Patent Application
20140336141
Late-onset Alzheimer's disease (LOAD) is the most prominent form of dementia among the elderly (>65 yrs). The only known genetic risk factor is the Apolipprotein E4 allele. In contrast to the early-onset variant, LOAD patients do not carry any mutations in the genes of amyloid precursor protein (APP), presenilin 1 and presenilin 2. The disease is characterized by progressive cognitive dysfunction, and various behavioural and neuro-psychiatric disturbances such as agitation, psychosis, depression, apathy, disinhibition, anxiety, purposeless behaviour, and disorders of sleep and appetite. Histopathological hallmarks in the brain are extracellular amyloid plaques and neurofibrillary tangles in neurons. Amyloid plaques are formed by aggregation of amyloid beta (1-42) that is derived from proteolytic processing of APP by beta and gamma secretases. Neurofibrillary tangles are found in cell bodies and apical dendrites as neurofibrillary tangles, in distal dendrites as neuropil threads, and in the abnormal neurites that are associated with some plaques (neuritic plaques).They are mainly composed of hyperphosphorylated aggregates of the protein tau.
Approximately 70% of annotated gene promoters are associated with CpG islands, including most of the housekeeping genes, many tissue-specific genes and developmental regulator genes. CpG islands are sites of transcriptional initiation and CpG island methylation leads to silencing of the associated promoter, and to decreased expression levels of the respective protein. Global DNA hypomethylation has been observed in the entorhinal cortex of LOAD patients. It has also been shown that several proteins such as presenilin 1 and APP that are involved in the beta amyloid formation in LOAD are regulated by promoter methylation. Additionally, APOE, the only known genetic LOAD-risk factor, and genes involved in methylation homeostasis (MTHFR, DNMT1) show a significant inter-individual epigenetic variability in LOAD, which may contribute to LOAD predisposition.
DNA methyltransferases (DNMTs) use S-adenosylmethionine (SAM), the main methyl donor in eukaryotes, as substrate for methylating cytosines on genomic DNA. The resulting S-adenosylhomocysteine (SAH) is a competitive inhibitor of DNMTs. SAH is hydrolyzed to adenosine and homocysteine, which can be re-methylated to methionine. These compounds are connected via the One-carbon cycle which is the only means of SAM production (
Administration of folic acid, vitamin B12 and vitamin B6 to AD patients with mild cognitive impairment for two years reduced brain atrophy and improved cognitive function when compared to the placebo group (1). Vitamin B12 therapy significantly reduced platelet MAO activity (Mao-B is the predominant isoform in platelets) in LOAD patients to apparently normal levels (2). The Inventors showed that increased meat consumption improves cognitive functions in young volunteers and reduces the platelet Mao-B level and that platelet Mao-B concentration inversely correlates with the plasma level of vitamin B12, which is a cofactor in the One-carbon cycle (3).
The flavoenzyme monoamine oxidase B (MAO-B) is located in the outer membrane of mitochondria and catalyses the oxidative deamination of amine neurotransmitters and dietary amines using O2 as the electron acceptor. MAO-B preferentially degrades phenylethylamine and benzylamine, as well as the cognition enhancing neurotransmitter dopamine. Oxidative deamination of primary monoamines by MAO-B results in the species with proven or potential toxicity, namely ammonia, aldehydes and hydrogen peroxide. Furthermore, MAO-B inhibition reduces dopamine inactivation enabling the neurotransmitter's increased availability in dopaminergic neurons. The activity of MAO-B in brain and in platelets is significantly increased in LOAD (EP1891445) and it has been demonstrated that MAO-B mRNA and MAO-B protein levels are elevated in platelets of LOAD patients (PCT/GB2010/052023). MAO-B inhibitor drugs currently find use in the treatment of hypertension, Parkinson's disease, senile dementia and depression. Thus evidence suggests the potential use of MAO-B inhibitors for the treatment of AD.
The search for therapeutic drugs for the treatment of debilitating pathologies is an ongoing and relentless process. However, it is also a lengthy and extremely costly exercise, with most candidate drug development programs being terminated before drug approval. The elucidation of possible pathophysiological pathways is an extremely difficult but highly desirable goal as it enables a more targeted approach to candidate drug choice, expedites the drug development process and increases the chances of a drug being approved for therapy. Diseases such as hypertension, depression, Parkinson's disease and dementias can be treated using MAO-B inhibitors. Described herein are novel compounds of the MAO-B inhibitor class identified through the elucidation of the connection of MAO-B to the One-carbon cycle.
(1) Smith A. D. et al. (2010). PLoS One, 8; 5(9): e12244.
(2) Regland B. et al. (1991). Eur. Arch. Psychiatry Clin. Neurosci., 240(4-5): 288-91.
(3) Zellner M. et al. (2011). J Neural Transm., 118(5): 653-62.
The invention, underpinned by the finding that the concentration of MAO-B in patient samples is affected by the SAM methylation cycle, describes the utility of SAM and compounds in the One-carbon cycle in disease treatment therapies in which the disease treatment depends upon MAO-B inhibition. Elucidation of the relationship between MAO-B concentrations and the components of the SAM one-carbon methylation cycle also enables methods of measuring the efficacy of a drug of or a drug that affects the SAM one-carbon methylation cycle in in vitro samples taken from patients, especially patients suspected of having or suffering from Alzheimer's Disease, especially late onset Alzheimer's disease.
In a first aspect, the invention describes the use of one or more of the compounds SAM, vitamin B6, vitamin B12, and folate, individually or in any combination, in the treatment or prevention of diseases which are mediated by monoamine oxidase B inhibitors. In a preferred embodiment the compound(s) is/are one or more of SAM, vitamin B6 and B12; SAM is an especially preferred embodiment. A combination of vitamin B6 and B12 is also a preferred embodiment.
A second aspect of the invention is the use as adjunct therapy of one or more of the compounds SAM, vitamin B6, vitamin B12 and folate, individually or in any combination, in the treatment and prevention of diseases which are mediated by monoamine oxidase B inhibitors. A preferred disease to be treated or prevented by the compounds of the invention when used as adjunct therapy is Alzheimer's disease; treatment of late onset Alzheimer's disease is an especially preferred embodiment. Adjunct therapy implies that the one or more compounds can be used in conjunction with other recognised forms of therapy for a specified diseased.
A preferred disease to be treated or prevented by the compounds of the invention whether alone or as adjunct therapy is Alzheimer's disease; treatment of late onset Alzheimer's disease is an especially preferred embodiment.
A further aspect of the invention is a method of determining the efficacy of a drug or drugs of the SAM one-carbon methylation cycle for use in therapy comprising measuring in an in vitro sample taken from a patient or from cultured or harvested cells the concentration of MAO-B following administration of a drug or drugs of the SAM one-carbon methylation cycle to the patient or to the cultured or harvested cells and comparing said concentration to a control concentration of MAO-B. The control MAO-B concentration can be a statistical measure of central tendency such as the median or mean of a suitable patient population. Preferably, the control concentration of MAO-B is the concentration of MAO-B in an in vitro sample taken from the patient or from cultured or harvested cells before administration of a drug or drugs. In an embodiment of the invention the MAO-B concentrations measured are normalised using a suitable compound such as GAPDH or 14-3-3 gamma. This aids in minimising the effect of differences in the inter-sample and inter-cellular MAO-B concentrations and can increase result accuracy. In a preferred embodiment the method is used to test the efficacy of drugs of the SAM one-carbon methylation cycle that are used or are to be used in Alzheimer's disease therapy, especially late onset Alzheimer's disease. A concentration of MAO-B in an in vitro sample taken from the patient or from cultured or harvested cells following drug or drugs administration that is lower than that of the control is indicative of an efficacious drug or drugs. What is meant by drug(s) of the SAM one-carbon methylation cycle is a chemical compound that is mentioned in
Alternatively, for pathologies in which an increase in MAO-B concentration is sought, it might be clinically desirable to associate the SAM one-carbon methylation cycle with an increase in MAO-B concentration in a sample taken from a patient. An increase in MAO-B would be expected if the SAM one-carbon cycle were to be interrupted by a drug or drugs causing a decrease in DNA-methylation. A further embodiment of the invention is a method of determining the efficacy of a drug or drugs in a patient to inhibit the SAM one-carbon methylation cycle comprising measuring in an in vitro sample taken from the patient or from cultured or harvested cells the concentration of MAO-B following administration of a drug or drugs to the patient or to the cultured or harvested cells and comparing said concentration to a control concentration of MAO-B. An increase in the concentration of MAO-B in the in vitro patient sample or the cultured or harvested cells compared to control indicates that the SAM one-carbon cycle has been inhibited and that the drug or drugs is/are efficacious and achieve the desired effect.
An additional aspect of the invention is a method of determining the effect of a drug or drugs upon the SAM one-carbon methylation cycle comprising
The neuroblastoma cell line SH-SY5Y was seeded at 110,000 cell/6-well. Neuronal differentiation treatment was started on the next day (=Day 0) by addition of 10 μM retinoic acid every second day until Day 6. On Day 8 differentiation treatment was switched to 4 μM uridine (Ur), 5 μM 5-fluoro-2′-deoxyuridine FdUr), and 1 μM cytosine beta-D-arabinofuranoside (araC). During mitotic inhibitor addition (=Days 8 to 12) cells were left untreated or treated with 625 nM 5-aza-2′-deoxycytidine (Aza), 25 μM S-adenosylmethionine (SAM), 5 μg/ml vitamin B6 (Vit B6), 7.5 μg/ml vitamin B12 (Vit B12), or 5 μg/ml vitamin B6/7.5 μg/ml vitamin B12. On Day 12 cells were harvested. Cells were washed twice with chilled PBS, scraped off in PBS/protease inhibitors, and centrifuged at 15000×g at 4 C for 10 min. The pellet was reconstituted in 150 μl PBS/protease inhibitors and precipitated with 50 μl trichloroacetic acid/80 mMDTT for 1 hour at 4 C. After centrifugation at 15000×g at 4 C for 10 min, precipitated protein was washed four times with 0.6 ml acetone/20 mM DTT and stored at −80 C until analysis. The dried total protein pellet was reconstituted in 7M urea/2M thiourea/4% CHAPS buffer and the protein concentration was determined using the Coomassie Plus (Bradford) Protein Assay (Pierce, Thermo Scientific). Equal protein amounts were loaded onto an 11% SDS polyacrylamide gel and proteins were blotted onto nitrocellulose. Mao-B was detected using a polyclonal Goat anti-Mao-B antibody (sc18401, Santa Cruz) and DyLight 649 AffiniPureF(ab′)2 Fragment Donkey Anti-Goat IgGgamma (Jackson ImmunoResearch Laboratories, Inc.), and a Typhoon scanner (GE Healthcare Life Sciences). Results were analysed with the software ImageJ. The signals of the Mao-B bands of SAM treated and untreated differentiated SH-SY5Y cells were compared and the latter was set 100%.
HepG2 cells were grown in DMEM low glucose/10% FBS and were treated with 10 μM 5-aza-2-deoxycytidine for 1, 2, or 3 days. Untreated HepG2 cells served as controls. Cells were scraped in precipitation buffer (PBS/25% trichloroacetic acid/20 mM DTT/protease inhibitors) at 4 C and proteins were precipitated for 1 hour at 4 C. Precipitated proteins were pelleted at 15000 rpm for 10 min (4 C) and washed 4 times with 700 μl acetone/20 mM DTT. Before analysis by SDS PAGE and Western blot, the precipitated samples were reconstituted in DIGE buffer/1% pH 4-7 ampholyte. Six parallel experiments were performed. Each sample was run on two different Western blots and the results are displayed as averages. Two normalisation compounds were used: GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and 14-3-3 gamma (protein kinase C inhibitor protein 1). Each Mao-B band signal was divided by the respective GAPDH or 14-3-3 gamma band signal to achieve normalisation. As band signals on different Western blots were not comparable due to the use of different antibody solutions, all normalised Mao-B signals of treated cells were compared to the normalised Mao-B signals of the untreated cells, which were set at 100%.
HepG2 cells were grown in DMEM low glucose/2% FBS and were treated with vitamin B6 (300 μg/ml) and vitamin B12 (74.4 μg/ml) for 1, 3, 4, 5 and 7 days. Untreated HepG2 cells served as controls. Cells were harvested and analysed by Western blot.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1122227.0 | Dec 2011 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/076815 | 12/21/2012 | WO | 00 | 6/20/2014 |
