CN1241941A - Treatment of neurodegenerative conditions with nimesulide - Google Patents

Abstract

The present invention relates to the application of nimesulide and structurally related compounds in the prevention and/or treatment of neurodegenerative diseases. This is based at least in part on the finding that nimesulide exhibits neuroprotective effects against β-amyloid-induced cell death. Without being bound by any particular theory, nimesulide appears to inhibit non-inflammatory mechanisms of neurodegeneration.

Description

Treatment of Neurodegenerative Diseases with Nimesulide

1 Introduction

The present invention relates to the application of nimesulide and structurally related compounds in the prevention and/or treatment of neurodegenerative diseases such as Alzheimer's disease. This is based at least in part on the finding that nimesulide inhibits cell death at effective concentrations.

2. Background of the invention

2.1. Alzheimer's disease

Sporadic Alzheimer's disease is the main neurodegenerative disease associated with aging. Between the ages of 65 and 85, the risk of developing this disease increases exponentially, doubling every 5 years. Histologically, the hallmarks of Alzheimer's disease are amyloid deposits on senile plaques and cerebral blood vessel walls; nerve fiber tangles, and neurodegeneration. However, the etiology of Alzheimer's disease remains unclear. Genetic factors have been proposed to play a role, including trisomy 21: amyloid beta-protein precursor ("APP") gene, presenilin-1 (PS1) and presenilin-2 (presenilin-2, PS2) genes Mutations in , and the presence of apolipoprotein E type 4 alleles (Younkin, 1995, Ann. Neurol. 37: 287-288; Lendon et al., 1997, JAM A 277: 825). Some studies have shown that β-amyloid induced apoptosis in cultured neurons (Loo et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 7951-7955; Li et al., 1996, Brain Res. .738:196-204). The induction of this apoptosis may involve immediate early gene proteins: c-jun and fos (Anderson et al., 1995, J.Neurochemistry65:1487-1498; Anderson et al., 1994, Experimental Neurology 125:286-295; Anderson et al., 1994, Experimental Neurology 125:286-295; et al., 1996, J. Neurosci. 16:1710-1719).

It has been suggested that apoptosis may be involved in the pathogenesis of Alzheimer's disease (Smale et al., 1995, Exp.Neurol.133:225-230; Anderson et al., 1996, J.Neurosci.16:1710-1719; Anderson et al. ., 1994, Exp.Neurol.125:286-295), and inflammatory mechanisms may also be involved (Pasinetti, 1996, Neurobiol.Ageing 17:707-716), which is supported by the following observations: Acute-phase proteins are elevated in serum and deposited in amyloid plaques; activated microglia tend to localize near senile plaques, and complement components localize around dystrophic axons and neurofibrillary tangles (Aisen & Davis , 1994, Am. J. Psychiatry 151: 1105-1113). Anti-inflammatory agents have been suggested as potential therapeutic agents (Aisen et al., 1996, Dementia 7:201-206; McGeer et al., 1996, Neurology 47:425-432). Selective inhibitors of cyclooxygenase-2 have been developed as therapeutic agents for this inflammation due to their fewer adverse side effects (International Publication No. WO 94/13635 by Merck Frosst Canada Inc.). However, prior to the present invention, no one believed that these drugs could be used to inhibit the non-inflammatory processes of neurodegenerative disease in the course of Alzheimer's disease.

2.2. Cyclooxygenase-2

Cyclooxygenases (" _COXs ") are enzymes that catalyze the formation of prostaglandin ("PG")- _H2 from arachidonic acid (AA). PG-H ₂ is further metabolized into physiologically active PGs (such as PG-D ₂ , PG-E ₂ and PG-F _2α ), prostacyclin (PG-I ₂ ) and thromboxane. Specific PGs have various, often antagonistic, effects on different tissues. For example, PG-I ₂ and PG-E ₂ are strong vasodilators that cause inflammation, while PG-F _2α is a vasoconstrictor.

There are two known COX isozymes: COX-1 and COX-2, which, although different physiologically, have similar amino acid sequences and enzymatic functions. COX-1 is expressed at substantially different levels in different cell types. However, COX-2 is basically not expressed and generally cannot be detected in normal peripheral tissues (Kujubu et al., 1991, J. Biol. Chem. 266: 12866-12872; O'Banion et al. 1992, Proc. Natl. Acad. Sci. U.S.A. 89:4888-4892). COX-2 expression is inducible (eg, with mitogens), and COX-2 mRNA levels are observed to increase rapidly in response to inflammatory stimuli such as interleukin-1β and lipopolysaccharide, while decreasing in response to glucocorticoids. When affected by these factors, the level of COX-1 mRNA remains basically unchanged, indicating that COX-2 is an isoenzyme that mediates inflammation (Cao et al., 1995, Brain Res. 697: 187-196; O'Banion et al. al. 1992, Proc. Natl. Acad. Sci. U.S.A. 89:4888-4892).

Recent evidence suggests that COX-2 may play a role in cell survival and cell adhesion mechanisms in peripheral cells (Lu et al. 1996, Proc. Natl. Acad. Sci. U.S.A. 92: 7961-7965; Tsujii et al., 1995, Cell 83: 493-501). Tsujii et al. reported that epithelial cells genetically engineered to express elevated levels of COX-2 were resistant to butyrate-induced apoptosis, showed elevated BCL2 protein expression, and reduced transforming growth factor β2 receptor levels (Tsujii et al ., 1995, Cell 83:493-501). Lu et al pointed out that non-steroidal anti-inflammatory drugs can induce apoptosis mechanisms involving the COX system.

The role of COX-1, COX-2 and PG synthesis in the normal brain and in the course of Alzheimer's disease has not yet been fully characterized. The importance of PG in brain physiology may be independent of inflammatory mechanisms. In the brain, PG receptors are found in the hypothalamus, thalamus and limbic system (Watanabe et al., 1989, Brain Res. 478: 143-148). PG is involved in hypothalamic-pituitary hormone secretion (Kinoshita et al., 1982, Endocrinol. 110: 2207-2209), thermoregulation and sleep-wake cycle (Hayaishi, 1988, J. Biol. Chem. 263: 14593-14596). Recent evidence suggests that COX-2 mRNA is expressed and regulated in the rat brain by synaptic activity and glucocorticoids (Adams et al., 1996, J. Neurochem. 66:6-13; Kaufmann et al., Proc. Natl. Acad. Sci. U.S.A. 93:2317-2321; Yamagata et al., 1993, Neuron 11:371-386). These studies demonstrate that COX-2 is regulated in the brain as an immediate early gene and suggest that PG may be important in transsynaptic signaling and long-term potentiation. Chang et al. (1996, Neurobiol.Of Aging17: 801-808) reported that the expression of COX-2 mRNA in Alzheimer's disease decreased.

3. Summary of invention

The present invention relates to the application of nimesulide and structurally related compounds in the prevention and/or treatment of neurodegenerative diseases. This is based at least in part on the findings that (i) COX-2 expression is increased in neurons but not glia in models of neurodegeneration (consistent with a non-inflammatory mechanism), and (ii) that nimesulide has an effect on β- Amyloid-induced neuronal cell death shows neuroprotective effects. This latter finding is particularly unexpected since COX inhibitors can increase apoptosis in non-neuronal cells. It appears that nimesulide inhibits non-inflammatory mechanisms of neurodegeneration without being bound by any particular theory.

4. Description of drawings

Figure 1. Regional expression of COX-2 mRNA in the brain of undamaged male rats in the control group

COX-2 mRNA expression was assessed by in situ hybridization and visualized by X-ray autoradiography. Abbreviations: DG, dentate gyrus; CA1, CA2, and CA3 subregions of the neurogenic pyramidal layer of the hippocampal formation; PAC, parietal cortex; PYC, piriform cortex; AC, amygdaloid ; THAL, ventroposterior nucleus of the thalamus. Adapted from Paxinos & Watson Plate 24, 1986 (The Rat Brain in Steriotaxic, Coordinates, Academic Press, NY).

Figure 2. Mature Regulation of COX-2 mRNA Expression in Rat Brain

Densitometric quantification was performed from autoradiographic images. Abbreviations are as above and as follows: PYC/AC, piriform area and amygdala complex as individual brain regions for quantification of COX-2 mRNA expression. N=5-8/time point.

Figure 3. Maturation effects on COX-2 mRNA expression in response to KA-induced convulsions

Micrographs prepared from autoradiographic images. See Figure 1 for the anatomical distribution of the changes. Control group, uninjured rats injected with vehicle; rats treated with KA 8H, KA 8 hours before sacrifice; postnatal days, P-7, P-14 and P-21.

Figure 4A–D. Time course of COX-2 mRNA changes in rat brain in response to KA treatment: mature effects and regional distribution of changes

Densitometric quantification was performed from autoradiographic images. Data are expressed as mean±SEM, N=4-6/group. p<0.01 (compared to OH group (saline injection group)); 4 hours, 8 hours, 16 hours, 30 hours, 120 hours, time after seizure induced by KA, in hours.

Figure 5A-D. Maturation effects on distribution of COX-2 mRNA expression and induction in rat hippocampus

COX-2 mRNA in P21 (A, B) and adult (C, D) hippocampal structures assessed by in situ hybridization assay and emulsion autoradiography with darkfield illumination. A and C, COX-2 mRNA expression in control rats (injected with vehicle); B and D, COX-2 mRNA 8 hours after KA-induced convulsive onset. Arrows point to the superficial layer of the DG (granular layer). Scale bar = 200 μm.

Figure 6. Selective induction of hippocampal COX-2 but not COX-1 mRNA in response to KA-induced convulsions assessed by agglutination blot hybridization assay

CTL, control rats injected with saline; KA, KA-treated rats, 12 hours after lesion.

Figure 7A-I. KA-induced COX-2 and apoptosis in adult rat brain

(A, B), the expression of COX-2 mRNA in the CA3 hippocampal pyramidal neurons of the control group and KA-treated rats; (C), the hippocampal structure of KA-lesioned rats showing neurons with apoptotic features (arrows) The CA3 subregion. (D, E), COX-2 mRNA expression in the piriform cortex of control and KA-treated rats, respectively. (F), Arrows point to apoptotic cells in the piriform cortex of KA-lesioned rats. (G「H), the expression of COX-2 mRNA in the amygdala cells of the control group and KA-treated rats, respectively. (I), the arrow points to the apoptotic cells of the amygdala-like complex of the KA lesioned rats. By in situ Evaluation of hybridization assays and determination of COX-2 mRNA by emulsion autoradiography with darkfield illumination. In situ 3′-end labeling was used to evaluate the apoptotic signature after KA treatment. Scale bars: in A, B, D, E, G, and H In = 200 μm; in C, F and I = 40 μm.

Figure 8A-D. Immunocytochemical Evidence of COX-2 Expression/Regulation in Glutamate-Responsive Neurons in In Vitro Assays

COX-2-like immunoreactivity in monotypic cultures of (A) control and (B) rat primary hippocampal neurons after glutamate exposure (12 hours). (C, D), Immunoreactivity of control and glutamate-treated cultures with immunoadsorbed COX-2 antibody, respectively. Scale bar = 50 μm.

Figure 9A-C. Effects of Nimesulide on Endotoxin-Mediated Cytokine and Nitrite Synthesis/Secretion in Glia

The effects of 10 ^-9 M nimesulide on the synthesis/secretion of (A) TNF, (B) NO intermediate (Griess reaction) and (C) PGE ₂ were evaluated in immortalized microglial cells of BV2 mice effect. Mean ± SEM, n = 8-10/group. Lipopolysaccharide ("LPS") = 5 μg/ml. LPS and nimesulide were added to the cultures in combination; incubation time was 24 hours. ANOVA was used for statistics, p<0.05.

Figure 10A-E. Time course of COX-2 mRNA changes in P19 cells in response to conditions leading to apoptosis

(A), Quantitative analysis of COX-2 induction in P19 cells, n=4-6/group, p<0.05 (compared with t=0); (B), Western analysis and evaluation of changes with chemiluminescence detection; (C, D) The morphological appearance of apoptotic nuclei in P19 cells was evaluated with Hoechst H33258 24 hours after serum removal (replaced with N2 medium); (E), the DNA electrophoresis pattern showing step-like degradation of DNA was evaluated 14 hours after serum removal (1st lane, control cells at t=0; 2nd lane, DNA ladder 14 hours after serum depletion; 3rd lane, DNA marker). The COX-2 polyclonal antibodies used in these studies are described in Section 6. COX-2-specific antibodies recognize two major bands with estimated molecular weights of approximately 70 and 65 kDa in total homogenates of mouse, rat and human brain.

Figure 11A-E. Regulation of COX-2 in response to Aβ1-40-mediated oxidative stress in SH-SY5Y neuronal cells

(A) Bar graph showing the amount of COX-2 immunoreactivity relative to the control group (CTL), the measure of immunoreactivity from the data shown in (B); Western blotting of proteins prepared from cells prepared from peptides for 48 or 72 hours reacting with COX-2 antiserum; (C) MTT test results of SH-SY5Y cells treated with control group or Aβ; (D) stripped and Western blot shown in (B) of protein antiserum immunoreaction; (E) examination of regulatory mechanisms in SH-SY5Y cultures.

Figure 12. Evaluation of the protective effect of nimesulide against Aβ1-40-mediated neurotoxicity by MTT assay using SH-SY5Y neuronal cells.

Abbreviations: CTL: control; NIM nimesulide; AB-beta-amyloid (1-40). ^* p<0.01 (compared to CTL); ^** p<0.05 (compared to CTL).

Figure 13. Micrographs of induction of COX-2 immunoreactivity in the temporal cortex of AD and age-matched neurological controls

This photomicrograph shows selective induction of COX-2 immunostaining in neuron-rich gray matter ("gm") but not glia-rich white matter ("wm") regions.

Figure 14A-D. COX-2 immunostaining of neurons in AD brain frontal cortex and co-localization of COX-2 immunostaining and AD plaques in AD brain frontal cortex

(A, B), COX-2 immunostaining showing AD neurons (A, low power; B, high power magnification); (C), immunostaining of diffuse plaques; (D) assessed by Aβ immunostaining in adjacent tissue sections COX-2 in neurogenic plaques.

Figure 15A-D. COX-2 expression in frontal cortex of AD brain

(A) Northern blot of COX-2 and COX-1 mRNA in AD frontal cortex relative to control group, quantitative analysis of data in (B); (C) Western blot of COX-2 protein in AD frontal cortex and control group, Quantitative analysis of data is in (D).

Figure 16. COX-2 mRNA regulation in the anterior and posterior horns of the ALS spinal cord and neurons of the neurological control group.

Autoradiographic images are displayed on X-ray film. Arrows pointing to the front corners show strong induction of COX-2 mRNA signal in ALS cases.

Figure 17A-D. Elevation of COX-2 mRNA in biopsies of human cortical epileptic foci assessed by in situ hybridization assay

Autoradiographic images are displayed on X-ray film. Arrows pointing to the cortex show strong COX-2 mRNA signal in epileptic brain (A) relative to control (B); sections (C) and (D) show analysis of compiled data on COX-2 and COX-1 mRNA levels.

Figure 18. Enhancement of cyclooxygenase and peroxidase activity by aggregated Aβ peptides

Figure 19A-B. Transient expression of COX-2 in neuronal cells enhances Aβ-mediated impairment of redox activity

SH-SY5Y neuronal cells were transfected with human COX-2 (A) or COX-1 (B) gene and treated with Aβ _25-35 , and then the redox activity was assessed by MTT assay.

Figure 20A-B. Generation of transgenic mice expressing COX-2

(A) In situ hybridization demonstrates that COX-2 mRNA is expressed in TgNHC32 mice but not in wild-type mice (one-month-old mice); (B) hCOX-2 mRNA is expressed in regions on NHC32 and NHC5 relative to wild-type, Quantification was performed using Bioquant image analysis.

Figure 21. Overexpression of hCOX-2 enhances Aβ-mediated oxidative damage in primary mouse neuronal cultures.

Figure 22. Nimesulide protects B12 neuronal cells against glutamate-mediated oxidative stress.

Figure 23. Control Study of Figure 22

The dose (A) and time course (B) associated with glutamate-mediated oxidative damage was determined using the LDH assay. (C) shows that nimesulide is not toxic to B12 cultures.

5. Detailed description of the invention

The present invention relates to the use of cyclooxygenase-2 ("COX-2") selective inhibitor nimesulide and structurally related compounds in the prevention and/or treatment of neurodegenerative diseases. This is based at least in part on the finding that increased expression of COX-2 parallels increased neuronal damage produced by kainic acid and increased induction of neuronal apoptosis in established in vitro systems (see Sections 6 and 8 below ). Contrary to prior art reports that COX-2 plays a major role in inflammation and may be involved in neurodegeneration (e.g. in the course of Alzheimer's disease), the expression of COX-2 in the human brain was further found Appears to be restricted to neurons rather than glial cells (whereas expression in glial cells is expected if inflammatory mechanisms are at work, see section 9 below). A correlation has been observed between COX-2 expression and Alzheimer's disease, the characteristic pathological changes associated with amyloid plaques (see section 9). Furthermore, it was demonstrated that nimesulide exerts a neuroprotective effect against β-amyloid-induced cell death in neuronal cultures. In view of these findings, according to the present invention, nimesulide and related compounds can be used to intervene in the process of neuronal apoptosis associated with Alzheimer's disease and other neurodegenerative diseases. The action of nimesulide and related compounds, without being bound by any particular theory, may act by reducing the apoptotic effects of oxidative stress.

The term "nimesulide" as used herein refers to a compound having the structure described in Formula I below: 4-nitro-2-phenoxy-N-methanesulfonanilide.

Compounds considered to be structurally related are those with similar bicyclic structures and selectivities for use as COX-2. For example, substituted compounds, analogs and enantiomers of nimesulide are considered to be structurally related compounds. As another example, a structurally related compound can compete with nimesulide for binding to COX-2, or can bind to substantially the same site on COX-2, as determined by crystallography. Furthermore, nimesulide cross-linked with other compounds as defined herein are also considered structurally related compounds.

Nimesulide or a structurally related compound can be administered to a patient to provide an effective concentration in the nervous system of the subject. Herein, an effective concentration is defined as a concentration capable of inhibiting at least 20% of neuronal cell death. In a non-limiting embodiment of the present invention, the concentration of nimesulide is at least 1 nanomolar, preferably at least 1 micromolar, at the site of the nerve cells to be treated. Ideally, the concentration at the neuronal site is below 10 ^-3 molar to avoid toxicity. In one such embodiment, wherein the neurodegenerative disease to be treated is Alzheimer's disease, the concentration of nimesulide in the hippocampus of the subject is at least 1 picomolar, preferably at least 1 nanomolar, more preferably at least 1 micromol. Corresponding serum concentrations may be at least 10 picomolar, preferably at least 10 nanomolar, more preferably at least 10 micromolar. Structurally related compounds can also be used in equal amounts (adjusted to compensate for differences in intensity).

Nimesulide or a structurally related compound may be administered in such a manner that the desired effective concentration is achieved. For example, suitable routes of administration include oral, intravenous, subcutaneous, intramuscular, transdermal, and intrathecal injections.

Nimesulide or a structurally related compound can be contained in a suitable pharmaceutical carrier. Such formulations are available for sustained release.

For example (but not limitation), nimesulide can be administered orally at a dose of 2-800 mg/day, preferably 50-400 mg/day, most preferably 200 mg/day.

Neurodegenerative diseases that may be treated in accordance with the present invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, neurodegeneration with convulsions, amyotrophic lateral sclerosis, spinal cord injuries, and conditions not conventionally recognized as mediators of inflammation. other induced or autoimmune diseases.

6. Example: Maturation Regulation and Regional Induction of COX-2 in Rat Brain

6.1. Materials and methods

Animals and Excitotoxicity Injuries: Male adult Sprague/Dawley rats of different ages were maintained in a light and temperature controlled environment with free access to food and water. Adult rats (250-300 g) were induced excitotoxic lesions in the hippocampus by subcutaneous injection of kainic acid ("KA", 10 mg/kg, Sigma). Since KA uptake is higher in young rats than in adult rats (Berger et al., 1986, in Schwartz and Ben-Ari, Advances in Experimental Medicine and Biology, Plenum, NY, pp. 199-209), the KA dose was adjusted to produce The maximum excitotoxicity did not reach the lethal dose (2mg/kg on the 7th day after birth to 6mg/kg on the 25th day after birth). Rats injected with saline served as the control group (0 hour time point).

COX-1 and COX-2 cDNA probes: linearize the Bluescript plasmid (product of Stratagene) containing the full-length (2.7 kb) of rat COX-1 cDNA by ClaI digestion; digest the rat COX-2 coding sequence by pfMI The PCRII plasmid (product of Invitrogen) was linearized (Feng et al., 1993, Arch. Biochem. Biophys. 307: 361-368). After agarose gel electrophoresis, the linearized plasmid was purified with Elu-Quick (Schleicher & Schuell).

In situ hybridization: At different intervals after the convulsions caused by KA, the rats were sacrificed, and the brains were quickly removed, rinsed in cold phosphate buffer (PBS, 10mM, pH7.4), and kept at -25°C. Dip in methylbutane for three minutes. Brains were sectioned into 10 micron sections, frozen, and the resulting cryosections were mounted on polylysine-coated glass slides and stored at -70°C. For immunocytochemistry ("ICC") or in situ hybridization ("ISH"), cryosections were post-fixed in 4% paraformaldehyde in PBS (30 minutes at room temperature) and rinsed with PBS. For ISH, tissue sections were rinsed with 0.1M triethanolamine ("TEA") (pH 8.0), incubated in acetic anhydride ("AAH", 0.25% v/v TEA solution, 10 min), washed with TEA and Rinse with PBS. After AAH treatment, tissue sections were hybridized with a [ ³⁵ S]-cRNA probe (0.3 μg/ml, 2×10 ⁹ dpm μg ^-1 ) prepared from a COX-2 linearized cDNA transcription vector (Feng et al. , 1993, Arch. Biochem. Biophys. 307:361-368). Sense strand hybridization was used as a control, which gave a negative result. After hybridization at 50° C. for 3 hours, stringent washing (0.1×SSC, 60° C.) and dehydration, slides were exposed to radiographs for seven days for quantification. Slides were then exposed to NTB-2 emulsion (Kodak, Rochester, NY) for microscopic analysis of COX-2 mRNA distribution. After development, tissue sections were counterstained with cresyl violet. Autoradiographic films were quantitatively analyzed using an image analysis system equipped with Drexel University software (Tocco et al., 1992, Eur. J. Neurosci. 4: 1093-1103). ANOVA was used for statistical calculation, followed by posthoc analysis.

In situ end labeling: In a parallel study, paraformaldehyde-fixed brain tissue sections were dehydrated, air-dried, and treated with dATP, dCTP, dGTP (0.2 mM), dTTP (13 μM), digoxigenin-11-dUTP, and DNA Polymerase I (Boehringer Mannheim) 10 units/100 μl, incubated at 37°C for 2 hours. Add 20mM EDTA (pH8.0) to terminate the reaction. The section was incubated overnight at room temperature with an alkaline phosphatase-crosslinked digitoxin antibody (Genius System, BoehringerMannheim), which was prepared in 150mM Nacl, 100mM TRIS-HCl (pH7.5) solution (containing 5% Sheep serum) was diluted 1:200. Colorimetric assay with nitroblue tetrazolium and 5-bromo-4-chloro-3-indole-phosphate was carried out with the Genius system according to the manufacturer's protocol (Sakhi et al., Proc. Natl. Acad. Sci. U.S.A. 91 :7525-7529).

Northern blot hybridization test: RNA extraction was performed as follows. After the rats were sacrificed, the brains were removed and stored at -75°C until the experiment. Total RNA was extracted from pooled hippocampal tissues (Pasinetti et al., 1994, J. Comp. Neurol. 339:387-400). Briefly, tissues were homogenized in 4M guanidine thiocyanate, 25mM sodium citrate (pH 7.5), 0.5% sodium lauryl sarcosine, and 0.1M β-mercaptoethanol (final volume 0.5ml). pulp. After extraction with acidic phenol/chloroform and ethanol precipitation, the RNA pellet was washed sequentially with 70% and 100% ethanol. Purified RNA was then dissolved in 0.5% sodium dodecyl sulfate ("SDS") and stored at -75°C. Total RNA was quantified in a UV spectrophotometer. Total RNA (5 μg) from the tissue was electrophoresed on a denaturing (0.2M formaldehyde) agarose gel. and transferred to a nylon membrane (Nylon 66+; Hoeffer, San Francisco CA) at 2×SSC. 10 ⁶ cpm/ml antisense COX-1 or COX-2 prepared with 50% formamide, 1.5×SSPE, 1% SDS, 0.5% milk powder, 100 μg/ml yeast total RNA and 500 mg/ml salmon sperm RNA [ ³² P]-cRNA probe, 53 ° C for 15 hours blot hybridization. Blots were washed at 72°C to a final stringency of 0.2 x SSC, 0.2% SDS. Blots were exposed to Kodak X-ray film (XAR-5) at -70°C using an intensifying screen.

Cell Culture: Hippocampal neuron cultures were obtained from rat embryonic mouse brain. E16-E18 embryos were segmented and cultured in Hank's balanced salt solution (Pasinetti et al., 1994, J.Comp.Neurol.339:387-400; Peterson et al., 1989, Dev.Brain Res.48: 187-195). Medium was changed every 3 days. For glutamate neurotoxicity studies, 8-day-old cultures were treated with glutamate (250 [mu]M, Sigma) in the presence of 2.4 mM calcium ions and 0.8 mM magnesium ions. Six hours after exposure to glutamate, the medium was replaced with fresh medium; 12 hours later, COX-2-like immunoreactivity was assessed in neurons.

Immunocytochemical assay of COX-2 in monotypic cultures of primary rat neurons: Control and glutamate-treated cultures were post-fixed in PBS containing 4% paraformaldehyde (30 min, room temperature), rinsed with PBS, treated with normal serum and incubated overnight at 4°C with the primary antibody. COX-2 antiserum (rabbit IgG) was raised against a synthetic peptide comprising the mouse COX-2 C-terminal region (CNASASHSRLDDINPT; SEQ ID NO: 1). The antisera reacted with human and rat COX-2 but not with COX-1 as assessed by Western blot analysis. Diaminobenzene staining (Pasinetti et al., 1994, J. Comp. Neurol. 339:387-400) was accomplished in a later step using the Vectastain ABC kit (Vector, Burlingame, CA). COX-2 antiserum was immunoadsorbed with synthetic COX-2 peptide to control specificity; 30 μg/ml of synthetic COX-2 peptide was used to absorb overnight at 4°C.

6.2. Results

COX-2 expression in the adult rat brain: Figure 1 shows by ISH that the regional distribution of COX-2 mRNA is most prominent in limbic structures, but also present in the neocortex (with Kaufmann et al., Proc. Natl. Acad. Sci.U.S.A.93:2317-2321 and Yamagata et al., 1993, Neuron 11:371-386 report). In the hippocampal formation, COX-2 mRNA is selectively expressed in cells of the granular layer and pyramidal neuron layer. COX-2 mRNA expression was also found in the outer layers of the parietal cortex, piriform cortex, and amygdala complex cells.

Mature regulation of hippocampal COX-2 mRNA

Expression: ISH results demonstrated differential regulation of COX-2 mRNA expression in subgroups of cells in the structural neuronal layer of the hippocampus during maturation (Figure 2, Figure 3 top). From P7 to P14 days after birth, the COX-2 mRNA in the granule cell layer of the dentate gyrus and the CA3 subregion of the pyramidal cell layer increased by more than 2 times (p<0.001, Figure 2). Although COX-2 mRNA expression was lower in the other brain regions examined, the pattern of mature expression was similar (Fig. 2). At postnatal day P21, COX-2 mRNA expression approached adult levels in all subregions examined (Fig. 2, top of Fig. 3).

Response to KA-induced convulsions: To further reveal the mature regulation of COX-2 in the brain, COX-2 mRNA expression in response to KA-induced convulsions was examined postnatally. Despite strong convulsive activity after KA treatment, no detectable changes in COX-2 mRNA were found in all brain regions examined at P7 (Fig. 3, Fig. 4A). Changes in COX-2 mRNA expression at 120 h after KA treatment in the P7 group indicated developmental maturation rather than a response to KA toxicity (Fig. 4A). In contrast to the P7 group, COX-2 mRNA increased in all hippocampal subregions examined within 4-8 hours after the onset of KA-induced convulsions on days P14 and P21 (Fig. 4B and 4C). COX-2 mRNA expression levels in the brains of P14, P21 and adult rats returned to control levels within 120 hours after treatment.

In the dentate gyrus, the expression of COX-2 in the rat control group at P14 and P21 was asymmetric and selectively localized to the neurons in the shallower layers of the granulosa rather than the deeper granule cells in the lobes of the dentate gyrus (Fig. 5A). In response to KA treatment, COX-2 mRNA induction showed similar expression asymmetry (Fig. 5B). In contrast, asymmetry within the dentate gyrus was less pronounced after KA induction in control animals and adults (Fig. 5C and 5D).

In a parallel experiment, Northern blot hybridization of total RNA obtained from the hippocampus confirmed the induction of COX-2 mRNA 12 hours after KA-induced convulsions in adult rats (Fig. 6). Induction of COX-1 mRNA was not detected in the same rat brain.

KA-induced COX-2 and programmed cell death in adult rats: 8 hours after KA-induced convulsive onset, hippocampal CA3 (Fig. 7B), piriform cortex (Fig. 7E) and amygdala complex In cells from the body (Fig. 7H), the induction of COX-2 mRNA transiently paralleled and anatomically overlapped the apoptosis occurring in the same brain region (assessed with in situ end labeling) (Fig. 7C, CA3 of the hippocampus area; Figure 7F, piriform cortex; Figure 7I, amygdaloid complex). Intracellular COX-2 mRNA expression was identified in adult mice, control mice (Figures 7A, 7D and 7G) and KA-treated (Figures 7B, 7E and 7H) rats by ISH assay by emulsion autoradiography.

Immunocytochemical evidence of neuronal COX-2 expression/regulation in response to glutamate in vitro: In an in vitro assay, primary cultures of rat hippocampal neurons were exposed to glutamate. Constitutive COX-2 expression was confirmed at baseline by immunocytochemistry (Fig. 8B). An increase in COX-2 immunoreactivity was observed after 12 hours of glutamate exposure, which coincided with a marked reduction in neuron numbers (Fig. 8D).

COX-2 expression in human epilepsy: Increased COX-2 mRNA expression was detected by ISH in epileptic foci in human brain biopsies (Fig. 17A-D). found that COX-2 mRNA but not COX-1 mRNA was significantly elevated in the cortex of epileptic patients. P<0-05.

7. Example: Nimesulide inhibits microglial cells

Production of cytokines and nitrite in culture

In experiments conducted in vitro, it was found that low concentrations of nimesulide (1 nanomolar) effectively inhibited endotoxin-mediated tumor necrosis factor ("TNF") in brain-derived immortalized microglia (BV-2) and astrocytes. Induction of TNF") production (Fig. 9A). Similarly, nimesulide was also effective in blocking nitrite production (Griess reaction; Figure 9B). The latter observation is particularly relevant since evidence suggests that blocking neurogenic nitric oxide ("NO") synthase protects against glutamate neurotoxicity. Nimesulide was also found to effectively block endotoxin-mediated induction of the prostaglandin _PGE2 in brain-derived microglia (Fig. 9C).

8. Example: Expression of COX-2 in programmed cell death cells

Regulation of COX-2 expression was studied using an established in vitro model of apoptosis. Specifically, the regulation of COX-2 in the response of P19 embryonal carcinoma cells to serum deprivation was studied. Under such conditions, P19 cells undergo programmed cell death, displaying characteristic DNA fragmentation and nuclear morphological changes. As shown in Figure 10, with this system, simultaneous apoptosis and COX-2 expression were observed.

These studies were extended to the human neurogenic cell line SH-SY5Y. Beta-amyloid has been shown to play a role in inducing neurogenic degeneration in SH-SY5Y cells (Oda et al., Alzheimers Res. 1:29-34). SH-SY5Y cells were treated with synthetic aggregated Aβ _1-40 peptide at a concentration of 20 μM for different times. Western blot analysis of cell extracts was performed using COX-2 antiserum or actin antiserum as controls (see Figures 1 IB and D, respectively). In FIG. 11A , COX-2 protein was quantified from control or Aβ-treated SH-SY5Y from the immunoblot shown in FIG. 11B (at the 72 hr time point; quantification was only for the 70 kDa mw species). The COX-2 signal was abolished by immunoadsorption of COX-2 antiserum with purified human recombinant COX-2 ("hrCOX-2") peptide. Figure 11 shows that attenuation of cellular redox activity was observed after 72 hours of treatment with aggregated A[beta _]1-40 (20 [mu]m), as assessed by the parallel culture MTT assay. Bar graphs represent mean ± SEM, n = 4-5/group, p < 0.05 (t-test). COX-2 integrated optical densities were analyzed from digitized images using Bioquant image analysis (Biometric, Nashville, TN). It was also found that the same expression of COX-2 as the DNA ladder occurs (Fig. 1 IE), which is a hallmark of apoptosis. Figure 12 shows that nimesulide can block Aβ _1-40 toxicity at 10 ^-6 and 10 ^-9 molar as assessed by MTT assay.

Interestingly, co-incubation of agglutinated synthetic Aβ _1-40 peptide (150 μm) with purified hr-holoCOX-2 (COX-2 + heme cofactor, 50 nM) in 1× PBS at 37°C for 16 hours determined the Oxygenase and peroxidase activity (Murphy et al., 1989, Neuron 2:1547-1558), increased cyclooxygenase and peroxidase activity (relative to the level of enzyme activity in the absence of Aβ; Figure 18) . Cyclooxygenase and peroxidase levels gave negative results in the absence of heme.

9. Example: Expression of COX-2 in Alzheimer's disease

The expression of COX-2 was studied in normal human brains and in the brains of Alzheimer's disease ("AD") patients. Immunocytochemical data showed that COX-2 expression was predominantly localized to cells with neurogenic morphology in the human brain; cells with glial morphology were found to have the least localized COX-2 immunostaining in all brain regions examined. These results tend to suggest that the role of COX-2 is in a non-inflammatory function. In the temporal cortex of neurological control cases, COX-2 immunocytochemical signal was only at the limit of detection, but COX-2 showed elevation in AD brains (Fig. 13A, 13B). This selective induction of COX-2 immunoreactivity in gray matter is consistent with the finding that the rat brain exhibits neuronal induction of COX-2 in response to an injury leading to neuronal death (see Section 6. ).

The cellular immune distribution of COX-2 in AD brains was also examined. It was observed that neuronal COX-2 was distributed not only in perikarya (Fig. 14A) but also in neuronal processes (Fig. 14B). In addition, dense immunostaining was also found in diffuse plaques (Fig. 14C) and AD neuritic plaques identified by Aβ immunostaining in tissue sections adjacent to the hippocampus. These findings suggest a role for COX-2 in neuronal death or survival mechanisms.

A study was performed to quantify COX-2 expression in the brains of AD and age-matched controls. Quantitative dot blot analysis and chemiluminescent detection were used. Quantitative evaluation was performed by Western analysis. COX-2 levels were found to be elevated more than 2-fold in hippocampal homogenates of AD brains compared to neurologically age-matched controls.

Since immunocytochemical evidence suggested that COX-2 immunoreactivity was in AD plaques, we compared COX-2 levels and total Aβ in AD cases and found a direct correlation.

Increased COX-2 mRNA and protein were found in AD brain frontal cortex. Figure 15A shows Northern blots of neurological control (C) and AD frontal cortex total RNA hybridized to [ ^32P ]COX-2 mRNA and COX-1 mRNA. These mRNA species detected by the COX-2 cRNA and COX-1 cRNA probes represent RNAs of the same size found in peripheral tissues. Figure 15B shows the quantitative results of the Northern study. Specifically, the prevalence (prevalence) of COX-2 mRNA in the AD frontal lobe cortex increased relative to the control group, and it was confirmed by normalizing to 28S rRNA on the hybridization membrane that there was an equal amount of RNA in the two lanes. The absence of 28S rRNA and 18S rRNA in the post-gel transfer demonstrates the complete transfer of RNA to the membrane. Integrate optical density from digitized image analysis with Biquant image analysis. The bar graph indicates that n=9/group, and the results were tested by t, showing significant significance (p<0.05).

Similarly, Western blot analysis of the AD frontal cortex versus the control group revealed an increase in the amount of COX-2 protein. Figure 15C shows the results of this Western blot. Lanes 1 and 2 represent the COX-2 protein content of the same tissue used for Northern analysis. Lanes 3-4 show that when COX-2 antiserum was immunosorbed with hrCOX-2 peptide, there was no binding of COX-2, demonstrating the specificity of the signal.

The expected new species of 70 kDa hrCOX-2 did not appear when antisera were pre-adsorbed with hrCOX-2 peptide (lane 5). Figure 15D quantifies these results (for the 70 kDa species). The immunospots were peeled off and immunoreacted with actin antiserum to confirm the specificity of the changes (actin values: control group 100±8; AD control bar graph 94±7%; n=9/group; t The test value is p<0.05). The inset of Figure 15D shows the correlation between COX-2 content and number of spots/ ^mm2 , n=8, r=0.72, p<0.03. From the digitized images, COX-2 and actin integration optical densities were analyzed using Biquant image analysis.

10. Example: COX-2 expression in amyotrophic lateral sclerosis

In situ hybridization demonstrated increased COX-2 mRNA in spinal cord anterior horn cells from patients with amyotrophic lateral sclerosis ("ACS") (Figure 16).

11. Example: Transient COX-2 in Neurogenic Cells

Expression enhances Aβ-mediated redox activity impairment

The role of COX-2 in neuronal death and/or survival was investigated in cultured human SH-SY5Y neuronal cells. Mammalian expression vectors (pRc/CMV/ hCOX, Invitrogen) transiently transfected cells. After treatment with Aβ25-35, the impairment of redox activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure 19 ). Overexpression of hCOX-2 in SH-SY5Y cells transfected with pRc/CMV/hCOX-2 was confirmed by immunocytochemical analysis on the same tissue culture tank slides using anti-COX-2 antibodies as previously described. In control cells transfected with pRc/CMV2/CAT, the same anti-COX antibody elicited minimal immunocytochemical signal. We found that transient overexpression of hCOX-2 in SH-SY5Y neurogenic cells enhanced Aβ25-35 (25 μM, 48 h treatment)-mediated redox activity compared with SH-SY5Y cultures transfected with control vector ( ^±± p<0.01 compared to Aβ/control vehicle; ^* p<0.001 compared to CTL). These data are consistent with evidence showing that impairment of redox activity co-exists with induction of COX-2 mRNA in SH-SY5Y neuronal cells (Fig. 11).

                                                                  

Based on the evidence that overexpression of COX-2 in neurogenic cells enhances Aβ-mediated redox damage and the fact that neuronal COX-2 is elevated in AD brain and in experimental neurodegeneration, we produced a Transgenic mouse model with human (h)COX-2 neuron overexpression. To obtain overexpression of COX-2 in neurons, a hybrid gene was prepared in which expression of the cDNA sequence containing the entire hCOX-2 coding region was regulated by the rat neuron-specific enolase (NSE) promoter.

We obtained 4 strains of mice with high expression of hCOX-2 mRNA. In one of them (NHC32), COX-2 mRNA was assessed by in situ hybridization analysis (Fig. 20A). We found high levels of hCOX-2 mRNA in the hippocampus, cerebral cortex, and other neuronal layers of the NHC32 mouse line (FIG. 20B). In situ hybridization on the same tissue sections using combined immunocytochemical analysis for NSE and hCOX-2 revealed no white matter hCOX-2 mRNA levels, confirming the selectivity of hCOX-2 transgene expression for neuroblasts.

Using neurogenic cultures from transgenic mice, it was further shown that hCOX-2 overexpression enhanced Aβ-mediated responses. As shown in Figure 21, the study showed that transgenic mice with neurogenic COX-2 overexpression (NHC32, Primary neuron cultures (n=3) were the most sensitive to the redox-active injury mediated by the aggregated Aβ25-35 peptide (MTT assay, Aβ25-35 μM, 48 hours). Furthermore, when examined morphologically, Aβ25-35-treated neurons from transgenic hCOX-2 mice showed exacerbated degeneration of neuronal processes compared to wild-type control Aβ25-35-treated neuronal cultures (A, inset). (B, inset).

13. Example: Nimesulide protects B12 neuronal cells against glutamate toxicity We tested nimesulide, indomethacin and NS398 (another preferred COX-2 inhibitor) against glutamate-mediated toxic effects. In this assay, we used B12 neuronal cells and assessed toxicity by increasing the levels of lactate dehydrogenase LDH in the conditioned medium following treatment. LDH is an indicator of cytotoxicity, and its elevation is generally considered a sign of neurotoxicity. As shown in Figures 22-23, we found that nimesulide was pretreated (24 hours ago) and co-treated (co-treated with glutamate for 24 hours) at 10-6, 10-9 and 10-12M, with Protection against glutamate toxicity. Indomethacin has a protective effect at 10-6M, but loses its protective effect at 10-9 and 10-12M. Borderline protection against glutamate-mediated toxicity was observed with indomethacin at 10-9M (24 hours pretreatment). NS398 pretreatment had no protective effect. In the case of pretreatment, the drug remained in the medium when treated with glutamate.

Various publications are cited in this article, the contents of which are incorporated in their entireties here.

Sequence Listing (1) General Information (i) Applicants: Pasinetti, Giulio M. and Aisen, Paul S. (ii) Title of Invention: Treatment of Neurodegenerative Diseases with Nimesulide (iii) Sequence Number: 1 (iv) Communication address:

(A) Addressee: Baker & Botts, L.L.P.

(B) Street: 30 Rockefeler Plaza

(C) City: New York

(D) State: NY

(E) Country: USA

(F) ZIP: 10112-0228(v) computer readable form

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(D) Software: Fast SEQ version 1.5 (vi) This application material

(A) Application number:

(B) Filing date:

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(A) Application No.: 08/831,402

(B) Filing date: April 1, 1997 (viii) Agent information

(A) Name: Clark, Richard S.

(B) Registration number: 26,154

(C) Reference/Application Number: (ix) Telecom Information

(A) Tel: 212-705-5000

(B) Fax: 212-765-2519

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(2) SEQ ID NO: 1 information (i) SEQUENCE CHARACTERISTICS:

(A) Length: 16 amino acids

(B) Type: amino acid

(C) chain: single chain

(D) Topology: Linear (ii) Molecular Type: Peptide (iii) Hypothetical: NO (iv) Antisense: NO (v) Fragment Type: (vi) Source: (xi) Sequence Description: SEQ ID NO: 1 :

Cys Asn Ala Ser Ala Ser His Ser Arg Leu Asp Asp Ile Asn Phe Thr 16

5 10 15

Claims (10)

CLAIMS 1. A composition comprising an effective amount of nimesulide for use in a method of protecting a patient suffering from a neurodegenerative disease from non-inflammatory neuronal cell death. 2. The composition of claim 1, wherein the neurodegenerative disease is Alzheimer's disease. 3. A composition comprising an effective amount of nimesulide for use in a method of protecting a patient suffering from amyotrophic lateral sclerosis from neuronal cell death. 4. A composition comprising an effective amount of nimesulide for use in a method of protecting a patient suffering from neurodegeneration due to chronic epilepsy from neuronal cell death. 5. The composition of claim 1, wherein neurons affected by the neurodegenerative disease are exposed to nimesulide at a concentration of at least 1 micromolar. 6. The composition of claim 2, wherein neurons affected by the neurodegenerative disease are exposed to nimesulide at a concentration of at least 1 micromolar. 7. The composition of claim 3, wherein neurons affected by the neurodegenerative disease are exposed to nimesulide at a concentration of at least 1 micromolar. 8. The composition of claim 4, wherein neurons affected by the neurodegenerative disease are exposed to nimesulide at a concentration of at least 1 micromolar. 9. A transgenic animal carrying a gene encoding human cyclooxygenase-2. 10. A neurogenic cell culture obtained from the transgenic animal of claim 9.

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