peptide sequence, subsequence, composition, pharmaceutical composition, nucleic acid molecule, vecto

专利摘要:
PEPTIDE SEQUENCE, SUBSEQUENCE, COMPOSITION, PHARMACEUTICAL COMPOSITION, NUCLEIC ACID MOLECULE, VECTOR, TRANSFORMED OR HOSPEDIC CELL, METHOD OF TREATING AN INDIVIDUAL, METHOD OF IMPROVING A METHODOLOGYThe invention relates to variants and fusions of fibroblast growth factor 19 (FGF19), variants and fusions of fibroblast growth factor 21 (FGF21), fibroblast growth factor 19 (FGF19) fusions and / or growth factor of fibroblasts 21 (FGF21), and variants or fusions of fibroblast growth factor 19 (FGF19) and / or fibroblast growth factor 21 (FGF21) of proteins and peptide sequences (peptidornyretic) with one or more activities, such as activity hypoglycemic agents, and methods and use for the treatment of hyperglycemia and other disorders.
公开号:BR112013033175A2
申请号:R112013033175-5
申请日:2012-06-29
公开日:2020-10-20
发明作者:Lei Ling；Darrin A. Lindhout
申请人:Ngm Biopharmaceuticals, Inc.；
IPC主号:

专利说明:

PEPTIDE SEQUENCE, SUBSEQUENCE, COMPOSITION, PHARMACEUTICAL COMPOSITION, NUCLEIC ACID MOLECULE, VECTOR, TRANSFORMED OR HOSPEDER CELL, METHOD OF TREATING AN INDIVIDUAL, METHOD OF IMPROVING THE METABOLIS
INDIVIDUAL AND METHOD FOR IDENTIFYING A PEPTIDE SEQUENCE Related Orders This order claims the priority benefit of application No. 61 / 504,128, filed on July 1, 2011 and application No. 61 / 515,126, filed on August 4, 2011, both The 5 orders are expressly incorporated herein by reference in their entirety.
Field of the Invention The invention relates to variants of fibroblast growth factor 19 (FGF19) proteins and sequences of 10 peptide (and peptidomyretic) and fibroblast growth factor 19 (FGF19) and / or protein fusions. fibroblast growth factor 21 (FGF21) and peptide (and peptidomyrrietic) sequences, and variants of fibroblast growth factor 19 (FGF19) and / or fibroblast growth factor 21 (FGF21) protein sequences and sequences of peptide (and peptidomimetics) that have glucose-lowering activity and methods and uses in the treatment of hyperglycemia and other disorders.
Introduction 20 Diabetes mellitus is a debilitating metabolic disease caused by the absence of insulin production (type 1) or insulin resistance or insufficient production of insulin (type 2) from pancreatic β-cells. cell-j3 are
2/116, / specialized endocrine cells that manufacture and store insulin for release after a meal. Insulin is a hormone that facilitates the transfer of glucose from the blood to the tissues where it is needed. Diabetes 5 patients must frequently monitor blood glucose levels and many require multiple injections of insulin daily to survive. However, these patients rarely reach ideal glucose levels by insulin injection (Turner, R.C. et al. JAMA 281: 2005 (1999)). In addition, prolonged elevation of insulin levels can result in harmful side effects, such as hyperglycemic shock and desensitization of the body's response to insulin.
As a result, diabetic patients still develop long-term complications, such as cardiovascular disease, kidney disease, blindness, nerve damage and healing healing disorders (UK Prospective Diabetes Study (UKPDS) Group, Lancet 352: 837 (1998)) .
Bariatric surgery has been proposed as a potential treatment for diabetes. It has been postulated that changes in the secretion of the gut from the intestine after surgery are responsible for the resolution of diabetic conditions. The underlying molecular mechanism has yet to be elucidated, although the peptide similar to glucagon type 1 (GLP-1) has been considered as a possible candidate (Rubino, F. 25 Diabetes Care 32 Suppl 2: S368 (2009)). FGF19 is highly expressed in the distal small intestine and- over transgenic expression of FGF19 improves glucose homeostasis (Tomlinson, E. Endocrinology 143 (5): 1741-7 (2002)). Serum levels of FGF19 in humans are elevated after gastric bypass surgery. Expression and increased secretion of FGF19 could explain, at least partially, the remission of the 'r.
diabetes experienced after surgery.
3/116 - Consequently, there is a need for alternative treatments of hyperglycemic conditions such as diabetes, pre-diabetes, insulin resistance, hyperinsulinemia, glucose intolerance or metabolic syndrome 5 and other disorders and diseases associated with high levels of glucose eui human beings. The present invention satisfies this need and also provides related advantages.
Summary of the Invention 010 the invention is based, in part, on variant of fibroblast growth factor 19 (FGFI9) peptide sequences, fibroblast growth factor 19 (FGF19) fusions and / or growth factor peptide sequences of fibroblasts 21 (FGF21) and the merger variants (chimeras) of fibroblast growth factor 19 (FGF19) and / or fibroblast growth factor 21 (FGF21) peptide sequences having one or more activities, such as activity of glucose reduction. These variants and fusions (chimeras) of FGF19 and / or FGF21 peptide sequences include 20 sequences that do not increase or induce carcinoma and hepatocellular (HCC) or HCC formation, tumorigenesis. Such variants and fusions (chimeras) of FGE'19 and / or FGF21 peptide sequences also include sequences that do not induce a substantial elevation or increase in lipid profile.
In one embodiment, a chimeric peptide sequence includes or consists of: an N-terminal region having at least seven amino acid residues, the N-terminal region having a first amino acid position and a last amino acid position, where the N-terminal region has a DSSPL or DASPH sequence, and a C-terminal region that has a FGF19 portion, where the C-terminal region has a first amino acid position and a last amino acid position, where the C region -terminal includes the amino acid residues 16-29 of FGF19 (WGDPIRLRHLYTSG), and where residue W corresponds to the first amino acid position in the C-terminal region. / 5 In another embodiment, a chirric peptide sequence includes or consists of: an N-terminal region that has a FGF21 portion, where the N-terminal region has a first amino acid position and a last amino acid position, where the N-terminal region has a GQV sequence and, where (! 3 10 residue V corresponds to the last amino acid position of the N-terminal region, and a C-terminal region including a FGF19 portion, the C- terminal having a first amino acid position and a last amino acid position, where the C-terminal region includes amino acid residues 21 to 15 29 of FGF19 (RLRHLYTSG), and where residue R corresponds to the first position of region C -terminal.
In another embodiment, a chemical peptide sequence includes or consists of any: an N-terminal region comprising a portion of SEQ ID NO: 100 [FGF21], the N-terminal region comprising a first amino acid position and a last amino acid position, where the N-terminal region comprises at least 5 (or more) contiguous amino acids of SEQ ID NO: 100 [FGF21], including amino acid residues GQV, and where residue V corresponds to the last 25 amino acid position of the N-terminal region, and a C-terminal region comprising a portion of SEQ ID NO: 99 [FGF19], the C-terminal region which has a first amino acid position and, a last position of the amino acid, in which the C-terminal region, comprises amino acid residues 21 to 29 of SEQ ID NO: 99 30 [FGF19], RLRHLYTSG, and where the residue R corresponds to the first position of the C-terminal region. In certain respects, the N-terminal region comprises at least 6 amino acids (eg. 7, 8, 9, 10, 11, 12, 13, 14, 1.5, 16, 17, 18) , 19, 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100 contiguous amino acids) of SEQ ID NO: 100 [FGF21], including GQV amino acid residues.
In a further embodiment, a peptide sequence includes or consists of any of: a variant sequence of fibroblast growth factor 19 (FGF19) having one or more substitutions, insertions or deletions of amino acids in comparison to a reference or type wild 10 of FGF19; a variant sequence of èj fibroblast growth factor 21 (FGF21) that has one or more amino acid substitutions, insertions or deletions compared to a FGF21 reference or wild type; a portion of a FGF19 sequence fused to a portion of a 15 FGF21 sequence, or a portion of a FGF19 sequence fused to a portion of a FGF21 sequence, wherein the 3eq 'portion (s) FGF19 and / or FGF21 has one or more amino acid substitutions, insertions or deletions compared to a reference or wild-type FGF19 and / or FGF21.
In yet other embodiments, a sequence of O peptide or a chimeric peptide sequence includes or, consists of amino-terminal amino acids 1 to 16 of SEQ ID NO: 100 [FGF21] fused to carboxy-terminal amino acids 21 to 194 of SEQ ID NO: 99 [FGF19], or the peptide sequence has 25 amino-terminal amino acids 1 to 147 of SEQ ID NO: 99 [FGF19] fused to carboxyland amino acids 147 to 181 of SEQ ID NO: 100 [FGF21 ] (M41), or the peptide sequence has amino-terminal amino acids 1 to 20 of SEQ ID NO: 99 [FGF19] fused to carboxy-terminal amino acids 17 to 181 of SEQ ID 30 NO: 100 [FGF21] (M44), or the peptide sequence has amino-terminal amino acids 1 to 146 of SRQ ID NO; 1OO [FGF21] fused to carboxy-terminal amino acids 148 to 194 of SEQ ID
NO: 99 [FGF19] (M45), or the peptide sequence has ahiino-terminal amino acids 1 to 20 of SEQ ID NO: 99 [FGF19] fused to internal amino acids 17 to 146 of SEQ ID NO: 100 [FGF21] fused to carboxy-terminal amino acids 148 to 194 of 5 SEQ ID NO: 99 [FGF19] (M46).
In still further embodiments, a peptide sequence or a chimeric peptide sequence has a WGDPI sequence pattern corresponding to the WGDPI amino acid sequence 16 to 20 of SEQ ID NO: 99 [FGF19], or has an e 10 sequence pattern Substituted, mutated or missing WGDPI corresponding to the FGF19 amino acid sequence WGDPI 16 to 20 of FGF19, or the standard. of sequence WGDPI has one or more, substituted, mutated or missing amino acids, or is different from a variant sequence of FGF 19 having either GQV, GDI, WGPI, 15 WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI , WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI replaced by FGF19 WGDPI sequence in amino acids 16 to 20.
In still other embodiments, a peptide-sequence or a chimeric peptide sequence has an N-terminal 20 region that includes or consists of amino acid and VHYG residues, amino acids, where the DASPHVHYG, N-terminal region or comprises the N-terminal residues region of comprises the DSSPLVHYG amino acid residues, or where the N-terminal region comprises DSSPLLQ amino acid residues, whereas the N-terminal region comprises DSSPLLQFGGQV amino acid residues. In particular aspects, G corresponds to the last position of the N-terminal region, or residue Q is the last position of amino acids in the N-terminal region, or residue V corresponds to the last position of the N-terminal region.
In further embodiments, a peptide sequence or a chimeric peptide sequence has an N-terminal region that includes or consists of RHPIP, where R is the first amino acid position in the N-terminal region, or HPIP 5 (for example , where HPIP is the first 4 amino acid residues of the N-terminal region), where H is the first amino acid position of the N-terminal region, or RPLAF, where R is the first amino acid position of the N-terminal region, or PLAF, where P is the first amino acid position of the N-10 terminal region, (jü R, where R is the first amino acid position is the N-terminal region, or t-in the N-terminal region any of the sequences following: MDSSPL, MSDSSPL, SDSSPL, MSSPL or SSPL.
In other embodiments, a peptide sequence or a chimeric peptide sequence has, in the first position of the N-terminal region, an "M" residue, an "R" residue, an "S" residue, an " H ", a" P "residue, an" L "residue or a" D "residue. In alternative embodiments, a peptide sequence or a quinoic peptide sequence 20 does not have an "M" residue or an "R" residue at the first amino acid position in the N-terminal region.
6 In yet other embodiments, a peptide sequence or a chimeric peptide sequence has the first and second positions of the N-terminal region of an MR sequence, 25 or the first and second positions of the N-'terminal region of a RM sequence, either at the first and second positions of the N-terminal region of an RD sequence, or at the first and second positions of the N-terminal region of a DS sequence, or at the first and second positions of the N-terminal region of a sequence DM, either in the first and second positions of the N-terminal region of an MS sequence, or in the first and third positions of the N-terminal region of an MDS sequence, or in the first and third positions of the N-terminal region of an RDS sequence, either in the first and third positions of the N-terminal region of an MSD sequence, or in the first and third positions of the N-terminal region of an MSS sequence, or in the first and third positions of the N-terminal region of an MSD sequence. sequence of DSS, or in the first and fourth positions of the N-terminal region of an RDSS sequence, or in the first and fourth positions of the N-terminal region of an MDSS sequence, or in the first and fifth positions of the N-terminal region a 10 MRDSS sequence, or in the first and fifth positions of the N-terminal region, is an MSSPL sequence, or in the first and sixth positions of the N-terminal region of an MDSSPL sequence, or in the first and seventh positions of the N-terminal region an MSDSSPL sequence.
In still other embodiments, a 15 peptide sequence or a chimeric peptide sequence, an addition of amino acids residues 30 to 194 of SEQ ID NO: 99 [FGF19] at the C-terminus, resulting in a chimeric polypeptide having at the last position of the C-terminal region that corresponds approximately to residue 194 of SEQ ID NO: 99 [FGF19]. In 20 other embodiments, a chimeric peptide sequence or peptide sequence comprises all or a portion of a FGF19 sequence (e.g., SEQ ID NO: 99), positioned at the C-terminal end of the peptide, or wherein the amino terminal "R" residue is excluded from the peptide. ..) In more particular embodiments, a chimeric peptide sequence or peptide sequence includes or consists of any of the M1 to M98 variant peptide sequences, or a subsequence or fragment of any of the M1 to M98 variant peptide sequences .
In additional particular embodiments, the chimeric peptide sequence or peptide sequence has a region
N-terminal- or C-terminal from about 20 to about 200 amino acid residues in length. In particular embodiments, a chimeric peptide sequence or peptide sequence has at least one 5-amino acid deletion. In even more particular embodiments, a chimeric peptide sequence or peptide sequence or a subsequence or fragment thereof has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid deletions at the amino, carboxy-terminal or 10 internally. In a particular non-limiting aspect, EJ amino acid substitution or deletion is at any of the positions of amino acids 8 to 20 of FGF19 (AGPHVHYGWGDPI).
In more particular embodiments, a chimeric peptide sequence or peptide sequence includes or consists of an amino acid sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids. In more particular forms of reaction, a chimeric peptide sequence or peptide sequence includes or consists of an amino acid sequence 20 of about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids of C) FGF19 or FGF21.
In other particular embodiments, the chimeric peptide sequences and peptide sequences have particular functions or activities. Err one aspect, a chimeric peptide sequence or peptide sequence maintains or enhances a mediated FGFR4 activity. In additional aspects, a chimeric peptide sequence or peptide sequence binds to fibroblast growth factor 4 receptor (ÊGFR4) or activates FGFR4, or does not bind FGFR4 in a detectable way to the growth factor 4 receptor. fibroblasts (FGFR4) or activates FGFR4, or binds to FGFR4 with a lower affinity, comparable to or greater than the binding affinity of FGF19 ccun FGFR4, or activates FGFR4 to a lesser extent or amount, comparable 'or greater than' active FGF19 FGFR4. In other respects, '' a chimeric peptide sequence 5 OL1 'peptide sequence reduced hepatocellular carcinoma (HCC) formation compared to FGF19, or a FGF 19 variant sequence having any of the GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADE'I, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI replaced by the sequence WGDPI in 10 amino acids 16 to 20 of FGF19 and / or has greater Ç hypoglycemic activity compared to " FGF19, or a sequence .. FGF 19 variant having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, - WDPI, WGDI, WGDP or FGDPI substituted- by the WGDPI sequence in 15 amino acids 16 to 20 of FGF19 and / or has less increased lipid activity compared to "FGF19, or a FGF 19 variant sequence having any of the GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDE'A, WDPI, WGDI, WGDP or FGDPI replaced by the 20 WGDPI sequence in amino acids 16 to 2 0 of FGF19, and / or has less activity to increase triglycerides, cholesterol, non-HDL or HDL compared to FGF19, or a variant sequence '; of FGF 19 having any of the GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, "WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, 25 WGDI, WGDP 'or FGDPI replaced by the sequence WGDPI in 16 amino acids to 20 of FGF19, and / or has less lean mass reduction activity compared to FGF21. Such functions and activities can be determined in vitro or in vivo, for example, in db / db rats.
.
In still further embodiments, the chimeric peptide sequences and isolated or purified peptide sequences, and / or chimeric peptide sequences and peptide sequences can be included in the compositions.
In one embodiment, a chimeric peptide sequence or peptide sequence is included "in a pharmaceutical composition. Such compositions include combinations of inactive or other active ingredients. In one embodiment, a composition, such as a pharmaceutical composition, includes the sequence of the chimeric peptide or a peptide sequence and a glucose reducing agent.
In still other embodiments, nucleic acid molecules encoding the chimeric peptide sequence or € 3 10 peptide sequence are provided. Such molecules may further include an operable linked expression control element that confers the expression of the nucleic acid molecule encoding the peptide in vitro, from a cell or in vivo, or a vector comprising the nucleic acid molecule 15 ( for example, a viral vector). Transformed and host cells that express the chimeric peptide sequences and peptide sequences are also provided.
Uses and methods of treatment that will not administer or release any chimeric peptide sequence or peptide sequence are also provided. In particular embodiments, a patient's use or method of treatment includes administering an invented chimeric peptide or a peptide sequence to a patient, such as a patient having, or at risk of, having a disease or condition. disorder that can be treated by a peptide sequence invented in an effective amount to treat the disease or disorder. In another embodiment, a method includes administering an invented chimeric peptide or a peptide sequence to a patient. such as a patient who has a hyperglycemic condition (for example, diabetes, such as insulin-dependent diabetes (type I), type II diabetes or gestational diabetes), insulin resistance, hyperinsulinemia,
glucose intolerance or metabolic syndrome is either obese or has an undesirable body mass.
In certain aspects of the methods and uses, a chimeric peptide sequence or peptide sequence is administered to a patient in an amount effective to improve glucose metabolism in the patient. In more specific aspects, a patient has a fasting plasma glucose level greater than 100 rng / dl or has a hemoglobin Alc (HbA1c) above 6% prior to administration.
010 In other embodiments, the use or method of * treating a patient is intended to or result in reduced glucose levels, increased insulin sensitivity, decreased insulin resistance, reduced glucagon, an improvement in tolerance glucose or glucose metabolism or homeostasis, improvement of pancreatic function or reduction of triglycerides, cholesterol, IDL, LDL and VLDL or a decrease in blood pressure, decrease in the thickening of the intimate layer of the blood vessel or decrease in body mass or weight gain .
e20 Methods of analysis and / or identification of a chimeric peptide sequence or peptide sequence are also provided, as are chirieric peptide sequences and peptide sequences that have glucose-lowering activity without substantial hepatocellular carcinoma activity 25 (HCC ). In one embodiment, a method comprises: a) providing a candidate chimeric peptide sequence or peptide sequence; b) administering the candidate peptide sequence to a test animal (for example, a db / db rat); c) measuring the animal's glucose levels after administration of the candidate peptide sequence to determine whether the candidate peptide sequence reduces glucose levels. In a particular aspect, the chimeric peptide sequence or peptide sequence is also analyzed for the induction of HCC in the animal (for example, evaluation of a liver tissue sample from the test animal) or expression of a correlator marker with HCC activity, in which a candi.dato peptide having glucose-reducing activity and not substantial HCC activity. Such methods identify the candidate as having glucose-lowering activity, optionally also without substantial hepatocellular carcinorn 10 (HCC) activity.
and Description of the Drawings FIG. 1 shows FGF19 and FGF21 protein sequences and representative variant sequences, that is, peptide sequences M5 variant, Ml variant, M2 variant, M69 variant, 15 M3 variant, M48 variant, M49 variant, M50 variant, M51 variant, M52 variant, variant M53 and variant M70. 3 additional allelic (polymorphic) forms of FGF21, ie M71, M72 and M73 are also shown.
FIG. 2 shows the representative domain changes between: 0 protein sequences FGF21 (without shading) and FGF19 (gray shading) and the resulting fused (chimeric) sequences. The amino acid regions of each of the FGF21 and FGF19 present in the fusion (chimera) are indicated by numbers. Glucose reduction and lipid elevation is shown for every 25 of the chimeric sequences.
FIG. 3À-3I shows blood glucose and body weight reduction data. Peptide sequences A) M5 variant; B) variant Ml; C) variant M2 and variant M69; D) varies M3; E) variant M48 and variant M49; F) variant M51 and variant M50; G) variant 30 peptide variant M52; H) peptide variant M53 and I)
M70 all having reduced blood glucose (ie, antidiabetic activity) in db / db rats. The rats were injected with AAV vector expressing FGF19, FGE'21, the selected variants "and saline and gep are negative controls.
FIG. 4A-4I shows a serum lipid profile (triglycerides, total cholesterol, HDL and non-HDL) from db / db mice injected with AAV vector expressing FGF19, FGF21 or peptide sequences A) variant M5; B) variant Ml; C) variant M2 and variant M69; D) variant M3; E) variant M48 and variant M49; F) variant @ 10 M51 and variant M50; G) M52 variant peptide; H) M53 variant peptide, and I) M70 variant. Sequence of the M5 variant peptide did not increase or elevate the lipids in contrast to FGF19, Ml, M2 and M69, which increase and elevate the lipids.
The serum levels of all variants were comparable. 15 Saline and GFP are negative controls.
FIG. 5A-5I shows data related to hepatocellular carcinoma (HCC) for peptide sequences A) variant M5: B) variant Ml; C) variant M2 and variant M69; D) variant M3; E) variant M48 and variant M49; F) variant M51 and variant 20 M50: G) variant M52; H) M53 variant peptide, and I) @ M70 variant. All variables did not increase significantly or induce the formation of hepatocellular carcinoma (HCC) or HCC tumorigenesis in contrast to FGF19. HCC score is recorded as the number of HCC nodules on the surface of the entire liver of mice with injected variants due to the number of HCC nodules of mice injected with wild-type FGF19.
fat mass = FIG. 6A-6I shows lean mass data or for peptide sequences A) variant M5; B) variant Ml; variant M48 30 C) variant M2 and variant M69; D) variant M3; E) and variant M49; F) variant M51 and variant M50; G) variant
M52; H) peptide variant M53 and I) variant M70. Except for M2, M5 and M69, the variant peptide sequences reduce lean mass or fat mass in contrast to FGF21.
FIG. 7A-7B shows graphical data demonstrating that injection of the recombinant polypeptides A) variant M5 and B) variant M69 reduces blood glucose in ob / ob mice.
FIG. 8 shows data indicating that the hepatic expression of family 1 aldo-keto reductase, members C18 (Akr1C18) and family 1 solute carrier, members 2 (slc1a2) appears to correlate - @IO if I ran HCC activity.
Detailed Description The present invention provides chimeric and peptide sequences that are capable of decreasing or reducing glucose levels. In one embodiment, a quinoline peptide sequence includes or consists of an N-terminal region having at least seven amino acid residues and the N-terminal region which has a first amino acid position and a last amino acid position, where the N-terminal region has a DSSPL or DASPH sequence and a C-terminal region that has a FGF19 portion and the C-terminal region that has a first amino acid position and a last amino acid position, where the C region -terminal includes amino acid residues 16 to 29 of FGF19 (WGDPIRLRHLYTSG) and residue W corresponds to the first arrtino acid position in the C-terminal region.
In another embodiment, a chemical peptide sequence includes or consists of an N-terminal region that has a FGF21 portion and an N-terminal region that has a first amino acid position and a last amino acid position, where the the N-terminal region has a GQV sequence and the V residue corresponds to the last amino acid position of the N-terminal region, and a C-terminal region that has a FGF19 portion and the C-terminal region that has a first amino acid position and last amino acid position in which the C-5 terminal region includes amino acid residues 21 to 29 of FGF19 (RLRHLYTSG) and the residue of R cor.responds to the first position of the C-terminal region.
In other embodiments, a peptide sequence includes or consists of a variant sequence of fibroblast growth factor 19 (FGF19) that has one or more amino acid substitutions, insertions or deletions compared to a wild-type FGF19 reference or type . In additional embodiments, a peptide sequence includes or consists of a variant sequence of fibroblast growth factor 21 (FGF21) that has one or more amino acid substitutions, insertions or deletions compared to a wild-type FGF21 reference or type. Still in additional embodiments, a peptide sequence includes or consists of a portion of a FGF19 "20 sequence merged with a portion of a FGF21 sequence. Still in embodiments it adds: U, a peptide sequence and includes or consists of a portion of a FGF19 sequence fused to a portion of a FGF21 sequence, where the (S) EGF19 and / or FGF21 sequence portion has one or more 25 amino acid substitutions, Y insertions or deletions compared to a reference or FGF19 wild type and / or FGF21.
The invention also provides methods and uses in the treatment of a patient with or at risk of having a treatable metabolic disorder using fibroblast growth factor 19 (FGF19) and / or fibroblast growth factor 21 peptide sequences and fusions. (FGF21). In one embodiment, a method includes contacting or administering to a patient one or more nucleic acids that encode variant peptide sequence or fusion of fibroblast growth factor 19 (FGF19) and / or 5 fibroblast growth factor 21 (FGF21) in an amount effective to treat the disorder. In another embodiment, a method includes contacting or administering to a patient one or more nucleic acid molecules that code for a variant peptide sequence or fibroblast growth factor 19 10 (FGF19) and / or growth factor fusion. fibroblasts 21 (FGF21) ® (for example, an expression control element operatively linked to the nucleic acid that encodes the peptide sequence, optionally including a vector) in an amount effective to treat the disorder.
15 Although the understanding of the underlying action of the peptides of the invention is not necessary for the practice of the invention, without being linked to any particular theory or hypothesis, it is believed that the peptides of the invention mimic, at least in part, the effect that bariatric surgery has about 20, for example, glucose homeostasis and weight loss. Changes3 in the secretion of gastrointestinal hormones (eg, the glucagon-like peptide type 1 (GLP-1)) after bariatric surgery are believed to be responsible for resolving, for example, diabetic conditions. FGF19 is highly expressed in the distal small intestine and transgenic overexpression of FGF19 improves glucose homeostasis. Since FGF19 levels in humans are also elevated after gastric bypass surgery, elevated FGF19 may be involved with the remission of diabetes seen after bariatric surgery.
30 A representative reference sequence or wild type FGF19 is presented as: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGE'HGLSSCFLRI RADgVVDCARgqSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSE, EDCAFE, E
EIRpDgyNVyRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESD MFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 99).
A representative reference or wild type FGF21 sequence is presented as HpIpDSSpLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGT VggAADqSpESLLqLKAT 5, KPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLED gyNVyqSEAHgLpLHLpgNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSD E'LSMVGPSQGRSPSYAS (SEQ ID NO: LOO). Allele variants FGF21 are illustrated in Figure 1 (for example, M70, M71 and M72).
The sequential terms of "peptides", "protein" and @ 10 "polypeptide" are used interchangeably herein to refer to two or more amino acids or "residues", including chemical modifications and amino acid derivatives, covalently linked by a amide or equivalent. Amino acids that form all or part of a peptide can be, among the known 21 naturally occurring amino acids, those which are referred to tar'.to by their one-letter abbreviation or common three-letter abbreviation. In the peptide sequences of the invention, conventional amino acid residues have their conventional meaning. So, "Leu" is leucine, "Ile" is isoleucine, and "Nle" is norleucine, and so on.
Exemplified here are sequences of peptides, polypeptides distinct from the reference FGF19 and FGF21 established here, which reduce or lower glucose, in vivo (Tables 25 1 to 8 and Figure 1). Non-limiting are particular exercises of a sequence peptide with amino terminal amino acids 1 to 16 of FGF21 fused to carboxy terminal amino acids 21 to 194 of FGF19; a peptide sequence with amino acids from amino-terminal 1 to 147 of FGF19 fused to carboxy-terminal 30 of amino acids 147 to 181 of FGF21; a peptide sequence with amino-terminal amino acids 1 to 20 of FGF19 fused to carboxy-terminal amino acids 17 to 181 of FGF21; a peptide sequence with amino-terminal amino acids 1 to 146 of FGE21 fused with carboxy-amino acids 148 to 194 of FGFI9, and a peptide sequence with amino-acid 5 amino acids 1 to 20 of FGF19 fused with the internal amino acids 17 to 146 of FGF21 fused at carboxy-terminal amino acids 148 to 194 FGF19.
Particular additional peptide sequences have a WGDPI sequence pattern corresponding to the FGF19 amino acid sequence 10 WGDPI 16 to 20, absence of a "3 WGDPI sequence pattern corresponding to the FGF19 amino acid sequence WGDPI 16 to 20 ~ or to have a substituted (i.e., mutant) WGDPI sequence corresponding to the FGF19 sequence WGDPI sequence of amino acids 16 to 20 of FGF19.
Specific peptide sequences of the invention also include distinct FGF19 and FGF21 sequences (for example, as set forth herein), and FGF19 variant sequences having any one of the GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or 'FGDE'I 20 replaced by FGF19 WGDPI sequence in amino acids 16 to 20. and "therefore, wild type FGF19 and FGF21 (for example, as set forth herein as SEQ ID NOS: 99 and 100, respectively) can be excluded strings and FGF19 with any of the GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, 25 WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted by the WGDPI sequence in amino acids 16 to 20 of FGF19 can also be excluded. This exclusion, however, does not apply where a sequence has, for example, three FGF21 residues fused to FGF19 having, for example, 30 any of the GQV, GQV, GDI or GPI, or two FGF21 residues fused to any of the WGPI , WGDI, GDPI, WDPI, WGDI or WGDP.
Particular non-limiting examples of peptide sequences include or consist of all or part of a sequence variant specified here as M1 to M98 (SEQ ID
NOS: 1 to 98). More particular non-limiting examples of
5 peptide sequences include or consist of all or part of a sequence shown as: HPIPDSSPLLQFGGQVRLRH LyTSgpFIgLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVFISVRYLCMGADGK MQgLLQYSEEDCAFEEKR
MVpEEpEDLRgHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (Strings
10 FGF21 can also include an "R" residue at the ainino-terminal),
€ 9 or DSSpLLqFggqVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV ALRTVAIKgVHSVRyLCMgADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSH
RSPSFEK Uina or subsequence or fragment thereof, or RPLAFSDASP 15 HVHygWgDpIRLRHLyTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKG VHSVRyLCMgADgKMqgLLqySEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKN RgFLpLSHFLpMLpMVpEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK or a subsequence or fragment thereof, or RPLAFSDSSPLVHYGWGDPI RLRHLyTSgpHgLSSCFLRIRADGVVDCARGQSARSLLEIKAVALRTVAIKGVμ.SVRYLCMG
20 ADgKMqgLLqySEEDCAF'EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFL pMLpMVpEEpEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK or a subsequence or fragment thereof, or DSSPLVHYGWGDPIRLRHLYTSG and pHgLsscFLRIwDgwDcARgQs-AHsLLEImvATjRTvAIKgvHsvRYLcMgADgKMQgLL qySEEDCAFEEEIRpDGYNVYRSEKHRLE'VSLSSAKQRQLYKNRGFLE'LSHFLPMLPMVPEE
PEDLRgHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 25 or a subsequence or fragment thereof, or RDSSpLVHygWgDpIRLRHLYTSGPHGLSSCFLRIRAD gVVDCARgqSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIR pDgyNVyRSEKIIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK (M69), or a subsequence or its
30 fragment, or RDSSpLLqWgDpIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS AHSLLEIKAVALRTVAIKGV "HSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE KHRLpVSLSSAKqRqLYKNRGFLPLSHFLPMLPMVPFEPEDLRGHLESDMFSSPLETDSMDP FGLVTGLEAVRSPSFEK (M52) or a subsequence or fragment thereof,
or FIpIpDSSpLLqFGgQVRLRHLYTSGPHGLSSCFLRIRADGWDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS SAKqRqLyKNRgFLpLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK (M5), or a subsequence or fragment thereof, or 5 HpIpDSSpLLqFggqVRQRYLYTDDAQQTEAH "LEIREDGTVGGAADQSPESLLQLKALKPGV IqILgVKTSRFLCqRpDgALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSP HRDpApRgpARFLpLpgI.pPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M71) or a subsequence or fragment thereof, or HPIPDSSPLL qFggqVRqRyLyTDDAqqTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSR FLCqRpDgALygSLHFDpE_ACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPA @ 10 RFLpLpgLppAppEppgILAPQE'PDVGSSDPLSMVGPSQGRSPSYAS (M72) or a subsequence or fragment thereof, or HPIPDSSPLLQFGGQVRQRYLYT DDAqqTW-HLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGS LHFDpEACSFRELLLEDgyNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALP 15 EppgILApqppDVgSSDpLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPVWDGI TGE (M73) or a subsequence or fragment thereof, or RpLAFSDASpHVHygWGDPIRI.RHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS SAKqRqLyKNRgFLpLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK 20 (M), or a subsequence or fragment thereof, or RpLAFSDSSpLVHygWGDPIP.LRHLYTSGPHGLSSCFLRIRADGVVDCARGQS-AHSLLEIKA
@ VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS SAKQRQTjYKNRGFLPLSHFLPMLPMVPEEPF, DLRGHLESDMESSPLETDSMDPFGLVTGLEA VRSPSFEK (M2), or a subsequence or fragment thereof, or 25 RE'LAFSDAgpHVHygWgDpIRLRHLYTSGPHGLSSCFLRIRADGWDCARGQSAHSLLEIKA VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF "EEILEDGYNVYRSEKHRLPVSLS SAKqRqLyKNRgFLpLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFE" K (M3), or a subsequence or fragment thereof, or RDSSpLLqFggqVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVA 30 IKgVHSVRyLCMgADgKMqgLLqySEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQL yKNRgFLpLSHFLpMLpMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K (M48); or a subsequence or its fragment, or RpLAFSDSSpLLqFggQVRLRHLYTSGPHGT, SSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSA KqRqLyKNRgFLpLSHFLpMLPMVPEEPEDLRGHLESDMFSSPLETDSMDFFGLVTGLEAVR SPSFEK (M49) or a subsequence or fragment thereof, or RHpIpDSSpLLqFgDqVRLRHLYTSGPHGL.SSCFLRIRADGVVDCARGQSAHSLLEIKAVAL 5 RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK qRqLyKNRgFLpLSHFLPMLPMVPREPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK (M50) or uina subsequence or fragment thereof, or RHpIpDsspLLQFggNvRLRHLYTsgpHgLsscFLRIRADgvvDcARgQsAHsLLEImvAL
10 and RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK qRqLyKNRgFLpLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK (M51) or a subsequence or fragment thereof, or MDSSpLLqWgDpIRLRHLyTSGPHGLSSCFLRIRADGVV "DCARGQSAHSLLEIKAVALRTVA IKgVHSVRyLCMgADgKMqgLLQYSEEDCAFEEEIRPDGYNvYRSEKHRLPVSLSSAKQRQL yKNRgFLpLSHFLpMLpMVpEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE 15 K (M53), o'u urn subsequence or fragment thereof, and MRDSSpLVHygWgDpIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR TVAIKgVHSVRyLCMgADgKMQGLLQYSEEDCAFEEEIRPDGvNVYRSEKHRLPVSLSSAKQ RqLyKNRgFLpLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK (M70) or its subsequence or urn fragment, or by any of the peptide sequences that precede the terminal residue can be deleted.
Non-limiting Exernplos and more particular peptide sequences comprise or consist of RNA: HPIPDSSPLLQFGGQVRLRHLYTS gpHgLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVFISVRYLCMGADGWQGL 25 LqySEEDCAFEEEIRpDGYNVYRSEKHRLPVSLSSAKQRQLYWRGFLPLSHFLPMLPMVPE EpEDLRgHLf, SDMFSSpLETDSMDPFGLVTGLRAVRSPSFEK, or a subsequence or fragment thereof, or DSSPLLQFGGQVRLRHLYTSGPHG LSSCFLRIRADgVVDCARgqSAIISLLEIKAVALRTVAIKGvHSVRYLCMGAdGKMQGLLQYS EEDCAFEEEIRpDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPED LRGHLESDMFSSPLETDSMDPFGLVTGLFAVRSPSFEK 30, or a subsequence or fragment thereof, or RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSS CFLRIRADgvvDCargqsAHslLEIKAValrtvAiKGVHSVRYLCMGADGKMQGLLQYSEEd CAFEEEIRpDgyNVyRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG
Z3 / 116 HLES.DMFSSPLETDSMDPFGLVTGLEAVRSPSFEK, or a subsequence or its fragment, or RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFL RIRADgvvDcARgQsAHsLLEImvALRTvAGYMG
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLE SDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 5, or a subsequence or your 'fragniento or DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ SAHSLLEI.KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMD PFGLVTGLEAVRSPSFEK, or a subsequence or its fragment.
Additional specific non-limiting examples of peptide sequences, having at the end N terminus, a peptide sequence including or consisting of all or part of any of the following: HPIPDSSPLLQFGGQVRLRHLYTSG (M5); DSSPLLQFGGQVRLRHLYTSG (M6): RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7); 15 HPIPDSSPLLQWGDPIRLRHLYTSG (M8); HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9); HPIPDSSPHVHYGWGDPIRLRHLYTSG (MlO); RPLAFSDAGPLL QWGDPIRLRHLYTSG (Mll); RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13); HPIPDSSPHVHYGGQVRLRHLYTSG (M14); RPLAFSDAGPHVHWGDPIRLRHLYTSG (M15); RPLAFSDAGPHVHWGDPI 20 RLRHLYTSG (M16): RPLAFSDAGPHVGWGDPI RLRHLYTSG (Mlj); RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18 ""): RPLAFSDAGPVYGWGDPIRLRHLYTSG @ (M19): RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20); RPLAFSDAGPVH YWGDPIRLRHLYTSG (M21); RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22); RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23); RPLAFSDAGPHHYWGDPIRLRHLYTSG 25 (M24); RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25); RPLAFSDSSPL VHWGDPIRLRHLYTSG (M26); RPLAFSDSSPHVHWGDPIRLRHLYTSG (M22); RPLAFSDAGPHVWGDPIRLRHLYTSG (M28); RPLAFSDAGPHVHYWGDPI RLRHLYTSG (M29); RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30); RHPIPDSSPLLQFGAQVRLRHLYTSG (M31); RHPIPDSSPLLQFGDQVRLRHLYTSG 30 (M32); RHPIPDSSPLLQFGPQVRLRHLYTSG (M33); RHPIPDSSPLL QFGGAVRLRHLYTSG (M34); RHPIPDSSPLLQFGGEVRLRHLYTSG (M35); RHPIPDSSPLLQFGGNVRLRHLYTSG (M36); RHPIPDSSPLT, QFGGQARLRHLYTSG (M37); RHPIPDSSPLLQFGGQIRLRHLYTSG (M38); RHPIPDSSPLLQ
FGGQTRLRHLYTSG (M39); RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40); DAGPHVHYGWGDPIRLRHLYTSG (M74); VHYGWGDPIRLRHLYTSG "(M75): RLRHLYTSG (M7J); RHPIDDSSPLLQFGWGDPIRLRHLYTSG; RHPIPDSSPLLQWGDPIRLRHLYTSG; RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; 5 RHPIPDSSPHVHYGWGDPIRLRHLYTSG; RPLAFSDAGPLLQFGGQVRLRHLYTSG: RHPIPDSSPHVHYGGQVRT.RHLYTSG; RPLAFSDAGPHVHYGGDIRLRHLYTSG; RDSS PLLQEGGQVRLRHLYTSG; RPLAFSDSSPLLQFGGQVRLRH.LYTSG; RHPIPDSSPLLQF GAQVRLRHLYTSG: RHPIPDSSPLLQFGDQVRLRHLYTSG; RHPIPDSSPLLQFG PQVRLRHLYTSG; RHPIPDSSPLLQFGGAVRLRHLYTSG: 10 RHPIPDSSPLLQFGGEVR LRHLYTSG; RHPIPDSSPLLQFGGNVRLRHLYTSG ; RHPIPDSSPLLQFGGQARL and RHLYTSG; RHPIPDSSPLLQFGGQIRLRHLYTSG: RHPIPDSSPLLQFGGQTRL RHLYTSG; RHPIPDSSPLLQFGWGQPVRLRHLYTSG, and for any urinary of the amino acid sequences that can be excluded from the amino acid sequences that precede the R-peptide sequences that are preceding amino acids
The peptide sequences of the invention additionally include those with induction or formation of hepatocellular carcinoma (HCC) compared to FGF19 or a variant sequence of FGF 19 with any of the GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, 20 WGDP or FGDPI replaced by the WGDPI sequence in amino acids 16 to 20 of FGF19. The peptide sequences of the invention also include those with greater hypoglycemic activity compared to FGF19 or a variant sequence of FGF 19 having any of GQV, GDI, WGPI, WGDPV, 25 WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI , WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence in amino acids 16 to 20 of FGF19. The peptide sequences of the invention further include those with less growth in lipid activity (g) e.g. triglycerides, cholesterol, non-HDL or HDL) compared to FGF19 or a variant sequence of FGF 19 having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, .'S
WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence in amino acids 16 to 20 of FGF19.
Typically, the number of, amino acids or residues of a peptide sequence of the invention will total less than about 250 (e.g., amino acids or mimetics thereof). In several particular embodiments, the number of residues comprises from about 20 to about 200 residues (for example, amino acids or mimetics thereof). In additional embodiments, the number of residues @iO comprises from about 50 to about 200 residues (for example, amino acids or mimetics thereof). In other embodiments the number of residues comprises from about 100 to about 195 residues (for example, amino acids or mimetics thereof) in length.
15 Amino acids or residues can be linked by amide or through unnatural and non-amide chemical bonds, including, for example, those formed with glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides or N, N'-t dicyclohexylcarbodiimide ( DCC). Non-amide bonds include, for example, cetomethylene, aminomethylene, olefin, ether, thioether and the like (see, for example, Spatpla in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, vol.
7, pp 267-357 (1983), "Peptide and Backbone Modifications'", Marcel Decker, NY). Thus, when a peptide of the present invention includes a portion of a FGF19 sequence and a portion of a FG21 sequence, the two portions do not need to be joined together by an amidav yas bond can be joined by any other chemical or conjugated together through a linker portion.
The invention also includes subsequences, variants and modified forms of exemplified peptide sequences
(including variants FGF19 and FGF21 and subsequences listed in Tables 1 to 8 and Figure 1 and the FGF19 / FGF21 and chimera mergers listed in Tables 1 to 8 and Figure 1), while the former maintains at least one activity or function 5 detectable or measurable. For example, certain embodiments exemplified peptides tern C-terminal sequence FGF19, pHgLsscFLRIRADgvvDcARgQsAHsLLEImvALRTvAIKgvHsvRYT.cMgADgKMQgLL qySEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE pRDLRgHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK the portion C lO terminal, e.g., following amino acid residues and "TSGs" variant.
In addition, certain exemplified variant peptides, for example, those that have all or a portion of the FGF21 sequence at the amino terminus, have a "R" residue positioned on the terminal N-15, which can be omitted. Likewise, certain exemplified variant peptides include an "M" residue positioned at the N-terminus, which can be added to or even replaced with an omitted residue, such as an "R" residue. More particularly, in various embodiments, 20 peptide sequences at the N-terminus include any of: RDSS, DSS, MDSS or MRDSS. In addition, in cells where an "M" residue is adjacent to an "S" residue, the "M" residue can be split in such a way that the "'M" residue is deleted from the peptide sequence , whereas when residue "M" is adjacent to residue "D", residue "M" may be. cleaved. Thus, by way of example, in various embodiments, peptide sequences include those with the following residues at the N-terminus: MDSSPL, MSDSSPL (cleaved to SDSSPL) and MSSPL (cleaved to SSPL).
Thus, the "peptide", "polypeptide" and "protein" sequences of the invention include subsequences, variants and modified forms of the FGF19 and FGF21 variants and subsequences shown in Tables 1 to 8 and Figure 1,. and FGF19 / FGF21 fusions and chimeras listed in Tables 1 to 8 and Figure 1, provided that the subsequence, variant or modified form (e.g., fusion or chimera) retains at least one detectable activity or function.
As used herein, the term "modify" and grammatical variations thereof, means that the composition deviates from a reference composition, such as a peptide sequence. These modified peptide sequences, e10 nucleic acids and other compositions may have a greater or lesser activity or function or have a different function or activity compared to an unmodified reference peptide sequence, nucleic acid or other composition or may have a desirable property of a protein formulated for therapy 15 (e.g., serum half-life), to induce antibodies for use in a detection assay and / or for protein purification. For example, a peptide sequence of the invention can be modified to increase serum half-life, to increase protein stability in vitro and / or in vivo, etc.
20 Particular examples of such subsequences, variants and and modified forms of the peptide sequences exemplified herein (for example, a peptide sequence listed in Tables 1 to 8 and Figure 1) include substitutions, deletions and / or insertions / additions of a or more amino acids, to or from the amino-terminal, carboxy-terminus or internally. An example is the replacement of an amino acid residue with another amino acid residue within the peptide sequence. Another is a deletion of one or more amino acid residues from the peptide sequence or an insertion or addition of one or more amino acid residues in the peptide sequence.
The number of residues replaced, eliminated or inserted / added are one or more amino acids (for example, 1 to 3, 3 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 5 120 to 130, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180 , 180 to 190, 190 to 200, 200 to 225, 225 to 250 or more) of a peptide sequence. Thus, a sequence of FGF19 cjü FGF21 may have few or many amino acids substituted, deleted or inserted / added (for example, 1 to 3, 3 to 5, 10 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70,, 6 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 1.30, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, 190 to 200, 200 to 225, 225 to 250 or more). In addition, an FCF19 amino acid sequence can include or consist of an amino acid sequence of about 1 to 3, 3 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 1'10 to 120, 120 to 130, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, 190 to 200, 200 to 225, 225 to 250 or more amino acids of FGF21 or an amino acid or FGF21 sequence may include or consist of an amino acid sequence of about 1 to 3, 3 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, and 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, 190 to 25 200, 200 to 225, 225 to 250 or more FGF19 amino acids.
Specific examples of substitutions include substitution of a D residue for an L residue. Therefore, although the residues are listed in the L-isomer configuration, D-amino acids in. particular or any of all positions of the 30 peptide sequences of the invention are included, unless a D-isomer leads to a sequence that has no detectable or measurable function.
Additional specific examples are non-conservative and conservative substitutions. A "conservative substitution" is the replacement of an amino acid with a biologically, chemically or structurally similar residue. 5 Biologically similar means that the substitution is compatible with a biological activity, for example, glucose-lowering activity. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine and serine, or having a similar size, or the structure of an additional first, second or peptide sequence is maintained. Chemical similarity means that the residues have the same charge or are hydrophilic and hydrophobic compounds. Particular examples include replacing a hydrophobic residue, such as isoleucine, valine, leucine or methionine with another, or replacing one polar residue with another, such as replacing arginine with lysine, glutamic acid with aspartic acid or glutamine with asparagine, serine to threonine, etc. Routine tests can be used to determine whether a subsequence, variant or modified form has activity, for example, glucose-lowering activity.
e The particular examples of subsequences, variants and modified forms of the peptide sequences exemplified herein (for example, a peptide sequence listed in Tables 1 25 to 8 and Figure 1) are 50% to 60%, 60% to 70%, 70 % to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95% or 96%, 97%, 98% or 99% identity with a reference peptide sequence ( for example, a peptide sequence in any of Tables 1 to 8 and Figure 1). The term "identity" and "homology" and 30 grammatical variations mean that two or more reference entities are the same. Thus, when two amino acid sequences are identical, they have the identical amino acid sequence. "Areas, regions or domains of identity"
they mean that part of two or more referenced entities are the same. Thus, when two amino acid sequences are identical or homologous over one or more regions of the sequence, they share identity in these regions.
The degree of identity between two sequences can be determined using a computer program and a mathematical algorithm known in the art. Such algorithms that calculate the percentage of sequence identity (homology) generally count sequence gaps and mismatches over the comparison region. For example, a BLAST search algorithm (eg BLAST 2.0) (See, for example, Altschul et al., J. Mol. Biol. 215: 403 (19 ° 0), publicly accessible through NCBI) has exemplary search parameters as follows: Incompatibility -2; open gap 5; 15 extension of gap 2. For peptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAMIOO, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA sequence comparison programs (for example- o, FASTA2 and FASTA3) and SSEARCH 20 are also used to quantify the degree of identity and (pearson et al., Proc. Natl. Acad. sci. USA 85: 2444 (1988); Pearson, Methods Mol. Biol. 132: 185 (2000); and Smith et al., J.
Mol. Biol. 147: 195 (1981)). Programs for the quantification of structural protein similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304: 320 (2003)).
In the present invention, peptide sequences, including subsequences, variants and modified forms of the peptide sequences exemplified herein (for example, sequences 30 listed in Tables 1 to 8 and Figure 1), an "amino acid" or "residue" include acids conventional alpha-amino, as well as beta-amino acids, alpha, alpha-disubstituted amino acids and N-substituted amino acids wherein at least one side chain is a portion of the side chain amino acid as defined herein. An "amino acid" also includes alpha-, + N-alkyl amino acids, wherein the N-terminal amino group has a linear or branched C 1 to C 6 alkyl group substituent. The term "amino acid" therefore includes stereoisomers and modifications of naturally occurring amino acids of the protein, non-protein amino acids, post-translational amino acids (for example, by glycosylation, phosphorylation, ester 10 or cleavage amide, etc.) enzyme amino acids -carnished and modified or synthesized, amino acids derived from constructions or structures that mimic amino acids, amino acids with a modified side chain portion, derived from naturally occurring or synthetic portions or that do not occur naturally, etc. Modified and unusual amino acids are included in the peptide sequences of the invention (see, for example, in Synthetic Peptides: A User's Guide; Hruby et al., Biochem J. 268: 249 (1990), and Toníolo C., Int. J Peptide Proteim Res. 35: 287 (1990)).
20 In addition, amino acid protection and modification groups are included. The term "6 amino acid side chain portion", as used herein, includes any side chain of any amino acid, as the term "amino acid" is defined herein. This therefore includes the naturally occurring portion of the side chain of amino acids. In addition, it includes side chain portions of naturally occurring amino acids modified as set forth herein and are known to those. experts in the art, such as side chain portions in stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically modified or synthesized amino acids, derived amino acids, constructions or structures that mimic amino acids, etc. For example, the side chain portion of any amino acid disclosed or known to a person skilled in the art is included within the scope of the definition.
The term "derived from a side chain amino acid moiety" is included within the definition of a side chain amino acid moiety. Non-limiting examples of portions of derived side chain amino acids include, for example: (a) adding one or more saturated or unsaturated carbon atoms to an existing chain, aryl or aralkyl e10 group, (b) replacing an atom carbon in the side chain with another atom, preferably oxygen or nitrogen, (c) adding a terminal group to a carbon atom in the side chain, including methyl (-C &), methoxy (- OCH3), nitro KnoÚ , hydroxyl (-OH) or cyano (-C = N), (d) 15 side chain portions, including a hydroxy, thiol or amine group, adding a suitable hydroxy, thiol or amino protecting group, or (e) side chain portions including a ring structure, the addition of one or more substituents on the ring, including hydroxyl, halogen, alkyl or aryl groups 20 directly linked or via an ether bond. For amino and amino groups, suitable protecting groups are known to those skilled in the art. As long as ta-derivation provides a desired activity in the sequence of the final peptide (eg reduced glycemia, glucose or improved lipid metabolism, the antidiabetic activity, absence of substantial HCC formation or tumorigenesis, absence of significant modulation of lean or fat mass, etc.).
A "side chain amino acid portion" includes all such derivations and in particular non-limiting examples 30 include: gamma-amino butyric acid, 12-amino dodecanoic acid, alpha-aminoisobutyric acid, 6-amino hexanoic acid, carboxylic acid 4 - (aminomethyl) -cyclohexane, acid 8-
aminQoctanoic, biphenyl-alanine, Boc-t-butoxycarbonyl, benzyl, benzoyl, citrulline, diaminobutyric acid, pyrrollisine, diaminopropionic acid, 3,3-diphenylalanine, ortonine,. citrulline, 1,3-dihydro-2H-isoindolcarboxylic acid, ethyl, 5 Fmoc-fluorenylmethoxycarbonyl, heptanoyl (CH3- (CH2) .sub.5-- C (= O) -), hexanoyl (CH3- (CH2) 4-C (= O) -), homoarginine, homocysteine, hornolysin, hornophenylalanine, homoserine, methyl, methionine sulfoxide, methionine sulfone, norvaline (VAL), phenylglycine, propyl, isopropyl, sarcosine (SAR), tert-butylalanine and benzyloxycarbonyl.
and A single amino acid, including stereoisomers and amino acid modifications of the naturally occurring protein, non-protein amino acids, post-translation modified amino acids, enzymatically synthesized amino acids, amino acids that do not naturally occur, including the derived amino acids, an alpha, alpha disubstituted amino acid derived from any of the above (for example, alpha amino acid, disubstituted alpha, where at least one side chain is the same as the residue from which it is derived), a beta-20 amino acid derived from any of the (ie, a beta-amino acid which, except for the presence of a beta- and carbon, is otherwise the same as the residue from which it is derived), etc., including all of the above can be here referred to as a "waste". Suitable substituents, in addition to the side chain portion of alpha-amino acid, include straight or branched C 1 to C 6 alkyl. Aib is an example of an alpha amino acid, a substituted alpha. While alpha, disubstituted alpha amino acids can be referred to using conventional L- and D- isomeric references, it is to be understood that these references are for convenience, and that when the substituents in the alpha position are different, such an amino acid can be referred to regardless. as an alpha, disubstituted alpha amino acid derived from L- or
D'-, as appropriate, from a residue with the designated side chain amino acid portion. Thus, (S) -2-Amino-2-methylhexanoic acid can be referred to either as an alpha, disubstituted alpha amino acid derived from L-5 Nle (Norleucine) or as an alpha, disubstituted alpha amino acid derived from O-Ala. Likewise, Aib can be referred to as an alpha, disubstituted alpha amino acid derived from Ala. Whenever an alpha, disubstituted alpha amino acid is provided, it is to be understood as 10 including all (R) and (S) configurations thereof.
€ 3 An "N-substituted amino acid" includes any amino acid, in which one fraction of side-chain arnirioacid is covalently attached to the amino group of the backbone, optionally, when there are no substituents other than 15 H in the alpha-carbon position. Sarcosine is an example of an N-substituted amino acid. By way of example, sarcosine can be referred to as an N-substituted amino acid derivative of Ala, where the side chain amino acid portion of sarcosine and Ala is the same, i.e., methyl.
The covalent modifications of the peptide sequences of the invention, including subsequences, variants and modified forms of the peptide sequences exemplified herein (for example, sequences listed in Tables 1 to 8 and Figure 1), are included in the invention. One type of covalent modification includes reacting target amino acid residues with an organic derivative agent that is capable of reacting with selected side chains or residues. N- or C-terminals of the peptide. Derivation with bifunctional agents is useful, for example, for the cross-linked peptide for a water-insoluble matrix matrix or surface surface for use in the method for purifying anti-peptide antibodies and vice versa. Commonly used cross-linking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including esters of d.issuccinimidyl such as 3,3'-5 dithiobis (succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3- [(p-azidophenyl) dithio] propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspart e10 residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amine groups of lysine, arginine, and side chains of histidine (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San 15 Francisco, pp 79-86 (1983)), acetylation of the N-terminal amine, the amidation of any C-terminal carboxyl group, etc.
Exemplified peptide sequences, and subsequences, variants and modified forms of the peptide sequences exemplified herein (for example, sequences listed in 20 Tables 1 to 8 and Figure 1), may also include changes in the spine for stability, derivatives and peptidomimetics . The "peptidomimetic" term includes a molecule that is a mimic of a residue (referred to as a "mimetic"), including, but not limited to, piperazine core molecules, ce'to-pipeEazine core molecules and core molecules diazepine. Unless otherwise specified, an amino acid mimetic of a peptide sequence of the invention includes both a carboxyl group and an amino group and a group that corresponds to a b30 amino acid side chain, or, in the case of a mimetic of gl: i [èl "n" a'Tji ' ' sem'-c "side sideline other than hydrogen.
As an example, these include compounds that mimic sterics, surface charge distribution, polarity, etc., of a naturally occurring amino acid, but it does not have to be an amino acid, which provide stability in the biological system. For example, proline can be replaced by other lactams or lactones of suitable size and substitution; Leucine can be substituted by an alkyl ketone urine, N-substituted amide, as well as the amino acid variations in length of the L side chain using an alkyl, alkenyl 10 or other substituents, others may be apparent to the person skilled in the art. The essential element for making such substitutions is to provide a molecule of approximately the same size and charge and configuration as the residue used to design the molecule. Refinement of these modifications will be done by analyzing the compounds in a functional (for example, lowering blood glucose) or another test, and comparing the activity relationship of the structure. Such methods are within the scope of work of the expert in the field of medicinal chemistry and drug development.
Another type of modification of the peptide sequences of the invention, including subsequences, sequence variants and and modified forms of the exemplified peptide sequences (including the peptides listed in Tables 1 to 8 and Figure 1), is glycosylation. As used herein, "glycosylation" 25 broadly refers to the presence, addition or insertion of one or more portions of sugar, (for example, carbohydrates) to proteins, lipids or other organic molecules. The use of the term "deglycosylation" here is generally intended to mean the elimination or suppression of one or more portions of sugar (for example, carbohydrates). In addition, the phrase includes qualitative changes in the glycosylation of native proteins involving a change in the type and proportions (quantity) of the various portions of sugars (for example, carbohydrates) present.
Glycosylation can be achieved by modifying an amino acid residue or by adding one or more glycosylation sites that may or may not be present in the native sequence. For example, a typically non-glycosylated residue 5 can be replaced by a residue that can be glycosylated. The addition of glycosylation sites can be accomplished by changing the amino acid sequence. Alteration of the peptide sequence can be done, for example, by adding or replacing one or more serine or threonine residues 10 (for O-linked glycosylation sites) or, and asparagine residues (for N- glycosylation sites) connected). The structures of N-linked and O-lkyis oligosaccharides and the sugar residues found in each type can be different. One type of sugar that is commonly found in both is N-acetyl-neuramic acid (hereinafter referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, due to its negative charge, can impart acidic properties to the glycoprotein.
The peptide sequences of the invention can optionally be altered by changes in the nucleotide level (for example, DNA), in particular by mutating the DNA encoding the peptide in pre-selected bases such that codons are generated that will translate into the amino acids desired. Another means of increasing the number of carbohydrate moieties in the peptide is by chemical or enzymatic coupling of glycosides to the polypeptide (see, for example, in WO 87/05330). Deglycosylation can be achieved by removing the underlying glycosylation site by deleting the glycosylation 30 by chemical and / or enzymatic means or by replacing ions that encode the amino acid residues that are glycosylated. Chemical deglycosylation techniques are known and enzymatic cleavage of carbohydrate moieties into polypeptides can be achieved through the use of a variety of endo- and exo-glycosidases.
Several cell lines can be used to produce proteins that are glycosylated. A non-limiting example is 5 dihydrofolate reductase (DHFR) - deficient Chinese hamster ovary (CHO) cells, which are from a host cell used for the production of recombinant glycoproteins. These cells do not express the beta-galactoside enzyme alpha-2,6-sialyltransferase and therefore do not add e10 sialic acid in the alpha-2,6 linkage of N-linked glycoprotein oligosaccharides produced in these cells.
Another type of modification is to conjugate (for example, link) one or more additional components or molecules at the N- and / or C-terminal of a peptide sequence of the invention, such as another protein (for example, a protein that has a heterologous acid amino group sequence for the patient protein) or a carrier molecule. Thus, an exemplary peptide sequence can be conjugated to another component or molecule.
20 Certain, amino- or carboxy-terminal embodiments of a peptide sequence of the invention can be fused to an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule) ). Fc fusion conjugates can increase the systemic half-life of biopharmaceuticals, and thus the biopharmaceutical may have prolonged activity or require less frequent administration. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels and, after binding, the Fc fusion molecule is protected against degradation and re-release into the circulation, keeping the molecule in circulation longer.
This linkage is believed to be the mechanism by which endogenous IgG keeps its plasma life longer for longer.
Well-known and validated Fc fusion drugs consist of two copies of a biopharmaceutical linked with the Fc region of an antibody to improve pharmacokinetics, solubility and production efficiency. More recently, Fc fusion technology links a single copy of a biopharmaceutical to an Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical, compared to traditional Fc fusion conjugates. a-0 A modification of the conjugate can be used to produce a peptide sequence that retains activity with an additional or complementary function or activity of the second molecule. For example, a peptide sequence can be conjugated to a 15 molecule, for example , to facilitate solubility, arma7, enarrlento, in vivo or "shelf or stability" half-life, reduced immunogenicity, controlled or delayed release in vivo, etc. Other functions or activities include a conjugate that reduces toxicity to an unconjugated peptide sequence, a conjugate that targets a cell type or organ more efficiently than an unconjugated peptide sequence, or a drug to combat it better. the causes or effects associated with a disorder or disease, as defined here (for example, diabetes).
The clinical effectiveness of protein therapy may be limited by its short plasma life and susceptibility to degradation. Studies of various therapeutic proteins have shown that various modifications, including the conjugation or binding of the peptide sequence of any of a variety of non-proteinaceous polymers, for example, polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes (see, for example, typically through a linkage of the covalently bound fraction to both the protein and the non-proteinaceous polymer (eg, a PEG) can extend the half-life, such PEG-conjugated biomolecules have been shown to have clinically useful properties, including better physical and thermal stability. , protection against su, susceptibility to enzymatic degradation, increased solubility, longer circulating in vivo half-life and decreased clearance, reduced immunogenicity and antigenicity and reduced toxicity., PEGS suitable for conjugation to a sequence of the invention is generally soluble in water at room temperature and have the general formula R (O-CH2-CH2) nO-R, where R is hydr ogen or a protecting group such as an alkyl group or an alkanol group and where 15 n is an integer from 1 to 1000. When R is a protecting group, it usually has 1 to 8 carbon atoms. The PEG conjugated to the peptide sequence can be linear or branched. The rarified PEG derivatives, "star-PEG" and multi-armed PEG are included in the invention. A molecular weight 20 of the PEG used in the present invention is not restricted to any particular range, but certain embodiments' K have a molecular weight between 500 and 20,000, while other embodiments have a molecular weight between 4,000 and
10,000.
The invention includes conjugate compositions in which PEGS have different "n '" values and, therefore, several different prg are present in specific proportions. For example, some compositions comprise a mixture of conjugates in which n = 1, 2, 3 and 4. In some compositions, the percentage of 30 conjugates in which n = 1 is 18 to 25%, the percentage of conjugates in that n = 2 is 50 to 66 ° s, the percentage of conjugates where n = 3 is 12 to 16% and the percentage of conjugates where n = 4 is up to 5%. Such compositions can be produced by reaction conditions and the purification methods known in the art.
PEG can directly or indirectly (for example, through an intermediary) Bind to the peptide sequences of the invention. For example, in one embodiment, the PEG is attached via a reactive terminal group (a "spacer"). The spacer is, for example, a terminal reactive group that mediates a bond between the free amino or carboxyl groups of one or more of the peptide sequences and is polyethylene glycol. The PEG that has the spacer, which can be attached to the free amino group, including polyethylenoglycol N-hydroxysuccinylimide, which can be prepared by activating the succinic acid ester of. polyethylene glycol with N-hydroxysuccinylimide. Another activated polyethylene glycol, which 15 can be attached to the free amino group, is 2,4-bis (O-methoxy polyethylene glycol) -6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride. The activated polyethylene glycol which is attached to the free carboxyl group, includes polyoxyethylene diamine.
The conjugation of one or more of the peptide sequences of the "invention to the PEG" which has a spacer can be carried out by several conventional methods. For example, the conjugation reaction can be carried out in solution at a pH of 5 to 10, at a temperature of 4 ° C to room temperature, for 30 25 minutes to 20 hours, using a molar ratio of protein reagent from 4: 1 to 30: 1. Reaction conditions can be selected to direct the reaction in order to predominantly produce a desired degree of substitution.
In general, low temperature, low pH (for example, pH = 5) and 30 short reaction times tend to decrease the number of PEGs>. bound, while at high temperature, a high to neutral pH (eg, pH27) and longer reaction time tend to increase the number of bound PEGs. Various methods known in the art can be used to terminate the reaction.
In some embodiments, the reaction is terminated by acidifying the reaction mixture and freezing at, for example, -20 ° C.
Peptide sequences of the invention, including subsequences, sequence variants and modified forms of the exemplified peptide sequences (including the peptides listed in Tables 1 to 8 and Figure 1), further include the conjugation of Ey0 large, slowly metabolized molecules, such as proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose spheres, polymeric amino acids, such as polyglutamic acid, polylysine, amino acid copolymers; inactive virus particles; inactivated bacterial toxins such as diphtheria toxoid, tetanus, cholera, leukotoxin molecules; inactivated bacteria and dendritic cells. Such conjugated forms, if desired, can be used to produce antibodies against the peptide sequences of the invention.
Additional suitable components and conjugation molecules C), include, for example, thyroglobulin, alburnins such as human serum albumin (HSA); tetanus toxoid; diphtheria toxoid, polyamino acids such as poly (O-lysine: D-glutamic acid); rotavirus VP6 polypeptides; hemagglutinin of influenza virus, the nucleoprotein of influenza virus; Keyhole Limpet hemocyanin (KLH) and hepatitis B virus core protein and surface antigen or any combination of the above.
Fusion of albumin to a peptide sequence of the invention can, for example, be achieved by genetic manipulation, such that the DNA encoding HSA (human serum alburnin) or a fragment thereof, is linked to the DNA encoding a peptide sequence. Thereafter, a suitable host can be transformed or transfected with the fused nucleotide sequence as, for example, a suitable plasmid 5, in order to express a fusion polypeptide. Expression can be carried out in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo, from, for example, a transgenic organism. In some embodiments of the invention, expression of the fusion protein is carried out on mammalian cell lines, for example, B CHO cell lines.
Other means of genetically fusing target proteins or albumin peptides include a technology known as Albufuse® (Novozymes Biopharma A / S, Denmark) and the sequences of conjugated therapeutic peptides often become much more effective with better absorption in the body. The technology has been used commercially to produce Albuferon "(Human Genome Sciences), a combination of alburnin and interferon cx-2B used to treat hepatitis C virus 20 infection.
and Another embodiment involves the use of one or more human domain antibodies (c1Ab). c1Abs are the smallest functional units of human antibody binding (IgGs) and have favorable stability and solubility characteristics.
25 The technology involves an HSA-conjugated dAb (s) (thus forming an "AlbudAb", see, for example, EP1517921B, WO2005 / 118642 and WO2006 / 051288) and a molecule of interest (for example, a sequence of peptides of the invention). Albuàbs are often smaller and easier to manufacture in 30 microbial expression systems, such as bacteria or fungi, than current technologies used to extend the half-life of peptides. Since HSA has a half-life,
of about three weeks, the resulting conjugated molecule improves the half-life. Use of dAb technology can also improve the effectiveness of the molecule of interest.
Additional suitable components and molecules for conjugation 5 include those that are suitable for isolation or purification. Particular non-limiting examples include binding molecules, such as biotin (specific binding pair avidin-biotin), an antibody, a receptor, a ligand, a lectin or molecules comprising a solid support, including, for example, plastic or polystyrene beads, pIacs or granules, magnetic beads, test strips and membranes.
Purification methods, such as cation exchange chromatography, can be used to separate conjugates by ¶ charge difference, which effectively separates conjugates 15 at their various molecular weights. For example, the cation exchange column must be loaded and then washed with -20mM ~ sodium acetate, pH-4 and then eluted with a linear gradient (OM at 0.5M) of pH-buffered NaCl from 3 to 5.5, preferably at pH-4.5. The content of fractions 20 obtained by cation exchange chromatography can 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. A fraction is then suitably identified, which contains the conjugate having the desired number of linked PEGs, purified free of unmodified protein sequences and conjugates with other linked PEGS numbers.
In still other embodiments, a sequence of the peptide of the invention is related, containing a chemical agent (for example, an immunotoxin or chemotherapeutic agent),
including, but not limited to, a cytotoxic agent, including taxol, cytochalasin B, gramicidin D, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin and the like or homologues thereof. 5 Other chemical agents include, for example, anti-metabolites (for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (for example, rnochlorethamine, carmustine and lomustine, cyclotosfamide, bussulfan , dibromomanitol, streptozotocin, 10 mitomycin C and cisplatin), antibiotics (eg, 0 bleomycin) and anti-mitotic agents (eg vincristine and vinblastine). Cytotoxins can be conjugated to a peptide of the invention using the ligand technology known in the art and described herein.
15 Other suitable components and molecules for conjugation include those that are suitable for the detection of an assay.
Particular non-limiting examples include detectable markers, such as a radioisotope (eg 125I, 35S, 32p, 33p), an enzyme that generates a detectable product (eg 20, luciferase, j3-galactosidase, horseradish and horseradish peroxidase and alkaline phosphatase), a fluorescent protein, a chromogenic protein, dye (for example, fluorescein isothiocyanate); fluorescence-emitting metals (eg 152Eu); chemiluminescent compounds (for example, 25 luminol and acridinium salts), bioluminescent compounds (for example, luciferin) and fluorescent proteins. Indirect markers include labeled or detectable antibodies that bind to a peptide sequence, where the antibody can be "tacted.
In certain embodiments, a peptide sequence of the present invention is conjugated to a radioactive isotope to generate a cytotoxic radiopharmaceutical (radioimmunoconjugates) useful as a diagnostic or therapeutic agent. Examples of such radioactive isotopes include, but are not limited to, i3'Indium1I ', yttrium90 and lutetium177. Methods for the preparation of radioimmunoconjugates are known to those skilled in the art. Examples of radioimmunoconjugates that are commercially available include ibritumomab, tiuxetan and tositumomab. q Other means and methods included in the invention for extending circulation half-life, increasing stability, reducing '0 clearance or changing the immunogenicity or allergenicity of a peptide sequence of the invention involves modifying the d' and peptide sequence by HESylation , which uses hydroxyethyl starch derivatives linked to other molecules, in order to modify the characteristics of the molecule Various aspects of HESylation are described in, for example, US Patent No. Nos. 2007/0134197 and 2006/0258607 .
Any of the above components and molecules used to modify the peptide sequences of the invention can optionally be conjugated via a linker. The. Suitable linkers include "flexible" linkers which are generally of sufficient length to allow some movement between the modified peptide sequences and the linked components and molecules. The binding molecules are generally about 6 to 50 atoms in length. The 25 binding molecules can also be, for example, aryl acetylene, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacids, amino acids or combinations thereof. Suitable linkers can be easily selected and can be of any suitable length, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 30, 30 to 50 amino acids (e.g., Gly).
Exemplary flexible binders include glycine (G) n polymers, glycine-serine polymers (eg (GSÁ, GSGGSn and GGGSn, where n is an integer of at least one), glycine-alanine polymers, alanine polymers —Serine and 5 other flexible binders. The glycine and glycine-serine polymers are relatively unstructured and therefore can serve as a neutral bond between components. Exemplary flexible binders include, but are not limited to GGSG, GGSGG, GSGSG, GSGGG, GGGSG and GSSSG.
6! 0 The peptide sequences of the invention, including the FGF19 and FGF21 variants and subsequences and the FGF19 / "GF21 fusions and chimeras listed in Tables I to 8 and Figure 1, as well as subsequences, sequence variants and modified forms of the listed sequences In Tables 1 to 8 and Figure 1 there is one or more activities as set forth here, an example of an activity is glucose-lowering activity Another example of an activity is the stimulation or reduced formation of hepatocellular carcinoma (HCC), for example exempj-o, compared to EGF 19. An additional example of an activity is decreased or reduced lipids (eg triglycerides, cholesteroi, non-HDL), increased HDL activity, for example, compared to C) with FGF21 Another example of an activity is a low or reduced lean muscle mass reducing mass activity, for example, compared to FGF21_ Yet another example of an activity is the binding to the fibrobla growth factor-4 receptor. stos (GFR4) or activating FGFR4, for example, peptide sequences that bind to FGFR4 with a comparable or greater affinity than the FGF19 binding affinity for FGFR4, and peptide sequences that activate 30 FGFR4 to an extent or equivalent amount or greater than FGF19 activates E'GFR4. Still other examples of activities include down-regulation or reduced expression of the aldo- keto reductase gene, for example, compared to FGFI9; on-
regulation or increased expression of the Slc1a2 gene compared to FGF21.
More particularly, the peptide sequences of the invention, including FGF19 and FGF21 variants and subsequences and the FGF19 / FGF21 fusions and chimeras listed in Tables 1 to 8 and Figure 1, as well as subsequences, variants and modified forms of the sequences listed in Tables 1 to 8 and in Figure 1 include those that have the following activities: peptide sequences having reduced hepatocellular carcinoma (HCC) formation, in
P 6Q compared to the formation of FGF19 or a sequence variant of FGF 19 having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16 to 20 of FGF19; peptide sequences with 15 higher blood glucose lowering activity compared to FGF19 or FGF 19 variant sequence with one of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI , WGDI, WGDP or FGDPI substituted for the WGDPI sequence in amino acids 16 to 20 of FGF19; peptide sequences 20 with less increasing lipid activity (eg less triglycerides, cholesterol, non-HDL) or greater increase in HDL activity compared to FGF19, or a variant FGF 19 sequence, with one of the GQV, GDI , WGPI, WGDPV, WGDI, GDPI, giÍ, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, 25 WGDI, WGDP or FGDPI substituted for the WGDPI sequence in amino acids 16 to 20 of FGF19, and peptide sequences with less lean mass reducing activity compared to FGF21.
More particularly, the peptide sequences of the invention, including FGF19 and FGF21 variants and subsequences and FGF19 / FGF21 fusions and chimeras listed in Tables 1 through 8 and Figure 1, as well as subsequences, variants and modified forms of the sequences listed in Tables 1 to 8 and in Figure 1 include those that have the following activities: sequences of peptides that bind to the fibroblast growth factor-4 receptor (FGFR4) or activate FGFR4, such as peptide sequences that bind FGFR4 with a comparable FGF19 affinity or greater than binding affinity for FGFR4; peptide sequences that activate FGFR4 to an extent or amount equivalent to or greater than FGF19 activates FGFR4; peptide sequences that down-regulate or reduce the expression of the aj-do-keto reductase gene, for example, compared to FGF19; peptide sequences that up-regulate or increase the carrier family of carrier 1, members 2 (Slc1a2) gene expression compared to FGF21.
Activities such as, for example, formation of hepatocellular carcinoma (HCC) or tumorigenesis, glucose-lowering activity, increased lipid activity or reduced lean mass activity can be seen in an animal, such as a db / db rat. Measurement of binding to FGFR4 or activation of F'GFR4 can be determined through assays described herein (see, for example, Example 1) or known to the person skilled in the art.
and The term "link" or "link", when used in reference to a peptide sequence, means that the peptide sequence interacts at the molecular level. Thus, a sequence of 25 peptides that bind FGFR4 binds all or part of the FGFR4 sequence. Specific and selective binding can be distinguished from binding using nonspecific assays, known in the art (for example, coupling, immunoprecipitation, ELISA, flow cytometry, Western blot).
30 Peptides and peptidomimetics can be produced and isolated using methods known in the art. Peptides can be synthesized, in whole or in part, using chemical methods (see, for example, Caruthers (1980) Nucleic Acids
Res.
Symp.
215, Horn (1980); and Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery
5 Systems (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be performed using various solid phase techniques (see, for example, Roberge Science 269: 202 (1995); Merrifield, Methods Enzymol 289: 3 (1997)) and automatic synthesis can be achieved, for example, using ABI
10 431A Peptide Synthesizer (Perkin Elmer), according to
@ manufacturer's instructions.
Peptides and peptide mimetics can also be synthesized using combinatorial methodologies.
Synthetic waste e. polypeptides incorporating mirnetics can be synthesized
15 using a variety of processes and methodologies known in the art (see, for example, Organic Syntheses Collective Volumes, Gilman, et al. (Eds) john Willey & Sons, Inc., New York). Modified peptides can be produced by chemical modification methods (see, for example, 20 Belousov, Nuc-Zeic Acids Res. 25: 3440 (1997); Frenkel, Free Radic.
Biol.
Med. 19: 373 (1995); and Blommers, Biochemistry
33: 7886 (1994)). Variations in the peptide sequence, derivatives, substitutions and modifications can be made using methods such as oligonucleotide-mediated (site-directed) 25, mutagenesis, alanine scan and PCR-based mutagenesis.
Site-directed m'-itagenesis (Carter 'et al., Nucl.
Acids Res., 13: 4331 (1986), Zoller et al.,
Nucl.
Acids Res. 10: 6487 (1987)), cassette mutagenesis (Wells et al., Gene 34: 315 (1985)), restriction selection mutagenesis (Wells et al. Phílos.
Trans. R.
Soc.
London SerA 317: 415 (1986)) and other techniques can be performed on cloned DNA to produce peptide sequences of invention,
variants, mergers, chimeras and variations, derivatives, substitutions and modifications thereof.
The "synthesized" or "manufactured" peptide sequence is a peptide made by any method that involves manipulation by the hand of man. Such methods include, but are not limited to, the above, such as chemical synthesis, recombinant DNA technology, biochemical or enzymatic fragmentation of larger molecules and combinations of the above.
The peptide sequences of the invention, including ELO subsequences, sequence variants and modified forms of the exemplified peptide sequences (for example, sequences listed in Tables 1 to 8 and in Figure I), can also be modified to form a chimeric molecule . According to the invention, there are predicted 15 peptide sequences that include a heterologous domain. These domains can be added to the amino terminus or the carboxyl terminus of the peptide sequence. Heterologous domains can also be positioned within the peptide sequence, and / or alternatively flanked by 20 FGF19 and / or FGF21-derived amino acid sequences.
and The term "peptide" also includes dimers or multimers (oligomers) of peptides. According to the invention, also are provided dimers or multimers (oligomers) of the exemplified peptide sequences, as well as 25 subsequences, variants and modified forms of the exemplified peptide sequences (for example, sequences listed in Tables 1 to 8 and in Figure 1 ).
The invention further provides nucleic acid molecules that encode peptide sequences of the invention, including 30 subsequences, sequence variants and modified forms of the sequences listed in Tables 1 to 8 and Figure 1 and vectors that include the nucleic acid encoding the peptide. Accordingly, "nucleic acids'" include those that encode the exemplified peptide sequences described herein, as well as those that encode for functional subsequences, sequence variants and the modified forms of the exemplified peptide sequences, as long as the above retain the detectable or measurable activity or function. For example, a subsequence, a variant or a modified 10 10 form of an exemplified sequence peptide disclosed herein (for example, a sequence listed in Tables 1 to 8 and Figure 1) that retains some ability to decrease or reduce glucose , provides normal glucose homeostasis or reduces the histopathological conditions associated with chronic or acute hyperglycernia 15 in vivo, etc.
Nucleic acid, which may also be referred to herein as a gene, polymicleotide, nucleotide sequence, primers, oligonucleotide or probe, refers to polymers containing purine and pyrimidine, natural or modified, of any length, whether polyribonucleotides or polydeoxyr- ribonucleotides or a mixture of polyribus-polydeoxyribus and nucleotides and a-anomeric forces of the same. Two or more polymers containing purine and pyrimidine are typically linked by a phosphoester bond or its analog. The terms can be used interchangeably to refer to all forms of nucleic acid, including deoxyribic) nucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids can be single, double or triplex, linear or circular. Nucleic acids include genomic DNA and CDNA. Nucleic acid RNA can be cut or uncut RNArrt, RNAr, RNAt or antisense. Naturally occurring nucleic acids include, synthetic, as well as nucleotide analogs and their derivatives.
As a result of the degeneracy of the genetic code, nucleic acid molecules that include sequences degenerate in relation to nucleic acid molecules that encode the peptide sequences of the invention. Thus, degenerate nucleic acid sequences encoding the peptide sequences, including subsequences, variants and modified forms of the peptide sequences exemplified herein (for example, sequences listed in Tables 1 to 8 and Figure 1), are provided. The term "complementary", when used 10 in reference to a nucleic acid sequence, means that the regions mentioned are 100% complementary, that is, they exhibit 100% base pairing without incompatibilities.
The nucleic acid can be produced using any of a variety of known cloning patterns and chemical synthesis methods, and can be intentionally altered by nutgenesis. site-directed or other recombinant techniques known to a person skilled in the art. Polynucleotide purity can be determined through sequencing, gel electrophoresis, UV spectrometry.
The nucleic acids can be inserted into a nucleic acid construct, in which the expression of the nucleic acid is influenced or regulated by an "expression control element", herein referred to as an "expression cassette" ". The term "expression control element" refers to one or 25 more elements of the nucleic acid sequence that regulate or influence the expression of a nucleic acid sequence to which it is operationally linked. An expression control element may include, as appropriate, promoters, enhancers, transcription terminators, 30-gene silencers, a start codon (eg, ATG) in front of one. gene encoding protein, etc.
An expression control element operably linked to a nucleic acid sequence controls transcription and, where appropriate, translation of the nucleic acid sequence. The term "operationally linked" refers to a juxtaposition 5 in which the referred components are in a relationship that allows them to function in their intended way. Typically, the expression control elements are juxtaposed at the 5 'or 3' ends of the genes, but they can also be intronic.
éL0 The expression control elements include elements that activate the transcription constitutively, that are inducible (that is, it requires an external signal or activation stimuli), or inactivation (that is, it requires a signal to deactivate the transcription, when the signal is no longer present, 15 transcription is enabled or "disabled"). Also included in the expression cassettes of the present invention are control elements sufficient to make gene expression controllable for specific cell or tissue types (e.g., tissue-specific control elements). Typically, such elements are located upstream or downstream (i.e., 5 'and 3') of the coding sequence. The prorotors are generally positioned 5 'from the coding sequence. Promoters produced by recombinant DNA or synthetic techniques can be used to provide transcription of the polynucleotides of the invention. A "promoter" usually means an element of a minimum sequence sufficient for direct transcription.
Nucleic acids can be inserted into a Lasmid for transformation into a host cell and for subsequent expression and / or genetic manipulation. A plasmid is a nucleic acid that can be stably propagated in a host cell; plasmids can optionally contain the elements of control of expEession in order to direct expression of the nucleic acid. For the purposes of this invention, a vector is synonymous with a plasmid. Plasmids and vectors generally contain at least one source of replication for the propagation of a cell and a promoter. Plasmids and vectors can also include an expression control element for expression in a host cell, and are therefore useful for the expression and / or genetic manipulation of nucleic acids encoding peptide sequences, 10 which express the sequences of peptides in cells and hosts and organisms (for example, a patient in need of treatment) or the production of peptide sequences, for example.
As used herein, the term "transgene" designates. a polynucl-eotide that has been introduced into a cell or organism by artifice. For example, in a cell having a transgene, transgene 7 was introduced by genetic manipulation or "transformation" of the cell. A cell or progeny into which the transgene has been introduced is referred to as a "transformed cell" or "transformant". Typically, the transgene is included in the progeny of the transformant or becomes one and part of the organism that develops from the cell. Transgenes can be inserted into chromosomal DNA or maintained as a self-replicating plasmid, YAC, minichromosome or the like.
Promoters of the bacterial system include T7 and inducible promoters such as pL of bacteriophage 2 , plac, ptrp, ptac (hybrid promoter ptrp-lac) and promoters that respond to tetracycline. Protoprotectors of the insect cell system 30 include constitutive or inducible promoters (e.g., ecdysone). Constitutive cellular mammalian promoters include SV40, RSV, pa virus, bovine piloma (BPV) and other virus promoters or inducible promoters derived from the mammalian cell genome. (e.g., metallothionein IIA promoter; heat shock promoter) or mammalian viruses (e.g., adenovirus late promoter, 5 long terminal repeat of the inducible rat tumor tumor virus). Alternatively, the retroviral- genome can be genetically modified to introduce and direct the expression of a peptide sequence in appropriate host cells.
e10 As the methods and uses of the present invention include in vivo distribution, expression systems also include vectors designed for in vivo use. Non-limiting particular examples include adenoviral vectors (US Patents No. 5,700,470 and 5,731,172), adeno-associated vectors 15 (US Patent No. 5,604,090), herpes simplex virus vectors (US Patent No. 5,501,979), retroviral vectors (US Patent No. 5,624,820, 5,693,508 and 5,674,703), BPV vectors (US Patent No. 5,719,054), CMV vectors (US Patent No.
5,561,063) and parvovirus, route, virus, Norwalk virus and 20 lentiviral vectors ( Ter, for example, U.S. Patent No. 6,013,516). Vectors include those that distribute genes to intestinal tract cells, including stem cells (Croyle et al., Gene Ther. 5: 645 (1998); SJ Flenning, Adv. Drug Deliv. Rev. 17: 341 (1997) , US Patent Nos. 5,821,235 and 6,110,456). 25 Many of these vectors have been approved for studies in humans.
Yeast vectors include constitutive and inducible promoters (see, for example, Ausubel et al., In: Current Protocols in Molecular Biology, Vol '. 2, Chap. 13, ed, Greene 30 Publish Assoc & Wiley Interscience, 1988; Grant et al. Methods in Enzymology, 153: 516 (1987), eds. Wu &Grossman; Bitter Methods in Enzymology, 152: 673 (1987), eds Berger & Kimmel,
Acad Press, N.Y .; and, Strathern et al., The Molecular Bio1ogy of the Yeast Saccharomyces (1982) eds. Cold Spring Harbor Press, Vols. I and II). A constitutive yeast promoter such as ADH or LEU2 or an induction promoter such as GAL can be used (R. Rothsteín In: DNA Cloning, A Practical Approach, Vol.11, Ch. 3, ed. DM Glover, IRL Press, Wash., DC, 1986). Vectors that facilitate the integration of foreign nucleic acid sequences into the yeast chromosome, through homologous recombination, for example, are known in the art.
10 Yeast artificial chromosomes (YAC) are typically used when the inserted polynucleotides are too large for more conventional vectors (for example, greater than about 12 Kb).
Expression vectors can also contain a selectable marker 15 that confers resistance to a selective pressure or identifiable marker (e.g., beta-galactosidase), thus allowing cells with the vector to be selected for cultivation and expanded. Alternatively, a selectable marker can be a "second vector co-transfected into a host cell by a first vector containing a nucleic acid that encodes a peptide sequence. Selection and selection systems include, but are not limited to, the herpes sirnplex virus of the gene. thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine gene (Szybalska et al. Proc.
25 Natl. Acad. Sci. USA 48: 2026 (1962)) and adenine phosphoribosyltransferase genes (Lowy et al., Cell 22: 817 (1980))) that can be employed in tk-, hgprt- or aprt- cells, respectively. In addition, anti-metabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (O'Hare et al., Proc. Natl.
Acad. Sci. USA 78: 1527 (1981)); the gpt gene, which confers resistance to mycophenolic acid (Mulligan et al., Proc.
Natl. Acad. Sci. USA 78: 2072 (1981)); neomycin gene, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin. et al., j. Mol. Biol. 150: 1 (1981)); puromycin; and hygromycin gene, which confers resistance to hygromycin (Santerre et al., Gene 30: 147 (1984)). Additional selectable 5 'genes include trpB, which allows cells to use indole instead of tryptophan; hisO, which allows cells to use histinol instead of histidine (Hartrnan et al., Proc. Natl.
Acad. Sci. USA 85: 8047 (1988)) and ODC (ornithine decarboxylase), which confers resistance to the ornithine-decarboxy-ase inhibitor, 2- (difluoromethyl) -DL-ornithine, DFMO @ (McConlogue (1987) In: Current Communications in Molecular, Biology, Cold Spring Harbor Laboratory).
According to the invention, transformed cells (in vitro, ex vivo and in vivo) and host cells that produce a variant or fusion of FGF19 and / or FGF21 as set forth herein are provided, where the expression of a FGFI9 and / or FGF21 variant or fusion is conferred by a nucleic acid encoding the FGF19 and / or FGF21 variant or fusion. Transformed and host cells expressing the 20 peptide sequences of the invention typically include a nucleic acid - which encodes the peptide sequence of the invention. In one embodiment, a transformed or host cell is a prokaryotic cell. In another embodiment, a transformed or host cell is an eukaryotic cell. In several respects, the eukaryotic cell is a yeast or mammalian cell (for example, human, prince, etc.).
As used herein, a "transformed" or "host" cell is a cell into which a nucleic acid is introduced, 1 which can be propagated and / or transcribed for the expression of a coded peptide sequence. The term also includes any progeny or subclones of the host cell.
Host and transformed cells include, but are not limited to microorganisms, such as bacteria and yeast and plants, insects and mammalian cells. For example, bacteria transformed with the recombinant nucleic acid of 5 expression vectors of bacteriophage, plasmid or cosmid nucleic acid; yeast transformed with recombinant yeast expression vectors, plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, IO CaMV; tobacco mosaic virus, TMV) or transformed with 6 recombinant vectors plasmid expression systems (for example, Ti plasmid), insect cell systems infected with recombinant virus expression vectors (for example, baculovirus) and animal cell systems infected with 15 recombinant virus expression vectors (for example, retrovirus, adenovirus, vaccinia virus) or transformed animal cell systems modified for transient or stable propagation or eXpression.
For gene therapy uses and methods, a transformed cell 20 may be from a patient. U-in a patient's cell can be transformed with a nucleic acid that encodes a peptide sequence of the invention, as established herein in vivo. Alternatively, a cell can be transformed in vitro with a transgene or polynucleotide urine, 25 and then transplanted into a patient's tissue in order to carry out the treatment. Alternatively, isolated primary cells or an established cell line can be transformed with a transgene or polynucleotide encoding a FGF19 and / or FGF21 variant or a chimeric fusion / sequence (or variant) thereof, such as a sequence of chimeric peptide, including all or a portion of FGE19 or 'including all or a portion of FGF21 and then, optionally, transplanted into a tissue of a patient.
Non-limiting target cells for the expression of peptide sequences, in particular for expression in vivo, include 5 cells of the pancreas (islet cells), muscle cells, mucous cells and endocrine cells. Such endocrine cells may provide inducible production (secretion) of a variant of FGF19 and / or FGF21 or a chimeric fusion / sequence (or variant) of the same, such as a 6Q chimeric peptide sequence, including all or a portion of FGF19 or including all or a portion of FGF21. Additional cells to transform include stem cells or other multipotent or pluripotent cells, for example, cells. progenitors differ in different cells of the pancreas 15 (islet cells), muscle cells, mucous cells and endocrine cells. Stem cell segmentation provides long-term expression of peptide sequences of the invention.
As used herein, the term "culture", when used in reference to a cell, means that the cell is cultured and in vitro. A particular example of such a cell is a cell isolated from a patient and cultured or adapted for growth in tissue culture. Another example is a genetically engineered cell, in vitro and transplanted back 25 to the same type or a different patient.
The term "isolated", when used in reference to a cell, means that a cell is separated from its natural environment in vivo. "Cultured" and "isolated" cells can be manipulated by the hand of man, such as genetically transformed 30. These terms include all of the cell's offspring, including offspring cells that may not be identical to the parental cell, due to mutations that occur during cell division. The terms do not include a complete human being.
Nucleic acids encoding peptide sequences of the invention can be introduced for stable expression in cells of an entire organism. Such organisms, including non-human transgenic animals, are useful for studying the effect of peptide expression on an entire animal and the therapeutic benefit. For example, as described herein, @! 0 production of a variant of FGF19 and / or FGF21 or one.
chimeric fusion / sequence (or variant) thereof, such as a chimeric peptide sequence, including all or a portion of FGF19 or including all or a portion of FGF21 as set forth herein, in reduced glucose rats and is antidiabetic. 7m --- Strains of mice that develop or are susceptible to the development of a particular disease (eg, diabetes, degenerative disorders, cancer, etc.) are also useful for the introduction of therapeutic proteins, as described here, in order to study the effect of the expression of the therapeutic protein of the rat su, sensitive to. disease. Models «transgenic and genetic animals that are susceptible to the disease in particular or under physiological conditions, such as induced diabetic rats (STZ). streptozotocin (STZ), there are 25 targets suitable for expressing FGF19 and / or FGF21 variants, chimeric fusions / sequences (or variant) thereof, such as a chimeric peptide sequence, including all or a portion of FGF19 or including all or a portion of FGF21, as set forth herein. Thus, according to the invention, there are provided non-human transgenic anirrials which produce a variant of FGF19 and / or FGF21 or a fusion / sequence, chiral (or variant) thereof, such as a peptide sequence chimeric, including all or a portion of FGF19 or including all or a portion of FGF21, the production of which does not occur naturally in the animal which is conferred by a transgene present in the animal's somatic or germ cells.
The term "transgenic animal" refers to an animal whose somatic or germline cells contain genetic information received, directly or indirectly, through deliberate genetic manipulation at the subcellular level, such COUlO € g10 by micro-injection or infection with recombinant virus. The term "transgenic" further includes cells or tissues (for example, "transgenic" cell "," transgenic tissue ") obtained from a genetically modified transgenic animal, as described herein. In the present context, a" transgenic animal "does not 15 covers animals produced by classical crossbreeding or in vitro fertilization, but indicates animals in which one or more cells receive a nucleic acid molecule. Transgenic animals of the invention can be heterozygous or homozygous with respect to the transgene. transgenic animals, including rats, sheep, pigs and frogs are well known in the art (see, for example, 6 'US Patent Nos. 5,721,367, 5,695,977, 5,650,298 and 5,614,396) and, as such, are additionally included.
Peptide sequences, nucleic acids encoding 25 for the peptide sequences, vectors and transformed host cells that express the peptide sequences include isolated and purified forms. The term "isolated", when used as an encoder for a composition of the invention, means that the composition is separated, substantially complete or at least in part, from one or more components of an environment. generally, compositions that exist in nature, when isolated, are substantially free of one or more materials with which they normally are. are associated with nature, for example, one or more proteins, nucleic acids, lipids, carbohydrates or cell membranes. The term "isolated" does not exclude alternative physical forms of the composition, such as variants, modifications or derived forms, fusions and chimeras, multimers / oligomers, etc., or forms expressed in host cells. C) "isolated" land also does not exclude forms (for example, pharmaceutical compositions, combination compositions, etc.), in which there are combinations, any of which is produced by the hand of man.
An "isolated" composition can also be "purified" when free of some, a substantial number of or most or all of one or more other materials, such as a contaminant or an unwanted substance or material. the peptide sequences of the invention are generally not known or believed to exist in nature. However, for a composition that does not exist in nature, an isolated composition will generally be free from some, a substantial number of or most or all other materials with which it typically associates in nature. Thus, a sequence of '0 isolated peptide, which' / also occurs in nature, does not include polypeptides or polynucleotides present among millions of other sequences, such as proteins from a library of 25 proteins or nucleic acids from a genomic or cDNA library , for example. A "purified" composition includes combinations with one or more other inactive or active molecules. For example, a peptide sequence of the present invention combined with another drug or agent, such as a glycemia-lowering drug or therapeutic agent, for example.
As used herein, the term "recombinant", when used as a modifier for peptide sequences, nucleic acids that encode peptide sequences, etc., means that the compositions have been manipulated (i.e., by engineering) from a way that normally does not occur in nature (for example, in vitro). A specific example of a recombinant peptide would be where a peptide sequence of the invention is expressed by a cell transfected with a nucleic acid that encodes the peptide sequence. A specific example of a recombinant nucleic acid would be where one is a nucleic acid (for example, genomic DNA or DNAC) that encodes a cloned peptide sequence in a plasmid, with or without 5 ', 3' or intron regions than the gene it is usually contiguous in the organism's genome. Another example of a recombinant peptide or nucleic acid is a hybrid or fusion sequence, such as a chimeric peptide sequence that comprises a FGF19 portion and a FGF21 portion.
According to the invention, compositions and mixtures of peptide sequences of the invention are provided, including 20 subsequences, variants and modified forms of the explified peptide sequences (including variants and 0 subsequences of FGF19 and FGF21 listed in Tables 1 to 8 and in Figure 1 provided and the FGF19 / FGF21 fusions and chimeras listed in Tables 1 to 8 and Figure 1). In one embodiment, a mixture includes one or more peptide sequences and a pharmaceutically acceptable carrier or excipient. In another embodiment, a mixture includes one or more sequences of peptides and a drug or therapeutic agent. adjunct, as an "anti-diabetic agent or drug 30 or blood glucose lowering therapeutic agent. Examples of drugs and therapeutic agents are presented below. Combinations, such as one or more peptide sequences in a pharmaceutically acceptable vehicle or excipient, with urn
65/116.
or more antidiabetic drugs or blood glucose lowering drugs or therapeutic agents are also provided. Such peptide sequence cordinations of the invention with gutro drug or agent, such as a blood glucose lowering drug or therapeutic agent, for example, are useful according to the methods and uses of the invention, for example, for the treatment of a patient.
The combinations also include the incorporation of peptide or nucleic acid sequences of the invention into particles or polymeric substances, such as polyesters, carbohydrates, polyamine acids, hydrogel, polyvinylpyrrolidone, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose, sulfate protamine or lactide / glycolide copolymers, polylactide / glycolide copolymers or ethylene vinyl acetate copolymers; imprisonment in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by using microcapsules of hydroximeÈylcellulose or gelatin or poly (methyl methacrolate microcapsules), 20 respectively; incorporation of release and dispersion systems in the colloid of. drugs, such as complexes of @ macromolecules, nanocapsules, microspheres, beads and lipid-based systems (for example, N-fatty acyl groups, such as N-lauroyl, N-oleoyl, fatty-amines, such as 25 amine dodecyl, oleoyl amine, etc., see US Patent No.
6,638,513), including oil-in-water emulsions, micelles, micelles.mixed and liposomes, for example.
Invention peptides, including subsequences, variants and modified forms of the exemplified peptide sequences 30 (including the FGF19 and FGF21 variants and subsequences listed in Tables 1 to 8 and Figure 1 and the FGF19 / FGF21 fusions and chimeras listed in Tables 1 to 8 and Figure 1) COUlO here
· - ~ m 66/116 established can be used to modulate glucose metabolism and facilitate the transport of glucose from the blood to metabolic organs, such as muscle, liver and adipose tissue. Such peptide sequences can be produced in sufficient or effective amounts to restore glucose tolerance and / or to improve or provide normal glucose homeostasis.
As disclosed herein, administration of various variant peptide sequences and fusion of FGF19 and FGF21 with <0 mice successfully reduced glucose levels. In addition, in contrast to FGF19, certain peptide sequences do not stimulate or induce the formation of HCC or tumorigenesis in rats. Thus, administration of the peptides of the invention, including subsequences, variants and modified forms of the 15 exemplified peptide sequences (including the FGF19 and FGF21 variants and subsequences listed in Tables 1 to 8 and Figure 1 and the FGF19 / FGF21 fusions and chimeras listed in Tables 1 to 8 and Figure 1), in an animal, either directly or indirectly in vivo or ex vivo methods (for example, administration of the fusion or peptide variant, a nucleic acid encoding the fusion variant or peptide or a transformed cell or gene therapy vector that expresses the variant or peptide fusion), can be used to treat various diseases.
. Consequently, the invention includes in vitro, ex vivo and in vivo (e.g., on or in a patient) methods and uses.
These methods and uses can be practiced with any of the peptide sequences of the invention set forth herein.
According to the invention, methods of treating a patient having or at risk for a disorder are provided. In various embodiments, a method includes administering a peptide sequence, such as a FGF19 or FGF21 variant, fusion or chimera listed in Tables 1 to 8 and in Figure 1 or a subsequence, variant or modified form of a variant, fusion or chimera of 5 FGF19 or FGF21 listed in Tables 1 to 8 and Figure 1 to a patient in an amount effective to treat the disorder.
Examples of treatable, preventable and similar disorders with the peptides of the invention and methods and uses, include diseases and metabolic disorders. Non-limiting examples of diseases and disorders include: 1. Disorders of glucose use and associated sequelae, including diabetes mellitus (type I and type-2), gestational diabetes, hyperglycemia, insulin resistance, abnormal glucose metabolism, "pre-diabetes" (impaired fasting glycemia (IFG) or glucose intolerance 15 (IGT)), and other physiological disorders associated with or resulting from hyperglycemic conditions, including, for example, histopathological changes such as cell destruction pancreatic j3. For treatment, the peptide sequences of the invention can be administered to patients who have a fasting plasma glucose (fpg) at a level greater than about 100 mg / dl. The peptide sequences of the invention and may also be useful in other hyperglycemic-related diseases, including damage to the kidneys (eg, tubule damage or nephropathy), liver degeneration, eye damage 25 (eg, diabetic retinopathy or cataracts) and disorders diabetic foot; 2. Dyslipidemias and their sequelae, such as, for example, atherosclerosis, artery and coronary disease, cerebrovascular diseases and the like; 3. Other conditions that may be associated with the metabolic syndrome, such as obesity and high body mass (including their comorbid conditions, such as, but not limited to, non-alcoholic fatty liver disease (NAFLD), non-fatty liver disease alcohol (NASH) and polycystic ovary syndrome (PCOS))
and also include thrombosis, hypercoagulable and prothrombotic states (arterial and venous), hypertension, cardiovascular disease, stroke and heart failure; 4. Disorders or conditions in which inflammatory reactions are involved, including atherosclerosis, chronic inflammatory bowel diseases (eg, Crohn's disease and ulcerative colitis), asthma, lupus erythematosus, arthritis or other rheumatic inflammatory disorders; 5. Disorders of cell cycle processes or cell differentiation, such as adipose cell tumors, carcinomas and lipomatous tumors, including, for example, liposarcoma, solid tumors and neoplastic diseases; 6. Neurodegenerative diseases and / or disorders of the central and peripheral nervous system and / or neurological diseases involving neuroinflammatory processes and / or other peripheral neuropathies, including Alzheimer's disease, multiple sclerosis, Parkinson's disease, progressive multifocal leukoencephalopathy and Guillain-8arre syndrome; 7. Disorders of the skin and / or diseases of wound healing processes, including erythematous-scaly dermatoses and 8. Other diseases such as syndrome X, osteoarthritis and acute respiratory distress syndrome.
and As used herein, the term "hyperglycemic" or "hyperglycemia", when used in reference to a patient's condition, an abnormally high transient or chronic level of glucose present in a patient's blood. The condition can be caused by a delay in glucose metabolism or absorption, such that the patient has glucose intolerance or a high glucose state not normally found in normal patients (for example, in 30 pre-diabetic, intolerant patients glucose at risk of 'developing diabetes or in diabetic patients). Fasting plasma glucose (FPG), normoglycemia levels are less than about 100 mg / dl, for impaired glucose metabolism, between about 100 and 126 mg / dl and for diabetics greater than about 126. wstg / d1.
As described herein, the invention includes methods of prevention, (for example, in patients predisposed to having a specific disorder (s)), delaying, delaying or inhibiting progression, at the beginning of or 'treatment (for example, example, improving) obesity or an undesirable body mass (for example, higher than normal body mass index or "BMI" in relation to an appropriate combined patient of comparable 6Q age, sex, race, etc.). Thus, in various embodiments, a method of the invention for, for example, q treatment of obesity or an undesirable body mass (including co-morbid conditions, such as obesity, such as obstructive sleep apnea, arthritis, cancer (for example, breast, endometrial and colon), gallstones or hyperglycemia, includes contacting or administering a peptide of the invention, as defined herein (for example, a variant of FGF19 and / or FGF21 as defined in Tables 1 to 8 or in Figure 1, for example) in an amount 20 effective to treat obesity or an undesirable body mass In particular aspects, a patient has a C) body mass index greater than 25, for example , 25 to 30, 30 to 35, 35 to 40 or greater than 40.
In addition, the invention includes methods of prevention (for example, in patients predisposed to have specific disorder (s)), slowing or inhibiting progression, delaying the onset or treatment of undesired or abnormally high levels of LDL in the serum / plasma, VLDL, cholesterol or triglycerides, which, alone or in combination, can lead, for example, to plaque formation, narrowing or blocking of blood vessels and increased risk of hypertension, stroke, and coronary artery disease. These disorders can be due to, for example, genetic predisposition or diet, for example.
The term "patient" refers to an animal. Usually the animal is a mammal that would benefit from treatment with a peptide sequence of the invention. Particular examples include primates (e.g., humans), dogs, cats, horses, cows, pigs and sheep.
Patients include those who have a disease, for example, a hyperglycemic disorder, such as diabetes or eL0 patients who do not have the disease, but can. be at risk of developing the disease, for example, pre-diabetic patients with FPG levels greater than 100 mg / dl, for example, between about 100 and 126 mg / dl. Patients at risk of developing a disease include, for example, those whose diet may contribute to the development of acute or chronic hyperglycemia (eg, diabetes), undesirable body mass or obesity, as well as those who may have a family history or genetic predisposition for the development of acute or chronic hyperglycemia or undesirable body mass or obesity.
and As described herein, methods of treatment include contacting or administering a peptide of the invention, as defined herein (for example, a variant or fusion of FGF19 and FGF21 or as defined in Tables 1 to 8 or in Figure 1 , for example) in an amount effective to achieve the desired goal or result. A treatment that results in a desired goal or result includes decreasing, reducing or preventing the severity or frequency of one or more symptoms of the patient's condition, for example , an improvement in the patient's condition 30 or a "beneficial" or "therapeutic effect." Therefore, treatment may increase or reduce or prevent the severity or frequency of one or more symptoms of the disease, stabilize or inhibit the progression or worsening disease, and in some cases, to reverse the disorder, temporarily (for example, for 1 to 6, 6 to 12 o'u 12 to 24 hours), for medium term (for example, 1 to 6, 6 to 12, 12 to 24 or 24 to 48 days) or long term (e.g. for 1 to 6, 6 to 12, 12 to 24, 24 to 48 weeks or greater than 24 to 48 weeks). Thus, in the case of a hyperglycemic disorder, for example, c) treatment can decrease or reduce blood glucose, increase glucose tolerance, improve glucose metabolism, provide normal glucose homeostasis, decrease or reduce resistance insulin, decrease or reduce insulin levels or decrease, prevent, improve or reverse metabolic syndrome or a histopathological disorder associated with or resulting from a hyperglycemic disorder, such as diabetes.
For example, a peptide sequence, method, or use can. decrease or reduce glucose in one or more patients with FPG levels greater than 100 rig / dl, for example, between about 20 out of 100 and 125 mg / dl or greater than 125 mg / dl, from about 5 to 10% , 10 to 20%, 20 to 30% or 30 to 50% or more, or, for example, Q from greater than 200 mg / dl and less than 200 mg / dl, more than 150 rng / dl and less than 150 mg / dl, greater than 125 mg / dl less than 125 rng / dl, etc. In addition, a peptide sequence, method or use can decrease or reduce glucose, for example, for patients with pre-diabetes or for diabetes (for example, type 2) with baseline HbAIc levels greater than about 5 ° 0, 6%, 7 ° 5, 8 ° 5, 9% or 10 ° :, especially 5 ° 5, 6 ° 0 or 7 ° ;.
30 Non-limiting examples of a me-hory of a histopathological alteration associated with a hyperglycemic condition include, for example, decreasing, inhibiting, reducing or stopping: the destruction or degeneration of cells in the pancreas (eg B-cells), damage to the kidneys, such as tubule calcification or nephropathy, liver degeneration, eye damage (eg, diabetic retinopathy, cataracts), diabetic foot, 5 mucosal ulcers such as the mouth and gums, peri.odontitis, excess bleeding, scarring slow or delayed lesions or wounds (for example, that churribo for diabetic carbuncles), skin infections and other skin diseases, coronary and cardiovascular diseases, peripheral vascular disease, stroke, dyslipidemia, and hypertension, obesity or risk to develop any of the precedents. Improvement in undesirable body mass or obesity may include, for example, a reduction in body mass (as reflected by BMI or the like) or an improvement in an associated pathology, such as a decrease in triglycerides, LDL or VLDL cholesterol , decrease in blood pressure, decrease in thickening of the intimal layer of the blood vessel or decrease in the reduced risk of cardiovascular disease or stroke, decrease in resting heart rate, etc.
An "effective amount" or a "sufficient amount" for and use and / or for the treatment of a patient refers to an amount that provides, in single or multiple doses, alone or in combination with one or more other compositions 25 (therapeutic agents such as a drug or hyperglycemia treatment), treatments, protocols or treatment regimen agents, a detectable response of any length of time (transient, medium or long term), a desired result or an objective or subjective benefit for a patient of any measurable or detectable degree or for any length of time (for example, for hours, days, iiieses, years or healed). These amounts are generally effective in ameliorating a disease, or one, several or all of the adverse symptoms, consequences or complications of the disease, to a measurable degree, although reducing or inhibiting the progression or worsening of the disease is considered a satisfactory result. .
5 As used herein, c) the term "improving" means an improvement in the patient's disorder, a reduction in the severity of the disease or an inhibition of the progression or worsening of the disease (for example, the stabilization of the disorder). In the case of 'a hypergemic disorder (eg diabetes, insulin resistance, glucose intolerance, metabolic syndrome, etc.), for example, an improvement could be a reduction or decrease in blood glucose, a reduction in resistance to insulin, a reduction in glucagon, an improvement in glucose tolerance or glucose metabolism or homeostasis. An improvement in a hyperglycemic disorder can also include an improvement in pancreatic function (for example, inhibiting or preventing the destruction of β-cell / islet or increasing the number and / or function of γ-cells), a decrease in an associated pathology a or resulting from disease, such as an improvement. in the histopathology of an affected tissue or organ, as set forth herein. In the case of undesirable body mass or @ obesity, for example, an improvement can be a reduction in weight gain, reduction in body mass (as reflected in the reduction in BMI, for example) OL1 an improvement in condition 25 associated with obesity of unwanted body mass, for example, as set forth herein (e.g., reduction or .decrease in blood glucose, triglycerides, LDL or VLDL cholesterol, decrease in blood pressure, decrease in thickening of the inner layer of the sariguous vessel, etc. ).
30 A therapeutic benefit or improvement, therefore, need not be complete ablation of any one, most or all of the symptoms, complications, consequences or causes associated with the disorder or disease. Thus, a satisfactory end point is achieved when there is a transient, medium or long term, an incremental improvement in a patient's condition or a partial reduction in the occurrence, frequency, severity, 5 progression or duration, or the inhibition or reversal of one or more associated adverse symptoms or complications or underlying consequences or causes, worsening or progression (for example, stabilization of one or more symptoms or complications of the disease, disorder or disease), disorder or disease over a period of time (hours, days, and weeks, months, etc.).
Thus, in the case of a disease treatable by a peptide sequence of the invention, the amount of peptide sufficient to alleviate a disorder will depend on the type, severity and extent or duration of the disease, the therapeutic effect or desired outcome and may be easily determined by the person skilled in the art. The appropriate amounts will also depend on the individual patient (eg, bioavailability within the patient, sex, age, etc.) For example, a temporary or partial restoration of normal glucose homeostasis in a patient may reduce the amount of 6 dosage or the frequency of injection of insulin, inhibits the complete release of insulin has not worked.
An effective amount can be determined, for example, by preventing one or more relevant physiological effects. Erri a specific non-limiting example, in the case of a hyperglycemic condition, a decrease or reduction in blood glucose or an improvement in the glucose tolerance test can be used to determine whether the amount of peptide sequence of the invention, including subsequences, sequence variants and modified forms of PePtídeOqq sequences (for example, exemplified, sequences Listed in Tables 1 to 8 and Figure
1) it is effective in treating a hyperglycemic condition.
In another particular non-limiting example, an effective amount is an amount sufficient to reduce or decrease any level
(eg, a baseline level) of FPG, where, for example, an amount sufficient to reduce c) an FPG level greater than 200 mg / dl to less than 200 mg / dl, an amount sufficient to reduce the FPG level between 175 mg / dl and 200 mg / dl less than the pre-administration level,
there was enough to reduce the level of FPG between
10 150 mg / dl and 175 mg / dl less than the pre-
and administration, an amount sufficient to reduce the FPG level between 125mg / dl and 150mg / dl less than the pre-administration level, and so on (for example, reducing FPG levels to less than 125 mg / dl, less than 120
15 mg / dl, less than 115 mg / dl, less than 110 mg / dl, etc.) In the case of HbAIc levels, an effective amount includes an amount sufficient to reduce or decrease levels of more than that about 10% to 9%, in more than about 9% to 8%, for more than about 8% to 7%, for more
20 than about 7% at 6 ° 5, by more than about 6% to 5%, and so on.
More particularly, the reduction or decrease in 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 an effective amount according to the invention.
In yet another particular non-limiting example, in the case of undesirable body mass or obesity, an effective amount is an amount sufficient to decrease or reduce a patient's body mass index (BMI), a decrease or reduction
30 of glucose, a reduction or reduction in serum / plasma levels of triglycerides, lipid, cholesterol, fatty acids, LDL and / or VLDL.
In still particular non-limiting examples, the amount is an amount sufficient to decrease or reduce any of the parameters mentioned above, for example, about 0.1%, 0.25%, 0.4%, 0 , 5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 ° 0, 1.5 ° ó, 2 ° 0, 3%, 4 ° 5, 5%, 10% , 20%, 30%, 33%, 35%, 40%, 45%, 50% or more.
The methods and uses of the invention for treating a patient are applicable for prophylaxis to prevent a disorder in a patient, such as a hyperglycemic disorder or development of undesirable body mass or obesity. Alternatively, the methods and uses can be practiced e10 during or after treating a patient. For example, before, during or after treating a patient to reduce glucose using insulin or another blood glucose lowering agent or therapeutic agent, for example, a method or use of the invention may, for example, a sequence of peptide from invention can be administered to the patient. In addition, a composition, such as a peptide sequence of the invention, can be combined with another drug or agent, such as a blood glucose lowering drug or therapeutic agent, for example.
Thus, the methods and uses of the invention for treating a patient can be practiced before, substantially contemporaneously with or following another treatment and can be complemented with other forms of therapy. Complementary therapies include other glucose-lowering treatments, such as insulin, an insulin sensitivity booster and other drug treatments, a change in diet (low sugar, fats, etc.), weight loss surgery (reducing the volume of stomach by gastric bypass, gastrectomy), gastric band, gastric balloon, gastric sleeve, 30 etc. For example, a method or use of the invention for treating a hyperglycemic disorder or insulin resistance can be used in combination with other drugs or pharmaceutical compositions that reduce glucose or increase insulin sensitivity in a patient. Medicines for the treatment of diabetes include, for example, biguanides and sulfonylureas (for example, tolbutamide, chloropropamide, 5 acetohexamide, tolazamide, glibenclamide and glipizide), thiazolidinediones (rosiglitazone, pioglitazone), GLP-i analogs, inhibitors dipeptidyl peptidase-4 (DPP-4), bromocriptine formulations (for example, bile acid scavengers and (for example, colesevelam)), and insulin IO (bolus and basal analogs), metformin (for example, hydrochloride and metformin) with or without a thiazolidinedione (TZD) and SGLT-2 inhibitors. Appetite suppressant medications are also well-known supplemental therapies and can be used in combination with the methods of the invention. They can be administered before, simultaneously or following methods of the invention and uses.
The peptide sequences of the invention, including subsequences, sequence variants and modified forms of exemplified peptide sequences (sequences listed in Tables 1 to 8 and Figure 1), can be formulated in a dosage unit or unit dosage form . In a particular embodiment, a peptide sequence is in an effective amount to treat a patient in need of treatment, for example, due to hyperglycemia. Exemplary 25 unit doses range from about 25 to 250, 250 to 500, 500 to 1000, 1000 to 2500 or 2500 to 5000, 5000 to 25,000, 25,000 to 50,000 ng, from around 25 to 250, 250 to 500, 500 to 1000, 1000 to 2500 or 2500 to 5000, 5000 to 25,000, 25,000 to 50,000 µg and about 25 to 250, 250 to 500, 500 to 1000, 1000 30 to 2500 or 2500 to 5000, 5000 to 25,000, 25,000 at 50,000ing.
The peptide sequences of the invention, including subsequences, sequence variants and modified forms of exemplified peptide sequences (sequences listed in Tables 1 to 8 and Figure 1) can be administered to provide the desired effect in single or multiple doses, for example example, in an effective or sufficient amount. Exemplary doses range from about 25 to 250, 250 to 500, 500 to 1000, 1000 to 2500 or 2500 to 5000, 5000 to 25,000, 25,000 to 50,000 pg / kg, from about 50 to 500, 500 to 500 " 0, 5000 to 25,000 or 25,000 to 50,000 ng / kg and from about 25 to 250, 250 to 500, 500 to 1000, 1000 to 2500 or 2500 to 5000, 5000 10 to 25,000, 25,000 to 50,000 µg / kg. or multiple and can be administered, for example, several times a day, on consecutive days, alternating days, weekly or intermittently (for example, twice a week, once every 1, 2, 3, 4, 5, 6, 7 or 8 weeks or once every 2, 15, 3, 4, 5 or 6 months).
The peptide sequences of the 'invention, including subsequences, variants and modified forms of the exemplified peptide sequences (sequences listed in Tables 1 to 8 and Figure 1), can be administered and methods can be practiced by systemic, regional or local administration, by any route. For example, a peptide sequence can be and administered parenterally (for example, subcutaneously, intravenously, intramuscularly or intraperitoneally), orally (for example, ingestion, buccally or sublingually), by inhalation, intradermally, intracavity , intracranial, transdermal (topical), transmucosal or rectal. The peptide sequences of the invention, including subsequences, variants and modified forms of the exemplified peptide sequences (sequences listed in Tables 1 to 8 and Figure 1) and those methods of the invention, including pharmaceutical co-compositions, can be administered via a system (micro) release capsules or packaged in an implant for administration.
The invention further provides "pharmaceutical compositions" that include a peptide sequence (or sequences) of the 'invention, including subsequences, variants and modified forms of the exemplified peptide sequences 5 (sequences listed in Tables 1 to 8 and Figure 1), and a or more pharmaceutically acceptable vehicles or physiologically acceptable diluent, vehicle or excipient. In certain embodiments, a peptide sequence or sequences are present in a therapeutically acceptable amount. Pharmaceutical compositions can and can be used in accordance with the methods and uses of the invention. Thus, for example, pharmaceutical compositions may be administered ex vivo or in vivo to a patient, in order to practice the methods of treatment and uses of the present invention.
The pharmaceutical compositions of the invention can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are presented here. In addition, pharmaceutical compositions can still contain other therapeutically active agents or compounds disclosed herein (e.g., glucose reducing agents) or known to those of skill in the art that can be used in the treatment or prevention of various diseases and disorders, such as established here.
Pharmaceutical compositions that typically comprise a therapeutically effective amount of at least one of the peptide sequences of the invention, including subsequences, variants and modified forms of the exemplified peptide sequences (sequences listed in Tables 1 to 8 and Figure 30 1 ) and one or more pharmaceutically and physiologically acceptable formulation agents. Suitable diluents,., Pharmaceutically acceptable or physiologically acceptable,
carriers or excipients include, but are not limited to, antioxidants (eg, ascorbic acid and sodium bisulfate), preservatives (eg, benzyl alcohol, methyl, ethyl or n-propyl parabens, p-hydroxybenzoate), emulsifying agents , suspending agents, emulsifying agents, solvents, fillers, bulking agents, buffers, vehicles, diluents and / or dispersion aids. For example, a suitable vehicle can be a physiological saline solution or citrate buffered saline, possibly supplemented with other materials common in compositions and pharmaceuticals for parenteral administration. Neutral buffered saline or saline mixed with chloro albumin are the most exemplary vehicles. Those skilled in the art will readily recognize a variety of buffers that can be used in the pharmaceutical compositions and dosage forms used in the invention. Typical buffers include, but are not limited to, weakly pharmaceutically acceptable acids, weak bases or mixtures thereof. Buffer components include water-soluble materials, such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid and its and salts., A primary solvent in a vehicle it can be either aqueous or non-aqueous in nature In addition, the vehicle can contain another 25 pharmaceutically acceptable excipients to modify or maintain the pH, osmolarity, viscosity, stability or sterility of the pharmaceutical composition. A pharmaceutically acceptable buffer is an aqueous buffer In other embodiments, a carrier comprises, for example, sodium chloride and / or sodium citrate.
The pharmaceutical compositions of the invention can also contain other pharmaceutically acceptable formulation agents for modifying or maintaining the rate of release of a peptide of the invention. Such formulation agents include those substances known to artisans specialized in the preparation of sustained release formulations. For other 5 references concerning pharmaceutically and physiologically acceptable formulation agents, see, for example, Remington's Pharmaceutical Sciences, 18th ed. {1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435 to 1712, The Merck Index, 12 "ed. (1996, Merck Publishing Group, Whitehouse, NJ) and 10 pharmaceutical Principles of Solid Dosage Forms (1993, and Technonic Publishing Co., Inc., Lancaster, Pennsylvania.) Additional suitable pharmaceutical compositions for administration are known in the art and are applicable to the methods and compositions of the invention.
15 The pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, dehydrated or lyophilized solid or powder. Such compositions can be stored either in a ready-to-use form, a lyophilized form that requires reconstitution before use, a liquid form that requires dilution before use or otherwise acceptable. In some form of realization, a pharmaceutical composition is provided in a single-use container (for example, a single üsc bottle), ampoule, syringe or auto-injector (similar to, for example, a 25 Epipen ")), while a multi-use container (eg, a multi-use vial) is provided in other embodiments Any drug delivery apparatus can be used to release the peptides of the invention, including implants (for example, implantable pumps ) and catheter systems, 30 both of which are known to those of skill in the art. Depot injections, which are generally administered subcutaneously or intramuscularly, can also be used to release the peptides of the invention over a defined period of time. deposits are generally solid or oil-based and generally comprise at least one of the formulation components set forth herein. The person skilled in the art is familiar with the possible formulations and uses of 5 deposit injections.
The pharmaceutical composition can be formulated to be compatible with your intended route of administration. Thus, pharmaceutical compositions include carriers, diluents or excipients suitable for parenteral 6L0 administration, including (for example, subcutaneously (SC), intravenously, intramuscular or intraperitoneal), intradermal, oral (for example, ingestion ), inhalation, intracavity, intracranial and transdermal (topical).
The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution or oleaginous suspension. This suspension can be formulated using suitable dispersing agents or wetting agents and suspending agents disclosed herein or known to the person skilled in the art. The sterile injectable preparation can 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-butanediol. Diluents, solvents and dispersion media that may be employed include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL "25 (BASF, Parsippany, NJ) or phosphate buffered saline (PBS), ethanol, polyol ( eg glycerol, propylene glycol and liquid polyethylene glycol) and their suitable mixtures. In addition, sterile fixed oils are conventionally used as a solvent or suspension medium. For this purpose, any mild fixed oil can be employed including - or synthetic diglycerides. In addition, fatty acids, such as oleic acid, find use in the preparation of
83/116 'injectable. Prolonged absorption of injectable formulations in particular can be achieved by including an agent that delays absorption (for example, aluminum monostearate or gelatin).
The pharmaceutical compositions may be in a form suitable for oral use, for example, in the form of tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups, solutions, micropellets or elixirs. Pharmaceutical compositions for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. Such compositions may contain one or more agents, such as sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and flavorful preparations. Tablets containing a peptide of the invention can be mixed with non-toxic pharmaceutically acceptable excipients suitable for making tablets. These excipients include, for example, 20 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.
Tablets, capsules and the like suitable for oral administration may be uncoated or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide sustained action over a longer period. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate can be loaded. They can also be coated by techniques - known in the art to form compressed osmotic therapeutics for controlled release. Additional agents include biodegradable or biocornable particles 5 or a polymeric substance such as pol, esters, polyamine acids, hydrogel, polyvinylpyrrolidone, polyanhydrides , polyglycolic acid, ethylene-vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide / glycolide copolymers, polylactide / glycolide copolymers or 10 ethylene vinyl acetate copolymers in order to supply a composition c) 0 For example, the agent orally can be trapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, using hydroxymethylcellulose or gelatin or poly microcapsules 15, respectively, or in a methanol administration system. colloidal drug. Colloidal dispersion systems include m complexes of micromolecules, nanocapsules, microspheres, micro-pearls and lipid-based systems, including oil-in-water emulsions, 20 micelles, mixed micelles and liposomes. Methods for preparing such formulations are known to those skilled in the art and are commercially available.
e Formulations for oral use can also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose or soft gelatine capsules in that the active ingredient is mixed with water or an oily meal, for example, peanut oil, liquid paraffin or olive oil.
Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of the samples. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone! tragacanth and acacia gum; dispersants or rinse agents can be a naturally occurring phosphatide, for example, lecithin or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate products or condensation of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxyethanol or condensation products of ethylene oxide with esters and partials derived from fatty acids and a hexitol, such as polyoxyethylene sorbitol monooleate or "ethylene oxide condensation products with partial esters derived from fatty acids and hexitol anhydrides , for example, polyethylene sorbitan monooleate 15. Aqueous suspensions may also contain one or more preservatives.
Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, for example, peanut oil, olive oil, sesame oil or coconut oil or in a mineral oil such as liquid paraffin. Oily suspensions can '. contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above and flavoring agents can be added to provide a palatable oral preparation.
Dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, for example, olive oil or peanut oil or a mineral oil, for example, liquid paraffin or mixtures thereof. Suitable emulsifying agents may be naturally occurring gorn, for example, gum arabic or tragacanth; naturally occurring phosphatides, for example, soybeans, lecithia and esters or partial esters derived from fatty acids; hexitol anhydrides, for example, 10 sorbitan monooleate and the condensation products of partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
Pharmaceutical compositions may 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. For example, a time delay material such as glyceryl monostearate or glyceryl stearate alone or in combination with a wax can be employed. The planned absorption of injectable pharmaceutical compositions can be and is achieved by including an agent that delays absorption, for example, aluminum monostearate or gelatin.
The prevention of the action of microorganisms can be achieved through various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
The invention also includes peptides of the invention in the form of suppositories for rectal administration. Suppositories can be prepared by mixing a peptide of the invention with a suitable non-irritating excipient, which is solid at normal temperatures, but liquid at the rectal temperature and therefore melts in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycols.
In accordance with the invention, methods are provided for identifying a peptide (or a subsequence, variant or modified form as set forth herein) having glucose-lowering activity without substantial hepatocellular carcinoma (HCC) activity. In one embodiment, a method includes: screening (for example, testing or measuring) an eL0. peptide sequence (or a subsequence, variant or modified form as set forth herein) for glucose-lowering activity and the screening (eg, assay or measurement) of a peptide sequence (or a subsequence, variant or form modified as set forth herein) for 15 HCC activity or expression of a marker that correlates with HCC activity. A peptide with reduced blood glucose activity and reduced or absent HCC activity thus identifies the peptide. In particular aspects, the marker correlating with HCC activity comprises a lipid profile - a peptide that has less increased activity than lipids compared to FGF19 indicates that the peptide has and reduced or absent HCC activity, or the marker correlating with activity HCC comprises expression of the aldo-keto reductase gene - a peptide that down-regulates or decreases the expression of the aldo-keto reductase gene compared to FGF19 indicates that the peptide has reduced or absent HCC activity, or the marker indicating HCC activity comprises expression Slc1a2 gene - a peptide that up-regulates or increases expression of the Slc1a2 gene compared to FGF19 30 indicates that the peptide has reduced or absent HCC activity.
The terms "test" and "measurement" and their grammatical variations' are used here interchangeably and refer to either qualitative or quantitative determinations or both qualitative and quantitative determinations. When the terms are used in reference to detection, any means of assessing the relative amount is contemplated, including the various methods described herein and known in the art. For example, gene expression can be assayed or measured by a Northern blot, Western blot, immunoprecipitation assay, or by measuring the activity, function or amount of the expressed protein (for example, aldo-keto 10 reductase or Slc1a2).
0 Risk factors for HCC, the most common type of liver cancer, include type 2 diabetes (probably exacerbated by Qbesity). The risk of HCC in type 2 diabetics is greater (from ~ 2.5 to -7 times the risk of non-diabetics), 15 depending on the duration of diabetes and the treatment protocol.
Various methodologies can be used in screening and diagnosing HCC and are well known to those skilled in the art.
Indicators of HCC include the detection of a tumor manufacturer, such as elevated levels of alpha-fetoprotein (AFP) or de-gamma carboxyprotrombi'na (DCP). A number of different scanning techniques and tarriban imaging are useful, including ultrasound, computed tomography and magnetic resonance imaging. In relation to the invention, the assessment of whether a peptide (eg, a candidate peptide) shows evidence of HCC induction can be determined in vivo by, for example, quantifying the formation of HCC nodules in an animal model, such as db mice / db, a peptide was administered, compared to the formation of HCC nodules by wild type FGF19. Macroscopically, liver cancer can be nodular, where the turrioral nodules (which are round-to-oval, gray or green, well circumscribed, but not encapsulated)
appear as a large mass or several smaller masses.
Alternatively, HCC may be present as an infiltrating tumor that is diffuse and poorly circumscribed and often infiltrates the portal veins.
5 Pathological assessment of liver tissue samples is usually performed after the results of one or more of the techniques mentioned above indicate the likely presence of HCC. Thus, the methods of the invention may further include evaluation of a liver tissue sample from an animal model in vivo (eg, a db / db urri rat) useful in HCC studies in order to determine whether a peptide sequence shows evidence induction of hepatocellular carcinoma. In microscopic evaluation, a pathologist can determine whether one of the four types of general architecture and cytology (standards) 15 of HCC are present (ie, fibrolamellar, pseudoglandular (adenoid), pleomorphic (giant cells) and clear cells).
The invention also includes the generation and use of antibodies and their fragments, which bind to the peptide sequences of the invention, including subsequence sequence variants and modified forms of the expressed peptide sequences and (including the peptides listed in Tables 1 to 8 and Figure 1).
As used herein, the terms "antibodies" (Ab) and "immunoglobulins'" (Ig) refer to glycoproteins having the same structural characteristics. While antibodies exhibit specificity of binding to an antigen, immunoglobulins include both antibodies and other antibody-like molecules, which may not have antigen specificity.
The term "antibody" includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) formed from "at least two intact antibodies, and binding fragments of 5 antibodies, including Fab and F (ab) fragments. '2, as long as they exhibit the desired biological activity, the structural base unit of the antibody comprises a tetramer and each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (about 25 10 kDa ) and a "heavy" chain (about 50 to 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. the "carboxy-terrninal portion of each chain defines a constant region 15 primarily responsible for the 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 silon, 12 define the antibody isotype as IgM, IgO, IgA and IgE, 20 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 a variable domain 25 (VH) at the end 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 variable domain of the light chain is aligned with the heavy chain variable domain. In light and heavy chains, 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. All antibody chains present the same general structure of relatively conserved structural regions (FR) joined by three hyper-variable regions, also called complementarity determining regions or CDRS 5. The CDRS of the two chains of each pair are aligned with the regions of structures, allowing binding to a specific epitope From N-terminal to C-terminal, both light and heavy chains comprise the FRI, CDRI, FR2, CDR2, FR3, 10 CDR3 and FR4 domains.
e An intact antibody has two binding sites and, except for bifunctional or bi-specific antibodies, the two binding sites are the same. A bi-specific or bifunctional antibody is an artificial hybrid antibody, having two different pairs of heavy / light chains and two different binding sites. Bi-specific antibodies can be produced by a variety of methods, including fusion of hybridomas or ligation of Fab 'fragments.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of antibodies and substantially homogeneous, that is, individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in smaller quantities. Monoclonal antibodies 25 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 in the antigen.
A "neutralizing antibody" is an antibody molecule that is capable of eliminating or significantly reducing an effector function of a target antigen to which it binds.
Antibody binding fragments can be produced by enzymatic or chemical cleavage of intact antibodies. Digestion of antibodies with the papain enzyme results in two identical antigen-binding fragments, also known as "Fab '" fragments and a "Fc" fragment, which has no antigen-binding activity. Digestion of antibodies with ®iO enzyme pepsin results in fragments of F (ab ') 2 in which the' two arms of the antibody molecule remain attached and form binding sites for two antigens. The F (ab ') 2 fragment has the ability to cross-link the antigen.
The term "Fab" refers to a fragment of an antibody that comprises the constant domain of the light chain and the CH1 domain of the heavy chain; The term "Fv", when used here, refers to the minimum antibody fragment that retains both recognition antigens and antigen binding sites.
In a two-chain Fv species, this region consists of a domain dimer of a heavy chain and a non-covalent association light chain variable. In a single-chain Fv species, a heavy-chain domain and a light-chain variable can be covalently linked by a flexible peptide linker, "in such a way that light and heavy villages 25 can associate in a" dimeric "structure. "'analogous to this in two-chain Fv species. It is in this configuration that the three CDRS of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. While the six CDRs, together, confer specificity of antigen binding to the antibody, even a 'single variable domain (or half an Ev comprising only three specific CDRS for an antigen) has the ability to recognize and bind to the antigen.
The term "complementarity determining regions" or "CDRS" 'refers to pieces of immune receptors that come into contact with a specific ligand and determine its specificity. The term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for binding to the antigen: The hypervariable region generally comprises amino acid residues of a ".10 complementarity determining region" or "CDRS" and / or the residues of a "back and hyper variable".
As used herein, the term "epitope" refers to antibody-binding sites of protein antigens. Epitopic determinants usually consist of chemically active surface clusters of molecules such as amino acids or sugar side chains, as well as three specific structural and dimensional load characteristics. An antibody is said to bind to an antigen when the dissociation constant is <1µM, preferably S 1OOµM and 20 more preferably <10µM. An increase in the equilibrium constant ("Kd") means that there is less affinity between the epitope and the antibody, whereas a decrease in the equilibrium constant means that there is a greater affinity between the epitope and the antibody. An antibody with a 25 Kd of "no more than" a certain amount means that the antibody binds to the epitope with q given Kd or more strongly.
While Kd describes the binding characteristics of an epitope and an antibody, "potency" describes the effectiveness of the antibody itself for an antibody function. There is not necessarily a correlation between the equilibrium constant and the power, so, for example, a P (relatively low d does not automatically mean a high power.
s'm. ==
The term "selectively binds", in reference to an antibody, does not mean that the antibody binds only to a single substance, but rather that the K [) Kd of the antibody with a first substance is less than Kd of the antibody to a second substance. An antibody that binds exclusively to an epitope binds only to that simple epitope.
When administered to humans, antibodies that contain variable and / or constant rodent (murine or rat) regions are often associated with, for example, rapid e10 clearance from the body or the generation of an immune response by 4 body parts. against the antibody. In order to avoid the use of antibodies derived from rodents, fully human antibodies cannot be generated by introducing human antibody function in a rodent, so that the rodent produces 15 fully human antibodies. Unless specifically identified here, "human" and "fully human" antibodies can be used here. The term "fully human" can be useful in distinguishing antibodies that are only partially human from those that are completely or fully human. The person skilled in the art is aware of several methods of producing completely Hurnan antibodies.
In order to resolve possible responses, human anti-mouse antibodies, chimeric or humanized antibodies can be used in another way. Chimeric antibodies have a human constant region and a murine variable region, and as such, human anti-chimeric antibody responses can be observed in some patients. Therefore, it is advantageous to provide fully human antibodies to 30 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 generating hybridoma cell lines by techniques known to those skilled in the art. Other methods of preparation involve the use of 5 sequences that encode specific antibodies for the transformation of a suitable mammalian host cell, such as a CHO cell. Transformation can be done by any known method of introducing polynucleotides into a host cell, including, for example, c) packaging the polynucleotide into a virus (or a vector and viral) 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 are well known in the art and include dextran mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, polynucleotide (S) encapsulation in liposorrtas. and direct microinjection of DNA into nuclei. Ornamental 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.
Antibodies can be used for diagnosis and / or therapy. For example, antibodies can be used as a diagnosis by detecting the level of one or more peptides of the invention in a patient and or comparing the level detected to the normal control level or to a baseline level in a subject. previously determined (for example, before any disease). Antibodies can be used as therapy to modulate the activity of one or more peptides of the invention, thus having an effect of a condition or disorder.
The present invention provides kits, including, but not limited to, peptide sequences of the invention, optionally in combination with one or more therapeutic agents, the compositions and their pharmaceutical compositions packaged in suitable packaging material. A kit optionally includes a label or packaging insert that includes a description of the components or instructions for in vitro, in vivo or ex vivo use of its components. Exemplary instructions include instructions for reducing or minimizing blood glucose, treating hyperglycemia, treating diabetes, etc.
A kit can contain a set of such components, for example, two or more peptide sequences alone or a combination of a peptide sequence with another therapeutically useful composition (for example, an antidiabetic drug, such as a gastrin compound ).
The term "packaging material" refers to a box of physical structure storing the components of the kit. the packaging material can keep the components sterile and can be made of material commonly used for such purposes (for example, paper, corrugated board, glass, and plastic, aluminum foil, ampoules, flasks, tubes, etc.). ).
Kits of the invention may include labels or inserts. Labels or inserts i.include "printed", for example, paper or cardboard, separated or affixed to a component or kit or packaging material (for example, a box), or attached to, for example, an ampoule, tube or tube bottle containing a kit component. Labels or inserts can also include a computer-readable medium, such as a disk (for example, hard disk, card, memory disk), optical disk, such as CD- or 30 DVD-ROM / RAM, DVD, MP3, tape magnetic or electric storage media such as RAM and ROM or hybrids of these as magnetic / optical storage media, flash media or type of memory cards.
Labels or inserts may include information to identify one or more components, dose values, 5 clinical pharmacology of the active ingredient (s) including mechanism of action, pharmacokinetics and pharmacokinetics. Labels or inserts may include identifying information, manufacturer information, batch number, manufacturer location and date.
The labels or inserts may include information about a condition, disorder, disease or symptom for c) which component of the kit can be used. Labels or inserts may include instructions for the clinician or an object for the use of one or more of kit components in a method, treatment protocol or therapeutic regimen. The instructions may not include dosage amounts, frequency or duration and instructions for the practice of any of the methods, treatment protocols or therapeutic regimens set forth herein. Exemplary instructions include instructions for the treatment or use of a peptide sequence as set forth herein. The kits of the invention, therefore, may additionally include labels or instructions for the practice of any of the methods and uses of the present invention described herein, including methods of treatment and uses.
25 Labels or inserts may include information about any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts may include information about potential adverse side effects, such as warnings to the patient or 30 medical situations in which it would not be appropriate to use a particular composition. Adverse side effects may also occur when the patient has, will be, or is currently taking one or more medications that may be incompatible with the composition, or the patient has, will be, or is going through another treatment protocol or therapeutic regimen that would be incompatible. with the composition and therefore the instructions may include information regarding such incompatibilities.
Kits of the invention may additionally include other components. Each component of the kit can be closed within an individual container and all of the various containers can be contained in a single package. Invention kits can be designed for cold storage. Kits of the invention may further be designed to contain sequences of peptides of the invention or that contain nucleic acids that encode. 15 peptide sequences. Kit cells can be kept under proper storage conditions until they are ready for use.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as the one normally understood by an ordinary person skilled in the art to which this and invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described here.
25 All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control. As used herein, the singular forms "one," and "and" O "include 30 plural referents unless the context clearly indicates otherwise. Thus, for example, the reference to" a peptide sequence "or" treatment " includes a plurality of such sequences, treatments, etc.
As used herein, numerical values are often presented in a strip format throughout 5 of this document. The use of a banner format is for convenience and brevity only and should not be interpreted as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Consequently, the use of a range expressly includes all possible sub-ranges, all individual numeric values within this range, and all numeric values or numeric ranges including whole numbers within those ranges and fractions of values or whole numbers within the standards unless the context clearly indicates otherwise. This construction is applicable regardless of the width of the gap and in all contexts throughout this patent document. Thus, for example, the reference to a range of 90 to 100% includes 91 to 99%, 92 to 98%, 93 to 95%, 91 to 98%, 91 to 97%, 91 to 96%, 91 to 95 %, 91 to 94%, 91 to 93%, and so on. Reference to a range of 90 to 100% also includes 91%, 92%, 93%, 94%, 95% ,, 95%, 97%, etc., as well as 91.1%, 91.2% , and 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on .
In addition, the reference to a range of 1 to 3, 3 to 5, 5 to 10, 25 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, 190 to 200, 200 to 225, 225 to 250 including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Ern plus a 30 example, the reference to a range of 25 to 250, 250 to 500, 500 to 1000, 1000 to 2500 or 2500 to 5000, 5000 to 25,000, 5000 to 50,000 includes any numerical value or range within or encompassing such values, for example, 25, 26, 27, 28, 29 ... 250, 251, 252, 253, 254 .... 500, 501, 502, 503, 504 ..., etc.
As used herein, a series of intervals are also described throughout this document. The use of a range of ranges includes combinations of the top and bottom ranges to provide a wider range. This construction is applicable regardless of the width of the range and in all contexts throughout this "patent document. Thus, for example g10, the reference to a series of ranges, such as 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100, 100 to 150, includes ranges such as 5 to 20, 5 to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150 and 10 to 30, 10 to '40, 10 to 50, 10 to 75, 10 to 100, 10 to 150 and 20 to 40, 20 to 50, 20 to 75, 20 to 100, 20 to 15 150 and so on.
For the sake of brevity, certain abbreviations are used here.
An example is the only letter of abbreviation to represent amino acid residues. The amino acids and their corresponding abbreviations "of three letters and a single letter are as follows: 20 and allan Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acid Asp (D) cysteine Cys (C) glutamic acid glu (E) glutamine .Gln (Q) glycine Gly (G) histidine His' (H)
isoleucine Ile (I) leucine Leu (L) E lysine Lys (K) methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser - (S) threonine Thr (T) tryptophan Trp (W) tyrosine Tyr ( Y) valine Val (V) e The invention is generally disclosed here using affirmative language to describe the various embodiments. The invention also specifically includes embodiments in which a particular subject is excluded, in whole or in part, as substances or materials, the method steps and conditions, protocols, procedures, tests or analyzes. Thus, although the general invention is not expressed here in terms of what the invention does not include, aspects that are not expressly included in the present invention are nevertheless here and disclosed.
A number of ways of carrying out the invention have been described.
However, it will be understood that various modifications can be made to depart from the spirit and scope of the invention. Therefore, the following examples are intended to illustrate, but not to limit the scope of the invention described in the claims.
Examples Example 1 The following is a description of the various methods and materials used in the studies described herein.
5 Animals. Cib / db mice were purchased from The jackson Laboratory (Bar Habor, ME). The rats were maintained according to well-being guidelines under controlled light (12 hours of light and 12 hours of dark cycle, dark 06:30 prn to 6:30 am), 6 temperature conditions (22 ± 4 ° C ) and humidity (50% ± 20%). They 10 had free access to water (autoclaved distilled water) and were fed ad libitum with a commercial diet (Harlan Laboratories, Indianapolis, IN, Irradiated 2018 Teklad Global 18% Protein Rodent Diet), containing 17 kcal% fat, 23 kcal ° protein and 60 kcal% c and carbohydrates. For diet-induced obesity 15, the C57BL6 / J rats (jackson Laboratory) were kept on a high-fat diet (D12492, Research Diet, New Brunswick, NJ. USA), containing 60 kcal% fat, 20 kcal% protein and 20 kcal% of carbohydrates for 16 to 20 weeks. All animal studies have been approved by the NGM and the Institutional Animal Care and Use Committee.
DNA and amino acid sequences. ORF CDNA encoding human FGF19 variants (Homo sapiens FGF19, Gen8ank Accession. No. NM 005117.2).
Sequence of proteins encoded by the cDNA (GenBank 25 Accession No. NP 005108.1) PCR. FGF19 ORF was amplified with polymerase chain reaction (PCR) using recombinant DNA (cDNA) prepared from human small intestine tissue. High-fidelity DNA polymerase PCR reagent kits were purchased from New
England 81oLabs (F-530L, Ipswich, MA). The following primers were used: forward PCR primer: 5 'CCGACTAGTCACCatgcggagcgggtgtgtgg and reverse PCR primer: 5' .ATAAGAATGCGGCCGCTTACTTCTCAAAGCTGGGACTCCTC.
5 Amplified DNA fragment was digested with the restriction enzymes Spe I and Not I (which restriction sites were included in the 5 'and 3' PCR primers, respectively) and was then ligated with AAV transgene vectors that had been digested with the same enzyme restriction. The eiL0 vector used for the expression contained a selectable marker and an expression cassette consisting of a strong eukaryotic promoter 5 'from a loc-al for the insertion of the cloned coding sequence, followed by a 3' untranslated and polyadenylation region of the bovine tail growth hormone.
The expression construct is also flanked by internal terminal repetitions at the 5 'and 3' end.
AAV production and purification. AAV293 cells (obtained from Agilent Technologies, Santa Clara, CA) were grown in Dulbeco's Modification of Eagle's Medium (DMEM, 20 Mediatech, Inc. Manassas, VA) supplemented with 10% bovine serum and fetal and antibiotic-antimycotic solution lx (Mediatech, Inc. Manassas, VA). The cells were plated at a density of 50% on day 1 in 150-millimeter cell culture plates and transfected on day 2. using the calcium phosphate precipitation method with the following three plasmids (20 µg / each plate):, transgenic AAV plasmid, pHelper plasmids (Agilent Technologies) and AAV2 / 9 pasmid (Gao et al., J. Virol. 78: 6381 (2004)). 48 hours after transfection, the cells were scraped from the 30 plates, sedimented by centrifugation at 300Oxg and resuspended in a tar containing 20 mM Tris pH 8.5, 100 mM NaCl and 1 mM MgC12. The suspension was frozen in a dry ice bath and alcohol was then thawed in a 37 ° C water bath.
The freeze and thaw cycles were repeated three times; Benzenase (Sigma-Aldrich, St. Louis, MO) was added at 50 units / ml; deoxycholate was added to a final concentration of 0.25%. After an incubation at 37 ° C for 30 minutes, cell debris was pelleted by cerrtrifugation at 500Oxg for 20. min. The viral particles present in the supernatant were purified using a discontinued iodixanal gradient (Sigma-Aldrich, St. Louis, 10 MO) as previously described (Zolotukhin S. et al (1999) € J Gene Ther. 6: 973). The viral stock was concentrated using Vivaspin 20 (MW cutoff 100,000 Dalton, Sartorius Stedim Bio "tech, Aubagne, France) and resuspended in phosphate buffered saline (" BS) with 10% glycerol and stored at 15 -80 ° C. To determine the number of copies of the viral genome, 2 µ1 of viral stock were incubated in 6 µ1 of solution containing 50 units / ml of Benzonase, 'iO mM Tris-HCl pH 7.5, MgC12 10 MM and CaCl2 10 mM at 37 ° C ° C for 30 minutes.
Then, 15 µl of solution containing 2 mg / ml of protein K, 20% 0.5 SDS and 25 mM EDTA were added and the mixture was incubated for another 20 min at 55 ° C to release the viral DNA. C) The viral DNA was cleaned with a DNeasy mini kit (Qiagen, Valencia, CA) and eluted with 40 µ1 of water. Copy of the viral genome (GC) was determined using quantitative PCR.
25 Viral stock was diluted with PElS to desirable GC / ml. Viral working solution (200 µ1) was delivered to rats by injection into the tail vein.
Blood glucose test. Glycemia in clipping of the rat tail was measured using ACCU-CHEK Active 30 test strips read by ACCU-CHEK Active meter (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instructions.
Lipidic assay. Whole blood from rat tail clippings was collected in smooth capillary tubes (BD Clay Adams SurePrep, Becton Dickenson and Co. Sparks, MD). The serum and blood cells were separated by centrifuging the 5 tubes in an Autocrit Ultra 3 (Becton Dickinson and Co. Sparks, MD). The serum samples were tested for the lipid profile (triglycerides, total cholesterol, HDL and non-HDL), using Integra 400 Clinical Analyzer (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instructions.
éQ FGF19 / FGF21 / variant exposure level assay. Whole blood (about 50 µ1 / mouse) from cutouts of the rat's tail was collected in smooth capillary tubes (BD Clay Adams SurePrep, Becton Dickenson and Co. Sparks, MD). The serum and Eoram blood cells were separated by centrifugation of the 15 tubes in an Autocrit Ultra 3 (Becton Dickinson and Co. Sparks, MD). Exposure levels of FGF19, FGF21 and variant in serum were determined using EIA kits (Biovendor), following the manufacturer's instructions.
"Hepatocellular careinoma (HCC) assay. Liver specimen 20 was collected from db / db mice 6 months after injection of AAV. HCC score is recorded as the number of HCC nodules on the surface of the entire liver of rats injected with variants divided by the number of HCC nodules of rats injected with wild type FGF19.
25 Hepatic genre expression test. Liver specimen was collected and homogenized in Trizol reagent (Invitrogerí). The total RNA was extracted following the manufacturer's instructions. The RNA was treated with DNase (Ambion), followed by quantitative analysis of RT-PCR using Taqman primers and 30 Applied Biosystems reagents. The relative levels of aldo-keto reductase and slc1a2 mRNA in the liver were calculated using the LlnàCt method.
FGFR4 activity and binding assays. Solid-phase ELISA (ligation) and ERK phosphorylation assay carried out using purified recombinant proteins. FGFR binding assay was performed using solid phase ELISA.
Briefly, the 96-well plate was coated with 2 µg / ml anti-hFc antibody and incubated with 1 µg / ml- of FGFR1-hFc or FGFR4-hFc. Binding to FGF19 variants in the presence of 1 µg / ml 10 of soluble b-klotho and 20 µg / ml of heparin was detected by and biotinylated anti-FGF19 antibodies (0.2ug '/ ml), followed by incubation with streptavidin-HRP ( 100ng / ml). For the FG "R4 activation assay, Hep3B cells were stimulated with FGF19 variants for 10 minutes at 37 ° C, then immediately 15 lysed and tested for ERK phosphorylation, using a kit commercially available from Cis-8io. Example 2 The following is a description of studies that show the glucose-lowering activity of various variants of the FGF19 and FGF21 sequences and FGF19 / FGF21 fusion constructs.
Figure 2 illustrates exemplary FGF19 / FGF21 fusion structures and the secrets of each of the FGF1 ° and FGF21 present in the fused peptides. These peptides were analyzed for glucose-lowering activity and, statistically, significant elevation or an increase in lipid activity (Tables 1 to 8 and Figure 1).
'Rats (db / db) were injected with a viral vector expressing FGE "19, FGF21 or variants, and analyzed after injection.
Glucose reduction activity for each sequence is represented by a "+" symbol (a "-" symbol means no glycemic reduction activity, a "+/-" symbol means that variants maintain minimal glucose reduction activity); lipid elevation activity is represented by a "+" sign (a "-" symbol means that there is no lipid elevation activity, a "+/-" symbol means that variants maintain minimal lipid elevation activity, Figure 2 ).
Two fusions of FGF21 and FGF19, denoted variant M5 and variant 6L0 45 (M45), exhibited glucose-lowering activity and a statistically significant absence of lipid elevation or increased activity. Variants denoted Ml, M2 and M69, respectively (Figure 1), also exhibited glucose-lowering activity (Figure 3B and 3C, Table 5). Data 15 comparing glucose-lowering and lipid-raising activity or increased activity of M5, Ml, M2 and M69 with FGF19 and FGF21 are illustrated in Figures 3A to 3C and 4A to 4C.
Example 3 The following is a description of studies that show that the e20 variants M5, Ml, M2 and M69 are not tumorgenic, as determined by the formation of hepatocellular carcinoma (HCC) and that the variants that M5, M2 and M69 also they do not reduce lean muscle mass and fat mass.
Anirnais (db / db) were injected with AAV vectors expressing 25 FGF19, FGF21, M5, M1, M2, or M69 or injected with saline and analyzed 6 months after '"the injection - The data indicate that the M5, Ml, M2, and M69 did not induce significant (HCC) formation (Figures 5A to 5C).
Animals (db / db rats) were also injected with a viral vector expressing FGF19, FGF21, M5, Ml, M2 or M69 or injected with saline and analyzed six months after the injection for the effect on lean mass and fat mass. The data indicate that 5 M5, M2 and M69 peptides did not cause a statistically significant reduction in lean mass or fat mass, in contrast to FGF21, and that M1 peptide reduces lean mass (Figures 6A to 6C).
Example 4 €) 10 The following is a summary of the data for 25 additional variant peptides analyzed for lipid-raising activity and tumorigenesis. The data clearly show a positive correlation between the elevation of lipids and tumorigenesis, as determined by the formation of hepatocellular carcinoma 15 (HCC) in db / db 'rats.
Tables 1 to 3 summarize data for 26 different variant peptides. Such exemplified variant peptides have FGF19 C-terminal sequence: PHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV èzo SLSSAKQRQT, YKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTG LEAVRS following, for example: Notably, variant peptides (total of 7, including M5) that do not cause a statistically significant increase in lipids' did not induce hepatocellular carcinoma (HCC) formation. In contrast, all variant peptides (17 in total) that caused a statistically significant increase in lipids also caused formation of hepatocellular carcinoma (HCC) in mice. These data indicate that there is a strong positive correlation between lipid-raising activity and the formation of hepatocellular carcinoma (HCC). Therefore, lipid-raising activity can be used as an indicator and / or prognosis for the formation of hepatocellular carcinoma (HCC) in animals.
Table 1: Elevated triglycerides and cholesterol in db / db rats seem to positively correlate with the formation of HCC.
Principal N-terminal Domain Elevation Formation - ^ - HCC lipids r> FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + + FGF21 HPIPDSSPLLQ - FGGQV RQRYLYTDD M5 · R-HPIPDSSPLLQ - FGGQV RLRHY + M75 R ------------- VHYGWGDPI RLRHLYTSG - M76 R ------------------ GDPI RLRHLYTSG - M77 R RLRHLYTSG M78 R ---- ---- AGPHVHYGWGDPI RLRHLYTSG + + M79 R ---------- GPHVHYGWGDPI RLRHLYTSG + + M80 R ----------- PHVHYGWGDPI RLRHLYTSG M81 R --------- - HVHYGWGDPI RLRHLYTSG - 5 Table 2: Triglyceride and high cholesterol ~ db / db rats seem to positively correlate with HCC formation €) N-term domain ^ Main Hypidic formation HCC formation -FGGQV RQRYLYTDD M82 + M83 + RPLAFSAAGPHVHYGWGDPI RLRHLYTSG RPLAFSDAAPHVHYGWGDPI RLRHLYTSG +/- + / + +/- RLRHLYTSG RPLAFSDAGAHVHYGWGDPI M84 / M85 RPLAFSDAGPHVHYGAGDPI RLRHLYTSG - RPLAFSDAGPHVHYGWGAPI RLRHLYTSG M86 + M87 + RPLAFSDAGPHVHYGWGDAI RLRHLYTSG
Table 3: Triglyceride and elevated cholesterol in db / db rats seem to positively correlate with HCC formation N-terminal domain Main Elevation Formation of HCC lipids FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + + FGF21 HPIPDSSPLLQ - FGGDDF + RYH + RY + H31A / H142A (M89) FGF19 + + K127A / R129A (M90) FGF19 + + K127A / S141A (M91) FGF19 + + K127A / H142A (M92) FGF19 + + êj R129A / S141A (M93) FGF19 + + S14A / H14A M94) FGF19 + + K127A / H142A (M95) FGF19 + + K127A / R129A / S141A (M97) FGF19 + 4- K127A / R129A / H142A (M98) FGF19 + + K127A / R129A / S141A / HI42A + MI + FG Example 5 5 The following is a summary of additional FGF19 € 3 variant peptide data analyzed for glucose-lowering and lipid-raising activity.
Table 4 illustrates the peptide "main sequences" of 35 additional FGF19 variants, denoted M5 to M40. Such 10 exenplified variant peptides have a C-terminal FGF19 sequence, PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYL
CMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPL SHFLPMLPMVPEEPEDLRGHLFSDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK in the C-terminal portion, for example, "following the amino acids" following the amino acid sequence. The data clearly shows that the M6, MJ, M8, rriM38 and M39 variants have the desired characteristics of glucose-lowering activity and not statistically significant lipid-raising activity in db / db rats.
Table 4 and 5 variants Additional N-terminal domain mapping the N-terminal primary GIícose Reduction Elevation of FGF19 Iípídeos RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + EGF21 HPIPDSSPLLQ - FGGQV RQRYLYTDD M5 + R-HPIPDSSPLLQ - FGGQV RI.RHLYTSG ~ M6 + R ------- DSSPLLQ - FGGQV RLRHLYTSG + + ê) M7 RPLAFSDSSPLLQ - FGGQV RLRHLYTSG + ~ M8 R-HPIPDSSPLLQ - WGDPI RLRHLYTSG + M9 R-FIPIPDSSPLLQFGWGDSI RLRHYGDSI RLRLYG WGDPI RLRHLYTSG N / AN / A RPLAFSDAGPLLQFGWGDPI RLRHLYTSG M12 + M13 RPLAFSDAGPLLQ - FGGQV RLRHLYTSG HPIPDSSPHVHYG R-M14 - M15 RPLAFSDAGPHVHYG GQV RLRHLYTSG ~ - + M16 GQV RLRHLYTSG RPLAFSDAGPHVH - WGDPI RLRHLYTSG N / AN / A RPLAFSDAGPHV M17 - GWGDPI RLRHLYTSG N / DN / D M18 RPLAFSDAGPH - YGWGDPI RLRHLYTSG N / DN / D M19 RPLAFSDAGP-V-YGWGDPI RLRHLYTSG N / DN / D M20 RPLAFSDAGP-VH-GWGDPI RLRHLYTSG R / RLHLYTSG NR DN / D M22 RPLAFSDAGPHVH-GWGDPI RLRHLYTSG N / DN / D M23 RPLAFSDAGPH-H-GWGDPI RLRHLYTSG N / DN / D M24 RPLAFSDAGPH-H Y-WGDPI RLRHLYTSG N / DN / D €) M25 RPLAFSDAGPHV-Y-WGDPI RLRHLYTSG N / DN / D M26 RPLAFSDSSPLVH - WGDPI RLRHLYTSG N / DN / D "M27 RPLAFSDSSPEIVH - WGDPI RLR - WGDPI RLR - 28G -WGDPI RLRHLYTSG N / DN / D M29 RPLAFSDAGPHVHY-WGDPI RLRHLYTSG N / DN / D M30 RPLAFSDAGPHVHYAWGDPI RLRHLYTSG N / DN / D M31 R-HPIPDSSPLLQ - FGAQQR-LLY-MGA-RQY -FGGQV RLRHLYTSG - ~ M34 'R-HPIPDSSPLLQ - FG7AV RLRHLYTSG +/- - M35 R-HPIPDSSPLLQ - FGGEV RLRHLYTSG +/- + / M36 R-HPIPDSSPLLQ - FGGQV RLRHLYTSG +/- - 37- RLRHLYTSG M38 R-HPIPDSSPLLQ - FGGQT RLRHLYTSG + ~ M39 R-HPIPDSSPLLQ - FGGQT RLRHLYTSG + M40. R-HPIPDSSPLLQFGWGQPO RLRHLYTSG ~ +
112, / 116 Table 4a: R ¢ duction of Election of the Training of N-temiinal Domain Glucose Lipids IiCC 'Main FGFI9 "RPL.AFSDAGPHVFIYGWGDPI RLRHLYTSG + + + FGF21 HPIPDSSPLLQ - FCGGQV RQRYYT - SHG. V RLRHLYTSG + -.
R M9-4- HPIPDSSPLLQFGWGDPI RLRHLYTSG '+ "R-M8 HPIPDSS'PLLQ - WGDPI RLRHLYTSG + + + ML2 RPLAFSDAGPLLQFGWGDPI RLRHLYTSG%." MLO R4IPIPDSSPHVHYGWGDPI RLRHLYTSG + W + ML3 RPLAFSDAGPLLQ-FGGQV RLRHLYTSG - + M15 RPLAFSDAGPHVHYG - GQV RIRHLYTSG '- + / · - MI4 RAPIPIJSSPHVHYG-GQV RLRHLYTSG - +/- - M43 RPLAFSDAGPHVHYG-GD-I RLRHLYTSG, - -' + / '- 0 M6 M7 R — DSSPLLQ - FGGQVQLLRHHLTS +
W 5 Table 4b: 'Reduction of N-terminal Domination Formulation Glucose - HCC lipids
I tt Main FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG "- + + FGF21 HPIPDSSPI.LQ - FGGQV RQRYLYTDD + b. MS R-HPIPDSSPL.LQ - FGGQV RLRHLYTSG" - M31 R-HPIPDSSPLLQ - RGA - FGA - FGA FGDQV RLRHLYTSG -r- 0. .
and M33 M34 M35 R-HPIPDSSPLLQ-FGPQV RLRHLYTSG R-HPIPDSSPLLQ - FGGAV RLRHLYTSG R-HPIPDSSPLLQ-FGGEV RLRHLYTSG m. . - + + + '' M36 R-HPIPDSSPLLQ - FGGNV RLRHI.YTSG + + / '-. R-IIPIPDSSPLLQ-FGGQA RLRHLYTSG - + M37 R-HPIPDSSPLLQ - FGGQI RLRHLYTSG + M38. RAIPIPDSSPLLQ - FGGQT RLRHLYTSG + M39 - RJIPTPDSSPLLQFGWGQPV RLRHLYTSG - + + "M4Q
Table 4c: Reduction of Ejevation of the N-terminal Doininio - Glycoscopic Lipids HCC tti Principaj FGFI9 RPTAFSDAGPI-IVHYGWGDPI RLRHLYTSO + "" FGF21 HPIPDSSPLLQ - FGGQV RQQHLTDL "RQRYLYTDD" RQRYLYTDDL " -DSSPLLQ-WGDPI RLRHLYTSG + - +. RPLAFSDAGPLLQ + M54 +-JVGDPI RLRHLYTSG RPLAFSDAGPH - YGWGDPI RLRHLYTSG + M55 + M56-V-RPLAFSDAGP YGWGDPI RLRHLYTSG + - M57 RPLAFSDAGP-VH-GWGDPI RLRHLYTSG - "" RPLAFSDAGPNHY M58-M59 + 4 WGDPI RLRHLYTSG RPLAFSDAGPH H-GWGDPI RLm-YTsg - + '+ M60 RPLAFSDAGPH-HY-WGDPI RLRHLYTSG m "+ + M61 RPLAFSDAGPHV - GWGDPI RLRHLYTSG m + + M62 RPLAFSDAGPHV-Y-WGDPI RLRHLYTSG RPLHRDS-MHL-WRHYRG * MH-R, * MR -.WGDPI RLRHLYTSG + _ + "+ MGS RPLAFSDSSPHVH-JVGDPI RLRHLYTSG '- + + Gl RPLAFSDAGPHLQ - WGDPI RLRHLYTSG% -r- + +' <> M66 M67 RPLAFSDAGPHV --- WGDPI RI ..% RPLAFSDAGPHVIIY-WGDPI RLRHLYTSG W +. ~ M4 RPLAFSDAGPHVHYAWGDPI RLRHLYTSG. + + + M69 R '--- DSSPLVHYGWGDPI RLRHLYTS "G + + - M70 MR --- DSSPLVHYGWD' - MPL MR- '- - RJ-RHLYTSG + "- Table 5 illustrates the peptide sequences of 3 additional FGF19 5 variants, denoted Ml, M2 and M69. The data clearly show that these three variants have the desired characteristics those of glucose-lowering activity in db / db rats (Figures 3B and 3C). These three variants appear to raise C) lipids in db / db mice (Figures 4B and 4C).
10 Table 5: Additional MI variables: RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCA-RGQSAHSLLE IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTG LEAVRSPSFEK. 15 M2: RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGWDCARGQSAHSLLE IKAVALRTVA-IKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV SLSSAKQRQLYKNRGFLPLSHELPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTG
LEAVRSPSFEK M69: RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV 20 ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSS AKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAV RSPSFEK
Example 6 The following is a summary of data showing that FGF19 reduces body weight in diet-induced obese rats and ob / ob rats and liver tumor formation activity and body weight 5 in db / db rats.
The rats were injected with FGF19 or FGF21 in AAV vector. Body weight was recorded 4 weeks after the injection.
Table 6: FGF19 reduces body weight in obese and 'diet-induced rats and exn ob / ob rats Reducing Weight Reducing Weight. Body N-terminal Domain in Body Dro in Ob / ob, P · main FGF19 RPLAFSDAGPHVHYGWGDPI RLREILYTSG + "RPIPDSSPLLQ - FGGQV RQRY.LYTDD + + FGE21 10 Table 7: Correlation between body weight and tumor formation in FGF liver and FGF19, selected variables in rats db / db 'Nodule Weight €). N-terminal Tumoral Domain in the Coiporal Liver j'. i Main FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + Increased FGF21 'HPIPDSSPLLQ - FGGQV RQRYGYL-RQRYLYTDL - RQRYLYTDL- Decreased M6 R --— DSSPLLQ-FGGQV RLRHLYTSG Decreased M32, R-HPIPDSSPLLQ - FGDQV RLRHLYTSG M52 R -—- DSSPLLQ - WGDPI RLRHLYTSG Decreased M69 R ---- DSSPLVHYGWGDPI RLRHLY
1l5l11'6 Example 7 The following is a study showing that peptides variant M5 and variant M69 reduce blood glucose.
Rats (ob / ob) were injected (subcutaneously) with M5 (0.15 and 1 mg / kg, sc) or FGF19 (1 mg / kg, sc) or M69 variant (0.1 and 1 mg / kg , Sc) o'u FGF19 (1 mg / kg, Sc). Plasma glucose levels were measured at 2, 4, 7 and 24 hours after injection and the healthy results shown in Figure 7. M5 (Figure 7A) and variant M69 (Figure 7B) showed similar glucose-lowering effects as wild type FGF19.
Example 8 This example describes a study showing that the hepatic expression of family 1 aldo-keto reductase, member C18 (Akr1C18) and family 1 carrying soIuto, month 2 (slc1a2) appears to correlate with HCC activity.
Rats (db / db) were injected with a viral vector that expresses FGF19 (HCC +), FGF21 (HCC-), dN2 (HCC-) or M5 (HCC-) or € 3 injected with GFP. Liver samples were collected and analyzed by quantitative RT-PCR 2 weeks after injection. 20 The data, shown in Figure 8, show that the liver expression of Akr1C18 and slc1a2 appears to correlate with HCC activity.
Table 8: Summary of FGF19 Variants ~ 3T3L1 Adipocyte Signaling Assay
P-Erk 3T3L1 adipocyte assay! FGF19 I FGF21) M5 M2 I M63 I M64 I Ml I M8 I Experiment € L:
I Emax I 3.67 I 4.33 l 3.52 4.19 i 3.21 I 3.67 l 4.24 | 4.16 I EC50 (nM) I 0.05 I 0.65 I 0.03 0.05 I 0.92 I 0.02 I 0.03 | 0.03 I Experiment # 2: I Emax! 4.52 I 4.83 | 4.01 5.56 i 4.17! 4.85: 5.30 j5.34 I EC50 (nM) I 0.33 I 1.48 I 0.14 0.15 I 0.66 I 0.12 I 0.09 | 0.09 I Experiment # 3 : I Ern.ax I 4.09 I 4.14 I 3.74 4.24 I 3.15 I 4.15 i 4.77 | 4.16 and I EC50 (nM) I 0.16 I 1.50 I 0.24 0.14 I () v28 I 0.14 I 0.07 | 0.14 and

权利要求:
Claims (21)
[1]
1. Chimeric peptide sequence characterized by comprising: (A) (a) an N-terminal region comprising at least seven amino acid residues, the N-terminal region having a first amino acid position and a last amino acid position, where the N-terminal region comprises DSSPL or DASPH; and (b) an e-terminal region comprising a portion of SEQ ID NO: 99 [FGF19], the e-terminal region having a first amino acid position and a last amino acid position, wherein the e-terminal region comprises residues amino acids 16 to 29 of SEQ ID NO: 99 [FGF19], WGDPIRLRHLYTSG, where residue W corresponds to the first amino acid position in the e-terminal region; or (B) (a) an N-terminal region comprising a portion of SEQ ID NO: 100 [FGF21], the N-terminal region having a first amino acid position and a last amino acid position, and where the N- terminal is comprised of amino acid residues GQV and where the radical V corresponds to the last amino acid position of the N-terminal region, and (b) an e-terminal region comprising a portion of SEQ ID NO: 99 [FGF19], the region and -terminal having a first amino acid position and a last amino acid position, where the e-terminal region comprises amino acid residues 21 to 29 of SEQ ID NO: 99 [FGF19], RLRHLYTSG, and where the radical R corresponds to first position in the e-terminal region.
[2]
2. Chimeric peptide sequence characterized by comprising: a) an N-terminal region comprising a portion of SEQ ID NO: 100 [FGF21], the N-terminal region having a first amino acid position and a last amino acid position, where the N-terminal region comprises at least five contiguous amino acids of SEQ ID N0: .100 [FGF21], including amino acid residues GQV, and where the radical V corresponds to the last amino acid position of the N-terminal region, and b) a e-terminal region comprising a portion of SEQ ID NO: 99 [FGF19], the e-terminal region having a first amino acid position and a last amino acid position, wherein the e-terminal region comprises amino acid residues 21 to 29 from SEQ ID NO: 99 [FGF19], RLRHLYTSG, and where the radical R corresponds to the first position in the e-terminal region.
[3]
3. Peptide sequence according to claim 2, characterized by the fact that the N-terminal region comprises at least 6 or at least 7 contiguous amino acids of SEQ ID NO: 100 [FGF21], including the GQV amino acid residues.
[4]
4. Peptide sequence characterized by comprising or consisting of any one of: a) a sequence variant of a fibroblast growth factor 19 (FGF19) having one or more amino acid substitutions, insertions or deletions in comparison with a reference or type wild FGF19; b) a sequence variant of a fibroblast growth factor 21 (FGF21) having one or more amino acid substitutions, insertions or deletions, in comparison with a FGF21 reference or wild type; c) a portion of a FGF19 sequence fused to a portion of a FGF21 sequence; or d) a portion of a FGF19 sequence fused to a portion of an FGF21 sequence, wherein the FGF19 and / or FGF21 sequence portion (s) have one or more amino acid substitutions, insertions or deletions compared to a reference or wild type FGFl 9 and / or FGF21.
[5]
5. Peptide sequence according to claim 4, characterized by the fact that the peptide sequence has (i) amino acids terminal 1 to 16 of SEQ ID NO: 100 [FGF21] fused to carboxy amino acids of 21 to 194 of SEQ ID NO: 99 [FGF19], (ii) amino-terminal amino acids 1 to 147 of SEQ ID NO: 99 [FGF19] fused with carboxy-earth amino acids 147 to 181 of SEQ ID NO: 100 [FGF21] (M41 ),
(iii) amino-terminal amino acids 1 to 20 of SEQ ID NO: 99 [FGFl9] fused with carboxy-terminal amino acids 17 to 181 of SEQ ID NO: 100 [FGF21] (M44), (iv) amino-terminal amino acids 1 to 146 of SEQ ID NO: 100 [FGF21] fused with carboxy-terminal amino acids 148 to 194 of SEQ ID NO: 99 [FGF19] (M45), or (v) amino-terminal amino acids 1 to 20 of SEQ ID NO: 99 [ FGF19] fused with internal amino acids 17 to 146 of SEQ ID NO: 100 [FGF21] fused with carboxy-terminal amino acids 148 to 194 of SEQ ID NO: 99 [FGF19] (M46).
[6]
6. Chimeric peptide sequence or peptide sequence according to claim 1 or 4, characterized in that (a) the peptide sequence has a WGDPI sequence motif corresponding to the WGDPI amino acid sequence 16 to 20 of SEQ ID NO: 99 [FGF19], wherein the peptide sequence optionally maintains or enhances FGFR4-mediated activity; (b) the peptide sequence has a substituted, mutated or missing WGDPI sequence motif corresponding to the FGF19 WGDPI sequence of amino acids 16 to 20 of FGF19; (c) the WGDPI sequence has one or more substituted, mutated or missing amino acids; (d) the peptide sequence is different from a variant sequence of FGF 19 having one of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI,
AGDPI, WAD PI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the FGF19 WGDPI sequence in amino acids 16 to 20; (e) the N-terminal region comprises amino acid residues DSSPLLQ and where residue Q is the last amino acid position of the N-terminal region; where optionally the N-terminal region further comprises: (i) RHPIP, where R is the first amino acid position of the N-terminal region, (ii) HPIP, where H is the first amino acid position of the N-terminal region , (iii) RPLAF, where R is the first amino acid position in the N-terminal region, (iv) PLAF, where P is the first amino acid position in the N-terminal region, (v) R, where R is the first amino acid position in the N-terminal region; or (f) said N-terminal region or said e-terminal region comprises or consists of an amino acid sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids.
[7]
Chimeric peptide sequence or peptide sequence according to one of claims 1, 2 or 4, characterized by the fact that
(a) the N-terminal or e-terminal region is about 20 to about 200 amino acid residues in length; (b) the peptide sequence comprises or consists of any of the M1 to M98 variant peptide sequences or a subsequence or fragment of any of the M1 to M98 variant peptide sequences; where the subsequence or fragment optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more deletions of amino acids at the amino, carboxy-terminal end or internally; (c) the HPIP amino acid residues are the first 4 amino acid residues in the N-terminal region; (d) the last position of the e-terminal region corresponds to residue 194 of SEQ ID NO: 99 [FGF19]; (e) the peptide sequence comprises or consists of: HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSeFLRIRADGVVOeARGQSAH SLLEIKAVALRTVAIKGVHSVRYLeMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; DSSPLLQFGGQVRLRHLYTSGPHGLSSeFLRIRADGVVDeARGQSAHSLLE IKAVALRTVAIKGVHSVRYLeMGADGKMQGLLQYSEEDeAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHL ESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDeARG QSAHSLLEIKAVALRTVAIKGVHSVRYLeMGADGKMQGLLQYSEEOeAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; or
DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG HLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; or a subsequence or its fragment; where the subsequence or fragment optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more deletion of amino acids at the amino, carboxy-terminal or internally; (f) said N-terminal region or said e-terminal region or said fast FGF19 sequence portion or said FGF21 sequence portion are joined via a linker or spacer; (g) the N-terminus of the peptide sequence comprises or consists of any of: HPIPDSSPLLQFGGQVRLRHLYTSG (MS); DSSPLLQFGGQVRLRHLYTSG (M6); RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7); HPIPDSSPLLQWGDPIRLRHLYTSG (MS); HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9); HPIPDSSPHVHYGWGDPIRLRHLYTSG (MlO); RPLAFSDAGPLLQWGDPIRLRHLYTSG (Mll); RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13);
HPIPDSSPHVHYGGQVRLRHLYTSG (M14);
RPLAFSDAGPHVHWGDPIRLRHLYTSG (MlS);
RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16);
RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17);
RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18);
RPLAFSDAGPVYGWGDPIRLRHLYTSG (Ml9);
RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20);
RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21);
RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22);
RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23);
RPLAFSDAGPHHYWGDPIRLRHLYTSG (M24);
RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25);
RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26);
RPLAFSDSSPHVHWGDPIRLRHLYTSG (M22);
RPLAFSDAGPHVWGDPIRLRHLYTSG (M28);
RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29);
RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30);
RHPIPDSSPLLQFGAQVRLRHLYTSG (M31);
RHPIPDSSPLLQFGDQVRLRHLYTSG (M32);
RHPIPDSSPLLQFGPQVRLRHLYTSG (M33);
RHPIPDSSPLLQFGGAVRLRHLYTSG (M34);
RHPIPDSSPLLQFGGEVRLRHLYTSG (M35);
RHPIPDSSPLLQFGGNVRLRHLYTSG (M36);
RHPIPDSSPLLQFGGQARLRHLYTSG (M3 7);
RHPIPDSSPLLQFGGQIRLRHLYTSG (M38); RHPIPDSSPLLQFGGQTRLRHLYTSG (M39); RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40); DAGPHVHYGWGDPIRLRHLYTSG (M74); VHYGWGDPIRLRHLYTSG (M75);
RLRHLYTSG (M77); or any of the foregoing peptide sequences, wherein the amino terminal R residue is eliminated; wherein optionally the peptide sequence further comprises the addition of amino acid residues 30 to 194 of SEQ ID NO: 99 [FGF19] at the C-terminus, resulting in a chimeric polypeptide; or where optionally the peptide sequence still comprises all or a portion of an FGFl ~ sequence defined as: PHGLSSCFLRIRADGVVDC
ARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCA
FEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK positioned at the e-terminal end of the peptide or where the "R" amino acid terminal is excluded from the peptide; _.
(h) the N-terminal of the peptide sequence comprises or consists of any of: RHPIPDSSPLLQFGWGDPIRLRHLYTSG; RHPIPDSSPLLQWGDPIRLRHLYTSG; RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; RHPIPDSSPHVHYGWGDPIRLRHLYTSG; RPLAFSDAGPLLQFGGQVRLRHLYTSG; RHPIPDSSPHVHYGGQVRLRHLYTSG; RPLAFSDAGPHVHYGGDIRLRHLYTSG; RDSSPLLQFGGQVRLRHLYTSG; RPLAFSDSSPLLQFGGQVRLRHLYTSG; RHPIPDSSPLLQFGAQVRLRHLYTSG;
RHPIPDSSPLLQFGDQVRLRHLYTSG; RHPIPDSSPLLQFGPQVRLRHLYTSG; RHPIPDSSPLLQFGGAVRLRHLYTSG; RHPIPDSSPLLQFGGEVRLRHLYTSG; RHPIPDSSPLLQFGGNVRLRHLYTSG; RHPIPDSSPLLQFGGQARLRHLYTSG; RHPIPDSSPLLQFGGQIRLRHLYTSG; RHPIPDSSPLLQFGGQTRLRHLYTSG; RHPIPDSSPLLQFGWGQPVRLRHLYTSG; or any one of the foregoing peptide sequences, wherein the amino terminal R residue is eliminated; wherein optionally the peptide sequence further comprises the addition of amino acid residues 30 to 194 of SEQ ID NO: 99 [FGF19] at the C-terminus, resulting in a chimeric polypeptide; or in which optionally the peptide sequence still comprises all or a portion of an FGF19 sequence defined as: PHGLSSCFLRIRADGVVDC
ARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCA
FEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK positioned at the e-terminal end of the peptide or where the "R" amino acid terminal is excluded from the peptide; (i) in which the subsequence optionally has at least one amino acid deletion; where the subsequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20 or more deletions of amino acids from the amino-terminal, the carboxy-terminal or internally;
(j) the position of the first amino acid in the N-
terminal is an "M" residue 'an "R" residue' an "S" residue, an "H" residue, an "P" residue, an "L" residue or an "D" residue or where the peptide sequence it does not have an "M" residue or an "R" residue at the position of the first amino acid in the N-terminal region; (k) the N-terminal region comprises any of the following sequences: MDSSPL, MSDSSPL, SDSSPL, MSSPL or SSPL; (1) the peptide sequence reduces the formation of hepatocellular carcinoma (HCC) compared to FGF19 or a variant sequence of FGF 19 having any of the GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI , WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence in amino acids 16 to 20 of FGF19; where optionally hepatocellular carcinoma (HCC) formation is determined in db / dB rats; (m) the peptide sequence has greater blood glucose lowering activity compared to FGF19 or a variant sequence of FGF19 having any of the GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence in amino acids 16 to 20 of FGF19;
where optionally hypoglycemic activity is determined in db / dB rats; (n) the peptide sequence has less lipid-raising activity compared to FGF19 or a variant sequence of FGF 19 having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence in amino acids 16 to 20 of FGF19; where optionally the lipid-raising activity is determined in db / dB mice; (o) the peptide sequence has less triglyceride, non-HDL or HDL cholesterol-raising activity compared to FGFl 9 or a sequence
1 • FGF 19 variant having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WAD PI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI replaced by WGDPI sequence in 16 amino acids to 20 of FGF19; (p) the peptide sequence has less activity in reducing lean mass compared to FGF21; where optionally the activity of reducing lean mass is determined in db / dB rats; (q) the peptide sequence binds to the fibroblast growth factor 4 (FGFR4) receptor or activates FGFR4 or does not detectably bind to the fibroblast growth factor 4 (FGFR4) receptor or activates FGFR4; ..
(r) the peptide sequence binds to FGFR4 with an affinity less than, comparable to, or greater than the binding affinity of FGF19 to FGFR4; (s) the active peptide sequence FGFR4 to an extent or amount less than, comparable to or greater than the active FGF19 FGFR4 (t) the peptide sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or insertions; wherein optionally the amino acid deletions are at the N- or e-terminal or internal; or where the amino acid substitution or deletion is in any of the positions of amino acids 8 to 20 of FGF19 (AGPHVHYGWGDPI) (u) the peptide sequence comprises one or more L-amino acids, D-amino acids, non-naturally occurring amino acids or mimetic amino acids , derivatives or the like.
[8]
Chimeric peptide sequence according to claim 1, characterized in that (a) the N-terminal region comprises amino acid residues (i) VHYG, (ii) DASPHVHYG, or (iii) DSSPLVHYG; where optionally G corresponds to the last position of the N-terminal region; where optionally the N-terminal region further comprises:
(i) RHPIP, where R is the first amino acid position in the N-terminal region; (ii) HPIP, where H is the first amino acid position in the N-terminal region; (iii) RPLAF, where R is the first amino acid position in the N-terminal region; (iv) PLAF, where p is the first amino acid position in the N-terminal region; or (v) R, where Ré is the first amino acid position in the N-terminal region; or (b) the N-terminal region comprises amino acid residues DSSPLLQFGGQV, and where residue V corresponds to the last position of the N-terminal region.
[9]
Chimeric peptide sequence or peptide sequence according to any one of claims 1, 2 or 4, characterized in that the peptide sequence comprises or consists of any one of:
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRL
PVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMD PFGLVTGLEAVRSPSFEK (M69);
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF GLVTGLEAVRSPSFEK (M52);
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHR
LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSM DPFGLVTGLEAVRSPSFEK (MS);
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKA
LKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSL
PLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGP SQGRSPSYAS (M71);
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKA
LKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGL
PLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGP SQGRSPSYAS (M72);
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKA
LKPGVIQILGVKTSRSLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGL
PLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQ DELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPVWDGITGE (M73);
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE TDSMDPFGLVTGLEAVRSPSFEK (Ml);
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE TDSMDPFGLVTGLEAVRSPSFEK (M2);
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE TDSMDPFGLVTGLEAVRSPSFEK (M3);
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF GLVTGLEAVRSPSFEK (M48);
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEK
HRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETD SMDPFGLVTGLEAVRSPSFEK (M49);
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS MDPFGLVTGLEAVRSPSFEK (M50);
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS MDPFGLVTGLEAVRSPSFEK (M51);
MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF GLVTGLEAVRSPSFEK (M53); and
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHR LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS1 6MDPFGLVTGLEAVRSPSFEK () where the subsequence or fragment optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20 or more deletions of amino acids at the amino, carboxy-terminal end or internally.
[10]
Chimeric peptide sequence or peptide sequence according to one of claims 1, 2, 4 OR 9f characterized by the fact that (a) the first position of the N-terminal region is (i) an R residue, or (ii) a residue of M; (b) the first and second position of the N-terminal region is (i) an MR sequence, (ii) an RM sequence, (iii) an RD sequence, (iv) an OS sequence, (v) a sequence MD, or (vi) a sequence of MS; (c) from the first to the third position of the N-terminal region is (i) a sequence of MOS, (ii) a sequence of RDS, (iii) a sequence of MSD, (iv) a sequence of MSS, or (v) · A DSS sequence, (d) from the first to the fourth position of the N-Earth region is (i) a ROSS sequence, or (ii) a MOSS sequence, (e) from the first to the fifth position of the N-Earth region is (i) an MRDSS sequence, or (ii) an
MSSPL (f) from the first to the sixth position of the N-Earth region is an MDSSPL sequence, or (g) from the first to seventh position of the N-Earth region is an MSDSSPL sequence.
[11]
11. Peptide sequence according to claims 2 or 4, characterized in that said portion of the FGFl 9 sequence or said portion of the FGF21 sequence, comprises or consists of an amino acid sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids of FGF19 or FGF21.
[12]
12. Peptide sequence according to claim 4, characterized in that (i) the FGF19 reference or wild type sequence is presented as:
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 99); or (ii) the FGF21 wild type reference or sequence is presented as:
RHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPE
SLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLE
DGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGI LAPQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID NO: 100).
[13]
Pharmaceutical composition or composition characterized by comprising the chimeric peptide sequence or peptide sequence according to any one of claims 1 to 12;
11 19/23 wherein the pharmaceutical composition optionally comprises a hypoglycemic agent.
[14]
Chimeric peptide sequence or peptide sequence according to any of claims 1 to 12, characterized in that the peptide sequence is isolated or purified.
[15]
Nucleic acid molecule characterized by the fact that it encodes the chimeric peptide sequence or a peptide sequence according to any one of claims 1 to 12 wherein the nucleic acid molecule further comprises an operable linked expression control element which confers the expression of the nucleic acid molecule encoding the peptide in vitro, in a cell or in vivo.
[16]
16. Vector characterized in that it comprises the nucleic acid molecule according to claim 15; wherein the vector optionally comprises a viral vector.
[17]
17. Transformed or host cell characterized by the fact that it expresses the chimeric peptide sequence or a peptide sequence according to any one of claims 1 to 12.
[18]
18. Use of the chimeric peptide sequence or a peptide sequence according to any one of claims 1 to 12, characterized in that it is in the manufacture of a medicament to treat an individual having, or at risk of having, a disease or disorder treatable by the sequence chimeric peptide or a peptide sequence;
where optionally
(a) the disease or disorder comprises a hyperglycemic condition, insulin resistance, hyperinsulinemia, glucose intolerance or metabolic syndrome; where occasionally the hyperglycemic condition comprises (i) diabetes; where optionally use results in reduced glucose levels, increased insulin sensitivity, decreased insulin resistance, reduced glucagon, improved glucose tolerance or glucose metabolism or homeostasis, improved pancreas function, reduced triglyceride, cholesterol, HDL, LDL and VLDL, decrease in blood pressure, decrease in the thickening of the intimate layer of the blood vessel or a decrease in body mass or weight gain; or (i) insulin-dependent diabetes (Type I), type II diabetes or gestational diabetes; or (b) the disorder comprises obesity or an undesirable body mass.
[19]
19. Use of the chimeric peptide sequence or a peptide sequence according to any one of claims 1 to 12, characterized in that it is in the manufacture of a medicament to improve glucose metabolism in an individual in need of it; where the individual optionally has a fasting plasma glucose level greater than 100 mg / dl or has a hemoglobin Ale (HbAlc) level above 6%; or where optionally the use results in reduced glucose levels, increased insulin sensitivity, decreased insulin resistance, reduced glucagon, improved glucose tolerance or metabolism. glucose or homeostasis, improved pancreas function, reduced triglyceride, cholesterol, HDL, LDL and VLDL, decreased blood pressure, decreased thickening of the intimal layer of the blood vessel or a decrease in body mass or weight gain.
[20]
20. Method to identify a peptide sequence having hypoglycemic activity without substantial hepatocellular carcinoma (HCC) activity, characterized by comprising: a) providing a candidate peptide sequence; b) administering the candidate peptide sequence to a test animal; e) measuring the glucose levels of the animal after administration of the candidate peptide sequence, to determine whether the candidate peptide sequence reduces glucose levels; and
I • I
D) analyzing the candidate peptide sequence for the induction of HCC in the animal or the expression of a marker correlated with HCC activity, in which a candidate peptide that has hypoglycemic activity and not substantial HCC activity thus identifies the candidate peptide sequence as a peptide sequence having hypoglycemic activity without substantial hepatocellular carcinoma (HCC) activity; wherein optionally (i) the test animal is a db / dB rat; (ii) the method further comprises evaluating a sample of liver tissue from the test animal to determine whether the candidate peptide sequence shows evidence of HCC induction; (iii) the marker correlated with HCC activity comprises lipid profile and in which less increase in lipid activity compared to FGF19 indicates that the peptide lacks substantial HCC activity (iv) the marker correlated with HCC activity comprises expression of the aldo gene -keto reductase, and where elevation or increase in the expression of the aldo-keto reductase gene compared to FGF19 indicates that the peptide lacks substantial HCC activity; or (v) the marker indicative of HCC activity comprises the expression of the S1cla2 gene, and where the expression of the down regulatory or diminishing S1c1a2 gene compared to FGF21 indicates that the peptide lacks substantial HCC activity.
[21]
21. Peptide sequence, subsequence, composition, pharmaceutical composition, nucleic acid molecule, vector, transformed or host cell, use of a chimeric peptide sequence or a peptide sequence, method of treatment for an individual, method for improving the metabolism of glucose in an individual and method for identifying a peptide sequence characterized by being as described herein and shown within the scope of the technical matter initially described, disclosed or illustrated in the present patent application.

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法律状态:
2020-11-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-04-20| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-09-08| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|Free format text: NOTIFICACAO DE DEVOLUCAO DO PEDIDO EM FUNCAO DA REVOGACAO DO ART. 229-C DA LEI NO 9.279, DE 1996, POR FORCA DA LEI NO 14.195, DE 2021 |

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2021-09-21| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161504128P| true| 2011-07-01|2011-07-01|

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US201161515126P| true| 2011-08-04|2011-08-04|

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