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WO2021226162A1 - Antagonistes de l'ep4 et leur utilisation dans le traitement des maladies prolifératives" - Google Patents

Antagonistes de l'ep4 et leur utilisation dans le traitement des maladies prolifératives" Download PDF

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Publication number
WO2021226162A1
WO2021226162A1 PCT/US2021/030771 US2021030771W WO2021226162A1 WO 2021226162 A1 WO2021226162 A1 WO 2021226162A1 US 2021030771 W US2021030771 W US 2021030771W WO 2021226162 A1 WO2021226162 A1 WO 2021226162A1
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Prior art keywords
pgem
patient
cancer
concentration
urine sample
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PCT/US2021/030771
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English (en)
Inventor
Jeffrey Ecsedy
Jason SAGER
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Arrys Therapeutics, Inc.
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Publication of WO2021226162A1 publication Critical patent/WO2021226162A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • EP4 ANTAGONISTS AND USES THEREOF TECHNICAL FIELD OF THE INVENTION The present invention relates to EP4 antagonists, and uses thereof for treating a proliferative disorder.
  • Prostaglandins are mediators of pain, fever and other symptoms associated with inflammation.
  • Prostaglandin E2 (PGE2) is the predominant eicosanoid detected in inflammation conditions.
  • PGE2 Prostaglandin E2
  • it is also involved in various physiological and/or pathological conditions such as cancer, hyperalgesia, uterine contraction, digestive peristalsis, awakeness, suppression of gastric acid secretion, blood pressure, platelet function, bone metabolism, angiogenesis or the like.
  • EP4 subtypes a Gs-coupled receptor
  • PI3K and GSK3 ⁇ signaling a Gs-coupled receptor
  • PI3K and GSK3 ⁇ signaling a Gs-coupled receptor
  • PI3K and GSK3 ⁇ signaling a Gs-coupled receptor
  • PI3K and GSK3 ⁇ signaling a Gs-coupled receptor
  • PI3K and GSK3 ⁇ signaling a major role in PGE 2 -mediated biological events
  • Various EP4 antagonists have been described previously, for example, in WO 2002/032900, WO 2005/021508, US 6,710,054, and US 7,238,714, the contents of which are incorporated herein by reference in their entireties.
  • PGE 2 is a major cyclooxygenase (COX) product that is important in human physiology and pathophysiology that is synthesized downstream of COX-1 and COX-2.
  • COX cyclooxygenase
  • PGEM or 11 ⁇ -hydroxy-9,15-dioxo-2,3,4,5- tetranor-prostane-1,20-dioic acid, is the major urinary metabolite of PGE 2 .
  • EP4 antagonists act to reduce or attenuate the effects of PGE2 signaling via the EP4 receptor, such as reduction of cAMP production, as well as PI3K and GSK3 ⁇ signaling.
  • urinary PGEM levels can be used, for example, to predict efficacy of treatments using EP4 antagonists and for patient selection purposes.
  • methods for predicting the efficacy of treatments using EP4 antagonists and/or selecting a patient for application or administration of a treatment comprising an EP4 antagonist comprise, in part, methods of identifying patients having elevated urinary concentrations of PGEM, and methods for treating patients having elevated urinary concentrations of PGEM using EP4 antagonists.
  • the present invention provides a method of identifying or selecting a patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of the patient. Such methods can comprise, for example, using an LC–MS/MS method as described herein, and selecting a patient having an elevated concentration of PGEM in the urine sample.
  • a method of treating a proliferative disorder in a patient having or identified as having an elevated urinary concentration of PGEM for example, as determined using a method described herein, comprising administering to the patient a therapeutically effective amount of an EP4 antagonist.
  • the present invention provides a method of treating a patient having a proliferative disorder, comprising selecting a patient having an elevated urinary concentration of PGEM, for example, using a method as described herein, and administering to the patient a therapeutically effective amount of an EP4 antagonist.
  • a proliferative disorder is a cancer.
  • an EP4 antagonist is selected from those as described herein.
  • FIG.1 depicts PGE 2 metabolism.
  • FIG.2 depicts (A): urinary concentrations of PGE-M of certain patients in study #1, and (B): urinary concentrations of PGE-M of all the patients in study #1.
  • FIG.3 depicts urinary concentrations of PGE-M of all patients in study #2.
  • DETAILED DESCRIPTION OF THE INVENTION 1 General Description of Certain Embodiments of the Invention
  • PGEM is the major urinary metabolite of PGE2.
  • PGE2 is a cyclooxygenase (COX) product biosynthesized from arachidonic acid by the cyclooxygenases COX-1 and COX-2 and PGE synthases.
  • COX cyclooxygenase
  • PGEM levels in urine can be used as an indirect indicator of systemic PGE2 levels given the stability of this analyte.
  • PGE2 can signal via the G-protein coupled receptor (GPCR) EP4.
  • GPCR G-protein coupled receptor
  • EP4 receptor leads to cAMP production, as well as PI3K and GSK3 ⁇ signaling.
  • PGE 2 establishes an immunosuppressive tumor microenvironment by binding to EP4, which is expressed on an array of immune cells.
  • the relationship between urinary PGEM and cancer risk have been reported in some retrospective exploratory analyses.
  • urinary PGEM levels are associated with the risk of, or poor prognosis of, colorectal cancer, gastric, breast, pancreatic cancer, and oral squamous cell carcinoma patients (Cai et al 2006 J Clin Oncol.; Johnson JC et al 2006 Clin.Gastroentero Hepatol; Shrubroke MJ et al 2012 Can Prev Res.; Davenport JR et al 2016 Mol Carcinog).
  • PGEM is significantly increased in patients with CRC compared to healthy individuals (Johnson JC et al 2006 Clin.Gastroentero Hepatol).
  • the present invention provides a method of measuring a urinary concentration of PGEM in a patient, comprising measuring the concentration of PGEM in a urine sample of the patient using, for example, an LC–MS/MS method, such as any of the various LC–MS/MS methods described herein.
  • the present invention provides a method of identifying or selecting a patient for treatment with an EP4 antagonist, comprising measuring the concentration of PGEM in a urine sample of the patient, for example, using an LC–MS/MS method as described herein, and selecting a patient having an elevated concentration of PGEM in a urine sample.
  • the present invention provides a method of treating a proliferative disorder in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM, for example, using a method as described herein, and administering to the patient a therapeutically effective amount of an EP4 antagonist, for example, as described herein.
  • the present invention provides a method of treating a proliferative disorder in a patient having an elevated urinary concentration of PGEM, for example, where the concentration of PGEM is determined using a method as described herein, and administering to the patient a therapeutically effective amount of an EP4 antagonist, such as any of the EP4 antagonists described herein.
  • an EP4 antagonist refers to an agent that reduces or attenuates the signaling activity or biological activity of an EP4 receptor.
  • agents can include proteins, such as anti-EP4 antibodies, nucleic acids, amino acids, peptides, carbohydrates, small molecules (organic or inorganic), or any other compound or composition which decreases the activity of an EP4 receptor, either by reducing the amount of EP4 receptor present in a cell, or by decreasing the binding or signaling activity or biological activity of the EP4 receptor.
  • EP4 receptor activity refers to one or more changes in EP4 receptor-mediated downstream signaling activity or biological activity, such as, for example, an EP4-mediated increase in cAMP levels upon PGE2 stimulation, an EP4-mediated change in PI3K signaling, an EP4-mediated change in GSK3 ⁇ signaling, an EP4-mediated change in one or more immune cell functions or inflammatory processes, such as modulating (e.g., decreasing or inhibiting) the activity of macrophages, neutrophils, cytotoxic lymphocytes, and/or NK cells, modulating (e.g., promoting or enhancing) the activity of tumor tolerant immune cells, such as myeloid derived suppressor cells and regulatory T cells, or any combination thereof of such EP4 receptor activities.
  • an EP4-mediated increase in cAMP levels upon PGE2 stimulation an EP4-mediated change in PI3K signaling
  • an EP4-mediated change in GSK3 ⁇ signaling an EP4-mediated change in one or more immune cell functions or inflammatory processes, such as modulating (e
  • the term "antagonist” refers to a molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native receptor or molecule disclosed herein (e.g., an EP4 receptor). In some embodiments, inhibition in the presence of the antagonist is observed in a dose-dependent manner.
  • the measured signal (e.g., signaling activity or biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the signal measured with a negative control under comparable conditions.
  • the potency of an antagonist is usually defined by its IC50 value (concentration required to inhibit 50% of the agonist response).
  • a selective EP4 antagonist is an agent that inhibits EP4 activity with an IC50 at least 10-fold less, at least 20-fold less, at least 30-fold less, at least 40- fold less, at least 50-fold less, at least 60-fold less, at least 70-fold less, at least 80-fold less, at least 90-fold less, preferably, at least 100-fold less or more, than the IC50 for inhibition of EP1, EP2, or EP3 activity, as determined by standard methods known in the art.
  • the terms “measurable affinity” or “measurably inhibit” refers to a measurable change in EP4 activity between a sample comprising an EP4 antagonist described herein, or a salt or a composition thereof, and EP4, and an equivalent sample comprising EP4, in the absence of said compound, or composition thereof.
  • the term “LC-MS/MS,” “LC/MS/MS,” or “liquid chromatography tandem mass spectrometry” refers to an analytical technique that combines the separating power of liquid chromatography (LC) with the highly sensitive and selective mass analysis capability of triple quadrupole mass spectrometry based on the unique mass/charge (m/z) transitions of each compound of interest.
  • a sample solution containing analytes of interest is pumped through a stationary phase (LC column) by a mobile phase flowing through at high pressure.
  • LC column stationary phase
  • Chemical interaction between the components of the sample, the stationary phase, and the mobile phase affects different migration rates through the LC column affecting a separation.
  • the wide variety of stationary phase and mobile phase combinations allow for customizing a separation to suit many complex solutions.
  • the effluent is directed to the mass spectrometer.
  • the mass spectrometer for an LC/MS/MS system has an ionization source where the LC column effluent is nebulized, desolvated, and ionized creating charged particles.
  • LC-MS/MS can be combined with other upstream processes such as solid phase extraction (SPE) and liquid phase extraction (LPE), as is known in the art, in some embodiments. Accordingly, as described herein, where a method of detecting the concentration of PGEM is said to use an “LC-MS/MS method,” that method can use any method known in the art involving LC-MS/MS.
  • SPE-LC-MS/MS solid phase extraction liquid chromatography tandem mass spectrometry
  • SPE or solid phase extraction is a method of sample preparation that concentrates and purifies analytes from solution by sorption onto a disposable solid-phase cartridge, followed by elution of the analyte with a solvent appropriate for instrumental analysis. This is then followed by performing the LC-MS/MS method on the eluted analyte from the SPE process dissolved in the appropriate buffer.
  • an “online SPE-LC- MS/MS” or “high-throughput SPE-LC-MS/MS” method can be used where a combined instrumentation setup allows for the SPE, LC, and MS steps to be performed without having to manually transfer the eluted analyte from the SPE to the LC-MS/MS instrument.
  • ELISA Enzyme-linked immunosorbent assay
  • ELISA methods including, but not limited to, direct ELISAs, indirect ELISAs, sandwich ELISAs, competitive ELISAs, multiplex ELISAs, ELISPOT technologies, and other similar techniques known in the art. Principles of these immunoassay methods are known in the art, for example John R. Crowther, The ELISA Guidebook, 1st ed., Humana Press 2000, ISBN 0896037282, the contents of which are incorporated herein by reference in their entirety. Typically, ELISAs are performed with antibodies, but they can be performed with any capture agents that bind specifically to one or more biomarkers, such as PGEM, that can then be detected.
  • biomarkers such as PGEM
  • Multiplex ELISA allows simultaneous detection of two or more analytes within a single compartment (e.g., microplate well) usually at a plurality of array addresses (Nielsen and Geierstanger 2004. J Immunol Methods 290: 107-20 (2004) and Ling et al.2007. Expert Rev Mol Diagn 7: 87-98 (2007)).
  • array addresses Naelsen and Geierstanger 2004. J Immunol Methods 290: 107-20 (2004) and Ling et al.2007. Expert Rev Mol Diagn 7: 87-98 (2007).
  • an EP4 antagonist described herein inhibits or reduces EP4 function and/or activity in a given system or assay or subject by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%, relative to a control or baseline amount of that function and/or activity.
  • the terms "increases,” “elevates,” or “enhances,” are used interchangeably and encompass any measurable increase in a biological function and/or biological activity and/or a concentration.
  • an increase can be by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 2-fold, about 3-fold, about 4- fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20- fold, about 25-fold, about 50-fold, about 100-fold, or higher, relative to a control or baseline amount of a function, or activity, or concentration.
  • the terms “elevated concentration,” “increased concentration,” “increased levels” of a substance (e.g., PGEM) in a sample refers to an increase in the amount of the substance of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 25-fold, about 50-fold, about 100- fold, or higher, relative to the amount of the substance in a control sample or control samples, such as an individual or group of individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control, as determined by
  • a subject can also be determined to have an “elevated concentration” of a substance if the concentration of the substance is increased by one standard deviation, two standard deviations, three standard deviations, four standard deviations, five standard deviations, or more relative to the mean (average) or median amount of the substance in a control group of samples or a baseline group of samples or a retrospective analysis of patient samples.
  • concentration of the substance is increased by one standard deviation, two standard deviations, three standard deviations, four standard deviations, five standard deviations, or more relative to the mean (average) or median amount of the substance in a control group of samples or a baseline group of samples or a retrospective analysis of patient samples.
  • control or baseline levels can be previously determined, or measured prior to the measurement in the sample, or can be obtained from a database of such control samples. In other words, the control and subject samples do not have to be tested simultaneously.
  • “reduced concentration,” “decreased concentrations,” “lowered levels,” or “reduced levels” refers to a decrease in concentration or a decrease in level by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% in a sample relative to a control.
  • a subject “in need of prevention,” “in need of treatment,” or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment or therapy.
  • an appropriate medical practitioner e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals
  • treatment refers to preventing, reversing, alleviating, reducing the severity of, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment can be administered after one or more symptoms have developed.
  • treatment can be administered in the absence of symptoms.
  • treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment can also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • the terms “patient” or “subject” refer to an animal, preferably a mammal, and most preferably a human.
  • a therapeutically effective amount of refers to the amount of an EP4 antagonist (e.g., compound A, or a pharmaceutically acceptable salt thereof), which is effective to reduce or attenuate the biological activity of an EP4 receptor in a biological sample or in a patient.
  • a therapeutically effective amount of refers to the amount of an EP4 antagonist (e.g., compound A, or a pharmaceutically acceptable salt thereof), which measurably reduces the amount of EP4 receptor present in a cell.
  • a therapeutically effective amount of refers to the amount of an EP4 antagonist (e.g., compound A, or a pharmaceutically acceptable salt thereof), which measurably decreases the binding or signaling activity of the EP4 receptor or any EP4 receptor-mediated activity.
  • an EP4 antagonist e.g., compound A, or a pharmaceutically acceptable salt thereof
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • suitable inorganic and organic acids and bases include those derived from suitable inorganic and organic acids and bases.
  • pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2– hydroxy–ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2–naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pec
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C1–4alkyl)4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
  • structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.
  • the terms “about” or “approximately” have the meaning of within 20% of a given value or range.
  • the term “about” refers to within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a given value.
  • the method further comprises administering an EP4 antagonist to the patient having the elevated concentration of PGEM.
  • the present invention provides a method of measuring a urinary concentration of PGEM in a patient, comprising measuring the concentration of PGEM in a urine sample of the patient.
  • an LC-MS/MS method is used.
  • an LC–MS/MS method is an SPE-LC-MS/MS method, such as an online SPE-LC– MS/MS method.
  • the method of measuring the concentration of PGEM in the urine sample comprises an ELISA method.
  • Methods of measuring or determining concentrations of analytes in biological samples, such as the urine are known in the art and include, for example, mass spectrometry approaches, such as MS/MS, LC-MS/MS, multiple reaction monitoring (MRM) or SRM and product-ion monitoring (PIM), as well as antibody-based methods such as immunoassays, including Western blots, enzyme-linked immunosorbant assay (ELISA), immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, and flow cytometry-based methods.
  • mass spectrometry approaches such as MS/MS, LC-MS/MS, multiple reaction monitoring (MRM) or SRM and product-ion monitoring (PIM)
  • antibody-based methods such as immunoassays, including Western blots, enzyme-linked immunosorbant assay (ELISA), immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, and flow
  • the method of measuring the concentration of PGEM in the urine sample comprises an LC-MS/MS method.
  • LC- MS/MS or “liquid chromatography tandem mass spectrometry” is an analytical technique that combines liquid chromatography (LC) and triple quadrupole mass spectrometry (MS).
  • a sample solution containing analytes of interest such as a urine sample or a processed urine sample from an SPE process, is pumped through a stationary phase (LC column) by a mobile phase flowing through at high pressure. After elution from the LC column, the effluent is directed to the mass spectrometer.
  • the mass spectrometer for an LC-MS/MS system has an ionization source where the LC column effluent is nebulized, desolvated, and ionized creating charged particles. These charged particles then migrate or transition from a precursor ion under high vacuum through a series of mass analyzers (quadrupole) by applying electromagnetic fields, resulting in a product ion that is highly specific to the structure of the compound of interest and therefore provides a high degree of selectivity. [0038] In some embodiments, an SPE-LC-MS/MS method is used.
  • SPE-LC-MS/MS or “solid phase extraction liquid chromatography tandem mass spectrometry” is a process or analytical technique in which a solid phase extraction (SPE) process is combined with the LC-MS/MS process.
  • SPE is a method of sample preparation that concentrates and purifies analytes from solution by sorption onto a disposable solid-phase cartridge, followed by elution of the analyte with a solvent appropriate for subsequent instrumental analysis.
  • the SPE process is then followed by performing the LC-MS/MS method on the eluted analyte from the SPE process, dissolved in the appropriate buffer.
  • an “online SPE-LC-MS/MS” or “high-throughput SPE-LC-MS/MS” method is used where a combined instrumentation setup allows for the SPE, LC, and MS steps to be performed without having to manually transfer the eluted analyte from the SPE process to the LC-MS/MS instrument.
  • Methods of performing SPE- LC-MS/MS and online SPE-LC-MS/MS are known in the art for detecting analytes like urinary PGEM and are described, for example, in Y Zhang, et al., J. Mass. Spectrom.2011, 46, 705-711, the contents of which are incorporated herein by reference in their entirety.
  • an LC–MS/MS method comprises a mobile phase at a pH of about 3.4-5.6. In some embodiments, an LC–MS/MS method comprises a mobile phase at a pH of about 3.4-4.0. In some embodiments, an LC–MS/MS method comprises a mobile phase at a pH of about 5.0-5.6. In some embodiments, an LC–MS/MS method comprises a mobile phase at a pH of about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. In some embodiments, an LC–MS/MS method comprises a mobile phase at a pH of about 4.5.
  • an LC–MS/MS method comprises a mass spectrometer operating in MRM mode.
  • a sample is introduced into a mass spectrometer through electrospray ionization in negative ion mode.
  • a mass spectrometer has a source temperature at about 400 o C, about 450 o C, about 500 o C, about 550 o C, about 600 o C, about 650 o C, about 700 o C, about 750 o C, or about 800 o C.
  • a mass spectrometer has a source temperature at about 600 o C.
  • a mass spectrometer has an ionization energy at about -2500 V, -2600 V, -2700 V, -2800 V, -2900 V, -3000 V, -3100 V, -3200 V, -3300 V, -3400 V, -3500 V. In some embodiments, a mass spectrometer has an ionization energy at about -3000 V. In some embodiments, a mass spectrometer has a declustering potential of about -50 V, -55 V, -60 V, -65 V, -70 V, -75 V, -80 V, -85 V, or -90 V.
  • a mass spectrometer has a declustering potential of about -70 V. In some embodiments, a mass spectrometer has a collision energy of about -18 V, -20 V, -22 V, -24 V, -26 V, -28 V, or -30 V. In some embodiments, a mass spectrometer has a collision energy of about - 24 V. [0042] In some embodiments, the method of measuring the concentration of PGEM in the urine sample comprises an ELISA method.
  • a non-limiting example of an ELISA method for use with the methods described herein includes a double antibody sandwich ELISA method using a monoclonal antibody specific against human PGEM that is detected using a labeled secondary antibody, such as a biotin-labeled secondary antibody, that can then be detected using an enzyme- conjugate, such as an avidin-peroxidase conjugate.
  • a labeled secondary antibody such as a biotin-labeled secondary antibody
  • an enzyme- conjugate such as an avidin-peroxidase conjugate.
  • a non-limiting example of such a PGEM ELISA that can be used with the methods described herein can be obtained from MyBioSource Cat. No. MBS773057.
  • a method of measuring a urinary concentration of PGEM in a patient comprises collecting a urine sample from a patient.
  • a urine sample is stored below room temperature until being analyzed. In some embodiments, a urine sample is stored at about 0 o C. In some embodiments, a urine sample is stored at about at about -10 o C, about -20 o C, about -30 o C, about -40 o C, about -50 o C, about -60 o C, about -70 o C, or about -80 o C until being analyzed. In some embodiments, a urine sample is frozen before being analyzed. In some embodiments, a urine sample is stored at about -80 o C before being analyzed. [0044] In some embodiments, a urine sample is diluted before being analyzed. In some embodiments, a urine sample is diluted in water.
  • a urine sample is diluted about 50-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, or about 1000-fold. In some embodiments, a urine sample is diluted less than 50-fold. In some embodiments, a urine sample is diluted about 1000-fold. In some embodiments, a urine sample is diluted more than 1000-fold. [0045] In some embodiments, a urine sample is spiked with an isotope-labeled creatinine as an internal standard.
  • a urine sample is diluted, for example with water as described herein, before being spiked with an isotope-labeled creatinine.
  • Isotope-labeled creatinine differs from creatinine only in the presence of one or more isotopically enriched atoms.
  • isotope-labeled creatinine can be prepared by replacement of hydrogen by deuterium or tritium, and/or by replacement of a carbon by a 13 C- or 14 C-enriched carbon.
  • an isotope labeled creatinine is d3-deuterated creatinine.
  • an isotope-labeled creatinine is spiked at about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, or about 600 ng/ml as an internal standard. In some embodiments, an isotope-labeled creatinine is spiked at about 400 ng/ml as internal standard. In some embodiments, an isotope-labeled creatinine is spiked at less than 50 ng/ml as an internal standard. In some embodiments, an isotope-labeled creatinine is spiked at more than 600 ng/ml as an internal standard.
  • any screening assay for measuring the urinary concentration of PGEM normalizes the obtained PGEM level or concentration against the level or concentration of creatinine in the same urine sample.
  • the level or concentration of a creatine in a given urine sample can be measured by a clinical urinary creatinine diagnostics test, such as those performed on Roche Modular or Cobas analyzers.
  • the terms “elevated urinary concentration of PGEM,” “increased urinary concentration of PGEM,” “increased concentration of PGEM in a urine sample,” and “elevated concentration of PGEM in a urine sample,” refer to a concentration of PGEM normalized to the concentration of creatinine in a urine sample of a patient or subject, which is higher than the normal normalized concentration of PGEM in the urine, or equal to or higher than a selected or prespecified or predefined normalized concentration of PGEM in the urine.
  • This higher value can be an increase in the amount of normalized PGEM of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 1.5 fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 25-fold, about 50-fold, about 100-fold, or higher, relative to the amount of normalized PGEM in a control sample or control samples, such as an individual or group of individuals who are not suffering from the disease or disorder (e.g., cancer), or a control sample databased based on retrospective patient sample analysis, or an internal control, as determined by techniques known in the art.
  • a control sample or control samples such as an individual
  • a urinary sample from a subject can also be determined or deemed to have an “elevated concentration of PGEM” if the normalized concentration of PGEM is increased by one standard deviation, two standard deviations, three standard deviations, four standard deviations, five standard deviations, or more, relative to the mean (average) or median amount of the substance in a control group of samples or a baseline group of samples.
  • control or baseline levels can be previously determined, or measured prior to the measurement in the sample, or can be obtained from a database of such control samples.
  • the normal concentration of PGEM in the urine is about 10.6 ng/mg normalized to creatinine for males and about 6.0 ng/mg normalized to creatinine for females.
  • an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 20% higher relative to the amount of normalized PGEM in a control sample or control samples.
  • an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 40% higher relative to the amount of normalized PGEM in a control sample or control samples.
  • an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 60% higher relative to the amount of normalized PGEM in a control sample or control samples.
  • an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 80% higher relative to the amount of normalized PGEM in a control sample or control samples. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 100% higher relative to the amount of normalized PGEM in a control sample or control samples. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 2-fold higher relative to the amount of normalized PGEM in a control sample or control samples.
  • an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 5-fold higher relative to the amount of normalized PGEM in a control sample or control samples. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 10-fold higher relative to the amount of normalized PGEM in a control sample or control samples. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 25-fold higher relative to the amount of normalized PGEM in a control sample or control samples.
  • an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 50-fold higher relative to the amount of normalized PGEM in a control sample or control samples. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized concentration of PGEM in a urine sample about 100-fold higher relative to the amount of normalized PGEM in a control sample or control samples. [0049] In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at more than about 10.6 ng/mg for a male patient.
  • an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at more than about 15, about 20, or about 25 ng/mg for a male patient. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 30 ng/mg or more for a male patient. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 35 ng/mg or more for a male patient.
  • an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 40 ng/mg or more for a male patient. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 45 ng/mg or more for a male patient. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 50 ng/mg or more for a male patient.
  • an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at more than about 55 ng/mg, about 60 ng/mg, 65 ng/mg, 70 ng/mg, or more for a male patient.
  • an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at more than about 6.0 ng/mg for a female patient.
  • an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at more than about 9, about 12, or about 15 ng/mg for a female patient. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 18 ng/mg or more for a female patient. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 21 ng/mg or more for a female patient.
  • an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 24 ng/mg or more for a female patient. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 27 ng/mg or more for a female patient. In some embodiments, an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at about 30 ng/mg or more for a female patient.
  • an elevated concentration of PGEM in a urine sample refers to a normalized to creatine concentration of PGEM in a urine sample at more than about 33 ng/mg, about 36 ng/mg, about 39 ng/mg, 42 ng/mg, or more for a female patient.
  • a method of identifying or selecting a patient having an elevated urinary concentration of PGEM is for selecting a patient having a proliferative disorder.
  • the proliferative disorder is a cancer.
  • a cancer is selected from those as described herein.
  • an elevated concentration of PGEM in a urine sample of a patient having a proliferative disorder refers to a normalized to creatine concentration of PGEM in a urine sample at about 15 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a patient having a proliferative disorder refers to a normalized to creatine concentration of PGEM in a urine sample at about 30 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a patient having a proliferative disorder refers to a normalized to creatine concentration of PGEM in a urine sample at about 40 ng/mg or more.
  • an elevated concentration of PGEM in a urine sample of a patient having a proliferative disorder refers to a normalized to creatine concentration of PGEM in a urine sample at about 50 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a patient having a proliferative disorder refers to a normalized to creatine concentration of PGEM in a urine sample at about 60 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a patient having a proliferative disorder refers to a normalized to creatine concentration of PGEM in a urine sample at about 70 ng/mg or more.
  • an elevated concentration of PGEM in a urine sample of a patient having a proliferative disorder refers to a normalized to creatine concentration of PGEM in a urine sample at about 80 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a patient having a proliferative disorder refers to a normalized to creatine concentration of PGEM in a urine sample at about 90 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a patient having a proliferative disorder refers to a normalized to creatine concentration of PGEM in a urine sample at about 100 ng/mg or more.
  • a method of identifying or selecting a patient having an elevated urinary concentration of PGEM is for selecting a patient having colorectal cancer. In some embodiments, a method of identifying or selecting a patient having an elevated urinary concentration of PGEM is for selecting a patient having metastatic colorectal cancer. In some embodiments, a method of identifying or selecting a patient having an elevated urinary concentration of PGEM is for selecting a patient having microsatellite- stable (MSS) metastatic colorectal cancer.
  • MSS microsatellite- stable
  • an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at about 14.65 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at about 20 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at about 25 ng/mg or more.
  • an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at about 30 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at about 35 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at about 40 ng/mg or more.
  • an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at about 45 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at about 50 ng/mg or more. In some embodiments, an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at about 55 ng/mg or more.
  • an elevated concentration of PGEM in a urine sample of a colorectal cancer patient refers to a normalized to creatine concentration of PGEM in a urine sample at more than about 60 ng/ml, about 65 ng/ml, or about 70 ng/mg.
  • the present invention provides a method of treating a proliferative disorder in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • an elevated urinary concentration of PGEM is as described herein.
  • a proliferative disorder is a cancer as described herein.
  • a cancer is colorectal cancer (CRC).
  • CRC colorectal cancer
  • a cancer is metastatic colorectal cancer.
  • a cancer is microsatellite-stable (MSS) metastatic colorectal cancer.
  • MSS microsatellite-stable
  • a cancer is advanced or progressive microsatellite-stable (MSS) CRC.
  • a cancer is non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • a cancer is advanced and/or metastatic NSCLC.
  • a cancer is ovarian cancer.
  • a cancer is breast cancer.
  • a cancer is head and neck cancer.
  • the present invention provides a method of treating a cancer in a male patient, comprising selecting a patient having an elevated urinary concentration of PGEM at more than about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, or about 25 ng/mg normalized to creatinine, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a male patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 30 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof. In some embodiments, the present invention provides a method of treating a cancer in a male patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 35 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a male patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 40 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a male patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 45 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a male patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 50 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a male patient, comprising selecting a patient having an elevated urinary concentration of PGEM at more than about 55 ng/mg, about 60 ng/mg, about 65 ng/mg, or about 70 ng/mg normalized to creatinine, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising selecting a patient having an elevated urinary concentration of PGEM at more than about 6 ng/mg, about 9 ng/mg, about 12 ng/mg, or 15 ng/mg normalized to creatinine, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 18 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 21 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 24 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 27 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 30 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising selecting a patient having an elevated urinary concentration of PGEM at more than about 33 ng/mg, about 36 ng/mg, about 39 ng/mg, or about 42 ng/mg normalized to creatinine, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a method of treating a proliferative disorder in a patient comprising selecting a patient having an elevated urinary concentration of PGEM at about 15 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the proliferative disorder is a cancer.
  • the cancer is selected from those as described herein.
  • a method of treating a proliferative disorder in a patient comprises selecting a patient having an elevated urinary concentration of PGEM at about 30 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a method of treating a proliferative disorder in a patient comprises selecting a patient having an elevated urinary concentration of PGEM at about 40 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a method of treating a proliferative disorder in a patient comprises selecting a patient having an elevated urinary concentration of PGEM at about 50 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof. In some embodiments, a method of treating a proliferative disorder in a patient comprises selecting a patient having an elevated urinary concentration of PGEM at about 60 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a method of treating a proliferative disorder in a patient comprises selecting a patient having an elevated urinary concentration of PGEM at about 70 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof. In some embodiments, a method of treating a proliferative disorder in a patient comprises selecting a patient having an elevated urinary concentration of PGEM at about 80 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a method of treating a proliferative disorder in a patient comprises selecting a patient having an elevated urinary concentration of PGEM at about 90 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof. In some embodiments, a method of treating a proliferative disorder in a patient comprises selecting a patient having an elevated urinary concentration of PGEM at about 100 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 15 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a colorectal cancer e.g., MSS CRC
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 20 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a colorectal cancer e.g., MSS CRC
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 25 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a colorectal cancer e.g., MSS CRC
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 30 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a colorectal cancer e.g., MSS CRC
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 35 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a colorectal cancer e.g., MSS CRC
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 40 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a colorectal cancer e.g., MSS CRC
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 45 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • a colorectal cancer e.g., MSS CRC
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising selecting a patient having an elevated urinary concentration of PGEM at about 50 ng/mg normalized to creatinine or more, and administering to the patient a therapeutically effective amount of an EP4 antagonist, or a pharmaceutical composition thereof.
  • an EP4 antagonist is selected from those described in WO 2002/032900, WO 2005/021508, and US Patent Nos. 6,710,054 and 7,960,407, the contents of each of which are incorporated herein by reference in their entireties.
  • an EP4 antagonist is a compound of formula I: or a pharmaceutically acceptable salt thereof, wherein each variable is as described in WO 2002/032900, the contents of which is incorporated herein by reference in its entirety.
  • an EP4 antagonist is a compound of formula I’: or a pharmaceutically acceptable s iable is as described in WO 2005/021508, the contents of which is incorporated herein by reference in its entirety.
  • an EP4 antagonist is compound A: (also known as grapiprant), or a pharmaceutically a ccep a e sa ereo . n some em odiment, compound A is in crystal form.
  • compound A is in polymorph Form A, as described in US Patent Nos.7,960,407 and 9,265,756, the contents of which are incorporated herein by reference in their entireties.
  • polymorph Form A of compound A is characterized by a powder X-ray diffraction pattern obtained by irradiation with Cu K ⁇ radiation which includes main peaks at 2- Theta° 9.8, 13.2, 13.4, 13.7, 14.1, 17.5, 19.0, 21.6, 24.0 and 25.7+/ ⁇ 0.2.
  • polymorph Form A of compound A is characterized by differential scanning calorimetry (DSC) in which it exhibits an endothermic event at about 160° C.
  • DSC differential scanning calorimetry
  • polymorph Form A of compound A exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at about 9.9, about 13.5, about 14.3, about 16.1, about 17.7, about 21.8, about 24.14, and about 25.8.
  • polymorph Form A of compound A exhibits a differential scanning calorimetry profile having showed an endotherm/exotherm at about 155-170° C.
  • polymorph Form A of compound A exhibits a thermogravimetric analysis showing a loss of mass of 0.5-0.6% when heated from about 30° to about 150° C.
  • the present invention provides a method of identifying or selecting a male patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC–MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 30 ng/mg or more.
  • the present invention provides a method of identifying or selecting a male patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC–MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 40 ng/mg or more.
  • the present invention provides a method of identifying or selecting a male patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC–MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 50 ng/mg or more.
  • the present invention provides a method of identifying or selecting a female patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC–MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 18 ng/mg or more.
  • the present invention provides a method of identifying or selecting a female patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC–MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 24 ng/mg or more.
  • the present invention provides a method of identifying or selecting a female patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC–MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 30 ng/mg or more.
  • the present invention provides a method of identifying or selecting a patient of a proliferative disorder having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 50 ng/mg or more.
  • the present invention provides a method of identifying or selecting a patient of a proliferative disorder having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 60 ng/mg or more.
  • the present invention provides a method of identifying or selecting a patient of a proliferative disorder having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 70 ng/mg or more.
  • the present invention provides a method of identifying or selecting a colorectal cancer patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 45 ng/mg or more.
  • the present invention provides a method of identifying or selecting a colorectal cancer patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 50 ng/mg or more.
  • the present invention provides a method of identifying or selecting a colorectal cancer patient having an elevated urinary concentration of PGEM, comprising measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein), and selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 60 ng/mg or more.
  • the present invention provides a method of treating a cancer in a male patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 30 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a cancer in a male patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 40 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a cancer in a male patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 50 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 18 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 24 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a cancer in a female patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 30 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a proliferative disorder in a patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 50 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a proliferative disorder in a patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 60 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a proliferative disorder in a patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 70 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 45 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • a colorectal cancer e.g., MSS CRC
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 50 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • a colorectal cancer e.g., MSS CRC
  • the present invention provides a method of treating a colorectal cancer (e.g., MSS CRC) in a patient, comprising: measuring the concentration of PGEM in a urine sample of a patient using an LC– MS/MS method (for example, as described herein); selecting a patient having a normalized to creatinine concentration of PGEM in a urine sample at about 60 ng/mg or more; and administering to the patient a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • Compound A, or a pharmaceutically acceptable salt thereof is administered once daily.
  • Compound A, or a pharmaceutically acceptable salt thereof is administered twice daily.
  • Compound A, or a pharmaceutically acceptable salt thereof is administered three times daily. [0089] In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered at a dosage of about 200-500 mg, about 250-450 mg, or about 300-450 mg per administration. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered at a dosage of about 200 or about 250 mg per administration. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered at a dosage of about 300 mg per administration. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered at a dosage of about 350 or about 400 mg per administration.
  • Compound A, or a pharmaceutically acceptable salt thereof is administered at a dosage of about 450 or about 500 mg per administration. [0090] In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered at a dosage of about 200-900 mg, about 250-900 mg, or about 300-900 mg per administration. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered at a dosage of about 600 or about 650 mg per administration. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered at a dosage of about 700 mg or 750 mg per administration. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered at a dosage of about 800 or about 850 mg per administration.
  • Compound A, or a pharmaceutically acceptable salt thereof is administered at a dosage of about 900 mg per administration.
  • Compound A, or a pharmaceutically acceptable salt thereof is administered twice a day, at a dosage of about 300 mg per administration. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered twice a day, at a dosage of about 450 mg per administration.
  • Compound A, or a pharmaceutically acceptable salt thereof is administered twice a day, at a dosage of about 600 mg per administration. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered twice a day, at a dosage of about 900 mg per administration. 4.
  • a method described herein comprises administering a pharmaceutical composition comprising an EP4 antagonist, as described herein, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.
  • the amount of an EP4 antagonist in a composition is such that is effective to measurably decrease the activity of an EP4 receptor in a biological sample or in a patient.
  • an EP4 antagonist composition is formulated for oral administration to a patient.
  • pharmaceutically acceptable carrier, adjuvant, or vehicle refers to a non- toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.
  • compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • the compositions are administered orally, intraperitoneally or intravenously.
  • Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • pharmaceutically acceptable compositions of this invention may be administered in the form of suppositories for rectal administration.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.
  • compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • provided pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • provided pharmaceutically acceptable compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
  • the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.
  • compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. [00104] Most preferably, pharmaceutically acceptable compositions of this invention are formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions of this invention are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food.
  • compositions of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration.
  • provided compositions should be formulated so that a dosage of between 0.01 - 100 mg/kg body weight/day of the antagonist can be administered to a patient receiving these compositions.
  • a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated.
  • the present invention provides a method of treating a proliferative disorder in a patient comprising selecting a patient having an elevated urinary concentration of PGEM normalized to creatinine, for example, using a method as described herein, and administering to the patient a therapeutically effective amount of an EP4 antagonist, for example, as described herein.
  • a treatment method further comprises measuring the concentration of PGEM in a urine sample of a patient, for example, using an LC– MS/MS method as described herein.
  • a proliferative disorder is a cancer, for example, as described herein.
  • the present invention provides a method of treating a proliferative disorder in a patient having an elevated urinary concentration of PGEM normalized to creatinine, determined, for example, using a method as described herein, the method comprising administering to the patient having an elevated urinary concentration of PGEM normalized to creatinine a therapeutically effective amount of an EP4 antagonist, for example, as described herein.
  • a treatment method further comprises measuring the concentration of PGEM in a urine sample of a patient, for example, using an LC–MS/MS method as described herein.
  • a proliferative disorder is a cancer, for example, as described herein.
  • the cancer or proliferative disorder or tumor to be treated using the methods and uses described herein include, but are not limited to, a hematological cancer, a lymphoma, a myeloma, a leukemia, a neurological cancer, skin cancer, breast cancer, a prostate cancer, a colorectal cancer, lung cancer, head and neck cancer, a gastrointestinal cancer, a liver cancer, a pancreatic cancer, a genitourinary cancer, a bone cancer, renal cancer, and a vascular cancer.
  • a cancer to be treated using the methods described herein can be selected from colorectal cancer, such as microsatellite-stable (MSS) metastatic colorectal cancer, including advanced or progressive microsatellite-stable (MSS) CRC; non-small cell lung cancer (NSCLC), such as advanced and/or metastatic NSCLC; ovarian cancer; breast cancer, such as inflammatory breast cancer; endometrial cancer; cervical cancer; head and neck cancer; gastric cancer; gastroesophageal junction cancer; and bladder cancer.
  • a cancer is colorectal cancer.
  • the colorectal cancer is metastatic colorectal cancer.
  • the colorectal cancer is microsatellite-stable (MSS) metastatic colorectal cancer.
  • a cancer is advanced or progressive microsatellite-stable (MSS) CRC.
  • a cancer is non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • a cancer is advanced and/or metastatic NSCLC.
  • a cancer is ovarian cancer.
  • a cancer is breast cancer.
  • a cancer is inflammatory breast cancer.
  • a cancer is endometrial cancer.
  • a cancer is endometrial cancer.
  • a cancer is head and neck cancer.
  • a cancer is gastric cancer. In some embodiments, a cancer is gastroesophageal junction cancer. In some embodiments, a cancer is bladder cancer.
  • Cancer includes, in some embodiments, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin’s disease or non-Hodgkin’s disease), Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcom
  • the cancer is glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.
  • the cancer is acoustic neuroma, astrocytoma (e.g.
  • GBM Glioblastoma
  • the cancer is a type found more commonly in children than adults, such as brain stem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic nerve glioma, pineal tumor, primitive neuroectodermal tumors (PNET), or rhabdoid tumor.
  • the patient is an adult human. In some embodiments, the patient is a child or pediatric patient.
  • Cancer includes, in another embodiment, without limitation, mesothelioma, hepatobilliary (hepatic and billiary duct), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and duodenal), uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymph
  • the cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatocholangiocarcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; anaplastic thyroid cancer; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal/stomach (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma, or brain cancer; neurofibromatosis-1 associated malignant peripheral nerve sheath tumors (MPN
  • the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatocholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumors (MPNST), Waldenstrom’s macroglobulinemia, or medulloblastoma.
  • HCC hepatocellular carcinoma
  • hepatoblastoma colon cancer
  • rectal cancer ovarian cancer
  • the cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma.
  • Solid tumors generally comprise an abnormal mass of tissue that typically does not include cysts or liquid areas.
  • the cancer is selected from renal cell carcinoma, or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma, or liver cancer; melanoma; breast cancer; colorectal carcinoma, or colorectal cancer; colon cancer; rectal cancer; anal cancer; lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatocholangiocarcinoma; soft tissue and bone synovial sarcoma; rhabdomyos
  • the cancer is selected from renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoblastoma, colorectal carcinoma, colorectal cancer, colon cancer, rectal cancer, anal cancer, ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatocholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, anaplastic thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, brain cancer, neurofibromatosis-1 associated malignant peripheral nerve sheath tumors (MPNST), Waldenstrom’s macroglobulinemia, or medulloblastoma.
  • HCC hepato
  • the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatocholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumors (MPNST), Waldenstrom’s macroglobulinemia, or medulloblastoma.
  • HCC hepatocellular carcinoma
  • hepatoblastoma colon cancer
  • rectal cancer ovarian cancer
  • ovarian cancer ova
  • the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is hepatoblastoma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is ovarian cancer, or ovarian carcinoma. In some embodiments, the cancer is ovarian epithelial cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is papillary serous cystadenocarcinoma. In some embodiments, the cancer is uterine papillary serous carcinoma (UPSC). In some embodiments, the cancer is hepatocholangiocarcinoma.
  • HCC hepatocellular carcinoma
  • the cancer is hepatoblastoma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is ovarian cancer, or ovarian carcinoma. In some embodiments, the cancer is ovarian epithelial cancer. In some embodiments,
  • the cancer is soft tissue and bone synovial sarcoma. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is anaplastic thyroid cancer. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer, or pancreatic ductal carcinoma. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is glioma. In some embodiments, the cancer is malignant peripheral nerve sheath tumors (MPNST). In some embodiments, the cancer is neurofibromatosis-1 associated MPNST.
  • MPNST peripheral nerve sheath tumors
  • the cancer is Waldenstrom’s macroglobulinemia. In some embodiments, the cancer is medulloblastoma. [00121] In some embodiments, the cancer is Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, Anal Cancer, Appendix Cancer, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Tumor, Astrocytoma, Brain and Spinal Cord Tumor, Brain Stem Glioma, Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Central Nervous System Embryonal Tumors, Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor, Carcinoma of Unknown Primary, Central Nervous System Cancer, Cervical Cancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia (C
  • the cancer is selected from bladder cancer, breast cancer (including TNBC), cervical cancer, colorectal cancer, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), esophageal adenocarcinoma, glioblastoma, head and neck cancer, leukemia (acute and chronic), low-grade glioma, lung cancer (including adenocarcinoma, non-small cell lung cancer, and squamous cell carcinoma), Hodgkin's lymphoma, non-Hodgkin lymphoma (NHL), melanoma, multiple myeloma (MM), ovarian cancer, pancreatic cancer, prostate cancer, renal cancer (including renal clear cell carcinoma and kidney papillary cell carcinoma), and stomach cancer.
  • CLL chronic lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • esophageal adenocarcinoma esophageal adenocar
  • the cancer is small cell lung cancer, non-small cell lung cancer, colorectal cancer, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), pancreatic cancer, liver cancer, hepatocellular cancer, neuroblastoma, other solid tumors or other hematological cancers.
  • the cancer is small cell lung cancer, non-small cell lung cancer, colorectal cancer, multiple myeloma, or AML.
  • the present invention further features methods and compositions for the diagnosis, prognosis and treatment of viral-associated cancers, including human immunodeficiency virus (HIV) associated solid tumors, human papilloma virus (HPV)-16 positive incurable solid tumors, and adult T-cell leukemia, which is caused by human T-cell leukemia virus type I (HTLV-I) and is a highly aggressive form of CD4+ T-cell leukemia characterized by clonal integration of HTLV- I in leukemic cells (See https://clinicaltrials.gov/ct2/show/study/ NCT02631746); as well as virus- associated tumors in gastric cancer, nasopharyngeal carcinoma, cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinoma of the head and neck, and Merkel cell carcinoma.
  • HCV human immunodeficiency virus
  • HPV human papilloma virus
  • HTLV-I human T-cell leukemia virus type I
  • the present invention provides a method for treating a cancer or a tumor in a patient in need thereof, comprising administering to the patient an agent that inhibits prostaglandin EP4 receptor (EP4) activity and an immuno-oncology agent as described herein, or pharmaceutical compositions thereof described herein.
  • the cancer or tumor comprises any of the cancers described herein.
  • the cancer comprises melanoma cancer.
  • the cancer comprises breast cancer. In some embodiments, the cancer comprises lung cancer. In some embodiments the cancer comprises small cell lung cancer (SCLC). In some embodiments, the cancer comprises non-small cell lung cancer (NSCLC). [00127] In some embodiments, the methods or uses described herein inhibit or reduce or arrest the growth or spread of a cancer or tumor. In some embodiments, the methods or uses described herein inhibit or reduce or arrest further growth of the cancer or tumor. In some embodiments, the methods or uses described herein reduce the size (e.g., volume or mass) of the cancer or tumor by at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90% or at least 99% relative to the size of the cancer or tumor prior to treatment.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • the methods or uses described herein reduce the quantity of the cancers or tumors in the patient by at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90% or at least 99% relative to the quantity of cancers or tumors prior to treatment.
  • the compounds and compositions, according to the methods of the present invention can be administered using any amount and any route of administration effective for treating or lessening the severity of a cancer or tumor. The exact amount required varies from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease or condition, the particular agent, its mode of administration, and the like.
  • the compounds and compositions, according to the methods of the present invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions is decided by the attending physician within the scope of sound medical judgment.
  • the specific effective dose level for any particular patient or organism depends upon a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts.
  • patient or “subject,” as used herein, means an animal, preferably a mammal, and most preferably a human.
  • compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, or the like, depending on the severity of the disease or disorder being treated.
  • the compounds of the invention can be administered orally or parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents,
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type can also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions examples include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.
  • the active compounds can also be in micro-encapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
  • Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention.
  • the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body.
  • Such dosage forms can be made by dissolving or dispensing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin.
  • the rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • EXEMPLIFICATION [00139] The following examples are provided for illustrative purposes only and are not to be construed as limiting this invention in any manner.
  • An EP4 antagonist described herein can be synthesized using the methods described in WO 2002/032900, WO 2005/021508, and US patent Nos.6,710,054 and 7,960,407, the contents of each of which are incorporated herein by reference in their entireties. Exemplary protocols for preparing polymorph form A of compound A are described in US Patent Nos.
  • the methoximated PGE- M was extracted with 10 ml water adjusted to pH 3, and the aqueous sample was applied to a C- 18 Sep-Pak (Waters, Milford, MA, USA) that had been preconditioned with 5 ml methanol and 5 ml water (pH 3). The Sep-Pak was washed with 20 ml water (pH 3) and 10 ml heptane. PGE-M was then eluted from the Sep-Pak with 5 ml ethyl acetate, and any residual aqueous material was removed from the eluate by aspiration.
  • Samples were separated by a gradient of 98–40% of mobile phase A over 15 min at a flow rate of 75 ⁇ l/min prior to delivery to a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer operating in the selected reaction monitoring (SRM) mode.
  • the ESI source used nitrogen for both sheath and auxiliary gas set at 60 psi and 7 L/min, respectively.
  • the mass spectrometer was operated in the negative ion mode with a capillary temperature of 210 °C, a spray voltage of 3.0kV, and a tube lens voltage of 118 V.
  • the source collision-induced dissociation (CID) was set at 10eV.
  • Precursor ions (m/z 385 and 391 for unlabeled PGE-M and the [ 2 H6]PGE- M internal standard, respectively) were collisionally activated at 22 eV under 1.5 mT argon gas.
  • the predominant product ion m/z 336 representing [M - (OCH 3 + H 2 O)]- and the analogous ion, m/z 339 [M - (OC[ 2 H3]+H2O)]- , for the deuterated internal standard were monitored in SRM mode.
  • Quantification of endogenous PGE-M used the ratio of the mass chromatogram peak areas of the m/z 336 and 339 ions.
  • Urinary 2,3-dinor-6-keto-PGF 1 ⁇ was determined as described previously [VC Daniel, et al., J. Chromatogr. B 653 (1994) 117–122]. Data are expressed after correction for urinary creatinine (Cr) concentrations and are reported as nanograms per milligram Cr. Urine Cr was measured by an Autoanalyzer technique (Technicon, BuValo Grove, IL, USA). Human studies [00148] Two studies were conducted involving patients with colorectal cancer (CRC). Compound A was administered at doses of 300 BID or 450 BID. The baseline, i.e., prior to treatment with Compound A, urinary concentrations of PGE-M of study #1 are shown in Table 1 and FIG.1 A and 1B.
  • the urinary concentrations of PGE-M of study #2 are shown in Table 2 and FIG.2.
  • Table 1 Baseline urinary concentrations of PGE-M in CRC patients.
  • Table 2. Baseline urinary concentrations of PGE-M in NSCLC patients.
  • Patient # Dose Sex PGE-M (ng/mg Cr)
  • Example 2. Quantification of PGE-M using a high-throughput online SPE-LC–MS/MS assay [00149] A description of this method can be found in Y Zhang, et al., J. Mass. Spectrom.2011, 46, 705-711, the content of which is incorporated herein by reference in its entirety.
  • tPGDM (9 ⁇ -hydroxy-11,15-dioxo-2,3,4,5-tetranor-prostan-1,20-dioic acid)
  • tPGDM- d6 (9 ⁇ -hydroxy-11,15-dioxo-2,3,4,5-tetranorprostan-1,20-dioic acid-17,17,18,18,19,19-d6)
  • tPGEM 11 ⁇ -hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostan-1,20-dioic acid
  • tPGEM-d6 11 ⁇ - hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostan-1,20-dioic acid-17,17,18,18,19,19-d6)
  • methoxyamine hydrochloride creatinine, creatinine-d3, acetonitrile, ammonium hydroxide, and ethyl acetate were obtained from commercial vendors.
  • Chromatographic separation was performed on a Kinetex 2.6 ⁇ m 50 mm ⁇ 3 mm C18 column using 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B with 5.00 ⁇ l of the sample injection volume. Chromatographic elution was performed using a gradient of 0–2.00% mobile phase B for the first 2 min followed by 0.6 min at 50% B at a flow rate of 0.300 ml/min. Detection was performed using an API 4000 Mass Spectrometer operating in multiple reaction monitoring (MRM) mode. Urine samples were diluted 1000-fold in water and spiked with a d3-deuterated creatinine at 400 ng/ml as internal standard (IS).
  • MRM multiple reaction monitoring
  • the main stock solution of analyte was prepared at 1.0 mg/ml in ethyl acetate and stored at ⁇ 80 o C in a glass vessel.
  • the calibration standards were prepared by adding appropriate amounts of the stock solution or subsequent calibration standards to blank artificial urine.
  • Artificial urine contains the following components: 24.2 g/l of urea, 10.0 g/l of sodium chloride, 6.0 g/l of potassium chloride, 6.4 g/l of sodium dibasic phosphate, 2.0 g/l of creatinine and 50 mg/l of albumin (pH 7.5 adjusted using 1M hydrochloric acid).
  • Nominal concentrations of calibration standards for tPGDM/tPGEM were 0.200/0.500, 0.400/1.00, 1.00/2.50, 2.00/5.00, 6.00/15.0, 10.0/25.0, 20.0/50.0 and 40.0/100 ng/ml in artificial urine.
  • QC samples were prepared at three concentration levels using pooled human urine.
  • Low-QC (LQC) contained only endogenous baseline levels of tPGDM/tPGEM, while both mid-QC (MQC) and high-QC (HQC) contained endogenous and spiked analytes.
  • Derivatization of analytes and ISs in biological samples, calibrator standards and QC samples was performed based on a previously published method [WL Song, et al., J. Biol. Chem.2008, 283, 1179]. Briefly, 500 ⁇ l of samples was mixed with 25 ⁇ l methoxyamine (1.0 g/ml in water) and 25 ⁇ l of IS solution (100 ng/ml of tPGDM-d6 and tPGEM-d6). Samples were then incubated at room temperature for 30 min. Finally, 100 ⁇ l of the above samples was injected to online SPE-LC–MS/MS system as described below.
  • Chromatographic separation of derivatized tPGDM and tPGEM was performed by back flushing the trapped analytes into a Kinetex C18 analytical column (2.6 ⁇ m, 50 mm ⁇ 3 mm) using a gradient of 40–50% mobile phase B for 4 min with a flow rate of 0.5 ml/min.
  • Detection was performed by an API 4000 Mass Spectrometer operating in MRM mode. Samples were introduced into the mass spectrometer through electrospray ionization in negative ion mode. The source temperature was 600 ⁇ C. The ionization energy was set at ⁇ 3000 V.
  • the transitions monitored for modified tPGDM or tPGEM and the modified IS were m/z 385.2 ⁇ 336.2 and 391.2 ⁇ 342.2, respectively, with a declustering potential of ⁇ 70 V and a collision energy of ⁇ 24 V.
  • the dwell time for each transition was 100 ms.
  • MRM data were acquired by Analyst 1.4.2 software.
  • the calibration curves were constructed based on the response ratio of peak area between analytes and their internal standards versus nominal standard concentrations by least-squares linear regression using a weighting factor of 1/x 2 . Concentrations of QC samples or unknown samples were determined using the response ratio from QC samples or unknown samples and the linear regression curve.
  • the surrogate matrix SURINE TM Negative urine control (Synthetic urine), will be used for the preparation of the calibration curve, as a diluent for any samples require dilution, and as matrix blanks.
  • Pre-Screen Blank Matrix Prior to spiking any QCs (quality controls) that contain a urine component, the urine is analyzed two different days and the average of two days is taken to determine the endogenous Tetranor-PGEM concentration. Endogenous concentration is determined by analyzing n ⁇ 6, extracted with ISTD in a batch containing calibration curves and batch acceptance QCs. Batch acceptance QCs can be surrogate matrix QCs, or previously prepared and qualified urine QCs.
  • Tetranor-PGEM-d6 Stock Solution 100 ⁇ g/mL
  • ISO1 Tetranor-PGEM-d6
  • Predissolved Tetrano-PGEM-d6 can be used directly or conducting appropriate dilution using Methanol to achieve the final concentration of 100 ⁇ g/mL.
  • Container Glass Storage temperature: Nominal -70°C
  • Expiry Assigned 365 days expiration
  • Preparation of Solutions for Comparison and/or Stability Evaluations [00170] Make a separate comparison solution for each stock solution being compared, as shown below. Solution Source Source Source Sol Diluent Final Final ID Solution ution Solution Diluent Concentration Volume Used Volume Concentration [00 p y.
  • ⁇ Volume or concentration can be determined based on urine endogenous level. The components are chosen to maximize the urine content while achieving the target final concentration and using workable volumes. [00175] The urine component is ⁇ 95% of the final volume for the HQC, MQC and DQC. [00176] The urine component can be ⁇ 95% for LQC QC due to endogenous levels. [00177] Ensure that the target final concentration is achieved to 3 significant figures.
  • HIS is the recovery solution for using the ISTD to match the HQC level.
  • MIS is the recovery solution for using the ISTD to match the MQC level.
  • LIS is the recovery solution for using the ISTD to match the LQC level.
  • IRS is the recovery solution for the internal standard.
  • Target final concentrations can be selected after endogenous level determination.
  • Parallelism A set of curves are prepared in one lot of artificial human urine. Three other sets of curves are prepared in three different lots of authentic human urine. Duplicate calibration curves are constructed in each lot of the above matrix. For artificial matrix curve, a linear regression is applied. For authentic matrix curves, linear regression and standard addition approach are used. Endogenous concentration is calculated based on slope and Y-intercept using this formula:
  • the surrogate matrix Surine Negative Urine Control [SUR] is used for matrix blanks as well as a diluent for samples requiring dilution.
  • Samples (including QC samples) which require dilution dilute with control matrix e.g. ,100-fold dilution is made by a two-step dilution in which 1) 100 ⁇ L of sample/QC to 900 ⁇ L of surrogate matrix [SUR], vortex mix (ca.10 seconds) 2) 200 ⁇ L of sample/QC to 1800 ⁇ L of surrogate matrix [SUR], vortex mix (ca.10 seconds) and take 200 ⁇ L for analysis. Alternate dilution scheme can be used. Note: All mixing times are approximate (unless otherwise stated). Automated liquid handling devices include, for example, a Nimbus96.
  • Exact mass ions can vary slightly ( ⁇ 0.5 Da) from instrument to instrument because of unit resolution of quadrupole mass spectrometers.

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Abstract

La présente invention concerne des méthodes de sélection d'un patient atteint d'une maladie proliférative avec concentration urinaire élevée de PGEM, des méthodes de traitement d'une maladie proliférative chez un patient comprenant la sélection d'un patient avec une concentration urinaire élevée de PGEM et l'administration au patient d'un antagoniste de l'EP4, et des méthodes de traitement d'une maladie proliférative chez un patient avec une concentration urinaire élevée de PGEM par l'administration au patient d'un antagoniste de l'EP4.
PCT/US2021/030771 2020-05-05 2021-05-05 Antagonistes de l'ep4 et leur utilisation dans le traitement des maladies prolifératives" WO2021226162A1 (fr)

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CN116350629A (zh) * 2021-12-27 2023-06-30 江苏美迪森生物医药有限公司 前列腺素e2受体ep4拮抗剂在制备预防和/或治疗肺动脉高压疾病的药物中的应用
CN116046930A (zh) * 2022-12-21 2023-05-02 丽珠集团新北江制药股份有限公司 一种检测格拉匹纶含量的高效液相色谱分析方法

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