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WO2003008632A1 - Conjugaison d'acides gras en tant que methode de criblage de substances potentiellement bioactives - Google Patents

Conjugaison d'acides gras en tant que methode de criblage de substances potentiellement bioactives Download PDF

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Publication number
WO2003008632A1
WO2003008632A1 PCT/SE2002/001391 SE0201391W WO03008632A1 WO 2003008632 A1 WO2003008632 A1 WO 2003008632A1 SE 0201391 W SE0201391 W SE 0201391W WO 03008632 A1 WO03008632 A1 WO 03008632A1
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Prior art keywords
acid
fatty acid
enzyme
conjugation
fatty
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PCT/SE2002/001391
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English (en)
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Edward HÖGESTÄTT
Peter Zygmunt
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase

Definitions

  • Paracetamol is a frequently used antipyretic and analgesic agent, which differs from most non-steroidal anti-inflammatory drugs (NS AEDs) in that it is a weak anti- inflammatory agent and does not produce the typical side effects related to cyclo-oxygenase (COX) inhibition (7, 15).
  • NNSAOs non-steroidal anti-inflammatory drugs
  • COX cyclo-oxygenase
  • AM404 is also a ligand at cannabinoid receptors and an inhibitor of the anandamide transporter, the inhibition of which leads to increased levels of endogenous cannabinoids (4, 24). As shown here, AM404 and structurally related fatty acid derivatives inhibit both COX-1 and COX-2 (Fig. 1). Thus, it is not surprising that AM404 has anti-nociceptive properties and potentiates the analgesic effect of anandamide in the mouse hot plate test (4, 6).
  • the endogenous fatty acid amide anandamide (arachidonoylethanolamide), which is an agonist at cannabinoid (22) and vanilloid (28) receptors, is hydrolysed to arachidonic acid and ethanolamine by a fatty acid amide hydrolase (FAAH) (9, 10).
  • FAAH fatty acid amide hydrolase
  • This enzyme may also act in the reverse direction, causing synthesis of anandamide from arachidonic acid and ethanolamine (10).
  • the structures of acetaminophen and AM404 differ only with regard to the length of the hydrocarbon chain. We have shown that acetaminophen, following deacetylation to its primary amine y -aminophenol, is conjugated with arachidonic acid to form AM404 (Fig. 3, 4 and 5).
  • AM404 as a metabolite of acetaminophen produced locally in the central nervous system provides an explanation for the mechanism of action of this widely consumed analgesic and antipyretic agent.
  • Other primary amines such as dopamine and serotonin, are also conjugated with arachidonic acid to form their respective arachidonoylderivatives (Fig. 6), indicating that this biochemical pathway represents a general mechanism for biotransformation of primary amines into fatty acid amides.
  • the invention provides a method for screening for compounds or molecules that undergo conjugation with fatty acids in the mammal body, including humans.
  • the method can further be used for testing whether a compound or molecule has the ability to undergo such a conjugation.
  • tissue homogenate preferably of liver or brain, or an enzyme, preferably an esterase or amidase, including fatty acid amide hydrolase (FAAH), or a mixture of enzymes is provided in a kit.
  • the homogenate, enzyme or mixture of enzymes can be present in the kit as the sole component, or it can be present as part of a composition, alone or in combination with a fatty acid or a mixture of fatty acids, or other compounds, solutions or devices necessary or desirable for use of the kit.
  • the enzyme is preferably an esterase or an amidase, including fatty acid amide hydrolase.
  • the fatty acid is saturated or unsaturated, having between 1 and 5 double bonds.
  • the fatty acid is a fatty acid present in the mammal body, including humans, whereby the fatty acid is preferably arachidonic acid, linolenic acid, linoleic acid, oleic acid, palmitoleic acid, lignoceric acid, behenic acid, arachidic acid, stearic acid, palmitic acid myristic acid and lauric acid.
  • the invention provides a kit for conjugation of a compound or molecule with a fatty acid according to existing knowledge in the art of organic chemistry.
  • the bond between the fatty acid and the compound or molecule can be an amide, ester or thioester bond.
  • the synthesis of such fatty acid derivatives can be carried out following known procedures, such as those described in refs 8, 13, 17 and 25.
  • the kit contains an enzyme, preferably FAAH, or a mixture of enzymes, a fatty acid or a mixture of fatty acids, or a combination of these.
  • the kit can contain modified or activated fatty acids or enzymes, according to existing knowledge in the arts of organic and combinatorial chemistry, to provide optimal substrates or conditions for the conjugation reaction to occur.
  • the kit for conjugation of a compound or molecule with a fatty acid to yield a fatty acid amide, ester or thioester comprises a fatty acid together with at least one conjugation promoting substance, or a modified (activated) fatty acid.
  • the promoting substance can be a chemical reagent and/or an enzyme, preferably an esterase and/or an amidase, including fatty acid amide hydrolase.
  • the fatty acid or modified fatty acid is saturated or unsaturated, having between 1 and 5 double bonds.
  • the fatty acid can also be a product of the cyclo-oxygenase, lipoxygenase and cytochrome P450 monoxygenase pathways, including prostaglandins, thromboxanes, leukotriens, lipoxins, and hydroxy-, epoxy- or ydroperoxyderivatives of eicosatetraenoic and eicosatrienoic acids.
  • COX-1 and/or COX-2 were also inhibited by the fatty acid derivatives arvanil (97% and 95%), arachidonoyldopamine (96% and 93%), arachidonoyl-3-methoxytyramine (94% and 77%), arachidonoylserotonin (71% and 2%), olvanil (97% and 93%), anandamide (14% and 44%) and 2- arachidonoylglycerol (11% and 67%), respectively (10 ⁇ M each; duplicate measurements).
  • COX activity was measured as prostaglandin formation in the presence of 10 ⁇ M arachidonic acid.
  • Dashed line indicates the basal tension level before addition of drugs
  • Broken line (triangles) shows the relaxant effect of "endogenous" AM404 from rat homogenates incubated withjtj-aminophenol (mean of 4 arterial segments from the same rat).
  • "Endogenous" AM404 was purified using LC and quantified by LC/MS-MS as described. Tension traces show relaxant responses to increasing concentrations of exogenous (upper trace) and "endogenous" (lower trace) AM404.
  • Acetaminophen is metabolised to the primary amine ?-aminophenol, which is further conjugated with arachidonic acid to form the bioactive fatty acid amide N-(4- hydroxyphenyl)-5,8,ll,14-eicosatetraenamide (AM404).
  • FIG. 4 Formation of AM404 and j?-aminophenol in rat after intraperitoneal injection of acetaminophen (30 - 300 mg kg "1 ) and its inhibition by PMSF (10 mg kg "1 ).
  • a,b Representative chromatograms of samples obtained from rat brains showing (a) the presence of AM404 (23.4 pmol g "1 ) in an animal treated with acetaminophen and (b) no AM404 in an animal injected with vehicle.
  • the tandem mass spectrometer was operated to select the protonated molecular ion of AM404 at m/z 396.1 in the first quadruple mass separator, while the mass fragment at 109.8 after fragmentation in the collision cell (corresponding to the protonated -aminophenol fragment) was selected by the second quadruple.
  • FIG. 5 The formation of AM404 in rat brain homogenates is dependent onp- aminophenol and is sensitive to .the enzyme inhibitor PMSF.
  • the 3-methoxytyramine (circles), dopamine (triangles) and tyramine (squares) derivatives of arachidonic acid also induced concentration-dependent relaxation (c). None of the agonists elicited a relaxation after pre-treatment with 10 ⁇ M capsaicin for 30 min (open symbols) to cause vanilloid receptor desensitisation and or depletion of sensory neuropeptides (49).
  • Acetaminophen (30, 100 or 300 mg kg "1 ) or vehicle (saline) at a volume of 2 - 3 ml was given to female Wistar-Hannover rats (200 - 300 g) by an intraperitoneal injection. Some rats were pretreated with PMSF (10 mg kg "1 ) or vehicle (saline:PEG 6000; 1:10 w/w) given subcutaneously (2 - 3 ml) 20 min before administration of acetaminophen. Approximately 20 min after injection of acetaminophen, the animals were killed to collect brain, liver, spinal cord and arterial blood. The tissues were homogenised in a Tris buffer (10 mM, pH 7.6) containing EDTA (1 mM).
  • the brain, liver, spinal cord and dorsal root ganglia from female Wistar-Hannover rats were homogenised in a Tris buffer (10 mM, pH 7.6), containing EDTA (1 mM), to give 90 - 330 mg tissue ml "1 .
  • a Tris buffer (10 mM, pH 7.6), containing EDTA (1 mM)
  • EDTA 1 mM
  • AM404 arachidonoyldopamine, arachidonoylserotonin and anandamide.
  • Sample aliquots of 5 ⁇ l were injected on a Genesis C 8 column (20 x 2.1 mm; Jones). Initially, the mobile flow was 25% water for 5.5 min. Then a linear gradient to 100% methanol was applied in 0.2 min and the mobile phase was kept at 100% methanol for 2.3 min, after which the column was reconditioned in 25% water for 2 min.
  • the electrospray interface was operating in the positive ion mode at 370°C, the ion spray voltage was 4500 volts and the declustering potential was 40 volts.
  • M/z 396.1/109.8 with a collision energy of 27 volts was used for the AM404 determinations.
  • M/z 440.2/153.5 with a collision energy of 25 volts, m/z 463.2/159.6 with a collision energy of 39 volts and m/z 348.2/61.6 with a collision energy of 35 volts were used for arachidonoyldopamine, arachidonoylserotonin and native anandamide, respectively.
  • M/z 356.4/62.2 with a collision energy of 35 volts was used for the internal standard [ 2 H 8 ] -labelled anandamide.
  • COX-1 and COX-2 assays were determined in the presence of 10 ⁇ M arachidonic acid using a COX (ovine) inhibitor screening assay (Cayman). Drugs were incubated with the enzyme preparation 8 min before application of arachidonic acid. Prostaglandin formation was used as a measure of COX activity and quantified via enzyme immunoassay (EIA).
  • EIA enzyme immunoassay
  • AM404 N-(4-hydroxyphenyl)-5,8,ll,14-eicosatetraenamide), capsaicin, capsazepine (all from Tocris); 1-arachidonoylglycerol, 2-arachidonoylglycerol, [ H 8 ] -anandamide, [ H 8 ]-arachidonic acid, arachidonoylserotonin, NS-398 (all from Cayman); anandamide (Biomol); arachidonoyldopamine, arachidonoyl-3-methoxytyramine and aracmdonoyltyramine (Syntelec) were all dissolved in and diluted with ethanol. DMSO substituted ethanol as a solvent in the COX assays.
  • the batch of acetaminophen contained no or less than 0.001% (w/w) of p-aminophenol as determined by LC/MS-MS.
  • Example 1 Fatty acid amides or esters as vanilloid receptor agonists.
  • capsaicin, arachidonoyldopamine, arachidonoyl-3-methoxytyramine, arachidonoyltyramine, 1- arachidonoylglycerol and 2-arachidonoylglycerol are all agonists at vanilloid receptors on vasodilator sensory nerves.
  • Acetaminophen andp-aminophenol in the presence of ascorbic acid to prevent its decomposition
  • Example 3 Fatty acid conjugation of acetaminophen in vivo. Since the structures of acetaminophen and AM404 differs only with regard to the length of the hydrocarbon chain, we hypothesised that acetaminophen, following deacetylation to its metabolite p-aminophenol (32), is conjugated with arachidonic acid to form AM404 (Fig. 3). To test this proposal, we measured the levels of AM404 and -aminophenol in various tissues of rat 20 min after intraperitoneal injection of acetaminophen at a commonly used dose (300 mg/kg), which produces a robust analgesic effect in rodents (18, 22, 46).
  • AM404 is formed via an enzyme-dependent process.
  • PMSF phenyl-methyl-sulphonylfluoride
  • a broad-spectrum protease, esterase and amidase inhibitor 13
  • N-acyl-dopamines novel synthetic C ⁇ cannabinoid- receptor ligands and inhibitors of anandamide inactivation with cannabimimetic activity in vitro and in vivo. BiochemicalJournal 351: 817-824, 2000.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Genetics & Genomics (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne une méthode de criblage de composés ou de molécules subissant une conjugaison avec des acides gras présents dans un corps de mammifère, notamment chez l'être humain. Selon cette invention, un composé à tester est mis en contact avec un acide gras et un homogénat de tissus ou une enzyme, et toute conjugaison entre le composé et tout acide gras présent dans ledit homogénat de tissus est déterminée en ce qui concerne la présence d'amides, d'esters ou de thioesters d'acides gras. L'invention concerne également un kit destiné à la conjugaison de tels composés ou molécules avec des acides gras.
PCT/SE2002/001391 2001-07-16 2002-07-16 Conjugaison d'acides gras en tant que methode de criblage de substances potentiellement bioactives Ceased WO2003008632A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US30515901P 2001-07-16 2001-07-16
SE0102549A SE0102549D0 (sv) 2001-07-16 2001-07-16 Fatty acid conjugation as a method for screenning of potentially bioactive substances
US60/305,159 2001-07-16
SE0102549-3 2001-07-16

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007047881A3 (fr) * 2005-10-19 2007-07-05 Allergan Inc Methode de traitement de la douleur
US7902251B2 (en) 2005-10-19 2011-03-08 Allergan, Inc. Method for treating pain
US9486441B2 (en) 2008-04-21 2016-11-08 Signum Biosciences, Inc. Compounds, compositions and methods for making the same
US10159268B2 (en) 2013-02-08 2018-12-25 General Mills, Inc. Reduced sodium food products

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994012466A1 (fr) * 1992-11-30 1994-06-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Derives d'acides gras et compositions pharmaceutiques les contenant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994012466A1 (fr) * 1992-11-30 1994-06-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Derives d'acides gras et compositions pharmaceutiques les contenant

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BISOGNO T. ET AL.: "Arachidonoylserotonin and other novel inhibitors of fatty acid amide hydrolase", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 248, no. RC988874, 1998, pages 515 - 522, XP002958407 *
BISOGNO TIZIANA ET AL.: "N-acyl-dopamines: novel synthetic CB1 cannabinoid-receptor ligands and inhibitors of anandamide inactivation with cannabimimetic activity in vitro and in vivo", BIOCHEM. J., vol. 351, 2000, pages 817 - 824, XP002958408 *
KAPHALIA B.S. ET AL.: "Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty aicd anilides", JOURNAL OF CHROMATOGRAPHY B, vol. 705, 1998, pages 269 - 275, XP004108718 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007047881A3 (fr) * 2005-10-19 2007-07-05 Allergan Inc Methode de traitement de la douleur
US7902251B2 (en) 2005-10-19 2011-03-08 Allergan, Inc. Method for treating pain
US9486441B2 (en) 2008-04-21 2016-11-08 Signum Biosciences, Inc. Compounds, compositions and methods for making the same
US10583119B2 (en) 2008-04-21 2020-03-10 Signum Biosciences, Inc. Compounds, compositions and methods for making the same
US10159268B2 (en) 2013-02-08 2018-12-25 General Mills, Inc. Reduced sodium food products
US11540539B2 (en) 2013-02-08 2023-01-03 General Mills, Inc. Reduced sodium food products

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