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Screening Methods of Parkinson's Disease

The document provides an overview of various screening methods for Parkinson's disease, emphasizing its etiology, pathophysiology, and classification of treatments. It details in vivo and in vitro preclinical methods used for research, highlighting specific models and techniques. The major gap identified in current studies is the lack of effective approaches for curing the disease.

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SCREENING METHODS OF PARKINSON’S DISEASE M. Pharm. (PTSM-1) PRESENTED BY NEERAJ RANA NOIDA INSTITUTE OF ENGINEERING AND TECHNOLOGY (PHARMACY INSTITUTE) GREATER NOIDA Under Guidance: Dr. Saumya Das Associate Professor
OUTLINE • Introduction • Etiology • Pathophysiology • Classification • In Vivo preclinical • In Vitro preclinical Methods • Gap of the study • References M. Pharm. (PTSM-1)
INTRODUCTION Parkinson's disease (PD), which is the second most common neurodegenerative disorder after Alzheimer's disease, is firstly defined after James Parkinson. M. Pharm. (PTSM-1)
PATHOPHYSIOLOGY Parkinson's disease is primarily associated with the gradual loss of cells in the substantia nigra of the brain. This area is responsible for the production of dopamine. Dopamine is a chemical messenger that transmits signals between two regions of the brain to coordinate activity.
ETIOLOGY The etiology of Parkinson's disease (PD) has long been thought to involve both genetic and environmental factors, but until recently there has been no direct evidence to support either one as a causative factor. However, in the past 8 years six different genes have been identified as causing familial PD.
CLASSIFICATION • Dopamine analogues: Levodopa • Peripheral deoxy decarboxylase inhibitor: carbidopa, benserazide • COMT- inhibitors: Tolcapone,Entacapone • MAO-B inhibitor: Selegiline, Clogyline • Dopamine agonists: Rosipirole, Pergoline • Centre anti-cholinergic: Benzotropine, biperiden • Antihistaminic: ophenadrine, promethazine M. Pharm. (PTSM-1
IN VIVO METHOD
SCREENING MODELS IN VIVO METHODS • Tremorineand Oxotremorine Antagonism • MPTP Model Of Parkinson’s Disease • Reserpine Antagonism • Circling Behavior In Nigrostriatal Lesioned Rats • Elevated Body Swing Test • Skilled Paw Reaching In Rats • Stepping Test In Rats • Gait Analysis • Rotenone Induced Parkinson’s M. Pharm. (PTSM-1
TREMORINE AND EXOTREMORINE ANTAGONISM • PURPOSE AND RATIONALE • The muscranic agonists Tremaine and oxotremorine induce parkinsonism-like signs such as tremor, ataxia, spasticity, salivation, lacrimation and hypothermia. • These signs are antagonized by anticholinergic drugs. PROCEDURE Groups of 6-10 male NMRI mice weighing 18-22g are used. They are dosed orally with the test compound or the standard ( 5 mg/kg benzatropine mesilate) 1 h prior the administration of 0.5 mg/kg oxotremorine s.c.
• Rectal temperature is measured before administration of the compound (Basal value) and 1,2and 3 h after oxotremorine injection. Tremor is scored after oxotremorine dosage in 10 s observation periods every 15 min for 1 h. Tremor Score Absent 0 Slight 1 Medium 2 Severe 3 M. Pharm. (PTSM-1
• Salivation and Lacrimation are scored 15 and 30 min after oxotremorine injection. Absent 0 Slight 1 Medium 2 Severe 3 EVALUATION Hypothermia The differences of the body temperature after 1,2 and 3 h versus basal values are summarized for each animals in the control groups. The average values are compared statistically.
• The scores for all animals in each group at the 3 observation periods are summarized. The numbers in the treated groups are expressed as percentage of the number of the control group. Salivation and Lacrimation The scores for the both symptoms for all animals in each group are summarized at the 2 observation periods. The numbers in the treated groups are expressed as percentage of the number of the control group TREMOR M. Pharm. (PTSM-1
MODIFICATIONS OF THE METHOD • Johnson et al. (1986) Developed a procedure for quantifying whole body tremors in mice. Displacement of a free floating platform by animal movement created a change in resistance across a strain gauge. Administration of oxotremorine, 2.5 mg/kg i.p. produced numerous high – frequency, high intensity peaks within 5 min. M. Pharm. (PTSM-1
• Clement and Dyck (1989) Constructed and tested a tremor monitor that quantitates soma and oxotremorine induced tremors. The device consisted of a force transducer, from which a plastic beaker was suspended containing a mouse. The signal from the force transducer was fed into a tremor monitor and quantitated using the Applecounter from Columus Instruments. Coward et al. (1977) Recommended N- carbamoyl-2- (2,6-dichlorophenyl) acetamidine hypo chloride (LON-1954), a tremor genic agent, as alternative to oxotremorine for the detection of anti Parkinson drugs.
B. MPTP model in Monkeys • PURPOSE AND RATIONALE N- MPTP ( N-methyl-4-phenyl-1,2,3,6- tetrahydopyridine) has been shown to cause symptoms of Parkinson’s disease in exposed individuals. When administered to primates this compound causes a partial destruction of basal ganglia and a syndrome that resembles Parkinson’s diseases. M. Pharm. (PTSM-1
PROCEDURE • Burns et al. (1983) treated 8 adult rhesus monkeys weighing 5-8 kg over a period of 5-8 days with cumulative intravenous doses of N-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (N-MPTP) up to 10-18 mg/kg. These animals showed a parkinsonism like disorder (Drooling, rigidity, akinesia, postural tremors), which was reversed by the administration of L-dopa. The pathological and biochemical changes produced by N- MPTP are similar to the well established changes in patients in patients with Parkinsonism. M. Pharm. (PTSM-1
EVALUATION • The severity of parkinsonian symptoms is rated trained observers. Movements (0: normal, 1: reduced; 2: sleepy) Checking movements (0: normal, 1: reduced; 2: sleepy) Attention and blinking ( 0: normal, 1: abnormal) Posture ( 0: normal, 1: abnormal trunk, 2: abnormal trun and tail and limbs, 4: flexed posture) Balance and Coordination ( 0: normal; 1: impaired; 2: unstable, 4 ; falls) Reactions (0: normal; 1: reduced; 2: slow; 3: absent) Vocalizations ( 0: normal; 1: reduced; 2: absent) M. Pharm. (PTSM-1
MODIFICATIONS OF THE METHOD • Kebanian et al. (1992) tested a selective D1 receptor agonist with antiparkinsonian activity in MTPT treated marmosets. Domino and Sheng (1993) studied the relative potency of some dopamine agonists with varying selectivities for D1 and D2 receptors in MPTP induced hemiparkinsonian monkeys. Asin et al. (1997) tested a selective D1 receptor agonist in rats previously given unilateral 6 hydroxydopamine injections and in macaques previously given unilateral, intracarotid infusions of MPTP. M. Pharm. (PTSM-1
IN VITRO METHOD
SCREENING MODELS IN VITRO METHODS • Experiment Using Rat Strial Slices • Dopamine Stimulated Adenyl Cyclase Activity • Culture Of Substantial Nigra • Inhibition of Apoptosis In Neuroblastoma SH-SY5Y Cells • Radioligant Binding Studies For D1 And D2 Dopamine Receptor • In Vitro Neuroprotective Efficacy M. Pharm. (PTSM-1
Experiment Using Rat Strial Slices • Striatum in brain is primarily affected in parkinsonism. The release of the neurotransmitter like dopamine and acetylcholine in response to test agent serve as a good in vitro marker of its activity.Male spargue dawley rats(150-250g)are decapitated,the skull is opened. • Right and left striata are removed & placed in ice-cold Krebs solution. • The striata is cut into 0.4mm thick slices using a tissue chopper.
• The slices are kept floating for 30 min in Krebs solution & gassed with 95% O2 & 5% Co2 at room temperature. • The slices are labelled by incubating for 30 min at 37C with[3H] dopamine (5ml) & [14c] choline (2ml) in the presence of 0.15mM pargyline chloride & 0.1 mm ascorbic acid. • Labeled slices are transferred to superfusion chambers & perfused with Krebs solutions at 37C at flow rate of 0.5ml/min. • After washing & stabilization 5 min fraction of superfast are collected.
• The perfusion buffer contains 1µM nomifensine to inhibit dopamine reuptake & 10µM hemicholinium to inhibit choline uptake. • The slices are subjected to field strength to the current strength of 10-15mA/cm2 & pulse duration of 2msec at stimulation frequency of 3 Hz for 5 min. • Drugs to be tested are percent in the superfusion fluid
• The radioactivity in the superfast samples & in the tissue is determined by liquid scintillation counting. • The radiolabelled choline method makes it possible to study Ach release in vitro without inhibiting cholinesterase, thus minimizing auto inhibition of transmitter release caused by accumulation of unhydrolised of Ach. M. Pharm. (PTSM-1
DOPAMINE STUMILATED ADENYLY CYCLASE ACTIVITY • Male sprege dawlety (150-250) are decapitated and left striata are removed • Striatal tissue homogenized in chilled buffer containing 10mm imidazole, 2mm EDTA and 10% source ph 7.3 • Homogenate is centrifuged at thousand g for 10min and supernatant is recentifuged at 27000g for 20min
• The pellet obtained is washed twice and suspended in 10mM imidazole pH. 7.3 membrane prate is determine by Bradford's method using bovine serum • Adenylyl cycles activity is measured by calculating the conversion rate of (32p) ATP to (32p) cAMP • The assay is perform in 250microaL solution containing imidazole ,mgcl2 papaverine dithiothretol ATP,GTP,phosphokinase
• The reaction mixture is preincubate at 30c for 5min the reaction is initiated by adding membrane proteins and incubated for 10min • The reaction is terminated by adding stopping solution (ATP,SDS,cAMP). Formed (32p)cAMP is separated from(32P)ATP by chromatography. M. Pharm. (PTSM-1
GAP OF THE STUDY The major gap of the studies is scientists could not have found exact approaches to cure this disease. M. Pharm. (PTSM-1)
REFERENCE •A. Schrage, P. Barone, R. G. Brown et al., “Depression rating scales in Parkinson’s disease: critique and recommendations,” Movement Disorders, vol. 22, no. 8, pp. 1077–1092, 2007. •A. F. G. Lenten's, K. Dujardin, L. Marsh et al., “Apathy and anhedonia rating scales in Parkinson’s disease: critique and recommendations,” Movement Disorders, vol. 23, no. 14, pp. 2004–2014, 2008. •H. F. Kaiser, “An index of factorial simplicity,” Psychometrical, vol. 39, no. 1, pp. 31–36, 1974. •F. Raudino, “Non motor off in Parkinson’s disease,” Acta Neurological Scandinavia, vol. 104, no. 5, pp. 312–315, 2001.
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