PROJECT REPORT

on

ASSOCIATION STUDY OF CYP2D6 GENE POLYMORPHISM IN HYPERTENSIVE PATIENTS

Submitted in partial fulfillment of the requirements for the award of the degree of BACHELOR OF TECHNOLOGY
in

BIOTECHNOLOGY
by

GEETHA SAARUNYA :( 10904556) DEEPMALA PANDEY :( 10904542) under the guidance of Mr. R. Balaji Raja (M.Tech) (Ph.D)
(Lecturer, Department of Biotechnology)

DEPARTMENT OF BIOTECHNOLOGY SCHOOL OF BIOENGINEERING FACULTY OF ENGINEERING AND TECHNOLOGY SRM UNIVERSITY KATTANKULATHUR 603 203 April 2008

CERTIFICATE

Certified that the project report entitled ASSOCIATION STUDY OF CYP2D6 GENE POLYMORPHISM IN HYPERTENSIVE PATIENTS submitted by GEETHA SAARUNYA (10904556) and DEEPMALA PANDEY (10904542) is a record of project work done by them under my supervision . This project has not formed the basis for the award of any degree, diploma, associateship or fellowship.

INTERNAL GUIDE

HEAD OF THE DEPARTMENT

For the purpose of viva voce

1.

2.

DECLARATION

I do hereby declare that the project report entitled ASSOCIATION STUDY OF CYP2D6 GENE POLYMORPHISM IN HYPERTENSIVE PATIENTS is a record of original work carried out by me under the supervision of Mr. R. Balaji Raja, Lecturer, Department of Biotechnology, SRM University, Kattankulathur. This project has not been submitted earlier in part or full for the award of any degree, diploma, associateship or fellowship.

Kattankulathur

GEETHA SAARUNYA

Date-

DECLARATION

I do hereby declare that the project report entitled ASSOCIATION STUDY OF CYP2D6 GENE POLYMORPHISM IN HYPERTENSIVE PATIENTS is a record of original work carried out by me under the supervision of Mr. R. Balaji Raja, Lecturer, Department of Biotechnology, SRM University, Kattankulathur. This project has not been submitted earlier in part or full for the award of any degree, diploma, associateship or fellowship.

Kattankulathur

DEEPMALA PANDEY

Date-

ACKNOWLEDGEMENT

The Success of any project largely depends on the guidance and encouragement of many people. We take this opportunity to thank our HOD, Dr. Kantha .D.Arunachalam, for giving us a chance to translate all that we have learnt during our course period into a practical project experience. We would also like to thank, Dr.A.Ganesh, Professor and Head, Department of Genetics, Madras University, Institute of Basic Medical Sciences, for giving us a chance to work on such an interesting project at his department. We would like to thank, Ms Padmaja, Research Scholar student at the Department of genetics, Madras University, for her time and efforts in guiding and helping us, complete the project on time. My heartfelt gratitude goes out to our internal guide, Mr. R. Balaji Raja, for pushing us to perform our best and trying to think on other different aspects of our project. The guidance and the support that we received from all the members were vital for the success of the project. We are grateful for their constant support and help.

Kattankulathur

GEETHA SAARUNYA

DEEPMALA PANDEY Date-

INDEX

SL.NO. 1 2 3 4 5 6 7 8

CONTENTS INTRODUCTION REVIEW METHODOLOGY PROCEDURE RESULT DISCUSSION SUMMARY REFERENCES

PAGE NO. 1 5 29 33 38 39 40 41

INTRODUCTION

High Blood Pressure, also called hypertension, is a risk factor for heart, kidney and disease. About one in every four Indian adults over 50 years has high blood

cerebral pressure.

Hypertension has been aptly called the silent killer, because it usually produces no symptoms. Sometimes breathlessness on exertion, headache or giddiness could be the only symptoms. Since blood is carried from the heart to all of your body's tissue and organs in vessels called arteries, blood pressure is the force of the blood pushing against the walls of those arteries. In fact, each time the heart beats (about 60-70 times a minute at rest), it pumps out blood into the arteries. The blood pressure is at its greatest when the heart contracts and is pumping the blood. This is called systolic pressure. When the heart is at rest, in between beats, your blood pressure falls. This is the diastolic pressure. Blood pressure is always given as these two numbers, systolic and diastolic pressures. Both are important. Usually they are written one above or before the other, such as 120/80mm Hg, with a top number the systolic, and the bottom the diastolic. Hypertension is a case of chronic (long lasting) elevation in either systolic or diastolic blood pressure. Though either systolic or diastolic blood pressure elevations can qualify someone for hypertension, systolic hypertension generally causes more serious problems, including heart, kidney, and vascular complications. Untreated BP gradually increases the increased BP and could suddenly lead to Brain hemorrhage, Heart attack or Heart failure. The chronically elevated BP damages the eyes, predispose as individual to coronary artery disease also causes cardiac enlargement. The most dreadful complication of untreated BP is renal failure (both kidneys fails to function). In rare cases (less than one percent of hypertensive patients), the blood pressure rises quickly (with diastolic pressure usually rising to 130 or higher), resulting in malignant or accelerated hypertension. This is a life threatening condition and must be treated immediately. Symptoms may include drowsiness confusion, headache, nausea, and loss of vision.

Some people have blood pressure that stays up all or most of the time. If untreated this can lead to serious medical problems like these. ARTERIOSCLEROSIS: ("hardening of the arteries"). High blood pressure harms the arteries by making them thick and stiff. This speeds the build up of cholesterol and fats in the blood vessels like rust in a pipe, which prevents the blood from flowing through the body, and in time can lead to a heart attack or stroke. HEART ATTACK: Blood carries oxygen to the body. When the arteries that bring blood to the heart muscle become blocked, the heart cannot get enough oxygen. Reduced blood flow can cause chest pain (angina). Eventually, the flow may be stopped completely, causing a heart attack. ENLARGED HEART: High blood pressure causes the heart to work harder. Over times, this causes the heart to thicken and stretch. Eventually the heart fails to function normally causing fluids to back up into the lungs. Controlling high blood pressure can prevent this from happening. KIDNEY DAMAGE: The kidney acts as a filter to get of the wastes in rid the body. Over a number of years, high blood pressure can narrow and thicken the blood vessels of the kidney. The kidney filters less fluid, and waste builds up in the blood. The kidneys may fail altogether. When this happens, medical treatment (dialysis) or a kidney transplant may be needed. STROKE: High blood pressure can harm the arteries, causing them to narrow faster. So less blood can get to the brain. If a blood clot blocks one of the narrowed arteries, a stroke (thrombolytic stroke) may occur. A stroke can also occur when very high pressure causes a break in a weakened blood vessel in the brain (hemorrhagic stroke). Blood Pressure may vary due to various reasons and making appropriate lifestyle changes under a Doctors guidance are the initial part of any treatment plan. For most people, there are no single known causes of high blood pressure. This type of high blood pressure is called "primary" or "essential" hypertension. This type of blood pressure can't be cured, although in most cases it can be controlled. That's why it's so important for everyone to take steps to reduce their chances of developing high blood pressure. In a few people, high blood pressure can be

traced to known cause like tumors of the adrenal gland, chronic kidney disease, hormone abnormalities, use of birth control pills and Pregnancy. This is called "secondary hypertension". Secondary hypertension is usually cured if its cause passes or is corrected. here are many choices of drugs available in the market. Many patients also require additional medications to bring BP under control They are ACE inhibitors, Beta Blockers, Calcium Blockers, Diuretics, Receptor Inhibitors, Blockers etc. The very fact that there are many drugs prove that non of them are suitable in all patients your doctor depending upon you co-morbid conditions viz diabetes, asthma, heart failure or kidney failure would choose a drug ideal to suit your hypertension. Please take the medications as advised. Do not heed to non-medical, neighborhood, free advises. If you missed on dose in advertently do not take double dose on that day. Try to take the medications at a given time of the day consistently. Ideally all BP medications are to be taken within one hour of getting up from bed in the morning with a glass of water or tea/coffee. Do not stop medications without consulting your doctor.BP cannot be cured, but it can only be controlled. If your BP is consistently normal your doctor might attempt to reduce your BP medication thats all, they cannot be stopped. Our study involves the identification of polymorphism in genes in patients, treated with METOPROLOL- a beta blocker. The greatest importance on the inter individual differences in drug response at present is exerted by the differences in capacity for drug metabolism caused by genetic polymorphism or by inhibition or induction of drug metabolism. Besides, drugdrug interactions, pathophysiological factors and environmental factors, the genetics of the drug metabolizing enzymes plays a critical role for understanding inter individual differences in drug response and adverse drug reactions. CYP2D6 is perhaps the most extensively studied polymorphically expressed drug metabolizing enzyme in humans; its polymorphism has a high clinical importance and was the first among the polymorphic P450s to be characterized at the molecular level. CYP2D6 is a polypeptide of 497 amino acids. The enzyme accounts for only a small percentage of all hepatic P450s, but its role in drug metabolism is extensively higher than its relative content. The CYP2D6 gene is localized on chromosome 22q13.1. Th elocus contains two neighboring pseudo genes, CYP2D7 and CYP2D8.30 The evolution of the human CYP2D locus has involved elimination of three genes and inactivation of two (CYP2D7P and CYP2D8P) and

partial inactivation of one (CYP2D6). At present, more than 46 known different major polymorphic CYP2D6 alleles are known. The presence of the highly similar closely located pseudo genes carrying detrimental mutations have through, for example, unequal crossover reactions led to the formation of many of the variant CYP2D6 alleles, which most commonly encode defective gene products. The activity in the CYP2D locus is high as compared to, for example, the CYP2C locus and as a result many variant alleles have been formed in a relatively short period of time. The most common variant alleles distributed in different ethnic groups. The variant CYP2D6 alleles can be classified into categories, which cause abolished, decreased, normal, increased or qualitatively altered catalytic activity. Among the most important variant ones are CYP2D6*2, CYP2D6*4, CYP2D6*5, CYP2D6*10, CYP2D6*17and CYP2D6*41.

REVIEW OF LITERATURE

DEFINITION
Hypertension is a state of sustained systolic blood pressure(SBP) of greater than 140 mm Hg or a sustained diastolic blood pressure(DBP) of greater than 90 mm Hg, it is directly associated with stroke, heart disease, renal failure and vascular disease.

CLASSIFICATION OF BLOOD PRESSURE

Based upon British Hypertension Society (BHS-IV); European Society of Hypertension (2003) and WHO (1999) guidelines Blood Pressure is classified as follows: TABLE-1 Category Optimal Normal High normal Hypertension Grade I (mild) Grade II (moderate) Grade III (severe) 140-159 160-179 180 90-99 100-109 110 Systolic Blood Pressure (mm Hg) Diastolic Blood Pressure (mm Hg) <120 <130-139 130-139 <80 <85 85-89

Isolated systolic hypertension Grade I Grade II : : 140-159 160 <90 <90

PREVALENCE
Hypertension is highly prevalent in both developed and developing countries , constituting more than 30% of adult population.In USA approximately 50 million (one in four) adults have hypertension ,it accounts for more than 5% of total deaths worldwide (chobanian et al., 2003) In India hypertension is present in 25% urban and 10% rural subjects. There are 31.5 million hypertensive in rural and 34 million in urban populations. A total of 70% of these would be Stage I hypertension (systolic BP 140-159 and/or diastolic BP 90-99 mm Hg) (Gupta et al., 2004) Hypertension affects one-fifth of Chennais population, according to the Chennai Urban Rural Epidemiology Study (CURES), which screened 26,000 individuals aged >=20 years.(V.mohan et al., 2007). In developing countries prevalence rate among urban population is 1.24% in 1949 to 36.4% in 2003 (nissinen et al., 1988). In western adult population the prevalence of hypertension exceeds 20%. In black Americans, the prevalence approaches 50% of the middle aged population. Large American surveys have shown about 60% of the entire elderly population to be hypertensive.

MORTALITY PATTERN
There is a consistent and continuous relationship between BP and risk of cardiovascular disease event. The higher the blood pressure reading, the greater are the chances of getting myocardial infarction, heart failure, leftventricular hypertrophy, Ischemic attack,

Stroke and kidney disease for individuals aged 40-70 years , each increment of 20mm Hg in SBP or 10mm Hg in DBP doubles the risk of Cardiovascular disease. CVD caused 2.3 million deaths in India during the year 1990. This is projected to double by the year 2020(Gupta et al.,2004)

ETIOLOGY

Essential hypertension
Essential hypertension is a common condition which accounts for 95% of hypertension. The cause is multifactorial, there are several factors whose combined effects produce hypertension. The basic causes or underlying defects are not known. Majority of patients with essential hypertension have an increased resistance (stiffness or lack of elasticity) of the arteries (medicine net).It develops only in groups or societies that have a fairly high * Intake of salt. * Obesity. * Hereditary (genetic) susceptibility. * Kidney failure (renal insufficiency). Approximately 30% cases of essential hypertension are attributable to genetic factors. In United States, the incidence is greater among African, American than Cacaucians or Asians. Current research is focused on the genetic factors that affect the renin-angiotensin-aldosterone system.(mednet.com)

Secondary Hypertension
It accounts for 5% hypertension. This may be associated with renovascular disease (artheromatous) in older patients, fibro muscular hyperplasia in younger individuals. Also Hyperaldosterism pheochromocytoma, coarction of the aorta (narrowing of the aorta) is the most common cause of hypertension in children i.e Cushing syndrome (excessive adrenal cortical production). Metabolic syndrome and obesity i.e insulin resistance, elevated blood lipids, vascular inflammation, endothelial dysfunction (abnormal reactivity of blood vessel) (Jhone pet ire et al.,).

MANAGEMENT OF HYPERTENSION

Blood pressure should be brought below 140/90 mm Hg in all patients and below 130/80 in those with diabetes mellitus or chronic kidney disease. Most patients require more than agent to achieve these blood pressure targets. In adults without compelling indications for other agents, the initial therapy should include thiazide diuretics. Other agents appropriate for first line therapy for diastolic and/systolic hypertension includes Angiotensin-Converting Enzyme (ACE) Inhibitors (except in black people) long acting Calcium Channel Blockers (CCBs), Angiotensin Receptor Blockers(ARBs) or beta blockers (in those less than 60 years of age). First line therapy for isolated hypertension includes long acting dihydro pyridines, CCBs or ARBs. Injectable drugs may be used for emergency treatment of hypertension. (Khan N A, et al .,2007) The most commonly used agents are sodium nitroprusside and labetolol. Pregnant women may develop hypertension or may have it already before conception. These patients have an increased risk of developing preeclampsia or eclampsia (toxemia of pregnancy). Antihypertensive agents used during pregnancy need to be safe for normal foetal development. -blockers, hydralazine (vasodilators), labetolol, alpha-methyldopa and more recently CCBs have been approved as suitable medication for hypertension during pregnancy. ACE inhibitors are the first line drugs used to treat high blood pressure in cases that also involves congestive cardiac failure, chronic kidney failure in both diabetes and non diabetes and myocardial infarction. A new class of drug called vasopeptidase blocker works on two different systems at the same time. It blocks that part of rennin- angiotensin- aldosterone hormonal system and part of the bodys loss of salt regulating system that conserves salt. This class of drug reduces the blood pressure by simultaneously dilating the peripheral arteries and increasing the bodys loss of salt. One such drug is Omapatrilat currently being studied in laboratory animals with high BP. This drug reduces BP and appears to protect the organs (heart, kidney and brain) from

damage by high BP. It has not yet been approved by FDA and is undergoing further testing to evaluate its effectiveness and safety. (Khan N A, et al., 2007) -blockers remain useful medications in treating hypertension, especially in patients with tachycardia (angina) or a recent heart attack (myocardial infarction). In the heart and blood vessels, the receptors for the sympathetic nervous system that are most important are the receptors. - blocking drugs acting on this receptors in the heart slow the heart rate and reduce the force of hearts contraction Among - blockers atenolol, metoprolol and propranalol are commonly used to treat mild to moderate hypertension. (Lyngstan O. et al., 1981).

DRUG PROFILE

METOPROLOL SUCCINATE

DESCRIPTION Metoprolol succinate is a -selective (cardio selective) adrenoceptor blocking agent, for oral administration, available as extended release tablets. Metoprolol succinate Extended-Release has been formulated to provide a controlled and predictable release of metoprolol for once-daily administration. The tablets comprise of a multiple unit system containing metoprolol succinate in a multitude of controlled release pellets.

AVAILABLE STRENGTHS: The tablets contain 23.75, 47.5, 95 and 190 mg of metoprolol succinate equivalent to 25, 50, 100 and 200 mg of metoprolol tartrate, USP, respectively.

STRUCTURAL FORMULA:

CHEMICAL NAME Its chemical name is () 1-(isopropyl amino)-3-[p-(2-methoxyethyl) phenoxy]-2-propanol succinate (2:1) (salt).

MOLECULAR WEIGHT

652.8.

SOLUBILITY It is freely soluble in water, soluble in methanol, sparingly soluble in ethanol, slightly soluble in dichloromethane and 2-propanol, practically insoluble in ethyl-acetate, acetone, diethyl ether and heptane.

CLINICAL PHARMACOLOGY

Metoprolol is a -selective (cardio selective) adrenergic receptor blocking agent. Metoprolol also inhibits 2-adrenoreceptors, chiefly located in the bronchial and vascular musculature Metoprolol has no intrinsic sympathomimmetic activity, and membrane-stabilizing activity. Animal and human experiments indicate that metoprolol slows down the sinus rate and decreases AV nodal conduction. Clinical pharmacology studies have confirmed the beta-blocking activity of metoprolol in man, is shown by (1) Reduction in heart rate and cardiac output at rest and upon exercise, (2) Reduction of systolic blood pressure upon exercise, (3) Inhibition of isoproterenol induced tachycardia, and (4) Reduction of reflex orthostatic tachycardia. The relative 1-selectivity of metoprolol has been confirmed by the following: (1) In normal subjects, Metoprolol is unable to reverse the beta2-mediated vasodilating effects of epinephrine. This contrasts with the effect of nonselective beta-blockers, which completely reverse the vasodilating effects of epinephrine. (2) In asthmatic patients, Metoprolol reduces FEV1 and FVC significantly less than a nonselective beta-blocker, propranolol, at equivalent 1-receptor blocking doses.

PHARMACOKINETICS

ABSORPTION
In man, absorption of metoprolol is rapid and complete. Plasma levels following oral administration of conventional metoprolol tablets, however, approximate 50% of levels following intravenous administration, indicating about 50% first-pass metabolism.

DISTRIBUTION
Metoprolol crosses the blood-brain barrier and has been reported in the CSF in a concentration 78% of the simultaneous plasma concentration.

METABOLISM
Plasma levels achieved are highly variable after oral administration. Only a small fraction of the drug (about 12%) is bound to human serum albumin. Metoprolol is a racemic mixture of Rand S- enantiomers, and is primarily metabolized by CYP2D6. When administered orally, it exhibits stereo selective metabolism that is dependent on oxidation phenotype.

ELIMINATION
Elimination is mainly by biotransformation in the liver, and the plasma half-life ranges from approximately 3 to 7 hours. Less than 5% of an oral dose of metoprolol is recovered unchanged in the urine; the rest is excreted by the kidneys as metabolites that appear to have no -blocking activity. Following intravenous administration of metoprolol, the urinary recovery of unchanged drug is approximately 10%. The systemic availability and half-life of metoprolol in patients with renal failure do not differ to a clinically significant degree from those in normal subjects. Consequently, no reduction in dosage is usually needed in patients with chronic renal failure.

MECHANISM OF ACTION

HYPERTENSION
The mechanism of the antihypertensive effects of -blocking agents has not been elucidated. However, several possible mechanisms have been proposed: (1) Competitive antagonism of catecholamines at peripheral (especially cardiac) adrenergic neuron sites, leading to decreased cardiac output; (2) A central effect leading to reduced sympathetic outflow to the periphery; (3) And suppression of rennin activity.

COMPETITIVE ANTAGONISM
The drug usually reduces the oxygen requirement of heart at any given level of effort. This in turn helps many patients in long term management of Angina Pectoris and also lowering BP.

REDUCED SYMPATHETIC OUTFLOW

METOPROLOL is indicated in the treatment of stable, symptomatic heart failure. Even when other drugs are used, METOPROLOL decreases rate of Mortality and Hospitalization

SUPRESSION OF RENNIN ACTIVITY

One of the characteristic feature of early stage hypertension is sympathetic nervous system overactivity, as reflected by high plasma concentration of norepinephrin. Plasma rennin activity is also elevated. METOPROLOL controls rennin activity.

INDICATIONS

HYPERTENSION
Metoprolol is indicated for the treatment of hypertension. It may be used alone or in combination with other antihypertensive agents.

ANGINA PECTORIS
Metoprolol is indicated for the treatment of hypertension. It may be used alone or in combination with other antihypertensive agents.

HEART FAILURE
Metoprolol is indicated for the treatment of stable, symptomatic (NYHA Class II or III) heart failure of ischemic, hypertensive, or cardiomyopathic origin. It was studied in patients already receiving ACE inhibitors, diuretics, and in the majority of cases, digitalis. In this population, Metoprolol decreases the rate of mortality plus hospitalization, largely through a reduction in cardiovascular mortality and hospitalizations for heart failure.

DOSAGE AND ADMINISTRATION

HYPERTENSION
The usual initial dosage is 25 to 100 mg daily in a single dose, whether used alone or added to a diuretic. The dosage may be increased at weekly (or longer) intervals until optimum blood pressure reduction is achieved. In general, the maximum effect of any given dosage level will be apparent after 1 week of therapy. Dosages above 400 mg per day have not been studied.

ANGINA PECTORIS
The dosage of Metoprolol should be individualized. The usual initial dosage is 100 mg daily, given in a single dose. The dosage may be gradually increased at weekly intervals until optimum clinical response has been obtained or there is a pronounced slowing of the heart rate. Dosages above 400 mg per day have not been studied.

HEART FAILURE
The recommended starting dose of metoprolol- extended release tablet is 25 mg once daily for two weeks in patients with NYHA Class II heart failure and 12.5 mg once daily in patients with more severe heart failure.

SIDE EFFECTS

CENTRAL NERVOUS SYSTEM

Tiredness and dizziness have occurred in about 10 of 100 patients. Depression has been reported in about 5 of 100 patients. Mental confusion and short-term memory loss have been reported. Headache, somnolence, nightmares, and insomnia have also been reported.

CARDIOVASCULAR SYSTEM
Shortness of breath and bradycardia has occurred in approximately 3 of 100 patients. Cold extremities; arterial insufficiency, usually of the Reynaud type; palpitations; congestive heart failure; peripheral edema; syncope; chest pain; and hypotension have been reported in about 1 of 100 patients

RESPIRATORY SYSTEM
Wheezing (bronchospasm) and Dyspnea have been reported in about 1 of 100 patients.

GASTROINTESTINAL SYSTEM
Diarrhea has occurred in about 5 of 100 patients. Nausea, dry mouth, gastric pain, constipation, flatulence, digestive tract disorders, and heartburn have been reported in about 1 of 100 patients.

HYPERSENSITIVE REACTIONS
Pruritus or rash has occurred in about 5 of 100 patients. Worsening of psoriasis has also been reported.

POST MARKETING EXPERIENCE

CARDIOVASCULAR SYSTEM
2nd and 3rd degree heart block, carcinogenic shock in patients with acute myocardial infarction.

GASTROINTESTINAL SYSTEM
Hepatitis, vomiting.

HEMATOLOGIC SYSTEM
Thrombocytopenia.

MUSCULOSKELETAL SYSTEM
Arthralgia.

NERVOUS SYSTEM/PSYCIATRIC
Anxiety/Nervousness, hallucinations, paresthesia.

REPRODUCTIVE-MALE
Impotence.

SKIN
Increased sweating, photosensitivity, urticar Special Sense Organs, Taste disturbances.

DRUG INTERACTIONS

Catecholamine-depleting drugs {e.g, reserpine, mono amine oxidase (MAO) inhibitors} may have an additive effect when given with -blocking agents. Patients treated with Metoprolol plus a catecholamine depletor should therefore be closely observed for evidence of hypotension or marked bradycardia, which may produce vertigo, syncope, or postural hypotension. Drugs that inhibit CYP2D6 such as quinidine, fluoxetine, paroxetine, and propafenone are likely to increase metoprolol concentration. In healthy subjects with CYP2D6 extensive metabolizer phenotype, co-administration of quinidine 100 mg and immediate release metoprolol 200 mg tripled the concentration of S-metoprolol and doubled the metoprolol elimination half-life. In four patients with cardiovascular disease, co-administration of propafenone 150 mg t.i.d. with immediate release metoprolol 50 mg t.i.d. resulted in two- to five-fold increases in the steadystate concentration of metoprolol. This increase in plasma concentration would decrease the cardio selectivity of metoprolol. Digitalis glycosides and beta-blockers slow atrioventricular conduction and decrease heart rate. Concomitant use can increase the risk of bradycardia. -blockers may exacerbate the rebound hypertension which can follow the withdrawl of clonidine.

WARNING
The use of metropolol is highly warned in case of patients with following diseases/disorders: Ischemic Heart Disease Bronchospastic disease Major surgery Diabetes and hypoglycemia Thyrotoxicosis Peripheral vascular diseases Calcium channel blockers

PRECAUTIONS
Metoprolol should be used with caution in patients with impaired hepatic function. In patients with pheochromocytoma, an alpha-blocking agent should be initiated prior to the use of any beta-blocking agent.

PREGNANCY CATEGORY C
Metoprolol tartrate has been shown to increase post-implantation loss and decrease neonatal survival in rats at doses up to 22 times, on a mg/m basis, the daily dose of 200 mg in a 60-kg patient. Distribution studies in mice confirm exposure of the fetus when metoprolol tartrate is administered to the pregnant animal. These studies have revealed no evidence of impaired fertility or teratogenicity. There are no adequate and well-controlled studies in pregnant women.

OVERDOSE
SIGNS AND SYMPTOMS
Over dosage of Metoprolol may lead to severe hypotension, sinus bradycardia, atrioventricular block, heart failure, cardiogenic shock, cardiac arrest, bronchospasm, impairment of consciousness/coma, nausea, vomitting, and cyanosis.

TREATMENT
In general, patients with acute or recent myocardial infarction or congestive heart failure may be more haemodynamically unstable than other patients and should be treated accordingly. When possible the patient should be treated under intensive care conditions. On the basis of the pharmacological actions of metoprolol, the following general measures should be employed:

Elimination of the Drug: Gastric lavage should be performed. Bradycardia: Atropine should be administered. If there is no response to vagal blockade,
isoproterenol should be administered cautiously.

Hypotension: A vasopressin should be administered, e.g., levarterenol or dopamine. Bronchospasm: A 2-stimulating agent and/or a theophylline derivative should be
administered.

Cardiac Failure: A digitalis glycoside and diuretics should be administered. In shock

resulting from inadequate cardiac contractility, administration of dobutamine, isoproterenol, or glucagon may be considered.

CONTRAINDICATION
Metoprolol is contraindicated in severe bradycardia, heart block greater than first degree, cardiogenic shock, decompensated cardiac failure, sick sinus syndrome and in patients who are hypersensitive to any single component of this product. PATIENT INFORMATION Patients should be advised to take Metoprolol regularly and continuously, as directed, preferably with or immediately after meals. If a dose should be missed, the patient should take only the next scheduled dose (without doubling it). Patients should not interrupt or discontinue Metoprolol without consulting the physician. Patients should be advised (1) to avoid operating automobiles and machinery or engaging in other tasks requiring alertness until the patients response to therapy with Metoprolol has been determined; (2) to contact the physician if any difficulty in breathing occurs; (3) to inform the physician or dentist before any type of surgery that he or she is taking Metoprolol. Heart failure patients should be advised to consult their physician if they experience signs or symptoms of worsening heart failure such as weight gain or increasing shortness of breath. The antihypertensive effect of metoprolol and atenolol were compared by a double blind placebo controlled trail on 53 patients with mild to moderate hypertension. The decrease in supine systolic/diastolic BP was 18/16 mm Hg on metoprolol and 20/16 mm Hg on atenolol. This result suggests that metoprolol and atenolol were equipotent following once daily administration in patients with mild to moderate hypertension. Once daily administration of metoprolol in management of mild to moderate hypertension was tested in 161 patients. Among those, 144 patients completed the study. Hypertension was fully controlled in 84 patients, but 33 failed to respond to metoprolol therapy. The efficacy of metoprolol was not influenced by a persons sex. (Fredman R M, et al.,1982). The efficacy, tolerability and compliance of once daily slow release formulation of metoprolol were compared with twice daily metoprolol in hypertension. Blood pressure control and apparent adverse effects were similar for both the groups. Patient compliance was significantly

improved in the group receiving once daily therapy. Simplification of the dosage regimen to once daily therapy appears to improve the patients willingness to comply with the physicians instructions. (Baird M G, et al., 1984). There are large inter individual response variation to drugs, including cardiovascular drugs. Some patient may achieve the desired therapeutic response from their drug therapy, others may have no therapeutic benefit, while others may experience toxicities. There is a subset of patients who will experience adverse effects, which can range from bothersome to life threatening (Julie A. Johnson et al., 2004) Variability in drug action may be Pharmacokinetic or Pharmacodynamic. Pharmacokinetic

variability refers to variability in delivery of drugs to removal from key molecular sites of action that mediate efficacy and /or toxicity. The molecules involved in these processes include both drug-metabolizing enzymes (such as Cytochrome -P450 or CYP super family) and drug transport molecules that mediate drug uptake into and efflux from intracellular sites. Pharmacodynamic variability refers to variable drug effects despite equivalent drug delivery to molecular sites of action. This may reflect variability in the function of the molecule that a drug targets to achieve its effects. (Dan M , 2003). Patients with extreme response (often toxicity) were found to have high plasma drug concentration, which is due to the variation in the gene for the enzyme involved in that drugs metabolism. A number of drug metabolizing enzymes have inactivating mutations, which leads to absent or nonfunctional protein and for drugs with high dependence on that particular enzyme for their elimination, plasma drug concentration can be 5-10 times normal. Genetic variability in P450 gene may be structural and regulatory. Variability in regulatory region (non-coding) of the CYP2D6 gene is therapeutically relevant. It is due to many clinically important drugs that are metabolized by these enzymes, which include -adrenergic blocking agents, antiarrhythmics, psycholeptics, antidepressants and narcotic analgesics. Inter-individual response to chemical exposure is a well documented phenomenon with a myriad of underlying causes including age, health, gender, nutritional status, concomitant drug therapy and genetic factors. (Dan M. 2003).

Pharmacogenetics is a term that can be defined as the study of genetically determined variations they are revealed by the effect on drugs and other Xenobiotics. (D.A.P.Evans, 1999) Genetic polymorphism in the gene encoding for drug metabolizing enzymes depends on both the type of reaction catalyzed and the type of substrate. With drugs the consequences of a polymorphism may be toxic plasma concentration if there is a deficiency in a metabolizing enzyme, or lack of response if activation by a polymorphic enzyme is required for biological activity(in pro drugs ) of if higher than normal levels of a metabolizing enzymes result in too rapid rate of elimination. (K, Brosen, et al., 1989). The absence of a metabolizing enzyme result in toxicity will depend on various factors including the therapeutic margin of safety versus activity. The Cytochrome -P450 family of heme- containing proteins is the most important enzyme system for Phase-I catalyzed oxidative biotransformation of biologically reactive metabolites. (K, Brosen et al.,1989).The P450 enzymes are hemoprotiens with approximately m.wt of 50,000 Da. These enzymes catalyze the mono oxidation of a wide variety of structurally unrelated compounds including endogenous steroids and fatty acids and an essentially unlimited number of lipophilic xenobiotics. (M.Lawton, et al.1990). The drug metabolizing P450 enzymes are primarily present in endoplasmic reticulum, they are found in high concentration in the liver, but are present in a variety of other tissues, including lung, kidney, gastrointestinal tract, nasal mucosa, skin and brain. The enzymes of this family exist in multiple forms and has different substrate selectivity, in addition to the diversity in enzyme composition, polymorphic variations in humans are also documented.(P.K.Srivatsava ,et al.,1991). Polymorphisms have been observed in humans in the oxidative metabolism of CYP2D6, CYP2C8-10 and CYP2C19. (Mahgoub A, et al.,).Gene polymorphism in the transcription regulatory region result in an increase in the rate of gene expression .( Rato S, et al.,1992) CYP2D6 is the only functionally active enzyme of the CYP2D6 subfamily in humans. The gene encoding its synthesis is located in the CYP2D6 locus at q 13.1 on the long arm of chromosome 22. (Eichelbaum M,et al.,1987)

Genetic variability in drug metabolizing enzymes based on the enzyme activity is generally bimodal distribution of activity are termed extensive and poor metabolizes (EM and PM) or fast and slow metabolizes. (Ross A McKinnon, et al.,).CYP2D6 (debrisoquine/spartiene hydroxylase) is involved in metabolism of 25% of drugs .CYP2D6 poor metabolizes phenotype is generally present in 5-10 % of Caucasian population and less than 1% in Japan. Poor metabolizers (PM) of debrisoquine, completely lacking CYP2D6 activity because of inactivating mutations in both the alleles of the CYP2D6 gene location (22q13.1) Christoph Sachse, et al.1977. studied the allele frequencies and phenotypic consequences of CYP 2D6 variants in Caucasian population. Frequency of alleles with complete deficiency of enzyme activity were 7% of the population and Poor Metabolizers (PM) have alleles*3, *4,*5, *6, *7,*15 and *16. Frequency of alleles with slightly (CYP 2D6 *2) or moderately (*9 and*10) reduced activity (Intermediate metabolizer phenotype (IM) having CYP 2D6 *2 and *9 and *10 allele variants respectively. The phenotypes of Extensive Metabolizer (EM) have 2D6 *1 allele frequency is 3% of the population. (Julia Kirchheiner, MD, et al., 2004) reported a linear relationship between the number of active CYP 2D6 genes and metabolic clearance of metoprolol and they found that the median clearances differed by more than 10 fold between the PM and UM groups. Metoprolol pharmacodynamics differed less than 2 fold between the EM and UM groups and only a marginal difference in metoprolol efficacy on heart rate between EM and UM were reported. Poor metabolizers(PM) had the alleles of 2D6 *3, *4, *5 and *6,alleles of 2D6 9*,*10, *17 and *41 are intermediate metabolizers(IM) , alleles with normal enzymatic activity having CYP2D6 *1,*2 and* 35 and ultra rapid metabolizer (UM) having the alleles of CYP2D6 *2x1 and *2x2.(Zanger UM, et al.,2001). (R.Ismail. et al.,2005) A 100 fold variation was noted for both metoprolol and hydroxy metoptolol. Patients homozygous for CYP 2D6 *4 had the metoprolol concentration / unit dose considered as poor metabolizer. (Toscana et al., 2006) investigated the molecular basis low expression and activity of CYP2D6 associated with the CYP 2D6 *41 allele in about 10-15 % Caucasians with intermediate

metobolizer phenotype and they explained the relationship between the intron 6 SNP 2988G>A and the low expression phenotype associated with allele 2D6 *41.

GENETIC INFLUENCES ON DRUG TARGET

Pharmacodynamic variability refers to variable drug effects despite equivalent drug delivery to molecular sites of action. This may reflect variability in the function of the molecule that a drug targets to achieve its effects. (Dan M .Roden, MD) It has been shown that genetic association between drug targets and antihypertensive response for beta-blockers, diuretics, ACE inhibitors and AT1 receptor blockers. (Julie A. Johnson). In case of beta-blockers, an association between polymorphism in the -1 adrenergic receptor gene and blood pressure lowering. The two functional polymorphism of in the ADRB1 is Ser49GLy and Arg389Gly (Deboarach A Mason) In the heart and blood vessels the receptors for the sympathetic nervous system, most important are the beta- receptors. Beta blocking drugs acting on the heart slow the heart rate and force of contraction. Beta blockers remain useful medication in treating hypertension, especially in patients with tachycardia, angina pectoris or with myocardial infarction (Medicinenet. Com) The beta-1-adrenergic, a 7 transmembrane Gs protein coupled receptor expressed in the heart and other organs, mediates the actions of catecholamine in the sympathetic nervous system (Manson DA, et al., 1999) Sympathetic nervous system plays a key role in the regulation of blood pressure. It acts via neurotransmitter neither (epinephrine, nor epinephrine) on adrenergic receptors. (Heart -2007) - adrenergic receptor gene is localized in chromosome 10. ADRB1R gene is located in 10 q 2426 and consists of 447 amino acids (Frielle, T, S. Collins, k, W.Daniel et al). Alterations in adrenergic beta receptors have a role in many clinical settings. Recent data suggest a role of the increased expression of G-protein-receptor kinases in hearts of patients with congestive heart failure and for genetic polymorphism in -adrenergic receptors in patients with obesity. (Heart, 2007).

A polymorphism in the intracellular cytoplasm tail near the 7 th transmembrane, spanning segment of human ADRB1R have been identified by Ligget et al., 1999. Two polymorphic sites located on theADRB1R gene at position A145G and C1165G and the two polymorphism of 1-receptor are Ser49Gly and Arg389Gly.These polymorphism have been associated with genetic susceptibility to several types of cardiovascular diseases and cardiovascular risk factors, including obesity, hypertension, diabetes, congestive heart failure and dilated cardiomyopathy.(Feldman, M D. 2001.) Invitro studies have shown that Gly49 1-receptors are more susceptible to down

regulation,whereas as Arg389 1-adrenoceptor shows increased responsiveness to agonist stimulation. (Stephen B, et al.,).In agonist competition studies shown that Gly389 receptor does not have high affinity than Arg389 receptor. (Ligget et al) Genetic variation of 1-adrenergic receptor at this locus may be the basis of interindividual differences in phathological characteristic or in the response to therapeutic agonists and antagonists in the cardiovascular diseases.( Massino et al.,2006) The 2b, -1 and -2 adrenergic are not associated with a family history of essential hypertension. Kristina et al.,2001 suggested that individuals homozygous for the Arg389 allele of the beta-1 adrenergic receptor gene are at increased risk to develop hypertension. Subjects with the argenine 389 genotype had a higher diastolic Blood Pressure& Heart rate than carriers of 1 or 2 Gly 389 alleles. (M. Borjessson, et al) A novel polymorphism in the in the gene coding for the -adrenergic receptor associated with the survival in patients with heart failure. (M.Borjesson et al had identified a mutation in the -adrenergic receptor was associated with a decreased mortality risk in patients with congestive cardiac failure. This data suggests that the beta-receptor Ser49Gly variation might be associated with altered receptor function, resulting in patients with heart failure. (Euro. Heart .J.2000). 1-Adrenergic receptor polymorphism was associated with different blood pressure responses to metoprolol therapy in patients with essential hypertension.

49Ser389Arg/49Ser389Arg 49Ser389Arg/49Gly389Arg

and patients

49Ser389Arg/49Gly389Arg did. 49Ser389Gly/49Gly389Arg

patients and

w49Ser389Arg/49Ser389Arg patients had a larger systolic blood pressure reduction than 49Ser389Gly/49Ser389Gly patients were non-responders to metoprolol antihypertensive therapy ere good responders to metoprolol therapy; (Jie Liu, et al., 2006).

METHODOLOGY

STUDY POPULATION
Study group: The study was conducted in newly diagnosed hypertensive patients and who are not currently on antihypertensive drug therapy. All the patients included in the study were attending hypertensive clinic, Government General Hospital, Madras Medical College, Chennai. Control group: Normotensive individuals who are attending the general Medical out patient unit, GGH, MMC, Chennai.

SAMPLE SIZE
119 patients with stage-I hypertension. 116 normotensive controls.

INVESTIGATIONAL PRODUCT
Metoprolol Succinate Extended Release Tablet 25mg (Embeta-XR 25 mg) once daily. The drug is manufactured by INTAS Pharmaceuticals Private Limited and was procured commercially from the retail drug store by the investigator.

CRITERIA USED FOR THE PATIENT INCLUSION IN THIS STUDY

Ambulatory patients of either sex. Age 30-65. Newly diagnosed hypertensive patients. Systolic blood pressure: 140-160mm Hg and Diastolic blood pressure: 90-100 mm Hg. Patients willing to give written informed consent. INCLUSION CRITERIA FOR CONTROL SELECTION: Healthy individuals matched to age and sex with study subjects. Systolic blood pressure: 90-130mm Hg and Diastolic blood pressure: 60-85mm Hg. Without clinically significant renal, hepatic, gastro enterology and metabolic problems.

CRITERIA EMPLOYED FOR EXCLUDING PATIENTS FOR THE STUDY

Patients with the following conditions were excluded from the study: History of bronchospasm or asthma. Diabetes mellitus, history of thyrotoxicosis and peripheral vascular diseases. Pregnant, breast feeding or women With sitting Diastolic blood pressure >110mm Hg or malignant hypertension. Secondary hypertension. Myocardial Infarction, Cardiac surgery and any other cardiac intervention or stroke in past three months and Congestive heart failure. Under treatment with Ca2+ channel blockers and ACE inhibitors and Digoxin or Lithium. Clinically significant hepatic, renal, neurological, gastrointestinal, metabolic, hematological and psychiatric problems. Known smokers and alcoholics. Known hypersensitive to metoprolol or beta blocker. Patients receiving catecholamine depleting drugs (e.g.: Reserpine, MOA inhibitors.) Quinidine, Methadone, Fluoxetine, Fluoxamine, Paroxetine, Sertaline and Propaphenone. SGOT/SGPT values >1.5 times upper limit of the normal range (10-40 unit/ml) alkaline phosphatase (13-39 IU/L) or total serum bilirubin >1.2 (0.3- 1.0 mg/dl) times upper limit of normal or creatinine 1.2mg/dl.

ADMINISTRATION OF DRUG

Before starting medication, the patients were asked to take rest for 20 to 30 minutes. Sitting blood pressure, pulse rate, respiratory rate and body temperature were measured. Sitting blood pressure was measured by sphygmomanometer. Patients were advised not to take any other medications without consulting the investigator. Patients were given metoprolol 25mg ER tablets and advised to take one tablet daily for two weeks. They were advised to take the medication after food around 8-10 a.m. Patient medication compliance card was given to all the patients and asked to mark X in the card after taking each dose. Patients were advised not to smoke and consume alcohol. At the end of two weeks, sitting blood pressure, pulse rate, body temperature, and respiratory rate were recorded as it was done earlier. Patient compliance card was collected from all the patients and verified to assess patient compliance. Any adverse reactions during the study period were recorded. Five milliliters blood was collected from all the patients and control subjects into containers with disodium EDTA and stored in a refrigerator for DNA studies. After completion of the study the patients were referred back to hypertensive clinic for regular management of hypertension.

ISOLATION OF DNA

GENOMIC DNA WAS ISOLATED FROM PERIPHERAL BLOOD BY AMMONIUM ACETATE METHOD (Miller SA et al., 1998)

The method involves:

Collection of Buffy white coat, lysis of RBCs using lysis buffer (Ammonium bicarbonate ((NH4) HCO ) Ammonium Chloride (NH4Cl) and double distilled water) and obtained WBC pellets. The pellet is suspended with WBC lysis buffer (1M Tris buffer (pH 8.5), 0.5M EDTA (pH 8), 10 percent SDS (Sodium Dodecyl Sulphate) and double distilled water) and added 2.5 ml of 5M Ammonium acetate. The tube was inverted several times and incubated for 10 minutes at room temperature. The precipitated proteins spinned down and the supernatant was collected in a fresh 1.5 ml microfuge tube, twice washed with 70% ethanol dried completely and dissolved in 1X TE or MilliQ water and quantified using UV spectrophotometer.

CYP 2D6 *4 PCR AMPLIFICATION

Genomic DNA was amplified using the following primer pairs. Forward primer: 5'-GCC TTC GCC AAC CAC TCC G-3'. Reverse primer: 5'-AAA TCC TGC TCT TCC GAG GC-3'. Under the following experimental condition:

TABLE 2- EXPERIMENTAL CONDITIONS Genomic DNA 50-100 ng

10X PCR Buffer

1l

10 mM dNTPs

0.02 l

50mM MgCl2

0.05 l

Forward primer

5 picomol

Reverse primer

5 picomol

Total

10.0 l

REACTION CYCLE

Initial Denaturation:-94C-5 min Denaturation: Annealing: Extension: Final Extension: 94C-45 sec 59C-30 sec 72C-30sec 72C-30sec

Number of cycles: - 34 PCR product: 355 bp

The PCR products were digested with MvaI (Micrococcus Varians I) restriction digestion respective buffers at suitable temperature. MvaI RESTRICTION ENZYME Restriction site 5'-CC^AGG-3', 3'-GGT^CC-5' OR 5'-CC^TGG-3', 3'-GGA^CC-5'

AGAROSE GEL ELECTROPHORESIS

The amplified PCR product was mixed with 6X loading buffer(0.25% bromophenol blue,30% glycerol), and run on 2% Agarose gel containing Ethidium bromide( EtBr)- mg/ml. Electrophoresis was carried out in 0.5 X TBE buffer for 25 minutes at 150 V using digest ladder for reference. The separated DNA band was visualized under UV light and documented by photography.

FIGURE 1- Agarose Gel Electrophoresis

FIGURE-2 Agarose Gel Electrophoresis

RESULT

CYP2D6*4 GENE POLYMORPHISM ANALYSIS

Presence of Mval restriction site generates 250 and 105 bp base pair fragments and its absence is indicated by the presence of undigested 355bp PCR amplicon. The restriction site is due the substitution of GA at the nucleotide position 1934 (Daly et al., 1996). The restriction fragment length polymorphism (RFLP) assay distinguished the wild type two DNA fragment of (250 and 105 bp) from the variant alleles (a single fragment of 355 bp), following 2 % Agarose gel electrophoresis.

DISCUSSION

A number of experimental approaches to 'genotypic' mutation systems have been developed (6). Several are based on Southern or Northern hybridization often with sequence amplification by polymerase chain reaction (PCR) and in combination with the specific cleavage of heteroduplexes at mismatched base pairs. Other protocols take advantage of differences in electrophoretic mobilities of hetero-duplexes or of mutated single stranded nucleic acids. The sensitivity of all these approaches are limited by backgrounds which arise from the presence of excess wild-type sequences. The RFLP/PCR approach to genotypic mutation analysis described here greatly reduces this problem. Base-pair (bp) mutations are detected which are located in a restriction recognition sequence and render this site resistant to cleavage by the corresponding endonuclease. The resistant DNA sequence containing the mutated site is amplified by PCR only after wildtpe DNA has been essentially eliminated by restriction digestion. Amplified DNA is cloned into XgtlO and mutants are quantitated by oligonucleotide plaque hybridization relative to an internal standard. The maximal sensitivity of the RFLP/PCR method isdetermined by the completeness of the removal of wild-typesequences together with the inherent error-rate at a particular base pair of the polymerase used in the PCR. The RFLP/PCR protocol for genotypic mutation analysis is applicable to any gene of known sequence. It is highly sensitive, but limited to the identification of point mutations (bp-changes, small deletions and insertions) which results in elimination of restriction sites. The aim of the present work was to test the feasibility to use the RFLP/PCR approach for the development of a genotypic mutation system. Such a system should be capable of measuring low frequency mutations in any gene of known sequence without the need for the phenotypic selection of mutated cells (6). In the RFLP/PCR approach wt-sequences are selectively removed by exhaustive restriction and resistant sequences are amplified by PCR. We have identifies that the restriction site is due the substitution of GA at the nucleotide position 1934 (Daly et al., 1996). The restriction fragment length polymorphism (RFLP) assay distinguished the wild type two DNA fragment of (250 and 105 bp) from the variant alleles (a single fragment of 355 bp), following 2 % Agarose gel electrophoresis.

SUMMARY

Hypertension, most commonly referred to as "High blood pressure", HTN or HPN, is a medical condition in which the blood pressure is chronically elevated. High blood pressure if left undetected and untreated will result in brain attack (stroke), heart attack, heart enlargement, heart failure and kidney failure. In India hypertension prevalence has steadily been increasing from 5% in 1960 to 12.15% in 1990s. Blood pressure varies naturally over the course of a day, and usually increases with age. In addition, activity affects blood pressure, which rises as a normal response to physical exertion and stress. Making appropriate lifestyle changes under a doctors guidance are an important initial part of any treatment plan for high blood pressure. However, many patients also require therapy with medications known as "antihypertensive drugs" to lower the blood pressure. There are various classes of antihypertensive agents that are commonly used to reduce high blood pressure. But the type of drug used for our study is METOPROLOL- a Beta Blocker. Blood samples were collected from patients suffering from hypertension and who were treated with the Beta- Blocker, from the Madras Medical College, Chennai. DNA was isolated from the sample, amplified using PCR and then restricted digestion was performed, to identify polymorphism induced in the isolated DNA samples

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URL REFERENCES

http://www.entrez gene.com. http://www.wikipedia.org/wiki/CYP2D6. http://en.wikipedia.org/wiki/Cytochrome_P450 http://www.dnadirect.com http://www.dnadirect.com/patients/resources/genesand_drugs/pro_active_drug.j sp. http:// www.medicinenet.com