Claims:1. A pharmaceutical composition for attenuating development of seizure comprising of:
a. a proteasome inhibitor, wherein the inhibitor is ubiquitin proteasome system inhibitor comprising Z-Leu-Leu-Phe-CHO, salts of Z-Leu-Leu-Phe-CHO or a combination thereof; wherein Z is N-benzyloxycarbonyl and CHO is phenylalanal.
b. one or more pharmaceutically acceptable excipients.
2. The pharmaceutical composition as claimed in claim 1, wherein the composition further comprises one or more additional therapeutic agents.
3. The pharmaceutical composition as claimed in claim 1, wherein the proteasome inhibitor is present in an amount ranges from 50% to about 75% by weight of the total composition.
4. The pharmaceutical composition as claimed in claim 4, wherein the additional therapeutic agent is selected from the group consisting of anti-inflammatory agents, anti-hypertensive agents, anti-hypotensive agents, anti-pyretic agents, anti-neoplasia agents, anti-psychotic agents, stimulants, anti-mycotic agents, anti-microbial agents, antibiotics, immunomodulating agents, anti-viral agents, bronchodilators, anti-thyroid agents, anti-hypoglycemic agents, anti-opioid agents, hormones, hormone antagonists, drugs affecting renal and/or cardiovascular function, drugs acting on blood forming organs and/or blood clotting mechanism(s), drugs acting on bone calcification and/or turnover, drugs affecting gastrointestinal function, drugs acting on the central and autonomic nervous system, anti-seizure agents or combinations thereof.
5. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutically acceptable excipient is selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, stabilizers, antimicrobial preservatives and combination thereof.
6. The pharmaceutical composition as claimed in claim 1, wherein the composition is administered to the subject before an onset of a seizure, during a seizure, and/or after a seizure to prevent further seizures.
7. The pharmaceutical composition as claimed in claim 1, wherein the composition is administered parenterally.
8. The pharmaceutical composition as claimed in claim 7, wherein the parenteral administration includes intramuscular (i.m.), intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), or intrathecal (i.t.) administration.
9. A method of preparing a pharmaceutical composition for attenuating development of seizure comprising the step of combining Z-Leu-Leu-Phe-CHO with at least one pharmaceutically acceptable excipient selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, stabilizers, antimicrobial preservatives, and combination thereof.

Description:FIELD OF THE INVENTION
[0001] The present disclosure pertains to pharmaceutical composition for attenuating seizure development. More specifically, the present disclosure pertains to a composition comprising N-benzyloxycarbonyl-leucyl-leucyl-phenylalanal (Z-Leu-Leu-Phe-CHO). This disclosure also relates generally a method of producing injectable formulation comprising Z-Leu-Leu-Phe-CHO.

BACKGROUND OF THE INVENTION
[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] The mainstay of treatment for seizure disorders has been the long-term and consistent administration of anticonvulsant drugs, and today many such drugs are well known. Several anti-epileptic drugs, such as phenytoin, phenobarbital, carbamazepine and valproic acid, are effective for the prevention of early PTS, but not late PTS or PTE. Unfortunately, despite the many available pharmacotherapeutic agents, a significant percentage of the population with epilepsy or related disorders are poorly managed. Moreover, none of the drugs presently available are capable of achieving total seizure control, do not treat the underlying cause of seizure activity which is both acute and persistent inflammation, and most have unfavorable side effects which limit their use. Available pharmacotherapeutic options have been found to effectively suppress the symptomatology of epilepsies and thus are quite successful in causing a remarkable improvement in the clinical condition of the patient. However, none of the available drugs have been found to attenuate the progression of epileptogenesis, which leads to the life time prevalence of the crippling disease.
[0004] Further, pharmacological and genetic studies have shown certain basic molecular signaling mechanisms underlying epileptogenesis. A number of ionotropic glutamate receptors, metabotropic glutamate receptors, neurotrophin receptors, calcium regulated enzymes and non-receptor tyrosine kinases have been documented to be involved in the progression of epilepsies.
[0005] Further, Ubiquitin–proteasome system (UPS) and other proteases are few of the principal enzyme systems responsible for protein degradation and has been reported to be abundantly expressed in the various cell types of the central nervous system. The addition of poly-ubiquitin chains to a protein has been shown to be cause its translocation to the proteasome, which then results in its degradation. The activation of UPS has been reported to be an important pathological change in neurons of subject having traumatic epilepsy.
[0006] Furthermore, microarray analysis of the hippocampal neurons of the epileptic mouse brain and brain samples obtained from pharmacoresistant temporal lobe epilepsy patients have demonstrated a substantial increase in the transcription of the enzymes of UPS. Furthermore, UPS based continued over-activation of voltage sensitive sodium channels has been shown to mediate a progressive increase in the severity of seizures.
[0007] Thus, there is a great need in the art for pharmaceutical composition that inhibits the UPS activation, which in turn attenuates the development of seizures.
[0008] The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS OF THE INVENTION
[0009] It is an object of the present disclosure to provide a pharmaceutical composition for attenuating the development of seizures.
[0010] It is another object of the present disclosure to provide a pharmaceutical composition for inhibiting the activation of Ubiquitin–proteasome system for attenuating the development of seizures.
[0011] It is another object of the present disclosure to provide a pharmaceutical composition for inhibiting the activation of Ubiquitin–proteasome system for attenuating the development of seizures including chemically induced kindled seizures, status epilepticus induced spontaneous recurrent seizures and rapid electrically induced kindled seizures.
[0012] It is another object of the present disclosure to provide a pharmaceutical composition for inhibiting the activation of Ubiquitin–proteasome system for treating epilepsy.

SUMMARY OF THE INVENTION
[0013] The present disclosure provides a pharmaceutical composition comprising of a ubiquitin proteasome system inhibitor, salts of said inhibitor or a combination thereof, wherein the said composition is, and may include further constituents, for example pharmaceutically acceptable carriers, adjuvants, solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, stabilizers or antimicrobial preservatives.
[0014] The present disclosure provides a pharmaceutical composition comprising Ubiquitin-Proteasome System Inhibitor for attenuating the development of seizures. The pharmaceutical composition may include further constituents, for example pharmaceutically acceptable carriers, adjuvants, solvents, solubility enhancers, suspending agents, buffering agents, is tonicity agents, stabilizers or antimicrobial preservatives.
[0015] The present disclosure provides a pharmaceutical composition comprising Z-Leu-Leu-Phe-CHO for attenuating the development of seizures. The pharmaceutical composition may include further constituents, for example pharmaceutically acceptable carriers, adjuvants, solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, stabilizers or antimicrobial preservatives.
[0016] The pharmaceutical combination described herein can be formulated as a parenteral formulation. Examples of suitable formulations include injectable formulations that can be administered through intramuscular (i.m.), intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), or intrathecal (i.t.) routes.
[0017] The present disclosure provides a pharmaceutical composition for attenuating the development of seizures. The present disclosure is based upon a surprising finding that none of the existing / available drugs have been found to attenuate the progression of epileptogenesis. The pharmaceutical composition allows the attenuation of the development of seizures, a faster onset of anti-seizure action, a longer-lasting action, better control of seizure activity with only one or a few applications, and allows significant reduction of neuronal damage in the brain of subjects.
[0018] The present disclosure provides a method for producing pharmaceutical composition comprising Z-Leu-Leu-Phe-CHO for attenuating the development of seizures.
[0019] The present disclosure provides a method for producing pharmaceutical composition comprising Z-Leu-Leu-Phe-CHO for attenuating the development of epilepsy.
[0020] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1A depicts pentylenetetrazole induced kindled seizure severity score assessment protocol.
[0022] FIG. 1B depicts pilocarpine induced spontaneous recurrent seizure severity score assessment protocol.
[0023] FIG. 1C depicts electrically kindled seizure severity score assessment protocol.
[0024] FIG. 2A depicts effect of Z-Leu-Leu-Phe-CHO on pentylenetetrazole-induced kindled seizures in mice in terms of Seizure severity score.
[0025] FIG. 2B depicts effect of Z-Leu-Leu-Phe-CHO on pentylenetetrazole-induced kindled seizures in mice in terms of Seizure frequency.
[0026] FIG. 3A depicts effect of Z-Leu-Leu-Phe-CHO on pilocarpine-induced spontaneous recurrent seizures in mice in terms of Seizure severity score.
[0027] FIG. 3B depicts effect of Z-Leu-Leu-Phe-CHO on pilocarpine-induced spontaneous recurrent seizures in mice in terms of Seizure frequency.
[0028] FIG. 4 depicts effect of Z-Leu-Leu-Phe-CHO on pilocarpine-induced spontaneous recurrent seizures in mice in terms of percentage non-viable neuronal area in mice brain.
[0029] FIG. 5A depicts effect of Z-Leu-Leu-Phe-CHO on electroshock-induced kindled seizures in mice in terms of Seizure severity score.
[0030] FIG. 5B depicts effect of Z-Leu-Leu-Phe-CHO on electroshock-induced kindled seizures in mice in terms of Seizure frequency.

DETAILED DESCRIPTION OF THE INVENTION
[0031] A pharmaceutical composition for inhibiting the activation of Ubiquitin–proteasome system for attenuating the development of seizures including chemically induced kindled seizures, status epilepticus induced spontaneous recurrent seizures and rapid electrically induced kindled seizures.
[0032] In some embodiments of the present invention, the compositions of this invention are suitable for treating one or more conditions selected from the group consisting of acute seizure, repetitive seizures, and status epilepticus in a human in need of such treatment. The term “treating” as used in this patent application means ameliorating, suppressing, eradicating, preventing, reducing the risk of, and/or delaying the onset of the condition being treated. In other words, the compositions of this invention are suitable for breaking an acute seizure, a repetitive seizures episode, and/or status epilepticus, and, optionally, also for preventing the occurrence of a subsequent seizure(s).
[0033] In some embodiments of the present invention, the seizure may be caused by any disease, disorder, or dysfunction of the subject including, but not limited to, epilepsy, infantile spasms, Lennox Gastaut syndrome, a rapidly increasing fever (febrile seizure), an extremely low blood sugar level, for example, in a person afflicted with diabetes, damage to the brain from a stroke, brain surgery, or a head injury, congenital disorders, such as those caused by a genetic mutation or an inborn error of metabolism, withdrawal from alcohol, prescription medicine, or illegal drugs, an infection, such as meningitis or encephalitis, a brain tumor or structural defect in the brain, such as an aneurysm, or parasitic infections, such as tapeworm or toxoplasmosis. In some embodiments, the seizure is caused by a disease, disorder, or dysfunction selected from the group consisting of epilepsy, a rapidly increasing fever, low blood sugar, damage to the brain from a stroke, brain surgery, or a head injury, a congenital disorder, withdrawal from alcohol, prescription medicine, or illegal drugs, an infection, a brain tumor or structural defect in the brain, and a parasitic infection. In other embodiments, the seizure is caused by epilepsy.
[0034] In some embodiments of the present invention, the compositions can treat the condition(s) being treated while reducing or eliminating the patient's risk of choking. The term “choking” as used in this patent application means obstruction of the flow of air from the environment into the patient's lungs.
[0035] In a preferred embodiment of the present invention, the compositions are suitable for treating status epilepticus.
[0036] In some embodiments of the present invention, symptoms of a seizure disorder may include, but are not limited to, episodes involving ataxia, gait impairment, speech impairment, vocalization, impaired cognition, abnormal motor activity, clinical seizure, subclinical seizure, hypotonia, hypertonia, drooling, and mouthing behavior, aura, repetitive movements, and unusual sensations. In embodiments, the methods and compositions provided may reduce or prevent one or more different types of seizures. Generally, a seizure can include repetitive movements, unusual sensations, and combinations thereof.
[0037] In a further exemplary embodiment of the present disclosure, the active ingredient of the pharmaceutical composition may consist essentially of Z-Leu-Leu-Phe-CHO.
[0038] In some embodiments of the present invention, the compositions comprise Z-Leu-Leu-Phe-CHO, a derivative of Z-Leu-Leu-Phe-CHO or a combination thereof. Alternatively, or in addition, the drug may correspond to a drug candidate identified in a method of screening for anti-seizure drugs, such as described in the preceding paragraph and elsewhere in the present teachings.
[0039] In some embodiments of the present invention, the term “derivative of Z-Leu-Leu-Phe-CHO”, as used herein, means a chemical structure formally related to Z-Leu-Leu-Phe-CHO and distinguished from Z-Leu-Leu-Phe-CHO by one or more substitutions, deletions, and/or insertions. A Z-Leu-Leu-Phe-CHO derivative may be produced de novo or, in some cases, may be synthesized by chemical modification of Z-Leu-Leu-Phe-CHO. Exemplary derivatives of Z-Leu-Leu-Phe-CHO may include deletions of one or more amino acids, insertions of one or more amino acids, substitution of one or more amino acids with other amino acids or with non-amino acids, or any combination thereof. A Z-Leu-Leu-Phe-CHO derivative may have an amino acid sequence with at least about 20%, 40%, 60%, 80%, or 90% amino acid identity with the amino acid sequence of Z-Leu-Leu-Phe-CHO, when the sequences are aligned.
[0040] In some embodiments of the present invention, pharmaceutical compositions described herein are suitable for parenteral administration, including, e.g., intramuscular (i.m.), intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), or intrathecal (i.t.).
[0041] In some embodiments of the present invention, parenteral compositions may be administered as needed, e.g., once, twice, thrice or four or more times daily, or continuously depending on the subject's needs. In a preferred embodiment, the compositions may be administered every other day or every alternative day (i.e. Q.O.D dosage).
[0042] In some embodiments of the present invention, the pharmaceutical compositions for parenteral administration are formulated as liquid formulations or as powder and/or lyophilized form for constitution with a suitable vehicle (e.g. sterile, pyrogen-free water, normal saline or 5% dextrose) before use.
[0043] In some embodiments of the present invention, the term “subject,” as used herein, means a many-celled vertebrate or invertebrate organism from the animal kingdom. The subject thus may be a person (also termed an individual or a human) or a non-human animal (hereafter, termed only an “animal”). Exemplary animals include but not limited to laboratory animals, farm animals, pets, or sport animals, among others. An animal subject thus may, for example, be a rodent (such as a mouse, rat, hamster, guinea pig), dog, cow, horse, non-human primate, bird, amphibian, reptile, fish, insect, or the like. Non-human subjects may be test species, that is, animals for testing the effect of a composition, chemical compound, or candidate drug, generally prior to human clinical trials and/or use as an approved drug in humans and/or animals. Alternatively, non-human: subjects may be drug recipients after a drug has been tested and/or approved, such as for treatment in a veterinary setting.
[0044] In some embodiments of the present invention, parenteral compositions must be sterile for administration by injection, infusion or implantation into the body of the subjects and may be packaged in either single-dose or multi-dose containers.
[0045] In some embodiments of the present invention, the pharmaceutical compositions for parenteral administration are formulated as a total volume of about but not limited to, e.g., 10 ml, 20 ml, 25 ml, 50 ml, 100 ml, 200 ml, 250 ml, or 500 ml. In embodiments, the compositions are contained in a bag, a glass vial, a plastic vial, or a bottle.
[0046] In some embodiments of the present invention, the compositions of the present invention may be used alone or in combination with any other pharmaceutically active compound or an excipient to treat the inflammation of epilepsy, seizures, and brain injury. Useful pharmaceutical compositions may comprise at least one further compound for the prevention, alleviation or/and treatment of seizures wherein the effect of this composition in the prevention, alleviation or/and treatment of seizures is synergistic as compared to the effect of the respective compounds given alone. Examples of combination drug include but not limited to a racetam, lacosamide, dibenzazepine, sulfamate, phenytoin, or barbiturate.
[0047] In some embodiments of the present invention, the pharmaceutical composition according to the invention may be formulated as controlled release formulations, for injection, for infusion, and may be presented in unit dose form in ampoules or in multidose vials or other containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions or gels, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
[0048] In some embodiments of the present invention, the compositions are administered in therapeutically or prophylactic effective does, i.e. 0.05-1000 mg of Z-Leu-Leu-Phe-CHO per day, in particular 5-250 mg per day. Very large doses may be used as the composition according to the invention is non-toxic. The dose administered daily of course depends on the degree of therapeutic response.
[0049] In a preferred embodiment of the present invention, a once-daily dose may be administered in a dosage comprising at least about 0.1 mg kg-1d-1 of Z-Leu-Leu-Phe-CHO. In another exemplary embodiment, a once-daily dose may be administered in a dosage comprising an amount of eslicarbazepine acetate ranging from about 0.05 mg kg-1d-1 to about 1.0 mg kg-1d-1. In further exemplary embodiments, a Q.O.D dose may be administered in a dosage comprising an amount of about 0.1 mg kg-1d-1, 0.3 mg kg-1d-1, 0.5 mg kg-1d-1 or more.
[0050] In a most preferred embodiment of the present invention, administration by injection or infusion of the compositions for treatment of adults of approximately 70 kg of body weight, will often range from 2-250 mg of active material, which may be administered in the form of 1 to 2 doses over each day.
[0051] In some embodiments of the present invention, the pharmaceutical composition comprising Z-Leu-Leu-Phe-CHO may optionally be administered by any route known to those skilled in the art, and may be in a form chosen from, for example, tablets or oral suspensions, or other forms.
[0052] In some embodiments of the present invention, the pharmaceutical compositions for parenteral administration provided herein may include one or more excipients, e.g., pharmaceutically acceptable carriers, adjuvants, solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, stabilizers or antimicrobial preservatives.
[0053] In some embodiments of the present invention, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and can include buffers, such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, BHA, and BHT; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, both L- and D-forms, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counter-ions, such as sodium; and/or nonionic surfactants, such as Tween, Pluronics, or PEG.
[0054] In another embodiment of the present invention, pharmaceutical composition comprising Z-Leu-Leu-Phe-CHO further comprises adjuvant that may include, but not limited to, monophosphoryl lipid A (MPL); LTK63, dimethyl dioctadecyl-ammonium bromide (DDA), lipophilic quaternary ammonium salt-DDA, Trehalose dimycolate and synthetic derivatives, DDA-MPL, DDA-TDM, DDA-TDB, IC-31, aluminum salts, aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate, Montanide ISA-51, ISA-720, microparticles, immuno stimulatory complexes, liposomes, virosomes, virus-like particles, CpG oligonucleotides, cholera toxin, heat-labile toxin from E. coli, lipoproteins, dendritic cells, IL-12, GM-CSF, nanoparticles, a combination of soybean oil, emulsifying agents, and ethanol to form a nanoemulsion; ASO4, ZADAXIN, or combinations thereof.
[0055] In some embodiments of the present invention, the compositions of this invention comprise one or more anti-seizure agents in addition to the other ingredient(s) of the composition. The term “anti-seizure agent” as used in this patent application means an agent suitable, either alone or together with additional medication(s), to treat one or more conditions selected from the group consisting of acute seizure, repetitive seizures, and status epilepticus. An anti-seizure agent can comprise a compound or a pharmaceutically acceptable salt of the compound (including solvate or hydrate). Suitable anti-seizure agents include the so-called classical anti-seizure agents as well as miscellaneous other anti-seizure agents. The classical anti-seizure agents are classified into four subclasses: barbiturates (e.g., methylphenobarbital, phenobarbital, hexibarbital), hydantoins (e.g., phenyloin, mephenyloin, ethotoin), oxazolidinediones (e.g., trimethdione), and succinimides (e.g., phensuximide, ethoxisuximide). Additional suitable anti-seizure agents include, for example, benzodiazepines (e.g., diazepam, lorazepam, midazolam, clonazepam, clorazepate), carbamazepine, primidone, valproic acid, fosphenyloin, felbamate, gabapentin, lamotrigine, tiagabine, zonisamide, topiramate, phenacetamide, and vigabatrin.
[0056] In additional embodiments of the present invention, the compositions of this invention can comprise one or more anti-seizure agents. In some embodiments, the anti-seizure agents are independently selected from the group consisting of barbiturates, hydantoins, oxazolidinediones, and succinimides. In some embodiments, the anti-seizure agents are independently selected from the group consisting of diazepam, lorazepam, midazolam, clonazepam, clorazepate, phenobarbital, methylphenobarbital, hexibarbital, phenyloin, mephenyloin, ethotoin, fosphenyloin, trimethdione, phensuximide, ethoxisuximide, carbamazepine, primidone, valproic acid, felbamate, gabapentin, lamotrigine, tiagabine, zonisamide, topiramate, phenacetamide, vigabatrin, and their pharmaceutically available salts. In some embodiments, the anti-seizure agents are independently selected from the group consisting of benzodiazepines, hydantoins, and pharmaceutically available salts thereof. In some embodiments, the anti-seizure agents are independently selected from the group consisting of diazepam, lorazepam, midazolam, clonazepam, clorazepate, phenobarbital, methylphenobarbital, hexibarbital, phenyloin, mephenyloin, ethotoin, fosphenyloin, and their pharmaceutically available salts. In some embodiments, the anti-seizure agents are independently selected from the group consisting of diazepam, lorazepam, midazolam, phenyloin, phenobarbital, and their pharmaceutically acceptable salts. In some embodiments, the anti-seizure agents are independently selected from the group consisting of diazepam, lorazepam, midazolam, clonazepam, and their pharmaceutically acceptable salts. In some embodiments, the anti-seizure agents are independently selected from the group consisting of diazepam, lorazepam, midazolam, and their pharmaceutically acceptable salts.
[0057] In some embodiments of the present invention, the compositions of this invention further comprise one or more therapeutic agents other than anti-seizure agents. The therapeutic agents can be used to (a) treat the condition(s) causing the patient's seizure(s); (b) treat any seizure-unrelated conditions that the patient has; and/or (c) to mitigate side-effect(s) of the administered anti-seizure agents. Suitable therapeutic agents include, for example, anti-inflammatory agents, anti-hypertensive agents, anti-hypotensive agents, anti-pyretic agents, anti-neoplasia agents, anti-psychotic agents, stimulants, anti-mycotic agents, anti-microbial agents, antibiotics, immunomodulating agents, anti-viral agents (e.g., acyclovir), bronchodilators (e.g., albuterol), anti-thyroid agents, anti-hypoglycemic agents, anti-opioid agents (e.g., naloxone), hormones, hormone antagonists, drugs affecting renal and/or cardiovascular function (e.g., diuretics, vasopressin, renin, anti-arrhythmic drugs), drugs acting on blood forming organs and/or blood clotting mechanism(s), drugs acting on bone calcification and/or turnover, drugs affecting gastrointestinal function, drugs acting on the central and autonomic nervous system (e.g., anticholinergics, adrenergics, anesthetics, parasympathomimetics, sympathomimetics, hypnotics, sedatives, serotonin-like drugs, serotonin blockers), and agents that mitigate the side-effects of the administered anti-seizure agents. Suitable anti-inflammatory agents include, for example, NSAIDs (e.g., aspirin, ibuprofen, naproxen, oxaprozin, celecoxib, refecoxib, valdecoxib), histamine antagonists, glucocorticoids (e.g., betamethazone, cortisone, dexamethasone, hydrocortisone, prednisone, prednisolone, triamcinolone), anti-inflammatory cytokines, and anti-histamines (e.g., diphenhydramine, chlorpheniramine, hydroxyzine, azelastine, levocabastine, ketotifen, cetirizine, levocetirizine, loratidine, desloratidine, acrivastine, ebastine, fexofenadine, mizolastine, cycloheptadine, azelastine, betahistine, perceptin, ciproxifan, thioperamide, burimamide, cimetidine, ranitidine, famotidine). Suitable anti-hypertensive agents include, for example, a1-adrenergic antagonists (e.g., prazocin), ß-adrenergic antagonists (e.g., propranolol, nadolol, timolol, metoprolol, pindolol, albuterol), angiotensin converting enzyme inhibitors (e.g., captopril, enalapril, lisinopril), calcium channel blockers (e.g., verapamil, diltiazem, nifedipine), and diuretics (e.g., hydrochlorothiazide, chlorthalidone, furosemide, triamterene). Suitable stimulants include, for example, caffeine and amphetamines (e.g., dextroamphetamine). Suitable anti-pyretic agents include, for example, aspirin and acetaminophen. Suitable anti-hypoglycemic agents include, for example, glucose and sucrose.
[0058] In some embodiments of the present invention, the effective concentration of each of the drugs, when administered in combination, to elicit a particular biological activity may be less than the effective concentration of each drug when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. In such combination therapies, the therapeutic effect of the first administered agent is not diminished by the sequential, simultaneous or separate administration of the subsequent agent(s).
[0059] The terms “co-administered with”, “administered in combination with”, “a combination of” or “administered along with” may be used interchangeably and mean that two or more agents are administered in the course of therapy. The agents may be administered together at the same time or separately in spaced apart intervals. The agents may be administered in a single dosage form or in separate dosage forms.
[0060] In another exemplary embodiment of the present invention, the pharmaceutical composition is administered in a dosage intended to maximize the total exposure to Z-Leu-Leu-Phe-CHO, as measured by the rate of exposure and extent of exposure (Cmax and AUC0-t).
[0061] In a further aspect of the present invention, Z-Leu-Leu-Phe-CHO may be administered to a patient in an amount resulting in a maximum plasma concentration (Cmax) of Z-Leu-Leu-Phe-CHO greater than about 7,400 ng/mL. In other exemplary embodiments, Z-Leu-Leu-Phe-CHO may be administered to a patient in an amount resulting in a Cmax of Z-Leu-Leu-Phe-CHO greater than about 12,000 ng/mL or greater than about 16,100 ng/mL. In further exemplary embodiments, Z-Leu-Leu-Phe-CHO may be administered to a patient in an amount resulting in an area under the concentration curve (which corresponds to the extent of systemic exposure) over the dosing interval (AUC0-t) of Z-Leu-Leu-Phe-CHO greater than about 22,700 ng/mL, such as greater than about 36,500 ng/ml, greater than about 45,200 ng/mL, or more.
[0062] In a further embodiment of the present invention, the present disclosure relates to a method of preparing a pharmaceutical composition comprising the combination of Z-Leu-Leu-Phe-CHO with at least one excipient, at least one auxiliary substance, at least one carrier material, or combinations thereof. Suitable excipients, carrier materials, and other auxiliary substances which would be useful in the present invention are known to those skilled in the art, and would be readily determined. Methods for preparing pharmaceutical compositions are also known to those skilled in the art.
[0063] The present disclosure provides a pharmaceutical composition for the treatment or prevention of epilepsy or seizures and a method for modulating, in particular reducing, an excessive immune response in an animal, such as a human or another mammal, specifically in the brain due to injury, trauma or infection resulting in activation of innate immune inflammatory pathways which cause excessive cytokine, chemokine, and TLR4/MyD88 receptor activation which leads to seizures, loss of normal function, loss of neurons, cognitive impairment, and even death.
[0064] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
[0065] The present disclosure is further explained in the form of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
[0066] Example 1: Attenuation of chemically kindled seizure
Induction of chemical kindling in mice
Pentylenetetrazole (PTZ) was administered in a sub-convulsant dose (40 mg/kg, i.p.) on alternate days for a total period of 15 days [9, 26, 28]. After each injection of PTZ, the severity of respective behavioral aberrations associated with seizures were recorded for 15 min by observing the animals in a Plexiglas chamber (30 X 24 X 22 cm). Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS) [26, 28]. Drug treatments were continued throughout the experimental procedure (once daily). KSSS quantification was performed 4 hr prior to the drug administration on the behavioral assessment day (every alternate day until day 15). On day 1, 7 and 15 of PTZ dosing protocol, the behavior of the animals was video recorded for a typical 24 hours period in order to assess the frequency of their daily seizures. Appearance of a typical cycle of generalized clonic-tonic seizure was recorded as a single episode, and frequency of the same, was thus obtained.
The test drugs were injected on every single treatment day throughout the experimental protocol. The dosing of the same was invariably done after the completion of grading the PTZ seizures like behavior in mice. This was done with a view of assessing their therapeutic usefulness in patients already suffering from progressive epileptogenesis instead of symptomatic cure of an epileptic patient that requires prior injection of test drugs before seizure induction.
Effect of Z-Leu-Leu-Phe-CHO (ZLLPC) on pentylenetetrazole (PTZ) induced kindled seizures in mice
Repeated treatment of sub-convulsive dose of PTZ (40 mg/kg, i.p.) on alternate days for a period of 15 days induced kindled seizures in mice as revealed by a progressive increase in kindled seizure severity score (KSSS) in control mice as compared to that of vehicle control mice (P<0.01). Similar results were obtained in terms of comparing the frequency of daily seizures in control mice as compared to that of vehicle control ones (P<0.01). Although vehicle did not have any effect, daily treatment with ZLLPC (0.1 or 0.3 mg kg-1 d-1, i.p.) for the 15-day period markedly and dose dependently attenuated (P<0.01) the development of kindled seizures as measured in terms of a significant suppression of the observed progressive increase in the KSSS as well as the frequency of daily seizures as compared to that recorded in the mice belonging to PTZ control group (Figure 2).
[0067] Example 2: Attenuation of electrically kindled seizure
Induction of electrical kindling in mice
In order to validate the efficacy of the test pharmacological agent(s), a relatively new model of rapidly evoked kindled seizures in mice was employed in the present study [28]. In this model, a quick train of consecutive stimuli induced the development of sustained and progressive seizures in mice. This model has been standardized and validated in the present authors’ laboratory and data relevant to this study is presented in the present manuscript.
Fifty electroshocks, each of 50 Hz and 3 seconds duration, were delivered to mice using corneal electrodes with continuously increasing intensities (the initial shock was of 100 µA and after every five shocks of similar intensity, the intensity of current for the next group of five shocks was increased by a value of 25 µA each time). Inter-shock interval that was allowed between two consecutive shocks was 40 seconds. After the completion of electroshocks, occurrence of central nervous system excitation was recorded for 15 min by observing the animals in a Plexiglas chamber (30 X 24 X 22 cm). Severity of kindled seizures was assessed in terms of a composite rapid electrical kindled seizure severity score (KSSS) [28]. Daily test drug treatment was continued throughout the 15 days observation period following the shock treatment. The behavioural assessments were done during the same 15 day period. On day 1, 7 and 15 of the experimental protocol, video recording for a 24 hours period was done to assess the frequency of daily seizures in the animals. Appearance of a typical cycle of generalized clonic-tonic seizure was recorded as a single episode, and frequency of the same during the 24 hour observation period was noted to grade the status of epileptic behaviour in mice.
The test drug administration was done immediately after the completion of actual/sham grading period of the kindled seizures severity score in mice. This was done with a view of assessing their therapeutic usefulness in subjects suffering from progressive epileptogenesis instead of symptomatic cure of an epileptic patient that requires prior injection of test drugs before seizure induction.
Results
Effect of ZLLPC on electroshock induced kindled seizures in mice
After the completion of electroshock procedure, the development of kindled seizures was observed as revealed by a progressive increase in electrically kindled seizure severity score (KSSS) and frequency of daily seizures in control mice as compared to that of the vehicle control animals (P<0.01). Although vehicle did not have any effect, treatment with ZLLPC (0.1 or 0.3 mg kg-1 d-1, i.p.) markedly attenuated (P<0.01) the development of kindled seizures as measured in terms of a significant suppression of the observed progressive increase in the KSSS and frequency of daily seizures as compared to that recorded in the mice belonging to the electroshock control group (Figure 5).
[0068] Example 3: Status epilepticus provoked spontaneous recurrent seizure activity
Induction of status epilepticus provoked spontaneous recurrent seizure activity in mice
Intraperitoneal application of pilocarpine (100 mg/ kg, i.p.) was repeated every 20 min until the onset of status epilepticus [13, 26, 28]. In order to avoid peripheral cholinergic side effects, hyoscine butylbromide (1 mg/kg, i.p.) was administered 20 min prior to the application of pilocarpine. Status epilepticus was defined as continuous seizure activity of the limbs, rearing, falling, Straub’s tail, repeated head twitches, jumping seizures and generalized clonic-tonic seizures. Status epilepticus was terminated, 40 min after the onset, using a single dose of diazepam (3mg/kg, i.p.). Normal saline was administered until complete recovery. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of the quantitative assessment of progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus (SE). Drug treatment was started 7 days after the SE episode and was continued until day 37. Behavioral assessment was done prior to the drug administration on each observation day (every third day until day 37). On day 1, 3, 7, 15, 21, 28 and 37 of the experimental protocol, the behavior of the animals was video recorded for a 24 hours period in order to assess their frequency of daily seizures. Appearance of a typical cycle of generalized clonic-tonic seizure was recorded as a single episode, and frequency of the same during the 24 hour observation period was noted to grade the status of epileptic behavior in mice.
The test drug was injected on every treatment day and immediately after the completion of the actual/sham grading period of the spontaneous recurrent seizures in mice, with a view of assessing their therapeutic usefulness in subjects suffering from progressive epileptogenesis instead of symptomatic cure of an epileptic that requires prior injection of test drugs before seizure induction.
Effect of ZLLPC on pilocarpine (PC) induced development of spontaneous recurrent seizures in mice
A single episode of pilocarpine induced status epilepticus for a period of 40 min followed by its pharmacological termination using diazepam, elicited a time dependent elicitation of the progressive spontaneous recurrent seizures as assessed in terms of spontaneous recurrent seizure severity score (SRSSS) and frequency of daily seizures throughout the observation period of 30 days in mice and percentage non-viable neuronal area in mice brain belonging to PC control group (P<0.01). However, daily treatment with ZLLPC (0.1 or 0.3 mg kg-1 d-1, i.p.) for the 30-day period significantly and dose dependently attenuated (P<0.01) the development of spontaneous recurrent seizures as assessed in terms of a marked suppression of the observed progressive increase in the SRSSS and percentage non-viable neuronal area in mice brain as compared to that noted in the mice belonging to the PC control group. Similar observations were made in terms of the frequency of daily seizures in the treated animals when compared to that of mice belonging to the PC control group (Figure 3, 4).
[0069] Example 4: Effect on cerebral infarct size
Assessment of cerebral infarct size
At the end of day 37 of pilocarpine status epilepticus-induced spontaneous recurrent seizures, animals were sacrificed by spinal dislocation and the brain was removed. Brain samples were immediately sliced into uniform coronal sections of about 1 mm thickness. The slices were incubated with 1% triphenyltetrazolium chloride (TTC) at 37?C in 0.2 M tris buffer (pH 7.4) for 20 min. TTC is converted to red formazone pigment by NAD and lactate dehydrogenase and thus stained the viable cells deep red. The dead cells lost the enzyme as well as cofactor and thus remain unstained dull yellow. The brain slices were placed over glass plate. A transparent plastic grid with 100 squares in 1 cm2 was placed over it. Average area of each brain slice was calculated by counting the number of squares on either side. Similarly, number of squares falling over non-stained dull yellow area was also counted. Non-viable neuronal area was expressed as a percentage of total brain volume [27].
Experimental protocol
In the present study, a total of eleven groups were employed and each group comprised of 10 animals.
Pentylenetetrazole induced kindled seizure severity score assessment protocol (Figure 1 (A)):
Group I (vehicle control group-a): vehicle (10 ml kg-1, i.p.) every alternate day for 15 days + vehicle (10 ml kg-1 d-1, i.p.) for 15 days.
Group II (pentylenetetrazole (PTZ) control group): PTZ (40 mg kg-1, i.p.) every alternate day for 15 days + vehicle (10 ml kg-1 d-1, i.p.) for 15 days.
Group III (low dose Z-Leu-Leu-Phe-CHO (ZLLPC) + PTZ group): PTZ (40 mg kg-1, i.p.) every alternate day for 15 days + ZLLPC (0.1 mg kg-1d-1, i.p.) for 15 days.
Group IV (high dose ZLLPC + PTZ group): PTZ (40 mg kg-1, i.p.) every alternate day for 15 days + ZLLPC (0.3 mg kg-1 d-1, i.p.) for 15 days.
Electrically induced kindled seizure severity score assessment protocol (Figure 1 (B)):
Group V (vehicle control group-b): vehicle (10 ml kg-1, i.p.) was administered on every alternate day after the electroshock procedure.
Group VI (low dose ZLLPC + electrical kindling group): ZLLPC (0.1 mg kg-1 d-1, i.p.) was administered on every alternate day after the electroshock procedure.
Group VII (high dose ZLLPC + electrical kindling group): ZLLPC (0.3 mg kg-1 d-1, i.p.) was administered on every alternate day after the electroshock procedure.
Pilocarpine induced spontaneous recurrent seizure severity score assessment protocol (Figure 1 (C)):
Group VIII (vehicle control group-c): vehicle (10 ml kg-1 d-1, i.p.) for the 30 days period starting 7th day after SE + vehicle (10 ml kg-1, i.p. every 20 min until the completion of 80 min period followed by diazepam (3 mg kg-1, i.p.) 40 min after last injection) on day 1.
Group IX (pilocarpine (PC) control group): vehicle (10 ml kg-1 d-1, i.p.) for the 30 days period starting 7th day after SE + PC (100 mg kg-1, i.p. every 20 min until the onset of SE followed by diazepam (3 mg kg-1, i.p.) 40 min after SE) on day 1.
Group X (low dose ZLLPC + PC group): ZLLPC (0.1 mg kg-1 d-1, i.p.) for the 30 days period starting 7th day after SE + PC (100 mg kg-1, i.p. every 20 min until the onset of SE followed by diazepam (3 mg kg-1, i.p.) 40 min after SE) on day 1.
Group XI (high dose ZLLPC + PC group): ZLLPC (0.3 mg kg-1 d-1, i.p.) for the 30 days period starting 7th day after SE + PC (100 mg kg-1, i.p. every 20 min until the onset of SE followed by diazepam (3 mg kg-1, i.p.) 40 min after SE) on day 1.
Statistical Analysis
Data obtained from the study was statistically analyzed using 2-way repeated measures ANOVA followed by post-hoc analysis. A value of p<0.05 was considered to be statistically significant.
[0070] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed examples will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.