Preclinical antihyperlipidemic screening models

1. PRECLINICAL SCREENING OF ANTIHYPERLIPIDEMIC DRUGS
Noida Institute of Engineering and Technology
(Pharmacy Institute)
Greater Noida
Presented by :-
ANMOL KANDA
M . Pharm (Pharmacology)
Submitted to :-
Dr. Saumya Das
Associate Professor
NIET (Pharmacy Institute)
Greater Noida

2. HYPERLIPIDEMIA
 It is a condition of abnormally elevated levels of any or all lipids or lipoproteins in the blood .
 Lipids are carried in plasma as lipoproteins .
 Plasma lipid concentration are dependent on the concentration of lipoproteins .
LIPOPROTIENS
 A lipoprotein is a biochemical assembly whose purpose is to transport lipid molecules in water , as
in blood or extracellular fluid .
 They have a single layer phospholipid and cholesterol outer shell with hydrophilic portion oriented
outward and lipophilic portion oriented inward .

https://healthjade.net/lipoprotein/

3. CLASSIFICATION OF LIPOPROTIENS
 Lipoproteins are classified on the basis
of their densities . Major classes of
lipoproteins are :
1. Chylomicrons or ultra low density
lipoproteins ( VLDL )
2. Very low density lipids ( VLDL )
3. Low density lipids ( LDL )
4. High density lipids ( HDL )
https://basicmedicalkey.com/lipids-lipoproteins-and-cardiovascular-disease/

4. 1. CHYLOMICRONS
 They carry triglycerides ( TGs) , from intestine
to liver , skeletal ,muscles and adipose tissue
where TG is hydrolyzed by lipoprotein lipase .
 After a large portion of TG is hydrolyzed ,
chylomicron remnants are formed and taken
up by liver thus transferring dietry fat also to
liver .
2. VERY LOW DENSITY LIPOPROTIEN ( VLDL)
 They carry TGs from liver to adipose tissue ,
and also transport endogenous ( in contrast
to chylomicrons ) phospholipids , cholesterol
and cholesteryl esters .
 They have a diameter between 30m- 80 mm
,Converted in to LDL and IDL in bloodstream
by removal of apoprotiens .

5. 3. LOW DENSITY LIPOPROTIEN ( LDL)
 Sometimes referred to as “ Bad lipoprotein “ as they are responsible for atherosclerosis progression
by oxidizing in the walls of arteries .
 Contains chiefly cholesterol .
 Carry fats and cholesterol from liver to tissue .
4. HIGH DENSITY LIPOPROTIEN (HDL)
 Referred as ‘” good lipoprotein “ as the high levels lead to low rates of atherosclerosis progression.
 Collect fat molecules from cells/tissues to the liver .
 Generally raised levels of VLDL , IDL and LDL are associated with atherogenic while HDL may be
protective as it facilitates removal of CH from tissue .

6.  ANTI-HYPERLIPIDEMIC DRUG CLASSIFICATION :Antihyperlipidemic agents or
hypolipidemic agents are a diverse group of pharmaceuticals that are used in the treatment
of high levels of fats (lipids) in the blood . They are also called lipid lowering drugs.
https://charanjeetsinghrar.blogspot.com/2017/11/pharmacological-classification-of-drugs.html

7. SCREENING MODELS
1. IN – VIVO MODELS
A. Tritan wistar rat induced hyperlipidemia
B. Cholesterol diet induced antherosclerosis in rabbit
C. Hereditary hyperlipidemia in rabbit
D. Hereditary hyper-cholesteromia in rats
E. Transgenic animal model
F. Hypolipidemic activity in Syrian hamster
2. IN – VITRO MODELS
A. Inhibition of isolated HMG-COA reductase inhibitions
B. ACAT inhibition model .

8.  IN-VIVO MODELS

9. TRITON WISTAR RAT INDUCED HYPERLIPIDEMIA
 PURPOSE : The systemic administration of the surfactant triton to rats , result in a biphasic
elevation of plasma cholesterol and triglycerides .
 REQUIREMENTS : chemicals = surfactant , triton .
animals = wistar strain male albino rats .
 PROCEDURE :
 Rat are divided into7 groups such that each group contains 6rats and kept in cages 5 days
prior to dosing .
 The animals are starved for 18 hrs with 10% aqueous solution of triton at 400mg/kg body
weight giving I.P . The test drugs and the solvent for control is administered simultaneously
with triton injection .
 After administration of triton , blood is collected by retro orbital puncture under either
anesthesia and sujected to centrifugation to obtain serum after 24 hrs or 48 hrs .

10. EVALUATION :
 Serum is analysed for
serum triglyceride ,
serum total cholesterol ,
serum high density
lipoprotein cholesterol ,
serum low density
lipoprotein cholesterol ,
serum vey low density
lipoprotein cholesterol ,
serum glucose .
 The result is evaluated by
ANOVA test and Dunnet
multiple comparison test
.

https://commons.wikimedia.org/wiki/File:Wista
r_rat.jpg

11. CHOLESTEROL-DIET INDUCED ATHEROSCLEROSIS IN RABBITS
 PURPOSE : rabbits are known to be susceptible to hypercholesterolemia and arteriosclerosis
after excessive cholesterol feeding . Therefore , this approach been chosen by to study the
effect of potential anti-arteriosclerotic drugs .
 REQUIREMENTS : Animals : White new Zealand male rabbit
 PROCEDURE :
 Group of male rabbits ( standard and control ) at an age of 8 to 10 weeks are used and
blood is withdrawn from the marginal ear vein for determination of total cholesterol , total
glycerides , and blood sugar
 The rabbits are switched from commercial food to a diet supplemented with 0.3-2%
cholesterol and kept on this regimen for a period of 10-12 weeks
 One group is kept on normal diet . During and at the end of the experiment blood is taken
for analysis .

12.  The animal sacrificed and the thoracic aorta is
removed , cleaned of surrounding tissues , and
longitudinally cut and opened for fixation with
formaldehyde .
 The tissue is strained with oil red . In animals fed a
normal diet , the aorta does not show any staining ,
where as in cholesterol – fed rabbits the aorta shows
severe atherogenic lesions .
 EVALUATION : The percentage of the intimal
surface covered by the oil red positive lesions is
calculated with a computerized plan meter.
 Statistically evaluation is performed by Dunnetts
or scheffes test .
 https://herebunny.com/care/new-zealand

13. HEREDITARY HYPERLIPEMIA IN RABBITS
 PURPOSE : To produce hereditary hyperlipidemia in rabbit. To study the effect of potential anti-
arteriosclerotic drugs .
 REQUIREMENTS :
 chemical = probucol
 animals = female DDY mice , homozygous wistar hereditary hyperlipidemic rabbits .
 PROCEDURE :
 Homozygous wistar hereditary hyperlipidemic rabbits are raised by mating heterozygous female
wistar hereditary hyperlipidemic rabbits with homozygous male wistar hereditary hyperlipidemic
rabbits.
 At 2 months of age , eight rabbits are divide into two groups ( group A and group B ). Rabbits in
group A ( two males , two females ) , are fed standard rabbit chow for 6 months .
 Rabbits in group B ( two males , two females ) were raised with rabbit chow enriched with 1 %
(wt/wt) probucol for 6 months .

14.  The amount of daily diet for each animal is restricted to 100 gm during the study period
 Sex months later ( at the age of 8 months ) , the rabbits are sacrificed and their blood and aortas
are taken for analysis.
 EVALUATION :
 Plasma levels of cholesterol is measured by the enzymatic method.
 Statistical significance is determined by the student t-test .
 The t-test compares two averages and tells if they are different from each other . The t-test also
tells how significant the difference are .

15. HYPOLIPIDEMIC ACTIVITY IN SYRIAN HAMSTERS
 PURPOSE : The lipoprotein and bile acid
metabolism of the hamster is closer to human .
 easy to handle and more human like .
 REQUIREMENTS :
 Animal : Syrian hamster
https://stock.adobe.com/in/se
arch?k=%22syrian+hamster%
22

16. PROCEDURE
 Male Syrian hamster weighting 95-125 gm at the start of experiment are randomly assigned to
form groups of 6 animals each .
 After 1 month of cholesterol rich diet they develop sub endothelial foam cells which are
precursors of fatty streaks that develops into complex plaques .
 The animals are anesthetized with diethyl ether , a blood sample is taken from the superior
venacava .
EVALUATION :
 The plasma is analyzed for total cholesterol using a colorimetric enzymatic assay .
 The cholesterol content of high density lipoprotein is determined using a precipitation kit .

17. INVITRO - MODELS

18. INHIBITION OF THE ISOLATED ENZYME HMG-COA-REDUCTASE
 PURPOSE : For screening purpose , studies on the inhibition of HMG-CoA reductase
obtained from rat liver microsomal fraction can be used .
 REQUIREMENTS : chemical : Dithiothreitol ( reducing agent ) , Animal : rats .
 The inhibitory activity of the test compound on HMGCo-A reductase is estimated with
soluble enzyme preparations obtained from the microsomal fraction of rat liver .
 HMG-CoA reductase is a rate controlling enzyme of mevalonate pathway that produces
cholesterol
 Inhibitors of HMG-CoA reduce the cholesterol production in body by inhibiting HMG-CoA .
 PROCEDURE :
 The inhibitory activity of the test compound on HMG-CoA reductase is estimated with
soluble enzyme preparations obtained from the microsomal fraction of rat liver (philipp and
Shapiro 1979).
 The enzyme reaction is carried out with 50uL partially purified HMG CoA reductase in buffer
containing Trid , EDTA , and dithiothreitol at Ph 7.5 , NADPH regenerating system .
 The final incubation volume is 200 ul .

19.  The main reaction is preceded by 20 min ,preincubation with the NADPH regeneration system at 37 Celsius , followed
by 20 min incubation at 37 Celsius of the complete samples with the test compound or the standard and stopped by
addition of 7 HCLo4 .
 After 60 min. , at room temperature , the samples are cooled in an ice –bath and neutralized by addition of 75uL 3N
POT . Acetate
 Supplementing the volume with water to 500uL , the precipitate is centrifuged and 250uL of the clear supernatant are
applied to a column ( o.6x8.0cm) OF BIORAD AG1-8 ( 100-200mesh).
 Mevalonolactone is eluted with water discarding the first 750uL and collecting the next 3, 500uL . Five hundred uL of
the eluate are used for measurement in duplicated , mixed in vials with 10 ml quickscint (Zinsser) and measured in a
liquid scintillation counter ( backman)
 The assay is generally performed in triplicate . Lovastatin sodium is used as standard .
 EVALUATION :
 The mean values with and without inhibitors are compared for the calculation of inhibition .
 IC50 values are calculated .

20. IN – VITRO ACAT INHIBITORY ACTIVITY .
 PURPOSE AND RATIONALE : IN-vitro ACAT inhibitory activity can be determined in
microsomal preparations from liver or intestine of rabbits .
 PROCEDURE :
 Hepatic or intestinal microsomes are prepared from rabbits . Prior to sacrifice , the animals
receive chow supplemented with 2% cholesterol and 10% safflower oil for 6 weeks.
 Each assay contain 0.2 mg of microsomal protein and fatty acid-poor bovine serum albumin
in KH2PO4 buffer , PH 7.4 , containing KCL , EDTA and sucrose .
 Drug dilutions are made in DMSO ( 5 ul DMSO/200ul total incubation volume ) . The
reaction is started by the addition of oleyl CoA .
 After 3 min the reaction is stopped by the addition of chloroform – methanol 2:1 [3H] ,
cholesteryl oleate is used as internal standard .
 Lipid extract are dissolved in chloroform , spotted on TCL plates (silica gel G) and developed
in hexane – petroleum ether –acetic acid 80:20:1

21.  Unlabeled , carrier cholesterol oleate is added to the internal standard to aid band visualization with iodine
vapor.
 The bands corresponding to cholesteryl esters is then scraped into scintillation vials and radioactivity is
determined by liquid scintillation spectroscopy .
 EVALUATION :
 For each compound four concentrations are evaluated in duplicate . IC50 values are
determined by performing a nonlinear least –squares fit of the data to a log dose – response
curve .

22. REFERENCE :
 https://www.hindawi.com/journals/ecam/2015/328545/
 https://www.ahajournals.org/doi/10.1161/atvbaha.107.147280
 Vogel H.G hard “Drug Discovery and Evaluation – Pharmacological Assay “ 2nd edition , spinger–verlag berlin
Heidelberg
 Tripathi K.D essentials of medical pharmacology (8th edition ) jaypee brothers medical .
 Goodman and gilman : the pharmacological basis of therapeutics (12th edition) .

23. 
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