CURCUMIN FROM TURMERIC AS A DRUG CANDIDATE FOR ALZHEIMER’S DISEASE

1. CURCUMIN FROM TURMERIC AS A DRUG CANDIDATE FOR ALZHEIMER’S DISEASE P.B. Tirupathi Pichiah

2. The Turmeric Rhizomatous herbaceous perennial plant Ginger family Used as a spice in Indian, Persian, Thai and Malay dishes, not only in curry, but also in masak lemak, rendang and many more. Used for dyeing fabrics, Active ingredient is curcumin and it has a distinctly earthy, slightly bitter, slightly hot peppery flavor and a mustardy smell. Most usage of turmeric is in the form of root powder. Erode (Yellow City), a city in the south Indian state of Tamil Nadu, is the world's largest producer and most important trading center of turmeric in Asia.

3. The curcumin solution or curcumin powder dissolved in alcohol is used for coloring products. Such as in pickles, relishes, and mustard. Turmeric has been used to color cheeses, yogurt, dry mixes, salad dressings, butter and margarine (Like Butter).

4. Uses in folk medicine Ayurvedic practices : antiseptic for cuts, burns and bruises. Turmeric mixed in milk for fever. Asian countries : dietary supplement, which allegedly helps with stomach problems and other ailments. Japan :Popular as a tea in Okinawa. Pakistan: Also use it as an anti-inflammatory agent, and remedy for gastrointestinal discomfort associated with irritable bowel syndrome, and other digestive disorders. Afghanistan , India and northwest Pakistan : Turmeric Paste applied in wound

5. COSMETICS SOAP FACE WASH SKIN CREAM

6. Importance of Turmeric in Indian Culture and Tradition: Used in various rituals wedding ceremony South Indian festival Pongal, Used to prepare special sweet dishes Patoleo In South Indian marriage ritual, a piece of dried turmeric tied with string is sometimes used to replace the Thali necklace temporarily.

7. Curcumin has a wide range of application:

8. PRELIMINARY MEDICAL RESEARCH Turmeric is currently being investigated for possible benefits in Alzheimer's disease cancer, arthritis, and other clinical disorders. The U.S. National Institutes of Health currently has registered 19 clinical trials underway to study use of dietary turmeric and curcumin for a variety of clinical disorders (dated February 2010).

9. What is Curcumin ? Curcumin is the principal curcuminoid of the popular Indian spice turmeric, which is a member of the ginger family (Zingiberaceae). Curcuminoids are Polyphenols, Responsible for yellow color of turmeric. Three major curcuminoids — 1.Curcumin, 2.Demethoxycurcumin and 3.Bisdemethoxycurcumin — occur naturally in these Curcuma species. Chemistry Aromatic ring systems, which are polyphenols are connected by two α,β-unsaturated carbonyl groups.  Two carbonyl groups form a diketone. The diketone form stable enols or are easily deprotonated and form enolates, while the α,β-unsaturated carbonyl is a good Michael acceptor and undergoes nucleophilic addition.

10. “DiKetone General Form” Curcumin in Ketoform Ketone General Form Enol General Form Curcumin in Enol Form: Enols are alkenes with a hydroxyl group affixed to one of the carbon atoms composing the double bond

11. Biological Properties of Curcumin : Anti-inflammatory effects by interrupting NF-κB signaling. Scavenging of ROS : Potent scavenger of ROS, including superoxide anions, hydroxyl radicals, singlet oxygen, 8 nitric oxide and peroxynitrite. Protect lipids, hemoglobin and DNA against oxidative degradation. Potent inhibitor of ROS-generating enzyme Cyclooxygenase and Lipoxygenase in mouse epidermis

12. Inhibition of Carcinogenesis Inhibits chemical carcinogenesis at different tissue sites in several experimental animal models. Curcumin in the diet decreased the number of azoxymethane (AOM) induced colon tumors in mice and rat. Topical application of curcumin strongly inhibited tumor production in the skin of mice applied with (7,12-dimethylbenz[α] anthracene (DMBA) [CARCINOGEN] . Induction of Apoptosis Induces apoptosis in several tumor cell lines. Curcumin-induced apoptosis is highly dependent on the origin and malignancy of the cell lines.

13. Curcumin treatment resulted in increase in the protein levels of Bax and Bak, and mitochondrial translocation and activation of Bax in fibroblast to trigger a drop in mitochondrial membrane potential, cytosolic release of apoptogenic molecules (cytochrome c, etc.), activation of caspase-9 and caspase-3, and ultimately induction of apoptosis. Neuroprotective Effects of Curcumin Neurodegenerative diseases result in the loss of functional neurons and synapses. Curcumin has an outstanding safety profile and a number of pleiotropic actions with potential for neuroprotective efficacy, including anti-inflammatory, antioxidant, and anti-protein-aggregate activities. Dietary curcumin is a strong candidate for use in the prevention or treatment of major disabling age-related neurodegenerative diseases like Alzheimer’s, Parkinson’s, and stroke.

14. MOLECULAR TARGETS OF CURCUMIN Curcumin possesses an anti-inflammatory activity and is a potent inhibitor of ROS-generating enzymes such as lipoxygenase, cyclooxygenase, xanthine oxidase and inducible nitric oxide synthase (iNOS). Potent inhibitor of protein kinase C (PKC), EGFR-tyrosine Ι-κB kinase. NF-κB And also, c-jun, c-fos, c-myc, NIK, MAPKs, ERK, ELK, PI3K, Akt, CDKs, and iNOS. It is considered that PKC, mTOR and EGFR tyrosine kinase are the major upstream molecular targets for curcumin

16. CLINICAL APPLICATION OF CURCUMIN Cancer. rheumatoid arthritis, atherosclerosis, chronic pancreatitis, psoriasis,hyperlipidemia, and neurodegenerative diseases. Curcumin significantly decreases serum cholesterol concentration. Curcumin treatments stimulated immune clearance of amyloidosis in AD brain, suggesting a potential neuropreventive role.

18. Neuroinflammation in Alzheimer’s disease: different molecular targets and potential therapeutic agents including curcumin Balmiki Ray and Debomoy K Lahiri

19. Born on the 14TH June 1864 in Marktbreit am Main (Germany) and died in Breslau (now Wroclaw, Poland). Fathers of neuropathology, Alois Alzheimer CLICK

20. Alzheimer’s disease (AD) : A brief Introduction

21. Degeneration of Central Nervous System and a common form of dementia. Incurable, degenerative, and terminal disease Alois Alzheimer in 1906 In 2006, there were 26.6 million sufferers worldwide. Alzheimer's is predicted to affect 1 in 85 people globally by 2050. Observable symptoms : inability to acquire new memories, difficulty in recalling recently , confusion, irritability and aggression, mood swings, language breakdown, long-term memory loss, and the general withdrawal of the sufferer as their senses decline

22. AD patients is caused by at least two distinct characteristic events, deposition of the amyloid beta (Ab) peptide in the intercellular space and formation of intra- neuronal tangle owing to hyperphosphorylation of axonal Tau protein . Amyloid beta and associated reactive oxygen species (ROS) mediated neuronal damage is one of the major hallmarks of AD, Pathological stages Stage I: Mild involvement that is confined to the transentorhinal region. Stage II: Lesion gradually increases and the pathology extends to entorhinal region. Stage III: Pathology in entorhinal region worsens and lesions extend to adjoining neocortex. Stage IV: Neurofibrillary pathology extends up to medial temporal gyrus. Stage V: Lesion extends up to occipital neocortex. Stage VI: Lesions visible in striate and parastriate areas of occipital neocortex.

23. Early and late onset AD Based on genetic etiology : 1.Familial form or Early Onset Alzheimer’s disease (EOAD) that occurs before 65 years of age 2.Late Onset Alzheimer’s Disease (LOAD) that occurs after 65 years of age and is usually sporadic. Amyloid β precursor protein (APP) and presenilin (PS) 1 and 2 are considered closely involved in the etiology of familial AD. Apolipoprotein E (APOE) gene has three common alleles, epsilon 2 (ε2), epsilon 3 (ε3), and epsilon 4 (ε4). The ε2 allele is considered protective but presence of the e4 is considered a risk factor for developing LOAD.

24. ε4 allele increases the risk for AD from 20% to 90% and decreases the age of onset from 84 to 68 years depending on gene dose of ε4 alleles. Key molecules in the pathogenesis of AD • Amyloid β precursor protein : • mammalian transmembrane protein • 695–770 amino acid residues • Large ectocytoplasmic N-terminus domain and a shorter intracytoplasmic carboxyl- • terminal region. • Cleaved by b-site APP cleaving enzyme 1 (BACE1) to produce sAPPb and a C- • terminal fragment containing 99 amino acid residues (C99)

25. C99 is further cleaved by γ secretase to produce Aβ peptides of varying numbers of amino acid residues. A β with a forty amino acid residue Aβ (1–40) is the most abundant form and is less pathogenic for the development of AD.

26. Aβ with 42 amino acid residue Aβ (1–42), which is produced in much lower quantities, is fibrilar in nature and forms aggregates that is pathognomonic in AD. BACE-1: Catalyzes the initial step of C99 cleavage to form Aβ peptide APO-E: lipoprotein that is part of chylomicrons, LDL and HDL and participates in the transport of lipids, particularly cholesterol. APOE e4 allele is related to an increased risk of AD. A potential function of ApoE in CNS is to decrease Aβ aggregation. APOEe4 has hyper-inflammatory properties leading to neuronal damage in AD brain

28. Aβ and concurrent ROS production synergistically increases the damage to the neurons. Glial (astrocyte) reactivation occurs around plaques to take up Aβ and neuronal debris. Activated astrocytes are also involved in plaque formation. interaction of microglia with Aβ peptide gives rise to ROS. activation of complement cascade forms membrane attack complexes, which not only cause substantial damage to the neurons but also can lead to phosphorylation of Tau protein leading to formation of neurofibrillary tangles Inflammations in AD Nature Medicine - 12, 885 - 887 (2006) 

29. Role of nuclear factor k beta (NFkB) Responsible for regulation in cytokine production. NFkB stays inactivated by an inhibitory protein IkB. Once activated, NFkB enters the nucleus and increases the transcription of different inflammatory mediators. TNFa, Aβ, and secreted APP activates NFkB. NFkB sites are present in the regulatory (promoter) region of APP, PS, and BACE-1 genes. An activation of NFkB increases transcription of APP and BACE-1, which eventually leads to increase in Aβ production. Presence of APOE e4 increases NFkB level

30. APOE gene promoter study showed that Aβ can stimulate APOE through NFkB-dependent pathway. Role of peroxisome proliferator-activated receptor-γ (PPAR γ) Ligand-dependent nuclear hormone receptor transcription factor. Regulates inflammatory responses in different organ systems including CNS. PPARg binds to peroxisome proliferator response element (PPRE) within the promoter regions of targeted genes of inflammatory mediators in T cells, such as TNFa, IL-10, IFN-gamma, and IL-4, and regulates their expression PPARg suppresses Aβ mediated induction of microglial cells from producing pro-inflammatory cytokines, also inhibits NFkB mediated inflammatory pathways by reducing its nuclear translocation.

31. Role of signal transducer and activator of transcription-1 (STAT-1) STAT-1 is a transcription factor - binding sites in the APP and BACE-1 gene promoter regions. Activated STAT-1 accelerated Aβ formation.

32. Current FDA approved drugs for the treatment of AD Role of cholinesterase inhibitors (ChEI) Lowers inflammatory infiltration caused by T cells and microglia in CNS of transgenic mouse and to decrease the production of inflammatory cytokines TNF-a and IFN-gamma, which are involved in inflammatory response followed by neuronal damage in AD brain. Donepezil treatment for 10mg/d (30d) reduce IL and TNF, measured in pheripheral blood mononuclear cells of AD patients. Cholinesterase promotes formation of Aβ peptide causing neuroinflammation. Acetylcholinesterase inhibitors reduces Aβ load and subsequent neuroinflammation. Some adverse effects nausea, vomiting, diarrhea, anorexia, headache, syncope, abdominal pain, and dizziness ,and therefore, the discovery of more novel drug targets is needed for AD treatment.

33. Role of NMDA (N-methyl D-aspartate) receptor and antagonists • -NMDA are ionotropic glutamate receptors in CNS. • Allows entry of calcium ions into the neurons and can cause excitatory • damage. • Memantine, a noncompetitive NMDA receptor antagonist. • Used for treatment of moderate to severe AD. • Protect neurons from neuroinflammation caused by activated macrophage and • glutamate excitotoxicity. • -Memantine also reduce the production of Aβ in rat primary cortical neurons. • Memantine has nicotinic antagonistic properties that restrict its use in early phase • of AD. • Rosi et al Study: • LPS Microglia Memantine treatment Rat 4th Ventricle neuroinflamation Control Microglia

34. Role of non steroidal anti-inflammatory drugs (NSAIDs) Prolonged use of NSAIDs with nonselective COX inhibition (both 1 and 2) can have several adverse effects such as gastrointestinal hemorrhage. Selective COX2 inhibitor (Celecoxib) were tested in transgenic mouse model of AD did not showed significant improvement. Role of statins Inhibit HMG-CoA, the rate-limiting enzyme in cholesterol biosynthesis. Anti-inflammatory, antiapoptotic, immunomodulatory, antithrombotic effects that are collectively termed as ‘pleotrophic effects’. In AD brain, reduced production of a potent vasodilator ‘endothelial nitric oxide synthase’ Statins upregulating eNOS, decrease the inflammation related to Aβ deposition. Clinical trial failure.

35. Role of PPARg agonists -Activation of PPARg can be beneficial in the treatment of neuroinflammation associated with AD. -PPARg agonists (e.g. pioglitazone and rosiglitazone) can be useful. -low penetration of blood brain barrier. Use of γ secretase inhibitor to block the Aβ -mediated amyloidogenic pathways The enzyme γ secretase is involved in the final cleavage of C99 fragment to produce Aβ peptide. γ secretase inhibitor LY450139 dose-dependently decreases Aβ production in human CNS. Another placebo control phase I clinical trial with a g secretase inhibitor MK0752 has recently been carried out by Merck & Co. Inc. But result not yet published.

36. Alternative strategies Herbal, natural products, and pineal hormone melatonin Ginkgo biloba ( 은행 ): Meta analysis of those studies revealed no convincing evidence that Ginkgo biloba is effective in the treatment of dementia and cognitive impairment Huperzine A: An alkaloid compound found in the plant Huperzia serrata. AChEI activity. Some preclinical studies showed that it can protect cells from ROS-mediated damage caused by Aβ aggregation. systematic review and meta analysis study found insufficient evidence

37. Garlic compounds: Aged garlic extract and a garlic derived compound ‘s-allyl cysteine’ showed significant improvement in hippocampal-based memory tasks in transgenic mice models. Protects ROS-mediated insults. Melatonin: Aβ lowering agent and free radical scavenger. Melatonin treatment decreases the levels of both secreted APP (sAPP) and APP mRNA in cell culture and potentiate neuronal differentiation. Melatonin was found to decrease the levels of cortical Ab in mice and was also shown to protect neuronal cells from NOS mediated generation of superoxide free radicals. Melatonin is in phase II clinical trial.

38. Recent Papers Published on “ Curcumin and Alzheimer”

39. Molecular targets of curcumin in the treatment of neuroinflammation in AD Lim et al. Study: 1.Curcumin decreases Aβ -ROS related inflammation and Ab burden in APP transgenic mice. 2. Decrease IL-1b level 3. Decreases activated glial marker GFAP. 4. Decreases microglial activation in hippocampal and cortical layers of the mouse brain. 5. Decreases the level of insoluble Aβ and plaque burden in cortex and hippocampus of the transgenic mice. Frautschy et al. Study: 1. Improves synaptic transmission in old female rats by retaining synaptophysin, in curcumin diet animal, which were lost in control animals when intracerebroventricular infusion of Aβ was given to both the group.

40. Yin et al. Aβ -induced mitochondrial damage and subsequent cell death (apoptosis) in mouse cerebral endothelial cell related to BIM overproduction. AP-1 regulated BIM expression, inhibition of AP-1 reduces BIM expression. Curcumin protects CEC from Ab mediated toxicity because of its potent role in AP-1 inhibition. Piper et al. Curcumin enhances the activity of detoxifying enzymes like glutathione-S-transferase in male rats. Motterlini et al. Curcumin upregulate endothelial HO-1 gene and protein expression and protect them from peroxide mediated toxicity.

41. Begum AN, et al: Inducible nitric oxide synthase (iNOS)-mediated production of reactive nitrogen species (RNS) and ROS cause neuronal damage in AD. curcumin and tetrahydrocurcumin (THC) significantly decrease production of both iNOS protein and mRNA in transgenic mouse brain. Baum L and Ng A : Increase in the concentration of copper, zinc, and iron in Aβ plaque, play an important role in the aggregation of Aβ and subsequent ROS production metal chelation activity of curcumin and they showed that curcumin can bind with copper and iron ions and act as a chelator and suggested reduced Aβ plaque and subsequent ROS generation . Becaria A, et al: induction of NFkB can occur in the presence of copper that can be prevented by the chelation activity of curcumin

42. Giri RK et al: Aβ and Aβ (1–42) treatment can increase mRNA expression of TNF-a, MIP-1b, IL-1b, MCP- 1, and IL-8 in monocytes. curcumin reduces Aβ mediated expression of the cyrochemokines, and it also decreases the phosphorylation of ERKs and expres- sion of Egr-1 in a dose-dependent manner. Atamna and Boyle study: Aβ binds with heme with high affinity and this Aβ -heme complex acts like a peroxidase, which can cause oxidative damage in the brain ,curcumin inhibits Ab-heme peroxidase in a dose-dependent way. Suh HW et al: Aβ and glutamate-induced neurotoxicity may perhaps result from JNK activation. By suppressing JNK activation, curcumin exerts its role in preserving Neurons.

43. Peschel et al. Curcumin dose dependently increases the expression of LDL-R mRNA in HepG2 cells, which would result in a higher net uptake of LDL-cholesterol to the liver from plasma .Excessive circulatory cholesterol increase brain APOE and as already discussed, APOE may play a role in the pathogenesis of AD. Kin et al. Low dose of curcumin stimulates proliferation of neural stem cells in mouse embryonic cortical culture as well as in the hippocampus of adult mouse Stimulation of neural stem cells is due to activation of ERK and p38 kinases by the action of curcumin.

44. Curcumin increases neuronal cell viability and neurite formation at a non-toxic dose Neuronal pheochromocytoma (PC12) cells 30 nM of nerve growth factor (NGF) [15d] neuronal differentiation. curcumin. 250 nM, 500 nM, 1 µM 48h Viability Test/ Toxicity Test/Morphology Study Cell Titer Glo Assay LDH assay fixed by using 4% paraformaldehyde

45. CTG assay showed a significant increase in viability of cells treated with 250 nM and 500 nM doses of curcumin where as 1 μM dose of curcumin did not show any change in cell viability compared to the vehicle. LDH assay was performed that revealed that 250 nM and 500 nM curcumin in conditioned media sample was non-toxic to the cells. However, secreted LDH level from the cells treated with 1 µM curcumin is insignificantly higher than that of vehicle and other two doses of curcumin

46. Curcumin increases neurite formation in cultured neuronal cells Morphology pictures of the cells showed more neurite outgrowth in the cells treated with 250 nM and 500 nM of curcumin (green represents a tubulin/ whole cell body and blue represents DAPI/nuclei) than the control [A neurite refers to any projection from the cell body of a neuron.]

47. Role of curcuminoid compounds in AD Major constituents of turmeric extract are: curcumin (~4.4%), = Already disscussed desmethoxycurcumin (~ 2.4%), = potent acetylcholine esterase property bisdesmethoxycurcumin (~3.6%). = potent acetylcholine esterase property, increases the transcription of genes for MGAT3 and TLRs [Downregulation of genes such as 1, 4-mannosyl-glycoprotein 4-N-acetylglu- cosaminyltransferase (MGAT3) and toll like receptors (TLRs) in mononuclear cells in AD patients is related to insufficient clearance of Aβ from brain interstitial space]

48. http://clinicaltrials.gov/ct2/show/study/NCT00099710 Curcumin in clinical studies

49. Curcumin in clinical studies Curcumin at dose of 2000 mg/day and 4000 mg/day in patients suffering from mild to moderate AD vs. age-matched healthy controls. Looked at different AD markers like levels of Aβ and tau proteins in CSF samples. Outcome of this study has not yet been published.