1 OriginalArticle

Evaluation of the Potential Interaction Between Tofacitinib and Drugs That Undergo Renal Tubular Secretion Using Metformin, an In Vivo Marker of Renal Organic Cation

Transporter 2†

Karen J. Klamerus,1 Christine Alvey,2 Lei Li,1* Bo Feng,1 Rong Wang,2 Irina Kaplan,2 Haihong Shi,2 Martin E. Dowty,3 and Sriram Krishnaswami2

1Pfizer Inc, San Diego, CA, USA; 2Pfizer Inc, Groton, CT, USA; 3Pfizer Inc, Andover, MA, USA * Currently at Cambridge, MA, USA Corresponding Author: Karen J. Klamerus, 10646 Science Center Drive (CB10), San Diego, CA 92121, USA Tel: +1 858 638 3848; fax: +1 877 481 3398 (email: karen.klamerus@pfizer.com) Financial disclosures: This study was sponsored by Pfizer Inc. Karen J. Klamerus, Christine Alvey, Bo Feng, Rong Wang, Irina Kaplan, Haihong Shi and Sriram Krishnaswami are employees of Pfizer Inc. Lei Li was an employee of Pfizer Inc at the time of the study. Editorial support was provided by Anne Marie Reid, PhD, at Complete Medical Communications and was funded by Pfizer Inc. Declaration of conflicting interests: Karen J. Klamerus, Christine Alvey, Bo Feng, Rong Wang, Irina Kaplan, Haihong Shi and Sriram Krishnaswami are employees of Pfizer Inc. Lei Li was an employee of Pfizer Inc at the time of the study. Keywords: drug-drug interactions, tubular secretion, metformin, organic cation transporter, tofacitinib †This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/cpdd.120]

Received 10 October 2013; Revised 13 March 2014; Accepted 20 March 2014 Clinical Pharmacology in Drug Development

© 2014 The American College of Clinical Pharmacology DOI 10.1002/cpdd.120

2

Abstract Tofacitinib is a novel, oral Janus kinase inhibitor. The potential for drug-drug interactions (DDIs) between

tofacitinib and drugs that undergo renal tubular secretion was evaluated using metformin as a probe transporter

substrate, and genotyping for organic cation transporter (OCT) 1, OCT2 and multidrug and toxin extrusion 1

polymorphisms.

Twenty-four healthy male subjects completed this open-label, fixed-sequence study. Subjects were

administered a single oral metformin 500 mg dose on Days 1 and 4, and multiple oral tofacitinib 30 mg twice

daily doses on Days 2, 3, and 4. Subjects underwent serial blood and urine samplings (Days 1 and 4) to estimate

metformin pharmacokinetics. A single blood sample for tofacitinib was collected 2 hours after the morning

dose (Day 4).

The 90% confidence intervals for the ratios of maximum plasma concentration, area under the curve and renal

clearance of metformin, with and without tofacitinib, were contained within the 80-125% acceptance range

commonly used to establish a lack of DDI. No deaths, serious adverse events (AEs), severe AEs or

discontinuations due to AEs were reported. The study confirms tofacitinib is unlikely to impact the

pharmacokinetics of drugs that undergo renal tubular secretion, and concurs with its weak in vitro OCT2

inhibitory profile.

3

Introduction Tofacitinib is a novel, oral Janus kinase (JAK) inhibitor, and preferentially inhibits JAK1 heterodimer cytokine

signaling over JAK2 homodimer signaling.1 The pharmacokinetic (PK) profile of tofacitinib is characterized by

rapid absorption, rapid elimination (terminal half-life [t1/2] of ~3 hours), and dose-proportional systemic

exposures. The tofacitinib PK profile has been evaluated in healthy volunteers2-5 and in patients with renal

transplantation,6 rheumatoid arthritis (RA),7 psoriasis,8 and ulcerative colitis.9 The clearance mechanisms for

tofacitinib in humans involve both hepatic (70%) and renal (30%) excretion of the parent drug.2 Of the

approximately 30% renal clearance, an estimate of 40% is active renal clearance, which equates to

approximately 11% of total clearance.

SLC22A2, or more commonly referred to as Organic Cation Transporter 2 (OCT2), is primarily a renal uptake

transporter that plays a key role in the renal clearance of drugs and endogenous compounds.10 OCT2 substrate

drugs have the potential for drug-drug interactions (DDIs) with co-administered therapeutics that are inhibitors

of this transporter.

Metformin (1,1-dimethylbiguanide) is an oral insulin-sensitizing agent commonly used, either alone or in

combination with other antihyperglycemic drugs, in patients with type 2 diabetes. Metformin has an absolute

bioavailability of approximately 50% to 60%, is rapidly distributed and is not highly protein bound.10

Metformin is eliminated primarily unchanged in the urine (98%),11 with active renal clearance being much

greater than glomerular filtration, indicating extensive transporter-mediated active secretion in the renal

proximal tubules.12,13 Estimates of metformin plasma t1/2 range from 1.5 to 4.9 hours.12,13

Metformin is a known substrate for the human major renal transporters OCT2 and multidrug and toxin

extrusion 1 (MATE1), as well as the hepatic uptake transporter organic cation transporter 1 (OCT1). Each of

the genes encoding OCT1, OCT2 and MATE1 have variations that affect transporter function.14-17 The

frequency of genetic polymorphisms in Caucasians is ~40% for OCT1, ~15% for OCT2 and 45% for

MATE1.18

Both creatinine and metformin are OCT2 substrates, although metformin is a more sensitive in vivo OCT2

substrate. This is because metformin is primarily cleared renally and transporter-mediated active renal secretion

4 is the major mechanism for renal clearance of metformin, whereas active renal secretion is only approximately

15% of total creatinine renal clearance.19

Although in vitro data indicated that the likelihood of a clinically relevant interaction was low ([I]/half maximal

inhibitory concentration [IC50] <0.1), patients with RA treated with tofacitinib have shown small (<0.1 mg/dL)

mean increases in serum creatinine levels.20 Since creatinine undergoes tubular secretion via OCT2, the present

study was designed to assess whether this observation could be explained by OCT2-inhibition. Howewer,

metformin was chosen for this study since it is a more sensitive probe for OCT2-mediated transport compared

to creatinine. The primary objective was to compare the PK profile of metformin with and without concomitant

tofacitinib. Secondary objectives were to evaluate the safety of tofacitinib and metformin co-administration, to

determine if pharmacogenetic markers could explain any observed PK effect, and to profile the in vitro OCT2

inhibition potential of tofacitinib. Finally, as there are several genetic variants in each transporter gene that may

change transporter functions, genotyping for OCT1, OCT2 and MATE1 polymorphisms that specifically affect

PK11,21,22 or pharmacodynamics (PD) were performed in order to predict transporter-related variability of

metformin PK parameters in this study.

Methods E t h i c s

The final protocol, any amendments, and informed consent documentation were reviewed and approved by the

Independent Ethics Committee of the investigational center participating in the study (Comite d'Ethique de

l'hopital Erasme, Route de Lennik 808, 1070 Bruxelles, Brussels, Belgium). This study was conducted in

compliance with the ethical principles originating in or derived from the Declaration of Helsinki and with all

International Conference on Harmonization Good Clinical Practice Guidelines. All subjects provided written

informed consent.

S u b j e c t s

Subjects eligible for enrollment into the study were male or female (of non-childbearing potential) aged 18 to

55 years, had no clinically relevant abnormalities as identified by a detailed medical history, and underwent full

physical examination (including blood pressure and pulse rate measurement), 12-lead electrocardiogram

(ECG), and safety laboratory tests. Subjects had a body mass index of 17.5 to 30.5 kg/m2, a total body weight

5 >50 kg, and no evidence of active, latent or inadequately treated infection with Mycobacterium tuberculosis

(TB; negative QuantiFERON® TB Gold In-Tube test or a negative Mantoux Purified Protein Derivation skin

test, and no history of either untreated or inadequately treated latent or active TB infection, or current treatment

for this).

Subjects were excluded if they had: an estimated creatinine clearance <80 mL/minute (derived using the

method of Cockcroft and Gault); used moderate to potent cytochrome P450 (CYP)3A4 inhibitors or inducers

within 14 days or five half-lives (whichever was longer) prior to dosing; used tobacco- or nicotine-containing

products in excess of the equivalent of five cigarettes per day; or a history of regular alcohol consumption

exceeding 14 drinks/week for females or 21 drinks/week for males within 6 months of screening.

S t u d y D e s i g n

This study was an open-label, fixed-sequence Phase 1 study designed to evaluate the potential effect of

tofacitinib on the PK of metformin in healthy volunteers (Study A3921143; ClinicalTrials.gov identifier

NCT01405118). A total of 24 healthy subjects were enrolled in the study to ensure 22 completers. Subjects who

withdrew for reasons unrelated to safety could be replaced at the discretion of the investigator upon

consultation with the sponsor. The study was conducted at a single center in Belgium (Pfizer Clinical Research

Unit, Hôpital Erasme, 808 Route de Lennik, Bruxelles, B-1070, Belgium).

Subjects were screened within 28 days of the first dose of study medication. Subjects reported to the Clinical

Research Unit (CRU) on Day 0, at least 12 hours prior to Day 1 dosing, and were required to stay in the CRU

for 6 days and 5 nights. Subjects were administered a single oral dose of metformin 500 mg on Days 1 and 4,

and multiple oral doses of tofacitinib 30 mg every 12 hours (BID) on Days 2, 3, and 4. On Day 4, the morning

dose included both metformin and tofacitinib; 12 hours later the second daily dose of tofacitinib was

administered alone.

On Days 1 and 4, subjects underwent serial blood and urine samplings for 24 hours after the metformin dose to

estimate the serum and urine PK of metformin. Metformin PK blood samples were collected on Days 1 and 4 at

0 hours (before the metformin dose) and at 0.5, 1.5, 2, 3, 4, 6, 8, 9, 10, 12, and 24 hours after the metformin

dose. Urine samples for metformin PK were collected on Days 1 and 4 at 0 hours (before the metformin dose)

6 and for the intervals 0-3, 3-6, 6-9, 9-12, and 12-24 hours after the metformin dose. Metformin PK sampling for

Day 1 was completed before the first dose of tofacitinib was administered on the morning of Day 2. On Day 4,

a single blood sample for tofacitinib was collected 2 hours after the tofacitinib morning dose.

Subjects were discharged from the CRU on Day 5, after the day’s assessments were completed.

B i o a n a l y t i c a l M e t h o d s

Plasma Analysis for Metformin

Human plasma samples were analyzed for metformin concentrations at Pharmaceutical Product Development

(PPD; Richmond, Virginia, USA) using a validated, sensitive, and specific high-performance liquid

chromatography tandem mass spectrometry (HPLC/MS/MS) method. Plasma specimens were stored at

approximately -20 °C until analysis and assayed within 339 days of established stability data generated during

validation.

Metformin was extracted from 50 μL human plasma by solid phase extraction with metformin-d6 as the internal

standard. Sample was eluted with formic acid in acetonitrile. The eluant was injected onto an HPLC column

(Betasil Silica 100; 2.1 mm × 50 mm; 5 µm pore size; Thermo Scientific, West Palm Beach, FL, USA) with a

gradient mobile phase containing acetonitrile, ammonium formate (10 mM), trifluoroacetic acid and acetic acid

(volume ratio: 95:5:0.05:1). Detection was performed using mass spectrometry (Sciex API 3000; AB SCIEX,

Framingham, MA, USA) in the positive ion mode. The multiple reaction monitoring ion transition was m/z

130→71 for metformin and m/z 136→77 for the internal standard.

The assay accuracy, expressed as percent relative error (%RE) for quality control (QC) concentrations, ranged

from -0.634% to 1.73% for the low, medium, high, and diluted QC samples. Assay precision, expressed as the

between-day percent coefficients of variation (%CV) of the mean estimated concentrations of QC samples, was

≤6.52 %CV for low (5.00 ng/mL), medium (45.0 ng/mL) and high (750 ng/mL) concentrations.

Selectivity of the method was acceptable with regards to endogenous compounds, potential interferences, and

possible impurities of the internal standard, as illustrated by chromatograms of a blank plasma sample with and

7 without internal standard (data not shown). Selectivity of the method was demonstrated in the presence of

tofacitinib 2000 ng/mL in human plasma.

Calibration standard responses were linear over the range of 2.00 to 1000 ng/mL using weighted

(1/concentration2) linear least squares regression. Those samples with concentrations above the upper limits of

quantification were adequately diluted into calibration range. The lower limit of quantification (LLOQ) for

metformin in plasma was 2.00 ng/mL.

Urine Analysis for Metformin

Human urine samples were analyzed for metformin concentrations at PPD using a validated, sensitive, and

specific HPLC-ultraviolet (UV) method. Human urine specimens were stored at approximately -20 °C until

analysis and assayed within 724 days of established stability data generated during validation.

A 50 μL human urine sample was aliquoted and diluted with mobile phase before being injected onto an HPLC

column (Spherisorb Cartridge Column; 4.0 mm × 125 mm; 5 μm pore size; Waters Corporation, Milford,

Massachusetts, USA). The mobile phase was composed of methanol, acetonitrile, and potassium phosphate

buffer (0.05 M), pH 2 (volume ratio: 28:28:44). Phenformin was used as the internal standard. Detection was at

wavelength 234 nm using a UV detector (Applied Biosystems 785A; Life Technologies Ltd, Paisley, UK).

The between-day assay accuracy, expressed as %RE for QC concentrations, ranged from -1.63% to 3.38% for

the low, medium, and high QC samples. Assay precision, expressed as the between-day %CV of the mean

estimated concentrations of QC samples, was ≤2.65 %CV.

Selectivity of the method was acceptable with regards to endogenous compounds, potential interferences, and

possible impurities of the internal standard, as illustrated by chromatograms of a blank plasma sample with and

without internal standard (data not shown). Selectivity of the method was demonstrated by the presence of

tofacitinib 35 μg/mL in human urine.

Calibration standard responses were linear over the range of 10 µg/mL to 10,000 µg/mL using weighted

(1/concentration2) linear least squares regression. Those samples with concentrations above the upper limits of

quantification were adequately diluted into calibration range. The LLOQ for metformin in urine was 10 µg/mL.

8

Plasma Analysis for Tofacitinib

Human plasma samples were analyzed for tofacitinib concentrations at BASi Laboratories (West Lafayette,

Indiana, USA) using a validated, sensitive, and specific HPLC/MS/MS method. Human plasma specimens were

stored at approximately -20 ºC until analysis and assayed within 693 days of established stability data generated

during validation.

Tofacitinib was extracted from plasma (300 µL aliquot) by 96-well solid-phase extraction with [13C15N]

tofacitinib as an internal standard. Samples were eluted with 13% ammonium hydroxide in methanol,

evaporated to dryness, and reconstituted with 50% methanol in water. The reconstituted sample was injected

onto an HPLC column (Synergi Polar-RP 4 Micro column; Phenomenex, Inc., Torrance, California, USA), with

a mobile phase of 40% 10 mM ammonium acetate and 60% methanol (with 0.05% formic acid). Detection was

performed using mass spectrometry (Sciex API 4000; AB SCIEX) in the positive ion mode. The multiple

reaction monitoring ion transition was m/z 313.4→173.2 for tofacitinib and m/z 316.3→173.1 for the internal

standard.

The between-day assay accuracy, expressed as %RE for QC concentrations, ranged from -5.3% to 8.7% for the

low, medium, and high QC samples. Assay precision was not calculated because only one batch of samples was

analyzed.

Selectivity of the method was acceptable with regards to endogenous compounds, potential interferences, and

possible impurities of the internal standard, as illustrated by chromatograms of a blank plasma sample with and

without internal standard (data not shown). Selectivity of the method was demonstrated in the presence of

metformin at 2000 ng/mL in human plasma.

Calibration standard responses were linear over the range of 0.100 ng/mL to 350 ng/mL using quadratic

weighted (1/concentration2) linear least squares regression. Those samples with concentrations above the upper

limits of quantification were adequately diluted into calibration range. The LLOQ for tofacitinib in plasma was

0.100 ng/mL.

Genotyping

9 Genotypes for OCT1, OCT2 and MATE1 polymorphisms that potentially affect PK11,21,22 were analyzed by

TaqMan® genotyping assays purchased from Life Technologies Ltd and validated in house.23 Genomic DNA

was extracted from whole blood using the QIASymphony platform (QIAgen, Venlo, Netherlands).

In Vitro OCT2 Inhibition

Human Embryonic Kidney (HEK 293) cells transfected with human OCT2 were obtained from Dr. Kathleen

Giacomini at UCSF. [14C]creatinine (55 mCi/mmol, 99% purity) was obtained from American Radiolabeled

Chemical Inc (St. Louis, MO, USA). Quinidine and cimetidine were obtained from Sigma-Aldrich (St. Louis,

MO, USA).

HEK 293 cells transfected with human OCT2 grown in Dulbecco's Modified Eagle Medium were seeded

(~5x105) on a 24-well poly-D-lysine coated plate (BiocoatTM, BD, Franklin Lakes, NJ, USA). Cells were

incubated with 5 μM [14C]creatinine and either tofacitinib or the two positive controls, quinidine and

cimetidine, and accumulated radioactivity was determined by scintillation counting. Percent uptake activity was

determined at increasing inhibitor concentration relative to baseline uptake in the absence of inhibitor. IC50

plots were generated for each compound using a variable slope symmetric sigmoid model y=100/(1+10(logIC50 -

x)*n), where y is the remaining uptake activity, x is the log of the inhibitor drug concentration, and n is the Hill

coefficient (GraphPad Prism, www.graphpad.com).

D a t a A n a l y s e s

Pharmacokinetic Analysis

PK parameters of metformin alone and in combination with tofacitinib were derived from the plasma

concentration-time profiles, urine concentrations and collection volumes using standard non-compartmental

methods. Samples below the LLOQ were set to zero and actual sample collection times were used for the PK

analysis.

The primary PK parameters were: area under the plasma concentration-time profile (AUC) from time zero to

infinity (AUCinf); maximum observed plasma concentration (Cmax); and renal clearance (CLr), determined as the

cumulative amount of drug recovered unchanged in urine from time zero to 24 hours post-dose (Ae24)/AUCinf.

10 The CLr calculation assumes that Ae24 represents complete urinary excretion since the 24-hour urine collection

represents approximately five half-lives for metformin.

Additional PK parameters included: AUC from time zero to the time of the last quantifiable concentration

(AUClast), the time point at which Cmax was observed (Tmax), terminal t1/2, and Ae24.

Statistical Analyses

Sample Size. A sample size of 22 subjects provided 80% coverage probability that the lower bound of the 90%

confidence intervals (CIs) for a 10% increase in AUC or Cmax exceeded 1.0. To allow for subjects potentially

discontinuing (other than for safety), 24 subjects were enrolled to this study to ensure 22 subjects completed the

study. The above calculations were based on the estimates of within-subject standard deviations (SDs) of 0.152

and 0.162 for the natural log of e (loge) AUCinf and loge Cmax of metformin, respectively; obtained as an average

from literature on metformin PK following single-dose administration.24,25

Statistical Methods. All PK parameters were summarized descriptively. Natural log-transformed AUCinf, Cmax

and CLr for metformin were analyzed using a mixed-effect model (SAS V9.2 Proc Mixed, with restricted

maximum likelihood estimation method and Kenward-Roger degrees of freedom algorithm) with treatment as a

fixed effect and subject as a random effect. Estimates of the adjusted mean differences (Test-Reference) and

corresponding 90% CIs were obtained from the model. The adjusted mean differences and 90% CIs for the

differences were exponentiated to provide estimates of the ratio of adjusted geometric means (Test/Reference)

and 90% CIs for the ratios. Metformin administered alone was the Reference treatment and metformin

co-administered with tofacitinib was the Test treatment.

Safety Analyses

Adverse events (AEs), ECGs, vital signs, and safety laboratory (hematology, clinical chemistry and urinalysis)

data were reviewed and summarized on an ongoing basis during the study. Baseline was defined as the last pre-

dose assessment taken on Day 0 for AEs, laboratory data and vital signs, and was defined as the last pre-dose

assessment taken on Day 1 for ECGs. Clinical safety laboratory tests (including serum creatinine) were

performed at baseline and on Day 5.

11

Results A total of 24 males, mostly Caucasian, with a mean age of 36 years (range: 20-55) and mean body mass index

of 25 kg/m2 (range: 22-29) were assigned to study treatment. All subjects received study treatment, completed

the study and were included in the PK analyses for metformin and tofacitinib. CLr was not reported for one

subject on Day 4 (metformin in combination with tofacitinib) due to incomplete urine collection. For another

subject on Day 4, an AE of vomiting occurred that could have affected the metformin PK. Vomiting occurred at

about the same time as the median metformin Tmax on Day 4; however, the data did not appear to be an outlier

based on either qualitative comparison with other subjects on Day 4 or on review of the diagnostic check of the

statistical analysis. Therefore, the subject’s PK parameters on Day 4 were included in all analyses.

M e t f o r m i n

Mean plasma metformin concentration-time profiles with and without tofacitinib were almost identical, with

slightly lower peak concentrations observed for the combination treatment (Figure 1). Metformin plasma and

urine PK parameters during tofacitinib co-administration were similar to those observed when metformin was

administered alone (Table 1). The ratios of adjusted means were 97.82% for AUCinf, 92.55% for Cmax, and

100.18% for CLr (Table 2). The corresponding 90% CIs for the ratios all fell within the 80-125% acceptance

range, which indicated that there was no interactive effect when metformin was given as a single dose with

multiple-dose tofacitinib compared with a single dose of metformin alone. Median Tmax and mean t½ were

similar for both treatments (Table 1).

T o f a c i t i n i b

Plasma tofacitinib concentrations for samples collected at 2 hours post-dose on Day 4 had a mean (%CV) of

125 ng/mL (24%) and individual values ranged from 53-174 ng/mL.

G e n o t y p i n g

Metformin PK parameters by genotyping subgroup were not visually different between treatments, which is

consistent with the primary analysis. Most differences in metformin CLr with tofacitinib versus metformin alone

were within 10%; the largest increase in metformin CLr with tofacitinib (26%) was observed in a subject with

no OCT1, OCT2 or MATE1 genotype variants.

12 In Vitro OCT2 Inhibition

In the concentration range tested, tofacitinib and the two positive controls, cimetidine and quinidine, inhibited

the uptake of creatinine mediated by OCT2 in a concentration-dependent manner. The calculated IC50 values

for tofacitinib, cimetidine, and quinidine were 150 μM, 406 μM, and 70 μM respectively (Figure 2).

S a f e t y

No deaths, serious AEs, severe AEs or discontinuations due to AEs were reported during the study. None of the

subjects had their dose reduced or temporarily discontinued due to AEs. There were no clinically significant

changes in individual safety laboratory parameters, vital signs or ECG findings. No individual laboratory and

vital signs, nor ECG changes, were reported as AEs.

All of the AEs in the study were categorized as mild or moderate. One subject experienced musculoskeletal

pain during the course of all three treatments (metformin alone, tofacitinib alone, and metformin plus

tofacitinib). Two subjects experienced treatment-related AEs of diarrhea while receiving metformin alone. Four

subjects experienced six treatment-related AEs while receiving metformin plus tofacitinib: two AEs of upper

abdominal pain and one AE each of abdominal distension, diarrhea, flatulence and vomiting.

Discussion This study was designed to use metformin as a probe transporter substrate and genotyping to identify any

potential DDI between tofacitinib and drugs that undergo renal tubular secretion. Our results revealed a lack of

a DDI between tofacitinib and metformin. The results show no changes in the plasma and urine PK of a single

oral dose of metformin 500 mg when administered alone or in combination with steady-state administration of

tofacitinib 30 mg BID. The 90% CIs for the ratios of adjusted geometric mean AUCinf, Cmax, and CLr values for

metformin were entirely contained within the 80-125% acceptance range commonly used to establish the lack

of a DDI.

These metformin DDI results were in agreement with the in vitro OCT2 results. In vitro transporter studies in

human OCT2 transfected cells suggested that tofacitinib inhibits the uptake of creatinine in a dose-dependent

manner, with an estimated IC50 of 150 µM. In the clinic, a dose of tofacitinib 30 mg results in a steady-state

total Cmax of approximately 1 µM (or unbound Cmax of 0.6 μM, considering a fraction unbound in plasma

13 protein of 0.61). The ratio of unbound drug at Cmax to its IC50 value is <0.01, which suggests a low potential for

tofacitinib to cause clinically relevant interactions with other drugs that undergo renal tubular secretion by

OCT2.26

These clinical and in vitro data are also supported by data from a study of healthy volunteers showing that

administration of tofacitinib 15 mg BID (free Cmax 0.3 μM) for 14 days did not alter serum creatinine

concentrations or creatinine clearance, as measured using 24-hour urine collection.27 In the present 5-day study,

mean serum creatinine increased 0.04 mg/dL from pre-dose to the end of the study (still within reference range

for all but one subject).

The tofacitinib 30 mg BID dose in the current study provided sufficient therapeutic concentrations to evaluate

any effect on metformin PK, with a non-Cmax 2-hour post-dose tofacitinib mean concentration of 125 ng/mL

(range: 53-174 ng/mL). In patients with RA, 2-hour mean plasma concentrations following therapeutic doses of

tofacitinib 5 mg BID and 10 mg BID were found to be 36 ng/mL and 71 ng/mL, respectively.28 This elevated

plasma concentration of tofacitinib in patients with RA was important since differences in systemic exposure

have been observed between healthy volunteers and patients with RA, and the choice of tofacitinib 30 mg BID

in our study ensured that plasma concentrations were at or above the therapeutic concentrations of up to 15 mg

BID that have been used in various patient populations. Three days of dosing was also considered sufficient

time to achieve steady-state concentrations of tofacitinib.

Genotyping samples were collected a priori to investigate if any effect of polymorphisms on transporter activity

was obscuring the results. Since there was no effect of tofacitinib on metformin PK, examination of renal

transporter polymorphisms to explain any potential tofacitinib-mediated effects on metformin PK was not

needed.

In summary, the preclinical results indicating that therapeutic concentrations of tofacitinib would weakly inhibit

OCT2 translated to our clinical study in healthy volunteers, which confirmed similar metformin plasma and

urine PK with and without tofacitinib. Overall, the results of this study confirm a lack of DDI between

tofacitinib and drugs that undergo renal tubular secretion, as shown with metformin.

14

Funding This study was sponsored by Pfizer Inc. Karen J. Klamerus, Christine Alvey, Bo Feng, Rong Wang, Irina

Kaplan, Haihong Shi, Martin Dowty, and Sriram Krishnaswami are employees of Pfizer Inc. Lei Li was an

employee of Pfizer Inc at the time of the study. Editorial support was provided by Anne Marie Reid, PhD, at

Complete Medical Communications and was funded by Pfizer Inc.

Declaration of Conflicting Interests Karen J. Klamerus, Christine Alvey, Bo Feng, Rong Wang, Irina Kaplan, Haihong Shi, Martin Dowty, and

Sriram Krishnaswami are employees of Pfizer Inc. Lei Li was an employee of Pfizer Inc at the time of the

study.

Acknowledgments The authors would like to thank the Study A3921143 investigators, study team, and subjects. In particular, they

would like to thank Constantino Kantaridis (investigator), Jennifer Reynolds (lab scientist), Maud Allard

(project manager), and Frances Tibbets (project manager).

15

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18

Table 1. Descriptive Summary of the Effect of Tofacitinib on the Pharmacokinetics of Metformin in 24

Subjects. Parameters Reported as Arithmetic Mean (Standard Deviation) for all Except Tmax Which is

Median (Range)

Parameter Metformin Alone Metformin + Tofacitinib

AUClast, ng·h/mL 6848 (1366) 6695 (1351)

AUCinf, ng·h/mL 6958 (1366) 6807 (1361)

Cmax, ng/mL 1107 (274) 1022 (240)

Tmax, h 3.0 (1.5-4.0) 2.5 (1.5-4.0)

t1/2, h 4.29 (0.50) 4.23 (0.45)

CLr, mL/min 606 (108) 604 (113)a

aNumber of subjects = 23 (CLr was not reported for one subject due to incomplete urine collection).

AUCinf, area under the plasma concentration-time profile from time 0 extrapolated to infinity; AUClast, area under the plasma

concentration-time profile from time 0 to the time of the last quantifiable concentration; CLr, renal clearance; Cmax, maximum

observed plasma concentration; Tmax, time to Cmax; t1/2, terminal half-life.

19 Table 2. Statistical Summary of Treatment Comparisons

Adjusted Geometric Means

Parameter units Metformin with

tofacitinib (Test)

Metformin alone

(Reference)

Ratio of adjusted

geometric means

(Test/Reference), %

90% CI for ratio,

%

AUCinf (ng·hr/mL) 6673 6821 97.82 97.25, 103.17

Cmax (ng/mL) 994 1074 92.55 86.91, 98.57

CLr (mL/min) 598 596 100.18 96.92, 103.55

Values have been back-transformed from the log scale. CLr for metformin with tofacitinib was not reported in one subject due to incomplete urine collection. AUCinf, area under plasma concentration-time profile from time zero extrapolated to infinity; CI, confidence interval; CLr, renal clearance; Cmax, maximum observed plasma concentration.

20

Figure 1. Effect of Multiple-dose Tofacitinib on the Mean (Standard Error) Plasma

Concentration-time Profile of Metformin: a) Non-log Transformed Data and b) Semi-log

Transformed Data.

21

Figure 2. Tofacitinib Inhibition of Creatinine Uptake Mediated by hOCT2. Cimetidine and

Quinidine were Positive Controls.