Anti dyslipidemic agents

Presented By – Gyanendra Kumar Prajapati
1st Year M.Pharm
Department Of Pharmacology
KLE University’s College Of Pharmacy, Bengaluru
1

Dyslipidemia is a disorder of elevated levels of lipids
and/or lipoproteins in the blood, characterized by
high cholesterol, triglycerides (TGs) or both, or low
HDL levels.
- a complex disease caused by the interplay of
genetic, dietary and physiologic factors
- LDL ≥ 130mg/dl (borderline high or higher)
Diagnosis - by using fasting lipoprotein profiles and
measuring plasma levels (total cholesterol, TGs,
Lipoproteins)
Dyslipidemia
2Department of Pharmacology
Introduction

Dyslipidemia Causes
 Primary Causes
 Secondary Causes

Other secondary causes are:
 Alcohol overuse Cigarette smoking
 Inactivity
 Diabetes mellitus
 Hypertension & Obesity
 Chronic kidney disease
 Hypothyroidism, Liver disease
 Low HDL < 40mg/dl
 Age and Gender ( Men >45ys, Women >55ys )
 Other cholestatic liver diseases and primary biliary cirrhosis.
 Drugs like thiazides, retinoids, estrogens and glucocorticoids,
among others.

Dyslipidemia Symptoms
 Dyslipidemia doesn’t have symptoms at all, but it can cause other
symptomatic vascular disease, like coronary artery disease.
 Eyelid xanthelasmas, tendinous xanthomas at the elbow, knee
tendons and Achilles and arcus cornea are caused by high levels of
LDL. Acute pancreatitis is caused by high levels of TGs.
 Patients that have familial hypercholesterolemia in homozygous form
can have the above findings with planar xanthomas. Patients that
have elevation of TGs in severe condition can expect having eruptive
xanthomas over their elbow, back, trunks, knees, buttocks, feet and
hands.
 Retinal arteries and veins can have a creamy white appearance due
to the severe hypertriglyceridemia. You can also have a milky
appearance in your blood plasma when you have high lipid levels.
You can expect symptoms like paresthesias, confusion and dypsnea.

Determining Goal
 Identify presence of clinical atherosclerotic dose
(high risk)
 Determine presence of major risk factors
 Cigarette smoking
 HTN (BP≥ 140/90 or uncontrolled or on meds)
 Low HDL (<40mg/dl)
 Family history of premature CHD
 Age ( men ≥ 45 , women ≥ 55)
 Determine Framingham risk

dyslipidemia Treatment
A. Non-Pharmacologic Treatment
 Therapeutic Lifestyle Change- diet plans include foods that are low
in cholesterol and calories and trans-fat free. Foods that are sugary
and fried must be avoided. Dairy products and red meat are taken in
moderation. In order to lower their cholesterol level, it is
recommended that patients should eat fish, vegetables, nuts and
fruits.
 -Eat in smaller portions and avoid their cravings.
- 3 months trial for all patients
 Smoking Cessation
 Physical Activity
 Weight Loss
 Dietary Modification
 Reduce saturated and “trans” fats
 Increase Fiber (25g/day) and complex carbohydrates

 Diet remains cornerstone of therapy
 LDL lowering of 25%
 Exercise to increase HDL
 Cardioprotective affect
 Non-prescription agents
 Garlic (-6%)
 Soy protien (-9%)
 Vitamin E reduces efficacy of statins and niacin

Regular Exercise-
 Regular exercises help the patients in losing weight,
improve the functions of their lungs and heart and to
stabilize their blood pressure.
 Exercise routines are adjusted to fit in the patient’s ability
level. If the patient is physically able, they are encouraged
to take walk regularly and ride bicycles. Other activities like
Pilates, Yoga, Workout classes and weightlifting are also
suggested.

Lipide Based
B. Pharmacologic Treatment
Adjuvant therapy Omega-3 fatty acids (fish oil)

Statin
 Synthesis of LDL cholesterol
 LDL lowering 10-70%
 Other benefits
Reduce plasma viscosity
Decrease platelet aggregation
Decrease C-reactive protein levels
Adverse effect
-Elevated LFT – obtain LFT at baseline, routine monitoring is
necessary
-Rhabdomyolysis w/ acute renal failure and or myopathy –patient
should be instructed to report unexplained muscle pain,
tenderness, weakness, brown urine.

Bile Acid Sequestrants
 Rarely used
 Bind bile acids in the intestinal lumen
 Increased clearance of cholesterol from blood
 Poorly tolerated (GI effects, Constipation, aggravate GI
conditions – IBS, Crohn’s)
 Mild LDL lowering
 Increase HDL

Nicotinic acid (Niacin)
 Modifies plasma lipoproteins and lipids favorably
 Effects on lipids
 LDL: dec 14%
 HDL: inc 25%
 TGs: dec 30%
 Preferred agent for patients with low HDL in whom therapeutic lifestyle
changes have already been tried.
 Niacin 500-1000mg PO QHS
 Pre-treat w/ ASA 325mg PO 1-2hrs before dose to avoid ADRs(flushing,
pruritus, Gi distress).
Adverse Effect
- Flushing – taking aspirin 30 mins before aspirin, taking niacin at bedtime w/
food, avoid hot beverages, spicy foods and hot shower at time of
administration
- Hyperglycemia – caution w/ diabetes
- Upper GI Distress

Fibrates
 Regulate genes that control lipid metabolism
 Indicated for hypertriglyceridemia with low LDL
 GI disturbances
Adverse Effect
- Gallstones – increase fluid intake, d/c if gallstones are
found
- Myopathy – baseline CPK
- - Increase hepatic transaminase – monitor LFT every 3
months

Fish Oil
 Omega-3 fatty acids
 Decrease triglycerides by 20-50%
 1-4g PO QD
 Chill capsules to increase palatability
Long-term intake results in increased HDL

Dyslipidemia in Diabetes
Diabetics with dyslipidemia are diagnosed to have low HDL
cholesterol and too many triglycerides. Patients of type 2 diabetes
have higher risk of having this condition. The risk factors are having
high blood glucose, being obese, and resistant to insulin. And with
the combination of these two conditions, there is a consequence of
having poor control of their diabetes.
Those diabetics with no dyslipidemia can develop one for no
apparent reasons like kidney diseases and hypothyroidism. Patients
with dyslipidemia can develop diabetes in the future, according to
studies. Female patients that have diabetes can have higher risk of
cardiac disease because of this form.

Screening Methods Of Antidyslipidemic
Drugs
Induction of Experimental Atherosclerosis
 Cholesterol diet induced atherosclerosis in rabbits and
other species.
 Hereditary hyper-cholesterolemia in rats.
 Hereditary hyper-lipidemia in rabbits .
 Evaluation of endothelial function in rabbits with
atherosclerosis.
 Intial reactions after endothelial injury.

Influence on Lipid Metabolism
 Triton induced hyperlipidemia .
 Fructose induced hypertryglyceridemia in rats
 IV lipid tolerance test in rats
 Influence of lipoprotein lipase activity
 Influence on cholesterol absorption.

 Inhibition of Cholesterol Biosynthesis
 Determination of HMG-CoA reductase inhibitory
activity
 Inhibition of the isolated enzyme HMG-CoA
reductace in-vitro.
 Effect of HMG-CoA reductace inhibitors in vivo.

Inhibition of Cholesterol Absorption
 Inhibition of ACAT
 In vitro ACAT inhibitory activity
 In vivo tests for ACAT inhibitory activity
Interruption of Bile Acid Recirculation
 Cholestyramine binding
Inhibition of Lipid Oxidation
 Inhibition of lipid per oxidation of isolated plasma
LDL2 cells.

1.Cholesterol diet induced atherosclerosis in
rabbits and other species
Rabbits are susceptible to hypercholesterolemia and
atherosclerosis after an excessive cholesterol feeding,
so this model is chosen for studying atherosclerotic
activity.

Procedure
 Male White Newzeland rabbits (8-10 weeks)
 Blood is withdrawn (marginal ear vein)
1. Total cholesterol
2.Toltal tri-glycerides
3. Blood sugar is detected
 2 Groups (10 animals in each group)
a) Control
b) Treatment with drug

 Rabbits feed with food containing cholesterol (3-2%) for
10-12 weeks
 One group is kept with normal diet
 At the end, blood is collected and tested for Total
cholesterol, and triglyceride levels.
 Animals are sacrificed, thoracic aorta is removed, cleaned
of surrounding tissue
 Cut opened longitudinally and fixed with HCHO
 Tissue is stained with oil red
If stains +ve lesion, %age of this is calculated using
computerized planimeter
 Animals with normal feed not show any of the lesions.

Evaluation
 Data are expressed as +/- S.D
 A p-value of <0.05 is regarded as statistically
significant drug .

Hereditary hyper-cholesterolemia in rats
 RICO – Rats with Increased Cholesterol
 RICO rats (genetically HC) compared in contrast to
Zuker-Rats (non obese)
 RICO rats decreased rate of catabolism of chylomicrons
and LDL and X’s production of these LP’s
 Drugs which decrease plasma levels of chylomicrons and
LDL are studied with this method
e.g.: β-cyclodextrin

Hereditary hyper-lipidemia in rabbits
 WHHL rabbits – Watamabe heritable hyperlipidemic rabbits
(hereditary hyper-lipemic)
 These animals are used to study the development of
atherosclerosis as well as histological and functional changes of
the aorta
 At the age of 10-14 months homozygous animals exhibit an
atheromatous plaques, distributed heterogeneously over the
luminal surface of the aorta
 Serum cholesterol is increased up to 400-600mg/dl
 This is studies with in the test drug and compared with standard
drugs.

Evaluation of endothelial function in
rabbits with atherosclerosis
Cholesterol feeding of rabbits impairs the endothelial
dependent relaxation evoked by Ach in the aorta. This
phenomenon can be studied influence of vasodilators
as well as the prevention by ACE-inhibitors.

procedure
 Male white NZL rabbits are used (3-4kg)
 2 Group of animals
Control- normal diet
Cholesterol diet- (0.25-1%) and coconut oil 3%
 After several weeks serum cholesterol levels
increases to 900-1000mg/dl in cholesterol feed
group
 End, iv anesthesia using pentobarbital,
 Sacrifice the animal and complete autopsy is taken
 Proximal of thoracic aorta sectioned in to 2mm
rings, strips are suspended in 25ml organ chamber
with 7.4 buffer (370c, carbogenated )

 After 2h a stable contractile tone is achieved
 NE is added in conc of 1*108 produces a sub
maximum isotonic contraction
 Ach is added (10 fold increase than NE) relaxation by
%age decrease in contraction is recorded
 Cholesterol feed rabbit strip of aorta shows an
impaired Ach induced conc dependent relaxation than
normal once
Evaluation:
The data is expressed as mean +/- SEM & compared by
student’s t-test for unpaired data

2.
Fructose induced hypertryglyceridemia in
rats
 Rats switched from diet low in CHO & high in protein
to high in take of fructose , develop an acute
hypertryglyceridemia, compounds are tested for this
phenomenon .

Procedure
 Male SD rats weighing 200-250g are fed for one week
a diet rich in protiens with reduced carbohydrate
content; eg:- Altromin C1080 or C1009.
 Groups of ten animals are treated for 3 days daily with
the test compound or the standard (clofibrate
100mg/kg) or the vehicle (polyethylene glycol) by oral
gavage.
 From 2nd to 3rd day water is with held for a period of
24h.

 Immediately afterwards, the animals are offered 20%
fructose solution ad libitum for a period of 20 h.
 After that the animals were anesthetised with ether and
1.2ml blood withdrawn by retroorbital puncture.
 The blood is centrifuged for 2 min at 1600 g. total
glycerol is determined in the serum and also total
cholesterol.
Evaluation
The average values of total glycerol of the treated groups
are compared with the control group using student’s t-
test.

Intravenous lipid tolerance in rats
PURPOSE AND RATIONALE
 Intravenous injection of a lipid emulsion results in an
increase of triglycerides in serum.
 The lipolytic activity can be determined by measuring
lipid elimination.

PROCEDURE
 Male Wistar rats weighing 200–240 g are treated daily
 with various doses of the test compound or the vehicle for 5 days.
 On the fifth day, 2 hrs after the last administration of the test
compound, the animals are anesthetized with 125 mg/kg sodium
hexobarbital i.p.
 Then they are injected intravenously with 2 ml/kg of a 10% lipid
emulsion .
 Prior to the injection and 10, 20, 30, and 40 min thereafter blood
is withdrawn by retro-orbital puncture for determination
of triglycerides.
EVALUATION
 Peak levels as well as elimination constant and half
life are determined.

3.Inhibition of isolated enzyme HMG-CoA
reductase in vitro
 PURPOSE AND RATIONALE
 For screening purposes, studies on the inhibition of
HMG-CoA reductase obtained from rat liver
microsomal fraction can be used.

 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 50 μl partially purified HMG CoA
reductase in buffer containing Tris, EDTA, and dithiothreitol at pH 7.5,
NADPH regenerating system .
 The final incubation volume is 200 μl.
 The main reaction is preceded by 20 min preincubation with the NADPH
regenerating system at 37 °C, followed by 20 min incubation at 37 °C of the
completed samples with the test compound or the standard and stopped by
addition of HClO4.
 After 60 min at room temperature, the samples are cooled in an ice-bath and
neutralized by addition of potassium acetate.

 Supplementing the volume with water to 500 μl, the precipitate is
centrifuged and 250 μl of the clear supernatant are applied to a
column (0.6 × 8.0 cm) of BIORAD AG 1-X8 (100–200 mesh).
 Mevalonolactone is eluted with water discarding the first 750 μl
and collecting the next 3 500 μl. Five hundred μl of the eluate are
used for measurement in duplicate, mixed in vials with 10 ml
Quickscint (Zinsser) and measured in a liquid scintillation
counter (Beckman).
 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.

Effect of HMG-CoA reductace
inhibitors in vivo
 A strain of rabbits with heritable hyperlipidemia, the WHHL
strain are used. These animals develop digital xanthoma and
aortic and coronary atherosclerosis already at an early age.
 This animal is considered to be a suitable model for the
evaluation of preventive or even regressive effects of drugs on
hyperlipidemia and atherosclerosis.

PROCEDURE
 Male heterozygous WHHL rabbits weighing 1.8 to 2.5 kg at an age
between 8 and 20 weeks are used.
 The test compounds are suspended in 0.5% methylcellulose and
are administered each day orally by gavage in the afternoon to
insure an increased plasma level at night, since in man HMG-CoA
reductase activity has been found to be higher at night than
during daytime similar to the enzyme in rodents.
 The treatment is continued for 14 days.
 Blood samples are taken in the morning without previous feeding.
2 ml of blood are drawn from the outer ear vein 5 days prior to the
beginning of treatment, on days 3 and 8 of treatment and 30 days
after the end of treatment for the determination of biochemical
parameters.

In addition, 6 ml blood are drawn at the first and the last day of
treatment and 10 days after the end of treatment for
determination of biochemical parameters and lipoprotein profile.
In order to obtain serum, blood is allowed to clot at room
temperature and then centrifuged twice at 10 000 rpm.
 The following biochemical parameters are determined in non-
frozen samples (kept at 4 °C): total cholesterol, HDL-cholesterol,
triacylglycerol, as well as creatinine, total bilirubin, alkaline
phosphates, alanine amino transferase (ALAT), aspartate amino
transferase (ASAT) using commercially available kits.
EVALUATION
 The data at 5 days before beginning of treatment and of day
0 of each animal are pooled and the mean is taken as
reference value.
 Student’s paired t-test is used to calculate for each group
the significance of difference between mean values.

4.In vitro ACAT inhibitory activity
 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 contains 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 μl DMSO/200 μl 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
an internal standard.
 Lipid extracts are dissolved in chloroform, spotted on TLC
plates (silica gel G) and developed in hexane-petroleum ether-
acetic acid 80 : 20 : 1.
 Unlabeled, carrier cholesterol oleate is added to the internal
standard to aid band visualization with iodine vapor.
 The band 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.

In vivo tests for ACAT inhibitory
activity
 Most authors test the in vivo anti-atherosclerotic and
antihyperlipemic effect of ACAT inhibitors in cholesterol- fed
hypercholesterolemic animals.
PROCEDURE
 Male Sprague-Dawley rats weighing 200–225 g are fed with a
diet containing peanut oil, cholic acid and cholesterol with or
without (controls) drugs for 1 week.
 On the last day, food is removed at 8:00 A.M. and the isotopes
are administered beginning at 2:00 P.M. [3H]cholesterol (13 μ)
is given by oral gavage and [14C]cholesterol (1.5 μ) is given is
given by tail vein injection.

 The [3H]cholesterol is prepared as an emulsion by dissolving
125 mg cholesterol in 1 625 mg olive oil. The oil phase is
suspended by sonication in 25 ml of water containing 156 mg
sodium salt.
 Each animal receives 1 ml. The intravenous dose is prepared
by drying the labeled cholesterol ,and then adding warm
ethanol followed by addition of saline.
 Each animal receives 0.5 ml of this colloidal suspension. The
rats are allowed to consume their respective diets at 3:00
P.M., and are sacrificed 48 h after the isotope administration.
EVALUATION
 The percentage of an oral dose of cholesterol absorbed is
calculated from the plasma isotope ratio

 The [3H]cholesterol is prepared as an emulsion by dissolving
125 mg cholesterol in 1 625 mg olive oil. The oil phase is
suspended by sonication in 25 ml of water containing 156 mg
(sodium salt).
 Each animal receives 1 ml. The intravenous dose is prepared
by drying the labeled cholesterol ,and then adding warm
ethanol followed by addition of saline.
 Each animal receives 0.5 ml of this colloidal suspension. The
rats are allowed to consume their respective diets at 3:00
P.M., and are sacrificed 48 h after the isotope administration.
EVALUATION
 The percentage of an oral dose of cholesterol absorbed is
calculated from the plasma isotope ratio

Cholestyramine binding
 Cholesterol is metabolized in the liver by oxidation to bile
acids which undergo enterohepatic circulation.
 In the untreated state, approximately 95% of the bile acids
that are secreted are reabsorbed and returned to the liver,
while the small loss is replaced by de novo biosynthesis
from cholesterol.
 Increased excretion of bile acids with the feces increases the
rate of oxidation of cholesterol in the liver leading to a
partial depletion of the hepatic cholesterol pool.

 A compensatory increase in uptake via the LDL receptors
results in lower serum LDL levels. This can be achieved by
addition of a bile acid binding resin, e.g., cholestyramine, to
the food.
 The binding of unconjugated and conjugated bile-salt anions
can be tested in vitro.
PROCEDURE
 Rabbits weighing 2.5–3 kg are switched from standard food
to a diet containing 10–20% polymeric basic- anion
exchanging resin, e.g. cholestyramine.
 Cholesterol levels in serum are measured at the beginning
and at the end of a 4 weeks feeding period.
EVALUATION
 Cholesterol levels as means ±SD are calculated for controls
and treated animals and compared by statistical analysis.

Inhibition of lipid peroxidation of
isolated plasma LDL
 Hypercholesterolemic Watanabe rabbits are considered to be a
suitable model to study the effect of antioxidants as anti-
atherosclerotic agents .
 Plasma of Watanabe heritable hyperlipidemic (WHHL) rabbits is
used to test the inhibition of Cu2+- induced lipid peroxidation of
isolated low density lipoproteins (LDL).
PROCEDURE
 Animals of a modified Watanabe heritable hyperlipidemic rabbit
strain (Gallagher et al. 1988) are used.
 The animals are fed over a period of 12 weeks with Purina rabbit
chow diet with or without 1% of test compound or standard .

 Plasma samples are collected in Na2EDTA (0.1% final
concentration). LDL are isolated from each rabbit plasma using a
sequential ultracentrifugation technique .
 LDL are then dialyzed against phosphate buffered saline at 4 °C for
24 h.
 Lipid peroxidation is initiated by addition of CuSO4 to a final
concentration of 5 μM followed by an incubation at 37 °C for 3 h.
The reaction is stopped by adding Na2EDTA. Fifty micrograms of
LDL from the reaction mixture are added to trichloroacetic acid
and vortexed.
 Finally, 1.5 ml of thiobarbituric acid (TBA) is added and the
mixture is incubated at 90 °C for 30 min.
 Samples are centrifuged at 1 500 rpm for 10 min. The absorbance of
the supernatant fractions is determined at 532 nm to estimate the
content of lipid peroxides

 A standard curve of malondialdehyde is generated using
malondialdehyde bis (dimethyl acetal) as reference to
determine the lipid peroxidation content in Cu2+-treated
LDL.
EVALUATION
 The content of lipid peroxide in LDL is plotted against the
drug concentration in LDL fractions. The extent of Cu2+-
induced peroxidation decreases with increasing drug
concentrations. The effects of test compounds are compared
to the standard.

Reference
2.Vogel H.Gerhard “ Drug Discovery and Evaluation-
Pharmacological Assay’’ 2nd edition, Spinger-Verlag.Berlin
Heidelberg,
3.Turner.r.A; Herborn.P. “Screening Methods in
Pharmacology”Academic Press New York,

Thank You!!!!!

Anti dyslipidemic agents

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