ANTIHPERLIPIDEMIC DRUGS

Ashwini Hariharan
Darshani Kansara
 WHAT IS HYPERLIPIDEMIA?
Common form of dyslipidemia
Abnormally elevated levels of lipids and
lipoproteins in the blood.
Two types:
1. PRIMARY : due to genetic causes
e.g. mutation in a receptor protein
2. SECONDARY: due to underlying causes like
diabetes etc.
Common in the general population
It is a risk factor for
Cardiovascular disease (atherosclerosis)
Acute pancreatitis
 ATHEROSCLEROSIS

Hardening of arteries
Causes by high lipid
levels
Normal arteries : smooth
and unobstructed on the
inside
Hyperlipidemia induces
plaque formation
Obstruction in blood
flow
 LIPOPROTEINS

Lipids are
transported in
a protein capsule.
The size of that
capsule,
or lipoprotein,
determines its
density.
Higher the proportion
of protein to choleste
rol, higher the
density.
 TYPES OF LIPOPROTEINS:
1. High density
lipoprotein (HDL)
2. Intermediate density
lipoprotein (IDL)
3. Low density
lipoprotein (LDL)
4. Very low density
lipoprotein (VLDL)
5. Chylomicrons
6. Chylomicron
Remnants
 ANTIHYPERLIPIDEMIC DRUGS

1. HMG CoA reductase

inhibitors
2. Fibrates
3. Bile acid
sequesterants
4. Cholesterol
absorption inhibitors
5. Niacin
 HMG COA REDUCTASE INHIBITORS
HMG CoA reductase converts HMG CoA to
mevalonic acid, which is a key step in the synthesis
of cholesterol.
Statins inhibit HMG CoA reductase enzyme
Hence, they decrease cholesterol concentrations
in the cell.
Increase LDL receptors
Hence, catabolism of LDL is increased.
Examples : Atorvastatin, Fluvastatin,Lovastatin,
Pravastatin, Rosuvastatin, Simvastatin.
 FIBRATES

Inhibit lipoprotein
lipase
The hydrolysis of
triglycerides in the
plasma is increased.
Inhibit synthesis and
secretion of VLDL
Examples :
fenofibrate,
gemfibrozil, clofibrate.
 BILE ACID SEQUESTERANTS

Highly positively charged

Bind negatively charged
bile acids
Because of the large size,
the resins are not
absorbed.
The bound bile acids are
excreted in the stool.
Examples:
Colesevelam,
Colestipol,
Cholestyramine
 CHOLESTEROL ABSORPTION
INHIBITORS

Selectively inhibits the absorption of dietary and

biliary cholesterol in the small intestine
Decrease in the delivery of intestinal cholesterol to
the liver.
Increase in clearance of cholesterol from the blood.
Example : Ezetimibe
 NIACIN

Inhibits the lipoprotein secretion by

the liver
Reduce the hepatic production of
VLDL
Consequent reduction in the serum
levels of triglycerides, VLDL and LDL.
Consistently increases hdl cholesterol
SCREENING METHODS FOR
ANTIHYPERLIPIDEMICS
IN-VITRO AND EX-VIVO MODELS
In-Vitro Models:
1. Isolated enzyme assays:
Isolate enzyme from liver and intestine microsomes.
E.g. Inhibition of isolated enzyme HMG-CoA reductase in-vitro.
2. Microsomal assays:
Directly use the liver and intestinal microsomes.
E.g. In-vitro acat inhibitory activity using liver and intestinal
microsomes.
3. Cell assays:
Assays are done using cells.
E.g. Inhibition of HMG-CoA reductase in isolated liver cells.

Ex-Vivo Models:
Organ is isolated and assay is performed
E.g. Inhibition of cholesterol biosynthesis in isolated rat liver slices
 INHIBITION OF THE ISOLATED
ENZYME HMG-COA REDUCTASE

PURPOSE AND RATIONALE :

For screening purposes, comparing
the activity with standard.
Lovastatin sodium is used as standard.
PROCEDURE:

HMG-CoA Reductase enzyme obtained from Rat

liver microsomal fraction

Incubation of enzyme with HMG-CoA under

suitable conditions with test or standard

Mevalonolactone produced from HMG-CoA is

eluted
 EVALUATION:

Mean values with and without inhibitors are

compared for the calculation of inhibition.
IC50 values are calculated.
IN VITRO ACAT INHIBITORY ACTIVITY

PURPOSE AND RATIONALE:

Acyl CoA:cholestrol acyl transferase
In vitro ACAT inhibitory activity can be determined in
microsomal preparations from liver or intestine of rabbits.
PROCEDURE:

intestinal microsomes are prepared from rabbits.

chow with: 2% cholesterol + 10% safflower oil for 6 weeks.

reaction is started by the addition of [14C]oleyl CoA

[3H]Cholesteryl oleate is used as an internal standard

The band corresponding to cholesteryl esters is scraped into

scintillation vials

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.
 IN-VIVO MODELS
Correlationbetween animal models and human
pathophysiology:
Animals exhibit total low lipid levels and a
higher percentage of HDL. Humans have high
percentage of LDL. So, it is difficult to depress
the already low lipid levels in animals.
Treatment in such animals is sensitive to HDL
and in contrast to LDL in humans.
 Animal Models used for In-Vivo Models:
Based on Methods used for Disease
Induction
Transgenic Animals
Normolipidemic Animals ANIMALS:
ANIMALS: Any animal, Hyperlipidemic Animals Apo E knockout mice
generally Rats, Cockerels Human Apo A-I transgenic
Apo E-deficient mice

Hereditary
Hyperlipidemia
Induced Hyperlipidemia
ANIMALS: RICO Rats and
Rabbits

Diet induced Chemical induced

-Fats & Cholesterol -Triton
-Fructose ANIMALS: Rats
ANIMALS: Weanling Rats, -Estrogen
Adult Rats, Rabbits, Birds, ANIMALS: Birds like Hens
Hamsters, Guinea Pigs and Cockerel
Animal Models used for In-Vivo
Models: Based on Drug to be
Evaluated

Fibrates: Rats and Mice

Drugs Interfering with lipid

metabolism: Hamsters and Guinea
pigs
 HYPOLIPIDEMIC ACTIVITY IN SYRIAN
HAMSTERS- DIET INDUCED
HYPERLIPIDEMIA
PURPOSE AND RATIONALE:
- Similar lipoprotein and bile acid metabolism to
humans, than that of rats and mice.
- Has CETP (Cholesteryl Ester Transfer Protein)
activity, similarly as seen in humans.
- Easy induction of hypercholesterolemia and
hyperlipidemia.
- Easy evaluation of various parameters.
 PROCEDURE:

Animals: Male Syrian Hamsters, Weight: 95125 g, Groups: 6-15 animals each

Controls: normal:lab chow hyperlipidemic:only cholesteramine; Other groups: test

drug or standard (cholestyramine 0.1 to 2%) for 1 week

Blood sample taken

Blood sample taken during and at end of experiment

Animals Sacrificed, Liver removed, Microsomes prepared

EVALUATION:

Total cholesterol , total triglycerides,

cholesterol content of HDL, LDL and VLDL
determined.
Dose-response curves for standard and test
are prepared using serum LDL cholesterol
value of standard chow feed as maximal effect
and cholesterol control data as zero.
 TRITON-INDUCED HYPERLIPIDEMIA
PURPOSE AND RATIONALE:

Systemic administration of surfactant TRITON to mice or

rats results in BIPHASIC elevation of plasma cholesterol and
triglycerides.
Phase I: Serum triglycerides levels increases in 1-8 Hrs
and serum cholesterol levels in 24 Hrs.
Phase II: Cholesterol level decreases to normal in next 24
Hrs.
Drugs interfering with hepatic triglyceride and cholesterol
biosynthesis were active in Phase I, while drugs interfering
with cholesterol clearance, excretion and metabolism were
active in Phase II.
PROCEDURE:
Animals: Male Sprague Dawley or Wistar Rats, Weight: 250-300 g

Starve the animals

Standard , test and vehicle injected simultaneously with triton for acute studies
Or added 1-3 days before triton is injected

IV Triton WR 1339 (isooctyl-polyoxyethylene phenol) injected

Serum triglyceride cholesterol analysis

Drugs interfering with triglyceride synthesis: at 0,2,4,6 and 8 Hrs. after triton injection
Drugs interfering with cholesterol synthesis: at 0,6,24 and 48 Hrs. after triton injection
EVALUATION:
Mean values standard deviation are calculated for each group
and time interval and compared statistically with the controls.

CRITICAL ASSESSMENT OF THE METHOD:

Rather simple and rapid for detection of compounds
interfering with the synthesis and excretion of cholesterol.
Since the test is rather artificial, the results have to be
validated by other methods.
 FRUCTOSE INDUCED
HYPERTRIGLYCERIDEMIA IN RATS

PURPOSE AND RATIONALE:

Rats switched from a diet low in carbohydrates and

high in protein to a high intake of fructose, develop
an acute hypertriglyceridemia.
Compounds are tested for inhibition of this
phenomenon.
PROCEDURE:
Male Sprague Dawley rats; 200250 g; Groups of 10

a protein diet with reduced carbohydrate content

test compound or the standard (clofibrate 100 mg/kg) or the vehicle (polyethylene
glycol) by oral gavage

20% fructose solution ad libitum for 20 h

anesthetized with ether; 1.2 ml blood withdrawn by retro orbital puncture.

The blood is centrifuged for 2 min at 16 000 g.

Total glycerol is determined in the serum

EVALUATION:
The average values of total glycerol of the treated
groups are compared with the control group using
Students t-test.
INFLUENCE ON SEVERAL STEPS
OF CHOLESTEROL ABSORPTION
AND FORMATION
 Lipids and Lipoproteins:
Serum or plasma are used.
Total Hepatic Cholesterol:
By extraction from liver.
Hepatic LDL Receptor Level:
Enzyme Immune Blotting.
Inhibition of HMG Co-A Reductase/
absorption of cholesterol
Decrease in hepatic cholesterol level
increase in hepatic LDL receptor
increase hepatic cholesterol and decrease
blood LDL.
Intestinal Cholesterol Absorption:
Dual isotope feces ratio method using 3H
sitostanol and 14C cholesterol.
Absorption ratio =
14C/3H application solution -
14C/3H feces
14C/3H application solution
Determination of Bile Acid in Feces:
Extraction from pooled sample of feces.
Individual bile acids are separated and
quantified.
 INFLUENCE ON LIPOPROTEIN LIPASE
ACTIVITY

Lipoprotein Lipase activity in post-heparin

plasma is measured.
Plasma lipolytic activity=
Hepatic Lipase + Lipoprotein Lipase
activity activity
Hepatic Lipase activity is selectively blocked
using antiserum to Hepatic Lipase.
Lipoprotein Lipase activity is measured using
Glycerol tri[1-14C] oleate as substrate.
 LYMPH FISTULA MODEL FOR CHOLESTEROL
ABSORPTION

PURPOSE AND RATIONALE:

Direct evidence for an inhibitory effect on

cholesterol absorption can be obtained.

Indicates:
the duration of inhibition
the relative selectivity of the compound on the
absorption of cholesterol versus triglyceride and
phospholipids.
PROCEDURE:
Silicon rubber cannulae placed in the main mesenteric lymph duct and the
duodenum

Test group: single bolus of ACAT inhibitor; Control : bolus injection of the
vehicle

After the drug dose, a lipid emulsion infused into the duodenal cannula

Lymph collections are obtained

Total and free cholesterol are quantitated by liquid gas

chromatography.
EVALUATION:

Esterified cholesterol of lymph is determined from

difference between total and free cholesterol.

CRITICAL ASSESSMENT OF THE METHOD:

Gallo et al. (1987) found normal cholesterol absorption in
rats in which intestinal ACAT activity was significantly
reduced by ACAT-inhibitors.
This challenges the value of ACAT-inhibition at least in
normal individuals.
 RESEARCH ARTICLE:
Antihyperlipidemic activity for Salacia
chinensis root extracts in triton induced
and atherogenic diet-induced
hyperlipidemic rats

SOURCE: Indian Journal Of Pharmacology

2012; 44:88-92
 Chloroform, ethanol, aqueous and petroleum ether
extracts of Salacia Chinensis root were evaluated
for antihyperlipidemic activity in triton induced and
atherogenic diet-induced hyperlipidemic rats.
Comparison was also made with a known
antihyperlipidemic drug Simvastatin.
Normal Control: Normal Diet, Hyperlipidemic
Control: High cholesterol diet, Positive Control:
Simvastatin.
Chloroform and ethanol extracts were found to
have significant antihyperlipidemic activity as
compared to aqueous and petroleum ether extract.
A. Results for triton induced hyperlipidemic rat
model
B. Results for atherogenic diet-induced
hyperlipidemic rat model
REFERENCES:
Goodman and Gilmans The Pharmacological
basis of Therapeutics
Rang and Dale Pharmacology
Lipincotts illustrated reviews of Pharmacology
Drug discovery and evaluation by H. Gerhard
Vogel
Screening methods in pharmacology by Robert
Arnold Turner
THANK YOU!