JP2002542280A - New treatment - Google Patents

Abstract

  (57) [Summary] Use of a Raf kinase inhibitor in the treatment of a neurotraumatic disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to the use of Raf kinase inhibitors in the treatment of neurotraumatic diseases.

[0002] Raf protein kinases are an important component of the signal transduction pathway, whereby certain extracellular stimuli elicit precise cellular responses in mammalian cells. The activated cell surface receptor activates ras / rap proteins on the inner surface of the plasma membrane,
It in turn recruits and activates the Raf protein. The activated Raf protein phosphorylates and activates intracellular protein kinases MEK1 and MEK2. Activation M
EK in turn catalyzes the phosphorylation and activation of p42 / p44 mitogen-activated protein kinase (MAPK). Various cytoplasmic and nuclear substrates of activated MAPK that contribute directly or indirectly to cellular responses to environmental changes are known. Raf protein; three different genes encoding A-Raf, B-Raf and C-Raf (also known as Raf-1) have been identified in mammals and obtained from differential splicing of mRNA. Isoform variants are known. Inhibitors of Raf kinase inhibit the growth of tumor cells and therefore cancer,
For example, it has been suggested to be useful in the treatment of histiocytic lymphoma, lung adenoma, small cell lung cancer, and pancreatic and breast cancer.

[0003] Disclosure of the invention [0003] Raf inhibitors have recently been associated with neurodegenerative disorders resulting from ischemic events, including cerebral ischemia after heart arrest, stroke and multiple infarct dementia, as well as surgery. And / or has been found to be useful in the treatment and / or prevention of disorders during childbirth. Thus, according to the present invention, there is provided a method of treating or preventing a neurotraumatic disease in a mammal, comprising administering to said mammal an effective amount of a Raf inhibitor. According to the present invention there is provided the use of a Raf inhibitor in the manufacture of a medicament for the prophylactic or therapeutic treatment of a condition, exacerbated or caused by a neurotraumatic event, in a human or non-human mammal.

[0004] The Raf inhibitor for use in the present invention is preferably a B-Raf inhibitor. Neurotraumatic diseases / events defined herein are both open or penetrating head trauma as caused by surgery, or non-open head trauma as caused by damage to the head area. Is included. In addition, ischemic stroke, especially ischemic stroke to the brain, transient ischemic stroke after coronary artery bypass and cognitive decline after other transient ischemic conditions are also included within this definition. Transient seizures can be defined as localized neurological disorders, usually resulting from an inadequate supply of blood to a particular brain, as a result of vascular embolism, thrombus or local atherosclerosis. Stressful stimuli in this area (eg oxygen starvation)
The role for redox damage, excessive neuroexcitatory stimulation and inflammatory cytokines has been identified, and the present invention provides potential means of treating these damages. Relatively little treatment was available for such acute injuries.

[0005] A model of non-open head injury and treatment with mixed 5-LO / CO drugs has been described by Shoham
i et al., J. Vaisc. & Clinical Physiology and Pharmacology (1992), 3 (
2), 99-107. Raf inhibitors suitable for use in the methods of the invention include small molecules and peptides. The definition of a Raf inhibitor also encompasses antisense constructs that reduce the expression of Raf kinase in target cells. Small molecule Raf inhibitors include GB2306108, WO98 / 22103, W
O98 / 50370, WO99 / 10325 and those described in WO99 / 17759. A particular Raf inhibitor that may be mentioned is 4- (4-chloro-3-hydroxyphenyl) -2-phenyl-5- (4-pyridyl) -1H-imidazole. The use of this compound for the treatment of Raf-mediated cancer is described in Poster 3793, Ame
rican Association of Cancer Research, New Orleans (April 1998).

[0006] Antisense Raf inhibitors are described in WO 97/10829, US Pat.
12, WO 99/02167, U.S. Pat. No. 5,872,232 and WO 96/02.
39415. To use a Raf inhibitor for therapy, the Raf inhibitor will usually be formulated into a pharmaceutical composition according to standard pharmaceutical procedures.

[0007] The inhibitors are conveniently administered by the routes normally used for drug administration, for example, parenterally, orally, topically or by inhalation. The inhibitor can be administered in conventional dosage forms prepared by combining it with standard pharmaceutical carriers by conventional procedures. Inhibitors can also be administered in conventional dosage forms in combination with other known therapeutically active compounds. These operations include mixing, granulating, compressing, or dissolving the ingredients as appropriate to the desired formulation. The form and properties of a pharmaceutically acceptable carrier are determined by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The carrier must be "acceptable" in the sense that it is compatible with the other ingredients in the formulation and is not harmful to the subject.

[0008] The pharmaceutical carrier used may be, for example, either a solid or liquid. Examples of the solid carrier include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Liquid carriers include, for example, syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include a time delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. A wide variety of pharmaceutical forms are available. That is, if a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule, in powder or pellet form, or in the form of a troche or lozenge. The amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation will be in the form of a sterile injectable liquid or non-aqueous liquid suspension such as a syrup, emulsion, soft gelatin capsule, ampoule or the like.

The inhibitor is preferably administered parenterally, ie, intravenously, intramuscularly, subcutaneously, intranasally, rectally, vaginally or intraperitoneally. Intravenous forms for parenteral administration are generally preferred. Suitable dosage forms for such administration can be prepared by conventional techniques. The inhibitors can also be administered orally. Suitable dosage forms for such administration can be prepared by conventional techniques. Inhibitors can also be administered by inhalation, ie, by nasal and buccal inhalation. Suitable dosage forms for such administration, such as an aerosol formulation, can be prepared by conventional techniques. Inhibitors can also be administered locally, ie, by non-systemic administration. This includes external application of the inhibitor to the epidermis or mouth and injection of such compounds into the ears, eyes and nose so that the compounds do not significantly enter the bloodstream.

For all uses disclosed herein for inhibitors, the daily oral dosage is preferably from about 0.1 to about 80 mg / kg body weight, preferably from about 0.1 to about 80 mg / kg body weight. 2 to about 30 mg / kg, more preferably about 0.5 to about 30 mg / kg.
mg to 15 mg / kg. The daily topical dose is preferably 0.1 m
g to 150 mg, administered 1 to 4 times, preferably 2 or 3 times a day. The daily inhalation dosage is preferably from about 0.01 mg / kg to about 1 mg / kg per day. The skilled artisan will determine the optimal amount and interval of individual administration of the inhibitor which will depend on the nature and extent of the condition to be treated, the form, route and site of administration, and the individual patient to be treated. Optimal conditions can be determined by conventional techniques. The skilled artisan will be able to ascertain the optimal course of treatment, i.e., the prescribed number of days, the number of doses of the inhibitor to be administered per day, using routine treatment course determination tests.

[0011] Without limitation, including, but not limited to, the patents and patent applications cited herein,
All publications are incorporated herein by reference. The present invention will be described with reference to the following examples, which are merely illustrative and do not limit the scope of the present invention.

Examples 4- (4-chloro-3-hydroxyphenyl) -2-phenyl-5- (4-pyridyl) -1H-imidazole a) 4- (4-chloro-3-methoxyphenyl)- 2-Phenyl-5- (4-pyridyl) -1H-imidazole The title compound was converted from 4-chloro-3-methoxybenzoic acid via its acid chloride via TFGallagher et al., Bioorg. Med. Chem. (1997). Prepared by a method analogous to the five-step procedure described in 5, 49 (General Method B). MS m / z
362/364 MH ^<+> (electrospray).

B) 4- (4-Chloro-3-hydroxyphenyl) -2-phenyl-5- (4-pyridyl) -1H-imidazole Boron tribromide (5.6 ml, 1 M in dichloromethane, 5.6 mmol) ) Was converted to the product of step a) (670 mg, 1.85 mmol) in dichloromethane (20 ml)
) Added to medium ice cold solution. The solution was warmed to ambient temperature and stirred for another 16 hours. 5M hydrochloric acid (5ml) was added and the mixture was heated at reflux for 30 minutes. After cooling, the solution was made alkaline with 40% aqueous sodium hydroxide and the solid product was collected by filtration. The residue was washed with ethyl acetate to give the title compound as a yellow solid: MS m / z
348/350 MH + (electronic injection).

Biological Examples The activity of compounds as Raf inhibitors was determined by the following in vitro assay.

Raf Kinase Assay The activity of human recombinant B-Raf protein was determined in vitro by an assay that incorporates radiolabeled phosphate into recombinant MAP kinase (MEK), a known physiological substrate of B-Raf. evaluated. By purifying sf9 insect cells infected with the human B-Raf recombinant baculovirus expression vector, a catalytically active human recombinant B-Raf protein was obtained. A catalytically inactive form of MEK was utilized to ensure phosphorylation of all substrates derived from B-Raf activity. This protein was purified from a bacterial cell expression mutant inactive MEK (GST-kdMEK) as a fusion protein with glutathione-S-transferase.

Method: The standard assay conditions of B-Raf catalytic activity utilized were: 3 μg GST-kdMEK, 10 μM ATP and 2 uCi ³³ P-ATP, 50 mM MOPS, 0. 3 in a total reaction volume of 30 μl. 1 mM EDTA, 0.1
M sucrose, 10 mM MgCl ₂ + 0.1% dimethyl sulfoxide (comprising compounds as appropriate). The reaction was incubated at 25 ° C. for 90 minutes and the reaction was terminated by adding EDTA to a final concentration of 50 μM. 10 μl of the reaction was dropped on P30 phosphocellulose paper and air dried. After washing four times in ice-cold 10% trichloroacetic acid and 0.5% phosphoric acid, the paper was air-dried, a liquid scintillation substance was added, and the radioactivity was measured with a scintillation counter.

Results: 4- (4-chloro-3-hydroxyphenyl) -2-phenyl-5- (4
- pyridyl)-1H-imidazole was found to be effective in inhibiting the phosphorylation of GST-kdMEK substrate B-Raf-mediated _(IC 50 24nm). In addition, the activity of the compounds as Raf inhibitors was determined by WO99 / 10325, McDona
ld, OB, Chen, WJ, Ellis, B., Hoffman, C., Overton, L., Rink, M., Smi
th, A., Marshall, CJ and Wood, ER (1999) "A scintillation prox
imity assay for the Raf / MEK / ERK kinase cascade: high throughput screen
ing and identification of selective enzyme inhibitors, '' Anal. Biochem.
, 268: 318-329 and AACR meeting New Orleans 1998 Post
It can also be measured by the assay described in er3793.

The neuroprotective properties of Raf inhibitors can be measured in an in vitro assay as follows. Neuroprotective Properties of 4- (4-Chloro-3-hydroxyphenyl) -2-phenyl-5- (4-pyridyl) -1H-imidazole in Rat Hippocampal Slice Cultures Organotypic cultures are: Intermediates between dissociated neuronal cell cultures and in vivo models of oxygen and glucose deficiency (OGD).
Glia-nerve interactions and neuronal circulation in cultured hippocampal slices.
Most of the circuitry has been maintained, which makes it easier to study the pattern of death between various cell types in a model that mimics the in vivo situation. These cultures allow for the study of delayed cell damage and death 24 hours or more post-injury, and can evaluate the consequences of long-term modification under culture conditions. Numerous laboratories have reported delayed nerve injury in response to OGD in organotypic cultures of hippocampal tissue (Vornov et al., Stroke (1994) 25: 457-465; New
ell et al., Brain Res. (1995a) 676: 38-44). EAA antagonists (Strasser et al., Brain Res. (1995) 687: 167-174), Na channel blockers (Tasker et al., J Neurosci. (1992) 12: 4298-430).
8) and Ca channel blockers (Pringle et al., Stroke (1996) 27: 2).
124-2130) showed protection in this model. To date, the role of intracellular kinase-mediated signaling pathways in neuronal cell death in this model is relatively poorly understood.

Methods: Stoppini et al., J. Neurosci. Methods. (1991) 37, 173-18.
Organotypic hippocampal slice cultures were prepared using Method 2 above. Briefly, after birth 7
400 micron fragments prepared from the hippocampus of Sprague-Dawley rats at -8 days were cultured on semi-porous membranes for 9-12 days. The OGD was then incubated for 45 minutes in a serum and glucose free medium in an anaerobic chamber.
Was triggered. The culture is then returned to the air / CO ₂ incubator 23 hours before analysis. Propidium iodide (PI) is used as an indicator of cell death. PI is not toxic to neurons and has been used in many studies to determine cell viability. PIs enter damaged neurons and bind to nucleic acids. The bound PI shows a large emission at 635 nm when excited at 540 nm. One PI fluorescence image and one white light image are captured and the percentage of cell death is analyzed. The portion of the area CA1 is determined from the white light image and is superimposed on the PI image. The PI signal is the threshold and fraction of PI damage expressed as a percentage of the CA1 fraction. The correlation between PI fluorescence and histologically confirmed cell death has been previously confirmed by the Nissl-staining method using cresylfast violet (Newell et al., J. Neurosci. (1995b) 15: 7702-7711). )
.

Results: When preincubated for 1 hour 45 minutes before OGD, 4- (4-chloro-3-hydroxyphenyl) -2-phenyl-5- (4-pyridyl) -1H-
Imidazole showed significant protection (FIG. 1). The IC ₅₀ for this protection is approximately 1
00 nM.

FIG. 1: 4- (4-chloro-3-hydroxyphenyl) -2-phenyl-5- (4
Effect of (pyridyl) -1H-imidazole on oxygen-glucose deprivation-induced neuronal cell death in organotypic hippocampal cultures. OGD duration 45 minutes. This graph shows the proportion of dead nerve CA1 portions as determined by PI staining. Control values are between 65-75 percent. The results were averaged ± SEM ^*** (P = <0
. 0005) n = 6-12.

Conclusion: B-Raf inhibitor is a physiological substrate of human B-Raf protein kinase M
It is an effective inhibitor of catalytic activity towards AP kinase (MEK). Besides, B
-Raf inhibitors are potent inhibitors of neuronal cell death resulting from oxygen-glucose deprivation in hippocampal tissue slice cultures. Such neuroprotective properties could make B-Raf inhibitors useful in protecting neuronal cell death associated with ischemic stroke and traumatic injury.

[Brief description of the drawings]

FIG. 1. 4- (4-Chloro-3-hydroxyphenyl) -2-phenyl-5
Figure 4 shows the effect of-(4-pyridyl) -1H-imidazole on oxygen-glucose deprivation-induced neuronal cell death in organotypic hippocampal cultures.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA , ZW (72) Inventor Elaine Allison Irving, C.M. 19.5 ADB, Essex, Harlow, Third Avenue, New Frontiers Science Park South, SmithKline Beecham Pharmaceuticals Tikals (72) Inventor Andrew A. Parsons, UK, CM 19.5 ADB, Essex, Harlow, Third Avenue, New Frontiers Science Park South, SmithKline Beecham Pharmaceuticals F-term (reference) 4C084 AA17 NA14 ZA012 ZA362 ZC202 4C086 AA01 AA02 BC17 BC38 GA07 GA08 NA14 ZA01 ZA36 ZC20

Claims (8)

[Claims] 1. A method of treating or preventing a neurotraumatic disease in a mammal in need thereof, comprising administering to the mammal an effective amount of a Raf inhibitor. 2. Use of a Raf inhibitor in the manufacture of a medicament for the prophylactic or therapeutic treatment of any condition aggravated or caused by a neurotraumatic event in a human or non-human mammal. 3. The method or use according to claim 1, wherein the Raf inhibitor is a small molecule Raf inhibitor. 4. The method or use according to any one of the preceding claims, wherein the Raf inhibitor is a B-Raf inhibitor. 5. The method or use according to claim 4, wherein the B-Raf inhibitor is 4- (4-chloro-3-hydroxyphenyl) -2-phenyl-5- (4-pyridyl) -1H-imidazole. . 6. The method or use according to any one of the preceding claims, wherein the neurotraumatic disease is ischemic stroke. 7. The method or use according to any one of the preceding claims, wherein the neurotraumatic disease is caused by surgery or is an open head injury. 8. The method or use according to any one of the preceding claims, wherein the neurotraumatic disease is a non-open head injury.