Conditions: Rifampicin Susceptible Pulmonary Tuberculosis
Keywords: Early Bactericidal Activity (EBA)
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 20
Healthy volunteers: no

Brief summary:
A Phase 2, Single-Centre, Open-Label, Parallel Control Arm, Randomised Clinical Study to Evaluate the Early Bactericidal Activity (EBA), Safety and Tolerability of Nebulised RESP301 in Adults with Newly Diagnosed, Rifampicin Susceptible Pulmonary Tuberculosis

Detailed description:
A total of approximately 15 participants will be recruited per treatment arm (total of approximately 75 participants in the study). Control arm participants will be split across sequential stages stages 1 and 2, with no stratification.

Stage 1: To determine the EBA of

* Treatment Arm 1 (Active; n = 15) - inhaled RESP301 6 ml dosed via nebulisation administered three times daily over 14 days
* Treatment Arm 2 (Control; n= 5) - HRZE taken orally once daily

On completion of Stage 1, recruitment will be paused and an interim analysis performed to determine whether the study should proceed to Stage 2.

Stage 2: To determine the EBA of

* Treatment Arm 2 (Control; n= 10) - HRZE taken orally once daily
* Treatment Arm 3 (Active; n = 15) - inhaled RESP301 6 ml dosed via nebulisation administered once daily over 14 days
* Treatment Arm 4 (Active; n = 15) - inhaled RESP301 6 ml dosed via nebulisation administered twice daily over 14 days
* Treatment Arm 5 (Active; n = 15) - inhaled RESP301 6 ml dosed via nebulisation administered three times daily in combination with HRZE taken orally once daily over 14 days

Arms:
arm 1: Inhaled RESP301 6ml via nebulisation three times daily arm 2: HRZE taken orally once daily arm 3: Inhaled RESP301 6 ml via nebulisation once daily arm 4: Inhaled RESP301 6 ml via nebulisation twice daily arm 5: Inhaled RESP301 6 ml via nebulisation three times daily plus HRZE taken orally once dialy

Interventions: intervention 1: RESP301 intervention 2: HRZE
Intervention descriptions: intervention 1: Nitric Oxide agent intervention 2: isoniazid 75 mg (H), rifampicin 150 mg (R), pyrazinamide 400 mg (Z), ethambutol 275 mg (E)

Protocol document:
Prot_000.pdf: 
Protocol No: RESP30X-EBA 
Version 1.0_25 May 2023 
Page 1 of 81 
 
 
 
CONFIDENTIAL 
 
 
 
Protocol Title: 
 
A Phase 2 Study to Evaluate the Early Bactericidal Activity, Safety and Tolerability 
of Nebulised RESP301 in Adults with Newly Diagnosed, Rifampicin Susceptible 
Pulmonary Tuberculosis 
 
Protocol Number: 
RESP30X-EBA 
Protocol Version: 
1.0 
Protocol Date: 
25 May 2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Version History: 
 
Version Number _ Date 
Incorporates 
v1.0 _ 06 April 2023 
Initial 
v1.0 _ 25 May 2023 
Administrative change – PI details 


Protocol No: RESP30X-EBA 
Version 1.0_25 May 2023 
 
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Sponsor Protocol Signature Page 
 
Protocol Title: A Phase 2 Study to Evaluate the Early Bactericidal Activity, Safety and Tolerability of 
Nebulised RESP301 in Adults with Newly Diagnosed, Rifampicin Susceptible Pulmonary Tuberculosis 
 
Protocol Number: RESP30X-EBA 
 
Protocol Date: 25 May 2023 
 
Sponsor: Thirty Respiratory Limited (30 Respiratory), 1 Red Place W1K 6PL, London, United Kingdom 
 
I agree to the terms of this study protocol. 
 
 


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Version 1.0_25 May 2023 
 
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Principal Investigator Protocol Signature Page 
 
Protocol Title: A Phase 2 Study to Evaluate the Early Bactericidal Activity, Safety and Tolerability of 
Nebulised RESP301 in Adults with Newly Diagnosed, Rifampicin Susceptible Pulmonary Tuberculosis 
 
Protocol Number: RESP30X-EBA 
 
 
Protocol Date: 25 May 2023 
 
Sponsor: Thirty Respiratory Limited (30 Respiratory), 1 Red Place W1K 6PL, London, United Kingdom 
 
Principal Investigator:   
 
I hereby confirm that I have read the above protocol and agree to conduct this clinical study as outlined 
in the above protocol. I will work in accordance with the ethical principles that have their origin in the 
Declaration of Helsinki and with the principles of Good Clinical Practice and local regulations. I will 
provide copies of the protocol and access to all the information required to conduct the clinical study 
according to the above protocol to the site personnel under my supervision. I will discuss this material 
with them and ensure they are fully informed on all study requirements. 


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Development Phase: 
2 
Funding by: 
Thirty Respiratory Limited (30 Respiratory) 
Sponsor: 
Thirty Respiratory Limited (30 Respiratory) 
1 Red Place W1K 6PL 
London 
United Kingdom 
Sponsor Representative(s): 
 
 
Thirty Respiratory Limited 1 Red Place 
London, W1K 6PL 
Principal Investigator 
  
TASK Clinical Research Centre  
1 Smal Street, Bellville 7531 
Cape Town, South Africa  
 
 
Email:  
 


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Table of Contents 
 
1. 
Protocol Synopsis .......................................................................................................................... 11 
1.1. 
Synopsis ................................................................................................................................. 11 
1.2. 
Evaluations ............................................................................................................................. 16 
1.3. 
Study Flow Chart for Stages 1 and 2 ...................................................................................... 18 
2. 
Introduction ................................................................................................................................... 20 
2.1. 
Background Information ......................................................................................................... 20 
2.1.1. 
Tuberculosis .................................................................................................................... 20 
2.1.2. 
Early Bactericidal Activity Clinical Studies ....................................................................... 21 
2.2. 
RESP301 ................................................................................................................................ 21 
2.2.1. 
Mechanism of Action....................................................................................................... 21 
2.2.2. 
Non-clinical studies ......................................................................................................... 22 
2.2.3. 
Clinical studies ................................................................................................................ 31 
2.3. 
HRZE ...................................................................................................................................... 40 
2.4. 
Known and Potential Risks and Benefits of the RESP301 ....................................................... 40 
2.5. 
Overall Benefit/Risk Assessment ............................................................................................ 41 
3. 
Study Rationale and Objectives ..................................................................................................... 42 
3.1. 
Study Rationale ...................................................................................................................... 42 
3.2. 
Dose Rationale ....................................................................................................................... 42 
3.3. 
Study Objectives .................................................................................................................... 42 
3.3.1. 
Primary Objectives .......................................................................................................... 42 
3.3.2. 
Secondary Objective ....................................................................................................... 43 
3.3.3. 
Exploratory Objective ...................................................................................................... 43 
4. 
Study Design ................................................................................................................................. 43 
4.1. 
Summary of Study Design ...................................................................................................... 43 
4.2. 
Study Population .................................................................................................................... 44 
4.2.1. 
Inclusion Criteria ............................................................................................................. 44 
4.2.2. 
Exclusion Criteria ............................................................................................................ 45 
4.3. 
Restrictions............................................................................................................................. 46 
4.3.1. 
Foods and Beverages ..................................................................................................... 46 
4.3.2. 
Type of Pulmonary Tuberculosis ..................................................................................... 46 
4.3.3. 
Concomitant Medications and Procedures ..................................................................... 47 
4.3.4. 
Pregnancy and Contraception ........................................................................................ 47 
4.3.5. 
HIV .................................................................................................................................. 49 


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4.4. 
Treatment Discontinuation and Participant Withdrawal........................................................... 49 
4.5. 
Study Stopping Rules ............................................................................................................. 51 
4.6. 
End of Study ........................................................................................................................... 51 
5. 
Investigational Medicinal Product (IMP) ......................................................................................... 51 
5.1. 
Study Treatments ................................................................................................................... 51 
5.2. 
Randomization and Blinding ................................................................................................... 52 
5.2.1. 
Screening ........................................................................................................................ 52 
5.2.2. 
Study enrolment and randomization ................................................................................ 52 
5.2.3. 
Blinding ........................................................................................................................... 52 
5.3. 
IMP Administration ................................................................................................................. 53 
5.3.1. 
Route and Dosage .......................................................................................................... 53 
5.3.2. 
Dosing Instructions ......................................................................................................... 53 
5.3.3. 
Participant Compliance ................................................................................................... 54 
5.3.4. 
Manufacturing, Packaging and Labelling......................................................................... 55 
5.3.5. 
Storage ........................................................................................................................... 55 
5.4. 
Dispensing and Accountability ................................................................................................ 55 
5.5. 
Returns and Destruction ......................................................................................................... 55 
6. 
Study Assessments ....................................................................................................................... 56 
6.1. 
Demographic and Baseline Assessments ............................................................................... 56 
6.2. 
Activity Assessments .............................................................................................................. 57 
6.3. 
Safety and Tolerability Assessments....................................................................................... 57 
7. 
Adverse Events .............................................................................................................................. 59 
7.1. 
Definitions............................................................................................................................... 59 
7.2. 
Reporting ................................................................................................................................ 62 
7.3. 
Follow up of Adverse Events ................................................................................................... 64 
7.4. 
Post-Study Serious Adverse Events ....................................................................................... 64 
7.5. 
Clinical Laboratory Adverse Events ........................................................................................ 64 
7.6. 
Disease under Study .............................................................................................................. 65 
7.7. 
Overdose ............................................................................................................................... 65 
7.8. 
Drug Interaction ..................................................................................................................... 65 
7.9. 
Pregnancy .............................................................................................................................. 65 
7.10. Monitoring and Safety for Specific Toxicities........................................................................... 65 
7.10.1. 
Allergy ......................................................................................................................... 66 
7.10.2. 
Methaemoglobinaemia ................................................................................................ 66 
7.10.3. 
Overdose .................................................................................................................... 67 


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7.10.4. 
Any other toxicities ...................................................................................................... 67 
7.11. Safety Review Committee ....................................................................................................... 67 
8. 
Statistical Analysis ......................................................................................................................... 67 
8.1. 
Analysis Population/s .............................................................................................................. 68 
8.2. 
Sample Size............................................................................................................................ 68 
8.3. 
Interim Review ........................................................................................................................ 68 
8.4. 
Endpoint Analysis ................................................................................................................... 68 
8.4.1. 
Primary Endpoint Analysis: Activity.................................................................................. 68 
8.4.2. 
Secondary Endpoints Analysis: ....................................................................................... 68 
8.4.3. 
Exploratory Endpoints Analysis: Activity .......................................................................... 69 
9. 
Records Management ................................................................................................................... 69 
9.1. 
Data Collection ....................................................................................................................... 69 
9.2. 
Source Documents ................................................................................................................. 70 
9.3. 
File Management at the Study Site ......................................................................................... 70 
9.4. 
Records Retention at the Study Site ....................................................................................... 70 
10. Quality Control and Assurance ...................................................................................................... 70 
10.1. Site Procedures ...................................................................................................................... 70 
10.2. Monitoring .............................................................................................................................. 71 
10.3. Auditing .................................................................................................................................. 71 
11. Ethics and Regulatory .................................................................................................................... 72 
11.1. Basic Principles ...................................................................................................................... 72 
11.2. Independent Ethics Committee/Institutional Review Board Review ......................................... 72 
11.3. Regulatory Authorities ............................................................................................................ 72 
11.4. Informed Consent ................................................................................................................... 72 
11.5. Confidentiality ......................................................................................................................... 73 
12. Incidental Findings ......................................................................................................................... 73 
13. Publication Policy ........................................................................................................................... 73 
14. Protocol Amendment Policy ........................................................................................................... 74 
15. Financial Aspects, Insurance and Indemnity .................................................................................. 74 
16. References .................................................................................................................................... 75 
 
 
 
 
 
 
 


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List of Abbreviations and Definitions of Terms 
AE 
Adverse Event 
AFB 
acid-fast bacteria 
ART 
Antiretroviral Therapy 
ALP 
alkaline phosphatase 
ALT 
Alanine Aminotransferase 
API 
Active Pharmaceutical Ingredient 
ASL 
Airway Surface Liquid 
AST 
Aspartate Aminotransferase 
AUC 
Area Under the Plasma Concentration Time Curve 
bd 
Twice daily 
BMI 
Body Mass Index 
CF 
Cystic Fibrosis 
CFU 
Colony Forming Units 
COPD 
Chronic Obstructive Pulmonary Disease  
(e)CRF 
(electronic)Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
(PA) CXR 
(Posteroanterior) Chest X-ray 
DBP 
Diastolic Blood Pressure 
DR 
Drug Resistant 
DS 
Drug Sensitive 
EBA 
Early Bactericidal Activity 
EC 
Ethics Committee 
EWD 
Early Withdrawal 
FEV1 
Forced Expiratory Volume in the first second 
FSH 
Follicle Stimulating Hormone 
GCP 
Good Clinical Practice 
GGT 
Gamma-Glutamyl transferase 
GMP 
Good Manufacturing Practice 
HIV 
Human Immunodeficiency Virus 
HRT 
Hormone Replacement Therapy 
HRZE 
Isoniazid/Rifampicin/Pyrazinamide/Ethambutol 
IB 
Investigator Brochure 
ICF 
Informed Consent Form 
IF 
Incidental Findings 
IMP 
Investigational Medicinal Product 
ISF 
Investigational Site File 
IV 
Intravenous 
IUATLD 
International Union Against Tuberculosis and Lung Disease 
MDR 
Multi Drug Resistant 
MGIT 
Mycobacterial Growth Indicator Tube 
MIC 
Minimum Inhibitory Concentration 
MRSA 
methicillin-resistant Staphylococcus aureus 
M.tb 
Mycobacterium Tuberculosis 
NTM 
Non tuberculosis mycobacteria 


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NEB 
Nebulisation 
NO 
Nitric oxide 
NO2 
Nitrogen dioxide 
NRTI 
Nucleoside Reverse Transcriptase Inhibitor 
OECD 
Organisation for Economic Co-operation and Development 
OCP 
Oral Contraceptive Pill 
PI 
Principal Investigator 
po 
per os/orally 
PoC 
proof of concept 
qd 
once daily 
QTcF 
QT interval corrected with the Fridericia formula 
PTB 
Pulmonary tuberculosis 
RR 
Rifampicin resistant 
SAE 
Serious Adverse Event 
SABA 
short-acting beta agonist 
SAP 
Statistical Analysis Plan 
SBP 
Systolic Blood Pressure 
SIFT-MS 
Selected Ion Flow Tube Mass Spectrometry 
SOC 
System Organ Class 
 SpMet 
Methaemoglobin saturation 
t 
Time 
TASK 
TASK Applied Science (Pty) Ltd 
TB 
Tuberculosis 
tds 
three times a day 
TEAEs 
Treatment-Emergent Adverse Events 
TMF 
Trial Master File 
TTP 
Time to Sputum Culture Positivity 
ULN 
Upper Limit of Normal 
WHO 
World Health Organization 
WOCBP 
Women of Child-Bearing Potential 
WONCBP 
Women of Non Child-Bearing Potential 
XDR 
Extensively Drug Resistant 
 
 
EBA Definitions 
EBA 
An agent’s ability to kill mycobacteria expectorated in sputum during the first weeks 
of treatment. 
EBA CFU 
Determination of EBA by quantification of viable mycobacteria as colony forming 
units (CFU) on a solid media culture. 
EBA TTP 
Determination of EBA by quantification of viable mycobacteria as time to culture 
positivity (TTP) in an automated liquid culture system. 
 


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1. Protocol Synopsis 
1.1. Synopsis 
Sponsor: 
Thirty Respiratory Limited (30 Respiratory) 
1 Red Place W1K 6PL 
London 
United Kingdom 
Funder: 
Thirty Respiratory Limited (30 Respiratory) 
Study Drug: 
RESP301 
Protocol Number 
RESP30X-EBA 
Protocol Title: 
A Phase 2 Study to Evaluate the Early Bactericidal Activity, Safety and 
Tolerability of Nebulised RESP301 in Adults with Newly Diagnosed, 
Rifampicin Susceptible Pulmonary Tuberculosis 
Treatment Indication: 
Rifampicin Susceptible Pulmonary Tuberculosis 
Test Product, Dose and 
Mode of Administration: 
The Investigational Medicinal Product (IMP) will be supplied as 
RESP301 6 mL solution, packaged in two differing 3.0 mL vials: 
• 
Active vial: Sodium nitrite 
 mannitol 
 
solution 
• 
Diluent vial: Citric acid 
  
These precursor solutions are mixed at point of use for inhaled 
administration via nebulisation (NEB), resulting in the release of Nitric 
oxide (NO) throughout the respiratory system, from the upper airways to 
deep into the lungs. 
Assuming a 100% conversion rate, theoretically 
 of NO is 
delivered. However due to intra- and inter-patient variations, the 
delivered dose is estimated to be ~40%-60% of this dose (section 3.2). 
Dosing is once daily (qd), twice daily (bd) or three times a day (tds) 
dependent on the study treatment arm, for 14 days. 
Control Product Dose and 
Mode of Administration: 
The Control Product will be supplied as isoniazid 75 mg (H), rifampicin 
150 mg (R), pyrazinamide 400 mg (Z), ethambutol 275 mg (E) (i.e., 
HRZE) combination tablets. Dosing is qd for fourteen days based on 
participants body weight rounded up to the nearest whole number: 
• 38 to 54 kg: 3 tablets 
• 55 to 70 kg: 4 tablets  
• 71 kg and over: 5 tablets 
The participants’ screening weight will be used to determine the HRZE 
dose and this dose will be administered for the duration of the study. 
Primary Study Objectives: 
Activity 
Stage 1: 
To determine the Early Bactericidal Activity (EBA) of tds dosing of 
inhaled RESP301 6 mL dosed via NEB over 14 days of treatment. 
Stage 2: 
To determine the EBA of inhaled RESP301 6 mL dosed via NEB 
administered qd, bd alone, or tds in combination with HRZE, over 14 
days of treatment to optimise the dosing schedule. 
Study Design: 
The study is a single-centre, open-label, parallel arm, randomised 
clinical study in two sequential stages, with no stratification. The study 




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Figure 1: Trial schematic 
 
 
HRZE: Isoniazid 75 mg /Rifampicin 150 mg /Pyrazinamide 400 mg /Ethambutol 275 mg. NEB: 
nebulisation, bd: twice daily, po: per os/orally, qd: once daily, tds: three times a day 
 
The participant will be admitted to the EBA unit during screening and 
discharged on day 16 of the study. During the treatment period daily 
study “visits” will occur where study assessments are performed, unless 
the participant is withdrawn early from the study. The follow-up visit will 
occur for all participants (completed treatment or early withdrawals).  
 
On discharge from the EBA unit, participants will be referred to the local 
community TB clinics to continue standard anti-tuberculosis 
chemotherapy according to national guidelines. The participants will be 
provided with a referral letter and sufficient TB medication to cover the 
time from attending their last visit at the EBA unit until their scheduled 
visit at the TB clinic. This will be documented in the participants source 
documents. 
Patient Population: 
A total of approximately 75 male and female participants, aged between 
18 and 65, inclusive, with sputum positive newly diagnosed, rifampicin 
susceptible pulmonary tuberculosis (PTB), will be enrolled onto the 
study. Approximately 20 participants will be enrolled in Stage 1 and 55 
participants enrolled in Stage 2. Participants who withdraw (section 4.4) 
may be replaced. Participants will be enrolled at the TASK centre in 
South Africa. 
Eligibility Criteria 
Inclusion Criteria 
Participants are required to meet all the following criteria to be 
randomized. 
1. Provide written, informed consent prior to all study-related 
procedures and agree to undergo all study procedures. 
2. Male or female, aged between 18 and 65 years, inclusive. 
3. Body weight (in light clothing and with no shoes) between 40 
and 90 kg, inclusive. 
4. Newly diagnosed pulmonary TB. 
5. Either GeneXpert Mycobacterium tuberculosis (M.tb) positive 
with a quantitative readout of medium or high, OR acid-fast 
bacteria (AFB) sputum smear positive with a grade of at least 
1+ on the IUATLD/WHO scale (Appendix 3: ), on at least one 
pre-treatment sputum sample. 


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6. Rifampicin susceptible pulmonary TB as determined by 
molecular testing. 
7. Ability to produce an adequate volume of sputum as estimated 
from a pre-treatment overnight sputum collection sample 
(estimated 10 mL or more). 
8. A chest X-ray taken during the screening period or up to 2 
weeks before screening which, in the opinion of the investigator, 
is consistent with pulmonary TB. 
9. Spirometry performed during screening with a Forced Expiration 
Volume in the first second (FEV1) of ≥ 40%. 
10. Be of non-childbearing potential or willing to use effective 
methods of contraception, as defined in section 4.3.4. 
 
Exclusion Criteria 
Participants will be excluded from participation if they fulfil any of the 
following criteria. 
 
Medical History: 
1. Evidence of clinically significant conditions or findings, other 
than TB, that might compromise safety or the interpretation of 
study endpoints, per discretion of the investigator. 
2. Clinical evidence of extra-thoracic TB, that might compromise 
safety or the interpretation of study endpoints, per discretion of 
the investigator. 
3. History of allergy to any of the study IMP as confirmed by the 
clinical judgement of the investigator. 
4. Alcohol or drug abuse, that in the opinion of the investigator, is 
sufficient to compromise the safety or cooperation of the 
participant. 
5. HIV positive AND: 
(a)  CD4 < 350 cells/mm3 
(b)  OR are receiving antiviral therapy (ART). 
6. Methaemoglobin saturation (SpMet) >3%. 
7. Female participant who is pregnant or breast-feeding. 
8. Participants planning to conceive a child within the anticipated 
period of study participation and for at least 90 days after the 
last dose of IMP in the study. 
 
Treatment History 
9. Participation in other clinical studies with investigational agents 
within 8 weeks prior to screening. 
10. Treatment received for this episode of TB with any drug active 
against M.tb [fluoroquinolones (including moxifloxacin, 
levofloxacin, ofloxacin and ciprofloxacin), bedaquiline, 
pretomanid, delamanid, ethionamide, isoniazid, ethambutol, 
amikacin, cycloserine, rifabutin, rifampicin, streptomycin, 


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kanamycin, pyrazinamide, para-aminosalicylic acid, rifapentine, 
thioacetazone, capreomycin, thioamides]. 
11. Treatment with immunosuppressive medications such as TNF-
alpha inhibitors within 2 weeks prior to screening, or systemic 
corticosteroids for more than 7 days within 2 weeks prior to 
screening. 
12. Treatment with nitric oxide and other nitric oxide donor agents 
(e.g., prilocaine, sodium nitroprusside and nitro-glycerine), 
phosphodiesterase inhibitors (e.g., sildenafil) and lung 
surfactant drugs (e.g., beractant, calfactant, poractant), within 
30 days prior to screening. 
 
Laboratory Safety Testing 
13. Participants with the following toxicities at screening as defined by 
the enhanced Common Terminology Criteria for Adverse Events 
(CTCAE) toxicity table version 5.0, 27Nov2017 ( 
14. Appendix 1) 
(a)  creatinine >1.5 times upper limit of normal (ULN) 
(b)  haemoglobin <8.0 g/dL 
(c)  platelets <50x109 cells/L 
(d)  serum potassium <3.0 mmol/L 
(e)  aspartate aminotransferase (AST) >3 x ULN 
(f) alanine aminotransferase (ALT) >3 x ULN 
(g)  alkaline phosphatase (ALP) ≥ 2.5 x ULN 
(h)  total bilirubin >1.5 x ULN 
(i) total white cell count <2 cells x 109/L. 
NOTE: Investigations may be repeated once during screening if the 
 
initial result is unfavourable. 
Study Duration 
Participants who complete the study will participate for a maximum of 
37 days (screening period of up to 9 days, followed by a treatment 
period of 16 days and a follow-up visit at Day 28 from first dose of IMP). 
 
The overall duration of participants involvement in the study is planned 
to be approximately 1.5 years (first participant in to last participant out, 
including the pause between stages 1 and 2 for the interim analyses and 
continuation decision). 
Sample Size 
This is a proof-of-concept study without hypothesis testing. The planned 
sample size of 15 participants per treatment group is in keeping with 
other phase 2 studies of this type and accounts for the possibility of up 
to 3 dropouts per arm which represents a conservative estimate of the 
expected drop-out rate. Previous EBA studies indicate that a sample 
size of 12 participants per arm allowed for the identification of proof-of-
concept i.e., change in TTP/CFU compared to no change in TTP/CFU 
for all evaluated arms. 
 








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A:  
Follow-up visit: a window of ±3 days allowed. 
B: 
Physical examinations: Full will be complete at screening and targeted (symptom-directed) thereafter. 
C:  
Vital signs, safety laboratory: on days where there is IMP dosing, to be performed pre-dose. 
D: 
IMP administration: The HRZE is to be administered fasted (1 hour before, or 2 hours after food) with approximately 240 mL of water. For study purpose this 
food should be breakfast. Patient identification is part of the IMP administration process. Experimental arms will not receive IMP on Day 15, only control arm 
will receive HRZE. 
E: 
Concomitant Medication: collected from 14 days prior to first dose of IMP. 
F:  
Adverse Events: Collected from first dose of IMP to Follow-Up visit. 
G:  
Eligibility Assessment: Continuous from screening to just prior to first IMP dose. 
H: 
EBA unit inpatient: admission, anytime from initial screening to day -2, whichever is in the participant’s best interest. 
I: 
EBA unit inpatient: discharge. 
J: 
Randomisation: May occur any time after eligibility has been confirmed to just prior to first IMP dose. 
K:        Spot or Overnight Sputum: A screening and the last positive sputum sample (if >7 days after treatment start) on M.tb culture will be retained by the 
mycobacteriology laboratory for possible future testing. These samples contain the mycobacterium genetic material only, no participant genetic material is 
retained. 
L: 
Spot or Overnight Sputum: For eligibility assessments. 
M:         Overnight sputum: Collection will start in the afternoons at approximately 15h00 and continue for 16 hours ± 1 hour overnight. The 16-hour sputum 
sampling for each of the sampling days must be finished prior to the administration of the next morning’s IMP.  Collection will be paused for 1 hour after any 
RESP301 dose that overlaps with the overnight sample collection window where applicable. If sputum collection is paused, participants to drink a cup of 
water before restarting (section 5.3.2). 
N: 
Methaemoglobin: Screening single measurement. Days 1, 8 and 14: continuous measurement for an hour ± 10 mins from start of treatment on the first dose 
of that day only. 
O:  
β-HCG Serum Pregnancy Test: All female participants (section 4.3.4). 
 


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2. Introduction 
2.1. Background Information 
2.1.1. Tuberculosis 
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb) infection, remains a concerning global 
health problem. TB is present in all countries and age groups. In South Africa, the estimated burden of 
bacteriologically confirmed pulmonary TB among people aged 15 years or older in 2017–19 was 852 
cases per 100 000 population. (Moyo S, 2022 Aug)  Worldwide a total of 1.6 million people died from TB 
in 2021, with TB the 13th leading cause of death and the second leading infectious killer after COVID-19 
(above HIV/AIDS). Eight countries in Africa and Asia accounted for more than two thirds of the global TB 
cases reported in 2021, with an estimated 10.6 million people falling ill with TB worldwide. However, the 
COVID-19 pandemic had a damaging impact on access to TB diagnosis and treatment and the burden 
of TB disease. The most obvious and immediate impact was a large global drop in the reported number 
of people newly diagnosed with TB. Reductions in the reported number of people diagnosed with TB in 
2020 and 2021 suggest that the number of people with undiagnosed and untreated TB has grown, 
resulting first in an increased number of TB deaths and more community transmission of infection and 
then, with some lag-time, increased numbers of people developing TB. The burden of drug-resistant TB 
(DR-TB) is also estimated to have increased between 2020 and 2021, with 450 000 new cases of 
rifampicin-resistant TB (RR-TB) in 2021. (WHO, 2022). Whilst official numbers have yet to be published, 
it is accepted in scientific circles that TB is once again the leading cause of death due to infectious 
disease. 
 
Whilst drug-sensitive (DS)-TB is a treatable and curable disease, it is treated with a standard 4-month or 
6-month course of four antimicrobial drugs. For this treatment to be effective and not lead to an increased 
likelihood of developing drug-resistance, adherence is essential. (WHO, 2022) However, the duration of 
treatment plus the treatment side effects often does result in poor adherence. Overall, the current long-
standing first-line antituberculosis agents are relatively ineffective in controlling the TB epidemic, 
especially in high-burden countries.  
 
Drug-resistant (DR)-TB also poses a major threat to the control of the TB epidemic. Strains that are 
resistant to one or more of the standard TB treatments have been documented in every country surveyed 
by the WHO, who note that multidrug resistant (MDR)-TB, resistance to rifampicin and isoniazid, remains 
a public health crisis and a health security threat. Globally, there were an estimated 450 000 incident 
cases of MDR/Rifampicin resistant (RR)-TB in 2021, up 3.1% from 437 000 in 2020. An estimated 191 
000 deaths occurred due to MDR/RR-TB in 2021. Globally in 2021, the estimated proportion of MDR/RR-
TB cases with even more extensive resistance, was 20%. (WHO, 2022) 
 
Significant progress has been made in recent years in identifying more efficacious, safer medicines and 
shorter treatment regimens. However current treatment regimens for MDR/RR-TB patients are far from 
satisfactory. Compared with treatments for DS-TB, these regimens require a longer course of treatment 
(minimum 6 months), a higher pill burden, and the use of medicines with a higher toxicity profile. In 
addition, patients may develop significant adverse events and have poorer treatment outcomes. Globally, 
although treatment success rates have increased, almost 15% of patients with MDR/RR-TB or more 
advanced forms of DR-TB die from the disease. (WHO, 2022). 
 
 




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At high concentrations in the micromolar range (≥1 M ) and above, NO: 
•
directly impairs bacterial enzyme activity by irreversibly binding iron sulphur cluster residues
and inhibit cellular respiration by binding heme groups within cytochrome oxidases. (Wink DA,
1998) (Thomas DD, 2008) (Radi R, 2018)
•
reacts with oxygen and superoxide spontaneously to generate further species that can cause
protein damage, through thiol and tyrosine nitrosylation, DNA damage through base
deamination, and damage to membranes and lipid structures through lipid peroxidation. (Hogg
N, 1999) (Toledo JC, 2012)
At low concentrations in the nanomolar range (≤1 M ), NO regulates and can promote the growth and 
activity of immune cells by e.g., guanylate cyclase activation. (Tripathi P, 2007 ) (Schairer DO, 2012) 
NO is thus an integral, highly conserved, part of the host immune response, with very limited evidence of 
bacterial resistance to its antimicrobial effect. (Andreakis N, 2011) (Joerink M, 2006) (Privett BJ, 2012) 
Inhaled Nitric Oxide gas is already marketed as a therapeutic agent in the US (INOmax) where it is 
indicated to improve oxygenation in neonates with hypoxic respiratory failure. (FDA, 2015)  
2.2.2. Non-clinical studies 
Nonclinical studies to assess the pharmacological, pharmacokinetic, and toxicological properties for 
sodium nitrite, which is the drug substance in RESP301 
In addition to the information available in the public domain, the sponsor has performed several supportive 
non-GLP nonclinical studies. Both these supportive sources are summarised below.  
 4 
Please refer to the RESP301 IB for full details. 
2.2.2.1. 
Primary Pharmacodynamics 
.












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Refer to IB for information on other routes of administration. 
 
Distribution  
Intratracheal and intravenous administration of radiolabelled 13N-nitrite to mice and rabbits showed an 
even distribution throughout the soft tissues and organs within 5 minutes after injection, most of it in the 
form of nitrate. (Parks N, 1981) Subsequent studies have determined the apparent volume of distribution 
to be 93 L (64–124 L), which suggests that nitrite distributes well into tissues when compared with blood. 
(Kortboyer JM, 1997)  
 
Metabolism  
In man, nearly all nitrite is converted to nitrate then excreted in urine. (Hunault CC, 2009) Further 
metabolites of nitrite are NO and reactive oxygen species formed during the conversion of nitrite to minor 
metabolites.  
 
Following inhalation of NO, the gas combines with haemoglobin, forming nitrosyl-haemoglobin (NOHb) 
from which, nitrites (NO2) and nitrates (NO3) are generated. In the presence of oxygen, rapid oxidation of 
NOHb into MetHb occurs; this is subsequently reduced by methaemoglobin reductase into ferrous Hb 
and nitrate. Following inhalation, a small amount is discharged in the oral cavity by way of the salivary 
glands. Nitrite is converted to N2 gas in the stomach. Nitrate in the intestine is partly reduced to ammonia 
(NH3), re-absorbed in the body, and converted to urea. (European Medicines Agency, 2021) 
 
Excretion  
There is evidence to suggest that nitrites and nitrates are primarily excreted via the urine. Nitrate has 
been identified as the predominant nitric oxide metabolite excreted in the urine, accounting for > 70 % of 
the nitric oxide dose inhaled. Nitrate is cleared from the plasma by the kidney at rates approaching the 
rate of glomerular filtration. (European Medicines Agency, 2021) 
 
2.2.2.6. 
Toxicology 
 
 
) In addition, repeated dose studies in the mouse and rat along with 
genotoxicity, carcinogenicity and reproductive toxicology studies have been performed under the 
direction of the National Toxicology Program (USA). Review of the respective publications suggests that 
these studies were performed in accordance with Good Laboratory Practice and conducted within an 
Organisation for Economic Co-operation and Development (OECD) member state. For the remaining 
publications, it cannot be verified whether the studies cited complied with GLP regulations; however, it is 
assumed that the studies conducted by the investigator would have been conducted in compliance with 
the standards prevailing at the time.  
 
 
.  
 
These are summarised below. 
 
Single-Dose Toxicity Studies  


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In the rat, inhalation of sodium nitrite at up to ~11 mg/kg (0.95 mg/L or 13.8 μM solution) for 4 hours was 
well tolerated with increased methaemoglobin. (OECD SIDS, 2005) The oral lethal dose at 50 percent 
(LD50) values for rats and mice are 180 and 215 mg/kg, respectively (Smyth HF, 1969)(OECD SIDS).  
 
Repeat-Dose Toxicity Studies  
Rats 
 
 
 It was found that methaemoglobin was increased in all 
dose groups as a function of dose. No cumulative effect of repetitive dosing on methaemoglobin levels 
was observed at the doses evaluated. In the decedents, mild lung oedema, moderate congestion as well 
as moderate vacuolation of the vomeronasal organ was observed. Among animals surviving the full 
treatment period, histopathology included minimal perivascular mixed cell infiltrates of the lung in both 
the control and animals at the highest dose (72/101 mg/kg, the only groups examined). Based mainly on 
the transient increases in methaemoglobin (methaemoglobin increased in all dose groups as a function 
of dose, with no cumulative effect observed), a NOAEL of 18 mg/kg/day was established. 
 
In a GLP compliant study described by Tepper et al. (Tepper J, 2014) sodium nitrite was administered 
with the aid of a nebuliser to Sprague Dawley rats for 26 weeks. The results showed that effects 
suggestive of chronic toxicity were not observed. Cyanosis and methaemoglobinaemia were observed 
primarily at the high dose. Lethality occurred in several of the high-dose rats (75/90 mg/kg) and was 
considered subsequent to acute hypoxia secondary to methaemoglobin levels greater than 50%. Other 
than acute methemoglobinemia, few significant findings were observed after 26 exposures. Given this 
data, the toxicity appears primarily due to methemoglobinemia; and therefore, the inhaled NOAEL, after 
26 weeks of dosing, was considered to be 30 mg/kg/d in rats. It should however be noted that no adverse 
respiratory tract findings occurred up to 75/90 mg/kg/day where the corresponding human equivalent 
doses are at least 4-fold higher than that proposed clinically. 
 
Dogs 
Nebulised sodium nitrite (acidified with citric acid; pH 5.5) was administered to dogs for 7 days with a 
NOAEL of 12 mg/kg/day (Aries Pharmaceuticals, 2009). No remarkable clinical observations were noted 
over the 7-day period. Methaemoglobin levels in blood were minimally above basal levels at 12 mg/kg/day 
and appreciably at 54 mg/kg/day. Gross necropsies, including histopathology of the lungs, of the treated 
groups were similar to controls. Inhalation of sodium nitrite up to 54 mg/kg/day produced only mild and/or 
transient changes in methaemoglobin; therefore, for this 7-day study, a NOAEL of 12 mg/kg/day was 
established. 
 
Tepper and colleagues (Tepper J, 2014) makes brief mention of a 28-day inhalation toxicity study using 
dogs restrained in slings for up to 4 hours. While mortality was observed at very high methaemoglobin 
levels (data not presented), no evidence of pulmonary toxicity was observed. The inhalation procedure 
was deemed too stressful to the dogs to be utilized for a study of 26 weeks duration. 
 
Further information on the above repeat dose toxicity studies, plus on studies of other routes of 
administration, are available in the IB. 
 
Genotoxicity 
In vitro studies  


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During a screening assay for a series of food additives, the potential for sodium nitrite was evaluated in 
the Ames test (at up to 5000 μg/per plate) using the S.Typhimurium tester strains TA92, TA94, TA100, 
TA1535 and TA1537 with and without rat liver microsome fraction (S9) (Ishidate M, 1984). No significant 
(<2-fold) increase in the number of revertant colonies was observed in treated plates when compared to 
controls; hence, sodium nitrite was not considered to be mutagenic.  
 
In a separate study, sodium nitrite at 100 to 10,000 μg/plate was evaluated and found to be mutagenic 
in the S. typhimurium strain TA100, with and without induced hamster and rat liver S9 enzymes but not 
mutagenic in strain TA98 (National Toxicology Program, 2001).  
 
In vivo studies  
In a rat micronucleus assay, sodium nitrite was administered by intraperitoneal injection at 6.25 to 200 
mg/kg to males three times at 24-hour intervals; on the basis of the observed trend towards to a small 
increase in the frequency of micronucleated polychromatic erythrocytes that was observed during this 
initial study, sodium nitrite was considered to be mutagenic. (National Institutes of Health, 2001) However, 
results of a subsequent rat bone marrow micronucleus test, demonstrated that sodium nitrite was not 
mutagenic in vivo at up to 50 mg/kg. A similar study in male mice also showed that doses of 7.81 to 250 
mg/kg were non-mutagenic. Moreover, in a separate study, no significant increase in the frequency of 
micronucleated normochromatic erythrocytes was observed following repeated administration of sodium 
nitrite via the drinking water (at 375 to 5,000 ppm) for 14 weeks. 
 
Carcinogenicity 
No evidence of carcinogenicity was reported from inhaled nitric oxide gas administered to rats at 20 ppm 
for 20 h/day for 2 years (0.02 mg/L) (European Medicines Agency, 2021). Based on the evidence 
provided, use of RESP301 during the clinical studies as proposed, does not pose a carcinogenic risk. 
 
Reproductive and Developmental Toxicity 
Taking findings from both mouse (National Toxicology Program, 2001)and rat (National Toxicity 
Programne, 1990) into consideration, no effects on male and female reproductive parameters were noted 
at human equivalent doses that are at least 4-and 6-fold higher respectively than the doses proposed 
clinically. 
 
Overall, no significant effects on embryofoetal development were noted in the mouse (Shimada T, 1989), 
hamster, or the rat (US Food and Drug Administration, 1972); the human equivalent doses evaluated 
were either below or similar to those proposed clinically. 
 
Local Tolerance  
 
 
 
 
 
 
 
 
 
 


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2.2.2.7. 
Conclusion 
The nonclinical studies have demonstrated the anti-M.tb activity, safety and tolerability of RESP301, 
making it a promising candidate for TB treatment and acceptable to move into human clinical studies. 
 
2.2.3. Clinical studies 
 
 
 
 
 
 
 
 
2.2.3.1. 
RESP301 development program 
 
 
 
 
 
 






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There is no PK data available for RESP301 as human clinical studies are still ongoing at the time of writing. 
 
 
 
 
 
 
 
 
 
 
 
 
Previous trials have shown once participants have experience with the nebuliser their tolerance improves. 
 
For further information refer to the IB. 
 
2.2.3.2. 
Inhaled Sodium Nitrite Published Literature  
Inhaled sodium nitrite, formulated similarly to RESP301, has been assessed during inhalation studies in 
man and was well tolerated based on clinical studies obtained from published literature. (Rix PJ, 2015) 
(Borlaug BA, 2016) (Parakaw T, 2017). Key points are noted in Table 10 with more detail in the IB and 
references. 
 
To support its use, safety reviews for both sodium nitrite and nitric oxide by the inhaled and/or oral route 
have been performed by regulatory agencies from the chemical, occupational and food safety industries 
e.g., ECHA, EFSA and NIOSH.  
 
 
 




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2.2.3.3. 
Inhaled Nitric Oxide Published Literature 
The active product of RESP301, NO gas, is a licensed product used: 
• 
in adults and neonates with pulmonary hypertension (Europen Medicines Agency, 2021).  
• 
for the treatment of acute cyanide poisoning in IV formulation (30 mg/mL) (Hope 
Pharmaceuticals, 2018)  
• 
as a food additive.  
 
To support its use, safety reviews for both sodium nitrite and nitric oxide by the inhaled and/or oral route 
have been performed by regulatory agencies from the chemical, occupational and food safety industries 
e.g., ECHA, EFSA and NIOSH.  
 
Inhaled NO has been assessed during inhalation studies in man and was well tolerated based on clinical 
studies obtained from published literature.  Key points are noted in Table 11 with more detail in the IB and 
references. 








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2.2.3.4. 
Conclusion 
The clinical studies have demonstrated safety and tolerability of RESP301 and, together with the 
nonclinical studies which demonstrate RESP301 expected anti-M.tb activity in humans, make RESP301 
a promising candidate for TB treatment and acceptable to move into human clinical studies. 
 
2.3. HRZE 
Participants randomized to treatment with the control product will receive standard, intensive phase 
pulmonary TB treatment as recommended in the South African National TB Treatment Guidelines, which 
is HRZE (H=isoniazid: R=rifampicin Z=pyrazinamide: E=ethambutol) 75/150/400/275 mg oral daily. The 
HRZE group is included as a control for the EBA quantitative mycobacteriology and to evaluate whether 
HRZE in this population gives similar EBA results to that demonstrated in prior studies with this 
combination. 
 
Please see a HRZE package insert e.g., Rifafour e-275® or other fixed dose combination, for HRZEs 
known and potential risks and benefits. (Sanofi-Aventis South Africa, 2018)  
 
2.4. Known and Potential Risks and Benefits of the RESP301 
The following are recognised as clinically significant potential risks of RESP301 treatment:  
• 
 
  
 
 
 
  
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 




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and immediately referred to the national TB treatment program at their local TB clinic. The investigators’ 
primary responsibility is to ensure participant safety. 
 
3. Study Rationale and Objectives 
3.1. Study Rationale 
As described in section 2.1.1, TB, caused by M.tb infection, remains a concerning global health problem. 
As a powerful antimicrobial agent as demonstrated in the nonclinical studies and literature to date (section 
2.2), the NO generated by RESP301, has shown anti-M.tb activity, safety and tolerability, making 
RESP301 a promising candidate for TB treatment. 
 
 
 
 
 
 
. 
 
3.2. Dose Rationale 
RESP301 is intended for inhaled administration via a nebuliser, 
 
 
 
 
 
 
 
 
 
 
 
 
 
82 healthy volunteers following six days of administration was 90 mg 
every 8 hours. (Rix PJ, 2015)  
 
RESP301 treatment will occur for 14 days as per standard EBA study format (section 2.1.2). However, 
overnight sputum sample collection will occur on Day 15 when no RESP301 dose is administered, to 
allow testing for any residual NO effect on M.tb in sputum samples from prior RESP301 dosing. 
 
3.3. Study Objectives 
3.3.1. Primary Objectives 
Stage 1 
To determine the EBA of tds dosing of inhaled RESP301 6 mL dosed via NEB over 14 days of 
treatment. 
Stage 2 






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7. Ability to produce an adequate volume of sputum 
 
 
8. A chest X-ray taken during the screening period or up to 2 weeks before screening which, in 
the opinion of the investigator, is consistent with pulmonary TB. 
9. Spirometry performed during screening with a FEV1 
 
 
10. Be of non-childbearing potential or willing to use effective methods of contraception, as defined 
in section 4.3.4.  
 
4.2.2. Exclusion Criteria 
Participants will be excluded from participation if they fulfil any of the following criteria. 
 
Medical History: 
1. Evidence of clinically significant conditions or findings, other than TB, that might compromise 
safety or the interpretation of study endpoints, per discretion of the investigator. 
2. Clinical evidence of extra-thoracic TB, that might compromise safety or the interpretation of 
study endpoints, per discretion of the investigator. 
3. History of allergy to any of the study IMP as confirmed by the clinical judgement of the 
investigator. 
4. Alcohol or drug abuse, that in the opinion of the investigator, is sufficient to compromise the 
safety or cooperation of the participant. 
5. HIV positive AND: 
 
 
6. Methaemoglobin saturation (SpMet)
 
7. Female participant who is pregnant or breast-feeding. 
8. Participants planning to conceive a child within the anticipated period of study participation and 
for at least 90 days after the last dose of IMP in the study. 
 
Treatment History 
9. Participation in other clinical studies with investigational agents within 
 
screening. 
10. Treatment received for this episode of TB with any drug active against M.tb 
 
 
 
 
 
11. Treatment with immunosuppressive medications 
 
 
 
12. Treatment with nitric oxide and other nitric oxide donor agents 
 
 
 
 
Laboratory Safety Testing 
13. 
 
 
 




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If the first spot sputum does not show a favourable result, the tests may be repeated on freshly collected 
spot sputum or overnight sputum and that result used for the eligibility assessment. If results are 
discordant, the tests may be repeated during screening at the investigator’s discretion. Overnight sputum 
may be collected for a number of screening days if the screening results are delayed. It will not be 
collected for more than the allowed pre-treatment time period. 
 
4.3.3. Concomitant Medications and Procedures 
Concomitant Medication and Procedures are defined as any medication taken/procedure performed 
within 14 days prior to first dose of IMP and during the study until the follow-up visit (all participants 
including early withdrawal/discontinuations). This includes for new and/or worsening medical conditions.  
 
Concomitant medications should be kept to a minimum during the study. However, if necessary for the 
participant’s welfare and are unlikely to interfere with the IMP, they may be given at the discretion of the 
investigator once the prescribing information has been consulted and reviewed carefully. The 
determinations listed in the respective contraindicated, warning, and precaution sections must be 
respected to prevent any potentially serious and/or life-threatening drug interactions. Drugs that may 
cause methaemoglobinaemia may be used with caution.  
 
The following concomitant medications are prohibited throughout the study. Please confirm any 
medication you are uncertain of with the Medical Monitor. 
• 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
Concomitant medications and procedures are to be reported on the concomitant medication and 
procedures page of the electronic case report form (eCRF). For concomitant medication the reported 
information is to include the drug name (trade name if known, otherwise generic), treatment period, 
dosing regimen, route of administration, and its indication (diagnosis if known, otherwise separate 
symptoms). Any changes in the dosage of concomitant medication must also be reported on the 
concomitant medication page of the eCRF. The corresponding diagnosis should be completed as medical 
history (before first dose of IMP) or as an AE (at or after first dose of IMP). 
 
4.3.4. Pregnancy and Contraception 
To be included in the study the following restrictions apply: 
• 
all participants to be of non-childbearing potential or using effective methods of birth control, as 
defined below and  
• 
women of childbearing potential (WOCBP) are to receive an injectable or other contraceptive 
method (Table 16) prior to or during screening, and at least 2 days prior to first dose of IMP. 
Should a WOCBP be receiving an Oral Contraceptive Pill (OCP) it is required that this is 
stopped for the duration of the study, and they transition to an injectable or other allowed 
contraceptive method (Table 16). The site staff will educate the participant on how the 


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transition should occur. It is recommended that female partners of male participants who are 
WOCBP also follow these guidelines. 
• 
all female participants are to have a negative β-HCG serum pregnancy test at screening (result 
available before first dose of IMP). 
 
During and after the study: 
Participants (female and male) should not conceive a child during IMP treatment or within 90 days after 
their last dose of IMP.  
 
Women in the following categories are considered WOCBP (fertile): 
• 
following menarche 
• 
from the time of menarche until becoming post-menopausal unless permanently sterile (see 
below). 
 
Women in the following categories are considered Woman of Non-childbearing Potential (WONCBP): 
• 
premenopausal female with permanent infertility or permanent sterilization due to one of the 
following (for the purpose of this study): 
• 
*Documented hysterectomy 
• 
*Documented bilateral salpingectomy 
• 
*Documented bilateral oophorectomy 
 
For individuals with permanent infertility due to an alternate medical cause other than the above, (e.g., 
Mullerian agenesis, androgen insensitivity, gonadal dysgenesis), investigator discretion should be applied 
to determining study entry. 
• 
postmenopausal females are defined as no menses for 12 months without an alternative 
medical cause. 
• 
A high follicle stimulating hormone (FSH) level in the postmenopausal range may be used to 
confirm a postmenopausal state in women not using hormonal contraception or hormonal 
replacement therapy (HRT). 
• 
Females on HRT and whose menopausal status is in doubt must discontinue HRT to allow 
confirmation of postmenopausal status before study enrolment. 
 
*Documentation can come from the site personnel’s review of the participants medical records, medical 
examination, or medical history interview. If fertility is unclear (e.g., amenorrhea in adolescents or 
athletes) and a menstrual cycle cannot be confirmed before first dose of IMP, additional evaluation should 
be considered. 
 
The following Highly Effective Contraceptive Methods are required for the study. 
 
Male participants: condoms must be used in addition to recommended female partner hormonal 
contraception, if applicable. Male condom and female condom should not be used together (due to risk 
of failure from friction). 
Female participants: Refer to Table 16. 
 




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A participant must immediately discontinue treatment, if applicable, and be prematurely withdrawn from 
the study for the following reasons: 
• 
withdrawal of informed consent 
• 
investigator considers it in the best interest of the participant that he/she be withdrawn 
• 
methaemoglobinaemia as defined as follows: 
o 
 
  
 
 
 
• 
pregnancy 
• 
serious adverse event (SAE) if probably or certainly (Table 21) related to the IMP  
• 
missed doses: 
o 
 
 
 
 
 
 
  
 
s 
• 
requires a medication that is prohibited by the protocol  
• 
at the specific request of the sponsor or termination of the study by the sponsor 
• 
lost to follow-up 
• 
failure to comply with protocol requirements as determined by the investigator or sponsor 
 
Participants who withdraw from the study before they have received all days of IMP, or whose study 
endpoints are not evaluable, may be replaced per the discretion of the sponsor. Sample size allows for 
up to 3 participant withdrawals per arm before replacement is necessary. Data collected on participants 
who withdraw prior to the final study visit will be entered on the eCRF for all visits conducted.  
 
Although a participant is not obliged to give his/her reason(s) for withdrawing prematurely from a study, 
the investigator should make a reasonable effort to ascertain the reason(s) and document it on the eCRF, 
while fully respecting the participants rights. If a participant requests to be withdrawn from the study, this 
request must be documented in the source documents and signed by the study staff.  
 
All early withdrawal/discontinued participants 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 and sufficient TB medication to cover the time from attending their last visit 
at the study clinic until their scheduled visit at the TB clinic. This will be documented in the participants 
source data. 
 
Early withdrawal/discontinued participants during treatment will have an early withdrawal visit and will be 
requested to attend the Follow-up visit within 14 days (±3 days) after withdrawal. The exceptions to this 
are those who withdraw their consent to do so, and those lost to follow-up. 
 
Lost to follow-up is defined as randomized participants who do not complete all study visits and cannot 
be contacted up to 7 days after Day 28 of the study. Before a participant is deemed lost to follow up, the 
investigator or designee must make every effort to regain contact with the participant (where possible, 
minimum of 3 telephone calls, plus email contact if applicable, plus attempt to contact the participant via 




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Dosing is once daily (qd), twice daily (bd) or three times a day (tds) dependent on the study treatment 
arm, for fourteen days. 
 
The Control Product will be supplied as isoniazid 75 mg (H), rifampicin 150 mg (R), pyrazinamide 400 mg 
(Z), ethambutol 275 mg (E) (i.e., HRZE) combination tablets. Dosing is qd for fourteen days based on 
participants body weight at screening rounded up to the nearest whole number: 
• 
38 to 54 kg: 3 tablets 
• 
55 to 70 kg: 4 tablets  
• 
71 kg and over: 5 tablets 
 
The participants’ screening weight will be used to determine the HRZE dose and this dose will be 
administered for the duration of the study. 
 
After completion of the 14 days of study treatment, participants on a RESP301 containing treatment arm 
will receive no treatment for one day (Day 15) and will begin standard HRZE od po dosing on the next 
day (Day 16). HRZE alone treatment arm participants will continue their HRZE treatment. Thereafter all 
participants will be referred to the local community TB clinics for standard anti-tuberculosis chemotherapy 
according to national guidelines (section 4.4). 
 
5.2. Randomization and Blinding  
5.2.1. Screening 
Participants who have given written, informed consent will be screened for the study during Visits 1, 2 
and 3 (days -9 to -1), and will be identified by a study generated participant identification code for 
anonymity (participant number).  
 
5.2.2. Study enrolment and randomization 
Randomisation by the delegated study personnel may occur once all the screening results are available 
and the investigator has determined that the participant is eligible for the study, until just prior to first IMP 
dose. On randomisation participants will be assigned a treatment number. The randomisation scheme 
will be generated by the study statistician/data manager. Randomisation will be conducted using 
permuted block randomisation.  
 
Consecutively numbered, sealed, opaque envelopes will be prepared and provided to the site. The site 
research pharmacist will maintain the randomisation envelopes in a secured location. After being notified 
that a participant is eligible for the study, the research pharmacist will write the participant's number on 
the next numbered envelope and open it to determine the participant's treatment group. The participant 
number, participant initials, the date and time the envelope was opened, and the signature of the person 
opening the envelope will be recorded on the treatment group card in the envelope at the time it is 
opened. Opened envelopes containing treatment group cards will then be filed in the participants’ source 
file. 
 
Participants who withdraw from the study before they have received all days of IMP, may be replaced.   
 
5.2.3. Blinding 


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This is an open label study and there is therefore no need for blinding or procedures to break the blind. 
The study endpoints are laboratory based and the microbiology staff will be blinded to treatment allocation 
until EBA database closure and release. 
 
5.3. IMP Administration 
5.3.1. Route and Dosage  
Refer to section 5.1. 
 
RESP301 
RESP301 is inhaled via nebulisation. 
 
 Participants will be trained in use 
of the nebuliser by the site personnel prior to using. The methodology will be detailed in the Pharmacy 
Manual. 
 
HRZE 
Will be administered orally. 
 
5.3.2. Dosing Instructions 
The methodology will be detailed in the Pharmacy Manual. 
 
On the days of IMP dosing: 
Inhaled RESP301 nebulisation: 
• 
all treatment regimens: 
o each dose is to be given using freshly prepared admix solution i.e., inhalation of the 
RESP301 should start with
 
o on Day 1 only: a short acting beta agonist is to be administered before starting the 
nebulisation for each dose, within 30 minutes of RESP301 is suggested 
o inhalation of RESP301 should be completed within 
 of starting NEB, pauses 
may be taken during NEB if not tolerated. The NEB should be stopped during a pause 
to preserve the remaining IMP until restarted. 
o the first dose should be given in the morning 
o All dosing should occur at approximately the same time each day (window of ± 1 hour of 
the IMP dose/s on Day 1).  
• 
once or twice daily: 
o When dosing overlaps with overnight sputum sample collection, sputum collection 
should be paused during and for 
 the end of the neb. A cup (
 
) of water should be taken after the neb is complete, and prior to continuation of 
overnight sputum sample to wash away any remaining RESP301 from the airway. 
o the first dose should be given at half dose (half of each vial) 
• 
three times a day (alone or with HRZE): 
o the first 2 doses should be given at half dose (half of each vial) 
o this tds dosing will be 
 to prevent overnight disruption to participants 
and limit overlap of dosing and sputum sampling 
• 
twice or three times a day (alone or with HRZE): 
o to ensure a dose does not affect any M.tb present in the overnight sputum sample, 
should a dose overlap with the overnight sample collection window, sputum collection is 




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Please consult the study medical monitor for guidance, caution should be given when considered 
repeat dosing. This is to be documented in source and in the eCRF. 
 
5.3.4. Manufacturing, Packaging and Labelling 
 
 
 
 
 
 
   
 
. 
 
5.3.5. Storage 
RESP301 vials will be stored within a temperature range of 
 
r.  
 
HRZE will be stored at or below a temperature of 25 °C, in well-closed containers, and protected from 
light.  
 
All investigational products will be kept securely stored by the site pharmacist/registered dispenser in a 
secured temperature-controlled area with limited access to designated site personnel only. Only qualified 
personnel will undertake the preparation, handling and safe disposal of investigational products. Logs will 
be kept for drug accountability (chain of custody) and temperature. 
 
5.4. Dispensing and Accountability 
The site pharmacist/delegated dispenser will be responsible for dispensing the IMP. The IMP will be 
prepared separately from drugs used for other participants and studies to prevent cross-contamination. 
Accurate accountability and participant identification records will be kept by the site to assure that the 
IMP will not be dispensed to any person who is not a participant under the terms and conditions set forth 
in this protocol i.e., delivery to site, inventory at site, use by participant, destruction etc. The 
investigator/designee will immediately inform the Sponsor of any quality issues arising with respect to the 
IMP. The Sponsor will take whatever action is required should such a situation arise. The investigator 
undertakes to use the study medication only as indicated in this protocol.  
 
5.5. Returns and Destruction 
Upon completion or termination of the study, and after IMP accountability and monitoring thereof has 
been completed, all unused and/or partially used: 
• 
RESP301 will be destroyed by the site according to their standard operational procedure or 
returned to the sponsor for use or destruction per their standard operational procedure. 
















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• 
outcome 
• 
relationship to IMP 
• 
occurrence 
• 
seriousness. 
 
Serious Adverse Events (SAE) 
SAE reporting begins from the time a participant enters screening through to the final visit.  
 
Initial Reporting 
• 
Must be communicated by the investigator to the sponsor, study monitor and medical monitor 
within 24 hours of the site first being aware of the SAE, whether or not the serious event is 
deemed associated with the use of the drug. 
• 
In addition, the investigator will provide a detailed SAE report form including estimates of the 
attribution/causality and the seriousness of the AE to the sponsor, study monitor and medical 
monitor and regulatory team within 24 hours of becoming aware of the SAE. The investigator 
will be requested to supply as much detailed information regarding the event that is available at 
the time of the initial contact (such as examinations carried out and laboratory results). 
 
ANY SERIOUS ADVERSE EVENT MUST BE REPORTED IMMEDIATELY (WITHIN 24 HOURS) TO TASK AT 
THE FOLLOWING EMAIL ADDRESS: RESP30XSAE@task.org.za 
 
Follow-up Reporting 
The Investigator is required to submit follow-up reports to sponsor, study monitor and medical monitor 
within 24 hours of becoming aware of additional information.  
• 
Additional information may include updated diagnosis, outcome, causality assessment, results 
of specific investigations and any new significant information that has not been previously 
reported.  
• 
Anonymized copies of reports should be sent when requested as applicable, such as additional 
laboratory tests, consultation reports, post-mortem reports, hospital progress reports and 
autopsy reports.  
• 
For submission of updated SAE report, the investigator will provide a newly completed serious 
adverse event form, designated as a follow-up report. This will be submitted to the study 
monitor, medical monitor, and regulatory team within 24 hours of the investigator receiving the 
information. 
 
The sponsor/investigator/designee will inform regulatory authorities and/or EC of all SAEs in accordance 
with local requirements and ICH guidelines for GCP. 
 
Clarification on reporting of Serious Adverse Events 
• 
Death is an outcome of an AE, not an AE in itself.   
• 
An AE/SAE may occur even if the participant was not being treated with the IMP at the 
occurrence of the event. 
• 
Life-threatening means that patient is at immediate risk of death.  
• 
Complications that occur during hospitalisations are typically AEs but can be an SAE. If a 
complication prolongs the hospitalisation, it is a SAE.  


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• 
Participant hospitalisation means that the participant stays overnight in the hospital. Pre-
planned hospital stays or hospital stays for non-medical (e.g., social) reasons will not be 
considered as a SAE.  
• 
A procedure for protocol / disease-related investigations should not be reported as SAE. 
Hospitalisation or prolonged hospitalisation for a complication such procedures should be 
reported as SAE.  
• 
Regarding a participant who experiences persistent or significant disability/incapacity, only a 
persistent or significant or incapacitating disability is implied. The item refers to a substantial 
disruption of a person’s ability to conduct normal life functions. Thus, disability is not intended to 
include experiences of relatively minor medical significance such as headache, nausea, 
vomiting, diarrhoea, influenza, and accidental trauma.  
• 
Congenital anomality / birth defect includes foetal malformations associated with spontaneous 
abortions or elective abortions.  
 
Sponsor Evaluation of SAEs 
The expected / unexpected status of the serious and related AE will be judged by the sponsor or its 
designated representative with regards to the reference document (IB). 
 
In all reporting to outside the study team it is important to ensure participant identifiers are removed from 
all notes, correspondence, results and reports when communicating with the study team noted below. 
 
7.3. Follow up of Adverse Events 
All AEs will be followed until: 
• 
Satisfactory clinical resolution or stabilization; or 
• 
Until the end of the follow-up period; and 
• 
Until all queries on these AEs have been resolved. 
 
Certain long-term AEs cannot be followed until resolution within the setting of this protocol. In these 
cases, follow-up will be the responsibility of the treating physician/referral clinic. This will be confirmed 
with the Sponsor. 
 
7.4. Post-Study Serious Adverse Events 
Any new SAEs reported by the participant to the investigator that occur up to 30 days after the last 
scheduled contact should be reported to the sponsor on an expedited basis. Any additional SAEs beyond 
this period that are determined by the investigator to be possibly, probably, or likely related to the use of 
the IMP will be reported to the sponsor, IEC/IRB and regulatory authorities on an expedited basis as 
required in accordance with local requirements and ICH guidelines for GCP. 
 
7.5. Clinical Laboratory Adverse Events 
Laboratory abnormalities are not necessarily reported as AEs or SAEs. Changes in the results of the 
clinical laboratory assessment results which the investigator feels are clinically significant will be reported 
as adverse events.  
 
It is the investigators’ responsibility to review the results of all laboratory tests as they become available. 
This review must be documented by the investigators’ dated signature on the laboratory report. For each 


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abnormal laboratory test result, the investigator needs to ascertain and document if this is a clinically 
significant change from baseline for that individual participant.  
 
This determination, however, does not necessarily need to be made the first time an abnormal value is 
observed. The investigator may repeat the laboratory test or request additional tests to verify the results 
of the original laboratory tests. If this laboratory value is determined by the investigator to be a clinically 
significant change from baseline for that participant, it is considered to be an adverse event. 
 
7.6. Disease under Study 
Symptoms of the disease under study (pulmonary TB) experienced by the participant while on the study 
will be assessed by the investigator. If the symptom has: 
• 
worsened while the participant is in the study; and 
• 
the investigator assesses it as clinically significant 
it will be recorded as an adverse event. If it does not meet these criteria, it will not be recorded as an AE 
and will be noted in the participant’s source documentation. All TB related symptoms that meet SAE 
criteria will be recorded and reported as a SAE. 
 
7.7. Overdose 
Overdose of IMP is defined as a dose exceeding the amount allocated within a one-day dosing interval. 
Overdose of IMP experienced by the participant while on the study, will be assessed by the investigator 
to determine whether the overdose led to an adverse event, including if the taking of the suspect medicine 
led to suicidal intention and subsequent overdose of the suspect medicine, or other medication. In this 
case it will be recorded as an adverse event. If it does not lead to an adverse event, it will not be recorded 
as an AE and this will be noted in the participant’s source documentation. 
 
7.8. Drug Interaction 
If the investigator becomes aware that the participant has experienced a drug interaction which has 
resulted in an adverse event, it will be recorded as an adverse event. 
 
7.9. Pregnancy 
If pregnancy is suspected while the participant is receiving IMP, the IMP will be withheld immediately and 
a pregnancy test performed. If pregnancy is confirmed, the IMP will be permanently discontinued in an 
appropriate manner and the participant withdrawn from the study. Protocol-required procedures for study 
discontinuation and follow-up will be performed unless contraindicated by the pregnancy. 
 
Pregnancy reporting will follow the same timelines and reporting structures as for a SAE (see above). 
Pregnancy will be followed up until the pregnancy outcome is known (birth, termination, etc). SAE 
reporting will also occur if the pregnancy outcome is a congenital anomaly. This will follow the reporting 
procedures described above for SAE reporting. 
 
7.10. Monitoring and Safety for Specific Toxicities 
Monitoring for specific toxicities is based upon target organs defined in preclinical toxicity studies and 
clinical studies (see IB). 
 




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7.10.3. 
Overdose 
Treatment of overdose should be as follows: 
RESP301 
• 
treatment should be supportive and symptomatic 
• 
 
 
. 
HRZE 
• 
gastric lavage should be performed as soon as possible. Following evacuation of the gastric 
contents, the installation of activated charcoal slurry into the stomach may help absorb any 
remaining medicine from the gastrointestinal tract  
• 
anti-emetic medication may be required to control severe nausea and vomiting  
• 
intensive support measures should be instituted, including airway patency and individual 
symptoms treated as they arise. 
 
7.10.4. 
Any other toxicities  
As defined by the CTCAE toxicity tables version 5.0 (Appendix 1): 
• 
Grade 1 or 2: Participants who develop grade 1 or 2 AE or laboratory toxicity may continue 
intake of IMP at the discretion of the investigator. 
• 
Grade 3 or 4: Participants who develop grade 3 or 4 AE or laboratory toxicity will be carefully 
evaluated by the investigator. Participants may either continue intake of IMP, or be withdrawn 
from the study if, in the opinion of the investigator, the AE or laboratory toxicity poses a 
significant risk for the participant to continue participation in the study. Participants should be 
followed as appropriate until resolution of the AE or toxicity. 
 
For AEs not described in the CTCAE table, the severity definitions defined in Table 22 are to be applied. 
 
7.11. Safety Review Committee 
Á Safety Review Committee will be formed which will meet if pre-determined events occur. This will be 
detailed in the Safety Review Committee Charter and Safety Management Plan which will be released 
before First Patient Screened. 
 
8. Statistical Analysis 
This section is an overview of the key elements of the statistical analysis for this study. Further details on 
statistical reporting and analyses will be contained in a separate statistical analysis plan (SAP).  
 
This SAP may be revised during the study only to accommodate protocol amendments and to make 
changes to adapt to unexpected issues in study execution and data collection that could affect planned 
analyses. In all circumstances, a final SAP should be issued prior to database lock.  
 
Descriptive summary statistics for continuous variables will include the number of participants, mean, 
standard deviation (SD), median and range. Descriptive summary statistics for categorical variables will 
include frequency count and percentages. Unless stated otherwise, the denominator for percentage 
calculations will be the number of participants with non-missing data. Descriptive statistics will be reported 
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Activity 
The EBACFU (0-14) as determined by the predicted rate of change in log10CFU per mL sputum over the 
periods day 0 - 14 will be described using
 
. 
 
 
. 
 
Safety and Tolerability 
Incidence of Treatment Emergent Adverse Events (TEAEs) will be presented by 
 
  
 
 
 
 
 
 
. 
 
8.4.3. Exploratory Endpoints Analysis: Activity 
Comparison of EBATTP(0-14) versus EBATTP(0-15) and the predicted EBATTP(14-15) versus observed 
EBATTP(14-15) analysed using 
 
 
 
9. Records Management 
9.1. Data Collection 
Electronic Data Capture will be used in this study. All eCRF pages will be completed for each participant 
who receives any amount of IMP, including EWD participants. For screen failure participants, certain 
predefined pages of the eCRF will be completed.  
 
The completed eCRFs are the sole property of the sponsor and should not be made available in any form 
to thirds parties, except for authorized sponsor’s representatives or appropriate regulatory authorities, 
without written permission from the sponsor.  
 
The eCRF being used for this study has been fully certified as being compliant with the ICH GCP guidance 
requirements.  
 
The investigator or designee will review and approve each completed eCRF. Should a correction be 
made, the corrected information will be recorded in the eCRF by the authorized person and explained (if 
necessary). All corrected data will be tracked through an electronic audit trail.  
 
It is the investigator’s responsibility to ensure documentation of all relevant data in the participants source 
file. All data recorded in the eCRF must be documented and referenceable in source data (source 
documents).  
 


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9.2. Source Documents 
Source documents are defined as all information in original records and certified copies of original records 
of clinical findings, observations, or other activities in a clinical study necessary for the reconstruction and 
evaluation of the study. Source documents will include, but are not limited to, progress notes, electronic 
data, screening logs, and recorded data from automated instruments. 
 
All source documents pertaining to this study will be maintained by the investigators and designees. The 
investigator will permit study related monitoring, audits, IEC/IRB review and regulatory inspections 
providing authorized persons direct access to source documents. 
 
9.3. File Management at the Study Site 
It is the responsibility of the investigator to ensure that the study site’s files are maintained in accordance 
with ICH and South African GCP Guidelines and the ethical principles that have their origin in the 
Declaration of Helsinki. 
 
9.4. Records Retention at the Study Site 
The investigator is obliged to retain records and data from the study for safety reasons and for audit and 
inspection subsequent to study completion. The sponsor, or other owners of the data, must retain all the 
sponsor-specific essential documents pertaining to the study for not less than 25 years or until at least 
two years have elapsed after the last marketing approval or since the formal discontinuation of clinical 
development of the IMP.  
 
The sponsor will give written instruction and make financial provisions for the investigator to deposit the 
documents at an external site for safekeeping for as long as required by regulations and the sponsor. 
 
10. Quality Control and Assurance 
10.1. Site Procedures 
The investigator undertakes to perform the clinical study in accordance with this protocol, ICH and South 
African GCP, and the ethical principles that have their origin in the Declaration of Helsinki. 
 
The investigator and designees undertake to complete the source and eCRFs according to the sponsor’s 
requirements, in a timely, accurate and legible manner. eCRF entries will be verifiable to source 
documentation other than the eCRF. 
 
Site standard operating procedures will be adhered to for all clinical and bio-analytical activities relevant 
to the quality of the study. Participant compliance will be monitored throughout the study. The investigator 
will sign and date any analysis results timeously (e.g., laboratory, ECG, etc.) to verify that the results have 
been reviewed. 
 
The principal investigator (PI) may appoint and delegate other qualified, approved and trained sub-
investigators and study staff (the team) to assist with the study. However, the PI maintains responsibility 
for the overall conduct of the study and will oversee the team and any vendors. Written IEC/IRB and 
Regulatory Authority approval will be obtained prior to involvement in the study. 
 


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The investigator will ensure that any party retained to perform study tasks and all site personnel are 
adequately trained in ICH and SA-GCP, the protocol, IMP, IB (as applicable) and all study procedures 
and requirements before commencing any study related duties. This training will be documented. 
 
10.2. Monitoring 
The PI is responsible for the validity of all data collected at the study site and must accept the various 
monitoring procedures employed by the sponsor. The purpose of monitoring is to verify that the rights, 
safety and well-being of human participants are protected; that study data are accurate, complete and 
verifiable with source data; and that the study is conducted in compliance with the protocol, International 
and SA GCP, the ethical principles that have their origin in the Declaration of Helsinki and the applicable 
regulatory requirements. 
 
Monitors assigned by the sponsor will conduct regular site visits for the purpose of monitoring various 
aspects of the study. The investigator and site staff will allow the study monitor, independent ethics 
committees, regulatory authorities, and authorized representatives of the sponsor to: 
(1) inspect all essential documents pertaining to the clinical trial (e.g., eCRFs, written informed consent 
documents and corresponding source documents i.e., original medical records, participant records and 
laboratory raw data) and  
(2) access clinical supplies, dispensing and storage areas.  
 
The investigator and site staff will also:  
 
(1) agree to assist with monitoring activities if requested and  
(2) provide adequate time, resources and space for monitoring visits.  
 
The monitor will query any missing, confusing, spurious, or otherwise ambiguous data with the PI or 
delegated staff. All queries should be resolved in a timely manner (preferably before the next monitoring 
visit). A monitoring log will be maintained recording each visit, the reason for the visit, the monitor’s 
signature and investigator or designee’s confirmation signature. 
 
10.3. Auditing 
To comply with International and SA GCP and regulatory agency guidelines, it may be necessary for 
sponsor-authorized quality assurance personnel and/or authorized personnel from an external regulatory 
agency to conduct an audit or inspection of the investigational site. The purpose of an audit is to assess 
participants safety, the quality of data accuracy, adequacy, and consistency, and to assure that studies 
are in accordance with the applicable guidelines and the protocol. Having the highest quality data from 
studies is an essential aspect of drug development. 
 
Unless it is an unannounced inspection, the investigator and site staff will be given sufficient notice to 
prepare for such visits which may be conducted at any stage during the study. The audit may involve the 
review of all study-related documentation required by GCP to be maintained by each site; drug storage, 
dispensing and return; all study-related supplies; and source documents against the eCRFs to assure the 
adequacy and accuracy of the information which has been recorded, including the verification of any AEs 
and SAEs which have occurred. 
 


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In the event of the site being notified of a regulatory inspection, the Sponsor will assist the 
investigational site with preparation. It is essential that the Sponsor be notified of the inspection as soon 
as possible. 
 
11. Ethics and Regulatory 
11.1. Basic Principles 
This research will be carried out in accordance with ICH and South African GCP, the ethical principles 
that have their origin in the Declaration of Helsinki and the applicable regulatory requirements. 
 
11.2. Independent Ethics Committee/Institutional Review Board Review 
The protocol and required study related documents will be reviewed by the site’s IEC/IRB. The study will 
not start until the IEC/IRB has approved the protocol, written informed consent, any written information 
to be provided to the participant or any modification thereof, plus any other study related documents 
required for review. The IEC/IRB shall be constituted and shall operate in accordance with ICH and South 
African GCP, the ethical principles that have their origin in the Declaration of Helsinki. The investigator 
will maintain an accurate and complete record of all submissions made to the IEC/IRB. The records should 
be filed in the ISF, and copies will be sent to the sponsor for their TMF. 
 
11.3. Regulatory Authorities 
The regulatory authorities will receive the protocol, amendments, reports on SAEs, and the integrated 
clinical study report according to national regulations. As required by local legislation, written approval 
will be obtained from the regulatory authorities prior to commencement of the study and implementation 
of e.g., amendments as applicable. 
 
11.4. Informed Consent 
Written informed consent will be obtained from all participants before any study-related procedures 
(including any screening or pre-treatment procedures) are performed. Investigators may discuss the 
availability of the study and the opportunity for entry with a potential participant without first obtaining 
consent. However, informed consent must be obtained and documented prior to initiation of any 
procedures that are performed solely for the purpose of determining eligibility for research, including 
withdrawal from current medication(s). When this is done in anticipation of, or in preparation for, the 
research, it is part of the research. Participants who are illiterate will make use of a witness but will still 
give verbal consent to participate. 
 
The investigators have both ethical and legal responsibility to ensure that each participant being 
considered for inclusion in this study is given a full explanation of the protocol by means of the informed 
consent process. This shall be documented on a written informed consent form that shall be approved 
by the same IEC/IRB responsible for approval of this protocol. Each informed consent form shall include 
the elements required by international and South African GCP and must adhere to the ethical principles 
that have their origin in the Declaration of Helsinki. 
 
Once the appropriate essential information has been provided to the participant and fully explained by 
the investigators (or qualified designees) and it is felt that the participant understands the implications of 
participating, the IEC/IRB approved written informed consent form will be signed and dated by both the 


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participant, and/or witness, and the person obtaining consent (investigators or designees), and by any 
other parties required by the IEC/IRB. 
 
The original signed informed consent form will be kept with the study records and a copy of signed 
informed consent form will be offered to the participant. Another copy of the signed informed consent 
form and a source document identifying the study and recording the dates of informed consent and 
participation will be placed in the participant’s medical record. 
 
The monitor will inspect the original completed consent form(s) as per the monitoring plan. 
 
11.5. Confidentiality 
All site staff, the sponsor, and any sponsor representatives will preserve the confidentiality of all 
participants taking part in the study, in accordance with ICH and South African GCP and applicable local 
legislation/regulations. All participants will agree to the requirement for source data verification with 
confidentiality of all participant identities maintained. Only participant study number will be used on the 
eCRF and in all study correspondence, as permitted. No material bearing a participant’s name will be 
kept on file by the sponsor. The written informed consent will contain a clause granting permission for 
review of the participants’ source data by the site staff, the monitor, auditors, inspectors from Regulatory 
authorities. 
 
12. Incidental Findings 
Incidental findings (IFs) in clinical research are findings with potential health or reproductive importance 
that are discovered while conducting the research but are beyond the aims of the study. IFs in this study 
may arise from the collecting and analysing of research images or in the tests or information collected to 
determine eligibility criteria or study endpoint assessments. 
 
The PI is responsible for handling an IF responsibly and promptly and members of the research team will 
promptly report a suspected IF. If an IF is discovered the PI will inform the participant and, as consented 
to by the participant, exercise his/her judgment on a case-by-case basis to determine the way forward 
that is in the best interest of the participant. The PI will make sure that all the necessary documentation 
for the referral of the participant is put together free of cost to the participant. The sponsor is not liable for 
the ongoing clinical management of any IFs. 
 
13. Publication Policy 
The definition of publication for this purpose is any public presentation of the data emerging from this 
study. 
 
Results of this research will be submitted for publication as soon as feasible upon completion of the study 
in the form of a joint publication(s) between compound owners, sponsor, and investigator(s), including 
site clinical and laboratory investigators, and statisticians as appropriate. 
 
The clinical study will be registered on the open access website https://clinicaltrials.gov and any other 
public websites required by regulation once approval by local regulators for the conduct of the study has 
been received.  
 


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14. Protocol Amendment Policy 
Any change to the protocol will be effected by means of a protocol amendment and will be submitted to 
the IEC/IRB and Regulatory Authorities prior to implementation as required by them. No amendment will 
be implemented until approved by the relevant authorities and/or IEC/IRB and signed by all required 
parties. Exceptions to this are when the investigator considers that the participant’s safety is 
compromised. 
 
Protocol amendments detailing minor administrative changes should be submitted by the investigator to 
the IEC/IRB and regulatory authorities, either for notification purposes or approval as appropriate. 
 
15. Financial Aspects, Insurance and Indemnity 
The study sponsor, who are funding the study, is Thirty Respiratory Limited. 
 
The participants will not receive any incentives for their involvement in the study. The sponsor has made 
provision to reimburse the participants for time, inconvenience, and out-of-pocket expenses such as 
travelling to and from the study site and other miscellaneous costs because of their study participation. 
 
Research participants should not bear any financial cost to rectify harms that occur because of study 
participation. The sponsor will indemnify (provide legal and financial cover for) the investigator against 
claims arising from the study, excluding claims that arise from malpractice or negligence. The sponsor 
certifies that it has liability insurance coverage for itself and will provide an associated certificate upon 
request. The participants taking part in the study will be covered by the insurance taken out by the 
sponsor for this study.  
 
The insurance does not relieve the investigators of the obligation to maintain their own professional liability 
insurance (e.g., MPS) as required by applicable law. The sponsor does not assume any obligation for the 
medical treatment of other injuries and illnesses not related to the study. 
 
 


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Hardwick, J. B. (2001). A novel method for the delivery of nitric oxide therapy to the skin of human 
subjects using a semi-permeable membrane. Clinical Science , 100 no. 4 : 395-400. 
Hogg N, K. B. (1999). Nitric oxide and lipid peroxidation. Biochim Biophys Acta., 1411(2-3), 378-384. 
Hope Pharmaceuticals. (2018). Sodium Nitrite Prescribing Information. Retrieved from 
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2018.pdf 
Hunault CC, v. V. (2009). Bioavailability of sodium nitrite from an aqueous solution in healthy adults. 
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Ishidate M, J. S. (1984). Primary mutagenicity screening of food additives currently used in Japan. Food 
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Jones ML, G. J. (2010). Antimicrobial properties of nitric oxide and its application in antimicrobial 
formulations and medical devices. Applied Microbiology and Biotechnology, 88, 401–407. 
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Kortboyer JM, O. M. (1997). The oral bioavailability of sodium nitirite investigated in healthy adult 
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Moyo S, I. F. (2022 Aug). Prevalence of bacteriologically confirmed pulmonary tuberculosis in South 
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Service. Retrieved Apr 01, 2023, from https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr495.pdf 


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National Toxicity Programne. (1990). Reproductive toxicity of sodium nitrite in CD-1 Swiss mice. 
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09b3f8725ef5 
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Tripathi P, T. P. (2007 ). The role of nitric oxide in inflammatory reactions. FEMS Immunol Med 
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WHO. (1996). NITRITE (and potential endogenous formation of N-nitroso compounds) and 
Environmental Protection, Bilthoven, Netherlands. Laboratory for Toxicology, National Institute 
of Public Health and Environmental Protection, Bilthoven, Netherlands. INCHEM. Retrieved Mar 
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Organisation: https://www.who.int/teams/global-tuberculosis-programme/tb-reports/global-
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WHO. (2022, December 15). WHO Consolidated Guideline on Tuberculosis Module 4 Drug-Resistant 
Tubercolosis Treatment. Retrieved March 04, 2023, from 
https://www.who.int/publications/i/item/9789240063129 
Wink DA, M. J. (1998). Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and 
cytoprotective mechanisms of nitric oxide. Free Radic Biol Med, 25:434-56. 
Yaacoby-Bianu K, G. M. (2018). Compassionate nitric oxide adjuvant treatment of persistent 
Mycobacterium infection in cystic fibrosis patients. . Pediatr Infect Dis J., 37(4), 336-338. 
 
 
 


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Appendix 1: Common Terminology Criteria for Adverse Events (CTCAE)  
 
Version 5.0 27Nov2017 
 
CTCAE v5.0 2017-11-27.xlsx (live.com) 
 
 




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Appendix 3: IUATLD/WHO Scale 
 
 
Reference:  
priorities for bacteriology services in low-income countries (2007).pdf (tbonline.info) 
Accessed 25March2023. 

SAP_001.pdf: 
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STATISTICAL ANALYSIS PLAN 
Early Bactericidal Activity, Demographics and Safety Analyses 
PROTOCOL NUMBER: RESP30X-EBA 
Phase 2 Study to Evaluate the Early Bactericidal Activity, Safety, and Tolerability of Nebulised 
RESP301 in Adults with Newly Diagnosed, Rifampicin Susceptible Pulmonary Tuberculosis 
Version 1.2 
Version and Date: 
Version 1.2 
22 Jul 2024 
Statements 
The statistical data analysis described in the present plan will be performed under Good Clinical 
Practice (GCP) principles and following the approved SOPs of TASK. 
The information contained in this document is provided in confidence. It is understood that this 
information will not be disclosed to others without prior agreement of TASK, in accordance with 
the signed confidentiality 




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CONTENTS 
TABLE OF RESPONSIBILITIES 
2 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
4 
1  DOCUMENT HISTORY _______________________________________________________ 6 
2  INTRODUCTION _____________________________________________________________ 7 
3 OBJECTIVES OF THE STUDY 
7 
4  DESIGN OF THE TRIAL ______________________________________________________ 8 
4.1 General ____________________________________________________________________ 8 
4.2 Study population ____________________________________________________________ 8 
4.3 Sample Size Determination 
9 
4.4 Treatment administration 
9 
4.5 Randomisation 
10 
4.6 Blinding __________________________________________________________________ 10 
4.7 Sampling of Observations 
10 
4.8 Data Collection and Integration _______________________________________________ 13 
4.9 Stopping Rules 
13 
 
5 ENDPOINTS 
14 
5.1 
Primary Endpoints 
14 
5.2 
Secondary Endpoints 
14 
5.3 Exploratory endpoints _______________________________________________________ 15 
6 ANALYSIS POPULATIONS 
16 
7  GENERAL PRINCIPLES _____________________________________________________ 17 
7.1 Analysis Datasets 
17 
7.2 Analysis Software/Statistical Packages _________________________________________ 19 
7.3 Changes in the Conduct of the Study or Planned Analysis _________________________ 19 
9 INTERIM ANALYSIS FOR SAFETY AND EBA 
19 
10 ANALYSIS METHODS 
21 
10.1 Demography , Safety , and Baseline Characteristics 
21 
10.2 Safety and Tolerability _____________________________________________________ 22 
10.3 Early Bactericidal Activity (EBA) ____________________________________________ 24 
11 REPORTING 
27 
11.1 Demography and Baseline 
27 
11.2 Safety and Tolerability _____________________________________________________ 28 
11.3 EBA 
31 
12 APPENDIX 
37 
12.1 Table Shells 
37 
12.2 Graphical exploration of early bactericidal activity data __________________________ 59 
13 REFERENCES 
67 


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List of Abbreviations and Definitions of Terms 
 
Abbreviations 
Stands for (Explanation) 
 
% 
Percentage 
ADaM 
Analysis Dataset Model 
AE 
Adverse Event 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
bd 
Twice daily 
BMI 
Body Mass Index 
CDISC 
Clinical Data Interchange Standards Consortium 
CRF 
Case Report Form 
CFU 
Colony Forming Units 
CS 
Clinically Significant 
eCRF 
electronic Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
DDT 
Data Definition Table 
DMP 
Data management plan 
EBA 
Early Bactericidal Activity 
EBEs 
Empirical Bayes Estimates 
EC 
Ethics Committee 
EWD 
Early Withdrawal 
FEV1 
Forced Expiratory Volume in the first second 
GCP 
Good Clinical Practice 
HIV 
Human Immunodeficiency Virus 
HRZE 
Isoniazid/Rifampicin/Pyrazinamide/Ethambutol 
IMP 
Investigational Medicinal Product 
NCS 
Not Clinically Significant 
NEB 
Nebulisation 
PI 
Principal Investigator 
po 
per os/orally 
Q1 
First quartile 
Q3 
Third quartile 
qd 
once daily 
PTB 
Pulmonary tuberculosis 
SAP 
Statistical Analysis Plan 
SD 
standard deviations 
SDTM 
Study data tabulation model 
SOC 
System Organ Class 
TASK 
TASK Applied Science (Pty) Ltd 
TB 
Tuberculosis 
tds 
three times a day 
TEAEs 
Treatment-Emergent Adverse Events 
TSC 
Trial Steering Committee 
TTP 
Time to Sputum Culture Positivity 


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ULN 
Upper Limit of Normal 
EBA Definitions 
EBA 
An agent’s ability to kill mycobacteria expectorated in sputum during the first 
weeks of treatment. 
EBA CFU 
Determination of EBA by quantification of viable mycobacteria as colony forming 
units (CFU) on a solid media culture. 
EBA TTP 
Determination of EBA by quantification of viable mycobacteria as time to culture 
positivity (TTP) in an automated liquid culture system. 


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1 Document History 
 
Version 
Date 
Reason for change 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
” 


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2 Introduction 
 
This Statistical Analysis Plan (SAP) describes the statistical methods to be used during the 
analysis and reporting of demographics, baseline characterstics, safety and tolerability, and 
early bactericidal activity (EBA) on data collected from the Protocol RESP30X-EBA 
(Phase 2 Study to Evaluate the Early Bactericidal Activity, Safety and Tolerability of 
nebulised RESP301 in Adults with Newly Diagnosed, Rifampicin Susceptible Pulmonary 
Tuberculosis (PTB)). Investigational medicinal products to be used in this study include 
RESP301 and Isoniazid/ Rifampicin/ Pyrazinamide/ Ethambutol (HRZE). All study 
treatments will be given orally either via inhalation or ingestion. 
 
This SAP should be read in conjunction with the Study Protocol. It has been developed 
using Protocol Version 1.0 dated 06 April 2023. 
 
The purpose of this SAP includes the outline of the planned analysis to assess the 
demographics (including baseline), safety and tolerability, and EBA. This analysis will 
support the completion of the clinical study report (CSR) and may be included in 
regulatory submissions and/or future manuscripts. Any deviations from the planned 
analyses will be documented in the CSR. 
 
3 Objectives of the Study 
Primary Objective 
Stage 1 
To determine the EBA of three times a day (tds) dosing of inhaled RESP301 6 ml dosed 
via nebulisation (NEB) over 14 days of treatment. 
 
Stage 2 
To determine the EBA of inhaled RESP301 6 ml dosed via NEB administered qd and bd 
alone, or tds in combination with HRZE, over 14 days of treatment to optimise the dosing 
schedule. 
 
Secondary Objectives 
To determine the safety and tolerability of inhaled RESP301 6 ml via NEB administered 
qd, bd, and tds alone or tds in combination with HRZE, over 14 days of treatment. 
 
Exploratory Objectives 
To explore the EBA variability due to potential M.tb sterilisation in sputum samples as a 
result of NO contamination in sputum samples. 






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4.5 Randomisation 
 
Eligible participants who have given written, informed consent will be screened on the 
trial during Visit 1 (Days -9 to -3) and will be identified by a study-generated participant 
identification code for anonymity (participant number). 
 
Participants who meet all the inclusion criteria and none of the exclusion criteria will be 
randomised and assigned a treatment number. The randomization scheme will be 
generated by the study statistician. Randomisation will be conducted using permuted block 
randomisation. Randomisation will be done with 3:1 (arm 1, arm 5) in stage 1. Once 
enrolment into stage 2 is opened, randomisation into the remaining arms will be 2:3:3:3 
(arm 5, arms 2-4) [Table 4]. 
Consecutively numbered, sealed, opaque envelopes will be prepared and provided to the 
site. The site research pharmacist will maintain the randomisation envelopes in a secured 
location. After being notified that a participant is eligible for the study, the research 
pharmacist will write the participant's number on the next numbered envelope and open it 
to determine the participant's treatment group. The participant number, participant initials, 
the date and time the envelope was opened, and the signature of the person opening the 
envelope will be recorded on the treatment group card in the envelope at the time it is 
opened. Opened envelopes containing treatment group cards will then be filed in the 
participants’ source file. 
Participants who withdraw from the study before they have received all days of IMP may 
be replaced per the discretion of the sponsor unless they are withdrawn for safety reasons. 
 
4.6 Blinding 
 
This is an open-label study and there is therefore no need for blinding or procedures to 
break the blind. The study endpoints are laboratory-based and the microbiology staff will 
be blinded to treatment allocation until the EBA database closure and release. 
 
4.7 Sampling of Observations 
 
Sampling of observation for fixed and random variables will be done over time per study 
procedure as shown in Table 5. Study Time and Study Treatment will be fixed factors 
(deterministic) in the study design while all other variables will be random (realised after 
sampling). 




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continue for 16 hours ± 1 hour overnight. The 16-hour sputum sampling for each of the 
sampling days must be finished prior to the administration of the next morning’s IMP. 
Collection will be paused for 30 minutes after evening RES301 dose where applicable. 
J: 
Spot or Overnight Sputum: For eligibility assessments. 
K: 
Blood collection for Haematology, Chemistry, and urinalysis on day 1 must be done 
prior to first dose. 
L: 
β-HCG Serum Pregnancy Test: All female participants. 
M: 
 Follow-up visit: a window of ±3 days allowed. 
N: 
EBA unit inpatient: discharge 


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4.8 Data Collection and Integration 
The sampled observational information from study procedures will be recorded in the study 
source documents. This information will then be captured electronically onto a Clinical Study 
Database that is developed based on the electronic Case Report Forms (eCRFs). The variables 
names, types, formats, domains/tables will be based on the annotated eCRFs. Annotation of the 
eCRFs will done according to the Clinical Data Interchange Standards Consortium (CDISC) 
study data tabulation model (SDTM) guidelines. These guidelines will ensure compliance with 
regulatory bodies and ensure that the FAIR principle applies to the clinical study data. The 
Clinical Study Database will be developed in a Clinical Data Management System (CDMS) 
compliant with the Protection of Personal Information Act, 2013, 21 Code of Federal Regulation 
(CFR) Part 11 and General Data Protection Regulation (GDPR). Laboratory results from the 
Safety, Mycobacteriology, and Pharmacokinetic laboratories will be imported into the Clinical 
Study Database and reconciled with blood and sputum sampling information. The study Data 
Management Plan describes the data collection and integration described in more elaborate 
detail. 
4.9 Stopping Rules 
 
If the Investigator, the Sponsor, or Sponsor’s Designee, or appropriate regulatory officials 
discover conditions arising during the study that indicate that the study should be halted or that 
the study centre should be terminated, this action may be taken after appropriate consultation 
among the Sponsor and Investigator/s. Conditions that may warrant termination of the study or 
discontinuation of a study site include, but are not limited to, the following: 
∙ 
The discovery of an unexpected, serious, or unacceptable risk to the participants enrolled in 
the study 
∙ 
A decision on the part of the Sponsor to suspend or discontinue testing, evaluation, or 
development of the product 
∙ 
Failure of the Investigator to enroll patients into the study at a rate commensurate with good 
clinical research practice and acceptable to the Sponsor 
∙ 
Failure of the Investigator to comply with pertinent regulations of appropriate regulatory 
authorities 
∙ 
Submission of knowingly false information from the research facility to the Sponsor, Study 
Monitor, or appropriate regulatory authority 
∙ 
Insufficient adherence to protocol requirements 
The Regulatory Authorities and Ethics Committees/Institutional Review Boards (EC/IRB) will 
be informed should the study be terminated. All study materials (except documentation that 
must remain stored at the site) will be returned to the Sponsor. The Investigator will retain all 
other documents until notification is given by the Sponsor for destruction. 
 
Participants currently on IMP will receive an appropriate TB regimen and all participants will be 
referred as soon as possible to the national TB programme. 


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5 Endpoints 
5.1 Primary Endpoints 
 
The following are the primary endpoints: 
 
The EBATTP0-14 in the Mycobacteria Growth Indicator Tube (BactecTM MGITTM 960) system 
as determined by the predicted rate of change in the TTP in sputum over the period day 0 to 14 
will be analysed 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5.2 Secondary Endpoints 
EBA 
 
The EBACFU (0-14) as determined by the predicted rate of change in log10CFU per ml 
sputum over the period day 0 to day 14 will be described 
 
 
 
 
 
 
 
 
 
Safety and Tolerability 
 
All adverse events will be coded using the Medical Dictionary for Regulatory Activities 
(MedDRA) and will be presented by preferred term within each MedDRA System Organ Class 
(SOC). Treatment-emergent adverse events (TEAEs) are defined as AEs which started at or 
after the first administration of IMP and includes those events which started prior to the first 
administration of IMP but which worsened after the first intake. Adverse events starting after 


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the first administration of IMP until the last scheduled visit/ assessment/ measurement will be 
regarded as treatment-emergent. 
The incidence of the following events will be summarized by treatment: 
• Incidence of treatment-emergent adverse events (TEAEs); 
• Incidence of TEAEs by Severity; 
• Incidence of drug-related TEAEs; 
• Incidence of Serious TEAEs; 
• Incidence of TEAEs leading to early withdrawal; 
• Incidence of TEAEs leading to death. 
 
Descriptive summary statistics will be presented for other safety variables: laboratory 
parameters, physical examination, vital signs, concomitant medication. Information on all 
adverse events will be collected and captured in the eCRF. 
 
Please refer to the Section 10.1 and 10.2 for additional information regarding analyses of these 
endpoints. 
 
5.3 Exploratory endpoints 
 
To explore the EBA variability due to potential M.tb 15terilization in sputum samples as a result 
of nitric oxide (NO) contamination in sputum samples. 
 
 
 
 
 
 
 
 
 


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6 Analysis Populations 
 
The Safety analysis population will consist of all randomised participants who took at least one 
dose of study treatment. 
 
The EBA population will consist of all participants who have baseline and at least one post 
baseline TTP or CFU observation. There will be one EBA population for TTP and one for CFU. 


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7 General Principles 
 
The SAP will be finalised and signed-off prior to database lock. Post-hoc analysis (analyses 
performed that are different from those described in the final version of the SAP) will be 
documented in the final CSR as changes to planned analysis. For the Demography, Safety and 
Tolerability, and EBA analyses, there will be two analysis periods, an interim analysis and an 
analysis at the end of stage 2 of the study. 
 
All data collected will be listed, and sorted by treatment, participant number, visit, and time. 
 
Descriptive summaries will be presented using summary statistics (e.g. number (n), mean, 
standard deviations (SD), median, first quartile (Q1), third quartile (Q3), minimum (min), 
maximum (max)) for continuous variables or number and percentage n (%) for 
categorical/discrete variables. Continuous variables will be expressed as median with inter- 
quartile range for skewed data and mean with SD for normal data. The percentages will not be 
provided for a zero count. The mean, median, SD, Q1, and Q3 will be presented to 1 more 
decimal place (dp) than the raw data, and min and max to the same number of dps as the raw 
data. Percentages will not be provided for a zero count and 100% will be provided to zero dps. 
 
All output will be generated using SAS version 9.4 for safety analysis, and R software Version 
4.3.1 or above for EBA analysis (including figures) and safety figures. 
 
Handling of Dropouts or Missing Data: No missing data will be replaced, and it will be shown in 
any summaries as missing for all analyses. 
 
Treatment of Outliers: TTP and CFU observations which are considered to be outliers by a team 
of microbiologist and clinical trialist, might be removed from the EBA analysis. Outliers and 
reason for removal will be documented. 
 
If the start date of an Adverse Event (AE) (or concomitant medication) is incomplete, the most 
conservative approach will be taken and the AE treated as treatment emergent (or concomitant, 
in the case of medication) to the last study treatment given. 
 
7.1 Analysis Datasets 
 
For the Demography, Safety and Tolerability analysis, the study data tabulation model (SDTM) 
datasets after soft-lock of the clinical database will be used. The soft-lock of the clinical 
database is described by the study data management plan (DMP). Analysis datasets will be 
generated during the analysis according to the ICH E3, E9, and Clinical Data Interchange 
Standards Consortium (CDISC) Analysis Dataset Model (ADaM) guidelines. 
 
For the EBA analysis, an EBA analysis dataset will be generated from the SDTM datasets 
according to the RESP30X-EBA Data Definition Table (DDT). The RESP30X-EBA DDT is a 
separate document that details the data specifications of the EBA analysis dataset and will be 
finalised prior to the first EBA analysis. In general, the EBA DDT will compromise at least the 
following variable: 


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CDSIC 
DOMAIN 
Variable 
Label 
Type/Format 
Leng 
th 
ALL 
#REF 
Consecutive record number. 
Numeric 
LONG 
ALL 
STUD 
Protocol number. Insert 
protocol number. 
Character 
200 
DS 
UID 
SUBJECT 
(RANDOMIZATION 
NUMBER). 
Character 
200 
ALL 
ID 
ID for NONMEM dataset 
Numeric 
LONG 
ALL 
TIME 
Nominal sample production 
day 
Numeric 
LONG 
LB MICRO 
DVCFU 
Dependent variable 
Demical/Float 
5.2 
LB MICRO 
DVTTP 
Dependent variable 
Numeric 
LONG 
LB MICRO 
SSCFU 
Sample status for CFU counts 
Numeric 
LONG 
LB MICRO 
SSTTP 
Sample status for TTP counts 
Numeric 
LONG 
LB MICRO 
REP 
Replicate number 
Numeric 
LONG 
DS 
GROUP 
Group 
Numeric 
LONG 
DS 
TRT 
Treatment 
Numeric 
LONG 
DS 
REGI 
Regimen 
Character 
200 
VS 
BMI 
BMI 
Demical/Float 
5.1 
VS 
HT 
Height 
Numeric 
LONG 
DM 
ETHNICITY 
Ethnicity 
Numeric 
LONG 
VS 
WT 
Weight 
Demical/Float 
5.1 
DM 
AGE 
Age 
Numeric 
LONG 
DM 
SEX 
Sex 
Numeric 
LONG 
LB MICRO 
BBLCFU 
Baseline bacterial load. 
(average log10 CFU on days - 
2 and -1) 
Numeric 
LONG 
LB MICRO 
BBLTTP 
Baseline bacterial load. 
(average TTP on days -2 and - 
1) 
Numeric 
LONG 
LB SAFETY 
HIV 
HIV 
Numeric 
LONG 
CX 
EXTENT 
Severity or extent of disease 
Character 
200 
CX 
CAV 
Presence of cavitation 
Numeric 
LONG 
LB MICRO 
SQUAL 
Sputum quality 
Numeric 
LONG 
LB MICRO 
SVOL 
Sputum volume 
Numeric 
LONG 
LB MICRO 
COMMENT 
Comments from the 
Microbiology Laboratory 
associated with sample 
observation 
Character 
200 
SU 
SMOKING 
Smoking 
Character 
200 
LB MICRO 
MBGENMTBULTRA 
Genexpert MTB/RIF ULTRA 
Detection. 
Numeric 
LONG 
LB MICRO 
MBGENMTBULTRAP 
os 
Genexpert MTB/RIF ULTRA 
Detection Positivity. 
Numeric 
LONG 
LB MICRO 
MBGENMTBULTRAC 
yc 
Genexpert MTB/RIF ULTRA 
Detection Cycle Threshold. 
Numeric 
LONG 
RE 
SPIROMETRY 
REFVC 
Spirometry Forced Vital 
Capacity Actual value. 
Numeric 
LONG 


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RE 
SPIROMETRY 
REFVC1 
Forced Expiratory Volume in 
1 Second Actual value. 
Numeric 
LONG 
RE 
SPIROMETRY 
REFEV1_FVC 
Modified Tiffeneau-Pinelli 
index Actual value. 
Numeric 
LONG 
CV SPMET 
CVSPMET 
SpMet Measurement Highest 
value. 
Numeric 
LONG 
LB SAFETY 
LBHGB 
Haemoglobin in Blood 
Plasma. 
Numeric 
LONG 
LB SAFETY 
LBBASOA 
Basophils Absolute (ABS) in 
Blood Plasma. 
Numeric 
LONG 
LB SAFETY 
LBEOSA 
Eosinophils Absolute (ABS) 
in Blood Plasma. 
Numeric 
LONG 
LB SAFETY 
LBIGA 
Imm Granulocyte Absolute 
(ABS) in Blood Plasma. 
Numeric 
LONG 
LB SAFETY 
LBLYMPHA 
Lymphocytes Absolute (ABS) 
in Blood Plasma. 
Numeric 
LONG 
LB SAFETY 
LBMONOA 
Monocytes Absolute (ABS) 
in Blood Plasma. 
Numeric 
LONG 
LB SAFETY 
LBNEUTA 
Neutrophils Absolute (ABS) 
in Blood Plasma. 
Numeric 
LONG 
LB SAFETY 
LBWBC 
White cell count in Blood 
Plasma. 
Numeric 
LONG 
 
7.2 Analysis Software/Statistical Packages 
 
All study analysis datasets will be generated using SAS version 9.4. SAS version 9.4 will also be 
used for the Demography, Safety and Tolerability analysis. The R software version 4.3.1 or 
above will be used for EBA analysis and for safety figures and statistical models. SAS version 
9.4 will be used to validate models to ensure consistent estimates from R software version. If 
updated versions of the above software become available these may be used and will be detailed 
in the Clinical Study Report. 
 
 
 
 
 
7.3 Changes in the Conduct of the Study or Planned Analysis 
 
Any deviations from the planned analysis will be provided and documented in the Statistical 
Reports and CSR (including Demographics, Safety and EBA). Information from these 
analyses will be combined into the Clinical Study Report. 
 
9 Interim Analysis for Safety and EBA 
 
The objective of stage 1 is to determine the EBA of inhaled RESP301, to inform ongoing 
development. When stage 1 is completed, recruitment will be paused, and an interim review will 
be conducted by a Trial Steering Committee (TSC) on the Safety and EBA. This will be detailed 


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in the Trial Steering Committee Charter. 
 
 
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In order to progress to Stage 2, the following minimum should be met: 
∙ 
RESP301 tds active treatment arm EBATTP(0-14): the predicted rate of change in the 
TTP in sputum over the period day 0 to 14 must be statistically different from zero at the 
5 % level (demonstrating acceptable EBA to continue to Stage 2). 
∙ 
≤50% of the participants experienced a grade 3 or higher AE. 
 
A protocol amendment will be considered if the following safety and tolerability criteria are 
met: 
∙ 
≥25% of the participants experienced a grade 2 or higher AE of any description 
considered probably or definitely related to IP. 
∙ 
≥20% of the participants experienced a grade 3 or higher AE reflecting intolerability to 
IMP (gastrointestinal: diarrhoea, nausea, or dyspepsia; respiratory: bronchospasm). 
 
The safety, tolerability, and EBA interim analysis will be conducted as described in Sections 
10.1, 10.2, and 10.3 of this SAP respectively. 


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10 Analysis Methods 
 
OVERVIEW OF THE ANALYSIS 
The analyses to be conducted in this study will include Demography, Baseline Characteristics, 
Safety and Tolerability, and EBA. 
10.1 Demography, Safety, and Baseline Characteristics 
The disposition of participants, completion of the study, and reasons for withdrawal will be 
summarised by treatment and overall. The number of participants withdrawing and the reason 
for withdrawal will also be summarised by the last treatment taken. 
 
In the event of more than a single data point (for a particular variable) prior to the administration 
of trial medication, “baseline” will be regarded as the first of these observations. 
 
Demographic data will be summarised by treatment and overall. Summary statistics will be 
calculated for age, race and gender, height, weight, and body mass index (BMI) for the Safety 
Population. 
 
Substance use of the safety population will also be summarised by treatment and overall. 
 
The results of the medical history (refer to Adverse Events for an explanation regarding the 
coding of medical history), vital signs, safety laboratory values, and physical examination at 
baseline/screening will be tabulated by treatment for the Safety Population. 
 
Prior medication is defined as any medication that ended prior to the first dose of trial 
medication. Incidence will be summarised by generic name and class by dose for the Safety 
Population. 
 
Listings for the data used in the tabulations will also be given. Listings containing the following 
information will also be included: 
1. 
Study Completion Status 
2. 
Visit Dates (including hospital admission and discharge) 
3. 
Eligibility and Inclusion and Exclusion Criteria 
4. 
Subject Eligibility (including Informed Consent, Initial Diagnosis and Randomization) 
5. 
Urine Drug Screen 
6. 
Serum Pregnancy 
7. 
Serology (HIV) and CD4 Count 
8. 
Study Drug Administration (including compliance) 
9. 
Adverse Events 
10. Physical Examination 
11. Vital Signs 
12. Chest X-ray 
 
The response to the eligibility assessments and inclusion/exclusion criteria will be listed. 
 
For each treatment exposure (EX), the dose/number of tablets/capsules (group dependent) taken 
overall will be calculated along with the percent compliance out of the total number of 


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tablets/capsules expected and will be summarised by dose for the Safety Population. 
 
10.2 Safety and Tolerability 
10.2.1 Adverse Events 
 
All adverse events will be coded using the Medical Dictionary for Regulatory Activities 
(MedDRA) and will be presented by Preferred Term within each MedDRA System Organ Class 
(SOC). 
 
An adverse event will be defined as Treatment Emergent (TEAE) if the onset date is after the 
first intake of study medication. 
 
Where dates are incomplete such that a decision cannot be made, it will be assumed that the 
adverse event is TEAE. 
 
The incidence of the following events will be summarised by treatment group: 
13. TEAEs by SOC and preferred term. 
14. TEAEs by maximum severity. 
15. TEAEs by strongest relationship to IP 
16. Related TEAEs. 
17. Potentially IMP-related (Related and Possibly Related) TEAEs. 
18. Potentially IMP- TEAEs by maximum severity. 
19. TEAEs with a fatal outcome. 
20. Serious TEAEs. 
21. Discontinuations due to TEAEs. 
The frequency of TEAEs, by severity and IMP relationship, will be presented by treatment. The 
sampled and recorded observations of AEs will be considered to be discrete random variables. 
AEs will be analysed based on absolute risk or crude incidence rates or exposure-adjusted 
incidence rates using frequency statistics. The AE variable will be modelled as a Bernoulli 
distribution with parameter (p) representing the probability of success/occurrence. The definition 
of absolute risk applied in this analysis will be the following: 
∙ 
The number of individuals experiencing an AE divided by the total number of 
participants in that dose treatment group. 
 
Applicable only in the AE summary table14.3.1.8, the following definition of absolute risk will 
be used for this table: 
∙ 
The number of AEs divided by the total number of AEs in that dose treatment group 
 
Furthermore, these rates do not adequately account for an individual participant’s profile of 
adverse events over the study period when a participant may remain in the trial after 
experiencing one or more events (i.e., occurrence of multiple events of the same kind or 
different kinds). Moreover, the required statistical assumptions (e.g., constant hazard rate over 
time) for valid estimates of incidence rates are not likely to be met in practice by adverse events 
data of clinical trials. To overcome this, mean cumulative function (MCF) will also be 
performed for the safety data. MCF is a nonparametric approach that provides a valid statistical 
inference on recurrent adverse event profiles of drugs in randomized clinical trials (RCT). The 


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estimate involves no assumptions about the form of MCF. As compared to the crude or 
exposure-adjusted incidence rates of adverse events, the MCF estimates facilitate more 
understanding of the safety profiles of a drug in an RCT. Survival analysis models to understand 
the probability of occurrence over time will be applied using the Kaplan-Meier, Cox-Proportion 
Hazzard, and Binary Logistical Regression models. A special focus will be placed on Adverse 
Events that are considered related to study treatment. 
 
10.2.2 Study Stopping and Amendment Criteria 
 
The study stopping and amendment criteria will be summarised as described in Section 9 in the 
form of frequency statistics per treatment group and time point. 
10.2.3 Other Safety Variables 
 
Laboratory parameters 
Laboratory safety tests are performed throughout the study. The data will be listed with values 
outside the normal range flagged and with the grade according to Common Terminology Criteria 
for Adverse Events (CTCAE) mentioned. The recorded values and the change from baseline will 
be summarized by treatment and time of collection. The assessment (normal/abnormal-NCS/ 
abnormal-CS as well as the grade according to CTCAE) will be summarized for haematology, 
blood chemistry, and urinalysis by treatment and time of collection. 
 
Physical examination 
The physical examination is repeated at each visit. The results (normal/abnormal-NCS/ 
abnormal-CS as well as the grade according to CTCAE, if applicable) will be summarized by 
body system by treatment and time of collection. 
 
Vital signs 
Vital signs are recorded at each visit. The change from the baseline will be calculated. The data 
will be summarized by treatment and time of collection. 
 
Concomitant medication 
Concomitant medication is defined as any medication taken within 14 days prior to IMP 
administration or during the trial until visit 19 (day 28). Where dates are incomplete such that a 
decision cannot be made, it will be assumed that a medication is concomitant. The incidence will 
be summarised by generic name, class, and treatment. 
 
Methemoglobin 
The assessment of Methemoglobin using Spmet will be summarized by single SpMet 
measurement and Highest SpMet measurement per treatment and time of assessment using 
descriptive statistics. 
 
Lung Function 
The assessment of lung function using Spirometry will be summarized by Forced Vital Capacity, 
Forced Expiratory Volume in 1 Second and Modified Tiffeneau-Pinelli index per treatment and 
time of assessment using descriptive statistics. 




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The models will include one intercept defined by A and one or two slopes defined by α and β 
and k the node parameter. 
3) Mono-exponential model: 
4) Bi-exponential model: 
 
The models will include one or two intercepts defined by A and B and one or two slopes defined 
by α and β. 
10.3.2 Stochastic model (Random Effects) 
1. Inter-individual variability 
During the course of the model development, the Inter-individual (IIV) parameters will be tested 
on the structural parameters of the EBA models. Inter-individual parameters will be included in 
an exponential manner to ensure that the individual EBA parameters have a lower bound of 
zero: 
 
P_i=TVP.eŋ 
 
where TVP is the typical (Expected) value of the parameter P, P_i is the individual value of the 
parameter and ŋ is a normally distributed random variable with mean 0 and standard deviation 
ɷ. Not all parameters need to be associated with IIV. 
 
If the distributions of the ŋs are markedly non-normal, transformations of ŋs may be considered. 
Terms to describe correlations between ŋs will also be considered if indicated by the data. 
Initially, IIV should be added to the baseline only. IIV in other parameters should be explored 
after the treatment effect evaluation. 
10.3.3 Residual unexplained variability (Random Error) 
The aim of the residual unexplained variability (RUV)/random error model is to ensure that the 
individual weighted residuals (IWRES) are approximately homoscedastically distributed across 
all predictors (e.g., time, occasion, and study). The RUV describes the differences between the 
observations and the individual predictions (IPRED)/estimates of the model. The RUV model 
can be either additive (homoscedastic), log-additive (exponential), proportional (heteroscedastic) 
or combined additive and proportional error model. The different RUV models will be evaluated 
for the models. 
10.3.4 Covariate model (Mixed Effects) 
After confirmation of the structural and stochastic EBA models a covariate analysis will be 
done. The median of baseline values of the covariate will be used for time-independent 
covariates. For time-dependent covariates, the actual value of the covariate at the time of the 
observation will be used (or closest in time to the observation). The inclusion of time-dependent 
covariates in the covariate analysis will be informed by the exploratory data analysis. For time- 
varying covariates to be included in a model there must be a clear rational for assuming that the 
time variation is independent of the drug treatment and not vice versa. Time-varying covariates 


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for which the causality is unclear may be investigated after the assessment of other covariates or 
drug effects. 
 
10.3.5 Model evaluation 
Model evaluation will include graphical analysis of goodness of fit (GOF) plots, relative 
standard errors (RSEs), visual predictive checks (VPCs), and possibly other simulation-based 
diagnostics. Stratifications will be used when appropriate to ensure that the models perform 
adequately across important subgroups of the data. 
10.3.6 Model discrimination 
The discrimination between models will mainly be made based on the inspection of graphical 
diagnostics and changes in the objective function value (OFV) and Akaike information criterion 
(AIC). The differences in OFVs are nominally X2 distributed and a difference of -3.64 
corresponding to approximately a p-value of < 0.05 for one degree of freedom, provided that the 
models are hierarchical. Preferably this should also demonstrate improvements in the graphical 
diagnostics and not lead to a high (>1000) conditional number. 
10.3.7 Evaluation of shrinkage 
Shrinkage in Empirical Bayes Estimates (EBEs) of model parameters used for diagnostic 
purposes will be evaluated. The shrinkage increases from 0 towards 100% as the EBEs becomes 
less informative, and EBE based graphs and diagnostics are more reliable when shrinkage is 
small. A shrinkage of <20% is generally considered as a low shrinkage. Shrinkage will be taken 
into account when EBE based graphs and diagnostics are evaluated. 
10.3.8 Final model 
A final model should provide a good description of the observed data, not have any important 
trends in the relevant GOF diagnostics, have a successful termination of the covariance step and 
preferably have a condition number less than 1000. Should a model be accepted as final without 
these criteria being met, it will be discussed and justified in the report. 
There will be one final model for each of the endpoints. 
 
Based on the final EBA models for TTP and CFU, mean bactericidal activity per treatment arm 
will be expressed as the daily change in TTP and CFU from day 0 to day 14. If required, periods 
from 0 to 2 days, 0 to 7 days, 7-14 days, or any other period will be predicted or calculated as 
well as the 95% Bayesian credibility interval using only the patient data included in the study. 
Clinical trial simulations (n= 1000) will be used to predict mean (95% prediction interval) 
bactericidal activity for different treatment arms and different characteristics depending on the 
covariates included in the final models. 
 
Early bactericidal activity data definition table (DDT): A separate data specification table will be 
created for EBA. The dataset should be in excel csv format. A readme file describing the dataset 
column names should be created with an example for 1 participant. 


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11 Reporting 
 
The Demography, Baseline Characteristics, Safety and Tolerability, and EBA analysis results 
will be reported. The CSR will incorporate these in one report. The statistical reports are 
independent and will be reported in parallel. The interim analysis report will be focused on 
safety and EBA. 
11.1 Demography and Baseline 
 
The results of the demographic and safety analysis will be reported as per the tables listed 
below: 
 
Table 
Number 
Population 
Table Title 
Shell/ 
Reference to 
Shell 
14.1 Demographic Data 
14.1.1 
All 
Participants 
Disposition of Participants 
S 
14.1.2 
Safety 
Demographics 
S 
14.1.3 
Safety 
Medical History 
S 
14.1.4 
Safety 
Vital Signs at Baseline 
14.1.2 
14.1.6.1 
Safety 
Physical Examination at Baseline 
S 
14.1.7 
Safety 
Prior Medications 
S 
14.1.8.1 
Safety 
Clinical Safety Laboratory Tests at Baseline – 
Haematology 
14.1.2 
14.1.8.2 
Safety 
Clinical Safety Laboratory Tests at Baseline – Serum 
Chemistry 
14.1.2 
14.1.8.3 
Safety 
Clinical Safety Laboratory Tests at Baseline – 
Urinalysis 
14.1.2 
14.1.9 
Safety 
Mycobacteriological Characteristics at Baseline 
14.1.2 
14.1.10 
Safety 
Exposure and Compliance 
14.1.2 
14.1.11 
Safety 
Substance Use 
14.1.2 
14.1.12 
Safety 
Methemoglobin Assessment 
14.1.2 
14.1.13 
Safety 
Lung Function Assessment 
14.1.2 


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11.2 Safety and Tolerability 
 
The results of the safety and tolerability analysis will be reported as per the tables, listings and 
figures listed below: 
 
Listing Number 
Listing Title / Summary 
16.2 Safety Listings 
16.2.1.1 
Study Completion Status 
16.2.1.2 
Visit Dates (including hospital admission and discharge) 
16.2.2 
Eligibility and Inclusion and Exclusion Criteria 
16.2.3 
Subject 
Eligibility 
(including 
Informed 
Consent, 
Initial 
Diagnosis 
and 
Randomization) 
16.2.4.1 
Demographic Data 
16.2.4.2 
Medical History 
16.2.4.3 
Urine Drug Screen 
16.2.4.4 
Serum Pregnancy 
16.2.4.5 
Serology (HIV) and CD4 Count 
16.2.4.6 
Prior and Concomitant Medication 
16.2.5 
Study Drug Administration (including compliance) 
16.2.6.1 
Adverse Events 
16.2.6.2 
Physical Examination 
16.2.6.3 
Vital Signs 
16.2.6.4 
Chest X-ray 
16.2.7 
Clinical Safety Laboratory Tests 
16.2.8 
Study Stopping and Amendment Criteria (for interim analysis) 
16.2.9 
Substance Use 


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Listing Number 
Listing Title / Summary 
16.2.10 
Methemoglobin Assessment 
16.2.11 
Lung Function Assessment 
 
 
 
 
14.3 Safety Data 
Table 
Number 
Population 
Table Title 
Shell/ 
Reference to 
Shell 
14.3.1.1 
Safety 
Summary of Incidence of Treatment Emergent Adverse 
Events 
S 
14.3.1.2 
Safety 
Incidence of Treatment Emergent Adverse Events by 
System Organ Class and Preferred Term 
S 
14.3.1.3.1 
Safety 
Incidence of IMP Related Treatment Emergent Adverse 
Events by System Organ Class and Preferred Term 
14.3.1.2 
14.3.1.3.2 
Safety 
Incidence of  Potentially  IMP  Related  Treatment 
Emergent Adverse Events by System Organ Class and 
Preferred Term 
14.3.1.2 
14.3.1.4 
Safety 
Incidence of Fatal Treatment Emergent Adverse Events 
by System Organ Class and Preferred Term 
14.3.1.2 
14.3.1.5 
Safety 
Incidence of Serious Treatment Emergent Adverse 
Events by System Organ Class and Preferred Term 
14.3.1.2 
14.3.1.6 
Safety 
Incidence of Treatment Emergent Adverse Events 
leading to discontinuation by System Organ Class and 
Preferred Term 
14.3.1.2 
14.3.1.7.1 
Safety 
Incidence of Treatment Emergent Adverse Events by 
System Organ Class, Preferred Term and Maximum 
Severity 
S 
14.3.1.7.2 
Safety 
Incidence 
of 
Potentially 
IMP-related 
Treatment 
Emergent Adverse Events by System Organ Class, 
Preferred Term and Strongest Relationship to IMP 
14.3.1.7.1 
14.3.1.7.3 
Safety 
Incidence of Treatment Emergent Adverse Events by 
System Organ Class, Preferred Term and Strongest 
Relationship to IMP 
14.3.1.7.1 
14.3.1.7.4 
Safety 
Incidence of Treatment Emergent Adverse Events by 
System  Organ  Class,  Preferred  Term,  Maximum 
Severity and Strongest Relationship to IMP 
S 
14.3.1.8 
Safety 
Summary of Frequency of Treatment  Emergent 
Adverse Events 
S 
14.3.2.1.1 
Safety 
Clinical Safety Laboratory Tests – Haematology over 
time 
S 
14.3.2.1.2 
Safety 
Clinical  Safety  Laboratory  Tests  –  Haematology 
Change from Baseline over time 
S 


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14.3.2.1.3 
Safety 
Clinical  Safety  Laboratory  Tests  –  Haematology 
Assessment over time 
S 
14.3.2.2.1 
Safety 
Clinical Safety Laboratory Tests – Serum Chemistry 
over time 
14.3.2.1.1 
14.3.2.2.2 
Safety 
Clinical Safety Laboratory Tests – Serum Chemistry 
Change from Baseline over time 
14.3.2.1.2 
14.3.2.2.3 
Safety 
Clinical Safety Laboratory Tests – Serum Chemistry 
Assessment over time 
14.3.2.1.3 
14.3.2.3 
Safety 
Clinical Safety Laboratory Tests – Urinalysis 
14.3.2.1.1 
14.3.4 
Safety 
Physical Examination 
14.1.6 
14.3.5 
Safety 
Vital Signs 
14.3.2.1 
14.3.6 
Safety 
Concomitant Medication 
14.1.7 
14.3.7 
Safety 
Summary  of  Assessment  of  Study Stopping and 
Amendment Criteria. (for interim analysis) 
S 
14.3.8 
Safety 
Methemoglobin Assessment Over Time 
14.3.2.1.1 
 
Figures 
Number 
Population 
Figure Title 
Shell/ 
Reference 
to Shell 
14.3.1 
Safety 
Summary of Incidence of Treatment Emergent 
Adverse Events 
S 
14.3.2 
Safety 
Clinically 
Significant 
Laboratory 
Tests 
by 
Treatment-Over Time 
S 
14.3.3 
Safety 
Clinically 
Significant 
Laboratory 
Tests 
by 
Treatment-Change from Baseline over Time 
S 


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11.3 EBA 
 
An EBA report will be written which will include 
∙ 
Figures for identification of outliers and data points to be omitted 
∙ 
Tables summarizing omitted data 
∙ 
Figures showing observed EBA 
∙ 
Figures showing predicted EBA 
 
Addition tables and figures will be generated during the modelling process. The general results 
of the EBA analysis will be reported as per the tables, listings, and figures listed below: 
14.4 EBA General Tables 
Table 
number 
Populati 
on 
Objective 
Table Title 
Shell 
References 
to Shell 
14.4.1 
EBA 
Missing 
values 
Positive and non-positive data in the dataset 
for CFU status at the different timepoints 
S 
14.4.2 
EBA 
Missing 
values 
Positive and non-positive data in the dataset 
for TTP status at the different timepoints 
14.4.1 
14.4.3 
EBA 
Outliers 
Listing of potential outliers and suspect 
values for CFU and TTP 
S 
14.4.4 
EBA 
Describing 
population 
Summary statistics of baseline discrete 
covariates for the treatment arms 
S 
14.4.5 
EBA 
Describing 
population 
Summary statistics of continuous covariates 
for the treatment arms at baseline 
S 
14.4.6 
EBA 
Describing 
population 
Summary statistics of continuous covariates 
for the treatment arms over time 
14.4.5 
14.4.7 
EBA 
Describing 
population 
Summary statistics of log 10 CFU over time 
14.4.5 
14.4.8 
EBA 
Describing 
population 
Summary statistics of log 10 TTP over time 
14.4.5 


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14.4.9 
EBA 
Model 
Median and interquartile range of observed 
EBA for 0-2, 0-7, 0-14 days, 0-15 and 14- 
15 days in CFU and TTP 
S 
14.4.10 
EBA 
Model 
Parameter estimates for the final CFU 
model 
S 
14.4.11 
EBA 
Model 
Expected model predicted EBA for 0-2, 0-7, 
0-14 days, 0-15 and 14-15 in CFU 
14.4.9 
14.4.12 
EBA 
Model 
Expected model predicted daily EBA in 
CFU 
S 
14.4.13 
EBA 
Model 
Individual model predicted EBA for 0-2, 0- 
7, 0-14 days, 0-15 and 14-15 in CFU 
14.4.9 
14.4.14 
EBA 
Model 
Individual model predicted daily EBA in 
CFU 
14.4.12 
14.4.15 
EBA 
Model 
Parameter estimates for the final TTP model 
14.4.10 
14.4.16 
EBA 
Model 
Expected model predicted EBA for 0-2, 0-7, 
0-14 days, 0-15 and 14-15 in TTP 
14.4.9 
14.4.17 
EBA 
Model 
Expected model predicted daily EBA in 
TTP 
14.4.12 
14.4.18 
EBA 
Model 
Individual model predicted EBA for 0-2, 0- 
7, 0-14 days, 0-15 and 14-15 in TTP 
14.4.9 
14.4.19 
EBA 
Model 
Individual model predicted daily EBA in 
TTP 
14.4.12 
14.4.20 
EBA 
Model 
Overview of the analysis dataset per 
treatment arm 
S 


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14.4 EBA General Figures 
Figure 
number 
Population 
Biomark 
er 
Objective 
Table Title 
Shell 
Referenc 
es to Shell 
14.4.1 
EBA 
CFU 
Outlier 
Log 10 CFU Cleveland 
dotplots 
S 
14.4.2 
EBA 
CFU 
Outlier 
Log 10 CFU Repetition 1 
vs Repetition 2 
S 
14.4.3 
EBA 
CFU 
Outlier 
Individual Mean CFU 
versus mean TTP 
S 
14.4.4 
EBA 
CFU and 
TTP 
Outlier 
Individual Log 10 CFU and 
DVTTP over Time per 
subject(to be added) 
S 
14.4.5 
EBA 
CFU 
Distributi 
on 
Distribution of log10CFU 
for HRZE and RESP301 
over 14 days 
S 
14.4.6 
EBA 
CFU 
Distributi 
on 
Distribution of log10CFU 
for HRZE over time 
14.4.5 
14.4.7 
EBA 
CFU 
Distributi 
on 
Distribution of log10CFU 
for RESP301 over time 
14.4.5 
14.4.8 
EBA 
CFU 
Trend 
Log 10 CFU versus time 
stratified for different 
regimens 
S 
14.4.9 
EBA 
CFU 
Trend 
Individual Log 10 CFU and 
CFU versus time for 
different regimens 
14.4.4 
14.4.10 
EBA 
CFU 
Multiple 
Colinearit 
y(baselin 
e) 
Correlation Matrix of 
Continuous Variables at 
baseline 
S 
14.4.11 
EBA 
CFU 
Multiple 
Colinearit 
y(overtim 
e) 
Correlation Matrix of 
Continuous Variables over 
TIME for HRZE 
14.4.10 
14.4.12 
EBA 
CFU 
Multiple 
Colinearit 
y(overtim 
e) 
Correlation Matrix of 
Continuous Variables over 
TIME for RESP301 
14.4.10 
14.4.13 
EBA 
CFU 
Multiple 
Colinearit 
y(baselin 
e) 
Log 10 CFU at baseline 
stratified for Sex 
S 


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14.4.14 
EBA 
CFU 
Multiple 
Colinearit 
y(overtim 
e) 
Log 10 CFU versus time 
for different regimens 
stratified by Sex 
S 
14.4.15 
EBA 
CFU 
Multiple 
Colinearit 
y(baselin 
e) 
Log 10 CFU at baseline 
versus AGE for different 
age groups 
14.4.13 
14.4.16 
EBA 
CFU 
Multiple 
Colinearit 
y(overtim 
e) 
Log 10 CFU versus time 
stratified for different age 
groups for all regimens 
S 
14.4.17 
EBA 
CFU 
Multiple 
Colinearit 
y(baselin 
e) 
Log 10 CFU at baseline 
versus BMI for different 
BMI categories 
14.4.13 
14.4.18 
EBA 
CFU 
Multiple 
Colinearit 
y(overtim 
e) 
Log 10 CFU overtime 
versus BMI for different 
BMI categories and 
regimen 
14.4.16 
14.4.19 
EBA 
CFU 
Multiple 
Colinearit 
y(baselin 
e) 
Log 10 CFU at baseline 
stratified for CAVITY 
14.4.13 
14.4.20 
EBA 
CFU 
Multiple 
Colinearit 
y(baselin 
e) 
Log 10 CFU at baseline 
stratified for EXTENT 
14.4.13 
14.4.21 
EBA 
CFU 
Multiple 
Colinearit 
y(baselin 
e) 
Log 10 CFU at baseline 
stratified for Sputum 
quality 
14.4.13 
14.4.22 
EBA 
CFU 
Multiple 
Colinearit 
y(overtim 
e) 
Log 10 CFU over TIME, 
stratified for TRT and 
Sputum quality 
14.4.16 
14.4.23 
EBA 
CFU 
Multiple 
Colinearit 
y(baselin 
e) 
Log 10 CFU at baseline vs 
MBGENMTBULTRAPos 
14.4.13 
14.4.24 
EBA 
TTP 
Outlier 
TTP Cleveland dotplots 
14.4.1 
14.4.25 
EBA 
TTP 
Outlier 
TTP Repetition 1 vs 
Repetition 2 
14.4.2 
14.4.26 
EBA 
TTP 
Distributi 
on 
Distribution of TTP for 
HRZE and RESP301 over 
14 days 
14.4.5 


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14.4.27 
EBA 
TTP 
Distributi 
on 
Distribution of TTP for 
HRZE over time 
14.4.5 
14.4.28 
EBA 
TTP 
Distributi 
on 
Distribution of TTP for 
RESP301 over time 
14.4.5 
14.4.29 
EBA 
TTP 
Trend 
TTP versus time stratified 
for different regimens 
14.4.8 
14.4.30 
EBA 
TTP 
Trend 
Individual TTP versus time 
for different regimens 
14.4.26 
14.4.31 
EBA 
TTP 
Multiple 
Colinearit 
y(baselin 
e) 
Correlation Matrix of 
Continuous Variables at 
baseline 
14.4.10 
14.4.32 
EBA 
TTP 
Multiple 
Colinearit 
y(overtim 
e) 
Correlation Matrix of 
Continuous Variables over 
TIME for HRZE 
14.4.10 
14.4.33 
EBA 
TTP 
Multiple 
Colinearit 
y(overtim 
e) 
Correlation Matrix of 
Continuous Variables over 
TIME for RESP301 
14.4.10 
14.4.34 
EBA 
TTP 
Multiple 
Colinearit 
y(baselin 
e) 
TTP at baseline stratified 
for Sex 
14.4.13 
14.4.35 
EBA 
TTP 
Multiple 
Colinearit 
y(overtim 
e) 
TTP versus time for 
different regimens stratified 
by Sex 
14.4.14 
14.4.36 
EBA 
TTP 
Multiple 
Colinearit 
y(baselin 
e) 
TTP at baseline versus 
AGE for different age 
groups 
14.4.13 
14.4.37 
EBA 
TTP 
Multiple 
Colinearit 
y(overtim 
e) 
TTP versus time stratified 
for different age groups for 
all regimens 
14.4.16 
14.4.38 
EBA 
TTP 
Multiple 
Colinearit 
y(baselin 
e) 
TTP at baseline versus 
BMI for different BMI 
categoriBMIes 
14.4.13 
14.4.39 
EBA 
TTP 
Multiple 
Colinearit 
y(overtim 
e) 
TTP overtime versus BMI 
for different BMI 
categories and regimen 
14.4.16 


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14.4.40 
EBA 
TTP 
Multiple 
Colinearit 
y(baselin 
e) 
TTP at baseline stratified 
for CAVITY 
14.4.13 
14.4.41 
EBA 
TTP 
Multiple 
Colinearit 
y(baselin 
e) 
TTP at baseline stratified 
for EXTENT 
14.4.13 
14.4.42 
EBA 
TTP 
Multiple 
Colinearit 
y(baselin 
e) 
TTP at baseline stratified 
for Sputum quality 
14.4.13 
14.4.43 
EBA 
TTP 
Multiple 
Colinearit 
y(overtim 
e) 
TTP over TIME, stratified 
for TRT and Sputum 
quality 
14.4.16 
14.4.44 
EBA 
TTP 
Multiple 
Colinearit 
y(baselin 
e) 
TTP at baseline vs 
MBGENMTBULTRAPos 
14.4.13 


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12 Appendix 
12.1 Table Shells 
 
Table 14.1.1: Disposition of Participants 
 
All participants 
RESP301tds 
RESP301qd 
RESP301bd 
 
 
Screened 
Completed Screening Period 
Withdrawn during Screening Period 
 
Reason for withdrawal during screening: 
Screening Failure 
Patient withdrew consent 
: : : 
 
Safety Population 
 
Completed Study 
Withdrawn 
 
Reason for Withdrawal 
Patient withdrew consent 
Loss to follow-up 
: : : 
 
 
 
 
Key: RESP301tds: Inhaled RESP301 6 ml via nebulisation (NEB) three times a day (tds); RESP301qd: Inhaled RESP301 6 ml via NEB once 
daily (qd); RESP301bd: Inhaled RESP301 6 ml via NEB twice daily (bd); RESP301tdsHRZE: Inhaled RESP301 6 ml via NEB tds plus 
HRZE po qd; HRZE po qd: Isoniazid 75 mg /Rifampicin 150 mg /Pyrazinamide 400 mg /Ethambutol 275 mg per os/orally (po) once 
daily (qd); 


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Table 14.1.2: Demographic Data 
Safety Population 
All 
RESP301tds 
RESP301qd n 
RESP301bd 
RESP301tdsHRZE 
participants 
n (%) 
n (%) 
(%) 
n (%) 
n (%) 
 
Gender 
Male 
Female 
Total 
Race 
Caucasian 
Black 
Coloured 
Other 
Total 


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Table 14.1.2: Demographic Data 
Safety Population 
All 
participants 
RESP301tds 
RESP301qd 
RESP301bd 
RESP301tdsHRZE 
Age (yr) 
n 
Mean 
SD 
Median 
Q1-Q3 
Min- Max 
Weight (kg) 
n 
Mean 
SD 
Median 
Q1-Q3 
Min- Max 
Height (m) 
n 
Mean 
SD 
Median 
Q1-Q3 
Min- Max 
BMI (kg/m^2) 
n 
Mean 
SD 
Median 
Q1-Q3 
Min- Max 


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Table 14.1.3: Medical History 
Safety Population 
 
All 
RESP301tds 
RESP301qd 
RESP301bd 
RESP301tdsHR 
participants 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
Subjects With Any History 
SOC1 
Preferred term 1 
Preferred term 2 
 
SOC2 
Preferred term 1 
Preferred term 2 
 
 
< repeat for all SOC and Preferred Terms > 


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Table 14.1.5.2: 12 Lead ECG at Baseline in Categories 
Safety Population 
 
All 
participants 
n (%) 
RESP301tds 
n (%) 
RESP301qd 
n (%) 
RESP301bd 
n (%) 
 
QT (msec) 
<= 450 msec 
> 450 msec and <= 480 msec 
> 480 msec and <= 500 msec 
> 500 msec 
Total 
 
< repeat for all QTcB, QTcF> 
QT (msec) 


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Table 14.1.5.3: 12 Lead ECG at Baseline – Overall Interpretation 
Safety Population 
All 
RESP301tds 
RESP301qd 
RESP301bd 
RESP301t 
participants 
n (%) 
n (%) 
n (%) 
n (%) 
n ( 
Normal 
Abnormal, not clinically significant 
Abnormal, clinically significant 
Total 


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Table 14.1.6.1: Physical Examination at Baseline 
Safety Population 
 
Body System 
All 
participants 
n (%) 
RESP301tds 
n (%) 
RESP301qd 
n (%) 
RESP301bd 
n (%) 
RESP301td
ZE 
n (%) 
Body System 1 
Not Done 
Normal 
Abnormal 
Body System 2 
Not Done 
Normal 
Abnormal 
< repeat for all body systems > 


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Table 14.1.7: Prior Medications 
Safety Population 
All 
RESP301tds 
RESP301qd 
RESP301bd 
RESP301td 
participants 
n (%) 
n (%) 
n (%) 
n (%) 
n (%
Subjects With Any Prior Medication 
Class 1 
Generic term 1 
Generic term 1 
 
Class 2 
Generic term 1 
Generic term 1 
 
 
< repeat for all classes and Generic Terms > 


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Table 14.3.1.1: Summary of Incidence of Treatment Emergent Adverse Events 
Safety Population 
All 
RESP301tds 
RESP301qd 
RESP301bd 
RESP30
participants 
n (%) 
n (%) 
n (%) 
n (%) 
n
Number of Subjects with TEAEs 
Number of Subjects with Serious TEAEs 
Number of Subjects with Fatal TEAEs 
Number of Subjects with Related TEAEs 
Number of Subjects with TEAEs Leading to 
Withdrawal 


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Table 14.3.1.2: Incidence of Treatment Emergent Adverse Events by System Organ Class and Preferred Term 
Safety Population 
 
All 
RESP301tds 
RESP301qd 
RESP301bd 
RESP301
System Organ Class/ 
Preferred Term 
participants 
n (%) 
n (%) 
n (%) 
n (%) 
n 
Subjects With Any Adverse Events 
SOC1 
Preferred term 1 
Preferred term 2 
 
SOC2 
Preferred term 3 
Preferred term 4 
 
< repeat for all SOC and Preferred Terms > 


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Table 14.3.1.7.1: Incidence of Treatment Emergent Adverse Events by System Organ Class, Preferred Term and Maximum Severity 
Safety Population 
 
 
Maximum Severity 
System Organ Class/ 
Preferred Term 
Grade 1/ 
Mild 
Grade 2/ 
Moderate 
Grade 3/ 
Severe 
Grade 4/Life- 
threatening 
n (%) 
n (%) 
n (%) 
n (%) 
RESP301tds (N=xx) 
Subjects With Any Adverse Event 
SOC1 
Preferred term 1 
Preferred term 2 
SOC2 
Preferred term 3 
Preferred term 4 
< repeat for all SOC and Preferred Terms > 
< repeat for other groups> 


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Table 14.3.1.7.4: Incidence of Treatment Emergent Adverse Events by System Organ Class, Preferred Term, Maximum Severity and 
Strongest Relationship to IMP 
Safety Population 
 
 
 
System Organ Class/ 
Grade 1/ 
Grade 2/ 
Grade 3/ 
Grade 4/Li 
Preferred Term 
Mild 
Moderate 
Severe 
 
 
 
RESP301tds (N=xx) 
 
Subjects With Any Adverse Event 
 
 
SOC1 
Preferred term 1 
Preferred term 2 
 
SOC2 
Preferred term 3 
Preferred term 4 
 
< repeat for all SOC and Preferred Terms > 
 
 
< repeat for other groups> 
Not Related 
Related 
Not Related 
Related 
Not Related 
Related 
Not Related 
n (%) 
 n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 


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Table 14.3.1.8: Summary of Frequency of Treatment Emergent Adverse Events 
Safety Population 
 
All 
RESP301tds 
RESP301qd 
RESP301bd 
RESP301tdsHRZ
participants 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
 
Number of TEAEs 
Number of Serious TEAEs 
 
Number of Grade 1 (Mild) TEAEs 
Number of Grade 2 (Moderate) TEAEs 
Number of Grade 3 (Severe) TEAEs 
Number of Grade 4 (Life-threatening) 
TEAEs 
 
Number of Unrelated TEAEs 
Number of Possibly related TEAEs 
Number of Related TEAEs 
 
< repeat for other groups > 


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Table 14.3.2.1.1: Clinical Safety Laboratory Tests – Haematology over time 
Safety Population 
 
All participants 
RESP301tds 
RESP301qd 
RESP301bd 
RESP 
 
Basophils (%) 
Baseline 
 
n 
Mean 
SD 
Median 
Q1-Q3 
Min- Max 
 
< repeat for all tests and days and parameters 
> 


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Table 14.3.2.1.2: Clinical Safety Laboratory Tests – Haematology Change from Baseline over time 
Safety Population 
 
 
 
Basophils (%) 
Day 1 
n 
Mean 
SD 
Median 
Q1-Q3 
Min- Max 
 
 
< repeat for all parameters > 
All participants 
RESP301tds 
n (%) 
RESP301qd 
n (%) 
RESP301bd 
n (%) 
RESP301tdsHR 
n (%) 


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Table 14.3.2.1.3: Clinical Safety Laboratory Tests – Haematology Assessment over time 
Safety Population 
 
All 
RESP301tds 
RESP301qd 
RESP301bd 
RESP301tdsHRZE
participants 
n (%) 
n (%) 
n (%) 
n (%) 
n (%) 
 
Basophils Abs (x10E9/L) 
Day 1 
High 
Normal 
Total 
 
 
< repeat for all parameters> 


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Table 14.3.7: Summary of Assessment of Study Stopping and Amendment Criteria. 
Safety Population 
 
All participants 
RESP301tds 
RESP301qd 
RESP301bd 
RESP
All Study Days 
AE Criteria 
Grade 3 or higher AEs 
Yes 
No 
 
Moderate (grade 2) or higher grade toxicity (CTCAE V5.0), that is 
probably or definitely related to IP 
Yes 
No 
 
Grade 3 or higher AEs (Gastrointestinal: diarrhoea, nausea, or 
dyspepsia; Respiratory: bronchospasm) that are at least possibly 
related to IP 
Yes 
No 


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Table 14.4.1: Positive and non-positive data in the dataset for CFU status at the different timepoints 
EBA 
All Participants 
HRZE 
RESP301tds 
RESP301qd 
 
Sample status 
Rep 1 
Rep 2 
Rep 1 
Rep 2 
Rep 1 
Rep 2 
Rep 1 
Rep 2 
R 
 
Day -1 
Contaminated 
No Growth/Negative 
No Result 
No Done 
Positive 
Repeat for each Study Day 


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Table 14.4.4: Summary statistics of baseline discrete covariates for the treatment arms 
EBA 
All 
RESP301tds 
RESP301qd 
RESP301bd 
participants 
N=XX, n (%) 
N=XX, n (%) 
N=XX, n (%) 
N=XX, n (%) 
Discrete covariate 
Outcome 1 
Outcome 2 
Outcome 3 
< repeat for all days and covariates> 


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Table 14.4.5: Summary statistics of continuous covariates for the treatment arms at baseline 
EBA 
 
All participants 
RESP301tds 
RESP301qd 
RESP301bd 
RESP 
 
Continuous covariate 
n 
Mean (SD) 
Median (IQR) 
Min-Max 
< repeat for all days and covariates> 


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Table 14.4.9: Median and interquartile range of observed EBA for 0-2, 0-7, 0-14 days, 0-15 and 14-15 days in CFU and TTP 
EBA 
Treatment arm 
All participants 
RESP301tds 
RESP301qd 
RESP301bd 
 
CFU log10 CFU/mL 
0-2 
Median (IQR) 
Repeat for study day differences and TTP (h) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 14.4.10: Parameter estimates for the final CFU model 
EBA 
Parameter 
Estimate 
RSE(%) 
Shrinkage 
 
 
 
 
 
 
 
 
 
 
 
 


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12.2 Graphical exploration of early bactericidal activity data 
Examples of graphical analysis plots are shown below. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 14.4.8: Log 10 CFU versus time stratified for different regimens. Solid black line is the 
median and red dashed lines from top to bottom are 5th and 95th percentiles of the observed 
data. 


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Figure 14.4.4: Individual log 10 CFU versus time for the HRZE treatment group. The lines 
connect the observations. The numbers represent the unique subject identification number in 
the analysis dataset. 


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Figure 14.4.26: Individual TTP versus time for the HRZE treatment group. The lines connect 
the observations. The numbers represent the unique subject identification number in the 
analysis dataset. 


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Figure 14.4.10: Correlation matrix for all continuous covariates. Scatterplot matrix with a 
loess smooth on the lower triangle. The diagonal illustrates the distribution for each covariate. 
Upper triangle displays the overall and per sex Pearson correlation coefficients, for all 
combinations of covariates. 


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Figure 14.4.13: Log 10 CFU at baseline stratified for sex. 


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Figure 14.4.14: Log 10 CFU versus time. Solid black and red lines are the median for males 
and females, respectively. 


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Figure 14.4.16: Log 10 CFU versus time stratified for different age groups. 


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Figure 6: Log 10 TTP at baseline versus BMI. Add info about what the different lines are etc. 


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13 References 
1. ICH Topic E9, Step 5 Note for guidance on statistical principles for clinical trials 
(CPMP/ICH/363/96). Final approval by CPMP: March 1998 / Date for coming into 
operation: September 1998 
2. Protocol for RESP301-EBA Phase 2 RESP301-EBA_Protocol_V1.0 Dated 31 March 
2023 Unapproved. 
 
3. Boeree, M. J., Diacon, A. H., Dawson, R., Narunsky, K., du Bois, J., Venter, A., 
Phillips, P. P., Gillespie, S. H., McHugh, T. D., Hoelscher, M., Heinrich, N., Rehal, S., 
van Soolingen, D., van Ingen, J., Magis-Escurra, C., Burger, D., Plemper van Balen, 
G., Aarnoutse, R. E., & PanACEA Consortium (2015). A dose-ranging trial to 
optimize the dose of rifampin in the treatment of tuberculosis. American journal of 
respiratory 
and 
critical 
care 
medicine, 
191(9), 
1058–1065. 
https://doi.org/10.1164/rccm.201407-1264OC