Effect of Oral Iron Therapy on Erythrocyte Protoporphyrin Levels in the Erythropoietic Protoporphyrias
Phase: NA
Conditions: Erythropoietic Protoporphyria, EPP, X-linked Protoporphyria, XLP
Sponsor: Icahn School of Medicine at Mount Sinai

Title: Effect of Oral Iron Therapy on Erythrocyte Protoporphyrin Levels in the Erythropoietic
Protoporphyrias 
PI: Manisha Balwani, MD, MS 
NCT02979249 
Document Date: June 14, 2017

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Iron for EPP 
 
 
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Rare Diseases Clinical Research Network 
 
Porphyrias Consortium 
 
 
Effect of Oral Iron Therapy on Erythrocyte 
Protoporphyrin Levels in the Erythropoietic 
Protoporphyrias 
 
 
 
 
 
Manisha Balwani, MD, MS 
Department of Genetics & Genomic Sciences 
Icahn School of Medicine at Mount Sinai 
One Gustave L. Levy Place, Box 1497 
New York, NY 10029 
Phone:  212-241-0915 
Fax:  212-426-9065 
Email:  manisha.balwani@mssm.edu 
 
 
This protocol is for research purposes only, and should not be copied, 
redistributed or used for any other purpose.  The procedures in this 
protocol are intended only for use by Consortium investigators in carefully 
controlled settings.  The Chair of this study should be consulted before 
using or attempting any procedure in this protocol. 

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Participating Institutions/Investigators Table (contact information) 
 
 
Principal Investigator: Manisha Balwani, MD, MS 
Contact:  Karli Hedstrom, MPH 
Institution: Icahn School of Medicine at Mount Sinai 
Address: One Gustave L. Levy Place, Box 1497 
New York, NY 10029 
Phone: 212-659-1450 
Fax: 212-426-9065 
Email: manisha.balwani@mssm.edu 
Sub-
Investigators: 
Lawrence Liu, MD; Robert J. Desnick, PhD, MD 
  
 
Principal Investigator: Ashwani Singal, MD, MS 
Contact: Angelia Johnson 
Institution: University of Alabama at Birmingham 
Address: Division of Gastroenterology and Hepatology Clinical Research Program  
 
Birmingham, AL  35294 
Phone: 205-934-0498 
Fax:  
Email:  asingal@uabmc.edu 
Sub-
Investigators: 
Joseph R. Bloomer, MD; Brendan M. McGuire, MD 
  
 
Principal Investigator: D. Montgomery Bissell, MD 
Contact: Yuvraaj Kapoor 
Institution: University of California at San Francisco  
Address: UCSF Porphyria Center 
 
San Francisco, CA 94143-0538 
Phone: 415-476-8405 
Fax: 415-476-0659 
Email: dwight.bissell@ucsf.edu 
Sub-
Investigator: 
Bruce Wang, MD; Jennifer Lai, MD, MPH 
  
 
Principal Investigator: John Phillips, PhD  
Contact: Sharada Dixit 
Institution: University of Utah 
Address: Clinical Trials Office 
 
Salt Lake City, Utah 84108 
Phone: 801-587-7525 
Fax: 801-585-9410 
Email: John.phillips@hsc.utah.edu 
Sub-
Investigators: 
Charles Parker, MD 
 
 

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Principal Investigator: Karl E. Anderson, MD 
Contact: Csilla Hallberg, MD 
Institution: University of Texas Medical Branch 
Address: Porphyria Center 
301 University Boulevard 
Galveston, TX 77555-1109 
Phone: 409-772-4661 
Fax: 409-772-6287 
Email: kanderso@utmb.edu 
Sub-
Investigators: 
 
  
 
Principal Investigator: Herbert L. Bonkovsky, MD 
Contact: Dee Faust 
Institution: Wake Forest School of Medicine 
Address: Department of Internal Medicine 
Section on Gastroenterology 
Medical Center Blvd 
Winston-Salem, NC 27157 
Phone: 336-713-1442 
Fax: 336-713-7322 
Email: hbonkovs@wakehealth.edu 
Sub-
Investigators: 
Pradeep Yarra, MD; Sara Taylor, MD 
  
 
  
 
Data Management and Coordinating Center Principal Investigator: Jeffrey Krischer, Ph.D. 
Contact:  Carrie Light, Research Compliance Administrator 
Institution/ 
Department: 
Data Management and Coordinating Center (DMCC); Pediatrics Epidemiology 
Center; University of South Florida 
Address: 
; Tampa, Fl 33612 
Phone: 813-396- 9248 
Fax: 813-910-5997 
Email: Carrie.Light@epi.usf.edu 
  
 
 
 

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Table of Contents 
Participating Institutions/Investigators Table (contact information) .......................................... 2 
1. Protocol Synopsis ............................................................................................................................... 6 
1.1 Overview ......................................................................................................................................... 8 
2. Specific Aims (Hypothesis and Objectives) ................................................................................ 8 
3. Background .......................................................................................................................................... 9 
4. Study Design and Methods ........................................................................................................... 13 
4.1 Inclusion Criteria ........................................................................................................................ 13 
4.2 Exclusion Criteria ....................................................................................................................... 14 
4.3 Recruitment of Participants ..................................................................................................... 14 
4.4 Retention Strategies ................................................................................................................. 15 
4.5 Data Elements ............................................................................................................................ 15 
4.6 Study Procedures ...................................................................................................................... 16 
4.7 Schedule of Events ................................................................................................................... 19 
4.8 Investigational Product ............................................................................................................. 20 
5.0 Data and Safety Monitoring Plan ............................................................................................... 20 
5.1 Study Oversight .......................................................................................................................... 20 
5.2 Definitions and Standards ....................................................................................................... 21 
5.3 Expected/Known Risks/Discomforts/Adverse Events Associated with Study 
Intervention and Procedures: Definition of Expected Adverse Events ............................. 21 
5.4 Reporting Timeline .................................................................................................................... 22 
5.5 RDCRN Adverse Event Data Management System (AEDAMS)................................. 22 
5.6 Study Discontinuation ............................................................................................................... 23 
5.7 Subject Discontinuation ........................................................................................................... 23 
5.8 Data Quality and Monitoring Measures ............................................................................... 24 
5.9 Quality Control: Study Related Procedures ....................................................................... 24 
6. Statistical Considerations ............................................................................................................... 24 
7. Data Management ............................................................................................................................ 27 
7.1 Registration .................................................................................................................................. 27 
7.2 Data Entry .................................................................................................................................... 28 
8. Human Subjects ................................................................................................................................ 28 
8.1. GCP Statement ......................................................................................................................... 28 
8.2. Risks ............................................................................................................................................. 28 
8.3. Benefits ........................................................................................................................................ 28 

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8.4. Recruitment ................................................................................................................................ 29 
8.5. Written Informed Consent: ..................................................................................................... 29 
8.6. Process of Consent: ................................................................................................................ 29 
8.7. Certificate of Confidentiality ................................................................................................... 30 
9. References ......................................................................................................................................... 31 
 
 
 
  
 
 
 

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1. Protocol Synopsis 
Interventional Synopsis 
Protocol Number: 7209 
 
Protocol Title: Effect of Oral Iron Therapy on Erythrocyte Protoporphyrin Levels 
in the Erythropoietic Protoporphyrias 
Study Chair: Manisha Balwani, MD, MS 
Statistician: Inga Peter, PhD; Icahn School of Medicine at Mount Sinai 
Jessica Overbey, MS; Icahn School of Medicine at Mount Sinai 
Consortium: Porphyrias Consortium 
Participating Sites: Icahn School of Medicine at Mount Sinai , New York, NY  
University of Alabama at Birmingham, Birmingham, AL  
University of California at San Francisco, San Francisco, CA  
University of Texas Medical Branch, Galveston, TX 
University of Utah, Salt Lake City, UT 
Wake Forest University Health Sciences, Winston-Salem, NC 
Activation Date:  
Current Status:  
Sample Size: 20 
Target Enrollment Period: 4 years 
Study Design: Interventional  
Primary Study Objective: To assess the potential efficacy of iron therapy for decreasing 
erythrocyte protoporphyrin levels in patients with EPP and XLP 
Secondary Study 
Objective(s): 
1) To assess whether oral iron therapy can improve iron 
status and decrease plasma porphyrin levels and clinical 
symptoms 
2) To assess if there in an improvement in the quality of life 
in treated patients 
 
Study Population and Main 
Eligibility/ Exclusion 
Criteria: 
Inclusion criteria:  
1. Enrollment in the Longitudinal Study of the Porphyrias 
(7201)  
2. Male or female age ≥18 years 
3. Willing to sign informed consent form and follow protocol  
4. History of nonblistering cutaneous photosensitivity 
5. Biochemical findings- A marked increase in erythrocyte 
protoporphyrin (total erythrocyte protoporphyrin >400 
ug/dL) with a predominance of metal-free protoporphyrin 
6. Serum ferritin ≤30 ng/mL at baseline 
7. Able to tolerate oral iron 
Exclusion criteria:  
1. History of liver or bone marrow transplant or 
clinically significant liver dysfunction as 
determined by the investigator 
2. Patient reported known or suspected allergy to 
oral iron  
3. Clinical evidence of active and ongoing GI 
bleeding 
4. Use of any other clinical or experimental therapy 
in the past 3 months which could confound study 
results 
5. Individuals with elevations of porphyrins in 
plasma or erythrocytes due to other diseases 
(i.e., secondary porphyrinemia) such as liver and 

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bone marrow diseases 
6. Pregnancy 
7. Patients with any clinically significant comorbid 
condition, which in the opinion of the investigator, 
precludes participation 
Treatment 
Agent- Ferrous sulfate USP  
Dosage, schedule, route of 
administration- 
325 mg capsule oral administered twice daily (total 650 mg daily) 
for 12 months 
Safety Issues- Gastrointestinal irritation, constipation, diarrhea, nausea, vomiting 
Primary Outcome 
Measures: 
The primary outcome is the relative difference in erythrocyte 
protoporphyrin levels between baseline and 12 months after 
treatment 
Secondary Outcome 
Measures: 
1) Erythrocyte protoporphyrin over time: Mean values and 
standard deviations will be computed at each time point and 
values will be compared descriptively.  Additionally, a random 
effects longitudinal model of erythrocyte protoporphyrin with 
time as the predictor variable will be fit and used to assess 
whether subjects’ erythrocyte protoporphyrin varied with time. 
 
2) EPP-specific QOL: Mean scores and standard deviations will 
be computed at each time point and values will be compared 
descriptively   
 
3) Safety: Rates of Adverse events will be computed and safety 
variables such as physical and other laboratory findings will 
be analyzed descriptively.   
Statistical Considerations 
(sample size and analysis 
plan): 
A Bayesian approach based on the posterior distribution that the 
mean relative difference between baseline and 12-month 
erythrocyte protoporphyrin levels is greater than 0 will be used to 
assess the strength of potential efficacy.  The posterior probability 
that the relative difference is greater than 0 will be computed.  A 
probability ≥ 80% (corresponding to an approximate quadrupling 
of the odds that iron is effective in lowering erythrocyte 
protoporphyrin) will be considered evidence that additional study 
of iron is warranted. 
 
Assuming the distribution of baseline erythrocyte protoporphyrin 
is gamma (2.3, 861) and that baseline and 1 year values are 
correlated with r = 0.7, simulations indicate that a total sample 
size of 20 patients will allow us to detect an approximate 
quadrupling of the odds that iron is effective in lowering 
erythrocyte protoporphyrin if the true effect size is 10%.   
Sponsors (federal, state, 
foundation and industry 
support): 
National Institutes of Health (NIH) 
 

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1.1 Overview 
 
Erythropoietic Protoporphyria (EPP) and X-Linked Protoporphyria (XLP) are inborn 
errors of heme biosynthesis, presenting clinically with severe, acute photosensitivity. 
Although the clinical presentation is similar, EPP results from autosomal recessive 
“loss-of-function” mutations in the ferrochelatase (FECH) gene, whereas XLP results 
from “gain-of-function” mutations in the erythroid-specific 5-aminolevulinate synthase 
(ALAS2) gene (1, 2). Mutations in the FECH gene reduce ferrochelatase activity to ~10-
30% of normal (3). This enzymatic deficiency leads to the accumulation of 
predominantly metal-free protoporphyrin in erythroid and hepatic cells. In XLP, gain of 
function mutations result in increased enzymatic activity of erythroid-specific 5-
aminolevulinate synthase 2, leading to accumulation of approximately equal amounts of 
metal-free and zinc-protoporphyrins (2).  EPP is the most common porphyria in children 
and the second most common porphyria in adults. Patients with EPP and XLP present 
with painful cutaneous photosensitivity, generally in early childhood, presenting 
immediately or soon after exposure to sunlight. This pain can be accompanied by some 
redness and swelling of the sun exposed skin. The pain is severe and not responsive to 
analgesics (1). Currently there is no FDA approved treatment for EPP or XLP.  
 
In the medical literature there are conflicting case reports on the effect of iron 
supplementation on EPP symptoms (4, 5, 6). In the case of an inherited deficiency of 
FECH activity, the lack of iron, a cosubstrate of FECH, could theoretically limit its 
capacity and thereby enhance the accumulation of protoporphyrin IX and in turn, 
aggravate the phototoxic symptoms. Iron supplementation in the case of iron deficiency 
is therefore hypothesized to improve EPP symptoms. 
 
Therefore, the purpose of this study is to determine the effect of oral iron therapy for 
EPP and XLP patients. 
The Office of Rare Diseases (ORD) of the National Institutes of Health (NIH) 
established a Rare Diseases Clinical Research Network (RDCRN) in collaboration with 
other NIH Institutes and has funded several rare diseases clinical research consortia 
and one Data Management and Coordinating Center. The Porphyrias Consortium was 
created as part of the RDCRN, to study the human porphyrias. The Porphyrias 
Consortium is a consortium of the academic institutions listed in the participating 
institutions table. All Centers in the Porphyrias Consortium are participating in this 
study.  Additional centers may be added if funding is available. 
2. Specific Aims (Hypothesis and Objectives) 
 
The overall objectives of this study are to determine the efficacy and safety of oral iron 
administration in the protoporphyrias.  Efficacy will be based on erythrocyte 
protoporphyrin levels, photosensitivity, other clinical symptoms, and quality of life in 
patients with EPP and XLP who have diminished iron stores (serum ferritin <30 ng/ml).  
 

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Primary Objective: To determine if oral iron therapy can decrease erythrocyte 
protoporphyrin levels in patients with EPP and XLP.   
 
Secondary Objectives:  
1) To assess whether oral iron therapy can improve iron status and decrease 
plasma porphyrin levels and clinical symptoms 
2) To assess if there is an improvement in the quality of life in treated patients 
 
This is an open label pilot study of the safety and efficacy of oral iron therapy in patients 
with EPP and XLP with diminished iron stores as defined by serum ferritin level.   
3. Background 
Erythropoietic Protoporphyria (EPP) is characterized by painful, non-blistering, 
cutaneous photosensitivity, with onset in early childhood.  EPP is the most common 
porphyria in children and the third most common in adults (after porphyria cutanea tarda 
and acute intermittent porphyria). Reports of prevalence vary between 5 and 15 cases 
per million population. (2-4)  
 
EPP is most often due to decreased activity of ferrochelatase (FECH), the enzyme that 
catalyzes the incorporation of ferrous iron into PPIX, the final step in the production of 
heme.  The pattern of inheritance is autosomal recessive. Homozygosity for a FECH 
mutation is rare. In most cases, the decreased activity is a consequence of a 
combination of an inherited inactivating mutation affecting one FECH allele and an 
intronic polymorphism that is common in the population and which alters splicing of the 
other allele. The alternative splice site, when used, produces a non-functional FECH 
mRNA.  The alternative splice site is used approximately 40% of the time.  Therefore, 
the polymorphic allele produces approximately 60% of normal FECH activity, and for 
this reason, is termed hypomorphic. When the hypomorphic FECH allele is in trans with 
the non-functional mutant allele, the result is 30% or less of the normal FECH enzyme 
activity.  This subnormal FECH activity becomes rate-limiting, resulting in accumulation 
of intracellular protoporphyrin IX (PPIX). Although the defect is presumably expressed in 
all tissues, the PPIX responsible for photosensitivity derives primarily from marrow 
reticulocytes. X-linked Protoporphyria is less common than EPP, occurring in ~10% of 
cases, but has the same phenotype, and results from gain of function mutations in the 
ALAS2 gene, leading to the overproduction of protoporphyrin in the bone marrow (2, 4). 
 
Significance 
There are no FDA-approved treatments for EPP and XLP other than pharmaceutical 
grade  carotene (Lumitene), which was FDA approved for EPP and later classified as a 
nutritional product and is now available without a prescription.  Cysteine and Vitamin C 
have been studied in these patients, but with little convincing evidence of efficacy. Most 
patients report minimal, if any improvement with any of these products (7).  There have 
been few multicenter trials, which are required for collecting high quality data, on safety 
and efficacy of new and existing treatments.  Afamelanotide (α-melanocyte stimulating 
hormone), one of the few drugs under investigation for the treatment of EPP and XLP 

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completed Phase 3 trials in August 2013. Although beneficial, afamelanotide acts by 
darkening the skin rather than by decreasing circulating protoporphyrin levels (5, 8). No 
treatments reported to date, other than marrow transplantation and Panhematin® 
infusions, given in the setting of acute liver failure, decreases erythrocyte protoporphyrin 
levels, in these patients.  A large proportion of patients with the EPP phenotype are 
mildly iron deficient, but that proportion is not well defined, and its significance and 
influence on disease severity is not known.  We believe that iron therapy in patients with 
the EPP phenotype and low iron stores has the potential to increase iron availability and 
thereby increase protoporphyrin utilization for heme synthesis. Additionally, we also 
believe, that this therapy, may sufficiently decrease protoporphyrin accumulation to be 
clinically beneficial. If so, this would provide significant and low cost benefit to these 
patients.   
 
Erythropoietic Protoporphyria and Iron: As noted above, EPP is caused by 
deficiency in ferrochelatase, the last enzyme in the heme biosynthetic pathway. The role 
of ferrochelatase is to catalyze the insertion of ferrous iron into protoporphyrin to form 
heme (1). Defective ferrochelatase activity results in the accumulation of protoporphyrin 
initially in the marrow and then in erythrocytes and plasma, which circulate to the skin, 
liver and other tissues leading to the clinical symptoms. Recent reports suggest that the 
availability of iron modulates ferrochelatase activity by regulating the amount of correctly 
spliced FECH mRNA (9), and thus iron appears to play an important role in disease 
expression.   
 
Many patients with EPP, also have mild anemia, with evidence of iron deficiency, 
including microcytosis and low serum ferritin and iron levels (10). In a Swedish cohort, 
of EPP patients, about 45% had low ferritin levels (11). In our longitudinal study, 52% of 
EPP and XLP patients had low ferritin at baseline Other than microcytosis, there is little 
evidence of impaired erythropoiesis or abnormal iron metabolism in these patients. 
Although inappropriately low iron absorption has been proposed to explain these 
findings, this has not been substantiated. 
 
The role of iron replacement in EPP and the clinical response to increased iron is 
controversial, some reports suggest clinical improvement (12, 13, 14, 15) while others 
describe an exacerbation of photosensitivity (16, 17). Notably, an increase in 
protoporphyrin levels, has never been documented, in EPP patients treated with iron.  
Based on these conflicting reports, clinical practice varies among physicians with some 
using oral iron replacement in EPP patients with low iron stores and others choosing no 
intervention. No results or follow up on the effectiveness of oral iron therapy have been 
reported. A recent report from the U.K. showed clinical and biochemical improvement in 
a single EPP patient that had been treated with low doses of intravenous iron (17). 
Hemin therapy has also been used in EPP patients with advanced liver disease prior to 
transplantation and decreases in erythrocyte and plasma protoporphyrin levels have 
been observed (18). Although the mechanism of action is unclear, a possible hypothesis 
is that hemin, which contains iron, improves iron stores in these patients by providing 
iron, as well as bypassing the deficient FECH enzyme, leading to a decrease in 
protoporphyrin levels. Results from previous studies of the natural history of EPP/XLP 

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suggest that there is little change in erythrocyte protoporphyrin levels over time and 
there is little intra-individual variation (11). 
 
 
Interestingly, oral iron replacement in one patient with XLP with diminished iron stores 
also led to a decrease in erythrocyte protoporphyrin levels (2). We also administered 
oral iron to a patient with XLP and observed a 20% decrease in erythrocyte 
protoporphyrin levels.  We have now enrolled a large number of patients in an EPP 
Natural History study, several of whom were treated in the past with oral or intravenous 
iron therapy and experienced improvements in iron status without adverse 
consequences before they entered our studies. Unfortunately, serial measurements of 
protoporphyrin levels were not done prior to enrollment in our longitudinal studies.  
While these reports and our experience to date suggest that iron replacement can 
improve iron status and may decrease erythrocyte protoporphyrin levels, the efficacy 
and safety of iron therapy in EPP and XLP have not been systematically investigated, 
and whether or not to use iron in these patients remains an unanswered clinical 
question.  
 
A recent pilot study (19) showed that patients with EPP and XLP absorb oral iron with 
post dose increases in serum iron. In addition, the serum and urine hepcidin were not 
inappropriately increased in these subjects at baseline and did not increase over time 
after oral ferrous sulfate. Thus, the basis of iron deficiency in EPP/XLP patients and the 
effectiveness and safety of iron therapy are not clear and warrant further investigation.  
 
This provides the rationale for a pilot therapeutic study with oral iron in EPP and XLP 
patients with well characterized phenotypes, genotypes, and iron status, with serial 
measurements of protoporphyrin levels in erythrocytes and plasma during treatment. 
 
 
Preliminary data:  
 
Laboratory Studies: To examine the additive effect of iron on ferrochelatase (FECH) 
activity, an in vitro assay was set up using lymphoblastoid cell lines. Blood samples 
were collected from the EPP patients, who carried the p.C411G mutation (no detectable 
enzymatic activity (20) in trans to IVS3-48C (genotype M/C) and from healthy donors 
(genotype T/T). Lymphoblastoid cell lines were established from these samples by 
transformation of the peripheral B lymphocytes with Epstein-Barr virus following a 
standard procedure (21). After treatment of cells with ammonium iron (III) citrate for 24 
hours, FECH activity was determined by fluorometric measurement of zinc-
deuteroporphyrin formation (nmol zinc-deuteroporphyrin/hour/mg protein at 37°C) (22) 
and compared to the value of lymphoblastoid (T/T) without any treatment. When 
lymphoblastoid cells (M/C) were treated with iron (III), FECH activity increased and 
reached 35.5%, up to 100µM, followed by a decrease at increased iron concentrations 
(Figure 1). Dissimilarly, treatment of lymphoblastoid cells (T/T) with iron (III) below 
100µM only resulted in a slight decrease in FECH activity. These results suggest that 

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Figure 1. Effect of Iron (III) on the 
ferrochelatase activity of lymphoblastoid cell 
lines established from healthy donors (IVS3-
48T/T) and patients (C411G/IVS3-48T>C). 
Data are expressed as mean ± standard 
deviation of four experiments.  
treatment of iron (III) at proper concentrations (below 100 µM) may help to increase the 
FECH activity in cells with loss-of-function mutations on FECH gene. 
 
 
 
This project derives from observations on the large number of patients with whom we 
have experience in the PC, and also from isolated experience with iron treatment in 
some patients.  For example, we administered oral iron (325 mg of ferrous sulfate daily) 
to a 13 year old male with XLP based on clinical indications including a low serum iron 
level of 29 mcg/dL (normal 50-200 mcg/dL); low ferritin levels 12 ng/ml (50-150 ng/ml); 
and low transferrin saturation, 9% (normal 15-50%). Erythrocyte protoporphyrin levels at 
baseline and at one year follow up are shown in Table 1.  A 20% decrease in 
erythrocyte protoporphyrin was seen at one year, and he reported increased tolerance 
to sunlight.   
 
 
100.00%
88.69%
97.81%
72.34%
0.00%
29.08%
35.47%
25.46%
0.00%
25.00%
50.00%
75.00%
100.00%
0
50
100
200
Iron (III) (µM)
Ferrochelatase Activity (%)
Lymphoblastoid (T/T)
Lymphoblastoid (M/C)

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Table 1: Erythrocyte protoporphyrin levels in an XLP affected male following oral 
iron supplementation 
 
Pre-Treatment 
Post-Treatment 
Normal Range 
PLASMA 
Total Porphyrins (mcg/dL) 
10.2 
9.7 
0-0.9 
Fluorescence Max (nm) 
634 
634 
No peak 
ERYTHROCYTES 
Protoporphyrin (mcg/dL) 
6,000 
4,700 
20-80 
Zinc protoporphyrin (%) 
32 
38 
 
Free protoporphyrin (%) 
68 
62 
 
Hematocrit (%) 
35.4 
36.1 
 
 
Thus, the preliminary data from the laboratory studies as well as anecdotal evidence 
from patient care provides support for this hypothesis.  
 
This study will test the hypothesis that successful iron replacement in these patients 
decreases erythrocyte protoporphyrin levels and clinical severity of disease.  If effective, 
this simple treatment may have significant clinical benefit and should decrease 
symptoms and improve quality of life in these patients. Decreases in circulating 
porphyrins after iron replacement might also decrease the risk for liver complications.  In 
addition, the cause of iron deficiency in these patients is unknown, and this study may 
provide insight into the mechanism of iron dysregulation in EPP and XLP. 
4. Study Design and Methods 
This is an open label study of ferrous sulfate USP 325 mg administered twice daily (total 
650 mg daily). All patients recruited for the study will be enrolled in the Longitudinal 
Study of the Porphyrias and have a confirmed diagnosis of EPP or XLP by biochemical 
testing. The sample size will be 20 patients with EPP or XLP. The study duration will be 
12 months. 
4.1 Inclusion Criteria 
1. Enrollment in the Longitudinal Study of the Porphyrias (7201)  
2. Male or female age ≥18 years 
3. Willing to sign informed consent form and follow protocol 
4. History of nonblistering cutaneous photosensitivity 
5. Biochemical findings - A marked increase in erythrocyte protoporphyrin (total 
erythrocyte protoporphyrin >400 ug/dL) with a predominance of metal-free 
protoporphyrin 
6. Serum ferritin ≤30 ng/mL at baseline 
7. Able to tolerate oral iron 

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4.2 Exclusion Criteria 
1. History of liver or bone marrow transplant or clinically significant liver 
dysfunction as determined by the investigator 
2. Known or suspected allergy to oral iron based on patient report 
3. Clinical evidence of active and ongoing GI bleeding 
4. Use of any other clinical or experimental therapy in the past 3 months 
5. Individuals with elevations of porphyrins in plasma or erythrocytes due to 
other diseases (i.e. secondary porphyrinemia) such as liver and bone 
marrow diseases 
6. Pregnancy 
7. Patients with any clinically significant comorbid conditions, which in the 
opinion of the investigator, precludes participation 
4.3 Recruitment of Participants 
The methods of recruitment will be: 
 
A. Patients followed by any of the Investigators in the Porphyrias Consortium.  
Each of the Investigators is a Porphyria specialist in a Porphyria Center, and 
provides clinical care to individuals suspected of and diagnosed with porphyria, 
providing diagnostic and follow-up evaluations, consultations, and/or treatment.  
For such patients who are likely to be eligible for the study, the Investigators will 
either discuss the study with the patient during a clinical visit or will contact the 
patient by telephone, email, or mail, as appropriate per site-specific IRB 
approval.  Any efforts to contact and recruit patients and families will be with 
IRB approval and adhere to standards for ethical conduct of research and be 
fully HIPAA compliant.   
B. Referrals from the American Porphyria Foundation (APF) to the Porphyrias 
Consortium centers.  The APF is an advocacy group that provides education 
about porphyria to patients, their families, healthcare professionals, and the 
public and supports porphyria research.  Their outreach program includes a 
website, as well as periodic newsletters and special announcements emailed 
and/or mailed to individuals who have registered with the APF with information 
about new developments in treatment, porphyria-related education 
opportunities, and research studies available through the Porphyria 
Consortium. Information about research studies includes contact information for 
the studies if they are interested in obtaining additional information or 
participating.  
C. Patients contacted through the Rare Diseases Clinical Research Network 
(RDCRN) Contact Registry. Individuals can register on-line or by faxing or 
mailing the registration form expressing an interest in receiving information 
about research studies for which they might qualify. The Contact Registry is 
managed by the Data Management Coordinating Center (DMCC) of the 
RDCRN.  The DMCC can refer them directly to the Porphyria Consortium for 
contact by their closest clinical site.  The DMCC sends information by email 

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about research studies along with contact information permitting registrants to 
obtain additional information about participating.   
D. Non-study Physician referrals.  Physicians providing clinical care to individuals 
suspected of or diagnosed with EPP who are not investigators in this study may 
refer patients who may be eligible for and express interest in the study to a 
Porphyrias Consortium center.  
E. Self-referrals, including family members of individuals diagnosed with EPP 
(proband) and other individuals who may have heard about the study from 
other subjects or prospective subjects.  In the case of family members, initial 
contact with the family member will not be made by the study team.  The 
proband will be asked to contact such family members, requesting that they 
contact the study coordinator or to register through the RDCRN-Porphyria 
Consortium if interested in obtaining additional information or participating.  
 
In the case of patients followed by one of the Consortium Investigators, the 
physician/Investigator or study coordinator will either: (a) contact the patient by 
telephone, email or mail informing him/her of the study and requesting that he/she 
contact the study coordinator if interested in obtaining additional information or 
participating, or; (b) discuss the study in-person during a clinical visit; informed consent 
may be obtained at this time. In all other cases, prospective subjects are instructed to 
contact the study coordinator, either the Project Manager or a site-specific study 
coordinator, by telephone, email or mail if interested in obtaining additional information 
or participating.  
 
4.4 Retention Strategies 
EPP and XLP are rare diseases.  Only a small number of specialists have the expertise 
to evaluate and treat affected individuals. Clinical relationships established by the 
investigators with patients who enroll in this pilot study will provide a context for 
increased communication with patients and their primary physicians that can facilitate 
retention. Furthermore, this study will provide a number of beneficial services to the 
participants and their primary physicians that will enhance retention. These include 
periodic evaluations, repeat laboratory testing, and information on new research 
developments in the field. Coordinators and other personnel at each site will also work 
to maintain close contact with participants, including regular clinic visits, newsletters and 
communication by phone, email and web sites. Counseling services will be provided so 
that patients and families are made aware of the results of studies and their correct 
interpretation. There will be no charge for the physician services or laboratory tests 
done as part of this pilot study. 
4.5 Data Elements 
The following laboratory tests and procedures will be used as the primary data 
elements: 
• Erythrocyte protoporphyrin 
• Plasma total porphyrins 
• A complete blood count with differential  
• Iron studies (serum iron, TIBC, Transferrin saturation) 

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• Serum ferritin 
• Hepatic function panel (serum ALT, AST, alkaline phosphatase, total and direct 
bilirubin, albumin, total protein) 
• EPP-specific Quality of Life Questionnaire 
 
These will be completed at each study visit, and an adverse event review will be 
completed bi-weekly.  
 
4.6 Study Procedures 
Baseline/Screening Visit: 
• Patients will be admitted to the Clinical Research Unit or a comparable facility for 
~2 hours. All patients will be enrolled in the Longitudinal Study and the diagnosis 
of EPP or XLP and inclusion/exclusion criteria will be reviewed prior to baseline 
visit.  
• A complete history and physical exam will be performed  
• All concomitant medications will be recorded- if the subject is currently taking an 
iron supplement they will be instructed to discontinue it for at least 4 weeks and 
return for re-screening. 
• Subjects taking Lumitene at screening will be asked to discontinue it for at least 4 
weeks and return for re-screening. 
• Patients will be asked to complete an EPP-specific quality of life questionnaire 
• Laboratory studies will include: 
o A complete blood count with differential 
o Serum iron 
o TIBC 
o Serum ferritin 
o Transferrin saturation 
o Hepatic function panel 
o Erythrocyte protoporphyrin levels with metal-free and zinc ratio 
o Plasma total porphyrins 
• Two yellow top ACD tubes will be collected and banked for future RNA and 
protein studies in this patient population 
• Subjects will be instructed to take ferrous sulfate 325 mg BID with water starting 
the next day on an empty stomach following discharge. 
Subjects will be discharged with a 3 month supply of medication with some 
surplus to account for dropped pills.  
 
The first dose of oral ferrous sulfate (two 325 mg tablets) will be administered within 30 
days of the screening visit. 
 
For patients with a borderline ferritin level, re-screening will be allowed within 60 days of 
the original screening date. Only ferritin and erythrocyte protoporphyrin levels should be 
done at the re-screening visit. If the ferritin level is ≤30 ng/mL on re-screening, the first 
dose of oral ferrous sulfate will be administered within 30 days of the re-screening visit.   
 

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Follow Up Phone Calls: 
• Coordinators will call the subject once a month (+/- 7 days) to review adverse 
events and concomitant medications.  
 
3 Month, 6 Month, and 9 Month Study Visits (+/- 7 days):  
• A review of systems will be performed, a physical exam will only be performed at 
the 6 Month visit  
• Adverse events will be reviewed 
• All concomitant medication changes will be recorded 
• Patients will be asked to complete an EPP-specific quality of life questionnaire 
• Laboratory studies will include: 
o A complete blood count with differential 
o Serum iron 
o TIBC 
o Serum ferritin 
o Transferrin saturation 
o Hepatic function panel 
o Erythrocyte protoporphyrin levels with metal-free and zinc ratio 
o Plasma total porphyrins 
• Two yellow top 10mL ACD tubes will be collected and banked for future RNA and 
protein studies in this patient population (at 6 month visit only) 
• Pill counts will be performed and recorded to monitor compliance 
• Old medication bottles will be collected  
• Subjects will be discharged with a 3 month supply of medication with some 
surplus to account for dropped pills. 
 
 
12 Month Study Visit (Or Early Termination Visit) (+/- 7 days):  
• Patients will be admitted to the Clinical Research Unit or a comparable facility for 
~2 hours  
• A review of systems and physical exam will be performed 
• Adverse events will be reviewed  
• All concomitant medication changes will be recorded 
• Patients will be asked to complete an EPP-specific quality of life questionnaire 
• Laboratory studies will include: 
o A complete blood count 
o Serum iron 
o TIBC 
o Serum ferritin 
o Transferrin saturation 
o Hepatic function panel 
o Erythrocyte protoporphyrin levels with metal-free and zinc ratio 
o Plasma total porphyrins 
• Two yellow top 10mL ACD tubes will be collected and banked for future RNA and 
protein studies in this patient population 

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• A single dose of oral ferrous sulfate (two 325 mg tablets) will be administered 
with water 
• Old medication bottles will be collected and pills will be counted for compliance  

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4.7 Schedule of Events 
 
Screeni
ng 
Da
y 0 
1 Month 
2 Month 
(via 
telepho
ne) 
3 
Mont
h 
4 Month 
5 Month 
(via 
telepho
ne)  
6 
Mont
h 
7 Month 
8 Month 
(via 
telepho
ne) 
9 
Mont
h 
10 
Month 
11 
Month 
(via 
telepho
ne) 
12 
Mont
h 
 
(Day -
30 to -1) 
 
(+ 7 
Days) 
(+ 7 
Days
) 
(+ 7 
Days) 
(+ 7 
Days
) 
(+ 7 
Days) 
(+ 7 
Days
) 
(+ 7 
Days) 
(+ 7 
Days
) 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
Inclusion/Exclu
sion Criteria 
Review 
X 
 
 
 
 
 
 
 
 
 
Labs (CBC, 
Iron, TIBC, 
Ferritin, 
Transferrin 
Sat, HFP) 
X 
 
 
X 
 
X 
 
X 
 
X 
Erythrocyte 
protoporphyrin 
levels & plasma 
porphyrins  
X 
 
 
X 
 
X 
 
X 
 
X 
2 Yellow top 
ACD tubes for 
banked samples 
X 
 
 
 
 
X 
 
 
 
X 
Urine/serum 
Pregnancy 
X 
 
 
 
 
X 
 
 
 
X 
Medical History 
review 
X 
 
 
 
 
 
 
 
 
 
Review of 
Systems 
X 
 
 
 
 
X 
 
 
 
X 
Physical Exam 
X 
 
 
 
 
X 
 
 
 
X 
Vital Signs 
X 
 
 
X 
 
X 
 
X 
 
X 
EPP QoL 
Questionnaire 
X 
 
 
X 
 
X 
 
X 
 
X 
Provide 3 
Month Supply 
of Medication 
 
X* 
 
X 
 
X 
 
X 
 
 
Pill Count 
 
 
 
X 
 
X 
 
X 
 
X 
Con Med 
Review 
X 
 
 
X 
 
X 
 
X 
 
X 
AE Review 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Review 
Compliance 
 
 
X 
 
X 
 
X 
 
X 
 

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4.8 Investigational Product 
All study drug will be stored and dispensed from individual centers’ research 
pharmacies. All study drug will be provided to each site Par Pharmaceuticals directly. 
 
Obtaining Study Drug: Each site is provided with an initial years’ supply of study drug for 
five patients.  
 
Dispensing, Dosage, and Accountability: All participants will receive treatment for a total 
of one year. Study drug will be dispensed with 3 month supplies. It is recommended that 
2 weeks of additional drug is provided with each 3 month supply. 
 
The dosage is 325 mg taken twice daily for a total of 650 mg daily. Tablets should not 
be broken or chewed. 
 
At each study visit the participant will bring all study drug with them. The site coordinator 
will count and reconcile all study medication at each visit. At each visit (3, 6, 9 and 12 
months) the subject will return all previous study drug and empty bottles and receive a 
new 3 month supply. At the final visit (12 months) a new supply will not be issued. 
 
Study Drug Destruction: After proper documentation, medication returned by subjects 
can be destroyed onsite per institutional SOP. 
 
If sites require additional study drug contact the PC Project Manager at least 2 months 
before it is needed on site. 
5.0 Data and Safety Monitoring Plan 
The study protocol will be reviewed and approved by the National Institutes of Health 
(NIH) and the Data Safety Monitoring Board (DSMB) before submission to individual 
center IRBs for approval.  Participant enrollment may only begin with IRB approved 
consent forms.  
 
This is an interventional pilot study that meets the federal definition of moderate risk. 
5.1 Study Oversight 
The Study Chair has primary oversight responsibility of this clinical trial. The NIH 
appointed Data Safety Monitoring Board (DSMB) has oversight responsibility of the 
Data Safety Monitoring Plan (DSMP) for this clinical trial. The DSMB will review accrual, 
patterns and frequencies of all adverse events, and protocol compliance every 6 
months. The DSMB makes recommendations to the NIH regarding the continuation 
status of the protocol. 
 
Each site’s Principal Investigator and their research team (co-Investigators, research 
nurses, clinical trial coordinators, and data managers) are responsible for identifying 
adverse events. Aggregate report- detailed by severity, attribution (expected or 
unexpected), and relationship to the study drug/study procedures – will be available 
from the DMCC for site review.  Adverse events will be reviewed at each study visit by 

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the research team.   A separate report detailing protocol compliance will also be 
available from the DMCC for site review on a monthly basis. The research team will 
then evaluate whether the protocol or informed consent document requires revision 
based on the reports. 
 
5.2 Definitions and Standards 
The Rare Diseases Clinical Research Network defines an adverse event as:  “…an 
unfavorable and unintended sign, symptom or disease associated with a participant’s 
participation in a Rare Diseases Clinical Research Network study.” 
 
Serious adverse events include those events that:  “result in death; are life-threatening; 
require inpatient hospitalization or prolongation of existing hospitalization; create 
persistent or significant disability/incapacity, or a congenital anomaly/birth defects.”  
 
An unexpected adverse event is defined as any adverse experience…the specificity or 
severity of which is not consistent with the risks of information described in the protocol.  
 
Expected adverse events are those that are identified in the research protocol as having 
been previously associated with or having the potential to arise as a consequence of 
participation in the study 
 
All reported adverse events will be classified by the site investigator where the AE 
occurred using the current version of the Common Terminology Criteria for Adverse 
Events (CTCAE) developed and maintained by CTEP at National Cancer Institute. 
 
As this study is considered an interventional trial, all adverse events, including those not 
directly related to study participation, will be reported as specified above. 
 
5.3 Expected/Known Risks/Discomforts/Adverse Events Associated with Study 
Intervention and Procedures: Definition of Expected Adverse Events 
Study Drug/Intervention: 
Drug Information for Ferrous sulfate USP 
 
Dosing: Adults- 325 mg capsule administered twice daily (total 650 mg daily)  
 
Human Toxicity/ Adverse Events (adapted from 
http://www.drugs.com/ferrous_sulfate.html): GI irritation, constipation, diarrhea, nausea, 
vomiting, and dark stools. In addition, as the effects of oral iron are unknown, patients 
may develop an increase in erythrocyte or plasma protoporphyrin levels and worsening 
clinical symptoms. 
 
Study Procedures:  
Physical Exam: A complete physical will be conducted every 6 months 
 

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Venipuncture:  The vein in which the needle has been inserted to draw blood may 
become sore and red.  A temporary bruise may develop, and rarely fainting may occur. 
The amount of blood, 24mL (~2 tablespoons) required for the tests described in this 
study is within the guidelines for blood collection for patients with porphyria. 
 
Urine Collection: A sample of urine will be collected. This procedure does not expose 
the patient to any risk. 
 
Safety Endpoints: The primary safety endpoints will be an increase in reported clinical 
symptoms or a 35% increase in erythrocyte protoporphyrin levels. If an increase of 35% 
over baseline erythrocyte protoporphyrin levels is observed this test will be repeated 
after 2-4 weeks without discontinuing iron. If the level remains elevated >35% on repeat 
testing then the study drug will be discontinued. If clinical symptoms increase to the 
point where the patient or treating physician is not comfortable continuing then the study 
drug should be discontinued. The secondary safety endpoints will be any increase in 
plasma porphyrins and side effects of oral iron therapy such as GI irritation, 
constipation, diarrhea, nausea, vomiting, and dark stools. If these secondary safety 
endpoints occur then discontinuing the oral iron will be at the discretion of the treating 
physician. 
 
5.4 Reporting Timeline 
• Within 24 hours (of learning of the event), investigators must report any reportable 
Serious Adverse Event (SAE) that: 
o 
Is considered life-threatening/disabling or results in death of subject 
-OR- 
o 
Is Unexpected/Unanticipated  
• Investigators must report all other reportable SAEs within 5 working days (of learning 
of the event). 
• All other (suspected) reportable AEs must be reported to the RDCRN within 20 
working days of the notification of the event or of the site becoming aware of the 
event. 
 
Local institutional reporting requirements to IRBs, any GCRC oversight committee and 
the FDA, if appropriate, remain the responsibility of the treating physician and the Study 
Chair. 
 
5.5 RDCRN Adverse Event Data Management System (AEDAMS) 
Upon entry of a serious adverse event, the DMCC created Adverse Event Data 
Management System (AEDAMS) will immediately notify the Study Chair, site PIs, the 
Medical Review Officer, and any additional agencies (if applicable- industry sponsor,) of 
any reported adverse events via email.  
 
Serious adverse events: The NIH appointed Medical Review Officer (MRO) reviews the 
site investigator’s report and determines causality of the adverse event. The MRO may 
request further information if necessary and possibly request changes to the protocol or 

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consent form as a consequence of the adverse event. A back-up notification system is 
in place so that any delays in review by the MRO beyond a specified period of time are 
forwarded to a secondary reviewer. Any follow up reports or requested additional 
participant data will be entered into the AEDAMS system by the reporting site and 
reviewed by the MRO. Completed AE reviews by the MRO will sent to Study Chair, site 
PIs, and the appointed NIH officers.  
 
If warranted, the MRO may request an ad hoc call with the DSMB to review the adverse 
event. All reported AE’s will be reviewed during the regularly scheduled DSMB call. 
 
The Adverse Event Data Management System (AEDAMS) maintains audit trails and 
stores data (and data updated) and communication related to any adverse event in the 
study.  
 
Non-serious expected adverse events: Except those listed above as immediately 
reportable, non-serious expected adverse events  that are reported to or observed by 
the investigator or a member of his/her research team will be submitted to the DMCC in 
a timely fashion (within 20 working days). The events will be presented in tabular form 
and given to the MRO and RDCRN DSMB on a bi-annual basis. Local site investigators 
are also required to fulfill all reporting requirements of their local institutions. 
 
The DMCC will post aggregate reports of all reported adverse events for site 
investigators and IRBs. 
5.6 Study Discontinuation  
The NIH, RDCRN DSMB and local IRBs (at their local site) have the authority to stop or 
suspend this trial at any time. This study may be suspended or closed if: 
• Early stopping rules have been met (see section 5.3) 
• The study objectives have been met 
• The Study Chair / Study Investigators believe it is not safe for the study to 
continue 
• The RDCRN DSMB suspends or closes the trial 
• The NIH suspends or closes the trial 
• The FDA suspends or closes the trial 
 
5.7 Subject Discontinuation 
An intent to treat approach will be used. If participants are unable to tolerate the 
study drug, the study drug will be stopped, but they will be followed at regular 
visits until the end of the trial unless they withdraw consent. All data acquired 
prior to termination for the reasons outlined below will be included in the primary 
analysis. If participants withdraw from the trial, every effort will be made to 
conduct a final study visit with the participant and participants will be followed 
clinically until, if applicable, all adverse events resolve. Reasons for withdrawal 
from the trial may include: 
 

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• Withdrawal of consent by the participant 
• Withdrawal by the investigator 
• Intercurrent illness or event that precludes further visits to the study site or ability 
to evaluate disease (e.g. mental status change, large pleural effusion).  
•  
 
5.8 Data Quality and Monitoring Measures 
As much as possible data quality is assessed at the data entry point using intelligent on-
line data entry via visual basic designed screen forms. Data element constraints, 
whether independent range and/or format limitations or ‘relative’ referential integrity 
limitations, can be enforced by all methods employed for data input. QA reports assess 
data quality post-data entry. As we note, data quality begins with the design of the data 
collection forms and procedures and incorporates reasonable checks to minimize 
transcription and omission errors.  Of the more important quality assurance measures 
are the internal validity checks for reasonableness and consistency.  
 
• Data Monitoring: The RDCRN DMCC identifies missing or unclear data and 
generates a data query to the consortium administrator contact.  
• Data Delinquency Tracking: The Data Management and Coordinating Center will 
monitor data delinquency on an ongoing basis.   
 
5.9 Quality Control: Study Related Procedures 
The standard laboratory measures (CBC, CMP, Iron panel, etc) will be performed at 
each sites’ central laboratory. Erythrocyte protoporphyrin measurements will be 
performed in the CLIA certified laboratory of Dr. Karl Anderson, University of Texas 
Medical Branch, Galveston, TX. 
 
The investigational pharmacy at each site will be responsible for drug accountability 
while the investigators/coordinators will be responsible for compliance of the subjects. 
The Investigational Drug Service at the Icahn School of Medicine at Mount Sinai will 
serve as the coordinating pharmacy for this study.   
6. Statistical Considerations 
The primary outcome is the relative difference in erythrocyte protoporphyrin levels 
between baseline and 12 months after treatment. A Bayesian approach based on the 
posterior distribution that the mean relative difference between baseline and 12-month 
erythrocyte protoporphyrin levels is greater than 0 will be used to assess the strength of 
potential efficacy of oral iron therapy. 
 
The procedure will be carried out as follows. Let X0 and X12 be the observed mean 
erythrocyte protoporphyrin level at baseline and 12 months.  Let X0 and X12 be 
correlated with a Pearson correlation coefficient of r and let d = 
X0−X12
X0
 and Ө = = 
µ0−µ12
µ0
  .   
Based on data observed in previous studies, we assume that the distribution of 

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erythrocyte protoporphyrin follows a gamma distribution   (see background data section 
below).  For an individual i, X0i ~ gamma (α0,β0) and X12i ~ gamma (α12,β12).   
 
X0 = 
∑
(X0i)
n
i=1
n
, therefore 
1
n X0 ~ n ∗gamma (α0, β0) = gamma (nα0, β0) and likewise 
1
n X12~ gamma (nα12, β12). 
 
Two correlated gamma distributions can be generated using a single parameter α, the 
means of each distribution and the standard deviation as detailed in Ronning et al (23).  
We specify a non-informative normal(n*0, 0.01) prior for α.  After data collection the 
posterior distribution of α|d ~ normal(n*
X0
σ02/X0 , 0.01). The posterior distribution for Ө|d 
can be generated by simulating baseline and 12 month mean values using the posterior 
distribution of α|d, the observed correlation between 12 month and baseline values and 
the observed standard deviations.  After generating the posterior distribution for Ө|d we 
will be able to compute the probability that Ө|d > 0.   
 
Prior to any data collection, equipoise implies that the probability that iron therapy will 
improve erythrocyte protoporphyrin levels (i.e. the relative difference between baseline 
and 12 months will be greater than 0) is 50%.  An increase in this probability to a level 
providing reasonable assurance of an efficacy signal (e.g. 80%) would warrant 
additional study of iron. A probability of 80% corresponds to an approximate 
quadrupling of the odds that iron is effective in lowering erythrocyte protoporphyrin (i.e., 
from 50% probability of irons efficacy, or 1:1 odds, to 80% or 4:1 odds). 
 
Sample size justification: 
Assuming the distribution of baseline erythrocyte protoporphyrin is gamma (2.3, 861) 
and that baseline and 12 month values are correlated with r = 0.7, simulations indicate 
that a total sample size of 20 patients will allow us to detect an approximate quadrupling 
of the odds that iron is effective in lowering erythrocyte protoporphyrin if the true effect 
size is 10%.  Table 2 lists simulated estimates (100,000 runs for each) of the probability 
that Ө|d > 0 across several sample size and effect size scenarios. 
 
Table 2:  Simulation results  
 
Effect Size 
r 
Sample 
Size 
0% 
5% 
10% 
15% 
20% 
.7 
10 
0. 50 
0.63 
0.74 
0.83 
0.90 
15 
0. 50 
0.65 
0.78 
0.88 
0.94 
20 
0. 50 
0.67 
0.81 
0.91 
0.96 
25 
0. 50 
0.69 
0.84 
0.93 
0.97 
30 
0. 50 
0.71 
0.86 
0.95 
0.98 
35 
0. 50 
0.72 
0.88 
0.96 
0.99 
40 
0. 50 
0.74 
0.89 
0.97 
0.99 
 
Background Data 
 

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As of March 17, 2015 there were 152 EPP and XLP patients in The Longitudinal Study 
of the Porphyrias who had an erythrocyte protoporphyrin level >400, were not on Iron 
therapy and had no reported liver disease (fibrosis, cirrhosis, steatosis) or liver 
transplant history. The distribution of these patients’ baseline erythrocyte protoporphyrin 
is pictured in figure 2. 
 
 
 
Data from The Longitudinal Study of the Porphyrias demonstrated that the correlation 
between erythrocyte protoporphyrin levels taken approximately 1 year apart was 0.74. 
 
Secondary Analyses: 
In addition to the primary outcome, the following secondary endpoints will be analyzed:  
 
1) Erythrocyte protoporphyrin over time: Mean values and standard deviations will 
be computed at each time point and values will be compared descriptively.  
Additionally, a random effects longitudinal model of erythrocyte protoporphyrin 
with time as the predictor variable will be fit.  Time will be treated as a continuous 
measure unless diagnostic plots suggest that a linear mean function for time is 
not the correct specification.  If this is the case, time (ie visit timepoint) will be 
treated as a categorical variable.  Using the fitted model, we will test the null 
hypothesis that the beta coefficient(s) for time = 0.  There have been no previous 
studies on the effect of iron therapy on erythrocyte protoporphyrin over time.  As 
such, no estimates of between-subject variability or effect size are availble to pre-
specify the power of this analysis.   
 
Figure 
2: 
Distribution 
of 
152 

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2) EPP-specific QOL overtime: To assess whether the subjects’ EPP-specific QOL 
changed during the duration of the study, the mean score and standard deviation 
will be computed at each time point and compared descriptively. 
 
3) Safety Endpoints: Rates of Adverse events will be computed. Safety variables 
such as physical and other laboratory findings will be analyzed descriptively.   
7. Data Management 
As much as possible, data quality is assessed at the data entry point using intelligent 
on-line forms via the DMCC. Data element constraints, whether independent range 
and/or format limitations or ‘relative’ referential integrity limitations, can be enforced by 
all methods employed for data input.QA reports assess data quality post-data entry. As 
we note, data quality begins with the design of the data collection forms and procedures 
and incorporates reasonable checks to minimize transcription and omission errors.  Of 
the more important quality assurance measures are the internal validity checks for 
reasonableness and consistency.  In addition to those described above, we propose to 
build these checks into the initial tables and cross tabulations that should reveal any 
remaining data quality issues. 
 
All study data will be collected via systems created in collaboration with the RDCRN 
Data Management and Coordinating Center and will comply with all applicable 
guidelines regarding patient confidentiality and data integrity. 
 
7.1 Registration 
Registration of participants on this protocol will employ an interactive data system in 
which the clinical site will attest to the participant’s eligibility as per protocol criteria and 
obtain appropriate informed consent. IRB approval for the protocol must be on file at the 
DMCC before accrual can occur from the clinical site. 
 
The DMCC will use a system of coded identifiers to protect participant confidentiality 
and safety.  Each participant enrolled will be assigned a local identifier by the enrollment 
site.  This number can be a combination of the site identifier (location code) and a serial 
accession number.  Only the registering site will have access to the linkage between 
this number and the personal identifier of the subject.  When the participant is registered 
to participate in the study, using the DMCC provided web-based registration system; the 
system will assign a participant ID number.  Thus each participant will have two codes: 
the local one that can be used by the registering site to obtain personal identifiers and a 
second code assigned by the DMCC. For all data transfers to the DMCC both numbers 
will be required to uniquely identify the subject.  In this fashion, it is possible to protect 
against data keying errors, digit transposition or other mistakes when identifying a 
participant for data entry since the numbers should match to properly identify the 
participant.  In this fashion, no personal identifiers would be accessible to the DMCC.  
 

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7.2 Data Entry 
Data collection for this study will be accomplished with paper source documentation and 
online electronic case report forms.  Using encrypted communication links, on-line forms 
will be developed that contain the requisite data fields.   
8. Human Subjects 
8.1. GCP Statement 
 
This clinical trial will be conducted in accordance with the ethical principles that have 
their origin in the Declaration of Helsinki, and that are consistent with Good Clinical 
Practice and all applicable regulatory requirements. 
8.2. Risks  
The potential risks of this study are: 
 
1) Side effects of Oral ferrous sulfate: GI irritation, constipation, diarrhea, nausea, 
vomiting, and dark stools. In addition, as the effects of oral iron are unknown, 
patients may develop an increase in erythrocyte or plasma protoporphyrin levels 
and worsening clinical symptoms. 
 
2) Blood Draw: Needle insertion can cause pain and bruising at the site of insertion. 
Rarely fainting caused by a vasovagal response to the phlebotomy procedure 
may occur. Because the skin is penetrated by the needle, there is a low risk for 
infection at the venipuncture site. The amount of blood needed for each study 
visit (8mL tubes) is within approved safety limits. For any participant who has a 
medical condition that restricts their capacity to provide adequate sample 
volume, the number of tubes of blood obtained will be modified on an individual 
basis. 
 
3) Loss of confidentiality: The risk of loss of confidentiality is low because as there 
are many safe guards in place. Conceivably, data including clinical and 
diagnostic information could be accidentally divulged. If such an event occurred, 
subjects might be at risk for discrimination by life or health insurance companies, 
by employers, and by adoption agencies. The Genetic Information Non-
Discrimination Act (GINA) is a federal law that protects Americans from being 
treated unfairly because of differences in their DNA that may affect their health. 
This law prohibits genetic discrimination in the workplace and by health insurers. 
However, it provides no such protection for life insurance and disability 
insurance. 
8.3. Benefits  
Participants may derive no direct benefit from being in this study. The potential benefits 
of this study are lowering the levels of erythrocyte protoporphyrins. Lower 
protoporphyrin levels may result in some improvement of sun sensitivity symptoms. 

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8.4. Recruitment  
Participants with EPP or XLP 18 years of age or older, including both genders and all 
ethnic groups can be enrolled in this interventional study should they meet 
inclusion/exclusion criteria. Participants potentially eligible will include 1) those known to 
the investigators who can be contacted and informed of the study; 2) participants who 
hear about the study through the American Porphyria Foundation, their physicians or 
other sources and contact one of the centers to express interest in the study; and 3) 
participants who contact the Data Management and Coordinating Center (DMCC) of the 
Rare Disease Clinical Research Network (RDCRN) and join the Contract Registry at the 
DMCC website  .   
 
Because XLP is an X-linked disorder, it does not affect both genders equally, but overall 
we expect to enroll an equal number of males and females. The cutaneous porphyrias, 
including the erythropoietic protoporphyrias, may be less manifest in African Americans 
due to protective skin pigmentation. Furthermore, the erythropoietic protoporphyrias are 
uncommon in Africa and in African-Americans because the low-expression allele 
associated with the disorder in >90% of EPP is rare in these groups.   
Inclusion of vulnerable groups: Children and pregnant women are excluded from 
participating in this interventional study. In addition subjects who are not able to give 
informed consent will be excluded.  
8.5. Written Informed Consent: 
Written informed consent will be obtained from each participant before any study-
specific procedures or assessments are done and after the aims, methods, anticipated 
benefits, and potential hazards are explained.  The participant’s willingness to 
participate in the study will be documented in writing in a consent form, which will be 
signed by the participant with the date of that signature indicated.  The investigator will 
keep the original consent forms and signed copies will be given to the participants.  It 
will also be explained to the participants that they are free to refuse entry into the study 
and free to withdraw from the study at any time without prejudice to future treatment.  
Written and/or oral information about the study in a language understandable by the 
participant will be given to all participants. 
 
8.6. Process of Consent:   
At each of the Porphryias Consortium sites the principal investigator and/or study 
coordinator will identify subjects with EPP/XLP who are enrolled in the Longitudinal 
Study of the Porphyrias and eligible for this study. The PI and/or coordinator will then 
approach the subject, either via a phone call or at a clinic visit, to fully describe the 
intent of the study and what the potential risks and benefits of participation in this study 
will be. The subject will have an opportunity to review the consent form and ask any 
questions of the study team, as well as discuss the study with their primary care 
physician or others of their choosing, prior to signing the consent form. 
 

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8.7. Certificate of Confidentiality 
To help protect participant privacy, a Letter of Confidentiality has been obtained from 
the National Institutes of Health (NIH) for the Longitudinal Study of the Porphyrias.  With 
this Certificate, the researchers cannot be forced to disclose information that may 
identify a study participant, even by a court subpoena, in any federal, state, or local civil, 
criminal, administrative, legislative, or other proceedings.  The researchers will use the 
Certificate to resist any demands for information that would identify a participant, except 
as explained below. 
 
The Certificate cannot be used to resist a demand for information from personnel of the 
United States Government that is used for auditing or evaluation of federally funded 
projects or for information that must be disclosed in order to meet the requirements of 
the federal Food and Drug Administration (FDA). 
 
Even with the Certificate of Confidentiality, the investigators continue to have ethical 
and legal obligations to report child abuse or neglect and to prevent an individual from 
carrying out any threats to do serious harm to themselves or others.  If keeping 
information private would immediately put the study participant or someone else in 
danger, the investigators would release information to protect the participant or another 
person. 
 
Department of Health and Human Services (DHHS) personnel may request identifying 
information for purposes of performing audits, carrying out investigations of DHHS grant 
recipients, or evaluating DHHS funded research projects. 
 
 
 

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