Effects of Broccoli Sprout Extract on Allergic Rhinitis
Phase: PHASE2
Conditions: Rhinitis,Allergic
Sponsor: VA Office of Research and Development

 
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Effects of Broccoli Sprout Extract on Allergic Rhinitis     
 
NCT02885025  
 
 
 
Study Protocol and Statistical Analysis Plan  
 
 
November 2, 2018  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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RESEARCH PLAN 
 
 
BACKGROUND AND SIGNIFICANCE 
 
Safe alternative nutritional supplementation is needed for atopic disorders 
suffered by US veterans:  
Allergic Rhinitis a major condition impairing nasal health, effects 10-30% worldwide and 8% 
nationwide (1). A 10 year retrospective review revealed the majority of patients receiving care at 
the VA Greater Los Angeles Healthcare System (VAGLAHS) suffered from Allergic Rhinitis (2). 
From suffering comes cost, both indirect and direct. This can manifest as lost days of work and 
medication expense. Among atopic disorders, completed surveys determined allergic rhinitis as 
the highest cost in regards to both presenteeism and absenteeism, with the average total 
productivity loss per employee surveyed at 593 dollars per year (3). Over 3.4 billion dollars is 
spent directly treating allergic rhinitis, with 46.6% attributed to prescription medications. Despite 
no cost effective treatment for allergic rhinitis, nasal corticosteroids appear to be the most 
beneficial.   Patients suffering from allergic rhinitis seek alternative therapies since 2/3rds report 
daily impairment from rhinitis symptoms despite the use of available medications (4). 
 
The Association between Pollutants and Allergy: 
The increase prevalence in allergic disorders may be attributable to pollutant factors (5). Bench 
Research led to a valid explanation of how pollutants can act as an adjuvant to allergens in 
susceptible patients. Original work discovered pollutant diesel exhaust particles (DEP) increase 
reactive oxygen species (ROS) in respiratory epithelium cells.  This overabundance of oxidative 
stress leads increases intracellular nuclear factor kappa beta (NfKB) and Mitogen Activated 
Protein Kinase (MAP-kinase) cascades causing atopic cytokine production (6-9).    
 
Animal Models investigating the association between Pollutants and Allergy: 
Mice models validated the association between pollutant exposure and allergic response 
increase. Intranasal administration of diesel exhaust particles (DEP) caused an enhanced IgE 
response (10). More specifically administering DEP to dust mite sensitized mice enhanced the 

 
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production of dust mite specific IgE (11). Further experiments on mice solidified the concept that 
pollutants cause an increase allergic response when mice given DEP plus allergen were later 
found to have increased levels of atopic cytokines IL4 and IL5 within lungs and lymph nodes 
(12, 13). 
 
Human Models investigating the association between Pollutants and Allergy: 
These findings were later replicated in human studies. Purified human B cells cultured with TH2 
cytokines and DEP significantly increased IgE production when compared to B cells cultured 
with cytokines alone (14).  Human studies demonstrated instillation of DEP through nasal 
lavage triggered an increase in nasal mucosa IgE production.  (15). Nasal instillation of both 
DEP and ragweed produced increased atopic cytokines IL4, 5,6,10 and 13 and ragweed IgE 50 
fold (16). Further review of these studies noted this increase in allergic biomarkers varied 
among individuals, with some having a drastic response and others no response (17).      
 
Phase II enzymes and Allergic Rhinitis: 
The exaggerated atopic response from DEP when present with allergen exposure can be 
prevented by antioxidant production. Genetic polymorphisms in phase 2 antioxidant enzymes 
among individuals can determine how pollutants influence atopy.  Endogenous anti-oxidant 
phase 2 enzymes GSTT1, GSTM1 and GSTP1 play a vital role in preventing the production of 
cytokines through restoration of redox balance between reduced and oxidized glutathione. Null 
genotypes for GSST1 and GSTM1 are more prevalent in atopic subjects (18), which could 
explain their diminished ability to produce phase II antioxidants leading to unchecked balance of 
oxidation. When exposed to pollutants, patients having null mutations in GST increase 
production of IgE and histamine levels respectively (19, 20).  Patients with GST null mutations 
suffering from allergic rhinitis may respond best to antioxidant therapy.   
  
The use of antioxidants in treating Allergic Conditions: 
Success rates in treating atopic conditions with antioxidants have been mixed (21-23). The 
importance of evaluating the patient for GST polymorphisms is supported by studies showing 
patients with GSTM1 null genotypes were more likely to have a greater detrimental loss of 
pulmonary function with exposure to ozone and a more positive response to consumption of 
Vitamin C/Vitamin E antioxidants supplementation (24, 25). 
 
Sulforaphane for treating allergic conditions: 
The antioxidant Sulforaphane increases phase II enzyme production through activation of the 
Nrf2 transcription factor and the anti-oxidant response element (ARE) leading to an increase 
production of phase II enzymes (26-30). Sulforaphane also interferes with the pro-inflammatory 
pathways NkKB and AP1 responsible for the production of TH2 cytokines and IgE.    
 
Sulforaphane has been shown to block B cell IgE production (31) and to inhibit IL8 production in 
human bronchial airway epithelial cell lines after exposure to DEP via inducing nuclear factor 
like 2, leading to enhanced Phase II enzyme production (32). A recent study showed that ova 
sensitized mice treated with SFN had a diminished inflammatory response after ova challenge 
leading to diminished hyper responsive airways (33). 
 
Broccoli Sprout for improving nasal health: 
Broccoli sprouts (BSE), members of the cruciferous vegetable family, have extremely high 
concentrations of sulforaphane (26). The first placebo controlled BSE human study performed at 
our partnering institution involved administering escalating doses of broccoli sprout homogenate 
(BSH) to subjects. Phase II enzymes were shown to increase proportionally to the dose of BSE 

 
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given. Furthermore the induction of individual Phase II enzymes was inversely correlated with 
the expression of phase II enzymes (glutathione-s-transferase M1, glutathione-s-transferase P1, 
NADPH quinone oxidoreductase, and hemoxygenase-1) as revealed by genetic testing. 
Patients with GSTM1 null or GSTP1 Ile/Ile105 polymorphisms known to endogenously produce 
the lowest Phase II enzyme levels responded best to BSE (34). 
 
PRELIMINARY STUDIES: 
 
Administering BSE to allergic subjects was first performed in a recent NIH sponsored study by 
Dr. Li and colleagues at the University of California, Los Angeles, a partnering institution to the 
VAGLAHS. Subjects with cat allergy through skin testing diagnosed allergic rhinitis were 
challenged with 300µg  DEP (equivalent to daily PM exposure levels on LA freeway) then 
underwent 4 weeks of washout, followed by daily BSE administered as a single oral morning 
dose (11umol sulforaphane equivalent to that formed from consuming 100 to 200 grams of 
broccoli) for four days. The diesel exhaust particle challenge was repeated after BSE 
consumption. The average nasal white blood cell counts increased by at least 4 fold (p<0.0005) 
within 24 hours post-DEP exposure and there was significant attenuation of cell count post-DEP 
exposure when patients were given daily BSE for four days prior to DEP challenge (p<0.01 at 6 
hours and p<0.0001 at 24 hours following DEP challenge). There was no correlation between 
the inflammatory response and the presence of GSTM1 null orGSTP1 Ile/Ile105 polymorphisms 
(35). Failure to determine a statistically significant difference in response among the genetic 
polymorphisms may have been due to the low subject number. 
 
We are proposing a double blinded randomized placebo controlled study to investigate the 
effectiveness of BSE on nasal health among US veterans with a diagnoses seasonal grass 
allergic rhinitis in an urban environment where DEP levels are high. 
 
During the study, GST genotype data will be collected in order to obtain data for future 
exploratory analysis.          
 
RESEARCH DESIGN AND METHODS 
 
Objectives: 
Primary Objective:   
To conduct a double-blind, randomized, placebo-controlled trial to compare clinical 
measurements for allergic rhinitis following Timothy, Bermuda or Johnson grass allergen 
(phleum pretense, cynodon dactylon, or songhum halepense) challenge before and after 
initiation of BSE supplementation. Clinical measurements will be derived from the total nasal 
symptoms score (TNSS) and peak nasal inspiratory flow (PNIF).   
 
Secondary Objectives: 
To conduct a randomized controlled trial to compare biomarker measurements for allergic 
rhinitis following grass allergen challenge before and after initiation of BSE supplementation. 
Biomarkers will include the following inflammatory cytokines: Interleukin 4, Interleukin 5, 
Interleukin 13 along with Tryptase and Eosinophil cationic protein (ECP) obtained through 
polyurethane sponges placed into both nostrils.   
 
To conduct an exploratory genetic analysis of the 3 glutathione S-transferase (GST) 
genes (GSTM1, GSTT1, and GSTP1) for future investigation into how genetic polymorphisms 
effect the response to BSE supplementation in atopic disorders.  

 
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Overview of Study Design 
The proposed study is a double-blind, randomized, placebo-controlled trial consisting of four 
arms. A total of 190 subjects diagnosed with grass (p. pretense, c. cynodon, and/or s songhum) 
specific allergic rhinitis will be randomly assigned to one of four groups: 
1. nasal corticosteroid + Placebo tablet  
2. placebo nasal spray + Placebo tablet 
3. nasal corticosteroid + Avmacol®  
4. placebo nasal spray + Avmacol®  
 
Avmacol is a supplement that supports sulforaphane production by providing glucoraphanin and 
an active myrosinase enzyme via our Sulforaphane Production System.  Subjects will be given 
4 tablets to take with their evening meal.   
 
Following consent, a list of antioxidant foods (including sulforaphane containing foods) and  
prohibited medications to avoid will be given to all subjects then subjects will be scheduled for 
visit 1 within <20 days.   
 
At visit 1, Subjects will undergo epicutaneous hypersensitivity skin testing to grass allergens 
prevalent in southern California: Johnson Grass (Sorghum Halepense), Bermuda Grass 
(Cynodon Dactylon), and Timothy Grass (Phleum Pretense), along with dog, cat and perennial 
allergens: dust mite (dermatophagoides pteronyssinus, dermatophagoides farina), and common 
mold allergens (alternaria, aspergillus, cladosporium, and penicillium) (36). Subjects with 
positive skin test results to one or more grass allergens and either negative skin test to 
perennial allergens (dust mite or mold) or positive tests to perennial allergens (dust mite or 
mold) without a correlating clinical history consistent with perennial allergic rhinitis  will undergo 
grass allergen nasal provocation testing. Subjects with a positive skin test to cat or dog and 
clinical symptoms consistent with cat or dog triggered allergic rhinitis will be asked to avoid cats 
and or dogs for the entire study. For subjects who have an epicutaneous allergen skin or blood 
test on file completed by the VA Allergy Clinic within <1 year that confirms a positive to grass 
pollens the results will be used to proceed onto the up-dosing nasal challenge. Subjects 
experiencing a positive nasal provocation test defined by a total nasal symptom score (TNSS) 
over 7, or PNIF is reduced by > 50% from baseline redaing will be tested for serum GSTM1 
polymorphism and baseline sulforaphane levels and will proceed to visit 2. Subjects failing to 
mount a positive response with the highest concentration (10,000 BU/cc for p. phleum and c. 
cynodon, or 1:100 w/v for s. sorghum) will be withdrawn from the study..     
 
Nasal challenge will repeat at visit 2, two weeks after visit 1, and visit 3, 3 weeks after beginning 
intervention at visit 2.  This challenge was developed and perfected by Guy W. Scadding PhD 
and colleagues and showed reproducibility in a 2012 study (36).  Prior to and following each 
challenge, subjects will undergo spirometry.  Challenges will not be performed in patients with 
forced expiratory volume in 1 second (FEV1) values less than 70%. The visit 1 nasal challenge 
will be considered a graded up-dosing challenge needed to determine the dose used at visits 2 
and 3.   
 
At visit 2 (2 weeks following visit 1) and visit 3 (5 weeks following visit 1), subjects will undergo a 
single nasal allergen challenge with the effective dose determined during the up-dosing 
challenge at visit 1. Total nasal symptom score and peak nasal inspiratory flow will be recorded 
throughout all nasal challenge procedures.   
  

 
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Biomarkers collected through sponges placed in both nasal passages during the challenge will 
include IL4, IL5, IL13, eosinophil cationic protein and tryptase. At the end of visit 2, patients will 
be randomized and will be instructed to begin the following evening, to return in 3 weeks for 
another nasal challenge.     
 
Throughout the study, serum sulforaphane levels for each subject will be checked at visits 1, 2 
and 3 to monitor adherence.   
 
All study procedures will be conducted at the VAGLAHS. Subjects will be assigned an 
appointment day and appointment time for each scheduled visit. All appointments will be at 
approximately the same time of the day for all subjects to avoid diurnal variation. 
 
Figure 1. Overview of Study Procedures 
 
Study Population 
General Considerations: 
Subjects participating in the study are followed at two VA medical centers and one academic 
medical center.  All subjects are non-smoking male or female US veterans aged 18 years and 
older with symptoms consistent with seasonal summer allergic rhinitis (rhinorrhea, nasal 
congestion, postnasal drainage sneezing, and/or nasal or eye pruritus). There will be no 
exclusion based on gender or ethnic/racial background.   
 
Inclusion Criteria 
1. 
Females and males 18 years or older.  
2. 
History consistent with seasonal allergic rhinitis consistent with grass allergy (symptoms               
during summer months, June through August, for at least two consecutive seasons).   
3. 
Not currently taking any medications for allergic rhinitis. 
4.         Provide written informed consent. 
5.  
Willing and able to comply with all aspects of the protocol.     
 
Exclusion Criteria 
1. 
The subject has any uncontrolled or serious disease, or any medical or surgical or 
condition that in the opinion of the investigator(s) could affect the subject’s safety and/or 
interfere with the study assessments. 
2. 
History of anaphylaxis to environmental allergens.or an unknown trigger.   
3. 
History of broccoli allergy. 
4. 
Recent upper respiratory infection (less than 4 weeks prior to study) or other active 
Infection. 

 
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5. 
Active smoker.   
6. 
Currently receiving allergy immunotherapy.  
7. 
History of rhinitis exacerbation within the past 2 weeks. 
8. 
Use of non-selective Beta-Blocker. 
9. 
Inability to give written informed consent. 
10. 
History or evidence of non-stable cognitive capacity within less than 1 year (i.e. 
Alzheimer’s disease, dementia, bipolar disorder) that in the opinion of the investigator(s) 
could affect the subject’s safety and/or interfere with the study assessments.    
11. 
Pregnancy. 
12.      Perennial rhinitis. 
13.     Uncontrolled asthma.   
14.     FEV1 (forced expiratory volume in 1 s) <70% predicted at screening.    
 
Recruitment: 
In order for 152 subjects to complete the study, 475 subjects will be recruited for enrollment 
under the assumption 50% (237) of subjects will have seasonal allergic rhinitis to grass 
confirmed on skin testing, 80% (190) of subjects will have a positive nasal challenge and 80% 
(152)  of subjects will complete the study. 
 
Subject recruitment will occur at three facilities:  VA Greater Los Angeles Health Care System 
(VA GLA) , VA Long Beach Health Care System (VA LB) and the University of California Los 
Angeles (UCLA) Medical Center.  Within each facility, recruitment will occur at primary and 
specialty (allergy) clinics.   
 
Recruitment at VA Greater Los Angeles and VA Long Beach Health Care Centers:   
Primary Care Clinic Recruitment:    
Investigators will work together with providers assigned to Patient Aligned Care Teams (PACT).  
On average, PACTs follow 44,000 patients at VA Long Beach and 63,000 at VA Greater Los 
Angeles.  Assuming involvement with primary care providers following 10,000 subjects at VA 
GLA and 10,000 subjects at VA LB, At each facility, 2,000 (20%) will should have a diagnoses 
of allergic rhinitis.  600 (30%) will experience allergic rhinitis symptoms during the summer 
months and 300 (50%) of these subjects will have symptoms limited to summer months.  We 
estimate at total of 600 potential recruitment subjects from primary care clinics at both VA GLA 
and VA LB.       
 
Allergy Clinic Recruitment: 
Within the past year, 40% (320) of new consultations at VA Greater Los Angeles Healthcare 
System and 68% (208) of new consultations at VA Long Beach Healthcare System were 
requested to evaluate allergic rhinitis. Based on these figures, we would anticipate a total of 
30% having allergic rhinitis associated with summer months and then 50% of these patients 
having allergic rhinitis limited to summer months or 79 (48 + 31) potential subjects at VA GLA 
and VA LB combined.  
 
Recruitment at the University of California Los Angeles Medical Center:   
In Southern California, patients are seen at 16 satellite primary care clinics affiliated with UCLA 
Medical Center.  Adult Allergists practice at four of these locations (Porter Ranch CA, Santa 
Monica CA, Thousand Oaks CA, Torrance CA.)  Investigators will work with providers at UCLA 
affiliated clinics in identifying potential subjects.  (see Contingency Plan)  
 
Use of Flyers at Institutions:  

 
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In addition to the above recruitment strategy, flyers will be placed in primary care, ear nose and 
throat, and dermatology clinics at VAGLAHS and VALBHCS with contact information for 
enrollment. Potential subjects who call will be preliminary screened via phone call screening 
script to determine possible eligibility. Those who pass the preliminary screening process will 
have an appointment scheduled. 
 
Recruitment from VA GLA Pharmacy Database: 
In addition, we will work with pharmacy to retrieve patients prescribed nasal corticosteroids 
limited to summer months.  These patients have a higher likelihood of suffering from grass 
pollen allergic rhinitis.      
 
Contingency Plan: 
Participation recruitment will be followed closely.  If needed, we will submit a request for 
expansion to involve non veterans.  The VA GLA is close to UCLA.  Students/staff/potential 
subjects attending UCLA may qualify for inclusion into the study.  
 
Medical History: 
A medical history will be obtained to confirm whether patient has clinical symptoms consistent 
with summer seasonal rhinitis (during grass season) allergic rhinitis. 
 
Procedures at Each Visit: 
 
Consenting (<20 days to Visit 1)  
-Affirm eligibility, check medication use. 
-Review study with patient and if subject agrees to enroll, obtain subjects written 
informedconsent. 
 -Instructions for avoidance of sulforaphane containing food products in diet. 
- Review concomitant medication use. 
-Instructions for avoidance of the following medications: oral antihistamines, nasal 
  corticosteroids, nasal mast cell stabilizers, nasal antihistamines, leukotriene antagonists. 
-Proceed onto visit 1, or schedule subject for visit 1 <20 days from providing written consent.   
 
Visit 1 (Day 1) 
 
-Spirometry performed (Note: subjects’ have up to 3 attempts to FEV1 of >70% to continue on 
with this study).  
-Collect blood pressure, heart rate, height and weight. 
- Check concomitant medication use since last visit.  
-General physical examination. 
-Nasal examination. 
-Epicutaneous Allergen Skin Testing. Patients who qualify based on skin test results will 
proceed  with up-dosing nasal challenge to the positive skin test grass allergen. Subjects that 
have completed epicutaneous allergen skin or blood test at the VA Allergy Service within < 1 
year with positive results to grass pollens will to proceed onto the up-dosing nasal challenge 
using the results on file.  
-Updosing nasal challenge. Patients with positive nasal challenge will continue as follows:   
-Obtain blood work to determine sulforaphane baseline level and genotype for  GSTM1 
polymorphisms. 
- Schedule subject for visit 2. 
 

 
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Visit 2 (Day 14) 
-Check concomitant medication use since last visit. 
-Collect blood pressure, heart rate and weight. 
-Spirometry performed 
-General physical examination. 
-Nasal  examination. 
-nasal lavage  
-Obtain blood work for sulforaphane level check. 
-Grass Nasal Challenge. 
-Randomize patients into one of four groups. 
-Medication Dispensing (to begin evening of Day 15).      
 
Visit 3 (Day 35) 
-Check concomitant medication use since last visit. 
- Collect medication dispensed at last visit (research team complete medication compliance).  
-Collect blood pressure, heart rate and weight. 
-Spirometry performed 
-General physical examination. 
-Nasal   examination. 
-Nasal lavage  
-Obtain blood work for sulforaphane level check.  
-Grass nasal Challenge.  
 
Study Schedule: 
 
The typical schedule is shown as an example for consenting.  
 
Consenting (<20 days prior to visit 1) Activitity (in sequence): 
 
Affirm eligibility, check medication use. 
 
Review study with patient and if subject agrees to enroll, obtain subjects written 
informedconsent. 
 
Instructions for avoidance of sulforaphane containing food products in diet prior to visit 1 and 
throughout the study. 
 
Instructions for avoidance of the following medications: oral antihistamines, nasal 
corticosteroids, nasal mast cell stabilizers, nasal antihistamines, leukotriene antagonists prior to 
visit 1 and throughout the study.  
 
Proceed onto visit 1, or schedule subject for visit 1 within < 20 days from providing written 
consent.   
 
The typical schedule is shown as an example for the enrollment visit (Visit 1).  
 
Visit 1 (Day 1) Activity (in sequence):  
 
Subject arrives at the Allergy/Immunology Clinic.  

 
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Spirometry will be performed before skin test. (Note: subjects’ have up to 3 attempts to FEV1 of 
>70% to continue on with this study.) 
 
Vital signs will be collected (heart rate, blood pressure, weight, height) and general physical 
examination along with nasal examination. 
 
Subjects undergo epicutaneous skin testing. (Subjects who qualify based on skin test will 
continue with nasal challenge). For subjects who have an epicutaneous allergen skin or blood 
test on file completed by the VA Allergy Clinic within < 1 year that confirms a positive to grass 
pollens the results will be used to proceed onto the up-dosing nasal challenge.   
 
Up-dosing nasal allergen challenge will be performed  
 
Subjects testing positive to nasal challenge will proceed with blood work to check sulforaphane 
Baseline levels and genotype for GSTM1 polymorphisms 
 
 
The typical schedule is shown as an example for a follow up evaluation day for visit 2.  
 
Visit 2 Activity (in sequence): 
 
Subject arrives at the Allergy/Immunology Clinic. 
                                                                 
Vital signs (heart rate, blood pressure and weight) , nasal examination , concomitant medication 
and diet checked since last visit.    
  
Spirometry will be performed. (Note: subjects’ have up to 3 attempts to FEV1 of >70% to 
continue on with this study.) 
 
general physical examination along with nasal examination. 
 
Nasal lavage performed.  
 
Blood work obtained for sulforaphane level    
 
Nasal allergen Challenge followed by monitoring explained below  
 
Randomization occurs.  Instructions for avoidance of  sulforaphane containing food products in 
diet and for avoidance of medications reiterated.   
  
The typical schedule is shown as an example for a follow up evaluation day for visit 3.  
 
Visit 3 Activity (in sequence):         
                                       
Subject arrives at the Allergy/Immunology Clinic. 
 
Study medication disepensed at visit 2 are returned by the subject and a compliance check is 
performed the study team.  
 

 
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Vital signs (heart rate, blood pressure and weight), nasal examination, concomitant medication 
and diet checked since last visit.   
 
Spirometry will be performed. (Note: subjects’ have up to 3 attempts to FEV1 of >70% to 
continue on with this study.) 
 
general physical examination along with nasal examination. 
 
Nasal lavage performed. 
 
Blood work obtained for sulforaphane level.   
 
Nasal Allergen Challenge followed by monitoring explained below      
 
Study completed.     
 
Study Outcome Measures: 
1. The Total Nasal Symptoms Score (TNSS), performed to determine adequate dosing for nasal 
challenge (visit 1) and during the nasal challenges (visit 2, 3).  During the challenge the TNSS 
will be taken 30 minutes after nasal lavage, and then repeated at 5, 15, and 30 minutes then 
hourly to 4 hours post-challenge.     
2. The peak nasal inspiratory flow (PNIF) performed at the up-dosing nasal challenge visit (visit 
1) and during nasal challenges (visit 2 and 3).  During the challenge, PNIF will be taken  30 
minutes after the nasal lavage, then repeated at 5, 15 and 30 minutes after the challenge, then 
hourly to 4 hours post-challenge.    
3. Biomarker measurements taken from nasal fluid collected by nasal sponge at visit 2 and visit 
3 will include:   
a) IL-4: 0 mintue, 5 minutes, 15 minutes, 30 minutes, then hourly to 4 hours post challenge  
b) IL-5: 0 minute, 5 minutes, 15 minutes, 30 minutes, then hourly to 4 hours post challenge 
c) IL-13: 0 minute, 5 minutes, 15 minutes, 30 minutes, then hourly to 4 hours post challenge   
d) Eosinophil Cationic Protein (ECP): 0 minute, 5 minutes, 15 minutes, 30 minutes, then hourly 
to 4 hours post challenge  
e) Tryptase: 0 minute, 5 minutes, 15 minutes, 30 minutes, then hourly to 4 hours post challenge   
 
Study Regimen: 
Study Medications and Placebo 
Nasal Corticosteroids: Fluticasone propionate nasal spray prescribed as 2 sprays in each nostril 
daily will be the nasal corticosteroid of choice for the current study. Placebo will be normal 
saline based. Both fluticasone and placebo will be ordered through the VAGLAHS Research 
Pharmacy.  For optimal control of nasal symptoms a treatment period of 1 week is required. 
Fluticasone will be given for 3 weeks in order to maximize effect.    
 
Active Avmacol Enteric Coated Tablets:Water, HPMC, Sodium Alginate, Purified Stearic Acid, 
Ethylcellulose, Medium Chain Triglycerides, Oleic Acid, Triactin, Mineral Oil, Sodium Lauryl 
Sulfate, Titanium Dioxide 
 
Avmacol, a blend of broccoli seed and sprout extracts contains the 2 essential ingredients 
needed for sulforaphane production: glucoraphanin and the myrosinase enzyme.   Avmacol 
contains ~ 30 - 35 umoles sulforaphane per 2/tablet recommended serving.  

 
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Placebo Avmacol Coated Tablets:Water, HPMC, Triactin, Mineral Oil, Sodium Lauryl Sulfate, 
Titanium Dioxide 
 
Concomitant Medications 
Concomitant medication is defined as any medication, which is taken during the study. All 
prescription, herbal supplements, and over-the-counter drugs which are being taken by subjects 
upon entry to the study or which are taken during the study are regarded as concomitant 
treatments and will be documented. Basic medication use information will be confirmed at the 
time of screening, in order to determine if subject meets exclusion criteria regarding 
medications. 
 
Rescue Medications 
Subjects in the study are atopic. During the course of the study, subjects may develop nasal 
symptoms. They will be allowed to treat their symptoms with saline nasal spray. If their 
symptoms persist and they require additional medications, they will be discontinued from the 
study. 
 
Prohibited Medications  
The days below indicate last dose received prior to visit 1 and throughout the subjects’ total 
participation time:  
 
1. Topical Nasal Steroid use in the past 14 days, 
2. Systemic steroid use in the past 14 days, 
3. Oral antihistamine use in the past 5 days,  
4. Intranasal antihistamine or cromolyn use in week prior to nasal challenge. 
6. currently receiving allergy immunotherapy, or use anytime during the protocol. 
7. short acting beta agonists in the past 6 hours. 
8. nasal cromolyn in the past 7 days, 
9. oral decongestants in the past 3 days,  
10. antihistamine-decongestant tablets/liquids in the past 3 days,  
 
Nasal Sprays: 
Both the active fluticasone proprionate (50mcg) and the placebo will be obtained from the VA 
Research pharmacy.     
 
Criteria for Premature Termination of the Study 
 
Participant Withdrawal Criteria 
In the best interest of the subject, participants may be prematurely terminated from the study for 
the following reasons: 
1. The participant elects to withdraw consent from all future study activities, including 
follow-up. 
2. The participant is “lost to follow-up” 
3. The participant takes any of the prohibited medications during the study. 
4. The participant develops an adverse event that, in the opinion of the Protocol Chair 
(Principal Investigator) or the Safety Monitoring Board of the study, increases his/her 
risks if study participation continues. 
5. The participant dies 
 

 
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Study Stopping Rules 
The Data Safety Monitoring Board (DSMB) will be provided summaries of all adverse events on 
a regular basis.  The DSMB will have the authority to terminate the trial at any time based on 
reported adverse events.   
 
Study enrollment and ongoing study treatments will be suspended pending expedited review of 
all pertinent data by the VAGLAHS Institutional Review Board (IRB), the Data Safety Monitoring 
Board (DSMB) if a significant adverse event as deemed by DSMB occurs  that is possibly 
related to the study procedures. The study may be terminated by the DSMB upon review of data 
with observations or findings that such action is necessary. (National Cancer Institute’s 
Common Terminology Criteria for Adverse EventsVersion 4 (published May 28, 2009)).   
 
Grade 1=Mild adverse event 
Grade 2=Moderate adverse event. 
Grade 3=Severe and undesirable adverse event 
Grade 4=Life-threatening or disabling adverse event 
Grade 5=Death 
 
Screening Visit 
At the screening visit, potential subjects are given a copy of the IRB-approved informed consent 
form for review and are invited to ask any questions or express concerns after reading the 
consent. The research study is clearly explained to the subject, including the rationale, risks, 
and benefits. The subject is asked to explain what he/she understands to be required in the 
study. The subject is informed that all participation is voluntary and that he/she is free to 
discuss the study with family or friends when considering enrollment. Subjects are told that only 
if the study protocol is fully understood should they sign the consent form. After the subject 
provides written consent, potential subjects’ eligibility will be confirmed based on the determined 
inclusion/exclusion criteria. Once the subject has signed the informed consent form, a study 
investigator will also sign it, and the subject will receive a copy. 
 
Procedures performed 
Skin Testing:  Epicutaneous skin testing is considered highly reliable, safe and widely used in 
diagnosing allergic rhinitis (37).   After application of the specific allergen to the sterile tip of the 
applicator, the subject’s skin will be gently lifted and the tip containing allergen will be applied.  
Along with application of the allergens mentioned, histamine and a saline control will be applied 
to subject’s skin. Results will be interpreted 20 minutes following and will be considered positive 
if the wheal and erythema are at least 3mm larger than the saline control. For subjects who 
have an epicutaneous allergen skin or blood test on file completed by the VA Allergy Clinic 
within < 1 year that confirms a positive to grass pollens the results will be used to proceed onto 
the up-dosing nasal challenge.   
 
Nasal Challenge: Nasal Challenge is considered a safe and useful diagnostic tool in diagnosing 
causes of both allergic and nonallergic rhinitis (38). The nasal challenge chosen was based on 
repeated reliability using the TNSS and PNIF scores along with the biomarkers chosen for this 
study. (36)       
 
Subjects will undergo an up-dosing nasal challenge performed at visit 1 in order to determine 
the threshold dose to be given at the subsequent nasal challenges (visit 2 and visit 3).  During 
the up-dosing nasal challenge, the TNSS will be recorded immediately before each subsequent 
dose.  Grass allergen extract (c. cynodon, p. pretense or s. sorghum,) will be used in the 

 
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challenge.  For c. cynodon (Bermuda grass), dosing will start at 30 BU/ml and will continue with 
the following concentrations until a TNSS score reaches 7 or greater or PNIF is reduced by 
>50% from baseline reading: 300, 1000, 3000 and 10,000 BU/ml. For p. pretense(Timothy 
grass), dosing will start at 300 and will continue with the following concentrations until a TNSS 
score reaches 7 or greater or baseline PNIF is reduced by > 50% from baseline reading: 
1,000,3,000,10,000 and 100,000 BU/ml.  For s. sorghum (Johnson grass) dosing will start at 
1:200,000 and will continue with the following concentrations until a TNSS score reaches 7 or 
greater or baseline PNIF is reduced by > 50% from baseline reading: 1:20,000, 1:2000, 1:200, 
and 1:20 w/v. Subjects will receive three sprays equivalent to 100 µl for each concentration into 
each nostril using an atomizer device, part of the standardized equipment at VAGLAHS for 
patients requiring nasal sprays during procedures applied every 10 minutes until maximal 
concentration or a TNSS score of 7 or PNIF reduction by > 50% from baseline reading is 
achieved.  Prior to subsequent challenges at visit 2 and 3, nasal lavage (SinusRinse) will be 
performed.  A baseline PNIF and TNSS will be recorded 30 minutes following nasal lavage for 
visits 2 and 3. Nasal challenge with 100 µl of the final dose achieved at visit 1 will then be 
applied into each nostril.  The PNIF and TNSS will be recorded following challenge at 5, 15, 30 
minutes following challenge and then at 1 hour, continuing to record each hour for 4 hours.  
During these intervals, nasal sponges (Plastocell & Co.) will be applied into each nostril onto the 
nasal mucosa, beyond the nasal vestibule, and alongside the inferior turbinate with the use of a 
crock  forceps (Phoenix surgical instruments, Harlow, UK)  and a Thuddicum’s nasal speculum 
(Phoenix surgical instruments, Harlow, UK).  After two minutes of placement, the sponges will 
be removed and then added to 2 ml centrifuge tubes with indwelling .22µm cellulose acetate 
filters (Costar Spin-X, Corning, NY, USA) and placed immediately into ice before centrifuging 
within the hour.  The liberated fluid will then be freezed at -80º until analysis occurs.    
  
General physical examination: A physical examination to determine general health to include 
measurement of vitals and a cardiopulmonary examination will be performed in the VA GLA 
Allergy/Immunology Clinic by the attending physician. A nasal examination will be performed at 
every visit before any nasal challenge or lavage is performed. On Day 1, a urine pregnancy test 
will be performed for women of child-bearing potential. 
  
The Immediate Total Nasal Symptom Score (TNSS): The TNSS has been used in prior 
interventional studies to evaluate different treatment modes for allergic rhinitis (39, 40, and 41). 
The score will consist of subjects rating 4 symptoms (nasal congestion/postnasal drainage, 
nasal pruritus, rhinorrhea, or sneezing) on a scale of 0 to 3, where 0=no symptoms present, 
1=mild symptoms that do not interfere with activity, 2=moderate symptoms that are slightly 
bothersome symptoms with slight interference with activity and/or nighttime sleep, or 3=severe 
symptoms with interference with activity and nighttime sleep. 
 
The Peak Nasal Inspiratory Flow (PNIF):  the PNIF (Alliance Tech Medical) has been used in 
prior interventional studies involving allergic rhinitis (42, 43, and 44).  Patients exhales fully and 
quickly after checking that an air tight seal covers the nose and seals the mouth.      
 
Nasal Examination:  Nasal examination will be performed at each visit prior to nasal challenge.   
Each nasal examination will include use of an otoscope with specula inserted into the nasal 
passage.  Nasal passages and nasal turbinates will be inspected for color, swelling, exudate or 
bleeding.    
  
Precautions: 

 
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Since allergen will be administered through nasal challenge, it is imperative that the study take 
place in a controlled environment that is prepared to treat hypersensitivity reactions. The 
VAGLAHS Allergy/Immunology clinic has the appropriate equipment and trained personnel to 
treat allergic reactions including anaphylaxis. A crash cart is available inside the clinic and 
epinephrine is readily available.  The clinic is located within the VAGLAHS hospital where there 
are physicians trained in advanced cardiac life support (ACLS). 
 
Visit Target Windows: 
Subject visit times during the treatment phase of the protocol should be consistent, within -+10 
days of targeted scheduled visit days. 
 
Randomization: 
This is a double blind randomized study. The subject randomization will be carried out based on 
a computer generated randomization list with a random blocked design of block sizes of 4 and 
8. Only the study statistician and pharmacist will maintain the randomization list, the treatment 
administrators and outcome evaluators are blind to the randomization. The investigator 
performing and recording the results of the challenge will be blinded to the subjects assigned 
treatment arm.    
 
PAYMENT OFFERED FOR PARTICIPATION 
 
Twenty dollars ($20.00) will be paid to participants who complete up to the skin test 
procedure at visit 1, and do not meet study qualifications (skin test results determine 
that you not allergic to grass) to continue participation in the study. 
Forty-five dollars ($45.00) will be paid to participants who meet study qualifications and  
complete visit 1.  
Ninety dollars ($90.00) will be paid to participants who complete visit 2. 
Ninety dollars ($90.00) will be paid to participants who complete visit 3. 
Additionally, those participants who will comply with study protocol and complete all three study 
visits will receive the completion of study payment in amount of $75 at the end of the study. 
 
Study Material and analysis: 
Choosing cytokines mediators:    
TH2 cytokines (IL4, IL15, IL13):  These cytokines direct production of IgE antibodies which latter 
attach to mast cells.   
 
Tryptase, Eosinophilic Cationic Protein (ECP):  Tryptase is immediately released from mast 
cells upon activation.  ECP marks presence of eosinophils which are involved in the delayed 
reaction of allergic rhinitis.   
 
Both the above cytokines, tryptase and ecp have been found in nasal mucus and levels have 
been replicated in prior nasal challenge (36)      
 
Analysis of biomarkers:  
All the following analysis will be performed at the biomarker lab, UCLA Center for Human 
Nutrition.  Measurements of IL-4, IL-5, and IL-13 will be  performed using MSD Human TH1/TH2 
7-Plex, Ultra-Sensitive Kit according to the manufacturer's instructions (MS6000 7 spot, Meso 
Scale Discovery, Maryland, USA). Plates will read using anMSD SECTOR® 6000 instrument. 
With this method, validation procedures demonstrated that the lower quantification limit for all 
cytokines was approximately 5 pg/ml, with an assay range of 5–5000 pg/ml in nasal secretions. 

 
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All measurements will be performed on undiluted nasal fluid samples in duplicate and reported 
as mean values, though cytokine levels will be standardized with ng/ml protein concentration.   
Tryptase and Eosinophilic Cationic Protein (ECP) will be measured through ImmunoCAP at the 
minimum level of detection of 2mg/ml.  Each plate/Immunocap carousel is run with standards 
and curve controls.   
 
Genomic DNA Isolation: 
Peripheral blood will be drawn into a BD Vacutainer K2 EDTA tube (BD Biosciences; San Jose, 
CA). Genomic DNA will be extracted from blood using the GenElute Mammalian Genomic DNA 
Miniprep Kit (Sigma-Aldrich; St. Louis, MO) according to manufacturer’s instructions. Briefly, 
200 μl blood will be treated with Proteinase K followed by RNase for 2 min before lysing cells in 
a 55 deg C water for 10 min. DNA will be precipitated with 100% ethanol and bound to a spin 
column. After two washes, DNA will be eluted from column with TE buffer. TaqMan Polymerase 
Chain Reaction Genotyping of GSTP1 Ile105Val Genotyping will be performed using SNP 
Genotyping Assays for GSTP1 (rs1695) (Applied Biosystems; Foster City, CA) on a 
StepOnePlus Real-Time PCR Machine (Applied Biosystems).  Real-time PCR will be performed 
with 25 ng DNA in a 20 μl reaction containing TaqMan Genotyping Master Mix and SNP 
genotyping assay. Reactions will be heated for 95 oC for 10 minutes following by 40 cycles of 
92 oC for 15 sec with 60 oC for 1 min. Alleles will be determined using the allelic discrimination 
plot in the StepOne software genotyping program.  Each sample will be performed in 
quadruplicate. 
 
GSTM1 Copy Number-GSTM1 copy number will be determined using Pre-designed TaqMan 
Copy Number Assays (Applied Biosystems). GSTM1 will run in a duplex assay with RNase P as 
a reference gene. Reactions will be heated for 95 oC for 10 minutes following by 40 cycles of 92 
oC for 15 sec with 60 oC for 1 min. Results will be analyzed using Copy Caller version 1.0 
software (Applied Biosytems). Each sample is performed in quadruplicate. 
 
Measurement of sulforaphane: 
HPLC-MS/MS identification of SFN metabolites in plasma R, S-sulforaphane will be purchased 
from LKT Laboratories, Inc. (Minnesota, USA). All solvents and other chemical used will be 
HPLC grade and were purchased from Fisher Scientific (USA). Water will be ultrafiltered using a 
Milli-Q pure water system. To the 4° C plasma sample (200 μL) in a 1.5mL micro centrifuge tube 
(Fisher Scientific, USA), pre-cooled (4° C) trifluoroacetic acid (20 μL) (TFA) will be added to 
precipitate plasma proteins (30 seconds) in the sample followed by brief mixing and refrigerated 
centrifugation at 15900×g for 10 min. The resulting supernatant will be filtered (0.2 μm pore 
size) and 20 μL injected for analysis. 
  
A LCQ Advance mass (Thermo Finnegan) and a Survey HPLC equipped with binary pump, 
degasser, cooled auto-sampler and a PDA detector (USA) were used for the LC–MS analysis. 
The HPLC column will be a Zorbax SB- will be 0.25 mL/min and gradient elution will be used: 
solution A (1% acetic acid solution): solution B (acetonitrile). The gradient started at 2% solution 
B increasing over 20 min to 30% B and 50% in 30 minutes, finally re-equilibrating to 2% B for 10 
min. The mobile phase solutions were filtered through 0.22 μm pore size cellulose nitrate or 
nylon membrane filters using a vacuum filtration apparatus. The LC eluent flow of 0.25 mL/min 
will be sprayed into the mass spectrometer interface without splitting. Electrospray ionization in 
the positive mode (ESI+) will be applied. The analytes are identified by molecular weight and 
their MS/MS, respectively. The identification of the MS/MS of SFN-Cys, SFN-Cys-Gly; SFN-
NAC; and SFN-GSH at m/z of 178; 179; 178 and 179, respectively were used to confirm 
consumption of the BSE supplement by subjects in the study. 

 
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Peak Nasal Inspiratory Flow (PNIF): 
PNIF will be performed on each subjectthroughout nasal allergy challenge testing.  
Measurements will be performed using the In-Check Inspiratory Flow Measurement Device 
(Alliance Tech Medical), a portable inspiratory flow meter that can monitor nasal patency.  Nasal 
peak inspiratory flow will be measured in L/min.  The subject will place the device over the nose 
and mouth.  The subject will be asked to take a deep, quick forced inspiration through the nose.  
The test will be repeated three times and the highest value will be recorded.      
 
Statistical Considerations and Analytical Plan: 
Summary statistics (mean, standard deviation, median, interquartile range and frequency 
distribution) will be generated for baseline demographic and clinical information to characterize 
the study population for subjects of 4 treatment arms, respectively. All outcomes of interest 
(TNSS, PNIF and biomarkers) will also be summarized by treatment arms and repeated 
measurement time points. Box plots and histograms will be generated to visualize the variable 
distributions, and scatter plot will be used to explore the relationship among variables. Primary 
analysis: outcomes of interest will be collected at different time points in visits 2 (baseline) and 3 
(after 3 weeks of treatment).  TNSS will be recorded before 30 minutes after nasal lavage, 5, 15, 
30, mintues then hourly to 4 hours after challenge initiation.  According to Bareille (figure 2), 
TNSS is stable after 3 hours following challenge, therefore we will average the TNSS at 3 and 
hour as the analysis outcome to stabilize the outcome measure and simplify the analysis.  
 
We are interested in detecting the difference in the outcomes among subjects receiving a 
placebo tablet  vs. active tablet  , with and without nasal corticosteroid.  The primary endpoint is 
the TNSS.  ANCOVA (Analysis of covariance) model will be used to evaluate the difference in 
TNSS after treatment between placebo tablet vs active tablet  , adjusting for their baseline 
values.  Analysis will be carried out based on intention to treat principal with and without 
covariate adjustment.  In addition to treatment assignment (4 groups), other fixed effects in the 
model may include baseline value, subject-level covariates (e.g., age, sex, baseline disease 
severity, medical history, etc.).  Estimates of difference between placebo vs. active tablet  will be 
calculated based on appropriate contrasts.  Variable transformation (e.g., log transformation) 
will be used if the outcome distribution is skewed and normality assumption is imperative or a 
better model fit is suggested with transformed covariates.  Secondary analysis will include the 
change in PNIF and biomarkers, and similar ANCOVA model will be used.  Correlation analysis 
will also be carried out to examine the associations among outcomes.     
 
Sample size consideration: Similar to the study design in Bareille 2012, we used standard 
deviation (SD) of TNSS of 2.08 to calculate the sample size needed.  With a total of 152 
subjects (36 subjects per arm) and the adjustment for multiple comparisons, we will have 80% 
power to detect a difference of 1.5 in TNSS between mango juice vs mango juice + BS groups 
at the 2.5% level with or without nasal corticosteroid.  This calculation is based on 2 sample t-
test with sample size of 36  per group, common standard deviation of 2.08 and alpha=0.025. 
The difference of 1.5 in TNSS is right in the range of difference between various treatments in 
Bareille 2012. Since TNSS outcome is taking average over 3, 4, 5 and 6 hours results, we 
expect the variability will be reduced and sample size calculation will be conservative.  Similar 
calculation can be carried out for PNIF and Biomarkers. According to (Lipworth 2012), we 
estimate the standard deviation % change in PNIF is about 12.7, with the sample size of 36 per 
arm, we will have 80% power to detect a difference of 9.1 in % change in PNIF between 
placebo tablet vs active tablet at the 2.5% level with or without nasal corticosteroid. According to 
(Baroody 2013), we estimate the SD of Tryptase is about 5 in their FF/PL (fluticasone 

 
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furoate/placebo) group, and we will have 80% power to detect a difference of 3.6 at the 2.5% 
level. 
 
We will use multiple imputation method to handle the missing data problem such that each 
missing value will be replaced with a set of plausible values that represent the uncertainty about 
the right value to impute.  There multiply imputed data sets will be analyzed by the proposed 
analytic methods and the results from these analyses will be combined based on Rubin’s 
formula (Rubin 1987, Multiple Imputation for Nonresponse in Surveys, New York: John Wiley & 
Sons, Inc.).    
 
We will recruit 475 subjects that meet the inclusion/exclusion criteria for screening.  We expect 
237 subjects will test positive to grass and negative to perennial allergens, 190 subjects will 
have adequate positive nasal challenge and will be randomized.  The expected dropout rate is 
20%.     
 
Scatterplots and Spearman correlation will be produced to assess correlations between 
increase/decrease levels of the above variables.  We are looking for change biomarker 
variables that reflect inflammation, TNSS and PNIF.  We will assume effective anti-inflammatory 
treatment will lead to a significant decrease in inflammatory markers (IL4, IL5, IL13 and ECP), a 
significant increase in Total Nasal Symptom Scores (TNSS) and Peak Nasal Inspiratory Flow 
(PNIF).