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  • C07D303/38—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
  • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
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  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
  • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
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  • A61K31/231—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having one or two double bonds
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  • C07C31/13—Monohydroxylic alcohols containing saturated rings
  • C07C31/133—Monohydroxylic alcohols containing saturated rings monocyclic
  • C07C31/1333—Monohydroxylic alcohols containing saturated rings monocyclic with a three-membered ring
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  • C07C53/132—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen containing rings
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  • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
  • C07C69/608—Esters of carboxylic acids having a carboxyl group bound to an acyclic carbon atom and having a ring other than a six-membered aromatic ring in the acid moiety
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
  • C07D303/02—Compounds containing oxirane rings
  • C07D303/38—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D303/40—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals by ester radicals
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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  • C12Y207/11013—Protein kinase C (2.7.11.13)

Definitions

• the present invention relates to compositions and methods to activate an isoform of protein kinase C (PKC).
• PLC protein kinase C
• the present invention also provides methods for reducing neurodegeneration and for treatment of neurological diseases including Alzheimer's disease and stroke.
• AD Alzheimer's disease
• SDAT Alzheimer's type
• AD is characterized clinically by progressive loss of memory, cognition, reasoning, judgment, and emotional stability that gradually leads to profound mental deterioration and ultimately, death.
• a ⁇ beta-amyloid
• AD Alzheimer's Disease Facts and Figures
• PKC Protein kinase C
• PKC isozymes are expressed in the brain, including PKC, PKC ⁇ l, PKC ⁇ ll, PKC ⁇ , PKC ⁇ , and PKC ⁇ .
• PKC is primarily a cytosolic protein, but with stimulation it translocates to the membrane.
• PKC has been shown to be involved in numerous biochemical processes relevant to Alzheimer's disease.
• PKC activation also has a crucial role in learning and memory enhancement and PKC activators have been shown to increase memory and learning.
• Sun and Alkon Eur J Pharmacol. 2005;512:43-51; Alkon et al., Proc Natl Acad Sci USA. 2005;102:l 6432-16437.
• PKC activation also has been shown to induce synaptogenesis in rat hippocampus, suggesting the potential of PKC-mediated antiapoptosis and synaptogenesis during conditions of neurodegeneration.
• Sun and Alkon Proc Natl Acad Sci USA. 2008; 105(36): 13620-13625.
• Postischemic/hypoxic treatment with bryostatin-1, a PKC activator effectively rescued ischemia-induced deficits in synaptogenesis, neurotrophic activity, and spatial learning and memory.
• This effect is accompanied by increases in levels of synaptic proteins spiniophilin and synaptophysin and structural changes in synaptic morphology.
• PKC also activates neurotrophin production.
• Neurotrophins particularly brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)
• BDNF brain-derived neurotrophic factor
• NGF nerve growth factor
• PKC ⁇ protect against neurological injury, most likely by upregulating the production of neurotrophins. Weinreb et al., FASEB Journal. 2004;l 8:1471- 1473).
• PKC activators are also reported to induce expression of tyrosine hydroxylase and induce neuronal survival and neurite outgrowth. Du and Iacovitti, J. Neurochem. 1997; 68: 564-69; Hongpaisan and Alkon, PNAS 2007; Lallemend et al., J. Cell Sci. 2005; 118: 451 1- 25.
• AD is also characterized by tau hyperphosphorylation.
• Tau is expressed mainly in the brain, where it regulates the orientation and stability of microtubules in neurons, astrocytes and oligodendrocytes.
• normal soluble tau is transformed into insoluble paired helical filaments. This is linked to the post-translational change in tau, primarily the hyperphosphorylation of tau by a number of protein kinases.
• synthetic A ⁇ promotes tau phosphorylation through activation of glycogen synthase kinase-3 GSK-3. Wang et al., Journal of N euro chemistry. 2006; 98(4): 1167-1175.
• Activation of PKC has been shown to protects rat primary hippocampal neurons from A ⁇ - mediated neurotoxicity, through inhibition of GSK-3 ⁇ . Garrido et al., FASEB J. 2002: 1982.
• TACE TNF-alpha converting enzyme
• ADAM 17 which is an enzyme that is involved in the proteolytic conversion of membrane- bound amyloid precursor protein (APP) to its non-pathogenic soluble form, known as soluble APP-alpha or sAPP ⁇ .
• APP membrane- bound amyloid precursor protein
• sAPP ⁇ soluble APP-alpha
• sAPP ⁇ -producing enzymes are referred to generically as alpha-secretases.
• Activation of TACE by PKC also reduces cellular levels of pathogenic A ⁇ , which is produced by cleavage of APP by the beta- secretase enzyme (BACE).
• a ⁇ levels are a major therapeutic goal in Alzheimer's disease. It has been speculated that inhibition of A ⁇ formation by PKC activators may be caused by competition of TACE and BACE for their common substrate, APP.
• AD patients already have reduced levels of PKC ⁇ / ⁇ -mediated phosphorylation of Erkl/2, a major downstream substrate of PKC. Khan and Alkon, Proc Natl Acad Sci USA. 2006; 103: 13203-13207.
• a ⁇ application to normal fibroblasts reduces PKC activity because A ⁇ directly down-regulates PKC ⁇ / ⁇ .
• PKC activators especially those specific for PKC ⁇ / ⁇ , would counteract the effect of A ⁇ and thereby reverse or prevent the A ⁇ -induced changes.
• Stroke is a leading cause of disability and death in the United States, yet limited therapeutic options exist.
• PKC isoforms have been shown to have 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 ⁇ is involved in induction of ischemic tolerance and prevents damage, while PKC ⁇ and ⁇ are implicated in injury. Takayoshi et al., Stroke. 2007; 38(2):375-380; and Bright et al., Stroke. 2005;36: 2781.
• PKC ⁇ ' s protective ischemic effect is that PKC ⁇ maintaining mitochondrial function via ERK activity and by mediating adenosine-induced mitochondrial ATP-sensitive potassium channels.
• Another potential mechanism is that PKC ⁇ elicits a neuroprotective effect via COX-2 induction.
• PGE2 Prostaglandin E2
• PGE2 the product of COX-2 activity, leads to neuroprotection in cerebral ischemia.
• bryostatin-1 a PKC activator, effectively rescued ischemia-induced deficits in synaptogenesis, neurotrophic activity, and spatial learning and memory.
• Circulating A ⁇ protein has been shown to be elevated in patients with acute ischemic stroke Circulating A ⁇ l— 40 level was markedly elevated in ischemic stroke patients, as compared to controls.
• Lee et al. Journal of Neural Transmission. 2005; 1 12(10): 1371- 79.
• a strong positive association between progressively accumulating vascular A ⁇ and augmentations in arteriole and frontal cortex wall thickness AD patients also has been shown, suggesting that the continually progressing A ⁇ -associated angiopathy, at the arteriolar level, harms the contractile apparatus and cerebral blood flow autoregulation, thereby making the downstream capillaries vulnerable to damage. Stopa et al., Stroke. 2008;39:814.
• a ⁇ cerebral amyloid angiopathy
• CAA congophilic amyloid angiopathy
• This disorder is a form of angiopathy in which the same A ⁇ 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 vessel to failure, increasing the risk of a hemorrhagic stroke.
• CAA is also associated with transient ischemic attacks, subarachnoid hemorrhage, Down syndrome, post irradiation necrosis, multiple sclerosis, leucoencephalopathy, spongiform encephalopathy, and dementia pugilistica.
• PKC ⁇ and ⁇ are the most important PKC isoforms in eliciting the aforementioned beneficial effects in AD, stroke, and depressive disorders.
• Antisense inhibition of PKC ⁇ has been shown to block secretion of sAPP ⁇ , while PKC ⁇ is the isozyme that most effectively suppresses A ⁇ production.
• isoform specific PKC activators are highly desirable as potential anti-Alzheimer's drugs. Specific activators are preferable to compounds such as bryostatin that show less specificity because non-specific activation of PKC ⁇ or ⁇ could produce undesirable side effects.
• PKC ⁇ is also expressed at very low levels in all normal tissues except for brain. Mischak et al., J Biol. Chem. 1993; 268: 6090-6096; Van Kolen et al., J. Neurochem. 2008;l 04: 1-13. The high abundance of PKCe in presynaptic nerve fibers suggest a role in neurite outgrowth or neurotransmitter release. Shirai et al., FEBS J. 2008; 275: 3988-3994). Therefore, effects of specific PKC ⁇ activators would be largely restricted to brain, and unlikely to produce unwanted peripheral side effects.
• PUFAs such as arachidonic acid (see Fig. 1)
• DHA Docosahexaenoic acid
• a ⁇ and tau proteins associated with the brain-clogging plaques and tangles implicated in AD. Sahlin et al., Eur J Neurosci. 2007; 26(4): 882-9.
• WO 2002/501 13 to Nishizaki et al. discloses carboxylic acid compounds and their corresponding salts having cyclopropane rings for LTP-like potentiation of synaptic transmission or for use as a cognition-enhancing drug or a drug to treat dementia.
• Their synthetic examples disclose preparation of esters but their experimental results teach the use of free acids. The reason is that the carboxylic acid group of the fatty acid starting material would react with the diethylzinc used in the Simmons- Smith reaction.
• the methyl ester acts as a protecting group and may be cleaved off by hydrolysis or allowed to remain as needed.
• omega-3 PUFAs can be beneficial for other mood disturbance disorders such as clinical depression, bipolar disorder, personality disorders, schizophrenia, and attention deficit disorders.
• Ross et al. Lipids Health Dis. 2007; 18;6:21.
• omega ⁇ 3 fatty acids particularly docosahexaenoic and eicosapentaenoic acids, and a healthy balance of omega-3 to omega-6 fatty acids, to lowering the risk of depression.
• Logan et al., Lipids Health Dis. 2004; 3: 25 Levels of omega-3 fatty acids were found to be measurably low and the ratio of omega-6 to omega-3 fatty acids were particularly high in a clinical study of patients hospitalized for depression.
• omega-3 fatty acids reduce inflammation and help prevent risk factors associated with chronic diseases such as heart disease, cancer, inflammatory bowel disease and rheumatoid arthritis.
• FIG. 1 Structures of and of molecules contemplated for use according to the present invention (BR-101 through BR-118).
• FIG. 2 shows the results of an in vitro PKC ⁇ activation by BR-
• DCP-LA DCP-LA
• BR-102 two less active derivatives
• FIG. 3 shows activation of PKC ⁇ with various concentrations of
• BR-111 DHA-CP6 methyl ester
• BR-114 EPA-CP5 methyl ester
• BR-115 AA-CP4 methyl ester
• FIG. 4 shows activation of PKC ⁇ with various concentrations of other cyclopropanated and epoxidized fatty acid methyl esters: cyclopropanated linolenyl alcohol (BR-104); cyclopropanated linoleyl alcohol (BR-105); epoxystearic acid (BR-116); vernolic acid methyl ester (BR-117); and cyclopropanated vernolic acid methyl ester (BR- 109).
• BR-104 cyclopropanated linolenyl alcohol
• BR-105 cyclopropanated linoleyl alcohol
• epoxystearic acid BR-116
• vernolic acid methyl ester BR-117
• cyclopropanated vernolic acid methyl ester BR- 109
• FIG. 5 shows a time course of PKC activation by various concentrations of bryostatin in H19-7/IGF-IR rat hippocampal neurons.
• FIG. 6 shows a time course of PKC activation in rat hippocampal primary neurons by bryostatin and DCP-LA.
• Figure 7. depict decreased levels of intracellular (7a) or secreted (7b) A ⁇ in neuro2a (N2A) cells exposed to PKC activators bryostatin, BR-IOl (DCP-LA), or BR-111 (DHA-CP6).
• FIG. 8 shows the effect of BR-111 (DHA-CP6) (0.1 to 10 ⁇ M) on degradation of exogenously applied A ⁇ in SH-S Y5 Y neuroblastoma cells.
• FIGs. 9a-c depict effects of PKC activators PKC activators bryostatin, BR-101 (DCP-LA), and BR-111 (DHA-CP6) on TACE activity in N2a neuroblastoma cells transfected with human APPSwe/PSID (9a); the effects of various concentrations of bryostatin on TACE activity in rat cortical primary neurons (9b) and the effects of BR-111 (DHA-CP6) on TACE activity in rat cortical primary neurons (9c).
• FIG. 10 shows the activation of endothelin converting enzyme
• ECE PKC activators bryostatin (0.27 nM), BR-101 (DCP-LA) (1 ⁇ M), BR-111 (DHA- CP6) (1 ⁇ M), or ethanol in SH-SY5Y neuroblastoma cells.
• FIG. 1 la-b depict the effect of BR-101 (DCP-LA) and BR-111
• the present invention provides a method for activating PKC ⁇ using certain derivatives of polyunsaturated fatty acids (PUFA) or monounsaturated fatty acids (MUFA). These compounds activate PKC ⁇ at nanomolar concentrations which makes them excellent candidates for the treatment of AD, stroke, and other neurological diseases in which PKC ⁇ is neuroprotective.
• PUFA polyunsaturated fatty acids
• MUFA monounsaturated fatty acids
• a "fatty acid” is a carboxylic acid with an unbranched aliphatic chain containing from about 4 to 30 carbons; most long chain fatty acids contain between 10 and 24 carbons.
• Fatty acids can be saturated or unsaturated. Saturated fatty acids do not contain any double bonds or other functional groups along the chain. Unsaturated fatty acids contain one or more alkenyl functional groups, i.e., double bonds, along the chain.
• the term "polyunsaturated fatty acid” or "PUFA” means a fatty acid containing more than one double bond. There are three classes of PUFAs, omega-3 PUFAs, omega-6 PUFAs, and omega-9 PUFAS.
• omega-3 PUFAs the first double bond is found 3 carbons away from the last carbon in the chain (the omega carbon).
• omega-6 PUFAs the first double bond is found 6 carbons away from the chain and in omega-9 PUFAs the first double bond is 9 carbons from the omega carbon.
• PUFAs are also called "polyenoic fatty acids.” As used herein, the term PUFA includes both naturally-occurring and synthetic fatty acids. A major source for PUFAs is from marine fish and vegetable oils derived from oil seed crops, although the PUFAs found in commercially developed plant oils are typically limited to linoleic acid and linolenic acid (18:3 delta 9,12,15).
• a "c/j-PUFA" is one in which the adjacent carbon atoms are on the same side of the double bond.
• X:Y indicates an acyl group containing X carbon atoms
• linoleic acid would be abbreviated 18:2.
• a "methylene-interrupted polyene” refers to a PUFA having two or more cis double bonds separated from each other by a single methylene group.
• non-methylene-interrupted polyene or “polymethylene-interrupted fatty acid,” refers to a PUFA having two or more cis double bonds separated by more than one methylene group.
• a "monounsaturated fatty acid” is a fatty acid that has a single double bond in the fatty acid chain and all the remaining carbon atoms in the chain are single- bonded.
• exemplary MUFAs include oleic acid, myristoleic acid and palmitoleic acid.
• a "c/s-monounsaturated fatty acid” means that adjacent hydrogen atoms are on the same side of the double bond.
• Conjugated fatty acids such as conjugated linoleic acid (9-cis, 11 -trans- octadecadienoic acid) possess a conjugated diene, that is, two double bonds on adjacent carbons. Some evidence suggests that conjugated linoleic acid has antitumor activity.
• Exemplary PUFAs include lineoleic acid (9,12-octadecadienoic acid); ⁇ - linolenic acid (GLA; 6,9,12-octadecatrienoic acid); ⁇ -linolenic acid (9,12,15-octadecatrienoic acid); arachidonic acid (5,8,11,14-eicosatetraenoic acid); eicosapentanoic acid (EPA; 5,8,1 1,14,17-eicosapentanoic acid); docosapentaenoic acid (DPA; 7,10,13,16,19- docosapentaenoic acid); docosahexaenoic acid (DHA; 4,7,10,13,16,19-docosahexanoic acid); and stearidonic acid (6,9,12,15-octadecatetraenoic acid).
• GLA 6,9,12-octadecatrien
• cyclopropane refers to a cycloalkane molecule with the molecular formula C3H6, consisting of three carbon atoms linked to each other to form a ring, with each carbon atom bearing two hydrogen atoms.
• An "epoxide” refers to a cyclic ether with three ring atoms.
• PUFA derivative refers to a PUFA, or alcohol or ester thereof, in which at least one of the double bonds has been cyclopropanated or epoxidized.
• a "MUFA derivative” refers to a MUFA, or alcohol or ester thereof, in which the double bond has been cyclopropanated or epoxidized.
• Selective activation of PKC ⁇ means that the PUFA derivative compound of the present invention activates PKC ⁇ to a greater detectable extent than any other PKC isozyme.
• the PUFA derivative activates PKC ⁇ at least 1-fold, 2- fold or 5-fold over the other PKC isozymes as measured using e.g., the PKC activation assay described herein.
• protein kinase C enzymes are translocated to the plasma membrane by RACK proteins (membrane-bound receptor for activated protein kinase C proteins).
• RACK proteins membrane-bound receptor for activated protein kinase C proteins.
• protein kinase C enzymes are translocated to the plasma membrane by RACK proteins.
• PKC activation include phosphorylation at specific C-terminal serine/threonine residues by phosphatidylinositol- trisphosphate-dependent kinase (PDKl), with at least two additional phosphorylations and/or autophosphorylations of well-conserved sequences in each enzyme of the PKC family.
• PDKl phosphatidylinositol- trisphosphate-dependent kinase
• Neurodegeneration refers to the progressive loss of structure or function of neurons, including death of neurons.
• a "neurological disease” refers to any central nervous system (CNS) or peripheral nervous system (PNS) disease that is associated with the ⁇ -amyloidogenic processing of APP. This may result in neuronal or glial cell defects including but not limited to neuronal loss, neuronal degeneration, neuronal demyelination, gliosis (Le. , astrogliosis), or neuronal or extraneuronal accumulation of aberrant proteins or toxins (e.g., A ⁇ ).
• CNS central nervous system
• PNS peripheral nervous system
• AD congophilic angiopathy
• CAA cerebral amyloid angiopathy
• AD The term "Alzheimer's Disease” or “AD” refers to any condition where A ⁇ deposition will eventually in the cells of the central nervous system.
• a ⁇ particularly A ⁇ l-42, peptide is formed from the ⁇ -amyloidogenic metabolism of APP.
• AD may be heritable in a Familial manifestation, or may be sporadic.
• AD includes Familial, Sporadic, as well as intermediates and subgroups thereof based on phenotypic manifestations.
• a ⁇ 42 is the earliest form of this protein deposited in Down syndrome brains, and may be seen in subjects as young as 12 years of age, and that soluble A ⁇ can be detected in the brains of DS subjects as early as 21 gestational weeks of age, well preceding the formation of A ⁇ plaques. Gyure et al., Archives of Pathology and Laboratory Medicine. 2000; 125:. 489-492.
• the term "subject" includes a mammal.
• pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject.
• pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• pharmaceutically acceptable carrier means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject and can refer to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
• terapéuticaally effective dose and "effective amount” refer to an amount of a therapeutic agent that results in a measurable therapeutic response.
• a therapeutic response may be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy, including improvement of symptoms and surrogate clinical markers.
• a therapeutic response will generally be an amelioration or inhibition of one or more symptoms of a disease or condition e.g., AD.
• a measurable therapeutic response also includes a finding that a symptom or disease is prevented or has a delayed onset, or is otherwise attenuated by the therapeutic agent.
• the terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2- fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
• the present invention includes use of cyclopropanated and epoxidized derivatives of PUFAs or MUFAs in which one, some, or all of the double bonds are replaced by a cyclopropane group or an epoxide group.
• the terminal function may be a free carboxylic acid, or a methyl ester, ethyl ester, or some other alkyl ester with an aliphatic or aromatic alcohol. This alcohol specifically may also include glycerol and derivatives thereof. Glycerol derivatives are biologically important because the fatty acids are most frequently found conjugated to glycerol in the form of phosphatidylcholine, phosphatidylserine, or phosphatidic acids.
• triacylglycerols are compounds in which the carboxyl groups of fatty acids are esterified to the hydroxyls of all three carbons found in glycerol are referred to as triacylglycerols or triglycerides.
• the purpose of esterifying the carboxylic acid is to facilitate transport across the blood-brain barrier by eliminating the negative charge.
• the purpose of an alcohol group is also to facilitate transport across the blood-brain barrier.
• the fatty acid which forms the basis for the compounds used in the present invention is a polyunsaturated fatty acid having the following structure:
• X is between 2 and 6, and Y is between 2 and 6, and include methylene- or polymethylene-interrupted polyenes.
• exemplary polyunsaturated fatty acids include linoleic acid, ⁇ -linoleic, arachidonic acid, and adrenic acid having the following structures:
• Adrenic CH 3 (CH 2 ) 4 (CH CHCH 2 ) 4 (CH 2 ) 4 COOH
• omega-6 PUFAs are omega-6 PUFAs.
• the fatty acid which forms the basis for the compounds used in the present invention is a polyunsaturated fatty acid having the following structure:
• X is between 2 and 6
• Y is between 2 and 6 and include methylene- or polymethylene-interrupted polyenes.
• Exemplary polyunsaturated fatty acids include ⁇ - lineoleic acid, docosahexaenoic acid, eicosapentaenoic acid, eicosatetraenoic acid having the following structures:
• the compound of the present invention is an ester of a c/5-PUFA, in which the hydroxyl group is replaced by an alkoxy group, and in which at least one of the double bonds has been cyclopropanated.
• the starting material for this embodiment has the following structures:
• R is the alkyl group from an alcohol including monohydric alcohols and polyhydric alcohols including but not limited to methanol, ethanol, propanol, butanol, pentanol, glycerol, mannitol, and sorbitol.
• the compound contains at least three cyclopropanated double bonds.
• the fatty acid which forms the basis for the compounds used in the present invention is a monounsaturated fatty acid having the following structure:
• Exemplary monounsaturated fatty acids that can be the basis for the compounds used in the present invention include cis- and trans- monounsaturated fatty acids such as oleic acid, elaidic acid, obtusilic acid, caproleic acid, iauroleic acid, linderic acid, myristoleic acid, palmitoleic acid, vaccenic acid, gadoleic acid, erucic acid, and petroselinic acid,
• An ester means a monoester or a polyester.
• Esters of fatty acids include methyl, propyl, and butyl esters, and also esters resulting from more complex alcohols such as propylene glycol.
• R' is straight or branched and includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, and tetradecyl.
• An ester may also be formed from a fatty acid linked to a fatty alcohol in an ester linkage.
• the ester can be a alcohol ester, including but not limited to an aliphatic alcohol ester.
• the alcohol ester is a glycerol ester.
• Glycerol esters of fatty acids include glycerol fatty acid ester, glycerol acetic acid fatty acid ester, glycerol lactic acid fatty acid ester, glycerol citric acid fatty acid ester, glycerol succinic acid fatty acid ester, glycerol diacetyl tartaric acid fatty acid ester, glycerol acetic acid ester, polygiycerol fatty acid ester, and poiyglycerol condensed ricinoleic acid ester.
• the compound is an alcohol of a cw-PUFA in which at least one of the double bonds has been cyclopropanated.
• the compound is an alcohol of a cw-PUFA which contains at least three cyclopropanated double bonds.
• These compounds include but are not limited to linoleic alcohol dicyclopropane (BR-105), or linolenic alcohol tri cyclopropane (BR-104).
• R' can be a normal or branched chain alcohol or a phenolic alcohol.
• the compound of the present invention is a c/s-polyunsaturated fatty acid, or derivative thereof, in which at least one of the double bonds is replaced with an epoxyl group.
• the compound contains at least three enoxidized double bonds.
• the compound is an epoxidized ester of a c/s-PUFA, including but not limited to a fatty alcohol ester.
• the esters can be the same esters as described above for the cyclopropanated PUFAS.
• the alcohol can be an aliphatic alcohol ester, such as glycerol.
• the compound is an epoxidized cis- polyunsaturated fatty alcohol such as linoleic alcohol dicyclopropane or linolenic alcohol tricyclopropane.
• the alcohols can be the same as described above for the cyclopropanated PUFAS.
• the compound includes cyclopropanated or epoxidized lipids derived from cw-monounsaturated fatty acids (cw-monoenoic acids), such as oleic acid, elaidic acid, elaidic alcohol, oleyl alcohol, and 1-monolinoleyl rac-glycerol.
• cw-monoenoic acids such as oleic acid, elaidic acid, elaidic alcohol, oleyl alcohol, and 1-monolinoleyl rac-glycerol.
• Exemplary compounds include eliadic alcohol cyclopropane (BR-106), eliadic acid cyclopropane (BR-107), and oleyl alcohol cyclopropane (BR-108).
• a further embodiment includes cyclopropanated lipids derived from cis- monounsaturated fatty acids or unsaturated fatty acids, fatty acid esters, or fatty acid alcohols, containing one or more epoxide residues, such as vernolic acid methyl ester cyclopropane O.g., BR-109).
• the PUFAs which forms the basis of the cyclopropanated compounds used in the present invention include but are not limited to arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA).
• exemplary compounds for use in the method of the present invention include docahexaenonic acid methyl ester hexacyclopropane (BR-Il 1); eicosapentaenoic acid methyl ester pentacyclopropane (BR-114); and arachidonic acid methyl ester tetracyclopropane (BR-115).
• the compound is a cyclopropanated PUFA derivative of docosahexaenoic acid having the following structure:
• R is H or an alkyl group
• R is CH3 (BR-111 or DHA- CB6 methyl ester or methyl-3-(2-((2-((2-((2-((2-((2-((2-((2- ethy Icy clopropy l)methyl) eye lopropy l)methy l)cycl opropy l)methy 1) - cyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)propanoate.
• the PUFA derivative has the following structure:
• This compound is BR-114 (EPA-CP5 or methyl 4-(2((2-((2-((2- ethylcyc lopropyl)methyl)cy clopropy l)methyl)cyc lopropyl)methy 1) cycl opropy l)methy I)- cyclopropyl)butanoate methyl ester).
• the PUFA derivative has the following structure:
• This compound is BR-1 15 (AA-CP4 or methyl 4-(2-((2-((2-((- pentylcyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)butanoate methyl ester).
• the PUFA derivative has the following structure:
• R is H or an alkyl ester.
• R is CH3.
• Naturally cyclopropanated or epoxidized MUFAS or ester or alcohol derivatives thereof contemplated for use in the present invention include malvenic acid, vernolic acid, and sterculic acid.
• An exemplary compound is vernolic acid methyl ester (BR- 117).
• Fatty acids, and esters and alcohols thereof can be obtained or made from purification from natural sources, e.g., fish oil, flaxseed oil, soybeans, rapeseed oil, or algae, or synthesized using a combination of microbial enzymatic synthesis and chemical synthesis.
• natural sources e.g., fish oil, flaxseed oil, soybeans, rapeseed oil, or algae
• fatty acid methyl esters can be produced by the transesterification of triglycerides of refined/edible type oils using methanol and an homogeneous alkaline catalyst.
• the present invention contemplates treatment of neurological diseases associated with pathogenic A ⁇ such as AD and stroke using the PUFA derivatives disclosed herein.
• the present invention also contemplates prevention of neurological diseases associated with pathogenic A ⁇ using the PUFA derivatives disclosed herein.
• selective activation of PKC ⁇ may result in increased activation of TACE, with a concomitant decrease in production of A ⁇ . However, this appears to occur mainly in non-neuronal cells such as fibroblasts.
• Activation of PKC ⁇ may also reduce the hyperphosphorylation of the pathogenic tau protein in AD.
• Activation of PKC ⁇ may also induce synapto gene sis or prevent apoptosis in AD or following stroke.
• PKC ⁇ activators may also protect rat neurons from A ⁇ -mediated neurotoxicity through inhibition of GSK-3 ⁇ .
• PKC ⁇ activators may also counteract the effect of A ⁇ on the downregulation of PKC ⁇ / ⁇ , and thereby reverse or prevent the A ⁇ -induced changes.
• Another possible mechanism of action is the activation of A ⁇ -degrading enzymes such as endothelin- converting enzyme. The results of experiments presented in the Examples suggest that this may be the mechanism of action.
• Yet another mechanism may be by stimulation of PKC-coupled Ml and M3 muscarinic receptors, which is reported to increase nonamyloidogenic APP processing by TACE. Rossner et al. , Prog. Neurobiol. 1998; 56: 541-569. Muscarinic agonists rescue 3 x- transgenic AD mice from cognitive deficits and reduce A ⁇ and tau pathologies, in part by activating the TACE/ AD AMI 7 nonamyloidogenic pathway. Caccamo et al., Neuron. 2006; 49:671-682. Muscarinic receptor signaling is closely tied to PKC.
• Muscarinic receptor mRNA is regulated by PKC and neuronal differentiation produced by Ml muscarinic receptor activation is mediated by PKC. Barnes et al., Life Sci. 1997; 60:1015-1021; Vandemark et al., J. Pharmacol. Exp. Ther. 2009; 329(2): 532-42.
• disorders contemplated for treatment by the methods of the present invention include, mood disorders such as depressive disorders and bipolar disorder, schizophrenia, rheumatoid arthritis, cancer, cardiovascular disease, type 2 diabetes, and any other disorder in which PUFAs or MUFAs have been shown to be beneficial, including but not limited to those mention in the background.
• the PUFA derivatives may be produced in useful dosage units for administration by any route that will permit them to cross the blood-brain barrier. It has been demonstrated PUFAs from plasma are able to cross into the brain. Rapoport et al., J. Lipid Res. 2001. 42: 678-685. Exemplary routes include oral, parenteral, transmucosal, intranasal, inhalation, or transdermal routes. Parenteral routes include intravenous, intra-arteriolar, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, intrathecal, and intracranial administration.
• the compounds of the present invention can be formulated according to conventional methods.
• the PUFA derivative compounds can be provided to a subject in standard formulations, and may include any pharmaceutically acceptable additives, such as excipients, lubricants, diluents, flavorants, colorants, buffers, and disintegrants.
• Standard formulations are well known in the art. See e.g., Remington's Pharmaceutical Sciences, 20th edition, Mack Publishing Company, 2000.
• the compound is formulated in a solid oral dosage form.
• the pharmaceutical composition may take the form of a tablet or capsule prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
• binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
• fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
• lubricants e.g., magnesium stearate, talc or silica
• disintegrants e.g., potato starch or sodium starch
• Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
• Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
• the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
• the drug Omacor® contains concentrated combinations of ethyl esters of an omega-3 PUFAS.
• Each 1-g capsule contains at least 900 mg of the ethyl esters of omega-3 fatty acids, primarily EPA (465 mg) and DHA (375 mg), according to the drug's label, Omacor® is administered up to 4 times per day as 1-gram transparent soft gelatin capsules filled with light-yellow oil.
• a similar composition can be used to administer the PUFA compounds of the present invention, although the present invention contemplates use of a lower dose of the PUFA derivatives.
• Stable wax-ester formulations of PUFAs have also been described by transesterifi cation of stoichiometric amounts of ethyl esters enriched with n-3 PUFA and long-chain alcohols (18-22 carbon atoms) by transesterifi cation of stoichiometric amounts of ethyl esters enriched with n-3 PUFA and long-chain alcohols (18TM 22 carbon atoms).
• the PUFA compound is formulated for parenteral administration.
• the compound may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions, dispersions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the PUFA derivatives of the present invention are administered with a hydrophobic carrier.
• Hydrophobic carriers include inclusion complexes, dispersions (such as micelles, microemulsions, and emulsions), and liposomes.
• Exemplary hydrophobic carriers are inclusion complexes, micelles, and liposomes. These formulations are known in the art (Remington's: The Science and Practice of Pharmacy 20th ed,, ed. Gennaro, Lippincott: Philadelphia, PA 2003).
• the PUFA derivatives of the present invention may be incorporated into hydrophobic carriers, for example as at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the total carrier by weight.
• other compounds may be included either in the hydrophobic carrier or the solution, e.g., to stabilize the formulation,
• the PUFA derivative may also be formulated as a depot preparation.
• Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• the PUFA derivative can be delivered in a vesicle, particularly a micelle, liposome or an artificial LDL particle as described in U.S. patent application serial no. 11/648,808 to Alkon et al.
• the doses for administration may suitably be prepared so as to deliver from 1 mg to 1O g, preferably from 10 mg to 1 g and very preferably from 250 mg to 500 mg of the compound per day.
• the optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient and other clinically relevant factors.
• the PUFA compound can be used to treat patients with AD or other neurological disorders associated with A ⁇ in combination with other drugs that are also used to treat the disorder.
• exemplary non-limiting pharmacological agents approved in the United States for the treatment of AD include cholinesterase inhibitors such as Aricept® (donepezil), Exelon® (rivastigmine), Reminyl® (galantamine), and NMDA receptor antagonists such as Namenda® (memantine).
• Other potential therapeutic agents include protease inhibitors (see e.g., U.S. Patent Nos. 5,863,902; 5,872,101; inhibitors of A ⁇ production such as described in U.S. Patent Nos.
• the present invention contemplates combination therapy with other PKC activators, including but not limited to benzolactam macrocyclic lactones.
• Bryostatin-1 is a macrocyclic lactone that has been shown to modulate PKC and result in an increase in cleavage of APP by TACE into the non-amyloidogenic pathway.
• Bryostatin was able to increase the duration of memory retention of the marine slug Hermissenda crassicomis by over 500%, and was able to dramatically increase the rate of learning in rats. See U.S. patent application 10/919,110; Kurzirian et al., Biological Bulletin. 2006; 210(3): 201-14; Sun and Alkon, European Journal of Pharmacology. 2005;512(l): 43- 51.
• Other non-limiting PKC activators are described in pending U.S. patent application serial number 12/068,742 to Alkon et al.
• Combinations with drugs that indirectly increase TACE such as by inhibiting endogenous TACE inhibitors or by increasing endogenous TACE activators.
• An alternative approach to activating PKC directly is to increase the levels of the endogenous activator, diacylglycerol.
• Diacylglycerol kinase inhibitors such as 6-(2-(4-[(4- fluorophenyl)phenylmethylene]-l-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin- 5-one (R59022) and [3-[2-[4-(bis-(4-fluorophenyI)methylene]piperidin-l-yl)ethyl]-2,3- dihydro-2-thioxo-4(lH)-quinazolinone (R59949) enhance the levels of the endogenous ligand diacylglycerol, thereby producing activation of PKC. Meinhardt et al. (2002) Anti-Cancer Drugs 13: 725. [00100] Still another embodiment is combination therapy with BACE inhibitors.
• BACE inhibitors are known and include CTS-21166, owned by CoMentis Inc., which has shown positive results in a human clinical trial.
• Other BACE inhibitors are described in published International PCT application WO2007/019080 and in Baxter et al., Med. Chem. 2007; 50(18): 4261-4264.
• Compounds used in combination therapy can be administered in the same formulation as the PUFA compound of the present invention, where compatible, or can be administered in separate formulations.
• Evaluation of treatment with the PUFA derivatives of the present invention can be made by evaluation improvement in symptoms or clinical surrogate markers of the disease. For example, improvement in memory or cognitive skills in a treated AD subject may suggest that there is a reduction of pathogenic A ⁇ accumulation.
• cognitive phenotypes include, but are not limited to, amnesia, aphasia, apraxia and agnosia.
• psychiatric symptoms include, but are not limited to, personality changes, depression, hallucinations and delusions.
• DSM-IV-TR Diagnostic and Statistical Manual of Mental disorders, 4th Edition (DSM-IV-TR) (published by the American Psychiatric Association) contains criteria for dementia of the Alzheimer's type.
• Phenotypic manifestations of AD may also be physical, such as by the direct
• imaging or indirect (biochemical) detection of A ⁇ plaques.
• In vivo imaging of A ⁇ can be achieved using radioiodinated flavone derivatives as imaging agents (Ono et al., J Med Chem. 2005;48(23):7253-60) and with amyloid binding dyes such as putrescine conjugated to a 40- residue radioiodinated A peptide (yielding 125I-PUT-A 1-40), which was shown to cross the blood-brain barrier and bind to A ⁇ plaques. Wengenack et al., Nature Biotechnology. 2000; 18(8): 868-72.
• AD Current measures for evaluation AD include observation of a clinical core of early, progressive and significant episodic memory loss plus one or more abnormal biomarkers (biological indicators) characteristic of AD, including atrophy (wasting) of the temporal lobe as shown on MRI; abnormal A ⁇ protein concentrations in the cerebrospinal fluid; a specific pattern showing reduced glucose metabolism on PET scans of the brain; and a genetic mutation associated with within the immediate family.
• biomarkers biological indicators
• a 100 ml 3 -neck round bottom flask with a stirring bar and a temperature probe was surrounded by an ice-dry ice mixture and filled with 1.25g (4.24 mmol) linoleic acid methyl ester or docosahexaenoic acid methyl ester in 25 ml dichloromethane and bubbled with N 2 .
• a IM solution of diethylzinc (51 ml, 54.94 mmol) in hexane was added anaerobically using a 24-inch-long 20-gauge needle and the solution was cooled to -5 0 C.
• Diiodomethane (8.2 ml, 101.88 mmol) or chloroiodomethane (C1CH2 I) was added dropwise, one drop per second, with constant stirring. The rate of addition was decreased if necessary to maintain the reaction mixture below 2 0 C. The reaction mixture became cloudy during the reaction and an insoluble white zinc product was liberated. The flask was sealed and the mixture was allowed to react for 1 hr and then allowed to come to room temperature gradually over 2 hr.
• diethylzinc was not evaporated off.
• the mixture was slowly poured into 100 ml of water under stirring to decompose any excess diethylzinc. Ethane was evolved.
• the mixture was centrifuged at 5000 rpm in glass centrifuge tubes and the upper aqueous layer discarded.
• the white precipitate was extracted with CH 2 Cl 2 and combined with the organic phase. The organic phase was washed with water and centrifuged.
• the product was analyzed by silica gel G TLC using hexane plus 1% ethyl acetate and purified by chromatography on silica gel using increasing concentrations of 1-10% ethyl acetate in n-hexane and evaporated under nitrogen, leaving the methyl ester as a colorless oil.
• the epoxide groups can be introduced by conventional means, e.g., by oxidation of the appropriate alkene with m-chloroperbenzoic acid or t-butylhydroperoxide.
• Recombinant PKC (1 ng of alpha or epsilon isoform) was mixed with the BR-101 (DCP-LA) in the presence of 10 micromolar hi stones, 5 mM CaCl 2 , 1.2 ⁇ g/ ⁇ l phosphatidyl-L-serine, 0.18 ⁇ g/ ⁇ l 1,2-dioctanoyl-sn-glycerol (DAG), 10 mM MgCl 2 , 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4 mM EGTA, 4% glycerol, 8 ⁇ g/ml aprotinin, 8 ⁇ g/ml leupeptin, and 2 mM benzamidine.
• DCP-LA 1,2-dioctanoyl-sn-glycerol
• DHA methyl ester was purchased from Cayman Chemical (Ann Arbor, ME).
• PKC isozymes were from Calbiochem (San Diego, CA). Purified PKC ⁇ was purchased from Calbiochem.
• Rat hippocampal Hl 9-7/IGF-IR cells (ATCC, Manassas, VA) were plated onto poly-L-lysine coated plates and grown at 35°C in DMEM/ 10% FCS for several days until about 50% coverage was obtained. The cells were then induced to differentiate into a neuronal phenotype by replacing the medium with 5 ml N 2 medium containing 10 ng/ml basic fibroblast growth factor at 39°C and grown in T ⁇ 75 flasks at 37 0 C. Human SH-SY5Y neuroblastoma cells (ATCC) were cultured in 45% F12K / 45% MEM / 10% FCS.
• Mouse N2A neuroblastoma cells were cultured in DMEM/ 10% FCS without glutamine.
• Rat hippocampal neurons from 18-day-old embryonic [00117] Sprague Dawley rat brains were plated on 12- or 96-well plates coated with poly-D-lysine (Sigma-Aldrich, St. Louis, MO) in B-27 neurobasal medium containing 0.5 mM glutamine and 25 ⁇ M glutamate (Invitrogen, Carlsbad, CA) and cultured for three days in the medium without glutamate.
• the neuronal cells were grown under 5% CO 2 in an incubator maintained at 37 0 C for 14 days.
• 0.2 ml homogenization buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaF, 1 ⁇ g/ml leupeptin, and 0.1 mM PMSF) and homogenized by sonication in a Marsonix micro-probe sonicator (5 sec, 10W).
• PKC protein kinase
• 10 ⁇ l of cell homogenate or purified PKC isozyme purchased from Calbiochem was incubated for 15 min at 37 0 C in the presence of lO ⁇ M histones, 4.89 mM CaCl 2 , 1.2 ⁇ g/ ⁇ l phosphatidyl-L-serine, 0.18 ⁇ g/ ⁇ l 1 ,2-dioctanoyl-sn-glycerol, 10 mM MgCl 2 , 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4 mM EGTA, 4% glycerol, 8 ⁇ g/ml aprotinin, 8 ⁇ g/ml leupeptin, and 2 mM benzamidine.
• O.S ⁇ Ci [ ⁇ "32 P]ATP was added and 32 P-phosphoprotein formation was measured by adsorption onto phosphocellulose as described previously. Nelson and Alkon, J. Neurochemistry. 1995; 65: 2350-57.
• PKC activity was measured in the absence of diacylglycerol and phosphatidylserine, as described by Kanno et al., and PKC ⁇ , ⁇ , ⁇ , and ⁇ were measured in the absence of added EGTA and CaC12 , as described by Kanno et al., J. Lipid Res. 2006; 47: 1146-50.
• BR-101 DCP-LA
• BR-IOl DCP-LA
• BR-111 DHA-CP6
• BR-114 EPA-CP5
• BR-115 AA-CP4
• AA-CP4 cyclopropanated derivatives of docosahexaenoic acid.
• eicosapentaenoic acid, and arachidonic acid, respectively activated purified PKC ⁇ to a similar extent (Fig. 3)
• the concentration needed to activate PKC was approx. 100 times lower than for BR-101 (DCP- LA), suggesting higher affinity.
• Cyclopropanated linolenyl and linoleyl alcohols (BR-104 and BR-105), epoxystearic acid (BR-116), and vernolic acid methyl ester (BR-117) had iittle or no effect on PKC (Fig. 4).
• Cyclopropanated vernolic acid methyl ester (BR-109) inhibited PKC ⁇ at concentrations above l ⁇ M (Fig. 4).
• PKC activators that bind to the diacyl glycerol binding site including bryostatin, gnidimacrin, and phorbol esters, produce a transient activation of PKC activity, followed by a prolonged downregulation.
• bryostatin binds to the diacyl glycerol binding site
• gnidimacrin binds to the diacyl glycerol binding site
• phorbol esters produce a transient activation of PKC activity, followed by a prolonged downregulation.
• PKC activity is strongly downregulated by phorbol ester at 8h, while PKC in bryo statin-treated cells is at or near the baseline (data not shown). This difference may explain the increases in A ⁇ produced by PdBu reported by da Cruz e Silva et al. J Neurochem. 2009: 108: 319-30. These investigators applied l ⁇ M PdBu to cultured COS cells for 8h and observed an increase in A ⁇ . This increase was attributed to downregulation of PKC by the phorbol ester, which is consistent with these results. Downregulation could not be measured for DCP-LA and related compounds.
• mice neuro2a N2a neuroblastoma cells transfected with human APPSwe/PSID, which produce large quantities of A ⁇ . Petanceska et al., J Neurochem. 1996; 74: 1878-84. Incubation of these cells for 24h with various concentrations of PKC activators, bryostatin, BR-101 (DCP- LA) and BR-111 (DHA-CP6) markedly reduced the levels of both intracellular (Fig. 7a) and secreted (Fig. 7b) A ⁇ .
• Etcheberrigaray et al. found that activation of PKC in human fibroblasts by 10 pM to 100 pM bryostatin increased the initial rate of ⁇ -secretase activity by 16-fold and 132-fold, respectively (Etcheberrigaray et al., Proc. Natl. Acad. Sci. USA. 2006).
• PKC activators bryostatin, BR- 101 (DCP-LA) and/or BR-111 (DHA-CP6) only produced small increases in TACE activity. This suggests that any reduction of A ⁇ levels in neurons by PKC activators must be caused by some other mechanism besides activation of TACE.
• ECE Endothelin- converting enzyme
• a ⁇ can be degraded in vivo by a number of enzymes, including insulin degrading enzyme (insulysin), neprilysin, and ECE. Because PKC ⁇ overexpression has been reported to activate ECE (Choi et al, Proc. Natl Acad. Sci. USA. 2006; 103: 8215-20), we examined the effect of PKC activators on ECE. Bryostatin, BR-101 (DCP-LA), and BR-Ul (DHA-CP6) all produced a sustained increase in ECE activity (Fig. 10).
• An advantage of compounds such as the PUFA derivatives of the present invention which specifically activate PKC ⁇ is that they produce less down-regulation than phorbol esters and similar 1 ,2-diacylglycerol (DAG) analogues.
• DAG 1 ,2-diacylglycerol
• the biphasic response of PKC to DAG-based activators means that a PKC activator may reduce A ⁇ levels at one time point and increase them at another, da Cruz e Silva et al., J. Neurochem. 2009; 108: 319-330. Careful dosing and monitoring of patients would be required to avoid effects opposite to those that are intended. Because of the relative inability of this new class of PKC activators to downregulate PKC, this problem can be avoided.

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