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PERFORMANCE SITE(S) (organization, city, state)

_____ of _____

_____, _____, _____, _____ _____

________________________________________________________________________________________________________________
KEY PERSONNEL. See instructions on Page 11. Use continuation pages as needed to provide the required information in the format shown below.


Name                                                                           Organization                                                                 Role on Project

_____                                                                           _____                                                                         Principal Investigator

_____                                                                           _____                                                                        Mentor
_____                                                                           _____                                                                        Cosponsor
_____                                                                           _____                                                                        Cosponsor
_____                                                                           _____                                                                        Collaborator
_____                                                                           _____                                                                        Co-Investigator
_____                                                                           _____                                                                        Co-Investigator
_________________________________________________________________________________________________________________________
 

BB

Principal Investigator/Program Director (Last, first, middle) _____
__________________________________________________________________________________________________________________________
 

 
_____                                                                             _____                                                                                                          _____                                                                             _____                                                                      Consultant  
                                                                                       _____
_____                                                                             _____                                                                      Collaborator
_____                                                                             _____


Advisory Committee:
_____                                                                            University of _____                                                   Advisory Committee
_____                                                                            University of _____                                                   Advisory Committee
_____                                                                            University of _____                                                   Advisory Committee
_____                                                                            University of _____                                                   Advisory Committee
 

__________________________________________________________________________________________________________________________
Principal Investigator/Program Director (Last, first, middle) _____


Use this substitute page for the Table of Contents of Research Career Awards
Type the name of candidate at the top of each printed page and continuation page
 

RESEARCH CAREER AWARD
TABLE OF CONTENTS

(Substitute Page)

Page Numbers

Section I: Basic Administrative Data

Section II: Specialized Information

1. Introduction to Revised Application (Not to exceed pages).................................................

2. Letters of Reference (Attach to Face Page)*

3. The Candidate

4. Statements by Sponsor(s), Consultant(s), and Collaborator(s)*

5. Environment and Institutional Commitment to Candidate
   

6. Research Plan

7. Appendix

(___) Permanent resident of U.S.         

(___) If a permanent resident of the U.S., a notarized statement is

___________________________________________________________________________________________________________________________________
FACILITIES: Specifiy the facilities to be used for the conduct of the proposed research. indicate the performance sites and describe capacities, pertinent capabilities, relative proximity, and extent of availability to the project. Under "Other," identify support services such as machine shop, electronics shop, and specify the extent to which they will be available to the project. Use continuation pages if necessary.

RESOURCES

Laboratory:

_____. All facilities capabilities and equipment described below are now available at this _____ site. The laboratories at the institution are equipped for biochemistry, hematology, cell biology, molecular biology, mass spectrometry, and microscopy (electron and light). All essential equipment is backed by emergency power; critical systems, including freezers, are monitored 24 hours/day. _____ Co-Directs the _____ of the institution. This laboratory contains the equipment for the measurement of biophysical and biochemical properties of red cell membranes, density profiles of subpopulations of red cells, and reactive nitric oxide species analysis through the Griess method.

Clinical:

Human subjects will be recruited from the _____ patients followed by the _____. The use of human subjects at the institution is
governed by the _____ under DHHS Assurance No. _____.

Animal:         N/A
 

Computer:

_____ computers are available throughout the laboratories and offices, and are linked in a network which includes seven laser
printers as well as a Graphics Center. The Graphics Center includes two large-screen _____ computers, a flatbed scanner, slide scanner, slide maker, and color laser printer. Also provided on the internet network are weekly access to current journal titles, and two CD-ROM-based systems for online searching of medical databases and protein/DNA sequences.

Office:

All investigators are equipped with private offices on site. Secretarial assistance is available to all investigators. Computers are supported by full-time computer specialists. Staff are available for grants management technology transfer, and facilities support.

Other:

Mass Spectroscopy:

This core facility is a resource for mass spectrometry in the institution’s hemoglobin and red cell membrane research program. Its services include mass spectroscopy of proteins, peptide mapping by HPLC/MS, and metabolic studies based on stable isotope incorporations into biomolecules.

Hemoglobin Diagnostics:

This laboratory _____assists in the diagnostic evaluation of variant hemoglobins. Using standard electrophoresis, thin-layer isoelectric focusing, HPLC, mass spectroscopy and molecular diagnostic procedures, a complete molecular and structural analysis of abnormal hemoglobins can be performed.

Darkrooms:

The institution’s darkrooms contain a photographic enlarger and equipment for printing, as well as a stand for UV illumination and photography of DNA sequencing gels. It also has a copy stand and high-quality camera for production of publication-quality photographs. An X-ray developer for autoradiographs is also available.

________________________________________________________________________________________________________________________________
MAJOR EQUIPMENT: List the most important equipment items already available for this project, noting the location and pertinent capabilities
of each. The mass spectrometry equipment _____

Section II: Specialized Information

1. Introduction to Revised Application
2. Letters of Reference
3. The Candidate

 a. Candidate’s Background

b. Career Goals and Objectives (Future Investigations): Scientific Biography

c. Career Development Activities during Award Period

                Co-Mentor _____.
    b. Consultants:
        1) _____
        2) _____
        3) _____
 

5. Environmental and Institutional Commitment to Candidate
    a. Description of Institutional Environment

    The institution ranks among _____, and houses _____. The emergency department of the institution sees more than 40,000 children a year, and provides an excellent arena for clinical research. The environment at the institution provides a unique opportunity for the collaboration of the ED with the Department of Hematology/Oncology, as the emergency department is often the first place that children with sickle cell disease present to with vaso-occlusive complications. During _____ fellowship, the candidate developed a commitment to clinical investigation, and was able to incorporate _____ ambitions with _____ interests in sickle cell disease. A Research Career Award will allow the candidate to attain the fund of knowledge necessary to become an independent clinical investigator, and provide _____ with the skills necessary to continue _____ research interests in the _____ department.

  b. Institutional Commitment to Candidate’s Research Career Development

    1) Didactic Plan: The institution, and Departments of Emergency Medicine and Hematology/Oncology at _____ are committed to the candidate’s
development as an academic _____ and an independent investigator.

        The Research Career Award will allow the candidate to pursue a didactic phase of study, which is essential to _____ development as an independent investigator. During _____ fellowship, the candidate had the opportunity to attend the annual Pediatric Emergency Medicine Fellows’ Conference which provided workshops on grant writing, research design in pediatric emergency medicine and on research ethics. The candidate also completed the Clinical Research Workshop of the ORACLE course, Outcomes Research and Clinical Epidemiology, at the _____, _____ under the direction of _____ and _____. The course provided the candidate with the essential basic skills necessary to plan and conduct a clinical research project. The candidate was instructed in study design, sample size and power calculations, data management and data analysis. This class was crucial to the successful completion of _____ first research project that resulted in the manuscript “_____”, recently published in the _____. The candidate also had the opportunity to present these results at several national meetings and at Grand Rounds at _____. The candidate’s workshop mentor for this course was _____, who holds a position as an _____ in the _____ at the _____, _____, _____. The mentor was of great assistance during the course, and was also of significant help in developing the biostatistical plan for the candidate’s initial arginine proposal to the _____ that resulted in the preliminary results for this grant application, and the candidate will continue to work with him closely.

In the first year of the Research Career Award, the candidate plans to enroll in the second phase of the _____ course that is offered at _____, _____. This course will provide the candidate with an overview of large database manipulation, meta-analysis, risk assessment and decision-making analysis. This will complete the prerequisites for the _____(_____) course, also offered at _____. It is a comprehensive 10 month course created by _____, that covers epidemiology, biostatistics, design methodology, bioethics and the implementation of clinical research projects and clinical trials. This course involves a 20 hour per week combined lecture, lab, and seminar series. Specific topics that are covered by the ATCR course include:

1. Introduction to Biostatistics
2. Biostatistical Methods for Clinical and Epidemiological Research
3. Epidemiologic Methods
4. Clinical epidemiology
5. Clinical Trials
6. Conduct and Presentation of Clinical Research
7. Ethical Issues in Clinical Research: Teaches responsible conduct in research
8. Health Services Research

____________________________________________________________________________________________________________________________
        In addition to the ethics education the candidate will obtain from the _____course, _____ will enroll in an additional course, _____ (_____), offered at _____. Directed by _____, _____, it is a forum for scientists to familiarize themselves with institutional policies/practices and professional standards that define scientific integrity. It gives an overview of ethics in research, authorship, patents, and human interest at the academic-commercial interface, and small group sessions for more extended discourse between students and faculty. Completion of this course fulfills the NIH requirement for instruction in the ethical conduct of research.

        During the course of this award, the candidate also plans to improve upon _____ statistical background by enrolling in additional biostatistical courses offered at _____:
 

1. _____, a course on bivariate correlation, simple linear regression, multiple regression, analysis of variance and the interpretation of results
2. _____, a course covering modeling of categorical responses
3. _____, including logistic regression and log-linear models, with a focus on interpretation of results
4. _____, covering statistical methods for analyzing repeated measurement data including classical ANOVA and ANOVA-RM approaches, likelihood approaches and semi-parametric approaches.

       In conjunction with _____ coursework, the candidate will also attend weekly research conferences within the institution, and will participate in quarterly _____ research meetings at which time updates of _____ research progress will be presented and discussed with the senior investigators. The candidate will also present her data to her colleagues in the _____ department monthly, at fellows’ conferences. The candidate will continue to present _____ results to the members of the _____, as well as two national conferences, annually. The candidate will also have the opportunity present her work during Grand Rounds at _____.

    2) Mentoring: The candidate’s primary mentor for this project will be _____. The mentor is the _____ at the institution, and the _____ at the institution, and an _____at the _____, _____. The candidate will be working closely with the mentor. The mentor has been a great source of encouragement and advice during the candidate’s previous research endeavors, and the mentor will continue to guide the candidate’s career development. The mentor has supervised many junior researchers and hematology fellows, and has successfully served as a mentor to former Clinical Associate Physician award recipients. The mentor has published extensively on the complications of sickle cell disease, and is well suited to mentor the candidate. The candidate will have weekly discussions with the mentor concerning the clinical aspects of this project and the relationship between laboratory findings and the clinical outcome of patients with VOC. The mentor will closely monitor the candidate’s progress, and will continue to assist in problem solving, and provide direction through any pitfalls that may arise during the completion of this project.

    The candidate will also be working closely with _____, who is a _____ at the institution, and an expert in red cell membrane biology. _____ will act as a cosponsor for the candidate, supervising the laboratory component of this application. His laboratory is one of the few labs in the country that measures red cell deformability through ektacytometry, and he has helped the candidate with the successful determination of nitric oxide measurements that are accurate and reproducible. His expertise and laboratory supervision is essential for the candidate’s project. Under the cosponsor’s laboratory guidance, the candidate will be studying arginine’s effect on nitric oxide metabolism and its impact on platelet function. The candidate will build upon _____ previous 2 years of laboratory experience, applying _____ skills to the performance of ELISA assays, use of mass spectrometry, and learning new laboratory skills as outlined in this proposal. The candidate will also become proficient in the use of the nitric oxide analyzer.

    Director of the institution, _____ is also the Co-Director of the _____ and the Principal Investigator of the satellite _____ at the institution. _____ and _____ have been working together for over 25 years, and have successfully collaborated on many projects. The candidate will benefit from their many years of experience, as _____ will also serve as a cosponsor. The environment at the institution is supportive of junior investigators and _____ has demonstrated a commitment to supporting the candidate in _____ endeavors.

    The candidate will learn techniques involved in mass spectrometry under the guidance of _____, _____, _____, _____ at the institution. As a _____ and a world-renowned expert in mass spectrometry, _____ will be a valuable resource, as the candidate explores the potential deleterious effects of nitric oxide activity by measuring oxidative damage.

        In addition to this impressive group of clinical and scientific investigators, the candidate will be consulting with several other individuals whose expertise is pertinent to _____ project. Dr. _____, from the _____ at _____, has done extensive nitric oxide research with a particular interest in the relationship of No_x and endothelin-1 in pulmonary disease. With expertise in nitric oxide metabolism, Dr._____ is a valuable resource. Interpretation of pain score data will be done under the guidance of _____, _____ from the _____ at _____. Dr. _____ is an expert in sickle cell pain evaluation, and supervises the pain management program for sickle cell disease at the institution. Dr. _____’s assistance will be helpful in measuring a significant outcome measure for this project. The candidate also has the support of _____, _____ at the institution, and a current Clinical Associate Physician Award recipient. Dr. _____ is a successful clinical researcher, and _____will help to identify patients for enrollment, and will serve as an additional consultant within the Department of Hematology. The
candidate is also collaborating withDr. _____, _____, who is the _____ at the institution. Dr. _____ will oversee the randomization and distribution of oral arginine and placebo in this clinical trial.

The collaborative efforts and talents of these consultants and mentors will promote the candidate pursuit to become an independent clinical investigator.

        3) Role for the _____: The support from the _____ has enabled the candidate to gather _____ preliminary data, and has introduced _____ to the
many levels of expertise that are available within this research establishment. The candidate will continue to work closely with the institution _____ nurses and staff, as in _____ preliminary study. Once the clinical trial is underway, the candidate will rely on the support of members of the _____ on a daily basis. Together they will identify and enroll eligible patients, obtain consent, collect blood samples, administer the arginine or placebo, and utilize the exhaled NO analyzer. The candidate will expand _____ study to include outpatient follow-up in order to obtain baseline exhaled No_x, serum No_x and arginine levels on enrolled patients, which will take place at the _____. The candidate has already established a relationship with Dr. _____, _____ at the _____, _____, and will continue to utilize _____ statistical expertise throughout the duration of this giant.

        The candidate has also had extensive involvement with _____ lab group at the institution, as they have been essential to perfecting the assay to measure nitric oxide metabolites. They have also been very supportive of this project, and _____ has been particularly helpful in providing guidance through the very complex biochemistry of nitric oxide as it may pertain to sickle cell disease. _____ will provide space in _____ laboratory for the candidate to perform _____ investigations, and _____ will continue to oversee _____ work, serving as a valuable
resource.

6. Research Plan

a. Statement of Hypothesis and Specific Aims

1. To institute a prospective, double-blind, placebo controlled phase II/III trial in SCD patients hospitalized with VOC to determine if oral
   arginine therapy will decrease the length of hospitalization for VOC.
    

2. To characterize the biochemical and cellular effects of oral arginine administration in SCD patients with VOC.

b. Background, Significance, and Rationale

        1. Nitric Oxide and Vaso-Occlusion

Some mechanisms believed to influence vaso-occlusion include abnormal adherence of sickle erythrocytes (SRBC) to the vascular endothelium, cytokine upregulation, increased platelet activation and altered vasomotor tone. ^1,2 Factors involved in vaso-regulation are likely to include nitric oxide, as NO is one of the most potent vasodilators known.³ The actions of NO are not confined to regulation of vascular smooth muscle tone. The diversity of biological functions include regulation of platelet reactivity⁴, nonadrenergic, noncholinergic neurotransmission, and the cytotoxic and cytostatic actions of inflammatory cells. It decreases endothelial cell adhesion receptor expression, and is involved in the maintenance of blood pressure,^3,5-10 and release of growth hormone. ^11,12. With these vital functions, NO is crucial in maintaining physiologic homeostasis.

An imbalance of NO production has been implicated in several disease processes and inflammatory conditions that can lead to tissue damage. Hence, the biological response of NO, whether beneficial or deleterious is determined by timing, location and amount of NO present under a given circumstance.^4,13

        2. L-Arginine and Nitric Oxide

      3.  Nitric Oxide in Sickle Cell Disease

Because most of the morbidity of SCD results from vaso-occlusion, NO production is likely to play an important role in the pathophysiology of SCD. In support of this, plasma NO metabolite levels (No_x, measured as nitrite and nitrate) have recently been correlated with pain scores experienced in SCD patients with vaso-occlusive crisis ²⁷. Plasma No_x levels inversely correlate with pain scores with higher No_x levels associated with lower pain scores. My preliminary data also demonstrates that children >10 years of age with VOC requiring hospitalization had presenting No_x levels that were significantly lower than baseline.

Since adults with SCD are significantly deficient in L-Arg ^15,16, and children with VOC demonstrate substantially reduced arginine levels upon initial evaluation ^26,28, my hypothesis is that low levels of arginine may contribute to complications in SCD, by becoming the rate-limiting substrate for NO production.

The hypothesis that a state of upregulated NO production as a compensatory mechanism in SCD is similar to what has been reported for asthma. An expression of inducible NO synthase (NOS) has been found in the epithelium of asthmatic patients but not in healthy non-asthmatic patients.^29,30 No_x can also be measured in exhaled air^31,32, and asthmatics have exhaled air No_x levels that are 3.5 times higher than non-asthmatics.³³ This initially led to the assumption that NO was associated with bronchoconstriction, but when NO production was blocked by L-Arg analogues, an increase in allergen-induced bronchoconstriction occurred. This suggests a protective role for NO in asthma. Thus, NO may be acting as a feedback against bronchoconstriction³⁴, just as NO may be acting as a feedback against vasoconstriction in SCD. The involvement of NO in asthma may also be relevant in SCD as many patients also have hyperreactive airways. As with asthma, baseline exhaled No_x concentrations are also significantly higher in anemic animal models³⁵. Since exhaled No_x has never been monitored sequentially in sickle cell patients, it may represent another means to measure and follow nitric oxide levels.

Nitric oxide therapy by inhalation has been used for years to manage pulmonary hypertension and severe respiratory distress syndrome in newborns. Recent studies have demonstrated that inhaled NO normalizes oxygen P50 in SCD⁵⁶, and it has also shown promise by improved oxygenation in several children with ACS57. Thus, the upregulation of nitric oxide production may be beneficial in SCD.

        4. Peroxynitrite

        5. Endothelin-1 (ET-1)

       6. L-Arginine Therapy

    Recent studies have demonstrated that oral L-Arg significantly increases serum arginine levels and No_x levels (in both serum and in exhaled air) in healthy non-SCD adults.⁵³. L-Arginine has already demonstrated potential for therapeutic utility. In animal studies, inhalation of low doses of L-Arg completely blocks hyper-responsiveness of reactive airways ^29,30. Infusions of L-Arg initiate release of growth hormone due to NO ^11,12. Supplemental dietary arginine accelerates wound healing⁴⁷, and as a result, L-Arg is now routinely added to many commercially available enteral and parenteral nutritional formulas. Inhaled L-Arg improves pulmonary functions of cystic fibrosis (CF) patients⁴⁸, and there is also growing evidence that arginine has some benefits for diabetes-associated abnormalities¹⁸ and cardiovascular disease²³. It has been suggested that L-Arg has a stabilizing effect on sickle-hemoglobin⁵⁴. Another interesting finding is that rapid healing of chronic leg ulcers during arginine butyrate therapy was noted in several patients with SCD. Leg ulcer healing occurred in patients treated with arginine butyrate despite little change in fetal hemoglobin,⁵⁵ which is thought to be butyrate’s mechanism of action.^{50, 51} It is possible that it was butyrate’s arginine component that may have been the therapeutic agent, by causing increased perfusion to the damaged tissue through NO generation.

    As the precursor to nitric oxide, supplemental arginine has demonstrated benefits in many disease processes that involve endothelial dysfunction^{18,b 23}, and may also have therapeutic value SCD.

 

   C. Preliminary Studies and Results

Figure 1:

    Low L-Arg levels in patients with VOC returned to baseline during convalescence in the hospital (Fig 2A). It is possible that low arginine levels during VOC reflect a poor nutritional state during illness, which resolves with hydration, pain therapy and an improved appetite during the hospitalization. My sequential data on patients who developed ACS during their hospitalization, however, implicates another process independent of diet. Patterns of L-Arg (Fig 2) and No_x (Fig 3) appear to be different under the circumstances of VOC when compared to patients who develop ACS. L-Arg levels were near baseline at presentation for patients with VOC who developed ACS, but dropped during hospitalization within 24 hours of the chest radiograph becoming positive (Fig 2B), This drastic change in L-Arg concentration occurs despite adequate hydration, pain control and diet. Arginine levels return to baseline as ACS resolves.

Figure 2 :

Figure 3: Serum NO x levels in SCD patients with uncomplicated VOC (n = 10) and those who developed ACS (n = 9). Low = lowest

No_x level during admission.

The drop in No_x levels during hospitalization in patients developing ACS corresponds to changes in L-Arg levels (Fig 4). An initial rise in No_x is noted as its arginine substrate is utilized. It is possible that as L-Arg becomes depleted, No_x production decreases, or is shunted towards other reactive nitric oxide species (RNOS).
 

Figure 4: L-Arginine and nitric oxide levels in a representative patient admitted for VOC who subsequently developed ACS. Day 0= day ACS diagnosed. TXN = transfusion. Lowest levels of L-Arg and No_x are demonstrated within 24 hours of the discovery of ACS on chest x-ray.

Four patients who developed ACS also had baseline No_x levels determined. Steady-state No_x levels were nearly double that of the values obtained during ACS, confirming that No_x is significantly reduced in ACS (11.2±1.6 vs. 22.1±1.6 μmol/L, p = 0.003).

This data suggests that there may be a relationship between the L-arginine-nitric oxide pathway and vaso-occlusion in SCD.

These results lead me to hypothesize that low arginine levels during VOC reflect a
state of acute substrate depletion, possibly impeding the compensatory increase in NO production that would be expected from vaso-occlusion.

Similar No_x responses were found after both low and high doses of L-Arg in patients at baseline, despite differences in peak L-Arg levels (mean peak L-Arg levels"±SD: 170.7±41.2 vs. 278.0±116.7 μM), indicating that L-Arg concentration is not a rate limiting factor for No_x production in SCD patients at baseline.


Figure 8: Percent change of serum No_x levels from baseline (Time 0) in SCD patients at steady-state after oral arginine administered at low dose (0.01g/kg, n=6) vs. high dose (0.25g/kg, n=3), vs. normal non-SCD controls given low dose (0.01g/kg, n=4).

In crisis, however, L-Arg may become rate limiting, as we have previously reported low L-Arg and No_x levels in SCD patients with VOC. Low dose L-Arg (0.1g/kg) was administered to 4 SCD patients with VOC (Fig 9). Unlike SCD patients at baseline, patients with VOC had a dramatic increase in No_x production after oral L-Arg (median % change: –18.0% at baseline vs. ±65.5% during VOC). This response was even greater than in non-SCD normal controls, (median % change: ±65.5% in VOC patients vs. ±26.4% in controls).

Figure 9: Percent change of serum No_x levels from baseline (Time 0) after oral arginine administration in SCD patients with VOC vs. steady-state. VOC patients were given low dose L-Arg (0.1g/kg, n=4), compared to SCD patients at steady-state, who received both low dose (0.1g/kg, n=6) and high dose (0.25g/kg, n=3) L-Arg supplementation.

Significant increases in No_x levels in SCD patients during VOC suggests that L-Arg concentration becomes the rate limiting factor in VOC.

Interestingly, peak L-Arg levels were significantly lower in the VOC group when compared to baseline patients receiving the same low L-Arg dose(mean±SD: 93.8±26.6 vs. 170.7±41.8:µM, p=.01), suggesting an accelerated metabolism of arginine under the conditions of VOC. This data also suggests that, in contrast to SCD patients at baseline, L-Arg is the rate-limiting substrate for NO production in patients with VOC. Subsequently, L-Arg may become depleted as a result of increased utilization of the amino acid in order to meet a greater demand for NO,
leading to an acute arginine deficiency.

Pharmacokinetics of L-Arginine

Figure 10: Maximum percent change in No_x production in SCD patients with VOC after oral L-Arg low dose (0.05g/kg) vs. intermediate dose (0.1g/kg).

Although there is significant variability between patients, data I have gathered on one representative patient strongly supports the use of
the intermediate L-Arg dose (0.1g/kg). At steady-state, the patient experiences a paradoxical decrease in No_x levels after oral arginine
(0.1g/kg) that is sustained throughout the 4 hours without much fluctuation, (Fig 11). The same dose of oral L-Arg produces a significant increase in No_x production during VOC that persists with continued acceleration 2 hours after L-Arg administration. Interestingly, a paralleling decrease in endogenous No_x occurs during a separate VOC event after supplementation of oral L-Arg at the lower dose (0.05g/kg).
 

Figure 11: Percent change in No_x levels From baseline (Time 0) in a representative SCD patient after oral L-Arg administration at steady-state (0.1g/kg), and during VOC (0.05g/kg and 0.1g/kg).

The cause of decreasing endogenous No_x levels after low dose oral L-Arg is uncertain. My data reveals that peak L-Arg levels are significantly lower in VOC patients when compared to SCD patients at steady-state receiving the same dose of arginine, suggesting an accelerated metabolism of the amino acid during VOC. It is possible that supplementation with a lower L-Arg dose may provide enough substrate to activate nitric oxide synthase, (NOS), but is insufficient to sustain increases in No_x production. Previous investigations have shown that NOS will synthesize superoxide in lieu of NO under conditions of lower L-Arg concentrations³⁸. It is possible that superoxide plays a role in this paradox, but regardless of the mechanism, low-dose L-Arg appears tobe subtherapeutic in SCD under conditions of VOC.

In summary, my preliminary findings lead me to hypothesize that an acute arginine deficiency plays a role in VOC, possibly by affecting NO production. Arginine supplementation, a therapy with low toxicity even at high doses, may prove beneficial for SCD patients by resulting in an increase in No_x production during VOC. I propose a prospective, blinded, placebo control trial of supplemental arginine in SCD patients with VOC designed to test this hypothesis.

Significance

d. Research Design and Methods

SPECIFIC AIM #1: To institute a prospective, double-blind, placebo controlled trial in SCD patients hospitalized with VOC to determine if oral arginine therapy will decrease the length of hospitalization for VOC, and if this occurs by virtue of its effect on NO.

Primary Outcome Measure:

Secondary Outcome Measures:

1. Length of parenteral narcotic use (in hours)
2. Amount of parenteral narcotic use
3. Pain scores

General Study Design

    I propose a double-blinded, placebo controlled trial of the effects of oral arginine on SCD patients hospitalized with VOC. A total of 56 patients will be enrolled (see statistical analysis at the end of this section). Patients will be randomized to receive either placebo or arginine. Physicians and staff involved in patient care will be unaware of who receives placebo vs. arginine. Patients will be treated for 5 days or until discharge, whichever is shorter. In light of my preliminary data, oral arginine will be administered to SCD patients with VOC at dose of 0.1g/kg TID, a dose and schedule that is consistent with recent clinical trials of oral arginine ^{14,18,20,21,23}.

    Sickle cell patients admitted to the hospital with vaso-occlusive crisis will be approached about enrollment into the trial according to the following criteria:

Inclusion Criteria

• Established diagnosis of sickle cell disease (Hb SS)
• >8 years of age, < 21 years of age
• Pain requiring hospitalization for parenteral narcotics (Morphine or Demerol), not attributable to non-sickle cell causes.

Exclusion Criteria

• Hemoglobin less than 5 gm/dL or immediate need for red cell transfusion
• Evidence of acute chest syndrome
• Hepatic dysfunction: increase in SGPT to > 2X normal value
• Renal dysfunction: increase in creatinine to >2X normal value or >1.5
• Mental status or neurological changes
• Pregnancy
• >10 hospitalizations per year or history of dependence to narcotics
• Inability to take oral medications or allergy to arginine
• Inability to use a PCA device

For patients meeting the above criteria, average length of hospitalization will be calculated for the last 5 admissions for VOC. Only patients whose mean length of hospitalization for uncomplicated VOC is 6 ± 3 days and who have not had a hospitalization for uncomplicated VOC greater than 14 days will be eligible for enrollment. This will help ensure the enrollment of a population of VOC patients whose history of VOC is similar and who do not have a history of prolonged admissions. Patients meeting these criteria and who agree to participate will be enrolled and receive their first dose of arginine or placebo within 24 hours of being admitted.

Randomization and Arginine/Placebo Administration

Administration of Analgesics to Study Patients

Monitoring During Study

Pain Scores

Justification of laboratory tests

CBC: Following possible changes in Hb levels, also following NO effects on platelet count.

Chem 20: Toxicity monitoring of potential changes in BUN/creatinine as a result of a high protein load and following liver function tests as arginine is metabolized by the liver.

Urine analysis: Following potential development of protein in the urine. Urine will also be used to determine presence of peroxynitrite (ONOO-) and NO_x.

Urinary ONOO- (peroxynitrite): Nitrotyrosine is also excreted in the urine and will be correlated with serum nitrotyrosine levels as a marker for potential toxicity.

Urinary No_x: Nitrites and Nitrates are also excreted in the urine. Such information, together with serum No_x, and exhaled No_x will help to determine total body No_x.
 

Hb Electrophoresis: Confirm SCD status, to determine %S and %fetal hemoglobin before/after arginine supplementation

Research Labs: See Specific Aim 3

Patient Removal from Study/Toxicity Monitoring

Patients will be removed from the study if they meet the following criteria:

1) Development of acute chest syndrome

2) Neurological dysfunction

3) Increase in SGPT to > 2X normal value

4) Increase in creatinine to >2X value at study entry or >1.5

5) Decrease in hemoglobin to < 5 gm/dL or need for transfusion

6) Increase in met-hemoglobin > 2X normal value

7) Development of priapism

8) Refusal or inability to take study medication (arginine/placebo)

9) Patient’s request Data from patients removed from the study will be collected and analyzed for potential side effects or toxicities of arginine. Blood analysis and exhaled No_x measurements will continue per protocol, but arginine/placebo supplementation will be terminated.

Sample Size and Statistical Analysis

Over a 1-year period (1998), 209 children > 8 years old were seen in the ED for VOC. Of these, 137 (66%) were admitted to the hospital for treatment of their pain crisis. The average length of stay was 6±3 days. Approximately 10% of these patients would be excluded due to histories of prolonged hospitalization. Nearly 120 patients/year with SCD admitted for VOC will be eligible for this study.

Discharge information on 150 recent patients admitted to _____ for VOC was used to develop a power calculation. Five thousand datasets were simulated from the distribution of length of stay for VOC. A placebo group was simulated from the historical data. A No_x group was simulated with the same shape but with a median stay decreased by 35%. Using the Wilcoxon rank-sum test to compare the groups, a sample size of 28 subjects per group had 82% power to detect a treatment difference on a two-sided 0.05 level (Wilcoxon) test.

    SPECIFIC AIM #2: To characterize the biochemical and cellular effects of oral arginine administration in SCD patients with VOC.

    Primary Objective:

1. Follow patterns of plasma amino acids (arginine, citrulline, and lysine) and serum No_x during the course of hospitalization

2. Observe effects of oral arginine on exhaled No_x during VOC, and determine correlation to serum No_x and clinical course

3. Monitor for toxicity/side effects

    The studies outlined under this specific aim will be undertaken throughout the first specific aim. Blood for the biochemical investigations will be drawn at the time of the clinical labs as indicated in the tables above (“RESEARCH LABS”). Namely, preliminary investigations into the physiologic effects of increased nitric oxide production will be assessed. Subsequently, changes seen with arginine administration in VOC patients will be compared with the changes seen in the patients who receive placebo. Investigations into the biochemical and cellular effects of arginine administration and subsequent nitric oxide upregulation will center on four areas:
 

1. Oxidative Balance and Damage
2. Vasoregulatory Changes
3. RBC Effects
4. Platelet Function

1. Oxidative Balance and Damage

Nitric oxide can have _____ favorable or deleterious effects, depending on the environment in which it is produced. It becomes crucial,
therefore, to measure other components which play an active role in maintaining oxidative balance.
 

• serum nitric oxide metabolites: nitrite and nitrate (No_x)
• exhaled nitric oxide
• peroxynitrite (nitrotyrosine)
• 8-iso-PGF_2α

     Serum and Urine Nitric Oxide Metabolites: Although measurements of serum No_x do not necessarily represent biologically active nitric oxide, this assay is routinely used to measure estimated nitric oxide levels in human studies^9,67. No_x can also be measured in urine68. The combination or serum, exhaled and urinary No_x may give us a better idea of total body nitric oxide metabolism.

My preliminary serum No_x data was obtained using the Griess reaction method. Measurements of serum and urine No_x by the Sievers NOAnalyzer
offers a significant advantage over the Griess reaction by immediate results. My preliminary data demonstrates that low arginine levels may be predictive for need of hospitalization during VOC ²⁸. I have also demonstrated that children >10 years old with VOC had No_x levels that were significantly lower than baseline upon presentation to the ED. Such information is useful to the emergency medicine physician only if it is available rapidly enough to impact the evaluation and treatment plan. The availability of real time serum No_x levels may create a role for the measurement of serum No_x levels in older children or adults with VOC in the future.
   

Exhaled Nitric Oxide:

    Peroxynitrite:

8-iso-PGF_2α:

2. Vasoregulatory Changes

Endothelin-1 (ET-1):

• P50 — measures changes in oxygen affinity

• Met Hemoglobin production

• Ektacytometry--measures red cell deformability

• H3 Technicon—measures red cell hydration

    P50:

   Met hemoglobin:

Ektacytometry and H3 Technicon:

Specific Laboratory Methods

  Arginine, citrulline, and lysine: Quantitative plasma amino acids are routinely done on patients being evaluated for metabolic disorders. L-Arginine, citrulline, and lysine are 3 of a total of 20 amino acids that are often evaluated. Plasma levels will be measured in μmol/L through ion exchange chromatography, done by Quest Diagnostics, Nichols Institute Biochemical Genetics Laboratory, San Juan Capistrano, CA. Amino acid reference ranges are available on normal children, run by Quest Diagnostics.

    II. Oxidative Balance and Damage

  

 NO_x:

NO Analyzer:

Exhaled Nitric Oxide:

Peroxynitrite:

    8-iso-PGF_2α:

  Endothelin-1: ET-1 levels will be determined in triplicate in serum by enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer’s instructions (Amersham, UK). Briefly, each kit uses a quantitative immunometric sandwich ELISA performed in a 96 well plate coated with monoclonal antibody against the ET-1 peptide and is incubated overnight at 4 degrees Celsius. A monospecific antibody conjugated to horseradish peroxidase is added. During an additional incubation of 30 minutes, the conjugate antibody binds to the affixed antibody/peptide. Upon addition of the substrate, a peroxidase-dependent color reaction occurs that is proportional to the amount of bound ET-1. The sample can be read at 630 nm, or the samples can undergo a 30 minute acidification by the addition of sulphuric acid into each well (preferred method, and the one I will employ), and the optical density can be read at 450 nm.

IV. RBC Effects
   

P50:  P50 is the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen, and is a test routinely done using a Hemox-Analyzer (TCS Medical Products, PA), to measure the oxygen dissociation curve. ⁷⁸ Met-hemoglobin production: Met-Hb levels are routinely run on venous blood using a CO-Oximeter in the pulmonary function laboratory at CHO by trained respiratory therapists.

  

 Ektacytometry: _____ is one of the few laboratories in the country performing this analysis.

Intact red blood cells (RBCs) are studied using osmotic gradient ektacytometry by a previously described method. ⁷⁹ RBC deformability is measured as a function of osmolality of the suspending medium. For each sample, the degree of maximal deformability (DI_max), the hypotonic osmolality at which minimal deformability is found and hypertonic osmolality at which the DI = ½ Di_max is recorded .

   

H3 Technicon: H3 Technicon measurements will be done following manufacturer’s protocol. Briefly, whole blood will be collected and stored refrigerated. Samples will be run within 72 hours of collection. Samples and controls are mixed with an oxazine Working Dye solution, which stains cellular RNA. They are then incubated at room temperature for 15 minutes. Samples are run on the H3 analyzer (Technicon, NY), and analyzed using a special computer program available at CHORI. This instrument will identify changes in red cell hydration status.

V. Platelet function

PFA-100:

Research Timetable:

e. Human Subjects

    Human subjects, specifically children and young adults 8 -21 years old with SCD will be included in this study. Patients will be recruited from patients followed regularly at the _____. Nationally, SCD is a disease seen almost entirely in the African-American population and affects both male and female patients equally. The population of SCD at the _____ is overwhelmingly African-American and equally represented by both genders. There is no reason to assume differences in outcomes according to gender or ethnicity. Although equal recruitment of both males and females will be attempted, all data will be analyzed together. Also, non-African American SCD patients who meet eligibility criteria will be included in the study and every attempt will be made to recruit these patients.
 

1. Peripheral blood will be required from study patients for serum, plasma and whole blood studies. This blood will generally be obtained at the time of other routine phlebotomies so the patient should experience no additional discomfort. Patients enrolled for Specific Aims 1 of this proposal will, however, need additional samples that would not routinely be drawn.
    
2. Patients with SCD at baseline and those with vaso-occlusive crisis meeting entry criteria will be approached concerning entrance into the clinical trial. The principal investigator will explain the study to the patients and their families and will obtain consent on all patients. Patients and/or parents will sign an IRB-approved consent form. Patients are free to exit the study at any point.
    
3. Blood samples for this study will be drawn at the time of other phlebotomies whenever possible to limit discomfort to the patient. For patients enrolled in the Specific Aims 1 portion of this proposal, discomfort from an venipuncture during placement of the intravenous catheter will be experienced, and bruising from the blood draws are a potential risk to the patient. For patients enrolled in the clinical trial, the risks of the study are confined to those associated with the oral arginine administration. As with any food or medication, the risk of an allergic reaction is a small but possible complication. Arginine is a nutritional supplement with a low toxicity and has been safely used in many human studies. Mild stomach discomfort may be experienced at higher doses. Patients with compromised liver or kidney functions may not tolerate the high protein load, and are therefore excluded from participation. Priapism is also a theoretical toxicity of increased nitric oxide production in male participants, as nitric oxide is an important component in erection physiology. All patients enrolled in the study will also receive the standard treatment for vaso-occlusive crisis offered at _____.
    
4. Parents will be informed that they and their child may decline to participate in this study, and that their decision will have no effect on their child’s care. Alternative treatments include the standard treatment for vaso-occlusive crisis without arginine or placebo.
    
5. Patient confidentiality will be protected as much as possible during the on-going clinical trial, and strictly for inclusion in grant proposals, abstracts and publications. Samples will be assigned a code that is known only to the investigator, _____.
    
6. The risks of this study are reasonable because of the significant benefits it may provide for patients with sickle cell disease. As a safe, nontoxic dietary supplement, oral arginine may prove beneficial for SCD patients by resulting in an increase in nitric oxide production, and is potentially a new therapy for the treatment of vaso-occlusive crisis. This trial could result in a new treatment that shortens hospital days, and decreases pain during vaso-occlusive events. It may be the foundation for future studies expanding arginine therapy as a prophylactic measure. The benefits of this study may impact significantly on the SCD population as a whole.

GENDER AND MINORITY INCLUSION TABLES--SICKLE CELL DISEASE

A. National Demographics for Disease
 

B. _____ Patient Population Available (%)

 

There is no difference with respect to ethnicity or gender between the national and _____ populations.

C. Planned Enrollment
 

Participation of Children

f. Vertebrate Animals

g. Literature Cited

h. Consortium/Contractual Arrangements

i. Consultants

7. Appendix