodiments of the present disclosure comprise an antibody that binds specifically to one of the aforementioned peptides.
  • the antibody comprises a light chain variable region and a heavy chain variable region present in separate polypeptides or in a single polypeptide.
  • An antibody of the present disclosure binds the peptide with an affinity of from about 10 7 M - " 1 to about 1012 M - " 1 in certain embodiments.
  • the antibody comprises a heavy chain constant region of the isotype IgGl, IgG2, IgG3, or IgG4.
  • the antibody is detectably labeled, while it is a Fv, scFv, Fab, F(ab')2, or Fab' in other embodiments.
  • the present disclosure also contemplates antibodies that comprise a covalently linked non-peptide polymer (e.g., a poly(ethylene glycol) polymer).
  • the antibody comprises a covalently linked moiety selected from a lipid moiety, a fatty acid moiety, a polysaccharide moiety, and a carbohydrate moiety.
  • the antibody is a single chain Fv (scFv) antibody in some embodiments, and the scFv is multimerized in others.
  • scFv single chain Fv
  • the antibodies of the present disclosure may be, but are not limited to, monoclonal antibodies, polyclonal antibodies, or humanized antibodies.
  • compositions comprising an antibody as described above formulated with at least one pharmaceutically acceptable excipient, carrier or diluent.
  • Such pharmaceutical compositions may also contain at least one additional prophylactic or therapeutic agent.
  • a sterile container that contains one of the above-mentioned pharmaceutical compositions and optionally one or more additional components.
  • the sterile container may be a syringe.
  • the sterile container is one component of a kit; the kit may also contain, for example, a second sterile container that contains at least one prophylactic or therapeutic agent.
  • the present disclosure also contemplates a method of treating or preventing a glucose metabolism disorder in a subject (e.g., a human) by administering to the subject a therapeutically effective amount of an Inhibitor (as defined herein).
  • the Inhibitor is an antibody as described above, a small molecule antagonist compound, or an antagonistic peptide.
  • the treating or preventing results in a reduction in plasma glucose in the subject, a reduction in plasma insulin in the subject, or an increase in glucose tolerance in the subject.
  • the glucose metabolism disorder is diabetes mellitus.
  • the subject is obese.
  • the present disclosure contemplates a method of treating or preventing a glucose metabolism disorder in a subject (e.g., a human) by administering to the subject a therapeutically effective amount of an Activator (as defined herein).
  • the Activator may be, for example, one of the aforementioned peptides, a small molecule agonist compound, an agonistic peptide distinguishable from the peptides described herein, or an antibody.
  • the administering is by parenteral (e.g., subcutaneous) injection.
  • FIG. 1 shows Tor2a gene expression in enteroendocrine cells (EECs) and enterocytes (ECs) isolated from the indicated segments of the mouse gastrointestinal tract. Hashed bars represent expression in EECs, while solid bars represent expression in ECs.
  • Figure 2 is a table depicting the murine peptides (ms pl-ms plO) and the human peptides (hu pl-hu plO).
  • ms pi SEQ ID NO:7); ms p2 (SEQ ID NO:8); ms p3 (SEQ ID NO : 11 ); ms p4 (SEQ ID NO : 12); ms p5 (SEQ ID NO : 13); ms p6 (SEQ ID NO : 14); ms p7 (SEQ ID NO: 15); ms p8 (SEQ ID NO: 16); ms p9 (SEQ ID NO: 17); ms plO (SEQ ID NO: 18); hu pl (SEQ ID NO:7); hu p2 (SEQ ID NO:8); hu p3 (SEQ ID NO: 11); hu p4 (SEQ ID NO: 12); hu p5 (SEQ ID NO: 19
  • Figure 3 shows the three human isoforms of Tor2a and the two murine isoforms of Tor2a that contain the hu pl-hu plO peptides and the ms pl-ms plO peptides, respectively.
  • the regions encompassing the Peptides are highlighted in gray.
  • Nucleic acid molecules encoding the polypeptides are also set forth.
  • the regions of the nucleic acid molecules representing the coding regions are highlighted in gray.
  • AAQ88547.1 SEQ ID NO:9;
  • AY358180.1 (SEQ ID NO: 10); CAI41187 (SEQ ID NO:23); NM 130459.3 (SEQ ID NO:24); NP 001127902.1 (SEQ ID NO:25); NM 001134430.2 (SEQ ID NO:26); NP 690013.1 (SEQ ID NO:27); NM 152800.3 (SEQ ID NO:28); P0C7W3.1 (SEQ ID NO:29);
  • ENSMUST00000167540 (SEQ ID NO:30).
  • FIG. 4 Panel A shows a sequence alignment of Tor2a amino acid sequences from the indicated species. Amino acid sequences from humans, mice and other species are highlighted in gray. Residue positions that are fully conserved are indicated by (*), whereas residue positions that are semi-conserved are indicated by Q.human (SEQ ID NO:9);
  • FIG 4 Panel B shows a sequence alignment of a central region of Tor2a amino acid sequences from the indicated species.
  • the region encompassing particular Human Peptide sequences, particular Murine Peptide sequences, and related sequences from other species is highlighted in gray. Residue positions that are fully conserved are indicated by (*), whereas residue positions that are semi-conserved are indicated by Q.human (SEQ ID NO:34);
  • Figure 5 is a table identical to that in Figure 2 except that it also indicates activity of the particular Murine Peptides and the particular Human Peptides in elevating blood glucose levels and reducing glucose-stimulated insulin secretion. "+” indicates that the peptide caused a significant (p ⁇ 0.05) elevation in blood glucose levels and a significant (p ⁇ 0.05) reduction in insulin levels during an oral glucose tolerance test, while “-” indicates that the peptide did not cause significant changes in blood glucose and insulin levels.
  • FIG 6 Panel a shows fasted plasma glucose concentration following a single, bolus i.p. injection of Tor2a-pl (10 mg/kg) [Murine Peptide-pl] or Vehicle control in high-fat fed mice.
  • FIG 6 Panel b shows the percent change in fasted blood glucose concentration normalized to basal glucose concentration (i.e., hr 0) following a single, bolus i.p. injection of Tor2a-pl (10 mg/kg) [Murine Peptide-pl] or Vehicle control in high-fat fed mice.
  • FIG. 6 Panel c shows fasted plasma insulin concentration following a single, bolus i.p. injection of Tor2a-pl (10 mg/kg) [Murine Peptide-pl] or Vehicle control in high-fat fed mice. These data indicate that Murine Peptide-pl reduces fasted plasma insulin
  • FIG. 7 Panel a shows the impact of a single, bolus i.p. injection of Tor2a-pl
  • FIG 7 Panel b shows the percent change in plasma glucose concentration normalized to basal glucose concentration (i.e. min-30) following a single, bolus i.p. injection of Tor2a- l (10 mg/kg) [Murine Peptide- l] or Vehicle control in high-fat fed mice following a 4 hr fast.
  • FIG 7 Panel c shows plasma insulin concentration following a single, bolus i.p. injection of Tor2a-pl (10 mg/kg) [Murine Peptide-pl]) or Vehicle control in high-fat fed mice following a 4 hr fast.
  • FIG 7 Panel d shows the percent change in plasma insulin concentration normalized to basal concentration (i.e. min -30) following a single, bolus i.p. injection of Tor2a- pl (10 mg/kg) [Murine Peptide-pl] or Vehicle control in high-fat fed mice following a 4 hr fast.
  • FIG 8 Panel a shows the impact of single, bolus i.p. injection of Tor2a-pl (10 mg/kg) [Murine Peptide-pl] or Vehicle control on insulin tolerance in high-fat fed mice.
  • FIG 9 Panel a shows the impact of single, bolus i.p. injection of Tor2a-pl (10 mg/kg) [Murine Peptide-pl] on pyruvate tolerance in high- fat fed mice.
  • FIG 9 Panel b shows the percent change in plasma glucose concentration in response to a pyruvate challenge, normalized to baseline (i.e. min-30) following a single, bolus i.p. injection of Tor2a-pl (10 mg/kg) [Murine Peptide-pl] or Vehicle control in high- fat fed mice.
  • Tor2a-pl 10 mg/kg
  • n 6 mice per group; p-values determined by a student's t-test comparing Murine Peptide-pl and Vehicle-treated mice are indicated above the graph).
  • FIG 9 Panel d shows the percent change in concentration of insulin normalized to baseline (i.e. min-30) in response to a pyruvate challenge following a single, bolus i.p. injection of Tor2a-pl (10 mg/kg) [Murine Peptide-pl] or Vehicle control in high-fat fed mice.
  • the present disclosure contemplates the use of the Modulators described herein, and compositions thereof, to treat and/or prevent various diseases, disorders and conditions, and/or the symptoms thereof.
  • the diseases, disorders and conditions, and/or the symptoms thereof pertain to metabolic-related disorders, while in other embodiments they pertain to glucose metabolism disorders.
  • the Modulators, and compositions thereof can be used for the treatment and/or prevention of diabetes (e.g., Type 2 diabetes), insulin resistance and diseases, disorders and conditions characterized by insulin resistance, decreased insulin production, hyperglycemia,
  • the Modulators, and compositions thereof, may also be useful in, for example, subjects who may be overweight or obese.
  • Torsin-2A also known as “Torsin family 2 member A”, “Torsin- related protein 1”, or “Prosalusin” encompasses peptides and variants thereof that are encoded by the Tor2a gene or homologs thereof.
  • Tor2a is found in many mammals, including humans, non-human primates, rodents (e.g., mice) and canines.
  • the peptides encoded by the Tor2 gene are the salusin peptides, which have previously been described in the literature.
  • Peptide subsequences predicted to be derived from the Tor2a gene product are described herein. More particularly, ten murine peptides (ms pl-ms plO) and ten human peptides (hu pl-hu pi 0) have been identified (see Figure 2), and these peptides are distinct from the salusin peptides (i.e., neither the murine peptides nor the human peptides overlap with the murine and human amino acid sequences of the salusin peptides (salusin-a and salusin- ⁇ ). While an understanding of all of the characteristics of these murine peptides and human peptides is not required in order to practice embodiments of the present disclosure, they are believed to be secreted peptides.
  • patient or “subject” are used interchangeably to refer to a human or a non-human animal (e.g., a mammal).
  • a Modulator (such as administering a Modulator or a pharmaceutical composition comprising a Modulator) initiated after a disease, disorder or condition, or a symptom thereof, has been diagnosed, observed, and the like so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of a disease, disorder, condition afflicting a subject, or at least one of the symptoms associated with a disease, disorder, condition afflicting a subject.
  • treatment includes inhibiting (i.e., arresting the development or further development of the disease, disorder or condition or clinical symptoms association therewith) an active disease (e.g., so as to decrease the level of insulin and/or glucose in the bloodstream, to increase glucose tolerance so as to minimize fluctuation of glucose levels, and/or so as to protect against diseases caused by disruption of glucose homeostasis).
  • an active disease e.g., so as to decrease the level of insulin and/or glucose in the bloodstream, to increase glucose tolerance so as to minimize fluctuation of glucose levels, and/or so as to protect against diseases caused by disruption of glucose homeostasis.
  • in need of treatment refers to a judgment made by a physician or other caregiver that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of the physician's or caregiver's expertise.
  • prevent refers to a course of action (such as administering a Modulator or a pharmaceutical composition comprising a Modulator) initiated in a manner (e.g., prior to the onset of a disease, disorder, condition or symptom thereof) so as to prevent, suppress, inhibit or reduce, either temporarily or
  • a subject's risk of developing a disease, disorder, condition or the like as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof, generally in the context of a subject predisposed to having a particular disease, disorder or condition.
  • the terms also refer to slowing the progression of the disease, disorder or condition or inhibiting progression thereof to a harmful or otherwise undesired state.
  • in need of prevention refers to a judgment made by a physician or other caregiver that a subject requires or will benefit from preventative care. This judgment is made based on a variety of factors that are in the realm of a physician's or caregiver's expertise.
  • the phrase "therapeutically effective amount” refers to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to a patient.
  • the therapeutically effective amount can be ascertained by measuring relevant physiological effects. For example, in the case of a hyperglycemic condition, a lowering or reduction of blood glucose or an improvement in glucose tolerance test can be used to determine whether the amount of an agent is effective to treat the hyperglycemic condition.
  • a therapeutically effective amount is an amount sufficient to reduce or decrease any level (e.g., a baseline level) of FPG, wherein, for example, the amount is sufficient to reduce a FPG level greater than 200 mg/dl to less than 200 mg/dl, wherein the amount is sufficient to reduce a FPG level between 175 mg/dl and 200 mg/dl to less than the starting level, wherein the amount is sufficient to reduce a FPG level between 150 mg/dl and 175 mg/dl to less than the starting level, wherein the amount is sufficient to reduce a FPG level between 125 mg/dl and 150 mg/dl to less than the starting level, and so on (e.g., reducing FPG levels to less than 125 mg/dl, to less than 120 mg/dl, to less than 115 mg/dl, to less than 110 mg/dl, etc.).
  • a baseline level e.g., a baseline level
  • the effective amount is an amount sufficient to reduce or decrease levels by more than about 10% to 9%, by more than about 9% to 8%, by more than about 8% to 7%), by more than about 7% to 6%, by more than about 6%> to 5%, and so on. More particularly, a reduction or decrease of HbAIc levels by about 0.1%, 0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more is contemplated by the present disclosure.
  • the therapeutically effective amount can be adjusted in connection with dosing regimen and diagnostic analysis of the subject's condition and the like.
  • the phrase "in a sufficient amount to effect a change” means that there is a detectable difference between a level of an indicator measured before (e.g., a baseline level) and after administration of a particular therapy.
  • Indicators include any objective parameter (e.g., level of glucose or insulin) or subjective parameter (e.g., subject's feeling of well-being).
  • glucose tolerance refers to the ability of a subject to control the level of plasma glucose and/or plasma insulin when glucose intake fluctuates.
  • glucose tolerance encompasses the subject's ability to reduce, within about 120 minutes, the level of plasma glucose back to a level determined before the intake of glucose.
  • the terms “diabetes” and “diabetic” refer to a progressive disease of carbohydrate metabolism involving inadequate production or utilization of insulin, frequently characterized by hyperglycemia and glycosuria.
  • pre-diabetes and pre- diabetic refer to a state wherein a subject does not have the characteristics, symptoms and the like typically observed in diabetes, but does have characteristics, symptoms and the like that, if left untreated, may progress to diabetes. The presence of these conditions may be determined using, for example, either the fasting plasma glucose test (FPG) or the oral glucose tolerance test (OGTT). Both require a subject to fast for at least 8 hours prior to initiating the test.
  • FPG fasting plasma glucose test
  • OGTT oral glucose tolerance test
  • a subject's blood glucose is measured after the conclusion of the fasting; generally, the subject fasts overnight and the blood glucose is measured in the morning before the subject eats.
  • a healthy subject would generally have a FPG concentration between 90 and about 100 mg/dl
  • a subject with "pre-diabetes” would generally have a FPG concentration between about 100 and about 125 mg/dl
  • a subject with "diabetes” would generally have a FPG level above about 126 mg/dl.
  • OGTT a subject's blood glucose is measured after fasting and again two hours after drinking a glucose-rich beverage.
  • a healthy subject Two hours after consumption of the glucose-rich beverage, a healthy subject generally has a blood glucose concentration below about 140 mg/dl, a pre-diabetic subject generally has a blood glucose concentration about 140 to about 199 mg/dl, and a diabetic subject generally has a blood glucose concentration about 200 mg/dl or above. While the aforementioned glycemic values pertain to human subjects, normoglycemia, moderate hyperglycemia and overt hyperglycemia are scaled differently in murine subjects.
  • a healthy murine subject after a four-hour fast would generally have a FBG concentration between 100 to 150mg/dl
  • a murine subject with "pre-diabetes” would generally have a FPG concentration between 175 to 250 mg/dl
  • a murine subject with "diabetes” would generally have a FPG concentration between 250 to >600mg/dl.
  • insulin resistance refers to a condition where a normal amount of insulin is unable to produce a normal physiological or molecular response.
  • a hyper-physiological amount of insulin either endogenously produced or exogenously administered, is able to overcome the insulin resistance in whole or in part and produce a biologic response.
  • metabolic syndrome refers to an associated cluster of traits that includes, but is not limited to, hyperinsulinemia, abnormal glucose tolerance, obesity, redistribution of fat to the abdominal or upper body compartment, hypertension, dysfibrinolysis, and dyslipidemia characterized by high triglycerides, low HDL-cholesterol, and small dense LDL particles.
  • Subjects having metabolic syndrome are at risk for development of Type 2 diabetes and, for example, atherosclerosis.
  • glucose metabolism disorder encompasses any disorder
  • Elevated levels of glucose and/or insulin may be manifested in the following diseases, disorders and conditions: hyperglycemia, type II diabetes (e.g., insulin-resistance diabetes), gestational diabetes, type I diabetes, insulin resistance, impaired glucose tolerance, hyperinsulinemia, impaired glucose metabolism, pre-diabetes, metabolic disorders (such as metabolic syndrome which is also referred to as syndrome X), hypoglycemia, and obesity, among others.
  • type II diabetes e.g., insulin-resistance diabetes
  • gestational diabetes type I diabetes
  • type I diabetes insulin resistance
  • impaired glucose tolerance e.g., hyperinsulinemia
  • impaired glucose metabolism e.g., pre-diabetes
  • metabolic disorders such as metabolic syndrome which is also referred to as syndrome X
  • hypoglycemia e.g., obesity, obesity, among others.
  • the Modulators of the present disclosure, and compositions thereof, can be used to, for example, achieve and/or maintain glucose homeostasis, e.g., to reduce glucose level in the bloodstream and/or to reduce insulin level to a range found in a healthy subject.
  • the term "hyperglycemia”, as used herein, refers to a condition in which an elevated amount of glucose circulates in the blood plasma of a subject relative to a healthy individual. Hyperglycemia can be diagnosed using methods known in the art, including measurement of fasting blood glucose levels as described herein.
  • hyperinsulinemia refers to a condition in which there are elevated levels of circulating insulin when, concomitantly, blood glucose levels are either elevated or normal.
  • Hyperinsulinemia can be caused by insulin resistance which is associated with dyslipidemia such as high triglycerides, high cholesterol, high low-density lipoprotein (LDL) and low high-density lipoprotein (HDL); high uric acids levels; polycystic ovary syndrome; type II diabetes and obesity.
  • Hyperinsulinemia can be diagnosed as having a plasma insulin level higher than about 2 ⁇ /mL.
  • Inhibitors refers to agents that, for example, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down-regulate the function or activity of one or more Peptides, such as, for example, antagonists including antibodies, small molecule antagonist compounds, and antagonistic peptides distinguishable from the peptides disclosed herein; other examples of Inhibitors are described below.
  • Activators refers to agents that, for example, stimulate, increase, activate, facilitate, enhance activation, sensitize or up-regulate the function or activity of one or more Peptides, such as, for example, agonists including antibodies, small molecule agonists, the peptides disclosed herein, and other agonistic peptides distinguishable from the peptides disclosed herein; other examples of Activators are described below.
  • Modulators collectively refers to Inhibitors and Activators.
  • Inhibitors and the Activators to decrease or increase, respectively, the function or activity of one or more Peptides (or the nucleic acid molecules encoding them), either directly or indirectly. Modulation may occur in vitro or in vivo.
  • homologues or “variants” are used interchangeably to refer to amino acid or DNA sequences that are similar to reference amino acid or nucleic acid sequences, respectively.
  • homologues may refer to nucleic acid or amino acid sequences in one species that are similar to nucleic acid or amino acid sequences in another species.
  • homologues may refer to nucleic acid or amino acid sequences in one species that are similar to nucleic acid or amino acid sequences in the same species.
  • homologues or variants encompass naturally occurring DNA sequences and proteins encoded thereby and their isoforms.
  • the homologues also include known allelic or splice variants of a protein or gene.
  • Homologues and variants also encompass nucleic acid sequences that vary in one or more bases from a naturally-occurring DNA sequence but still translate into an amino acid sequence that corresponds to the naturally-occurring protein due to degeneracy of the genetic code. Homologues and variants may also refer to those that differ from the naturally- occurring sequences by one or more conservative substitutions and/or tags and/or conjugates.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the terms include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. While the aforementioned terms can be used interchangeably, in general the term “peptide” refers to a polymeric form of amino acids of less than about 50 amino acids in length.
  • DNA DNA
  • nucleic acid nucleic acid molecule
  • polynucleotide polynucleotide
  • Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA
  • mRNA complementary DNA
  • cDNA complementary DNA
  • recombinant polynucleotides vectors, probes, primers and the like.
  • Probe refers to a fragment of DNA or RNA corresponding to a gene or sequence of interest, wherein the fragment has been labeled radioactively (e.g., by
  • a probe can be used to, for example, label viral plaques, bacterial colonies or bands on a gel that contain the gene of interest.
  • a probe can be cloned DNA or it can be a synthetic DNA strand; the latter can be used to obtain a cDNA or genomic clone from an isolated a protein by, for example, microsequencing a portion of the protein, deducing the nucleic acid sequence encoding the protein, synthesizing an oligonucleotide carrying that sequence, radiolabeling the sequence and using it as a probe to screen a cDNA library or a genomic library.
  • heterologous refers to two components that are defined by structures derived from different sources.
  • a “heterologous” polypeptide may include operably linked amino acid sequences that are derived from different polypeptides (e.g., a first component comprising a recombinant peptide and a second component derived from a native Tor2a peptide).
  • a heterologous polynucleotide may include in operably linked nucleic acid sequences that can be derived from different genes (e.g., a first component from a nucleic acid encoding a peptide according to an embodiment disclosed herein and a second component from a nucleic acid encoding a carrier polypeptide).
  • heterologous nucleic acids include expression constructs in which a nucleic acid comprising a coding sequence is operably linked to a regulatory element (e.g., a promoter) that is from a genetic origin different from that of the coding sequence (e.g., to provide for expression in a host cell of interest, which may be of different genetic origin than the promoter, the coding sequence or both).
  • a T7 promoter operably linked to a polynucleotide encoding a Tor2a polypeptide or domain thereof is said to be a heterologous nucleic acid.
  • heterologous can refer to the presence of a nucleic acid (or gene product, such as a polypeptide) that is of a different genetic origin than the host cell in which it is present.
  • operably linked refers to linkage between molecules to provide a desired function.
  • “operably linked” in the context of nucleic acids refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second polynucleotide, wherein the expression control sequence affects transcription and/or translation of the second polynucleotide.
  • “operably linked” refers to a functional linkage between amino acid sequences (e.g., of different domains) to provide for a described activity of the polypeptide.
  • N-terminus As used herein in the context of the structure of a polypeptide, "N-terminus" (or
  • amino terminus and “C-terminus” (or “carboxyl terminus”) refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while the terms “N-terminal” and “C-terminal” refer to relative positions in the amino acid sequence of the polypeptide toward the N-terminus and the C-terminus, respectively, and can include the residues at the N-terminus and C- terminus, respectively.
  • "Immediately N-terminal” or “immediately C-terminal” refers to a position of a first amino acid residue relative to a second amino acid residue where the first and second amino acid residues are covalently bound to provide a contiguous amino acid sequence.
  • “Derived from”, in the context of an amino acid sequence or polynucleotide sequence is meant to indicate that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid (e.g., a naturally occurring Tor2a polypeptide or a Tor2a-encoding nucleic acid), and is not meant to be limiting as to the source or method in which the protein or nucleic acid is made.
  • the term “derived from” includes homologues or variants of reference amino acid or DNA sequences.
  • isolated refers to a peptide of interest that, if naturally occurring, is in an environment different from that in which it may naturally occur. "Isolated” is meant to include peptides that are within samples that are substantially enriched for the peptide of interest and/or in which the peptide of interest is partially or substantially purified. Where the peptide is not naturally occurring, “isolated” indicates the peptide has been separated from an environment in which it was made by either synthetic or recombinant means.
  • Enriched means that a sample is non-naturally manipulated (e.g., by a scientist or a clinician) so that a peptide of interest is present in a) a greater concentration (e.g., at least 3- fold greater, at least 4-fold greater, at least 8-fold greater, at least 64-fold greater, or more) than the concentration of the peptide in the starting sample, such as a biological sample (e.g., a sample in which the peptide naturally occurs or in which it is present after administration), or b) a greater concentration than the environment in which the peptide was made (e.g., as in a bacterial cell).
  • a biological sample e.g., a sample in which the peptide naturally occurs or in which it is present after administration
  • a greater concentration than the environment in which the peptide was made e.g., as in a bacterial cell.
  • substantially pure indicates that a component (e.g., a polypeptide) makes up greater than about 50% of the total content of the composition and typically, greater than about 60%) of the total polypeptide content. More typically, “substantially pure” refers to compositions in which at least 75%, at least 85%, at least 90% or more of the total composition is the component of interest. In some cases, the polypeptide will make up greater than about 90%>, or greater than about 95% of the total content of the composition.
  • a component e.g., a polypeptide
  • antibodies refer to glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Antibodies are described in detail hereafter.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • An "isolated" antibody is one which has been separated and/or recovered from contaminant components of its natural environment; such contaminant components are materials which might interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • Torsin-2A is one which has been separated and/or recovered from contaminant components of its natural environment; such contaminant components are materials which might interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • Torsin-2A (Tor2a)
  • Tor2a also known as "Torsin family 2 member A", “Torsin- related protein 1", or “Prosalusin”
  • Tor2a encompasses human and murine peptides and variants thereof that are encoded by the Tor2a gene or homologs thereof.
  • Tor2a is found in many other mammals, including non-human primates and canines.
  • Torsin-2A (“Tor2a”), encompasses peptides and variants thereof that are encoded by the Tor2a gene or homologs thereof.
  • Tor2a is found in many mammals, including humans, non-human primates, rodents (e.g., mice) and canines.
  • peptides encoded by the Tor2 gene are the salusin peptides, which have previously been described.
  • Salusin-a and Salusin- ⁇ two related peptides, have been shown to cause hypotension and bradycardia in rats (Shichiri et al., Nature Medicine 9(9): 1166-72 (2003).
  • a Peptide of the present disclosure specifically excludes salusin-a and salusin- ⁇
  • Tor2a gene expression is prevalent in enteroendocrine cells (EECs) and enterocytes (ECs) of the gastrointestinal tract.
  • EECs enteroendocrine cells
  • ECs enterocytes
  • Figure 4 Panel a and Figure 4, Panel b depict a sequence alignment of Tor2a amino acid sequences from the indicated species and a sequence alignment of a central region of Tor2a amino acid sequences from the indicated species, respectively.
  • ms pi is identical to ms p2 except that the carboxy amino acid of ms pi is a threonine having a C-terminal amide, and the carboxy amino acid of ms p2 is a glycine having a C-terminal carboxylic acid.
  • Pairs of the other murine peptides i.e., ms p3 and ms p4, ms p5 and ms p6, ms p7 and ms p8, and ms p9 and ms plO
  • pairs of the other murine peptides i.e., ms p3 and ms p4, ms p5 and ms p6, ms p7 and ms p8, and ms p9 and ms plO
  • hu pi is identical to hu p2 except that the carboxyl amino acid of hu pi is a threonine having a c-terminal amide, and the carboxyl amino acid of hu p2 is a glycine having a c-terminal carboxylic acid.
  • Pairs of the other human peptides i.e., hu p3 and hu p4, hu p5 and hu p6, hu p7 and hu p8, and hu p9 and hu plO
  • pairs of the other human peptides i.e., hu p3 and hu p4, hu p5 and hu p6, hu p7 and hu p8, and hu p9 and hu plO
  • hu p3 and hu p4 hu p5 and hu p6, hu p7 and
  • ALVVKALKAFVRDPAPTKPLVLSLHGWTGT-CONH2 (SEQ ID NO:2)
  • Figure 5 indicates the effect of Murine Peptides ms pi - ms plO and Human
  • Human Peptides p5 and p6 are identical to Murine Peptides p5 and p6, respectively, except for two conservative or semi-conservative amino acid substitutions; and Human Peptides p7 - plO are identical to Murine Peptides p7 -plO, respectively, except for three conservative or semi- conservative amino acid substitutions. While the activity of Human Peptides p5 - plO has not been determined, it is predicted to be comparable to that of the corresponding Murine Peptides in view of the conservative and semi-conservative nature of the amino acid substitutions.
  • pancreatic beta-cells reduced insulin production and/or secretion due to a secondary messenger (e.g., glucagon or somatostatin); and increased insulin clearance by peripheral tissues, such as the liver.
  • a secondary messenger e.g., glucagon or somatostatin
  • peptide pi increases glucose levels and reduces glucose-stimulated insulin secretion.
  • the peptide designated pi increases circulating glucose concentration ( Figure 6, Panel a), decreases circulating insulin concentration ( Figure 6, Panel c), exacerbates glucose tolerance (Figure 7, Panel a), decreases glucose-stimulated insulin secretion (Figure 7, Panel c), exacerbates insulin tolerance ( Figure 8, Panel a), and increases glucose production (gluconeogenic capacity) ( Figure 9, Panel a); pi also appears to acutely increase insulin clearance ( Figures 8, Panesl c and d and 9, Panel c). Peptide pi does not significantly modulate body weight. Comparable activity to pi was also observed for p5.
  • Tor2a refers to Peptides of the present disclosure, or fragments thereof, or Nucleic Acid Molecules of the present disclosure, which include their naturally-occurring and non-naturally occurring isoforms, allelic variants and splice variants.
  • a Peptide also refers to peptides that have one or more alterations in the amino acid residues (e.g., at locations that are not conserved across variants or species) while retaining the conserved domains and having the same biological activity as the naturally- occurring Peptides.
  • Tor2a also encompasses nucleic acid sequences that vary in one or more bases from a naturally-occurring DNA sequence but still translate into an amino acid sequence that corresponds to a Peptide due to degeneracy of the genetic code.
  • Tor2a may refer to amino acid sequences that differ from the naturally-occurring sequences (e.g., Peptides) by one or more conservative substitutions, tags, or conjugates.
  • the present disclosure contemplates having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 usually no more than 20, 10, or 5 amino acid substitutions, where the substitution is usually a conservative amino acid substitution.
  • conservative amino acid substitution generally refers to substitution of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E.
  • Conservative amino acid substitutions preserve the activity of the protein by replacing an amino acid(s) in the protein with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or size of the side chain.
  • Guidance for substitutions, insertions, or deletions may be based on alignments of amino acid sequences of different variant proteins or proteins from different species.
  • the Peptides are active fragments containing contiguous amino acid residues derived from the full-length Tor2a polypeptide.
  • Tor2a There are three human isoforms of Tor2a and two murine isoforms of Tor2a that contain the hu pl-hu plO peptides and ms pl-ms plO peptides, respectively (see Figure 3); in Figure 3, the regions encompassing the human peptides and the murine peptides are highlighted in gray.
  • peptides and polypeptides may be from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, from about 40 amino acids to about 50 amino acids, from about 50 amino acids to about 75 amino acids, from about 75 amino acids to about 100 amino acids, from about 100 amino acids to about 150 amino acids, from about 150 amino acids to about 200 amino acids, or from about 200 amino acids up to the full-length peptide or polypeptide.
  • the Peptides have a length that is less than the full length of the naturally-occurring polypeptide.
  • the Peptides can have a length of from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 40 amino acids to about 45 amino acids, from about 45 amino acids to about 50 amino acids, or from more than about 50 amino acids.
  • the peptides contemplated by the present disclosure are less than about 35 amino acids in length (see Figure 2).
  • the Peptides can have a defined sequence identity compared to a reference sequence over a defined length of contiguous amino acids (e.g., a "comparison window").
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci.
  • a suitable Peptide can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%), at least about 98%>, or at least about 99%, amino acid sequence identity to a contiguous stretch of from about 5 amino acids to about 10 amino acids, from about 10 amino acids to 12 amino acids, from about 12 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to 22 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 27 amino acids, from about 27 amino acids to about 29 amino acids, or from more than about 29 amino acids of one of the following reference amino acid sequences:
  • PLVLSLHGATGT-CONH2 (SEQ ID NO: l 1)
  • PLVLSLHGATGTG-COOH (SEQ ID NO: 12)
  • ALVVKALKAFVRDPAPTKPLVLSLHGWTGT-CONH2 (SEQ ID NO:2)
  • a suitable Peptide can comprise an amino acid sequence having at least about 75%, at least about 80%>, at least about 85%>, at least about 90%), at least about 95%>, at least about 98%>, or at least about 99%>, amino acid sequence identity to a contiguous stretch of from about 5 amino acids to about 10 amino acids, from about 10 amino acids to 12 amino acids, from about 12 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to 22 amino acids, or from more than about 22 amino acids of the following sequence:
  • the Peptide has a length of from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 12 amino acids, from about 12 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 22 amino acids, from about 22 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 45 amino acids, from about 45 amino acids to about 50 amino acids, or from more than about 50 amino acids.
  • the carboxyl terminal amino acid can be amidated.
  • the Peptide comprises the amino acid sequence
  • the Peptides may be isolated from a natural source (e.g., in an environment other than its naturally-occurring environment) and also may be recombinantly made (e.g., in a genetically modified host cell such as bacteria; yeast; Pichia; insect cells; and the like), where the genetically modified host cell is modified with a nucleic acid comprising a nucleotide sequence encoding the peptide.
  • a genetically modified host cell such as bacteria; yeast; Pichia; insect cells; and the like
  • the Peptides may also be synthetically produced (e.g., by cell- free chemical synthesis). Methods of productions are described in more detail below.
  • a Peptide may be generated using recombinant techniques to manipulate different Tor2a - related nucleic acids known in the art to provide constructs capable of encoding the Peptide. It will be appreciated that, when provided a particular amino acid sequence, the ordinary skilled artisan will recognize a variety of different nucleic acid molecules encoding such amino acid sequence in view of her background and experience in, for example, molecular biology. .
  • Tor2a - related nucleic acid sequences and amino acid sequences encoding a variety of different Peptides are known and available in the art and include the following (listed with their corresponding GenBank accession nos).: 1) Homo sapiens: amino acid sequence: NP 001078816.1; nucleotide sequence: NM 001085347.2; 2) Homo sapiens: amino acid sequence: NP 001127902.1; nucleotide sequence: NM 001134430.2; 3) Homo sapiens: amino acid sequence: NP 001127903.1; nucleotide sequence: NM 001134431.2; 4) Homo sapiens: amino acid sequence: NP 001238947.1; nucleotide sequence: NM 001252018.1; 5) Homo sapiens: amino acid sequence: NP 001238950.1; nucleotide sequence: NM 001252021.1; 6) Homo sapiens: amino acid sequence: NP 001238952.1;
  • NM 001252023.1 Homo sapiens: amino acid sequence: NP 569726.2; nucleotide sequence: NM 130459.3; 8) Mus musculus: amino acid sequence: NP 690013.1; nucleotide sequence: NM 152800.3; 9) Rattus norvegicus: amino acid sequence: NP 001007745.1; nucleotide sequence: NM 001007744.1; 10) Pan troglodytes: amino acid sequence: XP 520274.2; nucleotide sequence: XM 520274.3; 11) Pan troglodytes: amino acid sequence:
  • Modemators refers to both “Inhibitors” and “Activators” of the
  • the Peptides e.g., peptides pi and p5
  • the Inhibitors e.g., peptides pi and p5
  • contemplated by the present disclosure counteract these effects by, for example, acting to partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or antagonize or down-regulate the effects of the Peptides.
  • the Inhibitor is an antibody that is capable of eliminating or significantly reducing an effector function of a target antigen (e.g., an Invention Peptide) to which it binds.
  • a target antigen e.g., an Invention Peptide
  • An antibody useful according to the present disclosure can be generated by a skilled artisan using routine techniques. For example, one or more antibodies to known antigens may be generated and then evaluated to select at least one with the desired properties.
  • one or more antibodies can be produced and formulated in a manner suitable for administration to a subject.
  • the Inhibitor is a small molecule antagonist compound, antibody-related molecule, or other bioorganic molecule that binds or otherwise blocks (e.g., at or around a Peptide binding site) a Peptide from having its normal endogenous effect.
  • the Inhibitor is an antagonistic peptide structurally distinguishable from the Peptides (e.g., a peptide variant having one or more amino acid deletions, insertions or non-conservative substitutions compared to the Peptides) capable of binding to a receptor (or other effector of activity), but deficient in its ability to mediate a biological response.
  • a receptor or other effector of activity
  • amino acid sequences of the Peptides can be systematically modified by making one or more amino acid deletions, insertions or non-conservative substitutions and then evaluating the activity of the resulting peptides as described in the Examples that follow or in other experimental settings known to the skilled artisan.
  • Activators that include, for example, agents that stimulate, increase, activate, facilitate, enhance activation, sensitize or agonize or up-regulate the function or activity of one or more Peptides.
  • the Activator is a Peptide described herein.
  • the Activator is an agonistic peptide structurally distinguishable from the Peptides (e.g., a variant peptide having one or more amino acid deletions, insertions or non- conservative substitutions compared to the Peptides) but having comparable activity.
  • the skilled artisan is able to identify agonistic peptides having desired properties using
  • the Activator is a small molecule agonist compound having activity similar to glucagon (GLUCAGEN), a linear peptide of 29 amino acids.
  • glucagon activates hepatic gluconeo genesis and stimulates the breakdown of glycogen stored in the liver, causing an increase in blood glucose levels.
  • a skilled medicinal chemist is able to utilize known techniques to identify and optimize a small molecule agonist or other bioorganic molecule having the desired properties.
  • the present disclosure envisions agonists that are both structurally related and unrelated to glucagon and analogs and derivatives thereof.
  • the Activator is an antibody that mimics, in whole or in part, the activity of the Peptides. The generation and use of such antibodies is known in the art (see, e.g., Kifor et al, JCEM (2004) 89(2): 548-56; Dragun et al, NEJM (2005) 352:558- 69).
  • a Peptide includes one or more linkages other than peptide bonds, e.g., at least two adjacent amino acids are joined via a linkage other than an amide bond.
  • linkages other than peptide bonds e.g., at least two adjacent amino acids are joined via a linkage other than an amide bond.
  • one or more amide bonds within the backbone of a Peptide can be substituted.
  • One or more amide linkages in an Invention Peptide can also be replaced by, for example, a reduced isostere pseudopeptide bond. See Couder et al. (1993) Int. J. Peptide Protein Res. 41 : 181-184. Such replacements and how to effect are known to those of ordinary skill in the art.
  • One or more amino acid substitutions can be made in a Peptide.
  • alkyl-substituted hydrophobic amino acids including alanine, leucine, isoleucine, valine, norleucine, (S)-2-aminobutyric acid, (S)-cyclohexylalanine or other simple alpha-amino acids substituted by an aliphatic side chain from CI -CIO carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions;
  • aromatic-substituted hydrophobic amino acids including phenylalanine, tryptophan, tyrosine, sulfotyrosine, biphenylalanine, 1-naphthylalanine, 2- naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, including amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy (from Cl- C4)-substituted forms of the above-listed aromatic amino acids, illustrative examples of which are: 2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4- methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5- methoxytry
  • amino acids containing basic side chains including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, including alkyl, alkenyl, or aryl-substituted (from Ci-Cio branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms (such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position for example.
  • heteroatoms such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon
  • N-epsilon-isopropyl-lysine 3-(4-tetrahydropyridyl)- glycine, 3-(4-tetrahydropyridyl)-alanine, ⁇ , ⁇ -gamma, gamma'-diethyl-homoarginine.
  • compounds such as alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid, alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl group occupies the pro-R position of the alpha-carbon.
  • amides formed from alkyl, aromatic, heteroaromatic where the hetero aromatic group has one or more nitrogens, oxygens or sulfur atoms singly or in combination
  • carboxylic acids or any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives
  • activated derivatives such as acid chlorides, active esters, active azolides and related derivatives
  • lysine, ornithine, or 2,3-diaminopropionic acid any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives
  • substitution of acidic amino acids including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of 2,4- diaminopriopionic acid, ornithine or lysine and tetrazole-substituted alkyl amino acids;
  • a Peptide comprises one or more naturally occurring non- genetically encoded L-amino acids, synthetic L-amino acids or D-enantiomers of an amino acid.
  • a Peptide can comprise only D-amino acids.
  • a Peptide can comprise one or more of the following residues: hydroxyproline, ⁇ -alanine, o-aminobenzoic acid, m- aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2,3-diaminopropionic acid, a-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine,
  • naphthylalanine pyridylalanine 3-benzothienyl alanine, 4-chlorophenylalanine, 2- fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, ⁇ -2-thienylalanine, methionine sulfoxide,
  • homoarginine N-acetyl lysine, 2,4-diamino butyric acid, rho-aminophenylalanine, N- methylvaline, homocysteine, homoserine, ⁇ -amino hexanoic acid, ⁇ -aminohexanoic acid, ⁇ - aminoheptanoic acid, ⁇ -aminooctanoic acid, ⁇ -aminodecanoic acid, ⁇ -aminotetradecanoic acid, cyclohexylalanine, ⁇ , ⁇ -diaminobutyric acid, ⁇ , ⁇ -diaminopropionic acid, ⁇ -amino valeric acid, and 2,3-diaminobutyric acid.
  • a cysteine residue or a cysteine analog can be introduced into a Peptide to provide for linkage to another peptide via a disulfide linkage or to provide for cyclization of the Peptide.
  • Methods of introducing a cysteine or cysteine analog are known in the art; see, e.g., U.S. Patent No. 8,067,532.
  • a Peptide can be cyclized.
  • One or more cysteine or cysteine analogs can be introduced into a Peptide, where the introduced cysteine or cysteine analog can form a disulfide bond with a second introduced cysteine or cysteine analog.
  • Other means of cyclization include introduction of an oxime linker or a lanthionine linker; see, e.g., U.S. Patent No. 8,044,175. Any combination of amino acids (or non-amino acid moiety) that can form a cyclizing bond can be used and/or introduced.
  • a cyclizing bond can be generated with any combination of amino acids (or with amino acid and -(CH 2 ) n -CO- or -(CH 2 ) n -C 6 H 4 -CO-) with functional groups which allow for the introduction of a bridge.
  • Some examples are disulfides, disulfide mimetics such as the - (CH 2 ) n - carba bridge, thioacetal, thioether bridges (cystathionine or lanthionine) and bridges containing esters and ethers.
  • n can be any integer, but is frequently less than ten.
  • hydroxymethyl derivatives O-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides.
  • one or more L-amino acids in a Peptide is replaced with a D- amino acid.
  • a Peptide is a retroinverso analog.
  • Retro-inverso peptide analogs are isomers of linear peptides in which the direction of the amino acid sequence is reversed (retro) and the chirality, D- or L-, of one or more amino acids therein is inverted (inverso) e.g., using D-amino acids rather than L-amino acids. See, e.g., Jameson et al. (1994) Nature 368:744; and Brady et al. (1994) Nature 368:692.
  • a Peptide can include a "Protein Transduction Domain” (PTD), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.
  • PTD Protein Transduction Domain
  • a PTD attached to another molecule facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle.
  • a PTD is covalently linked to the amino terminus of a Peptide.
  • a PTD is covalently linked to the carboxyl terminus of a Peptide.
  • Exemplary protein transduction domains include, but are not limited to, a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV- 1 TAT comprising
  • YGRKKRRQRRR SEQ ID NO:41
  • a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21 : 1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLMKR (SEQ ID NO:42); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:43);
  • RQIKIWFQNRRMKWKK (SEQ ID NO:45).
  • exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO:41), RKKRRQRRR (SEQ ID NO:46); an arginine
  • exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO:41); RKKRRQRRR (SEQ ID NO:46); YARAAARQARA (SEQ ID NO:47); THRLPRRRRRR (SEQ ID NO:48); and GGRRARRRRRR (SEQ ID NO:49).
  • the carboxyl group can also be esterified with primary, secondary or tertiary alcohols such as, e.g., methanol, branched or unbranched Cl-C6-alkyl alcohols, e.g., ethyl alcohol or tert-butanol.
  • the carboxyl group can also be amidated with primary or secondary amines such as ammonia, branched or unbranched Cl-C6-alkylamines or C1-C6 di-alkylamines, e.g., methylamine or dimethylamine.
  • primary or secondary amines such as ammonia, branched or unbranched Cl-C6-alkylamines or C1-C6 di-alkylamines, e.g., methylamine or dimethylamine.
  • the amino group can be present in a form protected by amino-protecting groups conventionally used in peptide chemistry such as, e.g., Fmoc, Benzyloxy-carbonyl (Z), Boc, or Alloc.
  • Alkyl residues can be straight- chained, branched or cyclic (e.g., ethyl, isopropyl and cyclohexyl, respectively).
  • a Peptide can include one or more modifications that enhance a property desirable in a protein formulated for therapy (e.g., serum half-life), that enable the raising of antibodies for use in detection assays (e.g., epitope tags), that provide for ease of protein purification, etc.
  • a Peptide is modified by conjugating (e.g., linking) it to one or more additional elements at the N- and/or C-terminus of the peptide, such as another protein (e.g., a protein having an amino acid sequence heterologous to the subject peptide) or a carrier molecule.
  • another protein e.g., a protein having an amino acid sequence heterologous to the subject peptide
  • a carrier molecule e.g., an exemplary protein can be provided as a fusion protein with a polypeptide(s) derived from a Peptide.
  • Glycosylation is another type of modification of a protein.
  • glycosylation is meant to broadly refer to the enzymatic process that attaches glycans to proteins, lipids or other organic molecules. The use of the term
  • glycosylation in conjunction with the present disclosure is generally intended to mean adding or deleting one or more carbohydrate moieties (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that may or may not be present in the native sequence.
  • the phrase includes qualitative changes in the glycosylation of the native proteins involving a change in the nature and proportions of the various carbohydrate moieties present.
  • Glycosylation can dramatically affect the physical properties of proteins and can also be important in protein stability, secretion, and subcellular localization. Proper
  • glycosylation can be essential for biological activity.
  • some genes from eucaryotic organisms when expressed in bacteria (e.g., E. coli) which lack cellular processes for glycosylating proteins, yield proteins that are recovered with little or no activity by virtue of their lack of glycosylation.
  • Addition of glycosylation sites can be accomplished by altering the amino acid sequence.
  • the alteration to the peptide sequence may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites).
  • the structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type may be different.
  • One type of sugar that is commonly found on both is N-acetylneuraminic acid (hereafter referred to as sialic acid).
  • sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycoprotein.
  • the peptide sequences of the present disclosure may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the peptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the peptide polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Removal of carbohydrates may be accomplished chemically or enzymatically, or by substitution of codons encoding amino acid residues that are glycosylated. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases.
  • DHFR Dihydrofolate reductase
  • CHO Chinese Hamster Ovary
  • Another type of modification is to conjugate (e.g., link) one or more additional components or molecules at the N- and/or C-terminus of a peptide sequence, such as another protein (e.g., a protein having an amino acid sequence heterologous to the subject protein), or a carrier molecule.
  • a peptide sequence such as another protein (e.g., a protein having an amino acid sequence heterologous to the subject protein), or a carrier molecule.
  • another protein e.g., a protein having an amino acid sequence heterologous to the subject protein
  • a carrier molecule e.g., a protein having an amino acid sequence heterologous to the subject protein
  • the amino- or carboxy- terminus of a peptide sequence of the present disclosure can be fused with an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule).
  • Fc fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, and thus the biopharmaceutical product may require less frequent administration.
  • a conjugate modification may result in a peptide sequence that retains activity with an additional or complementary function or activity of the second molecule.
  • a peptide sequence may be conjugated to a molecule, e.g., to facilitate solubility, storage, in vivo or shelf half-life or stability, reduction in immunogenicity, delayed or controlled release in vivo, etc..
  • Other functions or activities include a conjugate that reduces toxicity relative to an unconjugated peptide sequence, a conjugate that targets a type of cell or organ more efficiently than an unconjugated peptide sequence, or a drug to further counter the causes or effects associated with a disorder or disease as set forth herein (e.g., diabetes).
  • nonproteinaceous polymer e.g., a PEG.
  • PEG-conjugated biomolecules have been shown to possess clinically useful properties, including better physical and thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulating half-life and decreased clearance, reduced immunogenicity and antigenicity, and reduced toxicity.
  • PEGs suitable for conjugation to a peptide sequence are generally soluble in water at room temperature, and have the general formula R(0-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons.
  • the PEG conjugated to the peptide sequence can be linear or branched. Branched PEG derivatives, "star- PEGs" and multi-armed PEGs are contemplated by the present disclosure.
  • a molecular weight of the PEG used in the present disclosure is not restricted to any particular range, but certain embodiments have a molecular weight between 500 and 20,000 while other embodiments have a molecular weight between 4,000 and 10,000.
  • compositions of conjugates wherein the
  • Such compositions can be produced by reaction conditions and purification methods know in the art.
  • PEG may be bound to a peptide of the present disclosure via a terminal reactive group (a "spacer").
  • the spacer is, for example, a terminal reactive group which mediates a bond between the free amino or carboxyl groups of one or more of the peptides and polyethylene glycol.
  • the PEG having the spacer which may be bound to the free amino group includes N- hydroxysuccinylimide polyethylene glycol which may be prepared by activating succinic acid ester of polyethylene glycol with N-hydroxysuccinylimide.
  • Another activated polyethylene glycol which may be bound to free amino group is 2,4-bis(0-methoxypolyethyleneglycol)-6- chloro-s-triazine which may be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride.
  • the activated polyethylene glycol which is bound to the free carboxyl group includes polyoxyethylenediamine.
  • Conjugation of one or more of the peptide sequences of the present disclosure to PEG having the spacer may be carried out by various conventional methods.
  • the conjugation reaction can be carried out in solution at a pH of from 5 to 10, at temperature from 4°C to room temperature, for 30 minutes to 20 hours, utilizing a molar ratio of reagent to protein of from 4: 1 to 30: 1.
  • Various means known in the art may be used to terminate the reaction. In some embodiments the reaction is terminated by acidifying the reaction mixture and freezing at, e.g., -20°C.
  • a Peptide may also be conjugated to large, slowly metabolized macromolecules such as proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads;
  • polymeric amino acids such as polyglutamic acid, polylysine; amino acid copolymers;
  • conjugated virus particles inactivated virus particles
  • inactivated bacterial toxins such as toxoid from diphtheria, tetanus, cholera, leukotoxin molecules
  • inactivated bacteria inactivated bacteria
  • dendritic cells dendritic cells
  • conjugated forms if desired, can be used to produce antibodies against a peptide of the present disclosure.
  • Additional suitable components and molecules for conjugation include, for example, thyro globulin; albumins such as human serum albumin (HAS); tetanus toxoid;
  • Diphtheria toxoid polyamino acids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses; influenza virus hemaglutinin, influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B virus core protein and surface antigen; or any combination of the foregoing.
  • polyamino acids such as poly(D-lysine:D-glutamic acid)
  • VP6 polypeptides of rotaviruses rotaviruses
  • influenza virus hemaglutinin influenza virus nucleoprotein
  • KLH Keyhole Limpet Hemocyanin
  • hepatitis B virus core protein and surface antigen or any combination of the foregoing.
  • Fusion of albumin to one or more peptides of the present disclosure can, for example, be achieved by genetic manipulation, such that the DNA coding for HSA, or a fragment thereof, is joined to the DNA coding for the one or more peptide sequences.
  • a suitable host can be transformed or transfected with the fused nucleotide sequences in the form of, for example, a suitable plasmid, so as to express a fusion polypeptide.
  • the expression may be effected in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo from, for example, a transgenic organism.
  • the expression of the fusion protein is performed in mammalian cell lines, for example, CHO cell lines. Transformation is used broadly herein to refer to the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).
  • Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells.
  • Additional suitable components and molecules for conjugation include those suitable for isolation or purification.
  • Particular non-limiting examples include binding molecules, such as biotin (biotin-avidin specific binding pair), an antibody, a receptor, a ligand, a lectin, or molecules that comprise a solid support, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.
  • cation exchange chromatography may be used to separate conjugates by charge difference, which effectively separates conjugates into their various molecular weights.
  • the cation exchange column can be loaded and then washed with ⁇ 20 mM sodium acetate, pH ⁇ 4, and then eluted with a linear (0M to 0.5M) NaCl gradient buffered at a pH from 3 to 5.5, e.g., at pH ⁇ 4.5.
  • the content of the fractions obtained by cation exchange chromatography may be identified by molecular weight using conventional methods, for example, mass spectroscopy, SDS-PAGE, or other known methods for separating molecular entities by molecular weight.
  • immunogenicity or allergenicity of a peptide of the present invention involve modification of the peptide sequences by hesylation, which utilizes hydroxyethyl starch derivatives linked to other molecules in order to modify the molecule's characteristics.
  • hesylation utilizes hydroxyethyl starch derivatives linked to other molecules in order to modify the molecule's characteristics.
  • Various aspects of hesylation are described in, for example, U.S. Patent Appln. Nos. 2007/0134197 and 2006/0258607.
  • Suitable linkers include "flexible linkers" which are generally of sufficient length to permit some movement between the modified peptide sequences and the linked components and molecules.
  • the linker molecules are generally about 6-50 atoms long.
  • the linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.
  • Suitable linkers can be readily selected and can be of any suitable length, such as 1 (e.g., Gly) , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 amino acids (e.g., Gly).
  • Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (for example, (GS)n, GSGGSn (SEQ ID NO:50) and GGGSn (SEQ ID NO:51), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • Glycine and glycine-serine polymers are relatively unstructured, and therefore may serve as a neutral tether between components.
  • Exemplary flexible linkers include, but are not limited to GGSG (SEQ ID NO:52), GGSGG (SEQ ID NO:53), GSGSG (SEQ ID NO:54), GSGGG (SEQ ID NO:55), GGGSG (SEQ ID NO:56), and GSSSG (SEQ ID NO:57).
  • a peptide of the present disclosure can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis).
  • SPPS Solid-phase peptide synthesis
  • Fmoc and Boc are available for synthesizing peptides of the present disclosure. Details of the chemical synthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8).
  • Solid phase peptide synthesis may be performed as described hereafter.
  • the a functions (Na) and any reactive side chains are protected with acid-labile or base-labile groups.
  • the protective groups are stable under the conditions for linking amide bonds but can be readily cleaved without impairing the peptide chain that has formed.
  • Suitable protective groups for the a-amino function include , but are not limited to, the following: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), o-chlorbenzyloxycarbonyl, bi-phenylisopropyloxycarbonyl, tert- amyloxycarbonyl (Amoc), a, a-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl, 2- cyano-t-butoxy-carbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), l-(4,4-dimethyl-2,6- dioxocylohex-l-ylidene)ethyl (Dde) and the like.
  • 9-fluorenylmethoxycarbonyl can be used as the Na-protective group.
  • Suitable side chain protective groups include, but are not limited to: acetyl, allyl
  • the C-terminal amino acid is coupled to a suitable support material.
  • suitable support materials are those which are inert towards the reagents and reaction conditions for the step-wise condensation and cleavage reactions of the synthesis process and which do not dissolve in the reaction media being used.
  • Examples of commercially- available support materials include styrene/divinylbenzene copolymers which have been modified with reactive groups and/or polyethylene glycol; chloromethylated
  • TentaGel® derivatized with 4-benzyloxybenzyl-alcohol (Wang-anchor) or 2-chlorotrityl chloride can be used if it is intended to prepare the peptidic acid.
  • polystyrene (1%) divinylbenzene or TentaGel® derivatized with 5-(4'-aminomethyl)- 3',5'-dimethoxyphenoxy)valeric acid (PAL-anchor) or p-(2,4-dimethoxyphenyl-amino methyl)- phenoxy group (Rink amide anchor) can be used.
  • the linkage to the polymeric support can be achieved by reacting the C-terminal
  • Fmoc-protected amino acid with the support material with the addition of an activation reagent in ethanol, acetonitrile, ⁇ , ⁇ -dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N- methylpyrrolidone or similar solvents at room temperature or elevated temperatures (e.g., between 40°C and 60°C) and with reaction times of, e.g., 2 to 72 hours.
  • an activation reagent in ethanol, acetonitrile, ⁇ , ⁇ -dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N- methylpyrrolidone or similar solvents at room temperature or elevated temperatures (e.g., between 40°C and 60°C) and with reaction times of, e.g., 2 to 72 hours.
  • PAL Wang or Rink anchor can, for example, be carried out with the aid of coupling reagents such as ⁇ , ⁇ '-dicyclohexylcarbodiimide (DCC), ⁇ , ⁇ '-diisopropylcarbodiimide (DIC) or other carbodiimides, 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU) or other uronium salts, o-acyl-ureas, benzotriazol-l-yl-tris-pyrrolidino-phosphonium
  • DCC ⁇ , ⁇ '-dicyclohexylcarbodiimide
  • DIC ⁇ , ⁇ '-diisopropylcarbodiimide
  • TBTU 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate
  • hexafluorophosphate PyBOP or other phosphonium salts, N-hydroxysuccinimides, other N- hydroxyimides or oximes in the presence or also in the absence of 1-hydroxybenzotriazole or 1- hydroxy-7-azabenzotriazole, e.g., with the aid of TBTU with addition of HOBt, with or without the addition of a base such as for example diisopropylethylamine (DIEA), triethylamine or N- methylmorpholine, e.g., diisopropylethylamine with reaction times of 2 to 72 hours (e.g., 3 hours in a 1.5 to 3-fold excess of the amino acid and the coupling reagents, e.g., in a 2-fold excess and at temperatures between about 10°C and 50°C, e.g., 25°C in a solvent such as dimethylformamide, N-methylpyrrolidone or dichloromethane, e.g.,
  • the Na-protected amino acid e.g., the Fmoc amino acid
  • the Na-protected amino acid can be coupled to the 2-chlorotrityl resin in dichloromethane with the addition of DIEA with reaction times of 10 to 120 minutes, e.g., 20 minutes, but is not limited to the use of this solvent and this base.
  • the successive coupling of the protected amino acids can be carried out according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer.
  • the next protected amino acid in a 3 to 10-fold excess is coupled to the previous amino acid in an inert, non-aqueous, polar solvent such as dichloromethane, DMF or mixtures of the two and at temperatures between about 10°C and 50°C, e.g., at 25°C.
  • the peptide is cleaved from the support material while simultaneously cleaving the side chain protecting groups. Cleavage can be carried out with trifluoroacetic acid or other strongly acidic media with addition of 5%-20% V/V of scavengers such as dimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m- cresol, anisole ethanedithiol, phenol or water, e.g., 15% v/v dimethylsulfide/ethanedithiol/m- cresol 1 : 1 : 1, within 0.5 to 3 hours, e.g., 2 hours. Peptides with fully protected side chains are obtained by cleaving the 2-chlorotrityl anchor with glacial acetic acid
  • the protected peptide can be purified by chromatography on silica gel. If the peptide is linked to the solid phase via the Wang anchor and if it is intended to obtain a peptide with a C-terminal alkylamidation, the cleavage can be carried out by amino lysis with an alkylamine or fluoroalkylamine. The amino lysis is carried out at temperatures between about -10°C and 50°C, e.g., about 25°C and reaction times between about 12 and 24 hours, e.g., about 18 hours. In addition the peptide can also be cleaved from the support by re-esterification, e.g., with methanol.
  • the acidic solution that is obtained may be admixed with a 3 to 20-fold amount of cold ether or n-hexane, e.g., a 10-fold excess of diethyl ether, in order to precipitate the peptide and hence to separate the scavengers and cleaved protective groups that remain in the ether.
  • a further purification can be carried out by re-precipitating the peptide several times from glacial acetic acid.
  • the precipitate that is obtained can be taken up in water or tert- butanol or mixtures of the two solvents, e.g., a 1 : 1 mixture of tert-butanol/water, and freeze-dried.
  • the peptide obtained can be purified by various chromatographic methods, including ion exchange over a weakly basic resin in the acetate form; hydrophobic adsorption chromatography on non-derivatized polystyrene/divinylbenzene copolymers (e.g., Amberlite® XAD); adsorption chromatography on silica gel; ion exchange chromatography, e.g., on carboxymethyl cellulose; distribution chromatography, e.g., on Sephadex® G-25;
  • HPLC high pressure liquid chromatography
  • the peptide may be produced as an intracellular protein or as an secreted protein, using any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E. coli) or a yeast host cell, respectively.
  • a prokaryotic or eukaryotic cell such as a bacterial (e.g., E. coli) or a yeast host cell, respectively.
  • Other examples of eukaryotic cells that may be used as host cells include insect cells, mammalian cells, and/or plant cells.
  • mammalian host cells may include human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells (e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos 7 and CV1) and hamster cells (e.g., Chinese hamster ovary (CHO) cells).
  • human cells e.g., HeLa, 293, H9 and Jurkat cells
  • mouse cells e.g., NIH3T3, L cells, and C127 cells
  • primate cells e.g., Cos 1, Cos 7 and CV1
  • hamster cells e.g., Chinese hamster ovary (CHO) cells.
  • a variety of host- vector systems suitable for the expression of a peptide may be employed according to standard procedures known in the art. See, e.g., Sambrook et al, 1989 Current Protocols in Molecular Biology Cold Spring Harbor Press, New York and Ausubel et al. 1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The method for transfer can be selected so as to provide for stable expression of the introduced polypeptide-encoding nucleic acid.
  • the polypeptide-encoding nucleic acid can be provided as an inheritable episomal element (e.g., a plasmid) or can be genomically integrated.
  • Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome.
  • the expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression where the coding region is operably-linked under the transcriptional control of the
  • transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7, and the like).
  • Expression constructs generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest.
  • a selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector.
  • the expression construct may include additional elements.
  • the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selectable genes are well known in the art and will vary with the host cell used.
  • Isolation and purification of a protein can be accomplished according to methods known in the art.
  • a protein can be isolated from a lysate of cells genetically modified to express the protein constitutively and/or upon induction, or from a synthetic reaction mixture by immunoaffinity purification, which generally involves contacting the sample with an anti- protein antibody, washing to remove non-specifically bound material, and eluting the specifically bound protein.
  • the isolated protein can be further purified by dialysis and other methods normally employed in protein purification methods.
  • the protein may be isolated using metal chelate chromatography methods. Proteins may contain modifications to facilitate isolation.
  • the peptides may be prepared in substantially pure or isolated form (e.g., free from other polypeptides).
  • the peptides can present in a composition that is enriched for the peptide relative to other components that may be present (e.g., other polypeptides or other host cell components).
  • Purified peptide may be provided such that the peptide is present in a composition that is substantially free of other expressed proteins, e.g., less than 90%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1%, of the composition is made up of other expressed proteins.
  • antibody encompasses intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody binding fragments including Fab and F(ab)' 2 , provided that they exhibit the desired biological activity.
  • the basic antibody structural unit comprises a tetramer, and each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” chain (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • each chain defines a constant region primarily responsible for effector function.
  • Human light chains are classified as kappa and lambda light chains, whereas human heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively.
  • Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab') 2 , Fv, and single-chain antibodies.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids.
  • the antibody chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper-variable regions, also called complementarity-determining regions or CDRs.
  • both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments.
  • binding fragments may be produced by enzymatic or chemical cleavage of intact antibodies. Digestion of antibodies with the enzyme papain results in two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc” fragment which has no antigen-binding activity. Digestion of antibodies with the enzyme pepsin results in a F(ab') 2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab') 2 fragment has the ability to crosslink antigen.
  • Fab refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.
  • Fv refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. In a two-chain Fv species, this region includes a dimer of one heavy-chain and one light-chain variable domain in non-covalent association. In a single-chain Fv species, one heavy-chain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species.
  • variable domain interacts to define an antigen-binding site on the surface of the VH-VL dimer. While the six CDRs, collectively, confer antigen- binding specificity to the antibody, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen.
  • CDRs complementarity determining regions
  • hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a “complementarity determining region” or "CDR" and/or those residues from a “hypervariable loop”.
  • epitopic determinants refers to binding sites for antibodies on protein antigens.
  • Epitopic determinants usually comprise chemically active surface groupings of molecules such as amino acids or sugar side chains, as well as specific three dimensional structural and charge characteristics.
  • An antibody is said to bind an antigen when the dissociation constant is ⁇ 1 ⁇ , ⁇ 100 nM, or ⁇ 10 nM.
  • An increased equilibrium constant means that there is less affinity between the epitope and the antibody, whereas a decreased equilibrium constant means that there is a higher affinity between the epitope and the antibody.
  • An antibody with a KD of "no more than" a certain amount means that the antibody will bind to the epitope with the given KD or more strongly.
  • KD describes the binding characteristics of an epitope and an antibody
  • potency describes the effectiveness of the antibody itself for a function of the antibody. There is not necessarily a correlation between an equilibrium constant and potency; thus, for example, a relatively low KD does not automatically mean a high potency.
  • the term "selectively binds" in reference to an antibody does not mean that the antibody only binds to a single substance, but rather that the KD of the antibody to a first substance is less than the KD of the antibody to a second substance.
  • An antibody that exclusively binds to an epitope only binds to that single epitope.
  • antibodies that contain rodent i.e., murine or rat
  • rodent i.e., murine or rat
  • fully human antibodies can be generated through the introduction of human antibody function into a rodent so that the rodent produces fully human antibodies.
  • human and “fully human” antibodies can be used interchangeably herein.
  • the term “fully human” can be useful when distinguishing antibodies that are only partially human from those that are completely, or fully human. The skilled artisan is aware of various methods of generating fully human antibodies.
  • Chimeric or otherwise humanized antibodies can be utilized. Chimeric antibodies have a human constant region and a murine variable region, and, as such, human anti-chimeric antibody responses may be observed in some patients. Therefore, it is advantageous to provide fully human antibodies against multimeric enzymes in order to avoid possible human anti-mouse antibody or human anti-chimeric antibody responses.
  • Fully human monoclonal antibodies can be prepared, for example, by the generation of hybridoma cell lines by techniques known to the skilled artisan. Other preparation methods involve the use of sequences encoding particular antibodies for transformation of a suitable mammalian host cell, such as a CHO cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene -mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to CHO cells, HeLa cells, and human hepatocellular carcinoma cells.
  • the antibodies of the present disclosure can be used diagnostically and/or therapeutically.
  • the antibodies can be used as a diagnostic by detecting the level of one or more peptides of the present disclosure in a subject, and either comparing the detected level to a standard control level or to a baseline level in a subject determined previously (e.g., prior to any illness).
  • the antibodies can be used as a therapeutic to modulate the activity of one or more peptides of the present disclosure, thereby having an effect on a condition or disorder associated with the one or more peptides.
  • dAbs are the smallest functional binding units of human antibodies (IgGs) and have favorable stability and solubility characteristics.
  • the technology entails a dAb(s) conjugated to HSA (thereby forming a "AlbudAb”; see, e.g., EP1517921B, WO2005/118642 and WO2006/051288) and a molecule of interest (e.g., a peptide of the present disclosure).
  • AlbudAbs are often smaller and easier to manufacture in microbial expression systems, such as bacteria or yeast, than current technologies used for extending the serum half- life of peptides. As HSA has a half-life of about three weeks, the resulting conjugated molecule improves the half-life of the molecule of interest.
  • Use of the dAb technology may also enhance the efficacy of the molecule of interest.
  • the present disclosure provides methods for treating or preventing
  • Such methods may also have an advantageous effect on one or more of symptoms associated with a disease, disorder or condition by, for example, decreasing the severity or the frequency of a symptom.
  • a subject may be a candidate for the treatment or prevention of hyperglycemia, hyperinsulinemia, glucose intolerance, and/or glucose disorders by the methods provided herein.
  • various diagnostic methods known in the art may be utilized. Such methods include those described elsewhere herein (e.g., fasting plasma glucose (FPG) evaluation and the oral glucose tolerance test (oGTT)).
  • FPG fasting plasma glucose
  • oGTT oral glucose tolerance test
  • a subject may be considered obese or overweight by assessment of the subject's Body Mass Index (BMI).
  • BMI Body Mass Index
  • An adult having a BMI in the range of 18.5 to 24.9 kg/m is considered to have a normal weight; an adult having a BMI between 25 and 29.9 kg/m may be considered overweight (pre -obese); an adult who has a BMI of 30 kg/m or higher may be considered obese.
  • the Modulators of the present disclosure may be in the form of compositions suitable for administration to a subject.
  • compositions are "pharmaceutical compositions" comprising one or more Modulators and one or more pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients.
  • the Modulators are present in a therapeutically acceptable amount.
  • the pharmaceutical compositions may be used in the methods of the present disclosure ; thus, for example, the pharmaceutical compositions can be administered ex vivo or in vivo to a subject in order to practice the therapeutic and prophylactic methods and uses described herein.
  • compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein.
  • pharmaceutical compositions may be used in combination with other therapeutically active agents or compounds (e.g., glucose lowering agents) as described herein in order to treat or prevent the diseases, disorders and conditions as contemplated by the present disclosure.
  • compositions typically comprise a therapeutically effective amount of at least one of the Modulators contemplated by the present disclosure and one or more pharmaceutically and physiologically acceptable formulation agents.
  • pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, fillers, bulking agents, detergents, buffers, vehicles, diluents, and/or adjuvants.
  • a suitable vehicle may be physiological saline solution or citrate buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Typical buffers include, but are not limited to pharmaceutically acceptable weak acids, weak bases, or mixtures thereof.
  • the buffer components are water soluble materials such as phosphoric acid, tartaric acids, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof.
  • Acceptable buffering agents include, for example, a Tris buffer, N-(2-aminoethyroxine, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium
  • HPES Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)
  • 2-(N-Morpholino)ethanesulfonic acid MES
  • 2-(N-Morpholino)ethanesulfonic acid sodium salt MES
  • 3-(N- Morpholino)propanesulfonic acid MOPS
  • N-tris[Hydroxymethyl]methyl-3- aminopropanesulfonic acid TAPS
  • a pharmaceutical composition After a pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in a ready to use form, a lyophilized form requiring reconstitution prior to use, a liquid form requiring dilution prior to use, or other acceptable form.
  • the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, ampoule, syringe, or autoinjector (similar to, e.g., an EpiPen®)), whereas a multi-use container (e.g., a multi-use vial) is provided in other embodiments.
  • a single-use container e.g., a single-use vial, ampoule, syringe, or autoinjector (similar to, e.g., an EpiPen®)
  • a multi-use container e.g
  • Any drug delivery apparatus may be used to deliver the Peptides, including implants (e.g., implantable pumps) and catheter systems, both of which are well known to the skilled artisan.
  • Depot injections which are generally administered subcutaneously or intramuscularly, may also be utilized to release the peptides disclosed herein over a defined period of time. Depot injections are usually either solid- or oil-based and generally comprise at least one of the formulation components set forth herein.
  • implants e.g., implantable pumps
  • catheter systems both of which are well known to the skilled artisan.
  • Depot injections which are generally administered subcutaneously or intramuscularly, may also be utilized to release the peptides disclosed herein over a defined period of time. Depot injections are usually either solid- or oil-based and generally comprise at least one of the formulation components set forth herein.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents been mentioned herein.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3- butane diol.
  • Acceptable diluents, solvents and dispersion media that may be employed include water, Ringer's solution, isotonic sodium chloride solution, Cremophor ELTM (BASF,
  • Parsippany NJ or phosphate buffered saline (PBS), ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
  • PBS phosphate buffered saline
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables. Prolonged absorption of particular injectable formulations can be achieved by including an agent that delays absorption (e.g., aluminum monostearate or gelatin).
  • compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microbeads or elixirs.
  • Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets, capsules and the like suitable for oral administration may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time-delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by techniques known in the art to form osmotic therapeutic tablets for controlled release.
  • Additional agents include biodegradable or biocompatible particles or a polymeric substance such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides, polyglycolic acid, ethylene -vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers,
  • the oral agent can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, by the use of hydroxymethylcellulose or gelatin-microcapsules or poly (methylmethacrolate)
  • colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Methods of preparing liposomes are described in, for example, U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. Methods for the preparation of the above-mentioned formulations will be apparent to those skilled in the art and are commercially available.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose
  • water or an oil medium for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture thereof.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxy-ethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, ka
  • the pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth; naturally-occurring phosphatides, for example, soy bean, lecithin, and esters or partial esters derived from fatty acids; hexitol anhydrides, for example, sorbitan monooleate; and condensation products of partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
  • Formulations can also include carriers to protect the composition against rapid degradation or elimination from the body, such as a controlled release formulation, including implants, liposomes, hydrogels, prodrugs and microencapsulated delivery systems.
  • a time delay material such as glyceryl monostearate or glyceryl stearate alone, or in combination with a wax, may be employed.
  • the present disclosure contemplates the administration of a Peptide in the form of suppositories for rectal administration of the drug.
  • the suppositories can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter and polyethylene glycols.
  • Modulators contemplated by the present disclosure may be in the form of any other suitable pharmaceutical composition (e.g., sprays for nasal or inhalation use) currently known or developed in the future.
  • suitable pharmaceutical composition e.g., sprays for nasal or inhalation use
  • concentration of a peptide or fragment thereof in a formulation can vary widely (e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50%) or more by weight) and will usually be selected primarily based on fluid volumes, viscosities, and subject-based factors in accordance with, for example, the particular mode of administration selected.
  • Routes of Administration e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50%
  • Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral
  • intraparenchymal and intracerebro ventricular
  • oral nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), sublingual and inhalation.
  • Depot injections which are generally administered subcutaneously or intramuscularly, may also be utilized to release the peptides disclosed herein over a defined period of time.
  • Depot injections are usually either solid- or oil-based and generally comprise at least one of the formulation components set forth herein.
  • One of ordinary skill in the art is familiar with possible formulations and uses of depot injections.
  • an antibody or antibody fragment of the present disclosure is stored at 10 mg/ml in sterile isotonic aqueous saline solution for injection at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to
  • the antibody is administered by intravenous infusion over the course of 1 hour at a dose of between 0.2 and 10 mg/kg. In other embodiments, the antibody is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via subcutaneous bolus injection.
  • the present disclosure contemplates the use of the Modulators in combination with one or more active therapeutic agents or other prophylactic or therapeutic modalities.
  • the various active agents frequently have different mechanisms of action.
  • Such combination therapy may be especially advantageous by allowing a dose reduction of one or more of the agents, thereby reducing or eliminating the adverse effects associated with one or more of the agents; furthermore, such combination therapy may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder, or condition
  • kits therapies that can be administered separately, e.g., formulated separately for separate administration (e.g., as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a "co-formulation").
  • the Modulators are administered or applied sequentially, e.g., where one agent is administered before or one or more other agents. In other embodiments, the Modulators are administered simultaneously, e.g., where two or more agents are
  • the two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether the two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for purposes of the present disclosure.
  • Modulators of the present disclosure can be used in combination with other agents useful in the treatment, prevention, suppression or amelioration of the diseases, disorders or conditions set forth herein, including those that are normally administered to subjects suffering from hyperglycemia, hyperinsulinemia, glucose intolerance, and other glucose metabolism disorders,.
  • insulin insulin mimetics and agents that entail stimulation of insulin secretion, including sulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) and meglitinides (e.g., repaglinide (PRANDIN) and nateglinide (STARLIX)); 2) biguanides (e.g., metformin (GLUCOPHAGE)) and other agents that act by promoting glucose utilization, reducing hepatic glucose production and/or diminishing intestinal glucose output; 3) alpha-glucosidase inhibitors (e.g., acarbose and miglitol) and other agents that slow down carbohydrate digestion and consequently absorption from the gut and reduce postprandial hyperglycemia; 4) thiazolidinediones (e.g., rosiglitazone (AV
  • the present disclosure contemplates combination therapy with agents and methods for promoting weight loss, such as agents that stimulate metabolism or decrease appetite, and modified diets and/or exercise regimens to promote weight loss.
  • the Modulators of the present disclosure may be used in combination with one or more other agent in any manner appropriate under the circumstances.
  • treatment with the at least one active agent, and at least one Modulator of the present disclosure is maintained over a period of time.
  • treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable), while treatment with the Modulator of the present disclosure is maintained at a constant dosing regimen.
  • treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable), while treatment with the Modulator of the present disclosure is reduced (e.g., lower dose, less frequent dosing or shorter treatment regimen).
  • treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable), and treatment with the Modulator of the present disclosure is increased (e.g., higher dose, more frequent dosing or longer treatment regimen).
  • treatment with the at least one active agent is maintained and treatment with the Modulator of the present disclosure is reduced or discontinued (e.g., lower dose, less frequent dosing or shorter treatment regimen).
  • treatment with the at least one active agent and treatment with the Modulator of the present disclosure are reduced or discontinued (e.g., lower dose, less frequent dosing or shorter treatment regimen).
  • the Modulators of the present disclosure may be administered to a subject in an amount that is dependent upon, for example, the goal of the administration (e.g., the degree of resolution desired); the age, weight, sex, and health and physical condition of the subject to be treated; the nature of the Modulator, and/or formulation being administered; the route of administration; and the nature of the disease, disorder, condition or symptom thereof (e.g., the severity of the dysregulation of glucose/insulin and the stage of the disorder).
  • the dosing regimen may also take into consideration the existence, nature, and extent of any adverse effects associated with the agent(s) being administered. Effective dosage amounts and dosage regimens can readily be determined from, for example, safety and dose-escalation trials, in vivo studies (e.g., animal models), and other methods known to the skilled artisan.
  • dosing parameters dictate that the dosage amount be less than an amount that could be irreversibly toxic to the subject (i.e., the maximum tolerated dose,
  • MTD pharmacokinetic and pharmacodynamic parameters associated with absorption, distribution, metabolism, and excretion
  • An effective dose is the dose or amount of an agent that produces a therapeutic response or desired effect in some fraction of the subjects taking it.
  • the "median effective dose” or ED50 of an agent is the dose or amount of an agent that produces a therapeutic response or desired effect in 50% of the population that takes it.
  • the ED50 is commonly used as a measure of reasonable expectance of an agent's effect, it is not necessarily the dose that a clinician might deem appropriate taking into consideration all relevant factors. Thus, in some situations the effective amount is more than the calculated
  • the effective amount is less than the calculated ED50, and in still other situations the effective amount is the same as the calculated ED50.
  • an effective dose of the Modulators of the present disclosure may be an amount that, when administered in one or more doses to a subject, produces a desired result relative to a healthy subject.
  • an effective dose may be one that, when administered to a subject having elevated plasma glucose and/or plasma insulin, achieves a desired reduction relative to that of a healthy subject by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than 80%.
  • An appropriate dosage level will generally be about 0.001 to 100 mg per kg patient body weight per day which can be administered in single or multiple doses. In some embodiments, the dosage level will be about 0.01 to about 25 mg/kg per day, and in other embodiments about 0.05 to about 10 mg/kg per day. A suitable dosage level may be about 0.01 to 25 mg/kg per day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day. Within this range, the dosage may be 0.005 to 0.05, 0.05 to 0.5 or 0.5 to 5.0 mg/kg per day.
  • compositions can be provided in the form of tablets, capsules and the like containing from 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • the compounds may be administered on a regimen of 1 to 4 times per day, and often once or twice per day.
  • the dosage of the Modulators of the present disclosure may be repeated at an appropriate frequency which may be in the range once per day to once every three months, depending on the pharmacokinetics of the Modulators (e.g. half-life of the antibody in the circulation) and the pharmacodynamic response (e.g. the duration of the therapeutic effect of the Modulator).
  • the Modulator is an antibody or a fragment thereof, or a peptide or variants thereof
  • dosing is repeated between once per week and once every 3 months.
  • the antibody is administered approximately once per month.
  • the dosage of the disclosed Modulators is contained in a
  • unit dosage form refers to physically discrete units, each unit containing a predetermined amount of a Modulator of the present disclosure, either alone or in combination with one or more additional agents, sufficient to produce the desired effect. It will be appreciated that the parameters of a unit dosage form will depend on the particular agent and the effect to be achieved. Kits
  • kits comprising the disclosed
  • kits are generally in the form of a physical structure housing various components, as described below, and may be utilized, for example, in practicing the methods described above (e.g., administration of a Modulator to a subject in need of restoring glucose homeostasis).
  • a kit can include one or more of the Modulators disclosed herein (provided in, e.g., a sterile container), which may in the form of a pharmaceutical composition suitable for administration to a subject.
  • the Modulators can be provided in a form that is ready for use or in a form requiring, for example, reconstitution or dilution prior to administration.
  • the kit may also include buffers, pharmaceutically acceptable excipients, and the like, packaged with or separately from the Modulators.
  • the kit may contain the several agents separately or they may already be combined in the kit.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package.
  • a kit of the present disclosure can be designed for conditions necessary to properly maintain the components housed therein (e.g., refrigeration or freezing).
  • a kit may contain a label or packaging insert including identifying information for the components therein and instructions for their use (e.g., dosing parameters, clinical pharmacology of the active ingredient(s), including mechanism of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.). Labels or inserts can include manufacturer information such as lot numbers and expiration dates.
  • the label or packaging insert may be, e.g., integrated into the physical structure housing the components, contained separately within the physical structure, or affixed to a component of the kit (e.g., an ampoule, tube or vial).
  • Exemplary instructions include those for reducing or lowering blood glucose, treatment of hyperglycemia, treatment of diabetes, etc. with the disclosed Modulators, and pharmaceutical compositions thereof.
  • Labels or inserts can additionally include, or be incorporated into, a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.
  • a computer readable medium such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided.
  • MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF);
  • BW body weight;
  • U unit;
  • FPG fasting plasma glucose;
  • FPI fasting plasma insulin;
  • ITT insulin tolerance test;
  • PTT pyruvate tolerance test;
  • oGTT oral glucose tolerance test;
  • GSIS glucose-stimulated insulin secretion;
  • PBS phosphate -buffered saline;
  • ECCs enteroendocrine cells;
  • ECs enterocytes; and
  • FPKM fragments per kilobase of exon per million fragments mapped.
  • Tor2a-pl refers to both Human Peptide- p 1 and Murine Peptide -p 1.
  • Peptide Prediction Peptides derived from the full-length Tor2a precursor were predicted using bioinformatic prediction of processing sites as described previously. (See Samson et al, J. Biol. Chem. 283:31949 (2008)). Briefiy, Tor2a sequences from all mammalian species were aligned, and processed peptides were identified based on conservation of flanking mono- or di-basic processing sites.
  • mice Eight-week old male C57BL/6 mice were purchased from the Charles River Laboratory (Wilmington, MA). Mice were kept in accordance with welfare guidelines under controlled light (12 hr light and 12 hr dark cycle, dark 6:30 p.m. - 6:30 a.m.), temperature (22 ⁇ 4°C) and humidity (50% ⁇ 20%) conditions. Mice were maintained on a high-fat diet (D 12492, Research Diets, New Brunswick, NJ) containing 60 kcal% fat, 20 kcal% protein and 20 kcal% carbohydrate for a minimum of 12 weeks to induce obesity and systemic insulin resistance.
  • D 12492 Research Diets, New Brunswick, NJ
  • Body Weight was determined prior to all metabolic tests using an automated digital scale (Sartorius LE5201, Sartorius Mechatronics Corporation, Bohemia, NY) equipped with Software Wedge (Sartorius YSW05, Sartorius Mechatronics Corporation, Bohemia, NY).
  • mice received a single bolus LP. injection of peptide or vehicle control at minO.
  • blood was sampled from the tail-vein. Plasma glucose and insulin concentration were determined at 0, 30, 60, 120 and 180 min. FPG was determined using the mutarotase-GOD enzymatic assay enzymatic assay (Wako Diagnostics Inc,
  • FPI concentration was determined by a mouse ultra-sensitive enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH).
  • mice received a single bolus i.p. injection of peptide or vehicle control at -30 min.
  • glucose lg/kg
  • PBS PBS
  • Plasma glucose concentration was determined at (-30, 0, 15, 60 and 120 min) by the mutarotase-GOD enzymatic assay (Wako Diagnostics Inc, Richmond, VA). oGTT was used to assess glucose tolerance.
  • mice received a single bolus i.p. injection of peptide or vehicle control at -30 min. At min 0 mice received glucose (lg/kg) orally. Plasma insulin concentration was determined at (-30, 0, 15 and 60 min) by a mouse ultra-sensitive enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH). GSIS was used to assess insulin secretion in response to a glucose challenge.
  • mice received a single bolus i.p. injection of peptide or vehicle control at -30 min.
  • Plasma glucose concentration was determined at (-30, 0, 15, 30, 60 and 120 min) by the mutarotase-GOD enzymatic assay (Wako Diagnostics Inc, Richmond, VA).
  • Insulin concentration following administration of vehicle control was determined at (-30, 0, 15, 30, 60 min), while insulin concentration following administration of Humalin® was determined at (15, 30, 60 and 120 min). Insulin concentration was assayed by a mouse ultra-sensitive enzyme-linked
  • immunosorbent assay and human-specific enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH, USA), respectively. ITT was used to assess systemic insulin sensitivity.
  • mice received a single bolus i.p. injection of peptide or vehicle control at -30 min. At min 0, mice received a single bolus i.p. injection of sodium pyruvate (lg/kg) in PBS. Plasma glucose concentration was determined at (-30, 0, 15, 30, 60 and 120 min) by the mutarotase-GOD enzymatic assay (Wako Diagnostics Inc, Richmond, VA). PTT was used to assess hepatic gluconeogenic capacity in response to the gluconeogenic precursor, pyruvate.
  • EECs and ECs were isolated from the mouse duodenum, jejunum, ileum and colon. Tor2a gene expression was then measured in FACS-sorted cells using SOLiD deep sequencing technology as directed by the manufacturer (Applied Biosystems, Foster City, CA, USA).
  • Tor2a gene expression (as measured by FPKM) data are set forth in Figure 1. As set forth in Figure 1, Tor2a gene expression in EECs is most prevalent in the jejunum and the ileum, whereas Tor2a gene expression in ECs is most prevalent in the colon and the jejunum. No Tor2a gene expression in EECs was detected in the colon, and no Tor2a gene expression in ECs was detected in the ileum.
  • Example 2 Acute In Vivo Effect of Tor2a-pl on Glucose Homeostasis in High-Fat Fed Insulin-Resistant Mice
  • Tor2a-pl glucose homeostasis
  • twenty-two week old high-fat fed, insulin-resistant mice weighing approximately 50 g received a single bolus i.p. injection of Tor2a-pl (10 mg/kg) [Murine Peptide-pl] or Vehicle control.
  • Tor2a-pl 10 mg/kg
  • Vehicle control Following a four hr fast, plasma glucose and insulin concentration were measured over a three hr period, and the glucose-raising (pro-diabetic) effect of Murine Peptide-pl was then evaluated.
  • n 6 and p-values were determined by a student's t-test comparing Murine Peptide-p 1 and Vehicle-treated mice.
  • Example 3 Acute In Vivo Effect of Tor2a-Pl on Glucose Tolerance in High-Fat Fed Insulin-Resistant Mice
  • n 6 and p-values were determined by a student's t-test comparing Murine Peptide-pl and Vehicle-treated mice.
  • Example 4 Acute Effect of Tor2a-pl on Insulin Tolerance in High-Fat Fed Obese, Insulin- Resistant Mice
  • n 6 and p-values were determined by a student's t-test comparing Murine Peptide-pl and Vehicle-treated mice.
  • Example 5 Acute Effect of Tor2a-pl on Gluconeogenic Capacity in High-Fat Fed Obese, Insulin-Resistant Mice
  • a PTT was conducted to determine whether the exacerbation in fasted glycemia and glucose tolerance were attributed to decreased hepatic function in Murine Peptide-pl - treated mice.

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  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des procédés de traitement d'individus atteints d'un trouble du métabolisme du glucose et des compositions appropriées pour une utilisation dans les procédés. Des sous-séquences peptidiques dérivées du produit génétique Tor2a ont été identifiées. Plus précisément, dix peptides murins (ms p1-ms p10) et dix peptides humains (hu p1-hu p10) ont été identifiés (Figure 2) et ces peptides sont différents des peptides salusines (c'est à dire que ni les peptides murins ni les peptides humains ne chevauchent les séquences d'acides aminés murines et humaines des peptides salusines).
PCT/US2013/026879 2012-02-24 2013-02-20 Procédés de traitement de troubles du métabolisme du glucose WO2013151627A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053343A1 (fr) * 2000-01-18 2001-07-26 Human Genome Sciences, Inc. Polynucleotides, polypeptides et anticorps humains
US20030194704A1 (en) * 2002-04-03 2003-10-16 Penn Sharron Gaynor Human genome-derived single exon nucleic acid probes useful for gene expression analysis two
EP1743940A1 (fr) * 2004-04-02 2007-01-17 Japan Science and Technology Agency Nouveau peptide endogene cardio-inhibiteur/antihypertenseur physiologiquement actif
US20070037206A1 (en) * 1997-03-07 2007-02-15 Rosen Craig A Human secreted proteins

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070037206A1 (en) * 1997-03-07 2007-02-15 Rosen Craig A Human secreted proteins
WO2001053343A1 (fr) * 2000-01-18 2001-07-26 Human Genome Sciences, Inc. Polynucleotides, polypeptides et anticorps humains
US20030194704A1 (en) * 2002-04-03 2003-10-16 Penn Sharron Gaynor Human genome-derived single exon nucleic acid probes useful for gene expression analysis two
EP1743940A1 (fr) * 2004-04-02 2007-01-17 Japan Science and Technology Agency Nouveau peptide endogene cardio-inhibiteur/antihypertenseur physiologiquement actif

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