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Commensal gut bacteria convert the immunosuppressant tacrolimus to less potent metabolites

Yukuang Guo, Camila Manoel Crnkovic, Kyoung-Jae Won, Xiaotong Yang, John Richard Lee, Jimmy Orjala, Hyunwoo Lee, Hyunyoung Jeong
doi: https://doi.org/10.1101/426197
Yukuang Guo
1Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago
5Center for Biomolecular Sciences
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Camila Manoel Crnkovic
1Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago
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Kyoung-Jae Won
2Department of Pharmacy Practice, University of Illinois at Chicago
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Xiaotong Yang
4Department of Biopharmaceutical Sciences, University of Illinois at Chicago
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John Richard Lee
3Division of Nephrology and Hypertension, Department of Medicine, Weill Cornell Medicine
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Jimmy Orjala
1Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago
5Center for Biomolecular Sciences
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Hyunwoo Lee
1Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago
5Center for Biomolecular Sciences
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Hyunyoung Jeong
2Department of Pharmacy Practice, University of Illinois at Chicago
4Department of Biopharmaceutical Sciences, University of Illinois at Chicago
5Center for Biomolecular Sciences
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Abstract

Tacrolimus exhibits low and variable drug exposure after oral dosing, but the contributing factors remain unclear. Based on our recent report showing a positive correlation between fecal abundance of Faecalibacterium prausnitzii and oral tacrolimus dose in kidney transplant patients, we tested whether F. prausnitzii and other gut abundant bacteria are capable of metabolizing tacrolimus. Incubation of F. prausnitzii with tacrolimus led to production of two compounds (the major one named M1), which was not observed upon tacrolimus incubation with hepatic microsomes. Isolation, purification, and structure elucidation using mass spectrometry and nuclear magnetic resonance spectroscopy indicated that M1 is a C-9 keto-reduction product of tacrolimus. Pharmacological activity testing using human peripheral blood mononuclear cells demonstrated that M1 is 15-fold less potent than tacrolimus as an immunosuppressant. Screening of 22 gut bacteria species revealed that most Clostridiales bacteria are extensive tacrolimus metabolizers. Tacrolimus conversion to M1 was verified in fresh stool samples from two healthy adults. M1 was also detected in the stool samples from kidney transplant recipients who had been taking tacrolimus orally. Together, this study presents gut bacteria metabolism as a previously unrecognized elimination route of tacrolimus, potentially contributing to the low and variable tacrolimus exposure after oral dosing.

Abbreviations
BEI
Biodefense and Emerging Infections
BrdU
5-Bromo-2′-deoxyuridine
COSY
Homonuclear 1H-1H Correlation Spectroscopy
DEPTQ
Distorsionless enhancement by polarization transfer quaternary HMBC Heteronuclear multiple bond correlation spectroscopy
HPLC
High performance liquid chromatography
HRMS
High resolution mass spectrometry
HSQC
Heteronuclear single quantum coherence spectroscopy
IR
Infrared spectroscopy
MS/MS
Tandem mass spectrometry
NMR
Nuclear magnetic resonance spectroscopy
PBMC
Peripheral blood mononuclear cells
P-gp
P-glycoprotein
PHA
Phytohemagglutinin
TMB
3,3′,5,5′-Tetramethylbenzidine
TOCSY
Total correlated spectroscopy

1. Introduction

Tacrolimus is a commonly used immunosuppressant for kidney transplant recipients as well as patients with glomerular diseases like membranous nephropathy and focal segmental glomerulosclerosis. However, due to its narrow therapeutic index, under-exposure or over-exposure to tacrolimus in kidney transplant recipients increases the risks for graft rejection or drug-related toxicity, respectively.1 Maintaining therapeutic blood concentrations of tacrolimus has been difficult in part because tacrolimus pharmacokinetics shows large inter- and intra-individual variability.2, 3 For example, tacrolimus oral bioavailability in individual patients ranges from 5 to 93% (average ~25%).1 A better understanding of the factors responsible for the variability is crucial for maintaining target concentrations of tacrolimus and improving kidney transplant outcomes.

The human gut is home to over trillions of microbes that can influence multiple aspects of host physiology.4 In particular, intestinal bacteria can promote diverse chemical reactions such as hydrolysis and reduction of orally administered drugs, ultimately affecting the efficacy and/or toxicity of drugs.5-7 For example, digoxin is converted to the pharmacologically inactive metabolite, dihydrodigoxin, by the gut bacterium Eggerthella lenta.5 The expression of the enzyme responsible for digoxin metabolism in E. lenta is influenced by dietary protein content,5 indicating that in addition to the abundance of drug-metabolizing bacteria, diet composition may also govern the extent of drug metabolism in the gut and alter systemic drug exposure. For most clinically used drugs, the involvement of gut bacteria in their metabolism and/or disposition remains unknown.

Faecalibacterium prausnitzii is one of the most abundant human gut bacteria (108-109 16S rRNA gene copies/g mucosal tissue in ileum and colon), taxonomically belonging to the Order Clostridiales.8, 9 Because of its anti-inflammatory effects, F. prausnitzii has been investigated as a potential preventative and/or therapeutic agent for dysbiosis.10, 11 We have recently shown that in 19 kidney transplant patients, fecal F. prausnitzii abundance positively correlates with oral tacrolimus dosage required to maintain therapeutic blood concentrations, independent of gender and body weight.12 It remains unknown, however, whether F. prausnitzii is directly involved in tacrolimus elimination in the gut. Herein, we tested a hypothesis that gut bacteria, including F. prausnitzii, metabolize tacrolimus into less potent metabolite(s).

2. Materials and Methods

2.1. Reagents

Tacrolimus was purchased from AdipoGen (San Diego, CA). Casitone and yeast extract were purchased from HIMEDIA (Nashik, MH, IN) and BD (Sparks, MD), respectively. Other components for media were purchased from Thermo Fisher Scientific (Waltham, MA) or Sigma-Aldrich (St. Louis, MO).

Peripheral blood mononuclear cells (PBMC; C-12907) were purchased from PromoCell (Heidelberg, Germany). Phytohemagglutinin (PHA; L1668) and 5-bromo-2′-deoxyuridine (BrdU; B9285) were purchased from Sigma-Aldrich. 3,3′,5,5′-Tetramethylbenzidine (TMB; 34022) was purchased from Thermo Fisher Scientific.

2.2. Bacterial strains and growth

F. prausnitzii A2-165 was obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). F. prausnitzii VPI C13-20-A (ATCC 27766), and F. prausnitzii VPI C13-51 (ATCC 27768) were from American Type Culture Collection (ATCC). Other gut bacteria were from Biodefense and Emerging Infections (BEI) Research Resources Repository (Supplemental Table 1). Unless stated otherwise, all the bacterial strains were grown anaerobically (5% H2, 5% CO2, 90% N2) on YCFA agar or broth at 37ºC in an anaerobic chamber (Anaerobe Systems, Morgan Hill, CA), and colonies from the agar plate were inoculated into pre-reduced YCFA broth for preparation of overnight cultures. Optical density at 600 nm (OD600) was measured for estimation of bacterial concentration.

2.3. Tacrolimus metabolism by gut bacteria

To examine tacrolimus metabolism by gut bacteria, cells of a bacterial strain grown as described above were incubated tacrolimus. Typically, tacrolimus (100 μg/ml) was incubated with bacterial cells in the anaerobic chamber at 37ºC for 24-48 h. Reaction was terminated by adding the same volume of ice-cold acetonitrile. After vortexing for 30 sec, samples were centrifuged at 16,100×g for 10 min, and the supernatant was collected for HPLC/UV analysis as described below.

2.4. M1 detection

The reaction mixture was analyzed by using HPLC (Waters 2695) coupled with a UV detector (Waters 2487). Typically, 50 μL of a sample was injected and resolved on a C8 column (Eclipse XDB-C8;4.6 x 250 nm; 5 μm) using water (0.02 M KH2PO4, pH 3.5; solvent A) and acetonitrile (solvent B) as mobile phase with the following gradient: 0-12 min (50% B), 12-17 min (50%-70% B), 17-23 min (70% B), 24-30 min (90% B), and 30-40 min (50% B). Eluates were monitored at 210 nm.

For further verification of M1 production by gut bacteria, the supernatant was also analyzed by HPLC tandem mass spectrometry (HPLC/MS/MS), Agilent 1200 HPLC interfaced with Applied Biosystems Qtrap 3200 using an electrospray ion source. The mobile phase consisted of water with 0.1% formic acid and 0.1% ammonium formate (v/v; solvent A) and methanol (solvent B), and the following gradient was used: 0-2 min (40% B), 2-6 min (95% B), and 6-12 min (40% B). Separation was performed on a Xterra MS C18 (2.1x50mm, 3.5μm; Waters) column at a flow rate of 0.3 ml/min, and M1 was detected at m/z 828.5/463.5 in the multiple reaction monitoring mode.

2.5. Infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy

IR spectra were acquired on neat samples using a Thermo-Nicolet 6700 with Smart iTRTM accessory. 1D and 2D NMR spectra were obtained on a Bruker AVII 900 MHz spectrometer equipped with a 5 mm TCI cryoprobe. NMR chemical shifts were referenced to residual solvent peaks (CDCl3 δH 7.26 and δC 77.16). NMR experiments included 1H NMR, Distorsionless Enhancement by Polarization Transfer Quaternary (DEPTQ), Homonuclear 1H-1H Correlation Spectroscopy (COSY), Heteronuclear Single Quantum Coherence Spectroscopy (HSQC), Heteronuclear Multiple Bond Correlation Spectroscopy (HMBC), and 1H-13C HSQC-Total Correlated Spectroscopy (1H-13C HSQC-TOCSY).

2.6. Kidney transplant recipients’ stool samples

Stool samples were collected from ten kidney transplant recipients during the first month after transplantation at Weill Cornell Medicine and stored at -80°C until analysis. Tacrolimus dosing in each patient was adjusted to achieve a target therapeutic level of 8 to 10 ng/ml. The study protocol for kidney transplant stool sample collection was approved by the Institutional Review Board at Weill Cornell Medicine (protocol number 1207012730).

The microbiota composition of the stool samples was determined using 16S rRNA gene deep sequencing as previously described.13 In brief, DNA from stool samples was isolated using a phenol chloroform bead-beater extraction method. The V4-V5 hypervariable region was amplified by PCR and the fragments were sequenced on an Illumina MiSeq (250 x 250 bp). 16S rRNA gene paired-end reads were analyzed using UPARSE14 and taxonomic classification was performed using a custom Python script incorporating BLAST15 with NCBI RefSeq16 as a reference training set.

For the measurement of baseline levels of tacrolimus and M1 in stool samples, an aliquot of stool samples was suspended in PBS (final concentration 20 mg/ml). Also, to measure the capacity of stool samples to produce M1, an aliquot of stool samples was suspended in PBS (10 mg/ml) and incubated with tacrolimus anaerobically for 24 h at 37°C. These samples were mixed with 5 volumes of acetonitrile containing ascomycin as an internal standard. An aliquot (10 µl) was injected into Agilent 1290 UPLC coupled with Applied Biosystems Qtrap 6500. The mobile phase consisted of water with 0.1% formic acid and 10 mM ammonium formate (solvent A) and methanol (solvent B), and the following gradient was used: 0-2 min (20% B), 2-5 min (90% B), and 5-8 min (20% B). Separation was performed on an Xterra MS C18 column (2.1x50 mm, 3.5 µm: Waters) at a flow rate of 0.3 ml/min, with the column temperature set at 50°C. M1, tacrolimus, and ascomycin were detected at m/z 828.5/463.4, 821.6/768.6, and 809.5/756.5, respectively, in the multiple reaction monitoring mode. Standard curve (2–100 ng/ml for both tacrolimus and M1) was prepared by spiking tacrolimus and M1 into the stool samples of healthy volunteers.

2.7. Statistical analysis

Statistical analyses for comparison between two groups were performed using Student’s t-test. A p-value ≤ 0.05 was considered statistically significant.

3. Results

3.1. F. prausnitzii potentially metabolizes tacrolimus

To determine whether F. prausnitzii is capable of metabolizing tacrolimus, cells of F. prausnitzii A2-165 strain were grown to mid-exponential phase in YCFA media, and incubated with tacrolimus (100 µg/ml; 124 µM) anaerobically at 37°C. After 24 h incubation, the mixture was resolved using HPLC and analyzed by a UV detector. The HPLC chromatogram of intact tacrolimus showed multiple peaks, demonstrating tautomer formation as previously reported17 (Fig. 1A). For estimation of a concentration of intact tacrolimus, the area of the largest peak at retention time 19.7 min was used. After 24 h incubation with F. prausnitzii, the concentration of tacrolimus was decreased by ~50% (Fig. 1B), which was accompanied by appearance of two new peaks (designated M1 and M2, Fig. 1A). The M1 and M2 peaks were not observed when tacrolimus was incubated with boiled F. prausnitzii cells (Fig. 1A), indicating that the production of M1 and M2 requires live bacterial cells. Similarly to strain A2-165, two additional strains of F. prausnitzii (ATCC 27766 and ATCC 27768) were found to produce M1 and M2 (Supplemental Fig. 1), suggesting that this function is likely conserved in different strains of F. prausnitzii.

Fig 1.
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Fig 1. F. prausnitzii metabolizes tacrolimus.

A, F. prausnitzii (OD600 2.6) cultured in YCFA media was incubated with tacrolimus (100 µg/ml) anaerobically at 37°C for 48 h. The mixture was analyzed by using HPLC/UV. B, Time profiles of tacrolimus disappearance and M1 appearance upon anaerobic incubation of tacrolimus (100 µg/ml) with F. prausnitzii.

3.2. M1 is a C9 keto-reduction metabolite of tacrolimus

To gain insight into the chemical identity of M1 and M2, high resolution mass spectrometry (HRMS) and HPLC/MS/MS experiments were performed. The m/z values of M1 and M2 were [M+Na]+ 828.4846 and 846.4974, respectively, which are consistent with the formulas C44H71NO12Na (with calculated mass of 828.4874 Da) for M1 (Supplemental Fig. 2) and C44H73NO13Na (with calculated mass of 846.4980 Da) for M2. The calculated formulas suggested M1 to be a reduction product of tacrolimus (i.e., addition of 2H to the parent tacrolimus) and M2 to be a tautomer of M1. The fragmentation pattern of M1 as compared to that of tacrolimus indicated that M1 is likely a keto-reduction product of tacrolimus (Supplemental Fig. 2 and 3).

For structural elucidation, we focused on the major product M1. M1 was mass produced by incubating large amounts of tacrolimus with F. prausnitzii, followed by purification using preparative HPLC (Supplemental Methods). The chemical structure of M1 was then determined using various spectroscopic methods. Of note, when the purified M1 was re-injected into HPLC/UV, it resolved into multiple peaks (including one corresponding to M2), indicative of isomerization and/or tautomerization of M1 into M2 (Supplemental Fig. 4). IR spectroscopy further supported that M1 is a product of a carbonyl reduction from tacrolimus (Supplemental Fig. 5). Major differences were observed in the C=O and O-H stretch regions of the IR spectra. NMR spectra showed three major isomers of M1 in CDCl3, for which all resonances were assigned (Supplemental Table 3-5). Detailed analysis of 1D and 2D NMR spectra revealed the site of carbonyl reduction at C-9 and the identity of M1 to be 9-hydroxy-tacrolimus (Supplemental Fig. 6-12). In particular, analysis of the DEPTQ spectrum of M1 revealed the absence of the resonances associated with the carbonyl carbon C-9 found in tacrolimus (δC 196.3 for the major isomer, 192.7 for the minor isomer) (Supplemental Fig. 13). Instead, three resonances consistent with the reduction of the carbonyl at C-9 to an alcohol were observed at δC 73.0 (isomer I), 68.4 (isomer II), and 69.7 ppm (isomer III). These resonances were associated with protons at δH 4.02, 4.51, and 4.37 ppm, respectively, in the HSQC spectrum. In turn, the latter resonances showed COSY correlations to exchangeable protons (δH 4.23, 3.21, and 3.58, respectively). HMBC correlations from H-9 to C-8 and C-10 were observed (Supplemental Tables 3-5), supporting the assignment of M1 as 9-hydroxy-tacrolimus. These results establish the structure of M1 as the C-9 keto-reduction product of tacrolimus (Fig. 2).

Fig 2.
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Fig 2. Chemical structures of tacrolimus and F. prausnitzii-derived metabolite M1.

M1 structure was identified using mass spectrometry and nuclear magnetic resonance spectroscopy.

3.3. M1 is a less potent immunosuppressant than tacrolimus

We compared the activities of M1 and tacrolimus by measuring PBMC proliferation after treatment with T-lymphocyte mitogen PHA.18 The 50% inhibitory concentration (IC50) of M1 was 1.97 nM whereas IC50 of tacrolimus was 0.13 nM, demonstrating that M1 was ~15-fold less potent than the parent tacrolimus in inhibiting T-lymphocyte proliferation (Fig. 3A). Tacrolimus is known to exhibit antifungal activity via the same mechanism for immunosuppression.19 To further examine the pharmacological activity of M1, an antifungal assay was performed. An aliquot of M1 or tacrolimus was placed onto a lawn of the yeast Malassezia sympodialis, and their antifungal activities were estimated based on the size of halo formed. M1 was about 10 to 20-fold less potent than tacrolimus in inhibiting the yeast growth (Fig. 3B), consistent with the results obtained from the PBMC proliferation assay. Taken together, these results demonstrate that M1 is less potent as an immunosuppressant and antifungal agent than the parent drug tacrolimus is.

Fig 3.
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Fig 3. M1 is less potent than tacrolimus as an immunosuppressant and antifungal agent.

A, Immunosuppressant activities of tacrolimus and M1 were examined in PBMCs by measuring cell proliferation after treatment with a T-lymphocyte mitogen in the presence of tacrolimus or M1. B, Antifungal activities of tacrolimus and M1 were examined using Malassezia sympodialis. The yeast was inoculated on mDixon agar plate. After 1 h incubation, an aliquot of tacrolimus or M1 at different concentrations was placed on the plate as shown on the left panel, and incubated at 37°C for 2 days.

3.4. Tacrolimus is metabolized by a wide range of commensal gut bacteria

To determine whether other gut bacteria can produce M1/M2 from tacrolimus, we obtained 22 human gut bacteria from BEI Research Resources Repository (Supplemental Table 1) and tested them for potential tacrolimus metabolism. The tested bacteria include those belonging to major Orders that are known to be highly abundant in the human gut.8, 9 Bacteria grown overnight in YCFA media anaerobically were incubated with tacrolimus (100 µg/ml) for 48 h, and the mixtures were analyzed by HPLC/UV. Apparently, gut bacteria in the Orders of Clostridiales and Erysipelotrichales, but not those in Bacteroidales and Bifidobacteriales produced M1 (Table 1 and Fig. 4A). To further verify the results, the mixtures were re-analyzed by HPLC/MS/MS which exhibits higher sensitivity than HPLC/UV. M1 production by bacteria in Clostridiales was verified (a representative chromatogram of Clostridium citroniae shown in Supplemental Fig. 14). M1 production by bacteria in Bacteroidales was detectable by HPLC/MS/MS albeit at ~100-fold lower levels than that by bacteria in Clostridiales (Supplemental Fig. 14). M1 peak was not detected upon tacrolimus incubation with Bifidobacterium longum (Supplemental Fig. 14). The formation of M1 was not observed when tacrolimus was incubated with either human or mouse hepatic microsomes (Fig. 4B; also verified by HPLC/MS/MS, data not shown), suggesting that M1 is uniquely produced by gut bacteria.

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Table 1.

Screening gut bacteria for tacrolimus conversion to M1 in YCFA culture

Fig 4.
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Fig 4. Multiple commensal gut bacteria convert tacrolimus to M1.

A, Representative chromatograms of bacteria incubated with tacrolimus. M1 non-producer (B. longum) or producers (C. aldenense, C. citroniae, and Erysipelotrichaceae sp.) cultured overnight in YCFA media was incubated with tacrolimus (100 µg/ml) anaerobically at 37°C for 48 h. The mixture was analyzed by using HPLC/UV at 210 nm. B, Mouse or human hepatic microsomes (HM; 3 mg microsomal protein/ml) were incubated with tacrolimus (100 µg/ml) at 37°C for 2 h aerobically. The mixture was analyzed by using HPLC/UV.

To examine whether tacrolimus metabolism is indeed mediated by human gut microbiota, fresh stool samples from two healthy adults were incubated with tacrolimus, and M1 production was assessed. Both stool samples produced M1, whereas the control stool samples that were boiled prior to tacrolimus incubation did not (Fig. 5). Taken together, these results show that commensal gut bacteria belonging to different genera metabolize tacrolimus into the less potent M1 metabolite.

Fig 5.
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Fig 5. Human gut microbiota convert tacrolimus to M1.

Tacrolimus (100 µg/ml) was incubated anaerobically with human stool samples from two different subjects (100 mg wet weight/ml) for 48 h at 37°C. A separate set of sample was boiled for 10 min before incubation with tacrolimus. The incubation mixtures were analyzed by HPLC/UV.

3.5. M1 is detected in transplant patients stool samples

F. prausnitzii is one of the most abundant human gut bacteria species,8, 9 and its fecal abundance was shown to have a positive correlation with oral tacrolimus dosage.12 To explore a potential role of F. prausnitzii in tacrolimus metabolism in kidney transplant recipients, we evaluated 10 stool samples from kidney transplant recipients who were taking oral tacrolimus (demographic information provided in Table 2 and Supplemental Table 2). Based upon the deep sequencing results of the V4-V5 hypervariable region of the 16S rRNA gene in stool samples, we selected 5 kidney transplant recipients whose stool samples had a relative gut abundance of F. prausnitzii greater than 25% (designated as “high F. prausnitzii” group) and 5 kidney transplant recipients whose stool samples showed no to little (if any) presence of F. prausnitzii (“low F. prausnitzii” group). We first determined the baseline levels of tacrolimus and M1 in the stool samples. M1 was detected in three of the five samples from the high F. prausnitzii group and one of the five samples from the low F. prausnitzii group (Table 2). Because M1 levels were below detection limit for the majority of samples, statistical analysis was not performed. Next, we tested the stool samples of both high and low F. prausnitzii groups for the capability of M1 production by incubating each of them with exogenously-added tacrolimus (10 µg/ml) for 24 h. M1 was detected in all 10 samples, but its amount produced was similar between the high and low F. prausnitzii groups. The 16S rDNA sequencing analysis of the stool samples revealed that gut bacteria belonging to the Order Clostridiales (a main group of bacteria that are expected to produce the majority of M1) were highly abundant in all 10 samples (Table 2). No statistically significant correlation between Clostridiales abundance and M1 production was observed (data not shown). Oral tacrolimus dose (to maintain therapeutic blood concentrations) was similar between the two groups (Table 2).

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Table 2.

M1 levels in kidney transplant patients’ stool samples.

4. Discussion

In this study, we have demonstrated that a wide range of commensal gut bacteria can metabolize tacrolimus into a novel metabolite M1 (9-hydroxy-tacrolimus). To the best of our knowledge, this represents the first experimental evidence for commensal gut bacteria being involved in the metabolism of tacrolimus.

The extent of M1’s contribution to overall immunosuppression by tacrolimus therapy is unclear. M1 is ~15-fold less potent than tacrolimus in suppressing the proliferation of activated T-lymphocytes and the growth of yeast. This result is consistent with the currently available structure-activity relationships of tacrolimus analogs; modifications at the C-9 position affect the interaction of tacrolimus with its effector protein (i.e., FK506 binding protein 12) and lead to decreased immunosuppressant activities.20 While the systemic concentrations of M1 after oral tacrolimus dosing remain to be measured, results from previous tacrolimus disposition studies using a radiolabeled compound21 indicate that the blood concentrations of metabolites are likely lower than that of tacrolimus. These results suggest that pharmacological activity originated from circulating M1 is likely less than that from tacrolimus. Of note, certain tacrolimus metabolites (e.g., 13-O-demethyltacrolimus), independently of their immunosuppressive activities, cross-react with the antibodies used in the immunoassays for measurement of tacrolimus blood concentrations, leading to overestimation of tacrolimus concentrations.1, 22 Interestingly, such overestimation could not be fully explained by the cross-reactivity of currently known tacrolimus metabolites.22 Whether the novel metabolite M1 cross-reacts with the antibodies, accounting in part for the overestimation of tacrolimus concentrations, is currently being investigated.

Multiple factors have been reported to contribute to the low and variable bioavailability of orally administered tacrolimus. These include differential expression and/or activity levels of cytochrome P450 enzymes (especially CYP3A4 and CYP3A5 isoforms) and drug transporter P-glycoprotein (P-gp) in the intestine and liver.1 Previous pharmacokinetics studies in healthy volunteers and renal transplant recipients have shown that hepatic extraction of tacrolimus is very low (i.e., 4-8%),23, 24 suggesting that low oral bioavailability of tacrolimus is mainly due to drug loss in the gut. P-gp-mediated drug efflux and intestinal CYP3A-mediated metabolism were proposed as major contributors to the loss. However, results from drug-drug interaction studies with ketoconazole (a potent inhibitor of CYP3As and P-gp) have shown that oral bioavailability of tacrolimus increases to at most ~30% when co-administered with ketoconazole,23, 24 indicating that 70% of oral dose is lost (not reaching systemic circulation) even when intestinal CYP3A and P-gp activities are blocked by ketoconazole. Our results suggest that tacrolimus conversion to M1 in the gut may represent a previously unrecognized pathway of tacrolimus elimination in the gut, and the extent of tacrolimus conversion to M1 in the gut may determine tacrolimus bioavailability. To estimate its overall contribution to systemic tacrolimus exposure, detailed understanding of M1 disposition (e.g., its intestinal absorption and elimination) is warranted.

Our results obtained from testing commensal gut bacteria for tacrolimus metabolism suggest that differences in gut bacterial composition may lead to altered tacrolimus exposure in kidney transplant recipients. Gut bacteria that extensively metabolized tacrolimus into M1 (including F. prausnitzii) belong to the Clostridiales Order. On the other hand, bacteria in Bacteroidales were found to be weak producers of M1 (i.e., detectable only by sensitive HPLC/MS/MS), and B. longum in Bifidobacteriales did not produce detectable amounts of M1. F. prausnitzii is the most abundant gut bacterium at the bacterial species level.9 However, we observed no differences in M1 production between high and low F. prausnitzii stool samples. Also, we did not observe correlation between Clostridiales abundance and M1 production in the stool samples. This may be due to the small number of samples used for this exploratory study and/or the quality of samples non-optimal for enzymatic assays. The presence of multiple factors affecting gut bacterial gene expression in vivo such as nutritional status of the gut may further explain why we do not observe a correlation between our in vitro culture-based results and in vivo abundance of gut bacteria. For example, the amino acid arginine represses the expression of the gene encoding digoxin-metabolizing enzyme in E. lenta, thus reducing digoxin elimination by gut bacteria.5 Obviously, in vitro culture-based systems do not fully reflect the bacterial functions activated in the physiological gut ecosystem. In this regard, our follow-up study is focused on the identification of the bacterial gene(s) responsible for tacrolimus metabolism. Such information will enable us to examine the prevalence and expression of tacrolimus-metabolizing enzymes in gut bacterial community and identify factors such as certain diet or drugs that alter gut bacterial composition and/or gene expression that are specific for tacrolimus metabolism.

In summary, we present the evidence of tacrolimus metabolism by gut bacteria, providing potential explanations for its low oral bioavailability. Tacrolimus metabolism into M1 likely represents a novel elimination pathway that occurs before intestinal absorption of tacrolimus. While the extent of gut metabolism of tacrolimus on variable tacrolimus exposure remains to be determined, our data provide a novel understanding of tacrolimus metabolism and may explain variability in tacrolimus exposures in kidney transplant recipients and patients with glomerular diseases on tacrolimus therapy.

Author Contributions

All authors designed the study; Y.G., C.M.C., K.-J. W., X.Y. carried out experiments; all authors analyzed the data; all authors made the figures and tables; all authors drafted and revised the paper; all authors approved the final version of the manuscript.

Supplemental Materials

  • Supplemental Methods

  • Supplemental Fig 1-14

  • Supplemental Table 1-5

Acknowledgements and Financial Disclosures

This work was supported by National Institute of Health K23 AI 124464 (J.R.L.) and a grant from the Chicago Biomedical Consortium Catalyst Award C-066 (H.J.).

J.R.L. receives research support from BioFire Diagnostics, LLC. The other authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. 1.↵
    Staatz, CE, Tett, SE: Clinical pharmacokinetics and pharmacodynamics of tacrolimus in solid organ transplantation. Clin Pharmacokinet, 43: 623-653, 2004.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    Press, RR, Ploeger, BA, den Hartigh, J, van der Straaten, T, van Pelt, J, Danhof, M, de Fijter, JW, Guchelaar, HJ: Explaining variability in tacrolimus pharmacokinetics to optimize early exposure in adult kidney transplant recipients. Ther Drug Monit, 31: 187-197, 2009.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    Shuker, N, van Gelder, T, Hesselink, DA: Intra-patient variability in tacrolimus exposure: causes, consequences for clinical management. Transplant Rev (Orlando), 29: 78-84, 2015.
    OpenUrl
  4. 4.↵
    Schroeder, BO, Backhed, F: Signals from the gut microbiota to distant organs in physiology and disease. Nat Med, 22: 1079-1089, 2016.
    OpenUrlCrossRef
  5. 5.↵
    Haiser, HJ, Gootenberg, DB, Chatman, K, Sirasani, G, Balskus, EP, Turnbaugh, PJ: Predicting and manipulating cardiac drug inactivation by the human gut bacterium Eggerthella lenta. Science, 341: 295-298, 2013.
    OpenUrlAbstract/FREE Full Text
  6. 6.
    Wallace, BD, Wang, H, Lane, KT, Scott, JE, Orans, J, Koo, JS, Venkatesh, M, Jobin, C, Yeh, LA, Mani, S, Redinbo, MR: Alleviating cancer drug toxicity by inhibiting a bacterial enzyme. Science, 330: 831-835, 2010.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Koppel, N, Maini Rekdal, V, Balskus, EP: Chemical transformation of xenobiotics by the human gut microbiota. Science, 356, 2017.
  8. 8.↵
    Qin, J, Li, R, Raes, J, Arumugam, M, Burgdorf, KS, Manichanh, C, Nielsen, T, Pons, N, Levenez, F, Yamada, T, Mende, DR, Li, J, Xu, J, Li, S, Li, D, Cao, J, Wang, B, Liang, H, Zheng, H, Xie, Y, Tap, J, Lepage, P, Bertalan, M, Batto, JM, Hansen, T, Le Paslier, D, Linneberg, A, Nielsen, HB, Pelletier, E, Renault, P, Sicheritz-Ponten, T, Turner, K, Zhu, H, Yu, C, Li, S, Jian, M, Zhou, Y, Li, Y, Zhang, X, Li, S, Qin, N, Yang, H, Wang, J, Brunak, S, Dore, J, Guarner, F, Kristiansen, K, Pedersen, O, Parkhill, J, Weissenbach, J, Meta, HITC, Bork, P, Ehrlich, SD, Wang, J: A human gut microbial gene catalogue established by metagenomic sequencing. Nature, 464: 59-65, 2010.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    Arumugam, M, Raes, J, Pelletier, E, Le Paslier, D, Yamada, T, Mende, DR, Fernandes, GR, Tap, J, Bruls, T, Batto, JM, Bertalan, M, Borruel, N, Casellas, F, Fernandez, L, Gautier, L, Hansen, T, Hattori, M, Hayashi, T, Kleerebezem, M, Kurokawa, K, Leclerc, M, Levenez, F, Manichanh, C, Nielsen, HB, Nielsen, T, Pons, N, Poulain, J, Qin, J, Sicheritz-Ponten, T, Tims, S, Torrents, D, Ugarte, E, Zoetendal, EG, Wang, J, Guarner, F, Pedersen, O, de Vos, WM, Brunak, S, Dore, J, Meta, HITC, Antolin, M, Artiguenave, F, Blottiere, HM, Almeida, M, Brechot, C, Cara, C, Chervaux, C, Cultrone, A, Delorme, C, Denariaz, G, Dervyn, R, Foerstner, KU, Friss, C, van de Guchte, M, Guedon, E, Haimet, F, Huber, W, van Hylckama-Vlieg, J, Jamet, A, Juste, C, Kaci, G, Knol, J, Lakhdari, O, Layec, S, Le Roux, K, Maguin, E, Merieux, A, Melo Minardi, R, M’Rini, C, Muller, J, Oozeer, R, Parkhill, J, Renault, P, Rescigno, M, Sanchez, N, Sunagawa, S, Torrejon, A, Turner, K, Vandemeulebrouck, G, Varela, E, Winogradsky, Y, Zeller, G, Weissenbach, J, Ehrlich, SD, Bork, P: Enterotypes of the human gut microbiome. Nature, 473: 174-180, 2011.
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.↵
    Rossi, O, van Berkel, LA, Chain, F, Tanweer Khan, M, Taverne, N, Sokol, H, Duncan, SH, Flint, HJ, Harmsen, HJ, Langella, P, Samsom, JN, Wells, JM: Faecalibacterium prausnitzii A2-165 has a high capacity to induce IL-10 in human and murine dendritic cells and modulates T cell responses. Sci Rep, 6: 18507, 2016.
    OpenUrlCrossRefPubMed
  11. 11.↵
    Miquel, S, Leclerc, M, Martin, R, Chain, F, Lenoir, M, Raguideau, S, Hudault, S, Bridonneau, C, Northen, T, Bowen, B, Bermudez-Humaran, LG, Sokol, H, Thomas, M, Langella, P: Identification of metabolic signatures linked to anti-inflammatory effects of Faecalibacterium prausnitzii. MBio, 6, 2015.
  12. 12.↵
    Lee, JR, Muthukumar, T, Dadhania, D, Taur, Y, Jenq, RR, Toussaint, NC, Ling, L, Pamer, E, Suthanthiran, M: Gut microbiota and tacrolimus dosing in kidney transplantation. PLoS One, 10: e0122399, 2015.
    OpenUrlCrossRefPubMed
  13. 13.↵
    Lee, JR, Magruder, M, Zhang, L, Westblade, LF, Satlin, MJ, Robertson, A, Edusei, E, Crawford, C, Ling, L, Taur, Y, Schluter, J, Lubetzky, M, Dadhania, D, Pamer, E, Suthanthiran, M: Gut microbiota dysbiosis and diarrhea in kidney transplant recipients. Am J Transplant, 2018.
  14. 14.↵
    Edgar, RC: UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods, 10: 996-998, 2013.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    Altschul, SF, Gish, W, Miller, W, Myers, EW, Lipman, DJ: Basic local alignment search tool. J Mol Biol, 215: 403-410, 1990.
    OpenUrlCrossRefPubMedWeb of Science
  16. 16.↵
    Tatusova, T, Ciufo, S, Fedorov, B, O’Neill, K, Tolstoy, I: RefSeq microbial genomes database: new representation and annotation strategy. Nucleic Acids Res, 42: D553-559, 2014.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    Namiki, Y, Kihara, N, Koda, S, Hane, K, Yasuda, T: Tautomeric phenomenon of a novel potent immunosuppressant (FK506) in solution. I. Isolation and structure determination of tautomeric compounds. J Antibiot (Tokyo), 46: 1149-1155, 1993.
    OpenUrlCrossRefPubMed
  18. 18.↵
    Messele, T, Roos, MT, Hamann, D, Koot, M, Fontanet, AL, Miedema, F, Schellekens, PT, Rinke de Wit, TF: Nonradioactive techniques for measurement of in vitro T-cell proliferation: alternatives to the [(3)H]thymidine incorporation assay. Clin Diagn Lab Immunol, 7: 687-692, 2000.
    OpenUrlPubMed
  19. 19.↵
    Steinbach, WJ, Reedy, JL, Cramer, RA, Jr.., Perfect, JR, Heitman, J: Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections. Nat Rev Microbiol, 5: 418-430, 2007.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    Goulet, M, Rupprecht, K, Sinclar, P, Wyvratt, M, Parsons, W: The medicinal chemistry of FK-506. Perspectives in Drug Discovery and Design, 2: 145, 1994.
    OpenUrl
  21. 21.↵
    Moller, A, Iwasaki, K, Kawamura, A, Teramura, Y, Shiraga, T, Hata, T, Schafer, A, Undre, NA: The disposition of 14C-labeled tacrolimus after intravenous and oral administration in healthy human subjects. Drug Metab Dispos, 27: 633-636, 1999.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    Dubbelboer, IR, Pohanka, A, Said, R, Rosenborg, S, Beck, O: Quantification of tacrolimus and three demethylated metabolites in human whole blood using LC-ESI-MS/MS. Ther Drug Monit, 34: 134-142, 2012.
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    Tuteja, S, Alloway, RR, Johnson, JA, Gaber, AO: The effect of gut metabolism on tacrolimus bioavailability in renal transplant recipients. Transplantation, 71: 1303-1307, 2001.
    OpenUrlCrossRefPubMed
  24. 24.↵
    Floren, LC, Bekersky, I, Benet, LZ, Mekki, Q, Dressler, D, Lee, JW, Roberts, JP, Hebert, MF: Tacrolimus oral bioavailability doubles with coadministration of ketoconazole. Clin Pharmacol Ther, 62: 41-49, 1997.
    OpenUrlCrossRefPubMedWeb of Science
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Commensal gut bacteria convert the immunosuppressant tacrolimus to less potent metabolites
Yukuang Guo, Camila Manoel Crnkovic, Kyoung-Jae Won, Xiaotong Yang, John Richard Lee, Jimmy Orjala, Hyunwoo Lee, Hyunyoung Jeong
bioRxiv 426197; doi: https://doi.org/10.1101/426197
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Commensal gut bacteria convert the immunosuppressant tacrolimus to less potent metabolites
Yukuang Guo, Camila Manoel Crnkovic, Kyoung-Jae Won, Xiaotong Yang, John Richard Lee, Jimmy Orjala, Hyunwoo Lee, Hyunyoung Jeong
bioRxiv 426197; doi: https://doi.org/10.1101/426197

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