WO2008083454A1 - Procédé de radiomarquage de flavonoïdes et son application au diagnostic in vivo de dysfonctionnements cérébraux liés aux sites de réception de benzodiazépines. - Google Patents

Procédé de radiomarquage de flavonoïdes et son application au diagnostic in vivo de dysfonctionnements cérébraux liés aux sites de réception de benzodiazépines. Download PDF

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Publication number
WO2008083454A1
WO2008083454A1 PCT/BR2008/000005 BR2008000005W WO2008083454A1 WO 2008083454 A1 WO2008083454 A1 WO 2008083454A1 BR 2008000005 W BR2008000005 W BR 2008000005W WO 2008083454 A1 WO2008083454 A1 WO 2008083454A1
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WIPO (PCT)
Prior art keywords
flavonoids
radiolabeling
benzodiazepine
application
brain
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PCT/BR2008/000005
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English (en)
Inventor
Sibila Roberta Marques Grallertr
Leoberto Costa Tavares
Elaine Bortoleti De Araujo
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Comissão Nacional De Energia Nuclear
Universidade De São Paulo
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Publication of WO2008083454A1 publication Critical patent/WO2008083454A1/fr

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    • G—PHYSICS
    • G21—NUCLEAR PHYSICS; NUCLEAR ENGINEERING
    • G21H—OBTAINING ENERGY FROM RADIOACTIVE SOURCES; APPLICATIONS OF RADIATION FROM RADIOACTIVE SOURCES, NOT OTHERWISE PROVIDED FOR; UTILISING COSMIC RADIATION
    • G21H5/00—Applications of radiation from radioactive sources or arrangements therefor, not otherwise provided for 
    • G21H5/02—Applications of radiation from radioactive sources or arrangements therefor, not otherwise provided for  as tracers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones

Definitions

  • the present invention relates to the development of new agents for in vivo imaging and diagnosis, comprising flavonoids deriving from flavone fundamental nucleus, properly- labeled with a radioisotope suitable to perform images for in vivo diagnosis, related to benzodiazepine receiving sites, by using tomographic techniques .
  • brain radiotracers are molecules labeled with positron-emitting or photon-emitting and gamma-emitting isotopes, which are able to selectively bind to specific receiving sites in the central nervous system. For in vivo use, those radiotracers shall have no toxicity or pharmacological activity in the given dose.
  • neuroreceptors imaging in in vivo studies is applied basically to three major areas, namely: (i) to establish the role of the specific receiving system in physiopathology of neuropsychiatric malfunctions, such as schizophrenia, epilepsy, Alzheimer' s-like dementia, anxiety and depression, among others, as well as in cases of drug abuse, (ii) to assess the interaction of pharmacological agents and receiving systems, with the purpose of explaining psychotropic agents mechanism of action, as well as assisting in the development of alternative treatments for neuropsychiatric diseases, and (iii) to help the development of new drugs for use in diagnosis or therapy.
  • neuropsychiatric malfunctions such as schizophrenia, epilepsy, Alzheimer' s-like dementia, anxiety and depression, among others, as well as in cases of drug abuse
  • pharmacological agents and receiving systems with the purpose of explaining psychotropic agents mechanism of action, as well as assisting in the development of alternative treatments for neuropsychiatric diseases
  • iii to help the development of new drugs for use in diagnosis or therapy.
  • Neuroreceptors imaging consists of recording radioactivity spatial distribution, after injecting the radiotracer in the bloodstream, which crosses the hematoencephalic barrier and binds to a neuroreceptor for a time fraction.
  • flavonoids as a new class of compounds having selectivity related to benzodiazepine receiving sites in the central nervous system.
  • Figure 1 shows flavone fundamental nucleus, also known as ⁇ -benzopirane, which is the precursor in biosynthesis of all flavonoids.
  • Flavonoids usually are not considered toxic substances, and several pharmaceutical specialties describe them as toxicity-free.
  • the current literature validates that in publication U.S. Patent number 5,756,538, by exempting them from toxicity, mainly in the doses required for application as radiotracers.
  • GABA- benzodiazepine receiving complex plays an essential role as an inhibition triggering agent.
  • GABA A -benzodiazepine receptors subtypes concerning their composition, density and regional distribution in the central nervous system allow the development of new specific ligand agents, which, on their turn, help explaining the relevance of those receptors subtypes. Refer, for example, to Katsifis et al . , Mini-Reviews in Med. Chem. , v. 4, p. 909-921 , 2004 .
  • PET and SPECT studies using 11 C-flumazenil allowed mapping the density of benzodiazepine receiving sites in human brain, showing change in the density of these receptors in experimental apoplexy, Huntington's disease, Alzheimer's disease, anxiety disorders, ethanol dependence, hepatic encephalopathy, Angelman syndrome, schizophrenia and epilepsy.
  • Ishibashi et al . J. Nucl. Med., v. 39, p. 1518-1520, 1998 and Moriwaki et al . , J " . Nucl. Med., v. 39, p. 1348-1353, 1998.
  • Iomazenil a radioiodized flumazenil analogue, shown on Figure 2, has subnanomolar affinity to benzodiazepine receiving sites in vivo. This compound is used as a radioligand for SPECT imaging of benzodiazepine receiving sites in primates.
  • Iomazenil features favorable to in vivo access to benzodiazepine receiving sites are high brain uptake, small non-specific binding, high affinity to benzodiazepine receiving sites and not promoting intrinsic pharmacological effects in trace doses .
  • 123 I-iomazenil is extremely selective and has high affinity, about ten times greater than 11 C-Ilumazenil, for being more lipophilic than the latter.
  • NNC 13-8241 [3' -5-cyclopropyl-l, 2, 4- oxadiazol-3-il) -7-iodine-5, 6-dihydro-5-methyl-6-oxo-4H-imidazo [1, 5- a] [1,4] benzodiazepine] , Figure 3, is a partial agonist selectively binding and having subnanomolar affinity to benzodiazepine receiving sites in tissues homogenates .
  • NNC 13-8241 analogues have been synthesized, and a higher agonist efficacy has been observed for 3-oxadiazolyl imidazol than for the analogue esters. Refer, for example, to Kuika et al . , Eur. J. Nucl.
  • t-butyl ester group and of the pyrrol ring blended into the benzodiazepine nucleus in bretanezil improved the molecule metabolic stability, when compared to iomazenil and flumazenil, and it also allows SPECT and PET imaging, when labeled with iodine-123 and bromine-76, respectively.
  • the inconvenience associated to S-bretanezil use as a radiotracer is the relatively slow brain clearance, with 3% D/g uptake after 6 hours from injection, resulting in high radiation exposure for the patient.
  • the object of the present invention is to obtain a brain radiotracer with appropriate features for performing in vivo diagnosis of disorders associated to benzodiazepine receiving sites, by using tomographic techniques, such as PET and SPECT. That and other objects and advantages of the present invention are obtained by means of radiochemical development of flavone biodistribution compounds and assays in Swiss mice, by checking the radiotracer concentration in the animal brain, as well as its distribution in the other tissues of interest and in blood.
  • Figure 6 represents flavone or analogues radiochemical development diagram and;
  • Figure 7 represents a graph illustrating the brain uptake rate of the radiolabeled flavone.
  • the compounds in the present invention may be clinically used in tomographic techniques such as PET and SPECT, to detect disorders related to benzodiazepine receiving sites, such as depression, epilepsy, schizophrenia, panic disorder, among others.
  • duly radiolabeled compounds in this invention are novel, and other compounds are known, however, the radiopharmaceutical use of these compounds has not been described in literature as diagnosis agents in brain disorders related to benzodiazepine receiving sites .
  • Flavonoids comprised in the present invention are polyphenolic molecules deriving from flavone fundamental nucleus, and they may be obtained by- means of the techniques described in Example 1, having the following structural formula: or its pharmaceutically acceptable salt, wherein:
  • R 5 is a hydrogen or hydroxy1
  • R 6 is methyl or halogen (preferably 1-123, 1-125, 1-124, I, Br-76, F-18) ;
  • R 7 is hydrogen, hydroxy1, -COOR, Br, Cl, F, -OR,
  • R 8 is hydrogen, hydroxyl, -COOR, Br, Cl, F, -OR, -NH, -NHR, -NR, -CN or -COOH;
  • R' 2 is a hydrogen
  • R' 3 is a hydrogen, halogen (preferably F, F-18,
  • R' 4 is a hydrogen, halogen (preferably F, Br, I, Cl) , methyl, ethyl or propyl;
  • R' 5 is a hydrogen and;
  • R' 6 is a hydrogen
  • the representative compounds that may be used in the present invention include the following: 6-iodine-123-flavone, 6-iodine-123-3' -F-flavone, 6-iodine-123-3' -NO 2 -flavone and 6- methyl-3' -fluorine-18-flavone.
  • Compounds representing any of the above compounds are also included as representative compounds, which have the hydrogens from R 5 and R 7 positions substituted by hydroxyl or other substitute having hydrophilic feature similar to this group, simultaneously or separately.
  • the flavonoids proposed in this work may be prepared, in a first step, by solving 20 mmol of 2-hydroxy- acetophenone in 5 ml of pyridine. Then, 30 mmol of duly substituted benzoyl chloride are added, and the vial is shaken to ensure proper reagent mixing. The reaction shall occur for about 20 minutes, and at the end, the solution is poured into a beaker containing 120 ml hydrochloric acid (3%) and 40 g chopped ice, under shaking. The product formed by filtration is collected under reduced pressure, washed with methanol and, on a second step, with water. The product shall be filtered and methanol recrystallized.
  • the mixture shall be maintained under heating between 40 and 65 0 C, for about 50 to 70 minutes, under shaking.
  • the reacting mixture is poured over chopped ice with manual shaking. When all ice is melt, the product is collected by filtration under reduced pressure, the compound obtained from petroleum ether is recrystallized.
  • Radioiodized flavonoids may be prepared by solving 1 mg of the compound to be labeled in about 40 to 50 ⁇ l sodium phosphate buffer 0.1 to 0.3 M, in neutral pH labeled with Na 123 I or Na 131 I in the presence of about 30 to 50 ⁇ l chloramine-T solution, incubated for about 10 to 25 minutes at room temperature, under shaking. After this time, about 30 to 50 ⁇ l sodium metadisulfide solution were added to the mixture. Radiochemical control was performed with Instant Thin Layer Chromatography-Silica Gel (ITLC-SG), and hexane:ethyl acetate (1:1) was used as eluent.
  • ITLC-SG Instant Thin Layer Chromatography-Silica Gel
  • the Rf was determined for the reactive iodine ( 123 I ' ou 131 I " ) and for the iodide, by using an Na 123 I solution. Radiochemical purity: flavone: iodine-123 : 99.3%; 3' -NO 2 - flavone-iodine: 98.5%; 3 ' -F-flavone-iodine-123 : 98.3%; 4'-F- flavone-iodine-123 : 97.1%.
  • Example 3 Radiolabeled flavonoids biodistribution studies were performed on male Swiss mice, whose weight varied between 33- 44 g, by using an invasive method. 25-30 mCi of the compound labeled with iodine-131 at 0.5 ml volume were injected in the caudal vein of each animal. The animals were sacrificed by thoracostomy at 5, 10, 15, 30, 60 minutes and 24 hours timepoints after the injection, and the organs of interest were removed, weighed and had their radioactivity assessed by an automatic counter with scintillator NaI (Tl) , calibrated for the iodine-131 nuclide. For each timepoint in the study at least 5 animals were used.
  • % activity/tissue cpm tissue . 100 Equation 1 standard cpm
  • % activity/gram tissue % dose/tissue Equation 2 tissue weight
  • ligands for benzodiazepine receiving sites labeled with radioactive iodine in SPECT studies includes the use of iodine-123, a radioisotope having the appropriate physical features for diagnosing use, such as low power gamma monoemission (159 keV) , absence of particulate emissions and 13- hour physical half-life time, which is considered relatively short.
  • Such features together, allow obtaining images with excellent resolution, by using the detecting systems available, and also minimize the radiation dose absorbed by the patient.
  • Iodine-131 was used almost exclusively for two decades for thyroid gland imaging and uptake quantification. Its physical half-life time is about 8 days, main detectable gamma emission of 364 keV and its beta minus-type particulate emission is associated to high power, maximum 660 keV. This radioisotope nuclear properties make it useful for therapeutic applications.
  • Iodine-131 physical features are not optimal for diagnosing studies use, however, this radioisotope is routinely used in the investigation step, due to its low cost, when compared to iodine- 123.
  • Figure 6 Flavone radiochemical development diagram
  • Figure 7 Graph illustrating the brain uptake rate for 6-iodine-131-flavone compound.

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  • Health & Medical Sciences (AREA)
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Abstract

La présente invention se rapporte à un procédé de radiomarquage de flavonoïdes, et à son application au diagnostic in vivo de dysfonctionnements cérébraux liés aux sites de réception de benzodiazépines. Le procédé selon l'invention consiste en l'élaboration radiochimique de flavonoïdes radiomarqués et en leur application in vivo en tant qu'agents d'imagerie cérébrale associés aux sites de réception de benzodiazépines, à l'aide de techniques de tomographie. Le marquage de flavonoïdes par substitution électrophile aromatique et la biodistribution de flavonoïdes radiomarqués chez des souris suisses ont été étudiés dans le cadre dudit procédé. Les résultats ont révélé une forte concentration de l'indicateur radioactif dans le cerveau, principalement jusqu'à 30 minutes après l'injection du radiocomposé, ce qui rend ledit agent compatible avec un usage clinique pour la réalisation d'images diagnostiques et particulièrement adapté à une application en médecine nucléaire.
PCT/BR2008/000005 2007-01-10 2008-01-08 Procédé de radiomarquage de flavonoïdes et son application au diagnostic in vivo de dysfonctionnements cérébraux liés aux sites de réception de benzodiazépines. WO2008083454A1 (fr)

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BRPI0702640-4 2007-01-10
BRPI0702640-4A BRPI0702640A (pt) 2007-01-10 2007-01-10 processo de radiomarcação de flavonóides e sua aplicação em diagnóstico in vivo de disfunções cerebrais relacionadas aos sìtios receptores benzodiazepìnicos

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009102498A1 (fr) * 2008-02-14 2009-08-20 Siemens Medical Solutions Usa, Inc. Nouveaux agents d’imagerie pour la détection d’une dysfonction neurologique
US8420052B2 (en) 2008-07-24 2013-04-16 Siemens Medical Solutions Usa, Inc. Imaging agents useful for identifying AD pathology
US8491869B2 (en) 2009-03-23 2013-07-23 Eli Lilly And Company Imaging agents for detecting neurological disorders
US8691187B2 (en) 2009-03-23 2014-04-08 Eli Lilly And Company Imaging agents for detecting neurological disorders
US8932557B2 (en) 2008-02-14 2015-01-13 Eli Lilly And Company Imaging agents for detecting neurological dysfunction

Citations (1)

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Publication number Priority date Publication date Assignee Title
US4450149A (en) * 1981-06-15 1984-05-22 Research Corporation Radiohalogenation method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4450149A (en) * 1981-06-15 1984-05-22 Research Corporation Radiohalogenation method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ONO M. ET AL.: "Radioiodinated flavones for in vivo imaging of beta-amyloid plaques in the brain", J. MED. CHEM., vol. 48, no. 23, 17 November 2005 (2005-11-17), pages 7253 - 7260, XP002635976, DOI: doi:10.1021/jm050635e *
SCHRÖDER-VAN DER ELST J.P. ET AL.: "Different tissue distribution, elimination, and kinetics of thyroxine and its conformational analog, the synthetic flavonoid EMD 49209 in the rat", ENDOCRINOLOGY, vol. 138, no. 1, January 1997 (1997-01-01), pages 79 - 84 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009102498A1 (fr) * 2008-02-14 2009-08-20 Siemens Medical Solutions Usa, Inc. Nouveaux agents d’imagerie pour la détection d’une dysfonction neurologique
US8318132B2 (en) 2008-02-14 2012-11-27 Siemens Medical Solutions Usa, Inc. Imaging agents for detecting neurological dysfunction
EP2599763A1 (fr) * 2008-02-14 2013-06-05 Siemens Molecular Imaging, Inc. Nouveaux agents d'imagerie pour la détection d'un dysfonctionnement neurologique
US8932557B2 (en) 2008-02-14 2015-01-13 Eli Lilly And Company Imaging agents for detecting neurological dysfunction
US8420052B2 (en) 2008-07-24 2013-04-16 Siemens Medical Solutions Usa, Inc. Imaging agents useful for identifying AD pathology
US8491869B2 (en) 2009-03-23 2013-07-23 Eli Lilly And Company Imaging agents for detecting neurological disorders
US8691187B2 (en) 2009-03-23 2014-04-08 Eli Lilly And Company Imaging agents for detecting neurological disorders

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