This document describes preclinical screening models used to evaluate antihyperlipidemic drugs. It discusses in vivo models like triton-induced hyperlipidemia in rats and cholesterol-diet induced atherosclerosis in rabbits. It also discusses in vitro models like inhibition of the enzyme HMG-CoA reductase and ACAT inhibitory activity assays. The purpose of these preclinical models is to test potential lipid-lowering drugs for their ability to reduce elevated lipid levels or inhibit key enzymes prior to clinical trials.

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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