WO2000066725A1

WO2000066725A1 - Use of inducible no-synthase antisense oligonucleotides for preventing and treating cerebral ischemia

Info

Publication number: WO2000066725A1
Application number: PCT/FR2000/001191
Authority: WO
Inventors: Sophie Parmentier; Andrees Bohme; Michel Plotkine
Original assignee: Aventis Pharma S.A.
Priority date: 1999-05-04
Filing date: 2000-05-03
Publication date: 2000-11-09
Also published as: AU5081200A

Abstract

The invention concerns the use of antisense oligonucleotides of an inducible isoform of nitrogen monoxide synthase for preventing and treating cerebral ischemia. The invention also concerns vectors containing a nucleic acid coding for said antisense oligonucleotide sequences and compositions containing them.

Description

USE OF ANTISENSE NO-SYNTHASE OLIGONUCLEOTIDES

INDUCTIBLE IN THE PREVENTION AND TREATMENT OF

THE CEREBRAL ISCHEMY.

The present invention relates to the therapeutic field of the central nervous system and more particularly the use of antisense oligonucleotides of an isoform inducible of nitric oxide synthase (NO-synthase, NOS, EC 1.14.13.39) in the prevention and the treatment of cerebral ischemia. The invention also relates to vectors containing a nucleic acid encoding said antisense oligonucleotides and compositions containing them.

Cerebral ischemia is characterized by a decrease in cerebral blood flow resulting in a deficit in energy substrates essential for the brain (oxygen and glucose).

The consequences of ischemia depend on its severity (intensity of the drop in blood flow) and its duration. The decrease in blood flow leads to major cellular dysfunctions including the increase in cytosolic calcium, the activation of numerous enzymes which cause a loss of cellular integrity and the release of excitatory amino acids such as glutamate. and aspartate, leading to neuronal death.

At present, ischemic attacks of the brain are still one of the main causes of mortality and morbidity, due to the lack of effective therapies. The ^ischemia experimental models by occlusion of the middle cerebral artery (MCA) developed in various animal species have revealed the neuroprotective effect of strategies consisting or block different kinds of glutamate receptors or inhibiting release of glutamate.

However, it has been described by Margaill et al. (1996) that "anti-glutamate" strategies, effective in models of permanent focal ischemia, only present a narrow window of therapeutic opportunity in certain models of transient focal ischemia. This could be at the origin of the current difficulties in demonstrating an activity of these "antiglutamate" treatments during clinical trials. Furthermore, Margaill et al. (1997) demonstrated that a strategy aimed at inhibiting the production of nitric oxide (NO) retains protective activity even when treatment is started 9 hours after ischemia. This window of therapeutic opportunity, considerably greater than that observed following the administration of agents acting on glutamate, has prompted further exploration of the late mechanisms occurring following ischemia. Indeed, it can be hypothesized that a therapeutic strategy aimed at opposing a late deleterious phenomenon could have a clinical interest greater than that of "anti-glutamate" strategies.

The deleterious role of NO synthase and in particular of an inducible isoform of this enzyme has been established in models of permanent and transient focal ischemia thanks to the use of transgenic animals in which a gene coding for an inducible NOS has been invalidated (Iadecola et al., 1997) or of pharmacological substrates that inhibit inducible NOS (Parmentier et al, 1999).

The effect of inducible NOS antisense oligonucleotides has already been studied in the case of renal ischemia (Noiri et al, 1996). The results of these studies demonstrate an alleviation of kidney dysfunction in rats subject to experimental ischemia of these organs and an improvement in the viability of renal epithelial cells (Peresleni et al, 1996).

The effect of inducible NOS antisense oligonucleotides has also been studied in the case of a nervous system disorder of an immune nature such as multiple sclerosis (Ding et al., 1998). The results of this study demonstrate the inhibition of the induction of experimental autoimmune encephalomyelitis in mice.

Although studies have been conducted with antisense, the fact remains that no study has used this strategy to specifically block the synthesis of inducible NO-synthase and demonstrate the beneficial role of this inhibition in ischemia cerebral.

The Applicant has now demonstrated a surprising therapeutic effect for the protection of very specific cells, the nerve cells, in a disorder of the metabolic nervous system such as cerebral ischemia by the administration of antisense oligonucleotides. The present invention therefore relates to the use of antisense of an inducible isoform of NO synthase for the protection of nerve cells oligonucleotides against deleterious effects of overproduction monoxide ^nitrogen during cerebral ischemia. In particular, the invention relates to the use of antisense oligonucleotides of an inducible isoform of NO synthase for the preparation of a medicament intended for the prevention and treatment of cerebral ischemia.

We classically distinguish two groups of NO synthase. The so-called "constitutive" NO synthases (cNOs) are present in a physiological and calmodulin dependent state. These enzymes produce nitrogen monoxide (NO) for short periods (Bredt and Snyder, 1990; Fôrsterman et ai, 1991). This NO intervenes as mediator in many physiological processes such as vasodilation and neurotransmission. By purification and then cloning techniques, a neuronal constitutive isoform of NO-synthase (Bredt et al, 1991) and an endothelial constitutive isoform of NO-synthase (Lamas et al, 1992) have thus been identified. Inducible NO synthases (iNOs) are generally not present in the physiological state but are induced following stimulation by biological messengers such as cytokines such as interferon-gamma or rinterleukin-1, or bacterial products like lipopolysaccharides. These enzymes produce NO in large quantities and over long periods and their activation is calmodulin-independent. The NO produced plays a deleterious role in various pathophysiological processes. An inducible NO synthase has been cloned from mouse macrophages (Xie et al., 1992). It was then shown that NO synthase activity could also be induced in a large number of cell types such as smooth muscle cells, hepatocytes, cardiomyocytes, neutrophils, microglia, astrocytes, chondrocytes (Nathan, 1992) and also neurons (Minc-Golomb et ai, 1992).

Thus, in the context of the invention, the NO synthase against the expression of which the oligonucleotides of the invention are used is preferably an inducible NO synthase (iNOs) or also called type 2. The antisense oligonucleotides according to the invention constitute antisense sequences of the messenger RNAs of all or part of the enzyme NO synthase. As described above, the antisense oligonucleotides of the invention are preferably directed against all or part of an inducible NO synthase.

According to a first embodiment of the invention, the sequence of the antisense oligonucleotides according to the invention corresponds to the sequence SEQ ID No. 1 or one of the derived sequences.

According to a variant of the invention, the sequence of the antisense oligonucleotides corresponds to the sequence SEQ ID No. 2 or one of the derived sequences.

The term “derived sequence” means any sequence obtained by modification and coding for a product retaining the property of inhibiting the synthesis of an inducible isoform of NO synthase. This derived sequence also shares 90% and preferably 80% homology with the native sequence.

By modification is meant any mutation, deletion, substitution addition or modification of a genetic and / or chemical nature. These modifications can be carried out by techniques known to those skilled in the art. These derivatives can in particular be molecules having a great affinity for their binding sites, sequences allowing an improved expression in vivo, molecules having a greater resistance to proteases, molecules having a greater therapeutic efficacy.

Among the preferred derivatives, there may be mentioned more particularly the natural variants, the molecules in which one or more residues have been substituted, the derivatives comprising, relative to the native sequence, additional residues such as, for example, secretion and / or junction.

The sequence of the antisense oligonucleotide as described above comprises between 10 and 30 seas and preferably between 15 and 25 seas.

Another subject of the invention relates to a vector comprising in its genome a nucleic acid coding for the antisense oligonucleotide according to the invention. The vector of the invention can be for example a plasmid, a cosmid or any DNA encapsulated by a virus, a phage, an artificial chromosome, a recombinant virus, etc. It is preferably a plasmid or a recombinant virus.

By way of viral vectors in accordance with the invention, mention may very particularly be made of vectors of the adenovirus type, retroviruses, adeno-associated viruses, herpes virus or vaccinia virus as well as baculoviruses.

According to a preferred embodiment of the invention, the viral vector is defective, that is to say devoid of the sequences necessary for its autonomous replication in the target cells. Obtaining defective viral vectors such as those mentioned above is now well known to those skilled in the art.

Generally, the nucleic acid sequence includes a promoter region for functional transcription. It may be a promoter region of eukaryotic or viral genes.

Furthermore, the vector may include in its genome, in particular upstream of the nucleic acid sequence coding for the oligonucleotide according to the invention, a signal sequence directing the product synthesized in the secretory pathways of the target cell. This signal sequence can be a natural signal sequence or an artificial signal sequence.

The vector according to the invention can be used both in an ex vivo or in vivo approach. Within the framework of a gene therapy applicable to humans, an ex vivo approach can be envisaged by grafting cells genetically modified by a vector described above. These cells can be of various origins: for example nervous (human or non-human). The combination of certain drugs can be considered with a view in particular to improving the maintenance of the transplant or the expression of the transgene (immunosuppressants, anti-complement ...). It is also possible to envisage the implantation of genetically modified cells by a vector of the invention after packaging in an inert system. For their use according to the present invention, the antisense oligonucleotides or the vector containing them are preferably associated with one or more pharmaceutically acceptable vehicles to be formulated for administration by topical, oral, parenteral, intranasal, intravenous, intramuscular, sub- cutaneous, intraocular, transdermal, stereotaxic, etc. Preferably, the antisense oligonucleotides or the vector containing them are used in an injectable form. They may in particular be saline solutions (monosodium phosphate, disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition, as appropriate, of sterilized water or physiological saline, allow the constitution of injectable solutes. The doses of virus used for administration can be adapted as a function of various parameters, and in particular as a function of the site of administration considered, the number of injections, the gene to be expressed, or also the duration of the treatment sought. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 ^ and 10 -4 pfu, _e t preferably 10 "to 10 ^ pfu. The term pfu (" plaque forming unit ") corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 15 days, the number of plaques of infected cells. Techniques for determining the pfu titer of a viral solution are well documented in the literature.

This is why another object of the invention resides in a pharmaceutical composition comprising at least one viral or plasmid vector comprising in its genome a nucleic acid coding for the antisense oligonucleotide defined above.

Furthermore, another subject of the invention resides in a pharmaceutical composition characterized in that it comprises at least one antisense oligonucleotide of an isoform inducible of NO synthase for protection against cerebral ischemia.

Another subject of the invention is a medicament comprising at least one antisense oligonucleotide of an isoform inducible of nitric oxide synthase for the prevention and treatment of cerebral ischemia. Yet another object of the invention relates to the use of a vector described above for the manufacture of a medicament intended for the prevention and treatment of cerebral ischemia.

Thus, the invention also consists of a medicament comprising at least one vector as described above and preferably comprising at least one viral or plasmid vector.

LEGEND OF FIGURES

Figure 1: Realization of the transient focal ischemia model. Installation of the temporary occlusion device of the common carotid artery (A) and maneuver thereof (B).

Figure 2: Realization of the transient focal ischemia model. Installation of the temporary occlusion device (microclip) of the middle cerebral artery.

Figure 3: Quantification of histological damage by staining with triphenyl-tetrazolium.

Figure 4: Installation of the guide for intracerebroventricular injection

MATERIAL AND METHODS

All the experiments are carried out on male Sprague-Dawley rats (Iffa Credo, France) weighing between 300 and 330 grams.

1. TRANSIENT FOCAL CEREBRAL ISCHEMIA

The transient focal ischemia model used requires a craniectomy in order to clear the middle cerebral artery (MCA). The subtemporal approach we use is that originally described by Tamura et al. ( nineteen eighty one). 1.1. Surgical procedure

1.1.1. Surgical approach to the middle cerebral artery

The rats are anesthetized by intraperitoneal injection (i.p.) of chloral hydrate (400 mglkg in a volume of 10 ml / kg). The animal is then placed on the right flank under a binocular magnifier (Zeiss). The scalp is incised along an axis starting from the left ear using an electric scalpel (ME 80®, Martin). Along this same axis, the temporal muscle is incised and the zygomatic arch removed. A craniectomy 3 to 4 mm in diameter is then carried out around the oval foramen using a dental bur (Minigyr®, Anthogyr, France). The dura is incised to release the left MCA.

Throughout the operation, the rectal temperature of the animals is monitored using thermal probes (Digi-Sense®, Cole-Palmer, Chicago, USA) and maintained at 37.5 ± 0.5 ° C thanks to to a heating blanket.

1.1.2. Transient focal ischemia

The transient focal ischemia model used consists of reversibly occluding the left MCA using a microclip. Associated with this occlusion is the clamping of the common ipsilateral carotid artery (ACCi). For this, the rats, anesthetized by intraperitoneal injection of chloral hydrate (400 mg / kg in a volume of 10 ml / kg), are placed in supine position. A longitudinal incision is made in the tracheal region, the ACC is released and isolated from the vagus nerve using a silicone tube (Silastic®, ref. 602-7, Sigma), on which is inserted a lozenge d '' about 5 mm in diameter also made of silicone (Silastic®, ref. 500-135, Sigma) previously drilled with two holes. The free ends of the silicone tube are knotted as shown in Figure 1A. The ACC is thus ready for clamping which will be carried out immediately after the microclip is placed on the ACM.

The ACM surgical approach is that described in paragraph 1.1.1.

This is then occluded at its proximal part, upstream of the lenticulo-striated artery, by a microclip (zen type temporary clip, 15 mm x 0.4 Ohwa Tsusho Co., Ltd Tokyo, Japan) inserted perpendicularly on the surface of the brain, using special pliers (Zen clip applier type, 15 cm, Ohwa Tsusho Co., Ltd Tokyo, Japan) (Figure 2). The incision of the superficial skin planes is then closed with a surgical clip.

Finally, the ACC is clamped by threading onto the silicone tube a blue Gilson® pipette tip (Polylabo) previously cut at its two ends and split in its upper part; by pulling on the wire and wedging it in this slot, the carotid circulation is interrupted (Figure 1B).

At the end of the desired duration of occlusion, the animals are again anesthetized with chloral hydrate. The incision is reopened and the microclip and the carotid clamping are removed. The temporal and tracheal incisions are then permanently closed using staples. The animals are placed in enclosures thermostatically controlled at 29 ° C until they wake up and then returned to the animal facility until they are euthanized. The animals, having difficulty chewing, are fed with granules previously softened in water.

1.2. Measurement of physiological variables

For the measurement of mean arterial pressure (mean BP), partial O ₂ pressure (pO ₂ ), partial CO ₂ pressure (pCO ₂ ) and pH, a polyethylene catheter (Biotrol®, ref. E03403) is introduced from anesthesia of the animals into the artery of the tail. The catheter is connected to a three-way stopcock; one is used for drawing blood for the analysis of pO, pCO ₂ and pH by a blood gas analyzer (ABL 330, Radiometer®, Copenhagen); the other is connected to a sensor (EMKA Technologies, Paris), connected to a recorder (SEFRAM, Paris), for the measurement of average BP.

This device requires heparinization of animals. A heparin solution (Choay® heparin solution for injection) at 50 IU / ml of physiological solution is prepared. Two hundred microliters of this solution are injected through the tap as soon as the catheter is placed, then each time blood is drawn. The animals are kept in normothermia for the entire duration of the measurements using a heating blanket. Three measurements of the physiological variables are carried out, one immediately before the clamping of the arteries, the second 30 minutes after the clamping of the arteries, finally the third is carried out 30 minutes after the clogging of the arteries. Simultaneously, the rectal temperature of the animals is raised, in order to check their normothermia. At the end of the experiment, the catheter is removed and the tail artery is occluded using a wire.

It should be noted that the animals are kept anesthetized during the measurement of physiological variables.

1.3. Measurement of cerebral blood flow

The measurement of the cerebral blood flow is carried out using a laser-Doppler (Laseflow BPM2 (E Vasamedics). Animals anesthetized with a solution of chloral hydrate (400 mg / kg in a volume of 10 ml / kg ) are placed in stereotaxic restraint. The surface of the skull is shaved and an incision is made in order to clear the calvariwn. An orifice is drilled to allow the introduction of the laser-Doppler probe into the chosen region. Cerebral blood flow (DSC) is measured in an area corresponding to the darkness of the infarction The coordinates of the measurement were determined using Atlas Paxinos and Watson (1986):

- Antero-posteriority: 4.2 mm from the interaural line.

- Laterality: -3 mm compared to bregma.

The probe is placed on the surface of the cortex in contact with the dura mater. The probe is held at its measurement site thanks to a polyethylene support glued to the skull bone with a cyanolit® type glue. The animal is then removed from the stereotaxic apparatus and the surgical procedure of ischemia can be started. 1.4. Quantification of functional impairment

1.4.1. Weightloss

The weight loss of the animals is evaluated by the difference between the weight before the ischemia and the weight on the day of euthanasia.

1.4.2. Neurological deficit

The neurological examination consists of an evaluation of the presence of different reflexes and behavioral reactions.

Neurological disturbances are assessed using the following tests:

- The grip reflex at each of the forelegs. Score = 1: able to grip / 0: does not grip.

Total = 1 for each side.

- Visual placement reactions on each of the forelegs. Score = 1: able to place / 0: does not place.

Total = 1 for each side.

- Reactions of loss of support in each of the anterior and posterior legs.

Score = 1: takes support / 0: does not take support. Total = 2 for each side.

- The righting reflex with the rotation test. Score = 1: rotation in the opposite direction / 0: no rotation. Total = 1 for each side.

The overall neurological score is therefore for each side equal to 5.

1.5. Quantification of histological damage

Euthanasia of rats is carried out by injection of a massive dose of sodium pentobarbital (120 mg / kg, ip) (Sanofi). Their brains are removed and cut into 7 coronal sections 2 mm thick using a matrix for rat brains. They are taken from anteriority level 13.7 with respect to the inter-aural line and this every 2 mm up to anteriority level 1, 7.

1) Coloring

The freshly removed sections are immersed in a 2% solution of triphenyl tetrazolium hydrochloride (TTC) (Sigma) for 20 minutes at room temperature. This solution causes the healthy areas to be colored pink while the necrotic areas do not stain. The sections are then fixed in a 4% solution of paraformaldehyde (PFA, Sigma) in a sodium phosphate buffer at 50 mM (pH 7) and placed in the dark. The areas of infarction can then be measured 24 hours after staining (Figure 3).

2) Quantification of histological damage

The sections are examined using an image analysis system (IMSTAR®, Paris, France). The cortical and striatal infarction zones are delimited using a cursor and their area measured by the computer. We also determine the surface of the right hemisphere and the left hemisphere in order to evaluate the cerebral edema for each of the digitalized frontal sections. The areas of necrosis measured are then corrected as a function of this edema (that is to say multiplied by the ratio of the area of the right hemisphere to the area of the left hemisphere). The volume of the infarction can then be calculated according to the values of corrected areas of necrosis and the distance separating each of the cutting levels.

The edema is calculated as follows: (area of the left hemisphere - area of the right hemisphere / area of the right hemisphere)

2. INTRACEREBROVENTRICULAR INJECTION IN THE RAT

The route of administration chosen for the study of the effect of the inducible anti-sense anti-NOS oligonucleotides required the installation of a guide, 5 days before the first treatment (FIG. 4). 2.1. Guide installation

An 8 mm long guide, made from a 25G gauge needle (Terumo®), is attached to the micromanipulator of a stereotaxic device (David Kopf®, Roucaire establishment).

The rats are anesthetized with chloral hydrate (400 mg / kg, i.p.) and then placed in stereotaxic restraint. The surface of the skull is shaved and an incision is made to uncover the skull; the calvarium is clear. An opening is pierced vertically in the left ventricle according to the coordinates determined using Atlas Paxinos and Watson (1986):

- Anteroposteriority: 0.8 mm compared to bregma - Laterality: 1.5 mm compared to bregma.

The guide is then brought using the micromanipulator to the surface of the dura mater so as to induce as little damage as possible.

The guide is then fixed to the cranial box with dental cement (AP9,

AtlanticCodental) and two anchor screws. We can then detach the micromanipulator guide. The animal is removed from the stereotaxic device and returned to the animal house under normal conditions of housing.

2.2. Inducible anti-NOS antisense oligonucleotide injection protocol

Inducible anti-NOS antisense was synthesized using an automatic synthesizer. Treatments begin 5 days after the guide is put in place. The injection needle consists of a 30G caliber dental needle (Sofijet®), on which a stopper, consisting of a 23G caliber needle section (Terumo®) was previously glued using cyanolit® type glue, 1 1 mm from the end of the bevel of the dental needle. In this way, when the needle is inserted in its guide to the stopper, the middle of its bevel is 3.5 mm from the surface of the skull, which according to the Atlas of Paxinos and Watson (1986 ) corresponds to the center of the ventricle. EXAMPLES

EXAMPLE A Administration of the Antisense Oligonucleotides of the Invention

The oligonucleotides are administered in solution in artificial cerebrospinal fluid (Dulbecco, Sigma, ref. D8662) at a concentration of 1 nmole / μl. Three nanomoles of the different sequences (SEQ ID No. 1 and No. 2) are injected in a volume of 3 μl in 2 minutes using an infusion pump (flow rate of 1.5 μl / min), 12 hours before ischemia, just before ischemia, then every 12 hours until the animals are euthanized, 3 days after ischemia. The animals serving as controls for the solvent receive, according to the same protocol, an injection of artificial cerebrospinal fluid.

The antisense (AS) or antisense oligonucleotide sequence, its control sequence or the solvent are administered according to the protocol described above. Three days after ischemia, the animals are euthanized and their brains removed and cut into

7 coronal sections 2 mm thick using a matrix. The fourth section is isolated and dissected on ice. In this section, a sample containing both cortical infarction (except the clip area) and striatal infarction is taken and frozen at -40 ° C for the assay of NOS activities and nitrotyrosine production. These assays are also carried out in control-operated, but untreated animals, and in non-operated control rats. The other 6 sections are immersed in a 2% TTC solution for the measurement of infarct surfaces. Despite the absence of the fourth section, the total volume of the infarction can be determined, since the area of the infarction corresponding to the fourth section, is identical to that measured on the back of the third section.

EXAMPLE B Effect of the Antisense AS Oligonucleotide and Its Control Sequence on Histological and Functional Impairments

The purpose of this example is to demonstrate a histological and functional improvement with respect to the control sequence, by the action of the oligonucleotide of the invention, measured through parameters indicative of functional and histological damage. The treatments are injected intracerebroventricularly as described in example A (dose of 3 nmoles (1 nmole / μl) 12 hours before ischemia, just before ischemia, then every 12 hours until euthanasia of the animals, 3 days after ischemia).

1) Volumes of infarction

Three days after ischemia, the control animals which received the solvent have cortical and striatal infarction volumes of 1 12 ± 7 and 42 ± 2 mm ^{3 respectively} . These volumes are not significantly modified by the control sequence. On the other hand, the rats treated with the sequence AS have volumes of cortical infarction reduced by 25 to 50% compared to those of the animals treated with the solvent, and those of the rats treated with the control sequence. The volumes of striatal infarctions are not significantly affected by the AS sequence. nor by its control sequence. The reduction of the infarction is exerted on the levels of the sections ranging between 1 1, 7 and 9,7 mm compared to the interaural line. which corresponds to the anterior part of the infarction.

2) Neurological score

Control-operated animals have a neurological score of 5 for the left and right sides. Ischemia induces a marked neurological deficit on the right side. Treatment with the control sequence does not modify this neurological deficit. On the other hand, treatment with the AS sequence significantly reduces the deficit on the right side compared to that of the animals treated with the solvent, and compared to that of the rats treated with the control sequence.

3) Weight loss

The evolution of weight loss during the days following the ischemia of the animals treated, either by the AS sequence, or by its control sequence, or by the solvent was measured as well as in control-operated animals.

Control-operated animals lose an average of 7 grams during the two days after the intervention, then they regain their initial weight. After ischemia, the animals lose an average of 36 ± 3 g the first day after the intervention, 52 ± 2 g at 2 days and 66 ± 3 g at 3 days. Control sequence processing does not affect this gradual drop in weight. On the other hand, the rats treated with the AS sequence exhibit, 2 days after ischemia, a reduced weight loss of between 10 and 30% compared to those induced in the animals treated with the solvent or in the rats treated with the sequence control. Three days after ischemia, weight loss is no longer changed by treatment with the AS sequence.

By measuring the various parameters above, this example demonstrates that the animals treated with the antisense oligonucleotides present, 3 days after ischemia, a reduction of between 25 and 50% in the volumes of cortical infarction and a marked improvement in sensorimotor performance. This example also made it possible to demonstrate that treatment with the control sequence of the antisense has no effect, either on the size of the lesions or on the functional disorders. This absence of effect of the control sequence clearly demonstrates that the effect of the antisense oligonucleotide is not linked to a non-specific activity, but results from the inhibition of the synthesis of an inducible NO synthase.

EXAMPLE C Effect of the antisense oligonucleotide AS and of its control sequence on the assay of the NO-synthase activities

The purpose of this example is to demonstrate that the administration of antisense oligonucleotides according to the invention allows a reduction in the calcium-independent NOS activity induced by ischemia.

The calcium-dependent and calcium-independent NOS activities of the animals treated, either by the AS sequence, or by its control sequence, or by the solvent were evaluated as well as in control-operated rats and in non-operated control rats.

1) Calcium-dependent NO-synthase activity

Ischemia induces a decrease in calcium-dependent NOS activity compared to control-operated and non-operated control animals. Treatments with the AS sequence and its control sequence have no effect on this activity. 2) Calcium-independent NO-synthase activity

Ischemia induces an increase in calcium-independent NOS activity by a factor of 6 to 10 compared to control-operated and non-operated control animals.

Treatment with the control sequence does not significantly modify this activity.

On the other hand, treatment with the AS sequence results in a reduction of 25 to 50% in the calcium-independent NOS activity induced by ischemia.

The results of this example demonstrate that the administration of antisense oligonucleotides according to the invention advantageously makes it possible to reduce the calcium-independent NOS activity induced by ischemia in nerve cells.

These results further show that the treatment with antisense reduces between 25 and 50% the inducible NOS activity three days after ischemia, whereas its control sequence has no significant effect on this enzymatic activity. On the other hand, the antisense as its control does not modify the constitutive NOS activity. These data reveal the inactivity of the control sequence and the specific inhibitory effect of the antisense of the invention on the synthesis of NOS 2. In addition, the reduction in NOS 2 activity associated with the neuroprotective effect, observed in animals treated with antisense, suggests that the antisense exerts its beneficial effect via the inhibition of the synthesis of NOS 2.

EXAMPLE D Effect of the Antisense Oligonucleotide AS and Its Control Sequence on the Production of Nitrotyrosine

The nitrotyrosine production of the animals treated, either by the AS sequence, or by its control sequence, or by the solvent was thus evaluated in control-operated rats and in non-operating control rats.

Three days after ischemia, the production of nitrotyrosine is increased by a factor of 2 compared to that of control-operated and non-operated control animals. The control sequence does not modify this production. On the other hand, treatment with the AS sequence reduces by about a third the increase in the quantities of nitrotyrosine induced by ischemia. This example demonstrates the effect of the antisense of the invention on the production of peroxynitrites, the main toxic metabolites of NO. This production was evaluated by the nitrotyrosine assay. The results show that the treatment with antisense reduces by about 1/3 the increase in the production of nitrotyrosine induced by ischemia, while the control sequence has no significant effect on this production.

In addition, it should be noted that the importance of reducing the formation of peroxynitrites under the effect of the antisense is of the same order as the inhibition of the inducible NOS activity. This data seems to indicate that peroxynitrites come from

NO produced by the activation of an inducible NOS. Thus, antisense by inhibiting the synthesis of an inducible NOS reduces the formation of peroxynitrites. This effect associated with neuroprotection further suggests that peroxynitrites are responsible for the deleterious effects of activating an inducible NOS.

EXAMPLE E Effect of the antisense oligonucleotide AS and of its control sequence on physiological variables

The physiological variables of the animals treated, either by the AS sequence, or by its control sequence, or by the solvent were measured.

The control animals have physiological values of mean arterial pressure, pO ₂ , pCO ₂ , pH and rectal temperature which are not modified either by ischemia or by reperfusion. Treatments with the AS sequence and its control sequence have no effect on these physiological variables.

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Claims

1. Use of antisense oligonucleotides of an isoform inducible of nitric oxide synthase (iNOS) for the preparation of a medicament intended for the prevention and treatment of cerebral ischemia.

2. Use according to claim 1, characterized in that the antisense oligonucleotides are antisense for all or part of the inducible NO-synthase.

3. Antisense oligonucleotides as described according to claim 2, characterized in that the sequence of the oligonucleotides corresponds to the sequence presented in SEQ ID No. 1 or one of the derived sequences.

4. Antisense oligonucleotides as described according to claim 2, characterized in that the sequence of the oligonucleotides corresponds to the sequence presented in SEQ ID

N ° 2 or one of the derived sequences.

5. Antisense oligonucleotides as described according to claim 2, characterized in that the sequence of the oligonucleotides comprises between 10 and 30 seas.

6. Antisense oligonucleotides as described in claim 2, characterized in that the sequence of the oligonucleotides comprises between 15 and 25 seas.

7. Composition characterized in that it comprises at least one antisense oligonucleotide of an isoform inducible of nitric oxide synthase for the prevention and treatment of cerebral ischemia.

8. Vector comprising in its genome a nucleic acid coding for the antisense oligonucleotides as described according to claim 1.

9. Vector according to claim 8, characterized in that it is a plasmid vector.

10. Vector according to claim 8, characterized in that it is a recombinant virus.

1 1. Virus as described in claim 10, characterized in that it is defective.

12. Virus according to claim 11, characterized in that it is an adenovirus, a retrovirus, an adeno-associated virus, a herpes virus, the vaccinia virus.

13. Use of a vector as described according to one of claims 8 to 12 for the manufacture of a medicament intended for the prevention and treatment of cerebral ischemia.

14. Composition comprising at least one vector as described according to claims 8 to 12.

15. Medicament comprising at least one vector as described according to claims 8 to 12.

16. A medicament comprising at least one antisense oligonucleotide of an isoform inducible of nitric oxide synthase for the prevention and treatment of cerebral ischemia.