Dyslipidemia is a medical condition that refers to an abnormal level of blood lipids. The most common type of dyslipidemia is hyperlipidemia or high lipid levels. less common form of dyslipidemia: hypolipidemia, abnormally low lipid levels. Dyslipidemias can affect any lipid parameters including LDL cholesterol levels, HDL cholesterol levels,  triglycerides, or a combination of these lipids. Two categories: Primary dyslipidemia Secondary dyslipidemia

1. SCREENING OF DYSLIPIDEMIC
DRUGS
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Presented by,
Pavana K A
HSC18MPL004
M.Pharm,Pharmacology

2. What is Dyslipidemia?
• Dyslipidemia is a medical condition that refers to an abnormal level
of blood lipids.
• The most common type of dyslipidemia is hyperlipidemia or high
lipid levels.
• less common form of dyslipidemia: hypolipidemia, abnormally low
lipid levels.
• Dyslipidemias can affect any lipid parameters
including LDL cholesterol levels, HDL cholesterol
levels, triglycerides, or a combination of these lipids.
• Two categories:
– Primary dyslipidemia
– Secondary dyslipidemia
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3. CAUSES
• Primary dyslipidemia : abnormal lipid levels that are caused by
a mutated gene or genes inherited from one or both parents.
• Individuals with primary dyslipidemias involving increased LDL
are at a high risk of developing atherosclerosis early in life,
which can lead to premature cardiovascular disease.
• Secondary dyslipidemia: is more common and occurs due to a
variety of factors involving certain aspects of your lifestyle or
certain medical conditions.
– Poor or high fat, high sugar diet, Lack of exercise
– Alcohol abuse, cigarette smoking
– Uncontrolled diabetes, Liver disease
– Beta blockers, Oral contraceptives, Anti HIV drugs
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4. TREATMENT
• Statins/HMG-COA reductase inhibitors: Rosuvastatin
Atorvastatin, Simvastatin , Lovastatin, Pravastatin, Fluvastatin.
• Bile acid resins : Colestipol, cholestyramine, colesevelam.
• Fibrates: Gemfibrozil, Fenofibrate.
• Absorption inhibitors: ezetimibe.
• Omega 3 fatty acids: Epanova (omega-3 carboxylic acids),
Lovaza (omega-3 free fatty acids), Vascepa (icosapent)​.
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5. Screening of dyslipidemics
• Triton Wistar Rat 1339 Induced hyperlipidemia
• Hypolipidemic activity in rats
• Cholesterol-diet induced atherosclerosis in rabbits
• Hereditary hyperlipemia in rabbits
• Hereditary hypercholesteromia in rat
• Transgenic animal model
• Fructose induced hypertriglyceridemia in rat
• Hypolipidemic activity in Syrian hamsters
• Effect of HMG-CoA-reductase inhibitors in vivo
• Lymph fistula model for cholesterol absorption
• Inhibition of the isolated enzyme HMG-CoA-reductase in vitro
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6. Triton Wistar Rat 1339 Induced
hyperlipidemia
PURPOSE AND RATIONALE :
The systemic administration of the surfactant Triton to mice or rats
results in a biphasic elevation of plasma cholesterol and triglycerides.
Triton inhibits lipoprotein lipase (LPL) activity and so VLDL particle
clearance.
PROCEDURE
• Male Sprague Dawley or Wistar rats weighing 200–350 g were
starved for 18 h and then injected intravenously with 200 mg/kg
Triton WR 1339 (isooctylpolyoxy-ethylene phenol)
• Serum triglycerides increases 20 times linearly within 1-8 h, serum
cholesterol levels increased sharply 2–3 times within 24 h (phase I).
• The hypercholesterolemia decreased nearly to control levels within
the next 24 h(phase II)
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7. • Acute effect of drug investigation: Test or controls administered
simultaneously with triton, if not 1-3 days before.
• Blood collection: retro orbital puncture under ether anesthesia
centrifuged to obtain serum.
• Serum triglyceride cholesterol analyses: 0, 2, 4, 6, and 8 h after Triton
injection to investigate drugs interfering with triglyceride synthesis,
and 0,6, 24, and 48 h for investigation of drugs that are effective
against cholesterol synthesis.
• hyperlipidemia is induced by inhibition of triglyceride hydrolysis by
lipoprotein lipase. Since then, lipolysis inhibition has been used to
determine hepatic VLDL secretion rates. The VLDL secretion rate can
be calculated from the increase in triglyceride and cholesterol after
the Triton injection. Drugs interfering with hepatic triglyceride and
cholesterol biosynthesis were shown to be active in phase I, and
drugs interfering with cholesterol clearance, excretion and
metabolism were active in phase II.
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8. EVALUATION:
Mean values ± standard deviation are calculated for each group
and time interval and compared statistically with the controls.
CRITICAL ASSESSMENT OF THE METHOD :
The method employing Triton hypercholesterolemia is rather simple
and rapid for detection of compounds interfering with the synthesis of
triglycerides or synthesis and excretion of cholesterol. Since the test is
rather artificial, the results have to be validated by other methods,
e.g., ex vivo measurement of liver cholesterol synthesis.
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9. Hypolipidemic Activity in Rats
PURPOSE AND RATIONALE
• Hyperlipoproteinemia with increased concentrations of cholesterol-
and triglyceride-carrying lipoproteins is considered to be the cause of
arteriosclerosis with its dual sequelae of thrombosis and infarction.
Lipoproteins are divided into 6 major classes: chylomicrons,
chylomicron remnants, VLDL , IDL (intermediate density lipoproteins),
LDL , and HDL. HDL promotes the removal of cholesterol from
peripheral cells and facilitates its delivery back to the liver. Therefore,
increased levels of HDL are desirable.
• On the contrary, high levels of VLDL and LDL promote arteriosclerosis.
LDL, especially in its oxidized form, is taken up by macrophages via a
scavenger mechanism. Therefore, anti-arteriosclerotic drugs should
reduce VLDL and LDL and/or elevate HDL.
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10. PROCEDURE
• Groups of ten male Wistar or Sprague Dawley rats weighing 180–200
g are used. For 5-8 days ,daily once in the morning the test or the
standard given (ranging from 1 to 100 mg/kg , stomach tube ,volume
of 5 ml/kg)
• The control group is given the solvent (e. g., 0.5% HEC with 0.4%
Tween 80) only.
• The body weight of each animal is recorded at the beginning and at
the end of the experiment.
• If the focus is on the effect of VLDL and triglycerides then the
animals should be non-fasted and food should be withdrawn for no
longer than 4 h before blood sampling.
• On the morning of the first day, blood samples are taken under light
isoflurane or CO2 anesthesia by retro-orbital puncture. Then, the first
dose is applied.
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11. • During the whole period, the animals have free access to food and
water. At the end of the experiment, blood samples are taken by
retro-orbital puncture. Immediately thereafter, the animals are
sacrificed and the liver removed, blotted free from blood and
weighed.
• Samples of liver are frozen in liquid nitrogen and stored at –25°C for
lipid analysis. The blood samples are centrifuged for 2 min at
16,000g. Total cholesterol and triglycerides in each blood sample are
determined.
• To estimate the serum lipoproteins two methods are available:
ultracentrifugation and liquid chromatography.
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12. • Frozen samples of liver are thawed, homogenized and aliquots are
extracted with chloroform/methanol or dichloromethane/methanol
2:1 (v/v). The extracts are purified . Solvents of aliquots of the
extracts are evaporated and dissolved in isopropanol for
determinations of cholesterol and triglycerides, using enzymatic
assays for serum diagnostics .
EVALUATION:
• Average values of body weight, cholesterol and triglycerides are
expressed as percentage of initial value for each group at the end of
the experiment. Statistical differences between the controls and the
treatment groups are evaluated by covariance analysis.
• Cholesterol is determined using Boehringer Mannheim test
combinations by the CHOD-PAP high performance method and
triglycerides by means of an enzymatic assay.
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13. MODIFICATIONS OF THE METHOD
Similar tests can be performed in various species, e. g.:
• male NMRI mice, weighing 25–30 g,
male or female C57/bl6 mice weighing 18–24 g,
• male New Zealand obese (NZO) mice, weighing 35–40 g,
• male Syrian golden hamsters, weighing 80–120 g,
• male Pirbright guinea pigs, weighing 200–250 g,
• male miniature pigs, weighing 14–22 kg.
To study long term effects, the experiments are extended for 4 weeks or
3 months.
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14. Hereditary hyperlipemia in rabbits
• Purpose and Rational: To produced hereditary hyperlipidemia in
rabbit. To study the effect of potential anti-arteriosclerotic drugs.
Procedure:
• Homozygous wistar hereditary hyperlipidemic rabbits were raised in
Kyoto by mating heterozygous or homozygous female wistar
hereditary hyperlipidemic rabbits with homozygous male Wistar
hereditary hyperlipidemic rabbits.
• At 2 months of age, eight rabbits were divided into two groups
(group A and group B). Rabbits in group A (two males, two females)
were 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.
• The amount of daily diet for each animal was restricted to 100 g
during the study period.
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15. • Water was supplied ad lib. Six months later (at the age of 8 months),
the rabbits were sacrificed and their blood and aortas were taken for
analysis.
• Serum cholesterol is increased up to 400–600 mg/dl
• The increased levels of LDL have been studied in detail
Evaluation:
• Plasma levels of cholesterol were measured by the enzymatic
method. Statistical significance was determined by the student’s t
test.
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16. Cholesterol-Diet-Induced Atherosclerosis in Rabbits
PURPOSE AND RATIONALE
• Rabbits are known to be susceptible to hypercholesterolemia and
arteriosclerosis after excessive cholesterol feeding. Therefore, this
approach has been chosen by many to study the effect of potential
anti-arteriosclerotic drugs.
PROCEDURE
• male rabbits from an inbred strain e. g., white New Zealand, at an age
of 8–10 weeks are used. Body weight variation should be as low as
possible.
• At the beginning of the experiment, blood is withdrawn from the
marginal ear vein for determination of total cholesterol, total
glycerides, and blood sugar.
• Groups of 10 animals are used for treatment with drugs or as
controls.
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17. • diet supplemented with 0.3–2% cholesterol 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. Usually, cholesterol and
triglyceride levels increase several-fold over the original values.
• The animals are sacrificed and the thoracic aorta is removed, cleaned
of surrounding tissues, and longitudinally cut and opened for fixation
with formaldehyde. The tissue is stained with oil red.
• The percentage of the intimal surface covered by the oil red positive
lesions is calculated with a computerized planimeter. In animals fed
with normal diet, the aorta does not show any staining, whereas in
cholesterol-fed rabbits the aorta shows severe atherogenic lesions.
• EVALUATION: Data are expressed as mean ± standard deviation.
Statistical evaluation is performed by Dunnett’s or Scheffé’s test. Ap-
value of < 0.05 is regarded as statistically significant.
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18. Transgenic animal model
Purpose and Rational:
• Transgenic mice lacking apolipoprotein E showed severe
hypercholesterolemia and atherosclerosis.
Procedure:
• The widely used model is the Apo E knockout mouse. These Apo E
knockout mice have spontaneously elevated plasma cholesterol
levels, and develop atherosclerosis even on regular chow within
3–4 months.
• The time dependent progression of atherosclerosis leads to
lesions similar in histopathology to those observed in humans.
This animal model is used as background for atherosclerosis
research and target validation.
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19. • Transgenic mice lacking apolipoprotein E showed severe
hypercholesterolemia and atherosclerosis over expression of
apolipoprotein E in transgenic mice reduced plasma cholesterol
and triglyceride levels, prevented hypercholesterolemia and
inhibited the formation of fatty streak lesions.
Evaluation:
• For evaluation of the anti-atherosclerotic effect, the mice were
orally treated with GW501516 for 18 weeks and atherosclerosis at
the aortic valves was determined by cross sectional lesion analysis
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20. Fructose induced hypertriglyceridemia in rat
Purpose and Rational:
• 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 weighing 200–250 g are fed over a period
of one week a diet enriched in protein with reduced carbohydrate
content.
• Groups of 10 animals are treated for 3 days daily with the test
compound or the standard or the vehicle (polyethylene glycol) by oral
gavage.
• From the second to the third day water is with held for a period of 24
h. immediately afterwards, the animals are offered 20% fructose
solution ad libitum for a period of 20 h.
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21. • 20 hr after the last application of the test compound, the animals are
anesthetized with ether and 1.2 ml blood is withdrawn by retro
orbital puncture.
• The blood was centrifuged at 6000g for 5 min and the serum
removed. The serum was analyzed for total cholesterol and
triglycerides and glycerol using a colorimetric enzymatic assay for
clinical diagnostics.
EVALUATION
• The average values of total glycerol for the treated groups are
compared with those for the control group using ANOVA.
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22. Inhibition of the isolated enzyme HMG-CoA-reductase
in vitro
Purpose and Rational:
• 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.
• The enzyme reaction is carried out with 50 μl partially purified
HMG-CoA reductase in buffer containing 25 mM Tris, 10 mM EDTA,
and 10 mM dithiothreitol at pH 7.5, 20 μl of 910 μM HMG-CoA
solution containing 100 nCi (3.7 KBq) of 14C-HMG-CoA and 20 μl of
NADPH regenerating system (5.2 × 10–2 M glucose- 6-phosphate, 1
unit glucose-6-phosphate dehydrogenase, 5.3 × 10–3 M NADP), with
the actual concentration of 50 mM NADPH. The final incubation
volume is 200 ul.
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23. • 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 75 μl 2 N HClO4.
• After 60 min at room temperature, the samples are cooled in an ice-
bath and neutralized by addition of 75 μl 3 N 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 AG1-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.
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24. Evaluation:
• The mean values with and without inhibitors are compared for the
calculation of inhibition. IC50 values are calculated.
CONCLUSION:
There is no perfect animal model that completely replicates all stages of
human atherosclerosis, yet these small animal models are a promising
entity in exploring the etiopathogenesis and regression of
atherosclerosis.
Animal model are use to find new chemical moiety of drug for
treatment of disease and find out adverse drug reaction, side effects of
drug. Animal screening is very important for developing new drug.
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26. REFERENCES
• Drug Discovery and Evaluation : Pharmacological Assays , 3rd Edition,
by H. Gerhard Vogel.
• Asian Journal of Biomedical and Pharmaceutical Sciences 1 (2) 2011,
01-14, Preclinical Evaluation Methods for Screening of Anti-
Atherosclerotic Drugs: An Overview.K.H.Bibave, P.A.Shenoy*,
S.P.Mahamuni, D.D.Bandawane, S.S.Nipate, P.D.Chaudhari .
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