Single Dose Administration of Alpha-1 Anti-Trypsin for the Amelioration of Organ Injury in Patients Undergoing Cardiac Surgery
Single Dose Administration of Alpha-1 Anti-Trypsin for the Amelioration of Organ Injury in Patients Undergoing Cardiac Surgery
Protocol Summary STUDY DESIGN A pilot, prospective, double blind, randomized, placebo
 controlled study.

 STUDY POPULATION Patients assigned to elective CABG with cardiopulmonary bypass (CPB) at the
 Department of Cardiothoracic Surgery, Soroka University Medical Center.

 OBJECTIVE To evaluate anti-inflammatory effects, effects on organ function preservation, and
 postoperative blood loss reduction following AAT-1 administration in patients undergoing CABG
 with CPB.

 PRIMARY ENDPOINT Postoperative organ function preservation and blood loss following
 preoperative single-dose AAT-1 administration.

 SAMPLE SIZE CONSIDERATIONS A cohort of 20 patients will be recruited. Patients will be
 randomized to receive either AAT-1 or placebo prior to surgery. Whereas this is a proof of
 concept pilot study, statistical significance is not the primary objective.

 INCLUSION CRITERIA 1. The study population will comprise patients between 40 and 70 years of
 age, irrespective of gender, at low or intermediate operative risk (calculated Logistic
 Euroscore stratification of 5% or less), assigned to elective CABG with CPB. Recruitment
 depending on patients informed consent.

 EXCLUSION CRITERIA Co-existing conditions including:

 1. Coagulation abnormalities

 2. Severe pulmonary disease defined by blood oxygen saturation of 90% or less or FEV1 of
 less than 60% of predicted.

 3. Renal dysfunction defined be serum creatinine levels higher or equal to 1.8 mg%,

 4. Abnormal liver function tests

 5. Uncontrolled diabetes mellitus,

 6. Severe peripheral vascular disease

 7. Prior cerebrovascular neurological event.

 8. Abnormal left or right ventricular function.

 9. Treatment with warfarin or thienopyridine class of anti platelet agents.
3 Background The use of cardiopulmonary bypass (CPB) during cardiac surgery elicits
 generalized non-specific systemic inflammatory response syndrome (SIRS) and subsequent
 activation of the cytokine, complement, and coagulation-fibrinolytic cascades (1). In
 approximately 11% of the patients SIRS may deteriorate to severe multi-organ failure (MOF)
 resulting in mortality rate of 40-98%.

 Inflammatory modulation by intraoperative administration of anti-inflammatory substances has
 been advocated to attenuate the effects of SIRS. Attempts have subsequently focused on
 aprotinin which inhibits proteases that are key mediators in the complement, coagulation, and
 fibrinolytic system. Related anti-inflammatory properties of aprotinin include the inhibition
 of platelets, neutrophils and kallikrein activation. Reduction in blood loss and reduced need
 for allogenic blood transfusions have been proposed as additional mechanisms by which
 aprotinin limits the inflammatory response (2). The use of aprotinin during cardiac surgery,
 however, was discontinued following alarming results in terms of higher rate of bypass graft
 occlusion and overall inferior postoperative outcome.

 AAT-1 mechanism of action:

 Similar to aprotinin, α1-Antitrypsin (AAT) is a 52-kDa circulating serine protease inhibitor
 classified as a SERPIN protein. Besides its ability to inhibit serine proteases, accumulating
 data suggest that AAT-1 possesses independent anti-inflammatory and tissue-protective effects
 (3). AAT-1 modifies dendritic cell maturation and promotes regulatory T-cell differentiation,
 induces interleukin (IL)-1 receptor antagonist and IL-10 release, protects various cell types
 from cell death, inhibits caspases-1 and -3 activities and inhibits IL-1 production and
 activity (4). Contradictory to classic immunosuppressants, AAT-1 allows undeterred isolated
 T-lymphocyte responses (5).These effects have been repeatedly corroborated.

 Unlike aprotinin, AAT-1 is produced from human plasma, and does not have strong pro coagulant
 characteristics.

 AAT-1 in different clinical settings:

 Circulating AAT-1 levels increase by 4-fold during acute-phase response (3). Patients with
 low circulating levels of AAT-1 are at increase risk for lung, liver and pancreatic
 destructive diseases, particularly emphysema (3). Preclinical and clinical studies have shown
 that AAT-1 therapy for non-deficient individuals is safe, and may modify disease progression
 in type 1 and type 2 diabetes, acute myocardial infarction, rheumatoid arthritis,
 inflammatory bowel disease, cystic fibrosis, transplant rejection, graft versus host disease
 and multiple sclerosis (6-17, 32,33,37,38) . AAT-1 treatment has subsequently advanced from
 replacement therapy to potential treatment for a broad spectrum of inflammatory and
 immune-mediated diseases. AAT-1 also appears to be antibacterial and an inhibitor of viral
 infections, such as influenza and human immunodeficiency virus (HIV) (18).

 Human pharmacokinetic data:

 In patients with AAT-1 deficiency, intravenous administration of AAT-1 in a dose of 60 mg per
 kg body weight maintain plasma levels resembling acute phase response immediately after
 administration, and retain an appropriate level of serum AAT above the protective threshold
 (50 mg/dl) a week afterwards (39).

 Rational of AAT-1 dosage:

 Based on previous studies and clinical practice, the administration of multiple intravenous
 dosage of 60 mg per kg body weight of AAT-1 is safe and is associated with low incidence of
 benign side-effects.

 Derived from pharmacokinetic studies (39), AAT-1 plasma levels immediately following the
 administration of this external dosage resembles acute phase response. A 30% reduction in
 AAT-1 plasma levels is anticipated with gradual return to preoperative levels after
 termination of CPB (40).

 Risk/ benefit of the study:

 The role of AAT-1 in mitigating inflammatory response has been established. CPB is a potent
 stimulator of inflammatory cascades and associated with bleeding diatheses, coagulation
 abnormalities, potentially leading to organ dysfunction. To date, the administration of AAT-1
 in patients undergoing cardiac surgery with CPB has not been explored. It may be postulated
 that inherent AAT-1 effects may offset deleterious CPB effects. This may clinically result in
 improved preservation of organ function, reduced postoperative bleeding and reduced hospital
 stay. The benefits of other protease inhibitors in corresponding settings have been recently
 demonstrated (41-43).

 As mentioned above, administration of AAT-1 in various clinical settings is considered safe
 with low rate of side effects (see section 6.1 - potential risks).

 4 Methods 4.1 Primary Objective

 1. To evaluate the efficacy and safety of preoperative AAT-1 administration in patients
 undergoing CABG with CPB.

 2. Postoperative blood loss and organ function will be assessed.

 To date, no data exists with regard to AAT-1 effects in the settings of CPB. The primary
 goals of this study are to evaluate anti-inflammatory effects, organ function preservation
 (1-2) and postoperative blood loss reduction following AAT-1 administration in patients
 undergoing cardiac surgery with CPB.

 4.2 Determination of Study Eligibility 4.2.1 Inclusion Criteria

 1. Male/female patients 40-70 years of age.

 2. Candidates for isolated CABG with CPB.

 3. Calculated logistic Euroscore risk stratification of 5% or less.

 4. Signed patient's written informed consent.

 4.2.2 Exclusion Criteria

 Co-existing conditions including:

 10. Coagulation abnormalities 11. Severe pulmonary disease defined by blood oxygen saturation
 of 90% or less or FEV1 of less than 60% of predicted.

 12. Renal dysfunction defined be serum creatinine levels higher or equal to 1.8 mg%, 13.
 Abnormal liver function tests 14. Uncontrolled diabetes mellitus, 15. Severe peripheral
 vascular disease 16. Prior cerebrovascular neurological event. 17. Abnormal left or right
 ventricular function. 18. Treatment with warfarin or thienopyridine class of anti-platelet
 agents. 4.2.3 Withdrawal Criteria Patients will preserve the right to withdraw from the study
 at any time during treatment without prejudice.

 If medically indicated, the principal investigator or the surgeon poses the right to
 discontinue patient enrollment at any stage of the study.

 Discontinuation (failure to complete the study) will be documented. The reason for
 discontinuation will be recorded.

 Potential causes that may lead to discontinuation include:

 -  Adverse event(s)

 -  Protocol violation

 -  Patient withdrew consent

 -  Administrative problems 4.3 Enrollment Prior to participation in this study, the
 Investigator will obtain written appropriate approval for the protocol and the informed
 consent form the Ethics Committee (EC) and other local regulatory organizations. The
 approved consent form will clearly reflect the EC approval date.

 Failure to obtain a signed and hand dated informed consent prior to the procedure constitutes
 a protocol violation, and subsequently reportable to the EC.

 4.4 Subject Screening Patients that have been already scheduled for isolated CABG operation
 will be addressed by a member of the research team during the preoperative waiting period
 (either in the Department of Cardiothoracic Surgery, the Department of Cardiology or the
 Department of Internal Medicine). The goal of the study, procedure and incentive will be
 explained by a research staff member to each potential participant. Consenting subjects will
 be asked to provide written consent.

 4.4.1 Randomization The study participants will be randomized to receive either single dose
 AAT-1 60 mg per kg or placebo.

 4.4.2 Trial medication administration: Preparation and dosing of AAT-1 will be performed by
 an unblinded pharmacist. The medication will be diluted just prior to administration. The
 placebo solution will comprise human albumin that resembles the color and consistency of
 AAT-1 solution. The medication will be given 3-5 hours prior to surgery (skin incision).
 Administration rate of the drug will not exceed 0.04 ml per kg per minute (approximately
 60-80 minutes). Vital signs including blood pressure, pulse rate and body temperature will be
 correspondingly monitored.

 The patients, research staff, laboratory personnel and data analysts will remain blinded to
 the identity of the treatment from the time of randomization and until database lock. Data
 unblinding will commence in the case of patient's emergency.

 A randomization list will be produced by the pharmacist, however, will be secured and
 confidential until time of unblinding.

 4.4.3 Surgical technique Consistent with our routine policy, fentanyl citrate (20-50mcg/kg),
 midazolam (2-3mg) and isoflurane (0.5-2%) will be used for induction and maintenance of
 anesthesia.

 Standard median sternotomy will be followed by procurement of the conduits. Heparin loading
 dose will be administered to achieve a kaolin activated coagulation time (ACT). Ascending
 aorta - right atrial cannulation will be performed to institute CPB and ACT will be monitored
 at 480 seconds or more. Standard centrifugal pump and a membrane oxygenator will be used for
 extracorporeal circulation (cardiopulmonary bypass). Compatible with the standard technique,
 systemic active cooling will be avoided and patients core temperature will remain range
 between 32 and 37˚C. Distal anastomoses will be performed during single aortic cross-clamp
 and blood cardioplegic arrest. Proximal anastomoses will be performed during aortic
 cross-clamp. Cold (10˚C) blood cardioplegic solution will be delivered in a 4:1 ratio.
 Cardioplegia will be delivered antegrade via the aortic root with or without additional
 retrograde administration via the coronary sinus. After cardioplegic induction (10 ml / Kg),
 intermittent doses (300 - 500 ml) will be administered following completion of each distal
 anastomosis. Heparin will be reversed using protamine sulphate in a ratio of 1:1 after
 weaning from CPB.

 4.4.4 Data collection Preoperative data: Demographic, morphological and clinical descriptors
 including age, gender, body mass index (BMI), body surface area (BSA) co-morbidity, Euroscore
 risk-stratification, medication, etc. will be recorded. Preoperative laboratory analysis will
 include complete blood count, coagulation profile, serum creatinine levels and creatinine
 clearance, liver function test and arterial blood gases test and serology for HIV,HCV,HBC.
 Compatible with our routine policy, all patients will undergo preoperative echocardiography,
 coronary angiography, chest x-ray, lung function tests (spirometry) and carotid artery duplex
 study.

 Study participants will be assigned to undergo preoperative brain MRI; subjected to the
 protocol described below.

 Intraoperative: The type of surgery will be classified. The following data will be recorded:
 heparin dose given prior to bypass initiation; activated clotting time (ACT) counts during
 the operation (prior, during and after CPB); operative time, cross-clamp time and CPB time;
 number of trials to wean from CPB, type of inotropes and dosage used during weaning from CPB;
 blood products utilization during surgery. Allergic reactions or adverse events observed by
 the surgeon or anesthesiologist will be documented. The individual surgeon's impression
 regarding bleeding tendency will be recorded.

 Postoperative organ function and blood loss evaluation:

 The occurrence and magnitude of systemic inflammatory response and organ dysfunction will be
 recorded and quantified by laboratory markers. Related laboratory markers will be monitored
 on a daily basis during the recovery period (in the intensive care unit and at the ward). The
 following organs and corresponding markers will be monitored:

 Pulmonary function: Pulmonary function will be evaluated by measured overall mechanical
 ventilation time, peak inspiratory pressures (PIP), plateau pressures, physiologic dead space
 and static and dynamic lung compliance. Bronchoalveolar lavage (BAL) will be performed 3
 hours after operation (while the patient is anesthesized and intubated) and extracted fluid
 will be analyzed for inflammatory markers. A-a DO2 calculation [AaDO2 = (713 x FiO2) - (pCO2
 / 0.8) - (paO2)] will be measured daily.

 Complete pulmonary function test will be performed before and 4 days after the operation.
 Chest radiographs will be evaluated and quantified by an independent radiologist for the
 occurrence of atelectasis, pulmonary edema, or pleural changes.

 Renal function: Daily measurements of urine output, serum creatinine levels, creatinine
 clearance and urinary albumin levels. Acute kidney injury (AKI) markers will be sampled in
 the ICU.

 Brain injury assessment: The degree of insult to the brain will be measured by plasma S-100
 proteinlevels. Assessment of damage to the blood-brain barrier (BBB) will be performed by
 magnetical resonance imaging (MRI) modality (see technique protocol below).

 Hepatic function: Daily measurements of serum hepatic enzymes levels. Cardiac function:
 Monitoring of cardiac enzymes levels; need and magnitude of required inotrope treatment;
 occurrence of low cardiac output syndrome (defined as systolic blood pressure of 90 mmHg or
 less coupled with central venous pressure (CVP) of 15 mmHg or more) and incidence of cardiac
 arrhythmias. Transthoracic echocardiography examination will be performed on postoperative
 day 5 and assessed by an independent cardiologist.

 Blood loss: Operative and postoperative blood loss will be monitored as well as daily
 hemoglobin levels. Daily platelet counts and thromboelastograms will be performed. The
 distribution of blood products and total administered will be recorded daily. Postoperative
 CRP levels will be evaluated daily.

 Blood sampling and laboratory analysis methods for cytokine levels:

 Ten mL whole blood venous EDTA samples will be collected from radial artery catheter at five
 specified occasions: before induction of anaesthesia, 30 minutes after aortic cross-clamp
 positioning and 3, 6, and 9 hours after aortic cross clamp positioning. The blood samples
 will be subsequently centrifuged at 4 °C for 15 min and the serum stored at -70 °C. Samples
 will be analyzed for the following markers: Polimorphonuclear Neutrophil Elastase (PMNE),
 Interleukin-1α (IL-1α), Interleukin-1ß (IL-1ß), Interleukin-2 (IL-2), Interleukin-4 (IL-4),
 Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interferon-γ (IFN-γ),
 Tumor Necrosis Factor-α (TNF-α), Vascular Endothelial Growth Factor (VEGF), Monocyte
 Chemoattractant Protein-1 (MCP-1), and Endothelial Growth Factor (EGF).

 Daily blood samples will be collected postoperatively for platelet count, renal function,
 liver function, CRP levels S-100 protein and troponin.

 Early postoperative adverse events will be documented. These include 30-day mortality, new
 neurological events, myocardial infarction, renal dysfunction, need for re-exploration for
 bleeding and deep sternal wound infection.

 Blood-Brain Barrier (BBB) assessment by MRI The imaging modality used for BBB assessment will
 be MRI scanner (Philips 3T or General Electric 1.5T). The examination format will include 24
 cm FOV, 35 contiguous interleaved slices, 3.5-4 mm thick and co-localized across series.
 Trace-weighted DWI images will be obtained at b=1000 from a 13-15 direction DTI sequence with
 an in-plane resolution of 2.5×2.5mm and TR/TE=10s/58ms at 3T or TR/TE=10s/72ms at 1.5T.
 T2-FLAIR images will be obtained with an in-plane resolution of 0.94×0.94 mm, TR/TE=9000/120
 ms and TI=2600 ms at 3T or TR/TE=9000/140 ms and TI=2200 ms at 1.5T. (25)
Inclusion Criteria:

 1. The study population will comprise patients between 40 and 70 years of age, irrespective
 of gender, at low or intermediate operative risk (calculated Logistic Euroscore
 stratification of 5% or less), assigned to elective CABG with CPB. Recruitment depending on
 patients informed consent.

 Exclusion Criteria:

 Co-existing conditions including:

 1. Coagulation abnormalities

 2. Severe pulmonary disease defined by blood oxygen saturation of 90% or less or FEV1 of
 less than 60% of predicted.

 3. Renal dysfunction defined be serum creatinine levels higher or equal to 1.8 mg%,

 4. Abnormal liver function tests

 5. Uncontrolled diabetes mellitus,

 6. Severe peripheral vascular disease

 7. Prior cerebrovascular neurological event.

 8. Abnormal left or right ventricular function.

 9. Treatment with warfarin or thienopyridine class of anti platelet agents -