AAC Accepts, published online ahead of print on 24 March 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.02278-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Title
2
Comparative study of the effects of anti-tuberculosis drugs and antiretroviral drugs on
3
CYP3A4 and P-glycoprotein
4 5
Running title: Effects of anti-TB and ARV drugs on CYP3A4 and P-gp
6 7
Keywords: Anti-tuberculosis drugs, drug-drug interaction, CYP3A4
8 9
Authors
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Yasuhiro Horita1 : [email protected]
11
Norio Doi1# : [email protected]
12 13
Affiliation
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1. Department of Pathophysiology and Host Defense, Japan Anti-Tuberculosis
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Association, Research Institute of Tuberculosis, 3-1-24 Matsuyama, Kiyose, Tokyo
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204-8533, Japan
17 18
#
Corresponding author: [email protected]
19
Abstract
20
Predicting drug-drug interactions (DDIs) related to cytochrome P450 (CYP) such as
21
CYP3A4 and one of the major drug transporters, P-glycoprotein (P-gp), is crucial for
22
the development of future chemotherapeutic regimens to treat tuberculosis (TB) and
23
TB/AIDS co-infection cases. We evaluated the effects of 30 anti-TB drugs, novel
24
candidates, macrolides, and representative antiretroviral drugs on human CYP3A4
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activity using a commercially available screening kit for CYP3A4 inhibitors and a
26
human hepatocyte, HepaRG. Moreover, in order to estimate the interactions of these
27
drugs with human P-gp, screening for substrates was performed. For some substrates,
28
P-gp inhibition tests were carried out using P-gp-expressing MDCK cells. As a result,
29
almost all the compounds showed the expected effects on human CYP3A4 both in the
30
in vitro screening and HepaRG cells. Importantly, the unproved mechanisms of DDIs
31
caused by WHO group-five drugs, thioamides, and p-aminosalicylic acid were
32
elucidated. Intriguingly, CFZ exhibited weak inductive effects on CYP3A4 at more than
33
0.25 μM in HepaRG, while the inhibitory effect was observed at 1.69 μM in the in vitro
34
screening, suggesting that CFZ autoinduces CYP3A4 in the human liver. Our method
35
based on one of the pharmacokinetics parameters in humans provides more practical
36
information associated with not only DDIs but also drug metabolism.
37
Introduction
38
In the development of combination regimens for tuberculosis (TB) and co-infection
39
with TB and human immunodeficiency virus (HIV), the prediction of drug-drug
40
interactions (DDI) and drug-food interactions relevant to drug-metabolizing enzymes
41
such as cytochrome P450 (CYP) and membrane transporters, especially P-glycoprotein
42
(P-gp), helps to avoid the risk of adverse reactions caused by DDIs and to maintain
43
inherent medicinal effects. According to the World Health Organization (WHO) and
44
recent reports, 12 compounds have been evaluated in clinical trials for the treatment of
45
TB, especially multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB (1,
46
2). Of those, bedaquiline was approved by the U.S. Food Drug Administration (FDA)
47
last year (3). The other compounds are likely to be introduced in turn as orphan drugs
48
into the clinical setting to treat refractory drug-resistant TB in the foreseeable future. In
49
general, M/XDR-TB has been treated with a second-line anti-TB drug combination,
50
which is less potent, more toxic, and requires a longer duration of treatment. However,
51
the combination therapy increases the frequency of adverse reactions and DDIs, and the
52
effects of some second-line drugs both on CYPs and P-gp remain unclear. For instance,
53
the effects of p-aminosalicylic acid (PAS) and clofazimine (CFZ), both of which have
54
been used for more than half a century, on CYP3A4 and P-gp are still unclear and
55
remain controversial (4, 5). In addition, the effect on CYP3A4 of thiacetazone (TAC),
56
which is preferentially metabolized by flavin-containing monooxygenase, has not been
57
officially published (6). Moreover, the mechanisms of all the unexpected DDIs between
58
a novel candidate linezolid (LZD) and the well-known CYP3A4 modulators rifampicin
59
(RIF) and clarithromycin (CLR) remain unknown (7-10). As for antiretroviral (ARV)
60
drugs, the interactions between nevirapine (NVP) and drug-metabolizing enzymes,
61
namely, CYP3A4 and CYP2B6, are not completely understood (11, 12). For these
62
reasons, there is an urgent need to clarify the hidden mechanisms of DDIs involved in
63
these compounds. On the other hand, according to post-marketing surveillance, the
64
fixed doses of some antimycobacterial drugs have been adjusted due to frequent side
65
effects, inadequate efficacy, or the extent of adverse reactions (13-16). Hence, in vitro
66
or animal studies regarding DDIs should be reassessed more practically, after drugs
67
have been released on the market.
68
In the present study, according to the guidelines on the investigation of DDIs
69
published by the FDA and European Medicines Agency (EMA), the effects of anti-TB
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drugs, novel candidates, anti-Mycobacterium avium-intracellulare complex (MAC)
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agents, and representative ARV drugs on CYP3A4 at clinically achievable
72
concentrations were evaluated in vitro by using human enzymes and transporters
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followed by ex vivo studies using a human hepatoma cell line, HepaRG cells (17, 18).
74
Furthermore, to estimate the interactions of those drugs with P-gp, substrates were
75
screened using a commercially available kit. For some substrates, P-gp inhibition tests
76
were carried out using P-gp-expressing MDCK cells, an epithelial cell line of canine
77
kidney origin.
78
Materials and Methods
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Drugs and Chemicals
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RIF, isoniazid (INH), pyrazinamide (PZA), ethambutol (EMB), PAS, ethionamide
81
(ETH), cycloserine (DCS), streptomycin (STR), amikacin (AMK), capreomycin (CAP),
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CFZ, LZD, roxithromycin (RXM), ampicillin (AMP), metronidazole (MTZ),
83
ketoconazole (KTC), carbamazepine (CMZ), ofloxacin (OFX), cyclophosphamide
84
(CPA), trypan blue (0.4%), and Eagle’s Minimum Essential Medium (EMEM) were
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purchased from Sigma-Aldrich (MO, USA). Prothionamide (PRO) and azithromycin
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(AZM) were purchased from LKT Laboratories (MN, USA). TAC and erythromycin
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(ERY) were purchased from Acros Organics (Geel, Belgium). Rifabutin (RFB),
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rifapentine (RFP), and efavirenz (EFV) were purchased from Toronto Research
89
Chemicals (Ontario, Canada). NVP and ritonavir (RIT) were purchased from United
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States Pharmacopeial Convention (MD, USA). Raltegravir (RAL) was purchased from
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Selleckchem.com. CLR, kanamycin (KAN), phenytoin (PHN), dexamethasone (DEX),
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ifosfamide (IFA), phenobarbital sodium (PB), verapamil hydrochloride (VER),
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quinidine sulfate dihydrate (QD), thiazolyl blue tetrazolium bromide (MTT),
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hydrochloric acid, sodium hydroxide, sodium dodecyl sulfate, dimethyl sulfoxide
95
(DMSO), trypsin-EDTA solution with Phenol Red and Dulbecco’s phosphate buffered
96
saline (D-PBS) were purchased from Wako Pure Chemical Industries (Osaka, Japan).
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Spironolactone (SL) was purchased from Tokyo Chemical Industry (Tokyo, Japan).
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Moxifloxacin (MXF) was obtained from Kemprotec Limited (Middlesbrough, United
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Kingdom). Rifalazil (RLZ) was obtained from Kaneka Corporation (Hyogo, Japan).
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Levofloxacin (LVX) and DC-159a were obtained from Daiichi Sankyo Corporation
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(Tokyo, Japan). Gatifloxacin (GAT) was obtained from Kyorin Pharmaceutical (Tokyo,
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Japan). Ciprofloxacin hydrochloride monohydrate (CIP) was obtained from Bayer
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HealthCare (Osaka, Japan). Amoxicillin (AMX), meropenem (MEM), and potassium
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clavulanate (CLA) were supplied by Meiji Seika Pharma (Osaka, Japan). Enviomycin
105
(EVM) was obtained from Asahi Kasei Pharma Corporation (Tokyo, Japan).
106 107
Screening for CYP3A4 inhibitors
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CYP3A4/BFC High Throughput Inhibitor Screening Kit was purchased from BD
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Biosciences (MA, USA). This experiment was performed to measure the inhibitory
110
effects of each agent on the metabolism of a non-fluorescent CYP3A4 substrate
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7-benzyloxy-trifluoromethylcoumarin
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7-hydroxyl-trifluoromethylcoumarin
113
instructions, except for the plate-reading conditions which were set at an excitation of
(BFC) (HFC)
to
a
according
fluorescent to
the
metabolite
manufacturer’s
114
405 nm and an emission of 535 nm. The half maximal inhibitory concentration (IC50)
115
values of each drug were calculated by linear interpolation. The standard curve of HFC
116
was prepared over the range from 2000 to 0.9 pmol. KTC and VER were used as
117
positive control inhibitors.
118 119
Cytotoxicity test. Cytotoxicity testing was implemented by the modified MTT assay
120
(19, 20). DMSO and sodium hydroxide with high concentrations were used as positive
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control agents. Briefly, after the end of a differentiation period, HepaRG cells (7.2×104
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cells/well for a 96-well plate) were treated daily with and without test compound in
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triplicate and were incubated in a humidified 5% CO2 atmosphere at 37°C for 48 hours.
124 125 126
CYP3A4 induction and inhibition test using HepaRG cells
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An HepaRG culture kit for 50 plates, working growth medium 710, working
128
differentiation medium 720, and induction medium 640 were purchased from Biopredic
129
International (Rennes, France). Trypan blue (0.4%) was diluted with D-PBS at a final
130
concentration of 0.05% before use. The procedures of thawing, seeding, maintenance,
131
differentiation, and freezing of HepaRG cells were performed according to the
132
instructions for this kit. After the end of the differentiation period, cells were treated
133
daily with and without test compound at three concentrations in triplicate and were
134
incubated in a humidified 5% CO2 atmosphere at 37°C for 48 hours. In principle, the
135
highest concentration of each drug was set at more than two times higher than the
136
maximum concentration value achieved in the human blood (Cmax) at the generally
137
recommended dosage, because the drug concentration in the portal vein during the
138
process of absorption is generally greater than Cmax values after oral administration (18).
139
Cmax values were obtained from pharmaceutical package inserts, Handbook of
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Anti-Tuberculosis Agents, and previously published reports (21). In brief, cells were
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incubated in the presence of 50 μM midazolam in EMEM for one hour at 37°C. Each
142
supernatant was transferred into a new glass tube, followed by 100 μl of methanol and
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100 μl of 0.05 μg/ml diazepam dissolved in acetonitrile. The mixed suspension was
144
evaporated to dryness at 60°C for 80 minutes. Thereafter, a 200-μl aliquot of methanol
145
was added, sonicated for 2 seconds, and vortexed for 5 seconds. Finally, the solution
146
was filtered through a 0.45-μm syringe filter and injected in a high-performance liquid
147
chromatography-mass spectrometry (LC/MS) system.
148
The average percentage of 1-hydroxymidazolam production in wells treated with the
149
test compound was divided from that in wells treated with 0.1% DMSO. Each value
150
was expressed as the mean and standard deviation (n=6). Statistical analysis was
151
conducted by one-way layout regression analysis using Microsoft Office Excel 2010
152
software. Statistical significance was set at P values of 10 μM) as well as LZD (IC50 > 500 μM) (data not shown). These
309
results indicate that CFZ is a substrate for human P-gp. As for ARV drugs, NVP, EFV,
310
and RIT were found to be substrates for P-gp with Vmax values of 36.2, 14.2, and 20.6
311
nmol/minute, respectively (Table 5).
312
Discussion
313
CYP3A4 is by far the most abundant isoform of CYPs in the human liver and is
314
reckoned to be a key enzyme related to DDIs occurring in the treatment of TB,
315
especially drug-resistant TB, and co-infection with TB/AIDS (31). To date, the primary
316
human hepatocyte has been generally used as a gold standard to determine the effects of
317
test compounds on drug-metabolizing enzyme activities during preclinical development.
318
Their usage, however, is limited for several reasons: high cost, insufficient availability
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of donor organs, interdonor functional variability, and limited life span (32). Meanwhile,
320
the well-established human hepatoblastoma, HepG2, can also be used for the same
321
application and cytotoxicity testing because of its good proliferation, immortality, and
322
stable viability, but intrinsic enzyme activities and the reactivity of CYPs, especially
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CYP3A4 and CYP2B6, to CYP modulators are entirely inadequate, even in the
324
three-dimensional culture of HepG2 cells (33-35). Similarly, while immortalized human
325
hepatic cell lines, i.e. Fa2N-4 cells and ADV-1 cells that are derived from BC2 cells,
326
have been reported since 2000, the inherent characteristics of those lines restrict their
327
routine application in the drug-discovery setting (36, 37). For instance, the former does
328
not respond to a prototypical inducer of CYP2B6 and so far, the functions of the latter
329
have not been validated in detail as compared with HepaRG cells. In contrast, the
330
HepaRG cell line has been reported to be useful for evaluating the effects of drugs on
331
CYP3A4 superior to the cell lines (38). For these reasons, we evaluated the effects of 30
332
anti-TB drugs, novel candidates, macrolides, and ARV drugs on human CYP3A4
333
activity using HepaRG cells in a single experimental system. There were no cytotoxic
334
activities at any given concentration, except for EFV at the concentration of 50 μM with
335
and without RIF (data not shown). Throughout the ex vivo studies using HepaRG,
336
almost all compounds showed expected effects both on intrinsic and RIF-mediated
337
CYP3A4 activity. Among the CYP3A4 inhibitors determined by the in vitro screening,
338
INH, PAS, and macrolides showed less potent CYP3A4 inhibitory effects in HepaRG
339
than in HLMs. Thioamides showed little or no influence on CYP3A4 activity in
340
HepaRG in contrast with the results in HLMs. Importantly, in the in vitro screening
341
using HLMs, drug-metabolizing enzymes were hardly induced within the incubation
342
period (30 minutes), which indicates that autoinducers are likely to be mistaken for
343
inhibitors. Indeed, the well-established CYP3A4 inducers such as rifamycins and EFV
344
were determined as CYP3A4 inhibitors (Table 2). Nevertheless, this screening assay is
345
useful to find inhibitors quickly in practice and is far superior to ex vivo experiments in
346
terms of time required, simple procedures, and total cost.
347
Secondly, based on our results, we attempted to clarify the unrevealed mechanisms of
348
DDIs in the treatment of TB and AIDS. A previous clinical report regarding DDIs
349
between CFZ and INH (39) indicated that INH caused the increase of CFZ plasma and
350
urine concentration by up to 3-fold and 1.9-fold, respectively, with the exception of one
351
patient, suggesting that INH inhibits the CYP3A4-mediated metabolism of CFZ and
352
consequently leads to the accumulation of unchanged CFZ in the human body. Similarly,
353
in an experimental mouse model, a modest increase in the plasma concentration of CFZ
354
when combined with INH has been demonstrated (40). In addition, the DDI between
355
CFZ- and RIF-based regimens for leprosy has been reported (41). Notably, CFZ
356
delayed the absorption of RIF and decreased the Cmax value and area under the
357
time-concentration curve (AUC) for RIF, which might result from the additional
358
induction of intestinal and hepatic CYP3A4 by CFZ as shown in Table 4. This inference
359
could apply to other reports regarding DDIs between CFZ and physiologically active
360
substances such as steroid hormone and vitamin A, both of which are known as
361
CYP3A4 substrates (5). These results could support the interpretation of unknown
362
mechanisms on DDIs between CFZ and anti-TB drugs, i.e. INH and RIF (39, 42, 43). In
363
regard to the novel drugs and candidates undergoing preclinical and/or clinical trials,
364
bedaquiline, PA-824, TBA-354, and sutezolid have been reported to be substrates for
365
CYP3A4 (44, 45). In particular, PA-824 and TBA-354 have been shown to have weak
366
inhibitory effects on CYP3A4 (46). Therefore, it should be kept in mind that the
367
pharmacological influence of CFZ on the metabolism of co-administered drugs is
368
inevitable and careful clinical monitoring might be warranted throughout the
369
development of CFZ-containing regimens for the treatment of M/XDR-TB and other
370
mycobacterial infections.
371
Previously, the effects of NVP on CYP3A4 mRNA expression and CYP enzyme
372
activities have been examined using three lots of cryopreserved human hepatocytes (47).
373
While NVP has caused up to 21-fold changes in CYP3A4 mRNA expression, no
374
obvious induction of testosterone 6β-hydroxylase activity has been detected in two out
375
of three lots. In spite of the differences in experimental design, these results accord with
376
our findings. Hence, we inferred that the DDIs between NVP and CYP3A4 modulators
377
such as azoles, HIV protease inhibitors, and oral contraceptives would depend on
378
CYP2B6 induction by NVP, because these CYP3A4 modulators have been proved to be
379
substrates for CYP2B6 (29, 48, 49). However, the increased metabolism of ERY
380
mediated by NVP cannot be explained with this hypothesis, because macrolides have no
381
bearing on CYP2B6 activities (data not shown) (50). In the light of previous studies,
382
NVP might exhibit both inductive and inhibitory effects on CYP3A4 activity on a
383
case-by-case basis (47). Further studies are needed to solve this issue.
384
Nowadays, unexpected DDIs between LZD and CYP3A4 modulators have caused
385
considerable concern in the long-term treatment of infections, encompassing
386
M/XDR-TB (7, 10, 51). In the present study, LZD showed an inhibitory effect on
387
CYP3A4 with the IC50 value of 1.2 mM (406 μg/ml) in HLMs, while no inductive and
388
inhibitory effects on CYP3A4 were observed even at high concentrations (200 μM) in
389
HepaRG, regardless of the presence of RIF (Table 2, 3, S3). Additionally, there was no
390
inhibitory effect on P-gp even at the concentration of 500 μM both in the in vitro
391
screening and P-gp-expressing MDCK cells (Table S4). These results support the
392
previous report using human primary hepatocytes (52). We also assessed the effect of
393
LZD on CYP2B6 using an in vitro screening kit, but no inhibition was observed at 500
394
μM (data not shown). Taken together, LZD is a potential substrate with weak affinity to
395
CYP3A4, similar to sutezolid. With the aforementioned assumption, the following
396
mechanisms of 1) increased clearance of LZD both in healthy volunteers and patients
397
co-administrated with RIF, 2) increased serum AUC0-12h and decreased the elimination
398
of LZD in patients receiving CLR, 3) potential DDI between LZD and aztreonam, and
399
4) inhibition of LZD metabolism in the presence of a well-known CYP3A4 inhibitor
400
KTC might be elucidated (7, 10, 53, 54). Based on this concept, both thioamides and
401
PAS could be substrates for CYP3A4 enzymes.
402
Unexpectedly, CFZ exerted no inhibitory effect on P-gp activity in human
403
P-gp-expressing MDCK cells with concentrations as high as 10 μM, like LZD, whereas
404
the opposite data have been reported using either human lung cancer cells or
405
erythroleukemia cells (data not shown) (55, 56). This discrepancy would be due to the
406
differences in the origin of cells tested. If so, similar experiments should be performed
407
using cells derived from the human liver and small intestine.
408
One limitation of this study is that we didn’t assess the DDIs related to organic
409
anion-transporting polypeptides (OATPs) that play an important role in hepatic uptake
410
of pharmaceutical drugs. Recently, some drugs available for the treatment of TB and
411
AIDS, e.g. rifampicin and ritonavir, have been reported to be OATP1B1 inhibitors
412
and/or substrates (17, 57-59).
413
SLCO2B1, and SLCO1B3 genes have been demonstrated in differentiated HepaRG cells
414
as well as in primary hepatocytes, implying that OATP1B1, OATP2B1, and OATP1B3
415
are active in HepaRG cells (60). Hence, assessing the interactions between the drugs
416
and OATPs is of importance in understanding the drug transport across the biological
417
membrane in HepaRG cells. Further studies are warranted to explore hidden DDIs
418
between anti-TB drugs and OATPs.
419
Actually, the mRNA expression levels of the SLCO1B1,
Recently, mouse models using PXB-mouse and PXR-humanized mouse have been
420
proved to be powerful tools for investigating the DDIs relevant to human
421
drug-metabolizing enzymes such as CYP3A4 and CYP2B6 (61-63). In accordance with
422
these studies, the inductive effect of RIF and inhibitory effect of KTC on CYP3A4
423
activity were observed in HepaRG cells, implying that the ex vivo studies reflect
424
experiments using mouse models (62, 64) (Table 3, S3). Further investigation for
425
assessing the data correlation among these models is desired.
426
In conclusion, HepaRG is useful for estimating the inductive and/or inhibitory effects
427
of drugs and chemicals on CYP3A4 activity. Whereas the ex vivo study itself is
428
inconclusive, it sheds light on the pathway for developing safety regimens for the
429
treatment of TB, MAC, and AIDS. When officially determining a suitable dose in
430
humans, the effects of pharmaceutical drugs on drug-metabolizing enzymes and
431
membrane transporters which are likely to be the causative factors involved in DDIs
432
should be re-evaluated at clinically achievable concentrations.
433 434
Acknowledgments
435
Amoxicillin, meropenem, and potassium clavulanate were kindly provided by Meiji
436
Seika Pharma Co., Ltd. We also thank Dr. Helen Gill and coworkers (Cyprotex, UK) for
437
performing part of the experiments on this work and their instructive comments.
438
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658 659
660
Figure Legends
661
Figure 1. Differences in the inhibitory effect of nevirapine (NVP) on CYP3A4 induction
662
mediated by various CYP3A4 inducers. The inhibitory effects of NVP were estimated
663
by the amount of production for 1-hydroxymidazolam. Experiments were carried out in
664
triplicate and representative data of two separate experiments are shown. RIF,
665
rifampicin; RFB, rifabutin; EFV, efavirenz; PB, phenobarbital; CMZ, carbamazepine.
666
The numbers in parentheses represent the exposed micromolar concentrations for each
667
CYP3A4 inducer.
668
669
670
671
672
1
Title
2
Comparative study of the effects of anti-tuberculosis drugs and antiretroviral drugs on
3
CYP3A4 and P-glycoprotein
4 5
Running title: Effects of anti-TB and ARV drugs on CYP3A4 and P-gp
6 7
Keywords: Anti-tuberculosis drugs, drug-drug interaction, CYP3A4
8 9
Authors
10
Yasuhiro Horita1 : [email protected]
11
Norio Doi1# : [email protected]
12 13
Affiliation
14
1. Department of Pathophysiology and Host Defense, Japan Anti-Tuberculosis
15
Association, Research Institute of Tuberculosis, 3-1-24 Matsuyama, Kiyose, Tokyo
16
204-8533, Japan
17 18
#
Corresponding author: [email protected]
19
Abstract
20
Predicting drug-drug interactions (DDIs) related to cytochrome P450 (CYP) such as
21
CYP3A4 and one of the major drug transporters, P-glycoprotein (P-gp), is crucial for
22
the development of future chemotherapeutic regimens to treat tuberculosis (TB) and
23
TB/AIDS co-infection cases. We evaluated the effects of 30 anti-TB drugs, novel
24
candidates, macrolides, and representative antiretroviral drugs on human CYP3A4
25
activity using a commercially available screening kit for CYP3A4 inhibitors and a
26
human hepatocyte, HepaRG. Moreover, in order to estimate the interactions of these
27
drugs with human P-gp, screening for substrates was performed. For some substrates,
28
P-gp inhibition tests were carried out using P-gp-expressing MDCK cells. As a result,
29
almost all the compounds showed the expected effects on human CYP3A4 both in the
30
in vitro screening and HepaRG cells. Importantly, the unproved mechanisms of DDIs
31
caused by WHO group-five drugs, thioamides, and p-aminosalicylic acid were
32
elucidated. Intriguingly, CFZ exhibited weak inductive effects on CYP3A4 at more than
33
0.25 μM in HepaRG, while the inhibitory effect was observed at 1.69 μM in the in vitro
34
screening, suggesting that CFZ autoinduces CYP3A4 in the human liver. Our method
35
based on one of the pharmacokinetics parameters in humans provides more practical
36
information associated with not only DDIs but also drug metabolism.
37
Introduction
38
In the development of combination regimens for tuberculosis (TB) and co-infection
39
with TB and human immunodeficiency virus (HIV), the prediction of drug-drug
40
interactions (DDI) and drug-food interactions relevant to drug-metabolizing enzymes
41
such as cytochrome P450 (CYP) and membrane transporters, especially P-glycoprotein
42
(P-gp), helps to avoid the risk of adverse reactions caused by DDIs and to maintain
43
inherent medicinal effects. According to the World Health Organization (WHO) and
44
recent reports, 12 compounds have been evaluated in clinical trials for the treatment of
45
TB, especially multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB (1,
46
2). Of those, bedaquiline was approved by the U.S. Food Drug Administration (FDA)
47
last year (3). The other compounds are likely to be introduced in turn as orphan drugs
48
into the clinical setting to treat refractory drug-resistant TB in the foreseeable future. In
49
general, M/XDR-TB has been treated with a second-line anti-TB drug combination,
50
which is less potent, more toxic, and requires a longer duration of treatment. However,
51
the combination therapy increases the frequency of adverse reactions and DDIs, and the
52
effects of some second-line drugs both on CYPs and P-gp remain unclear. For instance,
53
the effects of p-aminosalicylic acid (PAS) and clofazimine (CFZ), both of which have
54
been used for more than half a century, on CYP3A4 and P-gp are still unclear and
55
remain controversial (4, 5). In addition, the effect on CYP3A4 of thiacetazone (TAC),
56
which is preferentially metabolized by flavin-containing monooxygenase, has not been
57
officially published (6). Moreover, the mechanisms of all the unexpected DDIs between
58
a novel candidate linezolid (LZD) and the well-known CYP3A4 modulators rifampicin
59
(RIF) and clarithromycin (CLR) remain unknown (7-10). As for antiretroviral (ARV)
60
drugs, the interactions between nevirapine (NVP) and drug-metabolizing enzymes,
61
namely, CYP3A4 and CYP2B6, are not completely understood (11, 12). For these
62
reasons, there is an urgent need to clarify the hidden mechanisms of DDIs involved in
63
these compounds. On the other hand, according to post-marketing surveillance, the
64
fixed doses of some antimycobacterial drugs have been adjusted due to frequent side
65
effects, inadequate efficacy, or the extent of adverse reactions (13-16). Hence, in vitro
66
or animal studies regarding DDIs should be reassessed more practically, after drugs
67
have been released on the market.
68
In the present study, according to the guidelines on the investigation of DDIs
69
published by the FDA and European Medicines Agency (EMA), the effects of anti-TB
70
drugs, novel candidates, anti-Mycobacterium avium-intracellulare complex (MAC)
71
agents, and representative ARV drugs on CYP3A4 at clinically achievable
72
concentrations were evaluated in vitro by using human enzymes and transporters
73
followed by ex vivo studies using a human hepatoma cell line, HepaRG cells (17, 18).
74
Furthermore, to estimate the interactions of those drugs with P-gp, substrates were
75
screened using a commercially available kit. For some substrates, P-gp inhibition tests
76
were carried out using P-gp-expressing MDCK cells, an epithelial cell line of canine
77
kidney origin.
78
Materials and Methods
79
Drugs and Chemicals
80
RIF, isoniazid (INH), pyrazinamide (PZA), ethambutol (EMB), PAS, ethionamide
81
(ETH), cycloserine (DCS), streptomycin (STR), amikacin (AMK), capreomycin (CAP),
82
CFZ, LZD, roxithromycin (RXM), ampicillin (AMP), metronidazole (MTZ),
83
ketoconazole (KTC), carbamazepine (CMZ), ofloxacin (OFX), cyclophosphamide
84
(CPA), trypan blue (0.4%), and Eagle’s Minimum Essential Medium (EMEM) were
85
purchased from Sigma-Aldrich (MO, USA). Prothionamide (PRO) and azithromycin
86
(AZM) were purchased from LKT Laboratories (MN, USA). TAC and erythromycin
87
(ERY) were purchased from Acros Organics (Geel, Belgium). Rifabutin (RFB),
88
rifapentine (RFP), and efavirenz (EFV) were purchased from Toronto Research
89
Chemicals (Ontario, Canada). NVP and ritonavir (RIT) were purchased from United
90
States Pharmacopeial Convention (MD, USA). Raltegravir (RAL) was purchased from
91
Selleckchem.com. CLR, kanamycin (KAN), phenytoin (PHN), dexamethasone (DEX),
92
ifosfamide (IFA), phenobarbital sodium (PB), verapamil hydrochloride (VER),
93
quinidine sulfate dihydrate (QD), thiazolyl blue tetrazolium bromide (MTT),
94
hydrochloric acid, sodium hydroxide, sodium dodecyl sulfate, dimethyl sulfoxide
95
(DMSO), trypsin-EDTA solution with Phenol Red and Dulbecco’s phosphate buffered
96
saline (D-PBS) were purchased from Wako Pure Chemical Industries (Osaka, Japan).
97
Spironolactone (SL) was purchased from Tokyo Chemical Industry (Tokyo, Japan).
98
Moxifloxacin (MXF) was obtained from Kemprotec Limited (Middlesbrough, United
99
Kingdom). Rifalazil (RLZ) was obtained from Kaneka Corporation (Hyogo, Japan).
100
Levofloxacin (LVX) and DC-159a were obtained from Daiichi Sankyo Corporation
101
(Tokyo, Japan). Gatifloxacin (GAT) was obtained from Kyorin Pharmaceutical (Tokyo,
102
Japan). Ciprofloxacin hydrochloride monohydrate (CIP) was obtained from Bayer
103
HealthCare (Osaka, Japan). Amoxicillin (AMX), meropenem (MEM), and potassium
104
clavulanate (CLA) were supplied by Meiji Seika Pharma (Osaka, Japan). Enviomycin
105
(EVM) was obtained from Asahi Kasei Pharma Corporation (Tokyo, Japan).
106 107
Screening for CYP3A4 inhibitors
108
CYP3A4/BFC High Throughput Inhibitor Screening Kit was purchased from BD
109
Biosciences (MA, USA). This experiment was performed to measure the inhibitory
110
effects of each agent on the metabolism of a non-fluorescent CYP3A4 substrate
111
7-benzyloxy-trifluoromethylcoumarin
112
7-hydroxyl-trifluoromethylcoumarin
113
instructions, except for the plate-reading conditions which were set at an excitation of
(BFC) (HFC)
to
a
according
fluorescent to
the
metabolite
manufacturer’s
114
405 nm and an emission of 535 nm. The half maximal inhibitory concentration (IC50)
115
values of each drug were calculated by linear interpolation. The standard curve of HFC
116
was prepared over the range from 2000 to 0.9 pmol. KTC and VER were used as
117
positive control inhibitors.
118 119
Cytotoxicity test. Cytotoxicity testing was implemented by the modified MTT assay
120
(19, 20). DMSO and sodium hydroxide with high concentrations were used as positive
121
control agents. Briefly, after the end of a differentiation period, HepaRG cells (7.2×104
122
cells/well for a 96-well plate) were treated daily with and without test compound in
123
triplicate and were incubated in a humidified 5% CO2 atmosphere at 37°C for 48 hours.
124 125 126
CYP3A4 induction and inhibition test using HepaRG cells
127
An HepaRG culture kit for 50 plates, working growth medium 710, working
128
differentiation medium 720, and induction medium 640 were purchased from Biopredic
129
International (Rennes, France). Trypan blue (0.4%) was diluted with D-PBS at a final
130
concentration of 0.05% before use. The procedures of thawing, seeding, maintenance,
131
differentiation, and freezing of HepaRG cells were performed according to the
132
instructions for this kit. After the end of the differentiation period, cells were treated
133
daily with and without test compound at three concentrations in triplicate and were
134
incubated in a humidified 5% CO2 atmosphere at 37°C for 48 hours. In principle, the
135
highest concentration of each drug was set at more than two times higher than the
136
maximum concentration value achieved in the human blood (Cmax) at the generally
137
recommended dosage, because the drug concentration in the portal vein during the
138
process of absorption is generally greater than Cmax values after oral administration (18).
139
Cmax values were obtained from pharmaceutical package inserts, Handbook of
140
Anti-Tuberculosis Agents, and previously published reports (21). In brief, cells were
141
incubated in the presence of 50 μM midazolam in EMEM for one hour at 37°C. Each
142
supernatant was transferred into a new glass tube, followed by 100 μl of methanol and
143
100 μl of 0.05 μg/ml diazepam dissolved in acetonitrile. The mixed suspension was
144
evaporated to dryness at 60°C for 80 minutes. Thereafter, a 200-μl aliquot of methanol
145
was added, sonicated for 2 seconds, and vortexed for 5 seconds. Finally, the solution
146
was filtered through a 0.45-μm syringe filter and injected in a high-performance liquid
147
chromatography-mass spectrometry (LC/MS) system.
148
The average percentage of 1-hydroxymidazolam production in wells treated with the
149
test compound was divided from that in wells treated with 0.1% DMSO. Each value
150
was expressed as the mean and standard deviation (n=6). Statistical analysis was
151
conducted by one-way layout regression analysis using Microsoft Office Excel 2010
152
software. Statistical significance was set at P values of 10 μM) as well as LZD (IC50 > 500 μM) (data not shown). These
309
results indicate that CFZ is a substrate for human P-gp. As for ARV drugs, NVP, EFV,
310
and RIT were found to be substrates for P-gp with Vmax values of 36.2, 14.2, and 20.6
311
nmol/minute, respectively (Table 5).
312
Discussion
313
CYP3A4 is by far the most abundant isoform of CYPs in the human liver and is
314
reckoned to be a key enzyme related to DDIs occurring in the treatment of TB,
315
especially drug-resistant TB, and co-infection with TB/AIDS (31). To date, the primary
316
human hepatocyte has been generally used as a gold standard to determine the effects of
317
test compounds on drug-metabolizing enzyme activities during preclinical development.
318
Their usage, however, is limited for several reasons: high cost, insufficient availability
319
of donor organs, interdonor functional variability, and limited life span (32). Meanwhile,
320
the well-established human hepatoblastoma, HepG2, can also be used for the same
321
application and cytotoxicity testing because of its good proliferation, immortality, and
322
stable viability, but intrinsic enzyme activities and the reactivity of CYPs, especially
323
CYP3A4 and CYP2B6, to CYP modulators are entirely inadequate, even in the
324
three-dimensional culture of HepG2 cells (33-35). Similarly, while immortalized human
325
hepatic cell lines, i.e. Fa2N-4 cells and ADV-1 cells that are derived from BC2 cells,
326
have been reported since 2000, the inherent characteristics of those lines restrict their
327
routine application in the drug-discovery setting (36, 37). For instance, the former does
328
not respond to a prototypical inducer of CYP2B6 and so far, the functions of the latter
329
have not been validated in detail as compared with HepaRG cells. In contrast, the
330
HepaRG cell line has been reported to be useful for evaluating the effects of drugs on
331
CYP3A4 superior to the cell lines (38). For these reasons, we evaluated the effects of 30
332
anti-TB drugs, novel candidates, macrolides, and ARV drugs on human CYP3A4
333
activity using HepaRG cells in a single experimental system. There were no cytotoxic
334
activities at any given concentration, except for EFV at the concentration of 50 μM with
335
and without RIF (data not shown). Throughout the ex vivo studies using HepaRG,
336
almost all compounds showed expected effects both on intrinsic and RIF-mediated
337
CYP3A4 activity. Among the CYP3A4 inhibitors determined by the in vitro screening,
338
INH, PAS, and macrolides showed less potent CYP3A4 inhibitory effects in HepaRG
339
than in HLMs. Thioamides showed little or no influence on CYP3A4 activity in
340
HepaRG in contrast with the results in HLMs. Importantly, in the in vitro screening
341
using HLMs, drug-metabolizing enzymes were hardly induced within the incubation
342
period (30 minutes), which indicates that autoinducers are likely to be mistaken for
343
inhibitors. Indeed, the well-established CYP3A4 inducers such as rifamycins and EFV
344
were determined as CYP3A4 inhibitors (Table 2). Nevertheless, this screening assay is
345
useful to find inhibitors quickly in practice and is far superior to ex vivo experiments in
346
terms of time required, simple procedures, and total cost.
347
Secondly, based on our results, we attempted to clarify the unrevealed mechanisms of
348
DDIs in the treatment of TB and AIDS. A previous clinical report regarding DDIs
349
between CFZ and INH (39) indicated that INH caused the increase of CFZ plasma and
350
urine concentration by up to 3-fold and 1.9-fold, respectively, with the exception of one
351
patient, suggesting that INH inhibits the CYP3A4-mediated metabolism of CFZ and
352
consequently leads to the accumulation of unchanged CFZ in the human body. Similarly,
353
in an experimental mouse model, a modest increase in the plasma concentration of CFZ
354
when combined with INH has been demonstrated (40). In addition, the DDI between
355
CFZ- and RIF-based regimens for leprosy has been reported (41). Notably, CFZ
356
delayed the absorption of RIF and decreased the Cmax value and area under the
357
time-concentration curve (AUC) for RIF, which might result from the additional
358
induction of intestinal and hepatic CYP3A4 by CFZ as shown in Table 4. This inference
359
could apply to other reports regarding DDIs between CFZ and physiologically active
360
substances such as steroid hormone and vitamin A, both of which are known as
361
CYP3A4 substrates (5). These results could support the interpretation of unknown
362
mechanisms on DDIs between CFZ and anti-TB drugs, i.e. INH and RIF (39, 42, 43). In
363
regard to the novel drugs and candidates undergoing preclinical and/or clinical trials,
364
bedaquiline, PA-824, TBA-354, and sutezolid have been reported to be substrates for
365
CYP3A4 (44, 45). In particular, PA-824 and TBA-354 have been shown to have weak
366
inhibitory effects on CYP3A4 (46). Therefore, it should be kept in mind that the
367
pharmacological influence of CFZ on the metabolism of co-administered drugs is
368
inevitable and careful clinical monitoring might be warranted throughout the
369
development of CFZ-containing regimens for the treatment of M/XDR-TB and other
370
mycobacterial infections.
371
Previously, the effects of NVP on CYP3A4 mRNA expression and CYP enzyme
372
activities have been examined using three lots of cryopreserved human hepatocytes (47).
373
While NVP has caused up to 21-fold changes in CYP3A4 mRNA expression, no
374
obvious induction of testosterone 6β-hydroxylase activity has been detected in two out
375
of three lots. In spite of the differences in experimental design, these results accord with
376
our findings. Hence, we inferred that the DDIs between NVP and CYP3A4 modulators
377
such as azoles, HIV protease inhibitors, and oral contraceptives would depend on
378
CYP2B6 induction by NVP, because these CYP3A4 modulators have been proved to be
379
substrates for CYP2B6 (29, 48, 49). However, the increased metabolism of ERY
380
mediated by NVP cannot be explained with this hypothesis, because macrolides have no
381
bearing on CYP2B6 activities (data not shown) (50). In the light of previous studies,
382
NVP might exhibit both inductive and inhibitory effects on CYP3A4 activity on a
383
case-by-case basis (47). Further studies are needed to solve this issue.
384
Nowadays, unexpected DDIs between LZD and CYP3A4 modulators have caused
385
considerable concern in the long-term treatment of infections, encompassing
386
M/XDR-TB (7, 10, 51). In the present study, LZD showed an inhibitory effect on
387
CYP3A4 with the IC50 value of 1.2 mM (406 μg/ml) in HLMs, while no inductive and
388
inhibitory effects on CYP3A4 were observed even at high concentrations (200 μM) in
389
HepaRG, regardless of the presence of RIF (Table 2, 3, S3). Additionally, there was no
390
inhibitory effect on P-gp even at the concentration of 500 μM both in the in vitro
391
screening and P-gp-expressing MDCK cells (Table S4). These results support the
392
previous report using human primary hepatocytes (52). We also assessed the effect of
393
LZD on CYP2B6 using an in vitro screening kit, but no inhibition was observed at 500
394
μM (data not shown). Taken together, LZD is a potential substrate with weak affinity to
395
CYP3A4, similar to sutezolid. With the aforementioned assumption, the following
396
mechanisms of 1) increased clearance of LZD both in healthy volunteers and patients
397
co-administrated with RIF, 2) increased serum AUC0-12h and decreased the elimination
398
of LZD in patients receiving CLR, 3) potential DDI between LZD and aztreonam, and
399
4) inhibition of LZD metabolism in the presence of a well-known CYP3A4 inhibitor
400
KTC might be elucidated (7, 10, 53, 54). Based on this concept, both thioamides and
401
PAS could be substrates for CYP3A4 enzymes.
402
Unexpectedly, CFZ exerted no inhibitory effect on P-gp activity in human
403
P-gp-expressing MDCK cells with concentrations as high as 10 μM, like LZD, whereas
404
the opposite data have been reported using either human lung cancer cells or
405
erythroleukemia cells (data not shown) (55, 56). This discrepancy would be due to the
406
differences in the origin of cells tested. If so, similar experiments should be performed
407
using cells derived from the human liver and small intestine.
408
One limitation of this study is that we didn’t assess the DDIs related to organic
409
anion-transporting polypeptides (OATPs) that play an important role in hepatic uptake
410
of pharmaceutical drugs. Recently, some drugs available for the treatment of TB and
411
AIDS, e.g. rifampicin and ritonavir, have been reported to be OATP1B1 inhibitors
412
and/or substrates (17, 57-59).
413
SLCO2B1, and SLCO1B3 genes have been demonstrated in differentiated HepaRG cells
414
as well as in primary hepatocytes, implying that OATP1B1, OATP2B1, and OATP1B3
415
are active in HepaRG cells (60). Hence, assessing the interactions between the drugs
416
and OATPs is of importance in understanding the drug transport across the biological
417
membrane in HepaRG cells. Further studies are warranted to explore hidden DDIs
418
between anti-TB drugs and OATPs.
419
Actually, the mRNA expression levels of the SLCO1B1,
Recently, mouse models using PXB-mouse and PXR-humanized mouse have been
420
proved to be powerful tools for investigating the DDIs relevant to human
421
drug-metabolizing enzymes such as CYP3A4 and CYP2B6 (61-63). In accordance with
422
these studies, the inductive effect of RIF and inhibitory effect of KTC on CYP3A4
423
activity were observed in HepaRG cells, implying that the ex vivo studies reflect
424
experiments using mouse models (62, 64) (Table 3, S3). Further investigation for
425
assessing the data correlation among these models is desired.
426
In conclusion, HepaRG is useful for estimating the inductive and/or inhibitory effects
427
of drugs and chemicals on CYP3A4 activity. Whereas the ex vivo study itself is
428
inconclusive, it sheds light on the pathway for developing safety regimens for the
429
treatment of TB, MAC, and AIDS. When officially determining a suitable dose in
430
humans, the effects of pharmaceutical drugs on drug-metabolizing enzymes and
431
membrane transporters which are likely to be the causative factors involved in DDIs
432
should be re-evaluated at clinically achievable concentrations.
433 434
Acknowledgments
435
Amoxicillin, meropenem, and potassium clavulanate were kindly provided by Meiji
436
Seika Pharma Co., Ltd. We also thank Dr. Helen Gill and coworkers (Cyprotex, UK) for
437
performing part of the experiments on this work and their instructive comments.
438
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Figure Legends
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Figure 1. Differences in the inhibitory effect of nevirapine (NVP) on CYP3A4 induction
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mediated by various CYP3A4 inducers. The inhibitory effects of NVP were estimated
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by the amount of production for 1-hydroxymidazolam. Experiments were carried out in
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triplicate and representative data of two separate experiments are shown. RIF,
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rifampicin; RFB, rifabutin; EFV, efavirenz; PB, phenobarbital; CMZ, carbamazepine.
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The numbers in parentheses represent the exposed micromolar concentrations for each
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CYP3A4 inducer.
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