WO2002000665A1 - Utilisation d'elements d'ouverture du canal a potassium, pour le traitement de l'insulite - Google Patents

Utilisation d'elements d'ouverture du canal a potassium, pour le traitement de l'insulite Download PDF

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Publication number
WO2002000665A1
WO2002000665A1 PCT/DK2001/000444 DK0100444W WO0200665A1 WO 2002000665 A1 WO2002000665 A1 WO 2002000665A1 DK 0100444 W DK0100444 W DK 0100444W WO 0200665 A1 WO0200665 A1 WO 0200665A1
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WO
WIPO (PCT)
Prior art keywords
thiadiazine
thieno
chloro
dioxide
potassium channel
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Ceased
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PCT/DK2001/000444
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English (en)
Inventor
John Bondo Hansen
Anders Karlsson
Mikael Kullin
Stellan Sandler
Elisabeth BJÖRK
Zhanchun Li
Birgitte Michelsen
Jesper Svendstorp Lund
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Novo Nordisk AS
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Novo Nordisk AS
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Priority to AU2001265841A priority Critical patent/AU2001265841A1/en
Publication of WO2002000665A1 publication Critical patent/WO2002000665A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame

Definitions

  • the present invention relates to the use of potassium channel openers, which are able to protect the beta cells against toxic damage, for treating or preventing diseases related to autoimmune destruction of human beta cells, such as different types of diabetes, and methods of using these compounds.
  • BACKGROUND OF THE INVENTION Streptozotocin and alloxan are beta cell toxins.
  • the toxic effect of these compounds on rat pancreatic islets in vitro and in vivo mimics the beta-cell death associated with Type 1 and late state Type 2 diabetes.
  • the compounds of the present invention are able to inhibit strep- tozotocin and alloxan induced beta cell degeneration and death.
  • the compounds of the present invention act as activators of ATP regulated potassium channels (Katp-channels) of the beta cell and the Katp- channels of mitochondria. They may also act by antagonising the depletion of NAD induced in the islets by these toxins. Cytokines are known to reduce beta cell viability and to induce apoptosis. Cytokines have been proposed to be involved with the autoimmune degeneration of beta cells in Type 1 diabetes. The compounds of the present invention antagonize the effects of cytokines on beta cells.
  • the compounds of the present invention can be used in the treatment of insulitis associated with various forms of diabetes.
  • IDDM Type 1 or Insulin Dependent Diabetes Mellitus
  • NIDDM Non Insulin Dependent Diabetes Mellitus
  • SPIDDM slowly progressive IDDM
  • LADA latent autoimmune diabetes in adults
  • gestational diabetes due to underlying IDDM.
  • potassium channel openers are compounds disclosed in PCT Publication No. WO 97/26265 (see for instance from page 14, line 5 to page 19, line 9) and WO 99/03861 (see for instance from page 17, line 20 to page 19, line 5) as well as the following com- pounds: 3-tert-Butylamino-6-chloro-4H-thieno[3,2-e]-1 ,2,4-thiadiazine 1 ,1-dioxide; 6-Chloro- 3-cyclobutylamino-4H-thieno[3,2-e]-1 ,2,4-thiadiazine 1 , 1 -dioxide; 6-Chloro-3-(1 , 1 -dimethyl- propylamino)-4H-thieno[3,2-e]-1 ,2,4-thiadiazine 1 , 1 -dioxide; 6-Chloro-3-(1 -methylcyclo- propyl)amino-4H-thieno[3,2-e]-1 ,2,4-thiadia
  • the remaining islets showed reduced insulin content and secretion and a reduced insulin biosynthesis, amounting to 50%, 60% and 35%, respectively of control.
  • the STZ islets also displayed a lowered rate of glucose oxidation - 16% of control.
  • islets pre-incubated with diazoxide or 6-Chloro-3-isopropylamino-4H-thieno[3,2-e]-1 ,2,4-thiadiazine 1 ,1-dioxide maintained higher insulin content and insulin secretion compared to islets incu- bated with STZ alone.
  • K A TP channel openers can protect insulin-producing cells from being damaged by a beta-cell toxin and suggest that such an effect might be applicable in subjects with ongoing insulitis.
  • Diazoxide and other K ATP channel openers such as cromakalim and pinacidil, have been employed in experimental studies of ischemic heart. A beneficial, cardioprotective effect was observed (Garlid KD et al., Circulation Res 1997; 81 :1072-82). Although the mechanism of this phenomenon is not understood, an opening of mitochondnal potassium channels seems to be involved, resulting in dissipation of the inner mitochondnal membrane potential. This in turn leads to net oxidation of the mitochondria with an apparent reduction of energy wastage.
  • Diazoxide is known to act on K ATP channels in the plasma membrane of beta cells. It hyper- polarizes the membrane and reduces the entry of Ca 2+ , essential for the exocytosis of secre- tory granulaes. Recently, exposure of beta cells to diazoxide was found to engage also mitochondnal K AT p channels (Grimmsmann T et al., Br J Pharmacol 1998; 123:781-788). In the present study, we examined the influence of potassium channel openers on experimental beta-cell damage induced by streptozotocin, an agent known to cause energy depletion, on damage induced by alloxan, a generator of reactive oxygen species and on damage induced by cytokines.
  • Pancreata from Sprague-Dawley rats were collagenase digested and islets collected with a braking pipette as previously described (Sandier S et al., Endocrinology, 1987;121 :1424-31). Islets were precultured free floating in RPM1 1640 medium with 10 % (v/v) fetal calf serum (FCS) and 11 mM glucose for 3 days in 5% CO 2 at 37°C before experiments. Medium was changed two times during preculture. Islets were then transferred to sterile Petri dishes in KRBH (Krebs-Ringer bicarbonate with HEPES) medium with 2 mg/mL bovine serum albumin (BSA) and 5.6 mM glucose.
  • KRBH Kerata-1
  • the islets were then washed twice in KRBH and studied for morphology and insulin secretion, or cultured for 2 or 24 hours in RPMI with 10% FCS and 11 mM glucose prior to morphological and biochemical examinations. Morphology and Islet Recovery
  • Triplicates of five islets were transferred to 200 ⁇ l of KRBH with 2 mg/mL BSA and 16.7 mM glucose and incubated for 60 minutes in 5% CO 2 at 37°C. Islets from each condition were then pooled and sonicated in 200 ⁇ l of redestilled water. A 50 ⁇ l aliquot of the homogenate was mixed with 125 ⁇ l acid ethanol (0.18 M HCI in 95% ethanol) and insulin extracted over- night. Insulin concentration in the sonicate and the culture medium was determined with ra- dioimmunoassay.
  • Groups of 10 islets were transferred to glass vials with 100 ⁇ l KRBH supplemented with D- [U 14 C]glucose and nonradioactive glucose to a final concentration of 16.7 mM glucose. Triplicate samples were used. The vials were suspended in scintillation flasks, gassed with 5% CO 2 and sealed airtight. The flasks were then shaken for 90 minutes at 37°C. Metabolism was stopped by injection of 100 ⁇ l of 0.05 mM antimycin A into the center vial. Immediately thereafter 250 ⁇ l hyamine hydroxide was injected into the outer flask.
  • CO 2 was released from the incubation medium by injecting 100 ⁇ l of 0.4 M Na 2 HPO 4 solution (pH 6.0) into the center vial. To allow the CO 2 to be trapped by the hyamine hydroxide the vials were incubated for another 120 minutes at 37°C. Scintillation fluid was then added to each flask and the radio- activity counted in a liquid scintillation counter.
  • the islets exposed to Streptozotocin for 30 minutes showed degranulation, and in some islets numerous pyknotic nuclei, at the 0 hour timepoint. No signs of recovery but a further destruction and also disintegration of islets was found at 2 and 24 hours.
  • islets incubated with test compounds + STZ appeared morphologically intact at the 0 hour timepoint. During the subsequent 24 hour culture a toxic effect of STZ became noticeable. At 2 hours the surface of these islets were somewhat irregular and this was more apparent at 24 hours.
  • the numerous pyknotic nuclei as seen in the STZ group were not found in the group of islets treated with test compounds.
  • Islets examined at the 0 hour timepoint ie after a 60 minutes incubation in 5.6 mM glucose, showed a stronger stain for insulin than the islets examined after 2 and 24 hours.
  • the latter islets had been cultured in 11 mM glucose.
  • the difference in insulin staining reflects a higher stimulation of insulin secretion at 11 mM compared to 5.6 mM glucose.
  • the insulin staining of the islets treated with test compounds + streptozotocin were stronger at both 2 and 24 hours than that seen with the islets incubated with medium alone.
  • the STZ treatment also had lowered the insulin and total protein biosynthesis as well as impaired the glucose oxidation rate.
  • the inhibitory effect of the K ATP channel openers on insulin secretion was seen in islets treated with test compounds + streptozotocin at 0 and 2 hours, but not after 24 hours.
  • a partial protection of the islet function was observed when compared with islets incubated with STZ alone.
  • the proinsulin and total protein biosynthesis in the recovered STZ islets were reduced to 35% and 51 % of control, respectively.
  • the proinsulin and total protein biosynthesis did not differ from the biosynthesis found in the recovered STZ.
  • PCO compounds The effect of PCO compounds on cell viability was analysed in 51 Cr-release cytotoxicity assays using either primary islet preparations (e.g. from newborn rats) or islet tumour cell lines (e.g. mouse transgenic ⁇ -cell lines ⁇ TC-3 or Min6, or rat insulinoma lines RIN5AH or MSLG2).
  • the assay has been used to measure toxic effects of e.g. cytokines or glucose, and to address the protective effect of PCO compounds on ⁇ -cell viability, e.g. during cytokine exposure.
  • the islets were then resuspended in 10 ml Islet media and 100 ⁇ l of the islet suspension were added to each well in a flat bottom 96 well plate (approximately 35 islets in each well).
  • Mixture of cytokines and test compounds or dimethyl sulphoxide were prepared in 100 ⁇ l media in each well. All test compounds were dissolved in dimethyl sulphoxide and prepared in stock solutions at a concentration of 100mM.
  • Viability assay using rodent adherent ⁇ -cell lines e.g. RIN cells, MIN6 cells, lns-1 cells and others.
  • HBSS life tech without Ca ++ and Mg ++ Cat 14185-045
  • 1 x trypsin in HBSS was used to split the cells.
  • the cells were seeded in a flat-bottomed 96 well plate in the desired media at a density of 40000 cells/well in 100 ⁇ l media and incubated overnight to secure proper adherence.
  • 2,5 ⁇ Ci/ml Na 51 Cr (Dupont, Nez 030S) was added to the labeling media (the desired media).
  • 1 x washing of the cells with HBSS 200 ⁇ l of media with Na 51 Cr were added to each well and incubated overnight.
  • the rodent adherent ⁇ -cell lines were incubated for 24h at 37 °C and 5% CO 2 .
  • the plates were centrifuged for 5 min at 1000 rpm, and 100 ⁇ l supernatant samples were harvested from each well.
  • 100 ⁇ l 1 % triton-X were added to each well in order to lyse the cells and 100 ⁇ l were harvested to get a maximum Na 51 Cr release from the cells of each well. All the samples and the maximum samples were counted on a cobra ⁇ -counter (Packard).
  • the effects on mitochondnal Katp channels kan be evaluated as described by e.g. Grimms- mann and Rustenbeck (Br. J. Pharmacol. 1998, 123, 781-788). Routinely the effects of the compounds of the present invention can be determined measuring changes in fluorescence of the dyes JC-1 or Rhodamine 123 when incubating beta cells or pancreatic islets in a medium containing the fluorencence indicators and the test compounds.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne l'utilisation d'agonistes du canal à potassium, pour le traitement de l'insulite associée à différentes formes de diabète telles que IDDM, NIDDM, SPIDDM (LADA) et le diabète gestationnel.
PCT/DK2001/000444 2000-06-26 2001-06-25 Utilisation d'elements d'ouverture du canal a potassium, pour le traitement de l'insulite Ceased WO2002000665A1 (fr)

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AU2001265841A AU2001265841A1 (en) 2000-06-26 2001-06-25 Use of potassium channel openers for the treatment of insulitis

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DKPA200000988 2000-06-26
DKPA200000988 2000-06-26

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003045955A1 (fr) * 2001-11-30 2003-06-05 Novo Nordisk A/S Utilisation d'activateurs selectifs d'ouverture de canaux potassiques
WO2003045954A1 (fr) * 2001-11-30 2003-06-05 Novo Nordisk A/S Utilisation d'activateurs d'ouverture de canaux potassiques
WO2004005299A1 (fr) * 2002-07-04 2004-01-15 Novo Nordisk A/S Formes polymorphiques d'un derive de 4h-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxyde
WO2020130836A1 (fr) * 2018-12-21 2020-06-25 ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n. LUMC) Composé destiné à être utilisé dans le traitement et la prévention du diabète de type i
NL2022291B1 (en) * 2018-12-21 2020-07-21 Academisch Ziekenhuis Leiden Compound for Use in Treatment and Prevention of Type I Diabetes

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1368948A (en) * 1970-11-11 1974-10-02 Manuf Prod Pharma Pyridine derivatives
EP0618209A1 (fr) * 1993-03-26 1994-10-05 Adir Et Compagnie Nouvelles pyridothiadiazines, leurs procédés de préparation et les compositions pharmaceutiques qui les contiennent
US5889002A (en) * 1996-01-17 1999-03-30 Novo Nordisk A/S Fused 1,2,4-thiadiazine and fused 1,4-thiazine derivatives, their preparation and use
US5972894A (en) * 1997-08-07 1999-10-26 Cytran, Inc. Peptides having potassium channel opener activity
WO2000037474A1 (fr) * 1998-12-18 2000-06-29 Novo Nordisk A/S Derives de 1,2,4-thiadiazine fusionnes, leur preparation et utilisation
US6225310B1 (en) * 1996-01-17 2001-05-01 Novo Nordisk A/S Fused 1,2,4-thiadiazine derivatives, their preparation and use

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1368948A (en) * 1970-11-11 1974-10-02 Manuf Prod Pharma Pyridine derivatives
EP0618209A1 (fr) * 1993-03-26 1994-10-05 Adir Et Compagnie Nouvelles pyridothiadiazines, leurs procédés de préparation et les compositions pharmaceutiques qui les contiennent
US5889002A (en) * 1996-01-17 1999-03-30 Novo Nordisk A/S Fused 1,2,4-thiadiazine and fused 1,4-thiazine derivatives, their preparation and use
US6225310B1 (en) * 1996-01-17 2001-05-01 Novo Nordisk A/S Fused 1,2,4-thiadiazine derivatives, their preparation and use
US5972894A (en) * 1997-08-07 1999-10-26 Cytran, Inc. Peptides having potassium channel opener activity
WO2000037474A1 (fr) * 1998-12-18 2000-06-29 Novo Nordisk A/S Derives de 1,2,4-thiadiazine fusionnes, leur preparation et utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
STEFAN GLOCKER ET AL: "Binding and effects of P1075, an opener of ATP-sensitive K+ channels, in the aorta from streptozotocin-treated diabetic rats", NAUNYN-SCHMIEDEBERG'S ARCH PHARMACOL, vol. 356, 1997, pages 210 - 215, XP002901885 *
T C HOHMAN ET AL: "ATP-sensitive K+ channel effects on nerve function, Na+, K+ ATPase, and glutathione in diabetic rats", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 397, 2000, pages 335 - 341, XP002901886 *
W D VLAHOS ET AL: "Diabetes prevention in BB rats by inhibition of endogenous insulin secretion", METABOLISM, vol. 40, no. 8, August 1991 (1991-08-01), pages 825 - 829, XP002901887 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003045955A1 (fr) * 2001-11-30 2003-06-05 Novo Nordisk A/S Utilisation d'activateurs selectifs d'ouverture de canaux potassiques
WO2003045954A1 (fr) * 2001-11-30 2003-06-05 Novo Nordisk A/S Utilisation d'activateurs d'ouverture de canaux potassiques
WO2004005299A1 (fr) * 2002-07-04 2004-01-15 Novo Nordisk A/S Formes polymorphiques d'un derive de 4h-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxyde
WO2020130836A1 (fr) * 2018-12-21 2020-06-25 ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n. LUMC) Composé destiné à être utilisé dans le traitement et la prévention du diabète de type i
NL2022291B1 (en) * 2018-12-21 2020-07-21 Academisch Ziekenhuis Leiden Compound for Use in Treatment and Prevention of Type I Diabetes

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