A Novel B1- blocker based on antisense oligonucleotides

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Title:
A Novel B1- blocker based on antisense oligonucleotides a new gene therapy approach in hypertension, cardiac hypertrophy and myocardial ischemia
Alternate title:
New gene therapy approach in hypertension, cardiac hypertrophy and myocardial ischemia
Physical Description:
vii, 126 leaves : ill. ; 29 cm.
Language:
English
Creator:
Zhang, Yuan Clare, 1971-
Publication Date:

Subjects

Subjects / Keywords:
Gene Therapy   ( mesh )
Oligonucleotides, Antisense -- therapeutic use   ( mesh )
Hypertension -- therapy   ( mesh )
Myocardial Ischemia -- therapy   ( mesh )
Hypertrophy, Left Ventricular -- therapy   ( mesh )
Adrenergic beta-Antagonists -- therapeutic use   ( mesh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 2000.
Bibliography:
Includes bibliographical references (leaves 104-125).
Statement of Responsibility:
by Yuan Clare Zhang.
General Note:
Typescript.
General Note:
Vita.

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Source Institution:
University of Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
oclc - 53949396
ocm53949396
System ID:
AA00011165:00001

Full Text









A NOVEL 11-BLOCKER BASED ON ANTISENSE OLIGONUCLEOTIDES:
A NEW GENE THERAPY APPROACH IN HYPERTENSION,
CARDIAC HYPERTROPHY AND MYOCARDIAL ISCHEMIA
















By

YUAN CLARE ZHANG


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2000































Dedicated to my husband Ryan, and my parents.













ACKNOWLEDGEMENTS


I would like to thank Dr. M. lan Phillips, Chairman of my supervisory

committee, for everything he has done for me in the past four years. I like his

hands-off style which makes me an independent thinker; I appreciate his faith in

me and enjoy the feeling of being backed up wherever I go; I cherish the

moments we talked about antisense, sense and nonsense. I am grateful for

having the best mentor who is a scientist, teacher, poet, friend and like a father.

I would like to thank Dr. Stephen Baker for his insightful comments and

providing the cell culture for in vitro experiments, and Dr. Jeffrey Hughes for

providing liposomes throughout my study. I also enjoyed the discussions with Dr.

Craig Gelband and his frank remarks.

I would like to thank Dr. William Hauswirth, Dr. Collin Sumners and Dr.

Luiz Belardinelli for their tremendous support and valuable suggestions.

I would like to thank my fellow student, Jon Bui, for his unselfish help in

my research and personal life. I sincerely appreciate Dr. Sara M. Galli, Leping

Shen, Dr. Keping Qian, Birgitta Kimura and Harold Snellen for making this

laboratory a wonderful place to study and stay.

Finally, my deepest thanks go to my parents, my husband and my big

family who have made everything possible.


















TABLE OF CONTENTS


ACKOWLEDGEMENTS


ABSTRACT

CHAPTER


1 INTRODUCTION


0 0 0 0 0 0 0 *


1.1 pi-Adrenoceptors *
1.2 Hypertension: Definition, Prevalence and Risks
1.3 pl-Adrenoceptors and Cardiovascular Diseases
1.4 Why Antisense Gene therapy for pi-Blockade?
1.5 Gene Delivery Vehicles (Viral vs. Nonviral Vectors)
1.6 Specific Aims *


2 GENERAL METHODOLOGY


S 0


2.1 Biochemical Methods 0 0 0 .
2.2 Molecular Biology Methods *
2.3 Pharmacological Methods .0 *. 0 0
2.4 Physiological Methods .
2.5 Clinical Pathology Methods *
2.6 Statistical Analysis .

3 OPTIMIZATION OF ANTISENSE DESIGN AND DELIVERY


3.1 Introduction
3.2 Results
3.3 Discussion


* 0 0 5 0 5
* S S S S 5
* S S 5 0 0


4 EFFECTS ON BLOOD PRESSURE AND HEART RATE:
pi-AS-ODN VS. ATENOLOL


4.1 Introduction
4.2 Results
4.3 Discussion


* 0 .


. 22


34

*. 34
* 36
*. 43


S. 48

*. 48
. 49
* 50


* 0 0


0 0 0 & 0 0 0 0 0 0 0


& 0 0 0 0 a 0 0 0 0 0 0 0 0 0 0 0










5 MECHANISMS FOR THE ANTIHYPERTENSIVE
EFFECTS OF pl-AS-ODN .

5.1 Introduction .
5.2 Results .
5.3 Discussion 0 .

6 REPEATED ADMINISTRATION OF p3-AS-ODN
AND SAFETY PROFILE 9

6.1 Introduction 0 .
6.2 Results .
6.3 Discussion .

7 PROTECTION BY pl-AS-ODN AGAINST MYOCARDIAL
DYSFUNCTION INDUCED BY ISCHEMIA-REPERFUSION

7.1 Introduction 0 0 0
7.2 Results 0 0 .
7.3 Discussion .

8 CONCLUSIONS AND FUTURE DIRECTIONS .

8.1 Conclusions .* .
8.2 Limitations of the Current Approach .
8.3 Future Directions .. *

PATENT AND PUBLICATIONS *

REFERENCE LIST .

BIOGRAPHICAL SKETCH *


S88

* 88
*..* 89
S *89
* 91


* 97
* 99
*.. *99
* 100

* 102

* 104

* 126


* *












Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

A NOVEL pl-BLOCKER BASED ON ANTISENSE OLIGONUCLEOTIDES:
A NEW GENE THERAPY APPROACH IN HYPERTENSION,
CARDIAC HYPERTROPHY AND MYOCARDIAL ISCHEMIA

By

Yuan Clare Zhang

August 2000

Chairman: M. lan Phillips

Major Department: Physiology

Objectives: p-Adrenoceptor antagonists (p-blockers) are effective in treating

hypertension, heart failure and myocardial infarction, but they have poor patient

compliance because of the daily regimen and side effects such as central

nervous system (CNS) reactions and nonspecific inhibition of p2-adrenoceptor

(P2-AR). To overcome these problems, I have developed a novel and specific pl-

blocker by targeting 31-adrenoceptor (pl-AR) mRNA with antisense

oligonucleotides (p1-AS-ODN) and optimized systemic delivery with cationic

liposomes. The effects of pi-AS-ODN on blood pressure, left ventricular

hypertrophy (LVH) and myocardial ischemia were tested in spontaneously

hypertensive rats (SHR) and isolated rat hearts subjected to

ischemia/reperfusion. The antihypertensive mechanisms were investigated and

the safety profile of repeated administration of p3-AS-ODN was studied.









Results: A single intravenous injection of 0.5-1 mg/kg l1-AS-ODN with cationic

liposomes significantly decreased blood pressure by 25-35 mmHg for 20-33 days

in SHR (P<0.05), with no effect on heart rate. 11-AS-ODN significantly reduced

the pl-AR levels in the heart and kidney, without affecting p2-AR. The

mechanisms for blood pressure reduction appeared to involve a rapid decrease

in cardiac contractility and a delayed but marked reduction in plasma renin

activity. Quantitative autoradiography revealed no changes in p-AR in the brain,

indicating the absence of CNS effects. pl-AS-ODN is effective with repeated

injections. It not only produced a sustained reduction in blood pressure, but also

significantly reduced LVH in SHR. No toxicity (assessed by liver transaminases,

hematology, and tissue histopathology) or immune response was observed

during long-term treatment. In addition, pretreatment with p1-AS-ODN

significantly attenuated cardiac dysfunction and lipid peroxidation of isolated rat

hearts induced by ischemia-reperfusion.

Conclusion: Systemic injection of pl-AS-ODN delivered by cationic liposomes

can reduce high blood pressure and cardiac hypertrophy, and can protect the

heart from ischemic injury. It is clearly longer-lasting than currently available p-

blockers and likely to have fewer side effects because of its p1-specificity and

lack of CNS effects and bradycardia. Repeated administration is effective without

causing toxicity or immune stimulation. Therefore 1p-AS-ODN is viable as a

potentially new prolonged antihypertensive agent with benefits for cardiac

remodeling and myocardial protection from ischemia.















CHAPTER 1
INTRODUCTION


1.1 31-Adrenoceptors

The sympathetic nervous system (SNS) plays an important role in the

regulation of a wide variety of physiological functions that are essential for

survival and quality of life. Most pbstganglionic sympathetic nerve terminals

release norepinephrine as a neurotransmitter, which exerts its effects through

interaction with adrenoceptors. Adrenoceptors (AR) were first subdivided into a

and p subtypes by Ahlquist in 1948 (1). p-AR was further classified into P, and 13

subtypes in 1967 (2) based on their relative potencies for the endogenous

agonists adrenaline (epinephrine) and noradrenaline (norepinephrine). A third

subtype P3 was discovered in 1989 (3). 3,-AR is the predominant AR subtype

expressed in the myocardium and renal cortex. When activated, P,-AR induces

positive inotropic, chronotropic and lusitropic responses to catecholamines in the

heart, i.e. an increase in myocardial contractility, heart rate and relaxation speed.

It also stimulates renin release from the juxtaglomerular apparatus in the kidney

and upregulates the peripheral renin-angiotensin system (4,5). Therefore the

combined effect of p,-adrenergic activation is an elevation in cardiac output and

blood pressure. P,-AR is essential for normal cardiac performance. Its







2

importance is manifested by the prenatal death of P,-AR knockout mice (6) and

by heart failure associated with impaired P,-AR functions (7,8).

p,-AR belongs to the superfamily of G-protein-coupled receptors that have

seven hydrophobic transmembrane domains. The ligand-binding site is located

in the transmembrane regions (9), and the third intracellular hydrophilic loop

appears to be most important for coupling 3,-AR to G protein (10). The coding

sequence of human P,-AR is 1434 bp long and contains no introns (11).

Although P,-AR has been characterized for many years, the understanding of its

intracellular signal transduction has largely derived from the study of P,-AR. The

putative signaling pathway for cardiac 3,-AR involves stimulatory G protein (Gs),

activation of adenylyl cyclase (AC), accumulation of cAMP, stimulation of cAMP-

dependent protein kinase A (PKA), and phosphorylation of key target proteins

(including L-type calcium channel [IcL], phospholamban and troponin).

P,-AR undergoes desensitization and down-regulation when exposed to

the agonists. Rapid desensitization occurs after acute activation (within minutes)

which appears to be through receptor phosphorylation and sequestration (12).

Chronic activation leads to receptor down-regulation which involves reduced

mRNA levels and enhanced degradation of P,-AR (13). Diminished density and

functional responsiveness of cardiac 3,-AR are common features of chronic

heart failure and myocardial infarction that result from an excessive adrenergic

drive in these diseases (7,14).









1.2 Hypertension: Definition, Prevalence and Risks

Hypertension, manifested by chronic high blood pressure, affects a large

population and is a silent killer leading to severe cardiovascular problems. The

conceptual definition of hypertension, according to Kaplan in his Clinical

Hypertension (15), is "the level of blood pressure at which the benefits (minus

the risks and costs) of treatment exceed the risks and costs (minus benefits) of

inaction." Numerically, hypertension is defined by the World Health Organization

(WHO) as the blood pressure (BP) higher than 160/95 mmHg (systolic/diastolic

BP). However, hypertension is treated more aggressively in the United States

with the clinical end point <140/90 mmHg. It is estimated that hypertension

afflicts one fourth of adults in America, i.e. 50 million people (16,17), and is the

No. 1 reason for office visits to specialists in internal medicine (18). However,

due to the asymptomatic nature of this disease, many patients are largely

unaware of high BP and therefore have little motivation to seek or follow

treatment. As a result, only about 50% of hypertensives in the U.S. get their BP

under control.

The danger of hypertension does not lie in high BP itself, but in the

complications of high BP. Hypertensives generally suffer premature disability or

death from cardiovascular diseases, because chronic untreated high BP

increases the incidence of deadly events such as stroke, heart failure,

myocardial infarction and renal dysfunction (16,19). The mechanisms are

complex and may involve the structural changes in blood vessels and cardiac







4

tissues termed hypertrophy and remodeling, which eventually lead to organ

damage and functional insufficiency (20).


1.2.1 Hypertension: a Major Risk Factor for Heart Failure

Hypertension and heart failure are closely associated. Heart failure, a

pathophysiological state whereby the heart fails to pump sufficient blood to meet

the oxygen requirements of the body, is life-threatening and affects 4 million

Americans (21). The prevalence is rising with 400,000 new cases every year,

mostly in patients over 65 years old. The best available data on hypertension as

a prelude to heart failure were derived from the Framingham study (22). This

study involved follow-up of more than 5,000 patients for a median of 14 years,

and found that hypertension is the largest single risk factor for the development

of heart failure, accounting for 39% of attributable risk in men and 59% in

women. How hypertension degenerates into heart failure is not completely

understood. However, it is well accepted that the heart responds to a prolonged

pressure overload in three major ways (23): 1) left ventricular hypertrophy (LVH),

2) adrenergic stimulation of heart rate and contractility, and 3) volume expansion.

Mild hypertension increases the risk of LVH by 2-3 times, and severe

hypertension leads to tenfold elevated risk (24). LVH is initially a compensatory

response to hypertension which helps maintain normal cardiac performance, but

gradually progresses to cardiac remodeling and dysfunction. An overactive

adrenergic system, indicated by an increase in norepinephrine (NE) release (25)

and sympathetic nerve traffic (26,27), has been observed in the early stage of







5

essential hypertension and results in the diminished responsiveness of cardiac

P-adrenergic receptors in failing hearts (7).


1.2.2 Hypertension: a Major Risk Factor for Myocardial Infarction

Hypertension is also a major risk factor for myocardial infarction.

Myocardial infarction occurs when there is an abrupt decrease in the coronary

blood flow after the thrombotic occlusion of a coronary artery previously

narrowed by atherosclerosis. Approximately 1.5 million myocardial infarctions

occur in America every year, with about 30% mortality rate. In the Framingham

Heart Study, 30-year follow-up data revealed that the incidence of myocardial

infarction was proportional to the level of systolic BP in a wide range from

normotension to severe hypertension (28-30). Active treatment of isolated

systolic hypertension in the elderly resulted in a 27% reduction in myocardial

infarction and coronary heart disease (CHD)-related mortality (31). The

mechanisms by which high BP increases the risk of CHD are yet unclear.

Extensive studies on hypertensive rats revealed that Ca2+ is elevated excessively

in myocytes and coronary artery wall of these animals. The Ca2+ overload

increases the incidence of coronary spasm, which occurs in the early phase of

acute myocardial infarction (32). Systemic hypertension also induces LVH, which

reduces coronary flow reserve and renders the heart vulnerable to ischemia-

induced damage (33). In addition, chronic sympathetic activation leads to

endothelial dysfunction (34) and vascular smooth muscle hypertrophy (20) that

further promote the development of atherosclerosis.











1.3 pl-Adrenoceptors and Cardiovascular Diseases

1.3.1 Overactive SNS in Cardiovascular Diseases

Overactive sympathetic activity plays a crucial role in the pathogenesis of

cardiovascular diseases, including essential hypertension, congestive heart

failure and myocardial infarction (35-37). Nearly 30 years ago, Julius et al. (38)

observed that a noticeable fraction of subjects with borderline hypertension had

an increased sympathetic and a decreased parasympathetic drive. This finding

has since been supported by a large body of evidence. A meta-analysis of all

published data (25) showed that plasma NE levels as an indirect marker of

sympathetic tone were significantly elevated in younger patients with essential

hypertension than in normotensive individuals, which was accompanied by an

increase in heart rate and cardiac output (39). Concomitantly, NE spillover rate

(40-42) and muscle sympathetic nerve traffic (27,43) rise progressively with the

development of essential hypertension. Likewise, elevated plasma NE levels

were observed universally in symptomatic and asymptomatic heart failure

(44,45). Myocardial infarction is also associated with reduced baroreflex and

increased sympathetic activation (46). These data indicate the sympathetic

overactivation as a key participant in the initiation and maintenance of these

cardiovascular problems. PI-AR, a central mediator of SNS activity, is therefore

unequivocally involved in these deleterious events.










1.3.2 P3-Adrenoceptors and Hypertension

Pi-adrenergic activation induces positive inotropic and chronotropic

responses in the heart and stimulates renin release in the kidney, thereby

increasing cardiac output and upregulating the renin-angiotensin system (47). As

a result, P,-agonists such as dobutamine can rescue acute cardiac failure by

increasing myocardial contractility and heart rate (48). Pharmacological

interventions by p-blockers are effective in reducing mortality in high risk

arrhythmic patients and in lowering BP (49,50). All p-blockers can inhibit 31-AR

and share structural similarity to p-agonist isoproterenol. Since the introduction of

propranolol in 1964 (51), p-blockers have become the first line therapy for

treating hypertension as recommended by the Joint National Committee and

WHO. They are now proposed as the initial monotherapy for younger and

middle-aged hypertensives, especially white males, and for patients with high

levels of stress (52). The precise mechanisms by which these agents reduce BP

remain unclear (53). However it is generally agreed that the primary effect of p-

blockers is a reduction in cardiac output by 15-20%, resulting from the blockade

of cardiac p1-AR, thereby reducing both heart rate and myocardial contractility

(54). Besides, the inhibition of pi-AR in renal cortex suppresses renin production

and release, which may lead to a diminished peripheral renin-angiotensin system

(55). These concepts are supported by the observation that p-blockers are more

effective for patients with high cardiac output and heart rate (52) and high renin

profiles (56,57).










1.3.3 pl-Adrenoceptors and Heart Failure

The importance of fully functional pi-AR in the maintenance of normal

heart performance is apparent, as the diminished functional responsiveness of

P3-AR in chronic heart failure is closely correlated with the severity of impaired

cardiac functions (7,58). Therefore pf-blockade was considered to contraindicate

heart failure for a long period of time. However, early medical attempts to treat

heart failure with p-adrenergic agonists showed only short-term benefits, and

then precipitated symptoms with chronic use (59). This observation challenged

the prevailing wisdom that p-adrenergic activation improved cardiac functions

and therefore led to the pioneering trial by Dr. Hjalmarson's group in Sweden

treating heart failure with p-blockers in 1975 (60). Since then, their success has

been confirmed by overwhelming clinical and experimental evidence. These data

suggest that in contrast to acute supportive effects, chronic p-adrenergic

activation is detrimental to the heart (37,61).

Several recently completed multi-national, multi-center, large-scale clinical

trials achieved clinical end points in heart failure patients on P-blockers, including

improvement of systolic functions, exercise capacity and quality of life; and

reduction in morbidity, hospitalization and mortality (62-67). In the Metoprolol in

dilated Cardiomyopathy (MDC) trial (n=383), metoprolol treatment in patients

with IDC resulted in a 34% reduction of the primary combined endpoint, total

number of deaths and need for cardiac transplantation (62). In the US Carvedilol

studies (n=1094), carvedilol reduced mortality by 65% in patients with ischemic







9
and idiopathic cardiomyopathy, which was highly significant (67). This led the

Food and Drug Administration (FDA) to approve carvedilol for chronic heart

failure. p-blockers are also effective in the prevention of heart failure in

hypertensive patients. In the STOP trial (n=1627), old hypertensive patients

treated with p-blockers (atenolol, metoprolol or pindolol) for an average of 25

months showed a significant reduction in heart failure rate and total death

(68,69).

Many mechanisms have been proposed for the protective role of p-

blockers in heart failure [reviewed in (70,71)]. They include the following:

Improvement in cardiac function by restoring the density and functional

sensitivity of p-AR that are desensitized and down-regulated after chronic

sympathetic activation (62).

Reduction in oxygen consumption and metabolic demand by decreasing

heart rate (72,73), cardiac contractility and thereby ventricular workload.

Regression of left ventricular hypertrophy (74,75).

Inhibition of the renin-angiotensin system (23).

Attenuation of cytotoxic and apoptotic effects of catecholamine (76,77).

Furthermore, recent advances in physiological genomics corroborate

these clinical observations. Cardiac overexpression of human p1-AR (78), Gas

(79) or Gaq proteins (80) in transgenic mice improved contractile force only at a

young age, then developed ventricular hypertrophy and systolic dysfunction,

which ultimately resulted in a cardiomyopathic phenotype and premature death.

The mechanisms are not clear, but growing evidence suggests that p1-AR







10
mediates the proliferative, cytotoxic (76) and apoptotic (77) effects of excessive

NE on cardiomyocytes.


1.3.4 pl-Adrenoceptors and Myocardial Infarction

During acute myocardial infarction, excessive reflex activation of SNS

kicks in and increases platelet aggregation, myocardial ischemia and arrhythmia

(81). Elevated SNS activity has been considered an independent predictor for

sudden cardiac death after myocardial infarction (46). p-blockers, by inhibiting

cardiac pI-AR, can attenuate the detrimental effects of sympathetic activation

and thus protect the heart from severe damage and ventricular fibrillation. Three

clinical trials published in 1981 (82-84) demonstrated for the first time that 3-

blockers started after the onset of myocardial infarction reduced total mortality.

Since then, over 55 randomized trials have been carried out in more than 55,000

patients. A summary of 24 long-term trials including about 25,000 patients

showed that p-blockers reduced total mortality by 20% and sudden cardiac death

by 34% over 2 years of follow-up after myocardial infarction (85). They also

significantly decreased the incidence of recurrent infarction (83). It is now

suggested that patients (especially older patients) with acute myocardial

infarction, who have no contraindications to p-blockers, should be treated with

early intravenous p-blockade followed by oral p-blockers indefinitely (81).

Many types of p-blockers including timolol, metoprolol, propranolol,

acebutolol, pindolol, carvedilol and bisoprolol have been found to significantly

reduce total mortality after myocardial infarction to similar extent. The property







11

they have in common is pi-blockade. Therefore pl-blockade appears to be the

essential life-saving ingredient.

In animal models of ischemia/reperfusion, the mRNA level and density of

pi-AR increased during acute infarction and decreased after chronic infarction

(14,86-88). Pretreatment with p-blockers significantly reduced infarct size and

prevented ventricular dysfunction compared with control animals (89).

The theoretical benefits of p-blockade in myocardial infarction include a

decrease in oxygen demand, an antiarrhythmic effect, and a positive influence

on myocardial metabolism (90). The decrease in oxygen demand results

primarily from a reduction in heart rate and contractility. Excessive release of

catecholamine during myocardial infarction can be arrhythmogenic. Sudden

cardiac death accounts for almost half of mortality after myocardial infarction,

which is due to myocardial tachyarrhythmia and fibrillation (91). p-blockers can

prevent arrhythmia and are the only drugs that have consistently reduced

sudden death in large trials (85). In addition, p-blockers prevent the

derangement of cardiac energy metabolism. The cellular mechanisms underlying

the beneficial effects of p-blockade in myocardial infarction are being studied

extensively, which seem to involve the protection of intracellular Ca2+

homeostasis and protein phosphorylation (92), antioxidant changes (93),

reduction in inflammatory cytokines (94), and favorable energy metabolism

(95,96).












1.4 Why Antisense Gene therapy for pi-blockade?

1.4.1 Drawbacks of Current p-blockers

It is evident that p-blockers are reliable, successful and relatively safe in

the control of hypertension, heart failure, myocardial infarction and arrhythmia. In

spite of the success, this traditional drug therapy has many shortcomings. For

example, the compliance for p-blockers in hypertensive patients has been less

than 50% by some estimates (97). The reasons are multiple. First, the obvious

asymptomatic nature of the initial phases of hypertension renders this disorder

underdiagnosed and undertreated. Second, p-blockers are associated with a

number of side effects due to nonspecific p2-antagonism (98) and central

nervous system (CNS) reaction (99). Another problem with p-blockers, which is

also true for all the available antihypertensive drugs, is short action, requiring a

mandatory daily regimen. This further discourages patient compliance and

produces a potentially dangerous peak-and-trough fluctuation of 24-hr BP (97).

f2-blockade

Most of the unwanted effects of p-blockers are caused by the blockade of

p2-mediated functions, including bronchodilation, vasodilation, and mobilization

of glucose and free fatty acids (98). Limited by pharmacological drug design,

truly pi-specific antagonists have not been discovered, making these adverse

effects inevitable even with highly Pi-selective blockers at therapeutic doses

(100).







13
Bronchodilation and vasodilation: Pulmonary bed and blood vessels

express high levels of 12-AR, activation of which leads to bronchodilation and

vasodilation. The suppression of this receptor leaves the al-mediated

vasoconstriction unopposed and results in elevated airway and vascular

resistance (101,102). Therefore nonselective 3-blockers are generally

considered to contraindicate asthma and peripheral vascular diseases.

Blood sugar. p2-adrenergic activation promotes glycogenolysis and

gluconeogenesis in the liver and thereby helps maintain normal glucose levels in

the plasma. Nonselective p-blockers tend to decrease glucose tolerance. pi-

selective agents are preferred over nonselective agents for patients with insulin-

dependent diabetes mellitus, because nonselective p-blockers can increase the

duration and severity of insulin-induced hypoglycemia, and pi-selective agents

have a less pronounced effect on blood glucose levels (103,104).

Quality of life: Nonselective p-blockers such as propranolol markedly

decrease quality of life when compared with the ACE inhibitor captopril (105). In

contrast, pi-selective drugs cannot be differentiated from ACE inhibitors in terms

of overall quality of life scores (105) and are superior to the calcium channel

blocker nifedipine (106). In patients with chronic heart failure, pi-selective

blockers showed significant advantages over nonselective agents in improving

quality of life and NYHA class (62,63,65,66,107). The reason is that p2-mediated

metabolic functions are largely unimpaired with pi-selective blockade (108,109).

These functions, including mobilization of glucose and free fatty acids, are

important in providing energy supply to and removing unwanted by-products from







14

exercising muscles. Therefore, post-exercise hypoglycemia is unlikely to occur,

which contributes to lesser fatigue with p3-selective agents (110).

Blood lipid profile: Serum levels of total cholesterol and low-density

lipoprotein (LDL) are generally not affected by p-blockers. But nonselective

agents such as propranolol significantly increase triglyceride (TG) concentration

and decrease high-density lipoprotein (HDL) levels (104,111), which have been

associated with a higher risk of atherosclerosis (112-114). These effects are due

to 32-blockade, as the changes are less apparent with p1-selective agents such

as metoprolol and atenolol (110). With highly p1-selective agents such as

bisoprolol, the changes appear to be minimal or absent (115).

CNS effects

In addition to the side effects induced by p2-inhibition, common complaints

about p-blockers include depression, impotence, vivid dreams and insomnia

(99). Because these effects are more pronounced with lipophilic agents that are

able to cross the blood-brain barrier and enter the brain (116,117), they are

generally attributed to the suppression of central p-AR functions (116).

Short action

Most p-blockers have short half-lives in plasma between 3 and 10 hr (90).

Therefore a daily regimen is necessary. This problem is not unique to p-blockers,

and all the antihypertensive drugs available today have to be taken at least once

a day (97). Compulsory daily medication, along with adverse effects, discourages

patients from staying on antihypertensive therapy.







15

These issues led us to conclude that the traditional pharmacological

approach for p-blockade has reached a plateau. A new and innovative way must

be considered to overcome these problems and to achieve better control of BP

and heart dysfunction. Because hypertension and its cardiovascular

complications are chronic disorders that affect a large population, any

improvement in patient compliance may lead to significant reductions in

morbidity and mortality in the long run and carry with it potential economical

benefits to society.


1.4.2 Antisense Gene Therapy

Ideally, such a new therapy should provide 31-specific inhibition, and

stable and persistent lowering of BP without CNS side effects or toxicity.

Antisense gene therapy for pi-blockade holds promise for achieving all these

goals.

The idea of using antisense oligonucleotides (AS ODN) to inhibit gene

expression was first proposed and performed by Zamecnik and Stephenson in

1978 (118). Antisense oligonucleotides are short (typically 15-25 bases in length)

synthesized and chemically modified DNA molecules that are complementary to

the mRNA sequences encoding proteins of interest. They bind to mRNA

transcripts in a specific manner by Watson-Crick base pairing and inhibit the

expression of target proteins.

Several mechanisms of action have been proposed for AS ODN and

demonstrated to work in different systems [reviewed by Crooke (119)]. One

theory is translational arrest by assuming a steric hindrance to prevent ribosomal







16

assembly and read-through (120-122). A second mechanism works through

activation of RNase H that degrades the RNA strand of an RNA-DNA duplex

(123-125). Thus the mRNA is cleaved irreversibly and cannot be translated into

proteins. In addition, AS ODNs have been designed to inhibit preRNA slicing

(126) and to form triple helix with DNA (127), but these appear to be possible

only in special cases.

In principle, an oligo of 12-15 bases long can provide sufficient affinity and

specificity to its complementary mRNA (128). The specificity of AS ODN is so

great that even two genes that differ by a mutation at a single base can be

inhibited differentially (129). Therefore gene targeting specificity is the most

obvious virtue of antisense strategy, as it is based on individual gene sequences.

This also endows antisense with another advantage, which is versatility.

Antisense can be designed to specifically intervene with the expression of any

gene whose sequence is known. The design of AS ODN is rational and the

production is inexpensive. Besides, AS ODNs are hydrophilic molecules that

cannot cross the blood-brain barrier to cause CNS effects (130).

Specificity, simplicity and easy availability of synthesized oligos, along with

developments in the human genome project, have made antisense technology a

powerful tool for basic and clinical applications. It has been used increasingly to

study gene functions in recent years (131-135). For example, transgenic mice

expressing an antisense RNA against angiotensinogen (AGT) mRNA in the brain

provided convincing data on the role of central AGT in the regulation of BP and

fluid homeostasis (136). AS ODNs have also been explored as therapeutic







17

agents to suppress harmful components involved in disease development (137),

including cancer (138,139), AIDS (140), autoimmune disorders (141,142) and

restenosis (143-145). Its success is manifested by the recent approval of the first

antisense drug, Vitravene, by FDA to treat cytomegalovirus-induced retinitis.

Furthermore, antisense can be delivered to produce long-term gene inhibition

and therapeutic benefits for treating cardiovascular diseases. Studies in our

laboratories have established that systemic delivery of antisense cDNA directed

at AT1-receptor or angiotensin-converting enzyme by adeno-associated virus

(AAV) results in a long-term attenuation (6 months) of hypertension in

spontaneously hypertensive rats (146) as well as in double transgenic mice

expressing human angiotensinogen and renin (147). The hypertension-

associated ventricular hypertrophy and endothelial dysfunction are concomitantly

reduced. Raizada's group (148,149) used retrovirus to deliver ATI-receptor

antisense which prevented the development of hypertension in SHR, and the

normotensive phenotype was passed to the second generation. In addition,

clinical trials are underway to evaluate the efficacy of AS ODN for treating

cardiovascular problems such as restenosis (narrowing of blood vessels) after

coronary angioplasty. These results indicate that antisense gene therapy is

conceptually sound and technically feasible for the treatment of cardiovascular

diseases including hypertension. Therefore a specific p,-adrenergic inhibition

without CNS effects should be achieved by antisense molecules targeted to P,-

AR.











1.5 Gene Delivery Vehicles (Viral vs. Nonviral Vectors)

It is evident that once a therapeutic target is defined, the success of gene

therapy largely depends on the choice of the delivery system. An ideal vector

should possess the following properties: (i) high efficiency of gene transfer; (ii)

low toxicity, non-immunogenicity and non-mutagenesis; (iii) simplicity to produce

on a large scale; (iv) maintained efficacy on repeated administration. Currently,

numerous systems have been developed for gene therapy, which fall into two

categories: viral and nonviral vectors.


1.5.1 Viral Vectors

The four viruses that have been broadly used for gene therapy are

retrovirus, adenovirus, adeno-associated virus (AAV) and herpes simplex virus

(HSV). The characteristics of these viral vectors are summarized in Table 1-1.

Although viral vectors are highly efficient in gene delivery, they suffer

several significant drawbacks that make them currently inappropriate for clinical

use, such as high acute toxicity, immunogenicity, risk for mutagenesis and

insufficient pharmaceutical quantities (viral titers) (150-152). The safety issue

has remained a major concern for viral vectors (153), and the critique became

more intense after the tragic death of a young patient after receiving adenovirus-

delivered gene therapy in 1999 (154,155). This incident alerts scientists of the

immature status of current viral gene therapy and warrants more scrupulous and

extensive study and improvement for viral vectors.











Table 1-1. Characteristics of presently available viral vectors for gene therapy.


Insert size

Viral titer
Viral production

Infectivity


Integration

In vivo risks


Latency


Retrovirus

Small (6.5kb)

Low
Helper-free

Dividing cells
only (except
Lentivirus)
Chromosomal
(random)
Mutagenesis


No


AAV

Small (5kb)

Low
Helper

Broad


Chromosomal
(not random)


No

No


Adenovirus

Intermediate
(8kb)
High
Helper-free

Broad


Episomal

Immune
response,
cytotoxicity
No


HSV

Large (36kb)

Low
Helper/helper-
free
Broad


Episomal

Cytotoxicity


Yes


1.5.2 Nonviral Vectors

The latest development in nonviral vectors includes gene gun (156,157),

cationic liposomes (158,159), polymer (160) and liposome/polymer complex

(161). Some drawbacks are associated with these vectors, i.e. lower efficiency

than viral vectors in gene transfer and transient gene expression. In spite of

these limitations, nonviral vectors are now being pursued vigorously as a

promising alternative for viral vector gene therapy, because of their low toxicity,

non-immunogenicity and feasibility to be produced in large quantities [reviewed

in (162,163)]. Liposomes, a traditional drug carrier used in pharmaceutics, have

established safety and pharmacological properties (164,165). They can also

reduce the toxicity of many compounds such as doxorubicin and anthracycline







20

(166,167). In addition, cationic liposomes are effective vehicles for gene delivery

(168) by facilitating cellular uptake of DNA, protecting DNA from degradation and

extending the circulation time (169). For example, cationic liposomes are used

routinely for in vitro transfection. Their therapeutic potentials as gene therapy

vectors have been demonstrated in clinical trials of a variety of diseases such as

cystic fibrosis (170) and cancer (171-173). Currently 75 clinical trials are using

liposomal vectors for gene delivery which involve approximately 21% of all

patients treated by gene therapy (174).



1.6 Specific Aims

3-blockers are used successfully to treat hypertension, heart failure and

myocardial infarction. However they have poor patient compliance due to the

mandatory daily regimen and side effects resulting from nonspecific 12-

antagonism and CNS reactions. Antisense gene therapy has the potential to

overcome these problems. The objective of this dissertation is to develop an

antisense oligonucleotide specifically against 1,-AR (P,-AS-ODN) and deliver it

systemically by nonviral vector cationic liposomes. Its antihypertensive effects

and underlying mechanisms will be studied in an animal model of human

essential hypertension. The potential benefits on inhibiting hypertension-

associated left ventricular hypertrophy and protecting the heart from myocardial

infarction will be explored. The safety profile of chronic treatment eith p,-AS-ODN

will be established.







21

AIM 1: To develop a P,-AS-ODN that can inhibit P,-AR expression specifically

and effectively without affecting other receptor subtypes.

AIM 2: To achieve efficient systemic delivery of P,-AS-ODN by cationic

liposomes. A commercially available liposome formula with established efficacy

and safety, DOTAP/DOPE, will be used to improve antisense delivery in vivo.

AIM 3: To test the antihypertensive effects of p,-AS-ODN in spontaneously

hypertensive rats (SHR). BP and HR will be measured by indirect and direct

methods, i.e. tailcuff and radiotelemetry.

AIM 4: To elucidate the mechanisms by which P,-AS-ODN reduces

hypertension. The effects of antisense on cardiac output, peripheral renin-

angiotensin system and CNS will be studied.

AIM 5: To investigate the effects of repeated administrations of P,-AS-ODN

on high BP and left ventricular hypertrophy of SHR. Three repeated

injections will be given to adult SHRs with established hypertension.

AIM 6: To study the safety profile of p,-AS-ODN after long-term treatment.

AIM 7: To explore the protective effects of p,-AS-ODN in an animal model of

myocardial infarction. Four days after antisense treatment, rat hearts will be

isolated and subjected to 30 min global ischemia followed by 30 min reperfusion.















CHAPTER 2
GENERAL METHODOLOGY


2.1 Biochemical Methods


Cell Culture

C6-2B cells and 1-15-5B cells were cultured in DMEM supplemented

with 5% fetal bovine serum (FBS), 100 U/ml penicillin, 0.1 mg/ml streptomycin

and 2.5 tg/ml amphotericin-B at 370C with 5% CO2 for 3-4 days until cells grew

to 80% confluency. The medium was changed to OPTI-MEM I (Life

Technologies, reduced serum medium). pl-AS-ODN or saline (as control) was

added to the medium.


Preparation of Liposomes and ODN/liposome Complex

The cationic lipid 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane

(DOTAP) was mixed with the helper lipid L-a dioleoyl phosphatidylethanolamine

(DOPE, Avanti Polar Lipids) at 1:1 mole ratio. The mixture was evaporated to

dryness with a rotary evaporator at 30C for 10 min. The lipid film was further

dried by nitrogen for 10 min to evaporate residual chloroform and resuspended

in sterile water to a concentration of 5 mg DOTAP/ml. The resultant mixture was

shaken in a water bath at 30-40C for 30 min and stored at 4C overnight. The







23

suspension was then briefly sonicated, stored at 40C and used within 3 months

(175). The average diameter of DOTAP/DOPE was 170nm.

ODN/liposome complex was prepared on the day of use by mixing desired

amount of ODNs with DOTAP/DOPE to the final DNA concentration of 300.ig/ml

in 5% (w/v) dextrose in water. The mixture was incubated at room temperature

for 40-60 min to facilitate the maturation of the complex. Rats were anesthetized

by inhalation of 6% isofluorene in a drop jar and injected intravenously through

tongue vein.


Plasma Renin Activity and Plasma Ang II Levels

Plasma renin activity (PRA) was determined using angiotensin I

radioimmunoassay kit (DuPont). Plasma Ang II levels were measured by radio-

immunoassay as previously described (176).


Determination of MDA Levels in the Myocardium

Malondialdehyde (MDA) levels in the myocardium were measured in

duplicate by a modified method of Ohkawa (177). Briefly, the ventricular tissues

were homogenized. The reaction mixture consisted of 0.1 ml tissue homogenate,

0.4 ml 0.9% NaCI, 0.5 mi 3% sodium dodecylsulfate and 3 ml thiobarbituric acid

reagent (containing equal amount of 0.8% aqueous thiobarbituric acid and acetic

acid) and was heated for 75 min at 95C. Thereafter the mixture was cooled by

addition of 1 ml cold 0.9% NaCI and extracted with 5 ml n-butanol. After

centrifugation at 3,000 rpm for 15 min, the butanol phase was assayed

spectrophotometrically at 532 nm. Tetramethoxypropane (in amounts of 0, 0.1,







24

0.2, 0.4, 0.8, 1.0 nmole) served as external standard. MDA levels were

expressed as .tmole/g tissue.



2.2 Molecular Biology Methods


Antisense Design

AS-ODN were designed according to the basic principles (178),

synthesized and modified by backbone phosphorothioation (Gemini).


RT-PCR

At different time points after the single injection of p1-AS-ODN or inverted-ODN

(as control), rats were sacrificed, renal cortex was dissected from left kidneys,

dipped into RNAlater tissue storage buffer (Ambion) immediately and stored at -

200C. Total RNA was extracted using RNAwiz reagent (Ambion) and quantified

by spectrophotometer. RNA samples from 4-5 rats of each time point were

pooled. 5ptg RNA was digested by DNase I, reverse transcribed by Superscript

reverse transcriptase (Life Technologies) at 420C for 50 min, and 1/20 of RT

product was used to run PCR for 20 cycles. PCR primers for renin were 5'-

AGGCAGTGACCCTCAACATTACCAG-3' (forward) and 5'-CCAGTATGCA-

CAGGTCATCGTTCCT-3' (reverse) and yielded products of 362 bp. Primers for

GAPDH were 5'-ATCAAATGGGGTGATGCTGGTGCTG-3' (forward) and 5'-

CAGGTTTCTCCAGGCGGCATGTCAG-3' (reverse) and yielded products of 505

bp (179).









Southern Blot

RT-PCR products were subjected to Southern blotting, hybridized with

psoralen-biotin labeled cDNA probes and detected using nonisotopic kits

(Ambion). After exposing membranes to X-ray films, the intensity of renin

mRNAs was quantified by densitometry and normalized with GAPDH mRNA

levels. The experiments were repeated twice.



2.3 Pharmacological Methods


Membrane Preparation and p-AR Binding Assay

Membrane preparation and binding assay were carried out as previously

described (180). Briefly, cells were detached and scraped from the cell culture

plates. Rats were decapitated, and hearts and kidneys were removed. Atria,

valves and fat were dissected from the ventricles. Cells and tissues were

homogenized in ice-cold 50mM Tris-HCI buffer at pH 7.5. Then the suspensions

were centrifuged at 48,000xg for 10 min at 40C. The pellet was resuspended in

ice-cold 50mM Tris-HCI at pH 7.5 with 5mM MgCI2, passed through a mosquito

net to remove large pieces of connective tissues and centrifuged at 48,000xg for

10 min. The resulting pellet was washed once more and diluted in 50mM Tris-

HCI, 5mM MgC12, pH 7.5. Protein content was determined by Lowry's method.

For saturation binding experiments, 100.tg membrane protein was incubated with

six concentrations of 1251-(-)iodocyano-pindolol (I-CYP, NEN Life Science, 6.25-

100pM) in a total volume of 250pl containing 50mM Tris-HCI (pH7.4), 5mM

MgCI2 at 360C for 60 min. For the single point binding, assay was performed with







26

100pM 1251-CYP. The nonspecific and 32-AR binding levels were determined in

the presence of 1l.M ()-alprenolol and 150nM CGP20712A (RBI), respectively.

Then the reaction mixture was passed through Whatman GF/B glass fiber filter

using Brandel harvester and the bound radioactivity was counted for 1 min. All

binding assays were performed in triplicate, with the results varying by less than

10%.


Tissue Preparation and Quantitative Autoradiography

Rats were anesthetized and perfused with 0.9% saline before euthanasia.

Tissues were removed and frozen in dry ice. Coronal sections of brain, horizontal

sections of heart and sagittal sections of kidney (20tm) were cut on a cryostat

(Microm) at -20oC and mounted on microscope slides. Every seventh slide was

stained with hematoxylin and eosin for histology. Autoradiography was

performed according to the previously published protocol (181). Tissue sections

were preincubated in Krebs buffer (NaCI 118.4mM, KCI 4.7 mM, MgS04 1.2mM,

CaCl2 1.27mM, NaH2PO4 10.0mM, pH 7.1) containing 0.1mM guanosine

triphosphate (GTP), 0.1mM ascorbic acid and 10iM phenylmethylsulfonylfluoride

(PMSF) for 30 min at 250C. Sections were then incubated in Krebs buffer

containing 0.1mM ascorbic acid and 10iM PMSF with 100pM I-CYP at 25C for

150 min, in the presence of 1pLM (-)propranolol, 100nM ICI118,551 (p2-selective

antagonist) or 100nM CGP20712A (pi-selective antagonist) to distinguish non-

specific, pi- and P2-bindings. Labeled sections were rinsed in the same buffer,

followed by two 15-min washes at 370C in the buffer and rinsed in distilled water

at 250C. Dried sections were then exposed to X-ray films. The images were







27

quantitated with a computerized image analysis system (MCID, Imaging

Research) and normalized using 1251-standards. Non-specific binding was less

than 10% of total binding.



2.4 Physiological Methods


Blood Collection

Blood samples were collected from the tail. Serum was obtained by

collecting blood with no anticoagulant, incubating on ice for 1 hr followed by

centrifugation at 4000rpm for 15 min. Plasma was obtained by collecting blood in

tubes containing EDTA as the anticoagulant and centrifuged at 4000rpm for 15

min immediately.


Animal Surgery

Male SHRs (250-350g, Harlan) and Sprague-Dawley rats (200-250g,

Harlan) were kept in cages in a room with a 12-hr light-dark cycle. Animals were

fed standard laboratory rat chow and tap water ad libitum. All care and surgical

conditions were approved by the UF Animal Care Committee.

Telemetric Sensor Implantation. Before implantation, the zero of each

radiotransmitter (TA11PA-C40, Data Sciences) was verified to be < 4 mmHg.

SHR were anesthetized with 100mg/kg ketamine and 15mg/kg xylazine and a

midline abdominal incision was made. A fluid-filled sensor catheter was then

inserted into the right femoral artery and the tip of the catheter was in the

abdominal aorta caudal to the renal arteries. The sensor battery was sewed to







28

the skin in the peritoneal cavity. The implanted rats were allowed to recover for 1

week.

Jugular Vein Cannulation. One week after telemetric implantation, rats

were anesthetized and a curved catheter made of PE 50 and vinyl tubing was

inserted into the jugular vein. The tubing was led under the skin of the neck and

exposed on the back to allow for drug infusion. Rats were allowed to recover for

24 hrs before experimentation. The catheters were flushed with 100U heparin

everyday to prevent clogging.


Determination of Effects of PI-AS and Atenolol on Cardiovascular
Parameters in Response to p-stimulation

Langendorff Heart Perfusion. 48hrs after injection of 1mg/kg pi-AS-ODN

(n=9) or inverted-ODN (n=6), SHR were anesthetized and sacrificed. Heart

perfusion was performed according to the protocol described previously (182).

Hearts were quickly removed and perfused via the aorta with oxygenated Krebs

buffer (118mM NaCI, 18.75mM NaHCO3, 1.2mM KH2PO4, 4.7mM KCI, 1.2mM

MgSO4, 1.25mM CaCI2, 11.1mM Glucose, 0.01mM EDTA) at a constant flow of

7.0ml/min at 360C. Coronary perfusion pressure (CPP) was measured via a

catheter placed proximal to the aorta and connected to a pressure transducer

(Gould Statham P231D). A latex balloon filled with water and connected to the

pressure transducer was inserted into the left ventricle through the left atrium to

measure left ventricular end-diastolic pressure (LVEDP), left ventricular systolic

pressure (LVSP), and developed left ventricular pressure (dLVP) (dLVP=LVSP-

LVEDP). LVEDP during equilibration was set at 5 to 7mmHg. CPP, LVEDP and







29

LVSP were continuously recorded on a 4-channel recorder (Astro-Med). After

baselines for dLVP and heart rate (HR) were stable for 5 min, isoproterenol (ISO,

non-specific p-agonist) was given at 0.01, 0.025, 0.05, 0.12pM at 10 min

intervals so as to avoid the effect of tachyphylaxis.

Telemetric Monitoring of Live Animals. The effects of p3-AS-ODN and

atenolol on cardiac dP/dtmax, HR and systolic BP (SBP) were compared in the

same group of SHR (n=4). Two days after catheterization of jugular vein, control

values were taken and 1mg/kg p3-AS-ODN was injected. 48 hrs later, rats were

tested for the effect of p1-AS-ODN. The rats were allowed to recover until all the

cardiovascular parameters returned to control values. Then 1mg/kg atenolol (1i-

selective antagonist) was injected and rats were tested 30 min later. For 1i-

stimulation, SHR were infused with dobutamine (pi-selective agonist) through

jugular vein catheter at 5, 10, 20, 40pg/kg/min. Each dose was given for 5 min

continuously and at 1 hr intervals until all the cardiovascular parameters returned

to baseline so as to avoid the effect of tachyphylaxis. BP and HR were sampled

every 1 min. dP/dtmax was calculated from the slope of the rising pulse pressure

curve and determined every 1 min. The difference between values at each dose

and baseline was denoted as A.


BP and HR Monitoring

Telemetry. Each rat cage was placed on a receiver (RLA1020, Data

Sciences) for measurement of cardiovascular parameters. Data were collected

with a computer-based data acquisition program (Dataquest LabPRO3.0; Data







30

Sciences). BP and HR were measured every 10 min and averaged every 1-24

hrs. Before treatment, SHR were monitored for a week to get a stable baseline.

Tailcuff. Rats were warmed for 20-30 min in cages on heating pads. The

temperature was controlled at 35-370C. Then rats were placed in a plastic

restrainer kept at 370C. A pneumatic pulse sensor was attached to the tail. After

cuff inflation, systolic BP was determined as the first pulsatile oscillation on the

descending side of pressure curve. Heart rate (HR) was determined by manual

counting of pulse numbers per unit time. BP and HR were recorded by a Narco

physiograph. Data values of each rat were taken as an average of at least four

stable readings. Baseline was determined by averaging three days of

measurements before antisense administration.



Myocardial Ischemia-reperfusion

SD rats were anesthetized with sodium pentobarbital (40 mg/kg)

intraperitoneally. The hearts were rapidly excised and placed in ice-cold Krebs-

Henseleit buffer (118 mM NaCI, 25 mM NaHCO3, 1.2 mM KH2PO4, 4.7 mM KCI,

1.2 mM MgSO4, 1.25 mM CaCI2, 11 mM Glucose, pH 7.4). Within 1 min, the

hearts were transferred to an Langendorff apparatus and perfused via the aorta

with oxygenated (95% 02 + 5% CO,) Krebs-Henseleit buffer at 370C using a

MasterFlex pump (model 7015-21, Cole-Palmer Instrument). The heart was

placed in a semiclosed circulating water-warmed (370C) air chamber, paced

atrially at a rate of 300 bpm with a Medtronic 530 pacemaker, and perfused at a

constant flow (5.5-6.0 ml/min). LVEDP during equilibration was set at 5-7 mmHg.







31

Sham controls were continuously perfused with Kreb-Henseleit buffer for 80 min.

Heart from other groups, were equilibrated for 20 min, subjected to 30 min global

ischemia followed by 30 min reperfusion. After completion of the experiment,

hearts were frozen in liquid nitrogen for p-AR radioligand binding assay and

measurement of MDA.



2.5 Clinical Pathology Methods


Liver Transaminase Levels

The plasma levels of alanine aminotransferase (ALT) and aspartate

aminotransferase (AST) were determined by a colormetric method using a kit

(Sigma, Cat. #505-OP). Briefly, plasma samples were incubated with proper

substrates for ALT or AST at 370C for 1 hr and subjected to color reactions. The

absorbency was read at the wavelength of 500 nm and the concentrations of

ALT and AST were calculated from the calibration curve obtained under the

same conditions.


Hematology

Hematology measurements were carried out in the Clinical Laboratory of

Shands Hospital at University of Florida. The hematology parameters tested

included erythrocyte count (RBC), hematocrit, platelet count and total leukocyte

count (WBC).









C-Reactive Protein (CRP)

CRP values were obtained on blood serum in the Clinical Laboratory of

Shands Hospital at University of Florida.


Histopathology

Rats were euthanized by a peritoneal injection of overdose pentobarbital.

Selected tissues, including heart, liver, spleen and kidney, were removed,

trimmed of fat and contiguous tissues and weighed. These tissues were fixed in

10% neutral buffered formalin, embedded in paraffin (Diagnostic Referral

Laboratory, University of Florida), sectioned at the thickness of 5.im, mounted on

gelatin-coated slides, stained with hematoxylin and eosin, and examined under

light microscopy.


Gel Immunodiffusion Assay

Specific antibodies to ODN/liposome complex were measured with double

immunodiffusion (Ouchterlony) method on premade discs purchase from ICN

Biomedicals. Briefly, the ODN/liposome complex was loaded in the center well,

and serum samples in the surrounding wells. They were allowed to diffuse at

room temperature in a moisturized and sealed container for up to 7 days. The

occurrence of precipitation bands indicated the presence of antibody-antigen

reaction.


2.6 Statistical Analysis

All experiments were repeated at least 2 times unless the situation and results

called for more experiments. Various statistical test were performed depending







33
on the experimental design using Jandel Sigmastat Software. Values were

expressed as mean SEM. Student t test was used to evaluate significant

differences when two groups were used. One way analysis of variance (ANOVA)

and appropriate multiple range test were used when more than two groups were

compared. Pearson product moment correlation was used to assess the

relationship between two parameters. In some studies, a two-way ANOVA were

utilized to evaluate the effects of individual treatments and any interactions

between the treatments. For analysis of parameters (e.g. dP/dt, BP, HR by

telemetry) obtained from the same animal over time, repeated measure of

ANOVA was incorporated. Significance for all tests was set at the 95%

confidence limit. All molecular biology experiments dealing with the comparison

of mRNA by RT-PCR were carried out in the linear range of the assay. The data

were normalized by GAPDH and subjected to appropriate statistics.














CHAPTER 3
OPTIMIZATION OF ANTISENSE DESIGN AND DELIVERY

3.1 Introduction

Antisense oligodeoxynucleotides (AS-ODN) are generally 15-20 bases in

length. Theoretically the length of 12-15 bases is able to provide sufficient

specificity and binding affinity for a target mRNA (128) while short enough to gain

entry into cells (183). The specificity of antisense molecules is so great that even

two genes that differ by a single base mutation can be differentially inhibited

(129). There is a wide range of potential sites within an mRNA sequence for

antisense targeting, the best of which is considered to be in the vicinity of AUG

translation initiation codon. The availability of these sites largely depends on the

folding of the mRNA (184). However currently there is no computer program

sophisticated enough to predict the secondary structure of the target mRNA.

Lacking a sure-fire method of oligo prediction, antisense development has to go

through a trial-and-error test.

The basic principles of antisense design are described below, which are

now published in Methods in Enzymology (178).

1. Identify the sequence: Check GenBank for the mRNA sequence of the target

protein in the species to be studied. If there is more than one lab cloning the

same protein, compare the homology of different reports. These sequences

will point out the controversial bases, which can be due to natural mutations

34







35
in variant strains of the same species or sequencing errors. Try to avoid

these debatable regions. Target the ODN to the identical regions, which

ensures reliability of the sequence being used.

2. Target the sites: Although there are several possible sites within the DNA

sequence to target for antisense design, the regions that are considered to be

the best targets for constructing effective AS-ODNs are the 5' cap region and

the area around the AUG translation initiation codon. In our experience, AUG

start codon and nearby bases downstream of AUG in the coding region are

most promising sites for antisense inhibition (185). Usually the length of 15-

20 mer is sufficient to guarantee sequence specificity, which must be

confirmed by checking with GenBank for existing sequences to avoid any

significant homology with other mRNAs.

3. Modify the backbone: AS-ODNs are chemically modified to increase nuclease

resistance and stability. The most commonly adopted modification is

backbone phosphorothioation, in which one non-bridging oxygen of the

phosphodiester linkage is replaced by sulfur (137). Compared to natural

DNA, phosphorothioated ODNs have a greatly extended half-life of 42-56 hrs

in the plasma of rat, monkey and human (186-188). They are also better

substrates to induce RNase H cleavage of mRNA. But phosphorothioation

significantly decreases the melting temperature (Tm) of ODNs and reduces

binding affinity and stability of DNA/DNA and DNA/RNA duplexes. For 15-25

mer, Tm of phophorothioate ODN is usually 7-120C lower than normal ODN,

with AT bases showing more Tm depression than GC (189,190). This fact







36
must be taken into account in the design of short AS-ODN, because 15-mer

phophorothioate ODN /RNA hybrids may have Tm close to body temperature

of 370C.

4. Avoid pitfalls: Some other basic rules applicable in the design of polymerase

chain reaction (PCR) primers also hold true for AS-ODN (191), e.g.

avoidance of palindromic structure and primer-dimer formation, and choosing

sequence with a balance AT/GC ratio to minimize toxicity and non-specific

binding caused by high GC content. Although a high GC/AT ratio should be

avoided generally, the danger of toxicity and non-specific binding can be

reduced by phosphorothioation.


3.2 Results

3.2.1 Design of pi-AS-ODN

My plan was to develop a 15 mer Pi-AS-ODN specific to rat (192) and

human (11) pi-AR mRNA. Thirteen sequences were picked within the conserved

regions between rat and human pi-AR mRNA, which covered a variety of sites

including upstream of AUG, spanning AUG, CDS and 3'-UTR. Seven were

discarded after specificity checking by Blast search and secondary structure

analysis of the ODNs. The remaining six candidates are listed below. They are

modified by backbone phosphorothioation. Their positions refer to rat p1-AR

mRNA (gene accession number: J05561) (192).

#1: 5'- ccg cgc cca tgc cga -3' (64-78, spanning AUG start codon)

#4: 5'- ggt cgt tgt agc agc -3' (691-705, in the CDS)









#6: 5'- cgg tct tga gta aac -3' (1602-1616, in the 3'-UTR)

#7: 5'- ggc cga cga cag gtt -3' (111-125, in the CDS)

#8: 5'- atg agc agc acg atg -3' (269-283, in the CDS)

#10: 5'- ttg gtg agc gtc tgc -3' (335-349, in the CDS)


3.2.2 In vitro and in vivo Screening

In vitro

The effects of pi-AS-ODN on the expression of P1-AR were examined in

C6-2B and i1-15-5B cell lines. C6-2B is a rat glial tumor cell line. P1-15-5B is a

mouse connective tissue cell line NCTC 2071 permanently transfected with

human p3-AR cDNA. Both cell lines express abundant p1-AR. Cells were treated

with 0.1-10 pM p -AS-ODN or saline (as control) for 3-48 hrs. P1-AR levels on the

cell membrane were measured with radioligand binding assay. Many

experiments were performed testing different doses and time course, but all

yielded negative results. None of the antisense candidates shown above

reduced PI-AR levels in either cell line. A representative experiment was

summarized in Table 3-1 and 3-2.

Reasons for the absence of antisense action were searched for. Although

efficient uptake of 15-18 mer S-ODN in cell cultures was shown in early studies

(183), the possibility still existed that AS-ODN did not enter cells. To answer this

question, cells were treated with fluorescin-labeled AS-ODN and the ODN

uptake was examined under confocal microscopy. 30 min after addition into the

medium, fluorescence was readily observed and widely distributed in the

cytoplasm of the majority of the cells. Another reason could be that cells were








38
not treated with an optimal dose of AS-ODN for enough time. Therefore,

experiments on dose response (0.1LiM, 1tiM, 2jLM, 5jiM, 10iM) and time course

(3hr, 6hr, 12hr, 24hr, 48hr) were performed and repeated, but still showing no

significant effects of AS-ODN on pj-AR expression. It was noticed that high

doses (5pM, 10p.M) of phophorothioate ODN seemed to be toxic because cell

viability and total protein content per plate were significantly reduced compared

to saline control in some experiments. More experiments were planned, including

liposomal delivery of AS-ODN and effects of AS-ODN on cellular responses to p

1-agonists. However, despite continuous efforts, pi-AS-ODN failed to reduce pi-

AR levels in vitro.


Table 3-1. Effects of p1-AS-ODN on C6-2B cells.

C6-2B Cell Cell count total protein /plate P-AR binding P-AR binding

/plate (mg) (fmol/mg) (% of Control)

Saline 5.4 x 106 0.71 57 100

p1 AS 1 4.4 x 106 0.56 68 119

P1 AS 7 4.8 x 106 0.80 48 84

p1 AS 8 5.2 x 106 0.54 53 93

P1 AS 10 4 x106 0.60 54 95

Cells were grown in DMEM / 5%FBS until 80% confluency and the
medium was changed to OPTI-MEM I (GIBCO BRL, reduced serum medium).
5pM p~-AS-ODN or saline were added to the medium 24hr before a single point
binding assay on membrane proteins at 100 pM 1251-CYP. Non-specific binding
was obtained in the presence of 1pM alprenolol.









Table 3-2. Effects of pf-AS-ODN on p1-15-5B Cells.

p1-15-5B Cell count total protein/plate P-AR binding P-AR binding

Cell /plate (mg) (fmol/mg) (% of Control)

Saline 5.2 x 106 0.53 156 100

p1 AS 1 5.2 x 106 0.50 175 113

p1 AS 7 5.0 x 106 0.49 151 97

31 AS 8 4.8 x 106 0.49 165 106

P1 AS 10 4.8 x 106 0.50 163 104


In vivo

It was hypothesized that an effective p1-AS-ODN, by suppressing the pi-

AR expression and function in heart and kidney, could efficiently inhibit

hypertension. Hence, the effectiveness of pi-AS-ODN was evaluated based on

the extent of reduction of Pi-AR expression and high BP in rats. To facilitate the

antisense delivery, ODNs were completed by cationic liposomes DOTAP/DOPE

at a (+/-) charge ratio of 0.5.

Antisense efficacy was first tested in normotensive Sprague-Dawley (SD)

rats, which received a single intravenous injection of pi-AS-ODN through tongue

vein. The inhibition of p1-AR levels in the heart was assessed by saturation

receptor binding assay two days after treatment. All antisense candidates

significantly reduced the cardiac p-AR levels to different extents as compared

with saline or inverted-ODN (INV-ODN) control (Table 3-3). Based on the

receptor level, ODN-1 and ODN-7 were better than other candidates.










Table 3-3. Effects of pi-AS-ODNs on cardiac P-AR in rats.


Treatment R KD (pM) Bmax Decrease in
(fmol/mg) percentage

Saline (n=3) 0.9716 553 42.11.6

INV-ODN-1 (n=3) 0.9217 562 38.71.2

AS-ODN-1 (n=3) 0.8650 341 27.50.8* 31%

AS-ODN-7 (n=3) 0.9500 322 26.01.5* 35%

AS-ODN-8 (n=3) 0.9712 363 28.92.1* 27%


Male SD rats (250-300g) were treated with a single i.v. injection of 200 pg
P1-AS-ODNs or INV-ODN for 48 hrs. Membrane proteins were prepared from
heart ventricles. Affinity and density of p-AR were measured by radioligand
binding assay. P<0.01 versus saline or INV-ODN.


The antihypertensive effects of 31-AS-ODNs were then tested in

spontaneously hypertensive rats (SHR) monitored by radiotelemetry. A single

injection of ODN-1 significantly diminished mean arterial pressure by up to 14

mmHg for 8 days (P<0.05), whereas the drop in BP produced by ODN-7 was

less marked (8 mmHg) and for a shorter duration (4 days). The hypotensive

impact of pi-AS-ODN was dose-dependent, with the order 800pg > 400pg > 200

lig.

Based on these results, ODN-1 was determined to be the optimal

antisense sequence for pi-AR which reduced Pi-AR expression and high BP

most effectively.











3.2.3 Optimization of Antisense Delivery by Cationic Liposomes

Although chemical modifications like phosphorothioation endow AS-ODN

with nuclease insensitivity and longer half-life, the stability and delivery of AS-

ODN must be further improved to be effective in vivo. Cationic liposomes are

known for their ability to facilitate cellular uptake of nucleic acids and protect

them from degradation in circulation and tissues (165,169). Numerous lipid

formulations have been developed to enhance the transfection of plasmid DNA

and oligonucleotides (168,193). Dioleoyl trimethyl ammoium propane (DOTAP)

was introduced in 1980s and remains one of today's favorite lipids for its

effectiveness and biodegradability (163,194,195). Not only has its usefulness

been proven in bench studies but also explored in clinical trials, e.g. to deliver

CFTR gene for cystic fibrosis. Therefore we chose to use DOTAP coupled with

the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) to deliver p3-AS-ODN

in our study.

However, high transfection efficiency that was seen in vitro could not be

readily achieved in vivo with the same liposome/DNA preparation. This is in part

attributed to the serum resistance of liposome/DNA complex because negatively

charged plasma proteins can bind to cationic lipids and destabilize

liposome/DNA complex (196). It was recently shown that increasing the +/-

charge ratio of liposomes to DNA and inducing the maturation of liposome-DNA

complex by prolonging incubation time could overcome this problem

(169,197,198). The optimal charge ratio of DOTAP:DNA was found to be ;2.0







42
(198,199). Thus I chose to test five charge ratios ranging from 0 to 3.5 to

optimize the delivery of p3-AS-ODN. In addition, DOTAP/DOPE and ODN were

carefully mixed and incubated at room temperature for 40-60 min before use to

facilitate stabilization of the complex.

Figure 3-1 shows the effect of different (+/-) liposome:ODN charge ratios

on systolic BP of SHR measured by tail cuff (n=6 for each ratio) in a

representative experiment. 0.5 mg/kg pi-AS-ODN alone, i.e. at ratio 0, did not

change SBP, whereas ratio 0.5 significantly reduced SBP by up to 38+5 mmHg

for 7-8 days. When the ratio was increased, the duration of the hypotensive

impact was drastically prolonged to 18-20 days at ratio 1.5, 20-33 days at ratio

2.5 and 3.5, varying with liposome preparations. However the maximum drop in

SBP was greater at ratio 1.5 and 2.5 (-35 mmHg) than at ratio 3.5 (-25 mmHg)

(Table 3-4). Accordingly, the optimal charge ratio of DOTAP:ODN was

determined as 2.5 and employed in the subsequent experiments. It was worth

noting that the antihypertensive effect of pi-AS-ODN varied slightly with different

batches of liposome mixture, probably due to the variation in particle size and

structure. Furthermore, aggregation and precipitation of liposome/ODN must be

avoided to ensure the successful delivery.








43
Table 3-4. Amplitude and duration of reduction in blood pressure of SHR after a
single i.v. injection of 0.5 mg/kg pi-AS-ODN delivered at different charge ratios of
liposome/ODN.

Charge ratio Maximum reduction in Range of reduction Duration

of liposome/ODN blood pressure (mmHg) (mmHg) (day)

0 2 --

0.5 35-38 24-38 7-8

1.5 28-35 18-35 18-20

2.5 30-34 20-34 20-33

3.5 20-24 15-24 20-33





3.3 Discussion

The purpose of this study is to develop an effective p1-AS-ODN targeted

to rat and human pi-AR mRNA that can reduce pi-AR expression and BP in

hypertensive models. After in vitro and in vivo screening, the best 31-AS-ODN

candidate was selected which was 15 mer in length and spanned the AUG site. It

significantly reduced the p3-AR levels in the heart and lowered high BP in SHR.

The delivery of AS-ODN was improved by cationic liposomes DOTAP/DOPE and

the condition was optimized.

These results support the notion that the region around the translation

initiation codon may be the best target for antisense design. ODN binding to this

area may interfere with the ribosomal assembly and/or form a structural hurdle to

ribosome sliding along the mRNA strand. Assuming that all the six p1-AS-ODN

candidates can bind to pi-AR mRNA, it is conceivable that interrupting the initial








44
phase of translation may be more effective than later blockade. However, this

assertion is more or less arbitrary, because I do not have evidence to show that

all candidates have assess to 11-AR mRNA at the same affinity. In spite of this,

since it is unlikely to screen every possible region in an mRNA for economical

reasons, I believe the first site to consider for AS targeting is the vicinity of AUG

codon if other criteria can be met.

Efficient gene delivery is vital to the therapeutic application of AS-ODN in

vivo. Among nonviral vectors, cationic liposomes are most widely used. They are

safe, nonimmunogenic and easy to produce on a large scale. However, relatively

low transfection efficiency has been obtained after intravenous administration,

mainly due to the inactivation of cationic liposome by serum. It was shown

recently that increasing the charge ratio (+/-) of liposome to DNA and inducing

the maturation of liposome-DNA complex can overcome this problem (197,198).

The optimal charge ratio of DOTAP:DNA was demonstrated to be =2 (198,199).

Based on these studies, the delivery of AS-ODN was optimized by testing five

charge ratios from 0-3.5. As shown in the results, increasing the charge ratio not

only improved the delivery efficiency but prolonged the duration of p3-AS-ODN

action. The best antihypertensive result (35 mmHg for 20-33 days) was achieved

at ratio 2.5 consistently.

The prolonged effect of ,3-AS-ODN on BP may be attributed to several

reasons. The half-life of phosphorothioate ODN is ~50hrs in rat plasma after

intravenous injection. Li et al. (183) showed that S-ODN could be found in its

intact form for at least 5 days after entry into cells. It has been shown that








45
liposome encapsulation not only improves the cellular uptake of DNA, but also

protects DNA from degradation and extends its circulation time in vivo (165,169).

In addition, Baker's group (180,200) and other researchers (201) have shown

that p-ARs had a slow turnover rate which took 200-250 hrs to recover in the

heart after irreversible blockade.

Then a question arose, i.e. why pi-AS-ODN reduced P3-AR density on the

cell membrane so rapidly (within 2 days), provided that p-ARs had a slow

turnover rate. It is possible that p3-AS-ODN not only inhibited the translation of p

1-AR, but also decreased the intracellular pool of pi-AR and accelerated the

internalization of the membrane receptors. The recycling of receptors between

cell membrane and cytoplasm is a much faster process than receptor

metabolism. This may explain the discrepancy between the rapid reduction in pi-

AR density and the slow turnover. However to test this hypothesis requires

further experiments.

It is still not understood why p1-AS-ODN did not decrease p1-AR levels in

vitro. Similar protocols had been successfully used in our lab to screen for AS-

ODN against AT1-receptor in cell culture. The possibility that AS-ODN failed to

enter cells was ruled out by the visualization of fluorescin-labeled AS-ODN in the

cytoplasm 30 min after addition into the medium. Further experiments on dose

response and time course did not improve the effects. We stopped in vitro study

after 6 months of pursuing. However, it is possible that AS-ODN delivery in cell

culture could have been improved by liposomes, as the case in vivo. It is also







46
likely that AS-ODN, after the endocytosis, did not escape endosomes to interact

with target mRNA.






















7" / p 7.

.f /


i^. ^. /**- d^
\ A IA' A J

\I A


-o

A


--- control
-- ratio 0
--W ratio 0.5
--0- ratio 1.5
-A ratio 2.5
--0 ratio 3.5


I I I I I I
0 10 20 30 40 50
Days after injection



Figure 3-1. Improving the antihypertensive effect of pi-AS-ODN by
optimization of liposome:ODN charge ratios.

SHR received a single iv injection of pi-AS-ODN or INV-ODN. 1mg/kg 31-AS-
ODN was delivered by DOTAP/DOPE at charge ratios from 0 to 3.5. 1mg/kg
INV-ODN delivered by DOTAP/DOPE at charge ratio 2.0 served as control (0).
Data represent mean values of each group (n=6). Standard errors were omitted
for clarity.


10


0-


-10 -


-20


-30


-40














CHAPTER 4
EFFECTS ON BLOOD PRESSURE AND HEART RATE:
l1-AS-ODN vs. ATENOLOL


4.1 Introduction

Pi-AR plays a crucial role in the regulation and maintenance of

hypertension. p1-adrenergic activation increases cardiac contractility and heart

rate, and stimulates renin production from the kidney. Thus the inhibition of this

receptor can lead to decreased cardiac output and renin-angiotensin activity and

thereby reduce hypertension. This concept has been proven correct by the

antihypertensive action of p-blockers in clinical practice. In developing an

antisense agent for pi-inhibition to reduce hypertension, the rationale borrows

from the success of p-blockers because both approaches can suppress Pi-AR

and therefore p1-AS should be able to lower high BP if p-blockers do. However

these two strategies adopt different pathways for pi-inhibition. Antisense reduces

the total numbers of Pi-AR by interfering with translation, while p-blockers

dampen the function of p-receptors by competing with endogenous p-agonists

like norepinephrine. Due to the intrinsic drawback of pharmacological

intervention, pi-specific antagonists have not been developed which inevitably

results in undesirable antagonism of p2-receptor subtype. This accounts for a

large portion of side effects caused by p-blockers including contraindication of

asthma, peripheral vascular disease and insulin-dependent diabetes mellitus,

48








49
and reduced exercise capacity. In contrast, antisense can be designed specific

to its target based on gene sequence. This will preclude adverse effects of

nonspecific antagonism by p-blockers. Furthermore, CNS reaction can be

avoided by antisense owing to the inability of antisense molecules to enter the

brain. Here the effects of pi-AS-ODN on BP and HR of spontaneously

hypertensive rats (SHR) were tested and compared with those of a hydrophilic p

i-selective blocker, atenolol.



4.2 Results

4.2.1 Effects of 31-AS-ODN on BP and HR Measured by Tail cuff

Figure 4-1 shows a typical experiment in which SHR was treated with a

single i.v. injection of 1mg/kg pi-AS-ODN delivered by the optimal ratio of

cationic liposomes determined in Chapter 3. BP and HR were measured by the

traditional tail cuff method. Antisense significantly decreased systolic BP for 4

weeks. The maximum drop was 30 mmHg. However, HR was not changed by 1i-

AS-ODN. Inverted-ODN (INV-ODN) as control had no effect on BP or HR.



4.2.2 Effects of 31-AS-ODN on BP and HR Measured by Radiotelemetry

In order to compare the effects of pi-AS-ODN and atenolol, radiotelemetry

was used to monitor BP and HR on a regular basis. Radiotelemetry allows for

accurate and continuous hemodynamic monitoring without inducing stress or

artificial factors. 1mg/kg pi-AS-ODN delivered with liposomes at the (+/-) ratio

0.5 produced a maximum drop of 15mmHg in mean blood pressure (Fig. 4-2).








50
The antihypertensive effect lasted for 8 days, which was consistent with the

results measured with tailcuff. HR was not significantly altered. In contrast to

AS-ODN, although the onset of hypotensive effect caused by 1mg/kg atenolol

occurred as early as 20 min after injection, it lasted for only 10 hrs (Fig. 4-3). In

addition, atenolol caused considerable bradycardia up to an average of -75 bpm.

All these hemodynamic changes with i.v. atenolol were not detectable within a

day.



4.3 Discussion

The results indicate that the antihypertensive effect of p3-AS-ODN is

significantly longer lasting than that of atenolol. A single iv injection can reduce

BP for 20-30 days with the maximum drop of 30 mmHg. In contrast, the

hemodynamic response to bolus iv injection of atenolol lasts only for hours. As a

result, atenolol must be taken every day to achieve chronic control of BP.

Moreover, atenolol causes the peak-and-trough fluctuation in BP, in contrast to

the steady hypotensive action of antisense. Greater than normal variation in 24-

hr BP is potentially dangerous and associated with early morning cardiovascular

catastrophe. Finally, 31-AS-ODN does not decrease HR as shown by both tail

cuff and telemetry, whereas atenolol induces appreciable bradycardia.

Bradycardia is a common complaint by patients taking p-blockers.

The most surprising finding of this study is the lack of effects on HR of 1i-

AS-ODN. The mechanisms are still under investigation. Several possibilities can

be considered. First, 31-AS-ODN did not affect P2-AR, which played an important







51
role in the regulation of HR (202,203), while not involved in the cardiac

contraction of rats. Second, antisense inhibition of 31-AR expression is gradual

and to a lesser extent than p-blockers. It is also possible that 11-AR may have a

larger reserve for controlling HR than contractility. However, to answer this

question requires more extensive studies and electrophysiology techniques

which are beyond the scope of this dissertation.

Both tailcuff and telemetry measures of BP showed a significant drop after

antisense treatment. SHR responded to p3-AS-ODN to a greater degree of

hypotension when subjected to tailcuff versus telemetry. In addition, the baseline

measured with tailcuff was consistently higher than that with telemetry by 20-

30mmHg in the same rats. Bazil et al. (204) reported a similar phenomenon.

They compared the cardiovascular parameters recorded by telemetry, tailcuff

and arterial catheter and observed a more sensitive hypotensive effect of

captopril with tailcuff. Tailcuff measurement of BP involves warming and restraint

of rats. It is conceivable that p1-AS, through the suppression of sympathetic

activity, can decrease BP of animals under stress more effectively.











A
210


200
E
S200190


180

ST --- AS-ODN
170**
170 -o- Saline
-- INV-ODN
160
0 5 10 15 20 25 30
B 480


460


440 -

420

400


380

360
0 5 10 15 20 25 30 35

Days after injection


Figure 4-1. Effects of P,-AS-ODN on BP and HR of SHR.

16-week old SHR were treated with a single iv injection of 1mg/kg P,-AS-ODN
delivered by DOTAP/DOPE at a (+/-) charge ratio of 2.5. BP (A) and HR (B) were
measured by tail cuff method. Data represent mean SEM (n=6-9 for each
group). *** P<0.001, ** P<0.01, P=0.059 vs. saline or INV-ODN.















A 10

5

r 0


Time after treatment (day)


Figure 4-2. Effects of pI-AS-ODN on mean BP
monitored by telemetry.


and HR of SHR


1mg/kg INV-ODN (0) or P,-AS-ODN (0) was injected with liposomes at a (+/-)
charge ratio 0.5. BP (A) and HR (B) were recorded every 10 min and averaged
every 24 hrs. Data represent mean SEM of each group (n=4). P<0.05 vs.
INV-ODN.


I 1 I j / /


I I / I 1 :
















A
20


10



-10o i
I I *






-30 -


-40 ,--
0 5 10 15 20
B 60-
-20 -

30 -





20 -
0 5 10 15 20


40

20

E o
0.

-20

-40 *

-60

-80 -

0 5 10 15 20

Time after treatment (hr)


Figure 4-3. Effects of atenolol on mean BP and HR of SHR monitored by
telemetry.

Saline (0) or 1mg/kg atenolol (0) was injected intravenously. BP (A) and HR (B)
were taken every 10 min and averaged every 1 hr. Data represent mean SEM
of each group (n=4). P<0.05 vs. saline.















CHAPTER 5
MECHANISMS FOR THE ANTIHYPERTENSIVE EFFECTS OF Pi-AS-ODN


5.1 Introduction

In the preceding studies, a pl-AS-ODN was developed which lowered BP

of SHR effectively and persistently with a single i.v. injection. The current study is

carried out to understand the mechanisms underlying its antihypertensive

effects. Pf-AR activation induces positive inotropic and chronotropic responses in

the heart, and stimulates renin production and release in the kidney. p-blockers

decrease cardiac output and plasma renin activity, which contribute to the BP

reduction. Therefore I hypothesize that:

i) p1-AS-ODN, through specific inhibition of p1-AR expression, will decrease the

functional sensitivity of pi-AR-mediated responses in the face of sympathetic

activation, and thereby achieve an antihypertensive effect.

ii) Since the pi-AS-ODN is designed specifically to p1-AR, it should not affect

the expression of 12-AR subtype.

iii) p1-AS-ODN cannot cross blood-brain barrier to affect p-AR in the CNS.









5.2 Results

5.2.1 Inhibition of Cardiac Contractility

Effects on cardiac p-AR levels

Fig. 5-1 shows that a single i.v. injection of 1mg/kg pi-AS-ODN delivered

with cationic liposomes at a (+/-) charge ratio of 2.5 significantly reduced the p1-

AR density in the SHR hearts for 18 days (P<0.01). The drop was at its

maximum of 47% on day 4, 33% on day 10 and maintained at 29% on day 18. In

contrast, there was no significant change in the Bmax of p2-AR. KD of both

subtypes remained unaltered (Table 5-1). Consequently, the 11/P2 subtype ratio

in the ventricles was diminished from -70/30 to -50/50 by pi-AS-ODN. INV-ODN

had no effect on either subtype.


Table 5-1. Effects of pi-AS-ODN on Bmax and KD of p,- and p2-AR in cardiac
ventricles of SHR.


Total (l1+32)

KD (pM) Bmax (fmol/mg)

55.46.7 27.6+0.9

57.13.5 26.3+3.0

37.01.0* 19.81.8*


131

KD (pM) Bmax (fmol/mg)

64.75.4 18.70.9

66.0+1.0 18.1+2.3

60.53.4 10.01.2*


02

KD (pM) Bmax (fmol/mg)

29.60.5 9.1+0.6

32.02.4 8.7+0.5

30.7+3.3 9.9+0.5


p,- and p2-AR levels were measured 4 days after
saline, INV-ODN or p,-AS-ODN. Data represent mean
(n=6-10). P<0.01 versus saline control.


a single i.v. injection of
+ SEM of each group


Saline

INV-ODN

3P-AS-ODN


--








57
Effects on cardiac contractility and heart rate in response to p-stimulation

48 hrs after injection of 1mg/kg p3-AS-ODN, the cardiac inotropic and

chronotropic responses to p-stimulation were determined in SHR in vitro and in

vivo.

First, isolated hearts were perfused with incrementing doses of

isoproterenol (ISO, nonselective p-agonist), which enhanced HR and contractility

via activating p-AR. The dLVP-ISO dose-response curve, which reflected the

positive inotropic effect of ISO, was significantly shifted down by p1-AS-ODN

(P<0.05). HR was not significantly decreased, except at one point, i.e. 0.01 PM

ISO (P<0.05) (Fig. 5-2).

In vivo test was performed in conscious SHR monitored by radiotelemetry.

11-AS-ODN significantly (P<0.02) dampened the increase in dP/dtmax (index for

cardiac contractility) in the face of dobutamine (p1-selective agonist) (Fig. 5-3A).

The change in HR was not significantly reduced (Fig. 5-3B), which echoed the

results in isolated perfused hearts. On the other hand, the response of BP to

dobutamine was biphasic (Fig. 5-3C). Dobutmaine elevated systolic BP by 5-

8mmHg at low infusion speed and reduced systolic BP at higher speed. This is

probably due to the partial 32-agonistic activity of dobutamine at high doses and

the consequent vasodilatory effect on BP. Fig. 5-3 also compares the results with

11-AS-ODN and atenolol (1p-selective antagonist). Relative to p3-AS-ODN,

1mg/kg atenolol produced a more profound decline in AdP/dtmax and AHR during

5-hr period of time after injection. Moreover, it reduced basal dP/dtmax (2450295

mmHg/sec versus control 2937277 mmHg/sec) and caused bradycardia (295







58
12 bpm versus control 365+8 bpm) (P<0.05), while p3-AS did not change basal

contractility (2922 249 mmHg/sec) or HR (36512 bpm). However the effects

of atenolol on AdP/dtmax and AHR were transient. Within 24hrs after atenolol

administration, the inotropic and chronotropic effects of dobutamine had returned

to control level.


5.2.2 Suppression of Peripheral Renin-Angiotensin System

Effects on renal p-AR levels

Scatchard analysis of p-AR binding in renal cortex (Fig. 5-4) indicated that

P1-AR was the major subtype in the control rats, composing 70% of total p-AR.

After p1-AS-ODN injection, the Bmax of pi-AR was significantly diminished by 35%

on day 4 (P<0.05), 29% on day 10 (P<0.05), 23% on day 18 and completely

resumed on day 40. p1-AR reduction in kidney coincided with that in heart, which

were accompanied by the significant drop in BP (P<0.01). In contrast, the p2-AR

level was not affected, nor the affinity of either subtype (data not shown). INV-

ODN had no effect on either subtype.

Kidney slices from the same rats were subject to quantitative

autoradiography in order to display the structural distribution of p-AR (Fig. 5-5). p

i-subtype composed -60% of p-AR levels, which was predominantly localized in

renal cortex and outer band of medulla. p2-subtype was more diffusely

distributed in the kidney at a lower level. This result was consistent with previous

reports(205). Four days after pi-AS-ODN treatment, the overall density of pi-

subtype in kidney was significantly reduced from 23.5+2.1 to 15.4+3.3 fmol/mg







59
(P<0.05). The diminution in renal cortex was particularly conspicuous due to the

higher basal level. As expected, the distribution and concentration of p2-subtype

remained unchanged in accord with binding results. This further confirms the

specificity of the inhibitory effect of 31-AS-ODN on p3-subtype.



Effects on the peripheral renin-angiotensin system

RT-PCR (Fig. 5-6) revealed that the renin mRNA level in renal cortex was

transiently decreased to 62% of control 4 days after p3-AS-ODN injection. It was

completely reversed by day 18 (Fig. 5-7). On the other hand, PRA and plasma

Ang II levels showed different patterns of reduction, which were significantly

decreased on day 10 and day 18 (P<0.01), but not on day 4. Thus PRA and Ang

II seemed to have a delayed action relative to the reduction in renin mRNA (Fig.

5-7).



5.2.3 Absence of CNS Effects

Quantitative autoradiography of 6-18 tissue slices in brain, heart and

kidney was analyzed four days after intravenous pi-AS-ODN administration (Fig.

5-8). No changes in the distribution of p-AR in forebrain and brainstem regions

were detected. This indicated the absence of antisense effect on the p-AR

expression in CNS. However, PI-AS-ODN significantly (P<0.05) reduced P1-AR

density in cardiac ventricles (from 30.22.1 to 20.62.5 fmol/mg) and renal

cortex (from 26.43.1 to 17.43.3 fmol/mg). This was consistent with the binding

results. 32-AR was not affected in any tissues.











5.3 Discussion

The data presented above demonstrate that pI-AS-ODN effectively

decreases p3-AR density in heart and kidney, which led to a significant reduction

in cardiac contractility and plasma renin activity. These findings are consistent

with the hypothesis that P3-AS, through the inhibition of p3-AR expression, is able

to render heart, kidney and other tissues less sensitive to sympathetic activation,

which is a major contributing factor in high BP.

A single injection of pi-AS-ODN effectively decreases the cardiac 31-AR

density, which produces a rapid reduction in ventricular contractility and thereby

in cardiac output in response to p-stimulation. Unlike p-blockers, 31-AS-ODN

does not decrease HR. Hence bradycardia is unlikely to be involved in the

antihypertensive mechanisms of pi-AS-ODN.

The inhibition of renin release is regarded as a primary mechanism

second to a decrease in cardiac output for the antihypertensive effects of p-

blockers. Many p-blockers can reduce PRA in patients and experimental animals

(55,206). They are found to be more effective in patients with higher renin

profiles (56,57). In my experiments, 3p-AS-ODN effectively decreased PRA and

Ang II chronically. But the decrease in PRA and Ang II did not occur until -10

days after p1-AS-ODN injection (Fig. 5-7), in contrast to the rapid drop in cardiac

output two days after injection (Fig. 5-2 & 5-3). Thus it appears that the effects of

Pi-AS-ODN on the kidney renin and the circulating renin-angiotensin system are

more delayed than cardiac action. Therefore suppression of cardiac output may







61
account for the early phase of the antihypertensive effect of 3p-AS-ODN while

the inhibition of renin-angiotensin activity acts as the secondary mechanism

underlying the sustained reduction of BP in SHR.

Plasma renin activity (PRA) is regulated in several steps: synthesis of

renin in kidney, conversion of prorenin into active renin, and secretion of renin

into the circulation. By decreasing p3-AR levels in kidney, p3-AS-ODN

significantly reduced PRA and the subsequent plasma Ang II levels. This is

unlikely through the inhibition of renin expression, because there is no chronic

diminution of renin mRNA levels. Instead, pi-AS-ODN may exert its inhibitory

impact on renin secretion or the conversion of inactive renin to active renin. This

hypothesis is consistent with the observation that p-adrenergic stimulation of

renin expression had a different time course than that of renin secretion

(207,208). Further, p-blockers have been shown to reduce prorenin processing

to active renin without changing total renin (prorenin + PRA) levels in plasma

(55). A flaw in this study is the omission to measure the local renin concentration

in kidney cortex, where the circulating renin comes from. This assay would have

provided an insight in the differential effects of p1-AS-ODN on renin production

and secretion.

In addition to pi-adrenergic activation, renin expression is also regulated

by the negative feedback of ANG II. Therefore the reversal of renin mRNA levels

on day 10 and day 14 after p1-AS-ODN treatment may be explained by the

decreased plasma ANG II concentrations that exerted less inhibition on renin

expression.







62
Antisense is known for the gene targeting specificity. Radioligand binding

assay showed that p1-AS-ODN is specific in inhibiting 31-AR without any effect

on 32-AR. Therefore pi-AS-ODN may be free of the side effects that are caused

by non-specific p2-antagonism of p-blockers.

The blood-brain barrier (BBB) formed by capillary endothelia is only

permeable to small lipophilic molecules with molecular weight <600 dalton

[reviewed by Pardridge (209)]. Owing to their high hydrophilicity, ODNs undergo

negligible transport through BBB and have very limited access to CNS (130).

Coupling with liposomes increases the lipophilicity of DNA, but the large size of

DNA/liposome complex (100-300nm) makes it unable to cross BBB. The cellular

and organ distributions of DNA/liposome complexes with fluorescent labeling

were previously studied in mice after intravenous injection and the results

indicated that the complexes were primarily taken up by capillary endothelial

cells in most of the peripheral organs including lung, heart, kidney and spleen,

but absent in the brain (210). Although the brain retention of liposomes after

peripheral administration was observed in some cases, it is due to entrapment

within the brain microvasculature (211). In our study, autoradiography in brain

revealed no detectable changes in the expression and distribution of p-AR after

intravenous 31-AS-ODN injection. This provided further evidence that our

antisense approach did not have CNS effects. It also suggested that the

inhibition of central p-adrenergic system is unlikely to contribute to the

antihypertensive action of p1-AS.














* 13p
o P3_


5 10
Time after treatment (day)


Figure 5-1. Pi-AS-ODN decreased the density of PI-AR but not p2-AR in
cardiac ventricles of SHR.

A). Time course of P,-AS-ODN effects on Bmax of pi-AR (0) and P2-AR (0).
B). Bmx of p-ARs 4 days after iv injection of saline (solid bar), 1mg/kg INV-ODN
(open bar) or 1mg/kg p3-AS-ODN (shaded bar). Data represent mean SEM of
each point (n=6). *P<0.01, **P<0.001 vs. saline or INV-ODN.








64


A 300

250-





E 150


100


50 -


0
0.00 0.02 0.04 0.06 0.08 0.10 0.12
B 300
290

280

270

E 260 o INV-ODN
250
S250 p1-AS-ODN
240

230

220 -

210

200
0.00 0.02 0.04 0.06 0.08 0.10 0.12

Isoproterenol (pM)


Figure 5-2. P,-AS-ODN reduced ISO-stimulated positive inotropic more than
chronotropic response in isolated perfused SHR hearts.

48 hrs after iv injection of 1mg/kg INV-ODN (0) or Pi-AS-ODN (0), hearts were
perfused with Krebs buffer containing increasing concentrations of ISO. Shown is
the effect of P,-AS-ODN on ISO-induced elevation in developed left ventricular
pressure (A) and heart rate (B). The data represent mean SEM of each group
(n=6-9). P<0.05, ** P<0.01 vs. INV-ODN.









65



A 2000


1500

I I
1000 T






010 20 30 40
B 140
120

100

80
OC 60

40



0







E


-8 ,

-12

-16
0 10 20 30 40

Dobutamine (Lg/kglmin)



Figure 5-3. In vivo effects of p1-AS-ODN and atenolol on cardiovascular
hemodynamics of SHR in response to pi-stimulation.


The same group of SHR (n=4) was tested for dobutamine-induced hemodynamic
alteration at control levels (0), 48 hrs after p1-AS-ODN injection (0), and 1 hr
after atenolol injection (V). The rats were allowed to fully recover between
treatments. During dobutamine infusion, cardiac dP/dtmax (A), heart rate (B) and
systolic BP (C) of SHR were monitored by telemetry system. Data represent
mean SEM. P<0.05, ** P<0.01 versus control.

























Yr~L_


40


Days after injection


10


0


Pi 32


Figure 5-4. Effects of 11-AS-ODN on p-AR levels in renal cortex.

SHR were injected with 0.5 mg/kg pi-AS-ODN or INV-ODN with liposomes at
charge ratio 2.0. A) Time course of the changes in Bmax of P1-AR (*) and 32-AR (
0). B) Bmax of p-AR 4 days after iv injection of saline (solid bar), INV-ODN (open
bar) or 11-AS-ODN (shaded bar). Data represent mean SEM of each point
(n=6). P<0.05, ** P<0.01 vs. saline or INV-ODN.




















Control


P3-AS-ODN


P,-AR


.., I ~t' .
II
' 7.

11


Figure 5-5. Representative autoradiography of p-AR levels in the kidney of
control (n=6) or pi-AS-ODN (n=6) treated SHR 4 days after
injection.
























renin


GAPDH


1 2 3 4 5 6

wwq' ms w -- a


W rwmm


Figure 5-6. RT-PCR and DNA blot of renin and GAPDH mRNA in renal
cortex of SHR.

Lane 1, saline control; 2, INV-ODN; 3, pf-AS-ODN day 4; 4, p3-AS-ODN day 10;
5, pi-AS-ODN day 18; 6, p1-AS-ODN day 40. Every point represents a pool of
total RNA extracted from 4-5 rats.









69



A 120

E 100
o0
o 80

< 60
z
Cr
E 40
C
0


B E
0
12
0)






C
S10

> 8

r 6 1







S40






E 30
E 2












10
80 -
= 50 \
< 40
E 30 A'
E- 20
101


0 10 20 30 40

Days after injection



Figure 5-7. pf-AS-ODN at a single injection exerts a delayed suppression on
renin-angiotensin system.

A, Effect on renin mRNA levels in renal cortex. B, Effect on plasma renin activity.
C, Effect on plasma angiotensin II levels. Data represent mean + SEM of each
time point (n=6). P<0.01, ** P<0.001 vs. baseline.












AS-ODN


Figure 5-8. Autoradiographic illustration of pf-AR levels in different tissues
of SHR.

A, forebrain; B, brainstem; C, heart; D, kidney. Autoradiography was performed 4
days after iv injection of INV-ODN (n=6) or Pf-AS-ODN (n=6).


Control


ta,.. ~t.~'l
r B















CHAPTER 6
REPEATED ADMINISTRATION OF p3-AS-ODN AND SAFETY PROFILE


6.1 Introduction

To be therapeutically useful, the pl-AS-ODN has to be effective with

repeated administration and non-toxic. Currently there are at least ten clinical

trials testing AS-ODNs in a variety of diseases [reviewed by Agrawal (137)].

These clinical data have convincingly demonstrated the sustained efficacy with

repeated iv injection of AS-ODNs. Nucleic acids are poor epitopes. Human

bodies usually do not develop immune response to nucleic acids except in rare

autoimmune diseases. This may explain why AS-ODNs maintain effectiveness

during chronic treatment.

Hypertension is associated with pathophysiological changes such as left

ventricular hypertrophy (LVH) (212,213). Long-term antihypertensive therapies

including angiotensin-converting enzyme inhibitors and P-blockers can prevent

and reverse LVH (214). Therefore, if repeated treatment with p1-AS-ODN is

effective in sustained reduction in high BP, it may as well inhibit the development

of LVH.

Safety and pharmacokinetic profiles of several AS-ODNs have been

reported. In these studies, phosphorothioate-ODN without any drug carriers were

given through continuous iv infusion over 3-11 weeks and a wide range of doses








72
were tested for toxicity. Phosphorothioate-ODNs are well tolerated in humans at

the doses < 2.5 mg/kg/day without any pathological or functional changes

(215,216). The commonly seen side effects at higher doses are transient

thrombocytopenia and fatigue (216,217). Likewise, studies in mice and monkeys

revealed no adverse effects at doses < or = 10 mg/kg/day. Toxicity with large

doses (>10 mg/kg/day) include splenomegaly, lymphoid hyperplasia and

mononuclear cell infiltrates in mice, and complement activation and

thrombocytopenia in monkeys (218,219). However, these adverse events are

transient, reversible and only associated with high dosage (219-221). Lack of

genetic toxicity has been confirmed by different researchers (164,219).

The polyanionic property of phosphorothioate-ODN due to the addition of

sulfur atoms is responsible for some side effects such as thrombocytopenia

(137).Due to the limited cellular uptake without appropriate vectors, AS-ODNs

have to be given at high doses and frequency in these clinical studies. This in

part accounts for the toxicity discussed above.

Liposomes are used as carriers for a wide variety of drugs. They are safe

and non-immunogenic. They can also reduce the acute toxicity of many

compounds such as amphotericin B and doxorubicin. Generally speaking,

liposome formulations tend to protect tissues from the toxic effects of

encapsulated compound and are usually better tolerated than the free drug

(222). With improved uptake by liposomes, AS-ODN can be given at a lower

dose to elicit the same therapeutic benefits. Cationic liposomes will also

decrease the net negative charge of phosphorothioate-ODN and thus attenuate








73
its nonspecific interaction with serum proteins. Therefore cationic liposomes

should be able to improve the delivery as well as reduce the toxicity of

phosphorothioate-ODN.

This study is designed to test the effects of repeated injections of 31-AS-

ODN on BP and cardiac hypertrophy in hypertensive rats. Toxicity and immune

response will be assessed.



6.2 Results

6.2.1 Effects of Repeated pI-AS-ODN Treatment on BP

The efficacy of three repeated iv administrations of 1 mg/kg pi-AS-ODN

(n=9) was tested in adult SHR with high BP. Animals treated with three repeated

injections of 1mg/kg INV-ODN (n=7) or saline (n=7) served as controls. Systolic

BP was measured every three days. A second injection of P1-AS-ODN was given

before the BP returned to baseline after the previous treatment.

Three repeated injections of pi-AS-ODN at 16, 20 and 26 days

maintained SBP of SHR at 20-30 mmHg lower than controls for more than 2

months (Fig. 6-1). There was no loss in the antihypertensive efficacy with

repeated treatments. Instead, the 2nd and 3rd injections were more effective than

the 1st one in the extended duration of BP reduction. Control animals treated with

saline or INV-ODN had no change in BP with repeated injections. HR was not

altered in any groups.









6.2.2 Effects of Repeated 31-AS-ODN Treatment on Cardiac Hypertrophy

At the end of 2-month treatment, SHR were sacrificed and the organs

were weighed in a blind manner. The left ventricle of the heart was dissected

free of fat and connective tissues, and weighed by a colleague who was not

aware of the group assignment. Left ventricular hypertrophy (LVH) was indicated

by the ratio of left ventricle weight to body weight (LV/BW). p3-AS-ODN treatment

produced a small but significant (P<0.05) reduction in hypertension-associated

LVH in SHR compared to saline control (Fig. 6-2).



6.2.3 Safety Profile

Liver Transaminases

Liver is a major organ for uptake and clearance of both liposomes and

phosphorothioate-ODN when they are administered separately (222-224).

Therefore large doses of liposome/ODN complex may overload the liver and

cause liver toxicity. Alanine aminotransferase (ALT) and aspartate

aminotransferase (AST) are routinely used as a clinical index for liver function.

Their levels increase substantially upon liver damage. Plasma concentrations of

ALT and AST were measured after each injection, which revealed no significant

difference between AS-ODN and control groups (Fig. 6-3).

Hematology

Phosphorothioate-ODN has increased negative charges due to sulfur

atoms and may interact with positively charged proteins in the blood and cause

hematological alterations. Reduced platelet counts were reported with high








75
doses of systemic injection of phosphorothioate-ODN (>5mg/kg/day for weeks)

(225). At the end of 2-month treatment, hematological parameters were

measured. There was no difference in total leukocyte count (WBC), platelet

count, hematocrit and mean platelet volume (MPV) among three groups (Table

6-1).


Table 6-1. Clinical biochemical and hematological parameters after repeated
administration of P3-AS-ODN.

p31-AS-ODN INV-ODN Saline


Liver Enzymes

AST (unit/ml)

ALT (unit/ml)

Hematology

hematocrit (%)

WBC (103/mm3)

platelet (103/mm3)

MPV (fL)

C-Reactive Protein

Antibody to ODN


74.0 + 3.7

25.7 1.1



46.2 0.63

5.48 0.41

685 12

6.93 0.08

ND

ND


83.0 8.3

30.5 3.8



46.0 0.1

5.75 1.15

706 28

7.2 0.20

ND

ND


82.3 3.8

30.0 3.2



46.6 0.33

5.86 1.12

685 22

7.0 0.09

ND

ND


n = 7-9 for each group, ND: non-detectable.


Immune Response

In order to investigate whether animals develop immune responses

against repeated administration of pi-AS-ODN, gel immunodiffusion assay was

employed to detect possible antigen-antibody reaction between liposome/ODN








76
complex and serum. If the body produces specific antibodies against a certain

antigen, the increase of IgG levels will have different time courses after the initial

and following inoculations. Second encounter with the antigen will mount a more

rapid and greater elevation in IgG production than the first response. Considering

this immune memory, serum samples for antibody detection were collected at

13, 7 and 7 days after the 1st, 2nd and 3rd injection, respectively.

No antibodies to ODN/liposome were detected in immunodiffusion

analysis. Presence of C-reactive protein (CRP) in the blood is a clinical indication

for immune reaction after infection. This parameter was found to be negative in

all animals before and after repeated injections of PI-AS-ODN, INV-ODN or

saline. It provided further evidence for the absence of immune responses.

Tissue Histopathology

High doses of phosphorothioate-ODN were associated with hyperplasia of

mononuclear/phagocytic cells in the reticuloendothelial (RE) system (225). Heart,

liver, spleen and kidney were subjected to pathological examination at the end of

the study, which revealed no significant sign of immunopathology or organ

damage (Fig. 6-4 to 6-7). Mononuclear cell infiltration was occasionally observed

in small areas of the heart, but this occurred in all three groups. It is probably due

to the higher background immune response in SHR, compared to WKY rats.

Organ Weights

Enlargement of spleen and kidney has been reported in rats receiving

high doses of phosphorothioate-ODN (219,225). In this study, no significant

change was found in the weight of spleen and kidney of all groups (Table 6-2).










Table 6-2. Organ weights after repeated administration of p3-AS-ODN.

pl-AS-ODN INV-ODN Saline

Tissue Weight

LV/BW (g/kg) 3.10 + 0.07* 3.26 0.06 3.33 + 0.04

spleen (g) 0.64 0.03 0.65 + 0.01 0.62 + 0.1

kidney (g) 1.32 + 0.03 1.34 + 0.08 1.34 0.06

n = 7-9 for each group
P < 0.05 vs. saline control.


6.3 Discussion

Maintained Efficacy and Lack of Immune Response after Repeated
Administration of pJ-AS-ODN by Cationic Liposomes
Any therapy that induces immune response cannot be truly useful in clinic,

because immune response not only increases the danger of acute treatment, but

also renders repeated administration ineffective. Gene therapy is no exception.

Tremendous efforts have been made to avoid immune reaction in gene therapy.

These efforts have mainly focused on the choice and development of

appropriate vectors. Among viral vectors, adenovirus and HSV are known for

acute toxicity and immune response, albeit new generations of vectors have

minimized viral genomes and are better tolerated. Retrovirus has high

transduction efficiency, but is usually limited in dividing cells. AAV is considered

a safe vector, because wild type AAV has not been associated with any

pathology in humans. It has a broad infectivity and can produce long-term

expression of therapeutic genes due to the ability to integrate into the host







78
genome. However it has its own limitations, i.e. difficulty in production and

relatively low expression [reviewed in (152-154)].

Liposomes, as a nonviral vector, are safe and non-immunogenic. In this

study, liposomal delivery of p3-AS-ODN maintained its antihypertensive

effectiveness upon repeated administration. No immune response or significant

toxicity was observed. These results suggest that the liposome formulation is

technically feasible to achieve sufficient gene delivery and produce prolonged

therapeutic benefits, which are considerably longer lasting than traditional

medicines.


Inhibition of LVH by Repeated Treatment with 31-AS-ODN

LVH, the disproportionate overgrowth of muscle in the left ventricle, has

historically been considered the natural evolution of hypertension. It is estimated

that the structural remodeling of the heart, including LVH, develops in about 70%

of persons with sustained hypertension (212,213). Hypertrophied cardiac muscle

not only increases myocardial mass and workload, but also contains excess

collagen and increased fibrosis. LVH is associated with higher cardiac risks and

viewed as a strong predictor for heart failure and myocardial infarction (226-229).

In light of the importance of LVH in cardiovascular physiology and patient

prognosis, much attention has been paid to the use of therapies to reverse the

process. Among the antihypertensive agents, angiotensin-converting enzyme

inhibitors are found to be the most potent for reversal of LVH, followed in the

order by calcium channel antagonists and P-blockers (214,230,231). pi-selective

blockade is highly effective in reversing LVH during chronic treatment (232).








79
My results indicate that repeated injection of 11-AS-ODN is not only able

to reduce high BP, but also attenuate the cardiac remodeling associated with

hypertension. 2-month treatment results in a small but significant reduction in

LVH of SHR. Increasing the treatment period may exert more marked effects, as

greater LVH regression has been observed after longer treatment with p-

blockers (232). Since LVH is a risk factor for heart failure and myocardial

infarction, 31-AS-ODN may as well protect the heart from these cardiac events

by inhibiting LVH. However, whether the beneficial effects of i1-AS-ODN on LVH

is due to direct inhibition of myocyte growth or secondary to the BP reduction is

not known.


Limitations of the Current Study

Generally, the clinical assessment of toxicity requires more extensive

measurements of pathological and biochemical parameters than what were

determined in this study. Therefore further experiments should be performed to

provide a complete safety profile of p1-AS-ODN including blood sugar, lipid

profile, and liver and kidney functions. In addition, the toxicity data presented

here were obtained at least 7 days after each injection. Although there was no

adverse effect beyond this time point, it is still possible that ODN/liposome

treatment may cause some acute toxicity.

Conclusion

The results of this study demonstrate that p -AS-ODN can be given

repeatedly to achieve a sustained reduction of hypertension and reduces cardiac








80
hypertrophy without causing toxic effects or immune stimulation. These data,

together with early results, show that pi-AS-ODN is viable as a potentially new

prolonged antihypertensive agent with benefits on cardiac remodeling.








81






--- AS-ODN
210 INV-ODN
---i- saline

200



E 190
E


180



170


0 10 20 30 40 50 60
Days

blood blood blood blood Sacrifice
blood, tissues

Figure 6-1. Sustained antihypertensive effects of repeated administrations
of 3,-AS-ODN in SHR.

Three repeated iv injections of 1 mg/kg P,-AS-ODN (n=9) delivered by liposomes
were given to adult SHR as indicated by upward arrows. Repeated injection of
saline (n=7) and 1 mg/kg INV-ODN (n=7) served as controls. Systolic BP was
measured by tail cuff method. Blood samples were collected at different time
points as indicated by downward arrows for safety profile analysis. Data
represent mean SEM. P<0.05 vs. saline or INV-ODN.























3.5






3.0 -






2.5






2.0


saline


INV-ODN


AS-ODN


Figure 6-2. Repeated administration of P,-AS-ODN reduced
hypertrophy in SHR.


left ventricular


SHR were treated with repeated injections of p,-AS-ODN (n=9), saline (n=7) or
INV-ODN (n=7) for 2 months. P<0.05 vs. saline.


T

I


I __ __ L __ i __ __














Plasma AST


100


AS-ODN INV-ODN saline


Plasma ALT


baseline
E2222 1st injection
K2. 2nd injection
M 3rd injection


AS-ODN INV-ODN saline


Figure 6-3.


Plasma levels of liver transaminases ALT and AST after
repeated injections of P,-AS-ODN.


Saline and INV-ODN serve as controls. Data represent mean SEM of each
group (n=7-9).









O
m~o
Q) 0
(D8
3
m 0










I -n
3
0 S


0

. O
co
o a

=r i

~Co O
( c3

a. a
oQ 3


x
0 0
















0
I I

S
o


0



(fD






0
5




o
z



0
Z



(A
ma
53
0p










I 3I

SEc
0 0
0
x *



CD
.0



StoI
Ue
-CCI







ow 0










PI




.z
x 0
w *
o -













0
5~ z









(A
I





o
-s
(D














0L










I "
CD 4*

x
-i


a 0
^-0-




13



































0
o



0) o








0)
S 0








o
03


o

0




3
I






6
0







O

o)















CHAPTER 7
PROTECTION BY 31-AS-ODN AGAINST MYOCARDIAL DYSFUNCTION
INDUCED BY GLOBAL ISCHEMIA-REPERFUSION


7.1 Introduction

Acute myocardial ischemia causes a significant increase in plasma

catecholamine levels, which leads to exacerbation of the myocardial injury and

dysfunction. Acute myocardial ischemia is also characterized by increased

sensitivity of p-ARs to catecholamines (233). P,-AR, being the predominant

subtype in the myocardium, forms an interface between sympathetic nervous

system and heart function. Several experimental studies show that the density

and mRNA of P,-AR are augmented in the myocardium after acute ischemia

(86,234). p-blockers, especially p,-selective blockers, can protect myocardium

against ischemic injury and dysfunction (235), decrease infarct size (236,237)

and reduce the incidence of sudden cardiac death after myocardial infarction

(85).

The previous results have shown that P,-AS-ODN can inhibit P,-AR

expression specifically and reduce BP in SHR for a prolonged period with a

single iv injection. This study is designed to test the hypothesis that the use of p,-

AS-ODN may protect myocardium against dysfunction in ischemia-reperfusion in

the isolated rat heart, an in vitro model of myocardial infarction.











7.2 Results

7.2.1 Cardiac Dysfunction during Ischemia-Reperfusion

Four parameters were measured as the major indices for cardiac

functions, including coronary perfusion pressure (CPP), left ventricular end-

diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP) and

developed left ventricular pressure (dLVP = LVSP LVEDP). Myocardial

ischemia results in cardiac dysfunction, which is indicated by increased CPP and

LVEDP and decreased LVSP and dLVP. An increase in CPP may result from an

impaired coronary endothelium-dependent relaxation. The increased LVEDP and

decreased LVSP represent insufficient contraction and relaxation of the

myocardium. The net result of these changes is a diminished dLVP whereby the

heart fails to pump enough blood to meet the elevated oxygen demand.

Five groups of SD rats were studied. Sham controls (n=6) were injected

with saline and perfused continuously for 80 min without ischemia/reperfusion

(I/R). In other groups, hearts were perfused for 20 min and subjected to global

ischemia (30 min) followed by reperfusion (30 min). Saline + I/R (n=6) or atenolol

+ I/R (n=7) groups were injected iv with saline or 2mg/kg atenolol 6 hr before the

hearts were excised. AS-ODN + I/R (n=7) or INV-ODN + I/R (n=7) were injected

iv with 1 mg/kg p,-AS-ODN or INV-ODN 4 days before the I/R experiment.

The basal levels of CPP, LVEDP and dLVP were similar in all groups of

rat hearts. In the sham controls, there were minimal (-5%) changes in the

indices of cardiac function. In saline group, 30 min ischemia followed by 30 min







90
reperfusion resulted in marked cardiac dysfunction, indicated by a significant

increase in CPP and LVEDP, and a decrease in dLVP (P<0.01 vs. pre-ischemia

values.)

Treatment with P,-AS-ODN markedly attenuated the ischemia-reperfusion

induced ventricular abnormality, manifested by the preservation of dLVP and

minimization of increase in CPP and LVEDP (P<0.05 vs. saline group). Overall,

AS-ODN treatment appeared to be equivalent to or even better than atenolol in

these effects. AS-ODN restored dLVP to a greater extent than atenolol. INV-

ODN showed no effect on ischemia-induced dysfunction and cannot be

differentiated from saline group. Data on cardiac function parameters from these

experiments are summarized in Fig. 7-1.


7.2.2 Malondialdehyde Levels in Myocardium

Malondialdehyde (MDA), a product of lipid peroxidation, has been used as

an index of tissue injury after ischemia-reperfusion by several investigators

(238;239). MDA levels in myocardium increased significantly after ischemia-

reperfusion (P<0.05 vs. sham control). Pretreatment of rats with AS-ODN and

atenolol significantly attenuated this increase (P<0.05 vs. saline treatment). As

expected, INV-ODN did not affect MDA levels (Fig. 7-2).


7.2.3 P,-AR Levels in Myocardium

p,-AR levels in the left ventricle were measured after ischemia-reperfusion

by radioligand saturation binding assay (Table 7-1). The Bmax of P,-AR was

slightly elevated during ischemia-reperfusion, compared to sham controls. P,-AS-







91

ODN treatment significantly reduced the 3,-AR density by nearly 50% (P<0.05),

while atenolol or INV-ODN had no effect. p1-AR levels were not significantly

changed in any group. The affinity of both i,- and p1-ARs remained unaltered.



Table 7-1. Bmax of P,- and 3,-ARs in myocardium during ischemia/reperfusion.

ischemia-reperfusion

Sham Saline INV-ODN AS-ODN Atenolol

Pi-AR 21.52.0 25.44.4 26.53.2 13.80.9* 27.14.0

12-AR 11.20.5 9.00.5 10.20.7 10.70.7 9.70.6


Data represent mean SEM of 7 rats per group. P<0.05 vs. sham, saline, INV-
ODN or atenolol groups.


7.3 Discussion

There is a generalized stimulation of sympathetic nervous system during

myocardial ischemia, manifested by an increase in catecholamine levels in both

plasma and myocardium (240-242), coupled with hypersensitized cardiac p-ARs

(233). These changes are viewed as a compensatory response in an attempt to

preserve cardiac function. However the consequence is detrimental, because the

sympathetic activation increases the chance of tachyarrhythmia and workload of

the heart which in fact exacerbates cardiac injury induced by ischemia.

Consistent with this concept, pharmacological intervention of p-ARs can protect

the heart and reduce mortality and morbidity during and after myocardial

infarction (85), and prevent recurrent infarction (243). It is noteworthy that all the







92

P-blockers that have shown benefits in myocardial infarction patients can inhibit

p,-AR, and no difference in the therapeutic efficacy has been found between p,-

selective and non-selective blockers. This suggests that 3,-AR is the major

subtype mediating the deleterious effects of overactive sympathetic system on

the heart. However, as discussed earlier, the use of p-blockers is limited by their

intrinsic drawbacks including undesirable p2-antagonism and short action. A new

strategy for specific P,-AR inhibition by antisense may overcome these problems

and provide an effective and longer-lasting protection against myocardial

ischemia.

Therefore, the present study was designed to examine the protective role

of 3,-AS-ODN against cardiac dysfunction after a brief period of ischemia-

reperfusion. The effects of 3,-AS-ODN was compared with those of a selective

pi-blocker atenolol in this process. The results indicate that 30 min ischemia

followed by 30 min reperfusion resulted in significant cardiac dysfunction and

lipid peroxidation in the saline-treated rat hearts. Pretreatment with both 3,-AS-

ODN and atenolol preserved cardiac performance and reduced lipid peroxidation

following ischemia-reperfusion. The protective effects of P,-AS-ODN on cardiac

function were at least equivalent to those of atenolol, and AS-ODN seemed to

provide a better restoration of dLVP than atenolol.

p,-AR, the predominant p-AR subtype in the myocardium, is the major

mediator of sympathetic activation of cardiac functions. Several experimental

studies have shown an upregulation of P,-AR, but not P,-AR levels, during acute

ischemia (86,234). Ihl-Vahl et al. (86) convincingly demonstrated a subtype-







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specific and time-dependent increase in mRNA levels of P,-AR during acute

myocardial ischemia. This is consistent with the clinical data that point to the

major role of 3,-AR in ischemia-induced cardiac dysfunction. In the current

experiments, a slight elevation in P,-AR levels was observed after brief ischemia-

reperfusion, which did not reach the statistical significance. Reperfusion may

explain the discrepancy here, because acute ischemia unequivocally increased

3,-AR expression but the following reperfusion usually restored P,-AR to normal

levels in several studies (87,234).

Both 3,-AS-ODN and atenolol suppress p1-AR functions and protect the

heart from severe injury after ischemia/reperfusion. However, these two

approaches are different in several aspects. First, p,-AS-ODN reduced the total

number of functional P,-AR on the cell membrane. Atenolol competed with

endogenous catecholamines and did not affect 3,-AR expression. Second, AS-

ODN was given 4 days prior to ischemia-reperfusion to allow for sufficient

reduction in P,-AR expression. Atenolol was given 6 hrs before excising the heart

because of its rapid onset of action and short half-life. Therefore, 3,-AS-ODN

may provide a prophylactic protection from myocardial infarction in addition to its

antihypertensive benefit. However it cannot replace the intravenous injection of

P-blockers as the acute postinfarction rescue because of its slow effects.

Isolated rat, rabbit or guinea pig heart model provides an inexpensive and

reproducible method to evaluate cardiac function and myocardial metabolic

alterations during ischemia-reperfusion. It has been used extensively as an in

vitro model for acute myocardial ischemia (182,239). In an isolated and