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BIOLOGICAL SCREENING OF ANTI-ALZHEIMER HERBAL DRUGS : 

p Presented by: CHANDRESH DWIVEDI BIOLOGICAL SCREENING OF ANTI-ALZHEIMER HERBAL DRUGS

Contents: : 

1. INTRODUCTION: 2. SCREENING METHODS: In vitro methods : In vitro inhibition of acetylcholine-esterase activity in rat stratum In vitro inhibition of butyrylcholine-esterase activity in human serum [3H]-N-Methyl scopolamine binding in the presence and absence of Gpp(NH)p Inhibition of respiratory burst in Microglial cells/macrophages Contents:

Contents: : 

In vivo methods : Step-down Step-through Scopolamine-induced amnesia in mice. Two compartment test Up-hill avoidance 3.REFERENCES Contents:

1. INTRODUCTION: : 

German physician Alois Alzheimer, 1906. He found abnormal clumps (now called amyloid plaques) and tangled bundles of fibres (now called neurofibrillary tangles). Today, these plaques and tangles in the brain are considered signs of AD. A progressive and fatal brain disease 5 million Americans Suffer Sixth-leading cause of death in USA The brain has 100 billion nerve cells (neurons). Nerve cell networks have special jobs. Some are involved in thinking, learning and remembering 1. INTRODUCTION:

Alzheimers versus normal brain : 

Alzheimers versus normal brain

Normal versus degenerating neuron : 

Normal versus degenerating neuron

Drug therapy: : 

Cholinesterase inhibitors prevent the breakdown of acetylcholine(Donepezil) huperzine, Muira puama Memantine: regulating the activity of glutamate Vitamen E Herbal treatment: Ginkgo biloba ,Melissa officinalis & Salvia officinalis Chinese herbal medicine(Ba Wei Di Huang Wan) Bacopa monnieri: Bacomind Green tea Drug therapy:

Screening methods of Alzheimer's: : 

In vitro methods: In vitro inhibition of acetylcholine-esterase activity in rat striatum: PURPOSE AND RATIONALE : To screen drugs for inhibition of AChE(True) activity. AChE distribution in brain highest level in nerve terminals Physiological role of AChE is the rapid hydrolysis and inactivation of Ach. Inhibitors of this enzyme may be useful for the treatment of AD. Screening methods of Alzheimer's:

B. PROCEDURE: : 

Reagents: 0.05 M Phosphate buffer, pH 7.2 a) 6.85 g NaH2PO4 · H2O/100 ml distilled H2O b) 13.40 g Na2HPO4 · 7H2O/100 ml distilled H2O c) Add a) to b) until pH reaches 7.2 d) Dilute 1: 10 2. Substrate (198 mg acetylthiocholine chloride) in buffer 3. 19.8 mg 5, 5-dithiobisnitrobenzoic acid (DTNB) in buffer 4. A 2 mM stock solution of the test drug IC50 values are determined from the inhibitory activity of subsequent concentrations. B. PROCEDURE:

Tissue preparation : 

Male Wistar rats are decapitated, brains rapidly removed Corpora striata dissected free, weighed and homogenized in 19 volumes (7 mg protein/ml) of 0.05 M NaH2PO4, pH 7.2 using a Potter- Elvejhem(PE) homogenizer. A 25 µl of this suspension is added to 1 ml of the vehicle or various concentrations of the test drug and Reincubated for 10 min at 37 °C. Tissue preparation

Assay : 

Blank: 0.8 ml PO4 buffer/DTNB 0.8 ml buffer/Substrates Control: 0.8 ml buffer/DTNB/Enzyme 0.8 ml PO4 buffer/Substrate Drug: 0.8 ml buffer/DTNB/Drug/Enzyme 0.8 ml PO4 buffer/Substrate This program (Beckman DU-50 series spectrophotometer) calculates the rate of absorbance change for each cuvette. Enzyme activity is measured with the BDS. This method can be used for IC50 determinations Assay

C. EVALUATION : 

% inhibition = [(Slope control-Slope drug)/Slope control] x100 IC50 values are calculated C. EVALUATION

2. In vitro inhibition of butyrylcholine-esterase activity in human serum : 

PURPOSE AND RATIONALE : This assay can be used in conjunction with the AChE assay to determine the enzyme selectivity of various cholinesterase inhibitions. Butyrylcholine-esterase (BChE), which is sometimes called pseudo cholinesterase, preferentially hydrolyzes butyrylcholine. This enzyme is found in the highest amounts in serum, but its physiological role is not known 2. In vitro inhibition of butyrylcholine-esterase activity in human serum

B. PROCEDURE: : 

Reagents: 0.05 M phosphate buffer, pH 7.2 2. Substrate in buffer  a) 225.8 mg s-butyrylthiocholine chloride b) q.s. to 100 ml with 0.05 M phosphate buffer, pH 7.2 3. DTNB in buffer 4. Prepared stock solution of the test drug   Enzyme Preparation: Lyophilize human serum This suspension is added to 1 ml of the vehicle or various conc. of the test drug and pre-incubated for 10 min at 37°C. B. PROCEDURE:

Slide 17: 

Assay: Enzyme activity is measured with the Beckman DU-50 spectrophotometer. 3. EVALUATION: % inhibition = [(Slope control-Slope drug)/Slope control] x100 IC50 values are calculated

Responding and Interacting to Persons with Alzheimer’s Disease : 

Responding and Interacting to Persons with Alzheimer’s Disease Patient with Alzheimer’s

3. [3H] –N-Methyl scopolamine binding in the presence and absence of Gpp (NH) p : 

A. PURPOSE AND RATIONALE: G-protein-linked muscarinic receptors are converted by nucleotides from a high-affinity binding state to a low-affinity binding state for muscarinic agonists, while the binding of muscarinic antagonists to the receptor is not affected. Therefore incubation of cerebellar membranes with 5’-guanylylimidophosphate (Gpp(NH)p), the non hydrolysable analog of GTP, causes a decreased affinity of the muscarinic agonist inhibition curves when 3H-NMS is used as the ligand. The assay differentiates the interaction of muscarinic agonists and muscarinic antagonists with 3H-N-methylscopolamine (3H-NMS)-labeled receptors in cerebellar tissue 3. [3H] –N-Methyl scopolamine binding in the presence and absence of Gpp (NH) p

B. PROCEDURE: : 

Based on 3H-NMS rat brain binding assay Reagents: 1. 0.5 M Tris-HCl buffer, pH 7.4 2. 0.05 M Tris-HCl buffer, pH 7.4 3. 0.05 M Tris-HCl buffer, pH 7.4 + 2 mM MgCl2 4. 0.05 M Tris-HCl buffer, pH 7.4 + 2 mM MgCl2 + 100 μM phenylmethylsulfonyl fluoride   5. Atropine sulfate used for nonspecific binding. B. PROCEDURE:

Continue………………… : 

6. 5’-Guanylylimidodiphosphate (Gpp (NH) p) is made up to 2 mM in distilled water (50 μM). 7. 3H-N-Methylscopolamine (NMS) and diluted to 4 nM in distill water (0.1 nM). 8. Test compounds for most assays, a 10 mM stock solution is made up in a suitable solvent and serially diluted, such that the final conc. in the assay ranges from 10–4 to 10–7 M.   9. Seven conc. are used for each assay. Higher or lower conc. may be used depending on the potency of the drug. Continue…………………

Tissue preparation : 

Male Wistar rats are decapitated and their brains rapidly removed The cerebella are dissected, weighed Homogenized in 10 volumes of 0.05M Tris-HCL buffer, pH 7.4+2 mM MgCl2 +100 µM phenylmethylsulfonyl fluoride , using a potter-elvehjem glass homogenizer fitted with a Teflon pestle. The homogenate is centrifuged The pellet is resuspended in 10 volumes of 0.05M Tris-HCL buffer + 2mM MgCl2 (Buffer 3). Tissue preparation

Binding Assay : 

Tubes are incubated at 20 °C for 90 min. Bound [3H] NMS is captured by vacuum filtration.   The filters are washed three times with 5 ml aliquots of 0.05 M Tris buffer, pH 7.4. Filters are counted in 10 ml Liquiscint scintillation fluid. Binding Assay

C. EVALUATION: : 

Specific binding of [3H] NMS is the difference between total bound (in the presence of vehicle) and that bound in the presence of 1 mM atropine. Percent inhibition of specific [3H] NMS binding is calculated for each concentration of test drug and IC50 values are determined C. EVALUATION:

4. Inhibition of respiratory burst in microglial cells/macrophages : 

PURPOSE AND RATIONALE: Activated phagocytes can produce large amounts of oxygen intermediates An increased production of free radicals, which also seems important in primary degenerative dementia (Alzheimer’s disease). Inhibition of this process by drugs may indicate therapeutic value in Alzheimer’s disease. 4. Inhibition of respiratory burst in microglial cells/macrophages

B. PROCEDURE : 

Isolated cerebral cortices from new-born albino rats are stripped of the meninges, minced in 0.25% trypsin solution. Cells are plated with 10% fetal bovine serum After 7 days, cultures are vigorously agitated on a rotary shaker at 37 °C for 15 h. Glial fibrillary acid protein positive astroglia remain adherent to the flasks. The resulting cell suspension, allowed to adhere at 37 °C. B. PROCEDURE

PROCEDURE………….. : 

After a 1–3 h adhering interval, loosely adhering and suspended cells (most of which are oligodendroglia) are removed by gently shaking the flasks at room temp. The strongly adherent microglia cells are then released by vigorous shaking in medium with 0.2% trypsin. ↓ Once the majority of microglia is suspended, fetal bovine serum is added (15% final volume). ↓ After a second 1–3 h interval to allow adhesion, the medium is removed, and adhering microglia are suspended using trypsin. Final preparation shows a nearly homogeneous population of nonspecific esterase positive cells. PROCEDURE…………..

C. Flow cytometric measurement of respiratory burst: : 

Cells are suspended in Hank’s buffered saline supplemented with N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES; 5 mM, pH 7.35 ;) (HBS-HEPES) and ↓ Stored at 4 °C for a maximum of 2 h. ↓ Dihydrorhodamine 123 (DHR) is dissolved in N, N-dimethylformamide (DMF) to obtain a 1 mM stock solution. ↓ Stained for 5 min at 37 °C with DHR solution in HBS (1 mM Stock solution in DMF). ↓ C. Flow cytometric measurement of respiratory burst:

Continue……………. : 

The DHR-loaded cells are incubated with the test drug at various concentrations for 15, 25, 35, 45, and 60 min with and without Con A (100μg/ml) stimulation. ↓ The DNA of dead cells is counter stained with 10 μl of 3 mM propidium iodide solution in HBS . ↓ Two fluorescence of at least 10 000 cells/sample are measured. Rhodamine 123 green fluorescence (515–545 nm) and Propidium iodide red fluorescence (650 nm) are measured with the light from an argon laser of 488-nm wavelength Continue…………….

D. EVALUATION : 

Calculate the differences of respiratory burst activities by t-test. Note: Before each experiment the flow cytometer is calibrated with standardized yellow-green fluorescent micro spheres of 4.3 μl diameter thus ensuring the compatibility of the fluorescence values from different experimental series. D. EVALUATION

In vivo methods : : 

Step-down Method: PURPOSE AND RATIONALE: An animal (mouse or rat) placed on an elevated platform in the center of a rectangular compartment, it steps down almost immediately to the floor to explore the enclosure and to approach the wall. PROCEDURE: Mice or rats of either sex are used. A rectangular box (50 × 50 cm) with electrifiable grid floor . The grid floor is connected to a shock device which delivers foot shocks.  Three phases: In vivo methods :

Procedure………….. : 

1) Familiarization The animal is placed on the platform, released after raising the cylinder, and the latency to descend is measured. After 10 s of exploration, it is returned to the home cage. 2) Learning: foot shock is applied (50 Hz; 1.5 mA; 1 s) and the animal is returned to the home cage, 3) Retention Test: 24 h after the learning trial the animal is again placed on the platform and the step-down latency is measured. The test is finished when the animal steps down or remains on the platform (cut-off time: 60 s). Procedure…………..

Evaluation: : 

The time of descent during the learning phase and the time during the retention test is measured. A prolongation of the step-down latency is defined as learning. Evaluation:

B. Step-through : 

PURPOSE AND RATIONALE: This test uses normal behavior of mice and rats. These animals avoid bright light and prefer dim illumination When placed into a brightly illuminated space connected to a dark enclosure, they rapidly enter the dark compartment and remain there. b) PROCEDURE: Mice and rats of either sex are used Small chamber connected to a larger dark chamber via a guillotine door & illuminated with a 7W/12 V bulb. B. Step-through

c) EVALUATION: : 

The time to step-through during the learning phase is measured and the time during the retention test is measured. An increase of the step-through latency is defined as learning. c) EVALUATION:

c) Scopolamine-induced amnesia in mice : 

PURPOSE AND RATIONALE: Scopolamine produces to transient memory deficits. It has been shown to impair memory retention when given to mice shortly before training in a dark avoidance task PROCEDURE: 10 male NMRI mice weighing 26–32 g in a one-trial Five min after i.p. administration of 3 mg/kg scopolamine hydro bromide (placed in the bright part) c) Scopolamine-induced amnesia in mice

Procedure…… : 

After a brief period, the mouse enters the second darker chamber. Once inside the second chamber, the door is closed and a 1 mA, 1-s foot shock is applied through the grid floor. 24 hours later Placing the animal again in the bright chamber. The latency is measured electronically. Whereas untreated control animals enter the darker chamber in the second trial with a latency of about 250 s, treatment with scopolamine reduces the latency to 50 s. Test compounds are administered A prolonged latency indicates that the animal remembers that it has been punished and, therefore, does avoid the darker chamber. Procedure……

EVALUATION: : 

Using various doses latencies after treatment with test compounds are expressed as percentage of latencies in mice treated with scopolamine only. In some cases, straight doses-response curves can be established. EVALUATION:

REFERENCES : 

Vogel H. G. ,Drug Discovery And Evaluation:Pharmacological Assay, Springer London, 2nd Edition,2002, Pp 601-623 www.alz.org./alzheimers_disease_what_is_alzheimers.asp www.en.wlklpedla.org/wlkl/Alzheimer’s_dlsease   www.psy.fsu.edu/faculty/glendenning/14Alzheimer%92s.ppt   http://users.ipfw.edu/Blumenth/Aging/Alzheimers.pdf   http://www.raysahelian.com/alzheimer.html REFERENCES

Slide 40: 

Thank you

Procedure…………. : 

Animals that do not step through the door within a cut-off time: 90 s (mice) or 180 s (rate) are not used. Immediately after the animals enters the dark compartment, the door is shut automatically and an footshock ( 1 mA; 1 s-mice; 1.5 mA; 2 s-rate) is delivered.   The animal is then quickly removed (within 10 s) from the apparatus and put back into its home cage.  The test procedure is repeated with or without drug. The cut-off time on day 2 is 300 s (mice) or 600 s (rate), respectively. Procedure………….

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