Pharmacokinetics of Drugs Used to Treat Drug-sensitive Tuberculosis in Breastfeeding Mother-infant Pairs: An Observational Pharmacokinetic Study
Conditions: Tuberculosis Active, Breastfeeding, Tuberculosis
Sponsor: University of Liverpool

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PHARMACOKINETICS OF DRUGS USED TO TREAT DRUG SENSITIVE-TUBERCULOSIS IN 
BREASTFEEDING MOTHER-INFANT PAIRS:  
AN OBSERVATIONAL PHARMACOKINETIC STUDY  
Area:  
Active-TB 
Type: 
Observational 
 
List of investigators and co-investigators: 
 
 
 
 
Principal Investigator/ Protocol Chair 
 
Catriona Waitt MRCP, PhD 
Professor of Clinical Pharmacology, University of 
Liverpool and Wellcome Clinical Research Career 
Development Fellow 
Infectious Diseases Institute,  
Makerere University College of Health Sciences 
(MakCHS), Kampala, Uganda 
Phone: +256778288217 
E-mail:  cwaitt@liverpool.ac.uk 
Co-investigators 
 
Barbara Castelnuovo MMed, PhD 
Head of Research 
Infectious Diseases Institute,  
Makerere University College of Health Sciences 
(MakCHS), Kampala, Uganda 
 
Ritah Nakijoba 
Study Co-ordinator and PhD Scholar 
Infectious Diseases Institute,  
Makerere University College of Health Sciences 
(MakCHS), Kampala, Uganda 
 
Allan Buzibye 
Laboratory Technologist and PhD Scholar 
Infectious Diseases Institute,  
Makerere University College of Health Sciences 
(MakCHS), Kampala, Uganda 
 
 
 
Phone: +256 786 623 613  
 
E-mail:  
bcastelnuovo@idi.co.ug 
 
 
 
 
Phone: +256789314632 
Email: rnakijoba@idi.co.ug 
 
 
 
 
Phone: +256777095518 
Email: abuzibye@idi.co.ug 

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Document History 
 
Document 
Version Date 
Summary of Changes 
Original protocol 
 27 OCT 2021 
N/A 
Pre-ethics version  
27 OCT 2021 
N/A 
Response to initial IDI REC, V 
2.0 
23 NOV 2021 
Clarifications as required by IRB. Added 
exclusion that breastfed infants should 
not be aged over 12 months. 
Clarification from Sponsor 
23 NOV 2021 
Clarification on safety reporting (DAIDS 
criteria to be used) 
Minor amendment, 3.0 
14 APR 2022 
Provision for participants to return for a 
second PK visit in the continuation 
phase of TB treatment if they are first 
sampled during the intensive phase of 
TB treatment 
Minor clarifications 
 
Sponsor:  
University of Liverpool / Liverpool Joint Research Office 
2nd Floor Block D Waterhouse Building 
3 Brownlow Street 
Liverpool L69 3GL 
sponsor@liv.ac.uk 
 
Authorised by: 
 
  
 
Name:     
Dr. Catriona Waitt 
 Role: 
Principal Investigator 
 
 
 
  
 
Signature: 
 
 Date: 
 
14 MAR 2022 

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PROTOCOL SUMMARY 
 
Hypothesis 
 
Active tuberculosis drug exposure to the breastfed infant may be 
clinically important. 
 
Aim 
 
To define transfer of Isoniazid, Rifampicin, Pyrazinamide and 
Ethambutol to the breastfed infant. 
To determine the area under the concentration-time curve (AUC), 
clearance and volume of distribution of these drugs. 
 
Rationale 
Existing data on anti-TB drug penetration into breastmilk is very sparse 
and information on clinically relevant infant exposure to anti-TB drugs 
is even more limited. This is an important knowledge gap both for 
safety, and because therapeutic concentrations could be 1) protective 
in exposed infants, obviating the need for TB preventive therapy or 2) 
sub- therapeutic concentrations could select for resistance in those 
infants infected with Mycobacterium tuberculosis. 
 
 
Study design 
Observational pharmacokinetic study 
 
Inclusions 
1. A personally signed and dated informed consent document 
indicating that the participant has been informed of all 
pertinent aspects of the study. 
2. Participants who are willing and able to comply with scheduled 
visits, treatment plan, laboratory tests, and other study 
procedures. 
3. Woman is aged 18 years or older 
4. Receiving treatment for active TB 
5. Pregnant or breastfeeding at enrolment 
 
Exclusions 
 
• Severe maternal or infant illness which in the opinion of the 
patient’s clinician would interfere with her participation in the 
study 
• Breastfed infant is aged over 12 months 
 
Primary 
Endpoints 
1) Concentrations of anti-TB drugs in maternal plasma and 
breastmilk at 0, 2, 4, 6, 8 and, in some cases, 24-hours post-dose  
2) Concentrations of anti-TB drugs in infant bloodat maternal pre-
dose, and up to 8 hours post maternal dose. 
3) Area under the concentration-time curve (AUC) of anti-TB drugs 
in maternal plasma and breastmilk  

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4) Breast milk to maternal plasma (M:P) ratio of TB drugs 
 
Secondary 
Endpoints 
1. Maximum concentration (Cmax) and time to maximum 
concentration (Tmax) of the TB drugs in maternal plasma and 
breastmilk  
2. Infant development (using Gross Motor Development Score) 
3. Depression and anxiety assessments for breastfeeding mothers 
 
Secondary 
Objectives 
1) To describe covariates influencing drug exposure in maternal 
plasma, breast milk and infant plasma 
2) To develop a population pharmacokinetic model including the 
breast and the infant as compartments, which will both enable 
optimal use of sparse data from future studies, and also enable 
simulations of different doses or combinations. 
3) To assess depression and anxiety levels among breastfeeding 
mothers on first line anti-TB drugs. 
4) Beliefs about medicines in breastfeeding mothers receiving TB 
treatment 
 
 
Number 
recruited 
20 mother-infant pairs 
 
Approximate 
timelines 
Study to commence as soon as approvals in place. 
Recruitment estimated to take two years. 
End of study on completion of analysis. 
 
 
 
 
 
 
 
 
 
 
 
 

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List of Abbreviations 
AE 
 
 
Adverse event 
ALT 
 
 
Alanine aminotransferase 
AUC 
 
 
Area under the concentration-time curve 
BMQ                              Beliefs and Medicines Questionnaire 
Cmax  
 
Maximum concentration 
CRF 
 
 
Case report form 
DBS 
 
 
Dried blood spot 
FDA 
 
 
United states food and drug administration 
GAD                                Generalised Anxiety Disorder 
HCT 
 
 
Haematocrit 
HIV 
 
 
Human immunodeficiency virus 
IDI                                   Infectious Diseases Institute 
M:P ratio 
 
Milk to Plasma ratio 
PK 
 
 
Pharmacokinetic 
PHQ                                Patient Health Questionnaire 
TB 
 
 
Tuberculosis 
Tmax  
 
Time to maximum concentration 
WHO  
 
World Health Organisation 
 
 
 
 
 
 
 
 
 
 
 

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Table of content 
Contents 
INTRODUCTION ....................................................................................................................................... 8 
1.1. Background .................................................................................................................................. 8 
1.2. Problem Statement ...................................................................................................................... 8 
1.3. Disease Setting/Patient Population ............................................................................................. 8 
2. STUDY OBJECTIVES AND ENDPOINTS .................................................................................................. 8 
2.1. Objectives..................................................................................................................................... 8 
3. STUDY DESIGN ..................................................................................................................................... 9 
4. PARTICIPANT SELECTION .................................................................................................................... 9 
4.1. Inclusion Criteria .......................................................................................................................... 9 
4.2. Exclusion Criteria ........................................................................................................................ 10 
5. TREATMENTS of Participants ............................................................................................................ 10 
5.1. Allocation to Treatment/Group ................................................................................................. 10 
5.2. Drug Supplies ............................................................................................................................. 10 
5.2.1. Drug Storage and Drug Accountability ................................................................................ 10 
5.3. Concomitant Medication(s) ....................................................................................................... 10 
6. STUDY PROCEDURES ......................................................................................................................... 11 
6.1. Informed Consent ...................................................................................................................... 11 
6.2. Screening .................................................................................................................................... 11 
6.3. Study Period ............................................................................................................................... 12 
6.3.1. Enrolment ........................................................................................................................... 12 
6.3.2. Pharmacokinetic Study Day ................................................................................................ 12 
6.4. End of TB treatment outcomes .................................................................................................. 12 
6.5. Participant Withdrawal .............................................................................................................. 13 
7. ASSESSMENTS ................................................................................................................................... 13 
7.1. Safety ......................................................................................................................................... 13 
7.2. Pharmacokinetics Assessments ................................................................................................. 13 
7.2.1. Blood for pharmacokinetic analysis .................................................................................... 13 
7.2.2. Sample Handling ................................................................................................................. 13 
7.2.3. Justification for PK/other sample shipment ....................................................................... 14 
8. DATA ANALYSIS/STATISTICAL METHODS .......................................................................................... 14 
8.1. Sample Size Determination ........................................................................................................ 14 
8.2. Analysis of Endpoints ................................................................................................................. 14 

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8.2.1. Analysis of Primary Endpoint .............................................................................................. 15 
8.3. Safety Analysis ........................................................................................................................... 15 
8.4. Interim Analysis .......................................................................................................................... 15 
8.5. Data Safety and Monitoring Committee .................................................................................... 15 
9. QUALITY CONTROL AND QUALITY ASSURANCE ................................................................................ 15 
10. DATA HANDLING/Record retention ................................................................................................ 16 
10.1. Case Report Forms (CRF) ......................................................................................................... 16 
10.2. Record Retention and Archiving .............................................................................................. 16 
10.3. Confidentiality .......................................................................................................................... 16 
11. ETHICS ............................................................................................................................................. 17 
11.1. Institutional Review Board (IRB) .............................................................................................. 17 
11.2. Ethical Conduct of the Study .................................................................................................... 17 
11.3. Participant Information and Consent ...................................................................................... 17 
12. DEFINITION OF END OF STUDY ....................................................................................................... 17 
13. PUBLICATION and Dissemination OF STUDY RESULTS .................................................................... 17 
14. Funding ........................................................................................................................................... 18 
 
 
 
 
 

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INTRODUCTION  
1.1. Background 
Worldwide, ~50% of women take medication during breastfeeding(1). Data surrounding the 
exposure of the breastfed infant to drugs and any associated risks are sparse (2). Despite 
longstanding recommendations from the US Food and Drug Administration (FDA) for lactation 
studies to be performed close to licensing for drugs anticipated to be widely used in women 
of childbearing age (3), such studies are rarely undertaken. Drugs taken by the breast feeding 
mother on TB treatment can be passed from the maternal circulation to her milk and then to 
the breastfed infant, a concern of effects of anti-tuberculosis drugs on nursing infants (4). 
Most TB drugs are metabolized by the liver, triggering  a potential risk of drug accumulation 
in infants due to their immature liver function particularly in premature infants (5), (6).  
Drugs are transferred to milk in small quantities, and many have been used without obvious 
infant toxicity for many years hence the large gaps in the data. Pharmacokinetic (PK) 
information of anti- TB drugs transfer to breast milk and breastfed infant is crucial to limit the 
development of drug resistance and understand the safety of prolonged exposure through 
breast milk. 
1.2. Problem Statement 
Whilst data on TB drug penetration into breastmilk is limited, information on clinically 
relevant infant exposure to TB drug-sensitive is even more limited and is an important 
knowledge gap both for safety, and because therapeutic concentrations could be 1) 
protective in exposed infants, obviating the need for TB preventive therapy or 2) sub- 
therapeutic concentrations could select for resistance in those infants infected with 
Mycobacterium tuberculosis. 
1.3. Disease Setting/Patient Population 
Breastfeeding women, including those living with HIV, treated for TB will be recruited 
prospectively from Infectious Diseases clinic (IDI) and IDI Kampala City Council Authority 
(KCCA) affiliated clinics. The clinics treat pregnant and breast-feeding women infected with 
TB. We anticipate that 80% of women will be living with HIV. Drug-drug interactions between 
antiretroviral therapy (ART) and TB drugs could affect the exposure of TB drugs. Data on 
concomitant medication will be recorded and included as a covariate in the pharmacokinetic 
analysis. In accordance with national protocol, the infants of mothers diagnosed with TB 
treatment will receive isoniazid preventive therapy for six months (the duration of the 
mothers’ TB treatment). 
2. STUDY OBJECTIVES AND ENDPOINTS 
2.1. Objectives 
To define transfer of isoniazid, Rifampicin, Pyrazinamide and Ethambutol to the breastfed 
infant. 
To determine the AUC, clearance and volume of distribution of these drugs. 

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Primary Endpoints 
1.  Concentrations of TB drugs in maternal plasma and breastmilk at 0, 2, 4, 6, 8 and, in 
some cases, 24-hours post-dose  
2. Concentrations of drugs in infant blood at maternal pre-dose, and up to 8 hours post 
maternal dose. 
3. Area under the concentration-time curve (AUC) of TB drugs in maternal plasma and 
breastmilk  
4. Breast milk to maternal plasma (M:P) ratio of TB drugs 
 
Secondary endpoints 
1. 
Maximum concentration (Cmax) and time to maximum concentration (Tmax) of the 
TB drugs in maternal plasma and breastmilk  
2. 
Infant development (using Gross Motor Development Score) 
3. 
Depression and anxiety assessments for breastfeeding mothers 
4. 
Beliefs about medicines in breastfeeding mothers receiving TB treatment 
3. STUDY DESIGN 
Pregnant or lactating women requiring treatment for drug sensitive-TB will be identified and 
invited for sampling. If they are pregnant when identified, they will be invited for sampling 
after delivery. Plasma and breastmilk samples will be obtained pre-dose and at 2, 4, 6, and 8 
hours post-dose. If logistics permit (for example living close to the research unit), she will be 
invited for a further sample at 24 hours post-dose. A heelprick sample will also be obtained 
from their breastfed infants at maternal trough (prior to maternal dose) and at a random 
timepoint (once per infant) over the 8-hour pharmacokinetic sampling visit in order to 
characterize concentrations of these drugs over an 8-hour dosing interval. We will 
determine concentrations of total plasma and breastmilk Isoniazid, Rifampicin, 
Pyrazinamide and Ethambutol.  
If a participant has her first pharmacokinetic profile in the intensive phase of TB treatment 
(whilst on all four of isoniazid, rifampicin, pyrazinamide and ethambutol), she will be invited 
for a subsequent sampling day with the same time points when she is on the continuation 
phase of therapy (rifampicin and isoniazid). 
4. PARTICIPANT SELECTION 
4.1. Inclusion Criteria 
Participants must meet all of the following inclusion criteria to be eligible for enrollment 
into the study: 

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1. A personally signed and dated informed consent document indicating that the 
participant has been informed of all pertinent aspects of the study. 
2. Participants who are willing and able to comply with scheduled visits, treatment 
plan, laboratory tests, and other study procedures. 
3. Woman is aged 18 years or older 
 
4.  Receiving treatment for drug sensitive TB 
 
5. Pregnant or breastfeeding at enrolment 
4.2. Exclusion Criteria  
Participants presenting with any of the following will not be included in the study: 
• Severe maternal or infant illness which in the opinion of the patient’s clinician would 
interfere with her participation in the study. 
• Breastfed infant is aged over 12 months 
5. TREATMENTS OF PARTICIPANTS 
This is an observational study whereby no changes will be made to the administered 
medication. 
5.1. Allocation to Treatment/Group 
Not applicable 
5.2. Drug Supplies 
All women (and infants) will continue to receive their TB treatment as prescribed by the clinic 
where they receive clinical care.  
5.2.1. Drug Storage and Drug Accountability 
Drug will be stored and dispensed from the hospital pharmacy, with no special procedures 
relating to this observational protocol.  
5.3. Concomitant Medication(s) 
At every study visit, women will be asked about concomitant medication and the results noted 
on the case report from (CRF). 

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6. STUDY PROCEDURES  
6.1. Informed Consent 
Women will be identified as they attend the clinic for TB treatment at the IDI and KCCA clinics 
Hospital in Uganda. Should a woman express willingness to participate, once her eligibility for 
enrolment in the study has been determined, informed consent will be obtained.  
 
Each potentially eligible participant must sign an Informed Consent Form prior to the conduct 
of any screening procedures.  Participants will be given the opportunity to ask any questions 
regarding the trial at this stage.  Screening evaluations will be used to determine the eligibility 
of each candidate for study enrolment.   
If the participant is unable to read and/or write, an impartial witness should be present during 
the informed consent discussion.  The witness should be able to read the consent form and 
participant information leaflet in the participant’s chosen language. After the written 
informed consent form is read and explained to the participant, and after they have orally 
consented to their participation in the study, and have either signed the consent form or 
provided their fingerprint, the witness should sign and personally date the consent form.  By 
signing the consent form, the witness attests that the information in the consent form and any 
other written information was accurately explained to, and apparently understood by, the 
patient and that informed consent was freely given by the patient. 
 
6.2. Screening 
At the screening visit, the participant will be evaluated against the eligibility criteria. Some 
women who intend to breastfeed will be identified in late pregnancy but will still be on TB 
treatment during breastfeeding. In this situation, consent will be sought, and details of how 
to contact her will be recorded with the aim to bring her for the pharmacokinetic sampling 
detailed in Section 6.3.2 at approximately 6 weeks postpartum. The exception to this will be 
where a mother will change from the four-drug intensive to the two-drug continuation phase 
of TB treatment prior to 6 weeks postpartum, in which case attempts to schedule the 
sampling visit whilst still on the intensive phase will be made. 
The study is being performed because there are no clear data that describe how much drug 
transfers from mother to breastfed infant. As this is an observational study, it has already 
been decided by the treating clinician that the mother requires the medication for her health 
and that benefits outweigh risks. Exclusive breastfeeding complies with WHO guidelines. 
There are no specific harms relating to the TB treatment about which we should specifically 
inform the mother prior to participation in the study, but rather the study aims to increase 
understanding. 

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6.3. Study Period 
6.3.1. Enrolment 
At enrolment, the mother will be assessed clinically.  
6.3.2. Pharmacokinetic Study Day 
On arrival, an intravenous cannula will be inserted into her antecubital fossa, and samples 
taken for trough drug measurement. After a standardized breakfast she will be administered 
her medication. Blood samples will be collected at 2, 4, 6, 8 and ideally* 24 hours. She will be 
advised to freely breastfeed her baby. She will be asked to provide a 2-5 ml sample of 
expressed breast milk pre-dose, and at 2, 4, 6 and 8 hours post dosing. A blood sample from 
the infant will be collected at maternal trough (pre-dose) and at a 3-8 hours post maternal 
dose (the second time point will be allocated sequentially to ensure spread of datapoints). 
The mother will be administered a standard lunch.  
*Due to the logistic considerations of sampling a postpartum mother and her infant who may 
have travelled a long distance to the clinic, the 24-hour sample may not be collected in all 
cases.   
Maternal albumin and creatinine will be sampled as they are important for isoniazid exposure. 
Maternal questionnaires will be filled on each visit to assess depression and anxiety; 
Generalised anxiety disorder questionnaire (GAD-7), Patient health questionnaire (PHQ-9), 
and the Beliefs about medicines questionnaire (BMQ). Infant clinical assessment will include 
use of the Gross Motor Development (GMD) checklist, see appendix. 
 
 
 
6.4. End of TB treatment outcomes  
End of TB treatment outcomes (cured, treatment completed, died, defaulted or transferred 
out) will be collected from the TB registry. 
Subject
Study Procedure
On arrival
0h
2h
4h
6h
8h
24h^
Mother
Confirm willingness to proceed
x
Clinical assessment
x
BMI 
x
Blood for creatinine and albumin
x
Blood PK sample 
x
x
x
x
x
x
BM PK sample
x
x
x
x
x
x
Standard meal
x
x
Observed dosing
x
PHQ9, GAD7 and BMQ questionnaires
X~
Infant
Clinical assessment
x
Weight
x
PK sample 
x
x*
*Time of second infant DBS to be recorded, between 3 and 8h
^Due to logistic constraints, the 24 hour may not be collected in every participant
~can be any time during PK day
Study Period (8h)^, time relative to maternal dose

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6.5. Participant Withdrawal 
Participants may withdraw from the study at any time at their own request, or they may be 
withdrawn at any time at the discretion of the investigator or sponsor for safety or 
behavioral reasons, or the inability of the participant to comply with the protocol required 
schedule of study visits or procedures. 
If a participant does not return for a scheduled visit (for example, has given consent but 
then does not return for the pharmacokinetic sampling day detailed in 6.3.2), every effort 
should be made to contact the participant. On enrolment participants will have been asked 
to provide contact numbers and directions to their home. Initial attempts will be made to 
speak to the participant by telephone, ascertaining the reasons for not attending clinic. In 
any circumstance, every effort should be made to document participant outcome, if 
possible.  The investigator should inquire about the reason for withdrawal and follow-up 
with the participant regarding any unresolved adverse events (AEs). 
If the participant withdraws from the study, and also withdraws consent for disclosure of 
future information, no further evaluations should be performed, and no additional data 
should be collected.   
7. ASSESSMENTS 
7.1. Safety 
Given that this is an observational study, we do not have concerns about the safety of the 
medication received. However, we will appropriately record and respond to adverse events 
related to the study procedures. These events might include adverse events relating to 
sampling of maternal blood, breastmilk or infant blood; these will be detailed on the case 
report form, and the participant followed until the event has resolved. Any events that do 
occur will be listed and analysed according to DAIDS criteria. Given that this is not a clinical 
trial, and the medication used is not an investigational product, there is no formal 
requirement for notification of adverse events to the regulatory authorities. 
The potential risk to healthcare workers is not considered higher than routine clinical work 
in this environment, where incidence and prevalence of HIV and TB are both high. 
Appropriate universal precautions will be taken. 
7.2. Pharmacokinetics Assessments  
7.2.1. Blood for pharmacokinetic analysis  
All efforts will be made to obtain the pharmacokinetic samples at the scheduled nominal time 
relative to dosing.  If a scheduled blood sample collection cannot be completed for any 
reason, the missed sample time may be re-scheduled with agreement of clinical investigators 
7.2.2. Sample Handling  
Maternal plasma and breast milk will initially be processed and stored in a -80Celsius 
freezer at IDI core laboratory.  

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Drug concentrations will be determined in plasma, infant DBS and total breastmilk using 
liquid chromatography-tandem mass spectrometry.  
7.2.3.  Justification for PK/other sample shipment  
This protocol aims to contribute to bioanalytical capacity in Uganda and all methods will be 
developed and performed at IDI. No shipment is therefore necessary. 
8. DATA ANALYSIS/STATISTICAL METHODS 
8.1. Sample Size Determination 
This study is exploratory, as no prior study has characterized the exposure of these drugs in 
maternal plasma, breastmilk and infant plasma. There are no prior data upon which to build 
a sample size calculation, and there is no comparison between groups which requires 
statistical analysis with a pre-specified certainty.  
Since no information is available about the penetration of these drugs into breastmilk, we 
used the following approach, described in detail for rifampicin. 
We modified a previously published pop-PK model of rifampicin in plasma, adding a 
compartment to describe breastmilk concentrations. This was characterised using an 
approach similar to an effect compartment (7) described by a time delay and an accumulation 
ratio between breastmilk and plasma. The half-life of the delay was fixed to 1 h and the 
accumulation ratio to 1.5, with 30% between-subject variability in both parameters. These 
were chosen to mimic a PK profile similar to Waitt et al (8). We assumed 15 individuals 
(considering a mother-infant dyad as a single unit) with an intensive PK sampling at 0, 1, 2, 4, 
6 and 8 hours post-dose of paired plasma and breastmilk (30% error in the breastmilk 
measurements was assumed) and performed Stochastic Simulations and Estimations (SSEs) 
(9) to evaluate trial design. Our design can characterise all the typical values of the plasma PK 
parameters with precision of better than 11% RSE, and all the breastmilk parameters are well 
characterised with a precision of 1.14% and 0.591% RSE on delay and accumulation ratio, 
respectively. 
Interim analysis after five participants is part of study design 
8.2. Analysis of Endpoints 
Pharmacokinetic data will be analysed using a population pharmacokinetic approach to 
estimate pharmacokinetic parameters and produce modelled fits to exposure data (10, 11) 
Inter-individual variability will be quantified in relation to the covariates. 
Non-compartmental methods will be used to assess correlations between maternal breast 
milk drug concentrations and measures of drug exposure in the infant (eg AUC) and 
pharmacodynamic factors. 
 

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8.2.1. Analysis of Primary Endpoint 
8.2.1.1. Non-Compartmental Analysis 
Plasma pharmacokinetic parameters including the maximum plasma concentration (Cmax), 
time to maximum plasma concentration (Tmax), and area under the plasma concentration 
versus time curve (AUClast, AUCτ)  for (drugs) will be estimated using non-compartmental 
analysis.  If data permit or if considered appropriate, area under the plasma concentration 
versus time curve to infinity (AUCinf), terminal elimination half-life (t1/2), plasma clearance 
(CL or CL/F), apparent volume of distribution (Vd or Vd/F) will be also estimated, as data 
allow. The single dose and/or steady-state PK parameters will be summarized descriptively 
by dose, cycle and day. 
8.2.1.2. Population Pharmacokinetic (pop-PK) analysis 
The population pharmacokinetic models will be applied to evaluate whether the drug 
concentrations are consistent with the expected exposures reported previously. If 
discrepancies between the observed exposure and the model-expected levels are observed, 
the models will be adjusted by re-estimating the values of the pharmacokinetic parameter 
values, with the use of Bayesian priors.  
Drug concentrations in breastmilk will be incorporated in the maternal population 
pharmacokinetic models using an effect compartment strategy, which will allow accurate 
estimation of the accumulation in breastmilk compared to plasma. Exposure to isoniazid and 
rifampicin in breastfed infants will be compared with the range of concentrations achieved 
in adults given therapeutic doses, assuming a minimal duration of exposure from delivery 
and a maximal duration from the time the mother started the drug (to account for placental 
transfer) 
8.3. Safety Analysis 
Safety events relating to the study procedures will be graded and analyzed. 
8.4. Interim Analysis  
Not applicable 
8.5. Data Safety and Monitoring Committee  
Not applicable 
9. QUALITY CONTROL AND QUALITY ASSURANCE 
During study conduct periodic monitoring may be conducted to ensure that the protocol 
and Good Clinical Practices (GCPs) are being followed.  The monitors may review source 
documents to confirm that the data recorded on CRFs is accurate.  Additionally, the study 
site may be participant to review by the Institutional Review Board (IRB) and/or to 
inspection by appropriate regulatory authorities. 

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10. DATA HANDLING/RECORD RETENTION 
10.1. Case Report Forms (CRF) 
A CRF is required and should be completed for each included participant.  
The investigator has ultimate responsibility for the collection and reporting of all clinical, 
safety and laboratory data entered on the CRFs and any other data collection forms (source 
documents) and ensuring that they are accurate, authentic / original, attributable, 
complete, consistent, legible, timely (contemporaneous), enduring and available when 
required.  The CRFs must be signed by the investigator or by an authorized staff member to 
attest that the data contained on the CRFs is true.  Any corrections to entries made in the 
CRFs, source documents must be dated, initialed and explained (if necessary) and should not 
obscure the original entry.  
10.2. Record Retention and Archiving 
To enable evaluations and/or audits, the investigator agrees to keep records, including the 
identity of all participating patients (sufficient information to link records, eg, CRFs and clinic 
records), all original signed informed consent documents, copies of all CRFs, safety reporting 
forms, source documents, and detailed records of treatment disposition, and adequate 
documentation of relevant correspondence (eg, letters, meeting minutes, telephone calls 
reports). 
Investigator records must be kept for as long as required by applicable local regulations (10 
years in Uganda). When more than one requirement can be applied, records must be 
maintained for the longest period provided. 
10.3. Confidentiality 
Clinical data will be entered into a study specific database by designated staff on a regular 
basis from completed Case Record Forms (CRF). Case Record Forms and other source 
documents will be kept in locked cabinets. Data will be entered on a regular basis to ensure 
that it is up to date. The database will be entered on regular basis on a secure PC, as will the 
pharmacokinetic data that will be received by the laboratories. Access to database will be 
given to authorized personnel only (members of the immediate study team) and a log of 
authorized personnel will be stored in the trial master file. CRF and trial documents will be 
kept in locked cabinets. No participant identifying information will be disclosed in any 
publication or at any conference activities arising from the study. 
10.4 Insurance 
This observational study will be covered under the University of Liverpool’s existing policy 
with Newline Insurance. 

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11. ETHICS 
11.1. Institutional Review Board (IRB) 
It is the responsibility of the investigator to have prospective approval of the study protocol, 
protocol amendments, informed consent documents, and other relevant documents from the 
IRB.  All correspondence with the IRB should be retained in the regulatory or trial master file. 
Copies of IRB approvals should be filed with other study documents. 
11.2. Ethical Conduct of the Study 
The study will be conducted in accordance with legal and regulatory requirements, as well as 
the general principles set forth in the International Ethical Guidelines for Biomedical Research 
Involving Human Participants and the Declaration of Helsinki. 
In addition, the study will be conducted in accordance with the protocol, GCP guidelines, and 
applicable local regulatory requirements and laws. 
11.3. Participant Information and Consent 
All parties will ensure protection of participant personal data and will not include participant 
names on any forms, reports, publications, or in any other disclosures, except where required 
by laws.  
The informed consent document used in this study, and any changes made during the course 
of the study, must be prospectively approved by the IRB. 
The investigator, or a person designated by the investigator, will obtain written informed 
consent from each participant or the participant's legal representative before any study-
specific activity is performed.  The investigator will retain the original of each participant's 
signed consent document. 
12. DEFINITION OF END OF STUDY 
The study will be considered complete when the target number of participants has been 
enrolled, and have completed the study period and the data analysis is complete. 
13. PUBLICATION AND DISSEMINATION OF STUDY RESULTS 
Study findings will be disseminated to researchers at scientific conferences and peer-
reviewed infectious disease and pharmacology journals. Specifically, for Uganda, study results 
will be communicated to the National Tuberculosis and Leprosy programme in the National 
Department of Health through meetings and reports. The Community Engagement Plan will 
detail the engagement with communities at all stages of the protocol from inception through 
dissemination, and activities under the related Wellcome Public Engagement Enrichment 
award Attaining Equity of Access to Research (At The EQUATOR) will ensure meaningful two-
way dialogue throughout. 

MILK Drug-Sensitive TB, Version 3.0, 14 APR 2022 
 
18 
 
Specifically, through At The EQUATOR, the following activities are proposed with regard to 
the MILK protocols: 
During Study Set-Up 
Early consultation with CAB to determine which community members to approach for 
dialogue 
Initial community meeting with religious leaders, women’ s local representative and 
community members, involving oral discussion and written flyers with pictures and simplified, 
translated text.  
Identification of key community members who are interested in co-creation of materials and 
resources.  
Establishment of social media channels relating to study activities. 
Engagement with journalists/ media – identify interested parties.  
At Site Initiation 
Community meeting at site for Q&A and more specific information about recruitment 
Radio broadcast or presentation of drama and song relating to activities. 
Tailor-made flyers and posters including photos with key, simplified text  
During Study  
Community meetings every 1-2 months for updates on study progress 
Social media updates 
Media updates 
At Dissemination 
Dissemination meeting/ event  
Listen to priorities about ongoing research, start to consider future work 
 
14. FUNDING  
This study is funded by a Wellcome Trust Clinical Research Career Development Fellowship 
awarded to Catriona Waitt. 
 
 
 
 

MILK Drug-Sensitive TB, Version 3.0, 14 APR 2022 
 
19 
 
References 
1. 
Saha MR, Ryan K, Amir LH. Postpartum women's use of medicines and breastfeeding 
practices: a systematic review. Int Breastfeed J. 2015;10:28. 
2. 
Byrne JJ, Spong CY. "Is It Safe?" - The Many Unanswered Questions about 
Medications and Breast-Feeding. N Engl J Med. 2019;380(14):1296-7. 
3. 
FDA. Clinical Lactation Studies: Considerations for Study Design. Guidance for 
Industry. US Department for Health and Human Services; Food and Drug Administration; 
Center for Drug Evaluation and Research (CDER); Center for Biologics Evaluation and 
Research (CBER); 2019.  Contract No.: 24840658dft.docx. 
4. 
Ito S, Lee AJAddr. Drug excretion into breast milk—overview. 2003;55(5):617-27. 
5. 
Berlin CM, Briggs GG, editors. Drugs and chemicals in human milk. Seminars in fetal 
and neonatal medicine; 2005: Elsevier. 
6. 
Tran JH, Montakantikul PJJoHL. The safety of antituberculosis medications during 
breastfeeding. 1998;14(4):337-40. 
7. 
Denti P, Martinson N, Cohn S, Mashabela F, Hoffmann J, Msandiwa R, et al. 
Population Pharmacokinetics of Rifampin in Pregnant Women with Tuberculosis and HIV 
Coinfection in Soweto, South Africa. Antimicrob Agents Chemother. 2015;60(3):1234-41. 
8. 
Waitt C, Olagunju A, Nakalema S, Kyohaire I, Owen A, Lamorde M, et al. Plasma and 
breast milk pharmacokinetics of emtricitabine, tenofovir and lamivudine using dried blood and 
breast milk spots in nursing African mother-infant pairs. J Antimicrob Chemother. 
2018;73(4):1013-9. 
9. 
Keizer RJ, Karlsson MO, Hooker A. Modeling and Simulation Workbench for 
NONMEM: Tutorial on Pirana, PsN, and Xpose. CPT Pharmacometrics Syst Pharmacol. 
2013;2:e50. 
10. 
Ette EI, Williams PJJAoP. Population pharmacokinetics I: background, concepts, and 
models. 2004;38(10):1702-6. 
11. 
Vozeh S, Steimer J-L, Rowland M, Morselli P, Mentré F, Balant LP, et al. The use of 
population pharmacokinetics in drug development. 1996;30(2):81-93. 
 

MILK Drug-Sensitive TB, Version 3.0, 14 APR 2022 
 
 
Study ID of child:_____________ 
Page 1 
15. APPENDIX 1: INFANT GROSS MOTOR DEVELOPMENT SCORE 
 
 
 
Examination of the baby/child at the time of PK visit    
 
 
Ist examination 
 
 
Date: 
 
Name of examining doctor or nurse 
 
1 
Age of child (in months) 
 
2 
Weight (kgs) 
 
3 
Length of child (cm)? 
 
4 
Head circumference (cm)? 
 
5 
Middle Upper Arm Circumference (cm)? 
 
7 
Is the child well at the moment? (Yes/ No)* 
 
8 
Is the baby/child picking up weight as he/she should?  
(Normal trajectory/ staying the same weight/losing weight)* 
 
9 
Any congenital abnormalities identified postnatally? (Yes/No)* 
 
16 
Is the baby being breast-fed or bottle-fed, or both? 
 
17 
If the baby is breast-fed, is milk from a donor used or partly used? 
 
* If the answer for any of these questions is the red option provide details on the last page of this assessment form. 
Development of the baby/child:  
 
Examination date: 
 
Age of child (in months) 
 
Turn his head to hear your voice? 
 
Recognise his caregiver? (Smile) 
 
Sits with support 
 
Laughs and squeals 
 
Explores objects 
 
Sits unsupported 
 
Rolls both ways 
 
Babbles 
 
Stranger anxiety 
 
Gets from sitting into crawling or kneeling 
 
Says ‘mama’, ‘dada’ 
 
Gestures ‘bye bye’ 
 
Walks 
 
Responds to own name 
 
Points at wanted objects 
 

MILK Drug-Sensitive TB, Version 3.0, 14 APR 2022 
 
 
Page 2 
Details for questions on page 1 of this assessment form or any other details on the child 
Question no 
Details 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
List of drugs the baby has been on since birth 
Name of drug 
Dosage 
Date started 
Drug stopped 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

MILK Drug-Sensitive TB, Version 3.0, 14 APR 2022 
 
 
16. APPENDIX 2: PHQ-9 AND GAD-7 TOOL 
Patient Health Questionnaire and General Anxiety Disorder  
(PHQ-9 and GAD-7)  
  
Date_______________ Patient ID: ________________________________ Date of Birth: 
______________  
  
Over the last 2 weeks, how often have you been bothered by any of the following 
problems?  Please circle your answers.  
PHQ-9  
Not at 
all  
Several 
days  
More than half 
the days  
Nearly 
every day  
1. Little interest or pleasure in doing things.  
0  
1  
2  
3  
2. Feeling down, depressed, or hopeless.  
0  
1  
2  
3  
3. Trouble falling or staying asleep, or sleeping too much.  
0  
1  
2  
3  
4. Feeling tired or having little energy.  
0  
1  
2  
3  
5. Poor appetite or overeating.  
0  
1  
2  
3  
6. Feeling bad about yourself – or that you are a failure or have let 
yourself or your family down.  
0  
1  
2  
3  
7. Trouble concentrating on things, such as reading the newspaper 
or watching television.  
0  
1  
2  
3  
8. Moving or speaking so slowly that other people could have 
noticed. Or the opposite  
– being so fidgety or restless that you have been moving around a 
lot more than usual.  
0  
1  
2  
3  
9. Thoughts that you would be better off dead, or of hurting yourself 
in some way.  
0  
1  
2  
3  
Add the score for each column  
  
  
  
  
  
Total Score (add your column scores): ______________  
  
Depression severity:0-4 None, 5-9 Mild, 10-14 Moderate, 15-19 Moderately severe, 20-27 Severe 
If you checked off any problems, how difficult have these made it for you to do your work, take care of 
things at home, or get along with other people? (Circle one)  
  
 
Not difficult at all  
  
Somewhat difficult  
  
Very Difficult  
Extremely 
Difficult  
  
---------------------------------------------------------------------------------------------------------------------------------------
---------------------------  
  
Over the last 2 weeks, how often have you been bothered by any of the following 
problems?  Please circle your answers.  
GAD-7  
Not at all 
sure  
Several 
days  
Over half 
the days  
Nearly 
every day  
1. Feeling nervous, anxious, or on edge.  
0  
1  
2  
3  
2. Not being able to stop or control worrying.  
0  
1  
2  
3  
3. Worrying too much about different things.  
0  
1  
2  
3  
4. Trouble relaxing.  
0  
1  
2  
3  
5. Being so restless that it’s hard to sit still.  
0  
1  
2  
3  

MILK Drug-Sensitive TB, Version 3.0, 14 APR 2022 
 
 
6. Becoming easily annoyed or irritable.  
0  
1  
2  
3  
7. Feeling afraid as if something awful might happen.  
0  
1  
2  
3  
Add the score for each column  
  
  
  
  
  
Total Score (add your column scores): ______________  
  
If you checked off any problems, how difficult have these made it for you to do your work, take care of 
things at home, or get along with other people? (Circle one)  
  
 
Not difficult at all  
  
Somewhat difficult  
  
Very Difficult  
Extremely 
Difficult   
  
  
  
UHS Rev 4/2020  
  Developed by Drs. Robert L. Spitzer, Janet B.W. Williams, Kurt Kroenke and colleagues, with an educational grant from Pfizer 
Inc.   
  
No permission required to reproduce, translate, display or distribute, 1999.  
 
 

MILK Drug-Sensitive TB, Version 3.0, 14 APR 2022 
 
 
17. APPENDIX 3: BELIEFS ABOUT MEDICINES QUESTIONAIRE  
 
Questions relating to medicine use in general 
 
This section explores your views and concerns (if any) about taking medicines in general. 
 
Please answer every question by ticking the box that best describes your views to each statement. 
 
Please remember, there are no “right” answers to the questions, we are simply interested in your 
views. 
 
 
 
 
Strongly 
agree 
Agree 
Uncertain 
Disagree 
Strongly 
disagree 
G1 
Doctors use too many medicines 
 
 
 
 
 
 
G2 
Patients who take medicines should stop their 
treatment for a while every now and again 
 
 
 
 
 
G3 
Most medicines are addictive 
 
 
 
 
 
 
G4 
Natural remedies are safer than medicines  
 
 
 
 
 
 
G5 
Medicines do more harm than good 
 
 
 
 
 
 
G6 
All medicines are poisons  
 
 
 
 
 
 
G7 
Doctors place too much trust on medicines  
 
 
 
 
 
 
G8 
If doctors had more time with patients they would 
prescribe fewer medicines  
 
 
 
 
 
 
 
 
 
 
 

MILK Drug-Sensitive TB, Version 3.0, 14 APR 2022 
 
 
Specific questions relating to TB TREATMENT  
 
The following questions explore your views and concerns (if any) around taking TB treatment 
specifically. 
 
Please answer every question by ticking the box that best describes your views to each statement. 
 
Please remember, there are no “right” answers to the questions, we are simply interested in your 
views. 
 
 
 
Strongly 
agree 
Agree 
Uncertain 
Disagree 
Strongly 
disagree 
S1 
My health at present depends on TB treatment 
 
 
 
 
 
 
S2 
Having to take TB treatment worries me 
 
 
 
 
 
 
S3 
My life would be impossible without TB treatment 
 
 
 
 
 
 
S4 
Without TB treatment I would be very ill 
 
 
 
 
 
 
S5 
I sometimes worry about the long term effects of 
TB treatment 
 
 
 
 
 
 
S6 
The TB treatment is a mystery to me 
 
 
 
 
 
 
S7 
My health in the future depends on TB treatment 
 
 
 
 
 
 
S8 
The TB treatment disrupts my life 
 
 
 
 
 
 
S9 
I sometimes worry about becoming too dependent 
on TB treatment 
 
 
 
 
 
 
S10 
TB treatment protects me from becoming worse