Bradykinin Receptor Antagonism During Cardiopulmonary Bypass- Specific Aim 3

Morbidity of cardiopulmonary bypass. Each year more than a million patients worldwide
undergo cardiac surgery. Nearly all cardiac surgeries are performed on unbeating hearts
supported by CPB. Although the use of off-pump coronary artery bypass surgery procedures are
increasing, concerns regarding incomplete revascularization and reduced venous graft patency
limit the use of this technique to specific patients. CPB activates various humoral
cascades including the coagulation cascade, the KKS, the fibrinolytic cascade, and causes a
systemic inflammatory response syndrome. Activation of these systems can lead to
hypotension, fever, disseminated intravascular coagulation, diffuse tissue edema, or, in
extreme cases, to multiple organ failure. Activation of the KKS contributes to the
hemodynamic perturbations, fibrinolysis and inflammatory response observed in patients
undergoing CPB. Aprotinin, a non-specific serine protease inhibitor, that works in part by
decreasing bradykinin generation, decreases fibrinolysis, hypotension and the systemic
inflammatory response associated with CPB. Aprotinin decreases blood loss and transfusion
requirements, however, its use is mainly limited to redo-cardiac surgery because of cost.
Other factors that may limit the widespread use of aprotinin include an increased risk for
renal dysfunction, allergic reaction and non-specificity of the drug. Bradykinin mediates
most of the effects of the KKS. Thus, bradykinin receptor antagonism has the potential to
modulate the effects of KKS activation during CPB. The purpose of this proposal is to test
the hypothesis that endogenous bradykinin contributes to the hemodynamic, fibrinolytic and
inflammatory response to CPB and that bradykinin receptor antagonism will reduce
hypotension, inflammation and transfusion requirements. The proposed studies promise to lead
to novel therapies to reduce morbidity associated with CPB.

Cardiopulmonary bypass activates the kallikrein-kinin system (KKS). Several groups,
including ours, have reported that bradykinin concentrations increase during CPB. For
example, Campbell et al demonstrated that bradykinin levels increase 10 to 20-fold during
the first 10 minutes of CPB, returned to basal levels by 70 minutes of CPB and remained 1.7
to 5.2-fold elevated after CPB. Plasma and tissue kallikrein were reduced by 80 and 60%
respectively, during the first minute of CPB. Similarly, we have demonstrated that
bradykinin increases significantly during CPB and that ACE inhibition and smoking potentiate
the kinin response during CPB.

Fibrinolytic response to cardiopulmonary bypass. CPB increases t-PA antigen and activity in
a time-dependent manner. The fibrinolytic response during CPB is heterogeneous, with t-PA
levels varying as much as 250-fold. The mechanism of t-PA release during CPB is likely
multifactorial. As outlined above, we and others have shown that CPB increases bradykinin, a
potent stimulus to t-PA release. In addition, thrombin or complement generated during CPB
may stimulate the release of t-PA from endothelium. In addition to the changes in t-PA
concentrations during CPB, PAI-1 activity falls because of hemodilution and the rise in t-PA
release which consumes active PAI-1. Plasmin generation increases over 100-fold while
D-dimer generation increases 200-fold within 5 minutes of CPB initiation. For the remainder
of the CPB, average plasmin and D-dimer levels remain 20-fold to 30-fold above baseline
levels. The postoperative period is marked by a systemic inflammatory response caused by a
combination of CPB and surgery producing an acute -phase response that results in increased
PAI-1 production. PAI-1 levels begin to rise about 2 hours after surgery. Once CPB is over,
PAI-1 levels continue to rise and peak during the first 12-36 hours postoperatively and
return to normal by the second postoperative day. Thus, the fibrinolytic response to CPB is
characterized by an initial hyperfibrinolytic phase that begins with a rapid rise in t-PA,
plasmin, and D-dimer concentrations followed by a postoperative hypofibrinolytic phase
associated with a rise in PAI-1 secretion and a fall in t-PA concentrations.

Interaction between the renin-angiotensin system (RAS), the KKS and fibrinolytic system.
There is evidence that fibrinolytic balance is regulated by the RAS and the KKS. ACE is
strategically poised to control fibrinolytic balance by promoting the breakdown of
bradykinin and the conversion of Ang I to Ang II. Ang II causes the release of PAI-1 thus
inhibiting fibrinolysis. Bradykinin stimulates t-PA release through its B2 receptor. ACE
inhibition decreases PAI-1 antigen levels and increases endothelial t-PA release through
endogenous bradykinin. In addition, ACE inhibition enhances exogenous bradykinin-mediated
vasodilation and t-PA release. The augmentation of bradykinin-induced vasodilation, the
increase in t-PA and the decrease in PAI-1 described with ACE inhibition in patients with
ischemic heart disease may contribute to the primary mechanism of the anti-ischemic effects
associated with chronic ACE inhibitor therapy. We have demonstrated that inpatients
undergoing coronary artery bypass grafting (CABG) requiring CPB, not only did ACE inhibition
increase fibrinolytic activity by decreasing PAI-1 antigen and increasing t-PA activity, but
also enhanced the kinin response. Increased PAI-1 concentrations in the perioperative period
are associated with acute vein graft thrombosis. Thus, ACE inhibitors have a potential to
reduce the risk of acute graft thrombosis through their effects on Ang II generation by
attenuating the PAI-1 response after CABG. As opposed to the beneficial effects of ACE
inhibition on PAI-1, the augmentation of the kinin response during CPB may have detrimental
effects including increased fibrinolysis with consequent bleeding and hypotension. The
effect of angiotensin II type 1 (AT1) receptor antagonist on the fibrinolytic response to
CPB is not known. Inpatients with essential hypertension AT1 receptor antagonist decreases
PAI-1 antigen in some but not other studies. In Specific Aim 1 we will test the hypothesis
that angiotensin-converting enzyme inhibitors and AT1 receptor antagonist modulate the
fibrinolytic and inflammatory response to CPB differently.

Bradykinin receptor antagonism could reduce the hypotensive response to CPB. Low systemic
vascular resistance (SVR) commonly occurs during and early after CPB. It is usually
transient and easy to treat. Occasionally, patients have a more severe and persistent fall
in SVR, referred to postoperative vasodilatory shock. Risk factors for vasodilatory shock
includes the preoperative use of ACE inhibitors, low left ventricular ejection fraction and
heart failure syndrome. Treatment is frequently required to maintain adequate perfusion
pressure during CPB and to establish satisfactory hemodynamics when ready to separate the
patient from bypass. This usually entails counteracting the effect of the vasodilatory
mediators by administration of drugs such as norepinephrine or phenylephrine. Although
usually effective and safe, these drugs can redistribute blood flow in such a way as to
compromise the splanchnic and renal circulation. Several mediators are thought to be
responsible for producing postoperative shock, including bradykinin. For example, there is
an inverse correlation between bradykinin concentrations and mean arterial pressure during
CPB, suggesting that bradykinin is an important mediator in the decrease in SVR. We and
others have shown that bradykinin induces vasodilation through its B2 receptor. In contract,
B1 receptor stimulation does not cause vasodilation. As outlined under PRELIMINARY STUDIES,
we have demonstrated that endogenous bradykinin contributes to protamine-related hypotension
following CPB and that bradykinin receptor antagonism administered just prior to protamine
attenuates this hypotensive response. In Specific Aim 2 we will test the hypothesis that
bradykinin receptor antagonism modulate the hemodynamic changes observed during CPB.

Bradykinin receptor antagonism could reduce hyperfibrinolysis and CPB-associated blood loss.
Inhibiting hyperfibrinolysis during CPB reduces blood loss and blood product requirements.
On the other hand, modulating the hypofibrinolytic phase after CPB has the potential to
reduce thrombotic complications. We and others have shown that bradykinin stimulates t-PA
release from human forearm vasculature and the coronary circulation through a NO
synthase-independent, and cyclooxygenase-independent pathway. As with vasodilation,
bradykinin-stimulated t-PA release is mediated via the B2 receptor. Several groups have
reported that bradykinin concentrations increase during CPB. We demonstrated a direct
correlation between bradykinin and t-PA concentrations during CPB suggesting that bradykinin
plays an important role in activating the fibrinolytic response during CPB. As outlined
under PRELIMINARY STUDIES we have shown that HOE 140 (a B2 receptor antagonist) administered
prior to CPB blunts the increase in D-dimer similar to e-aminocaproic acid. Thus, B2
receptor antagonism has the potential to reduce bradykinin-mediated fibrinolysis during CPB.
In Specific Aim 2 we will test the hypothesis that bradykinin receptor antagonism modulate
the fibrinolytic response observed during CPB.

Bradykinin receptor antagonism could reduce the inflammatory response to CPB. During CPB,
exposure of blood to bioincompatible surfaces of the extracorporeal circuit, as well as
tissue ischemia and reperfusion associated with the procedure, induce the activation of
several major humoral pathways of inflammation. Bradykinin produces many of the
characteristics of the inflammatory state, such as changes in local blood pressure, edema,
and pain, resulting in vasodilation and increased microvessel permeability. Bradykinin
activates NF-kB and upregulates interleukin(IL)-1b and TNFa-stimulated IL-8 production
through the B2 receptor. In addition, bradykinin stimulates the release of IL-6 from a
variety of cells. The growing knowledge of the biological role of kinins, in particular in
inflammation, has fueled the development of potent and selective kinin receptor antagonist
as potential therapeutics. For example, the bradykinin antagonist, deltibant (CP-0127)
showed a significant improvement in the 28-day risk-adjusted survival of patients with
gram-negative sepsis. In an animal model of intestinal ischemia-reperfusion injury, B2
receptor antagonism inhibited reperfusion induced increases in vascular permeability,
neutrophil recruitment and expression of B1 receptor mRNA. The role of B2 receptor
antagonist in myocardial ischemia-reperfusion injury is more controversial. Kumari et al
demonstrated a protective effect of HOE 140 during in vivo ischemia-reperfusion injury,
whereas in isolated rabbit heart studies, CP-0127 impaired recovery from acute coronary
ischemia. This contradictory results may be the result of different antagonist used,
differences in species sensitivity or different experimental protocols. The role of B1
receptor antagonist in inflammation is unclear. In contrast to the constitutively expressed
bradykinin B2 receptor, bradykinin B1 receptor expression is upregulated following an
inflammatory insult or ischemia-reperfusion injury. It appears that each kinin receptor
subtype mediates different aspects of the inflammatory response. However, B1 receptor
antagonism administered prior to CPB may be detrimental. For example, Siebeck et al
demonstrated that B2 receptor blockade attenuates endotoxin-induced mortality in pigs,
whereas additional B1 receptor blockade seemed to reverse these beneficial effects. Taken
together, B2 receptor antagonism may decrease the acute inflammatory response whereas
additional B1 receptor blockade may be harmful. These studies, and also the fact that
aprotinin exerts part of its beneficial effects through a reduction in bradykinin
concentrations, suggest the hypothesis that pharmacological strategies to block the
bradykinin B2 receptor may be superior to reducing bradykinin concentrations in modulating
the inflammatory response to CPB.

The RAS, KKS and inflammation. Activation of the RAS exerts proinflammatory effects. For
example, Ang II activates the transcription factor nuclear factor (NF)-kB, which in turn
regulates genes involved in cellular recruitment and the inflammatory cytokine cascade. Ang
II induces the synthesis and secretion of the inflammatory interleukin (IL)-6. As mentioned
above, bradykinin produces many of the characteristics of the inflammatory state and
upregulates IL-1b and TNFa-stimulated IL-8 and stimulates the release of IL-6. Thus, both
Ang II and bradykinin stimulates the release of IL-6. ACE inhibitor treatment is associated
with a reduction in IL-6 response to CPB. In a randomized non-blinded study, Trevelyan and
colleagues20 demonstrated that ACE inhibition produced a highly significant decrease of 51%
in the release of IL-6 in patients identified as high producers of IL-6 by the -174 G/C
polymorphism, whereas losartan had a similar but less marked effect. Potential mechanisms
for this variation in IL-6 response between ACE inhibitors and angiotensin receptor blocker
may be due to their differential effect on Ang II formation and bradykinin degradation.
Furthermore, bradykinin-induced increases in IL-6 protein and total mRNA are inhibited by
the selective B2 receptor antagonist HOE-140 but not by a selective B1 receptor antagonist.
In Specific Aim 1 we will test the hypothesis that angiotensin-converting enzyme inhibitors
and angiotensin II type 1 (AT1) receptor antagonist modulate the fibrinolytic and
inflammatory response to CPB differently.

Inclusion Criteria:

1. Subjects, 18 to 80 years of age, scheduled for elective CABG requiring CPB

2. For female subjects, the following conditions must be met:

postmenopausal for at least 1 year, or status-post surgical sterilization, or if of
childbearing potential, utilizing adequate birth control and willing to undergo urine
beta-hcg testing prior to drug treatment and on every study day

Exclusion Criteria:

1. Evidence of coagulopathy (INR greater than 1.7 without warfarin therapy)

2. Preoperative hematocrit less than 30%

3. Preoperative platelet count less than 100X109ml-1

4. GPIIb/IIIa antagonist within 48 hours of surgery

5. Emergency surgery

6. Impaired renal function (serum creatinine >1.6 mg/dl)

7. Pregnancy

8. Breast-feeding

9. Any underlying or acute disease requiring regular medication which could possibly
pose a threat to the subject or make implementation of the protocol or interpretation
of the study results difficult

10. History of alcohol or drug abuse

11. Treatment with any investigational drug in the 1 month preceding the study

12. Mental conditions rendering the subject unable to understand the nature, scope and
possible consequences of the study

13. Inability to comply with the protocol, e.g. uncooperative attitude and unlikelihood
of completing the study