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WO1998003178A2 - Pharmaceutical compositions containing p2y purinergic receptor antagonists - Google Patents

Pharmaceutical compositions containing p2y purinergic receptor antagonists Download PDF

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Publication number
WO1998003178A2
WO1998003178A2 PCT/EP1997/003844 EP9703844W WO9803178A2 WO 1998003178 A2 WO1998003178 A2 WO 1998003178A2 EP 9703844 W EP9703844 W EP 9703844W WO 9803178 A2 WO9803178 A2 WO 9803178A2
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Prior art keywords
atp
cells
receptor antagonist
release
demylinating
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PCT/EP1997/003844
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French (fr)
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WO1998003178A3 (en
Inventor
Frank Brown
Davina Elizabeth Mitchell
Ariyan Tufiq Rahim
Brian Robert Stewart
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Smithkline Beecham Plc
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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
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • 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
    • A61K31/4415—Pyridoxine, i.e. Vitamin B6

Definitions

  • the present invention relates to the therapeutic use of compounds which act as pharmacological receptor antagonists.
  • Alzheimer's disease is the most common cause of dementia in old age. It afflicts approximately 5% of the population over the age of 65 years in developed parts of the world such as the U.S. and Europe (Cross, A.J., Eur. J. Pharmacol. ( 1982)
  • AD Alzheimer's disease
  • McGeer and Rogers Two cell types believed to play a role in the progression of AD, and in the promotion of the CNS inflammatory response, are microglia and astrocytes. This assumption is based on the the fact that both cell types are associated intimately with the hallmark of AD pathology-senile plaques, and both types of cell are able to synthesise and secrete inflammatory mediators such as cytokines (e.g.
  • Tumour necrosis factor alpha [TNF- ⁇ ] interleukin- 6, [IL-6], granulocyte macrophage colony stimulating factor [GM-CSF]), gluta ate and reactive oxygen intermediates.
  • IL-6 interleukin- 6,
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Microglia are derived from macrophages which cross into the brain early in development and then (probably under the influence of astrocytes) undergo differentiation into microglia. Electrophysiological studies have shown that microglia activated by inflammatory stimuli, such as Lipopolysaccharide (LPS) and Interferon- ⁇ (IFN- ⁇ ) express an outwardly rectifying potassium channel; the opening of the potassium channel can be viewed as a marker of their activated state (Fischer et al., 1995). Recent findings suggest that ATP is able to generate outward potassium channels in microglial cells (Ischler and Kettenman, 1994; Kettenman et al., 1994; Walz et al., 1993).
  • inflammatory stimuli such as Lipopolysaccharide (LPS) and Interferon- ⁇ (IFN- ⁇ ) express an outwardly rectifying potassium channel; the opening of the potassium channel can be viewed as a marker of their activated state (Fischer et al., 1995).
  • P2Y1 receptors on astrocytes have been linked to the activation of phospholipase A2 (Bruner and Murphy, 1993) and phospholipase C (Salter and Hicks, 1995); the secretion of inflammatory eicosanoids (Bruner and Murphy, 1990) ; proliferation of astrocytes (Ciccarelli et al, 1994) and the stimulation of mitogen-activated protein Kinases (MAP-kinase) (Neary, J.T. and Zhu, Q. (1994).
  • MAP-kinase mitogen-activated protein Kinases
  • microglia and astrocytes that lead to an inflammatory condition
  • the two cell types respond differently to different stimuli and, depending on the stimuli, appear to release different inflammatory mediator; the type of response is also species dependent, for instance, human and pig microglia, unlike rodent microglia, do not produce nitric oxide.
  • astrocytes and microglia release cytokines which mutually influence their activation and proliferative state.
  • P2Y purinergic G protein coupled P2 receptor
  • the present invention provides the use of a P2Y receptor antagonist, in particular, a P2Y1 receptor antagonist, in the manufacture of a medicament for use in the treatment of CNS neurodegenerative disorders where an inflammatory component has been suggested, such as Alzheimer's disease.
  • CNS neurodegenerative disorders include multiple sclerosis, the effects of stroke, and CNS trauma (head injury).
  • the present invention provides the use of a P2Y receptor antagonist, in particular, a P2Y1 receptor antagonist, in the manufacture of a medicament for use in the treatment of peripheral demylinating diseases, such as Guillain Barre syndrome and central demylinating diseases, such as multiple sclerosis.
  • a P2Y receptor antagonist in particular, a P2Y1 receptor antagonist
  • a P2Y receptor antagonist may be identified according to the references given herein for P2Y receptor antagonists, in particular, those given for P2Y1 receptor antagonists.
  • Compounds which have been identified as P2Y1 receptor antagonists include suramin, Cibacron Blue (reactive blue 2), pyridoxal-phosphate-6-azophenyl-2',4'- disulphonic acid (PPADS), and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS).
  • the P2Y receptor antagonist may be employed in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the P2Y receptor antagonists of the present invention may be employed in conjunction with other therapeutic agents.
  • compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
  • the polypeptides or polynucleotides of the present invention is administered in an amount which is effective for treatment and/or prophylaxis of the specific indication.
  • the amounts and dosage regimens of active agent administered to a subject will depend on a number of factors such as the mode of administration, the nature of the condition being treated and the judgment of the prescribing physician.
  • the inhibitors of the invention are formulated in accordance with standard pharmaceutical practice.
  • the P2Y receptor antagonist when given orally can be formulated as a liquid, for example a syrup, suspension or emulsion, tablet, capsule or lozenge.
  • a liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
  • a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
  • a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.
  • suitable pharmaceutical carrier(s) include magnesium stearate, starch, lactose, sucrose and cellulose.
  • a composition in the form of a capsule can be prepared using routine encapsulation procedures.
  • pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
  • suitable pharmaceutical carrier(s) for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
  • Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
  • the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.
  • a typical suppository formulation comprises a compound which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
  • composition is in unit dose form such as a tablet or capsule.
  • dosage unit for oral administration contains preferably from 1 to 250 mg
  • the daily dosage regimen for an adult patient may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the P2Y receptor antagonist, the compound being administered 1 to 4 times per day.
  • the compound will normally be administered for a period of continuous therapy.
  • IL-6 interleukin-6
  • TNF- ⁇ Tumour Necrosis Factor-alpha
  • IL-l ⁇ and P2 agonists were washed with serum free media and left for at least two hours before the concomitant addition of IL-l ⁇ and P2 agonists; UTP, ATP, ADP and 2Me-S- ATP (10-7 to 10-4M).
  • the agonists were left in contact with the cells overnight and the media collected the following day.
  • Samples were stored at - 20oC until assayed for IL-6 by ELISA.
  • the data show that the P2 agonists enhanced the secretion of IL-6 from IL-l- ⁇ activated human microglia.
  • 2-Me-S-ATP (10 ⁇ to 10'4 M) was co-incubated with lOuM of the P2 antagonist PPADS.
  • the data suggest that the effect of 2-Me- S-ATP on the enhancement of IL-6 secretion is reduced by the P2 receptor antagonist, PPADS.
  • Table 5a The potentiation of GM-CSF (pg/ml)by P2 agonists from P2Y1 transfected 1321N1 astrocytoma cells activated by IL-l ⁇ (lOOpg/ml). (n values in parentheses; mean values ⁇ standard error of the mean)
  • Table 10 The potentiation of TNF- ⁇ release (pg/ml) from P2Y1 transfected 1321N1 astrocytoma cells activated by IL- ⁇ (lOOpg/ml).
  • Astrocyte cells were prepared from the brains of aborted human foetuses of- 17 week gestation. Tissue was subjected to trypsin dissociation (0.25%, 30 min) followed by mechanical disruption (trituration with a flamed fire pasteur pipette) to achieve a single cell suspension. Cells were plated onto tissue culture plastic (Nunc) and incubated at 37°C, 5% CO2 in DMEM (D-Valine), 10% Foetal Bovine Serum, 2mM Glutamine, 50U/ml Penicillin, 50ug/ml Streptomycin, for approx. 3 weeks until confluent, with the media being changed every 3-4 days.
  • Contaminating cells were removed from confluent cultures by shaking on an orbital shaker, lOOrpm, at 37°C for 4 hours. The cells still adhered to the flask could be passaged using trypsin upto 4 times and were positive for GFAP (Glial Fibrillary Acidic Protein) by immunocytochemistry.
  • GFAP Glial Fibrillary Acidic Protein
  • Cells were seeded on to 24 well plates (70,000 or 150,000 cells /well) on day 1 in 1 ml of DMEM 10% foetal calf serum and glutamine (2mM) at incubated 37°C, 5% CO2. On day 2 the cells were washed and the media replaced with serum free media (0.9mls). The cells were left for 2hrs before the addition of drugs (0.1ml). The cells were incubated overnight and the media collected the following day(24hrs). The media was harvested and assayed immediately for cytokines by ELISA or frozen at - 20°C for assessment by ELISA at a later date.
  • Human microglial cells (Clone 3) were obtained from Dr Janabi (Laboratoire de Neurovirologie et Neuroimmunologie, Universite Paris Xi, 63 rue Gabriel Peri, 94276 Le Kremlin Bicetre Cedex, France) and maintained in Dulbecco's modified Eagle's medium (DMEM plus 2mM glutamine; 10% heated inactivated FCS (Gibco) and penicillin/streptomycin).
  • DMEM plus 2mM glutamine 10% heated inactivated FCS (Gibco) and penicillin/streptomycin
  • DMEM plus 2mM glutamine 10% heated inactivated FCS (Gibco) and penicillin/streptomycin
  • cells were plated into 24 well Nunc plates at a density of 5x1 ⁇ cells/well for 24hrs, and changed into serum free media 2hrs before the addition of IL-l ⁇ 500pg/ml and purinergic agonists.
  • P2Y1 receptor human transfected 1321N1 astrocytoma
  • the cells were maintained in DMEM containing 5% foetal calf serum,
  • G418 (600ug/ml) and glutamine (2mM).
  • the cells were plated into 24 well plates, 150,000 cells/ well on day 1. On day 2 the medium was replaced with fresh medium (0.4mls). The cells were left for 2hrs before the addition of compound (0.05ml) and vehicle control (0.05ml). For antagonist studies, the compound was added lhr prior to the agonist. The cells were incubated overnight and the media collected the following day (24hrs). The media was harvested and assayed immediately for cytokines by ELISA or frozen at -20°C for assessment by ELISA at a later date. References
  • Oxidized ATP An irreverisible inhibitor of the macrophage P2Z receptor. J-Biol-Chem.; 268(11): 8199-8203.:

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Abstract

The use of a P2Y receptor antogonist, in particular, a P2Y1 receptor antagonist, in the manufacture of a medicament for use in the treatment of CNS neurodegenerative disorders where an inflammatory component has been suggested (such as Alzheimer's disease) or peripheral demylinating diseases, such as Guillain Barre syndrome and central demylinating diseases, such as multiple sclerosis.

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING P2Y PURINERGIC RECEPTOR ANTAGONISTS
The present invention relates to the therapeutic use of compounds which act as pharmacological receptor antagonists. Alzheimer's disease is the most common cause of dementia in old age. It afflicts approximately 5% of the population over the age of 65 years in developed parts of the world such as the U.S. and Europe (Cross, A.J., Eur. J. Pharmacol. ( 1982)
82: 77-80; Terry, R.D.. et al., Ann. Neurol. (1983) 14: 497-506) where it is estimated be the fourth most common cause of death after heart disease, cancer and stroke. There are currently around 20 million patients worldwide with this disease (Schenk, D.B.. et al., J. Med. Chem. (1995) 38: 4141-4154), the consequences of which are devastating to the individuals and families involved. In the final stages of Alzheimer's disease, the patients are severely demented, and often completely unable to speak, or to take care of themselves. There are presently no treatments available that are directed at the underlying causes of Alzheimer's disease.
Based on retrospective studies with anti-inflammatory agents and immunocytochemistry (ICC) pathological studies, there is an increasing evidence to suggest that Alzheimer's disease (AD) is the manifestation of a chronic inflammatory response of the brain (McGeer and Rogers, 1992 ). Two cell types believed to play a role in the progression of AD, and in the promotion of the CNS inflammatory response, are microglia and astrocytes. This assumption is based on the the fact that both cell types are associated intimately with the hallmark of AD pathology-senile plaques, and both types of cell are able to synthesise and secrete inflammatory mediators such as cytokines (e.g. Tumour necrosis factor alpha, [TNF-α] interleukin- 6, [IL-6], granulocyte macrophage colony stimulating factor [GM-CSF]), gluta ate and reactive oxygen intermediates. In addition, the pro- inflammatory role of microglia and astrocytes has been implicated in other CNS inflammatory disorders such as stroke and multiple sclerosis.
Microglia are derived from macrophages which cross into the brain early in development and then (probably under the influence of astrocytes) undergo differentiation into microglia. Electrophysiological studies have shown that microglia activated by inflammatory stimuli, such as Lipopolysaccharide (LPS) and Interferon- γ (IFN-γ) express an outwardly rectifying potassium channel; the opening of the potassium channel can be viewed as a marker of their activated state (Fischer et al., 1995). Recent findings suggest that ATP is able to generate outward potassium channels in microglial cells (Ischler and Kettenman, 1994; Kettenman et al., 1994; Walz et al., 1993). The promotion of the outward K+ channel in microglial cells by GTPγS, and its enhancement by ATP, suggests that ATP may promote outward channel opening through activation of a G-protein- coupled receptor. Limited pharmacological evidence suggests that the effect of ATP on microglia is mediated via a P2 purinergic receptor, which is most probably of the P2Y1 subtype (Norenberg et al., 1994; Langosch, J.M. et al 1994).
The effect of P2 purinergic receptors on astrocytes have been described. P2Y1 receptors on astrocytes have been linked to the activation of phospholipase A2 (Bruner and Murphy, 1993) and phospholipase C (Salter and Hicks, 1995); the secretion of inflammatory eicosanoids (Bruner and Murphy, 1990) ; proliferation of astrocytes (Ciccarelli et al, 1994) and the stimulation of mitogen-activated protein Kinases (MAP-kinase) (Neary, J.T. and Zhu, Q. (1994).
The interactions between microglia and astrocytes, that lead to an inflammatory condition, have not been fully elucidated. The two cell types respond differently to different stimuli and, depending on the stimuli, appear to release different inflammatory mediator; the type of response is also species dependent, for instance, human and pig microglia, unlike rodent microglia, do not produce nitric oxide. In addition astrocytes and microglia release cytokines which mutually influence their activation and proliferative state.
It is believed that ATP, released from compromised cells, activates microglial and/or astrocytes via P2Y receptors and leads directly to the release of inflammatory mediators or enhances/potentiates actions of other inflammatory mediators. The purinergic G protein coupled P2 receptor (P2Y) subtype on microglia has not, thus far, been identified on other inflammatory cell types (see table below). The P2Y approach potentially therefore offers central versus peripheral selectivity of action. The latter should be a property of any potential treatment for neurodegenerative disorders since the objective of this inflammatory approach is to halt the neurodegeneration without compromising the patient's immunological capability. Inflammatory cell type P2 receptor subtype References
Macrophages P2Z(P2X7), P2Y2 Murgia et al., 1993; Greenberg et al., 1988 lymphocytes P2Z(P2X7) Wiley et al., 1994 neutrophils P2Y2 Merritt and Moores, 1991 microglia P2Y1 Norenberg et al., 1994; Langosch,. et al 1994 astrocytes P2Y1 , P2Y2 Bruner and Murphy, 1990, 1993
Accordingly therefore, the present invention provides the use of a P2Y receptor antagonist, in particular, a P2Y1 receptor antagonist, in the manufacture of a medicament for use in the treatment of CNS neurodegenerative disorders where an inflammatory component has been suggested, such as Alzheimer's disease.
Other CNS neurodegenerative disorders include multiple sclerosis, the effects of stroke, and CNS trauma (head injury).
The present invention provides the use of a P2Y receptor antagonist, in particular, a P2Y1 receptor antagonist, in the manufacture of a medicament for use in the treatment of peripheral demylinating diseases, such as Guillain Barre syndrome and central demylinating diseases, such as multiple sclerosis.
A P2Y receptor antagonist may be identified according to the references given herein for P2Y receptor antagonists, in particular, those given for P2Y1 receptor antagonists. Compounds which have been identified as P2Y1 receptor antagonists include suramin, Cibacron Blue (reactive blue 2), pyridoxal-phosphate-6-azophenyl-2',4'- disulphonic acid (PPADS), and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS).
It will be appreciated that the use of antagonists of P2 Y G-protein coupled receptors other than P2Y1 are also included as an aspect of this invention.
The P2Y receptor antagonist may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutical ly effective amount of the active agent, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the P2Y receptor antagonists of the present invention may be employed in conjunction with other therapeutic agents.
The pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The polypeptides or polynucleotides of the present invention is administered in an amount which is effective for treatment and/or prophylaxis of the specific indication. The amounts and dosage regimens of active agent administered to a subject will depend on a number of factors such as the mode of administration, the nature of the condition being treated and the judgment of the prescribing physician. When used in therapy, the inhibitors of the invention are formulated in accordance with standard pharmaceutical practice.
The P2Y receptor antagonist when given orally can be formulated as a liquid, for example a syrup, suspension or emulsion, tablet, capsule or lozenge.
A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule. Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration. A typical suppository formulation comprises a compound which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
Preferably the composition is in unit dose form such as a tablet or capsule. Each dosage unit for oral administration contains preferably from 1 to 250 mg
(and for parenteral administration contains preferably from 0.1 to 25 mg) of the active ingredient.
The daily dosage regimen for an adult patient may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the P2Y receptor antagonist, the compound being administered 1 to 4 times per day. The compound will normally be administered for a period of continuous therapy.
The following biological test results and methods illustrate the invention.
Biological test results
The effect of P2 agonists on IL-lα-induced IL-6 secretion from human immortalised microglia
A number of immortalised human microglial cell lines generated by transformation of foetal microglial by S V40 were described by Janabi et al, 1995. The microglia had the characteristics of macrophages, but lacked the monocytic markers for CD 14, CD4, CD68/Ki-M6 and CD1 lc. In addition, the constitutive production of interleukin -6 (IL-6) could be increased by interleukin- lα (IL-lα) stimulation; however, the cells did not produce Tumour Necrosis Factor-alpha (TNF-α) or IL-6 in response to lipopolysaccharide. During routine screening by PCR we found cultures of clone 3 to be infected with mycoplasma fermentans (classified by Mycoplasma Experience, U.K.) and the high constitutive secretion of IL-6 could be ascribed to the infection. Nonetheless, following the removal of the infection, the cells still secreted IL- 6 in response to IL-lα. (fig 1 and 2). Using the mycoplasma minus cell line, we observed the effects of P2 agonists on the secretion of IL-6-induced by IL- lα [500pg/ml (Sigma)]. Nunc 24 well plates were seeded with 70,000 cells/well of clone 3 microglial cells . Twenty four hours later, the cells were washed with serum free media and left for at least two hours before the concomitant addition of IL-lα and P2 agonists; UTP, ATP, ADP and 2Me-S- ATP (10-7 to 10-4M). The agonists were left in contact with the cells overnight and the media collected the following day. Samples were stored at - 20oC until assayed for IL-6 by ELISA. The data show that the P2 agonists enhanced the secretion of IL-6 from IL-l-α activated human microglia. In two subsquent studies, 2-Me-S-ATP (10~ to 10'4 M) was co-incubated with lOuM of the P2 antagonist PPADS. The data suggest that the effect of 2-Me- S-ATP on the enhancement of IL-6 secretion is reduced by the P2 receptor antagonist, PPADS.
Figure imgf000009_0001
1000 300 100 30 IL-1 alpha (pg/ml)
Fig l .
The induction of Il-6 secretion from clone 3 mycoplasma fermentans (plus) immortalised microglia cells by IL-lα. The data represent the mean of two determinations from a single experiment.
Figure imgf000009_0002
1000 500 250 125 IL-1a (pgM)
Fig 2.
The induction of 11-6 secretion from clone 3 mycoplasma (minus) immortalised microglia cells by Il-lα. The data represent the mean (± S.E.M.) of three separate experiments The effect of P2 agonists on IL-6 secretion from Clone 3 immortalised human microglia
Cone Mean Std Mean Std. Mean Std. Mean Std. M UTP. Dev. ATP Dev. ADP Dev. 2-me-S- Dev.
Mean Mean Mean ATP Mean
10-4 204.57 52.30 259.28 61.50 248.58 65.29 309.80 63.20
10-5 203.41 20.21 249.10 37.32 223.81 23.55 21 1.06 35.17
10-6 180.74 31.04 210.13 64.30 127.20 19.10 133.66 24.46
10-7 183.60 38.09 176.81 46.64 156.42 27.49 135.47 6.42
Data represent the mean % increase above IL-lα .500pg/ml) stimulated IL-6 secretion from human immortalised microglia cells (n=3) induced by the P2 agomsts UTP, ATP, ADP and 2-Me-0S-ATP (10'7 to 10'4 M).
The mean (± S.E.M.) increase in IL-6 induced by IL-lα(500pg/ml) was 535.6 ± 79.84 pg/ml (n=7).
DAY Il-lbeta Il-lbeta (2ng/ml)+ (2ng ml) 2MeS-ATP(10-6M)
1 108.303 195.606
2 87.382 241.003
5 81.949 131.31 1
6 164.394 236.335
7 191.585 195.55
8 161.545 225.048
Table la. The effect of 2me-S-ATP (10-6° M) on IL-lβ( 2ng/ml) -induced release of GM-CSF (pg/ml) from human foetal astrocytes on days 1-8 after the start of incubation. 150,000 cells/ well.(n=l , values quoted in pg/ml). Cell media collected on the days indicated above. Samples were frozen and assayed for IL-6 and GM-CSF by ELISA.
DAY Il-lbeta Il-lbeta (2ng/ml)+
(2ng/ml) 2MeS-ATP(10-6M)
1 78.7 212.837
2 236.78 559.108
5 379.04 793.092
6 578.634 600
7 792.93 690.705
8 751.522 721.923
Table lb. The effect of 2me-S-ATP (106 M) on IL-lβ( 2ng ml) -induced release of IL-6 (pg/ml) from human foetal astrocytes on days 1-8 after the start of incubation. 150,000 cells/ well.(n=l, values quoted in pg/ml). Cell media collected on the days indicated above. Samples were frozen and assayed for IL-6 by ELISA. CONC (M) 2-Me-S-ATP UTP α,β-me-ATP
10-5 220.87 179.48 166.38
10-6 223.41 173.34 157.15
10-7 203.39 158.51 158.72
0 157.72 156.42 174.01
Table 2. Potentiation of Il-lβ (2ng/ml)-induced GM-CSF (pg/ml) release from human foetal astrocytes by 2me-S-ATP, UTP and α, β-me-ATP. 150,000 cells/ well.(n=l, values quoted in pg/ml). Cell media collected after 24hrs samples were frozen and assayed for GM-CSF by ELISA.
GM-CSF (pg ml)
Concentration (M) 2-me-S-ATP ADP UTP ATP
10-4 240.54 288.44 286.25 269.80
10"3 244.49 235.82 171.1 1 234.09
10-O 204.03 220.61 193.22 202.44 lo-/ 230.53 158.31 208.04 174.96
Table 3. The effect of P2 agonists, 2-Me-S-ATP, ADP, UTP and ATP on GM- CSF release from human foetal astrocytes activated by IL-lβ, 2ng/ml (70,000 cells/well, n=2). The mean ( ± S.E.M.) concentration (pg/ml) of GM-CSF produced by IL-lβ in the absence of P2 agonists was 156.33 ± 15.22 (n=8 determinations) ; the values ranged from 87 to 214 pg/ml.
Figure imgf000013_0001
Table 4. The effect of PPADS (lOuM) on ATP and ADP-induced increases in GMCSF release from primary human astrocytes cultures activated by IL-lbeta (2ng/ml). Values represent the mean of two experiments.
Collectively, the data suggest that the effect of the P2 agonists on the Il-beta- induced GM-CSF (and IL-6) secretion from human astrocytes-may be mediated through a P2Y receptor. This is-supported by the following experiments with 1321N1 astrocytoma cells transfected with the human P2Y1 receptor, where evidence for at least the involvement of a P2Y1 receptor in this mechanism is implied.
Effect of P2Y agonist on cytokine release in human P2Y1 transfected 1321N1 astrocytoma cells
The effect of P2Y agonists on the release of cytokines from P2Y1 (human) transfected 1321N1 astrocytoma cells (obtained under licence from the University of North Carolina) was investigated. 2 me-S-ATP, ATP, α-β-me-ATP and UTP (10-7M to 10-4M), produced concentration-related enhancement of the release of GM-CSF and IL-6 induced by IL-Iβ(100pg/ml) from hP2Yl transfected 1321N1 atrocytoma cells (Tables 5a, b) ; 2me-S-ATP and ATP also induced increases in TNF- α release across the same concentration range (Table 10). 2-me-S-ATP and ATP appeared to produce increases in GM-CSF release at ~10X lower concentrations than those required to increase IL-6 and TNF-α release. This suggests that the effect of the purinergic agonists on IL-6 secretion may be mediated indirectly through the release of another cytokine such as GM-CSF. In addition, 2me-S-ATP and ATP also induced much smaller increases in GM-CSF secretion, per se, without prior activation by IL- 1 β ( Table 9). The release of IL-6 and TNF-α from the transfected P2Y1 cells was observed only in the presence of the the P2Y agonists; Il-β had no effect on basal release of IL-6 and TNF-α cytokines. In contrast, IL-lβ (lOOpg/ml) induced the release of approximately lOOpg/ml of GM-CSF.
The rank order of potency for IL-6 and GM-CSF release, 2me-S- ATP> ATP>=α,β-me- ATP>UTP, is indicative of a P2 Y 1 receptor mediated effect. These data are supported by the following facts: P2Y agonists had no effect on IL-lβ stimulated cytokine release in the wild type 1321N1 astrocytoma cells; 2-me-S-ATP - induced potentiation of IL-1 β mediated cytokine release (GM-CSF, IL-6) was inhibited by the non-selective P2 antagonists, suramin (lOOuM), cibacron blue (lOOuM) and DIDS (lOOuM) (Tables 6a,b). This was confirmed in a subsequent study where suramin (10-100uM) and DIDS (10-100uM) produced concentration related inhibition of the 2-me-S-ATP effect (Tables. 7a, 7b, 8a, 8b). CONC 2me-S-ATP ATP UTP a,b-me-ATP
(M) (4) (4) (2) (1)
10-4 239.5± 10.1 251.5 ± 12.9 1 1 1.072 160.282
10-5 181.4 ± 21.3 102.9± 28.0 5.2955 1 10.828
10-6 107.1 ± 44.5 3.1 ±2.6 0.038 2.596
10-7 33.9 ± 29.5 0.09±0.09 7.87 0.00
Table 5a. The potentiation of GM-CSF (pg/ml)by P2 agonists from P2Y1 transfected 1321N1 astrocytoma cells activated by IL-lβ (lOOpg/ml). (n values in parentheses; mean values ± standard error of the mean)
CONC (M) 2-Me-ATP ATP TP α,β-me-ATP
(4) (3) (1) (1)
10-4 179.45± 68.6 161.39±93.73 0.52 16.81
10-5 57.0 ±12.8 3.24±3.05 0.00 8.87
10-6 6.0 ±2.8 O.OO±O.OO 0.00 0.00
10-7 0.00 2.27±2.27 0.00 0.00
Table 5b. The potentiation of IL-6 release (pg/ml) by P2 agonists from P2Y1 transfected 1321N1 astrocytoma cells activated by IL-lβ (100pg/ml)(n values in parentheses;mean values ± standard error of the mean)
CONC (M) 2me-s-ATP Cibacron Suramin DIDS blue
10-4 304.87 143.85 36.51 98.05
10-5 302.02 185.13 45.61 150.37
10-6 201.57 87.01 45.45 154.40
10-7 94.36 61.90 25.83 76.11
0 78.21 30.75 17.00 58.18
Table 6a. T le inhibition of 2-Me-S-ATP mediatec increase in GM
(pg/ml)release from P2Y1 transfected 1321N1 astrocytoma cells by Cibacron blue (lOOum), suramin (lOOuM) and DIDS (100uM)(n=l).
CONC (M) 2-me-s- Cibacron Suramin DIDS ATP blue
10-4 83.40 15.47 2.15 2.64
10-5 21.31 1 1.42 0.00 0.00
10-6 2.32 2.33 0.00 0.00
10-7 0.00 0.00 0.00 0.00
Table 6b The inhibition of 2-Me-S-ATP mediated increase in IL-6 release (pg/ml)from P2Y1 transfected 1321N1 astrocytoma cells by Cibacron blue (lOOum), suramin (lOOuM) and DIDS (lOOuM) (n=l).
CONC (M) 0 lOuM 33uM lOOuM
10-4 124.83 1 18.39 74.40 6.48
10-5 1 1 1.36 100.77 64.72 8.06
10-6 51.58 52.47 36.63 11.27
10-7 27.32 25.00 22.77 5.83
0 18.60 33.35 16.32 5.70 Table 7a Effect of suramin (10-100uM) on 2-me-S-ATP (10-7M-10-4M)-induced potentiation of GM-CSF release( pg/ml ) from P2Y1 transfected 1321N1 astrocytoma cells activated by IL-lβ (lOOpg/ml). CONC (M) 0 lOuM 33uM lOOuM
10-4 142.86 104.22 87.74 30.55
10-5 136.37 121.35 92.99 46.20
10-6 63.12 42.72 46.80 23.87
10-7 27.90 36.44 38.97 14.24
0 16.01 34.40 32.34 13.35
Table 7b Effect of DIDS (10-100uM) on 2-me-S-ATP (10-7M-10-4M)-induced potentiation of GM-CSF release( pg/ml ) from P2Y1 transfected 1321N1 astrocytoma cells activated by IL-lβ (lOOpg/ml) (n=l).
CONC(M) 2me-SATP lOuM 33uM lOOuM
10-4 71.63 49.08 18.18 3.59
10-5 12.43 7.38 5.79 1.82
10-6 0.00 8.37 1.41 0.00
10-7 0.00 0.00 1.92 0.00
0 0.00 0.00 0.00 0.00
Table 8a. Effect of suramin (10-100uM) on 2-me-S-ATP (10-7M-10-4M)-induced potentiation of IL-6 release( pg/ml ) from P2Y1 transfected 1321N1 astrocytoma cells activated by IL-lβ (lOOpg/ml) (n=l).
CONC(M) 0 lOuM 33uM lOOuM
10-4 79.06 52.70 17.93 3.65
10-5 1 1.65 5.45 0.56 0.00
10-6 0.00 0.00 0.00 0.00
10-7 0.00 0.00 0.00 0.00
0 0.00 0.00 0.00 0.00 Table 8b. Effect of DIDS (10-100uM) on 2-me-S-ATP (10-7M-10-4M)-induced potentiation of IL-6 release( pg ml ) from P2Y1 transfected 1321N1 astrocytoma cells activated by IL-lβ (lOOpg/ml) (n=l). CONC(M) 2-me-S-ATP ATP
10-4 1 1.10 9.53
10-5 9.35 1.85
10-6 4.75 0.00
10-7 0.00 1.30
Table 9. The effect of 2-Me-S-ATP and ATP, per se, on GM-CSF(pg/mI) release from P2Y1 transfected 1321N1 astrocytoma cells( Values quoted arc the mean of two experiments)
CONC(M) 2-me-S-ATP ATP
10-4 21.22 20.38
10-5 8.84 0.00
10-6 0.26 0.00
10-7 0.00 0.00
Table 10. The potentiation of TNF-α release (pg/ml) from P2Y1 transfected 1321N1 astrocytoma cells activated by IL-β (lOOpg/ml).
Biological test methods
Human foetal astrocyte cell preparation
Astrocyte cells were prepared from the brains of aborted human foetuses of- 17 week gestation. Tissue was subjected to trypsin dissociation (0.25%, 30 min) followed by mechanical disruption (trituration with a flamed fire pasteur pipette) to achieve a single cell suspension. Cells were plated onto tissue culture plastic (Nunc) and incubated at 37°C, 5% CO2 in DMEM (D-Valine), 10% Foetal Bovine Serum, 2mM Glutamine, 50U/ml Penicillin, 50ug/ml Streptomycin, for approx. 3 weeks until confluent, with the media being changed every 3-4 days. Contaminating cells were removed from confluent cultures by shaking on an orbital shaker, lOOrpm, at 37°C for 4 hours. The cells still adhered to the flask could be passaged using trypsin upto 4 times and were positive for GFAP (Glial Fibrillary Acidic Protein) by immunocytochemistry.
Primary culture experiments
Cells were seeded on to 24 well plates (70,000 or 150,000 cells /well) on day 1 in 1 ml of DMEM 10% foetal calf serum and glutamine (2mM) at incubated 37°C, 5% CO2. On day 2 the cells were washed and the media replaced with serum free media (0.9mls). The cells were left for 2hrs before the addition of drugs (0.1ml). The cells were incubated overnight and the media collected the following day(24hrs). The media was harvested and assayed immediately for cytokines by ELISA or frozen at - 20°C for assessment by ELISA at a later date.
METHODS
Human microglial cells (Clone 3) were obtained from Dr Janabi (Laboratoire de Neurovirologie et Neuroimmunologie, Universite Paris Xi, 63 rue Gabriel Peri, 94276 Le Kremlin Bicetre Cedex, France) and maintained in Dulbecco's modified Eagle's medium (DMEM plus 2mM glutamine; 10% heated inactivated FCS (Gibco) and penicillin/streptomycin). For all studies, cells were plated into 24 well Nunc plates at a density of 5x1 θ cells/well for 24hrs, and changed into serum free media 2hrs before the addition of IL-lα 500pg/ml and purinergic agonists. Supernatants were harvested ~18hrs later and assayed for IL-6 using a modified luminescent ELISA described by Carlson and Aschimies , 1995. The ELISA has a detection limit of < 2.4pg/ml and is linear up to 2500pg/ml.
P2Y1 receptor (human) transfected 1321N1 astrocytoma The cells were maintained in DMEM containing 5% foetal calf serum,
G418(600ug/ml) and glutamine (2mM). The cells were plated into 24 well plates, 150,000 cells/ well on day 1. On day 2 the medium was replaced with fresh medium (0.4mls). The cells were left for 2hrs before the addition of compound (0.05ml) and vehicle control (0.05ml). For antagonist studies, the compound was added lhr prior to the agonist. The cells were incubated overnight and the media collected the following day (24hrs). The media was harvested and assayed immediately for cytokines by ELISA or frozen at -20°C for assessment by ELISA at a later date. References
Bruner G and Murphy, S. (1990) ATP-evoked arachidonic acid mobilization in astrocytes is via a P2Y-purinergic receptor. J.Neurochem. 55,5,1569-1575.
Bruner G and Murphy, S.(1993) Purinergic P2Y receptors on astrocytes are directly coupled to phospholipase A2. Glia, 7, 219-224.
Ciccarelli, R., Di Iorio, P., Ballerini, P., Ambrosini, G., Giuliani, P., Tiboni, G.M. and Caciagli, F. (1994) Effects of exogenous ATP and related analogues on the proliferation rate of dissociated primary cultures of Rat astrocytes. J. Neurosci.Res. 39, 556-566 Fischer H.-G., Eder, C, Hadding, U. and Heinemann, U. (1995) Cytokine-dependent K+ channel profile of microglia at immunologically defined functional states. Neuroscience, 64, 1 , 183-191.
Greenberg S., Virgilio, F.D, Steinberg, T.H. and Silverstein, S.C.(1988) Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms. J. Biol.Chem., 263, 21, 10337-10343.
Ischler S. and Kettenmann, H. (1994) Regulation of K+-currents in cultured mouse microglia by ATP and TNF by Ca2+ and G proteins. Soc Neurosci abs abstract no. 305.16
Janabi, N., Peudenier, S., Heron, B., Ng, K.H. and Tardieu, M. (1995) Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen. Neurosci-Lett. 195(2): 105-8
Kettenmann H., Banati, R. and Walz, W. (1993) Electrophysiological Behaviour of
Microglia. Glia 7: 93-101
Langosch, J.M., Gebicke-Haerter, P.J., Norenberg, W. and Illes, P. (1994) Characterization and transduction mechanisms of purineroceptors in activated rat microglia. Br. J. Pharmacol., 113, 29-34.
Lee, S.C., Liu, W., Dickson, D.W., Brosnan, C.F. and Berman, J.W. (1993) Cytokine production by human fetal microglia and astrocytes: Differntial induction by lipopolysacchride and IL-l β. J. Immunol, 150, 2659-2667 McGeer P.L. Rogers, J. (1992) Anti-inflammatory agents as a therapeutic approach to
Alzheimer's disease. Neurology. 42. 447-449 Merritt J.E. and Moores, K.E. (1991) Human neutrophils have a novel purinergic P2-type receptor linked to calcium mobilization. Cell Signal 3, 3, 243-249.
Murgia M., Hanaus, S., Pizzo, P., Rippa, M. and Virgilio, F.D.. (1993) Oxidized ATP: An irreverisible inhibitor of the macrophage P2Z receptor. J-Biol-Chem.; 268(11): 8199-8203.:
Neary, J.T. and Zhu, Q. (1994) Signaling by ATP receptors in astrocytes . Neuroreport 5, 1617-1620
Norenberg W., Langosch, J.M., Gebicke-Haerter, P.J and Illes, P.(1994) Characterization and possible function of adenosine 5'-triphosphate receptors in activated rat microglia. Br. J. Pharmacol. I l l, 942-950
Salter M.W. and Hicks, J.L.(1995) ATP causes release of intracellular Ca2+ via the phospholipase Cb/IP3 pathway in astrocytes from the dorsal spinal cord. J. Neurosci., 15, 2961-2971.
Wiley J.S., Chen, J.R., Snook, M.B., and Jamieson, G.P. (1994) The P2Z- purinoreceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidised ATP. Br. J. Pharmacol., 112, 946-950.

Claims

Claims
1. The use of a P2Y receptor antagonist, in the manufacture of a medicament for use in the treatment of CNS neurodegenerative disorders where an inflammatory component has been suggested.
2. A method of treatment of CNS neurodegenerative disorders where an inflammatory component has been suggested, which method comprises the administration of a P2Y receptor antagonist.
3. A pharmaceutical composition for use in the treatment of CNS neurodegenerative disorders where an inflammatory component has been suggested, which comprises a P2Y receptor antagonist and a pharmaceutical carrier.
4. A use, method, or composition acccording to claim 1, 2, or 3, wherein the neurodegenerative disorder is Alzheimers disease.
5. The use of a P2 Y receptor antagonist, in particular, a P2 Y 1 receptor antagonist, in the manufacture of a medicament for use in the treatment of peripheral demylinating diseases and central demylinating diseases.
6. A method of treatment of peripheral demylinating diseases and central demylinating diseases, which method comprises the administration of a P2 Y receptor antagonist.
7. A pharmaceutical composition for use in the treatment of peripheral demylinating diseases and central demylinating diseases which comprises a P2Y receptor antagonist and a pharmaceutical carrier.
8. A use, method, or composition acccording to claim 5, 6, or 7, wherein the peripheral demylinating disease is Guillain Barre syndrome and the central demylinating disease is multiple sclerosis.
9. A use, method, or composition acccording to claim 1 to 8, wherein the P2Y receptor antagonist is a P2Y1 receptor antagonist.
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