Statin Adjunctive Therapy for TB (StAT- TB)
Phase: PHASE2
Conditions: Tuberculosis
Sponsor: Johns Hopkins University

1 
StAT- TB (Statin Adjunctive Therapy for TB): A Phase 2b Dose-finding 
Study of Pravastatin in Adults with Tuberculosis 
U.S. Investigators:  
Petros Karakousis - Johns Hopkins University- Principal Investigator 
Richard Chaisson - Johns Hopkins University- Co-Investigator 
South Africa Investigators: 
Neil Martinson – PHRU Chris Hani Baragwanath Academic Hospital, Soweto, South Africa- 
Co-Investigator 
IND:  143350 
IND Sponsor:  DAIDS 
ES Number:  38558 
Version 3.0 
June 1  2019 
NCT: 03456102

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TABLE OF CONTENTS 
STUDY TEAM .................................................................................................................................................. 6 
SIGNATURE PAGE ........................................................................................................................................... 9 
1.  Abstract ................................................................................................................................................... 10 
2. Objectives ................................................................................................................................................ 12 
Primary Objective .................................................................................................................................................... 12 
3. Background .............................................................................................................................................. 12 
Statins: Lipid-modulating agents with pleiotropic effects ....................................................................................... 13 
Statins in the adjunctive treatment of infectious diseases ........................................................................................ 14 
Statins as adjunctive HDT for TB ............................................................................................................................ 14 
Screening of statins for in vivo testing based on cytotoxicity in macrophages ....................................................... 14 
Statin efficacy testing in macrophages ..................................................................................................................... 15 
Intra-macrophage concentrations of statins ............................................................................................................. 16 
Tolerability, pharmacokinetics (PK), and dose proportionality studies ................................................................... 16 
In vitro synergy .................................................................................................................................................... 16 
Tolerability and PK of statins in uninfected C3HeB/FeJ mice ............................................................................ 16 
Drug-drug interaction (DDI) and PK studies in mice .............................................................................................. 17 
Anti-TB activity of statin adjunctive therapy in mice .............................................................................................. 18 
Statin adjunctive therapy for TB in a mouse necrotic granuloma model of TB ...................................................... 19 
Statin adjunctive therapy for TB in mice with cell-mediated immune deficiency................................................... 20 
Potential modes of action of statins as adjunctive HDT against TB ........................................................................ 21 
Cholesterol-lowering effect .................................................................................................................................. 21 
Statin effects on cytokine expression ....................................................................................................................... 22 
Pravastatin adjunctive therapy favorably alters immune cell profiles in mouse lungs ........................................ 23 
Effects of statins on anti-TB functions of macrophages .......................................................................................... 23 
Rationale for conducting the StAT-TB clinical trial ................................................................................................ 24 
4. Study Procedures ..................................................................................................................................... 25 
Protocol Registration ................................................................................................................................................ 25 
Screening .................................................................................................................................................................. 25 
Day 1 (Baseline) ....................................................................................................................................................... 26 
Day 2 ........................................................................................................................................................................ 27 
Days 3-6 ................................................................................................................................................................... 27 
Day 7   (Week 1) ...................................................................................................................................................... 27 
Days 8-14 ................................................................................................................................................................. 27 
Day 15 (Week 2) ...................................................................................................................................................... 27 
Day 21 ...................................................................................................................................................................... 28 
Day 30 (Month 1) ..................................................................................................................................................... 28 
Unscheduled visit ..................................................................................................................................................... 28 

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TB treatment continuation........................................................................................................................................ 28 
Targeted symptom assessment ................................................................................................................................. 28 
Concomitant medications ......................................................................................................................................... 29 
Administration of study drug ................................................................................................................................... 29 
Obtaining sputum specimens ................................................................................................................................... 29 
Laboratory aspects of sputum analysis .................................................................................................................... 29 
Steady State Pharmacokinetic Assessments............................................................................................................. 30 
Procedures to maximize compliance with study visits ............................................................................................ 30 
Informed Consent ..................................................................................................................................................... 30 
Contraception ........................................................................................................................................................... 31 
Safety and Tolerability Endpoints ............................................................................................................................ 31 
Safety Evaluation ..................................................................................................................................................... 31 
5. Adverse Events ......................................................................................................................................... 32 
Definitions ................................................................................................................................................................ 32 
Relationship of AE to Study .................................................................................................................................... 32 
Reporting of Serious Adverse Events, and Unanticipated Problems ....................................................................... 33 
Association and Attribution/causality ...................................................................................................................... 33 
Severity* ................................................................................................................................................................... 34 
Reporting .................................................................................................................................................................. 34 
Pregnancy ................................................................................................................................................................. 35 
Clinical Management of Adverse Events ................................................................................................................. 36 
Liver toxicity ........................................................................................................................................................ 36 
Creatinine Kinase Elevation ................................................................................................................................. 36 
Myalgias and Myopathy ....................................................................................................................................... 36 
Other toxicities ..................................................................................................................................................... 37 
6. Blinding, including justification for blinding or not blinding the trial, if applicable. ............................... 38 
7. Justification of why participants will not receive routine care or will have current therapy stopped. .. 38 
8. Justification for inclusion of a placebo or non-treatment group. ........................................................... 38 
9. Definition of treatment failure or participant removal criteria. ............................................................. 38 
Description of what happens to participants receiving therapy when study ends or if a participant’s participation in 
the study ends prematurely....................................................................................................................................... 39 
10. Monitoring and data handling ............................................................................................................... 40 
Source Documents and Access to Source Documents ............................................................................................. 40 
Data Management Responsibilities .......................................................................................................................... 40 
Data Capture Methods.............................................................................................................................................. 40 
Study Records Retention .......................................................................................................................................... 41 
Protocol Deviations .................................................................................................................................................. 41 
Quality Management ................................................................................................................................................ 41 

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11. Inclusion/Exclusion Criteria ................................................................................................................... 41 
Inclusion criteria: ..................................................................................................................................................... 41 
Exclusion criteria: .................................................................................................................................................... 41 
12. Special Populations ................................................................................................................................ 42 
Children, Pregnant Women, and Breast-Feeding Women ....................................................................................... 42 
Prisoners ................................................................................................................................................................... 43 
HIV-Infected Individuals ......................................................................................................................................... 43 
Ethical Issues in Doing This Trial in South Africa .................................................................................................. 43 
Management of participants deemed ineligible ....................................................................................................... 43 
13. Drugs/ Substances/ Devices .................................................................................................................. 43 
Study drug descriptions ............................................................................................................................................ 43 
Pravastatin ................................................................................................................................................................ 44 
Pharmacokinetics ................................................................................................................................................. 44 
Distribution .......................................................................................................................................................... 44 
Metabolism ........................................................................................................................................................... 44 
Excretion .............................................................................................................................................................. 45 
Drug-drug interactions ......................................................................................................................................... 45 
Tolerability ........................................................................................................................................................... 45 
Dosage .................................................................................................................................................................. 46 
Rifampin ................................................................................................................................................................... 46 
Pharmacokinetics ................................................................................................................................................. 46 
Drug-drug interactions ......................................................................................................................................... 46 
Tolerability ........................................................................................................................................................... 46 
Dosage .................................................................................................................................................................. 47 
Isoniazid ................................................................................................................................................................... 47 
Pharmacokinetics ................................................................................................................................................. 47 
Drug-drug interactions ......................................................................................................................................... 47 
Toxicity ................................................................................................................................................................ 47 
Dosage .................................................................................................................................................................. 48 
Pyrazinamide ............................................................................................................................................................ 48 
Pharmacokinetics ................................................................................................................................................. 48 
Drug-drug interactions ......................................................................................................................................... 48 
Toxicity ................................................................................................................................................................ 48 
Dosage .................................................................................................................................................................. 48 
Ethambutol ............................................................................................................................................................... 48 
Pharmacokinetics ................................................................................................................................................. 49 
Drug-drug interactions ......................................................................................................................................... 49 
Toxicity ................................................................................................................................................................ 49 
Dosage .................................................................................................................................................................. 49 

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Pharmacy: Product Supply, Distribution, and Accountability ................................................................................. 49 
Study Product Accountability .............................................................................................................................. 50 
14. The rationale for choice of statin and dose ........................................................................................... 50 
15. Justification and safety information if FDA-approved drugs will be administered for non-FDA 
approved indications or if doses or routes of administration or participant populations are changed. .... 50 
16. Justification and safety information if non-FDA approved drugs without an IND will be administered.
 ..................................................................................................................................................................... 51 
17.  Study statistics ...................................................................................................................................... 51 
Primary outcome variable. ....................................................................................................................................... 51 
Secondary outcome variables ................................................................................................................................... 51 
18. Statistical Analysis Plan .......................................................................................................................... 51 
Hypothesis ................................................................................................................................................................ 51 
Sample size............................................................................................................................................................... 51 
Data Analysis ........................................................................................................................................................... 52 
Decision-making algorithm for pravastatin safety/PK study: .................................................................................. 52 
Decision-making algorithm for initiating new dosing cohorts ................................................................................ 53 
Recruitment strategy ................................................................................................................................................ 54 
19. Risks ....................................................................................................................................................... 54 
Institutional Review Board Involvement ................................................................................................................. 54 
Medical risks, listing all procedures, their major and minor risks and expected frequency. ................................... 54 
Steps taken to minimize the risks ............................................................................................................................. 55 
Plan for reporting unanticipated problems or study deviations. .............................................................................. 55 
Legal risks such as the risks that would be associated with breach of confidentiality. ........................................... 55 
Financial risks to the participants. ............................................................................................................................ 55 
20.  Benefits ................................................................................................................................................. 55 
21.  Payment and Re-numeration................................................................................................................ 55 
22. Costs ....................................................................................................................................................... 55 
References ................................................................................................................................................... 56 
Appendix 1 ................................................................................................................................................... 60 
 
 

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STUDY TEAM 
 
Principal Investigator, United States 
Petros C. Karakousis, MD 
Professor of Medicine, International Health 
Division of Infectious Diseases 
Johns Hopkins University Center for Tuberculosis Research 
1551 E. Jefferson St., Room 110 
Baltimore, MD 21231 
Phone 410-502-8233 
Fax 410-614-8173 
Email petros@jhmi.edu 
 
Co- Investigator, United States 
Richard E. Chaisson, MD 
Professor of Medicine and International Health 
Johns Hopkins University Center for Tuberculosis Research 
1550 Orleans Street, Room 1M-06 
Baltimore, MD 21231 
Phone 410-502-2717 
Fax 410-955-0740 
Email rchaiss@jhmi.edu 
 
Co-Investigator, Study Coordinator, United States 
Grace Barnes, BSN, MPH 
Johns Hopkins University Center for Tuberculosis Research 
1550 Orleans Street 
CRB-2, 1M 
Baltimore, MD 21287-0014 
Phone 410-614-3812 
Email glbarnes@jhmi.edu 
 
Co-Investigator, Biostatistician, United States/India 
Nikhil Gupte, MSc, MPhil, PhD 
Center for Global Health and Education, Johns Hopkins University School of Medicine & 
BJ Medical College and Sassoon General Hospitals 
Jai Prakash Narayan Road 
Pune, India 411001 
Phone +91 98220-70853 
Email ngupte1@jhmi.edu 
 
Co-Investigator, United States 
Maria L. Gennaro, MD 
Professor of Medicine 
Public Health Research Institute Center 
New Jersey Medical School - Rutgers, The State University of New Jersey 
225 Warren Street 
Newark, New Jersey 07103 
Phone: (973) 854-3210  
Email: gennarma@njms.rutgers.edu 
 

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Co-Investigator, United States  
Veronique DARTOIS 
Associate Professor 
Public Health Research Institute 
NJMS Rutgers University 
225 Warren St 
Rm RBL 2232 
Newark, NJ 07103 
973 854 3160 
Email veronique.dartois@rutgers.edu 
 
Co-Investigator, Scientific Study Coordinator, United States 
Noton K. Dutta, Ph.D. 
Research Associate, Medicine 
Division of Infectious Diseases 
Johns Hopkins University Center for Tuberculosis Research 
1553 E. Jefferson St., Bond St. Annex, Room 122 
Baltimore, MD 21231 
Phone 410-502-0023 
Fax 410-614-8173 
Email ndutta1@jhmi.edu 
 
 
Co-Investigator, United States 
Kelly E. Dooley, MD, PhD 
Associate Professor of Medicine, Pharmacology & Molecular Sciences 
Divisions of Clinical Pharmacology and Infectious Diseases 
Johns Hopkins University Center for Tuberculosis Research 
600 N. Wolfe Street, Osler 527 
Baltimore, MD 21287 
Phone 443-287-0517 
Fax 410-614-9978 
Email kdooley1@jhmi.edu 
 
Co-Investigator, United States 
William Checkley, MD, PhD     
Associate Professor of Medicine, International Health, and Biostatistics 
Associate Director, UMJT Fogarty Global Health Fellowship 
Division of Pulmonary and Critical Care 
Johns Hopkins University     
1800 Orleans Avenue, Suite 9121 
Baltimore, MD 21287                       
Telephone: (443) 287-8741 
Fax:  (410) 367-2014 
Email wcheck1@jhmi.edu; 
 
Co-Investigator, United States  
Todd T. Brown, MD, PhD 
Professor of Medicine and Epidemiology  
Division of Endocrinology, Diabetes, and Metabolism 
Johns Hopkins University 

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1830 East Monument Street, Suite 333 
Baltimore, MD 21287 
Phone 410-955-3921 
Fax 410-367-2042 
Email tbrown27@jhmi.edu 
 
Site Principal Investigator, Soweto, South Africa 
Neil Martinson, MD, MPH 
Perinatal HIV Research Unit 
University of Witwatersrand 
South Africa 
Phone +27 11 989 9836 
Fax +27 11 989 9862 
Email martinson@phru.co.za  
 
Medical Officer : 
Richard Hafner, MD  
Senior Scientific Officer, Tuberculosis Clinical Research Branch 
Division of AIDS, NIAID, NIH, DHHS  
5601 Fishers Lane Room 9E30, MSC 9830 
Rockville, MD 20852 (for express delivery 20852)  
Phone: 301 435 3766 
E-mail: rhafner@nih.gov  
 
Irene Rwakazina, PharmD, CCRP, RPh 
Pharmacist, Pharmaceutical Affairs Branch 
Office of Clinical Site Oversight (OCSO) 
Division of AIDS/NIAID/NIH 
5601 Fishers Lane 
Room 9E14  MSC 9830 
Rockville, MD 20852 
Office: +1-301-761-7269 
Fax: +1-240-627-3112 
irene.rwakazina@nih.gov 
 
 
Medical Officer 
Neha Shah, 
 5601 Fishers Ln, Rockville, MD 20852,  
Rm 9E39,  
Phone: 240-292-0384,  
E-Mail: neha.shah@nih.gov  

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SIGNATURE PAGE 
 
StAT- TB (Statin Adjunctive Therapy for TB): A Phase 2b Dose-finding Study of Pravastatin in 
Adults with Tuberculosis, Version 3.0, Dated June 1 2019  DAIDS-ES  38558 
 
 
 
I will conduct the study in accordance with the provisions of this protocol and all applicable protocol-
related documents. I agree to conduct this study in compliance with United States (US) Health and 
Human Service regulations (45 CFR 46); applicable U.S. Food and Drug Administration regulations; 
standards of the International Conference on Harmonization Guideline for Good Clinical Practice (E6); 
Institutional Review Board/Ethics Committee determinations; all applicable in-country, state, and local 
laws and regulations; and other applicable requirements (eg, US National Institutes of Health, Division of 
AIDS) and institutional policies. 
 
 
 
 
 
Principal Investigator: _____________________________________________________ 
 
 
 
 
 
Signed: _______________________________________________Date: _____________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1.  Abstract 
 
Protocol Synopsis 
Protocol Title: 
StAT- TB (Statin Adjunctive Therapy for TB): A Phase 2b Dose-finding 
Study of Pravastatin in Adults with Tuberculosis 
 
Treatment Indication: 
Pulmonary Tuberculosis (TB) 
Trial Objective: 
To assess the safety, tolerability, and pharmacokinetics of pravastatin 
adjunctive therapy when combined with the standard TB treatment regimen. 
 
Trial Design: 
Phase IIB clinical trial: A two-week safety/PK study to determine pravastatin  
exposures over 24 hours when given together with first-line treatment (HRZE) 
and ensure the combination is safe and well-tolerated.  
 
Patient Population: 
Up to 35 evaluable participants aged 18 years or older will be enrolled. 
Treatment arms: 
 
5-10 participants per arm will be sequentially recruited to receive one of the 
following: 
Arm 1: Pravastatin 40 mg and Rifafour (fixed-dose combination of isoniazid, 
rifampin, pyrazinamide, and ethambutol) daily for 14 days (pravastatin will 
be given alone on Day 1, and pravastatin + Rifafour will be given on Days 2-
15) (5 participants if well tolerated) 
Arm 2:  Pravastatin 80 mg and Rifafour (fixed-dose combination of 
isoniazid, rifampin, pyrazinamide, and ethambutol) daily for 14 days 
(pravastatin will be given alone on Day 1, and pravastatin + Rifafour will be 
given on Days 2-15) (10 participants) 
Arm 3 will only be recruited if pravastatin 80 mg is well tolerated and safe, 
yet drug exposures are significantly reduced due to the known interaction 
with rifampin. 
Arm 3:  Pravastatin 120 mg, and Rifafour (fixed-dose combination of 
isoniazid, rifampin , pyrazinamide, and ethambutol) daily for 14 days 
(pravastatin will be given alone on Day 1, and pravastatin +, Rifafour will be 
given on Days 2-15) (10 participants) 
Arm 4 will only be recruited if pravastatin 120 mg is well tolerated and safe, 
yet drug exposures are significantly reduced due to the known interaction 
with rifampin. 
Arm 4:  Pravastatin 160 mg, and Rifafour (fixed-dose combination of 
isoniazid, rifampin , pyrazinamide, and ethambutol) daily for 14 days 
(pravastatin will be given alone on Day 1, and pravastatin + Rifafour will be 
given on Days 2-15) (10 participants) 
 
Vitamin B6 will be added to each of the regimens.  

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Criteria for evaluation: 
Primary Endpoint:  
 
Safety and tolerability of pravastatin co-administered with rifampin yielding 24-hour drug exposures expected 
with the recommended high intensity dose for lowering cholesterol (80 mg daily). 
 
Study site:  Study participants will be recruited from clinics in Soweto, South Africa by the study team at the 
PHRU, Chris Hani Baragwanath Academic Hospital, Soweto, South Africa.   
Study duration: 
It is estimated that 6 months will be required for recruitment and enrolment. The duration of participation for 
each study participant is 30 days; 14 days (two weeks) of experimental treatment with one follow-up visit at 30 
days (four weeks).  
  
 
 
 

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Study Scheme 
 
 
2. Objectives 
 
Primary Objective 
 
To assess the safety, tolerability, and pharmacokinetics of pravastatin adjunctive therapy when combined 
with the standard TB treatment regimen. 
 
Our findings will inform the selection of the appropriate dose for an anticipated larger follow-up study, in 
which the TB treatment-shortening potential and immunomodulatory effects of pravastatin adjunctive 
therapy will be assessed. 
 
 
3. Background  
 
Despite more than a century of research, tuberculosis (TB) kills millions of people every year. Multidrug-
resistant and extensively drug-resistant (MDR and XDR) strains of Mycobacterium tuberculosis (Mtb) are 
on the rise (1, 2), and the scientific consensus seems to be that the discovery of new antibiotics is not the 
only solution to this threat (3-5). A recent study (6) indicated that the number of new “druggable” targets 
in pathogens is relatively small and that almost all of these targets belong to evolutionarily conserved 
metabolic pathways that are already inhibited by current antibiotics. This could in part explain why so few 
new classes of antibiotics have been developed in the past decades (3) and why only one new drug against 
TB has been approved by the Food and Drug Administration during the last 40 years (7). 
 
Many host factors can trigger clinical disease in a small proportion of individuals latently infected with Mtb 
(8). Although the immune system is responsible for clearing the invading bacilli, Mtb has evolved various 

13 
 
strategies to evade host defense mechanisms (9, 10), which often result in excessive inflammation, tissue 
destruction and morbidity (11). These observations have led to the concept that host immunity may be 
manipulated through host-directed therapy (HDT) (12-15) to augment bacillary killing while minimizing 
tissue inflammation in response to conventional anti-TB drugs, thereby improving clinical outcomes. 
 
Statins (HMG-CoA reductase inhibitors) are among the most promising candidates as adjuvant, host-
directed therapy (HDT) against TB (16). We propose to test statins in combination with first-line TB drugs 
as adjunctive therapy aimed at optimizing, in a graduated and safe manner, the host’s treatment response to 
TB disease. Our study is motivated by several lines of investigation. First, several studies have shown that 
statins have anti-TB properties. We found that statin adjunctive therapy enhances the first-line anti-TB 
regimen’s antimicrobial activity and shortens the time required to achieve cure in a standard mouse model 
of chronic TB infection ((17, 18) and unpublished data). Recently, we found that 8 weeks of adjunctive 
therapy with simvastatin or pravastatin significantly reduced lung bacterial burden in Mtb-infected 
C3Heb/FeJ mice, which develop necrotic lung granulomas histologically resembling their human 
counterparts(19-21). Parihar et al. showed that peripheral blood mononuclear cells (PBMCs) and monocyte-
derived macrophages from patients with familial hypercholesterolemia receiving statin therapy were more 
effective in controlling Mtb growth compared with those of healthy donors, and statin therapy protected 
mice against TB-induced pathology (22). In addition, statins are synergistic with the key sterilizing drug, 
rifampin (23). Although controversial (24), recent data from observational studies suggest that statin use 
among patients with type 2 diabetes mellitus may reduce the frequency of incident TB cases (25, 26). 
Second, statins are among the most widely used drugs worldwide, and their safety profile is well 
documented and highly favorable (27). Third, statins and antiretroviral drugs can be co-administered (with 
drug and dose selection) (28), so patients with HIV infection on antiretroviral treatment (ART) may also 
benefit. Fourth, statins are attractive adjunctive agents for TB treatment due to the widespread availability 
of less costly generics. This is particularly important for TB, which is endemic primarily in resource-limited 
settings. 
 
Statins: Lipid-modulating agents with pleiotropic effects 
 
Statins are a class of drugs used to lower cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl 
coenzyme A ( HMG-CoA) reductase, a crucial enzyme in the cholesterol biosynthesis pathway. In addition, 
statins possess important immunomodulatory and inflammatory properties, which reduce mortality and 
multiple organ dysfunction in patients with bacteremia, the risk of lung cancer, and even virus replication 
(29-32). 
Currently, 
a number 
of 
statins are 
on 
the 
market, 
including atorvastatin, 
fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin. 
 
Recent reports suggest that statin effects are not limited to cardiovascular diseases, and that these drugs 
may confer additional benefits in patients with osteoporosis, Alzheimer disease, rheumatoid arthritis, acute 
lung injury, and COPD. These pleiotropic effects of statins may be mediated by: 
 
(i) 
Reducing the stability of lipid raft formation with subsequent effects on immune activation 
and regulation, and preventing the prenylation of signaling molecules with subsequent 
down-regulation of gene expression (33). 
(ii) 
Endothelial cell (EC) cytoskeletal rearrangement, NADPH oxidase, and nitric oxide 
activity, as well as effects on differential EC gene expression, are relevant to the 
pathobiology of acute lung injury (34). 
(iii) 
Inhibition of isoprenoid synthesis, which leads to the inhibition of intracellular signaling 
molecules Rho, Rac and Cdc42 (35).  
(iv) 
Altering the gene expression and function of cells of both the innate and adaptive immune 
systems, including endothelial cells, macrophages, dendritic cells and T cells (36)  

14 
 
(v) 
Inhibition of IκB degradation, MMP-9 activity, TNF-α production, and cell spreading, and 
by the upregulation of tetraspanins, especially CD9 (37) 
(vi) 
Activation of sirtuins (38, 39), leading to induction of autophagy 
(vii) 
Induction of HO-1-mediated processes (40) 
(viii) 
Inhibition of TGF-β (41, 42) 
 
Statins in the adjunctive treatment of infectious diseases 
 
Statin treatment improves the outcome of various infections. Statin treatment is associated with a 41% 
reduction in 30-day mortality in patients hospitalized with laboratory-confirmed influenza (43). Statins also 
reduce mortality in patients with bacteremia and multiple organ dysfunction (44, 45) and kill Chlamydia 
pneumoniae and Salmonella enterica within macrophages and in animal models of infection (46, 47). The 
use of statins was strongly and independently associated with a reduction in the risk of sepsis events in 
patients who were receiving dialysis due to chronic kidney disease (48). Prior statin therapy reduces the 
incidence of community-acquired pneumonia (CAP) (49, 50) and increases the survival of patients with 
CAP (51). 
 
Although the mechanism underlying the antimicrobial activity of statins remains unknown, recent reports 
indicate there may be multiple mechanisms of action (52), including reduced release of pro-inflammatory 
cytokines from mononuclear cells or monocytes (53). 
 
Statins as adjunctive HDT for TB 
 
Following infection by Mtb, macrophages accumulate lipid bodies, acquiring a “foamy” phenotype through 
an ESAT-6-dependent pathway (54). Although their precise role remains poorly characterized, these lipid 
bodies may serve as a food source for intracellular 
bacilli. In addition, their accumulation has been 
associated with restriction of Mtb growth, and 
phenotypic tolerance to front-line drugs (55). 
 
Cholesterol biosynthesis and transport may play a 
crucial role in the adaptation of Mtb within host 
tissues (56, 57) and its inhibition by statins could 
potentially alter protective immunity, thus altering 
disease outcome in the infected host (22). In ex vivo 
studies, statins reduced the accumulation of lipid 
droplets (22, 58), as well as the Mtb burden in 
human macrophages and in mice by enhancing 
phagosome maturation and autophagy (22). The 
process of autophagy is important for control of Mtb 
growth in vivo, as well as for preventing excessive inflammation in the host (59). Previous reports have 
highlighted the importance of autophagy for the full activity of isoniazid and pyrazinamide against 
intracellular Mtb through a mechanism based on release of reactive oxygen species (ROS) by host cells 
(mitochondria and NADPH oxidase) (60, 61). A recent study demonstrated the anti-TB activity of statins 
in the lungs of Mtb-infected mice (54). We recently showed simvastatin increases the bactericidal activity 
of the first line regimen against chronic TB in mice (62) and reduces the duration of curative treatment in 
this model (18).  
 
Screening of statins for in vivo testing based on cytotoxicity in macrophages 
 
Table 1. Statin cytotoxicity in macrophages 

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We showed that, although statins lack direct antimicrobial activity, they promote intracellular Mtb killing 
in vitro (17). Others have shown that statins enhance macrophage killing of Mtb by promoting phagosomal 
maturation and autophagy (22). Moreover, simvastatin and atorvastatin increase rifampin-mediated killing 
of Mtb in macrophages (23). 
 
For seven marketed statins and mevastatin (one of the original 
statins, which was never marketed), we first studied the cytotoxicity 
in THP-1 and Vero cells. Next, we characterized: (1) in vitro 
potency against intracellular Mtb in THP-1 cells and in murine 
macrophages, with and without companion anti-TB 
drugs, and (2) in vivo PK, drug-drug interactions and 
tolerability in mice.  
 
To determine the 50% cytotoxic concentration (CC50) of 
each statin, human monocytic THP-1 (ATCC TIB-202) 
and Vero cells (ATCC CCL-81) were treated with a range 
of doses of each statin (with lowest dose corresponding 
to the inhibitory dose (IC50) reported in the literature) 
and toxicity was measured at 24 hours post-treatment. 
The results are summarized in Table 1 (drugs are listed 
according to decreasing toxicity in THP-1 cells). 
Pravastatin was fpund to be the least toxic statin in this 
assay. 
 
Statin efficacy testing in macrophages 
 
 
 In order to determine the activity of statins against 
intracellular bacilli, we used a bioluminescent Mtb 
strain expressing luciferase to infect THP-1 cells. The 
luciferase strain was used to monitor growth of 
mycobacteria in real-time using a luminometer by 
measuring the relative light units (RLU), which serves 
as a reliable surrogate for colony-forming units (CFU) 
(16). The concentration of statin required to reduce 
bacterial colony counts by 50% after 6 days of exposure 
was defined as the effective concentration 50% (EC50). 
At non-toxic doses, atorvastatin and mevastatin were 
found to have no Mtb growth inhibition (not shown), 
while rosuvastatin, pitavastatin and lovastatin gave 
<50% inhibition. EC50 values could be obtained only 
for simvastatin, pravastatin and fluvastatin (Figure 1 
and Table 2).  
 
 
Table 2: Statin EC50 values 
Simvastatin 
0.2 μM 
Pravastatin 
7.8 μM 
Fluvastatin 
0.032 μM 
Figure 1: M. tuberculosis H37Rv-lux growth in 
THP-1 cells following 6 days of exposure to statins 
at the EC50 concentration.   
Figure 2. Ratio of intracellular to extracellular 
concentration (I/E) of statins.  

16 
 
Intra-macrophage concentrations of statins 
 
In order to determine whether cytotoxicity or anti-
TB activity of individual statins was associated with 
their intracellular accumulation, we studied their 
concentration inside THP-1 cells by liquid 
chromatography/tandem mass spectroscopy (LC-
MS/MS) (18) following 30-minute exposure to the 
following concentrations of statins:  
 
5x EC50 for the three statins in Table 2; for the 
remaining 
compounds 
we 
used 
5-fold 
the 
concentration 
giving 
~30-35% 
inhibition 
(atorvastatin, 24 μM; lovastatin, 4.35 μM; 
mevastatin, 0.47 μM; pitavastatin, 0.3 μM); 
rosuvastatin, 5.75 μM). Our data suggest that the 
relatively low CC50 of lovastatin and mevastatin 
may be related to their relatively high intracellular 
accumulation, while pravastatin, which showed a 
highly favorable CC50/EC50 ratio (161.3) had the 
lowest intracellular accumulation of the 8 statins tested (Figure 2). 
 
Tolerability, pharmacokinetics (PK), and dose proportionality studies 
 
In vitro synergy 
 
In order to determine whether statins can enhance the activity of anti-TB drugs against intracellular bacilli, 
we used the RLU assay described above. We first determined the EC50 of each of the three first-line drugs 
separately and then in combination. When we tested the 3-drug combination (each drug dosed at the 
EC50—see Figure 3 legend) alone or with simvastatin, pravastatin or fluvastatin (each tested at the 
respective EC50—see Table 2), we observed an additive effect of each of these three statins with the 3-
drug combination (Figure 3).   
 
Tolerability and PK of statins in uninfected C3HeB/FeJ mice 
 
Mtb residing in necrotic mouse lung lesions may be more akin to persisters in human lesions with reduced 
response to direct-acting anti-TB drugs; further these areas represent relative pharmacological sanctuaries. 
Because of these favorable features, we and other groups have begun to use C3HeB/FeJ mice to test the 
efficacy of various antitubercular regimens and novel anti-inflammatory therapies (21, 63-66). In steady-
state studies, separate groups of mice were given one of the 7 statins (mevastatin was not tested due to 
cytotoxicity) orally at a dose of 90 mg/kg together with isoniazid 10 mg/kg, rifampin 10 mg/kg, 
pyrazinamide 150 mg/kg and ethambutol 100mg/kg (HRZE refers to the 4-drug combination) for 5 
successive days. Rifampin doses were separated from the accompanying drugs by 1 hour to minimize drug–
drug interactions at the absorption stage (67, 68). In single-dose studies, mice were given one dose of 
simvastatin alone (20 mg/kg, 60 mg/kg and 90 mg/kg) or pravastatin (90 mg/kg and 180 mg/kg). Plasma 
drug concentrations were measured by LC-MS/MS. 
 
Each statin given at 90 mg/kg was well tolerated for 5 successive treatments. To select a mouse dose for 
efficacy studies, we aimed to reproduce the human AUC at the highest clinically licensed dose, and twice 
that dose for selected compounds exhibiting the most attractive cytotoxicity profile and anti-TB activity in 
 
Figure 3: In vitro synergy of statin with the 
combination treatment of rifampin (0.0055 μM), 
isoniazid (0.006 μM) and pyrazinamide (81.23 μM) 
(RHZ). SIM (simvastatin), PRAVA (pravastatin) and 
FLUVA (fluvastatin). The statin concentrations are 
shown in Table 2. 

17 
 
macrophages (simvastatin and pravastatin). In the case of inactive prodrugs for which the acid conversion 
product is the active molecule (simvastatin and lovastatin), we matched the human AUC of the acid. Note 
that for most statins, matching the AUC resulted in Cmax significantly higher than in humans (Table 3). 
 
Table 3. Comparison of statin pharmacokinetics in mice and humans 
MOUSE 
HUMAN 
Active drug dose 
AUC[0-2] Cmax (SD) 
Highest 
licensed 
dose 
AUC[0-24]  at  
highest licensed 
 dose 
Cmax at highest  
licensed dose 
Liabilities/ 
characteristics 
 
mg/kg ng*h/ml 
ng/ml 
mg 
ng*h/mL  
(SD) 
ng/mL (SD) 
 
Atorvastatin 90 
1,464 
2,131  
(3,610) 
80 
630-650 
81-84 
 
Fluvastatin 
90 
6,028 
4,201  
(1,090) 
80 
1400 
1000 
CYP2C9  
mainly 
Lovastatin 
-OH 
90 
1,081 
967 (896) 
80 
75-150 
18-25 
Prodrug 
Pitavastatin 
90 
3,398 
2,110 (449) 
4 
269 
 
 
Pravastatin 
90 
1,391 
1,959  
(2,498) 
80 
96 
 (40 mg) 
45-55  
(40 mg) 
 
Rosuvastatin 90 
7,704 
13,993 (8,383) 
40 
300 
40 
 
Simvastatin 
90 
SIM BLQ SIM BLQ 
80 
SIM 94 
SIM 18 
CYP3A substrate, prodrug 
Simvastatin- 
OH 
SIM-OH  
240 
SIM-OH 310 
SIM-OH 63  
(63) 
SIM-OH 6 (7) 
 
 
Drug-drug interaction (DDI) and PK studies in mice 
 
Significant effects of rifampin co-administration on simvastatin or pravastatin exposures were not observed 
in mice (data not shown), indicating that the standard mouse model does not recapitulate the known drug-
drug interactions observed between rifampin and simvastatin and pravastatin seen in humans (69, 70). 
Therefore, the mouse PK interaction data were not used directly as a criterion for selecting pravastatin doses 
for the Stage I study in the UH3 phase.  
 
On the basis of their favorable profiles (EC50, CC50, PK parameters), simvastatin and pravastatin were the 
2 statins selected to progress to dose-response efficacy studies in mice. 

18 
 
 
Anti-TB activity of statin adjunctive therapy 
in mice 
 
We showed previously that statins lack direct 
antimicrobial 
activity 
but 
promote 
intracellular Mtb killing in vitro (17). Others 
have 
suggested 
that 
statins 
enhance 
macrophage killing of Mtb by promoting 
phagosomal maturation and autophagy (22). 
Moreover, 
simvastatin 
and 
atorvastatin 
increase rifampin-mediated killing of Mtb in 
macrophages (23). This potential synergy 
between statins and the key sterilizing anti-TB 
drug may help explain our in vivo findings. 
 
We studied the role of adjunctive treatment 
with simvastatin 60 mg/kg on the time required to achieve stable cure in Mtb-infected BALB mice. After 
6 weeks of treatment, simvastatin adjunctive therapy led to a 1.4 log10 greater reduction in lung bacillary 
counts relative to the standard regimen alone (rifampin/isoniazid/pyrazinamide, RHZ) given at human-
equivalent doses (Figure 4). The addition of simvastatin shortened the time required to achieve lung culture 
negativity from 4.5 months to 3.5 months. After 3.5 months of treatment relapse rates were 50% (5/10 mice) 
and 20% (2/10 mice) in the RHZ and RHZ + simvastatin groups, respectively. No relapses were observed 
in either group after 4.5 months of treatment. These encouraging results suggest that adjunctive statin 
treatment may help to reduce the duration of treatment required to cure TB. 
 
Next, we conducted a preclinical study aimed at comparing the bactericidal activities of the standard TB 
regimen (rifampin, isoniazid, pyrazinamide and ethambutol; RHZE) with or without escalating doses of 
pravastatin against chronic TB in BALB/c mice. Antibiotics were given five times weekly for 8 weeks 
(continuous phase) beginning 6 weeks after infection.  
 
Treatment with RHZE plus pravastatin at doses ranging from 30 to 180 mg/kg demonstrated a dose-
dependent increase in bactericidal activity, reducing lung bacillary counts by 0.2–0.6 log10, 0.3–0.6 log10 
and 0.3–0.8 log10 compared to RHZE alone at weeks 2, 4 and 8, respectively (Figure 5).  
 
 Figure 4: Adjunctive treatment with simvastatin reduces
the time required to achieve culture-negative lungs in Mtb-
infected BALB/c mice. RHZ= rifampin 10 mg/kg+ isoniazid 
10 mg/kg + pyrazinamide 150 mg/kg. Sim= simvastatin 60 
mg/kg. CFU= colony-forming units. 

19 
 
 
 
After 8 weeks of treatment, the degree of lung inflammation correlated with the bactericidal activity of each 
drug regmen (Figure 6)  
 
 
Statin adjunctive therapy for TB in a mouse necrotic granuloma 
model of TB 
 
Mtb residing in necrotic mouse lung lesions may be more akin to 
persisters in human lesions with reduced response to direct-acting 
anti-TB drugs; further these areas represent relative pharmacological 
sanctuaries. Because of these favorable features, we and other groups 
have begun to use C3HeB/FeJ mice to test the efficacy of various 
antitubercular regimens and novel anti-inflammatory therapies (21, 
63-66). C3HeB/FeJ mice were infected with ~50 bacilli of Mtb 
H37Rv and 6 weeks later were treated with one of the following 
regimens: 1) No treatment (negative control); 2) Human-equivalent 
doses of the first-line regimen (RHZE) (positive control); 3) RHZE + 
simvastatin 90 mg/kg; 4) RHZE + pravastatin 50 mg/kg; 5) RHZE + 
pravastatin 90 mg/kg. The pravastatin 50 mg/kg dose was selected in 
Figure 6: Lung inflammation 8 weeks after initiation of treatment. At least one entire H&E-stained cross section per 
animal lung (5animals/group) was analyzed for degree of inflammation. The surface area occupied by granulomatous 
inflammation was determined by ImageJ software-based morphometry of digitized images of lung sections and results 
are represented as percentage of lung surface area involved. 
Figure 5: Adjunctive treatment with pravastatin augments bactericidal activity of the first-line TB regimen in  
BALB/c mice. RHZE= rifampin 10 mg/kg+ isoniazid 10 mg/kg + pyrazinamide 150 mg/kg+ Ethambutol 100 mg/kg. P=Prav
CFU= colony-forming units. . *p<0.05, **p<0.01, ***p<0.001 
W2 
W4 
W8 
Figure. 7. Adjunctive anti-TB activity 
of pravastatin and simvastatin in 
C3HeB/FeJ mice. Data represent 
log10 colony-forming units (cfu)/lung 
on week 8 after initiation of treatment. 
Prava= pravastatin; SIM= simvastatin. 

20 
 
this study to match the 24-hr AUC achieved with a human dose of pravastatin 80 mg. After 8 weeks of 
treatment, mice receiving statin adjunctive therapy had significantly reduced lung bacillary burdens relative 
to control mice receiving RHZE alone. Thus, relative to the control regimen, adjunctive therapy with 
simvastatin 90 mg/kg, pravastatin 90 mg/kg, and pravastatin 50 mg/kg further reduced lung cfu by 1.28 
log10 (p< 0.0001), 1.16 log10 (p< 0.01), and 0.78 log10 (p< 0.05), respectively (Figure 7).  
 
Statin adjunctive therapy for TB in mice with cell-mediated immune deficiency 
 
In order to determine the contribution of cellular 
immunity to the anti-TB effect of statins, we tested the 
activity of pravastatin in combination with the first-
line anti-TB regimen in Mtb-infected athymic NU/NU 
(nude) mice (71). 
 
Nude mice were aerosol-infected with Mtb H37Rv and 
treatment was initiated 4 weeks later with RHZE or 
RHZE + pravastatin 90 mg/kg (P90) or pravastatin 180 
mg/kg (P180) administered once daily 5 days a week 
(5/7) for a total of 8 weeks. As expected, nude mice 
treated with RHZE showed significantly reduced lung CFU counts compared to the untreated control group. 
There is a trend toward reduced lung CFU in the RHZE+ P90 or P180 groups to RHZE alone, but this is 
not statistically significant (Figure 8). Thus, one may speculate the lack of bactericidal effect of statins in 
nude mice is probably due to deficient adaptive immunity (72), which may be required for proper activation 
of macrophages.  
Figure 8. Adjunctive treatment with pravastatin 
(P) in Mtb-infected NU/NU mice. 

21 
 
Potential modes of action of statins as adjunctive HDT against TB 
The target enzyme of statins is HMG-CoA reductase, which catalyzes the conversion of HMG-CoA into 
mevalonate, a rate-limiting step in the cholesterol biosynthesis pathway (73). The mevalonate pathway is a 
multi-branched pathway leading to the biosynthesis of cholesterol, lipid hormones, including vitamin D, 
isoprenoids, 
and 
isoprenoid 
intermediates, 
such 
as 
farnesylpyrophosphate 
(FPP) 
and 
geranylgeranylpyrophosphate (GGPP), which are required for protein prenylation. The prenylation state of 
proteins, including the Rho, Rac and Ras protein families (74), results in their subcellular redistribution and 
membrane anchoring. In particular, Rac transduction signaling modulates generation of reactive oxygen 
species (ROS), which is an important 
mechanism by which macrophages 
control Mtb infection (75). Given the 
multiple functions of the mevalonate 
pathway, 
statins 
possess 
anti-
inflammatory activities in addition to 
their 
well-known 
lipid-lowering 
properties. 
For 
example, 
statins 
reduce 
expression 
of 
pro-
inflammatory 
chemokines 
and 
cytokines in animal models of 
atherosclerosis and in patients with 
rheumatic diseases (76-81). Indeed, 
the cardioprotective effect of statins is likely related to their immunomodulatory, rather than lipid-lowering, 
properties (74). In addition, statins appear to activate peroxisome proliferator-activated receptors (PPAR) -
α and –γ in inflammatory cells and other cell types (82). PPAR-γ regulates lipid uptake, adipogenesis, and 
glucose metabolism, and 
has been implicated in 
the pathology of obesity, 
diabetes, atherosclerosis, 
and cancer (83). 
 
Cholesterol-lowering 
effect 
It has been postulated 
that the lipid-lowering 
properties of statins may 
play a role in their anti-
TB 
activity. 
Mtb 
infection 
induces 
intracellular 
lipid 
accumulation 
in 
macrophages in the form 
of lipid droplets (LD), 
quasi-organelles 
consisting of cholesterol 
esters and triglycerides 
surrounded 
by 
a 
phospholipid monolayer 
(84). 
Lipid-laden 
macrophages acquire a 
“foamy” 
phenotype, 
which is associated with 
Figure 10. Changes in immune cell profiles in the lungs following TB treatment and 
adjunctive statin therapy. RHZE plus pravastatin 90 mg/kg increased the % of lung 
alveolar macrophages (A), decreased TNFα in alveolar macrophages (B), and 
increased the ratio of CD4+/CD8+ T cells in Mtb-infected lungs (C).  
# p<0.05 compared to no Tx (untreated). * p<0.05 compared to RHZE. 
Table 4. Genes differentially regulated after treatment with 
pravastatin with and without RHZE 

22 
 
delayed phagolysosomal maturation, enhanced IL-10 induction, alternative macrophage polarization, and 
blunting of innate immunity (85). Although their precise role remains poorly characterized, LD may serve 
as a food source for intracellular bacilli (85). In addition, their accumulation has been associated with 
slowed Mtb division and phenotypic tolerance to front-line drugs (55). Therefore, foamy macrophages 
appear to represent a stable reservoir for Mtb, facilitating persistence of the organism in the host.  
 
We have shown that there is a correlation between the antimycobacterial and lipid-lowering activities of 
statins, since statin treatment of Mtb-infected macrophages reduces bacterial burden, LD formation, and 
intracellular cholesterol levels (17). We found that the antimicrobial effect of statins is reproduced by 
treatment with inhibitors of 7-dehydrocholesterol reductase (DHCR7, an enzyme required for cholesterol 
biosynthesis), but not with inhibitors of prenylation enzymes (FPP transferase and type I and II GGPP-
transferases). However, prenylation-associated effects of statins cannot be excluded, since the inhibitory 
effects of statins are partially reduced by addition of GGPP (22).  
 
In order to determine if the anti-TB activity of statins correlated with their cholesterol-lowering properties 
in vivo, we measured plasma total cholesterol and low-density lipoprotein (LDL), as well as the content of 
cholesterol and cholesterol esters in lung lesions using MALDI-MSI, following adjunctive therapy of Mtb-
infected mice with  simvastatin 60 mg/kg. No significant differences were observed between the control 
group and simvastatin arm with respect to cholesterol levels in plasma or lung lesions (data not shown). 
These results suggest that cholesterol depletion is unlikely to be the major mechanism by which simvastatin 
potentiates anti-TB drugs, although an effect on intracellular cholesterol levels cannot be excluded. 
 
Statin effects on cytokine expression 
 
In order to gain insight into the anti-TB mechanism of action of statins in vivo, the lungs of mice treated 
with pravastatin adjunctive therapy and those treated with RHZE alone were analyzed by whole-genome 
microarrays and RT-PCR. Mouse sera were studied by multiplex enzyme-linked immunosorbent assays.  
 
Treatment with pravastatin had a profound effect on the global transcription in mouse lungs (Table 4).  
Major pathways that were altered after statin treatment included the following: differential regulation of 
cytokine production in macrophages and T helper cells by IL-17A and IL-17F, the role of JAK family kinases 
in IL-6-type cytokine signaling pathway, and hematopoiesis from pluripotent stem cells. 
Interestingly, global gene expression profiling of the lungs following Mtb infection revealed increased 
expression of IL-6, and increased IL-6 levels were detected by ELISA in the serum of Mtb-infected mice 
when compared with uninfected controls. RHZE reduced IL-6 gene expression in mouse lungs and 
Figure 9. Pravastatin adjunctive therapy for tuberculosis reduces IL-6 in the lungs and serum of mice 

23 
 
circulating IL-6 (by ELISA) in the serum relative to infected untreated controls (Figure 9). Pravastatin 
adjunctive therapy further reduced IL-6 expression in the mouse lungs and serum relative to RHZE controls. 
 
Pravastatin adjunctive therapy favorably alters immune cell profiles in mouse lungs 
 
Female BALB/c mice were aerosol-infected with Mtb H37Rv and, 6 weeks after infection, were randomized 
to receive not treatment, human-equivalent doses of the standard firs-line regimen (RHZE), or RHZE + 
 pravastatin 90 mg/kg once daily for 28 days. After harvesting the lungs, the intrapulmonary lymphocyte and 
macrophage populations were measured by FACS. RHZE treatment was found to restore the ratio of alveolar 
macrophage (AM) to interstitial macrophage (IM) relative to uninfected (naïve) mice. RHZE plus pravastatin 
90mg/kg further increased the ratio of AM to IM (Fig. 10A). Using intracellular cytokine staining, we found 
that TNFα levels were significantly lower in AM of mice treated with RHZE and pravastatin 90 mg/kg 
compared to those of control mice or those treated with the control regimen (Fig. 10B). Similar to the 
findings with AM, RHZE treatment restored the ratio of CD4+ to CD8+ T cells in mouse lungs and adjunctive 
therapy with pravastatin 90 mg/kg further increased the ratio of CD4+ to CD8+ T cells (Fig. 10C). 
 
Effects of statins on anti-TB functions of macrophages  
 
Treatment of macrophages with 100 nM simvastatin inhibits Mtb growth and supplementation with 
mevalonate was able to partially overcome the anti-TB effect, suggesting that statin treatment was not 
exerting an off-target effect (Figure 11A). Moreover, we found that exposure of primary murine 
macrophages to increasing concentrations of simvastatin resulted in phagosome maturation in a dose-
dependent manner. Two parameters of phagosome maturation were tested: proteolytic capacity (Figure 
11B) and acidification (Figure 11C). Recently, we found that pravastatin 125 μM similarly promotes 
phagosome-lysosome fusion in THP-1 cells (data not shown). The mechanism underlying the statins’ 
antimycobacterial effect and how it links with the observed phagosome/lysosome fusion is being actively 
investigated. One step is to determine whether the antibacterial activity of the statin can be assigned to a 
branch of the mevalonate pathway. Our preliminary results rule out a role for farnesylation and type I 
geranylgeranylation (data not shown). Under current investigation is the role of cholesterol biosynthesis 
and of the geranylgeranylation type II. The latter is promising because Rab GTPases (which are prenylated 
by GGT type II) are involved in phagosome maturation and vesicle trafficking. The effects of statins on 
other immune mediators affecting phagosome/lysosome fusion are also being investigated.  
 

24 
 
 
 
 
 
 
 
 
 
Rationale for conducting the StAT-TB clinical trial 
 
Our clinical study to test the adjunctive anti-TB activity of statins is motivated by several lines of 
investigation. First, several studies have shown that statins have anti-TB properties. We found that statin 
adjunctive therapy enhances the first-line anti-TB regimen’s antimicrobial activity and shortens the time 
required to achieve cure in a standard mouse model of chronic TB infection ((17, 18) and unpublished data). 
Recently, we found that 8 weeks of adjunctive therapy with simvastatin or pravastatin significantly reduced 
lung bacterial burden in the highly clinically relevant C3Heb/FeJ mouse model of chronic TB, which 
develops human-like, necrotic TB lung granulomas(19-21). Parihar et al. showed that peripheral blood 
mononuclear cells and monocyte-derived macrophages from patients with familial hypercholesterolemia 
receiving statin therapy were more effective in controlling Mtb growth compared with those of healthy 
donors, through induction of autophagy and promotion of phagosome maturation (22). In addition, statins 
are synergistic with the key sterilizing drug, rifampin (23). Recent data from observational studies suggest 
that statin use among patients with type 2 diabetes mellitus may reduce the frequency of incident TB cases 
(25, 26). Second, statins have immune-modulatory properties and statin use improves mortality in patients 
with sepsis (44, 45) or with community-acquired pneumonia (51). Several studies have shown that statins 
reduce lung inflammation in the mouse model of chronic TB (22) (and unpublished data). Our data suggest 
that these effects may be mediated by modulation of lung cytokine expression, including reduction of levels 
of IL-6 and TNFα, and an increase in the ratio of CD4+/CD8+ T cells in the lungs of Mtb-infected mice. 
Third, statins are among the most widely used drugs worldwide, and their safety profile is well documented 
and highly favorable (27). Fourth, statins and antiretroviral drugs can be co-administered (with drug and 
dose selection) (28), so patients with HIV infection on antiretroviral treatment (ART) may also benefit. 
Fifth, statins are attractive adjunctive agents for TB treatment due to the widespread availability of less 
costly generics. This is particularly important for TB, which is endemic primarily in resource-limited 
settings. 
 
Of 8 statins tested, pravastatin was selected for testing in the current study based on its favorable 
CC50/EC50 values and demonstration of adjunctive activity against chronic TB in the standard mouse 
model. Specifically, pravastatin demonstrated the least toxicity of all statins in our assays (CC50> 1.25 
mM and > 10 mM in THP-1 and Vero cells, respectively) and was one of 3 statins for which an EC50 
0
500
1000
1500
2000
-
-
+
+
-
-
+
+
-
+
+
-
mevalonate
simvastatin
RLU
DQ-BSA/AF594
0
40
80
120
160
200
240
0.0
0.5
1.0
1.5
2.0
2.5
Time (minutes)
DQ-BSA/AF594
Acidification
0
20
40
60
80
100
120
0
1
2
3
4
5
6
DMSO - resting
SIM 50 nM
SIM 200 nM
SIM 800 nM
SIM 2 
DMSO - beads only
SIM 50 nM - beads only
SIM 2 M - beads only
Time (minutes)
490/450nm ratio
A 
B 
C 
Figure 11. Reversal of the anti-Mtb effect of simvastatin in THP-1 cells by addition of mevalonate (A). 
Proteolysis bead assay (B): Beads were coated with a fluorescently tagged protein (DQ-BSA) and added to 
cultured macrophages.  Substrate hydrolysis in phagocytosed beads results in loss of fluorescence signal (for each 
dose tested, the increase of signal with time is a function of bead uptake). Acidification bead assay (C): beads 
were coated with pH-sensitive carboxyfluorescein and added to cultured macrophages.  Emission of fluorescence 
by the phagocytosed beads following excitation at two wavelengths (490nm and 450 nm) varies with phagosomal 
pH (for each dose tested, ratio decrease with time is a function of bead uptake). Beads only = beads in assay buffer 
without macrophages. 

25 
 
could be obtained within the tested range in THP-1 cells (7.8 μM), yielding a highly favorable 
CC50/EC50 >160.3. Importantly, pravastatin showed dose-dependent activity against chronic TB in the 
standard mouse model, and improved the percent surface area of lung involved by inflammation. 
Adjunctive therapy with pravastatin 50 mg/kg, which yields human-like drug exposures following an 80-
mg dose, significantly reduced the lung bacillary burden in a clinically relevant mouse model of necrotic 
TB granulomas (C3Heb/FeJ mice) relative to controls receiving the first-line regimen alone.  Although 
simvastatin also showed a highly favorable CC50/EC50 ration in macrophages and adjunctive activity in 
both mouse models of chronic TB, several factors dampened our enthusiasm for this agent relative to 
pravastatin. Based on our PK data, simvastatin is almost entirely converted to its acid metabolite in mice, 
whereas in humans metabolism to simvastatin acid is ~1:1. Since we do not know if the parent drug or 
acid metabolite is responsible for the anti-TB effect, it is possible that the mouse data overestimate the 
adjunctive activity of simvastatin. Perhaps more importantly, peak concentrations of both simvastatin and 
simvastatin acid are greatly reduced (by 90%) by rifampin (69). On the other hand, pravastatin exposures 
are reduced by only 30-50% during co-administration with rifampin (70). Our PK data suggest that drug 
clearance and exposures differ markedly between mice and humans. Given that we observed a dose-
responsive anti-TB effect with pravastatin adjunctive therapy in mice and the known drug interactions 
between pravastatin and rifampin in humans, we will study the safety/tolerability and PK of the highest 
clinically recommended dose of pravastatin (80 mg daily) in this clinical trial. Due to the anticipated 
~50% reduction in 24-hour pravastatin exposures when the drug is co-administered with rifampin, dose 
escalation (if tolerated) will continue up to 160 mg daily. The anti-TB effect of adjunctive statin therapy 
observed in mice was accompanied by a reduction in serum IL-6 (Figure 9) to levels seen in uninfected 
mice. If safety, tolerability, and PK of pravastatin adjunctive therapy are deemed acceptable following 
completion of this study, we will proceed to a larger follow-up study in which we will also explore the 
potential treatment-shortening and immunomodulatory effects of pravastatin adjunctive therapy for TB.   
 
4. Study Procedures 
 
Protocol Registration 
 
Prior to implementation of this protocol, and any subsequent full version amendments, each site must have 
the protocol and the protocol informed 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 and approved by the DAIDS PRO and sites 
will receive an Initial Registration Notification from the DAIDS PRO that indicates successful completion 
of the protocol registration process. 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 approval(s) 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. Site-specific ICF(s) WILL NOT be reviewed and approved by the DAIDS 
PRO and sites will receive an Amendment Registration Notification from the DAIDS PRO that approves 
the site specific ICFs and indicates successful completion of the amendment protocol registration process. 
A copy of the final amendment Registration Notification issued by the DAIDS PRO should be retained in 
the site's regulatory files. 
 
Screening 
 

26 
 
Participants with a presumptive diagnosis of sputum GeneXpert positive pulmonary TB will be invited to 
participate. Because culture confirmation is rarely available when TB treatment is initiated, patients will be 
recruited based on having a sputum specimen that is positive for TB by GeneXpert MTB/RIF.   
 
Informed written consent, using IRB-approved consent forms, will be obtained by trained study personnel 
prior to performing any study-specific procedures. Informed consent is a process that will be initiated prior 
to the individual’s agreeing to participate in the study and will continue throughout the individual’s study 
participation.   
 
The following will be performed after obtaining written informed consent: 
• 
Eligibility criteria will be confirmed 
 
• 
All participants will undergo HIV testing and counseling regardless of whether they have had a 
previous HIV test or not. HIV testing will be carried out with two rapid tests (the second test will 
be to confirm the result of the first); only one test need be carried out if the participant is known to 
have HIV, the single test will be confirmatory. Should there be a discrepancy between the two rapid 
tests, blood will be sent to the laboratory for enzyme-linked immunosorbent assay ELISA and 
Western Blot test in order to confirm the diagnosis. 
 
• 
For HIV-positive participants, a CD4 count should be performed unless written results are available 
from a test done within the preceding 3 months or less. 
 
• 
Participants will be asked questions related to demographics, current and past medical history, 
concomitant medications, alcohol and drug use and TB symptoms. 
   
• 
Participants will have heart rate, blood pressure, respiratory rate, temperature and weight measured. 
 
• 
An expectorated sputum sample will be obtained for GeneXpert MTB/RIF (as per inclusion 
criterion d), smear, culture, and drug susceptibility testing at the study laboratory (or documentation 
of results). 
 
• 
Blood will be drawn for liver function tests (AST, ALT, total bilirubin), Hepatitis B and C serology, 
CK, serum creatinine, CBC (WBC, hemoglobin, and platelets). 
   
• 
A posterior anterior chest radiograph will also be taken, unless a posterior anterior chest   
radiograph done within the previous 14 days or less is available for review.  
 
• 
Urine will be collected for pregnancy testing if a woman is of child-bearing potential. 
 
• 
Complete physical exam and vital signs 
• 
 
 
• 
Targeted symptom assessment 
 
The Screening evaluation process itself may occur over a few days with more than one visit, however, all 
Screening evaluations will be obtained within 3 days of study entry. 
Day 1 (Baseline) 
 

27 
 
• 
Treatment Assignment 
• 
DOT of pravastatin 
• 
Adverse event assessment  
• 
Chemistry (Comprehensive metabolic panel: total cholesterol, LDL, , C-reactive protein, uric 
acid)                                         
• 
Intensive PK for pravastatin  
• 
Lung inflammation assessment (PFT, St. George’s respiratory questionnaire, 6-minute walk test); 
+/- 3 days. 
• 
Targeted symptom assessment 
• 
Sputum 
 
Day 2 
 
• 
Intensive PK for pravastatin 
• 
DOT of pravastatin and TB treatment 
• 
Adverse event assessment 
      
Days 3-6 
 
• 
DOT 
• 
Adverse event assessment 
     
Day 7   (Week 1) 
 
• 
Targeted symptom assessment  
• 
Concomitant medications 
• 
DOT 
• 
Adverse event assessment 
• 
Sputum 
• 
Liver function tests 
• 
Chemistry (Comprehensive metabolic panel:, total cholesterol, LDL, , C-reactive protein, uric 
acid) 
• 
CK, serum creatinine 
Days 8-14 
 
• 
DOT 
• 
Adverse event assessment 
 
Day 15 (Week 2) 
 
• 
Targeted symptom assessment  
• 
Concomitant medications 
• 
DOT 
• 
Adverse event assessment 
• 
Sputum 
• 
Liver function tests 
• 
Chemistry (Comprehensive metabolic panel:, total cholesterol, LDL, , C-reactive protein, uric 
acid)                                         

28 
 
• 
Urine pregnancy test 
• 
Intensive PK for pravastatin  
• 
Lung inflammation assessment (PFT, St. George’s respiratory questionnaire, 6-minute walk test); 
+/- 3 days. 
• 
CK, serum creatinine 
 
Day 21 
• 
Chemistry,(Comprehensive metabolic panel: total cholesterol, LDL,  C-reactive protein, uric 
acid)                                         
• 
Liver function tests 
• 
Targeted symptom assessment 
• 
Adverse event assessment 
• 
CK, serum creatinine 
 
 
Day 30 (Month 1) 
 
• 
Targeted symptom assessment 
• 
Liver function tests 
• 
Chemistry (Comprehensive metabolic panel: total cholesterol, LDL, C-reactive protein, uric acid)                            
• 
Lung inflammation assessment (PFT, St. George’s respiratory questionnaire, 6-minute walk test); 
+/- 3 days 
• 
Urine pregnancy test 
• 
Adverse event assessment 
• 
CK, serum creatinine 
• 
 
     
Unscheduled visit 
 
• 
Exam and vital signs 
• 
Targeted symptom assessment 
• 
Adverse event assessment 
• 
Concomitant medications 
• 
Liver function tests 
• 
Chemistry 
 
TB treatment continuation 
 
All study participants will be referred appropriately to continue standard TB treatment at study completion. 
 
Targeted symptom assessment 
 
Participants will be asked whether they have experienced any of the following since the last study visit: 
fevers, sweats, cough, rash, itching, jaundice, nausea, vomiting, diarrhea, loss of appetite, vision problems, 
numbness/tingling of extremities, headache, joint pain, eye pain.  In addition, patients will be asked whether 
they have had other symptoms (not listed above); if yes, then these symptoms will be recorded and graded. 
In addition, targeted diagnostic evaluations may be indicated to determine the underlying cause of 
symptoms and their association with study drug use. 
 

29 
 
Concomitant medications 
 
The use of all non-study drugs (including over-the-counter medications) from 14 days before starting study 
therapy through the end of experimental treatment will be monitored and recorded. In addition, if a grade 3 
or higher AE or SAE occurs within 28 or fewer days after completion of experimental phase therapy, then 
the use of all non-study drugs (including over-the-counter medications) during the interval from study drug 
completion to AE/SAE onset will be recorded.  The use of all non-study drugs (including over-the-counter 
medications) will be recorded at this study visit.  
 
Concomitant antimicrobials with known antituberculous activity (amoxicillin/clavulanic acid, amikacin, 
kanamycin, streptomycin, moxifloxacin, ofloxacin, ciprofloxacin, levofloxacin, sparfloxacin, gatifloxacin, 
gemifloxacin, linezolid, capreomycin, cycloserine, ethionamide, para-aminosalicylate sodium) should not 
be used during the study phase therapy. A second statin should not be used during study phase therapy. Any 
patient who receives more than 2 doses of any of the medications listed above will be classified as being 
on a non-study regimen.  Antimicrobials with no known antituberculous activity may be prescribed for 
intercurrent infections at the discretion of the investigator.   
 
Ethanol can exacerbate the potential hepatotoxicity of isoniazid, rifampin, and pyrazinamide. Participants 
will be counseled to abstain from alcohol while on TB therapy. 
 
Administration of study drug 
 
Patients should start on assigned study drug (pravastatin)_as soon as possible after recruitment. Doses of 
study drug will be given as directly observed therapy (DOT) by study personnel, or by a health care worker 
or lay treatment supervisor who is aware of the study protocol and trained regarding the study protocol. 
Alternatively, doses of study drug can be given via DOT by a family member or employer who has been 
trained by the study team. Adherence will be confirmed by review of the participant’s treatment card, and 
retraining of the DOT provider will be provided if needed. Adherence will be defined as the number of 
prescribed doses taken. DOT may be administered at the TB clinic or other health care facility, or, with the 
participant’s permission, at the participant’s residence, workplace, or other mutually agreed upon location 
convenient for the participant.  
HRZE will not be provided by the study team. 
 
Obtaining sputum specimens 
 
Sputa should be obtained by spontaneous expectoration whenever feasible.  Sputa should be refrigerated at 
approximately 4 degrees Celsius after collection, and transported to the laboratory as soon as possible but 
within 2 working days of collection (i.e. received in the laboratory within 2 working days of collection). 
 
Laboratory aspects of sputum analysis 
 
Laboratory evaluation of sputa will be performed at a qualified mycobacteriology reference laboratory and 
will be done according to detailed written lab SOPs. Briefly, sputa will be homogenized and decontaminated 
using the NALC-NaOH method. Fluorescence smear microscopy will be performed on an aliquot of the 
concentrated processed specimen. Aliquots will be added to Lowenstein Jensen (solid) media and MGIT 
(liquid) media. Nucleic acid and/or biochemical methods will be used for mycobacterial species 
determination. Quality monitoring procedures will be documented, and laboratory activities will be 
participant to regular study monitoring. Sputum collection will not occur after Friday of each week, as the 
protocol requires transport of specimens to the laboratory within 2 working days of sputum collection. 
 

30 
 
Steady State Pharmacokinetic Assessments 
 
Intensive PK sampling: Each participant will have initial PK assessments for pravastatin at Day 1 
(immediately prior to dose administration and at 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours post-dose) as well as 
single point PK assessment on Day 2 (trough level immediately before the 2nd dose and 4-6 hours after the 
2nd dose of pravastatin). The post-dose PK assessment on Day 2 will be completed prior to the 
administration of the first dose of TB medications. Each participant will also have steady-state PK 
assessment at Day 15 immediately prior to administration of study medications and then at 0.5, 1, 1.5, 2, 3, 
4, 6, and 8 hours post-dose. Samples will consistent of 4cc of blood collected via an indwelling catheter or 
by direct venipuncture.  
 
For participants who miss or vomit the first dose of pravastatin on Day 1, that visit should be rescheduled 
>= 1 day later and study procedures recommenced. For participants who miss or vomit pravastatin dose on 
Day 2, that visit should be rescheduled within 2 days if possible, holding the first dose of TB medications 
until after these Day 2 PK draws. For participants who miss doses on one of the two days preceding the PK 
collection on Day 15 (steady state) or who vomit that day’s doses, the Day 15 visit should be rescheduled 
for >=2 days later. 
 
Specimens will be obtained, processed, and assayed according to the PK standard operating procedures 
manual.    
 
Procedures to maximize compliance with study visits 
 
Several methods will be used to maximize the proportion of participants who routinely keep their study 
visit appointments. First, only individuals with an expressed interest in participating and keeping 
appointments will be enrolled.  Second, at enrollment, information will be collected including the 
participant’s name, address, phone number, and, with the permission of the participant, names and 
telephone numbers of friends/relatives who would normally know the participant’s whereabouts. Each 
participant will be contacted on the day prior to the scheduled clinic visit. If the participant cannot be 
reached at the primary phone number listed, every effort will be made to contact him/her at the secondary 
numbers listed for friends/relative. Third, participants will be given written appointment cards. Fourth, 
participants with telephones may be called the day before the scheduled visit to remind them. Fifth, 
participants who miss appointments will be contacted either by phone or by domiciliary visit or through 
friends/family whose names were provided by the participant. Finally, study participants will be 
compensated for their time and travel to and from study visit. The form and amount of compensation will 
be in accordance with local guidelines. 
 
Informed Consent  
 
Participants enrolled in the study will be approached and invited to participate in the study when they 
present for TB evaluation and care at the CHBH, or local clinics in Soweto. Individual informed consent 
will be obtained by trained study personnel using Ethics Committee/IRB-approved consent forms. The 
study will be explained to potential participants using an information sheet available in the common local 
languages (e.g., Sesotho, isiZulu). If a participant is unable to read or write, witnessed verbal consent will 
be obtained. Informed consent is a process that will be initiated prior to the individual’s agreeing to 
participate in the study and will continue throughout the individual’s study participation. Discussion of 
risks and possible benefits of study participation will be provided by designated study staff to potential 
participants. Potential participants will receive counseling about study objectives and procedures, potential 
risks and benefits, and the informed consent process. The participants will have the opportunity to discuss 
the study with their surrogates or think about it prior to agreeing to participate. A copy of the signed and 

31 
 
dated informed consent document will be offered to the participants for their records. The rights and welfare 
of the participants will be protected by emphasizing to participants that the quality of their medical care 
will not be adversely affected if they decline to participate in this study, and that they may withdraw consent 
at any time. The investigator will retain a copy of the signed consent forms, which may be inspected at the 
monitor’s/auditor’s request. The investigator will promptly report to the Ethics Committee/IRB of all 
changes in the research activity and all unanticipated problems involving risks to human subjects or others, 
and will not make changes in the research without Ethics Committee/IRB approval, except where necessary 
to eliminate apparent immediate hazards to human subjects. 
 
In South Africa, patients are provided free access to TB care, and HIV testing and treatment through the 
public sector. According to current South African National Department of Heath guidelines, HIV-
seropositive patients with a CD4+ count <500 cells/μL should be advised and encouraged to start ART 
without delay. The current first line ART regimen in South Africa is a fixed-dose combination containing 
tenofovir, emtricitabine, and efavirenz.  
 
Diagnosis and treatment of TB is provided for free through public sector TB Clinics and hospitals in South 
Africa. GeneXpert testing, which can rapidly and accurately detect Mycobacterium tuberculosis and 
rifampin resistance, is available at public health facilities in South Africa, including at PHRU and 
SoMCHAT. GeneXpert will be used at study sites to diagnose pulmonary TB and exclude patients with 
rifampin resistance, which is a surrogate for multi-drug resistance (MDR). Phenotypic drug susceptibility 
testing will be routinely performed on all isolates from participants enrolled in the study to confirm 
susceptibility to all first-line drugs. 
 
Contraception  
 
Females may participate if they are: 1) of non-childbearing potential (have had a bilateral oophorectomy, 
tubal ligation and/or hysterectomy or have been postmenopausal for at least 12 consecutive months), 2) if 
they are using effective barrier birth control methods and are willing to continue practicing birth control 
methods throughout treatment or 3)  have a vasectomized male partner (confirmed sterile), or have a female 
partner. Therefore, to be eligible for this study women of childbearing potential should either: 1) use a 
double barrier method to prevent pregnancy (i.e. use a condom with either diaphragm or cervical cap) or 2) 
use an intrauterine device in combination with a barrier contraceptive. They must also be willing to continue 
these contraceptive measures until one week after the last dose of study medication or one week after 
discontinuation from study medication in case of premature discontinuation. (Note: Hormone-based 
contraception alone may not be reliable when taking rifampin-containing TB regimens; therefore, hormone-
based contraceptives alone cannot be used by female participants to prevent pregnancy). 
 
Safety and Tolerability Endpoints 
 
The primary endpoint for analysis of safety and tolerability is Grade 3 or higher adverse events.  Secondary 
endpoints include permanent discontinuation of assigned study regimen for any reason (other than new 
recognition of participant ineligibility based on absence of M. tuberculosis growth in baseline sputum 
cultures, or growth of M. tuberculosis resistant to rifampin by GeneXpert. Other aspects of safety and 
tolerability that will be assessed include mortality and the rates and types of toxicity thought related to study 
drugs by the Investigator. 
 
Safety Evaluation 
 
Participants will be carefully monitored to assure safety and detect and manage adverse events. Since 
treatment will be directly observed, a trained member of the team will assess participants daily for adverse 

32 
 
events. Specific tests to identify statin activity and potential toxicity will be performed at baseline 
(screening or Day 1) and Days 7, 15, and 30, and will include a serum chemistry panel and CK. A Data and 
Safety Monitoring Board (DSMB) will review the study protocol and oversee progress of the trial. The 
DSMB will review safety data after the recruitment of each dosing cohort. Since this is an open-label study, 
the DSMB will have access to treatment group assignment. The DSMB will be comprised of at least the 
following: an expert in statistics, clinical trials, clinical TB and statins. No early stopping rules will be 
formally adopted.  
 
5. Adverse Events 
 
The Investigators are responsible for eliciting adverse events by observing study participants and recording 
all adverse events observed by him/her or reported by study participants during the trial.   
 
Definitions 
 
Adverse event (AE):  Any untoward medical occurrence in a study participant administered a 
pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An 
adverse event can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not 
related to the medicinal product. 
 
Serious adverse event (SAE): Any untoward medical occurrence that at any dose: 
• 
results in death; 
• 
is life threatening (any event in which the subject was at risk of death at the time of the event; it does 
not refer to an event, which hypothetically might have caused death if it were more severe); 
• 
requires inpatient hospitalization or prolongation of existing hospitalization; 
• 
results in persistent or significant disability/incapacity;  
• 
is a congenital anomaly/birth defect; or 
• 
is a medically important event. 
Note:  
 
Medical and scientific judgment should be exercised in deciding events are “medically important.”  
These events are those that may not be immediately life-threatening or result in death or hospitalization, 
but may jeopardize the study participant or may require medical or surgical intervention to prevent one 
of the outcomes listed above.  Examples of such events are intensive treatment in an emergency room 
or at home for allergic bronchospasm, blood dyscrasias or convulsions that do not result in 
hospitalization, or the development of drug dependency or drug abuse.   
 
Unexpected Adverse Event (UAE):  An adverse reaction, the nature or severity of which is not consistent 
with the applicable product information (e.g., Investigator’s Brochure for an unapproved investigational 
product or package insert/summary of product characteristics for an approved product). 
 
Relationship of AE to Study  
 
The relationship of each adverse event (AE) to the study procedures will be characterized. The PI will make 
this determination and will classify AEs as related or unrelated. “Related” will mean that there is a 
reasonable possibility that the AE may be related to the study agent(s). “Not Related” will mean that there 
is not a reasonable possibility that the AE is related to the study agent(s). When a SAE is assessed as “not 
related” to study agent(s), an alternative etiology, diagnosis, or explanation for the SAE should be provided. 
If new information becomes available, the relationship assessment of any AE should be reviewed again and 

33 
 
updated, as required. The study drug (pravastatin) and standard-of-care regimen, Rifafour (isoniazid, 
rifampin, pyrazinamide, ethambutol) will be assessed for relationship with the AE. 
 
Reporting of Serious Adverse Events, and Unanticipated Problems 
 
Requirements, definitions and methods for expedited reporting of adverse events are outlined in Version 
2.0 of the DAIDS EAE Manual, which is available on the DAIDS RSC website at:  
https://rsc.niaid.nih.gov/clinical-research-sites/manual-expedited-reporting-adverse-events-daids. This study will 
use the SAE form of expedited adverse event reporting, as defined in Version 2.0 of the DAIDS EAE 
Manual. Expedited reporting is required for the study drug (pravastatin) and the standard-of-care regimen, 
Rifafour ( isoniazid, rifampin, pyrazinamide, ethambutol). The EAE reporting period for this study will be 
30 days, which is the day of the final study visit. During these 30 days, all SAEs will be reported to DAIDS 
in an expedited fashion, within 3 days of occurrence, in accordance with DAIDS EAE Manual. Any of the 
study and non-study drugs which a participant has taken will be assessed for relationship with the SAE. 
 
After the protocol-defined EAE reporting period, unless otherwise noted, only SUSARs as defined in 
Version 2.0 of the DAIDS EAE Manual will be reported to DAIDS if the study staff become aware of the 
events on a passive basis (from publicly available information). 
 
The DAIDS Adverse Experience Reporting System (DAERS), an internet-based reporting system, must be 
used for EAE reporting to DAIDS.  In the event of system outages or technical difficulties, EAEs will be 
submitted using the DAIDS EAE Form. This form is available on the DAIDS RSC website at  
https://rsc.niaid.nih.gov/clinical-research-sites/paper-eae-reporting 
 
For 
questions 
about 
DAERS, 
please 
contact 
NIAID 
CRMS 
Support 
at 
CRMSSupport@niaid.nih.gov.  Please note that site queries may also be sent from within the DAERS 
application itself. 
 
For questions about expedited reporting, please contact the DAIDS RSC Safety Office at 
DAIDSRSCSafetyOffice@tech-res.com.  
 
All AEs will be graded using the Division of AIDS table for Grading Severity of Adult and Pediatric 
Adverse Events, Corrected Version 2.1, July 2017. The clinical course of each event will be followed until 
resolution, stabilization, or until it has been determined that the study intervention or participation is not 
the cause. Serious AEs that are still ongoing at the end of the study period will be followed up to determine 
the final outcome. Any serious AE which we become aware of through a passive basis (participant reporting 
back to the study team, or through publicly available information), which occurs after the study period (after 
day 30) and is considered to be possibly related to the study intervention (suspected, unexpected serious 
adverse reactions, or SUSARs) or study participation will be recorded and reported immediately. 
 
Association and Attribution/causality 
 
An adverse event is considered associated with the use of the drug (Adverse Drug Reaction) if the 
attribution is possible, probable or definite. 
 
The following definitions for rating attribution/causality will be: 
Relatedness Rating 
Definition 
Not Related 
There is not a reasonable possibility that the AE is related to the study 
agent (pravastatin) or Rifafour (isoniazid, rifampin, pyrazinamide, 
ethambutol) 

34 
 
Related 
There is a reasonable possibility that the AE may be related to the study 
agent (pravastatin).or one or more components of Rifafour (isoniazid, 
rifampin, pyrazinamide and ethambutol) 
 
 
Severity* 
 
With the exception of creatinine kinase elevation, all symptoms and laboratory findings will be graded 
according to severity using the Division of AIDS Table for Grading Severity of Adult and Pediatric Adverse 
Events, Corrected Version 2.1, July 2017, which, in our view is the most comprehensive grading scale for 
patients with infectious diseases, including TB or HIV. 
 
The protocol-specific criteria for grading elevated CK are as follows: 
 
PARAMETER 
GRADE 1 
MILD 
GRADE 2 
MODERATE 
GRADE 3 
SEVERE 
GRADE 4 
POTENTIALLY  LIFE-
THREATENING 
Creatinine kinase, 
high 
3 to < 6 x ULN  
6 to <10 x ULN  
10 to <20 x ULN  
>20 x ULN  
 
The DAIDS toxicity table criteria for grading liver function tests are as follows: 
 
PARAMETER 
GRADE 1 
MILD 
GRADE 2 
MODERATE 
GRADE 3 
SEVERE 
GRADE 4 
POTENTIALLY  LIFE-
THREATENING 
AST or SGOT, 
High 
 
1.25 to < 2.5 x 
ULN 
2.5to < 5.0 x ULN  5.0 to < 10.0 x 
ULN 
≥10.0 x ULN 
ALT or SGPT, 
High 
1.25 to < 2.5 x 
ULN 
2.5to < 5.0 x ULN 5.0 to < 10.0 x 
ULN 
≥10.0 x ULN 
 
Total bilirubin 
1.1 to < 1.6 x ULN 
1.6 to < 2.6 x 
ULN 
2.6 to < 5.0 x ULN ≥  5.0 x ULN 
 
*The DAIDS AE Grading Table is available at: https://rsc.niaid.nih.gov/clinical-research-sites/daids-
adverse-event-grading-tables. 
 
Reporting 
 
AE:  Adverse events (AE) will be collected by the Investigators from the time a study participant receives 
the first dose of study drug through the Day 30 study visit. Any AE (serious or non-serious) observed by 
the Investigator or reported by the study participant that meets the AE reporting requirements (see below) 
will be recorded on an Adverse Event Case Report Form. The Investigator will review each AE and assess 
its relationship to drug treatment based on all available information at the time of the completion of the case 
report form. The following information will be recorded for each AE reported: 
• 
Diagnosis of the AE, if possible. In the case where an overall diagnosis cannot be made, each 
specific sign and/or symptom will be recorded as individual AEs; 
• 
Date of onset; 
• 
Stop Date (duration) if applicable; 
• 
Severity;   

35 
 
• 
Action taken with study drugs; 
• 
Other action taken; 
• 
Outcome; 
• 
Relationship to study drugs; 
• 
Seriousness. 
 
The following adverse events must be reported on an Adverse Event Report Form: 
• 
New medical diagnosis (at the time of enrollment, if the patient already has a medical diagnosis 
whose signs or symptoms worsen during the study to a Grade 3 or 4, this is an adverse event that 
must be reported) 
• 
Any grade 3 or higher adverse event 
• 
Study drug discontinuation due to an adverse event 
• 
Pregnancy   
• 
Lab values that are considered clinically significant by the investigator 
 
The standard-of-care drugs are Rifafour (Ethambutol, Isoniazid, Rifampin, and Pyrazinamide) and the study 
drug is pravastatin. The most common adverse effects associated with the standard-of-care drugs and the 
study drug are specified in the "Study Drugs" section. The Investigator is responsible for monitoring all 
adverse events that are observed or reported during the study, regardless of whether they are related to the 
study drug.  Participants presenting with AEs to other facilities during the study period will be referred back 
to the study site in order to complete all study visits and data collection. The participant will be given 
instructions to notify the study team if any AEs develop following the study period. 
In accordance with the FDA’s Code of Federal Regulations, the study Sponsor and the participating 
Investigators are responsible for reviewing all information relevant to the safety of the study drug and 
standard-of-care therapy. Reporting and monitoring of SAEs is required to alert the FDA, Sponsor, 
institutional review boards, and the clinical Investigators of real and potential safety issues. The 
Investigators will carefully review the AE Reports and use this information to monitor the investigational 
drug's toxicity profile and patient safety. Any AE associated with the use of the study drug and/or the 
standard of care regimen that is both serious and unexpected will be reported to the FDA in the form of a 
written Safety Report.   
The responsible institutional review boards, FDA and South African Regulatory body South African Health 
Products Regulatory Authority will be notified of all SAEs and AEs according to their individual guidelines. 
 
Pregnancy 
 
Since HMG-CoA reductase inhibitors decrease cholesterol synthesis and possibly synthesis of other 
biologically active substances derived from cholesterol, they are contraindicated during pregnancy and in 
nursing mothers. 
 
The Investigator will immediately notify the Sponsor of any pregnancy that is discovered during study drug 
administration or which started during study drug administration. Pregnancy forms will be completed for 
all pregnancies reported during the clinical trial. In addition, with the permission of the study participant, 
or female partner of a male participant, the investigator will report follow-up information regarding the 
outcome of the s, including perinatal and neonatal outcome. Infants will be followed for 6 months.   
 
All women of childbearing potential will be instructed to contact the study team immediately if they suspect 
they might have become pregnant (for example, missed or late menses) during study treatment or within 
one month of receiving their last dose of study medication. 
 

36 
 
If pregnancy is suspected for a participant in the experimental arm while the participant is receiving 
pravastatin, the pravastatin will be withheld immediately until the result of the pregnancy test is known. If 
pregnancy is confirmed, the pravastatin will be permanently discontinued in an appropriate manner and the 
participant withdrawn from the trial. Protocol-required procedures for trial discontinuation and follow-up 
will be performed unless contraindicated by the pregnancy. Patients will be referred to prenatal care and to 
the local TB program for continuation of their TB treatment. Women will be asked questions about 
pregnancy history as well as information about the current pregnancy and birth. Permission will also be 
requested to follow the infant for six months. 
 
Should the female partner of a male participant enrolled in the pravastatin arm become pregnant during the 
study or in the 4 weeks after the completion of pravastatin (Week 16 after study entry) and the Investigator 
becomes aware that this situation has occurred, consent will be requested from the female partner for 
collection of information on her pregnancy history and for information on the current pregnancy and birth. 
Permission will also be requested to follow the infant for six months. 
 
Pregnancy reporting will follow the same time lines and reporting structures as for a SAE (see above), but 
by using Pregnancy forms to be sent to the data management center. SAE reporting will also occur if the 
pregnancy outcome is a congenital anomaly. This will follow the reporting procedures described above for 
SAE reporting plus an additional clinical report.  
 
Clinical Management of Adverse Events 
 
Liver toxicity 
 
Study treatment will be permanently discontinued if any of the following criteria are met:   
 
• 
ALT > 5x the upper limit of normal (ULN) without symptoms 
• 
ALT > 3x ULN with signs or symptoms suggestive of clinical hepatitis including one or more of 
the following:  nausea, vomiting, abdominal pain, dark colored urine or clay colored stools, 
unexplained fever, jaundice, liver tenderness, or hepatomegaly 
• 
ALT > 3x ULN and Total Bilirubin ≥ 2x ULN  
 
If a patient develops hepatic toxicity meeting the above stopping criteria, the following evaluation will be 
undertaken: assessment for history of injection or non-injection drug use, alcohol ingestion, use of other 
hepatotoxic drugs, and performance of serologic tests for viral hepatitis (IgM antibody for Hepatitis A, 
Hepatitis B surface antigen, IgM antibody to Hepatitis B core antigen, antibody to Hepatitis C).  In addition, 
an AST and INR should also be checked, if not already done. 
Participants will be followed until resolution (return to baseline) or stabilization of the ALT elevation. 
 
Creatinine Kinase Elevation 
 
For Grade 1 (3 to < 6 x ULN) or Grade 2 (6 to < 10 x ULN) creatinine kinase elevation, participants may 
continue study drug treatment and should be carefully evaluated and followed closely. For Grade 3 (10 to 
< 20 x ULN) or higher creatinine kinase elevation, participants will permanently discontinue statin but will 
be requested to attend follow-up visits until study end. 
 
Myalgias and Myopathy 
 
Significant myalgias (Grade ≥3, i.e., muscle pain causing inability to perform usual social and functional 
activities) should be evaluated with a clinical assessment that includes an evaluation of CK, serum 

37 
 
creatinine, potassium, and urinalysis. Myopathy is defined as muscle aches, soreness, tenderness, or 
weakness with creatinine kinase (CK) >10 x ULN not related to exercise or other causes, including trauma. 
If the symptoms are associated with Grade 3 elevation in CK (10 x ULN) that is not related to exercise or 
other cause, study medications should be permanently discontinued. Participants will be followed on study, 
off study drug treatment through the study termination visit per protocol. 
 
Other toxicities 
 
Grade 1 toxicities: In general, for grade 1 toxicities, the patient will be followed carefully, and the study 
drugs will be continued.   
 
Grade 2 toxicities: For grade 2 toxicities, the patient will be followed more carefully, with additional 
laboratory and/or clinic visits as necessary, and the study drugs temporarily held at the investigator's 
discretion.  
 
Grade 3 toxicities: For any grade 3 toxicity involving creatinine kinase elevation, with or without myopathy, 
myalgias, hepatitis, nausea/vomiting, and/or diarrhea, that, in the principal investigator’s judgment is due 
to study drug, the study drug will be discontinued. The clinician should rule out other possible causes of 
the symptoms before discontinuing study medication. When possible, concomitant medications should be 
held first at the discretion of the principal investigator if he/she suspects they are contributing to the toxicity. 
In the event of study product discontinuation, management of the standard of care TB drugs will revert to 
the site clinician as per local practice.  
 
Grade 4 toxicities: Any patient with grade 4 renal, hepatic, cardiac or hematological toxicity will be 
immediately discontinued from study therapy. The laboratory test or clinical finding in question will be 
reassessed as soon as possible. The repeat test will guide management of the event as follows: 
• 
If the repeat assessment shows toxicity of grade 3 or lower, and if the patient has continued to 
receive study drugs between the two testing dates, then the patient will be managed according to 
the appropriate toxicity level of the repeat test. 
• 
If the repeat test shows toxicity of grade 3 or lower, and if the participant has not received study 
drugs between the two testing dates, then the participant will be managed at the discretion of the 
investigator with regard to the re-administration of study drugs, and otherwise according to the 
toxicity level of the repeat test. 
• 
If the repeat test shows grade 4 toxicity, then the participant will be permanently discontinued from 
study medications.  Further treatment of TB will be directed by the investigator on an individualized 
basis.  The participant will continue to be followed for study monitoring purposes (as are other 
participants who make a permanent departure). 
 
For other grade 4 toxicities, the study drugs will be permanently discontinued. Toxicities graded 3 or 4 and 
occurring during study therapy will be documented on the AE Form, according to the criteria described in 
the following sections. The maximum level of toxicity reached will be clearly indicated.   
For all toxicities that are treatment-emergent and that require permanent discontinuation of study treatment, 
relevant clinical and laboratory tests will be obtained as clinically indicated and repeated as needed until 
final resolution or stabilization of the toxicity. 
 
Permanent discontinuation of study drugs:  Certain events or conditions may necessitate permanent 
discontinuation of the statin study drug. Participants who experience such events or conditions, however, 
will still be "on study" and will be followed until study completion. Study drug will be discontinued with 
continued study follow-up for participants in whom treatment-emergent drug toxicity warrants 
discontinuation of study therapy and for participants who become pregnant. If study drug is permanently 

38 
 
discontinued, continued standard-of-care therapy for tuberculosis may be administered at the investigator's 
discretion. If Rifafour is discontinued, the individual components (isoniazid, rifampin, pyrazinamide, and 
ethambutol) may be gradually re-introduced, as per national guidelines. 
  
 
6. Blinding, including justification for blinding or not blinding the trial, if applicable. 
Not applicable 
 
7. Justification of why participants will not receive routine care or will have current therapy stopped. 
Not applicable 
 
8. Justification for inclusion of a placebo or non-treatment group. 
Not applicable 
 
9. Definition of treatment failure or participant removal criteria. 
 
Participants who prematurely discontinue study treatment after having received one or more doses of 
pravastatin will be replaced.  
 
Study treatment must be immediately discontinued as a result of the following: 
 
• 
Withdrawal of informed consent. 
• 
Investigator considers it for safety reasons in the best interest of the participant that he/she be 
withdrawn. 
• 
If a participant develops a new onset stage 4 illness after enrollment, they would meet the criteria for 
withdrawal from the study and, if HIV-positive, for fast track initiation of ART per the South African 
National Department of Health ART guidelines. The Interim WHO Clinical Staging of HIV/AIDS and 
HIV/AIDS Case Definitions for Surveillance, African Region (2005) will be used for guidance on 
definitions of stage 4 illnesses. 
• 
Resistance to rifampin, as determined by GeneXpert. 
• 
Participant experiences specific toxicities (including liver toxicity, CK elevation and myalgias and 
myopathy, as outlined in the “Clinical Management of Adverse Events” section) 
• 
Participant becomes pregnant. 
• 
Termination of the study by the Sponsor (US FDA, OHRP, or other relevant regulatory agencies) 
• 
Non-compliance with the study protocol (missing more than 2 doses of study drugs or not completing 
one of the PK sampling visits) 
 
All participants discontinuing study drug due to severe AE will remain in the study and continue to be 
monitored until the final scheduled clinic visit/laboratory evaluation. Their treatment will be referred to the 
local community TB clinics for standard anti-tuberculosis chemotherapy according to National Guidelines. 
The participants will be provided with a referral letter to take with them to the TB Clinic. Participants will 
be transported by the study staff to the clinic, or a follow-up call will be made by the study site staff to the 
clinic to determine if the participant attended the clinic on the date as arranged. Every effort will be made 
to continue to follow participants who discontinue study medicine prematurely until the final study visit, if 
they are agreeable. Any participant who received at least one dose of pravastatin and is withdrawn or 
withdraws early from study treatment will undergo an early treatment discontinuation visit and will be 
requested to return for the applicable follow up visits. Evaluations at these visits will not include PK 
sampling.  

39 
 
Patients will be encouraged at each visit to report new symptoms or illness experienced using a 
standardised checklist of questions which will be recorded in the source document that will also include 
participant attending other non-study health care workers or facilities since their last study visit.  
All participants  are provided with a 24 hour study phone number and are requested to report any adverse 
events to the site first before consulting any other health care facilities. 
In the event that there has been a clinical consultation with a non-study health care worker or facility, the 
site will request a copy of the clinical records including results of investigations done and treatment 
prescribed. As soon as it is clinically appropriate, and in conjunction with the attending physician, the 
management of patients will be transferred to the study site. Any missed visit will trigger an assessment 
of a possible adverse event.   
The most likely referral site for an admission to hospital is the adult wards at the Chris Hani Baragwanath 
Academic Hospital on the same campus as the research site.  There is a good relationships with staff there 
and the site is frequently notified of study participant admissions. As soon as the site is notified of an 
admission either by the participant, their family or the attending doctor, they visit the patient in the ward 
at least daily.   
Description of what happens to participants receiving therapy when study ends or if a participant’s 
participation in the study ends prematurely. 
 
Participants who withdraw from the trial after having received one or more doses of pravastatin  or who are 
discontinued from the trial because of new recognition of participant ineligibility based on absence of M. 
tuberculosis growth in baseline sputum cultures, or growth of M. tuberculosis resistant to rifampin will be 
replaced. A participant may be prematurely withdrawn from the trial as a result of the following: 
 
• 
At their own request or at the request of their legally acceptable representative. 
• 
If, in the investigator’s opinion, continuation in the trial would be detrimental to the well-being of 
the participant. 
• 
At the specific request of the Sponsor (FDA, OHRP, or other regulatory agencies). 
 
Every effort will be made to reduce loss to follow-up. Participants will be contacted by phone if they do 
not return for study visits.  
To ascertain the reason why patients have not returned for a scheduled study visit we will do the 
following: 
Participants will be contacted telephonically as soon as it becomes apparent, on the day of the scheduled 
visit, that they will not be attending the study visit, usually by noon of the day of the visit. The site’s usual 
practice is to identify at least three contact numbers which are tested at least once at each face-to face 
visit. The site will routinely call or text participants a day prior to their scheduled visit to remind them 
thereof. If they are able to contact the patient on any of the three numbers, they will ascertain the reason 
for the visit non-attendance, and if it is an issue with transport they will attempt to resolve this by making 
an offer for the study site to collect the participant. The site will also ask if there are any other reasons for 
non-attendance. A script for telephonic contacts of this nature will be drafted to ensure that questions are 
asked about possible adverse events that may have occurred and prevented a participant from attending 
the scheduled study visit.   
In the event that the telephonic contact, that day is unsuccessful, study staff will call the next day, on at 
least two occasions following the same process as above. 
If the patient is uncontactable a day after the visit, a home visit will be made later that day to contact the 
patient.  

40 
 
 
All participants withdrawn will be referred to the local community TB clinics for standard anti-tuberculosis 
chemotherapy according to National Guidelines. The participants will be provided with a referral letter to 
take with them to the TB Clinic. The participants will either be transported to the clinic by the study staff, 
or else a follow-up call will be made by the study site staff to the clinic to determine if the participant 
attended the clinic on the date as arranged.  
 
10. Monitoring and data handling 
 
Site Monitoring will be conducted to ensure that human subject protection, study procedures, laboratory 
procedures, study intervention administration, and data collection processes are of high quality and meet 
Sponsor, JHU/PHRU, and regulatory guidelines, and that the study is conducted in accordance with the 
protocol and site SOPs. Site monitoring will performed according to details in a written monitoring plan. 
 
Data handling and record keeping will be performed by the site, under the supervision of the site PI in 
accordance with procedures that are documented in the site’s detailed Data Management Operations 
Manual. Each participant will be assigned a unique study ID number. This number will be recorded on each 
data collection form and clinical specimen. Personally identifiable information including names will not be 
used on data collection forms. All study records will be stored in locked files in a secure area and access 
will be limited to study personnel and designated regulatory personnel. All forms will be reviewed prior to 
data entry for accuracy, consistency, and completeness by designated study staff.  Statistical analysis will 
be done as a joint effort between JHU Investigators and the site PI, using JHU statisticians. 
 
Source Documents and Access to Source Documents 
 
Appropriate records will be maintained for this trial, in compliance with ICH E6 GCP, and regulatory and 
institutional requirements for the protection of confidentiality of participants. The following will have 
access to study records: members of the study team; IRBs that review the study, the Office of Human 
Research Protections, the US FDA, EMEA, the NIAID, and other local, US, and international regulatory 
entities. Authorized representatives of the Sponsor and regulatory agencies will be permitted to examine 
study records for QA reviews, audits, and evaluation of the study safety and progress. 
  
Data Management Responsibilities 
 
The on-site Principal Investigator and data manager will be responsible for accuracy, completeness, and 
storage of source records and data collection forms according to site quality procedures. The study team 
and data entry staff will review source documents to ensure accuracy and completeness.  
 
Data Capture Methods 
 
Data capture will be accomplished via a set of study-specific paper source documents which have been 
developed for use in direct recording of data and observations during the patient visits. These records will 
be included in the patient chart as source records. Data captured during the patient visits will be reviewed 
to assure accuracy and completeness according to site quality procedures. Once reviewed, these data will 
be entered into the study database by manual data entry.  The study database will be maintained on JHU 
servers. Data in the study database will be reviewed by site personnel and monitored according to site 
procedures. All data management for this study will be performed manually, without the aid of a 
computerized data management system, for which computer system validation would be required. 
 

41 
 
Reports. The timing of reports will be detailed in the Data Management Operations Manual. Briefly, safety 
data will be reviewed by the DSMB after 50% of the participants have been enrolled and annually thereafter; 
reports for the DSMB will be prepared for the DSMB according to a schedule determined at the first 
convened DSMB meeting.  
 
Study Records Retention 
 
Study records will be maintained by the investigator following study discontinuation in accordance with all 
applicable NIH and local South African regulatory requirements. The NIH will be notified prior to 
destruction of study records. 
 
Protocol Deviations 
 
A protocol deviation is any noncompliance with the clinical trial protocol or Manual of Procedures. As a 
result of deviations, corrective actions are to be developed and implemented promptly.  The site will identify 
and report deviations according to the guidelines of the Sponsor and IRBs. 
 
Quality Management 
 
Procedures for quality management will be detailed in a separate Quality Management Plan. The study site 
will provide direct access to all trial-related facilities, source documents, and reports for the purpose of 
monitoring and auditing by the Sponsor, and inspection by regulatory authorities. Following written 
procedures, study monitors will verify that the clinical trial is conducted and data are generated, documented 
(recorded), and reported in compliance with the protocol and the applicable regulatory requirements. 
 
11. Inclusion/Exclusion Criteria  
 
Inclusion criteria: 
 
a) 18 years of age or older 
b) Clinical signs and symptoms of pulmonary tuberculosis 
c) Abnormal chest radiograph consistent with pulmonary tuberculosis  
d) At least one sputum positive for M. tuberculosis by Xpert MTB/RIF with a cycle threshold (Ct) <28. 
e) Documentation of HIV status 
f) Weight ≥45 kg 
g) Karnofsky score of at least 60  
h) Ability to provide informed consent 
i) Ability to adhere to study follow-up visits 
j) Negative pregnancy test in women of child-bearing age 
k) Ability to adhere to contraceptive requirements and willing to use two forms of contraception: 1) a 
double barrier method to prevent pregnancy (i.e. use of a condom with either diaphragm or cervical 
cap) or 2) use of an intrauterine device in combination with a barrier contraceptive. The participant 
must be willing to continue these contraceptive measures throughout the duration of the study and until 
one week after the last dose of study medication or one week after discontinuation from study 
medication in case of premature discontinuation.  
l) Five days or fewer of anti-tuberculosis treatment within the previous 3 months 
 
Exclusion criteria: 
 

42 
 
a) A history of severe adverse reactions to any statin or any other study agent or contraindications to use 
of statins. 
b) Current use of statins or other lipid-lower agents; 
c) Clinical indication for statin therapy based on cardiovascular risk: 
1) Familial hypercholesterolemia 
2) Previous history of myocardial infarction or stroke 
d) For HIV-positive individuals, a CD4+ T-cell count <350/mm3 
e) Use of antiretroviral drugs    
f) Hemoglobin concentration less than 8 g/dL; 
g) Baseline creatinine kinase elevation more than three times the upper limit of normal 
h) Abnormal baseline laboratory values 
1) Baseline alanine aminotransferase (ALT) concentration more than 2.5 times the upper limit of 
normal (Grade 1) 
2) Serum creatinine concentration more than twice the upper limit of normal; 
3) Serum total bilirubin level greater than twice the upper limit of normal 
4) Platelet count < 100,000/mm3 
5)  ANC <1,000/mm3 
i) Pregnant or breastfeeding; 
j) Silico-tuberculosis.  
k) Currently receiving TB treatment 
l) Serologies or PCR positive for viral hepatitis (Hepatitis, B, C) 
m) Concomitant disorders or conditions for which isoniazid, rifampin, pyrazinamide, or ethambutol is 
contraindicated. These include cirrhosis, acute liver disease of any cause, acute uncontrolled gouty 
arthritis and peripheral neuropathy. 
n) Any medical or psychological condition which, in the view of the study investigator, makes study 
participation inadvisable. 
o) Infection with an isolate determined to be resistant to rifampin by GeneXpert. 
p) More than five days of anti-tuberculosis treatment within the previous 3 months 
q) Planned or current use of cyclosporine, tacrolimus, erythromycin or colchicine 
r) CNS TB 
s) Extra-pulmonary TB only, not in combination with pulmonary TB 
t) History of TB 
 
12. Special Populations 
 
There will be no exclusion based on race, gender or ethnicity.  
 
Children, Pregnant Women, and Breast-Feeding Women 
 
Pregnant or breast-feeding women will be excluded from this study because statins are contraindicated 
during pregnancy based on their potential teratogenic effects. According to the NIH definition, a child is 
“any individual under the age of 21”. We will enroll participants ≥18 years of age with pulmonary 
tuberculosis, and, therefore, children are eligible for enrollment. It is anticipated that approximately 40% 
of study participants will be women, since, historically, approximately 60% of patients with tuberculosis at 
the clinical sites in our study are males. The gender, ethnicity, and socioeconomic background of study 
participants are expected to mirror that of the population of the hospital and clinics served, and that of the 
population most affected by tuberculosis. It is anticipated that the majority of study participants will be 
black South Africans. 
 

43 
 
Prisoners 
 
This study will not enroll prisoners. However, it is possible that a participant will be incarcerated after 
enrollment. If an enrolled individual is incarcerated, then study medications will be stopped and the 
participant will be treated for active TB according to the standards of the institution in which s/he is 
incarcerated. While incarcerated, individuals will not be followed in the study.  When the individual is no 
longer incarcerated, study follow-up may continue, at the discretion of the Investigator. 
 
HIV-Infected Individuals  
 
HIV-infected individuals and HIV-uninfected individuals will be included in this study. HIV-infected 
individuals with CD4 <350 cells/mm3 will be excluded in this study, as these patients are at increased risk 
of death and may be better served by individualized (non-study) TB and HIV therapy. If one or both of the 
proposed experimental regimens is demonstrated to be effective and safe in this study, then subsequent 
future studies including individuals with advanced AIDS may be warranted. This study will not enroll 
participants with HIV infection who are taking antiretroviral therapy. For patients with active TB and CD4 
cell counts >50/mm3 who are not already taking ART, initiation of ART is recommended after 2 months of 
TB treatment. HIV-infected individuals in South Africa are routinely treated with fixed-dose combination 
pills containing efavirenz, tenofovir, and emtricitabine. Efavirenz is known to lower the mean AUC of 
pravastatin by ~40%. Therefore, HIV-infected individuals receiving antiretroviral drugs will be excluded 
from the study. If a participant is already enrolled in the study, and they require antiretotroviral therapy, 
they will be taken off of study medicines and referred to the clinics for treatment with TB and HIV therapy.   
 
Ethical Issues in Doing This Trial in South Africa 
 
The vast majority of the global TB burden occurs in developing countries. South Africa has an exceedingly 
high TB case rate of approximately 600/100,000 population/year, and ranks #7 in the world based on 
number of incident cases. New treatment regimens that substantially improve TB treatment (shorten and/or 
simplify therapy) are critically needed in this setting. Therefore, it is appropriate to evaluate a new host-
directed treatment for TB in South Africa.   
 
Management of participants deemed ineligible 
 
Because of the relatively slow growth of M. tuberculosis in culture, the results of sputum mycobacterial 
cultures and drug susceptibility testing are rarely available at the time of initiation of TB treatment.  
Therefore, for this study we plan to enroll patients on the basis of a positive sputum test for TB on 
GeneXpert testing. We anticipate that approximately 10% of enrollees subsequently will be deemed 
protocol ineligible based on culture results.   
 
Management of individuals having either a) NO baseline sputum culture that is positive for M. tuberculosis 
OR b) a baseline M. tuberculosis isolate that is determined to be resistant to rifampin by GeneXpert: these 
individuals will be taken off of their assigned regimen, and treated with an individualized regimen at the 
discretion of the treating clinician.   
 
13. Drugs/ Substances/ Devices 
 
Study drug descriptions 
 
Pravastatin 40 mg tablets 
 

44 
 
Pravastatin  
 
Pravastatin (marketed as Sandoz Pravastatin) is a member of the drug class of statins, used in combination 
with diet, exercise, and weight loss for the treatment of dyslipidemia and the prevention of cardiovascular 
disease. 
 
Mechanism of action: Pravastatin acts as a lipoprotein-lowering drug through two pathways. In the major 
pathway, pravastatin inhibits the function of hydroxymethylglutaryl-CoA (HMG-CoA) reductase. As a 
reversible competitive inhibitor, pravastatin sterically hinders the action of HMG-CoA reductase by 
occupying the active site of the enzyme. Taking place primarily in the liver, this enzyme is responsible for 
the conversion of HMG-CoA to mevalonate in the rate-limiting step of the biosynthetic pathway for 
cholesterol. Pravastatin also inhibits the synthesis of very-low-density lipoproteins, which are the precursor 
to low-density lipoproteins (LDL). These reductions increase the number of cellular LDL receptors, thus 
LDL uptake increases, removing it from the bloodstream. Overall, the result is a reduction in circulating 
cholesterol and LDL. A minor reduction in triglycerides and an increase in high-density lipoproteins (HDL) 
are common. 
 
Pharmacokinetics  
 
Pravastatin is administered orally in the active form. In studies in man, peak plasma pravastatin 
concentrations occurred 1 to 1.5 hours upon oral administration. Based on urinary recovery of total 
radiolabeled drug, the average oral absorption of pravastatin is 34% and absolute bioavailability is 17%. 
While the presence of food in the gastrointestinal tract reduces systemic bioavailability, the lipid-lowering 
effects of the drug are similar whether taken with or 1 hour prior to meals. Pravastatin plasma 
concentrations, including area under the concentration-time curve (AUC), Cmax, and steady-state minimum 
(Cmin), are directly proportional to administered dose. Systemic bioavailability of pravastatin administered 
following a bedtime dose was decreased 60% compared to that following an AM dose. Despite this decrease 
in systemic bioavailability, the efficacy of pravastatin administered once daily in the evening, although not 
statistically significant, was marginally more effective than that after a morning dose. The coefficient of 
variation (CV), based on between-subject variability, was 50% to 60% for AUC. The geometric means of 
pravastatin Cmax and AUC following a 20 mg dose in the fasted state were 26.5 ng/mL and 59.8 ng*hr/mL, 
respectively. Steady-state AUCs, Cmax, and Cmin plasma concentrations showed no evidence of pravastatin 
accumulation following once or twice daily administration of PRAVACHOL tablets.  
 
Distribution 
 
Approximately 50% of the circulating drug is bound to plasma proteins. 
 
Metabolism 
 
Importantly, pravastatin is not metabolized by the CYP450 system in the liver (86, 87). Instead, the major 
metabolites are produced by chemical degradation in the stomach and the intact drug and its metabolites 
are cleared through both hepatic and renal routes, reducing the need for dosage adjustment if the function 
of either the liver or kidney is impaired, and also reducing the possibility of drug interactions compared 
with other statins (87). Therefore, increased toxicity due to potential reduced clearance of pravastatin by 
TB meds is not expected. 
The major biotransformation pathways for pravastatin are: (a) isomerization to 6-epi pravastatin and the 
3α-hydroxyisomer of pravastatin (SQ 31,906) and (b) enzymatic ring hydroxylation to SQ 31,945. The 3α-
hydroxyisomeric metabolite (SQ 31,906) has 1/10 to 1/40 the HMG-CoA reductase inhibitory activity of 

45 
 
the parent compound. Pravastatin undergoes extensive first-pass extraction in the liver (extraction ratio 
0.66). 
 
Excretion 
 
Approximately 20% of a radiolabeled oral dose is excreted in urine and 70% in the feces. After intravenous 
administration of radiolabeled pravastatin to normal volunteers, approximately 47% of total body clearance 
was via renal excretion and 53% by non-renal routes (i.e., biliary excretion and biotransformation). 
 
Drug-drug interactions 
Medications that may cause drug interactions with pravastatin include fibrates, erythromycin, niacin, and 
many others. These drug interactions may result in side effects or complications such as increased levels of 
medicine in the blood and an increased risk of developing serious muscle problems. The metabolism of 
pravastatin is not significantly mediated by CYP enzymes. However, pravastatin is a substrate of MRP2 
and the uptake transporter OATP2. Recent studies have illustrated differences in the interaction potential 
of the different statin drugs. Thus, itraconazole increased the AUC of simvastatin acid at least tenfold but 
had no significant effect on that of pravastatin. Furthermore, combination therapy with the HIV protease 
inhibitors ritonavir and saquinavir increased the median AUC(0–24) of simvastatin 30-fold, but decreased 
that of pravastatin by 50%. Pravastatin is a substrate of both influx and efflux transporters, and although 
significant interactions mediated by induction of pravastatin metabolism are not likely, the in vivo effects 
of induction of transporters are largely unknown.  
 
Rifampin caused a statistically significant decrease in the plasma concentration of pravastatin given as a 
single oral dose to healthy subjects. However, the effect of rifampin varied greatly between subjects. The 
mean rifampin-induced decrease in pravastatin concentration was considerably smaller than that observed 
previously for simvastatin. Unlike simvastatin and its metabolite (simvastatin acid), in which steady-state 
AUC is reduced by ~90% by rifampin (69), requiring co-administration with rifabutin to obtain adequate 
drug exposures, pravastatin AUC is reduced by only 30-50% during co-administration with rifampin (70). 
 
Tolerability 
 
Recent prospective (Prospective Pravastatin Pooling (PPP) Project) analysis indicates that during prolonged 
exposure, 40 mg of pravastatin is well tolerated, with no excess of noncardiovascular serious adverse 
events, including liver function abnormalities and laboratory and clinical evidence for myositis.  
 
The safety and tolerability of pravastatin at a dose of 80 mg in 2 controlled trials with a mean exposure of 
8.6 months was similar to that of pravastatin at lower doses except that 4 out of 464 patients taking 80 mg 
of pravastatin had a single elevation of CK >10 times ULN compared to 0 out of 115 patients taking 40 mg 
of pravastatin. 
 
In the PRAVACHOL placebo-controlled clinical trials database of 21,483 patients (age range 24-75 years, 
10.3% women, 52.3% Caucasians, 0.8% Blacks, 0.5% Hispanics, 0.1% Asians, 0.1% Others, 46.1% Not 
Recorded) with a median treatment duration of 261 weeks, 8.1% of patients on PRAVACHOL and 9.3% 
patients on placebo discontinued due to adverse events regardless of causality. 
 
Adverse event data were pooled from 7 double-blind, placebo-controlled trials (West of Scotland Coronary 
Prevention Study [WOS]; Cholesterol and Recurrent Events study [CARE]; Long-term Intervention with 
Pravastatin in Ischemic Disease study [LIPID]; Pravastatin Limitation of Atherosclerosis in the Coronary 
Arteries study [PLAC I]; Pravastatin, Lipids and Atherosclerosis in the Carotids study [PLAC II]; 
Regression Growth Evaluation Statin Study [REGRESS]; and Kuopio Atherosclerosis Prevention Study 

46 
 
[KAPS]) involving a total of 10,764 patients treated with pravastatin 40 mg and 10,719 patients treated 
with placebo. The safety and tolerability profile in the pravastatin group was comparable to that of the 
placebo group. Patients were exposed to pravastatin for a mean of 4.0 to 5.1 years in WOS, CARE, and 
LIPID and 1.9 to 2.9 years in PLAC I, PLAC II, KAPS, and REGRESS. In these long-term trials, the most 
common reasons for discontinuation were mild, non-specific gastrointestinal complaints. Collectively, 
these 7 trials represent 47,613 patient-years of exposure to pravastatin. All clinical adverse events 
(regardless of causality) occurring in ≥2% of patients treated with pravastatin in these studies are identified 
in the package insert for Pravastatin. 
 
Dosage 
 
The recommended starting dose of pravastatin for cholesterol-lowering is 40 mg once daily. If a daily dose 
of 40 mg does not achieve desired cholesterol levels, 80 mg once daily is recommended. In patients with 
significant renal impairment, a starting dose of 10 mg daily is recommended. Pravastatin sodium tablets 
can be administered orally as a single dose at any time of the day, with or without food. 
 
Rifampin 
 
Rifampin is a semi-synthetic rifamycin derivative that is highly active against mycobacteria, most gram-
positive bacteria, and some gram-negative bacteria. It is bactericidal for both intracellular and extracellular 
microorganisms. By inhibiting prokaryotic DNA-dependent RNA polymerase, it suppresses the early 
elongation of the nucleotide chain in RNA synthesis.  
 
Pharmacokinetics 
 
Rifampin is normally absorbed completely when taken orally, but food delays absorption. After 1.5 to 2 
hours, a 600-mg dose yields a peak blood level of 8-20 μg/mL. The half-life of rifampin varies from 2 to 5 
hours, and it is shortened by approximately 20-40% after the first week of daily treatment because of the 
induction of hepatic microsomal enzymes. The half-life is unaffected by renal impairment but is increased 
by liver disease or biliary obstruction. Rifampin is deacetylated to an enterohepatically recirculated active 
metabolite, and 50% to 60% is excreted in the feces. Up to 30% of a dose is excreted in the 
urine. Approximately 85% of circulating rifampin is bound to plasma proteins, and is widely distributed 
throughout the body.  
 
Drug-drug interactions 
 
Rifampin is an inducer of a number of hepatic enzymes involved in the metabolism of drugs and some 
hormones. This enzyme induction causes more rapid elimination (and potential loss of efficacy) of many 
drugs. Medications for which concomitant rifampin is contraindicated include: HIV-1 protease inhibitors 
(other than ritonavir), delavirdine, cyclosporine, tacrolimus, itraconazole, and ketoconazole. For many 
other medications, the dose can be increased to compensate for the effect of rifampin. Rifampin 
significantly lowers the peak levels of most statins. Specifically, pravastatin exposures are reduced by 30-
50% during co-administration with rifampin (70).  
 
Tolerability 
 
In the usual daily dose of 600 mg, rifampin is well tolerated. It often causes harmless but disconcerting red-
orange discoloration of tears, sweat, saliva, feces, and urine. Less than 4% of TB patients experience 
significant adverse reactions to rifampin. Gastrointestinal AEs are the most common, and they include 
epigastric distress, anorexia, nausea, vomiting, cramps, and diarrhea. Hepatitis rarely occurs in persons who 

47 
 
have normal baseline hepatic function. The incidence of hepatitis may be increased for older persons and 
those who have chronic liver disease or alcoholism, but remains substantially lower than that for 
pyrazinamide or isoniazid. Rifampin can cause a flu-like syndrome of fever, chills, and myalgia, although 
this is uncommon using the 600-mg dose given daily or thrice-weekly. In a very small proportion of patients 
the flu-like syndrome is associated with interstitial nephritis, acute tubular necrosis, thrombocytopenia, 
hemolytic anemia, and shock. There may be changes in menstruation. 
 
Dosage 
 
According to South African guidelines the recommended dose of rifampin for the treatment of TB is 450 
mg (weight < 50 kg) or 600 mg (weight>50 kg).  
 
Isoniazid 
 
Isoniazid is the hydrazide of isonicotinic acid and is one of the primary drugs for TB treatment.  The activity 
of isoniazid is limited to the mycobacteria of the M. tuberculosis complex; it is bactericidal for rapidly 
dividing organisms and bacteriostatic for “resting” bacilli. Its mechanism of action is the inhibition of the 
biosynthesis of mycolic acids, a component of the mycobacterial cell wall.   
 
Pharmacokinetics 
 
Isoniazid is generally well absorbed; food and antacids decrease the rate, but not the extent of absorption. 
The peak blood levels of isoniazid, 3 to 5 μg/ml, are obtained 30 minutes to 2 hours after ingestion of 
routine doses. It diffuses into all body fluids and cells and penetrates into the caseous material of a 
tuberculoma or pulmonary cavity. In the liver, it is acetylated to inactive metabolites, and 75% to 95% of 
the dose is excreted as inactive metabolites in the urine within 24 hours. Isoniazid clearance rates depend 
on 2 metabolic phenotypes, slow and fast acetylation. The isoniazid AUC among persons who have fast 
acetylation is 30% to 50% of that among persons who have slow acetylation. Because isoniazid is well 
tolerated over a wide range of therapeutic doses, a single dose per body mass is recommended. Persons 
who have rapid acetylation achieve effective concentrations, while persons who have slow acetylation do 
not experience increased toxicity. Half-life (t1/2) may vary from 1 hour in fast acetylators (t1/2 < 90min) to 
3 hours in slow acetylators (t1/2 >90min). 
 
Drug-drug interactions 
 
Isoniazid decreases the clearance of some medications that are metabolized in the liver (particularly 
carbamazepine, phenytoin, diazepam).   
 
Toxicity 
 
The total incidence of all adverse effects from isoniazid is approximately 5%, many of which do not require 
discontinuation of the drug.  Peripheral neurotoxicity is dose dependent and it is uncommon (<0.2%) at 
conventional doses. The risk of peripheral neuritis increases for persons who are malnourished or 
predisposed to neuritis by other illnesses. Concomitant administration of pyridoxine (vitamin B6) is 
recommended for HIV positive person in South Africa. It will be given to every participant in this trial. 
Other nervous system reactions are rare at normal doses, and they include convulsions, encephalopathy, 
optic neuritis, memory impairment, and psychosis. Gastrointestinal adverse effects include nausea, 
vomiting, and epigastric distress. Asymptomatic elevation of aminotransferases is common and occurs in 
10-20% of persons receiving isoniazid. However, idiosyncratic severe hepatic reactions are uncommon but 
are more likely in older persons (up to 2.3% hepatitis incidence in persons more than 50 years old), and 

48 
 
may be life threatening. Daily consumption of alcohol increases the risk of isoniazid-associated 
hepatotoxicity by approximately 4-fold.  The risk of isoniazid-induced hepatotoxicity may also be increased 
in the postpartum period. The prodromal symptoms of hepatotoxicity are anorexia, nausea, vomiting, 
fatigue, malaise, and weakness; persons who take isoniazid and have these symptoms should stop therapy 
and be evaluated immediately. 
 
 
Dosage 
 
According to South African guidelines the recommended dose of isoniazid for the treatment of TB is 300 
mg.  
 
Pyrazinamide 
 
Pyrazinamide is an analog of nicotinamide and has unique activity against M. tuberculosis, allowing the 
duration of treatment to be decreased from 9 months to 6 months (assuming rifampin is used throughout).  
The mechanism of action of pyrazinamide remains unknown. 
 
Pharmacokinetics 
 
Pyrazinamide is well-absorbed from the gastrointestinal tract and widely distributed into all tissues. Usual 
doses are 15-30 mg/kg/d, up to 2 gm/d. Peak serum concentrations of about 45 μg/ml are achieved 
approximately 2-3 hours after a dose. Food and antacids do not significantly affect the absorption of 
pyrazinamide. The half-life of pyrazinamide is approximately 9-10 hours, and is prolonged in the presence 
of hepatic insufficiency.  Pyrazinamide is metabolized to pyrazinoic acid by the hepatic microsomal enzyme 
pyrazinamide deamidase.  Approximately 40% of a dose is recovered in the urine as pyrazinoic acid and an 
additional 4% is excreted in the urine as the unchanged parent drug. The remaining drug is thought to be 
excreted in the bile.   
 
Drug-drug interactions 
 
There are no known clinically significant drug-drug interactions involving pyrazinamide. 
 
Toxicity 
 
The most frequent side effects are skin rash, gastrointestinal intolerance, hepatotoxicity (1.3%), arthralgias 
(1-7%), hyperuricemia due to blockade of urate excretion (up to 66%), and rarely acute gouty arthritis.  
These side effects are seldom dose-limiting. Asymptomatic elevations in serum uric acid are frequent, 
usually occur during the first or second month of treatment, and are self-limited and require no specific 
treatment. Minor arthralgias also may occur during pyrazinamide treatment and can usually be treated with 
salicylates or non-steroidal inflammatory agents such as indomethacin while continuing the drug.  The most 
common serious side effect of pyrazinamide is hepatotoxicity.   
 
Dosage 
 
According to South African guidelines the recommended dose of pyrazinamide for the treatment of TB is 
20-25 mg/kg.  
 
Ethambutol 
 

49 
 
Ethambutol is an ethylene derivative of butane that interferes with cell wall synthesis in mycobacteria; other 
bacteria are uniformly resistant to ethambutol.  In the treatment of human TB, ethambutol is effective in 
preventing the emergence of drug resistant strains, although it has no sterilizing activity at clinically-
tolerated doses. 
 
Pharmacokinetics 
 
Ethambutol is well absorbed from the gastrointestinal tract, reaching peak serum concentrations of 3-5 
mcg/ml in normal volunteers 2-4 hours after a dose.  Food slows absorption and decreases the peak serum 
concentration by 10-20%, but has no effect on the total systemic exposure (AUC).  Antacids decrease both 
the peak serum concentration and AUC, and so should not be administered at the same time.  Ethambutol 
is primarily eliminated by the kidneys as unchanged drug; the serum half-life averages 4 hours.   Patients 
with renal insufficiency are prone to accumulation of the drug and the resultant toxicity. 
 
Drug-drug interactions 
 
There are no known drug–drug interactions involving ethambutol. 
 
Toxicity 
 
Ethambutol is usually well-tolerated with low rates of skin rash, nausea, vomiting, or diarrhea. Fever, 
allergic reactions, abdominal pain, mental status changes, peripheral neuropathy, and increased liver 
function tests have rarely been associated with ethambutol. Adverse events occur in less than 2% of patients 
receiving ethambutol at the 15 mg/kg dose and include decreased visual acuity (0.8%), rash (0.5%) and 
asymptomatic hyperuricemia. The most common serious side effect of ethambutol is retinal toxicity, often 
first perceived as a decrease in color perception. Patients receiving ethambutol should be instructed about 
symptoms of ocular toxicity. If stopped promptly, permanent visual loss is rare among patients with 
ethambutol-related retinal toxicity. Rates of retinal toxicity are very low when the drug is given for 
relatively short periods, as is the case in this study. 
 
Dosage 
 
According to South African guidelines the recommended dose of ethambutol for the treatment of TB is 15-
20 mg/kg. This is the dosage that will be used for this study. 
 
Pharmacy: Product Supply, Distribution, and Accountability 
 
Pravastatin, Rifafour, and vitamin B6 will be supplied through the study. An FDA-approved preparation of 
Pravastatin 40 mg tablets will be made available through the National Institute of Allergy and Infectious 
Diseases (NIAID) Clinical Research Products Management Center (CRPMC). The site pharmacist can 
obtain the study product for this protocol by following the instructions in the manual Pharmacy Guidelines 
and Instructions for DAIDS Clinical Trials Networks in the section Study Product Management 
Responsibilities. Rifafour and vitamin B6 will be obtained in South Africa; Rifafour will be obtained from 
the National TB Program (purchased from Sanofi-Aventis) and B6 from a commercial distributor. Rifafour 
will be given in doses consistent with South African guidelines. 
Rifafour dosage by pre-treatment body weight 
Rifafour = Rifampin/isoniazid/pyrazinamide/ethambutol (150 mg/75 mg/400 mg/275mg)  
 
30-37 kg 2 tabs  
38-54 kg 3 tabs  

50 
 
55-70 kg 4 tabs  
>70 kg 5 tabs  
 
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 must be returned to the NIAID CRPMC 
(or as otherwise directed) after the study is completed or terminated. The procedures to be followed are 
provided in the manual Pharmacy Guidelines and Instructions for DAIDS Clinical Trials Networks in the 
section Study Product Management Responsibilities. 
 
14. The rationale for choice of statin and dose 
 
Pravastatin was selected for testing in this safety/PK study based on its favorable CC50/EC50 values and 
demonstration of adjunctive activity against chronic TB in the standard mouse model. Specifically, 
pravastatin demonstrated the least toxicity of all statins in our assays (CC50> 1.25 mM and > 10 mM in 
THP-1 and Vero cells, respectively) and was one of 3 statins for which an EC50 could be obtained within 
the tested range in THP-1 cells (7.8 μM), yielding a highly favorable CC50/EC50 >160.3. Importantly, 
pravastatin showed dose-dependent activity against chronic TB in the mouse model, and improved the 
percent surface area of lung involved by inflammation. Adjunctive therapy with pravastatin 50 mg/kg, 
which yields human-like drug exposures following an 80-mg dose, significantly reduced the lung bacillary 
burden in a clinically relevant mouse model of necrotic TB granulomas (C3Heb/FeJ mice) relative to 
controls receiving the first-line regimen alone. Although simvastatin also showed a highly favorable 
CC50/EC50 ratio in macrophages and adjunctive activity in the two different mouse models of chronic TB, 
several factors dampened our enthusiasm for this agent relative to pravastatin. Based on our PK data, 
simvastatin is almost entirely converted to its acid metabolite in mice, whereas in humans metabolism to 
simvastatin acid is ~1:1. Since we do not know if the parent drug or acid metabolite is responsible for the 
anti-TB effect, it is possible that the mouse data overestimate the adjunctive action of simvastatin. Perhaps 
more importantly, peak concentrations of both simvastatin and simvastatin acid are greatly reduced (by 
90%) by rifampin (69). On the other hand, pravastatin exposures are reduced by only 30-50% during co-
administration with rifampin. Our PK data suggest that drug clearance and exposures differ markedly 
between mice and humans. Given that we observed a dose-responsive anti-TB effect with pravastatin 
adjunctive therapy in mice and the known drug interactions between pravastatin and rifampin in humans, 
we will study the highest clinically recommended dose of pravastatin in this clinical trial. The anti-TB effect 
of adjunctive statin therapy observed in mice was accompanied by a reduction in serum IL-6 (Figure 9) to 
levels seen in uninfected mice. In addition to IL-6, a recent thesis project at JHU investigating predictors 
of respiratory impairment in TB identified levels of the cytokines IL-1β and TGF-β as being directly 
proportionate and levels of the cytokines IL-12 and TIMP-2 as being inversely proportionate to changes in 
St. George’s respiratory questionnaire scores during TB treatment (Akshay Gupte, unpublished).  
 
15. Justification and safety information if FDA-approved drugs will be administered for non-FDA 
approved indications or if doses or routes of administration or participant populations are changed. 
 
The goal of this study is to evaluate pravastatin as host-directed therapy for TB, a non-approved indication.  
Pravastatin has favorable, dose-dependent activity against chronic TB in two different mouse models. 
Pravastatin exposures are reduced by 30-50% during co-administration with rifampin (70). Therefore, we 
anticipate that a dose of pravastatin 160 mg daily when co-administered with rifampin will give similar 
pravastatin exposures as monotherapy with pravastatin 80 mg daily. However, to address safety concerns 
regarding the unknown effect of co-administering pravastatin with the other anti-TB drugs (isoniazid, 
ethambutol, and pyrazinamide) and to minimize study risk to participants, we will initially recruit a cohort 

51 
 
of 5 participants to assess safety/tolerability of pravastatin 40 mg daily in combination with standard of care 
(Rifafour) prior to further study drug dose escalation. If this dose is well tolerated when given with Rifafour, 
we will recruit 10 participants to receive pravastatin 80 mg daily and Rifafour. If it is determined that this 
dose, when co-administered with Rifafour is well-tolerated, yet the mean pravastatin exposures during 
adjunctive therapy are reduced by > 40% relative to pravastatin monotherapy in the same patients (as 
assessed prior to initiating anti-TB drugs), we will increase the dose of pravastatin incrementally up to a 
maximum of 160 mg/d, in order to achieve drug exposures equivalent to those of pravastatin 80 mg/d given 
as monotherapy. 
 
16. Justification and safety information if non-FDA approved drugs without an IND will be 
administered.  
 
 
Not applicable 
 
17.  Study statistics 
 
   Primary outcome variable.  
 
Safety and tolerability of pravastatin adjunctive therapy relative to standard treatment 
 
  Secondary outcome variables 
Steady-state PK of pravastatin among patients with pulmonary TB receiving co-administered 
Rifafour (isoniazid, rifampin, pyrazinamide and ethambutol) 
 
18. Statistical Analysis Plan 
 
Hypothesis 
 
Pravastatin given at the standard clinically recommended dose will be safe and well tolerated, and 
pravastatin drug exposures will not be significantly altered following co-administration with rifampin in 
the 14-day study. 
 
Sample size 
 
The sample size will be between 5and 35 evaluable participants, who will be recruited into the following 
arms: 1) Standard-of-care regimen (Rifafour) + pravastatin 40 mg daily (5 evaluable participants); 2) 
Standard-of-care regimen (Rifafour) + pravastatin 80 mg daily (10 evaluable participants); 3) Standard-of-
care regimen (Rifafour)  + pravastatin 120 mg daily (10 evaluable participants); 3) Standard-of-care 
regimen (Rifafour) + pravastatin 160 mg daily (10 evaluable participants). Participants in Arms 2, 3, and 4 
(10 participants each) will only be recruited if necessary, i.e., if pravastatin 40 mg is well tolerated and safe, 
and if escalated doses are also tolerated and safe, yet drug exposures are reduced by > 40% in participants 
due to the known interaction with rifampin.  
A sample size of 10 evaluable participants for pravastatin PK analysis was chosen based on within-subject 
variability (CV%) of statin AUC in interaction studies ranging from 6-60% and a within-subject coefficient 
of variation (CVw) of 33%. With 10 evaluable participants per arm, the precision (i.e., half width of the 
90% confidence interval on the log scale) for the treatment difference will be within 26% of the point 
estimate for AUC. If the point estimate of the ratio of geometric mean is 1, then 90% confidence interval 
will be approximately (0.74, 1.35). 
 

52 
 
Data Analysis 
 
The current study is a 2-week, 4-arm dose-escalation trial to determine the safety/tolerability and PK of 
pravastatin when co-administered with the standard drug regimen for drug-susceptible TB (Rifafour). 
Participants will receive standard anti-TB therapy and pravastatin beginning at a dose of 40 mg once daily. 
Each subsequent arm (pravastatin 80 mg, 120 mg, and 160 mg) of this study will continue to recruit 
participants until at least 10 participants in each group are evaluable for full PK analysis.  
 
The primary endpoints in this study are safety/tolerability and pravastatin exposures. A regimen will be 
deemed safe and well tolerated if none of the participants require treatment discontinuation for a 
pravastatin-related adverse reaction and <3 participants experiences a pravastatin-related grade 3 (or higher) 
adverse event during the 14-day study period and follow up. PK parameters (e.g., area under the 
concentration-time curve over 24 hours (AUC0-24), minimum concentration (Cmin), maximum concentration 
(Cmax), and oral clearance (Cl/F)), will be estimated for pravastatin from intensive sampling data after a 
single dose of pravastatin (baseline PK, prior to initiation of TB treatment) and after 14 days of co-
administration with the first-line anti-TB regimen. Based on PK data of pravastatin when co-administered 
with rifampin (Tmax = 1 hour, T1/2 = 1.5 hours), plasma samples will be collected for pravastatin 
measurements by LC-MS/MS at the following time points: pre-dose, 0.5 hrs, 1 hr, 1.5 hrs, 2 hrs, 3 hrs, 4 
hrs, 6 hrs, and 8 hrs. Pravastatin exposures will be calculated using a noncompartmental approach using 
standard PK software, e.g., Phoenix WinNonLin. Using all PK data, a PK/PD model will be developed to 
evaluate the relationship between drug exposures (e.g., Cmax, T>MIC or AUC0-24) and outcomes (e.g., 
safety/tolerability). Cmax and tmax will be taken directly from the original data. The terminal log-linear phase 
of the plasma concentration-time curve will be identified visually for each curve. 
 
The elimination rate constant (ke) will be determined by a linear regression analysis using the last 3–8 points 
on the plot of the natural logarithm of the plasma concentration-time curve. The t1/2 value will be calculated 
from the equation t1/2 = ln2/ke. AUC values will be calculated by the linear trapezoidal rule for the rising 
phase and the log-linear trapezoidal rule for the descending phase, with extrapolation to infinity, when 
appropriate, by dividing the last measured concentration by ke. Results will be expressed as mean values (± 
s.d.). 95% confidence intervals will be calculated for the mean differences between the baseline pravastatin 
and rifampin co-administration phases for AUC(0–∞), Cmax and t1/2. Statistical comparisons of the 
pharmacokinetic variables (except tmax) will be carried out by the Student t-test for paired values (two-
tailed), and the Wilcoxon signed-rank test will be used for analysis of tmax. Logarithmic transformation of 
Cmax and AUC values will be done prior to statistical analysis. The effect of rifampin (and other anti-TB 
drugs) on the AUC(0–∞) of pravastatin will be assessed with a t-test of log-transformed values. The analysis 
will be performed with Systat for Windows, V6.0.1 (SPSS Inc., Chicago, Ill). Statistical significance will 
be taken as P <0.05. 
 
Decision-making algorithm for pravastatin safety/PK study: 
 
 
Scenario/Pravastatin 
Dose 
Safe/tolerable? 
AUC0-24 
reduction 
Decision 
1 
40 mg daily 
Y 
- 
Increase dose to 
prava 80 mg and 
enroll 
additional 
participants 
2 
40 mg daily 
N 
- 
Enroll 5 additional 
participants 
(10 
total) 
and 
discontinue study 

53 
 
3 
80 mg daily 
N 
- 
Discontinue study  
4 
80 mg daily 
Y 
< 40% 
Discontinue study  
5 
80 mg daily 
 
Y 
 
> 40% 
Increase dose to 
prava 120 mg and 
enroll 
additional 
participants 
6 
120 mg daily 
N 
- 
Discontinue study  
7 
120 mg daily 
Y 
<40% 
Discontinue study  
8 
120 mg daily 
Y 
> 40% 
Increase dose to 
prava 160 mg and 
enroll 
additional 
participants 
9 
160 mg daily 
N 
- 
Discontinue study  
10 
160 mg daily 
Y 
- 
Study complete  
 
Safety/tolerability:  
Y= Yes; <3 participants with > one grade 3 (or higher) event of musculoskeletal 
symptoms, creatine kinase elevation, or liver function test elevation attributed to study drug (probable or 
definite) 
 
 
 
N= No; For Arm 1 (pravastatin 40 mg daily): > 1 participant with > one grade 3 
(or higher) event of musculoskeletal symptoms, creatine kinase elevation, or liver function test elevation 
attributed to study statin medication (probable or definite).  
For Arms 2, 3, and 4: > 3 participants with > one grade 3 (or higher) event of musculoskeletal symptoms, 
creatine kinase elevation, or liver function test elevation attributed to study statin medication (probable or 
definite) 
 
AUC0-24= Area-under-the-plasma-concentration-time curve for 24-hour exposure 
 
Decision-making algorithm for initiating new dosing cohorts  
 
The study will be monitored by a DSMB, which will review the safety data for each cohort and will give 
approval before initiation of each possible dose escalation. Specifically, study drug dose will not be 
escalated and the study if any three participants develop grade 3 or higher musculoskeletal/CK-related 
adverse events (AEs), LFT-related AEs (> 5 x UNL without symptoms, but > 3 x UNL with symptoms or 
bilirubin > 2 x ULN) or any type of grade 4 event regardless of causal attribution. Study drug dosage will 
be escalated only if the dose is deemed safe/tolerable based on the above rules and the mean pravastatin 
AUC is reduced by > 40% relative to the pre-Rifafour value. Study participants in whom the study drug has 
been discontinued will remain in the study until the final clinical visit and laboratory evaluation. In the 
event of study product discontinuation, management of the standard-of-care TB drugs will revert to the site 
clinician as per national guidelines.  
 
Early stopping rules 
Early stopping of the study will be considered if adverse events occur in a great proportion or with greater 
severity than would be expected at the dosage of the study drug being used based on the safety profile in 
patients with  cardiovascular disease. Upon reviewing the AE data, the DSMB will make a formal 
determination about early stopping of the study. 
 

54 
 
Recruitment strategy 
 
Eligible patients (who meet all of the inclusion criteria and none of the exclusion criteria) will be recruited 
in a step-wise fashion to the study arms.  
 
Although rifampin is known to reduce pravastatin plasma levels by 30-50% in healthy volunteers, the PK 
of pravastatin has not been studied during co-administration with the combination regimen Rifafour in 
patients with TB. Therefore, out of caution, we will begin to study pravastatin dosed at half the highest 
clinically recommended dose of pravastatin (40 mg/day). Drug exposures are expected to be lower 
following standard-of-care TB treatment (Rifafour) in the experimental group receiving pravastatin relative 
to pre-Rifafour pravastatin exposures.  
 
Participants meeting entry criteria will be recruited sequentially into each study arm for 10 -30 evaluable 
participants. 
 
This will be an open-label trial in which neither the participants nor the study staff will be blinded as to 
treatment assignment.  
 
19. Risks 
 
Institutional Review Board Involvement 
 
This research will be conducted in compliance with the requirements of the US DHHS regulations to protect 
human subjects from research risk (45 CFR Part 46), in compliance with South African regulations 
protecting human subjects, and with oversight by the IRBs of Johns Hopkins University School of Medicine 
(FWA #00005752) and the University of Witwatersrand (FWA #00000715). All study personnel will 
complete a course in protection of human subjects prior to participating in the study.   
 
The Investigator will ensure that the purpose of the study is explained to the participant and that written 
consent is obtained prior to participation in the study. The participant, Investigator or designee, and others 
as required by local regulatory guidelines will sign the consent prior to entry into the study. The participant 
will receive a copy of his/her signed consent form.  The Investigator will retain a copy of the signed consent 
forms, which may be inspected at the monitor’s or auditor’s request.  
 
The investigator will promptly report to the Ethics Committee/IRB and regulatory body of all changes in 
the research activity and all unanticipated problems involving risks to human subjects or others, and will 
not make changes in the research without Ethics Committee/IRB and regulatory body approval, except 
where necessary to eliminate apparent immediate hazards to human subjects. 
 
Written informed consent will be obtained by trained study personnel prior to screening for inclusion and 
exclusion criteria. Informed consent is a process that will be initiated prior to the individual’s agreeing to 
participate in the study and will continue throughout the individual’s study participation.  
 
Medical risks, listing all procedures, their major and minor risks and expected frequency. 
 
Risks associated with blood specimen collection include those related to venipuncture and blood loss. The 
risks of drawing blood from a vein include discomfort at the site of puncture; possible bruising and swelling 
around the puncture site; rarely an infection; and, uncommonly, faintness from the procedure. 
  

55 
 
Steps taken to minimize the risks 
 
Appropriate clinical practices will be performed by trained clinical and study staff to minimize 
complications from the risk mentioned above. 
 
Plan for reporting unanticipated problems or study deviations. 
 
The Investigator will promptly report to the Ethics Committee/IRB and regulatory body of all changes in 
the research activity and all unanticipated problems involving risks to human subjects or others, and will 
not make changes in the research without Ethics Committee/IRB and regulatory body approval, except 
where necessary to eliminate apparent immediate hazards to human subjects. 
 
Legal risks such as the risks that would be associated with breach of confidentiality. 
 
Risks to confidentiality will be minimized by the following procedures: study records will be stored in 
locked cabinets in secure locations, study records will be accessible only by authorized study staff, and 
databases will be password-protected and accessible only to authorized study staff. Participant names will 
not be stored in the study database. All electronic communications involving study data will be encrypted.  
Laboratory results will be maintained in a password-protected file accessible only to study personnel. The 
study monitor(s) or other authorized representatives of the Sponsor may inspect all documents and records 
required to be maintained by the investigator.  
Financial risks to the participants. 
 
There are no financial risks to the participants. Treatment for TB is routine and free in South Africa. 
Participants will receive reimbursement for study visits in accordance with SAHPRA guidelines. There will 
be no other costs to the participants. 
 
20.  Benefits 
 
The proposed research may not have direct benefit to individual study participants. The South African 
government provides HIV medicines, TB medicines, and prenatal care to its citizens free of charge. For 
individuals in this trial, follow-up visits and counseling may enhance these services. In addition, study-
related tests, such as viral load and resistance testing, may provide information regarding appropriate 
treatment that may benefit study participants directly. A better knowledge of the impact of statin adjunctive 
therapy for pulmonary TB is anticipated to inform public health practices and individual health activities to 
better treat drug-susceptible TB in HIV-infected and HIV-uninfected individuals. This will be especially 
pertinent in South Africa, a country that bears a disproportionate burden of HIV/TB co-infection.  
 
21.  Payment and Re-numeration 
 
Participants will receive reimbursement in line with South African guidelines and approved by the local 
Ethics Committee or SAHPRA. 
 
22. Costs 
 
There are no costs to the participants. 

56 
 
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60 
 
 
Appendix 1 
 
Screening9 
 (Visit Window Days 1-15) 
 
 
 
 
Unscheduled 
Visit 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
21 
30 
 
Informed consent 
 
      x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confirm eligibility 
 
      x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV testing/documentation 
      x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CD4 count (if applicable) 
      x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Treatment Assignment 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Complete Physical Exam  
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
Vital signs (heart rate, 
blood pressure, 
temperature, respiratory 
rate 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Targeted symptom 
assessment 
 
x 
x 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
x 
x 
 
x 
x 
Weight 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse event assessment 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
x 
x 
x 
x 
Concomitant Medications 
 
x 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
x 
 
 
x 
Liver Function Tests1  
x 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
x 
 
x 
 
x 
x 
Viral hepatitis serologies10 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

61 
 
 
Appendix 1 
 
Screening9 
 (Visit Window Days 1-15) 
 
 
 
 
Unscheduled 
Visit 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
21 
30 
 
CK and serum creatinine 
x 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
x 
 
x 
 
x 
 
Chemistry2 
 
 
x 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
x 
 
x 
 
x 
x 
Sputum  Xpert  
 
MTB/RIF 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sputum AFB Smear and 
Culture In Liquid/Solid 
Media3 
(with DST) 
x 
x 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
x 
 
 
 
PK Sampling (intensive)  
 
X4 
X8 
 
 
 
 
 
 
 
 
 
 
 
 
 
X4 
 
 
 
Urine pregnancy test 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
x 
 
Chest X-ray 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
DOT 
 
 
X5 
X6 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
x 
 
 
 
Lung Inflammation 
Assessment7 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
x 
 
 
1Liver function tests (AST/ALT, T. bilirubin)  
2 Chemistry (Comprehensive metabolic panel, total cholesterol, LDL, C-reactive protein, uric acid) 
3Two sputum samples for AFB smear microscopy and culture in liquid and solid media will be collected at each time point; sputum specimens and culture isolates will be stored 

62 
 
for culture-based drug-susceptibility testing and targeted deep sequencing in the event of TB treatment failure or TB recurrence. 
4 PK (intensive sampling) 
5 DOT of statin only 
6 DOT of statin and first dose of TB treatment 
7Pulmonary function test, St George’s Respiratory Questionnaire, 6-minute walk test; +/- 3 days 
8  Sparse PK 
9   The Screening evaluation process itself may occur over a few days; all Screening evaluations will be obtained within 3 days of study entry. 
     
10 Viral hepatitis serologies;  IgM antibody for Hepatitis B surface antigen, IgM antibody to Hepatitis B core antigen, antibody to Hepatitis C