WO2014195847A2 - Nouvelle utilisation du l-tryptophane marqué par un isotope stable - Google Patents

Nouvelle utilisation du l-tryptophane marqué par un isotope stable Download PDF

Info

Publication number
WO2014195847A2
WO2014195847A2 PCT/IB2014/061889 IB2014061889W WO2014195847A2 WO 2014195847 A2 WO2014195847 A2 WO 2014195847A2 IB 2014061889 W IB2014061889 W IB 2014061889W WO 2014195847 A2 WO2014195847 A2 WO 2014195847A2
Authority
WO
WIPO (PCT)
Prior art keywords
tryptophan
stable isotope
isotope labeled
tryp
serotonin metabolism
Prior art date
Application number
PCT/IB2014/061889
Other languages
English (en)
Other versions
WO2014195847A3 (fr
Inventor
Oliver Nayler
Magali VERCAUTEREN
Richard Welford
Original Assignee
Actelion Pharmaceuticals Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Actelion Pharmaceuticals Ltd filed Critical Actelion Pharmaceuticals Ltd
Publication of WO2014195847A2 publication Critical patent/WO2014195847A2/fr
Publication of WO2014195847A3 publication Critical patent/WO2014195847A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0402—Organic compounds carboxylic acid carriers, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders

Definitions

  • the invention relates to the use of L-tryptophan (L-Tryp) comprising one or more stable isotope labels for in vivo monitoring of the tryptophan-serotonin metabolism, especially for the characterization of compounds modulating tryptophan-serotonin metabolism, for detecting diseases associated with altered tryptophan-serotonin metabolism; and for monitoring therapeutic treatment of diseases associated with altered tryptophan-serotonin metabolism.
  • L-tryptophan L-tryptophan
  • the biogenic amine serotonin (5HT) is a biochemical messenger and regulator that signals through 13 receptors which are distributed throughout the nervous system and peripheral organs. 5HT is synthesized in 2 steps from the dietary amino acid L-tryptophan (L-Tryp). The first and rate limiting step in the tryptophan-serotonin metabolism is the hydroxylation of L- Tryp by the non-heme pterin dependent oxygenase tryptophan hydroxylase (TPH).
  • kynurenine e.g. melatonin
  • 5HT is further metabolized to 5-hydroxyindole acetic acid (5HIAA) by a combination of monoamine oxidase-A (MAO-A) and , subsequently, an aldehyde dehydrogenase. 5HIAA is excreted in the urine.
  • MAO-A monoamine oxidase-A
  • An additional 5HT metabolic pathway in the pineal gland leads to production of melatonin which is involved in the circadian regulation of the sleep-wake cycle.
  • TPH comprises two isoforms: TPH2 is mainly expressed in neuronal cell types in the central nervous system (CNS), while TPH1 is mainly expressed in peripheral tissues, including the entro chromaffin cells (EC) in the gut, where it is responsible for synthesizing 5HT that is stored in circulating blood platelets.
  • CNS central nervous system
  • TPH1 is mainly expressed in peripheral tissues, including the entro chromaffin cells (EC) in the gut, where it is responsible for synthesizing 5HT that is stored in circulating blood platelets.
  • TPH1 and thus altered tryptophan-serotonin metabolism has been implicated as a potential drug target in a number of pathophysiologies such as lung diseases including e.g. chronic obstructive pulmonary disease (COPD), pulmonary embolism, interstitial lung disease such as lung fibrosis (Konigshoff, M. et al.
  • COPD chronic obstructive pulmonary disease
  • cancer including e.g breast cancer (Pai VP et al. (2009) "Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival.”
  • breast cancer Panai VP et al. (2009) "Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival.”
  • prostate cancer Shinka T et al. (2011) “Serotonin synthesis and metabolism-related molecules in a human prostate cancer cell line.” Oncol Lett. Mar;2(2):211 -215) and neuroendocrine tumors (Hicks RJ. (2010) "Use of molecular targeted agents for the diagnosis, staging and therapy of neuroendocrine malignancy.” Cancer Imaging.
  • TPH2 has been implicated as a potential drug target in a number of neurological health disorders including depression; anxiety including generalized anxiety disorder and social phobia; emetic disorders; migraine; substance abuse; attention deficit disorder (ADD); attention deficit hyperactivity disorder (ADHD); bipolar disorder; suicidal behavior; behavioral disorder; schizophrenia; Parkinson's disease; Huntigton ' s disease; autism; dyskinesia; eating disorders; type 2 diabetes; pain; Alzheimer ' s disease; sexual dysfunction; and brain tumors.
  • neurological health disorders including depression; anxiety including generalized anxiety disorder and social phobia; emetic disorders; migraine; substance abuse; attention deficit disorder (ADD); attention deficit hyperactivity disorder (ADHD); bipolar disorder; suicidal behavior; behavioral disorder; schizophrenia; Parkinson's disease; Huntigton ' s disease; autism; dyskinesia; eating disorders; type 2 diabetes; pain; Alzheimer ' s disease; sexual dysfunction; and brain tumors.
  • TPH activity and modulators thereof e.g. TPH inhibitors
  • evaluation of TPH activity and modulators thereof in vivo was hampered by the lack of a reliable pharmacodynamic marker of target engagement after a single administration of drug.
  • simple measurement of 5HT in the blood cannot be used due to the long half-life (t 1 ⁇ 2 ) of platelet 5HT which prevents the observation of 5HT concentration changes after administration of a single dose of drug.
  • Brain 5HT is produced rapidly after uptake of circulating L-Tryp from the plasma (Hyyppa, M. T., et al. (1973) "Rapid accumulation of H3-serotonin in brains of rats receiving intraperitoneal H3- tryptophan: effects of 5,6-dihydroxytryptamine or female sex hormones", J Neural Transm 34, 1 1 1 -124).
  • the production of brain 5HT was extensively probed in the 1990s and 2000s, with the most prominent tool being intra venous (i.v.) administration of 14 C-1 -methyl- tryptophan which is taken-up into the brain (Diksic, M.
  • 5HT is predominantly produced by TPH1 in a number of organs.
  • the gut enterochromaffin cells are often cited to be the primary peripheral site of 5HT synthesis, where it plays roles amongst others in gut motor activity, visceral sensation and intestinal secretion (Bertrand, P. P., and Bertrand, R. L. (2010) “Serotonin release and uptake in the gastrointestinal tract", Auton Neurosci 153, 47-57; Hasler, W. L. (2009) “Serotonin and the Gl tract", Curr Gastroenterol Rep 11, 383-391 ). Serotonin secreted from the EC eventually finds its way out of the tissue into the blood.
  • 5HT is actively taken up by blood platelets, where it is stored. Activated platelets disgorge 5HT and it subsequently serves as a vasoconstrictor and helps to regulate hemostatis and blood clotting.
  • Linder et al. (2009) recently characterized 5HT concentrations in a number of organs in the rat (Linder, A. E., et al. (2009) "Body distribution of infused serotonin in rats", Clin Exp Pharmacol Physiol 36, 599-601).
  • the lung was found to have a similar 5HT concentration to the gut.
  • Other researchers have measured TPH1 gene expression by qPCR and the results suggest that TPH1 is probably active in other organs including the thymus and the spleen (Walther, D.
  • PCA p-chlorophenylalanine
  • LP533401 has been further characterized in both mouse and rat models of osteoporosis (Yadav, V. K., et al. (2010) “Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis", Nat Med 16, 308-312).
  • LX1 031 ((S)-2-Amino-3-(4- ⁇ 2-amino-6- [(R)-2,2,2-trifluoro-1 -(3'-methoxy-biphenyl-4-yl)-ethoxy]-pyrimidin-4-yl ⁇ -phenyl)-propionic acid, WO2007/089335) was the first TPH inhibitor from Lexicon Pharmaceuticals Ltd to enter clinical trials and similar to LP533401 lowers 5HT in the jejunum, with only a minor reduction observed in the colon and no effect on brain 5HT.
  • LX1031 qid did not affect blood 5HT and had very modest effects on urinary 5HIAA (up to 30% reduction) (Brown , P.
  • LX1031 shows clinical benefit in patients with nonconstipating irritable bowel syndrome", Gastroenterology 141, 507-516.
  • Another small molecule inhibitor of TPH1 is LX1032 ((S)-2-Amino-3-[4-(2-amino-6- ⁇ (R)-1 -[4-chloro-2-(3-methyl-pyrazol-1 -yl)-phenyl]-2,2,2-trifluoro-ethoxy ⁇ -pyrimidin-4-yl)- phenyl]-propionic acid ethyl ester, WO2008/073933), which is disclosed to be in clinical studies for carcinoid syndrome.
  • LCMS analysis is superior to radioactive- HPLC, where there is a potential risk of interference and loss of specificity due to overlapping peaks from the complex metabolism of L-Tryp.
  • a final advantage of the LCMS analysis is the simultaneous monitoring of both naturally occurring and labeled Tryp and 5HT, which results in improved data quality due to the extra data normalization possibilities (i.e. analytical variability can be largely removed by reporting isotope ratios vide infra).
  • Stable isotope labeled Tryp has mainly been used in NMR studies of proteins (see for example: Moseley, H. N., et al.
  • the present invention relates to novel uses of stable isotope labeled L- Tryp for monitoring the tryptophan-serotonin-metabolism, by means of mass spectrometry after having administered a stable isotope labeled L- Tryp to a subject.
  • the methods described herein are especially suitable for the in vitro testing of the in vivo modulatory effect of inhibitors of TPH after a single oral dose, including but not limited to the in vitro testing of the in vivo modulatory effect of small molecule inhibitors of TPH1 , and for the diagnosis of pathologies associated with altered 5HT metabolism.
  • Fig. 1 illustrates the formation of heavy 5HT (h5HT) and heavy 5HIAA (h5HIAA) from heavyTryp (hTryp) in blood from a rat.
  • Fig. 2 illustrates the formation of mid-weight 5HT (mw5HT) from mid-weight Tryp (mwTryp) in blood from a rat.
  • Fig. 3 illustrates the kinetics of h5HT and h5HIAA appearence in the blood of a rat during continuous i.v. infusion of hTryp.
  • Fig. 4 illustrates inhibition of h5HT appearence in rat blood by administration of the TPH1 inhibitor LX1032.
  • Fig. 5 illustrates the incorporation of label from Tryp into 5HT and 5HIAA in different organs and body fluids of a rat, after a single oral dose of hTryp.
  • the invention relates to a stable isotope labeled L-Tryp for use in a method for identifying , and / or for monitoring the pharmacological action of, a modulator of the tryptophan-serotonin metabolism in vivo; the method comprising:
  • said candidate compound may have been administered using a single dose or multiple dosing, wherein at least one dose of said candidate compound has been administered at a time point before, after, or simultaneously with respect to the administration of said stable isotope labeled L- Tryp that allows identifying and / or monitoring said candidate compound as a modulator of the tryptophan-serotonin metabolism; preferably at least one dose of said candidate compound has been administered within 5 hours before or after, or simultaneously, with respect to the administration of said stable isotope labeled L- Tryp;
  • sample(s) has / have been obtained at (a) time point(s) that allow(s) identifying and / or monitoring said candidate compound as a modulator of the tryptophan-serotonin metabolism; wherein in case more than one sample has been obtained, preferably each sample has been obtained at a different time point with respect to the administration of the stable isotope labeled L-Tryp.
  • the invention relates to a method for identifying, and / or for monitoring the pharmacological action of, a modulator of the tryptophan-serotonin metabolism in vivo; the method comprising:
  • said candidate compound may be administered using a single dose or multiple dosing, wherein at least one dose of said candidate compound is administered at a time point before, after, or simultaneously with respect to the administration of said stable isotope labeled L- Tryp that allows identifying and / or monitoring said candidate compound as a modulator of the tryptophan-serotonin metabolism; preferably said candidate compound is administered within 5 hours before or after, or simultaneously, with respect to the administration of said stable isotope labeled L-Tryp;
  • each sample is obtained at a different time point with respect to the administration of said stable isotope labeled L-Tryp; wherein it is well understood that said sample(s) is / are obtained at (a) time point(s) that allow(s) identifying and / or monitoring said candidate compound as a modulator of the tryptophan-serotonin metabolism; and d) determining in said sample(s) the respective amount(s) of
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiments 1 ) or 2), wherein • the respective amount(s) of
  • sample(s) is / are determined by mass spectrometry in one or more sample(s) (preferably more than one sample);
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiments 1 ) or 2), wherein
  • sample(s) is / are determined by mass spectrometry in one or more sample(s) (preferably more than one sample);
  • sample(s) are / have been obtained in an interval of about 0.5 h to about 36 h, especially at time points at about 1 h, 2 h, 4 h, 6 h, and / or 24 h, after the administration of said stable isotope labeled L-Tryp / after said stable isotope labeled L-Tryp has been administered;
  • a further embodiment relates to a method according to any one of embodiments 1 ) to 4), wherein at least one dose of said candidate compound is / has been administered within 5 hours before, or at least at the same time as, said stable isotope labeled L-Tryp; preferably 0.5 h to 24 h (notably 0.5 to 12 h, especially 0.5 to 6 h) before the administration of said stable isotope labeled L-Tryp / before said stable isotope labeled L-Tryp has been administered .
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1 ) to 5), wherein said candidate compound is a compound suspected to inhibit the tryptophan-serotonin metabolism in vivo, wherein especially said candidate compound has been identified as an inhibitor of the tryptophan- serotonin metabolism using an in vitro assay.
  • a further embodiment relates to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1) to 6), wherein the rate limiting step of said tryptophan-serotonin metabolism is the hydroxylation of L-Tryp catalyzed by TPH; and wherein said candidate compound has been identified as an inhibitor of TPH using an in vitro assay.
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1 ) to 6), wherein the rate limiting step of said tryptophan-serotonin metabolism is the hydroxylation of L-Tryp catalyzed by TPH1 ; and wherein said candidate compound has been identified as an inhibitor of TPH1 using an in vitro assay.
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1) to 8), wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism is / are determined; wherein said stable isotope labeled metabolite of the tryptophan-serotonin metabolism is stable isotope labeled 5HT or stable isotope labeled 2-(5-hydroxy-1 /-/-indol-3-yl)acetic acid (5HIAA).
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1) to 8), wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism is / are determined; wherein said stable isotope labeled metabolite of the tryptophan-serotonin metabolism is stable isotope labeled 5HT.
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1) to 8), wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism is / are determined; wherein said stable isotope labeled metabolite of the tryptophan-serotonin metabolism is stable isotope labeled 5HIAA.
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1 ) to 11 ), wherein said stable isotope labeled L-Tryp contains at least one stable isotope label independently selected from deuterium, 13 C and 15 N.
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1 ) to 11 ), wherein said stable isotope labeled L-Tryp contains at least one stable isotope label independently selected from 13 C and 15 N (wherein such stable isotope labeled L-Tryp especially is hTryp, mwTryp, or 13 C i L- tryptophan).
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1 ) to 11 ), wherein said stable isotope labeled L-Tryp is
  • hTryp heavy L-tryptophan
  • mwTryp mid-weight L-tryptophan
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to embodiment 1 1 ), wherein said stable isotope labeled L-Tryp is heavy tryptophan (hTryp).
  • a further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to embodiment 1 1 ), wherein said stable isotope labeled L-Trypis mid- weight tryptophan (mwTryp). 17) A further embodiment relates to a method or to a stable isotope labeled L-Tryp for use in a method according to any one of embodiments 1) to 16), wherein the suitable amount of stable isotope labeled L-Tryp is / has been administered using oral (p.o.) administration.
  • a further embodiment relates to a method according to embodiment 17), wherein the suitable amount of stable isotope labeled L-Tryp is between 1 mg and 300 mg per kg, notably between 1 mg and 100 mg per kg; especially between 2 mg and 50 mg per kg.
  • a further embodiment relates to a method according to any one of embodiments 1) to 16), wherein the suitable amount of stable isotope labeled L-Tryp is / has been administered using i.v. administration.
  • a further embodiment relates to a method according to embodiment 19), wherein the suitable amount of stable isotope labeled L-Tryp is between 0.1 mg and 50 mg per kg, notably between 0.1 mg and 20 mg per kg; especially between 0.2 mg and 10 mg per kg.
  • a further embodiment relates to a method according to any one of embodiments 1) to
  • a further embodiment relates to a method according to embodiment 21 ), wherein the suitable amount of said candidate compound is between 0.1 mg and 500 mg per kg , notably between 1 mg and 300 mg per kg; especially between 10 mg and 100 mg per kg.
  • a further embodiment relates to a method according to any one of embodiments 1) to 22), wherein the subject is a laboratory animal, especially a rat.
  • a further embodiment relates to a method according to any one of embodiments 1) to
  • the sample is a tissue sample or, especially, a body fluid.
  • a further embodiment relates to a method according to any one of embodiments 1) to
  • the sample is a tissue sample selected from gut (including sections of the gut such as duodenum and colon) and lung; or, especially, a body fluid selected from blood and urine.
  • a further embodiment relates to a method according to any one of embodiments 1 ) to 24) wherein Tryp, metabolites of the tryptophan-serotonin metabolism, stable isotope labeled tryp and / or stable isotope labeled metabolites of the tryptophan-serotonin metabolism are / have been extracted from said sample(s) (especially from tissue and / or body fluid).
  • a further embodiment relates to a method according to any one of embodiments 1 ) to 26) wherein said stable isotope labeled metabolite(s) of the tryptophan-serotonin metabolism and / or said stable isotope labeled L-Tryp is / are chemically derivatized prior to determination of their respective amount(s) by mass spectrometry.
  • a further embodiment relates to a method according to any one of embodiments 1 ) to 26) wherein the respective amount(s) of said stable isotope labeled metabolite of the tryptophan- serotonin metabolism and / or said stable isotope labeled L-Tryp is / are determined without chemical modification of said stable isotope labeled metabolite (i.e. said stable isotope labeled metabolite(s) is / are not chemically derivatized prior to determination of its / their respective amount(s) by mass spectrometry).
  • a further embodiment relates to a method according to embodiments 1 ) to 28) wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism; and / or said stable isotope labeled L-Tryp is / are determined using LCMS (especially using reverse phase HPLC and electrospray ionization mass spectrometry).
  • a further embodiment relates to a method according to any one of embodiments 1 ) to 28) wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan- serotonin metabolism; and / or said stable isotope labeled L-Tryp is / are determined using GCMS or MALDI.
  • a second aspect of the present invention relates to a stable isotope labeled L-Tryp for use in a diagnostic method for determining the activity of the tryptophan-serotonin metabolism in vivo, the method comprising:
  • each sample is obtained at a different time point with respect to the administration of said stable isotope labeled L-Tryp;
  • the invention relates to a diagnostic method for determining the activity of the tryptophan-serotonin metabolism in vivo, the method comprising: a) administering to a subject a suitable amount of a stable isotope labeled L-Tryp;
  • each sample is obtained at a different time point with respect to the administration of said stable isotope labeled L-Tryp;
  • the invention relates to a diagnostic method, or to a stable isotope labeled L-Tryp for use in a diagnostic method, for determining the activity of the tryptophan-serotonin metabolism in vivo, the method comprising:
  • each sample has been obtained at a different time point with respect to the administration of said stable isotope labeled L-Tryp;
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiments 31 ), 32), or 32), wherein the subject is suspected to have a disease or disorder characterized by an altered activity of the tryptophan-serotonin metabolism; wherein an altered activity (especially an elevated activity) as determined in the respective comparison step according to embodiments 26), 27), or 28) is indicative of such disease or disorder.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiment 34), wherein said disease or disorder characterized by an altered rate of the tryptophan-serotonin metabolism is a peripheral disease, wherein the rate limiting step of said tryptophan-serotonin metabolism is the hydroxylation of L-Tryp catalyzed by TPH1 .
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiment 34) or 35), wherein said disease or disorder characterized by an altered rate of the tryptophan-serotonin metabolism is selected from lung disease including: interstitial lung disease such as lung fibrosis, chronic obstructive pulmonary disease (COPD), pulmonary embolism, pulmonary hypertension, radiation pneumonitis (including that giving rise to or contributing to pulmonary hypertension), asthma, and adult respiratory distress syndrome (ARDS); osteoporosis; gastrointestinal disorders including inflammatory bowel disease, postinfectious irritable bowel syndrome, coeliac disease, idiopathic constipation , irritable bowel syndrome; ulcerative colitis; carcinoid syndrome; myxomatous valve disease; thrombosis; sleep disorders; pain; typel and type 2 diabetes; immune disorders; liver disease (including (viral-induced) hepatitis, fibrosis, transplantation,
  • breast cancer, prostate cancer, and neuroendocrine tumors with elevated serotonin secretion e.g carcinoid tumors); subarachnoid hemorrhage; abdominal migraine; CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia); Gilbert's syndrome; nausea; serotonin syndrome; functional anorectal disorders; functional bloating; and inflammatory diseases including e.g . multiple sclerosis, systemic sclerosis.
  • such disease or disorder characterized by an altered rate of the tryptophan-serotonin metabolism is selected from lung fibrosis; pulmonary hypertension; asthma; osteoporosis; ulcerative colitis; irritable bowel syndrome; carcinoid syndrome; cancer, including e.g breast cancer, prostate cancer, and neuroendocrine tumors with elevated serotonin secretion (e.g carcinoid tumors); and inflammatory diseases including e.g. multiple sclerosis, and systemic sclerosis.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31 ) to 36), wherein at least one sample is / has been obtained at least 15 minutes after the administration of said stable isotope labeled L-Tryp / after said stable isotope labeled L-Tryp has been administered (notably more than one sample is / has been obtained at different time points in an interval of about 0.5 h to about 36 h, especially at different time points at about 1 h, 2 h, 4 h, 6 h, and / or 24 h, after the administration of said stable isotope labeled L-Tryp / after said stable isotope labeled L- Tryp has been administered).
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31 ) to 36), wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism is / are determined; wherein said stable isotope labeled metabolite of the tryptophan-serotonin metabolism is stable isotope labeled serotonin 5HT or stable isotope labeled 5HIAA.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31 ) to 36), wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism is / are determined; wherein said stable isotope labeled metabolite of the tryptophan-serotonin metabolism is stable isotope labeled 5HT.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31 ) to 36), wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism is / are determined; wherein said stable isotope labeled metabolite of the tryptophan-serotonin metabolism is stable isotope labeled 5HIAA.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31 ) to 40), wherein said stable isotope labeled L-Tryp contains at least one stable isotope labels independently selected from deuterium, 13 C and 15 N.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31 ) to 40), wherein said stable isotope labeled L-Tryp contains at least one stable isotope labels independently selected from 13 C and 15 N (wherein such stable isotope labeled L-Tryp especially is hTryp, mwTryp, or 13 C i L- tryptophan).
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31 ) to 40), wherein said stable isotope labeled L-Tryp is
  • hTryp heavy L-tryptophan
  • mwTryp mid-weight L-tryptophan
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiment 43), wherein said stable isotope labeled L-Tryp is heavy tryptophan (hTryp).
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiment 43), wherein said stable isotope labeled L-Tryp is mid- weight tryptophan (mwTryp).
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31) to 45), wherein the suitable amount of stable isotope labeled L-Tryp is / has been administered using oral (p.o.) administration.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiment 46), wherein the suitable amount of stable isotope labeled L-Tryp is between 1 mg and 300 mg per kg , notably between 1 mg and 100 mg per kg; especially between 2 mg and 50 mg per kg .
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31) to 45), wherein the suitable amount of stable isotope labeled L-Tryp is / has been administered using i.v. administration.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiment 48), wherein the suitable amount of stable isotope labeled L-Tryp is between 0.1 mg and 50 mg per kg , notably between 0.1 mg and 20 mg per kg; especially between 0.2 mg and 10 mg per kg. 50)
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31 ) to 49), wherein the subject is a human.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31) to 50), wherein the sample is a tissue sample or, especially, a body fluid.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 31) to 50), wherein the sample is is a tissue sample selected from gut (including sections of the gut such as duodenum and colon) and lung; or, especially, a body fluid selected from blood and urine.
  • a further embodiment relates to a method according to any one of embodiments 31 ) to
  • tryptophan, metabolites of the tryptophan-serotonin metabolism, stable isotope labeled tryp and / or stable isotope labeled metabolites of the tryptophan-serotonin metabolism are / have been extracted from said sample(s) (especially from tissue and / or body fluid).
  • a further embodiment relates to a method according to any one of embodiments 31 ) to
  • a further embodiment relates to a method according to any one of embodiments 31 ) to 53) wherein the respective amount(s) of said stable isotope labeled metabolite of the tryptophan-serotonin metabolism and / or said stable isotope labeled L-Tryp is / are determined without chemical modification of said stable isotope labeled metabolite (i.e. said stable isotope labeled metabolite(s) is / are not chemically derivatized prior to determination of its / their respective amount(s) by mass spectrometry).
  • a further embodiment relates to a method according to embodiments 31 ) to 55) wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism; and / or said stable isotope labeled L-Tryp is / are determined using LCMS (especially using reverse phase HPLC and electrospray ionization mass spectrometry).
  • a further embodiment relates to a method according to any one of embodiments 31 ) to 55) wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism; and / or said stable isotope labeled L-Tryp is / are determined using GCMS or MALDI.
  • a third aspect of the present invention relates to a stable isotope labeled L-Tryp for use in a diagnostic method for determining the response of the disease state of a subject being diagnosed as having a disease or disorder characterized by an altered activity of the tryptophan-serotonin metabolism, to a therapeutic intervention (such as especially a drug treatment); comprising the steps of:
  • At least one sample is obtained at least 15 minutes after the administration of said stable isotope labeled L-Tryp (notably sample(s) are obtained in an interval of about 0.5 h to about 36 hours, especially at about 1 h, 2 h, 4 h, 6 h, and / or 24 hours, after the administration of said stable isotope labeled
  • each sample is obtained at a different time point with respect to the administration of said stable isotope labeled L-Tryp;
  • the invention relates to a diagnostic method for determining the response of the disease state of a subject being diagnosed as having a disease or disorder characterized by an altered activity of the tryptophan-serotonin metabolism, to a therapeutic intervention (such as especially a drug treatment); comprising the steps of:
  • At least one sample is obtained at least 15 minutes after the administration of said stable isotope labeled L-Tryp (notably sample(s) are obtained in an interval of about 0.5 h to about 36 hours, especially at about 1 h, 2 h, 4 h, 6 h, and / or 24 hours, after the administration of said stable isotope labeled L-Tryp);
  • each sample is obtained at a different time point with respect to the administration of said stable isotope labeled L-Tryp;
  • the invention relates to a diagnostic method, or to a stable isotope labeled L-Tryp for use in a diagnostic method, for determining the response of the disease state of a subject being diagnosed as having a disease or disorder characterized by an altered activity of the tryptophan-serotonin metabolism, to a therapeutic intervention (such as especially a drug treatment); comprising the steps of:
  • said stable isotope labeled L-Tryp has been administered at a time point before, after, or simultaneously with respect to the initiation of said therapeutic intervention (especially said drug treatment) that allows for determining the response of said disease state to said therapeutic intervention; preferably said stable isotope labeled L-Tryp has been administered within 5 hours before or after, or simultaneously, with respect to the initiation of said therapeutic intervention;
  • At least one sample has been obtained at least 1 5 minutes after the administration of said stable isotope labeled L-Tryp (notably sample(s) have been obtained in an interval of about 0.5 h to about 36 h, especially at time points at about 1 h, 2 h, 4 h, 6 h, and / or 24 hours, after the administration of said stable isotope labeled L-Tryp);
  • each sample has been obtained at a different time point with respect to the administration of said stable isotope labeled L-Tryp;
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to embodiments 58), 59), or 60), wherein
  • the sample(s) for said control(s) are / have been obtained from one or more subjects being diagnosed as not having a disease or disorder characterized by an altered rate of the tryptophan-serotonin metabolism.
  • a further embodiment relates to a diagnostic method, or to a stable isotope labeled L- Tryp for use in a diagnostic method, according to any one of embodiments 58) to 61 ), wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan- serotonin metabolism is / are determined; wherein said stable isotope labeled metabolite of the tryptophan-serotonin metabolism is stable isotope labeled serotonin (5HT).
  • a further embodiment relates to a diagnostic method, or to a stable isotope labeled L- Tryp for use in a diagnostic method, according to any one of embodiments 58) to 62), wherein said stable isotope labeled L-Tryp contains at least one stable isotope labels independently selected from deuterium, 13 C and 15 N.
  • a further embodiment relates to a diagnostic method, or to a stable isotope labeled L- Tryp for use in a diagnostic method, according to any one of embodiments 58) to 62), wherein said stable isotope labeled L-Tryp contains at least one stable isotope labels independently selected from 13 C and 15 N.
  • a further embodiment relates to a diagnostic method, or to a stable isotope labeled L- Tryp for use in a diagnostic method, according to any one of embodiments 58) to 62), wherein said stable isotope labeled L-Tryp is heavy tryptophan (hTryp).
  • a further embodiment relates to a diagnostic method, or to a stable isotope labeled L- Tryp for use in a diagnostic method, according to any one of embodiments 58) to 65), wherein the suitable amount of stable isotope labeled L-Tryp is / has been administered using oral (p.o.) administration.
  • a further embodiment relates to a diagnostic method, or to a stable isotope labeled L- Tryp for use in a diagnostic method, according to embodiment 66), wherein the suitable amount of stable isotope labeled L-Tryp is between 1 mg and 300 mg per kg, notably between 1 mg and 1 00 mg per kg; especially between 2 mg and 50 mg per kg.
  • a further embodiment relates to a diagnostic method, or to a stable isotope labeled L- Tryp for use in a diagnostic method, according to any one of embodiments 58) to 65), wherein the suitable amount of stable isotope labeled L-Tryp is / has been administered using intravenous (i.v.) administration .
  • a further embodiment relates to a diagnostic method, or to a stable isotope labeled L- Tryp for use in a diagnostic method, according to embodiment 68), wherein the suitable amount of stable isotope labeled L-Tryp is between 0.1 mg and 50 mg per kg, notably between 0.1 mg and 20 mg per kg; especially between 0.2 mg and 10 mg per kg.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 58) to 69), wherein the subject is a human.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 58) to 70), wherein the sample is a tissue sample or, especially, a body fluid.
  • a further embodiment relates to a method, or to a stable isotope labeled L-Tryp for use in a method, according to any one of embodiments 58) to 70), wherein the sample is is a tissue sample selected from gut (including sections of the gut such as duodenum and colon) and lung; or, especially, a body fluid selected from blood and urine.
  • a further embodiment relates to a stable isotope labeled L-Tryp for use in a method according to embodiment 72), wherein, in case said sample is a tissue sample; such sample is / has been obtained at a single time point with respect ot the respective administration of the stable isotope labeled L-Tryp.
  • a further embodiment relates to a stable isotope labeled L-Tryp for use in a method according to embodiment 72), wherein, in case said sample is a body fluid; more than one sample is obtained, at different time points with respect to the administration of the stable isotope labeled L-Tryp.
  • a further embodiment relates to a stable isotope labeled L-Tryp for use in a method according to embodiment 74), wherein said time point(s) is / are between 0.5 h to about 36 h
  • a further embodiment relates to a method according to any one of embodiments 58) to 75), wherein tryptophan, metabolites of the tryptophan-serotonin metabolism, stable isotope labeled tryptophan and / or stable isotope labeled metabolites of the tryptophan-serotonin metabolism are / have been extracted from said sample(s) (especially from tissue and / or body fluid).
  • a further embodiment relates to a method according to any one of embodiments 58) to 76), wherein said stable isotope labeled metabolite(s) of the tryptophan-serotonin metabolism and / or said stable isotope labeled L-Tryp is / are chemically derivatized prior to determination of their respective amount(s) by mass spectrometry.
  • a further embodiment relates to a method according to any one of embodiments 58) to 76), wherein the respective amount(s) of said stable isotope labeled metabolite of the tryptophan-serotonin metabolism and / or said stable isotope labeled L-Tryp is / are determined without chemical modification of said stable isotope labeled metabolite (i.e. said stable isotope labeled metabolite(s) is / are not chemically derivatized prior to determination of its / their respective amount(s) by mass spectrometry).
  • a further embodiment relates to a method according to embodiments 58) to 76), wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism; and / or said stable isotope labeled L-Tryp is / are determined using LCMS (especially using reverse phase HPLC and electrospray ionization mass spectrometry).
  • a further embodiment relates to a method according to any one of embodiments 58) to 76), wherein the respective amount(s) of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism; and / or said stable isotope labeled L-Tryp is / are determined using GCMS or MALDI.
  • a further embodiment relates to a method according to any one of embodiments 58) to 80), wherein said disease or disorder characterized by an altered rate of the tryptophan- serotonin metabolism is a peripheral disease, wherein the rate limiting step of said tryptophan-serotonin metabolism is the hydroxylation of L-Tryp catalyzed by TPH1 .
  • a further embodiment relates to a method according to any one of embodiments 58) to 81), wherein said disease or disorder characterized by an altered rate of the tryptophan- serotonin metabolism is selected from lung disease including: interstitial lung disease such as lung fibrosis, chronic obstructive pulmonary disease (COPD), pulmonary embolism, pulmonary hypertension, radiation pneumonitis (including that giving rise to or contributing to pulmonary hypertension), asthma, and adult respiratory distress syndrome (ARDS); osteoporosis; gastrointestinal disorders including inflammatory bowel disease, postinfectious irritable bowel syndrome, coeliac disease, idiopathic constipation, irritable bowel syndrome; ulcerative colitis; carcinoid syndrome; myxomatous valve disease; thrombosis; sleep disorders; pain; typel and type 2 diabetes; immune disorders; liver disease (including (viral- induced) hepatitis, fibrosis, transplantation, regeneration); acute and chronic hypertension; cancer inlcuding e.g.
  • breast cancer, prostate cancer, and neuroendocrine tumors with elevated serotonin secretion e.g carcinoid tumors); subarachnoid hemorrhage; abdominal migraine; CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia); Gilbert's syndrome; nausea; serotonin syndrome; functional anorectal disorders; functional bloating; and inflammatory diseases including e.g. multiple sclerosis, systemic sclerosis.
  • such disease or disorder characterized by an altered rate of the tryptophan-serotonin metabolism is selected from lung fibrosis; pulmonary hypertension ; asthma; osteoporosis; ulcerative colitis; irritable bowel syndrome; carcinoid syndrome; cancer, including e.g breast cancer, prostate cancer, and neuroendocrine tumors with elevated serotonin secretion (e.g carcinoid tumors); and inflammatory diseases including e.g. multiple sclerosis, and systemic sclerosis.
  • a fourth aspect of the present invention relates to a kit
  • kits comprising a stable isotope labeled L-Tryp, or a pharmaceutical composition comprising stable isotope labeled L-Tryp, and packaging materials therefore.
  • the kit further comprises instructions to use the stable isotope labeled L-Tryp for the methods according to embodiments 1 ) to 30); 31) to 57); or 58) to 82).
  • Definitions provided herein are intended to apply uniformly to any one of embodiments 1 ) to 83), and, mutatis mutandis, throughout the description and the claims unless an otherwise expressly set out definition provides a broader or narrower definition. It is well understood that a definition or preferred definition of a term defines and may replace the respective term independently of (and in combination with) any definition or preferred definition of any or all other terms as defined herein.
  • tryptophan, L-Tryp, and Tryp all refer to the natural non-dietary amino acid (2S)-2- amino-3-(1 /-/-indol-3-yl)propanoic acid.
  • serotonin, 5HT, and 5-hydroxytryptamine all refer to the compound 3-(2-aminoethyl)-1 /-/-indol-5-ol.
  • 5HIAA refers to the compound 2-(5-hydroxy-1 /-/-indol-3-yl)acetic acid.
  • any reference to compound such as a stable isotope labeled L-tryptophan, a stable isotope labeled metabolite of the tryptophan-serotonin metabolism, or an inhibitor of tryptophan- serotonin metabolism is to be understood as referring also to the pharmaceutically acceptable salts of such compound , as appropriate and expedient.
  • salts refers to salts that retain the desired biological activity of the subject compound and exhibit minimal undesired toxicological effects. Such salts include inorganic or organic acid and / or base addition salts depending on the presence of basic and / or acidic groups in the subject compound. For reference see for example "Handbook of Phramaceutical Salts. Properties, Selection and Use.”, P. Heinrich Stahl, Camille G. Wermuth (Eds.), Wiley-VCH, 2008; and “Pharmaceutical Salts and Co- crystals", Johan Wouters and Luc Quere (Eds.), RSC Publishing, 2012.
  • TPH refers to the enzyme non-heme pterin dependent oxygenase tryptophan hydroxylase encompassing the two isoforms TPH1 and TPH2.
  • the term preferably refers to the main peripheral enzyme tryptophan hydroxylase TPH1 .
  • TPH enzyme activity refers to the rate of TPH catalyzed metabolism of tryptophan to 5-hydroxy-tryptophan.
  • TPH activity may be monitored by measuring a detectable step in the metabolism cascade; e.g. by assaying 5HT or downstream metabolites such as melatonin or especially 5HIAA as shown in scheme 1 above.
  • 5-hydroxytryptophan may be measured as a surrogate for 5HT production, e.g. by including an aromatic aminoacid decarboxylase inhibitor to block the further action of TPH.
  • TPH activity preferably refers to the peripheral activity of TPH1 .
  • metabolic refers to any combination of the production, breakdown, modification, or clearance rate of a biomolecule (especially of Tryp, 5HT, 5HIAA) in a subject.
  • TPH inhibitory activity refers to specific binding of a compound, especially a small organic molecule, to the active site of the TPH polypeptide, thereby inhibiting the TPH activity.
  • the term preferably refers to inhibitory activity on peripheral TPH1 .
  • TPH inhibitory activity may be tested in standard in vitro assays well known in the art.
  • binding refers to the physical association of a modulator (e.g. a small molecule) with an enzyme (e.g. TPH1 ).
  • binding is "specific” if it occurs with an IC 50 or a K d of 500 nM or less.
  • IC 50 refers to that concentration of an agent at which a given activity of a TPH polypeptide is reduced to 50% of the maximum of TPH activity measurable using the same assay. Stated differently, the “ IC 50 " is the concentration of an agent that gives 50% inhibition, when 0% inhibition is set at the amount of activity that is observed in absence of an inhibitor using the same assay.
  • K d refers to the dissociation constant.
  • the dissociation constant has molar units (M), which correspond to the concentration of ligand at which the binding site on a particular protein is half occupied, i.e. the concentration of ligand, at which the concentration of protein with ligand bound, equals the concentration of protein with no ligand bound.
  • candidate compound and “candidate modulator” refer to a compound or a composition being evaluated for the ability to modulate TPH activity in vivo.
  • Candidate compounds can be natural or synthetic compounds, including, for example, small molecules, compounds contained in extracts of animal, plant, bacterial or fungal cells, as well as conditioned medium from such cells.
  • candidate compounds are small organic molecules.
  • a particular candidate compound which is a "compound suspected to inhibit the tryptophan-serotonin metabolism in vivo” preferably has been tested before its use in the present methods to have TPH (especially TPH1) inhibitory activity in an in vitro assay. Such in vitro assays are well known in the art (see for example WO2008/073933).
  • small molecule refers to a compound having molecular mass of less than 1500, preferably less than 1000, and more preferably less than 600 Daltons.
  • a "small molecule” preferably refers to a "small organic molecule” , i.e. a small molecule that comprises at least one carbon atom.
  • isotope refers to variants of a particular chemical element wherein all isotopes of a given element share the same number of protons and electrons, but each isotope differs from the others in its number of neutrons, i.e. each isotope refers to a different nuclide.
  • isotope labeled refers to a compound comprising at least one isotope which is not the most abundant naturally occurring nuclide for a given element, wherein said compound comprises such isotope(s) in an unnatural (enriched) amount.
  • the nuclide 12 C is the stable predominant (or most abundant naturally occurring) isotope of the element carbon
  • the nuclide 13 C is a stable (or naturally occurring) non-predominant isotope of the element carbon
  • 11 C and 14 C are examples of non-stable, radioactive isotopes of carbon.
  • the nuclide 13 C may be used as a stable isotope label, whereas the nuclides 11 C and 14 C may be used as non-stable radioactive isotope lables of carbon.
  • an example of a non- predominant stable isotope of the element nitrogen is the nuclide 15 N
  • an example of a non- predominant stable isotope of the element oxygen is the nuclide 18 0.
  • An example an example of a non-predominant stable isotope of the element hydrogen is deuterium.
  • preferred stable isotope labels are 13 C, 15 N, 18 0 and deuterium, especially preferred are deuterium, 13 C and 15 N; most preferred are 13 C and 15 N.
  • stable isotope labeled refers to such compounds comprising one or more, preferably two or more, stable isotopes in an unnatural abundance, wherein said stable isotopes are independently selected from 13 C, 15 N, 18 0 and deuterium (notably from 13 C, 15 N and deuterium; especially from 13 C and 15 N).
  • stable isotope labeling of L-Tryp will determine the isotope labeling of a metabolite of the tryptophan-serotonin metabolism.
  • stable isotope labeled L-tryptophan thus refers to L-Tryp containing at least one stable isotope which is not the most abundant naturally occurring nuclide for a given element, wherein said isotope is present in an unnatural abundance.
  • the term refers to L-Tryp containing at least one stable isotope label independently selected from deuterium, 13 C and 15 N (especially 13 C and 15 N) wherein said isotopes are present in an unnatural abundance.
  • the term refers to the compounds:
  • hTryp heavy L-tryptophan
  • mwTryp mid-weight L-tryptophan
  • the term refers to hTryp or mwTryp.
  • label incorporation or “amount of stable isotope labeled compound” refers to the amount of stable isotope labeled L-Tryp, or the amount of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism, determined at a certain timepoint (the time of obtaining the sample) with respect to the time of administration of stable isotope labeled L- Tryp. Such incorporation or amount is generally expressed or visualized either as percent isotope ratio (i.e. weight percent of stable isotope labeled compound with respect to total compound) or as amount of isotope labeled compound.
  • the term "activity of the tryptophan-serotonin metabolism” refers to the amount of a stable isotope labeled L-Tryp, or of a stable isotope labeled metabolite of the tryptophan- serotonin metabolism in vivo determined at a certain time point compared to a control, wherein said comparison is indicative of a change in activity, thus allowing a conclusion of the activity of the tryptophan-serotonin metabolism in vivo under the given conditions.
  • Such activity is generally expressed or visualized either as percent isotope ratio or amount of isotope labeled compound at one or more timepoints.
  • Such activity may also be expressed as a rate, i.e.
  • tryptophan-serotonin metabolism in vivo refers to the metabolism of L-Tryp in a subject comprising as first and rate-limiting step the TPH catalyzed hydroxylation of L- tryptophan to 5-hydroxy-L-tryptophan and leading further to the formation of 5HT and, subsequently, downstream metabolites such as melatonin and especially 5HIAA (see scheme 1 above).
  • the term preferably refers to the peripheral tryptophan-serotonin metabolism wherein the hydroxylation of L-tryptophan to 5- hydroxy-L-tryptophan is catalyzed by TPH1 .
  • stable isotope labeled L-Tryp is used to monitor the tryptophan-serotonin metabolism in vivo, it is well understood that the structu ral choice of said stable isotope labeled L-Tryp will determine the structure of the resulting "stable isotope labeled metabolite" that is formed through the tryptophan-seroton in metabolism.
  • the tryptophan-serotonin metabolism of heavy L-tryptophan (hTryp) will lead to heavy 5HT (h5HT) and , subsequently, for exam le to heavy 5HIAA (h5HIAA).
  • mid-weight L-tryptophan mwTryp
  • mid-weight 5HT mw5HT
  • mid-weight 5HIAA mw5HIAA
  • mass spectrometry refers to the process of ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. Molecules can be ionized using a number of different sources, including electrospray, MALDI , APCI . Molecules may be chemically derivatized prior to mass spectrometry. Mass spectrometry may also involve fragmentation of the generated ions and measu ring of the m/z of the resultant fragment ion , also known as tandem mass spectrometry. Mass spectrometry is generally associated with a separation method such as liquid chromatography (LCMS) or gas chromatography (GCMS).
  • LCMS liquid chromatography
  • GCMS gas chromatography
  • Such separation methods associated with mass spectrometry are intended to be encompassed in the term.
  • Preferred examples of mass spectrometry are LCMS especially with electrospray ionization and selected reaction monitoring detection .
  • the term "subject" refers to any vertebrate living organism. Especially, the subject is a mammal such as rodents including mice, rats, hamsters and guinea pigs; cats; dogs; rabbits; primates including marmosets; and humans. For avoidance of any doubt the term also includes transgenic animals.
  • the term preferably refers to a laboratory animal such as especially rodents.
  • the term preferably refers to humans.
  • transgenic animal refers to any non-human animal, preferably a non-human mammal, bird, fish or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation , such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • transgenic animal also includes those recombinant animals in which gene disruption of one or more genes is caused by human intervention, including both recombination and antisense techniques.
  • in vivo refers to "in a subject” wherein subject is defined as before.
  • sample refers to a body fluid or tissue obtained from a subject.
  • body fluid refers for example to cerebral spinal fluid (CSF); blood including whole blood, blood plasma, platelet poor plasma, platelet rich plasma, and blood serum (especially whole blood, and plasma); urine; saliva; perspiration; bronchoalveolar lavage (BAL) fluid; and tears.
  • CSF cerebral spinal fluid
  • BAL bronchoalveolar lavage
  • tissue refers to an aggregate of cells that perform a particular function in an organism. The term thus refers to cellular material from a particular physiological region.
  • tissue is also intended to encompass non-solid tissues, such as blood, comprised in such solid tissues.
  • Tissue samples may be collected by various invasive procedures, e.g., surgical excision, percutaneous biopsy, post-mortem analysis, or other sampling procedures.
  • Example are tissues collected from the periphery such as gut (including gut sections such as duodenum and colon), lung, thymus, and spleen; as well as brain tissue that further comprises neurons and glial cells; as well as capillary endothelial cells and blood cells contained in a given tissue section or sample.
  • control refers to a standard or reference value or activity obtained before or in absence of any intervention, wherein such control value or activity is of the same kind as the value or rate referred to.
  • the term generally refers to a (baseline) value or activity obtained from a sample obtained from one or more subjects not affected by a disease or disorder characterized by dysregulation of the tryptophan-serotonin metabolism, or from one or more subjects who are not characterized by a dysregulation of the tryptophan-serotonin metabolism in any other manner, whether caused by a modulator of said metabolism, or caused by a therapeutic intervention possibly altering said metabolism.
  • the term may also refer to a (positive) reference value or activity obtained from a sample obtained from one or more subjects who are affected by a disease or disorder characterized by dysregulation of the tryptophan-serotonin metabolism, wherein in such case the control is compared to a change caused e.g . by a therapeutic intervention possibly altering said metabolism.
  • a control may in certain circumstances be determined in the same subject before administration of a candidate compound, or before start of a therapeutic intervention.
  • a control may be used as a reference for the comparison of tryptophan-serotonin metabolism levels, or of amounts of a certain metabolite of the tryptophan-serotonin metabolism; as well as for the comparison of tryptophan-serotonin metabolism activity, or of the activity of production / the production rate of a certain metabolite of the tryptophan-serotonin metabolism.
  • the conditions used to measure a control e.g. the time point of sampling, the methods for analyzing the sample etc.
  • the conditions used to measure a control are as close as possible to (and preferably the same as) the conditions used to measure the value it is intended to compared to.
  • production rate refers to the rate at which the biomolecule of interest is produced.
  • elevated level or “elevated rate” means that a given level or rate is increased by 10% or more in a sample relative to a control.
  • the term “decrease in activity” refers to an at least 1 0% decrease in the tryptophan-serotonin metabolism rate, especially in TPH enzymatic activity relative to a control.
  • the term “disease or disorder characterized by an altered rate of the tryptophan-serotonin metabolism” refers to a neurological or peripheral disease or disorder characterized by an altered rate of the tryptophan-serotonin metabolism, wherein the rate limiting step of said tryptophan-serotonin metabolism is the hydroxylation of L-Tryp catalyzed by TPH.
  • the rate limiting step of said tryptophan-serotonin metabolism is the hydroxylation of L-Tryp catalyzed by TPH1 .
  • peripheral diseases or disorders are selected from lung disease including: interstitial lung disease such as lung fibrosis, chronic obstructive pulmonary disease (COPD), pulmonary embolism, pulmonary hypertension , radiation pneumonitis (including that giving rise to or contributing to pulmonary hypertension), asthma, and adult respiratory distress syndrome (ARDS); osteoporosis; gastrointestinal disorders including inflammatory bowel disease, postinfectious irritable bowel syndrome, coeliac disease, idiopathic constipation, and irritable bowel syndrome; ulcerative colitis; carcinoid syndrome; myxomatous valve disease; thrombosis; sleep disorders; pain; typel and type 2 diabetes; immune disorders; liver disease (including (viral-induced) hepatitis fibrosis, transplantation, regeneration); acute and chronic hypertension ; cancer inlcuding
  • COPD chronic o
  • breast cancer, prostate cancer, and neuroendocrine tumors with elevated serotonin secretion e.g carcinoid tumors); subarachnoid hemorrhage; abdominal migraine; CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia); Gilbert's syndrome; nausea; serotonin syndrome; functional anorectal disorders; functional bloating; and inflammatory diseases including e.g. multiple sclerosis, systemic sclerosis.
  • such disease or disorder characterized by an altered rate of the tryptophan-serotonin metabolism is selected from lung fibrosis; pulmonary hypertension ; asthma; osteoporosis; ulcerative colitis; irritable bowel syndrome; carcinoid syndrome; cancer, including e.g breast cancer, prostate cancer, and neuroendocrine tumors with elevated serotonin secretion (e.g carcinoid tumors); and inflammatory diseases including e.g. multiple sclerosis, and systemic sclerosis.
  • the term "effective amount" refers to that amount of a compound necessary to achieve the desired effect in vivo.
  • the effective amount of a stable isotope labeled L-Tryp is at least the minimal amount necessary to monitor the production of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism in vivo.
  • the effective amount of a TPH modulating agent is at least the minimal amount that results in a change in TPH enzyme activity as defined herein.
  • the effective amount of a drug is at least the minimal amount that results in a pharmacological effect.
  • compounds such as candidate compounds or L-Tryp may be administered as such, or in the form of pharmaceutical compositions, using enteral (such as especially oral) or parenteral administration [including bolus administration encompassing intravenous (i.v), intramuscular, intrathecal, and subcutaneous injection; topical application; and inhalation).
  • enteral such as especially oral
  • parenteral administration including bolus administration encompassing intravenous (i.v), intramuscular, intrathecal, and subcutaneous injection; topical application; and inhalation.
  • a stable isotope labeled L-Tryp when administered using oral (p.o.) administration is generally administered at a dose between 1 mg and 300 mg per kg, notably between 1 mg and 100 mg per kg; especially between 2 mg and 50 mg per kg.
  • administration is generally administered at a dose between 0.1 mg and 50 mg per kg, notably between 0.1 mg and 20 mg per kg; especially between 0.2 mg and 10 mg per kg.
  • a candidate compound when administered using oral (p.o.) administration is generally administered at a dose between 0.1 mg and 500 mg per kg, notably between 1 mg and 300 mg per kg; especially between 10 mg and 100 mg per kg.
  • a drug used in a therapeutic intervention when administered using oral (p.o.) administration is generally administered at a dose between 0.1 mg and 500 mg per kg, notably between 1 mg and 300 mg per kg; especially between 10 mg and 100 mg per kg.
  • an amount of a stable isotope labeled compound e.g. of a stable isotope labeled metabolite of the tryptophan-serotonin metabolism
  • determination of an amount is made in vitro, using the mass spectrometry techniques described herein.
  • the term "about” placed before a numerical value "X” refers in the current application to an interval extending from X minus 10% of X to X plus 10% of X, and preferably to an interval extending from X minus 5% of X to X plus 5% of X.
  • the term “about” (or alternatively the term “around") placed before a temperature ⁇ " refers in the current application to an interval extending from the temperature Y minus 10 °C to Y plus 10 °C, and preferably to an interval extending from Y minus 5 °C to Y plus 5 °C.
  • Rats may be maintained on a normal diet, or a aminoacid diet can be utilized to more strictly control Tryp intake.
  • Downstream analysis of Tryp metabolism can be performed in total blood and in platelet poor plasma (PPP) fraction.
  • Blood can be collected with the strong anti- coagulant EDTA and the milder anti-coagulant lithium/heparin, respectively.
  • the PPP usually is prepared with a 2 centrifugation steps protocol from lithium/heparin blood, as the milder anti-coagulant reduces the risk of platelet activation .
  • Blood sampling can be done under isofluran-induced slight narcosis (2.5-3%), allowing longitudinal blood sampling of animals, via sublingual venous puncture.
  • rats may be perfused by retrograde perfusion (for about 5 min) via abdominal aorta with a pre-warmed phosphate buffer solution (PBS 1 x + EDTA 333 mM).
  • Stable isotope labeled Tryp can be administered by oral gavage (5 ml_ per kg), e.g. from a solution of 0.5% (v/v) methyl-cellulose, 0.5% (v/v) Tween-80.
  • the potential interference of the 1 % natural abundance of 13 C with the signal coming from a tracer needs to be considered.
  • the M+1 and M+2 have natural abundances of 1 1 % and 1 .1 % respectively resulting in a significant background signal if the labeled tryptophan administered contains one or two labels.
  • This abundance combined with the comparatively slow turnover over of 5HT means that stable isotope labeled tryptophans with three mass labels or greater results in analytes that are more readily distinguished from the background signal.
  • the label incorporation can be determined by correcting for natural abundance of the M+1 or M+2 ion respectively (Hellerstein, M. K., and Neese, R. A. (1999) "Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations", Am J Physiol 276, E1 146- 1 170).
  • L-tryptophans are 13 C / 15 N 2 -L- tryptophan (heavy L-Tryp / hTryp) 15 N 2 -L-tryptophan (mid-weight L-Tryp / mwTryp) and 13 C - L-tryptophan as shown in Figure 2.
  • Organ samples are preferably homogenized under acidic conditions (for example using acetic acid) to avoid further biotransformation and thus preanalytical variation.
  • Samples may then be extracted, for example using solvent mediated (especially AcCN mediated) protein precipitation, followed by drying under a stream of heated N 2 , reconstitution in 5% AcCN (v/v) and LCMS analysis.
  • solvent mediated especially AcCN mediated
  • molecules may be chemically derivatized in one additional optional step using well known techniques (see for example Song, Y. et al. (2004).
  • suitable MRM transitions for quantitative LCMS analysis from the Collision Induced Dissociation (CID) fragmentation pattern can be determined from commercial or synthesized standards.
  • MRM transitions can be predicted from the fragmentation pattern of the naturally occurring form and confirmed experimentally using tissue and biofluid samples from animals which had received stable isotope labeled tryptophan.
  • the h5HT molecule can either be quantified using the MRM3 funtionality of the QTRAP®5500 or by standard MRM with insource fragmentation.
  • Transitions for all metabolites and their heavy equivalents are preferably combined into a single multiplexed LCMS method .
  • L-Tryp and 5HT may generally be quantified from the naturally occurring M+2 and M+1 ions, respectively, to avoid saturation of the detector resulting for example from the need to quantify analytes over a wide range of concentrations (5-6 orders of magnitude).
  • Samples from animals that had never been exposed to hTryp may be used to determine analytical selectivity of the method, control for interference in the LCMS chromatograms where the stable isotope labeled metabolites were produced and may be used as matrix for calibration curve for stable isotope labeled tryptophan and metabolites.
  • LCMS analysis is performed using a Dionex UltiMate® 3000 HPLC system with a QTRAP®5500 mass spectrometer (ABSciexTM).
  • a Luna® 3 ⁇ C18 (2) 100A, 1 00x2mm (Phenomenex®) column is used at a flow rate of 0.3mL/min with and a temperature of 35°C with buffer A: 5mM NH 4 formate + 0.01 % TFA and buffer B MeOH.
  • a gradient from 5 % buffer B to 55 % buffer B over 2 minutes is run followed by a second gradient from 55% to 80% buffer B over 2 minutes. Finally the column is cleaned and re-equilibrated.
  • the QTRAP®5500 is run in positive ion electrospray with the following settings curtain gas (20), CAD (medium), source temperature (550), gas 1 (50) gas 2 (50) ion source voltage (5500) EP (1 0) and CXP (10).
  • curtain gas (20) CAD (medium), source temperature (550), gas 1 (50) gas 2 (50) ion source voltage (5500) EP (1 0) and CXP (10).
  • MRM transitions Q1 /Q3
  • MRM transitions Q1 /Q3
  • quanititation 5HT 178/161
  • h5HT (171 /142.1 h5HT (171 /142.1
  • mw5HT 179.1/161 .1
  • Tryp 206/147
  • hTryp 21 8/156
  • mwTryp 5HIAA (192/146) and h5HIAA (203/156).
  • a second qualifier MRM transition can also be monitored .
  • the MRM and MRM3 chromatograms are processed using MultiQuantTM software (ABSciexTM). Calibration curves are generated either in surrogate matrix or in matrix for the stable isotope labeled analytes, fit to the simplest model possible and used to calculate concentrations. Concentration data can be reported as concentrations, peak area, peak height or isotope ratios, as is the norm for this kind of analysis (Magkos, F., and Mittendorfer, B. (2009) "Stable isotope-labeled tracers for the investigation of fatty acid and triglyceride metabolism in humans in vivo", Clin Lipidol 4, 215-230).
  • Example 1 Demonstration of h5HT and h5HIAA production by a rat
  • An EDTA-blood sample was taken from a Wistar rat. The rat was then administered with 30 mg per kg hTryp by oral gavage.
  • the MRM chromatograms demonstrate the production of h5HT and h5HIAA by the rat.
  • 5HT chromatograms of a blood sample from a rat before the administration of hTryp (1 A) a blood sample from 1 hour after the administration of hTryp (1 B) and commercial standard (1 C) are shown.
  • For h5HT chromatograms of a blood sample from a rat before the administration of hTryp (1 D) and a blood sample from 1 hour after the administration of hTryp (1 E) and a synthesized standard (1 F) are shown.
  • h5HIAA For h5HIAA a chromatogram of a blood sample from a rat before the administration of hTryp (1 1) and a blood sample from 1 hour after the administration of hTryp (1 J) are shown.
  • the MRM transition used for h5HIAA was that predicted based on the 5HIAA fragmentation pattern as no standard was available. Additionally the h5HIAA coeluted with the 5HIAA peak as would be expected.
  • Figure 1 shows the MRM (Q1 /Q3) chromatograms (intensity vs elution time (min)) generated by LCMS of 5HT (178/161) (Figure 1 A to Figure 1 C), h5HT (171/142.1 ) ( Figure 1 D to Figure 1 F) and h5HIAA (203/156) ( Figure 1 H and Figure 1 1).
  • 1 A, 1 D and 1 H are measured from a blood sample from a rat before hTryp administration.
  • 1 B, 1 E and 1 1 are measured from a blood sample from the same rat 1 hour after administration of 30 mg per kg hTryp.
  • 1 C and 1 F are the chromatograms of standards.
  • Example 2 Demonstration of mw5HT and mw5HIAA production by a rat
  • EDTA-blood sample was taken from a Wistar rat. The rat was then administered with 30 mg per kg mwTryp by oral gavage.
  • the MRM chromatograms demonstrate the production of mw5HT by the rat.
  • mw5HT chromatograms of a blood sample from a rat before the administration of mwTryp (2A) a blood sample from 2 h after the administration of mwTryp (2B) and a synthesised standard (2C), are shown.
  • the mw5HIAA contains only a single isotopic label, therefore there is considerable background signal from the naturally occurring M+1 ion of 5HIAA.
  • mw5HIAA can however still be determined by measuring the abundance of the [M+1 ] ion defined as (100*[M+1 ])/([M]+[M+1 )) in blood samples before and after the administration of mwTryp.
  • Figure 2 ( Figure 2A to Figure 2C) shows the MRM chromatograms (intensity vs elution time (min)) generated by LCMS of mw5HT monitoring the transition (Q1/Q3) 179.1/161 .1 .
  • Figure 2A is measured from a blood sample from a rat before mwTryp administration.
  • 2B is measured from a blood sample from the same rat 2 h after administration of 43 mg per kg mwTryp.
  • 2C is the chromatogram of a standard.
  • Example 3 time course of hTryp, h5HT and h5HIAA during continuous infusion
  • blood samples were taken and the concentrations of Tryp, hTryp, 5HT, h5HT, 5HIAA and h5HIAA were measured in the blood samples.
  • concentration of hTryp in the blood remained constant at around 1000 ng/mL or 10% hTryp.
  • the amount of h5HT increased linearly over the 12 hours at a rate of 5.8 nlWh of 0.26 % h5HT/h .
  • Example 5 Measurement of hTryp, h5HT and h5HIAA in different organs
  • Example 6 Measurement of half lifes of hTryp, h5HT and h5HIAA
  • a single oral dose of 43 mg per kg hTryp was administered to 2 Wistar rats by gavage.
  • EDTA-blood was sampled from the rats over a period of 10 days and the concentrations of Tryp, hTryp, 5HT, and h5HT were measured in the blood samples by LCMS. Fitting the determined amounts from specific time periods with suitable curves allowed calculation of half-life values of Tryp, and 5HT.
  • Example 7 Measurement of a change in 5HT synthesis caused by carcinoid syndrome
  • An oral dose of hTryp given to a healthy subject results in a certain h5HT level in the blood at a time point 8 hours after administration.
  • the same oral dose of hTryp is given to a subject having a carcinoid syndrome.
  • the blood h5HT level is detected at the same 8 hour time point.
  • the latter h5HT level is compared to the former.
  • Increased TPH activity indicates the presence of a carcinoid syndrome.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Optics & Photonics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Biophysics (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Cette invention concerne l'utilisation du L-tryptophane comprenant un ou plusieurs marqueurs isotopes stables pour surveiller in vivo le métabolisme tryptophane-sérotonine, notamment pour caractériser les composés modulant le métabolisme tryptophane-sérotonine, pour dépister les maladies associées à un métabolisme tryptophane-sérotonine altéré; et pour suivre le traitement thérapeutique des maladies associées à un métabolisme tryptophane-sérotonine altéré.
PCT/IB2014/061889 2013-06-03 2014-06-02 Nouvelle utilisation du l-tryptophane marqué par un isotope stable WO2014195847A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IBPCT/IB2013/054565 2013-06-03
IB2013054565 2013-06-03

Publications (2)

Publication Number Publication Date
WO2014195847A2 true WO2014195847A2 (fr) 2014-12-11
WO2014195847A3 WO2014195847A3 (fr) 2015-03-26

Family

ID=50977015

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2014/061889 WO2014195847A2 (fr) 2013-06-03 2014-06-02 Nouvelle utilisation du l-tryptophane marqué par un isotope stable

Country Status (1)

Country Link
WO (1) WO2014195847A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9199994B2 (en) 2013-09-06 2015-12-01 Karos Pharmaceuticals, Inc. Spirocyclic compounds as tryptophan hydroxylase inhibitors
WO2016141172A1 (fr) * 2015-03-03 2016-09-09 Laboratory Corporation Of America Holdings Procédés et systèmes de mesure de la sérotonine dans un échantillon
US9611201B2 (en) 2015-03-05 2017-04-04 Karos Pharmaceuticals, Inc. Processes for preparing (R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethanol and 1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethanone
US9765092B2 (en) 2013-11-19 2017-09-19 Actelion Pharmaceuticals Ltd Tricyclic piperidine compounds
US10189839B2 (en) 2013-11-19 2019-01-29 Actelion Pharmaceuticals Ltd Tricyclic imidazole compounds as inhibitors of tryptophan hydroxylase

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007089335A2 (fr) 2005-12-29 2007-08-09 Lexicon Pharmaceutical Inc. Derives d'acides amines multicycliques et procedes d'utilisation de ceux-ci
WO2008073933A2 (fr) 2006-12-12 2008-06-19 Lexicon Pharmaceuticals, Inc. Composés 4-phényle-6-(2,2,2-trifluoro-1-phényléthoxy) à base de pyrimidine et procédés concernant leur utilisation

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2930621B2 (ja) * 1989-11-10 1999-08-03 池田食研株式会社 標識化合物の製造方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007089335A2 (fr) 2005-12-29 2007-08-09 Lexicon Pharmaceutical Inc. Derives d'acides amines multicycliques et procedes d'utilisation de ceux-ci
WO2008073933A2 (fr) 2006-12-12 2008-06-19 Lexicon Pharmaceuticals, Inc. Composés 4-phényle-6-(2,2,2-trifluoro-1-phényléthoxy) à base de pyrimidine et procédés concernant leur utilisation

Non-Patent Citations (41)

* Cited by examiner, † Cited by third party
Title
"Handbook of Phramaceutical Salts. Properties, Selection and Use.", 2008, WILEY-VCH
"Pharmaceutical Salts and Co-crystals", 2012, RSC PUBLISHING
ALPINI, G. ET AL.: "Serotonin metabolism is dysregulated in cholangiocarcinoma, which has implications for tumor growth", CANCER RES, vol. 68, 2008, pages 9184 - 9193
BERTRAND, P. P.; BERTRAND, R. L.: "Serotonin release and uptake in the gastrointestinal tract", AUTON NEUROSCI, vol. 153, 2010, pages 47 - 57
BONI, R. L. ET AL.: "Quantification of L-tryptophan and L-kynurenine by liquid chromatography/electron capture negative ion chemical ionization mass spectrometry", BIOL MASS SPECTROM, vol. 23, 1994, pages 27 - 32
BROWN, P. M. ET AL.: "The tryptophan hydroxylase inhibitor LX1031 shows clinical benefit in patients with nonconstipating irritable bowel syndrome", GASTROENTEROLOGY, vol. 141, 2011, pages 507 - 516
CHANUT, E. ET AL.: "Metabolism of 6-fluoro-DL-tryptophan and its specific effects on the rat brain serotoninergic pathway", BIOCHEM PHARMACOL, vol. 45, 1993, pages 1049 - 1057
CIUCLAN, L. ET AL.: "Imatinib attenuates hypoxia-induced pulmonary arterial hypertension pathology via reduction in 5-hydroxytryptamine through inhibition of tryptophan hydroxylase 1 expression", AM J RESPIR CRIT CARE MED, vol. 187, no. 1, 2013, pages 78 - 89
DEES C ET AL.: "Platelet-derived serotonin links vascular disease and tissue fibrosis", J EXP MED., vol. 208, no. 5, 9 May 2011 (2011-05-09), pages 961 - 72
DIKSIC, M.: "Labelled alpha-methyl-L-tryptophan as a tracer for the study of the brain serotonergic system", J PSYCHIATRY NEUROSCI, vol. 26, 2001, pages 293 - 303
DIKSIC, M.; YOUNG, S. N.: "Study of the brain serotonergic system with labeled alpha-methyl-L-tryptophan", J NEUROCHEM, vol. 78, 2001, pages 1185 - 1200
DIKSIC, M.; YOUNG, S. N.: "Study of the brain serotonergic system with labeled alpha-methyl-L-tryptophan", NEUROCHEM, vol. 78, 2001, pages 1185 - 1200
DIKSIC, M: "Labelled alpha-methyl-L-tryptophan as a tracer for the study of the brain serotonergic system", J PSYCHIATRY NEUROSCI, vol. 26, 2001, pages 293 - 303
DURK, T. ET AL.: "Production of serotonin by tryptophan hydroxylase 1 and release via platelets contribute to allergic airway inflammation", AM J RESPIR CRIT CARE MED, vol. 187, no. 5, 2013, pages 476 - 485
ENGELMAN, K. ET AL.: "Inhibition of serotonin synthesis by para-chlorophenylalanine in patients with the carcinoid syndrome", N ENGL J MED, vol. 277, no. 21, 1967, pages 1103 - 1108
EVANS, J. ET AL.: "LC/MS analysis of NAD biosynthesis using stable isotope pyridine precursors", ANAL BIOCHEM, vol. 306, 2002, pages 197 - 203
GHIA, J. E. ET AL.: "Serotonin has a key role in pathogenesis of experimental colitis", GASTROENTEROLOGY, vol. 137, no. 5, 2009, pages 1649 - 1660
HASLER, W. L.: "Serotonin and the GI tract", CURR GASTROENTEROL REP, vol. 11, 2009, pages 383 - 391
HELLERSTEIN, M. K.; NEESE, R. A.: "Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations", AM J PHYSIOL, vol. 276, 1999, pages E1146 - 1170
HICKS RJ.: "Use of molecular targeted agents for the diagnosis, staging and therapy of neuroendocrine malignancy", CANCER IMAGING., vol. 10, no. A, 4 October 2010 (2010-10-04), pages S83 - 91
HYYPPA, M. T. ET AL.: "Rapid accumulation of H3-serotonin in brains of rats receiving intraperitoneal H3-tryptophan: effects of 5,6-dihydroxytryptamine or female sex hormones", J NEURAL TRANSM, vol. 34, pages 111 - 124
KONIGSHOFF, M. ET AL.: "Increased expression of 5-hydroxytryptamine2A/B receptors in idiopathic pulmonary fibrosis: a rationale for therapeutic intervention", THORAX, vol. 65, no. 11, 2010, pages 949 - 955
LACERDA, C. M. ET AL.: "Local serotonin mediates cyclic strain-induced phenotype transformation, matrix degradation, and glycosaminoglycan synthesis in cultured sheep mitral valves", AM J PHYSIOL HEART CIRC PHYSIOL, vol. 302, no. 10, 2012, pages H1983 - 1990
LEE, W. ET AL.: "Rapid corepressor exchange from the trp-repressor/operator complex: an NMR study of [ul-13C/15N]-L-tryptophan", J BIOMOL NMR, vol. 5, 1995, pages 367 - 375
LIN, R. C. ET AL.: "In vivo measurement of 5-hydroxytryptamine turnover rate in the rat brain from the conversion of C-tryptophan to 4 C-5-hydroxytryptamine", J PHARMACOL EXP THER, vol. 170, 1969, pages 232 - 238
LINDER, A. E. ET AL.: "Body distribution of infused serotonin in rats", CLIN EXP PHARMACOL PHYSIO, vol. 36, 2009, pages 599 - 601
LIU, Q. ET AL.: "Discovery and characterization of novel tryptophan hydroxylase inhibitors that selectively inhibit serotonin synthesis in the gastrointestinal tract", J PHARMACOL EXP THER, vol. 325, 2008, pages 47 - 55
MAGKOS, F.; MITTENDORFER, B.: "Stable isotope-labeled tracers for the investigation of fatty acid and triglyceride metabolism in humans in vivo", CLIN LIPIDOL, vol. 4, 2009, pages 215 - 230
MIWA, S ET AL.: "Determination of serotonin turnover in the rat brain using 6-fluorotryptophan", J NEUROCHEM, vol. 48, 1987, pages 1577 - 1580
MOSELEY, H. N. ET AL.: "Quantitative determination of conformational, dynamic, and kinetic parameters of a ligand-protein/DNA complex from a complete relaxation and conformational exchange matrix analysis of intermolecular transferred NOESY", BIOCHEMISTRY, vol. 36, 1997, pages 5293 - 5299
MULLER, A.; HILLEBRAND, H.; WEILER, E. W.: "Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana", PLANTA, vol. 206, 1998, pages 362 - 369
NOWAK EC ET AL.: "Tryptophan hydroxylase-1 regulates immune tolerance and inflammation", J EXP MED., vol. 209, no. 11, 12 October 2012 (2012-10-12), pages 2127 - 35
OSIM, E. E.; WYLLIE, J. H.: "oss of 5-hydroxytryptamine from mammalian circulating labelled platelets", J PHYSIOL, vol. 340, 1983, pages 77 - 90
PAI VP ET AL.: "Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival", BREAST CANCER RES., vol. 11, no. 6, 2009
SHINKA T ET AL.: "Serotonin synthesis and metabolism-related molecules in a human prostate cancer cell line", ONCOL LETT., vol. 2, no. 2, pages 211 - 215
SONG, Y. ET AL.: "Enhancing capillary liquid chromatography/tandem mass spectrometry of biogenic amines by pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole", RAPID COMMUN MASS SPECTROM, vol. 18, no. 9, 2004, pages 989 - 994
WALTHER, D. J. ET AL.: "Synthesis of serotonin by a second tryptophan hydroxylase isoform", SCIENCE, vol. 299, 2003, pages 76
WALTHER, D. J.; M. BADER: "A unique central tryptophan hydroxylase isoform", BIOCHEM PHARMACOL, vol. 66, no. 9, 2003, pages 1673 - 1680
WEBER, L. J.: "p-Chlorophenylalanine depletion of gastrointestinal 5-hyd roxytrypta mine", BIOCHEM PHARMACOL, vol. 19, 1970, pages 2169 - 2172
YADAV, V. K. ET AL.: "Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis", NAT MED, vol. 16, 2010, pages 308 - 312
ZHU, W. ET AL.: "Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry", ANAL BIOANAL CHEM, vol. 401, 2011, pages 3249 - 3261

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9199994B2 (en) 2013-09-06 2015-12-01 Karos Pharmaceuticals, Inc. Spirocyclic compounds as tryptophan hydroxylase inhibitors
US11759462B2 (en) 2013-09-06 2023-09-19 Altavant Sciences Gmbh Spirocyclic compounds as tryptophan hydroxylase inhibitors
US9512122B2 (en) 2013-09-06 2016-12-06 Karos Pharmaceuticals, Inc. Spirocyclic compounds as tryptophan hydroxylase inhibitors
US10946018B2 (en) 2013-09-06 2021-03-16 Roivant Sciences Gmbh Spirocyclic compounds as tryptophan hydroxylase inhibitors
US9750740B2 (en) 2013-09-06 2017-09-05 Kanos Pharmaceuticals, Inc. Spirocyclic compounds as tryptophan hydroxylase inhibitors
US10660893B2 (en) 2013-09-06 2020-05-26 ROIVANT SCIENCES, GmbH Spirocyclic compounds as tryptophan hydroxylase inhibitors
US10045988B2 (en) 2013-09-06 2018-08-14 Roivant Sciences Gmbh Spirocyclic compounds as tryptophan hydroxylase inhibitors
US10350208B2 (en) 2013-09-06 2019-07-16 Roivant Sciences Gmbh Spirocyclic compounds as tryptophan hydroxylase inhibitors
US10189839B2 (en) 2013-11-19 2019-01-29 Actelion Pharmaceuticals Ltd Tricyclic imidazole compounds as inhibitors of tryptophan hydroxylase
US9765092B2 (en) 2013-11-19 2017-09-19 Actelion Pharmaceuticals Ltd Tricyclic piperidine compounds
US10191070B2 (en) 2015-03-03 2019-01-29 Laboratory Corporation Of America Holdings Methods and systems for measuring serotonin in a sample
US10859586B2 (en) 2015-03-03 2020-12-08 Laboratory Corporation Of America Holdings Methods and systems for measuring serotonin in a sample
EP3889614A1 (fr) * 2015-03-03 2021-10-06 Laboratory Corporation of America Holdings Procédés et systèmes de mesure de la sérotonine dans un échantillon
US11442073B2 (en) 2015-03-03 2022-09-13 Laboratory Corporation Of America Holdings Methods and systems for measuring serotonin in a sample
WO2016141172A1 (fr) * 2015-03-03 2016-09-09 Laboratory Corporation Of America Holdings Procédés et systèmes de mesure de la sérotonine dans un échantillon
US10710948B2 (en) 2015-03-05 2020-07-14 Roivant Sciences Gmbh Processes for preparing (R)-1-(5-chloro-[1,1″-biphenyl] -2-yl)-2,2,2-trifluoroethanol and 1-(5-chloro-[1,1″-biphenyl]-2-yl)-2,2,2-trifluoroethanone
US9611201B2 (en) 2015-03-05 2017-04-04 Karos Pharmaceuticals, Inc. Processes for preparing (R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethanol and 1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethanone

Also Published As

Publication number Publication date
WO2014195847A3 (fr) 2015-03-26

Similar Documents

Publication Publication Date Title
Sonninen et al. Metabolic alterations in Parkinson’s disease astrocytes
Zhang et al. Glucose metabolic crosstalk and regulation in brain function and diseases
WO2014195847A2 (fr) Nouvelle utilisation du l-tryptophane marqué par un isotope stable
Bromek et al. Cytochrome P450 mediates dopamine formation in the brain in vivo
Babu et al. Serum metabolomics study in a group of Parkinson's disease patients from northern India
Liu et al. Altered urinary amino acids in children with autism spectrum disorders
Notarangelo et al. Gas chromatography/tandem mass spectrometry detection of extracellular kynurenine and related metabolites in normal and lesioned rat brain
US20150216845A1 (en) Ipa as a therapeutic agent, as a protective agent, and as a biomarker of disease risk
Blachnio‐Zabielska et al. Measuring long‐chain acyl‐coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring
Sun et al. Berberine ameliorates nonbacterial prostatitis via multi-target metabolic network regulation
Zhang et al. Simultaneous determination of multiple neurotransmitters and their metabolites in rat brain homogenates and microdialysates by LC-MS/MS
Van Kuilenburg et al. New insights in dihydropyrimidine dehydrogenase deficiency: a pivotal role for beta-aminoisobutyric acid?
Tang et al. Urinary metabolomics reveals alterations of aromatic amino acid metabolism of Alzheimer’s disease in the transgenic CRND8 mice
Xie et al. Development and validation of a sensitive LC–MS/MS method for the determination of d-serine in human plasma
Bannai et al. Oral administration of glycine increases extracellular serotonin but not dopamine in the prefrontal cortex of rats
Meng et al. Morphine-induced conditioned place preference in mice: Metabolomic profiling of brain tissue to find “molecular switch” of drug abuse by gas chromatography/mass spectrometry
Vetel et al. Extensive exploration of a novel rat model of Parkinson's disease using partial 6‐hydroxydopamine lesion of dopaminergic neurons suggests new therapeutic approaches
Lee et al. Active pyruvate dehydrogenase and impaired gluconeogenesis in orthotopic hepatomas of rats
Camuto et al. Urinary excretion profile of methiopropamine in mice following intraperitoneal administration: A liquid chromatography–tandem mass spectrometry investigation
JPWO2010104025A1 (ja) 筋変性疾患の検出方法、及び治療効果判定方法
Kapitsa et al. Characteristics of behavioral reactions and the profile of brain isatin-binding proteins of rats with the rotenone-induced experimental parkinsonism
Bannur et al. The differential metabolite profiles of acute lymphoblastic leukaemic patients treated with 6-mercaptopurine using untargeted metabolomics approach
Kondrat et al. In vivo microdialysis and gas-chromatography/mass-spectrometry for 13C-enrichment measurement of extracellular glutamate in rat brain
Takada et al. Measurement of plasma tryptophan metabolites: clinical and experimental application for depression and stress states assessment
Hamaue et al. Entacapone, a catechol-O-methyltransferase inhibitor, improves the motor activity and dopamine content of basal ganglia in a rat model of Parkinson's disease induced by Japanese encephalitis virus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14731385

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 14731385

Country of ref document: EP

Kind code of ref document: A2