Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
  • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/02—Linear peptides containing at least one abnormal peptide link
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format.
• the .txt file contains a sequence listing entitled“PRTH_036_0lWO_ST25.txt” created on July 10, 2019 and having a size of -172 kilobytes.
• the sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.
• the present invention relates to novel peptide inhibitors of the interleukin-23 receptor (IL-23R), and their use to treat or prevent a variety of diseases and disorders, including inflammatory bowel disease, Crohn’s disease and psoriasis.
• IL-23R interleukin-23 receptor
• IL-23R is expressed on various adaptive and innate immune cells including Thl7 cells, gd T cells, natural killer (NK) cells, dendritic cells, macrophages, and innate lymphoid cells, which are found abundantly in the intestine. At the intestine mucosal surface, the gene expression and protein levels of IL-23R are found to be elevated in IBD patients. It is believed that IL-23 mediates this effect by promoting the development of a pathogenic CD4 + T cell population that produces IL-6, IL-17, and tumor necrosis factor (TNF).
• TNF tumor necrosis factor
• IL-23 is enriched in the intestine, where it is believed to play a key role in regulating the balance between tolerance and immunity through T-cell-dependent and T- cell-independent pathways of intestinal inflammation through effects on T-helper 1 (Thl) and Thl7-associated cytokines, as well as restraining regulatory T-cell responses in the gut, favoring inflammation.
• Thl T-helper 1
• Thl7-associated cytokines T-helper 1
• IBDs inflammatory bowel diseases
• Psoriasis a chronic skin disease affecting about 2%-3% of the general population has been shown to be mediated by the body’s T cell inflammatory response mechanisms.
• 11-23 has one of several interleukins implicated as a key player in the pathogenesis of psoriasis, purportedly by maintaining chronic autoimmune inflammation via the induction of interleukin- 17, regulation of T memory cells, and activation of macrophages.
• Expression of IL-23 and IL- 23R has been shown to be increased in tissues of patients with psoriasis, and antibodies that neutralize IL-23 showed IL-23 -dependent inhibition of psoriasis development in animal models of psoriasis.
• IL-23 pathway which may be used to treat and prevent IL-23-asociated diseases, including those associated with autoimmune inflammation in the intestinal tract.
• compounds and methods for specific targeting of IL-23R from the luminal side of the gut may provide therapeutic benefit to IBD patients suffering from local inflammation of the intestinal tissue.
• novel peptide inhibitors that bind IL-23R to inhibit IL-23 binding and signaling and which are suitable for oral administration.
• the present invention provides inter alia novel peptide inhibitors of IL-23R and related methods of use.
• the present invention provides a peptide inhibitor of an interleukin-23 receptor, or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide inhibitor comprises or consists of an amino acid sequence of Formula (I):
• X7 is unsubstituted Trp, or Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy;
• X8 is Gln, alpha-Me-Lys, alpha-MeLeu, alpha-MeLys(Ac), beta-homoGln, Cit, Glu, Phe, Asn, Thr, Val, Aib, alpha-MeGln, alpha-MeAsn, Lys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), 1- Nal, 2-Nal, or Trp;
• X9 is Abu, Cys, (D)Cys, alpha-MeCys, (D)Pen, or Pen;
• XI 0 is unsubstituted Phe, or Phe substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carboxamido, 2-aminoethoxy, or 2-acetylaminoethoxy; and
• X7 and XI 1 are Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy;
• X4 is Abu, Cys, (D)Cys, alpha-MeCys, (D)Pen, Pen, or Pen(sulfoxide);
• X5 is Cit, Glu, Gly, Lys, Asn, Pro, alpha-MeGln, alpha-MeLys, alpha-MeLeu, alpha-MeAsn, Lys(Ac), alpha-MeLys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), Gln, Asp, or Cys;
• X6 is Thr, Aib, Asp, Dab, Gly, Pro, Ser, alpha-MeGln, alpha-MeLys, alpha-MeLeu, alpha- MeAsn, alpha-MeThr, alpha-MeSer, or Val;
• X7 is unsubstituted Trp, or Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy;
• X8 is Gln, alpha-Me-Lys, alpha-MeLeu, alpha-MeLys(Ac), beta-homoGln, Cit, Glu, Phe, Asn, Thr, Val, Aib, alpha-MeGln, alpha-MeAsn, Lys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), 1- Nal, 2-Nal, or Trp;
• X9 is Abu, Cys, (D)Cys, alpha-MeCys, (D)Pen, Pen, or Pen(sulfoxide); wherein if X4 is Abu then X9 is Cys, (D)Cys, alpha-MeCys, (D)Pen, Pen, or Pen(sulfoxide); and wherein if X9 is Abu, then X4 is Cys, (D)Cys, alpha-MeCys, (D)Pen, Pen, or Pen(sulfoxide); XI 0 is unsubstituted Phe, or Phe substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carboxamido, 2-aminoethoxy, or 2-acetylaminoethoxy; and
• XI 1 is 2-Nal, unsubstituted Trp, or Trp substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, Phe(2-Me), Phe(3-Me), Phe(4-Me), Phe(3,4-dimethoxy), or l-Nal;
• X7 and XI 1 are Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy;
• IL-2 interleukin-23
• the present invention provides a peptide inhibitor of an interleukin- 23 receptor, or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide inhibitor comprises or consists of an amino acid sequence of Formula (I):
• X8 is Gln, alpha-Me-Lys, alpha-MeLeu, alpha-MeLys(Ac), beta-homoGln, Cit, Glu, Phe, Asn, Thr, Val, Aib, alpha-MeGln, alpha-MeAsn, Lys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), 1- Nal, 2-Nal, or Trp;
• X9 is Abu, Cys, (D)Cys, alpha-MeCys, (D)Pen, or Pen;
• XI 0 is unsubstituted Phe, or Phe substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carboxamido, 2-aminoethoxy, or 2-acetylaminoethoxy; and
• XI 1 is 2-Nal, unsubstituted Trp, or Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, alkoxy, Phe(2-Me), Phe(3-Me), Phe(4-Me), Phe(3,4-dimethoxy), or l-Nal; and
• X7 is other than 5-F substituted Trp
• IL-2 interleukin-23
• the present invention provides a peptide inhibitor of an interleukin- 23 receptor, or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide inhibitor comprises or consists of an amino acid sequence of Formula (II):
• X4 is Abu, Cys, (D)Cys, alpha-MeCys, (D)Pen, Pen, or Pen(sulfoxide);
• X5 is Cit, Glu, Gly, Leu, Ile, beta- Ala, Ala, Lys, Asn, Pro, alpha-MeGln, alpha-MeLys, alpha- MeLeu, alpha-MeAsn, Lys(Ac), alpha-MeLys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), Gln, Asp, or Cys;
• X6 is Thr, Aib, Asp, Dab, Gly, Pro, Ser, alpha-MeGln, alpha-MeLys, alpha-MeLeu, alpha- MeAsn, alpha-MeThr, alpha-MeSer, or Val;
• X7 is unsubstituted Trp, or Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy;
• X8 is Gln, alpha-Me-Lys, alpha-MeLeu, alpha-MeLys(Ac), beta-homoGln, Cit, Glu, Phe, Asn, Thr, Val, Aib, alpha-MeGln, alpha-MeAsn, Lys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), 1- Nal, 2-Nal, or Trp;
• X9 is Abu, Cys, (D)Cys, alpha-MeCys, (D)Pen, Pen, or Pen(sulfoxide); wherein if X4 is Abu then X9 is Cys, (D)Cys, alpha-MeCys, (D)Pen, Pen, or Pen(sulfoxide); and wherein if X9 is Abu, then X4 is Cys, (D)Cys, alpha-MeCys, (D)Pen, Pen, or Pen(sulfoxide);
• XI 0 is unsubstituted Phe, or Phe substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carboxamido, 2-aminoethoxy, or 2-acetylaminoethoxy; and XI 1 is 2-Nal, unsubstituted Trp, or Trp substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, Phe(2-Me), Phe(3-Me), Phe(4-Me), Phe(3,4-dimethoxy), or l-Nal;
• X7 and XI 1 are Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy;
• X7 is other than 5-F substituted Trp
• IL-2 interleukin-23
• X5 is Cit, Glu, Gly, Lys, Asn, Pro, alpha-MeGln, alpha- MeLys, alpha-MeLeu, alpha-MeAsn, Lys(Ac), alpha-MeLys(Ac), Dab(Ac), Dap(Ac), homo- Lys(Ac), Gln, Asp, or Cys.
• X5 is Cit, Glu, Gly, Leu, Ile, beta-Ala, Ala, Lys, Asn, Pro, alpha-MeGln, alpha-MeLys, alpha-MeLeu, alpha-MeAsn, Lys(Ac), alpha- MeLys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), Gln, Asp, or Cys.
• X8 is Gln, alpha-Me-Lys, alpha-MeLeu, alpha-MeLys(Ac), beta-homoGln, Cit, Glu, Phe, Asn, Thr, Val, Aib, alpha-MeGln, alpha-MeAsn, Lys(Ac), alpha- MeLys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), l-Nal, 2-Nal, or Trp.
• X8 is Gln, alpha-Me-Lys, alpha-MeLeu, alpha-MeLys(Ac), beta-homoGln, Cit, Glu, Phe, Asn, Thr, Val, Aib, alpha-MeGln, alpha-MeAsn, Lys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), 1- Nal, 2-Nal, or Trp.
• X8 is Gln, alpha-Me-Lys, alpha-MeLeu, alpha-MeLys(Ac), beta-homoGln, Cit, Glu, Phe, Asn, Thr, Val, Aib, alpha-MeGln, alpha-MeAsn, Dab(Ac), Dap(Ac), homo-Lys(Ac), l-Nal, 2-Nal, Trp, or Lys(R’); and wherein R’ is Aib, bAla, IV A, Ala, cyclohexanoic, octanoic, -C(0)CH2Ph (-C(O)benzyl), trifluorpropionic, Gly, acetyl, valeric, or trifluoroacetyl.
• X8 is Asn, alpha-Me-Lys, alpha-MeLeu, Aib, Cit, or Lys(R’). In certain embodiments, X8 is Lys(R’). In one embodiment, R’ is acetyl. In another embodiment R’ is Gly, Aib, Ala, or bAla. In another embodiment, R’ is Gly, or Aib.
• X4 is Abu and X9 is Cys, (D)Cys, alpha-MeCys, (D)Pen, or Pen.
• X4 is Cys, (D)Cys, alpha-MeCys, (D)Pen, or Pen; and X9 is Abu.
• each X4 and X9 is independently Cys, (D)Cys, alpha-MeCys, (D)Pen, or Pen.
• each X4 and X9 is Cys, (D)Cys, alpha-MeCys, (D)Pen, or Pen.
• the terms“patient,”“subject,” and“individual” may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).
• mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).
• sequence similarity or sequence identity between sequences can be performed as follows.
• the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
• the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
• amino groups in the compounds of the present invention can be quatemized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides.
• acids which can be employed to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric.
• a pharmaceutically acceptable salt may suitably be a salt chosen, e.g., among acid addition salts and basic salts.
• acid addition salts include chloride salts, citrate salts and acetate salts.
• basic salts include salts where the cation is selected among alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, as well as substituted ammonium ions, such as ions of the type N(Rl)(R2)(R3)(R4)+, where Rl, R2, R3 and R4 independently will typically designate hydrogen, optionally substituted Cl -6-alkyl or optionally substituted C2-6-alkenyl.
• suitable base salts are formed from bases which form non-toxic salts.
• bases include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts.
• Hemisalts of acids and bases may also be formed, e.g., hemisulphate and hemicalcium salts.
• alkyl includes a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.
• Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, «-propyl, «-butyl, «-pentyl, «-hexyl, and the like, while saturated branched alkyls include, without limitation, isopropyl, vec-butyl. isobutyl, tert- butyl, isopentyl, and the like.
• saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, while unsaturated cyclic alkyls include, without limitation, cyclopentenyl, cyclohexenyl, and the like.
• haloalkyl includes alkyl structures in which at least one hydrogen is replaced with a halogen atom. In certain embodiments in which two or more hydrogen atoms are replaced with halogen atoms, the halogen atoms are all the same as one another. In other embodiments in which two or more hydrogen atoms are replaced with halogen atoms, the halogen atoms are not all the same as one another.
• An“aryloxy” group refers to an (aryl)O- group, where aryl is as defined herein.
• a“therapeutically effective amount” of the peptide inhibitor of the invention is meant to describe a sufficient amount of the peptide inhibitor to treat an IL-23/IL- 23R-related disease, including but not limited to any of the diseases and disorders described herein (for example, to reduce inflammation associated with IBD).
• the therapeutically effective amount will achieve a desired benefit/risk ratio applicable to any medical treatment.
• An“analog” of an amino acid e.g., a“Phe analog” or a“Tyr analog” means an analog of the referenced amino acid.
• a variety of amino acid analogs are known and available in the art, including Phe and Tyr analogs.
• an amino acid analog, e.g., a Phe analog or a Tyr analog comprises one, two, three, four or five substitutions as compared to Phe or Tyr, respectively.
• the substitutions are present in the side chains of the amino acids.
• Phe analogs include, but are not limited to: hPhe, Phe(4-OMe), a-Me-Phe, hPhe(3,4-dimethoxy), Phe(4-CONH2), Phe(4-phenoxy), Phe(4-guanadino), Phe(4-tBu), Phe(4-CN), Phe(4-Br), Phe(4-OBzl), Phe(4- Mf 2 ), BhPhe(4-F), Phe(4-F), Phe(3,5 DiF), Phe(CH 2 C02H), Phe(penta-F), Phe(3,4-Ch), Phe (3,4-F2), Phe(4-CF3), bb-diPheAla, Phe(4-N 3 ), Phe[4-(2-aminoethoxy)], 4-
• the present invention relates generally to peptides that have IL-23R antagonist activity, including both peptide monomers and peptide dimers.
• this invention demonstrates a new paradigm for treatment of IBD and other diseases and disorders by oral delivery of antagonists of IL-23.
• IBD represents a local inflammation of the intestinal tissue; therefore, advantageous therapeutic agents act from the luminal side of the intestine, yielding high drug concentrations in diseased tissue, minimizing systemic availability and resulting in improved efficacy and safety when compared to systemic approaches.
• the present invention relates to various peptides, or peptide dimers comprising hetero- or homo-monomer subunits, that form cyclized structures through disulfide or other bonds.
• the disulfide or other bonds are intramolecular bonds.
• the cyclized structure of the peptide monomer inhibitors and the monomer subunits of the peptide dimer inhibitors has been shown to increase potency and selectivity of the peptide inhibitors.
• a peptide dimer inhibitor may include one or more intermolecular bonds linking the two monomer peptide subunits within the peptide dimer inhibitor, e.g., an intermolecular bridge between two Pen residues, one in each peptide monomer subunit.
• the present invention provides peptide inhibitors that bind to IL-23R, which may be monomers or dimers.
• the peptide inhibitors inhibit the binding of IL-23 to IL-23R.
• the IL-23R is human IL-23R
• the IL-23 is human IL-23.
• a peptide inhibitor of the present invention reduces IL-23 binding to IL-23R by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to a negative control peptide.
• Methods of determining binding are known in the art and include ELISA assays, as described in the accompanying Examples.
• a peptide inhibitor of the present invention has an IC50 of > 1 mM, ⁇ 1 mM, 500 nM to 1000 nM, ⁇ 500 nM, ⁇ 250 nM, ⁇ 100 nM, ⁇ 50 nM, ⁇ 25 nM, ⁇ 10 nM, ⁇ 5 nM, ⁇ 2 nM, ⁇ 1 nM, or ⁇ 5 mM, e.g., for inhibiting binding of IL-23 to IL-23R (e.g., human IL-23 and human IL-23R).
• Methods of determining activity are known in the art and include any of those described in the accompanying Examples.
• a peptide inhibitor of the present invention has increased stability, increased gastrointestinal stability, or increased stability in stimulated intestinal fluid (SIF) or simulated gastric fluid (SGF), and/or under redox conditions (e.g., DTT) as compared to a control peptide.
• a control peptide is an unrelated peptide of the same or similar length.
• a control peptide is a peptide having the identical or a highly related amino acid sequence (e.g., > 90% sequence identity) as the peptide inhibitor.
• a control peptide is a peptide having the identical or a highly related amino acid sequence (e.g., > 90% sequence identity) as the peptide inhibitor, but which does not have a cyclized structure, e.g., through an intramolecular bond between two amino acid residues within the control peptide, or which is not dimerized, or which does not comprise a conjugate for stabilization.
• a highly related amino acid sequence e.g., > 90% sequence identity
• the only difference between the peptide inhibitor and the control peptide is that the peptide inhibitor comprises one or more amino acid substitutions that introduce one or more amino acid residues into the peptide inhibitor, wherein the introduced amino residue(s) forms an intrasulfide disulfide or thioether bond with another amino acid residue in the peptide inhibitor.
• a control for a peptide dimer inhibitor is a monomer having the same sequence as one of the monomer subunits present in the peptide dimer inhibitor.
• a control for a peptide inhibitor comprising a conjugate is a peptide having the same sequence but not including the conjugated moiety.
• a control peptide is a peptide (e.g., a naturally-occurring peptide) corresponding to a region of IL-23 that binds to IL-23R.
• the stability of a peptide inhibitor is determined using an SIF assay, e.g., as described in Example 3. In certain embodiments, the stability of a peptide inhibitor is determined using an SGF assay, e.g., as described in Example 3.
• a peptide inhibitor has a half-life (e.g., in SIF or SGF or DTT) under a given set of conditions (e.g., temperature) of greater than 1 minute, greater than 10 minutes, greater than 20 minutes, greater than 30 minutes, greater than 60 minutes, greater than 90 minutes, greater than 120 minutes, greater than 3 hours, or greater than four hours when exposed to SIF or SGF or DTT.
• the temperature is about 25 °C, about 4 °C, or about 37 °C
• the pH is a physiological pH, or a pH about 7.4.
• the half-life is measured in vitro using any suitable method known in the art, e.g., in some embodiments, the stability of a peptide of the present invention is determined by incubating the peptide with pre-warmed human serum (Sigma) at 37 0 C. Samples are taken at various time points, typically up to 24 hours, and the stability of the sample is analyzed by separating the peptide or peptide dimer from the serum proteins and then analyzing for the presence of the peptide or peptide dimer of interest using LC-MS.
• pre-warmed human serum Sigma
• a peptide inhibitor of the present invention exhibits improved solubility or improved aggregation characteristics as compared to a control peptide.
• Solubility may be determined via any suitable method known in the art.
• suitable methods known in the art for determining solubility include incubating peptides in various buffers (Acetate pH4.0, Acetate pH5.0, Phos/Citrate pH5.0, Phos Citrate pH6.0, Phos pH 6.0, Phos pH 7.0, Phos pH7.5, Strong PBS pH 7.5, Tris pH7.5, Tris pH 8.0, Glycine pH 9.0, Water, Acetic acid (pH 5.0 and other known in the art) and testing for aggregation or solubility using standard techniques.
• improved solubility means the peptide is more soluble in a given liquid than is a control peptide.
• improved aggregation means the peptide has less aggregation in a given liquid under a given set of conditions than a control peptide.
• the peptide inhibitors are stable to various pHs that range from strongly acidic in the stomach (pH 1.5 -1.9), trending towards basic in the small intestine (pH 6-7.5), and then weakly acidic in the colon (pH 5-7). Such peptide inhibitors are stable during their transit through the various GI compartments, a process that has been estimated to take 3-4 h in the intestine and 6-48 h in the colon.
• the peptide inhibitors of the present invention have less degradation, e.g., over a period of time (i.e., more degradation stability), e.g., greater than or about 10% less, greater than or about 20% less, greater than or about 30% less, greater than or about 40 less, or greater than or about 50% less degradation than a control peptide.
• degradation stability is determined via any suitable method known in the art.
• the degradation is enzymatic degradation.
• the peptide inhibitors have reduced susceptibility to degradation by trypsin, chhrmotrypsin or elastase.
• suitable methods known in the art for determining degradation stability include the method described in Hawe et al, J Pharm Sci, VOL. 101, No. 3, 2012, p 895-913, incorporated herein in its entirety. Such methods are in some embodiments used to select potent peptide sequences with enhanced shelf lifes.
• peptide stability is determined using a SIF assay or SGF assay, e.g., as described in PCT Publication No. WO 2016/011208.
• peptide inhibitors have increased redox stability as compared to a control peptide.
• assays that may be used to determine redox stability are known and available in the art. Any of these may be used to determine the redox stability of peptide inhibitors of the present invention.
• the peptide inhibitors inhibit rat IL-23R at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% as well as they bind or inhibit human IL-23R, e.g., as determined by an assay described herein.
• the peptide inhibitors preferentially bind and/or inhibit human and/or rat IL-23R as compared to mouse IL-23R.
• the peptide inhibitors preferentially bind to rat IL-23R as compared to mouse IL-23R.
• the peptide inhibitors preferentially bind to human IL-23R as compared to mouse IL-23R.
• the additional amino acids present in the mouse IL-23R are in the region corresponding to about amino acid residue 315 to about amino acid residue 340 of the mouse IL23R protein, e g., amino acid region NWQPWSSPFVHQTSQETGKR (SEQ ID NO: 106).
• the peptide inhibitors bind to a region of human IL-23R from about amino acid 230 to about amino acid residue 370.
• peptide inhibitors show Gl-restricted localization following oral administration.
• greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of orally administered peptide inhibitor is localized to gastrointestinal organs and tissues.
• blood plasma levels of orally administered peptide inhibitor are less than 20%, less than 10%, less than 5%, less than 2%, less than 1% or less than 0.5% the levels of peptide inhibitor found in the small intestine mucosa, colon mucosa, or proximal colon.
• the various peptide inhibitors of the invention may be constructed solely of natural amino acids.
• the peptide inhibitors may include non-natural amino acids including, but not limited to, modified amino acids.
• modified amino acids include natural amino acids that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid.
• the peptide inhibitors of the invention may additionally include one or more D-amino acids.
• the peptide inhibitors of the invention may include amino acid analogs.
• peptide inhibitors of the present invention include one or more modified or unnatural amino acids.
• a peptide inhibitor includes one or more non-natural amino acids shown in Table 1A.
• peptide inhibitors of the present invention include any of those described herein, including but not limited to any of those comprising an amino acid sequence or peptide inhibitor structure shown in any one of the tables herein.
• the present invention also includes any of the peptide inhibitors described herein in either a free or a salt form.
• embodiments of any of the peptide inhibitors described herein (and related methods of use thereof) include a pharmaceutically acceptable salt of the peptide inhibitor.
• the present invention also includes variants of any of the peptide inhibitors described herein, including but not limited to any of those comprising a sequence shown in any one of the tables herein, wherein one or more L-amino acid residue is substituted with the D isomeric form of the amino acid residue, e.g., an L-Ala is substituted with a D-Ala.
• Peptide inhibitors described herein include isotopically-labeled peptide inhibitors.
• the present disclosure provides peptide inhibitors identical to any of those having or recited in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
• isotopes that can be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as 2 H, 3 ⁇ 4, 13 C, 14 C, 15 N, 18 0, 17 0, 35 S, 18 F, 36 Cl, respectively.
• isotopically-labeled compounds described herein for example those into which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Furthermore, substitution with isotopes such as deuterium, i.e., 2 H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.
• the present invention also includes any of the peptide monomer inhibitors described herein linked to a linker moiety, including any of the specific linker moieties described herein.
• a linker is attached to an N-terminal or C-terminal amino acid, while in other embodiments, a linker is attached to an internal amino acid.
• a linker is attached to two internal amino acids, e.g., an internal amino acid in each of two monomer subunits that form a dimer.
• a peptide inhibitor is attached to one or more linker moieties shown.
• the present invention also includes peptides and peptide dimers comprising a peptide having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the peptide sequence of a peptide inhibitor described herein.
• peptide inhibitors of the present invention comprise a core peptide sequence and one or more N-terminal and/or C-terminal modification (e.g., Ac and NFh) and/or one or more conjugated linker moiety and/or half-life extension moiety.
• the core peptide sequence is the amino acid sequence of the peptide absent such modifications and conjugates.
• a peptide inhibitor or a monomer subunit of a peptide inhibitor of the present invention comprises, consists essentially of, or consists of 7 to 35 amino acid residues, 8 to 35 amino acid residues, 9 to 35 amino acid residues, 10 to 35 amino acid residues,
• a peptide inhibitor of the present invention is greater than 10, greater than 12, greater than 15, greater than 20, greater than 25, greater than 30 or greater than 35 amino acids, e.g., 35 to 50 amino acids. In certain embodiments, a peptide inhibitor (or a monomer subunit thereof) is less than 50, less than 35, less than 30, less than 25, less than 20, less than 15, less than 12, or less than 10 amino acids.
• a monomer subunit of a peptide inhibitor comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid residues.
• a monomer subunit of a peptide inhibitor of the present invention comprises or consists of 10 to 23 amino acid residues and, optionally, one or more additional non-amino acid moieties, such as a conjugated chemical moiety, e.g., a PEG or linker moiety.
• the monomer subunit comprises or consists of 7 to 35 amino acid residues, 7 to 20 amino acid residues, 8 to 20 amino acid residues, 9 to 20 amino acid residues, 10 to 20 amino acid residues,
• X comprises or consists of 7 to 35 amino acid residues, 8 to 35 amino acid residues, 9 to 35 amino acid residues, 10 to 35 amino acid residues, 7 to 25 amino acid residues, 8 to 25 amino acid residues, 9 to 25 amino acid residues, 10 to 25 amino acid residues, 7 to 18 amino acid residues, 8 to 18 amino acid residues, 9 to 18 amino acid residues, or 10 to 18 amino acid residues.
• Certain illustrative peptide inhibitors described herein comprise 12 or more amino acid residues.
• the present invention also includes peptide inhibitors comprising a fragment of any of the peptide sequences described herein, including peptide inhibitors having 7, 8, 9, 10, or 11 amino acid residues.
• peptide inhibitors of the present invention include peptides comprising or consisting of X4-X9, X4-X10, X4-X11, X4-X12, X4-X13, X4- X14, or X4-X15.
• amino acid sequences of the peptide inhibitors are not present within an antibody, or are not present within a VH or VL region of an antibody.
• the present invention includes a peptide inhibitor of an interleukin-23 receptor, or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide inhibitor comprises an amino acid sequence of Formula (I):
• X7 is unsubstituted Trp, or Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy;
• X8 is Gln, alpha-Me-Lys, alpha-MeLeu, alpha-MeLys(Ac), beta-homoGln, Cit, Glu, Phe, Asn, Thr, Val, Aib, alpha-MeGln, alpha-MeAsn, Lys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), 1- Nal, 2-Nal, or Trp;
• X9 is Abu, Cys, (D)Cys, alpha-MeCys, (D)Pen, or Pen;
• XI 0 is unsubstituted Phe, or Phe substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carboxamido, 2-aminoethoxy, or 2-acetylaminoethoxy; and
• XI 1 is 2-Nal, unsubstituted Trp, or Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, alkoxy, Phe(2-Me), Phe(3-Me), Phe(4-Me), Phe(3,4-dimethoxy), or l-Nal;
• i) at least one of X7 and XI 1 is Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy;
• the present invention includes a peptide inhibitor of an interleukin-23 receptor, or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide inhibitor comprises an amino acid sequence of Formula (II):
• X7 is unsubstituted Trp, or Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy;
• XI 0 is unsubstituted Phe, or Phe substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carboxamido, 2-aminoethoxy, or 2-acetylaminoethoxy; and
• X7 and XI 1 are Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy; ii) when X7 is 5-F substituted Trp, then XI 1 is other than 2-Nal; and
• the peptide inhibitor inhibits the binding of an interleukin-23 (IL-23) to an IL- 23 receptor.
• IL-23 interleukin-23
• X7 is 5-F substituted Trp
• XI 1 is not 2-Nal.
• X7 is 5-F substituted Trp
• XI 1 is unsubstituted Trp, or Trp substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, Phe(2-Me), Phe(3-Me), Phe(4- Me), Phe(3,4-dimethoxy), or l-Nal.
• X4 or X9 is Cys, (D)Cys, alpha-MeCys, (D)Pen, or Pen; and the bond between X4 and X9 is a disulfide bond.
• X4 is Cys, (D)Cys, or alpha-MeCys.
• X4 is (D)Pen, or Pen; or X4 is Pen(sulfoxide).
• X4 is Pen.
• X9 is Cys, (D)Cys, or alpha-MeCys.
• X9 is Pen or (D)Pen.
• X4 is Pen and X9 is Cys, and the bond is a disulfide bond.
• X4 is Abu and X9 is Cys, and the bond is a thioether bond.
• the peptide inhibitor is according to Formula (Ila) (lib), or (lie) or comprises a sequence of Formula (Ila) (lib), or (lie):
• X5 is Asn, Gln, or Glu.
• X5 is Asn, or Gln.
• X5 is Asn.
• X6-X8 and X10-X11 are as described for Formula (II); and the peptide inhibitor is cyclized via a Pen-Pen disulfide bond; or the peptide inhibitor is cyclized via a Abu-Cys or Abu-Pen thioether bond.
• X6 is Thr.
• the peptide inhibitor is according to Formula (IV a), (IVb), (IV c), or (IV d) or comprises a sequence of Formula (IV a), (IVb), (IV c), or (IV d):
• X8 is Gln. In certain embodiment, X8 is Cit.
• X7-X8 and X10-X11 are as described for Formula (II); and the peptide inhibitor is cyclized via a Pen-Pen disulfide bond; or the peptide inhibitor is cyclized via a Abu-Cys or Abu-Pen thioether bond.
• XI 0 is Phe, Phe[4-(2-aminoethoxy)] [F(4-2ae)], Phe[4-(2- acetylaminoethoxy)], or Phe(4-CONH2).
• X10 is Phe[4-(2-aminoethoxy)], or Phe[4-(2- acetylaminoethoxy)] .
• XI 5 is Asn, Leu, Aib, (D)Leu, beta- Ala, Cit, Gln, Asp, alpha-MeGln, alpha-MeAsn, Lys(Ac), (D)Lys, alpha-MeLys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), or absent; and the peptide inhibitor is cyclized via a Pen-Pen disulfide bond; or the peptide inhibitor is cyclized via a Abu-Cys or Abu-Pen thioether bond.
• each X12, X13, X14, or X15 is independently any amino acid.
• the amino acid is a natural amino acid.
• the amino acid is an unnatural amino acid.
• XI 4 is Asn, 2-Nap, Aib, Arg, Cit, Asp, Phe, Gly, Lys, Leu, Ala, (D)Ala, beta-Ala, His, Thr, n-Leu, Gln, Ser, (D)Ser, Tic, Trp, alpha-MeGln, alpha-MeAsn, alpha-MeLys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), or Lys(Ac).
• XI 5 is Asn, Aibminister beta- Ala, Cit, Gln, Asp, alpha-MeGln, alpha-MeAsn, Lys(Ac), alpha-MeLys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), or absent.
• XI 5 is Asn, Leu, Aib, (D)Leu, beta- Ala, Cit, Gln, Asp, alpha-MeGln, alpha-MeAsn, Lys(Ac), (D)Lys, alpha-MeLys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), or absent.
• X13 is Glu, Cit, Gln, Lys(Ac), alpha-MeArg, alpha-MeGlu, alpha-MeLeu, alpha-MeLys, alpha-Me-Asn, alpha-MeLys(Ac), Dab(Ac), Dap(Ac), homo- Lys(Ac), or Lys; or X13 is Lys, pegylated Lys, b-homoGlu, or Lys(Y2-Ac), wherein Y2 is an amino acid.
• XI 3 is Aib, Glu, Cit, Gln, Lys(Ac), alpha-MeArg, alpha- MeGlu, alpha-MeLeu, alpha-MeLys, alpha-Me-Asn, alpha-MeLys(Ac), Dab(Ac), Dap(Ac), homo-Lys(Ac), or Lys; or XI 3 is Lys, pegylated Lys, b-homoGlu, or Lys(Y2-Ac), wherein Y2 is an amino acid.
• X12 is 4-amino-4-carboxy-tetrahydropyran (THP), alpha- MeLys, alpha-MeLeu, Ala, cyclohexylAla, Lys, or Aib.
• X12 is 4-amino-4-carboxy-tetrahydropyran (THP), alpha- MeLys, or alpha-MeLeu.
• X12 is alpha-MeLeu. In certain embodiment, X12 is alpha- MeLys. In certain embodiment, X12 is 4-amino-4-carboxy-tetrahydropyran (THP).
• X13 is Glu, Gln, Lys(Ac), or Lys.
• X13 is Gln, Lys(Ac), or Lys.
• X13 is Lys(Ac), or Lys.
• XI 3 is Lys(Ac).
• the peptide inhibitor is according to Formula (Villa), (VUIb) or (VIII c) or comprises a sequence of Formula (Villa), (VUIb) or (VIIIc):
• XI 5 is Asn.
• the peptide inhibitor is according to Formula (IXa), (IXb), or (IXc) or comprises a sequence of Formula (IXa), (IXb), or (IXc):
• X7 is Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy; and XI 1 is as described herein.
• XI 1 is 2-Nal, Phe(2-Me), Phe(3-Me), Phe(4-Me), Phe(3,4- dimethoxy), or l-Nal.
• XI 1 is 2-Nal.
• X7 is unsubstituted Trp.
• the peptide inhibitor is according to Formula (XIa), (Xlb), (XIc), (Xld), (Xle), or (Xlf) or comprises a sequence of Formula (XIa), (Xlb), (XIc), (Xld), (Xle), or (Xlf):
• W’ is Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy; and the peptide inhibitor is cyclized via a Pen-Pen disulfide bond.
• W’ is Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy. In one embodiment, W’ is Trp substituted with alkyl. In certain embodiments, W’ is Trp substituted with Me, Et, n-Pr, or i-Pr.
• the peptide inhibitor comprises the structure of Formula (Z):
• R 1 is a bond, hydrogen, a C1-C6 alkyl, a C6-C12 aryl, a C6-C12 aryl-Cl-C6 alkyl, a C1-C20 alkanoyl, and including PEGylated versions alone or as spacers of any of the foregoing;
• X is the amino acid sequence of Formula (I), Formula (Il)-(XIf), or an amino acid sequence set forth in any of Table El, E2 or E3; and R 2 is OH or NH2.
• R 1 is H or C1-C20 alkanoyl.
• R 1 is H or Ac.
• R 1 is Ac.
• W’ is Trp substituted with cyano, halo, alkyl, haloalkyl, hydroxy, or alkoxy; and the substitution is at 4-, 5-, 6- or 7- position.
• W’ is Trp substituted with cyano, F, Cl, Br, I, Me, Et, i-Pr, n- Pr, n-Bu, t-Bu, CF3, hydroxy, OMe, or OEt; and the substitution is at 4-, 5-, 6- or 7- position. In a particular embodiment, the substitution is at 7- position.
• W’ is Trp substituted with 5- F, 6-F, 7-F, 5- Cl, 6-C1, 7-C1, 5- Me, 6-Me, 7-Me, 7-n-Pr, 7-i-Pr, 5-OH, 6-OH, 7-OH, 5-OMe, 6-OMe, or 7-OMe.
• W’ is Trp substituted with 7-Me, 5-F, 7-F, 6-C1, 6-Me, 4- OMe, 5-OMe, or 5-Br.
• W’ is Trp substituted with 7-Me, 6- Me, 4-OMe, or 6-C1.
• W’ is Trp substituted with 7-Me.
• the peptide inhibitor consists of about or less than 25 amino acids. In another embodiment the peptide inhibitor consists of about or less than 20 amino acids. In another embodiment the peptide inhibitor consists of about or less than 18 amino acids. In another embodiment the peptide inhibitor consists of about or less than 15 amino acids. In another embodiment the peptide inhibitor consists of about or less than 12 amino acids. In another embodiment the peptide inhibitor consists of about or less than 10 amino acids.
• the peptide comprises:
• the peptide comprises: -[Pen]-NTW-Gln-[Pen]-[Phe[4-(2-aminoethoxy)]-W(5-Cl)-[a-MeLeu]-[Lys(Ac)]-NN-(SEQ ID NO: 12);
• the peptide comprises or is:
• the peptide comprises:
• the peptide comprises:
• the peptide comprises:
• the peptide comprises or is:
• peptide inhibitor is cyclized via a Pen-Pen disulfide bond; or a pharmaceutically acceptable salt or solvate thereof.
• the peptide comprises or is: Ac-[Abu]-NTW(6-Cl)-Gln-Cys-[Phe[4-(2-aminoethoxy)]-[2-Nal]-[a-MeLeu]-[Lys(Ac)]-NN- NH 2 (SEQ ID NO: 107);
• the peptide comprises or is:
• the peptide comprises or is:
• the peptide comprises or is:
• the peptide comprises or is:
• the peptide comprises or is:
• the peptide comprises or is:
• the peptide comprises or is:
• the peptide comprises or is:
• the peptide inhibitor comprises or consists of:
• any of the peptide inhibitors of the present invention may be further defined, e.g., as described below. It is understood that each of the further defining features described herein may be applied to any peptide inhibitors where the amino acids designated at particular positions allow the presence of the further defining feature. In particular embodiments, these features may be present in any of the peptides of Formula (I)-(XIf).
• the peptide inhibitor comprises an N-terminus selected from hydrogen, a C1-C6 alkyl, a C6-C12 aryl, a C6-C12 aryl C1-C6 alkyl, or a C1-C20 alkanoyl, and including PEGylated versions alone or as spacers of any of the foregoing, e.g., acetyl.
• R 1 or the N-terminal moiety is hydrogen.
• R 1 is a bond, e.g., a covalent bond.
• R 1 or the N-terminal moiety is selected from methyl, acetyl, formyl, benzoyl, trifluoroacetyl, isovaleryl, isobutyryl, octanyl, and the conjugated amides of lauric acid, hexadecanoic acid, and g-Glu-hexadecanoic acid.
• R 1 or the N- terminal moiety is pGlu.
• R 1 is hydrogen.
• R 1 is acetyl, whereby the peptide inhibitor is acylated at its N-terminus, e.g., to cap or protect an N-terminal amino acid residue, e.g., an N-terminal Pen residue.
• R 1 or the N-terminal moiety is an alkylsulfonic acid selected from methanesulfonic acid, ethanesulfonic acid, l,2-ethane-disulfonic acid, and 2- hydroxyethanesulfonic acid.
• R 1 or the N-terminal moiety is an arylsulfonic acid selected from benzenesulfonic acid, 4- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4- toluenesulfonic acid, and camphorsulfonic acid.
• a peptide dimer inhibitor of the present invention may comprise two identical monomer subunits, resulting in a homodimer, or two non-identical monomer subunits, resulting in a heterodimer.
• a cysteine dimer comprises two peptide monomer subunits linked through a disulfide bond between a cysteine residue in one monomer subunit and a cysteine residue in the other monomer subunit.
• the peptide inhibitors of the present invention may be active in a dimer conformation, in particular when free cysteine residues are present in the peptide. In certain embodiments, this occurs either as a synthesized dimer or, in particular, when a free cysteine monomer peptide is present and under oxidizing conditions, dimerizes. In some embodiments, the dimer is a homodimer. In other embodiments, the dimer is a heterodimer.
• monomer subunits of the present invention may be dimerized by a suitable linking moiety, e.g., a disulphide bridge between two cysteine residues, one in each peptide monomer subunit, or by another suitable linker moiety, including but not limited to those defined herein.
• a suitable linking moiety e.g., a disulphide bridge between two cysteine residues, one in each peptide monomer subunit, or by another suitable linker moiety, including but not limited to those defined herein.
• Some of the monomer subunits are shown having C- and N-termini that both comprise free amine.
• the monomer subunit may be modified to eliminate either the C- or N-terminal free amine, thereby permitting dimerization at the remaining free amine.
• the C-terminal residues of the monomer subunits disclosed herein are optionally amides. Further, it is understood that, in certain embodiments, dimerization at the C-terminus is facilitated by using a suitable amino acid with a side chain having amine functionality, as is generally understood in the art. Regarding the N-terminal residues, it is generally understood that dimerization may be achieved through the free amine of the terminal residue, or may be achieved by using a suitable amino acid side chain having a free amine, as is generally understood in the art. PCT/U519/41665 10 October 2019 (10.10.2019)
• linker moieties connecting monomer subunits may incli
• a linker moiety is selected from the non-limiting group consisting of cysteine, lysine, DIG, PEG4, PEG4-biotin, PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, ADA, Boc- IDA, Glutaric acid, Isophthalic acid, l,3-phenylenediacetic acid, l,4-phenylenediacetic acid, l,2-phenylenediacetic acid, Triazine, Boc-Triazine, IDA-biotin, PEG4-Biotin, AADA, suitable aliphatics, aromatics, heteroaromatics, and polyethylene glycol based linkers having a molecular weight from approximately 400Da to approximately 40,000Da.
• PEG2 is HO2CCH2CH2OCH2CH2OCH2
• linker e.g., C- and N-terminal linker
• the present invention may include any suitable linker moiety.
• some embodiments of the present invention comprises a homo- or heterodimer peptide inhibitor comprised of two monomer subunits selected from the peptides shown in any of tables herein or comprising or consisting of a sequence presented in any of tables herein, wherein the C- or N-termini of the respective monomer subunits (or internal amino acid residues) are linked by any suitable linker moiety to provide a dimer peptide inhibitor having IL-23R inhibitory activity.
• a linker binds to the N- or C-terminus of one monomer subunit and an internal amino acid residue of the other monomer subunit making up the dimer. In certain embodiments, a linker binds to an internal amino acid residue of one monomer subunit and an internal amino acid residue of the other monomer subunit making up the dimer. In further embodiments, a linker binds to the N-or C-terminus of both subunits.
• one or both of the monomer subunits comprise the sequence or structure of any one of Formula (I)-(XIf), or shown in Table El, Table E2, or Table E3, or any of the peptides described herein.
• each R 2 is independently absent, a bond (e.g., a covalent bond), or selected from OH or NFh: L is a linker moiety; andeach X is an independently selected peptide monomer subunit comprising a sequence of Formula (I)-(XIf), as described herein.
• one or both peptide monomer subunit of a peptide dimer inhibitor is cycbzed, e.g., via an intramolecular bond between X4 and X9.
• one or both peptide monomer subunits is linear or not cycbzed.
• each R 1 is independently a bond (e.g., a covalent bond), or selected from hydrogen, a C1-C6 alkyl, a C6-C12 aryl, a C6-C12 aryl C1-C6 alkyl, a C1-C20 alkanoyl, and including PEGylated versions alone or as spacers of any of the foregoing.
• a bond e.g., a covalent bond
• the N-terminus of each subunit includes a moiety selected from hydrogen, a C1-C6 alkyl, a C6-C12 aryl, a C6-C12 aryl C1-C6 alkyl, a C1-C20 alkanoyl, and including PEGylated versions alone or as spacers of any of the foregoing.
• each R 1 (or N-terminal moiety) is selected from methyl, acetyl, formyl, benzoyl, trifluoroacetyl, isovaleryl, isobutyryl, octanyl, and the conjugated amides of lauric acid, hexadecanoic acid, and g-Glu-hexadecanoic acid.
• each R 2 (or C-terminal moiety) is independently a bond (e.g., a covalent bond), or selected from OH or NH2.
• the linker moiety (L) is any of the linkers described herein or shown in Table 1 or 7.
• L is a lysine linker, a diethylene glycol linker, an iminodiacetic acid (IDA) linker, a b-Ala-iminodiaceticacid (b-Ala-IDA) linker, or a PEG linker.
• each of the peptide monomer subunits is attached to a linker moiety via its N-terminus, C-terminus, or an internal amino acid residue.
• the N- terminus of each peptide monomer subunit is connected by a linker moiety.
• the C-terminus of each peptide monomer subunit is connected by a linker moiety.
• each peptide monomer subunit is connected by a linker moiety attached to an internal amino acid.
• peptide inhibitors of the present invention comprise one or more conjugated chemical substituents, such as lipophilic substituents and polymeric moieties, which may be referred to herein as half-life extension moieties.
• conjugated chemical substituents such as lipophilic substituents and polymeric moieties, which may be referred to herein as half-life extension moieties.
• lipophilic substituent binds to albumin in the bloodstream, thereby shielding the peptide inhibitor from enzymatic degradation, and thus enhancing its half-life.
• polymeric moieties enhance half-life and reduce clearance in the bloodstream.
• any of the peptide inhibitors e.g. peptides of Formula (I)- (Xlf) further comprise a linker moiety attached to an amino acid residue present in the inhibitor, e.g., a linker moiety may be bound to a side chain of any amino acid of the peptide inhibitor, to the N-terminal amino acid of the peptide inhibitor, or to the C-terminal amino acid of the peptide inhibitor.
• any of the peptide inhibitors e.g. peptides of Formulas (I)- (Xlf)
• further comprise half-life extension moiety attached to an amino acid residue present in the inhibitor e.g., a half-life extension moiety may be bound to a side chain of any amino acid of the peptide inhibitor, to the N-terminal amino acid of the peptide inhibitor, or to the C- terminal amino acid of the peptide inhibitor.
• any of the peptide inhibitors e.g. peptides of Formulas (I)- (Xlf)
• a peptide inhibitor of the present invention comprises a half- life extension moiety shown in Table 8.
• a half-life extension moiety is bound directly to a peptide inhibitor, while in other embodiments, a half-life extension moiety is bound to the peptide inhibitor via a linker moiety, e.g., any of those depicted in Tables 1, 2 or 4.
• a peptide inhibitor of the present invention comprises any of the linker moieties shown in Tables 2 or 4 and any of the half-life extension moieties shown in Table 3, including any of the following combinations shown in Table 5.
• linkers present between the peptide the conjugated moiety, e.g., half-life extension moiety, e.g., as depicted in Table 6.
• the half-life of a peptide inhibitor of the invention that includes a conjugated chemical substituent, i.e., a half-life extension moiety is at least 100%, at least 120%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% of the half-life of the same peptide inhibitor but without the conjugated chemical substituent.
• the lipophilic substituents and/or polypermic moieties enhance the permeability of the peptide inhibitor through the epithelium and/or its retention in the lamina limbal.
• the permeability through the epithelium and/or the retention in the lamina intestinal of a peptide inhibitor of the invention that includes a conjugated chemical substituent is at 100%, at least 120%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% of the half-life of the same peptide inhibitor but without the conjugated chemical substituent.
• a side chain of one or more amino acid residues (e.g., Lys residues) in a peptide inhibitor of the invention is conjugated (e.g., covalently attached) to a lipophilic substituent.
• the lipophilic substituent may be covalently bonded to an atom in the amino acid side chain, or alternatively may be conjugated to the amino acid side chain via one or more spacers. The spacer, when present, may provide spacing between the peptide analogue and the lipophilic substituent.
• the peptide inhibitor comprises any of the conjugated moieties shown in peptides disclosed in Tables 2-6.
• the lipophilic substituent may comprise a hydrocarbon chain having from 4 to 30 C atoms, for example at least 8 or 12 C atoms, and preferably 24 C atoms or fewer, or 20 C atoms or fewer.
• the hydrocarbon chain may be linear or branched and may be saturated or unsaturated.
• the hydrocarbon chain is substituted with a moiety which forms part of the attachment to the amino acid side chain or the spacer, for example an acyl group, a sulfonyl group, an N atom, an O atom or an S atom.
• the hydrocarbon chain is substituted with an acyl group, and accordingly the hydrocarbon chain may form part of an alkanoyl group, for example palmitoyl, caproyl, lauroyl, myristoyl or stearoyl.
• a lipophilic substituent may be conjugated to any amino acid side chain in a peptide inhibitor of the invention.
• the amino acid side chain includes a carboxy, hydroxyl, thiol, amide or amine group, for forming an ester, a sulphonyl ester, a thioester, an amide or a sulphonamide with the spacer or lipophilic substituent.
• the lipophilic substituent may be conjugated to Asn, Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys or Dbu, Dpr or Om.
• the lipophilic substituent is conjugated to Lys.
• An amino acid shown as Lys in any of the Formula provided herein may be replaced by, e.g., Dbu, Dpr or Om where a lipophilic substituent is added.
• the peptide inhibitors of the present invention may be modified, e.g., to enhance stability, increase permeability, or enhance drug like characteristics, through conjugation of a chemical moiety to one or more amino acid side chain within the peptide.
• a chemical moiety e.g., the N(epsilon) of lysine N(epsilon), the b-carboxyl of aspartic, or the g-carboxyl of glutamic acid may be appropriately functionalized.
• an amino acid within the peptide may be appropriately modified.
• the side chain is acylated with an acylating organic compound selected from the group consisting of: Trifluoropentyl, Acetyl, Octonyl, Butyl, Pentyl, Hexyl, Palmityl, Trifluoromethyl butyric, cyclopentane carboxylic, cyclopropylacetic, 4-fluorobenzoic, 4- fluorophenyl acetic, 3-Phenylpropionic, tetrahedro-2H-pyran-4carboxylic, succinic acid glutaric acid or bile acids.
• an acylating organic compound selected from the group consisting of: Trifluoropentyl, Acetyl, Octonyl, Butyl, Pentyl, Hexyl, Palmityl, Trifluoromethyl butyric, cyclopentane carboxylic, cyclopropylacetic, 4-fluorobenzoic, 4- fluorophenyl acetic, 3-Phenylpropionic,
• an amino acid with the peptide can be isosterically replaced, for example, Lys may be replaced for Dap, Dab, a-MeLys orOm. Examples of modified residues within a peptide are shown in Table 7.
• a side- chain of one or more amino acid residues in a peptide inhibitor of the invention is conjugated to a polymeric moiety, for example, in order to increase solubility and/or half-life in vivo (e.g. in plasma) and/or bioavailability.
• a polymeric moiety for example, in order to increase solubility and/or half-life in vivo (e.g. in plasma) and/or bioavailability.
• Such modifications are also known to reduce clearance (e.g. renal clearance) of therapeutic proteins and peptides.
• “Polyethylene glycol” or“PEG” is a polyether compound of general Formula H-(0-CH2-CH2)n-0H.
• PEGs are also known as polyethylene oxides (PEOs) or polyoxyethylenes (POEs), depending on their molecular weight PEO, PEE, or POG, as used herein, refers to an oligomer or polymer of ethylene oxide.
• PEOs polyethylene oxides
• POEs polyoxyethylenes
• the three names are chemically synonymous, but PEG has tended to refer to oligomers and polymers with a molecular mass below 20,000 Da, PEO to polymers with a molecular mass above 20,000 Da, and POE to a polymer of any molecular mass.
• PEG and PEO are liquids or low-melting solids, depending on their molecular weights. Throughout this disclosure, the 3 names are used indistinguishably .
• PEGs are prepared by polymerization of ethylene oxide and are commercially available over a wide range of molecular weights from 300 Da to 10,000,000 Da. While PEG and PEO with different molecular weights find use in different applications, and have different physical properties (e.g. viscosity) due to chain length effects, their chemical properties are nearly identical.
• the polymeric moiety is preferably water-soluble (amphiphilic or hydrophilic), non toxic, and pharmaceutically inert.
• Suitable polymeric moieties include polyethylene glycols (PEG), homo- or co-polymers of PEG, a monomethyl-substituted polymer of PEG (mPEG), or polyoxyethylene glycerol (POG).
• PEG polyethylene glycols
• mPEG monomethyl-substituted polymer of PEG
• POG polyoxyethylene glycerol
• PEGs that are prepared for purpose of half life extension, for example, mono-activated, alkoxy -terminated poly alky lene oxides (POA’s) such as mono-methoxy- terminated polyethyelene glycols (mPEG’s); bis activated polyethylene oxides (glycols) or other PEG derivatives are also contemplated.
• POA mono-activated, alkoxy -terminated poly alky lene oxides
• mPEG mono-methoxy- terminated polyethyelene glycols
• Glycols bis activated polyethylene oxides
• Suitable polymers will vary substantially by weights ranging from about 200 Da to about 40,000 Da or from about 200 Da to about 60,000 Da are usually selected for the purposes of the present invention.
• PEGs having molecular weights from 200 to 2,000 or from 200 to 500 are used.
• PEG poly(ethylene glycol)
• a common common initiator is a monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), abbreviated mPEG.
• mPEG methoxypoly(ethylene glycol)
• Lower-molecular-weight PEGs are also available as pure oligomers, referred to as monodisperse, uniform, or discrete. These are used in certain embodiments of the present invention.
• “PEGylation” is the act of covalently coupling a PEG structure to the peptide inhibitor of the invention, which is then referred to as a“PEGylated peptide inhibitor”.
• the PEG of the PEGylated side chain is a PEG with a molecular weight from about 200 to about 40,000.
• a spacer of a peptide of Formula I, Formula G, or Formula I” is PEGylated.
• the PEG of a PEGylated spacer is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11.
• the PEG of a PEGylated spacer is PEG3 or PEG8.
• polymeric moieties include poly-amino acids such as poly-lysine, poly- aspartic acid and poly-glutamic acid (see for example Gombotz, et al. (1995), Bioconjugate Chem, vol. 6: 332-351; Hudecz, et al. (1992), Bioconjugate Chem, vol. 3, 49-57 and Tsukada, et al. (1984), J. Natl. Cancer Inst., vol. 73, : 721-729.
• the polymeric moiety may be straight- chain or branched. In some embodiments, it has a molecular weight of 500-40,000 Da, for example 500-10,000 Da, 1000-5000 Da, 10,000-20,000 Da, or 20,000-40,000 Da.
• a peptide inhibitor of the invention may comprise two or more such polymeric moieties, in which case the total molecular weight of all such moieties will generally fall within the ranges provided above.
• the polymeric moiety is coupled (by covalent linkage) to an amino, carboxyl or thiol group of an amino acid side chain.
• an amino, carboxyl or thiol group of an amino acid side chain is coupled (by covalent linkage) to an amino, carboxyl or thiol group of an amino acid side chain.
• Certain examples are the thiol group of Cys residues and the epsilon amino group of Lys residues, and the carboxyl groups of Asp and Glu residues may also be involved.
• a PEG moiety bearing a methoxy group can be coupled to a Cys thiol group by a maleimido linkage using reagents commercially available from Nektar Therapeutics AL. See also WO 2008/101017, and the references cited above, for details of suitable chemistry.
• a maleimide-functionalised PEG may also be conjugated to the side-chain sulfhydryl group of a Cys residue.
• disulfide bond oxidation can occur within a single step or is a two step process.
• the trityl protecting group is often employed during assembly, allowing deprotection during cleavage, followed by solution oxidation.
• a second disulfide bond is required, one has the option of native or selective oxidation.
• Acm and Trityl is used as the protecting groups for cysteine. Cleavage results in the removal of one protecting pair of cysteine allowing oxidation of this pair.
• the second oxidative deprotection step of the cysteine protected Acm group is then performed.
• the trityl protecting group is used for all cysteines, allowing for natural folding of the peptide.
• suitable techniques which can be used to perform the oxidation step.
• One advantage of certain embodiments of the present invention includes the ability to add one or more chemical moiety (such as PEG) by incorporating one or more non-natural amino acid(s) that possess unique functional groups that react with an activated PEG by way of chemistry that is unreactive with the naturally occurring amino acids present in the peptide inhibitor.
• chemistry that is unreactive with all naturally occurring functional groups in a protein.
• a non-natural amino acid may be incorporated in one or more specific sites in a peptide inhibitor where PEG or another modification is desired without the undesirable non-specific reactions.
• the particular chemistry involved in the reaction results in a stable, covalent link between the PEG strand and the peptide inhibitor.
• such reactions may be performed in mild aqueous conditions that are not damaging to most peptides.
• the non-natural amino acid residue is AHA.
• Chemical moieties attached to natural amino acids are limited in number and scope.
• chemical moieties attached to non-natural amino acids can utilize a significantly greater spectrum of useful chemistries by which to attach the chemical moiety to the target molecule.
• any target molecule including any protein (or portion thereof) that includes a non-natural amino acid, e.g., a non-natural amino acid containing a reactive site or side chain where a chemical moiety may attach, such as an aldehyde- or keto-derivatized amino acid, can serve as a substrate for attaching a chemical moiety.
• azide moieties may be useful in conjugating chemical moieties such as PEG or others described herein.
• the azide moiety serves as a reactive functional group, and is absent in most naturally occurring compounds (thus it is unreactive with the native amino acids of naturally occurring compounds).
• Azides also undergo a selective ligation with a limited number of reaction partners, and azides are small and can be introduced to biological samples without altering the molecular size of significantly.
• the peptide inhibitors of the present invention may be synthesized by many techniques that are known to those skilled in the art.
• monomer subunits are synthesized, purified, and dimerized using the techniques described in the accompanying Examples.
• the present invention provides a method of producing a peptide inhibitor (or monomer subunit thereof) of the present invention, comprising chemically synthesizing a peptide comprising, consisting of, or consisting essentially of a peptide having an amino acid sequence described herein, including but not limited to any of the amino acid sequences set forth in any of Formulas I, II or tables herein.
• the present invention provides a method of producing a peptide inhibitor (or monomer subunit thereof) of the present invention, comprising introducing an intramolecular bond, e.g., a disulfide, an amide, or a thioether bond between two amino acids residues within a peptide comprising, consisting of, or consisting essentially of a peptide having an amino acid sequence described herein, including but not limited to any of the amino acid sequences set forth in any of Formulas (I) - (IX), the accompanying Examples or Tables.
• an intramolecular bond e.g., a disulfide, an amide, or a thioether bond between two amino acids residues within a peptide comprising, consisting of, or consisting essentially of a peptide having an amino acid sequence described herein, including but not limited to any of the amino acid sequences set forth in any of Formulas (I) - (IX), the accompanying Examples or Tables.
• the present invention includes polynucleotides that encode a polypeptide having a sequence set forth in any one of Formulas (I)-(IX), or the accompanying Examples or Table.
• the present invention includes vectors, e.g., expression vectors, comprising a polynucleotide of the present invention.
• the present invention includes methods of inhibiting IL-23 binding to an IL-23R on a cell, comprising contacting the IL-23 with a peptide inhibitor of the present invention.
• the cell is a mammalian cell.
• the method is performed in vitro or in vivo. Inhibition of binding may be determined by a variety of routine experimental methods and assays known in the art.
• the present invention includes methods of inhibiting IL-23 signaling by a cell, comprising contacting the IL-23 with a peptide inhibitor of the present invention.
• the cell is a mammalian cell.
• the method is performed in vitro or in vivo.
• the inhibition of IL-23 signalling may be determined by measuring changes in phospho-STAT3 levels in the cell.
• the present invention provides methods for treating a subject afflicted with a condition or indication associated with IL-21 or IL-23R (e.g., activation of the IL-23/IL-23R signaling pathway), wherein the method comprises administering to the subject a peptide inhibitor of the present invention.
• a method is provided for treating a subject afflicted with a condition or indication characterized by inappropriate, deregulated, or increased IL-23 or IL-23R activity or signaling, comprising administering to the individual a peptide inhibitor of the present invention in an amount sufficient to inhibit (partially or fully) binding of IL-23 to IL-23R in the subject.
• the inhibition of IL-23 binding to IL-23R occurs in particular organs or tissues of the subject, e.g., the stomach, small intestine, large intestine/colon, intestinal mucosa, lamina basement, Peyer’s Patches, mesenteric lymph nodes, or lymphatic ducts.
• methods of the present invention comprise providing a peptide inhibitor of the present invention to a subject in need thereof.
• the subject in need thereof has been diagnosed with or has been determined to be at risk of developing a disease or disorder associated with IL-23/IL-23R.
• the subject is a mammal.
• the disease or disorder is autoimmune inflammation and related diseases and disorders, such as multiple sclerosis, asthma, rheumatoid arthritis, inflammatory bowel diseases (IBDs), juvenile IBD, adolescent IBD, Crohn’s disease, sarcoidosis, Systemic Lupus Erythematosus, ankylosing spondylitis (axial spondyloarthritis), psoriatic arthritis, or psoriasis.
• IBDs inflammatory bowel diseases
• juvenile IBD juvenile IBD
• adolescent IBD Crohn’s disease
• sarcoidosis sarcoidosis
• Systemic Lupus Erythematosus ankylosing spondylitis (axial spondyloarthritis)
• psoriatic arthritis or psoriasis.
• the disease or disorder is psoriasis (e.g., plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, Palmo-Plantar Pustulosis, psoriasis vulgaris, or erythrodermic psoriasis), atopic dermatitis, acne ectopica, ulcerative colitis, Crohn’s disease, Celiac disease (nontropical Sprue), enteropathy associated with seronegative arthropathies, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis/esophagitis, colitis associated with radio- or chemo-therapy, colitis associated with disorders of innate immunity as in leukocyte adhesion deficiency-l, chronic granulomatous disease, glycogen storage disease type lb, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Wi
• the present invention provides a method of selectively inhibiting IL-23 or IL-23R signaling (or the binding of IL-23 to IL-23R) in a subject in need thereof, comprising providing to the subject a peptide inhibitor of the present invention.
• the present invention includes a method of selectively inhibiting IL- 23 or IL-23R signaling (or the binding of IL-23 to IL-23R) in the GI tract of a subject in need thereof, comprising providing to the subject a peptide inhibitor of the present invention by oral administration.
• exposure of the administered peptide inhibitor in GI tissues is at least lO-fold, at least 20-fold, at least 50-fold, or at least lOO-fold greater than the exposure in the blood.
• the present invention includes a method of selectively inhibiting IL23 or IL23R signaling (or the binding of IL23 to IL23R) in the GI tract of a subject in need thereof, comprising providing to the subject a peptide inhibitor, wherein the peptide inhibitor does not block the interaction between IL-6 and IL-6R or antagonize the IL-12 signaling pathway.
• the present invention includes a method of inhibiting GI inflammation and/or neutrophil infiltration to the GI, comprising providing to a subject in need thereof a peptide inhibitor of the present invention.
• methods of the present invention comprise providing a peptide inhibitor of the present invention (i.e., a first therapeutic agent) to a subject in need thereof in combination with a second therapeutic agent.
• the second therapeutic agent is provided to the subject before and/or simultaneously with and/or after the peptide inhibitor is administered to the subject.
• the second therapeutic agent is an anti-inflammatory agent.
• the second therapeutic agent is a non-steroidal anti-inflammatory drug, steroid, or immune modulating agent.
• the method comprises administering to the subject a third therapeutic agent.
• the second therapeutic agent is an antibody that binds IL-23 or IL-23R.
• the peptide inhibitor, or the pharmaceutical composition comprising a peptide inhibitor is suspended in a sustained-release matrix.
• a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
• a sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
• a biodegradable matrix is a matrix of one of either polylactide, polyglycobde, or polylactide co-glycobde (co-polymers of lactic acid and glycolic acid).
• the present invention includes pharmacetical compositions comprising one or more peptide inhibitors of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
• a pharmaceutically acceptable carrier, diluent or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or Formulation auxiliary of any type.
• Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.
• the compositions are administered orally, parenterally, intracistemally, intravaginally, intraperitoneally, intrarectally, topically (as by powders, ointments, drops, suppository, or transdermal patch), by inhalation (such as intranasal spray), ocularly (such as intraocularly) or buccally.
• parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous, intradermal and intraarticular injection and infusion. Accordingly, in certain embodiments, the compositions are Formulated for delivery by any of these routes of administration.
• compositions for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders, for reconstitution into sterile injectable solutions or dispersions just prior to use.
• suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, b- cyclodextrin, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
• Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• Thes compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
• Injectable depot forms include those made by forming microencapsule matrices of the peptide inhibitor in one or more biodegradable polymers such as polylactide-polyglycobde, poly(orthoesters), poly(anhydrides), and (poly )gly cols, such as PEG. Depending upon the ratio of peptide to polymer and the nature of the particular polymer employed, the rate of release of the peptide inhibitor can be controlled. Depot injectable Formulations are also prepared by entrapping the peptide inhibitor in liposomes or microemulsions compatible with body tissues.
• the injectable Formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
• Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye.
• Compositions for topical lung administration may involve solutions and suspensions in aqueous and non-aqueous Formulations and can be prepared as a dry powder which may be pressurized or non- pressurized.
• the active ingredient may be finely divided form may be used in admixture with a larger-sized pharmaceutically acceptable inert carrier comprising particles having a size, for example, of up to 100 micrometers in diameter.
• Suitable inert carriers include sugars such as lactose.
• the composition may be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant.
• a compressed gas such as nitrogen or a liquefied gas propellant.
• the liquefied propellant medium and indeed the total composition may bey such that the active ingredient does not dissolve therein to any substantial extent.
• the pressurized composition may also contain a surface active agent, such as a liquid or solid non-ionic surface active agent or may be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.
• a further form of topical administration is to the eye.
• a peptide inhibitor of the invention may be delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the peptide inhibitor is maintained in contact with the ocular surface for a sufficient time period to allow the peptide inhibitor to penetrate the comeal and internal regions of the eye, as for example the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera.
• the pharmaceutically acceptable ophthalmic vehicle may, for example, be an ointment, vegetable oil or an encapsulating material.
• the peptide inhibitors of the invention may be injected directly into the vitreous and aqueous humour.
• compositions for rectal or vaginal administration include suppositories which may be prepared by mixing the peptide inhibitorss of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at room temperature but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active compound.
• suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at room temperature but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active compound.
• Peptide inhibitors of the present invention may also be administered in liposomes or other lipid-based carriers.
• liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
• the present compositions in liposome form can contain, in addition to a peptide inhibitor of the present invention, stabilizers, preservatives, excipients, and the like.
• the lipids comprise phospholipids, including the phosphatidyl cholines (lecithins) and serines, both natural and synthetic. Methods to form liposomes are known in the art.
• compositions to be used in the invention suitable for parenteral administration may comprise sterile aqueous solutions and/or suspensions of the peptide inhibitos made isotonic with the blood of the recipient, generally using sodium chloride, glycerin, glucose, mannitol, sorbitol, and the like.
• the invention provides a pharmaceutical composition for oral delivery.
• Compositions and peptide inhibitors of the instant invention may be prepared for oral administration according to any of the methods, techniques, and/or delivery vehicles described herein. Further, one having skill in the art will appreciate that the peptide inhibitors of the instant invention may be modified or integrated into a system or delivery vehicle that is not disclosed herein, yet is well known in the art and compatible for use in oral delivery of peptides.
• Formulations for oral administration may comprise adjuvants (e.g. resorcinols and/or nonionic surfactants such as polyoxyethylene oleyl ether and n- hexadecylpolyethylene ether) to artificially increase the permeability of the intestinal walls, and/or enzymatic inhibitors (e.g. pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) or trasylol) to inhibit enzymatic degradation.
• adjuvants e.g. resorcinols and/or nonionic surfactants such as polyoxyethylene oleyl ether and n- hexadecylpolyethylene ether
• enzymatic inhibitors e.g. pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) or trasylol
• the peptide inhibitor of a solid-type dosage form for oral administration can be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffmose, maltitol, dextran, starches, agar, alginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, or glyceride.
• at least one additive such as sucrose, lactose, cellulose, mannitol, trehalose, raffmose, maltitol, dextran, starches, agar, alginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, or glyceride.
• These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, alpha- tocopherol, antioxidants such as cysteine, disintegrators, binders, thickeners, buffering agents, pH adjusting agents, sweetening agents, flavoring agents or perfuming agents.
• additives e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, alpha- tocopherol, antioxidants such as cysteine, disintegrators, binders, thickeners, buffering agents, pH adjusting agents, sweetening agents, flavoring agents or perfuming agents.
• oral dosage forms or unit doses compatible for use with the peptide inhibitors of the present invention may include a mixture of peptide inhibitor and nondrug components or excipients, as well as other non-reusable materials that may be considered either as an ingredient or packaging.
• Oral compositions may include at least one of a liquid, a solid, and a semi-solid dosage forms.
• an oral dosage form is provided comprising an effective amount of peptide inhibitor, wherein the dosage form comprises at least one of a pill, a tablet, a capsule, a gel, a paste, a drink, a syrup, ointment, and suppository.
• an oral dosage form is provided that is designed and configured to achieve delayed release of the peptide inhibitor in the subject’s small intestine and/or colon.
• a pharmaceutical composition which comprises a peptide inhibitor of the present invention and a protease inhibitor, such as aprotinin, in a delayed release pharmaceutical Formulation.
• pharmaceutical compositions of the instant invention comprise an enteric coat that is soluble in gastric juice at a pH of about 5.0 or higher.
• a pharmaceutical composition comprising an enteric coating comprising a polymer having dissociable carboxylic groups, such as derivatives of cellulose, including hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate and cellulose acetate trimellitate and similar derivatives of cellulose and other carbohydrate polymers.
• a polymer having dissociable carboxylic groups such as derivatives of cellulose, including hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate and cellulose acetate trimellitate and similar derivatives of cellulose and other carbohydrate polymers.
• a pharmaceutical composition comprising a peptide inhibitor of the present invention is provided in an enteric coating, the enteric coating being designed to protect and release the pharmaceutical composition in a controlled manner within the subject’s lower gastrointestinal system, and to avoid systemic side effects.
• the peptide inhibitors of the instant invention may be encapsulated, coated, engaged or otherwise associated within any compatible oral drug delivery system or component.
• a peptide inhibitor of the present invention is provided in a lipid carrier system comprising at least one of polymeric hydrogels, nanoparticles, microspheres, micelles, and other lipid systems.
• some embodiments of the present invention comprise a hydrogel polymer carrier system in which a peptide inhibitor of the present invention is contained, whereby the hydrogel polymer protects the peptide inhibitor from proteolysis in the small intestine and/or colon.
• the peptide inhibitors of the present invention may further be Formulated for compatible use with a carrier system that is designed to increase the dissolution kinetics and enhance intestinal absorption of the peptide. These methods include the use of liposomes, micelles and nanoparticles to increase GI tract permeation of peptides.
• bioresponsive systems may also be combined with one or more peptide inhibitor of the present invention to provide a pharmaceutical agent for oral delivery.
• a peptide inhibitor of the instant invention is used in combination with a bioresponsive system, such as hydrogels and mucoadhesive polymers with hydrogen bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA], cellulose, Eudragit®, chitosan and alginate) to provide a therapeutic agent for oral administration.
• a bioresponsive system such as hydrogels and mucoadhesive polymers with hydrogen bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA], cellulose, Eudragit®, chitosan and alginate)
• Other embodiments include a method for optimizing or prolonging drug residence time for a peptide inhibitor disclosed herein, wherein the surface of the peptide inhibitor surface is modified to comprise mucoadhesive properties through hydrogen bonds, polymers with linked mucins or/and hydrophobic interactions.
• modified peptide molecules may demonstrate increase drug residence time within the subject, in accordance with a desired feature of the invention.
• targeted mucoadhesive systems may specifically bind to receptors at the enterocytes and M-cell surfaces, thereby further increasing the uptake of particles containing the peptide inhibitor.
• Other embodiments comprise a method for oral delivery of a peptide inhibitor of the present invention, wherein the peptide inhibitor is provided to a subject in combination with permeation enhancers that promote the transport of the peptides across the intestinal mucosa by increasing paracellular or transcellular permeation.
• permeation enhancers and methods for the oral delivery of therapeutic agents is described in Bray den, D.J., Mrsny, R.J., 2011. Oral peptide delivery: prioritizing the leading technologies. Ther. Delivery 2 (12), 1567- 1573.
• compositions and Formulations of the present invention comprises a peptide inhibitor of the present invention and one or more permeation enhancer.
• absorption enhancers may include Bile salts, fatty acids, surfactants (anionic, cationic, and nonanionic) chelators, Zonular OT, esters, cyclodextrin, dextran sulfate, azone, crown ethers, EDTA, sucrose esters, and phosphotidyl choline, for example.
• absorption enhancers are not typically carriers by themselves, they are also widely associated with other carriers to improve oral bioavailability by transporting of peptides and proteins across the intestinal mucosa. Such substances can be added to the Formulation as excipients or incorporated to form non specific interactions with the intended peptide inhibitor.
• MCFAS medium chain fatty acids
• Dietary components and/or other naturally occurring substances affirmed as enhancing tight junction permeation and as Generally Recognized As Safe (GRAS) include, e.g., as glycerides, acylcamitines, bile salts, and medium chain fatty acids.
• MFAS medium chain fatty acids
• MCFAS medium chain fatty acids
• the most extensively studied MCFAS is sodium caprate, a salt of capric acid, which comprises 2-3% of the fatty acids in the milk fat fraction.
• sodium caprate is mainly used as an excipient in a suppository Formulation (DoktacillinTM) for improving rectal ampicillin absorption.
• a permeation enhancer is combined with a peptide inhibitor, wherein the permeation enhancer comprises at least one of a medium-chain fatty acid, a long-chain fatty acid, a bile salt, an amphiphilic surfactant, and a chelating agent.
• medium-chain fatty acid salts promote absorption by increasing paracellular permeability of the intestinal epithelium.
• a permeation enhancer comprising sodium N-[hydroxybenzoyl)amino] caprylate is used to form a weak noncovalent association with the peptide inhibitor of the instant invention, wherein the permeation enhancer favors membrane transport and further dissociation once reaching the blood circulation.
• a peptide inhibitor of the present invention is conjugated to oligoarginine, thereby increasing cellular penetration of the peptide into various cell types.
• a noncovalent bond is provided between a peptide inhibibitor of the present invention and a permeation enhancer selected from the group consisting of a cyclodextrin (CD) and a dendrimers, wherein the permeation enhancer reduces peptide aggregation and increasing stability and solubility for the peptide inhibitor molecule.
• a pharmaceutical composition or Formulation comprises a peptide inhibitor of the present invention and a transient permeability enhancers (TPEs).
• TPEs transient permeability enhancers
• Permeation enhancers and TPEs may be used to increase orally bioavailability or the peptide inhibitor.
• TPE transient permeability enhancers
• One example of a TPE that may be used is an oily suspension Formulation that disperses a powder containing sodioum caprylate and a therapeutic agent (Tuvia, S. et al, Pharmaceutical Research, Yol. 31, No. 8, pp. 2010-2021 (2014).
• composition and Formulations may include a peptide inhibitor of the present invention and one or more absorption enhancers, enzyme inhibitors, or mucoso adhesive polymers.
• peptide inhibors of the present invention are Formulated in a Formulation vehicle, such as, e.g., emulsions, liposomes, microsphere or nanoparticles.
• the present invention provides a peptide inhibitor having a half-life of at least several hours to one day in vitro or in vivo (e.g., when administered to a human subject) sufficient for daily (q.d.) or twice daily (b.i.d.) dosing of a therapeutically effective amount.
• the peptide inhibitor has a half-life of three days or longer sufficient for weekly (q.w.) dosing of a therapeutically effective amount.
• the peptide inhibitor has a half-life of eight days or longer sufficient for bi-weekly (b.i.w.) or monthly dosing of a therapeutically effective amount.
• the peptide inhibitor is derivatized or modified such that is has a longer half-life as compared to the underivatized or unmodified peptide inhibitor.
• the peptide inhibitor contains one or more chemical modifications to increase serum half-life.
• the total daily dose of the peptide inhibitors of the invention to be administered to a human or other mammal host in single or divided doses may be in amounts, for example, from 0.0001 to 300 mg/kg body weight daily or 1 to 300 mg/kg body weight daily.
• the peptide inhibitors of the invention may be used for detection, assessment and diagnosis of intestinal inflammation by microPET imaging, wherein the peptide inhibitor is labeled with a chelating group or a detectable label, as part of a a non-invasive diagnostic procedure.
• a peptide inhibitor is conjugated with a bifunctional chelator.
• a peptide inhibitor is radiolabeled.
• the labeled peptide inhibitor is then administered to a subject orally or rectally.
• the labeled peptide inhibitor is included in drinking water. Following uptake of the peptide inhibitor, microPET imaging may be used to visualize inflammation throughout the subject’s bowels and digestive track.
• Rink Amide MBHA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-tenminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid 'as used for peptide with C-terminal acids.
• the coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration.
• amino acids solutions were prepared at 100 mmol concentration.
• Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties. Assembly
• the peptide was cleaved from the resin by treatment with cleavage reagent, such as reagent K (82.5% trigluoroacetic acid, 5% water, 5% thioanisole, 5% phenol, 2.5% l,2-ethanedithiol).
• cleavage reagent such as reagent K (82.5% trigluoroacetic acid, 5% water, 5% thioanisole, 5% phenol, 2.5% l,2-ethanedithiol).
• cleavage reagent was able to successfully cleave the peptide from the resin, as well as all remaining side chain protecting groups.
• the peptide containing the free thiol (e.g., Cys) and hSer(OTBDMS) was assembled on a Rink Amide-MBHA resin following general Fmoc-SPPS procedure. Chlorination was carried out by treating the resin with PPh 3 (10 equiv.) and CbCCN (10 equiv.) in DCM for 2 h. The peptide was cleaved from the resin by treatment with cleavage reagent 90% trifluoroacetic acid, 5% water, 2.5% 1 ,2-ethanedithiol, 2.5% tri-isopropylsilane).
• cleavage reagent 90% trifluoroacetic acid, 5% water, 2.5% 1 ,2-ethanedithiol, 2.5% tri-isopropylsilane).
• the cleaved peptides were precipitated in cold diethyl ether followed by two washings with ethyl ether. The filtrate was poured off and a second aliquot of cold ether was added, and the procedure repeated. The crude peptide was dissolved in a solution of acetonitrile:water (7:3 with 1% TFA) and filtered giving the wanted uncyclized crude peptide.
• Separations were achieved using linear gradients of buffer B in A (Mobile phase A: water containing 0.15% TFA, mobile phase B: Acetonitrile (ACN) containing 0.1% TFA), at a flow rate of 1 mL/min (analytical) and 15 mL/min (preparative). Separations were achieved using linear gradients of buffer B in A (Mobile phase A: water containing 0.15% TFA, mobile phase B: Acetonitrile (ACN) containing 0.1% TFA), at a flow rate of 1 mL/min (analytical) and 15mL/min (preparative).
• Peptide monomers of the present invention were synthesized using standard Fmoc solid phase synthesis techniques on a CEM Liberty BlueTM microwave peptide synthesizer.
• the peptides were assembled using Oxyma/DIC (ethyl cyanohydroxyiminoacetate/diisopropylcarbodiimide) with microwave heating.
• Rink Amide- MBHA resin (100-200 mesh, 0 66 mmoi/g) was used for peptides with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C ⁇ terminal acids.
• Oxyma was prepared as a 1M solution in DMF with 0.1M DIEA.
• DIC was prepared as 0.5M solution in DMF.
• the Amino acids were prepared at 200mM.
• Peptide inhibitors of the present invention were identified based on medicinal chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.
• the peptides were made using standard CEM Liberty BlueTM protocols.
• the peptide sequences were assembled as follows: Resin (400 mg, 0.25 mmol) was suspended in 10 ml of 50/50 DMF/DCM. The resin was then transferred to the reaction vessel in the microwave cavity. The peptide was assembled using repeated Fmoc deprotection and Oxyma/DIC coupling cycles. For deprotection, 20% 4-methylpiperidine in DMF was added to the reaction vessel and heated to 90 °C for 65 seconds. The deprotection solution was drained and the resin washed three times with DMF.
• the peptide was then cleaved from the resin by treatment with a standard cleavage cocktail of 91 :5:2:2 TFA/H2O/TIPS/DODT for 2 hrs. If more than one Arg(pbf) residue was present the cleavage was allowed to go for an additional hour.
• the solvent mixture was then purified by first being diluted with water and then loaded onto a reverse phase HPLC Column (Luna® Cl 8 support, lOu, 100A, Mobile phase A: water containing 0.1% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA, gradient began with 15% B, and changed to 50% B over 60 minutes at a flow rate of l5ml/min). Fractions containing pure product were then freeze-dried on a lyophilizer.
• the peptide containing the free thiol (e.g., Cys) and hSer(OTBDMS) was assembled on a Rink Amide-MBHA resin following general Fmoc-SPPS procedure. Chlorination was carried out by treating the resin with Dichlorotriphenylphosphorane (5 eq, 0.5M) with Pinene (0.875M) and thioanisole (0.375M) scavengers at room temperature for 2 hours. The chloro- peptides were cleaved from the resin and precipitated as described above.
• reaction mixture was then purified by first being diluted lx with water and then loaded onto a reverse phase HPLC column (Luna® C18 support, lOu, 100 A, Mobile phase A: water containing 0.1% TFA, mobile phase B: Acetonitrile (ACN) containing 0.1% TFA, gradient began at 15% B, and changed to 50% B over 60 minutes at a flow rate of 20 ml/min). Fractions containing pure product as determined by RPHPLC were then freeze-dried on a lyophilizer.
• Swell Resin 10 g of Rink Amide MBHA solid phase resin (0.66mmol/g loading) was transferred to a 250 ml peptide vessel with filter frit, ground glass joint and vacuum side arm. The resin was washed 3x with DMF.
• Step 1 Coupling of FMOC-d-Lys(Boc)-OH: Deprotection of the FMOC group was realized by adding 2 resin-bed volumes of 20% 4-methyl piperidine in DMF to the swollen resin and shaking for 3-5 min prior to draining and adding a second, 2-resin-bed volume of the 4-methyl piperidine solution and shaking for an additional 20-30 min. After deprotection the resin was washed 3x DMF with shaking. FMOC-d-Lys(boc)-OH (3 eq, 9.3 g) was dissolved in 100 ml DMF along with Oxyma (4.5 eq, 4.22g).
• Preactivation of the acid was accomplished by addition of DIC (3.9 eq, 4 ml) and shaking for 15 min prior to addition to the deprotected resin. An additional aliquot of DIC (2.6 eq, 2.65 ml) was added after - 15 min of coupling. The progress of the coupling reaction was monitored by the colorimetric Kaiser test. Once the reaction was judged complete the resin was washed 3 x DMF with shaking prior to starting the next deprotection/coupling cycle.
• Step 2 Coupling of FMOC-Asn(Trt)-OH: FMOC Deprotection was again accomplished by adding two sequential, 2-resin-bed volumes of 20% 4-Methyl- Piperidine/DMF, one times 3-5 minutes and one times 20-30 minutes, draining in between treatments. The resin was then washed 3 times prior to coupling protected Asparagine. FMOC-Asn(Trt)-OH (3 eq, 11.8g) was dissolved in DMF along with Oxyma (4.5eq, 4.22g). Preactivation with DIC (3.9 eq, 4 ml) for 15 minutes was done prior to addition to the d-Lys- Rink- Amide resin.
• Step 3 Coupling of FMOC-Lys(Ac)-OH:
• the FMOC was removed from the N- terminus of the resin bound Asparagine and the resin washed as previously described.
• FMOC- Lys(Ac)-OH (2eq, 5.42g) was dissolved in lOOml of DMF along with Oxyma (3eq, 2.8lg).
• DIC 2.6 eq, 2.65 ml
• an additional aliquot of DIC 1.43 ml
• Step 4 Coupling of FMOC-a-Me-Lys(Boc)-OH: The FMOC was removed from the N-terminus of the resin bound Acetyl Lysine and the resin washed as previously described. FMOC-a-Me-Lys(Boc)-OH (2 eq, 6.27 g) was dissolved in lOOml of DMF along with Oxyma (3eq, 2.8lg). DIC (2.6 eq, 2.65 ml) was added for preactivation of the acid -15 minutes prior to addition to the Lys(Ac)-Asn-dLys-Rink-Amide resin.
• Step 6 Coupling of FMOC-4-[2-(boc-amino-ethoxy)]-L-Phenylalanine : The FMOC was removed from the N-terminus of the resin bound peptide and the resin washed as previously described. FMOC-4-[2-(boc-amino-ethoxy)]-L-Phenylalanine (3 eq, 10.8 g) was dissolved in lOOml of DMF along with Oxyma (4.5 eq, 4.22g).
• Step 8 Coupling of FMOC-Lys(Ac)-OH :
• the FMOC was removed from the N- terminus of the resin bound peptide and the resin washed as previously described.
• FMOC- Lys(Ac)-OH (2 eq, 5.4 g) was dissolved in 100 ml of DMF along with Oxyma (3 eq, 2.81 g).
• Oxyma (3 eq, 2.81 g).
• DIC 2.6 eq, 2.65 ml
• Step 11 Coupling of FMOC-Asn(Trt)-OH : The FMOC was removed from the N- terminus of the resin bound peptide and the resin washed as previously described. FMOC- Asn(Trt)-OH (4 eq, 15.8 g) was dissolved in 100 ml of DMF along with Oxyma (6 eq, 5.62 g). DIC (5.2 eq, 5.3 ml) was added for preactivation of the acid -15 minutes prior to addition to the Thr-7MeTrp-Lys(Ac)-Pen-AEF-2Nal-aMeLys-Lys(Ac)-Asn-dLys-Rink-Amide resin.
• Step 12 Coupling of FMOC-L-Pen(Trt)-OH : The FMOC was removed from the N- terminus of the resin bound peptide and the resin washed as previously described. FMOC-L- Pen(Trt)-OH (2 eq, 8.1 g) was dissolved in lOOml of DMF along with Oxyma (3 eq, 2.81 g).
• DIC (2.6 eq, 2.65 ml) was added for preactivation of the acid -15 minutes prior to addition to the Asn-Thr-7MeTrp-Lys(Ac)-Pen-AEF-2Nal-aMeLys-Lys(Ac)-Asn-dLys-Rink- Amide resin. After -15 minutes, an additional aliquot of DIC (2.6 eq, 2.65 ml) was added to the reaction. Once the reaction was complete as determined by the Kaiser test the resin was again washed 3x with DMF prior to the final deprotection and acetic acid capping of the constructed peptide.
• Step 13 Acetyl Capping: The FMOC was removed from the N-terminus of the resin bound peptide and the resin washed as previously described. 150 ml of Capping Reagent A (THF/Acetic anhydride/Pyridine, 80: 10: 10) was added to the constructed Pen-Asn-Thr- 7MeTrp-Lys(Ac)-Pen-AEF-2Nal-aMeLys-Lys(Ac)-Asn-dLys-Rink-Amide resin and shaken for 30 min. The resin was washed 3 x with DMF followed by 3x with DCM. The resin was divided into 5 - 50 ml centrifuge tubes and placed under vacuum for 1.5 hrs prior to cleavage with TFA.
• Step 14 TFA Cleavage and Ether precipitation: 200 ml of the TFA cleavage cocktail (90/5/2.5/2.5 TFA/water/Tips/DODT) was prepared. 40 ml of the cleavage cocktail was added to each of the 5 tubes containing the protected resin bound peptide and shaken for two hours. The spent resin was filtered away and the filtrate divided evenly into 18 - 50 ml centrifuge tubes for precipitation. Cold Diethyl Ether was added to each forming a white precipitate that was then centrifuged. The ether was decanted to waste and 2 more ether washes of the precipitate were performed. The resulting white precipitate cake was allowed to dry overnight in the hood to give ⁇ l0.4g of crude reduced peptide.
• Step 15 Disulfide Oxidation: The crude peptide was oxidized in four batches. ⁇ 2.5 g of crude peptide was dissolved in 50 ml of 50/50 acetonitrile/water and diluted to 1L 20% ACN/water. With swirling a saturated solution of Iodine in Acetic acid/Methanol was added dropwise to the 1L peptide solution until the yellow/brown color of the 12 remains and does not fade away. The slightly colored solution was allowed to sit for 5 min prior to quenching the excess 12 with a pinch of Ascorbic acid.
• Step 16 RP-HPLC purification: The RP-HPLC purification was performed in four batches immediately following 12 oxidation.
• the 1 L of quenched oxidized peptide was loaded onto the equilibrated column at 70 ml/min. After the solvent front eluted an elution gradient of 20-38% MPB over 60 min at 70ml/min was run.
• the desired oxidized peptide eluted at -26% MPB. Pure fractions were combined from all four purifications and lyophilized to give 3.83g the purified TFA salt ready for counterion exchange.
• the captured peptide was then washed with 5% MPB in MPC for 40 min at 70 ml/min to exchange the counterions from TFA to Acetate.
• the captured peptide was then washed with 5% MPB in MPA at 70ml/min for 10 min to clear all NH40Ac form system.
• the peptide is eluted with a quick gradient of 5-70% MPB in MPA over 60 minutes.
• Step 18 Final Lyophilization and Analysis: The collected fractions were analyzed by analytical RP-HPLC, and all fractions >95% purity were combined. Lyophilization of the combined fractions gave 3.24 g of Peptide #252 as a white powder with a purity of 97 %. Low resolution LC/MS of purified Peptide #252 gave 3 charged states of the peptide, M+3/3 of 621.8, M+2/2 of 932.1 and the molecular ion of 1863.5. The experimental mass agrees with the theoretical mass of 1863.3 da.
• Peptide optimization was performed to identify peptide inhibitors of IL-23 signalling that were active at low concentrations (e.g., ICso ⁇ 10 nM). Peptides were tested to identify peptides that inhibit the binding of IL-23 to human IL-23R and inhibit IL-23/IL-23R functional activity, as described below.
• Table E2 provides an illustrative structure depicting thioether cycbzation, which is indicated in the table by the term“cyclo,” with the cyclic region bracketed immediately following the term “cyclo.”
• the residue Abu is present where indicated, whereas in other embodiments, e.g., those related to the non-cyclized form, the Abu may be referred to as a hSer(Cl) or homoSer residue.
• IL-23 plays a central role in supporting and maintaining Thl7 differentiation in vivo. This process is thought to be mediated primarily through the Signal Transducer and Activator of Transcription 3 (STAT3), with phosphorylation of STAT3 (to yield pSTAT3) leading to upregulation of RORC and pro-inflammatory IL-17.
• STAT3 Signal Transducer and Activator of Transcription 3
• phosphorylation of STAT3 to yield pSTAT3 leading to upregulation of RORC and pro-inflammatory IL-17.
• This cell assay examines the levels of pSTAT3 in IL-23R-expressing DB cells when stimulated with IL-23 in the presence of test compounds.
• test peptides and IL-23 (Humanzyme #HZ-l26l) at a final concentration of 0.5 nM, were added to each well in a 96 well tissue culture plate (Coming #CLS3894) .
• DB cells (ATCC #CRL-2289), cultured in RPMI-1640 medium (Thermo Scientific #11875093) supplemented with 10% FBS, were added at 5 X 10E5 cells/well and incubated for 30 minutes at 37°C in a 5% CO2 humidified incubator.
• NK cells Natural killer (NK) cells, purified from human peripheral blood of healthy donors by negative selection (Miltenyi Biotech, Cat # 130-092-657), were cultured in complete media (RPMI 1640 containing 10% FBS, L-glutamine and penicillin-streptomycin) in the presence of IL-2 (RnD, Cat # 202-IL-010/CF) at 25 ng/mL. After 7 days, cells were centrifuged, and resuspended in complete media at 1E6 cells/mL.
• complete media RPMI 1640 containing 10% FBS, L-glutamine and penicillin-streptomycin
• Recombinant IL-23 at predetermined EC50 to EC75 and IL-18 (RnD, Cat # B003-5) at 10 ng/mL were mixed with varying concentrations of peptides, and added to NK cells seeded at 1E5 cells per well. After 20 to 24 hours, IFNy in the supernatant was quantified using Quantikine ELISA (RnD, Cat # DIF50).
• SIF was prepared by adding 6.8 g of monobasic potassium phosphate and 10.0 g of pancreatin to 1.0 L of water. After dissolution, the pH was adjusted to 6.8 using NaOH. DMSO stocks (2 mM) were first prepared for the test compounds. Aliquots of the DMSO solutions were dosed into 6 individual tubes, each containing 0.5 mL of SIF, which is pre- warmed to 37°C. The final test compound concentration was 20 mM. The vials were kept in a benchtop Thermomixer® for the duration of the experiment.
• Fecal homogenate (20%) was prepared by adding 4 mL of growth medium (1 liter contains 2 g peptone water powder, 2 g yeast extract, 0.1 g NaCl, 0.04 g KH2PO4, 0.01 g CaCh 6H2O, 0.01 g MgSCri 7H2O, 2 mL Tween 80, 0.5 g bile salts, 0.5 g L-cysteine HC1, 2 g NaHCCh, and 10 pL Vitamin K, pH adjusted to 6.8, and sterilized by filtration through a 0.22 pm filter) to every gram of feces (pooled freshly collected human or cynomolgus monkey feces).
• the suspension was vortexed to break up large clumps, and homogenized using a bead mill homogenizer. Centrifuged the homogenate at 2800 x g for 15 min. The supernatant is taken out and used for incubations.
• DMSO stocks (10 mM) were first prepared for the test compounds. Incubations were performed in an anaerobic chamber conditioned at 37°C. Aliquots of the DMSO solutions were dosed into 1.0 mL aliquots of 20% fecal homogenate, which are pre-warmed to 37°C. The final test compound concentration was 20 pM.
• Peptides of interest (20 mM) were incubated with pre-warmed rat plasma (SD rat, mixed gender pooled, EDTA, filtered through 0.22 pm, BioreclamationIVT) at 37°C. Aliquots were taken at various time points up to 24 hours (e.g. 0, 0.25, 1, 3, 6 and 24 hr), and immediately quenched with 4 volumes of organic solvent (acetonitrile/methanol (1 : 1) and 0.1% formic acid, containing 1 pM internal standard). Quenched samples were stored at 4 °C until the end of the experiment and centrifuged at 4,000 rpm for 10 minutes. The supernatant were diluted 1 : 1 with deionized water and analyzed using LC-MS. Percentage remaining at each time point was calculated based on the peak area ratio (analyte over internal standard) relative to the initial level at time zero. Half-lives were calculated by fitting to a first-order exponential decay equation using GraphPad.

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