Assessment of Metformin for Restoration of Immune Homeostasis in HIV+ and HIV- Individuals With a History of Injection Drug Use (MET-IH)
Phase: PHASE4
Conditions: Systemic Inflammation, Immune Dysregulation, Injection Drug Use, HIV
Sponsor: University of Alabama at Birmingham

 
 
PROTOCOL 
MET-IH 
Assessment of metformin for restoration of immune 
homeostasis in HIV+ and HIV- individuals with a history of 
injection drug use  
 
 
 
CLINICAL TRIAL SPONSORED BY 
National Institute on Drug Abuse (NIDA) 
Department of Health and Human Services (DHHS) 
Bethesda, Maryland, USA 
 
University of Alabama at Birmingham 
Birmingham, Alabama, USA 
 
 
 
 
March 25, 2026 
MET-IH  
Version 7.0 

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Protocol Signature Page 
Assessment of metformin for restoration of immune homeostasis in HIV+ and HIV- 
individuals with a history of injection drug use 
I will conduct the study in accordance with the provisions of this protocol and all 
applicable protocol-related documents. I agree to conduct this study in compliance 
with US Health and Human Service regulations (45 CFR 46); applicable US Food 
and Drug Administration regulations; standards of the International Council for 
Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) 
Guideline for Good Clinical Practice E6(R2); Institutional Review Board/Ethics 
Committee determinations; all applicable in-country, state, and local laws and 
regulations; and other applicable requirements (eg, US National Institutes of Health, 
NIDA) and institutional policies. 
 
Principal Investigator: _______Ellen F. Eaton, MD__________________________                            
 
                                
 
Signed: _____________________________ Date: ____________________ 
 
 
 
 
 
Protocol Number: NIDA-UAB-01 
Protocol Version: Version 7.0 
Protocol Date: March 25, 2026 

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Acronyms and abbreviations 
Ab 
 
antibody 
AE 
 
adverse event 
Ag 
 
antigen 
ALT 
 
alanine aminotransferase 
AFAB 
 
assigned female sex at birth 
AMAB 
 
assigned male sex at birth 
ANOVA  
analysis of variance 
AoU 
 
Assessment of Understanding 
BMI 
 
body mass index 
BP 
 
blood pressure 
CBC 
 
complete blood count 
CDM 
 
Clinical Data Manager 
CE 
 
European Conformity (Conformité Européenne) 
CI 
 
confidence interval 
CRF 
 
case report form 
DM 
 
diabetes mellitus 
EAE 
 
expedited adverse event 
eGFR 
 
estimated glomerular filtration rate 
EIA 
 
enzyme immunoassay 
FDA 
 
US Food and Drug Administration 
GCP 
 
Good Clinical Practice 
GINA 
 
Genetic Information Nondiscrimination Act 
GPP 
 
Good Participatory Practices 
HCV 
 
hepatitis C virus 
Hgb 
 
hemoglobin 
HHS 
 
United States Department of Health and Human Services 
ICH 
 
International Council for Harmonisation of Technical 
Requirements for Pharmaceuticals for Human Use 
IM 
 
intramuscular 
IND 
 
Investigational New Drug (application) 
INR 
 
international normalized ratio 
IRB 
 
Institutional Review Board 
MAAE 
 
medically attended adverse event 
MedDRA  
Medical Dictionary for Regulatory Activities 
MET 
 
Metformin 
MO 
 
Medical Officer 

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NIDA 
 
National Institute on Drug Abuse 
NIH 
 
National Institutes of Health 
NSAID 
 
nonsteroidal anti-inflammatory drug 
OUD 
 
opioid use disorder 
PBMC 
 
peripheral blood mononuclear cell 
PI 
 
Principal Investigator 
POPIA 
 
Protection of Personal Information Act 
PWID 
 
People who inject drugs 
PWH 
 
Person with HIV-1 
RA 
 
Regulatory Authority 
RCT 
 
Randomized Control Trial 
SAE 
 
serious adverse event 
SD 
 
standard deviation 
SICF 
 
sample informed consent form 
DSMB 
 
data safety monitoring board 
DSMP 
 
data safety monitoring plan 
SOP 
 
standard operating procedures 
SSP 
 
study specific procedures 
STI 
 
sexually transmitted infection 
SUSAR 
 
Suspected Unexpected Serious Adverse Reaction 
ULN 
 
upper limit of normal 
USP 
 
US Pharmacopeia 
WBC 
 
white blood cell 
 
 

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Protocol Team 
 
Principal investigator  
 
Ellen Eaton, MD, MSPH 
 
 
 
University of Alabama at Birmingham 
 
 
 
205-975-0661 
 
 
 
eeaton@uabmc.edu 
 
Co-principal investigator 
 
James Kobie, PhD 
 
 
 
University of Alabama at Birmingham 
 
 
 
205-975-2760 
 
 
 
jjkobie@uabmc.edu 
 
Clinic director 
 
 
Paul Goepfert, MD 
 
 
 
University of Alabama at Birmingham 
 
 
 
205-821-4998 
 
 
 
pgoepfert@uabmc.edu 
 
Medical monitor 
 
 
Sonya Heath, MD 
 
 
 
University of Alabama at Birmingham 
 
 
 
205-996-2371 
 
 
 
slheath@uabmc.edu 
 
Protocol pharmacist 
 
 
Deon Powell, Pharm.D., AAHIVP 
 
 
 
University of Alabama at Birmingham 
 
 
 
205-975-5009 
 
 
 
dpwhite@uabmc.edu 
 
Clinical trials manager  
 
Heather Logan, DNP, CRNP 
 
 
 
University of Alabama at Birmingham 
 
 
 
205-934-6774 
 
 
 
heatherlogan@uabmc.edu 
 
Statistician 
 
 
Anastasia Hartzes, PhD 
 
 
 
University of Alabama at Birmingham 
 
 
 
ahartzes@uab.edu 
 
Data safety monitoring board, chair  
Michael Saag, MD 
 
 
 
University of Alabama at Birmingham   
 
 
 
msaag@uabmc.edu 
 

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Contents 
1 Hypothesis and Study Objectives ................................................................................ 12 
1.1   Hypothesis...................................................................................................... 12 
1.2 
Primary Objective and Endpoint .................................................................. 12 
1.1 
Secondary Objectives and Endpoints ........................................................... 12 
1.2 
Exploratory Objective and Endpoints .......................................................... 13 
2 Introduction .................................................................................................................. 14 
2.1 
Study Rationale ............................................................................................ 14 
2.2 
Background .................................................................................................. 15 
2.3 
Metformin: Clinical use and potential adjunctive therapy for PWID. ......... 20 
3 Study Design ................................................................................................................ 26 
4 Selection and Enrollment of Participants ..................................................................... 27 
4.1 
Inclusion Criteria .......................................................................................... 27 
4.2 
Additional Inclusion Criteria for All Participants Providing Rectal Biopsy:28 
4.3 
Exclusion Criteria ......................................................................................... 29 
4.4 
Additional Exclusion Criteria for All Participants Providing Rectal Biopsy:30 
5 Study Treatment ........................................................................................................... 32 
5.1 
Regimens, Administration and Duration ...................................................... 32 
5.2 
Study Product Description, Storage, and Preparation .................................. 32 
5.3 
Pharmacy: Product Supply, Distribution, and Accountability ..................... 32 
5.4 
Concomitant Medications ............................................................................ 33 
6. Clinical and Laboratory Evaluations ........................................................................... 34 
6.1 
Schedule of Activities .................................................................................. 34 
6.2 
Timing of Study Activities ........................................................................... 37 
6.3   Instructions for Study Activities .................................................................... 39 
6.4 
Drug Use Measures ...................................................................................... 46 
7 Study Procedures ......................................................................................................... 48 
7.1 
Definition of “study day” and “study visit” ................................................. 48 
7.2 
Screening (V0) ............................................................................................. 48 
7.3 
Enrollment visit (V1) ................................................................................... 49 
7.4 
Post enrollment follow-up virtual visit (V2) ................................................ 49 
7.5 
Post enrollment follow-up virtual visit (V3) ................................................ 49 
7.6 
Post enrollment visit (V4) ............................................................................ 50 
7.7 
Post enrollment follow-up virtual visit (V5) ................................................ 50 
7.8 
Vaccination visit (V6) .................................................................................. 50 
7.9 
Post vaccination virtual visit (V7) ................................................................ 50 
7.10 Post vaccination virtual visit (V8) ................................................................ 51 
7.11 Vaccination visit (V9) .................................................................................. 51 
7.12 Post vaccination visit (V10) ......................................................................... 51 
7.13 Virtual visit (V11) ........................................................................................ 52 
7.14 Virtual visit (V12) ........................................................................................ 52 
7.15 In person visit (V13) .................................................................................... 52 

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7.16 Virtual visit (V14) ........................................................................................ 52 
7.17 Virtual visit (V15) ........................................................................................ 53 
7.18 Metformin cessation visit (V16) .................................................................. 53 
7.19 Post cessation virtual visit (V17) ................................................................. 53 
7.20 Post cessation virtual visit (V18) ................................................................. 53 
7.21 Final in person visit (V19) ........................................................................... 54 
7.22 Virtual visit (V20) ........................................................................................ 54 
7.23 Virtual visit (V21) ........................................................................................ 54 
7.24 Final virtual visit (V22) ................................................................................ 54 
7.25 Other visits ................................................................................................... 54 
7.26 Total blood volume ...................................................................................... 55 
7.27 Visit windows and missed visits .................................................................. 55 
7.28 Early termination visit .................................................................................. 55 
7.29 AE contact .................................................................................................... 55 
8 Adverse Events and Study Monitoring ........................................................................ 57 
8.1 
Definition of Adverse Events ....................................................................... 57 
8.2 
Adverse Event Collection Requirements for This Protocol ......................... 57 
8.3 
Expedited Adverse Event Reporting ............................................................ 59 
8.4 
Study Monitoring ......................................................................................... 59 
8.5 
Serious adverse events (SAEs) ..................................................................... 59 
8.6 
Safety monitoring ......................................................................................... 60 
8.7 
Safety reviews .............................................................................................. 61 
8.8 
Study termination ......................................................................................... 62 
9 Clinical Management Issues ........................................................................................ 63 
9.1 
Toxicity ........................................................................................................ 63 
9.2 
Hyperlactatemia /Lactic Acidosis ................................................................ 63 
10 Criteria for Discontinuation ......................................................................................... 65 
10.1 Permanent and Premature Study Product Discontinuation .......................... 65 
10.2 Premature Study Discontinuation ................................................................. 66 
11 Statistical Considerations ............................................................................................. 67 
11.1 General Design Issues .................................................................................. 67 
11.2 Outcome Measures ....................................................................................... 67 
11.3 Randomization and Stratification ................................................................. 68 
11.4 Sample Size and Accrual .............................................................................. 68 
11.5 Data and Safety Monitoring ......................................................................... 69 
11.6 Analyses ....................................................................................................... 69 
12 Pharmacology Plan ...................................................................................................... 71 
13 Data Collection and Monitoring .................................................................................. 72 
13.1 Records to Be Kept ...................................................................................... 72 
13.2 Role of Data Management ............................................................................ 72 
13.3 Clinical Site Monitoring and Record Availability ....................................... 72 
13.4 Reporting Protocol Deviations ..................................................................... 72 

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14 Participants ................................................................................................................... 73 
14.1 IRB Review and Informed Consent ............................................................. 73 
14.2 Participant Confidentiality ........................................................................... 73 
14.3 Study Discontinuation .................................................................................. 73 
15 Data Sharing, Public Access, and Publications ........................................................... 74 
16 Biohazard Containment ............................................................................................... 75 
17 References .................................................................................................................... 76 
18 Protocol Amendment Summary of Changes Table ..................................................... 87 
 
 
 
 

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Executive Summary  
 
Title 
Assessment of metformin for restoration of immune homeostasis in HIV+ and HIV- 
individuals with a history of injection drug use (MET-IH) 
 
 
 
Design 
This is a single site randomized controlled interventional trial. The purpose of this 
randomized clinical trial (RCT) is to determine the potential of metformin to reduce 
systemic inflammation and mitigate immune dysregulation among people who inject 
drugs (PWID). In the trial, adults with a history of recent injection drug including 
opioid and amphetamine use with (PWH) or without HIV and elevated systemic 
inflammation as determined by CRP  1 mg/L, will be recruited, and randomized 1:1 
to 16 weeks of metformin or placebo treatment, during which time they will receive 
Jynneos (MPOX) and Capvaxvie (PCV21, S. pneumoniae) vaccinations. All 
participants will be followed to 6 months post-enrollment. 
 
Study products and administration 
• Metformin: 500 mg metformin extended-release (ER) once a day for 1 week, 
followed by 1000 mg once a day for 15 weeks.  
 
Study participants 
100 volunteers with or without HIV, and a history of injection drug use in the 
Birmingham, AL metro area, aged 19 to 64 years 

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Study plan  
Figure 0-1 Study plan 
 
 
Eligible participants, ~1:1 stratified by HIV status, will be randomized 1:1 to receive either metformin (Group 1) or placebo (Group 2) starting at week 
(W) 0. At W1, participants will increase dosage and continue taking placebo or metformin for 112 days. At W5 participants will be immunized 
consistent with MPOX (Jynneos) and pneumococcal (Capvaxive, PCV21) vaccines.  At W 0 (enrollment), 9, 16, and 20 visits participants will have 
the option of providing rectal secretion samples.  Rectal biopsy samples will be collected at W16 from HIVneg participants that opt-in to this 
sampling. 

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Table 0-1 Treatment Schema 
Group 
N 
Week 0 - 1 (Day 0-7) 
Week 1 - 16 (Day 8-112) 
1 
(Metformin) 
50 
Metformin ER, 500 mg OD, orally 
Metformin ER, 1000 mg OD, 
orally 
2 
(Placebo) 
50 
Placebo, one tablet OD, orally 
 
Placebo, two tablets OD, orally 
 
Total 
100 
 
 
Both groups will open to enrollment concurrently.  
Duration per participant 
6 months of scheduled in-person clinic and virtual visits  
Estimated total study duration 
48 months  
Study site 
The study will be conducted at the University of Alabama at Birmingham. 
 
 
 

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1 
Hypothesis and Study Objectives 
1.1   Hypothesis 
Compared to placebo (Group 2), metformin (MET) treatment (Group 1) will 
result in a decrease in systemic inflammation and improvement in antibody 
responses to vaccination among people who inject drugs (PWID).  
 
1.2 Primary Objective and Endpoint 
Objective: To assess if metformin treatment results in reduction in serum C-
reactive protein (CRP) compared to placebo treatment. 
Endpoint: Change in serum C-reactive protein from baseline to week 16. 
Justification: CRP is a biomarker for systemic inflammation and is elevated in 
many PWID and PWH. Previous studies have demonstrated reduction of CRP 
with MET treatment. 
1.1 
Secondary Objectives and Endpoints 
a. Secondary Objective: To assess if metformin treatment results in a decrease in 
B cell exhaustion compared to placebo treatment. 
 
Secondary Endpoint: Change in peripheral blood exhausted B cells from 
baseline to week 16. 
 
 
b. Secondary Objective: To assess if metformin treatment results in a decrease in 
T cell exhaustion compared to placebo treatment. 
 
Secondary Endpoint: Change in peripheral blood exhausted T cells from 
baseline to week 16. 
 
c. Secondary Objective: To assess if metformin treatment improves antibody 
response to vaccination. 
 
Secondary Endpoint: Change in vaccine-specific IgG antibody titer from 
baseline to week 9 and baseline to week 13. 
 
 
Justification: Alterations in B cells, T cells, and antibody responses to vaccines are 
evident in many PWID and PWH. Previous studies have demonstrated improvement 
in B cell, T cell, and Ab responses with MET treatment 

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1.2 
Exploratory Objective and Endpoints 
 
 
 
 
 
 
Exploratory Objective: To assess if metformin treatment results in a decrease in 
mucosal inflammation compared to placebo treatment. 
Exploratory Endpoint: Change in oral and/or genital mucosal inflammatory 
cytokines from baseline to week 16. Differences in rectal lymphocyte 
characteristics at week 16. 
Justification: Substance use, including amphetamine use is associated with 
increased mucosal inflammation and related negative health consequences. As 
MET has been shown to decrease systemic inflammation, we seek to assess its 
effect on the mucosa.  
 
  
 
 
 
 
 

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2 
Introduction 
The aim of this study is to determine the potential of metformin (MET) to reduce 
systemic inflammation, mitigate immune dysregulation, and improve vaccine 
responses among people who inject drugs (PWID). 
To achieve these goals, this study will:  
1) Enroll adults with a history of injection drug including opioid and/or 
amphetamine use with or without HIV (PWH) and elevated systemic 
inflammation as determined by CRP  1 mg/L, who will then be treated with 
MET or placebo, and immunized with MPOX and Pneumococcal vaccines.  
2) Assess the ability of MET treatment to 1) reduce systemic inflammation, as 
measured by CRP, 2) reduce exhausted B cell and exhausted T cells, 3) improve 
antibody responses to vaccines, and 4) reduce mucosal inflammation.  
This study will determine the clinical potential of MET to mitigate 
immunopathology and improve immune responses to pathogens in PWID. 
2.1 
Study Rationale 
We and others have described significant immune alterations in people with HIV 
(PWH) and individuals that inject opioids and/or stimulants including increased 
systemic inflammation and lymphocyte dysregulation, including among B cells. 
This heighted inflammatory state and distorted B cell function likely contribute to 
increased risk and severity of infections and inflammatory comorbidities 
including cardiovascular, kidney, liver, and autoimmune diseases. Although anti-
retroviral therapy (ART) suppresses HIV, its ability to restore immune 
homeostasis is incomplete, resulting in accelerated immunological aging and 
associated co-morbidities; subsequently development of adjunctive interventions 
to mitigate these complications is ongoing. Among PWH, those who inject drugs 
are particularly vulnerable to inflammatory and infectious sequalae, driven by 
related factors including challenges in ART adherence, maintaining viral 
suppression, and engagement in care. Active substance use, intoxication, and 
related behaviors associated with injection drug use can impact HIV viral 
dynamics and immune competency. Among people who inject drugs (PWID) that 
do not have HIV, there is a high risk for acquiring HIV, clear indications of 
reduced effectiveness of HIV vaccines, and altered viral dynamics in the acute 
stage of HIV infection; these unique factors highlight the challenges of HIV 
vaccine development for PWID. Thus, there is an unmet need for interventions 
that can restore immune homeostasis in PWID to improve responses to pathogens 
and minimize inflammatory-related co-morbidities. 
Metformin (MET), the most widely prescribed drug for type 2 diabetes (T2D), 
may be such an intervention. FDA approved in 1994, MET is a first-line treatment 

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for T2D and is used off-label for a variety of metabolic-related conditions and 
used by >20 million people in the US annually. Animal and human studies have 
demonstrated the ability of MET to reduce inflammation and enhance vaccine 
responses, likely mediated in part through direct and indirect lymphocyte 
regulation through mTOR inhibition. Our central hypothesis is that MET can 
mitigate the inflammation and immune dysregulation that is prevalent in PWID 
including among PWH, restoring immune homeostasis and responsiveness. This 
will be addressed through a randomized, two-group, placebo controlled, clinical 
trial of MET, stratified by HIV status, in individuals with a history of injection 
drug use including opioids and stimulants and with elevated systemic 
inflammation. Assessment of MPOX and pneumococcal vaccine responses during 
this trial will allow examination of adaptive responses to pathogens of concern 
within these populations and serve as a surrogate for assessing their potential 
response to a future HIV vaccine. Vaccine outcomes will provide a metric to 
complement the integrated behavioral and high-resolution functional analysis of 
systemic and mucosal inflammation and immunity, providing mechanistic 
insights relevant to HIV prevention and pathogenesis. 
2.2     Background 
Immune dysregulation amplifies the HIV and opioid syndemic. Among PWH, 
HIV-associated co-morbidities driven in-part by accelerated inflammaging and 
not fully mitigated by anti-retroviral therapy (ART), continue to be a major health 
care burden(1, 2). These outcomes are further exacerbated in PWID(3, 4); with 
PWH that were presumed infected through injection drug use having the highest 
rates of all death among PWH even after adjustment for ART, including AIDS, 
non-AIDS infection, non-AIDS malignancy and heart/vascular deaths(5-7). 
Despite the increasing availability of ART for treatment and prevention, and 
encouraging declines in the incidence of new HIV infections overall, new HIV 
infections among PWID remain stable in the US(8). Particularly concerning is the 
current wave of the opioid epidemic is characterized by dominant synthetic opioid 
use (e.g. fentanyl) and its frequent co-use with illicit stimulants(9), primarily 
methamphetamine, and our understanding of immune dysregulation among PWID 
may now be underestimated. The differing pharmacology of fentanyl compared to 
heroin may further alter its impact on immunity, as it is more lipophilic(10) and is 
associated with a shorter intoxication compared to heroin(11), thus contributing to 
a higher frequency of injections per individual than was typically observed with 
heroin(11), and with the increased co-stimulant use(12, 13) may further drive 
inflammatory and infectious co-morbidities. In assessing the immunological and 
infectious complications associated with injection drug use, it has been 
challenging to disentangle the impact of the action itself and associated behaviors, 
from the substances themselves; however, it is clear that many of the co-
morbidities associated with PWID are driven in part by increased inflammation 
and immune dysregulation, suggesting that interventions to restore immune 
homeostasis may have the potential to improve outcomes for these individuals.  

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B cells: Critical mediators of pathogen-specific immunity compromised by 
HIV and injection drug use. B cells, defined in humans by the expression of 
CD19, are essential members of the adaptive immune response most notably for 
their production of antibody (Ab); responsible for protection from infectious 
pathogens, mediating autoimmune diseases, and driving inflammatory processes. 
Naive B cells develop from the bone marrow, and in response to antigen 
encounter (e.g., pathogen), undergo a differentiation process into various effector 
and memory subsets that have specialized localization and functions. This 
includes B cells that can produce inflammatory (e.g., TNF-α) or regulatory 
cytokines (e.g., IL-10, TGF-β), and subsets that are specialized in production of 
secreted Ab, including short-lived plasmablasts and long-lived plasma cells. The 
B cell deficits among PWH is well described by us and others, and although some 
peripheral B cells subsets return to near normal frequencies with ART, damage to 
the IgM memory/marginal zone-like B cell compartment, vital for responses to S. 
pneumoniae, persists(14-21). We have previously found that people who inject 
heroin have a remarkable 2-fold increase in total B cells (Fig 2-1A), including 
expansion of several activated B cell phenotypes such as cells expressing CD95 
Figure 2-1. B cell dysregulation 
among PWID. Peripheral blood B 
cells from HIV- HCV- PWID (n=19) 
and healthy controls (HC) (n=19) 
were analyzed by flow cytometry. 
(A) Representative samples gated 
on 
live, 
CD14-CD3-CD4- 
lymphocytes and frequency of 
total B cells. (B) B cell phenotypic 
profiles of individuals with heroin 
and cocaine use metric indicated. 
With severity of cocaine and 
heroin 
use 
indicated. 
(C) 
Activated B cell subsets among 
CD19+CD20low/neg B cells.      

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and CD183 (CXCR3) (Fig 2-1B)(22). The CD19+CD20low/neg population 
which is typically enriched for plasmablasts and pre-plasmablasts was 
phenotypically distinct, with increased CD27 and CD95 expression consistent 
with an activated B cell phenotype (Fig 2-1C). Although PWID are likely to have 
more frequent infectious insults and subsequent antigen-specific B cell expansion, 
the extent of B cell activation and expansion observed suggests that bystander 
activation, induced by inflammatory mediators (e.g., cytokines and TLR 
stimulation) is occurring. Such chronic bystander activation may deplete the naïve 
B cell pool and impact the ability of the B cells to respond appropriately upon 
encountering a pathogen. This B cell dysregulation likely underlies the numerous 
reports of decreased Ab response to vaccination, including Hepatitis A/B and S. 
pneumoniae among PWID and PWH(23-28).  
Systemic inflammation: a potential driver of B cell dysregulation and 
inflammatory diseases. Injection drug use has been associated with various 
inflammatory conditions including hypertension, cardiovascular disease, and 
kidney disease(29-32) as well as increased auto-reactive antibodies(33), 
suggestive of systemic inflammation and possible alteration in B cell tolerance. 
Our analysis of the plasma of individuals that inject heroin found significantly 
increased plasma TGF-α (~10-fold; an EGFR ligand), TNF-α (~5-fold, a potent 
inflammatory cytokine); IL-8 (~10-fold, a chemokine important for neutrophil 
migration, induction of phagocytosis and angiogenesis), and sCD40L (~5-fold; a 
potent inflammatory mediator produced primarily by platelets, which induces 
proliferation and class-switching of B cells) (Fig 2-2)(22). We also found plasma 
LPS (bacterial endotoxin) in several PWID, suggestive of bacterial infection or 
increased gut permeability. CRP was significantly increased in PWID, with 78% 
having CRP >3 mg/L (Fig 2-3). We performed global plasma proteomics analysis 
Figure 2-2. Plasma profile of PWID. Plasma cytokines, chemokines and growth factors were 
measured (A). (B) Select plots of individual analytes, each symbol represents an individual 
participant, the red lines indicate mean. 

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on a subset of PWID, those with the most 
distinct profiles from healthy controls 
(HC) to identify other factors and 
processes that may be altered and impact 
the B cell compartment. Our mass-
spectrometry based data-independent 
acquisition (DIA-MS) approach, which is 
a next generation quantitative proteomics 
technique that is ideally suited to analyze 
sample cohorts with minimal bias(34), 
determined the abundance of 721 
proteins, providing exceptional resolution 
of the plasma proteome. The plasma 
Figure 2-4. Plasma proteomics profile of PWID. Plasma
from healthy controls (HC n=5) and individuals who
inject heroin (PWID n=5) was analyzed by DIA-MS. (A)
PCA of samples based on expression of all proteins. (B)
Heatmap of significantly differentially expressed
proteins. .protein of interest 
Figure 2-3. Plasma CRP in PWID. Plasma high 
sensitivity (hs) CRP was determined by ELISA. 
Each 
symbol 
represents 
an 
individual 
participant. Dashed line = 3 mg/L. 

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proteomes of PWID were highly distinct from HC, and 86 proteins were 
significantly differently expressed (Fig 2-4). 
Many inflammatory factors were elevated including sCD14, sCD44, and LPS 
Binding Protein (LBP), consistent with previous reports(35, 36) (37). 
Additionally, many complement components and platelet/coagulation related 
factors including Pro-Platelet Basic Protein (PPBP) and Platelet Factor 4 (PF4), 
were elevated that have not been previously appreciated. These results are 
consistent with a chronic systemic inflammatory phenotype in PWID that may 
contribute to the observed B cell dysregulation. The elevated systemic 
inflammation in PWID that we observed is consistent with previous studies(15, 
38-42), and may contribute to co-morbidities as chronic inflammation is 
associated with cardiovascular disease, kidney disease, accelerated aging, and 
neurodegenerative diseases, all of which are increased among PWID(43-48). Our 
recent systematic review of opioid use disorder (OUD) related biomarkers(49) 
highlights that systemic, peripheral, and chronic inflammation is generally evident 
with OUD.  
 
 Need to understand and improve vaccine responses in PWID.  Previous 
studies have reported reduced vaccine immunogenicity among PWID, including 
for HCV, tetanus, influenza, and HIV (24, 50, 51). With the increased incidence 
of severe and chronic infections, inflammatory co-morbidities, and likelihood of 
diminished vaccine responses, a metric of B cell homeostasis and responsiveness 
would be advantageous in tailoring treatments to this population and monitoring 
clinical outcomes. In a small subset of PWID, using conventional univariate and 
multivariate machine learning analysis of 216 plasma and cellular features we 
identified 10 core systemic inflammatory and B cell parameters that distinguished 
PWID from healthy individuals (Fig 2-5). In a complementary meta-analysis of 9 
different Phase 1-2 HIV vaccine trials, to identify host features at baseline that 
were predictive of their antibody response following vaccination(52), we utilized 
SuperLearner, which estimates the performance of multiple machine learning 
Figure 2-5. PWID B cell and inflammatory core metric. A supervised recursive feature elimination approach 
was utilized to identify the top ten features (of 216 total) that when combined, classified the samples as PWID or 
HC with 100% accuracy using 10-fold cross-validation. Left. PCA plot of individual participants based on the ten 
features. Right. Identified features.  

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models, creating an ensemble analysis(53). We 
identified host features at baseline that were 
predictive of their Ab response following HIV 
vaccination.(52) This revealed several features 
associated with immune activation including 
white blood cells (WBC), neutrophils, and 
platelets that provide value in predicting 
vaccine response in this generally healthy 
population (Fig 2-6). It is anticipated these 
features may be further informative in PWID 
given their elevated systemic inflammation. An 
anticipated outcome of this study is to build 
upon the emergent data of PWID and vaccine 
responsiveness to monitor B cell homeostasis 
with the long-term goal of establishing 
clinically informative metrics that can be 
validated in future studies.  
This study will evaluate the impact of MET 
treatment on the immunogenicity of the FDA-
approved MPOX (Jynneos) and Pneumococcal conjugate (PCV21, Capvaxive) 
vaccines. Specifically, IgG antibody responses will be evaluated as secondary 
outcomes. Evaluation of responses to these vaccines enable the assessment of 
both T-cell dependent (Jynneos) and T-cell independent (PCV21, Capvaxive) B 
cell responses in PWID, and contribute to understanding of immunity to 
pathogens relevant to this population.  
2.3 
Metformin: Clinical use and potential adjunctive therapy for PWID. 
Numerous studies have cataloged the elevated inflammation and immune 
dysregulation in PWID and PWH, which was a critical first step in defining 
vulnerabilities in these populations; but now the field must advance to the 
development of interventions that can decrease inflammatory and infection-
related co-morbidities in these individuals. Metformin (dimethylguanide), the 
third most prescribed drug in the US, prescribed to >20 million individuals in the 
US annually, with an average cost of $0.17/day(54, 55), represents a potential and 
practical intervention to mechanistically define and treat the immune deficits in 
PWID and PWH. Its mechanism of action is not fully elucidated but appears to 
involve adenosine monophosphate–activated protein kinase (AMPK) activation 
and increased cytosolic redox due to inhibition of glycerol-3-phosphate 
dehydrogenase (GPD2), processes that can result in direct and indirect effects on 
lymphocyte regulation including through mTOR inhibition(56-59). Indeed, 
mTOR activation is a major regulator of T cell and B cell exhaustion(60-63), such 
as observed in PWID and PWH. Numerous human studies in diverse populations 
have shown MET can reduce systemic inflammation including decreasing 
Figure 2-6. Baseline determinants of antibody 
response to HIV vaccines. SuperLearner based 
analysis of 25 baseline features for predictors of 
IgG response to HIV vaccine components (gp120, 
gp140, gp41) among 831 individuals was 
performed and average rank of feature across 
analysis approaches indicated.  

MET-IH Version 7.0/ March 25, 2026 
MET-IH 7.0/ Page 21 of 88 
 
CRP(64-68), and within 4 weeks of MET start(68). Several human studies, 
although with small sample sizes (n=7-15) have demonstrated the ability of MET 
to enhance Ab responses to 
influenza vaccine in T2D and 
non-diabetic older adults, likely 
related to its ability to mitigate B 
cell and T cell exhaustion(69-
74). MET has been shown to 
improved COVID-19 
outcomes(75-79). Several studies 
have evaluated MET in PWH, 
although not in the context of 
substance use. The LILAC trial, 
which evaluated MET in non-
diabetic virologically suppressed 
and stable PWH, showed modest 
reduction in plasma analytes 
associated with systemic 
inflammation and peripheral 
blood activated T cells, and a 
more substantial MET effect in 
the gut, with reduction in 
activated mTOR and HIV-RNA/HIV-DNA ratios in colon-infiltrating CD4+ T 
cells, and increase of anti-inflammatory associated gut bacteria(56, 80). These 
were encouraging results, and it is likely that the beneficial effects of MET may 
have been more striking if the PWH cohort had been pre-selected for those with 
increased systemic inflammation. A reduction in exhausted peripheral blood 
CD4+ and CD8+ T cells occurred with MET treatment of non-diabetic PWH on 
ART(73), and perhaps is linked with the effects of MET on the increased gut 
inflammation and gut permeability that are thought to drive in-part the systemic 
inflammation observed in PWH and PWID(22, 81, 82). Our preliminary results 
indicate MET can improve B cell responsiveness to stimulation in vitro, including 
B cells from PWID, enhancing IgM and non-IgM (i.e. IgG/IgA) memory B cell 
development (Fig 2-7). Observing that metformin (MET) enhances memory B 
cell development in vitro (Fig 2-7), we evaluated the influence of MET on the 
transcriptome of B cells (Fig 2-8). MET treatment resulted in 44 differentially 
expressed genes, notably the upregulation of HIC1 which positively regulates 
plasma cell development. As expected MET resulted in significant inhibition of 
the mTOR pathway, but no significant effect on the AMPK pathway was 
Figure 2-7. MET enhances memory B cell 
development. B cells from HC (n=6), PWID (n=6), 
PWH (n=7), and PWH+PWID (n=3) were stimulated 
with anti-IgM & CD40L in vitro for 4 days in the 
presence of 1 mM MET and analyzed by flow 
cytometry.  

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observed (not shown). Further assessment of MET on the regulation of the B cell 
response is ongoing. 
Beyond the potential immune-related benefits of MET in PWID, emerging results 
from animal studies suggest MET may have beneficial effects in the setting of 
stimulant use, including decreasing cocaine seeking via an AMPK-mediated 
process in rats(83), neuroprotective and anti-inflammatory effects in 
methamphetamine treated rats(84), and reduction in methamphetamine-mediated 
gut microbiome dysbiosis, anxiety, and depression in mice(85). Subsequently, we 
will be testing MET in participants with a history of methamphetamine use. 
Overall, there is substantial evidence suggesting MET may have beneficial effects 
in PWID, justifying its evaluation in the proposed comprehensive bio-behavioral 
proof-of-concept mechanistic randomized clinical trial. Additionally, ongoing 
efforts to develop long-acting injectable MET(86, 87) could further elevate the 
long-term clinical potential impact of MET for at-risk populations.   
 
 
 
    Need to understand mucosal inflammation in PWID 
 
Increased mucosal inflammation and altered mucosal lymphocyte function in 
PWID remains poorly defined and likely contributes to increased risk of HIV 
acquisition(88), and in PWH exacerbation of associated co-morbidities(89). This 
mucosal inflammation seems to be in-part driven by the impact of infection and 
drug use on gut permeability and microbiome dysregulation(81, 82, 90-94). In 
addition to the well-established associations of rectal/genital inflammation and 
Figure 2-8. MET regulation of B cell responsiveness. Total PBMC from 3 PWID  (w/o HIV or HepC) were 
stimulated with IL-2, CD40L, and α-IgM for 4 d in the presence of 1mM MET and total B cells isolated 
by flow sorting and gene expression determined by RNA-Seq. (A) Volcano plot of RNA-Seq data. (B) 
Heatmap of differentially expressed genes. (C) mTOR signaling pathway IPA analysis.  

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HIV risk; oral inflammation, which is known to be substantial in individuals with 
methamphetamine use(95), a growing segment of PWID, likely drives the 
numerous oral pathologies observed. Oral inflammation is associated with the 
greater incidence and severity of periodontal disease(96, 97) and oropharyngeal 
cancers(98) that is observed in PWH. The potential for MET to reduce mucosal 
inflammation in PWID is substantiated by previous studies demonstrating its 
ability to reduce mucosal inflammation including in the oral cavity(99-102), and in 
PWH that MET reduces the frequency of CD4+ T cells and their expression of 
phosphorylated mTOR in the colon(56), and that it is suggested to mitigate gut 
microbiome dysbiosis to reduce mucosal inflammation(80, 81). Therefore, we 
posit that MET reduces oral and genital mucosal inflammation in PWID, which 
could mitigate HIV infectivity and associated co-morbidities.   
 
2.3.1 
The safety of study product administration 
Metformin was approved by the FDA in 1994 for the treatment of type 2 diabetes, 
and metformin ER (i.e. Glucophage XR) was approved by the FDA in 2000 for 
the treatment of type 2 diabetes. Additionally, it has been used off-label for 
treatment of various conditions (103) including polycystic ovarian syndrome, 
gestational diabetes, adjunct therapy in type 1 diabetes, and prevention of type 2 
diabetes in prediabetes.  It has been evaluated in numerous clinical trials in non-
diabetic populations including for lymphoma(104), schizophrenia(105), 
Alzheimer’s disease(106-108) (109-111), periodontal disease(112), and in people 
with HIV(73). 
We do not anticipate there are any additional safety risks of metformin to PWID 
and PWH who meet the inclusion and exclusion criteria for this study.  
The most common adverse reactions (>5%) associated with metformin ER are: 
diarrhea (9.6%) and nausea/vomiting (6.5%). And are more evident with doses of 
>1000 mg per day. 
Rare risks associated with metformin ER include lactic acidosis. Postmarketing 
cases of metformin-associated lactic acidosis have resulted in death, hypothermia, 
hypotension, and resistant bradyarrhythmias. The onset of metformin associated 
lactic acidosis is often subtle, accompanied only by nonspecific symptoms such as 
malaise, myalgias, respiratory distress, somnolence, and abdominal pain. 
Metformin associated lactic acidosis was characterized by elevated blood lactate 
levels (>5 mmol/Liter), anion gap acidosis (without evidence of ketonuria or 
ketonemia), an increased lactate/pyruvate ratio; and metformin plasma levels 
generally >5 mcg/mL. 
Risk factors for metformin-associated lactic acidosis include renal impairment, 
concomitant use of certain drugs (e.g. carbonic anhydrase inhibitors such as 
topiramate), age 65 years old or greater, having a radiological study with contrast, 
surgery and other procedures, hypoxic states (e.g., acute congestive heart failure), 

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excessive alcohol intake, and hepatic impairment. Subsequently, the 
inclusion/exclusion criteria, monitoring, and counseling of participants have been 
designed to exclude these risk factors and mitigate the potential risk of 
metformin-associated lactic acidosis within this trial.  
Other risks associated with metformin ER include decrease in vitamin B12 to 
subnormal levels ~7% of patients that appeared to be rapidly reversable with 
discontinuation of metformin ER. Subsequently, normal vitamin B12 levels at 
study enrollment are an inclusion criterion, and vitamin B12 monitoring are 
included in this trial to mitigate this potential risk. 
Hypoglycemia is a rare, but potential risk associated with metformin ER. 
Hypoglycemia does not occur in patients receiving metformin alone under usual 
circumstances of use, but could occur when caloric intake is deficient, when 
strenuous exercise is not compensated by caloric supplementation, or during 
concomitant use with other glucose-lowering agents (such as sulfonylureas and 
insulin) or ethanol. Elderly, debilitated, or malnourished patients, and those with 
adrenal or pituitary insufficiency or alcohol intoxication are particularly 
susceptible to hypoglycemic effects. Hypoglycemia may be difficult to recognize 
in the elderly, and in people who are taking beta-adrenergic blocking drugs. 
Subsequently, inclusion/exclusion criteria, monitoring, and counseling of 
participants have been designed to exclude these risk factors and mitigate the 
potential risk of metformin-associated hypoglycemia within this trial. 
 
2.3.2 
Other potential risks 
Blood collection (phlebotomy) may cause pain, bruising, fainting, and, rarely, 
infection at the site where the blood is taken. 
Optional rectal biopsy. Flexible sigmoidoscopy, the method used to take the rectal 
samples, may cause mild discomfort and pressure while the sigmoidoscope is 
moved along inside of the colon. There is a very slight risk of intestinal 
perforation, which carries the potential for peritonitis requiring additional medical 
intervention including surgery. These risks are associated with the sigmoidoscopy 
itself and are most commonly encountered when there is underlying colon 
abnormalities. The biopsies themselves pose minimal additional risk. 
Other risks: The medical tests performed as part of this research protocol may 
result in new diagnoses or abnormal values without clinical significance (“false 
positives”). Depending on the medical findings and consequences of being 
provided with the results of these tests, the study participant may view this as 
either a risk or a benefit. Any such information will be shared and discussed with 
the participant and, if requested by the participant, may be forwarded to the 
primary health care provider for further workup and management. 

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Participants in this study risk experiencing discrimination or other personal 
problems that may result from study participation itself; these are known 
collectively as negative social impacts. The study team is obliged to provide 
advocacy for and assistance to participants regarding these negative social 
impacts associated with trial.  
 
 

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3 
Study Design  
 
MET-IH is a 6 month, two-group, single blinded, placebo controlled, randomized 
study of MET treatment of people with a history of injection drug use, with or 
without HIV. Participants will be stratified ~1:1 with or without HIV, and 
randomized 1:1 to 16 weeks of treatment with MET (Group 1) or placebo (Group 2). 
Systemic and mucosal inflammation and antibody response to vaccines will be 
assessed.  
Persons aged 19-64 years with a history of injection drug use will be randomized to 
receive MET (Group 1, n=50) or placebo (Group 2, n=50), for a total of 100 
participants. 
 
 
 

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4 
Selection and Enrollment of Participants 
4.1 
Inclusion Criteria 
4.1.1  
Provision of signed and dated informed consent form 
4.1.2 
Stated willingness to comply with all study procedures and availability 
for the duration of the study 
4.1.3 
Aged 19 to 64 years old 
4.1.4 
Weight of at least 110 lbs 
4.1.5 
Body Mass Index 18.5-40. Enrollment of individuals with BMI >40, 
whom the study team assesses are in good health, may be considered 
by PSRT approval. 
4.1.6 
Willing to receive Jynneos and Capvaxive vaccines 
4.1.7 
Ability to take oral medication and be willing to adhere to the 
metformin treatment regimen 
4.1.8 
Injection opioid, amphetamine, and/or cocaine use with past 10 years 
(self-report) 
4.1.9 
Use of non-prescription opioid, amphetamine, and/or cocaine within 
the past 30 days (self-report) 
4.1.10 
Clinically confirmed urine drug screen for opioid, amphetamine, 
and/or cocaine within past 30 days 
4.1.11 
CRP  1 mg/L 
4.1.12 
glucose 70-180 mg/dL, non-fasting 
4.1.13 
HbA1c <6.4% 
4.1.14 
CD4 > 200 cells/ml 
4.1.15 
If PWH, HIV viral load < 200 copies / ml 
4.1.16 
If PWH, on anti-retroviral therapy (ART) for > 12 months 
 
 

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4.2 
Additional Inclusion Criteria for All Participants Providing Rectal 
Biopsy: 
1. HIV negative, as determined by fourth generation HIV screening test.  
2.  Willing to refrain from occasional over-the-counter use of aspirin and NSAID 
use for 72 hours before and after each study biopsy visit. NSAIDS should also 
be held for 72 hours after biopsy. 
3. Participants who were born female: negative serum or urine pregnancy test 
performed within 7 days prior to procedure 
4. Willing to abstain from insertion of anything (e.g., drug/medication, penis, 
object, sex toy, or enema including take-home enema) into the anorectum for 
72 hours before study drug dose and until 72 hours after each flexible 
sigmoidoscopy with biopsy collection, or one week after the study drug dose, 
whichever is later 
5. PT, PTT, and CBC within normal range performed within 30 days prior to 
procedure. 
6. Negative rectal swab for gonorrhea and chlamydia performed within 30 days 
prior to procedure 
7. Clinical abdominal exam performed within 42 days prior to the procedure and 
without any clinical findings which in the opinion of the clinician represents a 
contraindication to the biopsy procedure. 
 

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4.3 
Exclusion Criteria 
4.3.1 
Inability to give informed consent 
4.3.2 
Refusal or inability to have blood drawn 
4.3.3 
Bleeding disorder diagnosed by a doctor (eg, factor deficiency,      
coagulopathy, or platelet disorder requiring special precautions) 
4.3.4 
pregnant or nursing 
4.3.5 
diabetes mellitus 
4.3.6 
history of severe renal impairment and/or eGFR <60 mL/min/1.73m2 
 
 
> 10 days 
4.3.7 
creatine clearance <60 mL/min 
4.3.8 
history of liver disease 
4.3.9 
ALT/AST >3X upper limit of normal 
4.3.10 
Total Bilirubin >1.4 mg/dL 
4.3.11 
Albumin <3.5 g/dL 
4.3.12 
Prothrombin >1.5X upper limit of normal 
4.3.13 
AUDIT-C 8 
4.3.14 
hemoglobin <9.0 g/dL 
4.3.15 
absolute neutrophil <1,000/ml 
4.3.16 
platelet <100,000/ml 
4.3.17 
history of acute or chronic metabolic acidosis 
4.3.18 
anion gap >14 mEq/L OR 
anion gap >12 mEq/L when serum bicarbonate < 22 mEq/L 

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4.3.19 
serum lactate >2.2 mmol/L 
4.3.20 
serum vitamin B12 <250 pg/ml 
4.3.21 
history of chronic diarrhea 
4.3.22 
currently taking metformin or other diabetes medication 
4.3.23 
history of myocardial infarction, endocarditis, stroke, heart failure, 
chronic obstructive pulmonary disease, or sepsis 
4.3.24 
furosemide, nifedipine, ranolazine, vandetanib, or cimetidine use 
(current or within past 30 days) 
4.3.25 
active hepatitis B infection 
4.3.26 
Hepatitis C infection within 6 months of study entry. If previous 
history of hepatitis C, less than 6 months after completion of treatment 
with direct-acting antivirals 
4.3.27 
previous receipt of Jynneos or pneumococcal vaccine within past two 
years (self-report) 
4.3.28 
previous history of severe allergic reaction to metformin (self-report) 
4.3.29 
previous history of severe allergic reaction to previous dose of Jynneos 
or following exposure to any component of Jynneos (self-report) 
4.3.30 
previous history of severe allergic reaction to any component of 
Capvaxive or to diphtheria toxoid (self-report) 
4.3.31 
Donations of blood in the 8 weeks prior to enrollment which, 
combined with expected volumes to be drawn for this study, would 
exceed 450 mL in an 8 week period. 
4.3.32 
Participant has any medical, psychiatric, or social condition, or 
occupational or other responsibility that, in the judgment of the 
investigator, would interfere with, or serve as a contraindication to the 
planned procedure(s). 
 
4.4 
Additional Exclusion Criteria for All Participants Providing Rectal Biopsy: 
1. Abnormality of the colorectal mucosa, or significant colorectal symptom(s), 
which in the opinion of the clinician represents a contraindication to biopsy 

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(including but not limited to history of C. difficile colitis or enterocolitis within 3 
months prior to procedure, presence of unresolved injury, infectious or 
inflammatory condition of the local mucosa, and presence of hemorrhoids) 
2. Thrombocytopenia, platelets < 125,000 / mm3 
3. History of coronary artery disease, myocardial infarction, chronic obstructive 
pulmonary disease, chronic renal failure, decompensated cirrhosis, or any other 
condition that in the opinion of the investigator will compromise ability to 
participate in the study. 
4. Currently on anti-coagulation therapy (e.g. Coumadin/Warfarin). 
5. Receipt of any of the following medications within 30 days prior to procedure: 
systemic steroids (inhaled or nasal steroid therapy is permitted), interleukins, 
systemic interferons (e.g., local injection of interferon alpha for treatment of 
human papilloma virus is permitted) or systemic chemotherapy. 
6. History of intolerance, sensitivity, allergy, or anaphylaxis to benzodiazepines or 
other narcotics that may be used during the procedure 
7. Insertion of any foreign object into the anus within 3 days prior to procedure 
8. Participant has any medical, psychiatric, or social condition, or occupational or 
other responsibility that, in the judgment of the investigator, would interfere with, 
or serve as a contraindication to the planned procedure(s). 

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5 
Study Treatment 
5.1 
Regimens, Administration and Duration  
5.1.1 
Regimens/Administration 
Participants will be randomized 1:1 to: 
• Group 1: Metformin ER, 500 mg by mouth once per day with evening 
meal for Day 0 to Day 6, followed by Metformin ER, 1000 mg by mouth 
once per day with evening meal for Day 7 to Day 112 
• Group 2: Placebo, one tablet by mouth once per day with evening meal for 
Day 0 to Day 6, followed by Placebo, two tablets by mouth once per day 
with evening meal for Day 7 to Day 112 
5.1.2 
Duration 
Total study duration for all groups will be 6 months.  
5.2 
Study Product Description, Storage, and Preparation 
Metformin ER (MET) is supplied as white to off-white tablets, each containing 500 mg 
of metformin hydrochloride as the active ingredient. For the purposes of this study, MET 
will be over-encapsulated to accommodate a matching placebo. 
Store MET at 20°–25° C (68°–77° F); excursions permitted to 15°–30° C (59°–86° F). 
[See USP Controlled Room Temperature.] 
The placebo will be cellulose in identical capsules used to over-encapsulate MET. 
Store placebo at 20°–25° C (68°–77° F); excursions permitted to 15°–30° C (59°–86° F). 
Both MET and placebo will be dispensed in light-resistant containers 
5.3 
Pharmacy: Product Supply, Distribution, and Accountability  
5.3.1 
Study Product Acquisition/Distribution 
Metformin ER tablets, cellulose powder, and gelatin capsules are commercially 
available and will be purchased in bulk by UAB Hospital Investigational Pharmacy 
Services (IDS).  The metformin ER tablets and cellulose powder will be encapsulated 
using gelatin capsules by IDS and distributed to the site pharmacist. 

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5.3.2 
Study Product Accountability  
The site pharmacist is required to account for all study products.  An 
accountability record must be used to document receipt, management, and final 
disposal of study product.  The site pharmacist must also maintain a record of 
storage conditions for the study products. 
5.4 
Concomitant Medications 
Whenever a concomitant medication or study agent is initiated or a dose changed, 
investigators must review the concomitant medication’s and study agent’s most recent 
package insert, to obtain the most current information on drug interactions, contraindications, 
and precautions. 
5.4.1 
Prohibited Medications 
1. Non-study product diabetic medication  
2. Glyburide 
3. Furosemide 
4. Nifedipine 
5. Ranolazine 
6. Vandetanib 
7. Cimetidine 
 
5.4.2 
Precautionary Medications 
Interactions between the following medications and metformin have been 
previously noted, typically at metformin dose >1,000 mg/day, however 
commonly used together. Initiation and continuation of metformin for individuals 
taking these medications is at the discretion of study physicians.  
1. Dolutegravir  
2. Lamivudine 
 

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6. 
Clinical and Laboratory Evaluations 
6.1 
Schedule of Activities 
Table 6.1-1 Schedule of Activities 
*PWH only 
&PWH excluded 
^optional 
Visit number 
V0 
V1 
V2 
V3 
V4 
V5 
V6 
Study week 
 
0 
0.5 
1 
2 
3 
5 
Study Day  
(Extended Window Days) 
-14 to -3 
0 
2-4 
7-10 
12-16  
19-23 
30-40 
(+7) 
Procedure  
Screen 
Enrollment /MET start 
 
MET increase 
 
 
Vac 1 
In person (IP) / Virtual (V) 
IP 
IP 
V 
V 
IP 
V 
IP 
Study Procedures 
 
 
 
 
 
 
 
Assessment of 
Understanding 
 
x 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
Physical Exam 
X 
X 
 
 
 
 
X 
Concomitant medications 
X 
X 
 
 
X 
 
X 
ASSIST 
 
X 
 
 
 
 
X 
AUDIT-C 
X 
X 
 
 
X 
 
X 
BRAID 
 
X 
 
 
 
 
 
Health Survey 
Questionnaire 
X 
X 
X 
X 
X 
X 
X 
Adverse Events 
 
X 
 
 
X 
 
X 
MAAEs/SAEs 
 
X 
 
 
X 
 
X 
Adherence assessment/ 
metformin/placebo 
dispense 
 
X 
 
 
X 
 
X 
Acidosis / Hypoglycemia 
awareness counseling  
X 
X 
X 
X 
X 
X 
X 
Naloxone / overdose 
counseling 
 
X 
 
 
 
 
X 
Vaccination 
 
 
 
 
 
 
X 
Clinical Labs 
 
 
 
 
 
 
 
Pregnancy test (urine or 
serum) 
X 
X 
 
 
X 
 
X 
HIV-1/2 antibody& 
X 
 
 
 
 
 
 
HBsAg/anti-HCV 
X 
 
 
 
 
 
 
CRP 
X 
X 
 
 
X 
 
 
CD4 T cell count 
X 
 
 
 
X 
 
 
HIV PCR viral load* 
X 
X 
 
 
X 
 
X 
Complete blood count 
(CBC)/Differential 
X 
X 
 
 
X 
 
X 
Comprehensive metabolic 
panel 
X 
X 
 
 
X 
 
X 
HbA1c 
X 
 
 
 
X 
 
 
Serum vitamin B12 
X 
 
 
 
X 
 
 
Lactate  
X 
X 
 
 
X 
 
X 
Prothrombin 
X 
 
 
 
X 
 
 
Urine drug screen and Urine 
EtG 
X 
X 
 
 
X 
 
X 
Research Samples 
 
 
 
 
 
 
 
Blood (volume) 
 
X (51 ml) 
 
 
X (34 ml) 
 
X (34 ml) 
Saliva 
 
X 
 
 
 
 
 
Rectal secretion^ 
 
X 
 
 
 
 
 
Rectal biopsy^& 
 
 
 
 
 
 
 

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Table 6.1-1 Schedule of Activities- continued 
Visit number 
V7 
V8 
V9 
V10 
V11 
V12 
V13 
V14 
V15 
V16 
Study week 
6 
8 
9 
10 
11 
12 
13 
14 
15 
16 
Study Day  
(Extended Window Days) 
38-40 
54-58 
61-65 
(+ 7) 
68-72 
(+ 7) 
75-79 
82-86 
89-93 
(+ 7) 
96-100 
103-107 
110-114 
(+ 7) 
Procedure  
 
 
Vac 2 
 
 
 
 
 
 
MET 
stop 
In person (IP) / Virtual (V) 
V 
V 
IP 
IP 
V 
V 
IP 
V 
V 
IP 
Study Procedures 
 
 
 
 
 
 
 
 
 
 
Assessment of 
Understanding 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
 
 
 
 
 
 
 
 
 
 
Medical History 
 
 
 
 
 
 
 
 
 
 
Physical Exam 
 
 
X 
X 
 
 
X 
 
 
X 
Concomitant medications 
 
 
X 
X 
 
 
X 
 
 
X 
ASSIST 
 
 
 
X 
 
 
X 
 
 
 
AUDIT-C 
 
 
X 
X 
 
 
X 
 
 
X 
BRAID 
 
 
 
 
 
 
 
 
 
 
Health Survey 
Questionnaire 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events 
 
 
X 
X 
 
 
X 
 
 
X 
MAAEs/SAEs 
 
 
X 
X 
 
 
X 
 
 
X 
Adherence assessment/ 
metformin/placebo 
dispense 
 
 
X 
X 
 
 
X 
 
 
X 
Acidosis / Hypoglycemia 
awareness counseling  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Naloxone / overdose 
counseling 
 
 
 
X 
 
 
 
 
 
X 
Vaccination 
 
 
X 
 
 
 
 
 
 
 
Clinical Labs 
 
 
 
 
 
 
 
 
 
 
Pregnancy test (urine or 
serum) 
 
 
X 
 
 
 
X 
 
 
X 
HIV-1/2 antibody& 
 
 
 
 
 
 
X 
 
 
 
HBsAg/anti-HCV 
 
 
 
 
 
 
 
 
 
 
CRP 
 
 
X 
 
 
 
X 
 
 
X 
CD4 T cell count 
 
 
X 
 
 
 
 
 
 
X 
HIV PCR viral load* 
 
 
X 
 
 
 
 
 
 
X 
Complete blood count 
(CBC)/Differential 
 
 
X 
 
 
 
X 
 
 
X 
Comprehensive metabolic 
panel 
 
 
X 
 
 
 
X 
 
 
X 
HbA1c 
 
 
X 
 
 
 
X 
 
 
 
Serum vitamin B12 
 
 
X 
 
 
 
 
 
 
X 
Lactate  
 
 
X 
 
 
 
X 
 
 
X 
Prothrombin 
 
 
X 
 
 
 
 
 
 
X 
Urine drug screen and Urine 
EtG 
 
 
X 
X 
 
 
X 
 
 
X 
Research Samples 
 
 
 
 
 
 
 
 
 
 
Blood (volume) 
 
 
X (34 
ml) 
X (51 
ml) 
 
 
X (34 ml) 
 
 
X (51 ml) 
Saliva 
 
 
X 
 
 
 
 
 
 
X 
Rectal secretion^ 
 
 
 
 
 
 
 
 
 
X 
Rectal biopsy^& 
 
 
 
 
 
 
 
 
 
X 
*PWH only 
&PWH excluded 
^optional 
 
 

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Table 6.1-1 Schedule of Activities- continued 
Visit number 
V17 
V18 
V19 
V20 
V21 
V22 
VPD 
Study week 
17 
19 
20 
21 
22 
24 
 
Study Day  
(Extended Window Days) 
117-121 
131-135 
138-142 
(+ 7) 
145-149 
152-156 
166-170 
 
Procedure  
 
 
 
 
 
 
 
In person (IP) / Virtual (V) 
V 
V 
IP 
V 
V 
V 
IP 
Study Procedures 
 
 
 
 
 
 
 
Assessment of 
Understanding 
 
 
 
 
 
 
 
Informed Consent 
 
 
 
 
 
 
 
Medical History 
 
 
 
 
 
 
 
Physical Exam 
 
 
X 
 
 
 
X 
Concomitant medications 
 
 
X 
 
 
 
X 
ASSIST 
 
 
X 
 
 
 
X 
AUDIT-C 
 
 
X 
 
 
 
X 
BRAID 
 
 
X 
 
 
 
X 
Health Survey 
Questionnaire 
X 
X 
X 
X 
X 
X 
X 
Adverse Events 
 
 
X 
 
 
 
X 
MAAEs/SAEs 
 
 
X 
 
 
 
X 
Adherence assessment/ 
metformin/placebo 
dispense 
 
 
 
 
 
 
X 
Acidosis / Hypoglycemia 
awareness counseling  
X 
X 
X 
X 
X 
X 
X 
Naloxone / overdose 
counseling 
 
 
X 
 
 
 
X 
Vaccination 
 
 
 
 
 
 
 
Clinical Labs 
 
 
 
 
 
 
 
Pregnancy test (urine or 
serum) 
 
 
X 
 
 
 
X 
HIV-1/2 antibody& 
 
 
X 
 
 
 
X 
HBsAg/anti-HCV 
 
 
X 
 
 
 
X 
CRP 
 
 
X 
 
 
 
X 
CD4 T cell count 
 
 
X 
 
 
 
X 
HIV PCR viral load* 
 
 
X 
 
 
 
X 
Complete blood count 
(CBC)/Differential 
 
 
X 
 
 
 
X 
Comprehensive metabolic 
panel 
 
 
X 
 
 
 
X 
HbA1c 
 
 
X 
 
 
 
X 
Serum vitamin B12 
 
 
X 
 
 
 
X 
Lactate  
 
 
X 
 
 
 
X 
Prothrombin 
 
 
X 
 
 
 
X 
Urine drug screen and Urine 
EtG 
 
 
X 
 
 
 
X 
Research Samples 
 
 
 
 
 
 
 
Blood (volume) 
 
 
X (34 ml) 
 
 
 
X (34 ml) 
Saliva 
 
 
X 
 
 
 
X 
Rectal secretion^ 
 
 
 
 
 
 
 
Rectal biopsy^& 
 
 
 
 
 
 
 
*PWH only 
&PWH excluded 
^optional 
 

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6.2 
Timing of Study Activities 
6.2.1 Screening Activities 
Screening activities to determine eligibility must be completed within 45 days, and prior to 
study enrollment unless otherwise specified. In addition to data being collected on 
participants who enroll into the study, demographic, clinical, and laboratory data on 
screening failures will be captured in a Screening Failure Results form.  
6.2.2 Enrollment Activities 
Enrollment study activities must occur after screening activities unless otherwise specified. 
Study treatment should be initiated as soon as possible after enrollment activities have been 
completed and within 3 days of enrollment.  
6.2.3 Post-Enrollment Activities 
For all post-enrollment study activities, if a participant is unable to attend or complete a visit 
within the specified window, the visit can be completed during the extended window of an 
additional 7 days as indicated in SOA. If a visit cannot be completed within the specified 
window or extended window, every attempt should be made to bring or enable the participant 
in for an out-of-window visit rather than skip the visit, unless the next visit window has 
opened. 
If a participant experiences an acute inflammatory condition within 14 days prior to a 
scheduled vaccination visit (V6 and V9), the vaccination should be postponed for an 
additional 7 days from the initial scheduled visit. Examples of inflammatory conditions that 
would justify delaying vaccination visit include an infection requiring hospitalization, a 
systemic viral illness such as an influenza-like illness, a severe drug hypersensitivity 
reaction, myocardial infarction, fever on the day of visit (defined as T° >38.5°C), and major 
trauma. 
6.2.4 Event-Driven Activities 
Activities for Randomized Participants Who Do Not Start Study Treatment 
All eCRFs must be keyed for the period up to and including the enrollment visit. 
Premature Treatment Discontinuation Activities 
Participants who prematurely discontinue/modify any component of their study regimen 
should ideally complete the premature discontinuation study activities (listed in the (SOA)) 
prior to treatment discontinuation or within 7 days of discontinuation. The premature 
discontinuation study activities are listed in the SOA. Participants who discontinue the study 

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treatment will still be encouraged to remain in study follow-up through 24 weeks. Every 
reasonable effort should be made for the premature treatment discontinuation to include an 
in-person visit, however if not possible due to participant availability, completion of virtual 
visit elements should be attempted. 
 
Premature Study Discontinuation 
Participants who prematurely discontinue from the study will have the study discontinuation 
activities performed prior to being taken off the study (listed in the SOA). 
 
 

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6.3   Instructions for Study Activities 
All clinical and laboratory information required by this protocol is to be present in the source 
documents. 
All stated evaluations are to be recorded on the eCRF unless otherwise specified. 
The protocol team and/or study monitoring entity may determine that additional source data 
associated with procedures or evaluations performed per protocol should be entered into 
eCRFs so that the data can be used for analysis or to otherwise assist with interpretation of 
study findings. In such cases, sites will be officially instructed to enter the additional data 
into eCRFs from available source documentation. 
6.3.1 Documentation of HIV-1 status 
 
For participants with previous diagnosis of HIV-1, HIV-1 status should be confirmed 
from EHR, provider documentation, or confirmatory licensed rapid HIV or HIV 
enzyme or chemiluminescence immunoassay (E/CIA) test kit at any time prior to 
study enrollment.  
For participants without previous diagnosis of HIV-1, HIV-1 status should be 
determined by confirmatory licensed rapid HIV or HIV enzyme or 
chemiluminescence immunoassay (E/CIA) test kit, and as appropriate confirmed by a 
licensed Western blot or a second antibody test by a method other than the initial 
rapid HIV and/or E/CIA, or by HIV-1 antigen, plasma HIV-1 RNA viral load. 
6.3.2 Medical History 
The medical history must include all signs and symptoms regardless of grade for all 
clinical events and other diagnoses regardless of grade within the past 30 days. In 

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addition, the following diagnoses should be recorded regardless of when the diagnosis 
was made: 
• HIV status 
• Diabetes mellitus (exclusionary) 
• Bleeding disorder diagnosed by a doctor (e.g. factor deficiency, 
coagulopathy, or platelet disorder requiring special precautions) 
(exclusionary) 
• Pregnancy or nursing (exclusionary) 
• Severe renal impairment (exclusionary) 
• Liver disease (exclusionary) 
• Acute or chronic metabolic acidosis (exclusionary) 
• Chronic diarrhea (exclusionary) 
• Coronary heart disease, cardiovascular disease, myocardial infarction, 
stroke, heart failure (exclusionary) 
• Chronic obstructive pulmonary disease (exclusionary) 
• Sepsis (exclusionary) 
• Acute or chronic Hepatitis B virus (exclusionary) 
• HCV status, including history of spontaneous clearance or cure 
(exclusionary, if active) 
• Blood clots or clotting disorders (exclusionary) 
• Cerebrovascular accident 
• Orchiectomy or chemical castration 
• MPOX and Pneumococcal vaccination history (verbal history accepted) 
Any allergies to any medications and their formulations must also be documented. 
 
 

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6.3.3 Medication History  
A medication history must be present, including start and stop dates. The table below 
lists the medications that must be included in the history. 
Medication Category 
Complete History or 
Timeframe 
Diabetes-related medications 
Complete history, as available 
Antiretroviral therapy 
Complete ART history, as 
available 
Glyburide, Furosemide, nifedipine, ranolazine, 
vandetanib, ocimetidine 
Within 4 weeks prior to study 
enrollment 
Immune-based therapy 
Complete history, as available 
Study treatment 
Complete history 
Prescription drugs for treatment of opportunistic 
infections 
Within 4 weeks prior to study 
enrollment 
Prescription drugs for prophylaxis of opportunistic 
infections 
Within 4 weeks prior to study 
enrollment 
Anti-coagulation therapy 
Within 4 weeks prior to study 
enrollment 
Medications for opioid use disorder  
Complete history, as available 
Prescription drugs (other) 
Within 4 weeks prior to study 
enrollment 
Diet or weight loss therapy, either prescribed or 
over the counter 
Within 4 weeks prior to study 
enrollment 
Alternative therapies 
Within 4 weeks prior to study 
enrollment 
Dietary supplements 
Within 4 weeks prior to study 
enrollment 
Other category 
Within 4 weeks prior to study 
enrollment 
 

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6.3.4 Clinical Assessments 
 
Complete Physical Examination 
A complete physical examination at enrollment to include at a minimum an 
examination of the skin, head, mouth, and neck; auscultation of the chest; cardiac 
examination; abdominal examination; and examination of the lower extremities for 
edema. The complete physical examination will also include signs and symptoms, 
diagnoses, and vital signs (temperature, pulse, respiration rate, and blood pressure). 
Targeted Physical Examination  
A targeted physical examination at study visits after enrollment is to include vital 
signs (temperature, pulse, respiration rate, and blood pressure), and is to be driven by 
any previously identified or new adverse event, that the participant has experienced 
since the last visit or at this visit. 
Concomitant Medications 
Post-enrollment, all new or discontinued concomitant medications must be recorded, 
including but not limited to: 
• Glyburide, furosemide, nifedipine, ranolazine, vandetanib, and cimetidine 
• Anti-diabetic medications, antipsychotics, antidepressants, 
anticonvulsants/mood stabilizers, medications for opioid use disorder, 
dietary supplements, and weight loss agents (including over the counter) 
• Any drugs with anti-inflammatory properties 
• Any weight loss drugs (including non-prescription) 
• Antiviral therapy 
 
Study Treatment Modifications 
Record all study drug modifications including initial doses, participant-initiated, 
investigator-initiated, and/or protocol-mandated modifications, inadvertent and 
deliberate interruptions of more than 2 consecutive days since the last visit, and 
overdose. Study drug (metformin) dose may be decreased by study doctor due to 
tolerability.  Record any permanent discontinuation of treatment.  
 
6.3.5 Alcohol Use Disorders Identification Test - Consumption (AUDIT-C) 

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The AUDIT-C will be administered using Qualtrics at screening per the SOA.  
 
AUDIT-C  8 is exclusionary.  
 
6.3.6 Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) 
 
The ASSIST will be administered using Qualtrics at visits per the SOA. 
 
6.3.7 Behavioral Risk Assessment for Infectious Diseases (BRAID)  
 
The BRAID will be administered using Qualtrics at visits per the SOA. 
 
6.3.8  Health Survey Questionnaire (HSQ) 
 
The HSQ will be administered using Qualtrics at visits per the SOA. 
 
6.3.9 Adherence Assessment 
The adherence questionnaire will be administered per the SOA and the pill count 
eCRF will be completed per the SOA.  
NOTE: Site staff or investigators should review the results of the adherence 
assessments during the visit, and counsel participants on adherence if any concerns 
become apparent. 
 
6.3.10 Acidosis and Hypoglycemia Awareness Counseling  
Participants will be counseled on signs and symptoms of acidosis as indicated in SSP 
including confusion, difficulty talking, difficulty walking, memory loss, feeling intoxicated 
in the absence of drinking alcohol.  
Participants will be educated on signs and symptoms of hypoglycemia as indicated in SSP 
including tremor, palpitations, anxiety, sweating, hunger, and dizziness, weakness, 
drowsiness, confusion or altered mental status. 
 
6.3.11 Naloxone and Overdose Counseling 

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Participants will be provided naloxone and educated on overdose awareness as indicated in 
SSP including information on the Never Use Alone hotline and indications for naloxone for 
overdose reversal. Participants will be counseled on how to assess for signs of overdose, 
notify emergency medical services (911), and how to administer intranasal naloxone. 
Naloxone, and naloxone and overdose counseling can be provided outside of SOA at the 
discretion of study team.  
 
6.3.12 Vaccination  
At week 5 post enrollment visit (V6), participant will receive MPOX (Jynneos) 
vaccine and pneumococcal (Capvaxvie, PCV21) vaccine. 
Jynneos is administered subcutaneously.  
Capvaxvie is administered intramuscularly. 
At week 9 post enrollment visit (V9), participant will receive second MPOX 
(Jynneos) vaccine. 
 
6.3.13 Clinical Laboratory Evaluations 
At screening, enrollment, and post-enrollment all laboratory values must be recorded 
on the eCRF per the SOA. 
Hematology   
 
 
 
 
 
 
               
Complete blood count (CBC) with differential to include: hemoglobin, hematocrit, 
white blood cell count [WBC], differential WBC, absolute neutrophil count (ANC), 
platelets. Prothrombin, absolute CD4+ T cell count.  
Liver Function Tests   
 
 
 
 
 
 
          
Total and direct bilirubin, AST [SGOT], ALT [SGPT], alkaline phosphatase, total 
protein, albumin 
Chemistry  
 
 
 
 
 
 
 
 
     
Glucose, Sodium, potassium, chloride, phosphate, bicarbonate, serum creatinine, and 
blood urea nitrogen, anion gap, serum lactate, serum vitamin B12, HbA1c, C-reactive 
protein  
Calculated Creatinine Clearance 
 
 
 
 
 
 
Calculated creatinine clearance will be estimated by the CKD-EPI equation (a 
calculator is available at: https://qxmd.com/calculate/calculator_251/egfr-using-ckd-
epi). 

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Pregnancy Test  
 
 
 
 
 
 
 
 
 
For participants capable of becoming pregnant: serum or urine β-HCG (urine test 
must have a sensitivity of <25 mIU/mL) performed per SOA.  
Virological  
 
 
 
 
 
 
 
 
              
HBV surface antigen. HCV antibody followed by HCV RNA testing when HCV 
antibody testing is positive.  
For individuals without documented HIV infection, HIV enzyme immunoassay (EIA) 
or chemiluminescent microparticle immunoassay (CMIA).  
For individuals with documented HIV infect, plasma HIV-1 RNA.  
 
 
Urine Drug and Ethylglucuronide (EtG) Test 
The presence of the following substances: amphetamines, benzodiazepines, 
buprenorphine, cocaine, ecstasy (MDMA), marijuana (THC), methadone, 
methamphetamines, opiates/morphine, oxycodone, ethyl glucuronide (EtG), fentanyl, 
synthetic marijuana (K2), and tramadol will be recorded on eCRF. 
 
6.3.14 Stored Plasma/PBMC/Serum 
Per the SOA, plasma, viable PBMCs, and serum samples will be collected for clinical 
laboratory evaluations and research assays. Not shown is any additional blood volume 
that would be required if a safety lab needs to be repeated or if a serum pregnancy test 
needs to be performed. The additional blood volume would likely be minimal. The total 
blood volume drawn for each participant will not exceed 100 mL per visit, nor 500 mL 
in any 56-day (8-week) period, per American Red Cross guidelines for blood donation 
<https://www.redcrossblood.org/donate-blood/how-to-donate/eligibility-
requirements.html>. 
 
6.3.15 Stored Mucosal Secretion Samples 
Saliva  
 
 
 
 
 
 
 
 
 
  
Per the SOA, all participants will provide unstimulated saliva ~1 ml. Participants will 
be instructed to rinse their mouth using distilled water and after 2 minutes instructed to 
refrain from talking, sit comfortably, and accumulate saliva in their mouth before 
drooling into the collection tube until ~ 1 ml is collected.  
Optional Rectal secretions 
 
 
 
 
 
 
 
              
Per SOA, all participants will be offered the option of providing rectal secretion 
samples. For those participants Rectal fluid sampling (both sexes): For participants 
born female, a pregnancy test must be performed and be negative prior to any rectal 

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mucosal sampling. Persons who are NOT of reproductive potential due to having 
undergone total hysterectomy or bilateral oophorectomy (verified by medical records), 
are not required to undergo pregnancy testing. Rectal secretion sampling should be 
deferred if a participant is menstruating, but should be performed as soon as possible, 
if still within the visit window. In addition, rectal sampling will not be performed (or 
may be deferred to a later date if still within the visit window) if there is a 
contraindication to rectal secretion sampling, such as an active infection or 
inflammation of the colorectal area [such as a herpes simplex virus (HSV)-2 outbreak 
or inflamed hemorrhoids or colitis/diarrhea] or if the participant has any active genital 
tract infection (GTI).
For 72 hours prior to sample collection, participants should abstain from:
Receptive anal sex,
Insertion of any foreign object or substance into the anus (including but not limited 
to cleaning products [creams, gels, lotions, pads, etc.], lubricant, enemas, and douching 
even with water), and
Using perianal or intra-anal steroid or other anti-inflammatory cream in or around 
the anus.
Rectal fluid sampling will be performed by licensed and trained clinician using an 
anoscope and two pre-moistened sponges (e.g. Weck-Cel). The sponges will be 
inserted ~2-3 cm into the rectum and left in place for 5 minutes and then removed. 
Optional Rectal Biopsy
Per SOA, participants who have not been diagnosed with HIV will be offered the option 
to provide a rectal biopsy sample. A pre-procedural cleansing douche will be performed 
prior to baseline flexible sigmoidoscopies. The cleansing douche must be only tap 
water because it is demonstrated to be non-toxic to colorectal mucosa and doesn't have 
any impact on the ex-vivo explant HIV challenge assay. Colorectal tissue biopsy 
collection will be performed via flexible sigmoidoscopy by an experienced surgeon at 
UAB; up to 15 biopsies will be collected via a flexible sigmoidoscope. Collected 
biopsies will be used for the following:  tissue dissociation and isolation of mucosal 
mononuclear cells (MMC) for phenotypical analysis; ex vivo biopsy challenges with 
HIV-1 and subsequent p24 viral replication analysis and spatial transcriptomics. 
6.4
Drug Use Measures
6.4.1
Urine Drug Screen

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Urine Drug Screens (UDS) are collected at each in-person study visit. However, to 
minimize participant and staff burden we will accept non-study drug screen result 
from a test that was performed as part of standard of care or otherwise present in the 
participant’s medical record. Such extracted urine drug screen results may not include 
all substances tested using the study provide urine drug test cup. The study provide 
UDS will test for the presence of the following substances: amphetamines, 
benzodiazepines, buprenorphine, cocaine, ecstasy (MDMA), marijuana (THC), 
methadone, methamphetamines, opiates/morphine, oxycodone, ethyl glucuronide 
(EtG), fentanyl, synthetic marijuana (K2), and tramadol.  
6.4.2 Self-report and EHR Extracted Substances Use Measures 
 
 
Screening measures 
Eligibility is determined by either study-conducted UDS or EHR extracted UDS 
results.  Additional substance use measures for screening are collected by self-report. 
 
Enrollment and Post-Enrollment Substance Use Measures 
Alcohol and Drug use severity will be assessed with The Alcohol Use Disorders 
Identification Test-C (AUDIT-C) and the ASSIST. Results of AUDIT-C will be used 
to exclude those with unhealthy alcohol use as defined by an AUDIT C Score of 8 or 
greater. The ASSIST Survey will be used to identify substance use including opioids, 
stimulants, and other substances. 
 
 
 
 
 
 

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7 
Study Procedures 
All required clinic and laboratory procedures for each study visit are summarized 
in SOA. 
Optional rectal secretion sampling will be offered to all eligible participants. 
Optional rectal biopsy sampling will be offered to only eligible participants 
without HIV-1 infection. 
7.1 
Definition of “study day” and “study visit” 
Enrollment equals “study day 0” and is defined as the day of the first study-
product administration. A study visit may be conducted remotely, such as via 
phone, text message, email, or other electronic means, in lieu of, or in 
combination with, in-person visits. Procedures for a study visit can be completed 
over multiple days and should be completed within the visit window.  
7.2 
Screening (V0) 
Screening for eligibility will be performed after informed consent has been 
obtained and properly documented before enrollment. Screening evaluations and 
sample collection include medical history review, physical exam, review of 
concomitant medications, AUDIT-C, health survey, and any clinical laboratory 
tests that are needed to confirm eligibility, as detailed in the Schedule of 
Activities. Counseling on lactic acidosis and hypoglycemia awareness will be 
done. Urine drug screen and urine EtG will be performed. Persons of pregnancy 
potential will be given a pregnancy test. Screening procedures may occur over 
one or more visits. Additional assessments of health may be conducted at 
screening based on clinical judgment. 
Screening may be split into a two-step process over multiple visits. If a participant 
is deemed not eligible prior to completing all screening elements, remaining 
screening elements need not be completed.  
Individuals deemed not eligible will be informed that they do not meet the 
eligibility criteria for the study and will be referred for appropriate medical care, 
if necessary. 
A single out of range laboratory test may be repeated once 3-10 days following 
initial test at the discretion of the PI to determine eligibility.  
Potential participants may be rescreened once at the discretion of the PI.  

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7.3 
Enrollment visit (V1) 
An AoU will be completed and documented prior to enrollment. Records that 
document the reason screened participants did not enroll will be kept. 
If a participant has been screened and been found eligible, they will progress to 
the enrollment visit, to occur 3 to 28 days following the screening visit.  
The definition of enrollment in this study is the point of randomization.  That is, if 
a site successfully randomizes a participant in the randomization system, that 
participant is considered enrolled. 
All visit-specific administrative and regulatory, clinical, behavioral, and 
laboratory procedures outlined in the Schedule of Events (Table 6.1-1) should be 
conducted at this visit. 
Metformin (25 x 500 mg) or placebo (25 tablets) sufficient for 16 days will be 
dispensed by study pharmacist. Participants will be provided nutrition bars and 
reminded to take study drug with food at evening meal.       
Optional rectal secretion.  
7.4 
Post enrollment follow-up virtual visit (V2) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 2-3 days after enrollment visit that includes, abbreviated assessment of 
tolerance of study product, review of lactic acidosis and hypoglycemia awareness 
counselling, general health questions, and phone number of study coordinator and 
option to request call from study coordinator. If questionnaire is not completed 
within 2 days, study coordinator will call and text study participant.  
7.5 
Post enrollment follow-up virtual visit (V3) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 7 days after enrollment visit that includes, reminder to increase 
metformin (or placebo) dose, abbreviated assessment of tolerance of study 
product, review of lactic acidosis and hypoglycemia awareness counselling, 
general health questions, and phone number of study coordinator and option to 
request call from study coordinator. If questionnaire is not completed within 2 
days, study coordinator will call and text study participant.  

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7.6 
Post enrollment visit (V4) 
Two weeks following initiation of metformin (or placebo), participant will have 
an in-person visit.  
All visit-specific administrative and regulatory, clinical, behavioral, and 
laboratory procedures outlined in the Schedule of Activities should be conducted 
at this visit. 
Metformin (70 x 500 mg) or placebo (70 tablets) sufficient for 35 days will be 
dispensed by study pharmacist. Participants will be provided nutrition bars and 
reminded to take study drug with food at evening meal.            
7.7 
Post enrollment follow-up virtual visit (V5) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 19-23 days after enrollment visit that includes, abbreviated assessment 
of tolerance of study product, review of lactic acidosis and hypoglycemia 
awareness counselling, general health questions, and phone number of study 
coordinator and option to request call from study coordinator. If questionnaire is 
not completed within 7 days, study coordinator will call and text study participant.  
7.8 
Vaccination visit (V6)  
Five weeks following enrollment, participant will have an in-person visit.  
All visit-specific administrative and regulatory, clinical, behavioral, and 
laboratory procedures outlined in the Schedule of Activities should be conducted 
at this visit. 
Participant will receive Jynneos and Capvaxvie immunizations. 
Metformin (84 x 500 mg) or placebo (84 tablets) sufficient for 42 days will be 
dispensed by study pharmacist.  Participants will be provided nutrition bars and 
reminded to take study drug with food at evening meal.             
7.9 
Post vaccination virtual visit (V7) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 7 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product and vaccination, review of lactic acidosis and 
hypoglycemia awareness counselling, general health questions, and phone number 
of study coordinator and option to request call from study coordinator. If 

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questionnaire is not completed within 7 days, study coordinator will call and text 
study participant.  
7.10 
Post vaccination virtual visit (V8) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 21 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product and vaccination, review of lactic acidosis and 
hypoglycemia awareness counselling, general health questions, and phone number 
of study coordinator and option to request call from study coordinator. If 
questionnaire is not completed within 7 days, study coordinator will call and text 
study participant.  
7.11 
Vaccination visit (V9) 
Four weeks following first vaccination visit, participant will have an in-person 
visit.  
All visit-specific administrative and regulatory, clinical, behavioral, and 
laboratory procedures outlined in the Schedule of Activities should be conducted 
at this visit. 
Participant will receive Jynneos immunization. 
Metformin (34 x 500 mg) or placebo (34 tablets) sufficient for 17 days will be 
dispensed by study pharmacist. Participants will be provided nutrition bars and 
reminded to take study drug with food at evening meal.            
7.12 
Post vaccination visit (V10) 
On week following second vaccination visit, participant will have an in-person 
visit.  
All visit-specific administrative and regulatory, clinical, behavioral, and 
laboratory procedures outlined in the Schedule of Activities should be conducted 
at this visit. 
Metformin (70 x 500 mg) or placebo (70 tablets) sufficient for 35 days will be 
dispensed by study pharmacist. Participants will be provided nutrition bars and 
reminded to take study drug with food at evening meal.            

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7.13 
Virtual visit (V11) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 7 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product and vaccination, review of lactic acidosis and 
hypoglycemia awareness counselling, general health questions, and phone number 
of study coordinator and option to request call from study coordinator. If 
questionnaire is not completed within 7 days, study coordinator will call and text 
study participant.  
7.14 
Virtual visit (V12) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 14 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product and vaccination, review of lactic acidosis and 
hypoglycemia awareness counselling, general health questions, and phone number 
of study coordinator and option to request call from study coordinator. If 
questionnaire is not completed within 7 days, study coordinator will call and text 
study participant.  
7.15 
In person visit (V13) 
Four weeks following second vaccination visit, participant will have an in-person 
visit.  
All visit-specific administrative and regulatory, clinical, behavioral, and 
laboratory procedures outlined in the Schedule of Activities should be conducted 
at this visit. 
Metformin (70 x 500 mg) or placebo (70 tablets) sufficient for 35 days will be 
dispensed by study pharmacist. Participants will be provided nutrition bars and 
reminded to take study drug with food at evening meal.            
7.16 
Virtual visit (V14) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 7 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product, review of lactic acidosis and hypoglycemia 
awareness counselling, general health questions, and phone number of study 
coordinator and option to request call from study coordinator. If questionnaire is 
not completed within 7 days, study coordinator will call and text study participant.  

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7.17 
Virtual visit (V15) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 14 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product, review of lactic acidosis and hypoglycemia 
awareness counselling, general health questions, and phone number of study 
coordinator and option to request call from study coordinator. If questionnaire is 
not completed within 7 days, study coordinator will call and text study participant.  
7.18 
Metformin cessation visit (V16) 
Sixteen weeks following study enrollment, participant will have an in-person 
visit.  
All visit-specific administrative and regulatory, clinical, behavioral, and 
laboratory procedures outlined in the Schedule of Activities should be conducted 
at this visit. 
Any remaining study product will be collected.  
Optional rectal secretion.  
Optional rectal biopsy. Rectal biopsy can occur week 13 – week 16, prior to 
cessation of study product (metformin or placebo).  
7.19 
Post cessation virtual visit (V17) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 7 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product, review of lactic acidosis and hypoglycemia 
awareness counselling, general health questions, and phone number of study 
coordinator and option to request call from study coordinator. If questionnaire is 
not completed within 7 days, study coordinator will call and text study participant.  
7.20 
Post cessation virtual visit (V18) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 21 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product, review of lactic acidosis and hypoglycemia 
awareness counselling, general health questions, and phone number of study 
coordinator and option to request call from study coordinator. If questionnaire is 
not completed within 7 days, study coordinator will call and text study participant.  

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7.21 
Final in person visit (V19) 
Four weeks following metformin (or placebo) cessation, participant will have an 
in-person visit.  
All visit-specific administrative and regulatory, clinical, behavioral, and 
laboratory procedures outlined in the Schedule of Activities should be conducted 
at this visit. 
Optional rectal secretion.  
7.22 
Virtual visit (V20) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 7 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product, review of lactic acidosis and hypoglycemia 
awareness counselling, general health questions, and phone number of study 
coordinator and option to request call from study coordinator. If questionnaire is 
not completed within 7 days, study coordinator will call and text study participant.  
7.23 
Virtual visit (V21) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 14 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product, review of lactic acidosis and hypoglycemia 
awareness counselling, general health questions, and phone number of study 
coordinator and option to request call from study coordinator. If questionnaire is 
not completed within 7 days, study coordinator will call and text study participant.  
7.24 
Final virtual visit (V22) 
Participant will receive a link to Qualtrics-based questionnaire via text message 
and email 28 days after previous clinic visit that includes, abbreviated assessment 
of tolerance of study product, review of lactic acidosis and hypoglycemia 
awareness counselling, general health questions, and phone number of study 
coordinator and option to request call from study coordinator. If questionnaire is 
not completed within 7 days, study coordinator will call and text study participant.  
7.25 
Other visits 
Interim/unscheduled visits may be conducted to allow for repeat testing of 
laboratory results, conduct missed procedures/assessments, conduct safety 
assessments and safety labs.  

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7.26 
Total blood volume 
Required blood volumes per visit are shown in SOA. Not shown is any additional 
blood volume that would be required if a safety lab needs to be repeated or if a 
serum pregnancy test needs to be performed. The additional blood volume would 
likely be minimal. The total blood volume drawn for each participant will not 
exceed 500 mL in any 56-day (8-week) period, per American Red Cross 
guidelines for blood donation <https://www.redcrossblood.org/donate-blood/how-
to-donate/eligibility-requirements.html>. 
7.27 
Visit windows and missed visits 
The schedule of visits and evaluations performed at each visit and visit windows 
are shown in the Schedule of Activities. The procedures for documenting missed 
visits and out-of-window visits are described in the SSP. If the missed visit is one 
that requires safety assessments or safety labs, study staff should attempt to bring 
the participant in for an interim visit as soon as possible. If the participant does 
not respond to call or text, the study team will attempt to reach them via their 
emergency contact.   
7.28 
Early termination visit 
If a participant terminates participation in the study early for any reason, the PI 
should consider if the following assessments are appropriate: CBC with 
differential, serum chemistry, physical examination, and, if indicated, pregnancy 
test and assessment for new or unresolved AEs/intercurrent illnesses. If the PI has 
questions regarding a termination visit, they should consult with the PSRT. 
7.29 
AE contact 
If indicated, the participant may be asked to come in for a clinical assessment, 
which may also include referrals for an AE assessment.  
Record the participant’s responses to questions regarding any occurrence of the 
following events since the last visit/contact: 
• SAEs 
• AEs  
• Medically attended AEs (MAAEs), defined as any AEs leading to an 
unscheduled visit to a healthcare professional, which are reported regardless 
of their relationship to the study product(s) 

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• Pregnancies and outcomes, including congenital anomalies/birth defects. 
All such events will be recorded and AEs will be assessed for relationship to 
study products. 
If the site learns the participant is deceased, the site must attempt to learn the 
cause and date of death. 

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8 
Adverse Events and Study Monitoring 
8.1 
Definition of Adverse Events  
An adverse event (AE) is any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or diagnosis that occurs in a study 
participant during the conduct of the study REGARDLESS of the attribution (i.e., 
relationship of event to medical treatment/study product/device or 
procedure/intervention). This includes any occurrence that is new in onset or 
aggravated in severity or frequency from the baseline condition. 
8.2 
Adverse Event Collection Requirements for This Protocol  
All AEs must be recorded on the eCRFs if any of the following criteria have been 
met. 
• All Grade ≥3 AEs 
 
 
 
 
 
 
 
 
• All AEs that led to a change in study treatment/intervention regardless of grade 
• All MAAEs (defined as any AEs leading to an unscheduled visit with a 
healthcare professional in person or virtually/remotely) 
• All AEs meeting serious adverse event (SAE) definition or expedited adverse 
event (EAE) reporting requirement 
• The following AEs will be recorded at Grade ≥1: 
o 
Diarrhea 
o 
Nausea/vomiting 
o 
Change in laboratory values 
• The following AEs will be recorded at Grade ≥2: 
o Fatigue 
o Headache 
o Unintentional Weight Loss 
o Dyspnea 
• The following will be recorded regardless of grade: 

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o Coronary heart disease or other cardiovascular disease 
o Cancer (exclusive of basal/squamous cell skin cancer) 
o Diabetes mellitus or pre-diabetes 
o Any vascular events, including arterial events (strokes and myocardial 
infarctions) or deep venous thrombotic events (venous 
thromboembolism, deep venous thrombosis, retinal vein thrombosis, and 
pulmonary embolism) 
o Lactic acidosis 
o Hypoglycemia 
o New HIV diagnosis 
 
All AEs that are reported must have their severity graded. To grade AEs, refer to 
the Division of AIDS Table for Grading the Severity of Adult and Pediatric 
Adverse Events (DAIDS AE Grading Table), corrected Version 2.1, July 2017, 
which can be found on the DAIDS RSC website at 
https://rsc.niaid.nih.gov/clinical-research-sites/daids-adverse-event-grading-
tables. 
 
Serious Adverse Events (SAEs) 
 
An SAE is defined as any untoward medical occurrence that: 
• Results in death 
• Is life-threatening 
• Requires inpatient hospitalization or prolongation of existing hospitalization 
• Results in persistent or significant disability/incapacity 
• Is a congenital anomaly/birth defect 
• Is an important medical event that may not be immediately life-threatening or 
result in death or hospitalization but may jeopardize the patient or may require 
intervention to prevent one of the other outcomes listed in the definition above). 
 

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8.3 
Expedited Adverse Event Reporting  
All serious adverse events must be reported to the IRB according to the regulatory 
requirements. All serious adverse events must be followed to satisfactory 
resolution or until the investigator deems the event to be chronic or not clinically 
significant, the event is considered to be stable, or the participant is lost to follow-
up. 
8.4 
Study Monitoring 
 
Members of the protocol team will monitor the conduct and safety of the study via 
 
regular pooled summaries of accrual, baseline characteristics, treatment and study 
 
discontinuation, and data completeness. 
 
During the study, the safety and tolerability of the study medication will be monitored 
 
by toxicity reports presenting laboratory and clinical data. The subset of the team 
 
receiving these reports (as per the specifications in the DSMP) will discuss these 
 
reports on regularly scheduled conference calls or by e-mail.  
 
8.5 
Serious adverse events (SAEs) 
The term “serious adverse event” (SAE) is defined in 21 CFR 312.32 as follows: 
“An adverse event or suspected adverse reaction is considered ‘serious’ if, in the 
view of either the investigator or the sponsor, it results in any of the following 
outcomes: 
• Death 
• A life-threatening adverse event 
• Inpatient hospitalization or prolongation of existing hospitalization 
• A persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions 
• Congenital anomaly/birth defect 
• Important medical events that may not result in death, be life-threatening, or 
require hospitalization may be considered serious when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and 
may require medical or surgical intervention to prevent one of the outcomes 
listed in this definition. Examples of such medical events include allergic 

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bronchospasm requiring intensive treatment in an emergency room or at 
home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse.” 
“Life-threatening” refers to an AE that, at occurrence, represents an immediate 
risk of death to the participant. A hospital admission for an elective procedure is 
not considered an SAE. 
8.6 
Safety monitoring 
8.6.1 
Protocol Safety Review Team (PSRT) 
The PSRT comprises: 
• Medical Officer (MO), Sonya Heath, MD 
• Study PI:  Ellen Eaton, MD, MSPH 
• Study Co-PI:  James Kobie, PhD 
• Clinic Director: Paul Goepfert, MD 
• Lead Study Nurse: Heather Logan, DNP, CRNP 
The clinician members of the PSRT are responsible for decisions related to 
participant safety. The PSRT will review study safety information on a weekly 
basis through 6 weeks after the last participant completes study product. After this 
timepoint, less frequent safety reviews may be conducted at the discretion of the 
PSRT. 
8.6.2 
Data Safety Monitoring Board (DSMB) 
The DSMB is a multidisciplinary group consisting of biostatisticians, clinicians, 
and experts in HIV and substance use research that, collectively, has experience in 
the conduct and monitoring of trials. Members of the DSMB are not directly 
affiliated with the protocol under review. 
The DSMB reviews safety data (including cumulative AEs, laboratory safety data, 
and individual SAE reports) approximately every 4 months. The DSMB conducts 
additional special reviews at the request of the PSRT. 
Study investigators will receive DSMB summary minutes. DSMB summary 
minutes will be provided to Institutional Review Board (IRB)/Ethics Committee 
(EC) and any applicable Regulatory Authority (RA) as appropriate. 

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8.7 
Safety reviews 
8.7.1 
Safety evaluations  
Safety pause and prompt PSRT AE review 
The PSRT (see Section 8.6.1) will closely monitor participant safety. The trial can 
be paused at any time for any reason by the PSRT. When a trial is placed on 
safety pause, all enrollment will be held until further notice. The AEs that will 
lead to a safety pause or prompt the PSRT AE review are summarized in Table 8-
7. Enrollment and/or study product administration may be suspended for safety 
concerns other than those described in the table or before pause rules are met, if, 
in the judgment of the PSRT, participant safety may be threatened. Criteria for an 
individual participant’s departure from the schedule of vaccinations are listed in 
Section 10.1. 
Table 8-7 Pause Rules 
Event / relationship 
to study product 
Severity 
grade 
Study Team Actions 
PRST Actions 
SAE 
Related 
4 
Phone: 205-934-3411 and page Dr. 
Eaton and Dr. Heath immediately  
Email: metihpsrt@uabmc.edu  
Submit CRFs immediately 
Immediate pause 
for PSRT review 
SAE 
Related 
3, 2, or 1 
Email: metihpsrt@uabmc.edu  
Submit CRFs immediately 
Prompt PSRT AE 
review and 
consideration of 
pause 
AE 
Related 
4 or 3 
Email: metihpsrt@uabmc.edu 
Submit CRFs immediately 
Prompt PSRT AE 
review and 
consideration of 
pause 
 
Unrelated Participant Death: During working hours (local time) study team will 
email the PSRT upon learning of any unrelated participant deaths. The study team 
will also email the PSRT and immediately submit CRFs.  
 
8.7.2 
Plan for review of safety pause rules 
For all safety pauses, the PI will notify the PSRT, NIDA, DSMB, and IRB. 
Once a trial is paused, the PSRT will review safety data and decide whether the 
pause can be lifted or whether permanent discontinuation of study product is 
appropriate, consulting the DSMB if necessary. 

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If an immediate PSRT notification or prompt PSRT AE review is triggered, the 
study team will notify the PSRT as soon as possible during working hours (local 
time) or, if the information was received during off hours, by the morning of the 
next workday. If a prompt PSRT AE review cannot be completed within 72 hours 
of notification (excluding weekends and US federal holidays), an automatic safety 
pause will occur. 
The study team will submit to the IRB/EC and any applicable RA protocol-related 
safety information.  The study team must also follow all applicable RA reporting 
requirements. 
8.8 
Study termination 
This study may be terminated early by the determination of the PSRT, the 
National Institutes of Health (NIH), the United States Department of Health and 
Human Services (HHS) Office for Human Research Protections (OHRP), or the 
DSMB. In addition, the conduct of this study may be terminated by the 
determination of the IRB/EC and any applicable RA. 

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9 
Clinical Management Issues 
9.1 
Toxicity  
9.1.1 General instructions for management of Grades 1-4 AEs are provided in this section. 
Refer to section 9.2 and further for information on specific toxicities. 
9.1.2 Grade 1 or 2 
Participants who develop a Grade 1 or 2 AE or toxicity thought to be related to study-
supplied metformin may continue metformin at the discretion of the principal 
investigator and will be followed carefully until resolution or stabilization. 
If the principal investigator or participant chooses to discontinue any study-supplied 
metformin, the site should notify the medical monitor within 72 hours. The premature 
treatment/study discontinuation evaluations must be completed, and then the 
participant may remain in follow-up off study treatment. 
9.1.3 Grade 3 or 4 
For Grade 3 or 4 AE or toxicity that is related to study-supplied metformin, 
metformin must be discontinued, participants must be followed carefully, and the 
medical monitor should be notified within 72 hours. 
The participant should be followed carefully until resolution or stabilization. 
The premature study discontinuation evaluations must be completed, and then the 
participant may remain in follow-up off study treatment  
9.1.4 Serious Adverse Event 
For Grade 3 or 4 SAE (as defined in section 8.2) that is related to study-supplied 
metformin, metformin must be permanently discontinued and the medical monitor 
should be notified within 72 hours. The participant should be followed until the AE 
has decreased to Grade ≤2. The premature study discontinuation evaluations must be 
completed, and then the participant may remain in follow-up off study treatment 
9.2 
Hyperlactatemia /Lactic Acidosis 
The following case definition of symptomatic hyperlactatemia/lactic acidosis will be 
used in this protocol: 
New, otherwise unexplained and persistent (≥2 weeks) occurrence of one or more 
of the following symptoms: 

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• Nausea and vomiting 
• Abdominal pain or gastric discomfort 
• Abdominal distention 
• Elevated liver function tests 
• Unexplained fatigue 
• Dyspnea 
• Concern for hyperlactatemia/lactic acidosis should be confirmed by 
measurement of serum lactate level at the discretion of the principal 
investigator. If a lactate level >2.2 mmol/L is confirmed, participants 
should immediately discontinue their study medications and the medical 
monitor should be contacted to plan further treatment. 
 

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10 
Criteria for Discontinuation  
10.1 
Permanent and Premature Study Product Discontinuation 
Under certain circumstances, study product will be permanently discontinued for an 
individual participant. Specific events that will result in stopping product for an 
individual participant include: 
• Drug-related toxicity (see section 9.1) 
• Request by participant to terminate study product 
• Clinical reasons believed life-threatening by the physician, even if not addressed in 
section 9.0 of the protocol. 
• Requirement for prohibited concomitant medications. 
• At the discretion of the IRB, NIDA, Office for Human Research Protections (OHRP), 
other government agencies as part of their duties, medical monitor, or principal 
investigator. 
• SAE that is subsequently considered to be related to metformin 
• Grade 3 or Grade 4 AE assessed as related to metformin 
• glucose outside of range (70-180 mg/dL, non-fasting), that is not resolved within 1 
week 
• HbA1c >6.4% 
• eGFR <60 mL/min/1.73m2 
• creatine clearance <60 mL/min 
• ALT/AST >3X upper limit of normal, or >2X increase of ALT/AST from baseline 
• Total Bilirubin >1.4 mg/dL 
• Albumin <3.5 g/dL 
• Prothrombin >1.5X upper limit of normal 
• serum bicarbonate <22mEq/L 
• anion gap >14 mEq/L OR 

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anion gap >12 mEq/L when serum bicarbonate <22 mEq/L 
• serum lactate >2.2 mmol/L 
• Pregnancy (regardless of outcome). 
• PI or medical monitor assessment that it is not in the best interest of the participant to 
continue receiving study product. 
• A participant that was previously subjected to study product discontinuation due to 
previous (not current) criteria for discontinuation may re-enroll in the study at the 
discretion of the PI.  
10.2 
Premature Study Discontinuation 
• Failure by the participant to attend 3 consecutive in-person clinic visits. 
• Participant repeatedly does not adhere to study products as prescribed, per the 
principal investigator’s discretion. 
• Request by the participant to withdraw. 
• Failure/inability of participant to attend study visits. 
• Request of the primary care provider if she or he thinks the study is no longer in the 
best interest of the participant. 
• At the discretion of the IRB, NIDA, Office for Human Research Protections (OHRP), 
other government agencies as part of their duties, medical monitor, or principal 
investigator. 
 

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11 
Statistical Considerations 
11.1 
General Design Issues 
The purpose of this randomized clinical trial (RCT) is to determine the potential of 
metformin to reduce systemic inflammation and mitigate immune dysregulation among 
people who inject drugs (PWID). The study’s primary objective is to evaluate if in people 
with a history of injection drug use does MET result in a change (decrease in CRP) in 
systemic inflammation as measured by CRP compared to placebo over 20 weeks. It is of 
secondary interest to compare MET versus placebo for change in B cell exhaustion, T 
cell exhaustion and improvement in antibody responses to vaccination over 20 weeks.  
11.2 
Outcome Measures 
11.2.1 
Primary Outcome Measures 
Change (absolute) in CRP (mg/L) from enrollment to week 16. 
 
11.2.2 
Secondary Outcome Measures 
11.2.2.1 
Change (percentage) in peripheral blood exhausted B cells (% of total B cells) 
from enrollment to week 16. 
11.2.2.2 
Change (percentage) in peripheral blood exhausted T cells (% of total T cells) 
from enrollment to week 16. 
11.2.2.3     Change (absolute) in vaccine-specific IgG antibody titer from enrollment to week  
      9. 
11.2.2.4     Change (absolute) in vaccine-specific IgG antibody titer from enrollment to week 
13. 
 
11.2.3 
Exploratory Outcome Measures 
11.2.3.1 
Change in oral and/or genital mucosal inflammatory cytokines from enrollment to 
week 16. 
11.2.3.2 
Differences in rectal lymphocyte characteristics at week 16. 
 
 

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11.3 
Randomization and Stratification 
All eligible participants will be randomized using a 1:1 ratio to one of two study groups: 
MET or placebo, using permutated blocks. Participants will be stratified at randomization 
by sex assigned at birth and by HIV infection status. Participant enrollment targets are 
50% participants assigned female sex at birth and 50% persons with HIV.  
11.4 
Sample Size and Accrual 
The proposed sample size is 100 participants (50 per arm).  It is anticipated that the initial 
10 participants will be enrolled within 4 months, followed by a 2-month planned safety 
pause in new enrollment, and then commencing with enrollment of ~3-4 participants per 
month, with final enrollment expected to be completed by M36, and final study visit 
completed by M42. 
Based on previous studies that evaluated our planned endpoints in response to MET, a 
sample size was determined For the Primary Objective using a 2-way ANOVA with an 
interaction between treatment (MET/Placebo) and HIV status (+/-) with change in CRP 
of 1.56 from week 0 to 16(64, 65). Allowing for a significant interaction at the a=0.10 
level; an adjusted a=0.0167, for the 3 direct comparisons of interest: HIV+/MET vs 
HIV+/Placebo, HIV-/MET vs HIV-/Placebo, and HIV+/MET vs HIV-/MET; has 80% 
power to detect a change in CRP of 1.56 (SD 2.12) with 80.1% power with an n=80 
(n=100, with 20% attrition). Without the interaction, comparing only MET vs Placebo 
and HIV+ vs HIV-, an a=0.025, has power of 84.0%, or 80.3% power for a D=1.49, or 
D=1.56 with an increased SD=1.56 (JMP v17). Use of repeated measures mixed model 
will allow for use of results collected for all participants that terminate prior to week 16, 
the mixed model approach should also provide additional protection by leveraging within 
participant correlation. In addition, adjustment for a priori covariates known to be 
associated with CRP will also serve to increase power. Table 11-4-1 shows detectable 
differences for Secondary Outcomes: exhausted B cell(69) and T cell(73) subsets, from 
baseline to WK16, as well as differences in IgG plasma antibody response to 
vaccination(69, 70) from WK5 to WK9 (Capvaxvie, PCV21) or WK13 (Jynneos). 
Exploratory Objectives are not powered for confirmatory statistical comparisons, 
however, the goal with this research is to estimate variables associated with mucosal 
inflammation and immunity. The sample size of 20-50 for these analyses was selected 
based upon feasibility but aligns well with statistical recommendations for pilot trials to 
determine precise estimates(113). 
 
 
 
 
Table 11-4-1. Power considerations for 
secondary endpoints. α=0.05 

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11.5 
Data and Safety Monitoring 
The Data Safety Monitoring Board (DSMB) will undertake reviews of interim data to 
ensure safety of study participants and to possibly recommend termination or modification of 
the study. At DSMB reviews, data will be considered as detailed in section 8.6. 
At each interim review, the DSMB will review unblinded summaries, by randomized arm, of 
study status and, including loss to follow-up (LTFU), treatment discontinuations, and safety, 
including adverse events. Interim efficacy analyses are not planned. 
There are no pre-specified stopping guidelines for this study, but the DSMB should consider 
possible modifications or termination on bases of safety concerns or operational futility. With 
regards to safety, if 20% or more of participants in Group 1 (metformin treatment) have 
treatment-related Grade 2 AEs, this would be concerning to the study team. With respect to 
operational futility the DSMB should consider the overall and within arm LTFU rates. As a 
benchmark, an overall LTFU rate of 40% or higher would be cause for concern, particularly 
if a higher rate is observed in Group 1 (metformin treatment). 
 
11.6 
Analyses 
 
They study statistician, Dr. Hartzes will analyze the data using SAS or SPSS for quantitative 
analytical software.  
 
Participant characteristics will be presented as mean±SD, mean (median) and n (%), as 
applicable. Baseline comparability and early terminations will be assessed by parametric and 
nonparametric single-factor (treatment group) ANOVA or chi-square analyses, as applicable. 
Any baseline differences will be assessed for degree of imbalance and included as model 
covariates, as appropriate. The primary endpoints will be analyzed with linear mixed model 
regression, adjusting for CRP at baseline, HIV status (the stratification variable), with 
participant as a repeated effect. The effects considered will be treatment (MET/Placebo), 
HIV status, and the interaction between treatment and HIV status, creating 4 groups. If the 
significance level for the interaction does not correspond to an alpha value of 0.10 or less for 
Type I error, it will be removed to assess the differences between MET and placebo and HIV 
status as main effects (at an adjusted a=0.025). The study is not powered for the interaction 
but rather for the 2 main-effect comparisons. If the interaction is significant, the 3 direct 
comparisons of interest: HIV+/MET vs HIV+/Placebo, HIV-/MET vs HIV-/Placebo, and 
HIV+/MET vs HIV-/MET, will be compared at an adjusted a=0.0167. Fixed effects for 
primary endpoints will also include an interaction between treatment arm and time. This will 
allow estimation of the mean difference in change between arms and 95% confidence 
intervals. Using a unified analytic approach allows us to make sure that the structure of the 
different analyses is parallel, by including covariates in the same way, and reduces the 
complexity of comparing the results across different variables. If there are a large number of 
potential confounders at baseline, we will use the propensity score approach to ensure that 

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the estimates are unbiased while minimizing the degrees of freedom required. Sex will be 
considered as a biological variable, through efforts to equally recruit study participants and 
post-hoc analysis of the association of sex and immunological variables. While not powered 
for confirmatory subgroup analyses, the sample size of 100 should allow for investigation of 
binary covariates such as polysubstance use features, and exploratory endpoints such as 
PROs, and association with primary and secondary endpoints. The mixed model will also 
allow for estimation and assessment of time-vary covariates(113). Due to the exploratory 
nature of select advanced ‘omics analyses, a less conservative FDR of 0.01 will be used to 
generate hypotheses and identify areas for further investigation. Analyses will be carried out 
using SAS and R, p-values<0.05 will be considered meaningful, unless otherwise noted.  
 

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12 
Pharmacology Plan 
Blood will be stored to measure concentrations of MET at enrollment, week 5, week 16, and 
week 20 (or premature discontinuation) from Group 1 to inform study adherence 
interpretation.   
 
 
 
 
 
 
 
 

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13 
Data Collection and Monitoring 
13.1 
Records to Be Kept 
eCRF screens will be used for data entry. Participants must not be identified by 
name on any data submitted to the DSMB. Participants will be identified by the 
patient identification number (PID). 
13.2 
Role of Data Management 
 
The Principal Investigator is responsible for overseeing the data collection process, 
 
ensuring that all required information is captured accurately, efficiently, and timely 
 
throughout the trial. All source documents should be completed in a manner to ensure 
 
the Principles for Data Integrity are upheld. 
13.3 
Clinical Site Monitoring and Record Availability 
Clinical site monitoring is conducted to ensure that the rights and well-being of 
participants are protected, that the reported trial data are accurate, complete and 
verifiable, and the conduct of the trial is in compliance with the currently approved 
protocol/amendments and applicable regulatory requirements.  
 
Monitoring for this study will be performed by the UAB Center for Clinical and 
Translational Science (CCTS) Clinical Research Support Program and may be 
conducted onsite or remotely. Monitoring visits will occur after the first 5 enrollments 
and quarterly thereafter, and may include but are not limited to, review of regulatory 
files, accountability records, case report forms, consent documents, medical and 
laboratory reports, study product storage records, training records, and protocol and 
GCP compliance.  Upon conclusion of the monitoring visit, monitoring reports will 
be distributed to the Principal Investigator and relevant site staff. 
 
13.4 
Reporting Protocol Deviations 
The principal investigator and staff are responsible for identifying and reporting 
deviations. If protocol deviations are identified, corrective actions are to be developed by 
the site and implemented promptly. Protocol deviations must be reported to the IRB/EC 
per their guidelines. 
Refer to the SSP for the definition of protocol deviation and instructions for completing 
the study protocol deviation eCRF. 
 

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14 
Participants 
14.1 
IRB Review and Informed Consent 
This protocol and the informed consent document (Appendix A) and any subsequent 
modifications will be reviewed and approved by the IRB or PI for oversight of the study. 
A signed consent form will be obtained from the participant (or parent, legal guardian, or 
person with power of attorney for participants who cannot consent for themselves). The 
consent form will describe the purpose of the study, the procedures to be followed, and 
the risks and benefits of participation. A copy of the consent form will be given to the 
participant and this fact will be documented in the participant’s record. 
14.2 
Participant Confidentiality 
All laboratory specimens, evaluation forms, reports, and other records that leave the site 
will be identified by coded number only to maintain participant confidentiality. All 
records will be kept locked. All computer entry and networking programs will be done 
with coded numbers only. Clinical information will not be released without written 
permission of the participant, except as necessary for monitoring by the IRB/EC, NIDA, 
OHRP, or other local, US, and non-US regulatory entities as part of their duties. 
14.3 
Study Discontinuation 
The study may be discontinued at any time by the PI, IRB/EC, NIDA, OHRP, or other 
country-specific government agencies as part of their duties to ensure that research 
participants are protected. 
 
 

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15 
Data Sharing, Public Access, and Publications 
This study will comply with the NIH Data Sharing Policy and Implementation Guidance 
(https://sharing.nih.gov/data-management-and-sharing-policy).  Investigators will register 
and report results of the trial in ClinicalTrials.gov, consistent with the requirements of the 
Policy on the Dissemination of NIH-Funded Clinical Trial Information and the Clinical 
Trials Registration (https://grants.nih.gov/policy-and-compliance/policy-topics/clinical-
trials/reporting/nih-policy). 
Primary data for this study will be available to the public in the NIDA data repository, 
per NIDA policy (https://datashare.nida.nih.gov/). 
The primary outcome(s) publication will be included along with study underlying 
primary data in the data share repository, and it will also be deposited in PubMed Central 
(https://pmc.ncbi.nlm.nih.gov/) per NIH Policy (https://sharing.nih.gov/public-access-
policy). 
We will disseminate findings through abstracts for presentation at academic conferences, 
manuscripts for publication in interdisciplinary high-impact peer-reviewed journals, and 
grant proposals for subsequent studies. We anticipate this project will result in the 
production of at least three manuscripts. 
We will also disseminate findings from the study to members of the community so that 
trial results can inform policies and procedures. We will seek guidance from community 
stakeholders on the most effective ways to share findings with members of their 
organizations and the community at-large. 
 

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16 
Biohazard Containment 
As the transmission of HIV and other blood-borne pathogens can occur through contact 
with contaminated needles, blood, and blood products, appropriate blood and secretion 
precautions will be employed by all personnel in the drawing of blood and shipping and 
handling of all specimens for this study, as currently recommended by the US CDC and 
US National Institutes of Health. 
All dangerous goods and materials, including diagnostic specimens and infectious 
substances, must be transported using packaging mandated by CRF 42 Part 72. Please 
refer to instructions detailed in International Air Transport Association (IATA) 
Dangerous Goods Regulations.  

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18 
Protocol Amendment Summary of Changes Table 
A summary of amendment history is provided below. 
Protocol Amendment 6 (Protocol Version 7.0/25MAR2026) 
Key Changes Included in Amendment 6 
Section 4.1 Adjusted CRP inclusion criteria 
Clarifications throughout to reconcile referenced CRP inclusion criteria 
 
Protocol Amendment 5 (Protocol Version 6.0/03NOV2025) 
Key Changes Included in Amendment 5 
Section 18 Added section: Amendment Summary of Changes Table 
Section 6.1 SOA: Added extended window to V6, V9, V10, V13, V16, V19  
Section 6.2.3 added text to address extended window visits.  
Section 7.2 Screening (V0) added allowances and instructions for conducting a split screening visit   
Sections 7.6, 7.8, 7.11, 7.12, 7.15 Increased study drug product dispensed at V4, V6, V9, V10 to account for extended 
visit windows 
Minor clerical corrections and edits throughout 
Protocol Amendment 4 (Protocol Version 5.0/24JUL2025) 
Key Changes Included in Amendment 4 
Section 5.1 Corrected inconsistency in study drug product (metformin or placebo) treatment duration (120 days>112 
days (16 weeks)). 
Section 7.2 Added the ability to repeat a single out of range laboratory test once 3-10 days following initial test at the 
discretion of PI to determine eligibility. 
Section 7.25 Added section for clarification of interim/unscheduled visits. 
Protocol Amendment 3 (Protocol Version 4.0/23MAY2025) 
Key Changes Included in Amendment 3 
Executive Summary & Section 3 Corrected age range to 19-64 
Sections 4.1 & 4.3 Adjusted HbA1c and anion gap inclusion and exclusion criteria. 
Section 10.1 Adjusted permanent and premature study product discontinuation due to adjustment of 
inclusion/exclusion criteria. 
Section 10.1 Added criteria for re-enrollment of participants that met discontinuation criteria under prior protocol 
versions. 
 

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Protocol Amendment 2 (Protocol Version 3.0/01APR2025) 
Key Changes Included in Amendment 2 
Section 6.3.10 Added instruction regarding naloxone counseling and provision 
Minor clerical corrections and edits throughout 
 
Protocol Amendment 1 (Protocol Version 2.0/24FEB2025) 
Key Changes Included in Amendment 1 
Section 4.1.3 Inclusion Citeria: Age changed from 18-64 years old to 19-64 years old.   
Section 6.1 SOA: Updated blood volumes to – slightly reduced to match anticipated tubes to be used.   
Minor corrections and edits throughout