Field of the invention.

The present invention relates to the use of propylparaben (PPB) as neuroprotective agent to attenuate neuronal damage produced by Status epilepticus (SE) in mammals. Particularly relates to the administration of PPB, a compound widely used as antimicrobial, immediately after the occurrence of Status epilepticus to attenuate neuronal injury and prevent the possible development of epileptogenesis and neurocognitive disorders associated with this disease.

Background of the invention.

Epilepsy is a neurological disorder of the central nervous system (CNS) that is characterized by recurrent seizures with no apparent systemic cause or acute neurologic injury. It is associated with increased excitatory neurotransmission mediated principally by glutamate and reduced inhibition by gamma-aminobutyric acid (GABA). The worldwide prevalence of epilepsy is high (1 -2% of the population) with a cumulative lifetime incidence approaching 3%. Susceptibility to this disease is greater when individuals suffering therefrom have a history of Status epilepticus (SE).

SE is a life-threatening neurological disorder that essentially consists of an acute epileptic episode. It is characterized by repeated seizures (partial or generalized, convulsive or non-convulsive) for a period of more than 30 minutes without recovery between them. It can also be defined as a prolonged crisis lasting more than five minutes. SE is associated with increased morbidity and mortality more than any other neurological condition (22,000 to 42,000 deaths every year), while factors such as age, etiology, and seizure duration are determinants of the severity of this condition.

Several patent documents and reports describe the use of various compounds to control SE; however, none of these compounds has the ability to prevent neuronal damage subsequent to SE.

It is also well known that administration of propylparaben (PPB) attenuates convulsions caused by electroconvulsive shock and pentylenetetrazol.

The patent US2007244039 relates to the use of cyclic Prolyl Glycine ("cyclic PG" or "cPG") and analogs and mimetics thereof as neuroprotective agents for the treatment and/or prevention of neurological disorders including cerebral ischemia or cerebral infarction resulting from a range of phenomena such as thromboembolic or hemorrhagic stroke, cerebral vasospasms, hypoglycemia, cardiac arrest, Status epilepticus, perinatal asphyxia, anoxia such as from drowning, pulmonary surgery, and cerebral trauma, as well as to the treatment and prevention of chronic neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, and as anticonvulsants.

The patent US2013079336 relates to methods for terminating, preventing, and/or treating Status epilepticus. The method can include administering a prophylactically or therapeutically effective amount of stiripentol or a related compound thereof to an individual in need of treatment of Status epilepticus.

The patent WO201 1034849 relates to pharmaceutically active compounds of Formula I useful in the treatment of central nervous system (CNS) diseases and disorders such as anxiety, depression, insomnia, migraine headaches, schizophrenia, Parkinson's disease, Alzheimer's disease, and bipolar disorder.

Formula (I)

Despite the development of new anticonvulsant drugs, these do not prevent the neuronal damage produced by epileptic activity and SE. Recent studies of Virtual Screening (VS) suggest that molecules used on a daily basis produce anticonvulsant effects. However, to date, no documents have been found related to the use of PPB to decrease the expression and development of SE.

Therefore, it is necessary to repurpose available drugs to find additional molecular targets for developing new therapies to decrease the neuronal damage induced by Status epilepticus (SE) in mammals.

Brief description of the invention.

The present invention relates to the use of PPB, a compound generally used as an antimicrobial, to attenuate the neuronal damage produced by SE.

In the present invention, SE was induced in male Wistar rats by lithium-pilocarpine model. Animals were maintained for 2 hours in SE, and one hour after stopping SE with diazepam, they were given a sole dose of PPB (177.77 mg/Kg, i.p.). Animals surviving SE were sacrificed 7 days later, and their brains were processed for histological evaluation of neuronal damage in the dorsal hippocampus (Nissl and Fluoro-Jade staining). The result showed that, compared to control animals, the animals treated with PPB exhibited less neuronal damage in dentate gyrus, hilus, and CA3, with no changes in CA1 pyramidal cells. Brief description of the drawings.

Figure 1. Shows representative photomicrographs of coronal sections at the level of dorsal hippocampus level of brains processed for Nissl staining of an animal of the control group (A) with SE (B) and an animal of the SE+PPB group (C). Note the cell loss at hilus level, CA3 and CA1 (arrows) of panel B compared with panel C.

Figure 2. Shows representative photomicrographs of the regions of dorsal hippocampus (Nissl staining) at the dentate gyrus, hilus, CA3, and CA1 level. The upper panels correspond to an animal of the control group, the panels in the middle to sections of a rat of the SE group, and the lower panels to the sections of an animal of the SE+PPB group. Significant neuronal loss

(arrows) is observed in the animal of the SE group compared to animals of the SE+PPB group, except in CA1 pyramidal cells.

Figure 3. Shows representative photomicrographs of brain sections processed with

Fluoro-Jade staining in dentate gyrus, hilus, CA3, and CA1 of an animal of the SE group (upper panels) and the SE+PPB group. Cells undergoing apoptosis at 168 h after stopping SE are shown in phosphorescent green (note the white arrows in the panels of the dentate gyrus).

Figure 4. Shows the representation of the average number of cells undergoing apoptosis identified with Fluoro-Jade staining in the dentate gyrus (DG), the hilus (H), CA3, and CA1 , in the SE and SE+PPB groups. Values represent the average of neurons per mm ³ in the process of damage ± standard error. *p<0.05 for the SE group, based on t-Student analysis.

Detailed description of the invention.

The objective of the present invention is to provide the use of PPB to attenuate neuronal damage produced by SE.

Status epilepticus (SE) is an acute epileptic condition characterized by repeated crisis (partial or generalized, convulsive or non-convulsive) for a period of more than 30 minutes without recovery between them. According to its clinical manifestations, Status epilepticus can be divided in two categories: convulsive and non-convulsive.

In generalized convulsive SE, tonic-clonic seizures are more prominent than other type of seizures. The most common cause for status epilepticus in children is a febrile illness and meningoencephalitis, while in adults cerebral ictus (cerebrovascular accident), hypoxia, metabolic disorders, and alcohol intoxication or withdrawal precedes convulsive SE.

There are several subtypes of non-convulsive SE such as generalized SE with typical or atypical absence seizures, SE with complex partial seizures, and generalized non- convulsive SE, which are more difficult to diagnose clinically than convulsive SE. The first two subtypes show nystagmus, aphasia, automation, or abnormal limb postures. Generalized non-convulsive SE is characterized because the patient remains in a coma showing continuous electroencephalographic activity with or without convulsive movements.

Recurrent epileptic activity occurring in SE produces a cascade of fundamental molecular and cellular changes in the development of epileptogenesis. Some hypotheses propose that neuronal cell death and subsequent hippocampal plasticity are the cause for hyper excitability of the hippocampus. The death of GABAergic interneurons suggests decreased GABA-mediated inhibition resulting in pathological hyper excitability of the remaining neurons. It is known that the hilus and dentate granular cells are most vulnerable to neuronal damage. Also, the number of generalized convulsions and the duration of epileptic seizures are closely related to the severity of neuronal damage. It has been reported that a period between forty and sixty minutes of continuous seizures can cause selective neuronal damage in rat brain. The most vulnerable regions to neuronal damage are the hippocampus, cerebral cortex, cerebellum, thalamus regions, nuclei of the amygdale, and piriform cortex.

One embodiment of the present invention is to reduce the intensity of neurotoxic side effects of SE by administering PPB.

Animal models of SE allowed studying the mechanisms involved in the generation of seizures; in addition to design new methods of diagnosis and test the efficacy of antiepileptic drugs or other therapeutic alternatives. The SE model induced by Lithium- pilocarpine is commonly used for the evaluation of new anticonvulsants and antiepileptogenic strategies. The Lithium-pilocarpine model reproduces the clinical and neuropathological characteristics of SE. Pilocarpine is a muscarinic receptor agonist, which induces secondarily generalized limbic seizures that evolve to SE lasting up to 18 hours (acute period). SE is followed by a silent phase (without seizures) and a chronic stage that is characterized by the presence of recurrent spontaneous seizures (epilepsy).

In the lithium-pilocarpine model, the hippocampus is the primary structure involved in the generation of convulsive episodes, an effect associated with increased metabolic activity of structures such as CA1 , dentate gyrus, subiculum, and pre-subiculum. This condition leads to neuronal loss with the consequent reduction of GABAergic inhibition in the hippocampus ("dormant basket cell" hypothesis). It has been described that the ventral hippocampal region is highly susceptible to neuronal damage induced by SE. SE is followed by a latency period (days-weeks) in which no crises appear and electroencephalographic activity is apparently normal. Subsequent to the latency period, a gradual and progressive development of spontaneous recurrent seizures (epileptogenesis) is observed.

The treatment used in SE patients involves only the control and prevention of the seizures. By controlled trials, it has been proposed the use of long-acting benzodiazepines such as intravenous lorazepam for the initial control of SE. 35 to 50% of patients with SE have drug resistance to first-line treatments (benzodiazepines), while 20% respond to second-line drugs (phenobarbital, phenytoin, and valproic acid), and only 31 % of the patients respond to a combination of both groups of drugs.

Moreover, virtual screening (VS) is a computational technique used to explore the molecular structure of chemical compounds compiled in large libraries. This technique allows identifying potentially interesting drug candidates in pharmaceutical research. Among other advantages, VS is a timesaving and costly-effective approach, since it can scan databases including millions of chemical structures in a matter of weeks, it does not require a physical sample of the compounds in the data base to perform the scan, and exploration can even be performed on hypothetical structures. Also, VS eases the finding of new therapeutic indications for compounds already known and used in clinic. Additionally, this method of analysis reflects the philosophy of international applicable bioethics guidelines, which propose the use of computational models and in vitro assays previous to the performance of preclinical and clinical in vivo tests. VS techniques can be classified in two main groups: the first focused on the identification of compounds related to a specific molecular target (receiver), and the second to select the molecules depending on the ligand of interest. Propylparaben (PPB) is an ester of para-hydroxybenzoic acid used as an antimicrobial against moulds and yeasts. It has been reported to have inhibitory effects on membrane transport and mitochondrial processes. It meets the criteria of the ideal preservative: broad-spectrum antimicrobial activity, and safe and stable use in a wide range of pH. PPB is commonly used as a preservative in cosmetics, pharmaceuticals, and food industry.

PPB is completely absorbed along the gastrointestinal tract and its absorption is increased by the use of sodium chloride (NaCI). Twenty-four hours after oral administration in cats (168 mg/Kg), 90% of the drug is eliminated in urine and 3% in feces. Furthermore, it has been determined that p-hydroxybenzoic and p- hydroxyhippuric acids are the primary metabolites and do not have cumulative effects. PPB is metabolized by liver carboxylesterases from the β-esterases family. It was established by in vitro studies that the administration of PPB decreases testosterone release and induces a weak estrogenic activity and increased expression of the gene for the progesterone receptor. In female rats, it was found that repeated administration of PPB produces changes in the uterus histomorphology, while decreasing serum testosterone and sperm production in male rats. Evidence of acute toxicity of PPB has been reported since 1930. Over the years, it has been determined that lethal dose 50 (LD50) of PPB administered by various routes in different animal species is high but, to date there are no reports on carcinogenic or mutagenic activity induced by chronic administration of the compound.

Previous laboratory data established that intraperitoneal administration of PPB (60 mg/Kg) with sub effective doses of antiepileptic drugs (diazepam and phenobarbital) significantly increase the latency to convulsive crises in pentylenetetrazol (PTZ) model. Furthermore, it has been reported that the administration of 30 mg/Kg of PPB prevents tonic seizures by maximum electroconvulsive shock in mice.

According to the present invention, PPB can be administered via pharmaceutical compositions designed to this end to attenuate possible neuronal damage in individuals suffering from Status epilepticus (SE). Also, such compositions can be designed and administered according to known methods in the prior art for PPB as active principle to reach its target-binding site and exert its pharmacological activity to decrease neuronal damage caused by SE.

Additionally, PPB can be administered at a concentration of at least 177.77mg/Kg as described herein, with a dosage regimen depending on the patient's condition and according to what is known in the art regarding dosage regimen design; i.e. , considering the patient's characteristics such as sex, age, weight, etc.

The following examples are included with the sole purpose of illustrating the present invention and without implying any limitation to its scope.

Example 1. Materials and methods.

Male Wistar rats weighing 250-300 g were used for the experiment. Animals were housed in transparent acrylic boxes under controlled environmental conditions (light/dark cycles of 12 hours; 22-25°C) and maintained on food and given water ad libitum. The experimental protocol was performed according to the Mexican Official Norm (NOM-062-Z00-1999) and the guidelines (Protocol # 222/04) of the Internal Committee for the Care of laboratory Animals of CINVESTAV (Center for Research and Advanced Studies). SE Group.

Animals were habituated to handling by systemic administration of saline solution (1 ml/Kg, i.p.) for 5 days. They were injected (24-hours later) LiCI (3 mEq/Kg, i.p. Sigma- Aldrich), and 20-hours later, methylscopolamine (1 mg/Kg, s.c ; Sigma-Aldrich) followed by pilocarpine hydrochloride (25 mg/Kg, s.c ; Sigma-Aldrich) 30 minutes later. The onset of SE was considered after the animals showed continual seizures for more than 2 minutes without recovery between them. Two hours after starting SE, animals were injected with diazepam (2.5 mg/Kg i.m. Valium®, Roche) and immediately, they were maintained for one hour at 4°C to reduce hyperthermia achieved during SE. Eight hours after stopping of SE, the rats were administered an additional dose of diazepam (1 .25 mg/Kg, i.m.) and maintained at a temperature of 17°C overnight (n=8). Six samples were obtained from this group for the analysis of neuronal death.

SE+PPB Group.

Animals were handled in the same way as indicated in the previous protocol, except that 1 hour after stopping SE they received a sole administration of PPB (177.77 mg/Kg, i.p. n=8). Control animals (n=4) were handled in the same way as the experimental animals (see the previous example), except that 30% PEG (vehicle) was administered in place of PPB. Seven days after induction of SE, animals were anesthetized with a mixture of ketamine (100 mg/Kg, i.p. Ketalin®, Probiomed) and xylazine (20 mg/Kg, i.p. , Sigma-Aldrich), and LV myocardial reperfusion was done by infusing saline solution (0.9%) with heparin (1 mg/l) for 15 minutes (approximately 200-250 ml), followed by 4% paraformaldehyde + 0.2% glutaraldehyde in phosphate buffer (pH = 7.4). After perfusion, the brains were removed and placed in 4% paraformaldehyde for 168 hours at 4°C. Subsequently, the samples were embedded in paraffin and 5 pm thick coronal sections at the level of the dorsal hippocampus were obtained. The sections were mounted on lamellae pre coated with poly-L-lysine adhesive for further processing with cresyl violet of Fluoro-Jade.

Cresyl violet staining.

Brain sections at the level of the dorsal hippocampus were stained by cresyl violet technique. This dye is a basic aniline (positively charged) that binds to the basophilic regions of the cells, which allowed us to observe and compare the damage in various brain regions after inducing SE. The sections obtained were deparaffinized for 15 minutes at a temperature of 65°C to 70°C and then immersed in xylene (reagent grade) and alcohol (reagent grade) at 100%, 96% and 70% for 5 minutes in each solution. Subsequently, the sections were put in cresyl violet for 30 minutes. Lamellae were washed with distilled water for 1 minute, then immersed for 5 minutes in alcohol at 70%, 96% and 100% and finally in xylene for 10 minutes.

Fluoro-Jade staining.

Fluoro-Jade is a fluorochrome derivative of fluorescein that is used to identify neuronal damage in the nervous system.

Sections were handled according to the procedure described above, except that instead of incubating them in cresyl violate, they were immersed in basic alcohol (20 ml sodium hydroxide at 5% in 80 ml of absolute ethanol) for 5 minutes. They were thereafter placed in alcohol at 70% and distilled water for 2 minutes in each solution.

Subsequently, they were immersed in a potassium permanganate solution at 0.06% for 15 minutes and gently agitated on a rotating platform. Finally, they were washed for 2 minutes with distilled water. Samples were incubated in a Fluoro-Jade staining solution

(0.0001 %, Fluoro-Jade® B, Millipore) for 24 hours.

After staining, the sections were washed with distilled water and covered with synthetic resin. Samples were observed under the light microscope for the lamellae stained with cresyl violet; and under the fluorescence microscope including a refrigerated "Evolution" camera and using the image analysis software Image Pro Plus 5 (Media Cybernetics) for the lamellae stained with Fluoro-Jade. Example 2. Quantification of neuronal damage.

Quantification of neuronal damage was performed using the method of fractional count. It consisted of counting neurons in optical dissectors of one systematic sample, and constituting a volume fraction of the cerebral region analyzed. We assessed 5 pm hippocampal sections placed in series, evaluating one section of each series (3 series in total). Therefore, the sampling fraction (ssf) corresponded to 1/3. To perform the calculation, we took the volume fraction of each area of the hippocampus asf (box area/x, y area) corresponding to the counting area (0.460 x 0.600 mm). Dissector height, h, was calculated in relation to the section thickness t, (h/t). The number of positive neurons for the Fluoro-Jade staining was determined using the following formula:

N=[(EQ ^" )(t/h)(1/asf)(1/ssf)] (1 ) wherein E is summation and Q is the number of neurons in a known volume fraction of each area of the hippocampus.

Statistical analysis.

Statistical analysis of the effect induced by the administration of PPB was performed with unpaired t-Student test. Values are represented as the average of cells in process of neuronal damage per mm ³ ± standard error of the media.

Example 3. Effect of PPB in SE.

The evaluation of micrographs of animals with SE induced by the administration of lithium-pilocarpine showed significant neuronal loss in the entire hippocampal region compared with control animals (untreated, see figure 1A) and the group treated with PPB (figure 1 C). For a better analysis, zooms of photomicrographs were performed in different regions of dorsal hippocampus, wherein an important neuronal damage was observed in the animals of SE group, mainly in dentate gyrus, hilus, CA1 , and CA3 (see figure 2D).

By analyzing Fluoro-Jade staining, it was determined that the animals of the SE+PPB group receiving an administration of PPB (177.77 mg/Kg) after stopping SE had less neuronal damage in the dentate gyrus (62%, ^* p<0.05), hilus (39%, ^* p<0.05), and CA3 (25%, ^* p<0.05), while no significant changes were observed at CA1 level with respect to the control group (figures 3 and 4).

The present invention and the examples described above show that systemic administration of PPB immediately after the occurrence of SE considerably decreases neurotoxic side effects caused by this condition.