The Role of Endothelin-1 in Sickle Cell Disease

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