Preventing Life-Threatening Allergic Reactions With Acalabrutinib, an FDA-Approved Bruton's Tyrosine Kinase Inhibitor
Phase: PHASE2
Conditions: Food Allergy, Food Allergy Peanut
Sponsor: Johns Hopkins University

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PROTOCOL 
Study Title: 
Principal Investigator: 
Sub-Investigator(s): 
Biostatistician: 
Study Drug: 
 
Protocol 
Number: Version: 
NCT number
Preventing Life-Threatening Allergic Reactions with Acalabrutinib, 
an FDA-Approved BTK Inhibitor 
Melanie Dispenza, MD, PhD 
Johns Hopkins University School of Medicine 
Department of Medicine, Division of Allergy and Clinical Immunology 
Johns Hopkins Asthma and Allergy Center  
5501 Hopkins Bayview Circle, Room 3B.69 
Baltimore, MD 21224 
Phone: 410-550-1815 
Fax: 410-550-1733 
Email: mdispen1@jhmi.edu 
Donald MacGlashan, Jr, MD, PhD 
Johns Hopkins University School of Medicine  
Department of Medicine, Division of Allergy and Clinical Immunology 
Dhananjay Vaidya, MBBS, PhD, MPH 
Associate Professor, Medicine 
GeneSTAR Research Program 
1830 E. Monument St, Room 8028 
Baltimore, MD, 21287 
Phone: 410-614-2440 / 410-550-5032 
dvaidya1@jhmi.edu 
Acalabrutinib (Brand name: Calquence®) 
MD12042018 
February 9, 2022 
NCT05038904

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TABLE OF CONTENTS 
LIST OF ABBREVIATIONS ........................................................................................................................ 1 
STUDY SCHEMA ....................................................................................................................................... 2 
STUDY SUMMARY .................................................................................................................................... 2 
1.0 
BACKGROUND AND RATIONALE ............................................................................................... 4 
1.1 
Disease Background ................................................................................................................. 4 
1.2 
Study Drug Background and Associated Known Toxicities ......................................................... 4 
1.3 
Rationale ................................................................................................................................... 4 
2.0 
STUDY OBJECTIVES.................................................................................................................... 5 
2.1 
Primary Objective ...................................................................................................................... 5 
2.2 
Secondary Objectives ................................................................................................................ 5 
2.3 
Endpoints .................................................................................................................................. 5 
3.0 
SUBJECT ELIGIBILITY ................................................................................................................. 6 
3.1 
Inclusion Criteria ........................................................................................................................ 6 
3.2 
Exclusion Criteria ...................................................................................................................... 7 
3.3 
Study Stopping Criteria .............................................................................................................. 8 
3.4 
Recruitment ............................................................................................................................... 8 
4.0 
TREATMENT PLAN ...................................................................................................................... 8 
4.1 
Treatment Dosage and Administration ....................................................................................... 8 
4.2 
Toxicities and Dosing Delays ..................................................................................................... 9 
4.3 
Concomitant Medications/Treatments ........................................................................................ 9 
4.4 
Duration of Therapy ..................................................................................................................10 
4.5 
Duration of Follow Up ...............................................................................................................10 
4.6 
Subject Replacement ...............................................................................................................10 
5.0 
STUDY PROCEDURES ................................................................................................................10 
5.1 
Screening/Baseline Procedures ................................................................................................10 
5.2 
Procedures During Treatment ...................................................................................................11 
5.3 
Follow-up Procedures ...............................................................................................................13 
5.4 
Time and Events Table .............................................................................................................13 
5.5 
Removal of Subjects from Study ...............................................................................................13 
6.0        RESPONSE CRITERIA .................................................................................................................14 
6.1 
Safety/tolerability ......................................................................................................................14 
6.2  
Efficacy ....................................................................................................................................14 
7.0        ADVERSE EVENTS ......................................................................................................................14 
7.1 
Experimental Therapy...............................................................................................................14 
8.0 
ADVERSE EVENT MONITORING ................................................................................................16 
8.1 
Definitions ................................................................................................................................17 
8.2 
Steps to Determine If an Adverse Event Requires Expedited Reporting ....................................18 
8.3 
Reporting Requirements for Adverse Events .............................................................................19 
9.0 
DRUG INFORMATION ..................................................................................................................20 
9.1 
Acalabrutinib 100 mg tablets .....................................................................................................20 
9.2       Return and Retention of Study Drug ...........................................................................................20 
10.0 
STATISTICAL CONSIDERATIONS ..........................................................................................21 
10.1 
Study Design/Study Endpoints ..................................................................................................21 
10.2 
Sample Size and Accrual ..........................................................................................................21 
11.0 
STUDY MANAGEMENT ...........................................................................................................21 
11.1 
Conflict of Interest ....................................................................................................................21 
11.2 
Institutional Review Board (IRB) Approval and Consent ............................................................21 
11.3 
Data Management and Monitoring/Auditing...............................................................................22 
11.4 
Adherence to the Protocol ........................................................................................................22 
11.5 
Amendments to the Protocol .....................................................................................................23 
11.6  
Confidentiality ...........................................................................................................................23 
11.7 
Provisions to Protect the Privacy Interests of Subjects ..............................................................23 
11.8 
Record Retention .....................................................................................................................23 
11.9 
Obligations of Investigators .......................................................................................................24 
12.0 
REFERENCES .........................................................................................................................24 

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LIST OF ABBREVIATIONS 
 
AE 
Adverse Event 
ALC 
Absolute Lymphocyte Count 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
BAT 
Basophil Activation Test 
BTK 
Bruton’s Tyrosine Kinase 
BUN 
Blood Urea Nitrogen 
CBC 
Complete Blood Count 
CLL 
Chronic Lymphocytic Leukemia 
CMP 
Comprehensive Metabolic Panel 
CRU 
Clinical Research Unit 
SBPC 
Single-blind Placebo-controlled 
DLT 
Dose-Limiting Toxicity 
DSMB 
Data and Safety Monitoring Board 
EKG 
Electrocardiogram 
FC 
Food Challenge 
H&P 
History & Physical Exam 
HRPP 
Human Research Protections Program 
IgE 
Immunoglobulin E 
IV (or iv) 
Intravenously 
MCL 
Mantle Cell Lymphoma 
MTD 
Maximum Tolerated Dose 
OFC 
Oral Food Challenge 
PBMCs 
Peripheral Blood Mononuclear Cells 
p.o. 
per os/by mouth/orally 
PST 
Peripheral Blood Testing 
SAE 
Serious Adverse Event 
sIgE 
Surface Immunoglobulin E 
SGOT 
Serum Glutamic Oxaloacetic Transaminase 
SPGT 
Serum Glutamic Pyruvic Transaminase 
SPT 
Skin Prick Test 
WBC 
White Blood Cells 
WM 
Waldenstrom’s Macroglobulineia 
 
 

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This is a phase II open label study involving the use of acalabrutinib to prevent food-induced anaphylaxis 
in adults with food allergy.  Acalabrutinib (brand name Calquence®; manufacturer AstraZeneca [AZ]) is 
currently FDA approved for the treatment of mantle cell lymphoma (MCL).  We propose to administer this 
approved drug to adults with food allergy to inhibit anaphylaxis.  
 
STUDY SCHEMA 
This is an open-label study designed to determine the ability of acalabrutinib to prevent signs and 
symptoms of anaphylaxis during a food challenge in food-allergic subjects.    
   
At Visit 1, after undergoing informed consent, subjects will undergo procedures for assessment of 
baseline status and eligibility (clinical history and skin prick test [SPT] to peanut and/or tree nuts as well 
as histamine and saline SPT positive and negative controls, respectively).  After eligibility is confirmed, 
subjects will undergo a single-blind, placebo-controlled graded food challenge (SBPCFC) of up to 4 g of 
peanut or tree nut protein, administered over escalating doses while being monitored in a clinical 
research unit (CRU) setting.  Prior to the SBPCFC, 30 mL of peripheral blood will be collected for basophil 
activation testing (BAT), serum specific IgE to peanut/tree nuts and components, as well as toxicity lab 
testing monitoring.  During the food challenge, subjects will be assessed for signs and symptoms of food-
induced anaphylaxis between each dose per food challenge protocol, upon report of any symptoms and 
should any objective change in clinical status occur.  Once a confirmed reaction occurs, the food 
challenge will be terminated and the reaction treated until it has resolved. Subjects will be discharged 
from the CRU no sooner than 2 hours after completion of the food challenge. They may be observed 
longer should a reaction occur at the investigator’s discretion.  Additional blood will be drawn at fixed 
intervals for biomarkers of anaphylaxis including mast cell and basophil microparticle analysis.  This visit 
will establish each subject’s baseline threshold dose for peanut/tree nut reactivity.   
 
After a minimum of a two-week recovery period, subjects will be given acalabrutinib (four doses of 100 mg 
of acalabrutinib to be taken orally twice daily) and return for Visit 2 to undergo repeat SPT, SBPCFC, and 
phlebotomy in an identical manner to Visit 1 to establish each subject’s new threshold dose for 
peanut/tree nut reactivity while on acalabrutinib, which we hypothesize will be higher.  At this point of the 
protocol, acalabrutinib dosing will be stopped.  Follow up visits for up to 6 months after cessation of 
acalabrutinib will be conducted to repeat toxicity labs and skin testing.   
 
At each visit, subjects will be monitored for toxicities, including CBC w/diff, CMP, and pregnancy testing.  
Prior to drug initiation and at the end of the trial, an EKG will be performed.  
 
 
 
STUDY SUMMARY 
Title 
Preventing Life-Threatening Allergic Reactions with Acalabrutinib, an 
FDA-Approved BTK Inhibitor 
Protocol Number 
MD12042018 
Phase 
Phase 2 
Methodology 
Open label study to determine efficacy and safety of acalabrutinib for the 
treatment of food allergy 
Study Duration 
14 weeks  
Study Center(s) 
Single-center  
Objectives 
Primary:  To determine the ability of acalabrutinib to 1) suppress food 
skin prick test size and 2) prevent food-induced anaphylaxis and/or shift 
the threshold dose of food at which a food allergic subject will react to a 
higher level.  
 
Secondary:  To determine if blood mast cell and basophil microparticles 
can be utilized as markers for anaphylaxis. 
Number of Subjects 
10 

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Diagnosis and Main 
Inclusion Criteria 
Adults with history of food allergy to peanut and/or tree nut will be invited 
to participate.   
Study Product(s), Dose, 
Route, Regimen 
Acalabrutinib 100 mg, PO twice daily  
Duration of administration 
2 days  
Reference therapy 
There are currently no approved therapies for food allergy 
Statistical Methodology 
Primary efficacy endpoint:  Change in highest tolerated dose of the food 
allergen. This endpoint will be analyzed by paired Student’s t test. 
Secondary efficacy endpoints will include: 
1. change in severity of reaction during food challenge 
2. change in skin prick test wheal size 
3. change in BAT percent activation 
 
 
 
 

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1.0 
BACKGROUND AND RATIONALE 
 
1.1 
Disease Background 
 
Food allergy is a potentially life-threatening disease, and its prevalence continues to increase 
despite public health efforts (1, 2).  Newer studies have shown that it is less likely that affected 
persons will develop natural tolerance over time, and food allergies can also develop in 
adulthood, so more adults now have potentially life-threatening food allergies.  Severe and near 
fatal reactions can occur and can lead to devastating outcomes, including fatalities (3).  
Furthermore, the economic burden of childhood food allergy is nearly $25 billion per year, with as 
many as 200,000 emergency department visits annually (4).  Despite this significant disease 
burden, there are currently no approved therapies for food allergy.  Standard of care is constant 
vigilance in avoidance of allergen, and use of self-injected epinephrine after a reaction has 
occurred.  Therefore, there is a large unmet need for new therapeutics to prevent food-induced 
anaphylaxis.  FDA approved Bruton’s tyrosine kinase (BTK) inhibitors such as ibrutinib, 
acalabrutinib and others under development (5, 6) may be able to fill this void and offer a therapy 
to these patients.   
 
1.2 
Study Drug Background and Associated Known Toxicities 
 
Acalabrutinib is a twice daily, oral, highly selective, irreversible small-molecule inhibitor of BTK, a 
key component of B-cell receptor signaling (7, 8).  BTK is also essential for FcεRI signaling and 
triggering of mast cells and basophils, the primary cells activated during anaphylaxis (9, 10).  
Acalabrutinib is currently FDA approved for the treatment of Mantle Cell Lymphoma (MCL).   
 
Pharmacokinetic studies demonstrated the average bioavailability of acalabrutinib was 25%.  
Median time to peak acalabrutinib plasma concentrations (Tmax) was 0.75 hours.  Following a 
single oral dose of 100 mg acalabrutinib, the median terminal elimination half-life (t1/2) of 
acalabrutinib was 0.9 hours (range: 0.6 to 2.8). The t1/2 of the active metabolite, ACP-5862, was 
6.9 hours.  Acalabrutinib is predominantly metabolized in the liver by CYP3A enzymes, and to a 
minor extent, by glutathione conjugation and amide hydrolysis, based on in vitro studies.  
Following administration of a single 100 mg radiolabeled acalabrutinib dose in healthy subjects, 
84% of the dose was recovered in the feces and 12% of the dose was recovered in the urine, with 
less than 1% of the dose excreted as unchanged acalabrutinib. 
 
In healthy volunteers, co-administration with CYP3A inhibitors increased Cmax, and therefore the 
drug will not be concomitantly administered with strong or moderate inhibitors of CYP3A.  Among 
1029 pooled patients from clinical trials (820 patients taking acalabrutinib monotherapy across 6 
trials and 209 patients taking acalabrutinib plus obinutuzumab across 2 trials), most of whom 
(88%) were exposed to acalabrutinib for at least 6 months, adverse reactions in ≥30% of patients 
included anemia, neutropenia, upper respiratory tract infection, thrombocytopenia, headache, 
diarrhea, and musculoskeletal pain.  Acalabrutinib is also pregnancy category D.   
 
Notably, our prior work using ibrutinib (which  may have a less favorable safety profile) in adults 
with food allergy demonstrated no toxicity or adverse effects from up to 7 days of treatment (11), 
suggesting that adverse effects from BTK inhibitors may be mitigated with short-term use rather 
than chronic treatment.   
 
1.3 
Rationale 
 
There are currently no treatments for food allergy.  IgE uniquely arms mast cells and basophils 
via their surface expression of FcεRI, and when cross-linked by allergens, causes mediator 
release that then results in an allergic reaction.  Crosslinking of the receptor is known to activate 
a number of kinases.  BTK is a key component of FcεRI signaling and activation of mast cells and 

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basophils.  Since signaling mediated by FcεRI aggregation is thought to require the activity of 
BTK (8,9), MacGlashan et. al. was the first to test ibrutinib, a first generation BTK inhibitor, for its 
ability to prevent FcεRI activation of human basophils in vitro (9).  Ibrutinib inhibited IgE-mediated 
CD63, CD203c and CD11b upregulation, and release of histamine, LTC4 and IL-4 as well as the 
cytosolic calcium response (IC90 of ≈50 nM).  Ibrutinib did not inhibit basophil activation by fMLP 
or C5a and did not inhibit IL-13 release induced by IL-3, suggesting that BTK is required for IgE-
mediated activation, but not IgE-independent activation, of human basophils (12).  We recently 
completed an open label trial to test the hypothesis that ibrutinib would abrogate IgE-mediated 
BAT and food SPT responses in peanut and tree nut allergic adults (11).  We found that 2 doses 
of ibrutinib significantly reduced SPT wheal and flare area (77 and 86% reductions, respectively; 
overall, 44% of SPTs became negative).  Additional doses up to 7 days did not demonstrate 
further suppression compared to 2 days.  Anti-IgE induced BAT became negative in all subjects 
after 2 doses and remained negative during treatment.  The vast majority of SPTs and BAT 
returned to baseline within 1 week of cessation of ibrutinib.  No toxicity from treatment was 
observed, and ibrutinib was well-tolerated by subjects.   
 
We now propose to extend these promising findings in a subsequent open label study using 
acalabrutinib, a second generation BTK inhibitor, to investigate its ability to prevent clinical 
allergic reactions to oral food challenge.  Because acalabrutinib is more selective for BTK with 
fewer off-target effects, it has a more favorable safety profile (13).   
 
Peanut and tree nut allergies are among the most common adult food allergens, and ingestion 
reactions can often be the most severe and are the most likely to be life-threatening (1).  
Additionally, peanut and tree nut allergies are usually life-long.  Young adults and adolescents 
have a higher rate of accidental exposure and mortality as a result of food allergies than younger 
children, so there is an unmet need for therapies directed at food allergy, especially in this 
population (14).  Therefore, we have chosen peanut and tree nut allergies in an effort to 
investigate a treatment that has the potential to benefit the most individuals with a potentially 
severe allergy.  
 
2.0 
STUDY OBJECTIVES 
 
2.1 
Primary Objective 
 
2.1.1 
To determine if short term administration of acalabrutinib to adults with peanut or 
tree nut allergy will reduce allergen skin test reactions and raise oral challenge 
thresholds 
 
2.2 
Secondary Objectives  
 
2.2.1 
To describe frequency and severity of the adverse events associated with 
acalabrutinib when administered in a short course of 2 days 
 
2.2.2 
To determine if 2 days of acalabrutinib have any impact on blood levels of total 
and food-specific IgE, and numbers of circulating eosinophils and basophils. 
 
2.3 
Endpoints 
 
Primary Objective 2.1.1: The highest dose of food allergen that is tolerated (i.e. does not 
cause an objective clinical reaction) during a SBPCFC  both before and while taking 
acalabrutinib, as defined by objective clinical findings on physical exam before/after each 
dose of food ingested using a published graded scoring system (15).  Secondary 
endpoints include change in severity of clinical reaction as defined above SPT wheal size 
as defined by a decrease in wheal and/or flare diameter of skin prick tests to peanut/tree 

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nut extracts, and percent activation of basophils from BAT as assessed by the percent of 
basophils with CD63 surface upregulation by flow cytometry.   
 
Secondary Objective 2.2.1:  Adverse events, including bleeding, cytopenias, 
hepatotoxicity, and renal toxicity will be monitored using laboratory testing and clinical 
exam as outlined in Section 4.2.  
 
Secondary Objective 2.2.2:  Change in blood levels of total and food-specific IgE, and 
numbers of circulating eosinophils and basophils as assessed by laboratory testing of 
peripheral blood samples. 
 
Exploratory Objective:  Peripheral blood will be analyzed for mast cell and basophil 
microparticles before, during, and after SBPCFC.   
 
3.0 
SUBJECT ELIGIBILITY 
 
Subjects must meet all of the inclusion and exclusion criteria to be enrolled in the study.  Study 
procedures may not begin until a subject has signed the informed consent form. 
 
3.1 
Inclusion Criteria 
 
3.1.1 
History of IgE-mediated food allergy to peanut or tree nut.  
 
3.1.2 
Male or female age ≥ 18 years. 
 
3.1.3 
Positive skin prick test to the trigger food(s), either peanut and/or tree nut(s). 
 
3.1.4 
Objective clinical reaction to 1,044 mg or less of the food allergen during baseline 
oral food challenge  
 
3.1.5 
Adequate organ and marrow function as defined below: 
- leukocytes 
 
≥ 3,000/mcL 
- absolute neutrophil count ≥ 1,500/mcL 
- hemoglobin  
 
≥ 10 g/dL 
- platelets  
 
≥ 100,000/mcl 
- total bilirubin 
 
within normal institutional limits 
- AST(SGOT)/ALT(SPGT) within normal institutional limits 
- Creatinine  
 
within normal institutional limits 
 
 
3.1.6 
Women of child bearing potential must agree to two forms of highly effective 
contraception (hormonal, device, or barrier method of birth control; abstinence) 
prior to study entry, for the duration of study participation, and for 7 days 
following completion of acalabrutinib therapy.  Should a woman become 
pregnant or suspect she is pregnant while participating in this study, she should 
inform the Principal Investigator and her treating physician immediately. 
 
3.1.5.1 A female of child bearing potential is any woman (regardless of sexual 
orientation, having undergone a tubal ligation, or remaining celibate by 
choice) who meets the following criteria: 
• 
Has not undergone a hysterectomy or bilateral oophorectomy; or 
• 
Has not been naturally postmenopausal for at least 12 consecutive 
months (i.e., has had menses at any time in the preceding 12 
consecutive months). 
 
3.1.7 
Ability to understand and the willingness to sign a written informed consent. 

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3.1.8 
Ability to clearly understand and speak English at an 8th grade reading level. For 
safety reasons, subjects must speak English due to the anticipated need for clear 
and timely communication with investigators and the study team in emergency 
situations, since the investigators and study team are English speaking. 
 
3.2 
Exclusion Criteria 
 
3.2.1 
Subjects who have been on immunomodulatory therapies or oral corticosteroids 
within 1 month prior to study participation will be excluded, and those taking 
antihistamines must stop these drugs for one week prior to enrollment and must 
refrain from taking antihistamines during the duration of the study so as not to 
interfere with SPT responses or clinical responses during SBPCFC. 
 
3.2.2 
Subjects with symptoms consistent with food reactions other than type 1 
hypersensitivity (such as atopic dermatitis, eosinophilic esophagitis and any other 
non-IgE-mediated food sensitivities) will be excluded. 
 
3.2.3 
History of allergic reactions to study drug or reactions attributed to compounds of 
similar chemical or biologic composition to acalabrutinib, including active product 
or excipients. 
 
3.2.4 
Clinically significant cardiovascular disease such as symptomatic arrhythmias, congestive 
heart failure, or myocardial infarction within 6 months of screening, or any Class 3 or 4 
cardiac disease as defined by the New York Heart Association Functional Classification.  
 
3.2.5 
Psychiatric illness/social situations that would limit compliance with study requirements. 
 
3.2.6 
Subjects must not be pregnant or nursing due to the potential for congenital 
abnormalities and the potential of this regimen to harm nursing infants. 
 
3.2.7 
Subjects  requiring or receiving vitamin K antagonists, those with active bleeding, and 
those with history of bleeding disorder (e.g. Von Willebrand’s disease) or prothrombin 
time (PT)/INR or aPTT (in the absence of lupus anticoagulant) >2x upper limit of normal.   
 
3.2.8 
Subjects with history of idiopathic urticaria, dermatographism, idiopathic or 
unexplained anaphylaxis, or anaphylaxis (to foods or otherwise) resulting in 
intubation, prolonged hypotension, or neurological sequelae 
 
3.2.9 
Requires treatment with a strong cytochrome P450 3A4 (CYP3A4) inhibitor/inducer.or 
proton pump inhibitor.  Note: Subjects receiving proton pump inhibitors who switch to H2-
receptor antagonists or antacids are eligible for enrollment to this study. 
 
3.2.10 Subjects found to have active or latent Hepatitis B or C infection based on laboratory 
testing. Subjects who are hepatitis B core antibody (anti-HBc) positive and who are 
hepatitis B surface antigen (HBsAg) negative will need to have a negative polymerase 
chain reaction (PCR) and must be willing to undergo DNA PCR testing during the study 
to be eligible.  Those who are HBsAg positive or hepatitis B PCR positive will be 
excluded.  Subjects who are hepatitis C antibody positive will need to have a negative 
PCR result to be eligible.  Those who are hepatitis C PCR positive will be excluded. 
 
 
3.2.11 Known history of infection with HIV or any active significant infection (eg, bacterial, viral, 
or fungal). 
3.2.12 Major surgical procedure within 28 days of first dose of study drug.  Note:  If a subject 
had major surgery, they must have recovered adequately from any toxicity and/or 

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complications from the intervention before the first dose of study drug. 
3.2.13 History of significant cerebrovascular disease/event, including stroke or intracranial 
hemorrhage, within 6 months before the first dose of study drug. 
3.2.14 Presence of a gastrointestinal ulcer diagnosed by endoscopy within 3 months before 
screening. 
3.2.15 Uncontrolled AIHA (autoimmune hemolytic anemia) or ITP (idiopathic thrombocytopenic 
purpura). 
3.2.16 Prior malignancy (or any other malignancy requiring active treatment), except for 
adequately treated basal cell or squamous cell skin cancer, in situ cervical cancer. 
3.2.17 Has diffculty with or is unable to swallow oral medication, or has significant 
gastrointestinal disease that would limit absorption of oral medication. 
3.2.18 Concurrent participation in another therapeutic clinical trial.   
 
3.3 
Study Stopping Criteria 
 
The study will be stopped if 2 or more subjects experience a serious adverse event, 
excluding adverse events that would be expected within the context of skin testing or oral 
food challenge, including medical treatment of clinical reactions during food challenge.  
Toxicities (section 4.2) and adverse events (section 7.0) are listed below.  Monitoring is 
described in section 8.0.  
 
3.4 
Recruitment 
 
Adults with a diagnosis of peanut or tree nut allergy by a board-certified allergist-
immunologist will be identified as potential subjects from the allergy clinics and electronic 
medical record database search at the Johns Hopkins Hospital system.  The treating 
physician will ask potential subjects for permission to be contacted by study personnel.  
Potential subjects will then be contacted by authorized study personnel and given time to 
review the study information and consider participation.  If necessary, recruitment will be 
extended to include recruiting from local food allergy support groups and advertising on 
social media. 
 
4.0 
TREATMENT PLAN 
 
4.1 
Treatment Dosage and Administration 
 
Prior studies in food-allergic subjects suggest that two standard (420 mg) doses (two 
days) of ibrutinib, an irreversible BTK inhibitor, are sufficient to completely inhibit BAT 
and suppress food SPT wheal size by an average of 77% (44% of all food SPTs became 
negative) (16).  Additional doses of ibrutinib (up to 7 days) did not offer further 
suppression of SPTs, and BAT remained completely abolished for the duration of 
treatment.  Therefore, we propose to extend these findings to acalabrutinib, another 
selective irreversible BTK inhibitor.  Ten subjects will take acalabrutinib 100 mg twice 
daily by mouth (every 12 hours) for 2 days.  Each subject will be enrolled for a minimum 
of 3 months (including the follow-up visit for toxicity monitoring). 
 
Acalabrutinib is approved as a daily medication for another indication, and we propose to 
use the drug in adults only at its approved oral dosing of 100 mg twice daily for a 
maximum of two days.  Dose limiting events include grade 3 or 4 hematologic events, 
grade 3 or 4 hepatotoxicity, AST or ALT >3 times normal range, creatinine > 2.5 times 
normal range.  Dose-limiting toxicity (DLT) evaluation will be performed throughout the 
study.  
 
The capsules should be swallowed intact with water.  Subjects should not attempt to 
open capsules or dissolve them in water.  Acalabrutinib can be taken with or without food. 

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If a dose is missed, it can be taken up to 3 hours after the scheduled time with a return to 
the normal schedule with the next dose.  If it has been > 3 hours, the dose should not be 
taken and the subject should take the next dose at the scheduled time.  The missed dose 
will not be made up and must be returned to the site at the next scheduled visit. 
 
In healthy volunteers, co-administration with CYP3A inhibitors increased Cmax, and 
therefore the drug will not be concomitantly administered with strong inhibitors or 
inducers of CYP3A.  
 
4.2 
Toxicities and Dosing Delays 
 
Although toxicity is not expected given that subjects will only receive two days of drug, 
any subject who receives treatment on this protocol will be evaluable for toxicity.  Each 
subject will be assessed for the development of toxicity according to the Time and Events 
table (Section 5.4).  
 
At each visit, subjects will be monitored for toxicities.  This study will follow the FDA 
Guidance For Industry (“Toxicity grading scale for healthy adult and adolescent 
volunteers enrolled in preventative vaccine clinical trials”) grading system for adverse 
events (17).  If a subject develops a grade 3 or 4 nausea, vomiting, diarrhea, hematologic 
toxicity, renal toxicity, or hepatotoxicity, or any other Grade 4 toxicity or unmanageable 
Grade 3 toxicity, the medication will be stopped.  Protocols for specific toxicities are listed 
below.  
 
Hematological Toxicities:  ANC < 1,500/µL or platelets < 100,000/µL will result in 
discontinuation of protocol therapy.  
 
 
Renal Toxicity:  Creatinine will be monitored per protocol.  Creatinine levels >2.5 times 
the normal limit will result in discontinuation of protocol therapy.   
 
Hepatotoxicity:  Grade 3 hepatotoxicity, or an increase in AST or ALT >3 times upper limit 
of normal, will result in discontinuation in protocol therapy.  
 
4.3 
Concomitant Medications/Treatments 
 
At the systemic exposure levels expected in this study, acalabrutinib inhibition of CYP 
metabolism is not anticipated. 
 
However, acalabrutinib is metabolized by CYP3A.  Concomitant administration of 
acalabrutinib with a strong CYP3A and P-glycoprotein (P-gp) inhibitor, itraconazole 
increased exposure by approximately 5-fold.  Conversely, concomitant administration of 
acalabrutinib with a strong CYP3A inducer, rifampin, decreased acalabrutinib exposure 
and could reduce efficacy.  Consequently, the concomitant use of strong 
inhibitors/inducers of CYP3A should be avoided when possible.   
 
If medically justified, subjects may be enrolled if such inhibitors or inducers can be 
discontinued or alternative drugs that do not affect these enzymes can be substituted 
within 7 days before first dose of study drug.   
 
The effect of agents that reduce gastric acidity (eg, proton pump inhibitors or antacids) on 
acalabrutinib absorption was evaluated in a healthy volunteer study (ACE-HV-004).  
Results from this study indicate that subjects should avoid the use of calcium carbonate 
containing drugs or supplements for a period of at least 2 hours before and at least 2 
hours after taking acalabrutinib.  Use of omeprazole, esomeprazole, lansoprazole or any 

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other proton pump inhibitors while taking acalabrutinib is not recommended due to a 
potential decrease in study drug exposure.  However, the decision to treat with proton-
pump inhibitors during the study is at the investigator’s discretion, with an understanding 
of the potential benefit to the subject’s gastrointestinal condition and a potential risk of 
decreased exposure to acalabrutinib. 
 
Although the effect of H2-receptor antagonists (such as famotidine or ranitidine) on 
acalabrutinib absorption has not been evaluated, if treatment with an H2-receptor 
antagonist is required, the H2-receptor antagonist should be taken approximately 2 hours 
after an acalabrutinib dose. 
 
The following concomitant medications/treatments are not allowed: immunomodulatory 
therapies, oral corticosteroids within 1 month of study participation; those taking 
antihistamines must stop these drugs for one week prior to enrollment and must refrain 
from taking antihistamines during the duration of the study so as not to interfere with SPT 
responses; and any other prior investigational interventions in the past 6 months.   
 
4.4 
Duration of Therapy 
 
Treatment will last for 2 days.  Reasons the study medication may be discontinued 
include: 
• 
Intercurrent illness that prevents further administration of treatment; 
• 
Unacceptable adverse event(s); 
• 
Subject decides to withdraw from the study; or 
• 
General or specific changes in the subject’s condition render the subject 
unacceptable for further treatment in the judgment of the investigator. 
 
4.5 
Duration of Follow Up 
 
Subjects will be followed for 30 days after cessation of acalabrutinib treatment. Subjects 
will be removed from therapy when any of the criteria listed in Section 5.5 apply. The 
Principal Investigator will be notified.  The reason for study removal and the date the 
subject was removed will be documented in the Case Report Form.  Subjects removed 
from treatment for unacceptable adverse events will be followed until resolution or 
stabilization of the adverse event. 
 
4.6 
Subject Replacement 
 
Subjects who withdraw voluntarily from the study, but were tolerating the drug will be 
replaced.  Subjects who drop out or miss doses due to toxicity will not be replaced since 
these subjects will be considered to have experienced a dose limiting toxicity. 
 
 
5.0 
STUDY PROCEDURES 
 
5.1 
Screening/Baseline Procedures 
 
Assessments performed exclusively to determine eligibility for this study will be done only 
after obtaining written informed consent.  Assessments performed for clinical indications 
(not exclusively to determine study eligibility) may be used for baseline values even if the 
studies were done before informed consent was obtained.  
 
All screening procedures must be performed within 14 days prior to baseline SBPCFC. 
The screening procedures include: 

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5.1.1 Informed Consent 
 
Informed consent will be obtained per IRB and GCP guidelines.  
5.1.2 Medical history 
 
Complete medical and surgical history, including history of infections will be 
obtained.  
5.1.3 Demographics 
 
Age, gender, race, ethnicity will be obtained. 
5.1.4 Review subject eligibility criteria   
Subjects will be screened to ensure they meet inclusion and exclusion criteria, 
including history and prior laboratory testing consistent with a diagnosis of IgE-
mediated food allergy.  
5.1.5 Review previous and concomitant medications 
 
Medications will be reviewed and documented.  
5.1.6 Physical exam including vital signs, height, and weight 
Vital signs (temperature, pulse, respirations, blood pressure), height, weight and 
basic full physical exam will be performed by study physicians.  
5.1.7 Adverse event assessment 
Baseline kidney, liver, and heart function will be assessed. This will include a 
CMP, CBC, Hepatitis B and C testing, and EKG.  See section 6 for Adverse Event 
monitoring and reporting. 
5.1.8 Hematology 
 
Baseline CBC will be performed.  
5.1.9 Blood draw for correlative studies 
 
See Section 9.0 for details. 
5.1.10 Serum chemistries 
Comprehensive metabolic panel (CMP) to include: albumin, alkaline phosphatase, 
ALT/SGPT, AST/SGOT, BUN, creatinine, electrolytes (sodium, potassium, 
calcium, chloride, bicarbonate), glucose, and total bilirubin. 
5.1.11 Pregnancy test (for females of child bearing potential) 
 
See section 3.1.6.1 for definition.  
5.1.12 Immunoglobulin levels  
Total quantitative immunoglobulins (QUIGs), including IgG, IgA, IgM, and IgE, as 
well as serum specific IgE for peanut/tree nuts will be monitored  
5.1.13 Spirometry  
To be performed before SBPCFC if the patient has a history of asthma or 
exercise-induced bronchospasm. 
 
5.2 
Procedures During Treatment 
5.2.1 
Screening/Baseline visit (Visit 1) 
 
 
 
Screening: 
• Physical exam, vital signs 
• Urine pregnancy test (if applicable) 
• CBC w/diff 
• CMP 
• Hepatitis B and C testing 
• Total immunoglobulin levels  
• EKG 
• Spirometry (only if the subject has a history of asthma)  
• SPT to relevant foods, saline and histamine controls 
• BAT 
 
Procedures (to be done only if labs and EKG are within normal limits) 
• Single-blind placebo controlled oral food challenge (SBPCFC) as below 

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• Microparticle analysis  
 
All oral food challenges shall be performed as single (patient)-blinded, placebo-
controlled food challenges (SBPCFC) according to standard published guidelines (18, 
19).  Placebo challenge and active challenge will be performed in that order on the 
same day if the subject has no symptoms during the first (placebo) challenge.  If a 
reaction requiring treatment occurs during the placebo challenge, the next challenge 
should not be performed until the symptoms have subsided.  Food challenge to peanut 
or tree nut protein will be performed according to standard operating procedure as 
previously published in the food allergy field (15).   
 
Actions to minimize risk of severe allergic reactions during oral food challenge): 
Prior to the study visit, subjects will be told to contact study staff about asthma and 
seasonal allergy symptoms, fever, acute infections including upper respiratory 
infections, rashes, or change in status of chronic medical conditions.  Study visits will be 
rescheduled in the event that any of these occur.  Study staff will contact subjects 1-3 
days before study visits to inquire about recent symptoms. 
 
All food challenges will be conducted in the Clinical Research Unit within the Johns 
Hopkins Bayview Hospital.  Intravenous access will be obtained before the food 
challenge begins.  Emergency medications (epinephrine, diphenhydramine, cetirizine, 
albuterol, ranitidine) will be immediately accessible in the subject’s room.  All SBPCFCs 
will be conducted by trained personnel and supervised by a clinician with experience in 
performing SBPCFCs and trained in the treatment of anaphylaxis.  Spirometry 
(assessment of pulmonary function) will be performed prior to the onset of the 
SBPCFC.  Vital signs and pulse oximetry will be obtained at baseline and prior to each 
dose of the food allergen and q15 minutes thereafter with an observation period of at 
least 2 hours after the last dose is consumed (longer should a reaction occur).  The 
SBPCFC will begin with a low dose, which will increase incrementally.  At physician 
discretion, based upon history and possible subjective or very mild objective symptoms 
during a challenge, dosing intervals may be extended.  The procedure will be stopped 
when clear objective symptoms are noted.  
 
Subjects will be discharged after a period of observation appropriate to the nature of the 
symptoms.  They will receive information regarding possible delayed manifestations, 
emergency contact information and home treatment.  All subjects will be provided with 
an epinephrine auto-injector to take home (if they do not have one already) and 
instructions on how and when to use it.  
 
After a minimum recovery period of 2 weeks, subjects will then take acalabrutinib as 
described above, and return on the second day of treatment for Visit 2 (see below).   
 
5.2.2 
Visit 2 
• Physical exam, vital signs 
• Urine pregnancy test (if applicable) 
• CBC w/diff 
• CMP 
• Total immunoglobulin levels  
• EKG 
• Spirometry (only if the subject has a history of asthma)  
• SPT to relevant foods, saline and histamine controls 
• BAT 
• SBPCFC (performed as described above) 
• Microparticle analysis  
 

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5.3 
Follow-up Procedures 
 
Toxicity monitoring (including hematology, serum chemistries, QUIGs, ssIgE) and other 
study procedures will be repeated at 3 months after cessation of acalabrutinib; if at this 
time toxicity related lab results are not yet back to baseline, they will be repeated at 6 
months.   
• 
Physical exam, vital signs 
• 
CBC w/diff 
• 
CMP 
• 
Total immunoglobulin levels  
• 
SPT to relevant foods, saline and histamine controls 
• 
BAT 
 
 
5.4 
Time and Events Table 
 
 
Visit 
1 
2 
3 
 
 
Screening 
/ Day 0 
Day 2 
Follow up 
(30 days 
after Visit 
2) 
Informed 
Consent 
X 
 
 
Skin prick testing 
X 
X 
X 
Basophil 
activation test 
X 
X 
X 
SBPC food 
challenge 
X 
X 
 
History and PE 
X 
X 
X 
Toxicity (include 
DLT) Evaluations 
X 
X 
X 
CBC 
X 
X 
X 
CMP 
X 
X 
X 
EKG 
X 
X 
 
Urine pregnancy 
testb 
X 
X 
X 
Microparticle 
analysis 
X 
X 
X 
 
b Females of child-bearing potential only 
 
5.5 
Removal of Subjects from Study  
 
Subjects can be taken off the study treatment and/or study at any time at their own 
request, or they may be withdrawn at the discretion of the investigator for safety, 
behavioral or administrative reasons. The reason(s) for discontinuation will be 
documented and may include: 
5.5.1 
Subject voluntarily withdraws from treatment (follow-up permitted);  
5.5.2 
Subject withdraws consent (termination of treatment and follow-up); 
5.5.3 
Subject is unable to comply with protocol requirements; 
5.5.4 
Subject experiences toxicity that makes continuation in the protocol unsafe; 
5.5.5 
Treating physician judges continuation on the study would not be in the subject’s 
best interest; 

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5.5.6 
Subject becomes pregnant (pregnancy to be reported along same timelines as a 
serious adverse event); or 
5.5.7 
Lost to follow-up. If a research subject cannot be located after a period of 2 
months, the subject may be considered “lost to follow-up.” All attempts to contact 
the subject during the 2 months must be documented. 
 
6.0        RESPONSE CRITERIA 
 
6.1 
Safety/tolerability 
 
Analyses will be performed for all subjects having received at least one dose of study 
drug. CBC, CMP, EKG will be performed on all subjects to monitor for toxicities and 
safety. 
 
6.2  
Efficacy 
 
Increase in quantity of food that can be ingested on oral food challenge, decreased 
severity of reaction during oral food challenge, decrease in skin prick test responses and 
decreased basophil activation will be used to assess efficacy of the treatment, which will 
be assessed as described in Section 2.3.  
 
7.0        ADVERSE EVENTS 
 
7.1 
Risks Associated with Experimental Therapy 
 
The following summarizes the experience with acalabrutinib in hematologic cancer 
studies.  For the most recent safety update, please refer to the current Study Agent 
Prescribing Information.  (This drug is currently FDA approved for alternate indications. 
Sections below are from the current package insert).  
 
7.1.1 
Contraindications 
 
 
No contraindications are known for acalabrutinib.    
 
7.1.2  
Warnings and Precautions for Use 
 
As the subjects enrolled in this study will be generally healthy except for food allergy or 
other allergic disorders, and on acalabrutinib for short duration (2 days), we expect 
adverse events to be milder and less frequent, if at all, compared to prior studies of 
acalabrutinib in cancer trials.  However, for completeness, adverse events as 
documented with chronic use are detailed below.  
 
7.1.2.1 Hemorrhage 
 
Serious hemorrhagic events, including central nervous system, respiratory, and 
gastrointestinal hemorrhage, have been reported in clinical trials with 
acalabrutinib; some of these bleeding events resulted in fatal outcomes.  Grade 3 
or higher bleeding events, including gastrointestinal, intracranial, and epistaxis 
have been reported in 2% of patients. Overall, bleeding events including bruising 
and petechiae of any grade occurred in approximately 50% of patients with 
hematological malignancies.  
 
The mechanism for hemorrhage is not well understood.  Acalabrutinib may 
further increase the risk of hemorrhage in patients receiving antiplatelet or 
anticoagulant therapies and patients should be monitored for signs of bleeding.  

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Consider the benefit-risk of withholding acalabrutinib for 3-7 days pre- and post-
surgery depending on the surgery and the risk of bleeding.   
7.1.2.2 Infections 
 
Serious infections (bacterial, viral or fungal), including fatal events and 
opportunistic infections, have been reported in clinical studies with acalabrutinib.  
The most frequently reported Grade 3 or 4 infection was pneumonia.  Across the 
acalabrutinib clinical development program (including subjects treated with 
acalabrutinib in combination with other drugs), cases of hepatitis B virus (HBV) 
reactivation (resulting in liver failure and death in 1 case) and cases of 
progressive multifocal leukoencephalopathy have occurred in subjects with 
hematologic malignancies.  Monitor patients for signs and symptoms of infection 
and treat as medically appropriate. 
7.1.2.3 Cytopenias 
 
Treatment-emergent Grade 3 or 4 cytopenias including neutropenia, anemia, and 
thrombocytopenia have occurred in clinical studies with acalabrutinib.   Subjects 
should be closely monitored as appropriate. 
7.1.2.4 Second Primary Malignancies 
 
Events of second primary malignancies, including non-skin carcinomas, have 
been reported in clinical studies with acalabrutinib.  The most frequently reported 
second primary malignancy was skin cancer.  Advise protection from sun 
exposure. 
7.1.2.5 Atrial Fibrillation and Flutter 
 
Events of atrial fibrillation/flutter have been reported in clinical studies with 
acalabrutinib, particularly in subjects with cardiac risk factors, hypertension, 
diabetes mellitus, acute infections, and a previous history of atrial fibrillation. 
 
The mechanism for atrial fibrillation is not well understood. 
7.1.3 Embryo-Fetal Toxicity 
 
Based on findings in animals, acalabrutinib can cause fetal harm when administered to a 
pregnant woman.  In female rats, acalabrutinib administered orally ≥ 100 mg/kg/day 
produced maternal toxicity and 100 mg/kg/day resulted in decreased fetal body weights 
and delayed skeletal ossification.  Acalabrutinib and its active metabolite were present in 
the milk of lactating rats.  Advise women to avoid becoming pregnant while taking 
acalabrutinib.  If this drug is used during pregnancy or if the patient becomes pregnant 
while taking this drug, the patient will be apprised of the potential hazard to a fetus. 
 
7.1.4 
Interaction with other medications 
 
At the systemic exposure levels expected in this study, acalabrutinib inhibition of 
CYP metabolism is not anticipated. 
 
However, acalabrutinib is metabolized by CYP3A.  Concomitant administration of 
acalabrutinib with a strong CYP3A and P-glycoprotein (P-gp) inhibitor, 
itraconazole increased exposure by approximately 5-fold.  Conversely, 
concomitant administration of acalabrutinib with a strong CYP3A inducer, 
rifampin, decreased acalabrutinib exposure and could reduce efficacy.  
Consequently, the concomitant use of strong inhibitors/inducers of CYP3A be 
avoided when possible.   
 

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If medically justified, subjects may be enrolled if such inhibitors or inducers can 
be discontinued or alternative drugs that do not affect these enzymes can be 
substituted within 7 days before first dose of study drug.  If a subject requires a 
strong CYP3A4 inhibitor while on study, the subject should be monitored closely 
for any potential toxicities. 
 
The effect of agents that reduce gastric acidity (eg, proton pump inhibitors or 
antacids) on acalabrutinib absorption was evaluated in a healthy volunteer study 
(ACE-HV-004).  Results from this study indicate that subjects should avoid the 
use of calcium carbonate containing drugs or supplements for a period of at least 
2 hours before and at least 2 hours after taking acalabrutinib.  Use of 
omeprazole, esomeprazole, lansoprazole or any other proton pump inhibitors 
while taking acalabrutinib is not recommended due to a potential decrease in 
study drug exposure.  However, the decision to treat with proton-pump inhibitors 
during the study is at the investigator’s discretion, with an understanding of the 
potential benefit to the subject’s gastrointestinal condition and a potential risk of 
decreased exposure to acalabrutinib. 
 
Although the effect of H2-receptor antagonists (such as famotidine or ranitidine) 
on acalabrutinib absorption has not been evaluated, if treatment with an H2-
receptor antagonist is required, the H2-receptor antagonist should be taken 
approximately 2 hours after an acalabrutinib dose. 
 
7.1.5 
Adverse Reactions 
 
As per package insert (21), adverse reactions are listed in Section 7.1.2.  Among 
1029 pooled patients from clinical trials (820 patients taking acalabrutinib 
monotherapy across 6 trials and 209 patients taking acalabrutinib plus 
obinutuzumab across 2 trials), most of whom (88%) were exposed to 
acalabrutinib for at least 6 months, adverse reactions in ≥30% of patients 
included anemia, neutropenia, upper respiratory tract infection, 
thrombocytopenia, headache, diarrhea, and musculoskeletal pain. 
 
 
Of note, patients in the trial references took acalabrutinib for much longer 
duration that we propose in this study.  Our prior work using ibrutinib (which 
has a less favorable side effect profile) in adults with food allergy demonstrated 
no toxicity or adverse effects from up to 7 days of treatment (11), suggesting that 
adverse effects from BTK inhibitors may be mitigated with short-term use rather 
than chronic treatment.   
 
8.0 
ADVERSE EVENT MONITORING 
 
Adverse event data collection and reporting, which are required as part of every clinical trial, are 
done to ensure the safety of Subjects enrolled in the studies as well as those who will enroll in 
future studies using similar agents. Adverse events are reported in a routine manner at scheduled 
times during a trial. Additionally, certain adverse events must be reported in an expedited manner 
to allow for optimal monitoring of subject safety and care.  
 
All subjects experiencing an adverse event, regardless of its relationship to study drug/device, will 
be monitored until:  
• 
the adverse event resolves or the symptoms or signs that constitute the adverse event 
return to baseline;  
• 
any abnormal laboratory values have returned to baseline;  
• 
there is a satisfactory explanation other than the study drug for the changes observed; or 

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• 
death. 
 
8.1 
Definitions 
 
8.1.1 
Definition of Adverse Event 
 
An adverse event (AE) is any untoward medical occurrence in a subject receiving 
study treatment and which does not necessarily have a causal relationship with 
this treatment. An AE can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of an experimental intervention, whether or not related to 
the intervention.  
 
8.1.2  
Severity of Adverse Events 
 
This study will follow the FDA Guidance For Industry (“Toxicity grading scale for 
healthy adult and adolescent volunteers enrolled in preventative vaccine clinical 
trials”) grading system for adverse events (17).   
 
Briefly, the severity of an AE is graded as follows: 
 
Mild (grade 1): the event causes discomfort without disruption of normal daily 
activities. 
 
Moderate (grade 2): the event causes discomfort that affects normal daily 
activities. 
 
Severe (grade 3): the event makes the subject unable to perform normal daily 
activities or significantly affects his/her clinical status. 
 
Life-threatening (grade 4): the subject was at risk of death at the time of the 
event. 
 
Fatal (grade 5): the event caused death. 
 
8.1.3 
Serious Adverse Events 
 
A “serious” adverse event is defined in regulatory terminology as any untoward 
medical occurrence that: 
8.1.3.1       Results in death. 
8.1.3.2       Is life-threatening. 
(the subject was at risk of death at the time of the event; it does not 
refer to an event that hypothetically might have caused death if it were 
more severe). 
8.1.3.3       Requires in-subject hospitalization or prolongation of existing 
hospitalization for ≥ 24 hours. 
8.1.3.4       Results in persistent or significant disability or incapacity. 
8.1.3.5       Is a congenital anomaly/birth defect. 
8.1.3.6       Is an important medical event. 
 
Adverse Events (AEs) for malignant tumors reported during a study should 
generally be assessed as Serious AEs. If no other seriousness criteria apply, the 
‘Important Medical Event’ criterion should be used. In certain situations, however, 
medical judgement on an individual event basis should be applied to clarify that 
the malignant tumor event should be assessed and reported as a Non-Serious 

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AE. For example, if the tumor is included as medical history and progression 
occurs during the study, but the progression does not change treatment and/or 
prognosis of the malignant tumor, the AE may not fulfill the attributes for being 
assessed as Serious, although reporting of the progression of the malignant 
tumor as an AE is valid and should occur. Also, some types of malignant tumors, 
which do not spread remotely after a routine treatment that does not require 
hospitalization, may be assessed as Non-Serious; examples include Stage 1 
basal cell carcinoma and Stage 1A1 cervical cancer removed via cone biopsy. 
 
Any event that does not meet the above criteria, but that in the judgment of the 
investigator jeopardizes the subject, may be considered for reporting as a serious 
adverse event. The event may require medical or surgical intervention to prevent 
one of the outcomes listed in the definition of “Serious Adverse Event“. For 
example: allergic bronchospasm requiring intensive treatment in an emergency 
room or at home; convulsions that may not result in hospitalization; development 
of drug abuse or drug dependency. 
 
Pregnancy reporting:  Female subjects of childbearing age reporting conception 
on study and up to 2 days after last dose must stop taking study drugs and must 
notify study staff immediately.  Male subjects with partner conception on study 
must notify study staff immediately.  Any pregnancy that occurs with the subject 
or with the partner of a treated subject as described above during this study will 
be reported, followed to conclusion, and the outcome reported. Any occurrences 
of pregnancy should be reported to the IRB and Regulatory Authorities per 
institutional and/or regulatory guidelines, and to AZ per contractual guidelines. 
 
8.1.4   Adverse Events of Special Interest (AESI) 
 
The following events are adverse events of special interest (AESIs) for subjects 
exposed to acalabrutinib, and must be reported to the sponsors expeditiously 
(see Section 6.4 for reporting instructions), irrespective of regulatory seriousness 
criteria or causality:  Ventricular arrhythmias (e.g., ventricular extrasystoles, 
ventricular tachycardia, ventricular arrhythmia, ventricular fibrillation, etc).  
 
8.2 
Steps to Determine If an Adverse Event Requires Expedited Reporting 
 
Step 1: Identify the type of adverse event. 
 
Step 2: Grade the adverse event. 
 
Step 3: Determine whether the adverse event is related to the protocol therapy  
Attribution categories are as follows: 
- Definite – The AE is clearly related to the study treatment. 
- Probable – The AE is likely related to the study treatment. 
- Possible – The AE may be related to the study treatment. 
- Unrelated – The AE is clearly NOT related to the study treatment. 
Note: This includes all events that occur within 30 days of the last dose of protocol 
treatment. Any event that occurs more than 30 days after the last dose of treatment and 
is attributed (possibly, probably, or definitely) to the agent(s) must also be reported 
accordingly. 
 
Step 4:  Determine the prior experience of the adverse event.  
Expected events are those that have been previously identified as resulting from 
administration of the agent. An adverse event is considered unexpected, for expedited 
reporting purposes only, when either the type of event or the severity of the event is not 
listed in: 

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• 
the current known adverse events listed in the Agent Information Section of this 
protocol; 
• 
the drug package insert; 
• 
the current Investigator’s Brochure 
 
8.3 
Reporting Requirements for Adverse Events 
 
The Sponsor will submit appropriate reports to applicable local regulatory agencies and 
to ethics committees (ECs) as per local regulations. The Sponsor will notify AZ in parallel 
with submission to the IRB and concerned Regulatory Authority for Suspected 
Unexpected Serious Adverse Reactions (SUSARs) and within fifteen (15) calendar days 
of awareness for other SAEs or Special Situation Reports using individual unblinded or 
blinded case reports (Contingency SAE Report Form, Institution’s Standard SAE Report 
Form, MedWatch, or CIOMS). New information will be submitted to AZ within the same 
time frame as initial reports.  
Each recorded AE or SAE will be described by its diagnostic term, duration (eg, start and 
end dates), severity, regulatory seriousness criteria, if applicable, suspected relationship 
to the study drug, and any actions taken.  If study drug is discontinued because of an 
SAE, this information must be included in the SAE report. 
 
8.3.1 
Expedited Reporting 
 
• 
The Principal Investigator must be notified within 24 hours of learning of any 
serious adverse events, regardless of attribution, occurring during the study 
or within 30 days of the last administration of the study drug.  
• 
The Johns Hopkins University IRB must be notified within 5 business days 
of any unanticipated problems involving risk to subjects or others (UPIRSO). 
The following events meet the definition of UPIRSO: 
1. Any serious event (injuries, side effects, deaths or other problems), 
which in the opinion of the Principal Investigator was unanticipated, 
involved risk to subjects or others, and was possibly related to the 
research procedures. 
2. Any serious accidental or unintentional change to the IRB-approved 
protocol that alters the level of risk. 
3. Any deviation from the protocol taken without prior IRB review to 
eliminate apparent immediate hazard to a research subject. 
4. Any new information (e.g., publication, safety monitoring report, updated 
sponsor safety report), interim result or other finding that indicates an 
unexpected change to the risk/benefit ratio for the research. 
5. Any breach in confidentiality that may involve risk to the subject or 
others. 
6. Any complaint of a subject that indicates an unanticipated risk or that 
cannot be resolved by the Principal Investigator. 
• 
The FDA should be notified within 7 calendar days of any unexpected fatal 
or life-threatening adverse event with possible relationship to study drug, 
and 15 calendar days of any event that is considered: 1) serious, 2) 
unexpected, and 3) at least possibly related to study participation. 
• 
The Sponsor will submit appropriate reports to applicable local regulatory 
agencies and to ethics committees (ECs) as per local regulations. The 
Sponsor will notify AZ in parallel with submission to the concerned 
Regulatory Authority for Suspected Unexpected Serious Adverse Reactions 
(SUSARs) and other SAEs as individual unblinded or blinded case reports 
(Sponsor’s Standard SAE Report Form, MedWatch, or CIOMS) within 
fifteen (15) calendar days of awareness or quarterly SAE line listings. All 

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SAEs are to be submitted to the AstraZeneca Product Safety mailbox: 
AEMailboxClinicalTrialTCS@astrazeneca.com 
• 
New information will be submitted to AZ within the same time frame as initial 
reports 
 
8.3.2 
Routine Reporting 
 
All other adverse events - such as those that are expected, or are unlikely or 
definitely not related to the study participation - are to be reported annually to the 
FDA as part of regular data submission.  These adverse events will still be 
reported to the sponsor on a monthly basis.   
 
8.3.3 
Stopping Rules  
 
The study will be stopped if 2 or more subjects experience a serious adverse 
event, excluding adverse events that would be expected within the context of 
skin testing or oral food challenge, including medical treatment of clinical 
reactions during food challenge.   
 
9.0 
DRUG INFORMATION 
 
9.1 
Acalabrutinib 100 mg tablets 
• 
Other names for the drug(s)/device: Calquence® 
• 
Classification - type of agent/device: small molecule BTK inhibitor 
 
Mode of action: Acalabrutinib forms a covalent bond with a cysteine residue in the BTK 
active site, leading to inhibition of BTK enzymatic activity.  BTK is a signaling molecule of 
the B-cell antigen receptor (BCR) and cytokine receptor pathways.  BTK’s role in 
signaling through the B-cell surface receptors results in activation of pathways necessary 
for B-cell trafficking, chemotaxis, and adhesion.  Nonclinical studies show that 
acalabrutinib inhibits malignant B-cell proliferation and survival in vivo as well as cell 
migration and substrate adhesion in vitro.  
• 
Storage and stability:  Acalabrutinib is stored at room temperature and the capsules 
are stable.  
• 
Protocol dose: 100 mg twice daily for 2 days 
• 
Preparation: as supplied from the manufacturer 
• 
Route of administration for this study: by mouth 
• 
Incompatibilities:  N/A 
• 
Availability: commercially available by prescription.  Medication will be paid for 
through grant funding, and provided free of charge to subjects.  
• 
Side effects: Most common side effects are anemia, neutropenia, upper respiratory 
tract infection, thrombocytopenia, headache, diarrhea, and musculoskeletal pain .  
Please refer to package insert for comprehensive list of side effects.  
• 
Nursing implications:  Acalabrutinib is category D.  It should not be used in nursing 
mothers.   
 
 
9.2       Return and Retention of Study Drug 
 
Two bottles (120 pills) of study drug will be provided for free by its manufacturer, AZ, and 
obtained by the Research Pharmacy at Johns Hopkins University.  Study drug will 
dispensed as needed, until next follow up.  Therefore, we do not anticipate retention of 
study drug.  However, if there is unused study drug, it will be disposed of in an 
appropriate manner.  
 

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Compliance will be assessed by subject diary and self-report.   
 
10.0 
STATISTICAL CONSIDERATIONS 
 
As this is an open label study, each patient’s responsiveness will be examined compared to 
themselves prior to initiation of treatment.  
 
10.1 
Study Design/Study Endpoints 
 
This is an open label study of acalabrutinib in 10 food-allergic adults to determine the 
effect of pretreatment with acalabrutinib on food allergen reactivity in food-allergic 
subjects given acalabrutinib 100 mg twice daily for 2 days.  The primary endpoint is the 
threshold dose of food at which a subject reacts before (at baseline) and after 
acalabrutinib therapy.  Given the nature of the study, it will be stopped if 2 or more 
patients have a DLT.    
 
10.2 
Sample Size and Accrual 
 
10 subjects allows for 80% power to detect a 3-fold increase (1.1 natural log units; i.e. 1 
food dose escalation) in the threshold food dose assuming that difference in threshold 
from post to pre is normally distributed with a standard deviation of 1.1 natural log units 
using a paired t test with p<0.05 as an approximation (though non-parametric tests will be 
utilized in the final analysis if needed). Patients will be recruited from the allergy clinics at 
Johns Hopkins University with a diagnosis of peanut (or tree nut) allergy by a board-
certified allergist-immunologist.  
 
10.3 
Data Analysis  
 
As this is an open label trial, descriptive statistics will be used.  If appropriate, the primary 
outcome variable of threshold dose will be log transformed, and a paired t test will be 
used.  If assumptions for paired t test are violated, a Wilcoxon non-parametric sign test 
will be used.  Each patient will be compared to him/herself prior to beginning treatment 
and at each time point in the study to assess SPT size, BAT response, and food 
challenge dose threshold and response.  Similar methodologies will be used for analysis 
of adverse events.  
 
11.0 
STUDY MANAGEMENT 
 
11.1 
Conflict of Interest 
 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, 
or financial gain greater than the minimum allowable by their institution, etc.) must have 
the conflict reviewed Johns Hopkins University.   All investigators will follow the University 
conflict of interest policy. 
 
 
11.2 
Institutional Review Board (IRB) Approval and Consent  
 
It is expected that the IRB will have the proper representation and function in accordance 
with federally mandated regulations. The IRB should approve the consent form and 
protocol. 
 
In obtaining and documenting informed consent, the investigator should comply with the 
applicable regulatory requirement(s), and should adhere to Good Clinical Practice (GCP) 
and to ethical principles that have their origin in the Declaration of Helsinki. 
 
 

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Before recruitment and enrollment onto this study, the subject will be given a full 
explanation of the study and will be given the opportunity to review the consent form. 
Each consent form must include all the relevant elements currently required by the FDA 
Regulations and local or state regulations.  Once this essential information has been 
provided to the subject and the investigator is assured that the subject understands the 
implications of participating in the study, the subject will be asked to give consent to 
participate in the study by signing an IRB-approved consent form. 
 
Prior to a subject’s participation in the trial, the written informed consent form should be 
signed and personally dated by the subject and by the person who conducted the 
informed consent discussion. 
 
11.3 
Data Management and Monitoring/Auditing 
 
As has been done previously for the phase 1 study of acalabrutinib (13), adverse events 
will be monitored throughout treatment and toxicities will be assessed using the National 
Cancer Institute Common Toxicity Criteria for AEs, with its latest version, currently 
version 4.03.   
 
11.4 
Adherence to the Protocol 
 
Except for an emergency situation in which proper care for the protection, safety, and 
well-being of the study subject requires alternative treatment, the study shall be 
conducted exactly as described in the approved protocol.  
 
11.4.1 Emergency Modifications 
 
Investigators may implement a deviation from, or a change of, the protocol to 
eliminate an immediate hazard(s) to trial subjects without prior IRB approval.  For 
any such emergency modification implemented, an IRB modification form must 
be completed within five (5) business days of making the change.  
 
11.4.2 Other Protocol Deviations/Violations 
 
All other planned deviations from the protocol must have prior approval by the 
Principal Investigator and the IRB.  According to the IRB, a protocol deviation is 
any unplanned variance from an IRB approved protocol that:  
• 
Is generally noted or recognized after it occurs 
• 
Has no substantive effect on the risks to research participants 
• 
Has no substantive effect on the scientific integrity of the research plan or the 
value of the data collected  
• 
Did not result from willful or knowing misconduct on the part of the 
investigator(s).  
 
An unplanned protocol variance is considered a violation if the variance: 
• 
Has harmed or increased the risk of harm to one or more research 
participants. 
• 
Has damaged the scientific integrity of the data collected for the study. 
• 
Results from willful or knowing misconduct on the part of the investigator(s). 
• 
Demonstrates serious or continuing noncompliance with federal regulations, 
State laws, or University policies. 
 

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If a deviation or violation occurs without prior approval from the Principal 
Investigator, please follow the guidelines below: 
 
Protocol Deviations: Deviations should be summarized and reported to the IRB 
at the time of continuing review. 
 
Protocol Violations: Study personnel should report violations within two (2) 
week of the investigator becoming aware of the event using the same IRB online 
mechanism used to report Unanticipated Problems.  
 
11.5 
Amendments to the Protocol 
 
Should amendments to the protocol be required, the amendments will be originated and 
documented by the Principal Investigator.  It should also be noted that when an 
amendment to the protocol substantially alters the study design or the potential risk to the 
subject, a revised consent form might be required.  The written amendment, and if 
required the amended consent form, must be sent to the IRB for approval prior to 
implementation.  
 
11.6  
Confidentiality 
 
The specimens will be coded and data will be stored in the Allergy-Immunology Research 
Labs, Mezzanine, McGaw Pavilion, or in locked file cabinets and/or on password 
protected computers.  Only the study personnel will have access to the specimens and 
data generated.  The PI and the study team members will be responsible for receipt or 
transmission of the data and specimens. The specimens will be maintained until 
exhausted or until the study ends.  If there are remaining samples at the end of the study, 
any identifying information will be removed from the sample vial and the specimens will 
be treated with 10% bleach and discarded per standard practice.  Unused portions of the 
specimens may be shared with other members of the University community.  The original 
data will remain with the PI in the Allergy Division.  No PHI will be shared.  
 
11.7 
Provisions to Protect the Privacy Interests of Subjects 
 
Certain other people besides the PI and his research team may review the results of the 
study, such as administrative staff members from the Office of Research, Office of 
Research Integrity and members of the Institutional Review Board, and members of the 
Office for Human Research Protections.  There is a slight risk that a subject’s privacy 
might be at risk.  All of the above mentioned people in the respective capacities are 
obligated to protect a subject’s privacy and have training and certification in the 
safeguarding of the patient’s privacy.  All data and results will be maintained in the 
Allergy-Immunology Research Labs, Mezzanine, McGaw Pavilion, in locked file cabinets 
and/or on password protected computers. 
  
To help the subjects feel at ease, these safeguards are outlined in the consent form. 
  
As far as information in medical records, only licensed medical personnel would have 
permission to access the information. 
 
 
11.8 
Record Retention 
 
Study documentation includes all Case Report Forms, data correction forms or queries, 
source documents, Sponsor-Investigator correspondence, monitoring logs/letters, and 
regulatory documents (e.g., protocol and amendments, IRB correspondence and 
approval, signed subject consent forms). 

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Source documents include all recordings of observations or notations of clinical activities 
and all reports and records necessary for the evaluation and reconstruction of the clinical 
research study. 
 
Government agency regulations and directives require that the study investigator must 
retain all study documentation pertaining to the conduct of a clinical trial. In the case of a 
study with a drug seeking regulatory approval and marketing, these documents shall be 
retained for at least two years after the last approval of marketing application in an 
International Conference on Harmonization (ICH) region.  In all other cases, study 
documents should be kept on file until three years after the completion and final study 
report of this investigational study. 
 
11.9 
Obligations of Investigators 
 
The Principal Investigator is responsible for the conduct of the clinical trial at the site in 
accordance with Title 21 of the Code of Federal Regulations and/or the Declaration of 
Helsinki.  The Principal Investigator is responsible for personally overseeing the 
treatment of all study subjects.  The Principal Investigator must assure that all study site 
personnel, including sub-investigators and other study staff members, adhere to the 
study protocol and all FDA/GCP/NCI regulations and guidelines regarding clinical trials 
both during and after study completion. 
 
The Principal Investigator at each institution or site will be responsible for assuring that all 
the required data will be collected and entered onto the Case Report Forms. Periodically, 
monitoring visits will be conducted and the Principal Investigator will provide access to 
his/her original records to permit verification of proper entry of data.  At the completion of 
the study, all case report forms will be reviewed by the Principal Investigator and will 
require his/her final signature to verify the accuracy of the data. 
 
12.0 
REFERENCES 
 
1. 
Sicherer SH, Sampson HA. Food allergy: Epidemiology, pathogenesis, diagnosis, and treatment. 
The Journal of allergy and clinical immunology. 2014;133(2):291-307. 
2. 
Branum AM, Lukacs SL. Food allergy among U.S. children: trends in prevalence and 
hospitalizations. NCHS Data Brief. 2008(10):1-8. 
3. 
Sampson HA. Fatal food-induced anaphylaxis. Allergy. 1998;53(46 Suppl):125-30. 
4. 
Gupta R, Holdford D, Bilaver L, Dyer A, Holl JL, Meltzer D. The economic impact of childhood 
food allergy in the United States. JAMA Pediatr. 2013;167(11):1026-31. 
5. 
Burger JA. Bruton's tyrosine kinase (BTK) inhibitors in clinical trials. Curr Hematol Malig Rep. 
2014;9(1):44-9. 
6. 
Whang JA, Chang BY. Bruton's tyrosine kinase inhibitors for the treatment of rheumatoid arthritis. 
Drug Discov Today. 2014;19(8):1200-4. 
7. 
Kuehn HS, Swindle EJ, Kim MS, Beaven MA, Metcalfe DD, Gilfillan AM. The phosphoinositide 3-
kinase-dependent activation of Btk is required for optimal eicosanoid production and generation of 
reactive oxygen species in antigen-stimulated mast cells. Journal of immunology. 2008;181(11):7706-12. 
8. 
MacGlashan D, Jr., Honigberg LA, Smith A, Buggy J, Schroeder JT. Inhibition of IgE-mediated 
secretion from human basophils with a highly selective Bruton's tyrosine kinase, Btk, inhibitor. Int 
Immunopharmacol. 2011;11(4):475-9. 
9. 
Iyer AS, Morales JL, Huang W, Ojo F, Ning G, Wills E, et al. Absence of Tec family kinases 
interleukin-2 inducible T cell kinase (Itk) and Bruton's tyrosine kinase (Btk) severely impairs Fc epsilonRI-
dependent mast cell responses. The Journal of biological chemistry. 2011;286(11):9503-13. 
10. 
Wechsler JB, Schroeder HA, Byrne AJ, Chien KB, Bryce PJ. Anaphylactic responses to histamine 
in mice utilize both histamine receptors 1 and 2. Allergy. 2013;68(10):1338-40. 

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11. 
Dispenza MC, Pongracic JA, Singh AM, Bochner BS. Short-term ibrutinib therapy suppresses 
skin test responses and eliminates IgE-mediated basophil activation in adults with peanut or tree nut 
allergy. The Journal of allergy and clinical immunology. 2018;141(5):1914-6. 
12. 
Regan JA, Cao Y, Dispenza MC, Ma S, Gordon LI, Petrich AM, et al. Ibrutinib, a Bruton's tyrosine 
kinase inhibitor used for treatment of lymphoproliferative disorders, eliminates both aeroallergen skin test 
and basophil activation test reactivity. The Journal of allergy and clinical immunology. 2017;140(3):875-9 
e1. 
13. 
Zhang Z, Zhang D, Liu Y, Yang D, Ran F, Wang ML, et al. Targeting Bruton's tyrosine kinase for 
the treatment of B cell associated malignancies and autoimmune diseases: Preclinical and clinical 
developments of small molecule inhibitors. Arch Pharm (Weinheim). 2018;351(7):e1700369. 
14. 
Sampson HA, Aceves S, Bock SA, James J, Jones S, Lang D, et al. Food allergy: a practice 
parameter update-2014. The Journal of allergy and clinical immunology. 2014;134(5):1016-25 e43. 
15. 
Sampson HA, Gerth van Wijk R, Bindslev-Jensen C, Sicherer S, Teuber SS, Burks AW, et al. 
Standardizing double-blind, placebo-controlled oral food challenges: American Academy of Allergy, 
Asthma & Immunology-European Academy of Allergy and Clinical Immunology PRACTALL consensus 
report. The Journal of allergy and clinical immunology. 2012;130(6):1260-74. 
16. 
Dispenza MC, Pongracic JA, Singh AM, Bochner BS. Short-term ibrutinib therapy suppresses 
skin test responses and eliminates IgE-mediated basophil activation in adults with peanut or tree nut 
allergy. The Journal of allergy and clinical immunology. 2018. 
17. 
Toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventative 
vaccine clinical trials [Internet]. Center for Biologics Evaluation and Research, Food and Drug 
Administration. 2007. Available from: 
https://www.fda.gov/downloads/BiologicsBloodVaccines/ucm091977. 
18. 
Nowak-Wegrzyn A, Assa'ad AH, Bahna SL, Bock SA, Sicherer SH, Teuber SS, et al. Work Group 
report: oral food challenge testing. The Journal of allergy and clinical immunology. 2009;123(6 
Suppl):S365-83. 
19. 
Bindslev-Jensen C, Ballmer-Weber BK, Bengtsson U, Blanco C, Ebner C, Hourihane J, et al. 
Standardization of food challenges in patients with immediate reactions to foods--position paper from the 
European Academy of Allergology and Clinical Immunology. Allergy. 2004;59(7):690-7. 
20. 
Common Terminology Criteria for Adverse Events (CTCAE). (Version 4.0, May 28, 2009). 
National Institutes of Health, National Cancer Institute 
(<http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf>). 
21. 
CALQUENCE Prescribing Information [Internet]. U.S. Food and Drug Administration. Revised: 
11/2017. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/210259s000lbl.pdf. 
 
 
 
 
 

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Appendix 1:  Pill Diary  
 
 
 
 
 
 

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STATISTICAL ANALYSIS PLAN 
 
Study Design and Endpoints 
 
This is an open label study of acalabrutinib in 10 food-allergic adults to determine the effect of 
pretreatment (100 mg twice daily for 4 doses total) with acalabrutinib on food allergen reactivity in food-
allergic subjects. There are 3 planned study visits where endpoint assessments will be collected. The 
primary endpoint will be assessed at Visit 1 (baseline) and Visit 2 (after acalabrutinib). Secondary and 
exploratory endpoints will be assessed at Visits 1, 2, and 3 (follow-up).  
 
Primary endpoint:  Change in subjects’ threshold dose of peanut protein to elicit an objective 
clinical reaction 
 
Secondary endpoints:  
1. Change in size of skin prick test wheal  
2. Change in percent of ex vivo basophil activation to anti-IgE and peanut, as determined by 
CD63 expression 
 
Safety considerations and toxicity monitoring:  Laboratory testing (complete blood counts, serum 
chemistries) and electrocardiogram  
 
Given the nature of the study, it will be stopped if >50% of patients have a DLT as described in the 
protocol.  
 
Sample Size and Power Analysis 
 
Sample size was calculated based on the primary endpoint, assuming that the difference in threshold 
from baseline to after acalabrutinib is normally distributed with a standard deviation of 1.1 natural log 
units. Therefore, 10 subjects allows for 80% power to detect a 3-fold increase (1.1 natural log units; i.e. 1 
food dose escalation) in the threshold food dose, using a paired t test with p<0.05 as an approximation, 
though non-parametric tests will be utilized in the final analysis if needed.  
 
Data Analysis  
 
As this is an open label trial, each subject’s responsiveness will be examined compared to themselves (at 
baseline) prior to initiation of treatment. Descriptive statistics will be used to analyze endpoints. The 
primary endpoint will be analyzed by log transformation and paired t test with p value < 0.05, if 
appropriate. If assumptions for paired t test are violated, a Wilcoxon non-parametric sign test will be used.  
 
Secondary endpoints will be analyzed using ANOVA with repeated measures across all visits with p value 
< 0.05. For experiments with multiple dosing levels, we will first examine if there is a dose-level by 
treatment interaction. If there is an interaction, treatment effects in individual dosing levels will be 
examined. Non-parametric tests will be utilized in place of ANOVA if necessary. Corrections will be made 
for multiple comparisons, if applicable, using a Šídák or Bonferroni multiple comparisons correction with 
an alpha threshold of 0.05.  
 
For safety endpoints, ANOVA will be used for analysis of individual laboratory values (blood counts or 
chemistries).