Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/50—Fibroblast growth factor [FGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the identification of new muteins of fibroblast growth factor 21 that have reduced deamidati ⁇ n when compared to wild type fibroblast growth factor 21.
• Fibroblast growth factors are polypeptides widely expressed in developing and adult tissues (Baird et al, Cancer Cells, 3:239-243, 1991) and play crucial roles in multiple physiological functions including angiogenesis, mitogenesis, pattern formation, cellular differentiation, metabolic regulation and repair of tissue injury (McKeehan et al., Prog. Nucleic Acid Res. MoL Biol. 59:135-176, 1998).
• the FGF family now consists of at least twenty-three members, FGF-I to FGF- 23 (Reuss et al, Cell Tissue Res. 313:139-157 (2003).
• Fibroblast growth factor-21 has been reported to be preferentially expressed in the liver (Nishimura et al, Biochimica et Biophysica Acta, 1492:203-206, 2000); WO01/36640; and WO01/18172) and described as a treatment for ischemic vascular disease, wound healing, and diseases associated with loss of pulmonary, bronchia or alveolar cell function and numerous other disorders.
• FGF-21 has been shown to stimulate glucose-uptake in mouse 3T3-L1 adipocytes after treatment in the presence and absence of insulin, and to decrease fed and fasting blood glucose, triglycerides, and glucagon levels in ob/ob and db/db mice and 8 week old ZDF rats in a dose-dependant manner, thus, providing the basis for the use of FGF-21 as a therapy for treating diabetes and obesity (WO03/011213).
• the development of recombinant DNA technology has made possible the production of foreign products such as muteins of FGF-21 in host cells in which exogenous DNA sequences coding for those products have been introduced.
• the present invention solves the problem of deamidation associated with recombinant proteins by providing FGF-21 muteins that have a reduced amount of deamidation compared to wild type FGF-21.
• FGF-21 muteins with reduced deamidation can be produced in industrial fermentation conditions, can be pharmaceutically formulated in a solution form, and maintain the biological activity necessary to be useful to treat subjects with disorders including, but not limited to, type 2 diabetes, obesity, and metabolic syndrome.
• the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, comprising the substitution of any amino acid except Asn or GIn for Asn 121, wherein the numbering of the amino acids is based on SEQ ID NO:1 and wherein said mutein has reduced deamidation compared to wild-type human FGF-21.
• a second aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, comprising the substitution of any amino acid except Asn or GIn for Asn 121, in combination with the substitution of a cysteine for two or more of the following: arginine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27, glutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, glycine 42, glycine 43, glutamate 50, glutamine 54, leucine 58, valine 62, leucine 66, glycine 67, lysine 69, arginine 72, phenylalanine 73, glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81, leucine 82, glycine 84, serine 85
• a third aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, comprising the substitution of any amino acid except Asn or GIn for Asn 121 in combination with the substitution of a charged and/or polar but uncharged amino acid for one or more of the amino acids at positions: glycine 42, glutamine 54, arginine 77, alanine 81, leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine 126, proline 130, arginine 131,leucine 139, alaninel45, leucine 146, isoleucine .152; alanine 154; glutamine 156, glycine 161, serine 163, glycine 170, or serine 172, wherein the numbering of amino acids is based on SEQ ID NO:1 and wherein said mutein has reduced deamidation when compared to wild-type human FGF-21
• a fourth aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, comprising the substitution of any amino acid except Asn or GIn for Asn 121 in combination with he substitution of any amino acid except Ser or Thr for Ser 167, wherein the numbering of amino acids is based on SEQ ID NO:1 and wherein said mutein has reduced deamidation and reduced capacity of O-glycosylation when compared to wild-type human FGF-21.
• a fifth aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, selected from the group consisting of Leull ⁇ Cys- Alal34Cys-Asnl21Ala-Serl67Ala, Leull8Cys-Alal34Cys-Asnl21Val-Serl67Ala, Leull8Cys-Alal34Cys-Asnl21Ser-Serl67Ala, Leull8Cys-Alal34Cys-Asnl21Asp- Serl67Ala, and Leul 18Cys-Alal34Cys-Asnl21Glu-Serl67Ala wherein the numbering of amino acids is based on SEQ ID NO:1 and wherein said mutein has reduced deamidation and reduced capacity of O-glycosylation when expressed in yeast compared to wild-type human FGF-21.
• inventions are drawn to polynucleotides encoding the muteins of the first, second, third, fourth, and fifth aspects, vectors containing said polynucleotides and a host cell carrying said vector.
• Another embodiment is drawn to processes for producing a polypeptide, to produce cells capable of producing said polypeptide and to produce a vector containing DNA encoding said polypeptide.
• Yet another embodiment is drawn to methods of treating a patient exhibiting one or more of the following condition(s): obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, or metabolic syndrome comprising administering to said patient in need of such treatment a therapeutically effective amount of a human FGF-21 mutein of the first, second, third or fourth aspects.
• Human FGF-21 is a 208 amino acid polypeptide containing a 27 amino acid leader sequence. Human FGF-21 has -79% amino acid identity to mouse FGF-21 and -80% amino acid identity to rat FGF-21. Human FGF-21 is the preferred polypeptide template for the muteins of the present invention but it is recognized that one with skill in the art could readily make muteins based on an alternative mammalian FGF-21 polypeptide sequence.
• amino acid positions of the muteins of the present invention are determined from the mature human 181 amino acid FGF-21 polypeptide as shown below (SEQ ID NO:1): 1 10 20
• the corresponding DNA sequence coding for the mature human 181 amino acid FGF-21 polypeptide is (SEQ BD NO:2):
• Amino acids are identified using the three-letter code or alternatively are designated using the standard one letter code. Mutations are designated by the three-letter code for the original amino acid, followed by the amino acid number, followed by the three-letter code for the replacement amino acid. The numerical designations of each mutein is based on the 181 amino acid sequence of mature, wild-type, human FGF-21. For example, a substitution for serine at position 167 (i.e. Serl67) with the non- polar/hydrophobic amino acid, alanine (Ala), is designated as Serl67Ala or S 167 A.
• a "human FGF-21 mutein” is defined as comprising human FGF-21 in which at least one amino acid of the wild-type mature protein has been substituted by another amino acid. Examples of FGF-21 muteins are described in U.S. patent applications 60/528,582, 60/606,805, and 60/606,830, herein incorporated by reference. Generally speaking, a mutein possesses some modified property, structural or functional, of the wild-type protein. For example, the mutein may have enhanced or improved physical stability in concentrated solutions (e.g., less hydrophobic mediated aggregation), while maintaining a favorable bioactivity profile.
• the mutein may possess increased compatibility with pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl alcohol), thus enabling the preparation of a preserved pharmaceutical formulation that maintains the physiochemical properties and biological activity of the protein during storage.
• the mutein may have reduced O-glycosylation when expressed in yeast.
• the mutein may have less deamidation when compared to wild-type FGF-21.
• the process of deamidation is a well-recognized phenomenon that may impact the stability/activity of proteins and may occur at an Asn or GIn residue. Deamidation at Asn occurs more frequently and the rate of deamidation is highly dependent on the primary, secondary and tertiary structure of the protein.
• muteins with reduced deamidation when compared to wild-type FGF-21 have less structural alterations, while maintaining biological potency.
• biological potency As used herein, these terms are not limiting, it being entirely possible that a given mutein has one or more modified properties of the wild-type protein.
• a "biologically active peptide" is defined as a peptide of a mutein of the present invention that maintains the modified property(s) and the biological potency of the mutein.
• deamidation refers to the degradation of Asn or GIn residues in a protein/peptide (Robinson, et a (2001) Proc. Natl. Acad. ScL USA 12409- 12413).
• the intramolecular pathway for asparagine deamidation is via intermediate succinimide formation, resulting in a mixture of aspartyl and isoaspartyl residues (Harris, et al. (2001) /. of Chromatography 752:233-245).
• Deamidation introducing negative change, causes structurally and biologically important alterations in peptide and protein structures and may lead to a reduction of stability and/or the reduction or loss of activity of the protein. Deamidation can occur ex vivo during the preparation of the formulated therapeutic, negatively impacting the manufacturing and storage of the pharmaceutical agent. Moreover, the deamidation can occur in vivo effecting the protein's efficacy and duration of action.
• a “therapeutically-effective amount” is the minimal amount of an active agent necessary to impart therapeutic benefit to a patient.
• a “therapeutically- effective amount” to a patient suffering or prone to suffer or to prevent it from suffering from type 2 diabetes, obesity, or metabolic syndrome is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression, physiological conditions associated with or resistance to succumbing to the afore mentioned disorders.
• a "subject” or “patient” is preferably a human.
• Type 2 diabetes is characterized by excess glucose production in spite of the availability of insulin, and circulating glucose levels remain excessively high as a result of inadequate glucose clearance.
• Glucose intolerance can be defined as an exceptional sensitivity to glucose.
• Hyperglycemia is defined as an excess of sugar (glucose) in the blood. "Hypoglycemia” also called low blood sugar, occurs when your blood glucose level drops too low to provide enough energy for your body's activities.
• Hyperinsulinemia is defined as a higher-than-normal level of insulin in the blood.
• Insulin resistance is defined as a state in which a normal amount of insulin produces a subnormal biologic response.
• “Obesity”, in terms of the human subject, can be defined as that body weight over 20 percent above the ideal body weight for a given population (R.H. Williams, Textbook of Endocrinology, 1974, ⁇ .904-916).
• “Metabolic syndrome” can be defined as a cluster of at least three of the following signs: abdominal fat - in most men, a 40-inch waist or greater; high blood sugar - at least 110 milligrams per deciliter (mg/dl) after fasting; high triglycerides - at least 150 mg/dl in the bloodstream; low HDL - less than 40 mg/dl; and, blood pressure of 130/85 or higher.
• the present invention provides muteins with reduced deamidation wherein the site of deamidation is altered compared to native FGF-21.
• the deamidation site identified in the present invention is Asnl21. Applicants have discovered that substituting any amino acid except GIn or Asn for the Asnl21 site results in a significant reduction of deamidation of the expressed mutein when compared to wild- type FGF-21.
• the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, comprising the substitution of any amino acid except GIn or Asn for Asnl21, wherein the numbering of the amino acids is based on SEQ ID NO: 1 and wherein said mutein has reduced deamidation when compared to wild-type human FGF-21.
• Preferred muteins of the first embodiment are Asnl21Ala, Asnl21Val, Asnl21Ser, Asnl21Asp, and Asnl21Glu.
• a second aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, comprising the substitution of any amino acid except GIn or Asn for Asnl21, in combination with the substitution of a cysteine for two or more of the following: arginine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27, glutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, glycine 42, glycine 43, glutamate 50, glutamine 54, leucine 58, valine 62, leucine 66, glycine 67, lysine 69, arginine 72, phenylalanine 73, glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81, leucine 82, glycine 84, serine 85
• cysteines cysteine 75 and cysteine 93
• cysteine 75 and cysteine 93 could also be utilized as loci to introduce a novel disulfide bond that may impart improved properties.
• cysteine substitution at serine 85 or phenylalanine 73, coupled with a concomitant change at either cysteine 93 or cysteine 75, respectively, wherein the latter sites are replaced with any other amino acid.
• Muteins of FGF-21 with engineered disulfide bonds, in addition to the naturally occurring one at Cys75-Cys93 are described in U.S. patent application 60/528,582.
• the most preferred muteins of the second aspect are Leull8Cys-Alal34Cys-Asnl21Asp; Leu21Cys-Leu33Cys-Asnl21Asp; Ala26Cys-Lysl22Cys-Asnl21Asp; or Leu21Cys- Leu33Cys/Leull8Cys-Alal34Cys-Asnl21Asp.
• a third aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, comprising the substitution of any amino acid except GIn or Asn for Asnl21 in combination with the substitution of a charged and/or polar but uncharged amino acid for one or more of the amino acids at positions: glycine 42, glutamine 54, arginine 77, alanine 81, leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine 126, proline 130, arginine 131, leucine 139, alaninel45, leucine 146, isoleucine 152, alanine 154, glutamine 156, glycine 161, serine 163, glycine 170, or serine 172, wherein the numbering of the amino acids is based on SEQ ID NO:1 and wherein said mutein has reduced deamidation when compared to wild-type human FGF-
• a charged amino acid is defined as a positively- or negatively-charged amino acid.
• a positively charged amino acid is defined to include histidine, lysine, arginine, and non-naturally occurring analogs thereof (e.g., gamma aminobutyric acid, ornithine, etc.).
• a negatively charged amino acid is defined to included aspartate, glutamate, and non- naturally occurring analogs thereof (e.g., aminoadipic acid).
• a polar but uncharged amino acid is defined to include serine, threonine, asparagine, glutamine, and non- naturally occurring analogs thereof.
• Preferred muteins of the third aspect are Gln54Glu- Asnl21Asp, Leul39Glu-Asnl21Asp, Alal45Glu-Asnl21Asp, Leul46Glu-Asnl21Asp, Ilel52Glu-Asnl21Asp, Glnl56Glu-Asnl21Asp, Serl63Glu-Asnl21Asp, and Ilel52Gh> Serl63Glu-Asnl21Asp.
• a fourth aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, comprising the substitution of any amino acid except Asn or GIn for Asnl21 in combination with the substitution of any amino acid except Ser or Thr for Ser 167, wherein the numbering of amino acids is based on SEQ ID NO:1 and wherein said mutein has reduced deamidation and reduced capacity of O-glycosylation when expressed in yeast compared to wild- type human FGF-21.
• Preferred muteins of the fourth aspect are Asnl21 Asp-Serl67Ala, Asnl21Asp-Serl67Glu, Asnl21Asp- Serl67Asp, Asnl21Asp-Serl67Asn, Asnl21Asp-Serl67Gln, Asnl21Asp-Serl67Gly, Asnl21Asp-Serl67Val, Asnl21Asp, Serl67His, Asnl21As ⁇ -Serl67Lys, and Asnl21Asp-Serl67Tyr.
• muteins of human FGF-21 or a biologically active peptide thereof, comprising a combination of the first aspect of the present invention, the second aspect of the present invention, the third aspect of the present invention and the fourth aspect of the present invention wherein said mutein has reduced deamidation when compared to wild-type human FGF-21.
• Preferred muteins of this embodiment are Leull8Cys-Alal34Cys-Asnl21Ala-Serl67Ala, Leull8Cys- Alal34Cys-Asnl21Val-Serl67Ala, Leull8Cys-Alal34Cys-Asnl21Ser-Serl67Ala, Leull8Cys-Alal34Cys-Asnl21Asp-Serl67Ala, and Leull8Cys-Alal34Cys-Asnl21Glu- Serl67Ala.
• the biological potency of the muteins of the present invention is defined by the ability of the muteins to affect glucose uptake as measured in the in vitro 3T3-L1 cell assay (Example 2) and/or the lowering of plasma glucose levels, as well as, plasma triglycerides, as measured in vivo in the ob/ob mouse assay (Example 2)
• the muteins of FGF-21 administered according to this invention may be generated and/or isolated by any means known in the art.
• the most preferred method for producing the mutein is through recombinant DNA methodologies and is well known to those skilled in the art. Such methods are described in Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), which is incorporated herein by reference.
• the preferred embodiments include a biologically active peptide derived from the mutein described herein.
• a biologically active peptide derived from the mutein described herein.
• Such a peptide will contain at least one of the substitutions described and the mutein will possess biological activity.
• the peptide may be produced by any means known to those skilled in the art, examples of which include but are not limited to enzymatic digestion, chemical synthesis or recombinant DNA methodologies.
• DPP-IV dipeptidyl peptidase IV
• the N-terminus of FGF-21 contains two dipeptides that could potentially be substrates to DPP-IV, resulting in a fragment of FGF-21 truncated at the N-terminus by up to 4 amino acids.
• this fragment of wild- type FGF-21 has been demonstrated to retain biological activity (Table 1), thus, muteins of the present invention truncated at the N-terminus by up to 4 amino acids (des-HPIP) in combination with the amino acid substitutions of any of the aspects of the present invention are embodiments of the present invention.
• truncation of 5 amino acids or greater from the N-terminus negatively impacts biological activity.
• Preferred muteins of the present invention that are truncated at the N-terminus by up to 4 amino acids are des-HPIP-Leull8Cys-Alal34Cys-Asnl21Asp and des-HPJP- Leull8Cys-Alal34Cys-Asnl21Asp-Serl67Ala.
• the present invention also encompasses polynucleotides encoding the above- described muteins that may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
• the DNA may be double-stranded or single-stranded.
• the coding sequences that encode the muteins of the present invention may vary as a result of the redundancy or degeneracy of the genetic code.
• the polynucleotides that encode for the muteins of the present invention may include the following: only the coding sequence for the mutein, the coding sequence for the mutein and additional coding sequence such as a functional polypeptide, or a leader or secretory sequence or a pro-protein sequence; the coding sequence for the mutein and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mutein.
• polynucleotide encoding a mutein encompasses a polynucleotide that may include not only coding sequence for the mutein but also a polynucleotide, which includes additional coding and/or non-coding sequence.
• the present invention further relates to variants of the described polynucleotides that encode for fragments, analogs and derivatives of the polypeptide that contain the indicated substitutions.
• the variant of the polynucleotide may be a naturally occurring allelic variant of the human FGF-21 sequence, a non-naturally occurring variant, or a truncated variant as described above.
• the present invention also includes polynucleotides encoding the muteins described above, as well as variants of such polynucleotides, which variants encode for a fragment, derivative or analog of the disclosed mutein.
• Such nucleotide variants include deletion variants, substitution variants, truncated variants, and addition or insertion variants as long as at least one of the ! indicated amino acid substitutions of any of the aspects of the present invention is present.
• polynucleotides of the present invention may be expressed in mammalian, bacterial, fungal or yeast cells after the sequences have been operably linked to an expression control sequence.
• expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
• expression vectors will contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences.
• Yeast cells used for expressing the muteins of the present invention include Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia angust.
• the yeast host cells contain suitable vectors with expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
• the preferred yeast host of the present invention is Pichia pastoris wherein the expression vector is integrated into the host chromosomal DNA. Aspergillus niger, Trichoderma reesei; and Schizophyllum commune, are examples of fungi hosts, although others may also be employed as a matter of choice.
• the vectors containing the polynucleotide sequences of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts.
• the FGF-21 mutein-containing compositions should be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the patient, the site of delivery of the FGF-21 mutein composition, the method of administration, the scheduling of administration, and other factors known to practitioners.
• the therapeutically effective amount of the FGF-21 mutein for purposes herein is thus determined by such considerations.
• compositions of the FGF-21 muteins of the present invention may be administered by any means known in the art that achieve the generally intended purpose to treat type 2 diabetes, obesity, or metabolic syndrome.
• the preferred route of administration is parenteral, defined herein as referring to modes of administration that include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous, and intraarticular injection and infusion.
• the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
• Compositions within the scope of the invention include all compositions wherein an FGF-21 mutein is present in an amount that is effective to achieve the desired medical effect for treatment type 2 diabetes, obesity, or metabolic syndrome.
• the muteins of FGF-21 of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions.
• a desired formulation would be one that is a stable lyophilized product that is reconstituted with an appropriate diluent or an aqueous solution of high purity with optional pharmaceutically acceptable carriers, preservatives, excipients or stabilizers [Remington's Pharmaceutical Sciences 16th edition (1980)].
• the muteins of the present invention may be combined with a pharmaceutically acceptable buffer, and the pH adjusted to provide acceptable stability, and a pH acceptable for administration.
• the FGF-21 muteins are formulated generally by mixing one or more of them at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
• a pharmaceutically acceptable carrier i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
• a pharmaceutically acceptable anti-microbial agents may be added. Phenol, m-cresol, and benzyl alcohol are preferred pharmaceutically acceptable anti-microbial agents.
• one or more pharmaceutically acceptable salts may be added to adjust the ionic strength or tonicity.
• One or more excipients may be added to further adjust the isotonicity of the formulation. Glycerin, sodium chloride, and mannitol are examples of an isotonicity adjusting excipient.
• an FGF-21 mutein administered will result in lowering blood glucose levels and increasing energy expenditure by faster and more efficient glucose utilization, and thus is useful for treating type 2 diabetes, obesity and metabolic syndrome.
• a typical dose range for the FGF-21 muteins of the present invention will range from about 0.01 mg per day to about 1000 mg per day for an adult.
• the dosage ranges from about 0.1 mg per day to about 100 mg per day, more preferably from about 1.0 mg per day to about 10 mg per day. Most preferably, the dosage is about 1-5 mg per day.
• the appropriate dose of an FGF-21 mutein administered will result in lowering blood glucose levels and increasing energy expenditure by faster and more efficient glucose utilization, and thus is useful for treating type 2 diabetes, obesity and metabolic syndrome.
• Another aspect provides muteins of FGF-21 of the present invention, or biologically active peptides thereof, for use as a medicament. Yet another aspect provides for the use of an effective amount of a mutein of human FGF-21 of the present invention, or a biologically active peptide thereof, for the manufacture of a medicament to treat a subject with obesity, type 2 diabetes, or metabolic syndrome.
• FGF-21 muteins are expressed in yeast, such as Pichia pastoris, Pichia methanolica or Saccharomyces cerevisiae.
• yeast such as Pichia pastoris, Pichia methanolica or Saccharomyces cerevisiae.
• AOXl alcohol oxidase
• vectors that use the promoter from the GAP gene are available for high level constitutive expression.
• the multi-copy Pichia expression vectors allows one to obtain strains with multiple copies of the gene of interest integrated into the genome.
• yeast expression system is Saccharomyces cerevisiae.
• Expression vectors contain the promoter and enhancer sequences from the GALl gene.
• the GALl promoter is one of the most widely used yeast promoters because of its strong transcriptional activity upon induction with galactose.
• Analytical characterization indicates that the FGF-21 muteins expressed in Pichia pastoris are truncated (four amino acid removal at the wild- type N-terminus).
• this truncated variant of FGF-21 stimulates glucose uptake at the same level as wild-type FGF-21 (Table 1).
• GM growth medium
• DM differentiation medium
• Glucose Transport Assay-- Hexose uptake as assayed by the accumulation of 0.1 mM 2-deoxy-D-[ 14 C]glucose, is measured as follows: 3T3-L1 adipocytes in 12-well plates are washed twice with KRP buffer (136 mM NaCl, 4.7 mM KCl, 10 mM NaPO 4 , 0.9 mM CaCl 2 , 0.9 mM MgSO 4 , pH 7.4) warmed to 37 0 C and containing 0.2% BSA, incubated in Leibovitz's L-15 medium containing 0.2% BSA for 2 h at 37°C in room air, washed twice again with KRP containing, 0.2% BSA buffer, and incubated in KRP, 0.2% BSA buffer in the absence (Mg 2 SO only) or presence of wortmanninfor 30 min at 37 °C in room air.
• KRP buffer 136 mM NaCl, 4.7 mM
• Insulin is then added to a final concentration of 100 nM for 15 min, and the uptake of 2-deoxy-D-[ 14 C]glucose is measured for the last 4 min.
• Nonspecific uptake measured in the presence of 10 ⁇ M cytochalasin B, is subtracted from all values. Protein concentrations are determined with the Pierce bicinchoninic acid assay. Uptake is measured routinely in triplicate or quadruplicate for each experiment.
• the in vitro potency of muteins of FGF-21 of the present invention is compared to wild-type FGF-21 in Table 1. As indicated in Table 1, the muteins of the present invention maintained biological potency to various degrees compared to wild-type FGF- 21.
• Ob/ob Mouse Model A study in an obesity model using male ob/ob mice is done to monitor plasma glucose levels and triglyceride levels after treatment with FGF-21, compared to vehicle and insulin control groups.
• the ability of the FGF-21 muteins to lower plasma glucose levels and triglyceride levels as compared to the vehicle control is demonstrated with this model.
• the ability of the FHF-21 muteins to lower plasma glucose levels as compared to the vehicle control is shown in Table 2.
• IsoAspl21 is an unnatural amino acid, and hence, may have a less favorable PK than Asnl21.
• any naturally occurring amino acid other than Asn or GIn may be substituted for Asnl21 and result in a mutein that has less deamidation than wild-type FGF-21, although it is recognized that deamidation of GIn occurs at a reduced rate compared to deamidation at Asn.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Diabetes (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Hematology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Pharmacology & Pharmacy (AREA)
• Engineering & Computer Science (AREA)
• Obesity (AREA)
• Veterinary Medicine (AREA)
• Toxicology (AREA)
• Genetics & Genomics (AREA)
• Endocrinology (AREA)
• Zoology (AREA)
• Gastroenterology & Hepatology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Emergency Medicine (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Child & Adolescent Psychology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Publications (2)

Family

ID=36035700

Family Applications (1)

Country Status (4)

Cited By (37)

Families Citing this family (32)

Family Cites Families (5)

• 2005
  • 2005-12-07 EP EP05853126A patent/EP1831371A2/en not_active Withdrawn
  • 2005-12-07 US US11/718,636 patent/US7655627B2/en not_active Expired - Fee Related
  • 2005-12-07 WO PCT/US2005/044116 patent/WO2006065582A2/en not_active Ceased
  • 2005-12-07 JP JP2007545564A patent/JP2008522617A/ja not_active Withdrawn

Also Published As

Similar Documents

Legal Events