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Screening method of nootropics vikas malik

1. The document describes various methods for screening nootropic substances, including in vivo, in vitro, and molecular-level experiments. 2. Some key in vivo methods discussed are passive avoidance testing in rats/mice, active avoidance conditioning experiments, and electrophysiological studies like long-term potentiation experiments in hippocampal brain slices. 3. In vitro screening methods outlined involve measuring inhibition of acetylcholinesterase activity from rat brain tissue and butyrylcholinesterase activity from human serum. Molecular-level experiments involve analyzing effects on acetylcholine receptors and neurotransmitter release.

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SCREENING METHOD OF NOOTROPICS NOIDA INSTITUTE OF ENGINEERING AND TECHNOLOGY (PHARMACY INSTITUTE) GREATER NOIDA PRESENTED BY UNDER GUIDENCE VIKAS MALIK DR. SAUMYA DAS M.PHARMA.(PHARMACOLOGY) ASSOCIATE PROFESSOR PTSM-1 NIET,GR. NOIDA
CONTENT  INTRODUCTION 0F MEMORY  WHAT IS MEMORY ENHANCER/NOOTROPICS  CLASSIFICATION OF NOOTROPICS  IN VIVO METHOD  IN VITRO METHOD  EXPERIMENT AT MOLECULAR LEVEL
MEMORY •It is defined as the process by which information is encoded, stored and retrieved •“Memory” is a slippery concept because it is still not clear about complete, consensual notion about the physical nature of its trace. • The only sure way to grab at such phenomenon is by measuring behaviors and their modifications i.e. quantifying it in an indirect fashion. • Such approach is called “phenomenological”, and opposes itself to the so-called “mechanistic” vision, that departs from previously existent knowledge about the intrinsic machinery operating behind the phenomenon
MEMORY ENHANCERS/NOOTROPICS The substance used to enhance cognitive function. It can increase memory,motivation,mood or anything related to cognition and thought. They are referred to as “smart” drugs HISTORY - The term ‘nootropic’ was coined by DR CORNELEU GUERGEA in 1972. It is a Greek word with combination of ‘nous’-mind and ‘trepein’- to bend. In 1964,he was the first person to synthesize the first nootropic named PIRACETAM.
Classification of nootropics  RACETAM(piracetam,oxiacetam) –piracetam is first nootropic agent discovered  Choline (Acetylcholine Precursors)-(lecithin)  Acetyl cholinesterase Inhibitors (Over-The-Counter)  Huperzine A  Ampakines(aniracetam)  Herbal  Bacopa Monneri  Vinpocetine  Gingko biloba  Rhodiola rosea  Mucuna pruriens – precursor to dopamine  Gotu Kola  Other Nootropics  Sulbutiamine(vitamin b1 derivative)  Pyritinol  Citicholine  L-theanine  Picamilon  Phenylethylamine (PEA)  noopept(peptide)  Dihydroergotoxine  Piribedil
 IN VIVO METHODS  PASSIVE AVOIDANCE  ACTIVE AVOIDANCE  DISCRIMINATION LEARNING  CONDITIONED RESPONSE  STUDY IN MONKEYS  ELECTROPHYSIOLOGICAL METHODS  ANIMAL WITH MEMORY DEFICIT  IN VITRO METHOD  In vitro inhibition of acetylcholine-esterase activity in rat striatum  Inhibition of butyrylcholine-esterase activity in human serum  Cholinesterase inhibition  EXPERIMENTS AT MOLECULAR LEVEL  Molecular forms of acetyl cholinesterase from rat frontal cortex and striatum  [3H]Oxotremorine-M binding to muscarinic cholinergic receptors in rat forebrain  [3H]-N-Methylscopolamine binding in the presence and absence of Gpp(NH)p RELEASE OF [3H]ACH AND OTHER TRANSMITTERS FROM RAT BRAIN SLICES
1.PASSIVE AVOIDANCE(aversive task) It is usually employed to describe experiments in which animal learns to avoid a noxious event by suppressing a particular behavior STEP DOWN-Purpose and rationale- An animal(mouse or rat) spends most of the time close to walls and in corners. When placed on an elevated platform in the center of rectangular compartment, it steps down immediately to the floor to explore the enclosure and to approach the wall
PROCEDURE- Mice/rat of either sex used. A rectangular box (50x50cm) with electrifiable grid floor and 35 cm fits over the block. The grid floor is connected to a shock device which delivers scrambled foot shocks. A typical paradigm consists of 3 phases: Familiarization(animal is placed on platform, released after raising the cylinder and latency is measured. After 10s of exploration, it is returned to home cage) Learning(immediately after animal descends from platform an unavoidable foot shock applied- 50hz,1.5ma,1s and animal returned to home cage) 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). 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.
2.- ACTIVE AVOIDANCE Active avoidance learning is a fundamental behavioral phenomenon . As in other instrumental conditioning paradigms the animal learns to control the administration of the unconditioned stimulus by appropriate reactions to the conditioned stimulus preceding the noxious stimulus. The first stage of avoidance learning is usually escape, whereby a reaction terminates the unconditioned stimulus RUN AWAY AVOIDENCE PURPOSE AND RATIONALE A straightforward avoidance situation features a fixed aversive gradient which can be traversed by the animal. The shock can be avoided when the safe area is reached within the time allocated
PROCEDURE The same box as used in the step-through model can be used in this experiment. A loudspeaker mounted above the starbox serves for presenting the acoustic conditioned stimulus The animal is allowed to explore the whole apparatus for 5 min. The guillotine door is then closed and the animal is placed into the light starting area. After 10 s the acoustic CS is applied and the door is simultaneously opened. Shock is turned on after 5 s.The training is continued until the animal attains the criterion of 9 avoidances in 10 consecutive trials. EVALUATION •The time the animal needs to reach the safe area on is recorded. •Also, the number of errors committed is recorded
3- Electrophysiological methods LONG-TERM POTENTIATION IN HIPPOCAMPAL SLICES PURPOSE AND RATIONALE - This procedure is perhaps the most dramatic example of activity- dependent synaptic plasticity that has yet been identified in the mammalian brain . A brief tetanus to any one of a number of monosynaptic excitatory pathways in the hippocampus can enhance the amplitude of evoked responses in the tetanized pathway for hours or days thereafter. The fact that it occurs in the hippocampus has done much to stimulate interest in LTP as a synaptic model of memory PROCEDURE •Transverse slices, are cut from the hippocampus of guinea pigs which are incubated for 90–120 min in the recording chamber to allow equilibration with artificial cerebrospinal fluid. • They are submerged, placed on a nylon mesh and perfused at a flow rate of 2–2.5 ml/min with oxygenated cerebrospinal fluid having the following composition (in mM): NaCl 124, KCl 3.3, CaCl2 2.5, KH2PO4 1.25, MgSO4 2, NaHCO3 25,7,glucose 10. • The electrodes are placed into the stratum pyramidale of CA1 or CA3. • The signal is amplified and stored on magnetic discs for later analysis. •After the baseline is recorded for 10–20 min, LTP is induced by repetitive stimulation in CA1 and in CA3 at the same rate and are recorded 0, 10, 20 and 30 min after repetitive stimulation. EVALUATION The time course of LTP is registered for CA1 and CA3.The mean percent increase in the amplitude of the population spike from baseline responses after drug application is compared with controls.
4- 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.
PROCEDURE • The test apparatus consists of a small chamber connected to a larger dark chamber via a guillotine door. •In the acquisition trial the animal is placed in the illuminated compartment at a maximal distance from the guillotine door, and the latency to enter the dark compartment is measured. • Immediately after the animal enters the dark compartment, the door is shut automatically and an unavoidable foot shock is delivered. EVALUATION •The time to step-through during the learning phase is measured and the time during the retention test is measured. • In this test a prolongation of the step-through latencies is specific to the experimental situation. An increase of the step-through latency is defined as learning.
1. In vitro inhibition of acetylcholine-esterase activity in rat striatum PURPOSE AND RATIONALE The purpose of this assay is to screen drugs for inhibition of acetylcholine-esterase activity. Inhibitors of this enzyme may be useful for the treatment of Alzheimer’s disease. Acetyl cholinesterase (Ache) which is sometimes called true or specific cholinesterase, is found in nerve cells, skeletal muscle etc Its distribution in brain roughly correlates with cholinergic innervations and sub fractionation shows the highest level in nerve terminals. Recent studies have suggested that Ache inhibitors may also be beneficial in the treatment of Alzheimer’s dementia
PROCEDURE •A 2 mM stock solution of the test drug is made up in a suitable solvent and q.s. to volume with 0.5 mM DTNB (di thio nitro benzoic acid) •Drugs are serially diluted (1 : 10) such that the final concentration (in cuvette) is 10–4 M and screened for activity. •If active, IC50(half maximal inhibitory conc) values are determined from the inhibitory activity of subsequent concentrations TISSUE PREPARATION •Male Wistar rats are decapitated, brains rapidly removed, corpora striata dissected free, weighed and homogenized in 19 volumes. •A 25 μl aliquot of this suspension is added to 1 ml of the vehicle or various concentrations of the test drug and reincubate for 10 min
ASSAY •Enzyme activity is measured with the Beckman DU-50 spectrophotometer. •This method can be used for IC50 determinations and for measuring kinetic constants EVALUATION For IC50 determinations: Substrate concentration is 10 mM diluted 1 : 2 in an assay yielding a final concentration of 5 mM.DTNB concentration is 0.5 Mm yielding 0.25 mM final concentration %inhibition=slope control-slope drug x 100 slope control
2- In vitro inhibition of butyrylcholine-esterase activity in human serum PURPOSE AND RATIONALE This assay can be used in conjunction with the acetylcholine-esterase assay to determine the enzyme selectivity of various cholinesterase inhibitors. Butyrylcholine-esterase (BChE), which is called pseudocholinesterase, preferentially hydrolyzes butyrylcholine. PROCEDURE Enzyme Preparation - A vial of lyophilized human serum is reconstituted in 3 ml of distilled water. A 25 ml aliquot of this suspension is added to 1 ml of the vehicle or various concentrations of test drug and pre-incubated for 10 min at 37 °C. EVALUATION For IC50 determinations: Substrate concentration is 10 mM diluted 1 : 2 in an assay yielding a final concentration of 5 mM.DTNB concentration is 0.5 Mm yielding 0.25 mM final concentration %inhibition=slope control-slope drug x 100 slope control
EXPERIMENTS AT MOLECULAR LEVEL Molecular forms of acetyl cholinesterase from rat frontal cortex and striatum PURPOSE AND RATIONALE Different molecular forms of acetyl cholinesterase can be isolated from animal tissues. The number of forms isolated, their relative amounts and molecular characteristics depend on the tissue source and the conditions used for solubilization of the membrane bound enzyme [3H]Oxotremorine-M binding to muscarinic cholinergic receptors in rat forebrain PURPOSE AND RATIONALE The purpose of this assay is to determine the binding affinity of potential cholinomimetic drugs for muscarinic receptors in brain, using an agonist ligand
Screening method of nootropics vikas malik