Conditions: HIV Infections
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 60
Healthy volunteers: no

Brief summary:
The purpose of this study was to evaluate the safety and tolerability of ruxolitinib in HIV-positive adults who were virologically suppressed and who were on antiretroviral therapy (ART).

Detailed description:
Ruxolitinib is a medication approved by the U.S. Food and Drug Administration (FDA) to treat myelofibrosis, a disorder in which bone marrow is replaced by scar (fibrosis) tissue. Many of the cytokines affected by myelofibrosis are also affected by HIV. Because of this, ruxolitinib may also be a possible treatment for HIV. The purpose of this study was to evaluate the safety and tolerability of ruxolitinib in HIV-positive adults who were on ART and who were virologically suppressed. Researchers evaluated the effect ruxolitinib had on inflammation and immune activation.

This study enrolled HIV-positive adults who were on select ART regimens and who had viral suppression. ART was not provided by the study; participants continued to receive ART from their own health care providers. Participants were randomly assigned to receive either ruxolitinib (Arm A) or no study treatment (Arm B) in 2:1 ratio. Participants in Arm A received ruxolitinib twice a day for 5 weeks. All participants attended study visits at entry (Day 0) and Weeks 1, 2, 4, 5, 10, and 12. These visits included physical examinations, clinical assessments, blood collection, adherence assessments, oral swab collection, and pregnancy testing for female participants. At Weeks 1 and 4, participants in Arm A took part in pharmacokinetic (PK) sampling, which involved having blood drawn several times over 6 to 8 hours.

Arms:
arm 1: Participants received ruxolitinib twice a day for 5 weeks. Participants were required to remain on ART regimen (not provided by the study) for the duration of the study. arm 2: Participants did not receive any study treatment. Participants were required to remain on ART regimen (not provided by the study) for the duration of the study.

Interventions: intervention 1: Ruxolitinib
Intervention descriptions: intervention 1: 10 mg orally twice daily for 5 weeks

Protocol document:
Prot_001.pdf: 
 
 
 
 
 
A5336 
 
 
A Randomized, Pilot Study of Ruxolitinib in Antiretroviral-Treated HIV-Infected 
Adults 
 
 
A Multicenter Trial of the AIDS Clinical Trials Group (ACTG) 
 
 
 
Sponsored by: 
 
 
The National Institute of Allergy 
and Infectious Diseases 
 
 
 
IND # 125,580 
 
 
 
The ACTG End-Organ Disease and  
Inflammation Transformative Science Group Chair: 
Peter Hunt, MD, Chair 
 
Protocol Co-Chairs: 
Vincent Marconi, MD 
 
Jeffrey Lennox, MD 
 
Protocol Co- Vice Chairs:  
 
Steve Deeks, MD 
 
Carlos Del Rio, MD 
 
DAIDS Clinical Representatives: 
Randall L. Tressler, MD 
 
Grace Chen, MD, MPH 
 
Clinical Trials Specialist: 
Suria Yesmin, BS, CCRP 
 
 
 
 
FINAL Version 2.0 
May 21, 2015 


A5336 
 
 
FINAL Version 2.0 
 
 
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CONTENTS 
 
 
Page 
 
SITES PARTICIPATING IN THE STUDY .................................................................................... 4 
PROTOCOL TEAM ROSTER ..................................................................................................... 5 
STUDY MANAGEMENT ............................................................................................................. 8 
GLOSSARY OF PROTOCOL-SPECIFIC TERMS ..................................................................... 10 
SCHEMA .................................................................................................................................. 11 
1.0 
HYPOTHESES AND STUDY OBJECTIVES.................................................................. 14 
1.1 
Hypotheses ........................................................................................................ 14 
1.2 
Primary Objectives ............................................................................................. 14 
1.3 
Secondary Objectives ........................................................................................ 14 
1.4 
Exploratory Objective ......................................................................................... 15 
2.0 
INTRODUCTION ........................................................................................................... 15 
2.1 
Background ........................................................................................................ 15 
2.2 
Rationale ........................................................................................................... 17 
3.0 
STUDY DESIGN ........................................................................................................... 34 
4.0 
SELECTION AND ENROLLMENT OF PARTICIPANTS ............................................... 35 
4.1 
Inclusion Criteria ................................................................................................ 35 
4.2 
Exclusion Criteria ............................................................................................... 37 
4.3 
Study Enrollment Procedures............................................................................. 39 
4.4 
Coenrollment Guidelines .................................................................................... 40 
5.0 
STUDY TREATMENT ................................................................................................... 40 
5.1 
Regimens, Administration, and Duration ............................................................ 40 
5.2 
Study Product Formulation and Preparation ....................................................... 40 
5.3 
Pharmacy: Product Supply, Distribution, and Accountability .............................. 40 
5.4 
Concomitant Medications ................................................................................... 41 
6.0 
CLINICAL AND LABORATORY EVALUATIONS ........................................................... 42 
6.1 
Schedule of Events ............................................................................................ 42 
6.2 
Timing of Evaluations ......................................................................................... 44 
6.3 
Instructions for Evaluations ................................................................................ 46 
7.0 
CLINICAL MANAGEMENT ISSUES .............................................................................. 54 
7.1 
Toxicity .............................................................................................................. 54 
7.2 
Other Toxicities .................................................................................................. 55 
7.3 
Other Diseases .................................................................................................. 57 
7.4 
Pregnancy.......................................................................................................... 57 
7.5 
Confirmed Virologic Failure ................................................................................ 57 


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CONTENTS (Cont’d) 
Page 
 
8.0 
CRITERIA FOR DISCONTINUATION ........................................................................... 58 
8.1 
Premature Treatment Discontinuation ................................................................ 58 
8.2 
Premature Study Discontinuation ....................................................................... 58 
9.0 
STATISTICAL CONSIDERATIONS ............................................................................... 58 
9.1 
General Design Issues ....................................................................................... 58 
9.2 
Objectives .......................................................................................................... 59 
9.3 
Randomization and Stratification ........................................................................ 60 
9.4 
Sample Size and Accrual ................................................................................... 60 
9.5 
Monitoring .......................................................................................................... 62 
9.6 
Analyses ............................................................................................................ 62 
10.0 
PHARMACOLOGY PLAN .............................................................................................. 64 
10.1 
Pharmacology Objectives .................................................................................. 64 
10.2 
Pharmacology Study Design .............................................................................. 64 
10.3 
Primary and Secondary Data, Modeling, and Data Analysis .............................. 65 
10.4 
Anticipated Outcomes ........................................................................................ 66 
11.0 
DATA COLLECTION AND MONITORING AND ADVERSE EVENT REPORTING ........ 66 
11.1 
Records to Be Kept ............................................................................................ 66 
11.2 
Role of Data Management ................................................................................. 66 
11.3 
Clinical Site Monitoring and Record Availability .................................................. 66 
11.4 
Expedited Adverse Event Reporting to DAIDS ................................................... 66 
12.0 
PARTICIPANTS ............................................................................................................ 68 
12.1 
Institutional Review Board (IRB) Review and Informed Consent ........................ 68 
12.2 
Participant Confidentiality ................................................................................. 68 
12.3 
Study Discontinuation ........................................................................................ 68 
13.0 
PUBLICATION OF RESEARCH FINDINGS .................................................................. 68 
14.0 
BIOHAZARD CONTAINMENT ...................................................................................... 68 
15.0 
REFERENCES .............................................................................................................. 69 
 
APPENDIX I: SAMPLE INFORMED CONSENT 
 


 
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SITES PARTICIPATING IN THE STUDY 
 
A5336 is open to all U.S. ACTG clinical research sites (CRS). 
 
 
 
 
 
 


 
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PROTOCOL TEAM ROSTER 
 
Co-Chairs 
Vincent Marconi, MD  
Division of Infectious Diseases 
Emory University School of Medicine 
341 Ponce de Leon Avenue 
Atlanta, GA 30308 
Phone: (404) 616-2493 
FAX:    (404) 616-0592 
E-Mail: vcmarco@emory.edu 
 
Jeffrey Lennox, MD 
Division of Infectious Diseases 
Emory University School of Medicine 
341 Ponce de Leon Avenue 
Atlanta, GA 30322 
Phone: (404) 251-8784 
FAX:     (404) 616-0592 
E-Mail:  jlennox@emory.edu 
 
Vice Chairs 
Steve Deeks, MD 
UCSF School of Medicine 
SFGH Building 80 
995 Potrero Avenue 
San Francisco, CA  94110 
Phone: (415) 476-4082 Ext. 330 
FAX:     (416) 476-6953 
E-Mail:  sdeeks@php.ucsf.edu 
 
Carlos Del Rio, MD 
Division of Infectious Diseases 
Emory University School of Medicine 
1518 Clifton Road, NE Room 7011 
Atlanta, GA  30322 
Phone: (404) 727-1557 
FAX:     (404) 727-1278 
E-Mail:  cdelrio@emory.edu 
 
DAIDS Clinical Representatives 
Randall L. Tressler, MD 
TRP, DAIDS, NIAID, NIH 
5601 Fishers Lane, 9E47 
Rockville, MD  20852 
Phone: (240) 627-3072  
FAX:     (301) 435-9282 
E-Mail:  randall.tressler@nih.gov 
DAIDS Clinical Representatives (Cont’d) 
Grace Chen, MD, MPH 
TRP, DAIDS, NIAID, NIH 
5601 Fishers Lane 
Room 9E57 MSC 9832 
Rockville, MD  20892 
Phone: (240) 292-4836 
E-Mail:  grace.chen@nih.gov 
 
Clinical Trials Specialist 
Suria Yesmin, BS, CCRP 
ACTG Network Coordinating Center 
Social & Scientific Systems, Inc. 
8757 Georgia Avenue, 12th Floor 
Silver Spring, MD  20910-3714 
Phone: (301) 628-3493 
FAX:     (301) 628-3302 
E-Mail:  syesmin@s-3.com 
 
Statistician 
Carlee Moser, PhD 
Statistical & Data Analysis Center 
Center for Biostatistics in AIDS Research  
Harvard T. H. Chan School of Public Health 
FXB Building, Room 529A 
651 Huntington Avenue 
Boston, MA  02115 
Phone: (617) 432-2526 
FAX:     (617) 432-3163 
E-Mail:  cmoser@sdac.harvard.edu 
 
Data Manager 
David Nichols, BS 
Frontier Science and Technology Research 
Foundation, Inc. 
4033 Maple Road 
Amherst, NY  14226 
Phone:  (716) 834-0900 Ext. 7359 
FAX:      (716) 834-8432 
E-Mail:   nichols@fstrf.org 
 
 
 
 
 
 


 
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DAIDS Pharmacist 
Ana Martinez, RPh 
Pharmaceutical Affairs Branch 
DAIDS, NIAID, NIH 
5601 Fishers Lane, 9D32 
Rockville, MD 20852 
Phone:  (301) 435-3734 
FAX:      (301) 402-1506 
E-Mail:   amartinez@niaid.nih.gov 
 
Immunologist  
Michael Lederman, MD 
Case Western Reserve University 
The Foley Building, Room 401-A 
2061 Cornell Road 
Cleveland, OH 44106-5083 
Phone: (216) 844-8786 
FAX:     (216) 844-5523 
E-Mail:  mxl6@case.edu 
 
Pharmacologist 
Charles W. Flexner, MD 
Division of Clinical Pharmacology 
Johns Hopkins University 
600 North Wolfe Street-Osler 527 
Baltimore, MD 21287-5554 
Phone: (410) 955-9712  
FAX:     (410) 614-9978 
E-Mail:  flex@jhmi.edu  
 
Virologist 
Athe Tsibris, MD 
65 Landsdowne Street, Room 422 
Cambridge, MA 02139 
Phone: (617) 417-1162  
             (617) 768-8352 
FAX:     (617) 768-8738 
E-Mail: atsibris@partners.org 
 
Investigators 
Christina Gavegnano, PhD 
Veterans Administration Medical Center 
1760 Haygood Drive, NE 
Atlanta, GA 30322 
Phone:  (404) 727-1414 
E-Mail: cgavegn@emory.edu 
 
 
Investigators (Cont’d) 
Selwyn Hurwitz, PhD 
Veterans Administration Medical Center 
1760 Haygood Drive, NE 
Atlanta, GA 30322 
Phone:  (404) 727-1414 
FAX:      (404) 922-0397 
E-Mail:   shurwit@emory.edu 
 
Raymond F. Schinazi, PhD, DSc 
Emory Center for AIDS Research 
1760 Haygood Drive, Room E420 
Atlanta, GA 30322 
Phone:  (404) 727-1414  
FAX:      (404) 727-1330 
E-Mail:   rschina@emory.edu 
 
Javier Valencia, MD 
Asociacion Civil Impacta Salud y Educacion 
Av. Almirante Miguel Grau No. 1010  
Lima 4  
PERU 
Phone:  +(51-1) 206-7800 Ext. 209 
E-Mail:  jvalencia@impactaperu.org 
 
Sara Gianella Weibel, MD 
Center for Aids Research (CFAR) 
University of California San Diego  
9500 Gilman Drive 
La Jolla, CA 92093-0679 
Phone:  (858) 552-8585 Ext. 7193 
E-Mail:  gianella@ucsd.edu 
 
Field Representative 
Jay (John) Dwyer, RN, BA 
SFGH Positive Health Program 
995 Potrero Avenue, Ward 84 
San Francisco, CA 94110-2897 
Phone:  (415) 476-4082 Ext. 353 
FAX:      (415) 476-6736 
E-Mail:   jdwyer@php.ucsf.edu 
 
 
 
 
 
 
 


 
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05/21/15 
 
Laboratory Technologist 
Carmen M. Irizarry, MT 
Medical Science Campus 
Pathology 606-A 
San Juan, PR  00935 
Phone:  (787) 758-5815 
FAX:      (787) 751-9210 
E-Mail:   lab053.rcm@upr.edu 
 
Community Scientific Subcommittee (CSS) 
Representatives 
Graig Cote 
765 Kenwick Road, Apt #C 
Columbus, OH 43209 
Phone:  (614) 297-5667 
FAX:      (614) 293-4556 
E-Mail:   graigcote@yahoo.com 
 
William (Royce) Hardin 
University of North Carolina AIDS CRS 
1005 Iredell Street 
Durham,  NC 27705 
Phone:  (919) 286-4969 
E-Mail:   wrhardin05@gmail.com 
 
Laboratory Data Manager 
Adam Manzella, MA 
Frontier Science and Technology Research 
Foundation, Inc.  
4033 Maple Road 
Amherst, NY  14226 
Phone:   (716) 834-0900 Ext. 7418 
E-Mail:   manzella@fstrf.org 
 


 
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STUDY MANAGEMENT 
 
All questions concerning this protocol should be sent to actg.corea5336@fstrf.org via e-mail. 
The appropriate team member will respond with a "cc" to actg.corea5336@fstrf.org.  A response 
should generally be received within 24 hours (Monday-Friday). 
 
Protocol E-mail Group 
Sites should contact the Computer Support Group at the Data Management Center (DMC) as 
soon as possible to have the relevant personnel at the site added to the actg.protA5336 e-mail 
group. Include the protocol number in the e-mail participant line.  
 
Send an e-mail message to actg.user.support@fstrf.org  
 
Clinical Management  
For questions concerning entry criteria, toxicity management, concomitant medications, and 
coenrollment, contact the protocol team.  
 
Send an e-mail message to actg.corea5336@fstrf.org. Include the protocol number, patient 
identification number (PID), and a brief relevant history. 
 
Laboratory 
For questions specifically related to immunologic, virologic, or pharmacologic laboratory tests, 
contact the protocol immunologist, virologist, or pharmacologist.  
 
Send an e-mail message to actg.corea5336@fstrf.org (ATTN: Michael Lederman, Athe 
Tsibris and Charles Flexner). 
 
Data Management 
For nonclinical questions about transfers, inclusion/exclusion criteria, case report forms (CRFs), 
the CRF schedule of events, randomization/registration, delinquencies, and other data 
management issues, contact the data manager. CRFs can be downloaded from the FSTRF 
website at www.fstrf.org. 
 
For transfers, reference the Patient Transfer from Site to Site SOP 119, and contact Dave 
Nichols directly.  
 
For other questions, send an e-mail message to actg.corea5336@fstrf.org (ATTN: Dave 
Nichols).   
 
Include the protocol number, PID, and a detailed question. 
 
Randomization  
For randomization/participant registration questions or problems and study identification 
number SID lists.  
 
Send an e-mail message to rando.support@fstrf.org. Call the Statistical and Data Analysis 
Center (SDAC)/DMC Randomization Desk at 716-834-0900 x7301.    
 
Computer and Screen Problems  
Contact the SDAC/DMC programmers. 
 
Send an e-mail message to actg.support@fstrf.org  or call 716-834-0900 x7302. 
 


 
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Protocol Document Questions 
For questions concerning the protocol document, contact the clinical trials specialist.  
 
Send an e-mail message to actg.corea5336@fstrf.org (ATTN: Suria Yesmin). 
 
Copies of the Protocol 
To request a hard copy of the protocol, send an email message to ACTGNCC@s-3.com (ATTN: 
Diane Delgado). Electronic copies can be downloaded from the ACTG Web site 
(https://www.actgnetwork.org). 
 
Product Package Inserts and/or Investigator Brochures 
To request copies of product package inserts or investigator brochures, contact the DAIDS 
Regulatory Support Center (RSC) at RIC@tech-res.com or call 301-897-1708. 
 
Protocol Registration 
For protocol registration questions, send an e-mail message to Protocol@tech-res.com or call 
301-897-1707.  
 
Protocol Activation 
For questions related to protocol activation, contact the clinical trials specialist (Suria 
Yesmin, syesmin@s-3.com) or ACTG Site Coordination group at actgsitecoordination@s-
3.com.  
 
Study Product 
For questions or problems regarding study product, dose, supplies, records, and returns, call 
Ana Martinez, protocol pharmacist, at 301-435-3734.  
 
Study Drug Orders 
Call the Clinical Research Products Management Center (CRPMC) at 301-294-0741. 
 
IND (Investigational New Drug) Number or Questions  
The IND number will be available on the PSWP within 30 days of the submission to the FDA. 
For any questions related to the IND submission, contact the DAIDS RSC at Regulatory@tech-
res.com or call 301-897-1706. 
 
Expedited Adverse Event (EAE) Reporting/Questions  
Contact DAIDS through the RSC Safety Office at DAIDSRSCSafetyOffice@tech-res.com or call 
1-800-537-9979 or 301-897-1709; or fax 1-800-275-7619 or 301-897-1710.  
 
Phone Calls 
Sites are responsible for documenting any phone calls made to A5336 team members. 
 
Send an e-mail to actg.corea5336@fstrf.org. 
 
Protocol-Specific Web Page 
Additional information about management of the protocol can be found on the protocol-specific 
web page (PSWP). 


 
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GLOSSARY OF PROTOCOL-SPECIFIC TERMS 
 
3TC 
 
lamivudine 
ABC 
 
abacavir 
ACR 
 
American College of Rheumatology 
ALT  
 
alanine aminotransferase 
AML 
 
acute myeloid leukemia 
ANC 
 
absolute neutrophil count 
AST   
aspartate aminotransferase 
ATP 
 
adenosine triphosphate 
BBB 
 
blood brain barrier 
CMV  
cytomegalovirus 
CNS 
 
central nervous system 
CSF 
 
cerebrospinal fluid 
CVD 
 
cardiovascular disease 
CWRES 
conditional weighted residual 
EBV 
 
Epstein Barr virus  
EFV 
 
efavirenz 
ET 
 
essential thrombocythemia 
FOCE INT 
first order conditional estimation with - interactions  
FTC 
 
emtricitabine  
G-CSF  
granulocyte colony stimulating factor 
GM-CSF 
granulocyte-macrophage colony stimulating factor 
HBM  
human biological materials 
HHV 
 
human herpes viruses 
HSV 
 
herpes simplex viruses 
INSTI  
integrase strand transfer inhibitors 
JAK 
 
Janus activating kinase 
LTBI   
latent tuberculosis infection  
LTR 
 
long terminal repeat sequences 
mCSF  
macrophage colony stimulating factor 
MDM  
monocyte-derived macrophages 
NHP 
 
non-human primate 
NNRTIs 
nonnucleoside reverse transcriptase inhibitors  
NRTIs  
nucleoside reverse transcriptase inhibitors 
PML 
 
progressive multifocal leukoencephalopathy 
PV  
 
polycythemia vera  
RA 
 
rheumatoid arthritis 
ROS 
 
reactive oxygen species 
SAE 
 
serious adverse events 
SIV 
 
simian immunodeficiency virus 
STAT  
signal transducer and activator of transcription 
 
SUSARs 
serious unexpected serious adverse reactions 
TCR 
 
T cell receptor 
TDF 
 
tenofovir 
TNF-α  
tumor necrosis factor alpha 
Tscm 
 
stem-cell memory T-cells 
 
 


 
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SCHEMA 
A5336 
 
A Randomized, Pilot Study of Ruxolitinib in Antiretroviral-Treated HIV-Infected Adults 
 
DESIGN 
This is a multi-center, randomized, open-label, two arm (phase IIa) study. 
Eligible participants on select antiretroviral therapy (ART) with virologic 
suppression will be randomized to receive ruxolitinib or no study treatment 
to measure safety and tolerability and to determine whether there are 
changes in systemic immune activation and inflammation after 5 weeks of 
treatment with ruxolitinib.   
 
DURATION 
Participants will participate in the study for up to 12 weeks after 
randomization. 
 
SAMPLE SIZE 
A total of 60 participants will be enrolled in the study (40 on ruxolitinib 
[Arm A]; 20 on no study treatment [Arm B]). 
 
 
 
The study aims to enroll at least 15 participants on efavirenz (EFV) in 
order to have at least 8 ruxolitinib-treated participants to evaluate 
pharmacokinetic (PK) interactions with EFV. 
 
POPULATION 
HIV-1 infected males and females ≥ 18 and < 75 years of age on stable 
ART for at least 12 weeks prior to study entry containing either 
tenofovir/emtricitabine (TDF/FTC) or abacavir/lamivudine (ABC/3TC) plus a 
nonnucleoside reverse transcriptase inhibitor or integrase strand transfer 
inhibitor (NNRTI or INSTI, not containing cobicistat) who: 
 
 
Are on ART for at least 2 years 
 
Have virologic suppression defined as: (a) at least two HIV-1 RNA 
measurements < 50 copies/mL, one of which was obtained within 336 
to 31 days, inclusive, prior to study entry and one obtained between 337 
and 730 days, inclusive, prior to entry, (b) no HIV-1 RNA values ≥ 50 
copies/mL within 336 days, inclusive, prior to study entry, and (c) not 
more than one HIV-1 viral load equal to or greater than 50 and less than 
or equal to 200 copies/mL from a result obtained between 337 and 730 
days, inclusive, prior to study entry (and no HIV-1 viral load above 200 
copies/mL) 
 
Have a plasma HIV-1 quantitative RNA with a lower limit of 
quantification < 50 copies/mL (e.g., < 40 copies/mL) obtained ≤ 30 
days prior to study entry 
 
Have CD4+ T cell count > 350 cells/mm3 within 30 days prior to study 
entry 
 
Have no other medical conditions, or concomitant medications, 
prohibiting the use of a janus activating kinase-signal transducer and 
activator of transcription (Jak-STAT) inhibitor   
 
STRATIFICATION To ensure balance by treatment arm for EFV, participants will be stratified 
at randomization based on their EFV status (i.e., whether they are on an 
EFV-containing regimen). 
 
REGIMEN 
TDF/FTC or ABC/3TC + (NNRTI or INSTI, not containing cobicistat), with 
one of the following: 


 
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ARM A:  Treatment with ruxolitinib 10 mg orally twice daily for 5 weeks. 
 
ARM B:  No study treatment. 


 
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SCHEMA (Cont’d) 
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Arm A 
Study Week 
RUX+ART (ART not provided by 
study) 
   0              1                                      4               5                                                                                                                       12    
Follow-Up (only ART; ART not provided by study) 
Arm B 
No Study Treatment; only ART (ART not provided by study) 
PK Collection 
PK Collection 
Study Week 
   0                                                                                                                                                                                                12      
 
 
HIV+ on ART 
Suppressed VL 
2:1 


 
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1.0 
HYPOTHESES AND STUDY OBJECTIVES 
 
1.1 
Hypotheses 
 
1.1.1 The use of ruxolitinib for 5 weeks with antiretroviral therapy (ART) consisting of: 
tenofovir/emtricitabine (TDF/FTC) or abacavir /lamivudine (ABC/3TC) + a 
nonnucleoside reverse transcriptase inhibitor or integrase strand transfer inhibitor 
(NNRTI or INSTI) will be safe for virologically suppressed participants. 
 
1.1.2 The use of ruxolitinib for 5 weeks with ART will be tolerable for virologically 
suppressed participants.  
 
1.1.3 The use of ruxolitinib for 5 weeks with ART for virologically suppressed 
participants will reduce systemic immune activation and inflammation.   
 
1.2 
Primary Objectives 
 
1.2.1 To evaluate the safety of ruxolitinib in ART-treated HIV-1 infected virologically  
suppressed participants during 5 weeks of treatment. 
 
1.2.2 To evaluate the tolerability of ruxolitinib in ART-treated HIV-1 infected 
virologically suppressed participants during 5 weeks of treatment. 
 
1.2.3 To compare changes in interleukin-6 (IL-6) levels between baseline and week 5 
in ART-treated HIV-1 virologically suppressed participants who are randomized 
to ruxolitinib and those randomized to receive no study treatment. 
 
1.3 
Secondary Objectives 
 
1.3.1 To evaluate the safety of ruxolitinib in ART-treated HIV-1 infected virologically 
suppressed participants during all on-study visits through week 12. 
 
1.3.2 To compare changes in IL-6 levels between baseline and week 12 in ART-
treated HIV-1 virologically suppressed participants who are randomized to 
ruxolitinib to changes in those participants randomized to receive no study 
treatment. 
 
1.3.3 To assess whether the use of ruxolitinib will decrease levels of other measures of 
inflammation and immune activation including sCD14, tumor necrosis factor 
alpha (TNF-α), IL-1α, IL-1β, IL-18, macrophage colony stimulating factor (mCSF), 
neopterin, HLA-DR, CD38, and both the distribution of monocyte subsets 
(defined by expression of CD14+ and CD16+) and phenotypic indices of their 
activation during 5 weeks of treatment and at week 12. 
 
1.3.4 To examine whether the use of ruxolitinib is associated with changes in HIV-1 
plasma viral load levels (by a commercial clinical assay and single-copy assay) 
and CD4+ T cell counts in HIV-1 infected participants at baseline compared to 
12 weeks after initiation of treatment for those receiving 5 weeks of ruxolitinib 
versus those receiving no study treatment.  


 
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1.3.5 To assess changes from baseline in the HIV-1 reservoir, specifically plasma HIV-
1 RNA by single copy assay (as above), cellular HIV-1 RNA and total DNA, 2 
long-terminal repeat sequences [LTRs], and integrated DNA. 
 
1.3.6 To evaluate drug-drug interactions between ruxolitinib and antiretrovirals (ARVs) 
and to characterize the association between ruxolitinib pharmacokinetic (PK) 
parameters and changes from baseline in inflammatory biomarkers in 
participants on the ruxolitinib treatment arm.  
 
1.3.7 To evaluate whether the use of ruxolitinib is associated with more frequent 
reactivation of human herpes viruses (ie, cytomegalovirus [CMV], Epstein 
Barr virus [EBV], herpes simplex viruses [HSV], human herpes viruses 
[HHV] 6, 7, and 8) as measured in longitudinally collected oral swabs. 
 
1.4 
Exploratory Objective 
 
To derive and evaluate a summary measure of immune activation and inflammation 
and how changes in this measure differ between the study treatment arm and the no 
treatment arm. 
 
 
2.0 
INTRODUCTION 
 
2.1 
Background 
 
Although ART presents a viable and well-established strategy for treating individuals 
living with HIV-1, a limitation of current ART is the inability to completely reduce systemic 
inflammation to that seen in age-matched controls in the general population. This 
inflammation has several consequences for individuals on ART including: (1) 
establishment of a systemic pro-HIV-1 milieu resulting in an increase in HIV-1 target 
cells, and possibly thereby maintaining HIV-1 viral reservoirs and allowing infection to 
persist in various microenvironments; (2) less robust immunologic reconstitution [1]; and 
(3) increased end-organ damage.  
 
Primary myelofibrosis is a myeloproliferative disease whereby the bone marrow is 
replaced with collagen fibrosis. This fibrosis occurs due to stromal cell overproduction of 
collagen stimulated by abnormal megakaryocytes. In response to the subsequent 
pancytopenia, hematopoietic cells migrate out of the bone marrow resulting in 
extramedullary hematopoiesis and hepatosplenomegaly. A majority of patients with 
primary myelofibrosis have a somatic mutation in the gene encoding the cytoplasmic 
tyrosine kinase, Jak1/2, resulting in activation of Jak1/2 signaling. Activation of the janus 
activating kinase and signal transducer and activator of transcription (Jak-STAT) 
pathway leads to production of cytokines that cause the abnormal megakaryocytes to 
proliferate. Ruxolitinib, a commercially available, US Food and Drug Administration 
(FDA)-approved Jak2 inhibitor, disrupts this pathway. Many of the cytokines produced 
from Jak-STAT signaling are also central to the pathogenesis of HIV. It is from this 
observation that ruxolitinib was first considered as a potential therapeutic option for HIV-
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Ruxolitinib possesses unique qualities distinct from those of traditional ART, including 1) 
nanomolar in vivo inhibition of TNF-α, IL-6, and other pro-HIV inflammatory cytokines, 
which have been associated with increased risk of morbidity and mortality in ART-
suppressed HIV-1 individuals [2, 3], 2) sub-micromolar (0.1 µM) inhibition of IL-2, IL-6, 
IL-7, IL-15, or interferon (IFN)-α induced activation of the Jak-STAT pathway in primary 
CD4+ T cell lymphocytes, 3) sub-micromolar inhibition of HIV-1/2 replication in primary 
human lymphocytes and macrophages, 4) down-regulation of HIV-1 induced activation 
in primary human lymphocytes, and 5) direct inhibition of the Jak-STAT pathway, which 
is activated early in HIV-1 infection [4].  
 
These attributes of ruxolitinib could address many of the limitations of existing ART and 
provide a novel mechanism to 1) reduce reservoir seeding and homeostatic proliferation, 
which is driven by IL-7 and IL-15 orchestrated proliferation of stem-cell memory T cells 
(TSCM) and other latent T cell subsets; 2) reduce systemic inflammation by inhibition of 
IL-6 and other pro-HIV/pro-inflammatory cytokines, which would otherwise serve to 
promote pro-HIV-1 events including (a) priming of uninfected cells for infection, (b) 
recruitment of uninfected cells to the site of infection, (c) reservoir maintenance [5, 6], 
and to accelerate disease progression (including AIDS and serious non-AIDS events [7, 
8]; 3) reduce CD14+/CD16+ monocytes and CD163 macrophages, which can reduce 
trafficking of these activated, infected monocytes to the brain/central nervous system 
(CNS) where they would otherwise either differentiate into macrophage-like cells 
containing HIV-1 or transfer infection to perivascular macrophages within the CNS (this 
may also lead to a reduction in cardiovascular events via similar processes [8, 9]), and 
4) confer anti-HIV-1/2 potency across primary human lymphocytes and macrophages. 
Point 4 addresses persistent infection in macrophage-derived viral sanctuaries, which is 
not adequately controlled by current ART due to suboptimal intracellular concentration of 
ART in these cells [10]. Additionally, as macrophages are long-lived cells that harbor 
virus, efficient inhibition of viral replication in these cells, and down-regulation of HIV-1 
induced activation (CD163) in these cells by a mechanism distinct from direct inhibition 
of the viral replication cycle may provide a foundation to control inflammation-driven 
events including increased replication due to pro-inflammatory cytokines, induction of 
reactivation of virus from these cells, priming of uninfected macrophages by 
inflammation induced mCSF, cerebrospinal fluid production (which promotes viral 
replication), and trafficking of infected myeloid lineage cells to the brain (driven by HIV-1 
orchestrated activation of macrophages, which is marked by elevated CD163 levels). 
Together the potential ability of ruxolitinib to address these unmet needs could (a) 
contribute to control of persistent infection, (b) limit reservoirs maintenance, and (c) 
prevent ongoing infection across and within tissue compartments. Although adverse 
effects (AEs) exist, administration of ruxolitinib to patients with CD4+ T cell counts ≥ 350 
cells/mm3, platelet counts > 200,000/mm3, absolute neutrophil count (ANC) ≥ 750/mm3, 
and hemoglobin ≥ 12 g/dL with close monitoring for AEs should not present any greater 
risk to safety compared to HIV-1 infected healthy participants on ART. 
 
The HIV-1 reservoir is comprised of latently infected cells, predominantly central memory 
and transitional memory CD4+ T cell lymphocytes [11], that contain integrated HIV-1 
DNA. Integrated HIV-1 DNA can remain latent or be actively transcribed; the effect of 
ruxolitinib on this equilibrium is unknown. To assess the effects of ruxolitinib on the HIV-
1 reservoir, markers of HIV-1 production (plasma and cellular HIV-1 RNA levels, 2-LTR 
circles) and persistence (total and integrated HIV-1 DNA) can be measured and followed 
longitudinally. HIV-1 2-LTR circles are dead-end, non-integrated DNA species that are 


 
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formed when HIV-1 infects a cell but the linear HIV-1 DNA fails to integrate into the 
human genome. 2-LTR circles can remain detectable for at least 10-12 years during 
suppressive ART [12], and it remains unclear whether they are a marker of ongoing HIV-
1 replication or a byproduct of long-lived episomes maintained by latently-infected cell 
division. 
 
This study relies on the well-established anti-inflammatory profile of ruxolitinib in patients 
treated for primary myelofibrosis as a foundation to probe the effect of reducing 
inflammation on pro-HIV events described above in patients that are virologically 
controlled on ART, so that the role of inflammation in HIV-1 infection can be more clearly 
defined. Should ruxolitinib efficiently inhibit inflammation in the HIV-1 suppressed 
participant on ART, it may be feasible that individuals with well-controlled viremia 
receiving ART could undergo therapy interruption to determine whether viral rebound 
occurred post-ruxolitinib administration, either in the absence or presence of ruxolitinib. 
These data would ultimately define whether selective, systemic inhibition of pro-
inflammatory/pro-HIV cytokines could result in partial or total inhibition of viral rebound, 
or a functional cure. The study is designed to address the inflammation-driven facets of 
ART-suppressed HIV-1 infection, and to explore a novel therapeutic strategy that 
reduces cellular activation and systemic inflammation, targets Jak-STAT signaling 
across lymphocytes and macrophages, and ensures viable delivery across the blood-
brain barrier (BBB) to eliminate the HIV-1 reservoir of the CNS. 
 
2.2 
Rationale 
 
HIV-1 continues to be a global health crisis with more than 34 million people living with 
HIV-1 worldwide (UNAIDS/WHO). Despite the success of combination ART in 
suppressing viral replication, HIV-1 continues to persist. Although several mechanisms 
likely contribute to persistence during ART, the stable integration of HIV-1 DNA within 
resting CD4+ T cells and perhaps cells of the monocyte/macrophage lineage is generally 
assumed to be the most important mechanism [13]. All approved ART drugs target steps 
in actively replicating virus, rendering them ineffective for 1) eliminating latent virus, 2) 
preventing or reversing reactivation of latent virus, 3) preventing recruitment of 
uninfected cells to the site of infection, and 4) inhibiting pro-HIV/pro-inflammatory 
cytokine milieus that drive chronic infection, disease progression, and end-organ 
damage. To this end, in the era of ART, it has become apparent that inflammation will 
continue to result in end-organ damage and eradication of HIV-1 cannot occur without 
elimination of the latent viral reservoir. For these reasons, novel therapies are needed.   
 
Role of Jak-STAT Pathways in HIV-1 Infection  
The transcriptional status of HIV-1 is tightly coupled to the activation state of its host cell. 
The phosphorylation state of Jak-STAT pathways is known to influence HIV-1 infection 
in various target cells [14, 15]. HIV-1 orchestrated pro-inflammatory cytokines or 
envelope gp120 activates Jak and STAT proteins leading to expression of multiple 
receptors and/or cytokines and generalized immune dysfunction (including activation) in 
a variety of HIV-1 target cells, including primary human lymphocytes and macrophages 
[4, 8-9, 13-19]. Cytokines that are upregulated and expressed include IL-6, TNF-α, and 
IFN-1α/β [20-23]. Inflammation, most notably associated with IL-6, can contribute to 
reseeding and maintenance of the viral reservoir because IL-6 stimulates HIV-1 
replication by activating viral transcription in synergy with TNF-α and also by targeting a 
post-transcriptional step [24, 25]. 


 
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Blocking Jak-STAT signaling pathways could reverse or prevent HIV-induced activation 
of both macrophages and lymphocytes, thereby reducing HIV-orchestrated systemic 
immune activation and suppressing chronic inflammation. Consequently, HIV-1 
production from latently infected cells and HIV-1 infection of new target cells may 
decrease. Inhibition of these pathways may also reduce trafficking of virus and infected 
cells across the BBB, preventing CNS persistence and HIV-associated neurocognitive 
disease. Since HIV-1 infection of macrophages causes alterations in the cell cycle [26], 
chemotaxis [27], increased levels of TNF-α and other TH2-type cytokines [28], 
phagocytosis [29, 30], and antigen presentation [31], inhibition of Jak-STAT pathways 
might have profound beneficial effects on these cells.  
 
Consequently, we anticipate that by blocking Jak-STAT signaling, the CD4+ T cell count 
could also increase owing to two primary mechanisms: 1) a reduction in HIV-1 
replication-dependent cell death and 2) a reduction in bystander apoptosis-induced cell 
death independent of HIV-1 replication. The first mechanism relies upon the theory that 
there is ongoing HIV-1 replication in sanctuary sites despite suppressive ART [32-34]. In 
this setting, a reduction in pro-inflammatory cytokines could result in fewer target cells 
trafficking to sites of infection, decreased expression of chemokine receptor type 5 
(CCR5) and other cellular activation markers that would otherwise make uninfected cells 
more permissive to HIV-1 infection, and fewer virus particles would be produced per 
HIV-1 infected cell. 
 
Monocytes/Macrophages as a Reservoir for HIV-1 
Cells of the monocyte/macrophage lineage have, as of yet, a poorly defined role in HIV-1 
persistence during long-term ART. Several studies have shown a correlation between 
activated, CD14+/CD16+ monocytes and HIV-1-associated neurocognitive impairment, 
immune activation, higher viral loads, and disease progression [35-38]. In untreated HIV-
1 and possibly treated HIV-1 disease, carriage of HIV-1 across the BBB by infected 
monocytes, activated macrophages, and lymphocytes establishes a persistent infection 
primarily in perivascular macrophages and microglia as viral reservoirs in the immune-
privileged CNS (the “Trojan horse” hypothesis) [22, 36, 39]. In contrast to HIV-1 infected 
CD4+ T cells, infection in brain macrophages and microglia leads to an extended life 
span and increased survival in the presence of apoptotic stresses, while concomitantly 
orchestrating a pro-inflammatory cytokine milieu that drives viral replication in already 
infected cells, primes uninfected cells for infection, and results in production of reactive 
oxygen species and other factors that drive HIV-associated neurocognitive impairments 
[20, 40-44]. In addition, viral proteins expressed early in the viral replication cycle (such 
as Nef, Tat, and virion-associated Vpr) play a role in the formation of viral reservoirs in 
macrophages by activating transcription and interfering with apoptotic machinery [19].  
 
The BBB also consists of a number of unique elements that prevent passage of many 
drugs into the brain (as also seen in lymphoid tissue). Several drug physical properties 
influence the penetration of drugs through the BBB, including molecular weight, protein 
binding, lipophilicity, and the action of passive and adenosine triphosphate (ATP)-
dependent influx and efflux pumps [45]. Viral populations in the CNS can diverge from 
those of plasma due to the resulting differences in tissue drug exposures. Furthermore, 
the dynamic interaction between signaling pathways that promote virus replication, 
active/infiltrating macrophages, cytokines/chemokines, and secreted neurotoxic 
products, may interfere with the overall homeostatic regulation required to maintain 
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their role in inhibiting pro-HIV-1 events that are not addressed by current highly active 
ART are depicted below in Figure 1.   
 
 
Figure 1. The Jak-STAT pathway is activated by either HIV-orchestrated pro-inflammatory 
cytokines or HIV-1 gp120 engaging the host cell (upper left panel). Upon activation, multiple pro-
HIV-1 events transpire, including 1) increased production of virus in already infected cells; 2) 
priming of uninfected cells for infection; 3) recruitment of uninfected cells to the site of infection, 
therefore exposing them to infection and priming them for infection; 4) activation of monocytes 
and macrophages, which promotes trafficking of these infected cells across and to the CNS/brain; 
and 5) microbial translocation in the gut (boxes above). Use of Jak inhibitors prevents activation 


 
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of the Jak-STAT pathway, mitigating these pro-HIV-1 events (upper right panel) [46, 47]. 
 
Table 1: The Proposed Impact of Ruxolitinib on Various Biomarkers Relevant to HIV-1 
Pathogenesis, Immunologic Recovery, and the Reservoir 
 
Markers  
Role in HIV-1 infection in the 
absence of ruxolitinib 
CD14+/CD16+  monocytes 
Trafficking of infected monocytes to 
the CNS and across the BBB 
CD163 
A: Trafficking of infected 
macrophages to the CNS and across 
the BBB 
B: Recruitment of macrophages to 
privileged sites of low level 
replication (gut, lymph nodes, 
others)  
IL-6, IL-1, TNF-alpha, GM-CSF 
A: Prime uninfected cells for 
infection 
B: Produce more virus per infected 
cell 
C: Drive gut microbial translocation 
D: Impact end-organ disease such 
as cardiovascular, neurocognitive 
and malignancies 
E: Potential impact on collagen 
deposition in the bone marrow but 
this effect was  previously implicated 
in individuals with myelofibrosis 
IL-7, IL-15 
Drivers of homeostatic proliferation 
of latently infected memory 
lymphocytes 
pSTATs binding to HIV-1 LTR 
Promotes HIV-specific gene 
transcription  
 
Ruxolitinib  
The commercially available Jak 1/2 inhibitor, ruxolitinib ((3R)-3-cyclo-pentyl-3-[4-(7H-
pyrrolo [2, 3-d]pyrimidin-4-yl)pyrazol-1-yl]propanenitrile; Jakafi, Incyte Corporation and 
Novartis), acts by interfering with the binding of ATP to Jaks. Ruxolitinib was approved 
by the FDA for treatment of persons with intermediate and high-risk myelofibrosis [48] 
and polycythemia vera. Other clinical trials are currently underway to determine its 
efficacy and safety for the treatment of rheumatoid arthritis (RA), autoimmune psoriasis, 
and various malignancies.  
 
In vitro studies performed at Emory University demonstrate antiviral potency of ruxolitinib 
in HIV-infected monocyte-derived macrophages (MDM) and lymphocytes grown in 20% 
serum. The EC50/90 in macrophages was in the range of 0.09/1.3 to 0.3/4.8 µM with and 


 
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IC50 (cellular toxicity) of 13.4 to >100. The EC50/90 in lymphocytes ranged from 0.02 - 4.7 
µM with no effect on viability up to 50 µM [49]. Furthermore, ruxolitinib has a short 
plasma t1/2 (~3 hr), so that the duration of exposure to ruxolitinib is considerably shorter 
than in vitro exposure (5 days). Maximal plasma concentrations in humans (Cmax) are 
between 3.6-10.0 µM and persist for < 30 min [50, 51] demonstrating a wide therapeutic 
window of safety.  
 
Ruxolitinib is a nanomolar in vivo inhibitor of TNF-α, IL-6, and other pro-HIV 
inflammatory cytokines (see Figure 2, based on phase I/II and III clinical studies) [2, 3], 
and is a direct inhibitor of the Jak-STAT pathways activated early in HIV-1 infection [4]. 
Data provided by Incyte Corporation on participants treated with ruxolitinib for 
myelofibrosis (Study INCB 18424-256) demonstrated that inhibition of pro-inflammatory 
and activation markers were observed within 2 weeks of administration of ruxolitinib 25 
mg twice a day (BID), and in many cases were observed as early as 3 days post 
administration (Figure 4). Overall, ruxolitinib is a promising drug for further testing as a 
modulator of inflammation due to its well-documented safety profile and nanomolar anti-
inflammatory action in humans together with its sub-micromolar antiviral activity against 
HIV-1 in culture [50, 52].  
 
 
Figure 2 (adapted from [2, 3]). Changes in selected cytokines and C-reactive protein, an acute 
phase reactant and a marker for inflammation, show in patients with > 50% decrease those with < 
50% decrease, and those with no change or an increase in the composite symptom score after 6 
cycles of treatment as compared with baseline, where y-axis is reported as “points” which is a 
change cytokine level versus baseline (A), or median change in IL-6 levels versus baseline 
(percent) (B).   
 
Safety of Ruxolitinib 
Over 9,800 individuals have or are currently taking ruxolitinib, as reported by Incyte 
Corporation. Post-marketing surveillance for safety of this drug has continued since it 
was approved for myelofibrosis by the FDA in November 2011, with no recommended 
changes by the FDA. Additionally, Dr. Srdan Verstovsek, the first author of the clinical 
trials performed with ruxolitinib for myelofibrosis, stated that their longitudinal study has 
not identified any new toxicities [50]. In his review, all published reports have shown 
satisfactory safety data on 1,400 participants with no new toxicities [53]. 
 
In the initial dose-finding study of ruxolitinib in myelofibrosis (Study INCB 18424-251), 
the most common AEs observed included hematologic (thrombocytopenia and anemia) 
and non-hematologic AEs which were infrequent (bruising, dizziness, and headache) 
after a median of 14.7 months on therapy [2]. There were few Grade 3 or 4 events when 


 
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using FDA-approved doses (15 mg BID or 20 mg BID depending on the platelet count 
and adjusted as needed for efficacy or toxicity). Of the 153 participants in the study, 
serious adverse events (SAEs) occurred in 59 participants, of whom only 12 had SAEs 
that were considered to be possibly related to treatment. The hematologic events varied 
by the dosing regimen (Table 2a). Diarrhea, fever, and urinary tract infections were 
described in 5.9%, 2%, and 2.6%, respectively, for all grades with only 0.7% having a 
Grade 3 or 4 fever. Individuals with HIV-1 were excluded from these studies. 
 
In the phase III COMFORT-1 study in individuals with myelofibrosis (FDA dosing as 
above), there was a survival advantage when compared to placebo with 13 deaths in the 
ruxolitinib group (8.4%, N=155) and 24 deaths in the placebo group (15.6%, N=154) 
during a median follow-up period of 51 weeks [3]. Among the deaths, there was no 
detectable increase risk of serious infections; pneumonia occurred in two participants 
who received ruxolitinib and one who received placebo, and sepsis occurred in two 
participants from each arm. Although two participants developed acute myelogenous 
leukemia (AML) after receiving ruxolitinib, they were at increased risk for this disease 
prior to receiving the study drug. Grade 3 or 4 thrombocytopenia occurred in 12.9% of 
the ruxolitinib arm and 1.3% in the placebo arm with Grade 3 episodes of bleeding in 
2.6% of participants in the ruxolitinib arm and 2.0% in the placebo arm; 1.3% of 
participants in each arm had Grade 4 episodes of bleeding. There was one Grade 3 
event of bruising in the ruxolitinib arm. Thrombocytopenia rarely recurred at a Grade 3 or 
4 level after appropriate dose reduction and was not associated with an increase in 
bleeding events, although bruising was more common in the ruxolitinib group. Grade 3 
or 4 anemia peaked at weeks 8 to 12 and then subsequently declined to levels similar to 
those in placebo-treated participants.  
 
A longer term evaluation of 107 participants enrolled in the open label, non-randomized 
phase I-II study of ruxolitinib (INCB 18424-251) showed that 49 (46%) discontinued 
ruxolitinib by 3 years (24 by year 1, 36 by year 2, and 46 by year 3). Of those who 
discontinued treatment, only 3 (2.8%) did so for unacceptable toxicity. The others 
discontinued for progressive disease (12), withdrawal of consent (7), physician decision 
to discontinue (5), intercurrent illness (3), death (13), or other reasons (6). None of the 
deaths were considered by the investigators as related to ruxolitinib, see Table 2b. [52]. 
A cohort description in 120 Asian participants treated for myelofibrosis with ruxolitinib 
was published online in late 2014 [54]. The authors concluded that, “Adverse events 
were consistent with those seen in the COMFORT studies.” The principal AEs were 
anemia, thrombocytopenia and diarrhea. Of 7 deaths that occurred in the cohort, one 
death due to sepsis and one due to hepatorenal syndrome were suspected by the 
investigator to be related to ruxolitinib treatment. 
 
 
 
 
 
 
 
 
 
 
 


 
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Table 2. (a) Hematologic AEs [2] (*red box) indicates dose similar to that proposed for 
this study, (b) Rate and Reasons for Discontinuation in Study INCB18424-251 [52]. 
 
a. Hematologic Adverse Events 
Variable 
10 mg 
Twice 
Daily 
15 mg 
Twice 
Daily 
 
25 mg 
Twice 
Daily 
 
50 mg 
Twice 
Daily 
 
25 mg 
Once 
 Daily 
 
50 mg 
Once 
 Daily 
 
100 
mg 
Once 
 Daily 
 
200 mg 
Once 
 Daily 
 
 
Total 
Thrombocytopenia – no./total no. (%)* 
 
Grade 3 
3/29  
(10) 
1/35  
(3) 
11/47 
(23) 
3/5  
(60) 
0/6  
(0) 
6/22  
(27) 
2/6  
(33) 
0 
26/153 
(17) 
Grade 4 
0 
0 
3/47  
(6) 
1/5  
(20) 
0 
2/22  
(9) 
0 
1/3  (33) 
4/153  
(3) 
New anemia Ŧ, 
participants 
who were 
transfusion 
independent  
 
3/19 
(16) 
 
2/24  
(8) 
 
8/30 
(27) 
 
0/2  
(0) 
 
1/4  
(25) 
 
5/15 
(33) 
 
1/4  
(25) 
 
3/3 
(100) 
 
23/101 
(23) 
* Values for platelet count abnormalities were graded according to the Common Terminology Criteria for 
Adverse Events (CTCAE), version 3.0 (Grade 3 = 25 x 109/liter to < 50 x 109/liter; Grade 4 = < 25 x 
109/liter.   
Ŧ Anemia was defined as a decrease in the hemoglobin level of more than 20 g per liter to a Grade 3 or 4 
level (as defined according to the CTCAE, version 3.0) in patients who were not transfusion dependent.   
 
b. Rate and reasons for discontinuation in study INCB18424-251, An Open-Label, 
Nonrandomized Phase I-II Study of Ruxolitinib 
                                                                  MDACC                         Mayo Clinic Rochester 
Participants Enrolled, N 
107 
51 
Participants remaining on 
study, N (%) 
58 (54) 
5 (10) 
Participants who discontinued, 
N, (%) 
49 (46) 
46 (90) 
     Discontinued by 1 y., % 
24 
51 Ŧ 
     Discontinued by 2 y., % 
36 
72 Ŧ 
     Discontinued by 3 y., % 
46 Ŧ Ŧ 
89 Ŧ 
Primary reason for discontinuation,* N, (%) 
     Death 
13 (12.1) 
4 (7.8) 
     Progressive disease 
12 (11.2) 
10 (19.6) 
     Participant withdrawal of  
     Consent 
7 (6.5) 
15 (29.4) 
     Physician decision to       
     Discontinue 
5 (4.7) 
12 (23.5) 
     Intercurrent illness 
3 (2.8) 
0 
     Unacceptable toxicity 
3 (2.8) 
1 (2.0) 
     Other 
6 (5.6) 
4 (7.8) 
Data provided by Incyte Corporation upon MD Anderson Cancer Center request.  


 
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* As categorized by defined headings on case report forms (CRFs) filled out by the investigator.  
Ŧ Discontinuation rates as published in Tefferi et al in 2011[55].  
Ŧ Ŧ NOTE: not all ongoing participants have reached year 3; by Kaplan-Meier analysis, the 
     discontinuation ratio at year 3 was 53 %.  
 
Additional safety data on ruxolitinib from studies conducted for other indications are 
available online at “ClinicalTrials.gov”; however, causality of side effects listed was not 
determined in these reports. As an example, the “Study to Determine the Safety and 
Efficacy of Ruxolitinib (INCB018424) in Participants With Polycythemia Vera (PV) or 
Essential Thrombocythemia (ET)” had 13.7% (10/73) of participants with SAEs that 
varied depending on the dose (10 mg BID, 25 mg BID, and 50 mg QD) and underlying 
disease (PV or ET), and the SAE included one report each of pneumonia, 
thrombocytopenia, anemia, renal cancer, renal failure, congestive cardiac failure, atrial 
flutter, headache, variceal bleeding, gastrointestinal bleeding, cholecystitis and 
bronchitis. In a second metastatic prostate cancer study, out of the 22 participants who 
received ruxolitinib and experienced an SAE, two had anemia, and there was one report 
each of an overdose, pathologic fracture, cardiac arrest, sepsis, gastrointestinal 
bleeding, prostate cancer metastasis, metastatic pain, hydronephrosis, and renal failure. 
In a third multiple myeloma study, out of 20 participants who received either ruxolitinib 
(N=13) or ruxolitinib plus dexamethasone (N=7), there were three SAE reports of 
pneumonia, two reports each of gastrointestinal bleeding and disease progression, and 
one report each of anemia, congestive cardiac failure, pericardial effusion, pyrexia, 
fatigue, pain, pneumococcal sepsis, urinary tract infection, overdose, hyperglycemia, 
lung cancer, seizure, pulmonary embolism, and hematoma. The incidence of SAE was 
significantly higher in the ruxolitinib plus dexamethasone group versus ruxolitinib group. 
Progressive multifocal leukoencephalopathy (PML) [56], reactivation of latent 
tuberculosis (pulmonary [57, 58] and disseminated [59, 60]), hepatitis B infection [61, 
62], herpersvirus infections [63], Pneumocystis jiroveci pneumonia [64], toxoplasmosis 
retinitis [65], and Cryptococcus neoformans pneumonia [66] have been reported on 
participants who received ruxolitinib treatment for myelofibrosis. 
 
Incyte Corporation provided unpublished data presented at both the American College of 
Rheumatology (ACR) meeting as well as unpublished data obtained internally, 
demonstrating that 10 mg ruxolitinib BID has equivalent efficacy for cytokine reduction, 
but the average change in hemoglobin and platelet count is negligible. These data 
demonstrate that 10 mg BID is sufficient to inhibit production of pro-inflammatory and 
activation markers while concomitantly mitigating any potential detrimental side effects 
that were observed at higher dosages. Therefore, for this study the 10 mg BID dose has 
been selected for the treatment arm in order to minimize treatment-related AEs while 
maximizing efficacy. 
 
Dose Effects on Biomarkers 
Data on cytokine changes in participants treated with lower doses of ruxolitinib for 
myelofibrosis were provided by Incyte Corporation. Additionally, data on cytokine 
changes in participants treated with lower doses of ruxolitinib for RA have been 
obtained from an oral presentation at the 2008 American College of Rheumatology 
Annual Scientific Conference. Both of these data sources demonstrate reductions in 
levels of various biomarkers (see heatmap below, Figure 3), as well as RA and 
myelofibrosis symptom scores with doses as low as 5 mg BID.    
 


 
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Aggregate data have been provided by Incyte Corporation relative to the dose of 10 or 
15 mg BID ruxolitinib in Study INCB 18424-356, “Study of Efficacy and Safety in 
Polycythemia Vera Participants Who Are Resistant to or Intolerant of Hydroxyurea: JAK 
Inhibitor INC424 (INCB018424) Tablets Versus Best Available Care: (The RESPONSE 
Trial)”. Many participants in the study did not demonstrate levels of IL-6 above the 
lower limit of detection (LLD) of 6 pg/mL at baseline. Therefore, at endpoint 
measurement, it was not possible to determine whether their IL-6 levels had decreased 
(Figure 5 below). Considering only those participants with elevated levels of IL-6 at 
baseline, four of six participants demonstrated significant decreases in IL-6 levels 
(Figure 6 below). The participants with a low baseline IL-6 level could have had a 
decline in IL-6 while on ruxolitinib but those changes would not be detected by the assay 
used. A more sensitive assay for IL-6 will be employed in A5336, with a LLD of 0.2 log10 
pg/mL (SD=0.25 log10 pg/mL). This will allow us to measure an effect for any 
participants with a base line IL-6 above approximately 0.5 pg/mL. 
 
There are published data in myelofibrosis showing that symptomatic improvement for 
symptoms thought to be caused by elevated inflammatory cytokines, including night 
sweats, pruritus and bone pain are equally improved at doses of 10 mg BID as at higher 
doses up to 25 mg BID. There is a clear drop off in symptomatic benefit when the dose 
is lower than 10 mg BID (given as 5 mg BID), but even at that level there is improvement 
compared to placebo [67]. Reductions in phosphorylated-STATs and Jaks at 10 mg BID 
are also observed in myelofibrosis and RA studies, each of which are surrogate markers 
for down regulation of IL-6 levels.   
 
Figure 3. Ruxolitinib decreases levels of IL-6 biomarker 
 


 
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Figure 4. Changes in inflammatory markers demonstrate predominant effect at 4 weeks (28 days) 
for 21 participants with myelofibrosis receiving 25 mg BID (Study INCB 18424-256) 
 
 
 
 
Figure 5. Participants with and without elevated IL-6 at baseline who received 10-15 mg BID for 
polycythemia vera and essential thrombocythemia (Study INCB18424-356) 
 
 


 
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Figure 6: Participants with elevated IL-6 at baseline who received 10-15 mg BID for 
polycythemia vera and essential thrombocythemia (Study INCB18424-356) 
 
Pharmacology of Ruxolitinib 
The FDA-approved starting dose is 20 mg given orally (PO) BID for participants with a 
platelet count > 200,000/mm3, 15 mg PO BID for participants with a platelet count of 
100,000/mm3 to 200,000/mm3, and 5 mg PO BID for participants with a platelet count 
between 50,000/mm3 and 100,000/mm3. Data from Incyte Corporation have shown that 
ruxolitinib can be dosed at 10 mg BID with roughly equivalent myelofibrosis efficacy and 
significantly reduced AEs compared to higher doses. Dose reductions and subsequent 
dose increases in clinical trials and in the clinical setting are quite common with rapid 
resolution of AEs (and improvement in clinical indicators). Similarly, titrating dose to 
effect is both recommended and commonly practiced as well. Therefore, the protocol 
team has chosen to use a dose of 10 mg BID (without dose escalations) for all 
participants in this study and will exclude participants with platelet counts ≤ 
200,000/mm3.  
 
Ruxolitinib is a substrate of CYP3A4, but not an inhibitor of this enzyme (see package 
insert). It is recommended that when administering ruxolitinib with strong CYP3A4 
inhibitors, the total daily dose of ruxolitinib should be reduced to 5 mg BID (i.e., 
approximately 50% of the dose rounding up to the nearest dosage strength). No dose 
adjustment is necessary for mild or moderate CYP3A4 inhibitors (see package insert). 
Raltegravir and other INSTI are not substrates for CYP3A4. 
 
Safety and Coadministration of EFV with Ruxolitinib 
Efavirenz is classified as a moderate CYP3A4 inducer [68]. No dose adjustment is 
recommended when ruxolitinib is coadministered with a CYP3A4 inducer (see package 
insert). However, plasma AUC values for ruxolitinib metabolites increased with 
Upon removal of participants who did not have elevated levels of IL-6 at start of 
study, and consideration of only participants with elevated IL-6 at baseline, it is 
clear that 10-15 mg BID ruxolitinib reduces IL-6 levels by week 4 
 


 
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increasing severity of renal impairment.   
 
Metabolites of Ruxolitinib 
Metabolites of ruxolitinib act on the same pharmacological receptors, however at lower 
potency (1/5 to 1/2 of the parent compound) and at relatively low concentrations. 
Therefore, when ruxolitinib is metabolized, the effect of the metabolite is between 20-
50% of the original parent compound. Thus, the contribution of toxicity resulting from an 
increase in drug metabolites would be more than compensated for by a net decrease in 
concentration of the parent drug. The rates of elimination half-life of ruxolitinib and its 
metabolites are rapid and similar (approximately 3 – 5.8 hours). Therefore, steady-state 
accumulation is not expected to occur (see more at: 
http://www.incyte.com/sites/default/files/Jakafi_PI.pdf?time=20141230135651). The 
dose selected for this study is 10 mg BID, which is the recommended reduced dose and 
is below or equivalent to package insert guidelines when considering metabolites of 
ruxolitinib in the presence of EFV.  
 
The mass balance trial INCB 18424-134 reported that following a single 25 mg dose of 
ruxolitinib solution (100 μCi 14C-ruxolitinib) parent drug was the predominant entity in 
circulation, representing 58% to 74% of the total radioactivity between 1 and 6 h post-
dose. Metabolite M18 was observed at 17% of the total, circulating, drug-related material 
based on AUC and the other observed ruxolitinib mono- and di-hydroxylated and ketone 
metabolites represented less than 10% (Figure 7). Eight of these metabolites (M7, M8, 
M9, M11, M14, M16, M18, and M27) when added to parent drug, accounted for greater 
than 90% of the drug-related material in circulation (based on AUC). No metabolites 
were observed in human plasma after 12 h post-dose. 
 


 
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Figure 7: Metabolites of ruxolitinib. In summary, M18 is the major metabolite with 17%; sum of 8 
other metabolites ≤10%. 
 
Justification for Choice of IL-6 as a Primary Endpoint Marker  
IL-6 has been the most highly implicated biomarker for end-organ disease and mortality 
on ART. A number of inflammation-associated biomarkers have demonstrated 
prognostic significance in well-treated HIV-1 infection. These include IL-6, D-dimers, 
sCD14, sCD163, anti-TNF-receptor, CRP, monocyte phenotype, and T cell activation. 
Perhaps the most consistent and well-validated biomarkers are IL-6 and D-dimer. In the 
SMART study, IL-6 levels were most strongly associated with cardiovascular disease 
(CVD) and mortality (odds ratio for 4th/1st quartile of 3.5 and 11.8 for CVD and mortality, 
respectively) [69, 70]. These results were confirmed in a large analysis of all treated 
participants in the INSIGHT families (which included ESPIRIT and SILCAAT, n=3766), 
a single determination of IL-6 was associated with disease progression over the 
subsequent 5 to 8 years. The prognostic significance of IL-6 in treated HIV-1 disease 
was confirmed in a longitudinal analysis of participants in ALLRT and a case-control 
study from participants enrolled in SOCA/SCOPE [71, 72]. A number of other studies 
have confirmed that IL-6 is higher than expected in treated HIV-1 disease [73-75], and 
associated with poor prognosis [8, 76, 77]. The impact of IL-6 on prognosis is generally 
not attenuated after controlling for viral load, CD4+ T cell count, and other factors. To our 
knowledge, there is no other biomarker which has been so consistently associated with 
disease progression in the context of treated HIV-1 disease. Furthermore, mutations in 
the IL-6 gene have been associated with increased IL-6 levels coincident with an  
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increase in CVD, placing IL-6 in the casual pathway, not just as a surrogate marker of 
inflammation [7] but as an earlier predictor of disease progression [78]. 
 
In order to assess feasibility, we examined participants who were analyzed as part of 
NWCS 329, wherein participants from several ACTG trials were followed for 
cardiovascular and malignant endpoints. Cases and controls were selected based on 
these particular outcomes. Among the 171 controls with viral load < 50 copies/mL and 
CD4+ T cell > 350 cells/mm3 at 1 year after ART initiation, the following IL-6 quartiles 
were identified: 
 
Quartile 1 (25%):  
IL-6 < 0.86 
Quartile 2 (26%): 
IL-6 = 0.86 to 1.42 
Quartile 3 (20%): 
IL-6 = 1.43 to 2.0 
Quartile 4 (29%) 
IL-6 ≥ 2.1 
 
Based on these data, we believe that a reasonable number of participants will have 
levels of IL-6 within a range conducive for appreciating a reduction in the presence of 
ruxolitinib. 
 
Justification for the Choice of Assay to Measure Changes in IL-6  
We have measured plasma levels of IL-6 using the R&D high sensitivity assay. This is a 
solid-phase sandwich ELISA that can be performed on serum or plasma. In this assay, 
the LLD is usually around 0.16 pg/mL, and the assay range is 0.11 pg/mL-10 pg/mL. The 
manufacturer reports that there is < 0.5% cross-reactivity observed with molecules 
related to IL-6. Using this assay, it was reported [63] that among 60 participants with 
virologic control and immune failure (CD4+ T cell < 350/uL), 20 participants with 
virologic control and immune success (CD4+ T cell > 500/uL) and 21 healthy controls, 
none had IL-6 levels in plasma lower than 0.46 pg/mL. Samples are obtained in EDTA 
and processed and cryopreserved within 6 hours of blood draw. Assays are performed 
on cryopreserved samples using manufacturer’s instructions and inter-assay consistency 
is evaluated by running an aliquot of the same sample on each plate. Thus we are 
confident that our assay is sensitive enough to measure IL-6 levels in plasma in our 
study participants.  
 
HIV-1 Persistence  
Recent in vitro data have provided further insight into the potential role for ruxolitinib to 
affect the HIV-1 reservoir. 
 
Overall, the potential mechanisms impacted by ruxolitinib include the following: 
 
1. Seeding of the HIV-1 reservoir: Recent data (Figure 8) and others show that γ-C 
receptor cytokines (IL-2, IL-7, IL-15) which are triggered by chronic T cell receptor 
(TCR) activation and bystander immune activation (bacterial translocation, alarmins) 
can upregulate HIV-1 transcription and production. This is because the sequence of 
the HIV-1 LTR includes STAT binding sites [5, 79]. This ultimately results in 
increased numbers of infected cells, increased production of viral particles and an 
enhanced seeding of the HIV reservoir. Inhibition of Jak-STAT signaling pathways 
should decrease the number of HIV-1 infected cells and ultimately the HIV-1 
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2. Maintenance of the HIV-1 reservoir: We have shown that IL-7 (a γ-C receptor 
cytokine) maintains and expands the latent HIV-1 reservoir by triggering homeostatic 
proliferation [5]. Inhibition of homeostatic proliferation downstream of cytokines such 
as IL-7 and IL-15 should result in a decreased expansion/maintenance of the latent 
HIV-1 reservoir. 
 
3. Inhibition of cytokine induced inflammation driven HIV-1 replication and T cell  
dysfunction/exhaustion: Several pro-inflammatory cytokines, which are upregulated 
in HIV-1 infection, enhance HIV-1 replication as they can activate T cells thereby 
increasing expression of CCR5 and trafficking of these cells to sites of infection; they 
also inhibit T cell and innate immune function as these cytokines upregulate type I 
interferons (IL-7, IL-15) and several negative regulators of T cell activation (PD-1, 
Lag-3) and their ligands (PDL-1, MHC II) in individuals with HIV-1. This should 
induce HIV-1 latency in cells expressing these molecular factors [5, 22, 79, 80]. 
 
4. Inhibition of Jak-STAT may prevent the induction of an HIV-1 latent pool by  
preventing expression of negative regulators on T cells and their ligands on innate 
immune cells. This could result in the restoration of innate and adaptive immune 
function, two critical steps in controlling or eliminating latently infected cells and a 
functional cure [81]. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 8. Inhibition of HIV-1 Replication by Ruxolitinib. Viral production as measured by p24 
ELISA in primary CD4+ T cells isolated from a viremic donor stimulated for 3 (A) and 6 days 
(B) with CD3/28 in the presence of increasing concentration of ruxolitinib or DMSO (vehicle) 
only, reported in µM (x-axis).  
 
Infectious HIV-1 reservoir measurements using a quantitative virus outgrowth assay (Q-
VOA) may underestimate reservoir size [82] and require larger volume blood draws than 
are feasible in this study. We plan to use total and integrated HIV-1 DNA levels as 
markers of HIV-1 persistence and plasma HIV-1 RNA and peripheral blood mononuclear 
cells (PBMC)-associated HIV-1 RNA and 2-LTR circles as markers of HIV production. 
Measurement of plasma virus load, cellular RNA and 2-LTR circles will examine two 
different components of HIV-1 replication: the amount of virion production that may occur 
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form proviruses. STAT-5-like sequences are present in the HIV-1 LTR and suggest a 
mechanism whereby ruxolitinib treatment may modulate HIV-1 latency. 2-LTR circle 
quantification will corroborate changes that may be observed in total HIV-1 DNA, relative 
to integrated HIV-1 DNA, during ruxolitinib therapy. 
 
Activation of Viral Co-infections 
Previous studies reported reactivation of herpes viruses among participants 
receiving ruxolitinib for clinical indications; however, the use of other 
immunosuppressant therapies makes it impossible to assess the individual 
contribution of ruxolitinib [83, 84]. As part of this study we will determine whether 
the use of ruxolitinib is associated with more frequent reactivation of human HHV 
as measured in longitudinally collected oral swabs. Shedding of HHV DNA is 
frequently detected in saliva of healthy individuals and more frequently in HIV-
infected adults and persistent replication of HHV (in particular CMV) was 
repeatedly associated with increased systemic inflammation and cellular 
activation [85, 86, 87]. As an alternative hypothesis, since persistent inflammation 
can also be a trigger for HHV reactivation, reduction in inflammation after 
ruxolitinib administration might be associated with decrease in HHV replication.  
 
If treatment with ruxolitinib is associated with increased oral shedding of HHV, we 
will determine whether the presence of enhanced sub-clinical HHV replication 
might in turn affect the ability of this immuno-modulatory intervention to reduce 
inflammation during suppressive ART. Also, if increased reactivation occurs, then 
measures of asymptomatic HHV replication should be considered as a possible 
safety measure for future studies. 
 
Potential Impact of this Clinical Trial 
Chronic inflammation and immune activation promote end-stage organ disease leading 
to decreased survival in virologically suppressed individuals on ART. To date, 
interventions to mitigate this process are severely lacking. Inhibition of the Jak-STAT 
pathway using a FDA-approved agent, ruxolitinib, provides an unprecedented 
opportunity to significantly attenuate levels of inflammation and such an approach may 
restore the immunologic balance. We hypothesize that Jak-STAT inhibitor treatment will 
result in higher CD4+ T cell counts as well as lower levels of immune activation and 
chronic inflammation. We expect to observe decreased immune activation that will 
persist following discontinuation of ruxolitinib. We also posit that by “resetting” the 
immunologic balance in favor of less immune activation and inflammation, this 
immunologic milieu will persist in the absence of ruxolitinib. Since lower levels of 
immune activation and chronic inflammation have been associated with fewer AIDS and 
serious non-AIDS clinical events, we anticipate that long term use of ruxolitinib could 
provide a tangible clinical benefit, i.e., increased event-free survival. Furthermore, we 
have shown in vitro that ruxolitinib has specific anti-HIV-1 properties even in resting 
CD4+ T cells and monocyte/macrophages, and can reduce homeostatic proliferation, 
which we expect will result in a decline of the HIV-1 reservoir. If successful, this study 
will be the first of its kind to demonstrate both lasting immunologic enhancement and 
impact on the HIV-1 reservoir. 
 
Justification for Proceeding with Studies in Humans with HIV-1 
Presently studies of ruxolitinib are being conducted on non-human primates (NHP) 
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suppressed on ART). However, there is no guarantee that the PKs, safety profile, and 
efficacy of ruxolitinib in these models will correlate with humans infected with HIV-1.  
 
The dose selected for the NHP/SIV study is significantly lower than that which has 
already been dosed in other NHP studies (Incyte Corporation data). The dose being 
used in the NHP/SIV study is also calculated to mirror the 10 mg BID dosing in humans, 
which is well tolerated and lower than commonly prescribed doses of 15, 20, or 25 mg 
BID. To that end, studies with prolonged dosing periods of ruxolitinib in the NHP have 
not been conducted previously. The dose selected herein for the NHP study is designed 
to provide a balance between what we know is well tolerated in humans, and a safe level 
relative to what has been dosed in NHP previously. More specifically, the dose to be 
used in the NHP study has been calculated using the published single dose PK of 
ruxolitinib in NHP, allowing for mathematical scaling of NHP dosing to match plasma 
PKs observed in humans for 10 mg BID dosing. 
 
The data obtained could provide a proof-of-principle for reduction in SIV or HIV induced 
inflammation and activation, but species and virus differences dictate that results should 
not be used to modify human clinical studies. Differences between the SIV infected NHP  
model and humans, such as virus (HIV versus SIV), cellular factors, coreceptors/ 
receptors, and generalized cross-species differences, dictate that this pilot NHP study 
will probably not yield information resulting in modification of the human clinical protocol. 
  
The NHP pilot study included a total of four rhesus macaques with viral loads > 200,000 
copies/mL, which were treated with 2.2 mg/kg ruxolitinib BID via oral administration for a 
total of 6 weeks. Two monkeys were administered add-in HAART [(-)-FTC, PMPA, and 
Kaletra] starting at week 4 of ruxolitinib administration. During the first dose of ruxolitinib, 
blood samples were collected at baseline (before drug), and 1, 2, 4, and 12 hour post 
administration for PKs. Blood samples were tested once a week, thereafter, and plasma 
collected during the remainder of the study (6 weeks total). Samples (3 mL) were 
aliquoted to collect whole blood (for measuring of integrated viral DNA in PBMCs, 
monocytes and macrophages and cell associated immunology markers, e.g., D16, 
CD163, Ki67, HLA-DR, CD25, and CD38. Plasma samples have been stored at -70o C 
before being assayed to determine concentrations of ruxolitinib, (-)-FTC, PMPA, and 
Kaletra, and soluble activation markers, e.g., sCD14, IL-6, IL-1/b. CSF was collected 
once per week to measure drug levels and viral loads. 
 
The safety profile of ruxolitinib in individuals with myelofibrosis has been well-
documented and FDA-approved for this indication. Emerging data have shown that even 
fewer AEs have been reported in individuals without myelofibrosis (solid tumors and 
RA). We believe that the population of individuals living with HIV-1 who would be eligible 
for this study is immunologically and hematologically “healthier” than individuals with 
myelofibrosis and therefore should have few AEs. 
 
Justification for the Participant Population Selected 
 
Virologically Suppressed on ART 
In order to assess the impact of ruxolitinib on chronic inflammation and systemic immune 
activation, it was important to exclude the impact of ART on these same markers 
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Age and CD4+ T Cell Criteria 
In order to minimize AEs associated with ruxolitinib, we chose a participant population 
with an adequate CD4+ T cell -count and within a safe age range, ≥ 18 to < 75. 
Furthermore, the immune system of adults and children of ages outside of this range 
behave differently, which could introduce additional confounders into the efficacy 
assessment. 
 
Exclusion of Pregnant Women 
Ruxolitinib has been designated pregnancy category C. In animal studies, treatment with 
ruxolitinib resulted in an increase in late resorptions and reduced fetal weights at 
maternally toxic doses. There are no adequate and well-controlled studies of ruxolitinib 
in pregnant women. 
 
Exclusion of Participants with Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV) 
Infection 
According to Incyte Corporation, there are only five participants with HBV infection and 
no participants with HCV infection who received ruxolitinib. Additionally, two case 
reports of HBV reactivation in individuals receiving ruxolitinib for myelofibrosis were 
published [61, 62]. Therefore, it was recommended that participants with HBV infection 
or HCV infection be excluded from this study. 
 
Risk Benefit Assessment 
Ruxolitinib may have side effects, including thrombocytopenia, anemia, diarrhea, fever, 
infections, and fatigue and could result in changes of ARV concentrations. There are 
also risks associated with phlebotomy such as discomfort, bleeding, bruising and/or 
inflammation plus a small risk of infection. It is not known if ruxolitinib causes harm to the 
fetus. By using a low dose of ruxolitinib, short treatment duration, and choosing a 
population that has higher CD4+ T cell counts and that is not pregnant, we feel these 
risks have been minimized. This study is intended to explore how ruxolitinib could impact 
the immune system for individuals with virologic suppression on ART. There is no 
anticipated benefit for participants participating in this study. Information learned 
from this study may help others who have HIV-1 and are experiencing ongoing 
inflammation and systemic immune activation. For a further description of the potential 
impact of this study, refer to the section “Potential Impact of This Clinical Trial” (see 
above). 
 
 
3.0 
STUDY DESIGN 
 
A5336 is a randomized, open-label, prospective, 2-arm (phase IIa) study that will enroll 
60 HIV-1 infected participants (≥ 18 to < 75 years of age) who have been virologically 
suppressed for at least 2 years (defined below in section 4.1.3), have CD4+ T cells > 
350 cells/mm3 ≤ 30 days prior to study entry, and have plasma HIV-1 RNA below the 
limit of detection ≤ 30 days prior to entry. Participants will be randomized at a 2:1 ratio 
to either Arm A (treatment with ruxolitinib) or Arm B (no study treatment) (40 and 20 in 
the ruxolitinib and no study treatment arms, respectively). Participants in Arm A will 
receive treatment with ruxolitinib 10 mg BID for 5 weeks. Based upon the mechanism of 
action for ruxolitinib, it is not expected that the plasma viral load will increase. However, 
as a safety precaution, participants will be monitored for changes in HIV-1 viral load. If 
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which might provide further rationale for assessing the virologic endpoints in the 
absence of achieving the desired immunologic outcomes, we will assess for changes in 
the HIV-1 reservoir (specifically plasma HIV-1 RNA by single copy assay, cellular HIV-1 
RNA and total DNA, 2 LTRs, and integrated DNA).  Refer to section 6.1, Schedule of 
Events (SOE), for specific evaluations and time points. Participants in Arm A will be 
followed for 7 weeks after discontinuation of ruxolitinib, and will be on study for a total of 
12 weeks. Participants in Arm B will be followed on-study for 12 weeks. 
 
 
4.0 
SELECTION AND ENROLLMENT OF PARTICIPANTS 
 
4.1 
Inclusion Criteria 
 
4.1.1 HIV-1 infection, documented by any licensed rapid HIV test or HIV enzyme or 
chemiluminescence immunoassay (E/CIA) test kit at any time prior to study entry 
and confirmed by a licensed Western blot or a second antibody test by a method 
other than the initial rapid HIV and/or E/CIA, or by HIV-1 antigen, or historical 
plasma HIV-1 RNA viral load. 
 
NOTE: The term “licensed” refers to a US FDA-approved kit, which is required for 
all IND studies. 
 
WHO (World Health Organization) and CDC (Centers for Disease Control and 
Prevention) guidelines mandate that confirmation of the initial test result must 
use a test that is different from the one used for the initial assessment. A reactive 
initial rapid test should be confirmed by either another type of rapid assay or an 
E/CIA that is based on a different antigen preparation and/or different test 
principle (eg, indirect versus competitive), or a Western blot or a plasma HIV-1 
RNA viral load. 
 
4.1.2 CD4+ T cell count > 350 cells/mm3 ≤ 30 days prior to study entry at any US 
laboratory that has a CLIA (Clinical Laboratory Improvement Amendments) 
certification or its equivalent. 
 
4.1.3 Documentation of virologic suppression defined as:  
 
(a) at least two historical HIV-1 RNA < 50 copies/mL using FDA-approved assay 
performed by any laboratory that has a CLIA certification or its equivalent while 
on ART, one of which was obtained between 336 to 31 days, inclusive, prior to 
study entry and one obtained between 337 and 730 days, inclusive, prior to study 
entry,  
(b) no HIV-1 RNA values ≥ 50 copies/mL using a FDA-approved assay 
performed by any laboratory that has a CLIA certification or its equivalent within 
336 days, inclusive, prior to study entry, and  
(c) not more than one HIV-1 RNA value equal to or greater than 50 and less than 
or equal to 200 copies/mL from a result obtained between 337 and 730 days, 
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4.1.4 Screening plasma HIV-1 quantitative RNA below the limit of detection using an 
FDA-approved assay with a lower limit of quantification < 50 copies/mL (e.g., < 
40 copies/mL obtained by the Abbott Real-Time assay or 20 copies/mL by the 
Roche Taqman v2.0 assay) performed by any laboratory that has a CLIA 
certification or its equivalent obtained ≤ 30 days prior to study entry. 
 
4.1.5 Tuberculosis (TB) screening within 365 days of the screening visit 
diagnosed by tuberculin skin test or interferon gamma release assay. 
  
4.1.6 Currently on continuous ART for at least 730 days prior to study entry, defined as 
continuous ART for the 730 days period, inclusive, prior to study entry with no 
ART interruption longer than 7 consecutive days. 
 
NOTE: The current regimen must include tenofovir/emtricitabine (TDF/FTC) or 
abacavir/lamivudine (ABC/3TC) + a nonnucleoside reverse transcriptase inhibitor 
or integrase strand transfer inhibitor (NNRTI or INSTI, not containing cobicistat) 
for at least 60 days, inclusive, prior to study entry. 
 
4.1.7 The following laboratory values obtained ≤ 30 days prior to entry by any US 
laboratory that has a CLIA certification or its equivalent: 
 
 
Absolute neutrophil count (ANC) ≥ 1000/mm3 
 
Hemoglobin ≥ 12.0 g/dL 
 
Platelets ≥ 200,000/mm3 
 
Calculated creatinine clearance (CrCl) ≥ 80 mL/min (by Cockcroft Gault 
equation). NOTE: A calculator for estimating the CrCl can be found at 
www.fstrf.org/ACTG/ccc.html 
 
Aspartate aminotransferase (AST) (SGOT) ≤ 1.5x ULN 
 
Alanine aminotransferase (ALT) (SGPT) ≤ 1.5x ULN 
 
Alkaline phosphatase ≤ 1.5x ULN 
 
4.1.8 For females of reproductive potential (defined as women who have not been 
post-menopausal for at least 24 consecutive months, i.e., who have had menses 
within the preceding 24 months, or women who have not undergone surgical 
sterilization, specifically hysterectomy and/or bilateral oophorectomy or bilateral 
salpingectomy) must have a negative serum or urine pregnancy test with a 
sensitivity of 25 mIU/mL within 72 hours, inclusive, prior to study entry.  
 
4.1.9 All participants must agree not to participate in a conception process (e.g., 
active attempt to become pregnant or to impregnate, sperm donation, in vitro 
fertilization). 
 
4.1.10 All participants of reproductive potential, who are participating in sexual activity 
that could lead to pregnancy, must agree to use at least one reliable method of 
contraception while receiving the study drugs and for 7 weeks after stopping the 
medications. Acceptable forms of contraceptive include: 
 
 
Condoms (male or female) with or without a spermicidal agent 
 
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 
Intrauterine device (IUD) 
 
Hormone-based contraceptive (must contain at least 35 mcg of ethinyl 
estradiol) 
 
4.1.11 Women who are not of reproductive potential (women who have been post-
menopausal for at least 24 consecutive months or have undergone hysterectomy 
and/or bilateral oophorectomy or salpingectomy) or men who have documented 
azoospermia or undergone vasectomy are eligible to start study drugs without 
requiring the use of contraceptives. Acceptable documentation of sterilization and 
menopause is specified below. 
 
Written or oral documentation communicated by clinician or clinician’s staff of 
one of the following: 
 
 
Physician report/letter 
 
Operative report or other source documentation in the patient record (a 
laboratory report of azoospermia is required to document successful 
vasectomy)  
 
Discharge summary 
 
Follicle stimulating hormone-release factor (FSH) measurement elevated into 
the menopausal range as established by the reporting laboratory. 
 
4.1.12 Men and women age  18 and < 75 years. 
 
4.1.13 Ability and willingness of participant or legal representative to provide written 
informed consent and attend study visits as scheduled at a participating site. 
 
4.2 
Exclusion Criteria 
 
4.2.1 A current or past history of progressive multifocal leukoencephalopathy. 
 
4.2.2 Breastfeeding or pregnancy.  
 
4.2.3 Use of strong inhibitors or inducers of CYP3A4 including a protease inhibitor, 
cobicistat or entry inhibitors as part of the current ART regimen or other 
concomitant therapy.  
 
4.2.4 Known allergy/sensitivity or any hypersensitivity to components of study drug or 
their formulation. 
 
4.2.5 Active drug or alcohol use or dependence that, in the opinion of the site 
investigator, would interfere with adherence to study requirements. 
 
4.2.6 Acute or serious illness or infection requiring systemic treatment and/or 
hospitalization within 60 days prior to entry. 
 
4.2.7 Vaccinations (other than influenza) ≤ 30 days prior to the study entry visit.  
 


 
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NOTE: Influenza vaccine is permitted. Participants are encouraged to get this 
vaccine ≥ 7 days prior to the study pre-entry visit.  
 
4.2.8 Use of immunomodulators (e.g., interleukins, interferons, cyclosporine), systemic 
cytotoxic chemotherapy or investigational therapy ≤ 60 days prior to study entry.   
  
4.2.9 Any current diagnosis or past history of a significant pulmonary, 
neurologic, cardiac, renal, or hepatic disorder prior to study entry, excluding 
treated HIV or treated hypertension. Diagnoses that would lead to 
exclusion include, but are not limited to the following: 
 
CDC category C AIDS-indicator conditions 
NOTE A: Except HIV encephalopathy, HIV wasting, esophageal 
candidiasis, or pneumocystis pneumonia without dissemination. 
NOTE B: List available: 
http://www.cdc.gov/mmwr/preview/mmwrhtml/00018871.htm 
 
Varicella zoster 
 
Lymphoproliferative malignancy 
 
Chronic or severe psychiatric condition 
 
Chronic liver disease of any etiology and any degree of severity.  
 
Chronic hepatitis  
 
Disseminated fungal infection of any type or duration that is not limited 
to cutaneous or mucocutaneous surfaces 
 
A medical disorder that predisposes to bleeding 
NOTE: If a site investigator is unsure of whether a history of a 
significant medical or psychiatric condition should lead to participant 
exclusion, the investigator should err on the side of safety if s/he 
believes that an active or clinically resolved disorder may put the 
participant at risk from participation in the study, influence the results 
of the study, or affect the participant’s ability to participate. If still 
uncertain, then the site should contact the protocol team 
(actg.corea5336@fstrf.org) to assist in a determination. 
 
4.2.10 Change in the ART regimen within 12 weeks, inclusive, prior to study entry or 
intended modification of ART during the study. 
 
NOTE: Modifications of ART doses during the 12 weeks prior to study entry are 
permitted. In addition, the change in formulation (e.g., from standard formulation 
to fixed-dose combination) is allowed within 12 weeks prior to study entry. A 
within class single drug substitution (e.g., switch from nevirapine to efavirenz or 
from atazanavir to darunavir) is allowed within 12 weeks prior to study entry, with 
the exception of a switch between any other NRTI to/from abacavir. No other 
changes in ART within the 12 weeks prior to study entry are permitted. However, 
participants need to be receiving (and tolerating) an allowable (for study 
purposes) ART regimen for at least 4 weeks prior to study entry. 
 
4.2.11 History of untreated latent tuberculosis infection (LTBI) diagnosed by  
  
tuberculin skin test or interferon gamma release assay. LTBI treatment 
  
would consist of 9 months of isoniazid or an equivalent therapy completed at  
  
least 4 weeks prior to study entry.  


 
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4.3 
Study Enrollment Procedures 
 
4.3.1 Prior to implementation of this protocol, and any subsequent full version 
amendments, each site must have the protocol and the protocol consent form(s) 
approved, as appropriate, by their local institutional review board (IRB)/ethics 
committee (EC) and any other applicable regulatory entity (RE). Upon receiving 
final approval, sites will submit all required protocol registration documents to the 
DAIDS Protocol Registration Office (DAIDS PRO) at the Regulatory Support 
Center (RSC). The DAIDS PRO will review the submitted protocol registration 
packet to ensure that all of the required documents have been received.  
Site-specific informed consent forms (ICFs) WILL be reviewed or approved by 
the DAIDS PRO, and sites will receive an Initial Registration Notification when 
the DAIDS PRO receives a complete registration packet.  Receipt of an Initial 
Registration Notification indicates successful completion of the protocol 
registration process. Sites will not receive any additional notifications from the 
DAIDS PRO for the initial protocol registration. A copy of the Initial Registration 
Notification should be retained in the site's regulatory files.  
 
Upon receiving final IRB/EC and any other applicable RE approvals for an 
amendment, sites should implement the amendment immediately. Sites are 
required to submit an amendment registration packet to the DAIDS PRO at the 
RSC. The DAIDS PRO will review the submitted protocol registration packet to 
ensure that all the required documents have been received. Site-specific ICF(s) 
WILL NOT be reviewed and approved by the DAIDS PRO, and sites will receive 
an Amendment Registration Notification when the DAIDS PRO receives a 
complete registration packet. A copy of the Amendment Registration Notification 
should be retained in the site's regulatory files. 
 
For additional information on the protocol registration process and specific 
documents required for initial and amendment registrations, refer to the current 
version of the DAIDS Protocol Registration Manual.  
 
Once a candidate for study entry has been identified, details will be carefully 
discussed with the participant. The participant will be asked to read and sign 
the approved protocol consent form. For participants from whom a signed 
informed consent has been obtained, an ACTG Screening Checklist must be 
entered through the Data Management Center (DMC) Participant Enrollment 
System. 
  
4.3.2 Randomization/Participant Registration 
 
Participants who meet enrollment criteria will be registered to the study 
according to standard ACTG DMC procedures. For participants from whom 
informed consent has been obtained, but who are deemed ineligible or who do 
not enroll into the initial protocol step, an ACTG Screening Failure Results form 
must be completed and keyed into the database.  
 
 
 
 


 
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4.4 
Coenrollment Guidelines  
 
 
Sites are encouraged to coenroll participants in A5128, “Plan for Obtaining 
Informed Consent to Use Stored Human Biological Materials (HBM) for Currently 
Unspecified Analyses,” and A5351s, “Effect of Immune-Modulatory Interventions 
on Cytomegalovirus Replication during Suppressive Antiretroviral Therapy” 
 
Coenrollment in A5128 and A5351s does not require permission from the A5336 
protocol chairs.  
 
For specific questions and approval for coenrollment in other studies, sites should 
contact the protocol team via e-mail as described in the Study Management section. 
 
 
5.0 
STUDY TREATMENT  
 
5.1 
Regimens, Administration, and Duration 
 
Study treatment is defined as ruxolitinib. Participants will be randomized at a 2:1 ratio to 
either 5 weeks of treatment with ruxolitinib or no study treatment as follows:  
 
ARM A:  Ruxolitinib 10 mg PO BID with or without food. Participants must remain on 
ART regimen (not provided by the study) for the duration of the study. 
 
ARM B:  No study treatment. Participants should be encouraged to remain on ART 
regimen for the duration of the study. 
 
Participants in Arm A should be instructed not to drink grapefruit juice or eat 
grapefruit while taking ruxolitinib. Participants will remain on study treatment for five 
weeks and be followed for seven weeks after treatment discontinuation. Participants 
will be on study for a total of 12 weeks. 
 
5.2 
Study Product Formulation and Preparation 
 
Ruxolitinib tablets 10mg should be stored at room temperature between 20º and 25ºC 
(68º and 77ºF). Excursions permitted between 15ºC and 30ºC (59ºF and 86ºF). See 
USP controlled room temperature. 
 
5.3 
Pharmacy: Product Supply, Distribution, and Accountability 
 
5.3.1 Study Product Acquisition/Distribution 
 
Ruxolitinib, manufactured by Incyte Corporation, will be provided by Emory 
University and available through the NIAID Clinical Research Products 
Management Center (CRPMC). The site pharmacist should obtain the study 
product(s) for this protocol by following the instructions in the manual Pharmacy 
Guidelines and Instructions for DAIDS Clinical Trials Networks.  
 
 
 


 
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5.3.2 Study Product Accountability 
 
The site pharmacist is required to maintain complete records of all study products 
received from the NIAID CRPMC and subsequently dispensed. All unused study 
products in CRSs must be returned to the NIAID CRPMC (or as otherwise 
directed by the sponsor) after the study is completed or terminated. The 
procedures to be followed are in the manual Pharmacy Guidelines and 
Instructions for DAIDS Clinical Trials Networks.  
 
5.4 
Concomitant Medications 
 
Whenever a concomitant medication or study agent is initiated or a dose changed, 
investigators must review the concomitant medication’s and study agent’s most recent 
package insert, Investigator’s Brochure, or updated information from DAIDS to obtain the 
most current information on drug interactions, contraindications, and precautions. 
 
Additional drug information may be found on the updated ACTG Drug Interactions 
Database located at: http://tprc.pharm.buffalo.edu/home/di_search/    
 
5.4.1 Prohibited Medications 
 
A list of prohibited medications can be found on the protocol-specific web page 
(PSWP), https://member.actgnetwork.org/study/53292#profile=4. 
 
 
5.4.2 Precautionary Medications 
 
A list of precautionary medications is posted on the PSWP, 
https://member.actgnetwork.org/study/53292#profile=4. 
 
 
 


 
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6.0 
CLINICAL AND LABORATORY EVALUATIONS 
 
6.1 
Schedule of Events 
 
Evaluation 
Screening 
Pre-Entry 
Entry 
On-Study Evaluations 
(Weeks) 
Confirmation 
of Virologic 
Failure 
Premature 
Treatment/Study 
Discontinuation 
Evals. 
1 
2 
4 
5 
10 
12 
Window 
≤30 days 
before 
entry 
 
≥24 hours 
after 
screening and 
≤7 days 
before entry  
 
± 3 days 
± 7 days 
Documentation of HIV-1 
X 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
X 
 
 
 
 
 
 
 
 
Medication History 
X 
 
X 
 
 
 
 
 
 
 
 
Complete Physical 
Examination 
 
X 
 
 
 
 
 
 
 
 
 
Targeted Physical 
Examination 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Clinical Assessments 
 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Concomitant Medications 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Study Treatment 
Modifications 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
ART Modifications 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Hematology 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Liver Function Tests 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Blood Chemistries 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
Fasting Lipid Panel 
 
 
X 
 
 
 
X 
 
 
 
X (if prior to 
week 5) 
Pregnancy Testing 
X 
 
X1 
If suspected 
 
 
Nadir CD4+ T cell Count 
 
 
X 
 
 
 
 
 
Documentation of Pre-
ART CD4+ T cell Count 
 
 
X 
 
 
 
 
 
CD4+/CD8+ 
X 
X2 
X 
 
X 
 
X 
 
X 
 
X 


 
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Evaluation 
Screening 
Pre-Entry 
Entry 
On-Study Evaluations 
(Weeks) 
Confirmation 
of Virologic 
Failure 
Premature 
Treatment/Study 
Discontinuation 
Evals. 
1 
2 
4 
5 
10 
12 
Window 
≤30 days 
before 
entry 
 
≥24 hours 
after 
screening and 
≤7 days 
before entry  
 
± 3 days 
± 7 days 
Stored PBMCs for Other 
Immunologic Studies 
 
 
X 
 
 
 
X 
 
X 
 
X 
Stored Plasma Soluble 
Markers 
 
X 
X 
 
 
X 
X 
 
X 
 
X 
Stored Plasma Soluble 
Marker:IL-6 
 
X 
X 
 
 
X 
X 
X 
X 
 
X 
Documentation of Pre-
ART HIV-1 RNA 
 
 
X 
 
 
 
 
 
 
 
 
Plasma HIV-1 RNA  
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
Stored Plasma HIV-1 RNA 
for Single Copy Assay 
 
 
X 
 
 
 
X 
 
X 
 
X 
Stored PBMC Reservoir 
Assays 
 
 
X 
 
 
 
X 
 
X 
 
X 
Stored Plasma/PBMC 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
Stored Oral Swabs for 
HHV 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
Plasma  PK Collection for 
Arm A 
 
 
 
X 
 
X 
 
 
 
 
X (if prior to week 
4) 
Medication Diary for Arm A 
(D=Distribute; C=Collect) 
 
 
X (D) 
X (C) 
X 
(D) 
X (C) 
 
 
 
 
X (C) (if prior to 
week 4) 
Adherence Assessment  
 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
1Refer to section 6.2.2 for pregnancy testing at study entry. 
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6.2 
Timing of Evaluations 
 
6.2.1 Screening and Pre-Entry Evaluations  
 
Screening and pre-entry evaluations must occur prior to the participant starting 
any study medications, treatments, or interventions. 
 
Screening 
Screening evaluations to determine eligibility must be completed within 30 days 
prior to study entry unless otherwise specified. In addition to data being collected 
on participants who enroll into the study, demographic, clinical, and laboratory 
data on screening failures will be captured in a Screening Failure Results form 
and entered into the ACTG database.  
 
Pre-Entry  
Pre-entry evaluations must be completed at least 24 hours after screening 
evaluations have been completed and within 7 days prior to the entry visit. 
 
The pre-entry and screening visits may be combined in the event the participant 
presents with the following: Documentation of HIV-1 status and the results of an 
HIV-1 RNA level performed at a CLIA-certified laboratory ≤ 30 days prior to study 
entry. 
 
6.2.2 Entry Evaluations 
 
Entry evaluations must occur at least 24 hours after pre-entry/screening 
evaluations unless otherwise specified. All entry evaluations must be performed 
after successful randomization and completed prior to the initiation of study 
treatment. If needed, the pregnancy testing at screening can be accepted for the 
entry value if the screening value was obtained within 72 hours of entry. All 
participants must begin treatment within 72 hours after enrollment.  
 
6.2.3 Post-Entry Evaluations 
 
On-Treatment Evaluations  
Evaluations must occur after randomization. Study visits must be scheduled on 
the weeks indicated in section 6.1, within the visit windows. 
 
The weeks 1 through 5 study visits must be scheduled per section 6.1, the SOE 
table ± 3 days.  
 
Please note influenza vaccination is prohibited ≤ 7 days prior to all study visits. 
The study visit will need to be rescheduled if the influenza vaccine was 
given ≤ 7 days prior to a study visit. All other vaccinations are prohibited 
throughout the duration of the study. 
 


 
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Post-Treatment Evaluations  
There will be 7 additional weeks of follow-up after 5 weeks of study treatment. 
Participants will undergo evaluations as outlined at weeks 10 and 12 in section 
6.1. The week 10 and 12 study visits must be scheduled per the SOE ± 7days. 
Week 12 is the final study visit.   
 
NOTE: Participants who discontinue treatment prematurely are followed on 
study/off treatment until study completion, i.e., week 12.  
 
Virologic Failure 
For participants who have suspected virologic failure, based on an HIV-1 RNA 
level above the lower limit of quantification, the site should query the participant 
about intercurrent vaccination, acute illness, or identified periods of 
nonadherance. If one of these conditions is suspected to account for the 
increased HIV-1 RNA, then no further evaluation is necessary until the next study 
visit. If none of these factors are present, a confirmatory HIV-1 RNA should be 
done within 7 days of receipt of the results of the first test. If the repeat HIV-1 
RNA is below the assay limit of quantification, then no further evaluation is 
necessary. See section 7.5 for management of confirmed virologic failure.   
 
6.2.4 Discontinuation Evaluations 
 
Evaluations for Randomized or Registered Participants Who Do Not Start Study 
Treatment 
All CRFs must be completed and keyed for the period up to and including entry. 
No further follow up is required for participants randomized to Arm A who do not 
start study treatment. 
 
Premature Treatment Discontinuation Evaluations  
Participants who prematurely discontinue the study treatment prior to week 5 
should complete the premature treatment discontinuation evaluations within 14 
days after discontinuing treatment. Participants will then be followed on study/off 
study treatment.  
 
Site personnel should notify the protocol core team (actg.corea5336@fstrf.org) 
within 48 hours of any participant who prematurely discontinues the study 
treatment. 
 
Premature Study Discontinuation Evaluations  
 
Participants who prematurely discontinue the study will have the  
premature treatment/study discontinuation evaluations performed prior to being 
taken off the study. 
 
Site personnel should notify the protocol core team (actg.corea5336@fstrf.org) 
within 48 hours of any participant who prematurely discontinues the study. 
 


 
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Pregnancy 
Pregnancy will result in immediate and permanent discontinuation of the study 
treatment. Please see section 7.4 for detailed information regarding participant 
management. 
 
6.3 
Instructions for Evaluations 
 
All clinical and laboratory information required by this protocol is to be present in the 
source documents. Sites must refer to the Source Document Guidelines on the DAIDS 
Web site for information about what must be included in the source document: 
http://www.niaid.nih.gov/labsandresources/resources/daidsclinrsrch/documents/sourced
ocappndx.pdf. 
 
All stated evaluations are to be recorded on the CRF and keyed into the database 
unless otherwise specified. This includes events that meet the International Conference 
on Harmonisation (ICH) definitions for a SAE: 
 
 
Results in death 
 
Life-threatening 
 
Requires inpatient hospitalization or prolongation of existing hospitalization 
 
Results in persistent or significant disability/incapacity  
 
Congenital anomaly/birth defect 
 
Other important medical event (may not be immediately life-threatening or result in 
death or hospitalization but may jeopardize the patient or may require intervention to 
prevent one of the events listed above).   
 
To grade diagnoses, signs and symptoms, and laboratory results, sites must refer to the 
DAIDS Table for Grading the Severity of Adult and Pediatric Adverse Events (DAIDS AE 
Grading Table), Version 2.0, November 2014, which can be found on the DAIDS RSC 
Web site: http://rsc.tech-res.com/safetyandpharmacovigilance/.   
 
6.3.1 Documentation of HIV-1 
 
Section 4.1.1 specifies assay requirements for HIV-1 documentation. HIV-1 
documentation is not recorded on the CRF. 
 
6.3.2 Medical History 
 
The medical history must include all diagnoses identified by the ACTG criteria for 
clinical events and other diagnoses. In addition to reporting all diagnoses within 
the past 30 days, the following diagnoses should be reported regardless of when 
the diagnosis was made.  
 
 
AIDS-defining conditions 
 
Bone fractures (verbal history accepted) 
 
Coronary heart disease 
 
Cancer (exclusive of basal/squamous cell skin cancer) 
 
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 
Tuberculosis 
 
Chronic or acute HCV 
 
Chronic or acute HBV 
 
HIV encephalopathy 
 
HIV wasting 
 
Esophageal candidiasis 
 
Pneumocystis pneumonia without dissemination 
 
Any allergies to any medications and their formulations must also be 
documented.  
 
6.3.3 Medication History 
 
A medication history must be present, including start and stop dates. The table 
below lists the medications that must be included in the history.  
 
Medication Category 
Complete History or Timeframe 
Record on 
CRFs 
ART 
Complete history (estimated dates if the 
exact dates cannot be obtained) 
Yes 
Immune-based therapy 
Complete history (estimated dates if the 
exact dates cannot be obtained) 
Yes 
HIV-1-related vaccines 
Complete history 
Yes 
Prescription drugs for treatment 
of opportunistic infections 
Within 30 days prior to study entry 
Yes 
Prescription drugs for 
prophylaxis of opportunistic 
infections 
Within 30 days prior to study entry 
Yes 
Prescription drugs: All including 
statins, anticoagulation therapy, 
NSAIDS, anti-inflammatory 
drugs not considered immune 
based therapy  
Within 30 days prior to study entry 
Yes 
Alternative therapies 
Within 30 days prior to study entry 
Yes 
Dietary supplements 
Within 30 days prior to study entry 
Yes 
Aspirin use 
Documentation of any aspirin use over a 
period of more 
than 5 days in any given week, taken 
within 30 days prior to study entry 
Yes 
 
6.3.4 Physical Examinations 
 
Complete Physical Exam 
A complete physical examination is to include at a minimum an examination of 
the skin, head, mouth, and neck; auscultation of the chest; cardiac exam; 
abdominal exam; examination of the lower extremities for edema; and Karnofsky 
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symptoms, diagnoses, and vital signs (temperature, pulse, respiration rate, and 
blood pressure). 
 
Targeted Physical Exam 
A targeted physical examination is to include vital signs (temperature, pulse, 
respiration rate, and blood pressure) and is to be driven by any previously 
identified diagnoses or new signs or symptoms that the participant has 
experienced within 30 days prior to entry or since the last visit. 
 
6.3.5 Clinical Assessments 
 
Height 
Height (in cm) will be measured at the entry visit.  
 
Weight 
Weight (in kg) will be measured at screening, entry and at all subsequent study 
visits. 
 
Signs and Symptoms 
At entry, all grades that occurred 30 days before entry must be recorded; post-
entry, only signs and symptoms Grade  2 must be recorded except record all 
non-traumatic bruising regardless of grade. All Grade 3 or higher sign/symptom, 
any sign/symptom regardless of grade that led to a change in treatment, or that 
met ICH, EAE, or SAE guidelines, are defined by the protocol as reportable 
events that will require more detailed event reporting and must be reported on 
the CRF within 2 business days. 
 
Diagnoses 
At entry and on-study visits, record all diagnoses identified by the ACTG criteria 
and any of the following targeted diagnoses occurring since the last study visit: 
 
New onset seizures; or physician diagnosed CNS abnormality.  
 
Post entry diagnoses must be reported on the CRF within 2 business days. 
 
6.3.6 Concomitant Medications 
 
Record in source documents and CRFs additions or discontinuations of any 
prescription medications and any medication or supplement included in the 
precautionary medication list (See PSWP). Record on the source documents 
and CRFs use of aspirin (chronic use only, defined as daily aspirin use over a 
period of more than 5 days in any given week) since the last study visit, including 
actual or estimated start dates and stop dates.   
 
6.3.7 Study Treatment Modifications 
 
Record all study drug modifications, including initial doses, participant-initiated 
and/or protocol-mandated modifications, inadvertent and deliberate interruptions, 
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greater than 3 doses of study drug per week for two or more weeks results in a 
study treatment discontinuation and should be recorded on the CRF. The 
protocol core team (actg.corea5336@fstrf.org) must be notified of any permanent 
discontinuation of study drug.  
 
6.3.8 ART Modifications 
 
Record all modifications, including initial doses, participant-initiated and/or 
protocol-mandated modifications, inadvertent and deliberate interruption of 7 
days or more and permanent discontinuation. 
 
Arm A participants who change or discontinue ART must prematurely 
discontinue study treatment and be followed on study/off study treatment. Refer 
to protocol section 6.2.4 for premature treatment discontinuation evaluations. 
 
6.3.9 Laboratory Evaluations 
 
All laboratory evaluations at entry and week 5 study visit must be collected in a 
fasting state (8-hour fast with exception of water and medications). If 
participants are in a non-fasting state at the time of the study visit, they should 
return to the clinic within 72 hours for a fasting blood draw. 
 
 
At screening and entry all laboratory values must be recorded.  
 
For post-entry assessments, record all Grade  2 laboratory values, except 
for estimated CrCl, creatinine, AST (SGOT), ALT (SGPT), ANC, hemoglobin, 
and platelet count. These values should be recorded at every visit regardless 
of grade.  
 
All grade 3 or higher laboratory values, any laboratory value that led to a 
change in treatment or that met ICH, EAE, or SAE guidelines are defined by 
the protocol as reportable events that will require more detailed event 
reporting and must be reported on the CRF within 2 business days. 
 
Although specific virologic and immunologic assays are described below, newer 
technology may preclude the utility of these assays and, therefore, this protocol 
will allow for the more advanced assays to be performed instead.  
 
Hematology 
Hemoglobin, hematocrit, red blood cells (RBC), mean corpuscular volume 
(MCV), white blood cells (WBC), differential WBC, absolute neutrophil count 
(ANC), platelets. 
 
Liver Function Tests 
AST (SGOT), ALT (SGPT), alkaline phosphatase, indirect bilirubin, direct 
bilirubin, and total bilirubin.  
 
NOTE: Indirect bilirubin may be calculated from the total and direct bilirubin 
values.  
 


 
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Blood Chemistries 
Electrolytes (sodium, potassium, chloride, bicarbonate), glucose, creatinine,  
blood urea nitrogen (BUN) and CrCl as estimated by the Cockcroft-Gault 
equation.  
 
NOTE: A calculator for estimating the CrCl can be found at: 
https://www.fstrf.org/apps/cfmx/apps/common/Portal/index.cfm?event=postLogin. 
 
Fasting Lipid Panel  
Cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein 
(LDL). If the LDL cannot be calculated due to elevated triglyceride levels, a 
direct LDL should be measured. 
 
Pregnancy Testing 
For women with reproductive potential: Serum or urine -HCG (urine test must 
have a sensitivity of 25 mIU/mL). 
 
6.3.10 Nadir CD4+ T cell Count 
 
The participant’s prior nadir CD4+ T cell count (absolute value and date) should 
be documented when possible with a copy of the nadir CD4+ T cell count report. 
If this documentation is not available, then participant recollection will suffice. 
For participants who do not know the exact nadir value and for whom there is 
no source documentation, then recall of the categorical nadir (e.g., < 50, < 100, < 
200 cells/mm3) will suffice.   
 
6.3.11 Documentation of Pre-ART CD4+ T cell Count 
 
Record on the CRF the documented date and level of the participant’s pre-ART 
CD4+ T cell count that was obtained most immediately prior to a participant’s 
initiating ART. If documentation is not available, then participant recollection will 
suffice.  
 
6.3.12 Immunologic Studies 
 
CD4+/CD8+ 
At screening, pre-entry and for all on-study evaluations obtain absolute 
CD4+/CD8+ count and percentages from a laboratory that possesses a CLIA 
certification or equivalent and is certified for protocol testing by the DAIDS 
Immunology Quality Assurance (IQA) Program. Note only one CD4+/CD8+ 
value is needed if the screening and pre-entry visits are combined. 
 
Because of the diurnal variation in biomarkers that may be measured as part of 
the study (e.g., CD4+ and CD8+ T cell counts and other biomarkers that may be 
measured on stored samples), blood draws for individual participants should be 
performed consistently in either the morning or the afternoon throughout the 
study, if possible.  
 


 
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6.3.13 Stored PBMCs for Other Immunologic Studies 
 
PBMCs will be collected and stored at the indicated visits per section 6.1 (see 
Laboratory Processing Chart [LPC] for more information) for potential 
comparative assessment of:   
 
 
At specified time points, cellular (macrophage/monocytes and lymphocytes) 
markers of immune activation and inflammation in the peripheral blood 
including but not limited to HLA-DR, CD16+, CD38, CD56, CD69, and 
CD14+CD16+ monocyte levels will be determined using flow cytometry and 
using stored specimens at the end of the study.  
 
 
Lymphocyte markers of immune activation in the peripheral blood including 
but not limited to HLA-DR, CD38, CD69, and CD14+/CD16+ monocyte levels 
using microarray panels for both mRNA and protein expression.   
 
 
Macrophage/monocyte markers of immune activation and inflammation in the 
peripheral blood including but not limited to HLA-DR, CD38, CD69, and 
CD14+CD16+ monocyte levels using microarray panels for both mRNA and 
protein expression. 
 
6.3.14 Stored Plasma Soluble Markers 
 
Plasma for stored soluble markers will be collected and stored at the indicated 
visits per section 6.1 (see LPC for more information) for comparative assessment 
of: 
 
 
Soluble markers of immune activation and inflammation in the peripheral 
blood including but not limited to sCD14, TNF-α, IL-1α, IL-18, IL-1β, mCSF, 
and neopterin will be determined using enzyme-linked immunoassay using 
stored specimens at the end of the study.  
 
 
Soluble markers of immune activation and inflammation in the peripheral 
blood including but not limited to sCD14, TNF-α, IL-1α, IL-1β, mCSF, and 
neopterin using microarray panels. 
 
6.3.15 Stored Plasma Soluble Marker: IL-6 
 
 
Plasma for stored soluble marker IL-6 will be collected and stored at the  
 
indicated visits per section 6.1.  
 
6.3.16 Documentation of Pre-ART HIV-1 RNA 
 
Record on the CRF the documented date and level of the participant’s pre-ART 
viral load that was obtained immediately prior to a participant’s initiating ART. If 
documentation is not available, then participant recollection will suffice.  
 
 


 
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6.3.17 Virologic Studies 
 
Plasma HIV-1 RNA 
Screening HIV-1 RNA must be performed within 30 days prior to study entry. All 
HIV-1 RNA determinations must be done using an FDA-approved assay with a 
lower limit of quantification < 50 copies/mL (eg, < 40 copies/mL obtained by 
the Abbott real-time assay or < 20 copies/mL by the Roche Taqman v2.0 
assay) performed by a laboratory that possesses a CLIA certification or 
equivalent. Eligibility will be determined based on the screening value.  
 
After the screening visit, HIV-1 RNA quantification assay will be performed at the 
protocol-designated laboratory. See the LPC for processing, shipping, and 
storage information. 
 
6.3.18 Stored Plasma for HIV-1 RNA Single Copy Assay (SCA) 
 
Plasma will be obtained and stored for determination of plasma HIV-1 RNA by 
SCA at the indicated visits per section 6.1. This assay will be performed if 
significant changes in the immunologic markers are observed and/or if there are 
advances in the field that might provide further rationale for assessing the 
virologic endpoints in the absence of achieving the desired immunologic 
outcomes. See the LPC for details on processing plasma samples for the SCA 
test. 
 
6.3.19 Stored PBMC Reservoir Assays 
 
Samples will be collected and stored as indicated in section 6.1 for potential 
comparative assessments of change in HIV-1 reservoir size as measured by:  
a. Total and integrated HIV-1 DNA 
b. HIV-1 cell-associated RNA 
c. 2 LTR circles 
 
Reservoir assays will be performed if significant changes in the immunologic 
markers are observed and/or if there are advances in the field that might provide 
further rationale for assessing the virologic endpoints in the absence of achieving 
the desired immunologic outcomes.  
 
6.3.20 Stored Plasma/PBMC 
 
Stored Plasma/PBMC will be collected and stored as indicated in section 6.1 for 
future HIV-1 related studies and shipped according to the A5336 LPC. 
 
 
6.3.21 Stored Oral Swabs Collection 
 
Oral swabs will be collected as indicated in section 6.1 to measure levels of 
HHV DNA. It is recommended that participants abstain from drinking coffee 
1 hour prior to collection. Samples will be stored and shipped according to 
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6.3.22 Plasma PK Sample Collection for Arm A Participants 
 
Samples will be collected as indicated in section 6.1 for assessment of the PK of 
ruxolitinib and ARV medicines. Apart from arriving at the clinic in a fasted state, 
participants may eat and drink throughout the day as they wish. Each PK visit 
requires that the participants take their morning dose of ruxolitinib and their 
ARV medications as observed doses administered in the clinic. Participants 
should be contacted the night before and reminded not to take their morning 
dose of ruxolitinib or their ARV medications until instructed during their clinic visit, 
and to bring their study and ARV medications (including EFV) with them in the 
morning. The timing of the previous drug administration must be documented. If 
participant skipped previous day’s medication for the weeks 1 and 4 visits or 
took ruxolitinib before arriving at clinic, then baseline PK sampling should not be 
drawn and an alternative visit should be scheduled to measure PK. The exact 
time (hour and minute) of administration of the observed dose of ruxolitinib and 
ARV must be recorded on the CRF, along with the exact time at which each 
blood sample was obtained. Samples will be collected at the following time points 
during each visit: 
 
 
 
 
 
 
 
 
 
 
 
The use of an intravenous (IV) catheter is optional depending on participant 
preference. Please refer to the LPC for sample collection, processing, storage 
and shipping information.  
 
6.3.23 Medication Diary for Arm A Participants  
 
At the weeks before the PK assessment, a medication diary will be given to 
participants and collected from participants at the PK visits as indicated in 
section 6.1. Participants will be instructed to record the date and time they took 
their ruxolitinib and their ARV medications for the 3 days preceding the PK study 
visit.  
 
6.3.24 Adherence Assessment 
 
Self-reported adherence for ruxolitinib and ARV will be performed at the indicated 
visits per section 6.1.  
 
 
 
Hour 
Activity 
-0.5 – 0.0 
Pre-dose (baseline) PK sample  
0.0 
Observed Administration of study drug and 
ARV medications; start clock; record 
administration time 
1.0 – 1.5 
PK sample 
2.5 – 4.0 
PK sample 
4.0 – 6.0 
Lunch 
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7.0 
CLINICAL MANAGEMENT ISSUES 
 
7.1 
Toxicity  
 
Only toxicities related to the study drug, (ruxolitinib,) will be considered in the toxicity 
management section. The grading system is located in the Division of AIDS Table for 
Grading the Severity of Adult and Pediatric Adverse Events (DAIDS AE Grading Table), 
Version 2.0, November 2014, located at the DAIDS RSC Web Site: http://rsc.tech-
res.com/safetyandpharmacovigilance/ 
 
NOTE: The study core team must be notified by e-mail regarding toxicities that result in a 
change in regimen or discontinuation of study treatment (actg.corea5336@fstrf.org).   
 
7.1.1 Grade 1 or 2 
 
Participants who develop a Grade 1 or 2 AE or toxicity may continue study drug 
without alteration of the dosage, except as stated in sections 7.2.1 to 7.2.8. For 
participants experiencing Grade 1 or 2 AEs who choose to discontinue study 
drug, the site investigator should complete premature treatment discontinuation 
evaluations, and the participant should be encouraged to complete evaluations 
off treatment/on study. 
 
NOTE:  If participants discontinue study drug due to experiencing Grade 1 or 2 
AEs, this should be noted in the CRF as the reason for discontinuation.   
 
7.1.2 Grade 3 
 
Participants who develop a Grade 3 AE thought to be related to the study drug 
should have their study medication discontinued. If the investigator has 
evidence that the AE has NOT been caused by study drug, study drug should 
be held until the AE or clinically significant laboratory abnormality is Grade 
≤ 1 in severity, except as stated in sections 7.2.1 to 7.2.8 below.  
 
If the same Grade 3 AE recurs at any time within 3 weeks after restarting 
treatment, the study drug must be permanently discontinued.  
 
Participants experiencing Grade 3 AEs requiring permanent discontinuation of 
study drug should be followed weekly until resolution of the AE and should 
complete the premature treatment discontinuation evaluations and should be 
encouraged to complete off treatment/on study evaluations.  
 
7.1.3 Grade 4  
 
Participants who develop a Grade 4 symptomatic AE will have study drug 
permanently discontinued, except as stated in sections 7.2.1- 7.2.8 below. 
Participants experiencing Grade 4 AEs requiring permanent discontinuation of 
study drug should be followed weekly until resolution of the AE and should 
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participant should be encouraged to complete off treatment/on study 
evaluations. 
 
7.2 
Other Toxicities  
 
7.2.1 Platelet Count Reductions 
 
7.2.1.1 If a participant develops a platelet count decrease of >25% from 
baseline with an absolute value remaining above 200,000/mm3, then 
dosing can continue. 
 
7.2.1.2 If the platelet count decreases from baseline by ≥ 25%, and the absolute 
value is between 150,000-200,000/mm3, then dosing of the study drug 
should be reduced to 10 mg QD. The CBC should then be repeated 
weekly until the platelet count either returns to > 200,000/mm3 or 
remains less than 10% below the value that led to the dose reduction for 
two consecutive values. If the platelet count returns > 200,000/mm3, and 
is > 1.10 times the value that led to the dose reduction, then BID study 
drug dosing can resume. The participant will then have weekly 
monitoring until the platelet count remains at a value that is greater than 
the value at BID dose resumption for at least two consecutive values. 
After resumption of the BID dosing if the platelet count decreases to < 
200,000/mm3, then the dose will be decreased to 10 mg daily and 
remain at that dose except as noted in section 7.2.3.  
 
7.2.1.3 If the platelet count decreases by ≥ 25%, and an absolute value below 
150,000/mm3, then the study drug should be discontinued. The CBC 
should then be repeated weekly until the platelet count returns to > 
150,000/mm3.   
 
7.2.2 Anemia/Neutropenia 
 
If a participant experiences treatment-limiting Grade 3 or higher decrease (in 
the opinion of the site investigator) in anemia or neutropenia, then study drug 
should be withheld. Anemia should not be managed by transfusions or 
recombinant erythropoietin, and neutropenia should not be managed with 
granulocyte-colony stimulating factor (G-CSF) or granulocyte-macrophage 
colony-stimulating factor (GM-CSF). Once the anemia or neutropenia has 
returned to Grade ≤ 2, the study drug can be restarted. 
 
7.2.3 Creatinine Clearance 
 
Any participant experiencing a calculated CrCl < 60 mL/min should have the 
value confirmed within 7 days. For those participants who experience a 
confirmed (2 consecutive) CrCl < 60 mL/min, study drug should be withheld. If 
the site investigator determines that another condition caused the decrease in 
CrCl, then study drug may be restarted if approved by the protocol team. 
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returns to Grade ≤ 2 or baseline.   
 
7.2.4 Bruising and Bleeding 
 
For participants who report generalized bruising and/or symptoms of 
unexplained or abnormal bleeding, hematoma or epistaxis, the study drug 
should be discontinued. 
 
7.2.5 Nausea (with or without vomiting) and/or Diarrhea 
 
With the onset of Grade ≥ 1 nausea advise participants to take study drug with 
food. Participants may also be treated as needed with antiemetics given PO or 
by suppository. 
 
In participants with persistent nausea, consideration should be given for 
evaluation of pancreatitis, hepatitis, or hyperlactatemia/lactic acidosis, with 
evaluation for each if clinically indicated. 
 
For Grade 1 or 2 diarrhea, therapy should be continued with symptomatic 
treatment with antimotility agents. If Grade ≥ 3 diarrhea occurs and is 
unresponsive to antimotility agents, and for which an alternative etiology (e.g., 
infectious diarrhea) is not established and judged related to study drug, study 
drug should be withheld until resolution of diarrhea to Grade ≤ 1 or return to 
baseline. If Grade ≥ 3 diarrhea recurs upon the resumption of the study drug, 
study drug should be discontinued.   
 
7.2.6 Hypertriglyceridemia/Hyperlipidemia 
 
If elevated triglyceride or lipid levels are from a non-fasting blood draw, repeat 
the draw after an 8-hour fast. Only levels done in a fasting state should be used 
to determine toxicity management. Participants with asymptomatic Grade 3 or 
higher increases in triglyceride, total cholesterol, or LDL elevations must 
discontinue study drug. Please see the recent review adapted from the original 
recommendations of the ACTG Cardiovascular Disease Focus Group 
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753589) for full discussion of 
management of hyperlipidemia in the context of HIV-1 disease. 
 
7.2.7 Hyperglycemia 
 
Participants with Grade 3 or higher hyperglycemia must discontinue their 
study drug unless judged unrelated to the study drug. If judged unrelated, 
study drug must be held until the glucose abnormalities are Grade ≤ 1. Oral 
hypoglycemic and insulin should not be used in order to allow continued 
study drug administration. A confirmatory fasting glucose must be obtained 
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7.2.8 AST or ALT Elevations 
 
Study drug must be discontinued for asymptomatic Grade 3 or higher AST/ALT 
elevations unless judged unrelated to study drug. Careful assessments 
should be done to rule out the use of alcohol, non-study drug-related drug 
toxicity, or viral hepatitis as the cause of Grade 3 elevations. If judged 
unrelated, study drug should be held until AST and/or ALT levels are Grade ≤ 
1.  
 
7.3 
Other Diseases 
 
For unexplained confusion, seizures, or other CNS abnormality site investigators should 
assess the participant, in conjunction with the primary physician to determine the cause 
of the symptoms. If no cause can be identified, or other physician suspects PML, then 
study drug should be discontinued. The participant should then be followed on study 
until another cause is established, or PML is considered confirmed or probable 
according to criteria in Appendix 100, ACTG Criteria for Clinical and Other Events. For 
any infection, site investigators should assess the participant, in conjunction with the 
primary physician to determine relatedness to the study drug. If the infection is 
considered related to the study drug, then the study drug should be discontinued.  
 
7.4 
Pregnancy  
 
Female participants who become pregnant while on study treatment must immediately 
discontinue study treatment. Participants should have the Premature Treatment/Study 
Discontinuation Evaluations completed within 2 weeks after stopping study treatment. 
Pregnancies that occur on study should be reported prospectively to The Antiretroviral 
Pregnancy Registry. More information is available at www.apregistry.com. Phone: 800-
258-4263; Fax: 800-800-1052. 
 
Participants will be encouraged to remain in the study to be followed on study/off study 
treatment until study completion and will be followed by telephone contact thereafter to 
determine the pregnancy outcome. If a participant does not wish to remain on study 
follow-up at the time of study discontinuation, site staff should request permission to 
contact her regarding pregnancy and infancy outcomes at the end of the pregnancy. If 
the information is obtained, pregnancy outcomes for the participant and infant will be 
submitted on a CRF at the end of pregnancy.  
 
7.5 
Confirmed Virologic Failure 
 
In the event of confirmed virologic failure, defined as the occurrence of two sequential 
plasma HIV-1 RNA values above the lower limit of quantification in the absence of an 
interruption in ART, the study drug must be stopped for participants in Arm A. The 
participant will be followed on study with all evaluations performed through week 12. If 
the participant does not agree to continue on study, then the premature study 
discontinuation evaluations should be completed.  
 
  


 
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8.0 
CRITERIA FOR DISCONTINUATION 
 
8.1 
Premature Treatment Discontinuation 
 
 
Drug-related toxicity (see section 7.1 Toxicity). 
 
Requirement for prohibited concomitant medications (see section 5.4). 
 
Changes to or discontinuation of ART for Arm A participants. 
 
Participant repeatedly noncompliant (missing greater than 3 doses of study drug a 
week for 2 or more weeks). 
 
Confirmation of virologic failure (see section 7.5). 
 
Pregnancy or breastfeeding. 
 
Request by participant to terminate treatment. 
 
Clinical reasons believed life threatening by the physician, even if not addressed in 
the toxicity section of the protocol. 
 
8.2 
Premature Study Discontinuation 
 
 
Failure by the participant to attend 2 consecutive clinic visits. 
 
Request by the participant to withdraw. 
 
Request of the primary care provider if s/he thinks the study is no longer in the best 
interest of the participant. 
 
Participant judged by the investigator to be at significant risk of failing to comply with 
the provisions of the protocol as to cause harm to self or seriously interfere with the 
validity of the study results. 
 
At the discretion of the Institutional Review Board/Ethics Committee (IRB/EC), FDA, 
NIAID, Office for Human Research Protections (OHRP), other government agencies 
as part of their duties, or the investigator.  
 
 
9.0 
STATISTICAL CONSIDERATIONS  
 
9.1 
General Design Issues 
 
A5336 is a randomized (2:1), prospective, open-label, 2-arm pilot clinical trial designed 
to assess the safety and tolerability of ruxolitinib, and to evaluate whether 5 weeks of 
treatment with ruxolitinib decreases IL-6 levels in HIV-1 infected participants virally 
suppressed on ART. Participants will be randomized 2:1 to receive ruxolitinib or no 
study treatment. Participants receiving ruxolitinib will be followed on treatment for 5 
weeks, then for an additional 7 weeks off treatment, resulting in a total 12 weeks of 
follow-up; participants receiving no study treatment will be followed on-study for 12 
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9.2 
Objectives 
 
9.2.1 Primary Outcome Measures 
 
9.2.1.1 Safety 
 
Occurrence of any one of the following safety milestones (from entry to 
week 5).  
 
 
For participants with entry CD4+ T cell count < 700 cells/mm3, 
confirmed CD4+ decline > 33% of baseline and to < 350 cells/mm3. 
 
For participants with entry CD4+ T cell count ≥ 700 cells/mm3, a 
confirmed CD4+ T cell decline > 50 % of baseline. 
 
Confirmed HIV-1 RNA level above the lower limit of quantification 
in the absence of an interruption in ART. 
 
New or recurrent CDC category C AIDS-indicator condition. 
 
HIV-1 associated infection including Varicella zoster. 
 
Lymphoproliferative malignancies. 
 
Discontinuation of ruxolitinib due to thrombocytopenia. 
 
Grade 4 or recurrence of Grade 3 anemia/neutropenia. 
 
Any Grade 4 or recurrence of Grade 3 toxicity related to study drug. 
 
New diagnosis of pneumonia, sepsis, or bacteremia. 
 
9.2.1.2 Tolerability 
 
 
Occurrence of premature discontinuation of study treatment.  
 
9.2.1.3 IL-6 
 
Participant-specific change in the level of plasma IL-6 (using geometric 
means of two ELISA measurements at pre-entry and entry, and weeks 4 
and 5).  
 
9.2.2 Secondary Objective Measures  
 
9.2.2.1 Proportion of participants who experienced any one of the primary 
safety milestones during total follow-up (from entry to week 12). 
 
9.2.2.2 Summary of safety laboratories at baseline and weeks 1, 2, 4, 5, 10, and 
12. 
 
9.2.2.3 Change in the level of plasma IL-6 from baseline to weeks 4, 5, 10, and 
12.  
 
9.2.2.4 Changes in soluble markers of immune activation and inflammation in 
the peripheral blood (sCD14, TNF-α, IL-1α, IL-1β, mCSF, and neopterin) 
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9.2.2.5 Changes in cellular markers of immune activation and inflammation in 
the peripheral blood (HLA-DR, CD38, CD69 and CD14+/CD16+ 
monocyte levels) from baseline to weeks 5 and 12. 
 
9.2.2.6 Changes in plasma HIV-1 RNA and CD4+ T cell counts from baseline to 
weeks 2, 5, and 12. 
 
9.2.2.7 Changes in HIV-1 reservoir (HIV-1 RNA by SCA, cellular HIV-1 total 
RNA and DNA, 2-LTRs, integrated DNA) from baseline to weeks 5 and 
12.  
 
9.2.2.8 Level of HHV shedding (CMV, EBV, HHV 6, 7, 8) at pre-entry, entry, 
and weeks 1, 2, 4, 5, 10, and 12. 
 
9.2.3 Exploratory Objective Measures  
 
Summary measure of immune activation and inflammation and how changes in 
this measure differ between treatment groups. 
 
9.3 
Randomization and Stratification 
 
At study entry, participants will be randomized at a 2:1 ratio (ruxolitinib:no study 
treatment) using permuted blocks, without institutional balancing. Participants will be 
stratified based on whether they take EFV or do not take EFV at screening, in order to 
ensure balance by treatment arm for EFV. 
 
9.4 
Sample Size and Accrual 
 
The sample size for A5336 was determined based on the primary endpoint for change in 
IL-6, while at the same time ensuring the sample size was feasible for an early phase 
investigation of the safety of ruxolitinib. 
 
9.4.1 Changes in IL-6 
 
A total sample size of 60 participants will be randomized at a 2:1 ratio to 
ruxolitinib:no study treatment, allowing for 20% lost to follow-up and treatment 
discontinuation, is anticipated to yield 48 evaluable participants (see section 
9.6.1). This will provide 80% power to detect a treatment effect of 0.20 log10 
pg/mL, that is a difference between treatment groups in (change in plasma IL-6 
from entry to week 5), assuming that the standard deviation (SD) of within-
participant differences in IL-6 is 0.25 log10 pg/mL, using a two-sided t-test at a 
0.10 alpha level.   
 
To minimize variability and maximize statistical power, at each key time point 
markers will be measured and analyzed as the average of samples taken 1-2 
weeks apart (pre-entry and entry; at weeks 4 and 5; at weeks 10 and 12).  
 


 
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The treatment effect of 0.20 log10 scale pg/mL corresponds to average decreases 
of 37% and 0% in plasma IL-6 levels in the ruxolitinib and no study treatment 
arms, respectively. This choice of effect size is based on prior data examining 
plasma IL-6 levels in HIV-1 infected populations and changes in IL-6 levels in 
ruxolitinib treated populations. Specifically, a 27% (95% confidence interval [CI]: 
11% to 39%) reduction in IL-6 levels after 4 weeks and a 37% (95% CI: 13% to 
53%) reduction after 24 weeks of treatment was seen in the Phase III 
COMFORT-1 study [3]. The effect size of 37% is selected because it is contained 
in the 95% CI for IL-6 reduction at 4 weeks, and balances the interest in the 
efficacy endpoint with safety considerations as well as feasibility for this early 
phase investigation of the effects of the study treatment in the HIV-1 population. 
 
The assumed SD for within-participant differences in IL-6 of 0.25 log10 is a 
pooled estimate based on estimates of the between-participant SDs and within-
participant correlation from other ACTG studies. ACTG 5248 estimated the 
between-participant SD of IL-6 at weeks 24 and 48 (after starting ART 
treatment); the SD was 0.335 at week 24 (based on 35 participants) and 0.331 
at week 48 (based on 29 participants). The SD estimate from 311 controls from 
NWCS 329 at week 48 was 0.306 based on a single measure [71]. In addition, 
ACTG 5286 reported the within-participant correlation between baseline and 
week 4 IL-6 measures to be 0.68 (n=43). Two versions of the pooled SD were 
calculated assuming the same SD at baseline and weeks 5, one assuming both 
SDs = 0.306, and the other assuming both SDs = 0.331. Each set of SDs was 
then pooled by accounting for the correlation between time points (r = 0.68), 
which resulted in SD=0.2448 and SD = 0.2648, respectively. The pooled 0.25 SD 
is the average of these values.  
 
9.4.2 Safety 
 
The total sample size of 60 participants will be randomized at a 2:1 ratio to 
ruxolitinib:no study treatment ratio, results in 40 participants receiving ruxolitinib 
and 20 receiving no study treatment. The 40 ruxolitinib-treated participants will 
provide 90% probability of observing an AE that would occur in 6% or more of 
treated participants. 
 
We assume the rate of events (outlined in 9.2.1.1) in the no study treatment arm 
will be no more than 2%. Assuming this rate in the no study treatment arm, we 
will have 80% power to detect a significant difference between the two arms, via 
a one-sided Fisher’s exact test with 5% alpha, if the rate in the ruxolitinib arm is 
27%.  
 
9.4.3 Accrual 
 
It is anticipated that participants will accrue to the study at a rate of 7-8 
participants per month. At this rate, the study should fully accrue within 7-9 
months after the first participant enrolls. 
 
 


 
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9.5 
Monitoring 
 
Accrual and summary of all Grade ≥ 2 signs and symptoms, laboratory abnormalities 
and diagnoses, and all reported AEs will be reviewed by the team monthly. This 
summary will be pooled over the study arms. Baseline characteristics, early study 
treatment and study discontinuations, and virologic failures, pooled over study arms, will 
be reviewed regularly by the protocol team. In addition, data and specimen availability 
will be also regularly reviewed. The Statistical and Data Analysis Center (SDAC) will also 
prepare quarterly reports of all Grade ≥ 2 events, and all reported AEs by study arm, to 
be reviewed by the DAIDS clinical representative or designee.  
 
An ACTG-appointed Study Monitoring Committee (SMC) will review accrual, toxicity 
summaries, off-treatment and off-study rates, and reasons broken down by study arms.  
An interim safety review by the SMC is planned at the earliest of 6 participants with 3 
weeks of follow-up, 2 participants with Grade 3/4 toxicities, 3 participants 
discontinuing study treatment due to toxicities, or 3 months after the first participant 
enrolls. Subsequent SMC reviews are planned at approximately 6 month intervals, or in 
the event of 2 participants with Grade ≥ 3 toxicities or 3 participants discontinuing 
study treatment due to toxicities in the interim.  
 
In the event the SMC review is triggered due to toxicities (2 participants with 
Grade ≥ 3 toxicities or 3 participants discontinuing study treatment due to 
toxicities), accrual will be paused while the SMC review takes place. In addition, 
during the course of the study if any participant develops PML, or neurologic 
condition or dies of a drug-related adverse event, or if more than 2 participants, 
randomized to Arm A, experience confirmed virologic failure (see section 7.5 for 
definition) then dosing for all study participants will be stopped.  
 
The team has requested the SMC consider the grade of the events, the time to 
resolution (if any), the temporal relationship to drug administration, and the site 
clinician’s judgment of the relationship to treatment. The team would recommend 
particular review by the SMC if there were high occurrences of the safety events 
described in section 9.2.1.1.   
 
Complementing the SMC reviews, the team will also notify the SMC of all drug 
related SAEs, Grade 4 toxicities, as well as all drug-related Grade 3 toxicities.  In 
addition, the core team or the SMC may ask for the SMC, at any time, to 
independently review all available safety data (unblinded to treatment arms) and 
make a recommendation to the End-Organ Disease and Inflammation 
Transformative Science Group on whether to modify or stop the study. The team 
will also monitor (monthly) all Grade ≥ 2 signs and symptoms, laboratory 
abnormalities, and diagnoses, as well as all primary events with summaries 
pooled over study arms.  
 
9.6 
Analyses 
 
9.6.1 Primary Analyses 
 


 
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As this is a pilot study of biologic activity, our primary analysis of efficacy will be 
an as-treated analysis, limited to participants who meet all of the following 
conditions: (a) have data at baseline and week 5, (b) for the ruxolitinib arm, 
remain on study treatment through week 5 and miss no more than 6 doses 
cumulatively, (c) did not change ART, use prohibited medications, or have a 
virologic failure during this time period.  
 
Changes from baseline to week 5 in plasma IL-6 (on log10 scale) will be 
summarized with the mean and a 90% confidence interval (CI) for the mean 
change in each treatment arm, and compared between arms with a t-test. The 
baseline value will be calculated as the geometric mean of pre-entry and entry, 
and the week 5 value as the geometric mean of weeks 4 and 5. If missing one of 
the pre-entry or entry values, then the available measure will be used as the 
baseline value, and similarly, if missing one of the week 4 or week 5 values, then 
the available measure will be used as the week 5 value. 
 
Safety will be evaluated by estimating the proportion of participants in the 
ruxolitinib arm who experienceing the primary safety outcome measure 9.2.1.1. 
In addition, safety will be compared between the treatment arms via a one-
sided Fisher’s exact test at 5% alpha level, in terms of 9.2.1.1, but with this 
composite safety outcome modified to address the A5336 design where 
Arm B does not receive any study treatment. Specifically “discontinuation 
of ruxolitinib due to thrombocytopenia” will be modified to “occurrence of 
Grade 2 or higher thrombocytopenia” and “any Grade 4 or recurrence of 
Grade 3 toxicity related to study drug” will be modified to “any Grade 4 or 
recurrence of Grade 3 toxicity.” Tolerability will be evaluated by summarizing 
treatment modifications and discontinuations. 
 
9.6.2 Secondary Analyses 
 
Participant-specific changes from baseline to week 5 in other measurements of 
immune activation and inflammation, and CD4+ T-cell counts will be summarized 
similarly to the primary efficacy measure, via means and 90% CIs for the mean 
change for each of the study arms. The log10 transformation is planned for 
various soluble and cellular markers. Comparisons between study arms will be 
done via the Wilcoxon rank sum test. HIV-1 RNA will be summarized by the 
number of participants with confirmed HIV-1 RNA > 40 copies/mL and > 200 
copies/mL. Due to an anticipated high proportion of lower limit of detection 
censoring, levels of HHV shedding will be compared between arms at week 
5 using a two-group Wilcoxon rank-sum test with 2-sided alpha=0.10 level.  
Values below the lower limit of detection will be analyzed as the lowest 
rank. In the event of limited lower limit of detection censoring, repeated 
measure modeling will be used to explore longitudinal changes in HHV 
shedding. 
 
Safety events (signs and symptoms, laboratory values, diagnoses, and other 
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each study arm. Comparisons of overall and toxicity-specific grades between 
study arms will be shown descriptively.  
 
Longitudinal changes from baseline to week 12 in all measures will be examined 
via descriptive summaries and plots.   
 
Statistical significance will be tested using a two-sided alpha = 0.10 with no 
adjustment for multiple testing. 
 
 
10.0 
PHARMACOLOGY PLAN 
 
10.1 
Pharmacology Objectives 
 
10.1.1 Primary Objective 
10.1.1.1 To measure the PK of ruxolitinib in this population of participants with 
well-controlled HIV-1, and to characterize the relationship between 
ruxolitinib post-hoc estimated exposures (e.g., steady-state AUC = 
dose/CL/F) and immunological biomarkers of the study drug. 
 
10.1.2 Secondary Objectives 
 
10.1.2.1 To compare ruxolitinib PK parameters in study participants on specific 
ARV regimens to those reported for historical controls (no 
coadministered drugs) to assess the potential effect of ARV 
administration on ruxolitinib PK. 
 
 
10.1.2.2 To compare PK parameters of selected ARV medications (NNRTI’s and 
INSTI’s) when coadministered with ruxolitinib to historical controls (no 
coadministered drugs) in order to explore possible effects of ruxolitinib 
on ARV concentrations. 
 
10.1.2.3 To evaluate the relationship between ruxolitinib concentrations in 
individual participants with the time course of changes in biomarkers 
of immune response (hysteresis analysis). 
 
10.2 
Pharmacology Study Design 
 
Participants randomized to Arm A will undergo blood sampling during two study visits 
as indicated in section 6.1 for assessment of the PK of ruxolitinib and selected ARV 
drugs.  
 
Each PK visit requires that the study participant take their morning dose of ruxolitinib as 
a timed observed dose administered in the clinic. During each of the two PK visits, four 
blood samples will be collected for PK analysis.  
 
 


 
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10.3 
Primary and Secondary Data, Modeling, and Data Analysis 
 
The PK of ruxolitinib following oral administration will be modeled using population 
methods with the NONMEM computer program (7.3 or later, ICON Development 
Solutions, Leopardstown, Ireland) [88], using the PLT Tools statistical interface (5.1 or 
later, PLT Soft, San Francisco, CA). Resulting model parameters will be compared to a 
model recently reported for ruxolitinib without coadministered drugs in patients with 
myelofibrosis [89]. One and 2-compartmental models, assuming first-order absorption 
into the plasma compartment will be fitted as they were reported to adequately describe 
ruxolitinib PK in the myelofibrosis population PK study. Between-participant variances 
(IIV, 2) of PK parameters () will be modeled as log-normally distributed, and an 
appropriate residual error model (e.g., proportional and/or additive,) will be used. Also, 
since PK samplings are planned on two different occasions, we will also explore inter-
occasional variations in model parameters (e.g., oral systemic clearance (CL/F) and 
distribution volume (V/F, where F=bioavailability, assumed=1, no IV dose administered)), 
using established methods [90]. Only concentrations > LOQ were included in the 
analysis. Criteria for model convergence will include convergence to > 3 decimal places 
using an appropriate convergence method (e.g., First Order Conditional Estimation with 
- interactions. Model diagnostics of goodness of fit will include the minimum value the 
NONMEM objective function (OFV= -2 log-likelihood), and visual inspection of graphics, 
e.g., correlation/symmetry of observations versus predictions (population and post-hoc), 
conditional weighted residual versus time and predictions [91-93].  
 
Based on the planned 12 hour dosing regimen and reported ruxolitinib plasma 
concentrations over a 12 hour dose interval, we anticipate that a 1-compartment model 
will adequately describe the PK of ruxolitinib in this study. However, if the fit of the model 
to the data is improved using a 2-compartment model, based upon the dispersion of 
residuals about the respective lines of identity of observed versus predicted plasma 
concentrations (population and post-hoc), and by a decrease in OFV, then the 2-
compartment model will be selected for further modeling. We will also test the 
significance of body-weight as possible continuous covariate on V/F and CL/F and 
creatinine clearance (calculated using the Cockroft-Gault formula) as a covariate of CL/F 
[94]. The ability of the final model to simulate without bias will be tested using a “visual 
predictive check” method, in which > 1,000 individuals are simulated using the sampling 
scheme (to ensure reproducible predictions), and used to compute 5% - 95% percentile 
ranges of concentration versus time. Actual data will then be superimposed to visually 
assess model bias [95]. Post-hoc fitted parameters from participants administered 
ruxolitinib alone or with EFV will be compared using ANCOVA. A value < 0.05 will be 
considered statistically significant for all tests. A similar model will be attempted for EFV 
and other ARVs provided enough participants taking a particular ARV are recruited. 
Alternatively, their PK will be compared informally with published PK studies [50, 51, 96, 
97].  
 
Concentrations of ARVs (NNRTIs and INSTIs) obtained at the same time as ruxolitinib 
concentrations will be compared to historical controls in order to explore the possible 
effect of ruxolitinib on ARV concentrations. This is considered an exploratory analysis 
only. 
 


 
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10.4 
Anticipated Outcomes 
 
Based on what is known about the clearance of ruxolitinib, NNRTIs, and INSTIs, we do 
not expect a clinically significant effect of ruxolitinib on ARV concentrations, nor a 
clinically significant effect of INSTIs on ruxolitinib concentrations. EFV is a P450 enzyme 
inducer, and could decrease ruxolitinib concentrations as a result, although the 
magnitude of this effect is expected to be modest, and since concentrations of ruxolitinib 
would decrease rather than increase, this should not produce any safety concerns for 
participants. 
 
 
11.0 
DATA COLLECTION AND MONITORING AND ADVERSE EVENT REPORTING 
 
11.1 
Records to Be Kept 
 
CRF will be provided for each participant. Participants must not be identified by name 
on any CRFs. Participants will be identified by the patient identification number (PID) 
and study identification number (SID) provided by the ACTG DMC upon randomization. 
 
11.2 
Role of Data Management 
 
11.2.1 
Instructions concerning the recording of study data on CRFs will be provided by 
the ACTG DMC. Each CRS is responsible for keying the data in a timely 
fashion. 
 
11.2.2 
It is the responsibility of the ACTG DMC to assure the quality of computerized 
data for each ACTG study. This role extends from protocol development to 
generation of the final study databases. 
 
11.3 
Clinical Site Monitoring and Record Availability 
 
11.3.1 
Site monitors under contract to the NIAID will visit participating clinical research 
sites to review the individual participant records, including consent forms, 
CRFs, supporting data, laboratory specimen records, and medical records 
(physicians’ progress notes, nurses’ notes, individuals’ hospital charts), to 
ensure protection of study participants, compliance with the protocol, and 
accuracy and completeness of records. The monitors also will inspect sites’ 
regulatory files to ensure that regulatory requirements are being followed and 
sites’ pharmacies to review product storage and management. 
 
11.3.2 
The site investigator will make study documents (e.g., consent forms, drug 
distribution forms, CRFs) and pertinent hospital or clinic records readily 
available for inspection by the local IRB, the site monitors, the FDA, the NIAID, 
the OHRP, and designee for confirmation of the study data.  
 
11.4 
Expedited Adverse Event Reporting to DAIDS 
 
11.4.1 
Adverse Event Reporting to DAIDS 


 
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Requirements, definitions and methods for expedited reporting of AEs are 
outlined in Version 2.0 of the DAIDS EAE Manual, which is available on the 
RSC website at   http://rsc.tech-res.com/safetyandpharmacovigilance/. 
 
The DAIDS Adverse Events Reporting System (DAERS), an internet-based 
reporting system, must be used for expedited AE reporting to DAIDS.  In the 
event of system outages or technical difficulties, expedited AEs may be 
submitted via the DAIDS EAE Form. For questions about DAERS, please 
contact DAIDS-ES at DAIDS-ESSupport@niaid.nih.gov. Site queries may also 
be sent from within the DAERS application itself. 
Sites where DAERS has not been implemented will submit expedited AEs by 
documenting the information on the current DAIDS EAE Form. This form is 
available on the RSC website: http://rsc.tech-
res.com/safetyandpharmacovigilance/.  For questions about EAE reporting, 
please contact the RSC (DAIDSRSCSafetyOffice@tech-res.com). 
 
11.4.2 
Reporting Requirements for this Study 
 
 
The SAE Reporting Category, as defined in Version 2.0 of the DAIDS EAE 
Manual, will be used for this study. 
 
The study agent for which expedited reporting are required is ruxolitinib. 
 
In addition to the EAE Reporting Category identified above, other AEs that 
must be reported in an expedited manner are: 
o Platelet count decreases by ≥ 25% from baseline 
o Platelet count with an absolute value below 150,000/mm3 
o New onset leukemia 
 
11.4.3 
Grading Severity of Events 
 
The Division of AIDS Table for Grading the Severity of Adult and Pediatric 
Adverse Events (DAIDS AE Grading Table), Version 2.0, November 2014, 
must be used and is available on the DAIDS RSC Web site at http://rsc.tech-
res.com/safetyandpharmacovigilance/. 
 
11.4.4 
Expedited AE Reporting Period 
 
 
The expedited AE reporting period for this study is as per the EAE manual. 
 
After the protocol-defined AE reporting period, unless otherwise noted, only 
suspected, unexpected serious adverse reactions (SUSARs), as defined in 
Version 2.0 of the EAE Manual, will be reported to DAIDS if the study staff 
become aware of the events on a passive basis (from publicly available 
information). 
 
 


 
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12.0 
PARTICIPANTS 
 
12.1 
Institutional Review Board (IRB) Review and Informed Consent 
 
This protocol and the informed consent document (Appendix I) and any subsequent 
modifications will be reviewed and approved by the IRB or EC responsible for oversight 
of the study. A signed consent form will be obtained from the participant (or person with 
power of attorney for participants who cannot consent for themselves). The consent 
form will describe the purpose of the study, the procedures to be followed, and the risks 
and benefits of participation. A copy of the consent form will be given to the participant, 
or legal representative, and this fact will be documented in the participant’s record. 
 
12.2 
Participant Confidentiality 
 
All laboratory specimens, evaluation forms, reports, and other records that leave the site 
will be identified by coded number only to maintain participant confidentiality. All 
records will be kept locked. All computer entry and networking programs will be done 
with coded numbers only. Clinical information will not be released without written 
permission of the participant, except as necessary for monitoring by the ACTG, 
IRB/EC, FDA, NIAID, OHRP, other government agencies as part of their duties to 
ensure that research participants are protected, or designee.  
 
12.3 
Study Discontinuation 
 
The study may be discontinued at any time by the ACTG, IRB/EC, FDA, NIAID, OHRP, 
or government agencies as part of their duties to ensure that research participants are 
protected.  
 
 
13.0 
PUBLICATION OF RESEARCH FINDINGS 
 
Publication of the results of this trial will be governed by ACTG policies. Any 
presentation, abstract, or manuscript will be made available for review by the industry 
supporter(s) prior to submission.  
 
 
14.0 
BIOHAZARD CONTAINMENT 
 
As the transmission of HIV and other blood-borne pathogens can occur through contact 
with contaminated needles, blood, and blood products, appropriate blood and secretion 
precautions will be employed by all personnel in the drawing of blood and shipping and 
handling of all specimens for this study, as currently recommended by the Centers for 
Disease Control and Prevention and the National Institutes of Health. 
 
All dangerous goods and materials, including diagnostic specimens and infectious 
substances, must be transported using packaging mandated by CFR 42 Part 72. Please 
refer to instructions detailed in the International Air Transport Association (IATA) 
Dangerous Goods Regulations.  


 
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15.0 
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85. Gianella S, Massanella M, Richman DD, et al. Cytomegalovirus replication in  
semen is associated with higher levels of proviral HIV DNA and CD4+ T cell 
activation during antiretroviral treatment. J Virol 2014; 88(14): 7818-27. 
 
86. Gianella S, Strain MC, Rought SE, et al. Associations between virologic and  
immunologic dynamics in blood and in the male genital tract. J Virol 2012; 86(3): 
1307-15. 
 
87. Gianella S, Massanella M, Wertheim JO, et al. The Sordid affair between human 
herpesvirus and human immunodeficiency virus. J Infect Dis. 2015 Mar 6. pii: 
jiv148. [Epub ahead of print] Review. 
88. Beal S, Sheiner LB, Boekmann A, et al. NONMEM's User's Guides. ICON Development 
Solutions, Ellicott City, MD, USA, 2009. 
89. Chen X1, Williams WV, Sandor V, et al. Population pharmacokinetic analysis of orally-
administered ruxolitinib (INCB018424 Phosphate) in patients with primary myelofibrosis 
(PMF), post-polycythemia vera myelofibrosis (PPV-MF) or post-essential 
thrombocythemia myelofibrosis (PET MF). J Clin Pharmacol. 2013 Jul;53(7):721-30. 
90. Karlsson MO, Sheiner LB.The importance of modeling interoccasion variability in 
population pharmacokinetic analyses. J Pharmacokinet Biopharm. 1993 Dec;21(6):735-
50. 
91. Hooker A, Staatz CE, Karlsson MO. Conditional weighted residuals, an improved model 
diagnostic for the FO/FOCE methods. Annual Meeting of the Population Approach 
Group in Europe, 2012, Venice, Italy. Abstract #1001. 
92. Hooker AC, Staatz C, Karlsson MO. Conditional weighted residuals (CWRES): a model 
diagnostic for the FOCE method. Pharm Res. 2007;24:2187-97. 
93. Karlsson MO, Savic R. Diagnosing Model Diagnostics Pharmacology & Therapeutics. 
2007;82:17-20. 
94. Wurtz R, Itokazu G, Rodvolt K. Antibiotic dosing in obese patients. Clin Infect Dis. 
1997;25:112-8. 
95. Bergstrand M, Hooker AC, Wallin JE, et al. Prediction-corrected visual predictive checks 
for diagnosing nonlinear mixed-effects models. AAPS J. 2011 Jun;13(2):143-51.  
96. Kakuda T, Sekar V, Vis P, et al. Pharmacokinetics and pharmacodynamics of darunavir 
and etravirine in HIV-1-infected, treatment-experienced patients in the gender, race, and 
clinical experience (GRACE) trial. AIDS Res Treat. 2012:186987. 
97. Abdelhady AM, Desta Z, Jiang F, et al. Population pharmacogenetic-based 
pharmacokinetic modeling of efavirenz, 7-hydroxy- and 8-hydroxyefavirenz. J Clin 
Pharmacol. 2014 Jan;54(1):87-96. 


 
 
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APPENDIX I 
 
DIVISION OF AIDS 
AIDS CLINICAL TRIALS GROUP (ACTG) 
SAMPLE INFORMED CONSENT  
 
For protocol A5336:  A Randomized, Pilot Study of Ruxolitinib in Antiretroviral-Treated HIV-
Infected Adults 
 
SHORT TITLE FOR THE STUDY: (A5336) Trial of Ruxolitinib in Antiretroviral-Treated HIV-
Infected Adults (FINAL Version 2.0, Dated 05/21/15)  
 
 
INTRODUCTION 
 
When a person becomes infected with HIV (the virus that causes AIDS), his/her immune system 
(the system that helps fight infection) is weakened (partly because the number of CD4+ cells 
[the number of white blood cells that fight infection] goes down). Despite successful treatment 
with antiretroviral therapy (ART), latent reservoirs (infected cells that are not actively producing 
HIV) remain present in the blood and contribute to ongoing immune system activation (immune 
system becomes active in response to an infection) and inflammation (reaction to an infection or 
irritation) in the body. 
 
You are being asked to take part in this research study because you: 
 
 
are infected with HIV-1 
 
have a very low viral load (amount of virus in the blood) 
 
have CD4+ T cell count of more than 350 cells 
 
are on stable ART for at least 3 months 
 
This study is sponsored by the National Institutes of Health (NIH) for the AIDS Clinical Trials 
Group (ACTG). The doctor in charge of this study at this site is: (insert name of Principal 
Investigator). Before you decide if you want to be a part of this study, we want you to know 
about the study.  
 
This is a consent form. It gives you information about this study. The study staff will talk with you 
about this information. You are free to ask questions about this study at any time.  If you agree 
to take part in this study, you will be asked to sign this consent form. You will get a copy to 
keep. 
 
 
WHY IS THIS STUDY BEING DONE? 
 
The purpose of this study is to find out more about the safety and tolerability (how well your 
body accepts the study drug) of the use of ruxolitinib in persons with HIV-1 infection who are 
also being treated with ART. We want to learn whether ruxolitinib will decrease inflammation 
and immune activation in the body; whether ruxolitinib will change the level of HIV-1 in your 
blood; and how ruxolitinib interacts with ART in the blood.  


 
 
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Ruxolitinib is approved by the U.S. Food and Drug Administration (FDA) to treat myelofibrosis, a 
disorder in which bone marrow is replaced by scar (fibrosis) tissue. Many of the cytokines 
(regulators of the body’s reaction to infection, immune response, and inflammation) affected by 
myelofibrosis are also affected by HIV. Because ruxolitinib acts on these cytokines, it is 
proposed that it may also be a possible treatment for HIV by reducing inflammation. 
Inflammation has been associated with organ disease in people living with HIV. 
 
 
WHAT DO I HAVE TO DO IF I AM IN THIS STUDY? 
 
If you decide to take part in this research study, you will be asked to sign this consent form and 
schedule a screening visit to determine if you can join the study. If so, you will be randomly 
assigned (as if by the toss of a coin) to either one of the two groups, Group A or Group B 
(see below). The screening visit will take about 1.5 hours.  
 
Screening visit 
 
Your HIV-1 infection will be confirmed. If there is no record available, you will have another 
HIV-1 test. You may have to sign a separate consent form before having this test. 
 
You will have a physical exam and will be asked about your health and medicines you have 
taken in the past or are taking now. 
 
You will have about 1 tablespoon of blood drawn for routine safety blood tests and to 
measure HIV-1 viral load. You will be given the test results when they are available. 
 
If you are a woman able to become pregnant, you will be asked to give a urine or blood 
sample to see if you are pregnant. You will not be able to enroll in this study if you are 
pregnant. You will be told the result of the test when it becomes available.   
 
If you do not enroll into the study 
If you decide not to take part in this study or if you do not meet the eligibility requirements, we 
will still use some of your information. As part of this screening visit, some demographic (for 
example, age, sex, race), clinical (for example, disease condition, diagnosis), and laboratory (for 
example, CD4+ T cell count, viral load) information is being collected from you so that ACTG 
researchers may help determine whether there are patterns or common reasons why people do 
not join a study. 
 
Pre-Entry 
If you qualify for the study, you will come to the clinic at least one day after the screening visit 
for the pre-entry visit. Please note it is possible that the pre-entry and screening visits may be 
combined. If not, at the pre-entry visit you will have a complete physical exam. You will also 
have blood drawn for CD4+ T cell count and for samples to be stored for future HIV-related 
testing for this study. A total of 2 tablespoon of blood will be drawn at this visit. Saliva and cells 
from your cheeks will be collected with an oral swab and tested for the presence of 
human herpes viruses (HHV; a virus causing sores most often found around the mouth 
or the genitals). The visit will last about 30-60 minutes.  
 
Entry visit 
If you are eligible for the study, you will come in for an entry visit. You will need to fast before 
your entry visit. Fasting means nothing to eat or drink other than water and medication for 8 
hours before your visit. The visit will last about 60-90 minutes.  


 
 
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During this visit: 
 
You will have a physical exam performed. You will be asked about medical history and any 
medicines you have taken since the last visit.   
 
If you are a woman able to get pregnant, you will be asked to provide blood or urine for a 
pregnancy test. You cannot start treatment if you are pregnant. You will be given the results 
of your pregnancy test as soon as it becomes available. 
 
You will have 7 tablespoons of blood drawn for routine safety blood tests including lipid 
testing, CD4+ T cell count, viral load, immune tests along with other tests. In addition, some 
of your blood will be stored for future testing required by the study. 
 
Saliva and cells from your cheeks will be collected with an oral swab and tested for 
the presence of HHV. 
 
You will be asked questions about adherence to antiretroviral (ARV) medicines.  
 
If you are in Group A, you will be given a medication diary to record the date and time of 
ruxolitinib, and ARV medicines for the 3 days preceding the week 1 study visit. The 
medication diary will be collected at the week 1 study visit. 
 
Once all these tests are done, you will be randomly assigned to either one of the two groups 
(Group A or Group B). However, because of the way the study is designed, there will be a 
greater chance of your being in Group A.  
 
 
Group A: Takes ruxolitinib by mouth twice a day for the next 5 weeks. 
 
Group B: No study treatment.   
 
If you are randomized to Group A, at this visit study staff will give you enough study drug to last 
until the next study visit. You will be asked not to drink grapefruit juice or eat grapefruit while 
taking the study drug. At the week 5 visit, you must return any remaining study drug.  
 
On Study Visits after Entry 
Regardless of which group you are assigned to, you will come to the clinic one week after your 
initial entry visit (week 1) and then study weeks 2, 4, 5. For the week 5 visit, you will be asked to 
fast, which means that for eight hours before your visit, you cannot eat or drink anything except 
water and your medication. Your next visit will not be until week 10 on study and your final visit 
will be at week 12. Visits will take about 30-60 minutes except weeks 1 and 4 study visits if you 
are in Group A (see 5th bullet below). If you are in Group A, at the week 2 study visit you will be 
given a medication diary to record the date and time of ruxolitinib, and ARV medicines for the 3 
days preceding the week 4 study visit. The medication diary will be collected at the week 4 
study visit.  
 
During these visits: 
 
You will have a brief physical exam performed. 
 
You will be asked about any new medications since your last visit including your HIV-1 
medications.  
 
You will have 1 tablespoon of blood drawn for routine safety blood tests, including lipid 
testing at week 5, and immune tests. At weeks 2, 5, and 12 study visits, blood will be drawn 
for CD4+ T cells count and viral load tests.  
 
You will have approximately from 2 to 6 tablespoons of blood drawn and stored for future 
protocol-related testing (except at the week 1 visit).     


 
 
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 
Saliva and cells from your cheeks will be collected with an oral swab and tested for 
the presence of HHV. 
 
If you are in Group A, at weeks 1 and 4 study visits you will have about 2 tablespoons of 
blood drawn over 6-8 hours to measure ruxolitinib and ARV medicines levels in your blood. 
You will be contacted the night before each pharmacokinetic (PK) visit to remind you not to 
take your morning dose of ruxolitinib or ARV medicines (including efavirenz) until instructed 
during your clinic visit in the morning. You will be asked to bring your study drug and ARV 
medicines to the clinic. You will have blood drawn 4 times during this time period to 
determine the levels of ruxolitinib and ARV medicines in your blood. For the multiple blood 
samples required, an indwelling catheter (a small, thin tube) will be put into an arm vein and 
left in place during your stay in the clinic if preferred. An IV tube will be placed at the end of 
the catheter for the PK blood draws. This will allow the blood samples to be drawn without 
any additional needle sticks. You may eat or drink as you wish during this period. 
 
You will be asked questions about adherence to the study drug (Group A) and your ARV 
medicines. 
 
Virologic Failure 
If the study staff sees that your viral load has gone up, and there is no other known explanation 
such as the flu, you will be asked to have another test done within 7 days. If your viral load is 
still up and you are still taking study drug, you will be asked to stop the study drug and come to 
the clinic within 2 weeks. You will be followed on study/off study treatment until the final study 
visit, week 12. 
 
Pregnancy 
If you become pregnant, study drug will be discontinued immediately, and you will be 
encouraged to remain in the study to be followed on study/off treatment until study completion. 
You will be followed by telephone contact thereafter to determine the pregnancy outcome. 
 
Premature Treatment/Study Discontinuation 
If you stop taking the study drug before the study-defined 5-week treatment period, you will be 
asked to return to the clinic to complete some evaluations. 
 
You will have a brief physical examination performed. 
 
You will be asked about any new medications since your last visit including your HIV-1 
medications. 
 
You will have approximately 6 tablespoons of blood drawn for routine safety blood tests 
including lipid panel, CD4+ T cells count, viral load, immune tests along with other tests. 
Also, some of your blood will be stored for future protocol-related testing. 
 
Saliva and cells from your cheeks will be collected with an oral swab and tested for 
the presence of HHV. 
 
If you are in Group A, you will have 2 tablespoons of blood drawn for measuring ruxolitinib 
and HIV-1 drug levels in your blood if you are discontinuing study treatment prior to week 4. 
 
You will be asked questions about adherence to the study drug (Group A) and your ARV 
medicines.  
 
Other 
If you agree, some of your blood that is left over after all required study testing is done may be 
stored (with usual protectors of identity) and used for ACTG-approved HIV-1 related research. 
Please check the appropriate response if you agree to storing samples for future use. 


 
 
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Please indicate below “yes” or “no” and initial and date whether you approve the use of these 
stored samples. Note that you can withdraw your consent for research on stored specimens at 
any time you want and the specimens will be discarded. 
 
______________________ Yes, I agree.       ____________________ No, I do not agree. 
 
 
WHAT ARE THE RISKS OF THE STUDY? 
 
The drug used in this study may have side effects, some of which are listed below. Please note 
that this list does not include all the side effects seen with this drug. This list includes the more 
serious, life-threatening or common side effects with a known or possible relationship. If you 
have questions concerning the additional study drug side effects please ask the medical staff at 
your site. The most common side effects observed in the past ruxolitinib clinical studies include: 
 
 
Thrombocytopenia (decreased cells in the blood that are needed to stop bleeding) 
 
Neutropenia (an abnormally low count of white blood cells that help fight off 
infections) 
 
Diarrhea 
 
Fever 
 
Urinary tract infections 
 
Anemia 
 
Bruising 
 
Dizziness 
 
Headache 
 
Transaminase elevations (increased liver enzymes that may indicate liver disease)  
 
Cholesterol elevation 
 
Infections 
  
It is not known whether the study drug passes through breast milk and whether it can produce 
adverse effects in the infant. Additionally, it is not known whether taking the study drug will 
reduce the risk of passing HIV to the baby while breastfeeding. You must tell the study doctor or 
nurse whether you are breastfeeding before enrolling in the study. You cannot participate in 
the study if you are breastfeeding.  
 
There is a risk of serious and/or life-threatening side effects when non-study medications are 
taken with the study drug. For your safety, you must tell the study doctor or nurse about all 
medications you are taking before you start the study and also before starting any new 
medications while on the study. Also, you must tell the study doctor or nurse before enrolling in 
any other clinical trials while on this study. 
 
Risk of Blood Draw 
 
Taking blood may cause some discomfort, bleeding, bruising, and/or swelling where the needle 
enters the body and in rare cases it may result in fainting. There is a small risk of infection.  
 
 


 
 
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Risk of Social Harm 
Although the study site will make every effort to protect your privacy and confidentiality, it is 
possible that others could find out that you are participating in this study and that social harms 
may result (because you could become labeled as being infected with HIV). For example, you 
could be treated unfairly or discriminated against by family members, friends, and/or the 
community. 
 
 
HOW MANY PEOPLE WILL TAKE PART IN THIS STUDY? 
 
About 60 people will take part in this study 
 
 
HOW LONG WILL I BE IN THIS STUDY? 
 
You will be in this study for about 12 weeks.   
 
 
WHY WOULD THE DOCTOR TAKE ME OFF THIS STUDY EARLY? 
 
The study doctor may need to take you off the study early without your permission if: 
 
 
You miss 2 visits in a row. 
 
The study is canceled.  
 
The study doctor or your regular doctor thinks the study is no longer in your best interest. 
 
The study doctor may also need to take you off the study drug without your permission if: 
 
 
You have confirmed virologic failure if you are in Arm A. 
 
You miss more than 3 doses of study drug a week for at least two weeks. 
 
You change or stop your anti-HIV-1 medicines if you are in Arm A. 
 
Continuing the study drug may be harmful to you. 
 
You need a treatment that you may not take while on the study. 
 
You are not able to take the study medicine as required by the study. 
 
You become pregnant.  
 
If you must stop taking the study drug before the study is over, the study doctor may ask you to 
continue to be part of the study and return for some study visits and procedures. 
 
If you must permanently stop taking ruxolitinib before your study participation is over, the study 
staff will discuss other options that may be of benefit to you. After you have completed your 
study participation, the study will not be able to continue to provide you with ruxolitinib that you 
received on the study. If continuing to take this or a similar drug/agent would be of benefit to 
you, the study staff will discuss how you may be able to obtain ruxolitinib. 
 
 
 
 


 
 
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ARE THERE RISKS RELATED TO PREGNANCY? 
 
It is not known if the drug or drug combinations in this study harm unborn babies. If you are 
having sex that could lead to pregnancy, you must agree not to become pregnant or make a 
woman pregnant.  
 
Because of the risk involved, you and your partner must use one form of birth control that you 
discuss with the study staff. You must start one method of birth control when you are taking your 
study drug. You must continue to use this method until 7 weeks after you stop the study drug. 
 
 
Condoms (male or female) with or without a spermicidal agent. Condoms are recommended 
because their appropriate use is the only contraceptive method effective for preventing HIV-
1  transmission.  
 
Diaphragm or cervical cap with spermicide 
 
Intrauterine device (IUD) 
 
Hormone-based contraceptive 
 
If you can become pregnant, you must have a pregnancy test before you enter this study. The 
test must show that you are not pregnant. If you think you may be pregnant at any time during 
the study, tell your study staff right away. The study staff will talk to you about your choices. 
Pregnancy will result in immediate discontinuation of the study drug for the pregnant participant. 
You will be followed on study until study completion and will be followed by telephone contact 
thereafter to determine the pregnancy outcome. Pregnancies that occur on study will be 
reported prospectively to The Antiretroviral Pregnancy Registry.  
  
 
ARE THERE BENEFITS TO TAKING PART IN THIS STUDY? 
 
This study is intended to gather information about how ruxolitinib acts on your immune system, 
not to treat your HIV-1 infection. There is no anticipated benefit for taking part in this study. 
Information learned from this study may help others who have HIV. 
 
 
WHAT OTHER CHOICES DO I HAVE BESIDES THIS STUDY? 
 
Instead of being in this study you have the choice of: 
 
 
Treatment with prescription drugs available to you. 
 
Treatment with other experimental drugs, if you qualify.  
 
No treatment 
 
Please talk to your doctor about these and other choices available to you. Your doctor will 
explain the risks and benefits of these choices. 
 
 
 
 
 


 
 
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WHAT ABOUT CONFIDENTIALITY? 
 
We will do everything we can to protect your privacy. In addition to the efforts of the study staff 
to help keep your personal information private, we have gotten a Certificate of Confidentiality 
from the U.S. Federal Government. This certificate means that researchers cannot be forced to 
tell people who are not connected with this study, such as the court system, about your 
participation. Also, any publication of this study will not use your name or identify you 
personally.   
 
People who may review your records include the ACTG, Office for Human Research Protections 
(OHRP) or other government agencies as part of their duties, Food and Drug Administration 
(FDA), (insert name of site) Institutional Review Board (IRB) (a group that protects the rights 
and well-being of people in research), National Institutes of Health (NIH), study staff, study 
monitors, and their designees. Having a Certificate of Confidentiality does not prevent you from 
releasing information about yourself and your participation in the study. 
 
Even with the Certificate of Confidentiality, if the study staff learns of possible child abuse and/or 
neglect or a risk of harm to yourself or others, we will be required to tell the proper authorities. 
A description of this clinical trial will be available on www.ClinicalTrials.gov, as required by U.S. 
law. This Web site will not include information that can identify you. At most, the Web site will 
include a summary of the results. You can search this Web site at any time.  
 
 
WHAT ARE THE COSTS TO ME? 
 
Taking part in this study may lead to added costs to you and your insurance company. In some 
cases it is possible that your insurance company will not pay for these costs because you are 
taking part in a research study. There will be no cost to you for study drug, study-related visits, 
physical examinations, laboratory tests, or other study procedures. Note that ARV medicines will 
not be provided by the study. 
 
 
WILL I RECEIVE ANY PAYMENT? 
 
You will be compensated for participating in the study regardless of the group you were 
assigned to. 
 
 
WHAT HAPPENS IF I AM INJURED? 
 
If you are injured as a result of being in this study, you will be given immediate treatment for 
your injuries. The cost for this treatment will be charged to you or your insurance company. 
There is no program for compensation either through this institution or the National Institutes of 
Health. You will not be giving up any of your legal rights by signing this consent form. 
 
 
 
 


 
 
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WHAT ARE MY RIGHTS AS A RESEARCH PARTICIPANT? 
 
Taking part in this study is completely voluntary. You may choose not to take part in this study 
or leave this study at any time. Your decision will not have any impact on your participation in 
other studies conducted by NIH and will not result in any penalty or loss of benefits to which you 
are otherwise entitled. 
 
We will tell you about new information from this or other studies that may affect your health, 
welfare, or willingness to stay in this study. If you want the results of the study, let the study staff 
know. 
 
 
WHAT DO I DO IF I HAVE QUESTIONS OR PROBLEMS? 
 
For questions about this study or a research-related injury, contact: 
 
 
name of the investigator or other study staff 
 
telephone number of above  
 
For questions about your rights as a research participant, contact: 
 
 
name or title of person on the Institutional Review Board (IRB) or other organization 
appropriate for the site 
 
telephone number of above 
 


 
 
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SIGNATURE PAGE 
 
If you have read this consent form (or had it explained to you), all your questions have been 
answered and you agree to take part in this study, please sign your name below. 
 
 
 
____________________________ 
________________________________________ 
Participant’s Name (print) 
Participant’s Signature and Date 
 
 
____________________________ 
________________________________________ 
Participant’s Legal Representative (print) 
Legal Representative’s Signature and Date 
(As appropriate) 
 
 
____________________________ 
_________________________________________ 
Study Staff Conducting 
Study Staff’s Signature and Date 
Consent Discussion (print)  
 
 
____________________________ 
_________________________________________ 
Witness’s Name (print) 
Witness’s Signature and Date 
(As appropriate) 

SAP_000.pdf: 
Page | 1  
 
 
 
 
ACTG A5336 
 
 
 
A Randomized Pilot Study of Ruxolitinib in Antiretroviral-
Treated HIV-Infected Adults 
 
ClinicalTrials.Gov ID: NCT02475655 
 
 
 
 
 
Statistical Analysis Plan 
 
Version 2.0 
 
 
 
 
 
 
 
This is ACTG A5336 SAP Version 2.0 with names of authors, names of 
publication writing team members, and analysis timeline redacted. 
 
 
 
 
 
 
 
April 4, 2018 
 
 
 
 
 
 
 


A5336 Primary Statistical Analysis Plan (SAP) V2.0                                                           April 4, 2018 
Page | 2  
 
 
Table of Contents 
1 
Introduction ............................................................................................................................................ 3 
1.1 
Key Updates to the SAP ............................................................................................................... 3 
2 
Study Overview ..................................................................................................................................... 3 
2.1 
Design ........................................................................................................................................... 3 
2.2 
Hypotheses ................................................................................................................................... 4 
2.3 
Objectives ...................................................................................................................................... 4 
2.3.1 
Primary Objectives .................................................................................................................... 4 
2.3.2 
Secondary Objectives ............................................................................................................... 4 
2.4 
Outcome measures ....................................................................................................................... 5 
2.4.1 
Primary Outcome Measures ..................................................................................................... 5 
2.4.2 
Secondary Outcome Measures ................................................................................................. 6 
2.4.3 
Other Outcome Measures ......................................................................................................... 8 
3 
General Analysis Considerations .......................................................................................................... 8 
4 
Statistical Methods ................................................................................................................................ 9 
4.1 
Primary Outcome Measures ......................................................................................................... 9 
4.1.1 
Safety ........................................................................................................................................ 9 
4.1.2 
Tolerability ............................................................................................................................... 10 
4.1.3 
Efficacy .................................................................................................................................... 10 
4.2 
Secondary Outcome Measures................................................................................................... 10 
4.2.1 
Safety ...................................................................................................................................... 10 
4.2.2 
Longitudinal Summaries of CD4 Count and plasma HIV-1 RNA ............................................ 10 
4.2.3 
Immune Activation and Inflammation ...................................................................................... 11 
4.2.4 
HIV-1 Reservoir ....................................................................................................................... 11 
4.2.5 
CMV Co-Infection .................................................................................................................... 12 
5 
Key Report Elements .......................................................................................................................... 12 
6 
Core Manuscript Writing Team ........................................................................................................... 12 
7 
Citations .............................................................................................................................................. 13 
Appendix 1:  Analysis Timeline ................................................................................................................... 14 
Appendix 2:  Analysis Implementation Plan (AIP)....................................................................................... 15 
 
 
 


A5336 Primary Statistical Analysis Plan (SAP) V2.0                                                           April 4, 2018 
Page | 3  
 
 
1 
Introduction 
 
The Primary Statistical Analysis Plan (SAP) describes the proposed content for the primary 
statistical analysis report of ACTG A5336, which addresses the primary and secondary objectives 
of the study.  This document also describes the primary and secondary outcome measures for 
which results will be posted on ClinicalTrials.gov.  The Primary SAP outlines the general 
statistical approaches that will be used in the analysis of the study and has been developed to 
facilitate discussion of the statistical analysis components amongst the study team; and to 
provide agreement between the study team and statisticians regarding the statistical analyses to 
be performed and presented in the primary analysis report. 
   
Analyses for the primary analysis report will be initiated once the last participant has completed 
the Week 12 study visit; details on the analysis timeline are located in Appendix 1.  In addition, 
all primary and secondary outcomes outlined in this SAP will be submitted to ClinicalTrials.gov 
within 1 year of the PCD (5 weeks after the last participant enrolls).   
 
Additional specifications and details on other key elements of the primary analysis report are 
provided in the Analysis Implementation Plan (AIP) in Appendix 2. 
 
1.1 
Key Updates to the SAP 
 
The SAP was updated after the final interim review, but prior to the final analysis, to include the 
following changes: 
o 
Reformat to be compliant with new SOP requirements 
o 
Remove details on interim analyses 
o 
Update the statistical test for safety from a Fisher’s exact test to a mid-p Fisher’s 
exact test, which is a less conservative test, therefore more appropriate to compare 
safety 
 
2 
Study Overview 
 
2.1 
Design 
 
A5336 is a multi-center, randomized, open-label, two-arm, phase IIa study.  Eligible participants 
on approved ART with virologic suppression will be randomized to receive ruxolitinib or no study 
treatment to measure safety and tolerability and to determine whether there are changes in 
systemic immune activation and inflammation after 5 weeks of treatment with ruxolitinib.  
 
A total of 60 participants will be randomized 2:1 to ruxolitinib or no study treatment; 40 on 
ruxolitinib and 20 on no study treatment. The study aims to enroll at least 15 participants on 
efavirenz (EFV) in order to have at least 8 ruxolitinib-treated participants to evaluate 
pharmacokinetic (PK) interactions with EFV.   
 
The study population consists of HIV-1 infected males and females ≥ 18 and < 75 years of age on 
stable ART for at least 12 weeks prior to study entry containing TDF/FTC, TAF/FTC, TDF/3TC, or 
ABC/3TC, plus a NNRTI or INSTI (not containing cobicistat) who: 
 
o 
Are on ART for at least 2 years 
o 
Have virologic suppression (see protocol for definition) 
o 
Have CD4+ T cell count > 350 cells/mm3 within 45 days prior to study entry 
o 
Have no medical conditions, or concomitant medications, prohibiting the use of janus 
activating kinase-signal transducer and activator of transcription (Jak-STAT) inhibitor 
 


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All participants will be followed for up to 12 weeks after randomization.  Participants randomized 
to the ruxolitinib arm will receive 10 mg of ruxolitinib orally twice daily for 5 weeks and then will be 
followed off-treatment for an additional 7 weeks. 
 
 
2.2 
Hypotheses 
 
1) The use of ruxolitinib for 5 weeks with antiretroviral therapy (ART) consisting of: 
tenofovir/emtricitabine (TDF/FTC) or abacavir /lamivudine (ABC/3TC) + a nonnucleoside 
reverse transcriptase inhibitor or integrase strand transfer inhibitor (NNRTI or INSTI) will be 
safe for virologically suppressed participants.   
 
2) The use of ruxolitinib for 5 weeks with ART will be tolerable for virologically suppressed 
participants.  
 
3) The use of ruxolitinib for 5 weeks with ART for virologically suppressed participants will 
reduce systemic immune activation and inflammation.   
 
 
2.3 
Objectives 
 
The Primary SAP addresses the following primary and secondary objectives listed in the study 
protocol.  The secondary PK objectives will be analyzed outside of SDAC and their analyses will 
be outlined in a separate SAP.   
 
 
2.3.1 Primary Objectives  
 
1) To evaluate the safety of ruxolitinib in ART-treated HIV-1 infected virologically 
suppressed participants during 5 weeks of treatment [Protocol Objective 1.2.1]. 
 
2) To evaluate the tolerability of ruxolitinib in ART-treated HIV-1 infected virologically 
suppressed participants during 5 weeks of treatment [Protocol Objective 1.2.2]. 
 
3) To compare changes in interleukin-6 (IL-6) levels between baseline and week 5 in ART-
treated HIV-1 infected virologically suppressed participants who are randomized to 
ruxolitinib and those randomized to receive no study treatment [Protocol Objective 1.2.3].  
 
2.3.2 Secondary Objectives 
 
1) To evaluate the safety of ruxolitinib in ART-treated HIV-1 infected virologically 
suppressed participants during all on-study visits through week 12 [Protocol Objective 
1.3.1]. 
 
2) To compare changes in IL-6 levels between baseline and week 12 in ART-treated HIV-1 
virologically suppressed participants who are randomized to ruxolitinib to changes in 
those participants randomized to receive no study treatment [Protocol Objective 1.3.2]. 
 
3) To assess whether the use of ruxolitinib will decrease levels of other measures of 
inflammation and immune activation including sCD14, tumor necrosis factor alpha (TNF-
α), IL-1α, IL-1β, IL-18, macrophage colony stimulating factor (mCSF), neopterin, HLA-
DR, CD38, and both the distribution of monocyte subsets (defined by expression of 
CD14+ and CD16+) and phenotypic indices of their activation during 5 weeks of 
treatment and at week 12 [Protocol Objective 1.3.3]. 
 


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4) To examine whether the use of ruxolitinib is associated with changes in HIV-1 plasma 
viral load levels (by a commercial clinical assay and single-copy assay) and CD4+ T cell 
counts in HIV-1 infected participants at baseline compared to 12 weeks after initiation of 
treatment for those receiving 5 weeks of ruxolitinib versus those receiving no study 
treatment [Protocol Objective 1.3.4]. 
 
5) To assess changes from baseline in the HIV-1 reservoir, specifically plasma HIV-1 RNA 
by single copy assay (as above), cellular HIV-1 RNA and total DNA, 2 long-terminal 
repeat sequences [LTRs], and integrated DNA [Protocol Objective 1.3.5]. 
 
6) To evaluate whether the use of ruxolitinib is associated with more frequent reactivation of 
human herpes viruses (ie, cytomegalovirus [CMV], Epstein Barr virus [EBV], herpes 
simplex viruses [HSV], human herpes viruses [HHV] 6, 7, and 8) as measured in 
longitudinally collected oral swabs [Protocol Objective 1.3.7]. 
 
2.4 
Outcome measures 
 
2.4.1 Primary Outcome Measures 
 
Safety 
 
1) Proportion of participants on the Ruxolitinib arm who experience any of the following 
safety milestones while on-treatment: 
o 
For participants with entry CD4+ T cell count < 700 cells/mm3, confirmed CD4+ 
decline > 33% of baseline and to < 350 cells/mm3. 
o 
For participants with entry CD4+ T cell count ≥ 700 cells/mm3, a confirmed CD4+ 
T cell decline > 50 % of baseline. 
o 
Confirmed HIV-1 RNA level above the lower limit of quantification in the absence 
of an interruption in ART. 
o 
New or recurrent CDC category C AIDS-indicator condition. 
o 
HIV-1 associated infection including Herpes zoster. 
o 
Lymphoproliferative malignancies. 
o 
Discontinuation of ruxolitinib due to thrombocytopenia. 
o 
Grade 4 or recurrence of Grade 3 anemia/neutropenia. 
o 
Any Grade 4 or recurrence of Grade 3 toxicity related to study drug. 
o 
New diagnosis of pneumonia, sepsis, or bacteremia. 
 
2) Proportion of participants who experience any of the following safety milestones on-study 
from entry through week 5 by study arm: 
o 
For participants with entry CD4+ T cell count < 700 cells/mm3, confirmed CD4+ 
decline > 33% of baseline and to < 350 cells/mm3. 
o 
For participants with entry CD4+ T cell count ≥ 700 cells/mm3, a confirmed CD4+ 
T cell decline > 50 % of baseline. 
o 
Confirmed HIV-1 RNA level above the lower limit of quantification in the absence 
of an interruption in ART. 
o 
New or recurrent CDC category C AIDS-indicator condition. 
o 
HIV-1 associated infection including Herpes zoster. 
o 
Lymphoproliferative malignancies. 
o 
Occurrence of Grade 2 or higher thrombocytopenia. 
o 
Grade 4 or recurrence of Grade 3 anemia/neutropenia. 
o 
Any Grade 4 or recurrence of Grade 3 toxicity. 
o 
New diagnosis of pneumonia, sepsis, or bacteremia. 
 
 
 


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3) Proportion of each of the following components that occurred on-study from entry to week 
5 by study arm: 
o 
For participants with entry CD4+ T cell count < 700 cells/mm3, confirmed CD4+ 
decline > 33% of baseline and to < 350 cells/mm3. 
o 
For participants with entry CD4+ T cell count ≥ 700 cells/mm3, a confirmed CD4+ 
T cell decline > 50 % of baseline. 
o 
Confirmed HIV-1 RNA level above the lower limit of quantification in the absence 
of an interruption in ART. 
o 
New or recurrent CDC category C AIDS-indicator condition. 
o 
HIV-1 associated infection including Herpes zoster. 
o 
Lymphoproliferative malignancies. 
o 
Occurrence of Grade 2 or higher thrombocytopenia. 
o 
Grade 4 or recurrence of Grade 3 anemia/neutropenia. 
o 
Any Grade 4 or recurrence of Grade 3 toxicity. 
o 
New diagnosis of pneumonia, sepsis, or bacteremia. 
 
 
Tolerability   
 
4) Occurrence of premature discontinuation of study treatment, including reasons for 
discontinuations. 
 
 
Efficacy   
 
5) Participant-specific change in log10-transformed plasma IL-6 (using geometric means of 
two ELISA measurements at pre-entry and entry, and weeks 4 and 5). 
 
 
2.4.2 Secondary Outcome Measures 
 
1) Proportion of participants on Ruxolitinib who experienced any of the following safety 
milestones during study follow-up from entry through week 12. 
o 
For participants with entry CD4+ T cell count < 700 cells/mm3, confirmed CD4+ 
decline > 33% of baseline and to < 350 cells/mm3. 
o 
For participants with entry CD4+ T cell count ≥ 700 cells/mm3, a confirmed CD4+ 
T cell decline > 50 % of baseline. 
o 
Confirmed HIV-1 RNA level above the lower limit of quantification in the absence 
of an interruption in ART. 
o 
New or recurrent CDC category C AIDS-indicator condition. 
o 
HIV-1 associated infection including Herpes zoster. 
o 
Lymphoproliferative malignancies. 
o 
Discontinuation of ruxolitinib due to thrombocytopenia. 
o 
Grade 4 or recurrence of Grade 3 anemia/neutropenia. 
o 
Any Grade 4 or recurrence of Grade 3 toxicity related to study drug. 
o 
New diagnosis of pneumonia, sepsis, or bacteremia. 
 
 
 


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2) Proportion of participants who experienced any of the following safety milestones during 
study follow-up from entry through week 12 by study arm: 
o 
For participants with entry CD4+ T cell count < 700 cells/mm3, confirmed CD4+ 
decline > 33% of baseline and to < 350 cells/mm3. 
o 
For participants with entry CD4+ T cell count ≥ 700 cells/mm3, a confirmed CD4+ 
T cell decline > 50 % of baseline. 
o 
Confirmed HIV-1 RNA level above the lower limit of quantification in the absence 
of an interruption in ART. 
o 
New or recurrent CDC category C AIDS-indicator condition. 
o 
HIV-1 associated infection including Herpes zoster. 
o 
Lymphoproliferative malignancies. 
o 
Occurrence of Grade 2 or higher thrombocytopenia. 
o 
Grade 4 or recurrence of Grade 3 anemia/neutropenia. 
o 
Any Grade 4 or recurrence of Grade 3 toxicity. 
o 
New diagnosis of pneumonia, sepsis, or bacteremia. 
 
 
3) Proportion of each of the following components that occurred during study follow-up from 
entry to week 12 by study arm: 
o 
For participants with entry CD4+ T cell count < 700 cells/mm3, confirmed CD4+ 
decline > 33% of baseline and to < 350 cells/mm3. 
o 
For participants with entry CD4+ T cell count ≥ 700 cells/mm3, a confirmed CD4+ 
T cell decline > 50 % of baseline. 
o 
Confirmed HIV-1 RNA level above the lower limit of quantification in the absence 
of an interruption in ART. 
o 
New or recurrent CDC category C AIDS-indicator condition. 
o 
HIV-1 associated infection including Herpes zoster. 
o 
Lymphoproliferative malignancies. 
o 
Occurrence of Grade 2 or higher thrombocytopenia. 
o 
Grade 4 or recurrence of Grade 3 anemia/neutropenia. 
o 
Any Grade 4 or recurrence of Grade 3 toxicity. 
o 
New diagnosis of pneumonia, sepsis, or bacteremia. 
 
4) Occurrence of protocol-defined reportable adverse events (all diagnoses, Grade 3 or 
higher sign/symptoms or laboratory values, any signs/symptoms or laboratory values that 
led to a change in treatment or met ICH, EAE, or SAE guidelines) at any post-entry time 
point. 
 
5) Safety Laboratory values (estimated creatinine clearance, creatinine, AST (SGOT), ALT 
(SGPT), ANC, hemoglobin, platelet count) across all study weeks (at entry, week 1, and 
week 2, and arithmetic means at weeks 4 and 5, and at weeks 10 and 12). 
 
6) Change in safety laboratory values (estimated creatinine clearance, creatinine, AST 
(SGOT), ALT (SGPT), ANC, hemoglobin, and platelet count) across all study weeks (at 
weeks 1 and 2, and arithmetic means at weeks 4 and 5 and at weeks 10 and 12). 
 
7) Change in log10-transformed plasma IL-6 across all study weeks (geometric means of 
pre-entry and entry, and week 10 and 12). 
 
8) Changes in soluble markers of immune activation and inflammation in the peripheral 
blood (sCD14, TNF-α, IL-1α, IL-1β, IL-7, IL-10, IP-10, IL-18, mCSF, and neopterin) 
across study weeks from baseline (geometric mean of pre-entry and entry) to week 4/5 
(geometric mean of week 4 and week 5) and week 12. 
 
 


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9) Changes in cellular markers of immune activation and inflammation in the peripheral 
blood (HLA-DR, CD25, CD38, CD69, CD127, Ki67, BCL2, a4b7, CX3CR1, PAR-1 
measured on CD4 and CD8 cells; monocyte subsets (classical, inflammatory, and 
patrolling) defined by CD14 and CD16 expression; proportion of monocyte subsets 
(classical, inflammatory, and patrolling) expressing CCR2, CX3CR1, and CD163) from 
entry to weeks 5 and 12. 
 
10) Level of plasma HIV-1 RNA at baseline and weeks 2, 5, and 12. 
 
11) Change in CD4+ T cell counts from baseline (mean of pre-entry and entry) to weeks 2, 5, 
and 12. 
 
12) Changes in HIV-1 reservoir (HIV-1 RNA by SCA, cellular HIV-1 total RNA and DNA) from 
entry to weeks 5 and 12. 
 
13) Level of CMV shedding at pre-entry, entry, and weeks 1, 2, 4, 5, 10, and 12. 
 
 
2.4.3 Other Outcome Measures 
 
1) Changes in HIV-1 reservoir (2 long-terminal repeat sequences [LTRs], and integrated 
DNA) from entry to weeks 5 and 12. 
 
2) Level of EBV, HSV, HHV-6, HHV-7, and HHV-8 shedding at pre-entry, entry, and weeks 
1, 2, 4, 5, 10, and 12. 
 
 
3 
General Analysis Considerations 
 
Study entry is defined as the date of the entry visit recorded on study initiation case report form.  
 
The protocol-defined study week windows are modified for analysis purposes.  For all analyses, 
the following window definitions will be used: 
 
 
Pre-entry: 
The last visit that occurred at least 24 hours 
prior to entry, but not more than 7 days before 
Entry: 
The visit after randomization, and prior to 
initiation of study treatment: Days [0-3] 
Week 1: 
The first visit in the window: Days [4, 22] 
Week 2: 
The second visit in the window: Days [4, 22] 
Week 4: 
The first visit in the window: Days [25, 39] 
Week 5: 
The second visit in the window: Days [25, 39] 
Week 10: 
The first visit in the window: Days [56, 93] 
Week 12: 
The second visit in the window: Days [56, 93] 
 
Baseline values are the average of pre-entry and entry values, the week 4/5 values are the 
average of week 4 and week 5 values, and the week 10/12 values are the average of week 10 


A5336 Primary Statistical Analysis Plan (SAP) V2.0                                                           April 4, 2018 
Page | 9  
 
and 12 values; averages will be calculated using geometric means for log-transformed data and 
arithmetic mean for non-transformed data.  If missing one of the pre-entry or entry values, then 
the available measure will be used as the baseline value, and similarly, if missing one of the week 
4 or week 5 values or week 10 or week 12 values, then the available measure will be used as the 
week 4/5 value or week 10/12 value, respectively. 
  
Analyses will be done among those in the as-treated population or among those in the safety 
analysis population (Cameron 2008), depending on the outcome; see the Statistical Methods 
section for details on populations for specific outcomes.  The safety analysis population will 
consist of all participants randomized to the no-treatment arm and all participants who took at 
least one dose of ruxolitinib among those randomized to the ruxolitinib arm. The as-treated 
population is defined as participants who meet the following conditions:  (1) have data at baseline 
and week 4/5, (2) for the ruxolitinib arm, remained on study treatment through week 4/5 and 
missed no more than 6 doses cumulatively, (3) did not change ART, use prohibited medications, 
or had confirmed virologic failure in this time period.  Participants found to be ineligible after 
enrollment will be included in all safety analyses, but excluded from other analyses.  
 
 
Confirmed virologic failure is defined as the occurrence of two sequential plasma HIV-1 RNA 
values above the lower limit of quantification in the absence of an interruption of ART; the 
confirmatory HIV-1 RNA should occur within 7 days of receipt of the results for the first test.  In 
the event that a suspected virologic failure is not confirmed with a follow-up HIV-1 RNA test (i.e., 
there is no subsequent HIV-1 RNA available), this suspected virologic failure will be considered a 
confirmed virologic failure. Confirmed CD4 count decline is defined as two sequential CD4 counts 
meeting the respective criteria in the absence of an interruption in ART. Adverse events are 
classified as related (definitely, probably, possibly) or not related (probably not, not) by site 
investigators.   
 
Biomarker data distributions will be assessed for normality, and any non-normal data will be log-
transformed to the log-10 scale for analysis; it is anticipated that all soluble markers will be log-
transformed.  Descriptive summaries of continuous baseline characteristics will include 
frequency, frequency missing, mean, sd, median, IQR, 10th and 90th percentiles, and min/max; 
descriptive summaries of other continuous variables will use mean and sd, or median and IQR 
depending upon the corresponding analysis method. The frequency and percentage will be used 
for all categorical variables.  
 
 
4 
Statistical Methods  
 
4.1 
Primary Outcome Measures  
 
4.1.1 Safety 
 
Tabular summaries of all primary safety outcomes will be provided for primary outcomes (1), (2), 
and (3), which will include frequencies and percentages overall and by study arm.  Formal 
comparisons between study arms will contrast the proportion of participants who experienced any 
safety milestones on study from entry through week 5 as defined in primary outcome (2) using a 
one-sided mid-p Fisher’s exact test with 5% type-I error. 
 
All safety summaries will be done in the safety analysis population. 
 
 


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4.1.2 Tolerability 
 
All tolerability summaries will be done in the safety analysis population.  
 
Participant-level listings of occurrences of premature discontinuation of study treatment will be 
provided, including reasons for discontinuation.  If a given reason for premature discontinuation 
occurs in more than 5 participants, then tabular summaries including frequency and percentages 
will also be provided.  This summary will only be provided for those in the Ruxolitinib arm. 
 
 
4.1.3 Efficacy 
 
The primary efficacy analyses will be done in the as-treated population.  
 
Analyses addressing efficacy of ruxolitinib will contrast IL-6 changes from baseline to week 4/5 
between study arms.  Baseline and week 4/5 are defined based on the criteria mentioned in the 
General Analysis Considerations section and IL-6 will be transformed to the log-10 scale for all 
analyses.  
 
Formal study arm comparisons will contrast the mean change from baseline to week 4/5 between 
study arms.  The mean and 90% confidence interval (CI) for the mean change will be calculated; 
corresponding p-value based on a two-sided t-test with 10% alpha will also be calculated.  
 
Descriptive summaries of levels of IL-6 at baseline and week 4/5 will be presented by study arm 
using continuous summary statistics and longitudinal plots.  Changes in IL-6 (log10) from 
baseline to week 4/5 will be summarized with the mean and a 95% CI for the change separately 
within each study arm; change will be presented as absolute mean log10 change and 
corresponding geometric mean fold-change. 
 
4.2 
Secondary Outcome Measures 
 
4.2.1 Safety  
 
All summaries of secondary safety outcomes will be done in the safety analysis population.   
 
In a similar manner to the primary safety analyses, tabular summaries will be provided for 
secondary outcomes (1-4), which will include frequencies and percentages overall and by study 
arm.  Formal comparisons between study arms will contrast the proportion of participants who 
experienced any safety milestones on study from entry through week 12 as defined in secondary 
outcome (2) using a one-sided mid-p Fisher’s exact test with 5% type-I error.   
 
Levels and changes in safety laboratories (secondary outcomes 5 and 6) will be summarized with 
continuous descriptive summary statistics.  Changes in safety labs will be formally compared at 
week 4/5 and week 10/12 via two-sided t-tests with 10% alpha and corresponding 90% 
confidence intervals.   
 
4.2.2 Longitudinal Summaries of CD4 Count and plasma HIV-1 RNA  
 
Descriptive longitudinal summaries of levels and changes in CD4 cell counts and CD4 % will be 
provided by study arm and week.  Formal comparisons between study arms of change from 
baseline will occur at all post-entry visits (week 2, 5, and 12); two-sided t-tests with 10% alpha will 
contrast differences between groups.  In addition, change from week 5 (end of treatment) to week 
12 (end of study) will also be examined.  Participant narratives will be provided for those who had 
a protocol defined drop in CD4 cell counts (see primary safety endpoint). 
 


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Tabular summaries of categorized HIV-1 RNA levels (secondary outcome 10) by study week and 
arm will also be provided; no statistical inference will be done. HIV-1 RNA levels will be 
categorized as below or above the lower limit of quantification.  Participant narratives will be 
provided for those who had any viral loads above the lower limit of quantification while on study. 
 
All summaries will be done in the safety analysis and as-treated populations. 
 
 
4.2.3 Immune Activation and Inflammation 
 
The immunology markers outlined in secondary outcome measures (7-9) will be addressed in the 
following manner.  All analyses will be done in the as-treated population, and geometric means 
will be used to average pre-entry and entry, week 4 and 5, and week 10 and 12, as available.  As 
mentioned in the General Analysis Consideration section, biomarkers will be log-10 transformed 
as appropriate. 
 
Descriptive summaries of levels and changes (absolute changes and corresponding fold-
changes) will be done using continuous summary statistics and longitudinal plots by study week 
and arm.  Treatment group comparisons will contrast the change from baseline at each post-entry 
study week using two sided t-tests with 10% alpha; corresponding p-values and 90% CIs will also 
be calculated.  In addition, change from end of treatment to end of study will also be examined in 
a similar manner.   Results for log-10 transformed markers will be back transformed to fold-
changes for presentation as appropriate. 
 
4.2.4 HIV-1 Reservoir 
 
Tabular summaries of categorized HIV-1 RNA by iSCA (secondary outcome 12) will be provided 
by study week and arm.  To compare changes in iSCA over time and between study arms, a GEE 
model for binary data will be used since it is anticipated that a substantial proportion of the data 
will be below assay limit (Riddler 2016).   
 
Total HIV-1 DNA and cell-associated HIV-1 RNA will be log-10 transformed and summarized by 
arm and across study weeks with continuous summary statistics and longitudinal plots, imputing 
values below the assay limit as the lower limit of the assay; as with the immunology markers, 
results will be back-transformed to fold-changes for presentation. Total HIV-1 DNA and cell-
associated HIV-RNA will also be summarized categorically as the frequency and percent below or 
above the lower limit; if there are differential assay lower limits, an analytic lower limit will be 
applied for this summary.  
 
Results below assay limit are not anticipated for total HIV-1 DNA (Gandhi 2017).  Changes from 
baseline will be contrasted between study arms using a two-sided t-test with 10% alpha at each 
visit (week 5 and week 12); in addition, a comparison between week 5 (end of treatment) and 
week 12 (end of study) will also be done.   
 
Modest amounts of below assay limit observations are anticipated for cell-associated HIV-1 RNA 
(Gandhi 2017). To contrast changes over time between study arms, a longitudinal censored 
normal model approach will be used, which was developed for below assay limit HIV virology 
data and can handle different assay limits (Riddler 2016, Vaida 2009). 
 
In the event that a large proportion of participants have values below assay limits for total HIV-1 
DNA and cell-associated HIV-1 RNA, a binary analysis approach similar to the analysis of iSCA 
will be used. 
 
All analyses will occur in the as-treated population.  For GEE and longitudinal censored normal 
models, the analysis will also be restricted to those who have results for corresponding time 
points of interest.   


A5336 Primary Statistical Analysis Plan (SAP) V2.0                                                           April 4, 2018 
Page | 12  
 
4.2.5 CMV Co-Infection 
 
Level of CMV shedding (secondary outcome 13), will be summarized by study week and arm as 
the frequency and percentage of those above and below the assay limit of detection.  The 
proportion of participants with detectable CMV at any on-treatment time point (ever shedding at 
weeks 1, 2, 4, or 5) and any post-treatment time point (ever shedding at weeks 10 or 12) will be 
contrasted between study arms via using a two-sided mid-p Fisher’s exact test with 10% type-I 
error.  
 
This analysis will include all participants with available specimen. 
 
 
5 
Key Report Elements 
 
In addition to the previously mentioned primary and secondary analyses, the following elements 
will be included in the final analysis report: 
 
o 
CONSORT diagram 
o 
Description of ineligible enrollments 
o 
Screen failure rates with reasons 
o 
Accrual by month, site, and stratification factors 
o 
Baseline Characteristics 
o 
Study/Treatment Status 
o 
Duration of Follow-up 
o 
ARV Regimen Status 
o 
ART Adherence 
o 
Concomitant Medication Use 
o 
Additional safety summaries including EAEs, SAEs, deaths, and pregnancies 
 
Detailed descriptions of these elements are provided in the AIP in Appendix 2. 
 
6 
Core Manuscript Writing Team 
 
 
Redacted. 
 


A5336 Primary Statistical Analysis Plan (SAP) V2.0                                                           April 4, 2018 
Page | 13  
 
7 
Citations 
 
1. Cameron D, Casey M, Press M, Lindquist D, Pienkowski T, Romieu CG, Chan S, Jagiello-
Gruszfeld A, Kaufman B, Crown J, Chan A. A phase III randomized comparison of lapatinib plus 
capecitabine versus capecitabine alone in women with advanced breast cancer that has 
progressed on trastuzumab: updated efficacy and biomarker analyses. Breast cancer research 
and treatment. 2008 Dec 1;112(3):533-43. 
 
2. Riddler SA, Aga E, Bosch RJ, Bastow B, Bedison M, Vagratian D, Vaida F, Eron JJ, Gandhi RT, 
Mellors JW, ACTG A5276s Protocol Team. Continued Slow Decay of the Residual Plasma 
Viremia Level in HIV-1–Infected Adults Receiving Long-term Antiretroviral Therapy. The Journal 
of infectious diseases. 2015 Sep 2;213(4):556-60. 
 
3. Gandhi RT, McMahon DK, Bosch RJ, Lalama CM, Cyktor JC, Macatangay BJ, Rinaldo CR, 
Riddler SA, Hogg E, Godfrey C, Collier AC. Levels of HIV-1 persistence on antiretroviral therapy 
are not associated with markers of inflammation or activation. PLoS pathogens. 2017 Apr 
20;13(4):e1006285. 
 
4. Vaida F, Liu L. Fast implementation for normal mixed effects models with censored response. 
Journal of Computational and Graphical Statistics. 2009 Jan 1;18(4):797-817. 
 


Page | 14  
 
Appendix 1:  Analysis Timeline 
 
 
Redacted. 


Page | 15  
 
Appendix 2:  Analysis Implementation Plan (AIP) 
 
Redacted.