PKC is one of the largest families of protein kinase enzymes and is composed of a variety of isoforms. Conventional isoforms include α, βI, βII, γ; novel isoforms include δ, ε, η, θ; and atypical isoforms include ξ, and ι/λ.

PKC enzymes are primarily cytosolic but translocate to the membrane when activated. In the cytoplasm, PKC is phosphorylated by other kinases or autophosphorylates. In order to be activated, some PKC isoforms (e.g., PKC-ε) require a molecule to bind to the diacylglycerol (“DAG”) binding site or the phosphatidylserine (“PS”) binding site. Others are able to be activated without any secondary binding messengers at all.

PKC activators that bind to the DAG site include, but are not limited to, bryostatin, picologues, phorbol esters, aplysiatoxin, and gnidimacrin. PKC activators that bind to the PS site include, but are not limited to, unsaturated fatty acids and their derivatives.

Once activated and translocated, PKC is anchored into the membrane by the anchoring protein RACK1. See, e.g., Mochly-Rosen et al. (1991)Proc Natl Acad Sci USA88, 3997-4000; Nishizuka, Y. (1995)FASEB J9, 484-496; Sklan et al. (2006)Prog Neurobiol78, 117-134. RACK1 localizes PKC to its corresponding substrates for phosphorylation, thus making PKC functionally active and physiologically relevant.

Activated PKC participates in a variety of biological pathways. For example, PKC activates ELAV mRNA-stabilizing proteins and c-CAMP-response-element-binding (“CREB”) proteins. PKC isoforms also play a regulatory role in amyloid precursor protein (“APP”) processing and amyloid accumulation. For example, PKC-α and PKC-ε regulate APP processing by the non-amyloidogenic pathway, suggesting that decreases in these enzymes may lead to increases in A-beta synthesis and accumulation. Thus, PKC activators may be able to reduce levels of soluble A-beta and increase levels of soluble APP-α. PKC activators may also be able to reduce or eliminate amyloid plaques and neurofibrillary tangles.

PKC activators have been associated with prevention and treatment of various diseases and conditions. For example, PKC activators may allow for prevention and treatment of neurodegenerative diseases and conditions, neuroaffective diseases and disorders, cognitive impairments, and diseases and conditions associated with neuronal or synaptic loss. Indeed, PKC activators have been found to induce synapse formation. Moreover, PKC activators have been associated with improvement in, for example, memory and learning, including long-term memory.

In one specific example, PKC activators have demonstrated neuroprotective activity in animal models of Alzheimer's Disease (“AD”). See Etcheberrigaray et al.,Proc. Nat. Acad. Sci. USA,1992, 89: 7184-7188. AD is a neurodegenerative disorder that is characterized clinically by progressive decline of memory, cognition, reasoning, judgment, and emotional stability that gradually leads to profound mental deterioration and ultimately, death.

Pathologically, AD is associated with the accumulation of aggregated β-amyloid (“Aβ”), a 4 kDa peptide produced by the proteolytic cleavage of amyloid precursor protein (“APP”) by β- and γ-secretases. As disclosed herein, oligomers of Aβ are considered to be most toxic while fibrillar Aβ is largely inert. Interestingly, monomeric Aβ is found in normal patients and has an as-yet undetermined function.

PKC activators can reduce the levels of Aβ and prolong survival of AD transgenic mice. See Etcheberrigaray et al., 1992,Proc. Nat. Acad. Sci. USA,89: 7184-7188. PKC-ε was shown to be most effective at suppressing Aβ production. See Zhu et al.,Biochem. Biophys. Res. Commun.,2001, 285: 997-1006. Accordingly, isoform-specific PKC activators are highly desirable as potential anti-AD drugs and other conditions associated with Aβ production.

The earliest consistent cytopathological change in AD is loss of synapses. See Scheff et al.,Neurobiol. Aging,2006, 27: 1372-1384; and Marcello et al.,Eur. J. Pharmacol.2008, 585: 109-118. In fact, synaptic loss appears to be the only pathological finding in the brain that is closely correlated with the degree of dementia in AD patients. See Terry et al.,Ann. Neurol.,1991, 30: 572-580. To that end, evidence suggests that Aβ is involved in synaptic loss.

PKC activators may also be used to treat and prevent other diseases and conditions associated with synaptic loss and/or Aβ. For example, changes in dendritic spine density form the basis of learning- and memory-induced changes in synaptic structure that increase synaptic strength. Long-term memory, for example, is mediated, in part, by the growth of new dendritic spines to reinforce a particular neural pathway. By strengthening the connection between two neurons, the ability of the presynaptic cell to activate the postsynaptic cell is enhanced. Several other mechanisms are also involved in learning- and memory-induced changes in synaptic structure, including changes in the amount of neurotransmitter released into a synapse and changes in how effectively cells respond to those neurotransmitters (Gaiarsa et al., 2002). Because memory is produced by interconnected networks of synapses in the brain, such changes provide the neurochemical foundations of learning and memory.

Abnormalities in the number and morphology of dendritic spines have been observed in cognitive disorders, including attention deficit hyperactivity disorder, autism, mental retardation, and fragile X syndrome. For example, the brains of schizophrenic patients and people suffering from cognitive-mood disorders show a reduced number of dendritic spines in the brain areas associated with these diseases. In mental retardation and autism, the shape of the dendritic spines are longer and appear more immature. Similarly, the only microscopic brain anomaly found in fragile X syndrome, the most common inherited form of mental retardation and autism, is the presence of thin, elongated immature dendritic spines.

Fragile X Syndrome results from a mutation in the FMR1 gene found on the X chromosome, resulting in failure of the gene to express a protein required for normal neural development (fragile X mental retardation protein; FMRP). FMRP is a selective RNA-binding protein implicated in regulating transmission of mRNAs to dendrites. Delayed dendritic spine maturation was found in fragile X mental retardation patients as well as in Fmr1 knockout mice, indicating the functional requirement of FMRP in synaptic development. Lu et al.,Proc. Natl. Acad. Sci. U.S.A.,2008, 101(42):15201-06; and Comery et al.,Proc. Natl. Acad. Sci. U.S.A.,1997, 94 (10):5401-4. Autopsy results on several Fragile X patients have indicated that immature dendritic spine density (number per unit dendrite length) was higher in patient samples, suggesting a greater number of excitatory inputs to these neurons. Greenough et al.,Proc. Natl. Acad. Sci. U.S.A.2001, 98(13):7101-7106. This suggests that dendritic spine formation in Fragile X Syndrome fails to follow the normal maturational pattern of eliminating underused synapses and altering the retained synapses to a more mature-appearing form of shorter, fuller spines.

FMRP also has been linked to Alzheimer's Disease. Beta-amyloid, the predominant protein found in the senile plaques of Alzheimer's disease and Down syndrome, is elevated in Fragile X mice and patients. Recent studies indicate that FMRP associates with the same mRNA coding region element as the amyloid precursor protein (APP), i.e., the protein that is cleaved into beta-amyloid plaques, and silencing FMRP promotes APP protein expression. Lee et al.,Nat Struct Mol Biol.,2010, 17(6):732-9. In addition, two micro-RNAs (short non-coding RNAs that suppress translation of specific mRNAs) that strongly affect synaptic structure and function have been shown to interact with FMRP. Edbauer et al.,Neuron,2010, 65(3):373-84.

As another example, persons who have suffered a brain injury show increased synthesis and expression of APP and its proteolytic product Aβ. See, e.g., Zohar et al.,Neurobiology of Disease,2011, 41: 329-337; Roberts et al.,Lancet,1991, 1422-1423; Gentleman e al.,NeuroReport,1997, 8: 1519-1522; Iwata et al.,J. Neuropathol. Exp. Neurol.,2002, 61: 1056-1068. In animal models, the PKC activator Bryostatin-1 was shown to protect against traumatic brain injury-induced learning and memory deficits. See Zohar et al.,Neurobiology of Disease,2011, 41: 329-337. Thus, PKC activators may be able to enhance memory and other cognitive functions.

Additionally, some forms of stroke are caused by Aβ, such as those associated with cerebral amyloid angiopathy (“CAA”). See U.S. Patent Application Publication No. 2010/0022645 A1. This disorder is a form of angiopathy in which the same Aβ deposits as found in AD accumulate in the walls of the leptomeninges and superficial cerebral cortical blood vessels of the brain. Amyloid deposition predisposes these blood vessels to failure, increasing the risk of a hemorrhagic stroke. CAA is also associated with transient ischemic attacks, subarachnoid hemorrhage, Down's syndrome, post irradiation necrosis, multiple sclerosis, leucoencephalopathy, spongiform encephalopathy, and dementia pugilistica.

Both PKC-α and PKC-ε are important for synaptogenesis—i.e., the formation of synapses. The high abundance of PKC-ε in presynaptic nerve fibers suggests a role in neurite outgrowth, synaptic formation, and neurotransmitter release. See Shirai et al.,FEBS,2008, 29: 1445-1453. Nontoxic drugs activating PKC-α and PKC-ε can promote synaptogensis under non-pathological conditions and actually prevent synaptic loss under pathological conditions. See Nelson et al.,Trends Biochem. Sci.,2009, 34: 136-145; Hongpaisan et al.,Proc. Natl. Acad. Sci. USA,2007, 104: 19571-19576; Sun et al.,Proc. Natl. Acad. Sci. USA,2008, 105: 13620-13625; Sun et al.,Proc. Natl. Acad. Sci. USA,2009, 106: 14676-14680.

For example, PKC activators have demonstrated neuroprotective activity in animal models of stroke. See Sun et al.,Eur. J. Pharmacol.,2005, 512: 43-51. Several PKC isoforms play a central role in mediating ischemic and reperfusion damage following stroke. Studies with experimental stroke models, mouse genetics, and selective peptide inhibitors and activators have demonstrated that PKC-ε is involved in induction of ischemic tolerance and prevents damage, while PKC-δ and PKC-γ are implicated in injury. See Takayoshi et al.,Stroke,2007, 38(2): 375-380; and Bright et al.,Stroke,2005; 36: 2781. Postischemic/hypoxic treatment with Bryostatin-1 effectively rescued ischemia-induced deficits in synaptogenesis, neurotrophic activity, and spatial learning and memory. See Sun et al.,Proc. Natl. Acad. Sci. USA.,2008, 105(36): 13620-13625.

PKC activation has a crucial role in learning and memory enhancement and PKC activators have been shown to increase memory and learning. See Sun et al.,Eur. J. Pharmacol.2005, 512: 43-51; Alkon et al.,Proc. Natl. Acad. Sci. USA.,2005, 102: 16432-16437. For example, bryostatin increased the rate of learning in rodents, rabbits, and invertebrates. See Sun et al.,Eur. J. Pharmacol.,2005, 512: 43-51; Wang et al.,Behav. Pharmacol.,2008, 19: 245-256; and Kuzirian et al.,Biol. Bull.,2006, 210: 201-214. Additionally, bryostatin-induced synaptogenesis for long-term associative memory was shown to be regulated by PKC activation. Hongpaisan et al.,Proc. Natl. Acad. Sci. USA,2007, 104: 19571-19576.

PKC activation has been associated with a variety of other conditions. For example, PKC activators have demonstrated neuroprotective activity in animal models of depression. See Sun et al.,Eur. J. Pharmacol.,2005, 512: 43-51. PKC activators are also associated with prevention and treatment of Parkinson's disease, bipolar disorder, and schizophrenia, mental retardation (and related diseases like autism).

Niemann-Pick disease (NP) refers to a group of inherited metabolic disorders known as lipid storage diseases. Lipids (fatty materials such as waxes, fatty acids, oils, and cholesterol) and proteins are usually broken down into smaller components to provide energy for the body. In Niemann-Pick disease, harmful quantities of lipids accumulate in, for example, the spleen, liver, lungs, bone marrow, and the brain. Symptoms may include lack of muscle coordination, brain degeneration, eye paralysis, learning problems, loss of muscle tone, increased sensitivity to touch, spasticity, feeding and swallowing difficulties, slurred speech, and an enlarged liver and spleen. There may be clouding of the cornea and a characteristic cherry-red halo develops around the center of the retina.

The disease has four related types. Type A, the most severe form, occurs in early infancy. It is characterized by an enlarged liver and spleen, swollen lymph nodes, and profound brain damage by six months of age. Children with this type rarely live beyond 18 months. Type B involves an enlarged liver and spleen, which usually occurs in the pre-teen years. The brain is not affected. In types A and B, insufficient activity of an enzyme called sphingomyelinase causes the buildup of toxic amounts of sphingomyelin, a fatty substance present in every cell of the body. Types C and D may appear early in life or develop in the teen or adult years. Affected individuals have only moderate enlargement of the spleen and liver, but brain damage may be extensive and cause an inability to look up and down, difficulty in walking and swallowing, and progressive loss of vision and hearing. Types C and D are characterized by a defect that disrupts the transport of cholesterol between brain cells. Type D usually occurs in people with an ancestral background in Nova Scotia. Types C and D are caused by a lack of the NPC1 or NPC 2 proteins. NINDS Niemann-Pick Disease Information Page, available at http://www.ninds.nih.gov/disorders/niemann/niemann.htm.

It has been reported that the intermediate filament vimentin is hypophosphorylated in Niemann-Pick Diseased cells compared to Wt cells and that this hypophosphorylation results from reduced PKC activity, in particular the α, ε, and βII isoforms. Increased PKC α, ε, and/or βII expression can increase levels of soluble vimentin in Niemann-Pick Diseased cells, ameliorating the transport block of LDL-derived cholesterol from their lysosomes to the endoplasmic reticulum for esterification. Tamari et al., PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport, PLOS ONE, Vol. 8, Iss. 8 (2013).

PKC activators can be broad-spectrum activators, acting on multiple isoforms of PKC, or can be selective for certain isoforms. While all types of PKC activators are of interest, selective PKC activators may offer unique advantages because different isoforms perform different, and sometimes opposite, functions. For example, PKC-δ and PKC-θ are often regarded as having a pro-apoptotic function because they are components of the caspase apoptosis pathway. PKC-ε, by contrast, has an opposite role: its activation promotes proliferation and cell survival, and inhibits apoptosis. See Nelson et al.,Trends in Biochemical Sciences,2009, 34(3): 136-145.

PKC-ε activators have been shown in numerous animal models, as discussed above, to have neuroprotective activity and would be useful therapeutic agents against a multitude of neurological diseases, conditions, or disorders, including, but not limited to, Alzheimer's disease, schizophrenia, depression, and stroke.

Polyunsaturated fatty acids (“PUFAs”), such as arachidonic acid and 2-hydroxy-9-cis-octadecenoic acid (i.e., minerval), and monounsaturated fatty acids (“MUFAs”) are known PKC-ε activators. PUFAs and MUFAs are interesting molecules in that they are essential components of the nervous system. They are known to increase membrane fluidity, rapidly oxidize to highly bioactive products, produce a variety of inflammatory and hormonal effects, and are rapidly degraded and metabolized. In addition, they are of low molecular weight and are able to cross the blood-brain barrier. Further, PUFAs and MUFAs are stable to acid and base, making them potentially effective for oral administration.

Like PUFAs and MUFAs, certain derivatives of PUFAs and MUFAs have been shown to be PKC-ε activators. For example, cyclopropanated PUFAs such as DCPLA methyl ester (i.e., linoleic acid derivative), AA-CP4 methyl ester (i.e., arachidonic acid derivative), DHA-CP6 methyl ester (i.e., docosahexaenoic acid derivative), and EPA-CP5 methyl ester (i.e., eicosapentaenoic acid derivative) may be able to selectively activate PKC-ε. SeeJournal of Biological Chemistry,2009, 284(50): 34514-34521; see also U.S. Patent Application Publication No. 2010/0022645 A1; International Application Publication No. WO 2013/071281.

Potency, for example, is part of the criteria in determining the utility of a drug for treating neurological disorders. A highly potent drug implies that the desired effects may be achieved at lower doses as compared to the required dose of a less potent drug. Thus, a highly potent drug administered at lower doses may result in fewer side effects because of, e.g., nonspecific binding to other targets, and higher brain specificity. Many known PKC-ε activators, however, suffer from several disadvantages, including low specificity and/or low potency.

The present disclosure is directed to new chemical entities that possess high potency and specificity for PKC-ε. More specifically, these new chemical entities are halogenated esters of unsaturated fatty acids or derivatives thereof. As potent, specific activators of PKC-ε, the halogenated esters disclosed herein may be suited for use as therapeutic agents against neurological diseases, conditions, and disorders, such as Alzheimer's disease and other neurodegenerative diseases.

In one aspect, the present disclosure includes a compound chosen from a halogenated ester of a polyunsaturated fatty acid, monounsaturated fatty acid, or derivative thereof. In a further aspect, there is disclosed a halogenated ester of 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCPLA).

In another aspect, the present disclosure includes a composition comprising at least one halogenated ester of a polyunsaturated fatty acid, monounsaturated fatty acid, or derivative thereof, and a pharmaceutically acceptable carrier. In a further aspect, there is disclosed a composition comprising a halogenated ester of DCPLA and a pharmaceutically acceptable carrier.

In yet another aspect, the present disclosure includes a method for treating one or more diseases, conditions, and disorders, comprising administering to a patient in need thereof an effective amount of at least one halogenated ester of a polyunsaturated fatty acid, monounsaturated fatty acid, or derivative thereof, wherein the diseases, conditions and disorders are neurodegenerative diseases or conditions, neuroaffective disorders, stroke, mental retardation, and brain injury. In a further aspect, there is disclosed a method for treating one or more diseases, conditions, and disorders, comprising administering to a patient in need thereof an effective amount of a halogenated ester of DCPLA, wherein the diseases, conditions and disorders are neurodegenerative diseases or conditions, neuroaffective disorders, stroke, mental retardation, and brain injury.

In a further aspect, the present disclosure includes using the halogenated esters described herein as positron emission tomography (PET) agents for in vivo imaging of PKC levels, and hence neurological function.

