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Atenolol Exposure As a Risk for Adverse Metabolic Effects to Beta Blockers

Permanent Link: http://ufdc.ufl.edu/UFE0024564/00001

Material Information

Title: Atenolol Exposure As a Risk for Adverse Metabolic Effects to Beta Blockers
Physical Description: 1 online resource (54 p.)
Language: english
Creator: Navare, Hrishikesh
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: adverse, atenolol, beta, blockers, effects, glucose, hypertension, metabolic, oral, pharmacokinetics, test, tolerance
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cardiovascular disease (CVD) is the leading cause of mortality in the United States of America (USA) and hypertension (HTN) is an important CVD risk factor. It is also a risk factor for stroke, heart failure and renal failure. Beta-blockers (BB), along with diuretics have been among the consensus guideline recommended first-line agents for the treatment of HTN in the USA. BBs are widely used for treating HTN for many decades for their efficacy. However, many clinicians are concerned about the use of BB as a primary therapy for HTN due to the risk for adverse metabolic effects (AME) on glucose, insulin and lipids thereby the development of diabetes and dyslipidemia. However, these effects are observed only in certain fraction of the population. This suggests that there are other factors that predispose certain individuals to develop these AME. In our project, we determined if atenolol plasma concentration (Cp) as a factor may contribute to the inter-individual differences. We hypothesized, increasing systemic exposure to atenolol leads to increased risk for AME from atenolol therapy in HTN. We performed a 24-hour pharmacokinetic (PK) study in 15 hypertensive patients taking chronic atenolol 100 mg daily. We also measured their glucose and insulin levels by performing 2 hour oral glucose tolerance test (OGTT) and also measured their fasting lipids, glucose and insulin before the start of atenolol treatment and after 8 weeks of treatment. We did not found any association between atenolol Cp and glucose levels during the OGTT and between atenolol Cp and change in lipid levels during the study duration. We did not find atenolol Cp as a risk factor for the development of AME. However, our study has a small sample size and restricts our power. The information from this study may lead to future studies that will help us identify those people at risk of these AMEs from BB, so that we can prevent or avoid the AMEs. Ultimately, we can avoid or prevent the occurrence of AME, and these important anti-hypertensive agents can still be safely used rather than being rejected totally.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hrishikesh Navare.
Thesis: Thesis (M.S.P.)--University of Florida, 2009.
Local: Adviser: Johnson, Julie A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024564:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024564/00001

Material Information

Title: Atenolol Exposure As a Risk for Adverse Metabolic Effects to Beta Blockers
Physical Description: 1 online resource (54 p.)
Language: english
Creator: Navare, Hrishikesh
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: adverse, atenolol, beta, blockers, effects, glucose, hypertension, metabolic, oral, pharmacokinetics, test, tolerance
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cardiovascular disease (CVD) is the leading cause of mortality in the United States of America (USA) and hypertension (HTN) is an important CVD risk factor. It is also a risk factor for stroke, heart failure and renal failure. Beta-blockers (BB), along with diuretics have been among the consensus guideline recommended first-line agents for the treatment of HTN in the USA. BBs are widely used for treating HTN for many decades for their efficacy. However, many clinicians are concerned about the use of BB as a primary therapy for HTN due to the risk for adverse metabolic effects (AME) on glucose, insulin and lipids thereby the development of diabetes and dyslipidemia. However, these effects are observed only in certain fraction of the population. This suggests that there are other factors that predispose certain individuals to develop these AME. In our project, we determined if atenolol plasma concentration (Cp) as a factor may contribute to the inter-individual differences. We hypothesized, increasing systemic exposure to atenolol leads to increased risk for AME from atenolol therapy in HTN. We performed a 24-hour pharmacokinetic (PK) study in 15 hypertensive patients taking chronic atenolol 100 mg daily. We also measured their glucose and insulin levels by performing 2 hour oral glucose tolerance test (OGTT) and also measured their fasting lipids, glucose and insulin before the start of atenolol treatment and after 8 weeks of treatment. We did not found any association between atenolol Cp and glucose levels during the OGTT and between atenolol Cp and change in lipid levels during the study duration. We did not find atenolol Cp as a risk factor for the development of AME. However, our study has a small sample size and restricts our power. The information from this study may lead to future studies that will help us identify those people at risk of these AMEs from BB, so that we can prevent or avoid the AMEs. Ultimately, we can avoid or prevent the occurrence of AME, and these important anti-hypertensive agents can still be safely used rather than being rejected totally.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hrishikesh Navare.
Thesis: Thesis (M.S.P.)--University of Florida, 2009.
Local: Adviser: Johnson, Julie A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024564:00001


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1 ATENOLOL EXPOSURE AS A RISK FOR ADVERSE MET A BOLIC EFFECTS TO BETA BLOCKERS By HRISHIKESH A. NAVARE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY UNIVERSITY OF FLORIDA 2009

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2 2009 Hrishikesh A Navare

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3 To my parents

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4 ACKNOWLEDGMENTS I take this opp ortunity to thank my mentor Dr. Julie Johnson for her supp ort and guidance throughout the project. I sincerely thank her for giving me this opportunity to work on this project. The research project would not have been possible without her contributions and support. I want to express my gratitude to Dr. Hartmut D erendorf, Dr. Reginald Frye, Dr. Jonathan Shuster and Dr. Rhonda Cooper DeHoff for being part of my graduate committee and guiding me throughout this project. I want to acknowledge Dr. Frye for his help to develop the HPLC/MS/MS assay of atenolol and Dr. S huster for his guidance in statistical analysis and giving me valuable advice. T hank you Dr.Taimour Langaee, Dr.Yan Gong and other members of Center for Pharmacogenomics for their help, support and guidance over the years. I want to thank all the lab memb ers whom I had pleasure working with for past four years. Its been great experience to be part of such a diverse group. I express my gratitude to departments of Pha rmaceutics and Pharmacotherapy and Translational Research for admitting me in this program and providing me fi nancial support. I am obliged to American Heart Association for giving me pre -doctoral fellowship. I am grateful to the PEAR steering comm ittee for allowing me to re cruit PEAR patients in this study. This project would not have been pos sible without the participation of my volunteers and I want to sincerely thank every one of them for being a part of this project. This recruitment was the result of the support of the PEAR co -coordinators and I w ant to acknowledge their help Likewise, I want to show my appreciation to the staff of General Clinical Research Center for their inv aluable help in this project. There are no words to acknowledge my parents, my family for giving me the strength to stand on my feet for their constant motivation a nd continue to

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5 study further to enhance my knowledge. I sincerely express my gratitude from the bottom of my heart for all the sacrifices that my parents have done throughout their life so that all of their children get everything they desired, for inculca ting in us the virtues of hard work, honesty, compassion towards everyone which has resulted in developing my personality. I am very thankful to the Almighty for giving me such caring parents.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 LIST OF ABBREVIATIONS ............................................................................................................ 10 ABSTRACT ........................................................................................................................................ 11 CHAPTER 1. INTRODUCTION...................................................................................................................... 13 BB Usage and Occurrence of AME ........................................................................................... 15 Atenolol ....................................................................................................................................... 16 The Scope of Present Work ........................................................................................................ 17 2. ATENOLOL LC/MS/MS ASSAY ............................................................................................ 20 Introduction ................................................................................................................................. 20 Experimental ................................................................................................................................ 21 Chemicals and Reagents ...................................................................................................... 21 Preparations of Standards and Quality Control (QC) samples ......................................... 21 Sample Preparation .............................................................................................................. 22 LC/MS/MS Conditions ........................................................................................................ 22 Chromatographic Conditions .............................................................................................. 23 Standard Curve ..................................................................................................................... 23 Method Validation ............................................................................................................... 23 Application to Plasma Sampling ......................................................................................... 25 Results and Discussion ............................................................................................................... 25 Chromatographic Method ................................................................................................... 25 Method Validation ............................................................................................................... 26 Linearity, Precision, Accuracy and Dilution Integrity ............................................... 26 Selectivity, Matrix Effect, Recovery, and Stability ................................................... 26 Method Application .................................................................................................................... 27 Conclusions ................................................................................................................................. 27

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7 3. ATENOLOL PHARMACOKINETIC STUDY ........................................................................ 31 Introduction ................................................................................................................................. 31 Materials and Methods ................................................................................................................ 32 Study Design ........................................................................................................................ 32 PEAR Protocol ..................................................................................................................... 32 Laboratory Measurements .......................................................................................................... 33 Pharmacokinetic Data Analysis .................................................................................................. 34 Pharmacodynamic Data Analysis ............................................................................................... 34 Statistical Methods ...................................................................................................................... 34 Results .......................................................................................................................................... 35 Discussion .................................................................................................................................... 37 4. CONCLUSIONS AND FUTURE DIRECTIONS .................................................................... 47 LIST OF REFERENCES ................................................................................................................... 48 BIOGRAPHICAL SKETCH ............................................................................................................. 54

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8 LIST OF TABLES Table page 1 1 Effects of BB treatment on metaboli c risk factors ............................................................... 18 1 2 Studies assessing the effects of atenolol and other BB on lipid profile .............................. 18 1 3 Studies assessing the effec ts of atenolol and other BB on glucose and insulin ................. 19 2 1 Intra and inter run precision (%RSD) and accuracy (%RE) for atenolol quality control samples in human plasma ......................................................................................... 30 3 1 Clinical characteristics of patients ........................................................................................ 40 3 2 Atenolol AUC and glucose, insulin AUC during OGTT .................................................... 41 3 3 Pharmacokinetic estimates of atenolol .................................................................................. 42 3 4 Changes in lipids after atenolol treatment ............................................................................ 42 3 5 Changes in gl ucose, insulin after atenolol treatment ........................................................... 43 3 6 Pearsons correlation between atenolol AUCn and glucose AUC, insulin AUC ............... 43 3 7 P earsons correlation between atenolol AUCn and changes in lipids, glucose .................. 43 3 8 Multivariate analysis .............................................................................................................. 43

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9 LIST OF FIGURES Figure page 1 1 Atenolol chemical structure ................................................................................................... 19 2 1 The extracted LC -MS/MS chromatograms .......................................................................... 28 2 2 Mean ( SD) concentration time profile of atenolol after atenolol 100mg in ten patients .................................................................................................................................... 30 3 1 Atenolol plasma concentration .............................................................................................. 44 3 2 Plasma Levels During OGTT ................................................................................................ 45 3 3 Correlation Coefficients (r) between A) atenolol AUCn and glucose AUC 02, B) atenolol AUCn and insulin AUC 02 ....................................................................................... 46

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10 LIST OF ABBREVIATIONS CVD Cardiovascular disease HTN Hypertension BB Beta blockers BP Blood pressure B1AR Beta 1 adrenergic receptor AME Adverse metabolic effects PK Pharmacokinetic s HDL C High density lipoprotein -cholesterol TC Total cholestero l ISTD Internal standard LC/MS/MS Liquid chromatography/mass spectrometry/mass spectrometry HILIC H ydrophilic interaction chromatography QC Quality control Cmax P eak concentration Tmax T ime to achieve peak concentration Cp Plasma concentration T1/ 2 Elimination half life AUC Area under curve AUCn Area under curve, normalized OGTT Oral glucose tolerance test HOMA Homeostatic model assessment BMI Body mass index

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science in Pharmacy ATENOLOL EXPOSURE AS A RISK FOR ADVERSE MET A BOLIC EFFECTS TO BETA BLOCKERS By Hrishikesh A Navare May 2009 Chair: Julie A. Johnson Major: Pharmac eutical Sciences Cardiovascular disease (CVD) is the leading cause of mortality in the United States of America (USA) and hypertension (HTN) is an important CVD risk factor. It is also a risk factor for stroke, heart failure and renal failure. Beta block ers (BB), along with diuretics have been among the consensus guideline recommended first line agents for the treatment of HTN in the USA BBs are widely used for treating HTN for many decades for their efficacy. However, many clinicians are concerned about the use of BB as a primary therapy for HTN due to the risk for adverse met abolic effects (AME) on glucose, insulin and lipids thereby the development of diabetes and dyslipidemia However, these effects are observed only in certain fraction of the populat ion. This suggests that there are other factors that predispose certain individuals to develop these AME. In our project, we determined if atenolol plasma concentration (Cp) as a factor may contribute to the inter individual differences We hypothesized, i ncreasing systemic exposure to atenolol leads to increased risk for AME from atenolol therapy in HTN. We performed a 24 hour pharmacokinetic (PK) study in 15 hypertensive patient s taking chronic atenolol 100 mg daily. We also measured their glucose and ins ulin levels by performing 2 hour oral glucose tolerance test (OGTT) and also measured their fasting lipids, glucose and insulin before the start of atenolol treatment and after 8 weeks of treatment. We did not found any

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12 association between atenolol Cp and glucose levels during the OGTT and between atenolol Cp and change in lipid levels during the study duration. W e did not find atenolol Cp as a risk factor for the development of AME. However, our study has a small sample size and restricts our power. The in formation from this study may lead to future studies that will help us identify those people at risk of these AMEs from BB, so that we can prevent or avoid the AMEs. Ultimately, we can avoid or prevent the occurrence of AME, and these important anti -hypert ensive agents can still be safely used rather than being rejected totally.

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13 CHAPTER 1 INTRODUCTION Hypertension (HTN) is the most common chronic disease, affecting approximately 1 billion people worldwide and approximately 72 million Americans, with an ad ditional 25 million Americans considered to have prehypertensive blood pressure (BP) [120139/8089 mmHg] (Chobanian et al., 2003) Stroke, heart failure, ischemic heart disease (including acute myocardial infarction and angina), and chronic renal failure are adverse outcomes associated with HTN. The pathophysiology of HTN in the approximately 90% of patients with essential HTN is not completely understood. HTN has numerous potential causes, resulting from abnormalities in the kidneys, vasculature and/or neurohormonal systems. Epidemiologic evidence demonstrates that the risks of cardiovascular morbidity and mortality rise progressively with increasing blood pressure (Lloyd Jones et al., 2009) Diabetes is also one of the leading causes of morbidity and mortality in the U nited S tates of A merica (USA). Type 2 diabetes comprises approximatel y 90% of new cases of diabetes and is a common concomitant disease with HTN (Reaven et al., 1996) Specifically, HTN occurs in 24% of the adult population i n the USA, but in up to 60% of type 2 diabetics aged 45 75 years (Jacob et al., 1999) Notably, the risk of developi ng new onset diabetes is related to the antihypertensive treatment regimen (Elliott and Meyer, 2007) Beta adrenergic receptor blockers (BB) have been used to treat high BP for more than four decades and have proven to reduce morbidity and mortality. BB s along with diuretics have bee n recommended as first line drugs for treating HTN for several decades. However, results from various large clinical trials indicate that patients taking thiazide diuretics and BB have a higher risk of developing type 2 diabetes (Hansson et al., 1999; Brown et al., 2000; Gress et al., 2000; Lindholm et al., 2003; Nilsson and Berglund, 2006; Stump et al., 2006) These drug s

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14 decrease insulin sensitivity thereby causing type 2 diabetes (Pollare et al., 1989b; Reaven et al., 1996; Bonner et al., 1997; Giugliano et al., 1997; Reneland et al., 2000; Poirier et al., 2001; Sarafidis and Bakris, 2006) BB and thiazide diuretics are al so associated with adverse lipid effects particularly on triglycerides, high density lipoprotein cholesterol (HDL C) and total cholesterol (TC) (Day et al., 1979; Eliasson et al., 1981; Day et al., 1982; Weiner and Rossner, 1983; Otterstad et al., 1992; Rabkin et al., 1994) R ecently, the effe ctiveness of BB based treatment for HTN has been questioned and many clinicians have argued against the use of BB as a first line therapy in HTN (Beevers, 1998; Messerli et al., 2003; Carlberg et al., 2004; Pepine and Cooper Dehoff, 2004; Williams, 2007) Recent meta analysis have found that BB increased the ri sk for new -onset diabetes with no benefit on the end point of death or myocardial infarction and increased risk for stroke by 15% compared with other agents (Lindholm et al., 2005; Bangalore et al., 2007b) Evidence from several clinical outcome trials demonstrate that compared to other anti -hypertensive classes, BB were no more effective at preventing cardiovascular events, res ulting in BB being moved to fourth line treatment option for HTN in the latest UK and European guidelines (Chobanian et al., 2003; Mayor, 2006; Sever, 2006; Mancia et al., 2007) However, BB still hold a key role in treating patients with hypertension and other concomitant CVD, and remain a first line option in JNC 7 guidelines along with other drugs (Kendall, 1998; Cruickshank, 2000; Messerli et al., 2003; Fardoun, 2006; Nilsson and Berglund, 2006; Bangalore et al., 2007a; Cruickshank, 2007; Roberts et al., 2007) Given that the occurrence of adverse metabolic effects (AME) from BB, particularly diabetes, may offset their clinical benefit, it is impor tant to identify the factors that place a patient at risk for these adverse effects, such that BB therapy might be avoided in those patients who are at risk.

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15 Different BB s h ave variable effects on glucose, insulin and lipid levels. Nonselective BB s like pr opranolol have the most significant effects in decreasing insulin sensitivity, HDL -C and increasing triglyceride levels. Selective BB which specifically bind to the beta 1 adrenergic receptor (B1AR) have intermediate effects in reducing insulin sensitivity and HDL -C and increasing triglyceride levels. These drugs are prone to lose specificity at higher dose. Newer BBs like carvedilol, which have vasodilating properties, are found to improve insulin sensitivity and have a neutral effect on lipids (Jacob et al., 1998) (Table 1 1 ) The mechanisms by which BB treatme nt modifies insulin sensitivity and affect lipids and cardiovascular risk factors are not fully understood. Insulin secretion and insulin clearance is red uced by BB usage (Pollare et al., 1989a; Pollare et al., 1989b; Jacob et al., 1998) BB also have significant adverse effects on triglycerides and HDL C, which may result from their action in decreasing L ipoprotein lipase (L PL ) and L ecithin cholesterol acyl transferase (LCAT) activity and altering cholesterol synthesis (Grimm, 1991; Rabkin et al., 1994; Liggett et al., 2006) BB U sage and O ccurrence of AME The dose/concentration relationship between AME and thiazide diuretics is well established, but this is not the case for BB (Black, 1996; Savage et al., 1998) Low dose diur etics are found to have neutral effects on the development of new onset diabetes. There are many conflicting studies that have compared the effects of BB on lipid levels and insulin sensitivity. Some studies have found an association between short term use of BB and hypertriglyceridimia while others have not. Table 1 2 summarizes some of the studies which show the conflicting reports about BB effect on lipid levels, while T able 1 3 summ arizes some of the studies about BB effect on insulin sensitivity.

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16 Ateno lol Atenolol is a polar hydrophilic phenylacetamide [(4 2 -hydroxy 3 -isopropyl aminopropoxy) phenylacetamide] (Figure 1 1) It is a selective B1AR antagonist devoid of any intrinsic sympathomimetic and membrane stabilizing activity (Barrett et al., 1973; Mason et al., 1979) In humans, there is incomplete absorption after oral administration (Brown et al., 1976; Fitzgerald et al., 1978; Tabacova and Kimmel, 2002) Hence, the systemic bioavailability is approximately 50%. Peak plasma levels after oral administration are achieved after 2 4 hours. The absorbed drug is widely distributed in the body(Reeves et al., 1978b) but only a fraction of the administered dose rea ches the brain. It is minimally bound to plasma proteins, <5%, a bout 10% of the drug is m etabolized with the rest eliminated unchanged in urine by filtration (Reeves et al., 1978a; Reeves et al., 1978b) After intravenous administration, complete drug is eliminated in the urine whereas upon oral dosing, 4050% of the unchanged drug is recovered in urine and the res t is excreted through feces due to incomplete absorption (Tabacova and Kimmel, 2002) The elimination half life (t1/2) is between 6 8 hours. As atenolol is cleared by glome rular filtration, the elimination half life is prolonged in patients with renal disease. There is significant variability reported in atenolol pharmacokinetic parameters. There is a fourfold variation in the plasma con centrations, three fold in t1/2 and p eak plasma concentrations (Cmax) and two fold in time to achieve peak plasma concentration (Tmax) (Brown et al., 1976; Fitzgerald et al., 1978; Mason et al., 1979; Melander et al., 1979; Barnwell et al., 1993; Shiga et al., 1993; Sowinski et al., 1995; Sourgens et al., 2003). Some of the factors associated with this variability are age, disease status and presence of food. Young men had lower plasma concentrations and high er apparent oral clearance CL /F than old er men which can be due to age related decline in renal functio n, and changes in body composition (Greenblatt et al., 1982; Sowinski et al., 1995) Similarly, as renal function declines, the clearance decreases

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17 and the elimination half -life increases significantly (Sassard et al., 1977; McAinsh et al., 1980) Presence of food has also been shown to affect the absorption of atenolol. Food intake decreased th e plasma concentration by 20% (Melander et al., 1979) co administration of atenolol with orange juice also significantly decreased the plasma concentration by 40% (Lilja et al., 2005) The reasons behind the food associated reduction in bioavailabil ity are believed to be the release of bile acids after food intake and the complexation of the hydrophilic drug in the bile acid micelles (Barnwell et al., 1993) Atenolol pharmacokinetic variability might affect the development of the metabolic adverse effects on insulin sensitivity and lipids. The Scope of Present Work Despite the body of literature documenting the AME of BB, there are no studies that have addressed the relationship between the atenolol plasma concentration and the occurrence of the drug induced change in triglycerides or change in insulin sensitivity. Also, as mentioned above there is significant variability in the pharmacokinetic parameters o f atenolol which can potentially affect the development of the AME in different individuals. We hypothesize that the occurrence of the metabolic abnormalities with atenolol are concentration related and that people with higher atenolol area under the plasm a concentration time curve (AUC) are those who are more likely to develop these AME. For testing this hypothesis, we conducted a 24 hour pharmacokinetic (PK) study in hypertensive patients who were dose titrated to100mg atenolol for 4 weeks after taking a tenolol 50mg for 3 4 weeks. We also measured their glucose/insulin area under the curve by doing a 2 hour glucose tolerance test and their change in lipid levels from the time they started taking drug to the PK study visit.

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18 Table 1 1. Effects of BB tre atment on metabolic risk factors Compound Insulin sensitivity Triglycerides HDL C TC Propranolol 33% +25% 10% +9% Metoprolol 21% +30% 7% 1% Atenolol 22% +18% 9% = Pindolol 17% = = = Dilevalol +10% 22% = 6% Carvedilol +13% = = = Celiprolol +35% 15% +5% = Decreased, + increased, = no change (Adapted from Jacob et al. 1998) Table 1 2. Studies assessing the effects of atenolol and other BB on lipid profile Author Study duration n Results Day et al. 1979 6 8 months 49 Atenolol (50mg 100mg b id) had 24% increase, propranolol (40 160mg tid) had 60% increase in basal triglyceride levels Eliasson et al, 1981 15 months 15 Atenolol 100mg increased triglyceride concentrations by 30% Day et al. 1982 12 months 53 Increase in triglycerides were obse rved as follows: atenolol 100mg (24%), propranolol 160mg (51%), metoprolol 200mg (14%), oxeprenolol 160mg (26%) Weiner and Rossner, 1983 3 months 50 Atenolol 50 mg once daily did not change the serum lipoprotein levels while metoprolol 200mg increased t he lipoproteins Otterstad et al., 1992 3 12 months 100 Atenolol 50mg once daily increased triglycerides by 15% and decreased HDL by 5% compared with hydrochlorthiazide (HCTZ) 25mg amiloride 5mg combination Rabkin et al, 1994 6 months 131 Atenolol increas ed triglycerides by 18%, decreased HDL by 7% as compared to doxazosin

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19 Table 1 3. Studies assessing the effects of atenolol and other BB on glucose and insulin Author Study duration n Results Pollare et al., 1989b 16 weeks 60 Glucose uptake decreased for both atenolol and metoprolol causing increase in fasting plasma i nsulin, blood glucose, hemoglobin A1c Bonner et al., 1997 12 weeks 32 Glucose AUC in the IV glucose tolerance test increased during atenolol therapy Giugliano et al., 1997 24 weeks 45 Fasting plasma glucose concentrations and insulin levels increased with atenolol while they decreased with carvedilol Reneland et al. 2000 48 weeks 26 Insulin sensitivity decreased 23% by atenolol Poirier et al., 2001 16 weeks 25 Atenolol was a ssociated with 20% greater reduction in insulin sensitivity than with nebivolol Figure 1 1 Atenolol chemical structure

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20 CHAPTER 2 ATENOLOL LC/MS/MS ASSAY Introduction Atenolol is a selective B1AR antagonist that is commonly used to treat hypertension (Barrett et al., 1973; Mason et al., 1979) Numerous methods are reported in the literature for the determination of ate nolol concentration in biological fluids including gas chromatography with mass spectrometry (GC MS), high performance liquid chromatography (HPLC) or capillary zone electrophoresis (Abdel Hamid, 2000; Li et al., 2005) Although GC MS is a sensitive technique, the sample preparation requires derivatization, which is tedious and time consuming HPLC particularly reversed -p hase liquid chromatography is widely used for analysis of atenolol with mobile phases consisting of acetonitrile or methanol, buffer, an ion pairing reagent (e.g. alkyl sulphonates ) to provide adequate retention and organic am ines (e.g. triethyl amine) to reduce peak tailing (Verghese et al., 1983) But these additives can shorten life of reversed -phase packings such as C18 silica (Basci et al., 1998) Moreover, atenolol is a polar compound and hence it is difficult to retain on a C18 column, except when using a highly aqueous mobil e phase, which can collapse high density C8 or C18 columns (Naidong, 2003) Basic compounds can be separated on plain, unbonded silica with aqueous -rich mobile phases or with organic rich eluents employing hydrophilic int eraction chromatography (HILIC), (Jeong et al., 2007; Hsieh, 2008) In HILIC, analytes elute by passing a hydrophobic or mostly organic mobile phase across a neutral hydrophilic stationary phase causing solutes to elute in order of increasing hydrophilicity resulting in better separation of highly polar compounds (Li et al., 2005) We adapted a HILIC method reported by Li et al (Li et al., 2005) to analyze atenolol in human plasma

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21 Experimental Chemicals and R eagents Atenolol reference standard and atenolol -d7 were obtained from MP Biomedicals LLC, (Solon, OH, USA ) and Toronto Research Chemicals (North York, O N, Canada ) respectively. Acetic acid and trifluoro acetic acid were purchased from VWR, (West Chester, PA, USA ). Acetonitrile was obtained from Fisher Scientific, (Waltham, MA, USA ). Blank plasma was obtained from Shands hospital (Gainesville FL USA). H PLC grade deionized water was obtained from a Barnstead Nanopure Diamond UV Ultrapure Water system (Dubuque, IA, USA). Preparations of S tandard s and Q uality C ontrol (QC) samples A stock solution of atenolol was prepared at a concentration of 1mg/ mL in ace tonitrile and stored in a glass vial at 40C. Dilutions prepared in acetonitrile at concentrations of 100, 10 and 1 g/ mL were used to prepare calibration standards and quality control (QC) samples. Blank plasma was spiked wit h appropriate stock solutions. Eight calibration standards were prepared at concentrations of 5,10,25,50,100,250,500 and 1000 ng/ mL QC samples were prepared at concentrations of 15 ng/ mL (low QC), 200ng/ mL (medium QC) and 750ng/ mL (high QC). A dilution QC was prepared at a concentrati on of 2000 ng/ mL Standards and QC samples were prepared at the begi nning of the validation, transferred into 1.5 mL micro centrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA) and stored at 200C until analysis. A stock solution (1mg/ mL ) of the ate nolol d7, internal standard (ISTD) was prepared in acetonitrile and further diluted to 200 ng/ mL and used for all analyses. The ISTD stock solutions were stored in glass vials at 40C.

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22 Sample P reparation Frozen samples were thawed at room temperature. An a liquot (100l) of plasma was transferred to a 1.5 mL micro centrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA). The plasma was combined with 50l of ISTD (except for blank without ISTD) vortex mixed and 1 mL acetonitrile was added, except for blan k without ISTD and vortex for 1 minute. The tubes were centrifuged for 10 min ute s at 14000 rpm in an Eppendorf 5810R centrifuge. The supernatant (800 l) was transferred in a clean glass culture tube and briefly vortex -mixed. Then they were evaporated unde r nitrogen and reconstituted with 200 l acetonitrile. Samples were transferred to auto sampler vials with micro inserts and then injected into the system (20 l). The auto sampler temperature was maintained at 10oC. LC/ MS/MS C onditions The LC/MS/MS system consisted of a ThermoFinnigan Surveyor HPLC auto sampler, ThermoFinnigan Surveyor MS quaternary pump and a ThermoFinnigan TSQ Quantum Discovery triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, USA). The TSQ Quantum mass spectrometer was equipped with an electrospray (ESI) ion source and operated in the positive mode. The ESI source spray was set orthogonal to the ion transfer capillary tube. The mass spectrometer was calibrated with a solution of polytyrosine 1, 3, 6 according to the manufacturer. The MS/MS conditions were optimized by infusing atenolol in the mobile phase. The ESI source parameters were tuned for maximum abundance of [M H] ions of atenolol at the LC flow rate of 0.22 mL /min. For quantification, the TSQ Quantum wa s operated in high resolution multiple reaction monitoring mode. The acquisition parameters were: spray voltage 3.5V, source CID 10 V, and heated capillary temperature at 350oC. Nitrogen was used as the sheath and auxiliary gas and set to 35 and 15 (arbi trary units), respectively. The

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23 argon collision gas pressure was set to 1.5 mTorr. The collision energy was 3 3 eV for atenolol and atenolol -d 7. The selected reaction monitoring scheme followed transitions of the [M+H] + precursor to selected product ion s with the following values: m/z 267.12 144.50 for atenolol and 274.20 144.50 for atenolol -d 7. The instrument was operated in enhanced (high) resolution with peak width (FWHM) set to 0.2 m/z at Q1 and to 0.7 m/z at Q3. The scan time was 200 ms for e ach transition. MRM data were acquired and processed using ThermoFinnigan XCalibur software version 1.4, service release 1 (Thermo Electron Corporation, San Jose, CA, USA). Chromatographic C onditions Chromatographic separation was performed using a Bet asil Silica 100, 50 x 3 mm, 5 analytical column (Thermo Electron Corporation, Bellfonte, PA, USA). The mobile phase used for the analysis was 94% acetonitrile, 6% deionized water, 0.5% acetic acid and 0.04% trifluoro aceti c acid (TFA) The mobile phase was degassed and filtered through a 0.22 nylon membrane before use. The flow rate was 0.5 mL /min. Standard C urve Duplicate standard curves were analyzed in each run. For each standard calibration curve, the atenolol peak area to ISTD peak area ratio was calculated and plotted against nominal atenolol concentrations. Weighted (1/concentration2) linear regression analysis was used to construct calibration curves from the standards. The regression equation was used to calculate the concentrations in quality co ntrol ( QC ) and clinical samples. Method V alidation The current LC -MS/MS method was validated for precision, accuracy, linearity, dilution integrity, selectivity, carry over, matrix effect, recovery and stability. The accuracy and precision of the assay wa s determined by the analysis of atenolol QC samples at concentrations of 15.0,

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24 200.0 and 750.0 ng/ mL Six of each QC levels were analyzed daily for two runs and twelve of each QC le vels were analyzed for one run. The difference in the calculated mean conc entration relative to the spiked concentration was used to express accuracy (% deviation). Means, standard deviations and coefficients of variation were calculated from the QC values and used to estimate the intra and inter -run precision A dilution QC w as prepared at a concentration of 2000 ng/ mL Six replicates of the dilution QC were processed after being diluted 10-fold. Selectivity was evaluated by processing and analyzing blank plasma obtained from six sources. Carry -over was evaluated by placing vials of blank mobile phase at several locations in the analysis set. The potential for a matrix effect (suppression or enhancement of ionization) was evaluated by continuous infusion of atenolol post -column and injecting processed plasma sampl es from si x different sources. There was no evidence of a matrix effect in the region where atenolol and the deuterated internal standard elute. Extraction efficiency was determined by comparing the response obtained in the following samples: (A) quality control sam ples processed normally and (B) plasma samples extracted by the usual process and then spiked to contain the analyte and internal standard at concentration values corresponding to the QC conce ntrations (reference samples). This is done to eliminat e any pot ential matrix effect. Responses obtained from reference samples were defined as 100%. Processing and analysis of QC samples test the stability of the analytes (post -processing). The stability of the extracts in the auto sampler was evaluated after the e xtracts were left in the auto sampler at 10oC for at least 40 hours. The analytes were found to be stable under these conditions. The effect of freeze and thaw on the stability of the samples was tested using low and high concentration QC samples. Three al iquot tubes of each QC were stored at 40C as a reference

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25 and the other tubes were subjected to three freezethaw cycles prior to processing and analysis. The thawed samples were processed and analyzed as described above. Effects of freeze and thaw were mea sured by concentrations of each QC sample relative to the reference. Application to Plasma S ampling The method was used for the analysis of plasma samples obtained from hypertensive patients who were on atenolol 100mg for 4 weeks. The protocol was approve d by the University of Florida Institutional Review Board and all study subjects provided written informed consent. Atenolol 100mg (Sandoz Inc., Princeton, NJ, USA) was given orally at the General Research Center at the University of Florida after 4 weeks of pretreatment with atenolol 100mg once daily. Blood samples were collected in tubes containing sodium heparin at multiple time points within the dosing interval; plasma obtained by centrifugation was stored at 200 C until analyzed. Plasma concentrations of atenolol were determined as described above. A non -compartmental model was used to describe atenolol pharmacokinetics. The maximum atenolol concentration (Cmax), time at which Cmax occurred (Tmax), apparent oral clearance (Cl/F, where F is bioavailabil ity) and half life are reported. The pharmacokinetic parameter estimates were calculated using WinNonlin software (version 2.1, Pharsight Corporation, Mountain View, CA, USA). Results and Discussion Chromatographic M ethod Representative LC/ MS/MS chromatog rams of plasma samples are shown in Fig. 2 1. Fig. 2 1A shows a double blank plasma sample (no atenolol or ISTD), Fig. 2 1B is a plasma sample spiked with atenolol -d7 (ISTD; 50 ng/ mL ), Fig 2 1C is lower limit of quantification ( LLOQ ) of atenolol, Fig. 2 1D depicts a plasma sample obtained 2.5 h after the dose of atenolol 100mg.

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26 Retention time was approximately 1.6 min for atenolol. The peak of interest was well separated and free from interference with endogenous substances. Method V alidation Linearity, Precision, A ccu racy and Dilution I ntegrity Linear calibration curves ( n = 6) with a mean correlation coefficient of 0. 9872 were obtained for atenolol over the concentration range of 5 1000 ng/ mL The accuracy and precision data from QC samples demonstrate su itability of the method (Table 2 1). Intra and inter -run precision (% CV) was 7% and accuracy (% deviation) was within 13.3% (Table 2 1). The dilution QC (2,000 ng/ mL ) was processed after a 10 -fold dilution with blank plasma to determine dilution integrity. The mean concentration found for the dilution QC was 2726 ng/ mL The mean precision (%CV) was 6.3% and the accuracy (% deviation) was 3.2%, which were within the acceptance criteria (<15%). Selectivity, Matrix E ffect, R ecovery, and S tability No endogenous interference with atenolol was detected in six different sources of blank plasma and there was no evidence of sample carry -over. The post -column infusion experiments used to assess a matrix effect showed no evidence of a change in signal in the regions where atenolol eluted. Further, the matrix effect assessed by spiking samples post -processing showed <10% difference from spiked injection solvent. The mean extraction recoveries for atenolol were 102% and 119% at concentrations of 750 and 15 ng/ mL respectively. Atenolol was stable in processed samples held in the auto sampler at 10 C for at least 24 h with mean recoveries within 10%of the nominal concentration. No degradation of atenolol was observed in the samples subjected to three freeze thaw cycles.

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27 Method A pplication The mean atenolol concentrati on time profile obtained from 15 hypertensive patients after an oral dose of atenolol 100mg is shown in Fig. 2 2. The observed C max concentration of 1156 198 ng/ mL occurred at 2.8 0.9 h. The apparent oral clearance of atenolol after oral drug administration (CL/F ) was 176 54 mL /min and t he estimated half life was 6.9 1.7 h. The concentration of atenolol in all of the samples was greater than the lower limit of quantification, which was set at 5ng/ mL The results demonstrate that the assay is suitable for pharmacokinetic studies of atenolol in human subjects. Conclusions We have validated a LC MS/MS method developed by (Li et al., 2005) for atenolol using protein precipitation. The method requires only a small amount of plasma (100 ) and can be applied to quantitate concentrations of ateno lol in human plasma samples. The method was shown to be rapid, sensitive, selective, and reproducible. The method was used to determine atenolol concentrations in the pha rmacokinetic study reported in C hapter 3.

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28 A) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Time (min) 0 10 20 30 40 50 60 70 80 90 100 0.24 0.92 1.68 0.12 0.70 0.80 0.59 1.60 0.53 1.52 0.40 1.01 1.22 1.08 1.45 2.07 1.92 B) 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 1.69 0.93 0.11 0.41 0.28 1.41 1.15 0.86 1.01 0.78 1.32 0.57 1.89 2.05 024 Figure 2 1. The extract ed LC -MS/MS chromatograms of: A) double blank plasma (no atenolol or ISTD); B) Blank plasma with ISTD; C) atenolol lowest standard (5ng/ mL ); C) plasma sample from a patient obtained after 2.5hours after oral administration of atenolol 100mg and Blank with ISTD Time (min) Relative Absorbance

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29 C) 000 50 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 1.66 0.93 1.88 1.37 1.29 1.17 0.18 0.33 1.99 0.44 0.52 2.08 0.75 167 D) 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 1.67 0.92 1.32 0.67 0.61 0.87 1.19 0.45 1.44 1.03 0.15 0.37 1.98 2.02 2.09 169 Figure 2 1. Continued Time (min) Time (min)

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30 0.000 200.000 400.000 600.000 800.000 1000.000 1200.000 1400.000 1600.000 0 5 10 15 20 Time, hours Atenolol plasma concentration, ng/ml Figure 2 2. Mean ( SD) concentration time profile of aten olol after atenolol 100mg in fifteen patients Table 2 1. Intra and inter -run precision (%RSD) and accuracy (%RE) for atenolol quality con trol samples in human plasma Concentration (ng/ mL ) Precision (% CV) Accuracy (%Deviation) Nominal Observed (mean SD) Intra run (n=12) 15 .00 16.99 1.23 7.3 13.3 200 .0 182.6 10.1 5.5 8.7 750 .0 733 51.5 4.6 3.4 Inter run (n=24) 15 .00 16.78 1 1.9 7.8 11.9 200 .0 190.05 10.91 5.7 5.0 750 .0 733 51.53 7.0 2.3

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31 CHAPTER 3 ATENOLOL PHARMACOKINETIC STUDY Introduction Atenolol is a se lective B1AR blocker widely used for the treatment of HTN (Barrett et al., 1973; Mason et al., 1979) Despite the body of literature documenting the adverse metabolic effects (AME) of atenolol on glucose, insulin and lipids, there are no studies that have addressed the relationship between the atenolol plasma concentration and the occurrence of the drug induced change in triglycerides or change in insulin sensitivity (Day et al., 1979; Weiner and Rossner, 1983; Pollare et al., 1989b; Reaven et al., 1996; Bonner et al., 1997) Also, as mentioned earlier there is significant variability in the pharmacokinetic parameters of atenolol that can potentially affect the dev elopment of the AME in different individuals (Brown et al., 1976; Fitzgerald et al., 1978; Barnwell et al., 1993) The euglycemic hyperins ulinemic clamp is considered the gold standard for assessing insulin sensitivity (Uwaifo et al., 2002) However, it is a labor intensive, invasive and expensive method (Trout et al., 2007) Besides measuring simple fasting plasma glucose values, the oral glucose tolerance test (OGTT) is the most common method used for the di agnosis of t ype 2 diabetes by clinicians; it is cost effective and easy to perform, unlike the clamp method (American Diabetes, 2008) We hypothesize that the occurrence of the metabolic abnormalities with atenolol are concentration related and that people with higher plasma atenolol exposure are more likely to d evelop AME. For te sting this hypothesis, we conducted a 24 hour pharmacokinetic (PK) study in hypertensive patients who were dose titrated to 100 mg atenolol for 4 weeks after taking atenolol 50 mg for 3 4 weeks. We also measured glucose and insulin plasma concentrations af ter a 2 hour OGTT and the change in lipid levels from the start of therapy until the time of the PK study visit.

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32 Materials and M ethods Study D esign This was an open label study in hypertensive patients aged 18 to 65 years of any race ethnicity and gender Subjects were recruited from Pharmacogenomic Evaluation of Antihypertensive Response s (PEAR) study. The details of PEAR study are as follows: PEAR Protocol PEAR is an ongoing hypertension pharmacogenetics study. It is a prospective, open label, randomi zed study of the beta blocker, atenolol and diuretic, HCTZ given as monotherapy or combination to determine which genes are associated with either antihypertensive response or AME (Johnson et al., 2009) Patients are recruited from the U niversity of Florida (Gainesville, FL), Mayo Clinic (Rochester, MN) and Emory University (Atlanta, GA). These centers will recruit 800 mild to moderate essential hypertensive patients male or female, of any ethnicity and between the ages of 17 and 65. Pat ients, who have secondary forms of HTN, isolated systolic HTN, heart rate < 55 beats/min, known cardiovascular disease, diabetes mellitus (Type 1 or 2), renal insufficiency, primary renal disease, pregnancy or lactation are excluded from the PEAR study. Su bjects who are enrolled in PEAR and randomized to atenolol as their first study drug were asked by the PEAR study personnel about their interest in learning more about the PK study and if they express ed interest, they were contacted to have the study expla ined to them. Subjects from PEAR who are on atenolol 100 mg once daily were invited to participate. The protocol was approved by the University of Florida Institutional Review Board and all study subjects provided written informed consent. Inclusion criter ia: We enrolled all patients at the University of Florida who were randomized to atenolol monotherapy in the PE AR study, were on 100mg dose and consented to participate.

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33 PK Study protocol: Participants came to the University of Florida, General Clinical Re search Center (GCRC) as an outpatient after four weeks on 100 mg atenolol. They were asked to avoid any fruit juices for four days before the study visit. Patients fast ed for 8hours before reporting to the GCRC at 8am on the study day. After a basic clinic al examination including height, weight, blood pressure and heart rate; a forearm vein of the subject was cannulated w ith a plastic catheter Fasting lab measu rements of glucose, insulin, triglycerides, HDL -cholesterol, total cholesterol were done. At 8.30am they were given an atenolol 100mg tablet (Sandoz Inc., Princeton, NJ, USA). Blood was drawn at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 hours after atenolol dosing into heparinized vacutainers for analysis of atenolol. Blood samples for measuring H DL -cholesterol, triglycerides and to tal cholesterol were collected in vacutainers with EDTA (Becton Dickinson, Franklin Lakes, NJ, USA ) All patients underwent a two hour OGTT, one hour after atenolol dosing, whereby they were given 75g glucose solution (S /P lemon lime glucose tolerance beverage, Cardinal Health, Mcgaw Park, IL, USA) to drink. Glucose and insulin were measured during the OGTT at every 30 minutes by drawing blood in vacutainers with sodium fluoride for the measurement of glucose and in serum separator tubes for insulin. Following separation of the plasma, the samples were frozen and stored at 20 0C until assayed. Regular meals were served after the end of OGTT. Patients went home after the 12 hour blood sample and returned for the final 24 h our sample. Laboratory M easurements Atenolol was assayed by LC/MS/MS using a validated method as described in Chapter 2 Glucose was measured on YSI model 2300 STAT PLUS analyzer using glucose oxidase enzyme in the GCRC University of Florida Insulin was measured with Roche/Hitachi Modular by

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34 Shands laboratories, Gainesville, FL USA Lipids were measured using s pectrophotometric methods by Quest diagnostics, Tampa, FL USA. Pharmacokinetic D ata A nalysis The measured atenolol Cp was evaluated using non -co mpartmental pharmacokinetic data analysis using WinNonlin software (version 2.1, Pharsight Corporation, Mountain View, CA, USA) Terminal elimination half life (t1/2) was assessed by non linear regression. AUC within one dosing interval (AUC 024) at stead y state was calculated using the trapezoidal rule. Maximum concentration Cmax and time of maximum concentration Tmax was observed directly from the measured data The concentration time profile of the fifteen patients is shown in Figure 3.1. Pharmacodynami c Data Analysis Homeostatic model assessment (HOMA) of insulin resistance, HOMAIR was calculated as the product of basal glucose ( mmol/L cell function (HOMA BC) computed as product of 20 and basal insuli n ( /mL) divided by value of basal glucose ( mmol/L ) concentration minus 3.5. The AUC for glucose and insulin during the OGTT (AUC0 2) were calculated by the trapezoidal rule. Statistical M ethods With a sample size of 15 patients, we have 80% power at P=0.05 two-sided to detect a target population correlation of 0.62 between log atenolol AUC and log glucose AUC. The use of natural logarithms limits the effects of potential outliers We normalized atenolol AUC by body weight prior to log transformation. Des criptive statistics are given as means SD. We also fitted a multiple regression model of log atenolol AUC against log glucose AUC, log insulin AUC and change ( log atenolol AUC with ( (

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35 with stepwise adding weight, BMI, waist circumference and hip circumference in the model. The change from baseline lab values were compared with paired t test. All statistical analyses were conducted using SAS software ( ve rs ion 9.1 SAS Institute, Cary, NC, USA). Results A total of twenty patients were enrolled and fifteen patients completed the study (6 men, 9 women). Four patients declined to participate after consenting and one patient was consented while taking the 50 mg dose but was never titrated to the 100 mg dose. The patient characteristics are reported in Table 3 1. The mean age was 46 8.9 years, the majority of patients were women (60%), Caucasians (80%) and overweight with BMI of 28.5 5.9 kg/m2, waist circumfere nce was 35.93 6.45 inches. As there were many overweight patients and atenolol AUC024 was associated with body weight (r= 0.65, p=0.009) and BMI (r= 0.57, p=0.025), we normalized the atenolol AUC0 24 with respect to body weight (AUCn). Atenolol AUC0 24 and AUCn, glucose AUC0 2 and insulin AUC0 2 values are reported in Table 3 2. The atenolol pharmacokinetic estimates are reported in Table 3 3. The average atenolol AUC024 was 10,052 ng.hr/ mL, the maximum concentration (Cmax) achieved was 1195.7 ng/ mL time to achieve Cmax was 2.6 hours with a half life of 6.4 hours, the apparent oral clearance (Cl/F) was 182.3 mL /min and apparent volume of distribution (Vd/F) was 100.0 liters which is consistent with previous literature (Brown et al., 1976; Mason et al., 1979) We also calculated glucose and insulin AUC02 during the 2 hour OGTT. The mean glucose AUC02 was 241.4 mg.hr/dL and mean insulin AUC02 The glucose and insulin levels during the 2 hour OGTT are shown in Figures 32a and 3 2b respectively. Lipid data was not available for 3 patients and insulin data was not available for 6 patients and so they were not included in the a nalysis. There were no statistical significant

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36 changes in any of the metabolic parameters after 8 weeks of treatment. The changes in lipids and glucose, insulin after atenolol treatment are reported in Tables 3 4 and 3 5. The association between the atenol ol AUCn and other variables including log glucose AUC, log insulin AUC, body weight, BMI, waist circumference, hip circumference was evaluated. We also looked if the change in triglycerides, other lipids, glucose and insulin levels after atenolol treatment were associated with atenolol AUCn. Subsequently, we did a regression analysis in which we forced these variables in the model. The correlation coefficients are reported in Table 3 6 and Table 3 7. There was no relation between atenolol AUCn and glucose AUC (r= 0.078, p=0.78), however, there was a significant negative relation between atenolol AUCn and insulin AUC (r= 0.69, p=0.0043). We did not find any relationship between atenolol AUCn and ( <0.001, p=0.97), ( p=0.88). However, log atenolol AUC was inversely related to ( 0.61, p= 0.047). We performed a forward step wise regression of atenolol AUCn vs. log insulin AUC and (The analysis los t sig nificance for atenolol AUCn and log insulin AUC, ( once we adjust for other variables like weight and BMI (Table 3 8 ). We observed four patients (# 3,11,14,15) who had plasma glucose levels above 140 mg/dL at the end of 2 hour OGTT and hence were classified as having impaired glucose tolerance according to World Health Organizations and American Diabetes Associations criteria (American Diabetes, 2008) (Figure3 2a). The mean atenolol AUC024 for these four patients was 8697 ng.hr/ m L and mean glucose AUC02 was 303.6 mg.hr/dl and the mean atenolol AUC0 24 for rest was 10599.3 ng.hr/ mL and mean glucose AUC02 was 218.79 mg.hr/dl. T wo patients (# 5, 12) developed hyperinsulinemia, mean insulin AUC02 was 345.26 mU.hr/ mL and

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37 their mean atenolol AUC was 9195.8 ng.hr/ mL (Figure3 2b). Patients 4, 7, 10, 14 and 15 had low insulin levels and low insulin AUC02 and had high atenolol AUC024 (Table 3 2). Their mean atenolol AUC0 24 was 13613.01 ng.hr/ mL and mean insulin AUC0 2 was 45.92 .hr/ mL and mean atenolol AUC024 in remaining patients was 8331.86 ng.hr/ mL and mean insulin AUC0 2 was 156.87 .hr/ mL Discussion We hypothesized, the occurrences of the metabolic abnormalities with atenolol we re concentration related and that peop le with higher plasma atenolol exposure are mo re likely to develop these AME. These short term AME have the potential to subsequently develop into new cases of type 2 diabetes, dyslipidemia. Beta adrenergic receptor blockers decrease insulin sensitivity, an effect observed with nonselective beta blockers as well as selective beta blockers at higher doses when they lose their specificity and bind to beta 2 adrenergic receptors (Lundquist, 1971; Pollare et al., 1989b; Jacob et al., 1998) We did not find any association between atenolol AUCn and glucose AUC 0 2 and a significant negative correlation between atenolol AUCn and insulin AUC 02 and ( in was lost in the regression model after adjustment for weight and BMI indicating that the association was with these variables. Similarly, we did not observe any association with atenolol AUCn and change in triglyceride levels after atenolol treatment. T his lack of association may be is the result of small sample size and short follow up of the patients. We found patients who had delay in insulin response over the 2 hour OGTT, which can be due to decrease in insulin secretion (Lundquist, 1971; Pollare et al., 1989b; Jacob et al., 1998) and two patients (# 5, 12) who were hyperinsulinemic and had low glucose levels at the end of OGTT. Patients 4, 7, 10, 14 and 15 had low insulin levels and low insulin AUC0 2 during 2 hour OGTT and had high atenolol AUC024 ; however, this is not statistically significant. This can be

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38 explained by the fact that the beta 2 adrenergic receptors located in the pancreas being blocked by atenolol at higher doses and thereby reduce insulin secretion (William -Olsson et al., 1979) Insulin is secreted in a biphasic manner on glucose exposure; there is an instantaneous first phase insulin secretion followed by second lag phase. Beta blockers decrease the first phase of insulin secretion leading to more insulin secretion during the second phase thereby causing hyperinsulinemia. None of these observations we re statistically significant which may be due to a small sample size and other confounding variables. Atenolol AUCn and log glucose AUC were not associated with each other. Beta blockers have been found to increase blood glucose levels (Cerasi et al., 1972; Wright et al., 1979) We found four patients (# 3, 11 14 and 15) who had impaired glucose tolerance. Their glucose levels remained higher during the 2 hour OGTT but they had normal insulin levels and atenolol AUCn was not significantly different fr om the rest of the patients (American Diabetes, 2008) There was no associat ion between lipid changes after atenolol treatment and atenolol AUCn. There are studies that have observed neutral or opposite effects of BB on the exposure on lipids (Eliasson et al., 1981; Weiner and Rossner, 1983) This might be due to short period of treatment or lack of dietary control in the study period. We did no t have any restrictions on diet during the study period and we also did not maintain a food log or physical activity which can affect the lipid levels. Only one patient was on lipid lowering therapy (simvastatin), no other patient was on any other co medic ation which can potentially confound the observations. In our study, patients were taking atenolol 50mg for 3 4 weeks, after which they took atenolol 100mg for additional 4 weeks, so in all these patients were on atenolol treatment for average 8 weeks. In this short period of time, we were not able to see the development of AME on fasting glucose and triglyceride levels in this small cohort but more abnormalities were

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39 evident in the OGTT. There are no previous studies that have used plasma concentrations o f BBs to correlate with the glucose and change in triglyceride levels. We did not find plasma atenolol concentration is a risk factor for patients developing AME. However, our study has small sample size and our power was to detect a correlation coefficie nt of 0.62. It is possible that smaller correlations would be evident with a larger sample, although the data do not suggest this would be the case. Further studies should be conducted to determine the possible causes for development of AME because of BB treatment; if we can identify patients who will develop AME, we may be able to reduce the incidence of new onset diabetes in these patients. Our data do not suggest atenolol exposure is one of the important factors. If the factors that do place a patient at risk for the AMEs can be identified, subsequently these important anti hypertensive agents can continue to be used in the remaining population without bias relative to other anti -hypertensive agents due to these AME.

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40 Table 3 1. Clinical characteristics of patients (n = 15) Age, years (mean SD) 46 8.9 Gender: Men /Women (%) 40/60 Race: Caucasians / African Americans (%) 80/20 Systolic blood pressure mm Hg 133.13 13.17 Diastolic blood pressure mm Hg 81.86 6.79 BMI kg/m 2 28.5 5.9 Waist circu mference ,inches 35.93 6.45 Hip circumference, inches 38.21 12.09 Fasting glucose, mg/dL 81.8 7.79 Fasting insulin, I U / mL 8.26 7.5 Fasting triglycerides, mg/dL 149.93 84.97 Fasting HDL cholesterol, mg/dL 50.46 19.18 Fasting Total cholesterol, mg/dL 191.73 33.85 HOMA IR 1.73 1.56 HOMA BC 182.75143.04 Data are expressed as mean SD

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41 Table 3 2. A tenolol AUC and glucose, insulin AUC during OGTT Patient # ATN AUC0 24, ng.hr/ mL Log ATN AUC0 24, (AUCn) ng.hr/ mL .kg Glucose AUC0 2, mg.hr/dL Log g lucose AUC0 2, mg.hr/dL Insulin AUC0 2 IU.hr/ mL log Insulin AUC0 2 IU.hr/ mL A 0 1 7213.9 4.64 230.7 5.44 104.79 4.65 A0 2 7746.6 4.42 190.7 5.25 87.94 4.48 A0 3 6211.6 4.28 326.7 5.79 179.26 5.19 A0 4 13026. 1 5.16 209.5 5.34 36.61 3.60 A0 5 8969.6 4.41 268.3 5.59 280.47 5.64 A0 6 9127.8 4.76 202.5 5.31 113.65 4.73 A0 7 15189.1 5.55 268.7 5.59 28.86 3.36 A0 8 12345.9 5.09 161.5 5.08 128.33 4.85 A0 9 8060. 1 4.7 224.5 5.41 48.14 3.87 A 10 16434.0 5.63 193.3 5.26 43.54 3.77 A 11 5163.7 3.76 241. 3 5.49 156.63 5.05 A 12 9422. 1 4.57 214.5 5.37 410.06 6.02 A 13 9057. 2 4.82 242.5 5.49 59.46 4.09 A 14 10675 3 5.17 276.5 5.62 49.69 3.91 A 15 12740.6 5.21 369.7 5.91 70.84 4.26 M ean 10092.3 4.81 241.4 5.46 119.88 4.55 SD 3253 .7 0.5 54.3 0.22 104.67 0.77 CV 32.3 9.61 22.5 3.94 87.31 17.01 Minimum 5163.7 4.28 190.7 5.25 36.61 3.36 Maximum 16434.0 5.17 369.7 5.91 41 0.06 6.02 Fold diff. 3.2 1.21 1.9 1.12 11.20 1.79

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42 Table 3 3 Pharmacokinetic estimates of atenolol Patient # C max ng/ mL T 1/2 hou rs T max hours Vd/F, liters Cl/F, mL/min A 01 943.3 7.1 1.5 141.7 231.67 A02 1128.6 6.3 2.0 118.2 215 .00 A03 1034.7 4.7 3.0 110.3 268.33 A04 1182.8 7.3 4.0 81.2 128.33 A05 912.1 6.8 2.0 110.2 185.00 A06 1100.4 6.4 2.0 101.0 183.33 A07 1506.1 6.7 4.0 63.6 110.00 A08 1448.6 6.0 4.0 70.0 135.00 A09 1051.6 4.8 3.0 86.5 206.67 A10 1255.4 10.7 3.0 93.9 101.67 A11 577.1 6.8 1.5 191.5 323.33 A12 1359.1 5.7 3.0 87.7 176.67 A13 1326.4 4.7 3.0 75.8 183.33 A14 1751.9 7.2 2.5 97.0 156.67 A15 1233.2 6.4 1.5 71.9 130.00 Mean 1187.5 6.5 2.6 100.0 182.33 SD 280.9 1.5 0.9 32.7 60.77 C V 23.6 22.1 33.7 32.7 33.32 Minimum 577.5 4.7 1.5 63.6 101.67 Maximum 1751.9 10.7 4.0 191.5 323.33 Fold diff. 3.1 2.3 2.7 3.0 3.20 Table 3 4 Changes in lipids after atenolol treatment Triglycerides, mg/dL (n=12) HDL, mg/dL (n=12) TC, mg/dL (n=12) Baseline 133.10 88.13 46.1217.07 200.9134.69 After 8 weeks 133.8765.38 48.6620.95 201.8338.71 Change 0.8755.98 2.536.4 0.9121.81 Data are expressed as mean SD, groups compared by paired t -test, no statistical significant ch ange in any variable

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43 Table 3 5 Changes in glucose, insulin after atenolol treatment Glucose, mg/dL (n=11) Insulin, / mL (n=9) HOMA IR (n=8) HOMA BC (n=8) Baseline 83.728.20 8.365.09 1.791.23 143.1759.18 After 8 weeks 84.456.68 9.146.26 2.071.34 160.32102.77 Change 1.906.18 0.773.2 0.280.6 17.1375.0 Data are expressed as mean SD, groups compared by p aired t -test, no statistical significant change in any variable Table 3 6 Pearsons correlation between atenolol AUCn and glucose AUC, insulin AUC Variable Atenolol AUC n Log i ns ulin AUC Log g lu cose AUC Atenolol AUCn 1 r= 0.69, p= 0.004 r= 0.078, p=0.78 Log i ns ulin AUC r= 0.69, p= 0.004 1 r=0.004, p=0.98 Log g lu cose AUC r= 0.078 p=0.78 r=0.004, p=0.98 1 Table 3 7 Pearsons correlation between atenolol AUCn and changes in lipids, glucose Variable Trig lycerides g lu cose insulin HOMAIR HOMABC HDL Atenolol AUCn r= 0.01, p= 0.97 r=0.26 p=0.42 r= 0.67, p=0.04 r= 0.49, p=0.21 r= 0.59 p=0.11 r=0.09 p=0.77 r=0.04 p=0.8 Table 3 8 Multivariate analysis Dependent variable Covariat e entered Estimate Standard error p value Log insulin AUC Atenolol AUC n 1.03 0.32 0.008 Log insulin AUC Atenolol AUC n 0.32 0.35 0.38 BMI 0.087 0.03 0.014 Atenolol AUC n 0.42 3.36 0.90 Weight 0.13 0.09 0.22

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44 0.000 200.000 400.000 600.000 800.000 1000.000 1200.000 1400.000 1600.000 0 5 10 15 20 Time, hours Atenolol plasma concentration, ng/ml Figure 3 1 Atenolol plas ma concentration (ng/ mL ) vs. time (hours) profiles (mean SD) after oral administration of 100 mg atenolol once a day

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45 A) 0 50 100 150 200 250 0 0.5 1 1.5 2 2.5 Time,hours Glucose, mg/dl A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 A13 A14 A15 B) 0 50 100 150 200 250 300 350 0 0.5 1 1.5 2 2.5 Time, hours Insulin, mU/ml A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 A13 A14 A15 Figure 3 2 Plasma L evels D uring OGTT A) G lucose B) Insulin

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46 A ) 5 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 3.5 4 4.5 5 5.5 6 Atenolol AUCn, ng.hr/ml.kg Log glucose AUC, mg.hr/dl B) 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 3.5 4 4.5 5 5.5 6 Atenolol AUCn, ng.hr/ml.kg Log insulin AUC, mU.hr/ml Fig ure 3 3 Correlation Coefficients (r) between A ) atenolol AUCn and glucose AUC 02, B) atenolol AUCn and insulin AUC 02

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47 CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS CVD is leading cause of death in the world and HTN is one of important risk factors for CVD. There are several classes of anti hyperte nsive agents used clinically to control HTN. BBs have been widely used for several years for their safety and efficacy in reducing mortality and morbidity. However, there have been several concerns with the use of BB as first line therapy in HTN over the n ew agents in development of AME on glucose, insulin and lipids. However, unlike the reports on low dose thiazide diuretics to have neutral effect on development of these AME, there are conflicting studies on BB associations with AME. We had hypothesized occurrence of the AME are related with degree of atenolol exposure, and patients with higher atenolol AUC will be more likely to develop the AME. However, we did not found any association between plasma atenolol concentrations and glucose levels during the 2 hour OGTT or change in triglycerides, change in glucose levels and between plasma atenolol concentrations. There are several key points to note from this study. The occurrence of AME on BB therapy is a major factor in the preference of other anti -hype rtensive agents over BB and we were not able to associate plasma drug levels of atenolol with the AME. This leaves the question unanswered of why BBs are associated with AME and though there is high variability in the PK parameters as seen in our study as well as other studies, we cannot associate this PK variability in the development of AME. Thus, further studies to explore the reasons for development of AME by BBs like atenolol are needed. Ultimately, we can avoid or prevent the occurrence of AME, these important anti -hypertensive agents can still be safely used rather than being rejected totally.

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54 BIOGRAPHICAL SKETCH Hrishikesh A. Navare was born in Maharashtra, India. He received his Bachelor of Technology in Pharmaceuticals and Fine Chemicals from University of Mumbai, India in 2003. He further received Master of Technology in Pharmaceuticals and Fine Chemicals from University of Mumbai, India in 2005. Thereafter he came to University of Florida to continue his graduate studies in pharmacy. Under the supervision of Dr. Julie A. Johnson he conducted a clinical p harmacokinetic study in hypertensive patients with focus on studying development of adverse metabolic effects of beta blockers. He graduated with Master of Science in Pharmacy in May 2009.