Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/202—IL-3
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/36—Gynecology or obstetrics
  • G01N2800/364—Endometriosis, i.e. non-malignant disorder in which functioning endometrial tissue is present outside the uterine cavity

Definitions

• the present disclosure generally relates to compositions and methods for the diagnosis and treatment of endometriosis.
• the disclosure also relates to pre-implantation factor (PIF) mutants and methods of treatment , such as treating endometriosis.
• PPF pre-implantation factor
• Endogenous Pre-implantation Factor is a 15 amino acid peptide
• MVRIKPGSANKPSDD SEQ ID NO: l
• PIF exerts broad neurotrophic and neuroprotective effects. PIF regulates immunity, inflammation and transplant acceptance.
• PIF specifically reduces neural damage while it promotes neural development, protecting against maternal adverse environment.
• PIF precisely targets proteins in the embryo to reduce oxidative stress and protein misfolding. In vivo PIF reduces spontaneous and LPS induced pregnancy loss by decreasing the pro-inflammatory response in the placenta.
• PIF and synthetic PIF analogs have immune modulatory properties and such peptides are useful in the prevention and/or treatment of various immune-mediated diseases, including, but not limited to, endometriosis, a chronic inflammatory condition that affects reproductive age women.
• Compositions and methods for diagnosing, treating and/or preventing endometriosis are provided herein.
• the present disclosure relates to a method of treating or preventing endometriosis in a subject in need thereof, the method comprising administering to the subject at least one pre- implantation factor (PIF) peptide, a mimetic thereof, an analog thereof, or a pharmaceutically acceptable salt thereof.
• PEF pre- implantation factor
• the step of administering to the subject at least one PIF peptide, a mimetic thereof, an analog thereof, or a pharmaceutically acceptable salt thereof comprises administering a therapeutically effective dose of the at least one PIF molecule, an analog thereof, or a pharmaceutically acceptable salt thereof.
• the step of administering to the subject at least one PIF peptide, a mimetic thereof, an analog thereof, or a pharmaceutically acceptable salt thereof comprises administering a therapeutically effective dose of the PIF peptide, an analog thereof, or pharmaceutically acceptable salt thereof from about 0.001 mg/kg to about 200 mg/kg.
• the step of administering to the subject at least one PIF peptide, a mimetic thereof, an analog thereof, or a pharmaceutically acceptable salt thereof comprises administering a therapeutically effective dose of the PIF peptide, an analog thereof, or pharmaceutically acceptable salt thereof from about 0.5 mg/kg to about 5 mg/kg.
• the at least the PIF peptide, a mimetic thereof, an analog thereof, or pharmaceutically acceptable salt thereof comprises a chemical targeting moiety and/or a radioactive moiety.
• the at least one inhibitor of nuclear translocation of beta- catenin or pharmaceutically acceptable salt thereof comprises at least one radioactive moiety comprising at least one or a combination of the following isotopes: 2 H, H, 1 C, 14 C, 15 N, 16 0, 17 0, 31 P, 32 P, 35 S, 18 F, and 36 Cl.
• the method further comprises administering at least one analgesic and/or one anti-inflammatory compound.
• the method further comprises administering at least one analgesic and or one anti-inflammatory compound before, after, or simultaneously with the administration of a therapeutically effective dose of at least one PIF peptide, an analog thereof or pharmaceutically acceptable salt thereof.
• the therapeutically effective dose is from about 1.0 mg/kg to about 5.5 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is from about 1.0 mg/kg to about 5.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is from about 1.0 mg/kg to about 4.5 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose is from about 1.0 mg/kg to about 4.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is from about 1.0 mg/kg to about 3.5 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is administered subcutaneously, intravenously, intraperitoneally, topically, orally, sublingually, intranasally, or intramuscularly.
• the therapeutically effective dose is administered once a week, twice a week, three times a week, four times a week, or five days per week, and optionally, wherein the dose is administered once in two, three, four or five days in succession.
• the therapeutically effective dose is administered once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, or once every twelve weeks.
• the therapeutically effective dose is administered once a month, twice a month, three times a month, four times a month, or five times a month.
• the therapeutically effective dose is administered once a year, twice a year, three times a year, four times a year, five times a year, six times a year, seven times a year, eight times a year, nine times a year, ten times per year, or eleven times per year.
• the PIF peptide comprises SEQ ID NO: 1, SEQ ID NO:2, and/or SEQ ID NO:3. In some embodiments, the PIF peptide comprises SEQ ID NO:4 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 5 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:6 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:7 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 8 or a
• the PIF peptide comprises SEQ ID NO: 9 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 10 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 11 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 12 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 13 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 14 or a pharmaceutically acceptable salt thereof.
• the PIF peptide comprises SEQ ID NO: 15 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 16 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 18 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 19 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:20 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:21 or a pharmaceutically acceptable salt thereof.
• the PIF peptide comprises SEQ ID NO:22 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:23 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:24 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO: 25 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:26 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:27 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:28 or a pharmaceutically acceptable salt thereof. In some embodiments, the PIF peptide comprises SEQ ID NO:29 or a pharmaceutically acceptable salt thereof.
• a compound of the formula R1-R2-R 3 -R4 (SEQ ID NO:31) is provided, wherein Ri is Pro or a mimetic of Pro, R2 is Gly or a mimetic of Gly, R 3 is Ser or a mimetic of Ser, and R4 is Ala or a mimetic of Ala.
• the compound may comprise one or more of up to 11 additional amino acid residues.
• Embodiments include those peptides derived from pre-implantation embryos that induces TH2 type cytokines like IL-10 synthesis or secretion from lymphocytes or other white blood cells and
• pharmacophores that binds specifically to PIF receptors (such but not limited to PGSA (A)VRIKPGSANKPSDD or (Q)VRIKPGSANKPSDD) or by substituting with D amino acids or by adding PEG.
• PIF receptors such but not limited to PGSA (A)VRIKPGSANKPSDD or (Q)VRIKPGSANKPSDD
• peptides are from pre-implantation embryos and increases TH2/TH1 ratio through increased number of lymphocytes containing the desired cytokines and or by preferential secretion or TH2 over THI cytokines into the media.
• Such pre-implantation embryo-derived peptide may be used to cause a shift from proinflammatory to anti- inflammatory activities in lymphocytes.
• the pharmaceutical composition comprises a compound of the formula MVRIK (SEQ ID NO: 32).
• the pharmaceutically acceptable carrier is sterile and pyrogen- free water or aqueous buffer, such as saline or Lactated Ringer's solution.
• the therapeutically effective dose is about 1.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is about 2.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is about 3.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is about 4.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose is about 0.2 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is about 0.3 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is about 0.4 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is about 0.5 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose is about 0.6 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is about 0.7 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, the therapeutically effective dose is about 0.8 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the present disclosure also relates to a pharmaceutical composition
• a pharmaceutical composition comprising (i) a therapeutically effective dose of one or a combination of PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof; and (ii) a pharmaceutically acceptable carrier.
• the composition further comprises a therapeutically effective dose of one or a plurality of active agents.
• the one or plurality of active agents is one or a combination of compounds chosen from: an anti-inflammatory compound, alpha-adrenergic agonist, antiarrhythmic compound, analgesic compound, and an anesthetic compound, or a hormone therapy.
• the therapeutically effective dose of one or a combination of PIF peptide or mimetics thereof or thereof analogs thereof or pharmaceutically acceptable salts thereof is about 1.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose of one or a combination of PIF peptide or mimetics thereof or analogs thereof or pharmaceutically acceptable salts thereof is about 2.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose of one or a combination of PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 3.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose of one or a combination of PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 4.0 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose of one or a combination of PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 0.2 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 0.3 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose of one or a combination of PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 0.4 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose of one or a combination of PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 0.5 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose of one or a combination of PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 0.6 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the therapeutically effective dose of one or a combination of PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 0.7 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose. In some embodiments, wherein the therapeutically effective dose of one or a combination of PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 0.8 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• PIF peptide or analogs thereof or pharmaceutically acceptable salts thereof is about 0.9 mg/kg, wherein kg is kilograms of the subject and mg is milligrams of the therapeutically effective dose.
• the composition further comprises one or a plurality of stem cells.
• the stem cell is an autologous stem cell.
• the pharmaceutical composition is administered via parenteral injection, subcutaneous injection, intravenous injection, intramuscular injection,
• intraperitoneal inj ection transdermally, orally, buccally, ocular routes, intravaginally, by inhalation, by depot injections, or by implants.
• compositions further comprise one or a combination of active agents chosen from: an anti-inflammatory compound, alpha-adrenergic agonist, antiarrhythmic compound, analgesic compound, and an anesthetic compound.
• active agents chosen from: an anti-inflammatory compound, alpha-adrenergic agonist, antiarrhythmic compound, analgesic compound, and an anesthetic compound.
• the one or combination of active agents is selected from Table
• the present disclosure also relates to a method of improving the clinical outcome in a subject suffering with, diagnosed with or suspected of having endometriosis comprising administering to the subject at least one pharmaceutical composition comprising: pre- implantation factor (PIF) peptide, an analog thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
• PEF pre- implantation factor
• methods of diagnosing endometriosis in a human subject comprises measuring pre-implantation factor (PIF) protein or mRNA expression levels from an endometrial tissue sample from the subject; and comparing the PIF protein expression levels from the endometrial sample to the PIF expression levels in a control normal sample; wherein the patient is diagnosed with endometriosis if the PIF expression levels from the endometrial sample are greater than the PIF expression levels from the control normal sample.
• PIF pre-implantation factor
• methods of treating endometriosis in a human subject comprise measuring pre-implantation factor (PIF) protein or mRNA expression levels from a endometrial sample from the subject; comparing the PIF protein or mRNA expression levels from the endometrial sample to the PIF expression levels in a control normal sample, wherein the patient is diagnosed with endometriosis if the PIF expression levels from the endometrial sample are greater than the PIF expression levels from the control normal sample; and administering to the subject at least one pharmaceutical composition comprising a therapeutically effective amount of a PIF peptide, mimetics thereof, analogs thereof, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
• PIF pre-implantation factor
• FIG. 1 depicts a graph showing the dose dependent increase in the binding of PIF with
• CD4+/CD25+ cells in contract to a control scrambled-FITC-PIF.
• FIG. 2 depicts a graph showing the dose dependent increase in the binding of PIF with CD4+/CD25+/FoxP3+ cells.
• FIGs. 3A - 3D depict results showing that PIF imparts epithelial ectopic endometria.
• FIG. 3 A depicts PIF staining in epithelial and stromal cells.
• FIG. 3B depicts graphs showing the semi-quantitative evaluation of the PIF positive staining from FIG. 3A.
• FIG. 3C depicts a graph showing the cell viability with increasing sPIF concentration in epithelial cells compared to controls.
• FIG. 3D depicts a graph showing the cell viability with increasing sPIF concentration in stromal cells.
• FIG. 4A depicts results of a global gene array from sPIF treated epithelial ectopic cells lines.
• FIG. 4B depicts a heat map analysis of T-cell receptor genes identified in FIG. 4A.
• FIGs. 5A - 5E depict results showing that PIF interacts with FoxP3 positive cells.
• FIG. 5A depicts FoxP3 and PIF staining in eutopic and ectopic tissues.
• FIG. 5B depicts graphs showing the semi-quantitative evaluation of the PIF positive staining from FIG. 5A.
• FIG. 5C depicts the dose dependent increase in the binding of FITC-PIF to
• FIG. 5D depicts a graph showing the cell viability with increasing sPIF concentration in epithelial cells with TNFa pretreatment.
• FIG. 5E depicts a cartoon showing the hypothesis that PIF re-expresses in the epithelial compartment of ectopic endometria resulting in the recruitment of FoxP3+ cells into the stromal compartment creating a positive feed-back loop leading to cellular survival.
• compositions described herein may include D amino acids, L amino acids, a racemic backbone of D and L amino acids, or any mixture thereof at each residue. That is, at each position, the residue may be a D amino acid residue or a L-amino acid residue and each position can be independently D or L of each other position, unless context dictates otherwise.
• the phrase "in need thereof means that the animal or mammal has been identified or suspected as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis or observation. In any of the methods and treatments described herein, the animal or mammal can be in need thereof.
• the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by 10% and remain within the scope of the disclosed embodiments. Where a numerical value is used with the term “about” the numerical value without the term “about” is also disclosed and can be used without the term “about. "
• the terms “comprising” (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
• integer from X to Y means any integer that includes the endpoints. That is, where a range is disclosed, each integer in the range including the endpoints is disclosed. For example, the phrase “integer from X to Y” discloses about 1, 2, 3, 4, or 5 as well as the range from about 1 to about 5.
• the phrase "therapeutically effective amount” means the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician.
• the therapeutic effect is dependent upon the disorder being treated or the biological effect desired.
• the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and/or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or side-effects.
• the amount needed to elicit the therapeutic response can be determined based on the age, health, size and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject's response to treatment.
• treat can refer to therapeutic treatment and/or prophylactic or preventative measures wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results.
• beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease.
• Treatment can also include eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
• treatment of endometriosis means an activity that prevents, alleviates or ameliorates any of the primary phenomena or secondary symptoms associated with the endometriosis.
• preimplantation factor or “PIF” may also refer to synthetic PIF-1, which replicates the native peptide's effect and exerts potent immune modulatory effects on activated peripheral blood mononuclear cell (PBMC) proliferation and cytokine secretion, acting through novel sites on PBMCs and having an effect which is distinct from known immunosuppressive drugs.
• PBMC peripheral blood mononuclear cell
• preimplantation factor or "PIF” or “PIF analog” refers to an amino acid selected from SEQ ID NO: l, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO:32, or peptidomimetics or mutants thereof, and combinations thereof that are about 75, 80, 81, 82, 83, 84 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to any such amino acid.
• the terms "PIF,” “PIF peptide,” or “PIF analog” refers to an amino acid sequence of Table 1 or any peptidomimetics, mutant or analog thereof, that is from about 75% to about 100% homologous to any sequence of Table 1, optionally fused or unfused to one or more other amino acid sequences at its carboxy and/or its amino terminal ends.
• compositions of the present disclosure relate to any or all of the compounds or PIF peptides disclosed herein or their respective pharmaceutically effective salts or polymorphs.
• the compounds described herein may act as agonists of PIF-mediated signal transduction via the receptor or receptors of PIF.
• these compounds modulate signaling pathways that provide significant therapeutic benefit in the treatment of, but not limited to, endometriosis.
• the compounds of the present disclosure may exist in unsolvated forms as well as solvated forms, including hydrated forms.
• the compounds of the present disclosure also are capable of forming both pharmaceutically acceptable salts, including but not limited to acid addition and/or base addition salts.
• compounds of the present disclosure may exist in various solid states including an amorphous form (non-crystalline form), and in the form of clathrates, prodrugs, polymorphs, bio-hydrolyzable esters, racemic mixtures, non-racemic mixtures, or as purified stereoisomers including, but not limited to, optically pure enantiomers and diastereomers.
• amorphous form non-crystalline form
• prodrugs polymorphs
• bio-hydrolyzable esters racemic mixtures
• non-racemic mixtures or as purified stereoisomers including, but not limited to, optically pure enantiomers and diastereomers.
• all of these forms can be used as an alternative form to the free base or free acid forms of the compounds, as described above and are intended to be encompassed within the scope of the present disclosure.
• a "polymorph” refers to solid crystalline forms of a compound. Different polymorphs of the same compound can exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to stability (e.g., to heat or light), compressibility and density (important in formulation and product manufacturing), and dissolution rates (which can affect bioavailability). Different physical properties of polymorphs can affect their processing. The disclosure relates to a polymorph of any of the disclosed PIF peptides.
• the compounds of the present disclosure can be administered, inter alia, as pharmaceutically acceptable salts, esters, amides or prodrugs.
• salts refers to inorganic and organic salts of compounds of the present disclosure.
• the salts can be prepared in situ during the final isolation and purification of a compound, or by separately reacting a purified compound in its free base or acid form with a suitable organic or inorganic base or acid and isolating the salt thus formed.
• Representative salts include the
• the salts may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. See, for example, S. M. Berge, et al., "Pharmaceutical Salts," J Pharm Sci, 66: 1-19 (1977).
• salt refers to acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. Examples of these acids and bases are well known to those of ordinary skill in the art. Such acid addition salts will normally be pharmaceutically acceptable although salts of non-pharmaceutically acceptable acids may be of utility in the preparation and purification of the compound in question. Salts include those formed from hydrochloric, hydrobromic, sulphuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic, succinic, fumaric, maleic, methanesulphonic and benzenesulphonic acids.
• salts of the compositions comprising either a PIF or PIF analog or PIF mutant may be formed by reacting the free base, or a salt, enantiomer or racemate thereof, with one or more equivalents of the appropriate acid.
• pharmaceutical acceptable salts of the present disclosure refer to analogs having at least one basic group or at least one basic radical.
• pharmaceutical acceptable salts of the present disclosure comprise a free amino group, a free guanidino group, a pyrazinyl radical, or a pyridyl radical that forms acid addition salts.
• the pharmaceutical acceptable salts of the present disclosure refer to analogs that are acid addition salts of the subject compounds with (for example) inorganic acids, such as hydrochloric acid, sulfuric acid or a phosphoric acid, or with suitable organic carboxylic or sulfonic acids, for example aliphatic mono- or di-carboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or amino acids such as arginine or lysine, aromatic carboxylic acids, such as benzoic acid, 2-phenoxy- benzoic acid, 2-acetoxybenzoic acid, salicylic acid, 4-aminosalicylic acid, aromatic-aliphatic carboxylic acids, such as mandelic acid or cinnamic acid, heteroaromatic carboxylic acids, such as nicotinic acid
• salts may be formed.
• the reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, for example, water, dioxane, ethanol, tetrahydrofuran or diethyl ether, or a mixture of solvents, which may be removed in vacuo or by freeze drying.
• the reaction may also be a metathetical process or it may be carried out on an ion exchange resin.
• the salts may be those that are physiologically tolerated by a patient. Salts according to the present disclosure may be found in their anhydrous form or as in hydrated crystalline form (i.e., complexed or crystallized with one or more molecules of water).
• esters of the compounds of the present disclosure include Ci-Cg alkyl esters. Acceptable esters also include C5-C7 cycloalkyl esters, as well as arylalkyl esters such as benzyl. C1-C4 alkyl esters are commonly used. Esters of compounds of the present disclosure may be prepared according to methods that are well known in the art. Examples of pharmaceutically acceptable amides of the compounds of the present disclosure include amides derived from ammonia, primary Ci-Cg alkyl amines, and secondary Ci-Cg dialkyl amines.
• the amine may also be in the form of a 5 or 6 membered heterocycloalkyl group containing at least one nitrogen atom.
• Amides derived from ammonia, C1-C3 primary alkyl amines and C1-C2 dialkyl secondary amines are commonly used. Amides of the compounds of the present disclosure may be prepared according to methods well known to those skilled in the art.
• administering when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted.
• administering when used in conjunction with PIF, can include, but is not limited to, providing PIF peptide into or onto the target tissue; providing PIF peptide systemically to a patient by, e.g., intravenous injection whereby the therapeutic reaches the target; providing PIF peptide in the form of the encoding sequence thereof to the target (e.g., by so-called gene-therapy techniques).
• administering a composition may be accomplished by parenteral, oral or topical administration.
• the terms “pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, rash, or gastric upset.
• the therapeutic composition is not immunogenic when administered to a subject for therapeutic purposes.
• the terms “pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, rash, or gastric upset.
• the therapeutic composition is not immunogenic when administered to a subject for therapeutic purposes.
• the term "therapeutic” means an agent utilized to treat, combat, ameliorate, prevent or improve a subject with endometriosis or at risk of developing endometriosis.
• therapeutics include, but are not limited to, the PIF peptides described herein.
• a “therapeutically effective amount” or “effective amount” or “physiologically relevant amount” of a composition is an amount calculated to achieve a desired effect, i.e., to effectively inhibit or reduce symptoms and/or complications associated with endometriosis.
• Effective amounts of compounds of the present disclosure can objectively or subjectively reduce or decrease the severity or frequency of symptoms associated with endometriosis.
• the specific dose of a compound administered according to this disclosure to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated.
• the compounds are effective over a wide dosage range and, for example, dosages per day will normally fall within the range of from about 0.01 mg/kg to about 100 mg/kg, more preferably about 0.1 mg/kg to about 1 mg/kg.
• the therapeutically effective dose of PIF or PIF analog or peptide is about O. lmg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, and lmg/kg, where "mg" is milligram of PIF analog or peptide "kg" is kilogram of the subject.
• the pharmaceutical compositions comprise a therapeutically effective amount of PIF peptide or analog but the composition is free of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.
• a therapeutically effective amount of compound of this disclosure is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the tissue.
• the term "therapeutically effective amount” as used herein refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
• the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment.
• the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment. The specific
• therapeutically-effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient: the time of administration, route of
• the therapeutically effective amount is advantageously selected with reference to any toxicity, or other undesirable side effect, that might occur during administration of one or more of the compounds described herein.
• the co-therapies described herein may allow for the administration of lower doses of compounds that show such toxicity, or other undesirable side effect, where those lower doses are below thresholds of toxicity or lower in the therapeutic window than would otherwise be administered in the absence of a co-therapy.
• composition generally refers to any product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. It is to be understood that the compositions described herein may be prepared from isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and/or other morphological forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various hydrates and/or solvates of the compounds described herein. Accordingly, such pharmaceutical compositions that recite compounds described herein are to be understood to include each of, or any combination of, the various morphological forms and/or solvate or hydrate forms of the compounds described herein.
• compositions may include one or more carriers, diluents, and/or excipients.
• the compounds described herein, or compositions containing them may be formulated in a therapeutically effective amount in any conventional dosage forms appropriate for the methods described herein.
• the compounds described herein, or compositions containing them, including such formulations may be administered by a wide variety of conventional routes for the methods described herein, and in a wide variety of dosage formats, utilizing known procedures (see generally, Remington: The Science and Practice of Pharmacy, (21st ed., 2005)).
• beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease.
• Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
• Immuno-modulating refers to the ability of a compound of the present disclosure to alter (modulate) one or more aspects of the immune system.
• the immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.
• Auto-immune disease refers to various diseases that arise from an abnormal immune response of the body against substances and tissues normally present in the body. This may be restricted to certain organs or involve a particular tissue in different places. A large number of auto-immune diseases are recognized, including, but not limited to,
• Hashimoto's thyroiditis pernicious anemia, Addison's disease, type I (insulin dependent) diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and Grave's disease, alopecia greata, anklosing spondylitis, antiphospholipid syndrome, autoimmune hemolytic anemia, auto-immune hepatitis, auto-immune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), auto-immune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial
• Inflammatory response or “inflammation” is a general term for the local accumulation of fluid, plasma proteins, and white blood cells initiated by physical injury, infection, or a local immune response. Inflammation is an aspect of many diseases and disorders, including but not limited to diseases related to immune disorders, viral infection, arthritis, autoimmune diseases, collagen diseases, allergy, asthma, pollinosis, and atopy. Inflammation is characterized by rubor (redness), dolor (pain), calor (heat) and tumor (swelling), reflecting changes in local blood vessels leading to increased local blood flow which causes heat and redness, migration of leukocytes into surrounding tissues
• Inflammation is often accompanied by loss of function due to replacement of parenchymal tissue with damaged tissue (e.g., in damaged myocardium), reflexive disuse due to pain, and mechanical constraints on function, e.g., when a joint swells during acute inflammation, or when scar tissue bridging an inflamed joint contracts as it matures into a chronic inflammatory lesion.
• Anti-inflammatory refers to the ability of a compound to prevent or reduce the inflammatory response, or to soothe inflammation by reducing the symptoms of inflammation such as redness, pain, heat, or swelling.
• Inflammatory responses can be triggered by injury, for example injury to skin, muscle, tendons, or nerves. Inflammatory responses can also be triggered as part of an immune response. Inflammatory responses can also be triggered by infection, where pathogen recognition and tissue damage can initiate an inflammatory response at the site of infection. Generally, infectious agents induce inflammatory responses by activating innate immunity.
• Inflammation combats infection by delivering additional effector molecules and cells to augment the killing of invading microorganisms by the frontline macrophages, by providing a physical barrier preventing the spread of infection, and by promoting repair of injured tissue.
• “Inflammatory disorder” is sometimes used to refer to chronic inflammation due to any cause.
• immune disorder and "inflammatory response” are not exclusive. It is understood that many immune disorders include acute (short term) or chronic (long term) inflammation. It is also understood that inflammation can have immune aspects and non-immune aspects. The role(s) of immune and nonimmune cells in a particular inflammatory response may vary with the type of inflammatory response, and may vary during the course of an inflammatory response. Immune aspects of inflammation and diseases related to inflammation can involve both innate and adaptive immunity.
• Certain diseases related to inflammation represent an interplay of immune and nonimmune cell interactions, for example intestinal inflammation (Fiocchi et al, 1997 Am J Physiol Gastrointest Liver Physiol 273: G769-G775), pneumonia (lung inflammation), or glomerulonephritis.
• conservative amino acid substitutions may be defined as set out in Tables A, B, or C below.
• the PIF compounds of the disclosure include those wherein conservative substitutions (from either nucleic acid or amino acid sequences) have been introduced by modification of polynucleotides encoding polypeptides of the disclosure.
• Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure.
• a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
• the conservative substitution is recognized in the art as a substitution of one nucleic acid for another nucleic acid that has similar properties, or, when encoded, has similar binding affinities. Exemplary conservative substitutions are set out in Table A.
• inhibitors described herein are intended to include nucleic acids and, where the inhibitors include polypeptide, polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues.
• the peptides of the disclosure can be of any length.
• the peptides can have from about two to about 100 or more residues, such as, about 4 to about 15, about 12 to about 15, about 8 to about 18, about 18 to about 25, about 15 to about 50,about 50 to about 75, or about 75 to about 100 or more amino acids in length.
• peptides are from about 4 to about 18 residues in length.
• the peptides of the disclosure also include 1- and d-isomers, and combinations of 1 - and d-isomers.
• compositions or pharmaceutical compositions of the disclosure relate to analogs of any PIF sequence set forth in Table 1 that share no less than about 70%, about 75%, about 79%, about 80%, about 85%, about 86%, about 87%, about 90%, about 93%, about 94% about 95%, about 96%, about 97%, about 98%, about 99% homology with any one or combination of PIF sequences set forth in Table 1.
• PIF or PIF peptide may refer to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13 , 14 ,15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or a functional fragment thereof that is about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to any such amino acid sequence.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 20.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 21.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 22.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 23.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 24.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 25.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 26.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 27.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 28.
• PIF may refer to an amino acid sequence comprising, consisting essentially of, or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID. NO: 29.
• the PIF mutant comprises a sequence selected from: XVZIKPGSANKPSD, XVZIKPGSANKPS XVZIKPGSANKP XVZIKPGSANK
• XVZIKPGSAN XVZIKPGSA, XVZIKPGS, XVZIKPG, XVZIKP, XVZIK, XVZI, XVZ wherein X is a non-natural amino acid or a naturally occurring amino acid.
• the PIF mutant comprises a sequence selected from: XVZIKPGSANKPSD, XVZIKPGSANKPS XVZIKPGSANKP XVZIKPGSANK XVZIKPGSAN, XVZIKPGSA, XVZIKPGS, XVZIKPG, XVZIKP, XVZIK, XVZI, XVZ wherein X is a non-natural amino acid or a naturally occurring amino acid except that X is not methionine if Z is arginine, and Z is not arginine if X is methionine.
• the PIF analog or mutant is synthetic or synthetically made.
• Peptides disclosed herein further include compounds having amino acid structural and functional analogs, for example, peptidomimetics having synthetic or non-natural amino acids (such as a norleucine) or amino acid analogues or non-natural side chains, so long as the mimetic shares one or more functions or activities of compounds of the disclosure.
• the compounds of the disclosure therefore include "mimetic” and "peptidomimetic” forms.
• a "non-natural side chain” is a modified or synthetic chain of atoms joined by covalent bond to the a-carbon atom, ⁇ -carbon atom, or ⁇ -carbon atom which does not make up the backbone of the polypeptide chain of amino acids.
• the peptide analogs may comprise one or a combination of non-natural amino-acids chosen from: norvaline, tert-butylglycine, phenylglycine, He, 7-azatryptophan, 4-fluorophenylalanine, N-methyl-methionine, N-methyl- valine, N-methyl-alanine, sarcosine, N-methyl-tert-butylglycine, N-methyl-leucine, N- methyl-phenylglycine, N-methyl-isoleucine, N-methyl-tryptophan, N-methyl-7- azatryptophan, N-methyl-phenylalanine, N-methyl-4-fluorophenylalanine, N-methyl- threonine, N-methyl-tyrosine, N-methyl-valine, N-methyl-lysine, homocysteine, and Tyr;
• Xaa2 is absent, or an amino acid selected from the group consisting of Ala,
• the natural side chain, or R group, of an alanine is a methyl group.
• the non-natural side chain of the composition is a methyl group in which one or more of the hydrogen atoms is replaced by a deuterium atom.
• Non-natural side chains are disclosed in the art in the following publications: WO/2013/172954, WO2013123267, WO/2014/071241, WO/2014/138429,
• peptide mimetics include recombinantly or chemically modified peptides, as well as non-peptide agents such as small molecule drug mimetics, as further described below.
• analog refers to any polypeptide comprising at least one a-amino acid and at least one non-native amino acid residue, wherein the polypeptide is structurally similar to a naturally occurring full-length PIF protein and shares the biochemical or biological activity of the naturally occurring full-length protein upon which the analog is based.
• compositions, pharmaceutical compositions and kits comprise a peptide or peptidomimeic sharing share no less than about 70%, about 75%, about 79%, about 80%, about 85%, about 86%, about 87%, about 90%, about 93%, about 94% about 95%, about 96%, about 97%, about 98%, about 99% homology with any one or combination of PIF sequences set forth in Table 1; and wherein one or a plurality of amino acid residues is a non-natural amino acid residue or an amino acid residue with a non-natural sidechain.
• peptide or peptide mimetics are provided, wherein a loop is formed between two cysteine residues.
• the peptidomimetic may have many similarities to natural peptides, such as: amino acid side chains that are not found among the known 20 proteinogenic amino acids, non-peptide-based linkers used to effect cyclization between the ends or intemal portions of the molecule, substitutions of the amide bond hydrogen moiety by methyl groups (N- methylation) or other alkyl groups, replacement of a peptide bond with a chemical group or bond that is resistant to chemical or enzymatic treatments, N- and C-terminal modifications, and conjugation with a non-peptidic extension (such as polyethylene glycol, lipids, carbohydrates, nucleosides, nucleotides, nucleoside bases, various small molecules, or phosphate or sulfate groups).
• a non-peptidic extension such as polyethylene glycol, lipids, carbohydrates, nucleosides, nucleotides, nucleoside bases, various small molecules, or phosphate or sulfate groups.
• cyclic peptide mimetic or "cyclic polypeptide mimetic” refers to a peptide mimetic that has as part of its structure one or more cyclic features such as a loop, bridging moiety, and/or an intemal linkage.
• bridging moiety refers to one or a series of bonded atoms that covalently link one or a plurality of amino acid side chains to one another within an amino acid sequence.
• peptide or peptide mimetics are provided, wherein the loop comprises a bridging moiety selected from the group consisting of:
• the PIF peptides of the disclosure are modified to produce peptide mimetics by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains, for instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7 membered alkyl, amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7 membered heterocyclics.
• proline analogs can be made in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members.
• Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or nonaromatic. Heterocyclic groups can contain one or more nitrogen, oxygen, and/or sulphur heteroatoms. Examples of such groups include the furazanyl,furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino ), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g.
• the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.
• Peptidomimetics may also have amino acid residues that have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties.
• a compound of the formula R1-R2-R3-R4-R5-R6-R7-R.8- R9- R10-R11-R12-R13-R14-R15 wherein Ri is Met or a mimetic of Met, R2 is Val or a mimetic of Val, R 3 is Arg or a mimetic of Arg, or any amino acid, R4 is He or a mimetic of He, R5 is Lys or a mimetic of Lys, R6 is Pro or a mimetic of Pro, R7 is Gly or a mimetic of Gly, Rg is Ser or a mimetic of Ser, R 9 is Ala or a mimetic of Ala, Rio is Asn or a mimetic of Asn, Rn is Lys or a mimetic of Lys, R12 is Pro or a mimetic of Pro, R13 is Ser or a mimetic of Ser, R14 is Asp or a mimetic of As
• a compound comprising the formula R1-R2-R3-R4-R5-R6-R7-R8- R9-R10-R11-R12-R13-R14-R15, wherein Ri is a mimetic of the naturally occurring residue at position 1 of SEQ ID NO: 20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29 or the residue at that position of such sequences; wherein R 2 is a mimetic of the naturally occurring residue at position 2 of SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 , SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29 or the residue at that position of such sequences; wherein
• Ri 4 is a mimetic of the naturally occurring residue at position 14 of SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29 or the residue at that position of such sequences;
• Ri 5 is a mimetic of the naturally occurring residue at position 15 of SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29 or the residue at that position of such sequences.
• the pharmaceutical composition comprising the formula Ri- R2-R3-R4-R5-R0-R7-R8- R9-R10-R11-R12-R13-R14-R15-R16-R17-R18, wherein Ri is Ser or a mimetic of Ser, R 2 is Gly or a mimetic of Gly, R3 is He or a mimetic of He, R4 is Val or a mimetic of Val, R5 is He or a mimetic of He, R6 is Tyr or a mimetic of Tyr, R7 is Gin or a mimetic of Gin, R 8 is Tyr or a mimetic of Tyr, R 9 is Met or a mimetic of Met, Rio is Asp or a mimetic of Asp, Rn is Asp or a mimetic of Asp, R 12 is Arg or a mimetic of Arg, R13 is Tyr or a mimetic of Tyr, R M is Val or a mimetic of Val, R15
• Certain peptidomimetic compounds are based upon the amino acid sequence of the peptides of the disclosure. Often, peptidomimetic compounds are synthetic compounds having a three dimensional structure (i.e. a "peptide motif) based upon the three-dimensional structure of a selected peptide.
• the peptide motif provides the peptidomimetic compound with the desired biological activity, i.e., binding to PIF receptors, wherein the binding activity of the mimetic compound is not substantially reduced, and is often the same as or greater than the activity of the native peptide on which the mimetic is modeled.
• Peptidomimetic compounds can have additional characteristics that enhance their therapeutic application, such as increased cell permeability, greater affinity and/or avidity and prolonged biological half-life.
• Peptidomimetic design strategies are readily available in the art (see, e.g., Ripka & Rich, Curr. Op. Chern. Bioi. 2,441-452,1998; Hruby et al., Curr. Op.Chem. Bioi. 1,114- 119,1997; Hruby & Baise, Curr.Med. Chern. 9,945-970,2000).
• One class of peptidomimetics a backbone that is partially or completely non-peptide, but mimics the peptide backbone atom-for atom and comprises side groups that likewise mimic the functionality of the side groups of the native amino acid residues.
• peptidomimetics Several types of chemical bonds, e.g., ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics.
• Another class of peptidomimetics comprises a small non- peptide molecule that binds to another peptide or protein, but which is not necessarily a structural mimetic of the native peptide.
• Yet another class of peptidomimetics has arisen from combinatorial chemistry and the generation of massive chemical libraries. These generally comprise novel templates which, though structurally unrelated to the native peptide, possess necessary functional groups positioned on a nonpeptide scaffold to serve as "topographical" mimetics of the original peptide (Ripka & Rich, 1998, supra).
• PIF peptides A list of PIF peptides are provided below in Table 1. Antibodies to various PIF peptides and scrambled PIF peptides are also provided.
• a PIF peptide (or analog) is provided.
• the PIF analog binds or associates with human insulin degrading enzyme (IDE - SEQ ID NO: 30) at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or higher than native or wild-type PIF sequences.
• the PIF analog may have a binding affinity for insulin degrading enzyme (IDE) that has at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or higher than native or wild-type PIF sequences.
• the PIF analog may have a binding affinity for insulin degrading enzyme that has from about 1% to about 30% or higher than the affinity native or wild-type PIF sequences have for IDE. In some embodiments, the PIF analog may have a binding affinity for insulin degrading enzyme that has from about 1% to about 10% or higher than the affinity native or wild-type PIF sequences have for IDEIn some embodiments, the PIF analog may have a binding affinity for insulin degrading enzyme that has from about 1% to about 20% or higher than the affinity native or wild-type PIF sequences have for IDE.
• the PIF analog may have a binding affinity for insulin degrading enzyme that has from about 10% to about 20% or higher than the affinity native or wild-type PIF sequences have for IDE.
• PIF peptides in therapeutically effective amounts may be useful for treating any of the diseases or disorder disclosed herein.
• a pharmaceutical composition comprising a PIF peptide
• the pharmaceutical composition comprises a therapeutically effective amount of a PIF peptide or a pharmaceutically acceptable salt thereof.
• a method of treating endometriosis comprises administering an effective amount of a PIF peptide to a subject in need thereof.
• a method for treating endometriosis comprising administering an effective amount of a PIF peptide in combination with one or more immunotherapeutic, anti-epileptic, diuretic, or blood pressure controlling drugs or compounds to a subject in need thereof is provided. Such a combination may enhance the effectiveness of the treatment of either component alone, or may provide less side effects and/or enable a lower dose of either component.
• a PIF peptide is provided.
• Such PIF peptides may be useful for treating or ameliorating immune-mediated disorders, such as autoimmune diseases, such as endometriosis.
• the PIF peptide can be, for example, as described herein.
• a pharmaceutical composition comprising a PIF peptide or a pharmaceutically acceptable salt thereof.
• the pharmaceutical composition comprises a therapeutically effective amount of a PIF peptide or a pharmaceutically acceptable salt thereof.
• a PIF peptide may be administered as such, or can be compounded and formulated into pharmaceutical compositions in unit dosage form for parenteral, transdermal, rectal, nasal, local intravenous administration, or, preferably, oral administration.
• Such pharmaceutical compositions are prepared in a manner well known in the art and comprise at least one active PIF peptide associated with a pharmaceutically carrier.
• active compound refers to at least one compound selected from compounds of the formulas or pharmaceutically acceptable salts thereof.
• the active compound is known as "active ingredient.”
• the active ingredient will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier that may be in the form of a capsule, sachet, paper or other container.
• the carrier serves as a diluent, it may be a solid, semisolid, or liquid material that acts as a vehicle, excipient of medium for the active ingredient.
• the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, emulsion, solutions, syrups, suspensions, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.
• pharmaceutical preparation or “pharmaceutical composition” includes preparations suitable for administration to mammals, e.g., humans.
• pharmaceutical composition containing, for example, from about 0.1 to about 99.5% of active ingredient in combination with a pharmaceutically acceptable carrier.
• phrases "pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present disclosure to mammals.
• the carriers include, for example, liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
• Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
• materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;
• esters such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;
• Ringer's solution ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
• suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin, which is incorporated herein by reference in its entirety.
• the pharmaceutically acceptable carrier is sterile and pyrogen-free water.
• the pharmaceutically acceptable carrier is Ringer's Lactate, sometimes known as lactated Ringer's solution.
• wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
• antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), lecithin, propyl gallate, .alpha. -tocopherol, and the like; and metal chelating agents, such as citric acid,
• EDTA ethylenediamine tetraacetic acid
• sorbitol sorbitol
• tartaric acid tartaric acid
• phosphoric acid and the like.
• Formulations of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal, vaginal and/or parenteral administration.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
• the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety -nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
• suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate alginates, calcium salicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, tragacanth, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, tale, magnesium stearate, water, and mineral oil.
• the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
• the compositions may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
• a compound for oral administration, can be admixed with carriers and diluents, molded into tablets, or enclosed in gelatin capsules.
• the mixtures can alternatively be dissolved in liquids such as 10% aqueous glucose solution, isotonic saline, sterile water, or the like, and administered intravenously or by injection.
• the local delivery of inhibitory amounts of active compound for the treatment of immune disorders can be by a variety of techniques that administer the compound at or near the targeted site.
• Examples of local delivery techniques are not intended to be limiting but to be illustrative of the techniques available. Examples include local delivery catheters, site specific carriers, implants, direct injection, or direct applications, such as topical application.
• Local delivery by an implant describes the surgical placement of a matrix that contains the pharmaceutical agent into the affected site.
• the implanted matrix releases the pharmaceutical agent by diffusion, chemical reaction, or solvent activators.
• the disclosure is directed to a pharmaceutical composition comprising a PIF peptide, and a pharmaceutically acceptable carrier or diluent, or an effective amount of pharmaceutical composition comprising a PIF peptide.
• the compounds of the present disclosure can be administered in the conventional manner by any route where they are active. Administration can be systemic, topical, or oral. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, ocular routes, intravaginally, by inhalation, by depot injections, or by implants.
• modes of administration for the compounds of the present disclosure can be, but are not limited to, subligual, injectable (including short-acting, depot, implant and pellet forms inj ected subcutaneously or intramuscularly), or by use of vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams.
• Specific modes of administration will depend on the indication.
• the selection of the specific route of administration and the dose regimen is to be adjusted or titrated by the clinician according to methods known to the clinician in order to obtain the optimal clinical response.
• the amount of compound to be administered is that amount which is
• the dosage to be administered will depend on the characteristics of the subject being treated, e.g., the particular mammal or human treated, age, weight, health, types of concurrent treatment, if any, and frequency of treatments, and can be easily determined by one of skill in the art (e.g., by the clinician).
• compositions containing the compounds of the present disclosure and a suitable carrier can be solid dosage forms which include, but are not limited to, tablets, capsules, cachets, pellets, pills, powders and granules; topical dosage forms which include, but are not limned to, solutions, powders, fluid emulsions, fluid suspensions, semi-solids, ointments, pastes, creams, gels and jellies, and foams; and parenteral dosage forms which include, but are not limited to, solutions, suspensions, emulsions, and dry powder; comprising an effective amount of a polymer or copolymer of the present disclosure. It is also known in the art that the active ingredients can be contained in such formulations with
• diluents fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like.
• the means and methods for administration are known in the art and an artisan can refer to various pharmacologic references for guidance. For example, Modern Pharmaceutics , Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Oilman 's The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980) can be consulted.
• the compounds of the present disclosure can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
• the compounds can be administered by continuous infusion subcutaneously over a predetermined period of time.
• Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the compounds can be formulated readily by combining these compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
• Pharmaceutical preparations for oral use can be obtained by adding a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, alter adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, but not limited to, maize starch, wheat starch, rice starch, potato starch, gelatin, gum horrcanth, methyl cellulose,
• disintegrating agents can be added, such as, but not limited to, the cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores can be provided with suitable coatings.
• suitable coatings can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally include, but are not limited to, push-fit capsules made of gelatin, as well as soft, scaled capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with filler such as, e.g., lactose, binders such as, e.g., starches, and/or lubricants such as, e.g., talc or magnesium stearate and, optionally, stabilizers.
• the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.
• compositions can take the form of, e.g., tablets or lozenges formulated in a conventional manner.
• the compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
• the compounds of the present disclosure can also be formulated in rectal
• compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
• the compounds of the present disclosure can also be formulated as a depot preparation.
• Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• the compounds of the present disclosure can be applied to a plaster, or can be applied by transdermal, therapeutic systems that are consequently supplied to the organism.
• compositions of the compounds also can comprise suitable solid or gel phase carriers or excipients.
• suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivates, gelatin, and polymers such as, e.g., polyethylene glycols.
• analog can be, for example, formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
• parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used.
• the vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives).
• the formulation is sterilized by commonly used techniques.
• a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of analog in 0.9% sodium chloride solution.
• the pharmaceutical composition may be introduced by various means into cells that are removed from the individual.
• Such means include, for example, microprojectile bombardment and liposome or other nanoparticle device.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
• the analogs are generally admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, starch, or other generally regarded as safe (GRAS) additives.
• Such dosage forms can also comprise, as is normal practice, an additional substance other than an inert diluent, e.g., lubricating agent such as magnesium state.
• the dosage forms may also comprise a buffering agent. Tablets and pills can additionally be prepared with enteric coatings, or in a controlled release form, using techniques know in the art.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions and syrups, with the elixirs containing an inert diluent commonly used in the art, such as water. These compositions can also include one or more adjuvants, such as wetting agent, an emulsifying agent, a suspending agent, a sweetening agent, a flavoring agent or a perfuming agent.
• adjuvants such as wetting agent, an emulsifying agent, a suspending agent, a sweetening agent, a flavoring agent or a perfuming agent.
• the compounds and compositions described are used to treat a patient suffering from, or susceptible to endometriosis and an autoimmune disease.
• the appropriate dosage of the compositions and pharmaceutical compositions may vary depending on the individual being treated and the purpose. For example, the age, body weight, and medical history of the individual patient may affect the therapeutic efficacy of the therapy. Further, a lower dosage of the composition may be needed to produce a transient cessation of symptoms, while a larger dose may be needed to produce a complete cessation of symptoms associated with the disease, disorder, or indication. A competent physician can consider these factors and adjust the dosing regimen to ensure the dose is achieving the desired therapeutic outcome without undue experimentation. It is also noted that the clinician and/or treating physician will know how and when to interrupt, adjust, and/or terminate therapy in conjunction with individual patient response. Dosages may also depend on the strength of the particular analog chosen for the
• the dose of the composition or pharmaceutical compositions may vary.
• the dose of the composition may be once per day. In some embodiments, multiple doses may be administered to the subject per day.
• the total dosage is administered in at least two application periods. In some embodiments, the period can be an hour, a day, a month, a year, a week, or a two-week period. In an additional embodiment of the invention, the total dosage is administered in two or more separate application periods, or separate doses over the course of about an hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 or more hours, a day, a month, a year, a week, or a two-week period.
• subj ects can be administered the composition in which the composition is provided in a daily dose range of about 0.0001 mg/kg to about 5000 mg/kg of the weight of the subject.
• the dose administered to the subject can also be measured in terms of total amount of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof administered per day.
• a subject is administered from about 0.001 to about 3000 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per day.
• a subject is administered up to about 2000 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per day.
• a subject is administered up to about 1800 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per day. In some embodiments, a subject is administered up to about 1600 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per day. In some embodiments, a subject is administered up to about 1400 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per day. In some embodiments, a subject is administered up to about 1200 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per day. In some embodiments, a subject is administered up to about 1000 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per day.
• a subject is administered up to about 800 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per day. In some embodiments, a subject is administered from about 0.001 milligrams to about 700 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per dose. In some embodiments, a subject is administered up to about 700 milligrams of PIF peptide or PIF analog per dose. In some embodiments, a subject is administered up to about 600 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per dose. In some embodiments, a subject is administered up to about 500 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per dose.
• a subject is administered up to about 400 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per dose. In some embodiments, a subject is administered up to about 300 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per dose. In some embodiments, a subject is administered up to about 200 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per dose. In some embodiments, a subject is administered up to about 100 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per dose. In some embodiments, a subject is administered up to about 50 milligrams of PIF peptide or PIF analog or pharmaceutically acceptable salt thereof per dose.
• subjects can be administered the composition in which the composition comprising a PIF peptide or PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dose range of about 0.0001 mg/kg to about 5000 mg/kg of the weight of the subject. In some embodiments, the composition comprising a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 450 mg/kg of the weight of the subject. In some embodiments, the composition comprising a PIF peptide or PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 400 mg/kg of the weight of the subject.
• the composition comprising a PIF peptide or PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 350 mg/kg of the weight of the subject. In some embodiments, the composition comprising a PIF peptide or PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 300 mg/kg of the weight of the subject. In some embodiments, the composition comprising a PIF peptide or PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 250 mg/kg of the weight of the subject. In some embodiments, the composition comprising PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 200 mg/kg of the weight of the subject.
• the composition comprising PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 150 mg/kg of the weight of the subject. In some embodiments, the composition comprising a PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 100 mg/kg of the weight of the subject. In some embodiments, the composition comprising a PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 50 mg/kg of the weight of the subject. In some embodiments, the composition comprising PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 25 mg/kg of the weight of the subject.
• the composition comprising a PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 10 mg/kg of the weight of the subject. In some embodiments, the composition comprising PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 5 mg/kg of the weight of the subject. In some embodiments, the composition comprising a PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 2.0 mg/kg of the weight of the subject.
• the composition comprising a PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 3.0 mg/kg of the weight of the subject. In some embodiments, the composition comprising PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 1 mg/kg of the weight of the subject. In some embodiments, the composition comprising a PIF peptide or a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 0.1 mg/kg of the weight of the subject.
• the composition comprising a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 0.01 mg/kg of the weight of the subj ect. In some embodiments, the composition comprising a PIF analog or pharmaceutically acceptable salt thereof is administered in a daily dosage of up to about 0.001 mg/kg of the weight of the subject.
• the dose administered to the subject can also be measured in terms of total amount of a PIF peptide or PIF analog administered per day.
• a subject in need thereof is administered from about 1 ng to about 500 ⁇ g of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 1 ng to about 10 ng of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 10 ng to about 20 ng of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 10 ng to about 100 ng of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 100 ng to about 200 ng of analog or pharmaceutically salt thereof per day.
• a subject in need thereof is administered from about 200 ng to about 300 ng of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 300 ng to about 400 ng of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 400 ng to about 500 ng of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 500 ng to about 600 ng of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 600 ng to about 700 ng of analog or pharmaceutically salt thereof per day.
• a subject in need thereof is administered from about 800 ng to about 900 ng of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 900 ng to about 1 ⁇ g of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 1 ⁇ g to about 100 ⁇ g of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 100 ⁇ g to about 200 ⁇ g of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 200 ⁇ g to about 300 ⁇ g of analog or pharmaceutically salt thereof per day.
• a subject in need thereof is administered from about 300 ⁇ g to about 400 ⁇ g of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 400 ⁇ g to about 500 ⁇ g of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 500 ⁇ g to about 600 ⁇ g of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 600 ⁇ g to about 700 ⁇ g of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 800 ⁇ g to about 900 ⁇ g of analog or pharmaceutically salt thereof per day. In some embodiments, a subject in need thereof is administered from about 900 ⁇ g to about 1 mg of analog or pharmaceutically salt thereof per day.
• a subject in need thereof is administered from about .0001 to about 3000 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per day. In some embodiments, a subject is administered up to about 2000 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof day. In some embodiments, a subject is administered up to about 1800 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per day. In some embodiments, a subject is administered up to about 1600 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per day.
• a subject is administered up to about 1400 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per day. In some embodiments, a subject is administered up to about 1200 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per day. In some embodiments, a subject is administered up to about 1000 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per day. In some embodiments, a subject is administered up to about 800 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per day.
• a subject is administered from about 0.0001 milligrams to about 700 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 700 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 600 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 500 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose.
• a subject is administered up to about 400 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 300 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 200 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 100 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 50 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose.
• a subj ect is administered up to about 25 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 15 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 50, 60, 70, 80, 90, or 100 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose.
• a subject is administered up to about 10 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 5 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 1 milligram of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 0.1 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose. In some embodiments, a subject is administered up to about 0.001 milligrams of a PIF peptide or PIF analog or pharmaceutically salt thereof per dose.
• the dose administered to the subject can also be measured in terms of total amount of a PIF peptide or PIF analog or pharmaceutically salt thereof administered per ounce of liquid prepared.
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 2.5 grams per ounce of solution.
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 2.25 grams per ounce of solution.
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 2.25 grams per ounce of solution.
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 2.0 grams per ounce of solution.
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 1.9 grams per ounce of solution.
• the PIF analog or pharmaceutically salt thereof is at a concentration of about 1.8 grams per ounce of solution. In some embodiments, the PIF analog or pharmaceutically salt thereof is at a concentration of about 1.7 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 1.6 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 1.5 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 1.4 grams per ounce of solution.
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 1.3 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 1.2 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 1.1 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or
• pharmaceutically salt thereof is at a concentration of about 1.0 grams per ounce of solution.
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.9 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.8 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.7 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.6 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.5 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.4 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.3 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.2 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or
• the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.1 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.01 grams per ounce of solution. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.001 grams per ounce of solution prepared. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.0001 grams per ounce of solution prepared. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.00001 grams per ounce of solution prepared. In some embodiments, the PIF peptide or PIF analog or pharmaceutically salt thereof is at a concentration of about 0.000001 grams per ounce of solution prepared.
• Dosage may be measured in terms of mass amount of analog per liter of liquid formulation prepared.
• One skilled in the art can increase or decrease the concentration of the analog in the dose depending upon the strength of biological activity desired to treat or prevent any above-mentioned disorders associated with the treatment of subjects in need thereof.
• some embodiments of the invention can include up to 0.00001 grams of analog per 5 mL of liquid formulation and up to about 10 grams of analog per 5 mL of liquid formulation.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 1.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:2.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:3.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:4.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:5.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:6.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:7.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 8. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:9. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 10. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 11. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 12.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 13. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 14. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 15. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 16. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 17.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 18. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO: 19. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:20. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:21. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:22.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:23. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:24. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:25. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:26. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:27.
• the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:28. In some embodiments, the pharmaceutical compositions comprising a PIF analog or any of such compositions in any of the disclosed methods are free of SEQ ID NO:29.
• the pharmaceutical compositions of the claimed invention comprises at least one or a plurality of active agents other than the PIF peptide, analog or pharmaceutically acceptable salt thereof.
• the active agent is covalently linked to the PIF peptide or PIF analog disclosed herein optionally by a protease cleavable linker (including by not limited to Pro-Pro or Cituline-V aline di-a-amino acid linkers).
• the one or plurality of active agents is one or a combination of compounds chosen from: an anti-inflammatory compound, alpha-adrenergic agonist, antiarrhythmic compound, analgesic compound, and an anesthetic compound.
• anti-inflammatory compounds examples include:
• alpha-adrenergic agonists examples include:
• Clonidine (mixed alpha2-adrenergic and imidazoline-Il receptor agonist)
• Guanabenz (most selective agonist for alpha2-adrenergic as opposed to imidazoline-Il) Guanoxabenz (metabolite of guanabenz)
• antiarrhythmic compound examples include:
• Amiodarone Cordarone, Pacerone
• analgesic compound examples include:
• anesthetic compounds examples include:
• the compounds of the present disclosure can also be administered in combination with other active ingredients, such as, for example, adjuvants, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein.
• methods of diagnosing endometriosis in a human subject comprises measuring pre-implantation factor (PIF) protein or mRNA expression levels from an endometrial tissue sample from the subject; and comparing the PIF protein expression levels from the endometrial sample to the PIF expression levels in a control normal sample; wherein the patient is diagnosed with endometriosis if the PIF expression levels from the endometrial sample are greater than the PIF expression levels from the control normal sample.
• the endometrial sample is an endometrial epithelial tissue sample .
• the sample is a biopsy sample. That is the endometrial sample can, for example, be obtained by a biopsy.
• the measuring of PIF protein expression levels comprises contacting the sample with an anti-PIF antibody and detecting bound anti-PIF antibody to PIF in the sample.
• the detection can by staining methods or other type methods using modified antibodies to visualize and/or quantitate the amount of PIF in the sample.
• the amount of the bound antibody can be converted into the amount of PIF present in the sample by comparing to controls, which is routine in the art.
• the amount of bound antibody indicates the PIF protein expression levels.
• the sample can also be stained for PIF protein expression levels using available protocols.
• the method can comprise staining the sample with an anti-PIF antibody that binds to PIF in the sample.
• the measuring of PIF mRNA expression levels comprises contacting the sample with a probe that binds to PIF mRNA and detecting the bound probe in the sample.
• the probe can be detected by performing amplification methods, such as RT-PCR, PCR, and the like.
• the probe is linked to fluorescent or radioactive moiety and the probe is detected by measuring the fluorescence or radioactive signal to determine the presence or absence of PIF in the sample.
• a method of treating endometriosis is combined with a method of diagnosing endometriosis.
• the method comprises measuring pre- implantation factor (PIF) protein or mRNA expression levels from a endometrial sample from the subject; comparing the PIF protein or mRNA expression levels from the endometrial sample to the PIF expression levels in a control normal sample, wherein the patient is diagnosed with endometriosis if the PIF expression levels from the endometrial sample are greater than the PIF expression levels from the control normal sample; and administering to the subject at least one pharmaceutical composition comprising a therapeutically effective amount of a PIF peptide, mimetics thereof, analogs thereof, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
• the endometrial sample is an endometrial epithelial tissue sample .
• the endometrial tissue sample is a biopsy sample. The PIF protein expression levels and the PIF mRNA expression levels can be measured, for example, according to the methods described herein.
• the patient is diagnosed with endometriosis if the PIF expression levels in the endometrial epithelial tissue sample are detectable or greater than as compared to the control. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 1% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 5% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis.
• the PIF expression levels in the endometrial epithelial tissue sample are at least 10% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 15% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 20% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis.
• the PIF expression levels in the endometrial epithelial tissue sample are at least 25% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 30% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 50% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 100% greater than the PIF expression levels in the control sample, the patient is diagnosed with
• the PIF expression levels in the endometrial epithelial tissue sample are at least 200% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 300% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 200% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 300% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 200% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments
• the PIF expression levels in the endometrial epithelial tissue sample are at least 400% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 500% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis. In some embodiments, the PIF expression levels in the endometrial epithelial tissue sample are at least 1000% greater than the PIF expression levels in the control sample, the patient is diagnosed with endometriosis.
• the patient is diagnosed with endometriosis if the PIF expression levels from the endometrial epithelial sample are at least 10% greater than the PIF expression levels from the control normal sample. In some embodiments, the patient is diagnosed with endometriosis if the PIF expression levels from the endometrial epithelial sample are at least 50% greater than the PIF expression levels from the control normal sample. In some embodiments, the patient is diagnosed with endometriosis if the PIF expression levels from the endometrial epithelial sample are at least 100% greater than the PIF expression levels from the control normal sample.
• the patient is with endometriosis if the PIF expression levels from the endometrial epithelial sample are at least 200% greater than the PIF expression levels from the control normal sample.
• a control sample is a control normal sample unless otherwise indicated by context.
• the disclosure also relates to methods for treating endometriosis comprising administering an effective amount of the compositions described herein to a subject in need thereof.
• the composition is administered once a day to a subject in need thereof. In another embodiment, the composition is administered every other day, every third day or once a week. In another embodiment, the composition is administered twice a day. In still another embodiment, the composition is administered three times a day or four times a day. In a further embodiment, the composition is administered at least once a day for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 weeks. In still a further embodiment, the composition is administered at least once a day for a longer term such as at least 4, 6, 8, 10, 12 or 24 months. Administration in some embodiments includes but is not limited to a dosage of 10-50 mg of composition at a frequency of minimum 1 , 2, 3 or 4 times per day. Optionally, administration continues until all symptoms are resolved and cleared by medical personnel via standardized testing such as SCAT 2.
• the composition is administered at least once a day until the condition has ameliorated to where further treatment is not necessary.
• the composition is administered until all symptoms of endometriosis are resolved. In further embodiments, the composition is administered for at least 1 , 2, 3, 6, 8, 10 or 12 or 24 months after the subject is asymptomatic.
• compositions of the present disclosure are useful and effective when administered to treat endometriosis.
• the amount of each component present in the composition will be the amount that is therapeutically effective, i.e., an amount that will result in the effective treatment of the condition (e.g., endometriosis) when administered.
• the therapeutically effective amount will vary depending on the subject and the severity and nature of the injury and can be determined routinely by one of ordinary skill in the art.
• the disclosure relates to a method of treating or preventing any of the indications set forth in US Pat. Nos. 8,222,211 , 7,723,289, 7,723,290, 8,454,967, 9,097,725, (each of which are incorporated by reference in their entireties) comprising administering compositions or pharmaceutical compositions comprising any one or plurality of PIF peptides, analogs, or pharmaceutically acceptable salts thereof disclosed herein.
• the disclosure relates to a method of stimulating the differentiation and/or proliferation of stem cells in a subject in need thereof comprising administering compositions or pharmaceutical compositions comprising any one or plurality of PIF peptides, analogs, or pharmaceutically acceptable salts thereof disclosed herein.
• the disclosure relates to any of the methods disclosed in US Pat. Nos. 7,273,708, 7,695,977, 7,670,852, 7,670,851, 7,678,582, 7,670,850, 8,012,700 (each of which are incorporated by reference in their entireties) comprising administering compositions or pharmaceutical compositions comprising any one or plurality of PIF peptides, analogs, or pharmaceutically acceptable salts thereof disclosed herein.
• the disclosure relates to a method of treating endometriosis by administering at least one or a plurality of compositions disclosed herein comprising PIF peptide, an analog thereof, or a pharmaceutically acceptable salt thereof.
• the disclosure relates to a method of endometriosis by administering a therapeutically effective amount or dose of one or a plurality of compositions disclosed herein comprising at least one PIF peptide, an analog thereof, or a pharmaceutically acceptable salt thereof.
• the disclosure relates to a method of treating endometriosis by administration of a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount or dose of at least one PIF peptide, an analog thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the disclosure relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount or dose of at least one PIF peptide, an analog thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier for the treatment of endometriosis.
• the disclosure relates to the use of a therapeutically effective amount or dose of any one or plurality of compositions disclosed herein comprising at least one PIF peptide, an analog thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier in the manufacture of a medicament for the treatment of endometriosis.
• the disclosure relates to the use of a pharmaceutical composition comprising a therapeutically effective amount or dose at least one PIF peptide, an analog thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier in the manufacture of a medicament for the endometriosis.
• the disclosure relates to a method of inducing an
• the formulation may be supplied as part of a kit.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: l.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:2.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:3.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:5.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:6.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or a PIF analog or
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 8.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:9.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or a PIF analog or
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 11.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 12.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 13.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 14.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 15. In some embodiments, the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 16.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 17.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 18.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 19.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:20.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:21. In some embodiments, the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:22.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:23. In some embodiments, the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:24. In some embodiments, the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:26.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO:27.
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or
• the kit comprises comprising a PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, the PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof comprises a non-natural amino acid or is at least 70% homologous to SEQ ID NO: 29.
• the kit comprises a pharmaceutically acceptable salt of an analog with a rehydration mixture.
• the pharmaceutically acceptable salt of an analog are in one container while the rehydration mixture is in a second container.
• the rehydration mixture may be supplied in dry form, to which water or other liquid solvent may be added to form a suspension or solution prior to administration.
• Rehydration mixtures are mixtures designed to solubilize a lyophilized, insoluble salt of the invention prior to administration of the composition to a subject takes at least one dose of a purgative.
• the kit comprises a pharmaceutically acceptable salt in orally available pill form.
• the kit may contain two or more containers, packs, or dispensers together with instructions for preparation and administration.
• the kit comprises at least one container comprising the pharmaceutical composition or compositions described herein and a second container comprising a means for delivery of the compositions such as a syringe .
• the kit comprises a composition comprising an analog in solution or lyophilized or dried and accompanied by a rehydration mixture.
• the analog and rehydration mixture may be in one or more additional containers.
• compositions included in the kit may be supplied in containers of any sort such that the shelf-life of the different components are preserved, and are not adsorbed or altered by the materials of the container.
• suitable containers include simple bottles that may be fabricated from glass, organic polymers, such as polycarbonate, polystyrene, polypropylene, polyethylene, ceramic, metal or any other material typically employed to hold reagents or food; envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy.
• Other containers include test tubes, vials, flasks, and syringes.
• the containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components of the compositions to mix.
• Removable membranes may be glass, plastic, rubber, or other inert material.
• Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrates, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, zip disc, videotape, audio tape, or other readable memory storage device. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
• a packaged kit contains the pharmaceutical formulation to be administered, i.e., a pharmaceutical formulation containing PIF peptide and/or a PIF analog or pharmaceutically acceptable salt thereof, a container (e.g., a vial, a bottle, a pouch, an envelope, a can, a tube, an atomizer, an aerosol can, etc.), optionally sealed, for housing the formulation during storage and prior to use, and instructions for carrying out drug administration in a manner effective to treat any one or more of the indications disclosed herein.
• the instructions will typically be written instructions on a package insert, a label, and/or on other components of the kit.
• the kit may also include a device for administering the formulation (e.g., a transdermal delivery device).
• the administration device may be a dropper, a swab, a stick, or the nozzle or outlet of an atomizer or aerosol can.
• the formulation may be any suitable formulation as described herein.
• the formulation may be an oral dosage form containing a unit dosage of the active agent, or a gel or ointment contained within a tube.
• the kit may contain multiple formulations of different dosages of the same agent.
• the kit may also contain multiple formulations of different active agents.
• the kit may contain a number of pharmaceutically effective dosages in separate containers or syringes necessary to treat one or more symptoms of endometriosis, in some embodiments, the kit contains about 1, 2, 3, 4, or 5 or more dosages in 1, 2, 3, 4, or five containers (such as a syringe), that enable administration of any of the dosages into the subject in need thereof.
• kits will also typically include means for packaging the individual kit components, i.e., the pharmaceutical dosage forms, the administration device (if included), and the written instructions for use.
• packaging means may take the form of a cardboard or paper box, a plastic or foil pouch, etc.
• kits can be used for diagnosing subjects with endometriosis.
• the kits can, for example, comprises reagents that are used to detect PIF in the endometrial sample. These reagents can be buffers, antibodies to PIF, secondary antibodies, stains, probes to PIF mRNA and the like.
• the kit can also comprise instructions for performing methods described herein.
• the kit also comprises reagents for processing the endometrial sample before detecting PIF expression.
• Endometriosis is a chronic benign disease characterized by the presence of endometrial tissue, both stromal and epithelial components, outside the uterine cavity, affecting women in their reproductive age (Giudice LC 2010). Endometriosis lesions can be found mostly in the pelvis, where the most common sites are the ovaries, anterior and posterior cul-de-sac, broad and utero-sacral ligaments, uterus, fallopian tubes, sigmoid colon and appendix (Olive et al 1993). At the moment the most accepted hypothesis regarding its pathogenessis is the retrograde menstruation, even though stem cell origin as well as its genetic cause are gaining attention (Macer et al 2012).
• stem cells for the endometrium may been involved in endometriosis pathogenesis: apart from their of endometrial origin they may be mesenchymal stem cells derived from bone marrow.
• Pre-Implantation Factor or PIF is a fifteen amino acid linear peptide secreted by viable human, bovine and murine embryos (Stamatkin et al 2011 , Stamatkin et al 201 lb, Barnea et al 2012, Ramu et al 2013). In singly cultured bovine and murine embryos increased levels of PIF in the media correlate with embryos development whereas PIF is absent in nonviable embryos (Stamatkin et al 201 1).
• PIF plays an essential role in human pregnancy, as it primes the endometrium for implantation, promotes trophoblast invasion and regulates systemic immune response (Barnea et al 2012, Paidas et al 2010, Barnea et al 2012b, Duzyj et al 2010).
• PIF's immune regulatory features translational aspects to treatment of non-pregnant autoimmune and transplantation models were documented (Weiss et al 201 1, Weiss et al 2012, Azar et al 2013).
• PIF also promotes trophoblast invasion, and orchestrates maternal systemic immune response (Bamea et al 2012, Duzyj et al, Roussev et al 2013).
• Tregs are derived from the CD4 lineage of T cells and are produced naturally in the thymus, express IL-10 receptor (CD25+) and the forkhaed box P3 transcription factor (Foxp3+).
• CD4+ T cells become Tregs by the Foxp3 expression induced by the cytokines increase in the microenvironment.
• Activated Tregs suppress the response of effector T cell indirectly by inhibiting the dendritic cells or the other antigen presenting cells (APC) from triggering effector T cell proliferation (Tang et al 2006).
• Tregs induce immune tolerance through the production of IL-10 and Transforming Growth Factor- ⁇ , anti-inflammatory cytokines or Th2, which inhibit T helper cell activation, (von Boemer et al 2006).
• Tregs are critical for tumor growth, since they may provide an immunologically protected micro environment (Bergman et al 2007, Strauss et al 2007,Bergmann et al 2001 1, Wang et al 2012).
• the presence of Tregs in eutopic and ectopic endometrium of women affected by endometriosis has been reported (Budiu et al 2009, Berbic et al 2010).
• the aim of the study was to evaluate the possible expression of PIF and FOX-P3 in the ectopic endometrium in order to evaluate the expression of these antigens in the ectopic endometrium, and identify cells that are able to modulate immune competent cells response in endometriotic lesions.
• Synthetic PIF15 (MVRIKPGSANKPSDD) and a scrambled peptide same amino acid sequence but in random order (GRVDPSNKSMPKDIA) were synthesized by solid-phase peptide synthesis (Peptide Synthesizer, Applied Biosystems) employing Fmoc (9- fluorenylmethoxycarbonyl) chemistry at Bio-Synthesis, Inc. (Lewisville, TX). Final purification was carried out by reversed-phase HPLC and identity was verified by matrix- assisted laser desorption/ionization time-of-flight mass spectrometry and amino acid analysis at >95% purity.
• Fluorescein labeled FITC-PIF and scrambled PIF were also generated as previously reported (Barnea AJOG 2012).
• Anti-PIF monoclonal antibody against MVRIKPGSANKPSDD was generated in (Genway, SanDiego, CA). et al 2012).
• Tissue specimens were obtained from 25 women who underwent laparoscopic surgery for severe endometriosis according to the revised criteria of the American Society Reproductive Medicine. The surgical procedures were carried out in CERM-Hungaria Institute, Rome, Italy, from September 2014 through April 2015. The project had the approval of the Hospital's Ethical Committee. Samples were obtained from the ectopic endometrium, ovarian endometriomas and peritoneal implants. A total of 25 eutopic endometria, 25 ovarian endometriomas and 10 peritoneal implants were collected from patients. Furthermore, the endometria of 10 healthy women in different phases of the menstrual cycle were used as controls. Biopsy samples were fixed in 4% neutral buffered formalin overnight and were subsequently paraffin embedded.
• Incubation with the first antibody was carried out at 4° C overnight with a dilution of 1 to 100 for the monoclonal mouse anti-human PIF and with a dilution of 1 to 50 for the monoclonal mouse anti-human FOXP3.
• tissue sections were labeled with the avidin-biotin- peroxidase detection system Vectastain (Vector Laboratories, Burlingthon VT, USA). Each step was followed by meticulous washing with PBS.
• DAB 3,3'-diaminobenzidine
• AEC 3-amino-9-ethylcarbazole
• the positive controls were PIF and FOXP3 tissues. Negative controls were performed by replacing the primary antibody with mouse immunoglobulin at the same concentration as the primary antibody.
• the intra-observer and inter-observer coefficient of variation were 3.4% and 4.2% respectively.
• Three slides of each sample were observed for both antigens tested under the microscope and each observer was blind to which sample it was.
• the slides were numbered progressively by a technician, who reported on a separate worksheet the number of the slide and the corresponding name of the patient or control. Only after the slide was analyzed by the two different observers and scored for the HSCORE each value was reported on the worksheet against the corresponding name of the patient for each slide number.
• HSCORE analysis was performed separately for the component of endometrium, glandular and stromal cells, and for the ectopic tissue (each observer performed 4 different HSCOREs for each slide).
• FITC-PIF Flow Cytometry Studies Non-pregnant infertile and first trimester pregnant patients at Millenova Immunology Laboratories who were undergoing fertility treatments signed a standard informed consent (CARI, Institute, Chicago) as we have previously reported 17,18. All experiments were performed in accordance with the guidelines and regulations of CARI, Institute, Chicago and with the approval from the Institutional Review Board of the University of Illinois at Chicago in March 2006, Dr. R. Roussev, PI. The blood was drawn as part of their work-up process with the use of excess specimen without identifiers. We reported on FITC-PIF binding to CD14+ and CD3+, cells in both pregnant and non-pregnant patients.17 In addition binding to activated CD4+ cells were documented.
• PIF targets regulatory T-cells was examined by using specific anti-CD4+, anti- CD25+ and anti-FoxP3+ antibodies. Binding was compared with scrambled-FITC-PIF used as a negative control. Following separation using Ficoll-Hypaque, PBMC were isolated. Binding to Isotype control served as negative controls. Two- three color staining was done using conventional techniques. Fluorescence measurements (20,000-50,000 gated events per sample) were performed in a Coulter® Epics® XLTM Flow Cytometer using System II software for data acquisition and analysis (Beckman Coulter, Inc., Miami, FL).
• CD4+/CD25+/FoXP3+ systemic immune cells confirming partially a functional link between PIF and regulation of Treg cells in establishing a local immune privilege in endometriosis lesions.
• Tregs in eutopic and ectopic endometrium of women affected by endometriosis has been reported (Budiu et al 2009, Berbic et al 2010).
• Treg cells percentage was increased in the peritoneal fluid of these patients with respect to controls.
• autoimmune disorders might be due to a dysregulation of T regulatory cells function, in particular of CD4+-CD25+-FoxP3+ regulatory cells, which play a key role in the suppression of many types of effector cells such as macrophages, NK cells, dendritic cells, cytotoxic T cells (Sathaguchi et el 2008, Berbic & Fraser 201 1).
• Example 2 PIF in endometriosis :a potential role in in inducing immune privilege
• Endometriosis is a chronic inflammatory condition characterised by the growth of endometrial epithelial and stromal cells outside the uterine cavity. Besides Sampson's theory of retrograde menstruation, endometriosis pathogenesis is related to the privileged inflammatory microenvironment in these lesions.
• One of the pivotal factors are T regulatory FoxP3 + expressing T cells (Tregs).
• Prelmplantation factor (PIF) is a peptide essential for pregnancy recognition and development. Besides immune modulatory function the synthetic PIF analog (sPIF) was successfully tested in multiple animal models.
• Synthetic PIF 15 (MVRIKPGSANKPSDD) and a scrambled peptide sequence with the same amino acids in random order
• MVRIKPGSANKPSDD was generated in (Genway, SanDiego, CA, USA).
• Tissue specimens were obtained from 25 women who underwent laparoscopic surgery for severe endometriosis according to the revised criteria of the American Society Reproductive Medicine. The surgical procedures were carried out in CERM-Hungaria Institute, Rome, Italy, from September 2014 through April 2015. The project had the approval of the Hospital's Ethical Committee. Samples were obtained from the ectopic endometrium, ovarian endometriomas and peritoneal implants. A total of 25 eutopic endometria, 25 ovarian endometriomas and 10 peritoneal implants were collected from patients. Furthermore, the endometria of 10 healthy were used as controls. Biopsy samples were fixed in 4% neutral buffered formalin overnight and were subsequently paraffin embedded.
• tissue sections were stained with eosin and hematoxylin to select tissue with ectopic epithelial cells. Serial sections of 5 ⁇ thick were used for IHC. Commercially available monoclonal antibodies were used for the detection of PIF (Biolncept, LLC, NJ, USA) and Foxp3 (number: sc-53876, Santa Cruz, CA, USA). IHC was performed according to manuals instructions. Briefly, tissue sections were dewaxed and re-hydrated and endogenous peroxidase activity quenched by incubation with 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature. Sections were exposed to a non-immune block with normal horse serum for 30 minutes at room
• HSCORE DPi(i+l)
• i the intensity of staining with a value of 1, 2, or 3 (weak, strong or very strong respectively)
• Pi the percentage of stained cells for each intensity, varying from 0% to 100%.
• HSCORE analysis was performed separately for the component of endometrium, glandular and stromal cells, and for the ectopic tissue (each observer performed 4 different HSCOREs for each slide). The intra-observer and inter-observer coefficient of variation were 3.4% and 4.2% respectively.
• FITC-PIF Flow Cytometry.
• PIF targets regulatory T- cells was examined by using specific anti-CD4 + , anti-CD25 + and anti-FoxP3 + antibodies (BD, Pharmingen, USA). Binding was compared with scrambled-FITC-PIF used as a negative control. Following separation using Ficoll-Hypaque, peripheral blood mononuclear cells (PBMC) were isolated. Binding to Isotype control served as negative controls. Two- three color staining was done using conventional techniques. Fluorescence measurements (20,000- 50,000 gated events per sample) were performed in a Coulter ® Epics ® XLTM Flow Cytometer using System II software for data acquisition and analysis (Beckman Coulter, Inc., Miami, FL, USA).
• Primary endometrial stromal cells (ESC) from women with and without endometriosis were prepared via a collagenase digestion and size exclusion protocol as described previously (McKinnon et al, 2013).
• Isolated ESC were maintained in Iscoves's modified Eagle medium (IMEM)
• fcs fetal calf serum
• Epithelial cells were cultured in Dulbecco's modified Eagles medium (DMEM) (invitrogen) with 10% fcs and 1% antibiotic/antimycotic.
• DMEM Dulbecco's modified Eagles medium
• sPIF phosphate buffered saline
• Probe sets where redefined using the alternative chip definition file mogene20stmmentrezgcdf (Dai et al, 2005). Differential gene expression was calculated using the moderated t-test as described previously and implemented in the R/Bioconductor package limma (Ritchie et al, 2015).
• the Pathway analysis The output of limma was used to perform gene set enrichment analysis (GSEA) using the SetRank method.
• GSEA gene set enrichment analysis
• the key principle of this algorithm is that it discards gene sets that have initially been flagged as significant, if their significance is only due to the overlap with another gene set. It calculates the p-value of a gene set using the ranking of its genes in the ordered list of p-values as calculated by limma.
• the following databases were searched for significant gene sets: BIOCYC, Gene Ontology, KEGG, Pathway Interaction Database, REACTOME, and WikiPathways.
• PIF imparts epithelial ectopic endometria.
• tissue samples were obtained from women with or without endometriosis including the ectopic endometrium, ovarian endometriomas and peritoneal implants during the proliferative and secretory phases.
• PIF positive cells in the epithelial compartment of ectopic endometria documenting for the first time re-expression of PIF outside of pregnancy.
• PIF re-expression may be induced as a protective mechanism so we tested the effect of sPIF using an in-vitro system next.
• FoxP3+ cells were present in ectopic and eutopic epithelial cells, we hypothesize that FoxP3 may regulate epithelial transition and differentiation as a transcription factor instead of reflecting a reaction to the inflammatory process as in case of TNFa (Han et al, 2015).
• PIF has both a local and global integrated protective effects (Barnea et al, 2015). Furthermore, a potential for a bi-directional communication between PIF expressing epithelial cells and Treg cells exists. PIF increased cell viability of epithelial cells derived from the eutopic endometrium and decreased cell viability of cells derived from ectopic tissue (FIGs. 3C and 3D). These results indicate that PIF may have local paracrine effects on the epithelial cells within the endometriotic lesion. Furthermore, this effect may be modulated by the inflammatory microenvironment as in case of TNFa (FIG. 5E).
• the whole genome transcriptome analysis showed a significant influence on the expression of T cell receptor signalling pathways raising the possibility that not only does the paracrine effects of PIF influence the survival of ectopic epithelial cells, but that it also influences gene expression and the ability of these epithelial cells to respond to the immune regulating effects of infiltrating Tregs.
• the neuroprotective effects of epithelia derived PIF may stimulate an enhanced nerve presence and influence the interpretation of pain in endometriosis.
• peripheral sPIF and the ensuing reduced neuroinflammation may limit such neurotropic pain as well (Mueller et al, 2014; Weiss et al, 2012b).
• Example 3 sPIF has similar binding to lymphocytes compared to standard PIF.
• the sPIF sequences provided herein are non-naturally occurring mutants of PIF.
• FITC-PIF assays was performed to examine sPIF binding to monocytes.
• SEQ ID NO: 27 and SEQ ID NO: 29 were compared to natural PIF in binding to CDl lb (macrophage) and CD19 (B cell) monocytes.
• the sPIF sequences SEQ ID NOs: 27 and 29 showed higher binding affinity to both CD1 lb and CD19 monocytes. This result lends support to the idea that the sPIF sequences provided here (SEQ ID NOs: 20-29) may bind to PIF receptors at least as well as, if not better than, native PIF.
• Example 4 sPIF used as therapeutic in endometriosis animal model
• mice Animals Mature (approximately 8-week-old) female C57BL/6 mice will purchased from an animal supplier and will be allowed to acclimate for 1 week before surgery. Some experiments may be performed using transgenic Cfslr-EGFP mice (MacGreen) that were originally generated as described in detail elsewhere. Two heterozygous enhanced GFP+ males and two heterozygous enhanced GFP+ females were cross-bred with wild-type (WT) C57BL/6 mice to form a breeding colony and were bred under standard conditions. Offspring will be genotyped and classified as either MacGreen or WT. Mice will be maintained on standard chow and water available ad libitum and will be housed in environmentally controlled facilities illuminated between 7:00 am and 7:00 pm. All the animal procedures will be performed in accordance with legal requirements related to animal care and under licensed approval from the appropriate ethical authorities.
• MacGreen transgenic Cfslr-EGFP mice
• mice Menstruation will be induced in adult donor mice (approximately 8 weeks of age) using a protocol developed in-house set forth in PLoS One. 2014;9:e86378.
• ovariectomized mice (day 1) will be primed with s.c. injections of 100 ng of estradiol- 17 ⁇ (days 7 to 9), treated with progesterone (P4; Sigma-Aldrich, Dorset, UK) delivered via a SILASTIC implant (Dow Coming Corp, Midland, MI) from days 13 to 19, and injected with 5 ng of estradiol-17 in sesame oil on days 13, 14, and 15.
• Tissue from one decidualized donor horn will be used to inoculate each recipient mouse (approximately 40 mg tissue/0.2 mL PBS per mouse).
• recipient mice will be culled (photographs of the body cavity taken and were taken the lesions carefully dissected from surrounding tissue) and tissues will be either fixed in 4% normal buffered formaldehyde for histologic analysis or placed in RNA Save (Geneflow Ltd, Lichfield, UK) for RNA extraction.
• Endometiral tissues will be fixed to slides and stained using known immunostaining procedures. Briefly, after fixation, mouse lesions will be stained using H&Eosin; only lesions containing identifiable stromal and epithelial compartments will be used for further analysis.
• Single-color IHC analysis will be performed according to standard protocols24,26 with citrate antigen retrieval followed by blocking endogenous peroxidase with 3% H202 in methanol.
• a streptavidin-biotin block (Vector Laboratories, Peterborough, UK) will be performed, and any nonspecific staining will be reduced using species-specific blocking solution (1 :4 serum in Tris-buffered saline + 5% bovine serum albumin).
• Biotinylated secondary antibodies (dilution 1 :500) will be diluted in blocking solution and incubated at room temperature for 30 minutes.
• a streptavidin- horseradish peroxidase conjugate (dilution 1 : 1000; Sigma-Aldrich) will be diluted in Tris- buffered saline and incubated at room temperature for 1 hour, followed by visualization using ImmPACT diaminobenzidine peroxidase substrate (Vector Laboratories).
• Dual immunofluorescence was performed as previously herein with a secondary F (ab) polyclonal antibody to IgG (horseradish peroxidase), and antibody detection will be performed using a TSA system kit (PerkinElmer, Waltham, MA) labeled with either Cy3 (red) or fluorescein (green).
• TSA system kit PerkinElmer, Waltham, MA
• Cy3 red
• fluorescein green
• CD31 (Dako) Rabbit Mouse 1 :800
• Biotinylated anti-mouse Goat 1 :500
• Biotinylated anti-rabbit Goat 1 :500
• mice will be divided into two sets: those left treated with PIF and those left untreated. Immunohistochemistry will reveal the experimental factors disclosed in the table to the left. In mice that are treated with one or more PIF peptides, we anticipate that recovery rate of the animals will improve, lesion size will decrease, and the number of lesions will decrease as compared to mice untreated with PIF peptide. By using this study we can confirm that induction of a menses-like event in mice was associated with the presence of macrophages in the uterus and that lesions formed in mice contained tissue-resident macrophages and a proinflammatory microenvironment. We expect that therapeutically effective levels of PIF peptide or their pharmaceutically acceptable salts will be able to lessen the severity of the endometriotic state induced in the mice.
• Example 5 sPIF used as therapeutic for treatment of human endometriosis.
• sPIF will be administered to healthy female patients and/or female patients diagnosed with endometriosis.
• the purpose of this study is to examine the safety and tolerability of synthetic Prelmplantation Factor (sPIF) in female patients with endometriosis.
• PIF apparently initiates both maternal tolerance preventing the loss/rejection of the fetus.
• Synthetic PIF sPIF
• sPIF successfully translates PIF endogenous properties to treatment of pregnant and non-pregnant immune disorders. sPIF was found to be effective in preclinical models of autoimmunity and transplantation (published).
• sPIF administration to subjects diagnosed with autoimmune hepatitis followed by observation period demonstrated that at doses of sPIF ranging from 0.1 mg/kg and 1.0 mg/kg were tolerated safely.
• This study using sPIF a dose dependent design will evaluate the safety, tolerability and pain assessment of patients with endometriosis.
• the primary endpoint will be the total pain burden (dysmenorrhoea, deep dyspareunia and non-menstrual pain) rated on a daily basis by the patient using standard pain scales and analgesic intake as the outcome measures .
• Secondary endpoints will include the volume of the endometriotic nodule assessed clinically and on transvaginal ultrasound, the amount of pelvic tenderness on clinical examination and the presence of other endometriotic lesions at laparoscopy.
• Prelmplantation Factor will be randomized in a 2: 1 ratio (active drug : placebo) to
• Cohort 1 single dose 0.1 mg/kg sPIF Day 1-5 given SQ
• Placebo Comparator single ascending dose (SAD)
• Drug Placebo
• each cohort (at least 3 patients/cohort; at least 9 to mimic sPIF solution for subjects in total), subjects with abnormal uterine function injection
• Placebo Comparator MAD Normal Placebo Drug: Placebo
• Placebo Comparator MAD Abnormal Placebo Drug: Placebo
• the patients will be evaluated for changes in volume of the endometriotic nodule assessed clinically and/or on transvaginal ultrasound, degree of pelvic tenderness on clinical examination and the presence of other endometriotic lesions at laparoscopy.
• sPIF-treated cohorts will experience less pain than untreated patients presenting with abnormal uterine dysfunction.
• sPIF- treated cohorts will experience reduced volume of endometriotic nodules, less tenderness and fewer lesions or lesions with decreased size as compared non treated subjects.
• PEF Prelmplantation Factor
• Preimplantation factor negates embryo toxicity and promotes embryo development in culture. Reprod Biomed Online. 2011b Oct;23(4):517-24.
• PAF Protrofi Factor
• PEF Preimplantation factor
• PEF* Preimplantation factor reverses neuroinflammation while promoting neural repair in EAE model. J Neurol Sci.
• McCarty KS, Miller LS, Cox EB Estrogen receptor analysis, correlation of biochemical and immunohistochemical methods using monoclonal anti-receptor antibodies.

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