Best protocol competition: Second prize

Pharmacological investigation for prevention and /or treatment of Alzheimer's disease with investigational drug/s: A methodic study protocol






By:
Dr.Nishan Mathias
Post Graduate (MD Pharmacology)








Department of Pharmacology,
Father Muller Medical College,
Kankanady, Mangalore – 575002.

Introduction:
Alzheimer’s disease is an age-dependent, progressive, neurodegenerative disorder characterized by multiple cognitive deficits, gradual and an irreversible disturbance in calcium homeostasis, which is often, accompanied by behavioral disorders and mood changes (Qiu et al., 2009). Recent data indicates that in the year 2005, around 24 million people suffered from dementia. Further estimation are that the number may increase to 42 million by 2020 and 81 million by 2040; assuming no change in mortality, and any effective prevention strategies or curative treatment is invented (Ferri et al, 2005).
With enhance in longevity of most populations, the prevalence of Alzheimer's disease is likely to increase, imposing greater social and economic burdens on society and healthcare systems (Ferri et al, 2005). Alzheimer's and other dementias are projected to show a 66% increase of disability adjusted life years from 2005 to 2030 (WHO, 2009)
The cardinal features of Alzheimer’s disease (AD) include formation of extracellular protein deposits in the brain, consisting predominantly of aggregates of -amyloid protein (senile plaques of 39- 43 amino acid peptide), neurofibrilary tangles (hyperphosphorylated tau protein) in the intracellular compartments, disturbances in calcium homeostasis and degeneration/loss of synapses and neurons (Behl et al., 1994).
-Amyloid peptide has been shown to induce oxidative stress and inflammation in the brain, which are postulated to play important roles in the pathogenesis of Alzheimer's disease (Behl, 1999). -Amyloid plaque induces the production of hydrogen peroxide and lipid peroxide in neurons (Behl et al., 1994) and in turn the accumulation and precipitation of proteins may be aggravated by oxidative damage. This may in turn cause more oxidative damage by interfering with the function of the proteasome which then parallely increases the levels of oxidative damage not only to proteins but also to other biomolecules (Halliwell, 2001). A high incidence of RNA and DNA strand breaks are also reported in patients with Alzheimer's disease (Adamec et al., 1999; Nunomura et al., 2009).
Alzheimer's disease has tremendous impact on the affected individuals, caregivers, and society in both developed and developing nations. This has made it imperative to investigate the genetic and molecular basis for the causes and progression of Alzheimer's disease; and also on the search for pharmacological agent that can interfere with the pathological progress (Qiu et al., 2009). In clinics, the control of Alzheimer’s disease is ensued by enhancing the cholinergic activities of the brain (Brunto et al., 2006). Precursors of acetyl choline like choline chloride and phosphatidyl choline were used. However their clinical efficacies are limited. The use of anti-cholinesterase like physostigmine is also limited by its short duration of action and systemic cholinergic effects.
Recently, the use of lecithin, tacrine, donepazil, rivastigmine and galactamine is common. Donepazil has a long half life, hence once daily dosing is better in compliance than that of osfrivastigmine and galactamine which are to be given twice daily. Nephrotoxicity is not seen with rivastigmine, donepazil and galactamine. However, the use of tacrine is associated with adverse effects on the gastro intestinal tract like abdominal cramps, diarrhea, vomiting etc. Menantine causes dose dependent blockade of NMDA receptors with mild adverse effects (Brunto et al., 2006). In milieu of these observations it is realized that the present day therapies are far from the optimal justification and therefore studies are on to discover effective non toxic drug/s.

Objective of the study:
Dementia is a mental disorder characterized by loss of intellectual ability, sufficiently severe to interfere with one's occupational or social activities. Studies have shown that these disorders result form deterioration of neurons or their myelin sheath which over time lead to dysfunction and disabilities. Current estimates are that more than 25 million people in the world today are affected by dementia (Qiu et al., 2009).
Alzheimer's disease is the most common form of dementia, accounting for approximately 70% of the dementia cases in most industrialized countries (Qiu et al., 2009). The current treatment protocol and pharmacological agents are observed to be not uniformly effective and are associated with inherent toxicity. Therefore the call of time is to investigate and develop a drug that is uniformly active and is devoid of untoward effects. Here in the proposal the standard experimental models and the assay points needed to be evaluated would be enlisted with suitable justification.

In vitro models of Alzheimer's disease (Choi et al., 2007; Kumaria and Tolias, 2008; Apel et al., 2009): Organotypic explant (primary) cultures of hippocampal and ventral mesencephalic neurons from mice and rats have been used for studying the effect of investigational drugs. The cells are grown as per the standard tissue culture procedures and then treated with amyloid beta peptide.
The standard protective drug/s as well as the experimental investigational drug under inestigation is added either before (for preventive effective) or after the addition of amyloid beta peptide (for therapeutic effect). At the end of the experimental time schedule (1, 3, 5, 7 or 30 days) the assays need to be performed to understand both early and late effects. Assay end points like the cell viability, cytotoxicity, clonogenecity, microscopic observation, apoptosis and the biochemical and molecular markers/targets may be assayed as per the standard protocols (Apel et al., 2009).
Note: Immortalized neural cell lines and continuous primary cultures (like the neural stem cells; NSC-34, PC 12) may also be used instead of the primary cultures as it is experimentally less cumbersome and does not involve sacrifice of pregnant or new born mice/rats, which at times can be traumatic to the investigator/s (Choi et al., 2007; Kumaria and Tolias, 2008).

In vivo models of Alzheimer's disease (Manzano et al., 2009; Raghavendra et al 2009): Animal models aim to replicate the symptoms, the lesions or the cause(s) of Alzheimer's disease and also allow in designing new therapeutic approaches. After in vitro studies it is mandatory to observe similar results in animals and accordingly the study may be performed with lab mouse/rats. The most commonly used Alzheimer's disease animal model is by the intracerabral administration of -amyloid peptide in to mice. The pathogenesis and molecular cascade ensued by -amyloid peptide is akin to the clinico-pathological manifestations. Later, studies with transgenic animals will be more appropriate (explained later).

Experimental groups:
1. Normal animals (no treatment at all).
2. Animals + Saline only (vehicle of Alzheimer's-inducing agent -amyloid peptide)
3. Animals + -amyloid peptide dissolved in saline.
4. Animals + -amyloid peptide dissolved in saline + standard protective drug (Positive control, preferably a clinically used marketed drug).
5. Animals + -amyloid peptide dissolved in saline + Test drug/s (at least four concentrations, preferably 1/50th, 1/25th, 1/10th and 1/5th, of LD50)
6. Animals + Saline only + 1/5th, of LD50 (to check for whether experimental drug has any inherent toxic effects).

Note: Animal study/s is to be taken up after having the due permission from CPCSEA and the institutional ethical committee permission.


ASSAY END POINTS TO BE STUDIED:
Behavioral studies
1. Open-field test
2. Elevated plus-maze test
3. Porsolt's swim test
4. Learned helplessness test
(4a) Inescapable shock pretreatment
(4b) Conditioned avoidance training
5. Morris' water maze test
6. Y-maze test
7. Passive avoidance test

Biochemical analysis:
1. Assessment of antioxidant status: Total thiols, glutathione, protein thiol, vitamin
E levels, super oxide dismutase, catalase and glutathione peroxidase.
2. Assessment of Phase I enzymes: Cytochrome p450 and Cytochrome b5.
3. Assessment of Phase II enzymes: Glutathione S transferase, UDP-glucuronosyl
transferases, DT diaphorase and NAD(P)H-quinone oxidoreductase 1 (NQO1).
4. Assessment of levels of Nitric oxide and the levels of Nitric oxide synthase.
5. Assessment of macromolecular damage: Lipid peroxidation, Diene formation
and protein carbonyl contents.
6. Acetylcholinesterase activity.
7. Neuroamine studies by HPLC for GABA, serotonin, acetylcholine etc.


Cytological analysis:
1. Normal hemotoxylin and eosin histopathology studies
2. Immunohistochemistry for
The -Amyloid peptide (end point for quantitative reduction and
localization of plaques),
Apoptosis (end point for estimating the cell death)
Protein oxidation (DNPH reactive to understand protein oxidation)
Hydrogen peroxide (surrogate end point for quantitative reduction in
oxidative stress)
8-Oxo-7, 8-dihydroguanosine (Quantitative DNA damage).
3. Comet assay for quantative estimation of DNA damage.

Statistical analysis: The results of means ± S.D will be tabulated and data will be analyzed using Duncan’s multiple range tests. The statistical significance of differences among groups will be calculated by one-way ANOVA.

EXTENSION OF THE STUDIES:
If the experimental drug/s is observed to be effective in the chemical-induced model of Alzheimer's disease, the best dose may be proceeded with for detailed mechanistic studies in the transgenic animals adopting the earlier route and schedule. Literature studies show that there are genetic knockout mice based on the metabolism of the amyloid precursor protein; based on Presenilin (transgenic mice over expressing the wild-type or the mutant ones); the double mutants Presenilin / amyloid precursor protein that develop lesions earlier; based on tau protein (tau-JNPL3); the triple transgenic Presenilin / amyloid precursor protein/ tau-JNPL3; altered expression of neprilysin, the main degrading enzyme of Aß and models based on Apolipoprotein E (Manzano et al., 2009). The whole process of drug development is summarized in the preceding figure.



REFERENCES:
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Apel C, Forlenza OV, de Paula VJ, Talib LL, Denecke B, Eduardo CP, Gattaz WF (2009). The neuroprotective effect of dental pulp cells in models of Alzheimer's and Parkinson's disease. J Neural Transm. 116: 71-8.
Behl C (1999). Alzheimer's disease and oxidative stress: implications for novel therapeutic approaches. Prog. Neurobiol. 57: 301-323.
Behl C, Davis J, Lesley R and Schubert D (1994). Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77: 817-827.
Brunto LL, Lazo SJ, Parker KK (2006). The Pharmacological basis of therapeutics (Goodman and Gilman’s. Eleventh edition, McGraw Hill, Chicago USA.
Choi SJ, Kim MJ, Heo HJ, Hong B, Cho HY, Kim YJ, Kim HK, Lim ST, Jun WJ, Kim EK, Shin DH (2007). Ameliorating effect of Gardenia jasminoides extract on amyloid beta peptide-induced neuronal cell deficit. Mol Cells 24:113-8.
Ferri CP, Prince M, Brayne C, Brodaty M, Fratiglioni L, Ganguli M et al., (2005) Global prevelance of dementia: a Delphi concensus study. Lancet 366:2112-17
Halliwell B (2001). Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment. Drugs Aging 18: 685-716.
Kumaria A and Tolias CM (2008). In vitro models of neurotrauma. Br J Neurosurg. 22: 200-6.
Manzano S, González J, Marcos A, Payno M, Villanueva C, Matías-Guiu J (2009). Experimental models in Alzheimer's disease. Neurologia. 24: 255-62.
WHO (2009), Neurological disorders: Public health challenges – Chapter 2, Global burden of neurological disorders, estimates and projections.
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