JP4890759B2 - PKC activation as a means to enhance sAPPα secretion and improve cognition using bryostatin-type compounds - Google Patents

Description

Background of the Invention

(I) Field of the invention
The present invention relates to modulation of α-secretase and cognitive enhancement. The invention further relates to compounds for the treatment of conditions associated with amyloid processing (eg Alzheimer's disease) and compositions for the treatment of such conditions.

(Ii) Background of the invention
There are a variety of disorders and diseases that affect cognition. Cognition can generally be described as including at least three different components: attention, learning, and memory. Each of these components and their individual levels affect the overall level of the subject's cognitive ability. For example, Alzheimer's disease patients suffer from overall loss of cognition and associated degeneration of their individual characteristics, but the most commonly associated with the disease is loss of memory. In other diseases, patients experience cognitive impairment preferentially associated with different characteristics of cognition. For example, attention deficit hyperactivity disorder (ADHD) focuses on an individual's ability to maintain an attentional state. Other conditions include treatment of conditions that can produce a deleterious effect on other neurological diseases, aging, and mental capacity (eg, cancer therapy, stroke / ischemia, and mental retardation) ) Associated with general dementia.
Cognitive disorders are creating various problems in today's society.

  Therefore, scientists have put effort into efforts to develop cognitive enhancers or cognitive activators. Developed cognitive enhancers or activators include nootropics, vasodilators, metabolic enhancers, psychostimulants, cholinergic factors, biogenic amines It is generally classified as including drugs and neuropeptides.

  Vasodilators and metabolic enhancers (eg, dihydroergotoxins) are primarily effective in cognitive impairment induced by cerebral vascular ligation-ischemia; however, they are useful for clinical use. And ineffective for other types of cognitive impairment. Of the cognitive enhancers developed, typically only metabolic drugs are used for clinical use, while others are still in the research stage. Among nootropic factors, for example, piracetam activates the peripheral endocrine system and is not suitable for Alzheimer's disease due to the high concentration of steroids produced in patients, whereas tacrine (a cholinergic agent) Has various side effects, including vomiting, diarrhea, and hepatotoxicity.

  Methods for improving the cognitive ability of affected individuals have been the subject of various studies. In recent years, the cognitive state of Alzheimer's disease and the different methods of improving patient memory have been the subject of a variety of approaches and strategies. Unfortunately, these approaches and strategies only improved symptomatic and transient cognition in affected individuals and did not address disease progression. In the case of Alzheimer's disease, efforts to improve cognition, typically via the cholinergic pathway or via other brain transmitter pathways, have been studied. The main approach relies on inhibition of the acetylcholinesterase enzyme through drug therapy.

  Acetylcholinesterase is a major brain enzyme, and manipulating its level causes various changes in other neurological functions and causes side effects.

  Although these and other methods improve cognition (at least transiently), they do not correct disease progression and do not identify the cause of the disease. For example, Alzheimer's disease is associated with the formation of plaques, typically through the accumulation of amyloid precursor protein. Attempts to elicit an immunological response (via treatment for amyloid and plaque formation) have been carried out in animal models but have not been successfully applied to humans.

  Furthermore, cholinesterase inhibitors ameliorate several symptoms for a short time, which occurs only in some Alzheimer's disease patients with mid to moderate symptoms and thus overall It is a useful treatment only for a small portion of the patient population. More importantly, this attempt to improve cognition does not lead to treatment of the disease state, but merely ameliorates the symptoms. Current treatments do not affect disease progression. These therapies also include the use of “vaccines” to treat symptoms in Alzheimer's disease patients, which are theoretically valid and effective in mouse studies, but severe side effects in humans Has been shown to be the cause of

  As a result, the use of cholinergic pathways for the treatment of cognitive impairment (especially for Alzheimer's disease) has proven inadequate.

  Furthermore, current treatments for cognitive improvement are limited to certain neurodegenerative diseases and have not proven effective in the treatment of other cognitive conditions.

  Alzheimer's disease is associated with extensive loss of subpopulations of specific neurons in the brain, with memory loss being the most common symptom (Katzman, R. (1986) New England of Medicine 314. : 964). Alzheimer's disease has been well characterized for neuropathological changes. However, abnormalities have been reported in peripheral tissues, supporting the possibility that Alzheimer's disease is a systemic disorder (with the most prominent central nervous system pathology) (Connolly, G., Fibroblast models of neurodisorders: fluorescence measurement studies, Review, TiPS Vol. 19, 171-77 (1998)).

  For a discussion of Alzheimer's disease, link to genetic origin, chromosomes 1, 14, and 21 (St. George-Hyslop, PH, et al., Science 235: 885 (1987); Tanzi, Rudolph et al. ., The Gene Defects Responsible for Familial Alzheimer's Disease, Review, Neurobiology of Disease 3, 159-168 (1996); Hardy, J., Molecular Genetics of Alzheimer's disease, Acta Neurol Scand: Supplement 165: 13-17 See (1996). .

Although the cellular changes leading to neuronal loss and the etiology underlying the disease are under investigation, the importance of APP metabolism has been established. Two proteins that have been consistently identified in the brain of patients with Alzheimer's disease and that act in brain physiology or pathophysiology are β-amyloid and tau. (See Selkoe, D., Alzheimers Disease: Genes, Proteins, and Therapeutics, Physiological Reviews, Vol. 81, No. 2, 2001). Defects in β-amyloid protein metabolism and abnormal calcium homeostasis and / or calcium-activated kinases (Etcheberrigaray et al., Calcium responses are--impaired in fibroblasts from the circadian symbols) tool for early diagnosis, Alzheimer's Reports, Vol. 3, Nos. 5 & 6, pp. 305-312 (2000); , Regulation and role in regulating airways smooth muscle tone and mitogenesis, British Journal of Pharmacology, 130, pp 1433-52 (2000)).
Further matters regarding normal and abnormal memory have been demonstrated that both K ⁺ and Ca ²⁺ channels play a key role in memory storage and recall. It is that you are.

  For example, potassium channels have been found to change during memory conservation (Etcheberligaray, R., et al. (1992) Proceeding of the National Academy of Science 89: 7184; Sanchez-Andres, J. V. And Alkon, D. L. (1991) Journal of Neurobiology 65: 796; Collin, C., et al. (1988) Biophysics Journal 55: 955 (Alkon, D. L., et al. 1985 et al., Et al., 1985). Neural Biology 44: 278; Alkon, D.L. (1984) Science 226: 1037). This observation has been linked to almost all common symptoms of memory loss in Alzheimer's patients, which allows PKC modulation of potassium channel function and cognition as sites that may be associated with Alzheimer's disease pathology. A study of effects was led.

  PKC has been identified as one of the largest gene families of non-receptor serine-threonine protein kinases. The discovery of PKC in the early 80's by Nishizuka and collaborators (Kikkawa et al., J. Biol. Chem., 257, 13341 (1982)) and its identification as a major receptor for phorbol esters (Ashendel et al. al., Cancer Res., 43, 4333 (1983)), it is understood that the enzyme is involved in a number of physiological signal transduction mechanisms. The tremendous interest in PKC stems from its unique ability to be activated in vitro by calcium and diacylglycerol (and its phorbol ester mimetics). Diacylglycerol is an effector whose formation is coupled to phospholipid turnover by the action of growth and differentiation factors.

Currently, the PKC gene family consists of 11 genes, which are divided into four subgroups: 1) classical PKCα, β ₁ , β ₂ (β ₁ and β ₂ are alternative splicing of the same gene And γ, 2) novel PKCδ, ε, η and θ, 3) atypical PKCζ, λ, η and ι, and 4) PKCμ. PKCμ is similar to the novel PKC isoform, but differs by having a putative transmembrane domain (Blohe et al., Cancer Metast. Rev., 13, 411 (1994); Ilug et al., Biochem J., 291 329 (1993); reviewed by Kikkawa et al., Ann. Rev. Biochem. 58, 31 (1989)). The α, β ₁ , β ₂ , and γ isoforms are Ca ²⁺ , phospholipid, and diacylglycerol-dependent and represent the classic isoforms of PKC. Other isoforms are activated by phospholipids and diacylglycerol, but are not Ca ²⁺ dependent. All isoforms contain five variable (V1-V5) regions, and the α, β, and γ isoforms contain four (C1-C4) structural domains, which are highly conserved .

  All isoforms (except PKCα, β and γ) lack the C2 domain. Also, λ, η, and isoforms lack the 9 (nin) of the two cysteine-rich zinc finger domains in C1 to which diacylglycerol binds. The C1 domain also contains a pseudo-substrate sequence, which is highly conserved among all isoforms, and the pseudo-substrate sequence provides a self-regulating function by blocking the substrate binding site, thereby An inactive conformation is formed (House et al., Science, 238, 1726 (1987)).

  Because of these structural features, various PKC isoforms can be used for physiological stimulation (Nishizuka, Cancer, 10, 1892 (1989)), as well as for neoplastic transformation and differentiation (Nishizuka, Cancer, 10, 1892 (1989)) is thought to have a highly specialized role in responsive signaling. For a discussion of known PKC modulators, see PCT / US97 / 08141, US Pat. Nos. 5,652,232; 6,043,270; 6,080,784; 5,891,906; 5,962,498; 5 , 955,501; 5,891,870 and 5,962,504.

  In consideration of the central role that PKC acts on signaling, PKC has proven to be an interesting target for the regulation of APP processing. It has been established that PKC acts on APP processing. For example, phorbol esters have been shown to significantly increase the relative amount of non-amyloidogenic soluble APP (sAPP) secreted through PKC activation. However, PKC activation by phorbol esters does not appear to result in direct phosphorylation of APP molecules. Regardless of the exact site of action, phorbol-induced PKC activation results in enhancement or support of α-secretase, a non-amyloidogenic pathway. Thus, PKC activation is attractive to affect the production of non-deletious sAPP and to produce more beneficial sAPP and at the same time reduce the relative amount of Aβ peptide Approach. However, phorbol esters are not suitable compounds for final drug development because they have tumor promotion activity (lbarreta, et al., Benzolactam (BL ) Enhances APP section in fibroblasts and in PC12 cells, NeuroReport, Vol. 10, No. 5 & 6, pp 1035-40 (1999)).

  There is increasing evidence that individual PKC isozymes exert different (and in some cases opposite) actions in biological processes, which poses two directions for pharmacological exploration. One is the design of a specific (preferably isozyme specific) inhibitor of PKC. Given the fact that the catalytic domain is not the domain that primarily determines the isotype specificity of PKC, this approach is difficult. Another approach is to develop PKC activators that are isozyme selective and regulatory site-directed. These can provide a way to override the effects of other signaling pathways with the opposite biological effects. Alternatively, by inducing PKC down-regulation after acute activation, PKC activators can produce long-term antagonism. Currently, Bryostatin is in clinical trials as an anticancer agent. It is known that bryostatin binds to the regulatory domain of PKC and activates this enzyme. Bryostatin is an example of an isozyme selective activator of PKC.

  It has been discovered that compounds (in addition to bryostatin) modulate PKC (see, for example, WO 97/43268). There remains a need to develop therapeutic methods that improve overall cognition, either through specific characteristics of cognitive ability or general cognition. There also remains a need to develop methods to improve cognitive enhancement (whether it relates to a specific disease state or cognitive disorder). The methods and compositions of the present invention fulfill their needs and greatly improve clinical treatment for Alzheimer's disease and other neurodegenerative diseases, as well as improved cognitive enhancement I will provide a. The methods and compositions also provide for the treatment and / or enhancement of cognitive status through modulation of α-secretase.

Summary of the Invention

  The present invention relates to compounds, compositions and methods for the treatment of conditions associated with enhancement / improvement of cognitive ability. In preferred embodiments, the invention further relates to compounds, compositions, and methods for the treatment of conditions associated with amyloid processing (eg, Alzheimer's disease), which are improved / enhanced in treated subjects. Provided for cognitive ability. In particular, the compounds and compositions of the present invention are selected from bryostatin class and neristatin class macrocyclic lactones.

  In another aspect, the present invention relates to macrocyclic lactone compounds, compositions, and methods for modulating α-secretase activity. In particular, it relates to bryostatin and neristatin class compounds, and further to bryostatin-1.

  Another aspect of the present invention relates to bryostatin and neristatin class compounds as PKC activators, which are for altering conditions associated with amyloid processing, wherein α-secretase is It enhances to produce soluble α-amyloid precursor protein (αAPP) to prevent β-amyloid aggregation and improve / enhance cognitive ability. Such activation can be performed, for example, in the treatment of Alzheimer's disease (particularly bryostatin-1).

  In another aspect, the present invention provides a method for treating plaque formation (eg, associated with Alzheimer's disease), and a bryostatin or neristatin class compound to a subject to improve / enhance the cognitive status of the subject. It relates to a method comprising administering an effective amount. In a more preferred embodiment, the compound is bryostatin-1.

  Another aspect of the present invention is a composition for treating plaque formation and improving / enhancing cognitive ability, i.e .: (i) raising soluble [beta] -amyloid, producing soluble [alpha] APP, and [beta] -amyloid aggregation And (ii) a pharmaceutically effective carrier in an amount effective to inhibit In a preferred embodiment, the composition is used to improve / enhance cognitive abilities associated with Alzheimer's disease. Said macrocyclic lactone is preferably selected from bryostatin or neristatin class compounds, in particular bryostatin-1.

  In one aspect of the invention, activation of PKC isozymes results in improved cognitive ability. In one aspect, the improved cognitive ability is memory. In another aspect, the improved cognitive ability is learning. In another embodiment, the improved cognitive ability is attention. In another embodiment, PKC isozymes are activated by macrocyclic lactones (ie, bryostatin class and neristatin class). In particular, bryostatin-1 to 18 and neristatin are used to activate PKC isozymes. In a preferred embodiment, bryostatin-1 is used.

  In another aspect, the invention includes a PKC isozyme activator composition that is administered in an amount effective to improve cognitive performance. In a preferred embodiment, the PKC isozyme activator is selected from macrocyclic lactones (ie, bryostatin class and neristatin class). In a preferred embodiment, the amount of PKC isozyme activator administered is an amount effective to increase sAPP production. In a more preferred embodiment, the amount of composition administered does not cause myalgia.

  In a preferred embodiment, the PKC isozyme is activated in a subject, who is suffering from a neurological disease, strokes, or hypoxia (suffering or Have Suffered) subject. The examiner.

  In a more preferred embodiment, the PKC isozyme is activated in a subject or model of Alzheimer's disease.

  In another aspect of the invention, the PKC activation results in modulation of amyloid precursor protein metabolism. Furthermore, regulation by PKC activation results in an increase in the alpha secretase pathway. The alpha-secretase pathway produces non-toxic and non-amyloidogenic fragments associated with cognitive damage. As a result, the cognitive state of the subject is improved.

  In another aspect of the invention, the PKC activation reduces Abeta40 and Ab42, amyloidogenic and toxic fragments.

  Another aspect of the invention is a method for improving cognitive ability through activation of a PKC isoenzyme. In another embodiment of the invention, the PKC activation occurs in a “normal” subject. In another aspect of the invention, PKC activation occurs in a subject suffering from a disease that reduces cognitive capabilities or makes cognition dysfunctional. In a preferred embodiment, the method is a method for treating Alzheimer's disease.

  In another aspect of the invention, modulation of PKC is through the use of non-tumor promoting agents, resulting in improved cognitive ability. In a preferred embodiment, the PKC activator is selected from bryostatin-1 to bryostatin-18 and neristatin. In a more preferred embodiment, bryostatin-1 is used.

  In another embodiment, bryostatin-1 is used in combination with non-bryostatin class compounds to improve cognitive performance and reduce side effects.

  In another aspect of the invention, modulation of PKC via macrocyclic lactones (ie, bryostatin class and neristatin class) is used in vitro for the examination of conditions associated with Alzheimer's disease. In vitro use can include, for example, monitoring fibroblasts, blood cells, or monitoring ion channel conductance in cellular models.

  In a preferred embodiment of the invention, the compounds and compositions are administered in oral and / or injectable form, including intravenous and intraventricular administration.

  As described above, the present invention provides a method of treating impaired memory or learning disorder, wherein the method provides a subject with one therapeutically effective amount of the compound. Administration. The present compounds can be used in the therapeutic treatment of clinical conditions in which memory defects or impaired learning occurs. In this way memory and learning can be improved. The condition of the subject can be improved in this way.

  The compositions and methods have utility in the treatment of clinical symptoms and disorders, where impaired memory or learning disorders are occurring, either as central characteristics or associated symptoms. Examples of conditions in which the present compounds can be used for treatment include Alzheimer's disease, multi-infarct dementia, and a Laurie variant of Alzheimer's disease associated with or not associated with Parkinson's disease ( Lewy-body variant); Kreuzfeld-Jakob disease and Korsakow's disorder.

  The compositions and methods can be used to treat impaired memory or learning, which is age related, developed as a result of electro-convulsive therapy, or it is brain damage (e.g., , Stroke, anesthesia accident, head trauma, hypoglycemia, carbon monoxide poisoning, lithium intoxication or vitamin intoxication deficiency Developed as a result of brain damage caused by

  The compounds have an added advantage because they are non-tumor promoting and are already in phase II clinical trials.

  The present invention relates to pharmaceutical compositions for enhancing cognition and preventing and / or treating cognitive impairment. More specifically, the present invention relates to macrocyclic lactones (ie bryostatin class and neristatin class) and derivatives thereof as active ingredients for enhancing cognition and preventing and / or treating cognitive impairment. ).

  As described above, it is a major object of the present invention to provide a pharmaceutical composition for enhancing cognition and preventing and / or treating cognitive impairment. Said pharmaceutical composition comprises a macrocyclic lactone (particularly the bryostatin and neristatin class), or a pharmaceutically acceptable salt or derivative thereof, and a pharmaceutically acceptable carrier or excipient. )including.

  The pharmaceutical composition according to the present invention is useful for enhancing cognition, prophylaxis and / or treatment of cognitive impairment, wherein the cognitive impairment includes the learning acquisition described herein, This includes, but is not limited to, memory consolidation, and retrieval.

  The present invention is a method for the treatment of amyloidosis associated with neurological diseases, including Alzheimer's disease, which regulates or affects protein phosphorylation in mammalian cells. It relates to a method of treatment by administering to a patient an effective amount of one drug.

  The present invention also provides a method for treating Alzheimer's disease, comprising administering to a patient an effective amount of a macrocyclic lactone (ie, bryostatin class and neristatin class). .

  In another embodiment, the bryostatin or neristatin class compound may be used in the above methods in combination with a phorbol ester to block or reduce a tumorigenic response in a subject.

Detailed description of preferred embodiments

  Memory loss and impaired learning ability are characteristics of a range of clinical symptoms. For example, memory loss is the most common symptom of dementia, including Alzheimer's disease. Memory deficits are also associated with other types of dementia such as multiple infarct dementia (MID), senile dementia caused by cerebrovascular deficiency, and Alzheimer's with or without Parkinson's disease. It develops along with the Lowy-body variant of the disease, or Creutzfeldt-Jakob disease. Memory loss is a common feature of patients with brain injury. Brain damage can be, for example, after a classic stroke or after an anesthesia accident, head injury, hypoglycemia, carbon monoxide poisoning, lithium poisoning, vitamin (B1, thiamine and B12) deficiency, or It can develop as a result of excessive alcohol use or Karsakoff disorder. In addition, memory impairment may be the ability to recall information related to age-related; eg, names, places and words that appear to decrease with increasing age. Transient memory loss can also occur in patients suffering from a major depressive disorder after electroconvulsive therapy (ECT). Alzheimer's disease is actually the most important clinical entity with respect to progressive dementia in the aging population, while hypoxia / seizure is prominent not associated with neurological disorders This is a cause of memory defects.

  Individuals with Alzheimer's disease are characterized by progressive memory impairment, loss of language and visuospatial skills and behavioral defects (McHANn et al., 1986, Neurology, 34: 939-944). ). Cognitive damage in individuals with Alzheimer's disease is the result of degeneration of neuronal cells located in the cerebral cortex, hippocampus, basal forebrain, and other brain regions.

  Histological analysis of Alzheimer's disease brains collected at necropsy revealed neurofibrillary tangles (NFTs) in the perinuclear and axons of degenerating neurons, extracellular neuritis (senile) ) Demonstrated the presence of plaques and amyloid plaques inside and around the blood vessels in the affected brain area. The neurofibrillar tangle is an abnormal filamentous structure containing fibers (diameter, approximately 10 nm) paired in a helical fashion, with a pair of helical filaments. Also called. Neuritic plaques are localized to degenerating nerve endings (both axons and dendrites) and contain a core compound of amyloid protein fibrils. In summary, Alzheimer's disease consists of neuropathological properties, including intracellular neurofibrils composed primarily of cytoskeletal proteins and extracellular parenchymal and cerebral vascular amyloid. Tangle included). Further, currently in the art, to distinguish between Alzheimer's patients, normal aged people, and other neurodegenerative diseases such as Parkinson, Huntington's chorea, Wernicke Korsakoff or schizophrenia. For example, and are further described in US Pat. No. 5,580,748 and US Pat. No. 6,080,582.

  Alzheimer's disease is a brain disorder characterized by altered protein catabolism and typically develops with early memory loss. The most characteristic clinical symptom of AD is memory loss. Memory loss typically occurs early in the course of illness and mainly affects the learning of recent information. Molecular and cellular processes associated with normal associative memory storage and affected or disregulated in AD patient's cells to treat or alleviate AD And / or means for improving memory. Protein kinase C is the central and partly important locus of convergence between memory acquisition and memory loss in AD. Several molecular and molecular events related to associative memory in animal models have been shown to be altered or defective in AD. These include K + channels, calcium regulation, and protein kinase C (PKC). PKC is also involved in the processing of amyloid precursor protein (APP), a central element in AD pathophysiology. Altered protein phosphorylation has been linked to the formation of intracellular neurofibrillary tangles found in Alzheimer's disease. The role of protein phosphorylation in the catabolism of amyloid precursor protein (APP), from which the major components of amyloid plaque found in AD, are also being investigated. A central feature of Alzheimer's disease pathology is the deposition of amyloid protein within the plaque.

  Processing of amyloid precursor protein (APP) determines the production of the fragments, which later aggregate to form amyloid deposits known as senile or AD plaques characteristic of Alzheimer's disease (AD). Thus, APP processing is an early and key pathophysiological event in AD.

Three alternative APP processing pathways have been identified. Previously named “normal” processing involves the involvement of an enzyme that cleaves APP at residues Lys16 (or between Lys16 and Leu17; APP770 nomenclature) in the Aβ sequence, and is non-amyloidogenic Fragment: results in a large N-terminal ectodomain and a small 9 kDa membrane-bound fragment. This enzyme is an enzyme known as α-secretase that has not yet been fully identified. Two additional secretases are involved in APP processing. One alternative pathway involves the cleavage of APP outside the Aβ domain (between Met671 and Asp672 (by β-secretase)) and the endosome-lysosome system. An additional cleavage site occurs at the carboxyl terminus of the Aβ moiety, which is after amino acid 39 of the Aβ peptide in the plasma membrane. The secretase (γ) action produces an extracellular amino acid terminus (containing the entire Aβ sequence) and a cell-associated fragment that interacts with ˜6 kDa cells. Thus, processing with β and γ secretases produces potential amyloidogenic fragments (since they contain the complete Aβ sequence). Some evidence indicates that all alternative pathways occur in a given system and that soluble Aβ will be the “normal product”. However, there is also evidence that the amount of circulating Aβ in CSF and plasma is elevated in patients carrying the “Sweden” mutation. Moreover, cultured cells transfected with this mutation or the APP ₇₁₇ mutation secrete large amounts of Aβ. Recently, carriers of other APP mutations and PS1 and PS2 mutations have been shown to secrete large amounts of a particular form, long (42-43 amino acids) Aβ.

  Thus, although all alternative pathways can occur normally, an imbalance in favor of amyloidogenic processing occurs in familial and possibly sporadic AD. These enhanced amyloidogenic pathways ultimately result in the formation of fibrils and plaques in the brains of AD patients. Thus, non-amyloidogenic, interference in the direction of supporting the α-secretase pathway may impair the balance of APP processing, possibly a non-pathogenic process (this process is potentially toxic to the relative amount of sAPP). In the direction of increase) compared to the Aβ peptide.

  PKC isozymes are molecular targets that are critical, specific, and rate limiting (through this molecular target, biochemical, biophysical, and behavioral effectiveness). Can be demonstrated with a unique relevance of sex) and applied to subjects to improve cognitive ability.

The inventor has studied bryostatin as an activator of protein kinase (PKC). Changes in PKC (Alternations), as well as changes in calcium regulation and potassium (K ⁺ ) channels are included in changes in fibroblasts of Alzheimer's disease (AD) patients. PKC activation has been shown to restore normal K ⁺ channel function, as measured by TEA-induced [Ca ²⁺ ] elevation. Furthermore, patch clamp data substantiate the effect of PKC activators in restoring 113 pS K ⁺ channel activity. Thus, PKC activator-based recovery of the K ⁺ channel has been completed as an approach to AD pathophysiology studies, providing a useful model for AD therapeutics. (See pending application 09 / 652,656, which is incorporated herein in its entirety.)

The use of peripheral tissue and animal neuronal cells from Alzheimer's disease (AD) patients allows the identification of many cell / molecular changes that reflect corresponding processes in the AD brain, resulting in many pathophysiological factors. (Baker et al., 1988; Scott, 1993; Huang, 1994; Schuneer et al., 1996; Etchebrigary & Alkon, 1997; Gasparini et al., 1997).
Changes in potassium channel function have been identified in fibroblasts (Etcheberrigaray et al., 1993) and blood cells (Bondy et al., 1996) taken from AD patients. In addition, β-amyloid (Gandy & Greengard, 1994; Selkoe, 1994; Yankner, 1996), widely accepted as a major factor in AD pathophysiology, induces AD-like K ⁺ channel changes in control fibroblasts. (Etcheberrigaray et al., 1994). Similar or comparable effects of β-amyloid in the K ⁺ channel have been reported for experimental animal neurons (Good et al., 1996; also for a review see Fraser et al., 1997). Early observation of changes in the hippocampus of apamin-sensitive K ⁺ channels in AD brain (measured by apamin binding) further supports the suggestion that K ⁺ channels are pathophysiologically relevant in AD Data was provided (Ikeda et al., 1991).
Furthermore, protein kinase C (PKC) causes parallel changes to be presented in the peripheral and brain tissues of AD patients. The level (The levels) and / or activity of this enzyme was introduced into the brain and fibroblasts of AD patients (Cole et al., 1988; Van Huynh et al., 1989; Govoni et al., 1993; Wang et al., 1994). Studies using immunoblot analysis showed that, among various PKC isozymes, mainly alpha isoforms were significantly reduced in fibroblasts (Govoni et al., 1996), while alpha and beta isoforms Both were found to be decreased in the brains of AD patients (Shimohama et al., 1993; Masliah et al., 1990). These brain PKC changes may be early events in the disease process (Masliah et al., 1991). PKC activation appears to favor non-amyloidogenic processing of amyloid precursor protein (APP) (Buxbaum et al., 1990; Gillespie et al., 1992; Selkoe, 1994; Gandy & It is also interesting that Greengard, 1994; Bergamashi et al., 1995; Desdouits et al., 1996; Efimipooulus et al., 1996). Thus, both PKC and K ⁺ channel changes coexist in AD, with peripheral and brain expression in AD.

The link between PKC and K ⁺ channel changes was investigated. This is because PKC is known to control ion channels (including K ⁺ channels), and defective PKC produces defective K ⁺ channels. This is important not only for the regulation of APP, but also for the role of PKC and K ⁺ channels in establishing memory and recall. (See, eg, Alkon et al., 1988; Covarrubias et al., 1994; Hu et al., 1996) AD fibroblasts have been used to demonstrate both K ⁺ channels and PKC defects. Etcheberrigaray et al., 1993; Govoni et al., 1993, 1996). Studies have also shown that fibroblasts with known dysfunctional K ⁺ channels treated with PKC activators are present / absent in TEA-induced calcium elevations Restores the channel activity monitored by (absence). Furthermore, a functional 113pS K ⁺ channel (sensitive to TEA blockade) has been shown using an assay based on tetraethylammonium chloride (TEA) induced [Ca ²⁺ ] elevation (sensitive to TEA blockade) (Etcheberrigary et al. , 1993, 1994; Hirashima et al., 1996). The TEA-induced [Ca ²⁺ ] elevation and K ⁺ channel activity observed in fibroblasts from control individuals are substantially absent in fibroblasts from AD patients (Etcheberligaya et al., 1993; Hirashima) et al., 1996). These studies demonstrate that the use of PKC activators can restore the responsiveness of AD fibroblast cell lines to TEA challenge. Furthermore, immunoblot evidence from these studies demonstrates that this recovery is associated with preferential involvement of the alpha isoform.

In addition, the present inventor has also shown that activation of protein kinase C supports the α-secretase processing of Alzheimer's disease (AD) amyloid precursor protein (APP) and produces non-amyloidogenic soluble APP (sAPP). We also observed what happened. Consequently, the relative secretion of amyloidogenic A _1-40 and A _{1-42 (3)} is reduced. This is particularly important because fibroblasts and other cells expressing APP and presenilin AD variants secrete increased amounts of total Aβ and / or A _{1-42 (3)} / This is because an increase in the A _1-40 ratio occurs. Interestingly, PKC defects have been found in AD brains (α and β isoforms) and in fibroblasts (α-isoforms) of AD patients.

  Some studies have shown that other PKC activators (ie, benzolactam) with improved selectivity for α, β and γ isoforms enhance sAPP secretion beyond basal levels It shows that Also, sAPP secretion in benzolactam-treated AD cells was slightly higher compared to control benzolactam-treated fibroblasts, which showed only a significant increase in sAPP secretion after 10 μM BL treatment. Staurosporine (a PKC inhibitor) eliminates the effects of benzolactam in both control and AD fibroblasts, while related compounds give PC12 cells ˜3 fold sAPP secretion. It was further reported. The inventor has found that the use of bryostatin as a PKC activator in support of non-amyloidogenic APP processing has particularly therapeutic value, because bryostatin is non-tumor promoting, It is also because it is already in the stage II clinical trial.

Memory is thought to arise as a result of lasting synaptic modifications in the brain structures related to information processing. Synapses are thought to be critical sites in the final target, through which memory-related events achieve their functional expression (the events are altered gene expression and protein translation, altered Whether it is involved in kinase activity or a modified signaling cascade). A small number of proteins have been pointed out to be involved in associative memory, including Ca ²⁺ / calmodulin II kinase, protein kinase C, calexcitin, Ca ²⁺ associated with learning of 22-kDa. Binding proteins and type II ryanodine receptors are included. Modulation of PKC through administration of macrocyclic lactones provides a mechanism that affects synaptic modification.

  The area of memory and learning dysfunction is rich in animal models that can demonstrate different characteristics of memory and learning processes. (See, eg, Hollister, LE, 1990, Pharmacopychiat., 23, (SUPPL 11) 33-36). Animal models available for memory loss and impaired learning perform measurements of the animal's ability to memorize separate events. These tests include the Morris Water Maze and the passive aviation procedure. In the Morris water maze, animals are swam in tanks divided into four quadrants, only one of which has a safety platform under the surface of the water. The scaffold is removed and the animal is tested for how long it seeks the correct quadrant (compared to the wrong quadrant). In the passive avoidance procedure, the animal remembers a distinct environment (given a gentle lightning, which is avoided on the next occasion). A variant of the passive avoidance procedure takes advantage of rodent preference for a dark closed environment (as compared to a bright open environment). Further discussion can be found in the literature (Crawley, JN, 1981, Pharmacol. Biochem. Behav., 15, 695-699; Costall, B. et al, 1987, Neuropharmacol., 26, 195-200; Et al, 1989, Pharmacol.Biochem.Behav., 32,777-785; Barnes, J.M. et al, 1989, Br. J. Pharmacol., 98 (suppl) 693P; al, 1990, Pharmacol. Biochem. Behav., 35, 955-962.).

  The term “normal” may include individuals who have not been diagnosed or presently present with diminished or another impaired cognitive function Is meant. Different cognitive abilities can be tested and evaluated by known means established in the art, including complex memory recall tests from basic motor-spatial abilities However, it is not limited to these. Non-limiting examples of tests used for non-primate cognitive abilities include the Morris Water Maze, Radial Maze, T Maze, Eye Blink Conditioning, Delayed Regeneration (Delayed Recall), and Cue Recall, while primate subject testing includes Eye Blink, Delayed Replay, Cue Recall, Face Recognition, Minimental , And ADAS-Cog. Many of these tests are commonly used for mental status assessment of patients with AD. Similarly, assessments on animal models for similar purposes are described in the literature.

Of particular importance are macrocyclic lactones that stimulate PKC (ie bryostatin class and neristatin class). Of the bryostatin class compounds, bryostatin-1 has been shown to activate PKC and has proven to have no tumor promoting activity. Bryostatin-1 (as a PKC activator) is particularly useful because the dose response curve of bryostatin-1 is biphasic. In addition, bryostatin-1 exhibits different regulation in PKC isozymes, including PKCα, PKCδ, and PKCε. Bryostatin-1 has been tested for toxicity and safety in animals and humans and is actively being investigated as an anticancer agent. By using bryostatin-1 in the study, the main side effect in humans was determined to be myalgia, and the maximum dose was limited to 40 mg / m ² . The present invention utilized a bryostatin-1 concentration of 0.1 nM to produce a dramatic increase in sAPP secretion. Bryostatin-1 was compared to vehicle alone and another PKC activator, benzolactam (BL) (used at a 10,000-fold higher concentration). Also, the use of bryostatin at 0.01 nM has proven to be effective in increasing sAPP secretion. (See FIG. 1 (a)). PKC translocation shows that the measurement of activation is maximal at 30 min, then partially decays and remains higher than the basal translocation level for up to 6 hours (FIG. 1 (b)). 2, 8, and 9). The use of the PKC inhibitor staurosporine completely blocks the effect of bryostatin on sAPP secretion. Furthermore, the data demonstrate that PKC activation mediates the effect of bryostatin on sAPP secretion. (See Figures 1 and 2)

  Macrocyclic lactones, and in particular bryostatin-1, are described in US Pat. No. 4,560,774. Macrocyclic lactones and derivatives thereof may be obtained from other documents in the art (eg, US Pat. No. 6,187,568, US Pat. No. 6,043,270, US Pat. No. 5,393,897, US Pat. No. 5,072,004). U.S. Pat. No. 5,196,447, U.S. Pat. No. 4,833,257, and U.S. Pat. No. 4,611,066). The patent describes various compounds and various uses for macrocyclic lactones, including their use as anti-inflammatory or anti-tumor agents. Other discussions regarding bryostatin class compounds can be found in the following literature: Differential Regulation of Protein Kinase C Isozymes by Bryostatin 1 and Phorbol 12-Myristate 13 z Acetate in Nbet. , Journal of Biological Chemistry, Vol. 269, no. 3, pp. 2118-24 (1994); Preclinical Pharmacology of the Natural Product Antigen Agent Bryostatin 1, an Activator of Protein Kinase C, Zhang et al. , Caner Research 56, 802-808 (1996); Bryostatin 1, an activator of protein kinase C, inhibitors tumor promotion by bolsters in SENCARmouse. , Carcinogenesis vol. 8, no. 9, pp 1343-46 (1987); Phase II Trial of Bryostatin 1 in Patents with Relax Low-Grade Non-Hodgkin's Lymphoma and Chronic Lymphocet. , Clinical Cancer Research, Vol. 6, pp. 825-28 (2000); and review: Chemistry and Clinical Biology of the Bryostatins, Mutter et al. , Bioorganic & Medicinal Chemistry 8, pp. 1841-1860 (2000).

  Macrocyclic lactones (including the bryostatin class) refer to known compounds originally derived from Bugula neritina L. Multiple uses are known for macrocyclic lactones, particularly the bryostatin class, but the link between macrocyclic lactones and cognitive enhancement has previously been unknown.

  Examples of compounds that can be used in the present invention include macrocyclic lactones (ie, bryostatin class and neristatin class compounds).

  Although specific embodiments of these compounds are described in the examples and detailed description, it is understood that the compounds and derivatives thereof disclosed in the references may also be used in the compositions and methods of the present invention. Should.

  As will be appreciated by those having ordinary skill in the art, macrocyclic lactone compounds and derivatives thereof, particularly the bryostatin class, are applied to combinatorial synthetic techniques, and then the library of said compounds is Once created, pharmacological parameters (including but not limited to the efficacy and safety of the compounds) can be optimized. In addition, those libraries can be assayed to determine members that preferably modulate α-secretase and / or PKC.

  New drugs have been discovered through combinatorial libraries, high-throughput screening of natural products, and fermentation broths. Currently, creating and screening for chemical diversity is widely used as the main technique for discovering lead compounds, which is certainly a major important in the field of drug discovery. It is a great progress. Furthermore, even after “lead” compounds have been identified, combinatorial techniques provide valuable tools for optimizing the desired biological activity. As will be appreciated, subject reactions are easily combinatorial live for compounds for screening, of pharmaceutically or other biologically or medically relevant activity or material related quality. Applies to rally making. The combinatorial library for the subject of the present invention is a mixture of chemically related compounds, both of which may be screened for the desired properties; the library may be present in solution or a solid support It may be covalently linked to the body. By preparing many related compounds in one reaction, the number of screening processes to be performed is greatly reduced and simplified. Screening for the appropriate biological property can be performed by conventional methods. Accordingly, the present invention also provides a method for determining the ability of one or more inventive compounds to bind to and effectively modulate α-secretase and / or PKC.

  A variety of techniques are available for the techniques for generating the combinatorial library described below, but the invention is not limited by the examples and description given above. See, for example, the following document: Blondelle et al. (1995) Trends Anal. Chem. 14:83; Affymax U.S. Patents 5,359,115 and 5,362,899: Ellman U.S. Patent 5,288,514: Still et al. PCT Publication No. WO 94/08051; Chen et al. (1994) JACSl 1 6: 266 1: Kerr et al. (1993) JACSl 1 5: 252 :: PCT Publication Nos. W092 / 10098, W09 / 09668 and W09 / 07087; and Lemer et al. PCT Publication Number W093 / 20242). As a result, various libraries on the order of about 16 to over 1,000,000 diversomers can be synthesized and screened for specific activities or properties.

  The compounds can be administered by various routes and in various dosage forms, including oral, rectal, parenteral (eg, subcutaneous, intramuscular and intravenous), epidural, This includes those related to intrathecal, intra-articular, topical and buccal administration. The dose range for adult humans depends on many factors, including age, weight and patient condition and route of administration.

  For oral administration, fine powders or granules containing dilution, dispersion and / or surfactant-active agents are encapsulated in a draft, water or syrup, in a dry state. Or it may be present in sachets, in non-aqueous suspensions (which may include suspending agents), or in suspension in water or syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening or emulsifying may be included.

  Other compounds that may be included in the admixture include, for example, medically inert ingredients such as solid and liquid diluents, lactose, dextrose, sucrose, cellulose, starch or calcium phosphate tablets or capsules. For olive oil or ethyl oleate for soft capsules and water or vegetable oil for suspensions or emulsions; lubricants such as silica, talc ), Stearic acid, magnesium or calcium stearate and / or polyethylene glycol; gelling agents such as colloidal clays; thickening agents ), Eg tragacanth gum or sodium alginate, binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disintegrating agents such as starch, alginic acid, alginate or sodium starch glycolate (Sodium starch glycolate); effervescent mixings; dyestuffs; sweeteners; wetting agents such as lecithin, polysorbate or lauryl sulfate; Secondary ingredients such as humectants ts), preservatives, buffers and antioxidants, these are known additives for such formulations.

  Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrup may contain, for example, sucrose or sucrose (with glycerin added) and / or mannitol and / or sorbitol as a carrier. Syrups, particularly for diabetics, can contain, for example, sorbitol (not metabolized to glucose or a very small amount metabolized to glucose) as a carrier. The suspensions and emulsions can contain carriers such as natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.

  Suspensions or solvents for intramuscular injection can be combined with the active compound together with pharmaceutically acceptable carriers such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol and appropriate amounts as appropriate. Lidocaine hydrochloride may be included. Solvents for intravenous injection or infusion can include carriers, such as sterile water (generally water for injection). Preferably they may be in the form of a sterile, aqueous, isotonic saline solution. Alternatively, the present compound may be encapsulated in liposomes. The present compounds can also utilize other known active agent delivery systems.

  The compound may also be administered in a pure form unassociated with other additives, in which case capsules, sachets or tablets are suitable dosage forms.

  Tablets and other forms presented in separate units suitably contain one, a daily dose of the compound, or an appropriate fraction thereof. For example, a unit may contain 5 mg to 500 mg of one of the compounds, but more typically 10 mg to 250 mg.

  The pharmacological activity of the compounds of the invention can be demonstrated using standard pharmacological models known in the art. Furthermore, the inventive compositions can be introduced or encapsulated in a suitable polymer matrix or membrane for site-specific delivery, or the composition acts on site-specific delivery. It can be functionalized with specific targeting agents that have the ability. These techniques, as well as other drug delivery techniques, are well known in the art.

In summary, PKC activation affects memory acquisition, as well as promoting non-amyloidogenic, alpha-secretase, APP processing. A non-tumor promoter PKC activator (bryostatin 1) dramatically enhances secretion of α-secretase product (sAPPα) in fibroblasts of AD patients. The effect was prominent at the sub-nanomolar concentration of bryostatin. Bryostatin (injected intraventricularly) also enhanced the performance of rats tested in the Morris Water Maze paradigm. Recent In vivo studies to: indicated, i.e., a benzolactam (PKC activator, to enhance sAPPα in that and AD cells to reverse the ^{K +} channel defects ^(K + channels defects) (Shown previously) significantly increases the amount of sAPPα and decreases Aβ40 in the brains of transgenic mice carrying the London V717I APP mutation. These results demonstrated that PKC (and its activation) can be a tool for treating or alleviating symptoms associated with AD and memory loss. Bryostatin 1 is particularly important because it is not only potent but also lacks tumor-promoting activity and has already undergone clinical trials for cancer treatment in humans. The following examples provide the following evidence that bryostatin 1 dramatically and potently potentiates APP α-processing (which causes an increase in sAPPα levels) and the ability of rats in the Morris water maze task Is a significant improvement. The example also provides the following evidence that another PKC activator (benzolactam) results in a significant increase in sAPPα and a decrease in Aβ40 (in vivo).

  All books, articles, or patents in this specification are incorporated by reference to the extent they do not conflict with the present disclosure. Hereinafter, the present invention will be described by way of examples, but these are for illustrative purposes and do not limit the scope of the invention.

Example I
Cell culture
Cultured dermal fibroblasts were obtained from Coriell Cell Repositories and grown according to the general guidelines established for their culture with some modifications (Cristofalo & Carpentier) , 1988; Hirashima et al., 1996). As a culture solution for growing cells, Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Biofluids, Inc.) was used. Fibroblasts from control cell line (AC) cases AG07141 and AG06241 and familial AD (FAD) case (AG06848) were utilized.

PKC activators (Activators)
Different tissue distributions, clearly distinct roles of different isozymes, and differential involvement in disease states are important for the use of pharmacological tools that have the ability to preferentially target specific isozymes (Kojikowski et al., 1997; Hofmann, 1997). Recent research in the field of medicinal chemistry has developed several PKC activators, such as different benzolactams and pyrrolidinones. However, currently studied bryostatin PKC activators not only have the advantage of providing isospecific activity, but also of previously used PKC activators (eg, tumor-promoting) It is not affected by the problem (set back). Bryostatin competes with the regulatory domain of PKC and engages in a highly specific hydrogen bond interaction within this site.
Additional information regarding the organic chemistry and molecular modeling of this compound is described in the literature.

Treatment
Cells were grown for 5-7 days in 6 cm Petri dishes until confluence. On the day of the experiment, the medium was replaced with DMEM (without serum) and left for 2 h. Two hours of serum deprivation was achieved and treatment was achieved by applying Bryo, BL and DMSO directly to the media at the appropriate concentrations. (0.1 and 0.01 nM for bryostatin; 0.1 nM, 0.1 μM, and 1 μM BL). DMSO was less than 1% in all cases. In most cases, media was collected and processed 3 hours after treatment for sAPP secretion. In addition, the time course of secretion was investigated using other time points.

Immunoblot assay for PKC translocation
Immunoblot experiments were performed using established procedures (Dunbar, 1994). Cells were grown to confluence (˜90%) in 6 cm Petri dishes.
The level of isozyme in response to treatment with 0.1 nM bryostatin-1 (5, 30, 60, and 120 min) was quantified using a procedure that was slightly modified from the method established by Racchi et al. (1994). Fibroblasts were washed twice with ice-cold PBS, detached in PBS and collected by low speed centrifugation. Resuspend pellet in the following homogenization buffer: 20 mM Tris-HC1, pH 7.5, 2 mM EDTA, 2 mM EGTA, 5 mM DTT, 0.32 M sucrose, and protease inhibitor cocktail (Sigma) It became cloudy. The homogenate was collected by sonication and centrifuged at ˜12,000 g for 20 minutes, and the supernatant was used as the cytosolic fraction.
The pellet was homogenized in the same buffer (containing 1.0% Triton X-100), incubated in ice for 45 minutes, and centrifuged at ˜12,000 g for 20 minutes. The supernatant of this batch was used as a membrane fraction. After protein determination, 20 μg of protein is diluted in 2X electrophoresis sample buffer (Novex), boiled for 5 minutes, run on 10% acrylamide gel, and electrophoretically onto a PVDF membrane. Transcribed. The membrane was saturated with 5% milk blocker by incubation for 1 hour at room temperature. Primary antibodies against PKC isoforms (Transduction Laboratories) were diluted (1: 1000) in blocking solution and incubated with the membrane overnight at 4 ° C. After incubation with secondary antibody alkaline phosphatase anti-mouse IgG (Vector Laboratories), the membrane was developed with chemiluminescent substrate (Vector Laboratories) according to the manufacturer's instructions. Band intensity was quantified by densitometry, using a BioRad GS-800 calibration scanning densitometer and Multianalyst software (BioRad).

sAPP-determinations / sAPPα measurement
The concentration of secreted APP was measured using a protocol slightly modified from conventional immunoblotting techniques. The precipitated protein extract / treatment from each dish was loaded onto a freshly prepared 10% acrylamide Tris HCl minigel and separated with SDPAGE. The volume of the loaded sample was corrected for the total cell protein per dish. The protein was then electrophoretically transferred to a PVDF membrane. The membrane was saturated with 5% non-fat dry milk to block nonspecific binding. The blocked membrane was incubated overnight at 4 ° C. with the commercially available antibody 6E10 (1: 500), which recognizes sAPP-alpha in conditioned medium (SENETEK). After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Jackson's Laboratories) at room temperature.
The signal was then detected by exposure to Hyperfilm ECL (Amersham) using enhanced chemiluminescence. Band intensity was quantified by densitometry, using a BioRad GS-800 calibration scanning densitometer and Multianalyst software (BioRad).

  As shown in FIGS. 8 and 9, bryostatin-1 elicited a strong response and demonstrated activation of PKC. It should be noted that PKC activation is readily detectable 30 minutes after delivery of only 0.1 nM bryostatin-1 dose.

It is also useful to consider data relating to APP metabolism and the effects of its sub-products. PKC activation is non-amyloidogenic (soluble APP, possibly the product of the secretase) vs. Studies have demonstrated increasing the amount of the ratio of secreted fragments of amyloidogenic (Aβ1-40 and / or Aβ1-42) (Buxbaum et al., 1990; Gillespie et al., 1992; Selkoe, 1994). While not intending to hold on to this theory, AD cells (with low PKC) will have impaired secretion of sAPP and / or have an increased ratio of amyloidogenic fragments I could guess that. In fact, there is evidence that several AD cell lines exhibit both defective PKC and impaired sAPP secretion (Bermagashi et al., 1995; Govoni et al., 1996). In addition, β-amyloid induces AD-like K ⁺ channel defects in fibroblasts (Etcheberligaray et al., 1994) and blocks K ⁺ current in cultured neurons (Good et al., 1996) is shown. Thus, mechanistic links such that isozyme-specific PKC defects induce abnormal APP processing and, among other potentially harmful effects, this processing alters K ⁺ channel function. We advocate. Recent preliminary data also suggest that β-amyloid causes PKC reduction in turn, possibly in a vicious cyclical manner (Favit et al., 1997).

In summary, the data suggest that sAPP production is increased by strategies that target specific isozymes and up-regulate PKC function. It seems possible to expand these studies and such fibroblast models, and the effects of compounds (bryostatin, eg) that alter the underlying pathological potential processes that they underlie Could be used as a tool for monitoring. In addition, one of ordinary skill in the art will understand how to further test these samples with Ca ²⁺ imaging and electrophysiology. Such compounds could then be used as a basis for the rational design of pharmaceutical agents for this disorder.

Example II
Behavioral Studies (Behavioral Studies)
The Morris water maze paradigm (48) was used to study the effects of bryostatin 1 on learning and memory. Weighed Wistar albino rats (n = 20), which are between 220-250 g, were housed for one week and had free access to food and water. Stainless steel cannulas were placed bilaterally in each rat (49) according to the procedure described previously. All animals were given a 1 week recovery period prior to further experiments. Subsequently, the animals were randomly assigned to experimental and control groups. At least 24 h prior to treatment and training, all animals were pre-exposed by placing them in water and allowed to swim for 120 s to the MWM experimental setting. The training was followed by the standard procedure (49), with 2 trials / day consisting of 4 consecutive days. Treated animals received (icv) 1 μl / site of 2 μM solution of bryostatin 1 approximately 30 min before training trials 1 and 5. The control group received the same volume of vehicle alone on the same schedule. On the fifth day, the scaffold was removed and a retention test was performed. Animal movements and escape latencies were recorded with an automatic tracking system. Learning was measured as a reduction in trial-to-trial evacuation latency, which was significantly lower in treated animals.
Memory acquisition was measured as the time spent in the relevant quadrant (Day 5). Memory or retention was significantly enhanced in treated animals compared to sham injection animals (see FIGS. 3-4 (a) -4 (c)). Rats treated with bryostatin-1 showed improved cognition compared to control rats within 2 days of treatment (see FIG. 3). Bryostatin-1 can be used at concentrations that improve cognition, which is 300 to 300,000 times lower than that used to treat tumors. The above examples further show that cognitive ability can be improved in non-disseminated subjects (as compared to other unaffected subjects) by administration of bryostatin-1.

  The use of PKC activators (especially bryostatin-1) would be recognized as safe due to previously conducted cancer safety, toxicity, and phase II clinical trials, and AD treatment / cognition It can be concluded that phase II trials for enhancement could be facilitated. Furthermore, the lipophilic nature of bryostatin-1 provides increased blood brain barrier transport. The present invention will accept intravenous, oral, intraventricular, and other known methods of administration.

  Tests of sAPP secretion experiments, PKC activation experiments, and animal behavior experiments have shown that increased sAPP secretion occurs following increased PKC activation, resulting in improved cognition in animal behavior studies.

Example III
Transgenic animals and in vivo studies
Transgenic mice carrying the V717I mutation were treated with BL (1 mg / kg, ip; daily) for ~ 3 weeks (after weaning) to 17 weeks (n = 4). The control group (n = 4) received vehicle alone (Tween 20 1%, DMSO 25%, 74% PBS).
Another experimental group consisted of 5-6 month old animals and was treated for 7 weeks. Subgroups of these animals were treated with the following reagents: BL 1 mg / kg, daily (n = 5); BL 10 mg / kg, daily (n = 3; due to 2 individual deaths); BL 10 mg / kg, weekly (n = 4; 1 individual dead), LQ12 10 mg / kg, one day (n = 5); and LQ12 10 mg / kg, weekly (n = 5). Five additional animals received vehicle alone for the same period. After treatment, the animals are U. L. (Belgium) euthanized according to guidelines.
The brain was removed and prepared for biochemical analysis of APP species.
Biochemical analysis of APP processing in the brain of APP tg mice.

Immunoblot analysis. The biochemical analysis of intermediaries of APP metabolism has been described elsewhere by Dewachter et al. (Aging increased amyloid peptide and brain quotient in brain of liver cap APP / V7g. 1. J Neurosci. 2000; 20: 6452-8.). In summary, the brain is 6.5 vol. In ice-cold buffer (containing 20 mM Tris-HCl, pH 8.5, and a mixture of proteinase inhibitors (Roche, Darmstadt, Germany)). After centrifugation (135,000 × g, 4 ° C., 1 hr), the supernatant was centrifuged again (2 hr, 200,000 × g) before the soluble amyloid peptide was analyzed by a defined ELISA. The pellet from the first centrifugation was resuspended in TBS (containing 2% Triton X-100, 2% Nonidet P40 and proteinase inhibitor) and centrifuged (100,000 × g, 4 ° C., 1 hr). . This protein fraction was used for analysis of membrane-bound APP. A Western blot of membrane-bound APP was performed with monoclonal antibody 8E5 on this protein fraction (containing membrane-bound protein). Total secreted APP and secreted APP-α cleaved with α-secretase were detected by Western blot analysis on the supernatant of the first centrifugation (using monoclonal antibody 8E5 and monoclonal antibody JRF14, respectively) ). Proteins were denatured and reduced in sample buffer (containing a final concentration of 2% SDS, 1% 2-ME) and separated on an 8% TRIS glycine gel (Novex, San Diego, Calif.). After incubation with the appropriate secondary antibody, all Western blots were illuminated with an ECL detection system and recorded with photographic techniques. Quantification was possible with the application of a series of diluted samples.
Densitometric scanning and normalization of the film was performed using a flatbed optical density scanner and dedicated software for analysis and measurement (Image Master; Pharmacia, Uppsala, Sweden).

  The amyloid peptide ELISA protein extract was applied to a reverse phase column (C18-Sep-pack cartridge; Waters Corporation, Mild, Mass.) And added to a solution (5, 25, and 50%) with increasing acetonitrile concentration. Wash with a solution containing 1% trifluoroacetic acid. The last fraction contained amyloid peptide and was dried in vacuo overnight and dissolved for measurement by ELISA. Sandwich ELISAs against human Aβ40 and Aβ42 peptides were performed with capture antisera JRF / cAβ4O / 1O and 21F12, respectively, and they were developed with monoclonal antibodies JRFcAβtot / 14hrpo and 3D6, respectively (Vanderstichele H, Van Kerscher E, Hese C, Davidsson P, Buye MA, Andrewsen N, Minthon L, Wallin A, Blennow K, Vanmechel E. Standard boiled in 42. d and plasma Amyloid 2000; 7: 245-258).

  Standard general health assessment and open field were performed prior to biochemical assessment in all animals. In addition, a semi-quantitative ad hoc score was developed to measure the contraction of the abdominal cavity following injection (+ = weak, ≦ 2 min; ++: strong, ≧ min; +++ : Very strong, ≧ 1.2 min).

Example IV
In vivo studies using transgenic animals and bryostatin
A second transgenic study (using similar procedures / tests and protocols) was performed using double transgenic mice (holding the V717I mutation and the presenilin-1 (PS1) mutation). This resulted in accelerated amyloid formation, with the following major differences: Approximately 40 mice (including both treatment and control) were utilized. Treatment is started at about 3 weeks of age and 40 μg / kg. i. p. And consisted of treatment with bryostatin-1 three times a week.
Controls were given vehicle alone. Treatment was continued for about 7 months before the prevalence of untreated animals required termination of the experiment (see FIG. 10). The behavioral differences between treated and untreated animals were not significant in water testing (see FIG. 11), but treated animals were soluble Aβ-40 (see FIG. 12). And a decrease in soluble Aβ-42 (see FIG. 13). Furthermore, the treated mice had an overall low total APP (the total APP); the results are as shown in FIG. 14, where thioflavin S staining reduced percent plaque load. Is shown (compared to the control).

Consideration of the above experiment
sAPPα secretion: After 3 hours of treatment of AD cell line AG06848 with 0.1 nM bryostatin, a dramatic increase in sAPPα secretion was observed (compared to all other conditions) (overall ANOVA, p <0.0001 (FIG. 1 (a), solid bar). This effect was also significantly higher than when another PKC activator (BL) was used at the same (0.1 nM) concentration (p <0.01, Turkey post test). BL 0.1 nM did not have any realistic impact on secretion; nor was it different from DMSO alone. Pretreatment with 100 nM staurosporine (PKC blocker) abolished the effect of 0.1 nM bryostatin (FIG. 1A, rightmost bar). Two cell lines were also used from age-matched controls. In these cell lines (pooled), bryostatin (0.1 nM) was also significantly (p <0.05, Turkey's test compared to DMSO alone) enhanced sAPPα secretion, but AD strain To a significantly lower extent than in the case of the transformed cells (FIG. 1A, hatch bar; p <0.05, Turkey test). A time course experiment (FIG. 1 (b), inset) showed a marked increase in sAPPα secretion after 15 minutes incubation with 0.1 nM bryostatin. Progressive and proportional increases were observed at 30 and 60 min. The incubation period (2 and 3 h) was not substantially different from the 60 min incubation with respect to the amount of APPα secreted. With a lower concentration of bryostatin (0.01 nM), a robust enhancement of APPα secretion occurred after only 60 min incubation. However, the effect of the low concentration (0.01 nM) was indistinguishable from that of the higher concentration (0.1 nM bryostatin) incubated for 2 and 3 h (FIG. 1 (b)). A representative immunoblot (explaining various experimental conditions and sAPPα secretion in cell lines) is shown in FIG. 1 (c).

  PKC translocation: The level of cytosolic and membrane bound form of alpha isoenzyme was determined after incubation of bryostatin (various time points) at 0.1 and 0.01 nM. There was a relative increase in the membrane-bound component of PKC α-isoenzyme (compared to DMSO alone), which was measured as the ratio of particulate / soluble (P / S) immunoreactivity. . The increase was very consistent and was significantly different from DMSO alone after 30 min incubation (p = 0.411; t-test, two-sided). The P / S ratio declined progressively, but remained higher than DMSO alone (although not statistically significant) even after 180 min incubation (Figure 5a). Short incubation (5 min) did not induce consistent or significantly different translocation compared to DMSO alone (not shown). The effect of 0.01 nM bryostatin was insignificant and slow, with a maximum P / S ratio value at 120 min incubation. The level of translocation of other PKC isoenzymes was assessed with 0.1 nM bryostatin in a 30 min incubation. Clear immunoreactivity was detected with specific antibodies against ε, β and δ isoenzymes (both membrane bound and cytosolic). The ratio S / P was higher in all cases than DMSO alone and was comparable to the level of PKC-α (FIG. 5b).

  Behavior (MWM): The learning curve of the group receiving bryostatin was significantly faster than the control group. The evacuation latency was clearly reduced from early trials and lower than the control group from trial 3. The quadrant preference test showed retention in both groups, but was significantly enhanced (compared to the control) for the bryostatin treatment group. Figures 3-4 (c) summarize these results.

Transgenic animals: Transgenic animals (3 weeks old animals treated with BL and treated for 17 weeks) are proportional to a significant increase in sAPP-α and Aβ40 concomitant (FIGS. 6A to 6B). There was no difference in the amount of Aβ42, APP membrane binding, and total secreted sAPP (sAPPα + sAPPβ).
Animals showed no difference in general health and weight gain was similar in both groups. Injection resulted in variable abdominal contraction (reversible) (similar frequency in both groups). The intensity increased somewhat in the BL treatment group (data not shown). In addition, BL treated animals showed an increase in open field test scores (not reaching statistical significance) (not shown).

  Later in life (6 months old), short-term (~ 7 weeks) treated animals did not show dramatic changes associated with APP species. However, the general trend (small changes) was in the same direction as described for longer term treatment (previous section). A slight increase in sAPPα was observed in animals treated with BL 10 mg / kg (daily and weekly); also in animals treated with LQ12 10 mg / kg (daily) (FIG. 7 (b)). , Solid bars). BL 1 mg / kg (daily) and LQ12 10 mg / kg (weekly) had no effect (FIG. 5A, pattern bar). A slight decrease in Aβ40 was observed in animals treated with BL (n = 5) and LQ12 (n = 5) (both 10 mg / kg, weekly) (FIG. 7 (b), solid bar). There was no noticeable significant change in Aβ42 due to treatment. Similarly, no significant changes were observed in total soluble APP and membrane bound APP. In addition, abdominal contractions and hind limb relaxation were observed in old animals at the time of injection (reversible). They appeared to be dose related, but no clear system was arranged for a more accurate assessment. General health and weight were also normal. A few (2-3) animals died (7.8% of the total) during the course of the experiment for reasons that did not appear related to treatment. There was no difference in the open field test (not shown).

  However, treatment with bryostatin-1 showed observable changes in both (Aβ-40, Aβ-42, and total APP). (See FIGS. 12-14). In addition, the animals treated with bryostatin-1 showed a large life expectancy over time. (See FIG. 10).

  These results demonstrate the role of PKC in AD pathophysiology. These results further demonstrate that a common APP pool exists. Thus, an increase in one enzyme pathway results in a decrease in substrate for alternative enzymes. In this case, the reduction of amyloidogenic and toxic fragments (Aβ40) is achieved by increasing the non-pathogenic α-secretase processing of APP. The fact that the total secreted APP (α + β product) is not different between treated and untreated animals is consistent with and confirms such an interpretation. It is also clear that the increase in sAPPα is not the result of elevated total APP (or increased expression) because membrane-bound APP is similar in both groups.

The most striking “beneficial” effect was observed in animals that started treatment early (and for a long period of time). This suggests that preventing the long-term effects of toxic fragments should be an important therapeutic goal. Intervention at an older age and later in the course of the disease process (even without clinical symptoms) would have a very low impact in preventing damage from toxic fragments (obtained in older animals). As suggested by the results). It is also important to refer to the following, which means that this particular transgenic model first produces biochemical changes, then cognitive defects, and then (substantially Later) to produce amyloid deposits and plaques. Consistent with in vitro testing, this sequence indicates that amyloid species can be harmful (possibly in soluble form) (before any significant deposition occurs).

The results showing improved performance of normal rats in the MWM task after bryostatin administration (i.c.v.) demonstrated that PKC activation can produce cognitive enhancement as an additional therapeutic effect. In addition, secreted APP can itself improve memory in normal and amnestic mice. These experiments and models demonstrate that PKC regulation (especially via bryostatin-1) can result in increased sAPP and / or improved memory.
They have also demonstrated that regimens containing PKC activators can be used to prevent buildup of toxic fragments and to prevent memory declines.

FIG. 1 (a) shows the effect of different PKC inhibitors and concentrations on sAPPα secretion with bryostatin-1 (great efficacy is shown at lower concentrations than control and benzolactam). Bryostatin (0.1 nM, solid bar) also dramatically increased the amount of sAPP-α in the medium in AD cell lines that were well characterized and confirmed by autopsy after 3 h incubation. (P <0.0001, ANOVA). The graph unit is proportional to the vehicle (DMSO) alone. Bryostatin was significantly more potent (p <0.01, Turkey post test) than the same (0.1 nM) concentration of another PKC activator (BL). Pretreatment with staurosporine (100 nM) (rightmost bar) completely abolished the effect of bryostatin (0.1 nM). Bryostatin was also effective in enhancing secretion in the two control cell lines, but to a lesser extent (than in the AD cell line) (hatched bar). FIG. 1 (b) shows the effect of different concentrations of bryostatin-1 on sAPPα secretion over time; secretion was clearly almost enhanced with 15 min incubation (bryostatin 0.1 nM), 160 It is almost the maximum in the incubation and rises to 3h. Bryostatin at the lower concentration (0.01 nM) was very slow but had almost the same effect on secretion (after 120 min incubation). FIG. 1 (c) shows sAPPα secretion by Western blot presentation of sAPP-α in human fibroblasts using a variety of experimental conditions and cell lines; FIG. 2 shows the effect of different concentrations of bryostatin-1 on the PKCα isozyme. FIG. 3 shows the amount of time required to learn the treated rat vs. control water maze. The learning curve of the Morris water maze shows that bryostatin (i.v.c.) improves the animal's ability, which is evidenced by a decrease in the evacuation latency from early trials. FIG. 4 (a) shows the amount of time the control rats spent swimming in different quadrants. Both (control and treated animals) showed retention of priority over the target quadrant (see also FIG. 4 (b)). FIG. 4 (b) shows the amount of time the treated rats spent swimming in different quadrants. FIG. 4 (c) shows the difference between the amount of time that the treated rats spent in the target quadrant compared to control rats. Treated animals showed improved retention (compared to controls); FIG. 5 (a) shows PKC translocation in human fibroblasts as a bar graph. This graph shows the ratio between the immunoreactivity of membrane-bound PKC (P = particulate) (normalized with total protein content) and the immunoreactivity detected in the cytosolic fraction (S = soluble) . PKC-α translocation was significant after 30 min incubation with 0.1 nM bryostatin (solid bar). Translocation was still present in the 180 min incubation (rightmost bar) (P> S). FIG. 5 (b) shows that other PKC isoenzymes were detected and their translocation levels were comparable to those observed for PKC-α. FIG. 6 (a) shows an in vivo test using transgenic mice (young animals). The transgenic mice were treated for 17 weeks with BL 1 mg / kg (ip daily), starting immediately after weaning (3 weeks). There was a significant increase in sAPP-α in the brain of the treatment group (as compared to vehicle alone). FIG. 6 (b) shows that the same animal had a proportional reduction of Aβ40. FIG. 7 (a) shows an in vivo test using about 6 month-old transgenic mice (adult animals). The transgenic mice were treated with BL and LQ12 treatment for 7 weeks at the dose and schedule shown in the bar graph. A small increase in sAPP-α was observed with treatment (indicated by a solid bar). Figure (7b) shows a small Aβ40 decrease (no significant difference), which was observed in animals treated with BL and LQ12 (both 10 mg / kg weekly) (solid bars). An unexpected (hatchbar) increase in Aβ40 was observed in animals treated with BL 10 mg / kg (daily). FIG. 8 shows sAPPα secretion in human fibroblasts after administration of bryostatin 0.1 nM to both control and AD cells. FIG. 9 shows an immunoblot of sAPP after bryostatin administration to AD cells. FIG. 10 shows the positive effect of bryostatin and increased life span (compared to controls) in treated mice. FIG. 11 shows the time course duration in the water test for treated versus untreated animals. FIG. 12 shows a decrease in the concentration of soluble Aβ-40 in treated animals versus controls. FIG. 13 shows a decrease in the concentration of soluble Aβ-42 in treated animals versus controls. FIG. 14 shows the percent reduction in plaque (compared to control) observed in treated animals after thioflavin S staining.

Claims (12)

Use of a PKC activator in the manufacture of a medicament for enhancing cognitive performance in a subject having a neurological disease or disorder, using the PKC activator is bryostatin-1 through bryostatin 18.   2. The use according to claim 1, wherein the PKC activator selectively activates PKCα, PKCδ, or PKCε.   The use according to claim 1, wherein the enhanced cognitive ability is learning, memory, or attention.   The use according to claim 1, wherein the neurological disease is associated with or does not associate with Alzheimer's disease, multiple infarct dementia, Parkinson's disease; Lewy-body variant; Creutzfeldt Jacob Uses that are ill, Korsakow's disorder, or attention deficit hyperactivity disorder.   2. The use according to claim 1, wherein the disorder is related to age, electroconvulsive therapy, or brain injury.   6. The use according to claim 5, wherein the brain injury is caused by stroke, anesthesia accident, head injury, hypoglycemia, carbon monoxide poisoning, lithium poisoning or vitamin deficiency.   2. Use according to claim 1, wherein the PKC activator is administered in an amount effective to cause an increase in sAPP. Use of a PKC activator in the manufacture of a medicament for treating Alzheimer's disease, comprising an effective amount of bryostatin-1 to bryostatin 18 and a pharmaceutically acceptable carrier for treating Alzheimer's disease Including use.   Use of a PKC activator in the manufacture of a medicament for treating Alzheimer's disease, comprising an effective amount of bryostatin-1 and a pharmaceutically acceptable carrier for treating Alzheimer's disease.   Use of a PKC activator in the manufacture of a medicament for enhancing cognitive ability, comprising bryostatin-1 in an amount effective to enhance cognitive ability in a pharmaceutically acceptable carrier.   Use of a PKC activator in the manufacture of a medicament for reducing amyloid plaque formation, comprising an amount of bryostatin 1 and a pharmaceutically acceptable carrier effective to reduce amyloid plaque formation. Use according to claim 11, using the amyloid plaques comprising A [beta]-40 or A [beta]-42.

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