The Role of Endothelin-1 in Sickle Cell Disease
The Role of Endothelin-1 in Sickle Cell Disease
The primary goal of the study is to determine the safety and tolerability of ambrisentan. It
 is also expected that ambrisentan will improve blood flow in the lungs, decrease
 inflammation, and reduce pain in sickle cell patients. An additional goal is to evaluate the
 use of select biomarkers in evaluating sickle nephropathy.
The purpose of this study is to test the hypothesis that endothelin antagonist (ETA) receptor
 blockade using ambrisentan is safe, tolerable, and improves kidney function/albuminuria in
 patients with sickle cell disease (SCD). The investigators anticipate a reduction in
 proteinuria, a decrease in tricuspid regurgitation jet (TRJ) velocity, reduction in
 inflammatory markers, improvement in forearm blood flow, and improvement in nociception/pain.

 The primary efficacy endpoint was chosen as the effect of ETA receptor blockade on the
 reduction of microalbuminuria based upon findings in diabetic nephropathy, which has a
 similar pathogenesis to sickle nephropathy. A 30% reduction in proteinuria is rather
 conservative and realistic based upon the results of studies of ETA receptor blockade in
 diabetic nephropathy that consistently resulted in 40-45% reduction in proteinuria.
 Pre-clinical data has shown that changes can be detected in urinary nephrin excretion before
 overt proteinuria is observed in a model of chronic ET-1 elevation. Furthermore, tubular
 injury that can occur as a result of an intrarenal vaso-occlusive crisis (VOC) should be
 expected to increase the level of renal tubular injury markers, neutrophil
 gelantinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and netrin.
 Treatment with ambrisentan would be expected to attenuate these changes. Dr. Jennifer
 Pollock, has extensive experience with all of the biomarker assays including ET-1. Dr.
 Pollock runs bio-analytical core labs for two PO1s and has published extensively on these and
 similar methods in human and animal samples.

 Additional rationale for this project is based on recent 24-hour urine results for creatinine
 clearance and total protein excretion from 97 patients with sickle cell anemia (Hb SS) and
 sickle beta zero thalassemia (SB0-thalassemia) followed in the Augusta Sickle Cell Clinic.
 These patients ranged in age from 19-63 years (52 females, 45 males). Of these, 17 (18%) had
 microalbuminuria as defined by a 24-hour albumin excretion of 150-300 mg, 34 (36%) had
 macroalbuminuria (>300 mg albumin excretion/24-hours), thus 54% of the patients had
 microalbuminuria or macroalbuminuria and only 46% were normo albuminuric. 58 patients (58%)
 were on chronic hydroxyurea therapy. 9 patients were on angiotensin converting enzyme
 inhibitors (ACEi) and 20 were on angiotensin receptor blockers (ARBs) (total 30%). 55
 patients (57%) had glomerular hyperfiltration (creatinine clearance of >120 ml/min). There
 was an age related decline in the glomerular filtration rate (GFR) observed. The number of
 patients with normo albuminuria shows a sharp decline by age indicative of the progression of
 sickle nephropathy. Recent studies of pain in SCD have led to a change in perception.
 Approximately 50% of the patients reported experiencing daily, chronic pain. In addition,
 issues related to centralization and neuropathic pain in this patient population has begun to
 gain increasing attention. A large body of evidence suggests that endothelin-1 plays an
 important role in enhancing pain stimuli and nociception in various conditions. This is
 mediated by ETA receptor. Berkeley SCD transgenic mice do have hyperalgesia as shown in the
 rationale preliminary data section of Aim 2 and ETA receptor blockade reverses this
 hyperalgesia towards normal. As part of an ongoing National Institute on Minority Health and
 Health Disparities (NIMHD) funded study of pain and its genetic correlates (study reference:
 1 P20 MD003383-01), the investigators have found that SCD patients display significant
 hyperalgesia as measured by pressure pain algometer testing. The testing was done at three
 different anatomic sites in patients (n=38) and controls (n=20) and showed that SCD patients
 perceived pain at significantly lower pressures compared to controls (masseter: 157.7
 kilopascal (kPa) for patients, 214.4 kPa for controls, p=0.017; ulna: 299.1 kPa for patients,
 477.5 kPa for controls, p=0.0018; and trapezius: 290.1 kPa for patients, 462.8 kPa for
 controls, p=0.02). These data suggest that hyperalgesia is present in the vast majority of
 SCD patients and will constitute an objective parameter to monitor during the study.

 Protocol-required assessments performed at every study visit include: physical exam, vital
 signs, con medication review, study drug accountability, adverse events review, pain diary
 completion, and pregnancy testing if applicable.

 Protocol-required assessments performed at specified study visits include: collection of
 blood and urine for safety and efficacy testing; electrocardiogram (ECG); echocardiogram
 (echo); Quantitative Sensory Testing (pressure pain threshold via algometer, cutaneous
 mechanical pain via monofilament, and thermal testing via Q-Sense Small Fiber Test);
 transcranial Doppler (TCD); forearm blood flow measurement; skin blood flow measurement; and
 completion of quality of life questionnaires.

 Description of Procedures:

 Sensory Testing: Patients will also undergo sensory testing at baseline and at the end of the
 study period. Sensory testing will include: 1) Pressure pain threshold will be measured by a
 hand-held, digital algometer as described by Fillingim et al at three different anatomical
 sites: center of right upper trapezius, right masseter, and the right ulna; 2) Cutaneous
 mechanical pain testing- Assessment of temporal summation of mechanical pain will be done
 using nylon monofilaments tested on the dorsum of the hand.; and 3) Thermal sensory testing
 will be performed using Q-Sense Small Fiber Test, which offers a scientifically validated
 measure of warm, cool and heat-pain thermal sensory thresholds.

 Pain Diaries/Questionnaires: Patients will be asked to complete a daily pain diary as
 described in the PISCES study. The evaluation of pain diaries will be performed by Dr. Robert
 Gibson and the research staff. In addition, investigators will also incorporate two pre/post
 measures that will examine the effects of pain on functional performance and quality of life.
 The Assessment of Motor and Process Skills (AMPS) is a well-established functional
 assessment. This assessment has demonstrated the ability to discriminate between healthy
 controls and those with wide spread chronic pain. It also demonstrates the ability to measure
 change following intervention. The investigators will also measure patient's quality of life
 with the Adult Sickle Cell Quality of Life Measurement Information System (ASCQ-Me). This
 quality of life assessment has been specifically developed for sickle cell populations. Dr.
 Robert Gibson has training and experience with the ASCQ-Me and is certified to administer the
 AMPS.

 Echocardiogram for tricuspid regurgitation jet (TRJ) Velocity Measurement: A transthoracic
 echocardiogram for the measurement of TRJ velocity and pulmonary artery systolic pressure
 will be performed at baseline (unless obtained clinically within 6 months of Day 1) and at
 the end of the study.

 Electrocardiogram: An electrocardiogram (ECG) is a test that measures the electrical activity
 of the heart. An ECG will be done as a safety measure at baseline, Day 8 visit, and end of
 study, although ambrisentan is not known to prolong corrected QT interval (QTc).

 Forearm Blood Flow Measurement: Brachial artery flow-mediated dilation (FMD) will be
 performed in accordance to the recent tutorial for ultrasound assessment of FMD. Patients
 will be tested under fasting conditions in a controlled environment at baseline and at the
 end of the study period.

 Skin Blood Flow Measurement: Skin blood flow will be measured non-invasively using a low
 frequency laser imaging camera and/or a small skin probe. Ring-shaped probes that are about
 1.5 inches wide will be placed on the forearm with sticky tape. One ring will be filled with
 approximately 2 ml (about 1/2 teaspoon) of water for local heat and the others will be used
 for iontophoresis. For local heat, investigators will heat the water in the ring to between
 42°C (107°F) and 44°C (111°F) and wait approximately 30 minutes for the increase in skin
 blood flow to become stable. Iontophoresis is a technique that delivers a vasoactive drug
 about 1 mm deep into the skin using a low level electrical current. The advantage of this
 technique is that it is non-invasive and the drug does not go into the body, it just remains
 at the surface of the skin. We will use a 1-2% solution of either acetylcholine, sodium
 nitroprusside, or N-nitro-L-arginine methyl ester (L-NAME) (vasoactive substances) to
 increase or decrease skin blood flow. Multiple short currents (10-180 seconds) will be
 performed approximately 1 minute apart. Investigators will monitor skin blood flow through
 the ring during the forearm arterial blood flow test described above as well as during local
 heat and iontophoresis.

 Blood and Urine Measurement: Lab tests to be performed for safety/ efficacy assessments at
 specified study visits include complete blood count (CBC), complete metabolic panel (CMP),
 spot urine for albumin-creatinine ratio, 24-hour urine, pregnancy testing, biomarkers, and
 inflammatory markers.

 Transcranial Doppler (TCD): Pre- and post-treatment TCD will be performed to assess velocity
 of cerebral blood flow. This non-invasive test is performed while the subject is lying down
 and involves use of sound waves to assess macro-circulation in the head. A transducer is used
 to hear and record the results.
Inclusion Criteria:

 1. SS or Sβo-thalassemia

 2. Age 18-65 years

 3. Microalbuminuria (24-hour albumin 150-300 mg) or macroalbuminuria (24-hour albumin
 >300 mq) OR random urine albumin-creatinine ratio (MA Random) ≥ 30 µg/ mg creatinine

 4. Subjects can have Stage 1, II, or III chronic kidney disease (CKD)

 5. Subjects can be on hydroxyurea, ACE inhibitors (ACEi), or angiotensin receptor
 blockers (ARBs) for a period of 3 months or greater

 6. Females of child bearing potential must agree to use two forms of birth control with
 one being a barrier method; abstinence is an acceptable form of birth control

 Exclusion Criteria:

 1. Other genotypes of SCD

 2. History of renal transplant

 3. Chronic kidney disease (Stage IV and V including patients on hemo dialysis or
 peritoneal dialysis)

 4. Patients on chronic transfusion therapy

 5. Uncontrolled/poorly controlled hypertension or history of hypertension pre-dating
 proteinuria or

 6. Known history of HIV, Hepatitis C, and/or diabetes

 7. Peripheral edema

 8. History of congestive heart failure or pulmonary edema

 9. Recent history of coronary artery disease

 10. Pregnant or breast feeding

 11. Alanine Aminotransferase (ALT) or aspartate aminotransferase (AST) >3-fold upper limit
 of normal

 12. Albumin <2.5 gm/dl

 13. Hemoglobin < 6 gm/dL

 14. History of non-compliance with medications and clinic visits; or Inability to give
 informed consent; or Patient deemed ineligible or unsuitable in the judgment of
 investigators