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WO2014113893A1 - Utilisation de galantamine et de composés apparentés pour le traitement d'affections abdominales inflammatoires - Google Patents

Utilisation de galantamine et de composés apparentés pour le traitement d'affections abdominales inflammatoires Download PDF

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WO2014113893A1
WO2014113893A1 PCT/CA2014/050051 CA2014050051W WO2014113893A1 WO 2014113893 A1 WO2014113893 A1 WO 2014113893A1 CA 2014050051 W CA2014050051 W CA 2014050051W WO 2014113893 A1 WO2014113893 A1 WO 2014113893A1
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colitis
splenic
mice
dcs
agonist
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Jean-Eric Ghia
Valentin PAVLOV
Kevin Tracy
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University of Manitoba
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4468—Non condensed piperidines, e.g. piperocaine having a nitrogen directly attached in position 4, e.g. clebopride, fentanyl
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/27—Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system

Definitions

  • IBDs Inflammatory bowel diseases
  • CD Crohn's disease
  • UC ulcerative colitis
  • This vagus nerve- dependent and a7nAChR-mediated cholinergic anti-inflammatory pathway represents the efferent arm of an inflammatory reflex that controls immune responses and cytokine production 9 .
  • Electrical vagus nerve stimulation suppresses tumor necrosis factor (TNF) and other pro-inflammatory cytokine levels in endotoxemia and colitis 10 .
  • TNF tumor necrosis factor
  • IBDs are associated with an autonomic imbalance and up to 35% of patients with ulcerative colitis (UC) exhibit autonomic dysfunction with impaired efferent vagus nerve activity 11 .
  • UC ulcerative colitis
  • DCs Dendritic cells
  • the cholinergic anti-inflammatory pathway can be centrally activated by muscarinic acetylcholine receptor (mAChR) ligands or acetylcholinesterase (AChE) inhibitors 17 ' 18 .
  • Galantamine (GAL) is a reversible, competitive AChE inhibitor, which crosses the blood-brain barrier, increases brain cholinergic network activity 19 and is widely used in the treatment of Alzheimer's disease.
  • GAL activates efferent vagus nerve activity 20 and its anti-inflammatory activity has been associated with brain mAChR-mediated activation of the cholinergic anti-inflammatory pathway 18 .
  • a method of treating an individual suffering from an inflammatory bowel disease comprising administering to an individual in need of such treatment an effective amount of a M1 mAChR agonist.
  • M1 mAChR agonist for treating an inflammatory bowel disease.
  • a M1 mAChR agonist for use in a medicament for treating an inflammatory bowel disease.
  • FIG. 1 Galantamine (GAL) alleviates the severity of DSS-induced colitis.
  • GAL (6 days, i.p.) treatment was started one day prior to colitis induction.
  • A Disease activity index
  • B Macroscopic scores
  • C Myeloperoxidase (MPO) activity
  • D Serum C-reactive protein (CRP).
  • E Histological score.
  • F Appearance of a colon in mice with DSS-induced colitis (control group);
  • G Appearance of a colon in GAL-treated mice with DSS-induced colitis (Hematoxylin and eosin staining, 10X magnifications). Values are shown as means ⁇ SEM, n ⁇ 8. * P ⁇ 0.05 as compared to control DSS-treated group. # P ⁇ 0.05 as compared to water (H2O)-treated group.
  • FIG. 2 Galantamine effects in DSS-induced colitis are mediated through central mAChRs.
  • Atropine sulfate AS, a mAChR antagonist that crosses the blood- brain barrier
  • APN atropine methyl nitrate
  • A Macroscopic score
  • B Myeloperoxidase (MPO) activity
  • Values are shown as means+SEM, n>8.
  • FIG. 3 Central administration of a M1 mAChR agonist or a M2mAChR antagonist alleviates the severity of DSS-induced colitis.
  • McN-A-343 M1 mAChR agonist
  • M2 mAChR antagonist methoctramine
  • A Macroscopic score
  • B Myeloperoxidase (MPO) activity
  • Values are shown as means+SEM, n ⁇ 8.
  • VXP Vagotomy
  • SPX splenectomy
  • NRX splenic neurectomy
  • SPX splenectomy
  • *Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals;
  • FIG. 5 Cholinergic treatment with galantamine or McN-A-343 results in increased splenic ACh levels, mediated through vagus nerve and splenic nerve signaling.
  • Vagotomy (VXP) and/or splenic neurectomy (NRX) were performed prior to galantamine (GAL, 4mg/kg/day, i.p. for 6 days) or McN- A-343 (5ng/kg/day, i.c.v., for 6 days) treatment and spenic ACh levels analyzed as described in Material and Methods, 24h after the last injection.
  • Splenic ACh levels were determined as postmortem, Values are shown as means+SEM, n ⁇ 8. *P ⁇ 0.05 as compared to control Sham-DSS-treated group. # P ⁇ 0.05 as compared to the same treatment in sham- operated animals with DSS-induced colitis.
  • Figure 6 Effects of cholinergic treatment on splenic dendritic cell and T cell cytokine production in the context of colitis.
  • A Interleukin (IL)-12p40 production from dendritic cells.
  • Splenic dendritic cells were isolated from galantamine (GAL, 4 mg/kg/day, i.p. for 6 days)-treated groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX).
  • GAL galantamine
  • VXP vagotomy
  • NRX splenic neurectomy
  • B INF- ⁇ production from dendritic/T cell co-cultures. Galantamine (in vivo) and GTS-21 (ex vivo) treatments were performed as described above in A. See Material and Methods for details.
  • Figure 7 Effect of galantamine on the development of acute colitis induced by 2,4 dinitrobenzene-sulfonic acid (DNBS)-treatment. Influence of 4 days galantamine treatment (4 mg/kg/day, intraperitoneal (i.p.)) starting 1 day before injection of DNBS.
  • A Myeloperoxidase (MPO) activity.
  • B Interleukin ( ⁇ _)-1 ⁇ colonic level. A significant decrease of both markers was detected compared to the control group.
  • Pre-treatment with atropine sulfate (AS, 4 mg/kg, i.p.), but not atropine methyl nitrate (AMS, 4 mg/kg, i.p.) abolished the beneficial effect induced by galantamine.
  • Figure 8 Effect of muscarinic 1 agonist (McN-A343: 5ng/kg/day, intracerebroventricular (i.c.v.)) and Methoctramine (MTT: 5ng/kg/day, i.c.v.) on the development of colitis induced by 2,4 dinitrobenzen-sulfonic acid (DNBS)-treatment (5mg). Influence of 4 days of i.c.v. infusion starting 1 day before injection of DNBS. A: Myeloperoxidase (MPO) activity, B: Interleukin (IL)-1 ⁇ colonic level. A significant decrease of both markers is detected compared to the vehicle group.
  • FIG. 9 Effect of McN-A-343 intracerebroventricular ((5ng/kg/day, i.c.v.) infusion on the development of acute colitis induced by dextran sulfate sodium (DSS) 5% treatment in the presence or absence of the vagus nerve (vagotomy: VXP) or the spleen (SPX). Influence of a 6 days i.c.v. infusion starting 1 day before induction of DSS colitis. A: Myeloperoxidase (MPO) activity, B : Interleukin (ll_)-1 ⁇ colonic level. A significant decrease of both markers was detected compared to the sham vehicle group. VXP and SPX abolished the beneficial effect of M1 i.c.v. infusion. * vs sham vehicle group, # P ⁇ 0.05. The values are shown as mean ⁇ SEM, n ⁇ 8.
  • MPO Myeloperoxidase
  • ll_ Interleukin
  • Figure 10 I.C.V. infusions of the MlmAChR agonist McN-A-343.
  • FIG 11 Central administration of a MlmAchR agonist alleviates the severity of dextran sulfate sodium (DSS)-induced colitis through vagus and splenic nerve signaling to the spleen.
  • Vagotomy (VXP) and/or splenectomy (SPX), splenic neurectomy (NRX) and/or splenectomy (SPX) were performed 10 days prior to initiating McN-A-343 (5 ng/kg/day, i.c.v.) treatment and/or colitis induction as described in Material and Methods.
  • Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals;
  • A Serum amyloid protein (SAP);
  • B Colonic interferon- gamma (IFN-D);
  • C Colonic interleukin (IL)-17;
  • D Colonic IL-12p70;
  • E Colonic IL-23 and
  • F Colonic IL-4. Values are shown as means ⁇ SEM. Samples were collected on day 5 post-DSS induction; mice per group ⁇ 8.
  • a P ⁇ 0.05 as compared to sham-saline- DSS-treated group b P ⁇ 0.05 as compared to VXP-DSS-treated group or NRX-DSS- treated group respectively, C P ⁇ 0.05 as compared to sham-McN-A-343-DSS-treated group.
  • FIG. 12 Central administration of a Ml mAchR agonist alleviates splenic CD1 1c+ dendritic cells (DCs) interleukin (IL)-12p70 and IL-23 release in the context of dextran sulfate sodium (DSS)-induced colitis through vagus and splenic nerve signaling to the spleen.
  • DCs dendritic cells
  • IL-12p70 and B IL-23 production from splenic CD11c + DCs.
  • Splenic CD 1 c + DCs were isolated from McN-A-343 (5 ng/kg/day, i.c.v.
  • VXP vagotomy
  • NRX splenic neurectomy
  • Figure 13 Implication of the NF- ⁇ pathway in splenic CD 1 c + dendritic cells (DCs) cytokine release in the context of dextran sulfate sodium (DSS)-induced colitis.
  • Splenic CD1 1 c + DCs were isolated from McN-A-343 (5 ng/kg/day, i.e. v.
  • VXP vagotomy
  • NRX splenic neurectomy
  • FIG. 14 Role of the CD1 1 c + dendritic cells (DCs) in CD4 + CD25 " T cells priming in the context of dextran sulfate sodium (DSS)-induced colitis. Effect of McN- A343 (in vivo) and GTS-21 (in vitro) treatments on splenic CD1 1 c + DCs function and sequential CD4 + CD25 cell activation. Splenic CD1 1 c + DCs isolated from different colitic group were cultured in for 24h before being co-cultured with CD4 + T lymphocytes isolated from na ' ive mice.
  • DCs dendritic cells
  • A Interferon-gamma (IFN- ⁇ )
  • B Interleukin (IL)-17
  • C IL-4 were measured in media at 24hrs.
  • D CD4 + CD25- T cells proliferation.
  • Splenic CD1 1 c + DCs isolated from different colitic group were cultured in for 24h before being co- cultured with CD4 + T lymphocytes isolated from naive mice in the presence of absence of A; anti-pi 9 mAb (10ug/ml-1 ) or B: anti-p35 mAb (l Oug/ml 1 ), C: recombinant (r)IL-12p70 (25ng/mr 1 ) or D: rlL-23 (25ng/ml "1 ) protein.
  • GAL or McN-A-343 (a MlmAChR agonist) significantly ameliorate disease severity and inhibit inflammation in the context of experimental colitis.
  • This therapeutic efficacy is brain mAChR-dependent and mediated through a functional interaction between the vagus nerve and spleen and inhibition of splenic DCs cytokine production through an a7nAChR mechanism.
  • the cholinergic anti-inflammatory pathway is an efferent vagus nerve-based mechanism that regulates immune responses and cytokine production through a7- nicotinic-acetylcholine-receptor(a7nAChR) signaling. Decreased efferent vagus nerve activity is observed in inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the inventors determined whether central activation of this pathway alters inflammation in mice with colitis, the mediating role of a vagus nerve-to spleen circuit and a7nAChR signaling. Two experimental models of colitis were used in C57BL/6 mice.
  • compounds known for treating for example Alzheimer's Disease such as for example cholinesterase inhibitors such as Donepezil (Aricept), Rivastigmine (Exe!on) and Galantamine (Razadyne).
  • cholinesterase inhibitors such as Donepezil (Aricept), Rivastigmine (Exe!on) and Galantamine (Razadyne).
  • cholinesterase inhibitors that cross the blood-brain barrier can be used to treat intestinal diseases such as Crohn's disease and ulcerative colitis is highly surprising.
  • a medication for treating an inflammatory bowel disease must cross the blood-brain barrier and is not applied directly to the gut or the digestive tract, for example, intraperitoneally.
  • an immune cell can be regulated directly in this manner and an opposite effect induced.
  • AChE inhibitors or selective mAChR ligands results in a decreased susceptibility to experimental colitis. These protective effects are dependent on vagus nerve and splenic nerve signaling, which is mediated at a cellular level through cholinergic suppression of splenic DC activation.
  • a method of treating an individual suffering from an inflammatory bowel disease comprising administering to an individual in need of such treatment an effective amount of a 1 mAChR agonist.
  • an "effective amount” will depend on for example the age and general condition of the individual in need of such treatment or patient. However, such amounts can be determined through routine experimentation and/or by analysis of previous administrations of the compounds.
  • the 1 mAChR agonist crosses the blood-brain barrier.
  • the M1 mAChR agonist may be selected from the group consisting of McN-A-
  • the M1 mAChR agonist is McN-A-343.
  • an "effective amount" of these compounds may be similar to the effective amount for treatment of Alzheimer's Disease, that is, 8-24 mg/day.
  • a person in need of such treatment is an individual who suffers from or is suspected of suffering from or at risk of developing an inflammatory bowel disease, for example ulcerative colitis or Crohn's disease.
  • the effective amount of lmAChR agonist will accomplish at least one of the following: reduction in severity and/or frequency of associated symptoms, including but by no means limited to abdominal pain, vomiting, diarrhea, rectal bleeding, and intestinal cramps; and longer periods of remission and/or being symptom free.
  • Ml mAChR agonist for treating an inflammatory bowel disease.
  • the inflammatory bowel disease may be ulcerative colitis or Crohn's disease.
  • a Ml mAChR agonist for use in a medicament for treating an inflammatory bowel disease.
  • the results identify a vagus nerve - to spleen axis as an important mediator of the central cholinergic regulation of colitis severity.
  • the importance of the findings was additionally substantiated by the similar profile of results in a DNBS-model; this model closely resembles CD, which broadens the therapeutic implications of cholinergic modalities in the context of IBD.
  • the most widely used and characterized experimental model of UC is the DSS-induced colitis, which was developed by administration of DSS in the drinking water. DSS induces a very reproducible acute colitis characterized by superficial inflammation and infiltrations.
  • the most widely used and characterized experimental model of CD is the DNBS-induced colitis, which was developed by the intramural administration of 50% ethanol (barrier breaker) containing DNBS. DNBS is believed to haptenize colonic autologous or microbiota protein rendering them immunogenic to the host immune system. This model induces a very reproducible acute colitis characterized by ulceration and infiltration.
  • the inventors have further identified the mediating role of the a7AChR on DCs; a specific a7AChR agonist down-regulated ex vivo IL-12p40 release.
  • This corroborates data demonstrating that in vitro immature spleen DCs that mature in a nicotinic environment manifest lower endocytic and phagocytic activities 43 .
  • In vitro mature spleen DCs that are exposed to nicotine produce decreased levels of IL-12 and displayed reduced ability to induce T-Cell responses 44 .
  • the in vitro data do not support the possibility that galantamine may act as an allosteric modulator on the a7AChR on DCs to directly affect their activation.
  • this goes along with the surprising fact developed above, as normally a direct effect on this type of cell should have been seen, but in this case no effect was observed.
  • vagus nerve targets the proximal colon. Therefore, it is theoretically possible that alterations in the inflammatory state in the vagus nerve innervated proximal colon affect the enteric nervous system activity in the remainder of the colon 45 , which in turn may result in a reduction in neurotransmitters release and suppression of inflammation. This may also account for the effect of vagus nerve signaling on colitis observed in this and other studies 536 . Furthermore, the fact that inventors did not observe significant effects of GAL or McN-A-343 on IL- 0 levels suggests that alterations in antiinflammatory cytokine levels do not play a rote in mediating the beneficial effects of cholinergic modalities on colitis severity. This confirms previous data on sepsis and colitis 8,24 suggesting a minor role of IL-10. In addition, cholinergic treatments did not alter the stool consistency in control mice, indirectly pointing to a lack of significant effect on gut physiology.
  • IBD patients might be selected for novel treatment strategies including: 1) Centrally-acting AChE inhibitors, which are already clinically approved for the treatment of Alzheimer's disease; 2) highly selective muscarinic receptor agonists or antagonists 7 , which are being actively developed for the treatment of Alzheimer's disease and other neurodegenerative disorders, as discussed above; and 3) electrical vagus nerve stimulation, which is now being used for the treatment of refractory depression and epilepsy 50 .
  • Mucosal inflammation in IBD is accompanied by autonomic nervous system dysfunction and a decreased vagus nerve activity 58 . It is recognized that this autonomic dysfunction plays an important role in motility and immune response dysregulation during IBD 59 > 60 .
  • the vagus nerve-based CAIP has a major anti-inflammatory function in different inflammatory conditions including colitis 9 ' ° ⁇ 5 While the spleen plays an important mediating role in the cholinergic anti-inflammatory pathway 21 ' 56 ⁇ 57 , at certain experimental conditions of intestinal inflammation, this physiological mechanism does not require neural signalling to spleen 61 .
  • central muscarinic cholinergic activation results in a decreased susceptibility to experimental colitis; this effect is vagus nerve- and splenic nerve-dependent and mediated at a cellular level by a specific interaction between DCs and CD4 + CD25" T cells.
  • the DSS-induced colitis is a widely used model of ulcerative colitis 23 .
  • animals may develop acute or chronic colitis or even colitis-induced dysplastic lesions.
  • the innate and the adaptive immune response are shown to be an important component in colitis pathogenesis in this model.
  • Recently the inventors demonstrated a role of a CNS pharmacological mAChR-dependent stimulation of the CAIP in alleviating the disease seventy and suppressing colonic proinflammatory cytokine (IL- 6, IL- ⁇ and TNF-a) levels in experimental DSS-induced colitis 54 .
  • IL- 6, IL- ⁇ and TNF-a colonic proinflammatory cytokine
  • This effect was mediated through the vagus and splenic nerve, and the spleen, via the modulation of proinflammatory cytokines released by splenic dendritic cells 54 .
  • the inventors demonstrate that this effect is accompanied by a downregulation of IFN- Y and IL-17 levels in colonic tissues.
  • Th2 can downregulate the Th1 cytokines release and IL-4 is known to decrease the production of IL-12 in the context of T cell priming 62 .
  • NF- ⁇ an important transcription factor mediating the proinflammatory cytokine production, is attributed to the initiation and progression of colonic inflammation in mice and humans 68, 69 .
  • nAChRs are expressed on antigen presenting ceil including macrophages and DCs 70 , and ACh and nicotine inhibit TNF-a and NF- KB production from lipopolysaccharide (LPS)-stimulated human macrophages and splenocytes 55, 56 .
  • LPS lipopolysaccharide
  • GTS-2 can inhibit phosphorylation and subsequent degradation of I-KB, which is likely to suppress NF- ⁇ activation 71 .
  • NF- ⁇ pathway inhibitor As nAChRs are expressed on DC 70 , we tested the role of the intracellular NF- ⁇ pathway. Increased production of IL-12p70 and IL-23 by DCs in VXP- and NRX mice was significantly inhibited in the presence of the NF- ⁇ pathway inhibitor. Moreover, in the presence of NF-KB pathway activator the levels of both cytokines were increased in DCs isolated from stimulated groups. In vitro stimulation with GTS-21 decreased IL-12p70 and IL-23 production of DCs isolated from both group and this effect was abolished in the presence of the NF- ⁇ activator.
  • IBD is characterized by increased expression of innate (IL-12, IL- 23) and adaptive (IFN- ⁇ , IL-17) proinflammatory cytokines 41 , and Th1 , Th 7 polarization is mainly controlled by the IL-12p70 and IL-23 cytokine family produced by activated DCs 75 .
  • Increased numbers of MDC8 + monocytes, which are the precursors of mucosal DC populations, are found in patients with IBD and, hence, anti-TNF treatment results in reduced DC activation 76 ' 77 .
  • MDC8 + monocytes which are the precursors of mucosal DC populations
  • DCs are increased within the lamina limbal 78 and peripheral blood monocytes are potent immune response stimulators 79 .
  • the inventors also further identify the mediating role of IL-12p70 and IL-23 on
  • IL-23p19-deficient mice develop severe colitis in IL-10 deficient mice 84 and !L-12p35 deficient mice develop a mild colitis 85 .
  • vagus nerve-based CAIP signals in addition to the spleen the vagus nerve-based CAIP signals in other organs implicated in the development colonic inflammation 86 .
  • GAL Centrally-acting acetylcholinesterase inhibitor treatment ameliorates the severity of colitis.
  • Previous findings have characterized GAL as a central activator of the cholinergic anti-inflammatory pathway 20 18 ' 30 .
  • Daily administration of GAL 1-4 mg/kg/day, i.p.
  • dose-dependently reduced the severity of colitis ( Figure 1A, B, C, D).
  • GAL-treated mice with colitis showed a significantly lower DAI for the last 2 days as compared to saline-treated controls with the disease ( Figure A). This drug effect was dose-dependent and the highest reduction was achieved with a dose of 4mg/kg/day, which has been previously shown to inhibit mouse brain AChE activity by 43% 30 .
  • Huperzine-A a structurally distinct, highly selective, centrally-acting AChE inhibitor.
  • Huperzine-A administration (0.4 mg/kg/day, i.p.) significantly reduced the disease severity and inflammation in mice with DSS-colitis (Table 4).
  • GAL and Huperzine-A treatments decreased the severity of DNBS colitis.
  • Central muscarinic receptors are essential for alleviation of colitis and inhibition of colonic inflammation by an acetylcholinesterase inhibitor.
  • the inventors next studied whether central mAChRs, which critically mediate brain cholinergic pathways and vagus nerve activation 18 , are required for the anti- inflammatory effect of GAL.
  • AS a mAChR antagonist that penetrates the BBB
  • AMN a mAChR antagonist that does not cross the BBB
  • failed to alter the beneficial effects of GAL on colitis Figure 2 and Table 1 ).
  • VXP vagotomy
  • NRX splenic neurectomy
  • SPX splenectomy
  • Splenic CD1 1c + DCs were isolated from colitic mice subjected to sham-operation, VXP or NRX and a treatment with vehicle or GAL (i.p.). Then these cells were treated ex vivo with GTS-21 (a specific o7nAChR agonist). At all conditions the addition of GTS- 21 in the culture medium significantly decreased IL-12p40 production ( Figure 6A).
  • GAL has properties of an allosteric positive modulator of nicotinic, including a7nAChR34.
  • CD11c + DC isolated from non-colitic mice were activated with LPS and treated with GAL or vehicle ex vivo. The inventors did not observe any significant GAL effect on IL-12p40 (440 ⁇ 23 vs 435 ⁇ 41 pg/ml) levels in these settings.
  • GAL treatment could affect the ability of DCs to prime CD4 + T cells.
  • CD4 + T cells isolated from naive mice were co-cultured with splenic DCs isolated from colitic mice treated with GAL in the presence or absence of the vagus or splenic nerve for 24h.
  • Central muscarinic cholinergic activation decreases serum SAP level and colonic Th1/Th17 cytokine release via the vagus nerve and the splenic nerve.
  • VXP vagotomy
  • NRX splenic neurectomy
  • SPX splenectomy
  • NRX resulted in comparable to VXP exacerbation of disease severity (Figure 11). These effects were abolished in mice simultaneously subjected to SPX ( Figure 1 1 ). Furthermore, the beneficial effects of McN-A-343 were abrogated in mice with NRX, SPX or NRX and SPX ( Figure ). No effect of these manipulations was observed on colonic IL-4 levels. These results demonstrate that neural signaling through the vagus nerve and the splenic nerve is required for central cholinergic activation to alleviate colitis, by altering specifically the Th1/Th 7 cytokines and with no effect on IL-4.
  • CD4 + CD25 " T ceils To determine the contribution of CD4 + CD25 " T ceils to the decreased cytokines production observed in the spleen following activation of the CAIP during colitis, we isolated CD4 + CD25 _ T cells from the spleen of naive animals and determine the splenic CD1 1 c + DCs priming of CD4 + CD25 " T cells and their cytokine release.
  • CD4 + CD25 " T cells, isolated from naive mice were co-cultured for 24h with splenic CD11 c+ DCs isolated from colitic mice infused with McN-A-343.
  • DSS molecular weight [MW], 40 kilodaltons: ICN Biomedicals Inc
  • MW molecular weight
  • Controls were all time-matched and consisted of mice that received normal drinking water only. Mean DSS consumption was noted per cage each day.
  • Isoflurane Abbott, Abbott Park, IL
  • a 10cm long PE-90 tubing (ClayAdam, Parsippany, NJ), attached to a tuberculin syringe, was inserted 3.5cm into the colon.
  • Colitis was induced by administration of 100 ⁇ of 5mg of DNBS solution (ICN) in 50% ethanol and left for 3 days 24 .
  • Control mice received saline administration.
  • Mice with colitis were supplied with 6% sucrose in drinking water to prevent dehydration.
  • 2, 4-dinitrobenzen-sulfonic-acid (DNBS) colitis was induced by administration of 100 ⁇ of 4mg of DNBS solution (Sigma, Oakville, Canada) in 30% ethanol for 3 days.
  • mice were anaesthetized using ketamine (150 mg/kg, i.p) and xylazine (10 mg/kg, i.p).
  • I.c.v. implantation of the cannula, splenectomy (SPX), splenic neurectomy (NRX) or subdiaphragmatic bilateral vagotomy (VXP) was performed on the same day 5 .
  • SPX splenectomy
  • NRX splenic neurectomy
  • VXP subdiaphragmatic bilateral vagotomy
  • mice implanted with the cannula received vehicle; mice were anaesthetized and laparotomy performed but the spleen was not removed ; splenic nerve was exposed but not cut; vagal trunks were exposed but not cut, however, a pyloroplasty was performed.
  • vagotomy was verified during post-mortem inspection of vagal nerve endings using microscopic inspection associated with a Bielschowsky silver staining 25 .
  • the completeness of neurectomy was verified post-mortem by noradrenaline enzyme- linked immunosorbent assay in sham-operated and NRX animals. Mice were allowed to recover for 10 days.
  • GAL 1 -4 mg/kg/day, intraperitoneal (i.p.)
  • Huperzine-A Huperzine-A: (H-A, 0.4 mg/kg/day, i.p., Sigma, Oakville, ON)
  • Atropine Methyl Nitrate AMN, injected 20 min prior to GAL, 4 mg/kg/day, i.p., Sigma
  • Atropine sulfate AS, injected 20 min prior GAL, 4 mg/kg/day, i.p. Sigma).
  • Micro-osmotic pumps (Alzet, Cupertino, California, USA) were filled with vehicle (saline 0.2%), M1 mAChR agonist McN-A-343 (Sigma) or M2 mAChR antagonist methoctramine (MTT, 5 ng/day, Sigma) solution and placed as previously described 24 .
  • DAI Disease activity index
  • MPO Myeloperoxidase activity
  • CRP Serum C-reactive protein
  • cytokine levels were determined using ELISA commercial kit (R&D Systems, Minneapolis, Minnesota, USA). The amount of acetylcholine in the spleen was measured using the acetylcholine assay kit following manufacturer's recommendations (Molecular Probes Invitrogen).
  • samples were collected 5 days post-DSS activation and blood was collected by intracardiac puncture under isoflurane anesthesia.
  • Serum amyloid protein (SAP) and colonic cytokine levels were determined using ELISA commercial kit (R&D Systems, Minneapolis, MN).
  • the cells were then washed, resuspended in cell separation buffer (Dulbecco's Phosphate- Buffered Saline [D- PBS] without Ca21 and Mg21 containing 2% FBS and 2mM EDTA, Life Technology) and passed through magnetic columns for positive selection of DC. After passing consecutively through two columns, the collected DCs preparations showed greater than 95% purity.
  • DC isolated from different groups of mice were cultured in complete RPMI 1640 medium containing 10% heat-inactivated FBS, 25mg/m
  • lipopolysaccharide (LPS, Sigma) was added to the cultures at a final concentration of 100 ng/ml " .
  • GAL or the a7nAChR agonist GST-21 were added to medium at a final concentration of 10 _5 M.
  • CD1 c + DCs isolated from colitic mice receiving the different treatments were cultured for 24 hrs before being co-cultured with CD4 + T cell isolated from naive mice at ratio of 1 :3 (DC:T)29 in plate coated with 10 pg/ml " of anti-CD3 and 2 g/ml "1 of anti-CD28.
  • Cell culture supernatants were collected at 24 hrs, interferon (INF)-y and IL-17 levels were analyzed by ELISA (R&D Systems).
  • splenic CD11c+ cells and culture 5 days post-DSS activation, splenic CD1 1c + DC were isolated from different groups of mice and were cultured for 24hrs, the supernatant was measured for IL-12p70 and IL-23 by ELISA (R&D Systems).
  • o;7nAChR agonist GTS-21 100pM
  • betulinic acid a specific NF- ⁇ activator, 10 ⁇
  • BAY 11-7082 a specific NF- B inhibitor, 10 ⁇
  • CD1 1c+ DCs isolated from colitic mice receiving the different treatments were cultured for 24hrs before being co-cultured with CD4+/CD25- T cell isolated from naive mice at ratio of 1 :3 (DCs:T cells) 27 in plates coated with ⁇ g/ml '1 of anti-CD3 and 2 ⁇ g/m
  • Cell culture supernatants were collected at 24hrs, and interferon-gamma (IFN-v), IL-4 and IL- 17 levels were analyzed by ELISA (R&D Systems).
  • the cells were then washed, re- suspended in cell separation buffer (Dulbecco's Phosphate-Buffered Saline [D-PBS] without Ca21 and Mg21 containing 2% FBS and 2mM EDTA, Life Technology, Grand Island, NY) and passed through magnetic columns for positive selection. After passing consecutively through two columns, the collected splenic CD11c + cell preparations showed greater than 95% purity.
  • D-PBS Dulbecco's Phosphate-Buffered Saline
  • Splenic CD1 1c + cell isolated from different groups of mice were cultured in complete RPMI 1640 medium containing 10% heat-inactivated FBS, 25mg/ml-1 gentamicin, 2mM L-glutamine in 12-well plates at 1.10 + ⁇ cells/well for 24hrs, and the supernatants were measured for IL- 12p70 and IL-23 by ELISA (R&D Systems).
  • the a7nAChR agonist GTS-21 or betu!inic acid (a specific NF- B activator, 10DM) or Bay 1 1 -7085 (a specific NF- ⁇ inhibitor, 10 ⁇ ) were added to medium at a final concentration of 10 ⁇ 6 M.
  • CD4 + cells were isolated from freshly isolated spleen by magnetic microbeads selection according to manufacturer's protocol (MACS, Milteny Biotech). Briefly, splenocytes were incubated for 15 min in 4°C with microbeads and washed twice in washing buffer (10 min) and depleted using MACS LD column. The obtained CD4 + T cells were washed twice in washing buffer (10 min) resuspended and labeled with anti-CD25 microbeads. Subsequently, the cells were incubated for 5 min in 4°C. The cells were then incubated for the next 15 min at 4°C and washed twice in washing buffer.
  • MCS manufacturer's protocol
  • CD4 + /CD25 " T cells were isolated using MACS MS column. After the last centrifugation, the selected subpopulations of CD4 CD25 " T cells were resuspended in culture medium (RPMI 1640 supplemented with 10% FCS, l-Glutamine 200 mM, HEPES 1 M, antibiotic-antimycotic) and were immediately used in assay.
  • culture medium RPMI 1640 supplemented with 10% FCS, l-Glutamine 200 mM, HEPES 1 M, antibiotic-antimycotic
  • T-cell proliferation cells were labeled with CFSE 5x10 +4 of labeled cells were cultured for 24h, and then proliferation assay was assessed by CFSE dilution, using FACS and FlowJo program (TreeStar, Ashland, OR)
  • Van Der Zanden EP Boeckxstaens GE, de Jonge WJ.
  • the vagus nerve as a modulator of intestinal inflammation. Neurogastroenterol Motil 2009; 21 (1): 6-17.
  • Pavlov VA Ochani M
  • Gallowitsch-Puerta M Ochani K
  • Huston JM Huston JM
  • vagus nerve and nicotinic receptors modulate experimental pancreatitis severity in mice. Gastroenterology 2006; 130(6): 1822-1830.
  • Hasler WL Traditional thoughts on the pathophysiology of irritable bowel syndrome. Gastroenterol Clin North Am 2011 ;40:21-43.
  • Valdes-Ferrer SI Valdes-Ferrer SI, Rosas-Ballina M, Olofsson PS, et al. Vagus Nerve Stimulation Produces an Anti-Inflammatory Monocyte Phenotype in Blood. Journal of Immunology 2010;184:138.25.
  • Trinchieri G lnterleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3:133-46.
  • MacDermott RP Alterations in the mucosal immune system in ulcerative colitis and Crohn's disease. Med Clin North Am 1994;78:1207-31.
  • MTT metallocatechin gallate
  • *BLS lower the lowest standard; a ⁇ 0.05 as compared to non DSS-treated group (H20), Vo.05 as compared to vehicle DSS- treated group. See Material and Methods for details.
  • VXP vagotomy
  • NRX splenic neurectomy
  • SPX splenectomy
  • *Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals; a ⁇ 0.05 as compared to sham- vehicle-DSS-treated group, b ⁇ 0.05 as compared to VXP-DSS-treated group , C ⁇ 0.05 as compared to NRX-DSS-treated group d P ⁇ 0.05 as compared to sham-GAL-DSS-treated group.
  • Table 4 The centrally-acting AChE inhibitor huperzine-A reduces the severity of DSS-induced colitis and inflammation.
  • a MlmAchR agonist alleviates the severity of 2, 4 dinitrobenzene sulfonic acid (DNBS) -induced colitis.
  • Vagotomy (VXP) or splenic neurectomy (NRX) were performed 10 days prior to initiating McN-A-343 (5 ng kg/day, i.e. v.) treatment and/or colitis induction as described in Material and Methods.
  • Splenic CDl lc + DCs were isolated from McN- A-343 (5 ng/kg/day, i.c.v. for 6 days)-treated groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX) on day 5 post-colitis induction. Effect of McN-A343 (in vivo) treatment on splenic CDl lc + DCs function and sequential CD4 + CD25T cell activation. Splenic CDl lc "1" DCs isolated from different colitic group were cultured in for 24h before being co- cultured with CD4 + CD25 cells isolated from naive mice.
  • the levels of IFN- ⁇ and IL-17 and IL-4 were measured in media at 24hrs. a ⁇ 0.05 as compared to sham- saline-DNBS -treated group, b P ⁇ 0.05 as compared to VXP-DSS-treated group or NRX-DSS-treated group. P ⁇ 0.05 as compared to NRX- DSS-treated group.

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Abstract

La présente invention concerne le traitement d'une affection abdominale inflammatoire, y compris de la rectocolite hémorragique et de la maladie de Crohn, en utilisant des agonistes du récepteur muscarinique M1 (MlmAChR). Les agonistes sont capables de traverser la barrière hématoencéphalique et permettent l'activation cholinergique centrale d'un circuit nerf vague vers rate. L'activation centrale induite par les traitements avec des agonistes des récepteurs muscariniques ont entraîné une réduction de l'inflammation muqueuse associée à une réduction de la sécrétion de cytokines pro-inflammatoires par les cellules dendritiques (CD) de la rate, médiées par la signalisation de oc7nAChR.
PCT/CA2014/050051 2013-01-28 2014-01-24 Utilisation de galantamine et de composés apparentés pour le traitement d'affections abdominales inflammatoires Ceased WO2014113893A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110447787A (zh) * 2018-05-07 2019-11-15 中国科学院水生生物研究所 加兰他敏在防治豆粕饲料引起的鱼类肠炎中的应用
US11344534B2 (en) 2017-06-26 2022-05-31 The Trustees Of Columbia University In The City Of New York Cholinergic agonism for the treatment of pancreatic cancer

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2476896A1 (fr) * 2002-02-26 2003-09-04 North Shore-Long Island Jewish Research Institute Inhibition de la production d'une cytokine inflammatoire par la stimulation de recepteurs muscariniques cerebraux
CA2518324A1 (fr) * 2003-03-21 2004-11-04 Eli Lilly And Company Agonistes muscariniques
WO2010084499A2 (fr) * 2009-01-26 2010-07-29 Israel Institute For Biological Research Composés spiro hétérocycliques bicycliques
WO2011163539A2 (fr) * 2010-06-25 2011-12-29 The Johns Hopkins University Agonistes de nachrα7 et antagonistes de nachrα7 pour traiter la rectocolite hémorragique (uc) et la maladie de crohn (cd)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2476896A1 (fr) * 2002-02-26 2003-09-04 North Shore-Long Island Jewish Research Institute Inhibition de la production d'une cytokine inflammatoire par la stimulation de recepteurs muscariniques cerebraux
CA2518324A1 (fr) * 2003-03-21 2004-11-04 Eli Lilly And Company Agonistes muscariniques
WO2010084499A2 (fr) * 2009-01-26 2010-07-29 Israel Institute For Biological Research Composés spiro hétérocycliques bicycliques
WO2011163539A2 (fr) * 2010-06-25 2011-12-29 The Johns Hopkins University Agonistes de nachrα7 et antagonistes de nachrα7 pour traiter la rectocolite hémorragique (uc) et la maladie de crohn (cd)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11344534B2 (en) 2017-06-26 2022-05-31 The Trustees Of Columbia University In The City Of New York Cholinergic agonism for the treatment of pancreatic cancer
CN110447787A (zh) * 2018-05-07 2019-11-15 中国科学院水生生物研究所 加兰他敏在防治豆粕饲料引起的鱼类肠炎中的应用

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