Addition of Probenecid to Penicillin-V Therapy: an Open Label, Cross-over Study in Healthy Volunteers
Phase: PHASE1
Conditions: Infection, Bacterial
Sponsor: Imperial College London

 
ADDPROPEN STUDY 
 
 
 
 
Addition of probenecid to penicillin-V therapy: an open label, cross-over 
study in healthy volunteers 
 
 
 
 
<Version 2.2 23/07/2021> 
 
 
 
 
MAIN SPONSOR: Imperial College London 
 
FUNDERS: British Infection Association (BIA) 
 
STUDY COORDINATION CENTRE: Imperial College London 
 
NRES reference:  
21/LO/0558 
 
IRAS Project ID:  
299581 
 
EudraCT reference:  
2021-002800-11 
 
 
 
 
Protocol authorised by: 
 
Name & Role 
Date 
Signature 
 
 
 
Timothy M Rawson 
       23/07/2021 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Study Management Group 
 
Chief Investigator:  
Timothy Miles Rawson 
 
Co-investigators:  
Richard Wilson 
 
 
 
Alaa Riezk 
 
 
 
Paul Arkell 
Mark Gilchrist  
Alison Holmes 
 
Study Coordination Centre  
 
For general queries, supply of study documentation, and collection of data, please contact: 
 
Study Coordinator:  
Timothy Miles Rawson 
Address:  
 
Imperial College London 
 
 
 
 
Tel:  
 
 
07891750151  
 
 
 
 
 
E-mail:   
 
tmr07@ic.ac.uk 
Web address:  
http://www.imperial.ac.uk/people/timothy.rawson07 
 
 
Clinical Queries 
 
Clinical queries should be directed to Timothy M Rawson who will direct the query to the 
appropriate person. 
 
 
Sponsor 
 
Imperial College London is the main research Sponsor for this study.  For further information 
regarding the sponsorship conditions, please contact the Head of Regulatory Compliance at: 
 
 
 
Research Governance and Integrity Team 
Imperial College London and Imperial College Healthcare NHS Trust 
Room 215, Level 2, Medical School Building 
Norfolk Place 
London, W2 1PG 
Tel: 0207 594 9459 
 
Funder 
 
The British Infection Association (BIA) is funding this research.  
 
This protocol describes the named study and provides information about procedures for entering 
participants. The protocol should not be used as a guide for the treatment of other participants; every 
care was taken in its drafting, but corrections or amendments may be necessary. These will be circulated 
to investigators in the study, but centres entering participants for the first time are advised to contact the 
trials centre to confirm they have the most recent version.  
 
Problems relating to this trial should be referred, in the first instance, to the study coordination centre.  
This trial will adhere to the principles outlined in the Medicines for Human Use (Clinical Trials) 
Regulations 2004 (SI 2004/1031), amended regulations (SI 2006/1928) and the International 
Conference on Harmonisation Good Clinical Practice (ICH GCP) guidelines. It will be conducted in 
compliance with the protocol, the Data Protection Act and other regulatory requirements as appropriate.  

Table of Contents 
1. 
INTRODUCTION 
7 
1.1 
BACKGROUND 
7 
2.  STUDY OBJECTIVES 
7 
3.  STUDY DESIGN 
8 
3.1 
STUDY OUTCOME MEASURES 
10 
4. 
PARTICIPANT ENTRY 
10 
4.1 
PRE-RANDOMISATION EVALUATIONS 
10 
4.2 
INCLUSION CRITERIA 
10 
4.3 
EXCLUSION CRITERIA 
10 
4.4 
WITHDRAWAL CRITERIA 
11 
5. 
RANDOMISATION AND ENROLMENT PROCEDURE 
11 
5.1 
RANDOMISATION OR REGISTRATION PRACTICALITIES 
11 
5.2 
CONTRACEPTION REQUIREMENTS FOR FEMALE PARTICIPANTS 
11 
6. 
TREATMENTS 
12 
6.1 
TREATMENT ARMS 
12 
6.2 
DOSE MODIFICATIONS FOR TOXICITY 
13 
6.3 
PREMEDICATION 
13 
6.4 
INTERACTION WITH OTHER DRUGS 
13 
6.5 
DISPENSING AND ACCOUNTABILITY 
13 
6.6 
SAFETY MONITORING ASSESSMENTS 
13 
7. 
PHARMACOVIGILANCE 
14 
7.1 
DEFINITIONS 
14 
7.2 
CAUSALITY 
15 
7.3 
REPORTING PROCEDURES 
15 
8. 
ASSESSMENT AND FOLLOW-UP 
17 
8.1 
INCIDENTAL FINDINGS 
17 
8.2 
LOSS TO FOLLOW-UP 
17 
8.3 
TRIAL CLOSURE 
17 
9. 
STATISTICS AND DATA ANALYSIS 
17 
10. 
MONITORING 
18 
10.1 
RISK ASSESSMENT 
18 
10.2 
MONITORING AT STUDY CENTRE 
18 
11. 
REGULATORY ISSUES 
18 
11.1  
CLINICAL TRIALS APPROVAL 
18 
11.2 
ETHICS APPROVAL 
18 
11.3 
CONSENT 
18 
11.4 
CONFIDENTIALITY 
18 

11.5 
INDEMNITY 
18 
11.6 
SPONSOR 
19 
11.7 
FUNDING 
19 
11.8 
AUDITS AND INSPECTIONS 
19 
12. 
TRIAL MANAGEMENT 
19 
13. 
PUBLICATION POLICY 
19 
11. 
REFERENCES 
19 
APPENDIX 1. SUMMARY OF STUDY 
21 
APPENDIX 2. SUMMARY OF INVESTIGATIONS, TREATMENTS AND ASSESSMENTS
 
22 
 
 

GLOSSARY OF ABBREVIATIONS 
FBC 
Full Blood Count 
Hb 
Haemoglobin 
HBV 
Hepatitis B Virus 
HCV 
Hepatitis C Virus 
HIV 
Human Immunodeficiency Virus 
LFT 
Liver Function Tests 
PD 
Pharmacodynamics 
PK 
Pharmacokinetics 
QDS 
Four times a day / 6-hourly 
SmPC 
Summary of medicinal product characteristics 
ULN 
Upper Limit of Normal 
%fT>MIC 
Percentage time the free drug concentration spends above the minimum 
inhibitory concentration 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
KEYWORDS 
Beta-lactams, probenecid, pharmacokinetic-pharmacodynamic targets, healthy volunteer 
study, antimicrobial optimisation. 

STUDY SUMMARY 
 
TITLE Addition of probenecid to penicillin-V therapy: a randomised, open label, 
cross-over study in healthy volunteers 
DESIGN Randomised, open-label, cross-over study. 
HYPOTHESIS Addition of probenecid to oral phenoxymethylpenicillin (penicillin-V) has a 
clinically relevant influence of pharmacokinetic-pharmacodynamic (PK-
PD) target attainment.  
AIMS To describe the influence of concomitant probenecid administration on 
penicillin-V PK in healthy-volunteers.  
 
Estimate the influence probenecid driven PK variation on penicillin-V PK-
PD 
targets 
(i.e. free 
time 
over 
the 
minimum 
inhibitory 
concentration, fT>MIC).  
 
Develop a population PK-PD model that can support the design of future 
prospective studies exploring optimal methods for penicillin-V dose 
optimisation.  
OUTCOME 
MEASURES 
Free, unbound serum drug concentration at 45 & 180 minutes post 
antibiotic dose. 
POPULATION 50 healthy adult volunteers recruited to attend Imperial College Clinical 
Research Facility, Hammersmith Hospital, UK. 
ELIGIBILITY Healthy volunteers over 18 years old. 
TREATMENT Addition of probenecid to penicillin-V. 
DURATION 1 year (12 months) 
 
 
REFERENCE DIAGRAM 
 
See appendix 1. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
1. 
INTRODUCTION  
 
1.1 
BACKGROUND 
Probenecid, p-(di-n-propylsulfamyl)-benzoic acid, was developed in 1949 with the purpose of 
decreasing the renal clearance of penicillin.1 The development of probenecid stemmed from 
the requirement to ration supplies of penicillin during World War II. Its mechanism of action 
is thought to be to the competitive inhibition of organic anion transporters, which are 
responsible for the excretion of organic agents, such as penicillin.2 Reduction in renal 
clearance of penicillin with probenecid demonstrated significant increases in serum 
concentrations, meaning that lower doses of drug were required for similar pharmacokinetic-
pharmacodynamic (PK-PD) target attainment. Probenecid’s influence on penicillin clearance 
became mainly academic in the post-war era as our capability to produce more diverse, 
cheaper, and safer beta-lactam antibiotics rapidly expanded.1 Probenecid remains a 
recommended adjunct in the management of some sexually transmitted diseases to support 
therapeutic target attainment in compartments, such as cerebrospinal fluid.3 However, its 
potential important broader role in preserving the effectiveness of beta-lactams through the 
optimisation of beta-lactam PK needs to be considered.   
 
The 
World 
Health 
Organisation 
(WHO) AWaRe criteria 
require 
narrow 
spectrum 
antimicrobials, such as the penicillins, to be available in appropriate type, dose, and duration 
to treat common infections.4 With increasing drug-resistance within common causative 
organisms, such as in streptococcal infections, new methods to optimise the delivery 
of Access agents and protect the use of broader Watch and Reserve antimicrobials is 
required.4 It is not always possible to simply prescribe higher doses of an antibiotic to 
overcome problems such as increasing drug-resistance. In some instances, oral drug 
absorption or gastrointestinal side effects associated with high doses limit escalation of 
therapy. Some agents are not licenced for use at doses required to obtain acceptable PK-PD 
targets for high minimum-inhibitory concentration (MIC) organisms. Some settings, such as 
outpatient parenteral antibiotic therapy (OPAT) do not allow for multiple daily dosing schedules 
of an intravenous drug, meaning that an oral or once daily alternative intravenous option is 
required. Probenecid offers a means of optimising the delivery of oral and intravenous 
antimicrobial therapy through manipulation of beta-lactam PK without significant changes to 
antimicrobial dosing.   
 
1.2 
RATIONALE FOR CURRENT STUDY 
This study aims to build on previous work characterising the PK of penicillin-V to explore the 
potential impact of probenecid on PK-PD target attainment. Achievement of the aims of this 
study would provide data to support the design of experimental studies exploring the clinical 
impact of probenecid on treatment outcomes and also provide a rationale for exploration of 
probenecid’s effects on a larger number of beta-lactam antibiotics.   
 
Hypothesis: Addition of probenecid to oral phenoxymethylpenicillin (penicillin-V) has a 
clinically relevant effect on pharmacokinetic-pharmacodynamic (PK-PD) target attainment.   
  
 
2.  STUDY OBJECTIVES 
1. To describe the influence of concomitant probenecid administration on penicillin-V PK in 
healthy-volunteers.  
 
2. Estimate the influence probenecid driven PK variation on penicillin-V PK-PD targets 
(i.e. free time over the minimum inhibitory concentration, fT>MIC).  
 

3. Develop a population PK-PD model that can support the design of future prospective 
studies exploring optimal methods for penicillin-V dose optimisation.     
 
 
3.  STUDY DESIGN 
 
This study will link rich penicillin-V pharmacokinetic data from a prior healthy 
volunteer study5 to sparse PK data collected prospectively as part of a randomised, open-
label, cross-over study outlined below. This will support the development of a population PK 
model that describes probenecid’s influence on renal clearance of penicillin-V as a covariate.    
  
Population:    50 healthy adult volunteers recruited to attend Imperial College Clinical  
Research Facility, Hammersmith Hospital, UK.  
Design:   
Randomised, open-label, cross-over study.  
Intervention:  Oral penicillin-V plus oral probenecid.  
Comparator: Oral penicillin-V.  
Outcome:  
Free, unbound drug concentration at 45 & 180 minutes post antibiotic dose.  
Eligibility:  
Healthy volunteers over 18 years old.  
Inclusion:   
Adult, eGFR > 90, previously received penicillin-based antibiotic.  
Exclusion:  
Penicillin or probenecid allergy, eGFR < 90, pregnant (or likely to become), 
anaemic, abnormal liver function, lack capacity, history of gout or uric acid 
kidney stones, G6PD deficiency, symptoms indicative of active infection, 
medications that interact with probenecid. Recent involvement in other 
research (within prior 3 months). 
Randomisation:  
Simple randomisation will be performed aiming for 50% of individuals  
to commence on intervention and 50% comparator arm of the study.  
Blinding:  
This is an unblinded study.  
 
Sample size:  50 participants. 
Using sparse PK sampling in a cross-over design; previous estimates have suggested that a 
sample size of at least 13 participants are required to detect a binary covariate (i.e. 30% 
proportional increase in elimination clearance) with at least 80% statistical power and a 
significance of p=0.05. Prior studies, using different methodological approaches and penicillin-
based antimicrobials have suggested that probenecid has approximately 30-40% effect on 
drug clearance.    
 
Statistical analysis:  
PK modelling of data will be undertaken using Pmetrics in R. Non-linear mixed effects 
modelling will be used to estimate the posterior estimates for individual PK. Probenecid 
exposures influence on clearance will be explored as a covariate in the model. 
 
Probability of target attainment (40% fT>MIC) for common organisms (e.g. streptococci) will 
be estimated. Descriptive statistics will be applied to the estimated population parameters and 
simulated PTAs to facilitate comparison of the data.     
 
Summary of method:   
Participants will be screened and consented to attend Imperial College Clinical Research 
Facility (CRF) at Hammersmith Hospital on two study visits, at least 7 days apart. For one 
visit (randomised), participants will be required to take penicillin-V only. For their 
other visit, they will take penicillin-V plus probenecid at standard recommended dose. Prior to 
the study visits, participants may be required to have taken 36-hours of penicillin +/- 
probenecid, documenting this in a dosing diary. On arrival at the CRF, the participant will take 
an observed dose of penicillin +/- probenecid. They will undergo blood draw via needle 

phlebotomy or a cannula (participant choice) at 45 and 180 minutes post the observed. 
Samples will be spun down and frozen at -80oC. They will subsequently be analysed using an 
in-house HPLC-MS/MS methodology to determine total and free-unbound drug 
concentration.    
 
For analysis, data from this study will be pooled with rich PK data from a prior study that 
assessed plasma concertation of penicillin-V in healthy volunteers. Pmetrics in R will be used 
to model the data looking to explore the effect of probenecid on clearance of free-penicillin-V. 
Probability of target attainment for streptococci species will also be estimated to evaluate the 
potential clinical impact of the addition of probenecid to routine penicillin-V use. Rich PK data 
for intravenous benzylpenicillin will be used to estimate PK-PD target attainment and PTAs for 
intravenous formulations, allowing direct comparison of oral and IV regimes.  
 
Figure. Total plasm penicillin concentration – time profiles for healthy volunteers receiving 
oral penicillin-V.  
 
 
Medication used in the study: 
Penicillin-V and probenecid used in this study will be prescribed by a qualified doctor. It will 
be supplied by Imperial College Healthcare NHS Trust pharmacy. 
 
Probenecid will be dosed at 500mg four-time a day (QDS) for all participants. 
Note: probenecid is not licenced for use in the United Kingdom. It is licenced in the EU. The 
dose selected is based on the probenecid summary of product characteristics, known doses 
used safely in the treatment of neuro-syphilis with benzylpenicillin as part of British STI 
treatment guidance, and is in line with published literature and guidelines for unlicensed use 
in the treatment of infection within UK and international organisations. Probenecid will be 
sourced from BioPhausia AB, Sweeden via Imperial College Healthcare NHS Trust Pharmacy. 
Dosing will be in line with their SmPC. 
 
Penicillin-V within the study will be dosed as follows for participants: 
Group 1 (n=24) 
500mg QDS penicillin-V 
Group 2 (n=13) 
250mg QDS penicillin-V 
Group 3 (n=13) 
750mg QDS penicillin-V 
 
Dose escalation in increments of 250mg has been selected to provide a wide safety margin 
even at higher doses. Probenecid is estimated to reduce penicillin clearance by approximately 
30%. Given that the maximum licenced dose of penicillin-V in the UK is 4g / day, we estimate 
that for a 30% reduction in clearance, 750mg QDS should remain within this margin of safety. 

Furthermore, given that doses of intravenous penicillin achieve much greater concentrations 
with limited side effects, we do not anticipate issues with toxicity within this study.  
 
Both drugs will be dispensed by Imperial College Hospital NHS Trust pharmacy with labelling 
performed in-house in alignment with Annex 13 under the Reg 37 hospital exemption. 
 
3.1 
STUDY OUTCOME MEASURES 
Primary outcome measure:  
 
1. Total and unbound penicillin-V concentrations at 45 and 180 minutes post observed 
penicillin-V dose with and without probenecid.  
 
Secondary outcome:  
 
 
1. Probability of target attainment for common infections treated with penicillin (e.g. 
Streptococcus spp.) will be estimated.  
2. PK-PD model describing the influence of probenecid on penicillin-V in healthy 
volunteers.  
 
Other documented outcomes: 
1. Reported adverse events associated with penicillin-V +/- probenecid.  
 
 
 
 
 
4. 
PARTICIPANT ENTRY  
 
4.1 
PRE-RANDOMISATION EVALUATIONS  
Pre-registration evaluation will include:  
- Clinical assessment  
- Confirmation of prior penicillin use and no documented penicillin allergy.  
- Blood tests including FBC, LFT, Renal Profile, Blood Borne Viral Screen (HIV/HBV/HCV).  
- Pregnancy test in female participants. 
 
4.2 
INCLUSION CRITERIA 
- Adult healthy volunteers (>18 years old). 
- Previously taken penicillin-based antibiotics without allergic response. 
- Estimated Glomerular Filtration Rate (eGFR) > 90. 
 
4.3 
EXCLUSION CRITERIA 
- Lacking capacity to consent. 
- Documented allergy to penicillin, other beta-lactam antibiotics, or probenecid. 
- History of G6PD Deficiency. 
- Known blood dyscrasias. 
- Anaemia (Hb < 12g/dL female, 13g/dL males). 
- Abnormal liver function (ALT, AST, ALP or bilirubin > ULN).  
- eGFR < 90. 
- Pregnant or likely to become pregnant during study period. 
- Breastfeeding women.  
- Symptoms consistent with active infection. 
- History of gout or uric acid kidney stones. 
- Taking regular medication that may interact with probenecid including, but not limited to 
methotrexate, lorazepam, acetaminophen, oral hypoglycaemic medication, sulfa containing 
drugs, non-steroidal anti-inflammatory drugs. 
- History of evidence of any medical, neurological, or psychological condition that would 
expose the subject to an undue risk of a significant adverse event or interfere with study 
assessments during the course of the trial as determined by the clinical judgement of the 
investigator. 
- Recent involvement in other research (within prior 3 months). 

 
4.4 
WITHDRAWAL CRITERIA 
Participants can withdraw at any point from the study without providing a reason for their 
decision. If they choose to withdraw after entry into the study any samples provided to this 
point (with their consent) will be retained for use in the study.  
 
If a participant develops any exclusion criteria after enrolment, they will be withdrawn from the 
study immediately.  
 
Study stopping rules: 
 
If information were to arise during the course of this study that would negate the results or 
suggest any safety concerns, the study will be stopped immediately with participants 
withdrawn and an update provided to them explaining the rationale for this.  
 
IMP treatment will be discontinued individually if a study participant presents a severe or 
serious adverse event considered related to the study IMPs. 
 
The trial will be halted if any of the following is observed:  
• 
A serious adverse reaction (i.e. a serious adverse event considered at least possibly 
related to the IMP administration) in one subject. 
• 
Severe adverse reactions (i.e. severe adverse events considered as, at least, possibly 
related to the IMP administration) in two subjects, independent of within or not within the 
same system-organ-class.  
 
If the trial is halted due to any of the study stopping rules detailed above, the trial will only be 
re-started after regulatory authority approval via a substantial amendment. 
 
 
5. 
RANDOMISATION AND ENROLMENT PROCEDURE 
 
 
5.1 
RANDOMISATION OR REGISTRATION PRACTICALITIES 
Pre-randomisation screening will require clinical assessment, checking of eligibility, 
pregnancy testing if female, and screening blood tests (FBC, LFT, Renal Profile, HIV, HBV, 
& HCV).  
 
Randomisation will be performed using simple randomisation. This will aim for 50% of 
individuals to commence on intervention and 50% comparator arm of the study. A 
randomisation list will be generated prior to commencement of the study. Participants will then 
be added sequentially onto this list as they enter the study.  
 
This study will be unblinded.  
 
5.2 
CONTRACEPTION REQUIREMENTS FOR FEMALE PARTICIPANTS 
Probenecid crosses the placental barrier and appears in cord blood, and the use of this drug 
in patients that are women of childbearing potential requires that the anticipated benefit be 
weighed against possible hazards. As this clinical trial is in healthy volunteers whom are not 
deriving any benefit by taking part in the trial, female participants must follow adequate 
contraception advice during the full participation in the trial.  
 
This trial follows the contraception guidance for female participants set out in the Clinical Trial 
Facilitation Group (CTFG) guidance on ‘Recommendations related to contraception and 
pregnancy testing in clinical trials’. 
 

A woman of childbearing potential (WOCBP) is defined as a “female participant that is fertile, 
following menarche and until becoming post-menopausal unless permanently sterile. 
Permanent sterilisation methods include hysterectomy, bilateral salpingectomy and bilateral 
oophorectomy. A postmenopausal state is 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). However, in the absence of 
12 months of amenorrhea, confirmation with more than one FSH measurement is required”.  
 
For WOCBP, acceptable methods of contraception must be used for the total duration of 
enrolment in the trial. Acceptable methods of contraception to be included within this study 
are defined within section 4 of the CTFG guidance as: 
 
Acceptable and highly effective methods of birth control 
• 
Combined hormonal contraception (oestrogen and progesterone containing) associated 
with inhibition of ovulation (oral, intravaginal, or transdermal). 
• 
Progesterone only hormonal contraception associated with the inhibition of ovulation (oral, 
injectable, or implantable).  
• 
Intrauterine device (IUD). 
• 
Intrauterine hormone-releasing system (IUS).  
• 
Bilateral tubal occlusion. 
• 
Vasectomised partner.  
• 
Sexual abstinence* 
 
*NB: sexual abstinence is considered a highly effective method only if defined as refraining 
from heterosexual intercourse during the entire period of risk associated with the study 
treatments. The reliability of sexual abstinence needs to be evaluated in relation to the duration 
of the clinical trial (estimated at 48 hours for each dosing interval) and the preferred and usual 
lifestyle of the subject. Clear documentation of this decision must be made at the time of 
screening.  
 
Acceptable but not considered as highly effective methods of birth control 
• 
Progesterone-only oral hormonal contraception, where inhibition of ovulation is not the 
primary mode of action.  
• 
Male or female condom with or without spermicide.  
• 
Cap, diaphragm, or sponge with spermicide. 
• 
Double barrier methods (e.g. male condom with cap, diaphragm, or sponge with 
spermicide) are also considered as acceptable, but not highly effective methods of birth 
control.  
 
 
6. 
TREATMENTS 
 
6.1 
TREATMENT ARMS 
All treatments (penicillin-V and probenecid) used within this study will be supplied by Imperial 
College Healthcare NHS Trust pharmacy.  
 
Probenecid will be dosed at 500mg QDS for all participants. It will be supplied by Imperial 
College Healthcare NHS Trust pharmacy. The formulation will be tablet form (500mg tablets).  
 
Penicillin-V will be dosed as follows for participants: 
Group 1 (n=24) 
500mg QDS penicillin-V 
Group 2 (n=13) 
250mg QDS penicillin-V 
Group 3 (n=13) 
750mg QDS penicillin-V 

It will be supplied by Imperial College Healthcare NHS Trust pharmacy. The formulation will 
be tablet form (250mg tablets). 
 
All participants will be required to commence treatment 36 hours prior to the CRF visit. They 
will take a maximum of 6 doses of penicillin-V +/- probenecid prior to attending the CRF. They 
will then take a further observed dose of penicillin-V +/- probenecid during the CRF visit (at 
time-point 0 minutes). The requirement for 6 doses prior to attending the CRF visit is to ensure 
that drug concentration (penicillin-V +/- probenecid) is at steady state (normally requires 
approximately 5 doses) prior to drug level sampling. In total, throughout the study (both visits), 
participants will take a maximum of 14 doses of penicillin-V and 7 doses of probenecid. This 
treatment interval has been selected as it is the shortest feasible duration to ensure patients 
arrive for the study visit at steady-state, whilst minimising the potential risks to safety in terms 
of drug interactions and adverse events.  
 
6.2 
DOSE MODIFICATIONS FOR TOXICITY  
No dose reductions are anticipated in this study. Participants will be required to have normal 
renal function. The dose of penicillin-V is within its licensed range. The dose of probenecid 
used is widely used in clinical practice with minimal reported side effects.  
 
Dose escalation in increments of 250mg has been selected to provide a wide safety margin 
even at higher doses. Probenecid is estimated to reduce penicillin clearance by approximately 
30%. Given that the maximum licenced dose of penicillin-V in the UK is 4g / day, we estimate 
that for a 30% reduction in clearance, 750mg QDS should remain within this margin of safety. 
Furthermore, given that doses of intravenous penicillin achieve much greater concentrations, 
we do not anticipate issues with toxicity within this study.  
 
6.3 
PREMEDICATION 
No premedication will be required as part of this study. 
 
6.4 
INTERACTION WITH OTHER DRUGS 
Participants will be informed of potential interactions of probenecid and other drugs. These 
include but a not limited to: methotrexate, lorazepam, acetaminophen, oral hypoglycaemic 
medication, sulfa containing drugs, non-steroidal anti-inflammatory drugs. 
 
Participants will be asked not to take other medications if possible during the dosing visits. If 
they require paracetamol, they will be advised to take 500mg QDS maximum.  
 
6.5 
DISPENSING AND ACCOUNTABILITY 
All medications used within this study will be supplied via the NHS suppliers which will be 
overseen by Imperial College Healthcare NHS Trust Clinical Trials Pharmacy team. The 
investigation medicinal product (IMP) will be used in line with the summary of medicinal 
product characteristics obtained from the EU-licensed supplier. All medications will be 
prescribed by a physician within the research team at the doses required per participant.  
 
IMP 1: Probenecid (tablet form) – EU market authorisation BioPhausia AB (MA number: 4522) 
IMP 2: Penicillin-V (tablet form) – UK market authorisation Sandoz BmB (MA number: PL 
04520/0005) 
 
6.6 
SAFETY MONITORING ASSESSMENTS 
If there are concerns with potential participant safety as defined by investigator criteria or 
clinical indication at any time during the study a safety monitoring assessment will be 
performed by a clinician within the study team. This may include physical examination, blood 
testing, and measurement of participant vital signs.  
 
 

 
7. 
Pharmacovigilance 
 
7.1 
DEFINITIONS   
Adverse Event (AE): any untoward medical occurrence in a patient or clinical trial subject 
administered a medicinal product and which does not necessarily have a causal relationship 
with this treatment.  An AE can therefore be any unfavourable and unintended sign (including 
an abnormal laboratory finding), symptom, or disease temporally associated with the use of 
an investigational medicinal product (IMP), whether or not considered related to the IMP. 
 
Based on the investigator judgement, an AE will be defined as: 
 
Mild: An event that does not affect the activities of daily living. It is easily tolerated. The effect 
is mild and does not impact the individual.  
 
Moderate: Affects normal activities of daily living. Affects the ability of one to carry out tasks 
which they normally do. It causes discomfort and distress to the individual. 
 
Severe: An event that prevents normal daily activities. 
 
Adverse Reaction (AR): all untoward and unintended responses to an IMP related to any 
dose administered.  All AEs judged by either the reporting investigator or the sponsor as 
having reasonable causal relationship to a medicinal product qualify as adverse reactions.  
The expression reasonable causal relationship means to convey in general that there is 
evidence or argument to suggest a causal relationship. 
 
Unexpected Adverse Reaction: an AR, the nature or severity of which is not listed in the 
reference safety information (RSI) e.g. list of expected medical events within investigator’s 
brochure for an unapproved investigational product or section 4.8 of the summary of product 
characteristics (SmPC) for an authorised product.  When the outcome occurs this adverse 
reaction should be considered as unexpected. Side effects documented in the SmPC which 
occur in a more severe form than anticipated are also considered to be unexpected. 
 
Serious Adverse Event (SAE) or Serious Adverse Reaction: any untoward medical 
occurrence or effect that at any dose: 
• 
Results in death. 
• 
Is life-threatening – refers to an event in which the subject was at risk of death 
at the time of the event; it does not refer to an event which hypothetically might 
have caused death if it were more severe. 
• 
Requires 
hospitalisation, 
or 
prolongation 
of 
existing 
inpatients’ 
hospitalisation. 
• 
Results in persistent or significant disability or incapacity. 
• 
Is a congenital anomaly or birth defect. 
 
Medical judgement should be exercised in deciding whether an AE/AR is serious in other 
situations.  Important AE/ARs that are not immediately life-threatening or do not result in death 
or hospitalisation but may jeopardise the subject or may require intervention to prevent one of 
the other outcomes listed in the definition above, should also be considered serious. 
 
Suspected Unexpected Serious Adverse Reaction (SUSAR): any suspected adverse 
reaction related to an IMP that is both unexpected and serious.   
 

7.2 
CAUSALITY 
Most adverse events and adverse drug reactions that occur in this study, whether they are 
serious or not, will be expected treatment-related toxicities due to the drugs used in this study.  
The assignment of the causality should be made by the investigator responsible for the care 
of the participant using the definitions in the table below. 
 
If any doubt about the causality exists the local investigator should inform the study 
coordination centre who will notify the Chief Investigators.  The pharmaceutical companies 
and/or other clinicians may be asked to advise in some cases. 
 
In the case of discrepant views on causality between the investigator and others, all parties 
will discuss the case.  In the event that no agreement is made, the MHRA will be informed of 
both points of view.  
 
 
Relationship 
Description 
Unrelated 
There is no evidence of any causal relationship 
Unlikely 
There is little evidence to suggest there is a causal relationship (e.g. 
the event did not occur within a reasonable time after administration of 
the trial medication).  There is another reasonable explanation for the 
event (e.g. the participant’s clinical condition, other concomitant 
treatment). 
Possible 
There is some evidence to suggest a causal relationship (e.g. because 
the event occurs within a reasonable time after administration of the 
trial medication).  However, the influence of other factors may have 
contributed to the event (e.g. the participant’s clinical condition, other 
concomitant treatments). 
Probable 
There is evidence to suggest a causal relationship and the influence of 
other factors is unlikely. 
Definitely 
There is clear evidence to suggest a causal relationship and other 
possible contributing factors can be ruled out. 
Not assessable 
There is insufficient or incomplete evidence to make a clinical 
judgement of the causal relationship. 
 
7.3 
REPORTING PROCEDURES 
All adverse events should be reported. Depending on the nature of the event the reporting 
procedures below should be followed. Any questions concerning adverse event reporting 
should be directed to the study coordination centre in the first instance. A flowchart is given 
below to aid in the reporting procedures. All AE and SAEs must be recorded from the time of 
consent, until the end of the last study visit. 
 
7.3.1 Non serious AR/AEs 
All such toxicities, whether expected or not, should be recorded in the toxicity section of the 
relevant case report form and sent to the study coordination centre within one month of the 
form being due.   
 
7.3.2 Serious AR/AEs 
Fatal or life threatening SAEs and SUSARs should be reported on the day that the local site 
is aware of the event. The SAE form asks for nature of event, date of onset, severity, corrective 
therapies given, outcome and causality (i.e. unrelated, unlikely, possible, probably, definitely).  
The responsible investigator should sign the causality of the event. Additional information 
should be sent within 5 days if the reaction has not resolved at the time of reporting.   
 

SAEs 
An SAE form should be completed and faxed to the study coordination centre for all SAEs 
within 24 hours.   
 
SUSARs  
In the case of suspected unexpected serious adverse reactions, the staff at the site should:  
 
 
Complete the SAE case report form & send it immediately (within 24 hours,), 
signed and dated to the study coordination centre together with relevant 
treatment forms and anonymised copies of all relevant investigations. 
Or 
 
Contact the study coordination centre by phone and then send the completed 
SAE form to the study coordination centre within the following 24 hours as 
above. 
 
The study coordination centre will notify the MHRA, REC and the Sponsor of all SUSARs 
occurring during the study according to the following timelines; fatal and life-threatening within 
7 days of notification and non-life threatening within 15 days.   All investigators will be informed 
of all SUSARs occurring throughout the study. 
 
Local investigators should report any SUSARs and /or SAEs as required by their Local 
Research Ethics Committee and/or Research & Development Office. 
 
 
Contact details for reporting SAEs and SUSARs 
RGIT.ctimp.team@imperial.ac.uk 
CI email (and further details below). 
Please send SAE forms to: tmr07@ic.ac.uk 
Tel: 07891750151 (Mon to Fri 09.00 – 17.00) 

 
 
8. 
ASSESSMENT AND FOLLOW-UP  
 
After the second CRF visit there will be no further follow up of participants required. They will 
have completed their enrolment in the study at this time.  
 
The end of the study will be defined as when all 50 participants entered into the study have 
completed two planned study visits.  
 
8.1 
INCIDENTAL FINDINGS 
Incidental findings that arise as part of the study will be discussed with the patient. 
Permission will be sought to contact the patients GP to inform them of these findings.  
 
8.2 
LOSS TO FOLLOW-UP 
No follow up is required post the second dosing visit to the Imperial CRF.  
 
8.3 
TRIAL CLOSURE 
The definition for the end of the trial is when the last participant has completed their last visit. 
This is anticipated to be at participant number 50.  
 
 
9. 
STATISTICS AND DATA ANALYSIS 
 
Sample size:  50 participants. 
Using sparse PK sampling in a cross-over design; previous estimates have suggested that a 
sample size of at least 13 participants are required to detect a binary covariate (i.e. 30% 
proportional increase in elimination clearance) with at least 80% statistical power and a 
significance of p=0.05. Prior studies, using different methodological approaches and penicillin-
based antimicrobials have suggested that probenecid has approximately 30-40% effect on 
drug clearance.    
 
Statistical analysis:  
PK modelling of data will be undertaken using Pmetrics in R. Non-linear mixed effects 
modelling will be used to estimate the posterior estimates for individual PK. Probenecid 
exposures influence on clearance will be explored as a covariate in the model. 
 
Probability of target attainment (40% fT>MIC) for common organisms (e.g. streptococci) will 
be estimated. Descriptive statistics will be applied to the estimated population parameters and 
simulated PTAs to facilitate comparison of the data.     
 
Randomisation: 
Simple randomisation will be performed using the randomizeR package in R. Randomisation 
will be performed prior to commencement of recruitment. This will be stored on a central 
database with participants allocated and randomised sequentially upon entry into study visit 
1.  
 
Interim analysis plan: 
Formal interim analysis will be undertaken by members of the research team after every 10 
participants have completed two study visits. Drug concentration data and reported side 
effects / adverse events will be evaluated.  
 
Given the unblinded nature of this study, informal analysis will be iterative with researchers 
monitoring documented side effects / safety reporting per-participant visit to the CRF.  

 
Data and all appropriate documentation will be stored for a minimum of 10 years after the 
completion of the study, including the follow-up period.   
 
 
10. 
Monitoring 
 
10.1 
RISK ASSESSMENT 
The study has been reviewed by Imperial Clinical Research Facility (CRF) Protocol Review 
Board (PRB). A risk assessment and management plan for CTIMP has been completed with 
the study scoring low risk on risk assessment (appendix).  
 
10.2 
MONITORING AT STUDY CENTRE 
Routine monitoring will be performed for all participants throughout study visits. This will 
include data entry checking, consent form checking, and ensuring that all required data are 
input at the time of each participant visit. Transport and analysis logs for the processing, 
storage, and analysis of plasma samples will be kept to ensure that any abnormal results can 
be reviewed.  
 
 
11. 
REGULATORY ISSUES 
 
11.1  
CLINICAL TRIALS APPROVAL  
This study has Clinical Trials Authorisation from the UK Competent Authority; MHRA.  
Reference: xxx 
 
11.2 
ETHICS APPROVAL 
The Study Coordination Centre has obtained approval from the xxx Research Ethics 
Committee (REC) and Health Regulator Authority (HRA). The study must also receive 
confirmation of capacity and capability from each participating NHS Trust before accepting 
participants into the study or any research activity is carried out. The study will be conducted 
in accordance with the recommendations for physicians involved in research on human 
subjects adopted by the 18th World Medical Assembly, Helsinki 1964 and later revisions. 
 
11.3 
CONSENT  
Consent to enter the study must be sought from each participant only after a full explanation 
has been given, an information leaflet offered and time allowed for consideration.  Signed 
participant consent should be obtained.  The right of the participant to refuse to participate 
without giving reasons must be respected.  After the participant has entered the trial the 
clinician remains free to give alternative treatment to that specified in the protocol at any stage 
if he/she feels it is in the participant’s best interest, but the reasons for doing so should be 
recorded.  In these cases the participants remain within the study for the purposes of follow-
up and data analysis.  All participants are free to withdraw at any time from the protocol 
treatment without giving reasons and without prejudicing further treatment. 
 
11.4 
CONFIDENTIALITY 
Participants’ identification data will be required for the registration process.  The Study 
Coordination Centre will preserve the confidentiality of participants taking part in the study and 
is registered under the Data Protection Act. 
 
11.5 
INDEMNITY 
Imperial College London holds negligent harm and non-negligent harm insurance policies 
which apply to this study. 
 

11.6 
SPONSOR 
Imperial College London will act as the main Sponsor for this study.  Delegated responsibilities 
will be assigned to the NHS trusts taking part in this study 
 
11.7 
FUNDING 
The British Infection Association (BIA) are funding this study. 
 
Reimbursement of participants travel costs will be offered at up to £50.00 per participant per 
study visit. Reimbursement cannot be provided as part of screening visits.    
 
11.8 
AUDITS AND INSPECTIONS  
The study may be subject to inspection and audit by Imperial College London/Imperial College 
Healthcare NHS Trust (delete as applicable) under their remit as Sponsor, the Study 
Coordination Centre and other regulatory bodies to ensure adherence to GCP.  
 
 
12. 
Trial Management 
 
A Trial Management Group (TMG) will be appointed and will be responsible for overseeing 
the progress of the trial.  The day-to-day management of the trial will be co-ordinated through 
the Centre for Antimicrobial Optimisation (Dr Timothy M Rawson).  
 
 
13. 
PUBLICATION POLICY 
 
The results of the study will be published within 12 months of study completion in a peer-
reviewed journal. Preliminary results may also be presented at national and international 
academic research meetings, including ECCMID 2022 and FIS 2022.  
 
All publications and presentations relating to the study will be authorised by the Trial 
Management Group.  The first publication of the trial results will be in the name of the Trial 
Management Group, if this does not conflict with the journal’s policy.  If there are named 
authors, these will include at least the trial’s Chief Investigator, Statistician and Trial 
Coordinator.  Members of the TMG and the Data Monitoring Committee will be listed and 
contributors will be cited by name if published in a journal where this does not conflict with the 
journal’s policy.  Authorship of parallel studies initiated outside of the Trial Management Group 
will be according to the individuals involved in the project but must acknowledge the 
contribution of the Trial Management Group and the Study Coordination Centre. 
 
 
 
11. 
REFERENCES 
 
1. Robbins N, Koch SE, Tranter M, Rubinstein J. The History and Future of 
Probenecid. Cardiovasc Toxicol 2012; 12: 1–9.  
 
2.  Maeda K, Tian Y, Fujita T, Ikeda Y, Kumagai Y, Kondo T et al. Inhibitory effects of 
p-aminohippurate and probenecid on the renal clearance of adefovir and 
benzylpenicillin as probe drugs for organic anion transporter (OAT) 1 and OAT3 
in humans. Eur J Pharm Sci 2014; 59: 94–103.  
 

3.  Frieden TR, Harold Jaffe DW, Rasmussen SA, Leahy MA, Martinroe JC, Spriggs 
SR et al. Morbidity and Mortality Weekly Report Sexually Transmitted Diseases 
Treatment Guidelines, 2015. https://www.cdc.gov/std/tg2015/default.htm  
 
4.  Sharland M, Pulcini C, Harbarth S, Zeng M, Gandra S, Mathur S et al. Classifying 
antibiotics in the WHO Essential Medicines List for optimal use-be 
AWaRe. Lancet Infect Dis 2018; 18: 18–20.  
 
5.  Rawson TM, Gowers SAN, Freeman DME, Wilson RC, Sharma S, Gilchrist M et 
al. Microneedle biosensors for real-time , minimally invasive drug monitoring of 
phenoxymethylpenicillin : 
a 
first-in-human 
evaluation 
in 
healthy 
volunteers. Lancet Digit Heal 2019. 7(1);E335-E343  

Appendix 1. Summary of study 
 
 
 
        
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening & eligibility checking 
Screening visit with baseline 
blood tests 
Randomisation for inclusion 
Penicillin only visit 
 
Penicillin dose: 
n=12x500mg QDS 
n=6x250mg QDS 
n=7x750mg QDS 
Penicillin plus probenecid visit 
 
Penicillin dose: 
n=12x500mg QDS 
n=6x250mg QDS 
n=7x750mg QDS 
 
Probenecid dose: n=25x500mg QDS 
Penicillin plus probenecid visit 
 
Penicillin dose: 
n=12x500mg QDS 
n=7x250mg QDS 
n=6x750mg QDS 
Penicillin only visit 
 
Penicillin dose: 
n=12x500mg QDS 
n=7x250mg QDS 
n=6x750mg QDS 
 
Probenecid dose: n=25x500mg QDS 
Completion of study 
Washout period (>7 days) 
Washout period (>7 days) 

Appendix 2. Summary of investigations, treatments and assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening visit 
Study visit 1 
Study visit 2 
Screening questions 
X 
 
 
Consent obtained 
X 
 
 
History and examination 
X 
 
 
Screening bloods 
X 
 
 
Pregnancy test (if female) 
X 
 
 
Penicillin prescription 
 
X 
 
Penicillin + probenecid prescription 
 
 
X 
Diary card for dosing / side effects 
 
X 
X 
Phlebotomy 
 
X 
X 
Documentation and reporting of 
adverse events 
 
X 
X 
Documentation and reporting of 
serious adverse events 
 
X 
X