JP2018522812A - Caspase-6 modulator - Google Patents

Abstract

The present application relates to pharmaceutical compositions for the treatment or amelioration of nervous system diseases, wherein the composition comprises a therapeutically effective amount of a caspase-6 inhibitor that is an arylpropinamide derivative. The composition can be formulated for oral or topical administration, subcutaneous, intravenous, or intramuscular injection, infusion, inhalation, or intrathecal injection. In certain aspects, the present disclosure can be described as a pharmaceutical composition for the treatment of a neurodegenerative disease, the composition comprising a caspase-6 inhibitor and one or more pharmaceutical carriers or excipients. Including.

Description

Cross-reference of related applications Not applicable

  The present invention relates to the field of treatment for neurodegenerative diseases. More particularly, compounds and methods of using compounds that modulate caspase-6 activity.

  Huntington's disease (HD) is a progressive neurodegenerative disorder with an autosomal dominant mode of inheritance characterized by chorea, cognitive decline and behavioral changes that are usually manifested in mid adulthood. HD is caused by a mutated HD gene (mhtt) containing a tandem CAG repeat that encodes the polyglutamine region. The number of CAG repeats is inversely related to the age of onset of the disease. Neurodegeneration first occurs most intensely in the medium spiny neurons of the striatum and later in the cortex. There is strong evidence to suggest that apoptosis plays a role in the neurodegeneration observed in HD. Proteolytic cleavage of mhtt is a critical factor in the pathogenesis of HD.

  Htt is proteolytically cleaved by caspases, releasing an amino terminal fragment containing a glutamine chain (Wellington et al., 2000, J. Biol Chem, 275 (26): 19831-8). In particular, expression of mhtt fragments containing expanded polyglutamine repeats is toxic in vitro and in vivo, and accumulation of products truncated at the N-terminus of mhtt is observed in human and mouse HD brains . Caspase-6 (C6) is a cysteine-aspartic protease that, when activated, cleaves htt and mhtt. Studies in mice have shown that expression of (C6R) mhtt that is resistant to C6 cleavage significantly reduces htt toxicity and results in dramatic improvement in phenotype, while caspase-3 (C3) and caspase-2 It was shown that mice expressing mhtt resistant to cleavage by (C2) were not protected (Graham et al., 2006, Cell, 125 (6): 1179-91). Dramatically, mice expressing C6R mhtt do not show increased C6 activation. The absence of C6 activation in C6R mice suggests that the 586aa htt fragment may be part of the forward amplification cycle of C6 activation in HD (Graham, RK et al., J Neurosci, 2010, 30 (45): 15019-29). These data suggest that mhtt fragments generated by C6 cleavage may be required to initiate a toxic cycle leading to neuronal dysfunction and neuropathological abnormalities in HD (Graham et al., Trends Neurosci, 2011 34 (No. 12): 646-56).

  Caspase-6 (C6) mRNA is increased in early grade (0-2) human HD caudate nucleus and motor cortex compared to control tissues (Hodges et al., Hum Mol Genet, 2006, 15 ( 6): 965-77), activity C6 is also present in the HD brain of symptomatic and early grade human and mouse, and as other activators, caspases such as C3 are active in these stages It has not become. Interestingly, active C6 levels correlate with CAG size in human HD brain and inversely with age of onset. This evidence implies a relationship between C6 and htt, and the size of the CAG chain affects the level of C6 activation, thereby contributing to the disease process.

  Similar to htt, the amyloid precursor protein (APP), whose fragments accumulate and are thought to cause disease in Alzheimer's disease (AD), is also cleaved by caspases during apoptosis (Ayala-Grosso, C, Brain Pathol, 2002, 12 (4): 430-41). Two characterized sites at the amino terminus of Αβ include V (K or N) (M or L) D653 and VEVD664, both of which correspond to the C6 recognition motif (Gervais et al., Cell, 1999). 97 (3) L395-406). APP cleavage at the D664 site is detected early in human AD brain tissue, nuclear p21-activated kinase signaling (Nguyen et al., J. Neurochem, 2008, 22 (3): 703-12) and It is a requirement for defects in synaptic transmission, synaptic plasticity and learning observed in the pathophysiology of AD (Saganich et al., J. Neuroscience, 2006, 26 (52): 13428-36). Importantly, inhibiting the cleavage of APP at aa664, providing a proof of principle for the importance of cleavage at this site in AD pathogenesis, to some extent from behavioral defects and neuropathology in the AD mouse model (Zhang et al., Behav Brain Res, 2010, 206 (2): 202-207; Galvan et al., PNAS, 2006, 103 (18): 7130-5).

  The brain tissue of AD patients also shows a significant increase in C6 mRNA compared to control and active C6 (Pompl et al., Arch Neurol, 2003, 60 (3): 369-376). C6-cleaved tau and C6-cleaved α-tubulin are highly abundant in AD brain neurovilli, neurofibrillary tangles and senile plaques (Guo et al., Am J Pathol, 2004, 165 ( 2): 523-531; Klaiman et al., Cell Proteomics, 2008, 7 (8): 1541-55). Interestingly, the level of C6-cleaved tau is inversely correlated with global cognitive score, descriptive memory and semantic memory in the brain of elderly non-cognitively impaired individuals, which indicates that C6 activity is clinical in AD Suggests to precede diagnosis and pathological diagnosis (Albrecht et al., Am J Pathol, 2007, 170 (4): 1200-9; LeBlanc et al., Cell Death and Differentiation, 2014, 21: 696-706).

The role of C6 in neuronal dysfunction, particularly axonal degeneration, is supported by other studies and shows that C6 is activated in degenerated neurons and axons after trophic factor removal (Park et al., Nat Neurosci, 2010, 13 (5): 559-66). Furthermore, C6 is activated and is required for neurodegeneration following ischemic insult (Akpan et al., J Neurosci, 2011, 31 (24): 8894-904). Taken together, these studies support C6 inhibitors as therapeutic strategies for neurological diseases.
Most caspase inhibitors used to verify the role of caspases in disease processes are peptide-based and mimic the cleavage sites present in caspase substrates. Non-peptide inhibitors have been described for C6, and when tested in an in vitro model of HD, the pan-caspase inhibitors described by Levya and co-workers have shown protection from mhtt toxicity (Leyva et al., Chem Biol, 2010, 17 (11): 1189-200). More recently, Murray and colleagues have described small molecules that bind procaspase 6 at the allosteric site (Murray et al., Chem Med Chem, 2014, 9 (1): 73-77).

Wellington et al., 2000, J. MoI. Biol Chem, 275 (26): 19831-8

In certain aspects, the disclosure can be described as a pharmaceutical composition for the treatment of a neurodegenerative disease, the composition comprising a caspase-6 inhibitor and one or more pharmaceutical carriers or Contains excipients. The caspase-6 inhibitor can be a small molecule, eg, an arylpropamide derivative, can have one of the following structures I or II, or a pharmaceutically acceptable salt, stereoisomer thereof: Body, derivative or prodrug:

In which Ra and Rb are independently linear or branched C ₁ -C ₆ alkyl, aryl, or alkenyl, C ₁ -C ₉ alkyl aryl, C ₁ -C ₉ substituted alkyl aryl, or C _1- C ₉ alkylheteroaryl,

Phenyl is substituted with 0, 1 or 2 halogens;

Furthermore, the halogen is chloride, fluoride or bromide.

  The present disclosure also provides, in certain embodiments, a method of treating a nervous system disease wherein a subject in need of such treatment is treated with a therapeutically effective amount of Structure I or as described herein. Administering an effective dose of a pharmaceutical composition comprising a compound having any of the structures of II, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof, wherein the disease comprises Huntington's disease, Alzheimer's It can be described as a method of disease, dementia, mild cognitive decline, or memory loss. The composition can be administered alone or as an adjunct or combination therapy with administration of one or more drugs or agents useful in the treatment of nervous system diseases. The drug combinations can be administered in the same formulation or separately and can be administered simultaneously or sequentially. Exemplary drugs useful for the treatment of nervous system diseases are L-DOPA, rasagiline, memantine hydrochloride, donepezil hydrochloride, rivastigmine, galantamine, tetrabenazine.

  The methods of treatment described herein can begin after the onset of symptoms in a subject, or the treatment is susceptible or suspected of being sensitive to a nervous system disease Can be started before symptoms of nervous system disease in the subject are presented. A subject susceptible to or suspected of being susceptible to a nervous system disease is subject's neuronal cells to the expression of caspase-6 mRNA in a healthy subject due to the presence of expression of a mutant htt gene in the subject. Can be identified by the overexpression of caspase-6 mRNA in or by the presence of axonal degeneration in the subject as evidenced by loss of the white part on magnetic resonance imaging.

  It is understood that the term “subject” as used herein can refer to a human subject or a veterinary subject.

  In certain embodiments, the disclosed compositions can include pharmaceutical compositions for the treatment or amelioration of nervous system diseases, wherein the compositions are therapeutically effective having a structure as shown in FIG. A quantity of the active agent, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof, wherein the active agent has a structure designated as one of PG-3a-h or PG-3 .

  The present disclosure can be further defined as compositions and methods for the treatment of neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, dementia, mild cognitive decline, or memory loss. For the treatment of neurodegeneration, the disclosed compositions can be formulated and administered orally, topically, or parenterally. While systemic administration, eg, oral or parenteral routes, such as injection or infusion into the systemic circulation, is possible, the use of such routes for active agents targeted to the brain and central nervous system is therapeutic in the CNS Undesirably high serum levels may be required to achieve levels. Thus, the disclosed compositions can also be formulated for direct introduction into the brain by intraventricular or intraparenchymal injection or for intranasal delivery, for example via the olfactory bulb or trigeminal nerve.

  For injection, a therapeutic amount of the disclosed caspase-6 inhibitor or a pharmaceutically acceptable salt formulation thereof is prepared for subcutaneous, intravenous, or intramuscular injection, or the formulation is Prepared for direct introduction into the brain or CSF by intraventricular, intraparenchymal or intrathecal injection. In addition to the active ingredient, these pharmaceutical compositions can contain buffers that maintain the pH in the range of 3.5-7, and also salts, such as sodium chloride, and to adjust isotonicity Mannitol or sorbitol. In certain embodiments, DMSO or another organic solvent can be added.

  When the composition is formulated for intravenous, intraperitoneal or subcutaneous administration, eg, by infusion or injection, a solution of the active compound or salt thereof is prepared in water and optionally mixed with a surfactant. can do. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof, and in oils. The liquid carrier or vehicle is a solvent or liquid dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol and the like), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Good. Such compositions can also contain isotonic agents, for example, sugars, buffers or sodium chloride, and optionally absorption delaying agents, such as aluminum monostearate, cellulose ethers, and gelatin.

  It is another aspect of the formulation that the formulation can also include a lipid that facilitates penetration of the composition into the tissue of the CNS. Exemplary lipids useful for the disclosed compositions include, but are not limited to, phosphatidylcholine and cholesterol. The disclosed compositions can also be administered as oil-in-water emulsions, such as olive oil, soybean oil, cottonseed oil, soybean oil, sesame oil, sunflower oil, safflower oil, avocado oil, peanut oil, walnut oil Selected from synthetic oils or vegetable oils, including almond oil or hazelnut oil.

  In certain embodiments as a liposome preparation, the disclosed compositions can also be prepared, wherein the liposome is a large unilamellar vesicle (LUV), multilamellar vesicle (MLV), or small unilamellar vesicle. (SUV). LUVs have a particle system ranging from about 200 to about 1000 nm. MLV has a particle system ranging from about 400 to about 3500 nm. SUVs have a particle system ranging from about 20 to about 50 nm.

  Examples of lipids for forming liposomes include phospholipids, cholesterol, or nitrogen lipids. Phospholipids are naturally occurring phospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean lecithin, lysolecithin, or corresponding hydrogenated phospholipids Or synthetic phospholipids such as dicetyl phosphate, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, dipalmitoyl phosphatidylserine, eleostearoylphosphatidylcholine, eleostearoylphosphatidylethanolamine, and eleostearoylphosphatidylserine can do.

  It is well known that delivery of pharmaceuticals to the brain and central nervous system is challenging due to the blood-brain barrier (BBB) that inhibits or prevents the delivery of small and large molecules from the circulating blood to the brain. is there. Thus, the disclosed formulations can be formulated for inhalation or intranasal delivery to the brain. In order for the therapeutic agent to have proper absorption and bioavailability in the CNS, a solubilizing agent may be required. In certain embodiments, the active ingredient is encapsulated in a carrier for intranasal delivery to the CNS, such as a cyclodextrin, microemulsion, or nanoparticle. Cyclodextrin inclusion complexes containing a hydrophobic cavity surrounded by a hydrophilic shell can improve the solubility of poorly water-soluble drugs and thus enhance brain uptake after intranasal administration . Polymer nanoparticles having a hydrophobic core of polylactic acid (PLA) and a hydrophilic shell of methoxy-poly (ethylene glycol) (MPEG) also improve solubility and intranasal drug targeting to the CNS. Contemplated for use in the disclosed compositions.

  Since improving membrane permeability can enhance transport to the CNS along the olfactory and trigeminal nerves, efficient delivery to the CNS following intranasal administration is also dependent on membrane permeability. In addition, the composition may be used in conjunction with penetration enhancers such as surfactants, bile salts, lipids, cyclodextrins, polymers, and tight junction modifiers.

  In certain embodiments, the disclosed compositions can be formulated for oral administration. Such formulations may be, for example, in the form of tablets, capsules, or beads, or liquids, gels, or syrups. The form and strength of an oral delivery composition is determined by absorbency characteristics, acceptable or safe serum drug concentration and the ability to deliver an effective amount of drug to the CNS.

  Solid oral compositions can be prepared as tablets, powders or beads, for example, achieving release in selected portions of the gastrointestinal system, maintaining an effective dose for a longer period of time, or at specific times To deliver a selected dosage range, it can be formulated for immediate release, sustained or controlled release, delayed release, or a combination thereof. Sustained release, controlled release and delayed release formulations often require the use of a coating layer over an active pharmaceutical ingredient (API) core or layer.

  Orally administered tablets, troches, pills, capsules, etc. also contain binders, excipients, disintegrants, lubricants, flavoring or sweetening agents, buffers, salts, sugars, and polymeric Coatings (both water soluble and water insoluble), film formers and plasticizers can be included.

  Liquid formulations can contain the drug in a solution, emulsion, suspension, or colloidal suspension, eg, in a suitable solvent and excipient. A syrup or elixir can contain additional thickening or thickening agents, if desired.

  Compositions for topical application or infusion can be formulated as aqueous solutions, lotions, jellies or oily solutions or suspensions. A composition in the form of an aqueous solution is obtained by dissolving a caspase-6 inhibitor in a solvent and buffer solution and optionally a polymer binder. Oily formulations for topical application are obtained by suspending a caspase-6 inhibitor in oil and optionally adding a swelling agent such as aluminum stearate and / or a surfactant. Absorption enhancers, such as DMSO, can also be added to the topical composition for transdermal administration.

  Unless otherwise specified or defined in the context of use, all terms convey their ordinary meaning herein as used in the respective technical field and as discussed below. Means that.

  As used herein, “pharmaceutically acceptable carrier” includes, but is not limited to, solvents, dispersion media, coatings, antibacterial and antifungal agents, agents that affect isotonicity and absorption, and the like. Including any and all inactive or inactive ingredients. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional vehicle or agent is incompatible with the active ingredient, its use in a therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

  As used herein, the term “pharmaceutically acceptable salt” refers to a non-toxic pharmaceutically acceptable salt as described (Ref. International J. Pharm., 1986, 33, 201-217; J. Pharm. Sci., 1997 (January), 86, 1, 1). However, without limitation, hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, succinic acid, maleic acid, fumaric acid, Malic acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, hydroxyethanesulfonic acid, benzenesulfonic acid, oxalic acid, pamoic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, salicylic acid Other salts well known to those skilled in the art, including saccharic acid or trifluoroacetic acid, may be useful in preparing the compositions of the present disclosure. Representative organic or inorganic bases include, but are not limited to, basic or cationic salts such as benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium , Sodium and zinc.

  The compositions and active agents of the present disclosure are administered in an “effective amount”, “effective dose” or “therapeutically effective amount or dose”. By “effective” or “therapeutically effective amount” or dose of a drug or pharmacologically active agent is meant a non-toxic but sufficient amount of the drug or agent that achieves the desired effect. In this disclosure, an “effective amount” is the amount of the composition or active agent effective to ameliorate, ameliorate or prevent one or more symptoms of the condition being treated. The amount that is “effective” varies from subject to subject, depending on the age, weight and general condition of the individual, or the particular active agent. The therapeutic amount or effective dose or amount is often determined by the physician and is based on data obtained from experiments obtained by administering increasing doses until the best balance of benefits versus side effects is reached.

  The term “parenteral” as used herein includes transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection (directly into the CNS) or infusion techniques. Includes all parenteral delivery techniques.

  As used herein, “liposome” is meant to describe an amphiphilic lipid bilayer structure that encloses an aqueous or hydrophilic center, where the lipid “tail” forms a bilayer envelope. However, more hydrophilic “heads” are aligned at the media's internal and external interfaces. Typical phospholipids are phospholipids or cholesterol. Liposomes are very simple structures composed of one or more lipid bilayers of amphiphilic lipids, ie phospholipids or cholesterol. Both hydrophilic and hydrophobic drugs can be carried in liposomes, cores or bilayers, respectively, or negatively charged drugs can be carried by, for example, cationic liposomes Can do. The term liposome can be used to refer to liposomes having a size in the range of 20 nm to several μm.

  As used herein, “mixed micelles” are aggregates of surfactant particles dispersed in a liquid colloid. An exemplary detergent or surfactant structure can be an aggregation of bile salts, phospholipids, tri, di and monoglycerides, fatty acids, free cholesterol and fat soluble micronutrients. Micellar solutions are thermodynamically stable systems that form spontaneously in water and organic solvents. The interaction between the micelle and the hydrophobic / lipophilic drug results in the formation of mixed micelles (MM).

  As used herein, the term “lipid microparticle” is meant to include lipid nanospheres and microspheres. Microspheres are generally defined as small spherical particles in the range of about 0.2-100 μm made from any material. Smaller spheres less than 200 nm are usually referred to as nanospheres. Lipid microspheres are uniform oil / water microemulsions similar to commercially available fat emulsions and are prepared by intensive sonication procedures or high pressure emulsification methods (grinding methods).

  The term “polymer nanoparticle” is meant to refer to an active agent, eg, a caspase-6 inhibitor, dissolved in a nanopolymer matrix or trapped or adsorbed on the particle surface. Suitable polymers for the preparation of organic nanoparticles include cellulose derivatives and polyesters such as poly (lactic acid), poly (glycolic acid) and copolymers thereof. Due to their small size, their large surface area / volume ratio, and the possibility of functionalization of the interface, polymer nanoparticles are an advantageous carrier and release system. If the particle size is less than about 50 nm, the particles can avoid an immune response and can better deliver the drug across the membrane barrier.

  A variety of water soluble polymers can be used in the disclosed compositions. Such polymers include, but are not limited to, polyethylene oxide (PEO), ethylene oxide-propylene oxide copolymer, polyethylene-polypropylene glycol (eg, poloxamer), carbomer, polycarbophil, chitosan, polyvinyl pyrrolidone (PVP), polyvinyl Alcohol (PVA), hydroxyalkylcelluloses such as hydroxypropylcellulose (HPC), hydroxyethylcellulose, hydroxymethylcellulose and hydroxypropylmethylcellulose, sodium carboxymethylcellulose, methylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, polyacrylates such as carbomers, poly Acrylamide, polymethacrylate , Polyphosphazine, polyoxazolidine, polyhydroxyalkyl carboxylic acid, alginic acid and its derivatives such as carragnate alginate, ammonium and sodium alginate, starch and starch derivatives, polysaccharides, carboxypolymethylene, polyethylene glycol, natural Gums such as guar gum, gum arabic, gum tragacanth, karaya gum and xanthan gum, povidone, gelatin and the like are included.

  In certain embodiments in delayed release, or in the lower part of the gastrointestinal tract, at least the delayed release layer comprises one or more polymers, such as but not limited to EUDRAGIT® L100, EUDRAGIT® L100. Acrylic polymers, acrylic copolymers, methacrylic polymers or methacrylic copolymers, including -55, EUDRAGIT® L30D-55, EUDRAGIT® S100, EUDRAGIT® 4135F, EUDRAGIT® RS, acrylic acid And methacrylic acid copolymer, methyl methacrylate, methyl methacrylate copolymer, ethoxy ethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, Rylic acid, polymethacrylic acid, alkylamine methacrylate copolymer, polymethyl methacrylate, polymethacrylic anhydride, polymethacrylate, polyacrylamide, polymethacrylic anhydride and glycidyl methacrylate copolymer, alkylcelluloses such as ethylcellulose, methylcellulose, carboxy Methylcellulose calcium, certain substituted cellulose polymers such as hydroxypropylmethylcellulose phthalate, and hydroxypropylmethylcellulose succinate, cellulose acetate butyrate, cellulose acetate phthalate, and cellulose acetate trimaleate, polyvinyl acetate phthalate , Polyester, wax, shellac, zein Etc. can be included.

  Eudragits are well known polymers and copolymers that are useful for controlled release applications. EUDRAGIT® grade for enteric coatings is based on anionic polymers of methacrylic acid and methacrylate. These contain -COOH as a functional group. These dissolve in the range of pH 5.5 to pH 7. EUDRAGIT® FS30D is an aqueous dispersion of anionic copolymers based on methyl acrylate, methyl methacrylate and methacrylic acid. It is insoluble in acidic media but dissolves by the formation of salts above pH 7.0. EUDRAGIT® L100-55 and L30-55 dissolve at pH above 5.5. EUDRAGIT® L100 and S100 dissolve at pH above 6.0.

  Sustained release EUDRAGIT® formulations are used for many oral dosage forms to allow time-controlled release of the active ingredient. Drug delivery can be controlled throughout the entire gastrointestinal tract for increased therapeutic efficacy and patient compliance. The combination of EUDRAGIT® RL (easy permeable) and RS (poor permeable) grades of different polymers allows a tailored release profile to achieve a wide range of drug delivery performances Allow alternatives. EUDRAGIT® NE polymer is a neutral ester dispersion that does not require a plasticizer and is particularly suitable for granulation processes in the manufacture of matrix tablets and sustained release coatings.

  As used herein, “osmotic agent” means a compound that absorbs water from the environment, often used to cause swelling of the active ingredient and elimination of the active ingredient from the formulation. Intended. Exemplary osmagents or osmotic agents include organic and inorganic compounds such as salts, acids, bases, chelating agents, sodium chloride, lithium chloride, magnesium chloride, magnesium sulfate, lithium sulfate, potassium chloride, sulfite. Sodium, calcium bicarbonate, sodium sulfate, calcium sulfate, calcium lactate, d-mannitol, urea, tartaric acid, raffinose, sucrose, alpha-d-lactose monohydrate, glucose, combinations thereof, and extensively in the art Other similar or equivalent materials that are known are included.

  As used herein, the term “disintegrant” means a compound used in a solid dosage form to facilitate the perturbation of a solid mass (layer) into smaller particles that are more easily dispersed or dissolved. Intended to be. Exemplary disintegrants include, by way of example and without limitation, starches such as corn starch, potato starch, its pregelatinized and modified starches, sweeteners, clays, bentonite, microcrystalline cellulose (eg Avicel) , Carboxymethyl cellulose calcium, croscarmellose sodium, alginic acid, sodium alginate, cellulose polyacrylin potassium (eg, Amberlite ™), alginate, sodium starch glycolate, gum, agar, guar, carob, caraya, Pectin, tragacanth, crospovidone and other materials known to those skilled in the art are included. Super disintegrants are fast acting disintegrants. Exemplary superdisintegrants include crospovidone and low substitution HPC.

  In certain embodiments, a plasticizer is also included in the oral dosage form. Plasticizers suitable for use in the present composition include, but are not limited to, low molecular weight polymers, oligomers, copolymers, oils, small organic molecules, low molecular weight polyols with aliphatic hydroxyls, ester type plasticizers, Included are glycol ethers, poly (propylene glycol), multiblock polymers, single block polymers, low molecular weight poly (ethylene glycol), citrate ester type plasticizers, triacetin, propylene glycol and glycerin. Such plasticizers also include ethylene glycol, 1,2-butylene glycol, 2,3-butylene glycol, styrene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol and other poly (ethylene glycol) compounds, monopropylene glycol Monoisopropyl ether, propylene glycol monoethyl ether, ethylene glycol monoethyl ether, diethylene glycol monoethyl ether, sorbitol lactate, ethyl lactate, butyl lactate, ethyl glycolate, dibutyl sebacate, acetyl tributyl citrate, triethyl citrate, acetyl citric acid Triethyl, tributyl citrate and allyl glycolate can be included.

  The compositions of the present disclosure may also include one or more functional excipients, such as lubricants, thermal lubricants, antioxidants, buffers, alkalizing agents, binders, diluents. , Sweetener, chelating agent, colorant, flavoring agent, surfactant, solubilizer, wetting agent, stabilizer, hydrophilic polymer, hydrophobic polymer, wax, lipophilic material, absorption enhancer, preservative, absorbent , Crosslinkers, bioadhesive polymers, retarders, pore formers, and perfumes.

  Lubricants or thermal lubricants useful in the disclosed compositions include, but are not limited to, fatty esters, glyceryl monooleate, glyceryl monostearate, wax, carnauba wax, beeswax, vitamin E succinate, Stearates such as magnesium or calcium stearate, calcium hydroxide, talc, sodium stearyl fumarate, hydrogenated vegetable oils, stearic acid, glyceryl behenate, magnesium stearate, calcium and sodium, stearic acid , Talc, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycol, sodium oleate, or sodium lauryl sulfate.

  As used herein, the term “antioxidant” is an agent used to inhibit oxidation and thus prevent alteration of the preparation due to oxidation due to the presence of oxygen free radicals or free metals in the composition. Is meant to mean Examples of such compounds include, but are not limited to, ascorbic acid, ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), hypophosphorous acid, monothioglycerol, This includes sodium ascorbate, sodium formaldehyde sulfoxylate and sodium metabisulfite and others known to those skilled in the art. Other suitable antioxidants include, for example, vitamin C, sodium bisulfite, vitamin E and its derivatives, propyl gallate or sulfite derivatives.

  Suitable binders for use in the disclosed compositions include beeswax, carnauba wax, cetyl palmitate, glycerol behenate, glyceryl monostearate, glyceryl palmitostearate, glyceryl stearate, hydrogenated Includes castor oil, microcrystalline wax, paraffin wax, stearic acid, stearic alcohol, stearate 6000WL1644, gelluce 50/13, poloxamer 188, and polyethylene glycol (PEG) 2000, 3000, 6000, 8000, 10000 or 20000 It is.

  Buffers are used to resist pH changes due to dilution or addition of acid or alkali. Examples of such compounds include, but are not limited to, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate, inorganic acids or organic Acid salts, salts of inorganic or organic bases and others known to those skilled in the art are included.

  As used herein, the term “alkalizing agent” is intended to mean a compound used to provide an alkaline medium for product stability. Examples of such compounds include, but are not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine. And trolamine and others known to those skilled in the art.

  Exemplary binders for use in the disclosed compositions include: polyethylene oxide; polypropylene oxide; polyvinyl pyrrolidone; polyvinyl pyrrolidone-co-vinyl acetate; acrylate and methacrylate copolymers; polyethylene; polycaprolactone; Alkyl celluloses and cellulose derivatives such as low substituted HPC (L-HPC), methyl cellulose; hydroxyalkyl celluloses such as hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxybutyl cellulose; hydroxyalkyl alkyl celluloses such as hydroxy Ethylmethylcellulose and hydroxypropylmethylcellulose; starch, Cutin; PLA and PLGA, polyesters (shellac), waxes such as carnauba wax, beeswax; polysaccharides, e.g., cellulose, tragacanth, gum arabic, guar gum, and xanthan gum.

  Exemplary chelating agents include EDTA and its salts, alpha hydroxy acids such as citric acid, polycarboxylic acids, polyamines, derivatives thereof and others known to those skilled in the art.

  As used herein, the term “colorant” is intended to mean a compound used to impart color to a solid (eg, tablet) pharmaceutical preparation. Examples of such compounds include, but are not limited to, FD & C Red No. 3, FD & C Red No. 20, FD & C Yellow No. 6, FD & C Blue No. 2, D & C Green No. 5, D & C Orange No. 5, D & C Red No. 8 No., caramel, and iron oxide, red, other FD & C pigments and natural colorants such as grape skin extract, beet red powder, beta carotene, annatto, carmine, turmeric, paprika, and those skilled in the art Includes other known materials. The amount of colorant used will vary as desired.

  As used herein, the term “flavoring agent” is intended to mean a preferred flavor and often a compound used to impart an odor to a pharmaceutical preparation. Exemplary flavoring or flavoring agents include synthetic flavor oils and flavored aromatics and / or natural oils, extracts from plants, leaves, flowers, fruits, etc., and combinations thereof. These may also include cinnamon oil, winter green oil, peppermint oil, clove oil, bay oil, anise oil, eucalyptus, thyme oil, eucalyptus oil, nutmeg oil, sage oil, bitter peach oil and cassia oil. Other useful flavors include citrus oils including vanilla, lemon, orange, grape, lime and grapefruit, and fruit essences including apples, pears, peaches, strawberries, raspberries, cherries, plums, pineapples, apricots, etc. Including. Flavors that have been found to be particularly useful include commercially available orange, grape, cherry and bubble gum flavors and mixtures thereof. The amount of flavoring agent can depend on a number of factors including the desired organoleptic effect. The flavor is presented in any amount as desired by one skilled in the art. Particular flavors are grape and cherry flavors and citrus flavors such as orange.

  Suitable surfactants include polysorbate 80, sorbitan monooleate, polyoxymer, sodium lauryl sulfate or others known in the art. Soap and synthetic detergents can be used as surfactants. Suitable soaps include fatty acid alkali metal, ammonium, and triethanolamine salts. Suitable detergents include cationic detergents such as halogenated dimethyldialkylammonium halides, alkylpyridinium halides, and alkylamine acetates; anionic detergents such as alkyl, aryl and olefin sulfonates, alkyls, olefins, ethers and monoglyceride sulfates. Nonionic detergents such as fatty amine oxides, fatty acid alkanolamides, and poly (oxyethylene) -block-poly (oxypropylene) copolymers; and amphoteric detergents such as alkyl β-aminopropionates And 2-alkyl imidazoline quaternary ammonium salts; and mixtures thereof.

  A wetting agent is an agent that reduces the surface tension of a liquid. Wetting agents include alcohol, glycerin, protein, peptide water miscible solvents such as glycol, hydrophilic polymer polysorbate 80, sorbitan monooleate, sodium lauryl sulfate, fatty acid alkali metals, ammonium, and triethanolamine salts, halogenated Dimethyldialkylammonium, alkylpyridinium halides, and alkylamine acetates; anionic detergents such as alkyl, aryl and olefin sulfonates, alkyls, olefins, ethers and monoglyceride sulfates, and sulfosuccinates; nonionic detergents such as fats Amine oxides, fatty acid alkanolamides, and poly (oxyethylene) -block-poly (oxypropylene) copolymers; and amphoteric detergents For example, alkyl β- aminopropionates and 2-alkyl imidazoline quaternary ammonium salts; include and mixtures thereof.

  Solubilizing agents include cyclodextrins, povidone, combinations thereof and others known to those skilled in the art.

  Exemplary waxes include carnauba wax, beeswax, microcrystalline wax and others known to those skilled in the art.

  Exemplary absorption enhancers include dimethyl sulfoxide, vitamin E PGS, sodium cholate and others known to those skilled in the art.

  Preservatives include compounds that are used to prevent microbial growth. Suitable preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercury nitrate and thimerosal, and those skilled in the art. Other known ones are included.

  Examples of absorbents include sodium starch glycolate (Explotab ™, Primojel ™) and croscarmellose sodium (Ac-Di-Sol ™), cross-linked PVP (Polyplasmone ™ XL10), veegum, Clay, alginate, PVP, alginic acid, carboxymethylcellulose calcium, microcrystalline cellulose (eg, Avicel), potassium polacrilin (eg, Amberlite ™), sodium alginate, corn starch, potato starch, pregelatinized starch, processed Starches, cellulosic agents, montmorillonite clays (e.g., Bentonite), gum, agar, locust bean gum, karaya gum, pectin, tragacanth, and other disintegrants known to those skilled in the art.

  A crosslinker is defined as any compound that forms a crosslink between portions of a polymer. Crosslinkers can include, by way of example, but not limited to organic acids, alpha-hydroxy acids, and beta-hydroxy acids. Suitable crosslinkers include tartaric acid, citric acid, fumaric acid, succinic acid and others known to those skilled in the art.

  Bioadhesive polymers include polyethylene oxide, Klucel® (hydroxypropylcellulose), CARBOPOL, polycarbophil, GANTREZ, poloxamer, and combinations thereof and others known to those skilled in the art.

  A retarder is a glass transition temperature above 45 ° C. or above 50 ° C. before being plasticized by other drugs in the formulation, including other polymers and other excipients required for processing. A drug which is an insoluble or slightly soluble polymer having (Tg). Excipients include waxes, acrylic resins, cellulosic materials, lipids, proteins, glycols and the like.

  Exemplary pore formers include water soluble polymers such as polyethylene glycol, propylene glycol, poloxamer and povidone; binders such as lactose, calcium sulfate, calcium phosphate, etc .; salts such as sodium chloride, magnesium chloride, etc .; As well as other similar or equivalent materials widely known in the art.

  As used herein, the term “sweetening agent” is intended to mean a compound used to impart sweetness to a preparation. Such compounds include, by way of example, but not limited to aspartame, dextrose, glycerin, mannitol, sodium saccharin, sorbitol, sucrose, fructose and other such materials known to those skilled in the art.

  It should be understood that compounds used in the art of pharmaceutical formulation generally serve a variety of functions or purposes. Thus, when a compound listed herein is mentioned only once or is used to define a plurality of terms herein, its purpose or function is in addition to its stated purpose or function. It should not be interpreted exclusively.

  The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A shows a synthetic scheme for compounds 8a-f. FIG. 1B shows possible tautomeric forms of compounds 8a-f.

FIG. 2 shows hydrolyzable ring opening of compound 8 to compound 9 and decarboxylation of compound 9e to compound 10.

Figures 3 and 3a-h show the structure of the PG3 compound and its analogs. Figures 3 and 3a-h show the structure of the PG3 compound and its analogs. Figures 3 and 3a-h show the structure of the PG3 compound and its analogs. Figures 3 and 3a-h show the structure of the PG3 compound and its analogs. Figures 3 and 3a-h show the structure of the PG3 compound and its analogs. Figures 3 and 3a-h show the structure of the PG3 compound and its analogs. Figures 3 and 3a-h show the structure of the PG3 compound and its analogs. Figures 3 and 3a-h show the structure of the PG3 compound and its analogs. Figures 3 and 3a-h show the structure of the PG3 compound and its analogs.

FIG. 4 shows that PG3 compounds inhibit caspase 6 enzyme in a dose-dependent manner. Different concentrations of PG3 compound were incubated with Htt protein and caspase-6 enzyme. The Htt substrate from COS-7 cell lysate is cleaved by caspase 6, and the fragment is detected by FRET between the N-terminal BKP1 antibody and the neo-epitope antibody against amino acid 586. (A) shows dose response curves for compounds PG3, PG3a and PG3b. (B) shows dose response curves for compounds PG3c, PG3d and PG3e. (C) shows dose response curves for compounds PG3f, PG3g and PG3h.

FIG. 5 shows that the presence of PG3d compound inhibits cleavage of Htt by caspase-6 in a Western blot assay. COS-7 cells co-transfected with the htt-4C construct and human caspase-6 lacking the prodomain remained untreated, treated with 10 uM PG3d compound, or 3 uM pan-caspase inhibition Treatment with agent (Q-VD-Oph). (A-upper panel) Cleavage of the Htt protein resulting in a 586 amino acid fragment was analyzed by Western blot using the pan-HTT BKP1 antibody. The presence of PG3d inhibitor or pan-caspase inhibitor resulted in reduced cleavage of the Htt protein. (A, lower panel) The presence of PG3d compound or pan-caspase inhibitor (Q-VD-Oph) also resulted in reduced levels of active caspase-6 enzyme. (B) shows a graphical representation of the level of the 586 amino acid fragment compared to the actin level from Western blot analysis. Protein levels were quantified using Licor Odyssey imaging software. Expression of the Htt-586 fragment relative to actin expression is shown on the y-axis. The presence of 10 uM PG3d compound resulted in a significant reduction in the expression of the Htt cleavage fragment compared to untreated cells. Student t-test ^** : p <0.01

FIG. 6 shows that PG3 compounds inhibit the cleavage of lamin A by caspase-6 in HEK293 cells, as quantified by the Mesoscale ELISA method. (A) shows dose response curves for compounds PG3a, PG3b, PG3d and PG3d. (B) shows dose response curves for compounds PG3e, PG3g and PG3h.

FIG. 7 shows that PG3d inhibits intracranial activation of caspase-6. Primary cortical neurons from FVB / N mice were treated with 10 uM camptothecin for 30 hours in the presence or absence of 10 uM PG3d. Camptothecin treatment results in activation of caspase-6, which can be quantified by measuring the cleavage of lamin A (Ehrnhofer et al., PLoS One, 2011, 6 (11): e27680). The presence of PG3d in the neuronal medium significantly reduces caspase-6 activity. Student t test, ^** : p <0.01.

FIG. 8 shows that PG3d improves neuronal survival during excitotoxic stress. Primary cortical neurons from FVB / N mice were treated with different amounts of NMDA in the medium for 20 hours, which results in a loss of intracellular ATP levels, a measure of survival. The presence of 10 uM PG3d significantly improves neuronal survival in this paradigm. Two-way ANOVA: NMDA process p <0.0001, PG3d process p = 0.0001. Post hoc Bonferroni test: ^** = p <0.01.

FIG. 9 shows the results of cell studies showing the interaction between caspase-6 and Htt fragments in COS-7 cells. Following transfection, COS-7 cells were exposed to DMSO, pan-caspase inhibitor Q-VD-Oph (Q-VD-OPh) or PG3d compound (PG3d). After a 24 hour incubation period, cell lysates were processed by Western blot or immunoprecipitated with caspase-6 antibody followed by Western blot analysis. (A) Co-trans treated with DMSO (nt) and exposed to 3 uM pan-caspase inhibitor Q-VD-Oph (Q-VD-OPh) or treated with 10 uM PG3d compound (PG3d) Western blot analysis of lysates from infected COS-7 cells. The upper panel shows Western blot analysis with Htt antibody 2166 (Millipore). The lower panel shows Western blot analysis with caspase-6 antibody HD91, which detects the full length and active form of caspase-6. (B) Western blot analysis of cell lysate from FIG. 9A, following immunoprecipitation with caspase-6 antibody to identify binding partner of caspase-6 enzyme. The upper panel shows Western blot analysis with Htt antibody 2166 (Millipore) and the lower panel shows Western blot analysis with HD91 caspase-6 antibody.

FIG. 10 is a bar graph that quantifies the coprecipitation data shown in FIG. 9B by densitometric analysis. One-way ANOVA, p = 0.031, post-tukey test ^* : p <0.05.

Array:
SEQ ID NO: 1 (htt-4C). SEQ ID NO: 1 has an additional 10 amino acids at the N-terminus (relative to wild type huntingtin), including a His-tag that allows processing of the expressed polypeptide. Htt-4C is truncated at amino acid 1212 (numbering by wild type huntingtin sequence) and amino acids 513, 530, 552 and 589 (by wild type huntingtin sequence), marked by bold underlined text. Numbering) with four D to A amino acid substitutions. The IVLD caspase-6 cleavage site is marked with a double underline.

SEQ ID NO: 2 (caspase-6 delta prodomain). SEQ ID NO: 2 comprises amino acids 24-293 of human caspase-6 and the prodomain (aa1-23) is deleted. This deletion results in faster intracellular self-activation of the enzyme after transfection (Klaiman et al., BBA, 2009, 1793 (3): 592-601). The protein has an additional 31 amino acids at the C-terminus (relative to wild-type caspase-6) containing a DDK-tag that allows detection of the expressed polypeptide.

SEQ ID NO: 3 (wt htt). SEQ ID NO: 3 has an additional 10 amino acids at the N-terminus (relative to wild-type huntingtin), including a His-tag that allows processing of the expressed polypeptide. Wt Htt is truncated at amino acid 1212 (numbering by wild type huntingtin sequence).

SEQ ID NO: 4 (full length caspase-6). SEQ ID NO: 4 comprises amino acids 1-293 of human caspase-6. The protein has an additional 31 amino acids at the C-terminus (relative to wild-type caspase-6) containing a DDK-tag that allows detection of the expressed polypeptide.
Cell transfection and cell lysis

  COS-7 cells were grown in DMEM supplemented with 10% fetal calf serum, 1% penicillin / streptomycin and 0.5% glutamine. Transfections were performed using Fugene reagent (Roche) according to the manufacturer's instructions. The transfected DNA encodes amino acids 1-1212 of the human Htt protein with 15 glutamine and a C-terminal HA-tag under the control of the CMV promoter (Warby, 2008, supra; Wellington et al., 2000, J Biol Chem, 275: 19831-19838). The transfected construct is 4c Htt containing the D to A mutation at amino acids 513, 530, 552 and 589 (SEQ ID NO: 1). Twenty-four hours after transfection, cells were harvested by trypsinization, pellets were washed in PBS and 50 mM Tris, pH 8, 150 mM supplemented with lysis buffer (1 × complete protease inhibitor (Roche) and 4 mM Pefabloc). Dissolved in NaCl, 1% Igepal). Lysates were incubated on ice for 10 minutes, vortexed, sonicated for 4 seconds, and then centrifuged at 21,000 xg for 10 minutes at 4 ° C. The supernatant was saved and the protein concentration was determined by Biorad DC assay and stored at −80 ° C. until use.

HEK293 cells were grown in DMEM supplemented with 10% fetal calf serum, 1% penicillin / streptomycin and 0.5% glutamine. Transfections were performed using Fugene reagent (Roche) according to the manufacturer's instructions. The transfected DNA encodes amino acids 24-293 of human caspase 6 enzyme with a C-terminal DDK tag under the control of a CMV promoter (vector pCMVSport6) (SEQ ID NO: 2). Transfected HEK293 was used to measure the intracellular activity of caspase-6 as demonstrated by cleavage of the lamin substrate (described below).
Purification of Htt from COS-7 cell lysate

50 μl of HA-agarose beads (EZ-View, Sigma) was mixed with 1 ml of lysis buffer, centrifuged at 8200 × g for 30 seconds, and the supernatant was discarded. The beads were mixed with 200 μl of cell lysate diluted to 0.5 μg / μl in lysis buffer and incubated for 2 hours at 4 ° C. on a rotating wheel. An aliquot of diluted cell lysate was stored as the input fraction. The sample was centrifuged at 8200 × g for 30 seconds, the supernatant was saved, the beads were washed 3 times with 100 μl lysis buffer, and the supernatant was saved as a wash fraction. Add 100 μl HA peptide (100 μg / ml, Sigma) in RIPA buffer to beads (50 mM Tris, pH 8, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) Elution was performed by incubating at 37 ° C. for 10 minutes. The elution step was repeated 5 times and all eluates were saved. The beads were then mixed with SDS-added dye and manipulated with 10 μl aliquots of all fractions on a 3-8% NuPage Tris-Acetate gel (Invitrogen) after heat denaturation. Gels are blotted for detection with BKP1 (Kalchman et al., J Biol Chem (1996) 271 (32): 19385)) and HA antibody on the Odyssey Imaging System (Li-cor Biosciences) Or stained with Coomassie dye for total protein detection.
Assessment of Htt cleavage by Western blot

An aliquot of COS-7 lysate expressing 4c Htt corresponding to 25 μg of protein was added to cleavage buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% Glycerol) and the desired amount of recombinant caspase-6 (BioMol) was diluted to 10 μl. For experiments involving caspase-6 inhibitor compounds, the desired amount of inhibitor was mixed with caspase prior to addition to the COS-7 lysate. Samples were incubated at 37 ° C. for 1 hour and analyzed on 3-8% NuPage Tris-acetate gel (Invitrogen) followed by Western blotting and BKP1 and Neo- on Odyssey imaging system (Li-cor Biosciences). Detection with the 586 antibody was performed. BKP1 and Neo-586 antibodies have been previously described (Warby et al., Hum. Mol. Genet, 2008, 17 (15): 2390-404).
Quantification of Htt expression by FRET

A dilution series of cell lysate is prepared in sample buffer (PBS without 1 × CaCl 2 or MgCl 2, 0.4% Triton, 1 × complete protease inhibitor cocktail (Roche)), and Tb-labeled BKP1 antibody and D2-labeled HA antibody (Cisbio) was diluted to 1 ng / μl (Tb) and 10 ng / μl (D2) in antibody dilution buffer (50 mM NaH 2 PO 4, 0.1% BSA, 0.05% Tween) . The antibody was premixed at a 1: 1 ratio, then 10 μl of cell lysate and 2 μl of antibody mix were pipetted into each well of a white 384 well plate (Nunc). The plate was centrifuged briefly and FRET was measured with a Victor3 multilabel plate reader (Perkin Elmer) according to the following settings. Excitation: 340 nm, emission 1: 615 nm, emission 2: 665 nm, 50 μs delay, 200 μs window time, 2000 μs cycle time. To obtain the final FRET signal, the ratio between Emission 2 / Emission 1 (D2 / Tb signal) was calculated.
Simultaneous caspase-6 cleavage and 586 fragment detection by FRET

  A dilution series of cell lysate and caspase-6 (2.2 × final concentration) was added to FRET cleavage buffer (10 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% gelatin, 0.1% CHAPS, 2 mM). DTT). This is because it was previously found that this buffer best stabilizes the diluted form of caspase-6 at room temperature. For negative controls, 22 μΜ zVAD-fmk was added to the caspase-6 dilution.

  Tb-labeled BKP1 antibody and D2-labeled 586 antibody (Cisbio) were diluted to 1 ng / μl (Tb) and 10 ng / μl (D2) in FRET cleavage buffer and premixed at a 1: 1 ratio.

In each well of a white 384 well plate (Nunc), 10 μl of cell lysate is mixed with 10 μl of caspase and 2 μl of antibody mix, the plate is centrifuged briefly and 37 ° C. in a Victor3 multilabel plate reader (Perkin Elmer). Incubated at FRET was measured every 30 minutes up to 2 hours with the following settings. Excitation: 340 nm, emission 1: 615 nm, emission 2: 665 nm, 50 μs delay, 200 μs window time, 2000 μs cycle time. To obtain the final FRET signal, the ratio between Emission 2 / Emission 1 (D2 / Tb signal) was calculated. After 2 hours at 37 ° C., the assay plate was sealed and after further incubation at 4 ° C. for 20 hours, the FRET signal was read again.
Assessment of intracellular Htt cleavage by Western blot

COS-7 cells were co-transfected with the 4c htt fragment and human caspase-6 lacking the prodomain, which resulted in fast self-activation of the caspase-6 enzyme and generation of a 586aa Htt cleavage fragment. Co-transfected cells were exposed to 10 uM PG3d compound, 3 uM Q-VD-Oph pan-caspase inhibitor or left untreated. Untransfected cells were included as a negative control and purified 586aa Htt fragment was included as a positive control. The cell lysate was subjected to Western blotting, and the 586AA fragment generated in the cells was detected with Htt antibody 2166 (Millipore). Caspase-6 expression and activation was assessed by Western blotting using antibody HD91 (Ehrnhofer et al., HMG, 2014, 23 (3): 717-29).
Assessment of lamin cleavage in HEK293 cells overexpressing caspase-6

Quantitative assessment of intracellular lamin cleavage in caspase-6 transfected HEK293 cells was performed as previously described (Ehrnhofer et al., PLoS One, 2011, 6 (11): e27680). ). Briefly, HEK293 cell lysate was adjusted to 1 μg protein / μl in lysis buffer, diluted to 0.2 μg / μl in PBS, and 5 μl was added to a multi-array high binding 96 well plate (Mesoscale discovery). . After 1 hour incubation at room temperature, the wells were blocked by adding 150 μl 5% BSA in PBS followed by further incubation at room temperature for 1 hour. Wells were then washed 3 times with 150 μl PBS + 0.05% Tween and 25 μl antibody mix was added (Cell signaling # 2036 at 1: 100 dilution, Mesoscale at 1: 500 dilution in PBS with 1% BSA). discovery goat-anti-rabbit sulfo-tag). After 1 hour incubation at room temperature, the wells were washed 3 times with 150 μl PBS + 0.05% Tween and 150 μl / well 2 × reading reagent (Mesoscale discovery) was added. The plate was read with a Sector imager 6000 (Mesoscale discovery).
Assessment of lamin cleavage in primary neuronal cultures.

Cortical neuron cultures from FVB mice were prepared as described (Metzler et al., J Neurosci (2007) 27 (9): 2298). Cells were treated with camptothecin on day 10 in vitro and harvested after 30 hours by scraping in PBS supplemented with 1 × complete protease inhibitor (Roche) and 4 mM Pefabloc. Cells were lysed by suspension in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Igepal supplemented with 1 × complete protease inhibitor (Roche) and 4 mM Pefabloc). The lysate was incubated on ice for 10 minutes, vortexed and sonicated for 4 seconds, followed by centrifugation at 21000 × g for 10 minutes at 4 ° C. Supernatants are saved, protein concentration is determined by Biorad DC assay, and quantitative assessment of cleaved lamin A in neuronal lysates is performed by Ehrnhofer et al., PLoS One, 2011, 6 (11): e27680. By the Mesoscale ELISA method.
Assessment of neuronal survival during excitotoxic stress

Cortical neuron cultures from FVB mice were prepared as described (Metzler et al., J Neurosci (2007) 27 (9): 2298). Cells were treated in vitro on day 10 with 10 uM PG3d or DMSO as a negative control and then with 25 nM NMDA, 50 uM NMDA, or left untreated for 20 hours. Intracellular ATP measurements were used as an assessment of neuronal viability as previously described using the Cell-titer glo kit from Promega according to the manufacturer's instructions (Uribe et al., HMG, 2012, 21 (9): 1954-67).
Assessment of binding interaction between htt and caspase-6.

COS-7 cells were co-transfected with a 1212 amino acid HTT fragment (SEQ ID NO: 3) and full-length human caspase-6 enzyme (SEQ ID NO: 4). Co-transfected cells were exposed to 10 uM PG3d compound, 3 uM Q-VD-Oph pan-caspase inhibitor, or treated with DMSO as a control. An aliquot of the cell lysate was subjected to Western blotting, the HTT fragment was detected with Htt antibody 2166 (Millipore), and the presence of caspase-6 was detected using HD91 antibody (Ehrnhofer et al., HMG, 2014, volume 23 ( 3): 717-29). The remaining cell lysate (500 ug protein) was immunoprecipitated at 4 C with 5 ug caspase-6 antibody for 16 hours. The immunoprecipitated protein was then added to an acrylamide gel and immunoblotted to detect the presence of HTT fragments or active caspase 6 enzyme using the antibodies described above.
Synthetic methods for PG3 compounds and analogs thereof.

As described in Table 1, 3-phenylprop-2-inamide (1a) was prepared in high yield according to literature procedures by reaction of 3-phenylprop-2-ynoic acid ester with aqueous ammonia ( Struebing et al., Tetrahedron (2005) 61: 11333). According to this procedure, the corresponding arylpropyne amides 1b-e were obtained in good yield by ammonolysis of the crude arylpropionic acid esters, which was obtained from the corresponding arylpropionic acid with ethanol. Resulted from esterification.

  Treatment of 3-phenylprop-2-inamide (1a) with monosubstituted malonyl chloride (6a-f) or (chlorocarbonyl) ethylketene (7a, b) in diethyl ether at 0-5 ° C. Hydroxy-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8a-f) was delivered in 61-87% yield (as shown in Table 2 and FIG. 1A).

  Theoretically, heterocyclic ring 8 (R1 = C≡CPh) can take tautomeric forms AC (FIG. 1B). A similar mass spectrum of 4-hydroxy-6H-1,3-oxazin-6-one (R1 = aryl; R2 = Η, Me) showed the presence of 4-hydroxy-6-oxa, but zwitterionic And dioxo form a mixture in the gas phase. The presence of substituents at the para position of the benzene ring has a strong influence on the tautomeric ratio. Quantum chemical calculations revealed structure A as the most preferred 1,3-oxazine tautomer in the gas phase. These compounds exist predominantly as tautomers A, dissolved in tetrahydrofuran and dimethyl sulfoxide-d6. (Stanovnik et al., Adv. Heterocycl Chem (2006) 61: 1; Zaks et al., Khim. Geterotsikl. Soedin (1990) 552; Chem. Abstr (1990) 113: h, et al. Geterotsikl (1987) 386: Chem. Abstr. (1988) 108: 5938). In the case of 4-hydroxy-6H-1,3-oxazin-6-one (R1 = Bn, Me and R2 = Ph), Sheibani et al., By NMR spectroscopy, described the tautomer together with the major tautomer A. B could be detected. (Sheibani et al., ARKIVOC (2005) (xv), 88).

¹ H and ¹³ C NMR spectra of 4-hydroxy-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8a-f) described herein are consistent with tautomer A This signal was clearly demonstrated by X-ray crystal structure analysis in the case of 8d.

The prepared 4-hydroxy-2- (phenylethynyl) -6H-1,3-oxazin-6-one 8a-f was proved to be unstable in dimethyl sulfoxide solution. The NMR spectra of the 8a-f samples that have been held in dimethyl sulfoxide-d6 solution for 24 hours at ambient temperature show the hydrolysis product 9a in addition to the signal for 1,3-oxazin-6-one 8a-f. An additional set of signals belonging to ~ f was shown (Figure 2). In the case of 8e, intermediate 9e undergoes spontaneous decarboxylation to give imide 10 as the final product (FIG. 2). The imide 10 was also obtained by treatment of 4-hydroxy-5-phenyl-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8e) with boiling water. The signal of the isolated imide 10 is identical to the further signal in the 8e NMR spectrum that has been retained in the dimethyl sulfoxide-d6 solution. According to these results, it can be concluded that 1,3-oxazin-6-one 8a-f is hydrolyzed by a trace amount of water in a dimethyl sulfoxide solution (FIG. 2).
3-Phenylprop-2-ynamide (1a)

  Prepared from ethyl phenylpropinoate (8.71 g, 8.26 mL, 50 mmol) as colorless crystals by literature procedures; (Struebing et al., Tetrahedron (2005) 61: 11333)] Yield: 6. 70 g (92%); mp 100-102 ° C.

IR (KBr): 3383 and 3179 (NH ₂ ), 2223 (C≡C), 1654 cm ⁻¹ (C═O).

^{MS (EI, 70eV): m} / z (%) = 145 (64) [M] +, 129 (100) [M-NH 2] +, 75 (13), no other peaks> 10%.

_Analysis, calculated for _{C 9 H 7 NO: C,} 74.47; H, 4.86; N, 9.65. Found: C, 74.59; H, 4.84; N, 9.57.
3-Arylprop-2-ynamide (1b-e); general procedure

The corresponding arylpropionic acid (15 mmol) was heated to reflux with EtOH (60 mmol) in the presence of H ₂ SO ₄ for 5 hours. Excess EtOH was removed under reduced pressure and the residue was washed with H ₂ O and saturated aqueous NaHCO ₃ . The organic layer was separated and the aqueous layer was extracted with CHCl ₃ (3 × 50 mL). The combined organic layers were dried (MgSO ₄ ) and concentrated under reduced pressure to give ethyl 3-arylprop-2-inoate as an oily residue that was subsequently dissolved in 25% aqueous NH ₃ (60 mmol). And stirred at room temperature for 24 hours. The solid products 1b-d thus obtained were filtered off and recrystallized.
3- (2-Chlorophenyl) prop-2-ynamide (1b) (Mariella et al., Can J. Chem (1965) 43: 2426; Unangst et al., J. Heterocycl Chem. (1973) 10: 399 )

Colorless crystals; yield: 1.94g (72%); mp121~122 ℃ (EtOH-H 2 O, 1: 1).

IR (KBr): 3318 and 3162 (NH2), 2225 (C≡C), 1655 cm-1 (C = O).

MS (EI, 70 eV): m / z (%) = 181/179 (13/46) [M] ⁺ , 165/163 (34/100) [M-NH ₂ ] ⁺ , 136 (10), 99 ( 16), 75 (13), 74 (15), no other peaks> 10%.

_Analysis, calculated for _{C 9 H 6 ClNO: C,} 60.19; H, 3.37; Cl, 19.74; N, 7.80. Found: C, 60.07; H, 3.38; Cl, 19.75; N, 7.87.
3- (4-Chlorophenyl) prop-2-ynamide (1c) (Schmitt J. FR, 1305340 (1962); Chem Abstr. (1963) 58: 46538)

Colorless crystals; Yield: 2.10 g (78%); mp 186 ° -188 ° C. (MeCN).

IR (KBr): 3399 and 3172 (NH ₂ ), 2218 (C≡C), 1656 cm ⁻¹ (C═O).

MS (EI, 70 eV): m / z (%) = 181/179 (17/53) [M] ⁺ , 165/163 (32/100) [M-NH ₂ ] ⁺ , 99 (15), 75 ( 13) No other peaks> 10%.

_Analysis, calculated for _{C 9 H 6 ClNO: C,} 60.19; H, 3.37; Cl, 19.74; N, 7.80. Found: C, 60.29; H, 3.37; Cl, 19.64; N, 7.92.
3- (4-Bromophenyl) prop-2-ynamide (1d)

Colorless crystals; Yield: 2.55 g (76%); mp 202-204 ° C. (Celsius) (MeCN).

IR (KBr): 3386 and 3172 (NH ₂ ), 2216 (C≡C), 1655 cm ⁻¹ (C═O).

MS (EI, 70 eV): m / z (%) = 225/223 (64/65) [M] ⁺ , 209/207 (95/100) [M-NH ₂ ] ⁺ , 128 (21) [M− ^{_{NH 2 -Br] +, 99 (}} 15), 75 (29), 74 (50), no other peaks> 10%.

_Analysis, calculated for _{C 9 H 6 BrNO: C,} 48.25; H, 2.70; Br, 35.66; N, 6.25. Found: C, 48.04; H, 2.83; N, 6.24.
3- (4-Nitrophenyl) prop-2-ynamide (1e)

Colorless crystals; Yield: 1.91 g (67%); mp 192-194 ° C. (Celsius) (MeCN).

IR (KBr): 3406 and 3155 (NH ₂ ), 2220 (C≡C), 1662 cm ⁻¹ (C═O).

MS (EI, 70 eV): m / z (%) = 190 (84) [M] ⁺ , 174 (100) [M—NH ₂ ] ⁺ , 128 (70) [M—NH ₂ —NO ₂ ] ⁺ , 116 (26), 101 (16), 100 (28), 98 (13), 89 (50), 77 (15), 75 (30), 74 (54), 63 (19), 62 (20), 51 (23), 50 (23), no other peaks> 10%.

_Analysis, calculated for _{C 9 H 6 N 2 O 3} : C, 56.85; H, 3.18; N, 14.73. Found: C, 56.64; H, 3.24; N, 14.59.
Monosubstituted malonyl chlorides (6a-f); general procedure

Monosubstituted malonyl chlorides (6a-f) were obtained by refluxing the corresponding malonic acid with SOCl ₂ followed by distillation. In the case of phenyl-6e and benzylmalonyl chloride 6f, the corresponding (chlorocarbonyl) ethyl ketene 7a, b is formed as a byproduct, which is consistent with literature reports. (Nakanishi et al., Org Prep Proced. Int. (1975) 7: 155; Friedrichsen et al., Natureforsch. B. Chem Sci (1982) 37: 222; Chem. Abstr (1982) 96: 199624). page).
2- (Phenylethynyl) -6H-1,3-oxazin-6-one (8a-f); general procedure

The corresponding malonyl chloride [(chlorocarbonyl) ethylketene] 6a-f (7a, b) (3.1 mmol) was added to stirred 3-phenylprop-2-inamide (1a, 0.44 g, 3 mmol) in anhydrous Et ₂ O. (30 mL) was added to the solution at room temperature. The mixture was cooled in the refrigerator for 12-14 hours. During this time a solid deposited, which was filtered off, washed with Et ₂ O and dried to give pure 8a-f.

For a further portion of the product, the filtrate was evaporated at room temperature under reduced pressure and the remaining residue was recrystallized (MeCN).
4-hydroxy-5-methyl-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8a) (also referred to as compound PG-3a)

Yellowish crystals; Yield: 0.32 g (74%); mp 184-186 ° C.

IR (KBr): 2215 (C≡C), 1747 (C═O), 1635 cm ⁻¹ (C═N).

MS (EI, 70 eV): m / z (%) = 227 (14) [M] ⁺ , 171 (20), 130 (11), 129 (100) [PhC≡CCO] ⁺ , 128 (12), 83 (14), 77 (13) [Ph] ⁺ , 75 (29), 74 (14), 70 (12), 63 (11), no other peaks> 10%.

_Analysis, calculated for _{C 13 H 9 NO 3: C} , 68.72; H, 3.99; N, 6.16. Found: C, 68.72; H, 4.01; N, 6.26.
5-ethyl-4-hydroxy-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8b) (also referred to as compound PG-3b)

Yellowish crystals; Yield: 0.51 g (70%); mp 177-178 ° C.

IR (KBr): 2221 (C≡C), 1744 (C═O), 1633 cm ⁻¹ (C═N).

MS (EI, 70 eV): m / z (%) = 241 (28) [M] ⁺ , 129 (100) [PhC≡CCO] ⁺ , 128 (19), 75 (15), 51 (10), etc. No peak> 10%.

_Analysis, calculated for _{C 14 H 11 NO 3: C} , 69.70; H, 4.60; N, 5.81. Found; C, 69.42; H, 4.53; N, 5.84.
4-hydroxy-5-isopropyl-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8c) (also referred to as compound PG-3c)

Colorless crystals; Yield: 0.47 g (62%); mp 202-203 ° C.

IR (KBr): 2226 (C≡C), 1744 (C═O), 1627 cm ⁻¹ (C═N).

MS (EI, 70 eV): m / z (%) = 255 (35) [M] ⁺ , 240 (54) [M-CH ₃ ] ⁺ , 172 (11), 130 (10), 129 (100) [ PhC≡CCO] ⁺ , 128 (20), 75 (11), 69 (16), no other peaks> 10%.

_Analysis, calculated for _{C 15 H 13 NO 3: C} , 70.58; H, 5.13; N, 5.49. Found: C, 70.38; H, 5.32; N, 5.43.
5-Butyl-4-hydroxy-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8d) (also referred to as compound PG-3)

Yellow crystals; Yield: 0.53 g (65%); mp 157-159 ° C.

IR (KBr): 2222 (C≡C), 1743 (C═O), 1628 cm ⁻¹ (C═N).

MS (EI, 70 eV): m / z (%) = 269 (10) [M] ⁺ , 226 (18) [M−C ₃ H ₇ ] ⁺ , 165 (14), 130 (10), 129 (100 ) [PhC≡CCO] ⁺ , 128 (20), no other peaks> 10%.

_Analysis, calculated for _{C 16 H 15 NO 3: C} , 71.36; H, 5.61; N, 5.20. Found: C, 70.97; H, 5.46; N, 5.14.
4-Hydroxy-5-phenyl-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8e) (Komarov et al., Russ. J. Gen Chem (2005) 75: 770) Also referred to as compound PG-3e)

Yield: 0.76 g (87%); mp 187-189 ° C.

IR (KBr): 2220 (C≡C), 1747 (C═O), 1620 cm ⁻¹ (C═N).

MS (EI, 70 eV): m / z (%) = 289 (35) [M] ⁺ , 218 (14), 190 (12), 172 (12), 130 (10), 129 (100) [PhC≡ CCO] ⁺ , 118 (18), 89 (16), 77 (11) [Ph] ⁺ , 75 (13), 51 (10) [C ₄ H ₃ ] ⁺ , no other peaks> 10%.

_Analysis, calculated for _{C 18 H 11 NO 3: C} , 74.73; H, 3.83; N, 4.84. Found: C, 74.79; H, 4.01; N, 4.75.
5-Benzyl-4-hydroxy-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8f) (also referred to as PG-3d)

Yellow crystals; Yield: 0.55 g (61%); mp 179-181 ° C.

IR (KBr): 2221 (C≡C), 1746 (C═O), 1627 cm ⁻¹ (C═N).

MS (EI, 70 eV): m / z (%) = 302 (55) [M] ⁺ , 176 (28), 158 (74), 131 (15), 130 (65), 129 (100) [PhC≡ CCO] ⁺ , 128 (20), 103 (12), 102 (15), 91 (27) [PhCH ₂ ] ⁺ , 77 (22) [Ph] ⁺ , 75 (13), 51 (12) [C ₄ H ₃ ] ⁺ , no other peaks> 10%.

_Analysis, calculated for _{C 19 H 13 NO 3: C} , 75.24; H, 4.32; N, 4.62. Found: C, 75.27; H, 4.32; N, 4.65.
2-[(4-Chlorophenyl) ethynyl] -4-hydroxy-5-phenyl-6H-1,3-oxazin-6-one (also referred to as PG-3g)

Yellow crystals; Yield: 0.87 g (90%); mp 185-187 ° C. (Celsius).

IR (KBr): 2220 (C≡C), 1767 (C═O), 1620 cm ⁻¹ (C═N).

MS (EI, 70 eV): m / z (%) = 325/324/323 (15/9/47) [M] ⁺ , 254/252 (9/22), 225 (10), 224 (22), 206 (16), 165/163 (32/100) [4-Cl—C ₆ H ₄ C≡CCO] ⁺ , 118 (28), 99 (18), 90 (17), 89 (21), 77 ( 12) [Ph] ⁺ , no other peaks> 10%.

Analysis, calculated for C18H10ClNO3: C, 66.78; H, 3.11; N, 4.33. Found: C, 66.89; H, 3.10; N, 4.39.
2-[(2-Chlorophenyl) ethynyl] -4-hydroxy-5-methyl-6H-1,3-oxazin-6-one (also referred to as PG-3f)

Yellowish crystals; Yield: 0.46 g (59%); mp 175-178 ° C.

IR (KBr): 2225 (C≡C), 1747 (C═O), 1627 cm ⁻¹ (C═N).

MS (EI, 70 eV): m / z (%) = 263/262/261 (9/4/26) [M] ^<+> .

Analysis, calculated for C13H8ClNO3: C, 59.67; H, 3.08; N, 5.35.
3-oxo-3-[(3-phenylprop-2-inoyl) amino] propanoic acid (9a-f); general procedure

After _{DMSO-d 6} solution of compound 8A～f was kept for 24 hours at room temperature, ¹ of 4-hydroxy-2- (phenylethynyl) -6H-1,3-oxazine-6-one 8A～f H and ¹³ The C NMR spectrum showed new signals for the corresponding hydrolysis products 9a-d, f and 10 (shown in FIG. 2).
2-Methyl-3-oxo-3-[(3-phenylprop-2-inoyl) amino] propanoic acid (9a)

2-[(3-Phenylprop-2-inoyl) carbamoyl] butanoic acid (9b)

3-Methyl-2-[(3-phenylprop-2-inoyl) carbamoyl] butanoic acid (9c)

2-[(3-Phenylprop-2-inoyl) carbamoyl] butanoic acid (9d)

2-Benzyl-3-oxo-3-[(3-phenylprop-2-inoyl) amino] propanoic acid (9f)

3-phenyl-N- (phenylacetyl) prop-2-ynamide (10) (also referred to as compound PG-3h)

A suspension of 4-hydroxy-5-phenyl-2- (phenylethynyl) -6H-1,3-oxazin-6-one (8e, 0.3 mmol) in H ₂ O (20 mL) was added, and the color of 8e was yellow. The mixture was heated to reflux for 30 minutes until it turned white. The solid compound 10 was separated by filtration and recrystallized (toluene) to obtain colorless crystals. Yield: 71 mg (90%); mp 132-134 ° C.

IR (KBr): 3219 (NH), 2213 (C≡C), 1705 (C═O), 1683 cm ⁻¹ (C═O).

MS (EI, 70 eV): m / z (%) = 263 (26) [M] ⁺ , 147 (18), 251 (15), 130 (10), 129 (94) [PhC≡CCO] ⁺ , 118 (100), 117 (12), 105 (15), 91 (30) [PhCH ₂ ] ⁺ , 90 (22), 75 (13), 65 (11), no other peaks> 10%.

_Analysis, calculated for _{C 17 H 13 NO 2: C} , 77.55; H, 4.98; N, 5.32. Found: C, 77.49; H, 5.07; N, 5.25.

Example 1
PG3 Compound Inhibits Caspase-6 as Measured by FRET Assay FIG. 3 shows compounds (referred to as PG3a-h) that were tested in a dose response curve by the FRET assay. As shown in FIGS. 4A-C, the presence of increasing concentrations of several PG3 analogs resulted in increased inhibition of recombinant caspase-6 enzyme in the cell-free system. Table 3 shows the calculated IC ₅₀ values for inhibition of caspase-6 enzyme as measured by the FRET assay.
(Example 2)
PG3 compounds inhibit intracellular caspase-6 as measured by Western blot.

COS-7 cells were co-transfected with the 4c htt fragment and human caspase-6 lacking the prodomain, which resulted in fast self-activation of the caspase-6 enzyme and generation of a 586aa Htt cleavage fragment. As shown in FIG. 5B, co-transfected cells were exposed to 10 um PG-3d compound, 3 uM Q-VD-Oph pan-caspase inhibitor, or left untreated. Untransfected cells were included as a negative control and purified 586aa Htt fragment was included as a positive control. Cells exposed to the PG-3d compound show a significant reduction in the production of the 586AA fragment. Cell lysates were also assayed for levels of active caspase 6 enzyme, as shown in the lower panel of FIG. 5B. The presence of the PG-3d compound resulted in a decrease in the amount of active enzyme, which was also achieved in the presence of a pan-caspase inhibitor. FIG. 5A shows a graphical representation from the Western blot, which showed that the presence of PG-3d resulted in reduced production of the 586aa fragment.
(Example 3)
PG3 compounds inhibit intracellular caspase-6 as measured by lamin cleavage.

PG3 analogs were also tested for their ability to inhibit caspase-6 in cultured cells. HEK293 cells were transfected with human caspase 6 lacking the prodomain and incubated with increasing concentrations of PG3 analogs. After 24 hours of incubation, excess compound was washed away, cells were lysed, and the amount of cleaved lamin A protein was quantified by the Mesoscale ELISA method. As shown in FIGS. 6A and 6B, some of the PG3 compounds showed a dose-dependent ability to inhibit lamin caspase-6-mediated cleavage. Table 4 shows the calculated IC ₅₀ values for intracellular inhibition of caspase-6 for PG3 analogs.
Example 4
PG3 compounds inhibit neuronal caspase-6, as measured by lamin cleavage.

It was next determined whether PG-3d compounds can inhibit caspase-6 in neurons in the neuronal system in vitro, which is more relevant for testing therapeutic agents for neurodegeneration. There can be. Briefly, primary cortical neuron cultures from FVB / N mice were treated with 10 uM camptothecin for 30 hours starting with DIV10 in the presence or absence of 10 uM PG-3d compound. Camptothecin treatment resulted in activation of caspase-6, which was then quantified by cleavage of lamin A. As shown in FIG. 7, the presence of PG3d compound in the neuronal medium significantly reduces the level of caspase-6 activity, as evidenced by the reduction of cleaved lamin.
(Example 5)
PG3 compounds improve neuronal survival during excitotoxic stress.

In HD animal models, neurons expressing mHTT have increased vulnerability to excitotoxic stress (Graham et al., 2005, Neurobiol. Dis. 21: 444-455), which is caspase-6. (Graham et al., 2006, Cell, 6 (13): 1179-1191 and Urive et al., HMG, 2012; 21 (9): 1954-67). It has been. The link between NMDA-induced toxicity and other neurodegenerative diseases has been reviewed by Lipton et al., Nat Rev Drug Discovery, 2006, 5: 160-170. Therefore, the effect of the presence of PG-3d compound on the ability of neurons to survive during excitotoxic stress was investigated by the ability of neurons to inhibit the caspase-6 enzyme. As shown in FIG. 8, the presence of the PG-3d compound in the cell culture medium resulted in a significant improvement in cell viability in neurons exposed to NMDA (25 uM or 50 uM).
(Example 6)
PG3d inhibits the interaction between caspase-6 and Htt in mammalian cells.

  Cos-7 cells were cotransfected with Htt1-1212AA (SEQ ID NO: 3) and full-length human caspase-6 (SEQ ID NO: 6) and exposed to the pan-caspase inhibitor Q-VD-OPh, PG3d compound or DMSO as a negative control I let you. As shown in the upper panel of FIG. 9A, control cells that received DMSO alone (lane labeled nt) were Htt proteolytic cleavage, including the Htt1212AA protein, and 586, 552 and 513 amino acid fragments. Indicates the presence of a fragment. As expected, cells treated with the pan-caspase inhibitor Q-VD-OPh show a reduced amount of Htt-cleaved fragments, most notably 513AA fragments derived from caspase 3 activity. Cells treated with PG3d compounds show a reduced level of 586AA fragment derived from caspase 6 activity (FIG. 9A, upper panel).

  FIG. 9B shows the results following immunoprecipitation with caspase-6 antibody. As expected, the DMSO control lysate reveals various Htt fragments within the immune complex including 513AA fragments resulting from caspase-3 cleavage, full length 1212AA Htt and 586AA fragments resulting from caspase-6 cleavage. When cells are treated with the pan-caspase inhibitor Q-VD-OPh, the amount of Htt cleavage fragments that interact with caspase-6 is reduced and it is clear that caspase-6 interacts primarily with 1212AA Htt. . Treatment with 10 uM PG3d significantly reduces the interaction between 1212AA Htt and caspase-6, which also results in a reduction in the amount of co-immunoprecipitated Htt proteolytic fragments. These findings were analyzed by densitometric analysis of the immunoblot results in FIG. 9B, but their presence of PG3d compared to cells treated with the negative control DMSO or Q-VD-OPh peptide inhibitor. It is confirmed that this results in a significant reduction in the amount of Htt1212AA co-immunoprecipitated using the caspase-6 antibody.

  All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, the inventive concepts, spirits, and compositions and / or methods, and in the method steps or series of steps described herein. It will be apparent to those skilled in the art that variations can be applied without departing from the scope and scope. More specifically, it will be apparent that certain agents that are chemically or physiologically relevant may be substituted for the agents described herein while achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims (27)

A pharmaceutical composition for the treatment or recovery of a nervous system disease comprising a therapeutically effective amount of an active agent having caspase-6 inhibitory activity and one or more pharmaceutically acceptable excipients, said activity The agent has one of the following structures I or II, or is a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof:
In which Ra and Rb are independently linear or branched C ₁ -C ₆ alkyl, aryl, or alkenyl, C ₁ -C ₉ alkyl aryl, C ₁ -C ₉ substituted alkyl aryl, or C _1- C ₉ alkylheteroaryl,
The pharmaceutical composition, wherein the phenyl group is substituted with 0, 1 or 2 halogens, wherein the halogen is chloride, fluoride or bromide.   The pharmaceutical composition according to claim 1 formulated for oral or topical administration, subcutaneous, intravenous or intramuscular injection, infusion, inhalation, or direct intrathecal injection into the central nervous system. .   The pharmaceutical composition according to claim 1, wherein the nervous system disease is Huntington's disease, Alzheimer's disease, dementia, mild cognitive decline, or memory loss.   A method of treating a nervous system disease comprising administering to a subject in need of such treatment an effective dose of the pharmaceutical composition of claim 1, wherein the disease comprises Huntington's disease, Alzheimer's The method is disease, dementia, mild cognitive decline, or memory loss.   5. The method of claim 4, wherein the composition of claim 1 is administered in combination with one or more additional drugs useful in the treatment of nervous system diseases.   6. The method of claim 5, wherein the one or more additional drugs are selected from L-DOPA, rasagiline, memantine hydrochloride, donepezil hydrochloride, rivastigmine, galantamine and tetrabenazine.   5. The method of claim 4, wherein administration of an effective dose of the pharmaceutical composition of claim 1 is initiated prior to the appearance of symptoms of the nervous system disease in the subject.   8. The method of claim 7, wherein the subject's cells express a mutant htt gene.   The method according to claim 7, wherein the nerve cells of the subject overexpress caspase-6 mRNA.   The method of claim 4, wherein the subject is a human.   Treatment or recovery of nervous system diseases comprising a therapeutically effective amount of an active agent having the structure designated as PG-3a in FIG. 3A, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof Pharmaceutical composition for   In treating or ameliorating a nervous system disease comprising a therapeutically effective amount of an active agent having the structure designated as PG-3b in FIG. 3B, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof Pharmaceutical composition for   Treatment or recovery of nervous system diseases comprising a therapeutically effective amount of an active agent having the structure designated as PG-3c in FIG. 3C, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof Pharmaceutical composition for   In treating or ameliorating a nervous system disease comprising a therapeutically effective amount of an active agent having the structure designated as PG-3d in FIG. 3D, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof Pharmaceutical composition for   In treating or ameliorating a nervous system disease comprising a therapeutically effective amount of an active agent having the structure designated as PG-3e in FIG. 3E, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof Pharmaceutical composition for   In treating or ameliorating a nervous system disease comprising a therapeutically effective amount of an active agent having the structure designated as PG-3f in FIG. 3F, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof Pharmaceutical composition for   In treating or ameliorating a nervous system disease comprising a therapeutically effective amount of an active agent having the structure designated as PG-3g in FIG. 3G, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof. Pharmaceutical composition for   In treating or ameliorating a nervous system disease comprising a therapeutically effective amount of an active agent having the structure designated as PG-3h in FIG. 3H, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof Pharmaceutical composition for   3. Treatment or recovery of a nervous system disease comprising a therapeutically effective amount of an active agent having the structure designated as PG-3 in FIG. 3, or a pharmaceutically acceptable salt, stereoisomer, derivative or prodrug thereof. Pharmaceutical composition for   20. Use of any of the pharmaceutical compositions according to claims 1 to 3 or 11 to 19 for use as a medicament.   20. Use of any of the pharmaceutical compositions according to claims 1 to 3 or 11 to 19 for the treatment of a nervous system disease selected from Huntington's disease, Alzheimer's disease, dementia, mild cognitive decline or memory loss .   The use according to claim 21, wherein composition 1 is used in combination with one or more additional drugs useful in the treatment of nervous system diseases.   23. Use according to claim 21 or 22, wherein the one or more further drugs are selected from L-DOPA, rasagiline, memantine hydrochloride, donepezil hydrochloride, rivastigmine, galantamine and tetrabenazine.   24. Use according to any of claims 21 to 23, wherein treatment is initiated prior to the appearance of symptoms of the nervous system disease in the subject.   25. Use according to any of claims 21 to 24, wherein the treatment is directed to a subject expressing a mutated htt gene.   26. Use according to any of claims 21 to 25, wherein the treatment is directed to a subject having neurons that overexpress caspase-6 mRNA.   27. Use according to any of claims 21 to 26, wherein the treatment is directed to a human subject.