Clinical trial NCT05365451
Conditions: Interaction Endogenous Biomarkers
Keywords: Kidney Transporter Pharmacokinetics
Sponsor: Washington State University
Start date: 2022-04-11; Completion date: 2023-07-22
Enrollment: 16 participants; Allocation: NON_RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The objective of this study is to confirm the feasibility of using a panel of endogenous substrates/metabolites as a robust biomarker of OCTs and OATs by conducting a controlled, comprehensive clinical drug-drug interaction study in healthy adult volunteers. Metformin and furosemide will be used as probe drugs for OCTs and OATs, respectively; cimetidine and probenecid will be used as corresponding inhibitors. Results from this study will validate this novel approach, which will be extended to children by collaborators at Children's Mercy Hospital in Kansas City, MO.

Detailed description:
The kidneys are major organs responsible for the excretion of both endogenous and exogenous compounds, the latter including drugs and other xenobiotics. Excretion occurs via passive or active processes, the latter involving transporters such as organic cation transporters (OCTs) and organic anion transporters (OATs). Inhibition of these transporters, coupled with the large interindividual variability in transporter expression, can lead to toxic accumulation of compounds/xenobiotics cleared primarily by this route. During drug discovery and development, if in vitro evidence suggests renal transporters mediate excretion of a new chemical entity, the Food and Drug Administration recommends conducting a controlled clinical study to evaluate potential risks. These time-consuming and expensive clinical studies routinely involve adult participants and known substrates of renal transporters. However, such studies are not always feasible in children due to the enhanced potential for toxicities. This limitation led to the hypothesis that endogenous substrates could be used as surrogates, or biomarkers, of individual renal transporter function.

Endogenous OCT substrates, such as 1-methyladenosine (m1A) and 1-methylnicotinamide (MNA), as well as OATs, such as homovanillic acid (HVA) and pyridoxic acid (PDA), are promising biomarkers of renal transporters in adults. However, using one or few such endogenous substrates can be misleading due to factors other than variability in specific renal transporter function. We propose to address this knowledge gap by using a panel of endogenous substrates/metabolites that recently has been identified as a robust biomarker of rodent Octs and Oats. Validation of these substrates/metabolites as biomarkers of OCTs and OATs in humans, both adults and children, will aid in the development of physiologically-based pharmacokinetic models that can be used to predict renal transporter-mediated xenobiotic excretion, drug-drug interactions, and toxicity in children.

Arms: arm 1: Arm 1A will consist of administration of a single dose of metformin (50 mg) by mouth as a liquid to 16 subjects (8 males, 8 females). Plasma and urine will be collected from 0-24 hours. Participants may or may not elect to participate in Arms 2A and 2B. A washout of at least 7 days will occur between Arm 1A and Arm 1B. arm 2: Arm 1B will consist of administration of a single oral dose of cimetidine (400 mg) with water by mouth. One hour later, metformin (50 mg) will be administered by mouth as a liquid. Plasma and urine will be collected from 0-24 hours. Participants may or may not elect to participate in Arms 2A and 2B. A washout of at least 7 days will occur between Arm 1B and Arm 2A. arm 3: Arm 2A will consist of administration of a single dose of furosemide (5 mg) by mouth as a liquid. Plasma and urine will be collected from 0-24 hours. A washout of at least 7 days will occur between Arm 2A and Arm 2B. arm 4: Arm 2B will consist of administration of a single oral dose of probenecid (1,000 mg) with water by mouth. One hour later, furosemide (5 mg) will be administered by mouth as a liquid. Plasma and urine will be collected from 0-24 hours. Participants may or may not elect to participate in Arms 1A and 1B. A washout of at least 7 days will occur between Arm 2B and Arm 1A.
Interventions: intervention 1: MetFORMIN Oral Solution intervention 2: Cimetidine 400 MG intervention 3: Furosemide Oral Liquid Product intervention 4: Probenecid 500 MG
Intervention descriptions: intervention 1: liquid intervention 2: tablet intervention 3: oral solution intervention 4: tablet

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
 
 
NCT #05365451 
 
 
Pharmacokinetic drug-drug interaction study to identify 
biomarkers of kidney transporters 
 
 
PI: Mary F. Paine, RPh, PhD 
 
 
 
 
6/27/2025 
 
 
 
 
 
 
 
 
 
 
 
 


STUDY PROCEDURES FLOWCHART 
 
FIGURE 1. Overview of procedures. 
 
SUBJECTS 
Healthy non-smoking adult volunteers (8 men and 8 women) will be recruited from the local community to 
participate in this drug-drug interaction study (FIGURE 1). 
 
PRE-SCREENING 
Inclusion criteria:  
• Aged from 18-64 years and healthy 
• Not taking any medications (prescription and non-prescription) or dietary/herbal supplements known 
to alter the pharmacokinetics of any of the study drugs 
• Willing to abstain from consuming dietary/herbal supplements and citrus juices for several weeks 
• Willing to abstain from consuming caffeinated beverages or other caffeine-containing products the 
evening before and morning of the first day of a study arm 
• Willing to abstain from consuming any alcoholic beverages for one day prior to any study day, during 
the 14-hour inpatient days, and for the outpatient visit(s) following the 14-hour visit 
• Willing to use an acceptable method of contraception that does not include oral contraceptive pills or 
patches (such as abstinence, copper IUD, condom) 
• Have the time to participate  
YES 
YES 
NO 
 NO 
Meets 
inclusion/ 
exclusion 
criteria? 
Screening 
visit (2 h)  
Schedule 
study days 
Eligible to 
participate? 
ID potential 
subjects  
Do not 
screen  
Do not 
enroll 
Part 1 Arm A: 1 14-hour visit 
followed by 1 outpatient visit 
Part 1 Arm B: 1 14-hour visit 
followed by 1 outpatient visit 
Blood will be drawn 30 mins before and 0.33, 
0.67, 1, 1.5, 2, 2.5, 3, 3.5,4, 5, 6, 8, 10, 12, and 
24 h after administration of metformin. 
Blood will be drawn 30 mins before and 0.33, 
0.67, 1, 1.5, 2, 2.5, 3, 3.5,4, 5, 6, 8, 10, 12, and 
24 h after administration of metformin. 
Blood will be drawn 30 mins before and 0.33, 
0.67, 1, 1.5, 2, 2.5, 3, 3.5,4, 5, 6, 8, 10, 12, and 
24 h after administration of furosemide. 
Part 2 Arm B: 1 14-hour visit 
followed by 1 outpatient visit  
Part 2 Arm A: 1 14-hour visit 
followed by 1 outpatient visit 
Blood will be drawn 30 mins before and 0.33, 
0.67, 1, 1.5, 2, 2.5, 3, 3.5,4, 5, 6, 8, 10, 12, and 
24 h after administration of furosemide. 


• Written informed consent (and assent when applicable) obtained from subject or subject’s legal 
representative and ability for the subject to comply with the requirements of the study 
 
Exclusion criteria: 
• Under the age of 18 or over the age of 65 years 
• Smoke/vape/chew tobacco products 
• Use cannabis products, including marijuana, hemp, and other THC- or CBD-containing products 
• Have any current major illness or chronic illness such as (but not limited to) kidney disease, hepatic 
disease, diabetes mellitus, hypertension, coronary artery disease, chronic obstructive pulmonary 
disease, cancer, or HIV/AIDS 
• History of anemia or any other significant hematologic disorder 
• History of drug or alcohol addiction or major psychiatric illness 
• Pregnant or nursing or plan to become pregnant within 3 weeks after participation 
• History of allergy to metformin, cimetidine, furosemide, or probenecid 
• Taking concomitant medications, both prescription and non-prescription (including dietary 
supplements/herbal products), known to alter the pharmacokinetics of any study drug 
• Presence of a condition or abnormality that, in the opinion of the Investigator, would compromise 
participant safety or quality of the data 
• Out-of-range clinical laboratory value that the study physician considers participation in the study a 
health risk 
 
SCREENING VISIT 
Potential eligible subjects, based on the inclusion/exclusion criteria, will present for a screening visit at the 
Nursing or Health Sciences Building on the Spokane campus. Informed consent will be obtained after full 
explanation of the study protocol. Consented subjects will undergo the following: 
• A medical history and physical exam 
• CBC with differential, electrolytes, liver function tests, serum creatinine 
• Routine urine analysis 
• Urine pregnancy test for female subjects of child-bearing potential (prior to enrollment in the study and 
prior to each phase of the study using in-home urinalysis pregnancy screens) 
 
STUDY DESIGN  
This study will consist of two Parts, each with 2 Arms (FIGURES 2 and 3). Each Arm will include one 14-hour 
inpatient visit and one outpatient visit. Subjects will undergo a washout period of at least seven days after 
completing each Arm. Subjects will be administered the following: 
Part 1 Arm A: metformin (50 mg) by mouth as a liquid 
Part 1 Arm B: cimetidine (400 mg) by mouth as a tablet, then one hour later, metformin (50 mg) by mouth 
as a liquid  
Part 2 Arm A: furosemide (5 mg) by mouth as a liquid  
Part 1 Arm B: probenecid (1,000 mg) by mouth as a tablet, then one hour later, furosemide (5 mg) by mouth 
as a liquid 
 
 
 
FIGURE 2. Overview of study design. 
Screening 
≥1 week  
washout 
Part 1 
Arm A 
≥1 week  
washout 
Part 1  
Arm B 
Screening 
≥1 week  
washout 
Part 2 
Arm A 
≥1 week  
washout 
Part 2 
Arm B 


 
Study days. Subjects will be asked to present to the clinical research space in the Health Sciences Building or 
Nursing Building on the morning of each 14-hour inpatient visit. Vital signs will be checked and recorded on case 
forms. Subjects will be asked of any change to their health status; responses will be recorded on case forms. An 
intravenous line will be placed in one arm, and a blood sample (5 mL) will be drawn through the intravenous line. 
Subjects will then be administered a single dose of the object drug (metformin or furosemide) (Arm A) or the 
inhibitor drug (cimetidine or probenecid) followed one hour later by the object drug (Arm B). During all Arms, 
blood will be drawn from the intravenous line 0.5 hours before and 0.33, 0.67, 1, 1.5, 2, 2.5, 3, 3.5,4, 5, 6, 8, 10, 
12, and 24 after object drug administration. Urine will be collected into multiple jugs from 0-24 hours. Subjects 
will be given a urine jug to take home and void into until they return to the clinical research space for the blood 
draw 24 hours after object drug administration. After the 12-hour blood draw of all Arms, subjects will be allowed 
to leave once deemed safe by the study coordinator.  
All Arms will be separated by at least 7 days to ensure sufficient washout of all study drugs; probenecid has the 
longest average half-life (~6 hours), thus all drugs should be eliminated within 2-3 days. Subjects will be asked 
to refrain from consuming any dietary/herbal supplements or citrus juices for at least 1 week prior to each 14-
hour inpatient visit and for the duration of the study. They will be asked to refrain from alcoholic and caffeinated 
beverages the evening before and the morning of each inpatient and outpatient visit. 
Study drug acquisition. Dr. John White, a longstanding collaborator/co-investigator and registered pharmacist 
in WA, will oversee the acquisition and storage of metformin, cimetidine, furosemide, and probenecid. 
Pharmacokinetic and statistical analysis. Plasma and urine samples will be analyzed for the study drugs and 
endogenous transporter substrates by UPLC/MS/MS. The pharmacokinetics of each analyte will be determined 
using traditional noncompartmental analysis methods and the pharmacokinetics analysis software, Phoenix 
WinNonlinTM (v8.3, Certara, Princeton, NJ). The primary analysis will test overall differences in the Cmax, AUC0-
24h, and renal clearance (ClR) of furosemide, metformin, and all detected endogenous substrates between the 
control (object drug only) and treatment (inhibitor + object drug) groups. Cmax will be obtained directly from the 
plasma concentration-time profiles, and AUC0-24h will be calculated using the linear up-log down trapezoidal 
method. ClR will be calculated as the ratio of Xe,0-24h to AUC0-24h, where Xe denotes the cumulative amount of 
drug/endogenous substrate excreted into the urine. Cmax, AUC0-24h, Xe,0-24h, and ClR (with and without inhibitor) 
will be compared using the paired Student’s t-test or Wilcoxon test (depending on the data distribution). If an 
endogenous substrate is only detected in either blood or urine sample, the AUC0-24h or Xe,0-24h of the drug will be 
compared with the corresponding metrics of the endogenous substrates. 
FIGURE 3. Detailed timelines of the sequential arms. 
3.1. Part 1 Arm A 
3.2. Part 1 Arm B 
3.3. Part 2 Arm A 
3.4. Part 2 Arm B